Beginnings in Medicine
Andres:Well, I decided to study medicine, like almost all young people, at the end of childhood. The reasons I studied medicine are not very clear to me. I think it was the only real choice. I had some interest in marine biology because my grandfather, Angelo Andres, was a marine biologist, who, with Dohrn, founded the Marine Biology Institute in Naples, and then was a professor in Pavia after Golgi for many years. So there was a certain interest in my house, all these books, which all came to my father. So perhaps this was the reason.
Studying with Giuseppe Levi
Andres:I enrolled in the faculty of medicine in 1942, during the war. And at that time, Professor Levi, because the racial laws in Italy were in 1938, was not in Torino. He was in Biarritz because he was obliged to leave his chair and leave Italy. So in Torino when I enrolled in the medical school in the Institute of Anatomy, I remember only Professor Anprimo. And the influence of Professor Levi was there in everything but he was not present.
And then in ’43 there was the German invasion, and, because I was born in 1934, the class of 24 was supposed to be recruited for the Republique Social, the neo-fascist republic. So I went into hiding and did not attend medical school for two years.
It was only after the end of the war, in April ’45, that I was able to continue my work as an intern in the Institute of Anatomy. At that time professor Levi was back, and there was Rita Levi-Montalcini, Dulbecco, Anprimo, Filogamo. My direct supervisor was Professor Anprimo, but I have a perfect recollection of Rita Levi-Montalcini, who was working with the thermostat, and the electric power frequently was missing, so she was running around because she was taking care of the tissue culture of Professor Levi. So I have a clear recollection of Rita and we met again in Rome and went around, giving some lectures together for several months in the early 60’s. So the institute of Professor Levi – there was this atmosphere which is beautifully described in In Praise of Imperfection, this book, a beautiful book, of Rita Levi-Montalcini, and in the equally beautiful book, Lessico Famigliare, by Natalia Ginzburg who is the daughter of Professor Levi, describing the extraordinary personality of this man. I still remember this, he was a beautiful lecturer, but extremely demanding. And I have a recollection of my exam with Professor Levi. He asked me a question about an excretory system, Rita Montalcini was there, with Professor Anprimo, and at a certain point the electric power went out. We had a blackout, so my exam finished with candlelight. He gave me 27, which at that time was a very good mark. So this, I think, was very important for me – to be an intern in anatomy, because it was an institute where there was already, at that time, strict, very focused interest in research. And I remember very well those people.
Studying with Cataldo Cassano
Andres:I left Torino in ’46, because my family moved to Tuscany. I had already spent a year as a student in Florence. And so then I moved to the University of Pisa, where, as a consequence of the war, the destruction was still very visible all over the medical school. And my interest in being accepted in the institute of Professor Cassano was motivated by the fact that the lecture of Professor Cassano was mainly focused on pathogenesis. And I became interested in the pathogenesis of disease. I still remember Professor that Cassano was giving a lecture on pathogenesis, we were going on from one day to another day. And I think this was an important factor in my choice, despite the fact that I also took a specialization in Pediatrics, so I was also attending the pediatric clinic.
Beginnings in Nephrology
Andres:My recollection of Nephrology at that time is that we had patients in the pediatric clinic, children, with nephrotic syndrome and pneumococcal peritoneal disease, children with great, severe edema. And in the medical clinic, practically, the diagnosis of kidney disease was a diagnosis of death. There was not very much you could do. But despite the fact that there was not very much progress concerning treatment, there was a considerable interest in pathogenesis. And I remember that Professor Cassano was giving a lecture comparing the pathogenesis proposed by Volhard and Fahr, which maybe was focused on streptococcal infection, and the pathogenesis, and the classification of all this. So there was an interest in renal disease, but I think the two important factors in focusing the activity of the clinic, the Department of Internal Medicine, and Nephrology was one, the book of Smith, on the renal function which became known thanks to the work of Sergio Giovetti Angeli, which practically translated part of this book. So we became acquainted with the recent test of renal function for the measurement of glomerular filtration rate and tubular re-absorption. And the second important factor was the introduction of the percutaneous renal biopsy in the hospital in Santa Chiara. Using the test of renal function for measurement of the glomerular filtration rate, we were using sodium thiosulphate, and for the measurement of the tubular function, para-amino hippuric acid. We started to work with Professor Fiaschi and Bruno Grassi on patients with diabetes. I think it started to be interesting for us because there was this syndrome of polyuria, and there was not much information about the mechanism of this polyuria. So we started this work on renal function in diabetes and in metabolic conditions, metabolic compensation. This work went on for two or three years.
While I was involved in this study, Alder Torchialli introduced the retroperitoneum, with the visualization of the kidney, and with the urologist of Pisa, Professor Toni. The first real biopsy was performed in Pisa in ’51, practically, shortly after the paper of Iverson and Brun. And the renal biopsy was very important because for the first time it was possible to have information on the renal diseases at the initial stage of the disease and not only at the end, at the autopsy. So it started to be possible to have information about the morphology of renal disease already in ’52, despite the fact that the section in histology was not really first class. And the reason why I became involved in the work of the people who were doing the renal biopsy, especially Ernico Fiaschi, was that I was in charge of carrying these tissue fragments to Florence where there was a technician in the university who was able to cut a relatively thin section because at Pisa, many times the sections were probably ten microns and very difficult to read.
Coming to Columbia University
Andres:Professor Cassano was offered the possibility to go to Rome in 1955, so it became evident that he was going to Rome with a small group of people of his institute, including Professor Fiaschi and some others. I was not offered the opportunity to go to Rome, and so I thought I would have the opportunity to continue other work in Pisa, and I was offered the position of assistante voluntario in the institute of Professor Tronketti – this was my first academic appointment – who was the person who took the chair of Pathologie Medica at the University of Pisa. Profesoor Tronketti was the successor of Professor Cassano. This was my first appointment in Pisa. Then, again, in ’55, Professor Cassano became a member of the Commission of the Institute Nationale Currazione, for a fellowship, for an international fellowship. This was a single position. And Professor Cassano was moving to Rome, so he was leaving Pisa, and there was a little bit of uncertainty with the future of people working with him. Fiaschi for sure was going with Professor Cassano. So when Professor Cassano said, “I am in a commission, perhaps I will have a chance to help somebody to have this fellowship. Who wants to apply?” Practically I was the one who said that I wanted to apply, because I thought perhaps this would be an important experience for me. I will work with Professor Tronketti in Pisa and so on. So then for the first time, I must say, I studied very hard, and I went to Rome, and there was this kind of exam. I am sure Professor Cassano was a great help to me, so I was the winner of this concord, and I got this fellowship. It was one million lire, not really very much, including the ticket for the trip.
But there was not the slightest idea where I was going to go, because there was no information. So I remember I took a book, the most recent book with the name of American authors which I could find, in Pisa, which by the way was a book of Endocrinology, and I found there was a person in Philadelphia who had written a chapter on diabetes. So I, with my fellowship, applied to this position, and I was accepted. But then I had a problem because at that time very few people were going to the United States, especially without very clear objectives, and finally after many difficulties I got a visa from the American consulate in Florence, and I did not realize this visa was for a position of residency. When I arrived in the United States, I realized I was supposed to do clinical work. This was not my desire. I wanted to go to an institute to learn something about research. So then I had serious problems with the immigration office in New York, because I said that I did not really want to go to this hospital, which, after all, was not in Philadelphia. It was in New Jersey. It was a good hospital but there was no program of research. Fortunately because I was arriving in the United States one month in advance of the period I was supposed to start the job, I visited, in New York, all the medical schools.
