An HLA-G/SPAG9/STAT3 axis promotes brain metastases.

Brain metastases (BM) are the most common brain neoplasm in adults. Current BM therapies still offer limited efficacy and reduced survival outcomes, emphasizing the need for a better understanding of the disease. Herein, we analyzed the transcriptional profile of brain metastasis initiating cells (BMICs) at two distinct stages of the brain metastatic cascade-the "premetastatic" or early stage when they first colonize the brain and the established macrometastatic stage. RNA sequencing was used to obtain the transcriptional profiles of premetastatic and macrometastatic (non-premetastatic) lung, breast, and melanoma BMICs. We identified that lung, breast, and melanoma premetastatic BMICs share a common transcriptomic signature that is distinct from their non-premetastatic counterparts. Importantly, we show that premetastatic BMICs exhibit increased expression of HLA-G, which we further demonstrate functions in an HLA-G/SPAG9/STAT3 axis to promote the establishment of brain metastatic lesions. Our findings suggest that unraveling the molecular landscape of premetastatic BMICs allows for the identification of clinically relevant targets that can possibly inform the development of preventive and/or more efficacious BM therapies.

A chimeric anti-vascularization immunomodulator prevents high-risk corneal transplantation rejection via ex vivo gene therapy.

Corneal blindness affects more than 5 million individuals, with over 180,000 corneal transplantations (CTs) performed annually. In high-risk CTs, almost all grafts are rejected within 10 years. Here, we investigated adeno-associated virus (AAV) ex vivo gene therapy to establish immune tolerance in the corneal allograft to prevent high-risk CT rejection. Our previous work has demonstrated that HLA-G contributes to ocular immune privilege by inhibiting both immune cells and neovascularization; however, homodimerization is a rate-limiting step for optimal HLA-G function. Therefore, a chimeric protein called single-chain immunomodulator (scIM), was engineered to mimic the native activity of the secreted HLA-G dimer complex and eliminate the need for homodimerization. In a murine corneal burn model, AAV8-scIM significantly reduced corneal vascularization and fibrosis. Next, ex vivo AAV8-scIM gene delivery to corneal allografts was evaluated in a high-risk CT rejection rabbit model. All scIM-treated corneas were well tolerated and transparent after 42 days, while 83% of vehicle-treated corneas were rejected. Histologically, AAV-scIM-treated corneas were devoid of immune cell infiltration and vascularization, with minimal fibrosis at the host-graft interface. The data collectively demonstrate that scIM gene therapy prevents corneal neovascularization, reduces trauma-induced corneal fibrosis, and prevents allogeneic CT rejection in a high-risk large animal model.

HLA-G neo-expression modifies genetic programs governing tumor cell lines.

The development of immunotherapies has proved to be clinically encouraging to re-establish the immune function modified by the expression of immune inhibitory molecules in tumors. However, there are still patients with poor survival rates following treatment. The elucidation of molecular mechanisms triggered by the neo-expression of particular IC in tumors would constitute a major step toward better understanding tumor evolution and would help to design future clinical protocols. To this end, we investigate the modifications triggered by the neo-expression of the immune checkpoints HLA-G in ccRCC tumor cells. We demonstrate, for the first time, that HLA-G modifies key genes implicated mainly in tumor development, angiogenesis, calcium flow and mitochondria dynamics. The involvement of HLA-G on the expression of genes belonging to these pathways such as ADAM-12, NCAM1 and NRP1 was confirmed by the CRISPR/Cas9-mediated edition of HLA-G. The data reveal multifaceted roles of HLA-G in tumor cells which are far beyond the well-known function of HLA-G in the immune anti-tumor response. This warrants further investigation of HLA-G and these new partners in tumors of different origin so as to propose future new treatments to improve health patient's outcome.

Harnessing the potential of HLA-G in cancer therapy: advances, challenges, and prospects.

