Detection of soluble HLA-G molecules in plasma and amniotic fluid

Although the cDNA sequence of HLA-G antigens is compatible with their expression as soluble molecules (sHLA-G), the determination of native sHLA-G levels in body fluids has not yet been described. The lack of this information is likely to reflect the difficulties in developing an assay suitable to measure sHLA-G antigens in the presence of soluble HLA-A, -B and -C (sHLA-I) antigens, since most of the available anti-HLA-G mAb do not detect soluble beta2-m associated HLA-G antigens or crossreact with sHLA-I antigens. Therefore, we have developed a two-step assay which eliminates the interference of classical HLA class I antigens. In the first step, the sample is depleted of sHLA-I antigens and of HLA-E antigens with mAb TP25.99. Then, HLA-G antigens are captured with mAb W6/32 and detected with anti-beta2-m mAb in ELISA. Utilizing this assay, sHLA-G antigen levels were measured in EDTA plasma from 92 controls with known HLA types, 28 women at delivery and the corresponding cord bloods and in 50 amniotic fluids. Mean sHLA-G plasma levels did not differ between males (24.9+/-3.0 SEM ng/ml; n=42) and females (20.1+/-2.1 SEM ng/ml; n = 50). However, sHLA-G levels in HLA-A11 positive probands (mean: 13.0+/-4.4 SEM ng/ml; n=12) were significantly (P<0.05) lower than in HLA-A11 negative ones (mean: 24.5+/-2.0 SEM ng/ml; n=80). sHLA-G levels in women at delivery (mean: 22.9+/-2.2 SEM ng/ml; n=28) were in the range of controls but were significantly (P<0.001) reduced in the corresponding cord bloods (mean: 13.8+/-1.5 SEM ng/ml; n=28). sHLA-G levels in amniotic fluids (mean: 15.5 + 1.0 SEM ng/ml; n=50) were significantly (P<0.001) lower than in plasma. sHLA-G levels were 5 and 11% of those of sHLA-I antigens in plasmas and amniotic fluids, respectively. Individual sHLA-G levels were not correlated with sHLA-I levels. SDS-PAGE analysis of plasma sHLA-G antigens revealed two molecular variants with a 35 kD and a 27 kD MW corresponding to the sizes of sHLA-G1 and -G2 isoforms. In conclusion, our study has shown that the two-step assay we have developed is reliable in measuring sHLA-G antigen levels. This assay will facilitate the analysis of the biological and clinical significance of sHLA-G antigens in plasma.     

Placental abruption is associated with decreased maternal plasma levels of soluble HLA-G

During pregnancy the fetus represents a semi-allograft. Both membrane-bound and soluble forms of the nonclassic human leukocyte antigen (HLA)-G protect the fetus from maternal immune attack. To assess the relevance of soluble HLA-G (sHLA-G) levels in the maternal circulation for the occurrence of characteristic pregnancy disorders, we analyzed sHLA-G plasma levels of women with normal and pathological pregnancies. Compared to normal pregnancy, significantly increased sHLA-G levels were detected in women delivered preterm because of intrauterine activation (uncontrollable labor, rupture of fetal membranes, cervical insufficiency) and women with Hemolysis, Elevated Liver enzymes, Low Platelet count (HELLP) syndrome. Contrary to these disorders, the sHLA-G levels in women with placental abruption were more than three times lower than in normal pregnancy (p < 0.0001). Nonparametric discriminant analysis showed that women with sHLA-G levels below 9.95 ng/mL had a relative risk of 7.12 for the development of placental abruption during further course of pregnancy. These results suggest that the occurrence of pregnancy-associated diseases is strongly influenced by maternal sHLA-G plasma levels.     

sHLA-G1 and HLA-G5 levels are decreased in Tunisian women with multiple abortion

Pregnancy is associated with increased levels of soluble (s) human leukocyte antigen (HLA)-G molecules, while during abortion these molecules are decreased. To date, little is known about the role of sHLA-G isoforms during abortion. In this study, we investigated the levels of total sHLA-G and its isoforms: HLA-G1 (membrane shedded isoform) and alternative spliced HLA-G5 in plasma samples obtained from 55 women who had experienced spontaneous abortion, 108 pregnant healthy women and 56 non pregnant healthy women.We found that pregnant women exhibited higher amounts of sHLA-G compared to either non pregnant women or women with abortion. Among women who had experienced spontaneous abortion, women with recurrent abortions (RSA) had lower sHLA-G than women with only one abortion. In particular, RSA women were characterized by the absence of sHLA-G1 isoform, suggesting a possible implication in abortion event.     

