HLA-G 3’UTR polymorphisms & response to a yoga-based lifestyle intervention in rheumatoid arthritis: A randomized controlled trial

Background & objectives: Human leucocyte antigen (HLA)-G plays a vital role in immunomodulation in rheumatoid arthritis (RA). The mounting evidence suggests a link between HLA-G gene polymorphisms, disease susceptibility and methotrexate treatment response. Various environmental factors influence the onset and progression of RA and its treatment outcomes. The aim is to identify the treatment response of HLA-G 3’ untranslated region polymorphisms to yoga-based lifestyle intervention (YBLI). Methods: In this eight-week single-blinded randomized controlled trial (CTRI/2017/05/008589), patients with RA (n=140) were randomized into two groups namely, yoga group or non-yoga group. Baseline genomic DNA was isolated using salting-out method. PCR-based methods were used for genotyping. The levels of soluble (s) HLA-G and disease activity were assessed by ELISA and disease activity score-28–erythrocyte sedimentation rate (DAS28-ESR), respectively, at baseline (day 0) and after eight weeks of intervention. Results: Low-producing sHLA-G genotypes, i.e. +3142GG and 14 bp ins/ins, showed a significant increase in sHLA-G levels after YBLI. The association analysis between HLA-G polymorphisms and treatment for RA showed no considerable differential treatment remission in either of the groups (P>0.05). The percentages of improvement were higher in the yoga group as compared to the non-yoga group in both the HLA-G +3142G>C and 14 bp ins/del polymorphisms irrespective of their respective genotypes. No significant association was found between sHLA-G levels and disease activity with respect to genotypes. Interpretation & conclusions: Yoga intervention results in improvement and reduced severity of RA in patients irrespective of the HLA-G 14 bp ins/del or +3142G>C polymorphisms. YBLI may be used as an adjunct therapy in RA independent of the genotypes

Generation of genetic modified pigs devoid of GGTA1 and expressing the human leukocyte antigen-G5 / 生物工程学报

Pigs are considered as ideal donors for xenotransplantation because they have many physiological and anatomical characteristics similar to human beings. However, antibody-mediated immunity, which includes both natural and induced antibody responses, is a major challenge for the success of pig-to-primate xenotransplantation. Various genetic modification methods help to tailor pigs to be appropriate donors for xenotransplantation. In this study, we applied transcription activator-like effector nuclease (TALEN) to knock out the porcine α-1, 3-galactosyltransferase gene GGTA1, which encodes Gal epitopes that induce hyperacute immune rejection in pig-to-human xenotransplantation. Meanwhile, human leukocyte antigen-G5 gene HLA-G5, which acts as an immunosuppressive factor, was co-transfected with TALEN into porcine fetal fibroblasts. The cell colonies of GGTA1 biallelic knockout with positive transgene for HLA-G5 were chosen as nuclear donors to generate genetic modified piglets through a single round of somatic cell nuclear transfer. As a result, we successfully obtained 20 modified piglets that were positive for GGTA1 knockout (GTKO) and half of them expressed the HLA-G5 protein. Gal epitopes on the cell membrane of GTKO/HLA-G5 piglets were completely absent. Western blotting and immunofluorescence showed that HLA-G5 was expressed in the modified piglets. Functionally, the fibroblasts from the GTKO/HLA-G5 piglets showed enhanced resistance to complement-mediated lysis ability compared with those from GTKO-only or wild-type pigs. These results indicate that the GTKO/HLA-G5 pigs could be a valuable donor model to facilitate laboratory studies and clinics for xenotransplantation.

Níveis plasmáticos de HLA-G solúvel em pacientes com covid-19 / Plasma levels of soluble HLA-G in patients with covid-19

