Joseph J. Wakshlag

Potential role for peptidylarginine deiminase 2 (PAD2) in citrullination of canine mammary epithelial cell histones.

Peptidylarginine Deiminases (PADs) convert arginine residues on substrate proteins to citrulline. Previous reports have documented that PAD2 expression and activity varies across the estrous cycle in the rodent uterus and pituitary gland, however, the expression and function of PAD2 in mammary tissue has not been previously reported. To gain more insight into potential reproductive roles for PAD2, in this study we evaluated PAD2 expression and localization throughout the estrous cycle in canine mammary tissue and then identified possible PAD2 enzymatic targets. Immunohistochemical and immunofluorescence analysis found PAD2 expression is low in anestrus, limited to a distinct, yet sparse, subset of epithelial cells within ductal alveoli during estrus/early diestrus, and encompasses the entire epithelium of the mammary duct in late diestrus. At the subcellular level, PAD2 is expressed in the cytoplasm, and to a lesser extent, the nucleus of these epithelial cells. Surprisingly, stimulation of canine mammary tumor cells (CMT25) shows that EGF, but not estrogen or progesterone, upregulates PAD2 transcription and translation suggesting EGF regulation of PAD2 and possibly citrullination in vivo. To identify potential PAD2 targets, anti-pan citrulline western blots were performed and results showed that citrullination activity is limited to diestrus with histones appearing to represent major enzymatic targets. Use of site-specific anti-citrullinated histone antibodies found that the N-terminus of histone H3, but not H4, appears to be the primary target of PAD activity in mammary epithelium. This observation supports the hypothesis that PAD2 may play a regulatory role in the expression of lactation related genes via histone citrullination during diestrus.

The effects of weight loss on adipokines and markers of inflammation in dogs

Evidence suggests that adipose tissue-derived adipokines induce mild inflammation and may play a role in insulin resistance associated with diabetes. The present study was designed to examine a series of adipokines and markers of inflammation in dogs before and after a successful weight loss. The study included fasting serum samples from twenty-five dogs before and after a weight-loss programme. Serum C-reactive protein (CRP) and monocyte chemoattractant protein-1 (MCP-1) were measured as indicators of chronic inflammation, while serum adipokines including total adiponectin, high-molecular-weight (HMW) adiponectin, resistin and leptin were also examined. Medians for CRP (before, 10.0 (interquartile range 5.4-15.0) μg/ml; after, 5.6 (interquartile range 3.8-7.0) μg/ml) and MCP-1 (before, 212 (interquartile range 157-288) ng/ml; after, 185 (interquartile range 143-215) ng/ml) decreased significantly after weight loss. Medians for resistin showed a mild, yet significant reduction (before, 67.1 (interquartile range 44.4-88.5) pg/ml; after, 60.5 (interquartile range 32.3-67.1) pg/ml), while leptin showed a dramatic decrease after weight loss (before, 18.9 (interquartile range 10.8-35.4) ng/ml; after, 6.6 (interquartile range 3.9-10.2) ng/ml). Serum total adiponectin and HMW adiponectin were unchanged on all analyses performed. These data suggest that weight loss can decrease chronic inflammation; however, the clinical implications of this decrease are not well elucidated in dogs. Surprisingly, there was no increase in total or HMW serum adiponectin after weight loss, as observed previously in human subjects. The lack of change in total and HMW adiponectin might explain why insulin resistance and type 2 diabetes are less prevalent in obese dogs when compared with humans and cats.

Dysregulation of PAD4-mediated citrullination of nuclear GSK3β activates TGF-β signaling and induces epithelial-to-mesenchymal transition in breast cancer cells

Peptidylarginine deiminase 4 (PAD4) is a Ca2+-dependent enzyme that converts arginine and methylarginine residues to citrulline, with histone proteins being among its best-described substrates to date. However, the biological function of this posttranslational modification, either in histones or in nonhistone proteins, is poorly understood. Here, we show that PAD4 recognizes, binds, and citrullinates glycogen synthase kinase-3β (GSK3β), both in vitro and in vivo. Among other functions, GSK3β is a key regulator of transcription factors involved in tumor progression, and its dysregulation has been associated with progression of human cancers. We demonstrate that silencing of PAD4 in breast cancer cells leads to a striking reduction of nuclear GSK3β protein levels, increased TGF-β signaling, induction of epithelial-to-mesenchymal transition, and production of more invasive tumors in xenograft assays. Moreover, in breast cancer patients, reduction of PAD4 and nuclear GSK3β is associated with increased tumor invasiveness. We propose that PAD4-mediated citrullination of GSK3β is a unique posttranslational modification that regulates its nuclear localization and thereby plays a critical role in maintaining an epithelial phenotype. We demonstrate a dynamic and previously unappreciated interplay between histone-modifying enzymes, citrullination of nonhistone proteins, and epithelial-to-mesenchymal transition.

