BACKGROUND

Diffusion-weighted magnetic resonance (MR) imaging (DWI) has been suggested for staging liver fibrosis. The aim of this study was to evaluate the diagnostic accuracy of DWI for the noninvasive assessment of hepatic fibrosis.

METHODS

We retrospectively compared DWI from clinically acquired MR scans with histologic methods. Liver biopsy specimens were staged F0-F4 in accordance with the METAVIR score. Hepatic steatosis was classified on a 5-point scale. Hepatic iron was graded on a 3-point scale. Liver inflammation was scored according to the modified hepatic activity index. Nonparametric methods, linear regression models, and receiver operating characteristic analyses were used to determine diagnostic accuracy and apparent diffusion coefficient (ADC) cutoff values.

RESULTS

Liver ADC values were inversely correlated with fibrosis stage: P = -0.54 (P < 0.0001). Although there was substantial overlap in the ADC distributions, the differences in ADC values by METAVIR stages F0 versus (vs.) F1-4, F0-1 versus F>> 1, F0-2 versus F3-4 and F0-3 versus F4 were all significant. For prediction of fibrosis stage 1, stage 2, stage 3, and stage 4 area under the receiver operating characteristic curve of 0.79, 0.77, 0.77, and 0.79 were obtained, respectively. Inflammation also correlated significantly with ADC values (P = -0.23, P = 0.03), but iron content (P = 0.17) or steatosis (P = 0.63) did not correlate with ADC measurements.

CONCLUSIONS

Liver ADC can be used to predict liver fibrosis with acceptable diagnostic accuracy. DWI should be included in further prospective studies to validate a comprehensive MR imaging protocol for the noninvasive assessment of hepatic fibrosis.

The goal of the present study was to clarify whether the primary motor cortex (M1) and the supplementary motor cortex (SMA) both receive, via the motor thalamus, input from cerebellar and basal ganglia output nuclei. This is the first investigation that explores the problem by direct comparison, in the same animal, of thalamic zones that 1) project to M1 and SMA and 2) receive cerebellar-nuclear (CN) and pallidal (GP) afferents. These four zones were mapped in two monkeys by means of two retrograde tracers for M1 and SMA injections and of two anterograde tracers for CN and GP injections. All injections were performed under electrophysiological control (microstimulation and multiunit recordings). Injections in cortical areas were restricted to the hand/arm representation; in the SMA, the tracer deposit was within the "SMA-proper" (or "area F3") and did not include its rostral extension ("pre-SMA" or "area F6"). It was found that zones of all four types formed a number of highly complex patches of labeling that were usually not confined to one cytoarchitectonically defined thalamic nucleus. The overlap of clusters of labeled terminals and perikarya was evaluated morphometrically (area measurements) on a number of coronal sections along the anteroposterior extent of the motor thalamus. In line with previous studies, the thalamic territories innervated by CN and GP afferents rarely overlapped. However, zones projecting to M1 and/or to SMA included thalamic regions receiving CN as well as GP projections, providing the first evidence of such overlap from individual animals. The present observations support the previous conclusion from this laboratory (based on transsynaptic labeling) that the SMA receives, apart from its strong pallidal transthalamic input, a CN transthalamic input. These present findings that both M1 and SMA are recipients of transthalamic inputs from GP and CN thus support the concept that a mixed subcortical input consisting of weighted contributions from cerebellum, basal ganglia, substantia nigra, and spinothalamic tract is directed to each functional component of the sensorimotor cortex.

We recently reported a high divergence among African subtype F strains. Three well-separated groups (F1, F2, and F3) have been shown based on the phylogenetic analysis of the p24 gag and envelope sequences with genetic distances similar to those observed for known subtypes. In this study, we characterized the near-full-length genomes of two strains from epidemiological unlinked individual belonging to each of the subgroups: F1 (96FR-MP411), F2 (95CM-MP255 and 95CM-MP257), and F3 (96CM-MP535 and 97ZR-EQTB11). Phylogenetic analysis of the near-full-length sequences and for each of the genes separately showed the same three groups, supported by high bootstrap values. Diversity plotting, BLAST subtyping, and bootstrap plotting confirmed that the divergent F strains correspond to nonrecombinant viruses. The divergence between F1 and F2 is consistently lower than that seen in any other intersubtype comparison, with the exception of subtypes B and D. Based on all the different analyses, we propose to divide subtype F into two subclades, with F1 gathering the known subtype F strains from Brazil and Finland, and our African strain (96FR-MP411), and F2 containing the 95CM-MP255 and 95CM-MP257 strains from Cameroon. The F3 strains, 97ZR-EQTB11 from the Democratic Republic of Congo and 96CM-MP535 from Cameroon, meet the criteria of a new subtype designated as K. The equidistance of subtype K to the other subtypes of HIV-1 suggests that this subtype existed as long as the others, the lower distance between B and D, and between F1 and F2 suggest a more recent subdivision for these latter strains.