And I had also the opportunity to visit Columbia. And I was told by some Italian working there, especially the person who was a great help to me, Professor Lattis, professor of clinical pathology, who was one of the persons in Torino who was already a distinguished surgeon, who was obliged to leave Italy because of the racial law. Professor Lattis was very helpful to me. He gave me information. So I decided, really, I wanted to go to Columbia, and I asked Dr. Loeb, thanks to Dr. Lattis who was my intermediary to be accepted as a fellow, because I had my fellowship. So I became a fellow in Internal Medicine with Dr. Loeb, and this was kind of a tortuous trip, which brought me to Columbia. But in answering your question, it was really difficult, at least for me it was difficult, because I did not have any precise idea where to go, and at that time to have a fellowship was very difficult. I am still very grateful to Professor Cassano, who was instrumental in obtaining this fellowship for me.
Work with Dr. Loeb
Andres:At that time, Dr. Robert Frederick Loeb was really a leading clinician. Together with Walter Bauer at Harvard, they were leading clinicians in the United States. And he had formed probably one of the most authoritative departments in that country. Just to mention a few, there was Dickenson and Cournand, the Nobel prize for catheterization, Regan the rheumatologist, Rose, in Infectious Diseases, Chargraff who described the pairing of the bases which was instrumental for the description of the double helix of DNA, etc.
And Dr. Loeb was a great scientist in his own right. Dr. Loeb was the man who discovered that the adrenals controlled the metabolism of salt and water. And it was a kind of legend to tell to the students, how Dr. Loeb had discovered this association, because he had been a physician in the Balkan War, and he saw many people dying of cholera. They were dying from dehydration. So he observed that the patients with Addison’s coma also were dying from dehydration, suggesting that the adrenals – because Addison’s Disease at that time was considered a disease caused by tuberculosis – so the adrenals were controlling the metabolism of salt and water. So he did the classic experiment of bilateral adrenalectomy in a dog, demonstrating that the dog was dying from the loss of salt and water, and it was possible to correct the Addison’s coma in dogs by administration of salt and water. So Dr. Loeb was really a very prestigious scientist.
And then when he became chairman he said, “I really want to concentrate on building a strong department, and I will no longer work in research.” But he was a person who had great experience in research, and he was the person who was giving lectures in nephrology and diabetes at Columbia.
So I had the opportunity, several times, to ask him, concerning visiting patients with renal disease, some questions about pathogenesis of renal diseases, using my limited experience with renal biopsies, because at that time, in ’56, ’57, renal biopsies were not performed at Columbia. So I had a great advantage, and I was telling them, “But perhaps it will be necessary to perform a renal biopsy.” They were surprised that this could be done.
Work with Dr. Seegal on Streptococcal Disease and Renal Failure
Andres:The major influence… the major concept was that renal diseases were due to streptococcal infection. And I remember having asked Dr. Loeb, when there was a patient with renal disease, “What do you think it might be due to, this disease?” And due to fact that in the department, some people were really involved in the study of the association of streptococcal infection and renal disease, especially by measuring the anti-streptolysin titer, Dr. Loeb told me, “Well, perhaps the renal disease is due to the fact that when we have a streptococcal angina, the streptococcal toxin localizes in the kidney, because the streptococcal toxin has a certain polarity for the kidney. And then later on, after there is this famous period of latency, which was well described by von Pirquet, when there is this immune response to this antigen localizing in the kidney, then there is an inflammatory reaction in the kidney. And this is a mechanism similar to nephrotoxic nephritis, when we had this kidney serum, which localizes in the kidney and, as demonstrated by Kay, there is the autologous response, and the development of kidney disease.” And that the person who may tell more is Dr. Seegal, who is working, at that time medicine was on the ninth floor, is working on the twelfth floor. “So I think you should go there.”
So it was thanks to Dr. Loeb, that I made this connection, which was especially strong, because the wife of Dr. Loeb, Emily Loeb, was working with Dr. Seegal on experimental renal disease. I was introduced to Dr. Seegal, and Dr. Loeb was the link between the department of microbiology and the department of medicine.
So then I started to go to work at the laboratory of Dr. Beatrice Seegal. And I realized it was a very important laboratory because Dr. Seegal was the chairman of the department of microbiology, soon to be succeeded by Dr. Rose. And Dr. Seegal was deeply involved in the study of streptococcal infection and renal disease, because she had published, in ’53, a very important paper in the Journal of Experimental Medicine with Earle, who was one of her pupils. Then he became one of the chief nephrologists in Chicago with Janis, one of the groups doing renal biopsy in the United States. And she had published this paper with Emily Loeb, as I said, in ’53, on the elevation of anti-streptolysin titer in patients with renal disease. And so there was a great interest in this field, this association. In addition, at that time, there was in the laboratory of Dr. Seegal, John Zabriskie, who was a pupil of Lancefield and Maclyn McCarty, who were the leading experts, as you know, in streptococcal infection, the Lancefield classification of various types, various strains of strep. And John Zabriskie was sent to the laboratory of Dr. Seegal with the purpose to study the association between streptococcal infection and acute rheumatic fever. So they were using anti-streptococcal sera especially prepared against group A strep to study the hearts of patients who had died of rheumatic fever – at that time, this was still rather frequent – and for the study of antigenic mimicry between streptococcal antigen and myocardial tissue. So this work was one of the main interests of Dr. Seegal. And so going to this laboratory, I became involved in the study of streptococcal infection. Especially, I became acquainted with these people: I became a friend of John Zabriskie, and I started to go to the Rockefeller Institute. I met Dr. McCarty. I met Mrs. Lancefield. She was a technician, so she did this extraordinarily important work of classification of streptococcal strains. And I think going to work at the Rockefeller Institute at that time was a great experience for me, because I think it was practically, as you know, it was the institute that was instrumental in the progress of American medicine, at the beginning of the century. There was Flexner and Professor Rockefeller, and the American medical school started their reform, which brought them to their present position of leadership, thanks to the famous Flexner reform of the medical school. So I was going with them to this dining room and I was told, "This is Dr. Kunkel; this is Dr. McCarty." So these famous people, I still remember. It was quite an experience. So Dr. Seegal’s was a good place to study renal disease, and, in addition, Dr. Seegal gave me an assignment, “You should review what is the evidence of the immunological mechanisms produced in injured tissue.” So then I spent a lot of time in the library reviewing all the available evidence. And I came up with information that I published with Dr. Seegal. I think there were about twenty or thirty references, at the time, with special emphasis on the only model of experimental nephritis, which was available, which was nephrotoxic nephritis. And Dr. Seegal was very much involved in trying to use nephrotoxic nephritis for the localization of the antigenic antibodies.
