Sections Of Stomach Research Articles

‘Actin-reactive’ discriminated from ‘non-actin-reactive’ smooth muscle autoantibody by immunofluorescence reactivity with rat epithelial cell line

To compare smooth muscle antibody (SMA) patterns in tissue sections with patterns in an immunofluorescence assay (IFA) using a rat intestinal epithelial cell line and results from an F-actin IgG ELISA. SMA positive sera (n = 188) were classified by immunofluorescence staining of rodent kidney, stomach and liver sections as SMA-T (tubules) (n = 124) or SMA-V (vessels) (n = 64). The F-actin pattern on the rat epithelial cell line was identified by immunofluorescence staining of actin cables that was confirmed by dual immunofluorescence co-localisation with phalloidin. Of 124 SMA-T positive sera, 123 reacted with the epithelial cell line and 120 with F-actin by ELISA, giving sensitivity for detection of anti-F-actin antibody of 99% and 97%, respectively. Of 64 SMA-V positive sera, four reacted with the epithelial cell line (6%) and 41 with F-actin by ELISA (64%). Tests of 493 normal blood donors and 100 disease controls yielded specificities of 584/593 (98.5%) and 562/593 (94.8%) for the cell line IFA and F-actin ELISA, respectively. The rat epithelial cell line IFA is a robust diagnostic assay for anti-F-actin antibody that can either replace the routine screening for actin-reactive SMA-T antibody in tissue sections or be used as a confirmatory assay for anti-F-actin antibody after screening by F-actin ELISA or on rodent tissue sections by IFA.

Pathophysiological Classification of Functional Dyspepsia Using a Novel Drinking-Ultrasonography Test

Background: Functional dyspepsia (FD) is a heterogeneous disease characterized by various upper abdominal symptoms. The major mechanism of FD symptoms includes impaired fundic accommodation, delayed gastric emptying, and visceral hypersensitivity. We developed a novel drinking-ultrasonography test to combine a drink test with ultrasonography to assess gastric motility and sensory function of FD patients. Method: Subjects were 60 successive FD patients according to the Rome III criteria. A drinking-ultrasonography test was performed after subjects had fasted. The subjects ingested 200 ml of water at 2-min intervals 4 times (total 800 ml) through a straw. The maximum cross section of the proximal stomach was visualized before water intake, after each water intake, and 5 and 10 min after the completion of drinking using extracorporeal ultrasonography. Abdominal symptoms were evaluated using the visual analog scale (VAS) a total of 5 times. The normal range of cross-sectional area and VAS were set using average ± standard deviations of 33 healthy volunteers. Cases outside the normal range were diagnosed with a motor or sensory disorder. Results: The drinking-ultrasonography test classified FD patients into four groups without adverse effect or trouble. The distribution of each group was 27% in the normal group, 15% in the impaired relaxation group, 10% in the delayed emptying group, and 48% in the visceral hypersensitivity group. There was no significant correlation between the pathophysiological classification and subtypes of FD defined by the Rome III criteria. Conclusion: We developed a novel drinking-ultrasonography test that was effective in classifying FD patients according to pathophysiological features.

Effect of Chronic Consumption of Nutmeg on the Stomach of Adult Wistar Rats

Nutmeg is commonly used as a spice in various dishes; it is a component of teas and soft drinks, and can also be mixed in milk and alcohol. In this study, we investigated the effect of chronic consumption of nutmeg on the stomach of adult Wistar rats. Rats of both sexes (n = 24), with an average weight of 200 g, were randomly assigned into two treatment groups (A and B, n = 8 for each group) and one control group (C, n = 8). The rats in the treatment groups received 1 g of nutmeg in group A and 2 g of nutmeg in group B. The nutmeg was thoroughly mixed with feed and given daily for 32 days. Group C received an equal amount of feed daily without nutmeg for 32 days. The rats had free access to water. On day 33 of the experiment, the rats were sacrificed by cervical dislocation. The stomachs were carefully dissected out and immediately fixed in 10% formal saline for histological study. Hematoxylin and eosin staining showed that rats in groups A and B showed varying degrees of distortion in the epithelial cells of the stomach. Obvious signs of proliferation, hyperplasia, and atrophic changes in the treated stomach sections were more marked in group B than in the other groups. These findings indicate that nutmeg may have some deleterious effects on the microanatomy of the stomach of adult Wistar rats.

