Genetic variants in the HLA-G region are associated with Kawasaki disease

Kawasaki disease is an acute, self-limited vasculitis of infants and children, manifest as fever and signs of mucocutaneous inflammation. Treatment with high-dose immunoglobulin reduces systemic inflammation and prevents coronary artery lesions in Kawasaki disease. In this study, we investigated the possible association of the major histocompatibililty complex (MHC) region for the susceptibility to Kawasaki disease using an MHC panel of 2360 single nucleotide polymorphism (SNP) markers. Analysis of data obtained from screening MHC-specific SNP chips with 48 case and 90 control subjects revealed five candidate loci with significance levels of uncorrected p < 0.01. However, only one candidate locus (HLA-G) was confirmed to have a significant association with Kawasaki disease (rs2523790, odds ratio [OR] = 3.00, 95% confidence interval [95% CI] = 1.14–7.91, uncorrected p = 0.0263) in the replication study using 44 new case subjects and the previous 90 controls. In the fine mapping of the HLA-G locus, in particular, a nonsynonymous SNP (C/A) of the HLA-G gene (rs12722477, Leu134Ile) was significantly associated with Kawasaki disease (OR = 3.23, 95% CI = 1.12–9.32). A subgroup analysis showed that this association was more apparent in patients with coronary artery aneurysms (OR = 4.02, 95% CI = 1.23–13.19). Therefore, our results indicate that HLA-G may play a crucial role for the susceptibility to Kawasaki disease.     

A novel HLA-G allele, HLA-G*010111, in the Brazilian population

The human leukocyte antigen-G (HLA-G) gene plays an important role in pregnancy and is related to negative signals for natural killer cells and T lymphocytes. Herein a new HLA-G allele (HLA-G*010111) is described in the Brazilian population--one of the most heterogeneous populations in the world. The new allele is associated with the 14-bp deletion at exon 8 and is similar to the HLA-G*01010105 allele, except for a C to G transversion at codon 117 in exon 3.     

Expression of TAP1 by human trophoblast

Successful placentation in the human is dependent on the trophoblast evading recognition and destruction by the maternal immune system. However, invasive cytotrophoblast express HLA-G which may be able to present peptide to T cells. Transporter proteins are essential for peptide presentation and major histocompatibility complex (MHC) class I assembly. We have determined their expression by trophoblast in relation to HLA-G, using immunohistochemistry. Antitransporter protein antibody (TAP1) labeling closely paralleled that of MHC class I, but the intensity of its expression was much greater on the HLA-G⁺ extravillous cytotrophoblast than any other fetal or maternal tissue in the first trimester and at term. This suggests that the extravillous cytotrophoblast are very actively assembling MHC class I antigens with peptides. However, expression of MHC class I by the cytotrophoblast was not correspondingly elevated. This pattern could result from HLA-G being shed from the surface of the trophoblast, a process which may play a central role in protecting the fetus from maternal immune attack.     

原核表达、体外折叠的HLA-G5促进人外周血单核细胞产生TNF

通过原核表达获得HLA-G5重链和轻链(β2m),经初步纯化后,利用稀释法与人工合成的九肽(KGPPAALTL)一起进行体外复性折叠,形成HLA-G5-抗原肽复合物,经非变性PAGE/Western blot和夹心ELISA法鉴定,证实复性后成功获得天然构象的HLA-G5。利用PBMC受LPS刺激后产生TNF的特点,观察原核表达、体外折叠的HLA-G5对人PBMC分泌TNF的影响。TNF分泌量采用TNF敏感的L929细胞进行生物法测定,结果显示原核表达、体外折叠的HLA-G5促进人PBMC分泌TNF-α。     

