Gastric coinfection with thiopeptide-positive Cutibacterium acnes decreases FOXM1 and pro-inflammatory biomarker expression in a murine model of Helicobacter pylori-induced gastric cancer.

IMPORTANCE: H. pylori infects half of the world population and is the leading cause of gastric cancer. We previously demonstrated that gastric cancer risk is associated with gastric microbiota. Specifically, gastric urease-positive Staphylococcus epidermidis and Streptococcus salivarius had contrasting effects on H. pylori-associated gastric pathology and immune responses in germ-free INS-GAS mice. As gastritis progresses to gastric cancer, the oncogenic transcription factor Foxm1 becomes increasingly expressed. In this study, we evaluated the gastric commensal C. acnes, certain strains of which produce thiopeptides that directly inhibit FOXM1. Thiopeptide-positive C. acnes was isolated from Nicaraguan patient gastric biopsies and inoculated into germ-free INS-GAS mice with H. pylori. We, therefore, asked whether coinfection with C. acnes expressing thiopeptide and H. pylori would decrease gastric Foxm1 expression and pro-inflammatory cytokine mRNA and protein levels. Our study supports the growing literature that specific non-H. pylori gastric bacteria affect inflammatory and cancer biomarkers in H. pylori pathogenesis.

IL-1ß transgenic mouse model of inflammation driven esophageal and oral squamous cell carcinoma.

Chronic inflammation is integral to the development of esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC), although the latter has not been associated with reflux esophagitis. The L2-IL-1ß transgenic mice, expressing human interleukin (IL)-1ß in the oral, esophageal and forestomach squamous epithelia feature chronic inflammation and a stepwise development of Barrett's esophagus-like metaplasia, dysplasia and adenocarcinoma at the squamo-columnar junction. However, the functional consequences of IL-1ß-mediated chronic inflammation in the oral and esophageal squamous epithelia remain elusive. We report for the first time that in addition to the previously described Barrett's esophagus-like metaplasia, the L2-IL-1ß mice also develop squamous epithelial dysplasia with progression to squamous cell carcinoma (SCC) in the esophagus and the tongue. L2-IL-1ß showed age-dependent progression of squamous dysplasia to SCC with approximately 40% (n = 49) and 23.5% (n = 17) incidence rates for esophageal and tongue invasive SCC respectively, by 12-15 months of age. Interestingly, SCC development and progression in L2-IL-1ß was similar in both Germ Free (GF) and Specific Pathogen Free (SPF) conditions. Immunohistochemistry revealed a T cell predominant inflammatory profile with enhanced expression of Ki67, Sox2 and the DNA double-strand break marker, γ-H2AX, in the dysplastic squamous epithelia of L2-IL-1ß mice. Pro-inflammatory cytokines, immunomodulatory players, chemoattractants for inflammatory cells (T cells, neutrophils, eosinophils, and macrophages) and oxidative damage marker, iNOS, were significantly increased in the esophageal and tongue tissues of L2-IL-1ß mice. Our recent findings have expanded the translational utility of the IL-1ß mouse model to aid in further characterization of the key pathways of inflammation driven BE and EAC as well as ESCC and Oral SCC.

Evaluation and comparison of pharmacokinetic profiles and safety of two extended-release buprenorphine formulations in common marmosets (Callithrix jacchus).

While sustained-release buprenorphine (BSR) is used as a long-lasting opioid analgesic in common marmosets (Callithrix jacchus), there are no published studies on pharmaceutical-grade extended-release buprenorphine options such as Ethiqa XR (EXR) for this species. However, BSR is a compounded product and has been reported to cause injection site reactions in multiple species, including marmosets. Additionally, now with the availability of EXR, a pharmaceutical-grade veterinary product, the use of BSR in laboratory animals is not compliant with the Guide for the Care and Use of Laboratory Animals (Guide) unless scientifically justified and approved by the IACUC. We compared pharmacokinetic and safety profiles of BSR (0.15 mg/kg) and EXR (0.1-0.2 mg/kg) administered subcutaneously to adult marmosets. Blood was collected by venipuncture of the saphenous vein at multiple time points (0.25-72 h) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). EXR between 0.1 and 0.2 mg/kg resulted in a dose-dependent increase in Cmax (1.43-2.51 ng/mL) and were not statistically different from BSR (1.82 ng/mL). Tmax, lambdaz, and t1/2 were not statistically different between formulations. Mean plasma buprenorphine concentrations for BSR and EXR exceeded the therapeutic threshold (0.1 ng/mL) within 0.25 h and lasted for > 72 h. Mild sedation, but neither respiratory depression nor ataxia, was observed for both formulations. BSR injection sites had significantly higher histopathological scores compared to EXR. Video recordings for monitoring drug-induced behavioral changes showed increased animal activity levels after BSR and EXR versus saline controls. Norbuprenorphine, a buprenorphine metabolite associated with respiratory depression, was detected in the plasma after BSR and EXR administration as well as by in vitro liver microsome assays. In conclusion, we recommend using EXR over BSR as a long-lasting buprenorphine analgesic in marmosets because EXR is a pharmaceutical-grade formulation that is compliant with FDA guidelines and the Guide as well as exhibits comparable PK and safety profiles as BSR.

