Gene expression of prostaglandin EP4 receptor in three canine carcinomas.

BACKGROUND: Chronic inflammation mediated by the cyclooxygenase enzymes, specifically their product prostaglandin E2 (PGE2), can result in the development of cancer. PGE2 promotes cell proliferation, apoptosis, and angiogenesis through interaction with its specific receptors (EP1 receptor - EP4 receptor [EP1R-EP4R]). In multiple human cancers, the expression of EP4R is associated with the development of malignancy and a poor prognosis. The expression of EP4R has not yet been evaluated in canine tumors. The aim of this study was to characterize the mRNA gene expression of EP4R (ptger4) in canine squamous cell carcinoma (SCC), apocrine gland anal sac adenocarcinoma (AGASACA), and transitional cell carcinoma (TCC). Archived tumor samples of canine cutaneous SCC (n = 9), AGASACA (n = 9), and TCC (n = 9), and matched archived normal tissue controls were evaluated for mRNA expression of canine EP4R using RNA in situ hybridization (RNAscope®). Quantification of RNAscope® signals in tissue sections was completed with an advanced digital pathology image analysis system (HALO). Data was expressed as copy number, H-index, and percent tumor cell expression of EP4R. RESULTS: In all canine SCC, AGASACA, and TCC samples evaluated, strong universal positive expression of EP4R was identified. For SCC and AGASACA, mRNA EP4R expression was statistically higher than that of their respective normal tissues. The TCC tissues displayed significantly less mRNA EP4R expression when compared to normal bladder mucosa. CONCLUSIONS: These results confirm the mRNA expression of canine EP4R in all tumor types evaluated, with SCC and AGASACA displaying the highest expression, and TCC displaying the lowest expression. This study also represents the first reported veterinary evaluation of EP4R expression using the novel in situ hybridization technique, RNAscope®.

Alterations in Intestinal Innate Mucosal Immunity of Weaned Pigs During Porcine Epidemic Diarrhea Virus Infection.

In the small intestine, localized innate mucosal immunity is critical for intestinal homeostasis. Porcine epidemic diarrhea virus (PEDV) infection induces villus injury and impairs digestive function. Moreover, the infection might comprise localized innate mucosal immunity. This study investigated specific enterocyte subtypes and innate immune components of weaned pigs during PEDV infection. Four-week-old pigs were orally inoculated with PEDV IN19338 strain (n = 40) or sham-inoculated (n = 24). At day post inoculation (DPI) 2, 4, and 6, lysozyme expression in Paneth cells, cellular density of villous and Peyer's patch microfold (M) cells, and the expression of polymeric immunoglobulin receptor (pIgR) were assessed in the jejunum and ileum by immunohistochemistry, and interleukin (IL)-1ß and tumor necrosis factor (TNF)-α were measured in the jejunum by ELISA. PEDV infection led to a decrease in the ratios of villus height to crypt depth (VH-CD) in jejunum at DPI 2, 4, and 6 and in ileum at DPI 4. The number of villous M cells was reduced in jejunum at DPI 4 and 6 and in ileum at DPI 6, while the number of Peyer's patch M cells in ileum increased at DPI 2 and then decreased at DPI 6. PEDV-infected pigs also had reduced lysozyme expression in ileal Paneth cells at DPI 2 and increased ileal pIgR expression at DPI 4. There were no significant changes in IL-1ß and TNF-α expression in PEDV-infected pigs compared to controls. In conclusion, PEDV infection affected innate mucosal immunity of weaned pigs through alterations in Paneth cells, villous and Peyer's patch M cells, and pIgR expression.

Quantification of Macrophages and Mycobacterium avium Subsp. paratuberculosis in Bovine Intestinal Tissue During Different Stages of Johne's Disease.

