Trans-10, cis-12 conjugated linoleic acid modulates phagocytic responses of canine peripheral blood polymorphonuclear neutrophilic leukocytes exposed to Clostridium difficile toxin B

Trans-10, cis-12 conjugated linoleic acid (t10c12-CLA) has been reported to enhance phagocyte function. Clostridium difficile toxin B (TcdB) has been known to inhibit Ras-homologous (Rho) guanosine triphosphatases (GTPases) which play essential roles in neutrophil immune functions. Here, we examined whether in vitro treatment with t10c12-CLA modulates the filamentous actin (F-actin) polymerization, phagocytic capacity, and oxidative burst activity (OBA) of canine peripheral blood polymorphonuclear neutrophilic leukocytes (PMNs) exposed to TcdB. Treatment with t10c12-CLA, but not linoleic acid, enhanced PMN F-actin polymerization, phagocytic capacity, and OBA, while TcdB suppressed these functions. t10c12-CLA reversed the suppressive effects of TcdB on these PMN functions. t10c12-CLA stimulated F-actin polymerization regardless of whether phagocytosis was stimulated by microspheres but only elevated OBA when microspheres were added. We asked whether the effects of t10c12-CLA were associated with changes in the activation of the Rho GTPase Cdc42. Treatment with t10c12-CLA augmented Cdc42 activity in both TcdB-treated and TcdB-naive PMNs during phagocytosis. Thus, t10c12-CLA up-regulates PMN phagocytic responses attenuated by TcdB. This effect is associated with an increase in actin polymerization and may involve the activation of Cdc42.     

Cross-reactivity of commercially available anti-human monoclonal antibodies with canine cytokines: establishment of a reliable panel to detect the functional profile of peripheral blood lymphocytes by intracytoplasmic staining

Background

The process for obtaining monoclonal antibodies against a specific antigen is very laborious, involves sophisticated technologies and it is not available in most research laboratories. Considering that most cytokines remain partially conserved among species during evolution, the search for antibody cross-reactivity is an important strategy for immunological studies in veterinary medicine. In this context, the amino acid sequence from human and canine cytokines have demonstrated 49–96 % homology, suggesting high probability of cross-reactivity amongst monoclonal antibodies. For this, 17 commercially available anti-human monoclonal antibodies [IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8 (#1, #2), IL-10, IL-12, IL-13, IL-17A, IFN-γ (#1, #2), TNF-α (#1, #2) and TGF-β], were evaluated in vitro for intracellular cytokine detection in a stimulated canine blood culture by flow cytometry and confocal microscopy. Lymphocytes from peripheral blood of healthy and two unhealthy dogs were analyzed.

Results

Eleven anti-human mAbs [IL-1α, IL-4, IL-5, IL-6, IL-8 (#1, #2), IL-12, IL-17A, TNF-α (#1, #2) and TGF-β] cross-reacted against canine intracellular cytokines. The specificity of the assays was not affected after Fc-blocking. Three anti-human cytokine mAbs [IL-4, IL-8 (#2) and TGF-β] when evaluated by confocal microscopy also cross-reacted with intracellular canine cytokines. The identification of human mAbs that cross-reacted with canine cytokines may support their use as immunological biomarkers in veterinary medicine studies.

Conclusion

The identification of these 11 anti-human cytokine mAbs that cross-reacted with canine cytokines will be useful immunological biomarkers for pathological conditions by flow cytometry and fluorescence microscopy in dogs.     

Effect of parenteral l-alanyl-l-glutamine administration on phagocytic responses of polymorphonuclear neutrophilic leukocytes in dogs undergoing high-dose methylprednisolone sodium succinate treatment

Objective-To determine whether parenteral l-alanyl-l-glutamine (Ala-Gln) administration modulated phagocytic responses of polymorphonuclear neutrophilic leukocytes (PMNs) from dogs undergoing high-dose methylprednisolone sodium succinate (MPSS) treatment. Animals-15 healthy Beagles. Procedures-Dogs were randomly assigned to 3 treatment groups (n = 5/group): 38-hour IV infusion of saline (0.9% NaCl) solution (control group), saline solution with 8.5% amino acids (2.3 g/kg/d), or saline solution with 8.5% amino acids (1.8 g/kg/d) and 20% l-alanyl-l-glutamine (Ala-Gln; 0.5 g/kg/d). High-dose MPSS treatment was initiated at the same time that IV infusions began, such that a total dose of 85 mg of MPSS/kg was administered through multiple IV injections over a 26-hour period. The infusions were maintained until 12 hours after the last MPSS injection. Blood samples collected before MPSS injections began and 2, 12, and 24 hours after injections ceased were used to evaluate PMN function. Results-MPSS injections resulted in an increase in the total number of circulating leukocytes and increases in neutrophil and monocyte counts but did not affect lymphocyte, eosinophil, or basophil counts. Lymphocyte counts in the Ala-Gln group were higher than in the control group 12 hours after MPSS injections finished. Relative to preinfusion values, phagocytic capacity, oxidative burst activity, and filamentous actin polymerization of PMNs were suppressed in all dogs except those that received Ala-Gln. Conclusions and Clinical Relevance-Parenteral Ala-Gln administration in dogs resulted in an increase in PMN phagocytic responses that were suppressed by high-dose MPSS treatment.     

