Embryo morphokinetic score is associated with biomarkers of developmental competence and implantation.

PURPOSE: To study embryo morphokinetics in relation to release in spent media of molecules with possible roles in development and implantation (miR-20a, miR-30c, and sHLA-G). METHODS: Data were obtained from embryos generated in standard IVF and ICSI cycles. The Eeva system was used for embryo assessment, based on early morphokinetic parameters and producing a score (1-5, best-worst) corresponding to higher/medium/lower chances of development to blastocyst. miRNAs - mm miR-20a-5p and miR-30c-5p - and sHLA-G were quantified in 25 µl of spent blastocyst media (SBM) collected before vitrification or transfer. Statistical analyses were performed applying Kolmogorov-Smirnov, Shapiro-Wilk, and Spearman's correlation coefficient tests, where appropriate. RESULTS: SBM were collected from a total of 172 viable blastocysts. Their analysis showed that concentration of miR-20a was progressively lower as Eeva score increased and probability of development to blastocyst decreased (P = 0.016). The opposite trend was observed in the case of miR-30c, i.e., concentration was higher as score increased and chances of development to blastocyst decreased (P = 0.004). Analysis of sHLA-G revealed a negative correlation with Eeva score, i.e., levels were progressively lower as Eeva score increased and probability of development to blastocyst decreased (R = - 0.388, N = 141, P = 0.001). CONCLUSION: Our data suggest that morphokinetic algorithms that predict development to blastocyst stage, in fact, also identify embryos with molecular and cellular profiles more consistent with developmental functions.

Expression of classical human leukocyte antigen class I antigens, HLA-E and HLA-G, is adversely prognostic in pancreatic cancer patients.

The expression of classical human leukocyte antigen class I antigens (HLA-I) on the surfaces of cancer cells allows cytotoxic T cells to recognize and eliminate these cells. Reduction or loss of HLA-I is a mechanism of escape from antitumor immunity. The present study aimed to investigate the clinicopathological impacts of HLA-I and non-classical HLA-I antigens expressed on pancreatic ductal adenocarcinoma (PDAC) cells. We performed immunohistochemistry to detect expression of HLA-I antigens in PDAC using 243 PDAC cases and examined their clinicopathological influences. We also investigated the expression of immune-related genes to characterize PDAC tumor microenvironments. Lower expression of HLA-I, found in 33% of PDAC cases, was significantly associated with longer overall survival. Higher expression of both HLA-E and HLA-G was significantly associated with shorter survival. Multivariate analyses revealed that higher expression of these three HLA-I antigens was significantly correlated with shorter survival. Higher HLA-I expression on PDAC cells was significantly correlated with higher expression of IFNG, which also correlated with PD1, PD-L1 and PD-L2 expression. In vitro assay revealed that interferon gamma (IFNγ) stimulation increased surface expression of HLA-I in three PDAC cell lines. It also upregulated surface expression of HLA-E, HLA-G and immune checkpoint molecules, including PD-L1 and PD-L2. These results suggest that the higher expression of HLA-I, HLA-E and HLA-G on PDAC cells is an unfavorable prognosticator. It is possible that IFNγ promotes a tolerant microenvironment by inducing immune checkpoint molecules in PDAC tissues with higher HLA-I expression on PDAC cells.

Embryo sHLA-G secretion is related to pregnancy rate.

SummaryHLA-G expression has been detected in early preimplantation embryos and it has been postulated that a relationship between embryonic expression of this factor and successful pregnancy may exist. Forty-six patients were prospectively selected from our centre 'Unidad de Reproducción Humana, Hospital Universitario de Canarias' for conducting this study. In all cases, metaphase II (MII) oocytes were fertilized using intracytoplasmic sperm injection 2-4 h after retrieval. Embryos were cultured individually in 20 µl droplets of G-1 medium (VitroLife) under oil at 37°C and a 6% CO2 environment. Fertilization was assessed at 18 h postinsemination and all oocytes fertilized were passed into a new culture plaque individually in 300 µl culture medium until day 3 of culture. The culture medium was examined for the expression and secretion of sHLA-G with a sandwich enzyme-linked immunosorbent assay kit (BioVendor, Heidelberg, Germany) according to the manufacturer's instructions. We found statistical significance between higher levels of sHLA-G secretion and pregnancy rate. When both groups were compared there was no difference in embryo quality of transferred embryos, but a significant difference in the number of oocytes and the embryo quality of the cohort existed that was greater in the pregnant group. A standardized sHLA-G assay with a specifically defined range and standard units provides a non-invasive method to identify the most competent embryos for transfer.

