Chase, Chance, and Creativity

by James H Austin

  Henri Poincare

  His name was Tom. He was a handsome, intense, vigorous Brittany spaniel. As the name suggests, spaniels are a hunting breed originating in Spain. Almost 2,000 years ago, Sallust and Pliny described how their Roman compatriots brought pointing spaniels back from Spain and introduced them into France and Italy.' Down through the ages, pointing spaniels have always been dogs who show you where the game is.

  My longing for a dog of my own to accompany me in the field and forest dates back to early childhood. I waited until I was thirty-eight to own one. Were it not for my passion for running around in the out-ofdoors, we should never have acquired this dog. And without Tom, the following events would never have occurred.

  The sabbatical year on the dry plains of India was now over, and I was once again back among the green rolling hills of Oregon. We had meandered on from an interest in MLD into the field of other metachromatic molecules. These included hyaluronic acid (chapter 6) and other related sulfated mucopolysaccharides. These molecules are important constituents of the connective tissues which normally hold the body together. Now we needed techniques to identify, and then measure, these different sulfated molecules. The chemical journals described innumerable methods. (This invariably means that no one method is satisfactory.) Margo and I decided to devise some of our own methods. The thin-layer chromatographic methods we finally came up with could detect as little as five micrograms (five millionths of a gram) of sulfated polysaccharides. We originally used these methods to characterize the excess of sulfated polysaccharides in the two MLD children of the McLean family.' Would these same methods help us understand any other disease?

  I thought back to an illness called Lafora's disease. I had seen one patient with this disease during my training in New York. The disorder affects teenagers, causing epileptic seizures, abrupt muscular jerks (myoclonus), and a progressive deterioration of their mental abilities. Like MLD and globoid leukodystrophy, Lafora's myoclonus epilepsy is inherited as a recessive trait. In families with the disease it affects one out of four adolescents on an average. It is recognized microscopically by the presence of small round red-staining bodies inside the cytoplasm of nerve cells (figure 8). They look like tiny marbles and are called Lafora bodies after the Portuguese neuropathologist who first described them. I had seen them in books but never under the microscope.

  We started to study the disease on the wrong assumption. Our mistake was to think that Lafora bodies might contain acid mucopolysaccharides. We thought we could find these complex sugar molecules using our newer method, and were led into this false line of reasoning because existing histochemical techniques were not specific enough.

  Soon we were joined in this research by Susumu Yokoi. "Sum" was a neuropsychiatrist and the first of several outstanding Japanese coworkers who came to our laboratory. We had first met in Tokyo in 1962 and were drawn together by our mutual interest in leukodystrophies. I was delighted to find that he and his family wished to come to our laboratory.

  I was already partial to the Japanese. When I was stationed there in 1950-51, during the Korean War, l was struck by the beauty of the land, the ancient culture, the industry and friendliness of the people. I felt a deep kinship with their perception that the simplest elements of Nature-the drop of dew, the fallen blossom-were among the most sacred. I envied the way their gardens and homes created a tranquil atmosphere amid the hustle and bustle of their crowded islands. Traditional Japanese qualities of persistence and manual dexterity would soon also impress me in the laboratory.

  When Sum arrived, the obvious plan was to isolate Lafora bodies by the same general techniques we had used earlier to isolate globoid bodies. But Lafora's disease is rare. We had no autopsy material to start with. Then, I happened to encounter Dr. Richard Berry when we were both waiting for an elevator at a medical meeting. The elevator took longer than usual to arrive, and we struck up a conversation that continued for about ten minutes. I indicated that we had some new analytical methods to study Lafora's disease, and thereafter he obligingly sent us the material we needed. His kindness at this point was crucial.

  Figure 8

  The Lafora body and its chemical background. Under the microscope, the Lafora body is spherical and is found inside nerve cells. Chemically, it contains many sugar molecules linked together. In the chemical formula, each hexagonal-shaped link in the chain represents a molecule of glucose.