Aspectos da biologia da HLA-G, como as funções altamente imunoinibitórias são fundamentais para a compreensão da relevância dessa molécula em condições fisiológicas e patológicas, e podem auxiliar a projetar estratégias diagnósticas e terapêuticas em várias áreas da saúde humana. A covid-19 é uma doença viral que acomete o mundo. Por se tratar de uma doença viral, as moléculas de HLA-G solúveis (sHLA-G) são consideradas potentes imunomoduladores, e sua expressão desregulada têm sido implicadas em várias condições patológicas. O objetivo deste estudo foi quantificar os níveis plasmáticos de sHLA-G em pacientes com covid-19. Este é um estudo observacional, descritivo, do tipo transversal. O estudo foi conduzido na cidade de Ribeirão Preto. As análises estatísticas foram realizadas utilizando-se o programa GraphPad Prism versão 9. A análise de distribuição dos dados foi realizada pelo teste de normalidade de Shapiro-Wilk e a análise comparativa das variáveis entre os grupos foi realizada por meio de testes não paramétricos de Mann-Whitney e Kruskal-Wallis. Incluímos nesse estudo 262 indivíduos. Dentre os que apresentaram covid-19, 60 estavam em cuidados domiciliares e 129 eram pacientes hospitalizados. Como grupo controle saudável, foram incluídos 73 indivíduos. A mediana da idade foi 55 anos para o grupo com covid-19, já para o grupo controle foi de 33,5 anos. A maioria dos pacientes do grupo hospitalar era do sexo masculino (67,4%). Em relação ao gênero não houve diferenças estatisticamente significantes nos níveis plasmáticos de sHLA-G entre os grupos domiciliar e hospitalar. No entanto, ao classificar os pacientes de covid-19 de acordo com o seu estado de gravidade clínica, observamos que todos os tipos de gravidade clínica apresentaram diferenças estatisticamente significantes em relação ao controle saudável (controle vs leve: p=0,0004; controle vs moderado: p=0,0011; controle vs grave: p<0,0001 e controle vs crítico: p<0,0001), sendo que as concentrações de sHLA-G foram gradativamente aumentando nos pacientes com covid-19 de leve a grave. Contraditóriamente, os níveis de sHLA-G estavam significantemente menores entre pacientes com covid-19 crítico quando comparados aos com covid-19 grave (p=0,02). A partir do perfil plasmático de sHLA-G por gravidade, estratificamos os pacientes pelos desfechos clínicos de resolutividade da doença (com alta hospitalar) e óbito; e observamos maior concentração de sHLA-G no grupo de pacientes que morreram em relação ao controle saudável (p<0,0001), assim como comparado àqueles que tiveram a resolução da doença e receberam alta hospitalar (p=0,0166). Nossos resultados revelam que os níveis de sHLA-G, importante molécula de "checkpoint" imuno inibitória, estão maiores no início da infecção, e vai gradualmente crescendo conforme a gravidade clínica da doença progride, entretanto, quando os parâmetros clínicos avançam para a covid-19 crítica, os níveis de HLA-G caem, sugerindo a exaustão, após a superexpressão deste inibidor de controle imunológico. Estes dados destacam a importância da molécula HLA-G no contexto de infecções virais, como SARS-CoV-2, revelando sua potencial capacidade de interações para planejamento de terapias para gerenciar a infecção, progressão e prognóstico da covid-19.

Aspects of the biology of HLA-G, such as the highly immunoinhibitory functions, are fundamental for understanding the relevance of this molecule in physiological and pathological conditions and can help design diagnostic and therapeutic strategies in various areas of human health. Covid-19 is a viral disease that affects the world. As it is a viral disease, soluble HLA-G molecules (sHLA-G) are considered potent immunomodulators, and their unregulated expression has been implicated in several pathological conditions. The aim of this study was to quantify the plasma levels of sHLA-G in patients with covid-19. This is an observational, descriptive, crosssectional study. The study was conducted in the city of Ribeirão Preto. Statistical analyzes were performed using the GraphPad Prism version 9 program. Data distribution analysis was performed using the Shapiro-Wilk normality test and the comparative analysis of variables between groups was performed using nonparametric Mann tests. -Whitney and Kruskal-Wallis. We included 262 individuals in this study. Among those who had covid-19, 60 were in home care and 129 were hospitalized patients. As a healthy control group, 73 subjects were included. The median age was 55 years for the group with covid-19, while for the control group it was 33.5 years. Most patients in the hospital group were male (67.4%). Regarding gender, there were no statistically significant differences in sHLA-G plasma levels between the home and hospital groups. However, when classifying covid-19 patients according to their clinical severity status, we observed that all types of clinical severity showed statistically significant differences from the healthy control (control vs mild: p=0.0004; control moderate vs moderate: p=0.0011; control vs severe: p<0.0001 and control vs critical: p<0.0001), with sHLA-G concentrations gradually increasing in patients with mild to moderate COVID-19. serious. Contradictingly, sHLA-G levels were significantly lower among patients with critical covid-19 compared to those with severe covid-19 (p=0.02). Based on the sHLA-G plasma profile by severity, we stratified patients by clinical outcomes of disease resolution (with hospital discharge) and death; and we observed a higher concentration of sHLA-G in the group of patients who died in relation to the healthy control (p<0.0001), as well as in those who had the disease resolution and were discharged from the hospital (p=0.0166). Our results reveal that the levels of sHLA-G, an important immunoinhibitory checkpoint molecule, are higher at the beginning of the infection, and gradually increase as the clinical severity of the disease progresses, however, when the clinical parameters advance towards covid-19. 19 critical, HLA-G levels drop, suggesting exhaustion, following overexpression of this immune control inhibitor. These data highlight the importance of the HLA-G molecule in the context of viral infections, such as SARS-CoV-2, revealing its potential interactions for planning therapies to manage the infection, progression, and prognosis of covid-19.

Evaluation of HLA-G immune checkpoint molecule in melanocytic lesions / Avaliação da molécula de checkpoint imunológico HLA-G em lesões melanocíticas

ABSTRACT The human leukocyte antigen-G (HLA-G) is a non-classical molecule of the major histocompatibility complex. HLA-G has been associated with the process of tumorigenesis and tumor escape. In this study, we aim to evaluate the HLA-G expression in melanocytic lesions and in melanoma for determining when melanocytic lesions start its expression. Twenty-two skin biopsies samples were submitted to immunohistochemistry; HLA-G expression was detected in 63.6% of the samples. This expression in melanocytic cells was significantly higher in melanoma than in benign melanocytic lesions (p < 0.002). Our results suggest that HLA-G expression starts late in the process of tumorigenesis.

