How did malaria research invade my life, as malaria does the red blood cell? It had a great deal to do with several lucky breaks—meeting certain people who encouraged me---as well as a persistent desire on my part to see nature through the eyes of science.

My first brush with one-celled animals began in high school when I enrolled in an advanced biology class taught by Mrs. Marion Sweet. Though this course consisted of her "sweet-talking" us, the study of dead and pickled specimens, and the submission of a detailed set of lecture notes and drawings, I was entranced by the variety of life to which she introduced me. At age 16 I simply wanted to follow in Mrs. Sweet’s footsteps and teach high school biology. (An added bonus was that Mrs. Sweet was cute, blonde and sexy).

After graduating from James Monroe High School, I enrolled at the City College of New York (CCNY) where I majored in Biology and Education. CCNY was not known for its research-oriented faculty, but it was famous for its academic rigor and a student body filled with high achievers. As the time of graduation approached, several of my Biology professors, especially James Dawson (the Biology Department Chair and a protozoologist), William Tavolga (a behaviorist/histologist) and Herman Spieth (a Drosophila geneticist/behaviorist) encouraged me to go on to graduate school and abandon my career goal of high school teaching. One said, "Why not become a college faculty member like us?" Through personal contact between Tavolga and Spieth I was introduced to Libbie Hyman at the American Museum of Natural History and Dawson and Spieth arranged that W.C. Allee (who because of his age had been forced to retire from the University of Chicago and was now heading the Zoology Department at the University of Florida, Gainesville) accept me as a graduate student, and in turn Allee arranged a research assistantship for me with the protozoologist James B. Lackey in the Department of Sanitary Engineering.

My first independent research involved a survey of oligotrich ciliates in the Gulf of Mexico. Just as I was becoming proficient with ciliate taxonomy and behavior I received a letter from the Selective Service Board—I was drafted into the Army. Since the Korean War was at an end, and the need for draftees was diminishing, I resented this interruption of my graduate studies, but as luck would have it, this turned out to be an opportunity to learn some parasitology and to travel the world at Uncle Sam’s expense. After infantry basic training (Fort Dix, New Jersey) I was sent to Fort Sam Houston in Texas and then to Valley Forge Army Hospital in Pennsylvania to be trained as a medical technologist. I was then shipped overseas, where through good fortune I was assigned to medical laboratories in Salzburg, Austria and Darmstadt and Heidelberg, Germany and not made to drive a truck. In Germany and Austria I worked in clinical laboratories doing hematology, parasitology, microbiology, phlebotomy and blood chemistry. Upon my discharge from the Army I decided not to return to Florida to continue my graduate studies; instead I began to teach science and math in a junior high school in Yonkers,

At the same time I took a few graduate courses at CCNY and assisted in the teaching of the evening session introductory biology labs at CCNY. Knowing of my interests in research, William Etkin, an endocrinologist/behaviorist and a professor at CCNY, encouraged me to return to graduate school on a full-time basis. He said: "If you want to run with the hounds and do some original research get yourself an advanced degree." It was sage advice.

During the summer of 1957, thanks to a CCNY Biology Club Scholarship, I took the Invertebrate Zoology course at the Marine Biological Laboratory (MBL) in Woods Hole, MA. There I discovered what biology was all about. At the MBL I was able to spend all day, every day, for 10 weeks studying live invertebrates. The field trips were extraordinary, especially for a South Bronx slum kid, and the faculty were exceptional: Clark Read (Rice), Howard Schneiderman (Case Western), John Buck (NIH), Theodore Bullock (UCLA), Grover Stephens (Minnesota) and Ralph Smith (Berkeley. At the end of that summer I was invited to be the lab assistant for the course for the next two summers, I readily accepted and shared the work with another young parasitologists, Frank Friedl, from the University of Minnesota.

