The Microbiology of Cancer Compendium 978-0918816016

Table of contents :
To Add A Whisper
Dedication
TABLE OF CONTENTS
Table of Contents & Acknowledgments
Preface
Owen Webster Wheeler, M.D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Introduction
Virginia Livingston Wheeler, M.D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Contributions
Virginia Livingston Wheeler, M.D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Index of Reprints
Virginia Livingston Wheeler, M.D. . ........................... 14
Confirmatory Papers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Collaborative Investigations
Irene Cory Diller, Ph.D . ...................................... 176
Index of Independent Papers of Associates .... . .... . ............. 182
Abstracts Demonstrating Microbial Origin of Cancer ............. 287
Bibliography of Suggested Reading ............................... 3 15

Citation preview

THE MICROBIOLOGY Of CANCER

COMPENDIUM

LIVINGOTON-WI-Ii;i;Li;R

THE MICROBIOLOGY OF CANCER A Series of Five Books

*1-COMPENDIUM 2- PHYSICIANS

HANDBOOK

3-LABORATORY PROCEDURES 4-PATIENTS HANDBOOK 5- FOOD ALIVE

Edited and Compiled By Virginia Livingston Wheeler, and Owen Webster Wheeler,

M.D. M.D.

ISBN-0-918816-01-7

All rights reserved, including the right of reproduction in tchole or in all!/ form- Copyright® 1977 by The Livingston Wheeler Medical Clinic. A Livingston Wheeler Medical Clinic Publication Printed by Craftsman Graphics Manufactured in the United States of America

TO ADD A WHISPER

Sealed in each living cell a master plan.

I could have been a tulip or a lark, A stalking tiger, or a rose, or dark, Deep tunnel denizen that hides from man.

I could have been an emerald moss that ran In tendriled rivulet on stone, or shark! Or a firefly with incandescent spark, Or fish, whose /ins spread in a sequinned /an. But it was given to me to be aware Of finite things and of infinity, And one, long breath to breathe the eons in; A moment to be crowned, of all things, heir; To know the ecstasy of just to be! And add my whisper to the cosmic din.

Bessie F. Collins

TABLE OF CONTENTS

To Add A Whisper Dedication

Table of Contents & Acknowledgments Preface Owen Webster Wheeler, M.D.

................................. 5

Introduction Virginia Livingston Wheeler, M.D.

............................

8

. ...........................

12

. ...........................

14

Contributions Virginia Livingston Wheeler, M.D. Index of Reprints Virginia Livingston Wheeler, M.D.

Confirmatory Papers .............................................. 153 Collaborative Investigations

Irene Cory Diller, Ph.D. ...................................... 17 6

Index of Independent Papers of Associates

.... .... ............. 182 ............. 287 .

.

Abstracts Demonstrating Microbial Origin of Cancer

Bibliography of Suggested Reading ............................... 3 15

ACKNOWLEDGMENTS Grateful appreciation goes to the following: DR. MURRAY BLACK DR. JOHN BRANT MR. M.C. BOUCHARD DR. DEAN BURK DR. ART DAVIS, JR. MR. JOSEPH DE SILVA ARNOLD CHRISTEN MR. and MRS. WILLIAM KERR MR. and MRS. SHELLEY KRASNOW DR. J. KUMAMATO KUPPENHEIMER FOUNDATION SHIRLEY BOURDETTE DR. C. MARUYAMA JOSEPH NELSON DR. ROBERT NETTERBERG CAPTAIN NORVAL RICHARDSON MR. and MRS. LEO ROON MRS. RENATE VALOIS STAFF OF LIVINGSTON MEDICAL CLINIC FLEET FOUNDATION

In Loving Memory of AFTON MUNK LIVINGSTON, M.D.

4/19/1903-5/23/75

We who till now in thy shade Rested as under the boughs Of a mighty oak must endure Sunshine and rain as we might Lacking the shelter of thee.

0 strong soul,by what shore Tarriest thou now? For that force, Surely, has not been left vain! Somewhere, surely, afar In the sounding labor house vast Of being, is practiced that strength, Zealous. beneficent,firm! Yes, in some far-shining sphere, Still thou performest the word Of the spirit in whom thou dost live.

Matthew A mold

Preface to the MICROBIOLOGY OF CANCER Owen Webster Wheeler, M.D.

This Compendium is a distillation of the writings of a great many dedicated and competent scientists who have spent uncounted hours in the pursuit of knowledge concerning the cancer problem. Each one has left a noteworthy contribution which, if heeded, would have greatly advanced the battle against the common foe, cancer. Instead, their discoveries were rejected without scientific objectivity. Politics misguided and science wrongly directed are reaping the ugly reward of thousands of innocent deaths. In this Compendium we take you back many decades in some cases to the work of the early writers who meticulously demonstrated the presence of a pleomorphic microorganism to be the etiological agent in cancer. Since cancer does not behave like other infections, their discoveries were ignored. But the words of truth could not be stilled. There were always those in each decade who rediscovered the truth about cancer and tried to proclaim it anew to an unlistening world. Thirty years ago a physician, through a series of unpremeditated circumstances, was led to the rediscovery of the cancer microbe. She discovered it could be cultured in suitable media from malignant tumors fresh from surgery. This microbial growth was obtained hundreds of times, demonstrating that its presence could not be an accidental contamination as her skeptical peers insisted. She made a great contribution at this time which was to prove undeniably that the microbe belongs to the Actinomycetales, the family of tuberculosis and leprosy. It was demonstrated to be acid-fast, carbol-fuchsin dye retaining, and thus a new classification was established opening the door to fresh horizons in laboratory and clinical research. This person is Virginia Caspe Livingston Wheeler. The dedication on June 2, 1949 of the Presbyterian Hospital Branch of Rutgers University at Newark, N.J. for the Study of Proliferative Diseases with Dr. Virginia Wuerthele-Caspe (Livingston-Wheeler) as Director marked the beginning of the study of many animal tumors as microbial disease caused by an acid fast organism. The book, Cancer, A New Breakthrough, details the history of this laboratory and its research. It was at this time that Dr. E. A. Jackson, a microbiologist, and Dr. Lawrence Weld Smith, a pathologist, joined the staff. The grants were substantial and the work progressed well. Also close ties were established with Drs. William and Irene Diller, of the Institute for Cancer Research, Lankenau Hospital in Philadelphia. Dr. Irene Diller had previously noted the occurrence of fungus-like forms in tumors of animals. She was to confirm the etiology of the tumors, the P. Cryptocides in mice, their predictability, and their prevention. Always VLW was interested in the human host. Her great concern was for the helpless, dying patient. The long hours of laboratory work were but a means to discover ways to treat the cancer patient.

5

A large service of cancer patients at Presbyterian Hospital was a s s i g n e d t o h e r. T h e t o o l s f o r t r e a tme n t·w e r e l a r g e l y ineffectual, consisting only of surgery, radiation and early chem o thera py Day after day she attended the sick while they died, then returned in every spare moment to the laboratory to seek answers. The microbe was always there, in man and animals. There was nothing sure or promising for man except the knowledge that the cancer disease was an infection caused, in large part at least, by the ingestion of infected food. .

Initial methods for preparing the human vaccines were to come much later after Jackson visited Crofton in England. Also the association with George Clark and his reports of the Glover and Scott methods led to new hope. However, there were many flaws in these vaccine techniques which were not to be corrected for a number of years. Simplified culture media, recognition of the acid-fast staining properties, and the production of HCG by the P. Cryptocides now lead to certain identification of the microbe not previously obtainable. Prior to these years Virginia Livingston Wheeler reported a microbe similar to the cancer one in scleroderma and in Wilson's disease as well as in several other collagen diseases. Meanwhile the Newark Laboratory received a great deal of recognition marked by an award of $750,000 from the Black-Stevenson Trust for excellence in cancer research. However, when these funds were diverted from microbiological research and were misappropriated for the addition of a wing to the hospital in order to house a new and powerful cobalt machine, VLW closed the laboratory. VLW disheartened and discouraged left for California where she eventually went into practice in San Diego, California. In the intervening years, some reports reached the scientific and lay public concerning the infectious nature of cancer. Diller, Jackson, and VLW occasionally reported some of their separate findings. In 1969 the Livingston Medical Clinic was established. It was intended to be an Allergy-Immunology group but soon became largely devoted to the study of the microbiology of cancer and its primarily clinical applications. At long last VLW felt free to use her years of accumulated knowledge of the microbiology of cancer to help human beings. Fifteen years ago in 1962 a friend, complained to VLW that mice were treated but nothing was done for people, for instance her husband, a dentist, who had a malignant widespread inoperable thymoma. There must be something that could be done for him. VLW isolated the acid-fast microbe from his tissues and directed a local laboratory to make an autogenous vaccine for him. Along with a healthful diet provided by his wife, the doctor proceeded to recover completely and has had no recurrence of his tumor to the present day. A few patients, most with a poor prognosis, came from around the country,stayed in San Diego and received vaccine. Amazingly, a number of these people recovered. Then Dr. Don Nebeker of Covina, Calif. began to cooperate with VLW. A group of twenty patients were treated by vaccines made by VLW. The patients had a variety of tumors in various stages. Most of them greatly improved and many have remained well to the present time. 6

When the Livingstons first opened their San Diego Medical Clinic they were befriended by Mr. Joseph De Silva, head of the Retail Clerks Union of Los Angeles. After a little boy he had sent them recovered from a sarcoma of the wrist without the loss of his arm, Mr. De Silva hosted the Livingstons many times on his TV series on health which he presented every Sunday evening on Channel 13. From these beginnings the Clinic grew and continues to grow each month because there is a desperate need for cancer treatment based on sound physiological properties which promote the healing processes of the body rather than the destruction of the tumor itself. It was soon obvious that one cannot treat the result of a disease, the cancer, without treating the basic disease itself, the immunological failure in the face of an infectious process created by the microbical agent, the Progenitor Cryptocides. The following handbooks for physicians and patients amply present the present therapy for restoration of the immune status to the immune deficient patient.

In spite of the continued improvement of the patients treated at the Livingston Medical Clinic, there was still no marked acceptance of the microbial theory of cancer by the general medical public. Interested lay groups like Cancer Victims and Friends, the Metabology group, the Preventive Medicine symposiums kept alive the concepts. However, in 1972 VLW discovered that P. Cryptocides produces choriogonadotro­ pin (HCG) in the test-tube. This discovery was published in 1974 and confirmed by Cohen and Stramp in 1976. This proved to be a momen­ tous discovery which immediately gave credence and stature to the entire microbial theory. It is particularly significant in the light ofthe recent tremendous interest in recombinant biochemistry of nucleic acids. In addition to the source of production ofHCG in the cancer cell, without which it cannot exist, VLW was able to find an agent in nature which neutralizes HCG, the Vitamin analog Abscisic Acid, present in many natural foodstuffs, readily available to all. In addition, the mod­ ification of the P. Cryptocides to produce a universal vaccine for cancer of animals and man is now under investigation. The mouse studies indicate that vaccination plus Abscisic Acid virtually prevents implan­ tation and development of tumors in experimental mice. These studies carried out by John Majnarich of the MioMed Research Laboratory of Seattle are reported in the Laboratory Procedures. They have been confirmed in over 1800 mice. Field studies in chickens are in process at present. HCG can now be produced in large amounts for the induction and study of cancer and for immunization procedures in prevention of cancer. A new day of light and hope has now arrived. We can only express gratitude that our long-continued efforts are being blessed day by day by our Divine Creator who has guided us in the spirit of love and compassion to aid the helpless cancer patient who appears at our doorstep imploring humane and compassionate treatment.

7

INTRODUCTION TO THE MICROBIOLOGY OF CANCER VIRGINIA LIVINGSTON WHEELER, M.D.

It has been more than thirty years since the beginning of the research which led to this compilation of the Microbiology of Cancer intended to bring together sufficient information for the patient, student, and physician to interest and inform them so that each one may have the tools to repeat and to understand, to continue and to augment this work spanning a number of decades. We take from the hands of our predecessors the skills and knowledge which enable us to progress. It is my sincere desire in the following volumes to provide the means to go forward to a new understanding and a new treatment of cancer. Over the years I have watched with horror the ravages of the cancerous disease. With even more horror have I watched the therHpeutic measures inflicted upon the helpless victims. The physicians, many of them good and sincere men, have followed current concepts of cancer therapy like a parade of elephants, each one holding firmly to the tail of the one ahead, ponderously proceeding one after the other in a regimented order looking neither to the right nor to the left. They are caparisoned in the trappings of their profession as helpless to step aside as the poor victims of their misinformation. It has been my burden as well as my blessing for which I am deeply grateful, to have been granted the insight and the key to the imminent solution of this problem. No one person can solve it alone but the lamp posts along the road point to the logical, biological, humane treatment of neoplastic disease. It is my purpose to light the way so that you who come after me may have a brightness to shine on your path. It is my prayer that our Eternal Father and Creator will bless your efforts. The involvement of a lifetime can start with a very simple observation. All of my life's work started with the desire to help a school nurse who had ulcers of her fingertips and a perforated nasal septum. The pain was excruciating. The skin induration and ulcerations reminded me of the lepromatous lesions I had observed in the Contagious Disease Hospital of New York City when I was a resident physician there. I performed microscopic examinations of her tissues and found a strange, pleomorphic microbe that was acid-fast (retaining the red dye after decolorization) that rPsembled the tubercle and lepra bacilli. This led to the identification of a microbe I called Sclerobacillus. This finding has been confirmed many times since. However, as the experimental animals were injected with the Sclerobacillus, cancerous lesions developed at times. This event was totally unexpected. Using the techniques I had developed in the study of Scleroderma, I began to explore v a r i o u s c a n c e r o u s l e s i o n s of h u m a n p a t i e n t s . To my astonishment the same acid-fast microbes were present. This led to the keystone publication of my first paper, "Mycobacterial Forms Observed i n T u m o r C e l l s" which w a s p r e s e n t e d b e f o r e t h e New Y o r k Microscopical Society i n 1947. From this simple beginning began the years of association with the scientists both here and around the world 8

