Commensal Microbiota Promote Lung Cancer Development via γδ T Cells.

Lung cancer is closely associated with chronic inflammation, but the causes of inflammation and the specific immune mediators have not been fully elucidated. The lung is a mucosal tissue colonized by a diverse bacterial community, and pulmonary infections commonly present in lung cancer patients are linked to clinical outcomes. Here, we provide evidence that local microbiota provoke inflammation associated with lung adenocarcinoma by activating lung-resident γδ T cells. Germ-free or antibiotic-treated mice were significantly protected from lung cancer development induced by Kras mutation and p53 loss. Mechanistically, commensal bacteria stimulated Myd88-dependent IL-1ß and IL-23 production from myeloid cells, inducing proliferation and activation of Vγ6+Vδ1+ γδ T cells that produced IL-17 and other effector molecules to promote inflammation and tumor cell proliferation. Our findings clearly link local microbiota-immune crosstalk to lung tumor development and thereby define key cellular and molecular mediators that may serve as effective targets in lung cancer intervention.

Ketone Body Signaling Mediates Intestinal Stem Cell Homeostasis and Adaptation to Diet.

Little is known about how metabolites couple tissue-specific stem cell function with physiology. Here we show that, in the mammalian small intestine, the expression of Hmgcs2 (3-hydroxy-3-methylglutaryl-CoA synthetase 2), the gene encoding the rate-limiting enzyme in the production of ketone bodies, including beta-hydroxybutyrate (ßOHB), distinguishes self-renewing Lgr5+ stem cells (ISCs) from differentiated cell types. Hmgcs2 loss depletes ßOHB levels in Lgr5+ ISCs and skews their differentiation toward secretory cell fates, which can be rescued by exogenous ßOHB and class I histone deacetylase (HDAC) inhibitor treatment. Mechanistically, ßOHB acts by inhibiting HDACs to reinforce Notch signaling, instructing ISC self-renewal and lineage decisions. Notably, although a high-fat ketogenic diet elevates ISC function and post-injury regeneration through ßOHB-mediated Notch signaling, a glucose-supplemented diet has the opposite effects. These findings reveal how control of ßOHB-activated signaling in ISCs by diet helps to fine-tune stem cell adaptation in homeostasis and injury.

Tissue-specific abundance of interferon-gamma drives regulatory T cells to restrain DC1-mediated priming of cytotoxic T cells against lung cancer.

Local environmental factors influence CD8+ T cell priming in lymph nodes (LNs). Here, we sought to understand how factors unique to the tumor-draining mediastinal LN (mLN) impact CD8+ T cell responses toward lung cancer. Type 1 conventional dendritic cells (DC1s) showed a mLN-specific failure to induce robust cytotoxic T cells responses. Using regulatory T (Treg) cell depletion strategies, we found that Treg cells suppressed DC1s in a spatially coordinated manner within tissue-specific microniches within the mLN. Treg cell suppression required MHC II-dependent contact between DC1s and Treg cells. Elevated levels of IFN-γ drove differentiation Treg cells into Th1-like effector Treg cells in the mLN. In patients with cancer, Treg cell Th1 polarization, but not CD8+/Treg cell ratios, correlated with poor responses to checkpoint blockade immunotherapy. Thus, IFN-γ in the mLN skews Treg cells to be Th1-like effector Treg cells, driving their close interaction with DC1s and subsequent suppression of cytotoxic T cell responses.

Gremlin 1 identifies a skeletal stem cell with bone, cartilage, and reticular stromal potential.

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).

ILC3s select microbiota-specific regulatory T cells to establish tolerance in the gut.

Microbial colonization of the mammalian intestine elicits inflammatory or tolerogenic T cell responses, but the mechanisms controlling these distinct outcomes remain poorly understood, and accumulating evidence indicates that aberrant immunity to intestinal microbiota is causally associated with infectious, inflammatory and malignant diseases1-8. Here we define a critical pathway controlling the fate of inflammatory versus tolerogenic T cells that respond to the microbiota and express the transcription factor RORγt. We profiled all RORγt+ immune cells at single-cell resolution from the intestine-draining lymph nodes of mice and reveal a dominant presence of T regulatory (Treg) cells and lymphoid tissue inducer-like group 3 innate lymphoid cells (ILC3s), which co-localize at interfollicular regions. These ILC3s are distinct from extrathymic AIRE-expressing cells, abundantly express major histocompatibility complex class II, and are necessary and sufficient to promote microbiota-specific RORγt+ Treg cells and prevent their expansion as inflammatory T helper 17 cells. This occurs through ILC3-mediated antigen presentation, αV integrin and competition for interleukin-2. Finally, single-cell analyses suggest that interactions between ILC3s and RORγt+ Treg cells are impaired in inflammatory bowel disease. Our results define a paradigm whereby ILC3s select for antigen-specific RORγt+ Treg cells, and against T helper 17 cells, to establish immune tolerance to the microbiota and intestinal health.

