Chester M. Zmijewski

Gene dosage and susceptibility to insulin‐dependent diabetes

1. All members of 33 families in which two or more sibs have insulin‐dependent diabetes mellitus (IDDM) were HLA‐typed. The results strongly support the hypothesis that, closely linked to the HLA region, there is a locus (S) for susceptibility to IDDM. We use Sd for alleles at this locus which confer susceptibility to disease, and Sa for all other alleles.

A recent decrease in the time to development of monomorphous and polymorphous posttransplant lymphoproliferative disorder.

&NA; We have noted a decrease in the time to development of posttransplant lymphoproliferative disorder (PTLD) over the last two and one-half years in our multiorgan transplant program. From February 1965 until December 1990, 1622 transplants were performed including 1489 kidneys (KTxp), 87 livers (LTxp), and 46 pancreata. Between February 1965 and July 1988 (group 1), there were 1260 transplants performed and nine cases of either monomorphous PTLD (M-PTLD, n=8) or polymorphous PTLD (P-PTLD, n=1) were diagnosed. The mean time to development of PTLD was 163±128 weeks, all after KTxp. Five of these nine patients received haploidentical living-related grafts. All patients had presented with advanced disease, none had transplant nephrectomy, and all died of their disease. Between July 1988 and December 1990 (group 2), 362 transplants were performed, and four cases of M-PTLD and three cases of P-PTLD were recognized. Of the seven cases of PTLD in group 2, six developed within 90 days posttransplant (early PTLD). The mean time to development of PTLD was 11±16 weeks. This was significantly earlier than group 1 (P<.01). Four of the five cases after KTxp had a 1 or 2 DR-matched donor. Five of these seven patients had serological evidence of recent Epstein-Barr Virus infection, and four of these five had received OKT3 and then developed early PTLD. In group 2, three patients are alive 7–15 months after KTxp nephrectomy, the remaining four have died. We hypothesize that risk factors for the development of PTLD may include heavy immunosuppression, including the use of OKT3, good DR matching, and active EBV infection. Treatment should include graft removal, if applicable, and reduction or cessation of immunosuppression.

Genetic polymorphism of the human tumor necrosis factor region in insulin-dependent diabetes mellitus linkage disequilibrium of TNFab microsatellite alleles with HLA haplotypes

The TNF region within the MHC includes a number of immunologically important genes. Microsatellites TNFa and TNFb adjacent to TNF exhibit extensive polymorphism. Employing a PCR-based technique, we identified TNFab haplotypes and defined their distribution in 97 controls and 48 diabetics of Caucasoid origin in a search for other genes within the MHC potentially associated with IDDM. Twenty-five different TNFab haplotypes were identified. A significant difference (p < 0.0005) in frequency between patients and controls was found for TNFa1b5 (relative risk 53). However, no other TNFab microsatellites demonstrated significantly different frequencies. Among diabetics TNFa1b5 was found to be in linkage disequilibrium with HLA-DR3-B18, a haplotype known to be associated with IDDM. Thus the increased frequency of TNFa1b5 among diabetics could reflect a linkage disequilibrium with a gene within the TNF region or with other genes, including the HLAs, which characterize this haplotype. In both controls and diabetics TNFa2b3 and TNFa7b4 were in linkage disequilibrium with DR3-B8 and DR7, respectively. Among diabetics, TNFa2b1 and TNFa6b5 were in linkage disequilibrium with DR4-B62 and DR4-B44, respectively. It is intriguing that TNFab haplotypes, represented by a short piece of about 200 nucleotides in the untranslated region upstream of TNF beta gene, maintain strong linkage disequilibria with different HLA haplotypes extending over 1 million base pairs. The identification of TNFab microsatellites exhibiting a high polymorphic index in a region lacking known polymorphic markers may provide potentially important information regarding the association of HLA haplotypes with autoimmune diseases, as they are in close proximity to other genes of immunologic importance.

