CML is an hematopoietic stem cell disease characterized by the t(9;22) (q34;q11) translocation encoding the oncoprotein p210BCR-ABL. The effect of acadesine (AICAR, 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside) a compound with known antileukemic effect on B cell chronic lymphoblastic leukemia (B-CLL) was investigated in different CML cell lines. Acadesine triggered loss of cell metabolism in K562, LAMA-84 and JURL-MK1 and was also effective in killing imatinib-resistant K562 cells and Ba/F3 cells carrying the T315I-BCR-ABL mutation. The anti-leukemic effect of acadesine did not involve apoptosis but required rather induction of autophagic cell death. AMPK knock-down by Sh-RNA failed to prevent the effect of acadesine, indicating an AMPK-independent mechanism. The effect of acadesine was abrogated by GF109203X and Ro-32-0432, both inhibitor of classical and new PKCs and accordingly, acadesine triggered relocation and activation of several PKC isoforms in K562 cells. In addition, this compound exhibited a potent anti-leukemic effect in clonogenic assays of CML cells in methyl cellulose and in a xenograft model of K562 cells in nude mice. In conclusion, our work identifies an original and unexpected mechanism by which acadesine triggers autophagic cell death through PKC activation. Therefore, in addition to its promising effects in B-CLL, acadesine might also be beneficial for Imatinib-resistant CML patients.

The aim of this study was to prospectively measure liver stiffness with real-time tissue elastography in patients with nonalcoholic fatty liver diseases (NAFLD) and to compare the result with the clinical assessment of fibrosis using histological stage. One hundred and eighty-one prospectively enrolled patients underwent real-time tissue elastography, with the first 106 being analyzed as the training set and the remaining 75 being evaluated as the validation set. Hepatic and splenic elastic ratios were calculated and compared with stage of histological fibrosis. Portal hypertension (PH) was assessed. Real-time tissue elastography cut-off values by stage in the training set were 2.47 for F1, 2.67 for F2, 3.02 for F3, and 3.36 for F4. Using these cut-off values, the diagnostic accuracy of hepatic fibrosis in the validation set was 82.6%-96.0% in all stages. Only portal fibrosis correlated with the hepatic elastic ratio by multivariate analysis. The area under the receiver operating characteristic curve of elastic ratio better correlated than serum fibrosis markers in both early and advanced fibrosis stages. Patients with PH, defined by splenic elasticity, had early fibrosis. Patients with severe PH were found only in the group with cirrhosis.

CONCLUSIONS

Real-time tissue elastography is useful in evaluating hepatic fibrosis and PH in patients with NAFLD.

OBJECTIVE

Histological assessment of patients with chronic hepatitis C infection is no longer performed routinely; consequently, a simple test is needed to identify patients with significant hepatic fibrosis.

METHODS

Data were collected, retrospectively, on 923 consecutive patients undergoing percutaneous liver biopsy for chronic hepatitis C at King's College Hospital between 1 January 2000 and 30 June 2006; 602 patients were accepted to form the training set and a further 105 patients to form the validation set.

RESULTS

On liver biopsy, 132 (22%) had cirrhosis (Ishak F5-6) in the training set and 19 (18%) in the validation set. Factors found by multivariate analysis to be associated with fibrosis in the training set were used to construct the King's Score: age x aspartate aminotransferase x international normalized ratio / platelets. Area under receiver operating characteristic curves for predicting cirrhosis and significant fibrosis (F3-6) were 0.91 and 0.79, respectively. A King's Score of greater than or equal to 16.7 predicted cirrhosis in 34% of patients (odds ratio 36.2, 95% confidence interval, 22.0-59.6; P<0.0001) with sensitivity 86%, specificity 80% and a high negative predictive value of 96%; a score greater than or equal to 12.3 predicted F3-6 (odds ratio 33.9, 95% confidence interval, 15.2-34.4; P<0.001). The validation set confirmed the utility of this index, area under receiver operating characteristic curves 0.94 and 0.89 for cirrhosis and F3-6, respectively.

