Mouse Large Intestine Research Articles

Exploitation of the intestinal microflora by the parasitic nematode Trichuris muris.

The inhabitants of the mammalian gut are not always relatively benign commensal bacteria but may also include larger and more parasitic organisms, such as worms and protozoa. At some level, all these organisms are capable of interacting with each other. We found that successful establishment of the chronically infecting parasitic nematode Trichuris muris in the large intestine of mice is dependent on microflora and coincident with modulation of the host immune response. By reducing the number of bacteria in the host animal, we significantly reduced the number of hatched T. muris eggs. Critical interactions between bacteria (microflora) and parasites (macrofauna) introduced a new dynamic to the intestinal niche, which has fundamental implications for our current concepts of intestinal homeostasis and regulation of immunity.

Proteinase-activated receptors-1 and 2 induce electrogenic Cl−secretion in the mouse cecum by distinct mechanisms

Proteinase-activated receptors (PAR(1)-PAR(4)) belong to a family of G protein-coupled receptors that are cleaved by proteases. Previous in vitro studies on the mouse large intestine have indicated that PAR(1) and PAR(2) were involved in regulating epithelial ion transport, but that their roles were different between the proximal and distal colon. This present study was done to elucidate the roles of PAR(1) and PAR(2) in regulating anion secretion in the cecum, another segment of the large intestine. A mucosa-submucosal sheet of the mouse cecum was mounted in Ussing chambers, and the short-circuit current (I(sc)) was measured. The addition of a PAR(1)-activating peptide (SFFLRN-NH(2)) to the serosal surface increased I(sc). This increase in I(sc) induced by SFFLRN-NH(2) was partially suppressed by serosal bumetanide and substantially suppressed by mucosal 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and by the removal of Cl(-) from the bathing solution. The I(sc) increase was also substantially suppressed by serosal tetrodotoxin (TTX) and neurokinin-1 receptor antagonist L-703,606 and was partially inhibited by serosal atropine and hexamethonium. The addition of a PAR(2)-activating peptide (SLIGRL-NH(2)) to the serosal surface also induced an increase in I(sc); this increase was partially suppressed by bumetanide and substantially suppressed by NPPB and by the removal of Cl(-), but not by TTX. The expression of mRNA for PAR(1) and PAR(2) was confirmed in the mucosa as determined by RT-PCR. In conclusion, PAR(1) and PAR(2) both induced Cl(-) secretion in the mouse cecum. This secretion mediated by PAR(1) probably occurred by activation of the receptor on the submucosal secretomotor neurons, resulting mainly in the release of tachykinins and activation of the neurokinin-1 receptor, and partly in the release of ACh and activation of the muscarinic and nicotinic receptors. On the other hand, PAR(2)-mediated Cl(-) secretion probably occurred by activating the receptor on the epithelial cells. A variety of proteases would induce fluid secretion mediated by PAR(1) and PAR(2) in the cecum and thereby support bacterial fermentation and participate in mucosal inflammation.

Hyperimmune lipopolysaccharide antiserum mediated inhibition of the adherence of E. coli O157:H7 to HEP-2 cells and large intestine of mice

Summary Escherichia coli O157:H7 is found in cattle farms and can live in the intestine of healthy cattle. Most cases of human illnesses including nonbloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome can be traced, either directly or indirectly, to cattle. One strategy for reducing the risk of Enterohemorrhagic Escherichia coli (EHEC) infections in human is to reduce the prevalence of infection in cattle. Antiserum against whole cell of isolated E. coli O157:H7 from cattle showed inhibition of adherence of this strain to HEP-2 cells and intestine of model mice. The ability of this antiserum in blocking the attachment of bacteria to HEP-2 cells in 1:1280 titer and to intestine tissue of mice in 1:640 titer are significant. Histology of intestine tissue confirms our results. The difference between in vivo and in vitro titrations for blocking the attachment depends on these two different conditions.

