Rheumatoid Arthritis: Methods and Protocols [2 ed.] 1071636812, 9781071636817

Table of contents :
Preface
Contents
Contributors
Part I: Animal Models
Chapter 1: Collagen-Induced Arthritis Models
1 Introduction
2 Materials
2.1 Emulsion Preparation
2.2 Animal Immunization
3 Methods
3.1 Emulsion Preparation (See Notes 3 and 4)
3.2 Animal Immunization (See Note 7) (Fig. 1)
4 Notes
References
Chapter 2: Human Xenograft Model
1 Introduction
2 Materials
3 Methods
3.1 Isolation of PBMC from Peripheral Blood from RA Patients
3.2 Trimming Explanted Joint Tissue
3.3 Implantation (See Notes 4 and 5)
3.4 Evaluation of Invasion of Synovium
4 Notes
References
Chapter 3: Scaffolded Chondrogenic Spheroid-Engrafted Model
1 Introduction
2 Materials
2.1 3D Culture of Chondrogenic Spheroids
2.2 Implantation In Vivo
3 Methods (See Note 4)
3.1 3D Culture of Chondrogenic Spheroids
3.2 Implantation In Vivo (See Note 7)
4 Notes
References
Chapter 4: Denervation-Induced Sarcopenia Model
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 5: Long-Term Constant Subcutaneous Drug Administration
1 Introduction
2 Materials (See Note 1)
3 Methods (See Note 3)
3.1 Filling of ALZET Pump (See Note 4)
3.2 Implantation
3.3 Explanting Pumps
4 Notes
References
Chapter 6: Clinical Scoring of Disease Activity in Animal Models
1 Introduction
2 Materials
2.1 CIA Scoring System
2.2 Evaluation of Paw Volume
3 Methods
3.1 CIA Scoring System
3.2 Evaluation of Paw Volume by In Vivo MRI Scanning (See Note 3)
4 Notes
References
Chapter 7: Histological Analyses of Arthritic Joints in Collagen-Induced Arthritis Model Mice
1 Introduction
2 Materials
2.1 Sampling (Fixation and Decalcification)
2.2 Embedding
2.3 Sectioning
2.4 Staining
2.5 HE Staining
2.6 TRAP Staining
2.7 IHC for CRACM3 Antigen
3 Methods
3.1 Fixation and Decalcification
3.2 Embedding
3.3 Sectioning
3.4 HE Staining
3.5 TRAP Staining
3.6 Immunohistochemistry (IHC)
3.7 Observation
4 Notes
References
Chapter 8: Preparation of Joint Extracts
1 Introduction
2 Materials (See Note 1)
3 Methods
3.1 Harvesting Synovium from Mouse Knee Joint
3.2 Protein Extraction (See Note 6)
4 Notes
References
Part II: Therapeutic Approach
Chapter 9: Production of Immunizing Antigen Proteoliposome Using Cell-Free Protein Synthesis System
1 Introduction
2 Materials
2.1 Construction of Transcription Templates for Cell-Free Protein Synthesis
2.2 Cell-Free Proteoliposome Synthesis Using Bilayer-Dialysis Method
2.3 Preparation of Adjuvant-Containing Liposome
2.4 Large-Scale Proteoliposome Antigen Production
3 Methods
3.1 Construction of Expression Plasmid for Cell-Free Protein Synthesis
3.2 Small-Scale Test Proteoliposome Synthesis Using Bilayer-Dialysis Method
3.3 Preparation of Adjuvant-Containing Liposome
3.4 Large-Scale Proteoliposome Antigen Production
4 Notes
References
Chapter 10: Reconstruction of Protein/Liposome Complex
1 Introduction
2 Materials
2.1 Cell-Free Protein Synthesis
2.2 Membrane Protein Solubilization
2.3 Reconstituting the Membrane Protein
2.4 Sucrose Density Gradient Centrifugation and SDS-PAGE
3 Methods
3.1 Cell-Free Protein Synthesis
3.2 Solubilizing HRH1
3.3 Preparation of Bio-beads SM-2
3.4 Reconstituting HRH1
3.5 Sucrose Density Gradient Centrifugation
3.6 SDS-PAGE
4 Notes
References
Chapter 11: Production of Neutralizing Antibody
1 Introduction
2 Materials
2.1 Antigen Preparation (See Note 1)
2.2 Mouse Immunization
2.3 Preparation for Fusion
2.4 ELISA for Screening
2.5 Clone Selection and Expansion of Positive Clones
2.6 Purification and Storage of mAbs
3 Methods
3.1 Antigen Preparation
3.1.1 Homogenization Methods
3.1.2 Syringe-to-Syringe Method (Fig. 2)
3.2 Mouse Immunization
3.3 Cell Fusion
3.4 Hybridoma Screening (ELISA)
3.5 Expanding the Hybridomas and Freezing Positive Clones
3.6 Purification and Storage of mAbs
4 Notes
References
Chapter 12: Autoantibody Profiling Using Human Autoantigen Protein Array and AlphaScreen
1 Introduction
2 Materials
2.1 Construction of Transcription Templates for Cell-Free Protein Synthesis
2.2 Cell-Free Synthesis of Autoantigen Protein Array
2.3 AlphaScreen
3 Methods
3.1 Construction of Transcription Templates for Cell-Free Protein Synthesis
3.2 Cell-Free Synthesis of Autoantigen Protein Array
3.3 AlphaScreen
4 Notes
References
Chapter 13: Generation of Specific Aptamers
1 Introduction
2 Materials
2.1 Oligonucleotide Library and Primers
2.2 Selection
2.3 Amplification
2.4 Purification of Single-Strand DNA (ssDNA)
3 Methods
3.1 DNA Selection from Oligonucleotide Library
3.2 Amplification
3.3 Preparation of ssDNA
4 Notes
References
Chapter 14: Detailed Protocol for Predicting 3D Structure of DNA Aptamers and Performing In Silico Docking Calculations
1 Introduction
2 Methods
2.1 Software Installation and Environment Setup
2.2 Sequence Input and Structure Prediction
2.2.1 Sequence Modification
2.2.2 3D Structure Prediction
2.2.3 Structure Minimization
2.2.4 Preparing for Docking with Chimera
2.3 Docking with Hdock
3 Limitations
References
Chapter 15: RNA Interference Ex Vivo
1 Introduction
2 Materials
2.1 Isolation of T Cells from Peripheral Blood
2.2 Transfection of siRNA into Primary T Cells Using Oligofectamine
2.3 Lentiviral-Mediated shRNA Transfection
3 Methods (See Note 2)
3.1 Isolation of T Cells from Peripheral Blood
3.2 Transfection of siRNA into Primary T Cells Using Oligofectamine
3.3 Lentiviral-Mediated shRNA Transfection (See Note 6)
4 Notes
References
Chapter 16: Lentiviral-Mediated Systemic RNA Interference In Vivo
1 Introduction
2 Materials
2.1 Lentiviral Titration Using Quantitative Real-Time PCR (qRT-PCR)-Based Methods
2.2 Intraperitoneal Injection of shRNA-Encoding Lentiviral Particles
2.3 Determination of Integrated Lentiviral Copies in Tissues
3 Methods (See Note 1)
3.1 Lentiviral Titration
3.2 Intraperitoneal Injection of shRNA-Encoding Lentiviral Particles (See Note 3)
3.3 Determination of Number of Integrated Provirus Copies in Tissue
4 Notes
References
Chapter 17: Lentiviral Production Platform
1 Introduction
2 Materials (See Note 1)
2.1 Production of Lentiviral Particles
2.2 Preparation of Concentrated Viral Stock
3 Methods (See Notes 5 and 6)
3.1 Production of Lentiviral Particles
3.2 Preparation of Concentrated Viral Stock
3.2.1 Ultracentrifugation
3.2.2 PEG Precipitation
4 Notes
References
Chapter 18: Mesenchymal Stem Cell Engineering
1 Introduction
2 Materials (See Note 1)
2.1 Isolation of Synovium-Derived MSC
2.2 Knockout of Target Gene Using Via CRISPR/Cas9 (See Note 3)
3 Methods
3.1 Isolation of MSC from Human-Derived Synovium
3.2 Knockout Genes at Chromosomal Level in MSC
4 Notes
References
Part III: Evaluation of Immunological Status
Chapter 19: Screening of Ca2+ Influx in Lymphocytes
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 20: Single-Cell Ca2+ Imaging
1 Introduction
2 Materials
3 Methods
3.1 Single-Cell Ca2+ Imaging
3.2 Image Analysis (See Note 12)
4 Notes
References
Chapter 21: Electrophysiological Methods to Measure Ca2+ Current
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 22: Evaluation of Mitochondrial Respiratory Function in Murine Splenocytes
1 Introduction
2 Materials
2.1 Hydrate Cartridge
2.2 Cell Preparation for Seahorse XF Cell Mito Stress Test
2.3 Running Mito Stress Test
3 Methods
3.1 Hydrate Cartridge
3.2 Cell Preparation for XFp Mito Stress Assay
3.3 Run XFp Mito Stress Assay
4 Notes
References
Chapter 23: The Functional Assessment of T Cells
1 Introduction
2 Materials
2.1 Analysis of Cell Surface Markers
2.1.1 Lymphoid Cell Preparation
2.1.2 Enrichment of T Cells
2.1.3 Detection of Cell Surface Markers by Flow Cytometer (FCM)
2.1.4 Detection of the Cytoplasmic Molecules by FCM
2.2 Analysis of the mRNA
2.2.1 RNA Preparation
2.2.2 cDNA Synthesis
2.2.3 PCR
2.3 Measurement of the Cytokines
2.3.1 In Vitro Stimulation of T Cells
2.3.2 Intracellular Cytokine Staining (See Also Subheading 2.1.4)
2.3.3 ELISA (Enzyme-Linked Immunosorbent Assay)
2.4 Measurement of the Cytotoxic Activity
2.4.1 Measurement of NK Activity (51Cr Release Assay)
2.4.2 Measurement of CTL Activity (Non-RI CTL Assay)
3 Methods
3.1 Analysis of the Cell Surface Markers
3.1.1 Lymphoid Cells
3.1.2 Preparation of Single-Cell Suspension of the Spleen and Lymph Nodes
3.1.3 Enrichment of T Cells by Panning Method
3.1.4 Enrichment of T Cells by Magnetic Beads Method and by the Cell Sorting with FCM
3.1.5 Detection of Cell Surface Markers by FCM with Direct Staining Method
3.1.6 Detection of Cell Surface Markers by FCM with Indirect Staining Method
3.1.7 Detection of the Cytoplasmic Molecules by FCM
3.2 Analysis of the mRNA
3.2.1 Preparation of RNA from Cells
3.2.2 DNaseI Treatment of Cellular RNA
3.2.3 cDNA Synthesis
3.2.4 PCR
3.3 Measurement of the Cytokines
3.3.1 In Vitro Stimulation of T Cells
3.3.2 Measure Cytokines by ELISA
3.3.3 Intracellular Cytokine Staining
3.3.4 PCR Analysis of the Transcript Prepared from the In Vitro Stimulated Cells
3.4 Measurement of the Cytotoxic Activity
3.4.1 51Cr Release Assay (Fig. 7)
51Cr Labeling of Target Cells
Cytotoxicity Assay
3.4.2 Non-RI CTL Assay (Fig. 8)
CFSE Labeling of Target Cells
Measurement of Cytotoxicity of Effector Cells
4 Notes
References
Chapter 24: Release of Antibodies and Cytokines from B Cells
1 Introduction
2 Materials
2.1 Measurement of Total IgG Level Using Enzyme-Linked Immunosorbent Assay (ELISA) (See Note 1)
2.2 Rapid Latex Test for RF
2.3 Preparation of Pan B Cells for Cytokine Release Assay
3 Methods
3.1 Measurement of Total IgG Level Using ELISA
3.2 Rapid Latex Test for RF (See Note 10)
3.3 Preparation of Pan B Cells for Cytokine Release Assay
4 Notes
References
Chapter 25: Evaluation of Autoreactive Responses
1 Introduction
2 Materials
2.1 Preparation of Single-Cell Suspension of CIA Mice-Derived Splenocytes
2.2 Assessment of T-Cell Responses to CII
3 Methods
3.1 Preparation of Single-Cell Suspension of CIA Mouse-Derived Splenocytes (See Note 3)
3.2 Assessment of T-Cell Responses to CII
4 Notes
References
Chapter 26: Macrophage Polarization and Osteoclast Differentiation
1 Introduction
2 Materials
2.1 Isolation of Bone Marrow-Derived Macrophages
2.2 M1/M2 Polarization
2.3 Cellular Metabolism Assay
2.4 Osteoclast Differentiation
2.5 TRAP Activity Assay
2.6 TRAP Staining
3 Methods
3.1 Isolation of Bone Marrow-Derived Macrophages
3.2 M1/M2 Polarization
3.2.1 Cellular Metabolism Assay (Preparation from the Day before to the Day of Assay)
3.2.2 Cellular Metabolism Assay (Glycolysis Stress Test)
3.2.3 Cellular Metabolism Assay (Mito Stress Test)
3.3 Osteoclast Differentiation
3.4 TRAP Activity Assay
3.5 TRAP Staining
4 Notes
References
Chapter 27: Scanning Electron Microscopic Analysis of the Bone-Resorption Activity in Mature Osteoclasts
1 Introduction
2 Materials
2.1 Isolation of Mature Osteoclasts Using Collagen Films (See Note 1)
2.2 Hematoxylin Staining
2.3 FE-SEM Observation
3 Methods
3.1 Isolation of Mature Osteoclasts Using Collagen Films
3.2 Hematoxylin Staining
3.3 FE-SEM Observation
4 Notes
References
Chapter 28: Animal Models of Vasculitis
1 Introduction
2 Materials
2.1 Candida albicans Water-Soluble Glycoprotein (CAWS)
2.2 Induction of CAWS-Induced Vasculitis
2.3 Immunohistochemical Staining for Cell Proliferation, Hypoxia, and Angiogenesis
2.4 Dihydroethidium Staining
2.5 Real-Time RT-PCR
2.6 Immunoblot Analysis
3 Methods
3.1 Preparation of CAWS
3.2 Induction of CAWS-Induced Vasculitis
3.3 Immunohistochemical Staining for Cell Proliferation, Hypoxia, and Angiogenesis
3.4 Dihydroethidium Staining
3.5 Real-Time RT-PCR
3.6 Immunoblot Analysis
4 Note
References
Chapter 29: Evaluation of Skin Damage Under UV Exposure
1 Introduction
2 Materials
2.1 UVA-Photoaging Model
2.2 Silicone Replica Method for Detection of Reticular Pattern
2.3 Preparation for Histological Analyses Using Paraffin Section
2.4 HE Staining
2.5 Fontana-Masson Staining
2.6 Immunostaining for Ki67
2.7 Preparation for Histological Analyses by Frozen Section
2.8 Detection for Collagen Degradation on Frozen Section
2.9 Detection of DNA Damage
3 Methods
3.1 UVA-Photoaging Model
3.2 To Detect Visible Changes in the Reticular Pattern on the Dorsal Skin Surface
3.3 To Detect Histological Changing of Dorsal Skin Tissue (Embedding and Sectioning Process)
3.4 HE Staining for Epidermal Hyperplasia Analysis Using Paraffin Section
3.5 Fontana-Masson Silver Staining for Melanin Pigmentation Analysis Using Paraffin Section
3.6 Ki67 Immunostaining for Detection of Proliferating Cells in the Epidermal Basal Layer Using Paraffin Section
3.7 To Detect Collagen Degradation in the Dermis Using a Frozen Section
3.8 To Detect DNA Damage by Oxidative Stress in Dorsal Skin Tissue
3.9 To Detect DNA Damage by Oxidative Stress Using Urine Samples
4 Notes
References
Chapter 30: Bulk RNA-seq Assessment of Murine Spleen Using a Portable MinION Sequencing Device
1 Introduction
2 Materials (See Note 1)
2.1 Total RNA Extraction (See Note 1)
2.2 Library Preparation for RNA-seq
2.3 Computer Environment
2.4 Software Installation
2.5 Reference Database
3 Methods
3.1 Total RNA Extraction from Spleen
3.2 Library Preparation for RNA-seq (See Note 6)
3.3 Establishment of Bioinformatic Pipeline
3.3.1 Basecalling with Guppy Basecaller
3.3.2 Concatenating FASTQ Files
3.3.3 Generating Reference Data
3.3.4 Mapping to Reference Genome with Minimap2
3.3.5 SAM to BAM Conversion, Sorting, and Indexing
3.3.6 Counting Features with FeatureCounts
3.3.7 Data Frame Creation for Downstream Analysis
4 Notes
References
Part IV: Clinical Approach
Chapter 31: Institutional Review Board Considerations for Clinical Trials
1 Introduction of IRBs
2 How to Apply to IRB
2.1 Determine If Project Requires IRB Approval
2.2 Complete IRB Research Project Application
2.3 Prepare Informed Consent Document(s)
2.4 Submit Proposal Form
2.5 Make Adjustments as Necessitated by IRB Review Until Approved
2.6 Report Changes and Annual Renewal Authorization (If Needed)
3 Notes
Reference
Chapter 32: Design an Intervention Study
1 Introduction
2 Methods
2.1 Choice of Intervention and Control
2.2 Selection of Subjects
2.3 Informed Consent
2.4 Baseline Measurement
2.5 Bank Specimens
2.6 Randomized Allocation
2.7 Blinding
2.8 Outcome Measurements
2.8.1 Efficacy Outcomes
2.8.2 Safety Outcomes
3 Notes
References
Chapter 33: Assessment of Disease Activity, Structural Damage, and Function in Rheumatoid Arthritis
1 Introduction
2 Methods
2.1 Assessment of Disease Activity
2.1.1 Disease Activity Score in 28 Joints (DAS28) (See Note 1)
2.1.2 Simplified Disease Activity Index (SDAI) (See Note 2)
2.1.3 Clinical Disease Activity Index (CDAI) (See Note 3)
2.1.4 American College of Rheumatology (ACR) Core Set
2.1.5 2022 ACR/European Alliance of Associations for Rheumatology (EULAR) Remission Criteria
2.2 Functional Assessment
2.2.1 Health Assessment Questionnaire-Disability Index (HAQ-DI) (See Note 4)
2.2.2 Short Form-36 (SF-36) (See Note 5)
2.2.3 European Quality of Life-5 Dimensions (EQ-5D-5L) (See Note 6)
2.3 Assessment of Structural Damage of Joints
2.3.1 The van der Heijde Modification of the Sharp Method (the Sharp/van der Heijde Method)
2.3.2 The Larsen Method
3 Notes
References
Chapter 34: Assessment of Musculoskeletal Ultrasound of Rheumatoid Arthritis
1 Introduction
2 Methods
2.1 MSUS Findings
2.1.1 Synovitis
2.1.2 Bone Erosion
2.1.3 Tenosynovitis
2.2 Positioning of MSUS in the Management of RA
2.2.1 Early Diagnosis
2.2.2 Assessment of Disease Activity
2.2.3 Definition of Remission
2.2.4 Treatment Strategy Utilizing MSUS
3 Notes
References
Chapter 35: 16S rRNA Gene Amplicon Analysis of Human Gut Microbiota
1 Introduction
2 Materials
2.1 Stool Sampling (See Note 1)
2.2 DNA Isolation
2.3 Library Preparation
2.4 Sequencing
3 Methods
3.1 Stool Sampling
3.2 DNA Isolation
3.3 Library Preparation
3.4 Sequencing
4 Notes
References
Index

Citation preview

Methods in Molecular Biology 2766

Shuang Liu  Editor

Rheumatoid Arthritis Methods and Protocols Second Edition

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-bystep fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

Rheumatoid Arthritis Methods and Protocols Second Edition

Edited by

Shuang Liu Department of Pharmacology, Ehime University School of Medicine, Toon, Ehime, Japan

Editor Shuang Liu Department of Pharmacology Ehime University School of Medicine Toon, Ehime, Japan

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-3681-7 ISBN 978-1-0716-3682-4 (eBook) https://doi.org/10.1007/978-1-0716-3682-4 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A. Paper in this product is recyclable.

Preface Welcome to the second edition of Rheumatoid Arthritis: Methods and Protocols. This comprehensive volume represents a testament to the ever-evolving field of rheumatoid arthritis (RA) research, where groundbreaking advances continue to emerge on multiple fronts. As we embark on this journey through the labyrinthine world of RA investigation, we find ourselves at the forefront of a scientific endeavor that holds the promise of transforming the lives of millions afflicted by this debilitating autoimmune disease. Rheumatoid arthritis is a complex, systemic autoimmune disorder characterized by chronic inflammation, joint destruction, and a wide spectrum of associated comorbidities. Despite the significant progress made in understanding the pathogenesis and treatment of RA in recent years, there is an unceasing need for innovative approaches, methodologies, and therapies to further enhance our understanding and management of this disease. This second edition builds upon the foundations laid by its predecessor, expanding and refining the content to encompass the most cutting-edge laboratory and clinical protocols employed in RA research today. The book’s contents have been meticulously curated to provide a comprehensive roadmap for researchers, clinicians, and students who aspire to delve deep into the intricate world of rheumatoid arthritis. In this edition, we examine the multifaceted aspects of RA research, covering a wide range of methodologies and techniques. We explore the utilization of cell culture systems to decipher the intricate molecular pathways underlying RA pathogenesis. We navigate through the intricate realm of animal models that mirror the disease’s complexity and provide invaluable insights for translational research. Genetic modification techniques unveil the genetic underpinnings of RA susceptibility and offer potential avenues for therapeutic intervention. Furthermore, we delve into the development of novel therapeutics, including the promising realms of aptamers and antibodies. In silico docking and bioinformatics methods offer a computational approach to drug discovery, saving time and resources in the quest for effective treatments. We dive deep into the world of RNA sequencing, exploring bioinformatics techniques to unlock the secrets hidden within the vast transcriptomic landscape of RA. Finally, we navigate the challenging waters of clinical research, where the knowledge gleaned from laboratories meets the real-world complexities of patient care. We explore the latest clinical study designs, patient-centered approaches, and emerging trends in RA management. This second edition of Rheumatoid Arthritis: Methods and Protocols stands as a testament to the dedication and collaborative efforts of researchers worldwide. It is our hope that this book will serve as an indispensable resource, guiding you through the ever-evolving landscape of RA research and facilitating breakthroughs that will ultimately improve the lives of individuals living with this chronic condition.

v

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Preface

We extend our heartfelt gratitude to the authors, contributors, and the scientific community for their unwavering commitment to advancing our understanding of rheumatoid arthritis. May this book inspire and empower the next generation of researchers to continue pushing the boundaries of knowledge and innovation in the quest to conquer this challenging autoimmune disease. Toon, Ehime, Japan

Shuang Liu

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PART I

ANIMAL MODELS

1 Collagen-Induced Arthritis Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Maya Miyoshi and Shuang Liu 2 Human Xenograft Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Shuang Liu 3 Scaffolded Chondrogenic Spheroid-Engrafted Model . . . . . . . . . . . . . . . . . . . . . . . Shuang Liu 4 Denervation-Induced Sarcopenia Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Erika Takemasa and Shuang Liu 5 Long-Term Constant Subcutaneous Drug Administration . . . . . . . . . . . . . . . . . . . Shuang Liu and Maya Miyoshi 6 Clinical Scoring of Disease Activity in Animal Models . . . . . . . . . . . . . . . . . . . . . . . Maya Miyoshi and Shuang Liu 7 Histological Analyses of Arthritic Joints in Collagen-Induced Arthritis Model Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Takeshi Kiyoi 8 Preparation of Joint Extracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Shuang Liu and Erika Takemasa

PART II

v xi

3 9 17 25 31 37

43 55

THERAPEUTIC APPROACH

9 Production of Immunizing Antigen Proteoliposome Using Cell-Free Protein Synthesis System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Wei Zhou and Hiroyuki Takeda 10 Reconstruction of Protein/Liposome Complex. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Yasuyuki Suzuki 11 Production of Neutralizing Antibody. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Erika Takemasa and Shuang Liu 12 Autoantibody Profiling Using Human Autoantigen Protein Array and AlphaScreen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Hiroyuki Takeda 13 Generation of Specific Aptamers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Shuang Liu, Yasuyuki Suzuki, and Makoto Inui 14 Detailed Protocol for Predicting 3D Structure of DNA Aptamers and Performing In Silico Docking Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Yasuyuki Suzuki

vii

viii

Contents

15

RNA Interference Ex Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Shuang Liu 16 Lentiviral-Mediated Systemic RNA Interference In Vivo. . . . . . . . . . . . . . . . . . . . . Shuang Liu 17 Lentiviral Production Platform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Shuang Liu 18 Mesenchymal Stem Cell Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Shuang Liu

PART III 19 20 21 22 23 24 25 26 27

28 29 30

31 32

153 163 169

EVALUATION OF IMMUNOLOGICAL STATUS

Screening of Ca2+ Influx in Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Erika Takemasa and Shuang Liu Single-Cell Ca2+ Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Shuang Liu Electrophysiological Methods to Measure Ca2+ Current . . . . . . . . . . . . . . . . . . . . . Shuang Liu Evaluation of Mitochondrial Respiratory Function in Murine Splenocytes . . . . . Mochitsuki Marii and Shuang Liu The Functional Assessment of T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Saho Maruyama Release of Antibodies and Cytokines from B Cells . . . . . . . . . . . . . . . . . . . . . . . . . . Shuang Liu Evaluation of Autoreactive Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Shuang Liu Macrophage Polarization and Osteoclast Differentiation . . . . . . . . . . . . . . . . . . . . . Noritaka Saeki and Akihiro Nakata Scanning Electron Microscopic Analysis of the Bone-Resorption Activity in Mature Osteoclasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Takeshi Kiyoi Animal Models of Vasculitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Masaki Mogi and Shuang Liu Evaluation of Skin Damage Under UV Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . Takeshi Kiyoi Bulk RNA-seq Assessment of Murine Spleen Using a Portable MinION Sequencing Device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yasuyuki Suzuki and Shuang Liu

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177 183 191 199 207 233 241 247

263 271 281

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CLINICAL APPROACH

Institutional Review Board Considerations for Clinical Trials . . . . . . . . . . . . . . . . . 311 Masaki Mogi and Shuang Liu Design an Intervention Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317 Jun Ishizaki and Hitoshi Hasegawa

Contents

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Assessment of Disease Activity, Structural Damage, and Function in Rheumatoid Arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 Jun Ishizaki and Hitoshi Hasegawa Assessment of Musculoskeletal Ultrasound of Rheumatoid Arthritis. . . . . . . . . . . 335 Jun Ishizaki 16S rRNA Gene Amplicon Analysis of Human Gut Microbiota. . . . . . . . . . . . . . . 343 Noriyuki Miyaue

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors HITOSHI HASEGAWA • Department of Hematology, Clinical Immunology, and Infectious Diseases, Ehime University Graduate School of Medicine, Toon, Ehime, Japan MAKOTO INUI • Department of Pharmacology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan JUN ISHIZAKI • Department of Hematology, Clinical Immunology and Infectious Diseases, Ehime University Graduate School of Medicine, Toon, Ehime, Japan TAKESHI KIYOI • Division of Analytical Bio-medicine, Department of Pharmacology, Kanazawa Medical University, Kahoku, Japan SHUANG LIU • Department of Pharmacology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan MOCHITSUKI MARII • Department of Pharmacology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan SAHO MARUYAMA • Department of Basic Medical Research and Education, Ehime University Graduate School of Medicine, Toon, Ehime, Japan NORIYUKI MIYAUE • Department of Clinical Pharmacology and Therapeutics, Ehime University Graduate School of Medicine, Toon, Ehime, Japan MAYA MIYOSHI • Department of Pharmacology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan MASAKI MOGI • Department of Pharmacology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan AKIHIRO NAKATA • Department of Pathophysiology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan NORITAKA SAEKI • Division of Medical Research Support, Advanced Research Support Center, Ehime University, Toon, Ehime, Japan; Division of Integrative Pathophysiology, ProteoScience Center, Ehime University, Toon, Ehime, Japan YASUYUKI SUZUKI • Department of Anaesthesiology, Saiseikai Matsuyama Hospital Matsuyama, Ehime, Japan; Department of Pharmacology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan; Research Division, Saiseikai Research Institute of Health Care and Welfare, Tokyo, Japan HIROYUKI TAKEDA • Proteo-Science Center, Ehime University Matsuyama, Ehime, Japan ERIKA TAKEMASA • Department of Pharmacology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan WEI ZHOU • Proteo-Science Center, Ehime University Matsuyama, Ehime, Japan

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Part I Animal Models

Chapter 1 Collagen-Induced Arthritis Models Maya Miyoshi and Shuang Liu Abstract Due to the limitations of using patient-derived samples for systemic kinetic studies in rheumatoid arthritis (RA) research, animal models are helpful for further understanding the pathophysiology of RA and seeking potential therapeutic targets or strategies. The collagen-induced arthritis (CIA) model is one of the standard RA models used in preclinical research. The CIA model shares several pathological features with RA, such as breach of tolerance and generation of autoantibodies targeting collagen, synovial inflammatory cell infiltration, synovial hyperplasia, cartilage destruction, and bone erosion. In this chapter, a protocol for the successful induction of CIA in mice is described. In this protocol, CIA is induced by active immunization by inoculation with type II heterologous collagen in Freund’s adjuvant in susceptible DBA/1 mice. Key words Collagen-induced arthritis, Freund’s adjuvant, Type II collagen, Emulsion, Immunization

1

Introduction Rheumatoid arthritis (RA) is a chronic inflammatory disease that initially affects the joints, manifesting as pain, stiffness, and synovitis, leading to cartilage and bone erosion by invading fibrovascular tissue [1]. The central pathogenesis of RA is characterized by the activation of macrophages by autoreactive T cells, resulting in the release of a series of proinflammatory cytokines. However, how the systemic chronic inflammatory state triggers the onset of articular disorder is still poorly understood [2]. To further define the pathogenesis of RA, it is helpful to study human-derived cells and explanted tissues from patients who have undergone arthroscopic surgery or prosthetic replacement arthroplasty. However, this has significant limitations for systemic kinetic studies. Therefore, animal models are not only essential to facilitate understanding of the pathophysiology of RA and seek potential therapeutic targets or strategies but are also the starting point for in vivo application of new therapeutic agents. Based on the methods of induction, systemically induced models include those elicited by active immunization, such as

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collagen-induced arthritis model and proteoglycan-induced arthritis model, those elicited by passive immunization, such as collagen antibody-induced arthritis model and K/BxN antibodyinduced arthritis model, and those elicited by administration of irritant chemicals resulting in chronic inflammation [1, 3]. Each animal model is only an experimental tool that mimics a part of the disease and cannot reproduce the entire condition of human RA. The choice of model depends on the phase of the disease to be studied and the question to be addressed. The collagen-induced arthritis (CIA) model is a long-lasting and well-explored mouse model for RA. CIA and RA initial similarity in breach of tolerance and generation of autoantibodies targeting collagen, one of the important self-antigens that are also observed in human RA [2, 4]. CIA is induced by active immunization by inoculation with type II heterologous collagen (CII) in Freund’s adjuvant in susceptible strains of mice. DBA/1 mice are commonly used for the CIA model. The model requires at least 6–8 weeks for the accomplishment of clinical signs of disease, such as polyarthritis characterized by synovial inflammatory cell infiltration, synovial hyperplasia, cartilage destruction, and bone erosion [5, 6]. The autoreactive antibody observed in CIA mice is predominately IgG2 subclass, and high levels of both IgG2a and IgG2b are observed at the peak of CIA. Typical cytokine axes involving in human RA pathogenies, such as proinflammatory type 1 T help (Th1) cell-axis, anti-inflammatory cytokine interleukin (IL)-10 axis, and Th17 cell-axis, can be investigated using the CIA model [6, 7]. These characteristics of the CIA model make it the gold standard in vivo model for RA studies. In this chapter, a protocol for the successful induction of CIA in mice is described. Like any other antigen-induced model, certain technical skills and stable environmental factors are required. The highest arthritis incidence is obtained if the emulsion is correctly performed using bioactivity-qualified CII and appropriate intradermal immunization is performed.

2

Materials

2.1 Emulsion Preparation

1. Type 2 collagen (2 mg/mL, immunization grade) (see Note 1). 2. Incomplete Freund’s adjuvant (IFA). 3. Complete Freund’s adjuvant (CFA). 4. Glass syringes without needles (1 mL) (Hamilton). 5. Electronic homogenizer with a small blade (diameter of 5 mm or less). 6. T-shape stopcock. 7. 5-mL and 10-mL disposable plastic syringes.

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5

1. DBA/1 mice (male, 8–10 weeks old) (see Note 2). 2. 70% ethanol. 3. CII emulsion (CFA)/CII emulsion (IFA). 4. 25 and 27 gauge × 5/8″ needles.

3

Methods

3.1 Emulsion Preparation (See Notes 3 and 4)

1. Fill glass syringes with 500 μL of CFA (IFA for booster injection) and 500 μL of immunization grade CII, respectively. 2. Seal the tips of both syringes with a T-shape stopcock. 3. Connect the rest of the connector of the T-shape stopcock with a 5-mL or 10-mL plastic syringe without a plunger and cut halfway from the plunger opening. 4. Push the plunger of the glass syringes and let CFA (IFA for booster injection) and CII solution mix in the plastic syringe. Air bubbles should be avoided during solution mixing. 5. After sealing the plastic syringe with the T-shape stopcock, take off the glass syringes. 6. Clamp the syringe to a ring stand and place it in an ice water bath to keep the emulsion cool during mixing. 7. Homogenize the mixture to emulsify CFA (IFA for booster injection) with the collagen solution until the emulsion is stable (see Note 5). 8. Transfer the emulsion to a 1-mL glass syringe for animal injection (see Note 6). The prepared emulsion should be injected into animals as soon as possible (within 1 h). Keep the emulsion cool at 4 °C until use.

3.2 Animal Immunization (See Note 7) (Fig. 1)

1. DBA/1 mice are used for induction of CIA. Primary intradermal injection of CII and CFA emulsion is performed at a site 2 cm distal to the base of the tail on day 0. 2. Use a squirt bottle to apply 70% ethanol to the injection site and wipe with tissue. 3. Place a 25- or 27-gauge needle on the glass syringe. Wipe the needle to prevent leakage of emulsion. 4. Inject 100 μL (100 μg CII/ mouse) of CII and CFA emulsion intradermally at the base of the tail, with noticeable tissue resistance to the injection (see Note 8). 5. Put the mouse in a clean cage, and house the mice in specific pathogen-free (SPF) conditions.

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Fig. 1 Typical appearances of hindpaws of (a) non-arthritis control mouse and (b) collagen-induced arthritis (CIA) mouse. Erythema and edema are observed in CIA mouse

6. Administer a booster injection of emulsion of CII and IFA on day 21. The injection site is about 3 cm from the base of the tail. Choose a different location from the initial injection site. 7. Use a squirt bottle to apply 70% ethanol to the injection site and wipe with tissue. 8. Place a 25- or 27-gauge needle on the glass syringe. Wipe the needle to prevent leakage of emulsion. 9. Insert the needle 3 cm from the base of the tail until the tip reaches 1.5 cm from the base. Inject 100 μL (100 μg CII/ mouse) of CII and IFA emulsion intradermally at the base of the tail, with noticeable tissue resistance to the injection. 10. Put the mouse in a clean cage and house the mice in SPF conditions. The incidence of CIA should be 90–100% at 42–56 days. The CIA mice are ready for evaluation of arthritis severity.

4

Notes 1. Immunization-grade CII should be solubilized and stored in a diluted solution of acetic acid. 2. DBA/a (H-2q) and B10.RIII(H-2r) are highly susceptible to CIA. DBA/I mice respond to chick, bovine, and porcine type II collagen, while B10.RIII mice respond to bovine and porcine collagen, but poorly respond to chick and human collagen. 3. The procedures for emulsion preparation should be performed under sterile conditions. 4. A method using an electric homogenizer is highly recommended for preparing emulsion. Do not use syringe–syringe or sonication methods in the establishment of CIA.

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5. The highest arthritis incidence is obtained if the emulsion is correctly performed so that it has a consistency of dense whipped cream and it should not disperse quickly when a droplet of emulsion is placed on the surface of water. 6. It is sometimes difficult to move the plunger when a plastic disposable syringe is used. 7. The animal experiment protocols should be performed in accordance with the guidelines of the Animal Care Committee of the institute. 8. If the injection is rapid and easy without tissue resistance, it can result in a low incidence of CIA. References 1. Liu S, Kiyoi T, Takemasa E, Maeyama K (2015) Systemic lentivirus-mediated delivery of short hairpin RNA targeting calcium release-activated calcium channel 3 as gene therapy for collageninduced arthritis. J Immunol 194:76–83 2. McInnes IB, Schett G (2011) The pathogenesis of rheumatoid arthritis. N Engl J Med 365: 2205–2219 3. Bessis N, Decker P, Assier E, Semerano L, Boissier MC (2017) Arthritis models: usefulness and interpretation. Semin Immunopathol 39:469– 486 4. Trentham DE (1982) Collagen arthritis as a relevant model for rheumatoid arthritis. Arthritis Rheum 25:911–916

5. Caplazi P, Baca M, Barck K, Carano RA, DeVoss J, Lee WP et al (2015) Mouse models of rheumatoid arthritis. Vet Pathol 52:819–826 6. Miyoshi M, Liu S, Morizane A, Takemasa E, Suzuki Y, Kiyoi T et al (2018) Efficacy of constant long-term delivery of YM-58483 for the treatment of rheumatoid arthritis. Eur J Pharmacol 824:89–98 7. Mauri C, Williams RO, Walmsley M, Feldmann M (1996) Relationship between Th1/Th2 cytokine patterns and the arthritogenic response in collagen-induced arthritis. Eur J Immunol 26: 1511–1518

Chapter 2 Human Xenograft Model Shuang Liu Abstract Human-SCID grafting is a commonly used technique for the long-term investigation of rheumatoid arthritis (RA) explants. To establish a chimeric immunological system in NOD/SCID mice, RA patientderived pannus tissue from the synovial membrane, articular cartilage, and bone can be transplanted subcutaneously. Same patient-derived peripheral mononuclear cell chimerism can be successfully achieved by intraperitoneal engraftment. This xenograft model is able to be used for the initial screening of human target-specified biologics. Key words Xenograft rheumatoid arthritis model, NOD/SCID mouse, Peripheral mononuclear cell, Articular tissue, Synovial invasion

1

Introduction Several animal models, including antigen-induced models, such as collagen-induced arthritis, and spontaneous models, such as TNF-α transgenic mice and SKG mice, have been developed for the study of rheumatoid arthritis (RA). However, these models are not able to be used for in vivo screening of human target-specified biologics, especially for chimeric, humanized, and human-type monoclonal antibody or gene therapeutic products, which have been widely studied for clinical treatment of RA patients. Therefore, a xenograft model, in which human-derived explants are transplanted to a severe combined immunodeficiency (SCID) mouse, has been established for human target-specified biologics screening. It was first reported that RA synovial tissue could be transplanted into SCID mice, and this animal model was useful for studying the pathogenesis of RA and the development of antirheumatic drugs in the early 1990s [1]. The initial studies were conducted on small pieces of synovium transplanted beneath the renal capsule in the mice. The maintenance of human-derived lymphocytes was poor, and usage of the model was limited. Next,

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a challenge approach in which transplantation was changed to subcutaneous tissue on the back of SCID was conducted. In this model, tissue with a relatively large size, such as pannus tissue from the synovial membrane, articular cartilage, and bone, collected together from RA patients at the time of prosthetic replacement arthroplasty, was used for transplantation [2, 3]. The histologic features of human RA, such as pannus formation, proliferative synovial fibroblasts, osteoclasts and hyaluronic acid-positive articular cartilage, were observed. Based on the technique of the subcutaneous xenograft model, a chimeric human–mouse model was established using NOD/SCID mice, which are characterized by the absence of functional T cells and B cells, deficient NK function, lymphopenia, hypogammaglobulinemia, and a normal hematopoietic microenvironment. Patient-derived synovial tissue, bone, and articular cartilage were xenografted into NOD/SCID mice. To mimic the supporting inflammatory microenvironment of RA, peripheral blood mononuclear cells (PBMCs) derived from joint engrafts of the same patients were suspended in serum and engrafted into NOD/SCID mice [4]. Human multilineage hematocytes, including T lymphocytes, B lymphocytes, monocytes, myeloid maturation stages, and primitive progenitor cells, were sustained in xenografted mice for at least 8 weeks. Human rheumatoid factor was detected in the serum of xenografted mice, and invasion of synovium into the implanted cartilage was able to be scored. In this model, the maintenance of an inflammatory microenvironment is successfully achieved as a critical supportive factor for synovial invasion into cartilage.

2

Materials To obtain explants from RA patients, research protocols should be approved by the Institutional Ethics Committee. All animal experiment protocols should be performed in accordance with the guidelines of the Institutional Animal Care and Use Committee and approved by the committee. 1. Animals: Male NOD/ShiJic-scid (NOD/SCID) mice, 6–10 weeks of age, are used for xenograft experiments (see Note 1). 2. Explants from RA patients: Peripheral blood (20 mL), synovium, bone, and articular cartilage explants can be obtained from RA patients who have undergone prosthetic replacement arthroplasty for therapeutic purposes. All explants should be transferred between institutions or units in a biohazard and cooling container. Explants should be handled for the xenograft procedure as soon as possible after explantation.

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3. Inhalation anesthesia unit (see Note 2). 4. Centrifuges and centrifuge tubes. 5. Operating table. 6. Warming plate or heating pad. 7. Forceps (fine blunt) and scissors (fine dissection). 8. Syringes, 1 mL. 9. Wound clips and applier. 10. Pipettes and chips. 11. 70% ethanol. 12. Isoflurane or other anesthetics. 13. Histopaque 1077. 14. Cell suspension buffer: Phosphate-buffered saline (PBS), pH 7.2, and 2 mM EDTA. Sterilize the buffer by membrane filtration and keep it cold (2–8°C).

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Methods

3.1 Isolation of PBMC from Peripheral Blood from RA Patients

1. For serum collection, collect 2 mL of whole blood into a regular 1.5-mL Eppendorf tube and centrifuge the sample for 15 min at 1500× g at 4 °C. Harvested serum is ready for PBMC suspension. 2. Dilute the remaining whole blood with the same volume of cell suspension buffer. 3. Carefully layer 35 mL of diluted whole blood over 15 mL of Histopaque 1077 in a 50-mL conical tube. 4. Centrifuge at 400× g for 30 min at 20 °C in a swinging bucket rotor without a brake. 5. Harvest the mononuclear cell layer undisturbed at the interphase and carefully transfer the mononuclear cell layer to a new 50-mL conical tube. 6. Fill the conical tube with cell suspension buffer and mix gently. Centrifuge the tube at 300× g for 10 min at 20 °C and carefully remove the supernatant completely. 7. Wash the cells with cell suspension buffer and centrifuge the tube at 300× g for 10 min at 20 °C. Carefully remove the supernatant completely. 8. Resuspend PBMC (1 × 107) using 200 μL of the same patientderived serum for further transplantation.

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3.2 Trimming Explanted Joint Tissue

1. Keep the explants in saline-wet gauze at 4 °C and use as soon as possible. 2. Trim the explanted synovium and cartilage with bone to a block about 4–6 mm in diameter prior to implantation (see Note 3).

