Functional paralysis of GM-CSF-derived bone marrow cells productively infected with ectromelia virus.

Ectromelia virus (ECTV) is an orthopoxvirus responsible for mousepox, a lethal disease of certain strains of mice that is similar to smallpox in humans, caused by variola virus (VARV). ECTV, similar to VARV, exhibits a narrow host range and has co-evolved with its natural host. Consequently, ECTV employs sophisticated and host-specific strategies to control the immune cells that are important for induction of antiviral immune response. In the present study we investigated the influence of ECTV infection on immune functions of murine GM-CSF-derived bone marrow cells (GM-BM), comprised of conventional dendritic cells (cDCs) and macrophages. Our results showed for the first time that ECTV is able to replicate productively in GM-BM and severely impaired their innate and adaptive immune functions. Infected GM-BM exhibited dramatic changes in morphology and increased apoptosis during the late stages of infection. Moreover, GM-BM cells were unable to uptake and process antigen, reach full maturity and mount a proinflammatory response. Inhibition of cytokine/chemokine response may result from the alteration of nuclear translocation of NF-κB, IRF3 and IRF7 transcription factors and down-regulation of many genes involved in TLR, RLR, NLR and type I IFN signaling pathways. Consequently, GM-BM show inability to stimulate proliferation of purified allogeneic CD4+ T cells in a primary mixed leukocyte reaction (MLR). Taken together, our data clearly indicate that ECTV induces immunosuppressive mechanisms in GM-BM leading to their functional paralysis, thus compromising their ability to initiate downstream T-cell activation events.

In Vitro Influence of Mycophenolic Acid on Selected Parameters of Stimulated Peripheral Canine Lymphocytes.

Mycophenolic acid (MPA) is an active metabolite of mycophenolate mofetil, a new immunosuppressive drug effective in the treatment of canine autoimmune diseases. The impact of MPA on immunity is ambiguous and its influence on the canine immune system is unknown. The aim of the study was to determine markers of changes in stimulated peripheral canine lymphocytes after treatment with MPA in vitro. Twenty nine healthy dogs were studied. Phenotypic and functional analysis of lymphocytes was performed on peripheral blood mononuclear cells cultured with mitogens and different MPA concentrations- 1 µM (10(-3) mol/m(3)), 10 µM or 100 µM. Apoptotic cells were detected by Annexin V and 7-aminoactinomycin D (7-AAD). The expression of antigens (CD3, CD4, CD8, CD21, CD25, forkhead box P3 [FoxP3] and proliferating cell nuclear antigen [PCNA]) was assessed with monoclonal antibodies. The proliferation indices were analyzed in carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled cells. All analyses were performed using flow cytometry. The influence of MPA on apoptosis was dependent on the mechanism of cell activation and MPA concentration. MPA caused a decrease in the expression of lymphocyte surface antigens, CD3, CD8 and CD25. Its impact on the expression of CD4 and CD21 was negligible. Its negative influence on the expression of FoxP3 was dependent on cell stimulation. MPA inhibited lymphocyte proliferation. In conclusion, MPA inhibited the activity of stimulated canine lymphocytes by blocking lymphocyte activation and proliferation. The influence of MPA on the development of immune tolerance-expansion of Treg cells and lymphocyte apoptosis-was ambiguous and was dependent on the mechanism of cellular activation. The concentration that MPA reaches in the blood may lead to inhibition of the functions of the canine immune system. The applied panel of markers can be used for evaluation of the effects of immunosuppressive compounds in the dog.

Stability of immunophenotypic lymphoid markers in fixed canine peripheral blood for flow cytometric analysis.

BACKGROUND: Flow cytometric analysis of blood samples for immunophenotyping lymphoproliferative diseases has become popular in veterinary medicine. Unfortunately, the use of this technique has been limited by the necessity to test samples within a short time frame after blood collection. A possible solution to this problem is the use of fixative products to preserve the stability of lymphoid antigens. OBJECTIVES: The aim of this study was to evaluate the expression of 5 lymphoid surface markers (CD3, CD4, CD8, CD21, and CD45) in blood samples collected in K3-EDTA and Cyto-Chex BCT tubes from healthy dogs. METHODS: Blood from 8 dogs was collected in K3-EDTA and Cyto-Chex BCT tubes and analyzed by flow cytometry at 6 hours, one day, 3 days, and 7 days after collection. Lymphocyte percentage, lymphocyte mor-phology, expression of lymphoid surface markers and mean fluorescence intensity (MFI) were recorded at each time point and compared. RESULTS: Lymphocyte percentage and morphology were preserved up to 3 days in samples collected in Cyto-Chex BCT, and lymphocyte percentage was mildly decreased on day 7. CD4, CD8, and CD21 were stable in Cyto-Chex BCT up to 7 days, whereas CD3 and CD45 showed a significant decrease in expression from day 3, with a decrease on average of 21% and 2.4%, respectively, on day 7. MFI was significantly decreased on day 7 for all markers except CD21. CONCLUSIONS: These findings indicate that storage of samples in Cyto-Chex BCT affects lymphoid marker expression and caution should be exercised when interpreting data produced on such samples.

Blood phagocyte activity after race training sessions in Thoroughbred and Arabian horses.

Intensive exercise and exertion during competition promote many changes that may result in the impairment of immunity and increased susceptibility to infections. The aim of this study was to evaluate the activity of "the first line of defense": neutrophils and monocytes in racing Thoroughbred and Arabian horses after routine training sessions. Twenty-three (12 Thoroughbred and 11 Arabian) horses were examined. Routine haematological (number of red blood cells - RBC, haemoglobin concentration - HGB, haematocrit - HCT, total number of white blood cells - WBC), biochemical (creatine phosphokinase activity - CPK and total protein concentration - TP) parameters, cortisol concentration as well as phagocytic and oxidative burst activity of neutrophils and monocytes were determined. The values of basic parameters and the activity of phagocytes differed between breeds and distinct patterns of exercise-induced changes were observed. The training sessions did not produce the decrease in phagocyte activity that might lead to the suppression of immunity.