HLA-A and -B alleles in cornea donors as risk factors for graft rejection.

We determined the human leucocyte antigen (HLA)-A, -B and -DR allele frequencies in recipients and donors of 115 cornea transplants, for recipients who developed graft rejection and those who did not. No difference in HLA allele frequencies of the recipients was found. The frequencies of the HLA-A26, -B35 and -B44 alleles in cornea donors were increased in recipients who developed graft failure. The detrimental effect on corneal graft survival of these alleles was significant (p < 0.001). No such effect was observed in renal transplantation. Corneal graft survival was similar when one or two A26, B35 or B44 alleles were present on the donor cornea. The negative effect was similar in magnitude to the previously reported negative effect of an HLA-B locus match between donor and recipient. When both a B-locus match and an A26, B35 or B44 allele were present, the negative effect on graft survival was twice as strong, indicating that different immune mechanisms are responsible for these phenomena.

A unique African HLA haplotype may identify a population at increased risk for kidney graft rejection.

We determined HLA-A, -B, and -DR allele frequencies in kidney transplant recipients in relation to graft survival. Most recipients and donors were from African descent. The frequency of HLA-A30 was somewhat increased in recipients who rejected the graft. The frequency of HLA-B42 was significantly (P=0.002) increased in recipients who rejected the graft. Similar results were found for the HLA-DR3 allele; however, this effect was diminished when B42-, DR3+ individuals were analyzed. We further investigated the effect on transplant outcome of a unique African haplotype A30, B42, DR3, and of segments thereof, which have a high frequency in the local population. Log-rank analysis revealed that the negative effect on transplant outcome was least in A30-, B42+ recipients (P=0.417) and most pronounced in A30+, B42+ patients (P=0.006). We postulate that the negative effect on transplant outcome may reside in the A30, B42 segment of chromosome 6 and may be caused by a stronger than average immunoregulatory gene.

An adverse matching effect for the HLA-B locus in corneal transplantation.

The results of tissue typing on 115 recipient/donor pairs prior to corneal grafting were analyzed with the proportional hazard regression model for the incidence of the first rejection episode and for graft failure from rejection. Like other investigators, we found that a previously failed corneal graft and the degree of recipient corneal vascularization were significant risk factor for graft rejection. ABO blood group matching had no effect. The absence of mismatches in both the HLA-A and HLA-DR loci decreased the incidence of rejection. However, no difference was observed for the presence of one versus two mismatches. Regression results for the HLA-A and DR loci were not significant. Surprisingly, matching for one or both HLA-B alleles resulted in a significantly higher incidence of graft rejection episodes (P < 0.005) and of graft failure (P < 0.052). This adverse matching effect for the B locus was proportional to the number of mismatches.

C4 polymorphism and extended HLA haplotypes in Namibian San and Khoi and in South African Xhosa.

We studied C4A and C4B polymorphisms and HLA-B and -DR associations in the San, Khoi and Xhosa. C4A and C4B alleles were determined using conventional protein allotyping methods. The C4A*3, C4B*1 haplotype had a high frequency (30-55%) in all populations. The frequency of C4A*3, C4B*Q0 was 7-19%. The C4A*Q0, C4B*1 haplotype was frequent (15%) in the Khoi but very rare in the San (P < 0.001). C4A*12 A*91, C4B*Q0 was frequent in the Xhosa (15%) but rare in the San and Khoi (P < 0.001). Alleles C4A*5 and C4A*6, and the C4B*2 B*92 duplication were only found in the Xhosa. C4A alleles A*4, A*45, A*58, A*12, A*14, A*19 and the C4A*3 A*91 duplication were only found in the San/Khoi population group. In the San, fourteen extended haplotypes were found in a relatively high frequency (2-7%). In the Xhosa, one extended haplotype (B42, C4A*12 A*91, C4B*Q0, DR18) was found in a very high frequency (13%) and was characteristic for this group; five other extended haplotypes were found with a low frequency (< 3%).

DBP (vitamin D binding protein) and BF (properdin factor B) allele distribution in Namibian San and Khoi and in other South African populations.