RESUMEN El antígeno leucocitario humano G (HLA-G) es una molécula no clásica del complejo principal de histocompatibilidad que ha sido asociada al proceso de tumorigénesis y escape tumoral. En este estudio, nuestro objetivo es evaluar la expresión de HLA-G en lesiones melanocíticas y en el melanoma para determinar cuando las lesiones melanocíticas comienzan su expresión. Veintidós muestras de biopsias de piel se estudiaron mediante inmunohistoquímica; se detectó la expresión de HLA-G en el 63,6% de las muestras. Esa expresión en las células melanocíticas fue significativamente mayor en el melanoma que en lesiones melanocíticas benignas (p < 0,002). Nuestros resultados sugieren que la expresión de HLA-G empieza tardíamente en el proceso de tumorigénesis.

RESUMO O antígeno leucocitário humano G (HLA-G) é uma molécula não clássica do complexo principal de histocompatibilidade que tem sido associada ao processo de tumorigênese e escape tumoral. Neste estudo, objetivamos avaliar a expressão de HLA-G em lesões melanocíticas e no melanoma para determinar quando as lesões melanocíticas iniciam sua expressão. Vinte e duas amostras de biópsias de pele foram submetidas à imuno-histoquímica; a expressão de HLA-G foi observada em 63,6% das amostras. Essa expressão nas células melanocíticas foi significativamente maior no melanoma do que em lesões melanocíticas benignas (p < 0,002). Nossos resultados sugerem que a expressão de HLA-G se inicia tardiamente no processo da tumorigênese.

Effects of human leucocyte antigen-G expression on invasion and proliferation of chorionic trophoblastic cell line JEG-3 / 中华妇产科杂志

Objective To investigate the effects of human leukocyte-associated antigen-G (HLA-G) expression in silencing trophoblast cell line JEG-3 under normal and hypoxic conditions on invasion and proliferation of JEG-3 cells. Methods Inhibition of HLA-G expression in JEG-3 cells by transfection of small interfering RNA (siRNA),the transfected JEG-3 cells were divided into 4 groups: normoxia control group, hypoxia control group, normoxia inhibition group and hypoxia inhibition group. The levels of HLA-G mRNA and protein in 4 groups of cells were detected by real-time quantitive PCR and western blot. The proliferation activity and invasion ability of 4 groups of cells were determined by methylthiazolyl tetrazolium (MTT) assay and invasion assay.Results (1) Real-time quantitive PCR technology showed: the level of HLA-G mRNA in the hypoxic inhibition group (0.220±0.050) was significantly different (P<0.05), when compared with that in the hypoxic control group (0.630±0.030) and normoxic inhibition group (0.400± 0.020). (2) Western blot analysis showed: the expression level of HLA-G protein in the hypoxic inhibition group was 0.260±0.010, statistically different from that in the hypoxic control group (0.850±0.100) and the normoxic inhibition group (0.560±0.020; P<0.05).(3) MTT showed: proliferative activity of JEG-3 cells in the normoxic inhibition group was 0.490 ± 0.070, the ability of cell proliferation was reduced. When compared with that in the normoxic control group (0.850±0.050), the differences was statistically significant (P<0.05). The proliferative activity of JEG-3 cells in the hypoxic inhibition group (0.330±0.070) was lower than that in the normoxic inhibition group (0.490±0.070), and there was a significant difference (P<0.05). (4) Invasion assay showed: compared with the normoxic control group (98±7), the invasive ability of JEG-3 cells in the normoxic inhibition group (73 ± 7) was weakened, and the difference was statistically significant (P<0.05). The number of transmembrane cells (52±11) of JEG-3 cells in the hypoxic inhibition group was lower than that in the hypoxic control group (72±7), and the difference was statistically significant (P<0.05). Compared with the normoxic inhibition group, the invasion ability of JEG-3 cells in the hypoxic inhibition group decreased, and the difference was statistically significant (P<0.05). Conclusion Under hypoxia, using siRNA technology to down-regulate the expression of HLA-G may affect the proliferation and invasion ability of trophoblast cells, which may be involved in the occurrence of hypertensive disorder of pregnancy.

Expression and role of regulatory plasma cells in gravidas with systemic lupus erythematosus / 中华微生物学和免疫学杂志

Objective To investigate the expression and role of regulatory plasma cells in gravidas with systemic lupus erythematosus ( SLE) . Methods Gravidas with SLE were enrolled in Henan Provincial People's Hospital from April 2013 to April 2018. They were divided into three groups including pregnancy control group, SLE stable group and SLE deterioration group. The ratio of CD3-LAG-3+CD138high regulatory plasma cells was detected by flow cytometry. The concentrations of soluble human leukocyte antigen-G ( sHLA-G) and anti-nuclear antibody Ig were detected by ELISA. Lymphocytes in peripheral blood of SLE deterioration group were isolated, and then cultured in RPMI1640 medium containing 10％ fetal bovine ser-um and stimulated with HLA-G. Results Flow cytometry showed that the proportion of regulatory plasma cells in SLE stable group was (2. 483±0. 1318)％ and that in SLE deteriorating group was (1. 662± 0. 1304)％. There was a significant difference between the two groups (t=4. 431, P=0. 0013). The con-centrations of sHLA-G in SLE stable group and SLE deteriorating group were (36. 50±3. 510) ng/ml and (16. 50±2. 405) ng/ml, and the difference between the two groups was statistically significant (t=4. 701, P=0. 0008). Correlation analysis showed that the concentration of sHLA-G was positively correlated with the proportion of regulatory plasma cells (r=0. 7471, P=0. 0009). The results of in vitro experiment showed that the proportions of B cells and regulatory plasma cells were ( 7. 573 ± 0. 6539 )％ and ( 1. 593 ± 0.1879)％ in SLE deterioration group and (3. 732±0. 7178)％ and (2. 503±0. 2921)％ in HLA-G group with statistical differences between the two groups (t=3. 957, P=0. 0027;t=2. 620, P=0. 0256). Conclusions The proportion of regulatory plasma cells and the concentration of sHLA-G were significantly decreased in pregnant patients with SLE, which was closely related to disease severity. HLA-G played an important role in promoting the proliferation of regulatory plasma cells.