Evaluation of ascitic soluble human leukocyte antigen-G for distinguishing malignant ascites from benign ascites.

The overexpression of soluble human leukocyte antigen-G is associated with malignant tumours. The purpose of our study was to detect soluble human leukocyte antigen-G concentrations in ascites and to evaluate the value of ascitic soluble human leukocyte antigen-G for the diagnosis of malignant ascites. Enzyme-linked immunosorbent assay was used to detect soluble human leukocyte antigen-G levels in 64 patients with malignant ascites and 30 patients with benign ascites. Receiver operating characteristic curves were used to evaluate the diagnostic efficacy of ascitic soluble human leukocyte antigen-G for the detection of malignant ascites. Ascitic soluble human leukocyte antigen-G levels were significantly higher in the malignant ascites group than in the benign ascites group (20.718 ± 3.215 versus 12.467 ± 3.678 µg/L, t = 7.425, p < 0.001). The area under the receiver operating characteristic curve for ascitic soluble human leukocyte antigen-G was 0.957 (95% confidence interval, 0.872-0.992). At a cut-off value of 19.60 µg/L, the sensitivity and specificity of ascitic soluble human leukocyte antigen-G were 87.5% (95% confidence interval, 71.0%-96.5%) and 100% (95% confidence interval, 88.4%-100%), respectively. With respect to area under the receiver operating characteristic curve, sensitivity and specificity, ascitic carcinoembryonic antigen (0.810, 68.75% and 83.33%, respectively) and carbohydrate antigen 19-9 (0.710, 65.63% and 70%, respectively) significantly differed (all p < 0.05). In malignant ascites that were cytology-negative and biopsy-positive, the rate of positivity for ascitic soluble human leukocyte antigen-G was 75%, which was higher than the corresponding rates for ascitic carcinoembryonic antigen (31.25%) and carbohydrate antigen 19-9 (6.25%; both p < 0.05). In conclusion, The detection of ascitic soluble human leukocyte antigen-G exhibited good performance for diagnosing malignant ascites, and particularly those that were cytology-negative and biopsy-positive.

Follicular fluid concentration of soluble Human-G Leukocytic Antigen (sHLA-G) in in vitro fertilization cycles of women with and without peritoneal endometriosis.

OBJECTIVE: The objective of this research is to investigate the association between the concentrations of soluble human leukocyte G antigen (sHLA-G) in the follicular fluid (FF) in infertile patients with peritoneal endometriosis submitted to in vitro fertilization. METHODS: We performed a cross-sectional study, including ninety-six women undergoing in vitro fertilization (IVF) ageing ≤ 40 years. Infertile patients were classified into two groups: with endometriosis diagnosed by laparoscopy and without endometriosis due to tubal factor. ELISA measured soluble HLA-G in the FF of a pool of punctured (more than 17mm) follicles from women with endometriosis and without endometriosis who were subjected to ovulation induction for IVF. Embryos obtained after fertilization were classified according to the graduated embryo score (GES). RESULTS: Groups were comparables in terms of age, the number of follicles, AMH, FSH and all included reproductive outcomes. There was no association between sHLA-G concentrations and the average score of the generated embryos (p>0.05). Measurement of sHLA-G in the follicle fluid in women with endometriosis and without endometriosis (tubal factor) showed no significant difference (p>0.05). We also compared sHLA-G per follicle and per embryo, which were not different between both groups (p>0.05). CONCLUSIONS: Patients with peritoneal endometriosis submitted to IVF did not demonstrate an altered sHLA-G in the follicular fluid compared to the follicular fluid sHLA-G concentration in tubal factor patients. Also, this molecule was not linked to any other reproductive outcome.

Tumor immune subtypes distinguish tumor subclasses with clinical implications in breast cancer patients.