The genetic polymorphism of vitamin D binding protein (DBP) and of properdin Factor B (BF) was determined in unrelated Namibian San and Khoi, and in South African Blacks, Caucasoids and Cape Coloureds. Alleles have been confirmed by segregation patterns in family studies. The DBP phenotypes were identified by isoelectric focusing on ultrathin polyacrylamide gels and the BF phenotypes were identified by electrophoresis on 1% agarose gels; both methods were followed by immunofixation. The DBP and BF allele frequencies for all population groups were found to be in accordance with Hardy-Weinberg equilibrium. DBP*1S and BF*S allele frequencies in the San, Khoi and Blacks were similar; their frequency was far lower than in Caucasoids. The frequencies of the DBP*1F and BF*F were also similar in the San, Khoi and Blacks; however, the allele frequency was much higher in these groups than in Caucasoids. These differences were statistically significant (P < 0.001).

Folding of terminal histone-H1 peptides in the presence of the oligonucleotide 5'-(AT)6-3'.

Peptides H1(1-16) H1(204-218) of human histone H1, comprising the terminal parts of the N- and C-domain, and H1(120-210), comprising the entire C-domain of calf thymus H1, were studied using CD spectroscopy in the presence of trifluoroethanol (TFE) and the oligonucleotide 5'-(AT)6-3'. TFE induces a strong negative ellipticity at 220 nm, showing that the H1 fragments are capable of helical folding. The CD spectrum of free (AT)6 shows strong negative and positive absorptions in the 200-300 nm region resembling the psi-spectrum of DNA. Free (AT)6 showed no helix-coil transition and remained single stranded at room temperature. Combinations of the H1 peptides with increasing concentrations of (AT)6 in low-ionic-strength phosphate buffer developed a strong negative ellipticity at 235 nm. This ellipticity increased with rising (AT)6 concentration and diminished when the (AT)6 concentration exceeded the 1:1 molar ratio in H1(1-16) and H1(204-218) and the 2:1 molar ratio in H1(120-210). The 235 nm ellipticity is attributed to a complex of the H1 peptide with (AT)6 in which the protein is helical. Interaction between histone peptide and (AT)6 is also indicated by UV-absorption spectra which show that the 260 nm absorption is decreased and the 280 nm absorption is increased as compared to free (AT)6. The free peptides show no absorption in this window. The altered 260 and 280 nm absorption suggests that the single-stranded (AT)6 assumes a left-handed pitch and this is confirmed by the displacement of the 270 nm positive ellipticity of free (AT)6 towards 260 nm. Implications of a left-handed linker DNA for chromatin function are discussed.

The value of variable number of tandem-repeat polymorphisms in cases of disputed paternity not resolved by conventional markers: two case reports.

Disputed paternity cases are routinely tested in the authors' laboratory for red cell antigen, plasma protein, red cell enzyme, and HLA polymorphisms. This report concerns two cases in which the above test results made exclusion of paternity doubtful. In one case, exclusion of paternity was based on one discrepancy in the Duffy blood group system only, a unique situation in the investigators' experience of more than 2500 cases; the investigators were, therefore, reluctant to use this as the only evidence of exclusion. In the other case, it was necessary to postulate the presence of a rare haplotype, MSu, in the MNS blood group system to explain paternity. It was therefore decided to investigate allelic variable number of tandem-repeat (VNTR) DNA polymorphisms in these disputed paternity trios. VNTR DNA typing convincingly excluded these accused men from paternity.

Analysis of absolute T helper cell number and cellular immune defects in HIV antibody positive and negative homosexual men.

To investigate if serial measurement of T helper (CD4) lymphocyte number in peripheral blood is of prognostic value, we determined lymphocyte function in asymptomatic and symptomatic HIV antibody positive and negative homosexual males and related the results to absolute number of CD4 lymphocytes in peripheral blood. Lymphocyte function was determined by measuring streptolysin O (SLO)-induced proliferative responses of peripheral blood lymphocytes (PBL) and of PBL depleted of CD8 lymphocytes. Phytohemagglutinin (PHA) induced interleukin-2 (IL-2) production was also measured. In all functional tests values were significantly lower in HIV antibody-positive subjects than in HIV antibody-negative subjects. Results lower than the 95% confidence limit in HIV antibody-negative individuals were therefore defined as "decreased." Decreased functional responses were most frequent (83-100%) in individuals with a number of CD4 lymphocytes of less than 400/microliters, and were least frequent (3-21%) in subjects with a CD4 lymphocyte count of greater than 600/microliters. Frequency of decreased functional responses was intermediate in the population with 600-400/microliters CD4 lymphocytes. The magnitude of functional responses differed significantly between groups with less than 400, 400-600, and greater than 600 CD4 lymphocytes per microliter, indicating that T helper cell number decreases with loss of immune function.

