Individual and overlapping roles of BH3-only proteins Bim and Bad in apoptosis of lymphocytes and platelets and in suppression of thymic lymphoma development.

BH3-only proteins, such as Bim and Bad, contribute to tissue homeostasis by initiating apoptosis in a cell type- and stimulus-specific manner. Loss of Bim provokes lymphocyte accumulation in vivo and renders lymphocytes more resistant to diverse apoptotic stimuli and Bad has been implicated in the apoptosis of haematopoietic cells upon cytokine deprivation. To investigate whether their biological roles in apoptosis overlap, we generated mice lacking both Bim and Bad and compared their haematopoietic phenotype with that of the single-knockout and wild-type (wt) animals. Unexpectedly, bad(-/-) mice had excess platelets due to prolonged platelet life-span. The bim(-/-)bad(-/-) mice were anatomically normal and fertile. Their haematopoietic phenotype resembled that of bim(-/-) mice but lymphocytes were slightly more elevated in their lymph nodes. Although resting B and T lymphocytes from bim(-/-)bad(-/-) and bim(-/-) animals displayed similar resistance to diverse apoptotic stimuli, mitogen activated bim(-/-)bad(-/-) B cells were more refractory to cytokine deprivation. Moreover, combined loss of Bim and Bad-enhanced survival of thymocytes after DNA damage and accelerated development of γ-irradiation-induced thymic lymphoma. Unexpectedly, their cooperation in the thymus depended upon thymocyte-stromal interaction. Collectively, these results show that Bim and Bad can cooperate in the apoptosis of thymocytes and activated B lymphocytes and in the suppression of thymic lymphoma development.

A platelet tetraspanin superfamily member, CD151, is required for regulation of thrombus growth and stability in vivo.

BACKGROUND: This study was designed to determine the role of CD151 in platelet thrombus formation in vivo and define the contribution of platelet vs. endothelial CD151 in regulating platelet thrombus formation in vivo. METHODS AND RESULTS: Using intravital microscopy and ferric chloride (FeCl(3)) injury of mesenteric arterioles, we found that thrombi formed in CD151(+/-) and CD151(-/-) mice were smaller and less stable, than those formed in CD151(+/+) mice, with a tendency for embolization. Similarly, in Folt's FeCl(3)-induced carotid injury model, both CD151(+/-) and CD151(-/-) mice showed more prolonged times to 95% vessel occlusion than CD151(+/+) mice. In addition, laser-induced injury of cremaster muscle arterioles showed that thrombi formed in CD151(+/-) and CD151(-/-) mice were smaller and less stable than those formed in CD151(+/+) mice. Following platelet depletion/reconstitution with ex vivo-labeled donor platelets, platelet-depleted CD151(+/+) mice that received reconstitution with CD151(-/-) platelets had smaller thrombi that were unstable and embolized. In contrast, platelet-depleted CD151(-/-) mice that received reconstitution with CD151(+/+) platelets had normal thrombi that were stable. CONCLUSIONS: These data provide evidence that platelet CD151 is required for regulating thrombus formation in vivo.

Merozoite surface protein 4/5 provides protection against lethal challenge with a heterologous malaria parasite strain.

Immunization with merozoite surface protein 4/5 (MSP4/5), the murine malaria homologue of Plasmodium falciparum MSP4 and MSP5, has been shown to protect mice against challenge by parasites expressing the homologous form of the protein. The gene encoding MSP4/5 was sequenced from a number of Plasmodium yoelii isolates in order to assess the level of polymorphism in the protein. The gene was found to be highly conserved among the 13 P. yoelii isolates sequenced, even though many of the same isolates showed pronounced variability in their MSP1(19) sequences. Nonsynonymous mutations were detected only for the isolates Plasmodium yoelii nigeriensis N67 and Plasmodium yoelii killicki 193L and 194ZZ. Immunization and challenge of BALB/c mice showed that the heterologous MSP4/5 proteins were able to confer a level of protection against lethal Plasmodium yoelii yoelii YM challenge infection similar to that induced by immunization with the homologous MSP4/5 protein. To explore the limits of heterologous protection, mice were immunized with recombinant MSP4/5 protein from Plasmodium berghei ANKA and Plasmodium chabaudi adami DS and challenged with P. y. yoelii YM. Interestingly, significant protection was afforded by P. berghei ANKA MSP4/5, which shows 81% sequence identity with P. y. yoelii YM MSP4/5, but it was abolished upon reduction and alkylation. Significant protection was not observed for mice immunized with recombinant P. c. adami DS MSP4/5, which shows 55.7% sequence identity with P. y. yoelii YM MSP4/5. This study demonstrates the robustness of MSP4/5 in conferring protection against variant forms of the protein in a murine challenge system, in contrast to the situation found for other asexual-stage proteins, such as MSP1(19) and AMA1.

Comparison of the protective efficacy of yeast-derived and Escherichia coli-derived recombinant merozoite surface protein 4/5 against lethal challenge by Plasmodium yoelii.

The gene encoding the Plasmodium yoelii homologue of P. falciparum merozoite surface proteins 4 (MSP4) and 5 (MSP5) has been expressed in Escherichia coli and Saccharomyces cerevisiae. The protein contains a single epidermal growth factor (EGF)-like domain and is expressed in a form lacking the predicted N-terminal signal and glycosyl phosphatidylinositol (GPI) attachment sequences. The recombinant protein derived from E. coli (EcMSP4/5) was highly effective at protecting mice against lethal challenge with 10(5) parasites of the P. yoelii YM strain. In contrast, the protective efficacy of yeast-derived MSP4/5 (yMSP4/5) was considerably less. The antibody titres in both groups were significantly different with mice immunised with yeast-derived protein showing significantly lower pre-challenge antibody responses. There was a significant inverse correlation between antibody levels as measured by ELISA and peak parasitaemia. Mice immunised with EcMSP4/5 produced anti-PyMSP4/5 antibodies predominantly of the IgG2a and IgG2b isotypes, whereas, mice immunised with yMSP4/5 mainly produced antibodies of the IgG1 isotype. The differences in antibody titres and subtype distribution may account for the observed differences in protective efficacy of these protein preparations. Levels of protective efficacy of MSP4/5 were compared with that obtained using P. yoelii MSP1 produced in S. cerevisiae. Levels of protection induced by E. coli derived MSP4/5 were superior to those induced by MSP1 which in turn were better than those induced by yeast-derived MSP4/5.