Evaluating the cytokine profile of the WC1+ γδ T cell subset in the ileum of cattle with the subclinical and clinical forms of MAP infection.

In the present study, we evaluated expression of IFN-γ, IL-17, TNF-α, IL-10 and TGF-ß by mucosal cells, including WC1+ γδ T cells, in ileal tissues taken from non-infected cattle and cattle naturally infected with Mycobacterium avium subsp paratuberculosis (MAP). Infected cattle were either in the subclinical or clinical stage of infection. We hypothesized that the cytokine profile of the WC1+ γδ T cell subset would be different between subclinical and clinical cattle. Our data indicate a significant increase in the numbers of WC1+ γδ T cells expressing IL-10 in clinical cattle compared to subclinical and non-infected cattle. We observed a significant increase in TGF-ß expression by non-WC1+ cells in clinically infected cattle. Expression of IFN-γ, IL-17 and TNF-α in mucosal cells, including the WC1+ γδ T cell subset, was identified in all examined groups. However, our data indicate that the stage of infection did not significantly influence expression of these proinflammatory cytokines. This study demonstrates changes in the cytokine mRNA expression profile of mucosal cells in the ileum, and specifically WC1+ γδ T cells, as cattle progress to the clinical disease. The change is characterized by an increase in expression of anti-inflammatory cytokines.

WC1+ γδ T cells from cattle naturally infected with Mycobacterium avium subsp. paratuberculosis respond differentially to stimulation with PPD-J.

A role for γδ T cells in protection against mycobacterial infections including Johne's disease (JD) has been suggested. In neonatal calves where the risk to infection with Mycobacterium avium subsp. paratuberculosis (MAP) is high, the majority of circulating CD3+ lymphocytes are γδ TCR+. Bovine γδ T cells are divided into two major subsets based on the surface expression of workshop cluster 1 (WC1). The WC1+ subset, the predominant subset in periphery, is further divided into WC1.1+ and WC1.2+ subpopulations. The ability of γδ T cells to produce IFN-γ prior to CD4+ αß T cell activation could be crucial to the outcome of MAP infection. In the current study, cattle were naturally infected with MAP and were classified as either in the subclinical or clinical stage of infection. Compared to the control non-infected group, γδ T cell frequency in circulating lymphocytes was significantly lower in the clinical group. The observed decline in frequency was restricted to the WC1.2+ subset, and was not associated with preferential migration to infection sites (distal-ileum). γδ T cells proliferated significantly in recall responses to stimulation with purified protein derivative from MAP (PPD-J) only in subclinically infected cattle. These responses were a heterogeneous mixture of WC1.1 and WC1.2 subsets. Proliferation and IFN-γ production by the WC1.1+ γδ T cell subset was significantly higher in the subclinical group compared to the control and clinical groups. Our data indicates differences in MAP-specific ex-vivo responses of peripheral WC1+ γδ T cells of cattle with the subclinical or clinical form of JD.

Gamma-delta T-cell responses during subcutaneous Mycobacterium avium subspecies paratuberculosis challenge in sensitized or naive calves using matrix biopolymers.

We have developed a model to explore the early immune response against Mycobacterium avium subspecies paratuberculosis (Map) infection in the bovine calf using subcutaneously placed liquid gel matrix biopolymer (matrigel) containing live Map. Matrigel rapidly polymerizes in vivo, retains recruited cellular infiltrates and soluble immune mediators, and can be rapidly removed 48 hours later and depolymerized for analysis. In this study, we examined early host immune events at matrigel/Map sites; recruited cells were evaluated by histopathology and flow cytometry, and cytokines were measured by flow cytometry, enzyme-linked immunosorbent assay, and Luminex bead immunoassay. Our results demonstrate earlier recruitment of gamma-delta (γδ) T cells to matrigel/Map challenge sites compared to CD4+ T cells. We also show that significantly more γδ T cells were recruited to matrigel/Map sites postinfection day 7 compared to postinfection day 30 and that these cells produced significant amounts of the cytokine interferon gamma. We also provide evidence that peripheral blood-derived γδ T-cell subsets in cattle differentially generate interferon gamma, suggesting distinct roles for these cells. These data provide unique insight into initial antimycobacterial host cellular immune responses following Map infection in calves.

