OBJECTIVE

The impact of fibrosis stage on chronic hepatitis C virus (HCV) treatment response was explored in CHARIOT, a study of high dose peginterferon alfa-2a (PEG-IFNalpha-2a) induction therapy in treatment naïve genotype 1 infection.

METHODS

Eight hundred and ninety-six patients were randomised 1:1 to 360 microg (n=448) or 180 microg (n=448) PEG-IFNalpha-2a weekly with RBV 1000-1200 mg/day for 12 weeks followed by 36 weeks of 180 microg PEG-IFNalpha-2a weekly plus RBV 1000-1200 mg/day. Virological responses were assessed at week 4, 8, 12, 24, 48 (end of therapy), and 24 weeks following therapy (sustained virological response, SVR). As previously reported, there was no significant difference in SVR in the induction (53%) and standard (50%) arms, therefore the pooled study population was used for analysis of SVR and relapse.

RESULTS

A marked step-wise decline in SVR was evident by fibrosis stage: F0 (70%); F1 (60%); F2 (51%); F3 (31%); F4 (10%) (p<0.0001). Early virological responses were lower among F3/4 patients, including rapid virological response (RVR) (21% vs. 34% for F3/4 and F0-2, respectively) (p=0.0072), and the RVR positive predictive value was also lower (63% vs. 80%). Virological relapse rates were similar in early disease stages (F0, 16%; F1, 23%; F2, 26%), but increased markedly in advanced fibrosis (F3, 50%; F4, 80%) (p<0.0001). Cumulative PEG-IFNalpha-2a and ribavirin doses were similar among patients with F3/4 and F0-2 within treatment arms through week 4, 8, 12, and week 24.

CONCLUSIONS

Low virological response in hepatitis C genotype 1 patients with advanced fibrosis is not explained by inadequate cumulative PEG-IFN and ribavirin doses.

A study was initiated to determine the number, chromosomal location, and magnitude of effect of QTL (quantitative trait loci or locus depending on context) controlling protein and starch concentration in the maize (Zea mays L.) kernel. Restriction fragment length polymorphism (RFLP) analysis was performed on 100 F3 families derived from a cross of two strains, Illinois High Protein (IHP), X Illinois Low Protein (ILP), which had been divergently selected for protein concentration for 76 generations as part of the Illinois Long Term Selection Experiment. These families were analyzed for kernel protein and starch in replicated field trials during 1990 and 1991. A series of 90 genomic and cDNA clones distributed throughout the maize genome were chosen for their ability to detect RFLP between IHP and ILP. These clones were hybridized with DNA extracted from the 100 F3 families, revealing 100 polymorphic loci. Single factor analysis of variance revealed significant QTL associations of many loci with both protein and starch concentration (P < 0.05 level). Twenty-two loci distributed on 10 chromosome arms were significantly associated with protein concentration, 19 loci on 9 chromosome arms were significantly associated with starch concentration. Sixteen of these loci were significant for both protein and starch concentration. Clusters of 3 or more significant loci were detected on chromosome arms 3L, 5S, and 7L for protein concentration, suggesting the presence of QTL with large effects at these locations. A QTL with large additive effects on protein and starch concentration was detected on chromosome arm 3L. RFLP alleles at this QTL were found to be linked with RFLP alleles at the Shrunken-2 (Sh2) locus, a structural gene encoding the major subunit of the starch synthetic enzyme ADP-glucose pyrophosphorylase. A multiple linear regression model consisting of 6 significant RFLP loci on different chromosomes explained over 64 % of the total variation for kernel protein concentration. Similar results were detected for starch concentration. Thus, several chromosomal regions with large effects may be responsible for a significant portion of the changes in kernel protein and starch concentration in the Illinois Long Term Selection Experiment.

BACKGROUND

BBK32 is a surface expressed lipoprotein and fibronectin (Fn)-binding microbial surface component recognizing adhesive matrix molecule (MSCRAMM) of Borrelia burgdorferi, the causative agent of Lyme disease. Previous studies from our group showed that BBK32 is a virulence factor in experimental Lyme disease and located the Fn-binding region to residues 21-205 of the lipoprotein.