In-Vivo fluorescent nanosensor implants based on hydrogel-encapsulation: investigating the inflammation and the foreign-body response.

Nanotechnology-enabled sensors or nanosensors are emerging as promising new tools for various in-vivo life science applications such as biosensing, components of delivery systems, and probes for spatial bioimaging. However, as with a wide range of synthetic biomaterials, tissue responses have been observed depending on cell types and various nanocomponent properties. The tissue response is critical for determining the acute and long term health of the organism and the functional lifetime of the material in-vivo. While nanomaterial properties can contribute significantly to the tissue response, it may be possible to circumvent adverse reactions by formulation of the encapsulation vehicle. In this study, five formulations of poly (ethylene glycol) diacrylate (PEGDA) hydrogel-encapsulated fluorescent nanosensors were implanted into SKH-1E mice, and the inflammatory responses were tracked in order to determine the favorable design rules for hydrogel encapsulation and minimization of such responses. Hydrogels with higher crosslinking density were found to allow faster resolution of acute inflammation. Five different immunocompromised mice lines were utilized for comparison across different inflammatory cell populations and responses. Degradation products of the gels were also characterized. Finally, the importance of the tissue response in determining functional lifetime was demonstrated by measuring the time-dependent nanosensor deactivation following implantation into animal models.

Effects of chronic Helicobacter pylori strain PMSS1 infection on whole brain and gastric iron homeostasis in male INS-GAS mice.

Iron deficiency, the most common micronutrient deficiency in humans, is associated with long-term deficits in cognition and memory if left untreated. Infection with the gastric pathogen Helicobacter pylori has been linked to iron deficiency anemia (IDA). The H. pylori virulence factor cytotoxin-associated gene A (cagA) is proposed to be especially pertinent in iron deficiency. Male INS-GAS/FVB mice were infected with the CagA+ strain pre-murine Sydney strain 1 (PMSS1) for 12-13 or 27-29 weeks to investigate the role of chronic H. pylori infection in iron deficiency and neurological sequelae. Mice at both timepoints demonstrated significantly elevated gastric histopathology scores and inflammatory cytokines compared to sham-dosed controls. However, only mice at 27-29 weeks post infection had changes in hematological parameters, with significantly decreased erythrocyte count, hematocrit, serum hemoglobin, and increased serum total iron binding capacity. Gastric transcription of iron-regulatory genes Hamp and Bmp4 were significantly downregulated at both timepoints. In the brain, iron-dependent myelingergic and synaptic markers were significantly downregulated at 27-29 weeks. These results indicated that long-term infection of the CagA + PMSS1 strain of H. pylori in this study caused anemia, altered gastric iron homeostasis, and neurological changes similar to those reported in other rodent H. pylori CagA- strain infection models.

Maternal Microbiota Modulate a Fragile X-like Syndrome in Offspring Mice.