Opportunities and limitations of genetically modified nonhuman primate models for neuroscience research.

The recently developed new genome-editing technologies, such as the CRISPR/Cas system, have opened the door for generating genetically modified nonhuman primate (NHP) models for basic neuroscience and brain disorders research. The complex circuit formation and experience-dependent refinement of the human brain are very difficult to model in vitro, and thus require use of in vivo whole-animal models. For many neurodevelopmental and psychiatric disorders, abnormal circuit formation and refinement might be at the center of their pathophysiology. Importantly, many of the critical circuits and regional cell populations implicated in higher human cognitive function and in many psychiatric disorders are not present in lower mammalian brains, while these analogous areas are replicated in NHP brains. Indeed, neuropsychiatric disorders represent a tremendous health and economic burden globally. The emerging field of genetically modified NHP models has the potential to transform our study of higher brain function and dramatically facilitate the development of effective treatment for human brain disorders. In this paper, we discuss the importance of developing such models, the infrastructure and training needed to maximize the impact of such models, and ethical standards required for using these models.

PD-1 Signaling Promotes Tumor-Infiltrating Myeloid-Derived Suppressor Cells and Gastric Tumorigenesis in Mice.

BACKGROUND & AIMS: Immune checkpoint inhibitors have limited efficacy in many tumors. We investigated mechanisms of tumor resistance to inhibitors of programmed cell death-1 (PDCD1, also called PD-1) in mice with gastric cancer, and the role of its ligand, PD-L1. METHODS: Gastrin-deficient mice were given N-methyl-N-nitrosourea (MNU) in drinking water along with Helicobacter felis to induce gastric tumor formation; we also performed studies with H/K-ATPase-hIL1B mice, which develop spontaneous gastric tumors at the antral-corpus junction and have parietal cells that constitutively secrete interleukin 1B. Mice were given injections of an antibody against PD-1 or an isotype control before tumors developed, or anti-PD-1 and 5-fluorouracil and oxaliplatin, or an antibody against lymphocyte antigen 6 complex locus G (also called Gr-1), which depletes myeloid-derived suppressor cells [MDSCs]), after tumors developed. We generated knock-in mice that express PD-L1 specifically in the gastric epithelium or myeloid lineage. RESULTS: When given to gastrin-deficient mice before tumors grew, anti-PD-1 significantly reduced tumor size and increased tumor infiltration by T cells. However, anti-PD-1 alone did not have significant effects on established tumors in these mice. Neither early nor late anti-PD-1 administration reduced tumor growth in the presence of MDSCs in H/K-ATPase-hIL-1ß mice. The combination of 5-fluorouracil and oxaliplatin reduced MDSCs, increased numbers of intra-tumor CD8+ T cells, and increased the response of tumors to anti-PD-1; however, this resulted in increased tumor expression of PD-L1. Expression of PD-L1 by tumor or immune cells increased gastric tumorigenesis in mice given MNU. Mice with gastric epithelial cells that expressed PD-L1 did not develop spontaneous tumors, but they developed more and larger tumors after administration of MNU and H felis, with accumulation of MDSCs. CONCLUSIONS: In mouse models of gastric cancer, 5-fluorouracil and oxaliplatin reduce numbers of MDSCs to increase the effects of anti-PD-1, which promotes tumor infiltration by CD8+ T cells. However, these chemotherapeutic agents also induce expression of PD-L1 by tumor cells. Expression of PD-L1 by gastric epithelial cells increases tumorigenesis in response to MNU and H felis, and accumulation of MDSCs, which promote tumor progression. The timing and site of PD-L1 expression is therefore important in gastric tumorigenesis and should be considered in design of therapeutic regimens.

