Jolynne R. Tschetter

Summary: 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.

The cytochrome P450 CYP1B1 enzyme metabolically activates polycyclic aromatic hydrocarbons and is a major P450 isoenzyme in human monocytes and macrophages. We have shown previously that mice deficient in CYP1B1 were resistant to induced tumors after 7,12-dimethylbenz[a]anthracene exposure. The pathology of aging CYP1B1 null mice on a B6; 129 background was studied in groups of 29 males and 30 females. By 12 months, 50% of the female mice had developed a unusual progressive glomerulonephritis while males had similar renal lesions later in life. This disease followed a sequence of proliferative, membranoproliferative and sclerotic glomerulonephritis. Anti-DNA antibodies were found in the blood of the mice along with immune deposits containing immunoglobulins in subepithelial locations of the glomerular basement membrane. The lesions were unlike those found in aging wild-type B6;129 mice or mice of other strains. We found that macrophages from CYP1B1-null mice were impaired in the phagocytosis of apoptotic, necrotic, and opsonized cells. This suggests a generalized defect in the phagocytic activity of CYP1B1-null mouse macrophages. Male mice also developed a high incidence (62—64%) of histiocytic sarcomas. Our study provides evidence that deficiency of CYP1B1 can play a role in the development of glomerular disease, normal processing of catabolic DNA and tumors of the mononuclear phagocyte system. The function of CYP1B1 in histiocytes and macrophages may involve both self-tolerance and tumor suppression.

The parent-into-immunocompetent-F₁ 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-F₁ CTL precursor cells present in the inoculum. Injection of C57BL/6 (B6) splenocytes into (B6 × DBA/2)F₁ (B6D2F₁) 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 × B6)F₁ (CB6F₁) mice resulted in little engraftment of parental lymphocytes and the development of lupus as expected. Injection of B6 splenocytes into CB6F₁ initiated an initial burst of parental cell engraftment similar to that of B6 into B6D2F₁. However, the acute disease resolved, and the CB6F₁ 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 CB6F₁ and B6D2F₁, whereas DBA/2 and BALB/c splenocytes have a CTL precursor frequency of 1/20,000 for their respective F₁s. 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.