Binding of YY1/CREB to an Enhancer Region Triggers Claudin 6 Expression in H. pylori LPS-Stimulated AGS Cells.

Aberrant expression of the tight junction protein claudin 6 (CLDN6) is a hallmark of gastric cancer progression. Its expression is regulated by the cAMP response element-binding protein (CREB). In gastric cancer induced by Helicobacter pylori (H. pylori) there is no information regarding what transcription factors induce/upregulate the expression of CLDN6. We aimed to identify whether CREB and Yin Yang1 (YY1) regulate the expression of CLDN6 and the site where they bind to the promoter sequence. Bioinformatics analysis, H. pylori lipopolysaccharide (LPS), YY1 and CREB silencing, Western blot, luciferase assays, and chromatin immunoprecipitation experiments were performed using the stomach gastric adenocarcinoma cell line AGS. A gen reporter assay suggested that the initial 2000 bp contains the regulatory sequence associated with CLDN6 transcription; the luciferase assay demonstrated three different regions with transcriptional activity, but the -901 to -1421 bp region displayed the maximal transcriptional activity in response to LPS. Fragment 1279-1421 showed CREB and, surprisingly, YY1 occupancy. Sequential Chromatin Immunoprecipitation (ChIP) experiments confirmed that YY1 and CREB interact in the 1279-1421 region. Our results suggest that CLDN6 expression is regulated by the binding of YY1 and CREB in the 901-1421 enhancer, in which a non-described interaction of YY1 with CREB was established in the 1279-1421 region.

Chromosomally Unstable Gastric Cancers Overexpressing Claudin-6 Disclose Cross-Talk between HNF1A and HNF4A, and Upregulated Cholesterol Metabolism.

(1) Abnormally increased expression of claudin-6 in gastric cancer is considered a prognostic marker of the chromosomal unstable molecular subtype. However, a detailed molecular profile analysis of differentially expressed genes and affected pathways associated with claudin-6 increased (Cldn6high) expression has not been assessed. (2) The TCGA Stomach Adenocarcinoma Pan-Cancer Atlas Data was evaluated using Cytoscape's Gene Mania, MCODE, and Cytohubba bioinformatic software. (3) 96.88% of Cldn6high gastric cancer tumors belonging to the chromosomal unstable molecular subtype are associated with a worse prognosis. Cldn6expression coincided with higher mutations in TP53, MIEN1, STARD3, PGAP3, and CCNE1 genes compared to Cldn6low expression. In Cldn6high cancers, 1316 genes were highly expressed. Cholesterol metabolism was the most affected pathway as APOA1, APOA2, APOH, APOC2, APOC3, APOB-100, LDL receptor-related protein 1/2, Sterol O-acyltransferase, STARD3, MAGEA-2, -3, -4, -6, -9B, and -12 genes were overexpressed in Cldn6high gastric cancers; interestingly, APOA2 and MAGEA9b were identified as top hub genes. Functional enrichment of DEGs linked HNF-4α and HNF-1α genes as highly expressed in Cldn6high gastric cancer. (4) Our results suggest that APOA2 and MAGEA9b could be considered as prognostic markers for Cldn6high gastric cancers.

Claudin-6 increases SNAI1, NANOG and SOX2 gene expression in human gastric adenocarcinoma AGS cells.

BACKGROUND: Gastric cancer is a heterogeneous disease associated to deregulated gastric epithelia tight junction barrier function and di novo expression of claudin-6; these changes are associated with epithelial-mesenchymal transition, enhanced invasiveness, metastatic progression, resistance to chemotherapy, and poor prognosis. Gastric cancer stem cells represent a rare population of cells within the tumor implicated in tumor growth and higher tumorigenic capacity. The possible relation between claudin-6 expression and the expression of some markers associated to epithelial mesenchymal transition and cancer stem cells in gastric cancer cells have never been explored. METHODS AND RESULTS: CD44, CD24, Twist, Villin, DCLK1, claudin-6, NANOG, E-Cadherin, SOX2, and SNAI1 expression was evaluated by immunofluorescence and cytofluorometry in wild type and Claudin-6 transfected AGS cells. Cell migration assays were also performed. Differentially expressed genes and biological processes analysis was performed to determine gene preponderance. The results showed that claudin-6 overexpression enriched the CD44 + /CD24- subpopulation with an overall increase in the expression and the number of CD44 + cells. A significant increase in NANOG, SOX2 and SNAI1 expression and enhanced cell migration was observed in claudin-6 transfected cells. Transcriptome analysis revealed 271 genes involved in enhanced biological processes with only 31 with a significantly p value; thirteen of those genes are closely associated to epithelial mesenchymal transition processes and folding and unfolding processes of proteins in the endoplasmic reticulum. CONCLUSIONS: The pro-tumorigenic effect of claudin-6 in gastric cancer could be associated to dedifferentiation of epithelial cells and an increase in di novo cancer stem cell genesis.

