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摘要： Background:Malignant gliomas (MG) are the most common primary brain malignancies and are considered universally fatal. Oncolytic HSVs (oHSV) are promising immunotherapeutics capable of selectively lysing cancer cells, eliciting anti-tumor immunity, and providing local delivery of immune-activating transgenes. IL-27 is a pleiotropic cytokine capable of enhancing tumor-reactive cytotoxic T cell (CTL) function while also possessing neuroprotective properties. We hypothesized that IL-27 expression by oHSV would enhance CTL function and improve anti-glioma therapeutic activity. Methods:We developed an oncolytic herpes simplex virus (oHSV) that expresses IL-27 (C027). The anti-glioma efficacy of C027 was tested in three syngeneic orthotopic glioma models derived from both chemical (CT-2A) and genetic (SB28, KR158) glioma lines. Spectral flow cytometry was used to assess immunophenotypic and functional changes in the tumor infiltrates and systemically. To further investigate the C027-related CTL activity, we employed cell specific depletion and IL-27 blockade alongside in vitro T cell stimulation assays. Local and systemic antitumor memory was evaluated by both orthotopic and flank tumor rechallenge of C027-treated long-term survivors. Results:C027 significantly prolonged survival in syngeneic orthotopic glioma models derived from both chemical (CT-2A) and genetic (KR158, SB28) glioma lines. In the CT-2A model, IL-27-expressing oHSV treatment was associated with increased intratumoral multifunctional effector cytotoxic T lymphocytes (CTL) and functional T cell populations systemically. Mechanistically, both CD8 T cells and IL-27 were required for the C027 survival benefit and IL-27 enhanced CTL function . C027-treated mice that survived their initial tumors had local and systemic anti-glioma memory rejecting tumors on rechallenge. Conclusions:Our findings demonstrate that IL-27 expression by oHSV significantly improves anti-glioma therapeutic efficacy, enhances CTL effe

关键词： Mast cells   Organoids   Co-cultures   TNF-alpha   IL-33   Intestinal differentiation and architecture  

摘要： It is common knowledge that mast cells (MCs) exert different roles in the gastrointestinal tract, from the maintenance of homeostasis to the onset and propagation of different gut diseases such as food allergies, infections, inflammation, and cancer. However, the mechanisms through which MCs dialog and influence the intestinal tissue are not completely known. To get insight into the bidirectional crosstalk between MCs and the intestinal microenvironment, both in homeostatic and pathological settings, colon organoids from intestinal epithelium of healthy mice and adenomas from AOM/DSS-treated mice have been exploited and co-cultured with MCs. The influence of MCs on organoid architecture and the effect of healthy and tumoral organoids on the phenotype and responsiveness of MCs have been addressed. We observed that MCs interact with intestinal organoids and contribute to the differentiation of healthy organoids by upregulating the expression of mucin-2, chromogranin A, cadherin-1, and claudin 4. On the contrary, in co-culture with tumoral organoids a decrease in cell proliferation, chromogranin A, and lysozyme expression was observed. Tumoral organoids have been shown to activate MCs via the IL-33/ST2 axis leading to increased release of TNF-alpha which in turn was responsible for the observed effects on tumoral organoids. Our results indicate that MCs are important mediators of intestinal tissue homeostasis and that a different environment can shape and direct MCs toward the dampening or propagation of the inflammatory response. Ultimately, our MC-organoid co-cultures represent a valid in vitro tool to investigate the role of MCs in the gut.

关键词： Artificial antigen-presenting cell   Natural killer cell   Acute myeloid leukemia   Cord Blood NK cells   NK-92 cells  

摘要： Acute myeloid leukemia (AML) is an aggressive malignancy with limited treatment options. Enhancing natural killer (NK) cell functionality through artificial antigen-presenting cells (aAPCs) represents a promising immunotherapeutic strategy. This study evaluates the potential of genetically modified K562 cells, expressing CD137L and CD86, to enhance NK cell-mediated cytotoxicity against AML cell lines (HL-60, KG-1, and THP-1). Lentiviral transduction was used to generate aAPCs, confirmed by PCR, RT-PCR, and flow cytometry. Cord NK cells and the NK-92 cell line were co-cultured with aAPCs, and their cytotoxicity against cell lines was assessed using 7-AAD staining. The ability of transduced K562 cells to substitute for interleukin-15 (IL-15) was also evaluated. These cells significantly enhanced NK cell-mediated cytotoxicity, with greater effects observed at higher effector-to-target (E:T) ratios. The aAPCs partially replaced IL-15 in activating cord blood NK cells but were ineffective for NK-92 cells. The aAPCs effectively enhance NK cell cytotoxicity and may reduce cytokine dependence in therapeutic applications. These findings highlight the potential of aAPCs to improve NK cell-based immunotherapies.

