MICA, HLA-B haplotypic variation in five population groups of sub-Saharan African ancestry.

The human major histocompatibility complex (MHC) class I chain-related gene A (MICA), located 46 kb centromeric to HLA-B, encodes a stress-inducible protein, which is a ligand for the NKG2D receptor. In addition to its primary role in immune surveillance, data suggest that MICA is involved in the immune response to transplants and in susceptibility to some diseases. In this study, 152 subjects from the Yoruba (n=74), Efik (n=32), and Igbo (n=46) tribes of southern Nigeria, 39 nationwide African-American stem cell donors, and 60 African-American individuals residing in the metropolitan Boston area were studied for MICA, HLA-B allelic variation, haplotypic diversity, and linkage disequilibrium (LD). MICA and HLA-B exhibited a high degree of genetic diversity among the populations studied. In particular, MICA allele and HLA-B-MICA haplotype frequencies and LD in the Efik and Igbo tribes were significantly different from the other study groups. HLA-B and MICA loci demonstrated significant global LD in all five populations (P-values &<0.00001). LD also varied in a haplotype-specific manner. A novel MICA allele was detected in the Boston population. These findings are important from an anthropologic perspective, and will inform future HLA-linked disease association studies in related ethnic groups of African-derived ancestry.

TNF-alpha promoter single nucleotide polymorphisms are markers of human ancestry.

We present a map of single nucleotide polymorphisms (SNPs) in the human tumor necrosis factor (TNF)-alpha promoter based upon exploratory sequencing of 333 human TNF-alpha gene promoters from individuals of distinct ancestral backgrounds. We detect 10 TNF-alpha promoter SNPs that occur with distinct frequencies in populations of different ancestry. Consistent with these findings, we show that two TNF-alpha SNPs, the -243 SNP and the -856 SNP, are the first SNP markers of a sub-Saharan African-derived extended haplotype and an Amerindian HLA haplotype, respectively. Comparisons of TNF-alpha promoter SNP allele frequencies can thus help elucidate variation of HLA haplotypes and their distribution among existing ethnic groups and shed light into the history of human populations.

Hepatitis B surface antigen- and tetanus toxoid-specific clonal expansion of CD4+ cells in vitro determined by TCRBV CDR3 length and nucleotide sequence.

We demonstrate activation of primary human TCRBV-specific CD4+ cells in vitro towards hepatitis B surface antigen (HBsAg) and tetanus toxoid (TT) without the use of cell lines, clones or added cytokines. By multiplex PCR analysis and spectratyping, antigen-activated cells exhibited clonal T cell receptor expansion within specific and limited TCRBV families. The expanded CD4+ T cells were CD45RO. Three of four unrelated HBsAg responders showed CD4+ expansion within the TCRBV16 family. The response comprised predominantly single CDR3 sequences in all three donors and was completely monoclonal in one of them. However, the CDR3 lengths and sequences differed among the responders. Clonality induced by HBsAg in TCRBV16 was specific, reproducible and distinct from that induced by TT in terms of sequence, nucleotide addition and diversity (BD) or junctional (BJ) element usage. Thus, for the first time, we show monoclonal or oligoclonal expansion of primary human CD4- peripheral blood mononuclear cells (PBMC) in vitro in response to nominal protein antigen without manipulations utilizing exogenous IL-2. The ability to induce monoclonal/ oligoclonal responses to HBsAg now permits motif identification studies for determining the T cell role in nonresponsiveness to the HBsAg vaccine.

Complex cytokine responses to hepatitis B surface antigen and tetanus toxoid in responders, nonresponders and subjects naive to hepatitis B surface antigen.