RESULTS

Studies aimed at identifying interacting sites between BBK32 and Fn revealed an interaction between the MSCRAMM and the Fn F3 modules. Further analysis of this interaction showed that BBK32 can cause the aggregation of human plasma Fn in a similar concentration-dependent manner to that of anastellin, the superfibronectin (sFn) inducing agent. The resulting Fn aggregates are conformationally distinct from plasma Fn as indicated by a change in available thermolysin cleavage sites. Recombinant BBK32 and anastellin affect the structure of Fn matrices formed by cultured fibroblasts and inhibit endothelial cell proliferation similarly. Within BBK32, we have located the sFn-forming activity to a region between residues 160 and 175 which contains two sequence motifs that are also found in anastellin. Synthetic peptides mimicking these motifs induce Fn aggregation, whereas a peptide with a scrambled sequence motif was inactive, suggesting that these motifs represent the sFn-inducing sequence.

CONCLUSIONS

We conclude that BBK32 induces the formation of Fn aggregates that are indistinguishable from those formed by anastellin. The results of this study provide evidence for how bacteria can target host proteins to manipulate host cell activities.

Interleukin-12 (IL-12) is a heterodimeric cytokine composed of two disulfide-bonded subunits, p35 and p40, which has important regulatory effects on T cells and natural killer (NK) cells. In contrast to heterodimeric IL-12, a homodimer of the p40 subunit, designated (p40)2, has been shown to be a potent IL-12 antagonist. To study the interaction between (p40)2 and the known IL-12 receptor (IL-12R) subunits, IL-12Rbeta1 and IL-12Rbeta2, we directly measured the binding activity of mouse (p40)2 to ConA-activated lymphoblasts and purified B cells from splenocytes of C57BL/6J mice. These results demonstrated the presence of both high (Kd about 5 pM) and low affinity (Kd about 15 nM) binding sites for mouse 125I-labeled (p40)2. To elucidate which of the IL-12R subunits binds mouse (p40)2, binding studies of mouse 125I-labeled (p40)2 to Ba/F3 cells expressing recombinant mouse IL-12Rbeta1 and/or mouse IL-12Rbeta2 were carried out. Mouse IL-12Rbeta1 bound mouse 125I-labeled (p40)2 with high and low affinities, comparable to that observed on Con A blasts and B cells. In contrast, mouse IL-12Rbeta2 bound mouse 125I-labeled (p40)2 very poorly. These data demonstrate that similar to IL-12, mouse (p40)2 binds with both high and low affinity to Con A blasts and B cells, and that IL-12Rbeta1 is responsible for mediating the specific binding of mouse (p40)2.

Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199-314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73±12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-γ secretion, ratios of IFN-γ/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNFα/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5-88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-γ/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent vaccines against NHs-dependent pathogens.

OBJECTIVE

To study the clinical features of patients with autoantibodies to centromere protein CENP-F and the frequency of CENP-F autoantibodies in patients with various diseases.

METHODS

Retrospective clinical and serologic study.

METHODS

Thirty-six patients with anti-CENP-F were identified by a characteristic pattern of indirect immunofluorescence (IIF) on HEp-2 cells. Fifty patients with melanoma, 50 with breast cancer, 10 with lung cancer, 354 with systemic sclerosis, 120 with systemic lupus erythematosus and 50 with rheumatoid arthritis were also studied. Recombinant proteins were produced from 5 CENP-F cDNA clones representing amino acids 2192-3317 (p-F1), 5561-7126 (p-F2), 5892-6883 (p-F3), 7538-10,116 (p-F4) and 9242-10,096 (p-F5). The presence of CENP-F antigen was studied in a breast carcinoma cell line, cryosections of breast carcinoma, normal breast tissue and tonsils.

RESULTS

Twenty-two of 36 patients with CENP-F antibodies had neoplasms; breast (9/22) and lung (5/22) cancer were the most common diagnoses. Thirty-three sera were available for further study; when tested for reactivity to the recombinant peptides, the sera of 21 of 21 patients with neoplasms and 5 of 12 patients with other diseases bound the C-terminal p-F4 peptide. When the terminal third of the p-F4 peptide (p-F5) was studied, a significant difference in pattern of reactivity was not detected. By comparison, the frequency of reactivity with peptides representing other domains of CENP-F was less than that with p-F4 (p-F2>> p-F3>> p-F1). CENP-F autoantibodies were not found in any of the control sera from patients with systemic lupus erythematosus, rheumatoid arthritis or systemic sclerosis or in unselected sera from various malignancies. CENP-F antigens were identified in breast carcinoma tissue but were rarely observed in normal tissues.

CONCLUSIONS

A high proportion of individuals with CENP-F antibodies have neoplasia, and there is a bias among their sera for reactivity with determinants in the carboxy terminal domain of CENP-F. CENP-F antigens appear to be highly expressed in malignant tissues.