who had observed similar types of microbes in tuberculosis, leprosy, and cancer. It was my good fortune to form a lifelong friendship with two of them, Dr. Eleanor Alexander Jackson, a microb�ologist and Dr. Irene Corey Diller, a microbiologist and a skilled experimentalist in the field of genetically controlled mouse tumors. Many scientists are represented in this Compendium ,people who originated definitive experiments which helped to establish the etiology of cancer: the presence of a pleomorphic, Seitz filtrable, acid-fast microbe present in all cancers of man and animals, the Progenitor Cryptocides. One event Jed to another and soon we had an experimental laboratory in Newark, N.J.,a branch of Rutgers University. The history of these years is covered in the book Cancer a New Breakthrough. During this time we established many cancer cultures from animal tumors particularly the Rous sarcoma of chickens. It formed a vital link in the relationship of animal cancer transmitted to man. It was not until 1969 when A.M. and Virginia Livingston started the Livingston Medical Clinic in San Diego that the cancer research was applied to patients. A number of friends and acquaintances who knew that I had conducted animal research in cancer appealed to us for help. We were reluctant to enter the field of cancer therapy since we believed that present day methods of treatment were seldom effective. In the past, observations on animal models led to the conclusion that the very methods used to treat cancer were carcinogenic in themselves, that is, that radiation and chemotherapy not only induced cancer but also destroyed immunity to cancer. My studies had led me to the conclusion that cancer is an immune deficiency disease based on infection by a definite etiological agent, the Progenitor Cryptocides. On the basis of treating an immune deficiency in man we began to accept cancer patients. We made it clear that we were not treating cancer as such but that we were attempting to raise the immunity of the cancer patient so that he might overcome his disease, making sure at the same time that his inherent immune resistance was augmented and not compromised by deleterious agents. This concept is the one in use in the Livingston­ Wheeler Medical Clinic today. When Dr. A.M. Livingston died of cardiorenal failure May 23 1975 it seemed impossible to carry on the work without him. Yet he had worked to the end, always interested in the sick and dying who came to us. During the days of black despair the Clinic hung by a thread but the devotion of the staff, patients and many friends insured the continuation of the work. Dr. Owen Webster Wheeler, who had been successfully treated by us for malignant lymphoma, came to help after the death of his wife. In time the work and lives of Dr. Virginia Livingston and Dr. Owen Wheeler merged so that today the Livingston-Wheeler Medical Clinic continues with new doctors, new concepts, new hope for the future. It has been a long way and a long time since the first acid-fast microbes were observed in cancer, since the ultramicroscopic forms of the cancer microbe were first observed, since the first induction of neoplasms in the experimental animals were produced, since the transmission of the disease 9

from animals to man and man to man was demonstrated. Finally, the discovery

by

VLW

in

1972

that

the

Cryptocides

produces

choriogonadotropin (HCG) in the test tube, the first instance known of a mammalian

hormone

produced

by

a

microbe. This

discovery

is

indeed a momentous one since it is known that no cancer cell exists without HCG and that the nucleus of all cancer cells contains the imprint of a foreign antigen (agent). New modalities are available: the use of specific immunization, neutralization of HCG,

antibiotics, antisera,

specific skin tests, blood diagnostic studies, all of these methods are used and are proving effective. No longer does the cancer patient need to fear the surgeon's knife, the deadly radiation akin to the atomic bomb, and the horrendous

disability

induced

by

chemotherapeutic

agents. These

treatments, when indicated, must be moderated to a minimal and safe tolerance and preferentially the new modalities leading to immunological survival must be instituted. Freedom from fear will come. Ellen G. White states in the "Ministry of Healing" published in 1905 that "Those who eat flesh are but eating grains and vegetables at second hand; for the animal receives from these things the nutrition that produces growth. The life that was in the grains and vegetables passes into the eater. We receive it by eating the flesh of the animal. How much better to get it direct, by eating the food that God provi,ded for our Use. Flesh was never the best food; but its use is now doubly objectionable, since disease in animals is so rapidly increasing. Those who use flesh foods little know what they are eating. Often if they could see the animals when living and know the quality of the meat they eat, they would turn from it with loathing. People are continually eating flesh that is filled with tuberculosis and cancerous germs. Tuberculosis, cancer, and other fatal diseases are thus communicated." An amazing

observation considering it was written nearly 100 years ago.

There is no question but that nutrition plays a tremendous role in the

onset of cancer. Th� work of Irwin Stone and Linus Pauling prove that Vitamin C augments the healing process. Cell respiratory agents such as nicotinamide, riboflavin, B 12 are essential to cell life. The Vitamin A analogs, particularly Abscisic Acid, neutralize HCG, the hormone which blocks cancer antibodies. The avoidance of toxic food additives, the use of wholesome food grown in nutritious soil, the control of pesticides, the use of unprocessed food in its natural state all contribute to restorati.on of health. Minerals, vitamins, enzymes, body cleansing, all are necessary for regeneration. Immune stimulation by the use of splenic extracts and gamma globulin added to active immunization by specific and non­ specific agents are most vital but the total therapeutic effect lies in the combination of the whole.

10

The concept that only a few nucleic acids are necessary to initiate a disease has helped to explain what a virus is. It can be a living fragment of a larger aggregate of nucleic acids which may be part of a more complete microbe. Lewis Thomas' book, "Lives of a Cell" presents this astounding information. However, the question is, "What came first? Were the nucleic acids there to assemble into large entities or were the larger entities present first to break down into smaller ones? How do they combine?" These are the problems which. face the recombinant

scientists of our day. It appears that certain dissociated nucleic acid chains in the filterable stage of the Progenitor Cryptocides penetrate and attach to the nucleus of a normal cell as a foreign antigen trans­ forming it into a cancer cell by instructing the converted nucleus to produce choriogonodotropin, the inductive hormone of cancer. The P. C ryp tocides may become invasive into the normal cell nucleus when the protective factors of the host such as the immune bodies and essential nutritive elements become deficient and inoperative with the ('nd result that the metabolism of the cancer cell, anaerobic glycolysis known as the Warburg Cycle, is irreversibly produced. At any rate, man is but a small, however, select part of the flux and flow of Creation. We are privileged to be observers of the creation of living forces in the hand of the Great Architect. Regardless of the research yet to come, the fimndation and pillars of the etiology and treatment of cancer are firmly established.

11

Contributions of Virginia Caspe Livingston Wheeler. This is a list of the contributions made by VLW to the recognition of the etiology and treatment of various collagen and neoplastic diseases. ln 1946 isolated the Sclerobacillus, the cause of scleroderma.

In 1947 established the concept that the cancer microbe belongs to the same family as the tuberculosis and leprosy microbes, the Actinom!ycetales. In 1947 learned that the cancer microbe is Seitz filterable thus relating it in size to the viruses. From 1947 established the fact that tumors are neither species nor tissue specific, that is, the same microbe can be passed from one species of animals to another and that the microbe can be present in any tissue of the host whether skin, muscle, mucous membrane or any other. It is the soil on which the microbe falls that determines the kind of disease evoked. If the animal is resistant, then the resultant disease will be less severe, ranging from fibrosis to frank neoplasm when the host resistance is markedly lowered. 1950- 1953 These years at the Newark Laboratory with Drs. Jackson and Smith were devoted to culturing the microbes from many kinds of known animal tumors. The isolates were then passed into fresh animals and the tumors reestablished thus fulfilling Koch's postulates. There were no tumors that did not yield the specific microorganism. VLW at that time had a large tumor service at the Presbyterian Hospital

where she cared for twenty to thirty cancer patients daily. The human cultures were obtained from these patients for animal studies. Using human cultures, the incidence of cancer in the guinea pig could be increased from 11500,000 to 114. At this time, the Rous sarcoma was

extensively studied with Paul Little of the Lederle Laboratories. It was

proven that the Rous isolates could produce metastasizing tumors in mice. Microbial isolates were also obtained from patients with chronic collagen diseases such as sarcoidosis, lupus erythematosis, and some kinds of arthritis. These diseases yielded similar cultures, probably different strains of the same family.

1956 Isolation by VLW of a similar microbe but much larger in

size from two cases of hepatolenticular degeneration or Wilson's disease.

1965 VLW demonstrated mycobacterial forms in myocardial

vascular disease.

1968 Research at the Biomed Laboratory of San Diego under a

Fleet Foundation grant showed that the cancer microbe, when filtered and put into tissue culture produced degeneration of cells under certain conditions and cell proliferation in others. Study of several hundred

cultures showed that the specific microbes were sensitive to a number of antibiotics in their extracellular phase but markedly less or not at all when parasitized into the cell. San

1970 While under the Fleet and Kerr grants at the University of

Diego

VLW

et

al

showed

that

the

microbe

produced

an

actinomycin-like antibiotic as well as toxic materials which enhanced

12

the incidence of cancer in genetically controlled mice. The same year at the New York Academy of Sciences a number of papers were presented by the group, one of which proposed the classification of the cancer microbe. Henceforth it was called Progenitor Cryptocides.

1972 Following extensive studies of fresh blood from cancer and control patients by Darkfield microscopy. VLW developed a modern nomenclature for the forms seen in the blood. These forms had previously been observed but not described when compared to fresh blood cultures

so that the entire cycle was observed and named according to modern usage.

1 9 7 2 - 1 9 7 6 V L W p o s t u l a t e d t h e p r o d u c t i o n o f HC G ( choriogonodotropin) by the Cryptocides. Although HCG is a mammalian hormone she demonstrated its production b y Cryptocides. These results were published in the Transactions of the N. Y Academy of Sciences in 1974. In this paper the cross-reactivity of P. Cryptocides with other Mycobacteria was demonstrated. At the same time VLW discovered that Abscisic Acid, an analog of Vitamin A, widely distributed in nature, is cap · able of destroying HCG in the test tube. In July 1 976 Cohen and Stramp confirmed the production of HCG by the Cryptocides isolates. 1972 Owen Webster Wheeler made a momentous decision to treat

his malignant lymphoma of the neck by immunization alone. The l�·mphoma, the size of a tennis ball, was completely gone in six months. 1969- 1977 The Livingston Medical Clinic established the treatment

for immune deficient patients with vaccines, antibiotics, antibodies, diet, vitamins, minerals and certain conventional modalities as indicated.

1977 The Livingston Medical Clinic became the Livington Wheeler Medical Clinic.

13

Index of Reprints of Virginia Livingston Wheeler (Wuerthele-Caspe) Microorganisms associated with Neoplasms, New York Microscopical Society Bulletin, No. 2. Vol. 2, New York, August, 1948. Scleroderma treated with Promin - with report of a case -, . The Journal of the Medical Society of New Jersey, Vol. 44, p. 52, 1947. Etiology of Scleroderma - a preliminary clinical report -The Journal of the Medical Society of New Jersey, 1947. Cultural Properties and Pathogenicity of Certain Microorganisms obtained from Various Proliferative and Neoplastic Diseases, American Journal of the Medical Sciences, 220, 638 December, 1950. Microbiology of Cancer, Neoplastic Infection in Man and Animals,Atti Del VI Congresso Internazionale DiMicrobiologia, Roma, Vol. 6, Sez. XVII, pg. 3-9, Sept. 1953. Some Aspects of the Microbiology of Cancer, Journal of the American Medical Women's Association,Vol. 8, No. 1, pg.7-12, Jan. 1953. Neoplastic Infections of Man and Animals, Journal of the American Medical Women's Association, Vol. 10, No. 8, pg. 261-266, August 1955. Intracellular Acid-Fast Microorganism - Isolated from two cases of Hepatolenticular Degeneration, Journal of the American Medical Women's Association, Vol. II, No. 4, pg. 120-129, April 1956. An Experimental Biologic Approach to the Treatment of Neoplastic Disease, Journal of the American Women's Association, Vol. 20, No. 9, pg.859-866, September 1965. Mycobacterial Forms in Myocardial Vascular Disease, Journal of the American Medical Women's Association, Vol. 20, No. 5, pp. 449452, May 1965. A Specific Type of Organism Cultivated From Malignancy: Bacteriology and Proposed Classification, Annals of the New York Academy of Science, Vol. 174, Article 2, pg.636-654, Oct. 1970. Toxic Fractions Obtained From Tumor Isolates And Related Clinical Implications, Annals of the New York Academy of Scieces, Vol. 174, Article 2, pg. 675-689, October 30, 1970.

14

Demonstration of Progenitor Cryptocides in the Blood of Patients with Collagen and Neoplastic Diseases, in Transactions New York Academy of Sciences, Jan. 28, 1 972. Some

Cultural,

and Biochemical Properties of Transactions of the New York Academy of Sciences, Series II, Vol. 36, No. 6, pg.569-582, June 1 974. Progenitor

Immunological, Cryptocides,

Confirmatory Papers Pathology Lawrence W. Smith, (Professor of Pathology at Cornell University)

Pathologic Changes Induced in Animals by Microorganisms Recovered · ff'Om Cases of Human Cancer. From the Bureau of Biologic Research for t hi' Study of Proliferative Diseases Rutgers University Branch at P rl ' sbyterian Hospital, Newark, N.J. -

Scleroderma

N. Del motte

and L.

van der Meiren,

Recherches bacteriologiques

,.,,rH'Prnant la sclerodermie, in Dermatologica.

I >•·rrnatologische Zeitschrift-Separatum Vol. 107, No. 3, 1953. ,\l;tn R. Cantwell Jr., M.D. and Dan W. Kelso, Los Angeles, Acid-Fast I Ltd!' ria in Scleroderma and Morphea, reprinted from the Archives of

I >•·rrnatology, June 1971, Vol. 4, American Medical Association. Production of Choriotropin By P. Cryptocedes

ll•·rrnan Cohen and Alice Strampp, Bacterial Synthesis of Substance

Str11ilar to Human Chorionic Gonadotrophin in Proceedings of the Sowil'ty for Experimental Biology and Medicine, Academic Press, \',1. 152, No. 3, July 1976.

15

THE PRESEN C E OF C O N S I ST EN T L Y RECU RR I NG INVASIVE MYCROBACTERIA L FORMS IN TUM OR CEL LS• INTRODUCTION

S. J . ROSE, M . D., Pathologist ST . MICHAEL's HosPITAL, NEwARK, N . J .

T

HE complexity o f medicine and the seriousness o f many o f its

problems have led to so much theorizing and speculation that many of our present day concepts will of necessity be proven to be false and misleading unless there is endless application to investigation. In spite of extensive research, there still are numerous disease entities the etiology of which remains unknown. The riddle of cancer is rapidly becoming the most important of the etiologically unknown human afflictions with its tragic death toll of well over 170,0 0 0 persons annually in the United States alone. The conquest of many human infirmities must await a clearer understanding of the mechanisms and pathologies of the disease processes before we can be led to more successful means of prevention and treatment. This knowledge must be obtained through painstaking, expensive and prolonged application to research methods. Often, such methods fail to reveal the true facts and are apt to be abandoned in discouragement. Fortunately, persistent efforts occasionally yield successful results which constitute the chief incentive to all those interested in research. Cancer research had its beginning only fifty years ago, but during the past twenty-five years both public and professional interest has gained steady momentum. One who becomes interested in the problem of cancer research hears chiefly of the outstanding projects in large cities, in universities, and in research foundations. However, many worth-while projects are being carried out in smaller, less known laboratories. While it is not possible to consider all the concepts in the cancer problem, brief mention of those ideas which appear at present to be im­ portant and promising will be made. In the absence of a known causa­ tive factor for cancer, the following theories of carcinogenesis have been offered: 1. Hereditary, 2. Chemical, 3. Hormonal, 4. Dietary, 5. Aging, 6. Parasitic, 7. Radiation, 8. Enzymatic, 9. Bacterial, 1 0. Viql. All of these concepts have been derived from observations made on human tumors as well as on the spontaneous and induced tumors of animals. In the process of studying one problem, new experiments have suggested themselves, often leading to unexpected discoveries. This is what took place in the course of the investigation about to be re­ ported in this paper. In both scleroderma (generalized systemic

16

sc lerosis ) and c a ncer, Dr. Wuerthe le-Caspe found certain similar bac ­ teri a l forms to be consistently present in the lesions as well as i n the blood of individuals suffering from these diseases . Their recogni tion as forms of mycobacteria was subs t a n tiated by Dr. A lexander-Jac kson whose contribu tions to the study of mycobac teria are well known . Wi th the aid of a special staining tec hnic developed by A lexan der­ Jac kson, these heretofore unnoticed bodies were rendered conspic uous. C u l tural procedures and a n i mal passage led the way to further con­ fi rm a t ion. C l arification of these forms was made through the aid of Dr. Roy M . Allen, microscopist and photomicrographer, and Dr. James Hi llier, elec t ron microscopist. The presence of these organisms appears to be more than coinciden t a l . I t is hoped tha t this paper will serve as an i n t roduction to fu rther i nves tigations into the relationship between the;e mycobac teria and tumors .