CONCLUSIONS

The King's Score is a simple and accurate index for predicting cirrhosis in chronic hepatitis C. Patients with a score of less than 16.7 have a low risk of cirrhosis.

Transcranial direct current stimulation (tDCS) is a neuromodulation protocol that can facilitate or inhibit cortical excitability in particular areas of the brain. Although recent studies have reported that tDCS can successfully modulate the excitability of various brain sites, outcomes of tDCS were not consistent between subjects even when identical stimulation protocols were applied. Thus far, however, no studies have clearly verified the main cause of this individual variability. In this study, the main hypothesis was that individual variability in tDCS effects might be partly explained by anatomical differences among subjects. To verify our hypothesis, we investigated the relationship between the behavioral outcomes of a verbal working memory (WM) task and current density values at the dorsolateral prefrontal cortex (DLPFC) simulated using the finite element method (FEM). A 3-back verbal working memory task experiment was conducted in 17 healthy subjects before and after tDCS with cathode and anode electrodes located at the right supraorbital and F3 locations, respectively. The results showed that participants who showed evidence of enhanced WM task performance after tDCS had a significantly larger current density at the DLPFC than other participants, suggesting that inconsistent behavioral outcomes of tDCS might be partly due to individual anatomical differences.

Aberrant JAK2 signalling plays a central role in myeloproliferative neoplasms (MPN). JAK2 inhibitors have proven to be clinically efficacious, however, they are not mutation-specific and competent enough to suppress neoplastic clonal haematopoiesis. We hypothesized that, by simultaneously targeting multiple activated signalling pathways, MPN could be more effectively treated. To this end we investigated the efficacy of BEZ235, a dual PI3K/mTOR inhibitor, alone and in combination with the JAK1/JAK2 inhibitor ruxolitinib, in different preclinical models of MPN. Single-agent BEZ235 inhibited the proliferation and induced cell cycle arrest and apoptosis of mouse and human JAK2V617F mutated cell lines at concentrations significantly lower than those required to inhibit the wild-type counterpart, and preferentially prevented colony formation from JAK2V617F knock-in mice and patients' progenitor cells compared with normal ones. Co-treatment of BEZ235 and ruxolitinib produced significant synergism in all these in-vitro models. Co-treatment was also more effective than single drugs in reducing the extent of disease and prolonging survival of immunodeficient mice injected with JAK2V617F-mutated Ba/F3-EPOR cells and in reducing spleen size, decreasing reticulocyte count and improving spleen histopathology in conditional JAK2V617F knock-in mice. In conclusion, combined inhibition of PI3K/mTOR and JAK2 signalling may represent a novel therapeutic strategy in MPN.

Endocrine-disrupting chemicals (EDCs), among which methoxychlor (MXC), have been reported to affect the male reproductive system. This study evaluates the possible deleterious effects of MXC on imprinted genes. After administration of the chemical in adult male mice or in pregnant mice we analyzed by pyrosequencing possible methylation defects in two paternally imprinted (H19 and Meg3 (Gtl2)) and three maternally imprinted (Mest (Peg1), Snrpn, and Peg3) genes in the sperm and in the tail, liver, and skeletal muscle DNAs of the adult male mice and of the male offspring. MXC treatment of adult mice decreased the percentages of methylated CpGs of Meg3 and increased those of Mest, Snrpn, and Peg3 in the sperm DNA. MXC treatment of pregnant mice decreased the mean sperm concentrations by 30% and altered the methylation pattern of all the imprinted genes tested in the F1 offspring. In the latter case, MXC effects were transgenerational but disappeared gradually from F1 to F3. MXC did not affect imprinting in the somatic cells, suggesting that it exerts its damaging effects via the process of reprogramming that is unique to gamete development. A systematic analysis at the CpG level showed a heterogeneity in the CpG sensitivity to MXC. This observation suggests that not only DNA methylation but also other epigenetic modifications can explain the transgenerational effects of MXC. The reported effects of EDCs on human male spermatogenesis might be mediated by complex imprinting alterations analogous to those described in this study.