Ketamine inhibits the phagocytic responses of canine peripheral blood polymorphonuclear cells through the upregulation of prostaglandin E2 in peripheral blood mononuclear cells in vitro

Ketamine has been reported to decrease the immune functions of phagocytes. Previously, we observed that the phagocytic capacity and oxidative burst activity (OBA) of canine peripheral blood polymorphonuclear cells (PMNs) were inhibited by the supernatant from canine peripheral blood mononuclear cells (PBMCs) cultures treated with ketamine. In the present study, we examined whether in vitro treatment with ketamine modulates prostaglandin E₂ (PGE₂) production in PBMCs. Treatment with ketamine or with ketamine-treated PBMCs culture supernatant simultaneously decreased the phagocytic capacity and OBA of PMNs. Ketamine increased PGE₂ production by PBMCs. Recombinant PGE₂ decreased the phagocytic capacity and OBA of PMNs. AH-6809, an E-prostanoid 2 (EP2) antagonist, restored the phagocytic capacity and OBA of PMNs, decreased by either the ketamine-treated PBMCs culture supernatant or recombinant PGE₂. These results suggest that ketamine inhibits the phagocytic responses of canine PMNs, and that this results from the increase in PGE₂ produced by canine PBMCs.     

The immunophenotype of peripheral blood lymphocytes in clinically healthy dogs and dogs with lymphoma in remission

Lymphoma is a common cancer of dogs that frequently is treated with chemotherapy or radiation therapy. Response to therapy is variable and currently available diagnostic tests do not reliably predict response to therapy. Treatment for lymphoma often results in lymphopenia, but it is unknown whether the changes in circulating lymphocytes result from generalized or specific reduction of lymphocytes. In this study, blood lymphocytes from 12 clinically healthy dogs, 10 dogs in remission because of treatment for B-cell lymphoma, and 8 dogs in remission from T-cell lymphoma were analyzed by flow cytometry by using a panel of 20 antibodies reactive with canine leukocyte antigens. Results identified similar lymphocyte parameters in treated dogs regardless of the type of lymphoma. Treated dogs had >50% reduction in blood lymphocyte concentration, and an absolute decrease in most subsets of lymphocytes. Both groups of treated dogs had relative increases in the proportion of CD3+, T-cell receptor (TCR)αβ+, and CD90+ lymphocytes, and a decreased proportion of CD45RA+ cells. In addition, dogs with T-cell lymphoma in remission had a significant increase in the proportion of CD49d+ lymphocytes. These findings were interpreted as representing likely suppression of lymphocyte regeneration by chemotherapy, with a relative increase in the proportion of memory over naïve lymphocytes. Lack of correlation with the T- or B-cell origin of the initial lymphoma suggested that, by using flow cytometric methods, residual circulating neoplastic cells could not be detected. However, the changes in the lymphocyte profile of dogs treated with chemotherapy may have relevance to their immunocompetence.     

Effects of chemotherapy on immune responses in dogs with cancer

Chemotherapy is assumed to be immunosuppressive; yet to the authors' knowledge, the effects of common chemotherapy protocols on adaptive immune responses in dogs with cancer have not been fully evaluated. Therefore, a study was conducted to evaluate the effects of 2 common chemotherapy protocols on T- and B-cell numbers and humoral immune responses to de novo vaccination in dogs with cancer. Twenty-one dogs with cancer (12 with lymphoma, 9 with osteosarcoma) were enrolled in a prospective study to assess effects of doxorubicin versus multi-drug chemotherapy on adaptive immunity. Numbers of circulating T and B cells were assessed by flow cytometry, and antibody responses to de novo vaccination were assessed before, during, and after chemotherapy. The T- and B-cell numbers before treatment also were compared with those of healthy, age-matched, control dogs. Prior to treatment, dogs with cancer had significantly fewer (P < .05) CD4+ T cells and CD8+ T cells than did healthy dogs. Doxorubicin treatment did not cause a significant decrease in T- or B-cell numbers, whereas treatment with combination chemotherapy caused a significant and persistent decrease in B-cell numbers. Antibody titers after vaccination were not significantly different between control and chemotherapy-treated dogs. These findings suggest that chemotherapy may have less impact on T-cell numbers and ability to mount antibody responses in dogs with cancer than was previously anticipated, though dogs with lymphoma or osteosarcoma appear to be relatively T-cell deficient before initiation of chemotherapy.     