17

THE PRESENCE OF CONSISTENTLY RECURRING IN VASIVE MYCOBACTERIAL FORMS IN TUMOR CELLS ':· ( N umbers i n paren theses indic ate references.

Sec pp. 17, I8.)

VIRGINIA WuERTHELE-CASPE, M.D. series of more than flfty tu mors has been s t udied with particular a t ten tion given to the microscopic a n d cultural evidence of a definite m ycobacterium consis ten t l y found i n association with the m . This series i n c ludes a n i m a l tu mors s u c h a s Chicken Tumor #I 0, Rous' chic ken tumor, Mouse Sarcoma # 1 8 0 , spontaneous tumors of mice and chickens, and fuel oil induced t umors of mice and rabbits. The human tumors include Hodgkin's d isease, c arcinomas, sarcomas, melanomas, and many benign tumors. I n addition, many smears and sec tions of normal tissues were examined .

A

The rationale for the bac terial s t udy of tumors originated in the ini tial work done on the bac teriology of scleroderm a ( generalized sys­ temic sc lerosis ) . ( I7 ) . The similarity of the skin lesions of this disease to those of early sarcoma, as w e l l as the cystic lesions i n bone and lung which resemble the metas tases of tumors, led this w riter, in the s ummer of 1 947, to apply to the study of tu mors , the technics devised by A lex­ a nder-Jac kson and W uerthele-Caspe for the s t u d y of generalized scler­ osis. The d isease enti ties of tuberc ulosis, leprosy, genera lized sc lerosis , and c a ncer have certain fea tures i n common. A l l fou r diseases arc charac terized by a simultaneous process of produc tion and des truc tion of tissue and by a progressive, sys temic involvement of the host. The t uberc le bacillus, Mycobac teri um tuberc u losis, was first iso­ l a ted by Koc h in 1 8 8 2. Previous ly, i n 1 8 64, Villemin had infected ani mals w i t h t uberc u lous tiss ues. Much in I9 0 7, desc ribed the granu les oc c u rring i n tuberc ulous lesions. In 1 9 2 9 , Kahn w as able to demon­ s trate the developmen t of rods from these granu les. Alexander-Jac kson i n 1 94 5 demons trated the presence of non-bac i l l ary forms of the t uberc le and lepra bacilli in ac tive c ases i n which no acid-fast rods were found. ( 2) . The Triple Stain ( I ) was developed by A lexa nder-Jac kson to differ­ en tiate between the non-acid-fast forms of the mycobac teria and other non-ac id-fas t organisms prese n t in mixed i n fec tions. This s tain com ­ bines the Ziehl-Neelsen with a n additional technic devised for the penetra tion and fixation of methylene blue to the non-acid-fast forms by the use of sodium h y droxide. These non-acid-fas t forms, holding the me thylene blue, resis t bleaching with sodiu m hydros u lphi te. The *Original August

p a p er

23, 1947.

presented

before

Copyr i g h t Nos.

the

1835-37

Newark inclusive.

18

M edical

Wom e n ' s

j o urnal

Club.

counter-stain consis ts of a mixture of acid green and acid yellow . A smear s t ained wi th the Triple Stain shows the red acid-fas t forms and the blue non-ac id-fast forms of the mycobac teria against a green backg round. Organisms not be longing to the mycobac teri a l family will stain gree n with the bac kgrou n d . This differential stain is of great va lue in st udying pathological m a terial derived from sclerosis and tumor patien ts. By the usc of this stain as well as by the examination of suspensions of living, unstained organisms i n a hanging d rop, A lex­ ander-Jac kson has investiga ted the p leomorphism of the mycobac terial family. In addi tion to the wel l - k nown rods and granules, A lexander­ Jac kson has shown the presence of g loboid bodies and zoogleal forms ( 3) ( g ranules i n an amorphous ma terial) to be present as part of the generat ive process of the mycobac teria. The blood of pa tien ts suffering with a mycobac terial disease fre­ quently shows the presence of the organism on direc t blood s mear examination. The presence of petechiae in the skin and mucous mem­ b� anes of patients with generalized sys temic sclerosis, led A lexander­ Jackson and Wucrthele-Caspe to c u l ture the blood of these pa tients direc tly on Petrag nani and Lowenstein media, as well as to inject the blood and c u l t ures from these pa tients into the chorioallan toic sac of ' ten -day old chick embryos . The c u l ture materi a l was also derived from s k i n and nasa l u lcers as well as from the sputa of pa tients with lung lesions seen by xray. A n aci d - fast organism was recovered from the c u l t u res as well as from the infec ted chick tissues . The subcul tures from the c hicks appeared identic al with those made direc tly from the pa tien ts. However, examina tion of the uninocula ted chicks revealed the presence of an indigenous acid-fast organism, present in abou t ten percent of a l l uninoc ula ted control chicks examined . The part it plays i n the health or disease of the chick has not been determined . The unhatched chick is subject to other spontaneous diseases as welL An en tire experiment consis ting o f fi fty-six eggs had to be discarded bec ause a large percentage of the con trols showed marked lymphocytic i n fi l t ration into the tissues, resembling the pic ture of human leukemia . Still other controls were seen to have nests of ray fungus within the li ver as sho w n on liver sec tions . For further c onfirma tion, mice and guinea pigs were inoculated wi th blood and sputa from sc lerosis c ases. The animals were sacrificed .It suit able i n tervals, from three to nine mon ths after inoculation . A guinea pig examined after four months showed invasion by the sc lero b.tcillus into the inguinal nodes at the si te of injec tion . The organs were a lso in fec ted . The mice, partic ularly those sacrificed a t a later d.tte, showed not only the presence of the organism but also definite p.tt hological changes on sec tioning . Collagen stains indicated an in­ t"rease i n the amount of collagen present i n the tissues . After a partial hvdrolysis of the col lagen, the Ziehl-Neelsen s tain penetrated the or­ ,�.mi sms which were lyi ng on and within the collagen fi brils. Pa tients 19

and animals infec ted with the sc lerodetma organism we re t u berc ulin neg a t i v e . These experiments led to the c on c lusion that a previously unidentified mycobac teri u m might play an import ant part i n certain coll agen diseases, parti c u larly in generalized systemic sc lerosis. With the sclerosis study as a bac kground, this wri ter beg an the investiga tion of the bac teriology of tu mors . A smear ta ken from a freshly removed cervic a l metasta tic gland with the orig inal si te i n the thymus of a fifty-five year old m a le pa tien t , was fi xed and stained with the Triple Stain . The tumor cells appeared fresh and well delinea ted . N umerous acid-fast granules were seen not only be tween the cells but al� o wi thin the cytoplasm and nuc lei as wel l . Of ten, a s m a l l, c lear a rea was observed to s urround these granules as they lay within the cellular protoplasm. Larger g loboid bodies were also present . Several small acid ­ fast rods lying i n other vacuoles sugges ted t h a t they were deri ved from the globoid bodies . In other places, minute red rods could be seen lying free. Some of the tumor cells showing profound cy toplasmic disruption were fi l led with minute blue and red-s taining granules . The c ytoplasm appeared to be coagula ted into a homogeneous, mucin­ like mass fi l led with innumerable granules . These granules appeared more globular in other places . In still other areas, the roun d bodies seemed to duplicate themselves wi thin s m a l l rod forms which took either the red or blue s t ain, against a g reen background. This charac teris tic picture has been observed repea ted ly in many kinds of tumors . A mag­ nifi cation of one thousand to fi fteen hundred diameters w i th oil im­ mersion was mos t commonly used. Following this initial observation, bloods were obtained from two terminal c ases of m alignancy, one of the s tomach and one of the tongue . These b loods, o n examination, showed small acid-fast rods a n d gran­ u les that were highly refra c tile. With the s ame technic used in the sc lerosis s tudy, ten-day old chick embryos were inoc u l a ted into the c horioallantoic sac with .2 to .4 cc. of whole, fresh, s terile c itrated blood . Six chicks were used for each blood . Half of these died im­ media tely but i n those that s urvived, acid-fas t granules and rods oc­ curred i n great abundance i n the liver smears . There appeared to be a focal distribution of the rods and granules as islands within the normal liver tissue. There seemed to be an a t tempt at giant-cell formation around these islands which were necrotic a t the center. However, a bou t one in fifty of the normal controls showed a spontaneous gross t umor forma tion. Great care must be e xercised in the use of s ufficient m a terial for the duplica tion of results as well as for the careful s tudy of controls . Smears taken from the cervical glands of two patients with Hodg­ kin's disease were s tudied . The same gran ules, rods, and globoid forms were presen t . The bloods of seven c ases of Hodgkin's disease were

20

e x J m i n e d . E a c h of t hese b l oods W JS i nj e c ted i n to f o u r c h i c k e m b r y os . A l l t w en t y - ei g h t o f t h e c h i c k s s u rvived . They were des t r o y e d o n t h e t w e n t y - fi rs t d a y of i n c u b a t i o n . G ros s l y , t hey a p p e a r e d f a i r l y n o r m a l , e x c e p t f o r a som e w h a t m a r ked s o f te n i n g o f t he l i v e r a n d s p l ee n . M i c roscop i c e x a mi n a t i o n o f t h e c h i c k l i vers J l l s h o w e d t h e p rese n c e o f t h e c h a ra c t e ri s t i c g ra n u les J n d r o d s observed i n t h e origi n a l d i re c t s m e a rs . A C J re f u l s t u d y o f

t he fac e

w ;l s

a

p a t i e n t w i t h a l a rge e p i d e r m o i d t u m o r of 1 94 0

s t a r t e d at this t i m e . Th i s t u m o r :1 rose in

at t h e

m u c oc u t a n eous j u n c t u re o f t h e l o w e r l i p ne:1 r t he m i d l i ne o f t h e f a c e of

J

s i x t y y e a r o l d m a le p :1 t i e n t . I t W J S t reJ ted w i t h s i m p l e e x c i s i on .

It rec u rred i n less t h a n o n e y e a r . This p a t i e n t d i d n o t seck x r a y t re a t ­

men t u n t i l b t e w he n i t w J s g i v e n for p :1 l l i a tion o n l y . The t u mor l1:1 d d e s t royed the e n t i re m i d d le port i o n of the m a n d i b l e . B rea t h i n g a n d s w :1 1 lo w i n g w e r e d i ffi c u l t f o r t h e p : H i c n t . The redu n d a n t edges o f the t u mor which did not :lppe;u· t o be sec o n d a ri l y i n fe c ted , p r o v i d ed J n e x c e l l e n t oppor t u n i t y for o b t a i n i n g s m e a rs o f l i v i n g t u m o r c e l l s . S t e r i l e p u ze w a s used t o w i pe t h e s u rface. S te r i le, dry s w abs w e re used t o o b ­ t J i n m J. t e r i a l f r o m the i n t e rior o f t h e t u mor m a s s . S m e ;l rs o f t h e f a c e .m d o f t h e blood a g a i n re v e a led t h e s a m e m y coba c t e r i a obse r v ed i n t h e p re v i o u s l y s t u d i e d c ases. The b l ood w as i n oc u l a ted i n t o t h e a l l a n t o i c s .1 c s o f t w e l v e t e n - d a y o l d c h i c k e m bryos . S e v e n o f t h ese c h i c k s s u r­ v i v e d . E x a m i n J t i o n :1 n d c u l t u res w ere m ade from t h e l i v e rs of t h e s u r v i v i n g c h i c k s o n t h e t w e n t y - fi r s t d a y of i n c u b a t i o n . Seg m e n t s o f ' t cr i le l i v e r w e re s meued o n Pe t r a g n a n i s l a n ts . A f ter fi v e w e e k s , J

i n '' s m a l l , s c a n t g l a s s y c o l o n ies appeared . These showed t h e s a m e .1 n d g r a n u les obse r v e d on t h e d i rec t b l ood a n d t u m o r s m e a rs JS .1 s t h ose seen on t h e c h i c k e n l i v e r s m e a rs . A s u s pension o f t h i s t u n� w a s s t u d i e d b y e l e c t ron m i c rosc ope . C u l t u res m a d e from

rod s well cui­ the

t u m o r i t s e l f res u l ted i n a rapid g ro w t h o f l a rge, w h i t e c o l o n i e s c o n ­ s i q i n g o f b l u e rod s a n d w i n e - c o l ored g r a n u les . These w e re c o n s i dered

to be c o n t a m i n a n t s o f t h e d i p h t heroid f a m i l y .

S t u d y o f a d d i t i o n a l s m e a rs o b t J i ned from f resh t u mors o f m a n y

k i n d s g a v e f u r t h e r c o rroborJ t i o n . I t seemed possi b l e t h a t sec tions c u t fou r t o fi v e m i c ro n s i n t h i c k ness a n d s t a i ned w i t h t h e s a m e tec h n i c u s e d

No.

Plate !-K odachrome

I.

Ori ginal

direct

t u m or o f t h e thymus.

s mear

from

Photomicrographs

a

metastatic

cerv i c a l

node

of

, o n t a i n i n g ac id-fast bodi es·. a n d a c i d-fast a n d n o n -a c i d-fast globoid forms

1: r a n u les.

T r i ple s t a i n .

2 b l oo d

No. with

X 2.000.

