Knockdown of miR-26a in zebrafish leads to impairment of the anti-inflammatory function of TnP in the control of neutrophilia.

Our recent data show the valuable potential of TnP for the development of a new and safe anti-inflammatory drug due to its ability to control the traffic and activation of leukocytes in response to inflammation. Although there is considerable knowledge surrounding the cellular mechanisms of TnP, less is known about the mechanistic molecular role of TnP underlying its immunomodulatory functions. Here, we conducted investigations to identify whether miRNAs could be one of the molecular bases of the therapeutic effect of TnP. Using a zebrafish model of neutrophilic inflammation with a combination of genetic gain- and loss-of-function approaches, we showed that TnP treatment was followed by up-regulation of only four known miRNAs, and mature dre-miR-26a-1, herein referred just as miR-26a was the first most highly expressed. The knockdown of miR-26a ubiquitously resulted in a significant reduction of miR-26a in embryos, accompanied by impaired TnP immunomodulatory function observed by the loss of the control of the removal of neutrophils in response to inflammation, while the overexpression increased the inhibition of neutrophilic inflammation promoted by TnP. The striking importance of miR-26a was confirmed when rescue strategies were used (morpholino and mimic combination). Our results identified miR-26a as an essential molecular regulator of the therapeutic action of TnP, and suggest that miR-26a or its targets could be used as promising therapeutic candidates for enhancing the resolution of inflammation.

Neutrophilia is associated with a poorer clinical outcome in dogs with chronic hepatitis.

BACKGROUND: Liver disease is a common cause of morbidity and mortality in dogs. Currently, it is challenging to prognosticate in these cases. The aim of this study was to evaluate the utility of the haematological variables in dogs with chronic hepatitis. METHODS: Dogs with chronic hepatitis confirmed on histopathology had presenting haematological values retrospectively obtained and evaluated against survival time. Eighty-two dogs met the inclusion criteria and their data analysed. RESULTS: Neutrophilic patients, with a count greater than 12×109/l, controlled for sex and age, had a shorter survival time (P≤0.01). In dogs, neutrophilia at presentation predicted a poor outcome, whereas the other haematological parameters were not prognostically informative. When the dogs were split into even quarters on the basis of their neutrophil count, those within the higher quartiles had poorer survival times. Neutrophilia was associated with a poorer survival time in comparison to those patients with a lower count. CONCLUSION: The relationship between neutrophils, inflammation and clinical outcome is deserving of future study in dogs with chronic hepatitis.

Blood calcium, glucose and haematology profiles of parturient bitches diagnosed with uterine inertia or obstructive dystocia.

Bitches with dystocia most often present with clinical signs of uterine inertia (UI). The aetiology of myometrial dysfunction in most of these cases is still not elucidated. We compared blood ionized calcium (iCa) and glucose concentrations in bitches diagnosed with primary UI (PUI, n = 14), secondary UI (SUI, n = 6) or obstructive dystocia (OD, n = 6), and we described their haematology profiles. Bitches diagnosed with UI had a patent birth canal and delivered no puppies yet (PUI) or only part of the whole litter (SUI). The OD group had no UI and showed strong abdominal contractions. Blood iCa did not differ between the PUI, SUI and OD groups and was not influenced by litter size. There was a significant positive relationship (R2  = .241, p = .013) between iCa concentrations and the dam's body weight. Glucose concentrations were also not significantly different between dystocia groups or influenced by body weight and litter size. Hypocalcaemia was detected in 11 bitches, and hypoglycaemia in two bitches. Pregnancy-associated anaemia was seen in about one-third of the bitches. Eight of 12 dogs had increased platelet counts, and ten had leukocytosis with mature neutrophilia. Although iCa did not differ between dystocia groups, low concentrations may have contributed to the development of UI in some of the small size bitches. Hypoglycaemia was uncommon, and therefore, we consider low glucose concentrations not to have played an important role in the pathogenesis of UI in our study population. Pregnancy-associated anaemia, thrombocytosis, leukocytosis and mature neutrophilia were common findings in otherwise healthy bitches diagnosed with different forms of dystocia.

[Extreme neutrophilia in cats - aetiology and prognosis]. / Extreme Neutrophilie bei der Katze - Ätiologie und Prognose.

