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.

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.

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.

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.

Cytotoxic Escherichia coli strains encoding colibactin, cytotoxic necrotizing factor, and cytolethal distending toxin colonize laboratory common marmosets (Callithrix jacchus).

Cyclomodulins are virulence factors that modulate cellular differentiation, apoptosis, and proliferation. These include colibactin (pks), cytotoxic necrotizing factor (cnf), and cytolethal distending toxin (cdt). Pathogenic pks+, cnf+, and cdt+ E. coli strains are associated with inflammatory bowel disease (IBD) and colorectal cancer in humans and animals. Captive marmosets are frequently afflicted with IBD-like disease, and its association with cyclomodulins is unknown. Cyclomodulin-encoding E. coli rectal isolates were characterized using PCR-based assays in healthy and clinically affected marmosets originating from three different captive sources. 139 E. coli isolates were cultured from 122 of 143 marmosets. The pks gene was detected in 56 isolates (40%), cnf in 47 isolates (34%), and cdt in 1 isolate (0.7%). The prevalences of pks+ and cnf+ E. coli isolates were significantly different between the three marmoset colonies. 98% of cyclomodulin-positive E. coli belonged to phylogenetic group B2. Representative isolates demonstrated cyclomodulin cytotoxicity, and serotyping and whole genome sequencing were consistent with pathogenic E. coli strains. However, the presence of pks+, cnf+, or cdt+ E. coli did not correlate with clinical gastrointestinal disease in marmosets. Cyclomodulin-encoding E. coli colonize laboratory common marmosets in a manner dependent on the source, potentially impacting reproducibility in marmoset models.

Comparison of tyrosine kinase domain properties for the neurotrophin receptors TrkA and TrkB.

The tropomyosin-related kinase (Trk) family consists of three receptor tyrosine kinases (RTKs) called TrkA, TrkB, and TrkC. These RTKs are regulated by the neurotrophins, a class of secreted growth factors responsible for the development and function of neurons. The Trks share a high degree of homology and utilize overlapping signaling pathways, yet their signaling is associated with starkly different outcomes in certain cancers. For example, in neuroblastoma, TrkA expression and signaling correlates with a favorable prognosis, whereas TrkB is associated with poor prognoses. To begin to understand how activation of the different Trks can lead to such distinct cellular outcomes, we investigated differences in kinase activity and duration of autophosphorylation for the TrkA and TrkB tyrosine kinase domains (TKDs). We find that the TrkA TKD has a catalytic efficiency that is ∼2-fold higher than that of TrkB, and becomes autophosphorylated in vitro more rapidly than the TrkB TKD. Studies with mutated TKD variants suggest that a crystallographic dimer seen in many TrkA (but not TrkB) TKD crystal structures, which involves the kinase-insert domain, may contribute to this enhanced TrkA autophosphorylation. Consistent with previous studies showing that cellular context determines whether TrkB signaling is sustained (promoting differentiation) or transient (promoting proliferation), we also find that TrkB signaling can be made more transient in PC12 cells by suppressing levels of p75NTR. Our findings shed new light on potential differences between TrkA and TrkB signaling, and suggest that subtle differences in signaling dynamics can lead to substantial shifts in the cellular outcome.

Evaluating rectal swab collection method for gut microbiome analysis in the common marmoset (Callithrix jacchus).

The common marmoset (Callithrix jacchus) is increasingly used as an animal model for biomedical research; however, gastrointestinal diseases causing significant morbidity are endemic in many captive marmoset colonies. Establishing gut microbiome patterns in a marmoset colony may aid in clinical decision-making and model reproducibility. A standardized method of sample collection and storage is essential for proper interpretation of microbiome data. While microbiome studies commonly utilize fecal samples, the goal of this study was to determine whether the microbiome profile from a rectal swab performed on a sedated animal was comparable to the microbiome profile from a fecal sample. During routine physical exams, paired fecal and rectal swab samples were collected from each of 23 marmosets. DNA was extracted from all fecal and rectal swab samples and 16S ribosomal RNA gene sequences were amplified and analyzed. Initial comparison of the relative abundance of bacterial phyla between paired samples had a r2 value of 0.70 with S of 0.08 with no significant differences in α and ß diversity metrics between fecal and rectal samples. Initial analysis however, revealed 5 discordant fecal-rectal pairs which corresponded only with the 5 rectal swabs that were classified as free of visible fecal matter during collection. Exclusion of these 5 pairs resulted in an optimized fit of the data as evidenced by a r2 value of 0.91 with S of 0.05. These results demonstrate that rectal swabs are a reliable method for profiling the fecal microbiome in the marmoset since the bacterial composition from a rectal swab with visible fecal contents correlated well with the bacterial composition from a fecal sample from the same marmoset. This study highlights the importance of standardized sample collection methods and exclusion of inappropriate samples.

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 pylori CagA promotes epithelial mesenchymal transition in gastric carcinogenesis via triggering oncogenic YAP pathway.

