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Abstract: 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. [Copyright &y& Elsevier]

Abstract: Background: Adenosine, an endogenous purine nucleoside, is involved in several physiological functions. We have previously shown that A2BAR plays a pro-inflammatory role during colitis. Aims: Our goals were to determine if A2BAR expression was necessary on immune cells/non-immune cells during colitis and if A2BAR was a suitable target for treating intestinal inflammation. Methods: Wild-type and A2BAR knockout mice were utilized in bone marrow transplants to explore the importance of immune/non-immune A2BAR expression during the development of colitis. Additionally, a T-cell transfer model of colitis was used in Rag1 knockout or A2BAR/RAG1 double knockout recipients. Finally, A2BAR 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 A2BAR/RAG1 double knockout recipients. Animals receiving nanoparticles exhibited attenuated parameters of colitis severity compared to mice receiving control nanoparticles. Conclusions: Our results suggest that A2BAR on non-immune cells plays an important role for the induction of colitis and targeting A2BAR expression during colitis may be useful for alleviating symptoms of intestinal inflammation. [Copyright &y& Elsevier]

Background & Aims: Inflammatory bowel disease increases the risks of colon cancer and colitis-associated cancer (CAC). Epithelial cell–derived matrix metalloproteinase (MMP)-9 mediates inflammation during acute colitis and the cleavage and activation of the transcription factor Notch1, which prevents differentiation of progenitor cells into goblet cells. However, MMP-9 also protects against the development of CAC and acts as a tumor suppressor. We investigated the mechanisms by which MMP-9 protects against CAC in mice. Methods: C57/B6 wild-type mice were given a single dose of azoxymethane and 2 cycles of dextran sulfate sodium (DSS). Mice were also given the γ-secretase inhibitor difluorophenacetyl-l-alanyl-S-phenylglycine t-butyl ester (DAPT) or dimethyl sulfoxide (control) during each DSS cycle; they were killed on day 56. We analyzed embryonic fibroblasts isolated from wild-type and MMP-9 −/− mice and HCT116 cells that were stably transfected with MMP-9. Results: Wild-type mice were more susceptible to CAC following inhibition of Notch1 by DAPT, shown by increased numbers of tumors and level of dysplasia compared with controls. Inhibition of Notch1 signaling significantly reduced protein levels of active Notch1, p53, p21 WAF1/Cip1 , Bax-1, active caspase-3, as well as apoptosis, compared with controls. Similar results were observed in transgenic HCT116 cells and embryonic fibroblasts from MMP-9 −/− mice on γ-radiation–induced damage of DNA. Conclusions: MMP-9 mediates Notch1 signaling via p53 to regulate apoptosis, cell cycle arrest, and inflammation. By these mechanisms, it might prevent CAC. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Background & Aims: The A2B adenosine receptor (A2BAR) is the predominant adenosine receptor expressed in the colonic epithelia. We have previously shown that A2BAR mRNA and protein levels are up-regulated during colitis. In this study, we addressed the role of the A2BAR in the development of murine colitis and the potential mechanism underlying its effects. Methods: Dextran sodium sulfate (DSS), 2,4,6-trinitrobenzene sulfonic acid (TNBS), and Salmonella typhimurium were used to induce colitis in A2BAR-null mice (A2BAR−/−). Colitis was determined using established clinical and histologic scoring. Keratinocyte-derived chemokine (KC) measurements were performed using an enzyme-linked immunosorbent assay. Results: Colonic inflammation induced by DSS, TNBS, or S typhimurium was attenuated in A2BAR−/− compared with their wild-type counterparts. Clinical features, histologic score, and myeloperoxidase activity were significantly decreased in A2BAR−/− mice. However, A2BAR−/− showed increased susceptibility to systemic Salmonella infection. Tissue levels of the neutrophil chemokine, KC was decreased in colitic A2BAR−/− mice. In addition, flagellin-induced KC levels were attenuated in A2BAR−/− mice. Neutrophil chemotaxis in response to exogenous interleukin-8 was preserved in A2BAR−/− mice, suggesting intact neutrophil migration in response to appropriate stimuli. Conclusions: These data demonstrate, for the first time, that the A2BAR plays a proinflammatory role in colitis. A2B receptor antagonism may be an effective treatment for acute inflammatory intestinal diseases such as acute flare of inflammatory bowel disease. [Copyright &y& Elsevier]

