Any future programme of antenatal screening of pregnancies for risk of neonatal alloimmune thrombocytopenia will have as a major requirement the availability of cost-effective assays which can be applied to large numbers of samples. To address this, we developed a competitive ELISA to type whole blood samples for the platelet alloantigen HPA1a, based on the use of purified glycoprotein (GP) IIb/IIIa from donors of known HPA1 genotype along with well characterized anti-HPA1a antiserum. Microtitre plates were coated with purified GPIIb/IIIa from donors of genotype HPA1a1a/3a3a. Anticoagulated whole blood of unknown HPA1 type was added to each well followed by anti-HPA1a. Residual antiHPA1a antibody not bound to the platelets in the test blood sample, bound to the immobilized HPA1a on the plate and was quantitated by standard ELISA. 475 blood donors were typed by the assay and the results compared in a blinded comparison with typing in the Capture-Ptm assay. Concordance was 100% (468 HPA1a positive and seven HPA1a negative). The HPA1 type of control samples stored as whole blood could be discriminated by this assay for up to 23 d of storage at 4 degrees C. This assay should be suitable for use in large-scale population screening programmes.
Although low doses of tumor-derived stress protein gp96 elicit protective immunity to the tumor from which it is isolated, protection is lost at high doses because of the induction of immunoregulatory CD4 ϩ T cells. This study evaluated the influence of gp96 on resting rat bone marrow-derived dendritic cells (BMDCs) and purified CD3 ϩ T cells. In contrast to previous reports, gp96 had no effect on adhesion and costimulatory molecule expression by BMDCs, nor did it influence interleukin (IL)-10 and IL-12 secretion or their allostimulatory capacity. Gp96 did not bind to BMDCs but dose-dependently bound to CD4 ϩ and CD8 ϩ T cells. At low concentrations (1 and 25 g/mL), gp96 acted as a costimulator of CD3 ϩ T cells, inducing proliferation and the secretion of interferon (IFN)-␥ and IL-10. Gp96 also increased the proliferation of CD28-costimulated CD3 ϩ T cells and their secretion of IFN-␥, IL-4, and IL-10. Gp96 had no effect at higher concentrations (50 and 100 g/mL), despite the occurrence of cell surface binding at these concentrations. These findings indicate that gp96 can act as a costimulatory molecule for CD3 ϩ T cells, and an observed increase in the IL-10:IFN-␥ secretion ratio induced by gp96 suggests that it might, at appropriate concentrations, promote a regulatory T-helper 2 (Th2)-like phenotype.
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