BACKGROUND

Fibronectin-null cells assemble soluble fibronectin shortly after adherence to a substrate coated with intact fibronectin but not when adherent to the cell-binding domain of fibronectin (modules (7)F3-(10)F3). Interactions of adherent cells with regions of adsorbed fibronectin other than modules (7)F3-(10)F3, therefore, are required for early display of the cell surface sites that initiate and direct fibronectin assembly.

RESULTS

To identify these regions, coatings of proteolytically derived or recombinant pieces of fibronectin containing modules in addition to (7)F3-(10)F3 were tested for effects on fibronectin assembly by adherent fibronectin-null fibroblasts. Pieces as large as one comprising modules (2)F3-(14)F3, which include the heparin-binding and cell adhesion domains, were not effective in supporting fibronectin assembly. Addition of module (1)F3 or the C-terminal modules to modules (2)F3-(14)F3 resulted in some activity, and addition of both (1)F3 and the C-terminal modules resulted in a construct, (1)F3-C, that best mimicked the activity of a coating of intact fibronectin. Constructs (1)F3-C V0, (1)F3-C V64, and (1)F3-C Delta(V(15)F3(10)F1) were all able to support fibronectin assembly, suggesting that (1)F3 through (11)F1 and/or (12)F1 were important for activity. Coatings in which the active parts of (1)F3-C were present in different proteins were much less active than intact (1)F3-C.

CONCLUSIONS

These results suggest that (1)F3 acts together with C-terminal modules to induce display of fibronectin assembly sites on adherent cells.

The exposure of antigenic determinants of histones present in "native" chromatin was studied by: (1) testing their ability to elicit anti-histone antibodies and (2) measuring their ability to interact with anti-histone sera. To this end, antisera specific to purified histone fractions and to purified rat liver chromatin were elicited in rabbits. The anti-chromatin sera did not react with pure histone fractions and pure histone fractions F2b, F3, F2a1, and F2a2 failed to inhibit the complement fixation resulting from the binding of anti-chromatin to chromatin. These results suggest that in native chromatin, determinants in these histones are not immunogenic. Histone F1, however, inhibited the reaction between chromatin and anti-chromatin. Antisera elicited by histone fractions reacted weakly with "native" chromatin. The maximal complement fixations (obtained with 5-10 mug of chromatin DNA) were as follows: 60% with anti-F2b, 20% with anti-F1 and anti-F3, and less than 5% with either anti-F2a1 or anti-F2a2. Studies of the interaction between anti-histone antibodies and chromatin in which chromatin was used as an immunoadsorbent indicated that antibodies against different histones were adsorbed to a different degree by the same amount of chromatin. Differences in the immunoadsorbing capacity between sonicated and nonsonicated chromatin were found. Quantitative adsorbtion studies revealed that in the "native" chromatin structure, antigenic determinants of F1 and F2b were more available to interact with homologous antibody than those of F3 and F2a1 and that determinants in F2a2 were the least available. It could be calculated that the "equivalent antigenicity" of the histones in chromatin was 9.6% for F1, 3.2% for F2b, and 0.90% for F3 and F2a1. Upon sonication these values did not change for F1 but increased two-, three-, and fourfold for F2b, F3, and F2a1, respectively. Digestion of chromatin with trypsin totally abolished the ability of chromatin to adsorb anti-histone antibodies.

Distortion product otoacoustic emission (DPOAE) suppression measurements were made in 20 subjects with normal hearing and 21 subjects with mild-to-moderate hearing loss. The probe consisted of two primary tones (f2, f1), with f2 held constant at 4 kHz and f2/f1 = 1.22. Primary levels (L1, L2) were set according to the equation L1 = 0.4 L2 + 39 dB [Kummer et al., J. Acoust. Soc. Am. 103, 3431-3444 (1998)], with L2 ranging from 20 to 70 dB SPL (normal-hearing subjects) and 50-70 dB SPL (subjects with hearing loss). Responses elicited by the probe were suppressed by a third tone (f3), varying in frequency from 1 octave below to 1/2 octave above f2. Suppressor level (L3) varied from 5 to 85 dB SPL. Responses in the presence of the suppressor were subtracted from the unsuppressed condition in order to convert the data into decrements (amount of suppression). The slopes of the decrement versus L3 functions were less steep for lower frequency suppressors and more steep for higher frequency suppressors in impaired ears. Suppression tuning curves, constructed by selecting the L3 that resulted in 3 dB of suppression as a function of f3, resulted in tuning curves that were similar in appearance for normal and impaired ears. Although variable, Q10 and Q(ERB) were slightly larger in impaired ears regardless of whether the comparisons were made at equivalent SPL or equivalent sensation levels (SL). Larger tip-to-tail differences were observed in ears with normal hearing when compared at either the same SPL or the same SL, with a much larger effect at similar SL. These results are consistent with the view that subjects with normal hearing and mild-to-moderate hearing loss have similar tuning around a frequency for which the hearing loss exists, but reduced cochlear-amplifier gain.

