American journal of clinical and experimental immunology最新文献

Background: Hybridoma technology is an essential method used to produce monoclonal antibodies, providing equal specificity and scalability for biomedical applications. This methodology utilizes a hybridoma formation process through fusing short-lived, antibody-producing B lymphocytes with immortalized myeloma cells, thus providing hybridoma clones that produce monoclonal antibodies that are highly specific. Monoclonal antibodies produced through hybridoma technology have a consistent and reproducibility benefits over polyclonal forms of antibodies, making monoclonal antibodies an essential product in diagnostics and therapeutics. In this study, we attempted to produce monoclonal antibodies in order to target a synthetic peptide from venom of Buthotus Saulcyi, a medically important scorpion in the family Buthidae, native to Iran, known for having potent toxicity that is most dangerous in children and the elderly.

Methods: Balb/c mice were immunized with the synthetic peptide P4 before fusion to Sp2/0-Ag14 myeloma cells using polyethylene glycol at a 5:1 ratio. Hybridoma cells were cultured in HAT selective media with a single clone isolated using limiting dilution.

Result: Cell production was confirmed with an enzyme-linked immunosorbent assay (ELISA) and determined specificity to recognize B. Saulcyi venom. Neutralization was determined using MTT and SRB cell lines HepG2 and determined the monoclonal antibody treatment for B. Saulcyi venom had efficacy.

Conclusion: These findings highlight the potential of this mAb as a diagnostic tool for rapid detection of B. Saulcyi venom in clinical settings, paving the way for improved management of scorpion envenomation.

Background: DCUN1D3, a member of the DCNL (defective in cullin neddylation-like) protein family, has been implicated in ultraviolet (UV) radiation-induced cell cycle checkpoints, cell growth, survival, and neddylation. However, its specific function in male germ cells and potential involvement in spermatogenesis remain poorly understood.

Methods: To investigate the role of Dcun1d3 in male reproduction, we generated Dcun1d3 knockout (KO) mice. Sperm parameters were evaluated using computer-assisted sperm analysis (CASA), while histological and immunohistochemical analyses were performed to assess spermatogenesis.

Results: Dcun1d3-KO mice exhibited no significant differences in testicular histology, sperm quality, levels of germ cell apoptosis, or fertility outcomes compared to wild-type controls.

Conclusions: These findings indicate that Dcun1d3 is not essential for spermatogenesis or male fertility in mice. This study provides evidence to streamline future investigations by excluding Dcun1d3 as a critical regulator of male germ cell development and offers useful insights for human fertility gene research.

Background: This study aims to evaluate the diagnostic value of CD138 and CD20 immunohistochemical staining in chronic endometritis (CE).

Methods: A total of 131 patients with reproductive needs, treated at the Second Affiliated Hospital of Fujian Medical University between August 2020 and July 2021, were enrolled. Patients were divided into a CD138-positive group (n=91) and a CD138-negative group (n=40). All participants provided informed consent and underwent hysteroscopic examination using an Olympus 30° scope, following standard operating procedures. Lesion shape and size were recorded in detail. CE diagnosis was based on hysteroscopic findings of patchy bleeding and confirmed by endometrial biopsy of suspicious areas. Biopsy tissues were subjected to immunohistochemical staining for CD138 and CD20. Expression levels were assessed under low-power and high-power magnification (Original magnification: ×400. Scale bar: 20 μm).

Results: CD20 expression differed significantly between the CD138-positive and CD138-negative groups (χ²=45.440.160, P<0.05). The kappa coefficient for agreement between CD20 and CD138 was 0.530 (95% CI 2.072-4.684, P<0.05). A significant difference in CD20 expression at ×400 magnification was also observed between the two groups (χ²=12.520, P<0.05), with a kappa coefficient of 0.300(95% CI 0.594-1.164, P<0.05). The area under the ROC curve (AUC) for CD20 in predicting CE was 0.732, and 0.665 for CD20 at ×400 magnification, indicating reliable diagnostic performance for both.

Conclusions: In patients with chronic endometritis, both the positive expression of CD20 and its high-level expression at ×400 magnification provide valuable diagnostic indicators, demonstrating better diagnostic utility.

