Mouse Stomach Research Articles

Helicobacter pylori Uses the TlpB Receptor To Sense Sites of Gastric Injury

Helicobacter pylori is a pathogen that chronically colonizes the stomachs of approximately half of the world's population and contributes to the development of gastric inflammation. We demonstrated previously in vivo that H. pylori uses motility to preferentially colonize injury sites in the mouse stomach. However, the chemoreceptor responsible for sensing gastric injury has not yet been identified. In this study, we utilized murine gastric organoids (gastroids) and mutant H. pylori strains to investigate the components necessary for H. pylori chemotaxis. High-intensity 730-nm light (two-photon photodamage) was used to cause single-cell damage in gastroids, and repair of the damage was monitored over time; complete repair occurred within ∼10 min in uninfected gastroids. Wild-type H. pylori accumulated at the damage site after gastric damage induction. In contrast, mutants lacking motility (ΔmotB) or chemotaxis (ΔcheY) did not accumulate at the injury site. Using mutants lacking individual chemoreceptors, we found that only TlpB was required for H. pylori accumulation, while TlpA, TlpC, and TlpD were dispensable. All strains that were able to accumulate at the damage site limited repair. When urea (an identified chemoattractant sensed by TlpB) was microinjected into the gastroid lumen, it prevented the accumulation of H. pylori at damage sites. Overall, our findings demonstrate that H. pylori colonizes and limits repair at damage sites via chemotactic motility that requires the TlpB chemoreceptor to sense signals generated by gastric epithelial cells.

Probiotics Upregulate Trefoil Factors and Downregulate Pepsinogen in the Mouse Stomach.

Probiotics are used in the management of some gastrointestinal diseases. However, little is known about their effects on normal gastric epithelial biology. The aim of this study was to explore how the probiotic mixture VSL#3 affects gastric cell lineages in mice with a special focus on protective and aggressive factors. Weight-matching littermate male mice (n = 14) were divided into treated and control pairs. The treated mice received VSL#3 (5 mg/day/mouse) by gastric gavage for 10 days. Control mice received only the vehicle. Food consumption and bodyweight were monitored. All mice were injected intraperitoneally with bromodeoxyuridine (120 mg/Kg bodyweight) two hours before sacrificed to label S-phase cells. Stomach tissues were processed for lectin- and immunohistochemical examination. ImageJ software was used to quantify immunolabeled gastric epithelial cells. Real-time quantitative polymerase chain reaction was used to provide relative changes in expression of gastric cell lineages specific genes. Results revealed that treated mice acquired (i) increased production of mucus, trefoil factor (TFF) 1 and TFF2, (ii) decreased production of pepsinogen, and (iii) increased ghrelin-secreting cells. No significant changes were observed in bodyweight, food consumption, cell proliferation, or parietal cells. Therefore, VSL#3 administration amplifies specific cell types specialized in the protection of the gastric epithelium.

Study of toxicness of snails meat on biological model

Snails can be found in any part of the planet. They belong to the class of gastropods, which includes terrestrial, freshwater, marine snails. Their ancestors are some of the most ancient species of the fauna. Archaeologists say that the first lived on our planet 500 million years ago. There are about 90000 species of snails, but only 3 species have gained worldwide fame: gigantic African snail, grape and Helix аspersa. Snail’s meat improves bowel function and normalizes metabolism. It is believed that due to the content of magnesium, snail meat has calming effect on humans and helps to fight stress. To determine the level of toxicity of food snails meat on a biological model. Helix pomatia snails were collected in wet weather, after rain or in the morning, Helix aspersa maxima and Helix aspersa muller were recieved from the farm “SNAIL 2016” (Ukraine). As a model we used white mise. To determine the toxicity, the sampled meat preparation has been conducted. For this purpose an average sample of meat of each snail types of 10.0 g, carefully chopped. The milled sample of 5.0 g was introduced into a sterile glass, poured 50.0 cm3 of distilled water, stirred and filtered. There were formed experimental 6 and 2 control groups of mice were formed by six in each one, of 2 months of age (body weight 20 g). Subsequently, prepared samples of snails meat were injected into the mice stomach through a probe every day for 3 days by 0.5 cm3. As a probe, a thin, single-use micropipette was used. The white mice of each group experimental of all groups were placed in isolated cages and clinically monitored for five days. The mice didn’t recieved food, animals had free access to water. Mice of the control groups had free access to food. The meat of each type of snails in the experiments was used both fresh and after 7 days storage refrigerator at a temperature of 4 °С. It was found that filtrates from fresh meat of the three types of food snails Helix aspersa maxima, Helix aspersa muller and Helix pomatia were non-toxic, for the mice of I, II and III groups. We have noticed that the meat that was stored for 7 days in the refrigerator at a temperature of 4 °C was toxic. Mice of the V–VII groups that were fed such meat had a depressed state, muscle tremor, they refused of water and died on 2nd and 3rd days. The mise of control groups (IV and VIII) remained during all experience remained living. This work can be used as a method for determining the toxicity of snail’s meat. It is technologically simple, it requires only laboratory animals, namely white mice, does not require special equipment. Only 3 days can get the result. The method can be used in the work of scientific-experimental, production and regional laboratories of veterinary medicine.