Johne's disease is an enteric disease caused by the intracellular pathogen Mycobacterium avium subsp. paratuberculosis (MAP). Upon ingestion of MAP, it is translocated across the intestinal epithelium and may be killed by intestinal macrophages, or depending on the bacterial burden and immunological status of the animal, MAP may thwart innate defense mechanisms and persist within the macrophage. This study aimed to determine the numbers of macrophages and MAP present in bovine midileal tissue during different stages of infection. Immunofluorescent (IF) labeling was performed on frozen bovine midileal intestinal tissue collected from 28 Holstein dairy cows. The number of macrophages in midileal tissue sections was higher for clinically affected cows, followed by subclinically affected cows and then uninfected control cows. Macrophages were present throughout the tissue sections in clinical cows, including the tunica muscularis, submucosa, and the lamina propria around the crypts and in the villous tips, with progressively fewer macrophages in subclinically affected and control cows. Clinically affected cows also demonstrated significantly higher numbers of MAP and higher numbers of macrophages with intracellular MAP compared to subclinically affected cows. MAP IF labeling was present within the submucosa and lamina propria around the crypts, progressing into the villous tips in some clinically affected cows. Our findings indicate that number of macrophages increases with progression of infection, but a significant number of the macrophages present in the midileum are not associated with MAP.

Adaptation and evaluation of a monopolar electrosurgical triangle-tip knife for transendoscopic ventriculocordectomy in healthy horses.

OBJECTIVE: To evaluate the efficacy and safety of a transendoscopic monopolar electrosurgical triangle-tip knife as instrumentation to perform unilateral ventriculocordectomy (VC) in healthy adult horses. STUDY DESIGN: In vivo experimental study. STUDY POPULATION: Nine horses donated for medical conditions unrelated to respiratory system. METHODS: The triangle-tip knife was applied in contact fashion. Left VC was performed under standing sedation. Endoscopic images of the upper airway were graded for inflammation by 2 masked surgeons preoperatively and immediately, 24 hours and, in 2 cases, 7 and 14 days postoperatively. Four larynxes were examined for histological evidence of inflammation and collagen deposition at 24 hours (n = 2) and at 14 days (n = 2) after surgery. RESULTS: Ventriculocordectomy was successfully performed in all horses. Endoscopic evidence of inflammation was scored as normal (preoperatively), mild (immediately postoperatively), mild (24 hours postoperatively), mild (7 days postoperatively), and normal (14 days postoperatively). According to histopathology, inflammation of the surgical site and ventricularis muscle was generally increased (variable is common and is present in most high-power fields) 24 hours and 14 days postoperatively. Fibrosis and collagen deposition also seemed increased at the surgical site 14 days postoperatively. CONCLUSION: Ventriculocordectomy was successfully performed with an electrosurgical triangle-tip knife and resulted in acceptable short-term outcomes. CLINICAL SIGNIFICANCE: The use of an electrosurgical triangle-tip knife alternative instrumentation may be offer an alternative option to perform VC in practices when diode laser is not available or is cost prohibitive. Longer term evaluation of the VC site is required to determine the effect on rima glottic cross-sectional area.

Pulmonary biodistribution and cellular uptake of intranasally administered monodisperse particles.

PURPOSE: For the rational design of nanovaccines against respiratory pathogens, careful selection of optimal particle size and chemistry is paramount. This work investigates the impact of these properties on the deposition, biodistribution, and cellular interactions of nanoparticles within the lungs. METHOD: In this work, biodegradable poly(sebacic anhydride) (poly(SA)) nanoparticles of multiple sizes were synthesized with narrow particle size distributions. The lung deposition and retention as well as the internalization by phagocytic cells of these particles were compared to that of non-degradable monodisperse polystyrene nanoparticles of similar sizes. RESULTS: The initial deposition of intranasally administered particles in the lungs was dependent on primary particle size, with maximal deposition occurring for the 360-470 nm particles, regardless of chemistry. Over time, both particle size and chemistry affected the frequency of particle-positive cells and the specific cell types taking up particles. The biodegradable poly(SA) particles associated more closely with phagocytic cells and the dynamics of this association impacted the clearance of these particles from the lung. CONCLUSIONS: The findings reported herein indicate that both size and chemistry control the fate of intranasally administered particles and that the dynamics of particle association with phagocytic cells in the lungs provide important insights for the rational design of pulmonary vaccine delivery vehicles.

The effects of topical oxygen therapy on equine distal limb dermal wound healing.

