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It has been reported that CDK8 plays a key role in acute myeloid leukaemia. Here, a total of 40 compounds were rational designed and synthesised based on the previous SAR. Among them, compound 12 (3-(3-(furan-3-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide) showed the most potent inhibiting activity against CDK8 with an IC50 value of 39.2 ± 6.3 nM and anti AML cell proliferation activity (molm-13 GC50 = 0.02 ± 0.01 μM, MV4-11 GC50 = 0.03 ± 0.01 μM). Mechanistic studies revealed that this compound 12 could inhibit the phosphorylation of STAT-1 and STAT-5. Importantly, compound 12 showed relative good bioavailability (F = 38.80%) and low toxicity in vivo. This study has great significance for the discovery of more efficient CDK8 inhibitors and the development of drugs for treating AML in the future. [ABSTRACT FROM AUTHOR]

Abstract Lenvatinib, a multitarget kinase inhibitor, has been proven to be effective in the treatment of advanced hepatocellular carcinoma. It has been previously demonstrated that tumour associated macrophages (TAMs) in tumour tissues can promote HCC growth, invasion and metastasis. Furthermore, lenvatinib has certain immunomodulatory effects on the treatment of HCC. However, the role of lenvatinib in macrophage polarization during HCC treatment has not been fully explored. In this study, we used a variety of experimental methods both in vitro and in vivo to investigate the effect of lenvatinib on TAMs during HCC progression. This study is the first to show that lenvatinib can alter macrophage polarization in both humans and mice. Moreover, macrophages treated with lenvatinib in vitro displayed enhanced classically activated macrophages (M1) activity and suppressed liver cancer cell proliferation, invasion, and migration. Furthermore, during the progression of M1 macrophage polarization induced by lenvatinib, STAT-1 was the main target transcription factor, and inhibiting STAT-1 activity reversed the effect of lenvatinib. Overall, the present study provides a theoretical basis for the immunomodulatory function of lenvatinib in the treatment of HCC.

Lenvatinib, a multitarget kinase inhibitor, has been proven to be effective in the treatment of advanced hepatocellular carcinoma. It has been previously demonstrated that tumour associated macrophages (TAMs) in tumour tissues can promote HCC growth, invasion and metastasis. Furthermore, lenvatinib has certain immunomodulatory effects on the treatment of HCC. However, the role of lenvatinib in macrophage polarization during HCC treatment has not been fully explored. In this study, we used a variety of experimental methods both in vitro and in vivo to investigate the effect of lenvatinib on TAMs during HCC progression. This study is the first to show that lenvatinib can alter macrophage polarization in both humans and mice. Moreover, macrophages treated with lenvatinib in vitro displayed enhanced classically activated macrophages (M1) activity and suppressed liver cancer cell proliferation, invasion, and migration. Furthermore, during the progression of M1 macrophage polarization induced by lenvatinib, STAT-1 was the main target transcription factor, and inhibiting STAT-1 activity reversed the effect of lenvatinib. Overall, the present study provides a theoretical basis for the immunomodulatory function of lenvatinib in the treatment of HCC. [ABSTRACT FROM AUTHOR]

Programmed cell death protein‐1/programmed death‐ligand‐1 (PD‐1/PDL‐1) signalling pathway has gained attention in prostate cancer. The relationship between pSTAT‐1, pSTAT‐3 expressions and PTEN loss with PDL‐1 expression was assessed and the effects of the pathways on prostate cancer prognosis were evaluated. Patients who underwent radical prostatectomy between 2011 and 2017 were included in our study. Prostatectomy materials were evaluated using immunohistochemical staining of pSTAT‐1, pSTAT‐3, PTEN, and PDL‐1. The relationship between PDL‐1 and pSTAT‐1, pSTAT‐3 expressions and PTEN loss was evaluated. Additionally, factors affecting biochemical recurrence‐free survival and clinical progression‐free survival were analysed. Within100 patients, 9 of 11 patients with PDL‐1 expression also had intermediate‐high pSTAT‐1 staining intensity, and those with PDL‐1 expression had higher pSTAT‐1 staining intensity than those without (81.9% vs. 56.2%, p = 0.014). In univariate analysis, pSTAT‐1, pSTAT‐3 and PDL‐1 expressions had significant impact on biochemical recurrence‐free and clinical progression‐free survival. In multivariate analysis, pSTAT‐1 staining intensity with radical prostatectomy ISUP grade in terms of biochemical recurrence‐free survival and the pSTAT‐1 H‐score with radical prostatectomy ISUP grade in terms of clinical progression‐free survival were independent risk factors. Moderate‐high expression of pSTAT‐1 was closely associated with PDL‐1 expression, and pSTAT‐1 was also a predictor of biochemical recurrence and clinical progression. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi is the causative protozoan of Chagas' Disease, a neglected tropical disease that affects 6-7 million people worldwide. Interaction of the parasite with the host immune system is a key factor in disease progression and chronic symptoms. Although the human immune system is capable of controlling the disease, the parasite has numerous evasion mechanisms that aim to maintain intracellular persistence and survival. Due to the pronounced genetic variability of T. cruzi, co-infections or mixed infections with more than one parasite strain have been reported in the literature. The intermodulation in such cases is unclear. This study aimed to evaluate the co-infection of T. cruzi strains G and CL compared to their individual infections in human macrophages derived from THP-1 cells activated by classical or alternative pathways. Flow cytometry analysis demonstrated that trypomastigotes were more infective than extracellular amastigotes (EAs) and that strain G could infect more macrophages than strain CL. Classically activated macrophages showed lower number of infected cells and IL-4-stimulated cells displayed increased CL-infected macrophages. However, co-infection was a rare event. CL EAs decreased the production of reactive oxygen species (ROS), whereas G trypomastigotes displayed increased ROS detection in classically activated cells. Co-infection did not affect ROS production. Monoinfection by strain G or CL mainly induced an anti-inflammatory cytokine profile by decreasing inflammatory cytokines (IFN-g, TNF-a, IL-1b) and/or increasing IL-4, IL-10, and TGF-b. Co-infection led to a predominant inflammatory milieu, with reduced IL-10 and TGF-b, and/or promotion of IFN-g and IL-1b release. Infection by strain G reduced activation of intracellular signal transducer and activator of transcription (STAT) factors. In EAs, monoinfections impaired STAT-1 activity and promoted phosphorylation of STAT-3, both changes may prolong cell survival. Coinfected macrophages displayed pronounced activation of all STATs examined. These activations likely promoted parasite persistence and survival of infected cells. The collective results demonstrate that although macrophages respond to both strains, T. cruzi can modulate the intracellular environment, inducing different responses depending on the strain, parasite infective form, and co-infection or monoinfection. The modulation influences parasite persistence and survival of infected cells. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi is the causative protozoan of Chagas’ Disease, a neglected tropical disease that affects 6−7 million people worldwide. Interaction of the parasite with the host immune system is a key factor in disease progression and chronic symptoms. Although the human immune system is capable of controlling the disease, the parasite has numerous evasion mechanisms that aim to maintain intracellular persistence and survival. Due to the pronounced genetic variability of T. cruzi, co-infections or mixed infections with more than one parasite strain have been reported in the literature. The intermodulation in such cases is unclear. This study aimed to evaluate the co-infection of T. cruzi strains G and CL compared to their individual infections in human macrophages derived from THP-1 cells activated by classical or alternative pathways. Flow cytometry analysis demonstrated that trypomastigotes were more infective than extracellular amastigotes (EAs) and that strain G could infect more macrophages than strain CL. Classically activated macrophages showed lower number of infected cells and IL-4-stimulated cells displayed increased CL-infected macrophages. However, co-infection was a rare event. CL EAs decreased the production of reactive oxygen species (ROS), whereas G trypomastigotes displayed increased ROS detection in classically activated cells. Co-infection did not affect ROS production. Monoinfection by strain G or CL mainly induced an anti-inflammatory cytokine profile by decreasing inflammatory cytokines (IFN-γ, TNF-α, IL-1β) and/or increasing IL-4, IL-10, and TGF-β. Co-infection led to a predominant inflammatory milieu, with reduced IL-10 and TGF-β, and/or promotion of IFN-γ and IL-1β release. Infection by strain G reduced activation of intracellular signal transducer and activator of transcription (STAT) factors. In EAs, monoinfections impaired STAT-1 activity and promoted phosphorylation of STAT-3, both changes may prolong cell survival. Coinfected macrophages displayed pronounced activation of all STATs examined. These activations likely promoted parasite persistence and survival of infected cells. The collective results demonstrate that although macrophages respond to both strains, T. cruzi can modulate the intracellular environment, inducing different responses depending on the strain, parasite infective form, and co-infection or monoinfection. The modulation influences parasite persistence and survival of infected cells.