Expression and clinical significance of sHLA-G in plasma exosome in patients with colorectal cancer / 中华检验医学杂志

Objective To detect sHLA-G expression in plasma exosomes in patients with colorectal cancer and evaluate its clinical significance.Methods Retrospective study.Plasma was collected from 52 primary CRC patients,20 colorectal polyps patients,20 inflammatory bowel disease patients and 25 healthy donors in the Taizhou Hospital of Zhejiang Province from May 2017 to August 2018.The exosomes were extracted by exoEasyMaxikit and identified by nanoparticle tracking analysis (NTA) and Western blot.Exosomal sHLA-G was detected by flow cytometry (FCM) and enzyme-linked immunosorbent assay (ELISA).The diagnostic values of exosomal sHLA-G detected by FCM and ELISA were assessed,and their diagnostic performances were compared with carcinoembryonic antigen (CEA) and carbohydrate antigen CA19-9 by ROC curve and Youden index.Results The peak size of exosomes extracted from plasma in CRC patients was 101.1 nm and Western blot showed these exosomes expressed marker CD63,CDS1,and TSG101.Exosomal sHLA-G of CRC patients [28.0(21.5-35.1)U/ml] was significantly higher than that in healthy controls[19.6(16.8-21.3) U/ml,U=143.0,P＜0.001],colorectal polyps patients[19.7(16.2-22.5)U/ml,U=180.0,P＜0.001] as well as inflammatory bowel disease patients[19.9(16.7-25.2)U/ml,U=197,P＜0.001].The postoperative sHLA-G level[19.6(17.8-26.3)U / ml,U=325.5,P=0.015] was significantly lower than that in pre-operation.Exosomal sHLA-G was significantly different in different tumor status(U=64.0,P=0.006),lymph node metastasis (U=81.0,P=0.003) and TNM stage (U=105.0,P=0.015) in patients with CRC.ROC curve showed the area under the curve (AUC) of exosomal sHLA-G detected by FCM and ELISA,CEA and CA19-9 was 0.962±0.019,0.899±0.038,0.786±0.058,0.680±0.068,respectively.The difference of AUC was operated by Z test,and it showed that the exosomal sHLA-G detected by FCM was superior to CEA(Z=2.884,P=0.004)and CA19-9(Z=3.994,P＜0.001),and the exosomal sHLA-G detected by ELISA was superior to CA19-9(Z=2.811,P=0.005).Conclusion Plasma exosomal sHLA-G was associated with the progression of CRC and its diagnostic value was superior to the traditional tumor markers CEA and CA 19-9.

Experimental study on the in vitro induction of regulatory T cells by umbilical cord mesenchymal stem cells with positive human leukocyte antigen-G / 器官移植

Objective To explore the effect of umbilical cord mesenchymal stem cells with positive human leukocyte antigen(HLA)-G on inducing the production of regulatory T cells(Treg) in vitro.Methods Umbilical cord mesenchymal stem cells were isolated from umbilical cord of neonates. PEGFP-N1-HLA-G plasmid was transfected into the human umbilical cord mesenchymal stem cells by liposome transfection, as PEGFP-N1-HLA-G group. PEGFP-N1 empty vector plasmid was transfected into the human umbilical cord mesenchymal stem cells, as PEGFP-N1 group. The human umbilical cord mesenchymal stem cells without empty vector under the same conditions were set as blank control group. Markers of the umbilical cord mesenchymal stem cells were detected using flow cytometry. The expression of HLA-G protein in each group of cells was identified by Western Blot. After mixed-culturing with CD4+T cells in peripheral blood of healthy subjects for 24 h and 48 h, the proportion of CD4+CD25+Foxp3+Treg in total T cells of each group was detected by flow cytometry. Results CD45, CD34 and HLA-DR presented negative expression on umbilical cord mesenchymal stem cells, while CD29, CD44 and CD105 presented positive expression. HLA-G protein could be expressed in the PEGFP-N1-HLA-G group, which had statistically significant difference compared with the blank control group and PEGFP-N1 group (both P<0.01). After PEGFP-N1-HLA-G group and CD4+T cells were mixed-cultured for 24 h and 48 h, CD4+CD25+Foxp3+Treg accounted for (15.3±1.9)% and (14.3±2.1)% of the total T cells respectively, both of which presented statistically significant difference compared with the blank control group and PEGFP-N1 group (all P<0.05). Conclusions Umbilical cord mesenchymal stem cells with HLA-G gene modified can effectively induce the production of CD4+CD25+Foxp3+Treg in vitro.

