Albuquerque, NM, United States
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Al-Sadi R.,University of New Mexico | Al-Sadi R.,Albuquerque Veterans Affairs Medical Center | Guo S.,University of New Mexico | Guo S.,Albuquerque Veterans Affairs Medical Center | And 8 more authors.
Journal of Interferon and Cytokine Research | Year: 2012

Interleukin-1β (IL-1β) has been shown to play an essential role in mediating intestinal inflammation of Crohn's disease and other inflammatory conditions of the gut. Previous studies from our laboratory have shown that IL-1β causes an increase in intestinal tight-junction permeability in Caco-2 monolayers in vitro. However, the IL-1β effect on the intestinal epithelial barrier in vivo remains unclear. Aims: the major aims of this study were to examine the effect of IL-1β on mouse intestinal epithelial barrier in vivo and to delineate the mechanisms involved using an in vivo model system consisting of a recycling perfusion of mouse small intestine. Intraperitonial injection of IL-1β at varying doses (0-10μg) caused a concentration-dependent increase in mouse intestinal permeability to the paracellular marker dextran (10 KD), and the maximal increase in dextran flux occurred at IL-1β dose of 5μg. IL-1β treatment caused an increase in myosin light-chain kinase (MLCK) mRNA and protein expression in the small intestinal tissue starting at 24h, which continued up to 72h. Additionally, IL-1β did not cause an increase in intestinal permeability in MLCK-deficient mice (C57BL/6 MLCK-/-). MLCK inhibitor ML-7 (2mg/kg body weight) also inhibited the IL-1β-induced increase in small intestinal permeability. The IL-1β-induced increase in mouse intestinal permeability was associated with an increase in NF-κB activation. The intestinal tissue-specific silencing of NF-κB p65 inhibited the IL-1β-induced increase in intestinal permeability and increase in MLCK expression. These data show for the first time that IL-1β causes an increase in mouse intestinal permeability in vivo. These data suggested that the mechanism of IL-1β-induced increase in mouse intestinal permeability in vivo involved NF-κB p65-induced activation of the mouse enterocyte MLCK gene. © Copyright 2012, Mary Ann Liebert, Inc. 2012.


Al-Sadi R.,University of New Mexico | Ye D.,University of New Mexico | Said H.M.,University of California at Irvine | Ma T.Y.,University of New Mexico | Ma T.Y.,Albuquerque Veterans Affairs Medical Center
Journal of Cellular and Molecular Medicine | Year: 2011

Interleukin-1β (IL-1β) is a prototypical multifunctional cytokine that plays an important role in intestinal inflammation of Crohn's disease and other inflammatory conditions of the gut. Previous studies have shown that IL-1β causes an increase in intestinal epithelial tight junction (TJ) permeability both in in vivo animal and in vitro cell culture model systems. The IL-1β-induced increase in intestinal epithelial TJ permeability has been postulated to be an important pathogenic mechanism contributing to intestinal inflammation. However, the signalling pathways and the molecular processes that mediate the IL-1β modulation of intestinal epithelial TJ barrier remain unclear. Here, we show that the IL-1β-induced increase in Caco-2 monolayer TJ permeability was mediated by activation of extracellular signal-regulated kinases 1/2 (ERK1/2) signalling pathway and that inhibition of ERK1/2 activity inhibits the IL-1β-induced increase in Caco-2 TJ permeability. The activation of ERK1/2 pathway caused a downstream activation of nuclear transcription factor Elk-1. The activated Elk-1 translocated to the nucleus and binds to the cis-binding motif on myosin light chain kinase (MLCK) promoter region, triggering MLCK gene activation, MLCK mRNA transcription and MLCK protein synthesis and MLCK catalysed opening of the intestinal epithelial TJ barrier. These studies provide novel insight into the cellular and molecular processes that mediate the IL-1β-induced increase in intestinal epithelial TJ permeability. © 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.