Interactions between the dual Bcr/Abl and aurora kinase inhibitor MK-0457 and the histone deacetylase inhibitor vorinostat were examined in Bcr/Abl(+) leukemia cells, including those resistant to imatinib mesylate (IM), particularly those with the T315I mutation. Coadministration of vorinostat dramatically increased MK-0457 lethality in K562 and LAMA84 cells. Notably, the MK-0457/vorinostat regimen was highly active against primary CD34(+) chronic myelogenous leukemia (CML) cells and Ba/F3 cells bearing various Bcr/Abl mutations (ie, T315I, E255K, and M351T), as well as IM-resistant K562 cells exhibiting Bcr/Abl-independent, Lyn-dependent resistance. These events were associated with inactivation and down-regulation of wild-type (wt) and mutated Bcr/Abl (particularly T315I). Moreover, treatment with MK-0457 resulted in accumulation of cells with 4N or more DNA content, whereas coadministration of vorinostat markedly enhanced aurora kinase inhibition by MK-0457, and preferentially killed polyploid cells. Furthermore, vorinostat also interacted with a selective inhibitor of aurora kinase A and B to potentiate apoptosis without modifying Bcr/Abl activity. Finally, vorinostat markedly induced Bim expression, while blockade of Bim induction by siRNA dramatically diminished the capacity of this agent to potentiate MK-0457 lethality. Together, these findings indicate that vorinostat strikingly increases MK-0457 activity against IM-sensitive and -resistant CML cells through inactivation of Bcr/Abl and aurora kinases, as well as by induction of Bim.

T cell epitopes coupled to a lipid moiety (lipopeptides) may be superior immunostimulants compared to peptide antigens and are currently studied as potential vaccines. The cause of enhanced immunogenicity of lipopeptides is largely unknown but members of the novel family of Toll like receptors (TLR) such as TLR2 and TLR4 have been shown to mediate activation of cells in response to bacterial lipopolysaccharide (LPS) and other lipidated bacterial or viral components. We studied TLR-mediated activation by 14 synthetic lipopeptides corresponding to T cell epitopes on hepatitis C virus (HCV) core in human embryonic kidney cells (HEK293) transiently over-expressing TLR2 and in Ba/F3 mouse bone marrow cells stably transfected with TLR4 and the adaptor molecule MD-2. Stimulation of transfected HEK293 or Ba/F3 cells was measured via luciferase activity as a reporter of nuclear factor kappaB activation. Free peptides, a non-HCV-related lipopeptide as well as LPS and the lipopeptide SK4 were used as controls. Ten of the 14 HCV core lipopeptides stimulated luciferase activity in TLR2-transfected HEK293 cells but not in mock-transfected control cells. Nine of the 14 lipopeptides also stimulated luciferase activity in the TLR4/MD-2 double-transfected Ba/F3 cells but not Ba/F3 control cells. Overall, there was a close statistical correlation between TLR2 and TLR4/MD-2-mediated cell activation by the lipopeptides. In contrast, the corresponding free peptides had no stimulatory effect on TLR2 nor on TLR4/MD-2 transfected cells. Thus, lipopeptides but not their corresponding free peptides can activate cells via TLRs 2 and 4. This activation is apparently affected by the amino acid sequence of the peptide moiety.

