by Lewis Thomas
In this circumstance, anyone can become overwhelmed by his own true belief, and I confess here to mine. I am persuaded by the connection, thin as it is, to mycoplasma infection in animals, and by the gold story. I cannot count the hours that I have wasted in my own laboratory, during the last twenty years, concocting one baroque broth after another, trying to grow mycoplasmas from arthritic joint fluid, always with negative results. I have been obsessed with the possibility, unable to give it up. In any other circumstance, I suppose my behavior might be classed as paranoid. My laboratory notebooks contain an intermittent but endless harangue on this one topic; in between more or less respectable experiments on endotoxin, streptococci, papain, whatever, this business of mycoplasmas and arthritis keeps popping up, like King Charles’s head. I cannot leave it alone.
I have shifted my ground somewhat in the last several years. Very well, perhaps it is wrong to believe so ardently in mycoplasmas, but what about bacterial L-forms? These are quite different microorganisms in their genetic origin, but morphologically indistinguishable from mycoplasmas. They can be made in the laboratory by simply stripping off the walls of living bacteria and then growing the denuded fragile creatures in special media designed to prevent them from bursting. The outcome, once they are adapted to growing in cultures, are colonies that cannot be told apart from classical mycoplasmas.
I began working on wall-less bacteria ten years ago in my laboratory at Yale, while I was professor of pathology and dean, and brought them along to Sloan-Kettering. I have never worked on a problem with so many attractive, unpublishable diversions. L-forms are, for my taste, enormously interesting beings, leading the investigator down one garden path after another. Here is just one example:
A good way to make L-forms from normal streptococci is to grow the bacteria on agar plates containing a gradient of penicillin. Penicillin acts by preventing the bacteria from synthesizing the constituents of their walls, and when the concentration of penicillin is exactly right, you can produce wall-less organisms that are not killed off but abruptly change their mode of colony formation, and their growth requirements, to those characteristic of mycoplasmas. They are called L-forms because they were first described at the Lister Institute, in London. After cultivating such organisms in penicillin for a while, one can wean them away from the antibiotic, and then they will live forever in their wall-less form.
A blind garden path opened in my laboratory one winter day when I had occasion, for other reasons, to administer penicillin to some guinea pigs, who then died, to my surprise, within three days. I then learned something already known to some of my colleagues in infectious disease: penicillin is lethal for guinea pigs. Indeed, if Florey and Fleming had been using guinea pigs for their initial experiments with penicillin, we might never have entered the antibiotic era. The lethal action of penicillin in this single animal species has never been satisfactorily explained.
It occurred to me that there might be a connection between this and the capacity of penicillin to convert normal bacteria to L-forms. Guinea pigs are known to harbor their own species of streptococci and pneumococci, usually as latent infections in their lymph nodes, but sometimes flaring up in epidemics to produce overwhelming lethal infections. Perhaps penicillin might be setting up a gradient of antibiotic in these animals, with colonies of L-forms possessing some new toxic property as the result. It seemed a good enough idea to pursue, anyway, and I ordered several dozen guinea pigs and began drawing up protocols for the experiment. By the time I got around to it, because of the usual delays in academic purchasing offices, it was the middle of March.
We injected thirty guinea pigs with the properly lethal doses of penicillin and made elaborate preparations for culturing the blood and tissues of these animals at the moment of death. On the third day all was ready, but none of the guinea pigs was dead, not even perceptibly ill. More penicillin was injected, in higher and higher doses, and the animals thrived on it.
We must have gotten the wrong strain of guinea pigs, we decided. Others were ordered in, from other breeders in Connecticut and New York, and we set things up all over again. By now it was late April. We launched the experiment, a much bigger and more orderly one, and therefore, as it turned out, a much more impressive dud. Not a guinea pig died, or even sickened.
We went back to the literature and found a few remarks, here and there, about variability of the phenomenon. One laboratory, in the Netherlands, reported that its colony of guinea pigs had become resistant to penicillin, and their work had therefore been terminated. But most of the papers described 90–100 percent deaths from penicillin, even in very small doses.
We dropped the matter then, and became busy with other affairs through the following summer and early fall. Then, for reasons I’ve forgotten, we decided to try it again, and lined up a dozen guinea pigs for penicillin. It was now early in November. This time, all the animals were dead in three days. We went ahead with more guinea pigs, batch after batch, all vulnerable to penicillin, and for the next five months the laboratory was awash in cultures designed for the isolation of L-forms; although our results were still negative, we were optimistic and enthusiastic about the predicted outcome. And then, late in March, everything ground to a standstill. Pencillin was no longer lethal. From that time on, every two weeks we established as a laboratory routine the injection of six guinea pigs with penicillin. No deaths occurred through the summer or early autumn; then deaths began again in November and continued through March, then stopped again.
We did this for three years running, never having a long enough period of penicillin deaths to finish our planned experiments, and never able to figure out a reason for the regular seasonal occurrence, never even able to write a sufficiently illuminating account of these events for publication. The observations are still there in our notebooks, stuck, unexplained, waiting for next winter.
