A Genome-Wide Association Study Suggests New Susceptibility Loci for Primary Antiphospholipid Syndrome.

OBJECTIVE: Primary antiphospholipid syndrome (PAPS) is a rare autoimmune disease characterized by the presence of antiphospholipid antibodies and the occurrence of thrombotic events and pregnancy complications. Our study aimed to identify novel genetic susceptibility loci associated with PAPS. METHODS: We performed a genome-wide association study comprising 5,485 individuals (482 affected individuals) of European ancestry. Significant and suggestive independent variants from a meta-analysis of approximately 7 million variants were evaluated for functional and biological process enrichment. The genetic risk variability for PAPS in different populations was also assessed. Hierarchical clustering, Mahalanobis distance, and Dirichlet Process Mixtures with uncertainty clustering methods were used to assess genetic similarities between PAPS and other immune-mediated diseases. RESULTS: We revealed genetic associations with PAPS in a regulatory locus within the HLA class II region near HLA-DRA and in STAT1-STAT4 with a genome-wide level of significance; 34 additional suggestive genetic susceptibility loci for PAPS were also identified. The disease risk allele near HLA-DRA is associated with overexpression of HLA-DRB6, HLA-DRB9, HLA-DQA2, and HLA-DQB2 in immune cells, vascular tissue, and nervous tissue. This association is independent of the association between PAPS and HLA-DRB1*1302. Functional analyses highlighted immune-related pathways in PAPS-associated loci. The comparison with other immune-mediated diseases revealed a close genetic relatedness to neuromyelitis optica, systemic sclerosis, and Sjögren syndrome, suggesting co-localized causal variations close to STAT1-STAT4, TNPO3, and BLK. CONCLUSION: This study represents a comprehensive large-scale genetic analysis for PAPS and provides new insights into the genetic basis and pathophysiology of this rare disease.

The relationship between innate/adaptive immunity and gastrointestinal cancer : a multi-omics Mendelian randomization study.

BACKGROUND: Innate/adaptive immunity is the key to anti-tumor therapy. However, its causal relationship to Gastrointestinal (GI) cancer remains unclear. METHODS: Immunity genes were extracted from the MSigDB database. The Genome-wide association studies (GWAS) summary data of GI cancer were integrated with expression quantitative trait loci (eQTL) and DNA methylation quantitative trait loci (mQTL) associated with genes. Summary-data-based Mendelian randomization (SMR) and co-localization analysis were used to reveal causal relationships between genes and GI cancer. Two-sample MR analysis was used for sensitivity analysis. Single cell analysis clarified the enrichment of genes. RESULTS: Three-step SMR analysis showed that a putative mechanism, cg17294865 CpG site regulating HLA-DRA expression was negatively associated with gastric cancer risk. HLA-DRA was significantly differentially expressed in monocyte/macrophage and myeloid cells in gastric cancer. CONCLUSION: This study provides evidence that upregulating the expression level of HLA-DRA can reduce the risk of gastric cancer.

Changes in expression of VGF, SPECC1L, HLA-DRA and RANBP3L act with APOE E4 to alter risk for late onset Alzheimer's disease.

While there are currently over 40 replicated genes with mapped risk alleles for Late Onset Alzheimer's disease (LOAD), the Apolipoprotein E locus E4 haplotype is still the biggest driver of risk, with odds ratios for neuropathologically confirmed E44 carriers exceeding 30 (95% confidence interval 16.59-58.75). We sought to address whether the APOE E4 haplotype modifies expression globally through networks of expression to increase LOAD risk. We have used the Human Brainome data to build expression networks comparing APOE E4 carriers to non-carriers using scalable mixed-datatypes Bayesian network (BN) modeling. We have found that VGF had the greatest explanatory weight. High expression of VGF is a protective signal, even on the background of APOE E4 alleles. LOAD risk signals, considering an APOE background, include high levels of SPECC1L, HLA-DRA and RANBP3L. Our findings nominate several new transcripts, taking a combined approach to network building including known LOAD risk loci.

Evaluation of HLA-DR and HLA-DQ expression in gastric cancer tissues.