Maternal microbial dysbiosis has been implicated in adverse postnatal health conditions in offspring, such as obesity, cancer, and neurological disorders. We observed that the progeny of mice fed a Westernized diet (WD) with low fiber and extra fat exhibited higher frequencies of stereotypy, hyperactivity, cranial features and lower FMRP protein expression, similar to what is typically observed in Fragile X Syndrome (FXS) in humans. We hypothesized that gut dysbiosis and inflammation during pregnancy influenced the prenatal uterine environment, leading to abnormal phenotypes in offspring. We found that oral in utero supplementation with a beneficial anti-inflammatory probiotic microbe, Lactobacillus reuteri, was sufficient to inhibit FXS-like phenotypes in offspring mice. Cytokine profiles in the pregnant WD females showed that their circulating levels of pro-inflammatory cytokine interleukin (Il)-17 were increased relative to matched gravid mice and to those given supplementary L. reuteri probiotic. To test our hypothesis of prenatal contributions to this neurodevelopmental phenotype, we performed Caesarian (C-section) births using dissimilar foster mothers to eliminate effects of maternal microbiota transferred during vaginal delivery or nursing after birth. We found that foster-reared offspring still displayed a high frequency of these FXS-like features, indicating significant in utero contributions. In contrast, matched foster-reared progeny of L. reuteri-treated mothers did not exhibit the FXS-like typical features, supporting a key role for microbiota during pregnancy. Our findings suggest that diet-induced dysbiosis in the prenatal uterine environment is strongly associated with the incidence of this neurological phenotype in progeny but can be alleviated by addressing gut dysbiosis through probiotic supplementation.

The Epidemiology of Invasive, Multipleantibiotic-resistant Klebsiella pneumoniae Infection in a Breeding Colony of Immunocompromised NSG Mice.

Klebsiella pneumoniae (Kp) is a gram-negative opportunistic pathogen that causes severe pneumonia, pyelonephritis, and sepsis in immunocompromised hosts. During a 4-mo interval, several NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) breeders and pups in our facilities were diagnosed with Kp infections. An initial 6 adult and 1 juvenile NSG mice were submitted for necropsy and histologic examination because of acute onset of diarrhea and death. The evaluation revealed typhlocolitis in 2 of the mice and tritrichomoniasis in all 7. Escherichia coli positive for polyketide synthase (pks+) and Kp were isolated from the intestines. Given a history of sepsis due to pks+ E. coli in NSG mice in our facilities and determination of its antimicrobial susceptibility, trimethoprim-sulfamethoxazole (TMP-SMX) was administered to the colony in the drinking water for 4 wk. After this intervention, an additional 21 mice became ill or died; 11 of these mice had suppurative pneumonia, meningoencephalitis, hepatitis, metritis, pyelonephritis, or sepsis. Kp was cultured from pulmonary abscesses or blood of 10 of the mice. Whole-genome sequencing (WGS) indicated that the Kp isolates contained genes associated with phenotypes found in pore-forming Kp isolates cultured from humans with ulcerative colitis and primary sclerosing cholangitis. None of the Kp isolates exhibited a hyperviscous phenotype, but 13 of 14 were resistant to TMP-SMX. Antimicrobial susceptibility testing indicated sensitivity of the Kp to enrofloxacin, which was administered in the drinking water. Antibiotic sensitivity profiles were confirmed by WGS of the Kp strains; key virulence and resistance genes to quaternary ammonia compounds were also identified. Enrofloxacin treatment resulted in a marked reduction in mortality, and the study using the NSG mice was completed successfully. Our findings implicate intestinal translocation of Kp as the cause of pneumonia and systemic infections in NSG mice and highlight the importance of identification of enteric microbial pathogens and targeted antibiotic selection when treating bacterial infections in immunocompromised mice.

Translocation of Helicobacter hepaticus synergizes with myeloid-derived suppressor cells and contributes to breast carcinogenesis.

Microbial dysbiosis plays an important role in the development of intestinal diseases. Recent studies suggest a link between intestinal bacteria and mammary cancer. Here, we report that female ApcMin/+ mice infected with Helicobacter hepaticus exhibited an increased mammary and small/large intestine tumor burden compared with uninfected littermates. H. hepaticus DNA was detected in small/large intestine, mammary tumors, and adjacent lymph nodes, suggesting a migration pathway. CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) infiltrated and expressed high levels of Wnts, likely enhancing tumorigenesis through activation of Wnt/ß-catenin pathway. Our previous studies indicated that histidine decarboxylase (Hdc) marks a population of myeloid-biased hematopoietic stem cells and granulocytic MDSCs. Cytokines/chemokines secreted by IL-17-expressing mast cells and tumor tissues promoted Hdc+ MDSCs expansion and trafficking toward mammary tumors. Adoptive transfer of MDSCs isolated from H. hepaticus-infected mice increased MDSCs frequencies in peripheral blood, mesenteric lymph nodes, mammary gland, and lymph nodes in recipient ApcMin/+ mice. The adoptive transfer of H. hepaticus primed MDSCs also increased the size and number of mammary tumors. Our results demonstrate that H. hepaticus can translocate from the intestine to mammary tissues to promote mammary tumorigenesis with MDSCs. Targeting bacteria and MDSCs may be useful for the prevention and therapy of extraintestinal cancers. Abbreviations: Helicobacter hepaticus, Hh; myeloid-derived suppressor cell, MDSC; histidine decarboxylase, Hdc; Breast cancer, BC; T regulatory, TR; inflammatory bowel disease, IBD; fluorescence in situ hybridization, FISH; myeloid-biased hematopoietic stem cells, MB-HSCs; granulocytic MDSCs, PMN-MDSCs; Lipopolysaccharide, LPS; Toll-like receptors, TLRs; Mast cells, MCs; Granulocyte-macrophage colony-stimulating factor, GM-CSF; epithelial-mesenchymal transition, EMT; Intestinal epithelial cells, IECs.