The experiences in Florida, the Army and Woods Hole crystallized my interests in combining protozoology with clinical disease. Not wanting to return to Florida for graduate work (because I found the department to be too heavily oriented toward herpetology and ichthyology) I scoured University catalogs for those offering the greatest number of courses in parasitology and eventually settled on Northwestern University (or more accurately, they settled on me). I was awarded an Abbott Laboratory Fellowship with Robert Hull (a student of the eminent protozoologist R.R. Kudo) who was carrying out research with the bird malaria Plasmodium lophurae. The bug bit me, and from that time forward I was intellectually infected with malaria!

The winter before I completed my Ph.D. I spent the Christmas holidays in New York City visiting with my parents. During that time I dropped in to see Frank Friedl, with whom I had shared the Invertebrate Zoology lab assistant duties. Frank had finished his Ph.D. and was now a post-doctoral fellow at the Rockefeller University working in the laboratory of the distinguished helminthologist Norman R. Stoll, author of among many papers, "This Wormy World." Frank suggested that I stop in and see William Trager on the third floor of Theobald Smith Hall, just across from Stoll’s labs. Trager was the "dean" of malaria researchers and although I had no appointment, he graciously listened to my account of what I was doing and heard my future plans. This accidental encounter affected my scientific career for the next 40 years, when Trager offered me an NIH post-doctoral fellowship at Rockefeller beginning in the Fall of 1960.

For my doctoral thesis I had studied the changes in serum proteins in the host (chicken) infected with P. lophurae and also characterized malaria pigment (hemozoin), but when I arrived at Rockefeller my research interests shifted largely as a result of Trager having attended a conference in Florida where the other speakers were Julius Marmur (Brandeis) and Ernest Bueding (Johns Hopkins). Bueding was studying, among other things, enzyme heterogeneity in schistosomes, and Marmur was carrying out a survey of the base composition of DNA from a variety of organisms. Trager "volunteered" me to provide Marmur with large quantities of erythrocyte-free P. lophurae. When Marmur’s colleagues at Brandeis, Nathan Kaplan and Alan Wilson, heard of the availability of gram quantities of malaria parasites they too wanted some for their work on lactic dehydrogenase (LDH) heterogeneity. Trager, now my mentor, suggested that I concentrate on the LDH of P. lophurae and its duckling host. I was soon able to separate lophurae LDH from that of the red blood cell by starch gel and starch block electrophoresis with the help of Elliot Vessel and Philip D’Alesandro (at Rockefeller) and during a visit to Brandeis discovered that lophurae LDH had an exceptionally high affinity for the acetylpyridine analog of NAD. This proved to be the case for other species of Plasmodium and today LDH activity with acetylpyridine-NAD serves as the basis for the diagnostic test (OptiMal) for human malaria infections. I was now a budding biochemical parasitologist.

During my time at Rockefeller I studied red cell receptors for merozoite invasion and attempted to improve the in vitro culture medium. The duckling red cell and P. lophurae turned out to be poor experimental material since I found it impossible to block the invasion of lophurae merozoites into duckling red cells by their pre-treatment with enzymes in order to strip them of their merozoite receptors. This was due to the very high affinity the lophurae merozoites had for the duckling red cell. (Had I chosen to work with human red cells and P. knowlesi or P. falciparum I might have been able to identify the red cell receptors that Louis Miller did some years later). I learned an important lesson from this experience: choose the experimental system wisely.

Although I was quite happy at Rockefeller – truly a researchers paradise—I still longed to see whether I could carry out an independent research program and I also wanted an opportunity to teach. In 1962 I was offered the position of Assistant Professor of Biology at the University of California at Riverside (UCR) where one of my former CCNY professors (Herman Spieth) had moved in 1954 to head up the Department of Biology. By the time I arrived at UCR Herman was the Chancellor. At UCR I continued to work on enzyme heterogeneity (malate dehydrogenase, glutamic dehydrogenase, cathepsin D) and with a graduate student (Kenneth Yamada) and a post-doc (Christina Schimandle) isolated and characterized several P. lophurae purine salvage pathway enzymes (adenosine deaminase, purine nucleoside phophorylase, hypoxanthine phosphoribosyltransferase). We used P. lophurae growing in ducklings because it allowed us to obtain large quantities each week very inexpensively. At seminars, in discussing our research, I had a standard line that went: grams of parasites costing only a buck a duck. I was convinced (as was Bill Trager) that the lophurae-duckling system was an appropriate model for human malaria since P. falciparum was unavailable, having not yet been cultured continuously. Our work with lophurae provided a basis for subsequent work by others on the P. falciparum purine salvage pathway enzymes.

