A. Betancourt

Endometrial inflammatory markers of the early immune response in mares susceptible or resistant to persistent breeding-induced endometritis

Transient endometritis after breeding is necessary for clearance of bacteria and spermatozoa; however, in a subpopulation of mares, the inflammation fails to resolve in a timely fashion. The objective of this study was to describe the uterine inflammatory response in mares susceptible or resistant to persistent breeding-induced endometritis (PBIE) during the first 24 h after induction of uterine inflammation.Twelve mares were classified as susceptible (nZ6) or resistant (nZ6) to PBIE. Mares were inseminated over five estrous cycles and endometrial biopsies were collected at one time point per cycle before (0) and 2, 6, 12, and 24 h after insemination. qPCR analysis for IL1B, IL6, IL8, IFNG, TNF (TNFA), IL10, and IL1RN was performed, and endometrial inflammatory cells were counted for each sample. Relative quantification values reported fold changes in mRNA expression from 0 h values. A general pattern of expression post insemination was observed in both groups of mares. Cytokine mRNA increased at 2 h, peaked between 2 and 12 h, and then decreased.Differences were detected between groups of mares 6 h after challenge; resistant mares had higher mRNA expression of IL6, IL1RN,and IL10 than susceptible mares. Susceptible mares had an increased number of polymorphonuclear neutrophils in the endometrium 2 and 12 h after breeding when compared with resistant mares. These findings describe an inherent difference in the initial immune response to insemination and may help explain the transient nature of inflammation in resistant mares, whereas susceptible mares develop a persistent inflammation.

Granzyme B-mRNA expression by equine lymphokine activated killer (LAK) cells is associated with the induction of apoptosis in target cells

Lymphokine-activated killer (LAK) cells are a subset of cytotoxic cells capable of lysing freshly isolated tumor cells. While LAK activity is typically measured using the (51)Cr-release assay, here we used a non-radioactive flow cytometric method to demonstrate equine LAK activity. Equine peripheral blood mononuclear cells (PBMC) were stimulated in vitro with recombinant human interleukin 2 (hIL-2) to generate LAK cells. An equine tumor cell line, EqT8888, labeled with carboxyfluorescein succinimidyl ester (CFSE) was used as target cells. Following incubation of the targets with different concentrations of LAK cells, Annexin V was added to identify the early apoptotic cells. With increasing effector to target cell ratios, EqT8888 apoptosis was increased. We also measured interferon-gamma, granzyme B and perforin mRNA expression in the LAK cell cultures as possible surrogate markers for cytotoxic cell activity and found granzyme B mRNA expression correlated best with LAK activity. Also, we found that the reduced LAK activity of young horses was associated with decreased granzyme B mRNA expression. Our results indicate that fluorescence-based detection of LAK cell activity provides a suitable non-radioactive alternative to (51)Cr-release assays and mRNA expression of granzyme B can be used as surrogate marker for these cytotoxic cells.

Effects of advanced age on whole-body protein synthesis and skeletal muscle mechanistic target of rapamycin signaling in horses

OBJECTIVE To determine the effects of advanced age on whole-body protein synthesis and activation of the mechanistic target of rapamycin (mTOR) signaling pathway in skeletal muscle of horses. ANIMALS Six 22- to 26-year-old (aged) and six 7- to 14-year-old (mature) horses. PROCEDURES Whole-body protein synthesis was measured with a 2-hour primed constant infusion of (13)C sodium bicarbonate, followed by a 4-hour primed constant infusion of 1-(13)C phenylalanine. After the infusions, a biopsy specimen was obtained from a gluteus medius muscle and activation of protein kinase B (Akt), p70 riboprotein S6 kinase (S6K1), riboprotein S6 (rpS6), and eukaryotic initiation factor 4E binding protein 1 (4EBP1) was determined with western immunoblot analysis. For all horses, inflammatory cytokine expression in muscle and blood samples was measured with quantitative real-time PCR analysis. RESULTS Advanced age had no effect on whole-body protein synthesis or the phosphorylation of Akt, rpS6, and 4EBP1; however, muscle specimens of aged horses had 42% lower phosphorylation of S6K1 than did those of mature horses. Aged and mature horses had similar inflammatory cytokine expression in muscle and blood samples. CONCLUSIONS AND CLINICAL RELEVANCE The lower S6K1 activation for aged horses, compared with that for mature horses, could be indicative of low rates of muscle protein synthesis in aged horses. However, advanced age had no effect on any other indicators of whole-body or muscle protein synthesis or on measures of systemic or muscle inflammation, which suggested that protein metabolism and subsequently requirements may not differ between healthy mature and aged horses.

Effect of Propionibacterium acnes-containing immunostimulant on interferon-gamma (IFNγ) production in the neonatal foal.