BACKGROUND: Helicobacter pylori (H. pylori) delivers oncoprotein CagA into gastric epithelial cells via the T4SS and drives activation of multiple oncogenic signalling pathways. YAP, a core effector of the Hippo tumour suppressor pathway, is frequently overexpressed in human cancers, suggesting its potential tumor-promoting role. Although CagA is a casual factor in H. pylori induced gastric carcinogenesis, the link between CagA and YAP pathway has not been identified. In this work, we investigated the regulation of oncogenic YAP pathway by H. pylori CagA. METHODS: Expression of YAP and E-cadherin protein in human gastric biopsies were assessed by immunohistochemistry. H. pylori PMSS1 cagA- isogenic mutant strains were generated. Gastric epithelial cells were co-cultured with H. pylori wild-type cagA+ strains or isogenic mutants and were also treated by recombinant CagA expression. Immunofluorescence was performed for YAP localization. Immunoblot and quantitative PCR were performed for examining levels of YAP, downstream effectors and markers of epithelial-mesenchymal transition. Verteporfin and siRNA silencing were used to inhibit YAP activity. RESULTS: YAP is significantly upregulated in human gastric carcinogenesis. We generated PMSS1 CagA isogenic mutant strains with chloramphenicol resistance successfully. Our analysis indicated that H. pylori infection induced YAP and downstream effectors in gastric epithelial cells. Importantly, knockout of CagA in 7.13 and PMSS1 strains reduced the expression of YAP by H. pylori infection. Moreover, Inhibition of YAP suppressed H. pylori infection-induced Epithelial-mesenchymal transition (EMT). CONCLUSION: Our results indicated that H. pylori CagA as a pathogenic protein promotes oncogenic YAP pathway, which contributes to EMT and gastric tumorigenesis. This study provided a novel mechanistic insight into why cagA+ H. pylori infection is associated with a higher risk for the development of gastric cancer.

Lung Lobe Torsion in an Adult Male Common Marmoset (Callithrix jacchus).

A 6-y-old, intact, pair-housed male common marmoset (Callithrix jacchus) presented with acute onset dyspnea and tachypnea immediately after sedation with alfaxalone; a history of gradual weight loss initiated the examination under sedation. Thoracic radiographs revealed significant right-lung consolidation, with a vesicular gas pattern in the right caudodorsal lung field, pleural effusion, and dorsal displacement of the heart. The marmoset was euthanized due to his unstable condition and poor prognosis. At necropsy, the cranial and middle lobes of the right lung were homogenously dark red-brown, enlarged, edematous, and twisted around the longitudinal axis at the hilus. The left lung lobes were pale pink and slightly edematous. In light of the clinical and gross necropsy findings, acute torsion of the right cranial and middle lung lobes was diagnosed. Predisposing conditions for lung lobe torsion include trauma, neoplasia, pulmonary disease, previous thoracic surgery, and diaphragmatic hernia, but none of these applied to this case. Initial therapy for lung lobe torsion is to stabilize the patient and treat for underlying conditions, with prompt surgical resection as the treatment of choice. To our knowledge, this report is the first description of lung lobe torsion in an experimentally unmanipulated New World NHP.

Host-directed combinatorial RNAi improves inhibition of diverse strains of influenza A virus in human respiratory epithelial cells.

Influenza A virus infections are important causes of morbidity and mortality worldwide, and currently available prevention and treatment methods are suboptimal. In recent years, genome-wide investigations have revealed numerous host factors that are required for influenza to successfully complete its life cycle. However, only a select, small number of influenza strains were evaluated using this platform, and there was considerable variation in the genes identified across different investigations. In an effort to develop a universally efficacious therapeutic strategy with limited potential for the emergence of resistance, this study was performed to investigate the effect of combinatorial RNA interference (RNAi) on inhibiting the replication of diverse influenza A virus subtypes and strains. Candidate genes were selected for targeting based on the results of multiple previous independent genome-wide studies. The effect of single and combinatorial RNAi on the replication of 12 diverse influenza A viruses, including three strains isolated from birds and one strain isolated from seals, was then evaluated in primary normal human bronchial epithelial cells. After excluding overly toxic siRNA, two siRNA combinations were identified that reduced mean viral replication by greater than 79 percent in all mammalian strains, and greater than 68 percent in all avian strains. Host-directed combinatorial RNAi effectively prevents growth of a broad range of influenza virus strains in vitro, and is a potential therapeutic candidate for further development and future in vivo studies.

Cutaneous Dermatophilosis in a Meadow Jumping Mouse (Zapus hudsonius).