Guo S.,University of New Mexico | Guo S.,Albuquerque Veterans Affairs Medical Center | Al-Sadi R.,University of New Mexico | Al-Sadi R.,Albuquerque Veterans Affairs Medical Center | And 3 more authors.
American Journal of Pathology | Year: 2013

Bacterial-derived lipopolysaccharides (LPS) play an essential role in the inflammatory process of inflammatory bowel disease. A defective intestinal tight junction (TJ) barrier is an important pathogenic factor of inflammatory bowel disease and other inflammatory conditions of the gut. Despite its importance in mediating intestinal inflammation, the physiological effects of LPS on the intestinal epithelial barrier remain unclear. The major aims of this study were to determine the effects of physiologically relevant concentrations of LPS (0 to 1 ng/mL) on intestinal barrier function using an in vitro (filter-grown Caco-2 monolayers) and an in vivo (mouse intestinal perfusion) intestinal epithelial model system. LPS, at physiologically relevant concentrations (0 to 1 ng/mL), in the basolateral compartment produced a time-dependent increase in Caco-2 TJ permeability without inducing cell death. Intraperitoneal injection of LPS (0.1 mg/kg), leading to clinically relevant plasma concentrations, also caused a time-dependent increase in intestinal permeability in vivo. The LPS-induced increase in intestinal TJ permeability was mediated by an increase in enterocyte membrane TLR-4 expression and a TLR-4-dependent increase in membrane colocalization of membrane-associated protein CD14. In conclusion, these studies show for the first time that LPS causes an increase in intestinal permeability via an intracellular mechanism involving TLR-4-dependent up-regulation of CD14 membrane expression. © 2013 American Society for Investigative Pathology.


Al-Sadi R.,University of New Mexico | Khatib K.,University of New Mexico | Guo S.,University of New Mexico | Ye D.,University of New Mexico | And 3 more authors.
American Journal of Physiology - Gastrointestinal and Liver Physiology | Year: 2011

Defective intestinal epithelial tight junction (TJ) barrier has been shown to be an important pathogenic factor contributing to the development of intestinal inflammation. The expression of occludin is markedly decreased in intestinal permeability disorders, including in Crohn's disease, ulcerative colitis, and celiac disease, suggesting that the decrease in occludin expression may play a role in the increase in intestinal permeability. The purpose of this study was to delineate the involvement of occludin in intestinal epithelial TJ barrier by selective knock down of occludin in in vitro (filter-grown Caco-2 monolayers) and in vivo (recycling perfusion of mouse intestine) intestinal epithelial models. Our results indicated that occludin small-interfering RNA (siRNA) transfection causes an increase in transepithelial flux of various-sized probes, including urea, mannitol, inulin, and dextran, across the Caco-2 monolayers, without affecting the transepithelial resistance. The increase in relative flux rate was progressively greater for larger-sized probes, indicating that occludin depletion has the greatest effect on the flux of large macromolecules. siRNA-induced knock down of occludin in mouse intestine in vivo also caused an increase in intestinal permeability to dextran but did not affect intestinal tissue transepithelial resistance. In conclusion, these results show for the first time that occludin depletion in intestinal epithelial cells in vitro and in vivo leads to a selective or preferential increase in macromolecule flux, suggesting that occludin plays a crucial role in the maintenance of TJ barrier through the large-channel TJ pathway, the pathway responsible for the macromolecule flux. © 2011 the American Physiological Society.


Sanchez-Chardi A.,Autonomous University of Barcelona | Olivares F.,Autonomous University of Barcelona | Byrd T.F.,University of New Mexico | Byrd T.F.,Albuquerque Veterans Affairs Medical Center | And 3 more authors.
Journal of Clinical Microbiology | Year: 2011

In low-income countries some infections caused by nontuberculous mycobacteria are misdiagnosed as multidrug-resistant tuberculosis. In most of these settings the observation of microscopic cords is the only technique used to identify Mycobacterium tuberculosis in the laboratory. In this article we definitively demonstrate that Mycobacterium abscessus, an emerging pulmonary pathogen, also forms microscopic cords. Copyright © 2011, American Society for Microbiology. All Rights Reserved.


Ye D.,University of New Mexico | Guo S.,University of New Mexico | Alsadi R.,University of New Mexico | Alsadi R.,Albuquerque Veterans Affairs Medical Center | And 2 more authors.
Gastroenterology | Year: 2011