Dichotomous regulation of group 3 innate lymphoid cells by nongastric Helicobacter species.

Intestinal innate lymphoid cells (ILCs) contribute to the protective immunity and homeostasis of the gut, and the microbiota are critically involved in shaping ILC function. However, the role of the gut microbiota in regulating ILC development and maintenance still remains elusive. Here, we identified opposing effects on ILCs by two Helicobacter species, Helicobacter apodemus and Helicobacter typhlonius, isolated from immunocompromised mice. We demonstrated that the introduction of both Helicobacter species activated ILCs and induced gut inflammation; however, these Helicobacter species negatively regulated RORγt+ group 3 ILCs (ILC3s), especially T-bet+ ILC3s, and diminished their proliferative capacity. Thus, these findings underscore a previously unknown dichotomous regulation of ILC3s by Helicobacter species, and may serve as a model for further investigations to elucidate the host-microbe interactions that critically sustain the maintenance of intestinal ILC3s.

Helicobacter pylori Antimicrobial Resistance and Gene Variants in High- and Low-Gastric-Cancer-Risk Populations.

Colombia, South America has one of the world's highest burdens of Helicobacter pylori infection and gastric cancer. While multidrug antibiotic regimens can effectively eradicate H. pylori, treatment efficacy is being jeopardized by the emergence of antibiotic-resistant H. pylori strains. Moreover, the spectrum of and genetic mechanisms for antibiotic resistance in Colombia is underreported. In this study, 28 H. pylori strains isolated from gastric biopsy specimens from a high-gastric-cancer-risk (HGCR) population living in the Andes Mountains in Túquerres, Colombia and 31 strains from a low-gastric-cancer-risk (LGCR) population residing on the Pacific coast in Tumaco, Colombia were subjected to antibiotic susceptibility testing for amoxicillin, clarithromycin, levofloxacin, metronidazole, rifampin, and tetracycline. Resistance-associated genes were amplified by PCR for all isolates, and 29 isolates were whole-genome sequenced (WGS). No strains were resistant to amoxicillin, clarithromycin, or rifampin. One strain was resistant to tetracycline and had an A926G mutation in its 16S rRNA gene. Levofloxacin resistance was observed in 12/59 isolates and was significantly associated with N87I/K and/or D91G/Y mutations in gyrA Most isolates were resistant to metronidazole; this resistance was significantly higher in the LGCR (31/31) group compared to the HGCR (24/28) group. Truncations in rdxA and frxA were present in nearly all metronidazole-resistant strains. There was no association between phylogenetic relationship and resistance profiles based on WGS analysis. Our results indicate H. pylori isolates from Colombians exhibit multidrug antibiotic resistance. Continued surveillance of H. pylori antibiotic resistance in Colombia is warranted in order to establish appropriate eradication treatment regimens for this population.

High-Fat Diet Accelerates Carcinogenesis in a Mouse Model of Barrett's Esophagus via Interleukin 8 and Alterations to the Gut Microbiome.