关键词： ticks   saliva   cytokine-binding proteins   immunomodulation   Amblyomma sculptum  

摘要： The saliva of hematophagous arthropods, such as ticks and triatomines, contains bioactive ligands capable of modulating immune molecules, including cytokines. Cytokines play a critical role in immune regulation and have therapeutic relevance in inflammatory and immune-mediated diseases. Despite recent advances, identifying cytokine-binding molecules remains a significant challenge. Interferon-gamma (IFN-gamma), interleukin-4 (IL-4), and interleukin-17 (IL-17) are key cytokines involved in inflammation, adaptive immunity, and host defense. This study evaluated the ability of salivary components from Amblyomma sculptum and compared the results to the triatomine Rhodnius neglectus (used as control) to bind to IL-2, IL-4, IL-6, IL-10, IL-17, IFN-gamma, and TNF-alpha using ELISA assays with human cytokines. Saliva samples were tested at dilutions of 1:25, 1:50, and 1:100. Saliva from A. sculptum, which demonstrated significant anti-cytokine activity, was fractionated via HPLC to identify the active components. The results confirmed the inhibitory capacity of A. sculptum saliva on IFN-gamma, IL-4, and IL-17, with inhibition rates ranging from 30% to 70%, depending on the cytokine and dilution. No inhibitory activity was observed against IL-2, IL-6, IL-10, or TNF-alpha. These findings underscore the immunomodulatory role of A. sculptum saliva during tick feeding and suggest its potential for the development of novel immunobiologics to treat inflammatory and immune-mediated diseases.

摘要： The shared HLA-bound neoepitopes in pancreatic ductal adenocarcinoma (PDAC) represent a novel class of noncanonical antigens with single amino acid substitutions resulting from translational errors. These peptides, shared across patients with PDAC, showed higher immunogenicity than wild-type counterparts, offering potential candidates for specific immunotherapy development in PDAC. See related article by Zhang et al., p. 1956

摘要： Background:Patients living with Human Immunodeficiency Virus (PLWH) develop accelerated liver fibrosis, but the exact mechanism remains unknown. Hepatic stellate cell (HSC) activation is the cornerstone for liver fibrosis and influenced by multiple factors, such as viral infection, hepatocellular injury, chronic immune activation, gut mucosal barrier dysfunction, and microbial translocation. Unlike gram-negative bacterial products, the impact of gram-positive microbial products in Human Immunodeficiency Virus 1 (HIV-1) associated liver inflammation and fibrosis remains poorly understood. In this study, we investigated the effect of lipoteichoic acid (LTA), a major gram-positive bacterial component, on HSCs in the context of HIV-1 infection. Methods:Human HSCs were isolated from the livers of both non-HIV and HIV-infected patients undergoing hepatic resection. The inflammatory responses of HSCs to LTA stimulation were measured by ELISA before or after HIV-1BaL ex vivo expision. Western blotting, ChIP-qPCR and RNA-seq were used to reveal the mechanisms contributing to the IL-8 response to LTA stimulation and HIV-1BaL exposure in HSCs. Results:While LTA stimulation modestly increased IL-8 production in HSCs, this effect was significantly amplified in HIV-1-infected HSCs. Elevated IL-8 levels were detected in HIV-1+ liver sections, and IL-8 treatment of cultured HSCs significantly increased α-SMA and COL1A1 expression, supporting IL-8 as a key inflammatory driver of liver fibrosis in HIV-1 individuals. Transcriptomic analyses implicated histone acetylation as a regulator of the enhanced IL-8 response of HSCs to LTA in HIV-1 infection. Consistent with this, treatment with Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, further increased the IL-8 response in HIV-1-infected HSCs. ChIP-qPCR confirmed that histone H4K5 acetylation facilitated IL-8 promoter transactivation, rendering HIV-1-infected cells more responsive to LTA. Conclusions:HIV-1 infection sensi

关键词： Exosome   Anti-inflammation   Osteoarthritis   MicroRNA (miRNA)  

摘要： Background:This study investigated anti-inflammatory effects of exosomes derived from human fetal cartilage progenitor cells (hFCPC-Exo) and their microRNAs (miRNAs) on the osteoarthritis (OA) phenotype in vitro in comparison with exosomes from bone marrow mesenchymal stem cells (MSC-Exo).Methods:SW982 cells (synoviocytes) or hFCPCs (chondrocytes) were stimulated with 10 ng/mL IL-1 beta to mimic OA phenotypes. The effects of hFCPC-Exo and MSC-Exo were compared by measuring the expression of inflammatory cytokines and an anti-inflammatory protein. miRNA profiles of hFCPC-Exo and MSC-Exo were analyzed using a 2588 human miRNA dataset, and miRNAs potentially involved in the anti-inflammatory effect of hFCPC-Exo were selected. miRNA mimics and antisense inhibitors were used to investigate the role of selected miRNAs in the IL-1 beta signaling ***:Both hFCPC-Exo and MSC-Exo significantly decreased the expression of inflammatory cytokines (IL-1 beta, IL-6, and MCP-1), while slightly increased an anti-inflammatory protein (SOCS1) in IL-1 beta-treated SW982 cells. miRNA sequencing revealed anti-inflammatory miRNAs present in large amounts in both hFCPC-Exo and MSC-Exo. Among them, miR-125b-5p mimic significantly suppressed the expression of inflammatory cytokines induced by IL-1 beta, while anti-sense inhibitor of miR-125b-5p efficiently blocked anti-inflammatory effects of hFCPC-Exo. Both hFCPC-Exo and miR-125b-5p inhibited I kappa B alpha down-regulation and -NF-kappa B stabilization in IL-1 beta-treated SW982 cells. Additionally, hFCPC-Exo and miR-125b-5p showed similar effects on IL-1 beta-treated hFCPCs as an OA model in chondrocytes by down-regulating the expression of IL-1 beta, MMP13, and ADAMTS-5 and up-regulating the expression of aggrecan (ACAN) and type II collagen (COL2A1).Conclusion:This study demonstrated that hFCPC-Exo exhibits anti-inflammatory effects on IL-1 beta-treated synoviocytes and chondrocytes in vitro possibly by down-regulating the I

