Kamala Balakrishnan

Genetic, Immunologic and Biotransformation Studies of Patients on Procainamide

This report represents follow-up observations of a unique long-term study of patients on procainamide (PA) for various cardiac arrhythmias. Serologic and clinical evaluations associated with drug-related autoimmunity were assessed and patients were characterized for factors postulated to influence susceptibility to autoimmunity, including acetylator phenotype, oxidative metabolism of PA, HLA class profile, and production of interleukin-1 (IL-1) and tumor necrosis factor (TNF). Fifty-two percent had IgM and 70% IgG antibodies to total histones; 67% had IgG antibodies to histone H2A/H2B. Patients were equally divided between fast and slow acetylators. N-oxidative metabolism of PA was indicated by the presence of urinary nitroprocainamide, which correlated with elevated titers of antihistone antibodies. There was a significant incidence of the DQw7 split of DQw3 in PA patients when compared to controls, and the frequency of antibodies to total histones and H2A/H2B was significantly increased in the DQw7 patients. C4A*QO and C4B*QO alleles were more frequent in the PA patients than in controls. IL-1 and TNF production was not different in patients compared to controls. These data suggest that certain genetic factors may serve as markers for PA-related autoimmunity.

Recurrence of diabetic nodular glomerulosclerosis in a renal transplant.

A 36-year-old woman with a 26-year history of insulin-dependent diabetes mellitus developed chronic renal failure in 1974 and was started on dialysis. She received a kidney transplant from her HLA-identical brother. Her HLA typing showed the following antigens: A1, A28, B8, B12 (44), BW4, BW6 DR3, and DR4. Nephrectomy performed prior to transplantation showed advanced diffuse diabetic glomerulosclerosis. Her postoperative course was relatively uncomplicated, but within the next seven years she gradually developed symptoms of deteriorating renal function and hypertension. Two years later, a renal arteriogram showed 90% stenosis of the main renal artery. Biopsy of the kidney was obtained during surgical repair of this lesion and showed diffuse nodular diabetic glomerulosclerosis. Since the B8/DR3 form of diabetes is reported to have a predilection for diabetic microangiopathy and vascular complications, we are speculating that the patients antigenic composition might have enhanced the recurrence of the diabetic lesions in the transplanted kidney.

The effect of donor-specific transfusions on rat heart allograft survival

A rat heart allograft model employing donor-specific transfusions (DSTs) was used to investigate several questions relevant to their clinical usage. The effects of varying blood storage duration (stored vs. fresh), number and timing of DSTs as well as use of concomitant azathioprine and cyclosporine (CsA) were assessed in terms of allograft survival and recipient sensitization. A comparison of stored and fresh-blood DSTs revealed that blood stored for up to 5 weeks was as effective as fresh blood and that a 2-week storage was optimal. Increased storage appeared to be associated with decreased sensitization. Multiple DSTs were more effective than a single DST and the peak effect appeared after six. Transfusions given at the time of transplantation were ineffective. The addition of concomitant (preoperative) azathioprine or CsA resulted in a further decrease in sensitization but also resulted in a dosedependent diminution of the transfusion effect.

The Role of the Lymphocyte in an Immune Response

The immune system has evolved in the human being as an elaborate mechanism to distinguish itself from all else that is not self. This process serves in the defence against invaders. The cells of the immune system learn to tolerate all tissues, cells and proteins of the body. Failure to control the state of tolerance results in autoimmunity. The understanding of the role of T-cell receptors (TCR), the Major Histocompatibility Complex (MHC), adhesion molecules and growth factors in antigen recognition has lead to the exploration of various means to modulate the immune response. Safety measures exist to prevent the immune system from attacking its host. The antigen has to be recognized by the T-cell. This involves the TCR and the MHC. In addition it must receive a second signal to become activated. The second signal involves a protein such as B7 binding with CD28. Certain specialized cells, macrophages, dendritic cells and activated B-cells can deliver this second signal for activation; receipt of only one signal can prevent activation. The elucidation of the role of cell-to-cell interactions, the adhesion molecules involved and the accessory growth factors provides modalities for selectively modifying the immune response. This would be of great relevance in autoimmunity and transplantation.

Donor-specific blood transfusions with stored and fresh blood in a rat heart allograft model.