BACKGROUND & AIMS: Barrett's esophagus (BE) is a precursor to esophageal adenocarcinoma (EAC). Progression from BE to cancer is associated with obesity, possibly due to increased abdominal pressure and gastroesophageal reflux disease, although this pathogenic mechanism has not been proven. We investigated whether environmental or dietary factors associated with obesity contribute to the progression of BE to EAC in mice. METHODS: Tg(ED-L2-IL1RN/IL1B)#Tcw mice (a model of BE, called L2-IL1B mice) were fed a chow (control) or high-fat diet (HFD) or were crossbred with mice that express human interleukin (IL) 8 (L2-IL1B/IL8 mice). Esophageal tissues were collected and analyzed for gene expression profiles and by quantitative polymerase chain reaction, immunohistochemistry, and flow cytometry. Organoids were established from BE tissue of mice and cultured with serum from lean or obese individuals or with neutrophils from L2-IL1B mice. Feces from mice were analyzed by 16s ribosomal RNA sequencing and compared to 16s sequencing data from patients with dysplasia or BE. L2-IL1B were mice raised in germ-free conditions. RESULTS: L2-IL1B mice fed an HFD developed esophageal dysplasia and tumors more rapidly than mice fed the control diet; the speed of tumor development was independent of body weight. The acceleration of dysplasia by the HFD in the L2-IL1B mice was associated with a shift in the gut microbiota and an increased ratio of neutrophils to natural killer cells in esophageal tissues compared with mice fed a control diet. We observed similar differences in the microbiomes from patients with BE that progressed to EAC vs patients with BE that did not develop into cancer. Tissues from dysplasias of L2-IL1B mice fed the HFD contained increased levels of cytokines that are produced in response to CXCL1 (the functional mouse homolog of IL8, also called KC). Serum from obese patients caused organoids from L2-IL1B/IL8 mice to produce IL8. BE tissues from L2-IL1B mice fed the HFD and from L2-IL1B/IL8 mice contained increased numbers of myeloid cells and cells expressing Cxcr2 and Lgr5 messenger RNAs (epithelial progenitors) compared with mice fed control diets. BE tissues from L2-IL1B mice raised in germ-free housing had fewer progenitor cells and developed less dysplasia than in L2-IL1 mice raised under standard conditions; exposure of fecal microbiota from L2-IL1B mice fed the HFD to L2-IL1B mice fed the control diet accelerated tumor development. CONCLUSIONS: In a mouse model of BE, we found that an HFD promoted dysplasia by altering the esophageal microenvironment and gut microbiome, thereby inducing inflammation and stem cell expansion, independent of obesity.

Gamma-glutamyltranspeptidase expression by Helicobacter saguini, an enterohepatic Helicobacter species isolated from cotton top tamarins with chronic colitis.

BACKGROUND: Helicobacter saguini is a novel enterohepatic Helicobacter species isolated from captive cotton top tamarins with chronic colitis and colon cancer. Monoassociated H. saguini infection in gnotobiotic IL-10-/- mice causes typhlocolitis and dysplasia; however, the virulent mechanisms of this species are unknown. Gamma-glutamyltranspeptidase (GGT) is an enzymatic virulence factor expressed by pathogenic Helicobacter and Campylobacter species that inhibits host cellular proliferation and promotes inflammatory-mediated gastrointestinal pathology. The aim of this study was to determine if H. saguini expresses an enzymatically active GGT homologue with virulence properties. EXPERIMENTAL PROCEDURES: Two putative GGT paralogs (HSGGT1 and HSGGT2) identified in the H. saguini genome were bioinformatically analysed to predict enzymatic functionality and virulence potential. An isogenic knockout mutant strain and purified recombinant protein of HSGGT1 were created to study enzymatic activity and virulence properties by in vitro biochemical and cell culture experiments. RESULTS: Bioinformatic analysis predicted that HSGGT1 has enzymatic functionality and is most similar to the virulent homologue expressed by Helicobacter bilis, whereas HSGGT2 contains putatively inactivating mutations. An isogenic knockout mutant strain and recombinant HSGGT1 protein were successfully created and demonstrated that H. saguini has GGT enzymatic activity. Recombinant HSGGT1 protein and sonicate from wild-type but not mutant H. saguini inhibited gastrointestinal epithelial and lymphocyte cell proliferation without evidence of cell death. The antiproliferative effect by H. saguini sonicate or recombinant HSGGT1 protein could be significantly prevented with glutamine supplementation or the GGT-selective inhibitor acivicin. Recombinant HSGGT1 protein also induced proinflammatory gene expression in colon epithelial cells. CONCLUSIONS: This study shows that H. saguini may express GGT as a potential virulence factor and supports further in vitro and in vitro studies into how GGT expression by enterohepatic Helicobacter species influences the pathogenesis of gastrointestinal inflammatory diseases.

Helicobacter monodelphidis sp. nov. and Helicobacter didelphidarum sp. nov., isolated from grey short-tailed opossums (Monodelphis domestica) with endemic cloacal prolapses.

