Mutant isocitrate dehydrogenase 1 (IDH1) is common in gliomas, and produces D-2-hydroxyglutarate (D-2-HG). The full effects of IDH1 mutations on glioma biology and tumor microenvironment are unknown. We analyzed a discovery cohort of 169 World Health Organization (WHO) grade II-IV gliomas, followed by a validation cohort of 148 cases, for IDH1 mutations, intratumoral microthrombi, and venous thromboemboli (VTE). 430 gliomas from The Cancer Genome Atlas were analyzed for mRNAs associated with coagulation, and 95 gliomas in a tissue microarray were assessed for tissue factor (TF) protein. In vitro and in vivo assays evaluated platelet aggregation and clotting time in the presence of mutant IDH1 or D-2-HG. VTE occurred in 26-30 % of patients with wild-type IDH1 gliomas, but not in patients with mutant IDH1 gliomas (0 %). IDH1 mutation status was the most powerful predictive marker for VTE, independent of variables such as GBM diagnosis and prolonged hospital stay. Microthrombi were far less common within mutant IDH1 gliomas regardless of WHO grade (85-90 % in wild-type versus 2-6 % in mutant), and were an independent predictor of IDH1 wild-type status. Among all 35 coagulation-associated genes, F3 mRNA, encoding TF, showed the strongest inverse relationship with IDH1 mutations. Mutant IDH1 gliomas had F3 gene promoter hypermethylation, with lower TF protein expression. D-2-HG rapidly inhibited platelet aggregation and blood clotting via a novel calcium-dependent, methylation-independent mechanism. Mutant IDH1 glioma engraftment in mice significantly prolonged bleeding time. Our data suggest that mutant IDH1 has potent antithrombotic activity within gliomas and throughout the peripheral circulation. These findings have implications for the pathologic evaluation of gliomas, the effect of altered isocitrate metabolism on tumor microenvironment, and risk assessment of glioma patients for VTE.

Exercise has been widely related to changes in cortical activation and enhanced brain functioning. Quantitative electroencephalography (qEEG) is frequently used to investigate normal and pathological conditions in the brain cortex. Therefore, the aim of the present study was to observe absolute power alterations in beta and alpha frequency bands after a maximal effort exercise. Ten healthy young volunteers were submitted to an eight-minute resting EEG (eyes closed) followed by a maximal exercise test using a mechanical cycle ergometer. Immediately after the exercise, another identical eight-minute EEG was recorded. Log transformation and paired student's t-test compared the pre and post exercise values (p<0.05). Results indicated a significant absolute power increase in beta after exercise at frontal (Fp1, F3 and F4) and central (C4) areas, which might be related to increased cortical activation.

Our previous studies indicated that stromal cell-derived syndecan-4 might mediate some form of communication with pre-B cells in bone marrow. We now report additional aspects of this recognition and show that syndecan-4 is also present on pre-B cells. Indeed, the molecule is acquired at an early stage of differentiation and retained until mature B cells undergo Ig isotype switching. mAbs developed to two portions of the syndecan-4 protein core were used to probe possible functions on B lineage lymphocytes. Syndecan-4 ligation had no obvious influence on B lymphocyte formation or activation, but this treatment caused a dramatic morphological change in appropriately stimulated leukocytes. Extended filopodia appeared on transfected Ba/F3 or FDCP-1 cells, as well as activated B cell blasts that were placed on syndecan-4 Ab-coated surfaces. The dendritic processes contained polymerized actin as well as pp52(LSP1), a prominent F-actin binding protein in lymphocytes. The cytoplasmic domain of syndecan-4 was not required for this response. Shape changes of this type could facilitate interactions between B lymphocytes and other components of the immune system. Not only is syndecan-4 a useful marker for discriminating normal B lineage lymphocyte subsets, but our results suggest new ways for the syndecans to participate in immune responses.

