Steven H. Wright

DETECTION OF T. GONDII IN TISSUES OF SHEEP AND CATTLE FOLLOWING ORAL INFECTION

It has been reported in the literature that cattle are more resistant to toxoplasmosis than sheep. Congenital disease due to T. gondii infection is rarely reported in cattle whereas the parasite is a major cause of abortion and neonatal mortality in sheep. It is believed that sheep remain chronically infected for life. Undercooked meat from infected sheep is an important source of infection for man. In contrast cattle are thought to harbour fewer parasite tissue cysts which may not persist for the lifetime of the host. Therefore, cattle are believed to pose less of a risk for human infection. In this study we examined the presence of T. gondii within a range of tissues in sheep and cattle at 6 weeks and 6 months following oral infection with 10(3) or 10(5) sporulated oocysts of T. gondii. The presence of parasite was determined by bioassay in mice and using polymerase chain reaction (PCR). The results from this study show that T. gondii was more frequently and consistently detected in sheep, in particular within brain and heart tissues, whereas parasites were not detected in the samples of tissues taken from cattle. T. gondii was more frequently detected in sheep given the higher dose of T. gondii. Examination of tissues at either 6 weeks or 6 months after infection did not appear to affect the distribution of T. gondii. The polymerase chain reaction has more specificity and sensitivity when detecting the presence of T. gondii in large animals than histological detection.

Transforming growth factor‐bβ1 mediates coexpression of the integrin subunit aαE and the chymase mouse mast cell protease‐1 during the early differentiation of bone marrow‐derived mucosal mast cell homologues

Mucosal mast cells (MMC) play a central role in gut hypersensitivities and inflammation. They are morphologically, biochemically and functionally distinct from their connective tissue counterparts. Massive hyperplasia of MMC occurs 7–10 days after intestinal infection with nematodes but it has never been possible to replicate this phenomenon in vitro.

Expression Profiling Reveals Novel Innate and Inflammatory Responses in the Jejunal Epithelial Compartment during Infection with Trichinella spiralis

ABSTRACT Infection with intestinal nematodes induces profound pathological changes to the gut that are associated with eventual parasite expulsion. We have applied expression profiling as an initial screening process with oligonucleotide microarrays (Affymetrix MG-U74AV2 gene chips) and time course kinetics to investigate gene transcription triggered by the intraepithelial nematode Trichinella spiralis in jejunal epithelium from BALB/c mice. Of the 4,114 genes detected, 2,617 were present in all uninfected and T. spiralis-infected replicates, 8% of which were notably upregulated, whereas 12% were downregulated at the time of worm expulsion (day 14 postinfection). Upregulation of goblet cell mucin gene transcripts intestinal mucin gene 3 (MUC3), calcium chloride channel 5 (CLCA5), and goblet cell gene 4 (GOB4) is consistent with enhanced production and alteration of mucus, whereas a 60- to 70-fold upregulation of transcripts for mast cell proteases 1 and 2 (MCPT-1 and -2) is consistent with intraepithelial mucosal mast cell recruitment. Importantly, there was novel expression of sialyltransferase 4C (SIAT4C), small proline-rich protein 2A (SPRR2A), and resistin-like molecule β (RELMβ) on day 14 postinfection. In contrast, DNase I and regenerating protein 3 (REG3) transcripts were substantially downregulated. Time course analyses revealed early (within 48 h of infection) induction of Siat4c, Sprr2A, and Relmβ and later (within 120 h) induction of Mcpt-1 and -2. The findings demonstrate early innate responses and later inflammatory changes within the epithelium. The early epithelial responses may be associated both with repair (Sprr2A) and with the development of innate immunity (Siat4c and Relmβ).

