HLA-DRB1 alleles in juvenile-onset systemic lupus erythematosus: renal histologic class correlations

Human leukocyte antigens (HLA) DRB1*03 and DRB1*02 have been associated with systemic lupus erythematosus (SLE) in Caucasians and black populations. It has been observed that certain HLA alleles show stronger associations with SLE autoantibodies and clinical subsets, although they have rarely been associated with lupus renal histologic class. In the present study, HLA-DRB1 allele correlations with clinical features, autoantibodies and renal histologic class were analyzed in a cohort of racially mixed Brazilian patients with juvenile-onset SLE. HLA-DRB1 typing was carried out by polymerase chain reaction amplification with sequence-specific primers using genomic DNA from 55 children and adolescents fulfilling at least four of the American College of Rheumatology criteria for SLE. Significance was determined by the chi-square test applied to 2 x 2 tables. The HLA-DRB1*15 allele was most frequent in patients with renal, musculoskeletal, cutaneous, hematologic, cardiac, and neuropsychiatric involvement, as well as in patients positive for anti-dsDNA, anti-Sm, anti-U1-RNP, and anti-SSA/Ro antibodies, although an association between HLA alleles and SLE clinical features and autoantibodies could not be observed. The HLA-DRB1*17, HLA-DRB1*10, HLA-DRB1*15, and HLA-DRB1*07 alleles were significantly higher in patients with renal histologic class I, class IIA, class IIB, and class V, respectively. The present results suggest that the contribution of HLA- DRB1 alleles to juvenile-onset SLE could not be related to clinical or serological subsets of the disease, but it may be related to renal histologic classes, especially class I, class II A, class II B, and class V. The latter correlations have not been observed in literature.

Diabetes mellitus: new therapeutic approaches to treat an old disease

Diabetes mellitus is a widespread disease whose frequency increases constantly and is expected to reach alarming levels by the year 2025. Introduction of insulin therapy represented a major breakthrough; however, a very strict regimen is required to maintain blood glucose levels within the normal range and to prevent or postpone chronic complications associated with this disease. Frequent hyper- and hypoglycemia seriously affect the quality of life of these patients. Reversion of this situation can only be achieved through whole organ (pancreas) transplant or pancreatic islet transplant, the former being a high-risk surgical procedure, while the latter is a much simpler and may be accomplished in only 20-40 min. The advantages and perspectives of islet cell transplantation will be discussed, in the light of tissue engineering and gene therapy. Ongoing research carried out in our laboratory, aimed at developing clinical cell and molecular therapy protocols for diabetes will also be focused

Lack of evidence for the pathogenic role of iron and HFE gene mutations in Brazilian patients with nonalcoholic steatohepatitis

The hypothesis of the role of iron overload associated with HFE gene mutations in the pathogenesis of nonalcoholic steatohepatitis (NASH) has been raised in recent years. In the present study, biochemical and histopathological evidence of iron overload and HFE mutations was investigated in NASH patients. Thirty-two NASH patients, 19 females (59 percent), average 49.2 years, 72 percent Caucasians, 12 percent Mulattoes and 12 percent Asians, were submitted to serum aminotransferase and iron profile determinations. Liver biopsies were analyzed for necroinflammatory activity, architectural damage and iron deposition. In 31 of the patients, C282Y and H63D mutations were tested by PCR-RFLP. Alanine aminotransferase levels were increased in 30 patients, 2.42 + or - 1.12 times the upper normal limit on average. Serum iron concentration, transferrin saturation and ferritin averages were 99.4 + or - 31.3 g/dl, 33.1 + or - 12.7 percent and 219.8 + or - 163.8 æg/dl, respectively, corresponding to normal values in 93.5, 68.7 and 78.1 percent of the patients. Hepatic siderosis was observed in three patients and was not associated with architectural damage (P = 0.53) or with necroinflammatory activity (P = 0.27). The allelic frequencies (N = 31) found were 1.6 and 14.1 percent for C282Y and H63D, respectively, which were compatible with those described for the local population. In conclusion, no evidence of an association of hepatic iron overload and HFE mutations with NASH was found. Brazilian NASH patients comprise a heterogeneous group with many associated conditions such as hyperinsulinism, environmental hepatotoxin exposure and drugs, but not hepatic iron overload, and their disease susceptibility could be related to genetic and environmental features other than HFE mutations

Analysis of HLA-A antigens and C282Y and H63D mutations of the HFE gene in Brazilian patients with hemochromatosis