A laboratory-housed, wild-caught, subadult, male meadow jumping mouse (Zapus hudsonius) presented with extensive scaling of the face, limbs, and tail and severe edema of the paws. Postmortem examination revealed marked distal limb edema with focal digital hematomas and white scales, scabs, and crusts affecting the majority of nonhaired skin. Histopathologic analysis revealed severe, multifocal, chronic-active exudative and proliferative dermatitis characterized by multilaminated crusts covering the epidermis. The epidermis was expanded by hyperkeratosis, acanthosis, and hyperplasia. The superficial dermis contained moderate edema, hemorrhage, and pigmentary incontinence, and was infiltrated by granulocytes and mononuclear cells. The laminated crusts contained numerous branching filaments of gram-positive coccoid bodies arranged in parallel rows, consistent with cutaneous Dermatophilus congolensis infection. This diagnosis was confirmed through bacterial culture and 16S rRNA PCR analysis. In the presented case, factors that might have contributed to disease progression include climatic conditions at the capture site and stress associated with trapping and laboratory housing.

Assessing the range of kinase autoinhibition mechanisms in the insulin receptor family.

To investigate the range of autoinhibitory mechanisms used by TKDs (tyrosine kinase domains) from the insulin receptor family of RTKs (receptor tyrosine kinases), we determined crystal structures of TKDs from TrkA (tropomyosin receptor kinase A, a nerve growth factor receptor) and Ror2 (receptor tyrosine kinase-like orphan receptor 2, an unconventional Wnt receptor). TrkA autoinhibition closely resembles that seen for the insulin receptor, relying on projection of an activation loop tyrosine residue into the substrate-binding site and occlusion of the ATP-binding site by the activation loop. Ror2 employs similar mechanisms, but the unusual replacement of the phenylalanine residue in its Asp-Phe-Gly motif with leucine necessitates occlusion of the ATP-binding site by other means. The unusual Asp-Leu-Gly motif in Ror2 is displaced compared with other inactive kinases, allowing the activation loop to interact directly with the TKD's αC helix, in another mode of autoinhibition that is characteristic of the other extreme of this receptor family: ALK (anaplastic lymphoma kinase) and Met. These findings provide insight into the expected range of activating mutations in these TKDs in cancer. We also describe symmetrical dimers of the inactive TrkA TKD resembling those found in other RTKs, possibly reflecting an arrangement of kinase domains in a pre-formed TrkA dimer.

Immature CD4+CD8+ thymocytes and mature T cells regulate Nur77 distinctly in response to TCR stimulation.

The orphan steroid receptor, Nur77, is thought to be a central participant in events leading to TCR-mediated clonal deletion of immature thymocytes. Interestingly, although both immature and mature murine T cell populations rapidly up-regulate Nur77 after TCR stimulation, immature CD4+CD8+ thymocytes respond by undergoing apoptosis, whereas their mature descendants respond by dividing. To understand these developmental differences in susceptibility to the proapoptotic potential of Nur77, we compared its regulation and compartmentalization and show that mature, but not immature, T cells hyperphosphorylate Nur77 in response to TCR signals. Nur77 resides in the nucleus of immature CD4+CD8+ thymocytes throughout the course of its expression and is not found in either the organellar or cytoplasmic fractions. However, hyperphosphorylation of Nur77 in mature T cells, which is mediated by both the MAPK and PI3K/Akt pathways, shifts its localization from the nucleus to the cytoplasm. The failure of immature CD4+CD8+ thymocytes to hyperphosphorylate Nur77 in response to TCR stimulation may be due in part to decreased Akt activity at this developmental stage.

A structural basis for 14-3-3sigma functional specificity.

The 14-3-3 family of proteins includes seven isotypes in mammalian cells that play numerous diverse roles in intracellular signaling. Most 14-3-3 proteins form homodimers and mixed heterodimers between different isotypes, with overlapping roles in ligand binding. In contrast, one mammalian isoform, 14-3-3sigma, expressed primarily in epithelial cells, appears to play a unique role in the cellular response to DNA damage and in human oncogenesis. The biological and structural basis for these 14-3-3sigma-specific functions is unknown. We demonstrate that endogenous 14-3-3sigma preferentially forms homodimers in cells. We have solved the x-ray crystal structure of 14-3-3sigma bound to an optimal phosphopeptide ligand at 2.4 angstroms resolution. The structure reveals the presence of stabilizing ring-ring and salt bridge interactions unique to the 14-3-3sigma homodimer structure and potentially destabilizing electrostatic interactions between subunits in 14-3-3sigma-containing heterodimers, rationalizing preferential homodimerization of 14-3-3sigma in vivo. The interaction of the phosphopeptide with 14-3-3 reveals a conserved mechanism for phospho-dependent ligand binding, implying that the phosphopeptide binding cleft is not the critical determinant of the unique biological properties of 14-3-3sigma. Instead, the structure suggests a second ligand binding site involved in 14-3-3sigma-specific ligand discrimination. We have confirmed this by site-directed mutagenesis of three sigma-specific residues that uniquely define this site. Mutation of these residues to the alternative sequence that is absolutely conserved in all other 14-3-3 isotypes confers upon 14-3-3sigma the ability to bind to Cdc25C, a ligand that is known to bind to other 14-3-3 proteins but not to sigma.