BMAL1 is a core gene involved in the regulation of circadian rhythm; however, its role in sepsis remains incompletely understood. In this study, we investigated the molecular mechanisms by which BMAL1 influences sepsis. Sepsis models were established both in vivo using C57BL/6J mice and in vitro using THP-1-derived macrophages. We observed a significant downregulation of BMAL1 expression in peritoneal macrophages and hepatic Kupffer cells during sepsis. Overexpression of BMAL1 in macrophages via plasmid transfection suppressed LPS-induced inflammatory responses and promoted M2 macrophage polarization. Conversely, administration of STL1267, a BMAL1 inhibitor, reduced BMAL1 expression in mice and further exacerbated systemic inflammation and multi-organ injury. Moreover, we identified PGC-1α as a key downstream effector of BMAL1. Knockdown of PGC-1α using short hairpin RNA (shRNA) abrogated BMAL1-mediated anti-inflammatory effects. Collectively, these findings uncover a novel mechanism by which BMAL1 regulates acute inflammatory responses and organ damage in sepsis, highlighting its potential as a therapeutic target.

In the intensive care unit (ICU), patients often experience restricted mobility due to their critical condition, potentially leading to negative effects on both muscle strength and immune function. Previous research has highlighted the beneficial effects of early mobilization among patients, regardless of mechanical ventilation status. Hence, early bed cycling serves as a potential facilitator for early mobilization and is considered a feasible intervention for critically ill patients within the ICU. To mitigate this concern, we propose a randomized controlled clinical trial aiming to assess the efficacy of early bed cycling for patients undergoing mechanical ventilation and analgosedation. The study will encompass 56 participants randomly assigned to either the treatment or control group, each consisting of 28 patients. Participants in both groups will receive health education. However, the control group will not receive any therapeutic intervention throughout the study. In contrast, the experimental group will undergo passive bed cycling of their lower extremities for 20 minutes at a rate of 30 revolutions per minute. Primary outcomes will focus on changes in the rectus femoris muscle area and thickness, evaluated using ultrasound, interleukin-6 (IL-6), IL-10, and nitric oxide (NO) production function. Secondary endpoints will encompass the modified Barthel index score, Medical Research Council total score at 1, 2, and 4 weeks following the final treatment session, participants' mechanical ventilation duration, rate of extubation in the second week, 28-day survival rate, and occurrence of adverse reactions. Any encountered side effects will be duly documented. Statistical analysis will be employed to compare patient outcomes between the treatment and control groups.

Mistletoe extracts contain the ribosome inactivating protein viscumin, which exhibits effectiveness in alternative therapies but also presents considerable toxicity risks. Hence, specific and sensitive diagnostics for identifying viscumin exposure should be developed. This study aimed to develop monoclonal antibodies (mAbs) to viscumin and to test their protective capacity against its cytotoxic effects. A peptide epitope, representing A-chain of viscumin of 9 amino acids, was synthesized and conjugated to Bovine Serum Albumin (BSA) for the immunization of BALB/c mice. Spleen cells from immunized mice were fused with SP2/0 myeloma cells to obtain hybridomas. The generated mAbs for viscumin were selected through ELISA and further characterized. The cytotoxicity of mistletoe extract against Hep-G2 cells was conducted with the SRB assay, which revealed a reduction in cell viability, respectively: about 80% at 2.5 μg/mL, 64% at 5 μg/mL, and 46% at 10 μg/mL. Interestingly, it was observed that the mAbs significantly mitigated the cytotoxic activity of viscumin, causing the viability of about 86% at all tested concentrations. Hence, they showed potential for mAbs in developing sensitive diagnostic assays and therapeutic strategies to counteract the toxic effects of viscumin. Further mAb variants' characterization, epitope mapping, and determination of the affinity should be conducted to improve both diagnostic and therapeutic avenues of viscumin-induced toxicity.

Background: Systemic lupus erythematosus (SLE) and dilated cardiomyopathy (DCM) are closely linked biologically, especially regarding immune responses. However, key biomarkers mediating the onset and development of both diseases are still lacking. This study uses bioinformatic methods to analyse the immune microenvironment of the ventricles of DCM patients and to search for biomarkers related to DCM and SLE.

Methods: Single-cell and bulk transcriptomic data for DCM were obtained from the GEO database, while GWAS data for SLE were obtained from the FinnGen database. The SMR method was used to identify genetic variants in the ventricles associated with SLE. Differential analysis was used to detect genes specific to monocyte-macrophages. Subsequently, a combination of machine learning algorithms was employed to select hub genes. Finally, small molecule drugs targeting the hub genes were retrieved from the DGIdb database.