Determination of co-expression of activation antigens on proliferating CD4+, CD4+ CD8+ and CD8+ lymphocyte subsets by dual parameter flow cytometry.

The incidence of activation markers on proliferating CD4+, CD4+ CD8+ and CD8+ lymphocyte subsets was determined in a single laser Epics-C fluorescence-activated cell sorter system, using a series of double staining combinations. Experiments were performed after 3 days of culture with PHA on cell fractions enriched for CD4+ or CD8+ lymphocytes before initiation of culture. The percentage of CD4+, CD4+ CD8+ and CD8+ lymphocytes in the total population was determined using double staining with Leu3 PE for the detection of CD4+ cells, and Leu2 FITC for the detection of CD8+ cells. Next, double stainings with Leu3 and Leu2 antibodies conjugated with PE and antibodies directed against activation markers (M) IL-2 receptor, transferrin receptor, HLA-DR antigen and CALLA conjugated with FITC were performed, using the following combinations: Leu3 and Leu2/M, Leu3/M and Leu2/M. The expression of activation markers on CD4+ CD8+ lymphocytes was calculated from the results. Our findings indicate that CALLA is expressed on most CD4+ and all CD4+ CD8+ cells, and on a small percentage of CD8+ lymphocytes; the IL-2 receptor was expressed on most CD4+ cells, on approximately three-quarters of CD4+ CD8+ cells and half the CD8+ cells; HLA-DR was expressed on a small percentage of CD4+ cells, all CD4+ CD8+ cells and half of CD8+ cells. The transferrin receptor was almost exclusively expressed on CD4+ CD8+ cells. The standard deviation of the calculated values did not exceed 13% and this analysis can generally be applied to determine the co-expression of a third marker in a mixture of single and double stained cells using conventional methods.

Increased interleukin-2 receptor expression after mitogen stimulation on CD4- and CD8-positive lymphocytes and decreased interleukin-2 production in HTLV-III antibody-positive symptomatic individuals.

We investigated the expression of the interleukin-2 (IL-2) receptor on phytohaemagglutin-stimulated peripheral blood lymphocytes from homosexual men with persistent generalized lymphadenopathy, the prodrome of the acquired immune deficiency syndrome. The subjects were positive for antibody against human T-cell lymphotropic virus III. Using two-colour fluorescence flow cytometry, IL-2 receptor expression was determined on both the CD4- and CD8-positive lymphocyte subpopulations. After 48 hr of stimulation, expression of the IL-2 receptor on both T-cell subsets was significantly increased in lymphadenopathy patients as compared to values in heterosexual age-matched controls; this difference was less after 72 hr of stimulation. Results from two AIDS patients were within the normal range. IL-2 production was significantly reduced in both lymphadenopathy and AIDS patients as compared to values in heterosexual controls. We conclude that a defect in IL-2 production is associated with human T-cell lymphotropic virus III infection, but that the expression of the IL-2 receptor on T cells is not greatly affected.

Evaluation of natural killer cell activity in patients with persistent generalized lymphadenopathy and acquired immunodeficiency syndrome.

Natural killer (NK) cell activity was quantitated using 51Cr release from the human erythroleukemia cell line K562 in 39 heterosexual males, 60 asymptomatic homosexuals, 39 patients with persistent generalized lymphadenopathy (PGL), and 16 patients with acquired immunodeficiency syndrome (AIDS). PGL and AIDS patients showed a slight decrease in NK cell activity compared to control groups. Absolute numbers of Leu 11a-positive cells were decreased in PGL and AIDS patients, and this decrease correlated with a decrease in absolute number of both the T4+ and T8+ cell subsets. Autologous plasma inhibited NK cell activity in 48% of asymptomatic homosexuals, 63% of PGL patients, and 63% of AIDS patients, but in none of the heterosexual controls. NK cell responses in fetal calf serum, normal human plasma, or autologous plasma showed no correlation with absolute numbers of T4+ cells, or with T4/T8 ratio. We conclude that NK cell responses are not of prime importance in the pathogenesis of PGL and AIDS.