Case report: Control of intestinal nematodes in captive Chlorocebus sabaeus.

There are limited data on the efficacy of antiparasitic treatments and husbandry methods to control nematode infections in captive populations of African green monkeys (AGMs), Chlorocebus sabaeus. In faecal egg count (FEC) tests, 10 of the 11 (91%) adult male AGMs captured from the large feral population on the island of St Kitts had evidence of nematode infections, mostly Capillaria (8/11, 73%), Trichuris trichiura (7/11, 64%) and strongylid species (7/11, 64%) specifically (hookworm and Trichostrongylus, 50/50), but also Strongyloides fuelleborni (1/11, 9%). When kept in individual cages with cleaning and feeding regimens to prevent reinfections and treated concurrently with ivermectin (300 µg/kg, given subcutaneously) and albendazole (10 mg/kg, given orally) daily for 3 days, 60% (6/10) of the AGMs were negative at a follow-up FEC at 3 months and by FEC and necropsy at the end of the study 5-8 months later. One monkey appeared to have been reinfected with T. trichiura after being negative by FEC at 3 months post-treatment. Four AGMs were positive for T. trichiura at the 3 month FEC follow-up but were negative at the end of the study after one further treatment regimen. Although initially being cleared of Capillaria following treatment, three AGMs were found to be infected at the end of the study. The ivermectin and albendazole treatment regimen coupled with good husbandry practices to prevent reinfections effectively controlled nematode infections in captive AGMs.

Trichuris trichiura egg extract proteome reveals potential diagnostic targets and immunomodulators.

Embryonated eggs are the infectious developmental stage of Trichuris trichiura and are the primary stimulus for the immune system of the definitive host. The intestinal-dwelling T. trichiura affects an estimated 465 million people worldwide with an estimated global burden of disease of 640 000 DALYs (Disability Adjusted Life Years). In Latin America and the Caribbean, trichuriasis is the most prevalent soil transmitted helminthiasis in the region (12.3%; 95% CI). The adverse health consequences impair childhood school performance and reduce school attendance resulting in lower future wage-earning capacity. The accumulation of the long-term effects translates into poverty promoting sequelae and a cycle of impoverishment. Each infective T. trichiura egg carries the antigens needed to face the immune system with a wide variety of proteins present in the shell, larvae's surface, and the accompanying fluid that contains their excretions/secretions. We used a proteomic approach with tandem mass spectrometry to investigate the proteome of soluble non-embryonated egg extracts of T. trichiura obtained from naturally infected African green monkeys (Chlorocebus sabaeus). A total of 231 proteins were identified, 168 of them with known molecular functions. The proteome revealed common proteins families which are known to play roles in energy and metabolism; the cytoskeleton, muscle and motility; proteolysis; signaling; the stress response and detoxification; transcription and translation; and lipid binding and transport. In addition to the study of the T. trichiura non-embryonated egg proteome, the antigenic profile of the T. trichiura non-embryonated egg and female soluble proteins against serum antibodies from C. sabaeus naturally infected with trichuriasis was investigated. We used an immunoproteomic approach by Western blot and tandem mass spectrometry from the corresponding SDS-PAGE gels. Vitellogenin N and VWD and DUF1943 domain containing protein, poly-cysteine and histidine tailed protein isoform 2, heat shock protein 70, glyceraldehyde-3-phosphate dehydrogenase, actin, and enolase, were among the potential immunoactive proteins. To our knowledge, this is the first study on the T. trichiura non-embryonated egg proteome as a novel source of information on potential targets for immunodiagnostics and immunomodulators from a neglected tropical disease. This initial list of T. trichiura non-embryonated egg proteins (proteome and antigenic profile) can be used in future research on the immunobiology and pathogenesis of human trichuriasis and the treatment of human intestinal immune-related diseases.

No evidence for sylvatic cycles of chikungunya, dengue and Zika viruses in African green monkeys (Chlorocebus aethiops sabaeus) on St. Kitts, West Indies.