To develop the first heart-specific tetracycline (Tet)-On system in zebrafish, we constructed plasmids in which the cardiac myosin light chain 2 promoter of zebrafish was used to drive the reverse Tet-controlled transactivator (rtTA) and the green fluorescent protein (GFP) reporter gene was preceded by an rtTA-responsive element. In the zebrafish fibroblast cell-line, rtTA-M2, one of rtTA's derivatives, demonstrated the highest increase in luciferase activity upon doxycycline (Dox) induction. We then generated two germ lines of transgenic zebrafish: line T03 was derived from microinjection of a plasmid containing rtTA-M2 and a plasmid containing a responsive reporter gene, whereas line T21 was derived from microinjection of a single dual plasmid. Results showed that line T21 was superior to line T03 in terms of greater GFP intensity after induction and with of minimal leakiness before induction. The photographic images of induced GFP in the heart of F2 larvae showed that the fluorescent level of GFP was dose-responsive. The level of GFP expressed in the F3 3 days postfertilization larvae that were treated with Dox for 1 hr decreased gradually after the withdrawal of the inducer; and the fluorescent signal disappeared after 5 days. The GFP induction and reduction were also tightly controlled by Dox in the F3 adult fish from line T21. This Tet-On system developed in zebrafish shows much promise for the study of the gene function in a specific tissue at the later developmental stage.

Chronic myeloid leukemia (CML) is a malignant stem cell disease characterized by an expansion of myeloid progenitor cells expressing the constitutively activated Bcr-Abl kinase. This oncogenic event causes a deregulation of apoptosis and cell cycle progression. Although the molecular mechanisms protecting from apoptosis in CML cells are well characterized, the cell cycle regulatory event is poorly understood. An inhibitor of the cyclin-dependent kinases, p27, plays a central role in the regulation of growth factor dependent proliferation of hematopoietic cells. Therefore, we have analyzed the influence of Bcr-Abl in the regulation of p27 expression in various hematopoietic cell systems. An active Bcr-Abl kinase causes down-regulation of p27 expression in murine Ba/F3 cells and human M07 cells. Bcr-Abl blocks up-regulation of p27 after growth factor withdrawal and serum reduction. In addition, p27 induction by transforming growth factor-beta (TGF-beta) is completely blocked in Bcr-Abl positive M07/p210 cells. This deregulation is directly mediated by the activity of the Bcr-Abl kinase. A Bcr-Abl kinase inhibitor completely abolishes p27 down-regulation by Bcr-Abl in both Ba/F3 cells transfected either with a constitutively active Bcr-Abl or with a temperature sensitive mutant. The down-regulation of p27 by Bcr-Abl depends on proteasomal degradation and can be blocked by lactacystin. Overexpression of wild-type p27 partially antagonizes Bcr-Abl-induced proliferation in Ba/F3 cells. We conclude that Bcr-Abl promotes cell cycle progression and activation of cyclin-dependent kinases by interfering with the regulation of the cell cycle inhibitory protein p27. (Blood. 2000;96:1933-1939)

OBJECTIVE

Chronic hepatitis C is an insidiously progressive disease, in which repeated assessment of liver histology is required. Various serum fibrotic markers have now been introduced. Our present aim was to assess, by receiver operating characteristic analysis, the usefulness of serum fibrotic markers for diagnosing fibrotic staging and necroinflammatory grading in chronic hepatitis C.

METHODS

Serum levels of procollagen type III N-terminal peptide (PIIINP), 7S fragment of type IV collagen (PIVNP), hyaluronan (HA), matrix metalloproteinase (MMP)-1, MMP-2, and tissue inhibitor of metalloproteinases (TIMP)-1 were measured in 169 patients with chronic hepatitis C.

RESULTS

The accuracy of these tests for discriminating stages greater than F2 from stages less than F1 was superior to that for discriminating stage F3 from stages less than F2. The most useful test for predicting stages greater than F2 was the serum HA test (cutoff value, 50 ng/ml; sensitivity, 75%; specificity, 80%), and the next-most useful was the serum MMP-2 test (cutoff value, 550 ng/ml; sensitivity, 75%; specificity, 70%). The usefulness of these tests for discriminating moderate grade from grades less than mild was superior to that for discriminating grades more than mild from minimal grade. The most useful test for predicting moderate grade was the serum HA test (cutoff value, 60 ng/ml; sensitivity, 77%; specificity, 74%), and the second-most useful was the serum PIVNP test (cutoff value, 6.5 ng/ml: sensitivity, 74%; specificity, 75%). The combination of the most useful and next-most useful test results increased the accuracy of the diagnosis of staging and grading.

CONCLUSIONS

These serum fibrotic markers, especially the serum HA test, would be clinically useful for assessing staging and grading in patients with chronic hepatitis C.

