Month: September 2020

In addition, specific anti-apoptosis, cell adhesion and extracellular matrix proteins were only down-regulated in NEC patients, possibly revealing the different nature of bowel injury and/or severity of inflammation compared with SIP. Further validation of selected targets confirmed up-regulation of IL-6, Ang-2, sIL1-RII and suPAR in NEC infants compared with gestational age-matched controls, but the circulating level of sErbB3 was significantly decreased compared with SIP infants. mRNA expressions of IL-6, IL1-RII and uPAR were up-regulated in resected bowel tissues from NEC infants and in accordance with changes in plasma proteins. This indicated that immunomodulation had occurred at the tissue and cellular levels of the affected bowel, which could have contributed to their corresponding increased levels in the plasma. In a model of human fetal enterocytic cell line FHs-74 Int, Ang-2, IL1-RII and uPAR expressions were significantly induced by the combined treatment with LPS and PAF, again suggested that these genes could act synergistically and play a pivotal role in disease development and progression of NEC. Using cytokine array as a hypothesis-free approach, we provided specific profiles of plasma immunoregulatory proteins in NEC and SIP infants. Our findings are in line with other studies on cytokines, showing regulation of pro- and anti-inflammatory, and repair-associated proteins in NEC infants, TH-302 including IL-6, IL8, IL-10, TNF, ENA-78, matrix metalloprotease-9, GRO, MCP-1 and IGF-1. We have also discovered novel immunoregulatory proteins such as Ang-2, ErbB3 and uPAR, which have not been previously described in NEC or SIP. Importantly, there have not been any comprehensive data on immunoregulatory proteins reported in SIP. Our plasmatic protein profile thus represents the basic information platform for further mechanistic investigation of this disease. Despite sharing many common dysregulated immunomodulatory proteins in the two conditions, some appeared to be more specific to either NEC or SIP, thus reflecting differences regarding the etiology, pathophysiology and severity of inflammation of the two conditions. We validated IL-6, Ang-2, IL1-RII and uPAR in their soluble forms as well as in intestinal tissues and in a fetal enterocyte model, suggesting that the systemic dysregulation of these proteins not only occurred at the plasma level, but also at the cellular level of inflamed bowel tissues and enterocytes. The evidence suggested up-regulation of IL-6, Ang-2, sIL1-RII and suPAR in NEC infants, whereas sErbB3 was significantly higher in the plasma of SIP compared with NEC infants. Despite changes of immunoregulatory proteins were observed in the plasma of SIP patients, the targeted gene levels in the bowel specimens were not different from tissues of surgical control subjects. This might reflect minimal intestinal inflammation due to the localized nature of bowel perforation. The exaggerated up-regulation of immunomodulatory proteins in NEC infants represented imbalance of the inflammatory cascade, which could lead to necrosis, apoptosis and severe tissue damage. This could account for the high morbidity and mortality rate in NEC compared with SIP infants. Of clinical interest, further investigation on validating these dysregulated proteins in a cohort study, either individually or in combination as diagnostic biomarkers, might reveal their usefulness for early differentiation of NEC from SIP patients.

b-catenin is a multifunctional adaptor protein/transcription factor that is deregulated in many cancers. In the absence of Wnt ligand, cytosolic b-catenin levels are down-regulated via a degradation complex including CK1a, GSK3b, Axin, APC, and PP2A in which processive phosphorylation of b-catenin by CK1a and GSK3b leads to its ubiquitination and proteasomal degradation. In the presence of Wnt ligand, upon its binding to the frizzled/LRP5/6 receptor complex, Dishevelled is activated, at least in part by phosphorylation. Allowing the dephosphorylation and nuclear import of b-catenin, where it activates the TCF/LEF family of transcriptional CYT 11387 factors that control expression of various genes related to cell cycle and differentiation. Wnt/b-catenin pathway is strongly implicated in breast carcinogenesis, in addition to many other cancer types. Transgenic mice expressing degradation-resistant b-catenin in mammary gland tissue develop breast tumors. According to immunohistochemical analysis, nuclear and cytoplasmic b-catenin levels have been found to be elevated in about 60% of the breast tumors. Furthermore, reduced levels of extracellular Wnt-inhibitory molecules sFRP1 and WIF1 have been linked to 80% and 60% of breast carcinomas. Additionally, b-catenin has been associated with epidermal growth factor receptor family members and the stability of b-catenin and its TCF/LEFactivating function has been suggested to be regulated via tyrosine phosphorylation by the EGFR family, which may be significant for breast carcinogenesis, since human epidermal growth factor receptor 2 is overexpressed in about 30% of human breast tumors. Hence, identification of novel targets of Wnt/b-catenin pathway, serves an important purpose for cancer research field, particularly breast cancer, since target genes of the pathway are potential anti-cancer drug targets. Many actin-associated proteins play important roles in carcinogenesis of various types of cancers. MENA is an actinregulatory protein that belongs to ENA/VASP protein family. Members of this protein family are localized at the tips of protruding lamellipodia and filopodia and adhesion foci; and they are involved in control of cell motility and cell-cell adhesion, which are important subjects for development of metastatic potential. Di Modugno et al. showed that human MENA is overexpressed in,75% of primary breast cancers. In our previous study, we employed SAGE and genome-wide microarray approaches to screen for novel Wnt/b-catenin pathway targets by overexpressing degradation-resistant S33Y-b-catenin in Huh7 cell lines, which lacks detectable nuclear endogenous b-catenin levels. In these screens we found MENA to be differentially expressed and in this study we show that MENA is a transcriptional target of the Wnt/b-catenin pathway. Wnt and Notch pathways have essential roles in development with well-studied crosstalks, yet their interplay in cancer is not well understood. In order to investigate the role of MENA in tumorigenesis, we tested whether knock-down of the Drosophila homolog of MENA can affect tumor formation in the D. melanogaster eye cancer fly models “eyeful” and “sensitized”. Eyeful flies have a metastatic eye tumor phenotype induced by activated Notch signalling due to overexpression of the Notch ligand Delta and overexpression of polycomb genes lola and pipsqueak.