Localization of TRPV1 and contractile effect of capsaicin in mouse large intestine: high abundance and sensitivity in rectum and distal colon

We investigated immunohistochemical differences in the distribution of TRPV1 channels and the contractile effects of capsaicin on smooth muscle in the mouse rectum and distal, transverse, and proximal colon. In the immunohistochemical study, TRPV1 immunoreactivity was found in the mucosa, submucosal, and muscle layers and myenteric plexus. Large numbers of TRPV1-immunoreactive axons were observed in the rectum and distal colon. In contrast, TRPV1-positive axons were sparsely distributed in the transverse and proximal colon. The density of TRPV1-immunoreactive axons in the rectum and distal colon was much higher than those in the transverse and proximal colon. Axons double labeled with TRPV1 and protein gene product (PGP) 9.5 were detected in the myenteric plexus, but PGP 9.5-immunoreactive cell bodies did not colocalize with TRPV1. In motor function studies, capsaicin induced a fast transient contraction, followed by a large long-lasting contraction in the rectum and distal colon, whereas in the transverse and proximal colon only the transient contraction was observed. The capsaicin-induced transient contraction from the proximal colon to the rectum was moderately inhibited by an NK1 or NK2 receptor antagonist. The capsaicin-induced long-lasting contraction in the rectum and distal colon was markedly inhibited by an NK2 antagonist, but not by an NK1 antagonist. The present results suggest that TRPV1 channels located on the rectum and distal colon play a major role in the motor function in the large intestine.

Characterization of mucosa-associated bacterial communities of the mouse intestine by terminal restriction fragment length polymorphism: Utility of sampling strategies and methods to reduce single-stranded DNA artifacts

Terminal restriction fragment length polymorphism (T-RFLP) is a molecular technique used for comparative analysis of microbial community structure and dynamics. We evaluated three sampling methods for recovering bacterial community DNA associated with intestinal mucosa of mice (i.e. mechanical agitation with PBS, hand washing with PBS containing Tween 80, and direct DNA extraction from mucosal plugs). In addition, the utility of two methods (i.e. Klenow fragment and mung-bean nuclease) to reduce single-stranded DNA artifacts was tested. T-RFLP analysis indicated that diverse communities of bacteria are associated with mucosa of the ileum, cecum, and descending colon of mice. Although there was no significant difference in bacterial community structure between the mechanical agitation and direct DNA extraction methods regardless of intestinal location, community diversity was reduced for the hand wash method in the colon. The use of Klenow fragment and mung-bean nuclease have been reported to eliminate single-stranded DNA artifacts (i.e. pseudo-T-restriction fragments), but neither method was beneficial for characterizing mucosa-associated bacterial communities of the mouse cecum. Our study showed that the mechanical agitation and direct plug extraction methods yielded equivalent bacterial community DNA from the mucosa of the small and large intestines of mice, but the latter method was superior for logistical reasons. We also applied a combination of different statistical approaches to analyze T-RFLP data, including statistical detection of true peaks, analysis of variance for peak number, and group significance test, which provided a quantitative improvement for the interpretation of the T-RFLP data.

886 Do Mutations of NHE3 At the Site of Poorly Functioning NHE3 Polymorphisms Contribute to Diarrhea of Congenital Na Diarrhea?

of NHE3 from the membrane and diarrhea. Aim: We investigated the expression, brush border membrane (BBM) localization, and transport function of the major intestinal sodium absorptive transporter, the Na+/H+ exchanger NHE3, as well as the expression of its regulatory PDZ-adapter proteins NHERF1 and PDZK1, TNF-alpha, actin, and a variety of housekeeping genes, in the small and large intestine of mice after induction of a CD45RBhigh transfer colitis in the acute phase of diarrhea. Results: After colitis induction, TNF-alpha levels were increased both in the inflamed colon and microscopically normal-appearing small intestine. NHE3 mRNA expression was up regulated in the colon and unaltered in the small intestine, NHE3 protein expression and localization in the brush border membrane (BBM) was not altered, but acid-activated NHE3 transport rates in small intestinal villous and colonic surface cells were severely decreased, and fluid absorption In Vivo was decreased in the small intestine. PDZK1 mRNA expression was down regulated, whereas that of NHERF1 was not altered. Glucocorticoid treatment of colitic mice for 4 days decreased colonic mucosal cytokine expression and increased PDZK1 expression as well as fluid absorption. In order to investigate whether the down regulation of PDZK1 and the disturbed NHE3 transport activity is causally related, we studied PDZK1 protein expression and NHE3 transport activity in colonic surface enterocytes in the intestine of PDZK1 +/mice, and found PDZK1 content reduced by 60% and NHE3 transport activity significantly decreased. Conclusion: We suggest that during immune-mediated intestinal inflammation, NHE3 BBM protein abundance is normal, but the regulation of its transport activity is disturbed. PDZK1 down regulation during inflammation may be one factor responsible for inflammation-associated NHE3 dysfunction.

