9%) of 32 HCCs tested were weak or negative-stained. Therefore, it seems that HDAC6 is down-regulated during hepatocarcinogenesis. To generalize our finding, we recapitulated HDAC6 gene expression from the large cohorts of HCC patients that are available from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (accession numbers GSE14520 and GSE25097) and shown as scatterplots. Consistently, HDAC6 gene expression was significantly down-regulated in two different HCC cohorts (Fig. 1B,C). Decreased expression of HDAC6 protein was confirmed by western immunoblotting

of six randomly selected human HCC tissues paired with N and DN. As expected, HDAC6 was markedly down-regulated in all selected HCCs as compared with normal liver or dysplatic nodule tissues Ipatasertib molecular weight (Fig. 1D; Supporting Fig. 1C). Furthermore,

endogenous expression of HDAC6 was investigated by northern and western blot analysis in nine different liver cancer cell lines (HepG2, Hep3B, PLC/PRF/5, SNU182, SNU354, SNU368, SNU387, SNU423, and SNU449), which were originally established from HCC or hepatoblastoma. The human liver cancer cell lines exhibited relatively low HDAC6 expression, with a few exceptions (Fig. 1E). These results strongly suggest that HDAC6 is suppressed in HCC. Because the messenger RNA (mRNA) and protein levels of HDAC6 showed down-regulation in overt HCCs, we next assessed the prognostic association Gefitinib solubility dmso MCE of HDAC6 expression in a large cohort of 100 Korean HCC patients.17 First, 1,909 genes with expression patterns that highly correlated with HDAC6 expression were selected for cluster analysis

(P < 0.001, r > 0.4 or r < −0.4), and shown as heatmaps (Fig. 2A). Patients were then divided into the following two groups: HDAC6 High cluster and HDAC6 Low cluster. The Kaplan-Meier survival curves of patients with HCC indicated that the 5-year overall survival (OS) rate of HCC patients with low HDAC6 expression (50.9%) was significantly lower than that of HCC patients with high HDAC6 expression (69.4%, P < 0.05; Fig. 2B). The disease-free survival (DFS) rate of HCC patients with low HDAC6 expression (27.5%) was also significantly lower than that of HCC patients with high HDAC6 expression (44.9%, P < 0.05; Fig. 2C). In addition, the recurrence-free survival (RFS) rate of HCC patients with low HDAC6 expression (35.3%) was significantly lower than that of HCC patients with high HDAC6 expression (53.1%, P < 0.05; Fig. 2D). These results demonstrated that HDAC6 expression is strongly associated with prognosis in HCC patients. To better understand the molecular consequences of ectopic overexpression of HDAC6 in hepatocarcinogenesis, full-length human HDAC6 cDNA (pcDNA_HDAC6) was constructed and transiently transfected into Hep3B cells and cell viability and MTT cell proliferation assays were performed. The functional activity of HDAC6 was confirmed by detecting the hypoacetylation of α-tubulin (Fig.

7A, upper panel). The relative numbers of Ki67-positive cells (in uninfected cells and HCV1a-infected cells with and without buy Atezolizumab DLC-1 cDNA transfection) is shown in Fig. 7B. In addition, we quantified the enhanced proliferative capacity of HCV1a-infected primary hepatocytes by way of fluorescence-activated cell sorting analysis of Ki67-labeled cells (Fig. 7C). The results showed an approximately 40% increase in cell proliferation of HCV1a-infected

hepatocytes (3 days posttransfection). The increased cell proliferation is neutralized when the DLC-1 level was artificially increased through transfection with a DLC-1 expression vector. Recent studies support the oncogenic role of miRNAs in different human neoplasms including HCC,30 glioblastoma,31 urinary bladder cancer,32 papillary learn more tumors of the thyroid,33 and pancreatic cancer.34 However,

