Combining Bulk and Single Cell RNA-Sequencing Data to Identify Hub Genes of Fibroblasts in Dilated Cardiomyopathy.

Background: Dilated cardiomyopathy (DCM) is the second leading cause of heart failure, with intricate pathophysiological underpinnings. In order to shed fresh light on the mechanistic research of DCM, we combined bulk RNA-seq and single-cell RNA-seq (scRNA-seq) data to examine significant cells and genes implicated in the disease. Methods: This analysis employed publicly accessible bulk RNA-seq and scRNA-seq DCM datasets. The scRNA-seq data underwent normalization, principal component, and t-distribution stochastic neighbor embedding analysis. Cell-to-cell communication networks and activity analysis were conducted using CellChat. Utilizing enrichment analysis, the marker genes' role in the active cells was evaluated. After screening by limma software and weighted gene co-expression network analysis, the differentially expressed genes (DEGs) served as hub genes. Furthermore, these hub genes were subjected to immunological studies, transcription factor expression, and gene set enrichment. Lastly, the expression of the four hub genes and their connection to DCM were verified using the rat models. Results: Fibroblasts and monocytes were chosen as hub cells from among the eight identified cell clusters; their marker genes intersected with DEGs to yield six hub genes. In addition, the six hub genes and the essential module genes intersected to yield four essential genes (ASPN, SFRP4, LUM, and FRZB) that were connected to the Wnt signaling pathway and highly expressed in fibroblast. The four hub DEGs had an expression pattern in the DCM rat model experiment results that was in line with the findings of the bioinformatics study. Additionally, there was a strong correlation between decreased cardiac function and the up-regulation of ASPN, SFRP4, LUM, and FRZB. Conclusion: Ultimately, bulk RNA-seq and scRNA-seq data identified fibroblasts and monocytes as the main cell types implicated in DCM. The highly expressed genes ASPN, FRZB, LUM, and SFRP4 in fibroblasts may aid in the mechanistic investigation of DCM.

Polyhedral magnetic nanoparticles induce apoptosis in gastric cancer stem cells and suppressing tumor growth through magnetic force generation.

Gastric cancer is a prevalent malignant tumor worldwide, posing challenges due to its poor prognosis and limited treatment options. Cancer stem cells (CSCs) were demonstrated as a subset of cancer cells responsible for tumor initiation and progression, and their inherent resistance to conventional chemotherapy and radiotherapy critically contributes to tumor recurrence and metastasis. Promoting the eradication of cancer stem cells is crucial for enhancing the efficacy of cancer treatments. This study introduces a novel therapeutic strategy utilizing polyhedral magnetic nanoparticles (PMNPs) functionalized with CD44 antibodies and cell-penetrating peptides (CPPs) to improve uptake by gastric cancer stem cells (MCSCs). PMNPs, synthesized via thermal decomposition, exhibited a diameter of 90 nm ± 9 nm and a saturation magnetization of 79.9 emu/g. Functionalization enhanced their uptake capabilities. Under a rotating magnetic field (RMF) of 15 Hz, PMNPs disrupted cellular structure, leading to apoptosis and ferroptosis in MCSCs. The in vitro studies showed significant reduction in MCSCs viability, while in vivo studies demonstrated tumor growth suppression with minimal side effects and high biocompatibility. This work presents a novel strategy for designing magnetic nanoparticles to mechanically destroy cancer stem cells, offering a more efficient and safety treatment option for gastric cancer.

Identifying the mechanism of polysaccharopeptide against breast cancer based on network pharmacology and experimental verification.