3.3 Implantation (See Notes 4 and 5)

1. Put NOD/SCID mice in an anesthetic induction chamber. Initial induction can be performed using 2.5% isoflurane vaporized in 100% medical oxygen. Following induction, anesthesia should be maintained by placing the mice in front of a small face mask connected to the anesthetic machine using 1% isoflurane vaporized in 100% medical oxygen. 2. Weigh and put mice on the operating table. Place the mouse on its abdomen to expose the back. Shave the back. Use a squirt bottle to apply 70% ethanol to the back and wipe with tissue. 3. Cut the skin with fine dissection scissors, making a 1-cm vertical incision at a point level of the fourth to sixth lumbar vertebrae. 4. After exposing the subcutaneous tissue, the oblique external abdominal muscle is scraped with a scalpel until it bleeds. 5. Put the trimmed RA patient-derived synovium on the oblique external abdominal muscle, and let the connective tissue site of synovium attach to the bleeding muscle. 6. Put the articular cartilage and bone on the synovium (see Note 6) and let the smooth surface of the cartilage touch the articular luminal side of the synovium. 7. Clip the skin together with wound clips or sew up with two or three stiches. Clean the wound with 70% ethanol. 8. Inject serum-suspended PBMC (200 μL), prepared as described in Subheading 3.1, intraperitoneally (see Note 7). 9. At the end of the procedure, put the mouse in a clean cage and place the cage on a warming plate until the mouse recovers from the anesthetic.

3.4 Evaluation of Invasion of Synovium

1. Anesthetize the xenografted mice at 6–8 weeks after transplantation (see Note 8). 2. Remove the implanted tissues from xenografted mice, and immerse in 4% paraformaldehyde for tissue fixation. Decalcify and embed the tissues (the protocol can be found in Chap. 5). The sections should be stained with the methods as desired (e.g., hematoxylin and eosin) (Fig. 1). 3. For semi-quantification of synovial invasion into cartilage and bone tissues, sections can be scored from 0 to 4 based on the

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Fig. 1 Histological analysis of implants in xenografted mice. The engrafted tissues were explanted at 8 weeks after transplantation. Following fixation and decalcification, tissue sections were stained using hematoxylin and eosin (original magnification, ×400). Arrows indicate invasion of synovium into implanted cartilage. S synovium, C cartilage

number of invading cell layers and number of invasive sites [4, 5], as follows (see Note 9): • 0: no or minimal invasion • 0.5: invasion of 1–2 cells at three independent cartilage sites • 1: invasion of 3–5 cell layers • 1.5: invasion of 3–5 layers at three independent cartilage sites • 2: invasion of 6–10 cell layers • 2.5: invasion of 6–10 layers at three independent cartilage sites • 3: invasion of >10 cell layers • 3.5: invasion of >10 layers at two independent cartilage sites • 4.5: invasion of >10 layers at three or more independent cartilage sites

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Notes 1. NOD/SCID mice are characterized by the absence of functional T cells and B cells, deficient natural killer cells, lymphopenia, hypergammaglobulinemia, and a normal hemotopoietic microenvironment. To avoid any unexpected complications, the age of NOD/SCID mice used in the xenograft model should be under 10 weeks. Due to their severely immunocompromised state, NOD/SCID mice should be housed in maximum-barrier facilities. Below are the conditions that we recommend for housing NOD/SCID mice: • Use microisolator (filter bonneted) or pressurized, individually ventilated cages (PIV/IVC). • Sterilize or disinfect food, water, bedding, cages, and anything that will come in contact with the mice. • Only personnel involved in the care of the mice should have access to the mouse room, and caretakers should wear personal protective equipment. • Before accessing the housing room, operators or caretakers should pass the air shower unit. • Cages should be changed under a laminar flow hood. Change cages weekly to prevent the introduction of minimum-inoculating doses of opportunistic or commensal organisms into the cage environment. 2. All equipment should be used under sterile conditions. 3. For the evaluation of invasion of synovium, cartilage and bone with normal appearance rather than the lesion site should be chosen. 4. Implantation should be carried out under sterile conditions. 5. NOD/SCID mice could be pretreated by a single intraperitoneal cavity injection of 50 μL anti-asialo-GM1 serum to deplete natural killer cells 1 day before performing xenografting. In our experience, NOD/SCID mice can tolerate the engrafting procedure without any pretreatment. 6. Articular cartilage is always explanted with the bone beneath the cartilage. 7. PBMC can be engrafted by a single injection into the intraperitoneal cavity, intravenous, or intrasplenic injection [6]. The highest amount of human PBMC chimerism can be achieved by intrasplenic injection in NOD/SCID mice. Chimerism of human PBMC is poor using intravenous injection. Considering adverse effects, we chose intraperitoneal injection for engrafting human PBMC.

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8. The optimal timing of explantation is strain- and treatmentdependent. A pilot study is required for optimizing the end point of engrafting. 9. Quantification should be carried out on five high-power fields in each section and three sections for each specimen.

Acknowledgment This work was supported by a Japan Society for the Promotion of Science KAKEMHI Grant (15K19575). References 1. Rendt KE, Barry TS, Jones DM, Richter CB, McCachren SS, Haynes BF (1993) Engraftment of human synovium into severe combined immune deficient mice. Migration of human peripheral blood T cells to engrafted human synovium and to mouse lymph nodes. J Immunol 151:7324–7336 2. Matsuno H, Yudoh K, Uzuki M, Kimura T (2001) The SCID-HuRAg mouse as a model for rheumatoid arthritis. Mod Rheumatol 11: 6–9 3. Sakuraba K, Fujimura K, Nakashima Y, Okazaki K, Fukushi J, Ohishi M et al (2015) Brief report: successful in vitro culture of rheumatoid arthritis synovial tissue explants at the air-liquid interface. Arthritis Rheum 67:887– 892

4. Liu S, Hasegawa H, Takemasa E, Suzuki Y, Oka K, Kiyoi T et al (2017) Efficiency and safety of CRAC inhibitors in human rheumatoid arthritis xenograft models. J Immunol 199: 1584–1595 5. Maeshima K, Yamaoka K, Kubo S, Nakano K, Iwata S, Saito K et al (2012) The JAK inhibitor tofacitinib regulates synovitis through inhibition of interferon-gamma and interleukin-17 production by human CD4+ T cells. Arthritis Rheum 64:1790–1798 6. Zhou W, Ohdan H, Tanaka Y, Hara H, Tokita D, Onoe T et al (2003) NOD/SCID mice engrafted with human peripheral blood lymphocytes can be a model for investigating B cells responding to blood group A carbohydrate determinant. Transpl Immunol 12:9–18

Chapter 3 Scaffolded Chondrogenic Spheroid-Engrafted Model Shuang Liu Abstract Therapeutic approaches using mesenchymal stem cells (MSCs) for a cartilage regeneration strategy are based on their multipotent differentiation for skeletal regeneration. With the utilization of allergenic neutralized type I atelocollagen during the pre-formation of chondrogenic MSC spheroids, cellular condensation and chondrogenic differentiation can be easily achieved. It also benefits the recruitment of host MSCs, which differentiate into chondrocyte-like cells after implantation into the experiment model. Using pre-formed chondrogenic MSC spheroids, the efficacy of anti-rheumatoid agents for cartilage repair can be screened on a large scale ex vivo. Furthermore, atelocollagen-scaffolded chondrogenic spheroids can be utilized for in vivo transplantation into a humanized xenografted arthritis model. Thus, the ability of cartilage self-repair can be qualitatively and quantitatively evaluated. Key words Mesenchymal stem cell, Cartilage regeneration, Atelocollagen scaffold, Xenograft model, Chondrogenic spheroid

1

Introduction Because cartilage destruction directly causes joint pain and functional disability, the basic strategy of rheumatoid arthritis (RA) management is to alter the systemic immune status to prevent or delay cartilage destruction. After a prolonged period of time, cartilage and bone damage is almost irreversible, and very limited options remain other than considering prosthetic replacement arthroplasty. Since the regenerative capacity of cartilage is limited due to the slow metabolic rate of chondrocytes, lack of vascularity, and limitation of the number of progenitor cells, how to repair existing damage of cartilage has been challenging [1]. Autologous cellular implantation, including the employment of chondrocytes or multipotency mesenchymal stem cells (MSC), has emerged for joint regeneration [2, 3]. Therapeutic approaches using MSC for a regeneration strategy are based on their immunomodulatory capabilities to achieve systemic immunosuppression and multipotent differentiation for

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skeletal regeneration [4]. Human MSC utilized for cartilage regeneration can be obtained from many types of tissues, including bone marrow, synovial tissue, peripheral blood, periosteum, and adipose tissue [5]. Several MSC-based tissue engineering techniques for cartilage regeneration using a pellet culture system or threedimensional (3D) scaffold have been reported [6–8]. Encapsulating cells in a natural biomaterial-based scaffold with a loose framework and high water content, such as collagen, fibrin, hyaluronic acid, or agarose, has been widely studied for 3D spheroid formation [5]. Since collagen molecules are a major component of the cartilage extracellular matrix and are degraded by endogenous collagenases, after removing telopeptide, antigenic-neutralized collagen-based scaffolds provide a suitable background for the application for an articular cartilage repair strategy. Among diverse shapes of scaffolds such as sponge, hydrogel, fibers, and microparticles, collagen-sponge has been utilized for 3D-MSC-derived chondrogenic spheroid formation because of its biocompatibility and capability for maintaining the chondrogenic microenvironment for functional cartilage regeneration [9]. When MSCs are cultured on type I atelocollagen scaffolds, cellular condensation and chondrogenic differentiation are induced. The expression of cartilage-specific markers such as Sox9, type II collagen, and aggrecan was confirmed in atelocollagen-encapsulated MSCs along with a chondrocyte-like appearance [9]. Moreover, type I atelocollagen scaffolds are able to recruit host MSCs in vivo, which can differentiate into chondrocyte-like cells [10, 11]. In this chapter, a laboratory protocol for the establishment of an atelocollagen-scaffolded chondrogenic-MSC-spheroid-based anti-rheumatoid agent screening system is introduced. Using pre-formed chondrogenic MSC spheroids, the efficacy of anti-rheumatoid agents for cartilage repair can be screened on a large scale ex vivo. Furthermore, atelocollagenscaffolded chondrogenic spheroids can be easily utilized for in vivo transplantation into a humanized xenografted arthritis model [12]. Thus, the ability of cartilage self-repair can be qualitatively and quantitatively evaluated.

2

Materials

2.1 3D Culture of Chondrogenic Spheroids

1. MSC suspension, 2.5 × 105 cells for each spheroid (see Note 1). 2. Preconditioned Poweredby10 Medium, prewarmed (GlycoTechnica, Yokohama, Japan). 3. Human Mesenchymal Stem Cell Functional Identification Kit (#SC006, R&D Systems, Minneapolis, MN), including Chondrogenic Supplement 100× (PART# 390417) and ITS Supplement 100× (PART#390418) (see Note 2).

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4. Complete chondrogenic conditioned medium: Poweredby10 Medium containing chondrogenic Supplement 1× and ITS Supplement 1×. 5. AteloCell® Atelocollagen, Honeycomb Disc 96 (Koken, Tokyo, Japan). 6. 2% Atelocollagen Implant (Koken). 7. Round-bottom 96-well cell culture plates (see Note 3). 8. Forceps (fine blunt), sterile conditions. 9. Pipettor and tips, sterile conditions. 10. 37 °C and 5% CO2-incubator suitable for cell culture. 2.2 Implantation In Vivo

All animal experiment protocols should be reviewed and approved by the Institutional Animal Care and Use Committee. 1. Animals: Male NOD/ShiJic-scid 6–10 weeks of age.

(NOD/SCID)

2. MSC-atelocollagen-scaffolded chondrogenic (in round-bottom 96-well cell culture plate).

mice,

spheroids

3. Explants from patients used for human xenograft model establishment, including synovium, articular cartilage, and bone explants obtained from patients who underwent prosthetic replacement arthroplasty for therapeutic purposes (see Chap. 2, Subheading 2). Explants should be handled for the xenograft procedure as soon as possible after explantation. 4. Saline-wet gauze at 4 °C. 5. 70% ethanol. 6. Inhalation anesthesia unit. 7. Chondrogenic spheroids. 8. Operating table. 9. Forceps (fine blunt) and scissors (fine dissection). 10. Wound clips and applier. 11. Isoflurane. 12. Phosphate-buffered saline (PBS), pH 7.2.

3

Methods (See Note 4)

3.1 3D Culture of Chondrogenic Spheroids

1. Using fine pointed curved forceps, carefully set Atelocollagen Honeycomb Discs onto a round-bottom 96-well cell culture plate. 2. Immerse the Honeycomb sponge in 50 μL of 2% Atelocollagen Implant (see Note 5). 3. Incubate the matrix at 37 °C for 1 h.

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Fig. 1 Evaluation of chondrogenic micromass in vitro. Atelocollagen-scaffolded MSC spheroids were pre-formed by 21 days of culture in a chondrogenic conditioned medium containing methotrexate (MTX) at doses of 0, 0.01, 0.1, and 1 μM. Scanning of micromass was performed using an MR imaging and analytic system for small animals. T2-weighted scout images were acquired and reconstructed as a three-dimensional image. The volume of the region of interest could be easily achieved

4. Add 200 μL of prewarmed Poweredby10 Medium into the well. 5. Calibrate the formed matrix at 37 °C in a humidified atmosphere with 5% CO2 overnight. 6. Resuspend MSC (2.5 × 105) in prewarmed 200 μL complete chondrogenic differentiation medium. 7. Remove the Poweredby10 Medium in the well and carefully seed MSC on the pre-formed matrix. 8. Culture the atelocollagen-scaffolded spheroids for 21 days. Change the culture medium with freshly prepared complete chondrogenic differentiation medium every 3 days (see Note 6). 9. By directly applying drugs into the chondrogenic conditioned medium and quantifying the volume of 3D-formed micromass, atelocollagen-scaffolded chondrogenic spheroids could be directly utilized for evaluation of efficacy ex vivo (Fig. 1). 3.2 Implantation In Vivo (See Note 7)

1. Trim the synovium and cartilage with bone to a block about 4–6 mm in diameter prior to implantation. Keep the explants in saline-wet gauze at 4 °C. 2. Put NOD/SCID mice in an anesthetic induction chamber. Initial induction can be performed using 2.5% isoflurane vaporized in 100% medical oxygen. Following induction, anesthesia should be maintained by placing the mice in front of a small face mask connected to an anesthetic machine using 1% isoflurane vaporized in 100% medical oxygen.

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3. Put mice on the operating table. Place the mouse on its abdomen to expose the back. Shave the back. Use a squirt bottle to apply 70% ethanol to the back and wipe with tissue. 4. Cut the skin with fine dissection scissors, making a 1.5 cm longitudinal incision at the level of the fourth to sixth lumbar vertebrae. 5. After exposing the subcutaneous tissue, the oblique external abdominal muscle is scraped with a scalpel until it bleeds. 6. Put the trimmed synovium on the oblique external abdominal muscle, and let the connective tissue site of synovium attach to the bleeding muscle. 7. Briefly rinse a chondrogenic spheroid in PBS and carefully set the spheroid on the synovium (Fig. 2a).

Fig. 2 Implantation and evaluation of chondrogenic spheroid in a xenograft model. (a) Implants for xenograft model. Two pieces of in vitro pre-formed atelocollagen-scaffolded chondrogenic MSC spheroid are set on patient-derived synovium. The explanted articular cartilage and bone should be set on the spheroid, with the smooth surface of the cartilage touching the spheroid on the articular luminal side. A representative image of hematoxylin and eosin staining of xenografted-implants (b) without chondrogenic MSC spheroid and (c) with chondrogenic MSC spheroid 8 weeks after implantation is shown. Invasion of synovium into cartilage was observed in the xenograft model without chondrogenic spheroid implantation, while the cartilage damage was repaired and cartilage with a smooth surface was maintained in the chondrogenic MSCspheroid-implanted xenograft model. C patient-derived cartilage, CS chondrogenic MSC spheroid, S patient-derived synovium. Scale bar: 100 μm

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8. Put the articular cartilage and bone on the synovium and let the smooth surface of the cartilage touch the chondrogenic spheroid on the articular luminal side (Fig. 1). 9. Close the skin with wound clips or two or three sutures. Clean the wound with 70% ethanol. 10. At the end of the procedure, put the mouse in a clean cage and place the cage on a warming plate until the mouse recovers from the anesthetic. 11. The regenerative effectiveness of chondrogenic spheroids for cartilage repair is evaluated 8 weeks after implantation by histological analysis (Fig. 2b, c).

4

Notes 1. Focusing on chondrogenic potency, the efficacy of primary MSCs derived from bone marrow, synovium, peripheral blood, adipose tissue, skin, or periosteum is confirmed for the cartilage regeneration strategy. 2. In this kit, chondrogenic supplements dexamethasone, ascorbate-phosphate, proline, pyruvate, and recombinant tumor growth factor-β3, and ITS supplement including insulin, transferrin, selenious acid, bovine serum albumin, and linoleic acid were added to the basic Poweredby10 Medium. 3. The MSC spheroid formed in a round-bottom 96-well culture plate can be easily handled for subsequent implantation in vivo. 4. All procedures in this protocol should be carried out under sterile conditions. 5. The formation of honeycomb sponge seems collapsed at this step, but this would not affect 3D-matrix formation. 6. To detect chondrogenic markers, formed spheroids can be sectioned on a cryotome and subjected to aggrecan and CD44 detection. 7. Atelocollagen-scaffolded chondrogenic spheroids could also be directly implanted into animal models to evaluate drug effectiveness. Long-term subcutaneous infusion of anti-rheumatoid agents could be achieved by subcutaneous implantation of an osmotic pump encapsulating with the drug (Fig. 3) (see Chap. 5).

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Fig. 3 In vivo evaluation system for the efficacy of anti-rheumatoid agents for chondrogenic regeneration. Atelocollagen-scaffolded chondrogenic spheroids were directly implanted into NOD/SCID mice according to the protocol described in the current chapter. Methotrexate (MTX) was continuously subcutaneously infused at doses of 0, 5, or 10 mg/kg/day for 28 days using an ALZET® osmotic pump. Eight weeks later, the implants were exposed and subsequent evaluation, such as histological analysis or assessment of volume of micromass, was performed. Orange arrows: atelocollagen-scaffolded chondrogenic MSC spheroid; black arrows: ALZET® osmotic pumps

References 1. Inui A, Iwakura T, Reddi AH (2012) Human stem cells and articular cartilage regeneration. Cells 1(4):994–1009. https://doi.org/10. 3390/cells1040994 2. Anders S, Goetz J, Schubert T, Grifka J, Schaumburger J (2012) Treatment of deep articular talus lesions by matrix associated autologous chondrocyte implantation-results at five years. Int Orthop 36(11):2279–2285. https://doi.org/10.1007/s00264-0121635-1 3. Matsumoto T, Okabe T, Ikawa T, Iida T, Yasuda H, Nakamura H et al (2010) Articular cartilage repair with autologous bone marrow mesenchymal cells. J Cell Physiol 225(2): 291–295. https://doi.org/10.1002/jcp. 22223 4. El-Jawhari JJ, El-Sherbiny YM, Jones EA, McGonagle D (2014) Mesenchymal stem cells, autoimmunity and rheumatoid arthritis. QJM 107(7):505–514. https://doi.org/10. 1093/qjmed/hcu033

5. Yamagata K, Nakayamada S, Tanaka Y (2018) Use of mesenchymal stem cells seeded on the scaffold in articular cartilage repair. Inflamm Regen 38:4. https://doi.org/10.1186/ s41232-018-0061-1 6. Tsvetkova AV, Vakhrushev IV, Basok YB, Grigor’ev AM, Kirsanova LA, Lupatov AY et al (2021) Chondrogeneic potential of MSC from different sources in spheroid culture. Bull Exp Biol Med 170(4):528–536. https:// doi.org/10.1007/s10517-021-05101-x 7. Lam J, Bellayr IH, Marklein RA, Bauer SR, Puri RK, Sung KE (2018) Functional profiling of chondrogenically induced multipotent stromal cell aggregates reveals transcriptomic and emergent morphological phenotypes predictive of differentiation capacity. Stem Cells Transl Med 7(9):664–675. https://doi.org/ 10.1002/sctm.18-0065 8. Muttigi MS, Kim BJ, Choi B, Han I, Park H, Lee SH (2020) Matrilin-3-primed adiposederived mesenchymal stromal cell spheroids

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prevent mesenchymal stromal-cell-derived chondrocyte hypertrophy. Int J Mol Sci 2 1 ( 2 3 ) . h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / ijms21238911 9. Kim SA, Sur YJ, Cho ML, Go EJ, Kim YH, Shetty AA et al (2020) Atelocollagen promotes chondrogenic differentiation of human adipose-derived mesenchymal stem cells. Sci Rep 10(1):10678. https://doi.org/10.1038/ s41598-020-67836-3 10. Dahlin RL, Kinard LA, Lam J, Needham CJ, Lu S, Kasper FK et al (2014) Articular chondrocytes and mesenchymal stem cells seeded on biodegradable scaffolds for the repair of cartilage in a rat osteochondral defect model.

Biomaterials 35(26):7460–7469. https://doi. org/10.1016/j.biomaterials.2014.05.055 11. Yuan T, Zhang L, Feng L, Fan H, Zhang X (2010) Chondrogenic differentiation and immunological properties of mesenchymal stem cells in collagen type I hydrogel. Biotechnol Prog 26(6):1749–1758. https://doi.org/ 10.1002/btpr.484 12. Liu S, Kiyoi T, Ishida M, Mogi M (2020) Assessment and comparison of the efficacy of methotrexate, prednisolone, adalimumab, and tocilizumab on multipotency of mesenchymal stem cells. Front Pharmacol 11:1004. https:// doi.org/10.3389/fphar.2020.01004

Chapter 4 Denervation-Induced Sarcopenia Model Erika Takemasa and Shuang Liu Abstract Rheumatoid arthritis (RA) is an important risk factor for sarcopenia. Physical inactivity, systemic inflammatory factors, and medication directly or indirectly induce skeletal muscle loss in RA patients. The sarcopeniainduced systemic or local proinflammatory microenvironment also contributes to the onset and progression of autoimmune disease. Accumulated evidence suggests the importance of treatment and management of sarcopenia in patients with RA to improve their long-term prognosis. To elucidate the relationship between skeletal muscle and systemic immune homeostasis, a denervation-induced skeletal muscle-losing mouse model is introduced in this chapter. By developing local amyotrophy in the sciatic nerve-dominant area in a RA model, the underlying mechanism of sarcopenia in RA could be assessed. Also, an examination of the efficacy of anti-rheumatic regents on sarcopenia and the influence of sarcopenia management on RA improvement is also achievable. Key words Sarcopenia, Skeletal muscle, Sciatic nerve, Immune homeostasis, Autoimmune disease

1

Introduction The prevalence of impaired skeletal muscle quantity or quality along with low physical performance, which has been defined as sarcopenia [1], is high in rheumatoid arthritis (RA) patients, with a pooled estimate of 33% [2], while it is approximately 10% in both men and women in the general population [3]. Age-related sarcopenia is considered “primary” when no other specific cause is evident. Secondary sarcopenia can occur in systemic diseases, especially those that induce inflammatory processes, such as autoimmune diseases or organ failure. The sarcopenia status in RA patients could be due to several RA-specific and non-RA-specific factors, including malnutrition, physical inactivity, systemic inflammatory factors, and medication. It has been reported that functional limitations (Steinbrocker Stage) and a proinflammatory microenvironment (high C-reactive protein and rheumatoid factor seropositivity) could be risk factors associated with sarcopenia [2]. Chronic exposure to tumor necrosis factor-α and

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interleukin-6 impairs the function of myosynthetic enzymes and promotes muscle atrophy [4]. Furthermore, glucocorticoid myopathy could be an independent factor in skeletal muscle loss through activation of the transcription factor FOXO or repression of mTOR signaling. Prednisolone use at 3.25 mg/day or more has been reported to promote the onset of sarcopenia [5]. Baseline methotrexate usage is also considered to be a risk factor for RA, with an odds ratio of 0.70 and 95% confidence interval of 0.51–0.97 [6]. On the other hand, the status of skeletal muscle influences immune homeostasis, while atrophic skeletal muscle is considered a potential local inflammatory site. The results of immune cell proportion profiling and functional assessment demonstrated that the loss of skeletal muscle impaired the function of T cells and influenced the balance of helper T cell subsets. Consequently, a sarcopenic condition promoted the progression of autoimmune disease via enhanced function of the IL-23/IL-17 axis [7]. Dysfunctional mitochondria have also been observed in T cells in sarcopenia, and this contributes to the dominance of Th1 and Th17 differentiation in addition to the milieu of cytokines [8]. Although sarcopenia has been associated with the mortality of various chronic diseases such as solid cancers and cardiovascular disease, evidence on the long-term prognosis of patients with RA is limited [9, 10]. Moreover, studies that have examined the efficacy of anti-rheumatic agents on sarcopenia or the influence of sarcopenia management on RA improvement are still few and not detailed. Further studies to elucidate the underlying pathophysiology of chronic autoimmune disease-associated sarcopenia and its impact on disease outcomes are eagerly awaited. While abnormal immune function and sarcopenia occur coincidentally, the adverse effects on patient outcomes are particularly profound. In order to better capture molecular players responsible for skeletal muscle maintenance and determine how they alter the progression of RA, several disease models of sarcopenia, including mice and rats, have been utilized [11]. In this chapter, a denervation-induced amyotrophy model is introduced. Using this model, the systemic impact of sarcopenia was attenuated because the loss of skeletal muscle mass was limited to the local sciatic nerve-dominant area. It therefore does not perfectly reflect systemic aging-related sarcopenia. However, this model is easily established and reflects chronic inflammation-related sarcopenia by performing simple surgical procedures on an autoimmune disease model. Compared with the hindlimb-immobilization unloading-induced muscle atrophy model and dietary intake-induced model [12], potential confounding factors related to experimental animals were ameliorated.

Denervation-Induced Sarcopenia Model

2

27

Materials All animal experiment protocols should be reviewed and approved by the Institutional Animal Care and Use Committee. All materials should be prepared for utilization in a sterile operating field. 1. Animals: Male mice 8–10 weeks of age (see Note 1). 2. Inhalation anesthesia unit. 3. Stereo microscope. 4. Operating table. 5. Forceps (fine blunt) and scissors (fine dissection). 6. Warming plate or heating pad. 7. Non-absorbable surgical 8/0 silk sutures. 8. Wound clips and applier. 9. 70% ethanol. 10. Isoflurane.

3

Methods 1. Under anesthetic conditions of 1.5 vol% isoflurane vaporized in air, apply protective eye liquid gel (see Note 2). 2. Place the mouse on its left side and position the right hind limb on a small pillow. 3. Shave the right leg from the knee to the hip and wash the skin using 70% ethanol. 4. Confirm the position of the femur and make a 2-cm longitudinal skin incision. 5. Separate the lateral piriformis muscle and expose the sciatic nerve (see Note 3). 6. Separate the proximal sciatic nerve (see Note 4). 7. Place an 8-0 silk suture below the rostral sciatic nerve and clamp the nerve with the suture (see Note 5). 8. Place a second caudal ligature at least 0.5 cm from the rostral ligature (Fig. 1a). 9. Completely amputate an approximately 0.4-cm length of the proximal sciatic nerve (Fig. 1b). 10. Close the skin using three wound clips. 11. 11 Perform the same procedure on the sciatic nerve on the opposite side. 12. At the end of the procedure, put the mouse in a clean cage (see Note 6).

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Fig. 1 Amputation of sciatic nerve in mice. (a) A rostral ligature and caudal ligature were placed around the sciatic nerve using an 8-0 silk suture. (b) At least a 0.4-cm length of the proximal sciatic nerve is completely amputated

13. Denervation-induced loss of hind limb skeletal muscle can be confirmed 8 weeks after the operation by magnetic resonance imaging [7]. The maximum vertical distance between the surface of the tibia and the border of the attached muscles can be defined as the thickness of the muscle in the hind limb (Fig. 2).

4 Notes 1. A disease model, such as a collagen-induced arthritis model or a humanized xenograft model, can be used here. The timing of denervation in the disease model should be individually optimized according to the purpose of the study. 2. As an alternative anesthesia protocol, the animal can be anesthetized by intraperitoneal injection of a mixture of 0.75 mg/kg medetomidine, 4 mg/kg midazolam, and 5 mg/kg butorphanol, which would provide approximately 1 h of deep anesthesia. 3. The muscle layers can be easily separated without any bleeding. Never cut the skeletal muscle with scissors. 4. Mice in which the sciatic nerves were separated without ligation and axotomy were used as sham controls. 5. Ligation of both resection stumps is performed to prevent sciatic nerve regeneration. 6. The surgical procedure will cause long-term neuropathic pain in the hind limb of the mice. Putting the food and water in a place where the mouse can easily reach right after the operation is helpful for recovery. No significant difference in the general appearance and activity level was observed in between sham mice and denervated mice after surgery.

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Fig. 2 Typical MRI images of hind limbs. Imaging was performed using a three-dimensional T2-weighted flash sequence. The maximum vertical distance between the bone surface and the border of attached muscles can be defined as the thickness of the hind limb muscle References 1. Cruz-Jentoft AJ, Bahat G, Bauer J, Boirie Y, Bruye`re O, Cederholm T et al (2019) Sarcopenia: revised European consensus on definition and diagnosis. Age Ageing 48(1):16–31. https://doi.org/10.1093/ageing/afy169 2. Li TH, Chang YS, Liu CW, Su CF, Tsai HC, Tsao YP et al (2021) The prevalence and risk factors of sarcopenia in rheumatoid arthritis patients: a systematic review and metaregression analysis. Semin Arthritis Rheum 51(1):236–245. https://doi.org/10.1016/j. semarthrit.2020.10.002 3. Shafiee G, Keshtkar A, Soltani A, Ahadi Z, Larijani B, Heshmat R (2017) Prevalence of sarcopenia in the world: a systematic review

and meta- analysis of general population studies. J Diabetes Metab Disord 16:21. https:// doi.org/10.1186/s40200-017-0302-x 4. Torii M, Itaya T, Minamino H, Katsushima M, Fujita Y, Tanaka H et al (2023) Management of sarcopenia in patients with rheumatoid arthritis. Mod Rheumatol 33(3):435–440. https:// doi.org/10.1093/mr/roac095 5. Yamada Y, Tada M, Mandai K, Hidaka N, Inui K, Nakamura H (2020) Glucocorticoid use is an independent risk factor for developing sarcopenia in patients with rheumatoid arthritis: from the CHIKARA study. Clin Rheumatol 39(6):1757–1764. https://doi.org/10.1007/ s10067-020-04929-4

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6. Tam K, Wong-Pack M, Liu T, Adachi J, Lau A, Ma J et al (2023) Risk factors and clinical outcomes associated with sarcopenia in rheumatoid arthritis: a systematic review and metaanalysis. J Clin Rheumatol. https://doi.org/ 10.1097/rhu.0000000000001980 7. Liu S, Kiyoi T, Takemasa E, Mogi M (2020) Denervation-induced loss of skeletal muscle mass influences immune homeostasis and accelerates the disease progression of lupus nephritis. JCSM Clin Rep 5(4):12 8. Zhi L, Ustyugova IV, Chen X, Zhang Q, Wu MX (2012) Enhanced Th17 differentiation and aggravated arthritis in IEX-1-deficient mice by mitochondrial reactive oxygen species-mediated signaling. J Immunol 189(4):1639–1647. https://doi.org/10. 4049/jimmunol.1200528 9. Shuang L, Masaki M (2023) Impact of cancerrelated sarcopenia on systemic immune status.

Interdisciplinary Cancer Research. Springer, Cham 10. Atkins JL, Whincup PH, Morris RW, Lennon LT, Papacosta O, Wannamethee SG (2014) Sarcopenic obesity and risk of cardiovascular disease and mortality: a population-based cohort study of older men. J Am Geriatr Soc 62(2):253–260. https://doi.org/10.1111/ jgs.12652 11. Christian CJ, Benian GM (2020) Animal models of sarcopenia. Aging Cell 19(10):e13223. https://doi.org/10.1111/acel.13223 12. Speacht TL, Krause AR, Steiner JL, Lang CH, Donahue HJ (2018) Combination of hindlimb suspension and immobilization by casting exaggerates sarcopenia by stimulating autophagy but does not worsen osteopenia. Bone 110:29–37. https://doi.org/10.1016/j.bone. 2018.01.026

Chapter 5 Long-Term Constant Subcutaneous Drug Administration Shuang Liu and Maya Miyoshi Abstract In this chapter, a long-term drug delivery system for preclinical therapeutic research is introduced. By using a subcutaneously implanted ALZET® Osmotic Pumps osmotic pump, continuous zero-order delivery of drugs under investigation that need repeated oral or intravenous dosing is realizable. Compared to traditional delivery systems, implanted osmotic pumps present several advantages, such as that no external connections or researcher intervention is required during infusion and that it is possible to save time by eliminating the need for frequent animal handling and repetitive injection schedules. Most importantly, a stable peripheral concentration of a drug can be obtained using this constant drug delivery system, which would benefit researchers in verifying the efficiency of anti-rheumatoid drugs and establishing safety profiles in preclinical studies. Key words ALZET® osmotic pump, Long-term drug delivery, Implantation, Subcutaneous administration, Drug formulation

1

Introduction Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease and is generally thought to be due to increased inflammatory burden, which causes accelerated atherosclerosis. Long-term combination therapies utilizing disease-modifying anti-rheumatic drugs (DMARDs) with glucocorticoids have been widely used at the start of RA therapy [1]. These drugs are currently formulated as injectable solutions and tablets for oral administration for repeated dosing. The pharmacokinetic properties of these formulations may be unsatisfactory and result in inadequate clinical response. For example, methotrexate (MTX) is one of the most widely studied and effective therapeutic agents available to treat RA and other autoimmune diseases. The bioavailability of low-dose MTX is nearly complete, but at high doses it is 10–20%. This shows the presence of a saturatable intestinal active transport absorption mechanism with low-capacity characteristics. A large amount of the administered MTX is eliminated within a short period of time, resulting in a

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short plasma half-life of 5–8 h and a low drug concentration in target tissues [2–4]. To design an appropriate drug regimen, a shift of the dose– response curve to the right or left according to the time pattern of drug administration is critical. At equal total doses, compared to repeated dosing, the dose–response curve shifts to the right in the case of continuous infusion since the peak concentration often plays an important role in eliciting the responses [5]. Constant infusion in mice maintained drug and metabolite levels within a narrow range and therefore reduced the drug’s toxicity. To obtain a steady state of peripheral drug concentration, several therapeutic challenges of anti-rheumatoid drug delivery systems have therefore been developed for preclinical studies. The ALZET® osmotic pump is an available tool for continuous zeroorder delivery of compounds that would eliminate the need for repeated dosing [6, 7]. The operation of this osmotic pump is based on the osmotic pressure gradient developed between the salt layer compartment and the tissue environment in which the pump is implanted. As tissue fluid enters the salt layer compartment, it compresses the flexible drug reservoir and forces its contents through a delivery portal at a constant rate over a period of time for a maximum of up to 28 days. Compared to traditional delivery systems, implanted osmotic pumps present several advantages: (1) continuous delivery ensures constant compound levels in plasma or tissues for maximized therapeutic efficacy and reduced adverse effects; (2) no external connections or researcher intervention is required during infusion; and (3) time is saved by eliminating the need for frequent animal handling and repetitive injection schedules. In particular, during the drug discovery phase, scientists could gain reliable long-term efficiency of drugs and safety profiles by using constant ALZET osmotic pump delivery systems. In this chapter, the procedure of long-term constant subcutaneous delivery, which is the easiest and least invasive procedure using this system, is introduced. Besides subcutaneous implantation, ALZET® osmotic pumps are also available for intraperitoneal use as well as intravenous cannulation and brain infusion for longterm constant drug delivery. More information about surgical implantation is available in the guidelines on the ALZET® Osmotic Pumps site. Considering the current highlighted topics in the field of RA, examples of formulations for poorly water-soluble compounds and biologics are also introduced in the Notes.

2

Materials (See Note 1) All animal experiment protocols should be performed in accordance with the guidelines of the Institutional Animal Care and Use Committee and approved by the committee.

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1. Animals: Male DBA/1JNCrlj mice (CLEA Japan, Tokyo, Japan), 6–10 weeks of age, are used for the preparation of a collagen-induced arthritis model. 2. ALZET® osmotic pumps (Model #1004, 28-day delivery at 0.11 μL/h) (Durect Corporation). 3. Inhalation anesthesia unit. 4. Operating table. 5. Warming plate or heating pad. 6. Forceps (fine blunt) and scissors (fine dissection). 7. Hemostats. 8. Syringes, 1 mL. 9. Wound clips and applier. 10. 70% ethanol. 11. Isoflurane or other anesthetics (Sigma Aldrich). 12. Drug formulations (see Note 2).

3

Methods (See Note 3)

3.1 Filling of ALZET® Pump (See Note 4)

1. Before the filling procedure, ensure the drug formulation is at room temperature. 2. Weigh the empty pump together with its flow moderator. 3. Attach a 100-μL filling tube (supplied with each package of pump) to a 1-mL syringe and draw up the drug solution. Avoiding any air bubbles is essential in this step. Allow extra syringe volume for spillage. 4. Hold the pump in an upright position (with the exit port pointed vertically). Insert the filling tube through the opening at the top of the pump until it can go no further. This places the tip of the tube near the bottom of the pump reservoir. 5. Slowly push the plunger of the syringe. When the solution appears at the outlet, stop filling and carefully remove the tube (see Note 5). 6. Insert the flow moderator until the cap is flush with the top of the pump. Insertion of the flow moderator will displace some of the solution from the filled pump. This overflow should be wiped off. 7. Weigh the filled pump with the flow moderator in place. The loaded solution will give the net weight of the pump. For most dilute aqueous solutions, the weight in milligrams is approximately the same as the volume in microliters. The filled volume should be more than 90 μL of the reservoir volume. If not,

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there may be some air trapped inside the pump and the pump needs to be refilled. 3.2

Implantation

1. Mice before or after the final boosting of collagen emulsion, according to the study design, are used for implantation of filled ALZET® pumps. Put the mouse in an anesthetic induction chamber, and initial induction can be performed using 2.5% isoflurane vaporized in 100% medical oxygen. Following induction, anesthesia should be maintained by placing the mouse in front of a small face mask connected to the anesthetic machine using 1% isoflurane vaporized in 100% medical oxygen. 2. Shave and wash the skin over the implantation site, which is usually on the back of the mouse, for subcutaneous implantation. Use a squirt bottle to apply 70% ethanol to the back and wipe with tissue. 3. Cut the skin with fine dissection scissors, making a mid-scapular incision. 4. Insert a hemostat into the incision and spread the subcutaneous tissue to create a pocket for the pump. The pocket should be 0.8–1 cm longer than the pump. 5. Insert a filled pump into the pocket, delivery portal first. This minimizes interaction between the compound delivered and healing of the incision (see Note 6). 6. Clip the skin together using two clips of a wound clip. 7. At the end of the procedure, put the mouse in a clean cage and place the cage on a warming plate until the mouse recovers from the anesthetic.

3.3 Explanting Pumps

1. The pump should be explanted by day 42 after implantation. 2. Once the mice are anesthetized, make a simple skin incision on the site of implantation. 3. Sometimes, it is necessary to separate the surrounding connective tissue in order to release the pump. 4. If necessary, replace with a fresh filled pump, in order to infuse for a longer period (see Note 7).

4

Notes 1. All equipment should be used under sterile conditions. 2. ALZET osmotic pumps have proven to be extremely useful for the delivery of small molecular compounds that are watersoluble as well as those that are poorly water-soluble and biologics. The formulations of drugs should be optimized before

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the study, with consideration of the characteristics of drugs, the delivery period, and the site of implantation. • For poorly soluble investigational compounds, the use of hydrophilic, nonaqueous solvents (water miscible organic solvents) proved to be promising in meeting high delivery dose requirements. As an example, based on miscibility, clarity, and ability to solubilize the drug and keep it in solution against crystallization upon dilution with saline solution, a vehicle containing 25% w/w polyethylene glycol 300, 25% w/w cremophor ELP, 25% w/w glucofurol, 15% w/w ethanol, and 10% propylene glycol is suitable for solubilizing ELND006, an investigational compound [5]. Without considering the effects on the metabolism system, in our experience a poorly soluble investigational compound could be simply dissolved using various solvents and suspended using an intravenous lipid emulsion, such as Intralipid® 10%, which is a non-pyrogenic fat emulsion prepared for intravenous administration as a source of calories and essential fatty acids [8]. It is made up of 10% soybean oil, 1.2% egg yolk phospholipids, 2.25% glycerin, and water for injection. • For the preparation of a concentrate of neutralizing antibody, purified antibody elutes can be supplemented with 1.5 mg/mL polyoxyethylene (20) sorbitan monolaurate and dialyzed in an equilibration buffer consisting of 100 mM L-histidine, 50 mM L-arginine, 100 mM glutamic acid, and 150 mM trehalose. Finally, elutes are lyophilized in vials and stored at -20 °C until being used to fill pumps [9]. 3. All procedures should be performed under sterile conditions. 4. It is essential that each pump is completely filled with drug solution for accurate operation. Air bubbles trapped within the body of the pump or failure to insert the flow moderator into the pump may result in unpredictable fluctuations in the pumping rate. 5. Rapid filling of ALZET pumps should be avoided because it can introduce air bubbles into the reservoir. 6. The pump should not rest immediately beneath the incision, which could interfere with the healing of the incision. 7. The explanted pump cannot be reused. References 1. Abolmaali SS, Tamaddon AM, Dinarvand R (2013) A review of therapeutic challenges and achievements of methotrexate delivery systems

for treatment of cancer and rheumatoid arthritis. Cancer Chemother Pharmacol 71:1115–1130 2. Bleyer WA, Nelson JA, Kamen BA (1997) Accumulation of methotrexate in systemic tissues

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after intrathecal administration. J Pediatr Hematol Oncol 19:530–532 3. Creaven PJ, Hansen HH, Alford DA, Allen LM (1973) Methotrexate in liver and bile after intravenous dosage in man. Br J Cancer 28:589–591 4. Iqbal MP (1998) Accumulation of methotrexate in human tissues following high-dose methotrexate therapy. J Pak Med Assoc 48:341–343 5. Fara J, Urquhart J (1984) The value of infusion and injection regiments in assessing efficacy and toxicity of drugs. Trends Pharmacol Sci 5 6. Gullapalli R, Wong A, Brigham E, Kwong G, Wadsworth A, Willits C et al (2012) Development of ALZET(R) osmotic pump compatible solvent compositions to solubilize poorly

soluble compounds for preclinical studies. Drug Deliv 19:239–246 7. Theeuwes F, Yum SI (1976) Principles of the design and operation of generic osmotic pumps for the delivery of semisolid or liquid drug formulations. Ann Biomed Eng 4:343–353 8. Miyoshi M, Liu S, Morizane A, Takemasa E, Suzuki Y, Kiyoi T et al (2018) Efficacy of constant long-term delivery of YM-58483 for the treatment of rheumatoid arthritis. Eur J Pharmacol 824:89–98 9. Liu S, Hasegawa H, Takemasa E, Suzuki Y, Oka K, Kiyoi T et al (2017) Efficiency and safety of CRAC inhibitors in human rheumatoid arthritis xenograft models. J Immunol 199: 1584–1595

Chapter 6 Clinical Scoring of Disease Activity in Animal Models Maya Miyoshi and Shuang Liu Abstract Disease severity in murine arthritis models, such as collagen-induced arthritis (CIA), is commonly assessed by clinical scoring of paw swelling and histological examination of joints. Clinical scoring using a qualitative scoring system of paw inflammation (paw thickness, width, or volume) over time is the standard method used for subjective quantification of arthritis activity. To evaluate paw swelling status, a quantitative method using three-dimensional T2-weighted flash sequence magnetic resonance imaging (MRI) is introduced. The efficacy of a therapeutic approach can be semiologically quantified using a clinical scoring system and an index of paw inflammation in CIA mice. Key words Collagen-induced arthritis, Clinical scoring system, MRI, Paw volume, Disease activity

1

Introduction Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by synovial inflammation followed by progressive destruction of articular cartilage and bone. The collagen-induced arthritis (CIA) murine model is commonly used to gain further insights into the pathological mechanisms of joint inflammation and evaluate the efficacy of preclinical therapeutic approaches. The CIA model shares several similarities in pathology and immunological processes with human RA, as described in Chap. 1, such as systemic joint involvement, peripheral joints affected, synovitis, cartilage and bone erosions, and inflammatory cell infiltration of the synovium [1]. Disease severity in CIA mice is commonly assessed by clinical scoring of paw swelling and histological examination of joints [2]. Clinical scoring using a qualitative scoring system of paw inflammation (paw thickness, width, or volume) over time is the standard method used for quantification of arthritis activity, although it is subjective. Aiming at measurement of edema and erythema, the thickness and width of the paw can be assessed using a thickness gauge, such as a Mitutoyo loop handle dial

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thickness gauge with a round disc [2]. For volume quantification of the inflamed paw, hindpaw volume can be measured by a plethysmometer by dipping the paw into plain water up to a certain position of the ankle joint [3]. However, this method, which showed good performance in a rodent model, faces obstacles in a murine model because the mouse paw is too small to be measured precisely. Therefore, we attempted to gain the total volume of the paws, which reflects the edema of inflamed paws, using three-dimensional T2-weighted flash sequence magnetic resonance imaging (MRI) [4]. In this chapter, a qualitative clinical scoring system and a method for determining paw volume in CIA mice are introduced. Along with histological analysis as described in Chap. 5, arthritis severity in model mice can be evaluated, and therefore the efficacy of a therapeutic approach can be semiologically quantified.