CD38 and Regulation of the Immune Response Cells in Cancer.

Cancer is a leading cause of death worldwide. Understanding the functional mechanisms associated with metabolic reprogramming, which is a typical feature of cancer cells, is key to effective therapy. CD38, primarily a NAD + glycohydrolase and ADPR cyclase, is a multifunctional transmembrane protein whose abnormal overexpression in a variety of tumor types is associated with cancer progression. It is linked to VEGFR2 mediated angiogenesis and immune suppression as it favors the recruitment of suppressive immune cells like Tregs and myeloid-derived suppressor cells, thus helping immune escape. CD38 is expressed in M1 macrophages and in neutrophil and T cell-mediated immune response and is associated with IFNγ-mediated suppressor activity of immune responses. Targeting CD38 with anti-CD38 monoclonal antibodies in hematological malignancies has shown excellent results. Bearing that in mind, targeting CD38 in other nonhematological cancer types, especially carcinomas, which are of epithelial origin with specific anti-CD38 antibodies alone or in combination with immunomodulatory drugs, is an interesting option that deserves profound consideration.

Proteolytic Processing of CD44 and Its Implications in Cancer.

CD44 is a transmembrane glycoprotein expressed in several healthy and tumor tissues. Modifications in its structure contribute differently to the activity of this molecule. One modification that has provoked interest is the consecutive cleavage of the CD44 extracellular ectodomain by enzymes that belong mainly to the family of metalloproteases. This process releases biologically active substrates, via alternative splice forms of CD44, that generate CD44v3 or v6 isoforms which participate in the transcriptional regulation of genes and proteins associated to signaling pathways involved in the development of cancer. These include the protooncogene tyrosine-protein kinase Src (c-Src)/signal transducer and activator of transcription 3 (STAT3), the epithelial growth factor receptor, the estrogen receptor, Wnt/ßcatenin, or Hippo signaling pathways all of which are associated to cell proliferation, differentiation, or cancer progression. Whereas CD44 still remains as a very useful prognostic cell marker in different pathologies, the main topic is that the generation of CD44 intracellular fragments assists the regulation of transcriptional proteins involved in the cell cycle, cell metabolism, and most importantly, the regulation of some stem cell-associated markers.

Transcriptional and Epigenetic Bioinformatic Analysis of Claudin-9 Regulation in Gastric Cancer.

Gastric cancer is a heterogeneous disease that represents 5% to 10% of all new cancer cases worldwide. Advances in histological diagnosis and the discovery of new genes have admitted new genomic classifications. Nevertheless, the bioinformatic analysis of gastric cancer databases has favored the detection of specific differentially expressed genes with biological significance. Claudins, a family of proteins involved in tight junction physiology, have emerged as the key regulators of cellular processes, such as growth, proliferation, and migration, associated with cancer progression. The expression of Claudin-9 in the gastric cancer tissue has been linked to poor prognosis, however, its transcriptional and epigenetic regulations demand a more comprehensive analysis. Using the neural network promoter prediction, TransFact, Uniprot-KB, Expasy-SOPMA, protein data bank, proteomics DB, Interpro, BioGRID, String, and the FASTA protein sequence databases and software, we found the following: (1) the promoter sequence has an unconventional structure, including different transcriptional regulation elements distributed throughout it, (2) GATA 4, GATA 6, and KLF5 are the key regulators of Claudin-9 expression, (3) Oct1, NF-κB, AP-1, c-Ets-1, and HNF-3ß have the higher binding affinity to the CLDN9 promoter, (4) Claudin-9 interacts with cell differentiation and development proteins, (5) CLDN9 is highly methylated, and (6) Claudin-9 expression is associated with poor survival. In conclusion, Claudin-9 is a protein that should be considered a diagnostic marker as its gene promoter region binds to the transcription factors associated with the deregulation of cell control, enhanced cell proliferation, and metastasis.