Some human subjects vaccinated with hepatitis B surface antigen (HBsAg) do not produce antibodies to the vaccine (nonresponders). The mechanism for nonresponse is unknown. To understand the response and nonresponse to nominal antigens better, we determined the level and kinetics of cytokine secretion in response to HBsAg and tetanus toxoid (TT) by peripheral blood mononuclear cells (PBMC) in vitro from HBsAg vaccine responders and nonresponders and from individuals naive to HBsAg. Proliferating PBMC secreted peak levels of interleukin-2 (IL-2) at 2 days and peak levels of tumor necrosis factor-beta (TNF-beta), interferon-gamma (IFN-gamma), IL-4 and IL-10 at 3-6 days post-stimulation. In contrast, nonproliferating PBMC (whether from nonresponders, naive subjects or weak responders) did not produce detectable levels of TNF-beta or IFN-gamma, nor was IL-4 or IL-10 produced significantly, and that produced had a different kinetic profile from that of proliferating PBMC. HBsAg-specific cytokine production by PBMC from strong responders broadly paralleled their cytokine responses to TT. Cellular cytokine mRNA levels measured by reverse transcriptase-polymerase chain reaction corroborated the secreted cytokine results. The anti-HBsAg- and anti-TT-specific T cell cytokine responses were mixed Th(1/2)-like and donor-specific. An HBsAg-specific cytokine response, but not a TT-specific cytokine response, was completely missing in nonresponders. These data suggest that the T cell defect of HBsAg nonresponse is not due to a skewed cytokine profile.

HLA-DRB1 leprogenic motifs in nigerian population groups.

Amino acid residues involved in the peptide binding groove of HLA-DRB1 alleles were examined in three Nigerian ethnic groups with leprosy (n = 287) and 170 controls to determine the role of DRB1 alleles in disease outcome with Mycobacterium leprae. Nine positively charged motifs and two others with neutral charge to the binding groove were detected. These motifs occurred more frequently in leprosy (leprogenic) than was expected by chance (P < 0.0001). In contrast, five motifs with net negative or 'modified' neutral charges to the pocket were negatively associated with leprosy. We conclude that clinical outcome of infection with M. leprae is largely determined by a shared epitope in DRB1 alleles marked by several motifs. These motifs occur in otherwise normal DRB1 alleles, characterized by net positive or neutral charges in the binding groove. We hypothesize that these polarities cause poor binding of DRB1 to M. leprae. On presentation, the signal via the T cell receptor results in muted cell-mediated immunity. The resulting response translates to various forms of leprosy depending on degree of charge consonance between M. leprae and host DRB1 allele. Other factors within or without the HLA complex, such as the T cell receptor repertoire, may also influence the resulting disease.

CD4 TCRBV CDR3 analysis in prevalent SLE cases from two ethnic groups.

We examined CD4+ T cell TCRBV-CDR3 transcripts from 19 lupus patients and 16 controls to test the hypothesis that CD4+ TCRBV-CDR3 expression in SLE differs from normals. Within the disease group we also performed exploratory analyses to determine the association between risk of oligoclonality and HLA-DRB specificities and the duration of the CDR3 patterns. Oligoclonal patterns consistent with CDR3 restriction were three times more likely in SLE than in controls (OR = 3.7). TCRBV1, BV4, BV5.1, BV7, BV9, BV18 and BV22 gene segment CDR3 patterns of oligoclonality were seen exclusively among lupus patients. HLA-DRB3 increased the risk of oligoclonal expression in SLE. In four patients studied over time, the pattern of TCRBV-CDR3 expression was stable in a second sample obtained 6-14 months later. The increased frequency of CD4+ T cell TCRBV-CDR3 oligoclonal expression in SLE when compared to controls and the persistence of these patterns are consistent with an expanded pool of autoreactive CD4 T cells in SLE which recognize peptides derived from autoantigens. The association of HLA-DRB3 genes with increased risk of CDR3 oligoclonality among the SLE subjects is compatible with the hypothesis that molecules encoded by HLA-DRB3 may facilitate autoantigen recognition by CD4 T cells.

Carboxyhaemoglobin levels in some Lagos dwellers--a pilot study.