Applying Immunofluoresence to Renal Biopsy
Andres:When I arrived in the laboratory of Beatrice Seegal, she was involved, as I said before, already with studies of immunofluorescence, with John Zabriskie, for the study of rheumatic fever. In addition, there were several other people working there. It was a very active laboratory. There was Dr. Earle working with the streptococcal antigen. Dr. Middleton was working with nephrotoxic nephritis. Dr. Metzger who then went to the NIH, and he is a very important scientist on IgA and immediate hypersensitivity. And Dr. Seegal was one of the two laboratories in New York City trying to develop the immunofluorescence method. The other laboratory was the laboratory at the Sloan Kettering, the laboratory of Robert Mellors. And Robert Mellors had already published one paper on the localization of nephrotoxic sera in glomeruli of rats with nephrotoxic nephritis. This was in the tradition of the study of the department of Professor Fitzmuren, where there was Dr. Pressman, who was the first to show by using the radioimmunological method, that the nephrotoxic sera localize in the glomeruli, and then by using ultraradiography to confirm this localization. So this laboratory of Sloan Kettering was already involved in these studies. At Columbia, Dr. Seegal was the other person who was following the same trend of research. The reason why Dr. Seegal was doing this type of work was because she was trained at Harvard, together with David Seegal, her husband, who was professor of medicine at Columbia and director of the Goldwater hospital on Roosevelt Island. And Dr. Seegal at Harvard had been a classmate of Alfred Coons, so she was very aware of immunofluorescence, the importance of immunofluorescence. And she was still frequently going to Boston to discuss, with Dr. Coons, the progress of her work.
Actually I remember once Dr. Seegal took me with her to Boston, and I still remember this theory by Dr. Coons in the department. I remember if you asked Dr. Coons how he developed the immunofluorescence, he told us that as a student he went to Berlin, because at that time, before the war, Berlin was the center of medical research. And at that time, in Berlin, he developed the idea that it would be very important in order to show that the inflammatory reaction, studied especially by Conrad in Germany, were due to immunological mechanisms. It would be very important to show that the antigen and the antibody were present at the site of the lesions. So Dr. Coons, going back, after this fellowship, to Boston, met with some friend in Biochemistry and told him, “Would it be possible, perhaps, to conjugate antibody,” actually, at that time, antibody, this word, did not exist, only gamma globulin, so, “the serum, anti-serum with a tracer visible in the fluorescence microscope. So he did this first study before the war, labeling anti-pneumococcal sera, showing that this anti-pneumococcal sera were binding to pneumococci. And Dr. Coons had told us about the difficulty of using a fluoresence scope at Harvard, that there were only very rudimentary scopes, and that he wrote a manuscript. Then the second world war started, and he was in the Pacific, in the army, when he was told that this paper had been published, the first paper on immunoflourescence. After the war, it became evident, thanks to the work of Pressman and the work of Mellors, and Seegal, that it would be possible to study antigen-antibody reactions in tissue, not only in bacteria. And so this was the beginning of these studies.
Then when I arrived at Columbia and I told Dr. Seegal and Dr. Loeb that we were doing renal biopsy, and it would be possible to use human tissue, then Dr. Seegal was more than happy to try to use immunofluorescence in the study of renal biopsy. And this collaboration started, because I started to receive… I told this to Professor Fiaschi, who was in Rome with Professor Cassano. I told him about this possibility, so we started this collaboration between Rome and Columbia, and they were performing these biopsies in Rome, sending these biopsies with a thermos. I was going to Idlewilde Airport, at that time, it was not called Kennedy, to collect this thermos which was brought by the pilot of Alitalia, and that I knew well, because he came to ask me advice at Columbia, because his wife had leukemia. So he was really very, very important, this commandante italiano, in taking responsibility for carrying these biopsies.
And so we started the study of immunoflourescence on human renal biopsy, which was the first in the United States, because Dr. McCluskey, who was a couple of steps ahead of us, he had already started to study human tissue but autopsy tissue. But we were studying biopsy tissue. While we started to do this work, Dr. McCluskey who was in New York had already started to do this type of work on autopsy tissue. “How difficult?” you ask me. It was very difficult for two reasons. First, because we had to cut these very tiny fragments, so with every section it was very important not to lose any sections, and we had very good technicians, but I had already learned to cut sections. And the microtome was very rudimentary; it was a far cry from the modern microtome, cryostat. And the second was the fact that at Columbia, we were trying to use anti-human gamma globulin serum. So we were preparing with the help of Dr. Hsu this fraction, because you have to know that at Columbia, just across the corridor was the great laboratory of Elvin Kabat, the great father of immunochemistry, together with his teacher, professor Heidelberg. And Kabat was the person who had shown that the gamma globulin fraction contained antibodies. So, because there was the tradition that the work of one group was also discussed by all the other groups, the work we were doing was discussed by Kabat, by Rose, by Morgan, by Wisner, by Langer, by all these leading immunologists in the department. And Kabat told us, “Well, in order to show that you have an antigen-antibody reaction, you have to use the gamma globulin fraction.”
So I remember this separation of gamma globulin, the separation with the sodium thiosulfate into various Coombs’ fractions was a major endeavor. The other problem was that at Columbia, there didn’t exist an immunoflourescence microscope. The only microscope available, the one that they had, was Sloan Kettering’s. The one of Robert McCluskey was not available to us obviously; he was a competitor. The only other microscope was in the Roosevelt Hospital, in the department of David Seegal, Dr. Esson. So we were taking these dry, with the section already stained, and I was taking the A-train going to 97, then the cross-town bus, and then I was taking the elevator to the Roosevelt Island, and then there was this huge microscope. And only when there it worked, today we would say five plus positivity, it was possible to see something. So every time there was this definition. Is it positive? Is it negative? Is it little more than background staining? So it was a very difficult decision. A very difficult decision especially when we were coming to the question of localization of antigen, because at that time, always on the basis of the work on nephrotoxic nephritis, which was the bible, practically, of nephrology, it was necessary to show not only the immunoglobulin but also the antigen. So the antigen at that time was considered to be the streptococcal antigen by definition. And so we were using all these streptococcal sera, prepared at Rockefeller by Dr. Lancefield, Dr. McCarty, brought from the laboratory by John Zabriskie, for the localization of streptococcal antigen in glomeruli.
So one of the first major problems that we had, which led us to a major error that I still blush about… when I see Michael who is now dean of the medical school in Minneapolis we laugh about this, because Dr. Michael was one of the first in the famous group at Minneapolis to study renal biopsy. I will tell you why Minneapolis was important. And also Michael came up with the first paper, together with our paper, that perhaps streptococcal antigen was localized in glomeruli. I don’t know if perhaps in some biopsies, we may have been right, but you know very well that the issue of streptococcal antigen in glomeruli is still unsettled. Probably, we made some error and we published a paper saying there was streptococcal antigen in glomeruli, which I think was very wrong, because one of the patients had, I remember, it was glomerular nephritis, and probably his tissue was positive because of some rheumatoid factor which was picking up the anti-sera. So the difficulty was the technique was very rudimentary, and this, I think, did not tell you enough. Also, because we were too polarized on this concept that streptococcus caused all glomerular disease.
