Objective—To evaluate and compare the gene expression of interleukin(IL)-1β, IL-8, and interferon-γ during the first 72 hours after birth in healthy foals and during the first 72 hours after hospitalization in sick neonatal foals and investigate correlations of clinicopathologic variables with cytokine expressions in healthy and sick neonatal foals.
Animals—33 foals < 7 days old (10 healthy foals, 7 foals with sepsis, 6 foals with peripartum asphyxia syndrome, and 12 foals with other diseases [2 with failure of passive transfer of immunity only were not further evaluated]).
Procedures—A blood sample (15 mL) was collected from each foal immediately after birth or hospital admission (0 hours) and at 24 and 72 hours later. Clinicopathologic variables were evaluated, and cytokine gene expression in WBCs was measured with an absolute quantitative real-time reverse transcriptase PCR assay.
Results—At all time points, gene expression of interferon-γ was low in all groups. No time-dependent changes in cytokine expressions were detected in healthy or sick foals. Foals with sepsis had significantly higher IL-1β gene expression than did healthy foals, foals with peripartum asphyxia syndrome, or foals with other diseases. At 0 hours, IL-1β expression was correlated with plasma fibrinogen concentration in healthy foals and with the neutrophil-to-lymphocyte ratio in foals with sepsis; IL-8 expression was correlated with monocyte count in foals with sepsis and with arterial pH, plasma fibrinogen concentration, and plasma lactate concentration in foals with peripartum asphyxia syndrome.
Conclusions and Clinical Relevance—Data have suggested that evaluation of IL-1β expression in sick neonatal foals could help identify those with sepsis.
Objective—To determine the prevalence of perinuclear antineutrophil cytoplasmic autoantibodies (pANCA) in dogs with confirmed or suspected immune-mediated hemolytic anemia (IMHA) or dogs infected with various vector-borne pathogens, including Rickettsia rickettsii, Bartonella henselae, Bartonella vinsonii subsp berkhoffii, Ehrlichia canis, Borrelia burgdorferi, and Leishmania infantum.
Animals—55 dogs with confirmed or suspected IMHA, 140 dogs seroreactive for vector-borne pathogens, and 62 healthy dogs and dogs seronegative for vector-borne pathogens.
Procedures—Samples were allocated to subgroups on the basis of the health status of the dogs and the degree of seroreactivity against various vector-borne pathogens. Serum samples were tested retrospectively via indirect immunofluorescence assay to determine pANCA status.
Results—26 of 55 (47%) dogs with confirmed or suspected IMHA and 67 of 140 (48%) dogs seroreactive for vector-borne pathogens had positive results when tested for pANCA. Serum samples with the highest antibody concentrations against L infantum antigen had the highest proportion (28/43 [65%]) that were positive for pANCA. One of 20 (5%) dogs seronegative for tick-borne pathogens and 8 of 22 (36%) dogs seronegative for L infantum had positive results for pANCA. One of 20 (5%) healthy dogs had serum antibodies against pANCA.
Conclusions and Clinical Relevance—pANCA were detected in a high percentage of dogs with IMHA and vector-borne infectious diseases. Therefore, pANCA may be a relatively nonspecific marker for dogs with inflammatory bowel disease, although they could represent a biomarker for immune-mediated diseases and infections.
Objective—To compare composition and colony formation of bone marrow mononuclear cells (BMMCs) harvested from dogs by means of a new perfusion method and the conventional aspiration method.
Animals—7 healthy adult Beagles.
Procedures—BMMCs were collected from the humeri and femurs of Beagles via perfusion and aspiration methods. Flow cytometric analysis was performed to quantify the presence of contaminant cells from the peripheral blood and the percentage of CD34+ progenitor cells in the BMMCs. A CFU assay was conducted to determine the number of progenitor cells in the BMMCs.
Results—The perfusion method was safely performed in all 7 dogs. Flow cytometric analysis revealed that the percentages of contaminant CD3+CD4+, CD3+CD8+, and CD21 + lymphocytes in BMMCs obtained via perfusion were significantly lower than percentages obtained via aspiration. The percentage of CD34+ cells obtained via perfusion was significantly higher than that obtained via aspiration. In addition, perfusion yielded a significantly higher CFU count than did aspiration.
Conclusions and Clinical Relevance—The perfusion method used in this study can minimize the contamination of bone marrow samples with peripheral blood and was a more efficient means for collecting canine bone marrow progenitor cells than the conventional aspiration method. Therefore, the perfusion method can be more suitable than aspiration for harvesting bone marrow cells for transplantation in dogs.
Objective—To determine effects of infection with bovine leukosis virus (BLV) on lymphocyte proliferation and apoptosis in dairy cattle.