BACKGROUND AND OBJECTIVES: Despite recent advances in understanding the gastric cancer (GC) biology, the precise molecular mechanism of gastric carcinogenesis and role of deregulated immune responses in GC progression are still not well understood. In this study, mRNA levels of human leukocyte antigen (HLA)-DRA and -DQA1 were assessed in GC patients to find a potential association between expression of these HLA-II molecules and gastric carcinogenesis. METHODS: Using quantitative real-time (qRT)-PCR, mRNA levels of HLA-DRA and -DQA1 were assessed in 20 pairs of matched GC and normal tissues. RESULTS: Our results showed that overall mRNA level of HLA-DRA was decreased in the tumor samples relative to control tissues (median fold change [FC] = 0.693; P = 0.445). Overall HLA-DQA1 level was increased in the tumor samples relative to control tissues (median FC = 1.659; P = 0.5117). However, the mentioned data were not statistically significant. Meanwhile, using a ≥ 2.5 FC as the cutoff to determine upregulation or downregulation, 35% of patients showed a downregulated expression of HLA-DRA, while 10% of those showed upregulation in HLA-DRA expression. Upregulation and downregulation of HLA-DQA1 expression were detected, respectively, in 35% and 25% of samples. A strong positive correlation was determined between HLA-DRA and HLA-DQA1 levels in tumor tissues (r = 0.7298; P = 0.0003). CONCLUSION: The results reported here along with future studies can be useful to understand the interplay between immune system and GC, therefore, may be helpful to design an effective immune-based therapy.

Identification of gene signature markers in gestational hypertension and early-onset pre-eclampsia.

INTRODUCTION: Hypertensive disorders in pregnancy (HDP) are the leading cause of perinatal mortality worldwide. Inflammatory responses induced by insufficient placental perfusion have become a focal point in understanding the pathogenesis and aetiology of HDP and developing reliable and consistent biomarkers. Therefore, this study aims to identify gene signatures linked to the pathophysiology of HDP (gestational hypertension and early and late-onset pre-eclampsia). METHODS: RNA was extracted from the maternal serum from the blood samples collected from different groups of HDP patients. A multiplex inflammation panel (255 inflammatory and housekeeping genes) and further gene expression analysis using NanoString Digital Direct Detection were done. The prominent expressions of these genes were further validated through qPCR techniques. RESULTS: NanoString analysis identified nine unique, significantly expressed genes (MAPK1, MAPK3, MAFF, HLA-DRA, IL12B, RHOA, MASP2, MEF2A and NR3C1) between specific group comparisons of different HPD classes and the normotensive groups. The qPCR showed that the HLA-DRA gene was significantly upregulated in the early-onset pre-eclamptic and gestational hypertensive group compared to its respective normotensive group. In contrast, MAFF and MEF2A were significantly downregulated in both HDPs compared to their controls. The MAPK1 gene was significantly higher in the early-onset group compared to the gestational hypertensive and normotensive groups. DISCUSSION: The upregulation of these distinctive genes in hypertensive groups compared to normotensives confirmed their diagnostic potential. Therefore, HLA-DRA, MAFF and MEF2A could be candidate markers of HDP, while the MAPK1 gene could be a differentiating marker between early-onset pre-eclampsia and gestational hypertension.

Bioinformatics analysis of hub genes as osteoarthritis prognostic biomarkers.

Osteoarthritis (OA) is a progressive cartilage degradation disease, concomitant with synovitis, osteophyte formation, and subchondral bone sclerosis. Over 37% of the elderly population is affected by OA, and the number of cases is increasing as the global population ages. Therefore, the objective of this study was to identify and analyze the hub genes of OA combining with comprehensive bioinformatics analysis tools to provide theoretical basis in further OA effective therapies. Two sample sets of GSE46750 contained 12 pairs OA synovial membrane and normal samples harvested from patients as well as GSE98918 including 12 OA and non-OA patients were downloaded from the Gene Expression Omnibus database (GEO) database. Differentially expressed genes (DEGs) were identified using Gene Expression Omnibus 2R (GEO2R), followed by functional enrichment analysis, protein-protein interaction networks construction. The hub genes were identified and evaluated. An OA rat model was constructed, hematoxylin and eosin staining, safranin O/fast green staining, cytokines concentrations of serum were used to verify the model. The hub genes expression level in the knee OA samples were verified using RT-qPCR. The top 20 significantly up-regulated and down-regulated DEGs were screened out from the two datasets, respectively. The top 18 GO terms and 10 KEGG pathways were enriched. Eight hub genes were identified, namely MS4A6A, C1QB, C1QC, CD74, CSF1R, HLA-DPA1, HLA-DRA and ITGB2. Among them, the hub genes were all up-regulated in in vivo OA rat model, compared with healthy controls. The eight hub genes identified (MS4A6A, C1QB, C1QC, CD74, CSF1R, HLA-DPA1, HLA-DRA and ITGB2) were shown to be associated with OA. These genes can serve as disease markers to discriminate OA patients from healthy controls.

Abdominal surgery induces long-lasting changes in expression and binding of CTCF with impact on Major Histocompatibility Complex II transcription in circulating human monocytes.

