Publications by authors named "Hamed Laroui"

40 Publications

Internalization of Garlic-Derived Nanovesicles on Liver Cells is Triggered by Interaction With CD98.

ACS Omega 2020 Sep 2;5(36):23118-23128. Epub 2020 Sep 2.

Department of Chemistry, Center for Diagnostics and Therapeutics (CDT), Georgia State University, Atlanta, Georgia 30302, United States.

The mechanism of how plant-derived nanovesicles are uptaken by cells remains unknown. In this study, the garlic-derived nanovesicles (GDVs) were isolated and digested with trypsin to remove all surface proteins. Digested GDVs showed less uptake compared to undigested GDVs, confirming that the surface proteins played a role in the endocytosis. On the cell side (HepG2), interestingly, blocking the CD98 receptors significantly reduced the uptake of GDVs. During the cellular internalization of GDVs, we observed that some surface proteins of GDVs were co-localized with CD98. A total lysate of the GDV surface showed a high presence of a mannose-specific binding protein, II lectin. Blocking GDV II lectin (using mannose preincubation) highly reduced the GDV internalization, which supports that direct interaction between II lectin and CD98 plays an important role in internalization. The GDVs also exhibited anti-inflammatory effect by downregulating proinflammatory factors on the HepG2 cells. This work contributes to understanding a part of the GDV internalization process and the cellular anti-inflammatory effects of garlic.
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http://dx.doi.org/10.1021/acsomega.0c02893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7495725PMC
September 2020

Matrix metalloproteinase 9 (MMP9) limits reactive oxygen species (ROS) accumulation and DNA damage in colitis-associated cancer.

Cell Death Dis 2020 09 17;11(9):767. Epub 2020 Sep 17.

Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, United States.

Colitis-associated cancer (CAC) is a subtype of colon cancer that is driven by chronic inflammation and is prevalent in chronic ulcerative colitis patients. The development of CAC is associated with the inflammation-dysplasia-carcinoma pathway which is significantly different than adenoma-carcinoma pathway of sporadic colon cancer (CRC). Matrix Metalloproteinase 9 (MMP9) is a zinc-dependent endopeptidase against extracellular matrix (ECM) proteins expressed in the gastrointestinal tract during inflammation. We have previously shown that MMP9 plays a tumor suppressor role in CAC via "MMP9-Notch1-ARF-p53 axis" pathway. The aim of this study is to determine the role of MMP9 in maintaining genomic stability in CAC. Homozygous transgenic mice with constitutive-expression of MMP9 in the colonic epithelium (TgM9) with their wild-type littermates (WT) and stably transfected HCT116 cells with/without MMP9 were used for in vivo and in vitro experiments, respectively. As 'proof of concept' model, nanoparticles (NPs) loaded with MMP9 siRNA were used to examine the effect of MMP9 silencing in the colonic epithelium. In CAC, colonic epithelium of TgM9 mice exhibited lower amounts of reactive oxygen species (ROS), less DNA damage, and increased expression of mismatch repair genes compared to WTs. Our study showed that MMP9 expression correlates with the reduced ROS levels, decreased DNA damage, and upregulated mismatch repair pathway. This suggests that MMP9 expression is a natural biological way to suppress CAC by limiting ROS accumulation and DNA damage in the colon. Therefore, MMP9 inhibition could be deleterious for CAC patient.
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http://dx.doi.org/10.1038/s41419-020-02959-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7498454PMC
September 2020

Role of CD98 in liver disease.

Ann Hepatol 2020 Nov - Dec;19(6):602-607. Epub 2020 Jan 30.

Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, 145 Piedmont Ave SE, Atlanta, GA, USA; Department of Biology, Center for Diagnostics and Therapeutics, Georgia State University, 145 Piedmont Ave SE, Atlanta, GA, USA. Electronic address:

CD98 is a multifunctional glycoprotein that is involved in various biological processes such as amino acid transport, cell adhesion, diffusion, adhesion, and proliferation. The role of CD98 in liver disease has not thoroughly been examined and is limited reports in the literature. Among these reports, direct association for CD98 in nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC) have been reported. Our lab has reported that targeting CD98 in high fat diet mice reduced steatosis and inflammation in NAFLD. Other reports associate CD98 in HCC due in part to the role of CD98 in activating integrin signaling. Herein, we present CD98 staining on liver biopsies from NAFLD, chronic active hepatitis, cirrhosis, and 3 stages of HCC to demonstrate the upregulation of CD98 expression throughout liver disease progression. In addition, we analyze current literature to elucidate roles and potential roles of CD98 with each stage of liver disease.
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http://dx.doi.org/10.1016/j.aohep.2019.11.011DOI Listing
January 2020

CD98 siRNA-loaded nanoparticles decrease hepatic steatosis in mice.

Dig Liver Dis 2017 Feb 17;49(2):188-196. Epub 2016 Nov 17.

Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, USA; Department of Biology, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, USA. Electronic address:

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive lipid hepatic accumulation. Here, we investigated whether a reduction of CD98 expression mediated by CD98 siRNA-loaded nanoparticles (NPs) could attenuate liver disease markers in a mouse model of NAFLD. NPs were generated using a double emulsion/solvent evaporation technique. Mice fed a high fat diet for 8 weeks to induce fatty liver were treated with vein tail injections of CD98 siRNA-loaded NPs. In vitro, HepG2 treated with CD98 siRNA-loaded NPs showed significant downregulation of CD98 leading to a significant decrease of major pro-inflammatory cytokines and markers. In vivo, CD98 siRNA-loaded NPs strongly decreased all markers of NAFLD, including the blood levels of ALT and lipids accumulation, fibrosis evidence and pro-inflammatory cytokines. In conclusion, our results indicate that CD98 appears to function as a key actor/inducer in NAFLD, and that our NPs approach may offer a new targeted therapeutic for this disease.
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http://dx.doi.org/10.1016/j.dld.2016.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6475075PMC
February 2017

Epithelial derived-matrix metalloproteinase (MMP9) exhibits a novel defensive role of tumor suppressor in colitis associated cancer by activating MMP9-Notch1-ARF-p53 axis.

Oncotarget 2017 Jan;8(1):364-378

Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA.