A novel immunofluorescence test for f-actin-reactive smooth muscle antibody

<h3>Aim</h3> To compare smooth muscle antibody (SMA) immunofluorescence staining patterns with an immunofluore-scence assay (IFA) using a rat intestinal epithelial cell line and with an F-actin ELISA. <h3>Methods</h3> SMA sera (<i>n</i> = 188) were classified as SMA-T (<i>n</i> = 124) or SMA-V (<i>n</i> = 64) by immunofluorescence reactivity on composite rodent tissue sections of stomach, liver and kidney. Immunofluorsecence reactivity with the rat intestinal epithelial cell line was identified by staining of linear ‘actin cables' that was confirmed by dual immunof-luorescence co-localisation with F-actin specifically labelled by phalloidin. <h3>Results</h3> Of 124 SMA-T positive sera, 123 reacted with the rat intestinal epithelial cell line and 120 with F-actin by ELISA, giving sensitivities for detection of anti-F-actin antibody of 99% and 97%, respectively. In contrast, only four of 64 SMA-V positive sera reacted with the rat intestinal epithelial cell line (6%) while 41 reacted with F-actin by ELISA (64%). Tests of 493 normal blood donors and 100 disease controls gave specificities of 584/593 (98.5%) and 561/593 (94.6%) for the cell line IFA and F-actin ELISA respectively. <h3>Conclusions</h3> The rat intestinal epithelial cell line im-munofluorescence is a robust diagnostic assay for anti-F-actin antibody that can either replace the routine screening for anti-actin SMA-T antibody in tissue sections or be used as a confirmatory assay for anti-F-actin antibody after screening by F-actin ELISA or by immunofluorescence on rodent tissue sections.

A Human Domain Antibody and LewisbGlycoconjugate That Inhibit Binding ofHelicobacter pylorito LewisbReceptor and Adhesion to Human Gastric Epithelium

Increasing antibiotic resistance has prompted development of alternative approaches to antimicrobial therapy, including blocking microbial adhesion to host receptors. The BabA adhesin of Helicobacter pylori binds to fucosylated blood group antigens, such as the Lewis(b) antigens in human primate gastric mucosa. We have isolated a human domain antibody specific for BabA that inhibits binding of BabA to Lewis(b) and prevents adhesion of H. pylori to human gastric epithelium. In addition, Lewis(b) oligosaccharides covalently linked to poly-D-lysine inhibited BabA binding to Le(b). The poly-D-lysine-Le(b) hexasaccharide and an Le(b) human serum albumin conjugate not only inhibited adherence of H. pylori to gastric epithelium but also displaced adherent bacteria when added to human stomach sections. Combinations of Le(b) and sialyl Le(x) or domain antibody 25 and sialyl Le(x) acted synergistically. Domain antibody 25 inhibitor may have potential for prophylactic use and, in combination with Le(b) glycoconjugates, therapeutic use in treatment of drug-resistant H. pylori infection.

Orthosiphon stamineus Leaf Extract Protects Against Ethanol-Induced Gastropathy in Rats