The functions of wild-type and mutant mouse interleukin-10 receptors (mIL-10R) expressed in murine Ba/F3 cells were studied. As observed previously, IL-10 stimulates proliferation of IL-10R-expressing Ba/F3 cells. Accumulation of viable cells in the proliferation assay is to a significant extent balanced by concomitant cell death. Moreover, growth in IL-10 also induces a previously unrecognized response, differentiation of the cells, as evidenced both by formation of large clusters of cells in cultures with IL-10 and by induction or enhancement of expression of several cell surface antigens, including CD32/16, CD2, LECAM-1 (v-selectin), and heat-stable antigen. Two distinct functional regions near the C terminus of the mIL-10R cytoplasmic domain which mediate proliferation were identified; one of these regions also mediates the differentiation response. A third region proximal to the transmembrane domain was identified; removal of this region renders the cell 10- to 100-fold more sensitive to IL-10 in the proliferation assay. In cells expressing both wild-type and mutant IL-10R, stimulation with IL-10 leads to tyrosine phosphorylation of the kinases JAK1 and TYK2 but not JAK2 or JAK3 under the conditions tested.

The bovine papillomavirus E5 protein is a small, homodimeric transmembrane protein that forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor through transmembrane and juxtamembrane interactions, resulting in receptor activation and cell transformation. Glutamine 17 in the transmembrane domain of the 44-amino-acid E5 protein is critical for complex formation and receptor activation, and we previously proposed that glutamine 17 forms a hydrogen bond with threonine 513 of the PDGF beta receptor. We have constructed and analyzed mutant E5 proteins containing all possible amino acids at position 17 and examined the ability of these proteins to transform C127 fibroblasts, which express endogenous PDGF beta receptor. Although several position 17 mutants were able to transform cells, mutants containing amino acids with side groups that were unable to participate in hydrogen bonding interactions did not form a stable complex with the PDGF beta receptor or transform cells, in agreement with the proposed interaction between position 17 of the E5 protein and threonine 513 of the receptor. The nature of the residue at position 17 also affected the ability of the E5 proteins to dimerize. Overall, there was an excellent correlation between the ability of the various E5 mutant proteins to bind the PDGF beta receptor, lead to receptor tyrosine phosphorylation, and transform cells. Similar results were obtained in Ba/F3 hematopoietic cells expressing exogenous PDGF beta receptor. In addition, treatment of E5-transformed cells with a specific inhibitor of the PDGF receptor tyrosine kinase reversed the transformed phenotype. These results confirm the central importance of the PDGF beta receptor in mediating E5 transformation and highlight the critical role of the residue at position 17 of the E5 protein in the productive interaction with the PDGF beta receptor. On the basis of molecular modeling analysis and the known chemical properties of the amino acids, we suggest a structural basis for the role of the residue at position 17 in E5 dimerization and in complex formation between the E5 protein and the PDGF beta receptor.

Activating mutations in the juxtamembrane domain (FLT3-length mutations, FLT3-LM) and in the protein tyrosine kinase domain (TKD) of FLT3 (FLT3-TKD) represent the most frequent genetic alterations in acute myeloid leukemia (AML) and define a molecular target for therapeutic interventions by protein tyrosine kinase (PTK) inhibitors. We could show that distinct activating FLT3-TKD mutations at position D835 mediate primary resistance to FLT3 PTK inhibitors in FLT3-transformed cell lines. In the presence of increasing concentrations of the FLT3 PTK inhibitor SU5614, we generated inhibitor resistant Ba/F3 FLT3-internal tandem duplication (ITD) cell lines (Ba/F3 FLT3-ITD-R1-R4) that were characterized by a 7- to 26-fold higher IC50 (concentration that inhibits 50%) to SU5614 compared with the parental ITD cells. The molecular characterization of ITD-R1-4 cells demonstrated that specific TKD mutations (D835N and Y842H) on the ITD background were acquired during selection with SU5614. Introduction of these dual ITD-TKD, but not single D835N or Y842H FLT3 mutants, in Ba/F3 cells restored the FLT3 inhibitor resistant phenotype. Our data show that preexisting or acquired mutations in the PTK domain of FLT3 can induce drug resistance to FLT3 PTK inhibitors in vitro. These findings provide a molecular basis for the evaluation of clinical resistance to FLT3 PTK inhibitors in patients with AML.