Animals—27 adult Holstein cows.
Procedures—Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood from lactating Holstein cows seronegative for BLV (n = 9 cows), seropositive for BLV and aleukemic (aleukemic; 9), and seropositive for BLV and persistently lymphocytotic (PL; 9). Isolated PBMCs were assayed for mitogen-induced proliferation and were analyzed by means of flow cytometry. The PBMCs from a subset of each group were assayed for apoptosis, caspase-9 activity, and expression of selected genes related to apoptosis.
Results—PL cows had significantly higher total lymphocyte counts and significantly lower proportions of T-lymphocyte populations than did BLV-negative and aleukemic cows. Both groups of BLV-infected cows had significantly higher proportions of B cells and major histocompatibility complex II–expressing cells than did BLV-negative cows. Proliferation with concanavalin A was significantly lower for PL cows, compared with proliferation for BLV-negative cows. Pokeweed mitogen–induced proliferation was significantly higher for aleukemic and PL cows than for BLV-negative cows. Gene expression of apoptosis-inhibitory proteins BCL2 and BCL2L1 was significantly higher for aleukemic cows and expression of BCL2 was significantly higher for PL cows than for BLV-negative cows.
Conclusions and Clinical Relevance—Cattle infected with BLV had marked changes in PBMC populations accompanied by alterations in proliferation and apoptosis mechanisms. Because the relative distribution and function of lymphocyte populations are critical for immune competence, additional studies are needed to investigate the ability of BLV-infected cattle to respond to infectious challenge.
Objective—To evaluate effects of cyclosporine, dexamethasone, and the immunosuppressive agent human CTLA4-Ig on cytokine production by feline lymphocytes in vitro and to assess patterns of cytokine production for 5 immunosuppressed renal transplant recipient cats.
Animals—21 clinically normal cats and 5 immunosupressed renal transplant recipient cats.
Procedures—Peripheral blood mononuclear cells were isolated from clinically normal cats and stimulated with concanavalin A (Con A; 10 μg/mL) alone or Con A with cyclosporine (0.05 μg/mL), dexamethasone (1 × 10−7M), a combination of cyclosporine-dexamethasone, or human CTLA4-Ig (10 g/mL). Cells from transplant recipients were stimulated with Con A alone. An ELISA was performed to measure production of interferon (IFN)-γ, granulocyte macrophage–colony stimulating factor (GM-CSF), interleukin (IL)-2, IL-4, and IL-10. Proliferation of CD4+ and CD8+T cells from immunosuppressed cats were also evaluated. Pairwise comparisons were performed via a Wilcoxon signed rank test or Wilcoxon rank sum test.
Results—Cyclosporine, dexamethasone, cyclosporine-dexamethasone combination, and CTLA4-Ig caused a significant decrease in IL-2, IFN-γ, and GM-CSF production. Cyclosporine and cyclosporine-dexamethasone, but not human CTLA4-Ig, caused a significant decrease in IL-10 production. High basal concentrations of IL-2 and IL-10 were identified in transplant recipients, and IL-10 was significantly increased in stimulated cultures. In immunosuppressed cats, there was a decrease in frequency of responders and proliferative capacity of CD4+ and CD8+T cells.
Conclusions and Clinical Relevance—CTLA4-Ig successfully inhibited proinflammatory cytokines while sparing cytokines critical for allograft tolerance. These data may be useful for developing better strategies to prevent rejection while sparing other immune functions.
Objective—To determine whether rosiglitazone, an agonist of the peroxisome proliferator-activated receptor (PPAR) γ, could alleviate intestinal damage induced by Escherichia coli lipopolysaccharide (LPS) in weaned pigs.
Animals—18 weaned pigs (mean ± SD age, 28 ± 3 days).
Procedures—Pigs were allocated to 3 treatments (6 pigs/treatment). Control pigs were injected IP with dimethyl sulfoxide and then injected 30 minutes later with sterile saline (0.9% NaCl) solution, LPS-treated pigs were injected IP with dimethyl sulfoxide and then injected 30 minutes later with LPS (100 μg/kg, IP), and rosiglitazone plus LPS-treated pigs were injected with rosiglitazone (3 mg/kg, IP) and then injected 30 minutes later with LPS (100 μg/kg, IP). Pigs were euthanized 3 hours after challenge exposure, and samples of the small intestines were collected for histologic, biochemical, and immunohistochemical examination.
Results—Rosiglitazone alleviated LPS-induced intestinal damage, which was manifested as a lower crypt depth in the duodenum and a higher villus height-to-crypt depth ratio in the duodenum, jejunum, and ileum. Rosiglitazone also mitigated inhibition of crypt cell proliferation in the jejunum and ileum induced by LPS injection. Pretreatment with rosiglitazone significantly increased the number of cells that stained for PPARγ and significantly decreased the number of cells that stained for inducible nitric oxide synthase.