Orthosiphon stamineus Benth., which is used as a gastroprotective herbal remedy in Malaysia, was assessed for its anti-ulcerogenic activity against ethanol-induced ulcers in rats. Fifty percent methanol was used to extract the oven-dried O. stamineus leaves. The extract was then lyophilized with a rotary evaporator and freeze-dried. Oral administration of O. stamineus methanolic extract (OSME) (125, 250, 500, and 1,000 mg/kg) was found to significantly decrease the ulcer index (P < .01, P < .001, P < .001, and P < .001, respectively). Histological study of a section of the rat stomach also showed a marked improvement in the gastric mucosal damage in groups receiving OSME. In order to further investigate the gastroprotective mechanism of OSME, mucus secretion and lipid peroxidation level were estimated in vitro and ex vivo. OSME exhibited dose-dependent stimulation of mucus secretion (r = 0.718, P < .001) and inhibition of lipid peroxidation in rat gastric mucosal homogenates (both in vitro [r = 0.819, P < .05] and ex vivo [r = 0.981, P < .05]). It was concluded that the gastroprotective mechanism of OSME was partly due to its ability to inhibit lipid peroxidation and stimulate gastric mucus secretion.

Histologic changes in channel catfish, Ictalurus punctatus Rafinesque, fed diets containing graded levels of gossypol-acetic acid

A study was performed to evaluate the histologic changes among fingerling channel catfish, Ictalurus punctatus Rafinesque, fed purified diets containing gossypol from gossypol–acetic acid. The catfish were maintained on diets with 0, 300, 600, 900, 1200 or 1500 mg gossypol kg−1 diet for 12 weeks, and histologic samples from the stomach, proximal and distal intestines, pancreas, liver, and spleen were obtained from fish in all groups. Stomach sections exhibited significant gastric gland necrosis in fish fed 600 mg gossypol kg−1 diet or higher. Non-existent-to-mild enterocyte vacuolization loss, inflammatory cell infiltration, and hyperplastic lamina propria were noted in the intestinal sections from gossypol-fed fish, but no significant differences in severity scores were noted. The pancreas from fish fed doses of gossypol above 600 mg gossypol kg−1 diet exhibited significant mild-to-severe necrosis, and livers from fish fed 900 and 1500 mg gossypol kg−1 diet exhibited significantly higher pigment deposition. No other significant histologic differences were observed in the fish fed diets containing gossypol–acetic acid. The data in this study indicates that at least 600–900 mg gossypol kg−1 purified diet can cause statistically significant histologic changes in fingerling channel catfish, suggesting that gossypol should remain at concentrations below 600 mg gossypol kg−1 diet.

Colocalization of ghrelinO-acyltransferase and ghrelin in gastric mucosal cells

Ghrelin is a peptide hormone with many known functions, including orexigenic, blood glucose-regulatory, and antidepressant actions, among others. Mature ghrelin is unique in that it is the only known naturally occurring peptide to be posttranslationally modified by O-acylation with octanoate. This acylation is required for many of ghrelin's actions, including its effects on promoting increases in food intake and body weight. GOAT (ghrelin O-acyltransferase), one of 16 members of the MBOAT family of membrane-bound O-acyltransferases, has recently been identified as the enzyme responsible for catalyzing the addition of the octanoyl group to ghrelin. Although the initial reports of GOAT have localized its encoding mRNA to tissues known to contain ghrelin, it is as yet unclear whether the octanoylation occurs within ghrelin-producing cells or in neighboring cells. Here, we have performed dual-label histochemical analysis on mouse stomach sections and quantitative PCR on mRNAs from highly enriched pools of mouse gastric ghrelin cells to demonstrate a high degree of GOAT mRNA expression within ghrelin-producing cells of the gastric oxyntic mucosa. We also demonstrate that GOAT is the only member of the MBOAT family whose expression is highly enriched within gastric ghrelin cells and whose whole body distribution mirrors that of ghrelin.

Characterization of gene expression profiles for different types of mast cells pooled from mouse stomach subregions by an RNA amplification method