BACKGROUND: Postoperative immunosuppression has been recognized as an important driver of surgery-related morbidity and mortality. It is characterized by lymphocyte depression and impaired monocyte capability to present foreign antigens to T-cells via Major Histocompatibility Complex, Class II (MHC-II) molecules. In patients with postoperative abdominal sepsis, we previously detected a persisting differential binding of the CCCTC-Binding Factor (CTCF), a superordinate regulator of transcription, inside the MHC-II region with specific impact on human leucocyte antigen (HLA) gene expression. In this prospective exploratory study, we investigated to which extent major surgery affects the MHC-II region of circulating CD14+-monocytes. RESULTS: In non-immunocompromised patients undergoing elective major abdominal surgery, a postoperative loss of monocyte HLA-DR surface receptor density was accompanied by a decline in the transcription levels of the classical MHC-II genes HLA-DRA, HLA-DRB1, HLA-DPA1 and HLA-DPB1. The surgical event decreased the expression of the transcriptional MHC-II regulators CIITA and CTCF and led to a lower CTCF enrichment at an intergenic sequence within the HLA-DR subregion. During the observation period, we found a slow and only incomplete restoration of monocyte HLA-DR surface receptor density as well as a partial recovery of CIITA, HLA-DRA and HLA-DRB1 expression. In contrast, transcription of HLA-DPA1, HLA-DPB1, CTCF and binding of CTCF within the MHC-II remained altered. CONCLUSION: In circulating monocytes, major surgery does not globally affect MHC-II transcription but rather induces specific changes in the expression of selected HLA genes, followed by differential recovery patterns and accompanied by a prolonged reduction of CTCF expression and binding within the MHC-II region. Our results hint toward a long-lasting impact of a major surgical intervention on monocyte functionality, possibly mediated by epigenetic changes that endure the life span of the individual cell.

A role of inflammaging in aortic aneurysm: new insights from bioinformatics analysis.

Introduction: Aortic aneurysms (AA) are prevalent worldwide with a notable absence of drug therapies. Thus, identifying potential drug targets is of utmost importance. AA often presents in the elderly, coupled with consistently raised serum inflammatory markers. Given that ageing and inflammation are pivotal processes linked to the evolution of AA, we have identified key genes involved in the inflammaging process of AA development through various bioinformatics methods, thereby providing potential molecular targets for further investigation. Methods: The transcriptome data of AA was procured from the datasets GSE140947, GSE7084, and GSE47472, sourced from the NCBI GEO database, whilst gene data of ageing and inflammation were obtained from the GeneCards Database. To identify key genes, differentially expressed analysis using the "Limma" package and WGCNA were implemented. Protein-protein intersection (PPI) analysis and machine learning (ML) algorithms were employed for the screening of potential biomarkers, followed by an assessment of the diagnostic value. Following the acquisition of the hub inflammaging and AA-related differentially expressed genes (IADEGs), the TFs-mRNAs-miRNAs regulatory network was established. The CIBERSORT algorithm was utilized to investigate immune cell infiltration in AA. The correlation of hub IADEGs with infiltrating immunocytes was also evaluated. Lastly, wet laboratory experiments were carried out to confirm the expression of hub IADEGs. Results: 342 and 715 AA-related DEGs (ADEGs) recognized from GSE140947 and GSE7084 datasets were procured by intersecting the results of "Limma" and WGCNA analyses. After 83 IADEGs were obtained, PPI analysis and ML algorithms pinpointed 7 and 5 hub IADEGs candidates respectively, and 6 of them demonstrated a high diagnostic value. Immune cell infiltration outcomes unveiled immune dysregulation in AA. In the wet laboratory experiments, 3 hub IADEGs, including BLNK, HLA-DRA, and HLA-DQB1, finally exhibited an expression trend in line with the bioinformatics analysis result. Discussion: Our research identified three genes - BLNK, HLA-DRA, and HLA-DQB1- that play a significant role in promoting the development of AA through inflammaging, providing novel insights into the future understanding and therapeutic intervention of AA.

Identification of Potential Biomarkers of Septic Shock Based on Pathway and Transcriptome Analyses of Immune-Related Genes.

Immunoregulation is crucial to septic shock (SS) but has not been clearly explained. Our aim was to explore potential biomarkers for SS by pathway and transcriptional analyses of immune-related genes to improve early detection. GSE57065 and GSE95233 microarray data were used to screen differentially expressed genes (DEGs) in SS. Gene Ontology and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses of DEGs were performed, and correlations between immune cell and pathway enrichment scores were analyzed. The predictive value of candidate genes was evaluated by receiver operating characteristic (ROC) curves. GSE66099, GSE4607, and GSE13904 datasets were used for external validation. Blood samples from six patients and six controls were collected for validation by qRT-PCR and western blotting. In total, 550 DEGs in SS were identified; these genes were involved in the immune response, inflammation, and infection. Immune-related pathways and levels of infiltration of CD4 + TCM, CD8 + T cells, and preadipocytes differed between SS cases and controls. Seventeen genes were identified as potential biomarkers of SS (areas under ROC curves >0.9). The downregulation of CD8A, CD247, CD3G, LCK, and HLA-DRA in SS was experimentally confirmed. We identified several immune-related biomarkers in SS that may improve early identification of disease risk.