Abstract 48: Cellular dynamics and heterogeneity of active Kras induced-gastric pre-neoplasia in mouse

Abstract Cellular heterogeneity in cancer development is considered as a source of cancer cell generation and drug resistance. However, cellular heterogeneity has not been fully eluciated during metaplasia progression to gastric pre-neoplasia and neoplasia in gastric carcinogenesis. Recent investigations have suggested that Ras activation, rather than Ras mutation, may promote the gastric carcinogenesis. We have recently established several in vitro metaplastic/pre-neoplastic organoid lines; Meta3-metaplasia and Meta4-pre-neoplasia, derived from mouse stomachs induced active Kras expression in chief cells and investigated cellular heterogeneity and plasticity that may lead to the progression of metaplasia to gastric neoplasia. While both Meta3 and Meta4 initially formed spherical or spheroidal structures and generated complicated budding or cyst-like structures between 2 and 4 weeks, Meta4 cells displayed more disorganized structures and aggressive behaviors than Meta3. We also examined altered cellular behaviors, structural changes and survival of Meta4 organoids after MEK inhibition which is a downstream mediator of Kras signaling pathway. The Meta4 treated with Selumetinib for 3 days died or did not show an increase in size at various concentrations. However, some Meta4 organoids showed an ability to survive and retained a spheroidal structure despite the Selumetinib treatment. Single cell-RNA sequencing (scRNA-seq) data analysis demonstrated distinct characteristics of Meta4 cells as pre-neoplastic cells and a signature of presence and heterogeneity of pre-neoplasia stem cell (PNSC) populations. The scRNA-seq data analysis also revealed that cellular behavior and dynamics of pre-neoplastic cells can be controlled by MEK inhibition, but the PNSC populations still maintain the niche for stem-like cells in gastric pre-neoplasia. The isolated two PNSC populations showed functional differences and the stemness of the PNSCs in pre-neoplastic stage through high-throughput functional analysis. Therefore, our study concludes that putative gastric cancer stem cells in gastric pre-neoplasia might be the source for maintenance of heterogeneity and contribute to the progression of gastric pre-neoplasia to cancer. Citation Format: Eunyoung Choi, Paige N. Vega, Jimin Min, Amy C. Engevik, janice A. Williams, Qing Yang, Loraine M. Patterson, Alan J. Simmons, Ken S. Lau, Scott T. Magness, James R. Goldenring. Cellular dynamics and heterogeneity of active Kras induced-gastric pre-neoplasia in mouse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 48.

Oral administration of heat-killed Lactobacillus brevis SBC8803 elevates the ratio of acyl/des-acyl ghrelin in blood and increases short-term food intake

It is known that gastrointestinal microbiota, probiotics and heat-killed microbes can regulate intestinal immunity; however, their effect on the secretion of gastrointestinal hormones is unclear. The secretion of gastrointestinal hormones can be mediated by the elevation of intracellular Ca2+ concentration, suggesting that these hormones may act through common mechanisms. We have previously shown that heat-killed Lactobacillus brevis SBC8803 (hk-SBC8803) induced the secretion of serotonin and elevated intracellular Ca2+ concentration in serotonin-producing RIN-14B cells, suggesting that hk-SBC8803 could potentially cause the same effect on other gastrointestinal hormones, including hunger hormone ghrelin. Here, we tested this hypothesis by treating cultured cells and experimental animals with hk-SBC8803 and assessing ghrelin secretion, expression of ghrelin-related genes, and food intake. The results indicated that hk-SBC8803 treatment for 30 min significantly upregulated the secretion of acyl ghrelin (active form) (P=0.046) and mRNA expression of the Syt3 (synaptotagmin 3) gene related to ghrelin exocytosis (P<0.05) in primary mouse stomach cells. In addition, oral administration of 500 mg/kg hk-SBC8803 to rats tended to upregulate acyl ghrelin concentration (P=0.10) and significantly increased the ratio of acyl to des-acyl (inactive) ghrelin (P=0.027) in blood, which corresponded to a tendency of stimulating food intake (P=0.087) at 30 min post-treatment. However, when in order to minimise individual differences we normalised the data on food intake to those on one-day food intake prior to food deprivation, the resultant food intake ratio showed a significant increase (by 5% compared to control; P=0.032) at 30 min after hk-SBC8803 treatment, indicating that hk-SBC8803 administration stimulated rats to take more food during the first meal after fasting. These results suggest that hk-SBC8803 induces short-term ghrelin secretion and transiently increases appetite, which is an important effect for individuals with low energy intake.