There is strong evidence that the host's cellular immune response is linked to tumor progression, however its impact on patient outcome in breast cancer is poorly understood. The purpose of this study is to define tumor immune subtypes, focusing on cellular immune responses and investigate their prognostic effect in breast cancer patients. Our training (n = 440) and validation cohort (n = 382) consisted of all early breast cancer patients primarily treated with surgery in our center between 1985 and 1996. Tumor tissue sections were immunohistochemically stained for CD8 (CTL) and PEN5 (NK cells). Tumor expression of classical and non-classical human leukocyte antigen class I, and tumor-infiltrating Tregs were previously determined. Tumor immune subtypes were constructed based on quantification of these markers and biological rationale. High, intermediate, and low immune susceptible tumor immune subtypes were found, respectively, in 16, 63, and 20 % of patients in the training cohort and 16, 71, and 13 % in the validation cohort. The subtypes showed to be statistically significant prognostic in multivariate analyses for relapse free period (RFP) [p < 0.0001, intermediate versus high: hazard ratio (HR) 1.95; low versus high HR 2.98] and relative survival (RS) (p = 0.006, intermediate versus high HR 3.84; low versus high: HR 4.26). Validation of these outcome analyses confirmed the independent prognostic associations: RFP (p = 0.025) and RS (p = 0.040). The tumor immune subtypes that we present represent a prognostic profile with solid underlying biological rationale and with high discriminative power confirmed in an independent validation cohort. Our results emphasize the importance of tumor immune surveillance in the control of tumor development and, therefore, in determining patient prognosis. Tumor immune subtype profiling is promising for prognosis prediction and the achievement of tailored treatment for breast cancer patients.

Embryonic soluble human leukocyte antigen-G as a marker of embryo competency in assisted reproductive technology for Chinese women.

OBJECTIVE: To investigate whether embryonic soluble human leukocyte antigen-G (sHLA-G) could be a noninvasive marker for embryo competency in assisted reproductive technology (ART), which is still controversial due to the different detection assays used and the different culture conditions in laboratories. STUDY DESIGN: Based on the standardization of IVF procedures and the embryo culture condition, a total of 205 embryo culture supernatants (ESs) from 92 ART cycles were evaluated for sHLA-G contents by chemiluminescent ELISA assay. RESULTS: sHLA-G presence could be detected in 30.7% of the ESs tested. In the cycles where at least one of the embryos transferred was positive for sHLA-G, 51.9% of patients (27/52) achieved a clinical pregnancy. In cycles where none of the embryos transferred secreted detectable amounts of sHLA-G, the pregnancy rate was only 30.0% (12/40, p < 0.05). The implantation rate in sHLA-G-positive cycles was also significantly higher (31.5%) than that in sHLA-G-negative cycles (14.9%, p < 0.05). CONCLUSION: The results suggested sHLA-G in ESs as a potential marker of embryo competency in ART programs for the Chinese population.

Extracellular vesicles and their miRNA contents counterbalance the pro-inflammatory effect of air pollution during physiological pregnancy: A focus on Syncytin-1 positive vesicles.

The impact of exposure to respirable particulate matter (PM) during pregnancy is a growing concern, as several studies have associated increased risks of adverse pregnancy and birth outcomes, and impaired intrauterine growth with air pollution. The molecular mechanisms responsible for such effects are still under debate. Extracellular vesicles (EVs), which travel in body fluids and transfer microRNAs (miRNAs) between tissues (e.g., pulmonary environment and placenta), might play an important role in PM-induced risk. We sought to determine whether the levels of PM with aerodynamic diameters of ≤10 µm (PM10) and ≤2.5 µm (PM2.5) are associated with changes in plasmatic EV release and EV-miRNA content by investigating 518 women enrolled in the INSIDE study during the first trimester of pregnancy. In all models, we included both the 90-day averages of PM (long-term effects) and the differences between the daily estimate of PM and the 90-day average (short-term effects). Short-term PM10 and PM2.5 were associated with increased concentrations of all seven EV types that we assayed (positive for human antigen leukocyte G (HLA-G), Syncytin-1 (Sync-1), CD14, CD105, CD62e, CD61, or CD25 determinants), while long-term PM10 showed a trend towards decreased EV concentrations. Increased Sync-1 + EV levels were associated with the plasmatic decrease of sVCAM-1, but not of sICAM-1, which are circulating biomarkers of endothelial dysfunction. Thirteen EV-miRNAs were downregulated in response to long-term PM10 and PM2.5 variations, while seven were upregulated (p-value < 0.05, false discovery rate p-value (qFDR) < 0.1). Only one EV-miRNA (hsa-miR-221-3p) was downregulated after short-term variations. The identified PM-modulated EV-miRNAs exhibited putative roles in inflammation, gestational hypertension, and pre-eclampsia, as highlighted by miRNA target analysis. Our findings strongly support the hypothesis that EVs have an important role in modulating PM exposure effects during pregnancy, possibly through their miRNA cargo.

