As the years have passed, the tremendous contributions

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Oswald Avery and the Irving Kushner, MD, and David Samols, PhD Dr. Kushner (AΩA, Washington University in St. Louis, 1954) is professor emeritus of Medicine, and Dr.

1 Oswald Avery and the Irving Kushner, MD, and David Samols, PhD Dr. Kushner (AΩA, Washington University in St. Louis, 1954) is professor emeritus of Medicine, and Dr. Samols is professor of Biochemistry at Case Western Reserve University School of Medicine. As the years have passed, the tremendous contributions of Oswald T. Avery ( ) to biomedical science are fading from our collective memory. A modest and self-effacing man, Avery was one of the outstanding biological scientists of the first half of the twentieth century, 1 felt by some to be the most deserving scientist not to have received the Nobel Prize. His laboratory was responsible for three landmark contributions: 1. The demonstration that polysaccharides are antigenic 2. The discovery of C-reactive protein (CRP), which opened the door to study of the acute phase response 2 3. The demonstration that DNA conveys genetic information. Avery was born in Nova Scotia in 1877, the son of a Baptist minister. The family moved to New York City when Oswald was still a child. He graduated from Columbia University s College of Physicians and Surgeons in 1904, when medicine was just beginning to abandon its reliance on tradition, although medical practice was still largely empirical. Good outcomes largely depended on the healing power of nature and the power of faith. American physicians generally felt that laboratory science could never contribute anything of practical value to medical practice. Avery practiced medicine for three years, but apparently didn t find it intellectually or emotionally satisfying. At that time, infectious diseases were the major public health concern and bacteriology was the most glamorous and promising field in the nascent biomedical science of the day. Opportunities for research at that time were very few, but Avery did find a position at the Hoagland laboratory, a privately-endowed bacteriology laboratory in Brooklyn, where he worked for six years, performing largely unimaginative work. The Rockefeller Institute for Medical Research At the end of the nineteenth century, John D. Rockefeller was seeking guidance about how to deploy his philanthropies most effectively. He was the richest man in the world, perhaps the richest man in history. Rockefeller s principal philanthropic adviser was a Baptist minister, Frederick Taylor Gates, himself a physician s son, who had noticed during his ministry that physicians were rarely able to deal with serious medical problems. Gates read Sir William Osler s magisterial textbook, The Principles and Practice of Medicine, 3 in which Osler, something of a therapeutic nihilist, expressed his skepticism about prevalent forms of therapy. Gates was impressed. He later wrote: I had been a sceptic before... This book not only confirmed my scepticism, but its revelation absolutely astounded and appalled me.... I found... that the best medical practice did not, and did not pretend to cure more than four or five diseases.... about all that medicine up to 1897 could do was to nurse the patients and alleviate in some degree the suffering. Beyond this, medicine as a science had not progressed. 14 The Pharos/Spring 2011

The Rockefeller Institute for Medical Research.

In headline, Streptococcus pneumoniae (Diplococcus

2 The Rockefeller Institute for Medical Research. Left, Oswald Avery in his lab at the Rockefeller Institute, circa 1940s. Courtesy of the National Library of Medicine. In headline, Streptococcus pneumoniae (Diplococcus pneumoniae). Capsule stain light micrograph at 1000x. Visuals Unlimited/Corbis. The Pharos/Spring