The adaptor protein Grb10 plays important roles in mitogenic signaling. However, its roles in acute myeloid leukemia (AML) are predominantly unknown. Here we describe the role of Grb10 in FLT3-ITD-mediated AML. We observed that Grb10 physically associates with FLT3 in response to FLT3-ligand (FL) stimulation through FLT3 phospho-tyrosine 572 and 793 residues and constitutively associates with oncogenic FLT3-ITD. Furthermore endogenous Grb10-FLT3 association was observed in OCI-AML-5 cells. Grb10 expression did not alter FLT3 receptor activation or stability in Ba/F3-FLT3 cells. However, expression of Grb10 enhanced FL-induced Akt phosphorylation without affecting Erk or p38 phosphorylation in Ba/F3-FLT3-WT and Ba/F3-FLT3-ITD. Selective Grb10 depletion reduced Akt phosphorylation in Ba/F3-FLT3-WT and OCI-AML-5 cells. Grb10 transduces signal from FLT3 by direct interaction with p85 and Ba/F3-FLT3-ITD cells expressing Grb10 exhibits higher STAT5 activation. Grb10 regulates the cell cycle by increasing cell population in S-phase. Expression of Grb10 furthermore resulted in an increased proliferation and survival of Ba/F3-FLT3-ITD cells as well as increased colony formation in semisolid culture. Grb10 expression was significantly increased in AML patients compared to healthy controls and was also elevated in patients carrying FLT3-ITD mutants. The elevated Grb10 expression partially correlated to relapse as well as to poor prognosis. These results suggest that Grb10 binds to both normal and oncogenic FLT3 and induces PI3K-Akt and STAT5 signaling pathways resulting in an enhanced proliferation, survival and colony formation of hematopoietic cells.

The Philadelphia translocation commonly observed in chronic myeloid leukaemia (CML) and a proportion of cases of acute leukaemia results in the creation of a chimeric fusion protein, BCR-ABL. The fusion protein exhibits an elevated tyrosine kinase activity as compared to normal ABL. Using a temperature sensitive mutant of p210 BCR-ABL (ts-p210) we find that the primary effect of BCR-ABL expression in an IL-3 dependent cell line is to prolong survival following growth factor withdrawal; only a small proportion of cells remain viable and rapidly evolve to complete growth factor independence. During passage in the presence of IL-3 at the temperature permissive for kinase activity, ts-p210 expressing cultures become dominated by completely growth factor independent cells within 10-30 days. There is also a significant difference between BCR-ABL and IL-3 mediated signalling with respect to the MAP kinase pathway; in contrast to IL-3 stimulation or v-ABL expression, BCR-ABL does not signal ERK 2 (MAP 2 kinase) activation, underlining the apparent inability of BCR-ABL to deliver an immediate proliferative signal in Ba/F3 cells. Our data suggest that growth factor independence does not simply reflect the convergence of BCR-ABL and IL-3 mediated signalling pathways and its development, at least in Ba/F3 cells, requires prolonged exposure to BCR-ABL kinase activity. We suggest that the myeloid expansion characteristic of CML may result from the prolongation of survival of myeloid progenitor cells under conditions of limiting growth factor rather than their uncontrolled proliferation.