T1668 Mast Cell Proteases Stimulate Neurogenic and Non-Neurogenic Mucosal Chloride Secretion in Guinea Pig Distal Ileum

G A A b st ra ct s and involved in the various gut function. However little is known about the subpopulations of TRPV1-expressing nerves and sensory peptidergic nerves in gut. We previously demonstrated that TRPV1-positive nerve fibers in rectum were most abundant among the regions of mouse large intestine. In the present study, we investigated the immunohistochemical distribution of TRPV1 channels and sensory neuropeptides and their co-localization in mouse rectum. METHODS: Extensive TRPV1-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. Protein gene product 9.5 (PGP), CGRP, substance P (SP) and neurokinin A (NKA)-immunoreactivity was also detected by indirect staining with their specific antibodies. The longitudinal change in smooth muscle tone was isotonically measured using Magnus apparatus. RESULTS: The localization of TRPV1 was studied at both transverse and horizontal planes of sections in mouse rectum. Numerous TRPV1-immunoreactive nerve fibers were clearly detected in the mucosal, submucosal layer and myenteric plexus. In contrast, TRPV1 nerve fibers were sparsely distributed in circular and longitudinal muscle layers. Next, double labeling studies were carried out. In the mucosal layer, all the TRPV1 nerve fibers were found to colocalize with PGP and CGRP. SP and NKA positive nerve fibers were not observed. TRPV1 nerve fibers containing both CGRP and SP were observed running around blood vessels in the submucosal layer. In the myenteric plexus, most of the TRPV1 axons were found to colocalize with PGP and CGRP. Relatively small numbers of TRPV1 axons were immunopositive for SP. NKA and SP-immunoreactive axons were observed in the circular and longitudinal muscle, and the cell bodies were detected in the myenteric plexus. NKA and SP were almost colocalized at the axons and cell bodies in muscle layer. In the Magnus experiment, capsaicininduced contraction was significantly inhibited by NK1 and NK2 receptor antagonists. DISCUSION: The present results suggest that TRPV1 nerve fibers in mouse rectum are extrinsic primary afferents. TRPV1 fibers containing CGRP are distributed in mucosal, submucosal layer and myenteric plexus and play various roles in rectal function. TRPV1 fibers containing SP and/or NKA are distributed in myenteric plexus and involved in rectal motor function by stimulating intrinsic excitatory motor neurons.

T1667 Canonical Transient Receptor Potential Channels (TRPC Channels) in Regulation of Mucosal Chloride Secretion in Guinea Pig Ileum

G A A b st ra ct s and involved in the various gut function. However little is known about the subpopulations of TRPV1-expressing nerves and sensory peptidergic nerves in gut. We previously demonstrated that TRPV1-positive nerve fibers in rectum were most abundant among the regions of mouse large intestine. In the present study, we investigated the immunohistochemical distribution of TRPV1 channels and sensory neuropeptides and their co-localization in mouse rectum. METHODS: Extensive TRPV1-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. Protein gene product 9.5 (PGP), CGRP, substance P (SP) and neurokinin A (NKA)-immunoreactivity was also detected by indirect staining with their specific antibodies. The longitudinal change in smooth muscle tone was isotonically measured using Magnus apparatus. RESULTS: The localization of TRPV1 was studied at both transverse and horizontal planes of sections in mouse rectum. Numerous TRPV1-immunoreactive nerve fibers were clearly detected in the mucosal, submucosal layer and myenteric plexus. In contrast, TRPV1 nerve fibers were sparsely distributed in circular and longitudinal muscle layers. Next, double labeling studies were carried out. In the mucosal layer, all the TRPV1 nerve fibers were found to colocalize with PGP and CGRP. SP and NKA positive nerve fibers were not observed. TRPV1 nerve fibers containing both CGRP and SP were observed running around blood vessels in the submucosal layer. In the myenteric plexus, most of the TRPV1 axons were found to colocalize with PGP and CGRP. Relatively small numbers of TRPV1 axons were immunopositive for SP. NKA and SP-immunoreactive axons were observed in the circular and longitudinal muscle, and the cell bodies were detected in the myenteric plexus. NKA and SP were almost colocalized at the axons and cell bodies in muscle layer. In the Magnus experiment, capsaicininduced contraction was significantly inhibited by NK1 and NK2 receptor antagonists. DISCUSION: The present results suggest that TRPV1 nerve fibers in mouse rectum are extrinsic primary afferents. TRPV1 fibers containing CGRP are distributed in mucosal, submucosal layer and myenteric plexus and play various roles in rectal function. TRPV1 fibers containing SP and/or NKA are distributed in myenteric plexus and involved in rectal motor function by stimulating intrinsic excitatory motor neurons.