2

Materials

2.1 CIA Scoring System

1. CIA mice: According to the immunization protocol (Fig. 1) for CIA establishment, scoring should be started from the day when the mice receive the final booster injection (day 26). 2. Two independent examiners: Ankle circumference and articular indexes are measured in a blinded manner (see Note 1).

2.2 Evaluation of Paw Volume

1. CIA mice. 2. Anesthetic vaporizer (Fig. 2). 3. MR imaging and analytic system for small animals (Fig. 3). 4. Isoflurane. 5. Eye lubricating ointment.

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Methods

3.1 CIA Scoring System

Three types of joint, including interphalangeal joints, metacarpophalangeal joints, and carpal and tarsal joints, should be observed for each limb. The articular index (maximum score of 16 for four limbs) is scored from 0 to 4 as follows (see Note 2): 0: no swelling. 1: one joint type has slight welling and erythema. 2: two joint types have edema and swelling. 3: all three joint types have severe edema and swelling. 4: joint rigidity.

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Fig. 1 Time schedule of preparation of collagen-induced arthritis mice and evaluation of disease severity. CII type II collagen, CFA complete Freund’s adjuvant, IFA incomplete Freund’s adjuvant

Fig. 2 Anesthetic vaporizer used in our laboratory 3.2 Evaluation of Paw Volume by In Vivo MRI Scanning (See Note 3)

1. Anesthesia: Mice are anesthetized using vaporized isoflurane. A surgical level of anesthesia is obtained by initial induction using 4% vaporized isoflurane with a flow rate of 2.0 L/min for 2–3 min and maintained using 1.5% vaporized isoflurane at 0.8 L/min. 2. Place the mouse in an animal holder and put an anesthetic mask on its face (Fig. 4). 3. Apply eye lubricating ointment to both eyes of the mouse to keep them moist. 4. Set the animal holder in the center of an RF coil (see Note 4). 5. Insert the RF coil into an MRI scanner.

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Fig. 3 MR imaging and analytic system for small animals used in our facility

Fig. 4 Preparation for MRI scanning. After induction of anesthesia, the mouse is put in an animal holder and set in the center of an RF coil. The state of anesthesia is maintained by inhalation of vaporized isoflurane through a face mask

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Fig. 5 Reconstructed three-dimensional T2-wighted MRI image of hindpaw in (a) normal control mouse and (b) collagen-induced arthritis (CIA) mouse. Edema is observed in the paw of the CIA mouse

6. Turn on the MRI system. Conduct the initial shimming process using a single pulse sequence. This enables the magnetic field in the region of interest to be as homogeneous as possible (see Note 5). 7. Optimize the RF pulse by maximizing the one-dimensional image profile. 8. Acquire T2-weighted scout images along three orthogonal orientations to create axial, coronal, and sagittal images. Based on the scout images, adjust the position of the mouse to obtain the best filming condition. 9. Select proper sequence parameters (average = 3, recycle delay (TR) = 100–2000, echo time (TE) = 12, etc.). 10. Acquire the flash sequence. 11. Save the image as a *.shr file. 12. Reconstruct the images as a three-dimensional image (Fig. 5). 13. Measure the volume of the region of interest (see Note 6).

4

Notes 1. The researcher who performed the immunization procedure should not score the disease severity. 2. There are several ways to define the severity score from 0 to 4. The definition here refers to the CIA system introduced by Chondrex, Inc. More information can be found on the website www.chondrex.com/documents/Scoring-System.pdf.

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3. MRI uses a strong magnetic field that requires extreme caution. There is always a strong magnetic field, even when the MRI scanner is not being used. Any metallic object that comes into contact with such a strong magnetic field will be strongly and rapidly attracted to the magnet. Therefore, researchers who conduct MRI experiments should be careful to remove any metallic objects from their clothing before entering the proximity of the instrument and also maintain the surrounding environment free from such objects. 4. After set up, the region of interest, such as the hindpaw, should be placed in the center of the RF coil. 5. Each MRI scanner has its own way of performing the shimming process. 6. To compare the volume between mice, extraction of the region of interest should be performed with animals in the same position based on predefined biological markers, such as the carpal joint or inguinal ligament. References 1. Bessis N, Decker P, Assier E, Semerano L, Boissier MC (2017) Arthritis models: usefulness and interpretation. Semin Immunopathol 39:469– 486 2. Liu S, Kiyoi T, Takemasa E, Maeyama K (2015) Systemic lentivirus-mediated delivery of short hairpin RNA targeting calcium release-activated calcium channel 3 as gene therapy for collageninduced arthritis. J Immunol 194:76–83

3. Milici AJ, Kudlacz EM, Audoly L, Zwillich S, Changelian P (2008) Cartilage preservation by inhibition of Janus kinase 3 in two rodent models of rheumatoid arthritis. Arthritis Res Ther 10:R14 4. Liu S, Kiyoi T, Takemasa E, Maeyama K (2017) Intra-articular lentivirus-mediated gene therapy targeting CRACM1 for the treatment of collagen-induced arthritis. J Pharmacol Sci 133:130–138

Chapter 7 Histological Analyses of Arthritic Joints in Collagen-Induced Arthritis Model Mice Takeshi Kiyoi Abstract Histological analysis is a morphological technique and an effective method for understanding the pathology of rheumatoid arthritis (RA). RA is an inflammatory disease characterized by increased synovial tissue and osteoclasts, angiogenesis, infiltration of inflammatory cells, and pannus formation. These pathologies can be observed in a collagen-induced arthritis model mouse using formaldehyde-fixated paraffin-embedded (FFPE) samples. For the preparation of FFPE samples, the conditions of the fixation and decalcification process significantly affect tissue staining results. Since the lesion sites include bone tissue, a decalcification process is necessary when preparing an FFPE sample. Therefore, selecting an optimal condition for the fixating and decalcifying solution is important. In this chapter, we describe the procedures of preparing paraffin samples, including fixation, decalcification, embedding, and sectioning from the RA model mouse, as well as different staining methods (hematoxylin and eosin, tartrate-resistant acid phosphatase). Key words Histological analysis, Fixation, Decalcifying, Paraffin embedding, Sectioning, HE, TRAP, IHC, Antigen retrieval

1

Introduction Histological analysis has been used to investigate disease pathogenesis in clinical practice and research. Generally, the tissues taken from collagen-induced arthritis (CIA) model mice are visualized by histochemistry, immunohistochemistry (IHC), or other methods using a microscope after fixation, embedding, and sectioning processes. Depending on the research purpose, examining and choosing the more accurate method in each process is important. For example, staining results are significantly influenced by the choice of fixative and decalcifying solution. Adequate fixation is important for preserving morphology and antigen immunoreactivity [1]. Fixative solution using formaldehyde, such as 4% paraformaldehyde or periodate-lysine-paraformaldehyde, is favored when considering the method of IHC [2]. For hard tissues such as bone, a decalcifying process is typically needed. Although the decalcifying process

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attenuates some types of antigens, many antigens remain identifiable in IHC after the process [3–5]. There are several types of decalcifying solutions, such as acidic (plank-rychlo solution, formic acid, nitric acid) or neutral (EDTA), and the choice of the decalcifying solution affects the staining characteristics of the tissue [6]. Although formaldehyde-fixated paraffin-embedded (FFPE) samples are popular due to their excellent structural preservation, antigen attenuation remains an issue. However, the problem has been improved by developing antigen retrieval methods (heat or enzymatic treatment) and a highly sensitive detection system for IHC [7–11]. RA is an inflammatory disease characterized by increased synovial tissue and osteoclasts, angiogenesis, infiltration of inflammatory cells, and pannus formation. These characteristic features of RA joints can be evaluated using hematoxylin and eosin (HE) staining and tartrate-resistant acid phosphatase (TRAP) staining as primary and specific staining techniques, respectively. The increase and decrease in osteoclasts can be easily detected by TRAP staining. IHC can also be used to investigate the factors associated with RA. This chapter describes methods of HE, TRAP, and IHC (e.g., calcium-release-activated calcium channel 3 [CRACM3]) using FFPE specimens from RA model mice. CRACM3, a unique member of the CRAC family of Ca2+ selective channels, significantly increases cytosolic Ca2+ levels in lymphocytes from RA by activating CRACM3 [12].

2

Materials It is necessary to use fume hoods for histological experiments due to the use of paraformaldehyde and organic solvents.

2.1 Sampling (Fixation and Decalcification)

1. Mice; collagen-induced arthritis (CIA) model and negative control. 2. Surgical instruments: Forceps and scissors. 3. Phosphate-buffered saline (PBS) (pH 7.4): Dissolve 8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 in 1 L of distilled water. Then, adjust pH with HCl. 4. 4% paraformaldehyde (PFA) (see Note 1). 5. Decalcifying solution: 10% EDTA, pH 7.4. Add approximately 450 mL of water to a 500-mL glass beaker. Weigh 50 g of EDTA-2Na and transfer it to the beaker. Mix while heating it to approximately 45 °C until it is dissolved. Adjust pH with NaOH (see Note 2). 6. Embedding cassette (Murazumi Industrial).

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2.2

Embedding

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1. Auto tissue processor Vip5jr (Sakura Finetek Japan). 2. Ethanol: Fill the tanks of the auto tissue processor with 2 L of ethanol (70%, 80%, 90%, 95%, absolute × 3). 3. Xylene: Fill the tanks of the auto tissue processor with 2 L of xylene × 3. 4. Paraffin: Fill the tanks of the auto tissue processor with 2 L of paraffin × 4. Store at 60 °C (see Note 3). 5. Apparatus for paraffin block production: The apparatus comprises a paraffin bath at 60 °C, a paraffin dispenser at 60 °C, and a cooling plate at -5 °C. 6. Stemless mold for paraffin embedding.

2.3

Sectioning

1. Water bath. 2. Paraffin extension plate (Sakura Finetek Japan). 3. Blade for paraffin block (see Note 4). 4. MAS-coated slide glass (Matsunami Glass) (see Note 5). 5. Sliding microtome: REM710 (Yamato Kohki Industrial).

2.4

Staining

The slides with FFPE tissue were treated with xylene for deparaffinization and ethanol for hydration. In HE and IHC staining, after staining, the tissue is treated with ethanol for dehydration and xylene for permeation. In TRAP staining, ethanol is unnecessary for dehydration. 1. Xylene for deparaffinization: Prepare two Coplin Jars with xylene. 2. Ethanol for hydration: Prepare four Coplin Jars with absolute, 90%, 80%, and 70% ethanol. 3. Ethanol for dehydration: Prepare five Coplin Jars with 70%, 80%, 90%, and absolute × 2 ethanol. 4. Xylene for permeation: Prepare two Coplin Jars with xylene.

2.5

HE Staining

1. Mayer’s hematoxylin (see Note 6). 2. Eosin. 3. HSR mounting medium (see Note 7).

2.6

TRAP Staining

1. TRAP staining kit (FUJIFILM Wako Pure Chemical Corporation): Include tartaric acid solution (×10), acid phosphatase substrate solution A, and acid phosphatase substrate solution B. Mix 100 μL of tartaric acid solution (×10), 900 μL of phosphatase substrate solution A, and 10 μL of phosphatase substrate solution B to prepare the TRAP staining solution. Pre-warm the TRAP staining solution to 37 °C. 2. HSR mounting medium.

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2.7 IHC for CRACM3 Antigen

1. PBS (pH 7.4): Dissolve 8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 in 1 L of distilled water. Then, adjust pH with HCl. 2. PBST: 0.05% Tween 20 in PBS. 3. 3% hydrogen peroxide: Blocking solution for endogenous peroxidase. Dilute 30% hydrogen peroxide with distilled water to 3%. 4. Citrate buffer (0.01 M, pH 6.0) as an antigen retrieval buffer: Dissolve 2.1 g of citric acid monohydrate in 100 mL of distilled water (solution A) and 2.94 g of trisodium citrate dihydrate in 100 mL of distilled water (solution B). Mix 18 mL of solution A and 82 mL of solution B to prepare 10× citrate buffer and then dilute it with distilled water to 1× (see Note 8). 5. Blocking solution: 3% BSA in PBS. Store at 4 °C. 6. Antibody diluent buffer: 1% BSA in PBS containing 0.02% sodium azide. Store at 4 °C. 7. Primary antibody: Anti-mouse CRACM3 rabbit polyclonal antibody (see Note 9). 8. Anti-rabbit IgG secondary antibody: Mouse MAX-PO for rabbit IgG (Nichirei Biosciences) (see Note 10). 9. 3,3′-Diaminobenzidine (DAB): DAB kit (Nichirei Biosciences) (see Note 11). 10. HSR mounting medium.

3

Methods

3.1 Fixation and Decalcification

1. Anesthetize the model animal with isoflurane, followed by euthanasia. Then, dissect the lesion site tissue (knee joint) immediately. Fixate the tissue with 4% PFA at 4 °C for 48 h (see Note 12). 2. After the fixative process, remove the unnecessary region from the fixed tissue. Place the tissue into an embedding cassette and rinse with tap water for 2 h to remove paraformaldehyde. 3. Treat with 10% EDTA (pH 7.0) as a decalcifying solution at room temperature for 2 weeks and rinse for 2 h with tap water (see Note 13).

3.2

Embedding

1. Set up an auto tissue processor and start the paraffin embedding process. The detailed protocol is as follows: 70% ethanol (1 h), 80% ethanol (1 h), 90% ethanol (2 h), 95% ethanol (2 h), absolute ethanol 1 (3 h), absolute ethanol 2 (3 h), absolute ethanol 3 (6 h), xylene 1 (1 h), xylene 2 (1 h), paraffin 1 (1 h, 60 °C), paraffin 2 (1 h, 60 °C), paraffin 3 (1 h, 60 °C), and paraffin 4 (1 h, 60 °C) (see Note 14).

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2. Remove the cassette from the auto tissue processor and transfer it to the paraffin bath. Open the cassette and check the tissue size. Select a stemless mold that can fit the tissue. Embed the tissue with paraffin in the sagittal plane. 3. Cool the paraffin-embedded tissue on a cooling plate at -5 °C to fix it. 3.3

Sectioning

1. Set the paraffin-embedded block on the sliding microtome. 2. Trim the surface of the block to expose the lateral knee joint with RA symptoms (see Note 15). Cool the surface of the block using ice for 2–3 min, and cut sections (3–4 μm) using a new blade (see Note 16). 3. Pick up and float the sections in a water bath at room temperature. Mount the sections using MAS-coated slide glass (see Note 17). Stretch the mounted sections on a paraffin extension plate at 45–50 °C and stock them at 37 °C overnight.

3.4

HE Staining

1. Deparaffinize the sections in xylene twice for 10 min each. 2. Hydrate the sections in absolute ethanol, 90% ethanol, and 70% ethanol for 5 min each. 3. Rinse briefly in tap water. 4. Stain with Mayer’s hematoxylin for 15 min (see Note 18). 5. Rinse in tap water for 15 min. 6. Stain with eosin for 10 min. 7. Wash briefly in tap water. 8. Dehydrate the sections with 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, and absolute ethanol twice. Dehydration in the second absolute ethanol lasts at least 5 min (see Note 19). 9. Permeate in xylene three times for 10 min each. 10. Mount the cover slide with HSR mounting medium.

3.5

TRAP Staining

1. Deparaffinize the sections in xylene twice for 10 min each. 2. Hydrate in absolute ethanol, 90% ethanol, and 70% ethanol for 5 min each. 3. Rinse briefly in tap water. 4. Rinse in distilled water for 1 min. 5. Drop the TRAP staining regent to slide in a moist chamber at 37 °C for 30 min (see Note 20).

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6. Rinse in distilled water three times for 1 min each (see Note 21). 7. Dry the slides with cold air. 8. Permeate in xylene three times for 10 min each. 9. Mount the cover slide with HSR mounting medium. 3.6 Immunohistochemistry (IHC)

1. Deparaffinize sections in xylene twice for 10 min each. 2. Hydrate in absolute ethanol, 90% ethanol, and 70% ethanol for 5 min each. 3. Rinse briefly in tap water. 4. Block endogenous peroxidase activity with 3% hydrogen peroxide solution at room temperature for 10 min. 5. Rinse briefly in tap water. 6. For antigen retrieval, treat with citrate buffer (pH 6.0) at 120 ° C for 10 min in heat-resistant Coplin Jars, then gradually cool to room temperature (see Note 22). 7. Rinse in PBS (see Note 23). 8. Incubate the section with a blocking buffer in a moist chamber for 30 min (see Note 24). Do not wash the tissue with PBS after this process. 9. Incubate the primary antibody with diluted primary antibody at 4 °C overnight in a moist chamber (see Note 25). 10. Rinse in PBST for 5 min at least three times. 11. Incubate the secondary antibody at room temperature for 45 min in a moist chamber. 12. Rinse in PBST for 5 min at least three times. 13. DAB coloring (see Note 26). 14. Rinse in PBST and tap water. 15. Stain with hematoxylin for 1 min (see Note 27). 16. Rinse in tap water for 10 min. 17. Dehydrate the sections with 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, and absolute ethanol twice. Dehydration in the second absolute ethanol lasts for at least 10 min. 18. Permeate in xylene three times for 10 min each. 19. Mount the cover slide with HSR mounting medium.

3.7

Observation

Tissues on the slide were observed using a light microscope. The typical picture is shown in Figs. 1, 2, and 3.

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Fig. 1 HE staining. Histologically, control mice and mice with collagen-induced arthritis (CIA) were characterized by increased synovial tissue, angiogenesis, inflammatory cell infiltration, and bone destruction. The slides were observed using a BZ-9000 microscope (objective lens: ×10)

Fig. 2 TRAP staining for osteoclasts. An increase in osteoclast numbers is observed at the bone tissue margins in collagen-induced arthritis (CIA) mice compared with control mice. The slides were observed using a BZ-9000 microscope (objective lens: ×20)

Fig. 3 Immunohistochemistry (IHC) for CRACM3-expressed cells. CRACM3 expression was detected in the control and collagen-induced arthritis (CIA) mice. CRACM3 expression was increased in CIA joints. The slides were observed using a BZ-9000 microscope (objective lens: ×20)

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Notes 1. In the cases of self-prepared 4% PFA, the solution must be used as soon as possible. We recently purchased a commercial 4% PFA with inert gas because the solution can be stocked for approximately 6 months. 2. The selection of a decalcifying solution affects the staining characteristics of tissues. This chapter recommends using 10% EDTA (pH 7.0) as a decalcifying solution for histological analysis. Alternatively, the Kawamoto’s film method is an excellent method for hard tissue with high sensitivity of antigen detection because it requires no decalcification [13]. This technique is also beneficial for RA research. 3. Different types of paraffin are sold by different corporations. Histprep 580 (58–60 °C) (FUJIFILM Wako Pure Chemical Corporation) is routinely used in our facility. 4. Different types of blades are sold by different corporations. Histocutter super (Muto Pure Chemicals) is routinely used in our facility. 5. A coated slide glass is needed when using IHC. There are several types of coating agents (such as poly-L-lysine and silane). MAS-coated slide glass is used in our facility. 6. There are several types of hematoxylin solutions (such as Mayer’s, Carrazzi’s, Harris’s, and Gill’s). Mayer’s hematoxylin is often used for HE staining in our facility. 7. HRS solution (Sysmex Corporation, Japan) is a xylene-based mounting medium. 8. Heat treatment methods with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) are widely used for antigen retrieval. However, some cases used enzymatic treatment as an antigen retrieval method [14–17]. It is recommended to select proper antigen retrieval methods according to different antibody clones, antigens, or fixation methods. 9. CRACM3 rabbit polyclonal antibody (ProSci) is used as a primary antibody. Dilute the antibody to 1:200 with antibody diluent buffer. 10. There are some detection systems for IHC (such as PAP, ABC, LSAB, CSA, and polymer method) [9, 18–22]. The polymer method has been developed as a high-sensitivity and specificity detection system. Mouse MAX-PO (Nichirei Biosciences) for rabbit IgG as a secondary antibody is labeled by polymerbased HRP. 11. Prepare the DAB solution according to the instructions before use.

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12. Remove the skin and muscle tissue by dissection immediately. Preferably, for 4% PFA, use at least 10 times the wet weight of the tissue at 4 °C with slow shaking. If this process is insufficient, it will be challenging to obtain good results due to poor fixation. 13. Change the 10% EDTA solution every 3 or 4 days. Two weeks after initiation of decalcification, the extent of decalcification can be confirmed by inserting needles into the tibia and femur. If decalcification is sufficient, proceed to the following procedure after cutting the tibia and femur. 14. The time course of the procedure is fixed within the same experiment. 15. RA model mice showed increased synovial tissue angiogenesis, lymphocyte infiltration, and pannus formation in the lateral knee joint. Unifying the trimming direction on the sagittal plane between samples is necessary. 16. Cooled paraffin blocks facilitate thin sectioning. A thickness section of approximately 4 μm is recommended. 17. During antigen retrieval in IHC protocols, tissues, including bone, are more easily detached from slides. Coated glass slides must be used for this procedure. 18. The color levels of old or new hematoxylin are different. Adjust the staining time on a case-by-case basis. 19. During hydration, particularly in 70–90% ethanol, eosin dye fades out rapidly from the tissue. After confirmation of eosin contrast, the tissue is dehydrated in absolute ethanol. 20. The process is an enzymatic reaction; therefore, the staining characteristics are affected by temperature and time control. It is necessary to check the slides using a microscope. 21. If a lot of dye debris is observed on the tissue, wash the slide with PBS containing 0.05~0.1% Tween 20. 22. For many FFPE tissues, antigen retrieval is required due to the cross-linkage of antigen structures by formaldehyde [1, 11]. Our facility uses a pressure cooker for heat treatment. The same result can be obtained using a microwave instead of a pressure cooker [9]. 23. A waterproof pen, such as a Dako Pen (Dako), is better for drawing circles around the tissue. This procedure prevents the drying of the tissue and minimizes the amount of reagents. 24. The blocking buffer blocks nonspecific binding in the subsequent process through the hydrophobic interaction between the tissue and the antibody. 25. In some cases, samples were treated with the primary antibody for 1 h at room temperature.

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26. Prepare the DAB reagent prior to this procedure. Ensure reaction time under the microscope. 27. It is recommended to weakly stain the nucleus to observe DAB-reacted IHC slides. Also, a 0.01% methyl green solution is sometimes recommended for counterstaining. References 1. Berod A, Hartman BK, Pujol JF (1981) Importance of fixation in immunohistochemistry: use of formaldehyde solutions at variable pH for the localization of tyrosine hydroxylase. J Histochem Cytochem 29:844–850 2. McLean IW, Nakane PK (1974) Periodatelysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem 22:1077–1083 3. Miller RT, Swanson PE, Wick MR (2000) Fixation and epitope retrieval in diagnostic immunohistochemistry: a concise review with practical considerations. Appl Immunohistochem Mol Morphol 8:228–235 4. Mori S, Sawai T, Teshima T et al (1988) A new decalcifying technique for immunohistochemical studies of calcified tissue, especially applicable to cell surface marker demonstration. J Histochem Cytochem 36:111–114 5. Mukai K, Yoshimura S, Anzai M (1986) Effects of decalcification on immunoperoxidase staining. Am J Surg Pathol 10:413–419 6. Savi FM, Brierly GI, Baldwin J et al (2017) Comparison of different decalcification methods using rat mandibles as a model. J Histochem Cytochem. https://doi.org/10.1369/ 0022155417733708 7. Hashizume K, Hatanaka Y, Kamihara Y et al (2001) Automated immunohistochemical staining of formalin-fixed and paraffinembedded tissues using a catalyzed signal amplification method. Appl Immunohistochem Mol Morphol 9:54–60 8. Morgan JM, Navabi H, Schmid KW et al (1994) Possible role of tissue-bound calcium ions in citrate-mediated high-temperature antigen retrieval. J Pathol 174:301–307 9. Sabattini E, Bisgaard K, Ascani S et al (1998) The EnVision++ system: a new immunohistochemical method for diagnostics and research. Critical comparison with the APAAP, ChemMate, CSA, LABC, and SABC techniques. J Clin Pathol 51:506–511 10. Shi SR, Cote RJ, Taylor CR (1997) Antigen retrieval immunohistochemistry: past, present, and future. J Histochem Cytochem 45:327– 343

11. Shi SR, Liu C, Balgley BM et al (2006) Protein extraction from formalin-fixed, paraffinembedded tissue sections: quality evaluation by mass spectrometry. J Histochem Cytochem 54:739–743 12. Liu S, Kiyoi T, Takemasa E et al (2015) Systemic lentivirus-mediated delivery of short hairpin RNA targeting calcium releaseactivated calcium channel 3 as gene therapy for collagen-induced arthritis. J Immunol 194:76–83 13. Kawamoto T, Shimizu M (2000) A method for preparing 2- to 50-micron-thick fresh-frozen sections of large samples and undecalcified hard tissues. Histochem Cell Biol 113:331– 339 14. Curran RC, Gregory J (1978) Demonstration of immunoglobulin in cryostat and paraffin sections of human tonsil by immunofluorescence and immunoperoxidase techniques. Effects of processing on immunohistochemical performance of tissues and on the use of proteolytic enzymes to unmask antigens in sections. J Clin Pathol 31:974–983 15. Finley JC, Grossman GH, Dimeo P et al (1978) Somatostatin-containing neurons in the rat brain: widespread distribution revealed by immunocytochemistry after pretreatment with pronase. Am J Anat 153:483–488 16. Pileri SA, Roncador G, Ceccarelli C et al (1997) Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol 183:116–123 17. Reading M (1977) A digestion technique for the reduction of background staining in the immunoperoxidase method. J Clin Pathol 30: 88–90 18. Adams JC (1992) Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J Histochem Cytochem 40: 1457–1463 19. Hsu SM, Raine L, Fanger H (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577– 580

Histological Analyses of Arthritic Joints in Collagen-Induced Arthritis. . . 20. Kammerer U, Kapp M, Gassel AM et al (2001) A new rapid immunohistochemical staining technique using the EnVision antibody complex. J Histochem Cytochem 49:623–630 21. Shi ZR, Itzkowitz SH, Kim YS (1988) A comparison of three immunoperoxidase techniques for antigen detection in colorectal carcinoma tissues. J Histochem Cytochem 36:317–322

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22. Sternberger LA, Hardy PH Jr, Cuculis JJ et al (1970) The unlabeled antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidaseantihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 18:315–333

Chapter 8 Preparation of Joint Extracts Shuang Liu and Erika Takemasa Abstract Since mice are widely used to establish rheumatoid arthritis models, assessment of the pathogenesis of local arthritis is fundamental. Proteins are the most diverse group of biologically important molecules and are essential for cellular structure and function. The first step in pathogenesis-related protein analysis is joint tissue extraction. Unlike other large rodents, obtaining synovium from model mice is challenging since it is so small and fragile. In this chapter, methods for harvesting synovium through a quadriceps approach and preparing protein extracts are introduced. Key words Synovium, Joint tissue, Cell isolation, Protein extracts, Mouse

1

Introduction The major pathological changes that occur in rheumatoid arthritis (RA) include synovitis and resulting articular cartilage and bone damage, which ultimately lead to articular deformation. Morphological, cytological, and molecular assessment of arthritis joints is fundamental in preclinical studies of RA. The knee joint, between the femur and tibia, and the ankle joint, located in the lower limb and formed by the tibia, fibula, and talus, are often used for studies in mouse arthritis models [1–4]. Roughly dissected whole joints include cartilage, synovium, ligament, tendon, bone, and connective tissue. For cytological or molecular analysis, it is necessary to limit the study target to a specific tissue, such as synovium, which is especially utilized for studies of the inflammatory system and synovium-derived cells [4, 5], or cartilage, which facilitates the study of proteoglycans [6]. Evaluation of molecular components using synovial extracts enables assessment of the initial local pathological impact on arthritis joints. Proteins are the most diverse group of biologically important molecules and are essential for cellular structure and function. The first step in pathogenesis-related protein analysis is tissue

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extraction. Since protein extraction techniques vary depending on the source of the starting material, the location within the cell of the protein of interest, and the downstream application, there are various protocols for protein extraction [7]. Many techniques have been developed to obtain the best protein yield and purity for different types of cells or tissues, taking into account, where appropriate, the subcellular location of the protein and the compatibility of the protein extract with the next step in the experiment. The synovium is a thin membrane that lines the inside of synovial joints. To obtain a sufficient amount of synovium from mice is challenging since mice have a small amount of intra-articular synovial tissue. Unlike that in other large rodents, such as rats and rabbits, the mouse synovium is too tiny and fragile to be isolated by a common lateral approach to the knee joint. In this chapter, aiming at the assessment of the inflammatory status and establishment of the cytokine profile of joints, a method for harvesting mouse synovium through a quadriceps approach and preparation of synovial extracts in a mouse model is introduced [8, 9].

2

Materials (See Note 1) All animal experiment protocols should be performed in accordance with the guidelines of the Institutional Animal Care and Use Committee and approved by the committee. 1. Animals: Male DBA/1JNCrlj mice (6–10 weeks of age; CLEA Japan, Tokyo, Japan), which are available for the preparation of a collagen-induced arthritis model, are used in this protocol. 2. Operating table. 3. Stereomicroscope (for example, Stemi 305, Carl Zeiss Microscopy GmbH). 4. Microsurgical forceps (fine blunt) and scissors (fine dissection). 5. Homogenizer. 6. Round-bottom test tubes, 14 mL. 7. Syringes, 1 mL. 8. 100-mm Petri dishes. 9. Pentobarbital sodium (see Note 2). 10. 70% ethanol. 11. Saline. 12. Lysis buffer: A mixture of T-PER Tissue Protein Extraction Reagent (Thermo Scientific) and Halt™ Protease Inhibitor Cocktail (100×) (Thermo Scientific) at its final concentration should be prepared just before tissue lysis (see Notes 3 and 4).

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Methods

3.1 Harvesting Synovium from Mouse Knee Joint

This quadriceps approach method for harvesting synovium was originally described by Futami et al. [8]. 1. Perform euthanasia by intraperitoneal injection of an overdose of pentobarbital sodium (120 mg/kg). 2. Fix the mouse in the supine position with legs extended on the operating table. 3. Expose the knee joint through a midline skin incision. 4. Transversely resect the middle of the quadriceps (Fig. 1a, b) (see Note 5). 5. Under a stereomicroscope, reverse the quadriceps and distinguish the space between the patella and the patellar ligament (Fig. 1c). 6. Resect the synovium on the infra-patellar fat pad attached to the patellar ligament (Fig. 1d). 7. Transfer the harvested synovium to a 100-mm Petri dish and quickly wash it with saline.

3.2 Protein Extraction (See Note 6)

1. Weigh freshly harvested mouse synovium and put it into 14-mL round-bottom test tubes filled with an appropriate volume of pre-chilled lysis buffer (see Note 7). If T-PER Tissue Protein Extraction Reagent is used, the ratio of tissue and lysis buffer should be 1:20 (w/v) (see Note 8). 2. Vortex tubes briefly and proceed to homogenization using a homogenizer for 10 s. 3. Vortex samples and check the sample visually for disruption. If not present, repeat step 2 two or three times. The shortest homogenization time should be chosen to prevent protein degradation. 4. Transfer the supernatant to a new tube and centrifuge samples at 20,000 g for 15 min at 4 °C to remove any remaining insoluble material. 5. Take an aliquot for protein quantification (see Note 9). To avoid freeze-thaw damage, separate the synovial extracts into small aliquots in individual tubes and store at -80 °C until further analysis.

4

Notes 1. All equipment conditions should be up to the study purpose and following steps of the experiments. If synovial extracts are used for protein analysis, the equipment should be used under

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Fig. 1 Images of a mouse knee for harvesting synovium. The knee joint was exposed via a midline skin incision (a). The quadriceps was transversely resected in the middle (b) and reversed distally (c). At the distal femur, the reversed patella and patellar ligament were exposed. (d) The synovium on the infra-patella fat pad could be easily visualized for resection

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protease- and phosphatase-free conditions. If further nucleotide extraction is planned, RNase- and/or DNase-free conditions are required. 2. As the anesthetic used for euthanasia by over-dose, a combination anesthetic can also be prepared with 0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol. 3. It is possible to use RIPA buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% NP-40, and 0.25% Na-deoxycholate as a starting point for optimization. It is always recommended to optimize the buffer composition depending on the specific research project. 4. Sodium lauryl sulfate (SDS) can be added to the extraction buffer to maximize the yield of soluble proteins. SDS extracts can be used for SDS electrophoresis and western blotting. In the case of 2D electrophoresis, enzyme-linked immunosorbent assay, and mass spectrometry, the SDS concentration should be reduced. 5. Since the mouse synovium is very small and fragile, it is difficult to isolate it using a common lateral approach to the knee joint. 6. This protocol has been verified with up to 100 mg of tissue. For larger quantity preparation, cut the tissue up and proceed to disruption in separate tubes. 7. Harvested tissues can be snap-frozen in liquid nitrogen and stored at -80 °C until protein extraction. 8. The ratio of tissue and lysis buffer should be optimized according to the instructions for the extraction reagent being used. If RIPA buffer is used, the ratio of tissue and lysis buffer should be 1:10 (w/v). 9. There are many available protein quantification assays, such as absorbance at 280 nm, Lowry Assay, Bradford Assay, Bicinchoninic Assay (BCA), etc. It should be noted that measuring the protein concentration in SDS extract requires that the assay is compatible with the detergent and reducing agent in the solution. References 1. Caplazi P, Baca M, Barck K, Carano RA, DeVoss J, Lee WP et al (2015) Mouse models of rheumatoid arthritis. Vet Pathol 52:819–826 2. Herman S, Fischer A, Presumey J, Hoffmann M, Koenders MI, Escriou V et al (2015) Inhibition of inflammation and bone erosion by RNA interference-mediated silencing of heterogeneous nuclear RNP A2/B1 in two experimental

models of rheumatoid arthritis. Arthritis Rheum 67:2536–2546 3. Liu S, Kiyoi T, Takemasa E, Maeyama K (2017) Intra-articular lentivirus-mediated gene therapy targeting CRACM1 for the treatment of collagen-induced arthritis. J Pharmacol Sci 133:130–138 4. Liu S, Kiyoi T, Takemasa E, Maeyama K (2015) Systemic lentivirus-mediated delivery of short

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hairpin RNA targeting calcium release-activated calcium channel 3 as gene therapy for collageninduced arthritis. J Immunol 194:76–83 5. Roelofs AJ, Zupan J, Riemen AHK, Kania K, Ansboro S, White N et al (2017) Joint morphogenetic cells in the adult mammalian synovium. Nat Commun 8:15040 6. Rostand KS, Baker JR, Caterson B, Christner JE (1982) Isolation and characterization of mouse articular cartilage proteoglycans using preformed CsCl density gradients in the Beckman Airfuge. A rapid semi-micro procedure for proteoglycan isolation. J Biol Chem 257:703–707

7. Pena-Llopis S, Brugarolas J (2013) Simultaneous isolation of high-quality DNA, RNA, miRNA and proteins from tissues for genomic applications. Nat Protoc 8:2240–2255 8. Futami I, Ishijima M, Kaneko H, Tsuji K, Ichikawa-Tomikawa N, Sadatsuki R et al (2012) Isolation and characterization of multipotential mesenchymal cells from the mouse synovium. PLoS One 7:e45517 9. Zhao J, Ouyang Q, Hu Z, Huang Q, Wu J, Wang R et al (2016) A protocol for the culture and isolation of murine synovial fibroblasts. Biomed Rep 5:171–175

Part II Therapeutic Approach

Chapter 9 Production of Immunizing Antigen Proteoliposome Using Cell-Free Protein Synthesis System Wei Zhou and Hiroyuki Takeda Abstract Antibodies specifically recognizing integral membrane proteins are essential tools for functional analysis, diagnosis, and therapeutics targeting membrane proteins. However, developing antibodies against membrane proteins remains a big challenge because mass production of membrane proteins is difficult. Recently, we developed a highly efficient cell-free production method of proteoliposome antigen using a cell-free protein synthesis method with liposome and dialysis cup. Here, we introduce practical and efficient integrated procedures to produce a large amount of proteoliposome antigen for anti-membrane protein antibody development. Key words Proteoliposome, Membrane protein, Cell-free protein synthesis, Immunizing antigen, Adjuvant

1

Introduction Antibodies that specifically recognize integral membrane proteins are essential tools for the functional analysis of such proteins, including their temporal and spatial expression, stabilization, and functional regulation [1–3]. They are also gaining attention due to their potential applications in the diagnosis and therapeutics targeting membrane proteins [4, 5]. However, the development of antibodies against these proteins remains challenging. A primary reason is the difficulty associated with producing membrane proteins. For the generation of high-quality antibodies, several milligrams of antigen maintaining its natural conformation are needed for immunization and screening. Proteoliposomes, lipid vesicles onto which membrane proteins are anchored, are often used both for immunization and as screening antigens. It’s believed that membrane protein antigens on proteoliposomes, when highly concentrated and purified, can effectively stimulate the immune response and induce antibodies against the membrane protein antigen [5–

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7]. Nonetheless, preparing proteoliposomes suitable for immunization is labor-intensive. Overexpressing recombinant membrane proteins in cellular systems proves challenging due to the complex structure and translocation of these proteins. Optimizing cell culture conditions, stabilization, solubilization, purification of the target membrane protein, and reconstitution of the proteoliposome can be time-consuming and demanding [8–10]. In this chapter, we introduce a highly efficient method for in vitro (cell-free) production of proteoliposomes. When a lipid vesicle liposome is added to a cell-free translation system, the synthesized membrane protein spontaneously integrates with the liposome lipid membrane. A variety of integral membrane proteins, such as Gprotein-coupled receptors, transporters, ion channels, and claudins, can be synthesized using this cell-free system in conjunction with liposomes [11–17]. One advantage of cell-free membrane protein synthesis is the direct synthesis of the membrane protein on a liposome, eliminating the need for tedious steps such as the purification of membrane proteins and the reconstitution of proteoliposomes. Cell-free synthesized proteoliposomes can be easily purified through centrifugation with a buffer wash. Additionally, we have developed the bilayer-dialysis method, which employs a dialysis cup device to enhance translation efficiency [17]. In this method, a cup-type dialysis device is immersed in a substrate feeding buffer, with the translation reaction mixture and substrate feeding buffer forming a bilayer within the cup (Fig. 1). The feeding of translation substrates and the removal of byproducts proceed efficiently at both the top and bottom of the reaction mixture. The bilayer-dialysis method has significantly improved translation efficacy, allowing for the synthesis of a variety of membrane proteins with a high success rate (Fig. 2).

Fig. 1 Proteoliposome production by bilayer-dialysis method. (a) Schematic illustration of bilayer-dialysis method. (b) Large-scale proteoliposome production using a 2-mL dialysis cup and 25-mL centrifuge tube

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Fig. 2 Production of GPCRs using bilayer dialysis method. A total of 25 GPCRs were synthesized by the smallscale bilayer-dialysis method. Proteoliposomes were purified by centrifugation and buffer washing. Purified proteoliposomes were applied to SDS-PAGE and CBB staining. Arrowheads indicate recombinant GPCRs. (This figure was reproduced from Ref. 16, which licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0))

Cell-free membrane protein synthesis using the bilayer-dialysis method can produce several milligrams of membrane protein in a short time, sufficient for immunization, with a high success rate. We’ve successfully obtained monoclonal antibodies, including high-affinity, conformation-sensitive, flow-cytometry applicable, and inhibitory antibodies, using cell-free synthesized proteoliposomes as antigens [14, 15, 17]. This chapter details our integrated approach to proteoliposome production (Fig. 3). The initial step involves a small-scale expression test for the target membrane protein antigen to check the quality and productivity of the cell-free synthesized protein. Then, mass production is planned based on the productivity confirmed in small-scale trials. Both the small-scale test and mass production are achievable within 3 days, respectively. The wheat cell-free system offers both high reproducibility and scalability, maintaining consistent production efficiency from small to large scales. Therefore, a small-scale test is crucial to gauge production efficiency, assisting in protocol design and cost estimation for large-scale production. Additionally, we present a method for preparing adjuvant-containing liposomes, incorporating the lipid adjuvant monophosphoryl lipid A (MPLA) [15]. This enables the synthesis of the membrane protein antigen directly on these liposomes, making the resultant proteoliposomes ready for direct immunization. We hope our cell-free methods address challenges in developing antibodies against membrane proteins.