Roles of human leukocyte antigen-G ( HLA-G) in MDSC proliferation, M1/M2 macrophage differ-entiation and tumor immune evasion / 中华微生物学和免疫学杂志

Objective To investigate the roles of human leukocyte antigen-G ( HLA-G) in mye-loid-derived suppressor cell (MDSC) proliferation and M1/M2 macrophage differentiation in C57BL/6-NCI-H446-G tumor-bearing mice for better understanding the mechanisms of HLA-G involved in tumor immune evasion. Methods NCI-H446 ( human small cell lung cancer cells) and NCI-H446-G ( NCI-H446 cells ex-pressing HLA-G) cells were labeled with CFSE at a final concentration of 1μmol/L. CFSE fluorescence lev-els were measured by flow cytometry at different time points. Mouse tumor models were established by subcu-taneous injection of C57BL/6 mice with NCI-H446 and NCI-H446-G cells, respectively. PBS was used to set up negative control group. The mice in each group were sacrificed to collect tissue samples on 5 d, 10 d, 15 d and 20 d after injection. The percentages of splenic CD11b+Gr1+MDSCs, F4/80+CD80+M1 and F4/80+CD206+M2 macrophages were analyzed by flow cytometry. Results Steady expression of HLA-G in NCI-H446-G cells was confirmed by Western blot and flow cytometry. HLA-G enhanced the proliferation of NCI-H446 cells. Tumor size increased dramatically in tumor-bearing mice in the first five days and then de-creased over time. The tumor-bearing mice in the NCI-H446-G group had larger tumor than those in the NCI-H446 group in every time point (P<0. 05) and required longer time to fully reject the tumor. Compared with the PBS and NCI-H446 groups, the percentage of splenic MDSCs in tumor-bearing mice was significantly in-creased in the NCI-H446-G group (P<0. 05). Moreover, the ratio of M1/M2 in NCI-H446-G tumor-bearing mice was much lower than that in the other two groups (P<0. 05). Conclusion This study indicated that HLA-G could increase the percentage of MDSCs and decrease the ratio of M1/M2, which might illustrate the role of HLA-G in tumor immune evasion and its potential clinical significance in cancer immunotherapy.

Soluble HLA-G: A novel marker in acute myeloid leukemia patients

Objective: to study soluble HLA-G (sHLA-G) in Egyptian acute myeloid leukemia (AML) patients. HLA-G is speculated to be a tumor-driven immune escape mechanism. In addition, it might be a promising target for future immune therapeutic approaches. Methods: Thirty AML patients and 15 healthy controls of matched age and sex were the subject of the study. sHLA-G was done to all patients and controls by ELISA. Results: Statistically significant increase in sHLA-G level was present in AML patients compared to controls, being higher in relapsed cases. HLA-G levels was correlated to bone marrow blast percentages but not affected by age, gender, WBCs or response to chemotherapy. HLA-G had a sensitivity of 100% and a specificity of 62% to detect AML cases. Conclusion: HLA-G may be an additional marker for AML especially relapsed cases

Application value of combined detection of leukocyte antigen G and interleukin 10 in diagnosis of nfantile cytomegalovirus hepatitis / 国际检验医学杂志

Objective To explore the clinical value of combined detection of human leucocyte antigen G (HLA-G) and interleu-kin10(IL-10) in children patients with cytomegalovirus(CMV) hepatitis .Methods 122 children patients with CMV hepatitis in our hospital from January 2013 to January 2017 were selected as the children patients group and contemporaneous 116 healthy children were selected as the control group .The levels of plasma HLA-G and IL-10 were detected by adopting ELISA .Then their correlation and diagnostic value were analyzed .Results The levels of plasma HLA-G and IL-10 in the children patients group were significant-ly higher than those in the control group ,and the difference was statistically significant (P<0 .05) .In grouping according to the age of the month ,the levels of plasma HLA-G and IL-10 in the 1-6 months old patients group were highest .HLA-G and IL-10 had a positive correlation(r=0 .445) .Moreover ,the receiver operating characteristic curve analysis results showed that the area under the curve of combined detection of plasma HLA-G and IL-10 was maximal and its diagnostic efficiency was highest .Conclusion The combined detection of plasma HLA-G and IL-10 can increase the accuracy of CMV infection diagnosis and has an important clinical significance .

HLA-G 14 bp insertion/deletion polymorphism is a prognostic factor for non-small-cell lung cancer / 中华微生物学和免疫学杂志

