Recognition of the human antibody-mediated platelet destruction in adult ITP patients by C-reactive protein.

Immune thrombocytopenia purpura (ITP) is characterized by destruction of circulating platelets and the presence of antiplatelet IgG antibodies, which opsonize platelets for splenic clearance resulting in low levels of circulating platelets, and the disease severity can be predicted neither by antibody isotype nor by titer, indicating that other factors also play a role. Although the main cause of ITP remains unclear, but its relationship with some infection was demonstrated, including viral or bacterial infections. C-reactive protein (CRP), a member of the pentraxin family, is a major acute-phase protein in humans and is a clinical marker of infection. We aimed to investigate the correlation between the levels of CRP and the presence of antiplatelet IgG antibodies in adults with newly diagnosed ITP. CRP levels and platelet counts were measured in the blood samples from a 60 ITP patient (with confirmed anti-GPIIb/IIIa antibodies), 60 infection patients (all without anti-GPIIb/IIIa antibodies) and 60 normal individuals. The bleeding score, recover time of intravenous immune globulin (IVIg) therapy and the number of megakaryocytes in bone marrow were recorded in ITP patients. The platelet count, bleeding score, recover time of intravenous immune globulin (IVIG) therapy and the number of megakaryocytes in bone marrow and CRP concentrations were compared in ITP group using Spearman's correlation coefficient. We examined the influence of intraperioneal CRP administration on antibody-mediated platelet destruction in mice. There were no statistical differences in gender, age and body mass index among the three groups (P>0.05). Though CRP levels are significantly elevated in ITP patients and infection patients (P<0.05), the platelet count was markedly lower only in ITP patients. We found that CRP was inert toward platelets without antiplatelet antibodies in this study. There are a significant correlation between CRP levels and platelet counts, bleeding severity and the number of megakaryocytes in bone marrow aspiration (r=-0.5079, r=0.5498, r=0.4172, P<0.001, respectively). Moreover, a significant correlation was observed between the recovery time of platelet count and CRP levels (r=-0.5569, P<0.001). In mice, platelet count was lower in Anti-CD41 (0.75 µg)+, CRP (200 µg) group as compared with Anti-CD41 (0.75 µg)+, CRP(-) group and Anti-CD41 (0.75 µg)-, CRP (200 µg) group (P<0.05). In summary, this study indicated that CRP levels are significantly elevated in ITP patients all with confirmed anti-GPIIb/IIIa antibodies, which is able to predict the clinical bleeding severity of ITP patients. The slower CRP levels reduction after IVIg treatment predicted slower platelet count recovery in ITP.

Suppression of HSP27 increases the anti­tumor effects of quercetin in human leukemia U937 cells.

Quercetin, a natural flavonoid, inhibits the growth of leukemia cells and induces apoptosis. Heat shock protein 27 (HSP27) has been reported to promote the development of leukemia by protecting tumor cells from apoptosis through various mechanisms. The present study investigated the effects of small hairpin (sh)RNA-mediated HSP27 knockdown on the anti­cancer effects of quercetin in U937 human leukemia cells. Cells were transfected with recombinant lentiviral vector pCMV­G­NR­U6­shHSP27 (shHSP27), which expressed shRNA specifically targeting the HSP27 gene, alone or in combination with quercetin. The results showed that shHSP27 and quercetin synergistically inhibited U937 cell proliferation and induced apoptosis by decreasing the Bcl2-to-Bax ratio. Furthermore, this combined treatment significantly suppressed the infiltration of tumor cells and the expression of angiogenesis­associated proteins HIF1α and VEGF. Compared with shHSP27 or quercetin alone, shHSP27 plus quercetin markedly decreased the protein expression of cyclinD1 and thus blocked the cell cycle at G1 phase. The Notch/AKT/mTOR signaling pathway is important in tumor aggressiveness; quercetin plus shHSP27 significantly decreased Notch 1 expression and the phosphorylation levels of the downstream signaling proteins AKT and mTOR. The inhibitory effects of quercetin plus shHSP27 on this pathway may thus have been responsible for the cell cycle arrest, inhibition of proliferations and infiltration as well as enhancement of apoptosis. Therefore, these findings collectively suggested that suppression of HSP27 expression amplified the anti­cancer effects of quercetin in U937 human leukemia cells, and that quercetin in combination with shHSP27 represents a promising therapeutic strategy for human leukemia.