Polysaccharopeptide (PSP) is a potential active component in traditional Chinese medicine because of its anticancer effects on a variety of cancer cells and as immune enhancers of the immune system. Previous studies on the role of PSP in breast cancer have been limited, and the mechanism has not been clarified. This study is based on network pharmacology and molecular docking technology to predict the possible target of PSP treatment of breast cancer, and use experiments to verify the effect and mechanism of PSP on breast cancer. In this study, 287 PSP targets were obtained using SwissTargetPrediction database and PharmMapper database, and 183 breast cancer targets were obtained using DisGenNET database. By intersections of PSP targets and breast cancer targets, a total of 10 intersections were obtained. GO functional enrichment, KEGG pathway enrichment and molecular docking of these 10 target genes were performed to obtain the potential targets of PSP on breast cancer. In vitro experiments, we found that PSP significantly inhibited the proliferation and induced apoptosis of breast cancer cell lines MDA-MB-231, SUM-159 and MCF-7. Western Blot results showed that PSP could down-regulate the expression of p-JAK2 and p-STAT3 proteins. Similarly, the results of in vivo experiments showed that PSP can directly inhibit the tumor of MDA-MB-231 tumor-bearing mice, and the mechanism of action is mainly to inhibit the JAK2-STAT3 pathway. The above results were consistent with the results of network pharmacology, which provides a scientific basis for the clinical application of PSP in breast cancer patients.

A murine monoclonal antibody against H5N1 avian influenza virus cross-reacts with human kidney cortex cells.

To investigate the biological characteristics of monoclonal antibodies (mAbs) against avian influenza virus (AIV) and the possible mechanism of AIV-related kidney injury. BALB/c mice were immunized with inactivated H5N1 AIV to prepare monoclonal antibody H5-32, and its subtype, titer and cross-reactivity with other influenza viruses were identified. The reactivity of monoclonal antibody with normal human tissue was analyzed by immunohistochemistry. Immunofluorescence and confocal laser scanning technique were used to detect the binding sites between mAb and human renal cortical cells, and Western blotting was used to detect the size of binding fragments. Immunohistochemical analysis confirmed that monoclonal antibody H5-32 cross-reacted with normal human kidney tissue. In human kidney, mAb H5-32 was localized in the cytoplasm of human renal tubular epithelial cells, and its binding fragment size was about 43 kDa. H5N1 AIV appears to bind to human renal tubular epithelial cells, which may be one of the mechanisms of kidney injury caused by AIV infection.

The distribution of heterophilic antigens and their relationship with autoimmune diseases.

Introduction: Microbial infections are associated with the occurrence of autoimmune diseases, but the mechanisms of microbial infection inducing autoimmune diseases are not fully understood. The existence of heterophilic antigens between microorganisms and human tissues may explain part of the pathogenesis of autoimmune diseases. Here, we investigate the distribution of heterophilic antigens and its relationship with autoimmune diseases. Methods: Monoclonal antibodies against a variety of microorganisms were prepared. The titer, subclass and reactivity of antibodies with microorganisms were identified, and heterophilic antibodies that cross-reacted with human tissues were screened by human tissue microarray. The reactivity of these heterophilic antibodies with different individuals and different species was further examined by immunohistochemistry. Results: In this study, 21 strains of heterophilic antibodies were screened. The results showed that these heterophilic antibodies were produced due to the existence of heterophilic antigens between microorganism and human body and the distribution of heterophilic antigens had individual, tissue and species differences. Conclusion: Our study showed that heterophilic antigens exist widely between microorganisms and human body, and the heterophilic antigens carried by microorganisms may break the immune tolerance of the body through carrier effect and initiate immune response, which may be one of the important mechanisms of infection inducing autoimmune diseases.

Naringenin Impedes the Differentiation of Mouse Hematopoietic Stem Cells Derived from Bone Marrow into Mature Dendritic Cells, thereby Prolonging Allograft Survival.