S i n gle a c i d -fast of a p a t i e n t

o r g a n i s m from

w i t h an

Plate

I I,

( se e page

12)

a s m e a r of t h e l i ver o f a

epidermoid c a r c i n om a of t h e f a c e .

, · u l t u rc of t h i s l i ver t h at p i c t ures N o . of

a

mal i gnant

V a c uolated t u mor m a s s s h o ws a single a c i d - f a s t rod, a v a c u o l e

s h o ws a

I

an d N o .

ma•s

2

of P l a t e

V

of t h ese forms on

a n d s m aller

chick I t wa s

were o b t a i n e d . the

H

&

E

i n fe c t e d from No.

section

a

2

of

Triple s t a i n. X 2,000. N o . 3 . A c i d-fast i n t r a cellular o r g a n i s m s s e e n i n a se c t i o n of a H o d gk i n ' s gland. Z o c h l - N eels e n stain. X 2,500. No. 4. Duct C ar c i n o m a of the breast. Large a c i d-fa s t cell resembling iepra 1: l o h u s . w i t n small g r a n u k s M t h i n . S e m i - a c i d -fast or p urple g r a n u l e s are s e e n l > o c a k i n g o u t o f a s i m i la r cell. Z i e h l - N eelsen . X 1,50 0 .

t l 1 1 s t u mor.

21

for the study of tuberculosis and leprosy shou l d yield valu able infor­ ma tion . The services of Dr. Roy M . A l len were engaged in the latter part of Oc tober, 1 94 7, for this work . In the srcond part of this paper he discusses the microscopic and photographic evidence. for the pre­ sence of these organisms i n sec tions, his con c lusions confirming the evidence a lread y presented. In order to verify the fi ndi ngs obtained by the light microscope with s tained ma terial, Dr. James Hillier of RCA Labora tories, at Prince­ ton, N j . , kindly consented to study our pure c u l t ures w i t h the elec­ tron microscope . Sterile, aqueous s uspensions were made of the sc lcro­ bacillus , the chick organism, young forms of the tubercle bacillus, as wel l a.s two cultures m ade from chick livers infec ted w i t h human cancer blood . The organisms bore a c lose resemblance to one ano ther. Their morphology consis ted of one o r severa l round bodies or granules lying in a row, in pairs, and in pyramids , or of a larger spherica l body or globoid form surrounded by smaller spheres . Minute granu les oc­ c urred i n an amorphous m ass resembling the appearance of the zooglea l forms . Occasionally a group of two to several round bodies appeared to lie together in a kind of shea th, constitu ting a rod form . Dr. Hillier said the measurements of the spheri c a l bodies ;md the rods varied be­ t ween 70 and 3 00 millimicrons . The s m a l l partic les wou ld not be within the range of the light mic roscope while the l a rger ones appeared to correspond to the stained organisms previousl y observed . The q ues tion then arose a s t o how these bodies compare with the viru ses and submicroscopic forms observed by other in ves tiga tors such as C laude , Porter, and Gessler ( 6 ) , ( 1 5 ) , ( I 0 ) in the elec t ron micro­ scope examina tion of t umors . D r . Hillier has been willing to state that the organisms of our c u l t ures and the viruses observed by other workers are comparable in s i ze and in general morphology in so far as they can be compared . Much of the previous work has been done on single tumor cells containing the inclusion forms or viruses. Some­ ti mes these cells were treated with osmic acid fumes. Our m aterial has been in pure c u l t u re, un treated except for the making of sus­ pensions . There has a lso been a difference in the type of lens used m the elec t ron mic roscope as well as a difference in magnifica tion . The c lassifi cation of these bodies as viruses is unders tandable . The invasive, a c tive phase of this specific mycobac teri u m which we have been discussing, may take place as a small, refrac tile body which can be seen to be ac.id-fast with appropriate s t aining . It may break down to form minute granules w hich are a t or beyond the limits of the light microscope. The fac t that the sma ller forms arc fi l terable is not sur­ prizing. The fi l terable granu les can reproduce the disease in themselves as shown by the work on the chicken tumors . ( Claude e t a l . ) Viruses are believed to be c apable of growth only in the presence of living ma teria l . Yet the sc lcrobac i llus which appears s i m i l a r to the t u mor in­ c lusion bodies previously observed with the elec tron microscope will 22

grow on s o l i d m ed i a a l t ho u g h n o t so rea d i l y as t h e t u be r c l e ba c i l l u s . T w o s m a l l , poo r l y g ro w i n g c o l o n i es o n Pc t r a g n a n i m e d i a were ob­ tained d i rec t l y from the blood o f t w o t u mor p a t ie n t s . The g ro w t h appea r e d i n f o u r w e e k s b u t d i e d o u t . T h e e v i d e n c e for t h e prese n c e o f

t hese o rg a n i s m s o n d i rec t b l o o d c u l t u re f rom u n c c r p a t i e n t s is sho w n b y a n elec t ro n photograph i n t h i s p �t pe r . ( P h t e V . ) E l e c t ron m i c ro­ s c o p i c e x a m i n a t i o n o f y o u n g forms o f t he • u bnc le ba c i l l m , t h e c h i c k o r ­ g a n i s m , t h e s c l c roba c i l l us a n d t h e s u bc u l t u res f r o m c h i c k s w h i c h h a v e received t u mor bloods, s u g ges t s a c lose morpholog i c a l re l a t i o n s hi p .

Since considerable work h a s been done w i t h t h e elec t ron mic ros cope in the s t u d y o f c h i c k e n t u mors, i t w a s fel t a d v i s a ble to ob t a i n f resh Rou s ' c h i c ken tumor m a te ri a l for s t a i n i n g w i t h t h e Tri p l e and Ziehl­ Ncelsen s t a i n s . We req u e s t e d t h e oppor t u n i t y from D r . E p h r a i m Wol l of t h e Lederlc Labora tories a t Pea r l R i v e r to m a k e s m e a rs of several fresh Rou s ' t u mors . A c c o rd i n g l y , w e w ere p rese n t w h e n s i x c h i c k e n s were d es t royed a n d e x a m ined . F res h . o n t h e spot s m e a rs w e re t a k e n

of t h e t u m ors , fou r o f w h i c h h a d b e e n t re a t ed w i t h v a ri o u s a g e n t s . On s t ai n i n g , the t w o u n t re a ted t u mors s h o w e d t h e t ypic a l m y c obacterial forms i n g re a t n u mbers i n c l u d i n g many a c i d - fa s t rod s .

In the trea ted

t u mors , they w e re m u c h fewer in some c a ses, r a t h e r c lo u d y i n o u t l ine and d i d no t appc:tr t o be a s a c t i v e l y i n v a s i v e . I t i s outside the s c ope o f t h i s p a p e r to d i s c uss t he c h e m o t he r a p y o f t u mors a l t ho u g h i t is hoped t h a t s u c h a s t u d y may be u n d e r t a ke n at a l a te r d a t e . W h e t h e r t here a rc m a n y s u b - t y pes among these m y c ob a c teria t h a t a r c fou n d i n t u m ors is n o t k n o w n a t t h i s t i m e . Cer t a i n l y t h e org anisms

beh a v e d i ffe re n t l y in d i ffe re n t t u m ors. I n a h i g h l y i n v as i v e type of

Hodg k i n 's

disease,

no

rods

may

a p p e a r to be

prese n t .

Small,

fine

g r a n u les a p p e a r to e v o l v e i n t o t h e l a rg e r g loboid forms , whi c h i n t u rn , m a y bre a k d o w n t o form n e w g ra n u les . I n t u mors o f t h e skin, t h e

g r a n u l e i s o f t e n observed w i t h i n t h e epi t he l i oid c e l l s where it appears

to s w e l l a n d t o form a c le a r a re a a rou n d i t w h i c h m a y deve lop into a v a c u o l e . W i t h i n t h e v a c uole i n n u merable s m a l l bodies m a y form w h i c h

surrou nding

m a y b re a k o u t a n d s p r e a d t h ro u g h o u t t h e e n t i re c e l l a n d t i s s u e s , or

the

vac uole

may

rem a i n

a vac uole

within

which acid­

f a s t rod s m a y d e v e lop . I n c o n nec t i ve t i s s u e t u mors , a r o d m a y be seen to lie beside

s i n g l e cel l . The org a n i s m appears t o t h row ou t a s u b­

a

s t a n c e w h i c h s t a i ns as a n a c i d - fa s t h a ze , w h i c h , on e n v e lopi n g the c e l l , c a uses it t o lose i ts s h a r p o u t l i n e . The c e l l , i n t u r n , t a ke s o n the a c i d - f a s t s u i n a n d a ppears t o s w e l l . F i n e g r a n u les can be seen form-

No. o ft e n s t a g e,

C a p t i o n s for Pbte I I

I.

B a sal

greatly

e l o n g ated

c o m pared

X 2, 500. No.

2.

fi l l e d w i t h p r e p a ra t i o n

X 2, 500.

Cell

with

Carcinoma. in

t he

E p idermoid

o r g a n i sms in

wh i c h

one

and the

O r g a n isms

direction

globus

for m

C ar c i n o m a

and

of

of

c olored

occurring n u c le u s

t u be r c u l o s i s

Face.

devoid of n ucleus. p ur p l i s h

the

An

s!1own

d e n sely on

amoeboid·like

M icrograph taken

organ isms

23

in

destroyed.

are

not

from

packed

c e lls,

A non-aci d-fast Plate cell, H

b r i ll i a n t ly

I,

No.

6.

c o m pletely & E s ta i n e d

d i ffe re n t i at e d .

ing throughout, even in the nucleus . .A t this point, the cell may c losely rese mble the lepra bodies so charac teristic of Hansen's disease. The re lationship of the organism to collagen is striking. I n con­ nec t i v e tissue tu mors, the globoid forms can be seen lying along the collagen fi bri ls . A g lue-like ma teri al seems to surrou.nd the g loboid form to hold it to the fi bri l . The fibri l , at a l a ter stage, appears to break down , to swell, and to form a gelatinous or mucin-like m ass w i t h m a n y fi ne granu les in i t . I n the epi thelial cells of skin tumors, the minute intracel lular granu les can be seen lying in parallel rows, al most as if polarized . With collagen staining, - they c a n be seen lying a long the fi bri ls. The granules may evolve grad u a l ly into a large mass of g loboid forms or parallel rods before breaking ou t of the cel l . The vari a tions in the pleomorphism of these mycobac teria are n u merou s . In connection w i th the apparent proteolytic ac tion of these organ ­ isms, it is i n te resting to note the diagnos:·ic work done by the use of the Mucin Clot Prevention Tes t . ( 9 ) This test indica tes the presence of large amou nts of hyaluronidase in the urine of pa tients with m a lig­ n a n t diseases . Hyaluronidase is normally presen t in the body i n small amounts . Many bac teria produce large amou n ts of this en zyme . Com a n has postulated that t h e l a c k of cohesiveness of malignant c e l l s a n d their rapid spread may b e correla ted with t h e large amou n t of hyaluroni­ dase found in tu mors . ( 7 ) . Tumors have been produced experimentally by a n umber of in­ vestiga tors . Sugiura has induced tu mors with fuel oi l . L'Esperance cl aims to have prod uced Hodgkin's disease in the chicken with tu mor gland suspensions . G rand ( 1 2 ) reprod uced the Reed -Sternberg cell as well as the charac teristic cell inclusions on the allan toic membranes of the chick by using suspensions of Hodgkin 's glands . Lee ( 1 3 ) produced tu mors in the chick by the use of intravenous suspensions of tumor ma teria l . Green has implan ted tumors into the an�crior chamber of the rabbi t 's eye. Bittner ( 4 ) ( 5 ) has passed the mammary tu mor of mice through a number of genera tions . The t ransmissabili ty of the Rous' tu mor as well as the leukemias and lymphomatoses of chickens I S w e l l know n . Un less specific tu mor ma terial is used for t h e induc tion o f tumors in experimen tal ani m a ls, the occurrence of t umors in response to various irritants may require re-evaluation as to their postulated etiological signifi c a n c e . Tumors have been induced for many years with various phenanthrene compou nds. ( 8) I t is k nown that certain phenan threnes can block the · naturally occurring respiratory cell enzymes which arc largely responsible for cellular healing. ( 1 8 ) Once the reparative mechanism has been impaired, the indigenous organism might then be a ble to i n v a de. This state of a ffairs is true for the experimental i n ­ ocula tion of animals w i t h diphtheria and a n thra x . I t is necessary t o abrade t h e tissues of animals to permit t h e diphtheria organism to invade and to injec t a chemical or irritant such as c a lcium ch loride with the an thrax i n order that the animals may become diseased . When " defense 24

rupt ure " occurs, the organisms may then mu ltiply and prod uce the disease process. In the induc tion of phenanthrene tumors , there is a known difference in susceptibility of even c losely related anima l s . I t is often cus tomary to obtain a s train of s uscep tible animals by brother­ sister m a tings . It is concei vable t h a t the indigenous organism may be transferred by such inbreeding t o the offspring and become lethal only when the c e l l u l a r defenses are des t royed by a specific chemical irritant . I n conc lusion, i t m a y b e s t a ted t h a t a definite mycobac terium is observed i n many kinds of t umors . It appears to form a specific mor­ phologic a l and s taining p a t tern . I ts presence within the tu mor cells as well as within the blood of the pa tients suffering with the disease can be demons tra ted . Present evidence points to the fact that i t may be transferred to the chick embryo . Because of the many indigenous t umors and mycobac terial infec tions in c hic kens, tissue c u lture work mus t be d one for confirma tion . The rela tionship of this organism to the etiology of c ancer cannot be s ta ted at this time . This organism may be as ubiquitous as the tubercle bacillus. It may n ot invade until certain condi tions arise such as in the presence of cell res ts, i n chronic injury, in chemi c a l irrita tion, in arteriosc lerosis, in the poor nutrition of old age, in condi tions where the cell respiratory enzymes arc de­ ficient . The incuba tion period may be extremely long as it is in lepros y . W h a t i s i t s origin ? Is there a placental o r mammary transference t o the offspring ? Is i t ingested with infec ted animal products in o u r diet as the bovine tubercle bacillus is transmi t ted ? Is i t endemic in the soi l of some areas as the lepra bacillus is believed to be ? I t is apparent from this evidence that the time has come for us to look not only for endoc rine and metabolic changes in carci nogenesis, but a lso to give fresh a t ten tion to the bac terial aspects of the c a ncer problem ':- . *The q uestion as to whether t h e c h i c ke n and the e g g can serve as vectors for h uman mycoba cterial diseases i s a n open one. h umans.