Technical Validation and Utility of an HLA Class II Tetramer Assay for Type 1 Diabetes: A Multicenter Study.

CONTEXT: Validated assays to measure autoantigen-specific T-cell frequency and phenotypes are needed for assessing the risk of developing diabetes, monitoring disease progression, evaluating responses to treatment, and personalizing antigen-based therapies. OBJECTIVE: Toward this end, we performed a technical validation of a tetramer assay for HLA-DRA-DRB1*04:01, a class II allele that is strongly associated with susceptibility to type 1 diabetes (T1D). METHODS: HLA-DRA-DRB1*04:01-restricted T cells specific for immunodominant epitopes from islet cell antigens GAD65, IGRP, preproinsulin, and ZnT8, and a reference influenza epitope, were enumerated and phenotyped in a single staining tube with a tetramer assay. Single and multicenter testing was performed, using a clone-spiked specimen and replicate samples from T1D patients, with a target coefficient of variation (CV) less than 30%. The same assay was applied to an exploratory cross-sectional sample set with 24 T1D patients to evaluate the utility of the assay. RESULTS: Influenza-specific T-cell measurements had mean CVs of 6% for the clone-spiked specimen and 11% for T1D samples in single-center testing, and 20% and 31%, respectively, for multicenter testing. Islet-specific T-cell measurements in these same samples had mean CVs of 14% and 23% for single-center and 23% and 41% for multicenter testing. The cross-sectional study identified relationships between T-cell frequencies and phenotype and disease duration, sex, and autoantibodies. A large fraction of the islet-specific T cells exhibited a naive phenotype. CONCLUSION: Our results demonstrate that the assay is reproducible and useful to characterize islet-specific T cells and identify correlations between T-cell measures and clinical traits.

Genome-wide association study identifies multiple HLA loci for sarcoidosis susceptibility.

Sarcoidosis is a complex systemic disease. Our study aimed to (1) identify novel alleles associated with sarcoidosis susceptibility; (2) provide an in-depth evaluation of HLA alleles and sarcoidosis susceptibility and (3) integrate genetic and transcription data to identify risk loci that may more directly impact disease pathogenesis. We report a genome-wide association study of 1335 sarcoidosis cases and 1264 controls of European descent (EA) and investigate associated alleles in a study of African Americans (AA: 1487 cases and 1504 controls). The EA and AA cohort was recruited from multiple United States sites. HLA alleles were imputed and tested for association with sarcoidosis susceptibility. Expression quantitative locus and colocalization analysis were performed using a subset of subjects with transcriptome data. Forty-nine SNPs in the HLA region in HLA-DRA, -DRB9, -DRB5, -DQA1 and BRD2 genes were significantly associated with sarcoidosis susceptibility in EA, rs3129888 was also a risk variant for sarcoidosis in AA. Classical HLA alleles DRB1*0101, DQA1*0101 and DQB1*0501, which are highly correlated, were also associated with sarcoidosis. rs3135287 near HLA-DRA was associated with HLA-DRA expression in peripheral blood mononuclear cells and bronchoalveolar lavage from subjects and lung tissue and whole blood from GTEx. We identified six novel SNPs (out of the seven SNPs representing the 49 significant SNPs) and nine HLA alleles associated with sarcoidosis susceptibility in the largest EA population. We also replicated our findings in an AA population. Our study reiterates the potential role of antigen recognition and/or presentation HLA class II genes in sarcoidosis pathogenesis.

Evaluation of genetic risk, its clinical manifestation and disease management based on 18 susceptibility gene markers among West-Slavonic patients with sarcoidosis.