Immune checkpoint blockades have been prized in circumventing and ablating the impediments posed by immunosuppressive receptors, reaching an exciting juncture to be an innovator in anticancer therapy beyond traditional therapeutics. Thus far, approved immune checkpoint blockades have principally targeted PD-1/PD-L1 and CTLA-4 with exciting success in a plethora of tumors and yet are still trapped in dilemmas of limited response rates and adverse effects. Hence, unveiling new immunotherapeutic targets has aroused immense scientific interest in the hope of expanding the clinical application of immune checkpoint blockades to scale new heights. Human leukocyte antigen-G (HLA-G), a non-classical major histocompatibility complex (MHC) class I molecule, is enriched on various malignant cells and is involved in the hindrance of immune effector cells and the facilitation of immunosuppressive cells. HLA-G stands out as a crucial next-generation immune checkpoint showing great promise for the benefit of cancer patients. Here, we provide an overview of the current understanding of the expression pattern and immunological functions of HLA-G, as well as its interaction with well-characterized immune checkpoints. Since HLA-G can be shed from the cell surface or released by various cells as free soluble HLA-G (sHLA-G) or as part of extracellular vesicles (EVs), namely HLA-G-bearing EVs (HLA-GEV), we discuss the potential of sHLA-G and HLA-GEV as predictive biomarkers. This review also addresses the advancement of HLA-G-based therapies in preclinical and clinical settings, with a focus on their clinical application in cancer.

The correlation between soluble human leukocyte antigen (sHLA-G) levels and +3010 polymorphism.

Human leukocyte antigen-G (HLA-G) is classified as non-classical HLA, located in the short arm of chromosome 6 and composed of seven introns and eight exons. The HLA-G gene has a lower frequency polymorphism in the coding area and higher variability at the regulatory 5'- and 3'-untranslated regions linked to HLA-G microRNA regulation. HLA-G molecule is known to have an immunomodulatory and tolerogenic features role. In 199 Saudi individuals, we examined the association between plasma soluble HLA-G (sHLA-G) levels and eight polymorphic different sites, including 14 bp ins/del/+3003T-C/+3010C-G/+3027C-A/+3035C-T/+3142C-G/+3187A-G/+3196C-G single nucleotide polymorphisms (SNPs) in exon 8 in the HLA-G gene. Our results revealed higher frequency for rs17179101C (97%), rs1707T (92%) and rs9380142A (73%) alleles. Greater frequencies for the tested genotypes were observed in 3027C/C (rs17179101) (93%), 14 bp (rs1704) ins/del (92%), +3003T/T (rs1707) (85%) and +3035C/T (rs17179108) (79%) SNP genotypes. Moreover, we observed a significant association of sHLA-G with +3010G/C (rs1710) SNP. In conclusion, we showed a significant association between 3010G/C (rs1710) SNP and the sHLA-G level among our sample for Saudi populations. Our findings demonstrated that specific SNP within the HLA-G gene is linked to sHLA-G molecule secretion, suggesting sHLA-G levels may be regulated genetically.

HLA-G Expression/Secretion and T-Cell Cytotoxicity in Missed Abortion in Comparison to Normal Pregnancy.

The main role of HLA-G is to protect the semi-allogeneic embryo from immune rejection by proper interaction with its cognate receptors on the maternal immune cells. Spontaneous abortion is the most common adverse pregnancy outcome, with an incidence rate between 10% and 15%, with immunologic dysregulation being thought to play a role in some of the cases. In this study, we aimed to detect the membrane and soluble HLA-G molecule at the maternal-fetal interface (MFI) and in the serum of women experiencing missed abortion (asymptomatic early pregnancy loss) in comparison to the women experiencing normal early pregnancy. In addition, the proportion of T cells and their cytotoxic profile was evaluated. We observed no difference in the spatial expression of HLA-G at the MFI and in its serum levels between the women with missed abortions and those with normal early pregnancy. In addition, comparable numbers of peripheral blood and decidual total T and γδT cells were found. In addition, as novel data we showed that missed abortion is not associated with altered extravilous invasion into uterine blood vessels and increased cytotoxicity of γδT cells. A strong signal for HLA-G on non-migrating extravilous trophoblast in the full-term normal placental bed was detected. In conclusion, HLA-G production at the MFI or in the blood of the women could not be used as a marker for normal pregnancy or missed abortions.