rhIL-17A对人皮肤角质形成细胞和成纤维细胞活性和凋亡的影响

目的: 研究重组人白细胞介素17A (rhIL-17A)对人角质形成细胞和成纤维细胞活性、凋亡及成纤维细胞促纤维化因子分泌的影响,探讨IL-17在系统性硬皮病发病中的作用。方法: 用不同浓度的rhIL-17A作用于角质形成细胞和成纤维细胞,CCK-8测定其对角质形成细胞和成纤维细胞活性的影响;Western blotting 检测核转录因子NF-κB/p65及其抑制性蛋白IκBα的表达量;流式细胞术观察细胞凋亡变化;ELISA 检测rhIL-17A 处理组和对照组成纤维细胞培养液上清中IL-6和TGF-β₁的含量。结果: rhIL-17A处理组和对照组角质形成细胞活性没有明显差别;rhIL-17A 处理组成纤维细胞活性较对照组明显升高(P<0.05),rhIL-17A 处理组成纤维细胞NF-κB/p65表达量增加,而IκBα表达量降低;rhIL-17A 对2种细胞凋亡率无明显影响;rhIL-17A 处理组成纤维细胞IL-6 和TGF-β₁分泌量增加。结论: rhIL-17A对体外培养人角质形成细胞的活性没有明显影响,却可以显著增强体外成纤维细胞的活性,IL-17A对成纤维细胞活性增强的这种促进作用,可能是通过激活NF-κB实现。rhIL-17A可能通过刺激成纤维细胞分泌IL-6 和TGF-β₁,从而进一步引起成纤维细胞增殖和胶原合成。IL-17可能参与了系统性硬皮病的发病过程。     

Down-regulation of HLA-G1 cell surface expression in human cytomegalovirus infected cells

PROBLEM: Down-modulation of human leukocyte antigen (HLA)-G1 cell surface expression by human cytomegalovirus (HCMV) has only been studied in cellular models expressing independent unique short (US) recombinant proteins, but not in the context of viral infection. To explore the level of HLA-G1 cell surface expression after HCMV infection and to investigate the influence of US viral proteins, we infected HLA-G1 expressing cells by HCMV laboratory strains. METHOD OF STUDY: Human U373-MG astrocytoma cells were transfected with HLA-G1 cDNA. Following HCMV infection, HLA-G1 cell surface expression of these transfectants was evaluated by flow cytometry and confocal microscopy, using an HLA-G specific monoclonal antibody, and compared with that of uninfected cells. US-deleted viruses were then used to evaluate the implication of US proteins. RESULTS: Using flow cytometry, it was found that HCMV infection of U373-G1 cells decreased HLA-G1 cell surface expression. Similar results were obtained with two different HCMV strains, namely Towne and AD169. Two color confocal microscopy staining further confirmed such HLA-G down-modulation in HCMV-infected cells stained for immediate early (IE1/2) nuclear proteins expression. Infection of U373-G1 cells with US-deleted HCMV strain had no effect on the level of cell surface HLA-G1 expression, thus demonstrating the US dependency of the HCMV-mediated down-regulation of HLA-G1. CONCLUSION: HCMV infection down-modulates HLA-G1 expression at the cell surface. This is likely to have functional consequences in case of HCMV uterine infection during pregnancy.     

A study of complexes of class II invariant chain peptide: Major histocompatibility complex class II molecules using a new complex-specific monoclonal antibody

Complexes of major histocompatibility complex (MHC) class II molecules containing invariant chain (Ii)-derived peptides, known as class II-associated invariant chain peptides (CLIP), are expressed at high levels in presentation-deficient mutant cells. Expression of these complexes in mutant and wild-type antigen-presenting cells suggests that they represent an essential intermediate in the MHC class II antigen-presenting pathway. We have generated a monoclonal antibody, 30-2, which is specific for these complexes. Using this antibody, we have found quantitative differences in CLIP: MHC class II surface expression in mutant and wild-type cells. Our experiments also show that CLIP: MHC class II complexes are preferentially expressed on the cell surface similar to total mature MHC class II molecules. These complexes are found to accumulate in the endosomal compartment in the process of endosomal Ii degradation. Analysis of the fine specificity of the antibody indicates that these complexes have Ii peptide bound to the peptide-binding groove.     