Targeting CLDN18.2 by CD3 Bispecific and ADC Modalities for the Treatments of Gastric and Pancreatic Cancer

Human CLDN18.2 is highly expressed in a significant proportion of gastric and pancreatic adenocarcinomas, while normal tissue expression is limited to the epithelium of the stomach. The restricted expression makes it a potential drug target for the treatment of gastric and pancreatic adenocarcinoma, as evidenced by efforts to target CLDN18.2 via naked antibody and CAR-T modalities. Herein we describe CLDN18.2-targeting via a CD3-bispecific and an antibody drug conjugate and the characterization of these potential therapeutic molecules in efficacy and preliminary toxicity studies. Anti-hCLDN18.2 ADC, CD3-bispecific and diabody, targeting a protein sequence conserved in rat, mouse and monkey, exhibited in vitro cytotoxicity in BxPC3/hCLDN18.2 (IC50 = 1.52, 2.03, and 0.86 nM) and KATO-III/hCLDN18.2 (IC50 = 1.60, 0.71, and 0.07 nM) respectively and inhibited tumor growth of pancreatic and gastric patient-derived xenograft tumors. In a rat exploratory toxicity study, the ADC was tolerated up to 10 mg/kg. In a preliminary assessment of tolerability, the anti-CLDN18.2 diabody (0.34 mg/kg) did not produce obvious signs of toxicity in the stomach of NSG mice 4 weeks after dosing. Taken together, our data indicate that targeting CLDN18.2 with an ADC or bispecific modality could be a valid therapeutic approach for the treatment of gastric and pancreatic cancer.

Gastric Leptin and Tumorigenesis: Beyond Obesity

Leptin, an adipocyte-derived hormone and its receptor (ObR) expressed in the hypothalamus are well known as an essential regulator of appetite and energy expenditure. Obesity induces abundant leptin production, however, reduced sensitivity to leptin leads to the development of metabolic disorders, so called leptin resistance. The stomach has been identified as an organ that simultaneously expresses leptin and ObR. Accumulating evidence has shown gastric leptin to perform diverse functions, such as those in nutrient absorption and carcinogenesis in the gastrointestinal system, independent of its well-known role in appetite regulation and obesity. Overexpression of leptin and phosphorylated ObR is implicated in gastric cancer in humans and in murine model, and diet-induced obesity causes precancerous lesions in the stomach in mice. While the underlying pathomechanisms remain unclear, leptin signaling can affect gastric mucosal milieu. In this review, we focus on the significant role of the gastric leptin signaling in neoplasia and tumorigenesis in stomach in the context of hereditary and diet-induced obesity.

Pharmaceutically modified subtilisins withstand acidic conditions and effectively degrade gluten in vivo

Detoxification of gluten immunogenic epitopes is a promising strategy for the treatment of celiac disease. Our previous studies have shown that these epitopes can be degraded in vitro by subtilisin enzymes derived from Rothia mucilaginosa, a natural microbial colonizer of the oral cavity. The challenge is that the enzyme is not optimally active under acidic conditions as encountered in the stomach. We therefore aimed to protect and maintain subtilisin-A enzyme activity by exploring two pharmaceutical modification techniques: PEGylation and Polylactic glycolic acid (PLGA) microencapsulation. PEGylation of subtilisin-A (Sub-A) was performed by attaching methoxypolyethylene glycol (mPEG, 5 kDa). The PEGylation protected subtilisin-A from autolysis at neutral pH. The PEGylated Sub-A (Sub-A-mPEG) was further encapsulated by PLGA. The microencapsulated Sub-A-mPEG-PLGA showed significantly increased protection against acid exposure in vitro. In vivo, gluten immunogenic epitopes were decreased by 60% in the stomach of mice fed with chow containing Sub-A-mPEG-PLGA (0.2 mg Sub-A/g chow) (n = 9) compared to 31.9% in mice fed with chow containing unmodified Sub-A (n = 9). These results show that the developed pharmaceutical modification can protect Sub-A from auto-digestion as well as from acid inactivation, thus rendering the enzyme more effective for applications in vivo.

Expression of nutrient sensing mechanisms in the mouse stomach

Expression of nutrient sensing mechanisms in the mouse stomach

Oral immunization of BALB/c mice with Helicobacter pylori lysate combined with dmLT adjuvant for the induction of mucosal and system immune responses

Objective To observe the protective effects of oral immunization with Helicobacter pylori (Hp) lysates in combination with mucosal adjuvant dmLT (double mutant heat-labile toxin) against Hp infection in a BALB/c mouse model and to analyze the features of induced immune responses. Methods BALB/c mice were orally immunized with Hp lysate (Sydney strain 1, SS1 strain) and dmLT adjuvant, and then innoculated with live Hp strains through oral gavage. A control group was set up by oral administration of normal saline (200 μl/mouse). The colonization of Hp strains in the stomachs of mice was measured six weeks after bacterial inoculation. Samples of serum, spleen, mesenteric lymph node (MLN), small intestine, cecum and feces were collected from mice to analyze the features of induced immune responses. Results The colonization of Hp strains in the stomachs of the immunized mice was significantly decreased as compared with that of the control group. Increased specific IgG antibody responses which were predominantly of IgG1 subtype were detected in the serum samples of the immunized mice and the IgG1/IgG2a ratio was significantly higher than that of the control group. Elevated secretory IgA (sIgA) was detected in the samples of small intestine, cecum and feces in the immunization group, especially in the small intestine samples, while no significant change in sIgA secretion was observed in the control group. The percentages of IL-17+ CD4+ T cells in spleen and mesenteric lymph nodes of the immunization group were significantly higher than those of the control group. Conclusions Oral immunization with Hp lysates in combination with adjuvant dmLT induced mucosal and systemic immune responses and enhanced the resistance to Hp colonization in BALB/c mice, which was associated with the significantly increased Th17 immune responses and Th2 polarization. This study provided reference for further evaluation of dmLT as a mucosal adjuvant in the development of recombinant protein vaccines against Hp infection. Key words: Sydney strain 1 lysate; Double mutant heat-labile toxin (dmLT); BALB/c mouse; Immune response; Helicobacter pylori vaccine