Analysis of gut microbiome profiles in common marmosets (Callithrix jacchus) in health and intestinal disease.

Chronic gastrointestinal (GI) diseases are the most common diseases in captive common marmosets. To understand the role of the microbiome in GI diseases, we characterized the gut microbiome of 91 healthy marmosets (303 samples) and 59 marmosets diagnosed with inflammatory bowel disease (IBD) (200 samples). Healthy marmosets exhibited "humanized," Bacteroidetes-dominant microbiomes. After up to 2 years of standardized diet, housing and husbandry, marmoset microbiomes could be classified into four distinct marmoset sources based on Prevotella and Bacteroides levels. Using a random forest (RF) model, marmosets were classified by source with an accuracy of 93% with 100% sensitivity and 95% specificity using abundance data from 4 Prevotellaceae amplicon sequence variants (ASVs), as well as single ASVs from Coprobacter, Parabacteroides, Paraprevotella, Phascolarctobacterium, Oribacterium and Fusobacterium. A single dysbiotic IBD state was not found across all marmoset sources, but IBD was associated with lower alpha diversity and a lower Bacteroides:Prevotella copri ratio within each source. IBD was highest in a Prevotella-dominant cohort, and consistent with Prevotella-linked diseases, pro-inflammatory genes in the jejunum were upregulated. RF analysis of serum biomarkers identified serum calcium, hemoglobin and red blood cell (RBC) counts as potential biomarkers for marmoset IBD. This study characterizes the microbiome of healthy captive common marmosets and demonstrates that source-specific microbiomes can be retained despite standardized diets and husbandry practices. Marmosets with IBD had decreased alpha diversity and a shift in the ratio of Bacteroides:Prevotella copri compared to healthy marmosets.

Alterations in common marmoset gut microbiome associated with duodenal strictures.

Chronic gastrointestinal (GI) diseases are the most common diseases in captive common marmosets (Callithrix jacchus). Despite standardized housing, diet and husbandry, a recently described gastrointestinal syndrome characterized by duodenal ulcers and strictures was observed in a subset of marmosets sourced from the New England Primate Research Center. As changes in the gut microbiome have been associated with GI diseases, the gut microbiome of 52 healthy, non-stricture marmosets (153 samples) were compared to the gut microbiome of 21 captive marmosets diagnosed with a duodenal ulcer/stricture (57 samples). No significant changes were observed using alpha diversity metrics, and while the community structure was significantly different when comparing beta diversity between healthy and stricture cases, the results were inconclusive due to differences observed in the dispersion of both datasets. Differences in the abundance of individual taxa using ANCOM, as stricture-associated dysbiosis was characterized by Anaerobiospirillum loss and Clostridium perfringens increases. To identify microbial and serum biomarkers that could help classify stricture cases, we developed models using machine learning algorithms (random forest, classification and regression trees, support vector machines and k-nearest neighbors) to classify microbiome, serum chemistry or complete blood count (CBC) data. Random forest (RF) models were the most accurate models and correctly classified strictures using either 9 ASVs (amplicon sequence variants), 4 serum chemistry tests or 6 CBC tests. Based on the RF model and ANCOM results, C. perfringens was identified as a potential causative agent associated with the development of strictures. Clostridium perfringens was also isolated by microbiological culture in 4 of 9 duodenum samples from marmosets with histologically confirmed strictures. Due to the enrichment of C. perfringens in situ, we analyzed frozen duodenal tissues using both 16S microbiome profiling and RNAseq. Microbiome analysis of the duodenal tissues of 29 marmosets from the MIT colony confirmed an increased abundance of Clostridium in stricture cases. Comparison of the duodenal gene expression from stricture and non-stricture marmosets found enrichment of genes associated with intestinal absorption, and lipid metabolism, localization, and transport in stricture cases. Using machine learning, we identified increased abundance of C. perfringens, as a potential causative agent of GI disease and intestinal strictures in marmosets.