3 Oswald Avery and the pneumococcus... It became clear to me that medicine could hardly hope to become a science until medicine should be endowed and qualified men could give themselves to uninterrupted study and investigation, on ample salary, entirely independent of practice. To this end, it seemed to me an Institute of medical research ought to be established in the United States. Here was an opportunity, to me the greatest, which the world could afford, for Mr. Rockefeller to become a pioneer. 1p21 22 Rockefeller was receptive to this suggestion and the Rockefeller Institute for Medical Research was dedicated in Its small hospital followed in The Institute, now a university, still occupies its original site in New York City on the East River and 66th Street. When John D. Rockefeller, Jr., retired as president of its board of trustees in 1950, he stated that he had always regarded the institute as the most significant and the most permanent (philanthropy) of any that my father established. 4 Pneumonia In 1913, three years after its opening, Oswald Avery took the job of bacteriologist at the Hospital of the Rockefeller Institute, where a considerable effort was directed to the treatment of pneumonia and its most common cause, the pneumococcus. Avery became part of this effort. His entire subsequent career was driven by a search for an understanding of pneumococcal pneumonia and for its cure. At that time, pneumonia was the leading cause of death in the United States. Here is how Osler described it: Definition. An infectious disease characterized by inflammation of the lungs, toxaemia of varying intensity, and a fever that terminates abruptly by crisis.... Incidence. The most widespread and fatal of all acute illnesses, pneumonia is now the Captain of the Men of Death. 3p108 Lobar pneumonia was not limited to the infirm or elderly; people in the prime of life were affected. Entire lobes were consolidated, often more than one, resulting in little gas exchange. Most medical care was provided in the home. X-rays and blood counts were rare. The mortality rate ran between twenty and forty percent. Osler s section on treatment begins Pneumonia is a self-limited disease, which can neither be aborted or cut short by any known means at our command. 3p134 By the fourth day or so after the onset of symptoms, a hospitalized patient would typically be highly febrile, tachypneic, dyspneic, tachycardic, cyanotic. He was frequently delirious. Bacteremia occurred in about one-third of the patients. If infection of the meninges or of a heart valve ensued, it was invariably fatal. Patients, family members, and physicians would wait anxiously for the crisis, the characteristic feature of lobar pneumonia, which would not occur until a week to ten days after the onset of illness, if the patient survived that long. At that point the temperature, heart rate, and respiratory rate rapidly fell, and the patient recovered. Polysaccharides and type-specific serum therapy Avery started his career at the Rockefeller working on serological classification of the various pneumococcal types. This led to the finding that a soluble substance, specific for each pneumococcal type, was present in the serum and urine of patients. Avery and his collaborators identified these as polysaccharides and showed that they were antigenic, which had not been suspected previously. Most importantly, the polysaccharide made up the pneumococcal capsule, which was different for each pneumococcal type. It is the capsule that renders the organism resistant to phagocytosis. For the pneumococcus to be virulent, it must form a substantial capsule, while unencapsulated organisms are not virulent. The crisis is the result of the appearance, after about a week, of antibodies to that capsular polysaccharide, which opsonized the bacteria, leading to phagocytosis and clinical recovery. Type-specific serum therapy, made possible largely through the fundamental discoveries in Avery s lab, at that time consisted of administering horse serum prepared against the specific type of pneumococcus with which the patient was infected. 5 Some of the mechanics of its administration are illustrated in this tribute to Max Finland, who ran the pneumonia service at the Boston City Hospital for many years: you went to the hospital laboratory to get the [pneumococcal] isolate; if the house officer was not at the City Hospital, he got on the trolley and traveled to the Thorndike Memorial Laboratory, where Dr. Finland was always available; he would type the organism and hand you a bottle of type-specific serum; then, it was back on the trolley to your hospital; finally, you administered the serum to the patient. Whereas the mortality rate for untreated pneumococcal bacteremia was almost 90%, type-specific serum therapy resulted in survival of more than one-half of the patients. 6 This was the only way to treat pneumonia until the introduction of antimicrobial drugs in the late 1930s. C-reactive protein and the acute phase response Throughout his career, Avery s approach to biological elucidation of the pneumococcus involved understanding its immunochemistry. Pursuing this line of study, William Tillett, working in Avery s laboratory, prepared a polysaccharide fraction derived not from the capsule but from the cell wall. He called it the C fraction because it appeared to be analogous to the C polysaccharide of the hemolytic streptococcus studied by Rebecca Lancefield a few years previously. This 16 The Pharos/Spring 2011

$This chart compares the temperature course of pneumonia in a patient (top row) with anti-capsular antibodies (second row) and C-fraction precipitation (third row).$

4 Chart 3 adapted from Tillett and Francis, reference 7. This chart compares the temperature course of pneumonia in a patient (top row) with anti-capsular antibodies (second row) and C-fraction precipitation (third row). pneumococcal C-polysaccharide, it was later learned, was shared by all pneumococci, regardless of their type. To explore the serologic response to the C-polysaccharide, Tillett and his colleague Thomas Francis set up precipitin tests against sera from serial bleedings of pneumonia patients. The results were surprising completely the opposite of what they had expected. At the top of the figure is the temperature curve, starting at 104 F, with the patient undergoing a crisis on day six or seven. The second row shows the antibodies to the capsule, here called type specific agglutinins, which appear about the same time as the crisis and are responsible for it. When Tillett and Francis looked at the reaction with the C-polysaccharide, however, they got a surprise. A precipitate formed when the C substance was mixed with sera obtained at the time of admission and throughout the febrile period (the acute phase). This reaction diminished after the crisis and ultimately disappeared. This time course was the reciprocal of that seen with antibodies directed against the capsular polysaccharide, and was not how antibodies behaved. It took the investigators a while to digest this finding and for its implications to sink in. Ultimately they realized that the C-precipitation phenomenon differed from an immune reaction, and that it was something new, different from an antibody response. This phenomenon was not limited to pneumococcal pneumonia, nor to acute infections. For example, in a patient with bacterial endocarditis, who died on hospital day twenty-one, the C-precipitin never went away. They also observed precipitin reactions with sera from patients with acute rheumatic fever, lung abscess, and osteomyelitis, all believed to be caused by Gram-positive organisms. No reaction was observed with sera from normal individuals. These results were published in Avery was fascinated by these findings. He saw this C-reaction as an entrée to study of the host response to infection. Avery was a captivating raconteur, known for his inimitable monologues describing the various paths of research in his laboratory. In his biography of Avery, René Dubos recounts: Avery never discussed the C-reactive protein without turning the conversation to what he was wont to call the chemistry of the host. Although he never spelled out what he meant by that expression, he clearly had in mind all the unidentified body substances and mechanisms of a nonimmunological nature, both protective and destructive, that come into play in the course of infectious processes. 1p99 The Pharos/Spring

