Large Intestinal Mucosa Research Articles

Metoda fotodynamiczna w diagnostyce i terapii guzów głowy i szyi

The photodynamic phenomenon is based on the effect of tissue and cell fluorescence, which is excited by the light of a specified wavelength. Photodynamic diagnosing – PDD is based on the effect of ultraviolet rays on neoplastic and dysplastic tissues that emit light different from the healthy tissues. Tissue fluorescence spectrum can be obtained using fluorophoric substances that naturally occur in cells (autofluorescence) or applying photosensitizing substances before exposure (excited fluorescence – PDD). Fluorophores selectively accumulate in higher concentration in pathological than in normal tissues. An appropriate selection of the photosensitizer at an optimal dose, its high affinity to neoplastic cells with the best index of accumulation in the studied tissue seems essential for a good detection of lesions. In order to excite fuorescence, it is also necessary to provide energy using laser beams with the wavelength congruent with the absorption spectrum of the photosensitizer that has been accumulated in the neoplastic or dysplastic tissues. From all the presently available sources of light – lasers, which enable to obtain light with specifically defined parameters, have been applied most frequently. Photodynamic method as practical non invasive way was used in early diagnosis of precancerous lesion and cancer of skin and mucosa of oral cavity, bronchus, esophagus, large intestine, bladder and reproductive organ. Photodynamic therapy (PDT) is based on the cytotoxicity of light-activated photosensitizers accumulated in the cancer tissue PDT is minimally invasive treatment that uses light of a specific wavelength to activate a photosensitizing agent in the tumor and its microenvironment where pathological tissue is damaged. Photodynamic therapy proved effective to damage pathological tissue in cancer of skin and mucosa of oral cavity, bronchus, supper and upper part of digestive tract, bladder, vulva and uterine cervix. Small, last papers show ability of use photodynamic method to diagnosis and treatment head and neck cancer.

Role of Neuronal and Non-Neuronal Transient Receptor Potential Vanilloid Type 1 and Sensory Neuropeptides in DSS-Induced Peripheral Inflammatory Changes

BACKGROUND & AIMS: The activation of transient receptor potential vanilloid type 1 (TRPV1) leads to release of sensory neuropeptides such as substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) from the nerve endings, and these neuropeptides in turn initiate the biochemical cascade known as neurogenic inflammation. TRPV1 channels and the sensory neuropeptides are expressed not only in sensory nerve fibers but also in non-neuronal cells in the mucosa of large intestine. The aim of this study was to investigate alteration of the sensory neuropeptides and TRPV1 channels in mucosa of DSS-induced colitis mice and to characterize their non-neuronal cells. METHODS: Colitis was induced by 3% dextran sulfate sodium (DSS) solution given as drinking water for 7 days in C57BL/6 mice. To evaluate visceral nociception, colorectal distension was performed in mice treated with vehicle or BCTC on day 7 of DSS treatment. Immunohistochemical analysis was performed using rectal tissues of mice. SP, NKA, CGRP, 5-HT, F4/80, keratin, TNF-α, CD11c, and CD4 were detected by indirect staining with their specific antibodies. TRPV1-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. RESULTS: TRPV1 antagonist BCTC significantly attenuated the visceral hyperalgesia to control level in DSS-induced colitis model. We compared the number of the nerve fibers and non-neuronal cells expressing TRPV1 channels and sensory neuropeptides in normal and DSS-induced colitis model mice. The number of TRPV1and CGRP-expressing nerve fibers is significantly increased in colitis model. The non-neuronal TRPV1 cells were markedly increased but non-neuronal CGRP-expressing cells were not observed in colitis model. No significant alteration in the number of SP and NKA positive nerve fibers was detected. On the other hand, SP and NKA positive cells were drastically increased in colitis model. Next, double labeling studies were carried out to characterize TRPV1-, SP-, and NKA-expressing cells under inflammatory state. We found two types of non-neuronal TRPV1-expressing cells in mucosa of colitis model. One is keratin positive cells located upper part of mucosa. Another is TNF-α and/or F4/80 positive cells located middle part of the mucosa. Both types of TRPV1-expressing cells did not colocalize with CD4 and CD11c. SP and NKA positive cells almost completely colocalized with 5-HT in experimental colitis model mice. CONCLUSION: These results suggest that TRPV1immunopositive epithelial cells and macrophage are associated with visceral hyperalgesia in colitis.