Vanadium compounds and cellular death mechanisms in the A549 cell line: The relevance of the compound valence.

Non-small lung cell carcinoma has a high morbidity and mortality rates. The elective treatment for stage III and IV is cisplatinum that conveys serious toxic side effects. Vanadium compounds are metal molecules with proven antitumor activity that depends on its valence. Therefore, a better understanding of the mechanism of action of vanadium compounds is required. The aim of our study was to investigate the mechanisms of cell death induced by sodium metavanadate (NaVO3 [V(+5)]) and vanadyl sulfate (VOSO4 [(+4)]), both of which have reported apoptotic-inducing activity. We exposed the A549 cell line to various concentrations (0-100 µM) and to different exposure times to each compound and determined the cell viability and expression of caspases, reactive oxygen species (ROS) production, Bcl2, Bax, FasL and NO. Our results showed that neither compounds modified the basal expression of caspases or pro- and anti-apoptotic proteins. The only change observed was the 12- and 14-fold significant increase in ROS production induced by NaVO3 and VOSO4 , respectively, at 100 µm concentrations after 48 hours. Our results suggest that classical apoptotic mechanisms are not related to the cell death induced by the vanadium compounds evaluated here, and showed that the higher ROS production was induced by the [(+4)] valence compound. It is possible that the difference will be secondary to its higher oxidative status and thus higher ROS production, which leads to higher cell damage. In conclusion, our results suggest that the efficacy of the cell death mechanisms induced by vanadium compounds differ depending on the valence of the compound.

Vanadium pentoxide increased PTEN and decreased SHP1 expression in NK-92MI cells, affecting PI3K-AKT-mTOR and Ras-MAPK pathways.

Vanadium is an air pollutant that imparts immunosuppressive effects on NK cell immune responses, in part, by dysregulating interleukin (IL)-2/IL-2R-mediated JAK signaling pathways and inducing apoptosis. The aim of the present study was to evaluate effects of vanadium pentoxide (V2O5) on other IL-2 receptor-mediated signaling pathways, i.e. PI3K-AKT-mTOR and Ras-MAPK. Here, IL-2-independent NK-92MI cells were exposed to different V2O5 doses for 24 h periods. Expression of PI3K, Akt, mTOR, ERK1/2, MEK1, PTEN, SHP1, BAD and phosphorylated forms, as well as caspases-3, -8, -9, BAX and BAK in/on the cells were then determined by flow cytometry. The results show that V2O5 was cytotoxic to NK cells in a dose-related manner. Exposure increased BAD and pBAD expression and decreased that of BAK and BAX, but cell death was not related to caspase activation. At 400 µM V2O5, expression of PI3K-p85 regulatory subunit increased 20% and pPI3K 50%, while that of the non-pPI3K 110α catalytic subunit decreased by 20%. At 200 µM, V2O5 showed significant decrease in non-pAkt expression (p < 0.05); the decrease in pAkt expression was significant at 100 µM. Non-pmTOR expression displayed a significant downward trend beginning at 100 µM. Expressions of pMEK-1/2 and pERK-1/2 increased substantially at 200 µM V2O5. No differences were found with non-phosphorylated ERK-1/2. PTEN expression increased significantly at 100 µM V2O5 exposure whereas pPTEN decreased by 18% at 25 µM V2O5 concentrations, but remained unchanged thereafter. Lastly, V2O5 at all doses decreased SHP1 expression and increased expression of its phosphorylated form. These results indicated a toxic effect of V2O5 on NK cells that was due in part to dysregulation of signaling pathways mediated by IL-2 via increased PTEN and decreased SHP1 expression. These results can help to explain some of the known deleterious effects of this particular form of vanadium on innate immune responses.

