Search Results (31)

Attempts to activate an anti-tumor immune response in glioblastoma (GBM) have been met with many challenges due to its inherently immunosuppressive tumor microenvironment. The degree and mechanisms by which molecularly and phenotypically diverse tumor-propagating glioma stem cells (GSCs) contribute to this state are poorly defined. In this study, our multifaceted approach combining bioinformatics analyses of clinical and experimental datasets, single-cell sequencing, and the molecular and pharmacologic manipulation of patient-derived cells identified GSCs expressing immunosuppressive effectors mimicking regulatory T cells (Tregs). We showed that this immunosuppressive Treg-like (ITL) GSC state is specific to the mesenchymal GSC subset and is associated with and driven specifically by TGFβ type II receptor (TGFBR2) in contrast to TGFBR1. Transgenic TGFBR2 expression in patient-derived GBM neurospheres promoted a mesenchymal transition and induced a six-gene ITL signature consisting of CD274 (PD-L1), NT5E (CD73), ENTPD1 (CD39), LGALS1 (galectin-1), PDCD1LG2 (PD-L2), and TGFB1. This TGFBR2-driven ITL signature was identified in clinical GBM specimens, patient-derived GSCs, and systemic mesenchymal malignancies. TGFBR2^high GSCs inhibited CD4+ and CD8+ T cell viability and their capacity to kill GBM cells, effects reversed by pharmacologic and shRNA-based TGFBR2 inhibition. Collectively, our data identify an immunosuppressive GSC state that is TGFBR2-dependent and susceptible to TGFBR2-targeted therapeutics. Full article

Suicide is a major public health priority, and its molecular mechanisms appear to be related to imbalanced purine metabolism in the brain. This exploratory study investigates purinergic gene expression in the postmortem dorsolateral prefrontal cortex (DLPFC) tissue isolated from subjects with major depressive disorder (MDD) who died by suicide (MDD-S, n = 10), MDD subjects who did not die by suicide (MDD-NS, n = 6) and non-psychiatrically ill controls (CTL, n = 9–10). Purinergic system transcripts were assayed by quantitative polymerase chain reactions (qPCR) in superficial and deep gray matter as well as white matter DLPFC cortical layers using laser microdissection (LMD). Across all subjects, regardless of sex, P2RY12 (F_(2,23) = 5.40, p = 0.004) and P2RY13 (KW statistic = 11.82, p = 0.001) transcript levels were significantly greater in MDD-S compared to MDD-NS subjects. Several other perturbations were observed in the white matter tissue isolated from females: NT5E (F_(2,10) = 13.37, p = 0.001) and P2RY13 (F_(2,9) = 3.99, p = 0.011, controlled for age) transcript expression was significantly greater in MDD-S vs. MDD-NS female groups. ENTPD2 (F_(2,10) = 5.20, p = 0.03), ENTPD3 (F_(2,10) = 28.99, p < 0.0001), and NT5E (F_(2,10) = 13.37, p = 0.001) were among the transcripts whose expression was significantly elevated in MDD-S vs. CTL female groups. Transcripts that exhibited significantly altered expression in the superficial and deep gray matter included ENTPD2, NT5E, PANX1, and P2RY13 (p ≤ 0.05). Our medication analysis revealed that the expression of these transcripts was not significantly altered by antidepressants. This is the first study to holistically quantify the purinergic metabolic pathway transcripts in suicide and MDD utilizing human postmortem brain tissue. Our preliminary findings support evidence implicating changes in purinergic P2 receptors in the brain in suicide and provide support for broader purinergic system dysregulation in mood disorders. Full article