HLA-DQw3.2 allele of the DR4 haplotype is associated with insulin-dependent diabetes. Correlation between DQβ restriction fragments and DQβ chain variation

The pathophysiology of type I insulin-dependent diabetes (IDDM) is largely unknown, but an autoimmune origin of the disease has been suggested (Bottazzo et al. 1983). Population studies indicate that HLA antigens, especially the class II products, DR3 and DR4, are implicated in the susceptibility to the disease (Rubinstein et al. 1977, Svejgaard et al. 1983). However, because of linkage disequilibrium, it is unclear whether the HLA-DR antigens themselves or the products of other genes closely linked to HLA-DR, such as DP or DQ, are responsible for the disease susceptibility. Furthermore, DR and DQ molecules associated with the DR4 haplotype are heterogeneous (Groner et al. 1983, Holbeck et al. 1985, Bontrop et al. 1986). Therefore, we have used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Southern blotting to analyze the DQe alleles found among DR4-positive controls and IDDM patients. After establishing the correspondence of proteins to restriction fragment length polymorphisms (RFLPs), we studied a large number of DR4-positive controls and diabetics to determine the frequency of these polymorphic fragments in the two populations. We are thus able to examine polymorphic variation at the protein and DNA levels simultaneously, and to consider the possible significance of the DQ region for susceptibility to IDDM. • A panel of 12 DR4-positive cell lines has been generated by Epstein-Barr virus transformation of cells from IDDM patients and has been used for the present study. DQ¢ chains of the DR4 haplotype in DR3/4 heterozygotes were identified by using conditions tailored to exclude the DQ~ chain associated with the DR3 haplotype

SPECIES DISTRIBUTION OF HUMAN TISSUE ISOANTIGENS. I. DETECTION OF HUMAN TISSUE ISOANTIGENS IN CHIMPANZEES

Human tissue isoantigens were detected on chimpanzee leukocytes and on a continuous heteroploid cell line of chimpanzee origin. The antisera used to detect these isoantigens were taken from multiparous women, multiparous chimpanzees, and chimpanzees immunized with human leukocytes and platelets. These sera were known to contain antibodies to human leukocyte isoantigens. The methods used to demonstrate the isoantigens were leukocyte agglutination and inhibition of leukocyte agglutination, as well as mixed agglutination and inhibition of this reaction.

Mixed lymphocyte reactivity in cats

A method was developed to perform primary one-way mixed lymphocyte cultures (MLC) with cat cells. A polymorphic system of MLC reactivities was found within a cat population. In family studies, alloreactivity segregated as a single genetic locus with codominantly expressed products, designated feline lymphocyte defined (Fld)-although closely linked multigene control remains possible. The relationship of Fld to a putative feline major histocompatibility complex is discussed.

Analysis of DR and DQ gene products of the DR4 haplotype in patients with IDDM: Possible involvement of more than one locus

Fifteen DR4-bearing haplotypes from twelve patients with insulin-dependent diabetes mellitus (IDDM) were analyzed serologically, cellularly, and biochemically. The HLA-Dw composition of these DR4-positive haplotypes was Dw4 (46%), Dw14 (22%), and Dw10 (33%). The biochemical analysis by two-dimensional electrophoresis (2D-PAGE) of the DR beta chains showed that each Dw specificity is characterized by a specific DR4 beta chain that appears to be identical in normal and diabetic individuals. Analysis of DQ beta chains in the DR4-bearing haplotypes revealed that certain Dw specificities such as Dw4 are characterized by the presence of either the DQw7 (formerly DQw3.1) or DQw8 (formerly DQw3.2) alleles, which generate the Dw4.1 or Dw4.2 subtypes, respectively. Others such as Dw14 and Dw10 are characterized by the presence of the DQw8 allele. In our sample of 12 patients the Dw4.2 (Dw4, DR4 beta I-4 DQw8) and Dw10 (Dw10, DR4 beta I-1, DQw8) subtypes were predominant. It is concluded that individual DR beta and DQ beta gene products from the DR4-bearing haplotype of IDDM patients are identical to those of normal control subjects and that Dw14 as well as Dw10 are involved in disease susceptibility. We suggest that disease susceptibility may be influenced by more than one locus within the HLA-D region.