Human leukocyte antigen-G in the male reproductive system and in seminal plasma.

One of the non-classical human leukocyte antigen (HLA) class Ib proteins, HLA-G, is believed to exert important immunoregulatory functions, especially during pregnancy. The presence of HLA protein in paternal seminal fluid has been suggested to have an influence on the risk of developing pre-eclampsia. We have investigated whether HLA-G protein is present in human seminal plasma and in different tissue samples of the male reproductive system. Western blot technique and a soluble HLA-G (sHLA-G) assay were used to detect sHLA-G in human seminal plasma samples. Immunohistochemical staining was performed on paraffin-embedded tissue samples. We detected sHLA-G protein in seminal plasma, and HLA-G expression in normal testis and in epididymal tissue of the male reproductive system but not in the seminal vesicle. Furthermore, the results indicated a weak expression of HLA-G in hyperplastic prostatic tissue. In summary, several of the findings reported in this study suggest an immunoregulatory role of HLA-G in the male reproductive system and in seminal plasma.

Cytokines and Soluble HLA-G Levels in the Acute and Recovery Phases of Arbovirus-Infected Brazilian Patients Exhibiting Neurological Complications.

Severe neurological complications following arbovirus infections have been a major concern in seasonal outbreaks, as reported in the Northeast region of Brazil, where the same mosquito transmitted Zika (ZIKV), Dengue (DENV), and Chikungunya (CHIKV) viruses. In this study, we evaluated the levels of 36 soluble markers, including cytokines, chemokines, growth factors, and soluble HLA-G (Luminex and ELISA) in: i) serum and cerebrospinal fluid (CSF), during the acute phase and two years after the infection (recovery phase, only serum), ii) the relationship among all soluble molecules in serum and CSF, and iii) serum of infected patients without neurological complications, during the acute infection. Ten markers (sHLA-G, IL-10, IL-22, IL-8, MIP-1α, MIP-1ß, MCP-1, HGF, VEGF, and IL-1RA) exhibited differential levels between the acute and recovery phases, with pronounced increases in MIP-1α (P<0.0001), MCP-1 (P<0.0001), HGF (P= 0.0001), and VEGF (P<0.0001) in the acute phase. Fourteen molecules (IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-13, IL-15, IL-17A, IFN-α, TNF, and G-CSF) exhibited distinct levels between arbovirus patients presenting or not neurological complications. IL-8, EGF, IL-6, and MCP-1 levels were increased in CSF, while RANTES and Eotaxin levels were higher in serum. Soluble serum (IL-22, RANTES, Eotaxin) and CSF (IL-8, EGF, IL-3) mediators may discriminate putative risks for neurological complications following arbovirus infections. Neurological complications were associated with the presence of a predominant inflammatory profile, whereas in non-complicated patients an anti-inflammatory profile may predominate. Mediators associated with neuroregeneration (EGF and IL-3) may be induced in response to neurological damage. Broad spectrum immune checkpoint molecules (sHLA-G) interact with cytokines, chemokines, and growth factors. The identification of soluble markers may be useful to monitor neurological complications and may aid in the development of novel therapies against neuroinflammation.

Soluble HLA-G and TGF-ß in couples attending assisted reproduction - A possible role of TGF-ß isoforms in semen?