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Fig. 3 Overview of proteoliposome antigen production

2 Materials Prepare all solutions using analytical grade reagents and freshly prepared ultrapure water. We prepare ultrapure water using tandemly connected Elix UV and Milli-Q Direct (Merck Millipore). Prepare and store all reagents at room temperature (unless indicated otherwise). 2.1 Construction of Transcription Templates for CellFree Protein Synthesis

1. pEU-E01-MCS vector (CellFree Sciences, Ehime, Japan). 2. Forward primer for pEU vector linearization (Primer 1): 5′-tctggagctagtgctggaGGTACCTGTCCGCGGTCG-3′ Sequence indicated by uppercase is derived from pEU-E01MCS vector. Underlined part shows KpnI site. Lowercase shows T1 linker sequence. 3. Reverse primer for pEU vector linearization (Primer 2): 5′-tggtggtggtgggtggcgGATATCTTGGTGATGT AGATGGTGGTT-3′

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Sequence indicated by uppercase is derived from pEU-E01MCS vector. Underlined part shows EcoRV site. Lowercase shows S1 linker (complementary sequence) 4. PrimeStarMAX DNA polymerase (Takara Bio). 5. FastDigest DpnI (Thermo Fisher Scientific). 6. TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.3). 7. 1% agarose/TAE gel. 8. GeneRuler 1 kb DNA Ladder marker (Thermo Fisher Scientific). 9. SYBRsafe gel stain reagent (Thermo Fisher Scientific). 10. PCR product purification kit. 11. Synthetic DNA fragment. DNA fragment should contain S1 linker (cgc_cac_cca_cca_cca_cca), open reading frame with start codon and stop codon, and T1 linker (tct_gga_gct_agt_gct_gga) (see Note 1). 12. Plasmid harboring cDNA clone of interest. Human cDNA clones are available from several organizations and distributors such as ATCC, Thermo Fisher Scientific, Promega, Origene, Genecopoeia, and Addgene. 13. Gene-specific forward primer (Primer 3): 5′-cgccacccaccaccaccaNNNNNNNNNNNNNNNNNNNN-3′ Here, lowercase shows S1 linker, and NNNN. . . shows a genespecific nucleotide sequence (20–25 bp, Tm > 55 °C). 14. Gene-specific reverse primer (Primer 4): 5′-tccagcactagctccagaNNNNNNNNNNNNNNNNNNNN3′ Lowercase shows T1 linker (complementary), and NNNN. . . shows a gene-specific nucleotide sequence (20–25 bp, Tm > 55 °C). 15. Gibson Assembly master mix (NEB). 16. Chemical competent cells of E. coli strain JM109. 17. LB-ampicillin agar plate. 18. LB medium. 19. Sequencing primer from 5′ side of MCS in pEU plasmid (SPu-2, Primer 5): 5′-CAGTAAGCCAGATGCTACAC-3′ 20. Sequencing primer from 3′ side of MCS in pEU plasmid (SP-A1868, Primer 6): 5′- CCTGCGCTGGGAAGATAAAC-3′ 21. NucleoBond Xtra Midi (Takara Bio). 22. Phenol, chloroform, isoamyl alcohol (25:24:1).

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23. Chloroform. 24. Ethanol. 25. 7.5 M ammonium acetate. 26. 70% ethanol. 2.2 Cell-Free Proteoliposome Synthesis Using Bilayer-Dialysis Method

1. 5× Transcription Buffer LM, 25 mM NTP mixture (CellFree Sciences). 2. 80 U/μL RNase inhibitor (CellFree Sciences). 3. 80 U/μL SP6 polymerase (CellFree Sciences). 4. Freshly prepared ultrapure water (see Note 2). 5. Clean plastic tubes and chips (see Note 3). 6. Cooled incubator with temperature ranging from 0 to 40 °C or wider. 7. Wheat germ extract WEPRO7240 (CellFree Sciences) (see Note 4). 8. ×1 SUB-AMIX SGC feeding buffer. Dilute ×40 SUB-AMIX SGC stock solutions (S1–S4) (CellFree Sciences) with ultrapure water. 9. 20 mg/mL creatine kinase (see Note 5). 10. Lyophilized asolectin liposome (CellFree Sciences). 11. Slide-A-Lyzer MINI Dialysis Device, 10K molecular weight cut off, 10–100 μL (Thermo Fisher Scientific). 12. Phosphate-buffered saline (PBS). 13. ×3 SDS-PAGE 2-mercaptoethanol.

sample

buffer

containing

10%

14. SDS-PAGE gel. 15. SDS-PAGE running buffer. 16. BSA standards. BSA is dissolved in ×1 SDS-PAGE sample buffer at 1000 ng, 500 ng, 250 ng, and 125 ng/10 μL concentration. 17. Protein size marker. 18. CBB staining dye. 19. Kimwipes. 2.3 Preparation of Adjuvant-Containing Liposome

1. Lyophilized asolectin liposome (CellFree Sciences). 2. Monophosphoryl Lipid A, Synthetic (MPLA, Avanti Polar Lipids). 3. Chloroform. 4. Microman, M100 and M1000 (Gilson). 5. Evaporation flask, 100 mL.

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6. Rotary evaporator. 7. Vacuum desiccator and diaphragm pump. 2.4 Large-Scale Proteoliposome Antigen Production

In addition to the reagents shown in Subheading 2.2, the following materials are required. 1. Slide-A-Lyzer MINI Dialysis Device, 10K molecular weight cut off, 2 mL (Thermo Fisher Scientific). 2. 25-mL self-standing centrifuge tube.

3

Methods

3.1 Construction of Expression Plasmid for Cell-Free Protein Synthesis

Insert genes of interest into pEU-E01-MCS vector. You can conduct plasmid construction using either conventional restriction enzyme or Gibson Assembly (see Note 6). Here, we outline the procedure utilizing Gibson Assembly (Fig. 4) [18]. 1. Linearize pEU-E01-MCS vector and add S1 and T1 overlap linkers by inverse PCR. Prepare PCR reaction by mixing 25 μL of PrimeStarMax polymerase mix, 10 μL of 1 μM Primer 1, 10 μL of 1 μM Primer 2, and 5 μL of 0.5 ng/μL pEU-E01-MCS vector, and apply the reaction to thermal cycler (denaturing at 98 °C for 10 s, annealing at 55 °C for 5 s, extension at 72 °C for 30 s, 25 cycles). 2. Confirm the amplified inverse PCR product (3.7 kb) by gel electrophoresis. Apply the PCR product and DNA size marker to 1% agarose/TAE gel. After electrophoresis, visualize DNA using SYBRsafe gel stain reagent.

Fig. 4 Scheme of cell-free expression plasmid construction using Gibson Assembly

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3. Add 1 μL of FastDigest DpnI to the PCR reaction mixture, and incubate the reaction for 30 min at 37 °C to eliminate the PCR template plasmid (see Note 7). 4. Purify the PCR product using a PCR product purification kit. 5. Measure the concentration of the purified linearized vector by measuring absorbance at 260 nm, and adjust the concentration to 50 ng/μL. 6. Prepare insert DNA fragment of interest by PCR or gene synthesis. Insert DNA fragment codes mature peptide with start codon and stop codon (see Note 8). Remove intron or signal peptide sequences in advance if there are some. Add S1 and T1 overlap linker sequences at the 5′- and 3′- terminal of the open reading frame. 7. When a synthetic gene is used as an insert, dissolve the polynucleotide at 50 ng/μL by TE buffer. 8. When preparing an insert DNA by PCR reaction, mix 5 μL of PrimeStarMax polymerase mix, 2 μL of 1 μM primer 3, 2 μL of 1 μM primer 4, and 1 μL of 0.2 ng/μL template cDNA plasmid. Apply reaction mixture to thermal cycler (denaturing at 98 °C for 10 s, annealing at 55 °C for 5 s, extension at 72 °C for 10–60 s, 25 cycles). 9. After the PCR reaction, add 0.5 μL of DpnI to the PCR reaction and incubate at 37 °C for 30 min. Confirm amplification of the target gene by electrophoresis. 10. Purify the PCR product using a PCR product purification kit, and adjust concentration (see Note 9). 11. Assemble the plasmid by mixing 1 μL of insert DNA fragment, 1 μL of linearized vector, and 2 μL of Gibson Assembly enzyme mixture, and incubate at 50 °C for 15 min to 1 h. 12. Transform E.coli strain JM109 with the assembled plasmid. Add 2 μL of assembled plasmid to 40 μL of chemically competent cells. 13. Mix gently by inverting and place on ice for 20 min. Heat the tube at 42 °C for 30 s, and chill on ice for 1 min. Add 160 μL of LB medium, and spread the transformant on a LB-ampicillin agar plate. Incubate the plate at 37 °C overnight. 14. Confirm insertion of target DNA by colony direct PCR and sequencing using Primer 5 and Primer 6. 15. Culture the transformant E.coli in 150 mL of LB-ampicillin medium at 37 °C and 125 strokes per minute shaking overnight. 16. Collect E.coli by centrifugation at 8000 rpm for 5 min. Extract and purify plasmid using the NucleoBond Xtra Midi kit following the manufacturer’s instructions. Dissolve isopropanolprecipitated plasmid in 500 μL of TE buffer.

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17. Add 500 μL of phenol, chloroform, and isoamyl alcohol (25: 24:1) to the plasmid solution, shake vigorously for 5 min using a vortex mixer, and centrifuge at 15,000 rpm for 5 min. 18. Transfer the aqueous phase to a new tube, add 500 μL of chloroform, shake vigorously for 3 min using a vortex mixer, and centrifuge at 15,000 rpm for 5 min. 19. Transfer the aqueous phase to a new tube, add 1 mL of ethanol and 50 μL of 7.5 M ammonium acetate, and store at -30 °C for 1 h. 20. Centrifuge the tube at 15,000 rpm for 10 min, and wash the pellet with 500 μL of 70% ethanol. Remove 70% ethanol carefully and leave the pellet for 5 min to dry up. 21. Dissolve the plasmid in 100 μL of ultrapure water completely, and adjust the concentration to 1 mg/mL by adding ultrapure water. Store purified plasmid at -30 °C. 3.2 Small-Scale Test Proteoliposome Synthesis Using Bilayer-Dialysis Method

The purpose of this step is to test membrane protein synthesis and evaluate productivity. During cell-free protein synthesis, operators should wear disposable plastic gloves and masks to prevent RNase contamination. Only clean plastic tubes, plates, and chips should be used (see Note 3). It’s advisable to keep the plastic wares used for RNA manipulation separate from those used for DNA experiments. Do not use DEPC-treated water, as residual DEPC can strongly inhibit the reaction. Instead, the use of freshly purified ultrapure water is recommended for reagent preparation (see Note 2). 1. Turn on the air incubator and set the temperature to 37 °C. 2. Thaw ×5 Transcription Buffer LM and 25 mM NTP mixture. 3. Invert the tube several times to mix the reagents, ensuring even concentration, then briefly centrifuge. Keep all reagents and enzymes on ice during handling. 4. Dispense freshly prepared ultrapure water into a new plastic tube. 5. Prepare the transcription reaction mix by combining 14.4 μL of ultrapure water, 5 μL of ×5 Transcription Buffer LM, 2.5 μL of 25 mM NTP mixture, 2.5 μL of 1 mg/mL pEU expression plasmid, 0.32 μL of 80 U/μL RNase inhibitor, and 0.32 μL of 80 U/μL SP6 polymerase. Gently mix the reagents by inverting the tube, then briefly centrifuge. 6. Allow the transcription reaction to incubate at 37 °C for 6 h. 7. After incubation, gently mix the reaction by inverting the tube and then briefly spin down (see Note 10). 8. Verify mRNA synthesis through gel electrophoresis. Load 1 μL of the transcription reaction mixture onto a 1% TAE gel. Also, apply the DNA ladder marker to the same gel.

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9. After running the gel at 100 V for 20 min, stain the gel with SYBRsafe. 10. Examine the ladder band pattern of the mRNA. If you observe a smeared band less than 500 bp, this may indicate mRNA degradation. 11. Set the temperature of the air incubator to 15 °C. 12. Remove glycerol from the dialysis membrane of the dialysis cup. Place a new 1.5 mL tube on a tube rack. Add 1 mL of freshly made ultrapure water to the tube. Insert a 10–100 μL dialysis cup into the tube, then inject 500 μL of ultrapure water into the cup. Briefly cover the tube with its lid. 13. Incubate for 30 min at room temperature or at 4 °C overnight (see Note 11). 14. Quickly thaw the translation reagents by placing the tubes in a water bath at room temperature for a few minutes using a floater. After thawing, gently mix the reagents by inverting them, spin them down, and then place them on ice until used. 15. Prepare the ×1 SUB-AMIX SGC feeding buffer by mixing 1800 μL of ultrapure water and 50 μL of each of the ×40 SUB-AMIX SGC stock solutions (S1, S2, S3, and S4) in a tube. 16. Prepare the asolectin liposome. Remove both the outer plastic cap and the inner rubber cap from a lyophilized asolectin liposome vial (Fig. 5a). 17. Inject 200 μL of ×1 SUB-AMIX SGC, replace the inner rubber cap, and allow it to sit for 10 min at room temperature to hydrate the liposome.

Fig. 5 Materials for liposome preparation. (a) Lyophilized asolectin liposome provided from CellFree Sciences. (b) Thin layer of MPLA/asolectin mixed lipid. Empty evaporation flask (left), and evaporation flask with lipid thin layer (right)

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18. Vigorously mix the emulsion with a vortex mixer for 1 min to even out the liposome emulsion size. 19. Place the vial into a 50-mL centrifuge tube and centrifuge at 500× g for 1 min. Remove the vial and transfer the liposome suspension to a new 1.5-mL tube (see Note 12). 20. Discard the water from both the tube and the dialysis cup prepared in step 13. Inject 1.1 mL and 400 μL of ×1 SUB-AMIX SGC into the tube and dialysis cup, respectively. 21. Prepare the translation reaction by mixing 25 μL of WEPRO7240 wheat germ extract, 0.4 μL of 20 mg/mL creatine kinase, 20 μL of 50 mg/mL asolectin liposome, and 30.6 μL of ×1 SUB-AMIX SGC with 24 μL of mRNA prepared in step 7. Mix gently by inverting, and spin down. 22. Aspirate the entire translation reaction mixture (100 μL) using a pipette. 23. Insert the pipette tip into the surface of the SUB-AMIX SGC solution in the dialysis cup, and pipet out the reaction mixture slowly and gently. The reaction mixture will naturally sink and form a layer on the bottom of the well (Fig. 1a) (see Note 13). Do not mix the reaction and disturb the bilayer. Cover the dialysis cup with a lid to avoid evaporation. 24. Incubate the reaction at 15 °C for 20–24 h. 25. Transfer the proteoliposome suspension from the dialysis cup (500 μL) to a new 1.5-mL tube (see Note 14). Centrifuge the tube at 21,600× g at 4 °C for 10 min. 26. Remove the supernatant and resuspend the liposome pellet in 1 mL of PBS by pipetting. 27. Repeat centrifugation and washing steps two more times. 28. Resuspend the pellet in 100 μL of PBS. Transfer the suspension to a new 1.5-mL tube. 29. Mix 10 μL of synthesized and purified proteoliposome suspension, 10 μL of water, and 10 μL of ×3 SDS-PAGE sample buffer. Do not apply the sample to heat denaturation (see Note 15). 30. Load 6 μL of the liposome sample onto an SDS-PAGE gel (i.e., 2 μL of purified proteoliposome). In addition, load 2 μL of protein size standard and 10 μL of each BSA standard (1000 ng to 125 ng 10 μL/lane, respectively) to the same gel. 31. Electrophorese at 52 mA, 400 V for 30 min. 32. Remove the gel from the gel cassette, transfer the gel to a plastic box. Pour the CBB dye into the box to stain the gel. 33. Shake gently on a shaker for 1 h at room temperature.

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34. Remove the CBB dye and wash the gel with tap water several times. 35. Pour hot water (60–70 °C) into the box. Cover the gel with Kimwipes and gently shake the box. 36. Continue to replace the hot water while checking for decolorization. When the bands become obvious and the background is clear, stop decolorization and scan the gel image with a gel scanner. 37. Estimate the quantity of the target membrane protein applied to the gel by comparing the intensity of the target band with that of the BSA standard series. 38. Calculate the productivity of the target membrane protein using the bilayer-dialysis method. Protein synthesis efficiency is evaluated as the amount (mg) of synthesized protein per 1 mL wheat germ extract (WGE), which can be calculated from the band intensity in the CBB stained gel using the following format (synthesized on a small scale). y=a÷b÷c×x where, y: productivity (mg protein/mL WGE), x: amount of target membrane protein applied to SDS-PAGE gel (mg/lane) estimated in step 20, a: amount of purified proteoliposome (0.100 mL), b: amount of purified proteoliposome applied to SDS-PAGE (0.002 mL), and c: amount of WEPRO7240 WGE (0.025 mL) (see Note 16). 3.3 Preparation of Adjuvant-Containing Liposome

MPLA serves as a type of adjuvant lipid. A liposome can be formulated using a mixture of MPLA and asolectin. Utilizing the cellfree system, the membrane protein antigen can be synthesized on MPLA/asolectin liposomes. The resulting adjuvant-containing proteoliposomes can be directly injected into a host animal for immunization. This eliminates the need for vigorous and extended mixing with Freund’s adjuvant, which can be too harsh for sensitive membrane proteins. In a prior study, we confirmed that MPLAcontaining CLDN5 proteoliposomes efficiently stimulate the immune system of the host animal, eliciting antibodies against CLDN5 [15]. We recommend adding a weight of MPLA equal to the expected synthesis of the target protein to the liposomes. Here, we demonstrate the process of preparing MPLA liposomes to produce 1 mg of membrane protein antigen, leveraging the wheat cellfree system with a 1 mg/mL WEPRO7240 production efficacy. 1. Dissolve asolectin in chloroform. Add 200 μL of chloroform to each of the 5 vials containing 10 mg of lyophilized asolectin liposome (see Note 17). Once fully dissolved, combine the asolectin solutions into a single vial. The asolectin/chloroform solution can be stored at -30 °C.

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2. Add chloroform to the MPLA-containing vial to achieve a concentration of 10 mg/mL. Dissolve completely and store the MPLA/chloroform solution at -30 °C. 3. Combine 1000 μL of asolectin/chloroform solution with 125 μL of 10 mg/mL MPLA in an evaporation flask. 4. To prepare a lipid thin layer, use a rotary evaporator to evaporate the chloroform. Ensure that the asolectin/MPLA mixed lipid spreads evenly on the wall, about one-third up from the bottom of the flask (Fig. 5b). Avoid forming lipid clumps. 5. Place the flask in a vacuum desiccator and leave it overnight to ensure complete removal of chloroform. 6. Add 1000 μL of ×1 SUB-AMIX SGC to the evaporation flask. Swirl the flask to ensure the SUB-AMIX SGC covers the thin lipid layer. 7. Allow the flask to stand for 5 min to hydrate the lipid layer and facilitate liposome formation. 8. Shake the flask vigorously with a vortex. Sonicate the flask using an water-bath type ultrasonic cleaner. Repeat vortex mixing and sonication until the thin lipid layer detaches from the bottom, ensuring the emulsion becomes completely homogeneous (see Note 18). 9. Transfer the 50 mg/mL MPLA/asolectin liposome to a new 1.5 mL tube. If not using the liposome immediately for translation, flash freeze it with liquid nitrogen and store at -80 °C. 3.4 Large-Scale Proteoliposome Antigen Production

As an example, we present a protocol for producing 1 mg of membrane protein antigen with a production efficiency of 1 mg/ mL. Adjust the reaction volume and the number of dialysis cups based on the production efficiency of the target antigen membrane protein and the sample quantity needed for immunization. 1. Turn on the air incubator and set the temperature at 37 °C. Thaw reagents for transcription quickly. Prepare fresh ultrapure water in a new tube. 2. Prepare transcription reaction mix by mixing 610 μL of ultrapure water, 213 μL of Transcription Buffer LM, 106 μL of 25 mM NTP mixture, 106 μL of 1 mg/mL pEU expression plasmid, 13.5 μL of 80 U/μL RNase inhibitor, and 13.5 μL of 80 U/μL SP6 polymerase. Mix the reaction gently by inverting, and then spin down. Incubate the transcription reaction at 37 °C for 6 h. Confirm mRNA synthesis with gel electrophoresis. 3. Set the air incubator’s temperature to 15 °C.

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4. Thaw translation reagents quickly by floating the tubes in a floater in a water bath at room temperature. Place the reagents on ice. 5. Prepare 200 mL of ×1 SUB-AMIX SGC feeding buffer. Place 5 of the 50-mL tubes on a tube rack. In each tube, add 36 mL of ultrapure water and 1 mL of each ×40 SUB-AMIX SGC stock solution of S1–S4, respectively (totally 4 mL). Mix well by inverting. 6. Place 8 of the 25-mL tubes. Pour 22 mL of ×1 SUB-AMIX SGC into the tubes, respectively. Insert dialysis cups into the tubes, confirming the dialysis membrane immersed in the SUB-AMIX SGC buffer. Inject 2 mL of ×1 SUB-AMIX SGC in the cups (Fig. 1b) (see Note 19). Cover the tube with a lid. 7. Prepare translation reaction by adding 1062 μL of WEPTRO7240 wheat germ extract, 17 μL of 20 mg/mL creatine kinase, 850 μL of 50 mg/mL MPLA/asolectin liposome, and 1259 μL of ×1 SUB-AMIX SGC to 1062 μL of mRNA prepared in step 2. Mix gently by pipetting. 8. Aspirate 500 μL of the translation reaction mixture. Insert the pipet tip into the surface of SUB-AMIX SGC solution in a dialysis cup. Pipet out the reaction mixture slowly and gently (Fig. 1a). Do not mix the reaction and disturb the bilayer. 9. Repeat the injection process for the other cups. Cover the dialysis cup with a lid to avoid evaporation. 10. Incubate the reaction at 15 °C for 24 h. 11. Sterilize 50 mL of PBS using a 0.22-μm syringe filter. 12. Mix the reaction in the dialysis cups (2.5 mL) thoroughly using a pipette, and then transfer and combine all the proteoliposome suspensions into a new 50-mL tube. 13. Centrifuge the tube at 21,600× g, 4 °C for 10 min. Discard the supernatant and re-suspend the proteoliposome pellet in 10 mL of sterilized PBS by pipetting. 14. Repeat the centrifugation and washing of the proteoliposome two more times. 15. After washing, add 700 μL of PBS and thoroughly resuspend the proteoliposome pellet using a pipette. Measure the suspension’s volume with a micropipette, and then adjust to 1 mL with PBS. Transfer the suspension to a new 1.5-mL tube and store it at 4 °C. 16. Transfer 10 μL of proteoliposome suspension to a new 1.5-mL tube. Add 70 μL of water and 40 μL of ×3 SDS-PAGE sample buffer. 17. Load 3 μL, 6 μL, and 12 μL of the sample onto the SDS-PAGE gel (i.e., 0.25 μL, 0.5 μL, and 1 μL of proteoliposome

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suspension are applied to each lane, respectively). Also, load the protein size standard and BSA standard series. 18. Electrophorese at 52 mA, 400 V for 30 min. Stain the gel using CBB dye, decolorize it with hot water, and then scan the gel image. 19. Estimate the amount of the antigen membrane protein in each lane by comparing the intensity of the target band with those of the BSA standard series. Also, calculate the protein concentration in the proteoliposome suspension. 20. Aliquot the antigen proteoliposome suspension into several tubes based on the immunization plan. Flash-freeze the tubes in liquid nitrogen and store them at -80 °C until needed. Avoid repeated freeze-thaw cycles.

4

Notes 1. There is no need to insert the Kozak sequence before the start codon. Codon optimization is not required for the target gene because the wheat translation machinery doesn’t have a preference for specific codons. If the gene of interest has extremely high or low GC content, codon conversion effectively improves translation efficacy [15]. 2. Commercially-available DNase/RNase-free water is applicable for transcription and translation. However, pay attention to contamination after the bottle is opened, especially when stored for a long time. Do not use DEPC-treated water because residual DEPC strongly inhibits the reaction. We recommend using freshly purified ultrapure water. 3. We do not recommend autoclaving plastic wares for transcription and translation. Autoclave treatment cannot denature RNase. Furthermore, the autoclave of plastic ware may cause deformation, and aerosols from the autoclave can cause contamination. Sterilized disposable plastic ware is desirable. 4. WEPRO7240 should be stored at -80 °C. It can withstand several freeze/thaw cycles. 5. The wheat cell-free synthesis kit contains 20 mg/mL creatine kinase. However, repeated freezing and thawing of creatine kinase solution is not recommended. Lyophilized creatine kinase is available from Roche Diagnostics (catalog number: 04524977190). Add ultrapure water to a final concentration of 20 mg/mL, and solubilize completely. Dispense the solution into small portions in PCR tubes (10–50 μL each). Freeze the tubes using liquid nitrogen and store them at -80 °C. Avoid re-freezing after thawing.

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6. If a DNA fragment is inserted into the pEU-E01-MCS vector using a restriction enzyme, connect the 5′-terminal of the inserted DNA to one of the enzyme sites, EcoRV, SpeI, XhoI, SacI, and KpnI. Higher translation efficacy is expected by placing translation enhancer E01 sequence and start codon. Any restriction site in multiple cloning sites is applicable for connecting 3′- terminal of insert DNA. 7. DpnI recognizes and digests methylated GATC sites. Here, DpnI is used to digest and remove template plasmid in PCR reaction. PCR product is not digested because it is not methylated. There is no need to add DpnI reaction buffer because DpnI is active under common PCR buffer conditions. 8. Processing of mRNA or peptide is not conducted in wheat cellfree systems. 9. A 2–3 times higher concentration (in a mol:mol ratio) of insert DNA is recommended. 10. After transcription, a white precipitate is observed. This precipitate mainly consists of insoluble magnesium pyrophosphate, a byproduct of transcription, and also contains some amount of mRNA. Mix well supernatant and precipitate and add the mixture to the translation reaction, rather than removing the precipitate. 11. The dialysis membrane in Slide-A-Lyzer MINI Dialysis Device, 10–100 μL, is glycerol-treated. Glycerol should be washed out in advance because it suppresses cell-free translation. 12. The concentration of the liposome prepared in Subheading 3.2, step 16 is 50 mg/mL. Remaining liposome can be frozen with liquid nitrogen and stored at -80 °C. Repeated freezing and thawing cause aggregation. Sonication in water-bath type ultrasonic cleaner or long-time vortexing reduces the size of the aggregated liposomes. 13. Translation reaction mixture containing wheat germ extract and liposome has a much higher specific gravity than ×1 SUB-AMIX SGC. The mixture sinks to the bottom of the well and forms a bottom layer. 14. A flat-bottom 1.5-mL tube is preferable. After centrifugation, the liposome forms compact, easily visible pellet on the bottom. 15. Membrane protein is easily aggregated by oxidation and heat denaturation. Once membrane protein aggregates, it does not penetrate into acrylamide gel in electrophoresis. To prevent aggregation, add a sufficient amount of the reducing agent to the SDS-PAGE sample buffer (equal or more than 3% of 2-mercaptoethanol), and do not heat the sample.

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Fig. 6 An example of membrane protein production. A membrane protein (arrow head) was synthesized using the bilayer-dialysis method on a small scale

16. We introduce an example of the calculation of membrane protein production efficiency. The result of the small-scale test synthesis is shown in Fig. 6. Compared with the band intensity of the BSA standard series, we estimated the amount of the target protein at 800 ng/lane. Using the format above, we calculated the production efficiency and determined that 1 mL of WEPRO7240 WGE can produce 1.6 mg of membrane protein. 0:100 mL ÷ 0:002 mL ÷ 0:025 × 0:0008 = 1:6 mg protein=mL WGE 17. Microman (Gilson) with a capillary piston is helpful in handling a small amount of volatile organic solvent. 18. We use an ultrasonic homogenizer with a cup horn (Branson) to homogenize the liposome emulsion. Sonication makes liposome size monodisperse (average size is around 100 nm diameter). Do not use an ultrasonic disruptor with a probe horn, which is not suitable for homogenizing small amounts of liquid in evaporation flasks. If a sonicator with a cup horn is not available, a water-bath-type ultrasonic cleaner is applicable for homogenizing liposome emulsion. 19. The dialysis membrane of the dialysis cup (Slide-A-Lyzer MINI Dialysis Device, 10K molecular weight cut off, 2 mL) is not treated with glycerol. Pre-wash is not necessary.

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Acknowledgment The authors thank Mr. Tomio Ogasawara for his assistance in the technological development. They also thank Professor Tatsuya Sawasaki for his mentoring. This work was mainly supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED, Japan. This work was also partially supported by JSPS KAKENHI Grant Numbers 24710251, 26750375, and 16k01915. References 1. Wilkinson TCI (2016) Discovery of functional monoclonal antibodies targeting G-proteincoupled receptors and ion channels. Biochem Soc Trans 44:831–837. https://doi.org/10. 1042/bst20160028 2. Webb DR, Handel TM, Kretz-Rommel A, Stevens RC (2013) Opportunities for functional selectivity in GPCR antibodies. Biochem Pharmacol 85:147–152. https://doi.org/10. 1016/j.bcp.2012.08.021 3. Hino T, Iwata S, Murata T (2013) Generation of functional antibodies for mammalian membrane protein crystallography. Curr Opin Struct Biol 23:563–568. https://doi.org/10. 1016/j.sbi.2013.04.007 4. Ecker DM, Jones SD, Levine HL (2015) The therapeutic monoclonal antibody market. MAbs 7:9–14. https://doi.org/10.4161/ 19420862.2015.989042 5. Hutchings CJ, Koglin M, Marshall FH (2010) Therapeutic antibodies directed at G proteincoupled receptors. MAbs 2:594–606. https:// doi.org/10.4161/mabs.2.6.13420 6. Hino T, Arakawa T, Iwanari H, YurugiKobayashi T, Ikeda-Suno C, Nakada-Nakura Y, Kusano-Arai O, Weyand S, Shimamura T, Nomura N et al (2012) G-protein-coupled receptor inactivation by an allosteric inverseagonist antibody. Nature:1–5. https://doi. org/10.1038/nature10750 7. Pone EJ, Zhang J, Mai T, White CA, Li G, Sakakura JK, Patel PJ, Al-Qahtani A, Zan H, Xu Z et al (2012) BCR-signalling synergizes with TLR-signalling for induction of AID and immunoglobulin class-switching through the non-canonical NF-KB pathway. Nat Commun 3 : 7 6 7 . h t t p s : // d o i . o r g / 1 0 . 1 0 3 8 / ncomms1769 8. Bill RM, Henderson PJF, Iwata S, Kunji ERS, Michel H, Neutze R, Newstead S, Poolman B, Tate CG, Vogel H (2011) Overcoming barriers to membrane protein structure determination.

Nat Biotechnol 29:335–340. https://doi.org/ 10.1038/nbt.1833 9. Seddon AM, Curnow P, Booth PJ (2004) Membrane proteins, lipids and detergents: not just a soap opera. Biochim Biophys Acta Biomembr 1666:105–117. https://doi.org/ 10.1016/j.bbamem.2004.04.011 10. Milic´ DBVD (2015) Large-scale production and protein engineering of G protein-coupled receptors for structural studies. Front Pharmacol 6:394. https://doi.org/10.3389/fphar. 2015.00066 11. Nozawa A, Ogasawara T, Matsunaga S, Iwasaki T, Sawasaki T, Endo Y (2011) Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system. BMC Biotechnol 11:35. https://doi.org/10.1186/14726750-11-35 12. Suzuki Y, Ogasawara T, Tanaka Y, Takeda H, Sawasaki T, Mogi M, Liu S, Maeyama K (2018) Functional G-Protein-Coupled Receptor (GPCR) synthesis: the pharmacological analysis of Human Histamine H1 Receptor (HRH1) synthesized by a wheat germ cell-free protein synthesis system combined with asolectin glycerosomes. Front Pharmacol 9:38. https://doi. org/10.3389/fphar.2018.00038 13. Sackin H, Nanazashvili M, Makino S (2015) Direct injection of cell-free Kir1.1 protein into xenopus oocytes replicates single-channel currents derived from Kir1.1 MRNA. Channels 9: 1 9 6 – 1 9 9 . h t t p s : // d o i . o r g / 1 0 . 1 0 8 0 / 19336950.2015.1063752 14. Liu S, Hasegawa H, Takemasa E, Suzuki Y, Oka K, Kiyoi T, Takeda H, Ogasawara T, Sawasaki T, Yasukawa M et al (2017) Efficiency and safety of CRAC inhibitors in human rheumatoid arthritis xenograft models. J Immunol 199:1584–1595. https://doi.org/10.4049/ jimmunol.1700192

Production of Immunizing Antigen Proteoliposome Using Cell-Free Protein. . . 15. Hashimoto Y, Zhou W, Hamauchi K, Shirakura K, Doi T, Yagi K, Sawasaki T, Okada Y, Kondoh M, Takeda H (2018) Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5. Sci Rep 8:8383. https://doi.org/ 10.1038/s41598-018-26560-9 16. Nishiguchi R, Tanaka T, Hayashida J, Nakagita T, Zhou W, Takeda H (2022) Evaluation of cell-free synthesized human channel proteins for in vitro channel research. Membrane 13:48. https://doi.org/10.3390/ membranes13010048

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17. Takeda H, Ogasawara T, Ozawa T, Muraguchi A, Jih P-J, Morishita R, Uchigashima M, Watanabe M, Fujimoto T, Iwasaki T et al (2015) Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay. Sci Rep 5: 11333. https://doi.org/10.1038/srep11333 18. Gibson DG (2011) Enzymatic assembly of overlapping dna fragments. Methods Enzymol 498:349–361. https://doi.org/10.1016/ b978-0-12-385120-8.00015-2

Chapter 10 Reconstruction of Protein/Liposome Complex Yasuyuki Suzuki Abstract Most ion channels and receptors are distributed in cell membranes and are known as membrane proteins. These membrane proteins are folded in the cell membrane and become functional proteins. Here, we demonstrate a method of reconstructing membrane proteins into liposome membranes, which are commonly used as artificial cell membranes. Key words Membrane protein, Proteoliposome, Reconstitution, Detergent, Polystyrene beads

1

Introduction Many drugs bind to membrane-bound receptors and channels, which are commonly known as membrane proteins. Membrane proteins are key molecules for investigating drug functions. However, synthesizing functional membrane proteins in vitro is a challenging task. This is mainly because membrane proteins that are synthesized in hydrophilic reaction mixtures aggregate easily. Integrating membrane proteins into a hydrophobic environment such as liposomes, micelle with detergent, bilayer sheet, and nanodiscs is therefore essential to produce functional membrane proteins. Other methods were previously used to integrate membrane proteins into liposome membranes. These include sonication, freezing and thawing, liposome chaperone, and detergentmediated reconstitution. The sonication method integrates the membrane protein into a liposome membrane while reforming the liposome structure. This process has the advantage of being simple. However, this method has poor reproducibility and the extra heating used might cause protein denaturation. In contrast, the freezing and thawing method is protective to membrane proteins because it reduces the risk of protein denaturation. However, this method also has limited reproducibility.

Shuang Liu (ed.), Rheumatoid Arthritis: Methods and Protocols, Methods in Molecular Biology, vol. 2766, https://doi.org/10.1007/978-1-0716-3682-4_10, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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Liposome chaperon methods, which contain liposomes in the protein synthesis reaction mixture, integrate the membrane protein into the liposome membrane directly [1]. This integration process is simple. The mechanism underlying this process is however unclear. We hypothesize that some membrane proteins adhere to liposome membranes and do not integrate into the membrane correctly. In a common strategy, the detergent-mediated reconstitution method also integrates membrane proteins in vitro. After the solubilization of the membrane proteins by detergents, the removal of detergents from the micelles forms liposomes. Thereafter, membrane proteins integrate into liposome membranes during the reformation of the liposome structure. Detergent removal methods previously included the dilution method, dialysis, and the absorbing method. In the dilution method, the micelle mixture is diluted approximately 50 times with buffers to reform the liposome structure. Although this method is simple, we could not collect the reconstituted proteoliposomes efficiently using this method. The use of the dialysis method is widespread. This method requires a 10–24 h liposome reconstitution period. The long course results in the highly efficient integration of proteins into the liposome membranes. The high integration efficiency is due to the fact that high reconstitution speed might form liposomes from micelles without integrating membrane proteins. However, we experienced difficulties in reconstituting membrane proteins that are solubilized in detergents with low critical micelle concentration (CMC). This method is therefore dependent on the properties of the detergents. Cyclodextrin and polystyrene beads are commonly used to remove detergents. The affinity of detergents to cyclodextrin depends on the chemical structure of the detergent. For example, CHAPS, which has a steroid nucleus, has a high affinity to γ-cyclodextrin [2]. On the other hand, polystyrene beads such as Bio-beads can absorb various detergents. However, Bio-Beads might absorb a small amount of the proteins, but the absorption of this small amount of proteins is negligible as it presents very few problems [3]. Furthermore, adjusting the amounts of beads used could control the speed of detergent absorption [3, 4]. In this article, we demonstrate the reconstitution of histamine H1 receptor into liposome membranes with the detergentmediated reconstitution method combined with Bio-Beads SM-2. We are confident that this method is useful for the reconstitution of various membrane proteins.

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Materials

2.1 Cell-Free Protein Synthesis

1. Wheat germ cell-free system: WEPRO7240 expression kit (Cell-Free Sciences, Matsuyama, Japan). 2. Constructing cDNA: Gateway system (Thermo Fisher, Waltham, MA, United States). 3. Liposome: Asolectin from soybean mixture of phospholipids (Sigma-Aldrich, St. Louis, MO, United States) (see Note 1). 4. Glycerol. 5. 10-K MWCO Slide-A-Lyzer dialysis device (Thermo Fisher, Waltham, MA, United States).

2.2 Membrane Protein Solubilization

1. Detergent: Triton X-100 (Wako, Osaka, Japan). We recommend that you stock 10% Triton X-100. 2. Buffer A: 20 mM Bis-Tris propane, 1 M NaCl, 4 μM leupeptin, 4 mM dithiothreitol (DTT), KOH, at pH 7.5 (see Note 2).

2.3 Reconstituting the Membrane Protein

1. Polystyrene beads: Bio-Beads SM-2 (Bio-Rad Laboratories, Hercules, CA, United States). 2. Methanol. 3. 50-mesh sieve (we use the mesh designed for cooking). 4. Phosphate-buffered saline.

2.4 Sucrose Density Gradient Centrifugation and SDS-PAGE

1. Ultra speed centrifugation. 2. Sucrose. 3. Buffer B: 20 mM HEPES, 150 mM NaCl, 4 μM leupeptin, 4 mM DTT, KOH, at pH 7.5. 4. Tris–Glycine–SDS Buffer: 25 mM Tris, 192 mM Glycine, 0.1% SDS, pH 8.6. 5. 12.5% polyacrylamide gel (ATTO, Tokyo, Japan). 6. Molecular weight marker. 7. Quick CBB PLUS (Wako, Osaka, Japan).

3

Methods

3.1 Cell-Free Protein Synthesis

1. We synthesized HRH1 with a wheat germ cell-free system combined with asolectin liposome (Fig. 1). Details of HRH1 synthesis have been previously described [1]. Commonly, the E. coli cell-free system is used to synthesize various proteins. However, the E. coli cell-free system often produces aggregated membrane proteins. On the other hand, our

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Fig. 1 Synthesis of HRH1 by wheat germ cell-free system. (a) This schema shows the bilayer-dialysis method for synthesizing membrane proteins. Following consumption of amino acids and energy sources, these substances are supplied through the dialysis membrane. This suppling system can prolong the protein synthesis reaction and we can adapt a large amount of membrane proteins. (b) This image shows how the dialysis membrane cup was set on six-hole plastic plates

synthesis method produces a large amount of membrane protein that does not aggregate. The wheat germ cell-free system is therefore a convenient method to synthesize human membrane proteins. In our synthesis method, about 400 μg of HRH1 is synthesized in one synthesis reaction. Synthesized mixtures are dispensed into four tubes, and the synthesized HRH1 can be purified by centrifugation at 21,400 g for 20 min. 3.2 Solubilizing HRH1

1. The optimal concentration of the detergent in comparison to the targeted membrane protein should be determined. Too high an amount of detergent might prevent protein reconstitution. Furthermore, the optimal detergent concentration depends on the properties of the detergent and the amount of membrane proteins and lipids. In our previous study, we solubilized 100 μg of HRH1 containing 1 mg asolectin with 1–30 mM Triton X-100, which increased by 1 mM each. Each tube was rotated at 30 rpm, for 1 h, at 4 °C to solubilize the protein. 2. Absorbance at 540 nm was used to confirm the degree of solubilization. The degree of solubilization could be measured by quantitating absorbance because the solution becomes more transparent as the proteins solubilize [5]. We further determined the optimal concentration and minimum amount of Triton X-100 that is required to completely solubilize the targeted membrane protein. In our experiment, we solubilized 0.1 mg HRH1 with 20 mM Triton X-100 contained in 200 μL

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of Buffer A. To aid solubilization, the tube was rotated at 30 rpm, for 1 h, at 4 °C. 3. After solubilization, the aggregated protein was separated by centrifugation at 21,400 g for 20 min. The supernatant containing the solubilized HRH1 was used to reconstitute HRH1. 3.3 Preparation of Bio-beads SM-2

At this stage, it is essential to prepare Bio-Beads SM-2 for increasing the efficiency of proteoliposome reconstitution. 1. Add 20 g of dry beads to 150 mL of methanol in a plastic bottle. Rotate the bottle on the tube rotator for 20 min at about 30 rpm at room temperature. 2. Remove the methanol by suction. 3. Wash the beads with another 400 mL of methanol and remove the methanol by suction. 4. Wash the beads with deionized water. Use a total of 2500 mL of water. 5. Remove the crushed beads by a 50-mesh sieve using 5000 mL of water. Crushed beads absorb various hydrophilic substances easily and can cause various challenges in the bioassays (see Note 3). After this process, you need to take care not to crush the beads. 6. Suspend the washed beads in 50 mL of PBS to prevent them from drying.

3.4 Reconstituting HRH1

Triton X-100 contained in solubilized HRH1 is removed by Bio-Bead SM-2. 1. After adding 10 mg of Bio-Beads to solubilized HRH1, the solution is mixed with a tube rotator at 30 rpm for 1 h at 4 °C. 2. Subsequently, 10 mg of Bio-Beads are added three times into the mixture, every hour. 3. Finally, 40 mg of Bio-Beads are added into the tube, and the detergent is removed almost completely for overnight incubation (see Note 4). The absorbing sequence is carried out by rotating the tube at 30 rpm, 4 °C. The reconstituted HRH1 is separated using the following process. 1. Make a small hole on the bottom of the tube containing the reconstituted HRH1 with a 31G needle (Fig. 2a). Be careful not to injure your hands. 2. After puncturing the hole 2 or 3 times, the needle becomes dull and the hole is not uniform. Furthermore, a wide hole causes contamination of the Bio-Beads.