FibroTest (FT) is a biomarker of liver fibrosis initially validated in patients with chronic hepatitis C (CHC) and subsequently assessed in other frequent liver diseases, including chronic hepatitis B (CHB), alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD). The primary aim of the present study was to update a previous meta-analysis of FT diagnostic value, and to summarize its advantages and limitations. The secondary aim was to provide an overview of the prognostic value of FT in CHC, CHB and ALD. For diagnostic value, the main endpoint was the FT area under the ROC curves (AUROCs) for the diagnosis of bridging fibrosis (F2/F3/F4 vs F0/F1), standardized for the spectrum of fibrosis. Sensitivity analysis integrated the non-standardized observed AUROCs, the independency of authors, size (length) of biopsy, prospective design, correctness of procedures, co-morbidities, and timelag between biopsy and serum sampling. For prognostic value, the main endpoint was the FT AUROC for the prognostic value of liver complications or death related to liver disease. A total of 38 diagnostic studies were included, which pooled 7985 subjects who had undergone both FT and biopsy (4600 HCV, 1580 HBV, 267 NAFLD, 524 ALD and 1014 mixed). The mean standardized AUROC was 0.84 (95% CI, 0.83-0.86), with no differences in terms of causes of liver disease: HCV 0.84 (0.82-0.87); HBV 0.81 (0.78-0.83); NAFLD 0.84 (0.76-0.92); ALD 0.87 (0.82-0.92); and mixed 0.85 (0.81-0.89). Three prognostic studies were also included. FT was found to have higher or similar prognostic value compared with biopsy in patients with CHC, CHB or ALD. FibroTest is an effective alternative to biopsy in patients with chronic hepatitis C or B, ALD or NAFLD. Indeed, the prognostic performance of FibroTest was at least as accurate as that of biopsy in patients with chronic hepatitis C or B, or ALD.

BACKGROUND

The long-term efficacy and safety of endoscopic injection of N-butyl-2-cyanoacrylate (Histoacryl) were evaluated as the initial treatment for bleeding gastric varices.

METHODS

Fifty-two patients with bleeding gastric varices underwent endoscopic injections of Histoacryl for hemostasis over a 10-year period. Histoacryl was injected intravariceally. Among these 52 patients, 32 had active bleeding and 20 had recent bleeding. Most of the varices were large (F2 or F3, 48 cases). After Histoacryl injection, the patients were followed endoscopically with retreatment administered as necessary. The patients were followed for a mean 28.1 months.

RESULTS

The rate of initial hemostasis (no bleeding occurred for 48 hours after sclerotherapy) was 96.2%. Cumulative nonbleeding rates were 64.7%, 52.7%, and 48.2% at 1, 5, and 10 years, respectively. When rebleeding occurred, 80.0% was within 1 year after initial injection. Recurrent bleeding was easily stopped with the reinjection of Histoacryl in most patients. The treatment failure-related mortality rate was 4.0% (2 of 52). The cumulative survival rates were 66.9%, 60.4%, and 55.5% at 1, 5, and 10 years, respectively. The mortality depended on either malignancy or liver function (Child-Pugh classification).

CONCLUSIONS

These results suggest that Histoacryl injection sclerotherapy is highly effective for the treatment of bleeding gastric varices, with rare complications occurring both acutely and long-term. Therefore, Histoacryl injection sclerotherapy is considered to be the first choice of treatment for bleeding gastric varices, but the rate of recurrent bleeding is so high that further methods or devices still need to be developed in order to prevent gastric variceal rebleeding.

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.

By contrast to well-defined Fc gamma and Fc epsilon receptors, the structural and functional characteristics of Fc mu receptor are unclear. We have recently described a novel mouse Fc receptor, designated Fc alpha/mu receptor, and its human homologue, which bind both IgM and IgA. Here we show that the Fc alpha/mu receptor is expressed on mature, but not immature, B lymphocytes and acquires the ability to bind IgM and IgA antibodies after stimulation of B lymphocytes. Moreover, stimulation with phorbol 12-myristate 13-acetate increased endocytosis of IgM-coated microparticles mediated by the Fc alpha/mu receptor expressed on pro-B cell line Ba/F3 cells. We also show that the Fc alpha/mu receptor is expressed in secondary lymphoid organs, such as lymph node and appendix, kidney and intestine, suggesting an important role of the receptor for immunity in these organs.

In cortical neurons differentiating in vitro, transmembrane amyloid precursor protein (APP) is distributed in two pools. Whereas the first pool is present in all cell compartments, the second pool is highly enriched in the axon and cell body. In an earlier study we demonstrated that this second pool, referred to as axonal-APP (Ax-APP), is present in the vicinity of the plasma membrane and colocalizes only partially with clathrin (Allinquant, B., Moya, K.L., Bouillot, C., and Prochiantz, A. (1994) J. Neurosci. 14, 6842-6854). In this report, using immunocytochemical and fractionation techniques we demonstrate that Ax-APP is present in microdomains enriched in the glypiated glycoprotein F3. The F3/Ax-APP microdomains are resistant to nonionic detergents and sediment at low density on a sucrose gradient. The two latter properties are reminiscent of those of caveolae, a type of plasmalemmal vesicle found in several cell types, but not previously described in the nervous system due to the absence of caveolin in neurons. The presence of Ax-APP in caveolae-like vesicles raises the possibility that APP serves as a transmembrane signaling molecule for GPI-linked glycoproteins. In addition, our data support new hypotheses on the endocytic pathways leading to the production of the amyloidogenic betaA4 peptide.