关键词： Vasorin(VASN)   树鼩   单克隆抗体  

摘要： 目的 通过表达和纯化树鼩Vasorin（VASN）重组蛋白，免疫小鼠制备抗树鼩VASN单克隆抗体并探讨抗体的应用。方法 用反转录PCR（Reverse Transcription-Polymerase Chain Reaction，RT-PCR）法体外扩增树鼩Vasn基因全长序列，将树鼩Vasn基因插入pET30a载体中，构建树鼩pET30a-VASN重组载体，对重组质粒用BamHI酶和SalI酶进行双酶切和测序鉴定；将测序正确的重组载体转化至大肠杆菌BL21（DE3），异丙基-β-D-硫代半乳糖苷（Isopropyl β-D-thiogalactoside，IPTG）诱导表达树鼩VASN重组蛋白；通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis，SDS-PAGE）电泳分离蛋白，氯化钾（Potassium chloride，KCl）切胶纯化树鼩VASN重组蛋白；将纯化的树鼩VASN重组蛋白对BALB/c小鼠进行4次免疫，酶联免疫吸附实验（Enzyme Linked Immunosorbent Assay，ELISA）检测小鼠血清效价；取效价达到1:10 000以上的小鼠脾脏细胞进行细胞融合，通过含黄嘌呤、氨基蝶呤及胸腺嘧啶核苷（Hypoxanthine-Aminopterin-Thymidine，HAT）的培养基筛选杂交瘤细胞，ELISA法筛选出能够分泌特异性抗体的阳性杂交瘤细胞株，并通过有限稀释法获得单克隆细胞株；通过腹水诱生法大量制备VASN单克隆抗体并利用rProtein G纯化抗体，ELISA、实时定量反转录PCR （Real-Time Quantitative Reverse Transcription PCR，qRT-PCR）、Western blotting检测单克隆抗体的亲和力和特异性，以及树鼩永生化成纤维细胞和树鼩组织中VASN的表达情况。结果 成功扩增出树鼩Vasn基因全长序列并构建了树鼩pET30a-VASN重组质粒载体，重组质粒载体经测序得到的序列与树鼩Vasn目的基因序列一致；重组载体表达后的蛋白主要以包涵体的形式存在，重组树鼩VASN蛋白纯化后的纯度达到90%，符合后续免疫实验的要求；利用重组树鼩VASN蛋白4次免疫小鼠后，小鼠血清抗体效价达1:729 000；利用杂交瘤技术及有限稀释法获得单克隆阳性杂交瘤细胞株，通过腹水诱生法及纯化后得到的单克隆抗体，ELISA法测定单克隆抗体的亲和力常数值达2.59×107mol/L；Western blotting检测单克隆抗体特异性，结果显示，树鼩VASN单克隆抗体能与树鼩VASN重组蛋白结合，而与猪视黄醇结合蛋白4（Retinol-Binding Protein 4，RBP4）重组蛋白、人源VASN-富含亮氨酸重复序列（Leucine Rich Repeat，LRR）重组蛋白以及牛血清白蛋白（Bovine Serum Albumin，BSA）标准蛋白均无结合反应；Western blotting检测树鼩各主要组织VASN表达，结果显示抗树鼩VASN单抗能与树鼩的主要器官组织（心脏、肝脏、脾脏、肺脏、肾脏、肌肉）在相对分子量约70 000位置出现特异性的条带且背景清晰；qRT-PCR检测结果显示树鼩脾脏组织和肺脏组织Vasn的mRNA表达水平相对较高，利用树鼩VASN单克隆抗体进行免疫组化检测，发现该基因mRNA水平表达较高的树鼩脾脏组织、肺脏组织、树鼩永生化成纤维细胞中免疫组化呈现阳性结果，在相对低表达的组织中呈现阴性结果。结论 成功制备了抗树鼩VASN的单克隆抗体，该抗体可用于树鼩永生化成纤维细胞、树鼩脾脏组织和肺脏组织免疫组化检测，研究结果为进一步探索VASN在树鼩模型中功能研究提供了重要的工具。