Chronic exposure to 35% carbamide peroxide tooth bleaching agent induces histological and hematological alterations, oxidative stress, and inflammation in mice.

Previous studies have demonstrated the side effects of tooth whiteners on the gastric mucosa. However, the impact of dental bleaching products on the liver, kidney, and heart remains obscure. The present study investigated the toxic potential of 35% carbamide peroxide (CPO) containing tooth whitening product (TWP) on the liver, kidney, heart, and stomach of mice, pointing to the role of oxidative stress and inflammation. Mice received 250 or 500mg/kg body weight CPO-TWP orally for 3weeks and samples were collected for analyses. Both doses of CPO-TWP induced a significant increase in circulating liver, kidney, and heart function markers. CPO-TWP-administered mice showed several histological alterations and a significant increase in liver, kidney, heart, and stomach lipid peroxidation levels along with diminished glutathione, superoxide dismutase, and catalase. In addition, administration of CPO-TWP provoked anemia, leukocytosis, and a significant increase in circulating levels of pro-inflammatory cytokines. In conclusion, exposure to 35% CPO-TWP induced functional, histological, and hematological alterations, oxidative stress, and inflammation in mice. Therefore, the frequent use of tooth bleaching agents should be monitored very carefully to avoid the application of excess amounts as well as the intake.

Preventive Effect of Anji White Tea Flavonoids on Alcohol-Induced Gastric Injury through Their Antioxidant Effects in Kunming Mice.

Anji white tea (Camellia sinensis) is a traditional Chinese tea beverage, which is classified as green tea and contains an abundant amount of flavonoids. In this study, the preventive effect of Anji white tea flavonoids (AJWTFs) on ethanol/hydrochloric acid-induced gastric injury in mice was evaluated. The serum and gastric tissues of mice were analyzed using a biochemical kit and by quantitative polymerase chain reaction (qPCR). Observation of the appearance of the stomach indicated that AJWTFs could effectively reduce the area of gastric injury caused by ethanol/hydrochloric acid, and the inhibition rate of AJWTF on gastric injury increased with an increase in AJWTF concentration. The Anji white tea flavonoids could also reduce the volume and pH of gastric juice in mice with gastric injury. Biochemical results showed that AJWTFs could increase the superoxide dismutase (SOD) and glutathione (GSH) activities, as well as decrease the malondialdehyde (MDA) level, in the serum and liver of mice with gastric injury. Pathological observation confirmed that AJWTFs could inhibit the tissue damage caused by ethanol/hydrochloric acid in the stomach of mice. Further qPCR experiments also showed that AJWTFs could inhibit the decreases in neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), copper/zinc superoxide dismutase (Cu/Zn–SOD), manganese superoxide dismutase (Mn–SOD), catalase (CAT), and the increase in inducible nitric oxide synthase (iNOS) expression in the gastric tissue of mice caused by gastric injury. As observed, AJWTFs exerted a good preventive effect on alcohol-induced gastric injury in mice induced by ethanol/hydrochloric acid, and the effect is close to that of ranitidine. Anji white tea flavonoids present good antioxidant effect, which allows them to effectively prevent alcoholic gastric injury and be used as biologically active substances with a broad range of applications.

Biodistribution of Liposome-Encapsulated Bacteriophages and Their Transcytosis During Oral Phage Therapy.

This study sheds light on the biodistribution of orally administered, liposome-encapsulated bacteriophages, and their transcytosis through intestinal cell layers. Fluorochrome-labeled bacteriophages were used together with a non-invasive imaging methodology in the in vivo visualization of bacteriophages in the stomach and intestinal tract of mice. In those studies, phage encapsulation resulted in a significant increase of the labeled phages in the mouse stomach, even 6 h after their oral administration, and without a decrease in their concentration. By contrast, the visualization of encapsulated and non-encapsulated phages in the intestine were similar. Our in vivo observations were corroborated by culture methods and ex vivo experiments, which also showed that the percentage of encapsulated phages in the stomach remained constant (50%) compared to the amount of initially administered product. However, the use of conventional microbiological methods, which employ bile salts to break down liposomes, prevented the detection of encapsulated phages in the intestine. The ex vivo data showed a higher concentration of non-encapsulated than encapsulated phages in liver, kidney, and even muscle up to 6 h post-administration. Encapsulated bacteriophages were able to reach the liver, spleen, and muscle, with values of 38% ± 6.3%, 68% ± 8.6%, and 47% ± 7.4%, respectively, which persisted over the course of the experiment. Confocal laser scanning microscopy of an in vitro co-culture of human Caco-2/HT29/Raji-B cells revealed that Vybrant-Dil-stained liposomes containing labeled bacteriophages were preferably embedded in cell membranes. No transcytosis of encapsulated phages was detected in this in vitro model, whereas SYBR-gold-labeled non-encapsulated bacteriophages were able to cross the membrane. Our work demonstrates the prolonged persistence of liposome-encapsulated phages in the stomach and their adherence to the intestinal membrane. These observations could explain the greater long-term efficacy of phage therapy using liposome-encapsulated phages.