Topical oxygen therapy (TOT) has been used in human medicine to promote healing in chronic wounds. To test the efficacy and safety of TOT in horses, an experimental wound model was created by making 1 standardized dermal wound on each limb of 4 healthy horses (n = 16). Each wound was fitted with an oxygen delivery cannula and covered with a bandage. One limb of each front and hind pair was randomly assigned to the treatment group (fitted with an oxygen concentrator device), with the contralateral limb assigned to the control group (no device). Wound area, epithelial area, and contraction were measured every 3 to 4 d. Biopsy samples and culture swabs were taken on days 16 and 32 to evaluate angiogenesis, fibroplasia, epithelial hyperplasia, inflammation and bacterial growth. Mean healing time in treated wounds (45 d, range: 38 to 52 d) was not significantly different from that in the paired control wounds (50 d, range: 38 to 62 d). Topical oxygen therapy had little effect on dermal wound healing in this experimental wound model in healthy horses.

Effets de la thérapie à l'oxygène topique sur la guérison des blessures cutanées des membres distaux équins. La thérapie à l'oxygène topique (TOT) a été utilisée en médecine humaine pour traiter les blessures chroniques. Afin de tester l'efficacité et l'innocuité de la TOT chez les chevaux, un modèle de blessure expérimental a été créé en pratiquant une blessure cutanée normalisée chez 4 chevaux en santé (n = 16). Chaque blessure a été équipée d'une canule de distribution d'oxygène et couverte d'un pansement. Une jambe avant et une jambe arrière ont été assignées au hasard au groupe de traitement (équipée d'un dispositif de concentration d'oxygène) et la jambe controlatérale a été assignée au groupe témoin (aucun dispositif). La région de la blessure, la région épithéliale et les contractions ont été mesurées tous les 3 ou 4 jours. Des biopsies et des écouvillons pour culture bactérienne ont été prélevés aux jours 16 et 32 afin d'évaluer l' angiogenèse, la fibroplasie, l'hyperplasie épithéliale, l'inflammation et la croissance bactérienne. La durée moyenne de guérison des blessures traitées (45 jours, écart : de 38 à 52 jours) n'était pas significativement différente de celle des blessures témoins (50 jours, écart : de 38 à 62 jours). La thérapie à l'oxygène topique a eu peu d'effet sur la guérison des blessures dans ce modèle de blessure expérimentale chez des chevaux en santé.(Traduit par Isabelle Vallières).

Evidence for a Causal Role for Escherichia coli Strains Identified as Adherent-Invasive (AIEC) in Intestinal Inflammation.

Enrichment of adherent-invasive Escherichia coli (AIEC) has been consistently detected in subsets of inflammatory bowel disease (IBD) patients. Although some AIEC strains cause colitis in animal models, these studies did not systematically compare AIEC with non-AIEC strains, and causal links between AIEC and disease are still disputed. Specifically, it remains unclear whether AIEC shows enhanced pathogenicity compared to that of commensal E. coli found in the same ecological microhabitat and if the in vitro phenotypes used to classify strains as AIEC are pathologically relevant. Here, we utilized in vitro phenotyping and a murine model of intestinal inflammation to systematically compare strains identified as AIEC with those identified as non-AIEC and relate AIEC phenotypes to pathogenicity. Strains identified as AIEC caused, on average, more severe intestinal inflammation. Intracellular survival/replication phenotypes routinely used to classify AIEC positively correlated with disease, while adherence to epithelial cells and tumor necrosis factor alpha production by macrophages did not. This knowledge was then applied to design and test a strategy to prevent inflammation by selecting E. coli strains that adhered to epithelial cells but poorly survived/replicated intracellularly. Two E. coli strains that ameliorated AIEC-mediated disease were subsequently identified. In summary, our results show a relationship between intracellular survival/replication in E. coli and pathology in murine colitis, suggesting that strains possessing these phenotypes might not only become enriched in human IBD but also contribute to disease. We provide new evidence that specific AIEC phenotypes are pathologically relevant and proof of principle that such mechanistic information can be therapeutically exploited to alleviate intestinal inflammation. IMPORTANCE Inflammatory bowel disease (IBD) is associated with an altered gut microbiota composition, including expansion of Proteobacteria. Many species in this phylum are thought to contribute to disease under certain conditions, including adherent-invasive Escherichia coli (AIEC) strains, which are enriched in some patients. However, whether this bloom contributes to disease or is just a response to IBD-associated physiological changes is unknown. Although assigning causality is challenging, appropriate animal models can test the hypothesis that AIEC strains have an enhanced ability to cause colitis in comparison to other gut commensal E. coli strains and to identify bacterial traits contributing to virulence. We observed that AIEC strains are generally more pathogenic than commensal E. coli and that bacterial intracellular survival/replication phenotypes contributed to disease. We also found that E. coli strains lacking primary virulence traits can prevent inflammation. Our findings provide critical information on E. coli pathogenicity that may inform development of IBD diagnostic tools and therapies.