Association between HLA-G 14bp Gene Polymorphism and Serum sHLA-G Protein Concentrations in Preeclamptic Patients and Normal Pregnant Women

Background: Preeclampsia (PE) is a multisystem syndrome that is a primary source of fetal–maternal morbidity and mortality. Human leukocyte antigen-G (HLA-G) is a nonclassical Major histocompatibility complex (MHC) class-Ib molecule expressed on the extravillous trophoblast and seems to have immunomodulatory functions during pregnancy. The purpose of our study was to investigate whether HLA-G may be a vital marker in the modulation of the pregnancy.

Methods: In this case-control study, a number of 150 healthy pregnant women and 150 patients with PE had been genotyped for the 14 base-pair (bp) insertion/deletion polymorphism in exon 8 of the HLA-G gene, and the serum level of soluble HLA-G (sHLA-G) protein was measured using the enzyme-linked immunosorbent assay.

Results: Data showed that the PE syndrome was not related to the HLA-G 14 bp genotype. But, the serum level of sHLA-G in PE patients was significantly lower than that in healthy pregnant women in the third trimester (11.74 and 24.48 U/ml, respectively, p < 0.001). However, no significant association was observed between the HLA-G 14 bp genotype and serum sHLA-G level.

Conclusion: Our results demonstrate that measurement of sHLA-G protein level may be helpful as a primary diagnosis for the pathogenesis of PE. Overall, this study suggests that the association between HLA-G 14 bp polymorphism and serum sHLA-G level in different ethnic populations of PE should be taken into consideration.     

HLA-G alleles and HLA-G 14 bp polymorphisms in a Brazilian population

Brazilians represent one of the most heterogeneous populations in the world as the result of five centuries of interethnic crosses. We investigated the polymorphism of the HLA-G locus in a Brazilian urban sample. HLA-G alleles were determined by SBT analysis of exons 2-4 and 8 in 103 unrelated individuals. The genotyping analysis identified 11 different HLA-G alleles, including the recently described G*010110 allele, and four new variants in this population. These data indicate that this Brazilian population presents high HLA-G allelic polymorphism, suggesting increased levels of intrapopulational genetic diversity that may be because of the admixed nature of the sample. This is the first study regarding HLA-G genotyping that was performed at such refined resolution level in this heterogeneous population.     

Human leukocyte antigen (HLA)-G during pregnancy part II: Associations between maternal and fetal HLA-G genotypes and soluble HLA-G

Human leukocyte antigen (HLA)-G is a class Ib molecule with restricted tissue distribution expressed on the extra-villous trophoblast and seems to have immunomodulatory functions during pregnancy. Studies have linked HLA-G polymorphisms to pregnancy complications such as preeclampsia and recurrent miscarriage. Levels of soluble HLA-G (sHLA-G) in blood plasma from non-pregnant donors seem to be associated with these polymorphisms. In the current study, we have genotyped 246 mothers and their offspring for HLA-G polymorphisms in the 3′-untranslated region (3′UTR) and measured sHLA-G in maternal blood plasma samples from gestational week 20 and at term, as well as in fetal umbilical cord blood samples. This is the first large study simultaneously performing HLA-G genotyping of mother and offspring and measuring sHLA-G in both maternal and umbilical cord blood. The results showed that increasing numbers of 14 bp ins (rs66554220) alleles in the mother–child genotype combinations were associated with higher maternal sHLA-G levels at term when restricting the analysis to 14 bp ins/del heterozygous mothers (p = 0.015). Furthermore, increasing numbers of 14InsG haplotypes (14 bp ins/del and +3142C/G (rs1063320) polymorphism) in mother–child genotype combinations were associated with higher levels of sHLA-G at term in heterozygous 14DelC/14InsG mothers (p = 0.005). In conclusion, the results indicate that there is an association between combined feto-maternal HLA-G genotypes and sHLA-G levels in maternal blood plasma.     