Results: Mononuclear macrophages were found to be significantly infiltrated in dilated cardiomyopathy (DCM) samples. Seven key genes (HLA-DQB1, CD52, FCER1A, etc.) were identified by cross-tabulation analysis, of which FCER1A was the best-performing (AUC 0.8-0.9) among ten machine learning models. Validation of multiple datasets showed that FCER1A was highly expressed in the DCM group, was mainly involved in the immune cell activation pathway, and strongly interacted with other cells in the myocardial microenvironment through the MK/PROS pathway. The gene was highly expressed in the middle and late stages of monocyte-macrophage differentiation and was associated with drugs such as benzathine penicillin polylysine and omalizumab.

Conclusion: FCER1A was found to be a key differentially expressed gene in mononuclear macrophages in DCM myocardial tissue, and its significantly high expression was closely associated with immune cell activation in the myocardial microenvironment, which lays a theoretical foundation for immunotherapy of DCM and requires further clinical validation.

Objective: This study aimed to utilize single-cell RNA sequencing (scRNA-seq) to elucidate the autophagic landscape in breast cancer and to develop a prognostic model for breast cancer patients based on traditional high-throughput RNA sequencing (bulk RNA-seq).

Methods: We analyzed scRNA-seq data from the GSE75688 dataset to explore the expression patterns of autophagy-related genes (ARGs) across distinct cellular clusters. ARGs were retrieved from the GeneCards database, and bulk RNA-seq data were obtained from The Cancer Genome Atlas (TCGA). Cox proportional hazards regression was employed to construct a prognostic risk model based on ARGs. Patients were subsequently stratified into high-risk and low-risk groups according to their risk scores. For external validation, we used gene expression data from the GSE20685 and GSE48390 datasets. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the performance of the 3-gene signature.

Results: Using the FindClusters function in Seurat, all cells were grouped into four distinct clusters, highlighting the intratumoral heterogeneity within the samples. Significant differences in autophagy scores were observed among the clusters. Fifteen differentially expressed autophagy-related genes were identified, and a prognostic signature consisting of three autophagy-related genes - FEZ1, STX11, and ADAMTSL1 - was developed. Based on this model, patients were classified into high- and low-risk groups, with a statistically significant difference in survival between the two groups (log-rank test, P = 0.0011). The model demonstrated robust predictive performance with an AUC of 0.761 in the external validation dataset. A nomogram incorporating the 3-gene signature and clinical factors showed strong prognostic discrimination.

Conclusion: This study uncovered significant variation in autophagy levels among different breast cancer cell clusters. Furthermore, we established a novel 3-gene autophagy-related prognostic model that effectively stratifies patient risk and provides a potential tool for personalized prognosis in breast cancer.

Objective: The PYGO2 gene plays a significant role in various cancers. However, its prognostic significance and involvement in immune infiltration in liver cancer remain unclear. This study aimed to comprehensively evaluate PYGO2 expression and its associations with prognosis and clinicopathological features in liver cancer.

Methods: Data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases were analyzed. Functional enrichment analysis and immune cell infiltration assessments were performed to explore potential pathogenic mechanisms.

Results: PYGO2 was highly expressed in multiple cancer types, including bladder urothelial carcinoma, breast invasive carcinoma, cholangiocarcinoma, diffuse large B-cell lymphoma, and liver cancer. Analysis of 50 paired liver cancer tissues from TCGA revealed significant upregulation of PYGO2 expression. Moreover, high PYGO2 expression was significantly associated with pathological T stage, overall pathological stage, tumor status, and race. Kaplan-Meier survival analysis showed that low PYGO2 expression correlated with improved overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) in liver cancer patients. Functional enrichment analysis identified several enriched pathways, including the reactive oxygen species signaling pathway, MYC targets, interferon-alpha response, immune response regulation signaling pathway, and leukocyte migration. Additionally, PYGO2 overexpression was associated with lower proportions of cytotoxic cells, dendritic cells, immature dendritic cells, mast cells, neutrophils, plasmacytoid dendritic cell-like cells, Th17 cells, and regulatory T cells, but a higher proportion of Th2 cells. Furthermore, the high PYGO2 expression group exhibited increased immune checkpoint gene expression, particularly PDCD1.

Conclusion: PYGO2 is a promising prognostic biomarker for liver cancer, given its strong associations with clinicopathological features, survival outcomes, and immune-related characteristics.