Patterns of major histocompatibility complex class II expression by T cell subsets in different immunological compartments. 2. Altered expression and cell function following activation in vivo

This study characterizes antigen-induced phenotypic and functional aspects of major histocompatibility complex (MHC) class II expression on recirculating T cells in efferent lymph. In vivo secondary, but not primary challenge is associated with both kinetic and phenotypic alterations in class II expression by T cells. All three major T cell subsets, CD4⁺, CD8⁺ and T19⁺ (γδ T cell receptor), show an approximate four fold increase in the level of MHC class II expression during secondary responses. No changes in B cell expression of class II were seen. Resting efferent lymph T cells are predominantly either class II^? or DR⁺DQ^? but this changes to DR⁺DQ⁺ after antigenic challenge. The antigen-presenting function of these class II⁺ T cells was investigated at daily intervals after in vivo antigenic challenge. T cells from non-activated lymph nodes could not induce proliferation of antigen-specific T cells with soluble antigen but were weakly stimulatory in allo-mixed lymphocyte reaction (MLR) at high (> 2:1) stimulator cell ratios. Activated T cells isolated during secondary in vivo responses, and expressing increased quantities of MHC class II, were positive stimulator cells in the MLR. In contrast these cells could not present soluble antigen or trypsin-digested antigen to the T cell lines. In the MLR assays, the relative stimulation by class II⁺ T cells correlates with the levels of class II expression. We conclude from these experiments that both quantitative and qualitative changes in MHC class II, induced on T cells under physiological conditions, play a role in the regulation of the immune response in vivo but that that role is not simply one of presentation of soluble antigen.     

Wortmannin enhances lipopolysaccharide-induced inducible nitric oxide synthase expression in microglia in the presence of astrocytes in rats

There is evidence that morphological alterations concerning deficiency or abundance of thyroid hormones (TH) may be mediated by brain-derived neurotrophic factor (BDNF). It has been demonstrated that the mRNA-expression of BDNF is increased after TH-treatment during the first postnatal weeks. After transient treatment of newborn rats with thyroxine mRNA expression, protein concentration and number and size of BDNF-immunopositive neurons were quantified in the medial septum/vertical diagonal Band of Broca (MS/vDB). The number and size of BDNF-immunopositive neurons were estimated in young (P10) and adult (4 months). The amount of mRNA and protein are significantly increased in TH-treated rats at P10 compared to control animals. TH-treated animals showed a significant decrease of BDNF-immunopositive cell numbers in the adulthood. The results demonstrate a correlated increase of BDNF mRNA and protein in the septum at P10 which is an important stage of differentiation processes in the septohippocampal system. These results provide further evidence that BDNF is a possible candidate for the mediation the TH effects in the MS/vDB.     

HLA-G polymorphism and in vitro fertilization failure in a Polish population

To investigate whether human leukocyte antigen-G ( HLA-G ) gene polymorphism is associated with in vitro fertilization (IVF) failure, we sequenced exons 2–4 of the HLA-G gene in 50 couples with three or more IVFs (including 10 couples with five or more IVFs) and 58 control fertile couples from a Polish population. Of the 10 different HLA-G alleles identified in our study subjects, neither allele was found to be associated with IVF. We also genotyped 50 couples with IVF and 71 control couples for the −725C>G variant in the promoter region and the 14 bp insertion or deletion polymorphism in the 3' untranslated region of the HLA-G gene. The frequency of −725GG or GC genotype in women with IVF and in control fertile women was similar [26% vs 25.3%; odds ratio (OR) = 1.0; P  = 1.0]. The 14 bp ins/ins or ins/del genotype was more common in women with IVF than in control women (76.9% vs 59.1%; OR 2.4; P  = 0.03), but the difference was not significant after Bonferroni correction for multiple comparisons. The frequency of the ins/ins or ins/del genotype was particularly high (90%) in women who experienced five or more IVFs (OR = 6.2; P  = 0.08), but again, the excess was not statistically significant, possibly because of small sample sizes. These results are in line with functional studies that show lower levels of HLA-G mRNA and protein related to the HLA-G allele including the 14 bp sequence and suggest that the insertion allele may be associated with an increased risk of IVF.     

