Immune status assay (ISA): a noninvasive procedure for studying allograft rejection.

BACKGROUND: There is a need for a simple, sensitive, noninvasive technique for monitoring graft function. We report here on a simple assay called immune status assay (ISA) that determines the status of the graft by simply examining the activation status of blood T cells. METHODS: Graft-derived fibroblasts were used as a source of alloantigens and the recipient blood as a source of allograft-specific peripheral blood lymphocytes (PBL). PBL were added to wells containing donor or third-party graft-derived fibroblasts in the presence or absence of interleukin-2 (IL-2). On day 4 [(3)H]thymidine incorporation was quantified after the cells were incubated for 3 days at 37 degrees C, in a 5% CO(2) water-jacketed incubator. The results were analyzed using the following equation: %IL2 - /IL2+ = ((mean[(3)H]thymidine uptake in the absence of IL - 2) / (mean [(3)H]thymidine uptake in the presence of IL - 2)) x 100. RESULTS: The ISA score (%IL-2 - /IL-2+) correlated strongly with the outcome of the graft, as it had a sensitivity of 82% for detecting rejections (14/17), and a specificity of 81% (30/37) for detecting non-rejections. Notably, the ISA detected immune T cell activation in the blood of graft rejecting subjects, which were not detected by currently used techniques such as mixed lymphocytes reaction. CONCLUSION: The ISA is a straightforward procedure that detects allograft rejection with high specificity and sensitivity.

Genetics and type 1 diabetes: online resources for diabetes educators.

PURPOSE: Genetic education Internet sites and peer-reviewed medical literature were reviewed and critiqued to develop tables summarizing online resources for diabetes health professionals. METHODS: Using Internet search engines, each Web site identified for this project met the following criteria: (1) accurate and valid site content based on widely accepted genetic texts, (2) credibility of the organization that maintained the Web site, (3) ease of navigation, and (4) provision of continuing education credits. PubMed was used to find journal articles using similar criteria. RESULTS: There were 33 Web sites on genetic education for diabetes health professionals that met the inclusion criteria. The literature search identified 36 articles regarding the importance of genetic education for nurses and other health professionals, as well as information regarding genetics and diabetes. CONCLUSIONS: Valid and credible information on genetics and type 1 diabetes is available for diabetes health professionals on the Internet and in the medical literature.

Genetics and type 1 diabetes: online resources for patients.

PURPOSE: This Web-based review was undertaken to compile online resources for diabetes educators on genetics--specifically, the genetics of type 1 diabetes--and to provide helpful and accurate information for the public. METHODS: Keyword searches were performed to identify Web sites for genetics education for the lay public and for sites specifically geared toward children/young adults. Web sites were critiqued based on credibility (source, currency, relevance/utility), content (accuracy), and design (accessibility, logical organization). Additional keyword searches were conducted to find sites describing the genetics of type 1 diabetes, which were evaluated for content validity. RESULTS: The Web sites selected for general genetics education contain accessible, credible, and accurate information about basic genetics in an easy-to-follow format with both text and visual aides. Although these sites adequately educate the public about genetics, only diabetes-specific Web sites discussed the relationship between genetics and risk for type 1 diabetes associated with high-risk HLA alleles. CONCLUSIONS: In this genomic age, it is important for healthcare professionals to provide genetics information. Educational tools that specifically address the genetics of type 1 diabetes are urgently needed to fill the current information gaps on the Internet.

Rapamycin antagonizes cyclosporin A- and tacrolimus (FK506)-mediated augmentation of linker for activation of T cell expression in T cells.

The discovery of new immunosuppressive drugs such as rapamycin, cyclosporin A (CsA) and tacrolimus (FK506) has been very useful for preventing graft rejection and autoimmune disease. However, these drugs are not specific, and are associated with side-effects and toxicities. Therefore, understanding the molecular mechanisms of these drugs is important for designing specific immunosuppressants. Here, we show that in contrast to CsA and FK506, rapamycin blocks activation-induced expression of the linker for activation of T cells (LAT), a signaling molecule critical for initiating TCR signaling. Thus, whereas CsA and FK506 strongly enhanced TCR- and phorbol myristate acetate-induced LAT expression in T cells, rapamycin effectively inhibited activation-induced LAT expression. Importantly, these opposite effects were mutually antagonistic, as rapamycin acted as a potent antagonist for both CsA and FK506. Because CsA, unlike FK506 and rapamycin, does not bind to the intracellular immunophilin FK-binding protein (FKBP), the antagonism between these drugs is not simply due to competition for intracellular FKBP. Accordingly, RNA and protein stability analyses suggest inhibition by rapamycin at the translational level. Given the important role of LAT in initiating T cell activation, our data suggests that the effects of rapamycin, CsA and FK506 on T cell activation involve regulating early T cell signaling. These findings refine our understanding of the manifold effects of these immunosuppressants, thus providing insight into the drastic physiological contrasts observed between these drugs.

Modulation of the electrophoretic mobility of the linker for activation of T cells (LAT) by the calcineurin inhibitors CsA and FK506: LAT is a potential substrate for PKC and calcineurin signaling pathways.

The linker for activation of T cells (LAT) is essential for T cell activation. Cyclosporin A (CsA) and FK506, inhibitors of T cell proliferation, have been very useful for preventing autoimmune and inflammatory disease and graft rejection. However, both compounds are associated with side effects. We show that TCR ligation in the presence of FK506 or CsA induced rapid modifications in LAT that modulate the electrophoretic mobility of the molecule in SDS-PAGE. Calcineurin, a target for CsA and FK506, dephosphorylated LAT in vitro and restored its electrophoretic mobility. Stimulating T cells with the protein kinase C (PKC) activator PMA induced a shift in the mobility of LAT, whereas inhibitors of PKC blocked the effect of PMA. Thus, manipulating calcineurin or PKC activation alters the electrophoretic mobility of LAT. These results shed light on the molecular actions of CsA and FK506 in T cells and implicate LAT in mediating the drugs' actions.