OBJECTIVE: The aim of this retrospective study was to evaluate the aetiology and prognostic factors of extreme neutrophilia in cats. MATERIAL AND METHODS: Patient data over a 5-year period (January 2008  -  December 2013) were reviewed. Cats with a neutrophil count > 40 x 109/l were included. They were further assigned to four groups: "inflammation", "neoplasia", "immune-mediated diseases", "unknown aetiology". Clinical signs, rectal temperature, hospitalisation, duration of hospitalisation, survival, left-shift and toxicity of neutrophils were evaluated. RESULTS: In total, 28/5185 cats (0.5%) displayed extreme neutrophilia with a mean neutrophil count of 48.5 x 109/l (40.0-76.0 x 109/l). The most common aetiology was a severe inflammation, as seen in 16/28 cats (57%), whereby peritonitis (5/15 cats, 31%) predominated. In cats with neoplastic diseases (9/28 cats, 32%), intestinal neoplasia with subsequent peritonitis was the most common diagnosis (4/9 cats, 44%). Diseases of unknown aetiology (2/28 cats, 7%) and immune-mediated diseases (1/28, 3.6%) were rare. The most common clinical indications included lethargy, anorexia, fever, and gastrointestinal signs. Rectal temperature ranged between 33.9  °C and 40.2   °C, whereby in 2/24 cats (8%) hyperthermia (> 39.3°C) and in 5/24 cats (21%) hypothermia (< 38.0°C) was observed. Hospitalisation occurred in 21/28 cats (75%) with a median duration of 5.5 days (1-30 days). In 24/28 cats, a manual differential count was performed. A left-shift and toxicity of neutrophils were seen in 23/24 cats (96%) and 21/24 cats (88%), respectively. The overall median survival rate was 50%, whereby the survival rate was significantly lower in cats with neoplasia than in those with inflammatory diseases (22% vs. 56%, p < 0.0001). CLINICAL RELEVANCE: An extreme neutrophilia is rare. It is commonly caused by peritonitis due to foreign bodies or ruptured intestinal tumours (in particular, intestinal lymphomas) and is characterised by a high mortality.

Dexamethasone-induced eosinopenia is associated with lower progesterone production in cattle.

Eosinophilic cells accumulate in the capillaries of the bovine Graafian follicle shortly before ovulation and in the early developing corpus luteum (CL). Suppressing the migration of these eosinophilic cells by dexamethasone allowed us to evaluate their possible function in the CL development. Brown Swiss cows (n = 10) were randomly subdivided into two groups (n = 5). Every group was used once as control group and once as experimental group with two oestrous cycles between each treatment. Eighteen hours (h) after oestrus synchronization, dexamethasone or saline was given. Ovulation was induced 24 h later with gonadotropin-releasing hormone. Another injection of dexamethasone or saline was given 12 h later. Eosinophilic cells in the blood were counted daily until day 7 after the first dexamethasone injection. The collection of ovaries took place at days 1, 2 and 5. Gene expression, protein concentration and location of angiogenic factors, chemokines, insulin-like growth factor 1 (IGF1) and eosinophilic cells were studied. No eosinophilic cells were found in the CL of the treatment group. Blood progesterone decreased significantly in the dexamethasone group from day 8 to 17. The protein concentration of FGF2 increased significantly in CL tissue at day 2 and VEGFA decreased. Local IGF1 gene expression in the CL was not regulated. We assume from our data that the migration of eosinophilic cells into the early CL is not an essential, but an important stimulus for angiogenesis during early CL development in cattle.

Higher whole-blood selenium is associated with improved immune responses in footrot-affected sheep.

We reported previously that sheep affected with footrot (FR) have lower whole-blood selenium (WB-Se) concentrations and that parenteral Se-supplementation in conjunction with routine control practices accelerates recovery from FR. The purpose of this follow-up study was to investigate the mechanisms by which Se facilitates recovery from FR. Sheep affected with FR (n = 38) were injected monthly for 15 months with either 5 mg Se (FR-Se) or saline (FR-Sal), whereas 19 healthy sheep received no treatment. Adaptive immune function was evaluated after 3 months of Se supplementation by immunizing all sheep with a novel protein, keyhole limpet hemocyanin (KLH). The antibody titer and delayed-type hypersensitivity (DTH) skin test to KLH were used to assess humoral immunity and cell-mediated immunity, respectively. Innate immunity was evaluated after 3 months of Se supplementation by measuring intradermal responses to histamine 30 min after injection compared to KLH and saline, and after 15 months of Se supplementation by isolating neutrophils and measuring their bacterial killing ability and relative abundance of mRNA for genes associated with neutrophil migration. Compared to healthy sheep, immune responses to a novel protein were suppressed in FR-affected sheep with smaller decreases in FR-affected sheep that received Se or had WB-Se concentrations above 250 ng/mL at the time of the immune assays. Neutrophil function was suppressed in FR-affected sheep, but was not changed by Se supplementation or WB-Se status. Sheep FR is associated with depressed immune responses to a novel protein, which may be partly restored by improving WB-Se status (> 250 ng/mL).