Sarcoidosis is a heterogenous, multisystemic inflammatory disease that primarily affects lungs. In this study, we multiplex genotyped 18 single-nucleotide polymorphisms (SNPs) to replicate the findings from previous genome-wide association studies (GWAS) and candidate gene studies, and extended analyses to different clinical manifestations (Löfgren's syndrome and chest X-ray [CXR] stages) including treatment response among West-Slavonic subjects (564 sarcoidosis patients and 301 healthy controls). We confirm the replication (with Bonferroni's correction) of ANXA11 rs1049550 as protective variant for sarcoidosis (odds ratio [OR] = 0.71, p = 1.33 × 10-3), non-LS (OR = 0.66, p = 2.71 × 10-4) and CXR stages 2-4 (OR = 0.62, p = 7.48 × 10-5) compared to controls in West-Slavonic population. We also validate the association of risk variants C6orf10 rs3129927 (OR = 2.61, p = 2.60 × 10-8), TNFA rs1800629 (OR = 1.56, p = 6.65 × 10-4), ATF6B rs3130288 (OR = 2.75, p = 1.06 × 10-9) and HLA-DQA1 rs2187668 (OR = 1.74, p = 8.83 × 10-4) with sarcoidosis compared to controls. For sub-phenotypes compared to controls, risk variants C6orf10 rs3129927 (OR = 5.35, p = 1.07 × 10-12), TNFA rs1800629 (OR = 2.66, p = 5.94 × 10-7), ATF6B rs3130288 (OR = 5.24, p = 5.21 × 10-13), LRRC16A rs9295661 (OR = 2.97, p = 4.29 × 10-4), HLA-DQA1 rs2187668 (OR = 3.14, p = 1.09 × 10-6) and HLA-DRA rs3135394 (OR = 5.23, p = 8.25 × 10-13) were associated with LS while C6orf10 rs3129927 (OR = 1.96, p = 4.27 × 10-4) and ATF6B rs3130288 (OR = 2.15, p = 3.36 × 10-5) were associated with non-LS. For CXR stages compared to controls, C6orf10 rs3129927 (OR = 3.67, p = 3.63 × 10-11), TNFA rs1800629 (OR = 1.84, p = 1.32 × 10-4), ATF6B rs3129927 (OR = 3.63, p = 1.82 × 10-11), HLA-DQA1 rs2187668 (OR = 2.13, p = 9.59 × 10-5) and HLA-DRA rs3135394 (OR = 3.42, p = 3.45 × 10-10) were risk variants for early CXR stages 0-1 while C6orf10 rs3129927 (OR = 1.99, p = 5.51 × 10-4), ATF6B rs3129927 (OR = 2.23, p = 3.52 × 10-5) and HLA-DRA rs3135394 (OR = 1.85, p = 2.00 × 10-3) were risk variants for advanced CXR stages 2-4. The present findings nominate gene variants as plausible prognostic markers for clinical phenotypes, treatment response and disease resolution/progression and may form the basis for establishing genotype-phenotype relationships in patients with sarcoidosis among West-Slavonic population.

Biomarkers in cerebrospinal fluid for amyotrophic lateral sclerosis phenotypes.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease involving both upper and lower motor neurons. The motor phenotypes of ALS are highly clinically heterogeneous, and the underlying mechanisms are poorly understood. METHODS: A comparative proteomic analysis was performed in the cerebrospinal fluid (CSF) of bulbar-onset (BO) and spinal-onset (SO) ALS patients and controls (n = 14). Five biomarker candidates were selected from a differentially regulated protein pool, and further validation was performed in a larger independent cohort (n = 92) using enzyme-linked immunosorbent assay (ELISA). RESULTS: A total of 1732 CSF proteins were identified, and 78 differentially expressed proteins were found among BO-ALS patients, SO-ALS patients, and controls. Five promising biomarker candidates were selected for further validation, and lipopolysaccharide-binding protein (LBP) and HLA class II histocompatibility antigen, DR alpha chain (HLA-DRA) were validated. CSF LBP levels were increased in ALS patients compared with controls and higher in BO-ALS versus SO-ALS. The increased CSF LBP levels were correlated with the revised ALS Functional Scale (ALSFRS-R) score. CSF HLA-DRA levels were specifically elevated in BO-ALS patients, and there was no significant difference between SO-ALS patients and controls. Increased HLA-DRA expression was correlated with decreased survival. INTERPRETATION: Our data shows that elevated CSF LBP is a good biomarker for ALS and correlates with clinical severity, and increased HLA-DRA is a specific biomarker for BO-ALS and may predict short survival. It also suggests that the microglial pathway and HLA-II-related adaptive immunity may be differentially involved in ALS phenotypes and may be new therapeutic targets for ALS.

Characterization of genetic humanized mice with transgenic HLA DP401 or DRA but deficient in endogenous murine MHC class II genes upon Staphylococcus aureus pneumonia.