Signaling via the fibroblast growth factor receptor 1 (FGFR1, flg) was analyzed in Ba/F3 hematopoietic cells expressing either wild-type or a mutant FGF receptor (Y766F) unable to activate phospholipase C-gamma (PLC-gamma) and stimulate phosphatidylinositol (PI) hydrolysis. Stimulation of cells expressing wild-type or mutant FGFR with acidic FGF (aFGF) caused similar activation of Ras. However, an approximately 3-fold reduced activation of Raf-1 and MAP kinase was observed in aFGF-stimulated cells expressing mutant receptors as compared to cells expressing wild-type FGF receptors. Comparison of phosphopeptide maps of Raf-1 immunoprecipitated from the two cell types activated by either aFGF or the phorbol ester (12-O-tetradecanoylphorbol-13-acetate) suggests that Raf-1 is phosphorylated by both Ras-dependent and PLC-gamma-dependent mechanisms. In spite of the differential effect on Raf-1 and MAP kinase activation, aFGF stimulated similar proliferation of cells expressing wild-type or mutant receptors indicating that Ras-dependent activation of Raf-1 and MAP kinase is sufficient for transduction of FGFR mitogenic signals. Ras may also activate signal transduction pathways that are complementary or parallel to the MAP kinase pathway to stimulate cell proliferation.

Degradation of BCR-ABL oncoproteins by heat shock protein 90 (Hsp90) inhibitors in chronic myelogenous leukemia is expected to overcome resistance to ABL tyrosine kinase inhibitors. However, the precise mechanisms still remain to be uncovered. We found that while c-Cbl E3 ligase induced ubiquitin-dependent degradation of mature and phosphorylated BCR-ABL proteins, another E3 ligase CHIP (carboxyl terminus of the Hsc70-interacting protein) degraded immature BCR-ABL proteins and efficiently suppressed BCR-ABL-dependent leukemic growth. Interestingly, Bag1 (Bcl-2-associated athanogene-1), a nucleotide exchange factor for Hsc70, directly bound BCR-ABL with a high affinity, which was enhanced by CHIP and Hsp90 inhibitors, inhibited by imatinib and competed with Hsc70. Bag1 knockdown abrogated Hsp90 inhibitor-induced BCR-ABL degradation. Bag1 induced binding of immature BCR-ABL to proteasome. Expression of Bag1 induced BCR-ABL degradation and growth suppression in Ba/F3 cells when Hsc70 was knocked down with or without CHIP induction. CHIP appears to sort newly synthesized Hsp90-unchaperoned BCR-ABL to the proteasome not only by inhibiting Hsc70 and thereby promoting Bag1 to bind BCR-ABL, but also by ubiquitinating BCR-ABL. Bag1 may direct CHIP/Hsc70-regulated protein triage decisions on BCR-ABL immediately after translation to the degradation pathway.