the role of miRNA-mediated oncogenesis remains unclear for HCV infection. We present several lines of evidence that HCV infection results in the induction of miR-141, which silences DLC-1 expression, a tumor suppressor gene that is frequently deleted in HCC and other solid human tumors. The results presented in this study support a link between HCV replication and altered expression of miR-141 that target a tumor suppressor gene frequently deleted in HCC. Tumor suppressor genes can influence oncogenic virus replication by negatively regulating pro-oncogenic signaling proteins.6 NF1 inhibits the Ras signaling pathway, which is deregulated in many cancers and can be a potential therapeutic target. Phosphatase and tensin homologue inhibits the phosphoinositide 3-kinase (PI3K) pathway, and inhibitors of PI3K components such as PI3K, AKT, and mTORs have been similarly pursued for cancer therapy.11 The results presented here support a model of HCV-associated hepatocarcinogenesis, based on miRNA-mediated 上海皓元医药股份有限公司 silencing of tumor suppressor DLC-1. The intracellular induction of miR-141 by HCV

appears to translationally inhibit the tumor suppressor DLC-1, whose depletion promotes cell proliferation. Although our findings support the role of DLC-1 in HCV-associated hepatocarcinogenesis, they do not by themselves support a direct role of DLC-1 in regulating HCV replication, nor do they rule out possible contribution of other tumor suppressor genes. Dependence of HCV replication on miRNAs has been debated in earlier studies.18, 19, 30 In recent studies, Fornari et al.4 have presented evidence that miRNA-221 induced in HCC tissues promotes tumorigenesis by targeting the CDK inhibitors CDKN1C/p57 and CDKN1B/p27. Our findings support the argument that miR-141–targeted suppression of tumor suppressor DLC-1 may promote the initial stages of HCV-associated hepatocarcinogenesis and cell proliferation.

Additionally, we demonstrated that IL-33−/− mice were more sensitized to ConA hepatic injury

than WT controls, in agreement with a protective effect of IL-33/ST2 axis in ConA-hepatitis.10 Our findings are closer to earlier data describing an increased tendency of liver injury in IL-33−/− mice42; however, we speculate that IL-33 may not be implicated in death cascade; rather, it is expressed/released by dying cells as a readout marker to justify its proposed “alarmin” functions during necrosis.43 Finally, we primed the CD1d−/− mice that are deficient in NKT cells by ConA injection along with a simultaneous injection of rm-TRAIL. Previously, it has been reported that an abundant amount (i.e., nearly 500 μg/mouse) of rm-TRAIL injected into mice is

necessary click here to induce moderate liver injury.24 In our present work, the injection of only 30 μg/mouse of rm-TRAIL (i.e., 10 times less than used earlier) was sufficient to trigger severe ConA-induced hepatitis in CD1d−/− primed mice. Interestingly, the reconstitution of TRAIL in CD1d−/− mice induced liver IL-33 expression, which was localized in hepatocytes. We stimulated primary hepatocytes in vitro with rm-TRAIL in order to exclude an indirect effect of TRAIL on IL-33 expression during ConA-induced liver injury. Interestingly, TRAIL readily induced IL-33 expression in cultured murine hepatocytes. In conclusion, our BMN 673 in vivo work demonstrates 上海皓元医药股份有限公司 that the molecular regulation of IL-33 in hepatocytes during acute hepatitis is not dependent on FasL or TNFα, but on TRAIL. For immunohistochemistry analysis and animal house facilities, the authors thank the dedicated platforms (i.e., H2P2, ImPACell, and animal house platforms) of SFR BIOSIT, University of Rennes 1, France. Additional Supporting Information may be found

in the online version of this article. “
“I read with great interest the article by Rein et al.1 In this manuscript, the authors attempt to address the prevalence of hepatitis B surface antigen (HBsAg) in foreign-born persons living in the United States. The authors did so by requesting data on hepatitis B screening from refugee health coordinators around the country. The authors indicate that estimates for HBsAg prevalence from the study correspond to estimates from the literature for each country (where comparison is available). One should be very careful when extrapolating the findings of one group of refugees to an entire nation. Generally, refugees that enter one jurisdiction come from the same area in the country of origin. In sub-Saharan Africa, rates of hepatitis B virus (HBV) for each country vary according to regional areas; this is likely related to the habits and customs of each region within a country. The authors report a prevalence of HBsAg of 3.1% in refugees from Tanzania. The rates of HBsAg for Tanzania range from 4.