5 Oswald Avery and the pneumococcus The discovery of C-reactive protein and the acute phase response in the Avery lab opened the door to contemporary scientific pursuit of this phenomenon the metabolic, physiologic, nutritional and plasma protein changes that occur during inflammatory states 8 as well as to the clinical use of acute phase protein determination for diagnosis and management of patients. New insights into the biological and clinical significance of the acute phase response continue to emerge. 9 The transforming principle In 1928, Fred Griffith in London observed that heat-killed encapsulated Type I pneumococci (the donor) could transform living unencapsulated (hence non-virulent) Type II pneumococci (the recipient) into encapsulated virulent type I organisms. 10 The capsule of the organism that grew out consisted of the carbohydrate that had been expressed by the killed, donor organism. This characteristic persisted in all succeeding generations of daughter cells that is, it was heritable. At that time, the chemical basis for heredity was still unknown, and the vast majority of informed opinion considered it virtually certain that genes were composed of protein of some kind. Over the years Avery s interests increasingly shifted toward understanding basic biological questions relating to the pneumococcus, since they might bear on treatment. Over the next fifteen years, the Avery lab focused its attention on the nature of the transforming principle. 11 Since only encapsulated organisms could cause disease, Avery felt that an understanding of how capsule production is controlled might lead to treatment. This avenue of investigation ultimately led to one of the most important biomedical papers ever published. Avery, Colin MacLeod, and Maclyn McCarty reported in 1944 that hereditary information could be transmitted from one bacterium to another by purified DNA. 12 The report was greeted with considerable skepticism, and it took a few years before its validity was accepted. A feverish race to solve the structure of DNA then ensued, a race won by James Watson and Francis Crick nine years later, and described by Watson in his famous memoir The Double Helix. As we all know, the finding that genes are made of DNA revolutionized biology. Of note, in their paper in Nature describing the structure of DNA, Watson and Crick did not credit the Avery lab for their contribution. A long and winding road Several publications have recently indicated the public s impatience with the failure of recent basic science discoveries to lead to practical results something that helps patients. 13,14 However, as has recently been pointed out, The road from basic knowledge to successful technology is a long and winding one, usually taking decades, not weeks or months. 15 It is worthwhile to point out that the extremely consequential work of Avery s lab was not carried out under the aegis of translational research. Rather, it was carried out so that the biology of the pneumococcus could be better understood. The three major discoveries of the Avery laboratory have all led to major advances in diagnosis, disease prevention, and treatment, and the circumstances should remind us that accretion of sound basic biologic knowledge can be expected to ultimately lead to practical results. Without such knowledge, practical results will never ensue. References 1. Dubos RJ. The Professor, the Institute, and DNA. New York: Rockefeller University Press; McCarty M. Historical perspective on C-reactive protein. Ann NY Acad Sci 1982; 389: Osler W. The Principles and Practice of Medicine: Designed for the Use of Practitioners and Students of Medicine. New York: D. Appleton; The Rockefeller Archive Center JDR Jr. Biographical Sketch. rockarch.org/bio/jdrjr.php. 5. Podolsky SH. Pneumonia Before Antibiotics: Therapeutic Evolution and Evaluation in Twentieth-Century America. Baltimore (MD): Johns Hopkins University Press; Klein JO. Maxwell Finland: A Remembrance. Clin Infect Dis 2002; 34: Tillett WS, Francis T Jr. Serological reactions in pneumonia with a non-protein somatic fraction of pneumococcus. J Exp Med 1930; 52: Kushner I. The phenomenon of the acute phase response. Ann NY Acad Sci 1982; 389: Kushner I, Samols D, Magrey M. A unifying biologic explanation for high-sensitivity C-reactive protein and low-grade inflammation. Arthritis Care Res 2010; 62: Griffith F. The significance of pneumococcal types. J Hyg (Lond.) 1928; 27: McCarty M. The Transforming Principle: Discovering that Genes Are Made of DNA. New York: W. W. Norton; Avery OT, MacLeod CM, McCarty M. Studies on the chemical nature of the substance inducing transformation of pneumococcal types: Induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III. J Exp Med 1944; 79: Carmichael M, Begley S. Desperately seeking cures. Newsweek 2010 May 24 & 31: Wade N. A decade later, genetic map yields few new cures. NY Times 2010 Jun 13: A Krauss LM. The Gulf spill and the limits of science. Wall Street J 2010 Jun 11. Address correspondence to: Irving Kushner, MD MetroHealth Medical Center 2500 MetroHealth Drive Cleveland, Ohio ixk2@case.edu 18 The Pharos/Spring 2011

SACCHARIDES IN THE CONTROL OF SERUM DOSAGE

THE USE OF THE SKIN TEST WITH THE TYPE SPECIFIC POLY- SACCHARIDES IN THE CONTROL OF SERUM DOSAGE IN PNEUMOCOCCAL PNEUMONIA By COLIN M. MAcLEOD, CHARLES L. HOAGLAND, AND PAUL B. BEESON (From the Hospital