Na+/H+ exchanger isoform NHE3 and NHERF adaptor protein expression, localization and function in the small and large intestine of CD45RB high transfer colitis

BackroundThe molecular mechanisms for inflammatory diarrhea are multifactorial and incompletely understood.AimWe investigated the expression, localization and function of the major intestinal sodium absorptive transporter, the Na+/H+ exchanger NHE3, as well as its regulatory PDZ‐adapter proteins NHERF1 and PDZK1, in the small and large intestine of mice after induction of a CD45RBhigh transfer colitis.ResultsAfter colitis induction, TNF‐alpha levels were increased both in the inflamed colon and microscopically normal‐appearing small intestine. NHE3 mRNA expression was upregulated in the colon and unaltered in the small intestine, NHE3 protein expression and localization in the brush border membrane (BBM) was not altered, but acid‐activated NHE3 transport rates in small intestinal villous and colonic surface cells were severely decreased, and fluid absorption in vivo was decreased in the small intestine. PDZK1 mRNA expression was downregulated, whereas that of NHERF1 was not altered. Glucocorticoid treatment of colitic mice for 4 days decreased colonic mucosal cytokine expression and increased PDZK1 expression as well as fluid absorption.ConclusionWe suggest that during immune‐mediated intestinal inflammation, NHE3 BBM protein abundance is normal, but the regulation of its transport activity is disturbed. PDZK1 downregulation during inflammation may be one factor responsible for inflammation‐associated NHE3 dysfunction

Distribution and regulation of protein expression of the free fatty acid receptor GPR120

GPR120 is a G-protein-coupled receptor whose endogenous ligands have recently been identified as free fatty acids. It has been implicated as playing an important role in the control of lipid and glucose metabolism by regulating the secretion of glucagon-like peptide-1 and cholecystokinin. We have developed an antibody against the extracellular domain of GPR120. The specificity of the antibody was demonstrated by immunoprecipitation, Western blotting, flow cytometry, and immunocytochemistry using GPR120-transfected cells. Immunoreactivity for GPR120 was abundant in the mouse large intestine, lung, and adipose tissue. Furthermore, we found that the expression of GPR120 protein was up-regulated during the adipogenic differentiation of 3T3-L1 cells, which corresponded well with changes in mRNA expression. The anti-GPR120 antibody will be of value for the further study of the function of this nutrient-sensing receptor.

An Escherichia coli aer mutant exhibits a reduced ability to colonize the streptomycin-treated mouse large intestine