Production of the Th1 cytokine interferon gamma (IFNγ) is associated with resistance to intracellular pathogens, including Rhodococcus equi. While neonatal foals are initially deficient in IFNγ production, expression of this cytokine increases throughout their first year of life. This is presumably the result of stimulation by environmental antigens including pathogen associated molecular patterns (PAMPS) signaling through toll-like receptors (TLR). This increased expression of IFNγ is likewise associated with an age-related resistance to R. equi infection. While immunostimulants containing PAMPS have been administered to adult horses in an attempt to modify their immune response, the effect of these materials on IFNγ expression in foals is unknown. The main objective of this study was to determine the effect of administering a commercial immunomostimulant EqStim® (Propionibacterium acnes) on IFNγ production measured using intracellular staining (IFNγ) and RT-PCR.

The effect of age and exercise training on insulin sensitivity, fat and muscle tissue cytokine profiles and body composition of old and young Standardbred mares

This study tested the hypothesis that old and young mares exhibit different endocrine responses to a frequently sampled intravenous glucose tolerance test (FSIGT) and different cytokine profiles in blood, adipose and muscle tissues. It was also hypothesised that exercise training alters endocrine and tissue cytokine profiles. Pilot data from 15 mixed background horses indicated tissue differences in cytokine profiles. For the main study, six old (22.0±0.7 years) and six young (7.3±0.6 years; mean±SE) unfit Standardbred mares were tested pre- and post-training. Exercise training occurred three days/week for 15 weeks at ~60% maximum heart rate. Plasma insulin and glucose concentrations were measured via radioimmunoassay and enzyme-electrode interface, respectively. Samples of blood, middle gluteal muscle (RM), and subcutaneous adipose tissue from the neck (NF) were collected pre- and post-training for mRNA quantification. Minimal model analysis of FSIGT, repeated measures ANOVA and Pearson Product Moment we...

The role of proliferation in the regulation of interferon gamma (IFNγ) expression in foals.

Interferon-gamma (IFNγ) plays an important role against viral and intracellular bacterial infections and its production is deficient in foals. Cellular proliferation provides an opportunity for de novo gene expression, though little is known about its role in regulating IFNγ expression in foals. While stimulation of foal peripheral blood mononuclear cells (PBMCs) with concanavalin A (ConA) increased the frequency of IFNγ(+) cells, the overall percentage of IFNγ(+) cells remained below that of adults. By contrast, the proliferative response of foal PBMC was significantly greater than that of the adults. In foals, IFNγ production was predominantly associated with those T cells that underwent proliferation, whereas in adults non-dividing cells also produced IFNγ. While treatment with hydroxyurea inhibited cellular division, it failed to completely block IFNγ production. This residual IFNγ production likely represented memory cells as the proportion of these proliferation-independent IFNγ(+) cells increased with foal age. However, memory cells may not account for all of the IFNγ production as ConA stimulation likely provided additional signals that can control IFNγ expression.

The effect of environment on interferon-gamma production in neonatal foals

While interferon-gamma (IFNγ) plays an important role in protection against viral and intracellular bacterial infections, its production in neonates is deficient. Exposure to environmental antigens can promote the maturation of the immune system of neonatal humans and mice. We hypothesize that exposure to high level of microbial components would increase the production of IFNγ in neonatal foals. To test this hypothesis, one group of foals was placed into stalls three times a week for 8 weeks. A second group of foals remained on pasture. Air samples were collected from the barn and pasture for microbial culture. There were more bacteria and fungi in the air samples collected from the barn compared with those from the pasture. Bronchoalveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMC) were collected from both groups of foals at various times to assess IFNγ production. The frequency of IFNγ(+) lymphocytes in BAL cells and PBMC was higher for foals kept in the stalls.

Characterisation of the inflammatory cytokine response to anthelmintic treatment in ponies.

REASONS FOR PERFORMING STUDY Anthelmintic treatments have been associated with local inflammatory reactions. Since each class of anthelmintic has unique mechanisms of action affecting different subpopulations of parasites, we hypothesised that they will also induce characteristic proinflammatory responses. OBJECTIVES To determine the effect of anthelmintic class on the proinflammatory response post treatment. STUDY DESIGN Ponies naturally infected with cyathostomins and other parasites after pasture grazing were left untreated or treated with representatives of 3 different classes of anthelmintics: fenbendazole (benzimidazole); pyrantel tartrate (pyrimidine); and moxidectin (macrocyclic lactone). All were monitored for the expression of proinflammatory genes in the peripheral blood using real-time PCR. METHODS The ponies were divided into 4 treatment groups: Group 1 (n = 4) were untreated controls; Group 2 (n = 5) received 5 daily doses of fenbendazole (10 mg/kg bwt); Group 3 (n = 4) received daily treatment of pyrantel tartrate 2× (2.65 mg/kg bwt); and Group 4 (n = 5) received a single dose of moxidectin (400 μg/kg bwt). Blood samples were collected daily for 2 weeks to determine the effect of deworming on proinflammatory gene expression. Faecal egg counts were used to evaluate the efficacy of each drug. RESULTS While treatment with the benzimidazole significantly reduced egg counts up to 14 days post treatment, it also stimulated proinflammatory gene expression. Treatment with pyrantel salt also reduced faecal egg counts with less of a proinflammatory response. Treatment with the macrocyclic lactone was the most successful in reducing faecal egg counts and produced no signs of increased proinflammatory cytokine expression. CONCLUSIONS This study revealed pronounced differences in the cytokine responses to anthelmintic treatment. This inflammatory reaction may play a role in the development of parasitic disease post anthelmintic treatment.

