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关键词： Macrophage   Periapical lesions   Osteoclast differentiation  

摘要： Introduction and Aims: Existing research suggests that ARA290 may possess therapeutic potential for apical periodontitis (AP), although the mechanisms involved remain unclear. This study aims to investigate the protective effects and underlying mechanisms of ARA290 in mitigating pro-inflammatory responses of macrophages in the context of AP. Methods: Initially, in vivo models of AP were used to assess the aberrant activation of the SIRT1/NF-kB/IL-1/3 signalling pathway within periapical lesions. Furthermore, the expression levels of the SIRT1/NF-kB/IL-1/3 signalling pathway in ARA290-treated AP models, both in vivo and in vitro, were evaluated using immunohistochemistry (IHC), Western blotting, and immunofluorescence (IF) techniques to elucidate the role of ARA290 in modulating this signalling pathway during AP pathogenesis. Subsequently, the SIRT1/NF-kB/IL-1/3 signalling pathway was inhibited using selisistat, and the resultant changes in inflammatory levels and bone resorption in periapical lesions were assessed through haematoxylin and eosin staining, tartrate-resistant acid phosphatase staining, IHC and micro-computed tomography (Micro CT). Results: The in vivo AP model demonstrated a 50% reduction in SIRT1 expression within periapical lesions, accompanied by a 5-fold increase in the expression of acetylated NF-kB (p65) and IL-1/3. Additionally, the administration of ARA290 in the AP mouse model significantly reduced inflammatory infiltration, the number of osteoclasts and the extent of bone loss in the periapical region. Notably, ARA290 treatment enhanced SIRT1 expression by approximately 40% while concurrently decreasing the levels of acetylated NF-kB (p65) by around 75% and IL-1/3 by approximately 62.5% in both in vivo and in vitro AP models. Importantly, inhibition of the SIRT1/NF-kB/IL-1/3 signalling pathway negated the beneficial effects of ARA290 in attenuating the progression of AP. Conclusions: ARA290 plays a protective role by modulating the SIRT1/NF-k

关键词： Immunotherapy   T cell therapy   Chimeric antigen receptor   TCR-mimic CAR   TCR-mimic bispecific antibody   KRAS neoepitopes  

摘要： Mutations in the KRAS proto-oncogene, particularly at codon 12, are among the most frequent genetic alterations in various cancers, and KRASG12V accounts for about 25% of all KRAS mutations observed in lung, pancreatic, and colorectal adenocarcinomas. Despite improved treatment regimes using targeted therapy and checkpoint inhibitors, cellular immunotherapy options for KRAS-mutated cancers remain elusive. We therefore developed two TCR-mimic (TCRm) anti-KRASG12V/HLA-A*02:01 chimeric antigen receptors (CARs) containing different hinge regions and, alternatively, a TCRm anti-KRASG12V/HLA-A*02:01 bispecific T cell engager (BiTE) to explore immunotherapy to the highly prevalent KRASG12V neoantigen. CAR-redirected or BiTE-exposed JNL-reporter cells demonstrated potent signaling capacity upon recognition of KRASG12V. Moreover, human CAR T and NK cells elicited IFN-gamma release and cellular cytotoxicity upon encountering target cells pulsed with KRASG12V peptide, and the anti-KRASG12V Strep-tagII hinge CAR showed superior reactivity compared to a human IgG1-Fc hinge CAR. Similarly, a novel TCRm BiTE induced strong T cell immunity to KRASG12V. In contrast, we observed only very low CAR or BITE-mediated responses to naturally presented KRASG12V/HLA-A*02:01 complexes. In summary, this study demonstrates that the mutation-derived KRASG12V5-14 peptide can be effectively targeted by TCRm CAR and BiTE-redirected T cells, suggesting that TCRm anti-KRASG12V CAR or BiTE represent promising formats to advance immunotherapy to mutated KRAS *** messagesSuccessful development of TCRm CAR and BiTE targeting mutated KRAS/HLA-A*02:***-KRASG12V TCRm CAR and BiTE induce potent immunity to KRASG12V ***-KRAS/HLA-I TCRm CARs and BiTEs are novel therapeutics for cancer immunotherapy.