Lipid rafts are regions of the plasma membrane that are enriched in cholesterol, glycosphingolipids and acylated proteins, and which have been proposed as sites for the proteolytic processing of the Alzheimer's amyloid precursor protein (APP). Lipid rafts can be isolated on the basis of their insolubility in Triton X-100 at 4 degrees C, with the resulting low-density, detergent-insoluble glycolipid-enriched fraction (DIG) being isolated by flotation through a sucrose density gradient. The detergent-insolubility of APP in mouse cerebral cortex relative to a variety of DIG marker proteins (alkaline phosphatase, flotillin, F3 protein and prion protein) and non-DIG proteins (alkaline phosphodiesterase I, aminopeptidase A and clathrin) has been examined. Alkaline phosphatase, flotillin, F3 protein and the prion protein were present exclusively in the DIG region of the sucrose gradient over a range of protein/detergent ratios used to solubilize the membranes and displayed a characteristic enrichment in the low-density fraction as the protein/detergent ratio was decreased. In contrast, most of the APP, alkaline phosphodiesterase I, aminopeptidase A and clathrin was effectively solubilized at all of the protein/detergent ratios examined. However, a minor proportion of these latter proteins was detected in DIGs at levels which remained constant irrespective of the protein/detergent ratio. When DIGs were isolated from the sucrose gradients and treated with excess Triton X-100, both the DIG marker proteins and APP, alkaline phosphodiesterase I and clathrin were predominantly resistant to detergent extraction at 37 degrees C. These results show that, although a minor proportion of APP is present in DIGs, where it is detergent-insoluble even at 37 degrees C, it behaves as an atypical lipid raft protein and raises questions as to whether lipid rafts are a site for its proteolytic processing.

The erythropoietin receptor (EpR) belongs to a family of hematopoietin receptors whose members lack tyrosine kinase activity. Nonetheless, within minutes of binding Ep, a number of cellular proteins become transiently phosphorylated on tyrosine residues. One of these proteins, as we and others have shown previously, is the EpR itself. To identify the remaining protein substrates, we have examined the antiphosphotyrosine immunoprecipitates of lysates from Ba/F3 cells expressing high levels of cell surface EpRs. We now present data showing that, in response to Ep, the 85-Kd regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) becomes immunoprecipitable with antiphosphotyrosine antibodies. This appears to be due, in large part, to the specific association of PI 3-kinase with the tyrosine-phosphorylated EpR, either directly or through a 93- or 70-Kd tyrosine-phosphorylated intermediate. The activity of this EpR associated PI 3-kinase, assessed in anti-EpR immunoprecipitates, is maximal within 2 minutes of incubation with Ep and returns almost to baseline levels by 10 minutes. In vitro studies suggest that the interaction between PI 3-kinase and the activated EpR is mediated by the N- and C-terminal SH2 domains of p85 and tyrosine-phosphorylated motifs on the EpR.

Plant actins are involved in numerous cytoskeletal processes effecting plant development, including cell division plane determination, cell elongation, and cell wall deposition. Arabidopsis thaliana has five ancient subclasses of actin with distinct patterns of spatial and temporal expression. To test their functional roles, we identified insertion mutants in three Arabidopsis actin genes, ACT2, ACT4, and ACT7, representing three subclasses. Adult plants homozygous for the act2-1, act4-1, and act7-1 mutant alleles appear to be robust, morphologically normal, and fully fertile. However, when grown as populations descended from a single heterozygous parent, all three mutant alleles were found at extremely low frequencies relative to the wild-type in the F2 generation. Thus, all three mutant alleles appear to be deleterious. The act2-1 mutant allele was found at normal frequencies in the F1, but at significantly lower frequencies than expected in the F2 and F3 generations. These data suggest that the homozygous act2-1/act2-1 mutant adult plants have a reduced fitness in the 2N sporophytic portion of the life cycle, consistent with the vegetative expression of ACT2. These data are interpreted in light of the extreme conservation of plant actin subclasses and genetic redundancy.