L ' Esperance

i m portant role in tuberculin Whether

gave the

(14)

felt

H odgkin's disease. positive

organisms

bacillus is q uestionable.

the

Skin testing done b y L ' Espera n c e w i t h a v i a n

roactions whi c h

Avi an tuberc ulosis probably o c c urs rarely in . avian tubercle bacillus mi ght play an

�hat in

we

are

patients

aft"li cted

studyi n g

in

a

H od g k i n ' s

is

the

disease.

avi a n

tubercle

I t does not appear to grow with the usual rapidity of the

avian t u bercle bacillus whi c h may give m a cros copic weeks.

with

t u mors

colonies on solid

media i n two

I t seems possible that a group of closely related mycobacteria rather than

sin gle one may play a role i n the production of t umors.

M ycobacteria that c o u l d

n o t be cultured w i t h t h e usual met hods and w e r e therefore r u l e d out as being a v i a n t ubercle baci lli have been observed i n p a t h o l o g i c a l c o n d i t i o n s o c c ur r i n g i n birds.

W. B.

reported i n 1920 his discovery of a leprosy-l ike o r g a n i s m o c c urring i n

Wherry ( 1 6 )

t he lungs of a M e x i c a n parrot.

He did not report having c ult ured it.

The p athology

o f the lungs of t h e parrot i r. c l u d e d fi b rosis, giant c ell formation, and lep E , V . , C A S P E S. H e a l i n g of Metabolic Wo u n d s , A me ri c a n Jo u r n a l of Pharmacy, Vol . 1 2 0 , No. 1 , J a n . , ,

-

1 94 8 .

T H I S LI S T C O M P R I S F S

O N L Y A S M A L L P A RT O F T H E M A T E R I A L COVERED

28

THE MICROSCOPY OF MICRO-ORGAN ISMS ASSOCI A TED WITH N EOPLASMS RoY M. A L L E N , D. Sc.

H E advent of the microscope w as followed during the l a s t q u a rter of the 1 9 th century by great forw ard strides in the determi na tion of t h e c a uses o f many diseases . I t was inevitable, therefore, that i t would be a p p l i e d to the study of neoplasms, on the basis that tu mors were t h e res u l t of b a c t e ri a l ac tion . I n the early days a definite belief that � u c h w a s t he c ase was prevalent and numerous t y pes of orga nisms were repo r t ed by various in vestigators .

T

Failure of neop lasms to respond to various proofs usu a l l y adequate the c ase of other bac teri a l diseases brought about the abandonme n t of this concept a n d inquiry f o r t h e c a use of c a n c e r w a s tra n s ferred t o o t her l i nes of resea rc h . This res u l ted in c o n fi n i n g t h e u s c of t h e micro­ s c ope i n c a ncer work largely to the determi nation of specifi c types of t u m ors, their extent, and relation to su rrou nding tissues. Such s t u d i es a rc preferably made a t rel atively low magnifi c a t ions. Even when cell d e tails a rc of importance a n d higher powers employed , these usually do n o t g o be y o n d a 90x oil im mersion objec tive and l O x or 1 2 . 5 x e y e p i e c e , givmg a magnifi c a tion of arou nd 9 0 0 x to 1 1 O O x . This is not a deq u ate for the obse r v a n c e of any minute organisms assoc i a ted w i t h neoplasms, n o r i s the convention a l hematoxylin -eosin stain usually e m ­ p l oyed such as to yield a proper bacterial pic ture . in

Recently the virus concept of cancer origin h a s been recei ving a t ten tion . This implies re t u r n i n g to critical microscopi c a l work on tu mors once more, b o t h with t h e l i g h t mic rosc ope and the newer tool, the electron microscope.

considerable

With bo th smears and sec tions the idea l stain for the acid-fast ( t o which the organisms causing leprosy a n d t u ber­ c u losis belong ) is the Zichl-Neelsen stai n . This is a primary red stain of b a s i c f u c hs i n w i th phenol, dccolori zed until only the acid-fast or­ g a n i s m s re main colored, followed with an al kaline methy lene blue c o u n ters t a i n . This latter does not stain stric t l y acid -fast o r g a n i s m s . A f u r t h e r mod ifica tion of this stain deve loped by D r . A lexander­ J a c kson p rod u c e s a triple stain and serves to differentiate non- acid -fast forms of m y c obac teria from other blue-staining org a n i sm s . The c x ­ i \ t e n c c of non -acid-fast s tages o f both M . tuberculosis and M. leprac h .l \ bee n re co g n i z ed for some time. m y c oba c t e ri u m

S i n c e m y c o b a c t e r i a , as w e l l .1 s the c lose l y associa ted n o n - a c i d - L i s t c· o n · n e b a c t e ri .l , .1 re so n ea r l y re la ted to the fungi, it i s n o t s u r p r i s ­ i n g t h H t he �· nu n i fe � t c h a Ll c tcri s t i c s r 1 d i u l l y d i fferent f ro m t hose ot o t h e r b;J e t cr i .d g ro u p ' . These l a t ter a rc u s u a l l y rela t i v e l y u n i fo r m .

in

b o t h ' t .l i n 1 n g

n.: .H : t i o m .t n d g e n e r .1 l mor p ho l ogy---s i ze.

s

h a pe

.

re­

p r o d u .: t i o n , l' t c .. "· i r h i n t h l· i r rc , pe c t i v e g c n c Ll . M Y c o b .l c t e r i a , on t he

o t l w r h .m d

.

.

1 rc

c· x t rc m d y p l e o m o r p h i c . rep rod u c e· .1 c c o rd i n g to d i ffe re n t

29

laws, and respond differently to vanous staining reac tions and culture media . The pleomorphism extends all the way from adult rods ( fi la­ mentous forms, continuous and broken up into Much granules ) to coccus-like organisms, c lose-pac ked and isolated. The l a t ter may vary in size from around one micron in diameter, to those so small as to be in the virus class, certainly embracing a size order of a t least ten to one, possibly much g reater than this . This pleomorphism is well illus ­ trated by the tuberculosis organism, also by the corynebac teria . Even when specific acid -fast s taining technics are employed, recognition of these charac teris tic variables must be borne in mind i n the study of these organisms in smears and sec tions. for herein lies the key unlocking the evidence of their ubiquitous presence m neo­ plasms and other prolifera tive diseases . While study of smears suffices to demonstrate their presence in c arcinomatous tissues, i t tells but part of the s tory . The morpho­ logical pic ture must be completed from examination of tumor sec tions . In our s tudy to date, no tumor tissue blocks were prepared especiall y for the purpose. Undoubtedly special fixing methods will yield su­ perior results, but it is possible to use paraffin embedded blocks fixed by any standard technic provided the subsequent steps are c arefully c arried out. Blocks of a wide series of human tumors, embracing prac tically every general type, were secu red from v arious pa thological labora­ tories . In one instance a block of a Hodgkin's disease gland abou t 3 0 years old was used . Blocks of animal t umors, including chemically in­ d uced tumors, Rous ' chicken tumor and C hicken Tumor # 1 0 were secured from the foremost spec ia lis ts in these fields . Records of the fixation technics employed in these cases were not obtained. When micro-organisms are to be s tu died it is imperative that the sec tions be not over four to five microns thick and extreme c are must be taken to expand them on the s lide and in staining . In attention to these details lies success in demonstrating the presence of organisms and their morphology. For comparative purposes similar sec tions were stained with hema toxylin-eosin, Gram's stain,- Mallory's triple connec ­ tive tissue stain (in some cases ) , s traight Ziehl-Neelsen ( both s teamed and extended time at normal tempera ture ) , and modified Ziehi-Neelsen . The modified Ziehl-Neelsen consisted largely in varia tions in the stain-

Cap tions for Plate III No.

3.

F ibro-sarcoma.

A

group

o f organisms

o c c urring

free

in t h e tissues.

Partially acid-fast only, and a c cepting some of t h e c o u nterstain, hence are brilli antly p urple with Z1ehl-Neelsen stain. N o . 4. n u cleus.

X Z , SOO. 10. Ac id-fast organisms in a globus cell free of

C h i c k e n Sarcoma No.

I n this slide the greater portion of these cells show evidenc e of a residual

n u cleus, surrounded with the red-st ained organisms, been destroyed.

Ziehl- Neelsen stain.

X 2, 500.

30

but occasionly the n u c leus

has

mg times in both the carbol-fuchsin and methylene-blue, the degree of alkalinity of the latter ( up to at least four times the quantity of potassium hydroxide c alled for in Loeffler's formula being used in some c ases ) , and the degree of decolori zation of both s tains . Counters taining with Orange G was also tried but this proved undesirable. A lexander­ Jackson's triple stain was also used on sec tions, care being taken that the softening ac tion o f the sodi u m hydroxide on the tissues did not c ause loosening of the sec tion s . This can be avoided by c areful fi x­ ation and by d ipping rather than rinsing under running w a ter::· For visual examina tion of the s lides, magnifica tions of from 1 6 0 0 x t o 2 0 0 0 x were employed, while Kodachrome mic rographs o n Profes­ siona: I Type B film, 3 !i.l x 4 !i.l were taken a t 2 0 0 0 x, 2 5 0 0 x and 3 0 0 0 x, de­ pending on the area of field desired. t Where possible, a s tandard 2 5 O O x w a s adopted . The microscopical study brought o u t the following facts : 1 . Micro-organisms are presen t and can be demons trated with the proper technic in every type of human tu mor and in animal tumors we have studied . 2 . The general morphology of the organisms follows a similar pat­ tern in all. 3 . Hema toxylin-eosin, Gram's and Mallory's triple s tains are not sat­ isfac tory for a critica l demons tra tion of the organisms . 4 . The organisms are acid-fast in some phase of their life cycle, thus indic ating their c lose affinity with the mycobacteri a . 5 . O n t h e other h a n d , they a rc n o t a s re tentive of the carbol-fuchsin under decolori za tion as ei ther the mature forms of M. tubercu losis or M. lepra e . A weak solu tion of su lphuric acid works better than a lc ohol acidulated with hydrochloric acid . Semi-acid-fast stages retain some red and then take on some blue, resulting in a purple which contrasts with the greenish blue coun ters tain of the bac kgrou nd. 6. The acid- fast forms incl ude both rods ( frequently beaded ) and spherical forms . Non-acid-fast forms are more frequently of the spheric al type, densely packed , single, double, or chain-like, up to four *The triple stain has been used for the study and differentiation

of organisms

i n smears, and as employe d by Dr. W uerthele-Casp e, has proven i n va luable in t h i s p h ase

of

our

work.

Where

orga nisms

alone

are

to

be

r e d u c i n g t h e c o n c entration of the sodium hydrox i de and dec olorization,

the

obtained by the D.

sections a r e retained on

the slides.

studied

in

sections,

by

by e xer cising c are in t h e The overall pict ure

is

best

use of several different stains for d i fferent aspec t s of the problem.

t O n the taking of high pow"r m i c rographs, cf. A llen, Roy M . , Photomicro graphy. V a n Nostrand Co., p p . 1 7 3 - 1 7 6 .

Captions for Plate IV No. 5 . rupture of

F uel-o i l I n d u c ed T u mor in M o u se. the

cell

wall.

These

O r ganisms free i n t h e tissues after

are semi-ac i d-fast,

stai n i n g

a

reddish

p urple with

X Z,500.

Ziehl-N eelsen stain, while the organisms within c ells are bri ghtly a c id-fast. No.

6.

I n duced

Tumor

in

Rabbit.

prefere n c e for collagen fibers i n the skin.

A c i d-fast

organisms

Z iehl-Neelsen stain.

31

showin g

X Z, 500.

a

dec ided

or eight. The spherica l forms, both acid-fast and non-acid-fast are very highly refractile, more so, in fact, than either M. tuberculosis or M . leprae . This high refractility serves t o differentiate them from both staphylococci and s trep tococci, which possess a refrac tive index more nearly that of Canada balsam ( 1 . 5 3 ) . 7. The divergence in the size of the spheric a l forms runs between a maximum around 1 micron and a minimum of about . 2 micron. These can probably be considered adult forms . In addition, minute virus-like forms occur in profusion, but so near the limit of resolution of the microscope that ideal critic a l lighting and the bes t of apochromatic lenses are required to see them a t all.

8. M a ximum proliferation of the organisms apparently occurs within the confines of a wmor cell. The cytoplasm and frequently the nucleus are completely replaced by them. In some types of tumors the cell becomes greatly enlarged, especially in one dimension, until i t may be from five to ten times its normal length. I t then has the appearance of a fungoid ascus . Sooner or la ter, however, the organisms break out from the confines of the cell and migrate to surrounding tissues . ':· Multipli c a tion may take place outside of cells, especially when col­ lagenous tissue is present. The organisms show a decided preference for collagen. 9. Organisms have been found to be present in all animal tumors thus far examined, which include several fuel oil induced tumors in *

The

q uestion

has

been

eosinophilic granules or lipoids. low refra ct ive index

of lipoids

raised

as

to

and

their

u su al

organisms are distinguished from eosinophilic (a)

whether

these

bodies

might

not

The evidence i s conclusive that they are not. large size r ules

the m out.

be Th e

These

granules by the following d ifferen ces :

Eosinophilic granules do not stain by the Z i ehl- N eelsen technic ;

t he acid

destaining removes t h e color completely. (b)

These

(c)

Eosinophilic granules never attack or destroy the n u c leus of t h e cell con­

taining

them.

bodies are more highly refractile than eosinophilic The

nucleus

always

st ands

out

brilliantly

granules.

different i ated

from

the

cytoplasm. (d)

Eosinophilic granules do not stain with hematoxylin whereas t hese organisms

under some conditions will a c c ept it and become purple, hematoxylin sect ion . · (e)

e"specially

i n a n over-stained

I n a tumor section not over-stained with hematoxylin, eosinophilic granules

when present have their c haracteristic yellowi sh-red c olor.

U n der the same condition

Plate V

Electron Microscope Photographs No;

i.

inoculated

.

Single with

rod form derived from a p u re c ulture of

blood

from a term i n a l

case

of epidermoid

the liver

c arci noma

of a chick of

the

face.

C3 X 20,000. No. 2. S ame c ulture show i n g a more c o m m o n rod f o r m whi c h c onsists of two globoid bodies in a sheath. C3 X 20,000. No. 3. C ult ure derived from liver of chick inoculated with blood of terminal

c ase of carcinoma of the fundus uteri wit h lung metastases. virus-like forms can be seen. No.

4.

Wo X 20,000.

Poorly growi n g colony o n

Lowenstein's

media.

a blood c ulture of a t erminal case of carcinoma of the breast.