Colitis associated cancer (CAC) is chronic inflammation driven colon cancer, prevalent among individuals with Inflammatory Bowel Disease. Matrix-metalloproteinase (MMP9) is one of the essential regulators of extra cellular matrix components. We have shown that MMP9 is protective in CAC contrary to its inflammatory role in acute-colitis. Aim of our study is to identify the mechanism of the protective role of epithelial derived-MMP9 in CAC. We used homozygous transgenic mice constitutively-expressing MMP9 in colonic-epithelium (TgM9) and wild-type (WT) littermates for in vivo experiments. Stably-transfected HCT116 with/without MMP9, and mouse embryonic-fibroblasts (WT and MMP9-/-, MEFs) were used for in vitro experiments. TgM9 mice exhibited less tumor burden, increased apoptosis, and increased expressions of active-Notch1, p53, p21WAF1/Cip1, caspase-3 and cyclin E in CAC compared to WTs. These results were supported by MEFs data. HCT116-cells overexpressing MMP9 indicated decreased cell proliferation, S-phase cell-cycle arrest and less DNA damage compared to vector. MMP9-/- mice showed attenuation of MMP9 was directly associated with p19ARF. Our study identifies the tumor suppressor role of epithelial derived-MMP9 in CAC via novel mechanistic pathway "MMP9-Notch1-ARF-p53 axis" regulating apoptosis, cell-cycle arrest and DNA damage implying, that MMP9 expression might be a natural/biological way to suppress colonic ulceration due to chronic inflammation.
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http://dx.doi.org/10.18632/oncotarget.13406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5352126PMC
January 2017

Glial cell line-derived neurotrophic factor protects against high-fat diet-induced hepatic steatosis by suppressing hepatic PPAR-γ expression.

Am J Physiol Gastrointest Liver Physiol 2016 Jan 12;310(2):G103-16. Epub 2015 Nov 12.

Division of Digestive Diseases, Emory University School of Medicine, Atlanta, Georgia; Atlanta Veterans Affairs Medical Center, Decatur, Georgia;

Glial cell line-derived neurotrophic factor (GDNF) protects against high-fat diet (HFD)-induced hepatic steatosis in mice, however, the mechanisms involved are not known. In this study we investigated the effects of GDNF overexpression and nanoparticle delivery of GDNF in mice on hepatic steatosis and fibrosis and the expression of genes involved in the regulation of hepatic lipid uptake and de novo lipogenesis. Transgenic overexpression of GDNF in liver and other metabolically active tissues was protective against HFD-induced hepatic steatosis. Mice overexpressing GDNF had significantly reduced P62/sequestosome 1 protein levels suggestive of accelerated autophagic clearance. They also had significantly reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) and CD36 gene expression and protein levels, and lower expression of mRNA coding for enzymes involved in de novo lipogenesis. GDNF-loaded nanoparticles were protective against short-term HFD-induced hepatic steatosis and attenuated liver fibrosis in mice with long-standing HFD-induced hepatic steatosis. They also suppressed the liver expression of steatosis-associated genes. In vitro, GDNF suppressed triglyceride accumulation in Hep G2 cells through enhanced p38 mitogen-activated protein kinase-dependent signaling and inhibition of PPAR-γ gene promoter activity. These results show that GDNF acts directly in the liver to protect against HFD-induced cellular stress and that GDNF may have a role in the treatment of nonalcoholic fatty liver disease.
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http://dx.doi.org/10.1152/ajpgi.00196.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4719063PMC
January 2016

Longitudinal study of circulating protein biomarkers in inflammatory bowel disease.

J Proteomics 2015 Jan 16;112:166-79. Epub 2014 Sep 16.

Institute for Biomedical Sciences, Georgia State University, Atlanta, GA 30303, USA; Chemistry Department, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA; Veterans Affairs Medical Center, Decatur, GA, USA.

Unlabelled: Inflammatory bowel diseases (IBDs) are chronic and progressive inflammatory disorders of the gastrointestinal tract. In IBD, protein serological biomarkers could be relevant tools for assessing disease activity, performing early-stage diagnosis and managing the treatment. Using the interleukin-10 knockout (IL-10(-/-)) mouse, a model that develops a time-dependent IBD-like disorder that predominates in the colon; we performed longitudinal studies of circulating protein biomarkers in IBD. Circulating protein profiles in serum samples collected from 30-, 93-, to 135-day-old IL-10(-/-) mice were investigated using two-dimensional differential gel electrophoresis and MALDI-TOF/TOF tandem mass spectrometry. A total of 15 different proteins were identified and confirmed by ELISA and Western blot to be differentially accumulated in serum samples from mid- to late-stage IL-10(-/-) mice compared to early non-inflamed IL-10(-/-) mice. The use of another model of colitis and an extra-intestinal inflammation model validated this biomarker panel and demonstrated that comprised some global inflammatory markers, some intestinal inflammation-specific markers and some chronic intestinal inflammation markers. Statistical analyses using misclassification error rate charts validated the use of these identified proteins as powerful biomarkers of colitis. Unlike standard biomarker screening studies, our analyses identified a panel of proteins that allowed the definition of protein signatures that reflect colitis status.

Biological Significance: Crohn's disease (CD) and ulcerative colitis (UC) are the most common inflammatory bowel diseases (IBDs) occurring in humans. The major current diagnosis tool is colonoscopy, which is invasive and could lead to false diagnosis. The emergence of serological biomarkers enables the use of new diagnosis tools such as protein signatures for IBD diagnosis/management. Using 2D-DIGE coupled to mass spectrometry, our longitudinal study in a mouse model of colitis identified a signature of protein biomarkers for specific stages of disease.
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http://dx.doi.org/10.1016/j.jprot.2014.09.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4312267PMC
January 2015

A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion.

Proc Natl Acad Sci U S A 2014 Jul 14;111(30):11163-8. Epub 2014 Jul 14.

Emory Vaccine Center, andYerkes National Primate Research Center, Emory University, Atlanta, GA 30329;Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322

Clustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals.
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http://dx.doi.org/10.1073/pnas.1323025111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4121812PMC
July 2014

Fab'-bearing siRNA TNFα-loaded nanoparticles targeted to colonic macrophages offer an effective therapy for experimental colitis.

J Control Release 2014 Jul 5;186:41-53. Epub 2014 May 5.

Institute for Biomedical Sciences, USA; Department Chemistry and Biology, Center Diagnostics and Therapeutics, Georgia State University, 100 Piedmont Avenue, Atlanta, GA 30303, USA; Veterans Affair Medical Center, 1670 Clairmont Rd, Decatur, GA 30033, USA; Center for Inflammation, Immunity, & Infection, Georgia State University, 100 Piedmont Avenue, Atlanta, GA 30303, USA.