可溶性HLA-G1、-G2的原核表达及其蛋白质产物的功能检测

sHLA-G存在于整个妊娠期母胎接触面及母体血循环中,目前被认为是妊娠期一种特异性免疫抑制分子。实验将sHLA-G1、sHLA-G2基因克隆于原核表达载体pET-28a中,转化大肠杆菌BL21(DE3)宿主菌,经IPTG诱导实现目标融合蛋白的高效表达,并通过Ni2+-NTA柱纯化、蛋白质透析梯度复性获得纯化的His-sHLA-G1、His-sHLA-G2融合蛋白。融合蛋白再经凝血酶酶切、苯甲脒琼脂糖凝胶吸附去除6-His标签后,用于体外功能鉴定。结果显示:sHLA-G2与sHLA-G1一样,同样具有抑制T细胞增殖、抑制NK细胞杀伤活性的功能,提示sHLA-G2为一种免疫致耐分子。     

Human leukocyte antigen (HLA)-G during pregnancy part I: Correlations between maternal soluble HLA-G at midterm,at term,and umbilical cord blood soluble HLA-G at term

Human leukocyte antigen (HLA)-G is a class Ib molecule with restricted tissue distribution expressed on trophoblast cells and has been proposed to have immunomodulatory functions during pregnancy. Soluble HLA-G1 (sHLA-G1) can be generated by the shedding of membrane-bound HLA-G molecules; however, three soluble isoforms also exist (HLA-G5 to -G6). During pregnancy, it is unknown whether there is a correlation between sHLA-G levels in maternal and fetal blood. In 246 pregnancies, we have measured the levels of sHLA-G1/-G5 in maternal blood plasma samples from gestational week 20 (GW20) and at term, as well as in umbilical cord blood samples. Soluble HLA-G levels declined by 38.4% in maternal blood from GW20 to term, and sHLA-G levels were significantly lower in maternal blood at term than in GW20 (P < 0.001). At term, the sHLA-G levels were significantly higher in maternal blood than in umbilical blood (P < 0.001). HLA-G levels in maternal blood in GW20 and at term, and in maternal blood at term and umbilical cord blood, were correlated (P < 0.001 and P < 0.01, respectively). This is the first large study simultaneously measuring sHLA-G in both maternal and umbilical cord blood. The finding that sHLA-G levels are significantly lower in fetal compared with maternal blood at term documents for the first time that sHLA-G is not freely transferred over the placental barrier. Soluble HLA-G levels in maternal and fetal blood were found to be correlated, which may be due to shared genetic factors of importance for production of sHLA-G in the mother and child, or it may support the theory that sHLA-G in the pregnant woman and the fetus is partly derived from a “shared organ”, the placenta.     

Comparative reactivity of different HLA-G monoclonal antibodies to soluble HLA-G molecules

Different HLA-G monoclonal antibodies (mAbs) were first evaluated for their capability to identify soluble HLA-G (sHLA-G) in ELISA. Three of them, namely 87G, BFL.1 and MEM-G/9, when used as coating mAbs together with W6/32 capture mAb, identified beta2-microglobulin (beta2m)-associated-sHLA-G but not soluble HLA-B7 (sHLA-B7) in cell culture supernatants from transfected cells. By comparison, the anti-HLA class I mAb 90 did recognize both sHLA-G and sHLA-B7. By using these HLA-G mAbs, sHLA-G was identified in amniotic fluids as well as in culture supernatants of first trimester and term placental explants but not in cord blood. Intron 4-retaining sHLA-G isoforms were identified in some amniotic fluids by the use of an intron 4-specific mAb (16G1). Reactivity of these different HLA-G mAbs was then compared to determine their respective binding sites on soluble and membrane-bound HLA-G. Using both ELISA and flow cytometry analysis, we showed that they did not compete with each other, which suggested that they did not recognize the same determinants. Finally, we report that two mAbs directed against the alpha1 domain of HLA class I heavy chain (mAb 90 and YTH 862) did compete with 87G, therefore demonstrating that this latter mAb recognized an epitope localized on this external domain of HLA-G.     

