Translational Research最新文献

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by severe organ damage and lacking curative treatment. While various immune cell types, especially dysfunctional B and T cells and neutrophils, have been related with disease pathogenesis, limited research has focused on the role of monocytes in SLE. Increased DNA extracellular traps, apoptosis and necrosis have been related to lupus pathogenesis. Our goal is to analyze the contribution of P-selectin glycoprotein ligand 1 (PSGL-1) in SLE monocytes to disease pathogenesis by investigating the control exerted by PSGL-1 on monocyte apoptosis and DNA extrusion in extracellular traps (METs). Monocytes from active disease patients (aSLE) exhibited reduced levels of PSGL-1. Importantly, lower PSGL-1 levels in SLE monocytes associated with several clinical characteristics, including anti-dsDNA autoantibodies, lupus anticoagulant, clinical lung involvement, and anemia. Monocytes from SLE patients showed higher susceptibility to apoptosis than healthy donors (HD) monocytes and PSGL-1/P-selectin interaction decreased secondary necrosis in HD but not in aSLE monocytes. Regarding METs, aSLE monocytes exhibited higher susceptibility to generate METs than HD monocytes. The interaction of HD monocytes with P-selectin induced Syk activation and reduced the levels of DNA extruded in METs. However, in aSLE monocytes, PSGL-1/P-selectin interaction did not activate Syk or reduce the amount of extruded DNA. Our data suggest a dysfunctional PSGL-1/P-selectin axis in aSLE monocytes, unable to reduce secondary necrosis or the amount of DNA released into the extracellular medium in METs, potentially contributing to lupus pathogenesis.

Periodontitis is a chronic inflammatory oral disease that impaired the tooth-supporting apparatus, including gingival tissue destruction and alveolar bone resorption. The initiation of periodontitis is linked to the presence of oral bacteria, particularly P. gingivalis within pathogenic biofilms. Here, we demonstrated the central role of the autophagy regulator Transcription Factor EB (TFEB) in orchestrating autophagy activation and modulating the host immune response against P. gingivalis in periodontitis. Upregulation of TFEB expression at the protein level and heightened nuclear localization occurred during the progressive stages of periodontitis. Functionally, TFEB overexpression emerges as a potent alleviator of periodontitis-associated phenotypes, operating through the activation of autophagy and the inhibition of the NF-κB pathway in both in vivo and in vitro models. In addition, TFEB knockdown exacerbates the inflammatory response by upregulating pro-inflammatory cytokines. The dual regulatory role of TFEB in governing both autophagy and inflammatory responses unveils novel insights into periodontitis pathogenesis, positioning TFEB as a promising therapeutic target for periodontitis intervention.

Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed at metastatic stage and typically treated with fluorouracil, leucovorin, irinotecan and oxaliplatin (FOLFIRINOX). Few patients benefit from this treatment. Molecular subtypes are prognostic in particularly resectable PDAC and might predict treatment response. This study aims to correlate molecular subtypes in metastatic PDAC with FOLFIRINOX responses using real-world data, providing assistance in counselling patients. We collected 131 RNA-sequenced metastatic biopsies and applied a network-based meta-analysis using published PDAC classifiers. Subsequent survival analysis was performed using the most suitable classifier. For validation, we developed an immunohistochemistry (IHC) classifier using GATA6 and keratin-17 (KRT17), and applied it to 86 formalin-fixed paraffin-embedded samples of advanced PDAC. Lastly, GATA6 knockdown models were generated in PDAC organoids and cell lines. We showed that the PurIST classifier was the most suitable classifier. With this classifier, classical tumors had longer PFS and OS than basal-like tumors (PFS: 216 vs. 78 days, p = 0.0002; OS: 251 vs. 195 days, p = 0.049). The validation cohort showed a similar trend. Importantly, IHC GATA6^low patients had significantly shorter survival with FOLFIRINOX (323 vs. 746 days, p = 0.006), but no difference in non-treated patients (61 vs. 54 days, p = 0.925). This suggests that GATA6 H-score predicts therapy response. GATA6 knockdown models did not lead to increased FOLFIRINOX responsiveness. These data suggest a predictive role for subtyping (transcriptomic and GATA6 IHC), though no direct causal relationship was found between GATA6 expression and chemoresistance. GATA6 immunohistochemistry should be seamlessly added to current diagnostics and integrated into upcoming clinical trials.