Pathogen-derived oligosaccharides improve innate immune response to intracellular parasite infection.

Pathogen glycolipids, including Leishmania spp. lipophosphoglycan (LPG) and Mycobacterium tuberculosis mannosylated lipoarabinomannan (ManLAM), modulate essential interactions with host phagocytic cells. Polysaccharide and lipid components promote immunomodulation. Owing to the stereochemistry required to synthesize oligosaccharides, the roles for oligosaccharides in the pathogenesis of infectious diseases have remained largely unknown. Recent advances in carbohydrate chemistry allowed us to synthesize pathogen surface oligosaccharides to discern their immune response-altering activities. Trimannose cap carbohydrates from ManLAM and LPG altered the production of proinflammatory cytokines via a toll-like receptor (TLR2)-mediated mechanism in vitro and in vivo. In vivo treatment with trimannose led to increased Th1-polarizing, IL-12p40-producing cells from the draining lymph nodes of treated Leishmania major-infected mice compared with cells from untreated infected mice. Trimannose treatment increased the production of other Th1 proinflammatory cytokines (ie, interferon-γ, IL-6, and tumor necrosis factor-α) critical for a productive immune response to either pathogen. This significant difference in cytokine production between trimannose cap sugar-treated and control groups was not observed in draining lymph node cells from TLR2(-/-) mice. Type of inflammation and rate of bead entry into macrophages and dendritic cells were different for trimannose-coated beads compared with control oligosaccharide-coated beads, indicating selective lectin receptor/oligosaccharide interactions mediating cell entry and cytokine production. These novel findings may prompt the development of targeted oligosaccharide adjuvants against chronic infections.

Gastrointestinal biopsy in the horse: overview of collection, interpretation, and applications.

Evaluation of gastrointestinal (GI) biopsies is a multistep process that includes reviewing an appropriate history, determining sample quality, and evaluating histologic sections. Selected diagnostic parameters that, in combination with intestinal histopathology, can be useful to localize disease to the intestinal tract in the horse include hypoproteinemia and hypoalbuminemia, ultrasound evidence of increased thickness of the small intestinal wall, and alterations in glucose or D-xylose absorption tests. Biopsies may be acquired either endoscopically, or via laparoscopy or standing flank incisional approaches. GI sections should be evaluated using a systematic approach that includes both architectural changes and inflammatory cell infiltrates. Although strategies have been developed for assessment of GI biopsies from the dog and cat, a standardized approach to interpretation of the equine GI biopsy has yet to be developed. GI biopsies pose several challenges to the pathologist, especially for endoscopic biopsies in which the quality of the specimen and its orientation may vary greatly. Architectural changes are arguably the most critical changes to evaluate. In a horse with chronic GI inflammation, such as occurs in idiopathic inflammatory bowel disease (IBD), the cell types encountered frequently are macrophages, eosinophils, lymphocytes, and plasma cells. Increased numbers of these cell types are categorized loosely as mild, moderate, and severe. Specific forms of idiopathic IBD have been further classified by this infiltrate as granulomatous enteritis, eosinophilic enteritis, and lymphoplasmacytic enteritis; there is limited information on microscopic changes with each. Unfortunately, microscopic GI lesions are usually nonspecific, and determination of etiology requires further investigation.