可溶性HLA-G1-抗原肽复合物的体外折叠及其功能研究

目的:体外折叠形成可溶性HLA G1抗原肽复合物,并研究其生物学功能。方法:以原核表达的可溶性HLA-G1的重链及轻链β2m,与人工合成的抗原九肽体外进行共折叠复性。将折叠产物通过SephadexG75层析柱纯化,经Westernblot进行构象鉴定。探讨可溶性HLA-G1分子对NK细胞杀伤活性的影响,对混合淋巴细胞培养中T细胞增殖的影响,以及对活化T细胞凋亡的影响。结果:折叠产物可与mAbW6/32特异性结合,在Westernblot的鉴定中呈阳性反应。可溶性HLA-G1可有效地抑制NK细胞的细胞毒作用,抑制混合淋巴细胞培养中T细胞的增殖,促进活化T细胞发生凋亡。结论:体外折叠成功地形成可溶性HLA-G1抗原肽复合物,该复合物具有抑制NK细胞和T细胞的活性。     

Promoter methylation and mRNA expression of HLA-G in relation to HLA-G protein expression in colorectal cancer

Expression of human leukocyte antigen-G (HLA-G) is a suggested mechanism used by tumor cells to escape from host immune recognition and destruction. Advances in the field have made it evident that HLA-G is expressed in different types of malignancies including colorectal cancer (CRC). We analyzed HLA-G expression in 21 low passage CRC cell lines. The level of DNA methylation of the HLA-G gene and the presence of mRNA encoding HLA-G was measured. Moreover, HLA-G protein expression was determined by flow cytometry and immunohistochemistry (IHC). IHC was performed with three different monoclonal antibodies (mAbs) (4H84, MEM-G/1 and MEM-G/2). In addition, HLA-G protein expression was measured in matching primary tumor tissues.RNA analysis using RT-PCR followed by sequencing in 6 samples indicated strong homology of the PCR product with HLA-G3 in 5 samples. In accordance, in none of the cell lines, HLA-G1 expression was detected by flow-cytometry. Furthermore, no association between HLA-G DNA methylation patterns and HLA-G mRNA expression was observed. In addition, different immunohistochemical staining profiles among various anti-HLA-G mAbs were observed. In conclusion, the results of this study show that the HLA-G3 isoform was expressed in some of the CRC cell lines irrespective of the level of DNA methylation of HLA-G.     

Differential expression of HLA-G and ILT-2 receptor in human tuberculosis: Localized versus disseminated disease

Human leukocyte antigen-G (HLA-G) is an anti-inflammatory and immunosuppressive molecule that can modulate immune cell activation. The role of HLA-G in tuberculosis, an immune-mediated and chronic bacterial disease remains to be elucidated. We investigated the expression profile of soluble and membrane bound HLA-G in pulmonary TB (PTB), TB pleural effusion (TB-PE, localized disease) and Miliary TB (disseminated form). The expression of HLA-G receptor, ILT-2 was also determined on the immune cells. We observed that the plasma sHLA-G levels were significantly increased in Miliary TB than in TB-PE patients. In contrast, immunophenotyping revealed that the percent frequency of CD3⁺ T cells expressing HLA-G was significantly reduced in Miliary TB as compared to TB-PE, whereas frequency of CD14⁺ monocytes expressing HLA-G was significantly higher in TB-PE patients. Strikingly in the TB-PE cases, comparison of disease site, i.e. pleural effusion with peripheral blood showed increased expression of both soluble and surface HLA-G, whereas ILT-2 expressing cells were reduced at the local disease site. Furthermore, we demonstrated that in TB-PE cases, HLA-G expression on CD3⁺ T cells was influenced by broad spectrum MMP inhibitor. Thus, differential expression of HLA-G could potentially be a useful biomarker to distinguish different states of TB disease.