BACKGROUND: The use of immature dendritic cells (imDCs) to induce donor-specific immunotolerance following in vivo stimulation is limited by their low rate of induction and their tendency to undergo maturation. We derived imDCs from bone marrow hematopoietic stem cells (HSCs-imDCs). We then tested the ability of naringenin (Nar) to impede the maturation of HSCs-imDCs for inducing transplantation immune tolerance. METHODS: HSCs derived from bone marrow were collected and induced to differentiate into imDCs by treating with Nar (Nar-HSCs-imDCs). Flow cytometry was used to evaluate DC surface markers, apoptosis, and endocytic ability. The ability of DCs to influence the in vitro proliferation of T cells and of regulatory T cells (Tregs) was analyzed by mixed lymphocyte reaction assays. Enzyme-linked immunoassays were used to quantify cytokine levels in supernatants from co-cultured DCs and Tregs, as well as in the serum of experimental animals. The level of immunotolerance induced by Nar-HSCs-imDCs was evaluated by skin grafting in recipient Balb/c mice, while the Kaplan-Meier method was used to statistically evaluate graft survival. RESULTS: Compared with HSC-imDCs, Nar-HSCs-imDCs showed higher expression of cluster of differentiation 11c (CD11c), but lower expression levels of CD80, CD86, and major histocompatibility complex class II. Nar-HSCs-imDCs also showed stronger inhibition of T cells and higher Treg cell proliferation. Interleukin 2 (IL-2) and interferon gamma levels were downregulated in Nar-HSCs-imDCs, whereas IL-4, IL-10, and transforming growth factor beta levels were upregulated. The rate of apoptosis and endocytic capacity of Nar-HSCs-DCs increased significantly after treatment with lipopolysaccharide. HSCs-imDCs or Nar-HSCs-imDCs were injected into Balb/c mice via the tail vein 7 days before skin grafting. Significantly reduced donor-specific CD4+ T cells and induced proliferation of CD4+CD25+FoxP3+ Treg cells were observed in the spleen of mice from the Nar-HSCs-imDCs group, especially at a dose of 106 Nar-HSCs-imDCs. The latter group also showed significantly prolonged survival of skin grafts. CONCLUSIONS: Nar-HSCs-imDCs markedly improved the acceptance of organ allografts, offering a potentially new strategy for inducing immune tolerance in transplantation.

Identification and analysis of B cell epitopes of hemagglutinin of H1N1 influenza virus.

The frequent variation of influenza virus hemagglutinin (HA) antigen is the main cause of influenza pandemic. Therefore, the study of B cell epitopes of HA is of great significance in the prevention and control of influenza virus. In this study, the split vaccine of 2009 H1N1 influenza virus was used as immunogen, and the monoclonal antibodies (mAbs) were prepared by conventional hybridoma fusion and screening techniques. The characteristics of mAbs were identified by ELISA method, Western-blot test and hemagglutination inhibition test (HI). Using the obtained mAbs as a tool, the B cell epitopes of HA were predicted by ELISA blocking test, sandwich ELISA method and computer simulation method. Finally, four mAbs against HA antigen of H1N1 influenza virus were obtained. The results of ELISA and computer prediction showed that there were at least two types of epitopes on HA of influenza virus. The results of this study complemented the existing methods for predicting HA epitopes, and also provided a new method for predicting other pathogenic microorganisms.

Sinomenine promotes differentiation of induced pluripotent stem cells into immature dendritic cells with high induction of immune tolerance.