BACKGROUND: Dengue, chikungunya and Zika viruses (DENV, CHIKV and ZIKV) are transmitted in sylvatic transmission cycles between non-human primates and forest (sylvan) mosquitoes in Africa and Asia. It remains unclear if sylvatic cycles exist or could establish themselves elsewhere and contribute to the epidemiology of these diseases. The Caribbean island of St. Kitts has a large African green monkey (AGM) (Chlorocebus aethiops sabaeus) population and is therefore ideally suited to investigate sylvatic cycles. METHODS: We tested 858 AGM sera by ELISA and PRNT for virus-specific antibodies and collected and identified 9704 potential arbovirus vector mosquitoes. Mosquitoes were homogenized in 513 pools for testing by viral isolation in cell culture and by multiplex RT-qPCR after RNA extraction to detect the presence of DENV, CHIKV and ZIKVs. DNA was extracted from 122 visibly blood-fed individual mosquitoes and a polymorphic region of the hydroxymethylbilane synthase gene (HMBS) was amplified by PCR to determine if mosquitoes had fed on AGMs or humans. RESULTS: All of the AGMs were negative for DENV, CHIKV or ZIKV antibodies. However, one AGM did have evidence of an undifferentiated Flavivirus infection. Similarly, DENV, CHIKV and ZIKV were not detected in any of the mosquito pools by PCR or culture. AGMs were not the source of any of the mosquito blood meals. CONCLUSION: Sylvatic cycles involving AGMs and DENV, CHIKV and ZIKV do not currently exist on St. Kitts.

Survey of vector-borne agents in feral cats and first report of Babesia gibsoni in cats on St Kitts, West Indies.

BACKGROUND: As there is little data on vector-borne diseases of cats in the Caribbean region and even around the world, we tested feral cats from St Kitts by PCR to detect infections with Babesia, Ehrlichia and spotted fever group Rickettsia (SFGR) and surveyed them for antibodies to Rickettsia rickettsii and Ehrlichia canis. RESULTS: Whole blood was collected from apparently healthy feral cats during spay/ neuter campaigns on St Kitts in 2011 (N = 68) and 2014 (N = 52). Sera from the 52 cats from 2014 were used to detect antibodies to Ehrlichia canis and Rickettsia rickettsii using indirect fluorescent antibody tests and DNA extracted from whole blood of a total of 119 cats (68 from 2011, and 51 from 2014) was used for PCRs for Babesia, Ehrlichia and Rickettsia. We could not amplify DNA of SFG Rickettsia in any of the samples but found DNA of E. canis in 5% (6/119), Babesia vogeli in 13% (15/119), Babesia gibsoni in 4% (5/119), mixed infections with B. gibsoni and B. vogeli in 3% (3/119), and a poorly characterized Babesia sp. in 1% (1/119). Overall, 10% of the 52 cats we tested by IFA for E. canis were positive while 42% we tested by indirect fluorescent antibody (IFA) for R. rickettsii antigens were positive. CONCLUSIONS: Our study provides the first evidence that cats can be infected with B. gibsoni and also indicates that cats in the Caribbean may be commonly exposed to other vector-borne agents including SFGR, E. canis and B. vogeli. Animal health workers should be alerted to the possibility of clinical infections in their patients while public health workers should be alerted to the possibility that zoonotic SFGR are likely circulating in the region.

Detection of Leptospiral DNA in the Urine of Donkeys on the Caribbean Island of Saint Kitts.

Leptospirosis is a global zoonosis caused by pathogenic spirochetes classified within the genus Leptospira. Leptospires live in the proximal renal tubules of reservoir or chronic carrier animals, and are shed in the urine. Naïve animals acquire infection either when they come in direct contact with a reservoir or infected animals or by exposure to environmental surface water or soil that is contaminated with their urine. In this study, urine samples from a herd of donkeys on the Caribbean island of St. Kitts were screened using a TaqMan-based real-time quantitative polymerase chain reaction (qPCR) targeting a pathogen-specific leptospiral gene, lipl32. Out of 124 clinically normal donkeys, 22 (18%) tested positive for leptospiral DNA in their urine. Water samples from two water troughs used by the donkeys were also tested, but were found to be free from leptospiral contamination. Detection of leptospiral DNA in the urine of clinically healthy donkeys may point to a role that these animals play in the maintenance of the bacteria on St. Kitts.

