HLA-G and CD152 Expression Levels Encourage the Use of Umbilical Cord Tissue-Derived Mesenchymal Stromal Cells as an Alternative for Immunosuppressive Therapy.

Mesenchymal stromal cells (MSCs) have been used in immunosuppressive therapy due to their therapeutic effects, with the HLA-G molecule seeming to play a fundamental role. This work evaluated alternative MSC sources to bone marrow (BM), namely, umbilical cord tissue (UC), adipose tissue (AD) and dental pulp tissue (DP), and the influence of interferon-γ (IFN-γ) and hypoxia on the cultivation of these cells for use in immunosuppression therapies. Expression of costimulatory markers CD40, CD80 and CD86 and immunosuppressive molecules CD152 and HLA-G was analyzed. Lymphocyte inhibition assays were also performed. Sequencing of the HLA-G gene from exons 1 to 5 was performed using next-generation sequencing to determine the presence of alleles. UC-derived MSCs (UCMSCs) expressed higher CD152 and HLA-G1 under standard cultivation. UCMSCs and DP-derived MSCs (DPSCs) secreted similar levels of HLA-G5. All MSC sources inhibited the proliferation of peripheral blood mononuclear cells (PBMCs); growth under regular versus hypoxic conditions resulted in similar levels of inhibition. When IFN-γ was added, PBMC growth was inhibited to a lesser extent by UCMSCs. The HLA-G*01:04:01:01 allele appears to generate a more efficient MSC response in inhibiting lymphocyte proliferation. However, the strength of this conclusion was limited by the small sample size. UCMSCs are an excellent alternative to BM in immunosuppressive therapy: they express high concentrations of inhibitory molecules and can be cultivated without stimuli, which minimizes cost.

Novel HLA-A, HLA-B, and HLA-DRB1 alleles identified in Brazilian individuals.

Identification of 12 novel HLA class I and II alleles in Brazilian bone marrow donors.

HLA-E genotyping and its relevance in kidney transplantation outcome.

HLA-E, a class I nonclassical HLA molecule, is expressed in all tissues and is involved in the regulation of both innate (by interaction with the CD94/NKG2 receptor expressed mainly in NK cells) and adaptive immunity (by interaction with T CD8+ cells), suggesting a possible role in the solid organ transplantation context. Transplanted patients with chronic kidney disease and their respective donors (N = 107 pairs) were genotyped for exons 2 and 3 of the HLA-E locus by sequence-based typing (SBT). Groups' genotype frequencies were compared regarding episodes of clinical rejection by global G test, and binary logistic regression was made to demonstrate the contribution of genetic variables vs epidemiological variables. Comparisons of donors' genotype frequencies showed significant differences (P = .0230), revealing a protective profile of E*01:01/*01:01 compared to the other genotypes (P = .0099; OR = 0.3088; CI [95%] = 0.1333-0.7157). The same happened when the aforementioned genotype was combined with the E*01:01/*01:01 recipients' genotype (P = .0065; OR = 0.1760; CI [95%] = 0.0517-0.5987). A binary logistic regression analysis was performed, and, of all variables considered, only two were included in the resulting model (P = .007; R2 Cox and Snell = 0.243; R2 Nagelkerke = 0.328)- "End-Stage Renal Disease" and "HLA class II Mismatches." A protective profile (E*01:01/*01:01) was observed between the recipients and donors, suggesting a possible impact of the HLA-E genotype in rejection episodes.

MICA diversity and linkage disequilibrium with HLA-B alleles in renal-transplant candidates in southern Brazil.

The major histocompatibility complex (MHC) class I chain-related gene A (MICA) is located centromerically to the human leukocyte antigen (HLA)-B. The short distance between these loci in the MHC indicates the presence of linkage disequilibrium (LD). Similarly to the HLA, the MICA is highly polymorphic, and this polymorphism has not been well documented in different populations. In this study, we estimated the allelic frequencies of MICA and the linkage disequilibrium with HLA-B alleles in 346 renal-transplant candidates in southern Brazil. MICA and HLA were typed using the polymerase chain reaction-sequence-specific primer method (PCR-SSO), combined with the Luminex technology. A total of 19 MICA allele groups were identified. The most frequent allele groups were MICA*008 (21.6%), MICA*002 (17.0%) and MICA*004 (14.8%). The most common haplotypes were MICA*009-B*51 (7.8%), MICA*004-B*44 (6.06%) and MICA*002-B*35 (5.63%). As expected from the proximity of the MICA and HLA-B loci, most haplotypes showed strong LD. Renal patients and healthy subjects in the same region of Brazil showed statistically significant differences in their MICA polymorphisms. The MICA*027 allele group was more frequent in renal patients (Pc = 0.018, OR: 3.421, 95% CI: 1.516-7.722), while the MICA*019 allele group was more frequent in healthy subjects (Pc = 0.001, OR: 0.027, 95% CI: 0.002-0.469). This study provided information on the distribution of MICA polymorphisms and linkage disequilibrium with HLA-B alleles in Brazilian renal-transplant candidates. This information should help to determine the mechanisms of susceptibility to different diseases in patients with chronic kidney disease, and to elucidate the mechanisms involved in allograft rejection associated with MICA polymorphisms in a Brazilian population.