BACKGROUND: Immature dendritic cells (imDCs) play an important role in the induction of donor-specific transplant immunotolerance. However, these cells have limitations, such as rapid maturation and a short lifespan in vivo. In previous studies, induced pluripotent stem cells (iPSCs) differentiated into imDCs, and sinomenine (SN) was used to inhibit the maturation of imDCs. AIM: To study the capacity of SN to maintain iPSC-derived imDCs (SN-iPSCs-imDCs) in an immature state and the mechanism by which SN-iPSCs-imDCs induce immunotolerance. METHODS: In this study, mouse iPSCs were induced to differentiate into imDCs in culture medium without or with SN (iPSCs-imDCs and SN-iPSCs-imDCs). The imDC-related surface markers, endocytotic capacity of fluorescein isothiocyanate-Dextran and apoptosis were analyzed by flow cytometry. The effects of iPSCs-imDCs and SN-iPSCs-imDCs on T-cell stimulatory function, and regulatory T (Treg) cell proliferative function in vitro were analyzed by mixed lymphocyte reaction. Cytokine expression was detected by ELISA. The apoptosis-related proteins of iPSCs-DCs and SN-iPSCs-DCs were analyzed by western blotting. The induced immunotolerance of SN-iPSCs-DCs was evaluated by treating recipient Balb/c skin graft mice. Statistical evaluation of graft survival was performed using Kaplan-Meier curves. RESULTS: Both iPSCs-imDCs and SN-iPSCs-imDCs were successfully obtained, and their biological characteristics and ability to induce immunotolerance were compared. SN-iPSCs-imDCs exhibited higher CD11c levels and lower CD80 and CD86 levels compared with iPSCs-imDCs. Reduced major histocompatibility complex II expression, worse T-cell stimulatory function, higher Treg cell proliferative function and stronger endocytotic capacity were observed with SN-iPSCs-imDCs (P < 0.05). The levels of interleukin (IL)-2, IL-12, interferon-γ in SN-iPSCs-imDCs were lower than those in iPSCs-imDCs, whereas IL-10 and transforming growth factor-ß levels were higher (P < 0.05). The apoptosis rate of these cells was significantly higher (P < 0.05), and the expression levels of cleaved caspase3, Bax and cleaved poly(ADP-ribose) polymerase were higher after treatment with lipopolysaccharides, but Bcl-2 was reduced. In Balb/c mice recipients immunized with iPSCs-imDCs or SN-iPSCs-imDCs 7 d before skin grafting, the SN-iPSCs-imDCs group showed lower ability to inhibit donor-specific CD4+ T-cell proliferation (P < 0.05) and a higher capacity to induce CD4+CD25+FoxP3+ Treg cell proliferation in the spleen (P < 0.05). The survival span of C57bl/6 skin grafts was significantly prolonged in immunized Balb/c recipients with a donor-specific pattern. CONCLUSION: This study demonstrated that SN-iPSCs-imDCs have potential applications in vitro and in vivo for induction of immunotolerance following organ transplantation.

Monoclonal Antibody Targeting the HA191/199 Region of H1N1 Influenza Virus Mediates the Damage of Neural Cells.

Vaccination is the most effective mean of preventing influenza virus infections. However, vaccination-induced adverse reactions of the nervous system, the causes of which are unknown, lead to concerns on the safety of influenza A vaccine. In this study, we used flow cytometry, cell ELISA, and immunofluorescence to find that H1-84 monoclonal antibody (mAb) against the191/199 region of the H1N1 influenza virus hemagglutinin (HA) protein binds to neural cells and mediates cell damage. Using molecular simulation software, such as PyMOL and PDB viewer, we demonstrated that the HA191/199 region maintains the overall structure of the HA head. Since the HA191/199 region cannot be removed from the HA structure, it has to be altered via introducing point mutations by site-directed mutagenesis. This will provide an innovative theoretical support for the subsequent modification the influenza A vaccine for increasing its safety.

Biomechanical changes of freezer-storaged and decellularized pig tracheal scaffoldings.

BACKGROUND: As an excellent xenotransplant, the pig trachea can be decellularized and cryopreserved to reduce its immunogenicity. However, few reports are found on the changes of its mechanical properties after cryopreservation and decellularization. OBJECTIVE: To evaluate the structure and biomechanical properties in pig tracheal scaffolds resulting from decellularized and cryopreserved. MATERIAL AND METHODS: Twenty-five pig tracheal segments were separated into five groups: untreated (group A), only decellularized (group B), only cryopreserved (group C), decellularized after cryopreserved (group D) and cryopreserved after decellularized (group E). Tracheal segments were subjected to uniaxial tension or compression using a universal testing machine to determine structural biomechanical changes. RESULTS: It showed that there was no statistically significant difference in the tensile strength of the trachea in each group. The compressive strength of group B, C and D were same as the group A (P > 0.05), while the group E was lower than that of the group A (P < 0.05).Conclusions and significance: The histological examination of the decellularization after cryopreservation shows that the removal of epithelial cells and submucosal cells is more thorough, and the biomechanical structure of the trachea is better preserved. This proved to be a new method to prepare xenotransplantation of trachea graft.

Monoclonal antibody against H1N1 influenza virus hemagglutinin cross reacts with hnRNPA1 and hnRNPA2/B1.