The impact of cyclooxygenase‐2 selective and non‐isoform selective NSAIDs on the gut microbiota

Nonsteroidal anti‐inflammatory drugs (NSAIDs) exert their analgesic and anti‐inflammatory effects by inhibiting the cyclooxygenase (COX)‐1 and/or COX‐2, enzymes involved in biotransformation of arachidonic acid into prostanoids. It was previously reported that some NSAIDs alter populations of bacteria in the gut and modify the way that NSAIDs work in the body. Here we directly compared the impact of NSAIDs, selective (celecoxib) and non‐selective (naproxen) for COX‐2 inhibition, on the composition of the gut microbiota in mice. We conducted a longitudinal study in adult male C57BL/6 mice that were fed ad libitum with a regular chow or with a diet containing celecoxib or naproxen for three weeks. Naproxen caused significant COX‐1 inhibition as reflected by a ~70% inhibition of urinary thromboxane metabolite (TxM), an in vivo index of COX‐1 activity, and a 60% inhibition of a COX‐1/COX‐2 urinary prostaglandin (PG)E2 metabolite (PGEM), as measured by LC‐MS/MS. The treatment with celecoxib caused a significant 40% reduction of PGEM, but had no significant effect on TxM. Fecal samples were collected on day zero and day 21, DNA purified, 16S rRNA gene segments amplified using V1V2 primers, and amplicons sequenced. Community structure was compared using UniFrac, which generates distances between all pairs of samples as shared distances on a common phylogenetic tree, allowing assessment of clustering and gradients in community composition. For the unweighted analysis, the control group did not show a significant change over time (p=0.34; Permutational Multivariate Analysis of Variance Using Distance Matrices (‘Adonis’ function in R)), while a strong change was seen for naproxen (p=0.007; Adonis), and a lesser change was seen for celecoxib (p=0.023; Adonis). For the weighted analysis, the control group again did not change (p=0.31; Adonis), naproxen showed a trend (p= 0.089; Adonis), and no change was seen with celecoxib (p= 0.23; Adonis). Analysis of specific lineages showed strong longitudinal changes in some sequence clusters (operational taxonomic units), including Bacteroidetes S24‐7 and Clostridiales both p values &lt; 10−4). Moreover, we observed that naproxen caused gastroenteropathy in mice. The stomach of naproxen‐treated mice showed pyloric mucosal erosion and ulcers while the small intestine showed ulceration, inflammation in the lamina propria and epithelium, and thickening and blunting of the villi. Significantly fewer and less severe lesions were observed in mice treated with celecoxib both in the stomach and in the intestine. Thus, both NSAIDs have an impact on the composition of the gut microbiota, with the stronger effects seen for naproxen versus celecoxib. Effects were most evident in the unweighted analysis, indicating a greater effect on community membership than on their relative proportions. These data are consistent with the hypothesis that microbial dysbiosis is involved in the NSAID‐induced enteropathy that is more common with non‐isoform selective than COX‐2 selective NSAIDs.Support or Funding InformationThis work was supported by NIH grant U54 HL117798 to Garret A. FitzGerald.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sonic Hedgehog Acts as a Macrophage Chemoattractant During Gastric Epithelial Regeneration in Response to Injury