BackgroundMast cells (MCs) play pivotal roles in allergy and innate immunity and consist of heterogenous subclasses. However, the molecular basis determining the different characteristics of these multiple MC subclasses remains unclear.ResultsTo approach this, we developed a method of RNA extraction/amplification for intact in vivo MCs pooled from frozen tissue sections, which enabled us to obtain the global gene expression pattern of pooled MCs belonging to the same subclass. MCs were isolated from the submucosa (sMCs) and mucosa (mMCs) of mouse stomach sections, respectively, 15 cells were pooled, and their RNA was extracted, amplified and subjected to microarray analysis. Known marker genes specific for mMCs and sMCs showed expected expression trends, indicating accuracy of the analysis.We identified 1,272 genes showing significantly different expression levels between sMCs and mMCs, and classified them into clusters on the basis of similarity of their expression profiles compared with bone marrow-derived MCs, which are the cultured MCs with so-called 'immature' properties. Among them, we found that several key genes such as Notch4 had sMC-biased expression and Ptgr1 had mMC-biased expression. Furthermore, there is a difference in the expression of several genes including extracellular matrix protein components, adhesion molecules, and cytoskeletal proteins between the two MC subclasses, which may reflect functional adaptation of each MC to the mucosal or submucosal environment in the stomach.ConclusionBy using the method of RNA amplification from pooled intact MCs, we characterized the distinct gene expression profiles of sMCs and mMCs in the mouse stomach. Our findings offer insight into possible unidentified properties specific for each MC subclass.

Molecular characterization of the autochthonous microbiota in the gastrointestinal tract of adult yellow grouper ( Epinephelus awoara) cultured in cages

The present study used 16S rDNA PCR denaturing gradient gel electrophoresis (PCR-DGGE) technology to investigate the communities of autochthonous microbiota in the gastrointestinal (GI) tract; including stomach (ST), pyloric caeca (PC), proximal intestine (PI), mid intestine (MI) and distal intestine (DI) of the adult yellow grouper ( Epinephelus awoara) cultured in cages. Twenty-two DGGE bands were successfully sequenced. However, ten of these bands were classified as unculturable according to the phylogenetic analysis. The dominant autochthonous bacteria in the GI tract belonged to Proteobacteria, but other bacteria identified belonged to Firmicutes, Bacteroidetes, Deinococcus-Thermus, Spirochaetes and unclassified-bacteria. Pantoea sp. and uncultured Proteobacterium were ubiquitous in all five sections of the GI tract. Empedobacter sp. PH7-1 and Acinetobacter sp. N15 were unique for the ST section, while uncultured bacterium clone F6-37 and γ- Proteobacterium and Acinetobacter radioresistens Philippines-11 were only observed in PI, MI and DI sections. Furthermore, Lactococcus lactis, Bacteroides and uncultured Streptococcus sp. were also detected in the present study.

P.430 Changes in MRI flndings of the TMJ after simple irrigation

The present study used 16S rDNA PCR denaturing gradient gel electrophoresis (PCR-DGGE) technology to investigate the communities of autochthonous microbiota in the gastrointestinal (GI) tract; including stomach (ST), pyloric caeca (PC), proximal intestine (PI), mid intestine (MI) and distal intestine (DI) of the adult yellow grouper (Epinephelus awoara) cultured in cages. Twenty-two DGGE bands were successfully sequenced. However, ten of these bands were classified as unculturable according to the phylogenetic analysis. The dominant autochthonous bacteria in the GI tract belonged to Proteobacteria, but other bacteria identified belonged to Firmicutes, Bacteroidetes, Deinococcus-Thermus, Spirochaetes and unclassified-bacteria. Pantoea sp. and uncultured Proteobacterium were ubiquitous in all five sections of the GI tract. Empedobacter sp. PH7-1 and Acinetobacter sp. N15 were unique for the ST section, while uncultured bacterium clone F6-37 and γ-Proteobacterium and Acinetobacter radioresistens Philippines-11 were only observed in PI, MI and DI sections. Furthermore, Lactococcus lactis, Bacteroides and uncultured Streptococcus sp. were also detected in the present study.