BACKGROUND: Staphylococcus aureus can cause serious infections by secreting many superantigen exotoxins in "carrier" or "pathogenic" states. HLA DQ and HLA DR humanized mice have been used as a small animal model to study the role of two molecules during S. aureus infection. However, the contribution of HLA DP to S. aureus infection is unknown yet. METHODS: In this study, we have produced HLA DP401 and HLA DRA0101 humanized mice by microinjection of C57BL/6J zygotes. Neo-floxed IAß+/- mice were crossbred with Ella-Cre and further crossbred with HLA DP401 or HLA-DRA0101 humanized mice. After several rounds of traditional crossbreeding, we finally obtained HLA DP401-IAß-/- and HLA DRA-IAß-/- humanized mice, in which human DP401 or DRA0101 molecule was introduced into IAß-/- mice deficient in endogenous murine MHC class II molecules. A transnasal infection murine model of S. aureus pneumonia was induced in the humanized mice by administering 2 × 108 CFU of S. aureus Newman dropwise into the nasal cavity. The immune responses and histopathology changes were further assessed in lungs in these infected mice. RESULTS: We evaluated the local and systemic effects of S. aureus delivered intranasally in HLA DP401-IAß-/- and HLA DRA-IAß-/- transgenic mice. S. aureus Newman infection significantly increased the mRNA level of IL 12p40 in lungs in humanized mice. An increase in IFN-γ and IL-6 protein was observed in HLA DRA-IAß-/- mice. We observed a declining trend in the percentage of F4/80+ macrophages in lungs in HLA DP401-IAß-/- mice and a decreasing ratio of CD4+ to CD8+ T cells in lungs in IAß-/- mice and HLA DP401-IAß-/- mice. A decreasing ratio of Vß3+ to Vß8+ T cells was also found in the lymph node of IAß-/- mice and HLA DP401-IAß-/- mice. S. aureus Newman infection resulted in a weaker pathological injury in lungs in IAß-/- genetic background mice. CONCLUSION: These humanized mice will be an invaluable mouse model to resolve the pathological mechanism of S. aureus pneumonia and study what role DP molecule plays in S. aureus infection.

Blockade of endothelin-1 receptor B regulates molecules of the major histocompatibility complex in sickle cell disease.

Major Histocompatibility Complex (MHC) molecules have been proposed to play a role in Sickle Cell Disease (SCD) pathophysiology. Endothelial cells express MHC molecules following exposure to cytokines. SCD is characterized, in part, by vascular endothelial cell activation, increased oxidative stress, sickle cell adhesion, and excess levels of endothelin-1 (ET-1) contributing to vaso-occlusive crises. ET-1 activates endothelial cells, induces oxidative stress and inflammation, and alters erythrocyte volume homeostasis. However, the role of ET-1 on MHC regulation in SCD is unclear. We first studied two sickle transgenic knockout mouse models of moderate to severe disease phenotype, ßS-Antilles and Berkeley (BERK) mice. We observed significant increases in H2-Aa mRNA levels in spleens, lungs, and kidneys from transgenic sickle mice when compared to transgenic knockout mice expressing human hemoglobin A (HbA). Mice treated for 14 days with ET-1 receptor antagonists significantly reduced H2-Aa mRNA levels. We characterized the effect of ET-1 on MHC class II expression in the human endothelial cell line EA.hy926. We observed dose-dependent increases in the expression of MHC class II (HLA-DRA) and MHC transcription factor (CIITA) that were significantly blocked by treatment with BQ788, a selective blocker of ET-1 type B receptors. Chromatin immunoprecipitation studies in EA.hy926 cells showed that ET-1 increased Histone H3 acetylation of the HLA-DRA promoter, an event blocked by BQ788 treatment. These results implicate ET-1 as a novel regulator of MHC class II molecules and suggest that ET-1 receptor blockade represents a promising therapeutic approach to regulate both immune and vascular responses in SCD.

Targeted Quantitative Mass Spectrometry Analysis of Protein Biomarkers From Previously Stained Single Formalin-Fixed Paraffin-Embedded Tissue Sections.

Formalin-fixed, paraffin-embedded tissues represent a majority of all biopsy specimens commonly analyzed by histologic or immunohistochemical staining with adhesive coverslips attached. Mass spectrometry (MS) has recently been used to precisely quantify proteins in samples consisting of multiple unstained formalin-fixed, paraffin-embedded sections. Here, we report an MS method to analyze proteins from a single coverslipped 4-µm section previously stained with hematoxylin and eosin, Masson trichrome, or 3,3'-diaminobenzidine-based immunohistochemical staining. We analyzed serial unstained and stained sections from non-small cell lung cancer specimens for proteins of varying abundance (PD-L1, RB1, CD73, and HLA-DRA). Coverslips were removed by soaking in xylene, and after tryptic digestion, peptides were analyzed by targeted high-resolution liquid chromatography with tandem MS with stable isotope-labeled peptide standards. The low-abundance proteins RB1 and PD-L1 were quantified in 31 and 35 of 50 total sections analyzed, respectively, whereas higher abundance CD73 and HLA-DRA were quantified in 49 and 50 sections, respectively. The inclusion of targeted ß-actin measurement enabled normalization in samples where residual stain interfered with bulk protein quantitation by colorimetric assay. Measurement coefficient of variations for 5 replicate slides (hematoxylin and eosin stained vs unstained) from each block ranged from 3% to 18% for PD-L1, from 1% to 36% for RB1, 3% to 21% for CD73, and 4% to 29% for HLA-DRA. Collectively, these results demonstrate that targeted MS protein quantification can add a valuable data layer to clinical tissue specimens after assessment for standard pathology end points.