Background/HypothesisHedgehog (Hh) signaling not only plays a crucial role in embryonic development, but also controls gastric physiological events including epithelial cell differentiation, regeneration and acid secretion. Sonic Hedgehog (Shh), is secreted from gastric parietal cells in response to injury and contributes to the regeneration of the injured epithelium. Following injury, the recruitment of macrophages plays crucial role both in the regenerative process. The mechanism that regulates macrophage recruitment in response to injury is largely unknown. Thus, here we tested the hypothesis thatShh stimulates macrophage chemotaxis to the injured epithelium and contributes to gastric regeneration.MethodsA mouse model expressing a myeloid cell‐specific deletion of Smoothened (LysMCre/SmoKO) was generated using transgenic mice bearing loxP sites flanking exon 1 of the Smo gene (Smo loxP) and mice expressing a Cre recombinase transgene from the Lysozyme M locus (LysMCre). Acetic acid injury was induced in the stomachs of both control and KO mice and gastric epithelial regeneration and macrophage recruitment analyzed over a period of 7 days post‐injury. Bone marrow‐derived macrophages (BM‐Mø) were collected from control and KO mice. Migration of BM‐Mø was performed using ibidi μ‐Slide Chemotaxis chambers in response to recombinant murine Shh (rmShh). Murine‐ and human‐derived gastric organoid/macrophage co‐cultures were established and macrophage chemotaxis measured.Results1) LysMCre/SmoKO mice exhibit loss of macrophage recruitment and epithelial regeneration in response to injury: Compared to control mice, KO animals exhibited loss of ulcer repair and normal epithelial regeneration, that correlated with decreased macrophage infiltration at the site of injury. 2) In vitro, macrophages from LysMCre/SmoKO mice or organoid/macrophage co‐cultures treated with Vismodegib lose chemotaxis: Forward migration index, a measure of directed movement, was significantly elevated in control BM‐Mø migrating towards rmShh compared to media control. In contrast, Smo‐deficient BM‐Mø did not migrate towards rmShh. While control BM‐Mø migrated toward chemokine CCL2, this chemotactic response was not observed using Smo‐deficient BM‐Mø. In murine‐derived gastric organoid/BM‐Mø co‐cultures, epithelial Shh, stimulated with histamine, correlated with macrophage migration toward the organoid. This response was lost in organoid/BM‐Mø co‐cultures derived from KO mice. Human‐derived organoid/macrophage co‐cultures stimulated with histamine resulted in Shh secretion and macrophage migration toward the gastric organoid, a response that was blocked with Smo‐inhibitor Vismodegib.ConclusionsShh signaling acts as a macrophage chemoattractant via a Smo‐dependent mechanism during gastric epithelial regeneration in response to injury.Support or Funding InformationNIH (NIDDK) 5R01DK083402‐08 grant (YZ)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Defense against Reactive Oxygen Species during Gastric Wound Repair is Mediated by the Cystine‐Glutamate xCT Transporter in Response to Injury

Background/HypothesisCluster‐of‐differentiation gene 44 (CD44) is an alternatively spliced cell surface protein. CD44 variant isoform 9 (CD44v9) has been shown to mediate gastric repair, but the precise mechanism is unknown. Reactive Oxygen Species (ROS) driven p38/MAPK signaling leads to increased caveolin‐1, and re‐direction of β‐catenin from the nucleus to the membrane where it decreases fundamental events contributing to repair such as cellular proliferation and migration. CD44v9 stabilizes the cystine‐glutamate transporter, xCT, which provides defense against ROS. Thus, we hypothesized that CD44v9 stabilizes the xCT transporter to potentiate defense against ROS to establish an environment conducive to efficient wound repair.MethodsAcetic acid injury was induced in C57BL/6 (BL6) mice followed by vehicle, sulfasalazine (SSZ, an xCT inhibitor) or glutathione inhibitor (GSHinh). Acetic acid gastric injuries were induced in aged mice (&gt;8 months) and animals were treated with p38 inhibitor. Mice were analyzed between days 1 to 30 post‐injury. A novel scratch‐wound assay generated from organoids derived from human stomachs and transferred to a gastric epithelial cell monolayer (huGEM) was treated with vehicle, SSZ or GSHinh.Results1) CD44v9 and xCT co‐expressing cells emerge at the ulcer margin: CD44v9/xCT co‐expressing cells emerged at the ulcer margin within 3‐days post‐injury. Compared to control mice, animals treated with SSZ or GSHinh showed impaired wound healing and loss of normal epithelial regeneration in response to injury. Western blots showed loss of activated β‐catenin, increased expression of caveolin‐1 that coincided with loss in repair in SSZ‐treated mice. FACS sorted CD44v9+ve cells from the ulcer margin expressed markers of EMT. 2) SSZ and GSHinh inhibited wound healing in huGEM scratch wound assays: HuGEM cultures exhibited a cellular composition reflective of primary gastric tissue that included the expression of parietal, chief, and mucous neck and pit cells. CD44v9/xCT co‐expressing cells emerged at the wound edge in huGEM cultures in response to a scratch wound. HuGEM cultures treated with SSZ or GSHinh exhibited significantly delayed wound closure. 3) p38 inhibitor rescues wound healing in vivo in the stomachs of aged mice: In human gastric biopsies, CD44v9, xCT, and activated β‐catenin was significantly decreased in elderly patients. Aged mice treated with p38 inhibitor displayed increased cellular proliferation, CD44v9 expression, and rescued repair when compared to aged vehicle controls.ConclusionCD44v9 and xCT emerge during gastric regeneration which may potentiate defense against ROS to allow for effective healing.Support or Funding InformationR01 DK083402‐06A1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hydrogen Sulfide Improves Aberrant Gastric Smooth Muscle Function in Duchenne Muscular Dystrophy Mice