OBJECTIVE

Liver stiffness measurement (LSM) and FibroTest (FT) are frequently used as non-invasive alternatives for fibrosis staging to liver biopsy. However, to date, diagnostic performances of Enhanced Liver Fibrosis (ELF) test, which consists of hyaluronic acid, aminoterminal propeptide of procollagen type-III, and tissue inhibitor of matrix metalloproteinases-1, have not been compared to those of LSM and FT in Asian chronic hepatitis B (CHB) patients.

METHODS

Between June 2010 and November 2011, we prospectively enrolled 170 CHB patients who underwent liver biopsies along with LSM, FT, and ELF. The Batts system was used to assess fibrosis stages.

RESULTS

Areas under receiver operating characteristic curves (AUROCs) to predict significant fibrosis (F≥2), advanced fibrosis (F≥3), and cirrhosis (F = 4) were 0.901, 0.860, and 0.862 for ELF, respectively; 0.937, 0.956, and 0.963 for LSM; and 0.896, 0.921, and 0.881 for FT. AUROCs to predict F≥2 were similar between each other, whereas LSM and FT had better AUROCs than ELF for predicting F≥3 (both p<0.05), and LSM predicted F4 more accurately than ELF (p<0.05). Optimized cutoffs of ELF to maximize sum of sensitivity and specificity were 8.5, 9.4, and 10.1 for F≥2, F≥3, and F = 4, respectively. Using suggested ELF, LSM and FT cutoffs to diagnose F1, F2, F3, and F4, 91 (53.5%), 117 (68.8%), and 110 (64.7%) patients, respectively, were correctly classified according to histological results.

CONCLUSIONS

ELF demonstrated considerable diagnostic value in fibrosis staging in Asian CHB patients, especially in predicting F≥2. However, LSM consistently provided better performance for predicting F≥3 and F4.

KIT exon 8 mutations are located in the extracellular portion of the receptor and are strongly associated with core-binding factor (CBF)-acute myeloid leukemia (AML). To characterize the functional role of these mutants, we analyzed the proproliferative and antiapoptotic potential of 3 KIT exon 8 mutations in interleukin 3 (IL-3)-dependent Ba/F3 cells. All KIT exon 8 mutants induced receptor hyperactivation in response to stem cell factor (SCF) stimulation in terms of proliferation and resistance toward apoptotic cell death. A representative KIT exon 8 mutant showed spontaneous receptor dimerization, phosphorylation of mitogen-activated protein kinase (MAPK), and conferred IL-3-independent growth to Ba/F3 cells. MAPK and phosphatidylinositol 3-kinase (PI3-kinase) activation was essential for the phenotype of this mutant. Additionally, imatinib inhibited proliferation of KIT exon 8 mutant-expressing Ba/F3 cells. Our data show that KIT exon 8 mutations represent gain-of-function mutations and might represent a new molecular target for treatment of CBF leukemias.

The stem cell factor receptor/c-Kit plays an important physiological role in hematopoiesis, melanogenesis, and gametogenesis. It has also been implicated in numerous human malignancies. Signal transduction pathways shown to be of importance for c-Kit-mediated transformation include the phosphoinositide 3-kinase (PI3K)/Akt pathway. We have previously shown that two alternative splice forms of c-Kit, denoted GNNK(-) and GNNK(+), mediate distinctively different signals. In this study, we found that in the hematopoietic cell line Ba/F3, GNNK(-) c-Kit mediates a substantially stronger activation of PI3K/Akt than GNNK(+) c-Kit. This difference in signaling was shown to be dependent on the association of the scaffolding protein Gab2 with c-Kit, and Src-mediated phosphorylation of Gab2 was shown to be to be independent of the direct association of PI3K with c-Kit. Furthermore, proliferation and survival of Ba/F3 cells expressing a mutant of c-Kit that fails to bind to PI3K directly were slightly decreased compared with wild-type c-Kit-expressing cells. Using small interfering RNA technology, we further verified a role of Gab2 in inducing activation of PI3K/Akt downstream of c-Kit. To summarize, we show that PI3K activation by c-Kit is both splice form-dependent and cell type-specific. Furthermore, activation of PI3K by c-Kit is dependent both on the direct PI3K-binding site in c-Kit and on the phosphorylation of Gab2. The fact that c-Kit has been found mutated in numerous human malignancies, including acute myeloid leukemia, and that Gab2 is often overexpressed in acute myeloid leukemia suggests a potential role of Gab2-mediated PI3K activation in transformation.