TLR2 activation induced by H. pylori LPS promotes the differential expression of claudin-4, -6, -7 and -9 via either STAT3 and ERK1/2 in AGS cells.

Gastric carcinogenesis has been associated to H. pylori virulence factors that induce a chronic inflammation process. Lipopolysaccharides play a role in chronic inflammatory responses via TLR2- and TLR4-dependent signaling pathways. Similarly, cellular invasiveness, metastatic potential and prognosis are usually associated to claudin-4, -6, -7 and -9 expression in gastric carcinogenesis. Therefore, the aim of this study was to determine if H. pylori LPS exerts an influence on carcinogenesis-related claudin expression and if it was directly regulated through the TLR2 pathway. Human antrum gastric adenocarcinoma AGS cells exposed or not to H. pylori LPS were used. Polyclonal anti-claudin-4, -6, -7 and -9, anti-TLR2, anti-pERK1/2 as well as rabbit monoclonal anti-pNFκB p65 and mouse monoclonal anti-CdX2 were used. ERK1/2 inhibitor UO126 and STAT3 inhibitor Stattic were also used. Western blot, immunofluorescence and confocal experiments were performed in whole cells as well as total protein, nuclear and cell membrane fractions. The results showed that H. pylori LPS increased the expression of TLR2 in a time dependent bi-phasic manner (<12 and >12h exposure). Immunofluorescence using AGS monolayers corroborated the double phase TLR2 expression mainly on the cell membrane but a detectable signal was also determined in the cytoplasm of the cells. Activation of NFkB was downstream and depended on TLR2 expression as a statistically significant increase in pNFkB, that followed a pattern highly similar to the TLR2 expression was observed on the cell membrane fraction. The increase in TLR2 expression was accompanied by dramatically increased claudin-4 expression in cultures exposed from 30m to 8h to LPS. Increased expression of claudin-6, -7 and -9 also increases in >12h LPS exposure times. The increase in claudins expression was also dependent on NFkB activation. The results also showed an increase in pSTAT3 that followed a bi-phasic pattern that began 30min after stimulation and was compatible with the increase in TLR2 expression. The expression of the claudin-4 related CDX2 transcription factor did not followed the biphasic pattern. The results also showed that claudin-4 expression was STAT3 dependent whereas claudin-6, 7 and 9 expressions was ERK1/2 dependent. Our results suggest that H. pylori LPS induces TLR2 expression in the AGS cells, and that the longer the exposure to LPS, the greater the expression of TLR2 in the cell membrane. Consequently the expression of claudin-4, -6, -7 and -9 also increases.

¿Qué son los inflamosomas? El NLRP3 como por ejemplo / What are inflammasomes? The NLRP3 as an example

Resumen Una de las prácticas médicas más concurridas en la actualidad es el uso indiscriminado de medicamentos con actividad inhibidora de la inflamación. Sin embargo la inflamación es un proceso de reparación biológica fuertemente controlado por complejos intracelulares, conocidos como inflamosomas, que actúan como sensores y mediadores de la misma. Los inflamosomas forman parte de la familia de receptores tipo NOD que está formada de 3 subfamilias: NOD (nucleotide-binding oligomerization domain), NLRC (NOD-like receptor CARD domain containing) y NLRP (NOD-like receptor Pyrin domain containing), que es la que se relaciona con la formación de inflamosomas. Existen 14 diferentes tipos de NLRP. Los miembros de la familia NLRP responden a señales exógenas mediadas por PAMPs (pathogen-associated molecular patterns) o a señales endógenas mediadas por DAMPs (damage-associated molecular patterns, [también conocidas como alarminas]). Los componentes de los inflamosomas tipo NLRP, una vez activado, se ensamblan de acuerdo a un patrón determinado y forman un complejo que activa a la caspasa-1 que activa a los precursores de IL-1b, IL-18 e IL-33, favoreciendo la secreción de estas citosinas hacia el espacio extracelular. La IL-1 y la IL-18 son miembros de la misma familia y se les reconoce como reguladores de la respuesta inmune innata y adaptativa, la IL-33 también es miembro de la familia de IL-1 y se le considera una alarmina. A manera de ejemplo, en el presente manuscrito describimos la estructura y formación del inflamosoma NLRP3 y mencionamos algunas de las enfermedades en las que se activa, enfatizando de manera muy particular su participación en la enfermedad de Alzheimer.

