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2021 ◽  
Vol 12 ◽  
Author(s):  
Palmira Barreira-Silva ◽  
Rita Melo-Miranda ◽  
Claudia Nobrega ◽  
Susana Roque ◽  
Cláudia Serre-Miranda ◽  
...  

Disseminated infection with the high virulence strain of Mycobacterium avium 25291 leads to progressive thymic atrophy. We previously showed that M. avium-induced thymic atrophy results from increased glucocorticoid levels that synergize with nitric oxide (NO) produced by interferon gamma (IFNγ) activated macrophages. Where and how these mediators act is not understood. We hypothesized that IFNγ and NO promote thymic atrophy through their effects on bone marrow (BM) T cell precursors and T cell differentiation in the thymus. We show that M. avium infection cause a reduction in the percentage and number of common lymphoid progenitors (CLP). Additionally, BM precursors from infected mice show an overall impaired ability to reconstitute thymi of RAGKO mice, in part due to IFNγ. Thymi from infected mice present an IFNγ and NO-driven inflammation. When transplanted under the kidney capsule of uninfected mice, thymi from infected mice are unable to sustain T cell differentiation. Finally, we observed increased thymocyte death via apoptosis after infection, independent of both IFNγ and iNOS; and a decrease on active caspase-3 positive thymocytes, which is not observed in the absence of iNOS expression. Together our data suggests that M. avium-induced thymic atrophy results from a combination of defects mediated by IFNγ and NO, including alterations in the BM T cell precursors, the thymic structure and the thymocyte differentiation.


2021 ◽  
Vol 15 ◽  
Author(s):  
Xichang Liu ◽  
Gang Wu ◽  
Na Tang ◽  
Li Li ◽  
Cuimin Liu ◽  
...  

Objective: The “Glymphatic” system, a network of perivascular tunnels wrapped by astrocyte endfeet, was reported to be closely associated with the diseases of the central nervous system. Here, we investigated the role of the glymphatic system in intracerebral hemorrhage (ICH) and its protective mechanism.Method: Experimental ICH model was induced by type IV collagenase in rats. Cerebral lymphatic blockage was induced by ligation and removal of cervical lymph nodes. The experimental rats were divided into sham-operated (SO) group, ICH group, and cerebral lymphatic blocking and ICH (ICH + CLB) group. Neurological scores were measured using the Garcia scoring system on the third and seventh day after ICH. Active caspase-3 was immunostained to evaluate neuronal apoptosis. Brain water content was calculated using the dry-wet specific gravity method. The expression of inflammatory factors TNF-α, IL-1β, and IL-10 were detected using ELISA. Aquaporins-4 (AQP-4) and glial fibrillary acidic protein (GFAP) were detected using western blot analysis.Results: The neurological scores of rats in the CLB + ICH group were significantly lower than those in the in ICH group. The number of active caspase-3 neurons was significantly higher in the CLB + ICH group compared to the ICH group. CLB significantly aggravated ICH-induced brain edema 3 d after ICH. There was an increase in the expression of TNF-α, IL-1β, IL-10, AQP-4, GFAP after ICH. The expression of TNF-α was significantly higher in the CLB + ICH group compared to ICH group 3 d after ICH while there was no difference 7 d after ICH. There was no statistical difference in the expression of IL-1β between the ICH group and CLB + ICH group. However, the expression of IL-10 in the CLB + ICH group was significantly lower than that in the ICH group. Lastly, AQP-4 expression was significantly lower in the CLB + ICH group compared to the ICH group while the expression of GFAP was higher in the CLB + ICH group compared to the ICH group.Conclusion: CLB exacerbated cerebral edema, neuroinflammation, neuronal apoptosis and caused neurological deficits in rats with ICH via down-regulating AQP-4, up-regulating inflammatory TNF-α and inhibiting IL-10 expression. The glymphatic drainage system protects against neurologic injury after ICH induction in rats under normal physiological conditions.


2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Ya Qiu ◽  
Yan Ma ◽  
Min Jiang ◽  
Sulei Li ◽  
Jibin Zhang ◽  
...  

