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 hematopoietic cells upon cytokine deprivation. To investigate whether their biological roles in apoptosis overlap, we generated mice lacking both Bim and Bad and compared their hematopoietic 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 hematopoietic 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 demonstrate that Bim and Bad can cooperate in the apoptosis of thymocytes and activated B lymphocytes and in the suppression of thymic lymphoma development.

Summary. 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 FoltÕs 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 vivolabeled donor platelets, platelet-depleted CD151 +/+ mice that received reconstitution with CD151 )/) platelets had smaller thrombi that were unstable and embolized. In contrast, plateletdepleted 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.

It is widely believed that subunit vaccines composed of multiple components will offer greater protection against challenge by malaria, and yet there is little experimental evidence to support this view. We set out to test this proposition in the Plasmodium yoelii challenge system in rodents by comparing the degree of protection conferred by immunization with a mixture of merozoite surface proteins to that conferred by single proteins. We therefore examined a defined protein mixture made of the epidermal growth factor-like domains of P. yoelli merozoite surface protein 1 (MSP1) and MSP4/5, the homologue of P. falciparum MSP4 and MSP5. In the present study we demonstrate that this combination of recombinant proteins dramatically enhances protection against lethal malaria challenge compared to either protein administered alone. Many mice immunized with the MSP4/5 plus MSP1 19 combination did not develop detectable parasitemia after challenge. Combined immunization with MSP1 19 and yMSP4/5, a product characterized by lower protective efficacy, also greatly enhanced protection by reducing peak parasitemias and increasing the numbers of survivors. In some combination trials, levels of antibodies to MSP1 19 were elevated compared to the MSP1 19 alone group; however, improved protection occurred regardless of whether boosting of the anti-MSP1 19 response was observed. Boosting of anti-MSP1 19 did not appear to be due to contaminating endotoxin in the EcMSP4/5 material since enhanced protection was observed in C3H/HeJ mice, which are endotoxin insensitive. Collectively, these experiments show that multiantigen combinations offer enhanced levels of protection against asexual stage infection and suggest that combinations of MSP1, MSP4, and MSP5 should be evaluated further for use in humans.

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Oral immunization of mice with Escherichia coli-expressed Plasmodium yoelii merozoite surface protein 4/5 or the C-terminal 19-kDa fragment of merozoite surface protein 1 induced systemic antibody responses and protected mice against lethal malaria infection. A combination of these two proteins administered orally conferred improved protection compared to that conferred by either protein administered alone.

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Human platelets exhibit a circulating lifespan of ~10 days, mouse platelets ~5 days. This finite existence is circumscribed by members of the Bcl-2 family of proteins, which control the intrinsic apoptosis pathway. Pro-survival Bcl-xL is the critical regulator of platelet lifespan, functioning to keep pro-death Bak and Bax in check, thereby maintaining platelet viability. After 5–10 days in the circulation, platelets not consumed in hemostatic processes initiate a Bak and Bax-dependent cell death program and clearance from the bloodstream. Mutations in Bcl-xL reduce platelet lifespan in a dose-dependent fashion, while deletion of Bak and Bax extend it. Studies with the BH3 mimetic compound ABT-737, which inhibits pro-survival Bcl-xL, have shown that platelets induced to undergo cell death in vitro exhibit many of the hallmarks of apoptosis in nucleated cells, including mitochondrial damage, caspase activation and externalization of membrane phosphatidylserine (PS). Whether any of these features occur during physiological platelet clearance remains unclear. Certainly, mitochondrial damage can reduce the recovery of transfused platelets, but whether PS – which is known to promote the pro-coagulant activity of agonist-activated platelets – also acts as a clearance signal for dying platelets in vivo is yet to be established. Conversely, Bak and Bax may play a role in mediating PS exposure triggered by activation. Supporting the idea that there may be crosstalk between classical platelet signaling pathways and the intrinsic apoptosis pathway is recent evidence that platelet agonists can also activate caspases. Intriguingly, elements of the intrinsic pathway may also contribute to the generation of platelets by megakaryocytes. Several groups have demonstrated that megakaryocytes contain activated caspases and that their inhibition can block platelet shedding by cultured cells. Preliminary evidence we have generated suggests that Bcl-2 family proteins may be required for platelet production in vivo. Thus, it appears that there is much to be understood about the role of the intrinsic apoptosis pathway in the regulation of platelet biogenesis, function, and death.