Background & Aims: KPV is a tripeptide (Lys-Pro-Val), which possesses anti-inflammatory properties; however, its mechanisms of action still remain unknown. PepT1 is a di/tripeptide transporter normally expressed in the small intestine and induced in colon during inflammatory bowel disease (IBD). The aim of this study was to 1) investigate whether the KPV anti-inflammatory effect is PepT1-mediated in intestinal epithelian and immune cells, and 2) examine the anti-inflammatory effects in two models of mice colitis. Methods: Human intestinal epithelial cells Caco2-BBE, HT29-Cl.19A, and human T cells (Jurkat) were stimulated with pro-inflammatory cytokines in the present or absence of KPV. KPV anti-inflammatory effect was assessed using a NF-κB luciferase gene reporter, Western blot, real-time RT-PCR and ELISA. Uptake experiments were performed using cold KPV as a competitor for PepT1 radiolabelled substrate or using [3H]KPV to determine kinetic characteristics of KPV uptake. Anti-inflammatory effect of KPV was also investigated in DSS- and TNBS-induced colitis in mice. KPV was added to drinking water and inflammation was assessed at the histologic level and by proinflammatory cytokine mRNA expression. Results: Nanomolar concentrations of KPV inhibit the activation of NF-κB and MAP kinase inflammatory signaling pathways, and reduce pro-inflammatory cytokine secretion. We found that KPV acts via PepT1 expressed in immune and intestinal epithelial cells. Furthermore, oral administration of KPV reduces the incidence of DSS- and TNBS-induced colitis indicated by a decrease in pro-inflammatory cytokine expression. Conclusions: This study indicates tht KPV is transported into cells by PepT1 and might be a new therapeutic agent for IBD. [Copyright &y& Elsevier]

Background & Aims: We recently demonstrated that epithelial-derived matrix metalloproteinase (MMP) 9 up-regulated during inflammatory bowel disease is a critical mediator of tissue damage during colitis. MMP-9 null mice (MMP-9−/−) develop dramatically reduced inflammatory response to luminally administered colitic agents in the face of intact systemic immune response and inflammatory cell recruitment, suggesting protected epithelial barrier in these mice. In this study, we sought to address the role and mechanism by which MMP-9 influences barrier protective function. Methods: Wild-type and MMP-9−/− mice were used for in vivo studies, and the goblet cell line HT29-cl.16E and the enterocyte cell line Caco2-BBE were used for in vitro studies. Results: Compared with wild-type mice, MMP-9−/− mice had an increased number of goblet cells and MUC-2 expression. In addition, KLF-4 and Elf-3, transcription factors involved in terminal differentiation of goblet cells were up-regulated, whereas notch intracellular domain (NICD; activated Notch-1) was down-regulated in MMP-9−/− mice. These findings suggest altered epithelial cell differentiation in MMP-9−/− mice. Temporal expression of MMP-9 inversely correlated with MUC-2 expression during maturation of goblet cells. MMP-9 over expression inhibited goblet cell differentiation in vitro. Conversely, MMP-9 gene silencing in Caco2-BBE cells resulted in a change in their phenotype toward goblet cells. Finally, MMP-9 over expression or silencing in goblet cells increased or decreased Salmonella typhimurium adherence, respectively. Conclusions: MMP-9 regulates goblet cell differentiation in colon. The effect of MMP-9 on goblet cells could contribute to alteration in mucosal defense leading to inflammation. Together, our data uncover a novel function of MMP-9 in intestinal epithelial cells. [Copyright &y& Elsevier]

Background & Aims: There is mounting evidence that matrix metalloproteinases are the predominant proteinases expressed in the gut mucosa during active inflammatory bowel disease. We investigated the role of metalloproteinase 9 (MMP-9), a secreted gelatinase that is consistently up-regulated in both animal models and human inflammatory bowel disease and is associated with disease severity, in the pathogenesis of colitis by using mice containing a targeted deletion of the MMP-9 gene. Methods: Dextran sodium sulfate–induced colitis and Salmonella typhimurium–induced enterocolitis were used as animal models to study colitis. Results: MMP-9 activity and protein expression were absent from normal colonic mucosa but were up-regulated during experimental colitis. MMP-9−/− mice exposed to dextran sodium sulfate or salmonella had a significantly reduced extent and severity of colitis. Immunohistochemical studies showed that MMP-9 was localized to epithelial cells and granulocytes during active colitis. The immune response to systemic administration of Salmonella typhimurium was not affected in MMP-9−/− mice. Neutrophil transmigration studies and bone marrow chimeras showed that neutrophil MMP-9 is neither required for its migration nor sufficient to induce tissue damage during colitis and that epithelial MMP-9 is important for tissue damage. MMP-9 inhibited cell attachment and wound healing in the model intestinal epithelial cell line, Caco2-BBE. Conclusions: Taken together, our data suggest that MMP-9 expressed by epithelial cells may play an important role in the development of colitis by modulating cell–matrix interaction and wound healing. Thus, strategies to inhibit MMP-9 may be of potential therapeutic benefit. [Copyright &y& Elsevier]

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+CD45RBhigh 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. [Copyright &y& Elsevier]