Enteric Expression of the Integrin αvβ6 Is Essential for Nematode-Induced Mucosal Mast Cell Hyperplasia and Expression of the Granule Chymase, Mouse Mast Cell Protease-1

The immunoregulatory cytokine transforming growth factor (TGF)-β1 is secreted as a biologically inactive complex with latency-associated peptide, which must be modified by local factors to expose the functionally active cytokine. The epithelial integrin αvβ6 mediates local activation of TGF-β1 in the lung and β6−/− mice exhibit exaggerated pulmonary inflammation, but their response to inflammatory stimuli in the gut has not been investigated. We found that both β6 and TGF-β1 are constitutively expressed in the jejunal epithelial compartment in uninfected mice and during infection with the intestinal nematode Nippostrongylus brasiliensis. We also present data showing that β6−/− mice are seriously compromised in their ability to mount a mucosal mast cell response after infection, and there is a significant reduction in the expression and systemic release of the granule chymase, mouse mast cell protease-1. Because in vitro expression of this chymase is regulated by TGF-β1, these data indicate that in the absence of αvβ6 epithelially expressed TGF-β1 may not be activated, with a consequent absence of expression of mouse mast cell protease-1 and down-regulation of the mucosal mast cell response.

Purification and characterization of mouse mast cell proteinase-2 and the differential expression and release of mouse mast cell proteinase-1 and -2 in vivo.

Background Gastrointestinal nematode infection is associated with mucosal mast cell (MMC) hyperplasia. In the mouse, this is accompanied by the release of substantial quantities of the chymase mouse mast cell proteinase‐1 (mMCP‐1) into the gut lumen and peripheral bloodstream. Expression of mMCP‐1 is largely restricted to intraepithelial MMC and is thought to play a role in the regulation of epithelial permeability. MMCs also express mouse mast cell proteinase‐2 (mMCP‐2), but less is known about the expression or biological function of this proteinase.

Western blot analysis of the IgG responses of ruminants infected with Neospora caninum and with Toxoplasma gondii

The IgG antibody responses of sheep, goats and cattle inoculated subcutaneously with live Neospora caninum tachyzoites of the NC1 isolate were analysed by Western blotting. Antibodies were detected against a wide range of NC1 tachyzoite antigens (6.5 to 80 kDa). The dominant antibody responses were directed against proteins at 36.5-38, 45.5-48.5, 52-53.5, 58, 58.5, 59.5, 60.5, 62, 63.5, 64, 66.5, 67, 67.5, 68.5 and 69.5 kDa, with sera from all three species. These sera were also used to probe blots of Toxoplasma gondii antigen and, while a number of protein bands were recognized, there was no consistency within or between animal species. The IgG antibody responses of sheep, goats and cattle orally infected with T. gondii oocysts of the M3 isolate were analysed by the same methods. Antibodies were detected to a range of S48 toxoplasma tachyzoite antigens (11 to 83 kDa). The dominant antibody responses were directed against proteins at 11, 16-17, 21.5, 22.5-23.5, 26-28.5, 32-35, 49.5, 50.5, 53, 54.5, 60.5 and 61 kDa, with sera from all three species. These sera were also used to probe blots of N. caninum antigen; antibody responses to numerous antigens were detected but showed little consistency within or between animal species.

Diagnosis of Taenia saginata cysticercosis in Kenyan cattle by antibody and antigen ELISA

Sera from calves, either experimentally or naturally infected with Taenia saginata, were screened for an antibody response to T. saginata, and for parasite antigen, by enzyme-linked immunosorbent assays (ELISAs). An antibody response was detected by 3 weeks post infection (p.i.), rose to a peak at 10-12 weeks p.i., and was still in evidence 1 year p.i. Parasite antigen was first detected 4-7 weeks p.i. and persisted until the end of the experiment, over 1 year p.i. In the experimentally infected animals, cattle with 14 or more live cysticerci had detectable levels of parasite antigen in their sera at slaughter, while animals with live cyst burdens ranging from 0 to 4 were negative. Furthermore, levels of circulating antigen were positively correlated with live cysticercus burden in the experimental animals. In naturally infected cattle, 83% (5/6) of those with 30 or more live cysts, and 22% (5/23) of those with 1-29 live cysts, could be detected by the ELISA for parasite antigen, although no significant correlation between antigen level and live cyst burden could be detected. Antibody levels were not found to be associated with cyst burdens in either experimentally or naturally infected cattle. In slaughterhouse cattle, the antigen assay was almost three times as sensitive as meat inspection. However, there was no agreement between cattle found positive at meat inspection and those found positive by the antigen detection ELISA. One possible reason is that the ELISA only detects live cysts, while lesions left by dead cysts are more noticeable at meat inspection. The mouse monoclonal antibody-based antigen detection ELISA is of value for the diagnosis of naturally occurring, viable, T. saginata cysticercosis in live cattle and has an immediate application for field based epidemiological studies designed to determine prevalence.