A noninvasive method for the detection of foetal DNA in early pregnancy based on differential methylation pattern of Ras association domain family member 1A.

BACKGROUND: The detection of foetal DNA and extravillus trophoblasts (EVTs) in early pregnancy in cervical and uterine samples offers a potential pathway for non-invasive prenatal diagnostics. However, the challenge lies in effectively quantifying these samples. This study introduces a novel approach using the Ras association domain family 1 A (RASSF1A), which exhibits hypermethylation in foetal cells and hypomethylation in maternal cells. The differential methylation pattern of RASSF1A provides a unique biomarker for quantifying foetal cells in cervical and intrauterine samples. METHODS: This study was conducted between September 2022 and May 2023. In total, 23 samples (12 cervical cell samples and 11 intrauterine samples) were collected from women in the Sichuan Jinxin Women & Children Hospital, Jingxiu District, Chengdu, China. The cervical cell samples were collected via lavage and brush techniques, and the intrauterine cell samples were obtained via uterine lavage. These samples were collected as part of a broader effort to advance our understanding of foetal cell dynamics during early pregnancy. The sampling methods were chosen for their minimally invasive nature and their potential in capturing a representative cell population from the respective sites. After digestion of the cell samples using a methylation-sensitive restriction enzyme cocktail, a critical step to differentiate between maternal and foetal DNA, the quantitative polymerase chain reaction (qPCR) of RASSF1A and ß-actin (ACTB) were employed to measure foetal DNA and cell concentrations. Immunofluorescence techniques targeting histocompatibility complex, class I G (HLA-G) and GATA binding protein 3 (GATA-3) were employed to detect EVTs in the cell samples and in decidual tissue, with the latter providing an additional layer of confirmation for the presence of foetal cells. RESULTS: The results showed no hypermethylated RASSF1A was detected in any of the cervical samples, irrespective of whether the samples were obtained by brush or lavage. However, an average of 17,236 ± 7490 foetal cells per sample were detected in the uterine lavage samples. Foetal cells accounted for approximately 0.14% ± 0.10% of the total cell population in these samples. The presence of EVTs in these samples was confirmed by their expression of both HLA-G and GATA-3. CONCLUSION: The detection of foetal cells in uterine cavity samples based on hypermethylation of RASSF1A and quantification of foetal cells can be used to prenatal screening. GATA-3 can be used to label EVTs.

In the realm gestational foetal health, obtaining foetal cells or genetic information is important for detecting and managing potential genetic disorders. Although foetal cells can be obtained from the cervix or uterine cavity, methods to quantify the foetal cell and foetal DNA are lacking. We introduce an innovative technique that utilises DNA restriction endonucleases to selectively isolate foetal DNA and identify foetal cells. We used this technique to measure the number of foetal cells in a sample. Using this technique, we detected foetal cells collected via lavage in uterine but not cervical samples. This finding is significant as it paves the way towards early detection of chromosomal disorders in foetuses, potentially as early as the 10th week of pregnancy. Such early detection offers promising new screening options in early pregnancy, contributing to better prenatal care and outcomes.

Simplexviruses Successfully Adapt to Their Host by Fine-Tuning Immune Responses.

Primate herpes simplex viruses are species-specific and relatively harmless to their natural hosts. However, cross-species transmission is often associated with severe disease, as exemplified by the virulence of macacine herpesvirus 1 (B virus) in humans. We performed a genome-wide scan for signals of adaptation of simplexviruses to their hominin hosts. Among core genes, we found evidence of episodic positive selection in three glycoproteins, with several selected sites located in antigenic determinants. Positively selected noncore genes were found to be involved in different immune-escape mechanisms. The herpes simplex virus (HSV)-1/HSV-2 encoded product (ICP47) of one of these genes is known to down-modulate major histocompatibility complex class I expression. This feature is not shared with B virus, which instead up-regulates Human Leukocyte Antigen (HLA)-G, an immunomodulatory molecule. By in vitro expression of different ICP47 mutants, we functionally characterized the selection signals. Results indicated that the selected sites do not represent the sole determinants of binding to the transporter associated with antigen processing (TAP). Conversely, the amino acid status at these sites was sufficient to determine HLA-G up-regulation. In fact, both HSV-1 and HSV-2 ICP47 induced HLA-G when mutated to recapitulate residues in B virus, whereas the mutated version of B virus ICP47 failed to determine HLA-G expression. These differences might contribute to the severity of B virus infection in humans. Importantly, they indicate that the evolution of ICP47 in HSV-1/HSV-2 led to the loss of an immunosuppressive effect. Thus, related simplexviruses finely tune the balance between immunosuppressive and immunostimulatory pathways to promote successful co-existence with their primate hosts.