Signal transducer and activator of transcription (STAT)1 heterozygous gain‐of‐function (GOF) mutations are known to induce immune dysregulation and chronic mucocutaneous candidiasis (CMCC). Previous reports suggest an association between demodicosis and STAT1 GOF. However, immune characterization of these patients is lacking. Here, we present a retrospective analysis of patients with immune dysregulation and STAT1 GOF who presented with facial and ocular demodicosis. In‐depth immune phenotyping and functional studies were used to characterize the patients. We identified five patients (three males) from two non‐consanguineous Jewish families. The mean age at presentation was 11.11 (range = 0.58–24) years. Clinical presentation included CMCC, chronic demodicosis and immune dysregulation in all patients. Whole‐exome and Sanger sequencing revealed a novel heterozygous c.1386C>A; p.S462R STAT1 GOF mutation in four of the five patients. Immunophenotyping demonstrated increased phosphorylated signal transducer and activator of transcription in response to interferon‐α stimuli in all patients. The patients also exhibited decreased T cell proliferation capacity and low counts of interleukin‐17‐producing T cells, as well as low forkhead box protein 3+ regulatory T cells. Specific antibody deficiency was noted in one patient. Treatment for demodicosis included topical ivermectin and metronidazole. Demodicosis may indicate an underlying primary immune deficiency and can be found in patients with STAT1 GOF. Thus, the management of patients with chronic demodicosis should include an immunogenetic evaluation. [ABSTRACT FROM AUTHOR]

Interaction between thyroid hormones and the immune system is reported in the literature. Thyroid hormones, thyroxine, T4, but also T3, act non-genomically through mechanisms that involve a plasma membrane receptor αvβ3 integrin, a co-receptor for insulin-like growth factor-1 (IGF-1). Previous data from our laboratory show a crosstalk between thyroid hormones and IGF-1 because thyroid hormones inhibit the IGF-1-stimulated glucose uptake and cell proliferation in L-6 myoblasts, and the effects are mediated by integrin αvβ3. IGF-1 also behaves as a chemokine, being an important factor for tissue regeneration after damage. In the present study, using THP-1 human leukemic monocytes, expressing αvβ3 integrin in their cell membrane, we focused on the crosstalk between thyroid hormones and either IGF-1 or monocyte chemoattractant protein-1 (MCP-1), studying cell migration and proliferation stimulated by the two chemokines, and the role of αvβ3 integrin, using inhibitors of αvβ3 integrin and downstream pathways. Our results show that IGF-1 is a potent chemoattractant in THP-1 monocytes, stimulating cell migration, and thyroid hormone inhibits the effect through αvβ3 integrin. Thyroid hormone also inhibits IGF-1-stimulated cell proliferation through αvβ3 integrin, an example of a crosstalk between genomic and non-genomic effects. We also studied the effects of thyroid hormone on cell migration and proliferation induced by MCP-1, together with the pathways involved, by a pharmacological approach and docking simulation. Our findings show a different downstream signaling for IGF-1 and MCP-1 in THP-1 monocytes mediated by the plasma membrane receptor of thyroid hormones, integrin αvβ3.

The disease burden of sepsis continues to increase, with intraabdominal contamination being a significant source of infection. Sepsis is a syndrome involving both an increase in systemic inflammation as well as a regulatory component. We have previously demonstrated that neutrophils are significant IL-10 producers in the abdomen during sepsis. Here, we sought to further characterize these neutrophils and elucidate potential underlying mechanisms resulting in IL-10 generation. Using transcriptional reporter mice, we observed that IL-10 producing neutrophils were activated, non-apoptotic, and expressed C-X-C chemokine receptor type 4-expressing. Further, we observed that active Signal Transducer and Activator of Transcription 1 expression was significantly increased in IL-10 producing versus non-IL-10 producing neutrophils. During sepsis, IFN-γ blockade lead to a decrease of neutrophil IL-10 production, while peritoneal CD4 T cells were found to be the most numerous acute producers of IFN-γ. Altogether, this report demonstrates that during sepsis, mature neutrophils can potentially dampen local inflammation by IL-10 production and this can be orchestrated by CD4 T cells through an IFN-γ dependent manner. Image 1 • During sepsis, IL-10pos neutrophils are activated, non-apoptotic and express CXCR4. • Active STAT-1 expression is significantly increased in IL-10 producing neutrophils. • IFN-γ blockade leads to a decrease of neutrophil IL-10 production during sepsis. • Peritoneal CD4 T cells are the most numerous producers of IFN-γ in acute sepsis. [ABSTRACT FROM AUTHOR]

Acute lung injury is a major complication of hemorrhagic shock and the required resuscitation with large volumes of crystalloid fluids and blood products. We previously identified a role of macrophagederived chemokine (CCL22/MDC) pulmonary inflammation following hemorrhage and resuscitation. However, further details regarding the induction of CCL22/MDC and its precise role in pulmonary inflammation after trauma remain unknown. In the current study we used in vitro experiments with a murine alveolar macrophage cell line, as well as an in vivo mouse model of hemorrhage and resuscitation, to identify key regulators in CCL22/MDC production. We show that trauma induces expression of IFNγ, which leads to production of CCL22/MDC through a signaling mechanism involving p38 MAPK, NF-κB, JAK, and STAT-1. IFNγ also activates TNFα production by alveolar macrophages, potentiating CCL22/MDC production via an autocrine mechanism. Neutralization of IFNγ or TNFα with specific antibodies reduced histological signs of pulmonary injury after hemorrhage and reduced inflammatory cell infiltration into the lungs. [ABSTRACT FROM AUTHOR]

Fluconazole is first-line treatment for mucocutaneous candida infections in CMC patients regardless of subtype.6 CMC patients often require suppressive antifungal therapy and may develop azole-resistant infections.6 For patients with azole-unresponsive infections, immune modulators may be helpful. Keywords: chronic mucocutaneous candidiasis; genodermatosis; STAT-1 EN chronic mucocutaneous candidiasis genodermatosis STAT-1 e58 e60 3 12/23/21 20211101 NES 211101 CASE REPORT A 28-year-old Bangladeshi man was referred to dermatology for evaluation of a more than 25-year history of dorsal tongue pain that worsened with spicy or hot food and interfered with oral intake and brushing his teeth. DISCUSSION Chronic mucocutaneous candidiasis (CMC) represents a heterogeneous disorder characterized by chronic, non-invasive candida and dermatophyte infections of the mucous membranes skin, and nails. [Extracted from the article]

Lipid droplets (LD) are newly characterized dynamic cytoplasmic organelle which is the storehouse of different immunosuppressive cytokines and enzymes like cyclooxygenase and lipoxygenase. Tumors are known to modulate the immune system by immune-editing the microenvironment. Immuno-editing comprises of three steps namely cancer immune-surveillance, tumor dormancy and finally escape leading to tumor development. The latency of the tumor microenvironment is greatly contributed by the M2 polarized macrophages and TGF-β is a prime culprit. Modulating M2 macrophages to M1 can be a strategy against tumor progression. We found that tumor-conditioned medium or recombinant TGF-β was efficient to induce LD formation in Raw264.7 cells and the inhibition of LD was associated with the switch of M2 to M1 phenotype involving MEK1/2 axis. Signature molecules of M2 polarized macrophages like CD206 were also downregulated while co-stimulatory molecules like CD80, CD86 were up-regulated along with enhanced surface expression of MHCII when these macrophages were subjected to C75 treatment to reduce the LD formation. The level of pro-inflammatory cytokine, as well as ROS and NO generation, were also increased when TGF-β treated macrophages were subjected to C75 treatment. This study is probably the first report of this kind and can be used in the future in cancer treatment. Unlabelled Image • Tumor exudates and TGF-β can induce Lipid droplet(LD) formation in macrophages. • LD formation was found to be Erk mediated through MEK pathway. • Those cells developing LD are M2 biased. • M2 biasness is mainly contributed by down-regulation of Stat1. • Inhibition of LD formation shifted M2 macrophages to M1 phenotype. [ABSTRACT FROM AUTHOR]

