Herman Friedman is Professor and Chair of the Department of Medical Microbiology and Immunology, University of South Florida College of Medicine, Tampa. He was the recipient of the 1999 Abbott Laboratories Award in Clinical and Diagnostic Immunology; this article is adapted from the address he gave at the 1999 General Meeting upon receiving that award.

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From the Clinical to the Research Laboratory: a Reminiscence

This microbiologist encountered several problems in a clinical setting and pursued them as research challenges

Herman Friedman

My career as a medical microbiologist and immunologist began in the context of a diagnostic microbiology laboratory, where I served as a director for 20 years, and then changed contexts dramatically when I became a professor and chairman of an academic department of microbiology in a medical school, where I have spent the past two decades. This career path could be a model for others who are embarking in microbiology and immunology.

The journey along this path began more than 42 years ago when I received my Ph.D. from Hahnemann University School of Medicine in Philadelphia in the department then chaired by Amedeo Bondi, a clinical microbiologist. Bondi, early in his career while working with Earl Spaulding, developed the antibiotic paper disk test to replace what was then a cumbersome antibiotic susceptibility test using the Oxford cup technique. Bondi and Spaulding used small disks, made from filter paper with a paper punch, to detect on agar plates whether a bacterium was susceptible or resistant to an antibiotic.

The Hahnemann department was one of the few in the country that had the clinical microbiology laboratory in a basic science department. Along with all the other graduate students, I was required to rotate through the clinical microbiology laboratory to become proficient in diagnostic microbiology.

Early Experiences in Clinical Microbiology Laboratories

After I completed my Ph.D., I moved to my first professional position in an allergy research laboratory at the University of Pittsburgh VA Hospital, but stayed there less than one year. Next, I became the director of the clinical microbiology laboratory at Albert Einstein Medical Center in Philadelphia, with a joint appointment as a faculty member in the academic department of microbiology at Temple University School of Medicine, chaired by Earl Spaulding. Spaulding's department at Temple, like Bondi's at Hahnemann, included the clinical microbiology service. Thus all graduate students and faculty in that department had some affiliation with the diagnostic laboratory.

When I joined the hospital staff at Einstein Medical Center in 1959, the diagnostic microbiology and serology laboratory was a division of the department of pathology and clinical laboratories. We had five technical positions. By the time I left 20 years later for Florida, my diagnostic laboratory had grown to over 25 technical positions, including a serology and virology laboratory with three to five technicians.

When I joined the Einstein Medical Center, the one technician then working in serology used a request form that simply stated ``serology.'' In those days, syphilis serology was the only serology laboratory test available in that hospital, which was and remains one of the largest hospitals in Pennsylvania. Within a year we expanded the diagnostic testing to include serologic tests for autoimmune factors, including anti-nucleic acid tests for lupus and serology for virus infections. By the time I left that institution 20 years later, we were performing cytolytic tests for transplantation antigens, leukocyte culture tests, and mitogenic tests for immunodeficiency.

I believe an interaction between the clinical laboratory and a research laboratory such as the one we established at Einstein Medical Center, supported by grants from the National Institutes of Health and elsewhere, can be highly valuable. For one thing, it permitted us to train a relatively large number of graduate students and postdoctoral fellows. Moreover, it allowed us to interact with microbiologists and immunologists doing basic research at Temple University, elsewhere in Philadelphia, throughout the country, and indeed worldwide.

Early Series of Studies Point to Virus-Induced Immune Suppression

Although the principal duties of the diagnostic laboratory that I headed were to identify microbes that were causing clinical infections and to test bacteria for susceptibility to antibiotics, many of my laboratory studies in the 1960s and 1970s dealt with how pathogenic microorganisms avoid the host immune response. To address such issues, we studied animal models as well as human peripheral blood lymphocytes obtained from patients at the hospital.

One of the basic studies we undertook was to determine how a commonly investigated leukemia virus suppresses the immune response of the mice it infects. We considered this a good model for studying the broader problem of how tumor cells avoid host immunity. It was already known that patients with leukemia are markedly immunodeficient and often became infected with what we now call opportunistic pathogens. We began to study in the mid-1960s immunomodulation induced by a common murine leukemia virus, namely the Friend leukemia virus. When mice are infected with this virus, not only do they eventually develop leukemia within a few weeks, but within 2-3 days they show a marked suppression of their antibody response to a variety of antigens and become highly susceptible to infection, including to ordinarily poorly infectious bacteria such as Escherichia coli, Pseudomonas spp., and even Salmonella spp.