32

The ultra-microscopic D erived directly from

C h X 3 , 600.

mice and rabbits, the Rous ' chic ken tumor and Chic ken Tumor # 1 0 . The morphology, general charac teris tics and s taining reac tions cor­ respond to those of the human tumors . 1 0 . While in general the organisms found in all t umors, both human and animal appear to follow the same general pat tern, i t would not be surprising to find there is some difference between them, even those found in different types of neoplasms, corresponding to the difference be tween the tuberc u losis organism as occ urring in the human, bovine and avian strains . 1 1 . The exac t role these organisms play in the ongm, grow th and development of neoplasms is at present unknown, but that they have some defi nite role appe:lrs certain from the fac t that they are not presen:: in any norm al tissues, human or mammalian, so far examined . When found in tissues of individuals clinically free of disease, there has been mic roscopic evidence of a pa thological condi tion such as interstitial fi brosis and perivasc ular infil tra tion . I n some such c ases, a history of a previous tumor has been obtained . ( C f . elec tron mic roscope studies by Gessler, Grey and McCarty of the Research Labora tories of ln terchemical Corporation . ) The mic roscope findings may pressage the f u t ure development of a c linical disease process should " defense rupture " occ u r. The field of research is wide open in this respec t .

Con tinued fro m flagc

24.

t hese o r g a n i s m s show a brilliant pink. devoid o f a yellowi s h tint a n d stand o u t far more conspic uo usly as a consequen ce. (f)

Eosinophilic

granules d o not cause a n abnormal growth in the size o f the

cell c o n t a i n i n g them, whereas these organisms frequently cause an elon gation of the c ells i n whi c h they are developing, t o many times normal. (g)

W h i le cells c o n t a i n i n g eosinophilic gran ules can be r u p t ured i n the section

cutting and mounting proc esses. the granules do not permeate the surrounding tissues while t h e se organisms sooner or later always rupture the cell and sid'Orable

distances

remains,

only a mass o f the

origi nally.

within

t h e ti ssues.

In

late

stages

no

c l u stered organisms is prese nt

migrate to

evidence to

show

of

a

con­

c ell

wall

where i t

was

The outermost organisms are rapi dly m i g rating to other areas.

I t is proba ble that t hese organisms have been seen and noted by practic ally every pathologist.

but

recognized.

O nly t h «" Ziehl-N eelsen t e c h n i c re veals the difference.

interpreted

as

eosinophilic

33

gran ules,

hence

t heir

true

nat ure

not

L A T E R O B SE RVAT I O N S O N PLATES VI & V I I

FTER this paper had already gone to the publisher, two Lowens tein c u l tu res made February 2 3, 1 9 4 8 , direc tly from a patient i n the terminal stages of a carcinoma of the breast were found to have con­ siderable grow t h . These c u l tures had appeared negative after three months and were discarded . They remained tightly sealed and were left in a warm place. On re-examination, colonies consisting of mucoid, glassy, well-delineated areas were seen. On smear, both with the triple stain and the Zieh i-Neelsen, large numbers of organisms were fou nd to be acid-fas t . Those that were not acid-fast took up the differen tial alka linized methylene blue of the triple stain showing that they were non-acid-fast forms of mycobacteria. No contaminants were presen t . Cut No. 7 ( Plate VI ) , shows this smear with some branching forms, short, thick rods, globoid bodies and a fi ne bac k­ grou nd m a t of the viral form s . Two subcultures were m ade on Lowenstein media from each of the two original cultures . Cut No. 8 shows a smear taken from a subculture· These forms are practically all globoid and v i ral a t this s t age . The spear or palmet to forms are very prominent. These forms consist of an acid-fast matrix possibly of an organic, c rystalline nature within which the viral forms a re see n . Through the kindness and cooperation of Dr. Albert E . Gessler, Mr. Clifford E. Grey, and Mr. Kenneth McCarty of the Research Labora tories of the lnterchemical Corporation, New York City, elec­ tron s t udies and photographs were made. On comparing results, this group reported having found, independen tly of our work , the spear­ like, c rystalline forms containing viral bodies in one tenth of a micron sec tions s tudied with the elec tron microscope . To the lnterchemic al group they appear to be present in rapidly growing tumors. The same observations had been made by this w riter on tumor smears and egg c u l t u res of t u mor bloods but bec ause of the crystalline- like n a ture of the matrix, s tain artifac ts were suspected. They were not seen on the five micron thick sec tions of tumors . The presence of these forms in the unstained s t a te on one-tenth micron sec tions studied by the elec tron mic roscope show them to be a typic a l and integral part of a rapid ly growing tumor. ::· Their presence in great numbers in the rapidly growing subc u l t ures confirms these observations . Perhaps the ma trix ma teri a l is colloidal when moist and may assume the c rystal­ line form on drying. Hanging drop studies must be done to clarify this poi n t . Cut No. 9 ( Plate VII- ) is an elec tron photograph of one of the origi nal cultures showing the globoid, rod and viral forms as well as the matrix ma teria l all present i n one field . Cut No. 1 0 is a lso an elec tron photograph of an original culture showing globoid bodies wi thin a bac terial sheath . Two smaller rod forms can be seen

A

*The I n terchemical group h a s prepared a paper on the unst ained forms which will be published in the near future.

34

b u d d i n g from the l a r g er form . M a n y of t he rods s h o w " g host form s " w h i c h m a y b e t h e res u l t o f p ro t op l a s m i c c o n t rac t io n l e a v i n g t h e s h � a t h e x posed w he n d r y i n g o r e x pos u re to t h e e l ec t ro n ra y s oc c u r s . The \' i r a l forms in t he i r m a t ri x a re s i m i l a r t o t h e zoog lea l forms e x t r u d e d f ro m \1 . t u b e rc u l osis as des c r i b e d b y A le x a n der-J a c k so n . V e s i c u l a t i o n i s o b s e r v ed ;t s c h a r a c te r i s t i c o f the t u mor c e l l s i n b o t h t h e l i g h t a n d e l e c t ro n m i c ro g raphs of t u mors a n d occ u rs a l so on t h e s u rface o f the m ed i a . W h e t h e r t hese c u l t u res c a n b e used t o prod u c e t u m o rs i n c x pc ri ­ rn c n u l a n i m a l s a n d to p rod u c e c h a rac :eris t i c t u mo r m a sses i n t i ssue c u l t u re s rcnn i n s to be see n . The b l ood o f p a t i � n t s co n t a i n i n g t h ese m y c oba c teri ;t l forms appears to re p rod u c e t he p i c t u re in the i n j e c t e d chick embryo. E x pl·ri mcn t s to determ i n e w he t her a s u s pe n sion of t hes e c u l t u res c a n d u p l i c a te t he d iseas e p rocess a rc be i n g c a rried o u t . O n l y w i t h the f u lfi l l m e n t of a l l of Koc h 's pos t u l a tes c a n a n e w o rg a n i s m ( o r g ro u p o f orga n i s m s ) be p ro v e n t o be a spec i fi c p a t ho ­ l o g i c a l a g c :1 t . I f t hese pos t u l a tes c a n be s a t i s fi e d , w e m a y be able t o p l a c e t he m y c obac teri a oc c u rr i n g i n t u mors i n t o a g ro u p w h i c h m i g h t b e c a l led " tu mefac i e n t m y c obac teri a , "-a s u b d i v i s i o n o f a s t i l l l a rg e r g r o u p o f col bgenophi l i c m y cob a c teria . A ugust

1 9,

1 94 8 .

VI RG I N I A W u E R T H E L E - C A S P E

5 6 0 M t . P rospec t A v e n u e Ne w a r k , N . J

ACKN O W L E DG ME N T S G rateful a fllm•cia lio n by D r . W u atbclt·-Ca x f)(' ix n fm·sscd to :

D R . Juw ;-..n A u: x A N I> E R for hi s w i se c o u n se l a n d for h i s k i n d d o n a t i o n to a i d i n the reprod uc tion of the k o dachro m e s . D R . F u A :-.; o R A L E X A N I l l R ·J A c K S O N of Corne l l Me d i c a l C o l le g e, for h er i n d i s p e n s a b l e aid a n d ad v i c e i n t h e bac te riolog y o f t h i s w or k . for the nu t e r i ;t l on M . t u be rc u losis a n d for her c r i t i c a l h e l p i n t h e prcpar.H i o n o f t h i s p a per . :'\ e w Y o r k ,

M R . I . BA N N ER . DR. Rosi

BAss

of E i m e r and A mend, New Y o r k . for her e n c o u rageme n t a n d h e l p .

35

A C K N O W L E DG M E N TS-Crm t i n u l 'd D R . F . R . BEA UDETTE and D R . ]. A . BIVINS, D epa r t m e n t of Pathology, Experimental Pou l try Farm, Ru tgers Uni v e rsi t y , New Brunswick, N. J. Miss C L A R A B E E L E R , A merican Legion Hospi tal, Newark, N . J . , for the use of laboratory facili ties . M R . I L S L E Y Boo N E , P residen t of rhe N e w York M i c roscopical Society, for his help and advice in setting up and e d i ting this mono­ graph and for the dona tion of a k od a c h ro m e e n g r;1 v i n g . D R . EvA BRODK I N ,

N e w a rk ,

aspects of the d iseases stu died .

N . J., f o r

D R . J o s i . P H C A S P E for c h e m i c a l of c h e m i c a l l y ind u ced tumors.

;l i d i n t h �· Je r m .n o l o g i o l

a n d t ec h n i c a l h e l p i n t h e s t u d \·

M R . SA t: L C A S P E for help i n the s t u d y of c e l l the preparation of the bibliography. D R . N I N E C H O U C R O U N , Co r n e l l M edi c a l

e n z y mes

.m d for

C o l l e g e . N . Y . . f o r .ld \' i c c

o n r h e s t ud y o f controls. N.

D R . S . A . G o L DB E R G , Pat hologist, Presbyterian Hospi t a l , N e w a rk , ] . , for tu mor sec tions and other valuable a id . D R . C.

B . H E N L E , Rad i ologis t , City H o s p i t a l , N e w a r k , N . .J .,

for fresh t u mor m a teri a l . DR.

j A M ES

H I L L I E R , R C A - Vi c t or

Labora tories , Pri n c e t o n , N . .J .,

for the elec tron microscopy. DR . E LisE L'EsPE R A N C F ,

her k i nd a d v i ce and c r i t i c i s m . D R . C A M I L LE M E R M O D ,

Strang Memori a l C l i n i c ,

for her h e l p in the

egg

i n o c u lati o n

D R . J A M LS M u R P H Y of th e R o c k e fe l l e r I n s t i t u te , block of Chicken Tumor # 1 0 . D R . A . R oTTI N O , S t . V i ncen t 's H o s pi t a L N e w t h e material on Hodg k i n 's d i s e a s e .

New Y ork,

New

York ,

for

work .

Y o r k , for

for m u c h of

S i s T E R L u u o v i c A and M 1 ss N . T H o R F. N S TEI N S O N for thei r l a bora­ tory assis tance a t St. Michael's H o s p i t a l , Newark, N. J. DR. A N NA M1ss

S c H u L TS f o r h e r p e r so n a I hd p .1 n d �· n ( O ll r .l � l' l l l c n t .

S M I T H , Te c h n i c i a n , A m c r i c1 n Le � i o n ]., fo r her t e c h n i c a l .1 n d l a b o r a t o r y .l s s i s t .l ll ( C .

I DO R A

Newark, N .

D R . KA N E M A T S U

I J o, p i t .d .

S L' c l t: RO, N e w Y or k , for t he sc( t i o m o f i u l· l - oi l

i n duced tumors . D R . E P H R A I \1 W a L L ,

N . Y . . for m a teri a l a n d

Pa tholog i s t , Lcd e r l e L.1 bor.I t o r i �· s , l \· .1 r l R i w r . t h e t r e a t e d .1 n d u n t rl'.l l l' l l R o u , ·

s e c t i o ns o n

t u mors . 36

Reprin ttd !rom the February 194i hsue of

Tho Jou-' of The Medical Society of N- J..._ Vol.

44,

p. 52

SCLERODERMA TREATED WITH PROM IN

*

WITH REPORT OF .'\ CASE

VIRGINIA \VUF.IlTH ELE,. M . D . , Newark, N. J . Purpose o f t h i s report i s to record t h e dra­

t rade-name

of

hytakerol.

T h e s i m i larity in

matic effect of a new dmg on a well-established

chemical structure o f the two products i s evi ­

case o f scleroderma.

den t .

Scleroderma i s a d is­

order o f unproved etiology , involving chi e f l y

Chaulmoogra

oil

and

therapeusis are comparable.

hytakerol

as

On release o f the

t h e skin b u t a l s o , secondarily, t h e subcutaneous

material on prom i n . it seemed that since it was

t i : sues, blood vessels, and nerve supply to the

effect i ve in the treatment o f leprosy, it might

parts affected.

be

It is characterized by doughy

swel lings in the skin which gradua' ly merge

helpful

in

the treatment o f

scleroderma.

I t i s not suggested that these two diseases are

into the hard wooden indurations which give

of the same etiology , but the s i m i larity in the

the d i sease its name ·: scleroderma.

type of

becomes hard and

tight ;

The skin

the mucous me m ­

branes may also be involved ; nod u le s often ap­ pear i n circumscribed areas.

called sclerodactylia, i ·s the

A "common type,

result o f

sclero­

therapeutic oil -solubl e vi tami n prep­

arat i o n used i n both di seases . suggested the t t S Blelberg o f Newark

( fo r case 3 ) , to

s. L. Rosenstein of Vauxhall (for case 5 ) , to

DoctorH

Bernard

Slrottr of

Pau lsb o ro,

N. J., and

J. M. Schoenfeld of Norfolk, Virginia (for case 4 ) , to

Dr.

Joseph

photography

Caspe

and

of

Newark

microscopy ) ,

to

(for

the

micro­

Doctors

Henry

Brodkin and Allen Welkind of Newark (for x-rays) to

Mr.

Julius Weber of the Presbyter ian

Hospital

( for the photomicrography) and to Dr. Richard Dief­

fenbach or Newark ( for read ing the chest x�rays ) .

5 6 0 M t . Prospect Avenue 865 Osborne Terrace

16 Wasblngton Street

41

Extracted from the American Journal of the Medical Sciences, 220, 638---{) 4 8, December, 1 950

CULTURAL PROPERTIES AND PATHOGENICITY OF CERTAIN MICROORGANISMS OBTAINED FROM VARIOUS PROLIFERATIVE AND NEOPLASTIC DISEASES• BY VIRGINIA WUERTHELE-CASPE, M . D . ELEANOR ALEXANDER-JACKSON, JoHN A. ANDERSON, JAMES HILLIER, RoY

PH.D.

PH.D.

PH.D.