Down-regulated expression of AP-4, p21 and p53 in the meanwhile could make the cell cycle arrest caused knockdown of AP-4 disappearance. These results are in agreement with a model in which AP-4 induces cell cycle arrest by regulating the expression of cell cycle regulators, such as p53, p21 and cyclin D1. Apoptosis, or programmed cell death, is known to participate in various biological processes by two main apoptotic pathways, the mitochondrial pathway and the death receptor pathway. We found that silencing the AP-4 expression trigged cell apoptosis in our experiment, which demonstrated that AP-4 suppressed apoptosis in human gastric Oligomycin A ATPase inhibitor cancer cells. In our experiment, increasing levels of Caspase-9 and downregulation of Bcl-2 and Bcl-xL were detected in human gastric cancer cells, indicating that knockdown of AP-4 activated both intrinsic and extrinsic pathways to apoptosis in cancer cells. In summary, the data demonstrate that RNAi-mediated downregulation of transcription factor AP-4 effectively inhibited the cell proliferation, indicated cell cycle arrest, triggered apoptosis and enhanced chemo-sensitivity of human gastric cancer cells with the decreased expression of cyclin D1, Bcl-2 and Bcl-xL and activated p21, p53 and Caspase-9 expression, which suggested AP-4 may be a oncogene playing an important role in tumorigenesis. Although the precise mechanism of this role needs to be further investigated, the AP-4specific-siRNAs may be of potential values as novel therapeutic agents for human gastric cancer. Most Hepatitis C virus infections evolve in persistent infection, which may progress to fibrosis, cirrhosis, liver failure or even hepatocellular carcinoma. Current standard therapy is based on a combination of pegylated -IFN-a and ribavirin and treatment response may be influenced by several virusrelated factors such as HCV genotype and baseline titer of HCV RNA. A sustained virological response occurs in approximately 80% of patients infected with HCV genotypes 2 or 3, and in approximately 45% for genotypes 1 or 4. New antiviral strategies are currently in development for HCV infection and include drugs targeting key viral enzymes such as NS3-4A and the NS5B RNA-dependent RNA polymerase. Although effective, the use of these new antivirals seems associated to the selection of drug-resistant HCV variants, resulting in viral breakthrough. Thus, a combination between antivirals and standard treatment with IFNa and RBV is therefore necessary. HCV persistence is mainly due to the failure of the host’s immune system to effectively and definitively clear the infection and generate protective cellular immunity. Indeed, marked quantitative and qualitative defects of HCV-specific CD8 T-cells have been described in HCV patients, correlated with innate immune cell impairment such as dendritic cell and NK cells. In this context, immune modulation could represent a promising strategy aimed to restore protective immune response, inducing a long lasting immunity, necessary to obtain viral eradication. Among innate immune cells, Vc9Vd2 T-cells represent a good target for immunotherapy in infectious diseases for their multifaceted response capability. They may specifically be activated both in vitro and in vivo by using phosphoantigens and aminobisphosphonates without any MHC restriction.

