Contents Pyometra is a disease in dogs caused by bacterial infection of the uterus and resulting in SIRS (systemic inflammatory response syndrome) in nearly 6 of 10 cases. Clinical diagnostic criteria for SIRS are relatively unspecific, and biomarkers for the diagnosis of pyometra and SIRS in dogs are needed. Serum samples from 32 dogs were used in this study and grouped into dogs with pyometra and SIRS, dogs with pyometra without SIRS and healthy controls. The serum concentrations of IFN‐γ, IL‐4, IL‐6, IL‐7, IL‐8, IL‐10, IL‐15, IL‐18 and TNF‐α were measured using multiplex analyses. The serum concentrations of CRP (C‐reactive protein) were determined using sandwich ELISA. IL‐7, IL‐8, IL‐15, IL‐18 and TNF‐α were detected in >94% of samples. IL‐10 was detected in 28% of samples, and IL‐4, IL‐6 and IFN‐γ were undetectable. Higher serum concentrations of IL‐7 (p < 0.05) were detected in SIRS‐positive dogs with pyometra (n = 13) as compared with healthy controls (n = 11). The concentrations of IL‐8 were higher in SIRS‐positive dogs with pyometra compared to the SIRS‐negative group (n = 8; p < 0.05). Positive correlations of IL‐15 with IL‐18 (p < 0.0001) and with the concentrations of IL‐7 (p < 0.0001 for both) were found, although there was no significant difference between groups. Furthermore, IL‐15 correlated with concentrations of CRP (p < 0.05), which were higher in dogs with pyometra compared to controls (p < 0.0001). Our data suggest a role of several cytokines in the development of a systemic disease in dogs with pyometra and a possible diagnostic value for serum CRP, IL‐7, IL‐15 and IL‐18 in canine SIRS caused by pyometra.

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Autoimmune polyendocrine syndrome type 1 (APS-1) is a multiorgan autoimmune disease caused by mutations in the autoimmune regulator (AIRE) gene. Chronic mucocutaneous candidiasis, hypoparathyroidism and adrenal failure are hallmarks of the disease. The critical mechanisms causing chronic mucocutaneous candidiasis in APS-1 patients have not been identified although autoantibodies to cytokines are implicated in the pathogenesis. To investigate whether the Th reactivity to Candida albicans (C. albicans) and other stimuli was altered, we isolated PBMC from APS-1 patients and matched healthy controls. The Th17 pathway was upregulated in response to C. albicans in APS-1 patients, whereas the IL-22 secretion was reduced. Autoantibodies against IL-22, IL-17A and IL-17F were detected in sera from APS-1 patients by immunoprecipitation. In addition, Aire-deficient (Aire 0/0 ) mice were much more susceptible than Aire 1/1 mice to mucosal candidiasis and C. albicansinduced Th17-and Th1-cell responses were increased in Aire 0/0 mice. Thus an excessive IL-17A reactivity towards C. albicans was observed in APS-1 patients and Aire 0/0 mice.

: Mast cell (MC) granules contain large amounts of proteases of the chymase, tryptase and carboxypeptidase A (MC-CPA) type that are stored in complex with serglycin, a proteoglycan with heparin side chains. Hence, serglycinprotease complexes are released upon MC degranulation and may influence local inflammation. Here we explored the possibility that a serglycin-protease axis may regulate levels of IL-13, a cytokine involved in allergic asthma. Indeed, we found that wild-type MCs efficiently degraded exogenous or endogenously produced IL-13 upon degranulation, whereas serglycin −/− MCs completely lacked this ability. Moreover, MC-mediated IL-13 degradation was blocked both by a serine protease inhibitor and by a heparin antagonist, which suggests that IL-13 degradation is catalyzed by serglycin-dependent serine proteases and that optimal IL-13 degradation is dependent on both the serglycin and the protease component of the serglycin-protease complex. Moreover, IL-13 degradation was abrogated in MC-CPA −/− MC cultures, but was normal in cultures of MCs with an inactivating mutation of MC-CPA, which suggests that the IL-13-degrading serine proteases rely on MC-CPA protein. Together, our data implicate a serglycin-serine protease axis in the regulation of extracellular levels of IL-13. Reduction of IL-13 levels through this mechanism possibly can provide a protective function in the context of allergic inflammation.

Sepsis is difficult to diagnose and remains a common mortality cause worldwide in both humans and animals. The uterine infection pyometra causes sepsis in more than half of affected dogs and therefore allows the natural physiological development of sepsis to be studied. To find a sepsis-specific biochemical marker that could be combined with conventional clinical criteria for a more robust and quick diagnosis of sepsis, we measured systemic concentrations of high-mobility group box 1 (HMGB1) in 23 healthy control dogs and in 27 dogs with pyometra, 74% of which had sepsis.We also measured concentrations of the major acute phase protein C-reactive protein (CRP) and an indicator for endotoxaemia, prostaglandin F 2α metabolite (PGM) to assess the relative contribution of HMGB1 to the detection of systemic inflammation and endotoxaemia. We found that HMGB1 concentrations, in line with concentrations of CRP and PGM, were significantly increased in dogs with pyometra, and that concentrations of CRP, but not HMGB1, were significantly higher in dogs with sepsis compared to dogs without sepsis. Although serum HMGB1 did not differ between dogs with or without sepsis and was not correlated with either CRP or PGM concentrations, HMGB1 was correlated with the total white blood cell counts, suggesting an independent regulation and involvement in inflammation.