American Nephrology in the 1950's
Andres:It was in ’57 that I went around, visiting some of the renal groups. And in order to do so, the introduction of Dr. Seegal was really important, because Dr. Seegal was one of the few persons who was instrumental in organizing, with Jack Metcoff, who was a real leader, the first conference on the kidney, as we will discuss later on, the annual Conference on Nephrotic Syndrome. So she knew everybody, also because some of these people have been in her laboratory, like Earle. And so I remember having been in Washington to visit the group of Parish and Howe. I was in Chicago where I met Dr. Kark and Pirani. In Chicago also I met Earle, who was a pupil, as I said, of Dr. Seegal, who was chief of medicine of Northwestern University, and Dr. Jennings, who became a friend of mine, and who was the pathologist who was in the group with Kark, doing renal biopsy in the United States. Then I went to visit a friend of Dr. Seegal, Dr. Gutman, who was the head of the NIH, the immunology laboratory at the NIH, and Baxter and Gutman were working on nephrotoxic nephritis, trying to localize the nephrotoxic antigen. And I went to Boston and met Janeway, who was a friend, a classmate of Dr. Seegal. He was the chief of pediatrics in the children’s hospital. And in New York, obviously, Dr. Churg and Richman who were at the Mt. Sinai hospital. In addition, in Cleveland, I met Dr. Raymond who was a pediatrician, and he was very much interested in nephrology, and Dr. Raymond and I became friends, and I have continued to follow also the work of his pupils. So, also Dr. Raymond was a friend of Dr. Seegal.
What was my impression? Well, these people were mainly working on the renal biopsy, like the group of Kark, and Jennings, and Parish et. al., and the importance of renal biopsy in the diagnosis of renal disease. And I did not have the impression that they were very far ahead of us. We were doing this already in Pisa. And the quality of the sections were not really superior to ours. The technical program was of critical importance, so we were always looking if the section was readable or not. But I became interested in the work of Dr. Gutman who then became the chief of the WHO in Geneva, because when he was at the NIH he was trying to localize nephrotoxic antigen, and in the work of Dr. Raymond who was trying to study the pathogenesis of these nephrologies in rats, immunized with homogenate of rat tissue. So I learned a little bit concerning pathogenesis but not very much about the morphology.
I think at Columbia I was practically ahead of everybody else, for what concerned ideas and pathogenesis of the disease. Perhaps only the group of McCluskey was so much involved in the pathogenesis. The other group were more involved in the morphology and dialysis.
The Annual Conference on the Nephrotic Syndrome
Andres:Dr. Seegal was involved in the organization of the Annual Conference on the Nephrotic Syndrome. And while I as at Columbia I had the opportunity to very carefully study the proceedings of the previous meeting. Practically the Annual Conference started in 1940. And so I became acquainted for the first time, with the work of Vincent Hall. He was the director of the electron microscopy laboratory of the Argonne National Institute. And in ’53, and in ’54, at the Annual Conference, he presented the first study of electron microscopy of renal glomeruli. And these studies were, for me, extremely revealing, because it was evident that these new techniques would be of basic importance for understanding the pathogenesis of renal disease. In addition, in this volume of the Annual Conference on the Nephrotic Syndrome, I found the work of the Boston group of Hume, Murray, Merrill, concerning allogeneic transplantation in twins, and the first experience in allogeneic renal transplantation. Then there was the work of Goldblatt on hypertension, the work of Lewis Thomas who was a professor of pathology at that time of the NIU on the Schwartzman reaction, and lesion caused by the Schwartzman reaction and coagulation in the kidney. The work on BSA of Janeway – I would like to spend a few words about this, because Janeway, as I told you, was a pediatrician at the children’s hospital, was a friend, a classmate of Dr. Seegal. And with Dr. Seegal had been involved in the studies supported by the American government during the first World War, of trying to use bovine serum albumin as a substitute for human plasma in the people wounded on the battle field. So they did some experiments in injecting BSA in men, and then studying the immune response to BSA. This original work was published in The Zeigler, a classic book on microbiology, the first edition. And obviously they had to stop, because they were producing lesions which I think in a few people, resulted in the death of the patient.
And there was also in the Annual Proceedings of the Annual Conference, described the work of Gutman and Baxter on the identification of the nephrotoxic antigen by using serological methods, and also the work of the group of Kark, the group of Parish et. al. on renal biopsy. So I realized from the study of the first volume of this Annual Proceeding on Nephrotic Syndrome, which is very interesting to read, even today… because still, today, I find interesting observations and ideas which would serve perhaps to go back to the laboratory to do some work, in these Proceedings, which lasted until 1963. Then the Annual Proceedings, the Annual Conference on the Nephrotic Syndrome became the National Kidney Disease Foundation which was the forerunner of the American Society of Nephrology.
In 1957, the Proceedings of the Conference on the Nephrotic Syndrome was held at the NIH. And Dr. Seegal took me with her, and it was the first time in these conferences that the study of immunofluorescence was presented. So Dr. Seegal presented her work on localization of immunoglobulin and interaction with glomeruli with the nephrotoxic nephritis, and localization of the antigen. At the same conference there was Dr. Metcoff who presented the first use of steroid treatment in patients with renal disease. And I remember that visit to Washington also because it was my first visit to a medical library. I went to see this fabulous medical library of Walter Reed, which is in this place in the red building, which is in front of the Smithsonian Institute now. Obviously this was the library, which then became the national library of the NIH, one of the main buildings of the NIH. Well practically, today, there is everything. At that time, the medical library of the armed forces was very important for all the scientists, especially nephrologists working around the world, because it was very generous in providing, upon request, the microfilm of papers. In this was I came in possession of the microfilm of the original work of Masugi, the work of Smadele, the work of Oliver, the work of Volhard and Fahr, which, in Italy, were not available after the war. And so I remember this visit, which was my first visit to a medical library, with great pleasure. I still remember this balcony, people were going to collect the books, and I think it was a very interesting experience.
Interest in Electron Microscopy of the Kidney
Andres:In addition, I have to mention another thing, because we are talking about ’57. That was the year in which Farquhar, Vernier, and Good published the landmark paper on the ultrastructure of human renal disease, a classic paper, in the Journal of Experimental Medicine. That was the first application of electron microscopy to the study of renal disease, human renal disease, with the demonstration that foreign deposits were present in glomeruli. There are huge sub-endothelial deposits of electron-dense material in the picture published in that paper. And this paper was published because Bob Vernier was the first to start renal biopsy in Minneapolis, together with Good, who was the professor of pediatrics. And, at that time, Marilyn Farquhar, who was starting to do electron microscopy in San Francisco, with Professor Randall – she was a technician for Professor Randall – she was extremely gifted in sectioning and developing the best possible technique. And she moved to Minneapolis with her husband, and so the group of nephrologists in Minneapolis had the possibility to give the renal biopsies to Marilyn. Marilyn, at that time, had published, in Laboratory Investigations, the first usable technique for the fixation of tissue, using the osmium tetroxide, which was developed in ’55 by George Palade at the Rockefeller Institute, for the fixation of tissue. This is Dr. Palade, with me, but this is later on, in ’75 in Buffalo. And Dr. Farquhar was able to make this original observation. And this paper had a great influence on me, because, in addition to the original work of Vincent Hole, on the destruction of normal glomeruli in the rat, this was the first study showing that this new, powerful tool, the electron microscope, would be usable for the study of renal disease. So the possibility of investigation of localization of antigen-antibody by light microscope and the possibility of studying the renal lesions by the electron microscope, for me, at that time in ’57, became the two most important avenues for research, for the future. And from that point, I started to be rather sceptical of all these morphological studies which, yes, we were doing already in Pisa, the morphology of renal disease, but in order to learn something about the pathogenesis of renal disease, we had to use new tools. And this work of Farquhar was really very important to me. Probably, you can see the paper of Marilyn here. This was sitting on my desk. This is the group of Dr. Farquhar, this is Dr. Kariaski, and Dr. Nitin, and this is in San Antonio when Dr. Farquhar had the Wilson medal, the highest honor of the American Society of Nephrology, and I was in charge of introducing Marilyn. And this is Dr. Henry Movat, who was also, at that time, one of the scientists who gave a major contribution to the development of the study of ultrastructure of the kidney.