Extrachromosomal circular DNA (eccDNA) derived from linear chromosomes, are showed typical nucleosomal ladder pattern in agarose gel which as a known feature of apoptosis and demonstrated to be immunogenicity. In systemic lupus erythematosus (SLE) patients, elevated levels of cell-free DNA (cfDNA) can be found in either linear forms or circular forms, while circular ones are much less common and harder to detect. The molecular characteristics and function of circular forms in plasma SLE patients remains elusive. Herein, we characterized the hallmarks of plasma eccDNA in SLE patients, including the lower normalized number and GC content of eccDNA in SLE plasma than in the healthy, and SLE eccDNA number positively correlated with C3 and negatively with anti-dsDNA antibodies. The differential eccGenes (eccDNAs carrying the protein coding gene sequence) of SLE was significantly enriched in apoptosis-related pathways. The artificially synthesized eccDNA with sequences of the PRF1 exon region could promote transcriptional expression of PRF1, IFNA and IFIT3 and inhibit early-stage apoptosis. Plasma eccDNA can serve as a novel autoantigen in the pathogenesis of SLE.

Epithelial ovarian cancer is a significant global health issue among women. Diagnosis and treatment pose challenges due to difficulties in predicting patient responses to therapy, primarily stemming from gaps in understanding tumor chemoresistance mechanisms. Recent advancements in transcriptomic technologies like single-cell RNA sequencing and spatial transcriptomics have greatly improved our understanding of ovarian cancer intratumor heterogeneity and tumor microenvironment composition. Spatial transcriptomics, in particular, comprises a plethora of technologies that enable the detection of hundreds of transcriptomes and their spatial distribution within a histological section, facilitating the study of cell types, states, and interactions within the tumor and its microenvironment. Studies investigating the spatial distribution of gene expression in ovarian cancer masses have identified specific features that impact prognosis and therapy outcomes. Emerging evidence suggests that specific spatial patterns of tumor cells and their immune and non-immune microenvironment significantly influence therapy response, as well as the behavior and progression of primary tumors and metastatic sites. The importance of spatially contextualizing ovarian cancer transcriptomes is underscored by these findings, which will advance our understanding and therapeutic approaches for this complex disease.

Doxorubicin (DOX) is restricted due to its severe cardiotoxicity. There is still a lack of viable and effective drugs to prevent or treat DOX-induced cardiotoxicity(DIC). Vericiguat is widely used to treat heart failure with reduced ejection fraction. However, it is not clear whether vericiguat can improve DIC. In the present study, we constructed a DIC model using mice and neonatal rat cardiomyocytes and found that vericiguat ameliorated DOX-induced cardiac insufficiency in mice, restored DOX-induced mitochondrial dysfunction in neonatal rat cardiomyocytes, and inhibited the expression of inflammatory factors. Further studies showed that vericiguat improved mitochondrial dysfunction and reduced mtDNA leakage into the cytoplasm by up-regulating PRKG1, which activated PINK1 and then inhibited the STING/IRF3 pathway to alleviate DIC. These findings demonstrate for the first time that vericiguat has therapeutic potential for the treatment of DIC.

Bone malunion or nonunion leads to functional and esthetic problems and is a major healthcare burden. Activation of bone marrow mesenchymal stem cells (BMSCs) and subsequent induction of osteogenic differentiation by local metabolites are crucial steps for bone healing, which has not yet been completely investigated. Here, we found that lactate levels are rapidly increased at the local injury site during the early phase of bone defect healing, which facilitates the healing process by enhancing BMSCs regenerative capacity. Mechanistically, lactate serves as a ligand for the Olfr1440 olfactory receptor, to trigger an intracellular calcium influx that in turn activates osteogenic phenotype transition of BMSCs. Conversely, ablation of Olfr1440 delays skeletal repair and remodelling, as evidenced by thinner cortical bone and less woven bone formation in vivo. Administration of lactate in the defect area enhanced bone regeneration. These findings thus revealed the key roles of lactate in the osteogenic differentiation of BMSCs, which deepened our understanding of the bone healing process, as well as provided cues for a potential therapeutic option that might greatly improve bone defect treatment.