Fatal Autoimmune Hepatitis Induced by Concurrent Loss of Naturally Arising Regulatory T Cells and PD-1-Mediated Signaling

Because of the lack of animal models developing spontaneous autoimmune hepatitis (AIH), the molecular mechanisms involved in the development of AIH are still unclear. This study aims to examine the regulatory roles of naturally arising CD4(+)CD25(+) regulatory T (Treg) cells and programmed cell death 1 (PD-1)-mediated signaling in the development of AIH. To induce a concurrent loss of Treg cells and PD-1-mediated signaling, neonatal thymectomy (NTx), which severely reduces the number of Treg cells, was performed on PD-1(-/-) mice. After the NTx, we performed histologic examination, assessed autoantibody production and infiltrating cells in the liver, and conducted adoptive transfer experiments. In contrast to NTx mice and PD-1(-/-) mice, NTx-PD-1(-/-) mice produced antinuclear antibodies and developed fatal hepatitis characterized by a CD4(+) and CD8(+) T-cell infiltration invading the parenchyma with massive lobular necrosis. Induction of AIH in NTx-PD-1(-/-) mice was suppressed by transfer of Treg cells, even derived from PD-1(-/-) mice. Transfer of total but not CD4(+) T-cell-depleted splenocytes from NTx-PD-1(-/-) mice into RAG2(-/-) mice induced the development of severe hepatitis. In contrast, the transfer of CD8(+) T-cell-depleted splenocytes triggered only mononuclear infiltrates without massive necrosis of the parenchyma. NTx-PD-1(-/-) mice are the first mouse model of spontaneous fatal AIH. The concurrent loss of Treg cells and PD-1-mediated signaling can induce the development of fatal AIH. Autoreactive CD4(+) T cells are essential for induction of AIH, whereas CD8(+) T cells play an important role in progression to fatal hepatic damage.

Helicobacter pylori invades the gastric mucosa and translocates to the gastric lymph nodes

Helicobacter pylori has been considered to be non-invasive and to rarely infiltrate the gastric mucosa, even though there is an active Th1 immune response in the lamina propria of the H. pylori-infected stomach. To elucidate whether H. pylori invades the lamina propria and translocates to the gastric lymph nodes, we examined H. pylori in formalin-fixed and paraffin-embedded tissue sections of stomach and gastric lymph nodes obtained from 51 cancer patients using real-time PCR and immunohistochemistry (IHC) with a novel anti-H. pylori monoclonal antibody that recognizes lipopolysaccharides. Fresh gastric lymph nodes were used to culture for H. pylori. In 46 patients with H. pylori in the stomach, the bacterium was found in the lymph nodes from 21 patients by culture, 37 patients by PCR, and 29 patients by IHC. H. pylori captured by macrophages was found in the lamina propria of 39 patients. In the lymph nodes, the bacterium was found in many macrophages and a few interdigitating dendritic cells at the paracortical areas. H. pylori was also found in the intracellular canaliculi of parietal cells in 21 patients, but intracytoplasmic invasion into gastric epithelial cells was not identified. When compared to the commercially available anti-H. pylori antibodies, the novel antibody showed the highest sensitivity to detect H. pylori-positive macrophages, whereas no difference was found for H. pylori in the mucous layer. The H. pylori-positive macrophages in the lamina propria correlated with chronic gastritis as well as translocation of such cells to the lymph nodes. These results suggest that H. pylori-induced gastric epithelial damage allows the bacteria to invade the lamina propria and translocate to the gastric lymph nodes, which may chronically stimulate the immune system. The bacteria captured by macrophages, whether remaining alive or not, may contribute to the induction and development of H. pylori-induced chronic gastritis.

Sonic hedgehog is associated with H+-K+-ATPase-containing membranes in gastric parietal cells and secreted with histamine stimulation