So in ’57, my interests were focused on this new type of research. And as I told you I was already starting to know how fantastic an institute for research was the Rockefeller. And so at Rockefeller, there was notoriously the strongest group in electron microscopy, the group of Claude, Porter, and Palade. Porter was the first person who took a picture of a cell by electron microscopy. Palade had developed the fixative. They had discovered the endoplasmic reticulum, Porter, Palade, the ribosome. They were doing this work which led to the Nobel Prize, Palade and Claude. And so my interest would have been to go to Rockefeller, but when I asked Dr. Porter if I could go as a fellow in his laboratory, he had already two very good Italians, Giuseppe Milone, and Karl Brun, who did very important work at that time. Milone is the author of the Milone Fixative, and Brun was working on liver. And in addition, Marilyn Farquhar had already obtained a position in the laboratory of Dr. Palade. So it would have been difficult for me, and so the only place who was doing similar work, that in ’57 had published a paper on electron microscopy of the kidney was Andres Bergstrand in Stockholm. The two papers on electron microscopy of the kidney are Farquhar and Bergstrand.
Electron Microscopy Studies in Stockholm
Andres:And I told you that Rockefeller, it was difficult for me to go there, so the other possibility was to go to Karolinska. I applied and I got a fellowship from the Svenska Institute. And it was not a difficult choice, because the Rockefeller Institute and the Karolinska Institute were, at the time, the two lighthouses for ultrastructure studies. And obviously at Rockefeller, the group of Claude, Porter, and Palade had already provided very important contributions. But also in Sweden the school of electron microscopy was very advanced, because of the school of Professor Sjoestrand, and with Rodine, who was the first to study the structure of renal tubules, and Bergstrand, and many other pupils of Sjoestrand, in other universities. And there was a certain kind of competition between Rockefeller and Karolinska, especially because, it is easy to understand, at that time there was some controversy over the interpretation of the findings. One of the most celebrated was the one over the existence of the ribosome. Palade suggested that surely, the ribosome did exist, thanks to his work and work on the synthesis of protein, and Sjoestrand, who at that time was considering that the ribosomes were artifacts. And obviously the methods and the development of the various techniques were of key importance in order to obtain reliable results. And obviously the problem of fixation, the problem of sectioning, the problem of staining of the section, were all important for the interpretation of the findings. And in addition, the quality of the electron microscope was also important. So you have to consider that the electron microscope was developed in Germany in Berlin by Ruska, and so in Sweden they had the best electron microscope. They had this fantastic Siemens, while in the United States they had this RCA, which was not so good. But in the field of fixatives, and in the field of sectioning the group of Rockefeller was far ahead. Progress then was also made by the introduction by Harrison Latta of the glass knife, but Rockefeller’s Porter also developed the first rotary microtome. And so these two places, Rockefeller and Karolinska, were good places to learn electron microscopy.
Dr. Bergstrand was a pathologist, and he was studying with Dr. Bucht who was the clinical nephrologist who was taking the biopsies. He started to study the patients with amyloid and diabetes. These were two disease which were not so interesting for me, but practicing in his laboratory was very important, because I learned the basic techniques. And I did work with Jan Ericsson who then became professor of pathology in Uppsala. And I studied aminoglucoside nephropathy, so I published with Jan Ericsson in the American Journal of Pathology one of the first papers, with the one of Dr. Vernier on the ultrastructure of aminoglucosides nephropathy, especially showing that multiple injections produced chronic renal disease, which at that time, was not known, because it was considered the practical, experimental model of the nephrotic syndrome of children. This was called at that time.
So in addition, in Stockholm, I started receive from my connection with Dr. Nakarato and Dr. Fiaschi, some biopsies in Stockholm. And we studiedd also with Dr. Bergstrand some biopsies coming from Rome. So this was the period in which I suggested… we were frequently exchanging correspondence with Dr. Fiaschi. Dr. Fiaschi or I, I don’t remember who it was, but I think it was Dr. Fiaschi who wrote me a letter and said there is the possibility that Professor Cassano may be in charge of what is called in Italian Relatione al Congressio Nationale di Medicin Interna, a kind of report to the National Congress on Internal Medicine, and we could, on the basis of what you have told me, we could provide some new contribution in the field of renal disease. It was at that time, while I was in Stockholm, that we started to discuss this joint work. This was also important for me for going back to Rome, from Stockholm, and at that time, I left my appointment in Pisa, despite the fact that in Italy the situation was very difficult. The only position that I had was at the University of Pisa. I went to Rome in order to do this work with Dr. Fiaschi.
Intervention on Nephrotic Syndrome for the 61st Congress of the Italian Society of Internal Medicine
Andres:The majority of the writing work was done by Professor Fiaschi, and the clue of this presentation were the results of electron microscopy, and immunofluoresence of renal disease. And I realized that at the time, the meeting was held in Naples in October 1960, that there were other Italians doing work in electron microscopy, especially, from Milan, Folli Onida. They were doing very good work with electron microscopy. Folli had published an important paper with the collaboration of the Chicago group of Kark and Pirani on the restoration of the deposition of the foot processes in children with nephrotic syndrome, treated with steroids. So I met this Italian for the first time. And the meeting had special importance for me because the alternative was to stay in Italy as Professor Cassano proposed to me, or to return to the United States following my inclination to continue hunting for immune deposits and antigen-antibody reactions in tissue.
1960 ISN Congress
Andres:But in 1960, I think there were two important meetings, which I would like to mention, which I think left a mark on the development of Nephrology. One was the first congress of the International Society of Nephrology in Evian. And I vividly remember that meeting, especially two presentations.
One was professor Hamburger, who dramatically presented on the podium the first patient with renal transplant, which came from Paris. And everybody became aware that a new era was opening for Nephrology with kidney transplantation.
The second was another outstanding performance by Marilyn Farquhar with Palade, which presented all this beautiful work on vascular permeability using ferritin as the tracer. And the perfection of the technique, which was kind of a characteristic fingerprint of Rockefeller University, was such that everybody remained astonished to see how this molecule was slowly going through the glomerular capillary wall. So that presentation confirmed my idea that really, Marilyn Farquhar was the person to watch in order to follow the progress of Nephrology.