Aim: Imbalance of M1/M2 macrophages phenotype activation is a key point in diabetic nephropathy (DN). This study aimed to investigate whether active vitamin D (VD) suppresses macrophage transition to the M1 phenotype via inhibiting the high glucose‐induced STAT‐1 phosphorylation to reduce TREM‐1 expression. Methods: In vivo, pathological changes in kidney tissue were detected and the expression of CD68 TREM‐1, STAT‐1, M1 makers, and M2 makers were acquired in renal tissue of patients with DN and 18w DN rats. In vitro, RAW 264.7 cells were incubated in the presence of high glucose with or without VD. Silencing and overexpression of TREM‐1 and silencing and activate of STAT‐1 were explored to elucidate the underlying mechanism. The expression of TREM‐1 and STAT‐1 and the changes of macrophage phenotype were examined separately by western blot and immunofluorescence staining. Results: (a) Expression of TREM‐1, p‐STAT‐1, and M1 markers (iNOS and TNF‐α) were increased and positively correlated in kidneys from patients with DN. (b) In DN rats, the enlargement of glomerular surface area, expansion of glomerular mesangial matrix, the expression of CD68, TREM‐1, p‐STAT‐1, and M1 marker (iNOS) were significantly increased in comparison with the normal control group, whereas above changes were markedly decreased in the diabetic group treated with the VD group. (c) In vitro, VD significantly decreased high glucose‐induced CD68, TREM‐1, p‐STAT‐1, and M1 marker (iNOS) expression. However, above‐mentioned effects of VD are abolished when TREM‐1 is overexpressed or STAT‐1 is activated. Reductions in STAT‐1 expression decreased the TREM‐1 expression. Conclusion: VD can inhibit macrophage transition to the M1 phenotype through the STAT‐1/TREM‐1 pathway. Vitamin D can inhibit macrophage transition to the M1 phenotype through the STAT‐1/TREM‐1 pathway. [ABSTRACT FROM AUTHOR]

Sepsis is a major cause of neonatal morbidity and mortality. The current paradigm suggests that neonatal susceptibility to infection is explained by an innate immune response that is functionally immature. Recent studies in adults have questioned a therapeutic role for IFNβ in sepsis; however, the role of IFNβ in mediating neonatal sensitivity to sepsis is unknown. We evaluated the transcriptional regulation and expression of IFNβ in early neonatal (P0) and adult murine models of endotoxemia (IP LPS, 5 mg/kg). We found that hepatic, pulmonary, and serum IFNβ expression was significantly attenuated in endotoxemic neonates when compared to similarly exposed adults. Furthermore, endotoxemia induced hepatic p65/NFκB and IRF3 activation exclusively in adults. In contrast, endotoxemia induced immunotolerant p50/NFκB signaling in neonatal mice without evidence of IRF3 activation. Consistent with impaired IFNβ expression and attenuated circulating serum levels, neonatal pulmonary STAT1 signaling and target gene expression was significantly lower than adult levels. Using multiple in vivo approaches, the source of hepatic IFNβ expression in endotoxemic adult mice was determined to be the hepatic macrophage, and experiments in RAW 264.7 cells confirmed that LPS-induced IFNβ expression was NFκB dependent. Finally, treating neonatal mice with IFNβ 2 h after endotoxemia stimulated pulmonary STAT1 signaling and STAT1 dependent gene expression. Furthermore, IFNβ treatment of endotoxemic neonatal animals resulted in significantly improved survival following exposure to lethal endotoxemia. In conclusion, endotoxemia induced IFNβ expression is attenuated in the early neonatal period, secondary to impaired NFκB-p65/IRF3 signaling. Pre-treatment with IFNβ decreases neonatal sensitivity to endotoxemia. These results support further study of the role of impaired IFNβ expression and neonatal sensitivity to sepsis.

Background: Rilpivirine (RPV) is an antiretroviral drug characterized by good tolerability and a favorable liver safety profile. Recent research has shown that RPV ameliorates liver fibrosis in animal models of various chronic liver diseases. Our study aimed to analyze the effect of RPV on liver fibrosis by assessing changes in liver stiffness using transient elastography., Methods: Retrospective cohort study of HIV-infected patients who were exposed and not exposed to RPV. The change in liver stiffness during the period between two transient elastography measurements was analyzed and compared for patients exposed and not exposed to RPV., Results: We selected 118 RPV-exposed and 118 non-RPV-exposed HIV-infected patients. Median time between transient elastography (TE) measurements was 50 (29-68) months. A repeated-measures general linear model based on the main clinical characteristics revealed a significant decrease in the TE value of -0.8kPa in non-RPV-exposed patients (p=0.254) and -1.6kPa in the RPV-exposed group (p<0.001). The subgroup analysis showed a significant reduction in the TE value only patients cured of hepatitis C (RPV-exposed, -2.8kPa [p<0.001]; non-RPV-exposed, -1.1kPa [p=0.22])., Conclusion: RPV-based antiretroviral regimens significantly reduced liver stiffness, as measured by TE, in patients cured of chronic hepatitis C., (Copyright © 2022 Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. Published by Elsevier España, S.L.U. All rights reserved.)

The purpose of this study was to determine the efficiency of STAT-1 and STAT-3 proteins that exist in hepatic tissue of broilers as a basis for making synthetic STAT protein that would promote broiler growth and is safe for humans. This study used 25 male Lohman broilers. The broilers were placed in battery cages with capacity of one broiler per cage and fed twice daily (10% less than the standard feed) at 6:00 AM and 6:00 PM. At the age of 21 days, the broilers were sacrificed by cutting the esophagus, trachea, carotid artery and jugular vein, and a sample of 5 g hepatic tissue was obtained to examine the amino acid sequence of proteins STAT-1 and STAT-3 by MALDI-TOP method. Results showed that molecular weights of STAT-1 and STAT-3 were 59.3 kDa and 59.4 kDa, respectively. Identifying the amino acid structure of the STAT protein can allow for the creation of STAT synthetic protein, which is expected to prolong the action or effect of growth hormone, thus, promoting growth of broilers. [ABSTRACT FROM AUTHOR]

Sepsis is a major cause of neonatal morbidity and mortality. The current paradigm suggests that neonatal susceptibility to infection is explained by an innate immune response that is functionally immature. Recent studies in adults have questioned a therapeutic role for IFNβ in sepsis; however, the role of IFNβ in mediating neonatal sensitivity to sepsis is unknown. We evaluated the transcriptional regulation and expression of IFNβ in early neonatal (P0) and adult murine models of endotoxemia (IP LPS, 5 mg/kg). We found that hepatic, pulmonary, and serum IFNβ expression was significantly attenuated in endotoxemic neonates when compared to similarly exposed adults. Furthermore, endotoxemia induced hepatic p65/NFκB and IRF3 activation exclusively in adults. In contrast, endotoxemia induced immunotolerant p50/NFκB signaling in neonatal mice without evidence of IRF3 activation. Consistent with impaired IFNβ expression and attenuated circulating serum levels, neonatal pulmonary STAT1 signaling and target gene expression was significantly lower than adult levels. Using multiple in vivo approaches, the source of hepatic IFNβ expression in endotoxemic adult mice was determined to be the hepatic macrophage, and experiments in RAW 264.7 cells confirmed that LPS-induced IFNβ expression was NFκB dependent. Finally, treating neonatal mice with IFNβ 2 h after endotoxemia stimulated pulmonary STAT1 signaling and STAT1 dependent gene expression. Furthermore, IFNβ treatment of endotoxemic neonatal animals resulted in significantly improved survival following exposure to lethal endotoxemia. In conclusion, endotoxemia induced IFNβ expression is attenuated in the early neonatal period, secondary to impaired NFκB-p65/IRF3 signaling. Pre-treatment with IFNβ decreases neonatal sensitivity to endotoxemia. These results support further study of the role of impaired IFNβ expression and neonatal sensitivity to sepsis. [ABSTRACT FROM AUTHOR]

