Histocompatibility and Immunogenetics Laboratory
Transplantation Service, Department of Surgery
State University of New York At Stony Brook

Location:	19 Floor, Room 045, Health Sciences Center Building Stony Brook, New York 11794-8192
Telephons:	(631) 444-1789 or (631) 444-2209
Fax:	(631) 444-8907
E-mail:	Malinow@surg.som.sunysb.edu

The primary functions of the laboratory are: - histocompatibility testing for clinical transplantation, and - transplantation immunology research. Histocompatibility Testing for Clinical Transplantation The laboratory performs histocompatibility testing for clinical transplantation and disease association studies. The laboratory is licensed by the federal government under CLIA ‘88, the New York Department of Health, the American Society for Histocompatibility and Immunogenetics and the American College of Pathologists. The laboratory performs testing for kidney and bone marrow transplants. Dr. Kazimierz Malinowski is the Director of the laboratory. The laboratory also provides HLA typing and crossmatch services for the organ procurement agencies in New York.

The Laboratory is certified in the following areas: HLA Serology Typing Class I HLA Serology Typing Class II Disease Association Testing Cellular Testing - MLC DNA Testing of HLA Alleles Flow Cytometry for Transplantation Therapeutic Substances Monitoring/Quantitative Toxicology Clinical Cyclosporine Level Monitoring Clinical Talcrolimus Level Monitoring The Laboratory is certified to do histocompatibility testing for: - Solid Organ Transplantation Renal Cadaveric Renal Living - Bone Marrow Transplantation The Laboratory participates in the following national and international proficiency testing programs: - National - College of American Pathologists proficiency programs: HLA A, B, C typing, crossmatching Serology HLA ABC AB ID Serology HLA DR, DQ typing, crossmatching HLA-B27 typing Molecular - DNA - HLA ABC typing Molecular - DNA - HLA DR, DP, DQ typing Immunosuppressive Drugs - International -UCLA Tissue Typing Laboratory (Paul I. Terasaki, Ph.D. Program): HLA A, B, C Cell Exchange HLA DR B cell Line Exchange Serum Exchange for HLA A, B, C Antibody Identification

Histocompatibility Testing Vocabulary: Histocompatibility is: the state in which a donor and recipient share antigens so that a graft is accepted and remains functional. Immunogenetics is: the study of immunity to disease; and, the relationship to genetic make-up HLA antigens are: proteins on the surface of the cells in the body The function of HLA is: to help the immune system defend against invaders such as bacteria, viruses and parasites to recognize as foreign the histocompatibility antigens of other people’s cells and fight them, causing rejection of graft. HLA are polymorphic: 59 forms of HLA-A antigens 118 forms of HLA-B antigens 124 forms of HLA-DR antigens These different forms are caused by slight differences in the amino-acids that make up the proteins. HLA antigens: HLA is inherited as a "set" of the three HLA groups: A, B, DR This set is known as "haplotype" A child inherits one haplotype from each parent There are four different haplotype combinations from 2 parents What is the role of HLA antigens: play an important role in certain infectious diseases What is Tissue Typing? Tissue Typing is the name given to the test which identifies an individual’s HLA. This information is critical before a patient receives a donor organ. What is HLA matching? Because HLA antigens can be recognized as foreign by another person’s immune system, transplantation professionals try to match as many HLA antigens as possible between the donated organ and the recipient. HLA genes: A person’s genes are the blueprints that tell cells how to organize the amino-acids to make proteins. The HLA genes are on chromosome number six. The HLA genes are passed down from parent to child. Each person gets two copies of each gene, one copy from the father and one from the mother. A child will not match either parent exactly, but will be half of a match. Because each parent has two copies of each gene, there are four possible combinations of genes that could be passed down to their children. What is the Crossmatch? The crossmatch is the second test which will indicate if there is a specific immune reactivity between donor and recipient. The crossmatch is performed by mixing a small amount of the patient’s serum with a small amount of the potential donor’s white cells. If the patient has antibody to the donor’s HLA, the donor’s cells will be injured and this is referred to as a "positive crossmatch." A patient can develop antibody after a pregnancy, a transfusion, or a transplant. A positive crossmatch is a contraindication to transplant. What is Antibody Screening? A test procedure which will reveal if the patient has antibody to other Human Leukocyte Antigens (HLA). How is Antibody Screening performed? It is performed by mixing a very small amount of the patient’s serum with a small amount of cells from 60 different individuals, in separate tests. Remember the HLA are on the surface of these cells. What is Antibody Screening and PRA? PRA (Percent Reactive Antibody) is the amount of HLA antibody in a patient’s serum. If, for example, the patient’s serum reacts with 30 out of 60 HLA, then the patient’s PRA is 50%. The PRA is calculated for each monthly serum sample. In addition to determining how much or how little PRA a patient has, we need to know how specific the antibody is. Some patients have one or two antibody specificities, while others have numerous specificities. Since HLA antibody can "come and go," it is important to test the PRA regularly on a monthly basis. Histocompatibility Laboratory? HLA typing Crossmatch Antibody Screening DNA typing Immunosuppressive drug monitoring. Human Leukocyte Antigens (HLA): Cell surface structures recognized as foreign by one individual when exposed to tissue of another individual. HLA antigens play a key role in organ rejection Closer matched HLA may have greater graft survival