Objective To study the correlation between human leucocyte antigen-G (HLA-G) 14 bp insertion/deletion (I/D) polymorphism and susceptibility to non-small cell lung cancer (NSCLC) as well as poor prognosis in NSCLC.Methods A total of 113 patients with NSCLC and 150 age-and sex-matched healthy subjects were genotyped by PCR to analyze the HLA-G 14 bp insertion/deletion polymorphism in them.Epidermal growth factor receptor (EGFR) gene mutation in patients with NSCLC was detected by using amplification refractory mutation system (AMRS).Expression of HLA-G in NSCLC tissues was detected with immunohistochemistry.All patients with NSCLS were followed up to collect survival data, which were further analyzed with Kaplan-Meier method.Results The frequency of HLA-G 14 bp D/D genotype was significantly higher in the patients with NSCLC than that in the healthy subjects (x2=3.907, P=0.048, OR=1.66).Among the patients with NSCLC, HLA-G 14 bp I/I genotype carriers had a shorter overall survival time as compared with that of HLA-G 14 bp I/D or HLA-G 14 bp D/D genotype carriers (P=0.005).Patients who received chemotherapy or radiation had significantly shorter survival time than those received EGFR-targeted therapy (P=0.001).Among patients who were positive for EGFR mutation, HLA-G 14 bp D/D genotype carriers had longer survival time than those carrying HLA-G 14 bp I/I or HLA-G 14 bp I/D genotype (P=0.041).The expression of HLA-G was closely correlated with HLA-G 14 bp polymorphism in patients with NSCLC (P=0.001).Conclusion These data, reported for the first time, indicates that HLA-G 14 bp polymorphism might be a genetic factor related to the susceptibility to NSCLC and associated with survival in patient with NSCLC after excluding the interference of molecular targeted agents.

Association between 14bp Insertion/Deletion Polymorphism in Exon 8 of HLA-G gene and Oral Squamous Cell Carcinoma in Korean Population

Abnormal HLA-G expression occurs in various diseases such as melanoma, renal cell carcinoma, asthma, and classic Hodgkin's lymphoma. The purpose of this study was to determine whether HLA-G gene is linked with oral squamous cell carcinoma (OSCC). To investigate the possible link with susceptibility to OSCC, 54 OSCC patients and 120 healthy controls were enrolled in this study. HLA-G 14bp insertion/deletion polymorphism is in 3′-untranslated region of HLA-G gene. HLA-G 14bp insertion/deletion polymorphism was analyzed using the polymerase chain reaction (PCR) method. For the analysis of genetic data, SPSS18.0 program was used. Logistic regression models were performed for odds ratio (OR), 95 percent confidence interval (CI), and P value. There was a significant difference in distribution allele between OSCC patients and control subjects (OR=0.018, 95% CI=0.002-0.131, p<0.001). Our results suggest that HLA-G 14bp insertion/deletion polymorphism may be linked with susceptibility to OSCC in the Korean population.

Exosomes bearing HLA-G and tumor immunology / 中华微生物学和免疫学杂志

Human leukocyte antigen G ( HLA-G) is a member of the non-classical HLA classⅠb family. It is considered to play a crucial role in immune tolerance. A unique feature of HLA-G is the struc-tural diversity as surface expressed and as secreted molecules, which is mainly attributed to alternative spli-cing of the primary transcript. HLA-G can promote the invasion and metastasis of tumor cells through various ways. In addition, HLA-G has been described included in exosomes. Exosomes released by most cell types are nano-sized vesicular bodies that contain lipid bilayer and rich contents. As a new marker for diseases, exosomes are extensively involved in the occurrence and development of diseases. Recent studies have found that exosomes can express soluble HLA-G, which reveal a new way by which HLA-G regulates tumor micro-environment. In this review, we focus on the expression of HLA-G on exosomes to provide new thoughts for the early detection and treatment of tumors.

Clinical significance of HLA-G in patients with gastric cancer / 中华微生物学和免疫学杂志

Objective To investigate the clinical significance of CD14+HLA-G+ monocytes in pe-ripheral blood and the soluble form of HLA-G ( sHLA-G ) in plasma among patients with gastric cancer ( GC) . Methods Blood samples were collected from 135 patients with gastric cancer ( GC group) , 150 pa-tients with chronic gastritis ( CG group) and 80 healthy controls ( HC group) . Flow cytometry analysis and ELISA were used to detect the percentages of CD14+HLA-G+ monocytes in peripheral blood samples, the concentrations of sHLA-G in plasma samples and the levels of alpha fetoprotein (AFP), cacino-embryonic antigen ( CEA) , CA19-9 and CA125 in serum samples. Mann-Whitney U test was performed to analyze the differences between different groups. The feasibility of using CD14+HLA-G+ monocytes, sHLA-G, AFP, CEA, CA19-9 and CA125 as potential biomarkers to differentiate patients with GC from those with CG or healthy subjects was assessed by using receiver operating characteristic ( ROC ) curve analysis. Results The median percentages of CD14+HLA-G+ monocytes in subjects from GC, CG and HC groups were 18. 6% (12. 1%-26. 7%), 7. 3% (4. 2%-11. 0%) and 4. 6% (3. 6%-6. 3%), respectively. The percentages of CD14+HLA-G+monocytes in the peripheral blood of patients with GC were significantly higher than those in patients with CG and healthy subjects (P<0. 001). The concentrations of sHLA-G in plasma samples collected from patients with GC [(100. 6±61. 3) U/ml) were significantly higher than those in pa-tients with CG [(59.5±19. 9) U/ml) and healthy subjects [(45. 8±23. 3) U/ml] (P<0. 001). ROC curve analysis showed that in terms of GC diagnosis, the area under ROC curve ( AUC) , cutoff value, sensi-tivity and specificity for CD14+HLA-G+monocytes and sHLA-G in plasma were 0. 893 and 0. 720, 12% and 85 U/mL, 75. 8% and 50. 5%, 86. 7% and 95. 9% (P<0. 001), respectively, which indicated that CD14+HLA-G+ monocytes and sHLA-G were better than AFP, CEA, CA19-9 and CA125 in differentiating GC from CG and HC. Moreover, the multivariate logistic regression analysis revealed that the CD14+HLA-G+ monocytes, sHLA-G in plasma as well as CA19-9 and CA125 in serum were positively correlated with the risk of GC after excluding the differences caused by age and gender factors. Conclusion The levels of CD14+HLA-G+ monocytes in peripheral blood and sHLA-G in plasma increased dramatically in patients with gastric cancer, which suggested that CD14+HLA-G+monocytes and sHLA-G might be risk factors for GC and could be used as potential biomarkers for the diagnosis of GC.