My first graduate student Charles Walsh isolated P.lophurae DNA, found it to be exceedingly A+T rich (80%) and characterized the pyrimidine pathway. When in 1968 we published the base composition work (confirming Marmur’s unpublished findings 6 years earlier) we were told that it had no "real" significance since this was a bird malaria with little in common with mammalian malarias such as P. falciparum and P. vivax. It was gratifying to find that some 10 years later when the P. falciparum DNA base composition was described as 80% A+T that lophurae DNA was closer to the human parasite than other mammalian malarias. Our finding of the de novo pathway for the synthesis of pyrimidines provided a biochemical basis for the mechanism of action of anti-folate antimalarials that were studied by Robert Ferrone and George Hitchings.

From 1957 through 1980 I spent summers at the MBL reading in the library (a jewel) and teaching in the Invertebrate Zoology and Biology of Parasitism courses, where among others, I had an opportunity to rub shoulders (both on and off the beach and at many great Woods Hole parties) with one of the "giants" of parasitology, Clark P. Read. Read and his students were testing the hypothesis that parasites (especially dogfish and rat tapeworms) had low regulatory capacities and host homeostasis of amino acids was necessary for balanced protein synthesis. Stimulated by their work we began a 10 year study of amino acid transport (some of it with Ragunath Virkar, a student of Grover Stephens and an MBL colleague) as well as protein synthesis and ribosome characterization (with Jim Williamson and Bob Cox at the National Institute for Medical Research, Mill Hill England during a sabbatical year) in P. lophurae and P. knowlesi. We also disproved the hypothesis that malaria parasites were parasitic because they had to obtain a ribosomal subparticle from the host. Purine and pyrimidine transport studies were carried out with a graduate student, Susan Tracy, and we also studied glucose transport. During the course of the studies with purines we found hypoxanthine (not adenosine) to be the preferred purine for intracellular growth of P. falciparum. This explained why the parasites grow much better when the in vitro culture medium for P. falciparum is supplemented with hypoxanthine.

Knowing the value of good collaborators, I enlisted the support of a UCR colleague, Irwin P. Ting (a plant physiologist), who was working on carbon dioxide fixation in corn. It wasn’t long before we discovered that both P. lophurae and P. knowlesi fix carbon dioxide. Ting once assured me: all organisms fix carbon dioxide. In another collaboration with a UCR biochemist, Brian Mudd, we were able to document the striking changes in free amino acids in the lophurae-infected red cell.

Transport studies with P. lophurae led, almost naturally, to a characterization of the transport interface—the surface membranes of the parasitized red cell and the parasite itself. Work on membrane characterization had been undertaken earlier in collaboration with a post-doc from Japan, Yuzo Takahashi, we demonstrated by transmission electron microscopy and cytochemistry that there were distinct alterations in the membrane of erythrocytes infected with P. lophurae as well in the character of the membranes of the parasite itself.

We next moved on to membrane isolation and fractionation. Some of the basic techniques for the isolation and characterization of membranes were acquired during a summer at the MBL when Leonard Warren (Wistar Institute) and I shared a laboratory and worked on both and duckling dogfish red cell membranes. This work was continued at UCR using the duckling red cell and P. lophurae. We collaborated with George G. Holz (a colleague from MBL) on the fatty acid and lipid composition of the isolated membranes of the infected red cell and the free parasite. In 1976, after Trager and Jensen reported on their successful continuous in vitro cultivation of P. falciparum, the focus of our lab moved from the malaria of birds to humans. In some ways this shift in emphasis was regrettable since P. lophurae infections in young ducklings (unlike P. falciparum) provided exceptionally large amounts of material for biochemical analyses, but thankfully our decades of experience with bird malaria were of considerable benefit to us (and other researchers) in reaching a fuller understanding of the biochemical and molecular interactions of P. falciparum with the human erythrocyte.