HLA-G Alleles Impact the Perinatal Father-Child HPV Transmission.

The host factors that influence father-to-child human papillomavirus (HPV) transmission remain unknown. This study evaluated whether human leukocyte antigen (HLA)-G alleles are important in father-to-child HPV transmission during the perinatal period. Altogether, 134 father-newborn pairs from the Finnish Family HPV Study were included. Oral, semen and urethral samples from the fathers were collected before the delivery, and oral samples were collected from their offspring at delivery and postpartum on day 3 and during 1-, 2- and 6-month follow-up visits. HLA-G alleles were tested by direct sequencing. Unconditional logistic regression was used to determine the association of the father-child HLA-G allele and genotype concordance with the father-child HPV prevalence and concordance at birth and during follow-up. HLA-G allele G*01:01:03 concordance was associated with the father's urethral and child's oral high-risk (HR)-HPV concordance at birth (OR 17.00, 95% CI: 1.24-232.22). HLA-G allele G*01:04:01 concordance increased the father's oral and child's postpartum oral any- and HR-HPV concordance with an OR value of 7.50 (95% CI: 1.47-38.16) and OR value of 7.78 (95% CI: 1.38-43.85), respectively. There was no association between different HLA-G genotypes and HPV concordance among the father-child pairs at birth or postpartum. To conclude, the HLA-G allele concordance appears to impact the HPV transmission between the father and his offspring.

HLA-G 14 bp Ins/Del (rs66554220) Variant Is Not Associated with Breast Cancer in Women from Western Mexico.

HLA-G is a physiology and pathologic immunomodulator detrimentally related to cancer. Its gene is heavily transcriptionally and post-transcriptionally regulated by variants located in regulator regions like 3'UTR, being the most studied Ins/Del of 14-bp (rs66554220), which is known to influence the effects of endogen cell factors; nevertheless, the reports are discrepant and controversial. Herein, the relationship of the 14-bp Ins/Del variant (rs66554220) with breast cancer (BC) and its clinical characteristics were analyzed in 182 women with non-familial BC and 221 disease-free women as a reference group. Both groups from western Mexico and sex-age-matched (sm-RG). The rs66554220 variant was amplified by SSP-PCR and the fragments were visualized in polyacrylamide gel electrophoresis. The variant rs66554220 was not associated with BC in our population. However, we suggest the Ins allele as a possible risk factor for developing BC at clinical stage IV (OR = 3.05, 95% CI = 1.16-7.96, p = 0.01); nevertheless, given the small stratified sample size (n = 11, statistical power = 41%), this is inconclusive. In conclusion, the 14-bp Ins/Del (rs66554220) variant of HLA-G is not associated with BC in the Mexican population, but might be related to advanced breast tumors. Further studies are required.

Pemetrexed combined with dual immune checkpoint blockade enhances cytotoxic T lymphocytes against lung cancer.