Background & Aims: Defects in the intestinal epithelial tight junction (TJ) barrier contribute to intestinal inflammation. A tumor necrosis factor (TNF)-αinduced increase in intestinal TJ permeability contributes to the intestinal TJ barrier defect in inflammatory disorders. We investigated the mechanisms by which TNF-α induces occludin depletion and an increase in intestinal TJ permeability. Methods: We assessed intestinal TJ barrier function using intestinal epithelial model systems: filter-grown Caco-2 monolayers and recycling perfusion studies of mouse small intestine. Results: TNF-α caused a rapid increase in expression of microRNA (miR)-122a in enterocytes, cultured cells, and intestinal tissue. The overexpressed miR-122a bound to a binding motif at the 3′-untranslated region of occludin messenger RNA (mRNA) to induce its degradation; mRNA degradation depleted occludin from enterocytes, resulting in increased intestinal TJ permeability. Transfection of enterocytes with an antisense oligoribonucleotide against miR-122a blocked the TNF-αinduced increase in enterocyte expression of miR-122a, degradation of occludin mRNA, and increase in intestinal permeability. Overexpression of miR-122a in enterocytes using premiR-122a was sufficient to induce degradation of occludin mRNA and an increase in intestinal permeability. Conclusions: TNF-α regulates intestinal permeability by inducing miR-122amediated degradation of occludin mRNA. These studies show the feasibility of therapeutically targeting miR-122a in vivo to preserve the intestinal barrier. © 2011 AGA Institute.


Al-Sadi R.,University of New Mexico | Al-Sadi R.,Albuquerque Veterans Affairs Medical Center | Ye D.,University of New Mexico | Boivin M.,University of New Mexico | And 6 more authors.
PLoS ONE | Year: 2014

Defective intestinal epithelial tight junction (TJ) barrier has been shown to be a pathogenic factor in the development of intestinal inflammation. Interleukin-6 (IL-6) is a pleiotropic, pro-inflammatory cytokine which plays an important role in promoting inflammatory response in the gut and in the systemic circulation. Despite its key role in mediating variety inflammatory response, the effect of IL-6 on intestinal epithelial barrier remains unclear. The purpose of this study was to investigate the effect of IL-6 on intestinal epithelial TJ barrier and to delineate the intracellular mechanisms involved using in-vitro (filter-grown Caco-2 monolayers) and in-vivo model (mouse intestinal perfusion) systems. Our results indicated that IL-6 causes a site-selective increase in Caco-2 intestinal epithelia TJ permeability, causing an increase in flux of small-sized molecules having molecular radius <4 A&ring. The size-selective increase in Caco-2 TJ permeability was regulated by protein-specific increase in claudin-2 expression. The IL-6 increase in TJ permeability required activation of JNK signaling cascade. The JNK pathway activation of AP-1 resulted in AP-1 binding to its binding sequence on the claudin-2 promoter region, leading to promoter activation and subsequent increase in claudin-2 gene transcription and protein synthesis and TJ permeability. Our in-vivo mouse perfusion showed that IL-6 modulation of mouse intestinal permeability was also mediated by AP-1 dependent increase in claudin-2 expression. In conclusion, our studies show for the first time that the IL-6 modulation of intestinal TJ permeability was regulated by JNK activation of AP-1 and AP-1 activation of claudin-2 gene.


Al-Sadi R.,University of New Mexico | Guo S.,University of New Mexico | Ye D.,University of New Mexico | Ma T.Y.,University of New Mexico | Ma T.Y.,Albuquerque Veterans Affairs Medical Center
American Journal of Pathology | Year: 2013

Tumor necrosis factor (TNF-α) is a proinflammatory cytokine that plays a critical role in the pathogenesis of inflammatory bowel disease. TNF-α causes an increase in intestinal permeability; however, the signaling pathways and the molecular mechanisms involved remain unclear. The major purpose of this study was to investigate the role of MAP kinase pathways (ERK1/2 and p38 kinase) and the molecular processes involved. An in vitro intestinal epithelial model system consisting of Caco-2 monolayers and an in vivo mouse model system were used to delineate the cellular and molecular mechanisms involved in TNF-α effects on tight junction barrier. The TNF-α-induced increase in Caco-2 tight junction permeability was mediated by activation of the ERK1/2 signaling pathway, but not the p38 kinase pathway. Activation of the ERK1/2 pathway led to phosphorylation and activation of the ETS domain-containing transcription factor Elk-1. The activated Elk-1 translocated to the nucleus, where it bound to its binding motif on the myosin light chain kinase (MLCK) promoter region, leading to the activation of MLCK promoter activity and gene transcription. In addition, in vivo intestinal perfusion studies also indicated that the TNF-α-induced increase in mouse intestinal permeability requires ERK1/2-dependent activation of Elk-1. These studies provide novel insight into the cellular and molecular processes that regulate the TNF-α-induced increase in intestinal epithelial tight junction permeability. © 2013 American Society for Investigative Pathology.

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