The effect of progesterone and 17-β estradiol on membrane-bound HLA-G in adipose derived stem cells

Membrane-bound HLA-G (mHLA-G) discovery on adipose derived stem cells (ADSCs) as a tolerogenic and immunosuppressive molecule was very important. Many documents have shown that HLA-G expression can be controlled via some hormones such as progesterone (P4) and estradiol (E2). Therefore, this study was designed to evaluate progesterone and estradiol effects on mHLA-G in ADSCs at restricted and combination concentrations. Three independent cell lines were cultured in complete free phenol red DMEM and subcultured to achieve suffi cient cells. These cells were treated with P4, E2 and P4 plus E2 at physiologic and pregnancy concentrations for 3 days in cell culture conditions. The HLA-G positive ADSCs was measured via monoclonal anti HLA-G-FITC/MEMG-09 by means of flow cytometry in nine groups. Data were analyzed by one way ANOVA and Tukey's post hoc tests. There were no signifi cant values of the mean percentage of HLA-G positive cells in E2-treated and the combination of P4 plus E2-treated ADSCs compared to control cells (p value>0.05) but P4 had a signifi cant increase on mHLA-G in ADSCs (p value<0.05). High P4 concentration increased mHLA-G but E2 and the combination of P4 plus E2 could not change mHLA-G on ADSCs.

Greater expression of the human leukocyte antigen-G (HLA-G) and interleukin-17 (IL-17) in cervical intraepithelial neoplasia: analytical cross-sectional study / Aumento da expressão do antígeno leucocitário humano-G (HLA-G) e interleucina-17 (IL-17) em neoplasia intraepitelial cervical: estudo transversal analítico

CONTEXT AND OBJECTIVE:Impaired local cell immunity seems to contribute towards the pathogenesis and progression of cervical intraepithelial neoplasia (CIN), but the underlying molecular mechanisms promoting its progression remain unclear. Identification of new molecular markers for prognosis and diagnosis of early-stage CIN may aid in decreasing the numbers of CIN cases. Several novel immunoregulatory molecules have been discovered over the past few years, including the human leukocyte antigen G (HLA-G), which through interaction with its receptors exerts important tolerogenic functions. Several lines of evidence suggest that T-helper interleukin-17 (IL-17)-producing cells (Th17 cells) may play a role in antitumor immunity. However, recent reports have implicated Th17 cells and their cytokines in both pro and anti-tumorigenic processes. The aim of the study was to evaluate the roles of HLA-G and Th17 in the immunopathogenesis of CIN I.DESIGN AND SETTING:Analytical cross-sectional study with a control group using 58 cervical specimens from the files of a public university hospital providing tertiary-level care.METHODS:We examined HLA-G and IL-17 expression in the cervical microenvironment by means of immunohistochemistry, and correlated these findings with clinical and pathological features.RESULTS:There was a greater tendency towards HLA-G and IL-17 expression in specimens that showed CIN I, thus suggesting that these molecules have a contribution towards cervical progression.CONCLUSION:These findings suggest that HLA-G and IL-17 expression may be an early marker for assessing the progression of cervical lesions.

CONTEXTO E OBJETIVO:A deficiência na imunidade celular localizada parece contribuir para a patogênese e progressão das neoplasias intraepiteliais cervicais (NIC), no entanto, ainda não está totalmente esclarecido o mecanismo molecular fundamental nesse processo de progressão. A identificação de novos marcadores moleculares de prognóstico e diagnóstico das NIC em estágios precoces pode ajudar a diminuir a quantidade de casos de NIC. Várias novas moléculas com função imunorregulatória foram descobertas nos últimos anos, inclusive o antígeno leucocitário humano G (HLA-G), que, através de interação com os receptores, tem importantes funções tolerogênicas. Diversas linhas de evidência sugerem que as células T-ajudantes produtoras de interleucina-17 (IL-17, células Th17), podem desempenhar um papel na imunidade antitumoral. Porém, recentes relatos implicaram as células Th17 e suas citocinas tanto em processos pro- quanto anti-tumorigênicos. O objetivo do estudo foi avaliar o papel do HLA-G e Th17 na imunopatogênese das NIC I.TIPO DE ESTUDO E LOCAL:Estudo transversal analítico com grupo controle em 58 espécimes cervicais dos arquivos de um hospital universitário público com assistência prestada no nível terciário.MÉTODOS:Avaliamos a expressão de HLA-G e IL-17 por imunoistoquímica no microambiente cervical, associando esses achados com as características clínico-patológicas.RESULTADOS:Houve tendência aumentada da expressão de HLA-G e IL-17 em espécimes que apresentaram NIC I, sugerindo que essas moléculas têm contribuição na progressão cervical.CONCLUSÃO:Estes resultados sugerem que a expressão do HLA-G e da IL-17 pode ser um marcador precoce para avaliar a progressão das lesões cervicais.