The oxygen sensor Aer of Escherichia coli affects the expression level of genes that are involved in sugar acid degradation. Phenotypes of Aer mediated gene regulation, namely growth on sugar acids was tested 'in vitro' with Phenotype MicroArrays and colonization of the mouse large intestine was tested 'in vivo'. The aer mutant did not grow on the sugar acids D: -gluconate, D: -glucuronate, D: -galacturonate, as well as the sugar alcohol D: -mannitol. Since sugar acids are the predominant carbon source for E. coli in the intestinal mucosa, the ability of the aer mutant to colonize the streptomycin-treated mouse large intestine was tested. The mutant exhibited a decreased ability to colonize the intestine when compared to wild-type cells. This effect was more pronounced under competitive conditions. The colonization phenotype of the aer mutant was complemented with either of two plasmids. One of them expressed the Aer protein, whereas the other one expressed the sugar acid degradation enzymes that are encoded by edd and eda. The data support the interpretation that decreased expression of edd and eda along with the decreased ability to grow on sugar acids may contribute to the reduced capacity of the aer mutant to colonize the mouse intestine. While Aer seems to be important during the initiation phase of colonization, FlhD/FlhC appears to be of disadvantage during maintenance phase. FlhD/FlhC is the master regulator of all flagellar genes and required for Aer expression. Mutants in flhD exhibited an initial competitive disadvantage during the first 3 days of colonization, but recovered lateron.

Synbiotic effect of Lactobacillus helveticus M92 and prebiotics on the intestinal microflora and immune system of mice

The synbiotic effect of the oral treatment of Swiss albino mice with milk-based diets supplemented with Lactobacillus helveticus M92 and various kinds of prebiotics was investigated. Survival, competition, adhesion and colonization, as well as, immunomodulating capability of Lb. helveticus M92, in synbiotic combination, in the gastrointestinal tract (GIT) of mice, were monitored. After the mice were fed with synbiotics, the lactic acid bacteria (LAB) counts in faeces were increased and reduction of enterobacteria and sulphite-reducing clostridia was observed. Similar results were obtained in homogenates of small and large intestine of mice on the 1st and 14th day, after feeding with synbiotics. After the mice were orally given viable Lb. helveticus M92 cells, alone or in combination with prebiotic, the concentration of faecal SIgA and total serum IgA antibodies from all immunized mice were higher compared with the control. The specific humoral immune response was not evoked after oral administration, therefore their synbiotic application is suitable. Among inulin, lactulose and raffinose, Lb. helveticus M92 in combination with inulin, has shown the best synbiotic effect on intestinal and faecal microflora and immune system of mice.

The mannose receptor binds Trichuris muris excretory/secretory proteins but is not essential for protective immunity.

Trichuris muris is a natural mouse model of the human gastrointestinal nematode parasite Trichuris trichiura and it is well established that a T helper type 2-dominated immune response is required for worm expulsion. Macrophages accumulate in the large intestine of mice during infection and these cells are known to express the mannose receptor (MR), which may act as a pattern recognition receptor. The data presented here show for the first time that T. muris excretory/secretory products (E/S) induce bone-marrow-derived macrophages (BMDM) to produce several cytokines and have MR-binding activity. Using alternatively activated BMDM from MR knockout mice it is shown that the production of interleukin-6 partially depends on the MR. Infection of MR knockout mice with T. muris reveals that this receptor is not necessary for the expulsion of the parasite because MR knockout mice expel parasites with the same kinetics as wild-type animals and have similar cytokine responses in the mesenteric lymph nodes. Furthermore, despite acting to reduce serum levels of proinflammatory mediators, absence of the MR does not lead to increased gut inflammation after T. muris infection when assessed by macrophage influx, goblet cell hyperplasia and crypt depth. This work suggests that, despite binding components of T. muris E/S, the MR is not critically involved in the generation of the immune response to this parasite.

Imaging of goblet cells as a marker for intestinal metaplasia of the stomach by one-photon and two-photon fluorescence endomicroscopy

Goblet cells are a requirement for the diagnosis of intestinal metaplasia of the stomach. The gastric mucosa is lined by a monolayer of columnar epithelium with some specialization at the crypts, but there are no goblet cells in normal gastric epithelium. The appearance of goblet cells in gastric epithelium is an indicator of potential malignant progression toward adenocarcinoma. Therefore, in vivo three-dimensional imaging of goblet cells is essential for diagnoses of a premalignant stage of gastric cancers called intestinal metaplasia. We used mouse intestine, which has goblet cells, as a model of intestinal metaplasia. One-photon confocal fluorescence endomicroscopy and two-photon fluorescence endomicroscopy are employed for 3-D imaging of goblet cells. The penetration depth, the sectioning ability, and the photobleaching information of imaging are demonstrated. Both endomicroscopy techniques can three-dimensionally observe goblet cells in mouse large intestine and achieve an imaging depth of 176 microm. The two-photon fluorescence endomicroscopy shows higher resolution and contrast of the imaging sections at each depth. In addition, two-photon fluorescence endomicroscopy also has advantages of sectioning ability and less photobleaching. These results prove that two-photon fluorescence endomicroscopy is advantageous in diagnoses of a premalignant stage of gastric cancers.