Additionally, we demonstrated that IL-33−/− mice were more sensitized to ConA hepatic injury

than WT controls, in agreement with a protective effect of IL-33/ST2 axis in ConA-hepatitis.10 Our findings are closer to earlier data describing an increased tendency of liver injury in IL-33−/− mice42; however, we speculate that IL-33 may not be implicated in death cascade; rather, it is expressed/released by dying cells as a readout marker to justify its proposed “alarmin” functions during necrosis.43 Finally, we primed the CD1d−/− mice that are deficient in NKT cells by ConA injection along with a simultaneous injection of rm-TRAIL. Previously, it has been reported that an abundant amount (i.e., nearly 500 μg/mouse) of rm-TRAIL injected into mice is

necessary this website to induce moderate liver injury.24 In our present work, the injection of only 30 μg/mouse of rm-TRAIL (i.e., 10 times less than used earlier) was sufficient to trigger severe ConA-induced hepatitis in CD1d−/− primed mice. Interestingly, the reconstitution of TRAIL in CD1d−/− mice induced liver IL-33 expression, which was localized in hepatocytes. We stimulated primary hepatocytes in vitro with rm-TRAIL in order to exclude an indirect effect of TRAIL on IL-33 expression during ConA-induced liver injury. Interestingly, TRAIL readily induced IL-33 expression in cultured murine hepatocytes. In conclusion, our http://www.selleckchem.com/products/BKM-120.html work demonstrates MCE that the molecular regulation of IL-33 in hepatocytes during acute hepatitis is not dependent on FasL or TNFα, but on TRAIL. For immunohistochemistry analysis and animal house facilities, the authors thank the dedicated platforms (i.e., H2P2, ImPACell, and animal house platforms) of SFR BIOSIT, University of Rennes 1, France. Additional Supporting Information may be found

in the online version of this article. “
“I read with great interest the article by Rein et al.1 In this manuscript, the authors attempt to address the prevalence of hepatitis B surface antigen (HBsAg) in foreign-born persons living in the United States. The authors did so by requesting data on hepatitis B screening from refugee health coordinators around the country. The authors indicate that estimates for HBsAg prevalence from the study correspond to estimates from the literature for each country (where comparison is available). One should be very careful when extrapolating the findings of one group of refugees to an entire nation. Generally, refugees that enter one jurisdiction come from the same area in the country of origin. In sub-Saharan Africa, rates of hepatitis B virus (HBV) for each country vary according to regional areas; this is likely related to the habits and customs of each region within a country. The authors report a prevalence of HBsAg of 3.1% in refugees from Tanzania. The rates of HBsAg for Tanzania range from 4.

4B). Knockdown of SIRT2 also caused a redistribution of cytoplasmic and nuclear LY2606368 datasheet β-catenin to the membranous localization (Fig. 4C). Concordantly, TOPflash and FOPflash luciferase reporter analysis revealed that the transactivation of TCF reporter was inhibited

by the depletion of SIRT2 (Fig. 4D). To further determine whether SIRT2 exerts its function by β-catenin signaling, we ectopically expressed β-catenin or green fluorescent protein (GFP) in SIRT2-depleted SK-Hep-1 cells. Importantly, ectopic expression of β-catenin, but not GFP, significantly restored cell proliferation (Fig. 5A), as well as enhanced cell migration (Fig. 5B) and invasion (Fig. 5C). In contrast, ectopic expression of SIRT2 in nontumorigenic L02 cells promoted their migration and invasion that was inhibited by depletion of β-catenin (Fig. 5D). Collectively, these data suggested that SIRT2 regulates HCC cell growth and motility through regulating β-catenin signaling. To elucidate the underlying mechanism of SIRT2-dependent β-catenin inactivation,