Embryonic soluble HLA-G as a marker of developmental potential in embryos

BACKGROUND: In human reproduction, embryo implantation is complex and poorly understood. At present, no single markers are used in routine treatment to assay biochemical functions of the human embryo. Soluble human leukocyte antigen-G (sHLA-G) could be considered a possible marker of embryo developmental potential. It is localized primarily on the extravillous trophoblast, making this antigen a potential mediator of immune interaction at the maternal-fetal interface during gestation. METHODS: Soluble-HLA-G levels were evaluated by an enzyme-linked immunosorbent assay (ELISA) employing monoclonal antibody MEM-G9. It was evaluated in 318 media of single embryo cultures. We correlated the presence of sHLA-G with embryo morphology and the pregnancy obtained in that treatment cycle. RESULTS: No correlation was found between embryo morphology and sHLA-G levels. Pregnancy was observed only when the medium of at least one transferred embryo contained sHLA-G. In 26 out of 66 patients, none of the obtained embryos showed any detectable sHLA-G molecules and no pregnancy occurred. CONCLUSIONS: From our results, we propose sHLA-G as a potential marker of embryo development: the sHLA-G ELISA can be a useful biochemical assay in addition to embryo morphology in embryo selection for transfer in IVF treatment if there are other embryos with the same morphology.     

HLA-G14bp基因多态性与儿童Epstein-Barr病毒感染的相关性分析

目的 通过检测Epstein-Barr病毒(EBV)感染传染性单核细胞增多症(IM)患儿人类白细胞抗原G(HLA-G) 14 bp插入/缺失多态性及血浆可溶性HLA-G(sHLA-G)水平,探讨其与儿童EBV感染IM的相关性.方法 采用PCR结合聚丙烯酰胺凝胶电泳(PCR-PAGE)技术,对102例EBV感染IM患儿及165例正常对照儿童进行了HLA-G 14 bp插入/缺失多态性检测;采用ELISA检测了51例EBV感染IM患儿及146例正常对照儿童血浆sHLA-G水平.结果 EBV感染IM组与正常对照组HLA-G 14 bp插入/缺失基因型多态性分布频率差异有统计学意义(X2=6.742,P=0.034),两组等位基因分布频率差异亦有统计学意义(x2=6.672,P=0.01);EBV感染IM组血浆sHLA-G水平明显高于正常对照组,经检验差异有统计学意义(Z=-9.472,P＜0.01),EBV感染IM组不同基因型间血浆sHLA-G水平差异有统计学意义(H=6.09,P=0.048),14 bp+/+基因型组血浆sHLA-G水平明显低于另两组基因型(Z=-2.376,P=0.018).结论 HLA-G 14 bp插入/缺失基因型多态性与儿童EBV感染IM的发生有关,携带14 bp-/-基因型或缺失14 bp等位基因的儿童可能更易发生EBV感染.血浆sHLA-G水平可作为EBV感染IM的辅助诊断指标之一.     

High level of soluble HLA-G in amniotic fluid is correlated with congenital transmission of Toxoplasma gondii

The expression of human leukocyte antigen (HLA)-G on cytotrophoblast cells contributes to maternal-fetal tolerance. Soluble forms of HLA-G (sHLA-G) can be detected in amniotic fluid (AF) and a decrease of sHLA-G is known to be correlated to fetal loss. In this work we investigated the role of sHLA-G in the transplacental passage of the protozoan parasite Toxoplasma gondii, responsible for congenital toxoplasmosis in about 30% of fetuses when primary infection (PI) occurs during pregnancy. We determined the sHLA-G concentration in 61 AF from women with PI and 24 controls. Our results showed higher sHLA-G levels in AF from PI than in controls (p<0.001). Moreover sHLA-G level from congenitally infected fetuses (n=12) was higher than in fetus in whom congenital infection was ruled out (n=49, p<0.05). These data suggest that sHLA-G could participate in immunomodulation necessary to avoid fetal loss due to Toxoplasma infection, but that over-expression could favor congenital transmission.     