Microdispersed Oxidized Cellulose as a novel potential substance with hypolipidemic properties

Objective The aim of this study was to determine whether Microdispersed Oxidized Cellulose (MDOC) possesses a hypolipidemic effect in apolipoprotein-E/low-density lipoprotein receptor double-knockout (ApoE/LDLR-deficient) mice and the possible mechanism of this effect in mice. Methods Female ApoE/LDLR-deficient mice subdivided into two groups were fed with a Western-type diet for 8 wk, and the experimental group was supplemented with 5% MDOC for 8 wk. Female C57BL/6J mice were fed an atherogenic diet containing 5% MDOC or pectin for the determination of a possible hypolipidemic mechanism of MDOC action. Results Biochemical analysis showed that 5% MDOC treatment significantly decreased total cholesterol by 20% ( P = 0.0338) and very-LDL cholesterol by 21% ( P = 0.0110) and significantly increased the level of high-density lipoprotein cholesterol by 62% ( P = 0.0172) when compared with non-treated ApoE/LDLR-deficient mice. The results Association of Official Analytical Chemists method 991.43 revealed that MDOC contains 59.78 ± 5.0% of fiber. Furthermore, it was demonstrated that administration of MDOC did not affect cholesterol absorption in the small intestine. Using C57BL/6J mice, MDOC and pectin treatments decreased cholesterol content in liver and increased fermentation in the gut in vivo. In vitro experiments confirmed that MDOC is fermentable under conditions mimicking those in the large intestine. Conclusion We demonstrated hypolipidemic effects of MDOC in ApoE/LDLR-deficient mice. Moreover, we propose that MDOC is a hypolipidemic soluble fiber acting probably by increased fermentation and production of short-chain fatty acids in the large intestine in mice. We propose that MDOC might be a possible source of soluble fiber for use in dietary supplements.

Functions of p21 and p27 in the regenerating epithelial linings of the mouse small and large intestine

The epithelial linings of the small and large intestine are rapidly turned over and provide an ideal system for exploring links between differentiation and regulation of cell cycle exit. We utilized wild type, p21-/-, p27-/- and p21/p27-/- mice to address contributions of the Cdk inhibitors p21 and p27 to proliferation and differentiation in the mouse gastrointestinal tract. We did not detect any significant differences in proliferation, and all differentiated epithelial cell lineages were represented in all four genotypes. These data indicate that p21 and p27 do not play essential roles in the regulation of normal epithelial renewal in the intestine. These Cdk inhibitors are not needed in vivo for either assembly of Cdk/Cyclin complexes that drive active proliferation, or inhibition of Cdk/Cyclin complexes during cell cycle exit. However, expression of Cyclin D2 and to a lesser degree Cyclin D3 was reduced in p27-/- and p21/p27-/- mice, indicating a unique role for p27 in the regulation of these specific D-type Cyclins in vivo. In the absence of p27, reduced levels of Cyclin D2 and D3 may help to counteract increased proproliferative signals in the intestine.

T1463 Primary Afferent Neurons Innervating the Adult Mouse Small and Large Intestines Are Mostly Medium-Sized and Are Marked By TRPV1, NOS, SP and CGRP

T1463 Primary Afferent Neurons Innervating the Adult Mouse Small and Large Intestines Are Mostly Medium-Sized and Are Marked By TRPV1, NOS, SP and CGRP

Generation of T‐ and IgA‐secreting B cells in the murine large intestine is independent of retinoic acid