Conclusions and Clinical Relevance—Rosiglitazone alleviated intestinal damage induced by LPS injection in weaned pigs. The protective effects of rosiglitazone on the intestines may be associated with inhibition of intestinal proinflammatory mediators, such as inducible nitric oxide synthase. (Am J Vet Res 2010;71:1331–1338)
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.
Objective—To determine whether expression of equine platelet activation–dependent surface markers is influenced by phospodiesterase (PDE) isoenzyme activity and whether antigen challenge alters platelet PDE activity in horses with recurrent airway obstruction (RAO).
Procedures—7 healthy horses were used for in vitro experiments, 6 horses with RAO were used for antigen challenge, and 6 healthy horses were used as control animals. Three of the healthy horses had also been used in the in vitro experiments. Effects of PDE inhibition and activation of adenylyl cyclase on CD41/61 and CD62P expression on platelets and platelet-neutrophil aggregate formation in vitro were investigated via flow cytometry. Platelet PDE activity and sensitivity to inhibition of PDE3 and PDE5 isoenzymes were examined in horses with RAO and control horses before and after antigen challenge.
Results—Inhibition of PDE or activation of adenylyl cyclase significantly inhibited stimulus-induced expression of CD41/61 and CD62P (by approx 94% and 40%, respectively) and percentage of CD62P positive cells (by approx 30%). Only the PDE3 inhibitor, trequinsin, caused a significant (53%) reduction in platelet-neutrophil aggregate formation. Platelet PDE activity decreased following antigen challenge in RAO-affected horses and control horses. In horses with RAO, a significant increase in sensitivity of platelet PDE to inhibition by the PDE5 inhibitor zaprinast was observed after 5 hours.
Conclusions and Clinical Relevance—Results provided further evidence that PDE3 is an important regulator of equine platelet activation and suggested that changes in regulation of platelet PDE5 may contribute to antigen-induced response in horses with RAO.
Objective—To evaluate effects of a high dose of methylprednisolone sodium succinate (MPSS) on function of polymorphonuclear neutrophilic leukocytes (PMNs) in dogs.
Animals—7 healthy male Beagles (body weight, 10.5 to 15 kg; age, 2 to 4 years).
Procedures—All dogs were treated by IV administration of a high dose of MPSS (30 mg/kg). Additional doses of MPSS (15 mg/kg) were administered IV at 2 and 6 hours and then at 6-hour intervals until 48 hours after the initial dose. Blood samples were collected before and 1, 2, 4, 7, and 14 days after completion of the MPSS administrations and used for evaluation of PMN functions. Isolated PMNs were used for assessment of functions, such as adhesion, migration, phagocytosis, and oxidative burst.
Results—On days 1, 2, and 4 after completion of MPSS administration, there was a decrease in PMN expression of adhesion markers such as CD11b and CD18. There was a decrease in the phagocytotic ability of PMNs on days 1, 2, and 7 after completion of MPSS administration, with a reduction in the oxidative burst of PMNs detected on day 7. No significant changes were identified for migration. All functional changes returned to their pretreatment values by 14 days after completion of MPSS treatment.
Conclusions and Clinical Relevance—Treatment with a high dose of MPSS suppressed PMN functions in dogs. Analysis of these results suggested that treatment with a high dose of MPSS can suppress some of the major functions of PMNs for at least 7 days.
Objective—To determine whether skin-related clinical signs in cutaneous food hypersensitivity (CFH) coincide with immune reactivity in the intestine in dogs.
Animals—11 dogs with CFH without intestinal clinical signs and 8 healthy control dogs.
Procedures—After a provocation and elimination diet, the duodenal gene expression levels of Th1-, Th2- and Treg-related cytokines and transcription factors were investigated by means of quantitative PCR assay. The presence of CD3+, CD8+, CD4+, CD1c+, γδ T-cell receptor+, and major histocompatibility complex II+ cells in duodenal epithelium and lamina propria were determined.
Results—The expression of Th1-, Th2-, and Treg-related genes in dogs with CFH and healthy control dogs was similar. Although clinical signs disappeared, there was no effect of the elimination diet on cytokines, transcription factors, or cellular phenotypes.
Conclusions and Clinical Relevance—No change in T-cell phenotypes or a distinct Th1, Th2, or Treg profile was detected in the duodenum of dogs with only cutaneous clinical signs of food hypersensitivity. This suggested that the intestinal mucosa is not the primary site of T-cell activation that eventually leads to cutaneous food hypersensitivity.