Deficiency of dystrophin, a cytoskeletal protein localized in the inner face of the plasma membrane in skeletal, cardiac and smooth muscle, results in Duchenne Muscular Dystrophy (DMD). Through its interactions with extracellular matrix and plasma membrane proteins, dystrophin plays a role in contraction and signal transduction. In DMD, gastrointestinal disorders such as gastric dilation and intestinal pseudo‐obstruction resulting from altered motility have been reported. The role of dystrophin in the regulation of contractile protein expression and smooth muscle function, per se, is not known. Studies have suggested that inflammation contributes to the pathophysiology of DMD. Exogenous H2S had been shown to exert beneficial cardiovascular and gastrointestinal functions, probably via exerting anti‐inflammatory actions.AimTo test the hypothesis that a lack of dystrophin causes a decrease in contractile protein expression and smooth muscle function and that treatment with H2S restores the effects of dystrophin deficiency.MethodsThe role of dystrophin was examined using mice deficient in dystrophin alone (mdx) and mice deficient in dystrophin plus telomerase RNA (mdx/mTR), which exhibit increased disease severity. The effect of an orally‐active, slow releasing H2S agent (SG1002) was tested in mdx/mTR mice (40 mg/kg body weight in chow/every 3 days starting from 3 weeks to 9 months). Contraction in response to acetylcholine (ACh) was measured in gastric muscle strips isolated from mdx, mdx/mTR and SG1002‐treated mdx/mTR mice. Age‐matched control mice were used for each group. Contraction was also measured in muscle cells isolated from the stomach of control and mdx mice by scanning micrometry and expressed as the percent decrease in muscle cell length. Expression of contractile proteins such as smoothelin, caldesmon, calponin and tropomyosin was measured by qRT‐PCR and western blot.ResultsAcetylcholine‐induced contraction was reduced in muscle strips from mdx/mTR mice (19 ± 5 mN/100 mg tissue) compared to age‐matched 9‐month old control mice (34 ± 7 mN/100 mg tissue). Treatment of mdx/mTR mice with SG1002 restored contraction to above normal levels (56 ± 8 mN/100 mg tissue). Contraction was also reduced in mdx mice (22 ± 8 mN/100 mg tissue) compared to age‐matched 3‐month old control mice (40 ± 5 mN/100 mg tissue). Acetylcholine‐induced contraction was also decreased in gastric muscle cells from mdx mice (25 ± 3% decrease in cell length) compared to control mice (41 ± 5% decrease in cell length). Expression of contractile proteins was decreased in gastric smooth muscle from mdx and mdx/mTR mice compared to age‐matched controls. Treatment of mdx/mTR mice with SG1002 restored expression of contractile proteins to levels similar to control.ConclusionThe results support our hypothesis that dystrophin deficiency affects contractile protein expression leading to decreased muscle contraction. Furthermore, treatment with H2S restores gastric smooth muscle function and contractile protein expression suggesting therapeutic potential of H2S in the treatment of motility disorders in DMD.Support or Funding InformationSupported by DK15564, DK28300, DK34153, and HL133167.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Na+/HCO3− Cotransporter (Nbce1, Slc4a4) is Enriched in Interstitial Cells of Cajal Responsible for Generating Electrical Slow Wave Activity in the Mouse Gastrointestinal Tract

Electrical slow wave activity, generated by interstitial cells of Cajal (ICC), is essential for efficient gastrointestinal (GI) transit. Electrophysiology of slow waves has been extensively studied; inclusion of bicarbonate (HCO3−) in extracellular solutions is required for recording normal slow wave activity. HCO3− transport contributes to regulation of intracellular pH, which has multiple consequences for normal cellular function. Previous transcriptional profiling found that the Na+/ HCO3− cotransporter (NBCe1/Slc4a4) is enriched in pacemaker myenteric ICC (ICC‐MY) compared to non pacemaker ICC in the deep muscular plexus (ICC‐DMP) of the small intestine. Our aim was to determine the distribution and identity of Slc4a4/NBCe1 variants in ICC in the GI tract.NBCe1 immunoreactivity (IR) was detected by fluorescence labeling of parafomaldehyde‐fixed cross sections and whole mounts of adult mouse small intestine, colon and stomach using primary antibodies directed against a 329 amino acid sequence in the N terminus of the NBCe1 protein. Labeling of Kit receptor tyrosine kinase with a goat anti‐Kit polyclonal antibody was used to detect ICC. Slc4a4 knockout (KO) mice were bred from heterozygous founders. Slc4a4fl/fl mice were bred with ETV1CreERT2/− mice to allow targeted, conditional KO of Slc4a4 in ICC in response to tamoxifen treatment (130 mg/Kg). mRNA transcripts for Slc4a4 in Kit expressing cells were isolated from Kit‐ribotag mice, which were generated by breeding KitcreERT2 with Rpl22tm1.1Psam mice. The abundance of specific Slc4a4 transcripts was measured by quantitative real time PCR (qRT‐PCR). Green fluorescent protein (GFP)‐labeled ICC were dissociated from the small intestine of neonatal KitcopGFP mice and RNA expression levels were determined using the Single Cell‐to‐CT™ qRT‐PCR Kit from Ambion.NBCe1‐IR was absent in all cells in Slc4a4‐KO tissues but co‐localized with Kit in ICC‐MY, but not in ICC‐DMP, in small intestine of wild type mice. In the proximal and distal colon, pacemaker submucosal ICC (ICC‐SMP) but not intramuscular ICC (ICC‐IM) or ICC‐MY showed NBCe1‐IR. In the corpus and antrum of the stomach NBCe1‐IR was detected in ICC‐MY but not in ICC‐IM. NBCe1‐IR did not co‐localize with Kit in the gastric fundus. mRNA for variant C, but not variants A or B of Slc4a4, was detected in small intestinal ICC‐MY. Reduced NBCe1‐IR was restricted to small intestinal ICC‐MY of Slc4a4 conditional KO mice.NBCe1 is enriched in subtypes of ICC responsible for pacemaker activity in all regions of the GI tract indicating a role for this transporter in slow wave generation. The presence of the C variant of Slc4a4 in ICC‐MY suggests dynamic regulation of NBCe1 activity in ICC by the IP3R binding protein released with inositol 1, 4, 5‐trisphosphate (IRBIT).Support or Funding InformationSupported by NIH R01 DK57061.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