Vertical transmission of attaching and invasive E. coli from the dam to neonatal mice predisposes to more severe colitis following exposure to a colitic insult later in life.

The gastrointestinal microbiota begins to be acquired at birth and continually matures through early adolescence. Despite the relevance for gut health, few studies have evaluated the impact of pathobiont colonization of neonates on the severity of colitis later in life. LF82 is an adherent invasive E. coli strain associated with ileal Crohn's disease. The aim of this study was to evaluate the severity of dextran sodium sulfate (DSS)-induced colitis in mice following E. coli LF82 colonization. Gnotobiotic mice harboring the altered Schaedler flora (ASF) were used as the model. While E. coli LF82 is neither adherent nor invasive, it was been demonstrated that adult ASF mice colonized with E. coli LF82 develop more severe DSS-induced colitis compared to control ASF mice treated with DSS. Therefore, we hypothesized that E. coli LF82 colonization of neonatal ASF mice would reduce the severity of DSS-induced inflammation compared to adult ASF mice colonized with E. coli LF82. To test this hypothesis, adult ASF mice were colonized with E. coli LF82 and bred to produce offspring (LF82N) that were vertically colonized with LF82. LF82N and adult-colonized (LF82A) mice were given 2.0% DSS in drinking water for seven days to trigger colitis. More severe inflammatory lesions were observed in the LF82N + DSS mice when compared to LF82A + DSS mice, and were characterized as transmural in most of the LF82N + DSS mice. Colitis was accompanied by secretion of proinflammatory cytokines (IFNγ, IL-17) and specific mRNA transcripts within the colonic mucosa. Using 16S rRNA gene amplicon sequencing, LF82 colonization did not induce significant changes in the ASF community; however, minimal changes in spatial redistribution by fluorescent in situ hybridization were observed. These results suggest that the age at which mice were colonized with E. coli LF82 pathobiont differentially impacted severity of subsequent colitic events.

Helicobacter bilis colonization enhances susceptibility to Typhlocolitis following an inflammatory trigger.

BACKGROUND: Aberrant mucosal immune responses to antigens of the resident microbiota are a significant cause of inflammatory bowel diseases (IBD), as are genetic and environmental factors. Previous work from our laboratory demonstrated that Helicobacter bilis colonization of immunocompetent, defined microbiota mice induced antigen-specific immune responses to the resident microbiota, yet these mice failed to develop colitis, suggesting that the immunological provocation induced by H. bilis alone was insufficient to induce disease. AIM: The purpose of this study was to test the hypothesis that the introduction of a bacterial provocateur such as H. bilis enhances the host's susceptibility to IBD following an inflammatory event. METHODS: Defined microbiota (DM) mice colonized with H. bilis were administered low dose (1.5%) dextran sodium sulfate (DSS) in drinking water for 5 days followed by a 4-day restitution period. Severity of lesions was assessed grossly and microscopically. Differential expression of select mucosal genes and histopathologic lesions was characterized. RESULTS: Helicobacter bilis colonization increased the severity of intestinal inflammation induced by an inflammatory trigger in the form of low-dose DSS. An analysis of the molecular and cellular mechanisms associated with H. bilis colonization revealed significant increases in expression of mucosal genes associated with lymphocyte activation and inflammatory cell chemotaxis as well as increased infiltration of mucosal macrophages and T cells in mice colonized with H. bilis prior to DSS treatment versus DSS treatment alone. CONCLUSIONS: These results indicate that prior colonization with H. bilis heightens the host's sensitivity to enteric inflammation by altering mucosal homeostasis and initiating immune cell activation and migration.

Analysis of gene expression of prostaglandin EP4 receptor in canine osteosarcoma.

In many human cancers, the expression of the prostaglandin receptor EP4 (EP4R) is associated with the development of malignancy and a poor prognosis. The expression of EP4R has not yet been evaluated in canine tumors. The objective of this study was to characterize the messenger RNA (mRNA) expression of EP4R in canine osteosarcoma (OSA). Gene expression of EP4R was evaluated using RNA in-situ hybridization (RNAscope). In all canine OSA samples evaluated, strong universal positive expression of EP4R was identified. Gene expression was significantly higher in OSA tissue samples than in normal nasal turbinate bone, possibly implicating EP4R in the pathogenesis of canine OSA.