Patients suffering from inflammatory bowel disease (IBD) are currently treated by systemic drugs that can have significant side effects. Thus, it would be highly desirable to target TNFα siRNA (a therapeutic molecule) to the inflamed tissue. Here, we demonstrate that TNFα siRNA can be efficiently loaded into nanoparticles (NPs) made of poly (lactic acid) poly (ethylene glycol) block copolymer (PLA-PEG), and that grafting of the Fab' portion of the F4/80 Ab (Fab'-bearing) onto the NP surface via maleimide/thiol group-mediated covalent bonding improves the macrophage (MP)-targeting kinetics of the NPs to RAW264.7 cells in vitro. Direct binding was shown between MPs and the Fab'-bearing NPs. Next, we orally administered hydrogel (chitosan/alginate)-encapsulated Fab'-bearing TNFα-siRNA-loaded NPs to 3% dextran sodium sulfate (DSS)-treated mice and investigated the therapeutic effect on colitis. In vivo, the release of TNFα-siRNA-loaded NPs into the mouse colon attenuated colitis more efficiently when the NPs were covered with Fab'-bearing, compared to uncovered NPs. All DSS-induced parameters of colonic inflammation (e.g., weight loss, myeloperoxidase activity, and Iκbα accumulation) were more attenuated Fab'-bearing NPs loaded with TNFα siRNA than without the Fab'-bearing. Grafting the Fab'-bearing onto the NPs improved the kinetics of endocytosis as well as the MP-targeting ability, as indicated by flow cytometry. Collectively, our results show that Fab'-bearing PLA-PEG NPs are powerful and efficient nanosized tools for delivering siRNAs into colonic macrophages.
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http://dx.doi.org/10.1016/j.jconrel.2014.04.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4100604PMC
July 2014

Glycoprotein CD98 as a receptor for colitis-targeted delivery of nanoparticle.

J Mater Chem B 2014 Mar;2(11):1499-1508

Center for Diagnostics and Therapeutics, Department of Biology and Chemistry, Georgia State University, Atlanta, 30302, USA. ; Tel: +1-404-413-3597 ; Atlanta Veterans Affairs Medical Center, Decatur, 30033, USA.

Treatment strategies for inflammatory bowel disease have been constrained by limited therapeutic efficacy and serious adverse effects owing to a lack of receptor for targeted drug delivery to the inflamed colon. Upon inflammation, CD98 expression is highly elevated in colonic epithelial cells and infiltrating immune cells. To investigate whether CD98 can be used as a colitis-targeted delivery receptor, we constructed CD98 Fab'-bearing quantum dots (QDs)-loaded nanoparticles (Fab'-NPs). The resultant Fab'-NPs had desired particle size (~458 nm) with a narrow size distribution and zeta-potential (approximately +19 mV), low cytotoxicity, and excellent fluorescence properties. Electron microscopy images provided direct evidence for the well-dispersed distribution of QDs within spherical Fab'-NPs. Cellular uptake experiments demonstrated that Fab'-NPs were efficiently internalized into Colon-26 and RAW 264.7 cells through the CD98-mediated endocytosis pathway, and showed that the targeting effect of CD98 Fab' markedly increased their cellular uptake efficiency compared with control pegylated QDs-loaded NPs (PEG-NPs). Furthermore, studies showed much more effective accumulation of Fab'-NPs in colitis tissue than that of PEG-NPs. These findings suggest that because of inflammation-dependent over-expression of CD98, active colitis-targeted delivery can be accomplished using NPs decorated with CD98 antibody.
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http://dx.doi.org/10.1039/C3TB21564DDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3981968PMC
March 2014

Genetic deletion of Klf4 in the mouse intestinal epithelium ameliorates dextran sodium sulfate-induced colitis by modulating the NF-κB pathway inflammatory response.

Inflamm Bowel Dis 2014 May;20(5):811-20

*Department of Medicine, Stony Brook University, Stony Brook, New York; †Department of Biology and Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia; and ‡Veterans Affairs Medical Center, Decatur, GA.

Background: Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor expressed in the differentiated epithelial cells lining of the intestine. Under physiological conditions, KLF4 inhibits cell proliferation. Conversely, KLF4 mediates proinflammatory signaling in macrophages and its overexpression in the esophageal epithelium activates cytokines, leading to inflammation-mediated esophageal squamous cell cancer formation in mice. Here, we tested whether KLF4 has a proinflammatory activity in experimental colitis in mice.

Methods: Villin-Cre;Klf4 mice with intestine-specific Klf4 deletion (Klf4) and control mice with floxed Klf4 gene (Klf4) were treated or not with 3% dextran sodium sulfate (DSS) for 7 days to induce colitis. Additionally, WT mice were administered or not, nanoparticles loaded with scrambled or Klf4-siRNA, and concomitantly given DSS.

Results: Compared with DSS-treated Klf4 mice, DSS-treated Klf4 mice were significantly less sensitive to DSS-induced colitis. DSS treatment of Klf4 mice induced Klf4 expression in the crypt zone of the colonic epithelium. DSS-treated Klf4 mice had increased proliferation relative to DSS-treated control mice. DSS treatment induced NF-κB signaling pathway in Klf4 mice colon but not Klf4 mice. Additionally, WT mice given DSS and nanoparticle/Klf4-siRNA were less sensitive to colitis and had reduced Klf4 expression and while maintaining the proliferative response in the colonic epithelium.

Conclusions: Our results indicate that Klf4 is an important mediator of DSS-induced colonic inflammation by modulating NF-κB signaling pathway and could be involved in the pathogenesis and/or propagation of inflammatory bowel disease. Thus, Klf4 may represent a novel therapeutic target in inflammatory bowel disease.
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http://dx.doi.org/10.1097/MIB.0000000000000022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4091934PMC
May 2014

Nanoparticles with surface antibody against CD98 and carrying CD98 small interfering RNA reduce colitis in mice.

Gastroenterology 2014 May 4;146(5):1289-300.e1-19. Epub 2014 Feb 4.

Center for Diagnostics and Therapeutics, Institute for Biomedical Science, Departments of Biology and Chemistry, Georgia State University, Atlanta; Atlanta Veterans Affairs Medical Center, Decatur.

Background & Aims: Nanoparticles have been explored as carriers of small interfering RNAs (siRNAs) and might be developed to treat patients with inflammatory bowel disease (IBD). Overexpression of CD98 on the surface of colonic epithelial cells and macrophages promotes the development and progression of IBD. We developed an orally delivered hydrogel that releases nanoparticles with single-chain CD98 antibodies on their surface (scCD98 functionalized) and loaded with CD98 siRNA (siCD98). We tested the ability of the nanoparticles to reduce levels of CD98 in the colons of mice with colitis.