Abstract One highly common medical malpractice is the undiscriminatory use of inflammation inhibiting drugs. Inflammation is a biological repair process vastly controlled by intracellular complexes known as the inflammasome that act as sensors and mediators of the inflammation process. Inflammasomes are members of the NOD innate immune system family of receptors that consist of 3 closely related subfamilies: nucleotide-binding oligomerization domain (NOD), NOD-like receptor CARD domain containing (NLRC), and NOD-like receptor Pyrin domain containing (NLRP); the latter is the most directly related to the inflammasome. There are 14 different NLRPs all of which are activated by exogenous signals through pathogen-associated molecular patterns (PAMPs) or by endogenous signals via damage-associated molecular patterns, also known as alarmins, are endogenous molecules constitutively available and released upon tissue damage (DAMPs). Once activated, the components of the NLRP inflammasome begin an assembling process that follows a pre-established pattern so a caspase-1 activating complex is formed. This complex activates IL-1b, IL-18 and IL-33 precursors thus favoring the secretion of this cytokines to the extracellular milieu. IL-1 and IL-18 are members of the same cytokine family and their main function is to regulate the innate and adaptive immune response whereas IL-33, also a member of the IL-1 family of cytokines, is considered an alarmin. We emphasize the structure and formation of NLRP3, implicated on a host of inflammatory disorders, with special attention to its participation in Alzheimer´s diseases.

Activation of Janus kinase/signal transducers and activators of transcription pathway involved in megakaryocyte proliferation induced by vanadium resembles some aspects of essential thrombocythemia.

Vanadium (V) is an air pollutant released into the atmosphere by burning fossil fuels. Also, it has been recently evaluated for their carcinogenic potential to establish permissible limits of exposure at workplaces. We previously reported an increase in the number and size of platelets and their precursor cells and megakaryocytes in bone marrow and spleen. The aim of this study was to identify the involvement of Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway and thrombopoietin (TPO) receptor, and myeloproliferative leukemia virus oncogene (Mpl), in megakaryocyte proliferation induced by this compound. Mice were exposed twice a week to vanadium pentoxide inhalation (0.02 M) and were killed at 4th, 6th, and 8th week of exposure. Phosphorylated JAK2 (JAK2 ph), STAT3 (STAT3 ph), STAT5, and Mpl were identified in mice spleen megakaryocytes by cytofluorometry and immunohistochemistry. An increase in JAK2 ph and STAT3 ph, but a decrease in Mpl at 8-week exposure was identified in our findings. Taking together, we propose that the morphological findings, JAK/STAT activation, and decreased Mpl receptor induced by V leads to a condition comparable to essential thrombocythemia, so the effect on megakaryocytes caused by different mechanisms is similar. We also suggest that the decrease in Mpl is a negative feedback mechanism after the JAK/STAT activation. Since megakaryocytes are platelet precursors, their alteration affects platelet morphology and function, which might have implications in hemostasis as demonstrated previously, so it is important to continue evaluating the effects of toxics and pollutants on megakaryocytes and platelets.

Vanadium pentoxide prevents NK-92MI cell proliferation and IFNγ secretion through sustained JAK3 phosphorylation.