Chemotherapy, in combination with immune checkpoint blockade (ICB) targeting to programmed death-1 (PD-1) or its ligand PD-L1, is one of the first-line treatments for patients with advanced non-small-cell lung cancer (NSCLC). However, a large proportion of patients, especially those with PD-L1 negative tumors, do not benefit from this treatment. This may be due to the existence of multiple immunosuppressive mechanisms other than the PD-1/PD-L1 axis. Human leukocyte antigen-G (HLA-G) has been identified as an immune checkpoint protein (ICP) and a neoexpressed tumor-associated antigen (TAA) in a large proportion of solid tumors. In this study, we evaluated the induction of HLA-G as well as PD-L1 using sublethal doses of chemotherapeutics including pemetrexed in different NSCLC cell lines. Except for gefitinib, most of the chemotherapeutic agents enhanced HLA-G and PD-L1 expression in a dose-dependent manner, whereas pemetrexed and carboplatin treatments showed the most consistent upregulation of PD-L1 and HLA-G in each cell line. In addition to protein levels, a novel finding of this study is that pemetrexed enhanced the glycosylation of HLA-G and PD-L1. Pemetrexed potentiated the cytotoxicity of cytotoxic T lymphocytes (CTLs) to treat NSCLC. Both in vitro and in vivo experiments revealed that CTL-mediated cytotoxicity was most pronounced when both anti-PD-L1 and anti-HLA-G ICBs were combined with pemetrexed treatment. In conclusion, anti-HLA-G could be an intervention strategy in addition to the anti-PD-1/PD-L1 pathway for NSCLC. Moreover, dual targeting of PD-L1 and HLA-G combined with pemetrexed might have a better extent of CTL-based immunotherapy.

HLA-G expression in Merkel cell carcinoma and the correlation with Merkel cell polyomavirus infection.

Merkel cell carcinoma (MCC) is a rare aggressive neuroendocrine cutaneous carcinoma with a high mortality rate. The MCC etiology is not fully understood. Merkel cell-associated polyomavirus (MCPyV) was found in MCC patients, indicating a risk factor for the tumor. Caucasian, elderly, and immunocompromised individuals are more likely to develop this tumor. HLA-G consists of a non-classical class I (Ib) HLA molecule with an immunoregulatory function and was associated with tumor escape in different types of tumors, nonetheless, never been studied in MCC. The purpose of this study was to evaluate the HLA-G expression and also to detect the MCPyV in MCC patients and correlate it with the clinical course of the disease. Forty-five MCC patients were included in a retrospective study. Formalin-fixed paraffin-embedded cutaneous skin biopsies were used by immunohistochemistry and RT-PCR to verify the HLA-G expression and MCPyV infection. HLA-G expression was found in 7 (15.6%), while the presence of MCPyV was detected in 28 (62.2%) of the studied patients. No significant association was found between HLA-G expression and MCPyV infection (p = 0.250). The presence of MCPyV was associated with areas of low sunlight exposure (p = 0.042) and the HLA-G expression with progression to death (p = 0.038). HLA-G expression was detected in MCC patients, as well as the MCPyV presence was confirmed. These markers could represent factors with a possible impact on patient survival; however, further studies with a greater number of patients are needed, to better elucidate the possible role in disease progression.

The + 3010/C single nucleotide polymorphism (rs1710) at the HLA-G 3' untranslated region is associated with a short transcript exhibiting a deletion of 92 nucleotides.

The physiological expression of HLA-G is mainly observed in the placenta, playing an essential role in maternal-fetal tolerance. Among the HLA-G mRNA alternative transcripts, the one lacking 92 bases at the HLA-G 3' untranslated region (3'UTR), the 92bDel transcript, is more stable, is associated with increased HLA-G soluble levels, and was observed in individuals presenting a 14 bp insertion (14 bp+) at the 3'UTR. We investigated the presence of the 92bDel transcript in placenta samples, correlating its expression levels with the HLA-G polymorphisms at the 3'UTR. The 14 bp+ allele correlates with the presence of the 92bDel transcript. However, the polymorphism triggering this alternative splicing is the + 3010/C allele (rs1710, allele C). Most 14 bp+ haplotypes (UTR-2/-5/-7) present allele + 3010/C. However, 14 bp- haplotypes such as UTR-3 are also associated with + 3010/C, and the 92bDel transcript can be detected in homozygous samples for the 14 bp- allele carrying at least one copy of UTR-3. The UTR-3 haplotype is associated with alleles G*01:04 and the HLA-G lineage HG0104, which is a high-expressing lineage. The only HLA-G lineage that is not likely to produce this transcript is HG010101, associated with the + 3010/G allele. This functional difference may be advantageous, considering the high worldwide frequency of the HG010101 lineage. Therefore, HLA-G lineages are functionally distinct regarding the 92bDel transcript expression, and the 3010/C allele triggers the alternative splicing that produces this shorter and more stable transcript.