Methods: scCD98-functionalized siCD98-loaded nanoparticles were fabricated using a complex coacervation technique. We investigated the cellular uptake and lysosome escape profiles of the nanoparticles in Colon-26 cells and RAW 264.7 macrophages using fluorescence microscopy. Colitis was induced by transfer of CD4(+)CD45RB(high) T cells to Rag(-/-) mice or administration of dextran sodium sulfate to C57BL/6 mice. Mice were then given hydrogel (chitosan and alginate) containing scCD98-functionalized nanoparticles loaded with siCD98 or scrambled siRNA (control) via gavage.

Results: The scCD98-functionalized nanoparticles were approximately 200 nm in size and had high affinity for CD98-overexpressing cells. The scCD98-functionalized siCD98-loaded nanoparticles significantly reduced levels of CD98 in Colon-26 cells and RAW 264.7 macrophages, along with production of inflammatory cytokines (tumor necrosis factor α, interleukin-6, and interleukin-12). In mice with colitis, administration of the scCD98-functionalized siCD98-loaded nanoparticles reduced colon expression of CD98. Importantly, the severity of colitis was also reduced compared with controls (based on loss of body weight, myeloperoxidase activity, inflammatory cytokine production, and histological analysis). Approximately 24.1% of colonic macrophages (CD11b(+)CD11c(-)F4/80(+)) in the mice had taken up fluorescently labeled siRNA-loaded nanoparticles within 12 hours of administration.

Conclusions: Nanoparticles containing surface CD98 antibody and loaded with siCD98 reduce expression of this protein by colonic epithelial cells and macrophages, and oral administration decreases the severity of colitis in mice. This nanoparticle in hydrogel (chitosan/alginate) formulation might be developed to treat patients with IBD.
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http://dx.doi.org/10.1053/j.gastro.2014.01.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3992175PMC
May 2014

Targeting intestinal inflammation with CD98 siRNA/PEI-loaded nanoparticles.

Mol Ther 2014 Jan 12;22(1):69-80. Epub 2013 Sep 12.

1] Department of Chemistry and Biology, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia, USA [2] Veterans Affairs Medical Center, Decatur, Georgia, USA.

Intestinal CD98 expression plays a crucial role in controlling homeostatic and innate immune responses in the gut. Modulation of CD98 expression in intestinal cells therefore represents a promising therapeutic strategy for the treatment and prevention of inflammatory intestinal diseases, such as inflammatory bowel disease. Here, the advantages of nanoparticles (NPs) are used, including their ability to easily pass through physiological barriers and evade phagocytosis, high loading concentration, rapid kinetics of mixing and resistance to degradation. Using physical chemistry characterizations techniques, CD98 siRNA/polyethyleneimine (PEI)-loaded NPs was characterized (diameter of ~480 nm and a zeta potential of -5.26 mV). Interestingly, CD98 siRNA can be electrostatically complexed by PEI and thus protected from RNase. In addition, CD98 siRNA/PEI-loaded NPs are nontoxic and biocompatible with intestinal cells. Oral administration of CD98/PEI-loaded NPs encapsulated in a hydrogel reduced CD98 expression in mouse colonic tissues and decreased dextran sodium sulfate-induced colitis in a mouse model. Finally, flow cytometry showed that CD98 was effectively downregulated in the intestinal epithelial cells and intestinal macrophages of treated mice. Finally, the results collectively demonstrated the therapeutic effect of "hierarchical nano-micro particles" with colon-homing capabilities and the ability to directly release "molecularly specific" CD98 siRNA in colonic cells, thereby decreasing colitis.
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http://dx.doi.org/10.1038/mt.2013.214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3978807PMC
January 2014

Dextran sodium sulfate inhibits the activities of both polymerase and reverse transcriptase: lithium chloride purification, a rapid and efficient technique to purify RNA.

BMC Res Notes 2013 Sep 8;6:360. Epub 2013 Sep 8.

Department of Biology, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta GA 30303, USA.

Background: Dextran sodium sulfate (DSS) is commonly used in mouse studies to induce a very reproducible colitis that effectively mimics the clinical and histological features of human inflammatory bowel disease (IBD) patients, especially ulcerative colitis. However, the mechanisms of action of DSS remain poorly understood, and observations by our laboratory and other groups indicate that DSS contamination of colonic tissues from DSS-treated mice potently inhibits the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) amplification of mRNA.

Results: A prior study used poly-A-mediated mRNA purification to remove DSS from RNA extracts, but we herein report a second efficient and cost-effective approach to counteract this inhibition, using lithium chloride precipitation to entirely remove DSS from RNAs. We also explored how DSS interferes with qRT-PCR process, and we report for the first time that DSS can alter the binding of reverse transcriptase to previously primed RNA and specifically inhibits the enzymatic activities of reverse transcriptase and Taq polymerase in vitro. This likely explains why DSS-treated colonic RNA is not suitable to qRT-PCR amplification without a previous purification step.

Conclusion: In summary, we provide a simple method to remove DSS from colonic RNAs, and we demonstrate for the first time that DSS can inhibit the activities of both polymerase and reverse transcriptase. In order to reliably analyze gene expression in the colonic mucosa of DSS-treated mice, the efficiency rate of qRT-PCR must be the same between all the different experimental groups, including the water-treated control group, suggesting that whatever the duration and the percentage of the DSS treatment, RNAs must be purified.
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http://dx.doi.org/10.1186/1756-0500-6-360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3847706PMC
September 2013

Mannosylated bioreducible nanoparticle-mediated macrophage-specific TNF-α RNA interference for IBD therapy.

Biomaterials 2013 Oct 29;34(30):7471-82. Epub 2013 Jun 29.

Department of Biology and Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta 30302, USA.