139 A Novel Nanoparticle Approach to Induce Colorectal and Vaginal Mucosal Immunity

Many infections, including HIV, are transmitted through mucosal surfaces. Where T cell immunity is needed to protect, we have previously found that local mucosal immunity is most effective. Moreover, intrarectal immunization was the most effective route of several we compared to induce such immunity in the GI mucosa. However, the intrarectal route may not be the most acceptable for human vaccination. We hypothesized that we might mimic intrarectal immunization if we could deliver the vaccine to the colorectal mucosa, and might be able to do so by a more practical oral route of delivery if we could bypass destruction in the stomach and induction of oral tolerance in the small intestine. We have now accomplished this in mice by using a novel construct of pH-dependent microparticles encapsulating nanoparticles that contain the vaccine. The particles are designed to avoid uptake of the microparticles or release of the nanoparticles until they reach the large intestine. This oral vaccine induced a level of T cell immunity in the large intestinal mucosa that rivaled that induced by direct intrarectal delivery, and resulted in comparable protection against intrarectal or intravaginal challenge with a recombinant virus. In contrast, vaccine particles targeted to the small intestine resulted in greater T cell immunity in the small intestine but not in the large intestine, and failed to protect against intrarectal or intravaginal viral challenge. Thus, with these directed particles, we have been able to demonstrate functional compartmentalization of the gut mucosal immune system for T cell immunity, and to develop an orally delivered vaccine capable of providing protection against rectal or vaginal mucosal challenge.

CCL25/CCR9 Interactions Regulate Large Intestinal Inflammation in a Murine Model of Acute Colitis

Background & AimsCCL25/CCR9 is a non-promiscuous chemokine/receptor pair and a key regulator of leukocyte migration to the small intestine. We investigated here whether CCL25/CCR9 interactions also play a role in the regulation of inflammatory responses in the large intestine.MethodsAcute inflammation and recovery in wild-type (WT) and CCR9−/− mice was studied in a model of dextran sulfate sodium (DSS)-induced colitis. Distribution studies and phenotypic characterization of dendritic cell subsets and macrophage were performed by flow cytometry. Inflammatory bowel disease (IBD) scores were assessed and expression of inflammatory cytokines was studied at the mRNA and the protein level.ResultsCCL25 and CCR9 are both expressed in the large intestine and are upregulated during DSS colitis. CCR9−/− mice are more susceptible to DSS colitis than WT littermate controls as shown by higher mortality, increased IBD score and delayed recovery. During recovery, the CCR9−/− colonic mucosa is characterized by the accumulation of activated macrophages and elevated levels of Th1/Th17 inflammatory cytokines. Activated plasmacytoid dendritic cells (DCs) accumulate in mesenteric lymph nodes (MLNs) of CCR9−/− animals, altering the local ratio of DC subsets. Upon re-stimulation, T cells isolated from these MLNs secrete significantly higher levels of TNFα, IFNγ, IL2, IL-6 and IL-17A while down modulating IL-10 production.ConclusionsOur results demonstrate that CCL25/CCR9 interactions regulate inflammatory immune responses in the large intestinal mucosa by balancing different subsets of dendritic cells. These findings have important implications for the use of CCR9-inhibitors in therapy of human IBD as they indicate a potential risk for patients with large intestinal inflammation.

Immunomodulatory effect of mushrooms on cytotoxic activity and cytokine production of intestinal lamina propria leukocytes does not necessarily depend on β-glucan contents

We evaluated the effects of seven mushroom extracts (Grifola frondosa, Pholiota nameko, Panellus serotinus, Hypsizygus marmoreus, Pleurotus cornucopiae, Armillaria mellea, and Flammulina velutipes) on cytotoxic activity and cytokine production of lamina propria leukocytes (LPLs) isolated from rat small (S) and large (L) intestinal mucosa. Boiling water extracts from seven species of mushrooms showed no direct cytotoxicity against the YAC-1 target cells. However, prominent increases of cytotoxicity were observed in S- and L-LPLs co-cultured with P. serotinus extract. Cytokine production (TNFα, IFNγ, IL-12 p70, and IL-4) of S- and L-LPLs was stimulated in response to P. cornucopiae extract. Mushroom extracts contributed to target cell adhesion and/or cytokine production in the effector cells. The promotion of cytotoxic activity in S- and L-LPLs was not necessarily related to β-glucan content of the mushroom.