Glioma is the most common primary brain tumor in adults and the second most common malignant tumor in children. Aberrant expression of signal transducer and activator of transcription 1 (STAT‑1) and p53 are known to affect the occurrence and progression of malignant tumors. The aim of the present study was to investigate the expression of STAT‑1 and mutant p53 gene, as well as their correlation, in patients with glioma. The present study included 50 patients who underwent glioma resection at the First Affiliated Hospital of Inner Mongolia Medical University between December 2007 and December 2011, and 10 patients with acute cerebral contusion who underwent intracerebral hematoma removal at the same hospital between January 2013 and January 2014. The expression of STAT‑1 and mutant p53 protein in patients with different grades of glioma was assessed by immunohistochemistry. Spearman's correlation coefficient was employed to examine the correlation between STAT‑1 and the grade of glioma, and mutant p53 expression. The results demonstrated that the mean expression of STAT‑1 in glioma was significantly lower compared with normal brain tissue (P<0.05). However, there was no significant difference in the STAT‑1 positive expression rate between the two groups (χ2=1.38, P>0.05). The expression score (P<0.05) and positive expression rate (χ2=31.27, P<0.05) of mutant p53 in glioma was significantly higher compared with those in normal brain tissue. Statistical analysis revealed a negative correlation between STAT‑1 expression and the grade of glioma (r=‑0.767, P<0.05). In addition, mutant p53 expression was negatively correlated with STAT‑1 expression in glioma (r=‑0.876, P<0.05). The observed negative correlation between STAT‑1 and the pathological grade of glioma suggested an association between STAT‑1 and the occurrence and development of glioma, thus revealing the potential of STAT‑1 as a diagnostic biomarker and therapeutic target for glioma. [ABSTRACT FROM AUTHOR]

Background: In earlier studies, it was shown that ex vivo Mycobacterium tuberculosis-infected type II alveolar epithelial cells generate de novo nitric oxide (NO), but the mycobactericidal quantity of NO was released only by stimulation of these cells with proinflammatory cytokines, i.e. IFN-γ, TNF-α and IL-1β. In the present communication, it was demonstrated that M. tuberculosis-infected/mycobacterial antigens stimulated cells utilize both, JAK-STAT and NF-κB pathways for the induction of inducible Nitric Oxide Synthase (iNOS) mRNA and NO production. Methods: Alveolar epithelial cell line A549 were either infected with M. tuberculosis or stimulated with M. tuberculosis components. Confocal microscopy, NO estimation and EMSA were performed on the infected/stimulated A549 cells. Results: Nuclear extracts prepared from M. tuberculosis infected A549 cells alone or stimulated with IFN-γ or a combination of three cytokines (IFN-γ, TNF-α and IL-1β) formed DNA protein complexes with probes from both −5.2kb region (specific for binding of STAT-1 protein) and −5.8kb region (specific for binding of both STAT-1 and NF-κB) of the iNOS promoter. However, TNF-α or IL-1β stimulated M. tuberculosis-infected A549 cells showed no protein DNA complexes with construct from −5.2kb region. Conclusions: This differential response indicated that TNF-α/IL-1β does not allow STAT-1 production or its translocation to nucleus in M. tuberculosis-infected A549 cells in the absence of IFN-γ. This differential signaling of iNOS induction in M. tuberculosis-infected alveolar epithelial cells by cytokines may be responsible for controlled production of NO intracellularly.

Since interferon-γ (IFN-γ) tunes both innate and adaptive immune systems, it was expected to enter clinical practice as an immunomodulatory drug. However, the use of IFN-γ has been limited by its dose-dependent side effects. Low-dose medicine, which is emerging as a novel strategy to treat diseases, might circumvent this restriction. Several clinical studies have proved the efficacy of therapies with a low dose of cytokines subjected to kinetic activation, while no in vitro data are available. To fill this gap, we investigated whether low concentrations, in the femtogram range, of kinetically activated IFN-γ modulate the behavior of Jurkat cells, a widely used experimental model that has importantly contributed to the present knowledge about T cell signaling. In parallel, IFN-γ in the nanogram range was used and shown to activate Signal transducer and activator of transcription (STAT)-1 and then to induce suppressor of cytokine signaling-1 (SOCS-1), which inhibits downstream signaling. When added together, femtograms of IFN-γ interfere with the transduction cascade activated by nanograms of IFN-γ by prolonging the activation of STAT-1 through the downregulation of SOCS-1. We conclude that femtograms of IFN-γ exert an immunomodulatory action in Jurkat cells. [ABSTRACT FROM AUTHOR]

CDK8 plays a key role in acute myeloid leukemia, colorectal cancer and other cancers. Here, a total of 54 compounds were designed and synthesized. Among them, the most potent one compound 43 (3-(1H-pyrrolo[2,3-b]pyridin- 5-yl)benzamide), a novel CDK8 Ⅰ inhibitor, showed strong inhibitory activity against CDK8 (IC 50 = 51.9 nM), good kinase selectivity, good anti AML cell proliferation activity (molm-13 GC 50 = 1.57 ± 0.59 μM) and low toxicity in vivo (acute toxicity: 2000 mg/kg). Further mechanistic studies revealed that this compound could target CDK8 and then phosphorylate STAT-1 and STAT-5 thereby inhibiting of AML cell proliferation. In addition, compound 43 showed relatively good bioavailability (F = 28.00%) and could inhibit the growth of AML tumors in a dose-dependent manner in vivo. This study facilitates the further development of more potent CDK8 inhibitors for the treatment of the AML. [Display omitted] • New oral type Ⅰ CDK8 inhibitors were designed. • The structure-based optimization and SAR study were fully carried out. • Further mechanism against acute myeloid leukemia was explored. [ABSTRACT FROM AUTHOR]

Cystatin B is a cysteine protease inhibitor that induces HIV replication in monocyte-derived macrophages (MDM). This protein interacts with signal transducer and activator of transcription (STAT-1) factor and inhibits the interferon (IFN-β) response in Vero cells by preventing STAT-1 translocation to the nucleus. Cystatin B also decreases the levels of tyrosine-phosphorylated STAT-1 (STAT-1PY). However, the mechanisms of cystatin B regulation on STAT-1 phosphorylation in MDM are unknown. We hypothesized that cystatin B inhibits IFN-β antiviral responses and induces HIV replication in macrophage reservoirs through the inhibition of STAT-1 phosphorylation. Macrophages were transfected with cystatin B siRNA prior to interferon-β treatment or infected with HIV-ADA to determine the effect of cystatin B modulation in STAT-1 localization and activation using immunofluorescence and proximity ligation assays. Cystatin B decreased STAT-1PY and its transportation to the nucleus, while HIV infection retained unphosphorylated STAT (USTAT-1) in the nucleus avoiding its exit to the cytoplasm for eventual phosphorylation. In IFN-β-treated MDM, cystatin B inhibited the nuclear translocation of both, USTAT-1 and STAT-1PY. These results demonstrate that cystatin B interferes with the STAT-1 signaling and IFN-β-antiviral responses perpetuating HIV in macrophage reservoirs. [ABSTRACT FROM AUTHOR]