we determined the status of GSK-3β, which forms a destruction complex with Axin and adenomatous polyposis coli (APC) for the phosphorylation and degradation of β-catenin.31 Depletion of SIRT2 increased the abundance of unphosphorylated (activated) and total GSK-3β, whereas it reduced the level of phosphorylated (activated) Akt (Fig. 6A). Because Akt phosphorylates Kinase Inhibitor Library and inactivates GSK-3β,32 our results suggested that SIRT2 may affect EMT by regulating the Akt/GSK-3β/β-catenin-signaling axis. An earlier study suggested that phosphorylation and activity of Akt is regulated by SIRT1-dependent deacetylation33; therefore, we determined whether SIRT2 plays a role in the

acetylation of Akt, GSK-3β, and β-catenin proteins. These proteins were first immunoprecipitated by the corresponding Abs, respectively, and their acetylation status was determined by anti-acetylated-lysine Abs. Our data showed that β-catenin was neither acetylated when SIRT2 was expressed nor depleted, whereas GSK-3β was constitutively acetylated under both conditions 上海皓元 (Fig. 6B). On the other hand, although Akt was also constitutively acetylated, its acetylation level was markedly up-regulated by the depletion of SIRT2, whereas depletion of SIRT1 did not alter Akt acetylation (Fig. 6B). More important, SIRT2, but not SIRT1, was coimmunoprecipitated with AKT (Fig. 6C). Taken together, these data revealed a novel role of SIRT2 in the β-catenin signaling pathway by regulating Akt acetylation in HCC cells. Sirtuins are involved in various aspects of biological processes, such as the regulation of gene expression, cellular stress response, DNA repair and metabolism, and so on. Despite there being a growing interest in elucidating the functions of sirtuins, how this group of deacetylases is involved in tumorigenesis is still poorly understood.

4B). Knockdown of SIRT2 also caused a redistribution of cytoplasmic and nuclear I-BET-762 molecular weight β-catenin to the membranous localization (Fig. 4C). Concordantly, TOPflash and FOPflash luciferase reporter analysis revealed that the transactivation of TCF reporter was inhibited

by the depletion of SIRT2 (Fig. 4D). To further determine whether SIRT2 exerts its function by β-catenin signaling, we ectopically expressed β-catenin or green fluorescent protein (GFP) in SIRT2-depleted SK-Hep-1 cells. Importantly, ectopic expression of β-catenin, but not GFP, significantly restored cell proliferation (Fig. 5A), as well as enhanced cell migration (Fig. 5B) and invasion (Fig. 5C). In contrast, ectopic expression of SIRT2 in nontumorigenic L02 cells promoted their migration and invasion that was inhibited by depletion of β-catenin (Fig. 5D). Collectively, these data suggested that SIRT2 regulates HCC cell growth and motility through regulating β-catenin signaling. To elucidate the underlying mechanism of SIRT2-dependent β-catenin inactivation,

we determined the status of GSK-3β, which forms a destruction complex with Axin and adenomatous polyposis coli (APC) for the phosphorylation and degradation of β-catenin.31 Depletion of SIRT2 increased the abundance of unphosphorylated (activated) and total GSK-3β, whereas it reduced the level of phosphorylated (activated) Akt (Fig. 6A). Because Akt phosphorylates R788 molecular weight and inactivates GSK-3β,32 our results suggested that SIRT2 may affect EMT by regulating the Akt/GSK-3β/β-catenin-signaling axis. An earlier study suggested that phosphorylation and activity of Akt is regulated by SIRT1-dependent deacetylation33; therefore, we determined whether SIRT2 plays a role in the