Interaction of non-human primate complement and antibodies with hypermucoviscous Klebsiella pneumoniae.

Emergent hypermucoviscosity (HMV) phenotypes of Klebsiella pneumoniae have been associated with increased invasiveness and pathogenicity in primates. In this study, we investigated the interaction of African green monkeys (AGM) (Chlorocebus aethiops sabaeus) complement and antibody with HMV and non-HMV isolates as in vitro models of primate infection. Significantly greater survival of HMV isolates was evident after incubation in normal serum or whole blood (p < 0.05) of AGM donors when compared to non-HMV strains. Greater survival of HMV strains (p < 0.05) was found after incubation in whole blood and serum from seropositive donors when compared to seronegative donor samples. Additionally, significantly greater amounts of K. pneumoniae were phagocytozed by AGM leukocytes when complement was active (p < 0.05), but no difference in uptake was observed when serum from seropositive or seronegative animals was used in challenged cells utilizing flow cytometry. Results demonstrate that interaction of cellular and humoral immune elements play a role in the in vitro killing of K. pneumoniae, particularly HMV isolates. Neither AGM serum, nor washed whole blood effectively killed HMV isolates; however, assays using heparinized whole blood of seronegative donors significantly reduced viability of HMV and non-HMV strains. The lack of bacterial killing observed in seropositive donors treatments could be at least partially associated with low IgG2 present in these animals. A better understanding of the pathogenesis of klebsiellosis in primates and host immune response is necessary to identify surface molecules that can induce both opsonizing and bactericidal antibody facilitating killing of Klebsiella, and the development of vaccines in human and animals.

Effect of firocoxib on cyclooxygenase 2, microsomal prostaglandin E2 synthase 1, and cytosolic phospholipase A2 gene expression in equine mononuclear cells.

OBJECTIVE: To validate primer sets for use in reverse transcription quantitative PCR assays to measure gene expression of cytosolic phospholipase A2 (cPLA2) and microsomal prostaglandin E2 synthase 1 (mPGES1) in equine mononuclear cells and determine the effects of firocoxib, a selective cyclooxygenase 2 (COX-2) inhibitor, on COX-2, cPLA2, and mPGES1 gene expression following incubation of mononuclear cells with lipopolysaccharide (LPS). ANIMALS: 8 healthy adult horses. PROCEDURES: Peripheral blood mononuclear cells were isolated by density gradient centrifugation and incubated at 37°C with medium alone, firocoxib (100 ng/mL), LPS (1 ng/mL or 1 µg/mL), or combinations of firocoxib and both LPS concentrations. After 4 hours, supernatants were collected and tested for prostaglandin E2 (PGE2) concentration with an enzyme inhibition assay, and gene expression in cell lysates was measured with PCR assays. RESULTS: Primer pairs for cPLA2 and mPGES1 yielded single products on dissociation curve analyses, with mean assay efficiencies of 102% and 100%, respectively. Incubation with firocoxib and LPS significantly decreased PGE2 supernatant concentrations and significantly reduced COX-2 and mPGES1 gene expression, compared with values following incubation with LPS alone. CONCLUSIONS AND CLINICAL RELEVANCE: Primer sets for mPGES1 and cPLA2 gene expression in equine mononuclear cells were successfully validated. Firocoxib significantly decreased LPS-induced COX-2 and mPGES1 expression, suggesting that it may be useful in the control of diseases in which expression of these genes is upregulated.

Is it the systemic inflammatory response syndrome or endotoxemia in horses with colic?

