Defective intestinal epithelial tight junction (TJ) barrier is a key pathogenic factor of inflammatory bowel disease (IBD) and other inflammatory conditions of the gut. The defective TJ barrier allows increased intestinal permeation of bacterial antigens that induce inflammatory response. However, there are no currently available therapeutic agents that target the intestinal TJ barrier. Moreover, the mechanisms that lead to an enhancement of intestinal TJ barrier remain poorly understood. The major goals of this proposal are to introduce and develop a new therapeutic agent that targets the intestinal TJ barrier, which can be rapidly advanced for clinical usage;and to identify novel intracellular mechanisms that regulate the tightening of the intestinal TJ barrier. In our preliminary studies, we screened over 20 probiotic bacterial strains (that are widely used commercially) to identify a single strain, Lactobacillus acidophilus (LA), which has a remarkable intestinal TJ barrier augmenting properties and therapeutic efficacy in animal models of IBD. The over-arching goals of this application are to investigate the intestinal TJ barrier enhancing effects of LA and to determine the therapeutic efficacy of LA in animal models of IBD. Based on our preliminary studies, we advance a novel hypothesis that LA enhancement of intestinal TJ barrier is regulated by a nucleotide-binding oligomerization domain - containing protein 1 (Nod1) signal transduction pathway activation of occludin gene. Occludin is a trans-membrane protein which plays a central role in intestinal TJ barrier regulation. In this grant application, we challenge to well-established scientific paradigms 1) that Nod1 is a cytoplasmic pattern recognition receptor and 2) that primary cellular target of Nod1 is the activation of NF-?B. Based on our preliminary data showing that LA induces a rapid cytoplasmic-to-apical membrane translocation of Nod1 in intestinal epithelial cells in-vitro and in-vivo, we hypothesize that LA- induced augmentation of intestinal TJ barrier is regulated by apical membrane translocation of Nod1. We also hypothesize that LA activation of Nod1 signal transduction pathway leads to a suppression (not activation) of NF-?B, a key pro-inflammatory mediator that induces the TJ opening, and an activation of p38 kinase pathway, which signals occludin gene activation and occludin-dependent enhancement in intestinal TJ barrier function. The proposed specific aims are to: 1) delineate the role of Nod1 in LA-induced augmentation of intestinal epithelial TJ barrier;2) delineate the mechanism of LA-induced regulation of occludin gene and intestinal TJ barrier;and 3) delineate the therapeutic efficacy of LA in animal models of IBD. The successful completion of the proposed studies will provide the critical pre-clinical data necessary to support our future clinical trials that will examine the therapeutic efficacy of LA in intestinal barrier tightening in IBD patients and also in the prevention and treatment of IBD.

Public Health Relevance

Patients with inflammatory bowel disease, Crohn's disease and ulcerative colitis, have a leaky gut, characterized by an increase in intestinal permeability t harmful antigens in the intestine. The studies proposed in this grant application seek to introduce a new therapeutic agent that induces a tightening of the leaky gut and prevents intestinal inflammation. The data derived from these studies will be crucial in supporting the future clinical trials that will investigate the therapeutic efficacy of intestinal barrier tightenng in the prevention and treatment of inflammatory bowel disease.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Clinical, Integrative and Molecular Gastroenterology Study Section (CIMG)
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Hamilton, Frank A
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University of New Mexico Health Sciences Center
Internal Medicine/Medicine
Schools of Medicine
United States
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Guo, Shuhong; Al-Sadi, Rana; Said, Hamid M et al. (2013) Lipopolysaccharide causes an increase in intestinal tight junction permeability in vitro and in vivo by inducing enterocyte membrane expression and localization of TLR-4 and CD14. Am J Pathol 182:375-87
Al-Sadi, Rana; Guo, Shuhong; Ye, Dongmei et al. (2013) Mechanism of IL-1* modulation of intestinal epithelial barrier involves p38 kinase and activating transcription factor-2 activation. J Immunol 190:6596-606
Al-Sadi, Rana; Ye, Dongmei; Said, Hamid M et al. (2011) Cellular and molecular mechanism of interleukin-1* modulation of Caco-2 intestinal epithelial tight junction barrier. J Cell Mol Med 15:970-82
Al-Sadi, Rana; Khatib, Khaldun; Guo, Shuhong et al. (2011) Occludin regulates macromolecule flux across the intestinal epithelial tight junction barrier. Am J Physiol Gastrointest Liver Physiol 300:G1054-64
Ye, Dongmei; Guo, Shuhong; Al-Sadi, Rana et al. (2011) MicroRNA regulation of intestinal epithelial tight junction permeability. Gastroenterology 141:1323-33
Al-Sadi, Rana; Ye, Dongmei; Said, Hamid M et al. (2010) IL-1beta-induced increase in intestinal epithelial tight junction permeability is mediated by MEKK-1 activation of canonical NF-kappaB pathway. Am J Pathol 177:2310-22
Al-Sadi, Rana; Boivin, Michel; Ma, Thomas (2009) Mechanism of cytokine modulation of epithelial tight junction barrier. Front Biosci 14:2765-78
Boivin, Michel A; Roy, Praveen K; Bradley, Angela et al. (2009) Mechanism of interferon-gamma-induced increase in T84 intestinal epithelial tight junction. J Interferon Cytokine Res 29:45-54
Ye, Dongmei; Ma, Thomas Y (2008) Cellular and molecular mechanisms that mediate basal and tumour necrosis factor-alpha-induced regulation of myosin light chain kinase gene activity. J Cell Mol Med 12:1331-46
Al-Sadi, Rana; Ye, Dongmei; Dokladny, Karol et al. (2008) Mechanism of IL-1beta-induced increase in intestinal epithelial tight junction permeability. J Immunol 180:5653-61

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