  We then went on to modify our previous isolation method, first used in globoid leukodystrophy. Sum centrifuged out the Lafora bodies in modest yield, extracted them in a way which should yield mucopolysaccharides, and applied the extract for chromatography. No spot appeared. There were iio acid mucopolysaccharides. We were up a blind alley. I was not only chagrined; I felt very stupid.

  Tom, the spaniel, now came to the rescue. Always a spirited animal, Tom frequently coursed far afield. His white and rich rust-brown colored coat usually showed up well at a distance, but one day we were both going into thick cover. In order to know where he was, I took the precaution of tying a small bell to the front of his collar. The bell is worth noting.

  A few days later I noticed a rapidly growing mass on the front of Tom's neck. The growth was hard, slightly tender, not obviously warm, and seemed to be malignant, both in my judgment and in that of Thomas Fletcher, a surgical colleague who was doing research on malignant tumors in dogs. Dr. Fletcher suggested that the mass be removed at once. Unfortunately, I had to leave town to present a paper at a medical meeting and would have to miss the operation. I had a very empty feeling during this trip away from home, because a friend and companion in the field soon becomes one of the family. The thought that this vigorously active dog would be slowed by a cancer moved me to despair.

  On my return, Tom bounded up to greet me in his usual frisky manner. The microscopic diagnosis, to everyone's surprise, was not that of a malignant tumor after all. Instead, the slides showed a subacute inflammatory response involving some of the lymph glands in Tom's neck. We were baffled as to what might be the cause of this inflammation. Tuberculosis? Yeast? Fungi? I asked the technicians to make more sections of the tissue and stain them especially for these organisms. A few days later, I heard that the new sections from the mass showed an unusual round "fungus." I hurried over to surgical pathology to look. Round structures were indeed there, and they were red. Curiously, they lay around the outside of the mass. None lay inside. All the infectious disease specialists duly called in for sober-minded consultation solemnly shook their heads, for they had never seen a fungus quite like this before. Finally, someone on the surgical service brightened with a happy thought. Perhaps these were not fungi, but round spherules of starch!

  Starc{h? Starch, we all now recalled, is used to dust surgical gloves. Some of it could have remained on the gloves during Tom's operation and could have been transferred to the outside of the mass when it was resected.

  When I checked out this possibility and looked at starch dust under the microscope, I finally realized that starch is made up of round spherules. Moreover, because starch is composed of many sugar (glucose) molecules, the starch in Tom's biopsy turned red when stained with a special histochemical stain for sugars. When these elementary facts entered my awareness, they completely transformed our approach to Lafora's disease. Could Lafora bodies in humans be some kind of starch?

  To answer this question, I set out with dogged (so to speak) determination to obtain many different kinds of starch. I then studied their size, shape, and histochemical staining characteristics. Soon I could prove that starch spherules and Lafora body spherules shared several things in common: strong red staining for carbohydrate, dense staining with iodine and silver. In addition, both structures sometimes had a Y-shaped crack in the center. Collectively, these findings suggested a new hypothesis: Lafora bodies were not an acid mucopolysaccharide; instead, they were quite a different kind of molecule-one made up of many sugar units linked together in a long chain to form a polymer. Based on this line of re
asoning, we abruptly shifted the focus of our research. We turned toward the requisite techniques of chromatography and infrared spectrophotometry. These permitted us to confirm our hypothesis. Within a few weeks we finally knew that Lafora bodies, like starch, were essentially a glucose polymer (figure 8).'

  By hindsight, it seemed reasonable to postulate that Tom's original inflammation came when the lymph glands in his neck were irritated by repeated contact with his new bell. Next came the operation, and finally, the starch from the surgeon's glove. Without doubt, the pace of our study was accelerated enormously by the chance placement and chance finding of starch in his biopsy specimen. To me, the fragile web of circumstances involved in this whole episode will always remain fantastic, yet one can perceive some of the sequences by looking at them longitudinally and analyzing them from some different perspectives (figure 9).