JAK3 mutations have been reported in transient myeloproliferative disorder (TMD) as well as in acute megakaryoblastic leukaemia of Down syndrome (DS-AMKL). However, functional consequences of the JAK3 mutations in TMD patients remain undetermined. To further understand how JAK3 mutations are involved in the development and/or progression of leukaemia in Down syndrome, additional TMD patients and the DS-AMKL cell line MGS were screened for JAK3 mutations, and we examined whether each JAK3 mutation is an activating mutation. JAK3 mutations were not detected in 10 TMD samples that had not previously been studied. Together with our previous report we detected JAK3 mutations in one in 11 TMD patients. Furthermore, this study showed for the first time that a TMD patient-derived JAK3 mutation (JAK3(I87T)), as well as two novel JAK3 mutations (JAK3(Q501H) and JAK3(R657Q)) identified in an MGS cell line, were activating mutations. Treatment of MGS cells and Ba/F3 cells expressing the JAK3 mutants with JAK3 inhibitors significantly decreased their growth and viability. These results suggest that the JAK3 activating mutation is an early event during leukaemogenesis in Down syndrome, and they provide proof-of-principle evidence that JAK3 inhibitors would have therapeutic effects on TMD and DS-AMKL patients carrying activating JAK3 mutations.

The expression of a strongly immunomodulatory mannoprotein complex (GMP) in the different forms of growth of the human commensal and opportunistic pathogen Candida albicans was studied using a monoclonal antibody (mAb AF1) directed against an oligosaccharide epitope of GMP. Immunofluorescence revealed that the surface of the yeast cells was highly reactive with mAb AF1, but that the reactivity was greatly reduced or disappeared during mycelial conversion. This modulation was shared by a number of strains of C. albicans, and was not solely a temperature- or nutrition-dependent phenomenon. Hypha-deficient strains (A12 and CA2) did not show variations of surface fluorescence under environmental conditions which were permissive for hyphal conversion (incubation in N-acetylglucosamine or Lee's medium, at 37 degrees C). GMP extracts from yeast and mycelial forms of the fungus were separated into three chromatographically distinct, high molecular mass mannoprotein fractions (F1, F2 and F3), which were tested individually by indirect ELISA for mAb AF1 recognition. All yeast-derived constituents and two (F2 and F3) of the hyphal mannoproteins were recognized by the mAb. The low or absent reactivity of the F1 constituent from hyphal cells was confirmed by immunoblots. Irrespective of their source (yeast or mycelial), all fractions reacted to a similar extent with a polyclonal anti-Candida serum. Overall, the data suggest changes in epitope specificity and/or confinement of reactive constituents in the inner wall layers as possible mechanisms of modulated expression of mAb AF1-reactive epitope during mycelial conversion.

The erythropoietin receptor (EPO-R) is synthesized in transfected Ba/F3 cells as a major 64-kDa endoglycosidase H (Endo H)-sensitive species, with a single N-linked oligosaccharide, and a minor 62-kDa unglycosylated form. Approximately half of the newly made EPO-R is processed to a mature 66-kDa form with a Golgi-processed Endo H-resistant oligosaccharide, of which only a minor fraction is expressed at the cell surface. Both the Endo H-sensitive and the Endo H-resistant forms of the receptor have a half-life of 45-60 min (Yoshimura, A., D'Andrea, A. D., and Lodish, H. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4139-4143). The mature, Endo H-resistant form of the EPO-R appears to be degraded in lysosomes or in other acidic organelles, since receptor degradation is blocked by treatment with NH4Cl, chloroquine, or leupeptin. A fraction of the Endo H-resistant EPO-R molecules is cleaved, generating two fragments of 46 and 39 kDa. The sizes of these fragments and their reactivities with carboxyl-terminal-specific antibodies indicate that the receptor is cleaved at two sites in the exoplasmic domain, 7 kDa apart, and carboxyl-terminal to the N-glycosylation site. Both fragments are membrane anchored and are probably formed in a late or post-Golgi compartment, since their formation is blocked by incubation of cells at 20 degrees C or by incubation with brefeldin A. These membrane-anchored COOH-terminal fragments are probably degraded in lysosomes or in other acidic vesicles as cell fractionation demonstrates that they colocalize with lysosomes, and similar to the intact EPO-R, their degradation is inhibited by NH4Cl. Finally, double labeling immunofluorescence experiments demonstrate that in NH4Cl-treated cells both intact mature EPO-R and the 46- and 39-kDa fragments accumulate in lysosomes and presumably are normally degraded there. The sensitivity of the EPO-R to endoproteolytic cleavages in its exoplasmic domain may relate to its low surface expression and to its extreme metabolic instability.