关键词： Triple negative breast cancer   Anthracycline   Taxane   Interleukin-8   Feedback loop  

摘要： Triple-negative breast cancer (TNBC) is a subtype of breast cancer that is highly metastatic compared with other BC subtypes. Taxane- and anthracycline-based therapies remain the main clinical TNBC regimens. However, TNBC patients usually suffer through chemotherapy owing to the lack of valid biomarkers to predict therapeutic effectiveness. We found that IL-8 upregulation is detected in paclitaxel (PTX)-sensitive/doxorubicin (DOX)resistant TNBC cells. Moreover, transcriptional profiling revealed that, compared with non-TNBC and normal mammary tissues, TNBC tissues highly express IL-8;its upregulation predicted a favorable response to taxane treatment but poor sensitivity to anthracycline-based therapy in TNBC cohorts. Whereas IL-8 supplementation in HCC1937 cells, which exhibit lower IL-8 expression and a PTX-resistant/DOX-sensitive phenotype, enhanced the cellular response to PTX but reduced their sensitivity to DOX, IL-8 knockdown in Hs578t cells, which have higher IL-8 expression and a PTX-sensitive/DOX-resistant phenotype, desensitized those cells to PTX treatment but improved their sensitivity to DOX. Gene set enrichment analysis (GSEA) results indicated that IL-8 upregulation is likely associated with E2F-regulated cell cycle progression and the activation of the NF-kB and MTOC1 pathways in TNBC. IL-8 supplementation promoted but IL-8 knockdown in Hs578t cells suppressed the activity of the E2F, NF-kB and MTOC1 signaling pathways. Immunohistochemistry (IHC) revealed that higher and lower IL8/p-NF-kappa B protein levels predicted good therapeutic outcomes in TNBC patients who initially received taxane and anthracycline-based chemotherapy, respectively. This study revealed that the formation of an IL-8/NF-kB feedback loop is required for maintaining the PTX-sensitive/DOX-resistant phenotype in TNBC.

摘要： Objective To investigate the role and mechanism of the zinc finger protein Kruppel-like transcription factor 9 (KLF9) in the stimulation of type I interferon expression induced by herpes simplex virus type 1 (HSV-1) in macrophages. Methods Agarose Gel electrophoresis, quantitative real-time PCR (qRT-PCR) and western blot analyses were employed to detect the KLF9 relative expression in bone marrow-derived macrophages (BMDMs) from Klf9 (gKO) mice and wild-type (WT) mice. RNA-seq analysis was utilized to identify the potential targeted genes upon HSV-1 stimulation in BMDMs. ELISA was used to measure the potent of IFN-β in the supernatant of BMDMs derived from gKO and WT mice after HSV-1 stimulation. qRT-PCR analysis was employed to further confirm the changes of Ifnb1 and interferon-stimulated gene (ISG) such as interferon-induced protein with tetratricopeptide repeats 1 (Ifit1), interferon-stimulated exonuclease gene 20 (Isg20), cholesterol 25-hydroxylase (Ch25h) and 2'-5' oligoadenylate synthetase-like 1 (Oasl1). Western blot was used to detect the expression of phosphorylated interferon regulatory factor-3 (p-IRF3), IRF3, phosphorylated interferon regulatory factor-7 (p-IRF7), IRF7, phosphorylated nuclear factor-kappa B p65 (p-NF-κB p65) and NF-κB p65. CUT-Tag and ChIP-qPCR assay were utilized to confirm the binding region of KLF9 in Ifnb1. Results The KLF9 expression was significantly decreased in BMDMs from gKO mice compared with that from WT mice. The RNA-seq analysis showed that Klf9 deletion in BMDMs resulted in an impaired type I interferon signaling pathway. The qRT-PCR analysis revealed that Klf9 deletion in BMDMs led to a significant decrease of Ifnb1 and ISG such as Ifit1, Ch25h and Oasl1 except Isg20. Moreover, ELISA revealed that Klf9 knockout in BMDMs resulted in a significant decrease of IFN-β secreted from BMDMs. Mechanistically, KLF9 directly binds to the promoter of Ifnb1. Conclusion KLF9 is essential for macrophages to resist HSV-1 infection.