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Fig. 2 Separating the reconstituted proteoliposome from the Bio-Beads SM-2. (a) 31G needle was used to make a small hole on the bottom of the tube containing reconstituted proteolipsomes and Bio-Beads SM-2. (b) The tube with a small hole is placed on an empty tube. The two tubes are then taped together with Parafilm. Place this apparatus in a centrifuge. Centrifuge at 1000 g for 1 min to separate the proteoliposome solution from the Bio-Beads

3. Insert the tube containing the mixture into another tube of the same size. 4. Tape the two tubes together using parafilm (Fig. 2b). 5. You should set the jointed tubes into a centrifuge. Centrifugation at 1000 g for 1 min to separate the reconstituted HRH1 solution from the Bio-Beads. Absorbing detergents can promote the reconstitution of HRH1 into the liposome membrane. The reconstitution reactions are confirmed by the change in absorbance at 540 nm. We also observed the change in transparency when apparent turbidity was observed when the liposomes were reconstituted efficiently. Reconstituted HRH1 proteoliposomes are collected by centrifugation at 21,400 g for 20 min. You might need to centrifuge at ultra-high speed when the ratio of protein to liposome is low. If you need to separate proteoliposomes from the empty liposomes, you should separate the sample by sucrose density gradient centrifugation. 3.5 Sucrose Density Gradient Centrifugation

Sucrose density gradient centrifugation not only separates the proteoliposome, but it also confirms the integration of membrane proteins into the liposome membrane.

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Fig. 3 Separation of HRH1 proteoliposome by density gradient centrifugation. Tube A: Two different bands are separated by centrifugation. The band in the lower layer includes the HRH1 proteoliposomes

1. The density gradient ladder consisted (bottom to top) of a 45% (w/w) sucrose layer, a 20% (w/w) sucrose layer, and a 15% (w/w) sucrose layer containing Buffer B, in a volume ratio of 1: 1:1. 2. The reconstituted proteoglycerosome suspension was carefully layered on top of the gradient (see Note 5). 3. After centrifugation (18 h, 4 °C, 200,000 g), the proteoliposomes migrated into the middle sucrose layer [2]. The asolectin glycerosome with no integrated membrane protein was observed at a higher position in the density gradient than the proteoglycerosomes (Fig. 3). 3.6

SDS-PAGE

1. The collected 100 μL of each fraction from top to bottom of the density gradient after centrifugation can verify the distribution of HRH1. 2. Each fraction was separated by SDS-PAGE using a 12.5% polyacrylamide gel (ATTO, Tokyo, Japan) (see Note 6). Electrophorese at 25 mA till the dye front reaches the bottom of the gel. 3. Following electrophoresis, stain the gel with Quick-CBB PLUS (Wako, Osaka, Japan). The stained gel demonstrates that the turbid lower fraction contains the HRH1 integrated into liposome membranes (Fig. 4). If HRH1 is aggregated during the reconstitution process, the bottom fraction will show the HRH band in SDS-PAGE. In that case, you should plan to change various conditions, such as detergents, buffer, temperature, and so on, to achieve the desired results.

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Fig. 4 SDS-PAGE of the HRH1 fractions separated by sucrose density gradient centrifugation. The band containing HRH1 was distributed in the high-density area. Other fractions included no HRH1 protein

4

Notes 1. Commonly, asolectin liposomes are directly sonicated with ultrasound probes. However, direct sonication with a probe increases the temperature of the liposome solutions and might cause unnecessary oxidation of the lipid in liposomes. You should sonicate the liposome solution gently in an ultrasonication bath filled with ice-cold water. 2. A previous study showed that Bis-Tris propane buffer was suitable for reconstituting membrane proteins [6]. Furthermore, highly concentrated NaCl was also shown to be protective of membrane proteins [7]. On the other hand, highly concentrated NaCl changes the CMC of detergent. The changed CMC might inhibit the reconstitution of membrane proteins. You should therefore adjust the concentration of NaCl to be optimal for the membrane protein you want to reconstitute. 3. Our previous study demonstrated that small amounts of Bio-Beads SM-2 absorb radio-labeled ligands easily. Crashed Bio-Beads showed nonspecific ligand binding, and we could not estimate the specific binding to the reconstituted receptors. You should therefore remove as much of the unnecessary Bio-Beads as possible. 4. Absorbing detergents with Bio-Beads at a high speed often causes low membrane protein reconstitution efficiency. 5. You should sonicate the HRH1 proteoliposome solutions before applying the sample to the sucrose density gradient. If

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HRH1 proteoliposomes are not uniformly distributed in the solution, the separated bands will form lumps. 6. Do not boil the samples at 95 °C for 5 min before separating HRH1 by SDS-PAGE, as the membrane protein often forms large insoluble aggregates. References 1. Suzuki Y, Ogasawara T, Tanaka Y et al (2018) Functional G-protein-coupled receptor (GPCR) synthesis: the pharmacological analysis of human histamine H1 receptor (HRH1) synthesized by a wheat germ cell-free protein synthesis system combined with asolectin glycerosomes. Front Pharm 9:38. https://doi.org/10.3389/fphar. 2018.00038 2. De Grip JW, Van Oostrum J, Bovee-Geurts PH (1998) Selective detergent-extraction from mixed detergent/lipid/protein micelles, using cyclodextrin inclusion compounds: a novel generic approach for the preparation of proteoliposomes. Biochem J 330(2):667–674. https:// doi.org/10.1042/bj3300667 3. Rigaud JL, Levy D, Mosser G et al (1998) Detergent removal by non-polar polystyrene beads. Eur Biophys J 27(4):305–319. https:// doi.org/10.1007/s002490050138 4. Levy D, Bluzat A, Seigneuret M et al (1990) A study of liposome and systematic

proteoliposome reconstitution involving biobead-mediated triton X-100 removal. BBA-Biomem 1025(2):179–190. https://doi. org/10.1016/0005-2736(90)90096-7 5. Knol J, Sjollema K, Poolman B (1998) Detergent-mediated reconstitution of membrane proteins. Biochemist 37(46): 16410–16415. https://doi.org/10.1021/ bi981596u 6. Timothy AC (2008) Adv. topics – membrane proteins, U.S.-Canada winter school on biomolecular solid state NMR. http://web.mit.edu/ fbml/winterschool2008/talks/Fri2a%20-%20 Cross_membrane_proteins.pdf 7. Ratnala, VRP (2004). Ligand-protein interaction of histamine with its human histamine H1 receptor target: high yield expression and magicangle spinning NMR studies. Doctoral dissertation, thesis, University of Leiden

Chapter 11 Production of Neutralizing Antibody Erika Takemasa and Shuang Liu Abstract Techniques employing monoclonal antibodies (mAbs) are widely used in the initial development phase of biologics. The usefulness of mAbs in basic RA research has been established based on their characteristics, including specificity of binding, homogeneity, and ability to be produced on a large scale. MAb immunoglobulins are the starting material for the generation of smaller antibody fragments and other engineered immunomodulatory antibodies. In this chapter, the basic hybridoma technique, which is a well-established and feasible method for the production of mAbs involving animal immunization, cell fusion, hybridoma screening, expanding positive hybridomas, and purification, is introduced. Aiming at specific affinity to a membrane protein, synthetic proteoliposomes are used in the immunization and screening steps. Key words Monoclonal antibody, Proteoliposome, Cell fusion, Immunization, Hybridoma

1

Introduction Over the past two decades, biologic disease-modifying anti-rheumatoid drugs (b-DMARs) have markedly expanded the treatment options for rheumatoid arthritis (RA). Specific antibodies are the most rapidly growing class of b-DMARs. The therapeutic targets of these specific antibodies include tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6 receptor, CD20, and CD80/86 [1]. Since fewer than 30% of patients show a robust response to currently available treatment and a number of adverse events are reported, further therapeutic options are required for patients who show a poor response to initial treatment [2, 3]. Monoclonal antibodies (mAbs) specifically against a pathogenic cytokine or cellular component within the RA synovium or in the whole immune system have been the most common form of biologic development. Many mAbs against new therapeutic targets are undergoing clinical trials [4]. The usefulness of mAbs in basic RA research is established based on their characteristics, including specificity of binding, homogeneity, and ability to be produced on a large scale. Although the preparation of mAbs is usually more

Shuang Liu (ed.), Rheumatoid Arthritis: Methods and Protocols, Methods in Molecular Biology, vol. 2766, https://doi.org/10.1007/978-1-0716-3682-4_11, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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time-consuming and costly than that of polyclonal antibodies, once a hybridoma cell line is established, it can provide an unlimited supply of antibodies for RA research. Hybridoma technology has been a significant and essential platform for producing mAb products. Using this technique, antibodies are able to be generated in a naı¨ve form. The basic process begins with immunization of experimental animals, usually mice, with a targeted antigen. Currently, there are several choices of immunizing antigen, such as peptide, whole cell or membrane fraction of transfected cultured cells, DNA, virus-like particle, or purified protein reconstituted in proteoliposomes [5]. If the binding target is located on the plasma membrane, compared to synthetic peptides, which are often used in the sensitization process, proteoliposome would be the most promising antigen, considering the highly concentrated quantity and stabilized structure in lipid vesicles [6, 7]. Especially in the case of producing neutralizing mAb with biological function targeting a membrane protein, the correct transmembrane structure is fundamental to reconstitute binding sites on an extracellular loop. Using the technologies introduced in Chapters 7 and 8, a sufficient quality and quantity of proteoliposomes can be obtained for immunization. Then, the spleen or lymph nodes are obtained from immunized animals, and isolated B lymphocytes must be somatically fused with myeloma cells using various technologies. After cell selection, hybridoma cells producing the desired antibody are screened by an appropriate high-throughput screening system. Antibody capture assays are the easiest and most convenient screening method. A capture or sandwich enzyme-linked immunosorbent assay (ELISA) is often used for this purpose. For neutralizing antibodies, the following functional screen is required. The choice of functional screen method is based on the functional characteristics of the target protein. After expanding positive clones and purification, the characterization of mAbs is based on the determination of the physicochemical and immunochemical character, isotype, and concentration. By using computational and bioinformatics tools, antibody selection and epitope prediction are feasible. MAb immunoglobulins are the starting material for the generation of smaller antibody fragments and other engineered immunomodulatory antibodies. Hybridoma technology is a wellestablished feasible method. Because of the comparatively simple procedure with minimal cost for steady production of naı¨ve whole immunoglobulins, this technique is still widely used in laboratories that implement basic cell biological research [8]. In this chapter, the basic hybridoma techniques used in the production of neutralizing mAb targeting human calcium-releasing calcium-activated channel 1 (CRACM1), including animal immunization, cell fusion, hybridoma screening, expanding positive hybridoma, and

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purification, are introduced. Aiming at innovation in bDMRD discovery and development, these techniques could provide the basis for preclinical research in the field of RA.

2

Materials

2.1 Antigen Preparation ( See Note 1)

1. Synthetic CRACM1 proteoliposomes (10 mg/mL). 2. Incomplete Freund’s adjuvant (IFA). 3. Complete Freund’s adjuvant (CFA). 4. Glass syringes without needles (1 mL) (Hamilton). 5. 5- and 10-mL disposable plastic syringes. 6. T-shape stopcock. 7. Electronic homogenizer with a small blade (diameter of 5 mm or less).

2.2 Mouse Immunization

1. Animals: BALB/c mice (female, 8–10 weeks old). 2. 70% ethanol. 3. Antigen emulsion (CFA)/antigen emulsion (IFA). 4. 25- and 27-gauge × 5/8″ needles.

2.3 Preparation for Fusion

1. Dulbecco’s Modified Eagle Medium (DMEM) culture medium. Pre-warm the medium to 39 °C. 2. Fetal bovine serum (FBS) (see Note 2). 3. Hypoxanthine and thymidine (HT) culture medium: DMEM medium containing 10% FBS, 1% penicillin, and HT Media Supplement (1×) Hybri-Max™ (SIGMA ALDRICH). HT medium provides preformed purines and a pyrimidine to overcome the effects of residual intracellular aminopterin. Once the de novo biosynthesis pathway for nucleosides has been reestablished, HT is no longer needed in the culture medium. Pre-warm the medium to 39 °C. 4. Hypoxanthine aminopterin thymidine (HAT) culture medium: DMEM medium containing 10% FBS, 1% penicillin, and HAT Media Supplement (1×) Hybri-Max™. HAT medium selects only successfully fused hybridoma cells. Pre-warm the medium to 39 °C. 5. Sp2/0-Ag14 cells [9]. 6. Penicillin–streptomycin (×100). 7. Pre-warmed 50% polyethylene glycol (PEG) (39 °C). 8. 0.4% trypan blue solution. 9. 96-well flat-bottom plates. 10. Water bath (set to 39–40 °C).

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11. Fine-pointed forceps and scissors. 12. 70% ethanol. 13. Surgical drapes. 14. Pipets (5 and 10 mL). 15. 25-mL beakers. 16. 100-mm petri dishes. 17. Cell strainers (100 μm). 2.4 ELISA for Screening

1. PBS. 2. CRACM1 proteoliposomes. 3. Proteoliposome protein mock. 4. 0.1 M NaHCO3 buffer (pH 9.6). 5. Block & Sample 5× Buffer (Promega). 6. Washing buffer: 0.05% Tween in PBS. 7. 3,3′,5,5′-Tetramethylbenzidine (TMB) solution (Promega). 8. Positive control capture antibodies (e.g., polyclonal antihuman CRACM1 IgG). 9. Secondary antibodies (e.g., monoclonal anti-mouse IgG labeled with horseradish peroxidase (HRP)). 10. Nunc-Immuno™ MicroWell™ 96-well solid plates. 11. Multichannel pipette (10–200 μL) and chips. 12. Plate seals. 13. Plate shaker. 14. Plate reader (450 nm). 15. Paper towels.

2.5 Clone Selection and Expansion of Positive Clones

1. Cell culture flasks. 2. HAT culture medium (see Subheading 2.3, item 4). 3. CELLBANKER® cryopreservation medium. 4. Cell cryopreservation tubes.

2.6 Purification and Storage of mAbs

1. Saturated solution of ammonium sulfate ((NH4)2SO4): Dissolve 100 g of (NH4)2SO4 in 100 mL of miliQ by heating the solution to 45–50 °C. Keep the solution at room temperature for at least 2 days. 2. 50% saturated (NH4)2SO4. 3. Binding buffer: 0.02 M disodium phosphate buffer, pH 7.0. 4. Elution buffer: 0.1 M glycine–HCl buffer, pH 2.7–3.0. 5. Neutralizing buffer: 1.0 M Tris–HCl buffer, pH 9.0. 6. Polyoxyethylene (20) sorbitan monolaurate.

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7. Lyophilizing buffer: 100 mM histidine, 5 mM L-arginine, 100 mM glutamic acid, and 150 nM trehalose. 8. HiTrap Protein G HP (5 mL). 9. Syringes (20 mL). 10. Collection tubes. 11. Dialysis membrane (MWCO 10 K). 12. Lyophilizing vials. 13. Vacuum freeze drying apparatus.

3

Methods

3.1 Antigen Preparation

3.1.1 Homogenization Methods

The emulsion quality is critical for inducing a high rate of mAb production. The emulsion can be made using various methods. Here, we introduce the homogenization method and the syringeto-syringe method if a homogenizer is not available. Homogenization is highly recommended for emulsion preparation. 1. Sonicate the synthetic CRACM1 proteoliposomes just before preparation of emulsion by placing a test tube containing proteoliposomes in an ice water bath sonicator and sonicating for 5–10 min. This step typically produces small, unilamellar vesicles with diameters in the range of 15–50 nm. 2. Put 500 μL of CFA (IFA for booster injection) and sonicated CRACM1 proteoliposomes in respective glass syringes. 3. Seal the tips of both syringes with a T-shape stopcock. 4. Connect the rest of the connector of the T-shape stopcock with a 5- or 10-mL plastic syringe cut halfway from the plunger opening. 5. Push the plunger of the glass syringes and let CFA (IFA for booster injection) and CRACM1 proteoliposome solution mix in the plastic syringe. Air bubbles should be avoided during solution mixing. 6. After sealing the plastic syringe with the T-shape stopcock, take off the glass syringes. 7. Clamp the syringe to a ring stand and place it in an ice water bath to keep the emulsion cool during mixing (Fig. 1). 8. Homogenize the mixture to emulsify the CFA (IFA for booster injection) with the collagen solution until the emulsion is stable. 9. Test the stability of the emulsion by adding one drop of emulsion into a beaker of water. If the emulsion is stable, the drop will remain as a solid clump that does not dissipate (see Note 3).

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Fig. 1 Emulsion preparation using a homogenization method. Clamp a syringe filled with a mixture of adjuvant and antigen to a ring stand and place it in an ice water bath to keep the emulsion cool during mixing

10. Transfer the emulsion to a 1-mL glass syringe for animal injection (see Note 4). The prepared emulsion should be injected into the animals as soon as possible (within 1 h). Keep the emulsion cool at 4 °C until use. 3.1.2 Syringe-to-Syringe Method (Fig. 2)

1. Sonicate the synthetic CRACM1 proteoliposomes just before preparation of emulsion by placing a test tube containing proteoliposomes in an ice water bath sonicator and sonicating for 5–10 min. This step typically produces small, unilamellar vesicles with diameters in the range of 15–50 nm. 2. Put 500 μL of CFA (IFA for booster injection) and sonicated CRACM1 proteoliposomes in respective glass syringes. 3. Connect both syringes with a T-shape stopcock. During connecting, air bubbles should be avoided. 4. Slowly push the plunger of the syringe filled with CRACM1 proteoliposomes, and let the protein suspension mix with CFA (IFA for booster injection). 5. Slowly push back the plunger of the syringe filled with protein suspension and CFA (IFA for booster injection). 6. Repeat the mixing steps at least 200 times. Generally, an increase in the resistance of the plunger while pushing indicates that the emulsion is becoming stable.

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Fig. 2 Syringe-to-syringe method for emulsion preparation. (a) Put 500 μL of CFA (IFA for booster injection) and antigen in respective glass syringes. Connect both syringes with a T-shape stopcock. (b) Slowly push the plunger of the syringe filled with antigen solution and let the protein suspension mix with CFA (IFA for booster injection). (c) Slowly push the plunger back and repeat the steps. Generally, an increase in resistance while pushing the plunger indicates that the emulsion is becoming stable

7. Test the stability of the emulsion by adding one drop of emulsion into a beaker of water. If the emulsion is stable, the drop will remain as a solid clump that does not dissipate. 8. Transfer the emulsion to a 1-mL glass syringe for animal injection. The prepared emulsion should be injected into the animals as soon as possible (within 1 h). Keep the emulsion cool at 4 °C until use. 3.2 Mouse Immunization

The immunization route is based on the choice of animal species, adjuvant, concentration, and quality of the antigen. Immunization routes include subcutaneous, intradermal, intramuscular, intraperitoneal, and intravenous [10]. The subcutaneous route of injection for water-in-oil emulsions such as CFA and IFA is immunologically effective, with a low risk of infection and systemic influence. 1. The primary injection is performed subcutaneously using an emulsion containing CFA and CRACM1 proteoliposomes (day 0). 2. Five BALB/c female mice are used for the production of mAb. Use a squirt bottle to apply 70% ethanol to the injection site and wipe with tissue. 3. Place a 25- or 27-gauge × 5/8″ needle on the glass syringe filled with antigen emulsion (see Note 5). 4. Insert the needle, bevel side up and parallel to the tail, 2 cm from the base of the tail, and inject 0.1 mL (0.5 mg of CRACM1 proteoliposomes per mouse) of emulsion subcutaneously (see Note 6).

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5. Put the immunized mice in a clean cage. 6. On day 14 and day 21, freshly prepared emulsions containing IFA should be injected subcutaneously. Booster injections should be administered at a different location from that of the initial injection (see Note 7). 7. On day 30, determine the serum antibody titer for each mouse using the ELISA technique described in Subheading 3.4. Sample peripheral blood from immunized mice. The minimum serum antibody titer of the immunized mice prior to hybridoma preparation ideally should be about 50% reactivity at a dilution of 1/2500 (see Note 8). 8. A final boost is used to synchronize the maturation of the response on day 34. Inject 100 μL of synthetic CRACM1 proteoliposomes (10 mg/mL) without any adjuvant intravenously. 9. The immunized mice will be ready for cell fusion 4 days after final boosting. 3.3

Cell Fusion

1. Remove the spleen from immunized mice and put it in a petri dish filled with 10 mL of DMEM (Fig. 3). 2. Gently tease apart the spleen using a pair of fine forceps. 3. Pass the splenocytes through a cell strainer (100 μm). 4. Centrifuge the splenocytes at 100×g for 5 min. Discard the supernatant and resuspend the splenocytes in 5 mL of DMEM without antibiotics and FCS. 5. Take 50 μL of the suspension of splenocytes and dilute it to 1 mL using DMEM without antibiotics and FCS. 6. Count the cells using trypan blue solution with a hemocytometer. Viable cells do not take up impermeable dye. Approximately 1 × 108 viable splenocytes are expected from each mouse. 7. Harvest the pre-cultured Sp2/0 cells and centrifuge at 100×g for 5 min. Discard the supernatant and resuspend the Sp2/0 cells in 5 mL of DMEM without antibiotics and FCS. 8. Count the cell number of Sp2/0 cells using trypan blue solution. 9. Put 2 × 107 splenocytes and 10 × 107 Sp2/0 cells in a 50-mL Falcon tube. The ratio of splenocytes to Sp2/0 should be 5:1. 10. Centrifuge the cell mixture at 100×g for 5 min and resuspend the cells in 10 mL of DMEM without antibiotics and FCS. Perform this step twice. After the final centrifugation, discard DMEM and keep the cell pellet. 11. Gently tap the bottom of the 50-mL Falcon tube to loosen the pellet.

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Fig. 3 Position of the spleen (point by the arrow)

12. Slowly add 1 mL of pre-warmed PEG dropwise over 1 min with gentle mixing. The tube should be kept warm in a water bath (39–40 °C). 13. Add 1 mL of pre-warmed DMEM without antibiotics and FCS over 1 min, add 3 mL over the second minute, and add 16 mL over the third minute. 14. Stand the tube for 10 min in a water bath (39–40 °C). Then, bring up to 50 mL with complete DMEM containing 10% FCS and penicillin–streptomycin (×1). 15. Centrifuge the tube at 400×g for 10 min. Cell fusion will be achieved in this step. 16. Remove the supernatant carefully. Add 10 mL of pre-warmed complete DMEM without resuspending the cells, and centrifuge the tube at 200×g for 2 min. 17. Remove the supernatant. Gently resuspend the cells using 100 mL of pre-warmed complete HAT medium. 18. Add 100 μL of cell suspension to each well of 96-well flatbottom plates. Incubate at 37 °C in 10% CO2. 19. Feed the cells with a complete HAT medium every few days. Carefully change the medium since the cells are not adherent. 20. The hybridomas are ready for screening 7–21 days later. Once the culture medium starts to turn yellow, 100 μL of medium should be taken for further screening. 21. In a 96-well plate, a maximum of two changes of medium can be tolerated by hybridoma cells. After that, the cells need to be transferred to larger wells (48-well or 24-well plates). Otherwise, the hybridoma cells tend to die.

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22. Wells containing more than one hybridoma clone should be identified and sub-cloned. Aspirate hybridomas and add HAT medium to 10 mL. Gently mix the cells. Perform limiting dilution of the hybridomas by dispensing 100 μL of cell suspension into a new 96-well culture plate. 23. Once the culture medium starts to turn yellow, take 100 μL of medium for further screening. 3.4 Hybridoma Screening (ELISA)

Indirect ELISA is a widely used method for the detection of antibodies and is appropriate for screening hybridoma supernatants. The synthetic proteoliposomes used for animal immunization can be directly coated on a plate and antibody–antigen binding can be detected using a secondary antibody conjugated to an enzyme and a chromogenic substrate. 1. Coat the wells of a Nunc-Immuno™ MicroWell™ 96-well solid plate with CRACM1 proteoliposomes and proteoliposome protein mock. The concentration of proteoliposomes is 1–10 μg/mL in 0.1 M NaHCO3 buffer (pH 9.6). Dilute the proteoliposomes using 0.1 M NaHCO3 buffer to an appropriate final concentration and add 100 μL of the dilution to each well. Cover the plate with an adhesive plastic seal and incubate the plate overnight at 4 °C. 2. Remove the coating solution by patting the plate on a paper towel. Wash the plate twice by filling the wells with 200 μL of PBS. 3. Dilute Block & Sample 5× Buffer to the final concentration and add 100 μL of buffer to each well. Cover the plate with an adhesive plastic seal and shake the plate for at least 2 h at room temperature. 4. Dilute the positive control capture antibody appropriately using Block & Sample buffer. Remove the Block & Sample buffer by patting the plate on a paper towel. Add 50–100 μL of hybridoma supernatant or positive control capture antibody at the final concentration (see Notes 9–11). 5. Seal the plate and shake it at room temperature for 2 h. 6. Remove the solutions in the well by patting the plate on a paper towel. Wash the plate twice by filling the wells with 200 μL of washing buffer. 7. Dilute the secondary antibody (e.g., monoclonal anti-mouse IgG labeled with HRP) appropriately using Block & Sample buffer (see Note 12). Add 100 μL of secondary antibody dilutions to each well. 8. Seal the plate and shake it at room temperature for 1 h.

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9. Remove the solutions in the wells by patting the plate on a paper towel. Wash the plate five times by filling the wells with 200 μL of washing buffer. 10. As the detection step, add TMB solution to each well, incubate for 15–30 min, add an equal volume of stopping solution (2 M H2SO4), and read the optical density at 450 nm. 3.5 Expanding the Hybridomas and Freezing Positive Clones

1. According to the results of hybridoma screening, choose actively growing hybridomas of interest for cell expansion (Fig. 4). 2. Aspirate the hybridoma chosen for cell expansion, and add into 2 mL of HAT medium. Gently mix the cells and dispense them into a 24-well culture plate. 3. Feed the cells with 1 mL of complete HT medium twice a week and incubate the plates at 37 °C, in 5% CO2. 4. Select the best clone for further expansions using HT medium and transfer it into T-25 or even bigger size flasks. 5. Aliquoting into liquid nitrogen is one option for storing hybridomas of interest. Divide the cultured hybridomas at least 2 days before freezing while maintaining them at mid-log. Freshly divided cells have stronger membranes that withstand the freezing process better. 6. Aspirate 10 mL of hybridoma suspension and centrifuge the cells at 100 × g for 5 min. 7. Resuspend the pellet using 1 mL of CELLBANKER® cryopreservation medium. 8. Transfer the cells to a cell cryopreserve tube. Record the date, cell line, position, and antibody production of the cryopreserved cells. 9. Store in a freezing box at -80 °C overnight and then transfer to liquid nitrogen.

3.6 Purification and Storage of mAbs

1. Gather the cultured hybridoma supernatant as a sample for purification under sterile conditions. 2. Adjust the composition of samples by slowly adding an equal volume of saturated solution of (NH4)2SO4 (sample: (NH4)2SO4 = 1:1) with stirring (see Note 12). 3. Stir the solution at 4 °C for 1 h. 4. Centrifuge the solution at 10,000×g at 4 °C for 10 min. 5. Remove the supernatant and resuspend the pellet with 50% saturated solution of (NH4)2SO4. 6. Centrifuge the solution at 10,000×g at 4 °C for 10 min.

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a

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B5 C8 D7 D11 F3 H9 H10 H12 A1 A11 B1 B5 B8 B9 B10 C11 D3 D4 E2 E4 E10 F3 F8 F9 G4 G5 G6 G9 G10 H3 H11 H5 H11 E11 1A6 1H6 3A5 1H6 3D7 482 Positive1 Positive2

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B5 C8 D7 D11 F3 H9 H10 H12 A1 A11 B1 B5 B8 B9 B10 C11 D3 D4 E2 E4 E10 F3 F8 F9 G4 G5 G6 G9 G10 H3 H11 H5 H11 E11 1A6 1H6 3A5 1H6 3D7 482 Positive1 Positive2

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Fig. 4 Positive clone selection according to screening results. ELISA screening is performed for positive clone selection. (a) Raw absorbance ELISA data. (b) Calibrated ratio of target proteoliposome and mock. Arrow: positive clones that should be considered for further limited dilution and cell expansion

7. Remove the supernatant and resuspend the pellet with 10 mL of binding buffer. 8. Prepare collection tubes by adding 60–200 μL of 1 M Tris– HCl, pH 9.0, per mL of fraction to be collected. 9. Prepare collection tubes by adding 100 μL of neutralizing buffer per mL of fraction to be collected. 10. Fill the syringe with binding buffer. Remove the stopper and connect the column to the syringe (with the provided adaptor). Avoid introducing air into the column.

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11. Remove the snap-off end at the column outlet. Wash the column with 50 mL of binding buffer at 5 mL/min. 12. Apply the sample using a syringe fitted to a lure adaptor. 13. Wash the column with 25 mL of binding buffer. 14. Elute the column with 10 mL of elution buffer. Collect the eluted fractions using collection tubes with prefilled neutralizing buffer. 15. Add polyoxyethylene (20) sorbitan monolaurate to eluted fractions at a final concentration of 1.5 mg/mL. 16. Put the eluted fractions in a dialysis membrane tube and seal it tightly. 17. Dialyze the eluted fractions in lyophilizing buffer at 4 °C for 3 days. Change the dialysis solution with fresh lyophilizing buffer at 1, 4, 24, and 48 h after the start. 18. Collect the fractions and dispense them into lyophilizing vials under sterile conditions. 19. Set the lyophilizing vials on a vacuum freeze drying apparatus and lyophilize the antibody fractions. 20. Store the lyophilized antibodies at -20 °C. Dissolve it to the desired concentration using sterilized miliQ before further applications.

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Notes 1. All solutions and materials must be prepared under sterile conditions. 2. FBS should be qualified ahead of time, and suitable FBS with good performance for supporting hybridoma growth should be chosen. 3. If the emulsion dissipates on the water surface, then the emulsion is not stable. Add a few drops of adjuvant, mix again, and retest. 4. Injecting an accurate volume of emulsion is difficult with a plastic syringe. 5. Before each injection, wipe the needle to prevent leakage of the emulsion. 6. Intraperitoneal injection is not recommended because both CFA and IFA cause severe inflammatory reactions in the peritoneal and thoracic cavities. 7. Boosting injection at the same site as the initial injection will cause a severe inflammatory reaction.

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8. If the titer is not high enough, further boosting injection using emulsion containing IFA should be given to the mice. 9. Since the monoclonal antibodies in the hybridoma supernatant recognize an individual epitope of the antigen, a monoclonal antibody with a different binding epitope or a polyclonal antibody should be used as the positive control antibody. 10. This is only a semiquantitative assay. If a quantitative assay is wanted, it is necessary to dilute the primary antibody to a series of doses in a blocking solution. 11. The secondary antibody used for the positive control capture antibody may be different from the antibody used for hybridoma supernatants. 12. A rapid increase in concentration of (NH4)2SO4 will cause protein agglutination. References 1. Davis BP, Ballas ZK (2017) Biologic response modifiers: indications, implications, and insights. J Allergy Clin Immunol 139:1445– 1456 2. Evans CH, Ghivizzani SC, Robbins PD (2013) Arthritis gene therapy and its tortuous path into the clinic. Transl Res 161:205–216 3. Mandema JW, Salinger DH, Baumgartner SW, Gibbs MA (2011) A dose-response meta-analysis for quantifying relative efficacy of biologics in rheumatoid arthritis. Clin Pharmacol Ther 90:828–835 4. Kalden JR (2016) Emerging therapies for rheumatoid arthritis. Rheumatol Ther 3:31–42 5. Hutchings CJ, Koglin M, Marshall FH (2010) Therapeutic antibodies directed at G proteincoupled receptors. MAbs 2:594–606 6. Liu S, Hasegawa H, Takemasa E, Suzuki Y, Oka K, Kiyoi T et al (2017) Efficiency and safety of CRAC inhibitors in human

rheumatoid arthritis xenograft models. J Immunol 199:1584–1595 7. Takeda H, Ogasawara T, Ozawa T, Muraguchi A, Jih PJ, Morishita R et al (2015) Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay. Sci Rep 5:11333 8. Tomita M, Tsumoto K (2011) Hybridoma technologies for antibody production. Immunotherapy 3:371–380 9. Kohler G, Howe SC, Milstein C (1976) Fusion between immunoglobulin-secreting and nonsecreting myeloma cell lines. Eur J Immunol 6: 292–295 10. Apostolico Jde S, Lunardelli VA, Coirada FC, Boscardin SB, Rosa DS (2016) Adjuvants: classification, modus operandi, and licensing. J Immunol Res 2016:1459394

Chapter 12 Autoantibody Profiling Using Human Autoantigen Protein Array and AlphaScreen Hiroyuki Takeda Abstract Autoantibodies that recognize self-antigens are believed to have a close relationship with diseases such as autoimmune diseases, cancer, and lifestyle diseases. Analysis of autoantibodies is essential for investigating pathology mechanisms, diagnosis, and therapeutics of these diseases. We developed an autoantibody profiling assay using a cell-free synthesized protein array and high-throughput screening technology. Our assay system can sensitively detect interaction between recombinant antigen protein and autoantibody and efficiently analyze autoantibody profiling in patients’ sera. Key words Autoantibody, Autoantigen, Protein array, AlphaScreen, Autoantibody profiling, Wheat cell-free protein synthesis

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Introduction Numerous patients worldwide are affected by various autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, Hashimoto’s thyroiditis, and myasthenia gravis. Over the past century, autoantibodies targeting self-antigens, such as nucleotides or proteins, have been identified in these patients and are thought to play crucial roles in disease pathogenesis [1–5]. In addition, autoantibodies have been increasingly implicated in other diseases, such as cancer and lifestyle-related diseases [6– 8]. This expanding body of evidence linking autoantibodies to a wide range of diseases has significantly heightened the interest of researchers in the fields of autoantibodies and autoantigens. Gaining insight into the roles of autoantibodies and autoantigens in these diseases is essential for unraveling intricate disease mechanisms, creating innovative diagnostic methods, and investigating therapeutic strategies that have the potential to benefit a vast number of patients worldwide.

Shuang Liu (ed.), Rheumatoid Arthritis: Methods and Protocols, Methods in Molecular Biology, vol. 2766, https://doi.org/10.1007/978-1-0716-3682-4_12, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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Several approaches exist for analyzing autoantibodies and their antigens, including immunoaffinity methods, proteomics analysis, cell-based assays, and protein arrays [7]. However, profiling autoantibodies and identifying their specific antigens remain challenging tasks. With tens of thousands of protein species present in cells or serum and widely varying concentrations (ranging from fM to mM), detecting trace amounts of autoantigens in biological samples is difficult, even with recent advancements in the sensitivity and dynamic range of analytical methods. In this chapter, we introduce our approach for investigating autoantibody profiling using protein arrays and high-throughput screening technology. Protein arrays, also known as protein chips, protein microarrays, or protein functional arrays, are research tools developed to efficiently obtain interactions between proteins or between proteins and other biomolecules. Protein arrays consist of hundreds to tens of thousands of recombinant proteins densely arranged in parallel (arrayed) on a chip or titer well plate. The protein arrays we have developed consist of thousands of recombinant proteins stored in 96- or 384-well plates (Fig. 1). Each well contains one protein solution, which allows for a one-to-one reaction of a sample of interest with a large number of proteins. The location of each protein is recorded in a spreadsheet along with its ID, sequences, and other relevant information, enabling the easy identification and extraction of hit samples following a screening assay. Protein arrays offer several advantages, such as simplified hit identification and uniform protein abundance for interaction assays utilizing biological samples. In immunoprecipitation-mass spectrometry, a common method for analyzing interactions with biological samples, identifying proteins that have interacted with target molecules can be labor-intensive due to the vast range of protein concentrations present in cells and blood. For instance, plasma contains proteins with concentrations spanning from several hundred μM, like serum albumin, to peptide hormones with concentrations less than nM, representing a 106-fold or greater difference. To detect trace amounts of proteins, high sensitivity is necessary, while the abundance of certain proteins can also inhibit the detection and ionization of other proteins. Protein arrays can effectively address the limitations of cell-based systems, as the type and location of the loaded proteins are predetermined, and their concentrations are relatively consistent. We have constructed protein arrays using the wheat cell-free protein synthesis system (wheat cell-free system) [9, 10], an in vitro translation method utilizing eukaryotic translation machinery that is suitable for expressing a wide variety of proteins in small quantities. To date, tens of thousands of proteins from diverse species, including humans, animals, plants, bacteria, viruses, and even malaria, have been successfully expressed using the wheat cell-free system [11–15]. To apply our cell-free protein arrays to

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Fig. 1 Illustration of protein array. Each well contains different proteins

high-throughput screening assays, we adopted AlphaScreen technology (Fig. 2) [16], a bead-based protein–protein interaction assay featuring high sensitivity, a wide dynamic range, and a homogeneous assay format. Over the past decade, we have refined our screening procedure using protein arrays and AlphaScreen, built and updated screening facilities (Fig. 3), and are now capable of conducting screening assays with 60,000 AlphaScreens in a single day [17, 18]. For autoantibody profiling, we constructed protein arrays containing biotinylated antigen candidate proteins [19]. Each protein in the array has a bls tag (also known as Avi tag) at the N-terminus and is enzymatically biotinylated during translation [20]. The autoantibody–antigen assay using AlphaScreen is straightforward: mix a biotinylated recombinant protein, serum, and AlphaScreen detection beads, incubate, and measure the signal. In the reaction, the biotinylated antigen is captured by streptavidin-conjugated AlphaScreen donor beads, while antibodies in the serum interact with protein G-conjugated AlphaScreen acceptor beads. If autoantibodies recognizing the biotinylated antigen are present in the serum, the antibody–antigen–beads complex shown in Fig. 2a forms, bringing the donor and acceptor beads into close proximity. The donor bead is excited by 680 nm laser light, converting oxygen to singlet oxygen (1O2). The singlet oxygen then transfers energy to the acceptor bead, generating a strong chemiluminescence signal (520–620 nm). Conversely, when a serum sample lacks autoantibodies that recognize the antigen in the reaction, the donor and acceptor beads remain distant (Fig. 2b), causing 1O2 to be quenched before reaching the acceptor bead and yielding no signal.

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Fig. 2 Autoantibody-antigen interaction assay using AlphaScreen. (a) Reaction with a serum containing autoantibody. (b) Reaction with a serum without autoantibody. SA streptavidin, B biotin, Ag cell-free synthesized antigen, G Protein G

We constructed an autoantigen candidate array comprising 2181 biotinylated proteins and performed several autoantibody profiling assays targeting diseases such as rheumatoid arthritis, lupus nephritis, periodontitis, pancreatic cancer, and atherosclerotic diseases using this array [5, 8, 21–23]. We now have 29,000 cell-free synthesized human proteins, and even proteome-scale autoantigen screening is possible. Large-scale screening assays like this necessitate significant effort, cost, and access to numerous cDNA resources and screening facilities (Fig. 3). However, constructing small-scale protein arrays with tens of proteins for autoantibody profiling can be accomplished without excessive resource allocation or cost. In this chapter, we provide several laboratory-scale protocols for autoantibody profiling, including the preparation of expression plasmids, array format cell-free protein production, AlphaScreen bead preparation, and AlphaScreen assay execution.

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Fig. 3 Screening facilities. (a) Liquidator 96 manual dispenser. (b) Janus automated dispensing workstation with Nanohead. (c) FlexDrop dropper

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Materials Prepare all solutions using analytical-grade reagents and ultrapure water. We produce ultrapure water using tandemly connected Elix UV and Milli-Q Direct (Merck Millipore).

2.1 Construction of Transcription Templates for CellFree Protein Synthesis

1. pEU-E01-MCS vector (CellFree Sciences). 2. Forward primer for inverse PCR to modify pEU-E01-MCS vector (Primer 1): 5′-cctgaacgacatcttcgaggcccagaagatcgagtggcacgaaGATATC ACTAGTTCTCGAGCTCG-3′ The uppercase sequence is derived from the pEU-E01MCS vector. The underlined portion indicates EcoRV site. The lowercase sequence shows the tag fragment to be inserted. The underlined part with a broken line represents the overlap region in the Gibson Assembly reaction. 3. Reverse primer for inverse PCR to modify pEU-E01-MCS vector (Primer 2): 5′-aagatgtcgttcaggccatgatggtgatggtgatgacccatTTGGTGA TGTAGATAGGTGGTTAGTG-3′ The uppercase sequence is derived from the vector. The lowercase sequence shows the start codon and tag fragment to be inserted. The underlined part with a broken line represents the overlap region in the Gibson Assembly reaction. 4. PrimeStarMAX DNA polymerase (Takara Bio). 5. FastDigest DpnI (Thermo Fisher Scientific). 6. TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.3). 7. 1% agarose/TAE gel. 8. GeneRuler 1 kb DNA Ladder marker (Thermo Fisher Scientific).

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9. SYBRsafe gel stain reagent (Thermo Fisher Scientific). 10. PCR product purification kit. 11. Gibson Assembly master mix (NEB). 12. Chemical competent cells of E. coli strain JM109. 13. LB-ampicillin agar plate. 14. LB medium. 15. Sequencing primer from the 5′ side of MCS in pEU plasmid (SPu-2, Primer 3): 5′-CAGTAAGCCAGATGCTACAC-3′ 16. Sequencing primer from the 3′ side of MCS in pEU plasmid (SP-A1868, Primer 4): 5′- CCTGCGCTGGGAAGATAAAC-3′ 17. Plasmid mini prep kit. 18. cDNA plasmid of interest. Human cDNA clones are available from several organizations and distributors, such as ATCC, Thermo Fisher Scientific, Promega, Origene, Genecopoeia, and Addgene. 19. Gene-specific forward primer (Primer 5): 5′-cgccacccaccaccaccaNNNNNNNNNNNNNNNNNN NN-3′ The lowercase sequence shows the S1 linker (Fig. 4b), and NNNN... shows the gene-specific nucleotide sequence (20–25 bp, Tm > 55 °C). 20. Gene-specific reverse primer (Primer 6): 5′-tccagcactagctccagaNNNNNNNNNNNNNNNNNN NN-3′ The lowercase sequence shows the T1 linker (complementary). NNNN. . . shows the gene-specific nucleotide sequence (20–25 bp, Tm > 55 °C). 21. Synthetic DNA fragment. DNA fragments prepared by gene synthesis services are also applicable. The DNA fragment should contain the S1 linker (cgc_cac_cca_cca_cca_cca), an open reading frame with a stop codon, and the T1 linker (tct_gga_gct_agt_gct_gga). 22. Forward primer for pEU vector linearization (Primer 7): 5′-tctggagctagtgctggaGGTACCTGTCCGCGGTCG-3′ The uppercase sequence is derived from the pEU-E01His-bls-MCS vector. The underlined portion indicates the KpnI site. The lowercase sequence shows the T1 linker sequence. 23. Reverse primer for pEU vector linearization (Primer 8): 5′-tggtggtggtgggtggcgGATATCTTCGTGCCACTCGA TCT-3′ The uppercase sequence is derived from the pEU-E01His-bls-MCS vector. The underlined portion indicates the

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Fig. 4 Scheme of transcription template construction. (a) Insertion of tag sequences into pEU vector. (b) Subcloning of target cDNA into expression vector and transcription template preparation

EcoRV site. The lowercase sequence shows the S1 linker (complementary sequence). 24. Reverse primer for transcription template amplification (AODA2303, Primer 9): 5′-GTCAGACCCCGTAGAAAAGA-3′ This primer is used together with the SPu-2 primer (Primer 3, see Subheading 2.1, item 11). 25. 96-well PCR plate and PCR seal. 26. Aluminum foil sealing tape, applicable for -80 °C storage.