Soluble isoforms of the non-classical Human Leukocyte Antigen (HLA)-G as well as Transforming Growth Factor (TGF)-ß is expressed in seminal plasma possibly influencing the pregnancy potential. We wanted to examine the association of seminal plasma sHLA-G, TGF-ß1, TGF-ß2 and TGFß3 with pregnancy success in a cohort of 127 couples and 4 single women attending fertility treatment with the use of assisted reproduction technologies (ART). Soluble HLA-G, TGF-ß1, TGF-ß2 and TGF-ß3 in seminal plasma did not fluctuate significantly over time. We did not find any impact of seminal plasma sHLA-G, TGF-ß1, TGF-ß2 and TGF-ß3 on time-to-pregnancy measured as number of treatment cycles. There was a significant association between concentrations of seminal plasma sHLA-G and HLA-G variations in the 3'untranslated region (3'UTR) of the HLA-G gene, supporting and extending previous findings. Furthermore, by comparing seminal plasma concentrations of sHLA-G, TGF-ß1, TGF-ß2 and TGF-ß3 in male subjects with reduced semen quality, male subjects with normal semen quality, and sperm donors, we found that TGF-ß2 was significantly lower, and TGF-ß3 was significantly higher, in seminal plasma from sperm donors. These findings suggest that TGF-ß isoforms may influence semen quality and fertility.

Soluble HLA-G levels in seminal plasma are associated with HLA-G 3'UTR genotypes and haplotypes.

Soluble HLA-G (sHLA-G) levels in human seminal plasma (SP) can be diverse and may affect the establishment of maternal-fetal tolerance and thereby the outcome of pregnancy. We investigated whether sHLA-G levels in SP are associated with polymorphisms in the 3'-untranslated region (UTR) and UTR haplotypes of the HLA-G gene. Furthermore, we compared the HLA-G genotype distribution and sHLA-G levels between men, whose partner experienced unexplained recurrent miscarriage (RM), and controls. Soluble HLA-G levels (n = 156) and HLA-G genotyping (n = 176) were determined in SP samples. The concentration of sHLA-G was significantly associated with several single-nucleotide polymorphisms (SNPs): the 14 base pair (bp) insertion/deletion (indel), +3010, +3142, +3187, +3196, and + 3509. High levels of sHLA-G were associated with UTR-1 and low levels with UTR-2, UTR-4, and UTR-7 (P < .0001). HLA-G genotype distribution and sHLA-G levels in SP were not significantly different between the RM group (n = 44) and controls (n = 31). In conclusion, seminal sHLA-G levels are associated with both singular SNPs and 3UTR haplotypes. HLA-G genotype and sHLA-G levels in SP are not different between men whose partner experienced RM and controls, indicating that miscarriages are not solely the result of low sHLA-G levels in SP. Instead, it is more likely that these miscarriages are the result of a multifactorial immunologic mechanism, whereby the HLA-G 3'UTR 14 bp ins/ins genotype plays a role in a proportion of the cases. Future studies should look into the functions of sHLA-G in SP and the consequences of low or high levels on the chance to conceive.

Immunohistochemical evaluation of HLA-G and FoxP3+ T regulatory cells in oral cavity and lower lip squamous cell carcinomas.

Human Leukocyte Antigen G (HLA-G) is a molecule involved in the tumor immunosuppression and also in the generation of regulatory T (Treg) cells, thus leading to evasion to the immune system host, and consequently, contributing to tumor progression in several cancers. The aim of this study was to evaluate the immunoexpression of HLA-G by tumor cells and FoxP3+ Treg cells in 25 oral tongue squamous cell carcinomas (SCCs) and 25 lower lip SCCs and analyze their relationship with clinical parameters. HLA-G expression was higher in oral tongue SCCs than in lower lip SCCs. In oral tongue SCCs and lower lip SCCs, no association between HLA-G expression and clinical parameters (tumor size, lymph node status, distant metastasis, and clinical stage) was verified (P>0.05). FoxP3+ Treg cells were detected along the tumor invasive front in all cases of oral tongue and lower lip SCCs. In oral tongue SCC cases, the number of Treg cells tended to be higher in smaller tumors, tumors without regional lymph node metastasis, and tumors in early clinical stages, but the difference was not statistically significant (P>0.05). A significant positive correlation was found between the expression of HLA-G by neoplastic cells and Treg cells in lower lip SCCs (p = 0.008). Our findings suggest the involvement of HLA-G and Treg cells in the modulation of immune responses in oral tongue and lower lip SCCs. This interaction between HLA-G and Treg cells may represent an evasion mechanism in these malignancies.