In a search for potential causes of increased prolapse incidence in grey short-tailed opossum colonies, samples from the gastrointestinal tracts of 94 clinically normal opossums with rectal prolapses were screened for Helicobacter species by culture and PCR. Forty strains of two novel Helicobacter species which differed from the established Helicobacter taxa were isolated from opossums with and without prolapses. One of the Helicobacter species was spiral-shaped and urease-negative whereas the other Helicobacter strain had fusiform morphology with periplasmic fibres and was urease-positive. 16S rRNA gene sequence analysis revealed that all the isolates had over 99 % sequence identity with each other, and were most closely related to Helicobacter canadensis. Strains from the two novel Helicobacter species were subjected to gyrB and hsp60 gene and whole genome sequence analyses. These two novel Helicobacter species formed separate phylogenetic clades, divergent from other known Helicobacter species. The bacteria were confirmed as novel Helicobacter species based on digital DNA-DNA hybridization and average nucleotide identity analysis of their genomes, for which we propose the names Helicobacter monodelphidis sp. nov. with the type strain MIT 15-1451T (=LMG 29780T=NCTC 14189T) and Helicobacter didelphidarum sp. nov with type strain MIT 17-337T (=LMG 31024T=NCTC 14188T).

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.

Multi-Omics Characterization of Inflammatory Bowel Disease-Induced Hyperplasia/Dysplasia in the Rag2-/-/Il10-/- Mouse Model.

Epigenetic dysregulation is hypothesized to play a role in the observed association between inflammatory bowel disease (IBD) and colon tumor development. In the present work, DNA methylome, hydroxymethylome, and transcriptome analyses were conducted in proximal colon tissues harvested from the Helicobacter hepaticus (H. hepaticus)-infected murine model of IBD. Reduced representation bisulfite sequencing (RRBS) and oxidative RRBS (oxRRBS) analyses identified 1606 differentially methylated regions (DMR) and 3011 differentially hydroxymethylated regions (DhMR). These DMR/DhMR overlapped with genes that are associated with gastrointestinal disease, inflammatory disease, and cancer. RNA-seq revealed pronounced expression changes of a number of genes associated with inflammation and cancer. Several genes including Duox2, Tgm2, Cdhr5, and Hk2 exhibited changes in both DNA methylation/hydroxymethylation and gene expression levels. Overall, our results suggest that chronic inflammation triggers changes in methylation and hydroxymethylation patterns in the genome, altering the expression of key tumorigenesis genes and potentially contributing to the initiation of colorectal cancer.

Risk and characteristics of gastric carcinoma in the chow chow dog.

Gastric carcinoma is not commonly reported in dogs. There is an increased risk, however, in certain breeds such as the Belgian Tervuren. Review of the Veterinary Medical Database (VMDB) established an increase in risk for gastric carcinoma in the chow chow breed. In 106 chow chow dogs signs commenced, on average, 3 weeks before definitive diagnosis. The most common clinical signs were vomiting, loss of appetite, diarrhea, and melena. Most affected dogs were euthanized, without treatment, within 2 weeks of diagnosis. Two dogs which were treated aggressively (surgery and chemotherapy) survived a considerably longer time (12 and 36 months). Histologically, these chow chow dogs comprised a similar histologic type as familial gastric carcinoma in humans; diffuse-type carcinoma that was enriched in the signet ring and mucinous variants. Understanding the pathogenesis of diffuse gastric carcinoma in the chow chow dog may provide insight into the biology of this aggressive cancer in humans.

Risques et caractéristiques d'un carcinome gastrique chez le chien de race chow-chow. Le carcinome gastrique n'est par rapporté fréquemment chez les chiens. Il y a toutefois une augmentation du risque chez certaines espèces telle que le Tervuren belge. Une revue de la base de données Veterinary Medical Database (VMDB) a établi une augmentation dans le risque pour le carcinome gastrique chez la race chow chow. Chez 106 chiens chow chow les signes débutèrent, en moyenne, 3 semaines avant le diagnostic définitif. Les signes cliniques les plus fréquents étaient vomissement, perte d'appétit, diarrhée et méléna. La plupart des chiens affectés furent euthanasiés, sans traitement, à l'intérieur de 2 semaines du diagnostic. Deux chiens furent traités de manière agressive (chirurgie et chimiothérapie) ont survécu beaucoup plus longtemps (12 et 36 mois). Histologiquement, ces chiens chow chow comprennent un type histologique similaire aux carcinomes gastrique familiaux chez les humains; le carcinome de type-diffus qui s'est développé dans les variants de cellules en bague à chatons et mucineux. Comprendre la pathogénie du carcinome gastrique diffus chez le chien chow chow pourrait fournir des informations sur la biologie de ce cancer agressif chez l'humain.(Traduit par Dr Serge Messier).

