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关键词： antigen presentation   antigen processing   endoplasmic reticulum aminopeptidase   MHC class I   ovalbumin   peptide trimming  

摘要： Peptide presentation by major histocompatibility complex (MHC) class I molecules enables CD8+ T lymphocytes to monitor the intracellular proteome of tissue cells. CD8+ T cell priming and acquisition of effector functions is affected by cognate peptide-MHC-I complex density on the cell surface, which partly depends on the efficacy of intracellular proteolytic peptide generation. Peptide generation frequently requires final trimming by the human aminopeptidases ERAP1, ERAP2, and IRAP. All display genetic polymorphism associated with the risk of multiple autoimmune diseases but also some cancers. This finding has prompted interest in the development of small molecule inhibitors to enhance antitumor or conversely attenuate autoreactive T cell responses. However, efficient assays for assessment of inhibitor effects are wanting. We describe the development of an assay for quantitative assessment by flow cytometry of selective inhibitor effects on peptide trimming both in the endogenous MHC-I processing pathway and in cross-presentation. We use the assay to identify a selective ERAP2 inhibitor and show that inhibitor effects can be read out not only through assessing a specific peptide-MHC complexes but also by measuring cell surface levels of bulk MHC-I molecules. Next to its practical interest as tool for inhibitor testing, our assay highlights how ERAP1-dependent immunodominance of a single epitope processed with exceptional efficacy can have a massive effect on the immunopeptidomic identity of cells presented to CD8+ T cells. We propose that ERAP effects on the presentation of such rare and exceptionally immunodominant epitopes may underlie the epistatic genetic associations of ERAP polymorphism with HLA class I-linked autoimmune diseases.

关键词： Adaptive immunity   Cytokines and Chemokines  

关键词： Ovarian Cancer   Transcriptomics   Single-Cell Sequencing   Prognostic Model   Gene Expression  

摘要： Ovarian cancer represents a malignancy characterized by high incidence and mortality rates, necessitating further elucidation of its underlying mechanisms. We conducted an analysis using bulk transcriptomic data of ovarian cancer and normal ovarian tissues, as well as single-cell sequencing data according to publicly available databases. Through calculation of Gene Set Variation Analysis (GSVA) scores for TNF family genes, weighted gene co-expression network analysis (WGCNA) for hub genes identification, and subsequent Gene Ontology (GO) enrichment analysis, we delineated pathways crucial in ovarian cancer pathogenesis. Furthermore, differential expression gene analysis facilitated the identification of genes with pronounced expression levels in tumor tissues and their intersection with hub genes, followed by GO analyses across molecular functions (MF), cellular components (CC), and biological processes (BP). Utilizing multivariable Cox regression and LASSO analyses, we constructed a prognostic model comprising 14 genes (GFPT2, PDE4B, PODNL1, TGFBI, CSF1R, PTGIS, SFRP2, COL5A2, TRAC, SLAMF7, VCAN, GBP1P1, C2, TRBV28). Both training and validation sets demonstrated robust diagnostic and prognostic capabilities. Clinical information and immune cell infiltration analyses were further conducted based on the model. In the single-cell sequencing analysis, reducing dimensional complexity and classifying cell types were performed, followed by exploration of gene expression patterns within each subtype and investigation of temporal expression variations across cell subtypes. Biological functional exploration and drug sensitivity analyses were also conducted. Our study contributes novel insights and theoretical foundations for prognosis, treatment, and development of drugs in patients.

关键词： Clinical Trial   Randomised controlled trial   Tapering   Remission   Glucocorticoids  

关键词： Photobiomodulation   Spinal cord injury   Autophagy   Inflammation   TLR2  

摘要： After spinal cord injury (SCI), peripherally derived macrophages infiltrated the injury area to exert inflammatory effects, causing barriers to the repair of spinal cord injury. Our previous study confirmed that photobiomodulation (PBM) could promote the motor function recovery and inhibit the secretion of inflammatory cytokines after SCI, moreover, PBM also has a role in promoting autophagy, but the mechanism is not clear. Therefore, we aimed to investigate whether PBM promotes autophagy by regulating the inflammatory response of macrophages, which in turn regulates functional repair after SCI. Male C57/BL6 mice were used to prepare a model of clamped spinal cord injury, and PBM irradiation was performed for 28 consecutive days, which showed that motor function of the mice was improved. We observed that autophagy proteins (LC3, Beclin-1 and P62) were inhibited and inflammasome-related proteins (NLRP3, Caspase-1 and IL-1 beta) expression was significantly enhanced in SCI mice. We analyzed the RNA sequencing (RNA-seq) of SCI, SCI+PBM treated mice in combination with autophagy database. The results showed 25 differentially expressed genes (DEGs). Protein-protein interaction (PPI) network and Hub gene analysis revealed TLR2 as a key molecule in the regulation of autophagy levels by PBM after SCI. We performed preliminary analysis in macrophages cultured in vitro and observed that PBM suppressed the expression of TLR2 and inflammasome-related proteins in M1-type macrophages and promoted the expression of autophagy proteins. Subsequently, we used an agonist of TLR2 (CU-T12-9) to up regulate TLR2 expression and observed that macrophage autophagy was inhibited and inflammatory response was enhanced. After PBM irradiation, the effect of CU-T12-9 was counteracted. Taken together, PBM promotes autophagy and attenuates the inflammatory response by regulating TLR2, a key molecule of autophagy in spinal cord injury.