Interaction between anthelmintic treatment and vaccine responses in ponies naturally infected with cyathostomins

Anthelmintics and vaccines are commonly given concurrently in routine equine management, but it is unknown to what extent an interaction between the two exists. Cyathostomins can modulate the local immune response by stimulating a type 2 helper T cell (Th2) response. In addition, anti-inflammatory effects of ivermectin have been found in rodent models. It is unknown whether these anti-inflammatory effects affect the acute phase response elicited by commonly used vaccines. This study evaluated how the acute phase inflammatory response, leukocyte expression of pro-inflammatory cytokines, and vaccine-specific titers induced by simultaneous injection of three vaccines (West Nile Virus, Equine Herpes Rhinopneumonitis, and Keyhole Limpet Hemocyanin) were modulated by concurrent administration of ivermectin or pyrantel pamoate in ponies naturally infected with cyathostomins. Mixed-breed yearling ponies were blocked by gender and fecal strongyle egg count, then randomly assigned to three treatment groups: ivermectin (n=8), pyrantel pamoate (n=8), and control (n=7). All ponies received vaccinations intramuscularly on days 0 and 29, and anthelmintics were administered on the same days. Whole blood, serum and plasma samples were collected one, three and 14 days after each vaccination. Samples were analyzed for concentrations of acute phase reactants (haptoglobin, serum amyloid A, fibrinogen and iron), mRNA expression levels of cytokines (interleukin (IL)-1β, IL-4, IL-10, tumor necrosis factor (TNF)-α and interferon (IFN)-γ) in leukocytes, and vaccine-specific antibody titers. A marked acute-phase response was noted following both vaccinations. In contrast, the pattern of change in cytokine expression was less pronounced and more variable. Statistical differences were observed between groups for haptoglobin, fibrinogen, IL-1β, IL-4, and IL-10, but differences were generally small and none of the vaccine titers were different between the groups. Taken together, the study found some signs of modulation of immunologic or inflammatory responses to the administered vaccines, when anthelmintics were administered concurrently, but these are unlikely to have practical implications for vaccination routines.

Characterization of the in situ immunological responses to vaccine adjuvants.

Adjuvants are included with many inactivated and some modified live vaccines to enhance immune responses to specific antigens. While early vaccines relied exclusively upon aluminum salts, still the major adjuvant used in human vaccines, other adjuvant products are used in veterinary medicine. In addition to enhancing antigen presentation, adjuvants can also enhance the development of specific immune responses. Thus, alum adjuvants often preferentially stimulate humoral immune responses. By contrast, lipid-based adjuvants are often more effective at stimulating cell-mediated immune responses. Metastim(®) is a lipid-based adjuvant reported to elicit both humoral and cellular immune responses, though the mechanism responsible for this activity remains unclear. In this study, we compared the ability of equine influenza virus vaccines containing either saline or Metastim(®) or an aluminum phosphate adjuvant to stimulate antigen presenting cell function in vivo. Six ponies were intradermally inoculated with inactivated equine influenza (KY97) mixed with either adjuvant or saline. Multiple sites were injected so that biopsies could be collected at different times post injection. The 4mm punch biopsies were formalin-fixed, paraffin-embedded, and stained with hematoxylin and eosin (H&E). Total RNA was isolated from 2mm punch biopsies for the determination of gene expression by real-time PCR. H&E staining revealed a variety of cells recruited to the injection sites, including lymphocytes, neutrophils, eosinophils and macrophages. Real-time PCR analysis of the injection site confirmed this cellular infiltration and identified increased expression of activation markers. Both vaccines also stimulated gene expressions of pro-inflammatory cytokines. The vaccine containing Metastim(®) elicited significantly higher gene expression of interferon-γ, IL-12, CD4 and CD83 compared to alum (p<0.05). While the greater induction of IFNγ-related gene expression indicates that Metastim(®) can elicit Th-1 immune responses more effectively than the aluminum salt, there was also evidence of Th2 cytokine induction.