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.

Muc5ac null mice are predisposed to spontaneous gastric antro-pyloric hyperplasia and adenomas coupled with attenuated H. pylori-induced corpus mucous metaplasia.

Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide and is strongly associated with chronic Helicobacter pylori (Hp) infection. The ability of Hp to closely adhere to the gastric surface protective mucous layer containing mucins (MUC in humans and Muc in animals), primarily Muc5ac, is integral in the stepwise pathogenesis from gastritis to cancer. To probe the role of Muc5ac in Hp-induced gastric pathology, Muc5ac-/- and Muc5ac+/+ (WT) mice were experimentally infected with Hp Sydney strain (SS1). At 16 weeks and 32 weeks post infection (wpi), groups of mice were euthanized and evaluated for the following: gastric histopathological parameters, immunohistochemical expression of mucins (Muc5ac, Muc1, Muc2), Trefoil factor family proteins (Tff1 and Tff2), Griffonia (Bandeiraea) simplicifolia lectin II (GSL II) (mucous metaplasia marker) and Clusterin (Spasmolytic Polypeptide Expressing Metaplasia (SPEM) marker), Hp colonization density by qPCR and gastric cytokine mRNA levels. Our results demonstrate that Muc5ac-/- mice developed spontaneous antro-pyloric proliferation, adenomas and in one case with neuroendocrine differentiation; these findings were independent of Hp infection along with strong expression levels of Tff1, Tff2 and Muc1. Hp-infected Muc5ac-/- mice had significantly lowered gastric corpus mucous metaplasia at 16 wpi and 32 wpi (P = 0.0057 and P = 0.0016, respectively), with a slight reduction in overall gastric corpus pathology. GSII-positive mucous neck cells were decreased in Hp-infected Muc5ac-/- mice compared to WT mice and clusterin positivity was noted within metaplastic glands in both genotypes following Hp infection. Additionally, Hp colonization densities were significantly higher in Muc5ac-/- mice compared to WT at 16 wpi in both sexes (P = 0.05) along with a significant reduction in gastric Tnfα (16 wpi-males and females, P = 0.017 and P = 0.036, respectively and 32 wpi-males only, P = 0.025) and Il-17a (16 wpi-males) (P = 0.025). Taken together, our findings suggest a protective role for MUC5AC/Muc5ac in maintaining gastric antral equilibrium and inhibiting Hp colonization and associated inflammatory pathology.

Mutagenicity of Helicobacter hepaticus infection in the lower bowel mucosa of 129/SvEv Rag2-/- Il10-/- gpt delta mice is influenced by sex.

Inflammatory bowel disease and colonic tumors induced by Helicobacter hepaticus (Hh) infection in susceptible mouse strains are utilized to dissect the mechanisms underlying similar human diseases. In our study, infection with genotoxic cytolethal distending toxin-producing Hh in 129/SvEv Rag2-/- Il10-/- gpt delta (RagIl10gpt) mice of both sexes for 21 weeks induced significantly more severe cecal and colonic pathology compared to uninfected controls. The mutation frequencies in the infected RagIl10gpt males were 2.1-fold higher for the cecum and 1.7-fold higher for the colon than male RagIl10gpt controls. In addition, there was a 12.5-fold increase of G:C-to-T:A transversions in the colon of Hh-infected males compared to controls. In contrast, there was no statistical significance in mutation frequencies between infected female Rag2Il10gpt mice and controls. Moreover, Hh infection in RagIl10gpt males significantly up-regulated transcription of Tnfα and iNos, and decreased mRNA levels of cecal Atm compared to the infected females; there was no significant difference in mRNA levels of Il-22, Il-17A, Ifnγ and Atr between the infected males and females. Significantly higher levels of cecal and colonic iNos expression and γH2AX-positive epithelial cells (a biomarker for double-strand DNA breaks [DSB]) in Hh-infected Rag2Il10gpt males vs. Hh-infected females were noted. Finally, Hh infection and associated inflammation increased levels of intestinal mucosa-associated genotoxic colibactin-producing pks+ Escherichia coli. Elevated Tnfα and iNos responses and bacterial genotoxins, in concert with suppression of the DSB repair responses, may have promoted mutagenesis in the lower bowel mucosa of Hh-infected male RagIl10gpt mice.