We studied the effect of the methanol extract of garlic bulbs (EOG) and of three pure components isolated from it (F1, F2, F3), on human platelet aggregation induced by ADP, epinephrine, collagen, thrombin, arachidonate, PAF, and the ionophore A-23187. Incubation of PRP with EOG, either in methanol or in homologous PPP, inhibits platelet aggregation induced by all of the above mentioned agonists. F1, F2, and F3 also inhibit platelet aggregation, however, F3 was about four times more potent. Addition of EOG or F3 to platelets that have already been irreversibly aggregated by 10 microM ADP, induces rapid deaggregation. Inhibition of aggregation was still present after three hours. The inhibitory effect persisted even after the treated platelets were Gel-Filtered (GFP) or separated from plasma through a metrizamide gradient and resuspended in new homologous PPP. Thrombin-induced release of ATP from GFP was inhibited by 75-80% after EOG or F3 treatment. Incorporation of [3-H]-arachidonate by intact platelets was decreased by 50-60% in treated platelets. However, platelets incubated with the inhibitors after incorporation of radiolabeled arachidonate, although did not aggregate, produced, after thrombin activation similar amounts of radiolabeled TXB2 and lipoxygenase products as the controls. Electron microscopy of inhibited platelets, in the presence of thrombin, showed no degranulation but an increase of spherical forms. Our results suggest that the effects described might be mediate by a perturbation of the physicochemical properties of the plasma membrane rather than by affecting arachidonate or calcium metabolism in the cells. Chemical structures of F1, F2 and F3 have been provisionally assigned: F1 is diallytrisulfide, F2 is 2-vinyl-1,3-dithiene, and F3 is most probably allyl 1,5-hexadienyltrisulfide.

To study the effects of hydrogen peroxide, pig coronary artery smooth muscle subcellular fractions enriched in plasma membrane (F2) or sarcoplasmic reticulum (F3) were incubated in various concentrations of peroxide and 5 mM azide. ATP-dependent azide-insensitive oxalate-stimulated Ca2+ uptake was determined for F3 and phosphate-stimulated uptake for F2. Only 1.5-5 microM hydrogen peroxide was required for 50% inhibition of the Ca2+ uptake by F3, but the corresponding concentration for F2 was 10-50 microM. This effect was not prevented by superoxide dismutase. Hydrogen peroxide inhibited the Ca(2+)-dependent formation of a 115-kDa acylphosphate band in F3 and 140- and 115-kDa bands in F2. The inhibition of Ca2+ uptake in F3, however, exceeded the inhibition of the acylphosphate formation. Efflux of Ca2+ from F2 and F3 was enhanced by hydrogen peroxide but F3 was more sensitive than F2. We conclude that hydrogen peroxide has dual effect on Ca2+ dynamics in the coronary artery smooth muscle, i.e., it inactivates the Ca2+ pumps and increases membrane permeability to Ca2+. The effect is more pronounced on sarcoplasmic reticulum than on plasma membrane. Intrinsic catalase may, however, provide partial protection against such damage.

BACKGROUND

Interferon-beta (IFN-beta) has potent antitumor activity; however, systemic toxicity has limited its clinical use. We investigated the potential of targeted delivery using tumor-tropic neural progenitor cells (NPCs) transduced to express human IFN-beta (hIFN-beta).

METHODS

Disseminated neuroblastoma was established in SCID mice by tail vein injection of tumor cells. Fourteen days after tumor cell inoculation, systemic disease was confirmed with bioluminescence imaging (BLI). Mice were then treated by intravenous injection of human F3.C1 NPCs that had been transduced with a replication deficient adenovirus to overexpress hIFN-beta (F3-IFN-beta). Two injections were given: the first at 14 days and the second at 28 days following tumor cell injection. Control mice received NPCs transduced with empty vector adenovirus at the same time points. Progression of disease was monitored using BLI. At sacrifice, organ weights and histology further evaluated tumor burden.

RESULTS

After initiation of therapy, BLI demonstrated a significant decrease in the rate of disease progression in mice receiving F3-IFN-beta. At necropsy, control mice had bulky tumor replacing the liver and kidneys, as well as extensive retroperitoneal and mediastinal adenopathy. Impressively, these sites within mice receiving F3-IFN-beta therapy appeared grossly normal with the exception of small nodules within the kidneys of some of the F3-IFN-beta-treated mice. The accumulation of F3.C1 cells within sites of tumor growth was confirmed by fluorescence imaging. Importantly, systemic levels of hIFN-beta in the treated mice remained below detectable levels.

CONCLUSIONS

These data indicate that in this model of disseminated neuroblastoma, the tumor-tropic property of F3.C1 NPCs was exploited to target delivery of IFN-beta to disseminated tissue foci, resulting in significant tumor growth delay. The described novel approach for effective IFN-beta therapy may circumvent limitations associated with the systemic toxicity of IFN-beta.

OBJECTIVE

To assess the regression of liver fibrosis after interferon (IFN) treatment in patients with chronic hepatitis C, liver stiffness (LS) was measured repeatedly and the factors associated with reduction of LS were assessed.

METHODS

LS was measured by transient elastography before treatment, at end of treatment (EOT), and 1 year and 2 years after EOT in 145 patients with chronic hepatitis C treated by IFN with or without ribavirin.