These studies were published in a series of papers in the Journal of Immunology and the ASM journal Infection and Immunity. Later, others in the field told us that if we had called this series of papers ``acquired immunodeficiency'' instead of ``virus-induced immunosuppression,'' we would have been the first to report that a leukemia virus, which turned out to be a retrovirus, caused an AIDS-like disease and could have named the effect ``murine AIDS.'' In that way, we were at least 10-15 years ahead of our time.

We began this work only because many clinicians were sending us blood specimens from patients who had leukemias or other blood cell tumors. Moreover, these patients were immunosuppressed and this condition appeared to be related to their infections with opportunistic microorganisms. Because other experimental work, particularly with mice, indicated that viruses could induce leukemia and lymphoproliferative diseases, we thought that studying such viruses could elucidate the nature and mechanism of immunosuppression in leukemia among some of the patients in our medical center. Thus, work in a research laboratory, using animal models, correlated well with one of the obvious questions then being raised in the clinical diagnostic laboratory. In other words, clinical work led to projects for research studies. Indeed, much of my research addressed questions that arose from my clinical diagnostic work.

A Pneumonia Outbreak in Philadelphia Leads to Identification of a New Pathogen

Let us jump ahead approximately 10 years to 1976. Those who know Philadelphia, where I was born and grew up, should realize that many consider this city the birthplace of the United States. In 1976 the city was planning the bicentennial celebration of this country, with a major celebration scheduled for July 4th. Unfortunately, a few weeks before this scheduled event when more than 3 million visitors were expected to converge on Philadelphia, an unwelcome visitor appeared in the city.

This noteworthy event is indelibly a part of the convention of Pennsylvania Legionnaires who had gathered at the center city Bellevue Stratford Hotel. The convention quickly turned into a medical disaster for Philadelphia as well as for the United States. Approximately 3,000 Legionnaires were staying at the Bellevue Stratford Hotel, which was called the ``Grand Old Dame of Philadelphia.'' Built at the end of the 19th century and a mere three blocks from City Hall on Broad Street, it had the reputation of being the best in Philadelphia. Indeed, the first national ASM Meeting I attended was held there in 1961. As a microbiologist in Philadelphia, I was part of the organizing committee. How well I remember the beautiful crystal chandeliers that decorated the ballroom on the top floor of the hotel where the main ASM sessions were held.

Sometime following that ASM meeting, however, air conditioning equipment was installed on the roof of the hotel, and new air ducts were placed along the ceiling of the ballroom, where the Legionnaires' convention was also held. Within the first few days after that 1976 meeting, a serious illness began affecting many of the conventioneers. By the time the outbreak was over, more than 200 of them developed a serious pneumonia, and about 20 died.

Investigators from the federal Centers for Disease Control and Prevention (CDC) in Atlanta came to Philadelphia and linked all these cases to attendance at the convention. This and other information threw Philadelphians into a panic, particularly because no one could say what was causing the deadly outbreak. Meanwhile, some members of a special government task force, as well as the President of the United States, claimed that the outbreak was caused by swine flu. Such an outbreak had been predicted that year, following the tentative identification of four cases in nearby New Jersey. Other government officials and newscasters speculated that this disease was caused by poison gas or was the result of a biological warfare attack by Soviet agents against Legionnaires. Still others speculated that the outbreak stemmed from an unknown microorganism or a new toxin. Needless to say, instead of 3 million visitors, fewer than 100,000 showed up for the bicentennial celebrations in Philadelphia by the end of June 1976, making it more a ghost town than a prime site for celebrating the nation's birth.

As a clinical microbiologist in a hospital on Broad Street several miles from downtown Philadelphia, my laboratory was also experiencing an increase in atypical pneumonias among patients being hospitalized. Moreover, we could not isolate any specific bacterium or virus from these patients. Nevertheless, the epidemiologists and other public health officials categorically denied that this increase in pneumonias had anything to do with the disease at the Bellevue Stratford, since the outbreak there was exclusively a ``Legionnaires''' disease. For instance, CDC officials said that pneumomias among individuals who did not come in contact with the guests at the Bellevue Stratford were simply not Legionnaires' disease.