The hemochromatosis gene, HFE, is located on chromosome 6 in close proximity to the HLA-A locus. Most Caucasian patients with hereditary hemochromatosis (HH) are homozygous for HLA-A3 and for the C282Y mutation of the HFE gene, while a minority are compound heterozygotes for C282Y and H63D. The prevalence of these mutations in non-Caucasian patients with HH is lower than expected. The objective of the present study was to evaluate the frequencies of HLA-A antigens and the C282Y and H63D mutations of the HFE gene in Brazilian patients with HH and to compare clinical and laboratory profiles of C282Y-positive and -negative patients with HH. The frequencies of HLA-A and C282Y and H63D mutations were determined by PCR-based methods in 15 male patients (median age 44 (20-72) years) with HH. Eight patients (53 percent) were homozygous and one (7 percent) was heterozygous for the C282Y mutation. None had compound heterozygosity for C282Y and H63D mutations. All but three C282Y homozygotes were positive for HLA-A3 and three other patients without C282Y were shown to be either heterozygous (N = 2) or homozygous (N = 1) for HLA-A3. Patients homozygous for the C282Y mutation had higher ferritin levels and lower age at onset, but the difference was not significant. The presence of C282Y homozygosity in roughly half of the Brazilian patients with HH, together with the findings of HLA-A homozygosity in C282Y-negative subjects, suggest that other mutations in the HFE gene or in other genes involved in iron homeostasis might also be linked to HH in Brazil

Molecular typing of HLA class II antigens in a Säo Paulo population

Uma análise do perfil dos antígenos HLA de classe II numa amostra da populaçäo de Säo Paulo é descrita neste trabalho. Os dados foram obtidos através de técnicas de amplificaçäo gênica utilizando-se iniciadores seqüências-específicos para HLA-DRB (PCR-SSP) ou amplificaçäo gênica seguida de hibridaçäo com oligonucleotídeos específicos (PCR-SSOP) para HLA-DQA e DQB. Foram calculadas as freqüências gênicas e as freqüências haplotípicas DRB-DQB e DQA-DQB e a populaçäo mostrou estar em equilíbrio genético de acordo com a lei de Hardy-Weinberg. Finalmente, comparamos também os dados obtidos com os da populaçäo de Porto Velho, Rondônia, salientando a importância da obtençäo de dados regionais para os controles quando se estuda este complexo sistema genético.

Absence of linkage between MHC and a gene involved in susceptibility to human schistosomiasis

Six hundred million people are at risk of infection by Schistosoma mansoni, MHC haplotypes have been reported to segregate with susceptibility to schistosomiasis in murine models. In humans, a major gene related to susceptibility/resistance to infection by S. mansoni (SM1) and displaying the mean fecal egg count as phenotype was detected by segregation analysis. This gene displayed a codominant mode of inheritance with an estimated frequency of 0.20-0.25 for the deleterious allele and accounted for more than 50 percent of the variance of infection levels. To determine if the SM1 gene segregates with the human MHC chromosomal region, we performed a linkage study by the lod score method. We typed for HLA-A, B, C, DR and DQ antigens in 11 informative families from an endemic area for schistosomiasis in Bahia, Brazil, by the microlymphocytotoxicity technique. HLA-DR typing by the polymerase chain reaction with sequence-specific primers (PCR-SSP) and HLA-DQ were confirmed by PCR-sequence-specific oligonucleotide probes (PCR-SSOP). The lod scores for the different theta values obtained clearly indicate that there is no physical linkage between HLA and SM1 genes. Thus, susceptibility or resistance to schistosomiasis, as defined by mean fecal egg count, is not primarily dependent on the host's HLA profile. However, if the HLA molecule plays an important role in specific immune responses to S. mansoni, this may involve the development of the different clinical aspects of the disease such as granuloma formation and development of hepatosplenomegaly.

Molecular analysis of sex determination in sex-reversed and true hermaphroditism

The SRY (sex region of Y) gene determines testis formation but not all cases of sex reversal in humans can be explained by alterations in this gene. We studied on 46,XY female, four 46,XX males, and nine true hermaphrodites (TH): three with an XY and six with an XX chromosomal constitution. The SRY gene was identified in the XX males and the TH with a Y chromosome but was not demonstrated in the XY female and the six XX TH. The Y-heterochromatin region was also identified in one 46,XX male, indicating a low grade mosaicism undetected by cytogenetics. The amplification of the amelogenin gene showed the presence of a 977-bp band that belongs to the short arm of chromosome X in all patients but the absence of a 780-bp band of the short arm of chromosome Y in three 46,XX males and in all the 46,XX TH. These studies demonstrate that the molecular study of sex-reversed patients and TH will help to understand the complex mechanisms of sex determination. The SRY gene is involved but other genes on the X chromosome and autosomes still remain to be studied.