Lower HLA-G levels in extravillous trophoblasts of human term placenta in gestational diabetes mellitus than in normal controls.

The non-classical human leucocyte antigen (HLA) class I molecule HLA-G is widely known to play a major role in feto-maternal tolerance. We tested the hypothesis that HLA-G expression is altered in placentas of women with gestational diabetes mellitus (GDM) in a specific pattern that depends on fetal sex. HLA-G expression was analysed in a total of 80 placentas (40 placentas from women with GDM and 40 healthy controls) by immunohistochemistry using the semi-quantitative immunoreactive score (IRS). Double immunofluorescence staining identified the cells expressing HLA-G in the decidua and allowed evaluation of the expression pattern. We found a significant (p < 0.001) reduction of HLA-G expression in extravillous cytotrophoblasts (EVTs) in the placentas of women with GDM as compared to the healthy controls and were able to demonstrate that this downregulation was not due to a loss of cell number, but to a loss of expression intensity. A special change in the cell pattern of EVTs was observed, with these cells showing an obvious decrease in HLA-G expression on their cell surface. No significant differences according to fetal sex were found. These data show a possible association between decreased HLA-G expression and presence of GDM and provide new insights into altered placental function in women with GDM.

Prognostic significance of HLA-G in patients with colorectal cancer: a meta-analysis and bioinformatics analysis.

PURPOSE: Human leukocyte antigen-G (HLA-G) has been reported to be aberrantly expressed in colorectal cancer (CRC); however, its prognostic value remains controversial. Hence, our meta-analysis aims to assess the prognostic value of HLA-G in CRC patients based on published literature and The Cancer Genome Atlas (TCGA) datasets. METHODS: A systematic search was conducted on relevant studies retrieved from four electronic databases including PubMed, Embase, Web of Science and Cochrane Library. Hazard ratios (HRs) with 95% confidence intervals (CIs) were recorded to be applied as effective values. Fixed-effects models or random-effects models were applied on the basis of the value of heterogeneity (I 2). Publication bias was analyzed by Begg's and Egger's tests. In addition, the results were validated by using TCGA datasets. RESULTS: Thirteen studies comprising 3896 patients were incorporated into this meta-analysis. The pooled results showed that HLA-G expression was significantly associated with poor overall survival (OS) in both the univariate analysis (HR = 1.44, 95% CI: 1.14-1.83, P = 0.002) and the multivariate analysis (HR = 1.55, 95% CI: 1.23-1.95, P < 0.001). Nevertheless, the expression of HLA-G is not related to age, sex, tumor type, tumor differentiation, TNM stage, or distant metastasis but lymph node metastasis. Notably, the prognosis of colorectal cancer was not consistent with the analysis result from TCGA data. CONCLUSION: HLA-G expression was significantly related to poor OS in CRC according to the results of our meta-analysis. However, we found that the prognostic significance was inconsistent with our results according to the TCGA data in CRC. Hence, more research is still needed to further illustrate the prognostic role of HLA-G in CRC.

Opposing impacts of HLA-G haplotypes PROMO-G010104-UTR-3 and PROMO-G010101b/c-UTR-4 on risk of recurrent implantation failure.

RESEARCH QUESTION: The human leukocyte antigen (HLA) class Ib molecules HLA-F and HLA-G are implicated in pregnancy success, but how do HLA-G and HLA-F genetic polymorphisms impact recurrent implantation failure (RIF)? DESIGN: Prospective cohort study at a fertility clinic including a cohort of 84 women experiencing RIF and 35 IVF controls to assess the influence of HLA-G haplotypes and diplotypes and HLA-F single nucleotide polymorphisms (SNP) on RIF. RESULTS: Over-representation trends for HLA-F SNP genotypes rs1362126, rs2523405 and rs2523393, previously linked with a short time-to-pregnancy, were detected in female control groups compared with RIF patients with no identified pathology linked to infertility. The HLA-G promoter haplotype PROMO-G010101b/c linked with the HLA-G 3'-untranslated region (3'UTR) haplotype UTR-4, which previously has been associated with positive IVF outcome and pregnancy success, was less frequent in the RIF group. For RIF patients carrying the UTR-4 haplotype, the odds ratio (OR) was 0.27 (95% CI 0.12-0.66; P = 0.0044, Pc = 0.026). The HLA-G PROMO-G010104-UTR-3 haplotype was associated with an increased risk of RIF. For RIF patients carrying the UTR-3 haplotype, the OR was 5.86 (95% CI 1.52-26.23; P = 0.0115, Pc = 0.069). CONCLUSIONS: These results show that specific HLA-G haplotypes based on the promoter region and the 3'UTR are either associated with an increased risk of reduced fertility, including the manifestation of RIF, and lower chance of achieving pregnancy, or with a reduced risk of experiencing RIF.