Ongoing Research: SUPPRESSION OF ALLOGRAFT REJECTION BY GENISTEIN Animal Study Organ transplantation from one genetically different individual to another of the same species (allotransplantation) or from one species to another (xenotransplantation) require suppression of the immune responses if the graft is to survive. Although immunosuppression (Mitotic inhibitors, Corticosteroids, Cyclosporine A, Tacrolimus, Rapamycin, OKT3, humanized IL-2R antibodies, Mycophenolic Acid, Total Lymphoid Irradiation, Antilymphocyte serum) has prolonged human graft survival, these results are frequently not satisfactory due to the side effects of used immunosuppressants. Our recent study demonstrates that Genistein (4',5,7-Trihydroxy-isoflavone) has the capacity to inhibit two important activation mechanisms of organ rejection: (a) initiation of the proliferative phase of lymphocytes; and, (b) transition of lymphocytes through the cell cycle. These effects are Genistein concentration-specific, the latter effect, and especially the transition of normal lymphocytes from the quiescent state to actively prolifering cells, was particularly sensitive to the action of Genistein. An important observation may be that allo/xeno-stimulation of human lymphocytes may be abolished by Genistein, showing small differences in individual host sensitivity to the compound. In light of the observation that allo/xeno-lymphocytes stimulation is sensitive to Genistein, this compound may hold significant potential as a novel immunosuppressant for human and animal transplantation. In this regard, the possible therapeutic value of Genistein may be enhanced by very low toxicity in vitro. To ascertain whether the antiproliferative effects of Genistein observed in human lymphocyte cultures are also lymphocyte-selective in vivo and can prolong animal and allograft survival we plan to perform series of cardiac, kidney and pancreatic islet transplantations between LEW-ACI inbred rats. Adult LEW (RT11) will be used as recipients of ACI (RT1a). These two strains of rats are "strong responders" to each other and are the standard transplantation model. EFFECT OF DIFFERENT ANTI-REJECTION REGIMES UPON THE EXPRESSION OF DIFFERENTIATION AND ACTIVATION MARKERS ON THE SURFACE OF HOST LYMPHOCYTES. Human Clinical Study The aim of this study is to correlate characteristic changes in the expression of differentiation and activation markers on the surface of peripheral blood lymphocytes from kidney transplant recipients during the course of anti-rejection therapy. Our studies included three groups of patients treated with: (1) Cyclosporine A (CsA) in combination with mycophenolate mofetil (MMF), n= 22; (2) FK506 (FK) with MMF, n=20; (3) CsA with azathioprine (Aza), n=16, and the results were compared with a control group of ESRD patients on hemodialysis (n=71). There were no significant differences in the underlying primary disease, the length of treatment and the average age of the subjects tested in the three regimes groups and control group. Each group of patients included roughly the same number of males and females. The percentile differences in the subset levels were calculated by taking the mean subset level of control group as the 100%. The most significant differences in the subset level were observed between CsA/Aza and FK/MMF groups when compared with control group. The mean of cells expressing CD3 was higher in FK/MMF and CsA/Aza groups (by 16%, P<0.0001; and by 17%, P<0.0001; respectively) and cells expressing CD19 was lower (by 44%, P<0.0001, and by 72%, P<0.0001, respectively) when compared with control group. A higher average number of cells expressing CD3CD8, CD3DR, and CD8DR double markers was seen in the FK/MMF group (higher by 46%, P<0.0001; 87%, P<0.0001; and 105%, P< 0.001; respectively) and in the CsA/Aza group (higher by 58%, P<0.0001; 63%, P<0.001; and 72%, P<0.030; respectively) when compared with control group; and CD56-16 cells was lower in the both groups by 43%, P<0.0001, and 42%, P<0.001, respectively. When comparing CsA/MMF group with the control group, a lower average number of cells expressing CD19 markers (lower by 48%, P<0.0001) and higher average number of cells expressing CD8CD57 and CD3CD25 double markers (by 43%, P<0.031; and 33%, P< 0.035; respectively) was seen. Our data indicate that the CsA/MMF anti-rejection medication may avoid a decrease of NK cells observed in the course of FK/MMF and CsA/Aza treatment. IMMUNOINHIBITORY ACTIVITY OF TUMOR TISSUE Human Study Preliminary data from our laboratory suggest conversion of host lymphocytes to a hyporeactive state by exposure to renal cell carcinoma or its extracts. Renal cell carcinoma extract-treated normal volunteers’ lymphocytes showed a marked inhibition in the mitogenic response to Concanavalin-A, Pokeweed and alloantigens. The central objective of the ongoing study is to test the hypothesis that an effective immune response to tumors might be inhibited by suppressive factor(s) generated in the micro environment of the tumor cells. In a test of this hypothesis (1) the responses of prostate-, breast-, and colon-infiltrating lymphocytes to mitogenic lectins and alloantigens will be compared with the response of blood lymphocytes of the same patient to these agents; (2) the responses of lymphocytes obtained from normal donors exposed in vitro to prostate-, breast-, and colon-extracts will be compared with extracts of corresponding normal tissue; and, (3) an attempt will be made to isolate, identify and quantitate potential immunosuppressive factor(s) generated in the micro environment of tumor cells. The primary targets are lipoprotein-bound gangliosides (Gs). These immunosuppressive compounds were found to be shed into the environment by melanoma and neuroblastoma cells. This observation raises the question whether other tumors shed in vivo ganglioside(s) which exert immunomodulatory activity. The long term goals of the proposed research are: (1) expand prostate, breast, colon and RCC protocol to the study of other malignant tumors; (2) identify the potential gene(s) responsible for "immunosuppressive factor(s)" production; and, (3) engineer the strategy to suppress this gene or develop an approach to "neutralize" potential immunosuppressive factor(s) shed by tumors.

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