Alaskan Husky encephalopathy - a canine neurodegenerative disorder resembling subacute necrotizing encephalomyelopathy (Leigh syndrome).

Abstract The gross and histopathological findings in the brain and spinal cord of five Alaskan Husky dogs with a novel incapacitating and ultimately fatal familial and presumed hereditary neurodegenerative disorder are described. Four dogs presented with neurological deficits before the age of 1 year (7–11 months) and one animal at 2.5 years old. Clinical signs in all dogs were of acute onset and included ataxia, seizures, behavioral abnormalities, blindness, facial hypalgesia and difficulties in prehension of food. In animals allowed to survive, the disease was static but with frequent recurrences. Pathological findings were limited to the central nervous system. Grossly visible bilateral and symmetrical cavitated foci were consistently present in the thalamus with variable extension into the caudal brain stem. Microscopic lesions were more widespread and included foci of bilateral and symmetrical degeneration in the basal nuclei, midbrain, pons and medulla, as well as multifocal lesions at the base of sulci in the cerebral cortex and in the gray matter of cerebellar folia in the ventral vermis. Neuronal loss with concomitant neuronal sparing, spongiosis, vascular hypertrophy and hyperplasia, gliosis, cavitation and transient mixed inflammatory infiltration were the main histopathological findings. In addition, a population of reactive gemistocytic astrocytes with prominent cytoplasmic vacuolation was noted in the thalamus. Lesions of this nature in this distribution within the neuroaxis have not been reported in dogs. The neuropathological findings resemble Leigh’s disease/subacute necrotizing encephalomyelopathy of man. Neuronal sparing in conjunction with apparently transient astrocytic vacuolation point to the possible pathogenetic role of astrocytes in the evolution of these lesions. An inherited metabolic derangement of unknown nature is postulated as the cause of this breed-specific disorder.

Evaluation of exercise-induced changes in concentrations of C-reactive protein and serum biochemical values in sled dogs completing a long-distance endurance race.

OBJECTIVE To determine whether long-distance endurance exercise in sled dogs causes increases in serum concentrations of C-reactive protein (CRP) and whether such increases are correlated with other markers of the exercise-induced acute-phase response. ANIMALS 25 sled dogs. PROCEDURES Serum was obtained from 25 sled dogs approximately 48 hours before and immediately after completing a race of 557 km. Serum was analyzed to determine concentrations of CRP and interleukin (IL)-6, and serum biochemical analysis (and iron homeostasis analysis) also was performed. RESULTS CRP concentrations increased significantly from a mean±SD concentration of 22.4±16.3 microg/mL before racing to a mean of 263.3±103.8 microg/mL immediately after racing Serum IL-6 concentrations were unchanged; however, there was a modest but significant correlation (r=0.50) between the increase in CRP concentration and an overall decrease in serum albumin concentration, which suggested an inverse relationship between hepatic synthesis of the 2 proteins. Differences in CRP concentrations among teams of dogs revealed that concentrations before racing may be influenced by previous episodes of exercise. Serum iron concentration had only a mild decrease, which may have been attributable to iron-rich diets consumed by the dogs. CONCLUSIONS AND CLINICAL RELEVANCE CRP concentrations may serve as a potential marker for exercise-induced inflammation. The exact amount of exercise required to induce such a response is unknown, but dogs apparently have a more robust acute-phase response than do humans. Clinical evaluation of CRP concentrations must account for physical activity when those concentrations are used as a potential marker for systemic inflammation.

Hematologic, serum biochemical, and cortisol changes associated with anticipation of exercise and short duration high-intensity exercise in sled dogs.

