Wilma Shuman

Characterization of peripheral blood and pulmonary leukocyte function in healthy foals

Studies in infants and foals indicate an age-dependent maturation of peripheral lymphocyte subsets. The age-dependent relationship for maturation of cellular immune responses, such as phagocytosis and lymphocyte responses of the peripheral and pulmonary-derived leukocytes, has not been characterized in foals. Lymphocyte subpopulations, mitogen stimulation response of lymphocytes, lymphokine-activated killing cell activity, phagocytosis and oxidative burst activity, and serum immunoglobulin (Ig) classes G and M concentrations were determined in developing foals. This study illustrates age-dependent changes in immunoglobulin class concentrations, lymphocyte subsets, and EqMHC Class II expression in cells of the peripheral blood and lungs of developing neonatal-to-weanling foals. The increase in peripheral blood and BAL B-lymphocytes and serum immunoglobulins in developing foals suggests expansion of immune cell populations during a time in which environmental pathogen exposure is great. General immune function, mitogenic responses, LAK cell activity, opsonized phagocytosis, and oxidative burst activity of newborns was similar to the adult horse. Total immune-cell numbers, rather than function, seemed to be the limiting factor in the development of the equine neonatal immune system. There was an age-related percent increase in the appearance of pulmonary lymphocytes, but a percent decrease in macrophages. Although development of the respiratory immune system follows changes in the peripheral blood, cellular expansion, activation, and migration may occur at a slower pace, making the respiratory environment susceptible to pathogens prior to optimal immune system maturity.

Fusobacterium necrophorum Leukotoxin Induces Activation and Apoptosis of Bovine Leukocytes

ABSTRACT Fusobacterium necrophorum, a gram-negative, rod-shaped, anaerobic bacterium, is a primary or secondary etiological agent in a variety of necrotic, purulent infections in humans and animals. Its major virulence factor is leukotoxin, a high-molecular-weight secreted protein, primarily toxic to ruminant leukocytes. In this study, bovine peripheral blood leukocytes were exposed to various concentrations of immunoaffinity-purified leukotoxin and the cytotoxicity was analyzed by flow cytometry and scanning and transmission electron microscopy. At very low toxin concentrations, polymorphonuclear leukocytes (PMNs) showed activation, as indicated by translocation of primary and secondary granules to the periphery of the cytoplasm. Furthermore, these cells showed changes characteristic of apoptosis, including decreased cell size, organelle condensation, cytoplasmic membrane blebbing (zeiosis), and chromatin condensation and margination, and decrease in cellular DNA content. At moderately high concentrations of leukotoxin, bovine mononuclear cells were also induced to undergo programmed cell death. At very high concentrations, leukotoxin caused necrotic cell death of bovine peripheral leukocytes. The ability of F. necrophorum leukotoxin to modulate the host immune system by its toxicity, including cellular activation of PMNs and apoptosis-mediated killing of phagocytes and immune effector cells, represents a potentially important mechanism of its pathogenesis.

Immunologic function in horses after non-specific immunostimulant administration

Inactivated Propionibacterium acnes is a biologic response modifier for treatment of non-specific respiratory disease in horses. The objectives of this investigation were to determine alterations in phagocytic activity, phenotypic expression of lymphocyte subpopulations and lymphokine-activated killing cell response in healthy young horses. Samples were collected on day 0, 7 and 14 of the investigation. Blood samples were obtained via jugular venipuncture and pulmonary leukocytes were recovered via bronchoalveolar lavage (BAL). Commercially available P. acnes (Eqstim) was administered intravenously on days 7, 9 and 11 of the investigation. Fever was observed on days 8 and 10, indicating immune reaction. Total peripheral blood white cell count was increased (P < 0.05) on day 14 after P. acnes administration compared to values on days 0 and 7. Total BAL fluid cell count decreased (P < 0.01) on day 14 compared to values on days 0 and 7, which was characterized by a decrease in total lymphocyte (P < 0.01) and macrophage (P < 0.01) counts. The proportion of lymphocytes in BAL fluid decreased (P < 0.005) on day 14 compared to values on days 0 and 7, and the proportion of macrophages increased (P < 0.005) on day 14 compared to values on days 0 and 7. P. acnes administration increased the total (P < 0.05) and proportional (P < 0.05) counts of CD4+ T lymphocytes in peripheral blood. Bronchoalveolar lavage fluid proportion of CD4+ (P < 0.05), CD5+ (P < 0.001) and MHC II (P < 0.05) lymphocytes increased on day 14 after P. acnes administration compared to values on days 0 and 7. Nonopsonized phagocytic activity in peripheral blood increased (P < 0.0005) on day 14 after P. acnes administration compared to values on days 0 and 7. Lymphokine-activated killing cell activity in peripheral blood and BAL fluid leukocytes was enhanced (P < 0.005) on day 14 after P. acnes administration compared to values on days 0 and 7. Serum IgG and IgM concentrations were within laboratory reference values and were not altered by administration of P. acnes. This investigation demonstrated immunostimulant and immunomodulatory properties of P. acnes, characterized by increased CD4+ T lymphocyte expression and LAK activity in peripheral blood and BAL fluid, increased nonopsonized phagocytosis in peripheral blood leukocytes and decreased pulmonary cellularity.

Simultaneous flow cytometric analysis of phagocytosis and oxidative burst activity in equine leukocytes.

This paper describes a method for simultaneously measuring phagocytosis and oxidative burst activity in equine peripheral blood leukocytes by flow cytometry. Opsonized propidium iodide-labelled Staphylococcus aureus (PI-Sa) was used to measure the uptake of bacteria by equine phacocytes and the oxidative burst activity by oxidation of dihydrorhodamine 123. The requirements to achieve optimal activity of phagocytosis and oxidative burst are described. The advantage of the simultaneous technique is that it provides both independent and comparative values for phagocytosis and the oxidative burst, for the detection of impaired mechanisms of microbial destruction. Furthermore, the technique allows evaluation of opsonization activity in this context.

Detection of antiplatelet immunoglobulin in thrombocytopenic dogs

Indirect enzyme-linked immunosorbent assays (ELISA) were used to detect platelet-associated immunoglobulin in sera from dogs with immune-mediated thrombocytopenia (IMT) and/or other autoimmune syndromes. One ELISA, utilizing whole platelets as the antigen substrate, readily detected antibody associated with platelets, either as specific, antiplatelet antibody or as immune complexes. This assay apparently lacked specificity because of the position reactions with sera from dogs with miscellaneous autoimmune disorders and no concurrent thrombocytopenia. Although the second ELISA, utilizing immunoaffinity purified platelet antigens was not influenced as much by immune complexes, absorbance values apparently were slightly increased. However, a small number of dogs with non-thrombocytopenic autoimmune disease tested positive. Immunoadsorption and Western immuno-blotting techniques demonstrated a complex pattern of specificities for antiplatelet antibodies. Clinical significance of these findings is discussed.