Carboxyhaemoglobin (COHb) concentrations were determined by differential spectrophotometry in blood of 60 healthy adult subjects from various locations in Lagos. Half of these were either occasional or regular tobacco cigarette smokers. Our findings showed that the Lagos dweller has elevated COHb concentration ranging between 7.6%-9.9%, several folds higher than permitted by Air Quality Standards. The range and scatter of COHb in smokers were wider (7.4%-13.0%) than in non smokers. In particular, COHb concentrations were significantly higher in regular smokers than in non smokers by Fisher's exact test (p < 0.0006). Elevated COHb concentrations among smokers were related to frequency of tobacco use (p < 0.01). There was however no statistically significant difference in COHb concentrations when the regular and occasional smokers were taken as a group and compared with the non smokers. Haematocrit measurements showed that a degree of anaemia was present in most of the subjects tested irrespective of smoking status (mean packed cell volume = 36.1). It is inferred from this data that the Lagos dweller has high ambient concentrations of COHb and that these may be further aggravated by cigarette smoking.

HLA class I phenotype and gene frequencies in parts of southern Nigeria.

HLA-A, B and C antigens were determined in 101 healthy subjects from two major and several minor ethnic groups in some parts of Southern Nigeria. Compared to earlier data based on a panel of expatriate Nigerians, significant differences were observed in antigen phenotype and gene frequencies particularly at the HLA-A locus. At least three antigenic specificities not previously observed in the expatriate Nigerians were detected in the present study. These included HLA-B8. B14 and CW1. These antigens however occurred at low frequencies. The antigens A23 and B7 were in positive linkage disequilibrium along with others which involved CW4 with B53 or B35. It is concluded from our findings that HLA polymorphisms in Nigerians may not be completely reflected in major population group studies alone. It is possible that more specificities may be detected by continued testing of the minor ethnic groups. The importance of this could be immense in disease association studies involving HLA genes as well as in anthropology.

IgG2 associated hypergammaglobulinaemia in some Nigerians with HIV infection.

Concentrations of immunoglobulins (IgA, IgG and IgM) were measured in Nigerians with (HIV) infection. Considerable elevations up to two-fold the reference values were observed for IgG and IgM in the patient group as a whole but elevations in IgA concentration were least marked albeit significantly different from the healthy subjects. Elevation of a particular isotype was not always concomitant with elevation of the other major classes in the same patient. Overall, these elevations were observed in both symptomatic and asymptomatic infected subjects. Further analysis of IgG hyperglobulinemia showed that increases in this major class may be due to increased IgG2 subclass concentrations. It is suggested that elevation of IgG2 subclass in Nigerians with HIV infection and not IgG1 or IgG3 may be due to genetic and environmental factors rather than variation in the strain of the virus.

PIP: The major and subclass concentrations of immunoglobulins were examined in 27 Nigerians with HIV infection. 12 had definite HIV-1 infection, 2 had both HIV-1 and HIV-2, and the remaining 15 were included because of the reactivity of their sera. The reference group was drawn from four major Nigerian population groups that were part of a group of 238 healthy Nigerians. Individual increases in IgM and IgG concentrations in the patient group varied and was sometimes up to 7-fold above the mean of those in the control group. Overall, the increases were about twice the mean concentrations found in the reference group. The IgM concentration range was 0.6-9.7 g/l in the HIV group (n = 27) vs. 0.4-4.6 in the reference group (n = 157, p 0.02). The IgG concentration range was 10-70 g/l in the HIV group (n = 27) vs. 10-30 g/l in the reference group (n = 160, p .001). The highest IgG concentrations in cases were found in symptomatic patients, but this relationship was not observed for IgM and IgA. The scattergram of IgA concentrations was the least elevated. The increase was significant when those with HIV-1 infection alone were compared with the healthy subjects (p .05). IgG2 subclass concentrations were determined only in patients of Kanuri and Hausa populations. In comparison to their healthy counterparts, IgG2 concentrations were significantly higher in the patient group (p .001). Other IgG subclasses showed a bimodal distribution in both groups. There was no significant difference in distribution of IgG1, IgG3, and IgG4 concentrations between the reference and the HIV groups. In several ethnic groups polyclonal hypergammaglobulinemia has been reported to be a frequent feature of HIV infection with markedly increased IgA concentrations. The differences observed here do not reflect a variation in the strain of the virus in the Nigerian populations, but may be related to racial and environmental factors.