M . ALLEN, D. Sc. AND

LAWRENCE w. SMITH, M.D. NEWARK, NEW JERSEY ( From the Bureau of Bi o lo gical Research ( Presbyterian H ospital Branch ) , Rutgers University )

DBIONSTRATION of the presence of specific microorganisms consistently occurrin g in tumor cells was first re­ ported by Wuerthele-Caspe in 1947. These organisms exhibited cultural and morphologic features suggesting their possible relationship to the mycobac­ teria. A symposium reviewing these findings in some detail was published in 1 9489. Prior to the observation and isolation of these acid-fast organisms from n eoplastic tissues of animals and from man, the presence of similar microorganisms in generalized sys­ temic sclerosis ( scleroderma ) , a pro­ l i ferative collagen disease, had been obserwd and reported10. Following these initial observations, further iso­ lations were made from both blood and tissues of other scleroderma] and cancerou s patients. The nature and pathogenicity of these organisms were reported in a preliminary study7• The widespread occurrence of these micro­ organisms in several types of prolifera-

tive diseases, and their successful cul­ ture outside the body in artificial media, emphasized the importance of a more detailed study of their char­ acteristic�. The present paper is an extension and continuation of the pre­ vious work. SCLERODERMA STRAIN. The original c•.1ltures isolated on Petragnani and Lowenstein egg media from blood and lesions of patients with scleroderma revealed acid-fast granules of various sizes as well as rod-like forms. Elec­ tron-microscope studies suggest that the latter may be linear aggregates of the granular forms. Subcultures of the scleroderma organism taken from the original egg medium grew poorly on Sabouraud's agar. These organisms subsequently were isolated more satis­ factorily in an especially devised l iquid medium. These scleroderma strains have produced a consistent type of lesion when inoculated into white mice.t

0 Aided by grants from the American . Cancer Society, the Damon Runyon Fund, and the Abbott Laboratories of Chica�o. Ill. Acknowledgement is made also for the support received from the Presbyterian Hospital, Newark , N. f. f A report dealing more fullv with the cultural properties and pathogenicity of the sclero­ derm a organism as well as Alexander-Jackson's l iquid medium will be presented at a late;· date.

42

� I I C I! O O H G A X I S:\ I S

JX

l'fl O L J F E IL\TI VE

I Iu�rAN }. I ALIG NAX T STHAI:-1. Em­ plo�·i.ng the t echniqu e d e v elop e d in 1 1 11 ' scleroderma study by A l exan d e r­

J ac-kson

and

\Vu erthele-Caspe10,

the

! a l ter isol ated similar m i cro o rg anis m s f ro m th e blood of 18 cancer p a t i en ts, 7 1 1 ! whom had Ho d gk i n 's d i s e a s e , by

i t t ocu lating their blood into chick em­ S i n ce the chicken has many s i t ; t ilar appearing o r ga ni sm s occu rrin s pontaneously, direct isol ations were s t t bseq u e n tl y made on sol id egg media. l r n fo rtu n a tel y g ro wth o n thi s a rt i fic i a l m ed iu m pro ved to be feebl e and sc·anty. It w a s not until Dubos', Alex­ a n d e r -Jack so n 's, and brain-heart infu ­ s i o n l i q u i d media w e r e u s e d that the organ i s m s could be i s ol a ted from h u ma n p athologic blood and tissues , and tha t sufficient gr owth could be o bta i n e d for cultural and pa th oge n i c i ty s t n d ies. To date, n o g row th has been obtained from a similar control s e r ie s of 17 normal individual s. ! J I' \ ' O S .

g

S P O NTANEOUS A NI M A L TuMOR STRAINS. C o ncomitant with the sh1 dy

of isol a tes obtained from human can­ cer. isol ations were also made repeat­ edl v from Rou s chicken sarcoma and S a rcoma 180 of the mouse. These cul­ tu res l ikewise re v e a l a co n sisten t rn o r pho l ogv similar to that of the m i­ croorganisms foun d in tissues from cases · of human can cer . Al th o u gh all of th e se organisms we re morphologi­ cal l v similar, vet certain d i fferences, such a s rate o f gro w th a n d si ze o f g r anu l e s . were fou n d that may ai d in their d i fferen tiation from one an oth er. The Rous chicken sarcoma cul tures were obta ined on many media, either

from the blood of c h i ck e ns ·inoculated with tumor tissue o r from the tumor d i re ctl y . The Roll S c u l tu re s grew o n egg media, blood agar, Du bas' medi­ u m , Petroff's medium and other en­ riched agars. The growth app e ar ed in from 3 to 7 d a y s . Morphologically similar organi s m s isolated from mouse sarcoma 180 were grown in Dubos'

43

X :\ 1>

:\ E U PLASTIC

D I SI:: A SES

and Alexander-Jackson's l i quid media. granules are characteristically smaller, but otherwise present the s a m e g e n er a l features as those deriwd from other groups. M ore r ecen tl y , s u c­ cessful isolation of another similar strain of these organisms from a pri­ mary lung cancer in a mouse has been a cc o m p l is hed. \ 1oRPHOLOGIC AND C u LTURAL CH.\R­ ACTER J STJCS. I t has been di fficult to cl assi f y these organisms on the b.1 s i s of t h e u s u a l criteria, t h a t is. m o r ph o ­ lo � i c, staining, culturaL and phvsiol o gic cha racteristics. This di fficultv is chw in l a r ge me asu r e t o the e x t r em e l v s l o w r at e a nd s p a rs i t y o f g r o wth obta i n ed in the best of culture medi a . \\'h en obs erve d in tissues the o r ga ni s m s are p re s ent in both acid-fast and n o n -a ci d ­ fast forms as granules, sh or t rods. a n d globoid bodies. In culture media m i ­ nute acid-fast granules p r e d omi n a te in young cultures. As the c u l hu e a ges. these granules i nc rea se in size until they become as large as staph yl o co c ci, or even l arger, some a ppar e n tl v dewl ­ o pi n g into small a c id -fast r od-l i ke form s . S u bs equ ently, the l a r g e r gran­ u l es and rod-like forms appear to pass th rou gh the acid-fast state to one which retains the Ziehi-Neel sen fu cl1 sin only when decolorized with ethvl a l c o ­ h ol alone. Ultimately they miw l ose this acid-fast quality a l to g e ther and stain with ord in a rv d y e s. Certain of tht>se rod -l ike fon n s contain acid-fast granules wh ile the re m a i n in g stru c­ tu r e s fa il to hold the fu chsin stai n . Th i s i s par t icu l a rl v t r u e of ol d cultures which co n t ain manv of these irreg­ u l arly s ta in i n g form s. Tru e b r anc h i n g Hl anients have not been o b s e r n·d . al th ou gh u n u su al forms are often fou n d in old cul tures. The g r an u l es ar e g r a m - p o s i tive in ch a r a c te r , while the matrix m a t

NEOPLA:S'l'lC DI SEASES

REFE RE N CES

l. 2. 3. 4. 5. 1 948.

Alexander-Jackson, E.: Ann. N. Y. Acad. Sci. , 46, 127, 1945. Idem : Science, 101, 563, 1 9 45 . Cuttino, J. T., and McCabe, A. M . : Am. J. Path., 25 , 1, 194 9 . Diller, I. C. : Anat. Rec. , 105, 24 , 1949. Gerlach, F . : Krebs und obligater Pilzparasitismus, Wien, Urban & Schwartzenberg,

6. Grigoraki, L.: Edit. Lefrancois, Paris, 1944. From J . Alexander, Colloid Chemistry, Theoretical and Applied, Vol. 7-8, New York, Reinhold. In press. 7. Wuerthele-Caspe, V. : J . Am. Med. Women's Assn., 4, 135, 1949. 8 . Ide m : J. Med. Soc. New Jersey, 44, 52, 1947. 9 Wuerthele-Caspe, V., Alle n, R. M., and Rose, S . J.: Bull. N . Y. Micro. Soc. 2, 1, 1948. 10. Wuerthele-Caspe V., Brodkin, E., and Mermod, C.: J . Med. Soc. New Jersey, . 44, 256,

1 94 7 .

52

Estr3tto dagli

ATTI RO�IA,

DEL

\'!

CONGRESSO

6- 1 � Seuembre i 9 5 3

-

I :-.ITE R :-.I A Z ION A LE

Vo i . 6,

Scz. XVII

DI

MICROB IOLOG IA

A, pag. 3·9

MICROBIOLOGY OF CA NCER N EOPLAST I C I N FECT ION IN MAN A N D A N I MALS VIRGINIA W U ERTHELE· CASPE ( Cancu Ra�arch Laboratory , N�ttvark , N . J . , U . S . A . )

The degenerati1·e, proliferative, collagen diseases o f m a n a n d a n i m a l s have yielded by bacteriologic methods, a group o f pleomorph ic, fi l t rable m i croorga n isms probably belonging to the mycobacteria, a subdivision of the actinomycetales. These microorga nisms are pathogenic for men a n d a n i mals. The bactetiologic n atu re, the cultural properties and the pathology produced i n a n imals a s well as the results obta i ned by the study and usc o f spec rfic a n t isera will be dealt w i th i n a series of pa pers prese n ted by other members o f this group. The purpose of t h i s paper i s to relate the pleomorph ic ph a ses of this group of m ic roorga n i s m s to the pleomor­ phic group of collagen diseases present i n man and a n i mals. The pa thologic and c l i n ical respon ses i n t h e host to the pyoge n ic m i c roorga nisms a re

well konwn. The discovery o f the spi rocheta-pa l l i d a rel a ted the human etiology

of syph i l i s to a wide spectrum of pathologic m a n i festa t ions called by m a n y n ames prior to the recognition of the spec ific agent.

The same i s true o f Koch's bac illus.

Before the identification of the tubercle baci llus, the c l i n ical m:.111 i festa tions o f tu ber­ culosis were cal led by many n a mes such as Pott's d i sease, scrofu l a , l u pus, phthisis, depend i ng upon the local t issue reac tions.

So it i s with the wi despread spectrum

of d i sea ses of the col lagen grou p characteri zed by prol i fera tio n , fibros is and dege­ neratio!l of 1· a r ious degrees ca pable of i n vol v i n g a n y one or m a n y of the host ti ssues whether bra i n , m u scle. s k i n , splee n , mucous membra ne, bone, g l a n d , bone marrow, or connecti1·e tissue. The s i te of pred i lect i o n , the degree of i m·olveme n t or resista nce depend primarily upon host properties such

as

genetic suscept ibil ity, hormonal

balance, a n d degree of i m m u n i t y as wel l as upon biochemical a n d mech a n ical factors not e n t i rely u n derstood or demonstrated a t th i s time. T h e l i fe spa n o f man h a s greatl y i ncreased d ue to the victory of med i c i n e over

the c o m m o n i n fec tio ns which are now preve n t able by i m m u n ization o r a m e n a ble to treatment by a n t ibiot ics or a n tisera .

But now the la tter part of m::111s's l i fe is

made u ncerta i n by many dege nera tive and pro l i fera t i ve d i seases such as the 1·arious

53

kinds of cancer, scleoderma, sarcoidosis, Raynaud's and Berger's disease, arthritis and other members of the collagen group.

The evidence for the specific microbial

concept of the etiology o f these widely described pleomorphic but fundamentally related diseases will now be presented. The present speaker (VWC) first noted and published in 1947 the close similarity between the lesions of scleroderma (generalized systemic sclerosis) to leprosy - the ulcerations of the fi ngers and nasal septum , the a nesthetic ar�as, the nod ulations and h ard ness of the sk i n , the prol i ferative cha nges i n joints as well as i n so ft tissue such a s i n l u ng, kidney, and l i ver, poi n ted to a possible m icrobial agent related to

the mycobac teria .

microorganism,

S mears and biops ies of the lesio ns showed a pleomorphic acid-fast later

confi rmed

by

Alexander-J ackson

and

W uerthele-Caspe

by

cultural methods. Experime ntal a n i mals i noculated with the cultures derived from scleroderma revea led proli ferative lesion s bordering o n the neoplastic.

This observation led V\VC

to study smears of neoplast ic tissues using the acid-fast s tains i ncluding the triple

stain of Alexande r-} ackso n .

Confirmation of these early results was obtained by

later studies using many tech n ics appl ied to isolated cultures, pathologic sec tions and tissue cul tures.

Many sta i n s were u sed such as the Ziehl-Neelsen, the triple

sta i n o f Alexa nder- J ackson, the McManus, the Fite, the Supra-Vital (Toludin blue) and the tri-basic sta i n of Coidan as wel l as those stains demonstrating the pheno­ menon of bacterial flu orescence. Although

the most easi l y

recogn ized

forms are ac id-fast,

there are m a n y

non-acid-fast forms w h i c h are t h e same organisms but ar e not readily distinguished from common contamina nts.

Therefore, it was essential that a pure cul ture o f the

specific microorga n i sms be obta i ned.

The first .pure culture which recogn i zed thi�

microorga n i s m as a mycobacterium was made by \Vuerthele-Caspe i n the summer of 1 948.

A series o f Petragnani tubes were inoc u l a ted with material from living

cancer tissue.

M a n y were contaminated but one tube after six months after i nocu­

lation yielded a pure culture. Once the first culture was foun d the search for easier and more prol ific cultivation we n t o n with the cooperation and direction of Dr. Elea­ nor Alexa nder-J ackson .

It was a time-consu m i ng, back-breaking task requiring a

number of years for fu lfillment before sufficient amounts of culture could be obtained for a n i mal inoculation. Prior to obta i n ing s u fficient m aterial by d i rect cu lture, the blood of ter m i n a l ca ncer patie n ts was i noculated i n to the allantoic sac of te n day old chick embryos.

A rapid destructi\'e growth of the

m icroorganisms in the chick

embryo occ u rred i n a ma tter of hours. The search the n bega n to fi n d these bodies in the various wel l k nown neoplasms

of an imals k nown to be i n fectious in n a t ure.

M r . Paul Little o f the Lederle

Laboratories collaborating with our grou p made a great n u mber o f cultures from the Rous Sarcoma o n various med ia.

Lesio ns cou ld be produced with these cultu res

by serial passage th rough chick embryos.

54

The chemical l y i n d uced tu mors of the

rabbit

e p i the l ium of S uguira of Sloan-f\ettering Institute o f New York as wel l a s

t he sarcomas of the: wh i t e rat of M a r g are t Lew is at the Wistar Institute, Ph il a delph i a

showed the same bodies on appropriately stai ned sec tions.

Cultures made from

material contai n in g the B i t t ner factor as well as from S h ope ' s rabbit papilloma yi e l ded similar results.

Since these various agents are known to be fil trabk throu gh

the & i t z filter, the cultures were examined and found to be fil trable in all s t age s

of growth.