Expression of HLA-G in ovarian serous carcinoma and its clinical significance / 临床与实验病理学杂志

Purpose To investigate the expression and significance of HLA-G in ovarian serous carcinoma ( OSC) . Methods HLA-G antigen was immunohistochemically labeled on paraffin-embedded sections of 108 OSCs. The relationship between HLA-G expression and the clinicopathologic parameters was studied. Results The positive expression of HLA-G was observed in 58. 33% (63/108) of OSC tissues. Positive expression of HLA-G was significantly related with lymph node metastasis, recurrence, occurrence site, FIGO stage and MDACC grading system (P<0. 05). In survival analysis, the expression of HLA-G was significantly relevant to prognosis (P=0. 015). Multivariate Cox analysis showed the expression of HLA-G was an important prognosis factor of OSC (P=0. 01). Con-clusion The positive expression of HLA-G could predict high grade and advanced stage of OSC as well as poor prognosis. Also it could distinguish high-grade OSC from low-grade OSC.

Effect of HLA-G on proliferation in peripheral blood lymphocytes of childbearing age women and Treg cell subsets / 中国免疫学杂志

Objective:To study the effect of HLA-Gon proliferation in peripheral blood lymphocytes of childbearing age women and Treg cell subsets,and investigate the mechanisms of immune tolerance in pregnancy.Methods:The high expression of HLA-G cho-riocarcinoma cell lines JEG-3 cells with peripheral blood lymphocytes( PBLC) of healthy childbearing age women co-culture,using the HLA-G neutralizing antibodies(87G)and recombinant human tumor necrosis factor receptor typeⅡ-antibody fusion protein(rhTNFR:Fc)to intervene.Experiments were divided into seven groups:①JEG-3+PBLC culture group;②JEG-3+PBLC+87G culture group;③JEG-3+PBLC non-contact culture group;④JEG-3+PBLC+87G non-contact culture group;⑤The control group(PBLC group);⑥JEG-3+PBLC+rhTNFR:Fc culture group;⑦JEG-3+PBLC+87G+rhTNFR:Fc culture group.Detected the PBLC proliferation inhibition by CCK-8 method and the expression of TNF-αmRNA by RT-PCR in①-⑤groups.The proportion of Treg cells were detected by flow cytometry in①-⑦groups.Results:The assay of CCK-8 showed that the PBLC proliferation inhibition rate of JEG-3+PBLC culture group,JEG-3 +PBLC+87G culture group,JEG-3+PBLC non-contact culture group,and JEG-3+PBLC+87G non-contact culture group were(48.00±5.56)%,(14.67±4.04)%,(37.67±2.31)% and(8.33±3.21)%,there was a statistically significant difference on each group ( P0.05 ).Compared with the JEG-3+PBLC culture group,the proportion of Treg cells of JEG-3+PBLC+87G culture group was significantly decreased(P<0.05).Compared with JEG-3+PBLC culture group,the proportion of Treg cells of JEG-3+PBLC+rhTNFR:Fc culture group was significantly increased( P<0.05).Set JEG-3+PBLC+rhTNFR:Fc culture group as control,the proportion of Treg cells of JEG-3+PBLC+rh TNF:FC+87G culture was significantly decreased,but obviously higher than JEG-3+PBLC+87G culture group,there was a statistically significant difference on each group(P<0.05).Conclusion:HLA-G can inhibit peripheral blood lymphocyte proliferation of childbearing age women and inhibit the expression of TNF-α,and up-regulate the proportion of Treg cells.

Associations between HLA-G gene polymorphisms and the susceptibility to high risk HPV type 18 in-fection / 中华微生物学和免疫学杂志

Objective To investigate the associations between single nucleotide polymorphisms in the gene encoding human leukocyte antigen-G ( HLA-G) and the genetic susceptibility to high risk human papillomavirus ( HPV) type 18 infection in the subjects from Taizhou, Zhejiang province. Methods The genetic polymorphisms of HLA-G gene (14 bp In/Del and +3142C/G) in cervical samples collected from HPV 18-positive and healthy women were analyzed by PCR and gene sequencing technology. Statistical anal-ysis was performed by using SPSS version 16. 0. Chi-squared test was used to analyze the differences with HLA-G gene allele and genotype frequencies between healthy subjects and patients. Results Compared with healthy subjects, women with oncogenic HPV18 infection showed lower frequencies of -14 bp allele,-14 bp/-14 bp genotype and -14 bp/+3142C haplotype (P<0. 05). Moreover, lower frequencies of+3142C/C genotype were detected in HPV18-infected women with pathologically normal cervix (6. 3% vs 21. 1%, OR=0. 25, P<0. 05) and higher percentages of +3142G/G genotype were detected in women with CIN2/3 stage HPV18 infection as compared with those of the control group (68. 8% vs 35. 1%, OR=4. 06, P<0. 05). Conclusion The HLA-G gene 3′ UTR polymorphisms were closely associated with HPV18 in-fection and cervical intraepithelial neoplasia.