Inbred or outbred? An evaluation of the functional allogenicity of farm sheep used in cardiac valve studies.

OBJECTIVE: Cryopreserved allograft cardiovascular tissue elicits a strong cellular and humoral response in recipients; this may accelerate the deterioration of the allograft and complicate future heart transplantation. Juvenile sheep are the standard model for in vivo valve research and have been used to investigate the allogeneic immune response to cardiac valve and vascular tissue transplantation. Studies to date have not considered the extent of allogenicity of sheep used in transplantation studies. METHODS: Functional allogenicity was assessed by standard one-way mixed lymphocyte reaction assay using peripheral blood mononuclear cells. Responder cells were stimulated with irradiated stimulator cells and cultured at 37 degrees C in 95% air and 5% carbon dioxide for 3, 4, 5, and 6 days. Cultures were pulsed with tritiated thymidine for 24 hours and harvested onto filtermats. RESULTS: The allogeneic response, measured as counts per minute, demonstrated a bimodal distribution. Fifty-nine (36.9%) of 160 pairs fell within the first peak (counts per minute < 10,000) and were defined as weak responders. The remaining 101 (63.1%) of 160 pairs of animals demonstrated a strong allogeneic response (counts per minute > or = 10,000) that followed a normal distribution. CONCLUSIONS: More than 1 in 3 pairs of sheep is too closely related to elicit an immune response when cross-reacted. This finding may alter the interpretation of studies that base their findings on allogeneic transplantations in sheep without ascertaining the genetic similarities of the animals. Valve transplantation studies in a sheep model should assess the extent of allogenicity of donor and recipient sheep.

Identification of a novel human granzyme B inhibitor secreted by cultured sertoli cells.

Sertoli cells have long since been recognized for their ability to suppress the immune system and protect themselves as well as other cell types from harmful immune reaction. However, the exact mechanism or product produced by Sertoli cells that affords this immunoprotection has never been fully elucidated. We examined the effect of mouse Sertoli cell-conditioned medium on human granzyme B-mediated killing and found that there was an inhibitory effect. We subsequently found that a factor secreted by Sertoli cells inhibited killing through the inhibition of granzyme B enzymatic activity. SDS-PAGE analysis revealed that this factor formed an SDS-insoluble complex with granzyme B. Immunoprecipitation and mass spectroscopic analysis of the complex identified a proteinase inhibitor, serpina3n, as a novel inhibitor of human granzyme B. We cloned serpina3n cDNA, expressed it in Jurkat cells, and confirmed its inhibitory action on granzyme B activity. Our studies have led to the discovery of a new inhibitor of granzyme B and have uncovered a new mechanism used by Sertoli cells for immunoprotection.

Glutaraldehyde-fixed bioprosthetic heart valve conduits calcify and fail from xenograft rejection.

BACKGROUND: Glutaraldehyde fixation (G-F) decreases but likely does not eliminate the antigenicity of bioprosthetic heart valves. Rejection (with secondary dystrophic calcification) may be why G-F xenograft valves fail, especially in young patients, who are more immunocompetent than the elderly. Therefore, we sought to determine whether rejection of G-F xenograft occurs and to correlate this with graft calcification. METHODS AND RESULTS: Ascending aortas/valves (from rats [syngeneic] or guinea pigs [xenogeneic]) were transplanted (fresh or after 48 hour of G-F) into the infrarenal aortas of young rat recipients for 20 days. A xenogeneic group was also treated with steroids until graft harvest. The valves and media/adventitia were scored blindly for inflammation (0 to 4). Percent graft infiltration by T cells/macrophages was determined (immunohistochemistry), and rat IgG ELISAs were performed. There was >3 times more valve inflammation, >10 times more valve T-cell/macrophage infiltrate, and >3 times antibody rise in the G-F xenogeneic groups compared with the fresh syngeneic or the G-F syngeneic groups (P<0.05). There was >2 times more adventitial inflammation and T-cell/macrophage infiltrate in the xenogeneic groups (P<0.05). Steroid treatment decreased inflammation and antibody rise in the xenogeneic groups (P<0.05). Correlation analysis revealed media/adventitia inflammation (P=0.02) and percent macrophage (P=0.01) infiltration to be predictors of calcification. CONCLUSIONS: G-F xenografts have cellular/humoral rejection and calcify secondarily.

Metformin, but not leptin, regulates AMP-activated protein kinase in pancreatic islets: impact on glucose-stimulated insulin secretion.

Metformin, a drug widely used in the treatment of type 2 diabetes, has recently been shown to act on skeletal muscle and liver in part through the activation of AMP-activated protein kinase (AMPK). Whether metformin or the satiety factor leptin, which also stimulates AMPK in muscle, regulates this enzyme in pancreatic islets is unknown. We have recently shown that forced increases in AMPK activity inhibit insulin secretion from MIN6 cells (da Silva Xavier G, Leclerc I, Varadi A, Tsuboi T, Moule SK, and Rutter GA. Biochem J 371: 761-774, 2003). Here, we explore whether 1) glucose, metformin, or leptin regulates AMPK activity in isolated islets from rodent and human and 2) whether changes in AMPK activity modulate insulin secretion from human islets. Increases in glucose concentration from 0 to 3 and from 3 to 17 mM inhibited AMPK activity in primary islets from mouse, rat, and human, confirming previous findings in insulinoma cells. Incubation with metformin (0.2-1 mM) activated AMPK in both human islets and MIN6 beta-cells in parallel with an inhibition of insulin secretion, whereas leptin (10-100 nM) was without effect in MIN6 cells. These studies demonstrate that AMPK activity is subject to regulation by both glucose and metformin in pancreatic islets and clonal beta-cells. The inhibitory effects of metformin on insulin secretion may therefore need to be considered with respect to the use of this drug for the treatment of type 2 diabetes.