The Meyer-Overton hypothesis, predicting that the potency of an anesthetic correlates with its affinity for lipid, is a cornerstone of modern anesthetic theory. Several halogenated compounds were recently found to deviate from this prediction, whereas others did not. We tested the abilities of enflurane and five of these compounds to potentiate gamma-aminobutyric acid (GABA)A receptor responses in Xenopus oocytes expressing alpha 1 beta 2 or alpha 1 beta 2 gamma 2S GABAA receptors. Enflurane and the anesthetic 1-chloro-1,2,2-trifluorocyclobutane (F3) strongly potentiated chloride currents produced by 5 microM GABA with both alpha 1 beta 2 and alpha 1 beta 2 gamma 2S receptors. This potentiation decreased as the GABA concentration was raised. The transitional compound (less potent than predicted by its lipid solubility) 2-bromoheptafluoropropane produced modest enhancement, whereas three nonanesthetics (neither causing anesthesia in vivo nor decreasing the requirement for known anesthetics), 1,2-dichlorohexafluorocyclobutane, 2-chloroheptafluoropropane, and 2,3-chlorooctafluorobutane, did not affect GABAA receptor currents. Although all five compounds were predicted to be anesthetics by the Meyer Overton hypothesis, only F3 behaved as an anesthetic in vivo and only F3 markedly potentiated GABAA receptor responses in oocytes. These results strongly implicate the GABAA receptor in general anesthesia. Fluorescence polarization studies showed that anesthetics (enflurane and F3), but not nonasthetics (1,2 dichlorohexafluorocyclobutane and 2,3-chlorooctafluorobutane) disordered membrane lipids. Thus, for the compounds studied actions on both GABAA receptor function and lipid order distinguish between anesthetics and nonanesthetics.

We document the protocols and methods for the production of immortalized cell lines of human neural stem cells from the human fetal central nervous system (CNS) cells by using a retroviral vector encoding v-myc oncogene. One of the human neural stem cell lines (HB1.F3) was found to express nestin and other specific markers for human neural stem cells, giving rise to three fundamental cell types of the CNS: neurons, astrocytes, and oligodendrocytes. After transplantation into the brain of mouse model of stroke, implanted human neural stem cells were observed to migrate extensively from the site of implantation into other anatomical sites and to differentiate into neurons and glial cells.

BACKGROUND

Few data are available on chronic hepatitis C (CHC) in elderly patients. The aim of this study was to compare the features and severity of CHC and the efficacy/safety of antiviral therapy in patients<65, between 65 and 80, and >80 yr old, and to determine the usefulness of biochemical markers (Fibrotest-Fibrosure/ActiTest [FT-AT]) in aged patients.

METHODS

This was a retrospective study with two groups of patients: Group 1: prospective cohort including all hepatitis C virus patients from our institution (N=4,182); Group 2: all consecutive patients who had FT-AT performed in France between 2002 and 2004 (N=33,738).

RESULTS

A total of 6,865 patients>or=65 yr old was included (Group 1=881, Group 2=5,984). Group 1: patients>or=65 had a longer duration of and a higher age at infection, more genotype 1, and a history of transfusion (p<0.001). Among the 2,169 patients who underwent liver biopsy, bridging fibrosis (F2,F3,F4) was more frequent in patients>or=65 yr old, regardless of the duration of infection. In multivariate analysis, ages at biopsy and at infection were associated with F2,F3,F4. Discovery of CHC by a complication was more frequent in patients>or=65 yr (p<0.001). One hundred seventy patients>or=65 yr received antiviral therapy. A sustained virologic response was obtained in 45% of patients>or=65 yr treated with pegylated interferon/ribavirin. Group 2: At FT, 58% of patients>80 yr, 37% of patients between 65 and 80 yr, and 14% of patients<65 yr (p<0.001) had cirrhosis. Patients>80 yr (43%) with cirrhosis had nonelevated alanine amino transferase (ALT), compared with 31% of patients<65 yr (p<0.001).

CONCLUSIONS

In patients>or=65 yr, CHC is more severe and presents with lower ALT than in younger patients. Treatment is effective. Biochemical markers seem particularly useful as a noninvasive alternative to liver biopsy in this population.