Fluorene-9-bisphenol (BHPF) has been increasingly used as a bisphenol A substitute in the synthesis of various products. Previous studies have suggested that BHPF can be released from plastic bottles into drinking water, and BHPF accumulation has been reported to cause various adverse effects in humans. Nevertheless, the impact of BHPF exposure on endometrial epithelial cells remains largely unexplored. Here, we investigated the effects of exposure to different concentrations of BHPF on endometrial cells and used integrated metabolomic and transcriptomic methods to elucidate the underlying molecular mechanisms. Our results revealed significant associations between specific metabolites and genes, indicating that low-concentration exposure to BHPF affects endometrial epithelial cells by targeting pathways related to primary immunodeficiency, in which the key genes are IL7R and PTPRC. High-concentration exposure to BHPF decreased cell viability by regulating the purine metabolism pathway, as well as dysregulating the expression of PGM1, PDE3B, AK9, and ENTPD8. Our study highlights that the health risk of BHPF exposure to endometrial epithelial cells is concentration-dependent and that integrated analysis of metabolomic and transcriptomic data not only revealed the biological effects of BHPF and its underlying mechanisms, but also provided key candidate target genes for further exploration. Full article

Inhibitory receptors are critical for regulating immune cell function. In cancer, these receptors are often over-expressed on the cell surface of T and NK cells, leading to reduced anti-tumor activity. Here, through the analysis of 11 commonly studied checkpoint and inhibitory receptors, we discern that only HAVCR2 (TIM3) and ENTPD1 (CD39) display significantly greater gene expression in glioblastoma compared to normal brain and lower grade glioma. Cell surface TIM-3, but not ENTPD1, was also elevated on activated CD4⁺ and CD8⁺ T cells, as well as on NK cells from glioblastoma patients compared to healthy donor T and NK cells. A subsequent analysis of molecules known to co-ordinate TIM-3 function and regulation was performed, which revealed that BAT3 expression was significantly reduced in CD4⁺ and CD8⁺ T cells, as well as NK cells from glioblastoma patients compared to counterparts from healthy donors. These pro-inhibitory changes are also correlated with reduced levels of the activation marker CD69 and the pro-inflammatory cytokine IFNγ in CD4⁺ and CD8⁺ T cells, as well as NK cells from glioblastoma patients. Collectively, these data reveal that glioblastoma-mediated CD4⁺ and CD8⁺ T cell and NK cell suppression is due, at least in part, to dysregulated TIM-3 and BAT3 expression and the associated downstream immunoregulatory and dysfunctional effects. Full article

Schizophrenia is a neuropsychiatric illness characterized by altered neurotransmission, in which adenosine, a modulator of glutamate and dopamine, plays a critical role that is relatively unexplored in the human brain. In the present study, postmortem human brain tissue from the anterior cingulate cortex (ACC) of individuals with schizophrenia (n = 20) and sex- and age-matched control subjects without psychiatric illness (n = 20) was obtained from the Bronx–Mount Sinai NIH Brain and Tissue Repository. Enriched populations of ACC pyramidal neurons were isolated using laser microdissection (LMD). The mRNA expression levels of six key adenosine pathway components—adenosine kinase (ADK), equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2), ectonucleoside triphosphate diphosphohydrolases 1 and 3 (ENTPD1 and ENTPD3), and ecto-5′-nucleotidase (NT5E)—were quantified using real-time PCR (qPCR) in neurons from these individuals. No significant mRNA expression differences were observed between the schizophrenia and control groups (p > 0.05). However, a significant sex difference was found in ADK mRNA expression, with higher levels in male compared with female subjects (Mann–Whitney U = 86; p < 0.05), a finding significantly driven by disease (t₍₁₇₎ = 3.289; p < 0.05). Correlation analyses also demonstrated significant associations (n = 12) between the expression of several adenosine pathway components (p < 0.05). In our dementia severity analysis, ENTPD1 mRNA expression was significantly higher in males in the “mild” clinical dementia rating (CDR) bin compared with males in the “none” CDR bin (F_{(2, 13)} = 5.212; p < 0.05). Lastly, antipsychotic analysis revealed no significant impact on the expression of adenosine pathway components between medicated and non-medicated schizophrenia subjects (p > 0.05). The observed sex-specific variations and inter-component correlations highlight the value of investigating sex differences in disease and contribute to the molecular basis of schizophrenia’s pathology. Full article

