05) and uninfected controls (MFI 13240; P < 0.05; Fig. 4C), whereas activated buy GDC-0449 B cells did not exhibit differential expression levels of Bcl-2 (Fig. 4D). To confirm the functional relevance of differential Bcl-2 expression, B cells were isolated and immature B cells were removed via CD10-positive selection. The resulting B cell population

was cultured overnight without growth factors or cytokines, and mature B cell subsets were analyzed for apoptosis. Naïve B cells of HCV-infected patients with MC expressed higher levels of activated caspase-3 and caspase-8 than those of HCV-infected patients without MC and uninfected controls (P < 0.01; Fig. 4E). Apoptosis of naïve B cells from HCV patients with MC was confirmed by increased expression of D4-GD1, a substrate of active caspase-3, when compared with naïve B cells of HCV-infected patients without MC and uninfected

controls (P < 0.01; Fig. 4E). Furthermore, caspase-3, caspase-8, and D4-GD1 MFI were increased in naïve B cells of HCV-infected patients with MC compared with uninfected controls and HCV-infected patients without MC (data not shown). In contrast, caspase-3 and caspase-8 and the caspase-3 substrate D4-GD1 were not differentially expressed in CD27+ activated/memory B cells of HCV-infected patients with and without MC and uninfected controls (Fig. 4F). Of note, it was not possible to distinguish between naïve and

tissue-like memory B cells in this assay as dying cells down-regulate CD21. Wnt inhibitor However, even if we could not formally exclude the presence of apoptosis-prone, CD21- tissue-like memory cells, they were unlikely to account for the large percentage (≈70%) of apoptotic cells in the culture because they compromise only a small (<5%) fraction of CD19+ B cells, which translates to at most 10% of the cells in culture. Thus, naïve B cells from HCV patients with MC were more susceptible to apoptosis, which is reflected in their reduced percentage and number. Because naïve B cells make up the largest fraction of the 上海皓元医药股份有限公司 mature B cell compartment (≈75%) their reduced frequency may contribute to the observed reduction in CD19+ B cell numbers of HCV-infected patients with MC. To investigate whether apoptosis of naïve mature B cells caused compensatory changes in the immature subset, we studied immature transitional B cells, which link the pro-B cell compartment in the bone marrow to the mature B cell compartment in the spleen.9 Immature B cells that emigrate from the bone marrow to the spleen are defined as transitional B cells and can be distinguished from mature B cells by the presence of CD10 and absence of CD27 (Fig. 1B).