  The crucial point in the story is the intersection between the personal hobby (involving the dog) and the scientific problem (the Lafora body). The two lines meet by chance. A moment of closure occurred where the two joined. Thereafter, the solution almost suggested itself.

  If you've ever had an experience like this, you may also have emerged from it sobered and awed, and, one would hope, not left with a fatalistic attitude. Nothing in this story is intended to mean that you can blunder along to a fruitful conclusion, pushed there solely by external events and lacking any sense of your own mental or physical participation. No, the hard core of work involved in research is too much a reality to ignore. Rather, what seems to be superimposed on the conscious work are some personalized drives and sensibilities interacting with chance. Later, we will say more about the qualities of mind, temperament and instinct that underlie this kind of experience. For the moment, let me note that the episode with Tom the dog can he used as an example of the "barking up the right tree" phenomenon. It means that if you happen to be the kind of person who hunts afield, it may be, in fact, your dog who leads you up to the correct tree, and to a desirable conclusion.

  Figure 9

  Evolution of the research in Lafora's myoclonus epilepsy

  The story has unfolded still further. Once we had the new technical momentum developed during the Lafora body study, we could next plunge into an entirely new field-aging of the brain. For example, when we modified our earlier methods, we could then proceed to isolate and analyze other round structures such as amyloid bodies (corpora amylacea),' senile plaques and cores,' and finally Lewy bodies." Our hope is that when we know more about the chemistry of each of these structures, we may understand why some cerebral functions dwindle as the brain grows older. Now, we have become fully engaged in aging research. We are there not only because our own technical capabilities have carried us in, but also because a former graduate student, Donald Armstrong began studying the biological mechanisms of aging in relation to oxidative mechanisms in the retina.'

  11

  Finger Prints on the Window;

  Filling in the Hole

  Every child knows that prevention is not only better than cure, but also cheaper.

  Henry Sigerist

  Whether he is an archaeologist, chemist, or astronomer, at heart the researcher's goal is very much the same. Basically, he looks for facts that interest him and then tries to arrange them in meaningful sequence. The researcher in any field is an "adverbial man." He defines what happens, then figures out where, whets, bow, and why it happens. His adverbial search for cause and effect, for the basic order in things, is primal, compelling, and satisfying, quite apart from practical considerations.

  Who does research in medical schools? It depends on where you look. If you look in the laboratories of basic science departments (e.g., biochemistry), the vast majority, 73 percent, are Ph.D.'s, whereas 18 percent are M.D.'s and 9 percent hold both the Ph.D. and the M.D. degree. However, in the clinical departments (e.g., neurology), an even greater percentage, 89 percent, are M.D.'s, only 5 percent are Ph.D.'s, and 6 percent hold both degrees.' Overall, there are more than twice as many M.D.'s as Ph.D.'s in medical schools, but more Ph.D.'s (11 percent) are involved solely in research than are M.D.'s: 2 percent. Still, it is the rare Ph.D. or engineer who actually goes into the medical sciences. For example, only 5 percent of a total number of 265,500 doctoral scientists and engineers were, in fact, in the field of medicine.'

  A young physician makes a substantial financial sacrifice if he pursues an academic career in a medical school. If, for example, he takes a two-year fellowship to learn research after his residency training, his net loss could range from about $48,000 if he is a psychiatrist, to $137,000 if he is a radiologist.'

  To such a physician responsible for the daily care of his patients, his role as a laboratory researcher brings more than its share of conflicts. Can he really justify long hours spent in the laboratory, when caring for patients at the bedside has so many other advantages, both tangible and intangible?

  A favorite anecdote of Dr. Alan Gregg provides an illuminating answer. One day, Dr. Henry Forbes told Gregg of a vision he had of a long line of patients waiting to see him-a line extending far out of his office and into the street. He already knew what their diagnosis was: each had sprained an ankle stepping into a deep hole in the sidewalk outside. Forbes felt keenly the source of his own dilemma. It was simple: he was just so busy seeing patients in pain with sprained ankles that he never had the opportunity to go out and fill the hole.`

  Why research? To fill in the hole.