1960 CIBA Symposium and Return to Columbia University
Andres:I was aware of this symposium when I was in Stockholm because Dr. Bergstrand was invited, but we were not invited so I did not expect to go there. But when we were in Rome, for certain days I was in charge of the ward with renal patients. At that time there was no chair in Nephrology, but I was in charge of renal disease. And a certain day, Sir Gordon Wolstenholme, on short notice, came to see Dr. Fiaschi, and probably because he was impressed by talking with Dr. Fiaschi, we got this invitation, but not as a member of the official program, in fact, our name was not on the program. So then we went to London, I remember we were really like persons who were not invited, we were like auditors. And we attended all these presentations, and I think that at a certain point we were asked to summarize the experience, but in some informal way, telling them what we were doing, so the presentation did not have very much evidence.
But for me it was very important, that meeting, because it was a meeting of morphologists, and I had a clear impression that the progress of Nephrology was no longer in morphology, but was in other areas. And so I came back, and I remember I told Fiaschi, “Look, we know more than all of them.” And I think that this is not the time to insist on morphology, trying to classify renal disease. Yes, this is important, but let them do these things, we have to try to do something different. And this was especially true because I already had certain plans in my head, and the very important information that I had at the time were two. One, the classic publication of Johnson in Nature that it was possible to conjugate antibody with a tracer visible by electron microscopy. This was a paper on biochemistry showing that the conjugation of antibody with ferritin does not prevent the capacity of the antibody to bind to the respective antigen. This was very important because I was always in contact with Dr. Seegal, and she told me, “Look, now there is this paper. Our laboratory of electron microscopy, which was the leading laboratory in the United States, and in the world for Virology, with Morgan Rose, our laboratory will try to use this method, to apply for the first time, this method to the study of viruses, and their entry and exit from the cell. It would be wonderful if in the future we would try to develop a technique using this type of labeled antibody for the identification of antigen-antibody reaction in tissue. So this was one of the things I had in mind already, at the time of the London Conference. The second piece of information that I got from New York by my red line with Dr. Seegal was that in 1960 the Annual Conference on the Nephrotic Syndrome was organized by Dr. Seegal at Columbia. And it was in this meeting that Dixon presented his first data on the development of a new model for representing the spectrum of human glomerulonephritis by multiple immunization, the so called crowning serum sickness. Specially by showing, with the work of Feldman, who was doing electron microscopy at the Scripps clinic, by showing that in this type of glomerulonephritis there were electron dense deposits, similar to the one shown by Farquhar in renal biopsy. So for the first time we had a model for the Masugi nephritis, nephrotoxic nephritis, which is not characterized by presence of electron dense deposits, which, for me, were the hallmark of human disease, were clearly visible in the Dixon model. So I knew that there was a model, and there was the new method of John Singer.
So for me, this information were important for two reasons. One, for my decision to go back to Columbia. This was asked of me by Dr. Seegal and Dr. Morgan. And two, to decline this offer of professor Cassano to stay in Rome, because I thought that was the time to learn more in Nephrology, something which would be very difficult for me to do in Rome. And this was also the reason for the end of my collaboration with Fiaschi, who, at that time, got a chair in Internal Medicine, first at Cagliari and then at Padua, and then he moved there.
Work on Experimental Nephritis and Ferritin-Conjugated Antibodies
Andres:But really, the real issue was to try to convincingly show that the foreign deposits, shown for the first time by Marilyn Farquhar, were indeed, the same thing as the immune deposits detected by immunofluoresence. So we had the morphology and immunology but necessary in order to show convincingly to combine these two observations to show that they were really the same things. And because now, it seems to be obvious, but at that time, entire meetings were dedicated to this issue, this discussion, and people in favor, and people against. So this was the aim, and we though that the model provided by nephrotoxic nephritis, and the model of chronic serum sickness of Dixon would have been worth investigation with the new method of John Singer. At that time the immunoperoxidase technique didn’t exist.
So I went back to Columbia and this, as I told you before, was a very difficult decision, because I was practically kissing goodbye to my career in Italy. But I thought it was important to me, to continue this line of research. And I went back as a fellow of the John Wheatman foundation.
The method of Singer was working well for Morgan because he was taking cells with viruses, pellets with viruses, and was mixing with the conjugated antibody, and then embedding the pellets, and the problem was solved. But with tissue, we rapidly realized that there was almost insurmountable difficulty was the fact that the ferritin was 800,000 molecular weight, the Igg 160. So it’s almost one million and this huge molecule did not penetrate tissue, when we have renal tissue, or any other tissue, especially compact tissue, like muscle or even interstitial renal tissue. Practically there is no penetration. So for almost two years I was struggling with this program. Is the conjugation not working? Is it the fact that we did not have good penetration? We had in nature the fixation of antigen in tissue with fixation. And, practically, we thought there was no solution, until Dr. Gabriel Gardman, a great friend of mine, still emeritus professor of pathology at Columbia, suggested several tricks. And so by trial and error we were able to find certain combination between a mild fixative, the paraformaldehyde, and the mincing of tissue in order to allow penetration of the antibody conjugated with ferritin in structure like renal glomeruli. So finally we were able to obtain reliable results, because at certain point, we were also in doubt about the validity of our control, because we were exposing so much of the section to an old RCA microscope that we had a lot of contamination forming all these black spots, little black spots resembling ferriting, so the black spots were not in the right place. And so finally we adjusted the technique, and we had these reliable results, and we were able to publish the technique of localization of nephrotoxic sera in Nature. And this was a paper which was well received, because it became possible that the electron microscope would become usable for the investigation of antigen-antibody reaction in tissue. Something which then has been improved with the immunoperoxidase, with the colloidal gold in other, more simple methods that we are using today.
Then in 1961, there were two important events. One was the publication of Dixon, Feldman, and Vazquez of the paper that Dixon had presented at the Annual Conference on the Nephrotic Syndrome, the year before. It was officially published in the Journal of Experimental Medicine, and that paper was really very important, because that paper showed two things. One, that there were electron-dense deposits comparable to the ones of human disease. And second, the different types, this is very clearly stated in the paper, different types of morphologic lesions may be produced by the intensity and the quality of the antibody response. And this was, I think, the first time, that this was clearly stated, something that was reiterated by Dixon in his Harvard lecture of ’63 to the New York Academy of Medicine, that the quality and the quantity of the antibody, immune response may condition the morphology of the lesions.
In 1961, also for the first time we were invited, Dr. Seegal and me, to present our work to the Annual Conference on the Nephrotic Syndrome in Princeton. And so I presented my work on nephrotoxic nephritis, and Dr. Seegal presented her work to do with immunofluorescence of the human renal biopsies that we were receiving from Italy. And then in ’62, when I was still struggling with my technique, then there was another important fact for me, that Dixon came to Columbia to see me. He came, and Dr. Feldman came a few months later, and I have some pictures of Dr. Dixon who came to see me and discussed the picture of his model with me. And then this is a picture of La Jolla, of the laboratory of Dixon, which was set up by Dixon in La Jolla. And this is a recent picture of when Dr. Dixon retired. And I think I have also a picture of Dr. Feldman, which is this one, when he came to see me in ’61, and more recently in his house in La Jolla. Then in this meeting I remember we discussed, we went to play tennis and then on the Hudson River we discussed with Dr. Dixon, one, that he strengthened my idea that it would be important to study the pathogenesis of disease, renal disease, and therefore we should not pay great attention to the morphologies, the main point of the meeting, and Dixon was considered a little bit of an outcast. And second, he invited me to go to the Scripps clinic that he was developing as the head of the renal group, and he said think about this for one year, and then give me your answer.