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2.2 Cell-Free Synthesis of Autoantigen Protein Array

1. ×5 Transcription Buffer LM (CellFree Sciences). 2. 25 mM NTP mixture (CellFree Sciences). 3. 80 U/μL RNase inhibitor (CellFree Sciences). 4. 80 U/μL SP6 polymerase (CellFree Sciences). 5. Freshly prepared ultrapure water. 6. Clean plastic tubes and chips (see Note 1). 7. Multichannel reagent reservoir, 10 mL and 25 mL (Integra). 8. Discovery Comfort 8-channel pipette, 10 μL, 50 μL, and 200 μL (HTL). 9. Picus 8-channel electric pipette, 120 μL and 300 μL (Sartrius). 10. 96-well PCR plate. 11. Plate seal (polypropylene or PET). 12. Plate spinner. 13. Cooled incubator with temperature ranging from 0 to 40 °C or wider. 14. Wheat germ extract WEPRO7240 (CellFree Sciences) (see Note 2). 15. ×1 SUB-AMIX SGC feeding buffer. Dilute ×40 SUB-AMIX SGC stock solutions (S1–S4) (CellFree Sciences) with ultrapure water. 16. 20 mg/mL creatine kinase (Roche Diagnostics) (see Note 3). 17. Cell-free synthesized BirA biotin ligase (see Note 4). 18. 60 μM biotin solution. (see Note 5). 19. 96-well flat-bottom titer plate (TPP). 20. SDS-PAGE gel. 21. SDS-PAGE running buffer. 22. PVDF membrane. 23. Blotting buffer. 24. TBST buffer. 25. Skimmed milk. 26. Anti-biotin antibody, HRP conjugate (Sigma-Aldrich). 27. Chemiluminescence HRP substrate.

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AlphaScreen

1. Unconjugated AlphaScreen Donor beads (PerkinElmer). 2. 0.1 M MES buffer, pH 6.0. Sterilize using a 0.22-μm syringe filter. Store at 4 °C. 3. 10 mg/mL protein G solution. Add 5 mL of 0.1 M MES buffer, pH 6.0, to lyophilized recombinant Protein G (Thermo Fisher Scientific). Dissolve completely by rotation. Divide into small portions, and freeze with liquid nitrogen. Store at -80 °C.

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4. 1% Tween 20, BioXtra (Sigma-Aldrich). Dilute 10% Tween 20 (see Note 6) with ultrapure water 10 times before use. 5. 25 mg/mL sodium cyanoborohydride (Sigma-Aldrich). Prepare right before use. 6. 65 mg/mL O-(Carboxymethyl)hydroxylamine hemihydrochloride (CMO, Sigma-Aldrich). CMO should be dissolved in 2 M NaOH. Prepare just before use. 7. 0.1 M Tris–HCl, pH 8.0. 8. Storage buffer of protein G-conjugated acceptor beads, which contains 0.1 M Tris–HCl, pH 8.0, and 0.05% Proclin 300 (Sigma-Aldrich). 9. Streptavidin-conjugated donor beads (PerkinElmer). 10. 1 M Tris–HCl, pH 8.0. 11. 0.1% Tween 20. Dilute 10% Tween 20 (see Note 6) with ultrapure water. 12. 10 mg/mL BSA. Store at -30 °C. 13. OptiPlate 384 (PerkinElmer). 14. Deep well plate, 96-well. 15. TopSeal-A PLUS (PerkinElmer). 16. Plate reader with AlphaScreen measurement mode, for example, Envision or Enspire-Alpha (PerkinElmer).

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Methods This protocol is designed to construct an antigen protein array containing 92 proteins and 4 negative controls (mock, translation reaction without mRNA template) and to perform autoantigen screening using a single plate of the protein array with 10 sera in quadruplicate format. The protocol can be adapted to suit the specific goals of the assay, the number of proteins and sera being tested, and the desired level of replication.

3.1 Construction of Transcription Templates for CellFree Protein Synthesis

1. Insert a His tag and a bls tag into the pEU-E01-MCS vector using inverse PCR and Gibson Assembly [24] (Fig. 4a) (see Note 7). Mix 10 μL of PrimeStarMax polymerase mix, 4 μL of 1 μM Primer 1, 4 μL of 1 μM Primer 2, and 1 μL of 0.5 ng/μL pEU-E01-MCS vector. Perform the PCR reaction (denaturing 98 °C for 10 sec, annealing at 55 °C for 5 s, extension at 72 °C for 30 s, 25 cycles). 2. Verify the amplification of the 3.7 kbp product using electrophoresis. Apply 1 μL of the PCR reaction and a DNA size marker to a 1% agarose/TAE gel. After electrophoresis, visualize the DNA using the SYBRsafe gel stain reagent.

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3. Add 1 μL of DpnI to the remaining PCR reaction mixture and incubate for 30 min at 37 °C (see Note 8). Purify the DpnI-treated PCR product using a PCR product purification kit. 4. Connect the overlapping terminal sequences of the PCR product using the Gibson Assembly reaction; mix 2 μL of the PCR product and 2 μL of Gibson Assembly master mix, and then incubate the mixture at 50 °C for 15–60 min. 5. Transform E. coli strain JM109 with the assembled plasmid. Add 2 μL of the assembled plasmid to 40 μL of chemically competent cells. 6. Gently mix by inverting, place it on ice for 20 min. Heat the tube at 42 °C for 30 s, and chill on ice for 1 min. Add 160 μL of LB medium, and spread the transformant on an LB-ampicillin agar plate. Incubate the plate at 37 °C overnight. 7. Confirm the insertion of tags through sequencing using Primer 3 and Primer 4. Purify the pEU-E01-His-bls-MCS plasmid using a mini prep kit. 8. Insert the genes of interest into the pEU-E01-His-bls-MCS vector. For subcloning, either the conventional restriction enzyme method (see Note 9) or the Gibson Assembly method can be applied. Here, we introduce the method using Gibson Assembly. 9. Prepare the insert DNA fragment by PCR or gene synthesis (Fig. 4b). The insert DNA should consist of the S1 linker, gene of interest, stop codon, and T1 linker. The target DNA should not contain extra sequences such as introns or signal sequences, as mRNA or peptide processing is not conducted in the wheat cell-free system. Avoid frame shifts, considering the upstream start codon and tags in the pEU-E01-His-bls-MCS vector (Fig. 4a). 10. When using a synthetic gene as an insert, dilute the polynucleotide to 50 ng/μL with TE buffer. 11. When preparing an insert DNA by PCR, create a PCR reaction by mixing 5 μL of PrimeStarMax polymerase mix, 2 μL of 1 μM Primer 5, 2 μL of 1 μM Primer 6, and 1 μL of 0.2 ng/μL template DNA plasmid. Apply the reaction to a thermal cycler (denaturing at 98 °C for 10 s, annealing at 55 °C for 5 s, extension at 72 °C for 10 to 60 s, 25 cycles). 12. After the PCR reaction, add 0.5 μL of DpnI to the PCR reaction and incubate at 37 °C for 30 min (see Note 8). 13. Confirm amplification of the target by electrophoresis. Purification of the insert PCR product is not necessary (see Note 10).

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14. Linearize the pEU-E01-His-bls-MCS vector using inverse PCR. Prepare a PCR reaction by mixing 25 μL of PrimeStarMax polymerase mix, 10 μL of 1 μM Primer 7, 10 μL of 1 μM Primer 8, and 5 μL of 0.5 ng/μL pEU-E01-His-bls-MCS vector. Apply the reaction to a thermal cycler (denaturing 98 °C for 10 s, annealing at 55 °C for 5 s, extension at 72 °C for 30 s, 25 cycles). Linearize the pEU-E01-His-bls-MCS vector using inverse PCR. Prepare a PCR reaction by mixing 25 μL of PrimeStarMax polymerase mix, 10 μL of 1 μM Primer 7, 10 μL of 1 μM Primer 8, and 5 μL of 0.5 ng/μL pEU-E01-His-bls-MCS vector. Apply the reaction to a thermal cycler (denaturing at 98 °C for 10 s, annealing at 55 °C for 5 s, extension at 72 °C for 30 s, 25 cycles). 15. Add 1 μL of DpnI, incubate the reaction for 30 min at 37 °C, and purify the inverse PCR product using a PCR product purification kit. Measure the concentration of the purified linearized vector by measuring absorbance at 260 nm, and adjust it to 50 ng/μL concentration. 16. Assemble the plasmid by mixing 0.8 μL of an insert DNA fragment, 1.2 μL of the linearized vector, and 2 μL of Gibson Assembly master mix (see Note 10). Incubate the assembly reaction at 50 °C for 15 min to 1 h. 17. Transform E. coli strain JM109 with 2 μL of the assembled plasmid. Confirm target DNA insertion by colony direct PCR and sequencing using Primer 3 and Primer 4. 18. Purify plasmid using a mini prep kit. Store the purified plasmid at -30 °C. 19. Construct a plasmid plate. Add 200 μL of TE buffer to each well of a 96-well plate. Transfer 1 μL of pEU-E01-His-blstarget ORF plasmid to each well (approximately 0.1 to 2 ng plasmid/μL). Record the location information of each plasmid in the plate using spreadsheet software. Leave four wells (for example, wells H9–H12) empty to prepare mock translation reactions as negative controls in AlphaScreen. Seal the plate with aluminum foil sealing tape tightly, mix using a vortex mixer, and spin down. Store the plate at -30 °C. 20. Amplify transcription template DNA fragments from the plasmid plate constructed in Subheading 3.1, step 19 (see Note 11). To amplify 96-well samples, prepare PCR reaction master mix by mixing 1100 μL of PrimeStarMax polymerase mix, 440 μL of 1 μM SPu-2 (Primer 3), and 440 μL of 1 μM AODA2303 (Primer 9) in a reservoir. 21. Dispense 18 μL of the master mix into an empty 96-well PCR plate using an electric or manual 8-channel pipette.

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22. Transfer 2 μL of diluted pEU-E01-His-bls-target ORF plasmids to each well using an 8-channel pipette. Seal the plate with PCR plate sealing tape, mix gently using a vortex mixer, and spin down. 23. Apply the reaction plate to a PCR reaction (denaturing at 98 ° C for 10 s, annealing at 55 °C for 5 s, extension at 72 °C for 30 s, 30 cycles). 24. Confirm the amplified PCR products by gel electrophoresis. The length of the 5′ UTR and 3′ UTR is 0.2 and 1.6 kbp from the MCS, respectively. Seal the plate using aluminum sealing tape and store at -30 °C. 3.2 Cell-Free Synthesis of Autoantigen Protein Array

Protein synthesis includes transcription and translation reactions, with RNA manipulation occurring in both steps. To prevent RNA degradation, wear disposable plastic gloves and masks. Use clean plastic tubes, plates, and chips. It is recommended to separate plastic wares from those used for DNA experiments. Autoclaving these plastic wares is not necessary (see Note 1). Avoid using DEPC-treated water, as any remaining DEPC can inhibit reactions. It is recommended to use freshly purified ultrapure water for reagent master mix preparation. The reaction volumes introduced in this section are primarily for transcription and translation using one 96-well plate of PCR product as a template. The reaction volume should be adjusted according to the number of samples. 1. Set up transcription by turning on the air incubator and setting the temperature to 37 °C. Thaw the ×5 Transcription Buffer LM and 25 mM NTP mixture, mix by inverting, and spin down. Prepare fresh ultrapure water in a new plastic tube. Keep reagents and enzymes on ice until use. 2. Prepare transcription master mix in a reservoir by mixing 605 μL of ultrapure water, 275 μL of Transcription Buffer LM, 137.5 μL of 25 mM NTP mixture, 27.5 μL of 80 U/μL RNase inhibitor, and 55 μL of 80 U/μL SP6 polymerase. Gently mix the master mix by pipetting. Dispense 10 μL of master mix into new 96-well PCR plates using an 8-channel pipette. 3. Transfer 2.5 μL of transcription template DNA fragments to the master mix plate using an 8-channel pipette. Seal the plate with polypropylene sealing tape. Gently mix the reaction plate using a Vortex Mixer and spin down. 4. Incubate the transcription reaction at 37 °C for 6 h. After incubation, gently mix the reaction using a Vortex Mixer and spin down (see Notes 12 and 13). Keep the 96-well PCR plates containing mRNA at room temperature.

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5. Set up translation by setting the temperature of the air incubator to 15 °C. Thaw translation reagents by floating the tubes in a water bath at room temperature for several minutes. Gently mix the reagents, spin down, and keep on ice until use. 6. Prepare fresh ultrapure water in a new plastic tube. 7. Prepare ×1 SUB-AMIX SGC feeding buffer by mixing 14.4 mL of ultrapure water and 400 μL of each ×40 SUB-AMIX SGC stock solution (S1–S4, respectively) in a reagent reservoir. Mix the solution well by pipetting. 8. Dispense 125 μL of ×1 SUB-AMIX SGC into a 96-well flatbottom plate using an 8-channel pipette. Cover the plate with a lid or polypropylene sealing tape to prevent evaporation and contamination. 9. Prepare translation master mix in a reservoir by mixing 880 μL of WEPTRO7240 wheat germ extract, 55 μL of RNase inhibitor, 11 μL of 20 mg/mL creatine kinase, 110 μL of cell-free synthesized BirA, 132 μL of 60 μM biotin, and 187 μL × 1 SUB-AMIX SGC. Gently mix the master mix and spin down. 10. Spin down the 96-well PCR plates containing mRNA from Subheading 3.2, step 5. Transfer 12.5 μL of master mix into 12.5 μL of mRNA using a 50-μL 8-channel pipette, and mix gently by pipetting (avoid bubbling). 11. Aspirate the mixture using a 50-μL 8-channel pipette. 12. Insert the pipette tip into the surface of the SUB-AMIX SGC solution in the 96-well flat-bottom plate. Pipet out the reaction mixture slowly and gently. The reaction mixture naturally sinks and forms a layer at the bottom of the well (Fig. 5) (see Note 14). Do not mix or stir the reaction mix, as this will disturb the bilayer. 13. Seal the plate with polypropylene sealing tape to prevent evaporation. 14. Incubate the plate at 15 °C for 24 h.

Fig. 5 Cell-free protein synthesis using bilayer method

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15. After translation, gently mix the reaction, then spin down. Transfer 40 μL of synthesized proteins to three new 96-well PCR plates for assay. Additionally, transfer 10 μL of proteins to a 96-well PCR plate for Western blotting. Seal the plates tightly using aluminum foil sealing tape, freeze them using liquid nitrogen, and store them at -80 °C. 16. Confirm the production of biotinylated proteins by Western blotting. Add 5 μL of ×3 SDS-PAGE sample buffer to 10 μL of translation reaction mixture. Mix well, spin down, and heat the mixture at 70 °C for 10 min. 17. Load 6 μL of the samples and protein size marker onto an SDSPAGE gel. Electrophorese at 52 mA, 400 V for 30 min. 18. Transfer the proteins to a PVDF membrane using a semi-dry blotting apparatus according to the manufacturer’s instructions. 19. Block the blot in 5% milk-TBST for 1 h at room temperature with gentle shaking. 20. Rinse the membrane with TBST for 5 min at room temperature three times. Dilute anti-biotin antibody-HRP with TBST (1/1000) (see Note 15). Place the blot on a flat plastic film and add 2 mL of diluted anti-biotin antibody. Keep the blot in a plastic box with a lid to prevent evaporation and contamination, and incubate for 1 h. 21. Wash the blot with TBST for 5 min three times, and detect HRP-conjugated antibody using chemiluminescent HRP substrate and CCD imager. An example of a Western blotting image is shown in Fig. 6. 3.3

AlphaScreen

In this section, the procedure for autoantigen screening using one 96-well plate of protein array and 10 sera in a quadruplicate format is described. We use in-house protein G-conjugated AlphaScreen acceptor beads instead of commercially available protein A-conjugated acceptor beads because protein G binds to a wider range of immunoglobulin subclasses. We recommend using electric multichannel pipettes for dispensing in AlphaScreen assays due to their high efficiency and accuracy. 1. Prepare protein G-conjugated AlphaScreen acceptor beads by amine coupling. Conduct the following procedures in a dim lighting room (see Note 16). 2. Add the following reagents into a 1.5-mL tube: 612.5 μL of 0.1 M MES buffer, 62.5 μL of 1% Tween 20, 50 μL of 25 mg/ mL sodium cyanoborohydride, 200 μL of 10 mg/mL protein G, and 250 μL of 20 mg/mL unconjugated AlphaScreen acceptor beads.

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Fig. 6 Cell-free synthesized, biotinylated recombinant proteins. Immunoblot is proved with HRP conjugated anti-biotin antibody

3. Gently mix the reaction mixture by pipetting and wrap the tube with aluminum foil to shield light-sensitive beads from light. Incubate the tube at 37 °C for 66 h. 4. After incubation, add 50 μL of 65 mg/mL CMO and incubate the reaction at 37 °C for 1 h to terminate the coupling reaction. 5. Centrifuge the tube at 14,000 rpm at 4 °C for 15 min. Discard the supernatant and resuspend the pellet with 1 mL of ice-chilled 0.1 M Tris–HCl. Repeat the washing step once more. 6. Finally, resuspend the pellet with 1 mL of ice-chilled storage buffer. Transfer the protein G-conjugated acceptor bead suspension to a new 1.5-mL screw cap black tube and store at 4 °C until use (see Note 17). 7. Prepare dilution buffer. Mix 30 mL of 1 M Tris–HCl, pH 8.0, 30 mL of 0.1% Tween 20, 30 mL of 10 mg/mL BSA, and 210 mL of ultrapure water in a glass bottle. Use up the dilution buffer and diluted reagents within a day. 8. Dilute sera with dilution buffer and dispense into plates. Mix 15 μL of serum and 4985 μL of dilution buffer in a reservoir. Dispense 10 μL of diluted serum into each well of a 384-well optiplate384 using an electric 8-channel pipette (0.03 μL serum/reaction, 1/1000 dilution at final).

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9. Repeat serum dilution and dispensing, and prepare 10 plates. Seal the plates with polypropylene sealing tape and spin down. 10. Dilute and dispense protein array. Thaw the protein array plate completely at 4 °C. Add 570 μL of dilution buffer and 30 μL of cell-free synthesized protein in a 96-deep-well plate. Mix the diluted proteins by pipetting. 11. Using an electric 8-channel pipette, transfer 10 μL of diluted proteins to a 384-well plate, respectively, in a quadruplicate format. The final protein volume is 0.5 μL/reaction. Seal the 384-well plates using polypropylene sealing film, mix using a Vortex Mixer, and spin down. 12. Incubate the plates at 25 °C for 30 min. 13. Dilute and dispense AlphaScreen beads. Turn off the sealing light (see Note 16). Prepare the master mix in a reservoir, containing 38,612 μL of dilution buffer, 394 μL of protein G conjugate acceptor beads, and 394 μL of streptavidin donor beads (0.1 μL of acceptor bead and 0.1 μL of donor bead/ reaction). 14. Dispense 10 μL of diluted beads into the 384-well plates using an electric 8-channel pipette. Seal the plate using polyolefin sealing tape (see Note 18). Mix the plates using a vortex mixer and spin down. 15. Incubate the plates at 25 °C for 60 min. 16. Detect AlphaScreen signal using a plate reader. 17. Analyze data. Normalize AlphaScreen luminescence signals of each sample well by dividing by the average signal of mock wells in the same plate. To visualize and compare large amounts of data, we use the heat mapping and clustering function in Multiple Experiment Viewer (MeV, http://mev.tm4.org/). An example is shown in Fig. 7. (Option): For large-scale screening assays, using a plate-format compatible dispensing system is essential to achieve high speed and uniform results (Fig. 3). Once the protein array transfer template DNA is placed in 96 or 384 wells as described in Subheading 3.1, the subsequent process can be operated at high speed and convenience using a plate-format dispenser to efficiently handle the liquid samples on a plate-by-plate basis. The advantages of plate-format dispensers are speed, accuracy, and ease of use, enabling screening assays with large numbers of samples/plates and reducing human error. We use a motorized plate-format dispenser Viaflo 96/384 (Integra) that can be equipped with a 96-channel or 384-channel exchangeable head (Fig. 3a). This device can be operated manually or automatically for accurate microdispensing to build protein arrays and prepare AlphaScreen. We use a MultiDrop Combi nL (Thermo Fisher Scientific) to

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Fig. 7 Autoantigen profiling. Sera from breast cancer patients are reacted with each protein in antigen candidate protein array, respectively. Relative AlphaScreen signal is shown as heat map image

dispense AlphaScreen beads evenly onto the plate (Fig. 3b). The detection speed of AlphaScreen varies greatly depending on the plate reader. It is advisable to select a plate reader that is appropriate for the scale of screening to be performed. We use the EnVision multilabel reader (PerkinElmer), which has a fast and sensitive detection mode (Fig. 3c).

4

Notes 1. Autoclave treatment may not completely denature RNase. Additionally, autoclaving plasticware can cause deformation, and aerosols in a dirty autoclave apparatus can be a source of contamination. Using sterilized disposable plasticware is preferable. 2. WEPRO7240 should be stored at -80 °C. It withstands several freeze/thaw cycles. 3. Creatine kinase is sold in a lyophilized state. Add ultrapure water to lyophilized creatine kinase to achieve a 20 mg/mL concentration and dissolve completely. Dispense the creatine

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kinase solution into small portions in PCR tubes (10–50 μL each). Freeze the tubes using liquid nitrogen and store them at -80 °C. Avoid refreezing after thawing. 4. BirA biotin ligase is commercially available. However, the authors recommend synthesizing BirA using a cell-free system for the following reasons: (1) highly active BirA can be synthesized, and (2) cell-free synthesized BirA is RNase-free. The protocol for BirA preparation is described below: 4.1. Prepare the pEU-E01-BirA plasmid. Amplify the E. coli BirA gene (NM_31927) by PCR using E. coli cells as a template and subclone the fragment into the pEU-E01MCS vector. 4.2. Purify the plasmid from a 100-mL E. coli culture using the Nucleobond Xtra Midi kit (Takara Bio), followed by phenol-chloroform purification and ethanol precipitation. Dissolve the plasmid in ultrapure water at a concentration of 1 mg/mL. 4.3. Synthesize BirA using the bilayer method. Mix 115 μL of ultrapure water, 40 μL of transcription buffer LM, 20 μL of 25 mM NTP mix, 2.5 μL RNase inhibitor, 2.5 μL SP6 RNA polymerase, and 20 μL of 1 mg/mL pEU-E01-BirA plasmid. Incubate the reaction at 37 °C for 6 hours to synthesize mRNA. 4.4. Add 200 μL of WEPRO7240 and 1 μL of 20 mg/mL creatine kinase to the mRNA solution. Mix the reaction gently and spin down. Prepare 5.5 mL of 1× SUB-AMIX SGC (see Subheading 3.2, step 2) in a well of a 6-well flatbottom titer plate (TPP, 92406). 4.5. Aspirate the translation reaction mixture containing WEPRO7240 and mRNA using a pipet. Insert the pipet tip into the 1× SUB-AMIX SGC solution, and slowly and carefully pipet out the reaction mixture. Do not mix and disturb the bilayer. 4.6. Seal the plate with polypropylene sealing tape to avoid evaporation. Incubate the plate at 15 °C for 24 h. 4.7. After translation, mix the reaction gently. Dispense cellfree synthesized BirA into small portions (10–50 μL each), freeze the tubes using liquid nitrogen, and store them at 80 °C. Avoid refreezing after thawing. 5. Measure 14.6 mg of D-Biotin (Sigma-Aldrich) and add 800 μL of ultrapure water. Suspend the biotin by mixing. Add 1 M KOH dropwise and continue mixing until the biotin is completely dissolved. Adjust the volume to 1 mL (60 mM)

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by adding additional ultrapure water. Filter the biotin solution using a 0.22-μm syringe filter (Sartorius). Store the 60 mM biotin stock solution at -30 °C. Before use, prepare a 60-μM biotin solution by diluting the 60-mM biotin stock solution 1000 times with 1× SUB-AMIX SGC. 6. Prepare a 10% Tween 20 stock solution as follows. Pour 10 mL of Tween 20 into a 100-mL graduated cylinder. Add 90 mL of ultrapure water. Cover the cylinder with Parafilm and mix the solution thoroughly by inverting and rotating the cylinder. The 10% Tween 20 solution can be stored at room temperature. 7. The His tag can be used for confirmation of protein expression and purification if required. The bls (biotin ligation site) tag, which is a recognition sequence for the BirA biotin ligase, is necessary for the association of bls-tagged proteins with streptavidin donor beads. The lysine residue in the bls tag (Fig. 4a) is biotinylated by BirA. 8. DpnI recognizes and digests methylated GATC sites. In this case, DpnI is used to eliminate the template plasmid in the PCR reaction. The PCR product is not digested because it is not methylated. There is no need to add DpnI reaction buffer, as DpnI is active under common PCR buffer conditions. 9. If a DNA fragment is inserted into the pEU-E01-MCS vector using a restriction enzyme, connect the 5′-terminal of the insert DNA to one of the enzyme sites: EcoRV, SpeI, XhoI, SacI, and KpnI. Higher translation efficacy is expected by positioning the translation enhancer E01 sequence and start codon. Any restriction site in the multiple cloning site can be used for connecting the 3′-terminal of the insert DNA. 10. A high concentration of primers containing overlap sequences may interfere with the Gibson Assembly reaction. However, a small amount of unpurified PCR product (up to 20% of the Gibson Assembly reaction) can be added to the reaction without causing issues. In this protocol, the linearized pEU-E01His-bls-MCS is purified by column, while the insert DNA is not purified for convenience. 11. Plasmids purified with a mini prep kit are not suitable for transcription due to their low DNA concentration and the potential for RNase contamination from the mini prep kit. Midi or Maxi prep purification followed by phenol-chloroform purification can yield ideal transcription template plasmids; however, preparing multiple samples using maxi prep can be time-consuming and labor-intensive. Although it may not provide the same translation efficacy as a plasmid template, DNA fragments prepared by PCR can be used as transcription

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templates. In this protocol, PCR templates are used because they are more suitable for preparing multiple transcription templates. 12. After transcription, white precipitate particles may be observed in the reaction mixture. These precipitates primarily consist of insoluble magnesium pyrophosphate, a byproduct of transcription, and also contain a small amount of mRNA. Instead of removing the precipitate, mix the supernatant and precipitate well by pipetting, and then add the translation reaction mixture to it. 13. To confirm mRNA synthesis, perform electrophoresis by applying 1 μL of the RNA sample to a 1% agarose TAE gel. Also, apply a DNA ladder marker. Run electrophoresis at 100 V for 20 min and then stain the gel using SYBRsafe. If a smeared band smaller than 500 bp is observed, it may indicate the presence of degraded mRNA. RNase contamination of the reaction solution should be checked and reagents and equipment should be replaced as necessary. 14. The translation reaction mixture has a much higher specific gravity than ×1 SUB-AMIX SGC. Therefore, the slowly injected mixture spontaneously sinks and forms a layer at the bottom of the well, limiting diffusion. During incubation, the exchange of substrates and removal of byproducts between the reaction mixture and feeding solution occur slowly and effectively. 15. Skimmed milk contains biotin. Make sure to wash the blot thoroughly to remove any residual skimmed milk before treating it with the anti-biotin antibody. Do not dilute the antibiotin antibody with milk, as the presence of free biotin may block the antibody and prevent the detection of biotinylated proteins. 16. AlphaScreen beads are sensitive to intense light. The manufacturer recommends conducting AlphaScreen assays under dim light conditions of 100 Lux or less. Turn off direct lighting and use indirect lighting during the assay. 17. To confirm the performance of the protein G acceptor bead, conduct a control assay using biotinylated rabbit IgG that is originally included with the AlphaScreen IgG (Protein A) detection kit (PerkinElmer). 18. Avoid using polypropylene or PET sealing films, as they may cause high background noise. Be cautious of wrinkles or lifting in the sealing film, as these can affect the results.

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Acknowledgments The author would like to express gratitude to all collaborators and colleagues who have worked together on autoantibody profiling assays. Special thanks go to Dr. Yohei Miyagi for his bioresource collection, Professor Tatsuya Sawasaki for his mentorship, and Wei Zhou for proofreading and providing valuable comments. This work was performed using the facilities provided by the Division of Applied Protein Research Support, the Advanced Research Support Center (ADRES), Ehime University. This work was partially supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED, Japan, under Grant Number JP23ama121010. References 1. Scofield RH (2004) Autoantibodies as predictors of disease. Lancet 363:1544–1546 2. Jog NR, James JA (2017) Biomarkers in connective tissue diseases. J Allergy Clin Immunol 140:1473–1483 3. Sirotti S, Generali E, Ceribelli A, Isailovic N, Santis MD, Selmi C (2017) Personalized medicine in rheumatology: the paradigm of serum autoantibodies. Auto Immun Highlights 8:10 4. Lleo A, Invernizzi P, Gao B, Podda M, Gershwin ME (2010) Definition of human autoimmunity—autoantibodies versus autoimmune disease. Autoimmun Rev 9:A259–A266 5. Onishi S, Adnan E, Ishizaki J, Miyazaki T, Tanaka Y, Matsumoto T, Suemori K, Shudou M, Okura T, Takeda H et al (2015) Novel autoantigens associated with lupus nephritis. PLoS ONE (Bobe´, P., ed.) 10: e0126564 6. Wu J, Li X, Song W, Fang Y, Yu L, Liu S, Churilov LP, Zhang F (2017) The roles and applications of autoantibodies in progression, diagnosis, treatment and prognosis of human malignant tumours. Autoimmun Rev 16: 1270–1281 7. Meeusen E, Lim E, Mathivanan S (2017) Secreted tumor antigens – immune biomarkers for diagnosis and therapy. Proteomics (Mathivanan, S., ed.) 17:1600442 8. Ishigami T, Abe K, Aoki I, Minegishi S, Ryo A, Matsunaga S, Matsuoka K, Takeda H, Sawasaki T, Umemura S et al (2013) Antiinterleukin-5 and multiple autoantibodies are associated with human atherosclerotic diseases and serum interleukin-5 levels. FASEB J 27: 3437–3445

9. Takai K, Sawasaki T, Endo Y (2010) Practical cell-free protein synthesis system using purified wheat embryos. Nat Protoc 5:227–238 10. Sawasaki T, Hasegawa Y, Tsuchimochi M, Kamura N, Ogasawara T, Kuroita T, Endo Y (2002) A bilayer cell-free protein synthesis system for high-throughput screening of gene products. FEBS Lett 514:102–105 11. Endo Y, Sawasaki T (2006) Cell-free expression systems for eukaryotic protein production. Curr Opin Biotechnol 17:373–380 12. Goshima N, Kawamura Y, Fukumoto A, Miura A, Honma R, Satoh R, Wakamatsu A, Yamamoto J, Kimura K, Nishikawa T et al (2008) Human protein factory for converting the transcriptome into an in vitro–expressed proteome. Nat Methods 5:1011–1017 13. Takahashi H, Takahashi C, Moreland NJ, Chang Y-T, Sawasaki T, Ryo A, Vasudevan SG, Suzuki Y, Yamamoto N (2012) Establishment of a robust dengue virus NS3-NS5 binding assay for identification of protein-protein interaction inhibitors. Antivir Res 96:305–314 14. Nemoto K, Takemori N, Seki M, Shinozaki K, Sawasaki T (2015) Members of the plant CRK superfamily are capable of trans- and autophosphorylation of tyrosine residues. J Biol Chem 290:16665–16677 15. Morita M, Takashima E, Ito D, Miura K, Thongkukiatkul A, Diouf A, Fairhurst RM, Diakite M, Long CA, Torii M et al (2017) Immunoscreening of plasmodium falciparum proteins expressed in a wheat germ cell-free system reveals a novel malaria vaccine candidate. Sci Rep 7:46086

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16. Ullman EF, Kirakossian H, Wartchow CA, Pease J, Dafforn A, Davalian D, Skold C, Kurn N, Wagner DB (1996) Luminescent oxygen channeling assay (LOCI): sensitive, broadly applicable homogeneous immunoassay method. Clin Chem 42:1518–1526 17. Segawa R, Takeda H, Yokoyama T, Ishida M, Miyata C, Saito T, Ishihara R, Nakagita T, Sasano Y, Kanoh N et al (2021) A chalcone derivative suppresses TSLP induction in mice and human keratinocytes through binding to BET family proteins. Biochem Pharmacol. https://doi.org/10.1016/j.bcp.2021.114819 18. Nishiyama K, Maekawa M, Nakagita T, Nakayama J, Kiyoi T, Chosei M, Murakami A, Kamei Y, Takeda H, Takada Y et al (2021) CNKSR1 serves as a scaffold to activate an EGFR phosphatase via exclusive interaction with RhoB-GTP. Life Sci Alliance 4: e202101095 19. Matsuoka K, Komori H, Nose M, Endo Y, Sawasaki T (2010) Simple screening method for autoantigen proteins using the N-terminal biotinylated protein library produced by wheat cell-free synthesis. J Proteome Res 9:4264– 4273 20. Sawasaki T, Kamura N, Matsunaga S, Saeki M, Tsuchimochi M, Morishita R, Endo Y (2008)

Arabidopsis HY5 protein functions as a DNA-binding tag for purification and functional immobilization of proteins on agarose/ DNA microplate. FEBS Lett 582:221–228 21. Mizutani Y, Tsuge S, Takeda H, Hasegawa Y, Shiogama K, Onouchi T, Inada K, Sawasaki T, Tsutsumi Y (2014) In situ visualization of plasma cells producing antibodies reactive to Porphyromonas gingivalis in periodontitis: the application of the enzyme-labeled antigen method. Mol Oral Microbiol 29:156–173 22. Mizutani Y, Matsuoka K, Takeda H, Shiogama K, Inada K, Hayakawa K, Yamada H, Miyazaki T, Sawasaki T, Endo Y et al (2013) Novel approach to identifying autoantibodies in rheumatoid synovitis with a biotinylated human autoantigen library and the enzyme-labeled antigen method. J Immunol Methods 387:57–70 23. Nagayoshi Y, Nakamura M, Matsuoka K, Ohtsuka T, Mori Y, Kono H, Aso T, Ideno N, Takahata S, Ryo A et al (2014) Profiling of autoantibodies in sera of pancreatic cancer patients. Ann Surg Oncol 21(Suppl 3):S459– S465 24. Gibson DG (2011) Enzymatic assembly of overlapping DNA fragments. Meth Enzymol 498:349–361

Chapter 13 Generation of Specific Aptamers Shuang Liu, Yasuyuki Suzuki, and Makoto Inui Abstract Nucleic acid aptamers are therapeutic agents consisting of short single-strand DNA or RNA oligonucleotides, which have the ability to bind to target therapeutic molecules with high affinity and specificity and have been developed as potent drugs for the treatment of rheumatoid arthritis. Aptamers have unique and advantageous features over antibodies, such as superior affinity with nano- or pico-molar dissociation constants and ease of chemical synthesis, modification, and inactivation by designing antisense sequences. In this chapter, using a DNA-oligonucleotide pool, the technology of proteoliposome-systematic evolution of ligands by exponential enrichment (SELEX) is introduced. By using this technique, potential therapeutic agents with high affinity and specificity could be obtained. Key words Aptamer, Proteoliposome, SELEX, Single-strand DNA, PCR

1

Introduction Nucleic acid aptamers are biochemical or therapeutic agents consisting of short single-strand DNA or RNA oligonucleotides, which have the ability to bind to target therapeutic molecules with high affinity and specificity. Aptamers are also potent inhibitors of protein function and have thus been applied to the development of new drugs for the treatment of rheumatoid arthritis (RA) [1– 3]. Aptamers targeting interleukin (IL)-6 and IL-17 have been selected for inhibiting relative cell signaling pathways. The results of preclinical studies suggested that aptamers could be attractive therapeutic agents for the treatment of RA. An aptamer can be selected through an iterative selectionamplification process known as systematic evolution of ligands by exponential enrichment (SELEX) (Fig. 1). In 1990, the laboratories of G. F. Joyce (La Jolla), J.W. Szostak (Boston), and L. Gold (Boulder) independently developed a technique that allows simultaneous screening of more than 1015 individual nucleic acid molecules for different functionalities [4, 5]. The concept of SELEX is based on the ability of short sequences (up to 80 m) to

Shuang Liu (ed.), Rheumatoid Arthritis: Methods and Protocols, Methods in Molecular Biology, vol. 2766, https://doi.org/10.1007/978-1-0716-3682-4_13, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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Fig. 1 Schematic representation of proteoliposome-SELEX. The selection procedure is based on the following steps: (1) DNA selection from oligonucleotide library and (2) amplification of the enriched DNA pool. Negative selection should be performed before or after positive selection in order to remove nonspecific binding sequences

fold in the presence of a target into unique three-dimensional structures that bind the target with high affinity and specificity. During the selection process, a single oligonucleotide pool consisting of 1014–1015 variants of a random 30–100 nt sequence is incubated with a target molecule [6]. Variants with high binding activity are then harvested, followed by amplification of the enriched library by transcription-polymerase chain reaction (PCR). Finally, the single-strand pool is regenerated by templatestrand removal. Typically, this process is repeated for several rounds for the identification of aptamers. Due to their ability to bind to proteins with high affinity and selectivity, aptamers are often compared to therapeutic antibodies, which have become one of the fastest-growing classes of drugs in recent years and are approved for the treatment of RA. Aptamers have unique and advantageous features over antibodies, such as superior affinity with nano- or pico-molar dissociation constants and ease of chemical synthesis, modification, and inactivation by designing antisense sequences [7]. Antibodies have generally better pharmacokinetic and other systemic properties, often sufficient to support product development [1]. In contrast, aptamers have a relatively shorter circulating half-life, which needs to be improved by chemical modifications. A tunable short circulating half-life can be used in situations that do not require a long half-life, such as the need for an anticoagulant agent during cardiac surgery or when antibody therapy exerts an adverse effect due to its long half-life.

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In this chapter, using a DNA-oligonucleotide pool, the technology of proteoliposome-SELEX is introduced. The synthesis and optimization of proteoliposomes are described in Chapters 7 and 8. Proteoliposome-SELEX offers several advantages. This technology is specific to membrane protein targets. Mass production of highquality proteins that maintain potential functional structure by insertion into the lipid layer allows conformation-sensitive aptamer isolation. Also, using structure-modified target proteins could become an application in SELEX. Since synthetic proteoliposomes can be pellets by centrifugation, the selected oligonucleotides can be easily harvested and used for further PCR amplification.

2

Materials

2.1 Oligonucleotide Library and Primers

1. Starting pool of DNA-oligonucleotides: DNA-APT40: 3′- ATGACCATGACCCTCCACACNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCA GACTGTGGCAGGGAAAC-5′. 2. DNA-primer 1 (Tm 62 °C): 3′- ATGACCATGACCCTCCA CAC-5′. 3. DNA-primer 2 (Tm 62 °C): 3′- GTTTCCCTGCCA CAGTCTGA-5′. 4. DNA-primer 2-biotin (Tm 62 °C): 3′- GTTTCCCTGCCA CAGTCTGA-Biotin-5′.

2.2

Selection

1. Synthetic proteoliposomes (5 mg/mL): the target synthetic proteins are inserted into the lipid layer. 2. Mock liposomes without protein insertion. 3. 1 M hydroxymethyl-aminomethane (Tris)–HCl buffer (pH 7.4): Weigh 12.1 g of Tris base and dissolve in 80 mL of water. Mix and adjust pH with HCl to 7.4 (see Note 1). Make up to 100 mL with water. Autoclave it and store it at room temperature. 4. 1 M sodium chloride (NaCl) solution: Dissolve 5.84 g of NaCl in 50 mL water. Make up to 100 mL with water. Autoclave it and store it at room temperature. 5. Binding buffer: 20 mM Tris–HCl (pH 7.4) and 150 mM NaCl. Mix 20 mL of 1 M Tris–HCl (pH 7.4) and 15 mL of 1 M NaCl solution. Make up to 100 mL with water. Mix the buffer well and store at 4 °C. 6. Binding buffer supplied with 0.5% sodium dodecyl sulfate (SDS): Weigh 0.5 g of SDS and add to 50 mL of binding buffer. Mix and make up to 100 mL with additional binding buffer. Store the buffer at 4 °C.

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7. 0.5 M ethylenediaminetetraacetic acid (EDTA) buffer: Weigh 18.6 g ethylenediaminetetraacetic acid disodium salt, 2-hydrate and dissolve it in 80 mL of water. Mix and adjust pH to 8.0 using sodium hydroxide (NaOH) (see Note 2). Autoclave the buffer and store it at room temperature. 8. Tris–EDTA (TE) buffer (pH 8.0): Mix 1.0 mL 1 M Tris–HCl buffer (pH 8.0) and 0.5 mL 0.5 M EDTA (pH 8.0). Make up to 100 mL with water. Store at 4 °C. 9. Phenol:chloroform:isoamyl alcohol 25:24:1 (saturated with 10 mM Tris, pH 8.0, with 1 mM EDTA). 10. 70% ethanol: Mix 70 mL of ethanol and 30 mL of water. Mix and store at -20 °C. 11. 20 mg/mL glycogen. 12. 10 mg/mL bovine serum albumin (BSA): Dissolve 50 mg of BSA in 5 mL of water. Filter it using a 0.45-μm filter and dispense in small volumes. Store at -20 °C. 13. Water: Autoclave and cool down to room temperature. 2.3

Amplification

1. TaKaRa PCR Amplification Kit. 2. NucleoSpin® Gel and PCR Clean-up kit. 3. TBE solution (5×): Add 800 mL of water to 27.5 g of boric acid and 54 g of Trizma base. Mix and add 20 mL of 0.5 M EDTA solution (pH 8.0). Make up to 1 L and store at room temperature. 4. Ethidium bromide solution (10 mg/mL). 5. 3% agarose gel: Weigh 3 g of agarose and put into a 500-mL beaker. Add to 100 mL of TBE solution (1×). Heat the solution to 80 °C and mix until the solution becomes clear (see Note 3). Avoid air bubbles in the gel solution. Add one drop of ethidium bromide solution (10 mg/mL) to the gel solution and mix by gently shaking the beaker (see Note 4). Pour the agarose into a gel tray with the well comb in place (see Note 5). Keep the newly poured gel at 4 °C for 10–15 mins or let it sit at room temperature for 20–30 min, until it has completely solidified. 6. 20 mg/mL glycogen. 7. Casting tray. 8. Well combs. 9. Voltage source. 10. Gel box. 11. UV light source. 12. Microwave (optional).

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13. 0.2-mL PCR tubes. 14. Thermal cycler. 2.4 Purification of Single-Strand DNA (ssDNA)

1. Pierce™ high capacity streptavidin beads (Thermo Fisher). 2. TE buffer containing 1 M NaCl: Dissolve 5.84 g of NaCl in 100 mL of TE buffer and store at 4 °C. 3. 0.1 M NaOH: Dissolve 0.4 g of NaOH in 100 mL of water. Autoclave it and store it at room temperature. 4. 5 M NaCl: Dissolve 29.2 g of NaCl in 100 mL of water. Autoclave it and store it at room temperature. 5. Phenol:chloroform:isoamyl alcohol 25:24:1 (saturated with 10 mM Tris, pH 8.0, 1 mM EDTA). 6. 70% ethanol. 7. 20 mg/mL glycogen. 8. Binding buffer: 20 mM Tris–HCl (pH 7.4) and 150 mM NaCl.