Effect of olive oil and barley diets on the caecal mucosa histomorphology

Nutrition is an environmental factor of major importance in human health. Barley is a cereal, rich in dietary fibres (DF) such as β-glucan, arabinoxylans and cellulose. DF is rapidly fermented by the intestinal microflora, with the formation of short-chain fatty acids (SCFA). High levels of SCFA, particularly of butyrate, are important for a healthy large intestine mucosa. SCFA are rapidly absorbed by the colonic mucosa. Butyrate is the preferred energy source of the colonic epithelial cells and acts specifically as a signal metabolite, stimulating cell migration and proliferation. Both the significantly higher formation and the increased absorption of SCFA are important in the protection of the colon mucosa. Olive oil is an integral ingredient of the Mediterranean diet. It is known for its high levels of monounsaturated fatty acids and is also a good source of phytochemicals. Accumulated evidence suggests that it may have health benefits that include the reduction of several varieties of cancers affecting the mucosa in the colon. It might influence the polyamine metabolism in colonic enterocytes in ways that reduce the progression from normal mucosa to adenoma and carcinoma. Therefore, it is important to study the effects of olive oil and the barley on the colonic mucosa, because they are commonly combined in the diet of North African population. The aim of our study is to determine the effects of consumed olive oil and the barley on some histological colonic mucosa parameters as the proliferation, crypt depth and width in rats consuming these types of diets experimentally, the coecum was taken as an example of colonic portion for this study. Seventy-two female Wistar rats were considered to assess the effects of consumption of the olive oil and barley diet on colonic morphology. These diets (olive oil and barley) and a control diet were compared. The experimental period was 40 days. Eight rats from each group were killed at the, 20th and 40th day after consuming experimental diets, caecums were than extracted. Crypt depth and width, the average of mitoses per crypt in the caecal mucosa were determined, using a morphometric system (SAMBA, Grenoble, France). Our results showed that length of the crypts was very similar; this did not achieve statistical significance for olive oil or barley diet supplementation. However, the barley diet was associated with a statistically significant increase in the width of the crypt compared to the control diet (p < 0.05). Statistical differences were not observed for the number of mitoses per crypt, compared to the experimental diets. We concluded that Barley diet can have influence on the colonic crypt width. This might be explained by the high levels of SCFA. Olive oil and the barley diet do not influence on the number of mitoses per crypt and the crypt length in ceacal mucosa.

MicroRNAs Control Intestinal Epithelial Differentiation, Architecture, and Barrier Function

Whereas the importance of microRNA (miRNA) for the development of several tissues is well established, its role in the intestine is unknown. We aimed to quantify the complete miRNA expression profile of the mammalian intestinal mucosa and to determine the contribution of miRNAs to intestinal homeostasis using genetic means. We determined the miRNA transcriptome of the mouse intestinal mucosa using ultrahigh throughput sequencing. Using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP), we identified miRNA-messenger RNA target relationships in the jejunum. We employed gene ablation of the obligatory miRNA-processing enzyme Dicer1 to derive mice deficient for all miRNAs in intestinal epithelia. miRNA abundance varies dramatically in the intestinal mucosa, from 1 read per million to 250,000. Of the 453 miRNA families identified, mmu-miR-192 is the most highly expressed in both the small and large intestinal mucosa, and there is a 53% overlap in the top 15 expressed miRNAs between the 2 tissues. The intestinal epithelium of Dicer1(loxP/loxP);Villin-Cre mutant mice is disorganized, with a decrease in goblet cells, a dramatic increase in apoptosis in crypts of both jejunum and colon, and accelerated jejunal cell migration. Furthermore, intestinal barrier function is impaired in Dicer1-deficient mice, resulting in intestinal inflammation with lymphocyte and neutrophil infiltration. Our list of miRNA-messenger RNA targeting relationships in the small intestinal mucosa provides insight into the molecular mechanisms behind the phenotype of Dicer1 mutant mice. We have identified all intestinal miRNAs and shown using gene ablation of Dicer1 that miRNAs play a vital role in the differentiation and function of the intestinal epithelium.