Vanadium is a major air pollutant with toxic and carcinogenic effects; it also exercises immunosuppressive effects on the adaptive immune response. Its effect on the innate immune response is poorly explored. The aim of this study was to identify if vanadium pentoxide (V2O5) impairs the function of immunoregulatory NK cells and to determine possible mechanisms associated with this effect. Interleukin-2-independent NK-92MI cells were exposed to different V2O5 concentrations for 6, 12, or 24 h periods. Cell proliferation was then evaluated using CFSE staining, apoptosis by Annexin V binding, and necrosis by 7-AAD staining. The release of IL-2, -4, -6, -10, -17A, IFNγ, and TNFα by the cells were assessed using a human CBA kit. Expression of CD45, SOCS1, JAK3, pJAK3, STAT5, pSTAT5, IL-2R, IL-15R, Fas, and FasL in/on the cells was determined by flow cytometry; JAK3 and pJAK3 expression were also evaluated via confocal microscopy. The results indicated that V2O5 could inhibit NK-92MI cell proliferation and induce cell apoptosis in a dose- and time-related manner. V2O5 also inhibited IL-2, IL-10, and IFNγ secretion but mostly only after 24 h of exposure and with primarily the higher doses tested. V2O5 had no effect on expression of JAK3 and STAT5, but did cause an increase in pJAK3 and appeared to lead (trend) to reductions in levels of phosphorylated STAT5. V2O5 increased the expression of IL-2R, IL-15R, Fas, and FasL at concentrations above the 50-100 µM range. V2O5 had no effect on expression of the CD45 membrane phosphatase, but it did cause an increase in the expression of SOCS1. These results indicate that a key toxic effect of V2O5 on NK cells is a dysregulation of signaling pathways mediated by IL-2. These effects could help to explain the previously-reported deleterious effects on innate immune responses of hosts exposed to inhaled V2O5.

Inhibition of Helicobacter pylori growth and its colonization factors by Parthenium hysterophorus extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Parthenium hysterophorus (Asteraceae) is a traditional medicinal plant used to treat gastrointestinal disorders, such as gastritis. Helicobacter pylori have been described as the etiological agent of gastritis, peptic ulcer, as well as gastric adenocarcinoma. 50% of the world's population is infected with this bacterium and the current therapy fails due to the increment in antibiotic resistance; therefore, it is necessary to find new approaches to control H. pylori infection, either by its eradication or by preventing the bacterial colonization. AIM OF THE STUDY: To investigate the effect of P. hysterophorus extracts on H. pylori growth and upon its colonization-related factors. MATERIALS AND METHODS: Five different polarity extracts from roots and aerial parts of P. hysterophorus were evaluated in vitro against H. pylori growth by the broth dilution method. Anti-colonization activities were determined as follows: motility in soft agar plates, urease activity by ammonia colorimetrical quantification, and adherence of FITC labeled H. pylori to AGS cells by fluorometrical measurement. RESULTS: Organic extracts inhibited H. pylori growth. Particularly, the dichloromethane extract from roots showed a MIC of 15.6 µg/ml while the aqueous extracts showed low or null activity. There is a direct correlation between antibacterial activity and inhibition of motility. Urease activity was partially inhibited by organic extracts, at best 46%, except for the roots dichloromethane extract which reached 74% of inhibition with 500 µg/ml (IC50=136.4 µg/ml). Plant extracts inhibited adherence in different ranges but the dichloromethane-methanol ones possessed the highest effect, with a 70% maximal inhibition at 1mg/ml. CONCLUSION: The results indicate that some P. hysterophorus extracts have various biological activities that could act synergistically against H. pylori. This work contributes to the ethnomedical knowledge of this species and underlines the potential of some organic extracts as a good source for the isolation of bioactive compounds.

Unremitting cell proliferation in the secretory phase of eutopic endometriosis: involvement of pAkt and pGSK3ß.

OBJECTIVE: Endometriosis is linked to altered cell proliferation and stem cell markers c-kit/stem cell factor (SCF) in ectopic endometrium. Our aim was to investigate whether c-kit/SCF also plays a role in eutopic endometrium. DESIGN: Eutopic endometrium obtained from 35 women with endometriosis and 25 fertile eumenorrheic women was analyzed for in situ expression of SCF/c-kit, Ki67, RAC-alpha serine/threonine-protein kinase (Akt), phosphorylated RAC-alpha serine/threonin-protein kinase (pAkt), Glycogen synthase kinase 3 beta (GSK3ß), and phosphorylated glycogen synthase kinase 3 beta (pGSK3ß), throughout the menstrual cycle. RESULTS: Expression of Ki67 and SCF was higher in endometriosis than in control tissue (P < .05) and greater in secretory rather than proliferative (P < .01) endometrium in endometriosis. Expression of c-kit was also higher in endometriosis although similar in both phases. Expression of Akt and GSK3ß was identical in all samples and cycle phases, whereas pAkt and pGSK3ß, opposed to control tissue, remained overexpressed in the secretory phase in endometriosis. CONCLUSION: Unceasing cell proliferation in the secretory phase of eutopic endometriosis is linked to deregulation of c-kit/SCF-associated signaling pathways.