3

Methods The protocol includes the following steps: Step 1. DNA selection from oligonucleotide library; Step 2. Amplification; and Step 3. Preparation of ssDNA, consisting of one round of SELEX. This process should be repeated for 15–30 rounds or more for the identification of aptamers. After the selection process, the enriched oligonucleotide pool should be cloned into competent cells for sequencing. We do not describe the process of sequencing in detail here, but some information about the subsequential process after SELEX is introduced at the end of the protocols.

3.1 DNA Selection from Oligonucleotide Library

1. Dilute 3 nmol DNA-APT40 in 400 μL of binding buffer. 2. Heat DNA-APT40 (400 μL) to 100 °C for 3 min and immediately cool on ice for 5 min. 3. Negative selection: Mix 400 μL of boiled library, 100 μL of 10 mg/mL BSA, 20 μL of mock liposomes (see Note 6), and 2 mL of binding buffer. Incubate for 30 min at room temperature with occasional mixing. This step deletes the oligonucleotides nonspecifically binding to liposomes. 4. Centrifuge the mixture at ×16,000g for 15 min at 4 °C. 5. Recover the supernatant. 6. Positive selection: Add 20 μL of proteoliposomes to the supernatant (see Note 7). Incubate the mixture for 30 min at room temperature with occasional mixing. This step selects the oligonucleotides with conformation-sensitive binding to the target protein.

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7. Centrifuge the mixture at ×16,000g for 15 min at 4 °C. 8. Remove the supernatant and obtain the pellet (see Note 8). 9. Resuspend the pellet using 2.5 mL of binding buffer (see Note 9). 10. Centrifuge resuspended proteoliposomes at ×16,000g for 15 min at 4 °C. 11. Remove the supernatant and obtain the pellet. Repeat steps 9– 11 twice. 12. Resuspend the pellet with 100 μL of binding buffer containing 0.5% SDS. 13. Add 100 μL of phenol:chloroform:isoamyl alcohol (25:24:1) to the protoliposomes, and shake thoroughly by hand for approximately 20 s. 14. Centrifuge at room temperature for 5 min at 16,000×g. Remove the upper aqueous layer, and transfer the layer to a fresh tube. Do not to carry over any phenol during pipetting. 15. Add 500 μL of 100% ethanol and 1 μL of 20 mg/mL glycogen to the aqueous layer. Place the tube at -80 °C for at least 1 h. 16. Centrifuge at 16,000×g at 4 °C for 30 min to pellet the DNA. 17. Wash the DNA pellet by adding 1 mL of 70% ethanol. Centrifuge at 16,000×g at 4 °C for 30 min to pellet the DNA. 18. Completely remove the supernatant and dry the DNA. 19. Resuspend the DNA using 20 μL of autoclaved water. 3.2

Amplification

1. Mix the PCR reagent in a PCR tube according to the manufacturer’s instructions. Using a TaKaRa PCR Amplification Kit, the mixture should be prepared as follows: 10× PCR buffer (Mg2+ plus) 5 μL dNTP mixture (2.5 mM) 4 μL DNA-primer 1 (0.2 μM) 0.5 μL DNA-primer 2-biotin (0.2 μM) 0.5 μL TaKaRa Taq 0.25 μL DNA template 20 μL (obtained as described in Subheading 3.1, step 19) Autoclaved water 19:75 μL Total 50 μL 2. Set the PCR tubes filled with 50 μL of final reaction mixture in a thermal cycler. 3. Set the reaction conditions as follows:

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Step 1: 95 °C

2 min

Step 2 (10 cycles): 95 °C

30 s

56 °C

30 s

72 °C

30 s

Step 3: 72 °C

3 min

4 °C

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4. Electrophorese the whole volume of PCR production on 3% agarose gel at 100 V for 20 min. 5. Subject the gel to detection using a UV device and cut out the band with size around 80 bp. 6. Extract the DNA from the gel using a NucleoSpin® Gel and PCR Clean-up kit according to the manufacturer’s instructions. 7. Recover 20 μL of eluates of DNA from the purification process. 3.3 Preparation of ssDNA

1. Wash 5 μL of High Capacity Streptavidin beads by adding 500 μL of TE buffer containing 1 M NaCl. Centrifuge the beads at ×5000g for 1 min. 2. Remove the supernatant. Repeat the washing step three times. 3. Add 10 μL of purified PCR product to the beads. Gently mix for 30 min at room temperature. 4. Centrifuge the beads at 5000g for 1 min at 4 °C. 5. Wash the beads by adding 1 mL of TE buffer containing 1 M NaCl. Centrifuge the beads at 5000g for 1 min at 4 °C. Remove the supernatant. Repeat this wash step five times. 6. Add 100 μL of 0.1 M NaOH to the beads and incubate at room temperature for 5 min. 7. Centrifuge the beads at 5000g for 1 min at 4 °C. 8. Obtain the supernatant and fill in a new 1.5-mL tube. 9. Add an additional 100 μL of 0.1 M NaOH to the beads. 10. Centrifuge the beads at 5000g for 1 min at 4 °C. 11. Obtain the supernatant and combine it with the supernatant recovered in step 8. 12. Add 1 mL of 100% ethanol and 1 μL of 20 mg/mL glycogen to the combined supernatant. Keep the tube at -80 °C for at least 1 h. 13. Centrifuge at 16,000×g at 4 °C for 30 min to pellet the ssDNA.

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14. Wash the ssDNA pellet by adding 1 mL of 70% ethanol. Centrifuge at 16,000×g at 4 °C for 30 min to pellet the ssDNA. 15. Completely remove the supernatant and dry the ssDNA. 16. Resuspend the ssDNA using 50 μL of binding buffer. The selected ssDNA library is ready for the next round of SLEXE. Store the rest of the sample at -20 °C. By multiple cycles of selection and evolution, the complexity of the initial random DNA library is reduced, and potential aptamers with high affinity and specificity are enriched. The final selected pool, which is amplified by PCR using unmodified primers, should be cloned into a plasmid vector for sequencing. For sequencing, a TOPO® TA Cloning® Kit is recommended. The product information and manufacturer’s instructions can be found on the following website: https://www.thermofisher.com/order/catalog/product/ K457501. The final aptamer pool may be more complex than we thought. It is necessary to identify special sequence patterns and distinguish the real binding aptamer based on the sequencing information. A representative sequence that has a distinct sequence pattern should be synthesized, and further binding assay with the target protein should be performed.

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Notes 1. To adjust the pH, a lot of HCl is required. Start adjusting the pH using undiluted HCl, and use 1 N HCl to perform minor adjustments when pH is near the desired value. CAUTION: The solution is very hot. Be careful stirring, as eruptive boiling can occur. 2. Use solid NaOH at the start of pH adjustment, and use 5N NaOH when pH is near the desired value. 3. Agarose can also be dissolved by microwaving: Microwave for 30–45 s, stop and swirl, and then continue to boiling. Place cling film over the top of the flask to avoid the contents boiling over. 4. CAUTION: The gel solution is very hot. 5. Pour slowly to avoid bubbles that would disrupt the gel. Any bubbles can be pushed toward the edges of the gel with a pipette tip. 6. Always sonicate the mock liposomes in an ice-cooled bath for 15 min just before oligonucleotide binding. 7. Always sonicate the proteoliposomes in an ice-cooled bath for 15 min just before oligonucleotide binding.

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8. In this step, pellets should be harvested, which is different from step 5. 9. Violent pipetting may disturb the formation of proteoliposomes. The proteoliposomes should be resuspended gently. References 1. Ishiguro A, Akiyama T, Adachi H, Inoue J, Nakamura Y (2011) Therapeutic potential of anti-interleukin-17A aptamer: suppression of interleukin-17A signaling and attenuation of autoimmunity in two mouse models. Arthritis Rheum 63:455–466 2. Li W, Lan X (2015) Aptamer oligonucleotides: novel potential therapeutic agents in autoimmune disease. Nucleic Acid Ther 25:173–179 3. Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818–822

4. Hirota M, Murakami I, Ishikawa Y, Suzuki T, Sumida S, Ibaragi S et al (2016) Chemically modified interleukin-6 aptamer inhibits development of collagen-induced arthritis in cynomolgus monkeys. Nucleic Acid Ther 26:10–19 5. Takahashi M (2018) Aptamers targeting cell surface proteins. Biochimie 145:63–72 6. Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505–510 7. Ohuchi S (2012) Cell-SELEX technology. Biores Open Access 1:265–272

Chapter 14 Detailed Protocol for Predicting 3D Structure of DNA Aptamers and Performing In Silico Docking Calculations Yasuyuki Suzuki Abstract This paper presents a comprehensive protocol for predicting the three-dimensional (3D) structure of DNA aptamers and performing in silico docking calculations. The protocol includes steps for sequence input, structure prediction, sequence modification, structure minimization, and docking. The procedure is executed on a Mac environment utilizing bioinformatics tools such as mfold, RNA Composer, PyMOL, and Hdock. The protocol is intended to provide a guide for researchers in structural biology and drug design. Key words Docking, DNA aptamer, In silico, Structure prediction, 3D structure

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Introduction DNA aptamers are short, single-stranded DNA molecules that can bind to specific target molecules with high affinity and specificity. Their 3D structure is crucial for their binding properties, and predicting this structure can provide valuable insights into their function and potential applications. Furthermore, in silico docking calculations allow prediction of the interaction between the aptamer and its target, which is essential in drug design and development. This paper provides a detailed protocol for predicting the 3D structure of DNA aptamers and performing in silico docking calculations.

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Methods

2.1 Software Installation and Environment Setup

Before starting, ensure that the necessary software and packages are installed on your Mac. This includes mfold, RNA Composer, PyMOL, UCSF Chimera, and Hdock. Detailed instructions can

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Fig. 1 Overview of our protocol. (1) Start with a 40 bp sequence. (2) Input this sequence into mfold. (3) The output from mfold is then modified by replacing all instances of “T” with “U.” (4) This modified sequence is input into RNA Composer. (5) The output from RNA Composer is opened in PyMOL. (6) The structure is minimized in PyMOL. (7) Finally, docking is performed with Hdock using the minimized structure. This flowchart provides a visual representation of the steps in the protocol, making it easier to understand the process

be found on their respective websites. A flowchart outlining our protocol is shown below (Fig. 1).

Installing PyMOL To install PyMOL on a Mac, you can use the Homebrew package manager. If you don’t have Homebrew installed, you can install it using the following command in the terminal:

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‘‘‘ /bin/bash -c "$(curl -fsSL https://raw.githubusercontent.com/ Homebrew/install/HEAD/install.sh)" ‘‘‘

Then, you can install PyMOL using the following command: ‘‘‘ brew install pymol ‘‘‘

To install the Optimize plugin in PyMOL, download the plugin file from the official website and save it in the plugins directory of PyMOL. Then, in PyMOL, go to the Plugin menu and select “Manage Plugins.” Click on “Install” and select the downloaded plugin file. Installing UCSF Chimera UCSF Chimera can be downloaded from the official website (https://www.cgl.ucsf.edu/chimera/download.html) [1]. Choose the appropriate version for your operating system and follow the instructions provided on the website to install it. After downloading the installer, open it and follow the prompts to install UCSF Chimera. Once the installation is complete, you can open UCSF Chimera from your Applications folder. With PyMOL and UCSF Chimera installed, you are now ready to proceed with the protocol. 2.2 Sequence Input and Structure Prediction

Start by inputting your 40 bp sequence into the mfold web service (http://www.unafold.org/mfold/applications/dna-folding-form. php) under the following conditions: Na 0.15 mM, Mg 0.002 mM, 25 °C [2]. The output file from mfold will be in DNA format. Several candidates are presented, but the standard approach is to choose the calculation result that is most energetically stable. However, predictions that have a two-dimensional structure with a large loop structure should be avoided, as these structures are unstable. Small loops and long stem structures are stable and desirable candidates. The following sample data are presented for this study (Fig. 2). Download files in Vienna format. The file is in text format, and an example is shown below. ATGNNCNTGACCCTCCACACGCCCCCCTAGTGTTGACCACTCTGATCACCGTACTGTGGATCAGACTGTGGCAGGGAAACA . . . . . . . . . . ( ( ( ( . . ( ( ( ( . . . . . . . . . ) ) ) ) . . . (((((((((((...........)))))))..)))).))))..... (-9.54)

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Fig. 2 2D structure of sample DNA aptamer, as predicted by mfold 2.2.1 Sequence Modification

To use RNA Composer [3], the output file from mfold needs to be modified to replace all instances of “T” with “U.” You can edit the Vienna file directly with an editor, but it is easier to process it by referring to the following command: ‘‘‘ find ./ -type f | xargs sed -i.bak "s/T/U/g" ‘‘‘

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2.2.2 3D Structure Prediction

Input the modified sequence into RNA Composer (https:// rnacomposer.cs.put.poznan.pl/) to obtain the primary predicted 3D structure of the aptamer in PDB format.

2.2.3 Structure Minimization

Open the output file from RNA Composer in PyMOL. From the Display menu, select Sequence and choose “U.” Then, select Mutagenesis and change “U” back to “T” to revert back to DNA format. Install the Optimize plugin in PyMOL and use it to minimize the structure. The output will be a minimized PDB file of the aptamer. The minimization step is crucial as it allows the structure to reach a lower energy state, which is more stable and likely to occur in nature. This step is based on the energy minimization principle, which states that all systems tend to reach a state of minimum energy.

2.2.4 Preparing for Docking with Chimera

Before performing docking with Hdock, preparing the structure using the Dock Prep function in UCSF Chimera is necessary. This function adds hydrogens and assigns charges to the structure, which are essential for accurate docking calculations. To use the Dock Prep function, follow these steps: 1. Open the minimized PDB file of the aptamer in UCSF Chimera. 2. From the Tools menu, select Structure Editing and then Dock Prep. 3. In the Dock Prep window, click on the Preprocess button. This will add hydrogens and assign charges to the structure. 4. After preprocessing, click on the Write button to save the prepared structure in PDB format. The prepared PDB file can then be used for docking experiments with Hdock.

2.3 Docking with Hdock

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Perform docking between the prepared PDB file of the aptamer and a protein (e.g., “hoge.pdb”) using the Hdock web service (http:// hdock.phys.hust.edu.cn/) [4]. Follow the instructions on the Hdock website to input the PDB files and perform the docking calculation.

Limitations While this protocol provides a detailed guide for predicting the 3D structure of DNA aptamers and performing in silico docking calculations, certain limitations should be considered when interpreting the results.

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1. **Structural Fluctuations of Aptamers**: The protocol assumes that the structure of the aptamer is fixed at its energy-minimized state. However, aptamers exist in a state of constant fluctuation, which can influence their binding properties. Therefore, the docking data obtained from this protocol should be treated as a prediction and interpreted with caution. 2. **Conversion between RNA and DNA Formats**: The protocol involves converting the DNA sequence into an RNA format for structure prediction with RNA Composer and then converting it back to DNA format. This step could introduce errors or inaccuracies in the predicted structure. Future developments in the field will likely lead to the creation of software applications that can directly predict the 3D structure of DNA aptamers, eliminating the need for this conversion step. Despite these limitations, this protocol provides a valuable tool for researchers in the field of structural biology and drug design. It is important to consider these limitations when interpreting the results and continue refining and improving the protocol as new tools and techniques become available. References 1. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF chimera—a visualization system for exploratory research and analysis. J Comput Chem 25(13):1605–1612 2. Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31(13):3406–3415

3. Popenda M, Szachniuk M, Antczak M, Purzycka KJ, Lukasiak P, Bartol N, Adamiak RW (2012) Automated 3D structure composition for large RNAs. Nucleic Acids Res 40(14):e112 4. Yan Y, Zhang D, Zhou P, Li B, Huang SY (2017) HDOCK: a web server for protein–protein and protein–DNA/RNA docking based on a hybrid strategy. Nucleic Acids Res 45(W1): W365–W373

Chapter 15 RNA Interference Ex Vivo Shuang Liu Abstract RNA interference (RNAi) is a widely used technique to regulate the expression of genes and proteins with a high degree of specificity that is not easily accessed by traditional pharmacological approaches. For preclinical research on rheumatoid arthritis (RA), silencing of target genes in primary immune cells can be easily achieved by the application of small interfering RNA (siRNA) and synthetic short hairpin RNA (shRNA). Cellular and systemic administration of siRNA or shRNA has been a significant advance in preclinical research on RA. In this chapter, the basic techniques for gene silencing in human-derived peripheral T cells using liposome-dependent siRNA transfection and lentiviral-mediated shRNA delivery, aiming at gene silencing of therapeutic targets, are introduced. Key words RNA interference, Gene silencing, Lentiviral-mediated delivery, Transfection, T cells

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Introduction In 1998, Andrew Fire and Craig Mello discovered the process of RNA interference (RNAi) during their research on gene expression in the nematode worm C. elegans, which earned them a Nobel Prize in 2006, indicating the importance of RNAi technology [1]. RNAi has post-transcriptional gene silencing activity and cleaves homolog transcripts that modulate gene expression in different ways [2]. RNAi can be used to regulate the expression of proteins that are not easily accessed by traditional pharmacological approaches, such as molecules lacking ligand-binding domain proteins that share a high degree of structural homology [3]. RNAi-based therapeutic approaches especially appeal because they can achieve a high degree of specificity. Theoretically, RNAi, a small noncoding RNA, can exist as small interfering RNA (siRNA), Piwi interacting RNA (piRNA), and micro RNA (miRNA). Additionally, short hairpin RNA (shRNA) is synthetically created RNAi. siRNAs are 21–25 base pair (bp)-long

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single-stranded short linear RNAs. They act as blockers of the translation process by forming a complex with argonaute proteins (Ago) to help in interference and by endonuclease activity causing cleavage of transcripts, thus inducing the process of silencing [4, 5]. siRNA forms an RNA-induced silencing complex (RISC) that achieves silencing of target mRNA through repression of degradation. Sharing a similar mechanism with siRNAs, shRNAs are artificially created RNAi that require viral vectors or plasmids to be transcribed into cells. The effectiveness of shRNAs depends on their characteristics of being heritable, stable, and more potent in mammalian cells, on par with siRNA [6]. Cellular and systemic administration of siRNA or shRNA has been a significant advance in preclinical research on rheumatoid arthritis (RA) [7–11]. piRNAs form complexes with RNA protein through their interaction with piwi proteins, which act by targeting transposons. piRNAs usually act on germ cells, initially during spermatogenesis, by epigenetic and post-transcriptional mechanisms [3]. miRNAs are also small, single-stranded noncoding RNAs and bind to the partial complementary site of mRNA in three different regions, 3′, 5′, and the coding region, causing post-translational repression [12]. To deliver RNAis into cultured or primary cells, the barriers of the cell membrane and the endocytic pathway should be considered. Liposomes, micelles, and nanoparticles have been utilized for siRNA delivery [2]. Liposomes, which consist of a lipid bilayer with an internal aqueous core, are the most common tool to encapsulate siRNAs and are commercially available [13]. Micelles comprise a close spherical monolayer of phospholipids. They differ from liposomes in that they do not have an aqueous core. Because of their low toxicity, long half-life, and good tissue penetration, they are suitable for systemic delivery [14]. Nanoparticles are very small, generally with a diameter of 10–100 nm. They have several advantages over other delivery systems, including small size, high surface area, stability in physiological media, and immunologically inert surface, which give them especially good in vivo retention [15]. Here, aiming at gene silencing of a specific therapeutic target, the basic techniques for gene silencing in human-derived peripheral T cells using liposome-dependent siRNA transfection and lentiviral-mediated shRNA delivery are introduced. For primary T cells in RA studies, lentiviral-mediated shRNA is a very useful tool, while liposome-dependent methods show limitations in the rate of transfection. Delivery methods using RNAi should be optimized for further systemic administration.

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Materials

2.1 Isolation of T Cells from Peripheral Blood

1. Histopaque®-1077, sterile-filtered, density: 1.077 g/mL (Sigma Aldrich). 2. Conical tubes (50 mL). 3. Phosphate-buffered saline (PBS): Dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, 0.24 g of KH2PO4, 0.133 g of CaCl2•2H2O, and 0.10 g of MgCl2•6H2O in 800 mL of bi-distilled water. Adjust pH to 7.2 with HCl. Add water up to 1000 mL. Dispense the solution into aliquots and sterilize them by autoclaving or by filter sterilization. Store the solution at room temperature. 4. 0.5 M ethylenediaminetetraacetic acid (EDTA) solution (pH 8.0): Add 186.1 g of disodium EDTA•2H2O to 800 mL of bio-distilled water. Mix well by vigorously stirring. Adjust pH to 8.0 using NaOH. Dispense into aliquots and sterilize by autoclaving. Store the solution at room temperature. 5. Peripheral blood mononuclear cell (PBMC) separation buffer: PBS (pH 7.2) and 2 mM EDTA. Add 2 mL of 0.5 M EDTA solution (pH 8.0) to PBS (pH 7.2) and make the volume up to 250 mL. Keep buffer cold (2–8 °C). Bring it back to room temperature before PBMC separation. 6. Pan T cell isolation kit (Miltenyi Biotec). 7. T cell separation buffer: PBS (pH 7.2) with 0.5% bovine serum albumin (BSA) and 2 mM EDTA. Add 400 μL of 0.5 M EDTA solution (pH 8.0) and 500 mg of BSA to PBS (pH 7.2) and make the volume up to 250 mL. Keep buffer cold (2–8 °C). 8. LS separation column (Miltenyi Biotec). 9. Magnet and magnetic separation stand (Miltenyi Biotec). 10. Peripheral blood samples (see Note 1). 11. RPMI 1640 medium: Prepare a complete RPMI 1640 medium containing 10% fetal bovine serum (FBS). 12. Cell culture plates (96 well). 13. Pipettes (10 mL) and pipettors. 14. Centrifuge with swinging bucket rotor.

2.2 Transfection of siRNA into Primary T Cells Using Oligofectamine

1. Isolated human-derived T cells are seeded in a 96-well plate (104 cells/well). 2. siRNA/siRNA cocktail stock (20 μM). 3. Stealth™/siRNA Transfection Oligofectamine™ (Thermo Fisher Scientific). 4. RPMI 1640 medium without FBS and antibiotics.

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5. RPMI 1640 medium containing 30% FBS. 6. Centrifuge tubes (1.5 mL). 7. Pipettes and tips. 2.3 LentiviralMediated shRNA Transfection

1. shRNA lentiviral particles. 2. 4 mg/mL polybrene stock: Filter and aliquot into 1.5-mL microfuge tubes containing 100 μL each, and store at -20 °C. 3. Complete RPMI 1640 medium containing 10% FBS and 1% penicillin and streptomycin. 4. Transfection medium: Complete RPMI 1640 containing 10% FBS, 1% penicillin and streptomycin, and 8 μg/mL polybrene. 5. Pipette and tips.

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Methods (See Note 2)

3.1 Isolation of T Cells from Peripheral Blood

1. Dilute peripheral blood with a three-fold volume of PBMC separation buffer in 50-mL conical tubes (see Notes 3 and 4). 2. Add 15 mL of Histopaque®-1077 into new 50-mL conical tubes. 3. Carefully layer 35 mL of diluted peripheral blood on the top of 15 mL Histopaque®-1077. Carefully adjust the pressure in the pipette (to alter the speed of fluid uptake and dispensing). 4. Centrifuge tubes at 400×g for 30 min at 20 °C in a swinging bucket rotor without a brake. 5. After density gradient centrifugation, carefully take the tubes from the centrifuge and set them in a tube stand. The layers after centrifugation, from the top, are the plasma layer, PBMC layer, Histopaque layer, granulocyte layer, and erythrocyte layer. 6. Remove the upper plasma layer, leaving the PBMC layer undisturbed at the interphase. 7. Carefully transfer the PBMC layer to a new 50-mL conical tube. 8. Fill the tube with buffer and bring the volume up to 50 mL. Centrifuge the tube at 300×g for 10 min at 20 °C. 9. Remove the supernatant completely. Resuspend the PBMC in 10 mL pre-cooled T cell separation buffer. Determine the cell number. 10. Centrifuge the tube at 300×g for 5 min at 20 °C. Remove the supernatant carefully and resuspend the cell pellet in 40 μL of T cell separation buffer per 107 total cells. 11. Add 10 μL of Pan T cell biotin-antibody cocktail (Pan T cell isolation kit) per 107 total cells.

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12. Mix well and incubate for 5 min in a refrigerator (2–8 °C) (see Note 5). 13. Add 30 μL of Pan T separation buffer per 107 total cells. 14. Add 20 μL of Pan T cell microbeads cocktail (Pan T cell isolation kit) per 107 total cells. 15. Mix well and incubate for 5 min in a refrigerator (2–8 °C). 16. During incubation, place an LS column in the magnetic field of a magnetic stand. 17. Add 3 mL of T cell separation buffer to the column. Avoid any air bubbles in the column. Discard the flow-though. 18. Set a new collection tube under the column. 19. Apply the cell suspension to the column. Collect flow-through containing unlabeled cells, representing enriched T cells. 20. Wash the column by applying 3 mL of T cell separation buffer to the column. Collect the flow-through. Repeat the washing step three times. Combine all the eluent from step 19. 21. Centrifuge the tube at 300×g for 5 min at 20 °C. Remove the supernatant carefully and resuspend the cell pellet using a suitable volume of complete RPMI 1640 medium. 22. Determine the cell number. Culture enriched T cells at 104 cells/well in complete RPMI 1640 medium using a 96-well plate in a CO2 incubator overnight. The T cells are ready for RNAi transfection. 3.2 Transfection of siRNA into Primary T Cells Using Oligofectamine

1. Cells should be approximately 50% confluent on the day of infection. Replace the culture medium with 80 μL of pre-warmed RPMI 1640 medium without FBS and antibiotics. 2. Dilute 1 μL of 20 μM stock of single siRNA or siRNA cocktail in 16 μL of RPMI 1640 without FBS and antibiotics for each transfection sample in a 1.5-mL centrifuge tube. Mix gently. 3. Add 0.5 μL of oligofectamine into 2.5 μL of RPMI 1640 in a new 1.5-mL centrifuge tube for each sample. Mix gently and incubate for 10 min at room temperature. 4. Add oligofectamine mixture into siRNA mixture dropwise. Mix gently and incubate for 20 min at room temperature. 5. After complexes form, add the complexes to the cells. 6. Gently mix the plate and incubate the cells at 37 °C in a CO2 incubator for 4 h. 7. Add 50 μL of RPMI 1640 containing 30% FBS to the wells without removing the transfection mixture. 8. Assay for gene expression and protein expression at 24–72 h post-transfection.

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3.3 LentiviralMediated shRNA Transfection (See Note 6)

1. Cells should be approximately 50% confluent on the day of infection. Replace the culture medium with 100 μL of pre-warmed transfection medium containing polybrene. 2. Thaw lentiviral particles at room temperature and keep the viral particles on ice before transfection. Mix gently before use. 3. Add lentiviral particles to the culture medium (see Note 7). 4. Swirl the plate gently to mix and incubate overnight. 5. Remove the culture medium and replace it with 100 μL of complete RPMI 1640 medium (see Note 8). Assay for gene expression and protein expression at 24–72 h post-transfection.

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Notes 1. Before peripheral blood sampling, all subjects should give informed consent in accordance with the Declaration of Helsinki, and the research protocols should be approved by the Ethics Committee of the research institutes. 2. All experiments should be performed under sterile conditions. 3. Fresh peripheral blood should be used, and the samples should not be older than 8 h. 4. The more diluted the blood sample, the better the purity of the mononuclear cells. 5. Do not incubate PBMC on ice. 6. All procedures in this protocol should be performed under biosafety level 2 (BSL-2). 7. When transducing a shRNA lentiviral construct into a cell for the first time, we suggest using several amounts of shRNA lentiviral particle stock. 8. Polybrene is a polycation that neutralizes charge interactions to increase binding between the pseudoviral capsid and the cell membrane. Excessive exposure to polybrene (>12 h) can be toxic to T cells.

References 1. Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 16:948–958 2. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806–811

3. Simmer F, Buscaino A, Kos-Braun IC, Kagansky A, Boukaba A, Urano T et al (2010) Hairpin RNA induces secondary small interfering RNA synthesis and silencing in trans in fission yeast. EMBO Rep 11:112–118 4. Carthew RW, Sontheimer EJ (2009) Origins and mechanisms of miRNAs and siRNAs. Cell 136:642–655

RNA Interference Ex Vivo 5. Yousefpour Marzbali M, Yari Khosroushahi A (2017) Polymeric micelles as mighty nanocarriers for cancer gene therapy: a review. Cancer Chemother Pharmacol 79:637–649 6. Shi Q, Rondon-Cavanzo EP, Dalla Picola IP, Tiera MJ, Zhang X, Dai K et al (2018) In vivo therapeutic efficacy of TNFalpha silencing by folate-PEG-chitosan-DEAE/siRNA nanoparticles in arthritic mice. Int J Nanomedicine 13:387–402 7. Liu S, Kiyoi T, Takemasa E, Maeyama K (2017) Intra-articular lentivirus-mediated gene therapy targeting CRACM1 for the treatment of collagen-induced arthritis. J Pharmacol Sci 133:130–138 8. Liu S, Sahid MN, Takemasa E, Kiyoi T, Kuno M, Oshima Y et al (2016) CRACM3 regulates the stability of non-excitable exocytotic vesicle fusion pores in a Ca(2+)independent manner via molecular interaction with syntaxin4. Sci Rep 6:28133 9. Liu S, Watanabe S, Shudou M, Kuno M, Miura H, Maeyama K (2014) Upregulation of store-operated ca entry in the naive CD4 T cells with aberrant cytokine releasing in active rheumatoid arthritis. Immunol Cell Biol 92(9): 752–760

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10. Terenzi R, Manetti M, Rosa I, Romano E, Galluccio F, Guiducci S et al (2017) Angiotensin II type 2 receptor (AT2R) as a novel modulator of inflammation in rheumatoid arthritis synovium. Sci Rep 7:13293 11. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494–498 12. Ahmadzada T, Reid G, McKenzie DR (2018) Fundamentals of siRNA and miRNA therapeutics and a review of targeted nanoparticle delivery systems in breast cancer. Biophys Rev 10: 69–86 13. Willkomm S, Restle T (2015) Conformational dynamics of ago-mediated silencing processes. Int J Mol Sci 16:14769–14785 14. Chuang SY, Lin CH, Huang TH, Fang JY (2018) Lipid-based nanoparticles as a potential delivery approach in the treatment of rheumatoid arthritis. Nanomaterials (Basel) 15:8 15. Dhanapal R, Somasundarapandian S, Wihaskoro S, Kannan R, Rajkumar G, Chidambaram R (2017) Interference RNA in immunemediated oral diseases – minireview. Cent Eur J Immunol 42:301–304

Chapter 16 Lentiviral-Mediated Systemic RNA Interference In Vivo Shuang Liu Abstract The shRNA-encoding lentivirus has been widely used for gene manipulation in preclinical studies. It is a powerful tool for gene transfer and shows promise in its ability to efficiently transduce immune cells and hematopoietic stems cells, which are the initial therapeutic target of autoimmune diseases, and considering that gene manipulation of these cells is usually difficult to achieve using other techniques. In previous chapters, we have described how to produce concentrated shRNA-encoding lentiviral particles. Here, systemic in vivo application of lentivirus, including viral quantification prior to injection, intraperitoneal injection, and quantification of integrated provirus, is introduced. Key words Lentivirus, shRNA, Systemic delivery, Viral quantification, Integrated provirus

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Introduction Gene therapy has been discussed as a promising option for rheumatoid arthritis (RA) treatment and is slowly progressing on a trialand-error basis, though financial and social obstacles still remain [1, 2]. In the treatment of RA, both interference with the systemic immunological disorder and control of symptoms affecting individual joints are required. Nonviral and viral gene delivery techniques have been studied extensively. The characteristics of mammalian viruses allow them to be transferred and express exogenous genetic material via the natural life cycle of infection of host cells. Retroviruses and adenoviruses have been developed as vector platforms for gene therapy in preclinical studies and clinical trials. Several phase I/II trials have demonstrated that adenoviral-based therapies are safe with lack of adverse events following administration [3]. Lentivirus has not yet been trialed because of substantial biosafety concerns. However, it has been widely used for gene manipulation in preclinical studies. Lentivirus is a viral vector which is modified from human immunodeficiency virus, as previously introduced in Chapter 12. It is a powerful tool for gene transfer and shows promise in its ability to efficiently transduce

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immune cells and hematopoietic stems cells, which are the initial therapeutic target of autoimmune diseases [4]. We have successfully performed systemic and local knockdown of the expression of therapeutic targets using specific shRNA along with a lentiviral delivery system in a collagen-induced arthritis model [5, 6]. A decrease in severity of arthritis can also be achieved by single intravenous injection of lentiviral particles encoding the invariant chain for the immunodominant collagen type II peptide [7]. Insertional mutagenesis and transient immunological response to virus are always risks with gene manipulation via a lentiviral system. Though no evidence has demonstrated mutagenesis due to a therapeutic lentiviral system, the target cells that receive genetic modification will always be associated with an intrinsic risk of vector-induced genomic perturbations. Vector-induced systemic immunological responses are temporary, but could potentially be fatal [5, 8]. Updated design of lentiviral vectors has been conducted to optimize their usage in preclinical studies or even clinical trials, including the use of rapamycin to enhance the abilities of transduction, the inclusion of chromatin opening elements, and an ankyrin insulator for improved vector-derived expression [9–13]. Although some obstacles still exist to the use of a lentiviral delivery system in clinical trials, this system will no doubt undergo improvements toward studies on the treatment of RA.

2

Materials

2.1 Lentiviral Titration Using Quantitative Real-Time PCR (qRT-PCR)-Based Methods

1. Concentrated Chapter 12).

shRNA-encoding

lentiviral

stock

(see

2. Lenti-X™ qRT-PCR titration kit (Clontech). 3. 96-well PCR plates or 8-well PCR strips. 4. Pipettes and tips. 5. Quantitative real-time PCR thermocycler (e.g., ABI 7000, or equivalent).

2.2 Intraperitoneal Injection of shRNAEncoding Lentiviral Particles

1. Concentrated shRNA-encoding lentiviral stock. 2. Mice used for arthritis model or control. 3. Saline. 4. Syringes with gauge 27 needles. 5. 70% ethanol.

2.3 Determination of Integrated Lentiviral Copies in Tissues

1. Lenti-X™ provirus quantitation kit, including Lenti-X provirus quantitation components, NucleoSpin® Tissue Kit, SYBP Advantage qPCR Premix (Clontech).

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2. Forceps (fine blunt) and scissors (fine dissecting). 3. Operating table. 4. 1.5 mL microtubes for sample lysis and DNA elution. 5. Autoclaved bidistilled water. 6. Quantitative real-time PCR thermal cycler (e.g., ABI7000). 7. 96-well PCR plate or 8-well PCR strips. 8. Pipettes and tips.

3

Methods (See Note 1)

3.1 Lentiviral Titration

1. Dissolve concentrated lentiviral stock at room temperature and keep on ice. 2. Mix the virus by gently pipetting the viral solution. 3. Take 150 μL lentiviral stock. Purify RNA from the virus using a Neucleospin RNA Virus Kit, which is a component of the Lenti-X™ qRT-PCR titration kit, according to the manufacturer’s instructions. 4. At the end of RNA purification, elute the RNA in 50 μL RNase-free water. 5. Mix 2.5 μL DNase I buffer (10×), 4.0 μL DNase I (5 units/μ L), 6.0 μL RNase-free water, and 12.5 μL lentiviral RNA, in a total volume of 25 μL. All reagents are components of the kit. 6. Incubate the mixture at 37 °C for 30 min, followed by 70 °C for 5 min. Store the reaction solution on ice until ready to perform qRT-PCR (see Note 2). 7. Prepare four tenfold serial dilutions in duplicate for each viral RNA sample. Prepare three no-template controls (NTC) containing only dilution buffer without any RNA sample in duplicate. Prepare five tenfold serial dilutions using Lenti-X RNA Control Template supplied in the kit in duplicate as controls. Use EASY Dilution Buffer supplied in the kit to dilute the samples, controls, and NTCs. 8. Prepare PCR reaction mixture (total 25 μL) using the reagents supplied in the kit for each sample. RNase-free water

8.5 μL

Quant-X buffer (2×)

12.5 μL

Lenti-X forward primer (10 μM)

0.5 μL

Lenti-X reverse primer (10 μM)

0.5 μL

ROX reference dye (50×)

0.5 μL (continued)

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0.5 μL

RT-enzyme mix

0.5 μL

Sample (control or NTC)

2 μL

9. Mix well by tapping the PCR plate gently, and centrifuge the plates at 1500x g at 4 °C for 1 min to remove any bubbles. 10. Program the real-time qPCR instrument for the following reaction cycles RT reaction 42 °C

5 min

95 °C

10 min

qPCR × 40 cycles 95 °C

15 s

60 °C

30 s

Dissociation curve 95 °C

15 s

60 °C

30 s

All (75 °C) 11. Analyze the data according to the manufacturer’s instructions and obtain the number of viral copies. Briefly, the quantification includes the following steps. • Generate a standard curve using control template values. Determine the average concentration for each pair of duplicate control template amplifications, and plot the average concentration versus copy number on a log scale to generate a standard curve. • Calculate RNA copy numbers and infectivity coefficients; for example, 150 μL of viral stock was purified and eluted in 50 μL. The undiluted sample corresponded to a raw copy number of 1 × 107 copies on the qRT-PCR standard curve. Copies=mL =

1 × 107 copies ½1000 μL=mL]½2 × DNase]½50 μL elution] =ð½150 μL sample]½2 μL added to well]Þ

Copies=mL = 3:33 × 109 3.2 Intraperitoneal Injection of shRNAEncoding Lentiviral Particles (See Note 3)

1. Dilute the lentiviral concentrates using an appropriate volume of saline to make a final concentration of lentiviral particles of 109 copies/200 μL. 2. Use a squirt bottle to apply 70% ethanol to the back and wipe with tissue.

Lentivirus (copies/cell)

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157

3

2

1

Lymph node

Intestine

Heart

Bone marrow

Muscle

Spleen

Kidney

Liver

Lung

Blood

0

Fig. 1 Quantification of integrated provirus in mice. shRNA-encoding lentiviral particles (109 copies) were intraperitoneally injected to mice three times on each 7 days (MEANING NOT CLEAR at 7-day intervals). At 21 days after the first injection, genomic DNA was purified from tissues and integrated provirus was quantified. Results are expressed as mean ± SEM

3. Hold the mouse firmly with its head slightly downward, while a 27 G short needle is pushed into the left caudal area of the abdominal cavity. Systemic shRNA-delivery of 109 copies can be achieved by injection of three injections at 7-day intervals (Fig. 1). 4. Put the mouse in a clean cage and keep the injected mice under biosafety level 2. 3.3 Determination of Number of Integrated Provirus Copies in Tissue

1. Sacrifice the mice by cervical dislocation after three intraperitoneal injections of lentiviral particles (21 days after the first injection). 2. Cut about 25 mg tissue, which is required for proviral quantification, into small pieces. Put the tissues in 1.5 mL microcentrifuge tubes. 3. Purify genomic DNA from the tissue samples following the standard manufacturer’s protocols. 4. Obtain 100 μL eluted genomic DNA from tissues and keep it on ice. Measure OD260 to determine DNA yield and concentration (see Note 4). 5. Prepare four- of fivefold serial dilutions in duplicate for each genomic sample (50 ng/μL). Prepare three no-template controls (NTC) containing only dilution buffer without any RNA sample in duplicate. Prepare five ten-fold serial dilutions in duplicate as controls using Lenti-X Provirus Control Template supplied in the kit. Use EASY Dilution Buffer supplied in the kit to dilute the samples, controls, and NTCs.

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6. Prepare the PCR reaction mixture (total 20 μL) using the reagents supplied in the kit for each sample (see Note 5). Autoclaved bidistilled water

6.8 μL

Lenti-X provirus forward primer (10 μM)

0.4 μL

Lenti-X provirus reverse primer (10 μM)

0.4 μL

ROX reference dye (50×)

0.5 μL

SYBR advantage qPCR premix (2×)

10.0 μL

Sample (control or NTC)

2.0 μL

7. Mix well by tapping the PCR plate gently, and centrifuge the plates at 1500×g at 4 °C for 1 min to remove any bubbles. 8. Program the real-time qPCR instrument for the following reaction cycles (see Note 6). Initial denaturation 95 °C

30 s

qPCR × 40 cycles 95 °C

5s

60 °C

31 s

Dissociation curve 95 °C

15 s

60 °C

30 s

95 °C

15 s

9. Analyze the data according to the manufacturer’s instructions and obtain the number of viral copies. Briefly, the quantification steps include: • Generate a standard curve using control template values. Determine the average concentration for each pair of duplicate control template amplifications, and plot the average concentration versus copy number on a log scale to generate a standard curve. • Determine the qPCR copy number equivalent. Determine the average concentration for each pair of duplicate sample template amplifications. • Determine total qPCR copy number equivalent for the original sample using the following equation: DNA dilution factor = ([Total DNA extracted] μg)(1000 ng/μg) / ([100, 20, 4, or 0.8] ng). • Convert total qPCR copy number equivalent to provirus copy number using the following equation: Provirus copy number = (qPCR copy number)(62.84 provirus copies/ qPCR number).

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• Calculate the total genome number equivalent present in the original genomic DNA (gDNA) sample using the following equation: Cell number = ([gDNA] μg)(1 × 106 pg/ μg)/(6.6 pg/cell). • Obtain provirus copy number/cell using the following equation: Provirus copy number/cell = (provirus copy number)/(cell number).

4

Notes 1. All experiments should be performed under biosafety level 2. 2. A thermocycler should be used for this reaction. 3. All animal experiment protocols should be performed in accordance with the guidelines of the Institutional Animal Care and Use Committee and approved by the committee. 4. The desired yield of DNA in the extraction should be 20–30 μg at a concentration of 200–300 ng/μL. 5. The sufficient volume qPCR mixture should be specified for different qPCR instruments (Table 1).