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。     

Plasma soluble HLA-G levels in a cohort of heart failure patients exposed to chemicals

Heart failure (HF) is a syndrome caused by structural and/or functional cardiac abnormalities, resulting in a reduced cardiac output and/or elevated intracardiac pressures. Several studies reported a crucial role of immune activation and inflammation in the chronic heart failure (HF) pathogenesis, suggesting that pro-inflammatory and anti-inflammatory mediators could be predictive markers of the HF development and/or progression. Human Leukocyte Antigen-G (HLA-G), a tolerogenic and anti-inflammatory class I non-classical major histocompatibility complex molecule, was reported to be upregulated in patients diagnosed with HF, suggesting a tentative to regulate the inflammatory condition. We evaluated soluble (s)HLA-G plasmatic levels in patients with stable chronic heart failure at baseline visit and after 6 and 12 months. The 14 bp Insertion/Deletion polymorphisms of the HLA-G gene was also analyzed. We showed that in HF subjects, sHLA-G levels were higher in NYHA class II and III subjects (mild-severe symptoms) (6.11 ± 1.15 ng/ml; 8.25 ± 2.27 ng/ml, respectively) in comparison with NYHA class I subjects (no symptoms) (2.35 ± 0.43 ng/ml) (I vs II: p = 0.0156; I vs III: p = 0.0122). Moreover, the exposure to chemicals seems to affect sHLA-G levels, with higher sHLA-G levels in exposed patients (3.36 ± 5.12 ng/ml) in comparison with unexposed subjects (2.01 ± 2.84 ng/ml). The HLA-G 3′UTR 14 bp INS/DEL polymorphism correlated with sHLA-G, with the 14 bp INS/INS genotype associated with higher sHLA-G levels during the 12 months follow-up in unexposed subjects (p = 0.008). In conclusion, these results support a correlation between sHLA-G levels, genetics and HF disease in presence of work chemical exposition.     

Association of the maternal 14-bp insertion polymorphism in the HLA-G gene in women with recurrent spontaneous abortions

Human leukocyte antigen (HLA)-G has been postulated as an important immunotolerant molecule in maintaining fetal-maternal relationship. Recent reports indicated that the 14-bp deletion/insertion polymorphism in exon 8 of HLA-G gene influences HLA-G mRNA stability and isoform splicing patterns, thus modulating the levels of HLA-G expression. This might play an immunomodulatory role of HLA-G during implantation and pregnancy. In the present study, 109 unrelated fertile control women and 79 women who had experienced recurrent spontaneous abortion (RSA) were genotyped for the 14-bp insertion/deletion polymorphism. No significant difference was observed in the distribution of 14-bp insertion/deletion genotype between controls and the RSA group. However, a greater number of 14-bp insertion alleles exist in the RSA group than in the controls.     

Association of HLA-G alleles and 3' UTR 14 bp haplotypes with recurrent miscarriage in Brazilian couples

Human leukocyte antigen (HLA)-G expression is restricted, expressed on trophoblast, with a major role in fetus acceptance. In addition to the 46 HLA-G alleles, the presence or absence of a 14 bp polymorphism located in the 3' UTR contributes to gene polymorphism that may influence both HLA-G mRNA stability and HLA-G isoform's splicing and consequently could play an immunomodulatory function in pregnancy. To elucidate the role of HLA-G polymorphism in pregnancy, HLA-G allele frequencies and the 14 bp polymorphisms were analyzed and compared in 60 couples with recurrent miscarriage (RM) and 68 fertile control couples. Two haplotypes showed a significant elevated frequency in patients (HLA-G*01:01:08/+14, p(c) < 0.0001 and HLA-G*01:04:01/-14, p(c) < 0.0001). The haplotype HLA-G*01:01:A/+14 exhibited a significant protective effect against RM in women (p(c) = 0.0238). Remarkably, significant differences in linkage disequilibrium were observed between patient and control groups. Two alleles showed a positive association with the +14 bp segment in RM patients and a strong negative association with fertile controls (HLA-G*01:01:08 = patients D' = 0.295-0.371; controls D' = -0.715 to -1.000; HLAG* 01:05N = patients D' = 0.728-1.000; controls D' = -1.000). HLA-G*01:04:01 showed a negative association with the 14 bp segment in patients and a positive association in controls (patients D' = -0.249 to - 0.674; controlss D' = 0.688-1.000). Our results suggest that haplotypic combinations of HLA-G alleles and the 14 bp segment may be associated with RM.