The application of RNA interference (RNAi) for inflammatory bowel disease (IBD) therapy has been limited by the lack of non-cytotoxic, efficient and targetable small interfering RNA (siRNA) carriers. TNF-α is the major pro-inflammatory cytokine mainly secreted by macrophages during IBD. Here, a mannosylated bioreducible cationic polymer (PPM) was synthesized and further spontaneously assembled nanoparticles (NPs) assisted by sodium triphosphate (TPP). The TPP-PPM/siRNA NPs exhibited high uniformity (polydispersity index = 0.004), a small particle size (211-275 nm), excellent bioreducibility, and enhanced cellular uptake. Additionally, the generated NPs had negative cytotoxicity compared to control NPs fabricated by branched polyethylenimine (bPEI, 25 kDa) or Oligofectamine (OF) and siRNA. In vitro gene silencing experiments revealed that TPP-PPM/TNF-α siRNA NPs with a weight ratio of 40:1 showed the most efficient inhibition of the expression and secretion of TNF-α (approximately 69.9%, which was comparable to the 71.4% obtained using OF/siRNA NPs), and its RNAi efficiency was highly inhibited in the presence of mannose (20 mm). Finally, TPP-PPM/siRNA NPs showed potential therapeutic effects on colitis tissues, remarkably reducing TNF-α level. Collectively, these results suggest that non-toxic TPP-PPM/siRNA NPs can be exploited as efficient, macrophage-targeted carriers for IBD therapy.
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http://dx.doi.org/10.1016/j.biomaterials.2013.06.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3762508PMC
October 2013

Nanotechnology in diagnostics and therapeutics for gastrointestinal disorders.

Dig Liver Dis 2013 Dec 7;45(12):995-1002. Epub 2013 May 7.

Department of Biology, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, USA. Electronic address:

This review describes the state of the art in nanoparticle and nanodevice applications for medical diagnosis and disease treatment. Nanodevices, such as cantilevers, have been integrated into high-sensitivity disease marker diagnostic detectors and devices, are stable over long periods of time, and display reliable performance properties. Nanotechnology strategies have been applied to therapeutic purposes as well. For example, nanoparticle-based delivery systems have been developed to protect drugs from degradation, thereby reducing the required dose and dose frequency, improving patient comfort and convenience during treatment, and reducing treatment expenses. The main objectives for integrating nanotechnologies into diagnostic and therapeutic applications in the context of intestinal diseases are reviewed.
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http://dx.doi.org/10.1016/j.dld.2013.03.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3970315PMC
December 2013

Intestinal epithelial CD98 directly modulates the innate host response to enteric bacterial pathogens.

Infect Immun 2013 Mar 7;81(3):923-34. Epub 2013 Jan 7.

Department of Biology, Center Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia, USA.

CD98 is a type II transmembrane glycoprotein whose expression increases in intestinal epithelial cells (IECs) during intestinal inflammation. Enteropathogenic Escherichia coli (EPEC) is a food-borne human pathogen that attaches to IECs and injects effector proteins directly into the host cells, thus provoking an inflammatory response. In the present study, we investigated CD98 and EPEC interactions in vitro and ex vivo and examined FVB wild-type (WT) and villin-CD98 transgenic mice overexpressing human CD98 in IECs (hCD98 Tg mice) and infected with Citrobacter rodentium as an in vivo model. In vivo studies indicated that CD98 overexpression, localized to the apical domain of colonic cells, increased the attachment of C. rodentium in mouse colons and resulted in increased expression of proinflammatory markers and decreased expression of anti-inflammatory markers. The proliferative markers Ki-67 and cyclin D1 were significantly increased in the colonic tissue of C. rodentium-infected hCD98 Tg mice compared to that of WT mice. Ex vivo studies correlate with the in vivo data. Small interfering RNA (siRNA) studies with Caco2-BBE cells showed a decrease in adherence of EPEC to Caco2 cells in which CD98 expression was knocked down. In vitro surface plasmon resonance (SPR) experiments showed direct binding between recombinant hCD98 and EPEC/C. rodentium proteins. We also demonstrated that the partial extracellular loop of hCD98 was sufficient for direct binding to EPEC/C. rodentium. These findings demonstrate the importance of the extracellular loop of CD98 in the innate host defense response to intestinal infection by attaching and effacing (A/E) pathogens.
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http://dx.doi.org/10.1128/IAI.01388-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3584858PMC
March 2013

NaxD is a deacetylase required for lipid A modification and Francisella pathogenesis.

Mol Microbiol 2012 Nov 11;86(3):611-27. Epub 2012 Sep 11.

Department of Microbiology and Immunology, Microbiology and Molecular Genetics Program, Emory University, Atlanta, GA, USA; Emory Vaccine Center, Emory University, Atlanta, GA, USA.

Modification of specific Gram-negative bacterial cell envelope components, such as capsule, O-antigen and lipid A, are often essential for the successful establishment of infection. Francisella species express lipid A molecules with unique characteristics involved in circumventing host defences, which significantly contribute to their virulence. In this study, we show that NaxD, a member of the highly conserved YdjC superfamily, is a deacetylase required for an important modification of the outer membrane component lipid A in Francisella. Mass spectrometry analysis revealed that NaxD is essential for the modification of a lipid A phosphate with galactosamine in Francisella novicida, a model organism for the study of highly virulent Francisella tularensis. Significantly, enzymatic assays confirmed that this protein is necessary for deacetylation of its substrate. In addition, NaxD was involved in resistance to the antimicrobial peptide polymyxin B and critical for replication in macrophages and in vivo virulence. Importantly, this protein is also required for lipid A modification in F. tularensis as well as Bordetella bronchiseptica. Since NaxD homologues are conserved among many Gram-negative pathogens, this work has broad implications for our understanding of host subversion mechanisms of other virulent bacteria.
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http://dx.doi.org/10.1111/mmi.12004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841722PMC
November 2012

A(₂B)AR expression in non-immune cells plays an important role in the development of murine colitis.

Dig Liver Dis 2012 Oct 19;44(10):819-26. Epub 2012 Jun 19.

Center for Diagnostics & Therapeutics, Department of Biology, Georgia State University, Atlanta, GA, United States.

Background: Adenosine, an endogenous purine nucleoside, is involved in several physiological functions. We have previously shown that A(2B)AR plays a pro-inflammatory role during colitis.

Aims: Our goals were to determine if A(2B)AR expression was necessary on immune cells/non-immune cells during colitis and if A(2B)AR was a suitable target for treating intestinal inflammation.

Methods: Wild-type and A(2B)AR knockout mice were utilized in bone marrow transplants to explore the importance of immune/non-immune A(2B)AR expression during the development of colitis. Additionally, a T-cell transfer model of colitis was used in Rag1 knockout or A(2B)AR/RAG1 double knockout recipients. Finally, A(2B)AR small interfering RNA nanoparticles were administered to dextran sodium sulphate-treated mice.

Results: Wild-type mice receiving wild-type or knockout bone marrow developed severe colitis after dextran sodium sulphate treatment, whereas colitis was significantly attenuated in knockout mice receiving wild-type or knockout bone marrow. Colitis induced in Rag1 knockout animals was attenuated in A(2B)AR/RAG1 double knockout recipients. Animals receiving nanoparticles exhibited attenuated parameters of colitis severity compared to mice receiving control nanoparticles.