Sonic hedgehog (Shh) is found within gastric parietal cells and processed from a 45-kDa to a 19-kDa bioactive protein by an acid- and protease-dependent mechanism. To investigate whether Shh is associated with the parietal cell membrane compartment that becomes exposed to both acid and proteolytic enzymes during acid secretion, the cellular location of Shh within resting and stimulated gastric parietal cells was examined. Immunofluorescence microscopy of rabbit stomach sections showed that Shh colocalized predominantly with parietal and pit, not chief/zymogen or neck, cell markers. In resting and histamine-stimulated rabbit gastric glands Shh was expressed only in parietal cells close to H+-K+-ATPase-containing tubulovesicular and secretory membranes with some colocalizing with gamma-actin at the basolateral membrane. Gastric gland microsomal membranes were prepared by differential and sucrose gradient centrifugation and immunoisolation with an anti-H+-K+-ATPase-alpha subunit antibody. The 45- and 19-kDa Shh proteins were detected by immunoblot in immunopurified H+-K+-ATPase-containing membranes from resting and stimulated gastric glands, respectively. Incubating glands with a high KCl concentration removed Shh from the membranes. Histamine stimulated 19-kDa Shh secretion from gastric glands into the medium. In human gastric cancer 23132/87 cells cultured on permeable membranes, histamine increased 19-kDa Shh secretion into both apical and basolateral media. These findings show that Shh is a peripheral protein associated with resting and stimulated H+-K+-ATPase-expressing membranes. In addition, Shh appears to be expressed at or close to the basolateral membrane of parietal cells.

Emergence of spasmolytic polypeptide-expressing metaplasia in Mongolian gerbils infected with Helicobacter pylori

Spasmolytic polypeptide (TFF2)-expressing metaplasia (SPEM) is observed in mucosa adjacent to human gastric cancer and in fundic glands showing oxyntic atrophy in Helicobacter felis-infected mice. Mongolian gerbils infected with Helicobacter pylori (Hp) develop goblet cell intestinal metaplasia and adenocarcinoma, but the presence of SPEM has not been studied in gerbils. We therefore have sought to examine the development of metaplastic mucosal changes in Hp-infected Mongolian gerbils. Mongolian gerbils were assigned to either uninfected controls or infected with Hp at 17 weeks of age. The animals were killed at 17, 20, 26, 31, 41 and 56 weeks of age. Stomach sections were stained using antibodies for TFF2, intrinsic factor, H/K-ATPase, BrdU and MUC2. Dual immunofluorescence staining for TFF2 with intrinsic factor and for TFF2 with MUC2 was performed. In uninfected animals, no SPEM or intestinal metaplasia was observed. Infected gerbils developed SPEM initially in the intermediate zone along the lesser curvature and subsequently spread out towards the greater curvature. In the earlier stages of infection, SPEM glands demonstrated TFF2 and intrinsic factor double staining cells. However, after 35 weeks of infection, the number of double staining SPEM cells decreased. While early in infection SPEM organized in straight glands, in the later stages of infections, SPEM glands became distorted or dilated along with the development of gastritis cystica profunda that was TFF2 positive. Goblet cell intestinal metaplasia developed only late in the infection. Dual staining for TFF2 and MUC2 showed glands containing both SPEM- and MUC2-positive goblet cell intestinal metaplasia. SPEM develops early in Hp infection in Mongolian gerbils, and alterations in gland morphology arise from SPEM glands during the course of gastric infection with goblet cell intestinal metaplasia developing subsequent to SPEM.

Experimental infection of Haemonchus contortus strains resistant and susceptible to benzimidazoles and the effect on mast cells distribution in the stomach of Mongolian gerbils (Meriones unguiculatus)

Establishment rate of Haemonchus contortus in non-suppressed and immunosuppressed gerbils within 14 days post-infection was compared after inoculation with 1,000 third-stage larvae (L3), exsheathed BZ-susceptible larvae. Based on significantly higher number of larvae in gerbils receiving low doses of immunosuppressant agent hydrocortisone, development of benzimidazole (BZ)-susceptible and BZ-resistant strain of nematode in the stomach was studied on days 4, 7, 10, and 14 p.i. Sections of stomach from both groups of animals were examined for overall histopathological response and dynamics of mucosal mast cells (MMC) and connective tissue mast cells (CTMC). In the immunosuppressed gerbils, H. contortus L3 stage larvae developed to the L4 stage on days 10 and 14 p.i., and their sex ratio was higher toward female worms. Significantly higher ratios of establishment rate were recorded for BZ-susceptible than BZ-resistant strain. Infection elicited strong inflammation mainly in the lamina propria mucosae, where MMC numbers peaked on day 7 p.i., being present in a significantly higher numbers in gerbils infected with BZ-susceptible strain. Infection with BZ-susceptible strain of nematode also resulted in a higher number of CTMC in comparison with the effect of BZ-resistant strain, which were observed in the tela submucosa only. Thus, H. contortus infection in gerbils seems to be a suitable model to study host-parasite interactions. Our results indicate that BZ-resistant strain of H. contortus have a decreased capacity to establish infection in direct relation with lower mucosal and connective tissue MCs counts in the stomach.