SDD: don’t be selective in considering pros and cons

Dear Editor, We thank Dr. Zandstra and colleagues [1] for their interest in our work [2]. Although in their scientific careers they have amassed impressive data on selective digestive microbial decontamination (SDD), they have not fully appreciated what our paper adds to this large body of knowledge. The intestinal microbiota contains many more cells than the human body itself. Furthermore the intestinal microbial gene set is approximately 150 times larger than the human genome [3]. Last year marked the 150th anniversary of Darwin’s On the Origin of Species. How can we explain any genetic advantage for humans to carry so many microbes along? What benefit does our microbiome provide us during health? And what happens during critical illness? How does the intensive care unit (ICU) environment impact on our microbiota? What impact does SDD have on our microbiota? And what is the impact of SDD on the emergence of resistant bacteria in the ICU? Zandstra et al. rightly allude to the fact that Enterobacteriaceae increase during critical illness—we also found a more than tenfold relative increase (as a percentage of total bacteria) compared to data from healthy volunteers [4, 5]. To fight overgrowth of Enterobacteriaceae seems like a good idea—but is there a trade-off? Why did SDD—though providing a small but significant survival advantage compared to a control group—fail to further reduce mortality compared with patients receiving only oral topical antimicrobial products, even though these study participants had similarly increased percentages of Enterobacteriaceae? A potential advantage of having such massive numbers of anaerobic bacteria in the colon is that colonocytes feed on bacterial products. Indeed colonocytes feed on butyrate, produced by a limited number of colonic bacterial species. The Faecalibacterium prausnitzii group is predominant amongst these butyrate-producing groups. This group is already reduced during tube feeding, resulting in a significantly reduced concentration of butyrate [4]. We describe a significant reduction in two groups of microbiota that help in maintaining the integrity of the large intestinal mucosa. Quoting Vollaard’s and Donskey’s work, Zandstra et al. agree that SDD is a contradiction in terms. We apparently disagree in our concern that SDD might have a “dark side”, i.e. that some important beneficial microbiota are harmed by SDD. Besides, there is additional collateral damage: in the analysis of point-prevalence cultures from the de Smet study [6], an increase of intestinal colonisation by resistant organisms was observed after cessation of SDD [7]. None of the articles cited by Zandstra et al. describe the use of molecular methods for detection and/or enumeration of the intestinal microbiota. Bacteria from the F. prausnitzii group are highly sensitive to oxygen and require very specific growth media. Culture-based quantification yields a high degree of culture bias. These limitations of culture-based microbiological techniques have precluded reliable testing of the effects of antibiotics (i.e. SDD) on the intestinal microbiota. We are obviously not the first to claim that SDD is not selective. We are, however, the first to back up these claims using absolute numbers of faecal bacteria derived from a quantitatively reliable, molecular method. Referring to Wensinck as an argument that not F. prausnitzii but clostridia contribute to colonisation resistance (CR), Zandstra et al. display a misunderstanding of the composition of the intestinal microbiota. Although phenotypically Gram-negative, F. prausnitzii are genetically closely related to the Gram-positive clostridia Zandstra et al. refer to. In fact, they belong to clostridial cluster IV [8]. These phylogenetic relations have only been discovered since the dawn of the molecular era. We agree that the clinical impact of F. prausnitzii reduction in the critically ill is unclear—at least, that this should be further studied; we do not know whether there is a critical threshold of butyrate substrate for colonocytes to survive, or to maintain the integrity of the large intestinal mucosa. Recent studies have shown an anti-inflammatory effect of F. prausnitzii in Crohn’s disease that could also be beneficial for the critically ill [9]. We have, however, provided novel information that may explain the discrepancy of effective reduction of Enterobacteriaceae by SDD, which fails to translate into further survival benefit in comparison with selective oropharyngeal decontamination (SOD).