One of the most interesting aspects (at least to me) of the P. falciparum-infected red cell was the dramatic change in the surface composition and morphology (knobs) of red cells that had been described from the laboratories of Bill Trager and Masamichi Aikawa. Patricia Maguire and Shigetoshi Eda, post docs in the lab, documented alterations in membrane cholesterol, phospholipids and exposure of phosphatidylserine. Together with Jean Gruenberg (a post doc from Geneva, Switzerland) and a graduate student, David Allred, changes in the size and distribution of the knobs, as well as a clustering of intramembranous particles (IMPs) in the region of the knob were described. I became interested in determining the biochemical alterations in the red cell surface that contributed to the changes in antigenicity and adhesivity of the falciparum infected red cell after I spent a sabbatical at the Walter and Eliza Hall Institute in Melbourne, Australia. Enrique Winograd, a grad student and later a post doc in the lab, was able (after being taught the lore of hybridomas by a UCR plant virologist, David Gumpf) to prepare a monoclonal antibody that was anti-adhesive and the antigen recognized was not a parasite-encoded protein (i.e. PfEMP-1) but instead was related to the anion transporter of the red cell, band 3. Two post docs, Ian Crandall and Neil Guthrie, and a graduate student Kirkwood Land made other monoclonal antibodies, identified their epitopes and then used synthetic peptides based on the epitopes to block the adhesion of infected red cells to endothelial cells. In collaboration with Birte Hogh (Copenhagen, Denmark) the sera from adults living in an area where malaria is endemic (Liberia) and who are presumable immune, were found to have antibodies to these peptides. Again, thanks to a collaboration, Jurg Gysin (Pasteur Institut) and Bill Collins (CDC) infused falciparum-infected monkeys with synthetic peptides which resulted in a flood of mature forms into the peripheral blood; this result suggested the peptides were active as adhesion blockers in vivo. Enrique Winograd identified the endothelial cell receptor for one of the band 3 peptides as CD36, and Shigetoshi Eda and Jesus Lucas (both post docs) found another peptide sequence of band 3 to mediate binding to the type 3 repeat (RGD) of thrombospondin. We never found merozoite receptors, but did identify endothelial cell receptors that bind the falciparum-infected red cell. We hypothesized that sickle cells would adhere to endothelial cells through the band 3 epitopes, and were pleased by the findings by Stephen Shohet and Bernard Thevenin (UC San Francisco) that the peptides (based on band 3 amino acid sequences) blocked sickle cell adhesion to human umbilical vein endothelial cells. We continue to explore the possibility of using synthetic peptides, based on amino acid sequences in band 3, as well as peptidomimetics as "molecular teflons" and/or vaccine candidates.

Recently, Enrique Winograd was able to show that the adhesive sequences in band 3 are cryptic in the uninfected red cell, but with intra-erythrocytic growth of the parasite there is clustering of band 3--- evidenced by the IMP work by Allred---and this clustering is sufficient to expose the adhesive epitope and make the infected red cell "sticky".

My more than four decades of parasitological research has been peripatetic--involving biochemical, chemotherapeutic and immunological studies of malaria—sometimes in birds and sometimes with human red cells. Were it not for several perceptive professors during my undergraduate days at CCNY, who saw something in me that I did not know was there, I might have become a "talking head" high school teacher rather than leading a life posing questions to nature, answering some, and through it all enjoying the struggle to better understand the nature of parasitism. One of the great joys of being a malaria researcher has been the opportunity to share these voyages of discovery with others--- bright, talented and creative colleagues, and to see them launch their own research careers. Being a University professor has enabled me to have a professional life filled with productive research as well as teaching —but the instructional part of the story will have to wait for another time. Fiat lux!