关键词： TNF-alpha antagonist   Diabetic rat colon   Volume of colonic layers   Number of myenteric neurons   Death commitment points   Stereology   Physical disector  

摘要： Background: this study aimed to examine the impact of a TNF-alpha antagonist on markers of neuronal death, the total number of myenteric neurons, and the volume of each colonic layer in diabetic rats. Methods: thirty male rats were divided into normal control (NC), diabetic control (DC), and diabetic etanercept (DE) groups. Diabetes was induced using streptozotocin and nicotinamide. The DE group received twice-weekly injections of etanercept. Immunohistochemistry staining and western blotting detected activated caspase-3, Gasdermin D, and phosphorylated-MLKL. Stereological methods were performed in colon sections stained with Toluidine-blue to estimate the total number of neurons in the colonic myenteric plexus and the colonic layer volume. Results: immunohistochemistry showed etanercept reduced expression of apoptosis and pyroptosis markers, but not necroptosis, in the myenteric plexus of the colon in diabetic rats. Diabetic rats exhibited a lower total number of neurons in the colonic myenteric ganglions, and etanercept did not prevent this neuronal loss. Furthermore, colonic layer enlargement was observed in diabetic rats, with only the muscular layer hypertrophy being prevented by etanercept administration. Conclusions: the TNF-alpha antagonist did not prevent neuronal loss in the colonic myenteric plexus;it partially inhibited colonic muscular layer enlargement in diabetic rats.

关键词： biosimilar   CHO-K1   IL-6   IL-6R alpha   lentivirus   mAb   Tocilizumab  

摘要： Tocilizumab is a monoclonal antibody (mAb) that recognizes human Interleukin 6 receptor alpha (IL-6R alpha) and antagonizes IL-6 signaling. It is therefore used in the treatment of moderate to severe rheumatoid arthritis in patients with inadequate response to antirheumatic drugs, and in the treatment of systemic juvenile idiopathic arthritis. During the course of the Coronavirus Disease 2019 (COVID-19) pandemic, the potential application of Tocilizumab in severe inflammatory diseases was demonstrated. In addition, the drug is attracting increasing attention for its potential application in other pathologies, particularly cancers where the central role of Interleukin 6 (IL-6) has been described, such as pancreatic ductal adenocarcinoma. The necessity for further research in this area is far too great, which is why the development of additional biosimilars is both necessary and welcome. In this work, we describe the generation and biological characterization of a biosimilar candidate of Tocilizumab. A stable producing CHO-K1 cell line was obtained by lentiviral transduction. We demonstrated that the antibody generated, named 41TB, has similar size heterogeneity and electrophoretic profile as Tocilizumab, and high levels of purity. Moreover, 41TB exhibits comparable binding capacity to IL-6R alpha, as well as the ability to interrupt IL-6/IL-6R alpha binding in ELISA. Additionally, both antibodies were likewise able to inhibit DS-1 cell proliferation, IL-6 induced STAT3 phosphorylation, and C-Reactive Protein production in vitro. Galactose supplementation of the culture medium appears to be necessary to generate a glycosylation profile similar to the original antibody.

关键词： hPSC   HLA   gene editing   immune checkpoint  

摘要： Human pluripotent stem cells (hPSCs) hold great promise in regenerative medicine. However, immune rejections remain one of the major obstacles to stem cell therapy. Though conventional immunosuppressants are available in clinics, the side effects prevent the wide application of hPSCs derivatives, compromising both survival rate and quality of life. In recent years, a myriad of strategies aimed at inducing immune tolerance specifically by engineering stem cells has been introduced to society. One strategy involves human leukocyte antigen (HLA) deletion through gene editing, affording allografts the capability to evade the host immune system. Another strategy involves immune cloak, which is the focus of this review, with emphasis on the overexpression of immune checkpoints and the blocking of immune cytotoxic pathways. Nevertheless, co-transplantation with mesenchymal stem cells (MSCs) and enhanced MSCs confers immune privilege to engraftments. This review summarizes recent studies on the intricacies of immune tolerance induction by engineering stem cells. In addition, we endeavor to deliberate upon the safety and limitations associated with this promising and potential therapeutic modality.

关键词： activation   CD8(+) T cell   experimental cerebral malaria   heterogeneity   microglia   recruitment  

摘要： Aims: This study aimed to investigate the heterogeneity of microglia and their role in recruiting and activating CD8(+) T cells in experimental cerebral malaria (ECM). Methods: C57BL/6J mice were infected with Plasmodium berghei ANKA (PbA) to induce ECM. Morphology and distribution of microglia were assessed via immunofluorescence (IF) staining and electron microscopy. Single-cell RNA-sequencing (scRNA-seq) analyzed the activation characteristics of infiltrating CD8(+) T cells and the transcriptional heterogeneity of microglia in ECM. In vitro, ECM-associated microglia were induced by TNF alpha, IFN gamma, and parasite-infected red blood cells (pRBCs). The interaction between microglia and CD8(+) T cells was explored in co-culture systems through transwell assay, adhesion assay, and cytotoxicity test. Results: In vivo, microglia were aggregated in the brainstem and olfactory bulb in the ECM brain, regions that exhibited more severe pathological injury. The transcriptional characteristics of ECM microglia distinguished from physiological microglia and exhibit morphological heterogeneity in the ECM brain. Infiltrating CD8(+) T cells in the ECM brainstem exhibit sustained activation characteristics associated with microglia interaction. Based on subcluster analysis, a unique subtype of ECM-associated microglia was identified, characterized by CXCL10(high)TNF alpha(high)Ki67(+). These microglia mediated the recruitment and sustained activation of CD8(+) T cells through persistent interactions in co-culture systems. Conclusions: Our study identified an ECM-associated microglia subtype and explored its interaction with CD8(+) T cells, which deepened the understanding of the multifaceted role of microglia in the pathogenesis of CM neuroinflammation.