Objective: Current study was designed to determine the STAT-1 in co-infected patients of hepatitis B and C resistant to interferon therapy. Study Design: Cross-sectional analytical study. Place and Duration of Study: Department of Biochemistry & Molecular Biology & Gastroenterology departments of various hospitals of Rawalpindi. Material and Methods: The study included 15 co-infected patients of hepatitis B and C resistant to interferon therapy and 15 healthy individuals as control. Methodology: Detection of STAT-1 was done by conventional PCR technique. Results: Sixty seven percent of the patients were expressing STAT-1 in their blood while 33% of the patients did not have STAT-1. Controls showed 57% detection of STAT-1 and 43% did not exhibit STAT-1. Mean age of the patients and controls was 35.90 ± 8.95. Comparison between patients and controls was done by chi square test. Fisher exact probability value obtained was 0.287 which was not significant. Conclusion: Patients suffering from hepatitis B and C co-infection resistant to interferon therapy revealed higher detection of STAT-1 which indicate greater liver damage, fibrosis and an extensive and severer disease course in co-infection. [ABSTRACT FROM AUTHOR]

The cellular isoform of prion protein, PrPc, may confer neuroprotection in the brain, according to recent studies. To elucidate the role of PrPc in stroke pathology, we subjected PrPc-knockout (Prnp0/0), wild-type and PrPc-transgenic (tga20) mice to 30 min of intraluminal middle cerebral artery occlusion, followed by 3, 24 or 72 h reperfusion, and examined how PrPc levels influence brain injury and cell signaling. In immunohistochemical experiments and Western blots, we show that PrPc expression is absent in the brains of Prnp0/0 mice, detectable in wild-type controls and ∼4.0-fold elevated in tga20 mice. We provide evidence that PrPc deficiency increases infarct size by ∼200%, while transgenic PrPc restores tissue viability, albeit not above levels in wild-type animals. To elucidate the mechanisms underlying Prnp0/0-induced injury, we performed Western blots, which revealed increased activities of ERK-1/-2, STAT-1 and caspase-3 in ischemic brains of Prnp0/0 mice. Our data suggest a role of cytosolic signaling pathways in Prnp0/0-induced cell death.

Several primary immunodeficiency diseases affecting the interleukin 12/interferon gamma (IFN-gamma) pathway have been identified, most of them characterized by recurrent and protracted infections produced by intracellular microorganisms, particularly by several species of mycobacteria. In the present study we analyzed the expression of IFN-gamma receptor (IFN-gammaR) and signal transducer and activator of transcription 1 (STAT-1) in 4 children with Mycobacterium tuberculosis infection of uncommon clinical presentation. These molecules were evaluated by flow cytometry and Western blotting in B cells transformed with Epstein-Barr virus and mutations were scanned by single-strand conformational polymorphisms and DNA sequencing. The expression of IFN-gammaR1 was normal in all 4 patients. The genetic analysis of IFN-gammaR1 and IFN-gammaR2 coding sequences did not reveal any mutation. The expression of the STAT-1 molecule was similar in patients and healthy controls; however, when the phosphorylation of this transcription factor in response to IFN-gamma activation was evaluated by Western blot, a significant lower signal was evident in one patient. These data indicate that there are no alterations in the expression or function of the IFN-gammaR chains in these patients. However, the low level of STAT-1 phosphorylation found in one of these patients might be explained by a defect in one of the molecules involved in the signal transduction pathway after IFN-gamma interacts with its receptor. In the other three patients the inability to eliminate the mycobacteria may be due to a defect in another effector mechanism of the mononuclear phagocytes.

Alzheimer's disease (AD) is the most common neurodegenerative disorder worldwide, accounting for most cases of dementia in elderly individuals, and effective therapies are still lacking. This study was designed to investigate the anti-inflammatory properties of sulforaphane against Aβ 1–42 monomers in human THP-1 microglia-like cells. The results showed that sulforaphane preferentially inhibited cathepsin B- and caspase-1-dependent NLRP3 inflammasome activation induced by mostly Aβ 1–42 monomers, an effect that potently reduced excessive secretion of the proinflammatory cytokine interleukin-1β (IL-1β). Subsequent mechanistic studies revealed that sulforaphane mitigated the activation of signal transducer and activator of transcription–1 induced by Aβ 1–42 monomers. Sulforaphane also increased nuclear factor erythroid 2–related factor 2 (Nrf2) nuclear translocation, which was followed by upregulation of heme-oxygenase 1 (HO-1). The anti-inflammatory effect of sulforaphane on Aβ 1–42 -induced IL-1β production was diminished by small interfering RNA–mediated knockdown of Nrf2 or HO-1. Moreover, sulforaphane significantly attenuated the levels of microRNA-146a, which is selectively upregulated in the temporal cortex and hippocampus of AD brains. The aforementioned effects of sulforaphane were replicated by the tyrosine kinase inhibitor, herbimycin A, and Nrf2 activator. These results indicate that signal transducer and activator of transcription–1 dephosphorylation, HO-1 and its upstream effector, Nrf2, play a pivotal role in triggering an anti-inflammatory signaling cascade of sulforaphane that results in decreases of IL-1β release and microRNA-146a production in Aβ 1–42 -stimulated human microglia-like cells. These findings suggest that the phytochemical sulforaphane has a potential application in AD therapeutics. [ABSTRACT FROM AUTHOR]

Equine herpesvirus-1 (EHV-1) infects equine endothelial cells (EECs) lining the small blood vessels in the central nervous system. However, the effect of type I IFN on EHV-1 replication in the EECs is not well studied. Thus, the primary objective of this study was to investigate the effect of type-I IFN on the replication of the neuropathogenic T953 strain of EHV-1 in vitro in EECs. The initial data showed that the EHV-1 was partly resistant to the biological effect of exogenously supplied recombinant equine IFN-α. Subsequent investigation into the mechanism of resistance showed that EHV-1 infection of EECs interfered with the STAT-1 phosphorylation through which type-I IFN exerts its antiviral effect. Immunofluorescence staining showed interference with the translocation of STAT-1 molecules from cytoplasm to nucleus confirming the virus mediated suppression of STAT-1 activation. Downstream of the JAK-STAT signaling, EHV-1 infection inhibited expression of cellular antiviral proteins including IFN-stimulated gene 56 (ISG56) and viperin. Taken together these findings suggest that the neuropathogenic T953 strain of EHV-1 evades the host innate immune response by inhibiting IFN and this may provide some insight into the pathogenesis of EHV-1 infection. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the expression of STAT-1 (Signal Transducers and Activators of Transcription-1) in HCV patients non responder to interferon treatment. Study Design: Case control study. Place and Duration of Study: The research was carried out at Army Medical College Rawalpindi from January to July, 2012. Patients and Methods: The study after approved by institute's ethics committee was conducted on 15 HCV infected patients who were non responder to interferon therapy and 5 controls responder to interferon therapy. Their age, sex, body mass index (BMI) and marital status was noted. PCR based detection of STAT-1 mRNA was carried out in blood of HCV infected patients resistant to interferon therapy as well as controls. Data was presented in the form of frequencies and percentages and p values were calculated using Fisher exact test and student t-test. Results: Results showed that more males were resistant to interferon therapy as compared tofemales. The mean age was less in responders as compared to non responders. Forty percent of the HCV infected patients non responder to interferon therapy were positive for STAT-1 expression. Conclusion: STAT-1 blood expression can predict treatment response in HCV patients undergoing interferon treatment. [ABSTRACT FROM AUTHOR]

The aim of the present investigation was to delineate cytokine-induced signaling and death using the EndoC-βH1 cells as a model for primary human beta-cells. The cytokines IL-1β and IFN-γ induced a rapid and transient activation of NF-κB, STAT-1, ERK, JNK and eIF-2α signaling. The EndoC-βH1 cells died rapidly when exposed to IL-1β + IFN-γ, and this occurred also in the presence of the actinomycin D. Inhibition of NF-κB and STAT-1 did not protect against cell death, nor did the cytokines activate iNOS expression. Instead, cytokines promoted a rapid decrease in EndoC-βH1 cell respiration and ATP levels, and we observed protection by the AMPK activator AICAR against cytokine-induced cell death. It is concluded that EndoC-βH1 cell death can be prevented by AMPK activation, which suggests a role for ATP depletion in cytokine-induced human beta-cell death. [ABSTRACT FROM AUTHOR]