These data are consistent with our results, which demonstrate that the suppression of monocyte/macrophage infiltration acts as an important cellular mechanism for INCB3344 treatment in the current model. To determine the impact of INCB3344 on the downstream signaling of macrophages in CNV formation, we detected VEGF on the peak response day of macrophages. Our results demonstrated that VEGF significantly decreased with the suppression of infiltrating macrophages by INCB3344 treatment. After photocoagulation, VEGF is up-regulated, and acts as a promoting mediator in the development of CNV. The variation of VEGF levels correlates highly to that of macrophages after laser injury, and their peak responses are reported to coincide with each other. While in pharmacologically macrophage-depleted mice, VEGF production is reduced in proportion to the decrease in the number of macrophages. Moreover, enriched ocular-infiltrating macrophages from laser-induced model mice have shown angiogenic ability in a dorsal air sac assay, and express activation-surface markers and the mRNA for potential angiogenic factors including VEGF, which indicates that the infiltrating macrophages are a rich source of VEGF. Our results agree with these data and reveal that macrophages play an important role in the variation of intro-ocular VEGF after laser injury. Further more, our results demonstrate that elevated VEGF expression in infiltrating macrophages is suppressed by INCB3344. We reveal that INCB3344 can not only inhibit macrophage infiltration but also suppress the angiogenic ability of infiltrating macrophages, which results in the reduction of VEGF, and finally in suppression of CNV. In this study, we revealed that INCB3344 treatment inhibited CNV formation via the suppression of macrophage infiltration. Our study focused on CCR2, macrophages, and VEGF, although several other cytokines such as tumor necrosis factor-alpha –, interleukin -1beta, hypoxia inducible factors, IL-6 and tissue factor are reported to be involved in CNV formation. We cannot rule out a potential link between macrophages and other cytokines, however, VEGF is crucial in the pathogenesis of CNV formation because anti-VEGF drug therapy, for example, bevacizumab and ranibizumab have achieved an obvious effect in CNV due to age-related macular degeneration. We demonstrated that the ERK1/2 phosphorylation induced by laser treatment was significantly suppressed by INCB3344. We examined the phosphorylation of ERK 1/2 because the activation of ERK1/2 in macrophages can be induced through the CCR2 and their activation is thought to be a key component in the cellular events leading to the infiltration and activation of macrophages –. Our results are consistent with the results in vitro, and the Niltubacin previous reports that blockage of CCR2 by anti-CCR2 monoclonal antibodies inhibits phosphorylation of ERK1/2 in peritoneal macrophages. Our results on the inhibitory effect on VEGF secretion are also compatible with the data that ERK1/2 activation in macrophages or monocytes is reported to be responsible for VEGF production in these cells. We can conclude that INCB3344 inhibits the activation of ERK1/2 in macrophages by blocking CCR2, which results in the reduction of macrophage infiltration and VEGF production.

Alcoholic liver disease affects millions of people worldwide and is a major cause of morbidity and mortality. ALD encompasses varying degrees of hepatic injury progressing from steatosis to more advanced damage, including hepatic inflammation and cell death, fibrosis/cirrhosis and hepatocellular carcinoma. However, less than 10% of heavy drinkers progress to later stages of injury, suggesting other contributing factors in development of severe liver injury due to excessive alcohol consumption. Of these factors, health status, environmental exposures and genetics influence development and progression of ALD. Sex differences are observed in rodent models of ALD, with females displaying greater susceptibility to the detriments of alcohol than males. Since decreasing estrogen levels protects females from ethanol-induced liver injury, E2 is implicated in sex differences observed in ALD. However, in these reports, E2 manipulation was initiated post-pubertally or at 4 weeks of age. No studies to date have determined the contribution of cyclic E2 in the development of ALD. Additionally, OVX studies cannot exclude the possibility that other ovarian factors besides E2 may play a role in development and progression of ALD. Gonadal sex steroid hormone levels are regulated by the hypothalamic pituitary gonadal axis, which is disrupted by neonatal androgenization. Further, gonadal hormone production, regulated by the HPG axis, programs sex-specific expression of hepatic enzymes during pubertal development and can profoundly affect adult liver physiology. Perinatal hormonal imprinting of hepatic enzymes including members of the cytochrome P450 family and those involved in steroid metabolism has been shown. Alterations in expression of ethanol metabolizing enzymes have been implicated in predisposition to ALD. Following acute and chronic ethanol consumption, hepatocytes are the principal site of ethanol metabolism. Classically, ethanol metabolism occurs via alcohol dehydrogenase, the microsomal ethanol oxidizing system, comprised predominantly of BYL719 inducible cytochrome P450 2E1 and catalase. Metabolism by ADH and CYP2E1 generates reactive oxygen species, promoting lipid peroxidation, protein adduct formation and collagen synthesis, the major mechanisms of damage in ALD progression. The liver counteracts the deleterious effects of oxidative stress via increased antioxidant defense mechanisms, providing protection against ALD development. During chronic ethanol exposure, the balance between prooxidants and anti-oxidants may favor pro-oxidants, thus rendering the cell susceptible to oxidative stress. Despite increased oxidative stress by ethanol-induced CYP2E1, previous studies report CYP2E1 expression may not be the predominant mechanism of alcohol-induced damage, suggesting possible involvement of other CYP450 family members in ethanol metabolism and ALD progression. CYP1A2 is also capable of metabolizing ethanol, and interestingly, is induced in a sexspecific manner with potential regulation by E2. Therefore, sex-specific ethanol metabolizing enzymes may contribute to sexual dimorphism in ALD. In the present study, neonatal androgenization was used as a model to examine the contribution of adult cyclic E2 in the development and progression of ALD.