HISTOCHEMICAL STUDY OF NON-SPECIFIC ESTERASE ACTIVITY IN SMALL AND LARGE INTESTINE OF YOUNG RABBIT

The distribution of non-specific esterase in normal duodenal, jejunal, ileal and large intestinal mucosa of the rabbit has been studied using histochemical methods. Thirty three New Zealand White rabbits were used ranging from 26-day old fetuses to 43-day old young (3 rabbits / age). Distribution and intensity (strong to very strong) varied little throughout the small and large intestine from the 30th day of foetal life and as after birth up to 43 days. At the 26 or 28th days of foetal life only mild to positive reactions were seen. I In the caecum and in the upper colon, a low non-specific esterase activity was observed in the 7- and 15 day-old rabbits, although this activity was very strong in younger one-day-old rabbits, and in the 19-day-old and older rabbits.

The protective effect of oral colitis-derived proteins in a murine model of inflammatory bowel disease is associated with an increase in γδ T cells in large intestinal mucosa

Oral tolerance has previously been shown effective in preventing several immune-mediated disorders in animal models. The aims of this study were to investigate the effect of oral colitis-extracted proteins (CEP) on dextran sulfate sodium (DSS)-induced colitis in BALB/c mice and to explore the relative role of the intestinal mucosal gammadelta T cells. The effect of five low oral doses of CEP on colitis was evaluated by clinical manifestation and histological lesions. Serum cytokines were measured by enzyme-linked immunosorbent assay. The percentages of the intestinal mucosal gammadelta T cells were evaluated by flow cytometry. CEP-fed colitis mice showed less severe symptoms and histological injury than bovine serum albumin (BSA)-fed control mice. Tolerized mice developed an increase in TGF-beta1 and no change in IFN-gamma serum levels. Increases in TCRgammadelta(+) T cells and CD8alpha(+)TCRgammadelta(+) T cells in small intestinal mucosal lymphocytes and no quantitative change in large intestinal mucosal lymphocytes were demonstrated in colitis mice compared to untreated mice. The proportions of TCRgammadelta(+) T cells and CD8alpha(+)TCRgammadelta(+) T cells in large intestinal mucosal lymphocytes from CEP-fed colitis mice were significantly higher compared to BSA-fed controls. The disease activity index negatively correlated with the percentages of large intestinal mucosal gammadelta T cells. Furthermore, mucosal repair in repair-period mice was also accompanied by increases in TCRgammadelta(+) T cells and CD8alpha(+)TCRgammadelta(+) T cells in large intestinal mucosal lymphocytes. Improvement of DSS-induced colitis that resulted from oral administration of colitis-extracted proteins is associated with an increase in gammadelta T cells in large intestinal mucosa.

CD4+ T‐cell localization to the large intestinal mucosa during Trichuris muris infection is mediated by Gαi‐coupled receptors but is CCR6‐ and CXCR3‐independent

Infection of mice with the gastrointestinal nematode Trichuris muris represents a valuable tool to investigate and dissect intestinal immune responses. Resistant mouse strains respond to T. muris infection by mounting a T helper type 2 response. Previous results have shown that CD4(+) T cells play a critical role in protective immunity, and that CD4(+) T cells localize to the infected large intestinal mucosa to confer protection. Further, transfer of CD4(+) T cells from immune mice to immunodeficient SCID mice can prevent the development of a chronic infection. In the current study, we characterize the protective CD4(+) T cells, describe their chemokine receptor expression and explore the functional significance of these receptors in recruitment to the large intestinal mucosa post-T. muris infection. We show that the ability to mediate expulsion resides within a subpopulation of CD4(+) T cells marked by down-regulation of CD62L. These cells can be isolated from intestine-draining mesenteric lymph nodes (MLN) from day 14 post-infection, but are rare or absent in MLN before this and in spleen at all times post-infection. Among CD4(+) CD62L(low) MLN cells, the two most abundantly expressed chemokine receptors were CCR6 and CXCR3. We demonstrate for the first time that CD4(+) CD62L(low) T-cell migration to the large intestinal mucosa is dependent on the family of G alpha(i)-coupled receptors, to which chemokine receptors belong. CCR6 and CXCR3 were however dispensable for this process because neutralization of CCR6 and CXCR3 did not prevent CD4(+) CD62L(low) cell migration to the large intestinal mucosa during T. muris infection.