Pegylated interferon-α2b and ribavirin decrease claudin-1 and E-cadherin expression in HepG2 and Huh-7.5 cells.

BACKGROUND: Hepatitis C virus (HCV) infection usually results in long-term viremia. Entry of HCV into the hepatocyte requires claudin-1, -6, -9 and occludin. The efficacy of Pegylated interferon-α (PEG-IFN) treatment against HCV infection increased when ribavirin (RBV) was added to the therapeutic scheme. Our aim was to investigate if PEG-IFN plus RBV regulate claudin expression. MATERIAL AND METHODS: HepG2, Huh-7 and Huh-7.5 cells were treated with PEG-IFN-α2a or α2b and/or RBV at different times before obtaining the cytosolic, membrane and cytoskeletal fractions. Claudin-1, 3, 4, 6, and 9, E-cadherin and occludin expression was evaluated by Western blot analysis. Transepithelial electrical resistance (TER) was also determined. RESULTS: Claudin-1, 3, 4, 6, E-cadherin and occludin are constitutively expressed mainly in HepG2 cell membrane. Claudin-1 and E-cadherin cell membrane expression diminished after exposure to PEGIFNα2b (50 ng) + RBV(50 µg); the maximal decrease was observed with 200 ng of PEG-IFNα2b + 200 µg of RBV. The effect was less intense with PEG-IFNα2a. The inhibition of claudin-1 and E-cadherin expression in Huh-7 and Huh-7.5 cells was only observed with 200 ng of PEG-IFNα2b + 200 µg of RBV. TER diminished marginally in the HCV containing hepatoma cells with 200 ng of PEG-IFNα2b + 200 µg of RBV. Claudin-1 mRNA expression level was not affected by the combined treatment. CONCLUSION: The increased therapeutic efficacy of the PEG-IFNα2b plus RBV treatment could be secondary to the inhibition of claudin-1 and E-cadherin cell membrane expression.

Neuroprotective effect of the aminoestrogen prolame against impairment of learning and memory skills in rats injected with amyloid-ß-25-35 into the hippocampus.

Alzheimer's disease (AD) is a neurodegenerative disorder caused by the deposition of the amyloid-beta peptide (Aß) in senile plaques and cerebral vasculature. Its neurotoxic mechanisms are associated with the generation of oxidative stress and reactive astrogliosis that cause neuronal death and memory impairment. Estrogens reduce the rate of Azheimer's disease because of their antioxidant activity. Prolame (N-(3-hydroxy-1,3,5(10)-estratrien-17ß-yl)-3-hydroxypropylamine) is an aminoestrogen with estrogenic and antithrombotic effects. In our study we evaluated the role of prolame on Aß(25-35)-caused oxidative stress, reactive astrogliosis, and impairment of spatial memory(.) The Aß(25-35) (100 µM/µl) or vehicle was injected into the CA1 subfield of the hippocampus of the rat. The subcutaneous injection of prolame (400 µl, 50 nM) or sesame oil (400 µl) started 1 day before the Aß(25-35) injection and was continued for another 29 days. The results showed a significant impairment of spatial memory evident 30 days after the Aß(25-35) injection. The prolame treatment significantly reduced spatial-memory impairment and decreased lipid peroxidation, reactive oxygen species, and reactive gliosis. It also restored the eNOS and nNOS expression to normal levels. In conclusion the aminoestrogen prolame should be considered as an alternative in the treatment of Alzheimer's disease.

The effect of vanadium on platelet function.