The relationship between type 2 diabetes mellitus (T2DM) and colorectal cancer (CRC) has long been extensively recognized, but their crosstalk mechanisms based on gene regulation remain elusive. In our study, for the first time, bulk RNA-seq and single-cell RNA-seq data were used to explore the shared molecular mechanisms between T2DM and CRC. Moreover, Connectivity Map and molecular docking were employed to determine potential drugs targeting the candidate targets. Eight genes (EVPL, TACSTD2, SOX4, ETV4, LY6E, MLXIPL, ENTPD3, UGP2) were identified as characteristic comorbidity genes for T2DM and CRC, with EVPL and ENTPD3 further identified as core comorbidity genes. Our results demonstrated that upregulation of EVPL and downregulation of ENTPD3 were intrinsic molecular features throughout T2DM and CRC and were significantly associated with immune responses, immune processes, and abnormal immune landscapes in both diseases. Single-cell analysis highlighted a cancer-associated fibroblast (CAF) subset that specifically expressed ENTPD3 in CRC, which exhibited high heterogeneity and unique tumor-suppressive features that were completely different from classical cancer-promoting CAFs. Furthermore, ENTPD3⁺ CAFs could notably predict immunotherapy response in CRC, holding promise to be an immunotherapy biomarker at the single-cell level. Finally, we identified that droperidol may be a novel drug simultaneously targeting EVPL and ENTPD3. In conclusion, previous studies have often focused solely on metabolic alterations common to T2DM and CRC. Our study establishes EVPL and ENTPD3 as characteristic molecules and immune biomarkers of comorbidity in T2DM and CRC patients, and emphasizes the importance of considering immunological mechanisms in the co-development of T2DM and CRC. Full article

(1) Introduction: Despite documented clinical and pain discrepancies between male and female osteoarthritis (OA) patients, the underlying mechanisms remain unclear. Synovial myofibroblasts, implicated in synovial fibrosis and OA-related pain, offer a potential explanation for these sex differences. Additionally, interleukin-24 (IL24), known for its role in autoimmune disorders and potential myofibroblast production, adds complexity to understanding sex-specific variations in OA. We investigate its role in OA and its contribution to observed sex differences. (2) Methods: To assess gender-specific variations, we analyzed myofibroblast marker expression and IL24 levels in synovial tissue samples from propensity-matched male and female OA patients (each n = 34). Gene expression was quantified using quantitative polymerase chain reaction (qPCR). The association between IL24 expression levels and pain severity, measured by a visual analog scale (VAS), was examined to understand the link between IL24 and OA pain. Synovial fibroblast subsets, including CD45-CD31-CD39- (fibroblast) and CD45-CD31-CD39+ (myofibroblast), were magnetically isolated from female patients (n = 5), and IL24 expression was compared between these subsets. (3) Results: Females exhibited significantly higher expression of myofibroblast markers (MYH11, ET1, ENTPD2) and IL24 compared to males. IL24 expression positively correlated with pain severity in females, while no correlation was observed in males. Further exploration revealed that the myofibroblast fraction highly expressed IL24 compared to the fibroblast fraction in both male and female samples. There was no difference in the myofibroblast fraction between males and females. (4) Conclusions: Our study highlights the gender-specific role of myofibroblasts and IL24 in OA pathogenesis. Elevated IL24 levels in females, correlating with pain severity, suggest its involvement in OA pain experiences. The potential therapeutic implications of IL24, demonstrated in autoimmune disorders, open avenues for targeted interventions. Notwithstanding the limitations of the study, our findings contribute to understanding OA’s multifaceted nature and advocate for future research exploring mechanistic underpinnings and clinical applications of IL24 in synovial myofibroblasts. Additionally, future research directions should focus on elucidating the precise mechanisms by which IL24 contributes to OA pathology and exploring its potential as a therapeutic target for personalized medicine approaches. Full article