Chronic caloric restriction induces forestomach hypertrophy with enhanced ghrelin levels during aging

Caloric restriction (CR) is the only preventive intervention that has robust pro-longevity effects in experimental models. Various circulating hormones that regulate the state of negative energy balance may drive the multi-system beneficial effects of the CR phenomenon. Ghrelin, one such stomach-derived circulating peptide hormone stimulates food intake, promotes GH release and inhibits pro-inflammatory cytokines. We have recently demonstrated that ghrelin also reverses age-related thymic involution. Here, we report that chronic CR in aging mice results in reduction in body weight, and spleen size but remarkably, leads to a significant increase in the size and weight of stomach. The increased size of stomach was largely due to increased size of fundus (forestomach) and also smaller but statistically significant enlargement of antrum. The analysis of serial stomach sections revealed that chronic CR leads to a striking hypertrophy of lamina propria, stratum basale, stratum corneum and the stratified squamous epithelium of forestomach of the aged animals. We also report for the first time that chronic CR during aging significantly increases circulating ghrelin levels as well as total ghrelin production in the stomach and reverses age-related loss of ghrelin receptor expression in pituitary. Our data suggests that long-term CR-induced increased ghrelin production from hypertrophic stomach in mice may be an adaptive survival strategy in response to sustained negative energy balance that triggers heightened state of food seeking. Taken together, these data provide new insights into the underlying mechanism behind the salutary effects of chronic caloric restriction during aging process.

Gene expression analysis of multiple gastrointestinal regions reveals activation of common cell regulatory pathways following cytotoxic chemotherapy

Gastrointestinal mucositis involves many changes at the gene level, affecting epithelial/subepithelial interactions and leading to overt damage. The regional specificity and time course of these changes, and how they relate to subsequent mucositis development however remain unknown. The aim of this study was to determine the early time course of gene expression changes along the gastrointestinal tract of the DA rat following chemotherapy. Female DA rats were treated with a single dose of 200 mg/kg irinotecan to induce mucositis, and were killed at short intervals following treatment. Small sections of stomach, jejunum and colon were harvested for analysis of genetic profiles. RNA was hybridised to high density Affymetrix oligonucleotide microarrays. Data analysis was carried out with software package, TimeCourse, freely available through Bioconductor. As early as 1 hr following chemotherapy, expression of hundreds of genes was altered, including those for transcription factors, stress response proteins and protein turnover. These genes are involved in cell proliferation, differentiation and apoptosis along with other cellular processes. At early time points, there was a significant response involving the mitogen-activated protein kinase pathway, cell cycle regulation and cytokine receptor signalling. At later time points, changes to the complement cascade became prominent. We have shown that changes in gene expression following chemotherapy occur by 1 hr, and persist for at least 72 hr after treatment. Many of these changes are highly likely to be specifically related to the subsequent development of gastrointestinal mucositis.

