Role of CD98 in liver disease.

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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.

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.

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

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-ß-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.

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

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.

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

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.

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

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 ß-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 ß-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 ß-actin-hPepT1 and villin-hPepT1 transgenic mice, indicating that commensal bacteria are required to aggravate intestinal inflammation. Nod2-/-, ß-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.

MicroRNA-7 modulates CD98 expression during intestinal epithelial cell differentiation.

The transmembrane glycoprotein CD98 regulates multiple cellular functions, including extracellular signaling, epithelial cell adhesion/polarity, amino acid transport, and cell-cell interactions. MicroRNAs post-transcriptionally regulate gene expression, thereby functioning as modulators of numerous cellular processes, such as cell differentiation, proliferation, and apoptosis. Here, we investigated if microRNAs regulate CD98 expression during intestinal epithelial cell differentiation and inflammation. We found that microRNA-7 repressed CD98 expression in Caco2-BBE cells by directly targeting the 3'-untranslated region of human CD98 mRNA. Expression of CD98 was decreased, whereas that of microRNA-7 was increased in well-differentiated Caco2-BBE cells compared with undifferentiated cells. Undifferentiated crypt cells isolated from mouse jejunum showed higher CD98 levels and lower levels of mmu-microRNA-706, a murine original microRNA candidate for CD98, than well-differentiated villus cells. Importantly, microRNA-7 decreased Caco2-BBE cell attachment on laminin-1, and CD98 overexpression recovered this inhibition, suggesting that microRNA-7 modulates epithelial cell adhesion to extracellular matrix, which in turn could affect proliferation and differentiation during the migration of enterocytes across the crypt-villus axis, by regulating CD98 expression. In a pathological context, the pro-inflammatory cytokine interleukin 1-beta increased CD98 expression in Caco2-BBE cells by decreasing microRNA-7 levels. Consistent with the in vitro findings, microRNA-7 levels were decreased in actively inflamed Crohn disease colonic tissues, where CD98 expression was up-regulated, compared with normal tissues. Together, these results reveal a novel mechanism underlying regulation of CD98 expression during patho-physiological states. This study raises microRNAs as a promising target for therapeutic modulations of CD98 expression in intestinal inflammatory disorders.

MicroRNA-92b regulates expression of the oligopeptide transporter PepT1 in intestinal epithelial cells.

MicroRNAs (miRNAs), which are noncoding RNAs that posttranscriptionally inhibit expression of target genes, have recently emerged as important regulators of many cellular functions such as cell differentiation. The epithelial di/tripeptide membrane transporter PepT1 is expressed in highly differentiated cells (the villous tip) but not in undifferentiated cells (the crypt) of the small intestine. Here, we investigated the regulation of PepT1 expression by miRNAs and its functional consequences. We observed a reverse correlation between the expression levels of PepT1 and mature miRNA-92b (miR-92b) during the differentiation of intestinal epithelial Caco2-BBE cells, suggesting a miR-92b-mediated regulation of PepT1 expression. We demonstrate that miR-92b suppressed PepT1 expression at both mRNA and protein levels, with subsequent reduced PepT1 transport activity, in Caco2-BBE cells by directly targeting the PepT1 3'-untranslated region. In addition, miR-92b suppresses bacterial peptide-induced proinflammatory responses in intestinal epithelial cells by inhibiting PepT1 expression. Altogether, our study provides for the first time evidence for the regulation of PepT1 expression at a posttranscriptional level by miRNAs in intestinal epithelial cells during pathophysiological states.

Nanomedicine in GI.

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.

Drug-loaded nanoparticles targeted to the colon with polysaccharide hydrogel reduce colitis in a mouse model.

BACKGROUND & AIMS: One of the challenges to treating inflammatory bowel disease (IBD) is to target the site of inflammation. We engineered nanoparticles (NPs) to deliver an anti-inflammatory tripeptide Lys-Pro-Val (KPV) to the colon and assessed its therapeutic efficacy in a mouse model of colitis. METHODS: NPs were synthesized by double-emulsion/solvent evaporation. KPV was loaded into the NPs during the first emulsion of the synthesis process. To target KPV to the colon, loaded NPs (NP-KPV) were encapsulated into a polysaccharide gel containing 2 polymers: alginate and chitosan. The effect of KPV-loaded NPs on inflammatory parameters was determined in vitro as well as in the dextran sodium sulfate-induced colitis mouse model. RESULTS: NPs (400 nm) did not affect cell viability or barrier functions. A swelling degree study showed that alginate-chitosan hydrogel containing dextran-fluorescein isothiocyanate-labeled NPs collapsed in the colon. Once delivered, NPs quickly released KPV on or within the closed area of colonocytes. The inflammatory responses to lipopolysaccharide were reduced in Caco2-BBE (brush border enterocyte) cells exposed to NP-KPV compared with those exposed to NPs alone, in a dose-dependent fashion. Mice given dextran sodium sulfate (DSS) followed by NP-KPV were protected against inflammatory and histologic parameters, compared with mice given only DSS. CONCLUSIONS: Nanoparticles are a versatile drug delivery system that can overcome physiologic barriers and target anti-inflammatory agents such as the peptide KPV to inflamed areas. By using NPs, KPV can be delivered at a concentration that is 12,000-fold lower than that of KPV in free solution, but with similar therapeutic efficacy. Administration of encapsulated drug-loaded NPs is a novel therapeutic approach for IBD.