Induction of pulmonary HLA-G expression by SARS-CoV-2 infection.

The non-classical human leukocyte antigen (HLA)-G exerts immune-suppressive properties modulating both NK and T cell responses. While it is physiologically expressed at the maternal-fetal interface and in immune-privileged organs, HLA-G expression is found in tumors and in virus-infected cells. So far, there exists little information about the role of HLA-G and its interplay with immune cells in biopsies, surgical specimen or autopsy tissues of lung, kidney and/or heart muscle from SARS-CoV-2-infected patients compared to control tissues. Heterogeneous, but higher HLA-G protein expression levels were detected in lung alveolar epithelial cells of SARS-CoV-2-infected patients compared to lung epithelial cells from influenza-infected patients, but not in other organs or lung epithelia from non-viral-infected patients, which was not accompanied by high levels of SARS-CoV-2 nucleocapsid antigen and spike protein, but inversely correlated to the HLA-G-specific miRNA expression. High HLA-G expression levels not only in SARS-CoV-2-, but also in influenza-infected lung tissues were associated with a high frequency of tissue-infiltrating immune cells, but low numbers of CD8+ cells and an altered expression of hyperactivation and exhaustion markers in the lung epithelia combined with changes in the spatial distribution of macrophages and T cells. Thus, our data provide evidence for an involvement of HLA-G and HLA-G-specific miRNAs in immune escape and as suitable therapeutic targets for the treatment of SARS-CoV-2 infections.

Evolution and molecular interactions of major histocompatibility complex (MHC)-G, -E and -F genes.

Classical HLA (Human Leukocyte Antigen) is the Major Histocompatibility Complex (MHC) in man. HLA genes and disease association has been studied at least since 1967 and no firm pathogenic mechanisms have been established yet. HLA-G immune modulation gene (and also -E and -F) are starting the same arduous way: statistics and allele association are the trending subjects with the same few results obtained by HLA classical genes, i.e., no pathogenesis may be discovered after many years of a great amount of researchers' effort. Thus, we believe that it is necessary to follow different research methodologies: (1) to approach this problem, based on how evolution has worked maintaining together a cluster of immune-related genes (the MHC) in a relatively short chromosome area since amniotes to human at least, i.e., immune regulatory genes (MHC-G, -E and -F), adaptive immune classical class I and II genes, non-adaptive immune genes like (C2, C4 and Bf) (2); in addition to using new in vitro models which explain pathogenetics of HLA and disease associations. In fact, this evolution may be quite reliably studied during about 40 million years by analyzing the evolution of MHC-G, -E, -F, and their receptors (KIR-killer-cell immunoglobulin-like receptor, NKG2-natural killer group 2-, or TCR-T-cell receptor-among others) in the primate evolutionary lineage, where orthology of these molecules is apparently established, although cladistic studies show that MHC-G and MHC-B genes are the ancestral class I genes, and that New World apes MHC-G is paralogous and not orthologous to all other apes and man MHC-G genes. In the present review, we outline past and possible future research topics: co-evolution of adaptive MHC classical (class I and II), non-adaptive (i.e., complement) and modulation (i.e., non-classical class I) immune genes may imply that the study of full or part of MHC haplotypes involving several loci/alleles instead of single alleles is important for uncovering HLA and disease pathogenesis. It would mainly apply to starting research on HLA-G extended haplotypes and disease association and not only using single HLA-G genetic markers.

