Detection of differentially regulated genes in ischaemic equine intestinal mucosa.

REASONS FOR PERFORMING STUDY: Colic is a serious disease syndrome in horses. Much of the mortality is associated with ischaemic-injured intestine during strangulating obstruction, yet there is limited understanding of the associated molecular events. Identification of differentially expressed genes during ischaemic injury should expand our understanding of colic and may lead to novel targeted therapeutic approaches in the future. OBJECTIVE: To isolate and identify differentially expressed genes in equine jejunum following a 2 h ischaemic event compared to normally perfused jejunum. METHODS: Suppressive subtractive hybridisation was used to clone genes that are differentially expressed in equine jejunum injured by 2 h of complete ischaemia as compared to time-matched control jejunal tissues. Expression of selected clones was further evaluated by northern blot analysis. RESULTS: Of the 384 clones selected, 157 were confirmed to possess cDNAs corresponding differentially expressed genes by dot blot analysis. Two genes, fatty acid binding protein 2 and calcium-activated chloride channel 4 were further confirmed to be differentially expressed by northern blot analysis. CONCLUSIONS: Suppressive subtractive hybridisation can be used to detect changes in expression of a broad array of genes, as confirmed by northern blot analysis of selected genes. POTENTIAL RELEVANCE: These initial results have identified a pool of equine intestinal epithelial genes that are differentially expressed following a 2 h ischaemic event. In particular, genes indicative of deranged metabolic activity and those potentially involved in early repair events were identified and may ultimately provide clues as to the nature of epithelial ischaemic injury in horses.

Progression from acute to chronic disease in a murine parent-into-F1 model of graft-versus-host disease.

The parent-into-immunocompetent-F(1) model of graft-vs-host disease (GVHD) induces immune dysregulation, resulting in acute or chronic GVHD. The disease outcome is thought to be determined by the number of parental anti-F(1) CTL precursor cells present in the inoculum. Injection of C57BL/6 (B6) splenocytes into (B6 x DBA/2)F(1) (B6D2F(1)) mice (acute model) leads to extensive parental cell engraftment and early death, whereas injection of DBA/2 cells (chronic model) results in little parental cell engraftment and a lupus-like disease. This study demonstrated that injection of BALB/c splenocytes into (BALB/c x B6)F(1) (CB6F(1)) mice resulted in little engraftment of parental lymphocytes and the development of lupus as expected. Injection of B6 splenocytes into CB6F(1) initiated an initial burst of parental cell engraftment similar to that of B6 into B6D2F(1). However, the acute disease resolved, and the CB6F(1) mice went on to develop chronic GVHD with detectable Abs to ssDNA, dsDNA, and extractable nuclear Ags. Limiting dilution CTL assays determined that B6 splenocytes have CTL precursor frequencies of 1/1000 against both CB6F(1) and B6D2F(1), whereas DBA/2 and BALB/c splenocytes have a CTL precursor frequency of 1/20,000 for their respective F(1)s. The Th cell precursor frequency for B6 anti-DBA/2 was 3-fold higher than that for B6 anti-BALB/c determined by limiting dilution proliferation assays. These results indicate the importance of adequate allospecific helper as well as effector T cells for the induction and maintenance of acute GVHD in this model, and presents an unexpected model in which initial acute GVHD is replaced by the chronic form of disease.

CD8 dimer usage on alpha beta and gama delta T lymphocytes from equine lymphoid tissues.

Eight murine monoclonal antibodies (mAb) were used to identify the equine CD8 alpha or CD8 beta chains and to define the expression of these chains on lymphocytes from various lymphoid tissues. CD8 alpha was a 39 kDa protein and CD8 beta was a 32 kDa protein. Both chains were expressed on most of the CD8+ T lymphocytes in the peripheral blood, spleen, thymus, mesenteric lymph nodes and ileal intraepithelial lymphocytes (IEL), however, in each lymphoid compartment a percentage of lymphocytes expressed only the CD8 alpha chain. The largest percentage of CD8 alpha alpha expressing T lymphocytes was 37.7% of the IELs. Purified T lymphocytes from the ileum expressing CD8 alpha beta co-expressed the alpha beta T cell receptor (TCR). In contrast, purified CD8+ T lymphocytes from the PBMC co-expressed either the alpha beta or gamma delta TCR by RT-PCR. Use of pooled anti-CD8 alpha mAb of the murine IgG2a isotype and rabbit complement resulted in lysis of the entire CD8 expressing population in peripheral blood mononuclear cells (PBMC). These results indicated that CD8 dimer usage by equine T lymphocytes is similar to other species and that the mAb described can be further used to separate equine CD8+ T lymphocyte subsets from the lymphoid tissues to define their function in protection against viral and other infections.

Control of equine infectious anemia virus is not dependent on ADCC mediating antibodies.

Horses infected with equine infectious anemia virus (EIAV) have recurrent episodes of viremia which are eventually controlled, but the immune mechanisms have not been identified. Antibodies were detected to the surface of EIAV-infected cells within 1 month postinfection and remained for at least 3.5 years postinfection. These antibodies recognized cell surface-exposed envelope (Env) glycoproteins, but could not mediate antibody dependent cellular cytotoxicity (ADCC) using EIAV-WSU5-infected equine kidney (EK) cells as targets and peripheral blood mononuclear cells (PBMC) or polymorphonuclear cells (PMN) as effector cells. Furthermore, purified IgG antibodies from horses infected with either EIAV-WSU5 or EIAV-Wyo did not mediate ADCC of infected target cells. Armed effector cells could not be detected in infected horse blood nor could effector cells be prearmed by incubation with serum antibodies to cell surface antigens. The use of EIAV-WSU5-infected equine macrophages as target cells did not result in ADCC. In contrast, serum antibody from EHV-1 vaccinated horses and PBMC or PMN as effector cells caused ADCC of EHV-1-infected EK cells. These results indicate that ADCC is not involved in the control of EIAV in carrier horses.