Table 1 qPCR mixes recommended for different qPCR instrumentsa qPCR instrument

a

Reagent

Takara Bio Thermal Cycler Applied Stratagene Dicer™ Biosystems Mx3000P Real-time system Instruments Reagent volume (μL/well) for each instrument

Autoclaved H2O

9.0

9.5

6.8

7.2

Lenti-X provirus forward primer (10 μM)

0.5

0.5

0.4

0.4

Lenti-X provirus reverse primer (10 μM)

0.5

0.5

0.4

0.4

ROX reference dye LSR or LMP (50×)

0.5

–

0.4

–

SYBR advantage qPCR premix (2×)

12.5

12.5

10.0

10.0

Sample, control, or dilution buffer 2

2

2

2

Total (μL/well)

25

20

20

25

This figure is adopted from Lenti-XTM Provirus Quantitation Kit User Manual with minor changes

Roche LightCycler®

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Table 2 Recommended thermal cycling conditions for different qPCR instrumentsa qPCR instrument

Reaction cycles

Takara Bio Thermal Cycler Stratagene Dicer™ ABI7500 Mx3000P® Real-time system fast ABI7000 Thermal cycling conditions for each instrument

Roche LightCycler®

Initial denaturation (1 cycle)

95 °C 10 s 95 °C 30 s

95 °C 30 s 95 °C 30 s 95 °C 30 s

qPCR (40 cycles)

95 °C 5 s 95 °C 5 s 60 °C 20 s 60 °C 30 s

95 °C 5 s 95 °C 15 s 95 °C 5 s 60 °C 25 s 60 °C 31 s 60 °C 20 s

Dissociation curve (1 cycle)

95 °C 15 s 95 °C 60 °C 30 s 1 min 60 °C 10 s 95 °C 15 s

95 °C 15 s 95 °C 15 s 95 °C 0 s 60 °C 60 °C 15 s 60 °C 1 min 1 min 95 °C 0 s 95 °C 15 s 95 °C 15 s

a

This figure is adopted from Lenti-XTM Provirus Quantitation Kit User Manual

6. The programmable real-time qPCR instrument should be optimized according to Table 2. References 1. Evans CH, Ghivizzani SC, Robbins PD (2011) Getting arthritis gene therapy into the clinic. Nat Rev Rheumatol 7:244–249 2. Liu S, Maeyama K (2016) Gene therapy for rheumatoid arthritis. Crit Rev Immunol 36: 149–161 3. Clement N, Grieger JC (2016) Manufacturing of recombinant adeno-associated viral vectors for clinical trials. Mol Ther Methods Clin Dev 3:16002 4. Kotterman MA, Chalberg TW, Schaffer DV (2015) Viral vectors for gene therapy: translational and clinical outlook. Annu Rev Biomed Eng 17:63–89 5. Liu S, Kiyoi T, Takemasa E, Maeyama K (2017) Intra-articular lentivirus-mediated gene therapy targeting CRACM1 for the treatment of collagen-induced arthritis. J Pharmacol Sci 133:130–138 6. Liu S, Kiyoi T, Takemasa E, Maeyama K (2015) Systemic lentivirus-mediated delivery of short hairpin RNA targeting calcium releaseactivated calcium channel 3 as gene therapy for collagen-induced arthritis. J Immunol 194:76–83

7. Eneljung T, Tengvall S, Jirholt P, Henningsson L, Holmdahl R, Gustafsson K et al (2013) Antigen-specific gene therapy after immunisation reduces the severity of collagen-induced arthritis. Clin Dev Immunol 2013:345092 8. Marshall E (1999) Gene therapy death prompts review of adenovirus vector. Science 286:2244–2245 9. Arumugam PI, Urbinati F, Velu CS, Higashimoto T, Grimes HL, Malik P (2009) The 3′ region of the chicken hypersensitive site-4 insulator has properties similar to its core and is required for full insulator activity. PLoS One 4:e6995 10. Dighe N, Khoury M, Mattar C, Chong M, Choolani M, Chen J et al (2014) Long-term reproducible expression in human fetal liver hematopoietic stem cells with a UCOE-based lentiviral vector. PLoS One 9:e104805 11. Groth AC, Liu M, Wang H, Lovelett E, Emery DW (2013) Identification and characterization of enhancer-blocking insulators to reduce retroviral vector genotoxicity. PLoS One 8: e76528

Lentiviral-Mediated Systemic RNA Interference In Vivo 12. Phaltane R, Lachmann N, Brennig S, Ackermann M, Modlich U, Moritz T (2014) Lentiviral MGMT(P140K)-mediated in vivo selection employing a ubiquitous chromatin opening element (A2UCOE) linked to a cellular promoter. Biomaterials 35:7204–7213

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13. Wang CX, Sather BD, Wang X, Adair J, Khan I, Singh S et al (2014) Rapamycin relieves lentiviral vector transduction resistance in human and mouse hematopoietic stem cells. Blood 124:913–923

Chapter 17 Lentiviral Production Platform Shuang Liu Abstract Lentiviral-mediated transfection technique is a powerful tool for gene modification in preclinical studies. By using this technique, the desired gene modification can be achieved easily in immune cells, nondividing, and terminally differentiated cells, including hematopoietic stem cells, neurons, and even tumor cells, which other viral vectors cannot do. The main considerations of therapeutic gene delivery using a lentiviral system are the risk of insertional mutagenesis and the immune reaction elicited by infected cells. Although some biosafety concerns need to be addressed before clinical trials in rheumatoid arthritis, the lentiviral system targeting therapeutic targets has been widely used for in vivo gene transfer in animal models. In this chapter, the protocols for production of viral particles and viral concentration are provided. As an alternative utilization, this lentiviral production platform could also be employed to produce a pseudotype severe acute respiratory syndrome-related coronavirus 2 in which the spike glycoprotein of SARS-CoV-2 was incorporated into pseudovirions for viral study. Key words Lentivirus, Short-hairpin RNA, Gene silencing, Transfection, SARS-Cov-2

1

Introduction Due to their natural life cycle, mammalian viruses are suitable for the transfer and expression of exogenous genetic material during infection of host cells. The high efficacy of oncogenic retroviruses, adenoviruses, and lentiviruses has led to their being extensively used in preclinical research and clinical trials on the treatment of rheumatoid arthritis (RA) [1]. Lentivirus is a viral vector which is modified from human immunodeficiency virus (HIV)-1, with a stable property of ./BC$i.fastq done ‘‘‘

3.3.3 Generating Reference Data

This is a command to convert rat reference data for use with minimap. Download and convert the reference data for the experimental animals in FASTA format. In this case, Rattus_norvegicus. mRatBN7.2.dna.toplevel.fa was used. ‘‘‘bash minimap2 -x splice -d ./genome.mmi ./Rattus_norvegicus. mRatBN7.2.dna.toplevel.fa ‘‘‘

3.3.4 Mapping to Reference Genome with Minimap2

Execute the following shell script. Specify the number of CPU threads to use with the -t option. Adjust the reference data path as needed. ‘‘‘bash #sam.sh for i in ‘seq -w 1 12‘;do minimap2 -t 16 -ax splice ./hogehoge/ref/genome.mmi ./BC $i.fastq > ./barcode$i.sam done ‘‘‘

3.3.5 SAM to BAM Conversion, Sorting, and Indexing

After the mapping, the SAM files are converted to BAM, sorted, and indexed using samtools: The following scripts can be created and conveniently processed in batches. ‘‘‘bash #sam2bam.sh for i in ‘seq -w 1 12‘;do samtools view -bS -F 256 barcode$i.sam > barcode$i.bam done #sort.sh for i in ‘seq -w 1 12‘;do samtools sort -T barcode$i.sort -o barcode$i\_sort.bam barcode$i.bam

Bulk RNA-seq Assessment of Murine Spleen Using a Portable MinION. . .

305

done #index.sh for i in ‘seq -w 1 12‘;do samtools index barcode$i\_sort.bam done ‘‘‘

3.3.6 Counting Features with FeatureCounts

Finally, featureCounts is used to count the features: ‘‘‘bash #count.sh ~/subread-2.0.3-Linux-x86_64/bin/featureCounts ’exon’

-g

’gene_id’

-a

-L

-t

/hogehoge/Rattus_norvegicus.

mRatBN7.2.107.gtf -T 10 -o count_all.txt ./barcode01_sort.bam

./barcode02_sort.bam

barcode04_sort.bam sort.bam

./barcode07_sort.bam

barcode09_sort.bam

./barcode03_sort.bam

./barcode05_sort.bam

./barcode08_sort.bam

./barcode10_sort.bam

./

./barcode06_./

./barcode11_-

sort.bam ./barcode12_sort.bam ‘‘‘

3.3.7 Data Frame Creation for Downstream Analysis

Use the tidyverse package in R to create a data frame to be used for downstream analysis. Install the tidyverse package and run the following script; running in R studio is strongly recommended. Set the directory where the output count data and its summary are stored as a working directory and run it. ‘‘‘r count.R setwd("~/hogehoge") library(tidyverse) rm (list = ls()) options(max.print=10000000) dat_mini01 11

11 to >3.3

3.3≥

CDAI

>22

22 to >10

10 to >2.8

2.8≥

Table 4 Definition of 20% improvement (ACR20) measured with ACR core set Required

≥ 20% improvement in tender joint count ≥ 20% improvement in swollen joint count

≥20% improvement in 3 of following 5: Patient pain assessment Patient global assessment (PaGA) Physician global assessment (PhGA) Patient self-assessed disability (HAQ-DI) Acute-phase reactant (ESR or CRP)

therapy, ACR20, ACR50, and ACR70, are defined as 20%, 50%, and 70% improvement from baseline, respectively. ACR 50 and ACR 70 are essential rather than ACR 20 in response to biological agents. 2.1.5 2022 ACR/ European Alliance of Associations for Rheumatology (EULAR) Remission Criteria

Remission is a major therapeutic target in the clinical practice and can be achieved in a significant proportion of patients undergoing routine follow-up care. In 2011, ACR and EULAR endorsed remission criteria that included index-based and Boolean-based definitions [12]. Furthermore, in the revised ACR/EULAR remission criteria of 2022, the revised Boolean 2.0 criteria were endorsed [13]. In index-based definitions, an SDAI score ≤3.3 and CDAI score ≤2.8 indicate remission. The achievement of Boolean 2.0 remission requires all of the following: TJC28 ≤1, SJC28 ≤1, CRP (mg/dL) ≤1, and PaGA (0- to 10-cm VAS) ≤2. In contrast, DAS28 has not been recommended for remission criteria. DAS28 of 50% of the total SH area) surface convex (curved upward)a

No SH and no PD signal

Grade 2 SH and ≤grade 2 PD signal; or grade 1 SH and a grade 2 PD signal Grade 3 SH and ≤grade 3 PD signal; or grade 1 or 2 SH and a grade 3 PD signal

Adapted from ref. [5] a Independently of the presence of effusion. EULAR European League Against Rheumatism, OMERACT Outcome Measures in Rheumatoid Arthritis Clinical Trials, GS grayscale, PD power Doppler, SH synovial hypertrophy, M metacarpal head, P proximal phalanx

Fig. 2 Bone erosion in the fifth Metatarsophalangeal joint (B mode). Discontinuity of the bone surface in two perpendicular planes (*) MT, metatarsal bone

These classification criteria specifically describe the evaluation of joint involvement, which includes physical examination findings, as well as the confirmation of synovitis through MSUS and MRI. Due to its higher sensitivity for the detection of synovitis and bone erosions than a physical examination, MSUS has become a powerful tool for an early diagnosis [7].

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Fig. 3 (a) Transverse view of tenosynovitis of extensor carpi ulnaris (ECU) tendon. Left, synovial thickening around the ECU tendon (arrow heads) in the B mode; Middle, Power Doppler positive tenosynovitis; Right, normal ECU tendon. (b) Longitudinal view of tenosynovitis of the ECU tendon (PD mode) U ulna

Fig. 4 2010 ACR/EULAR classification criteria for RA ACR, American College of Rheumatology; EULAR, European League Against Rheumatism. (Adapted from ref. 6])

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2.2.2 Assessment of Disease Activity

The simplified disease activity index (SDAI) and clinical disease activity index (CDAI) are commonly used to assess disease activity in RA. These indices are composite measures that incorporate joint assessments, a patient’s and physician’s global assessments, and inflammation markers [8, 9]. However, in some cases in clinical practice, a disconnect exists between joint assessments, symptoms, and laboratory findings. MSUS is valuable for assessing disease activity in these cases because it allows for a direct evaluation of synovitis.

2.2.3 Definition of Remission

In cases in which progressive bone destruction progresses despite achieving clinical remission, residual synovitis is often detected by MSUS [10]. Synovitis that is only detected by MSUS, and not by a physical examination, is referred to as subclinical synovitis. Regardless of how strictly the criteria for clinical remission are defined, subclinical synovitis is inevitably present [11]. However, it is important to note that not all abnormal findings detected by MSUS lead to future joint damage. There is currently no established treatment guideline for subclinical synovitis in patients in remission, and thus, the accumulation of further evidence is required [12].

2.2.4 Treatment Strategy Utilizing MSUS

Positive PD findings in patients with RA in remission are associated with relapse and progressive bone destruction within 1 year [13]. On the other hand, in the ARCTIC study and TaSER study, which are two randomized clinical trials that examined the usefulness of tight control with interventions based on positive MSUS findings in addition to conventional disease activity assessments, no significant differences were observed in disease activity or imaging outcomes between the conventional evaluation group and the tightly controlled group using MSUS [14, 15]. Therefore, a discourse remains regarding whether MSUS needs to be restricted to its role as a diagnostic tool for RA or if it may be employed as an indicator to evaluate remission and guide treatment intensification.

3

Notes 1. High-frequency linear probes ranging between 7.5 and 14 MHz are used in MSUS. 2. In the B mode, gain, dynamic range, and focus need to be adjusted in order to accurately and clearly visualize the target joint. 3. In the PD mode, the aim is to detect low-velocity blood flow; therefore, the pulse repetition frequency (PRF) is set as low as possible. A range of between 500 and 1000 Hz is generally recommended. After maximizing the gain, it is gradually reduced until noise becomes imperceptible, and it is observed

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at the maximum point at which noise is no longer visible. By expanding the region of interest (ROI) to the top of the screen, the incorrect identification of abnormal vessels based on the presence of superficial reflections caused by superficial blood vessels is prevented. 4. To ensure the optimal visualization of PD signals and joint fluid, it is crucial to minimize or avoid applying pressure on the joint with the probe. Excessive pressure may impede the detection of these findings and hinder accurate assessments. 5. Under conditions where a more objective assessment is needed, such as in clinical trials or research studies, it is preferable for MSUS to be performed by a third party who is blinded to clinical information. References 1. Colebatch AN, Edwards CJ, Østergaard M et al (2013) EULAR recommendations for the use of imaging of the joints in the clinical management of rheumatoid arthritis. Ann Rheum Dis 72:804–814 2. Wakefield RJ, Balint PV, Szkudlarek M et al (2005) Musculoskeletal ultrasound including definitions for ultrasonographic pathology. J Rheumatol 32:2485–2487 3. Bruyn GA, Iagnocco A, Naredo E et al (2019) OMERACT definitions for ultrasonographic pathologies and elementary lesions of rheumatic disorders 15 years on. J Rheumatol 46: 1388–1393 4. D’Agostino MA, Terslev L, Aegerter P et al (2017) Scoring ultrasound synovitis in rheumatoid arthritis: a EULAR-OMERACT ultrasound taskforce – part 1: definition and development of a standardised, consensusbased scoring system. RMD Open 3:e000428 5. Terslev L, Naredo E, Aegerter P et al (2017) Scoring ultrasound synovitis in rheumatoid arthritis: a EULAR-OMERACT ultrasound taskforce – part 2: reliability and application to multiple joints of a standardised consensusbased scoring system. RMD Open 3:e000427 6. Aletaha D, Neogi T, Silman AJ et al (2010) 2010 rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Ann Rheum Dis 69: 1580–1588 7. Nakagomi D, Ikeda K, Okubo A et al (2013) Ultrasound can improve the accuracy of the 2010 American College of Rheumatology/European League Against Rheumatism

classification criteria for rheumatoid arthritis to predict the requirement for methotrexate treatment. Arthritis Rheum 65:890–898 8. Smolen JS, Breedveld FC, Schiff MH et al (2003) A simplified disease activity index for rheumatoid arthritis for use in clinical practice. Rheumatology (Oxford) 42:244–257 9. Aletaha D, Nell VP, Stamm T et al (2005) Acute phase reactants add little to composite disease activity indices for rheumatoid arthritis: validation of a clinical activity score. Arthritis Res Ther 7:R796–R806 10. Brown AK, Conaghan PG, Karim Z et al (2008) An explanation for the apparent dissociation between clinical remission and continued structural deterioration in rheumatoid arthritis. Arthritis Rheum 58:2958–2967 11. Nguyen H, Ruyssen-Witrand A, Gandjbakhch F et al (2014) Prevalence of ultrasounddetected residual synovitis and risk of relapse and structural progression in rheumatoid arthritis patients in clinical remission: a systematic review and meta-analysis. Rheumatology (Oxford) 53:2110–2118 12. D’Agostino MA, Terslev L, Wakefield R et al (2016) Novel algorithms for the pragmatic use of ultrasound in the management of patients with rheumatoid arthritis: from diagnosis to remission. Ann Rheum Dis 75:1902–1908 13. Han J, Geng Y, Deng X, Zhang Z (2016) Subclinical synovitis assessed by ultrasound predicts flare and progressive bone erosion in rheumatoid arthritis patients with clinical remission: a systematic review and metaanalysis. J Rheumatol 43:699–706

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14. Haavardsholm EA, Aga AB, Olsen IC et al (2016) Ultrasound in management of rheumatoid arthritis: ARCTIC randomised controlled strategy trial. BMJ 354:i4205

15. Dale J, Stirling A, Zhang R et al (2016) Targeting ultrasound remission in early rheumatoid arthritis: the results of the TaSER study, a randomised clinical trial. Ann Rheum Dis 75: 1043–1050

Chapter 35 16S rRNA Gene Amplicon Analysis of Human Gut Microbiota Noriyuki Miyaue Abstract The intestinal microbiota is associated with a variety of diseases, and there are a growing number of research reports on the gut microbiota. In addition, a new technique such as Nanopore sequencing has recently become available, making it easier to conduct research related to the gut microbiota. In this chapter, we introduce a technique used in gut microbiota analysis, from stool collection to sequencing with MinION. Key words 16S rRNA, Gut microbiome, MinION, PCR, Sequencing

1

Introduction The gut microbiota is associated not only with inflammatory bowel disease [1] but also with diabetes [2], obesity [3], and various other diseases, and has attracted considerable attention as a research field. The strong link between the gastrointestinal environment and central nervous system function has been termed the “brain–gut axis,” and disruption of this relationship has been shown to be associated with the etiology of several neurological disorders, including Parkinson’s disease [4]. A recent meta-analysis across countries showed a bias in the gut microbiota in patients with Parkinson’s disease compared to healthy controls [5]. Research on the intestinal microbiota may lead to the elucidation of various diseases and the development of new treatments. The analysis of gut microbiota is commonly performed by 16S ribosomal RNA (rRNA) analysis, in which DNA of the entire gut microbiome is purified from the collected stool and subjected to polymerase chain reaction (PCR) amplification targeting the 16S rRNA gene to comprehensively analyze the presence and ratio of bacterial species. Recently, MinION (Oxford Nanopore Technologies, Oxford, UK) with a new sequencing technology called Nanopore has become available and is expected to provide more accurate and sensitive identification of gut microbiota [6].

Shuang Liu (ed.), Rheumatoid Arthritis: Methods and Protocols, Methods in Molecular Biology, vol. 2766, https://doi.org/10.1007/978-1-0716-3682-4_35, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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This chapter introduces a technique used in gut microbiota analysis, from stool collection to sequencing with MinION. Note that this chapter does not include data analysis after sequencing.

2

Materials

2.1 Stool Sampling (See Note 1)

1. Stool collection kit includes a seat for collecting stools, a stoolcollecting spoon, a stool collection container, and a cap with preservative solution (Metabolokeeper®; TechnoSuruga Lab, Shizuoka, Japan).

2.2

1. ISOSPIN Fecal DNA kit includes FE1 Buffer, FE2 Buffer, FB Buffer, FW Buffer, TE (pH 8.0), RNase A, and Spin Column (Nippon Gene, Tokyo, Japan).

DNA Isolation

2. Isopropanol. 3. 1.5-mL microcentrifuge tubes. 4. 2.0-mL microcentrifuge tubes. 5. Microcentrifuge. 6. Vortex mixer. 7. Heat block. 8. Ultrasonic cleaner. 9. Pipettes and tips. 2.3 Library Preparation

1. DNA sample. 2. PCR Barcoding Kit including Barcode Primer (BP) 01–12 and Rapid Adapter (RAP) (SQK-PBK004; Oxford Nanopore Technologies). 3. Inner primer described by Matsuo et al. [7]: forward (5′-TTT CTGTTGGTGCTGATATTGCAGRGTTYGATYMTGGCTC AG-3′) and reverse (5′-ACTTGCCTGTCGCTCTATCTTCC GGYTACCTTGTTACGACTT-3′). 4. KAPA2G Robust HotStart Ready Mix (Kapa Biosystems, Wilmington, MA, USA). 5. Nuclease-free water. 6. Ethanol. 7. 10 mM Tris-HCl pH 8.0 with 50 mM NaCl. 8. 0.2-mL thin-walled PCR tubes. 9. 1.5-mL Eppendorf DNA LoBind tubes. 10. Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA). 11. Qubit fluorometer.

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12. Thermal cycler. 13. Microfuge. 14. Magnetic rack. 15. Hula mixer. 16. Ice bucket with ice. 17. Pipettes and tips. 2.4

Sequencing

1. Flow Cell Priming Kit including Flush Buffer (FB) and Flush Tether (FLT) (EXP-FLP002; Oxford Nanopore Technologies). 2. PCR Barcoding Kit including Sequencing Buffer (SQB) and Loading Beads (LB) (SQK-PBK004). 3. MinION Mk1B (Oxford Nanopore Technologies). 4. SpotON Flow Cell (Oxford Nanopore Technologies). 5. Nuclease-free water. 6. 1.5-mL Eppendorf DNA LoBind tubes. 7. Pipettes and tips.

3 3.1

Methods Stool Sampling

1. Place a seat for collecting stools in the toilet bowl. 2. Defecate on the sheet. 3. Using a stool-collecting spoon, scoop stool from multiple locations. 4. Place the spoon in a stool collection container and insert it all the way to the back. 5. Attach a cap with preservative solution to the container over the spoon. 6. Tighten the cap until it clicks. 7. Shake the container about 10 times (see Note 2). 8. Store at room temperature until DNA isolation.

3.2

DNA Isolation

1. Place in ultrasonic cleaner for 5–10 min to suspend stools more. 2. Transfer 200 μL of the fecal suspension in the stool collection container to a new 2–0 mL microcentrifuge tube (see Note 3). 3. Add 700 μL of FE1 Buffer and 10 μL of RNase A. 4. Tighten the cap securely and then mix the tube thoroughly by vortexing.

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5. Incubate the tube in a heat block at 65 °C for 60 min. During this time, mix well by vortexing every 15 min. 6. Allow to stand until room temperature. Spin down, add 90 μL of FE2 Buffer, and mix thoroughly by vortexing. 7. Centrifuge at 12,000×g for 15 min. 8. Transfer 500 μL of the supernatant to a new 1.5-mL microcentrifuge tube. 9. Add 200 μL of FB Buffer and 200 μL of isopropanol to the collected supernatant and mix well by inverting. 10. Apply the mixture to the Spin Column. 11. Centrifuge at 13,000×g for 30 s. 12. Discard the flow-through and return the Spin Column to the same Collection Tube. 13. Apply 600 μL of FB Buffer to the Spin Column. 14. Centrifuge at 13,000×g for 1 min. 15. Discard the flow-through and return the Spin Column to the same Collection Tube. 16. Apply 600 μL of FW Buffer to the Spin Column. 17. Centrifuge at 13,000×g for 1 min. 18. Discard the flow-through and Collection Tube, and place the Spin Column to a new 1.5-mL microcentrifuge tube. 19. Apply 50 μL of TE (pH 8.0) near the center of the column membrane, and incubate at room temperature for 3 min. 20. Centrifuge at 13,000×g for 1 min. 21. Remove the column from the tube and close the tube lid. 22. Store at -80 °C until analysis. 3.3 Library Preparation

Four-primer PCR method is applied, in which PCR is performed twice using the selected inner primer and outer primer (included in BP 01–12). 1. Thaw frozen DNA sample on ice. 2. Quantify DNA sample using a Qubit fluorometer. 3. Transfer DNA sample into a DNA LoBind tube and adjust the volume to 30 ng/μL with nuclease-free water. 4. In a 0.2-mL thin-walled PCR tube, mix the following: 30 ng of DNA sample, 50 nM of forward inner primer, 50 nM of reverse inner primer, 25 μL of KAPA2G Robust HotStart Ready Mix, and up to 50 μL of nuclease-free water. 5. Mix gently by flicking the tube and spin down. 6. Amplify with the following PCR conditions: initial denaturation at 95 °C for 1 min, 30 cycles of 95 °C for 10 s, 55 °C for

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15 s, and 72 °C for 2 min, followed by a final extension at 72 °C for 1 min. 7. Resuspend the AMPure XP beads by vortexing immediately before use. 8. Transfer the sample to a 1.5 mL DNA LoBind Eppendorf tube. 9. Add 40 μL of resuspended AMPure XP beads to the reaction and mix by flicking the tube. 10. Incubate on a Hula mixer for 5 min at room temperature. 11. Prepare 500 μL of fresh 70% ethanol in nuclease-free water. 12. Spin down the sample and pellet on a magnetic rack. 13. Keep the tube on the magnetic rack for 30 s and remove the supernatant using a pipette. 14. Keep the tube on the magnetic rack and wash the beads with 200 μL of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard. 15. Repeat the previous step. 16. Spin down and place the tube back on the magnetic rack. 17. Remove any residual 70% ethanol using a pipette and allow to dry briefly. 18. Remove the tube from the magnetic rack and resuspend pellet in 10 μL of 10 mM Tris-HCl pH 8.0 with 50 mM NaCl. 19. Incubate for 2 min at room temperature. 20. Pellet the beads on magnet until the eluate is clear and colorless. 21. Collect 10 μL of the eluate in a clean 1.5-mL Eppendorf DNA LoBind tube. 22. Quantify eluted DNA sample using a Qubit fluorometer. 23. Adjust the volume to 30 ng/μL with nuclease-free water. 24. In a 0.2-mL thin-walled PCR tube, mix the following: 30 ng of DNA sample, 1.5 μL of BP 01–12 from the SQK-PBK004 kit, 25 μL of KAPA2G Robust HotStart Ready Mix, and up to 50 μL of nuclease-free water. 25. Repeat steps 5–21. 26. Adjust samples to a total of 100 ng/10 μL without sample bias while diluting with 10 mM Tris-HCl pH 8.0 with 50 mM NaCl. 27. Add 1 μL RAP to the 10 μL amplified DNA library. 28. Mix gently by flicking the tube and spin down. 29. Incubate the reaction for 5 min at room temperature.

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Sequencing

1. Thaw SQB, LB, FLT, and one tube of FB, mix by vortexing, and spin down at room temperature. 2. Open the lid of the MinION Mk1B and slide the flow cell under the clip. 3. Slide the priming port cover clockwise to open the priming port. 4. Check for small air bubbles under the cover and if any, draw back a small volume to remove any bubbles as follows: set the P1000 pipette to 200 μL, insert the tip into the priming port, and rotate the wheel until you can see a small amount of buffer entering the pipette tip. 5. To prepare the flow cell priming mix, add 30 μL of FLT directly to the tube of FB, and mix by vortexing at room temperature. 6. Load 800 μL of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles and wait for 5 min (see Note 4). 7. Thoroughly mix the contents of the LB tubes by vortexing immediately before use. 8. In a new tube, prepare the library for loading as follows: 34 μL of SQB, 25.5 μL of LB, 4.5 μL of nuclease-free water, and 11 μL of DNA library. 9. Gently lift the SpotON sample port cover and load 200 μL of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles (see Note 4). 10. Mix the prepared library gently by pipetting up and down just prior to loading. 11. Add 75 μL of the library to the flow cell via the SpotON sample port in a dropwise fashion. Make sure each drop flows into the port before adding the next drop. 12. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port, and replace the MinION Mk1B lid (see Note 5).

4

Notes 1. Prior to collection of stool sampling, all subjects should give written informed consent in accordance with the Declaration of Helsinki. The research protocols should be approved by the ethics committee of the research institutes. 2. Ensure that the stool is away from the spoon and in the preservative solution. 3. The tip is cut at an angle to make it easier to suck up stool.

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4. The priming mix should be loaded in the priming port as soon as it is pipetted, and not in its entire volume to avoid further bubble contamination. 5. Starting a MinION sequencing run using MinKNOW software. This protocol includes only the Wet portion; please refer to other materials and literature for details on bioinformatics analysis, such as EPI2ME 16S workflow (https:// nanoporetech.com/resource-centre/epi2me-16s-workflowreal-time-identification-bacteria-and-archaea). References 1. Qiu P, Ishimoto T, Fu L, Zhang J, Zhang Z, Liu Y (2022) The gut microbiota in inflammatory bowel disease. Front Cell Infect Microbiol 12: 733992. https://doi.org/10.3389/fcimb. 2022.733992 2. Cunningham AL, Stephens JW, Harris DA (2021) Gut microbiota influence in type 2 diabetes mellitus (T2DM). Gut Pathog 13(1):50. h ttps://d oi.org/10.1186/s130 99-02100446-0 3. Geng J, Ni Q, Sun W, Li L, Feng X (2022) The links between gut microbiota and obesity and obesity related diseases. Biomed Pharmacother 147:112678. https://doi.org/10.1016/j.bio pha.2022.112678 4. Tiwari P, Dwivedi R, Bansal M, Tripathi M, Dada R (2023) Role of gut microbiota in neurological disorders and its therapeutic significance. J Clin Med 12(4). https://doi.org/10.3390/ jcm12041650

5. Nishiwaki H, Ito M, Ishida T, Hamaguchi T, Maeda T, Kashihara K et al (2020) Meta-analysis of gut dysbiosis in Parkinson’s disease. Mov Disord 35(9):1626–1635. https://doi.org/10. 1002/mds.28119 6. Szoboszlay M, Schramm L, Pinzauti D, Scerri J, Sandionigi A, Biazzo M (2023) Nanopore is preferable over Illumina for 16S amplicon sequencing of the gut microbiota when specieslevel taxonomic classification, accurate estimation of richness, or focus on rare taxa is required. Microorganisms 11(3). https://doi.org/10. 3390/microorganisms11030804 7. Matsuo Y, Komiya S, Yasumizu Y, Yasuoka Y, Mizushima K, Takagi T et al (2021) Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution. BMC Microbiol 21(1):35. https://doi.org/10. 1186/s12866-021-02094-5

INDEX A Adjuvant ...................................4, 39, 65, 72, 98, 99, 104 AlphaScreens ........................................................ 107–126 Anesthetic vaporizer..................................................38, 39 Antigen ................................................. 63–79, 94, 95, 98, 99, 101, 106, 109, 115, 177, 200, 207, 208, 241, 283, 287, 290 Aptamer ...............................................129–137, 142, 144 Atelocollagen.............................................................18, 19 Autoantibodies ....................................... 4, 107–126, 200, 233, 234, 241 Autoreactive responses......................................... 241, 242

B BAM...................................................................... 304–305 Bank specimens ............................................................. 320 Basecalling ............................................................ 301–303 Baseline measurement................................................... 188 B cells .................................... 10, 14, 164, 177, 178, 199, 200, 233–236, 238, 239 Bilayer-dialysis method .....................................64, 65, 67, 70–74, 79, 86 Blocking solution ...........................................46, 106, 283 Bone marrow..........................................18, 21, 170, 234, 252–254, 256, 258, 259 Bone resorption pit ....................................................... 263

C Ca2+ ...............................................................177–198, 234 Ca2+ imaging ........................................................ 183–190 Ca2+ indicator Fluo-4 .......................................... 184, 185 Ca2+ indicator Fura-2 ..................................178, 183–185 Ca2+ influx ............................................................ 177–182 Candida albicans water-soluble glycoprotein (CAWS).............................................271, 273–275 Cartilage ................................3, 4, 10, 12–14, 17–22, 37, 55, 169, 170, 241, 242 Cell-free protein synthesis ....................70, 108, 111, 115 Cell fusion ............................................... 94, 99–101, 178 Cell markers......................................... 208–209, 212–218 Chloroform ........................ 67, 68, 71, 72, 75, 132–134, 209, 210, 218, 220 Chondrogenic spheroids...........................................18–23

CIA scoring system ...................................................38, 39 Clinical Disease Activity Index (CDAI)......................321, 322, 325–329, 332, 340 Clinical score .............................................................37–42 Clinical trials ................................93, 153, 154, 163, 164, 233, 317–322, 330, 331, 336, 338, 340, 341 Clone expansion...............................................94, 96, 103 Clone selection ...............................................96, 103, 104 Collagen films....................................................... 264–266 Collagen-induced arthritis (CIA)...................3–7, 28, 33, 37–41, 56, 154, 164, 242–244, 268 Counting ....................................276, 283, 288, 302, 305 CRAC-like current ............................................... 196, 197 CRISPR/Cas9...................................................... 170, 171 Cytokine ................................................... 3, 4, 26, 56, 93, 177, 200, 207, 208, 210–211, 222–226, 233– 240, 242, 244, 245, 272, 278

D DBA/1 mice.................................................................. 4, 5 Decalcifying ................................................. 43, 44, 46, 50 Density gradient centrifugation ...................... 88–90, 148 Detergents ................................................. 59, 83–90, 217 Diagnosis ...............................................63, 271, 337, 338 Disease activity ................................37–42, 325–332, 340 Disease Activity Score in 28 joints (DAS28)................. 321, 322, 325–327, 329, 332 DNA ................................... 67, 69, 70, 78, 94, 111, 112, 115–117, 122, 125, 126, 133–136, 139–144, 155, 157–159, 164, 170, 172, 200, 210, 221, 231, 281, 283, 288–291, 293, 295, 296, 298– 301, 307, 320, 343–348 cDNA......................................... 67, 70, 85, 110, 112, 113, 210, 220, 221, 225, 294, 295, 298, 301 ssDNA.................................................... 133, 135, 136 Docking calculations............................................ 139–144

E 8-hydroxydeoxyguanosine (8-OHdG) ............... 289, 290 Electrophysiology.......................................................... 192 Enzyme-linked immunosorbent assay (ELISA) ........... 59, 94, 96, 99, 101, 104, 211, 222–223, 235–237, 239, 245, 289

Shuang Liu (ed.), Rheumatoid Arthritis: Methods and Protocols, Methods in Molecular Biology, vol. 2766, https://doi.org/10.1007/978-1-0716-3682-4, © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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Escherichia coli ................................................................. 70 European Quality of Life-5 Dimensions (EQ 5D-5L) ............................321, 322, 326, 330 Euthanasia ................................................. 46, 57, 59, 306

F Fixation ................................... 12, 13, 43, 44, 46, 50, 51, 217, 219, 226, 266, 268, 290 FlexStation 3 Multi-Mode Microplate Reader............ 180 Flow cytometry ..............................................65, 239, 245 Freund adjuvant ..........................................................4, 72 Freund adjuvant complete ..............................4, 5, 39, 95, 97–99, 104 Freund adjuvant incomplete...........................4–6, 39, 95, 97–99, 104, 106

G Gene silencing ...................................................... 145, 146 Gigaseal formation ............................................... 195–198 Gut ........................................................................ 343–349

H Health Assessment Questionnaire-Disability Index (HAQ-DI) ............................. 321, 322, 325, 327, 329, 330, 332 Hematoxylin and eosin (HE) staining .................. 44, 282 Histological analysis .............................13, 22, 23, 38, 43, 50, 278, 282, 283, 286, 290 Histopaque 1077 ............................................................ 11 Homogenizer ............................... 4, 6, 56, 57, 79, 95, 97 Hybridomas ..............................94, 95, 99, 101–104, 106

I IgG................. 46, 50, 96, 101, 126, 234–237, 239, 283 ImageJ................................ 184, 186, 187, 189, 285–287 Immunization.............................. 3–6, 38, 40, 63–65, 72, 75, 77, 94, 99, 101 Immunoblot analysis............................................ 274, 277 Immunohistochemical staining ........................... 273, 276 Implantation ......................................... 12, 14, 17, 19–22, 32, 34, 35, 170, 171 Informed consent (IC) ..................... 150, 173, 312, 313, 316, 319, 348 Institutional review board (IRB)......................... 311–316 Integrated provirus .............................................. 157–159 Intervention study ............................................... 317–323 Intracellular cytokine staining .....................211, 223–225 Invasion of synovium ...................................10, 12–14, 21 Isoflurane ..............................................11, 12, 19, 20, 27, 33, 34, 38–40, 46

J Jurkat cells ..................................................................... 185

K Ki67 ............................................282, 283, 286, 287, 290

L Larsen score ................................................. 321, 322, 325 Latex test .............................................................. 235, 237 Lentivirus .............................................153, 163, 164, 167 Liquid junction ............................................................. 195 Lymph nodes.........................................94, 212, 213, 229 Lymphocytes ................................. 9, 10, 44, 51, 94, 164, 177, 178, 180, 181, 183, 185, 186, 191, 192, 200, 201, 207, 212, 229, 233, 242, 245, 259

M Macrophages ..........................3, 170, 199, 200, 247–260 Macrophage polarization ..................................... 247–260 Magnetic resonance imaging (MRI)...................... 28, 29, 38–42, 335, 338 Mapping ............................................................... 122, 304 Membrane protein ............................... 63–65, 70, 72, 74, 75, 77–79, 83–86, 88–91, 94, 131 Mesenchymal stem cell (MSC).........................17, 18, 20, 21, 23, 169–173 Metabolism.......................... 35, 200, 247–250, 254, 255 Microbiota ............................................................ 343, 344 MinION ............................ 293–307, 343–345, 348, 349 Mito Stress Test..........................200, 201, 203, 204, 257 Mitochondria..................................................26, 199, 200 Monoclonal antibodies (mAbs).......................... 9, 65, 93, 94, 96, 103, 106, 233, 236 Monoclonal antibody production .................................. 99 Monoclonal antibody purification ..................94, 96, 103 Monoclonal antibody storage................................ 96, 103 Mounting medium................................... 45–48, 50, 282, 283, 286–288 mRNA.......................................... 71–73, 75, 76, 78, 115, 116, 118, 119, 124, 126, 146, 208, 209, 218, 225, 228, 272, 276, 278, 296 Musculoskeletal ultrasound GB mode........................335, 336, 338 Musculoskeletal ultrasound PD mode............... 335, 336, 338–341 Musculoskeletal ultrasound (MSUS) .................. 335–341

N NOD/SCID mice.................................10, 12, 14, 20, 23

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O

S

Osmotic pumps ............................................21, 23, 32–34 Osteoclasts ...............................10, 44, 49, 178, 247–260, 263–266, 268, 269 Oxidative stress.................................. 199, 274, 278, 281, 282, 288, 289 Oxygen consumption rate (OCR) ...................... 200, 256

Sarcopenia..................................................................25–29 Scaffolds........................................................................... 18 Scanning electron microscope (SEM) ............... 263, 264, 267–269 Sciatic nerves .............................................................27, 28 SDS-PAGE ...........................................65, 67, 73, 74, 76, 78, 85, 89–91, 114, 120, 277 Sectioning ............................................43, 45, 47, 51, 285 Sequencing .............................................67, 70, 112, 116, 117, 133, 136, 293–307, 343–345, 348, 349 Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) ...................................... 164–167 Sharp Score (modified) ............................... 321, 322, 325 Short Form-36 (SF-36) ............................. 321, 322, 325, 329, 330, 332 Simplified Disease Activity Index (SDAI).......... 321, 322, 325–329, 332, 340 Single cell........................................... 183, 184, 187, 188, 191, 192, 213, 214, 228 Single-strand DNA (ssDNA)..............129, 133, 135, 136 16S rRNA ...................................................................... 343 Sodium lauryl sulfate (SDS) ...........................59, 85, 131, 134, 274 Spike glycoprotein......................................................... 164 Splenocytes ................................................. 100, 201, 202, 205, 228, 243, 244 Stools ............................................................343–345, 348 Store-operated Ca2+ entry (SOCE) ........... 178, 181, 192 Synovium ................................. 10, 12, 19–21, 37, 55–57, 93, 170–173, 247, 248 Synovium human ................................................. 171, 172 Synovium mice ...............................................9, 56, 57, 59 Systematic evolution of ligands by exponential enrichment (SELEX)........................................... 129, 131, 133 Systemic delivery ........................................................... 146

P Paraffin embedding ................................. 45, 46, 282, 285 Patch-clamp ................................................. 192, 193, 195 Paw volume ...............................................................38, 39 PCR Real-time qPCR ................................. 156, 158, 160 Peripheral blood mononuclear cell (PBMC)..........10–12, 14, 147, 148, 150, 234, 235, 238, 239 Phenol..........................67, 132–134, 210, 220, 248, 249 Photoaging ........................ 281, 282, 284–286, 289, 291 Plasmid ............................... 67, 69–71, 75, 78, 110, 112, 116–118, 124, 125, 136, 146, 165–167 Polymerase chain reaction (PCR) ....................67, 69, 70, 75, 78, 111–120, 124, 125, 131–136, 155, 156, 158, 210, 220, 221, 225, 231, 274, 276, 295, 298–300, 343–347 Power Doppler (PD) mode................335, 336, 339, 340 Proliferating cell nuclear antigen (PCNA) .................. 273 Protease inhibitor................................................... 56, 274 Protein arrays........................................................ 107–126 Protein extraction .............................................. 56, 57, 59 Protein G ...................................... 97, 114, 119, 122, 126 Protein quantification assays........................................... 59 Protein solubilization...................................................... 85 Proteoliposome ....................... 63–67, 69, 70, 72–77, 84, 87–91, 94–99, 101, 104, 131, 133, 134, 136, 137 Puromycin ............................................................ 172, 173

R Randomized allocation ................................................. 317 Reconstitution ....................................... 64, 83, 84, 86–90 Rheumatoid factor (RF) ....................10, 25, 39–42, 234, 235, 237–239, 241 RNA .............................................70, 116, 124, 126, 129, 139, 140, 142–150, 153–160, 164, 170, 209, 210, 218–220, 222, 230, 273, 276, 293–298, 300, 306, 307 RNA mRNA ............................................... 70, 73, 75, 78, 115, 116, 119, 124, 126, 146, 208–210, 218–222, 225, 272, 276, 278, 296 RNA-seq ............................................................... 293–307 RNA shRNA............................................... 145, 146, 148, 150, 154, 157, 164–166

T Tartrate-resistant acid phosphatase (TRAP) staining................... 44, 45, 47, 49, 251, 258, 268 T cells HEK 293 T cells ................................................ 165 T cells Pan T cells ....................................... 147, 148, 204, 205, 209, 238 T cells Primary T cells .........................146, 147, 149, 192 Thapsigargin (TG) ............................. 179–182, 186–188, 194, 196, 197 3,3’,5,5’-Tetramethylbenzidine (TMB) solution........ 235 3,3’-Diaminobenzidine (DAB) ................. 46, 48, 50, 52, 273, 276, 283, 287 3D structure of DNA aptamers........................... 139–144

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Transcriptome analysis .................................................. 293 Transfection ................................ 146–150, 164, 172, 173 Type II collagen .......................................... 6, 18, 39, 242

U Ultraviolet rays ....................................132, 135, 281–291 UVA ................................... 281, 282, 284–287, 289–291

V Vasculitis ............................................................... 271–279 Vector (pEU).......................................................... 66, 112

X Xenograft rheumatoid arthritis model .......................9, 18