RESULTS

In the patients with sustained virological response (SVR) (n = 93) and relapsers (n = 28), LS significantly decreased at EOT (median, 5.4 [interquartile range, 4.0-8.6] kilopascals [kPa], P < 0.0001 and 6.8 [4.5-8.9] kPa, P = 0.0023) and 1 year after EOT (5.3 [4.2-7.0] kPa, P < 0.0001 and 6.8 [4.5-9.3] kPa, P = 0.0204) compared with baseline (8.0 [5.0-11.9] kPa and 10.6 [7.0-16.6] kPa). In SVR patients, LS significantly decreased 2 years after EOT (5.3 [4.1-6.3] kPa) compared with baseline (P < 0.0001) and LS at EOT (P = 0.0034). Two points or greater reduction of deduced stage at last LS measurement was observed in 78% of SVR patients, 59% of relapsers and 15% of patients with non-virological response whose pretreatment deduced stages were F3-F4. Fibrosis stage, hyaluronic acid levels, duration of treatment, response to treatment and alanine aminotransferase levels were associated with a 2-point or greater decrease of deduced fibrosis stage.

CONCLUSIONS

IFN treatment reduced LS in SVR patients and relapsers. Significant reduction of LS is associated with milder fibrosis stage, lower hyaluronic acid levels, longer IFN treatment, virological response of SVR or relapse and higher alanine aminotransferase levels.

The use of targeted nanoparticles (NPs) as a platform for loading photosensitizers enables selective accumulation of the photosensitizers in the tumor area, while maintaining their photodynamic therapy (PDT) effectiveness. Here two novel kinds of methylene blue (MB)-conjugated polyacrylamide (PAA) nanoparticles, MBI-PAA NPs and MBII-PAA NPs, based on two separate MB derivatives, are developed for PDT. This covalent conjugation with the NPs (i) improves the loading of MB, (ii) prevents any leaching of MB from the NPs and (iii) protects the MB from the effects of enzymes in the biological environment. The loading of MB into these two kinds of NPs was controlled by the input amount, resulting in concentrations with optimal singlet oxygen production. For each of the MB-NPs, the highest singlet oxygen production was found for an MB loading of around 11 nmol mg(-1). After attachment of F3 peptide groups, for targeting, each of these NPs was taken up, selectively, by MDA-MB-435 tumor cells, in vitro. PDT tests demonstrated that both kinds of targeted NPs resulted in effective tumor cell kill, following illumination, while not causing dark toxicity.

Increasing evidence has shown that the Notch signalling pathway regulates oligodendrogliogenesis. Upon binding to classical Delta/Serrate/Lag-2 ligands, Notch signalling promotes generation of oligodendrocyte precursor cells while inhibiting their further differentiation into myelinating oligodendrocytes. In our recent studies, we have found that two neural cell adhesion molecules, F3/contactin and NB-3 interact with Notch receptors and promote oligodendrocyte development. Remarkably, all these F3 and NB-3/Notch cascade-related events required Deltex1 as the intermediate element. Experiments using several animal models further imply the function of F3/Notch signalling in vivo, which designates Notch signalling as a ligand-dependent, multipotential cascade involved in oligodendrocyte development.

The mouse neuronal F3 glycoprotein and its chicken homolog F11 belong to a subclass of proteins of the immunoglobulin superfamily with preferential localization on axons and neurites. We have transfected F3 cDNA into CHO cells. Biochemical analysis establishes that the cDNA we have cloned codes for a 130 kd phosphatidylinositol-anchored polypeptide. F3-expressing transfectants exhibited enhanced self-adhesive properties, aggregating with faster kinetics and forming larger aggregates than F3-negative control cells. When used as a culture substrate for sensory neurons, F3-transfected cells showed a markedly enhanced ability to promote neurite outgrowth compared with nontransfected cells. The results support the idea that F3/F11 and other closely similar proteins function as cell adhesion molecules that play a role in axonal growth and guidance.