Following influenza A vaccination, certain individuals exhibit adverse reactions in the nervous system, which causes a problem with the safety of the influenza A vaccine. However, to the best of our knowledge, the underlying mechanism of this is unknown. The present study revealed that a monoclonal antibody (H1­84mAb) against the H1N1 influenza virus hemagglutinin (HA) protein cross­reacted with an antigen from brain tissue. Total brain tissue protein was immunoprecipitated with this cross­reactive antibody, and mass spectrometry revealed that the bound antigens were heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and hnRNPA2/B1. Subsequently, the two proteins were expressed in bacteria and it was demonstrated that H1­84mAb bound to hnRNPA1 and hnRNPA2/B1. These two proteins were expressed in three segments and the cross­reactivity of H1­84mAb with the glycine (Gly)­rich domains of hnRNPA1 (195aa­320aa) and hnRNPA2/B1 (202aa­349aa) was determined using ELISA blocking experiments. It was concluded that the Gly­rich domains of these two proteins are heterophilic antigens that cross­react with influenza virus HA. The association between the heterophilic antigen Gly­rich domains and the safety of influenza A vaccines remains to be investigated.

Antibodies against H1N1 influenza virus hemagglutinin cross-react with prohibitin.

Influenza virus infection is associated with type 1 diabetes (T1DM), but its pathogenesis remains unclear. Here, our study found that one of the monoclonal antibodies against H1N1 influenza virus hemagglutinin(HA) cross-reacted with human pancreatic tissue and further demonstrated that it binded to rat islet ß-cells. We immunoprecipitated islet protein with this cross-reactive antibody and identified the bound antigen as prohibitin by mass spectrometry. We then expressed the prohibitin protein in bacteria and confirmed the antibody binding to prohibitin by Western blot. We also verified the cross-reactivity of the antibody by prohibitin-siRNA transfection in islet beta cells. We conclude that prohibitin is an autoantigen that cross-reacts with influenza virus HA. The correlation between the autoantigen prohibitin and type 1 diabetes remains to be investigated.

Localization Analysis of Heterophilic Antigen Epitopes of H1N1 Influenza Virus Hemagglutinin.

Previous studies have indicated that two monoclonal antibodies (mAbs; A1-10 and H1-84) of the hemagglutinin (HA) antigen on the H1N1 influenza virus cross-react with human brain tissue. It has been proposed that there are heterophilic epitopes between the HA protein and human brain tissue (Guo et al. in Immunobiology 220:941-946, 2015). However, characterisation of the two mAbs recognising the heterophilic epitope on HA has not yet been performed. In the present study, the common antigens of influenza virus HA were confirmed using indirect enzyme-linked immunosorbent assays and analysed with DNAMAN software. The epitopes were localized to nine peptides in the influenza virus HA sequence and the distribution of the peptides in the three-dimensional structure of HA was determined using PyMOL software. Key amino acids and variable sequences of the antibodies were identified using abYsis software. The results demonstrated that there were a number of common antigens among the five influenza viruses studied that were recognised by the mAbs. One of the peptides, P2 (LVLWGIHHP191-199), bound both of the mAbs and was located in the head region of HA. The key amino acids of this epitope and the variable regions in the heavy and light chain sequences of the mAbs that recognised the epitope are described. A heterophilic epitope on H1N1 influenza virus HA was also introduced. The existence of this epitope provides a novel perspective for the occurrence of nervous system diseases that could be caused by influenza virus infection, which might aid in influenza prevention and control.

Antibodies against H1N1 influenza virus cross-react with α-cells of pancreatic islets.

Epidemiological studies have documented that the incidence of human type 1 diabetes was significantly increased after H1N1 epidemic. However, a direct link between human type 1 diabetes and virus infection remains elusive. We generated 84 clones of murine monoclonal antibodies against the H1N1, and carried out immunohistochemistry in normal human tissue microarray. The results showed that two clones specifically cross-reacted with human α-cells of pancreatic islets. Reverse transcription polymerase chain reaction and deoxyribonucleic acid sequencing showed that the amino acid sequences of light and heavy chains of these clones were different. Importantly, the expression profiles of two monoclonal antibodies were individual different. For the first time, we provide direct evidence that monoclonal antibodies against H1N1 can cross-react with human pancreas α-cells, another source of ß-cells, suggesting α-cells might be a novel target to be investigated in diabetes research.