Exploring the pathogenesis and immune infiltration in dilated cardiomyopathy complicated with atrial fibrillation by bioinformatics analysis.

Background: Atrial fibrillation (AF) is a serious complication of dilated cardiomyopathy (DCM), which increases the risk of thromboembolic events and sudden death in DCM patients. However, the common mechanism of DCM combined with AF remains unclear. This study aims to explore the molecular mechanism and analyze immune infiltration in DCM complicated with AF through comprehensive bioinformatics analysis. Methods: The gene expression datasets of DCM (GSE141910) and AF (GSE41177 and GSE79768) were obtained from the Gene Expression Omnibus database. Gene enrichment analyses were performed after screening the common differentially expressed genes (DEGs) of DCM and AF. Protein-protein interaction network was constructed in the STRING database and visualized in Cytoscape software, which helped to further screen the central functional modules of DEGs and hub genes. In addition, ImmuCellAI algorithm was performed to estimate immune infiltration patterns, and Spearman correlation was conducted to investigate the correlation between the abundance of multiple immune cells and the expression levels of hub immune-related genes after obtaining hub immune-related genes from the ImmPort database. The hub immune-related genes expression and immune infiltration patterns were additionally verified in the validation datasets (GSE57338, GSE115574, and GSE31821). The diagnostic effectiveness of hub immune-related genes was evaluated through Receiver Operator Characteristic Curve analysis. Results: A total of 184 common DEGs in DCM and AF were identified for subsequent analyses. The functions of hub genes were significantly associated with immune responses. We identified 7 hub immune-related genes (HLA-DRA, LCK, ITK, CD48, CD247, CD3D, and IL2RG) and a spectrum of immune cell subsets including Monocyte, Neutrophil, and follicular helper T (Tfh) cells were found to be concurrently dysregulated in both DCM and AF. 7 hub immune-related genes were predominantly favorably correlated with Tfh cells and were primarily negatively correlated with Neutrophil infiltrations in DCM and AF. CD48+CD3D were verified to diagnose DCM and AF with excellent sensitivity and specificity, showing favorable diagnostic value. Conclusions: Our study reveals that immune cells (Tfh cells) disorders caused by hub immune-related genes (CD48 and CD3D) may be the common pathogenesis of DCM combined with AF, which lays a foundation for further immune mechanism research.

Early peripheral blood MCEMP1 and HLA-DRA expression predicts COVID-19 prognosis.

BACKGROUND: Mass vaccination has dramatically reduced the incidence of severe COVID-19, with most cases now presenting as self-limiting upper respiratory tract infections. However, those with co-morbidities, the elderly and immunocompromised, as well as the unvaccinated, remain disproportionately vulnerable to severe COVID-19 and its sequelae. Furthermore, as the effectiveness of vaccination wanes with time, immune escape SARS-CoV-2 variants could emerge to cause severe COVID-19. Reliable prognostic biomarkers for severe disease could be used as early indicator of re-emergence of severe COVID-19 as well as for triaging of patients for antiviral therapy. METHODS: We performed a systematic review and re-analysis of 7 publicly available datasets, analysing a total of 140 severe and 181 mild COVID-19 patients, to determine the most consistent differentially regulated genes in peripheral blood of severe COVID-19 patients. In addition, we included an independent cohort where blood transcriptomics of COVID-19 patients were prospectively and longitudinally monitored previously, to track the time in which these gene expression changes occur before nadir of respiratory function. Single cell RNA-sequencing of peripheral blood mononuclear cells from publicly available datasets was then used to determine the immune cell subsets involved. FINDINGS: The most consistent differentially regulated genes in peripheral blood of severe COVID-19 patients were MCEMP1, HLA-DRA and ETS1 across the 7 transcriptomics datasets. Moreover, we found significantly heightened MCEMP1 and reduced HLA-DRA expression as early as four days before the nadir of respiratory function, and the differential expression of MCEMP1 and HLA-DRA occurred predominantly in CD14+ cells. The online platform which we developed is publicly available at https://kuanrongchan-covid19-severity-app-t7l38g.streamlitapp.com/, for users to query gene expression differences between severe and mild COVID-19 patients in these datasets. INTERPRETATION: Elevated MCEMP1 and reduced HLA-DRA gene expression in CD14+ cells during the early phase of disease are prognostic of severe COVID-19. FUNDING: K.R.C is funded by the National Medical Research Council (NMRC) of Singapore under the Open Fund Individual Research Grant (MOH-000610). E.E.O. is funded by the NMRC Senior Clinician-Scientist Award (MOH-000135-00). J.G.H.L. is funded by the NMRC under the Clinician-Scientist Award (NMRC/CSAINV/013/2016-01). S.K. is funded by the NMRC under the Transition Award. This study was sponsored in part by a generous gift from The Hour Glass.