The severity of intestinal inflammation in mammals can be profoundly impacted by weaning stress. Cathelicidins protect intestinal homeostasis by not only directly killing bacteria but also immune regulators. Here, we investigated the effects of cathelicidin-BF (C-BF) derived from the snake venoms of Bungarus fasciatus on weaning stress and intestinal inflammation and examined the mechanisms by which C-BF modulates intestinal immune responses in weanling piglets. We found that C-BF treatment significantly increased performance and reduced the diarrheal index in weanling piglets. Serum IL-6, IL-22 and TNF-α production was decreased by C-BF treatment. We demonstrated that C-BF inhibited the expression of the inflammatory cytokines TNF-α, IL-6 and IL-8 but increased the expression of the anti-inflammatory cytokine IL-10 in the intestine. We also demonstrated that C-BF suppressed inflammation by down-regulating the nuclear factor-κB (NF-κB) signaling pathway in the intestine and in LPS-induced macrophages in vitro. However, C-BF significantly induced the phosphorylation of signal transducer and activator of transcription 1 (STAT-1) to enhance the phagocytosis of macrophages when inflammation was suppressed. In summary, our study demonstrated that C-BF suppressed intestinal inflammation by down-regulating the NF-κB signaling pathway and enhancing the phagocytosis of immune cells by activating STAT-1 during weaning. [ABSTRACT FROM AUTHOR]

The control of Trypanosoma cruzi infection is related to interferon- γ ( IFN- γ) activation leading to intracellular clearance of parasites. The transcription factor signal transducer and activator of transcription 1 ( STAT-1) is a key mediator of IFN- γ intracellular signalling and knockout of this protein leads to susceptibility to several intracellular microbes. To determine the role of STAT-1 in host susceptibility to T. cruzi infection we compared the survival, parasite loads and balance of IFN- γ and interleukin-10 ( IL-10) responses between wild-type and STAT-1 knockout mice. We found that the lack of STAT-1 resulted in a more robust infection, leading to higher levels of blood and tissue parasites and markedly reduced survival. In addition, infected STAT-1 knockout mice had higher systemic levels of both IFN- γ and IL-10, suggesting that the absence of STAT-1 leads to a disequilibrium of pro-inflammatory and anti-inflammatory cytokines. Analysis of spleen cells indicates that CD4, CD8 cells generate IFN- γ and natural killer cells express IL-13 in STAT-1 knockout animals. The production of IL-17 is particularly enhanced in the absence STAT-1 expression but did not reduce mortality. Overall these results indicate that STAT-1 is important for the control of T. cruzi infection in mice. [ABSTRACT FROM AUTHOR]

Wild Chinese horseshoe bats have been proven to be natural reservoirs of SARS-like coronaviruses. However, the molecular characterization of key proteins in bats still needs to be explored further. In this study, we used cloning and bioinformatics to analyze the sequence of RIG-I, STAT-1 and IFN-β in the immortalized cell lines from Rhinolophus affinis and Rhinolophus sinicus . Then, we treated different bat cells, mouse embryonic fibroblasts (MEF) and splenocytes with polyinosinic–polycytidylic acid (polyI:C) and vesicular stomatitis virus (VSV) to assess and compare antiviral immune responses between bats and mice. Our results demonstrated that bat RIG-I, STAT-1 and IFN-β showed close homology with human, mouse, pig and rhesus monkey. RIG-I and STAT-1 were both highly expressed in bat spleen. Furthermore, IFN-β was induced by polyI:C and VSV in both bat and mouse cells. These findings have provided new insight into the potential characteristics of the bat innate immune system against viral infection. [ABSTRACT FROM AUTHOR]

Protein malnutrition (PM) is an important public health problem that affects resistance to infection by impairing a number of physiological processes. PM induces structural changes in the lymphoid organs that affect the roles of the immune and inflammatory responses in a crucial way. The activation of different transcription factors, including signal transducer and activator of transcription (STAT) family members, leads to the production of different cytokines, which are mediators essential to mounting adequate immune and inflammatory responses. In this study, malnourished animals presented anemia, leukopenia, and a severe reduction in spleen cellularity, with reduced numbers of most cell populations, as well as increased percentages of CD3 and CD4 cells. The proliferation rates were reduced, and cells were increasingly observed in the G0/G1 cell cycle phase; further, IL-2 production was reduced, while IL-10 production was increased. In spleen cells from malnourished animals, STAT-3 protein expression was increased, with a concomitant reduction in STAT-1 expression. Knowing that STAT-1 and STAT-3 are key transcription factors in both immunity and inflammatory pathways, these results infer, at least in part, a mechanistic pathway that affects the manner or intensity of the immune response in malnourished individuals, increasing susceptibility to infection. [ABSTRACT FROM AUTHOR]

More than 20% of chronic hepatitis C (CHC) patients receiving interferon-alpha (IFN-α)-based anti-hepatitis C virus (HCV) therapy experienced significant depression, which was relieved by treatment with fluoxetine. However, whether and how fluoxetine affected directly the anti-HCV therapy remained unclear. Here, we demonstrated that fluoxetine inhibited HCV infection and blocked the production of reactive oxygen species (ROS) and lipid accumulation in Huh7.5 cells. Fluoxetine facilitated the IFN-α-mediated antiviral actions via activations of signal transducer and activator of transcription (STAT)-1 and c-Jun amino-terminal kinases (JNK). Alternatively, fluoxetine elevated peroxisome proliferator-activated receptor (PPAR) response element activity under HCV infection. The inhibitory effects of fluoxetine on HCV infection and lipid accumulation, but not production of ROS, were partially reversed by the PPAR-β, -γ, and JNK antagonists. Furthermore, fluoxetine intervention to the IFN-α-2b regimen facilitated to reduce HCV titer and alanine transaminase level for CHC patients. Therefore, fluoxetine intervention to the IFN-α-2b regimen improved the efficacy of anti-HCV treatment, which might be related to blockades of ROS generation and lipid accumulation and activation of host antiviral JNK/STAT-1 and PPARβ/γ signals. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Smooth muscle cell (SMC) de-differentiation is a key step that leads to pathological narrowing of blood vessels. De-differentiation involves a reduction in the expression of the SMC contractile genes that are the hallmark of quiescent SMCs. While there is considerable evidence linking inflammation to vascular diseases, very little is known about the mechanisms by which inflammatory signals lead to SMC de-differentiation. Given that the Signal Transducers and Activators of Transcription (STAT) transcriptional factors are the key signaling molecules activated by many inflammatory cytokines and growth factors, the aim of the present study was to determine if STAT transcriptional factors play a role SMC de-differentiation. Methods and results: Using shRNA targeted to STAT-1 and STAT-3, we show by real time RT-PCR and Western immunoblots that STAT-1 significantly reduces SMC contractile gene expression. In contrast, STAT-3 promotes expression of SMC contractile genes. Over-expression studies of STAT-1 and STAT-3 confirmed our observation that STAT-1 down-regulates whereas STAT-3 promotes SMC contractile gene expression. Bioinformatics analysis shows that promoters of all SMC contractile genes contain STAT binding sites. Finally, using ChIP analysis, we show that both STAT-1 and STAT-3 associate with the calponin gene. Conclusion: These data indicate that the balance of STAT-1 and STAT-3 influences the differentiation status of SMCs. Increased levels of STAT-1 promote SMC de-differentiation, whereas high levels of STAT-3 drive SMC into a more mature phenotype. Thus, inhibition of STAT-1 may represent a novel target for therapeutic intervention in the control of vascular diseases such as atherosclerosis and restenosis. [Copyright &y& Elsevier]