  But not just any kind of research will do. There is a great call nowadays for research that is "relevant." If his studies in the laboratory become too impractical, the experimentalist is soon brought back to reality by the stark human tragedies he faces in the wards and clinics. The world seems all too close when we follow children who were previously normal and see them slowly crippled by diseases of the nervous system. The following lines taken from the letter of a mother whose child developed MLD illustrate the situation:

  Can you imagine the anguish connected with wiping little finger marks from a window-knowing that the hand which once put them there still wants to touch the window but now cannot? Can you imagine the impact of seeing one's own bright and beautiful child stop developing and then regress to nothing?

  I can imagine it, but not of course to the same degree. My encounters with pneumonia, accidents, meningitis, cancer, and myocarditis either in myself or in members of my family have been far too close for comfort. It is as the worried patient or relative that one learns best what illness really means in human terms. Indeed, I believe a physician who has never experienced a major illness in himself or in his family has missed a crucial part of his medical education. It pays to be sick sometimes, both as a reminder of what illness is and of how lucky we are to be healthy.

  12

  Overview: What Next? So What?

  Again I saw that under the sun the race is not to the swift, nor the battle to the strong, nor bread to the wise, nor riches to the intelligent, nor favor to the men of skill; but time and chance happen to them all.

  Ecclesiastes 9.11

  Time and chance have a way of deciding things: such as which project our laboratory is going to take on next and how far we're going to go with it. The future isn't clear. You can appreciate some of the reasons for this uncertainty by glancing at figure 10. Here is one schematic version of how in the past the growing points of research have budded out, grown into branches, and how one field of study proliferated into another. Clearly, there is a conceptual link between research in metachromatic and globoid leukodystrophy. But there is no rational way to link hypertrophic neuritis (lower left) with Lewy bodies (top right) except by tenuous meanderings and chance encounters through five other intervening disorders.

  What is all this work leading up to? What are the ultimate goals? Let's step out from among the branches and take a look at the tree as a whole. In general our laboratory has been concerned with inherited diseases caused by an inborn metabolic error. We have been working at the interfac
e between molecular biology and medicine. Three themes have been under investigation: polyneuropathies, deposition disorders, and aging of the brain (figure 10, at top). None of these themes or interrelationships was preordained; each has grown haphazardly by a certain curious, inconsistent, internal logic of its own. In broad outline, these have been more meanderings than searches, more peregrinations (wanderings) than trips.

  Figure 10

  Evolution of the studies starting with hypertrophic neuritis. My early interests in hypertrophic neuritis (lower left), proliferated into several new and seemingly unrelated lines of research. The headings at the top indicate the general category of molecule or disease that was studied.

  And none of our work is completed. Some of the task will be taken on by subsequent generations of investigators. How do we define completion? The pragmatic physician must define it in terms of total eradication or effective treatment of disease. Only in one disorder, recurrent polyneuropathy (figure 10, at left), has our research yet led to a form of therapy that could rapidly restore a patient to normal function. Even then, the treatment with adrenal cortical hormones has only suppressed the disease, not cured it. The discovery was empirical; the treatment just happened to work. It then took five long years of controlled study before we were certain it did work.' We still have no solid conceptual understanding of what causes this illness, why it recurs, nor do we understand, in precise molecular terms, why the treatment with corticosteroid hormones helps the nerves. Currently, the cause and ultimate treatment of hypertrophic neuritis is also unknown.

  What next, then, in the diseases in which molecules are depositedthe deposition disorders? Better to understand what must come next, it will first be useful to review some steps leading up to the causes of diseases that are genetically determined. Here an awesome realization confronts us: we must be dealing with an enormous number of diseases. For example, the standard compendium of inherited diseases lists almost 9,000 entries for diseases in its latest printed edition, dated 1998, and even then occupying three volumes.' Now that the human genome has been essentially "mapped," it should become easier to diagnose early, head off, delay, or reverse many of these metabolic disorders, to the enormous benefit of the affected families and of humankind in general.