Then beg a n a series of electron microscope st ud t e s w i th Dr. James

H i l l ier o f the RCA Victor Laboratories w h ic h showed the ra nge in size of t h ese

b a c te r i o log i cal bodies. From these stud ies it became evident that a w i d e l y d i stributed group of microo rga nis m s of a specific m yc o b a c ter i a l nature is etiologically i nvolved in the production of a l a r ge group o f prol i ferative and n e o p l a s t ic d i seases i n a n i mal and man. In

order to fac ilitate the recognition of these m yc ob a cte r ia in tissues, it

is

necessary to re v ie w th e i r pl eo m or ph ic form s . Th ey \·ary from m i n ute a c i d - fa st intracellular a n d i n t r a nuc lea r bodies to larger g l obo i d bod ies which may e n l a rge and contain w i t h i n them many m i n u te gra n ul es.

The f r ee l y grow i n g for m of th e

culture is a fla ge lla te d motil rod which o n agi n g converts to a m yc e l i a l networ k .

A l l these forms a re demonstrable i n t i ssue. D r. I rene C o re y D i l le r has d o n e confir­ matory work on this phase of cultu re . Once a n a d e q ua te amount o f c u l t a rc material was obta i ned, t he s t u d ies in n pe ri m en t a l a n i ma l s bega n . T h e blood c u l ture o f a 26 year old yo ung wo m an ( BK) w i t h wid e s p rea d m e t a s t a se s to bone w a s i nocu l a ted i n to a s eries o f 1 2 new l y wea ned S w i ss heterozygous wh ite m ice. O f t hese, four died w i t h i n s i x w ee k s o f the l y ti c f o r m of a neoplastic i n fection. T h e org: m s were s oi t , l yze d a n d s howe d w i d e s p r ea d spec i fi c m ycobacter ial i n vasion. Four m ice survi\·ed a p p r oxi m at e l y th ree months. These all showed caseous les i o n s . The fou r r e ma i n i n g m ice d i d not a ppear invaded but o f these,

two appare n t ly

in good condition

d ro ppe J dead

suddenly and two gra d u a l l y became emac i ated but survi ved a n u mber of mont hs

These results correspo11 d to D u r a n - Rey n :1 l s fi g ure s

1 11

chicb i n fected w i t h Rous

Sarcoma - one th i rd sh owing no resistance, w ith rapid i n vasion a n d death withou t

tumor form ation ; one th i rd show i ng pa rtial resista nce w i th tumor form ation and one th ird s h o w i n g al m ost c o m pl ete i m m u n i ty . \Ve fou n d , even i n the lat ter group

exhib i ti n g h igh i m m u n i ty, that there were m a n y t issue c h a n ge s of a chronic nature other than neopb s i a . These results will sh o r t l y be d i sc u sse d at greater le ngth .

I t was dec i ded to d i ssect o u t a c a seou s lesion a nd troca r it i nto a series o f

mice i n t h e s am e way as Sarcoma 1 80 tumor fragme n t is done.

T w e n t y s e r ia l passages c o n s i s t i ng of si xteen to eighteen m ice i n each p:1ssage were so treated .

There were of cou rse i n d i v i d u a l v a r i a t i o n s in t h e reJc tions of the m ice - the

fi rst two o r t hr e e p a ss J ge s sh o w in g fewer deaths a t the end of one week but

by the e n d of the 7th passage a l l m i ce so trocared d ie d w i thi n

s e v e n d a ys . When the tumor mass from these was passe d , by the fi fth to seve nth d a y a fte r i m p l a nati o n ,

55

95 % of the a n imals d ied .

to

a

Passage from a n animal survlVlng relativel y longer led

less i nvasive type of lesion .

Here aga i n immunity plays a v ital role.

These

results coincided quite closely to those fou nd in serial implantation of Sarcoma

18o in mice.

However, the implants done b y u s gave o n l y 1 0 /� incid�nce of gross

neoplasm over all in spite of 95 �1� death due to agran ulomatous i m·asi\'e lesion when rapid tra n s pl a n ta ti on was done. However, focal a t y p ic a l cell s could be seen in almost every animal.

The perce ntage of gross tu mors i s of relative u n im p ortan c e

in view of the wide m a n i festations of d i se a se in these a n i mals.

At this point, I wish to stress the fact that occurrence of gross ne o pl asia i n

th e neoplastic disease is o f no more i m portanc e re l a t iv el y to th e d i sease i n general than the occurrence of a gumma in s y ph i l i s.

It i s an obvious man i festation but

relativel y only small evidence o f the disease as a whole. There were 320 m ice i nject ed s e ria ll y with tumor ma sses o ng m ati ng from the initial inoculation of (BK) human s tra i n o f orga n t s m .

Ten hundred s i x ty n i n e

sections were exami ned by H & E and Ziehl Neelsen sta i n s a t 1 500 m agn i fi c a ti o n F o r c o n v e n t e n c e a n d bre v i t y a r epre s enta t i v e

using a n i ntense bea m o f l ight.

sampling of results obtained in the serial p a s s a g e s are given :

K x 5o- 1 34 Caseous lesions, n eopl asti c cells i n sma l l microscopic foc i t h ro ug h o u t

many tiss ues.

S pl een neoc rotic w i t h many areas of i n fi l tra t i o n .

A gr a n u l o m a to m

mass was prese n t i n the t e s t is . K3 5o- 1 64 S ite of i n oc u l at i on shows mass1ve gro w th o f the m ic roorga n i sm

with necros1s.

N eo p la s t i c cel l s

in l u n g s i n g l y a n d i n foc i .

W i de s p r ead gro\n..�

of microorgan isms i n k id ne y.

50- 1 93 A no ther a n i m a l

m

this group showed adenocarc i noma o f the l a rg e

intestine. K4 5 o- 1 96 Globoid bodies seen 1 11 con nective tissues o f almost e \·ery org a n .

Areas of degeneration o f cardiac m u scle w i th m a n y o f t h e s pec i fi c bacteri a prese nt. Spleen shows wide s pr e a d necrosis and massive gro w t h w i th n u c l e a r ch a n ge s in

the l i ver c el l s .

K5 50- 1 88 This one was dead fou r d a y s fol l ow i ng i m pl a n tat i o n . M u scle fiber of ab d om i n a l wall i n vaded a n d l arg e l y d e s t r o ye d a t site o f i nocu l a t i o n , marked i n v asi o n of heart musc l e , l arge bacterial foc i i n l i ver and k i d ne y . A reas i n lung

are n eopla s t i c . K6 50- 192 Sol i d g r o w t h i n lung.

into l i ver.

M a s s i ve

Growth o f t roc a r c d m a s s exte n d s d i recdv

i n mh- c m c nt of heart m u scle w i t h fi b r obla s ts re p l a c i ng l a r ge

areas of de s tr o y e d hear t muscle.

Sel ec t i ,· c destruct i o n of I s l e s of L a n g er h :m s .

Th i s

a n i m a l w o u l d proba b l y b e d iabet i c . K 10

so-288

Per ibronch i a l

chan g es,

the sk i n .

56

pleurisy

with

etTu s i o n ,

c a rc i noma

of

K r o so-287 Animal killed a fter fi fth day.

S i te of inoculation shows h igh

degree of resistance. Many macrophages engu l fi ng globoid bodies. t i ssue changes.

Other organisms relatively normal.

Little general

This type of growth coincides

with the observations of Michael Lev ine as to the role of the reticulo-endothelial .

system in resistance. W here there is

a

h igh activity o f the macrophages at the site

of i noculation of tumor fragments from Rous Sarcoma, the tumor has been obsen·ed to regress. Kr

1

so-265 I n fi ltrated a reas replacing one l u ng.

Adhesive pericarditis, peri­

hepatitis. It can be seen b y this sampling process what the wide rangt; of tissue cha nges A h uman example of the wide panorama of disease possible when

a re.

this

microorga n ism is presen t i n the blood stream a n d tissues i s demonstrated by the cas

e J . S . of Dr. Charles Crane of :1'\ewark, N. J . He had us see this white fe male

because he thought the lesions would be of interest to us. Her blood stream readily y ielded the specific culture. She had hard i n d uration of the skin of the anterior chest w a l l and neck resembl ing scleroderma, widespread fibrosis of the lu ngs, enlarged l i \ er, enlarged splee n , rheu matic heart disease .

The diagnoses were as follows : cor-pulmonale h ypertrophy o f right ,·entricle m itral stenosis, rheumatic poss ibly a nemia general ized malignant l ymphoma splenomegal y hepatomegaly sarcoidosis of the lu n g s splee n , l iver and lymph n odes ,

amyloidosis of the s p lee n

,

k i d nevs and l ymph nodes

rheumatic mitral \ alvuli t is '

bronch iectasis p u r ule n t bronch itis. Rece n tl y S p oe r ci Co i d a n of H a rlem Hospital has reported diagnosis a n d grad i n g o f h u m a n n e op l a sm by 2 4 h o u r tissue c u ltures. \Ve have exam i ned h e r t i ss u e e x p l a n ts s t :� i ne d by her tribasic sta i n a s wel l as by the triple sta i n of Alexander­ J ac·k son a n d Z i e hl-1\"eclse n .

These segme n t s a re a l l positive by d i rect exa m i n a t i o n .

It is of in terest to observe that i n the tissue ex plant of a hu ma n axill a r y metastatic node

from

c :� r c i n o m a

of the

breast

t he r e

arc

many

m ic rosco p i c

foc i

consisting

o f one or two to s e n: r a l n eopl a s t i c cells a r i s i ng arou nd a s m a l l a n d i n Y a s i Y e si te

where the s pec i fic nwcob;1 c te r i u m c a n be c l e a r l y Y i s u a l i z ed

57

.

The grad ing of the

t u m o r as to d e g ree of m a l i g n a n c y a n d the p r og n o s i s of the p a t i e nt b y t h e t i ss u e c u l t u re grow t h o f t h e e x pb nt n l .-\ t i ss u e c u l t ur e

remoYed.

in

w h ich

t h e re i s

c e l l s i n d i c a te s n e o pl a s i a b u t g o o d rt: s i s u n c e a n d

s :nn p l c o f t h e rapid a

Cl ll

be d e te r m i ned

IH:opla sm

prol i fe r a t i o n

good p r og n o s i s .

o f t he

surgically i nvolved

A n n ph n t sh ow­

i n g poor prol i fe r a t i o n w i th l i q u e h c t i o n a n d l y s i s o f the cel l s s h o w s poor re s i s t a nce and poor prog n o s i s . I n c o n c l u s i o n the fol l o w i n g s t a te m e n t s a r c m a d e : 1.

.-\

spec i fi c c l a s s of

m i c ro ( lrg:� n i s m

teri u m has been observed a n d i s g ro w n

a p p a re n t l y bel o n g i ng

to

the

m ycobac­

from the o nc c r o u s blood :111 d t i ssues o f

m a n a n d a n imals. 2.

They

:�rc fi l t rable a n d pleo m o r ph i c .

3 · l n oc u b t i o n s o f t h e s e m ic roorg:� n i s m s reproduce t h e s pec i fi c d i sease i n expe­ r i m e n t a l a n i m a l s t h u s fu l fi l l i n g K o c h ' s post u l a tc s . assoc i a ted i m m u n o l o g i c a l l y w i th

a

Th i s g r o u p of m i c roorga n i s m s i s

w i d e r a nge o f p h y s i c a l a n d c l i n i c a l m a n i fe s t a t i o n s

i n b o t h a n i r.1 a l s a n d m a n , •.h e s c m a n i fe s t : n i o n s i n c l u d i n g n o t o n l y v a r io u s d i seases o f n t:: o pl a s i a b u t a l s o a w i d e r a nge of o t h e r d i sea ses s u c h a s sclcrode r m :J , i n te r s t i ti a l m \· oc a rd i t i s . pc r i v a sc td a r i n ti l t r :J t i o n ; i n te r s t i t i a l fi b r os i s , pleu r i s y , pc r i cud i t i s , rheu­ m a t i c fn e r

n e ph r i t i s , h c p: n i t i s

and

a r t h r i t i s d e pe n d i n g u po n

t h e degree of host

res i s ta n c e , the i n va s i veness o f the spec i fi c m i c roorg a n i s m s , t h e s t ra i n spec i fi c i t y , a n d t h e s i t e o f t i ssue su sceptibi l it y . E . , A n clrr m s obt a i n ed f r o m v J r i o u s proi i fcrJ t i v c a n d ncoph>tic d i seases , A m . J . �1 . Sc . 220 : 6 ;� - 6 4 6 , Dec . 1 950. W u c r r h c l ·ic lded by bacteriologic method s of study, a group of pleomorphic, filtrable microorganisms probably belonging to the m ycobacteria, a subdivision of the actinomycetales, These microorganisms are patho­ genic for experimental animals. The purpose of this article is to relate the pleomorphic phases of this group of microorganisms to the multiple mani festa­ tions of collagen disease, including neoplasia, oc­ curring in man and animals. ' · " I n 1 947, while studying a case of generalized systemic sclerosis (scleroderma) , this author noted a marked similarity to leprosy. An organism, ap­ paren tly a mycobacterium, was described and named "Sclerobacillus Wuerthele-Casp ; " fol-

lowing cultural studies. Recent con firmation of this work is reported by Delmotte and Van Der Meiren.' In a series o f animals inoculated with the scleroderma cultures, not only degenerative and proli ferative changes occurred, but also disturb­ ances in nuclear pattern. Multinucleated cells con ­ taining acid-fast inclusion bodies were seen. This led to a study of tumors, and to the recovery of mycobacterial colonies from a culture o f breast cancer. (The bacteriology work was carried on with Dr. Eleanor Alexander-Jackson, Dr. Harriette Vera, Dr. Irene Corey Dilb, Dr. George Cbrk, and Dr. John Anderson.) The pattern of these d;s�a ;es lc j the writer to postulate the existence of a group of collagenophilic mycobacteria, similar in their gen­ eral morphology and cu ltural properties but differ­ ing in their serologic response.'· ' " Various animal neoplasms known t o b e in fectious

Dr. Wuerthele-Caspi, former Director of Research, Laboratory for the Study of Prolif­ erative Diseases, Presbyterian Hospital, New­ ark, New Jersey, is now practicing i n San D iego, Califo rnia.

T h i � work w a s dont· u m l t - r tlw a u s p i n·s of R u t gc rs * l ' u i v . -r s i t y a n d t h e· P rc·sh y t l ' r i :m H os p i t a l a t t lw P rc·s. h y t n · i ; m Hos p i t a l B r a n d l , B u r c · ; � t � o f n:olo_g i c a l Rc·­ �w :nT h . :\ nv a r k . F u n d s W l ' l " t ' l n ; H k ;J \ ' ; I i b h h · h\' :\hho t t Lahor:J t o r i c·s, C h a s . P f i z t T & t : o . , U ouln \ D i g n t . Rosc· u w a l d F o u n d a t i o n : as \\T i l a s hy p r i \ · a t e i n d i ,· i d u � ls a n d .g roups s u c h as t h e La d i «·s· .-\ u x i l i a ry or Prcshyt