In vitro evaluation of the effect of trans-10, cis-12 conjugated linoleic acid on phagocytosis by canine peripheral blood polymorphonuclear neutrophilic leukocytes exposed to methylprednisolone sodium succinate

OBJECTIVE: To examine whether in vitro treatment with trans-10, cis-12 conjugated linoleic acid (t10c12-CLA) restores the phagocytic capacity and oxidative burst activity (OBA) of canine polymorphonuclear neutrophilic leukocytes (PMNs) exposed to methylprednisolone sodium succinate (MPSS). SAMPLE POPULATION: Peripheral blood PMNs obtained from 12 healthy Beagles. PROCEDURES: The experimental design involved administration of a high dose of MPSS, which is the recommended protocol for dogs with acute spinal cord injury. To evaluate PMN function, blood samples were collected from dogs before IV injections of doses of MPSS or saline (0.9% NaCl) solution (time 0) and 2, 12, and 24 hours after injections ceased. Polymorphonuclear neutrophilic leukocytes were isolated from blood samples and incubated with t10c12-CLA alone or t10c12-CLA in combination with N-acetylcysteine (an antioxidant agent). Phagocytic capacity and OBA were measured simultaneously by use of flow cytometry. RESULTS: The phagocytic capacity and OBA of PMNs were suppressed by IV injection of MPSS and restored 12 hours after injection ceased. In vitro treatment with t10c12-CLA enhanced the phagocytic capacity and OBA of PMNs, regardless of whether dogs had been treated with MPSS. Effects of t10c12-CLA on OBA were detected only when phagocytosis was stimulated by microspheres. Use of N-acetylcysteine attenuated the stimulatory effects of t10c12-CLA. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure to t10c12-CLA enhanced the phagocytic capacity and OBA of canine PMNs, and this effect may have involved t10c12-CLA-induced generation of reactive oxygen species.     

Immunoenhancing effect of trans-10, cis-12 conjugated linoleic acid on the phagocytic capacity and oxidative burst activity of canine peripheral blood phagocytes

The effect of trans-10, cis-12 conjugated linoleic acid (t10c12-CLA) on the phagocytic capacity and oxidative burst activity (OBA) of canine peripheral blood phagocytes was examined. t10c12-CLA did not directly affect the phagocytic capacity and OBA of peripheral blood mononuclear cells (PBMC), monocytes or polymorphonuclear cells (PMN). However, the phagocytic capacity of PMN and monocytes was enhanced by the culture supernatant from t10c12-CLA-treated PBMC. This supernatant enhanced the latex bead-induced OBA of PMN and monocytes. t10c12-CLA also increased TNF-alpha production by PBMC. Recombinant canine (rc) TNF-alpha also increased the phagocytic capacity and OBA of PMN and monocytes. The ability of the culture supernatant from t10c12-CLA-treated PBMC to stimulate the phagocytic capacity and OBA of phagocytes was inhibited by anti-rcTNF-alpha pAb. These results suggest that t10c12-CLA has an immunoenhancing effect on the phagocytic capacity and OBA of phagocytes, and this effect may be mediated by TNF-alpha released from t10c12-CLA-treated PBMC.     

Effect of climatic factors on urinary cortisol and peripheral blood leukocytes in lambs grazing on a semi-natural grassland in the Hokuriku District of Japan

The effects of climatic factors on stress and immune functions of grazing lambs in summer and autumn in the Hokuriku District of Japan were evaluated by determining urinary cortisol (U-COR) levels and peripheral blood leukocyte populations and comparing those with lambs kept indoors. Two groups of five lambs, consisting of those grazed on a semi-natural grassland (GRL) and those housed indoors in a domestic animal shelter (INL), were maintained at from July to October. The temperature–humidity index at each location was indicative of heat stress during summer; however, the U-COR elevation was not observed in both groups. The elevation was observed in GRL in autumn and was higher than INL in October. Climatic conditions in autumn were characterized by high humidity and a sudden drop in temperature. U-COR was positively correlated with the relative humidity. The GRL was exposed to low-nutrient conditions for a relatively long time. The CD4⁺/CD8⁺ T cell ratio in GRL decreased in October. Subsequently, the total leucocyte, including granulocytes, monocytes, and lymphocytes, sharply increased. The responses indicated an immune deficiency caused by immunosuppression because of a low nutrition caused by grazing and high-stress conditions in autumn.