Bf S gene frequency in Nigerians with HIV infection.

Complement immunogenetic susceptibility to human immunodeficiency virus (HIV) infection was examined in 40 Nigerians with serological and/or clinical evidence of the infection. A mild increase in C4A null alleles (C4AQO) frequency was observed in the patient group compared to a group of healthy subjects (25 per cent vs 17 per cent) but overall the HIV infected and the reference groups did not differ significantly in the frequency of alleles of C4A or C4B. In contrast, properdin factor B (Bf) S gene frequency was significantly higher in the patients with HIV infection (p less than 0.025). There was a concomitant decrease in Bf F allele and gene frequencies (p less than 0.01, and p less than 0.05), respectively. Furthermore, blank Bf allotypes due to excessive complement consumption were detected in two asymptomatic patients. These findings suggest that Major Histocompatibility Complex (MHC) located complement genes may be important HIV infection. In particular Bf S gene or even C4AQO alleles may be permissive or influence outcome of infection with HIV.

Properdin factor B allotypes in diabetic Nigerians. A preliminary report on chromosome 6 markers.

Properdin factor B(Bf) allotypes were determined in patients with insulin dependent (type 1) diabetes mellitus (n = 15); in patients with non-insulin dependent diabetes mellitus n = 15); and in healthy Nigerians (n = 252) from various tribal groups. In all three groups only commonly reported Bf allotypes namely BfF, F1, S and S1 were observed. More important, BfF1 allele was significantly increased in patients with insulin dependent (type 1) diabetes mellitus (expected 1/15, observed 5/15), X2 = P less than 0.005). It is suggested that this allele is probably the same as that reported in caucasoids and is part of a supratype or ancestral haplotype defined by HLA-B18, C4A3, C4A3, BQo, BfF1, DR3 marking type 1 (insulin dependent) diabetes mellitus.

HLA, C4, Bf, Gm and autoantibodies in rheumatoid arthritis.

Sera from 48 subjects with rheumatoid arthritis (RA), more than 80% of which were seropositive, were examined for the presence of antibodies to intact nuclei (ANA) by immunofluorescence, extractable nuclear antigens (ENA), double-stranded DNA by immunoassay and immunoglobulins, i.e. rheumatoid factors (RF) by haemagglutination. The genetic markers of HLA, the fourth complement component (C4) and properdin factor (Bf), which are all coded for within the major histocompatibility complex and Gm immunoglobulin allotypic markers were all analysed in relation to various parameters of these autoantibodies. The only significant association to emerge was between low ENA antibody affinities and the Bf Fs phenotype (p less than 0.02). Although weak associations between individual autoantibody parameters and the other immunogenetic markers were detected, there were no significant HLA, C4 or Gm association with either ANA subsets or RF, a finding which agrees with, and also extends previous HLA studies of predominantly seropositive RA cohorts.

Reference ranges for serum C4 concentrations in subjects with and without C4 null alleles.

Serum C4 concentrations were measured in 102 healthy subjects and 90 subjects with type I diabetes mellitus. A wide range was observed in the group as a whole (0.08-0.67 g/l; mean = 0.26 g/l; SEM = 0.01 g/l). After C4 allotyping it was possible to subgroup 134 of these subjects according to the number of C4 null alleles present. C4 concentrations in the group with two null alleles were lower than in the group without null alleles (mean 0.2 v 0.37 g/l; p less than 0.001). C4 concentrations in the group with one C4 null allele were intermediate and significantly different from those of the group without null alleles (mean 0.24 v 0.37 g/l; p less than 0.001). Appropriate analysis has defined reference ranges for serum C4 concentrations in subjects with two, one, or zero C4 null alleles. Interpretation of low serum C4 concentrations should take account of the number of C4 null alleles present.