Vanadium pentoxide (V(2)O(5)) inhalation effect on platelet function in mice was explored, as well as the in vitro effect on human platelets. Mouse blood samples were collected and processed for aggregometry and flow cytometry to assess the presence of P-selectin and monocyte-platelet conjugates. Simultaneously, human platelets were processed for aggregometry(.) The mouse results showed platelet aggregation inhibition in platelet-rich-plasma (PRP) at four-week exposure time, and normality returned at eight weeks of exposure, remaining unchanged after the exposure was discontinued after four weeks. This platelet aggregation inhibition effect was reinforced with the in vitro assay. In addition, P-selectin preserved their values during the exposure, until the exposure was discontinued during four weeks, when this activation marker increased. We conclude that vanadium affects platelet function, but further studies are required to evaluate its effect on other components of the hemostatic system.

Claudin-6, 7, or 9 overexpression in the human gastric adenocarcinoma cell line AGS increases its invasiveness, migration, and proliferation rate.

Altered claudin expression is related to metastatic potential, poor prognosis, or tumor recurrence. We analyzed if the overexpression of claudin-6, claudin-7, or claudin-9 in AGS cells altered cell motility, invasiveness, or proliferation rate. Claudin-7, claudin-9, and claudin-6 enhanced their invasive potential by 3.4-fold, 1.6-fold, and 2.0-fold, respectively. Claudin-6 and claudin-9 enhanced cell migration, while the proliferation rate of claudin-6-, claudin-7-, and claudin-9-transfected cells increased by 12.7%, 9.0%, and 13.3%, respectively. Claudin-7 and claudin-9 overexpression increased claudin-1 and zonula occludens-1 levels. In summary, individual increased expression of claudin-6, claudin-7, or claudin-9 is sufficient to enhance tumorigenic properties of a gastric adenocarcinoma cell line.

Hypothyroidism in Noninterferon Treated-HCV Infected Individuals Is Associated with Abnormalities in the Regulation of Th17 Cells.

HCV-Ag-specific TH17 cells secrete IL17, a cytokine involved in autoimmune diseases and regulated by IL10 and TGF-b. 5-12% of patients with chronic HCV infection have hypothyroidism. We evaluated the role of these cytokines in this patients by determining serum concentration of TsH, T3, free T4, IL2, IL10, IL12, IL17, TGF-b, anti-TG, TPO, CCP, GBM, and cardiolipin antibodies in 87 chronically noninterferon treated HCV-infected patients. 20 patients (group A) had elevated TsH values (>5 µUI/ml) whereas the remaining 67 (group B) had normal values. The percentage of anti-TPO, TG, GBM, and cardiolipin antibodies in group A patients (33%, 41%, 5% and 5%, resp.) as well as IL17, IL2 and TGF-b concentrations (25 ± 23 pg/ml, 643 ± 572 pg/ml, and 618 ± 221 pg/ml, resp.) were significantly higher than group B. Abnormal Th17 regulation mediated by IL-2 and low TGF-b concentrations is associated with hypothyroidism in chronically-infected HCV patients.

Distribution and expression pattern of claudins 6, 7, and 9 in diffuse- and intestinal-type gastric adenocarcinomas.

INTRODUCTION: Intestinal- and diffuse-type gastric adenocarcinomas differ in clinical outcome and genetic profile. Abnormal claudin expression has been well documented in several malignancies. Our aim was to find specific claudin markers for each type. METHODS: Fifty paraffin-embedded tissue blocks of diffuse- and intestinal-type gastric adenocarcinomas and fresh gastric biopsies obtained endoscopically from 20 patients with a presumptive diagnosis of gastric cancer were analyzed. Claudin-specific polyclonal and monoclonal antibodies were used for immunohistochemistry and Western blot analysis in total lysate and subcellular fractions. RESULTS: Claudin-6 expression was high in both types. Claudin-7 was expressed mainly in the diffuse-type whereas claudin-9 was mainly found in the apical membrane of the gland cells in the intestinal-type. Strong claudin-9 expression was associated with higher mortality rate (66%) in the diffuse type vs the intestinal type (25%) after a 2-year follow-up. CONCLUSION: Claudins 6, 7, and 9 expressions are closely related to gastric carcinogenesis, and their detection is a useful prognostic marker in "intestinal-" and "diffuse-type" gastric adenocarcinomas.