摘要： Low or absent expression of major histocompatibility complex (MHC) on tumor cells is a presumed mechanism of resistance to immunotherapy, but evidence for this has largely been indirect. Likewise, whether immunotherapy can be effective without tumor MHC expression is also poorly understood. Using genetically-engineered mouse tumor cells expressing the model neoantigen ovalbumin (OVA), we found that MHC class I-deficient tumor cells, but not MHC class I-sufficient tumor cells, grew progressively when injected subcutaneously into syngeneic C57BL/6 mice. However, combination immunotherapy using agonistic anti-CD40 and dual immune checkpoint blockade (ICB) (anti-PD1 and anti-CTLA-4) was equally effective against tumors that did not express the MHC class I H-2Kb allele, MHC class II, or IFN-γ receptor across multiple pancreatic tumor lines (regardless of OVA). Moreover, CD4+ T cells, but not CD8+ T cells or perforin, were necessary to mediate immunotherapeutic responses. We excluded a role for CD4+ T cell-instructed macrophage-mediated tumor cell death but observed reprogramming of MHC class II-expressing stromal cells within the tumor after anti-CD40/ICB treatment. These data indicate that cancer immune surveillance by T cells does not absolutely require tumor-expressed MHC class I nor CD8+ T cells but instead can facilitate a clinically relevant remodeling of endothelial cells, further underscoring tumor-extrinsic roles for CD4+ T cells as mediators of tumor rejection and durable immune memory.

关键词： HLA-DPB1*1730:01   novel allele   sequencing-based typing  

摘要： HLA-DPB1*1730:01 differs from HLA-DPB1*107:01:01:01 by a single non-synonymous nucleotide change in exon 4.

关键词： Bilirubin   biological agent   creatinine   psoriasis   treatment  

摘要： INTRODUCTION:High serum creatinine to total bilirubin ratio has been associated with an increased risk of developing hypertension. We evaluated the effect of biological agents on serum creatinine to total bilirubin ratio in patients with psoriasis. METHODS:Serum creatinine and total bilirubin levels of patients were reviewed between June 2018 and October 2023. RESULTS:This study included 302 patients, 143 (47.4%) females and 159 (52.6%) males with a mean age of 49.46 ± 13.82 years. The creatinine to total bilirubin ratio was higher in males than in females (p = 0.028), and in patients with comorbidities compared to those without (p = 0.031). Six months after biological agent treatment, serum total bilirubin levels increased (p = 0.010), while the creatinine to total bilirubin ratio significantly decreased (p = 0.038) in all patients. Serum creatinine to total bilirubin ratio decreased (p = 0.031) in patients who received IL-17 inhibitors after 6 months. Among them, patients treated with ixekizumab showed a statistically significantly decrease in creatinine to total bilirubin ratio (p = 0.015). CONCLUSION:The decrease in creatinine to total bilirubin ratio after 6 months of treatment was statistically significant only in patients treated with IL-17 inhibitors, particularly ixekizumab. Therefore, we suggest that IL-17 inhibitors may have a protective role against hypertension in patients with psoriasis.