Conclusions: Our results suggest that A(2B)AR on non-immune cells plays an important role for the induction of colitis and targeting A(2B)AR expression during colitis may be useful for alleviating symptoms of intestinal inflammation.
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http://dx.doi.org/10.1016/j.dld.2012.05.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3436952PMC
October 2012

Intestinal epithelial cell-specific CD98 expression regulates tumorigenesis in Apc(Min/+) mice.

Lab Invest 2012 Aug 28;92(8):1203-12. Epub 2012 May 28.

Department of Biology, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, USA.

The transmembrane glycoprotein CD98 regulates integrin signaling that in turn controls cell proliferation and survival. CD98 expression is upregulated in various carcinomas, including colorectal cancer. Recently, by generating gain- and loss-of-function mouse models featuring genetic manipulation of CD98 expression specifically in intestinal epithelial cells (IECs), we have explored the crucial role of CD98 in the regulation of intestinal homeostasis and inflammation-associated tumorigenesis. In the present study, we investigated the contribution of CD98 to intestinal tumorigenesis in Apc(Min/+) mice and the underlying mechanism of action. Mice featuring IEC-specific CD98 overexpression (Tg animals) were crossed with Apc(Min/+) mice, and the characteristics of intestinal adenoma formation were assessed. Compared with Apc(Min/+) mice, Tg/Apc(Min/+) animals exhibited increases in both intestinal tumor incidence and tumor size; these parameters correlated with enhanced proliferation and decreased apoptosis of IECs. IEC-specific CD98 overexpression resulted in increased synthesis of the oncogenic proteins c-myc and cyclin-D1 in Apc(Min/+) mice, independently of the Wnt-APC-β-catenin pathway, suggesting the implication of CD98 overexpression-mediated Erk activation. IEC-specific CD98 overexpression enhanced the production of proinflammatory cytokines and chemokines that are crucial for tumorigenesis. We validated our results in mice exhibiting IEC-specific CD98 downregulation (CD98(flox/+)VillinCre animals). IEC-specific CD98 downregulation efficiently attenuated tumor incidence and growth in Apc(Min/+) mice. The reduction of intestinal tumorigenesis upon IEC-specific CD98 downregulation was caused by the attenuation of IEC proliferation and cytokine/chemokine production. In conclusion, we show that CD98 exerts an oncogenic activity in terms of intestinal tumorigenesis, via an ability to regulate tumor growth and survival.
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http://dx.doi.org/10.1038/labinvest.2012.83DOI Listing
August 2012

Gastrointestinal delivery of anti-inflammatory nanoparticles.

Methods Enzymol 2012 ;509:101-25

Department of Biology, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia, USA.

The concept of nanomedicine has risen to be the future of medicine. Advantages of using nanoobjects as vectors for drug delivery systems are numerous, such as fewer side effects due to a low drug dose, and high specificity between drug and target. Unlike systemic therapy, targeting a specific target is more efficient and less costly. In inflammatory bowel disease, including ulcerative colitis and Crohn disease, the colon represents the targeted organ. A large number of drugs are candidates for loading into nanoparticles (NPs). Small molecules, such as tripeptides and siRNA, or larger molecules, such as proteins (hormones, antibodies (Ab), etc.), can be encapsulated alone or in a complex form inside the NPs. In our studies, once NPs are synthesized and loaded with anti-inflammatory compounds, they are delivered to the colon. An efficient technique has been developed for specific NP targeting to digestive tract regions, including the colon, using a hydrogel based on electrostatic interactions between positive ions and negative polysaccharides. An in situ double cross-linking process, mediated by Ca²⁺ and SO₄²⁻, of chitosan and alginate administered to the mouse gastrointestinal (GI) tract by double gavage, is used for gel formation. When the drug is given in NPs, NPs are targeted to the colon, and NP degradation by aggressive environmental conditions in the GI tract is significantly reduced. Using a biomaterial (hydrogel) associated with nanotechnology, lower doses of drug can be loaded efficiently and delivered to the colon to reduce colonic inflammation.
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http://dx.doi.org/10.1016/B978-0-12-391858-1.00006-XDOI Listing
August 2012

Intestinal epithelial CD98 synthesis specifically modulates expression of colonic microRNAs during colitis.

Am J Physiol Gastrointest Liver Physiol 2012 Jun 12;302(11):G1282-91. Epub 2012 Apr 12.

Department of Biology, Center Diagnostics and Therapeutics, Georgia State University, Atlanta, USA.

The transmembrane glycoprotein CD98 is known to be involved in intestinal inflammation. In the present study, we found that CD98 overexpression in intestinal epithelial cells does not normally affect the expression of colonic (epithelial and immune cell) microRNAs (miRNAs), small noncoding RNAs that posttranscriptionally regulate a wide variety of biological processes. However, upon dextran sulfate sodium (DSS) treatment, the expression of several colonic miRNAs, but not miRNAs from other tissues such as liver and spleen, were differentially regulated in mice overexpressing CD98 in epithelial cells compared with wild-type (WT) animals. For example, the level of colonic miRNA 132 was not affected by DSS treatment in WT animals but was upregulated in mice overexpressing CD98 in intestinal epithelial cells. Other colonic miRNAs, including colonic miRNA 23a and 23b, were downregulated in WT animals after DSS treatment but not in colonic epithelial cell CD98-overexpressing mice. Interestingly, the expression of potential miRNA target genes affected intestinal epithelial cells that overexpress CD98 and cell types that did not overexpress CD98 but were in close proximity to CD98-overexpressing intestinal epithelial cells. Taken together, these observations show that the combination of an inflammatory context and intestinal epithelial cell expression of CD98 affects the regulation of miRNA expression in colonic epithelial and immune cells. This is new evidence that protein expression modulates miRNA expression and suggests the existence of regulatory crosstalk between proteins and miRNAs in diseases such as colitis.
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http://dx.doi.org/10.1152/ajpgi.00401.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3378169PMC
June 2012

Dextran sodium sulfate (DSS) induces colitis in mice by forming nano-lipocomplexes with medium-chain-length fatty acids in the colon.

PLoS One 2012 9;7(3):e32084. Epub 2012 Mar 9.

Department of Biology, Center Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia, United States of America.