Dans de nombreux cancers humains, l'expression du récepteur des prostaglandines EP4 (EP4R) est associée au développement d'une malignité et à un mauvais pronostic. L'expression d'EP4R n'a pas encore été évaluée dans les tumeurs canines. L'objectif de cette étude était de caractériser l'expression de l'ARN messager (ARNm) de l'EP4R dans l'ostéosarcome canin (OSA). L'expression génique de l'EP4R a été évaluée en utilisant l'hybridation in situ d'ARN (RNAscope). Dans tous les échantillons canins OSA évalués, une forte expression positive généralisée d'EP4R a été identifiée. L'expression génique était significativement plus élevée dans les échantillons de tissus OSA que dans l'os normal du cornet nasal, ce qui impliquait peut-être EP4R dans la pathogenèse de l'OSA canin.(Traduit par Docteur Serge Messier).

Comparison of Sheep, Goats, and Calves as Infection Models for Mycobacterium avium subsp. paratuberculosis.

Animal infection models to study Mycobacterium avium subsp. paratuberculosis (MAP) infection are useful for evaluating the efficacy of vaccines and other therapeutics for the prevention or treatment of infection. The goal of the present study was to compare smaller ruminants, sheep and goats, with calves as infection models. Neonatal sheep, goats, and calves (n = 4) received 109 cfu of a cattle isolate of MAP in milk replacer on days 0, 3 and 6 in a 12-month study and sampled monthly thereafter. Results demonstrated a robust antigen-specific IFN-γ response at 90 days post-inoculation for sheep and goats, with lower responses noted for calves. By 360 days, IFN-γ responses were 50 and 82% higher for calves than for goats and sheep, respectively. Although MAP-specific antibody responses were first observed in sheep at 90 days, calves had higher antibody responses throughout the remainder of the study. Following pass-through shedding on day 7, fecal shedding was fairly negligible across treatments but remained higher for calves throughout the study. Colonization of tissues was variable within treatment group and was higher for calves and sheep for the majority of tissues. Upon antigen stimulation of PBMCs, higher populations of CD4 + T cells cells and lower populations of γδ TCR + and NK cells were observed for goats and calves compared to sheep. Relative gene expression of IL-4, IL-12, and IL-17 in PBMCs was higher in goats, corresponding to lower tissue colonization with MAP. These data suggest that ruminant species are fairly comparable as infection models for MAP, but discrete differences in host responses to MAP infection exist between species.

Evaluation of Locally Injected Mycobacterium Cell Wall Fraction in Horses with Sarcoids.

A reformulation of Mycobacterium cell wall fraction immunotherapeutic can be used to successfully treat sarcoids in horses. Sarcoids are reported to be the most common equine skin tumors with tumor type and location influencing the choice of treatment. Wide surgical excision is curative for many tumors, but may not always be feasible. Previous studies have reported sarcoid regression after injection with mycobacterial cell wall immunotherapeutics. A new formulation of the Mycobacterium phlei cell wall fraction immunostimulant (Immunocidin Equine) was used to treat cutaneous tumors in horses. Equids with skin tumors diagnosed as sarcoids were enrolled in the study. Sarcoids were injected at the initial visit with Immunocidin Equine and subsequently at approximately 2-week intervals. Of 17 cases, nine cases were completely resolved at the end of the study period evaluation or at the time of final follow-up (52.9%). Three cases were reported as improved (smaller), but not resolved (17.6%). Three cases were discontinued from the study as the respective masses were growing larger or not resolving (17.6%). One case (5.8%) with two masses had resolution of one mass, whereas the other tumor had a small regrowth 5 months after the last treatment. One case (5.8%) was lost to follow-up. All cases had mild to moderate swelling of the injection site, and some cases had discharge after the second, third, or fourth injections. No serious systemic side effects or complications were encountered during the study.

Campylobacter jejuni persistently colonizes gnotobiotic altered Schaedler flora C3H/HeN mice and induces mild colitis.