Donor-specific transfusions ( DSTs ) using stored and fresh blood were investigated in a rat heart allograft model. Lewis rats were recipients of preoperative DSTs and cardiac allografts from ACI donors. Blood was stored for 0, 1, 2, 3, 4, and 5 weeks prior to transfusion. Allograft survival and recipient sensitization were recorded. In all DST groups, allograft survival was superior to nontransfused controls (P less than 0.05). Stored blood was equal or better than fresh blood, and a 2-week storage was optimal (P less than 0.05). Storage of 2 weeks or greater resulted in decreased sensitization compared to storage for 1 week or less (P less than 0.05). This study supports the use of stored blood to enhance allograft survival and reduce recipient sensitization.

Evaluation of Flowcytometric Crossmatching in Renal Allograft Recipients

Forty-four cadaver renal allograft recipients who had flowcytometric cross-match (FCXM) testing and sequential quadruple immunosuppression were studied with respect to the number of rejection episodes and the functional viability of the graft in the first year after transplantation. Fourteen of these patients had antibodies to donor T cells by FCXM. All were negative by conventional crossmatch. Multiple-regression analysis with HLA mismatches, panel-reactive antibody (PRA) percentage, flowcytometric channel shifts and transplant number as independent variables revealed that transplant number and high PRA (> 50%) impacted (p < 0.05) on serum creatinine at 1 month and 1 year, and graft survival at 1 year. In first transplants, a positive FCXM had no impact on 1-year graft survival rates; however, in retransplants, a positive FCXM and/or high PRA had a significant negative impact on 1-year graft survival. This study indicates that the FCXM should be utilized for retransplant patients, and in patients with a high PRA, in an attempt to improve graft survival for these high-risk recipients.

Genetic and Immunologic Studies of Patients on Procainamide

Forty (40) patients with cardiac arrhythmias receiving procainamide (PA) therapy and 24 patients who were receiving other drugs for their cardiac disorders were investigated for class II HLA phenotypes and their DRB1*04 and DQB1*03 subtypes. Other genetic marker evaluations in the PA patients included: 1) class III MHC C4A and C4B null alleles of complement; and, 2) acetylation phenotype. Twenty (20) of the PA patients were also tested for the ability of their stimulated cells to secrete Interleukin-1 (IL-1 beta) and tumor necrosis factor (TNF alpha). We also examined the spontaneous production of these cytokines by peripheral blood leukocytes (PBL) from patients who were receiving chronic PA treatment. The results revealed no association of acetylation phenotypes with the class II HLA phenotypes nor class III MHC C4 allotypes in these patients. The results did show a significant increase in class III C4 complement allotypes in the PA patients when compared to the controls. The results also showed a significant increase in autoantibodies and DQw3 phenotypes in the PA patient group when compared to control populations. Results of spontaneous IL-1 and TNF production suggested there may be an association of select class II HLA phenotypes in some patients and this may be relevant to host responsiveness to PA treatment.

Flowcytometric evaluation of cytotoxic peripheral blood lymphocytes in acute renal graft rejection

The presence of the S6F1+ epitope on the surface of CD8+ lymphocytes is believed to be uniquely representative of cytotoxic subpopulations. A preliminary study was conducted to evaluate the CD8+ S6F1+ peripheral lymphocytes by flow cytometry in patients undergoing renal allograft biopsy for allograft dysfunction. Lymphocytes, obtained at the time of biopsy, were analyzed by flow cytometry with CD8-FITC/S6F1-RD1 as the test monoclonal antibody and MsIgG-RD1/MsIgG-FITC as internal control. A 100% increase in S6F1+ cells over internal control was considered to be positive result. The results were correlated with the histopathologic findings in 14 instances of allograft dysfunction occurring 26.5 +/- 11.6 days posttransplantation. The histopathologic diagnosis was acute cellular rejection in eight cases, acute tubular necrosis in four, and cyclosporine nephrotoxicity in two. Flow cytometric detection of an increase in S6F1+ cells yielded a sensitivity of 87.5% and a specificity of 83.3% for the diagnosis of acute rejection. It would appear that the use of a monoclonal antibody to detect increases in the number of CD8+ S6F1+ peripheral lymphocytes is a valuable test for the detection of acute allograft rejection in the initial period after transplantation.