Osteoarthritis (OA) is a prevalent degenerative joint disorder characterized by cartilage erosion, structural changes, and inflammation. Synovial fibroblasts play a crucial role in OA pathophysiology, with abnormal fibroblastic cells contributing significantly to joint pathology. Fibrocytes, expressing markers of both hematopoietic and stromal cells, are implicated in inflammation and fibrosis, yet their marker and role in OA remain unclear. ENTPD1, an ectonucleotidase involved in purinergic signaling and expressed in specific fibroblasts in fibrotic conditions, led us to speculate that ENTPD1 plays a role in OA pathology by being expressed in fibrocytes. This study aimed to investigate the phenotype of ENTPD1+CD55+ and ENTPD1−CD55+ synovial fibroblasts in OA patients. Proteomic analysis revealed a distinct molecular profile in ENTPD1+CD55+ cells, including the upregulation of fibrocyte markers and extracellular matrix-related proteins. Pathway analysis suggested shared mechanisms between OA and rheumatoid arthritis. Correlation analysis revealed an association between ENTPD1+CD55+ fibrocytes and resting pain in OA. These findings highlight the potential involvement of ENTPD1 in OA pain and suggest avenues for targeted therapeutic strategies. Further research is needed to elucidate the underlying molecular mechanisms and validate potential therapeutic targets. Full article

This study investigates the immunomodulatory potential of Galium aparine L. (GAE) in immunodeficient animals. In this study, animals were categorized into five groups: the normal group, CYP group (cyclophosphamide intraperitoneal injection), GA5 group (cyclophosphamide + 5 μg GAE), GA50 group (cyclophosphamide + 50 μg GAE), and GA500 group (cyclophosphamide + 500 μg GAE). The CYP group exhibited significantly reduced spleen weights compared to the normal group, while the groups obtaining GAE displayed a dose-dependent increase in spleen weight. Furthermore, the GAE demonstrated dose-dependent enhancement of splenocyte proliferating activity, with significant increases observed in both LPS and ConA-induced assays. NK cell activity significantly increased in the GA50 and GA500 groups compared to the CYP group. Cytokine analysis revealed a significant increase in IL-6, TNF-α, and IFN-γ levels in ConA-induced splenocytes treated with GAE. Gene expression analysis identified 2434 DEG genes in the extract groups. Notable genes, such as Entpd1, Pgf, Thdb, Syt7, Sqor, and Rsc1al, displayed substantial differences in individual gene expression levels, suggesting their potential as target genes for immune enhancement. In conclusion, Galium aparine L. extract exhibits immunomodulatory properties. The observed gene expression changes further support the potential of Galium aparine L. extract as a natural agent for immune augmentation. Full article

Training has a significant effect on the physiology of blood coagulation in humans and in horses. Several hemostatic changes have been reported after exercise in the horse but data available are inconclusive. The aim of this study was to investigate platelet activation and primary platelet-related hemostasis modifications in young never-trained Thoroughbreds in the first incremental training period in order to improve knowledge on this topic. Twenty-nine clinically healthy, untrained, 2-year-old Thoroughbred racehorses were followed during their incremental 4-month sprint exercise training. Blood collection was performed once a month, five times in total (T-30, T0, T30, T60, and T90). Platelet aggregation was measured by light transmission aggregometry in response to various agonists: adenosine diphosphate (ADP), collagen, and calcium ionophore A23187. Platelet function was evaluated using a platelet function analyzer (PFA-100^®) using collagen/ADP and collagen/adrenaline cartridges. Nitrite-nitrate (NOx) plasma concentrations were measured via a colorimetric assay to assess in vivo nitric oxide bioavailability. Platelet activation was also investigated through gene expression analyses (selectin P-SELP, ectonucleotidase CD39-ENTPD1, prostaglandin I2 synthase-PTGIS, endothelial nitric oxide synthase 3-NOS3). Differences among the time points were analyzed and mean ± SEM were calculated. Significant modifications were identified compared with T-30, with an increase in platelet aggregation (collagen:32.6 ± 4.8 vs. 21.6 ± 4.9%; ADP: 35.5 ± 2.0 vs. 24.5 ± 3.1%; A23187: 30 ± 4.7 vs. 23.8 ± 4%) and a shorter closure time of C-ADP cartridges (75.6 ± 4.4 vs. 87.7 ± 3.4 s) that tended to return to the baseline value at T90. NOx concentrations in plasma significantly increased after 30 days of the training program compared with the baseline. The first long-term training period seems to induce platelet hyperactivity after 30 days in never-trained Thoroughbreds. Regular physical training reduces the negative effects of acute efforts on platelet activation. Full article