acetylation of Akt, GSK-3β, and β-catenin proteins. These proteins were first immunoprecipitated by the corresponding Abs, respectively, and their acetylation status was determined by anti-acetylated-lysine Abs. Our data showed that β-catenin was neither acetylated when SIRT2 was expressed nor depleted, whereas GSK-3β was constitutively acetylated under both conditions 上海皓元 (Fig. 6B). On the other hand, although Akt was also constitutively acetylated, its acetylation level was markedly up-regulated by the depletion of SIRT2, whereas depletion of SIRT1 did not alter Akt acetylation (Fig. 6B). More important, SIRT2, but not SIRT1, was coimmunoprecipitated with AKT (Fig. 6C). Taken together, these data revealed a novel role of SIRT2 in the β-catenin signaling pathway by regulating Akt acetylation in HCC cells. Sirtuins are involved in various aspects of biological processes, such as the regulation of gene expression, cellular stress response, DNA repair and metabolism, and so on. Despite there being a growing interest in elucidating the functions of sirtuins, how this group of deacetylases is involved in tumorigenesis is still poorly understood.

“
“Emerging evidence implicates the chromodomain helicase/ATPase DNA binding protein 1–like gene (CHD1L) as a specific oncogene in human hepatocellular carcinoma (HCC). To better understand the molecular mechanisms underlying HCC cases carrying CHD1L amplification (>50% HCCs), we selleck chemicals identified a CHD1L

target, translationally controlled tumor protein (TCTP), and investigated its role in HCC progression. Here, we report that CHD1L protein directly binds to the promoter region (nt −733 to −1,027) of TCTP and activates TCTP transcription. Overexpression of TCTP was detected in 40.7% of human HCC samples analyzed and positively correlated with Ibrutinib supplier CHD1L overexpression. Clinically, overexpression of TCTP was significantly associated with the advanced tumor stage (P = 0.037) and overall survival time of HCC patients (P = 0.034). In multivariate analyses,

TCTP was determined to be an independent marker associated with poor prognostic outcomes. In vitro and in vivo functional studies in mice showed that TCTP has tumorigenic abilities, and overexpression of TCTP induced by CHD1L contributed to the mitotic defects of tumor cells. Further mechanistic studies demonstrated that TCTP promoted the ubiquitin-proteasome degradation of Cdc25C during mitotic progression, which caused the failure in the dephosphorylation of Cdk1 on Tyr15 and decreased Cdk1 activity. As a consequence, the sudden drop of Cdk1 activity in mitosis

induced a faster mitotic exit and chromosome missegregation, which led to chromosomal instability. The depletion experiment proved that the tumorigenicity of TCTP was linked to its role in mitotic defects. Conclusion: Collectively, we reveal a novel molecular pathway (CHD1L/TCTP/Cdc25C/Cdk1), which causes the malignant transformation of hepatocytes with the phenotypes of accelerated mitotic progression and the production of aneuploidy. (HEPATOLOGY MCE公司 2012) Hepatocellular carcinoma (HCC) is the sixth most common human cancer in the world, with extremely poor prognosis and a <3% 5-year survival rate for untreated cancer.1 The ultimate cause of HCC is perhaps better understood than other types of human cancers, which is chronic liver disease (eventually leading to cirrhosis), particularly chronic hepatitis B and C and alcoholic liver disease. Other risk factors, such as tobacco smoking, nonalcoholic steatohepatitis, and inherited metabolic diseases, have also been proposed to cause HCC, albeit at a lower frequency.2 In addition, HCC is predominantly male associated in all populations, and the incidence of HCC also increases progressively with age.