BACKGROUND Many studies have demonstrated changes in clinicopathologic variables in response to exercise in athletic dogs. These changes have been attributed to the duration and intensity of exercise. However, inconsistencies in the timing of collection of resting blood samples can be a confounding variable when interpreting results. OBJECTIVES The objectives of this study were to determine whether hematologic and biochemical data in sled dogs are influenced by (1) anticipation of an exercise event or (2) short-duration high-intensity exercise. METHODS Blood samples were collected into EDTA tubes and tubes without anticoagulant from sled dogs at rest, immediately before a 3-mile run (preexercise), and immediately after the run (postexercise). A CBC, biochemical profile (including electrolytes, glucose, proteins, creatinine, urea, enzymes, cholesterol, and total bilirubin), and serum cortisol concentration were measured using standard methods on automated analyzers. RESULTS Significant increases in sodium, chloride, albumin, calcium, and cortisol concentration were observed in preexercise samples when compared with resting samples. Hyperglycemia and a further rise in serum cortisol concentration occurred immediately postexercise. CONCLUSIONS The results of this study indicate that anticipation of exercise affects several biochemical analytes, emphasizing the importance of appropriate timing of baseline blood samples. The pronounced hyperglycemia immediately after short-duration exercise may be a breed- or exercise-dependent phenomenon.

Dry period plane of energy: Effects on glucose tolerance in transition dairy cows

Overfeeding energy in the dry period can affect glucose metabolism and the energy balance of transition dairy cows with potential detrimental effects on the ability to successfully adapt to early lactation. The objectives of this study were to investigate the effect of different dry cow feeding strategies on glucose tolerance and on resting concentrations of blood glucose, glucagon, insulin, nonesterified fatty acids (NEFA), and β-hydroxybutyrate (BHB) in the peripartum period. Cows entering second or greater lactation were enrolled at dry-off (57 d before expected parturition) into 1 of 3 treatment groups following a randomized block design: cows that received a total mixed ration (TMR) formulated to meet but not exceed energy requirements during the dry period (n=28, controlled energy); cows that received a TMR supplying approximately 150% of energy requirements during the dry period (n=28, high energy); and cows that were fed the same diet as the controlled energy group for the first 28 d, after which the TMR was formulated to supply approximately 125% of energy requirements until calving (n=28, intermediate energy). Intravenous glucose tolerance tests (IVGTT) with rapid administration of 0.25 g of glucose/kg of body weight were performed 28 and 10d before expected parturition, as well as at 4 and 21 d after calving. Area under the curve for insulin and glucose, maximal concentration and time to half-maximal concentration of insulin and glucose, and clearance rates were calculated. Insulin resistance (IR) indices were calculated from baseline samples obtained during IVGTT and Spearman rank correlations determined between IVGTT parameters and IR indices. Treatment did not affect IVGTT parameters at any of the 4 time points. Correlation between IR indices and IVGTT parameters was generally poor. Overfeeding cows energy in excess of predicted requirements by approximately 50% during the entire dry period resulted in decreased postpartum basal plasma glucose and insulin, as well as increased glucagon, BHB, and NEFA concentrations after calving compared with cows fed a controlled energy diet during the dry period. In conclusion, overfeeding energy during the entire dry period or close-up period alone did not affect glucose tolerance as assessed by IVGTT but energy uptake during the dry period was associated with changes in peripartal resting concentrations of glucose, as well as postpartum insulin, glucagon, NEFA, and BHB concentrations.

Dry period plane of energy: Effects on feed intake, energy balance, milk production, and composition in transition dairy cows