For many weeks, the cause of this disease remained unknown, even though CDC sent an army of investigators to find out what was happening. However, a few months later during a quiet moment between Thanksgiving and Christmas, Joseph McDade at CDC reexamined slides of tissues from some of the Philadelphia patients and thought he saw some bacteria in them. Because he was director of a rickettsial laboratory, he injected autopsy material into eggs, then a standard procedure for isolating rickettsiae. Sure enough, something grew out. When he injected this cultured material into guinea pigs, most of the animals developed pneumonia and many of them died.

To isolate a bacterium, McDade used a special medium containing charcoal yeast extract, and found a microorganism that appeared new to him. Genetic isotyping, which was then under development, showed that the isolate's genome was different from any other known microbe. It came to be called Legionella pneumophila, with ``Legionella'' referring to the Legionnaires in whom the disease had first been identified and ``pneumophila'' alluding either to Philadelphia or meaning ``lung loving.''

Antiserum to this bacterium was quickly developed and used in an immunofluorescent test to identify the organism in blood and sputum of many patients who subsequently came down with the disease. The same bacterium was soon proved responsible for causing other epidemics of lung or systemic disease during the previous 20 to 30 years. We now know this organism is a ubiquitous, intracellular bacterium that preferentially grows in phagocytic cells. The legionellae usually infect protozoa or paramecia initially, and then incidentally move from their ordinary hosts and environmental habitats to infect humans.

The microorganism grows well in warm water, including the water used in air conditioner cooling towers such as those that were installed on the roof of the Bellevue Stratford Hotel. Epidemiologists from CDC performed an outstanding service in their follow-up analysis. They found that, of the approximately 3,000 Legionnaires who attended the convention, those most likely to develop clinical infection and pneumonia sat closest to an air conditioner vent, were about 10 years older than the average conventioneer, smoked more, drank more, and partied more--in short, those who stressed their immune response system more were more likely to succumb to infection.

Legionella Follow -Up Leads to Broader Research Interests

We now know that Legionella causes infection mainly in individuals who have a defect in their immune response either because of aging or environmental factors. Once the organism was isolated and became available for study, it turned out one could assess the immune response mechanism that confers resistance or immunity to the bacteria in animals such as rodents. The same mechanism appears to be at work in humans.

We found that the bacterium infects macrophages preferentially and that such cells from genetically susceptible A/J mice--but not from resistant mice--are highly permissive for these bacteria. Furthermore, lymphocytes, especially helper T cells, secrete cytokines when stimulated by Legionella. For instance, Th1 helper lymphocytes secrete cytokines such as interferon and interleukin-2 (IL-2) that can activate macrophages to evince resistance to infection with Legionella, even if macrophages are genetically susceptible. Proinflammatory monokines such as TNF-a and IL-1 are important in stimulating resistance of macrophages in vitro as well as in vivo when infected with Legionella.

These animal experiments not only helped to delineate the mechanisms of susceptibility to Legionella at both the genetic and molecular levels, they also provide insights as to why individuals may be either susceptibile or resistant to this ubiquitous bacterium and shed light on important immune system-related risk factors for infection. For instance, a defect in the immune system of an individual, either because of aging or from environmental factors, increases one's susceptibility to this organism as well as to other opportunistic pathogens.

These studies of the interactions between the immune system and a specific opportunistic pathogen led me into another area of research--namely, the impact of drugs of abuse on infectious diseases. In 1982, after I had been at the University of South Florida for only a few years, I received a call from a program director of an institute in the national public health system with which I was then unfamiliar--ADAMHA, the Administration for Drug Abuse, Mental Health and Alcoholism. A few years later the part of ADAMHA that administered research programs investigating drugs of abuse merged with NIH and became the National Institute on Drug Abuse. Other parts were incorporated into the National Institute of Mental Health and the National Institute of Alcoholism and Alcohol Abuse.