Cardiac fibrosis occurs following insults to the myocardium and is characterized by the abnormal accumulation of non-compliant extracellular matrix (ECM), which compromises cardiomyocyte contractile activity and eventually leads to heart failure. This phenomenon is driven by the activation of cardiac fibroblasts (cFbs) to myofibroblasts and results in changes in ECM biochemical, structural and mechanical properties. The lack of predictive in vitro models of heart fibrosis has so far hampered the search for innovative treatments, as most of the cellular-based in vitro reductionist models do not take into account the leading role of ECM cues in driving the progression of the pathology. Here, we devised a single-step decellularization protocol to obtain and thoroughly characterize the biochemical and micro-mechanical properties of the ECM secreted by activated cFbs differentiated from human induced pluripotent stem cells (iPSCs). We activated iPSC-derived cFbs to the myofibroblast phenotype by tuning basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGF-β1) signalling and confirmed that activated cells acquired key features of myofibroblast phenotype, like SMAD2/3 nuclear shuttling, the formation of aligned alpha-smooth muscle actin (α−SMA)-rich stress fibres and increased focal adhesions (FAs) assembly. Next, we used Mass Spectrometry, nanoindentation, scanning electron and confocal microscopy to unveil the characteristic composition and the visco-elastic properties of the abundant, collagen-rich ECM deposited by cardiac myofibroblasts in vitro. Finally, we demonstrated that the fibrotic ECM activates mechanosensitive pathways in iPSC-derived cardiomyocytes, impacting on their shape, sarcomere assembly, phenotype, and calcium handling properties. We thus propose human bio-inspired decellularized matrices as animal-free, isogenic cardiomyocyte culture substrates recapitulating key pathophysiological changes occurring at the cellular level during cardiac fibrosis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by inflammation in the synovial lining of the joints. Key inflammatory cytokines such as interleukin-6 (IL-6), TNF-α, and others play a critical role in the activation of local synovial leukocytes and the induction of chronic inflammation. Tocilizumab (TCZ), a humanized anti-IL-6 receptor monoclonal antibody, has demonstrated significant clinical efficacy in treating RA patients. However, similar to other inflammatory cytokine blockers, such as TNF-alpha inhibitors, Interleukin-1 inhibitors, or CD20 inhibitors, some patients do not respond to treatment. To address this challenge, our study employed a high-precision proteomics approach to identify protein biomarkers capable of predicting clinical responses to Tocilizumab in RA patients. Through the use of data-independent acquisition (DIA) mass spectrometry, we analyzed serum samples from both TCZ responders and non-responders to discover potential biomarker candidates. These candidates were subsequently validated using individual serum samples from two independent cohorts: a training set (N = 70) and a test set (N = 18), allowing for the development of a robust multi-biomarker panel. The constructed multi-biomarker panel demonstrated an average discriminative power of 86 % between response and non-response groups, with a high area under the curve (AUC) value of 0.84. Additionally, the panel exhibited 100 % sensitivity and 60 % specificity. Collectively, our multi-biomarker panel holds promise as a diagnostic tool to predict non-responders to TCZ treatment in RA patients.

In general, ensuring safety is the top priority of a new modality. Although oncolytic virus armed with an immune stimulatory transgene (OVI) showed some promise, the strategic concept of simultaneously achieving maximum effectiveness and minimizing side effects has not been fully explored. We generated a variety of survivin-responsive “conditionally replicating adenoviruses that can target and treat cancer cells with multiple factors (m-CRAs)” (Surv.m-CRAs) armed with the granulocyte-macrophage colony-stimulating factor (GM-CSF) transgene downstream of various promoters using our m-CRA platform technology. We carefully analyzed both therapeutic and adverse effects of them in the in vivo syngeneic Syrian hamster cancer models. Surprisingly, an intratumor injection of a conventional OVI, which expresses the GM-CSF gene under the constitutively and strongly active “cytomegalovirus enhancer and β-actin promoter”, provoked systemic and lethal GM-CSF circulation and shortened overall survival (OS). In contrast, a new conceptual type of OVI, which expressed GM-CSF under the cancer-predominant and mildly active E2F promoter or the moderately active “Rous sarcoma virus long terminal repeat”, not only abolished lethal adverse events but also prolonged OS and systemic anti-cancer immunity. Our study revealed a novel concept that optimal expression levels of an immune stimulatory transgene regulated by a suitable upstream promoter is crucial for achieving high safety and maximal therapeutic effects simultaneously in OVI therapy. These results pave the way for successful development of the next-generation OVI and alert researchers about possible problems with ongoing clinical trials.