Some veterinarians describe particularly sick horses or neonatal foals as being endotoxemic, whereas others refer to the same animals as having the systemic inflammatory response syndrome. This article reviews the basis for the use of each of these terms in equine practice, and highlights the mechanisms underlying the response of the horse's innate immune system to key structural components of the microorganisms that initiate these conditions, including how some of those responses differ from other species. Current approaches used to treat horses with these conditions are summarized, and caution advised on extrapolating findings from other species to the horse.

HJB-1, a 17-hydroxy-jolkinolide B derivative, inhibits LPS-induced inflammation in mouse peritoneal macrophages.

Jolkinolide B (JB) and 17-hydroxy-JB (HJB) are diterpenoids from plants and it has been reported that the presence of a C-17 hydroxy group in JB significantly enhances the anti-inflammatory potency of JB. In this study, two HJB derivatives HJB-1 and HJB-2 were generated by the chemical modification of a 17-hydroxy group of HJB. HJB-1 more effectively inhibited TNF-α, IL-1ß and IL-6 release in LPS-stimulated mouse peritoneal macrophages. In addition, HJB-1 reduced LPS-induced mRNA expression of TNF-α, IL-1ß, IL-6, COX-2 and iNOS in a concentration-dependent manner, but did not alter IL-10 mRNA expression. LPS-induced NF-κB activation and MAPK phosphorylation were also effectively inhibited by HJB-1. These results demonstrate that HJB-1 exerts anti-inflammatory effects on LPS-activated mouse peritoneal macrophages by inhibiting NF-κB activation and MAPK phosphorylation and modification of a 17-hydroxy group of HJB may enhance the anti-inflammatory potency of HJB derivatives.

Relationships between intrauterine infusion of N-acetylcysteine, equine endometrial pathology, neutrophil function, post-breeding therapy, and reproductive performance.

Persistent endometritis in the mare is associated with hypersecretion of mucus by endometrial epithelium and migration of neutrophils into the uterine lumen. This study examines the relationships between N-acetylcysteine (NAC), a mucolytic agent with anti-inflammatory properties, and endometrial architecture, serum neutrophil function, post-breeding therapy, and reproductive performance of NAC-treated mares in a clinical setting. In study 1, endometrial biopsies from mares receiving intrauterine saline (fertile-control, n = 6) or 3.3% NAC (fertile-treatment, n = 6; barren-treatment, n = 10) were evaluated by histology and image analysis. In study 2, phagocytic activity of serum-derived neutrophils was measured after adding 0.5% or 3% NAC. In study 3, pregnancy rates of repeat breeders (n = 44) receiving an intrauterine infusion of 3.3% NAC 24-36 hours before mating (group 1) was recorded, as was first cycle of the season pregnancy rates of reproductively normal mares (group 2, n = 85), and mares treated for bacterial endometritis the cycle before mating (group 3, n = 25). Intrauterine NAC did not adversely affect endometrial histology. Extracellular mucus thickness and staining intensity were reduced in fertile-treatment mares (P < 0.03). Neutrophil function was inhibited by 3% NAC solution, but not by 0.5% NAC (P < 0.05). In study 3, for groups 1, 2, and 3, respectively, the first-cycle pregnancy rates were 77%, 74%, and 56%, and early embryonic death rates were 15%, 13%, and 7%. In group 2 mares treated with uterine lavage and oxytocin post-mating, the pregnancy rate was 89% (39/44), whereas in mares treated with uterine lavage and 1 g ceftiofur, it was 60% (24/40). Of the oxytocin-treated mares, 18% (8/44) had ≥ 1 cm of intrauterine fluid or marked uterine edema, whereas 80% (32/40) of the antibiotic-treated mares did. In conclusion, intrauterine infusion of a 3.3% solution of NAC was not irritating and inhibited the oxidative burst of neutrophils. Repeat breeder mares, with evidence of mucus hypersecretion, but no uterine pathogens, when treated with NAC followed by post-mating uterine lavage and oxytocin (and in some cases intrauterine antibiotics), achieved a pregnancy rate of 77%.