HLA-G 3'UTR haplotype analyses in HCV infection and HCV-derived cirrhosis, hepatocellular carcinoma and fibrosis.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease. Chronic HCV infection is also an important cause of hepatic fibrosis, cirrhosis and hepatocellular carcinoma (HCC). HCV has the capacity to evade immune surveillance by altering the host immune response. Moreover, variations in immune-related genes can lead to differential susceptibility to HCV infection as well as interfere on the susceptibility to the development of hepatic fibrosis, cirrhosis and HCC. The human leucocyte antigen G (HLA-G) gene codes for an immunomodulatory protein known to be expressed in the maternal-foetal interface and in immune-privileged tissues. The HLA-G 3' untranslated region (3'UTR) is important for mRNA stability, and variants in this region are known to impact gene expression. Studies, mainly focusing in a 14 bp insertion/deletion polymorphism, have correlated HLA-G 3'UTR with susceptibility to viral infections, but other polymorphic variants in the HLA-G 3'UTR might also affect HCV infection as they are inherited as haplotypes. The present study evaluated HLA-G 3'UTR polymorphisms and performed linkage disequilibrium test and haplotype assembly in 286 HCV infected patients who have developed fibrosis, cirrhosis or HCC, as well as in 129 healthy control subjects. Haplotypes UTR-1, UTR-2 and UTR-3 were the most observed in HCV+ patients, in the frequencies of 0.276, 0.255 and 0.121, respectively. No statistically significant difference was observed between HCV+ and control subjects, even when patients were grouped according to outcome (HCC, cirrhosis or fibrosis). Despite that, some trends in the results were observed, and therefore, we cannot rule out the possibility that variants associated to high HLA-G expression can be involved in HCV infection susceptibility.

COVID-19 induced aorto duodenal fistula following evar in the so called "negative" patient.

OBJECTIVES: Since October 2019, SARS-CoV-2 pandemic represents a challenge for the international healthcare system and for the treatment and survival of patients. We normally focus on symptomatic patients, and symptoms can range from the respiratory to the gastrointestinal system. In addition, we consider patients without fever and respiratory symptoms, with both a negative RT nasopharyngeal swab and lung CT, as a "Covid-19 negative patient." In this article, we present a so called Covid-19 "negative" patient, with an unsuspected vascular clinical onset of the viral infection. METHODS: An 80 y.o. man, who previously underwent endovascular aortic repair for an infrarenal abdominal aortic aneurysm, presented to our department with an atypical presentation of an aorto-enteric fistula during the pandemic. While in hospital, weekly nasopharyngeal swab tests were always negative for SARS-CoV-2. However, the absence of aortic endograft complications, the gross anatomy of duodenal ischemic injury, and the recent history of the patient who lived the last months in Bergamo, the Italian city with the highest number of COVID-19 deaths, lead the senior Author to suspect an occult SARS-CoV-2 infection. The patient underwent to resection of the fourth portion of the duodenum and the first jejunal loop, with subsequent duodenum-jejunal latero-lateral anastomosis and the direct suture of the aortic wall. The intestinal specimen was investigated as suspected SARS-CoV-2 bowel infection by the means of immune-histochemistry (IHC). An ileum sample obtained in the pre-COVID-19 era was used as a control tissue. RESULTS: The histological analysis of the bowel revealed sustained wall ischemia and liponecrosis of the duodenal wall, with intramural blood vessels thrombosis. Blood vessel endotheliitis and neo-angiogenesis were also observed. Finally, the IHC was strongly positive for SARS-CoV-2 RNA and for HLA-G presence, with a particular concentration both in blood vessels and in the intestinal villi. The control tissue sample was not positive for both SARS-CoV-2 and HLA-G. CONCLUSIONS: Coronavirus pandemic continues to be an international challenge and more studies and trials must be done to learn its pathogenesis and its complications. As for thromboembolic events caused by SARS-COV-2, vascular surgeons are involved in treatment and prevention of the complications of this syndrome and must be ready with general surgeons to investigate atypical and particular cases such as the one discussed in this article.