Increased protein kinase B (PKB; c-Akt) activation is a hallmark of diverse neoplasias providing both proliferative and antiapoptotic survival signals. In this study, we investigated the effect of chronic PKB activation on cellular survival and proliferation using cytokine-dependent bone marrow-derived Ba/F3 cells, in which PKBalpha activation can be directly, and specifically, induced by addition of 4-hydroxytamoxifen (4-OHT). Direct activation of PKB rescued Ba/F3 cells from cytokine withdrawal-induced apoptosis; however, surprisingly, these antiapoptotic effects were short lived, cells only being protected for up to 48 hours. We observed that activation of PKB in survival factor-deprived cells led to a dramatic increase of Foxo3a on both the transcriptional and protein level leading to expression of its transcriptional targets Bim and p27(kip1). High levels of PKB activity result in increased aerobic glycolysis and mitochondrial activity resulting in overproduction of reactive oxygen species. To determine whether oxidative stress might itself be responsible for Foxo3a up-regulation, we utilized hydrogen peroxide (H(2)O(2)) as an artificial inducer of oxidative stress and N-acetylcysteine (NAC), a thiol-containing radical oxygen scavenger. Addition of NAC to the culture medium prolonged the life span of cells treated with 4-OHT and prevented the up-regulation of Foxo3a protein levels caused by PKB activation. Conversely, treatment of Ba/F3 cells with H(2)O(2) caused an increase of Foxo3a on both transcriptional and protein levels, suggesting that deregulated PKB activation leads to oxidative stress resulting in Foxo3a up-regulation and subsequently cell death. Taken together, our data provide novel insights into the molecular consequences of uncontrolled PKB activation.

Using electrophoretic mobility shift assays and DNA-protein cross-linking techniques, we demonstrate that IL-2 binding to functional forms of the human IL-2R activates nuclear expression of the eukaryotic transcription factor NF-kappa B. These inductive effects of IL-2 were observed in three different cellular systems including human Jurkat T cells stably transfected with IL-2R beta cDNA, mouse pro-B BA/F3 cells stably expressing human IL-2R beta chains either alone or in combination with human or murine IL-2R alpha chains, and purified primary resting human T cells constitutively displaying small numbers of IL-2R beta molecules. IL-2 activation of nuclear NF-kappa B expression is regulated in part at a post-translational level, involving the rapid translocation of both the 50- and 65-kDa subunits of NF-kappa B from the cytoplasm to the nucleus. However, IL-2 induction also produced an increase in mRNA for NF-kappa B p105, indicating an additional pretranslational component of regulation. In contrast, IL-2 exerts only minor effects on NF-kappa B p65 mRNA expression. IL-2 induction of NF-kappa B through the IL-2R beta subunit was both correlated with activation of the endogenous IL-2R alpha gene and critically dependent upon the presence of a serine-rich cytoplasmic domain within IL-2R beta (amino acid residues 267-312). This domain has previously been shown to be essential for IL-2-induced cell growth and may correspond to a binding site for an IL-2R beta-associated tyrosine kinase. Together, these findings suggest that NF-kappa B may play an important role as one intracellular second messenger relaying signals from the plasma membrane to cell nucleus that leads to IL-2-induced activation and growth.

This study was undertaken to evaluate an image processing method for assessing liver fibrosis in conventional computed tomography (CT) scans in patients with chronic hepatitis C. Two cohorts (designated "estimation," n = 34; and "validation," n = 107) of chronic hepatitis C patients were assessed using digitized conventional helical CT. Weighted CT mean fibrosis (Fibro-CT) was calculated as a nonlinear weighted mean F-score for each sample. Fibrosis was defined according to Scheuer on the F0 to F4 scale by 2 pathologists blinded regarding the Fibro-CT data. Fibrosis according to Fibro-CT correlated with histology-determined fibrosis (r = 0.69; P < 0.001) and with increasing F-stage: F0 = 0.23 +/- 0.39; F1 = 0.90 +/- 0.99; F2 = 1.41 +/- 0.94; F3 = 2.79 +/- 0.55; F4 = 3.15 +/- 0.35 [analysis of variance: P < 0.0001). The receiver operating characteristics curve to diagnose significant fibrosis >>/=F2) was 0.83; 95% confidence interval (95%CI), 0.75 to 0.91; and, to diagnose advanced fibrosis >>/=F3), was 0.86, 95%CI: 0.80 to 0.93. The correlation between Fibro-CT and fibrosis was higher in patients with homogeneous distribution of fibrosis than in patients with heterogeneous distribution (r = 0.77 versus r = 0.43; P < 0.05).

CONCLUSIONS

Optical digital analysis of CT images of the liver is effective in determining the stage and distribution of liver fibrosis in chronic hepatitis C. In patients with homogeneous fibrosis distribution, the correlation between Fibro-CT and histology was better than in patients with heterogeneous distribution. Fibro-CT is a simple to use, readily available, and useful method for the diagnosis of fibrosis in patients with chronic hepatitis C.