Characterization of cytokines associated with Th17 cells in the eyes of horses with recurrent uveitis.

OBJECTIVE: Equine recurrent uveitis (ERU) is a spontaneous disease that is the most common cause of blindness in horses, affecting up to 15% of the horse population. Th17 cells are a major cell population driving the pathogenesis in several mouse models of autoimmune inflammation, including experimental autoimmune uveitis. The purpose of this study is to investigate the role a Th17 cell-mediated response plays in the pathogenesis of ERU. PROCEDURE: Banked, Davidson's-fixed equine globes histopathologically diagnosed with ERU (n = 7) were compared immunohistochemically with healthy control globes (n = 7). Immunohistochemical staining was performed using a pan-Leptospira antibody and antibodies against IL-6, IL-17, and IL-23. Additionally, immunostaining was performed for T-cell (CD3) and B-cell (CD79α) markers. Specificity of immunoreactivity was confirmed by western blot analysis. RESULTS: Immunohistochemical staining was positive for IL-6, IL-17, and IL-23 within the cytoplasm of nonpigmented ciliary epithelial cells and mononuclear inflammatory cells infiltrating the iris, and ciliary body of ERU horses (n = 7) but negative in controls (n = 7). ERU-affected eyes were CD3 positive (n = 7) and CD79α negative (n = 7). Staining for Leptospira was negative in all ERU and control globes. CONCLUSIONS: Strong immunoreactivity for IL-6, IL-17, and IL-23, in conjunction with the fact that T lymphocytes are the predominating inflammatory cells present in ERU, suggests that IL-17-secreting helper T-cells play a role in the pathogenesis of ERU. These findings suggest that horses with ERU may serve as a naturally occurring animal model for autoimmune uveitis.

Disparities in TLR5 expression and responsiveness to flagellin in equine neutrophils and mononuclear phagocytes.

As sentinel cells of the innate immune system, neutrophils and mononuclear phagocytes use specific TLRs to recognize the conserved molecular patterns that characterize microbes. This study was performed to compare the responses of equine neutrophils and mononuclear phagocytes to LPS and flagellin, components of bacteria that are recognized by TLR4 and TLR5, respectively. Neutrophils and mononuclear phagocytes isolated from healthy horses were incubated in vitro with LPS, flagellin, or pronase-inactivated flagellin in the presence or absence of polymyxin B. Production of reactive oxygen species and expression of mRNA for proinflammatory cytokines were used as readouts for activation of neutrophils; production of TNF-α was used for the mononuclear cells. Western blot analysis and flow cytometry were used to detect TLR5 protein in both cell types. Although the neutrophils responded to both LPS and flagellin by producing reactive oxygen species and expressing mRNA for proinflammatory cytokines, flagellin had no stimulatory effect on monocytes or macrophages. Although both neutrophils and monocytes expressed mRNA for TLR5, it appeared to be translated into protein only by the neutrophils. Incubation with neither LPS nor IFN-γ altered TLR5 expression by the monocytes. These findings indicate that flagellin has disparate effects on neutrophils and mononuclear phagocytes isolated from horses, a species that is exquisitely sensitive to the TLR4 ligand, LPS, and that equine mononuclear phagocytes, unlike corresponding cells of other mammalian species, lack surface expression of TLR5 and do not respond to flagellin.

Effects of low-dose hydrocortisone therapy on immune function in neonatal horses.