Gastric Non-Helicobacter pylori Urease-Positive Staphylococcus epidermidis and Streptococcus salivarius Isolated from Humans Have Contrasting Effects on H. pylori-Associated Gastric Pathology and Host Immune Responses in a Murine Model of Gastric Cancer.

In populations with similar prevalence of Helicobacter pylori infection, cancer risk can vary dramatically. Changes in composition or structure of bacterial communities in the stomach, either at the time of exposure or over the course of H. pylori infection, may contribute to gastric pathology. In this study, a population of 37 patients from the low-gastric-cancer-risk (LGCR) region of Tumaco, Colombia, and the high-gastric-cancer-risk (HGCR) region of Túquerres, Colombia, were recruited for gastric endoscopy. Antral biopsy specimens were processed for histology and bacterial isolation. Fifty-nine distinct species among 26 genera were isolated by aerobic, anaerobic, and microaerobic culture and confirmed by 16S rRNA analysis. Urease-positive Staphylococcus epidermidis and Streptococcus salivarius were frequently isolated from gastric biopsy specimens. We asked whether coinfection of H. pylori with urease-positive S. salivarius and/or S. epidermidis had a demonstrable effect on H. pylori-induced gastritis in the germfree (GF) INS-GAS mouse model. Coinfections with S. salivarius and/or S. epidermidis did not affect gastric H. pylori colonization. At 5 months postinfection, GF INS-GAS mice coinfected with H. pylori and S. salivarius had statistically higher pathological scores in the stomachs than mice infected with H. pylori only or H. pylori with S. epidermidis (P < 0.05). S. epidermidis coinfection with H. pylori did not significantly change stomach pathology, but levels of the proinflammatory cytokine genes Il-1ß, Il-17A , and Il-22 were significantly lower than in H. pylori-monoinfected mice. This study demonstrates that non-H. pylori urease-positive bacteria may play a role in the severity of H. pylori-induced gastric cancer in humans. IMPORTANCE Chronic infection with H. pylori is the main cause of gastric cancer, which is a global health problem. In two Colombian populations with high levels of H. pylori prevalence, the regional gastric cancer rates are considerably different. Host genetic background, H. pylori biotype, environmental toxins, and dietary choices are among the known risk factors for stomach cancer. The potential role of non-H. pylori gastric microbiota in gastric carcinogenesis is being increasingly recognized. In this study, we isolated 59 bacterial species from 37 stomach biopsy samples of Colombian patients from both low-gastric-cancer-risk and high-gastric-cancer-risk regions. Urease-positive S. epidermidis and S. salivarius commonly cultured from the stomachs, along with H. pylori, were inoculated into germfree INS-GAS mice. S. salivarius coinfection with H. pylori induced significantly higher gastric pathology than in H. pylori-monoinfected mice, whereas S. epidermidis coinfection caused significantly lower H. pylori-induced proinflammatory cytokine responses than in H. pylori-monoinfected mice. This study reinforces the argument that the non-H. pylori stomach microflora play a role in the severity of H. pylori-induced gastric cancer.

Factors Affecting Hematologic and Serum Biochemical Parameters in Healthy Common Marmosets (Callithrix jacchus).

Physiologic changes during development, aging, and pregnancy may affect clinical parameters. Previously available reference values have been based on samples that may include wild and captive marmosets, with little representation of geriatric or pregnant animals. Establishing reference values under various conditions would support better recognition of pathologic conditions in marmosets. One hundred and forty-seven (70 males and 77 females) healthy marmosets from a research colony were included in this study. Exclusion criteria were abnormal physical exam findings at the time of blood sampling, chronic medications, or clinical or pathologic evidence of disease. Reference intervals were calculated for serum chemistry and hematology. Using metadata, samples were classified based on age, sex, colony source and pregnancy status. Multiple tests indicated significant differences with varying effect sizes, indicating that developing reference intervals based on metadata can be useful. Across all the comparisons, medium or large effect sizes were observed most frequently in blood urea nitrogen (BUN), calcium, total protein, alkaline phosphatase (ALP), weight and serum albumin. We report normative clinical pathologic data for captive common marmosets through all life stages and reproductive status. Significant differences were observed in most parameters when stratifying data based on age, sex, colony source, or pregnancy, suggesting that developing reference intervals considering this information is important for clinicians.