Subpopulations of lymphocytes in maternal peripheral blood during pregnancy

The fetus can be considered an allograft with up to one-half of its MHC antigens being potentially recognized by the mother as foreign. This study compares expression of OKT3, OKT4, OKT8, Kappa, Lambda and Ia antigens on lymphocytes in the peripheral blood of normal non-pregnant women, normal pregnant women, patients who are chronic spontaneous aborters and pregnant insulin-dependent diabetic women. Monoclonal antibodies and cytofluorometric analyses were used for these determinations. There were no significant differences (P = 0.01) between these groups for T-cell markers. A statistically significant (P = 0.001) increased ratio of cells bearing surface immunoglobulin to those expressing Ia antigen (K&L/Ia) was observed between normal non-pregnant controls and women with a history of chronic spontaneous abortion. It is concluded that T-lymphocytes in the peripheral blood do not demonstrate a phenotypic abnormality that would account for the non-rejection of the fetal allograft; however, women with chronic spontaneous abortion may have abnormal B-cell differentiation or T-cell activation that mediates chronic spontaneous abortion.

Renal transplantation despite a positive antiglobulin crossmatch with and without prophylactic OKT3.

The antiglobulin crossmatch (AGXM) is a sensitive technique employed by many transplant centers to enhance detection of preformed antibody to donor antigens that may cause hyperacute rejection. However, positive AGXM may detect irrelevant or very low titers of anti-HLA antibody precluding transplantation in suitable recipients. To investigate the significance of a positive AGXM, cadaveric renal transplantation was carried out despite a weakly positive AGXM (defined as cell killing above background but not greater than 20%) in 48 recipients. In an initial group (n = 10), maintained on triple therapy (cyclosporine, azathioprine, and prednisone), accelerated acute rejection occurred in 4 recipients and 3 grafts were lost. A subsequent group (n = 38) was treated with a prophylactic course of OKT3 then triple therapy. There were no episodes of accelerated acute rejection (P less than 0.01) although clinical hyperacute rejection claimed one graft and the incidence of delayed graft function was high (75%). The prophylactic OKT3 group had a reduced incidence of acute rejection (0.5 versus 1.0) per recipient and the onset of first episodes was delayed (mean onset: 13 versus 35 days after transplantation). One year actuarial primary graft survival was 88% in the prophylactic OKT3 group as compared with only 50% in the initial group. The outcome in the positive AGXM group was similar to a concurrent group (n = 32) with a negative AGXM and immediate graft function. On the other hand, the subset of positive AGXM regraft recipients treated with prophylactic OKT3 fared poorly, with a 36% (4/11) incidence of primary nonfunction. In summary, a positive AGXM, as defined in this report, is not a contraindication to primary renal transplantation--in fact, the use of the AGXM will identify recipients that would benefit from prophylactic OKT3.

Monoclonal rat anti-MHC alloantibodies detectHLA-linked polymorphisms in humans

Two monoclonal rat anti-MHC alloantibodies detect a polymorphic determinant expressed on the peripheral lymphocytes of normal human donors. The pattern of cytotoxicity observed with these antibodies correlated with theHLA type of the individual; no HLA-A-locus specificities showed significant associations, and all of the HLA-B-locus specificities showing significant association were members of the Bw6 supertype. Family studies established that the determinant detected by the monoclonal antibodies is linked toHLA. These studies therefore provide an alternative basis for the production of monoclonal antibodies to polymorphic HLA determinants based on the conservation of polymorphic MHC determinants between man and rats.