Elevated soluble human leukocyte antigen G levels in patients after allogeneic stem cell transplantation are associated with less severe acute and chronic graft-versus-host disease.

HLA-G is a non-classical class I molecule which induces tolerance in allogeneic situations by inhibition of cytotoxic NK and CD8 + T cells and by induction of regulatory T cells. Concordantly, in solid organ transplantation HLA-G is associated with a lower risk for acute and chronic rejection, whereas its role in allogeneic stem cell transplantation (allo-SCT) is less established. We here present detailed analyses of HLA-G-levels in patients after allo-SCT showing a correlation of elevated soluble HLA-G (sHLA-G) levels with less severe acute (p = 0.06) and chronic GvHD (p = 0.0025) and with a superior overall survival (p = 0.03). Soluble HLA-G levels are also positively correlated with the frequency of regulatory T cells in vivo. These clinical data are corroborated by in vitro analyses showing that patients-derived sHLA-G inhibit allogeneic immune responses. ATG-treatment of patients dominantly affects the sHLA-G levels post allo-SCT. Thus, this study highlights the association of elevated sHLA-G levels with less severe acute and chronic GvHD and provides additional functional analyses elucidating possible tolerance-inducing mechanisms of sHLA-G in the context of allo-SCT.

HLA-G expression in gastric carcinoma: clinicopathological correlations and prognostic impact.

To analyze expression of human leukocyte antigen-G (HLA-G) in gastric adenocarcinoma and correlate its expression with histological and clinical variables. A continuous series of 94 unselected patients with gastric adenocarcinoma (stage I to III) were selected. All histological and clinical variables were collected including the intensity of intra- and peri-tumor lymphocytic infiltration. HLA-G expression was investigated using immunohistochemistry. All histological samples analyzed for HLA-G expression were taken from the primary gastric lesion and included non-neoplastic mucosa. Evaluation of HLA-G expression was performed on the transition zone between tumor and non-neoplastic mucosa, and the invasive front of the tumor and assessment was performed as follows: percentage of positive (strong expression vs weak) cells. A variable amount of HLA-G-positive tumor cells was found in 24 out of 94 cases (25.5%). No significant correlation was found between HLA-G expression and other clinicopathological variables (sex, age, stage, grade, histotype). The overall median survival was worse in patients with HLA-G-positive adenocarcinoma (24.3 months, CI95% 7.7-41.0) compared to those with HLA-G-negative tumors (66.3 months, CI95% 53.0-79.7; p < 0.0001). Two- and 5-year survival rates of HLA-G-negative patients were 88 and 44%, respectively, while were 42 and 11% in those HLA-G-positive. This trend was observed in all stages but was more marked in stage III. HLA-G expression is associated with poor survival in stage III gastric cancer patients and represents a possible immunoescape mechanism of cancer cells.

HLA-G+ HIV-1-specific CD8+ T cells are associated with HIV-1 immune control.

OBJECTIVE(S): To assess the frequency and function of HIV-1-specific HLA-G (histocompatibility antigen class I, G) CD8 T cells in HIV-1 controllers and progressors. DESIGN: We performed an observational cross-sectional cohort analysis in untreated (n = 47) and treated (n = 17) HIV-1 patients with different rates of disease progression and n = 14 healthy individuals. METHODS: We evaluated the frequency, the proportion and the function of total and virus-specific HLA-G CD8 T cells by tetramer or intracellular cytokine staining, followed by flow cytometric analysis. Cytokine secretion of sorted CD8 T-cell subsets was evaluated by Luminex assays. RESULTS: The proportion and the absolute frequency of HLA-G HIV-1-specific CD8 T cells were directly associated with CD4 T-cell counts and inversely correlated with viral loads, whereas total or HLA-G-negative HIV-1-specific CD8 T cells were not. In functional assays, HLA-G CD8 T cells from HIV-1-negative individuals had higher abilities to produce the antiviral (C-C chemokine receptor type 5) ligands MIP-1ß (macrophage inflammatory protein-1ß), MIP-1α and Rantes. CONCLUSION: HLA-G HIV-1-specific CD8 T cells may represent a previously unrecognized correlate of HIV-1 immune control.

Novel landscape of HLA-G isoforms expressed in clear cell renal cell carcinoma patients.