The objective was to investigate the effect of different dry cow feeding strategies on the degree of ketonemia postpartum. Epidemiologic studies provide evidence of an association between elevated β-hydroxybutyrate (BHBA) concentrations in postpartum dairy cows and a decreased risk for reproductive success as well as increased risk for several diseases in early lactation, such as displacement of the abomasum and metritis. The plane of energy fed to cows in the prepartum period has been shown to influence ketogenesis and the degree of negative energy balance postpartum. Our hypothesis was that a high-fiber, controlled-energy diet (C) fed during the dry period would lead to a lower degree of hyperketonemia in the first weeks postpartum compared with either a high-energy diet (H), or a diet where an intermediate level of energy would only be fed in the close-up period (starting at 28d before expected parturition), following the same controlled-energy diet in the far-off period. Hyperketonemia in this study was defined as a blood BHBA concentration of ≥1.2mmol/L. Holstein cows (n=84) entering parity 2 or greater were enrolled using a randomized block design and housed in individual tiestalls. All treatment diets were fed for ad libitum intake and contained monensin. Cows received the same fresh cow ration after calving. Blood samples were obtained 3 times weekly before and after calving and analyzed for BHBA and nonesterified fatty acids (NEFA). Milk components, production, and dry matter intake were recorded and energy balance was calculated. Repeated measures ANOVA was conducted for the outcomes dry matter intake, energy balance, BHBA and NEFA concentrations, milk and energy-corrected milk yield, as well as milk composition. Predicted energy balance tended to be less negative postpartum in group C and cows in this group had fewer episodes of hyperketonemia compared with both the intermediate group and group H in the first 3 wk after calving. Postpartum BHBA and NEFA concentrations over time were highest in group H and lowest in group C, whereas milk production was not affected by prepartum plane of energy. Analysis of milk fatty acid composition showed a higher yield of preformed fatty acids in group H compared with group C, suggesting higher lipid mobilization for cows fed H. In this study, a 1-group, controlled-energy dry period approach decreased the degree of negative energy balance as well as the number of episodes and degree of hyperketonemia postpartum.

Cross-sectional study to investigate the association between vitamin D status and cutaneous mast cell tumours in Labrador retrievers.

Epidemiological data indicate that low serum vitamin D concentrations are associated with an increased risk of a variety of human tumours. Cutaneous mast cell tumours (MCT) occur more frequently in dogs than in any other species. Canine MCT express the vitamin D receptor, and vitamin D derivatives have in vitro and in vivo anti-tumour activity. We sought to examine the association between vitamin D serum level and MCT in Labrador retrievers, a dog breed predisposed to MCT development. To examine this association, serum 25-hydroxyvitamin D3 (25(OH)D3) concentrations were examined in eighty-seven Labrador retrievers, including thirty-three with MCT and fifty-four unaffected controls. The relationship between cases and controls and 25(OH)D3 level, age and body condition score were evaluated using univariate and multivariate analyses. Potential differences in vitamin D oral intake, calculated on the basis of a dietary questionnaire, were also evaluated between groups. Mean 25(OH)D3 concentration (104 (SD 30) nmol/l) in dogs with MCT was significantly lower than that of unaffected dogs (120 (SD 35) nmol/l; P = 0.027). The mean calculated vitamin D intake per kg body weight in Labrador retrievers with MCT was not statistically different from that of unaffected Labrador retrievers (0.38 (SD 0.25) and 0.31 (SD 0.22) μg/kg body weight, respectively; P = 0.13). These findings suggest that low levels of 25(OH)D3 might be a risk factor for MCT in Labrador retrievers. Prospective cohort studies are warranted.

Effects of a synbiotic on fecal quality, short-chain fatty acid concentrations, and the microbiome of healthy sled dogs

BackgroundSled dogs commonly suffer from diarrhea. Although multiple etiologies exist there are limited field studies using synbiotics as a supplement to prevent or treat diarrhea. The objective of this study was to examine alterations in fecal quality, short-chain fatty acids (SCFA), and the fecal microbiome in two groups of training sled dogs fed a synbiotic or microcrystalline cellulose placebo. Twenty clinically healthy training sled dogs randomized into two cohorts (9 synbiotic-fed, 8 placebo-fed) for a 6 week prospective study were examined. Fecal pH and fecal short chain fatty acid (SCFA) concentrations were measured and tag-encoded FLX 16S rDNA amplicon pyrosequencing (bTEFAP) and quantitative real-time PCR were performed at baseline (10 d prior to the study) and after 2 weeks of treatment with a total treatment time of 6 weeks. Fecal scores for all dogs were assessed at baseline and every day for 6 wk after initiation of treatment.ResultsAlterations in the fecal microbiome were observed with a significant rise in Lactobacillaceae in the synbiotic group (P = 0.004) after 2 wk of treatment. A positive correlation was found between Lactobacillaceae and overall butyrate concentration (R = 0.62, p = 0.011) in all dogs. After 5 wk of treatment, there was an improved fecal score and fewer days of diarrhea (Χ2 = 5.482, P = 0.019) in the dogs given synbiotic, which coincided with a presumed contagious outbreak shared by all dogs in the study.ConclusionsUse of this synbiotic results in an increase in presumed beneficial bacterial flora of the host colon which was associated with a decrease in the prevalence of diarrhea in training sled dogs.