Low-dose hydrocortisone (LDHC) therapy modulates inflammatory responses in adults and improves outcomes in some septic adults and neonates, but its immunologic effects have not been evaluated in neonates. The objective of this study was to evaluate effects of LDHC therapy on ex vivo immune function in neonatal horses (foals). We hypothesized that LDHC treatment would dampen proinflammatory responses without impairing neutrophil function. Hydrocortisone (1.3 mg/kg/d i.v.) was administered to foals in a tapering 3.5 d course. Peripheral blood leukocytes were collected from foals before, during, and after hydrocortisone treatment. A separate group of age-matched untreated foals served as controls. Endotoxin-induced peripheral blood mononuclear cell gene expression of inflammatory cytokines was measured by real-time quantitative RT-PCR. Neutrophils were incubated with labeled, killed Staphylococcus aureus or Escherichia coli for assessment of phagocytosis, and with phorbol myristate acetate, zymosan, or endotoxin for measurement of reactive oxygen species (ROS) production. Neutrophil phagocytosis and ROS production were similar in both groups. Foals receiving hydrocortisone had significantly decreased endotoxin-induced expression of TNF-α, IL-6, IL-8, and IL-1ß. These data suggest that this LDHC treatment regimen ameliorates endotoxin-induced proinflammatory cytokine expression in neonatal foals without impairing innate immune responses needed to combat bacterial infection.

Expression of Toll-like receptors 2, 3, 4, 6, 9, and MD-2 in the normal equine cornea, limbus, and conjunctiva.

OBJECTIVE: Human corneal cells have detectable levels of TLRs 1-10. TLRs 2 and 4 are the major corneal receptors, recognizing the PAMPs associated with fungal invasion in humans. The conjunctiva and limbus contain TLRs 2, 4, and 9. Our purpose was to determine the expression of TLRs 2, 3, 4, 6, 9, and MD-2 in the normal equine cornea, conjunctiva, and limbus. METHODS: Corneal, limbal, and conjunctival tissues were collected from seven euthanized horses having no evidence of ocular disease. RNA extraction with DNase-1 digestion was performed followed by RT-PCR to determine expression of TLRs 2, 3, 4, 6, 9, and MD-2. Products were resolved by electrophoresis on 1.5% agarose gels and visualized using ethidium bromide staining. RESULTS: Expression of TLRs 2, 3, 4, 6, 9, and MD-2 was present in the cornea, limbus, and conjunctiva of each horse, except one horse, where TLR3 expression was unable to be demonstrated in the dorsal and ventral conjunctiva. CONCLUSIONS: Confirming the expression of TLRs in equine ocular tissues is an initial step in identifying how they play a role in infectious keratitis, particularly fungal. The results further support the use of equine ocular tissues as a model for human fungal keratitis. Studies of the TLR expression together with their cytokine profile induced during equine fungal keratitis may help further clarify the pathogenesis of the disease and possibly lead to the development of new treatment protocols for both equines and humans.

Expression of genes associated with inflammation induced by ex vivo exposure to lipopolysaccharide in peripheral blood leukocytes from horses with gastrointestinal disease.

OBJECTIVE: To investigate the effect of ex vivo exposure to lipopolysaccharide (LPS) on the expression of inflammatory genes in leukocytes from horses with gastrointestinal (Gl) disease and determine whether the pattern or magnitude of the response to LPS correlated with the type of disease and outcome. ANIMALS: 49 horses with Gl disease and 10 healthy horses PROCEDURES: Leukocytes were isolated from blood samples and submitted to 3 protocols: immediate freezing, freezing after 4-hour incubation in medium, and freezing after 4-hour incubation in medium containing LPS. Expression of 14 genes associated with inflammation was assessed via PCR assay. Results were compared by disease type and outcome RESULTS: Horses with Gl disease had colic of unknown etiology (n=8), Gl inflammation or strangulation (18), or nonstrangulating Gl obstruction (23). Among the 44 horses receiving treatment, 38 were discharged from the hospital and 6 died or were euthanized. Incubation of leukocytes in medium alone changed the expression of several genes. Incubation with LPS resulted in increased expression of interleukin-10 and monocyte chemotactic protein-3 in leukocytes from healthy and sick horses. Leukocytes from horses with nonstrangulating obstruction and horses that survived had less pronounced LPS-induced increases in interleukin-10 expression than did cells from healthy horses. The opposite was evident for monocyte chemotactic protein-3. CONCLUSIONS AND CLINICAL RELEVANCE: No evidence existed for a reduced response of leukocytes from horses with gastrointestinal disease to ex vivo exposure to LPS. Leukocyte expression of inflammatory genes after ex vivo incubation with LPS appeared to be related to pathogenesis and prognosis.