OmpA variants affecting the adherence of ulcerative colitis-derived Bacteroides vulgatus.

To investigate the adhesion factor of Bacteroides vulgatus derived from ulcerative colitis (UC), we isolated B. vulgatus strains from the large intestinal mucosa of UC patients and non-UC individuals, and examined their adherence to tissue-cultured cells. The adherence to tissue-cultured cells in UC-derived strains (36.5 +/- 7.9 %) was higher than that in non-UC-derived strains (13.2 +/- 7.7 %). PCR and sequencing analysis of outer membrane proteins revealed that the strains derived from five of seven (71.4 %) UC patients had ompA genes belonging to either ompA variant type A or B. The adherence rates in Escherichia coli DH5 a transformants with ompA type A variants (33.3 +/- 4.6 %) and type B variants (34.6 +/- 7.1 %) were higher than the rate in those with non-UC ompA (16.4 +/- 4.0 %). Our results suggest that B. vulgatus isolates in the mucosal flora of the large intestine of UC patients have a high frequency of ompA variants and that the variation of ompA variants is one of the factor causing an increase in the adherence of the bacterium.

Luminal sulfide and large intestine mucosa: friend or foe?

Hydrogen sulfide (H(2)S) is present in the lumen of the human large intestine at millimolar concentrations. However, the concentration of free (unbound) sulfide is in the micromolar range due to a large capacity of fecal components to bind the sulfide. H(2)S can be produced by the intestinal microbiota from alimentary and endogenous sulfur-containing compounds including amino acids. At excessive concentration, H(2)S is known to severely inhibit cytochrome c oxidase, the terminal oxidase of the mitochondrial electron transport chain, and thus mitochondrial oxygen (O(2)) consumption. However, the concept that sulfide is simply a metabolic troublemaker toward colonic epithelial cells has been challenged by the discovery that micromolar concentration of H(2)S is able to increase the cell respiration and to energize mitochondria allowing these cells to detoxify and to recover energy from luminal sulfide. The main product of H(2)S metabolism by the colonic mucosa is thiosulfate. The enzymatic activities involved in sulfide oxidation by the colonic epithelial cells appear to be sulfide quinone oxidoreductase considered as the first and rate-limiting step followed presumably by the action of sulfur dioxygenase and rhodanese. From clinical studies with human volunteers and experimental works with rodents, it appears that H(2)S can exert mostly pro- but also anti-inflammatory effects on the colonic mucosa. From the available data, it is tempting to propose that imbalance between the luminal concentration of free sulfide and the capacity of colonic epithelial cells to metabolize this compound will result in an impairment of the colonic epithelial cell O(2) consumption with consequences on the process of mucosal inflammation. In addition, endogenously produced sulfide is emerging as a prosecretory neuromodulator and as a relaxant agent toward the intestinal contractibility. Lastly, sulfide has been recently described as an agent involved in nociception in the large intestine although, depending on the experimental design, both pro- and anti-nociceptive effects have been reported.

Ontogeny of FOXP3(+) regulatory T cells in the postnatal human small intestinal and large intestinal lamina propria.

FOXP3(+) regulatory T cells (Treg) suppress innate and adaptive immune responses and are critical for intestinal immune homeostasis. Our objective was to define the postnatal developmental regulation of Treg in relationship to other T cells in the human intestinal tract. We analyzed 41 small and 18 large intestinal paraffin-embedded tissue samples from preterm and term infants with and without necrotizing enterocolitis (NEC) for the presence of CD3(+), CD4(+), CD8(+), and FOXP3(+) cells by immunohistochemistry. We compared labeled cells against age, gestational age (GA), or (corrected) postmenstrual age (PMA). The GA ranged from 23 to 40 weeks, with a mean of 32 (standard deviation, 4.7) weeks. Independent of age, GA, or PMA, the numbers of CD4(+) cells were higher in the small intestine compared to the large intestine (P = 0.046), except in patients with NEC. FOXP3(+) cells could be detected as early as 23 weeks in GA in both large and small bowel, and similar quantities were detected at the highest GA examined (40 weeks). We saw no statistically significant effect of GA, age, or PMA on total number of FOXP3(+) cells or by comparing FOXP3(+) to CD4(+) or FOXP3(+) to CD8(+) ratios, indicating intact ontogeny of Treg in intestinal tissue early in gestation. Human infants exhibit presence of mucosal FOXP3(+) cells in the small and large intestinal mucosa at birth and as early as 23 weeks GA. The frequency of FOXP3(+) cells and the ratios of FOXP3(+) to CD4(+) or CD8(+) cells do not change with increasing intrauterine development or postnatal age.