Throughout all this time, in and out of the guinea pig experiments, my colleague Dorothy McGregor and I have continued trying to isolate L-forms from specimens of joint fluid and biopsies of synovial tissue from patients with rheumatoid arthritis, thoughtfully supplied by surgical colleagues at various hospitals in New York and New Haven. We are still at it, ten years after first having the notion that it was a good notion. Not once have we cultured anything resembling an L-form colony, but we have seen some things in the centrifuged pellets of joint fluid that had been incubated for twenty-four hours or so that look rather like L-forms, and we have grown bacteria called corynebacteria from most of the fluids and biopsies. These bacteria all look alike and behave alike, and they are not recoverable from joint fluid or synovial tissues from patients with other kinds of arthritis. It may turn out that the idea is right, after all. Perhaps there are L-forms in there, behaving like mycoplasmas, and perhaps they are the L-forms of corynebacteria, and perhaps they have something to do with the etiology of rheumatoid arthritis. I hope so, for it has been a long time on one problem.
* * *
• • •
In 1971 I was invited by the University of California at Davis to come for a two-week visit as Regents Professor. It was a wonderfully flattering invitation, involving such congenial and undemanding duties as a few lectures and several informal seminars, plus an opportunity to learn a lot at first hand about whatever research projects I happened to be interested in, and I accepted quickly. I would have accepted in any case, but the great attraction was the faculty issuing the invitation and the part of the university to which I would be assigned. It was the Veterinary School.
The different campuses of the University of California have each developed one or another particularly strong area, and Davis has for decades been distinguished for having one of the country’s best schools of veterinary medicine, rivaled only by Cornell, Iowa, and Pennsylvania. I had first become fascinated by this field because of the influence of Richard Shope. Shope became a good friend during our months in the same tent on Okinawa, and he used to tell me a
t length, during dark evenings, about the accomplishments of veterinary science and his deep respect for veterinarians. He was of course trained initially as a physician, but most of his experimental work had involved animal diseases (rabbit papilloma and swine influenza preoccupied much of his life), and he had many close friends and collaborators among animal doctors. He had an impressive list of honorary degrees, and the one he prized the most was a D.V.M. (hon.) from the University of Utrecht.
I was assigned an office with access to a small laboratory in the Davis Veterinary Sciences Center and allowed to roam. The undergraduates were a considerable surprise. As a teacher-physician I had thought of medical students as the very top of the line, and I was not prepared for young men and women as excellent as those Davis veterinary students. It saddens me to say so, but their intellectual quality and verve, their curiosity and skepticism, most of all the sheer fun they were having as students, made them a more interesting lot than I had been used to in medical school.
Watching the students and faculty on their rounds was another small shock. The animal clients were not, as I had rather expected, treated as interesting objects or technological problems to be solved. We rounded through the barnyard wards of sick cattle and horses, pens of ailing hogs, sheds containing scores of pet dogs and cats, cages of birds, even two locked wards for monkeys and chimpanzees, and all of these animals were known and recognized as individuals by the scholars and their professors. Moreover, they were handled with as high a level of affection and regard as I could wish for if I were bedded down in any New York City hospital.
Later on, in seminars with the students, I found that the competition for admission to the veterinary school at Davis was just as intense as for the medical school, maybe more so, but the reasons for competing were not the same. Individual students had different careers in mind—some were hoping to specialize in large farm animals, others in horse-breeding establishments, others in city-bound pet animals, a few were looking forward to research opportunities in the federal Department of Agriculture or faculty posts in veterinary colleges—but none of them seemed to have in mind high income or social status. They were there, having the best of times, because they liked animals.
One reason I had been invited was that I had been working with mycoplasmas, which are of special concern, scientifically and economically, within the veterinary world because of the formidable epidemics of lethal disease they produce: pleuropneumonia in cattle; arthritis in pigs, goats, and sheep; pneumonia and encephalitis in chickens, turkeys, and other birds; a long list of others. It is an odd anomaly that organisms with so wide a host range among animals, even extending to those Miami palm trees, seem to have, relatively anyway, so little interest in humans. One important lung disease, once known as virus pneumonia or primary atypical pneumonia, on which I had worked at the Rockefeller Institute, was finally proved to be caused by a mycoplasma now called M. pneumoniae, and several types of mycoplasmas are now known to be implicated in genital infections (perhaps also involved in the causation of spontaneous abortion), but except for these ills, human beings are not known to be prone to mycoplasma infection. However, because some of the animal diseases do resemble, in the details of the pathologic lesions in joints and blood vessels, such human diseases as rheumatoid arthritis, lupus, inflammation of the arteries, and encephalitis, and because I happened to be one of the interested parties in the field, my invitation to Davis made some sense.