Endotoxemia in sepsis remains a problem due to a lack of effective strategies. Our previous studies have demonstrated that melatonin (Mel) protects against ischemic heart injury and arteriosclerosis. However, its role in endotoxemia-exposed cardiomyocytes remains poorly understood. This study explored, for the first time, the protective effect of Mel on the pyroptosis of human stem cell-derived cardiomyocytes (hiPSC-CMs) exposed to lipopolysaccharide (LPS). Our results showed that treatment with 1 μM or 10 μM Mel for 12 h significantly improved 1 μg/ml LPS-induced hiPSC-CM injuries, as reflected by drastically decreased LDH release and increased cell viability, which was accompanied by the overt induction of autophagy. Specifically, Mel profoundly alleviated LPS-induced cell pyroptosis, as evidenced by decreased propidium iodide (PI) and active caspase-1 double-positive cell rates; suppressed the expression of NLRP3, cleaved caspase-1 (activated form of caspase-1), and GSDMD-NT (functional N-terminal fragment of GSDMD) expression; and inhibited the production of the cleaved IL-1β and cleaved IL-18 cytokines. Additionally, double-membrane autophagosomes were observed in LPS-injured hiPSC-CMs treated with 1 μM or 10 μM Mel. The hiPSC-CMs treated with LPS exhibited considerably fewer acidic vesicles (as revealed by LAMP1 staining) and autophagosomes (as revealed by LC3-II staining); however, Mel reversed this outcome in a dose-dependent manner. Furthermore, coincubation with rapamycin (an autophagy activator) or 3-MA (an autophagy inhibitor) accentuated and attenuated the antipyroptotic actions of Mel, respectively. Collectively, our findings demonstrate that Mel shields hiPSC-CMs against pyroptosis during endotoxemia by activating autophagy.


2021 ◽  
Author(s):  
Nermine Aly Moussa ◽  
Mahira Mohamed ◽  
Medhat Haroun ◽  
Maged Helmy Wasfy

Abstract Despite the tremendous efforts to implement new paradigms for breast cancer, the disease still remains a major challenge worldwide. Genetic deregulation is evident in all breast cancer subtypes and comprises a multitude of mutated genes and deregulated signaling cascades. In this sense, co-targeting Src and COX-2 signaling cascades have attracted fervent interest. This work explored the probable anti-carcinogenic effects of Dasatinib as a Src inhibitor, Celecoxib as a selective COX-2 inhibitor, and their combination in MDA-MB-231 triple-negative breast cancer cell line. Drug growth inhibition 50 (GI50) was determined using the MTT assay and the obtained results were analyzed using CompuSyn 3.0.1 software. MDA-MB-231 cells were divided into four treatment groups including a positive control, Dasatinib-treated, Celecoxib-treated, and combination-treated groups. Standard sandwich ELISA was used for the determination of the protein levels of c-Src, Bcl-2, p-AKT, FAK, PGE2, VEGF, and cyclin D1. Active caspase-3 was determined colorimetrically and the expression of COX-2 and c-Src genes was quantitatively determined via quantitative real-time polymerase chain reaction. The GI50 for Dasatinib was 0.05699 µM while that for Celecoxib was 69.0976 µM. Dasatinib up-regulated c-Src gene while Celecoxib and Dasatinib/Celecoxib combination down-regulated such expression level. COX-2 gene was down-regulated by Celecoxib while it was up-regulated by both Dasatinib and Dasatinib/Celecoxib combination. On one hand, Dasatinib, Celecoxib, and their combination significantly reduced the protein levels of c-Src, Bcl-2, p-AKT, FAK, PGE2, VEGF, and cyclin D1. On the other hand, they elevated active caspase-3. To sum up, Dasatinib/Celecoxib combination increased the capability for apoptosis and suppressed proliferation, angiogenesis, migration, and invasion suggesting a strong cross-talk between Src signaling cascade and COX-2/PGE2 via the intermediate PI3K/AKT/mTOR pathway. Further in-vitro and in-vivo studies are warranted to verify the present findings.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1180-1180
Author(s):  
Manuel Quadri ◽  
Claudia Saitta ◽  
Sonia Palamini ◽  
Chiara Palmi ◽  
Andrea Biondi ◽  
...  