Constitutive secretion of the granule chymase mouse mast cell protease‐1 and the chemokine, CCL2, by mucosal mast cell homologues

Background The mucosal mast cell (MMC) granule‐specific β‐chymase, mouse mast cell protease‐1 (mMCP‐1), is released systemically into the bloodstream early in nematode infection before parasite‐specific IgE responses develop and TGF‐β1 induces constitutive release of mMCP‐1 by homologues of MMC in vitro. Intraepithelial MMC may also express the chemokine CCL2 (monocyte chemotactic protein‐1) during nematode infection but the expression of this chemokine by MMC homologues has not been investigated.

Expression of Integrin-αE by Mucosal Mast Cells in the Intestinal Epithelium and Its Absence in Nematode-Infected Mice Lacking the Transforming Growth Factor-β1-Activating Integrin αvβ6

Peak intestinal mucosal mast cell (MMC) recruitment coincides with expulsion of Trichinella spiralis, at a time when the majority of the MMCs are located within the epithelium in BALB/c mice. Although expression of integrin-αEβ7 by MMCs has not been formally demonstrated, it has been proposed as a potential mechanism to account for the predominantly intraepithelial location of MMCs during nematode infection. Co-expression of integrin-αEβ7 and the MMC chymase mouse mast cell protease-1, by mouse bone marrow-derived mast cells, is strictly regulated by transforming growth factor (TGF)-β1. However, TGF-β1 is secreted as part of a latent complex in vivo and subsequent extracellular modification is required to render it biologically active. We now show, for the first time, that intraepithelial MMCs express integrin-αEβ7 in Trichinella-infected BALB/c and S129 mice. In S129 mice that lack the gene for the integrin-β6 subunit and, as consequence, do not express the epithelial integrin-αvβ6, integrin-αE expression is virtually abolished and recruitment of MMCs into the intestinal epithelium is dramatically reduced despite significant overall augmentation of the MMC population. Because a major function of integrin-αvβ6 is to activate latent TGF-β1, these findings strongly support a role for TGF-β1 in both the recruitment and differentiation of murine MMCs during nematode infection.

TGF-β1 Regulates Adhesion of Mucosal Mast Cell Homologues to Laminin-1 Through Expression of Integrin α7

Mucosal mast cells (MMC) or their precursors migrate through the intestinal lamina propria to reside intraepithelially, where expression of mouse mast cell protease-1 indicates the mature phenotype. Alterations in expression of integrins that govern cell adhesion to the extracellular matrix may regulate this process. As the key cytokine mediating differentiation of mouse mast cell protease-1-expressing MMC homologues in vitro, TGF-β1 was considered a likely candidate for regulation of the integrins that facilitate intraepithelial migration of MMC. Therefore, we examined adhesion of bone marrow-derived mast cells cultured with and without TGF-β1 to laminin-1, fibronectin, and vitronectin along with expression of integrins likely to regulate this adhesion. Adhesion of PMA-stimulated cultured mast cells to laminin-1 increased from 5.3 ± 3.6% (mean ± SEM) in the absence of TGF-β1 to 58.7 ± 4.0% (p < 0.05) when cultured mast cells had differentiated into MMC homologues in the presence of TGF-β1. Increased adhesion of MMC homologues to laminin-1 was also stimulated by FcεRI cross-linking and the calcium ionophore A23187. Expression of the laminin-binding integrin α7 by MMC homologues grown in the presence of TGF-β1 was demonstrated by RT-PCR and flow cytometry, and preincubation of MMC homologues with the α7-neutralizing Ab 6A11 inhibited adhesion to laminin-1 by 98% (p < 0.05), demonstrating a novel role for this molecule in adhesion of a hemopoietic cell to laminin-1.