Hierarchical cell-type-specific functions of caspase-11 in LPS shock and antibacterial host defense.

Caspase-11 sensing of intracellular lipopolysaccharide (LPS) plays critical roles during infections and sepsis. However, the key cell types that sense intracellular LPS and their contributions to the host responses at the organismal level are not completely clear. Here, we show that macrophage/monocyte-specific caspase-11 plays a dominant role in mediating the pathological manifestations of endotoxemia, including gasdermin D (GSDMD) activation, interleukin (IL)-1ß, IL-18, and damage-associated molecular pattern (DAMP) release, tissue damage, and death. Surprisingly, caspase-11 expression in CD11c+ cells and intestinal epithelial cells (IECs) plays minor detrimental roles in LPS shock. In contrast, caspase-11 expression in neutrophils is dispensable for LPS-induced lethality. Importantly, caspase-11 sensing of intracellular LPS in LyzM+ myeloid cells and MRP8+ neutrophils, but not CD11c+ cells and IECs, is necessary for bacterial clearance and host survival during intracellular bacterial infection. Thus, we reveal hierarchical cell-type-specific roles of caspase-11 that govern the host-protective and host-detrimental functions of the cytosolic LPS surveillance.

Cutaneous leiomyosarcoma with visceral metastases in a White Carneau pigeon and literature review.

Cutaneous leiomyosarcomas are malignant mesenchymal tumors of smooth muscle origin and are reported occasionally in avian species. A 14-y-old male laboratory White Carneau pigeon (Columba livia) was presented for surgical excision of a cervical soft tissue mass. Ultrasonography with color flow Doppler imaging revealed multiple cavitations of mixed echogenicity within the mass and vascularization. Histologically, the dermis and subcutis were expanded by a densely cellular multinodular mass comprised of fusiform cells forming haphazardly arranged broad streams and short interwoven bundles, often surrounding blood vessels and variably sized cavitations. Neoplastic cells were strongly immunopositive for desmin and α-smooth muscle actin, and negative for pancytokeratin, S100, and von Willebrand factor. Based on histopathology and IHC findings, the cutaneous mass was diagnosed as leiomyosarcoma (LMS). The pigeon died 312 d post-operatively. Postmortem examination revealed masses infiltrating the left and right pulmonary airways and one hepatic nodule, but no regrowth at the surgical site. Histologic and IHC evaluation of the pulmonary and hepatic masses were consistent with LMS, representing metastatic foci from the primary cutaneous LMS. Our case highlights the malignant behavior and histomorphologic features of cutaneous LMS in an avian species.

Helicobacter suis and Helicobacter pylori infection in a colony of research macaques: characterization and clinical correlates.

Introduction. Helicobacter suis (Helicobacter heilmannii type 1) commonly infects nonhuman primates but its clinical importance is in question.Aim. To characterize H. suis infection in a colony of rhesus macaques (Macaca mulatta) used in cognitive neuroscience research.Hypothesis/Gap Statement. Inquiries into the nature of Helicobacter suis in nonhuman primates are required to further define the organism's virulence and the experimental animal's gastric microbiome.Methodology. Animals with and without clinical signs of vomiting and abdominal pain (n=5 and n=16, respectively) were evaluated by histology, culture, PCR amplification and sequencing, fluorescent in situ hybridization (FISH) and serology. Three of the five animals with clinical signs, an index case and two others, were evaluated before and after antimicrobial therapy.Results. The index animal had endoscopically visible ulcers and multifocal, moderate, chronic lymphoplasmacytic gastritis with intraglandular and luminal spiral bacteria. Antimicrobial therapy in the index animal achieved histologic improvement, elimination of endoscopically visible ulcers, and evident eradication but clinical signs persisted. In the other treated animals, gastritis scores were not consistently altered, gastric bacteria persisted, but vomiting and abdominal discomfort abated.Nineteen of 21 animals were PCR positive for H. suis and five animals were also PCR positive for H. pylori. Organisms were detected by FISH in 17 of 21 animals: 16S rRNA sequences of two of these were shown to be H. suis. Mild to moderate lymphoplasmacytic gastritis was seen in antrum, body and cardia, with antral gastritis more likely to be moderate than that of the body.Conclusion. No clear association between the bacterial numbers of Helicobacter spp. and the degree of inflammation was observed. H. suis is prevalent in this colony of Macaca mulatta but its clinical importance remains unclear. This study corroborates many of the findings in earlier studies of H. suis infection in macaques but also identifies at least one animal in which gastritis and endoscopically visible gastric ulcers were strongly associated with H. suis infection. In this study, serology was an inadequate biomarker for endoscopic evaluation in diagnosis of H. suis infection.