Abstract: Background: In earlier studies, it was shown that ex vivo Mycobacterium tuberculosis-infected type II alveolar epithelial cells generate de novo nitric oxide (NO), but the mycobactericidal quantity of NO was released only by stimulation of these cells with proinflammatory cytokines, i.e. IFN-γ, TNF-α and IL-1β. In the present communication, it was demonstrated that M. tuberculosis-infected/mycobacterial antigens stimulated cells utilize both, JAK-STAT and NF-κB pathways for the induction of inducible Nitric Oxide Synthase (iNOS) mRNA and NO production. Methods: Alveolar epithelial cell line A549 were either infected with M. tuberculosis or stimulated with M. tuberculosis components. Confocal microscopy, NO estimation and EMSA were performed on the infected/stimulated A549 cells. Results: Nuclear extracts prepared from M. tuberculosis infected A549 cells alone or stimulated with IFN-γ or a combination of three cytokines (IFN-γ, TNF-α and IL-1β) formed DNA protein complexes with probes from both −5.2kb region (specific for binding of STAT-1 protein) and −5.8kb region (specific for binding of both STAT-1 and NF-κB) of the iNOS promoter. However, TNF-α or IL-1β stimulated M. tuberculosis-infected A549 cells showed no protein DNA complexes with construct from −5.2kb region. Conclusions: This differential response indicated that TNF-α/IL-1β does not allow STAT-1 production or its translocation to nucleus in M. tuberculosis-infected A549 cells in the absence of IFN-γ. This differential signaling of iNOS induction in M. tuberculosis-infected alveolar epithelial cells by cytokines may be responsible for controlled production of NO intracellularly. [Copyright &y& Elsevier]

Abstract: The induction of nitric oxide synthase (iNOS) expression via the signal transducer and activator of transcription 1 (STAT-1) is involved in the mechanism of oxidative/nitrosative stress. We investigated whether acetylcholine (ACh) generates oxidative/nitrosative stress in bronchial epithelial cells during airway inflammation of COPD and evaluated the effects of Tiotropium, a once-daily antimuscarinic drug, and Olodaterol, a long-acting β2-agonist on these mechanisms. Human bronchial epithelial cells (16-HBE) were stimulated (4h, 37°C) with induced sputum supernatants (ISSs) from healthy controls (HC) (n=10), healthy smokers (HS) (n=10) or COPD patients (n=10), as well as with ACh (from 1μM to 100μM). The activation of STAT-1 pathway (STAT-1Ser727 and STAT-1Tyr701) and iNOS was evaluated in the cell lysates by Western blot analysis as well as nitrotyrosine levels by ELISA, while reactive oxygen species (ROS) were evaluated by flow cytometry. Finally, the effect of Tiotropium (Spiriva®) (100nM), alone or in combination with Olodaterol (1nM), was tested in this model. ISSs from COPD patients significantly increased the phosphorylation of STAT-1Ser727 and STAT-1Tyr701, iNOS and ROS/Nitrotyrosine when compared with ISSs from HC or HS subjects in 16-HBE cells. Furthermore, synthetic ACh increased all these parameters in stimulated 16HBE when compared with untreated cells. Tiotropium and Olodaterol reduced the oxidative/nitrosative stress generated by ACh and ISSs. We concluded that ACh mediated the oxidative/nitrosative stress involving the STAT-1 pathway activation in human bronchial epithelial cells during COPD. β2-Long acting and antimuscarinic drugs, normally used in the treatment of COPD as bronchodilator, might be able to control these cellular events. [Copyright &y& Elsevier]

Introduction: Periodontal disease (PD) is one of the most common inflammatory diseases, affecting about 10 % of the world population. The establishment of PD is influenced by polymorphisms in genes involved with the inflammatory response. Signal Transducer and Activator of Transcription (STAT)-1 is a transcription factor that plays a key role in the intracellular signaling triggered by cytokines and, thus, its activation is critical in inflammatory diseases. Aim and methods: We aim to evaluate the occurrence of association between STAT- 1 (rs3771300) polymorphism and distinct clinical forms and severity of PD; we genotyped 180 subjects using realtime PCR. Results and conclusion: We observed that the presence of the G allele for STAT- 1 was associated with twice as high of a chance to develop aggressive periodontitis, and the most severe form of the disease. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus ( SLE) is a systemic autoimmune disease with abnormal T cell immune responses. We hypothesized that aberrant expression of micro RNAs ( miRNAs) in T cells may contribute to the pathogenesis of SLE. First, we analysed the expression profiles of 270 human mi RNAs in T cells from five SLE patients and five healthy controls and then validated those potentially aberrant-expressed mi RNAs using real-time polymerase chain reaction ( PCR). Then, the expression of m RNAs regulated by these aberrant-expressed mi RNAs was detected using real-time PCR. Finally, mi RNA transfection into Jurkat T cells was conducted for confirming further the biological functions of these mi RNAs. The initial analysis indicated that seven mi RNAs, including mi R-145, mi R-224, mi R-513-5p, mi R-150, mi R-516a-5p, miR-483-5p and mi R-629, were found to be potentially abnormally expressed in SLE T cells. After validation, under-expressed mi R-145 and over-expressed mi R-224 were noted. We further found that STAT1 m RNA targeted by mi R-145 was over-expressed and apoptosis inhibitory protein 5 ( API5) m RNA targeted by mi R-224 was under-expressed in SLE T cells. Transfection of Jurkat cells with mi R-145 suppressed STAT1 and mi R-224 transfection suppressed API5 protein expression. Over-expression of mi R-224 facilitates activation-induced cell death in Jurkat cells. In the clinical setting, the increased transcript levels of STAT1 were associated significantly with lupus nephritis. In conclusion, we first demonstrated that mi R-145 and mi R-224 were expressed aberrantly in SLE T cells that modulated the protein expression of their target genes, STAT1 and API5, respectively. These mi RNA aberrations accelerated T cell activation-induced cell death by suppressing API5 expression and associated with lupus nephritis by enhancing signal transducer and activator of transcription-1 ( STAT)-1 expression in patients with SLE. [ABSTRACT FROM AUTHOR]

Double-stranded RNA-dependent protein kinase (PKR) is an interferon-induced protein that has been identified and characterized as a translational inhibitor in an interferon-regulated antiviral pathway. PKR is also reported to play important roles in the regulation of cell growth and differentiation. We have previously demonstrated that PKR inactivation suppresses osteoblast calcification and osteoclast formation. However, reports concerning the roles of PKR in chondrogenesis are limited. In this study, we have demonstrated that PKR is required for the in vitro differentiation of the mouse clonal chondrogenic cell line ATDC-5. ATDC-5 cells treated with insulin differentiated into chondrocytes and produced an alcian-blue-positive cartilage matrix. The protein expression of signal transducers and activators of transcription (STAT) peaked at day 7 of differentiation, whereas the expression of SRY-box-containing gene 9 (Sox-9), which is a transcription factor for chondrocyte differentiation, increased gradually. When the cells were treated with a PKR inhibitor (2-aminopurine), the cartilage matrix formation decreased. The protein expression of STAT1 continued to increase up to day 21, whereas the expression of Sox-9 was low and did not increase. We also demonstrated that PKR was localized to a marginal region of the mandibular condyle cartilage in mouse embryos. Our findings suggest that PKR has important functions in the differentiation of chondrocytes through the modulation of STAT1 and Sox-9 expression. [ABSTRACT FROM AUTHOR]

Abstract: Artemisinin is a well-known anti-malarial drug and has been shown to inhibit nitric oxide (NO) production. In this study, we investigated the effect of artemisinin on lipopolysaccharide (LPS)-induced production of IFN-β and characterized the potential relationship between artemisinin-mediated inhibition of IFN-β and NO production. Artemisinin suppressed IFN-β production and mRNA expression in a dose-dependent manner in LPS-stimulated RAW 264.7 cells. LPS-induced phosphorylation of signal transducer and activator of transcription-1 (STAT-1) was also inhibited by artemisinin treatment in RAW 264.7 cells. In addition, artemisinin suppressed LPS-induced production of NO in RAW 264.7 cells. Further study demonstrated that artemisinin-mediated inhibition of NO production and STAT-1 phosphorylation was reversed by addition of exogenous IFN-β. Moreover, artemisinin does not affect IFN-β-induced STAT-1 phosphorylation in RAW 264.7 cells. Collectively, these results suggest that the inhibition of IFN-β production by artemisinin and concomitant attenuation of STAT-1 activation might be involved in artemisinin-mediated inhibition of NO production in macrophages. [Copyright &y& Elsevier]