BackgroundObese dogs risk poor life quality, creating a need for increased knowledge of metabolism in overweight dogs.ObjectivesInvestigate postprandial metabolic and hormonal responses to a high‐fat mixed‐meal in dogs and responses of lean versus overweight dogs.AnimalsTwenty‐eight healthy intact male Labrador Retrievers were included.MethodsProspective observational study. Twelve dogs were grouped as lean (body condition score (BCS 4–5), 10 as slightly overweight (BCS 6), and 6 as overweight (BCS 6.5–8) on a 9‐point scale. After an overnight fast, urine and blood samples were collected. Dogs were then fed a high‐fat mixed‐meal, and blood was collected hourly for 4 hours and urine after 3 hours.ResultsPostprandial concentrations of insulin and glucagon were increased at 1 hour (both P < 0.0001), triglycerides at 2 hours (P < 0.0001), and glucose at 3 hours (P = 0.004); and all remained increased throughout the feed‐challenge in all dogs. Postprandial urine cortisol/creatinine ratio was higher than fasting values (P = 0.001). Comparing between groups, there was an overall higher triglyceride response in overweight compared to lean (P = 0.001) and slightly overweight (P = 0.015) dogs. Overweight dogs also had higher fasting cortisol/creatinine ratio compared to lean dogs (P = 0.024).Conclusions and Clinical ImportancePostprandial responses of dogs to a high‐fat mixed‐meal were similar to those previously reported in people. The higher postprandial triglyceride response and fasting cortisol/creatinine ratio in the overweight dogs could be early signs of metabolic imbalance. Thus, although overweight dogs often appear healthy, metabolic alterations might be present.

SUMMARY 15Chemokines play a central role in cellular communication in response to bacterial 16 infection. However, the knowledge of the chemokine responses to bacterial infections in 17 dogs remains limited. Uterine bacterial infection (pyometra) is one of the most common 18 bacterial diseases in dogs and causes sepsis in most of the cases. We have shown 19 previously that dogs with pyometra have higher mRNA levels of chemokines in uterus. 20To assess whether the stromal part of endometrium express chemokines in response to 21 bacterial infection, we cultured endometrial stromal cells isolated from healthy dogs and 22 exposed them to either live pathogenic Escherichia coli (E. coli) isolated from uterus of a 23 dog with pyometra, or to lipopolysaccharide (LPS). Changes in the mRNA expression of 24 ELR + CXC chemokines IL-8, CXCL5, CXCL7, and ELR -CXC chemokine CXCL10 25 were measured after 24 h using quantitative real-time PCR. Levels of IL-8, CXCL5 and 26 CXCL10 were upregulated in endometrial stromal cells exposed to E. coli and to LPS 27 whereas the level of CXCL7 was decreased or unaffected. In addition, levels of IL-8 and 28CXCL5, but not CXCL7 or CXCL10, were significantly higher in dogs with pyometra as 29 compared to healthy dogs. Our findings show that pathogenic uterine-derived E. coli 30 induces a CXC chemokine response both in cultured endometrial stromal cells within 24 31 h and in pyometra-affected uteri from dogs. Stromal cells could therefore play an 32 important role in early neutrophil and T cell recruitment to the site of inflammation 33 during Gram negative bacterial infection of the uterus. Further study is needed to clarify 34 the role of chemokines in host response to bacterial infection in dogs and the possibility 35 of using chemokines as diagnostic parameters for bacterial infection in this species. 36 37 3

Mast cells contain large amounts of fully active proteases that are stored in complex with serglycin proteoglycan in their secretory granules. Upon degranulation, such serglycin:protease complexes are released to the extracellular space and can potentially have an impact on the local inflammatory reaction, either through direct effects of serglycin proteoglycan or through effects mediated by its bound proteases. The objective of this study was to address this scenario by investigating the possibility that serglycin‐associated proteases can regulate levels of pro‐inflammatory cytokines. Indeed, we show here that activated cultured peritoneal mast cells from wild type mice efficiently reduced the levels of exogenously administered IL‐6 and IL‐17A, whereas serglycin‐deficient mast cells lacked this ability. Furthermore, our data suggest that the reduction of IL‐6 and IL‐17A concentrations is due to proteolytic degradation mediated by serglycin‐dependent serine proteases. Moreover, we show that activated mast cells have the capacity to release IL‐6 and that the levels of this cytokine in supernatants were markedly higher in cultures of serglycin‐deficient versus serglycin‐sufficient mast cells, suggesting that serglycin‐dependent serine proteases also participate in the regulation of endogenously produced IL‐6. In summary, although the general consensus is that mast cells have a pathogenic impact on inflammatory settings, this study identifies a role for a mast cell‐derived serglycin:serine protease axis in down‐regulating levels of major inflammatory cytokines. These findings support the notion that mast cells could have a dual role in inflammatory settings, by both being able to secrete pathogenic compounds and being able to regulate their levels after release.