Establishing a Renal Group in Rome
Andres:Well, really, the alternatives were to remain in the United States or come back to Italy. My bend was to stay in Italy because I had my family, my education, and I was very attached to Italy. I would have preferred to stay in Italy.
At the same time the study of the pathogenesis of renal disease was making great strides, especially because Dixon, who became chairman of pathology in Pittsburg when he was 33 years old. He assembled this extraordinary group of people in Pittsburgh: Barry Pierce, Charlie Cork, and John Feldman, Bob Reigel, and others. I decided to leave academia and go to La Jolla, which is a kind of American Portofino, and to build on this peninsula a new center for the study of renal disease, the inflammatory reaction. Dixon was one of the first in the United States, with Pressman, to use a radioisotope in the study of the antibody-antigen reaction, and he had a clear idea what to do. He especially was a great manager. I think he would have been an extraordinary manager of General Motors, or any place. And he was assembling an extraordinary group of people in this new center, the Scripps clinic in La Jolla, especially Emil Unanue, who was studying renal disease. And quickly he became, with the application of radioisotope to the turnover of antibodies in experimental glomerulonephritis, what we used to call “Mr. Kidney.” He was an immigrant from Cuba, and extremely talented. He became one of the leading immunologists. Now he is the chairman of immunology and pathology at Washington University in St. Louis, but at that time, he was really one of the most advanced, young investigators, as were many others working with Dixon.
So at that time, Nephrology was making great advances in the United States. I am just talking about my field, because it was making progress in other fields. And so it was not really the right time to leave and to go set up a new group in Italy, but I made the decision. And soon, I did not regret this decision, because it was a new challenge, and I went there with new ideas, and quickly I was able to recruit very good, talented, young people. But to say ‘recruit’ is the wrong word, because I did not recruit anybody. They came, simply because I set up a laboratory, I started to work, and they were coming from every place. I could not really take all of them, but there were ones that we were able to accommodate in this small laboratory we tried to organize according to new ideas. And we had NIH money, so we were not really bound to the strict rules of the Italian university. It was difficult to pay the bills, etc.. We were paid directly. We had these extraordinarily good young people who then became my friends for life, I would like to mention all of them, because then my plan was to send them to some laboratory in the United States, for two, three years, to build up different kinds of expertise, in order to have overlapping experiences, in order to build a strong group, something similar to what Dixon was doing with a much larger scale in La Jolla, much larger scale, I have to say. I had Accinni, who then went to work at Columbia, with Dr. Seegal and Dr. Hsu, and she became an expert in the conjugation of antibodies, and then became a senior investigator of the Italian National Research Council. Dr. Tonietti, who went to the Scripps clinic with Dixon, and Tonietti did very important work with Dixon and Oldstone, showing that with the autoimmune disease of NCB mice, viral infection can exacerbate the course of the disease, increasing the production of anti-DNA antibody. And Tonietti is a professor of medicine, dean of the medical school at the University of Laguia in Italy. Mario Stefanini I sent to work in Los Angeles in the department of ultrastructure, and now is professor of anatomy in the University of Rome. It’s one of the best laboratories in Italy, where they are very advanced in the study of stem cells, and Mario is a very good investigator, and very good professor. And Karl Brun was sent to me by Professor Callifano who was head of the research council, who is now professor of pathology in Naples. And de Martino, who was my collaborator when I was in the States, who I sent also to Los Angeles, and was director of the laboratory of electron microcopy in the Ewan Institute in Rome. And Pere-Giorgio Natalie, who is now the scientific director of the cancer institute in Rome. And Pere-Giorgio went to work at the Scripps Clinic for three years working with them, and they did very nice work on the effect of ultra-violet light in the autoimmune response with Dr. Tam.
So all these people were of great stimulation, and I learned from them more than I was able to teach them. So we formed a group and I was very excited, and very happy to work with them, luckily, and I remember that period with great nostalgia.
Failure of Transplantation Studies
Andres:When I was in Rome, at that time I had an appointment of visiting professor at Columbia, so I was spending, every year, a certain number of months at Columbia. It was during one of these periods, and I think during a meeting of the Federation Society in Philadelphia, that I was approached by Dr. Kendric Porter. Dr. Kendric Porter was the head of pathology at St. Mary’s hospital in London. And I knew that was considered the leading study of the morphology of the human allograft. And he approached me and told me, “Look, we have a group, and the leading person is Tom Starzel, in Denver. And every year, we meet in Denver for a period of ten, fifteen days in order to collect material, because Dr. Starzel is calling back all his transplants. And renal biopsies are taken, a sample of blood, and all the various people interested, the group of Cherpolini, the group from Italy, the group of Amos from the United States, the group of Kendric Porter from London, meet there and collect all these materials, and then we study all these materials together. Then after a short while, the group of Denver, the group of London, and the group of Montreal, and the group of Toronto, they participated in this joint effort. So I started to go to Denver at the beginning, and then also other places with my bag, with my own fixative ingredients, because in this group I was in charge of taking the specimen and dividing part of the specimen obtained by biopsy, by surgical biopsy, which was performed by Tom Starzel, for morphology and they were going to London, and part for immunofluorescence and immunoferritin. And I was studying the specimen, in Rome and not at Columbia because I had a better laboratory in Rome, especially electron microscopy, I did not have at Columbia. So this was the beginning of the study.
And this study was remarkable for its failure. And it is still very painful to remember this failure, because the main issue after some time became the issue of pathogenesis of hyperacute rejection. You have to consider that allograft rejection, mainly on the basis of skin transplantation, was considered a cell-mediated phenomenon. There was no evidence whatsoever that antibodies were involved. And so there was no evidence. Practically, my work was supposed to provide information in this field. And very shortly after the beginning of this study, the cross-matching test was not yet performed, Starzel had two or three cases of hyperacute rejection of organs in patients who had received already a previous graft, or that probably had preformed antibodies. But at that time, we didn’t know. Terasaki was starting to know because he was starting to do his very advanced serology. So I had this tissue, and I found a lot of fibrinogen, but not immunoglobulin. So Starzel was calling and writing me at Columbia or in Rome, “What happen? I cannot continue to go on with the program because I need to know what is the pathogenesis of this,” what he called, “this disaster.” because the kidney was practically rejected under his eyes when the abdomen was still open. They had removed the kidney which was from a living donor.