Protective, lytic antibodies are believed to be correlated with active Trypanosoma cruzi infection. In patients with chronic infection, antibodies lysing trypomastigote forms recognize chiefly alpha-galactosyl structures at the parasite surface. The target molecules on cell-derived trypomastigotes that react with anti-alpha-galactosyl antibodies (anti-Gal) from patients with chronic Chagas disease were investigated. Glycoconjugates were isolated from trypomastigotes and shown to absorb purified Chagasic (Ch) anti-Gel effectively as well as lytic antibodies from Ch sera. Active fractions were F2 (74 kD and 95.6 kD) and F3 (120-200 kD). A differential reactivity with antibodies from untreated Ch patients (trypanolytic) and from treated, presumably cured, individuals (not trypanolytic) was evident using F2 and F3 antigenic fractions. No cross-reactivity with heterologous sera (other infections) was observed. The F2 glycoconjugate (mostly 74 kD) can be used in the diagnosis of active Chagas infection, replacing the quantitative determination of complement-mediated lysis. With the present sample of patients' sera and normal human sera, it showed 100% sensitivity and specificity.

OBJECTIVE

The aim of this study was to investigate the accuracy of noninvasive tests, e.g. serum fibrotic markers, transient elastography and real-time tissue elastography, in the diagnosis of hepatic fibrosis, and to determine whether they can replace liver biopsy.

METHODS

119 patients with chronic liver disease were included in this study. Serum fibrotic markers including hyaluronic acid, type IV collagen, type IV collagen 7S domain and type III procollagen-N-peptide were measured. Aspartate aminotransferase (AST) and platelet counts were also measured to calculate the AST to platelet ratio index (APRI). Liver stiffness was measured using FibroScan and real-time tissue elastography.

RESULTS

The fibrotic stage, determined by histopathological diagnosis of a liver biopsy sample, did not correlate as well with serum fibrotic markers although it was useful to diagnose liver cirrhosis. However, the stage of hepatic fibrosis correlated well with liver stiffness measured by FibroScan. FibroScan was also a much better predictor of liver cirrhosis than APRI. Furthermore, the levels of liver strain measured by real-time tissue elastography correlated well with liver stiffness (p < 0.05).

CONCLUSIONS

Serum fibrotic markers and FibroScan are useful for distinguishing liver cirrhosis (F4) from chronic hepatitis (F1-F3). In addition, real-time tissue elastography is a novel and promising method to determine the stage of hepatic fibrosis.

The cannabinoid CB1 receptor, a member of the Rhodopsin (Rho) family of G protein coupled receptors (GPCRs), exhibits high levels of constitutive activity. In contrast, Rho exhibits an exquisite lack of constitutive activity. In Rho, W6.48(265) on transmembrane helix 6 (TMH6) is flanked by aromatic residues at positions i-4 (F6.44) and i + 3 (Y6.51), while in CB1 the residues i-4 and i + 3 to W6.48 are leucines (L6.44 and L6.51). Based upon spectroscopic evidence, W6.48 has been proposed to undergo a rotamer switch (chi1 g+ ->>trans) upon activation of Rho. In the work reported here, the biased Monte Carlo method, Conformational Memories (CM) was used to test the hypothesis that the high constitutive activity exhibited by CB1 may be due, in part, to the lack of aromatic residues i-4 and i + 3 from W6.48. In this work, the W6.48 rotamer shift (chi1 g+ ->>trans) was used as the criterion for activation. Conformational Memories (CM) calculations on WT CB1 TMH6 and L6.44F and L6.51Y mutant TMH6s revealed that an aromatic residue at 6.44 tends to disfavor the W6.48 chi1 g+ ->>trans transition and an aromatic residue at 6.51 would require a concomitant movement of the Y6.51 chi1 from trans->>g+ when the W6.48 chi1 undergoes a g+ ->>trans shift. In contrast, CM calculations on WT CB1 TMH6 revealed that the presence of leucines at 6.44 and 6.51 provide W6.48 with greater conformational mobility, with a W6.48 transchi1 preferred. Conformational Memories calculations also revealed that the W6.48 chi1 g+ ->>trans transition in WT CB1 TMH6 is correlated with the degree of kinking in TMH6. The average proline kink angles for TMH6 were higher for helices with a W6.48 g+ chi1 than for those with a W6.48 transchi1. These results are consistent with experimental evidence that TMH6 straightens during activation. Transmembrane helix (TMH) bundle models of the inactive (R) and active (R*) states of CB1 were then probed for interactions that may constrain W6.48 in the inactive state of CB1. These studies revealed that F3.36 (transchi1) helps to constrain W6.48 in a g+ chi1 in the inactive (R) state of CB1. In the R* state, these studies suggest that F3.36 must assume a g+ chi1 in order to allow W6.48 to shift to a transchi1. These results suggest that the W6.48/F3.36 interaction may act as the 'toggle switch' for CB1 activation, with W6.48 chi1 g+/F3.36 chi1 trans representing the inactive (R) and W6.48 chi1 trans/F3.36 chi1 g+ representing the active (R*) state of CB1.