Systemic Adenovirus Infection in Bearded Dragons ( Pogona vitticeps): Histological, Ultrastructural and Molecular Findings

Three Inland Bearded Dragons (Pogona vitticeps) from two breeding groups were humanely destroyed following a period of anorexia. Two of the animals were 8-months old and related and one animal was approximately 2-weeks old. Necropsy examination revealed poor bodily condition but no other gross abnormalities. Microscopically there was non-suppurative hepatitis and interstitial nephritis. Multiple large, amphophilic, intranuclear inclusion bodies were present within hepatocytes and epithelial cells of the bile ducts, renal tubules, small and large intestinal mucosa, pancreatic acini and oral mucous membranes. Transmission electron microscopy (TEM) demonstrated that the inclusions comprised viral particles with morphology consistent with an adenovirus. A fragment of the adenoviral polymerase gene was amplified, sequenced and compared with other reptilian adenoviral sequences.

Distinct mucosal microenvironments differentially regulate memory T cell differentiation state and maintenance (39.20)

Abstract T cell location and differentiation state are critical factors involved in protection against viral infection. We found that the phenotype of LCMV-specific CD8 and CD4 T cells varied by location, even amongst different mucosal tissues. Virus-specific CD8 T cells upregulated αE integrin and downregulated Ly6C in the small intestine but this did not occur in the spleen, lung, large intestine, or vaginal mucosa. αE integrin was required for maintenance of CD8 T cells in the small intestine IEL (see Casey et al. abstract). TGF β was responsible for inducing this tissue-specific phenotype. Interestingly, virus-specific CD4 T cells did not modify αE integrin and Ly6c in response to TGF β. Virus-specific CD4 T cells exhibited a decrease in αE integrin expression compared to CD8 T cells and this correlated with decreased maintenance of memory antigen-specific CD4 T cells within this compartment. Taken together, these data may explain why the abundant CD4 T cell effector response was poorly maintained specifically within the small intestine epithelium and highlights a mechanism by which tissue microenvironment regulates maintenance and compartmentalization of T cells in mucosal tissues.

Biomarkers Correlate With Colon Cancer and Risks

We previously reported that gene expression analysis of biopsies of normal-appearing large intestinal mucosa can distinguish individuals with colonic cancer and many individuals at risk for colon cancer from controls. The purpose of this study was to determine whether noninvasively removed rectal swabs can identify individuals with colon cancer or risk of colon cancer as effectively as we previously demonstrated using biopsies. Rectal mucosa cells were removed by rectal swabs, and their gene expression profiles were compared with those of biopsies removed by colonoscopy. Expression of 16 genes in the rectal mucosa of 12 individuals with colon cancer, 25 with polyps, 37 with family or self-reported cancer history, and 23 controls was measured by real-time reverse transcription-polymerase chain reaction. We found similar results using rectal swabs and biopsies. Groups of individuals with or at risk for cancer showed an altered gene expression profile compared with controls. Moreover, each of the 12 cancer patients showed altered expression relative to the mean of controls. Gene expression analysis using rectal swabs may provide a convenient way to screen for colon cancer.