Campylobacter jejuni is a major cause of food-borne human bacterial gastroenteritis but animal models for C. jejuni mediated disease remain limited because C. jejuni poorly colonizes immunocompetent, conventionally-reared (Conv-R) mice. Thus, a reliable rodent model (i.e. persistent colonization) is desirable in order to evaluate C. jejuni-mediated gastrointestinal disease and mechanisms of pathogenicity. As the nature and complexity of the microbiota likely impacts colonization resistance for C. jejuni, Conv-R and gnotobiotic C3H/HeN mice were used to evaluate the persistence of C. jejuni colonization and development of disease. A total of four C. jejuni isolates readily and persistently colonized ASF mice and induced mild mucosal inflammation in the proximal colon, but C. jejuni did not stably colonize nor induce lesions in Conv-R mice. This suggests that the pathogenesis of C. jejuni is influenced by the microbiota, and that ASF mice offer a reproducible model to study the influence of the microbiota on the ability of C. jejuni to colonize the gut and to mediate gastroenteritis.

Temporal Dynamics of Chronic Inflammation on the Cecal Microbiota in IL-10-/- Mice.

The intestinal microbiota is a critical component of mucosal health as evidenced by the fact that alterations in the taxonomic composition of the gastrointestinal microbiota are associated with inflammatory bowel diseases. To better understand how the progression of inflammation impacts the composition of the gastrointestinal microbiota, we used culture independent taxonomic profiling to identify temporal changes in the cecal microbiota of C3Bir IL-10-/- mice concomitantly with the onset and progression of colitis. This analysis revealed that IL-10-/- mice displayed a biphasic progression in disease severity, as evidenced by histopathological scores and cytokine production. Beginning at 4 weeks of age, pro-inflammatory cytokines including TNF-α, IFN-γ, IL-6, G-CSF, and IL-1α as well as chemokines including RANTES and MIP-1α were elevated in the serum of IL-10-/- mice. By 19 weeks of age, the mice developed clinical signs of disease as evidenced by weight loss, which was accompanied by a significant increase in serum levels of KC and IL-17. While the overall diversity of the microbiota of both wild type and IL-10-/- were similar in young mice, the latter failed to increase in complexity as the mice matured and experienced changes in abundance of specific bacterial taxa that are associated with inflammatory bowel disease in humans. Collectively, these results reveal that there is a critical time in young mice between four to six weeks of age when inflammation and the associated immune responses adversely affect maturation of the microbiota.

Aerosol-Jet-Printed Graphene Immunosensor for Label-Free Cytokine Monitoring in Serum.

Graphene-based inks are becoming increasingly attractive for printing low-cost and flexible electrical circuits due to their high electrical conductivity, biocompatibility, and manufacturing scalability. Conventional graphene printing techniques, such as screen and inkjet printing, are limited by stringent ink viscosity requirements properties and large as-printed line width that impedes the performance of printed biosensors. Here, we report an aerosol-jet-printed (AJP) graphene-based immunosensor capable of monitoring two distinct cytokines: interferon gamma (IFN-γ) and interleukin 10 (IL-10). Interdigitated electrodes (IDEs) with 40 µm finger widths were printed from graphene-nitrocellulose ink on a polyimide substrate. The IDEs were annealed in CO2 to introduce reactive oxygen species on the graphene surface that act as chemical handles to covalently link IFN-γ and IL-10 antibodies to the graphene surfaces. The resultant AJP electrochemical immunosensors are capable of monitoring cytokines in serum with wide sensing range (IFN-γ: 0.1-5 ng/mL; IL-10: 0.1-2 ng/mL), low detection limit (IFN-γ: 25 pg/ml and IL-10: 46 pg/ml) and high selectivity (antibodies exhibited minimal cross-reactivity with each other and IL-6) without the need for sample prelabeling or preconcentration. Moreover, these biosensors are mechanically flexible with minimal change in signal output after 250 bending cycles over a high curvature (Φ = 5 mm). Hence, this technology could be applied to numerous electrochemical applications that require low-cost electroactive circuits that are disposable and/or flexible.

Fyn kinase mediates pro-inflammatory response in a mouse model of endotoxemia: Relevance to translational research.