Immune checkpoints are powerful inhibitory molecules that promote tumor survival. Their blockade is now recognized as providing effective therapeutic benefit against cancer. Human leukocyte antigen G (HLA-G), a recently identified immune checkpoint, has been detected in many types of primary tumors and metastases, in malignant effusions as well as on tumor-infiltrating cells, particularly in patients with clear cell renal cell carcinoma (ccRCC). Here, in order to define a possible anticancer therapy, we used a molecular approach based on an unbiased strategy that combines transcriptome determination and immunohistochemical labeling, to analyze in-depth the HLA-G isoforms expressed in these tumors. We found that the expression of HLA-G is highly variable among tumors and distinct areas of the same tumor, testifying a marked inter- and intratumor heterogeneity. Moreover, our results generate an inventory of novel HLA-G isoforms which includes spliced forms that have an extended 5'-region and lack the transmembrane and alpha-1 domains. So far, these isoforms could not be detected by any method available and their assessment may improve the procedure by which tumors are analyzed. Collectively, our approach provides the first extensive portrait of HLA-G in ccRCC and reveals data that should prove suitable for the tailoring of future clinical applications.

The association of HLA-G and immune markers in recurrent miscarriages.

OBJECTIVE: To determine role of human leukocyte antigen (HLA)-G, CD8, CD16, CD56, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α for recurrent miscarriages in feto-maternal interface. METHOD: Chorion and decidua samples were obtained from 11 women with unwanted pregnancies (healthy pregnancy, HP) and 10 women with missed abortion diagnosis after at least two pregnancy losses (recurrent miscarriage, RM). In addition, endometrial tissues were obtained from 10 non-pregnant women (NonP). The expressions of markers were evaluated using the Western blot analysis. The values obtained between different groups were compared. RESULTS: The highest protein expression of CD56 was found in the HP compared to NonP and RM. Meanwhile, the lowest protein expression of CD16 was observed in the NonP compared to HP and RM. The HLA-G expression exhibited the highest level in HP; however, there was no statistically significant difference between groups. CD8 and IFNγ expressions were lowest in the NonP group; however, TNF-α was highest in the RM group. CONCLUSIONS: The CD56 expression of uterine NK cells may be an indicator of a HP. However, not statistically significant, the increased expression of CD16, CD8, and also significantly increased expression of TNF may be associated with the predominant cytotoxic activity in the maternal immune system in patients with RM. Although there was no change in the expression of HLA-G, this finding may mean that the maternal immune system is unresponsive to HLA-G-mediated immunosuppressive signals originating from the fetus in these cases.

Soluble Human Leukocyte Antigen-G in the Bronchoalveolar Lavage of Lung Cancer Patients. / Antígeno leucocitario humano-G soluble en el lavado broncoalveolar de pacientes con cáncer pulmonar.

BACKGROUND: The main function of the HLA-G molecule in its membrane-bound and soluble forms is to inhibit the immune response by acting on CD4+ T cells, cytotoxic T cells, NK cells and dendritic cells. Lung cancer is a leading cause of death worldwide, and annual incidence is high in both women and men. Some studies have reported an increase of HLA-G serum levels in lung cancer, probably generated by tumor cells escaping the antitumor immune response. In this study the concentration of soluble HLA-G in bronchoalveolar lavage (BAL) in patients with primary and metastatic lung cancer was measured to determine its relation with tumor histological type and overall patient status according to the Karnofsky scale. METHODS: Thirty-one lung cancer patients were included. A tumor biopsy was obtained by bronchoscopy and the tumor type was determined by hematoxylin and eosin staining. BAL samples were obtained to measure soluble HLA-G concentrations in an ELISA sandwich assay. RESULTS: The average value of soluble HLA-G was 49.04ng/mL. No correlation between soluble HLA-G levels and age, gender or smoking was observed. A highly significant difference was observed in the levels of soluble HLA-G in BAL from patients with different histological types of lung cancer, especially in metastatic tumors. The Karnofsky index showed a significant and inverse correlation with soluble HLA-G levels in BAL. CONCLUSIONS: Soluble HLA-G protein is significantly associated with metastatic tumors and patients with lower Karnofsky index and may be useful as a prognostic marker in lung cancer.