关键词： Allele typing   Major Histocompatibility Complex   NGS  

摘要： Objective:Understanding the individual- and population-level polymorphisms of major histocompatibility complex (MHC) genes is crucial for identifying associations between MHC variations and immune phenotypes. To support this, we developed NGSMHC, a streamlined bioinformatics tool for efficient and accurate MHC genotyping using next-generation sequencing (NGS) data in non-human species. Methods:NGSMHC constructs phased haplotype contigs of selected MHC genes from BAM-format mapping data and determines the best matching MHC alleles and genotypes via nucleotide BLAST analysis against a user-provided reference set of MHC alleles. We evaluated NGSMHC using short-read whole-genome sequencing (WGS) data from 12 pigs, focusing on swine leukocyte antigen (SLA) genes. The typing results from NGSMHC were compared to those obtained using polymerase chain reaction sequence-based typing (PCR-SBT). In addition, we tested NGSMHC on a publicly available long-read WGS dataset with known SLA genotypes. Results:The short-read WGS data showed an average read depth of 20.9× across the SLA region, enabling typing of SLA-2, SLA-3, SLA-DRB1, and SLA-DQB1 using NGSMHC. The concordance rates between NGSMHC and PCR-SBT were 88% for SLA-3, 92% for SLA-DRB1, and 100% for SLA-DQB1. However, SLA-2 typing showed lower concordance (58%), likely due to its high sequence similarity with other SLA class I genes and complex intra-locus polymorphisms. In contrast, NGSMHC accurately identified all tested SLA genotypes-including SLA-1, SLA-2, SLA-3, SLA-DRA, SLA-DRB1, SLA-DQA, and SLA-DQB1-when applied to the long-read WGS data. Conclusion:NGSMHC is a simple and effective tool for MHC genotyping using NGS data, particularly for non-human species. Its accuracy is significantly improved by long-read sequencing, underscoring the importance of read length in precise MHC allele determination.

关键词： 芪黄健脾滋肾颗粒   系统性红斑狼疮   肾脏保护   补体C1q   巨噬细胞极化  

摘要： 目的：基于补体1q（C1q）探讨芪黄健脾滋肾颗粒对系统性红斑狼疮小鼠（MRL/lpr）肾脏巨噬细胞极化的影响。方法：将MRL/lpr小鼠随机分为模型对照组、芪黄健脾滋肾颗粒3.9g/kg组、醋酸泼尼松2.73mg/kg组、补体C1q抑制剂40U/kg组，每组6只，正常对照组为C57BL/6 小鼠。每日固定时间灌胃给药，连续 8 周。采用HE染色、PAS染色、Masson染色观察肾脏病理，计算胶原容积分数（CVF）；采用测定试剂盒检测肌酐（CREA）、尿素氮（BUN）、尿蛋白（PRO）含量；ELISA法检测小鼠血清抗双链DNA（dsDNA）抗体，以及肾脏匀浆中C1q、白细胞介素（IL）-10、转化生长因子β1（TGFβ1）、肿瘤坏死因子-α（TNF-α）、IL-6含量；RT-qPCR法、Western Blot法检测肾脏组织中精氨酸酶1（Arg1）、IL-10、IL-6、一氧化氮合酶（iNOS）的mRNA及蛋白的表达；流式细胞术检测肾组织中CD86+M1 和CD206+M2 型细胞比例；免疫荧光双标法检测F4-80+ iNOS M1型巨噬细胞、F4-80+Arg1 M2 型巨噬细胞的表达。结果：与正常对照组比较，模型对照组可见肾小球基底膜增厚、部分可见肾间质水肿及炎症细胞浸润等病理现象，模型对照组CVF、PRO、CREA、BUN、抗dsDNA 抗体、TNF-α和IL-6含量显著升高（P＜0.01），C1q、TGFβ1、IL-10 含量显著降低（P＜0.01），肾组织IL-6、iNOS的mRNA、蛋白表达显著上调，Arg1、IL-10的mRNA、蛋白表达下调（P＜0.01），M1型巨噬细胞占比显著升高，M2型巨噬细胞占比显著降低（P<0.01），M1/M2 型巨噬细胞比例显著升高（P<0.01）；与模型对照组比较，芪黄健脾滋肾颗粒 3.9 g/kg 组肾脏病理改变出现改善，肾小球结构、肾小管排列有所恢复正常，CVF、PRO、CREA、BUN、抗dsDNA抗体、TNF-α、IL-6含量显著降低（P＜0.01），C1q、TGFβ1、IL-10含量明显升高（P＜0.05），IL-6、iNOS的mRNA、蛋白表达显著下调（P＜0.01），Arg1、IL-10的mRNA、蛋白表达显著上调升高（P<0.01），M1型巨噬细胞百分率显著降低，M2型巨噬细胞百分率显著升高（P<0.01），M1/M2型巨噬细胞比例明显降低（P<0.01）。结论：芪黄健脾滋肾颗粒对MRL/lpr小鼠的肾脏有明显的保护作用，其机制可能与C1q调控巨噬细胞极化表型有关。