During Ca2+‐dependent gastric epithelial repair, Ca2+ is sourced from both Ca2+ uptake and intracellular Ca2+ release

BackgroundCalcium (Ca2+) is a known accelerator for gastric repair. However the mechanism by which Ca2+ mobilizes to promote repair remains unclear, and cannot be readily evaluated in vivo. Using gastric organoids derived from transgenic mice expressing a fluorescent Ca2+ reporter (yellow cameleon‐nano15; YC‐Nano), we previously observed intracellular Ca2+ increases in cells directly adjacent to a damaged cell. Using this Ca2+ sensor, we investigate the potential sources of intracellular Ca2+ essential for repair.MethodsGastroids generated from YC‐Nano mouse stomach corpus were cultured 4–5 days prior to experiments. Photodamage and resultant cell death was induced to 1–2 gastroid epithelial cells by ~3 sec high intensity 840 nm light. YC‐Nano reports Förster resonance energy transfer (FRET) from CFP to YFP in response to increased intracellular Ca2+. Change in intracellular Ca2+ was measured as FRET/CFP ratio, in cells adjacent to damaged cells. Inhibitors were used to test roles of Ca2+ channels (10 μM verapamil, 20 μM YM58483), intracellular Ca2+ (50 μM BAPTA/AM), Phospholipase C (10 μM U73122), and IP3R (50 μM 2‐APB).ResultsUnperturbed gastroid cells maintain a constant FRET ratio, indicating stable Ca2+ levels. In response to photodamage of the gastroid epithelium, increased levels of Ca2+ were observed specifically within the lateral membranes of cells neighboring the damaged cell. Chelation of intracellular Ca2+ by BAPTA/AM resulted in significant dampening of Ca2+ response, as well as blocking prompt repair. Inhibition of L‐type channels (verapamil) or store operated Ca2+ entry (YM58483) resulted in delaying repair and dampening intracellular Ca2+ response. Furthermore, inhibition of PLC (U73122) or IP3R (2‐APB) resulted in delayed repair and dampened Ca2+ response.ConclusionThese results suggest both extracellular and intracellular Ca2+ sources are essential for supplying the Ca2+ that stimulates repair. The findings implicate an intracellular Ca2+ raise mediated via Ca2+ uptake via plasma membrane Ca2+ channels and intracellular Ca2+ release from the ER. Collectively this work indicates the usefulness of YC‐Nano to further assess intracellular Ca2+ dynamics and further investigate the signaling cascade behind Ca2+‐mediated repair.Support or Funding InformationThis work was supported by the National Institutes of Health (NIH) R01DK102551 (Montrose; Aihara) and F31DK115126 (Engevik). This project was also supported in part by the NIH P30 DK078392; Live Microscopy Core and DNA Sequencing and Genotyping Core of the Digestive Disease Research Core Center in Cincinnati.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Clinical Pharmaceutical Research Based on New Proteome Analysis Based on Chromatographic Separation

Comprehensive identification of antigens in immune complexes (IC-antigens) is beneficial to provide insights into pathophysiology and could form the basis for novel diagnostic and treatment strategies for many immune-related diseases. Immune complexome analysis is a method for comprehensively identifying and profiling IC-antigens in biological fluids (such as serum and cerebrospinal fluid). We applied this strategy to the analysis of circulating ICs in autoimmune diseases (rheumatoid arthritis, Sjögren's syndrome, systemic scleroderma, and systemic lupus erythematosus), infectious diseases, and cancers. Fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) consists of fluorogenic derivatization of proteins, followed by HPLC of the derivatized proteins, isolation of the proteins differentially expressed in a certain group, enzymatic digestion of the isolated proteins followed by LC-tandem MS using a database-searching algorithm for protein identification. We have applied this method to understand the cardioprotective effect of pre-administration of docetaxel in adriamycin/docetaxel combination anti-cancer therapy, and the cellular processes that are affected by non-steroidal anti-inflammatory drugs (NSAIDs) in mouse stomach tissue during ulcer formation.