Extractable nuclear antigen (ENA) autoantibodies in SLE: an immunogenetic relationship with HLA, C4 and Bf alleles.

Sera from 36 subjects with systemic lupus erythematosus (SLE) were examined for extractable nuclear antigen (ENA) autoantibodies by immunoassay with the ribonucleoprotein (RNP) subset being determined by immunodiffusion. The prevalence of the genetic markers of HLA, the fourth complement component (C4) and properdin factor (Bf), which are all coded for within the major histocompatibility complex on chromosome 6 were analysed in relation to various parameters of these autoantibodies. The following associations were observed: The lowest ENA antibody titres of the RNP negative group were associated with HLA A9 (P less than 0.05), while the lowest RNA-ase sensitive ENA (RSE) subset antibody levels were associated with HLA Dr 1 (P less than 0.05). For the complement markers, C4 AQo was associated with the lowest affinity ENA antibodies (P less than 0.05), while the BF F allele and Fs phenotype had lower RSE antibody levels than did the S allele (P less than 0.05) and the SS phenotype (P less than 0.05) respectively. This study demonstrated diverse association between various MHC markers and ENA antibody parameters, indicating that there are distinctive immunogenetic influences over ENA autoantibodies in SLE.

Serum C4 concentration in the monitoring of systemic lupus erythematosus: requirement for C4 allotyping.

We have examined the influence of genetic and other factors on the serum concentration of the fourth component of complement (C4) in four patients with systemic lupus erythematosus (SLE) studied over 3 to 8 years. Complement allotyping was performed to determine the number of C4 null genes in each patient. Two patients with C4 null genes had relatively low serum C4 concentrations with normal serum anti-DNA binding and no evidence of active disease. By contrast two patients without null alleles appeared to be consuming C4 when the serum C4 concentrations were within the conventional reference range. We therefore propose the use of appropriate reference ranges adjusted for the number of null alleles. Such adjusted reference ranges may improve the utility of serum C4 concentration in monitoring disease activity.

CR1 deficiency in SLE: acquired or genetic?

Erythrocytes from 30 patients suffering from systemic lupus erythematosus (SLE) were tested for CR1 activity by an immune adherence haemagglutination technique. Defective CR1 activity (CR1D) was found in 11 (37%) of the patients on initial testing. On repeat testings, however, CR1 activity often varied from time to time and was shown to be inversely related to serum anti-DNA binding and apparent complement activation in vivo. Two of the 19 patients with normal CR1 activity acquired CR1D during the study. One patient with previously defective CR1 attained normal activity in the course of the study. The increased occurrence of CR1D in patients with SLE is largely or wholly acquired rather than genetically determined.

Secondary Sjögren's syndrome and chromosome six markers.

Patients with Sjögren's syndrome (SS) in association with either Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE) or Sclerodactyly were tested for HLA-A, B and Dr antigens and the phenotypes of the 4th component of complement (CA). Significant associations were identified between HLA-Bw62, Dr4, C4B2.9, C4A8 and females with SS-RA. The MHC antigen frequencies noted for secondary Sjögren's syndrome differed from those reported for their respective primary diseases and the implications of these observations are discussed.

Complement allotyping in SLE: association with C4A null.

Immunogenetic factors are important in systemic lupus erythematosus (SLE) and deficiency of a number of complement components is often associated with a lupus-like illness. The complement components Bf, C2 and C4 are encoded within the human major histocompatibility complex (MHC) and are polymorphic. A study of HLA and Bf and C4 polymorphism in 43 patients with SLE was undertaken firstly, to determine whether partial deficiency of C2 and C4 may predispose to disease and secondly, because it may allow the better definition of important supratypes associated with the disease and which may include the relevant disease gene(s). An increased frequency of C4A null alleles has been shown in SLE, with a minimal estimated C4A null gene frequency of 0.32 versus 0.20, but no case of partial C2 deficiency was identified. These results may indicate a direct role for partial C4 deficiency or that C4A null may be a marker for an important supratype which includes the relevant disease gene(s).