Characterization of the immune response in the placenta of cattle experimentally infected with Neospora caninum in early gestation.

A serial examination of three groups of cattle infected intravenously (iv) (Group 1, n=8) or subcutaneously (sc) (Group 2, n=8) with live Neospora caninum tachyzoites or with VERO cells (Group 3, n=8) at 70 days' gestation was carried out and the nature of the inflammatory responses in the placenta and the presence of parasite antigen were analysed. Immune cells expressing CD3, CD4, CD8, gamma delta (gammadelta) T-cell receptors (TCR), CD79alpha cytoplasmic (cy) (B cells) and NKp46 [natural killer (NK) cells] antigens were identified immunohistochemically and cells expressing mRNA for interferon-gamma (IFN-gamma) were labelled by in-situ hybridization. Intravenous inoculation caused mortality in all fetuses from 28 days post-inoculation (dpi) onwards. Subcutaneous inoculation caused mortality in 50% of the animals by 28dpi. Pathological changes in the placenta consisted of necrosis of fetal placental villi, necrosis and inflammation in adjacent areas of the maternal septum and inflammation at the base of the maternal caruncle. The inflammatory infiltrate consisted mainly of CD3(+) lymphocytes, dominated by CD4(+) and gammadelta TCR(+) cells, with CD8(+) cells present to a lesser extent. The results from the control group indicated fewer NK cells than those occurring in the placenta of human beings or mice. Infiltration of CD4(+) cells and NKp46(+) cells was observed in the caruncular base and septa 14 days after infection, whereas infiltration of gammadelta TCR(+) cells was observed from 28 dpi onwards. To our knowledge this is the first report on the presence and distribution of NK cells in the bovine placenta. Maternal inflammatory cells expressing mRNA for IFN-gamma were identified in animals inoculated with parasites iv or sc at 14 and 28 dpi, respectively. In the sc-inoculated dams with live fetuses at 28, 42 and 56dpi, there was no evidence of parasite antigen, infiltration of immune cells or production of IFN-gamma, suggesting that the parasite had not reached the placenta. The exact cause of fetal death was not established. Tissue destruction by the parasite may have occurred; in addition, there may have been a T helper 1 (Th-1) immune response to the neospora infection at the materno-fetal interface, resulting in infiltrations of CD4T cells, gammadelta T cells and NK cells and the subsequent production of IFN-gamma. It is possible that a pro-inflammatory Th-1 response early in gestation protects the dam by eliminating the parasite; however, it may lead to destruction of the placental tissues themselves and thus be incompatible with fetal survival.

The effects of recombinant ovine interleukin-3 and recombinant ovine stem cell factor on the growth and mediator expression of caprine and ovine bone marrow-derived mast cells.

The growth of ovine and caprine mast cells in bone marrow cultures has been achieved using recombinant ovine interleukin-3 (rOvIL-3) and recombinant ovine stem cell factor (rOvSCF). After approximately 2-3 weeks' growth in optimal concentrations of either rOvIL-3 alone or a combination of rOvIL-3 and rOvSCF, the majority of the cells produced in bone marrow culture from both species were mast cells. The significant increase in the total numbers of cells and survival times of the cultures when both cytokines were present compared to either alone, indicated synergy between rOvIL-3 and rOvSCF on mast cell growth. Ovine and caprine cells cultured in rOvIL-3 alone produced a four-fold increase in cell numbers compared with medium only controls. The resulting cultures contained up to 52% mast cells by day 18 and had a lifespan of 3-4 weeks. In contrast, cells from both species grown in both rOvIL-3 and rOvSCF produced up to six times more cells than the equivalent rOvIL-3 stimulated cultures, contained up to 69% mast cells by day 21 and could be maintained for at least 6 weeks. Ovine cells grown in rOvIL-3 alone or rOvIL-3 and rOvSCF contained significantly more aryl-sulfatase and serine protease but similar amounts of beta-hexosaminidase compared with caprine cells during the second week of culture. There were no significant differences in the granule-associated mediator content of cells from either individual species grown in rOvIL-3 alone compared with those grown in rOvIL-3 and rOvSCF during the first 21 days of culture.

The isolation and purification of a dual specific mast cell-derived protease from parasitised caprine jejunal tissue.

A mast cell granule protease has been isolated and purified from nematode-infected caprine jejunal homogenate by FPLC techniques and termed Goat Mast Cell Protease (GMCP). The purification steps were monitored for proteolytic activity against the synthetic substrate carboxybenzoyl-L-lysine thiobenzyl ester (BLT) and the presence of a homogenous protease preparation in the final sample was shown by SDS-PAGE electrophoresis. This protease was compared with enzymatic activity from isolated mucosal mast cells, which demonstrated the putative mast cell-derived source of the purified enzyme. Rabbit antiserum was raised against the protease and through the use of immunohistochemistry and Western blotting techniques the mast cell origin of the protease was confirmed. NH2-Terminal amino acid sequence analysis demonstrated a high degree of homology between GMCP and other previously isolated mast cell proteases including sheep mast cell protease (SMCP). Substrate analysis showed that GMCP also had an unusual dual chymotrypsin-like and trypsin-like activity similar to SMCP and bovine duodenase.

Local lung responses following local lung challenge with recombinant lungworm antigen in systemically sensitized sheep.

BACKGROUND: Chronic mast cell-mediated inflammation may contribute significantly towards the extensive tissue remodelling that is a feature of lungworm infection in ruminants. Understanding the factors that control tissue remodelling is a necessary step toward effective management and treatment of conditions that feature such pathology. OBJECTIVE: We sought to define in a novel ovine model system, the cellular, immune and mast cell phenotypic events that occur following local lung challenge with a recombinant protein antigen, DvA-1, derived from the ruminant lungworm nematode, Dictyocaulus viviparus. METHODS: Two spatially disparate lung segments in systemically sensitized sheep were challenged on three occasions with DvA-1 (3xDVA) and two further segments were challenged with saline (3xSAL). Two months after the third challenge, one of the two segments previously repeatedly challenged with DvA-1 was challenged again with DvA-1 (3xDVA:DVA) whilst the other was challenged with saline (3xDVA:SAL). A similar protocol was followed with the saline challenged segments (3xSAL:SAL and 3xSAL:DVA). Bronchoalveolar lavage fluid (BALF) (n = 16) and tissue (n = 3) were collected after the last challenge. RESULTS: Cellular changes 24 h after the fourth challenge were characterized by an increase in the absolute numbers of neutrophils and eosinophils in BALF from 3xDVA:DVA and 3xSAL:DVA segments. Local antibody production was implied through increased levels of antibody in both 3xDVA:DVA and 3xDVA:SAL segments, with the latter being unaffected by inflammation. Levels of active transforming growth factor beta-1 (TGF-beta(1)) were significantly increased in 3xDVA:SAL segments and a trend towards an increase was apparent in 3xDVA:DVA segments. Total TGF-beta1 levels were significantly correlated with eosinophil counts in all except the 3xDVA:SAL segments. Such changes in the bronchoalveolar space were complemented by increased ratios of sheep mast cell proteinase-1 expressing cells and tryptase expressing cells, to toluidine blue positive cells in airways from 3xDVA:DVA segments. CONCLUSION: Mast cell phenotypic events occurring as a consequence of antigen challenge were limited to segments in which changes in BALF were characterized by neutrophil influx and increased local antibody production.