Professor Henry Adler and his avian disease group at Davis had been studying for some years a respiratory disease of poultry caused by a mycoplasma called M. gallisepticum. One variant of this organism, labeled the S-6 strain, was unique for its capacity to produce an unusual neurological disease in turkeys. When a suspension of S-6 mycoplasmas was injected into young turkey poults, the birds became comatose on the sixth or seventh day and then, within twelve hours, died. Death was caused by the selective destruction and occlusion of the arteries in the brain; the only other affected vessels in the body were those in the connective tissues around the joints. The arterial lesions were of special interest to me, for they looked very much like those in a human disease known as polyarteritis nodosa. I had begun work with this mycoplasma several years before, in the pathology department at Yale. Among other things, I had learned that the S-6 mycoplasma possesses a special and unique affinity for the arteries of the turkey’s brain; mycoplasma antigen can be demonstrated in these vessels within a day or two of infection, lodged in dense masses beneath the lining cells and often extending through all the layers of the arterial wall. The organisms carry a neurotoxin of some sort (still to be chemically identified), which produces signs of extensive brain damage within less than an hour when they are injected intravenously. To be active, the toxin has to reach the brain by way of its blood vessels; when the same dose or larger doses of mycoplasmas are injected directly into the brain tissue itself, there is no evidence of toxicity. The species specificity of the toxin is remarkable: it affects only turkeys, not chickens or pigeons or ducks, not rats or mice or hamsters, and only young turkeys at that.
I had been working for several years before this with another type of mycoplasma, affecting mice and rats, called M. neurolyticum. This agent, a particularly fragile organism, hard to grow in any culture medium, elaborates another sort of neurotoxin which affects mice and rats but no other laboratory animal or bird. The toxin itself is extremely delicate and difficult to work with; it can be stored frozen, but if left around at room temperature for a couple of hours it becomes totally inactive. The brain lesions caused by the toxin do not involve the arteries; instead, there are widespread cystic cavities within and among the neurones in the cerebral cortex, rather like the so-called spongiform lesions seen in certain types of brain disease in humans.
Then there is that business of gold salts as a treatment for rheumatoid arthritis. A mycoplasma now known as M. arthritides, which produces extensive, chronic arthritis in mice and rats, was encountered in infected mice by Albert Sabin in the late 1930s, while he was engaged in an unrelated problem involving the serial passage of mouse tissue suspension from animal to animal. At that time, it was known that gold salts were therapeutically useful in the treatment of humans suffering from rheumatoid arthritis, so Sabin, on a mild flyer, treated his mice with gold. The effects were dramatic: joint swellings went down and the mycoplasmas vanished. Since that time, thanks to Sabin, animal infections by all forms of mycoplasma have been shown to respond to gold. It is an interesting but not particularly useful finding, since the mycoplasma diseases can be treated more cheaply and safely with antibiotics. But highly useful for purposes of speculation. It is another piece of indirect evidence for the possibility that rheumatoid arthritis may be caused by a mycoplasma. Parenthetically, in this series of chance observations, it is worth noting that the beneficial effect in arthritis was first observed by Forestier, a French clinician who was seeking a treatment for tuberculosis in the 1920s and decided to try various metal salts, gold among them. None of the tuberculous patients was improved, but several of them had rheumatoid arthritis as well, and these were relieved of their joint disease by gold.
M. arthritides infections in mice and rats remain an interesting disease model, but not as close to human arthritis as another disease, which occurs spontaneously in pigs, is readily transmissible from pig to pig (but not to any other species), and is caused by a species of mycoplasma found only in pigs.
These observations were the immediate and practical reasons for my laboratory’s interest in mycoplasmas, but there was another reason, quite a different one, for my obsession with these organisms over so many years. To put it briefly, mycoplasmas are incredibly beautiful creatures. I first saw them through a microscope in the early 1960s, when someone sent me a culture of M. pneumoniae which I wanted to use in order to compare its antigens with those of a streptococcus that I had isolated from patients with primary atypical pneumonia (it was first recovered in the lung tissue of a patient name
d McGinnis and accordingly called the McGinnococcus, later formally designated as Streptococcus MG). As it turned out, there is some sharing of antigens between the mycoplasma and the streptococcus, but this is not what caught, and held, my eye. I learned what mycoplasma colonies look like when they are grown for a few days on clear agar.
The technique for staring at mycoplasmas is the simplest of things. You need an agar culture with colonies growing on it—the colonies are themselves so small that you must use a hand lens to be sure they are there. Then you cut away a block of the agar, place it upright on a glass slide, and place on top a thin glass coverslip which has been immersed beforehand in a lovely blue dye and then dried. The colonies pick up the stain within less than a minute and can thus be examined under the highest power of an ordinary light microscope.
The colonies are about 20 micrometers in diameter, about twice the size of a white blood cell. They have perfectly round centers, which stain a very deep-blue color, and around each center is a sort of halo, paler blue, shading off to a grayish-blue, vaguely outlined, circular perimeter.
That’s all. I cannot say why they are so lovely to look at, but they are. The central, dark staining core is a solid mass of mycoplasmas that have somehow tunneled deep into the agar; the halo is produced by a thin layer of organisms growing on the surface. How the mycoplasmas at the center manage to grow with such force down into the agar is not known, but it is the characteristic and identifying structural feature of this class of organisms, and perhaps it is this geometric configuration, and the sense of something energetic going on in these quiet, motionless structures, that make them so pretty to look at. The color as well. Under the oil-immersion lens, magnified 1,200 times, each organism can be seen as a bright-blue speck, nothing more, but when you look at the millions of such sharp blue specks massed all together in great clouds, it is like looking at life itself.