Abstract Background. Although the Event Free Survival for Childhood Acute lymphoblastic leukemia (ALL) reaches 85%, the remaining 15% of patients relapse, and 25-40% of them die. Novel molecular targets may increase the efficacy of therapy and reduce treatment toxicity. Among B-other ALL patients, JAK/STAT pathway alterations represent about 7% of the 'Philadelphia-like' cases. JAK2 gene encodes for a non-receptor tyrosine kinase fundamental for hematopoiesis, cellular proliferation and survival. In last years, JAK2 mutations have been widely studied in leukemia and lymphoma, whereas JAK2 fusion genes are still poorly characterized. Aim. This study aims to identify JAK2 fusion genes among BCP-ALL pediatric patients and develop a target strategy in in vitro and in vivo preclinical models. Methods. A targeted capture RNA Next Generation Sequencing strategy was applied to discover JAK2 fusion genes in a large cohort of PCR-based MRD high risk (HR) BCP-ALL pediatric patients. Fusions were validated by RT-PCR and/or FISH. Primary patients' cells have been in vivo expanded in NSG mice. We performed ex vivo and in vivo drug treatments with JAK2 inhibitors; phosphoflow and apoptosis-viability assays were performed in patients' blasts in co-culture with human bone marrow stroma. Results. We identified 10 pediatric cases carrying a JAK2 fusion gene with different partners in single cases, such as ATF7IP, ZEB2, MPRIP, BCR, TLE4, GIT2 and RAB7, in addition to PAX5, which was the only recurrent in three cases. Cells were available from 3 cases, carrying PAX5-JAK2, ATF7IP-JAK2 and ZEB2-JAK2, respectively. After in vivo expansion, we demonstrated that the JAK2 signaling pathway was active at basal level, through phosphorylation on Y1007-1008 JAK2 residues inside the catalytic activation loop, compared to cases wild type for JAK2 and CRLF2 (+70%, two-tailed P value 0.0355); a positive trend was also shown compared to primary cells with P2RY8-CRLF2 rearrangements and JAK2 mutation, as positive controls (+40% two-tailed P value 0.158). The JAK2 downstream effectors pS727-STAT3 and pY694-STAT5 were also activated. We thus setup a JAK2 targeted drug treatment using CHZ868, a new class-II tyrosine kinase inhibitor (TKI) (Novartis, Basel, CH). After 30 minutes of treatment, we appreciated a mean inhibition of -62% of Y1007-1008 JAK2 residues in PAX5-JAK2, -22% in ATF7IP-JAK2 and -35% in ZEB2- JAK2. Contemporarily, we observed a decrease of pS727-STAT3 (-35-50%) and pY694-STAT5 (-15-50%). After 48h monotherapy treatment by CHZ868, we detected apoptosis induction and cell viability decrease between 20- 75% at IC50. In combination with dexamethasone, we assessed a further decrease of viability between 10 to 95%. A biological variability among the three different patients was appreciated, according to the different partner genes. Exclusively for the PAX5-JAK2 fusion, we also performed treatments with the kinase inhibitor BIBF1120/Nintedanib, targeting LCK, which is activated downstream PAX5 fusions and we observed a 20% reduction of cell viability. Importantly, combination of BIBF1120 and CHZ868 showed a synergistic effect (-45%, at IC50). Moreover, we found that ruxolitinib caused autophagy as observed by higher levels of LC3-II compared to untreated cells (+ 45%, p<0.01), with consequent reduction of apoptosis induction. Indeed, active caspase 3 increased when ruxolitinib was given in combination with chloroquine, an autophagy inhibitor (+20% vs ruxolitinib alone, p<0.01). CHZ868 alone or in combination with chloroquine instead does not induce autophagy as LC3-II and active caspase 3 levels are the same of untreated cells. Finally, we demonstrated the in vivo efficacy of CHZ868 in patient derived xenograft model in presence of PAX5-JAK2 fusion. After two weeks of 30mg/Kg daily treatment of CHZ868, we observed a significant reduction of leukemic CD10+/CD19+ cells both in bone marrow (p<0.01, -43%), spleen (p<0.001, -72%), central nervous system (-40%) and peripheral blood (p<0.05, -46%), compared to vehicle mice. Further in vivo experiments are ongoing in other JAK2 fusion settings. Conclusion. CHZ868 is a promising candidate for treatment of BCP-ALL carrying JAK2 fusions, showing high efficacy and specificity, both ex vivo and in vivo. Further studies will include combination with standard chemotherapy drugs with the aim to maintain its efficacy by reducing the intensity and toxicity of chemotherapy. Disclosures Biondi: Novartis: Honoraria; Bluebird: Other: Advisory Board; Incyte: Consultancy, Other: Advisory Board; Colmmune: Honoraria; Amgen: Honoraria.


2021 ◽  
Vol 22 (11) ◽  
pp. 3607-3613
Author(s):  
Ambreen Ashfaque ◽  
Farina Hanif ◽  
Shabana Simjee ◽  
Muhammad Bari ◽  
Shaheen Faizi ◽  
...  

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Vishnu Raman ◽  
Nele Van Dessel ◽  
Christopher L. Hall ◽  
Victoria E. Wetherby ◽  
Samantha A. Whitney ◽  
...  