So I was not able to provide information because I had only fibrinogen, and no immunoglobulin. So at a certain point Starzel was probably disappointed about my failure to provide information. So he asked Dixon to help. So Lerner was, at that time, still working on the kidney, started to collect specimens, so we were dividing part of the specimen for me, and part for Dick Lerner. And then we had another case of hyperacute rejection, and again, I found fibrinogen, and also Dick Lerner found fibrinogen. So then quickly, Dixon was very quick. Dr. Seegal was behind because, as I told you, at Columbia it was very hard to publish paper, because the paper had to be read and approved by all the members of the department. And we had to cross the forke cordina of Ervin Kabat, because if you did not show that the antibody is specific for the certain antigen present in the lesions, and therefore the lesions were ascribed exactly to that type of antigen-antibody reaction, then it was not possible to publish anything. So I had close scrutiny at Columbia with Ervin Kabat. So Dixon rapidly published a paper which made a big splash, in the New England Journal of Medicine, that hyperacute reaction was a kind of Schwartzman reaction. The same toxin was responsible for this massive intravascular coagulation and deposition. Do at the same time, Dr. Milgrom, in Portugal, by using the mixed agglutination technique, was able to show by working with Williams, in Baltimore at John Hopkins, to show that in kidney tissue which were rejected, it was possible to show that there was binding of autologous human antibody. And this was using this technique of using erythrocytes coated with Ripley cells, which were detecting, which were agglutinated by human IgG, or the human antibody, which, then, he had agglutination of erythrocytes. And then he published in the New England Journal of Medicine this first paper that showed in hyperacute rejection, there were antibodies bound to tissue. So then at Columbia, the paper of Schwartz and Dixon were cosidered completely wrong, that there was a big mistake, that it was not possible the Schwartzman reaction. But really, there was not convincing evidence, with the only evidence, serologic evidence, already published in Denmark by a group of investigators, and also by Starzel with Terasaki, that there was a decrease in level of transplantation antibody after rejection, because the antibody were fixed to tissue, and therefore the level was decreased in the circulation. I remember, but then, I had a patient who had rejected a kidney, but slowly, and then in this kidney there was a lot of immunoglobulin in the glomeruli, and I had these pictures, but I was not able really… they were precious because they were the first real evidence that in rejection the humoural mechanism was important. But I could not publish this because Dr. Kabat and Dr. Seegal said, “But you have immunoglobulin, but we have no proof that there are antibodies.” So one day, John Van Rood, who we say discovered the transplantation antibodies, came to Columbia to give a lecture and then came to visit, and I had my picture on the desk. And he saw this picture and said, “But you have immunoglobulin. This is really evidence that immunoglobulins participate in acute hyperrejection. So there is evidence of a humoural mechanism.” And so I had this evidence, but I did not publish it, so I lost. This was another disappointment.
Coming to State University of New York
Andres:But disappointment sometimes may be combined with some new openings, because I had a visit, after Van Rood, of a gentleman from Buffalo. It was Felix Milgrom, who was the chairman of microbiology who succeeded Dr. Witebsky who was the father of autoimmunity, the one who, with Rose, developed autoimmune thyroiditis, which was the first experimental autoimmune disease. And Dr. Milgrom came, and he was the man who did this paper on the mixed agglutination technique in allograft reaction, came to me and said, “Well, it would be very nice if you could join our group. Think about coming as professor, and we will do everything to organize a good group.” At that time the offer was very attractive, because Buffalo was, with La Jolla, the strongest group in immunology, much stronger than Columbia where there was practically no tradition of renal study, with the exception of some renal physiology with Dr. Bradley, and there was no real interest in renal transplantation. In contrast, in Buffalo, the group was organized, which was built by Dr. Witebsky, included Dr. Milgrom who discovered rheumatoid factor, and then was deeply involved in the study of transplantation with the group of John Hopkins. There was Dr. Rose, who was the one who did the work on the experimental thyroiditis. There was Tom Tomasi who was the one who discovered IgA, and the first line of defence, the mucosal immune system. There was Morris Reichlin, who was, with Crick, the leading expert on DNA, discovering all the antibodies to RNA, lupus, etc.. There was Allen, who was very strong in the study of immunologic infection diseases. So it was a very strong group, and I was supposed to build a renal group in Buffalo, with the appointment in the Buffalo General Hospital, and the university, and with the program of studying specially the pathogenesis of renal disease and allograft rejection in Buffalo. And so for reasons independent of my will, related to the situation of the university in Italy, and this offer from the State University of New York, in 1969, I left Italy for good, and with great regrets, I departed from my Roman group, and I went to Buffalo.
Studies on Pathogenesis of Glomerular Disease
Andres:We were involved with Dr. Milgrom and his group, collaborative work, in the study of immunologic mechanisms in renal diseases. Especially we were interested in the mechanism of the formation of immune deposits in membranous glomerulonephritis in the rat, in the Heymann model, the model of Dr. Heymann. And for several years, we studied various approaches in this model, sometimes in collaboration with Dr. Farquhar, and we came to the conclusion that this antigen which is expressed on the plasma membrane of the foot processes of the podocytes is shed after interaction with the antibody, between the podocytes and the basal membrane forming these subepithelial deposits, producing probably an hypersecretory reaction in the podocytes which gives rise to these spikes. So we studied these mechanisms, and then, in addition, we did several studies on the pathogenetic mechanism of tubulointerstitial nephritis, which led to the identification of tubular basal membrane antigen, which may be responsible for some rare cases autoimmune tubulointerstitial nephritis. Then we continued with Dr. Milgrom, the study of the mechanism of rejection. So we had the opportunity to continue especially with the involvement of several Italian and Japanese investigators, in Japan, the group of nephrology of Nagoya. That was a great pleasure to have him in Buffalo.
Reflections on Study of Renal Disease
Andres:I think I can only talk about my narrow field of interest. And when I started to attend the meetings of the Annual Conference on the Nephrotic Syndrome, the real issue was “Is the immune response involved in the pathogenesis of renal diseases?” And I think today, after forty years of work, we can categorically state that yes, the immune response is responsible for many types of renal diseases. In order to achieve this results, it was necessary to take many different roads, to recognize many mistakes, but I think there is convincing evidence that the immunological response, and also, today, we know this in detail, the various facets of this response, antibody-mediated, cell-mediated, combined, play a key role. But at the same time, despite awareness of this progress, there are many disappointments, because we still, especially in the western world group of Nephrology, which are involved in the study of autoimmune mechanisms of renal disease, we still don’t know which are the antigens involved. And so from this point of view, for example, glomerulonephritis, the antigen is still unknown after fifty years of investigation. So from this point of view, there is a certain dissatisfaction. On the other hand, I think probably most studies should be interested or involved in various types of nephropathies, which occur in third world countries, where there is less need for the study of renal diseases. Obviously in addition to the progress in understanding the aetiology and the pathogenesis of the renal disease, the major progresses were in the area of hemodialysis, peritoneal dialysis, and transplantation. When I started as a medical student, a young physician, renal diseases were not treatable. I still remember when we were trying to apply to expose our patient to uremia, to heat in order to promote the excretory function of the skin, to produce the release of urea from the skin. There was no other type of treatment that was available. Nowadays, we are able to give a second shot at life to most patients with chronic renal disease, so this progress has been fantastic. Now, with the advancement of modern technique in molecular biology, and in the possible use of stem cells, we certainly have opened new avenues of research, for young nephrologists, and new possibilities for treatment for our patients.