Bcr-Abl constitutes a deregulated tyrosine kinase involved in the pathogenesis of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL). Although activation of the transcription factor NF-kappaB/Rel has been demonstrated, mechanisms of NF-kappaB/Rel activation by Bcr-Abl remain obscure. In this paper we demonstrate activation of NF-kappaB/Rel by Bcr-Abl and for the first time by v-Abl. Furthermore, we investigated mechanisms of NF-kappaB/Rel induction by Bcr-Abl and v-Abl. Both Bcr-Abl and v-Abl induced NF-kappaB/Rel DNA binding in Ba/F3 cells. DNA binding was a result of nuclear translocation of p65/RelA, whereas p65/RelA expression was unaffected. Nuclear translocation of p65/RelA is at least partially due to increased IkappaBalpha degradation, which is independent of IkappaB kinase (IKK) activity. IKK activity is not deregulated by Bcr-Abl and v-Abl. NF-kappaB/Rel transactivation was dependent on abl kinase activity but independent of Grb2 and Grb10 binding tobcr sequences. In addition, NF-kappaB/Rel activation was dependent on Ras activity. Primary CML blasts showed constitutive p65/RelA NF-kappaB/Rel DNA binding activity. Thus NF-kappaB/Rel represents a potential target for molecular therapies in CML.

5-Fluorouracil (5-FU), 5-fluoro-2'-deoxyuridine (FdUrd) and 5-trifluorothymidine (F3(d)Thd) are antimetabolites which are metabolized to their corresponding active forms which inhibit DNA synthesis via inhibition of thymidylate synthase (TS). To investigate ways of overcoming 5-FU-resistance, we established acquired-resistant colorectal cancer cell lines against these three drugs by continuous and step-wise escalation of drugs, and analyzed the cytotoxicity and the mechanism of resistance to the drugs. When cells were incubated with the 3 drugs for 72 h, the resistance ratio to parental DLD-1 human colorectal tumor cells was 65.2 for DLD-1/5-FU, 9.7 for DLD-1/FdUrd and 448.6 for DLD-1/F3(d)Thd cells. DLD-1/5-FU cells did not show any cross-resistance against FdUrd and F(3)dThd. However, DLD-1/FdUrd cells showed 3- and 9-fold increased resistance to 5-FU and F3(d)Thd, respectively, and DLD-1/F3(d)Thd cells also showed about 90-fold resistance to FdUrd. Analysis of enzyme activities and gene expression associated with pyrimidine metabolism indicated that a significant decrease in orotate phosphoribosyltransferase activity in DLD-1/5-FU cells, a 7-fold increase of TS mRNA in DLD-1/FdUrd cells, and a 37-fold decrease in thymidine kinase activity of DLD-1/F3(d)Thd cells were the major mechanisms of drug resistance. These findings were closely associated with the cytotoxicity of 5-FU, FdUrd and F3(d)Thd against the established 5-FU-, FdUrd- or F3(d)Thd-resistant cells. When DLD-1/FdUrd cells expressing increased TS mRNA were treated with FdUrd and F3(d)Thd for only 4 h, the resistance ratios of DLD-1/FdUrd cells to parental DLD-1 cells were markedly different for FdUrd and F3(d)Thd, suggesting that the cytotoxicity with short-time exposure to F3(d)Thd is due to a mechanism other than TS inhibition, although the cytotoxicity of F3(d)Thd in the short-time is low compared to that of long-time exposure. In conclusion, F3(d)Thd, an antimetabolite that inhibits TS activity, may be effective against 5-FU and/or FdUrd-resistance in colorectal cancer cells caused by amplification of TS and/or deletion of orotate phosphoribosyltransferase.