Inflammatory bowel diseases (IBDs), primarily ulcerative colitis and Crohn's disease, are inflammatory disorders caused by multiple factors. Research on IBD has often used the dextran sodium sulfate (DSS)-induced colitis mouse model. DSS induces in vivo but not in vitro intestinal inflammation. In addition, no DSS-associated molecule (free glucose, sodium sulfate solution, free dextran) induces in vitro or in vivo intestinal inflammation. We find that DSS but not dextran associated molecules established linkages with medium-chain-length fatty acids (MCFAs), such as dodecanoate, that are present in the colonic lumen. DSS complexed to MCFAs forms nanometer-sized vesicles ~200 nm in diameter that can fuse with colonocyte membranes. The arrival of nanometer-sized DSS/MCFA vesicles in the cytoplasm may activate intestinal inflammatory signaling pathways. We also show that the inflammatory activity of DSS is mediated by the dextran moieties. The deleterious effect of DSS is localized principally in the distal colon, therefore it will be important to chemically modify DSS to develop materials beneficial to the colon without affecting colon-targeting specificity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0032084PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3302894PMC
August 2012

The role and pathophysiological relevance of membrane transporter PepT1 in intestinal inflammation and inflammatory bowel disease.

Am J Physiol Gastrointest Liver Physiol 2012 Mar 22;302(5):G484-92. Epub 2011 Dec 22.

Center for Diagnostics & Therapeutics, Department of Biology, Georgia State University, Atlanta, 30302-5090, USA.

Intestinal inflammation is characterized by epithelial disruption, leading to loss of barrier function and the recruitment of immune cells, including neutrophils. Although the mechanisms are not yet completely understood, interactions between environmental and immunological factors are thought to be critical in the initiation and progression of intestinal inflammation. In recent years, it has become apparent that the di/tripeptide transporter PepT1 may play an important role in the pathogenesis of such inflammation. In healthy individuals, PepT1 is primarily expressed in the small intestine and transports di/tripeptides for metabolic purposes. However, during chronic inflammation such as that associated with inflammatory bowel disease, PepT1 expression is upregulated in the colon, wherein the protein is normally expressed either minimally or not at all. Several recent studies have shown that PepT1 binds to and transports various bacterial di/tripeptides into colon cells, leading to activation of downstream proinflammatory responses via peptide interactions with innate immune receptors. In the present review, we examine the relationship between colonic PepT1-mediated peptide transport in the colon and activation of innate immune responses during disease. It is important to understand the mechanisms of PepT1 action during chronic intestinal inflammation to develop future therapies addressing inappropriate immune activation in the colon.
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http://dx.doi.org/10.1152/ajpgi.00477.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3311434PMC
March 2012

The PepT1-NOD2 signaling pathway aggravates induced colitis in mice.

Gastroenterology 2011 Oct 14;141(4):1334-45. Epub 2011 Jul 14.

Department of Medicine, Division of Digestive Diseases, Emory University, Atlanta, Georgia 30322, USA.

Background & Aims: The human di/tripeptide transporter human intestinal H-coupled oligonucleotide transporter (hPepT1) is abnormally expressed in colons of patients with inflammatory bowel disease, although its exact role in pathogenesis is unclear. We investigated the contribution of PepT1 to intestinal inflammation in mouse models of colitis and the involvement of the nucleotide-binding oligomerization domain 2 (NOD2) signaling pathway in the pathogenic activity of colonic epithelial hPepT1.

Methods: Transgenic mice were generated in which hPepT1 expression was regulated by the β-actin or villin promoters; colitis was induced using 2,4,6-trinitrobenzene sulfonic acid (TNBS) or dextran sodium sulfate (DSS) and the inflammatory responses were assessed. The effects of NOD2 deletion in the hPepT1 transgenic mice also was studied to determine the involvement of the PepT1-NOD2 signaling pathway.

Results: TNBS and DSS induced more severe levels of inflammation in β-actin-hPepT1 transgenic mice than wild-type littermates. Intestinal epithelial cell-specific hPepT1 overexpression in villin-hPepT1 transgenic mice increased the severity of inflammation induced by DSS, but not TNBS. Bone marrow transplantation studies showed that hPepT1 expression in intestinal epithelial cells and immune cells has an important role in the proinflammatory response. Antibiotics abolished the effect of hPepT1 overexpression on the inflammatory response in DSS-induced colitis in β-actin-hPepT1 and villin-hPepT1 transgenic mice, indicating that commensal bacteria are required to aggravate intestinal inflammation. Nod2-/-, β-actin-hPepT1 transgenic/Nod2-/-, and villin-hPepT1 transgenic/Nod2-/- littermates had similar levels of susceptibility to DSS-induced colitis, indicating that hPepT1 overexpression increased intestinal inflammation in a NOD2-dependent manner.

Conclusions: The PepT1-NOD2 signaling pathway is involved in aggravation of DSS-induced colitis in mice.
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http://dx.doi.org/10.1053/j.gastro.2011.06.080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3186842PMC
October 2011

L-Ala-γ-D-Glu-meso-diaminopimelic acid (DAP) interacts directly with leucine-rich region domain of nucleotide-binding oligomerization domain 1, increasing phosphorylation activity of receptor-interacting serine/threonine-protein kinase 2 and its interaction with nucleotide-binding oligomerization domain 1.

J Biol Chem 2011 Sep 12;286(35):31003-31013. Epub 2011 Jul 12.

Department of Medicine, Division of Digestive Diseases, Emory University, Atlanta, Georgia 30322; Veterans Affairs Medical Center, Decatur, Georgia 30033.

The oligopeptide transporter PepT1 expressed in inflamed colonic epithelial cells transports small bacterial peptides, such as muramyl dipeptide (MDP) and l-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) into cells. The innate immune system uses various proteins to sense pathogen-associated molecular patterns. Nucleotide-binding oligomerization domain (NOD)-like receptors of which there are more than 20 related family members are present in the cytosol and recognize intracellular ligands. NOD proteins mediate NF-κB activation via receptor-interacting serine/threonine-protein kinase 2 (RICK or RIPK). The specific ligands for some NOD-like receptors have been identified. NOD type 1 (NOD1) is activated by peptides that contain a diaminophilic acid, such as the PepT1 substrate Tri-DAP. In other words, PepT1 transport activity plays an important role in controlling intracellular loading of ligands for NOD1 in turn determining the activation level of downstream inflammatory pathways. However, no direct interaction between Tri-DAP and NOD1 has been identified. In the present work, surface plasmon resonance and atomic force microscopy experiments showed direct binding between NOD1 and Tri-DAP with a K(d) value of 34.5 μM. In contrast, no significant binding was evident between muramyl dipeptide and NOD1. Furthermore, leucine-rich region (LRR)-truncated NOD1 did not interact with Tri-DAP, indicating that Tri-DAP interacts with the LRR domain of NOD1. Next, we examined binding between RICK and NOD1 proteins and found that such binding was significant with a K(d) value of 4.13 μM. However, NOD1/RICK binding was of higher affinity (K(d) of 3.26 μM) when NOD1 was prebound to Tri-DAP. Furthermore, RICK phosphorylation activity was increased when NOD was prebound to Tri-DAP. In conclusion, we have shown that Tri-DAP interacts directly with the LRR domain of NOD1 and consequently increases RICK/NOD1 association and RICK phosphorylation activity.
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http://dx.doi.org/10.1074/jbc.M111.257501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3162459PMC
September 2011

Overexpression of Ste20-related proline/alanine-rich kinase exacerbates experimental colitis in mice.