Claudin-18 Loss Alters Transcellular Chloride Flux but not Tight Junction Ion Selectivity in Gastric Epithelial Cells.

BACKGROUND & AIMS: Tight junctions form a barrier to the paracellular passage of luminal antigens. Although most tight junction proteins reside within the apical tight junction complex, claudin-18 localizes mainly to the basolateral membrane where its contribution to paracellular ion transport is undefined. Claudin-18 loss in mice results in gastric neoplasia development and tumorigenesis that may or may not be due to tight junction dysfunction. The aim here was to investigate paracellular permeability defects in stomach mucosa from claudin-18 knockout (Cldn18-KO) mice. METHODS: Stomach tissue from wild-type, heterozygous, or Cldn18-KO mice were stripped of the external muscle layer and mounted in Ussing chambers. Transepithelial resistance, dextran 4 kDa flux, and potential difference (PD) were calculated from the chambered tissues after identifying differences in tissue histopathology that were used to normalize these measurements. Marker expression for claudins and ion transporters were investigated by transcriptomic and immunostaining analysis. RESULTS: No paracellular permeability defects were evident in stomach mucosa from Cldn18-KO mice. RNAseq identified changes in 4 claudins from Cldn18-KO mice, particularly the up-regulation of claudin-2. Although claudin-2 localized to tight junctions in cells at the base of gastric glands, its presence did not contribute overall to mucosal permeability. Stomach tissue from Cldn18-KO mice also had no PD versus a lumen-negative PD in tissues from wild-type mice. This difference resulted from changes in transcellular Cl- permeability with the down-regulation of Cl- loading and Cl- secreting anion transporters. CONCLUSIONS: Our findings suggest that Cldn18-KO has no effect on tight junction permeability in the stomach from adult mice but rather affects anion permeability. The phenotype in these mice may thus be secondary to transcellular anion transporter expression/function in the absence of claudin-18.

Male-Dependent Promotion of Colitis in 129 Rag2-/- Mice Co-Infected with Helicobacter pylori and Helicobacter hepaticus.

The prevalence of gastric Helicobacter pylori (Hp) infection is ~50% of the world population. However, how Hp infection influences inflammatory bowel disease in humans is not fully defined. In this study, we examined whether co-infection with Hp influenced Helicobacter hepaticus (Hh)-induced intestinal pathology in Rag2-/- mice. Rag2-/- mice of both sexes were infected with Hh, of which a subgroup was followed by infection with Hp two weeks later. Co-infected males, but not females, had significantly higher total colitis index scores in the colon at both 10 and 21 weeks post-Hh infection (WPI) and developed more severe dysplasia at 21 WPI compared with mono-Hh males. There were no significant differences in colonization levels of gastric Hp and colonic Hh between sexes or time-points. In addition, mRNA levels of colonic Il-1ß, Ifnγ, Tnfα, Il-17A, Il-17F, Il-18, and Il-23, which play important roles in the development and function of proinflammatory innate lymphoid cell groups 1 and 3, were significantly up-regulated in the dually infected males compared with mono-Hh males at 21 WPI. These data suggest that concomitant Hp infection enhances the inflammatory responses in the colon of-Hh-infected Rag2-/- males, which results in more severe colitis and dysplasia.

Anaplastic nephroblastoma with peritoneal metastasis in an adult female Sprague Dawley rat.