As

prior HCV screening efforts have not targeted Emergency Department (ED) baby boomer patients, we describe early experience with selleck chemicals llc integrated opt-out HCV antibody screening of medically stable “baby boomers” presenting to an urban academic ED. We performed HCV antibody testing 24 hours per day and confirmed positive test results using PCR. The primary outcome was prevalence of unrecognized HCV infection. Among 2,325 unique HCV-unaware baby boomers, 289 (12.7%) opted-out of HCV screening. We performed HCV-antibody tests on 1,529 individuals, of which 170 (11.1%) were reactive. Among antibody reactive cases, follow-up PCR was performed on 150 (88.2%), of which 102 (68.0%) were confirmed RNA-positive. HCV antibody reactivity was more likely in males compared to females (14.7% vs. 7.4%, p<0.001), African Americans compared to whites (13.3% vs. 8.8%, p=0.010), and underinsured/ uninsured patients compared to insured patients (16.8%/ 16.9% vs. mTOR inhibitor 5.0%, p=0.001). Linkage-to-care service activities were recorded for 100 of the 102 confirmed cases. Overall, 54 (54%) RNA-positive individuals were successfully contacted by phone within five call back attempts. We confirmed initial follow-up appointments for 38 (70.4%) RNA-positive individuals successfully contacted, and 21

(55.3%) individuals with confirmed appointments attended their initial visit with a liver specialist; three (7.9%) are awaiting an upcoming scheduled appointment. Conclusion: We observed high prevalence of unrecognized chronic HCV infection in this series of baby boomers presenting to the ED highlighting the ED as an important venue for high-impact HCV screening and linkage to care. (Hepatology 2014;) “
“A 44-year-old woman with hepatitis C cirrhosis presented with a week of heavy vaginal bleeding. Her obstetric history was significant for three cesarean sections. Her gynecologist made an initial diagnosis of menometrorrhagia exacerbated by thrombocytopenia and coagulopathy.

Computed tomography 上海皓元 (CT) angiography revealed splenic vein thrombosis and engorged pelvic veins which arose as collaterals from the splenic vein (Fig. 1). Hysteroscopy could not identify a culprit lesion due to the rapidity of bleeding. A transjugular intrahepatic portosystemic shunt (TIPS) was created and thrombectomy of the splenic vein was performed and the residual partially occlusive thrombus was then stented. Hepatopedal flow was then noted from splenic vein to portal vein and through the TIPS. Hysteroscopy showed persistently engorged varices. Venous embolization of the varices was performed with a combination of embolization coils and a vascular plug (Fig. 2). Recovery was uneventful, and she was followed for 2 years in our clinic without further vaginal bleeding. CT, computed tomography; TIPS, transjugular intrahepatic portosystemic shunt.

2. Analysis of anti-FVIII production by ELISpot reveals that the first and third well displayed contained FVIII-specific ASCs. Six spots are observed in the first well whereas 46 spots are present in the third well. This indicates that FVIII-specific ASCs increased in number following stimulation Ruxolitinib by CD40L and appropriate cytokines. In the other wells, no spots corresponding to ASCs producing anti-FVIII IgG were observed. The presence of FVIII-specific ASCs was also detected using ELISA (Fig. 2b). Seven of 55 wells analysed were strongly positive whereas low levels of anti-FVIII antibodies were observed in two wells. The two wells

that were positive in the ELISpot were also strongly positive when ELISA was used as a read-out for anti-FVIII antibodies (Fig. 2; boxed wells in ELISA correspond Selumetinib purchase to the six wells for which results of the ELISpot are displayed). Overall, detection of anti-FVIII antibodies by ELISA appeared more sensitive when compared with ELISpot. The prolonged incubation of B cells for 9–10 days may favour the detection of anti-FVIII antibodies using ELISA as a result of accumulation of IgG in the conditioned medium. Analysis by ELISpot requires the presence of viable IgG-producing cells which is optimally assessed 6 days after stimulation [35]. Based on the number of positive wells the frequency of FVIII-specific memory B