Selected pathological, immunohistochemical and ultrastructural changes associated with an infection by Diphyllobothrium dendriticum (Nitzsch, 1824) (Cestoda) plerocercoids in Coregonus lavaretus (L.) (Coregonidae)

The pathological changes induced by an infection of Diphyllobothrium dendriticum (Nitzsch, 1824) plerocercoids in powan, Coregonus lavaretus (L.), from Loch Lomond, Scotland, were assessed using immunohistochemical and ultrastructural techniques. In a sample of 26 powan, the occurrence of encysted plerocercoids of D. dendriticum on the outer surface of the stomach was 38.5% (n = 10) with the number of cysts ranging from 4 to 15 and measuring 4.2 +/- 1.0 mm x 3.4 +/- 0.9 mm (mean +/- SD). Histological examination of intestinal samples also revealed plerocercoids (2-21) encapsulated within a proliferation of mesenteric fibrous tissues of the gastric wall and, occasionally, by the gut lamina propria-submucosa and lamina muscularis. In section, cysts were tri-layered and were formed from a series of concentric whorls of fibroblast and collagen fibre-based connective elements. The extent of necrosis within each muscle layer and the serosa of the stomach differed, notably within the latter that was marked by a chronic inflammatory reaction and fibrosis. Within the cyst and around it, a large number of degranulating mast cell/eosinophilic granule cells were seen, in addition to melano-macrophage centres. Immunohistochemical staining of sections of infected stomach revealed a high density of elements, in close proximity to plerocercoids, staining positive for serotonin, bombesin, substance P and galanin. Uninfected material did not present the same levels of activity. Sections through both infected and uninfected tissue were also tested for elements containing vasoactive intestinal peptide, met-enkephalin, calcitonin gene-related peptide, neuropeptide Y and nitric oxide synthase, but these were absent.

MICE LACKING THE GENE FOR PEPSINOGEN F, A MARKER OF YOLK SAC AND GASTRIC ENDODERM, EXHIBIT LOW BIRTH WEIGHTS AND REMAIN SMALL AS NEONATES

Pepsinogen F (pepF) is an aspartic peptidase (AP) that is evolutionarily related to the placental Pregnancy-Associated Glycoproteins (PAGs) of ruminants and swine. Unlike the PAGs, pepF is widely distributed in eutherians, having been identified in four orders [horse (Perissodactyla), cat (Canivora), rabbit (Lagomorpha), rats and mice (Rodentia)]. The PAGs are products of the trophoblast. However, mouse pepF has been shown previously to have restricted expression in the parietal and visceral yolk sac (YS) and the neonatal stomach. The presence of pepF in the YS and the stomach of newborn pups suggested that pepF has specialized digestive functions that are vital to fetal and/or neonatal development of mice. To better define the physiological role of pepF, a mouse line was established that lacked a functional pepF gene. A Neomycin/Thymidine Kinase targeting vector was generated that allowed the elimination of exons 3–5 of the pepF gene by homologous recombination. This region of the gene encodes for parts of the protein responsible for stabilizing substrates in the binding cleft. It also contains one of the catalytic aspartates required in the catalytic mechanism. The loss of the gene product in homozygous null mice (−/−) was verified by immuno-histochemistry on sections of stomach and extraembryonic membranes. Those mice lacking pepF possessed no obvious morphological changes in the cellular architecture of the yolk sac or the glandular regions of the stomach. However, the absence of pepF in null pups did result in significantly lower birth weights as well as a significant retardation of weight gains during the first week after birth. For example, the −/− pups were 36% smaller than +/− and +/+ pups at day 7 after birth (p<0.001). There was no significant deviation between heterozygous and wild type mice. Although, there was recovery in the weights of the null pups during the second and third week, the difference in relative weights compared to wild type and heterozygous littermates was still significant at the time of weaning. The −/− pups were 27.8% smaller than +/− and +/+ pups on Day 21 (p<0.001). Finally, there was no skewing in the genotypes of pups from heterozygous crosses (i.e. each genotype was represented in a normal Mendelian distribution). There was no apparent loss of fertility in null mice, and the average litter size in null matings was comparable to wildtype matings. However, mice lacking pepF made up for a significant proportion of those pups that died during the first week after birth. Based on these results, it can be concluded that pepF plays an important role in fetal and early neonatal growth, but is not absolutely required for survival. These experiments were supported by a grant from the MU Research Board. (platform)