J Immunol 2011 Aug 24;187(3):1496-505. Epub 2011 Jun 24.

Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.

Inflammatory bowel disease, mainly Crohn's disease and ulcerative colitis, are characterized by epithelial barrier disruption and altered immune regulation. Colonic Ste20-like proline/alanine-rich kinase (SPAK) plays a role in intestinal inflammation, but its underlying mechanisms need to be defined. Both SPAK-transfected Caco2-BBE cells and villin-SPAK transgenic (TG) FVB/6 mice exhibited loss of intestinal barrier function. Further studies demonstrated that SPAK significantly increased paracellular intestinal permeability to FITC-dextran. In vivo studies using the mouse models of colitis induced by dextran sulfate sodium (DSS) and trinitrobenzene sulfonic acid showed that TG FVB/6 mice were more susceptible to DSS and trinitrobenzene sulfonic acid treatment than wild-type FVB/6 mice, as demonstrated by clinical and histological characteristics and enzymatic activities. Consistent with this notion, we found that SPAK increased intestinal epithelial permeability, which likely facilitated the production of inflammatory cytokines in vitro and in vivo, aggravated bacterial translocation in TG mice under DSS treatment, and consequently established a context favorable for the triggering of intestinal inflammation cascades. In conclusion, overexpression of SPAK inhibits maintenance of intestinal mucosal innate immune homeostasis, which makes regulation of SPAK important to attenuate pathological responses in inflammatory bowel disease.
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http://dx.doi.org/10.4049/jimmunol.1002910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3140558PMC
August 2011

Microbiota modulate host gene expression via microRNAs.

PLoS One 2011 Apr 29;6(4):e19293. Epub 2011 Apr 29.

Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, United States of America.

Microbiota are known to modulate host gene expression, yet the underlying molecular mechanisms remain elusive. MicroRNAs (miRNAs) are importantly implicated in many cellular functions by post-transcriptionally regulating gene expression via binding to the 3'-untranslated regions (3'-UTRs) of the target mRNAs. However, a role for miRNAs in microbiota-host interactions remains unknown. Here we investigated if miRNAs are involved in microbiota-mediated regulation of host gene expression. Germ-free mice were colonized with the microbiota from pathogen-free mice. Comparative profiling of miRNA expression using miRNA arrays revealed one and eight miRNAs that were differently expressed in the ileum and the colon, respectively, of colonized mice relative to germ-free mice. A computational approach was then employed to predict genes that were potentially targeted by the dysregulated miRNAs during colonization. Overlapping the miRNA potential targets with the microbiota-induced dysregulated genes detected by a DNA microarray performed in parallel revealed several host genes that were regulated by miRNAs in response to colonization. Among them, Abcc3 was identified as a highly potential miRNA target during colonization. Using the murine macrophage RAW 264.7 cell line, we demonstrated that mmu-miR-665, which was dysregulated during colonization, down-regulated Abcc3 expression by directly targeting the Abcc3 3'-UTR. In conclusion, our study demonstrates that microbiota modulate host microRNA expression, which could in turn regulate host gene expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019293PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084815PMC
April 2011

CD98 expression modulates intestinal homeostasis, inflammation, and colitis-associated cancer in mice.

J Clin Invest 2011 May 1;121(5):1733-47. Epub 2011 Apr 1.

Department of Medicine, Emory University, Atlanta, Georgia, USA.

Expression of the transmembrane glycoprotein CD98 (encoded by SLC3A2) is increased in intestinal inflammatory conditions, such as inflammatory bowel disease (IBD), and in various carcinomas, yet its pathogenetic role remains unknown. By generating gain- and loss-of-function mouse models with genetically manipulated CD98 expression specifically in intestinal epithelial cells (IECs), we explored the role of CD98 in intestinal homeostasis, inflammation, and colitis-associated tumorigenesis. IEC-specific CD98 overexpression induced gut homeostatic defects and increased inflammatory responses to DSS-induced colitis, promoting colitis-associated tumorigenesis in mice. Further analysis indicated that the ability of IEC-specific CD98 overexpression to induce tumorigenesis was linked to its capacity to induce barrier dysfunction and to stimulate cell proliferation and production of proinflammatory mediators. To validate these results, we constructed mice carrying conditional floxed Slc3a2 alleles and crossed them with Villin-Cre mice such that CD98 was downregulated only in IECs. These mice exhibited attenuated inflammatory responses and resistance to both DSS-induced colitis and colitis-associated tumorigenesis. Together, our data show that intestinal CD98 expression has a crucial role in controlling homeostatic and innate immune responses in the gut. Modulation of CD98 expression in IECs therefore represents a promising therapeutic strategy for the treatment and prevention of inflammatory intestinal diseases, such as IBD and colitis-associated cancer.
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http://dx.doi.org/10.1172/JCI44631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083801PMC
May 2011

Nanomedicine in GI.

Am J Physiol Gastrointest Liver Physiol 2011 Mar 9;300(3):G371-83. Epub 2010 Dec 9.

Department of Medicine, Division of Digestive Diseases, Emory University School of Medicine, Atlanta, GA 30322, USA.

Recent advances in nanotechnology offer new hope for disease detection, prevention, and treatment. Nanomedicine is a rapidly evolving field wherein targeted therapeutic approaches using nanotechnology based on the pathophysiology of gastrointestinal diseases are being developed. Nanoparticle vectors capable of delivering drugs specifically and exclusively to regions of the gastrointestinal tract affected by disease for a prolonged period of time are likely to significantly reduce the side effects of existing otherwise effective treatments. This review aims at integrating various applications of the most recently developed nanomaterials that have tremendous potential for the detection and treatment of gastrointestinal diseases.
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http://dx.doi.org/10.1152/ajpgi.00466.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064120PMC
March 2011
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