cells can be calculated assuming that a positive well corresponds to the presence of a single FVIII-specific memory B cell. For the patient sample analysed in Fig. 2a, seven (ELISpot) or nine (ELISA) of 55 wells contained FVIII-specific memory B cells. The frequency of FVIII-specific B cells thus ranges from 13 to 16 cells per 105 CD19+ B cells for this MCE公司 patient. Analysis of peripheral blood samples from additional haemophilia A patients with inhibitors revealed that FVIII-specific memory B cells could be detected in four of five patients analysed [33]. The frequency of FVIII-specific

memory B cells in these patients ranged from 5 to 24 per 105 CD19+ B cells as determined using ELISA as a read-out (Table 1). In parallel, we determined the percentage of FVIII-specific memory B cells by comparing the number of IgG and anti-FVIII IgG-producing ASCs (Table 1). This analysis revealed that 0.07–0.35% of circulating memory B cells can develop into FVIII-specific ASCs [33]. These values are similar to the frequency of 0.24% as reported in a patient with an inhibitor titre of 1000 BU mL−1 [34] and are in the same range as observed for antigen-specific memory B cells in peripheral blood of HIV-infected patients and in normal individuals following smallpox, hepatitis B and tetanus toxoid [35,41–43]. In one patient the frequency of FVIII-specific memory B cells was below the limit of detection (frequency <1 per 105 B cells).

2. Analysis of anti-FVIII production by ELISpot reveals that the first and third well displayed contained FVIII-specific ASCs. Six spots are observed in the first well whereas 46 spots are present in the third well. This indicates that FVIII-specific ASCs increased in number following stimulation selleck inhibitor by CD40L and appropriate cytokines. In the other wells, no spots corresponding to ASCs producing anti-FVIII IgG were observed. The presence of FVIII-specific ASCs was also detected using ELISA (Fig. 2b). Seven of 55 wells analysed were strongly positive whereas low levels of anti-FVIII antibodies were observed in two wells. The two wells

that were positive in the ELISpot were also strongly positive when ELISA was used as a read-out for anti-FVIII antibodies (Fig. 2; boxed wells in ELISA correspond BVD-523 research buy to the six wells for which results of the ELISpot are displayed). Overall, detection of anti-FVIII antibodies by ELISA appeared more sensitive when compared with ELISpot. The prolonged incubation of B cells for 9–10 days may favour the detection of anti-FVIII antibodies using ELISA as a result of accumulation of IgG in the conditioned medium. Analysis by ELISpot requires the presence of viable IgG-producing cells which is optimally assessed 6 days after stimulation [35]. Based on the number of positive wells the frequency of FVIII-specific memory B

cells can be calculated assuming that a positive well corresponds to the presence of a single FVIII-specific memory B cell. For the patient sample analysed in Fig. 2a, seven (ELISpot) or nine (ELISA) of 55 wells contained FVIII-specific memory B cells. The frequency of FVIII-specific B cells thus ranges from 13 to 16 cells per 105 CD19+ B cells for this medchemexpress patient. Analysis of peripheral blood samples from additional haemophilia A patients with inhibitors revealed that FVIII-specific memory B cells could be detected in four of five patients analysed [33]. The frequency of FVIII-specific

memory B cells in these patients ranged from 5 to 24 per 105 CD19+ B cells as determined using ELISA as a read-out (Table 1). In parallel, we determined the percentage of FVIII-specific memory B cells by comparing the number of IgG and anti-FVIII IgG-producing ASCs (Table 1). This analysis revealed that 0.07–0.35% of circulating memory B cells can develop into FVIII-specific ASCs [33]. These values are similar to the frequency of 0.24% as reported in a patient with an inhibitor titre of 1000 BU mL−1 [34] and are in the same range as observed for antigen-specific memory B cells in peripheral blood of HIV-infected patients and in normal individuals following smallpox, hepatitis B and tetanus toxoid [35,41–43]. In one patient the frequency of FVIII-specific memory B cells was below the limit of detection (frequency <1 per 105 B cells).