MicroRNAs determine human intestinal epithelial cell fate.

MicroRNAs (miRNAs) are small, non-coding RNA molecules that post-transcriptionally regulate gene expression. Evidence has shown that miRNAs play important roles in various cellular processes, including proliferation, differentiation and survival. The intestinal epithelium is regenerated throughout life, and enterocytes undergo differentiation during migration along the crypt/villus axis. Our study aimed at establishing the expression profiles of miRNAs during intestinal epithelial cell (IEC) differentiation and determining a miRNA "signature" that distinguishes between small and large IECs. MiRNA arrays were employed to profile miRNA expression in two IEC models: the enterocyte-like Caco2-BBE and the colonocyte-like HT29-Cl.19A cell lines. Microarray data showed that in both cell lineages, the differentiated stage exhibited a different miRNA expression profile from undifferentiated stage. Interestingly, Caco2-BBE cells were distinguished from HT29-Cl.19A cells by their unique miRNA expression profile. Notably, HT29-Cl.19A cells exhibited down-regulation of miR-1269 and up-regulation of miR-99b and miR-125a-5p compared with Caco2-BBE cells. Most importantly, transfection of Caco2-BBE cells with mature miR-99b, mature miR-125a-5p and antisense of mature miR-1269 decreased growth rate and trans-epithelial resistance of the cells, indicating their shift toward HT29-Cl.19A cell phenotype. In conclusion, our study shows that miRNAs might play a role in determining the unique physiological characteristics of IECs.

Doxorubicin Intracellular Release Via External UV Irradiation of Dextran-g-poly(o-nitrobenzyl acrylate) Photosensitive Nanoparticles.

In the present study, innovative doxorubicin-loaded nanoparticles (NPs) made of a photosensitive poly(o-nitrobenzyl acrylate) (PNBA) hydrophobic matrix and an hydrophilic dextran (Dex) shell were first formulated by the emulsion-solvent evaporation process. Doxorubicin (DOX), a very well-known anticancer drug, was herein chosen as the model. DOX-loaded NPs were successfully produced by covering the hydrophobic PNBA core with Dex chains either physically adsorbed or covalently linked by changing process parameters as the presence of a catalyst (CuBr or CuSO4/ascorbic acid). It was then proved that the neutralization of DOX optimized drug loading. DOX loading and release were independent of the coverage mechanism if the catalyst used to covalently link the shell to the core was correctly chosen. Second, the kinetics of DOX release were investigated by simple diffusion or light irradiation of the NPs. Experiments showed that less than 20% of DOX was released by simple diffusion after 48 h in PBS or DMEM media when 45% of DOX released after only 30 s of light irradiation of the NPs. Finally, the impact of the phototriggered DOX release on cell viability was investigated on various cell lines [Caco-2, HepG2, HCT-116, and HT-29 cells as well as murine macrophages (RAW 264.7)]. Cellular mortality was evaluated to be dependent on the cell lines tested. Our approach provided an improved DOX release toward the human liver cancer cell line, and a high internalization of the PNBA-based NPs into HepG2 cells was observed using fluorescence microscopy.

PepT1 mediates transport of the proinflammatory bacterial tripeptide L-Ala-{gamma}-D-Glu-meso-DAP in intestinal epithelial cells.

PepT1 is a di/tripeptide transporter highly expressed in the small intestine, but poorly or not expressed in the colon. However, during chronic inflammation, such as inflammatory bowel disease, PepT1 expression is induced in the colon. Commensal bacteria that colonize the human colon produce a large amount of di/tripeptides. To date, two bacterial peptides (N-formylmethionyl-leucyl-phenylalanine and muramyl dipeptide) have been identified as substrates of PepT1. We hypothesized that the proinflammatory tripeptide l-Ala-gamma-d-Glu-meso-DAP (Tri-DAP), a breakdown product of bacterial peptidoglycan, is transported into intestinal epithelial cells via PepT1. We found that uptake of glycine-sarcosine, a specific substrate of PepT1, in intestinal epithelial Caco2-BBE cells was inhibited by Tri-DAP in a dose-dependent manner. Tri-DAP induced activation of NF-kappaB and MAP kinases, consequently leading to production of the proinflammatory cytokine interleukin-8. Tri-DAP-induced inflammatory response in Caco2-BBE cells was significantly suppressed by silencing of PepT1 expression by using PepT1-shRNAs in a tetracycline-regulated expression (Tet-off) system. Colonic epithelial HT29-Cl.19A cells, which do not express PepT1 under basal condition, were mostly insensitive to Tri-DAP-induced inflammation. However, HT29-Cl.19A cells exhibited proinflammatory response to Tri-DAP upon stable transfection with a plasmid encoding PepT1. Accordingly, Tri-DAP significantly increased keratinocyte-derived chemokine production in colonic tissues from transgenic mice expressing PepT1 in intestinal epithelial cells. Finally, Tri-DAP induced a significant drop in intracellular pH in intestinal epithelial cells expressing PepT1, but not in cells that did not express PepT1. Our data collectively support the classification of Tri-DAP as a novel substrate of PepT1. Given that PepT1 is highly expressed in the colon during inflammation, PepT1-mediated Tri-DAP transport may occur more effectively during such conditions, further contributing to intestinal inflammation.