Identification of immune cell infiltration and diagnostic biomarkers in unstable atherosclerotic plaques by integrated bioinformatics analysis and machine learning.

Objective: The decreased stability of atherosclerotic plaques increases the risk of ischemic stroke. However, the specific characteristics of dysregulated immune cells and effective diagnostic biomarkers associated with stability in atherosclerotic plaques are poorly characterized. This research aims to investigate the role of immune cells and explore diagnostic biomarkers in the formation of unstable plaques for the sake of gaining new insights into the underlying molecular mechanisms and providing new perspectives for disease detection and therapy. Method: Using the CIBERSORT method, 22 types of immune cells between stable and unstable carotid atherosclerotic plaques from RNA-sequencing and microarray data in the public GEO database were quantitated. Differentially expressed genes (DEGs) were further calculated and were analyzed for enrichment of GO Biological Process and KEGG pathways. Important cell types and hub genes were screened using machine learning methods including least absolute shrinkage and selection operator (LASSO) regression and random forest. Single-cell RNA sequencing and clinical samples were further used to validate critical cell types and hub genes. Finally, the DGIdb database of gene-drug interaction data was utilized to find possible therapeutic medicines and show how pharmaceuticals, genes, and immune cells interacted. Results: A significant difference in immune cell infiltration was observed between unstable and stable plaques. The proportions of M0, M1, and M2 macrophages were significantly higher and that of CD8+ T cells and NK cells were significantly lower in unstable plaques than that in stable plaques. With respect to DEGs, antigen presentation genes (CD74, B2M, and HLA-DRA), inflammation-related genes (MMP9, CTSL, and IFI30), and fatty acid-binding proteins (CD36 and APOE) were elevated in unstable plaques, while the expression of smooth muscle contraction genes (TAGLN, ACAT2, MYH10, and MYH11) was decreased in unstable plaques. M1 macrophages had the highest instability score and contributed to atherosclerotic plaque instability. CD68, PAM, and IGFBP6 genes were identified as the effective diagnostic markers of unstable plaques, which were validated by validation datasets and clinical samples. In addition, insulin, nivolumab, indomethacin, and α-mangostin were predicted to be potential therapeutic agents for unstable plaques. Conclusion: M1 macrophages is an important cause of unstable plaque formation, and CD68, PAM, and IGFBP6 could be used as diagnostic markers to identify unstable plaques effectively.

HLA-DRA Gene Polymorphisms Are Associated with Graves' Disease as an Autoimmune Thyroid Disease.

Background: Autoimmune thyroid disease (AITD), one of the most prevalent organ-specific autoimmune diseases, mainly includes Graves' disease (GD) and Hashimoto's thyroiditis (HT). This study was aimed at researching the association between AITD and single nucleotide polymorphisms (SNPs) of the HLA-DRA gene. Methods: Using Hi-SNP high-throughput sequencing technology, we detected the distribution of three SNPs (rs3177928, rs7197, and rs3129878) of HLA-DRA genotypes in 1033 AITD patients (634 GD and 399 HT ones) and 791 healthy volunteers in Chinese Han Population. Chi-square test, multivariate logistic regression, and haplotype analysis were performed by SPSS and Haploview software to analyze the relationship between HLA-DRA gene polymorphisms and AITD. Results: The results show that allele frequency and genotype distribution of rs3177928 and rs7197 were correlated with AITD and GD compared with the healthy control group, but not with HT. Rs3177928 and rs7197 were correlated with AITD and HT in the allele model, dominant model, and overdominant model before and after gender and age adjustment, but not with HT. In addition, we found that two loci (rs3177928 and rs7197) constituted a linkage disequilibrium (LD) region, and haplotype AA was associated with AITD and GD. However, we found no association between rs3129878 and AITD. Conclusion: Our study is the first to find that rs3177928 and rs7197 of HLA-DRA are significantly correlated with AITD and GD in the Chinese Han population. This will help further reveal the pathogenesis of AITD and provide new candidate genes for the prediction or treatment of AITD.