Drugs of Abuse Suppress Immune System Responses to Pathogens

Long before those administrative changes came about, I was invited to undertake studies concerning the effects of drug abuse on immune responses to microorganisms. At the time, Congress mandated ADAMHA to investigate whether drugs of abuse affect the immune response. That interest arose mainly because of keen interest in the emerging AIDS epidemic and widespread recognition that a high proportion of AIDS patients were drug abusers.

Thus, we soon began experiments to study the effects of marijuana, especially its major active psychoactive component tetrahydrocannabinol (THC), on the immune response. We also asked whether THC affects the immune response to microorganisms such as Legionella. It was thought that other drugs of abuse, especially alcohol, may predispose an individual to infection with other opportunistic intracellular bacteria such as mycobacteria.

We soon determined that THC has profound suppressive effects on the immune response, affecting cellular immunity as well as antibody formation. THC suppressed B and T cell functions as well as cytokine production and macrophage activities. During the past decade or so, it became evident that there are specific receptors for marijuana not only on brain cells, since this is a psychoactive substance, but also on lymphocytes. Meanwhile, researchers learned that, in addition to alcohol and marijuana, other common drugs of abuse, especially morphine, enhance susceptibility to infection in humans as well as in animals being studied in laboratories.

My colleagues and I have focused on finding precisely how marijuana suppresses immunity. To do so, we used the L. pneumophila model of infection, which we studied ever since the outbreak of legionellosis in Philadelphia, to test the effects of THC on different lymphoid cell populations. The psychoactive effect of marijuana smoke is due primarily to the cannabinoid THC. In addition, this cannabinoid affects immunity, both cellular and humoral, thereby increasing susceptibility to common opportunistic pathogens, such as Legionella.

The adverse effects of marijuana cannabinoids and other drugs of abuse on immunity to infectious agents are of particular concern, especially since marijuana is widely used recreationally as well as medicinally. THC and various analogs produce a range of effects by binding to cannabinoid receptors in the brain and periphery. Findings from various laboratories show that cannabinoid receptor CB1 is expressed mainly in the brain while the cannabinoid receptor CB2 is expressed mainly on lymphoid and nonbrain cells. Besides binding these plant-derived drugs, the receptors also bind endogenous ligands that derive from arachidonic acid, a fatty acid building block. Thus, the body contains a complete endogenous cannabinoid system of receptors and ligands.

We investigated how THC compromises host resistance to infection with opportunistic bacterial agents, including Legionella in human cells and in mice, where the infection displays many of the cellular and cytokine features that are involved in immunity to other intracellular bacteria. For instance, such infections activate T helper cells, especially Th1 cells.

In one series of experiments, we injected THC into mice prior to a sublethal Legionella infection, which ordinarily induces specific immunity. However, the THC treatment increases the susceptibility of the animals to a subsequent sublethal challenge infection and, in addition, suppresses development of Th1-related activities, including production of interferon g and of Ig2a antibodies. This suppression of Th1 helper cell by THC is not due merely to an increase in production of immunomodulating IL-4, which is associated mainly with Th2-related activity, such as suppression of production of IL-12 as well as the IL-12 receptor. Instead, a cannabinoid receptor antagonist attenuates the drug effect on cytokine production, suggesting that a cannabinoid receptor subtype is involved in modulating Th1 cell activity during a Legionella infection.

Reflections on Combining Clinical and Research Laboratory Experiences

Thus, throughout my career, my clinical laboratory background enabled me to undertake both basic and applied studies on important questions, such as how drugs of abuse can affect health and alter the susceptibility of an individual to infectious agents, including Legionella. Of course, important questions for study through basic research arise from problems encountered in the clinical setting. Many such questions can be addressed through basic science and, in turn, basic research can benefit immensely from clinical studies.

As we have come to appreciate, there is little fundamental separation between basic and clinical research. The differences are more subtle, and reflect more how one approaches some of the questions being raised. For instance, those of us whose training came in the clinical diagnostic laboratory tend to define important immunological parameters which influence disease, including infectious diseases. Both research and clinical diagnostic laboratory studies sometimes use the same techniques to provide answers to the similar basic questions, and the findings are often quite practical. In fact, many of those practicing diagnostic microbiology and immunology already know there is very little difference separating basic from applied studies, since both types of studies require hard work, honest interpretation, and a willingness to face important medical challenges.