Effects of renal autograft ischemic storage and reperfusion on intraoperative hemodynamic patterns and plasma renin concentrations in clinically normal cats undergoing renal autotransplantation and contralateral nephrectomy.

OBJECTIVE: To evaluate the effect of the duration of cold Ischemia on the renin-angiotensin system during renal transplantation In cats and to define the potential Influence of vasoactive factors in renal tissue following cold ischemic storage versus warm ischemic storage. ANIMALS: 10 purpose-bred 6-month-old sexually Intact female cats. PROCEDURES: 10 cats underwent renal autotransplantation after 30 minutes (n=5) or 3 hours (5) of simple, ex vivo cold storage of renal autographs. Following autograft reperfusion, direct hemodynamic variables were measured with a telemetric Implant and samples were collected for plasma renin concentration. Activation of vascular-related genes (renin, endothelin, and angiotensin converting enzyme) relative to 2-hour simple cold or warm ischemia was also evaluated. RESULTS: No significant difference between groups was detected In any of the hemodynamic variables or postreperfusion plasma renin concentrations measured in this study relative to the duration of cold ischemic storage. There was also no difference between warm- and cold-stored kidneys in the expression of vascular-related genes. CONCLUSIONS AND CLINICAL RELEVANCE: Prolonged renal Ischemia for clinically relevant durations does not appear to predispose clinically normal cats to altered hemodynamics or high plasma renin concentrations following graft reperfusion. Activation of vasoactive genes does not appear to be Influenced by type of Ischemia over 2 hours.

Differential induction of Toll-like receptor gene expression in equine monocytes activated by Toll-like receptor ligands or TNF-α.

Toll-like receptors (TLRs) function as sentinels for the innate immune system, detecting microbial ligands during infection and inflammation. Previous studies indicate that activation of these receptors on equine monocytes leads to discrete pro- and anti-inflammatory responses that are mediated through the induction of specific cytokine genes. However, less is known regarding the regulation of TLR gene expression in these cells. Therefore, we investigated the effects of ligands recognized by TLR2, 3 or 4 upon TLR2, 3 and 4 gene expression by equine monocytes. We determined that incubation of monocytes with TLR2 and 4 ligands, which signal through the intracellular adaptor protein MyD88, induces expression of the TLR2 and 4 genes, but not the TLR3 gene. Conversely, incubation with a TLR3 ligand, which recruits the TRIF adaptor protein, selectively induces expression of the TLR3 gene, but not TLR2 or 4 genes. Furthermore, incubation of these cells with TNF-α, the pro-inflammatory cytokine that is a hallmark of TLR activation, does not affect expression of the three TLR genes. These findings suggest that exposure of equine monocytes to microbial ligands but not to endogenous inflammatory mediators may initiate responses that alter the horse's sensitivity to other microbial components during infections.

Expression of inflammation-associated genes in circulating leukocytes collected from horses with gastrointestinal tract disease.