A pilot study on Hla-G locus control region haplotypes and cervical intraepithelial neoplasias.

Human papillomavirus (HPV) can induce cervical intraepithelial neoplasias (CIN) grades 1, 2 and 3. Untreated, these lesions may progress to cervical cancer (CC) which is the third most common cancer in women worldwide. HLA-G plays an immunotolerant role in the immune response. The aim of this study was to characterize the configuration of SNPs located at the distal promoter of HLA-G in patients with CIN2 and CIN3 and control women. The study sample was composed of 207 women as follows: 73 diagnosed with CIN2 lesions, 56 with CIN3 and 78 healthy control women. Genotyping was performed by sequence base typing. Eleven haplotype configurations subdivided in two main haplogroups (H1dist and H2dist), were characterized and compared between patients and controls. The haplotypes H1.1Dist (GAGAACGC) and H2.1Dist (AGGTACAC) were more frequent in Euro-Descendants as well as in Brazilian Mixed. Nevertheless, the haplotype H2.1Dist standed out as a susceptibility haplotype in Brazilian Mixed patients while the H1.1Dist presented a protector effect in this same ethnic group. Whether such LCR haplotype configurations can impact on HLA-G gene expression levels in women who developed cervical intraepithelial neoplasia is still unknown and it is of utmost importance that more investigation on this field be pursued.

Absence of strong linkage disequilibrium between odorant receptor alleles and the major histocompatibility complex.

The odorant receptor (OR) genes constitute the largest gene family among vertebrates. While over 800 loci are present in the human genome, their allele diversity is still poorly characterized. It has been hypothesized that the products of OR genes can be relevant in the reproductive context, thereby interacting with products of genes of the major histocompatibility complex (MHC). Here we investigated the genetic diversity of the OR2W6P, OR2B8P, OR1F12 and OR12D2 genes, in order to define haplotypes and haplotype frequencies. We measured levels of linkage disequilibrium (LD) between these OR genes and the MHC genes HLA-A, HLA-B and HLA-DRB1. This was accomplished through the assessment of 30 single nucleotide polymorphisms (SNPs) in samples from 61 family trios. We characterized 26 alleles among the four OR genes and identified three SNPs that had not yet been reported. Based on our haplotype analysis, LD spanning the OR-HLA region is not very strong, and therefore not enough to enable selection regarding specific HLA-OR haplotypes.

Considerations on regulatory sequences of the distal promoter region of the HLA-G gene.

Gene expression in eukaryotic cells is accomplished via association of transcription factors, some of which directly bind to DNA regulatory sequences. HLA-G codes for an immunoregulatory protein with tissue-specific expression, its unique promoter regulatory region is responsible for this feature. The aim of the present study was to explore motif composition as well as identify haplotypes in the HLA-G 5' distal promoter region. The sample was composed by 176 euro-descendents individuals genotyped by Sequence Based Typing of HLA-G distal promoter, encompassing 16 SNPs. Haplotypes were inferred by the expectation maximization algorithm. Only haplotypes with frequency higher than 1% were aligned to check for similarities and differences and thirteen haplotypes remained. For a better understanding of the nucleotide diversity of the analyzed region our approach was to split the whole sequence into two regions. Two contrasting haplotype groups were found in both regions, allowing us to suggest the existence of different transcription factors capable of binding cis elements while the intra-group variations suggest the intensity modulation of binding with regulatory factors.

Analysis of HLA-G polymorphisms in couples with implantation failure.

PROBLEM: HLA-G expression is related as an immune modulator of fetal-maternal tolerance, and its levels was correlated with pregnancy outcome. In a case-control study, we investigate the association between the genetic variability of the HLA-G gene and serum levels of soluble HLA-G in cases of embryo implantation failure. METHOD OF STUDY: Forty couples with at least two unsuccessful fresh embryo transfers (implantation failure; IF) and 83 fertile couples with at least two successful pregnancies was genotyped by sequencing-based typing. HLA-G alleles were defined by nucleotide sequence variations at exon 2, 3, and 4, and the quantification of soluble HLA-G (sHLA-G) was performed by ELISA. RESULTS: There was a significant difference between the HLA-G allelic distributions between IF couples and the control couples. The HLA-G*01:03:01 allele was increased in the IF couples. There were no significant differences in the serum levels of sHLA-G in the IF and control groups. CONCLUSION: The results suggest that the distribution of HLA-G products may play a significant role in the modulation of maternal-fetal immune response.

Genetic profile characterization of ten X-STRs in a sample from Paraná, Brazil.

This work reports the allele frequencies for ten X-STRs (DXS8378, DXS7132, DXS9898, DXS6809, DXS9902, DXS6789, DXS7133, DXS7423, GATA172D05, GATA31E08) in a sample of 800 individuals from Paraná, Brazil. No deviations from the Hardy-Weinberg equilibrium were observed. Linkage disequilibrium analysis did not reveal association between the X-STRs. High overall power of discrimination was obtained for female and male samples, and high probability of exclusion was observed in father/mother/daughter trios and father/daughter duos. Genetic comparisons revealed significant differences between Paraná and other Brazilian populations.