In vitro/in vivo biorecognition of lectin-immobilized fluorescent nanospheres for human colorectal cancer cells

Peanut agglutinin (PNA)-immobilized polystyrene nanospheres with surface poly( N-vinylacetamide) (PNVA) chains encapsulating coumarin 6 were designed as a novel colonoscopic imaging agent. PNA was a targeting moiety that binds to β- d-galactosyl-(1-3)- N-acetyl- d-galactosamine, which is the terminal sugar of the Thomsen–Friedenreich antigen that is specifically expressed on the mucosal side of colorectal cancer cells. PNVA was immobilized with the aim of reducing nonspecific interactions between imaging agents and normal tissues. Coumarin 6 was encapsulated into nanosphere cores to provide endoscopically detectable fluorescence intensity. After incubation of imaging agents with human cells, the fluorescence intensity of imaging agent-bound cells was estimated quantitatively. The average fluorescence intensity of any type of colorectal cancer cell used in this study was higher than that of small intestinal epithelial cells that had not exposed the carbohydrate. The in vivo performance of imaging agents was subsequently evaluated using a human colorectal cancer orthotopic animal model. Imaging agent-derived strong fluorescence was observed at several sites of the large intestinal mucosa in the tumor-implanted nude mice after the luminal side of the colonic loop was contacted with imaging agents. In contrast, when mice that did not undergo tumor implantation were used, the fluorescence intensity on the mucosal surface was extremely low. Data indicated that imaging agents bound to colorectal cancer cells and the cancer cell-derived tumors with high affinity and specificity.

The effects ofStrongylus vulgarisparasitism on eosinophil distribution and accumulation in equine large intestinal mucosa

Eosinophilic granulocytes have been associated with parasite or immune-mediated diseases, but their functions in other disease processes remain unclear. Cause and timing of eosinophil migration into the equine gastrointestinal mucosa are also unknown. To determine the effects of intestinal parasitism on eosinophils in equine large intestinal mucosa. Large intestinal mucosal samples were collected from horses and ponies (n = 16) from the general veterinary hospital population, ponies (n = 3) raised in a parasite-free environment, ponies experimentally infected with 500 infective Strongylus vulgaris larvae and treated with a proprietary anthelmintic drug (n = 14), and a similar group of ponies (n = 7) that received no anthelmintic treatment. Total eosinophil counts and eosinophil distribution in the mucosa were determined by histological examination. A mixed model analysis was performed and appropriate Bonferroni adjusted P values used for each family of comparisons. P<0.05 was considered significant. There was no difference in large intestinal mucosal eosinophil counts and eosinophil distribution between ponies infected with S. vulgaris and those raised in a parasite-free environment. Experimental infection with S. vulgaris, with or without subsequent anthelmintic treatment, did not change eosinophil counts, and counts were similar to those for horses from the general population. Migration of eosinophils to the equine large intestinal mucosa appears to be independent of exposure to parasites. Large intestinal mucosal eosinophils may have more functions in addition to their role in defence against parasites.

Apoptosis and Surivivin gene expression during Holothuria glaberrima intestinal regeneration

The sea cucumber (Holothuria glaberrima) is an excellent model to study regeneration processes in deuterostomes. H.glaberrima has the ability to regenerate most of its viscera after the process of evisceration, where most of the viscera are expelled from the body. We have showed that apoptosis takes place during intestinal regeneration. Apoptosis, is the result of DNA internucleosomal cleavage, the morphological characteristics are: cell contraction, nuclear pyknosis, chromatin condensation and cell fragmentation. By using the TUNEL assay, where the TdT enzyme catalyzes the addition of Br‐dUTP, labelling the 3′‐OH terminal groups exposed during the internucleosomal cleavage; we identified apoptotic cells by fluorescence microscopy. We showed a peak in the number of apoptotic cells throughout the first week of regeneration and the distribution of apoptotic cells in the different layers of the large intestine (serosa, sub‐mucosa, mucosa and mesentery). Moreover we have now identified the holothurian homologue of the Survivin gene that in vertebrates functions as an inhibitor of apoptosis. Preliminary results have shown a differential expression of this gene during regeneration. Molecular experiments, PCR and In‐situ hybridization are under way to asses the temporal and spatial expression of apoptosis and survivin expression. Our results prove that similar to embryonic development, apoptosis plays an important role during intestinal regeneration. These studies provide a deeper understanding of the process of intestinal regeneration in the sea cucumber and the applications to studies in other animals, including humans.