BACKGROUND

Recent data suggest that the mammalian target of rapamycin (mTOR) is involved in the regulation of growth of neoplastic cells in chronic myeloid leukaemia (CML).

METHODS

We treated six patients with imatinib-resistant CML in haematological relapse (leukocytes>> 20,000 microL(-1)) with rapamycin at 2 mg per os daily for 14 consecutive days, with dose-adjustment allowed to reach a target rapamycin serum concentration of 10-20 pg mL(-1).

RESULTS

A major leukocyte response with decrease to less than 10,000 microL(-1) was obtained in two patients, and a minor transient response was seen in two other patients. In responding patients, we also observed a decrease in vascular endothelial growth factor (VEGF) mRNA levels in circulating leukaemic cells. Side effects during rapamycin treatment were mild in most patients. In one patient, pneumonia developed. Rapamycin was also found to counteract growth of CML cells in vitro as determined by (3)H-thymidine incorporation. Moreover, rapamycin inhibited the growth of Ba/F3 cells exhibiting various imatinib-resistant mutants of BCR/ABL, including the T315I variant that exhibits resistance against most currently available BCR/ABL kinase inhibitors.

CONCLUSIONS

Rapamycin shows antileukaemic effects in imatinib-resistant CML in vitro and in vivo. Larger trials with rapamycin or rapamycin-derivatives in combination with other targeted drugs are warranted to further determine clinical efficacy in CML.

Cellulosomes produced by Clostridium cellulolyticum grown on cellulose were purified and separated using anion-exchange chromatography. SDS/PAGE analysis of six fractions showed variations in their cellulosomal protein composition. Hydrolytic activity on carboxymethyl cellulose, xylan, crystalline cellulose and hatched straw differed from one fraction to another. Fraction F1 showed a high level of activity on xylan, whereas fractions F5 and F6 were most active on crystalline cellulose and carboxymethyl cellulose, respectively. Several cellulosomal components specific to fractions F1, F5 and F6 were investigated using MS analysis. Several hemicellulases were identified, including three xylanases in F1, and several cellulases belonging to glycoside hydrolase families 9 and 5 and, a cystein protease inhibitor were identified in F5 and F6. Synergies were observed when two or three fractions were combined. A mixture containing fractions F1, F3 and F6 showed the most divergent cellulosomal composition, the most synergistic effects and the highest level of activity on straw (the most heterogeneous substrate tested). These findings show that on complex substrates such as straw, synergies occur between differently composed cellulosomes and the degradation efficiency of the cellulosomes is correlated with their enzyme diversity.

Epigenetic regulation modulates gene expression without altering the DNA sequence to facilitate rapid adjustments to dynamically changing environmental conditions. The formation of an epigenetic memory allows passing on this information to subsequent generations. Here we propose that epigenetic memories formed by adverse environmental conditions and stress represent a critical determinant of health and disease in the F3 generation and beyond. Transgenerational programming of epigenetic regulation may represent a key to understand adult-onset complex disease pathogenesis and cumulative effects of life span and familial disease etiology. Ultimately, the mechanisms of generating an epigenetic memory may become of potentially promising diagnostic and therapeutic relevance due to their reversible nature. Exploring the role of environmental factors, such as stress, in causing variations in epigenetic profiles may lead to new avenues of personalized, preventive medicine based on epigenetic signatures and interventions.