Abstract: Veratric acid, a simple benzoic acid derived from plants and fruits, has been reported to have anti-oxidant, anti-inflammation, and blood pressure-lowering effects. This study was designed to evaluate the inhibitory effects of veratric acid on nitric oxide (NO) production in LPS-stimulated RAW264.7 cells. It was found that veratric acid inhibited NO production and inducible nitric oxide synthase (iNOS) expression in LPS-stimulated RAW264.7 cells. The inhibitory effects of veratric acid on the generation of interleukin-6 (IL-6) and interferon-γ (IFN-γ) was determined. Furthermore, veratric acid facilitated the inactivation of glycogen synthase kinase-3β (GSK-3β), STAT-1, and STAT-3 in dose-dependent manner. Notably, NF-κB and members of the mitogen activated protein kinase (MAPK) family including p38, ERK, and JNK were dephosphorylated by veratric acid. These findings suggest that the treatment of veratric acid might be effective in neutralizing the over-expression of NO in inflammatory disorders. [Copyright &y& Elsevier]

Abstract: Increased microvessel density in atherosclerotic plaques plays a major role in promoting plaque destabilization resulting in increased risk of stroke and myocardial infarction. Previously we have shown that expression of the inflammatory cytokine, Oncostatin-M (OSM), in human atherosclerotic plaques correlated with increased microvessel density, indicating a role for OSM in promoting plaque angiogenesis. The purpose of this study was to determine the mechanism by which OSM regulates Vascular Endothelial Growth Factor (VEGF) expression in human coronary artery smooth muscle cells. Using shRNA and overexpression studies, we have shown that the transcription factor, STAT-1 inhibited VEGF expression, while STAT-3 promoted the expression of VEGF. We further show that the mechanism by which STAT-1 and STAT-3 regulates VEGF expression is through modulation of Hypoxia Inducible Factor-1α (HIF-1α). STAT-1 suppresses HIF-1α expression, whereas STAT-3 positively regulates HIF-1α expression. These results provide evidence that activated STAT-1 and STAT-3 regulate VEGF expression indirectly, by modulating HIF-1α activity. [Copyright &y& Elsevier]

Rotavirus (RV), a leading cause of diarrhea, primarily infects intestinal epithelial cells (IEC). Rotavirus-infected IEC produce IFN-β and express hundreds of IFN-dependent genes. We thus hypothesized that type 1 IFN plays a key role in helping IEC limit RV replication and/or protect against cell death. To test this hypothesis, we examined IEC (HT29 cells) infected with RV (MOI 1) ± neutralizing antibodies to IFN-α/β via microscopy and SDS-PAGE immunoblotting. We hypothesized that neutralization of IFN would be clearly detrimental to RV-infected IEC. Rather, we observed that blockade of IFN function rescued IEC from the apoptotic cell death that otherwise would have occurred 24-48 h following exposure to RV. This resistance to cell death correlated with reduced levels of viral replication at early time points (< 8 h) following infection and eventuated in reduced production of virions. The reduction in RV replication that resulted from IFN neutralization correlated with, and could be recapitulated by, blockade of IFN-induced protein kinase R (PKR) activation, suggesting involvement of this kinase. Interestingly, pharmacologic blockade of caspase activity ablated RV-induced apoptosis and dramatically increased viral protein synthesis, suggesting that IFN-induced apoptosis helps to control RV infection. These results suggest non-mutually exclusive possibilities that IFN signaling is usurped by RV to promote early replication and induction of cell death may be a means by which IFN signaling possibly clears RV from the intestine. [ABSTRACT FROM AUTHOR]

Abstract: Studies of insulin producing β-cells have reported conflicting responses to NF-κB activation, encompassing both pro- and anti-apoptotic effects, possibly reflecting the use of β-cells from different species. Therefore, the aim of this study was to compare the temporal activation of NF-κB in rat and human insulin producing cells and relate this to the dynamics of cell death, STAT-1 activation and the production of nitric oxide (NO). Rat RIN5AH and human islet cells were exposed to the cytokines IL-1β and IFN-γ and the NOS inhibitor aminoguanidine. Cell death, NO production, IκBα phosphorylation, p65 methylation, STAT-1 phosphorylation and cIAP-2 levels were analyzed at different time-points. Cytokine-induced RIN5AH cell death occurred on day 1, and this was paralleled by NF-κB activation, STAT-1 phosphorylation and production of NO. On the other hand, the human islet cells instead died by an NO-independent mechanism on day 3 and 5. This later occurring cell death was associated with a gradual decrease in IκBα phosphorylation and p65 methylation, and a lowered expression of the NF-κB target genes IκBα and cIAP-2. STAT-1 phosphorylation was persistently high during the entire cytokine exposure period in human islet cells. The results favor a pro-survival role of NF-κB and a pro-apoptotic role of STAT-1 in human islet cells. Thus, rodent insulin producing cells may not be suitable as models for human β-cells in the context of cytokine-induced damage. [Copyright &y& Elsevier]

Background: Geranylgeranylacetone (GGA), an isoprenoid compound which includes retinoids, has been used orally as an anti-ulcer drug in Japan. GGA acts as a potent inducer of anti-viral gene expression by stimulating ISGF3 formation in human hepatoma cells. This drug has few side effects and reinforces the effect of IFN when administered in combination with peg-IFN and ribavirin. This study verified the anti-HCV activity of GGA in a replicon system. In addition, mechanisms of anti-HCV activity were examined in the replicon cells. Methods: OR6 cells stably harboring the full-length genotype 1 replicon containing the Renilla luciferase gene, ORN/C-5B/KE, were used to examine the influence of the anti-HCV effect of GGA. After treatment, the cells were harvested with Renilla lysis reagent and then subjected to a luciferase assay according to the manufacturer's protocol. Result: The results showed that GGA had anti-HCV activity. GGA induced anti-HCV replicon activity in a time- and dose-dependent manner. GGA did not activate the tyrosine 701 and serine 727 on STAT-1, and did not induce HSP-70 in OR6 cells. The anti-HCV effect depended on the GGA induced mTOR activity, not STAT-1 activity and PKR. An additive effect was observed with a combination of IFN and GGA. Conclusions: GGA has mTOR dependent anti-HCV activity. There is a possibility that the GGA anti-HCV activity can be complimented by IFN. It will be necessary to examine the clinical effectiveness of the combination of GGA and IFN for HCV patients in the future. [ABSTRACT FROM AUTHOR]

Background: Chikungunya virus (CHIKV) is an arthritogenic member of the Alphavirus genus (family Togaviridae) transmitted by Aedes mosquitoes. CHIKV is now known to target non hematopoietic cells such as epithelial, endothelial cells, fibroblasts and to less extent monocytes/macrophages. The type I interferon (IFN) response is an early innate immune mechanism that protects cells against viral infection. Cells express different pattern recognition receptors (including TLR7 and RIG-I) to sense viruses and to induce production of type I IFNs which in turn will bind to their receptor. This should result in the phosphorylation and translocation of STAT molecules into the nucleus to promote the transcription of IFN-stimulated antiviral genes (ISGs). We herein tested the capacity of CHIKV clinical isolate to infect two different human fibroblast cell lines HS 633T and HT-1080 and we analyzed the resulting type I IFN innate immune response. Methods: Indirect immunofluorescence and quantitative RT-PCR were used to test for the susceptibility of both fibroblast cell lines to CHIKV. Results: Interestingly, the two fibroblast cell lines HS 633T and HT-1080 were differently susceptible to CHIKV infection and the former producing at least 30-fold higher viral load at 48 h post-infection (PI). We found that the expression of antiviral genes (RIG-I, IFN-β, ISG54 and ISG56) was more robust in the more susceptible cell line HS 633T at 48 h PI. Moreover, CHIKV was shown to similarly interfere with the nuclear translocation of pSTAT1 in both cell lines. Conclusion: Critically, CHIKV can control the IFN response by preventing the nuclear translocation of pSTAT1 in both fibroblast cell lines. Counter-intuitively, the relative resistance of HT-1080 cells to CHIKV infection could not be attributed to more robust innate IFN- and ISG-dependent antiviral responses. These cell lines may prove to be valuable models to screen for novel mechanisms mobilized differentially by fibroblasts to control CHIKV infection, replication and spreading from cell to cell. [ABSTRACT FROM AUTHOR]