High frequency of specific T cell receptor rearrangements in rheumatoid arthritis: analysis of synovial fluid T lymphocytes

1. We have searched for rearrangements in the beta chain T cell receptor genes to identify clonal T lymphocyte populations in the synovial fluid of 10 patients with well established rheumatoid arthritis, using a T cell population unbiased by preselection. 2. Analysis of the restriction fragments with the beta chain constant region probe C beta 2 disclosed a rearranged band in 50 of cases (5/10). No significant differences in age, duration of the disease, treatment employed and presence of articular deformities or erosion upon X-ray examination were observed when patients with or without rearrangements were compared. 3. The rearranged band observed after BamH I digestion was of the same size in the 5 patients (14 kb). In addition, two patients presented a 10-kb rearranged band upon restriction with Hind III. 3. These data indicate that a significant number of rheumatoid arthritis patients probably present oligoclonal T cell proliferation of their synovial fluid lymphocytes.

Human leukocyte a and B antigen, gene and haplotype frequencies in the population of the city of Säo Paulo in Brazil

Antigen, gene and haplotype frequencies are imnportant data for population analysis, poaternity exclusion testing, genetic studies, and for organ transplantation selection. We have studied the class I histocompatibility antigens of 617 unrelated individuals from the city of Säo Paulo, Brazil, to determine antigen and gene frequencies of HLA-A and 28 HLA-B antigens. Estimated haplotype frequencies were also determined, as well as the genetic distances of this population from European Caucasian and Negro populations. A previously unknown linkage disequilibrium was detected for A23-B49 and a clear trend towards antigen frequencies intermediate between Caucasoid and Negro populations was observed

DNA analysis of histocompatibility antigens: fragments from the recently identified DR loci

The DR locus of class II histocompatibility antigens (HLA-DR) is routinely typed by microlymphocytotoxicity tests. However, the preparation of good antisera is a complex process which relies heavily on a well-defined cell panel. We have studied homozygous typing-cells representing the most common DR specificities in order to determine their typing pattern at the DNA level. Restriction fragment length polymorphisms obtained using six restriction enzymes permitted us to identify most of the DR specificities, including recently described splits of the DRw6. Specific fragments were tentatively allocated to the three DRw52 alleles. The number of beta genes present on DRw52 and DRw53 antigen carrying cells is also discussed

DNA analysis of histocompatibility antigens: identification of new DQw specificities and of DPw patterns

The HLA-D region of the major histocompatibility complex has several subregions, the most important of which are DR, DQ and DP. The genes coding for the beta chains of these proteins present most of the polymorphisms which result in the large variety of class II antigens observed. We have studied the restriction fragment length polymorphism (RFLP) of the DQ beta and DP beta genes in order to establish accurate typing patterns. The data show that DQ typing based on RFLP permits the identification of the recently described DQw1 splits (new antigenic specificities), DQw5 and DQw6. The TA 10-monoclonal antibody-positive split of DQw3, designated DQw7, is associated with specific DNA fragments after digestion with four different enzymes: Taq I, Hind III, Pvu II and Bg1 II. Furthermore, the recently reported specificity DQw4 (formerly typed as a blank) is associated with a specific 2.4-kb fragment when the DNA is digested with EcoRV. DP typing proved to be more difficult even though six enzymes were used, and only broad groups could be identified

DNA analysis of histocompatibility antigens of a sample of the Säo Paulo population

Human major histocompatibility complex (HLA) antigens have attracted great interest because of their prominent role in cell-mediated immune response and thus in graft rejection. An enormous variety of histocompatibility antigens has been demonstrated and the number of known antigens increases every year. The objective of the present study was to apply restriction fragment lengh polymorphism (RFLP) analysis to the normal population of Säo Paulo in order to confirm and extend the scope of our laboratory's cell panel for HLA typing. DNA typing of class I antigens has not been very productive since only some specificities correlate with DNA fragments demonstrable with Southern blots. In contrast, DNA typing of class II antigens is a valuable tool for reclassifying cells previously typed by anti-HLA sera. Since antisera for recently described HLA antigens are not easily available, the development of a well-characterized panel is essential for the correct screening of incoing sera. These sera, in turn, permit the accurate typing of donor-recipient pairs in kidney and bone marrow transplantation