F3/contactin (CNTN1) and TAG-1 (CNTN2) are closely related axonal glycoproteins that are differentially regulated during development. In the cerebellar cortex TAG-1 is expressed first as granule cell progenitors differentiate in the premigratory zone of the external germinal layer. However, as these cells begin radial migration, TAG-1 is replaced by F3/contactin. To address the significance of this differential regulation, we have generated transgenic mice in which F3/contactin expression is driven by TAG-1 gene regulatory sequences, which results in premature expression of F3/contactin in granule cells. These animals (TAG/F3 mice) display a developmentally regulated cerebellar phenotype in which the size of the cerebellum is markedly reduced during the first two postnatal weeks but subsequently recovers. This is due in part to a reduction in the number of granule cells, most evident in the external germinal layer at postnatal day 3 and in the inner granular layer between postnatal days 8 and 11. The reduction in granule cell number is accompanied by a decrease in precursor granule cell proliferation at postnatal day 3, followed by an increase in the number of cycling cells at postnatal day 8. In the same developmental window the size of the molecular layer is markedly reduced and Purkinje cell dendrites fail to elaborate normally. These data are consistent with a model in which deployment of F3/contactin on granule cells affects proliferation and differentiation of these neurons as well as the differentiation of their synaptic partners, the Purkinje cells. Together, these findings indicate that precise spatio-temporal regulation of TAG-1 and F3/contactin expression is critical for normal cerebellar morphogenesis.

OBJECTIVE

Circumstantial evidence links retroviruses (RVs) with human autoimmune diseases. The aim of the present study was to obtain direct evidence of RV gene expression in rheumatoid arthritis (RA).

METHODS

Synovial samples were obtained from patients with RA, patients with osteoarthritis (OA), and normal control subjects. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using synovial RNA and primers to conserved sequences in the polymerase (pol) genes of known RVs.

RESULTS

PCR products (n = 857) were cloned and sequenced. Multiple pol transcripts, many with open reading frames, were expressed in every sample. Sequences were aligned and classified into 6 families (F1-F6) that contained 33 groups of known and unknown endogenous RVs (ERVs), each distinguished by a specific, deduced peptide motif. The frequency of sequences in each family was similar between RA, OA, and normal synovial tissue, but differed significantly in RA synovial fluid cells. F1 sequences (undefined, but related to murine and primate type C RVs) were lower in frequency, F2 (ERV-9-related), F4 (HERV-K-related), and F6 (HERV-L-related) sequences were higher in frequency, and F3 (RTVL-H-related) sequences were not detected, in the RA synovial fluid cells compared with the RA synovial tissues.

CONCLUSIONS

Multiple ERVs are expressed in normal and diseased synovial compartments, but specific transcripts can be differentially expressed in RA.

Recurrent hepatitis C virus (HCV) infection occurs universally in the allograft in the absence of effective antiviral therapy before liver transplantation (LT). Antiviral therapy with sofosbuvir and simeprevir has proven to be highly effective and well tolerated in the nontransplant setting for treatment of HCV genotype 1 infection; therefore, we sought to evaluate the efficacy and safety of this regimen in LT recipients with recurrent HCV infection. This was a retrospective analysis of a single-center treatment protocol of patients with HCV genotype 1 infection who received a 12-week combination regimen of sofosbuvir and simeprevir. Sixty-one patients (35 with genotype 1a and 26 with genotype 1b) completed treatment with simeprevir and sofosbuvir. Three patients received additional ribavirin. Laboratory data and clinical assessments performed at the baseline, on treatment, at the end of treatment, and 12 weeks after the completion of antiviral therapy [sustained virological response at 12 weeks (SVR12)] were analyzed. The median time after LT was 5.4 years [interquartile range (IQR), 1.9-8.4 years], and tacrolimus was the most commonly used immunosuppressive agent (80.3%). Overall, SVR12 was achieved in 93.4% [95% confidence interval (CI), 84%-97%] of LT recipients treated with 12 weeks of sofosbuvir and simeprevir. When they were analyzed according to the HCV subtype, LT recipients with genotype 1b had a 100% SVR12 rate (95% CI, 87%-100%), whereas SVR12 was 89% (95% CI, 74%-95%) for those with genotype 1a. Advanced fibrosis (METAVIR F3-F4) was associated with diminished antiviral efficacy in LT recipients with genotype 1a [SVR12, 67% (95% CI, 39%-86%); P = 0.01]. Overall, the incidence of adverse events (AEs) was low, and no severe AEs occurred during treatment. In conclusion, treatment with a 12-week regimen of sofosbuvir and simeprevir was well tolerated and resulted in a high SVR12 rate for LT recipients with recurrent HCV genotype 1 infection. Genotype 1a patients with advanced fibrosis of the allograft were more likely to relapse.