Disease-modifying treatments have transformed the natural history of spinal muscular atrophy (SMA), but the cellular pathways altered by SMN restoration remain undefined and biomarkers cannot yet precisely predict treatment response. We performed an exploratory cerebrospinal fluid (CSF) proteomic study in a diverse sample of SMA patients treated with nusinersen to elucidate therapeutic pathways and identify predictors of motor improvement. Proteomic analyses were performed on CSF samples collected before treatment (T0) and at 6 months (T6) using an Olink panel to quantify 1113 peptides. A supervised machine learning approach was used to identify proteins that discriminated patients who improved functionally from those who did not after 2 years of treatment. A total of 49 SMA patients were included (10 type 1, 18 type 2, and 21 type 3), ranging in age from 3 months to 65 years. Most proteins showed a decrease in CSF concentration at T6. The machine learning algorithm identified ARSB, ENTPD2, NEFL, and IFI30 as the proteins most predictive of improvement. The machine learning model was able to predict motor improvement at 2 years with 79.6% accuracy. The results highlight the potential application of CSF biomarkers to predict motor improvement following SMA treatment. Validation in larger datasets is needed. Full article

NTPDase1/CD39, the major vascular ectonucleotidase, exerts thrombo-immunoregulatory function by controlling endothelial P2 receptor activation. Despite the well-described release of ATP from endothelial cells, few data are available regarding the potential role of CD39 as a regulator of arterial diameter. We thus investigated the contribution of CD39 in short-term diameter adaptation and long-term arterial remodeling in response to flow using Entpd1^−/− male mice. Compared to wild-type littermates, endothelial-dependent relaxation was modified in Entpd1^−/− mice. Specifically, the vasorelaxation in response to ATP was potentiated in both conductance (aorta) and small resistance (mesenteric and coronary) arteries. By contrast, the relaxing responses to acetylcholine were supra-normalized in thoracic aortas while decreased in resistance arteries from Entpd1^−/− mice. Acute flow-mediated dilation, measured via pressure myography, was dramatically diminished and outward remodeling induced by in vivo chronic increased shear stress was altered in the mesenteric resistance arteries isolated from Entpd1^−/− mice compared to wild-types. Finally, changes in vascular reactivity in Entpd1^−/− mice were also evidenced by a decrease in the coronary output measured in isolated perfused hearts compared to the wild-type mice. Our results highlight a key regulatory role for purinergic signaling and CD39 in endothelium-dependent short- and long-term arterial diameter adaptation to increased flow. Full article

The vascular endothelium is key target for immune and thrombotic responses that has to be controlled in successful xenotransplantation. Several genes were identified that, if induced or overexpressed, help to regulate the inflammatory response and preserve the transplanted organ function and metabolism. However, few studies addressed combined expression of such genes. The aim of this work was to evaluate in vivo the effects of the simultaneous expression of three human genes in a mouse generated using the multi-cistronic F2A technology. Male 3-month-old mice that express human heme oxygenase 1 (hHO-1), ecto-5′-nucleotidase (hE5NT), and ecto-nucleoside triphosphate diphosphohydrolase 1 (hENTPD1) (Transgenic) were compared to wild-type FVB mice (Control). Background analysis include extracellular nucleotide catabolism enzymes profile on the aortic surface, blood nucleotide concentration, and serum L-arginine metabolites. Furthermore, inflammatory stress induced by LPS in transgenic and control mice was used to characterize interleukin 6 (IL-6) and adhesion molecules endothelium permeability responses. Transgenic mice had significantly higher rates of extracellular adenosine triphosphate and adenosine monophosphate hydrolysis on the aortic surface in comparison to control. Increased levels of blood AMP and adenosine were also noticed in transgenics. Moreover, transgenic animals demonstrated the decrease in serum monomethyl-L-arginine level and a higher L-arginine/monomethyl-L-arginine ratio. Importantly, significantly decreased serum IL-6, and adhesion molecule levels were observed in transgenic mice in comparison to control after LPS treatment. Furthermore, reduced endothelial permeability in the LPS-treated transgenic mice was noted as compared to LPS-treated control. The human enzymes (hHO-1, hE5NT, hENTPD1) simultaneously encoded in transgenic mice demonstrated benefits in several biochemical and functional aspects of endothelium. This is consistent in use of this approach in the context of xenotransplantation. Full article