Retinoic acid (RA), the vitamin A (retinol) metabolite, generates gut‐homing specific T‐ and IgA‐secreting B cells in the small intestine (SI). We found that generation of T‐ and polyclonal IgA‐secreting B cells in the mouse large intestine (LI) are independent of RA. In contrast to the SI, the numbers of IgA+B cells and CD4+ or CD8+ T cells in the lamina propria of LI were not changed in mice fed a vitamin A‐deficient (−) diet. Further, the numbers of Ag‐specific IgA Ab‐secreting cells (ASC) in the LI induced by intrarectal vaccination with tetanus toxoid and cholera toxin were identical in mice fed vitamin A (+) and vitamin A (−) diets, whereas significant reduction of Ag‐specific IgA ASC was detected in the SI of mice fed vitamin A (−) diets after oral vaccination. Interestingly, in mice treated orally with 50 μg of RA from 1 week before oral immunization, the numbers of Ag‐specific IgA ASC were dramatically decreased in the lamina propria of the SI. However, the numbers of Ag‐specific IgA ASC were normally induced in the lamina propria of the LI with or without RA treatment before intrarectal vaccination. These data demonstrate that generation and/or homing of T‐ and IgA‐secreting B cells in the LI are independently regulated by RA.

Development of Novel Biodegradable Polymeric Nanoparticles-in-Microsphere Formulation for Local Plasmid DNA Delivery in the Gastrointestinal Tract

There is a critical need for development of novel delivery systems to facilitate the translation of nucleic acid-based macromolecules into clinically-viable therapies. The aim of this investigation was to develop and evaluate a novel nanoparticles-in-microsphere oral system (NiMOS) for gene delivery and transfection in specific regions of the gastrointestinal (GI) tract. Plasmid DNA, encoding for the enhanced green fluorescent protein (EGFP-N1), was encapsulated in type B gelatin nanoparticles. NiMOS were prepared by further protecting the DNA-loaded nanoparticles in a poly(epsilon-caprolactone) (PCL) matrix to form microspheres of less than 5.0 microm in diameter. In order to evaluate the biodistribution following oral administration, radiolabeled ((111)In-labeled) gelatin nanoparticles and NiMOS were administered orally to fasted Balb/C mice. The results of biodistribution studies showed that, while gelatin nanoparticles traversed through the GI tract fairly quickly with more than 54% of the administered dose per gram localizing in the large intestine at the end of 2 h, NiMOS resided in the stomach and small intestine for relatively longer duration. Following oral administration of EGFP-N1 plasmid DNA at 100 microg dose in the control and test formulations, the quantitative and qualitative results presented in this study provide the necessary evidence for transfection potential of NiMOS upon oral administration. After 5 days post-administration, transgene expression in the small and large intestine of mice was observed. Based on these results, NiMOS show significant potential as novel gene delivery vehicle for therapeutic and vaccination purposes.

Exacerbating Role of γδ T Cells in Chronic Colitis of T-Cell Receptor α Mutant Mice

T-cell receptor (TCR) gammadelta T cells are an important component of the mucosal immune system and regulate intestinal epithelial homeostasis. Interestingly, there is a significant increase in gammadelta T cells in the inflamed mucosa of patients with ulcerative colitis (UC). However, the role of gammadelta T cells in chronic colitis has not been fully identified. TCRalpha-deficient mice, which spontaneously develop chronic colitis with many features of human UC including an increase in gammadelta T-cell population, represent an excellent model to investigate the role of gammadelta T cells in UC-like colitis. To identify the role of gammadelta T cells in this colitis, we herein have generated TCRgamma-deficient mice through deletion of all TCR Cgamma genes (Cgamma1, Cgamma2, Cgamma3, and Cgamma4) using the Cre/loxP site-specific recombination system and subsequently crossing these mice with TCRalpha-deficient mice. An increase in colonic gammadelta T cells was associated with the development of human UC as well as UC-like disease seen in TCRalpha-deficient mice. Interestingly, the newly established TCRalpha(-/-) x TCRgamma(-/-) double mutant mice developed significantly less severe colitis as compared with TCRalpha-deficient mice. The suppression of colitis in TCRalpha(-/-) x TCRgamma(-/-) double mutant mice was associated with a significant reduction of proinflammatory cytokine and chemokine productions and a decrease in neutrophil infiltration. gammadelta T cells are involved in the exacerbation of UC-like chronic disease. Therefore, gammadelta T cells may represent a promising therapeutic target for the treatment of human UC.