Systemic mastocytosis (SM) is a myeloid neoplasm involving mast cells (MCs) and their progenitors. In most cases, neoplastic cells display the D816V-mutated variant of KIT. KIT D816V exhibits constitutive tyrosine kinase (TK) activity and has been implicated in increased survival and growth of neoplastic MCs. Recent data suggest that the proapoptotic BH3-only death regulator Bim plays a role as a tumor suppressor in various myeloid neoplasms. We found that KIT D816V suppresses expression of Bim in Ba/F3 cells. The KIT D816-induced down-regulation of Bim was rescued by the KIT-targeting drug PKC412/midostaurin. Both PKC412 and the proteasome-inhibitor bortezomib were found to decrease growth and promote expression of Bim in MC leukemia cell lines HMC-1.1 (D816V negative) and HMC-1.2 (D816V positive). Both drugs were also found to counteract growth of primary neoplastic MCs. Furthermore, midostaurin was found to cooperate with bortezomib and with the BH3-mimetic obatoclax in producing growth inhibition in both HMC-1 subclones. Finally, a Bim-specific siRNA was found to rescue HMC-1 cells from PKC412-induced cell death. Our data show that KIT D816V suppresses expression of proapoptotic Bim in neoplastic MCs. Targeting of Bcl-2 family members by drugs promoting Bim (re)-expression, or by BH3-mimetics such as obatoclax, may be an attractive therapy concept in SM.

Chronic myeloid leukemia (CML) is a myeloproliferative disease in which BCR/ABL enhances survival of leukemic cells through modulation of proapoptotic and antiapoptotic molecules. Recent data suggest that proapoptotic Bcl-2-interacting mediator (Bim) plays a role as a tumor suppressor in myeloid cells, and that leukemic cells express only low amounts of this cell death activator. We here show that primary CML cells express significantly lower amounts of bim mRNA and Bim protein compared with normal cells. The BCR/ABL inhibitors imatinib and AMN107 were found to promote expression of Bim in CML cells. To provide direct evidence for the role of BCR/ABL in Bim modulation, we employed Ba/F3 cells with doxycycline-inducible expression of BCR/ABL and found that BCR/ABL decreases expression of bim mRNA and Bim protein in these cells. The BCR/ABL-induced decrease in expression of Bim was found to be a posttranscriptional event that depended on signaling through the mitogen-activated protein kinase pathway and was abrogated by the proteasome inhibitor MG132. Interestingly, MG132 up-regulated the expression of bim mRNA and Bim protein and suppressed the growth of Ba/F3 cells containing wild-type BCR/ABL or imatinib-resistant mutants of BCR/ABL. To show functional significance of "Bim reexpression," a Bim-specific small interfering RNA was applied and found to rescue BCR/ABL-transformed leukemic cells from imatinib-induced cell death. In summary, our data identify BCR/ABL as a Bim suppressor in CML cells and suggest that reexpression of Bim by novel tyrosine kinase inhibitors, proteasome inhibition, or by targeting signaling pathways downstream of BCR/ABL may be an attractive therapeutic approach in imatinib-resistant CML.

Some tumors are dependent on the continued activity of a single oncogene for maintenance of their malignant phenotype. The best-studied example is the Bcr-Abl fusion protein in chronic myelogenous leukemia (CML). Although the clinical success of the Abl kinase inhibitor imatinib against chronic-phase CML emphasizes the importance of developing therapeutic strategies aimed at this target, resistance to imatinib poses a major problem for the ultimate success of CML therapy by this agent. We hypothesized a sequential blockade strategy that is designed to decrease the expression of the Bcr-Abl protein, with the goal of complementing the action of imatinib on kinase activity. In this study, flavopiridol, an inhibitor of transcription, homoharringtonine (HHT), a protein synthesis inhibitor, and imatinib were used singly and in combination against the Bcr-Abl-positive human CML cell line K562. Flavopiridol alone inhibited phosphorylation of the RNA polymerase II COOH-terminal domain, specifically reduced RNA polymerase II-directed mRNA synthesis, and decreased the Bcr-Abl transcript levels. HHT inhibited protein synthesis and reduced the Bcr-Abl protein level. Imatinib directly inhibited the kinase activity of Bcr-Abl. The combinations of flavopiridol and HHT and flavopiridol and imatinib synergistically decreased clonogenicity as evaluated by the median-effect method. Greater synergy was observed when HHT and imatinib were given sequentially compared with simultaneous administration. Imatinib-resistant Ba/F3 cells that were transfected to express the E255K and T315I mutations of Bcr-Abl were not cross-resistant to flavopiridol and HHT. These results provided a rationale for the combination of inhibitors of transcription and/or translation with specific kinase inhibitors.