The adenosine-5’ triphosphate (ATP)-gated, ion channel, P2X receptor superfamily has seven members expressed by many cancer types. Subtype 7 (P2X7 receptor) is expressed consistently at levels higher than in comparatively healthy tissues. Moreover, transcript variant heterogeneity is associated with drug resistance. We have previously described the role of the P2X7 receptor in myeloma, a rare blood disease that uniquely presents with aggressive bone destruction. In this study, we used known agonists of the P2X7 receptor to induce calcium influx and YO-PRO-1 uptake in murine MOPC315.BM myeloma cells as readouts of P2X7 receptor-mediated channel activation and pore formation, respectively. Neither ATP- nor BzATP-induced calcium influx and YO-PRO-1 indicated an absence of the P2X7 receptor function on MOPC315.BM cells. TaqMan revealed low (Ct > 35) P2rx7 but high P2rx4 gene expression in MOPC315.BM; the latter was downregulated with BzATP treatment. The concomitant downregulation of CD39/Entpd1, Icam-1, and Nf-kb1 and the upregulation of Casp-1 genes regulated during purinergic signaling and with established roles in myeloma progression suggest P2RX4-mediated survival adaptation by cancer cells. Further studies are needed to characterize the P2RX4 pharmacology on MOPC315.BM since transcriptional regulation may be utilized by cancer cells to overcome the otherwise toxic effects of high extracellular ATP. Full article

The ectonucleotidases CD39 and CD73 are present on immune cells and play important roles in cancer progression by suppressing antitumour immunity. As such, CD39 and CD73 on peripheral blood mononuclear cells (PBMCs) are emerging as potential biomarkers to predict disease outcomes and treatment responses in cancer patients. This study aimed to examine T and B cells, including CD39 and CD73 expressing subsets, by flow cytometry in PBMCs from 28 patients with head and neck squamous cell carcinoma (HNSCC) and to assess the correlation with the treatment modality, human papillomavirus (HPV) status, and relapse-free survival (RFS). The PBMCs were examined pre-, mid-, and post-radiotherapy with concurrent cisplatin chemotherapy or anti-epidermal growth factor receptor antibody (cetuximab) therapy. Combination radiotherapy caused changes to T and B cell populations, including CD39 and CD73 expressing subsets, but no such differences were observed between concurrent chemotherapy and cetuximab. Pretreatment PBMCs from HPV⁺ patients contained increased proportions of CD39⁻CD73⁻CD4⁺ T cells and reduced proportions of CD39^−/+CD73⁺CD4⁺ T cells compared to the equivalent cells from HPV⁻ patients. Notably, the pretreatment CD4⁺:CD8⁺ T cell ratios and CD39⁺CD73⁺CD19⁺ B cell proportions below the respective cohort medians corresponded with an improved RFS. Collectively, this study supports the notion that CD39 and CD73 may contribute to disease outcomes in HNSCC patients and may assist as biomarkers, either alone or as part of immune signatures, in HNSCC. Further studies of CD39 and CD73 on PBMCs from larger cohorts of HNSCC patients are warranted. Full article