Loss of Tight Junction Protein Claudin 18 Promotes Progressive Neoplasia Development in Mouse Stomach.

BACKGROUND & AIMS: Loss of claudin 18 (CLDN18), a membrane-spanning tight junction protein, occurs during early stages of development of gastric cancer and associates with shorter survival times of patients. We investigated whether loss of CLDN18 occurs in mice that develop intraepithelial neoplasia with invasive glands due to infection with Helicobacter pylori, and whether loss is sufficient to promote the development of similar lesions in mice with or without H pylori infection. METHODS: We performed immunohistochemical analyses in levels of CLDN18 in archived tissues from B6:129 mice infected with H pylori for 6 to 15 months. We analyzed gastric tissues from B6:129S5-Cldn18tm1Lex/Mmucd mice, in which the CLDN18 gene was disrupted in gastric tissues (CLDN18-knockout mice), or from control mice with a full-length CLDN18 gene (CLDN18+/+; B6:129S5/SvEvBrd) or heterozygous disruption of CLDN18 (CLDN18+/-; B6:129S5/SvEvBrd) that were infected with H pylori SS1 or PMSS1 at 6 weeks of age and tissues collected for analysis at 20 and 30 weeks after infection. Tissues from CLDN18-knockout mice and control mice with full-length CLDN18 gene expression were also analyzed without infection at 7 weeks and 2 years after birth. Tissues from control and CLDN18-knockout mice were analyzed by electron microscopy, stained by conventional methods and analyzed for histopathology, prepared by laser capture microdissection and analyzed by RNAseq, and immunostained for lineage markers, proliferation markers, and stem cell markers and analyzed by super-resolution or conventional confocal microscopy. RESULTS: CLDN18 had a basolateral rather than apical tight junction localization in gastric epithelial cells. B6:129 mice infected with H pylori, which developed intraepithelial neoplasia with invasive glands, had increasing levels of CLDN18 loss over time compared with uninfected mice. In B6:129 mice infected with H pylori compared with uninfected mice, CLDN18 was first lost from most gastric glands followed by disrupted and reduced expression in the gastric neck and in surface cells. Gastric tissues from CLDN18-knockout mice had low levels of inflammation but increased cell proliferation, expressed markers of intestinalized proliferative spasmolytic polypeptide-expressing metaplasia, and had defects in signal transduction pathways including p53 and STAT signaling by 7 weeks after birth compared with full-length CLDN18 gene control mice. By 20 to 30 weeks after birth, gastric tissues from uninfected CLDN18-knockout mice developed intraepithelial neoplasia that invaded the submucosa; by 2 years, gastric tissues contained large and focally dysplastic polypoid tumors with invasive glands that invaded the serosa. CONCLUSIONS: H pylori infection of B6:129 mice reduced the expression of CLDN18 early in gastric cancer progression, similar to previous observations from human gastric tissues. CLDN18 regulates cell lineage differentiation and cellular signaling in mouse stomach; CLDN18-knockout mice develop intraepithelial neoplasia and then large and focally dysplastic polypoid tumors in the absence of H pylori infection.

Characterization of Campylobacter jejuni, Campylobacter upsaliensis, and a novel Campylobacter sp. in a captive non-human primate zoological collection.

BACKGROUND: The aim of this study was to longitudinally investigate the prevalence and characterization of Campylobacter spp. from non-human primates primate (NHP) with a history of endemic diarrhea housed at Como Park Zoo. METHODS: Fecal samples from 33 symptom-free NHP belonging to eight different species were collected weekly for 9 weeks. Species-level characterization and phylogenetic analysis of isolates included biochemical testing and 16S rRNA sequencing. RESULTS: Campylobacter spp. were isolated from the feces of 42% (14/33) of the primates. Three Campylobacter spp. (C upsaliensis, C jejuni, and novel Campylobacter sp.) were identified from three NHP species. A possible positive host Campylobacter species-specificity was observed. However, no statistical association was observed between the isolation of Campylobacter spp. and age and sex of the animal. CONCLUSIONS: The study revealed the value of conducting repeated fecal sampling to establish the overall prevalence of Campylobacter in zoo-maintained NHP; it also importantly identifies a novel Campylobacter sp. isolated from white-faced saki monkeys.