AbstractCritical cancer pathways often cannot be targeted because of limited efficiency crossing cell membranes. Here we report the development of a Salmonella-based intracellular delivery system to address this challenge. We engineer genetic circuits that (1) activate the regulator flhDC to drive invasion and (2) induce lysis to release proteins into tumor cells. Released protein drugs diffuse from Salmonella containing vacuoles into the cellular cytoplasm where they interact with their therapeutic targets. Control of invasion with flhDC increases delivery over 500 times. The autonomous triggering of lysis after invasion makes the platform self-limiting and prevents drug release in healthy organs. Bacterial delivery of constitutively active caspase-3 blocks the growth of hepatocellular carcinoma and lung metastases, and increases survival in mice. This success in targeted killing of cancer cells provides critical evidence that this approach will be applicable to a wide range of protein drugs for the treatment of solid tumors.


2021 ◽  
Author(s):  
Mohamed youssef ◽  
Nermine Moussa ◽  
Maged W. Helmy ◽  
Medhat Haroun

Abstract Aberrant activation of several signaling pathways has been implicated in prostate cancer (PCa) progression to castrate-resistant prostate cancer (CRPC). Phosphoinositide-3-kinase/Protein Kinase B/mechanistic Target of Rapamycin (PI3K/AKT/mTOR) and Hedgehog/GLI (Hh/GLI) pathways are major participants in progression to CRPC. In this sense, the current work aims to assess the potential antitumor effects resulting from co-targeting the aforementioned pathways in PC3 cells with Dactolisib as a dual PI3K/mTOR inhibitor and GANT61 as a GLI1 antagonist. Three replica of PC3 cells were assigned for four treatment groups; vehicle control, Dactolisib-treated, GANT61-treated, and combination-treated groups. GLI1 gene expression was determined by quantitative real-time PCR while active caspase-3 was determined colorimetrically. P-AKT, p70 ribosomal s6 protein kinase 1 (pS6K1), cyclin D1, vascular endothelial growth factor 1 (VEGF1), and Microtubule-associated proteins 1A/1B light chain 3 (LC3) protein levels were determined by ELISA technique. GLI1 gene expression was down-regulated as a result of Dactolisib, GANT61, and their combination. Additionally, both drugs significantly reduced p-AKT, pS6K1, cyclin D1, and VEGF1 protein levels. Dactolisib elevated LC3 protein levels and GANT61 augmented Dactolisib effect on LC3. Moreover, only Dactolisib/GANT61combination significantly increased active caspase-3 level. To sum up, Dactolisib/GANT61 combination was shown to be promising in PCa treatment. Further in-vitro and in-vivo studies are warranted to support our findings.


PLoS Biology ◽  
2021 ◽  
Vol 19 (8) ◽  
pp. e3001304
Author(s):  
Yingying Zhang ◽  
Kai Huang ◽  
Yuxia Zhang ◽  
Tao Han ◽  
Lang Li ◽  
...  

Tumor necrosis factor receptor-1 (TNFR1) signaling, apart from its pleiotropic functions in inflammation, plays a role in embryogenesis as deficiency of varieties of its downstream molecules leads to embryonic lethality in mice. Caspase-8 noncleavable receptor interacting serine/threonine kinase 1 (RIPK1) mutations occur naturally in humans, and the corresponding D325A mutation in murine RIPK1 leads to death at early midgestation. It is known that both the demise of Ripk1D325A/D325A embryos and the death of Casp8−/− mice are initiated by TNFR1, but they are mediated by apoptosis and necroptosis, respectively. Here, we show that the defects in Ripk1D325A/D325A embryos occur at embryonic day 10.5 (E10.5), earlier than that caused by Casp8 knockout. By analyzing a series of genetically mutated mice, we elucidated a mechanism that leads to the lethality of Ripk1D325A/D325A embryos and compared it with that underlies Casp8 deletion-mediated lethality. We revealed that the apoptosis in Ripk1D325A/D325A embryos requires a scaffold function of RIPK3 and enzymatically active caspase-8. Unexpectedly, caspase-1 and caspase-11 are downstream of activated caspase-8, and concurrent depletion of Casp1 and Casp11 postpones the E10.5 lethality to embryonic day 13.5 (E13.5). Moreover, caspase-3 is an executioner of apoptosis at E10.5 in Ripk1D325A/D325A mice as its deletion extends life of Ripk1D325A/D325A mice to embryonic day 11.5 (E11.5). Hence, an unexpected death pathway of TNFR1 controls RIPK1 D325A mutation-induced lethality at E10.5.


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