OBJECTIVE: To investigate whether expression of inflammation-associated genes in leukocytes from horses with gastrointestinal tract (GIT) diseases correlated with the type of disease and outcome. ANIMALS: 10 healthy horses and 50 horses with GIT disease. PROCEDURES: A blood sample was collected from each healthy horse or horse with GIT disease (during admission to the hospital). Leukocytes were isolated, diluted to a standard concentration, and frozen until RNA extraction. Expression of 14 genes associated with inflammation was quantified by use of a real-time quantitative reverse transcription PCR assay. Results were grouped by GIT disease type and disease outcome for comparison. RESULTS: Horses with GIT disease had colic of unknown etiology (n = 8 horses), GIT inflammation or strangulation (19), or nonstrangulating GIT obstruction (23). Among the 45 horses receiving treatment, 38 were discharged from the hospital, and 7 died or were euthanized. Compared with healthy horses, horses with colic of unknown etiology had similar gene expression. Significant differences in expression of the interleukin-8, leukocyte-selectin molecule, matrix metalloproteinase-9, platelet-selectin molecule, mitochondrial superoxide dismutase, Toll-like receptor 4, and tumor necrosis factor-A genes were detected between healthy horses and horses with GIT disease. Significant differences in expression of the interleukin-1 receptor antagonist, interleukin-8, leukocyte-selectin molecule, matrix metalloproteinase-9, platelet-selectin molecule, mitochondrial superoxide dismutase, Toll-like receptor 4, and tumor necrosis factor-A genes were detected among healthy horses and horses grouped by disease outcome. CONCLUSIONS AND CLINICAL RELEVANCE: Inflammatory gene expression in leukocytes of horses with GIT disease appeared to be related to disease pathogenesis and prognosis.

Lipopolysaccharide and TNF-alpha modify adenosine A(2A) receptor expression and function in equine monocytes.

Stimulation of adenosine A(2A) receptors results in anti-inflammatory effects in a variety of cell types. Lipopolysaccharide (LPS) and pro-inflammatory cytokines, such as TNF-alpha and IL-1, have been reported to up-regulate the expression of adenosine A(2A) receptors and thereby enhance the functional activity of adenosine A(2A) receptors in human and murine monocyte/macrophage cell lines and in monocytes/macrophages isolated from those species. In this study, we investigated the effects of LPS and TNF-alpha on the expression and functional activity of adenosine A(2A) receptors in isolated equine peripheral blood monocytes. The results of this study indicate that LPS and TNF-alpha up-regulate the transcription of adenosine A(2A) receptors for up to 24h; the response to LPS was of greater magnitude than the response to TNF-alpha. In this study, incubation with LPS, but not with TNF-alpha, resulted in down-regulation of adenosine A(3) receptor mRNA expression. Furthermore, incubation of these cells with LPS significantly increases the surface density of adenosine A(2A) receptors, and incubation with low concentrations of either LPS or TNF-alpha significantly increases the potency of the adenosine A(2A) receptor agonist, ATL313, to inhibit LPS-induced production of TNF-alpha. These findings suggest that the increased expression of adenosine A(2A) receptors and the enhanced functional potency of adenosine A(2A) receptor agonists after exposure to pro-inflammatory substances such as LPS or TNF-alpha may render adenosine A(2A) receptor agonists particularly important in the treatment of the systemic inflammatory response syndrome that occurs secondary to endotoxemia and bacterial infections in adult horses and neonatal foals.

Innate immune responses of primary murine macrophage-lineage cells and RAW 264.7 cells to ligands of Toll-like receptors 2, 3, and 4.

Although studies have been performed to characterize responses of macrophages from individual anatomical sites (e.g., alveolar macrophages) or of murine-derived macrophage cell lines to microbial ligands, few studies compare these cell types in terms of phenotype and function. We directly compared the expression of cell surface markers and functional responses of primary cultures of three commonly used cells of monocyte-macrophage lineage (splenic macrophages, bone marrow-derived macrophages, and bone marrow-derived dendritic cells) with those of the murine-leukemic monocyte-macrophage cell line, RAW 264.7. We hypothesized that RAW 264.7 cells and primary bone marrow-derived macrophages would be similar in phenotype and would respond similarly to microbial ligands that bind to either Toll-like receptors 2, 3, and 4. Results indicate that RAW 264.7 cells most closely mimic bone marrow-derived macrophages in terms of cell surface receptors and response to microbial ligands that initiate cellular activation via Toll-like receptors 3 and 4. However, caution must be applied when extrapolating findings obtained with RAW 264.7 cells to those of other primary macrophage-lineage cells, primarily because phenotype and function of the former cells may change with continuous culture.