Direct inoculation of Mycobacterium avium subspecies Paratuberculosis into ileocecal Peyer's patches results in colonization of the intestine in a calf model.

The objective of this study was to develop an intestinal model of Mycobacterium avium subspecies paratuberculosis (Map) infection in the calf for evaluation of mucosal pathology and local and systemic immunologic responses. Map was inoculated into Peyer's patches of young calves using a right flank surgical approach in standing calves to exteriorize the ileocecal junction. Inoculum doses ranging from 10(3) to 10(9) colony-forming units of strain K10 Map were injected through the serosal surface into Peyer's patches of the distal ileum near the ileocecal valve. Fecal samples were collected for culture from each calf weekly until termination of the study. Calves were necropsied at 7, 30, 60, and 90 days after infection, when inoculation sites, lymph nodes, spleen, and peripheral blood were collected for evaluation. Ileocecal lymph nodes were consistently colonized by Map in the 10(5) to 10(9) groups. The ileocecal valve was also colonized in 10(7) and 10(9) groups. This correlated with fecal culture results as infected calves intermittently shed Map in their feces throughout the study. Granulomatous lesions with giant cells and acid-fast bacilli at the ileocecal junction, ileocecal lymph nodes, and lamina propria of high-dose animals (10(7) and 10(9)) were identified from each time point. Flow cytometry was used to detect antigen-specific production of interferon-γ and interleukin-4 locally (ileocecal lymph node) and systemically (peripheral blood mononuclear cells), which defined distinct immunologic profiles in low-dose and high-dose calves. This study demonstrates intestinal Map infection via Peyer's patch inoculation, a novel model with many shared features of natural Map infection.

Regulation of surfactant protein and defensin mRNA expression in cultured ovine type II pneumocytes by all-trans retinoic acid and VEGF.

Beta-defensins and surfactant proteins are components of the pulmonary innate immune system. Their gene expression is regulated by development, hormones, growth and immunoregulatory factors. It was our hypothesis that growth and differentiation factors such as all-trans retinoic acid (RA) and vascular endothelial growth factor (VEGF) may affect expression of selected innate immune genes by respiratory epithelial cells. Ovine JS7 cells (alveolar type II pneumocytes) were incubated in serum-free Dulbecco's modified Eagle's medium (DMEM) complete media that contained: no treatment (negative control), RA (500 nM), or VEGF (100 ng/ml) for 6, 12 or 24 h incubation. Total RNA was isolated, cDNA synthesized, and relative mRNA levels of surfactant protein A (SP-A) and SP-D, and sheep beta-defensin-1 (SBD-1) were determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Cells had significantly increased expression of SP-D mRNA at 6 h and 24 h, decreased expression of SP-A mRNA at 12 h, and unchanged levels of SBD-1 mRNA after the treatment with RA compared with their respective negative controls. VEGF did not alter the expression of the three innate immune genes. These findings suggest that SP-A and SP-D have different transcription regulation pathways, and that expression of SBD-1 is not inducible by RA similar to its human homolog HBD-1. The lack of changes induced by VEGF treatment suggests that VEGF does not have a direct effect on epithelial cells, but may affect gene expression indirectly.

Experiences with conversion of jejunoileal bypass to gastric bypass: its use for maintenance of weight loss.

Jejunoileal bypass for morbid obesity has been associated with a variety of long-term adverse sequelae, including excess diarrhea, electrolyte imbalance, kidney dysfunction, liver abnormalities, and polyarthropathy. Takedown of the bypass without providing a means of maintenance of weight reduction will lead to recurrence of morbid obesity. We studied 32 patients who underwent conversion of jejunoileal bypass to gastric bypass either in one procedure (27 patients) or more than one procedure (five patients). There were no operative deaths, but significant morbidity. Nevertheless, the conversion has maintained weight reduction and reversed the adverse effects of the small-bowel bypass. We concluded that conversion of the jejunoileal bypass to the gastric bypass, particularly with a Roux-en-Y anastomosis, in a single procedure is relatively safe, simple, and effective.