Spontaneous nephroblastomas are uncommon tumors of laboratory rats. This report describes a spontaneous nephroblastoma with peritoneal metastasis in an 11-month-old, female Sprague Dawley rat. The rat was part of a breeding program and presented 15 days post parturition with clinical signs including tachypnea, dyspnea and abdominal distension. At necropsy, the right kidney was markedly enlarged by an expansile pale-tan to white multinodular mass with extension into the retroperitoneal space, with multifocal variably sized nodules involving the mesentery, and surface of pancreas, liver, uterus, and ovarian bursa. The rat also had severe bicavitary effusion. Histologically, the renal parenchyma of the affected kidney was replaced by a moderately cellular, poorly-demarcated, non-encapsulated, multilobulated mass that appeared to compress the adjacent renal outer medulla and cortex. Three distinct neoplastic cell populations were identified in this renal tumor: epithelial cells (convoluted and dilated tubules / rare primitive glomeruloid structures), mesenchymal (neoplastic spindle cells in connective tissue), and blastemal cells (primitive neoplastic cells). The extrarenal nodular masses were predominantly composed of neoplastic mesenchymal and pleomorphic blastemal cells. Immunohistochemically, neoplastic epithelial cells in the renal mass were positive for pancytokeratin, and blastemal cells in both renal and extrarenal masses were positive for Wilms' tumor 1 protein (WT1) and vimentin. Neoplastic mesenchymal elements in both renal and extrarenal masses were positive for vimentin. The neoplasm was negative for chromogranin A and S100. The tumor was classified as an anaplastic nephroblastoma with metastasis to the mesentery and peritoneal organs.

Implantable Nanosensors for Human Steroid Hormone Sensing In Vivo Using a Self-Templating Corona Phase Molecular Recognition.

Dynamic measurements of steroid hormones in vivo are critical, but steroid sensing is currently limited by the availability of specific molecular recognition elements due to the chemical similarity of these hormones. In this work, a new, self-templating synthetic approach is applied using corona phase molecular recognition (CoPhMoRe) targeting the steroid family of molecules to produce near infrared fluorescent, implantable sensors. A key limitation of CoPhMoRe has been its reliance on library generation for sensor screening. This problem is addressed with a self-templating strategy of polymer design, using the examples of progesterone and cortisol sensing based on a styrene and acrylic acid copolymer library augmented with an acrylated steroid. The pendant steroid attached to the corona backbone is shown to self-template the phase, providing a unique CoPhMoRE design strategy with high efficacy. The resulting sensors exhibit excellent stability and reversibility upon repeated analyte cycling. It is shown that molecular recognition using such constructs is viable even in vivo after sensor implantation into a murine model by employing a poly (ethylene glycol) diacrylate (PEGDA) hydrogel and porous cellulose interface to limit nonspecific absorption. The results demonstrate that CoPhMoRe templating is sufficiently robust to enable a new class of continuous, in vivo biosensors.

Identification of a new strain of mouse kidney parvovirus associated with inclusion body nephropathy in immunocompromised laboratory mice.

Inclusion body nephropathy (IBN) and kidney fibrosis in aged immunodeficient mice and, to lesser extent, in immunocompetent mice have been recently linked to infection of mouse kidney parvovirus (MKPV), also known as murine chapparvovirus (MuCPV). Knowledge about its prevalence and the complete genome sequence of more MKPV strains is essential for understanding phylogenetic relationships and pathogenicity among MKPV strains. In the present study using PCR and genome walking, we determined the complete 4440-nucleotide genome of a new MKPV strain, namely MIT-WI1, which was identified in IBN-affected Il2rg-/-Rag2-/- c-Kit W-sh/W-sh mice housed in the vivarium at Whitehead Institute for Biomedical Research (WI). The overall nucleotide (>94%) and deduced amino acid sequences (>98%) of p10, p15, NS1 (replicase), NS2 and VP1 (capsid protein) within the MIT-WI1 genome, are closely related to MKPV/MuCPV strains described in laboratory and wild Mus musculus mice. In addition, PCR and qPCR assays using newly designed primers conserved among the known MKPV/MuCPV genomes were developed and utilized to assess MKPV status in selected laboratory mice. MKPV was also detected in immunodeficient (NSG) and immunocompetent (Crl:CD1(ICR), UTXflox) mouse strains/stocks. The abundance of the MKPV genome copies was significantly correlated with the severity of IBN. Our data indicate that MKPV is present in selected mouse strains/stocks, and provides new insights into the genome evolution of MKPV.