Systemic inflammation resulting from the release of pro-inflammatory cytokines and the chronic activation of the innate immune system remains a major cause of morbidity and mortality in the United States. After having demonstrated that Fyn, a Src family kinase, regulates microglial neuroinflammatory responses in cell culture and animal models of Parkinson's disease, we investigate here its role in modulating systemic inflammation using an endotoxic mouse model. Fyn knockout (KO) and their wild-type (WT) littermate mice were injected once intraperitoneally with either saline or 5 mg/kg lipopolysaccharide (LPS) and were killed 48 h later. LPS-induced mortality, endotoxic symptoms and hypothermia were significantly attenuated in Fyn KO, but not WT, mice. LPS reduced survival in Fyn WT mice to 49% compared to 84% in Fyn KO mice. Fyn KO mice were also protected from LPS-induced deficits in horizontal and vertical locomotor activities, total distance traveled and stereotypic movements. Surface body temperatures recorded at 24 h and 48 h post-LPS dropped significantly in Fyn WT, but not in KO, mice. Importantly, endotoxemia-associated changes to levels of the serum pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), splenocyte apoptosis and inducible nitric oxide synthase (iNOS) production in hepatocytes were also significantly attenuated in Fyn KO mice. Likewise, pharmacologically inhibiting Fyn with 10 mg/kg dasatinib (oral) significantly attenuated LPS-induced increases in plasma TNF-α and IL-6 protein levels and hepatic pro-IL-1ß messenger ribonucleic acids (mRNAs). Collectively, these results indicate that genetic knockdown or pharmacological inhibition of Fyn dampens systemic inflammation, demonstrating for the first time that Fyn kinase plays a critical role in mediating the endotoxic inflammatory response.

Gamma-delta T cell subsets are differentially associated with granuloma development and organization in a bovine model of mycobacterial disease.

The characteristic lesion in bovine tuberculosis is well-organized respiratory granulomas. This is typically associated with a strong T-helper 1 biased cell-mediated immune response and eventual containment of the infection. In bovine paratuberculosis, the classic lesion is unorganized granulomatous intestinal inflammation. Clinical paratuberculosis is associated with a T-helper 2 biased humoral immune response and eventual death because of inability of the host to contain the infection. Recent reports have suggested that gamma-delta (gammadelta) T cells play a significant role in granuloma development and/or maintenance during initial stages of infection and may influence the subsequent adaptive immune response. The objective of this study was to use an in vivo bovine model to evaluate gammadelta T cells during the early host immune response to mycobacterial infection. We used immunofluorescent staining, hyperspectral microscopy, and computerized assisted morphometry to evaluate staining and distribution of gammadelta T cells during development of organized and unorganized granulomas. Our data suggest that bovine gammadelta T cell subsets are differentially recruited to early infection sites, and may be instrumental during the initial antimycobacterial host immune response as well as for granuloma organization.

Cutaneous viral papilloma with local extension and subungual cyst formation in a dog.

A viral-induced digital cutaneous exophytic papilloma was diagnosed in a 2-year-old, spayed, female Siberian husky dog with lameness. Digital pain and lameness persisted after removal of the initial papilloma, and the fifth lateral digit was subsequently amputated. Upon histologic examination of the digit, a de novo digital, cutaneous, inverted, viral papilloma and subungual cyst were diagnosed. The inverted cutaneous papilloma, located at the junction of the digital paw pad and ventral nail, extended focally through the nail into the subungual space, where an expansile cyst was formed. Cellular changes suggestive of papillomavirus infection were present in the epithelium of the original exophytic papilloma, as well as the endophytic mass and subungual cyst. Cytopathic effects included ballooning degeneration of keratinocytes, koilocytosis, irregularity of keratohyalin granules, and margination of nuclear chromatin. Numerous faintly basophilic to eosinophilic intranuclear inclusions measuring 10-15 microm in diameter were present within keratinocytes of the exophytic, endophytic, and subungual cystic lesions. Electron microscopy was performed on tissues from all lesions and revealed numerous 40-45 nm diameter hexagonal virions characteristic of papillomavirus that were arranged in crystalline arrays and dense clusters within affected nuclei.