FLT3 is a member of the type III receptor tyrosine kinase (RTK) family. These receptors all contain an intrinsic tyrosine kinase domain that is critical to signaling. Aberrant expression of the FLT3 gene has been documented in both adult and childhood leukemias including AML, ALL and CML. In addition, 17-27% of pediatric and adult patients with AML have small internal tandem duplication mutations in FLT3. Patients expressing the mutant form of the receptor have been shown to have a decreased chance for cure. Our previous study, using a constitutively activated FLT3, demonstrated transformation of Ba/F3 cells and leukemic development in an animal model. Thus, there is accumulating evidence for a role for FLT3 in human leukemias. This has prompted us to search for inhibitors of FLT3 as a possible therapeutic approach in these patients. AG1296 is a compound of the tyrphostin class that is known to selectively inhibit the tyrosine kinase activity of the PDGF and KIT receptors. Since FLT3 is a close relative of KIT, we wanted to test the possible inhibitory activity of AG1296 on FLT3. In transfected Ba/F3 cells, AG1296 selectively and potently inhibited autophosphorylation of FL-stimulated wild-type and constitutively activated FLT3. Treatment by AG1296 abolished IL-3-independent proliferation of Ba/F3 cells expressing the constitutively activated FLT3 and thus, reversed the transformation mediated by activated FLT3. Inhibition of FLT3 activity by AG1296 in cells transformed by activated FLT3 resulted in apoptotic cell death, with no deleterious effect on their parental counterparts. Addition of IL-3 rescued the growth of cells expressing activated FLT3 in the presence of AG1296. This demonstrates that the inhibition is specific to the FLT3 pathway in that it leaves the kinases of the IL-3 pathway and other kinases further downstream involved in proliferation intact. Several proteins phosphorylated by the activated FLT3 signaling pathway, including STAT 5A, STAT 5B and CBL, were no longer phosphorylated when these cells were treated with AG1296. The activity against FLT3 suggests a potential therapeutic application for AG1296 or similar drugs in the treatment of leukemias involving deregulated FLT3 tyrosine kinase activity and as a tool for studying the biology of FLT3.

Talin is a large cytoskeletal protein that couples integrins to F-actin. Three actin-binding sites (ABS1-3) have been reported: one in the N-terminal head, and two in the C-terminal rod domain. Although the C-terminal ABS3 has been partially characterized, the presence and properties of ABS1 within the talin head are less well defined. We show here that the talin head binds F-actin in vitro and in vivo at a specific site within the actin filament. Thus, purified talin head liberated from gizzard talin by calpain cleavage cosediments with F-actin in a low salt buffer at pH 6.4 (conditions that are optimal for binding intact talin), and using recombinant polypeptides, we have mapped ABS1 to the FERM domain within the talin head. Both the F2 and F3 FERM subdomains contribute to binding, and EGFP-tagged FERM subdomains colocalize with actin stress fibers when expressed in COS cells. High-resolution electron microscopy of actin filaments decorated with F2F3 localizes binding to a site that is distinct from that recognized by members of the calponin-homology superfamily. Finally, we show that the FERM domain can couple F-actin to PIPkin, and by inference to integrins, since they bind to the same pocket in the F3 subdomain. This suggests that the talin FERM domain functions as a linker between PIPkin or integrins and F-actin at sites of cell-matrix adhesions.

Apical dendrites of pyramidal neurons in the neocortex have a stereotypic orientation that is important for neuronal function. Neural recognition molecule Close Homolog of L1 (CHL1) has been shown to regulate oriented growth of apical dendrites in the mouse caudal cortex. Here we show that CHL1 directly associates with NB-3, a member of the F3/contactin family of neural recognition molecules, and enhances its cell surface expression. Similar to CHL1, NB-3 exhibits high-caudal to low-rostral expression in the deep layer neurons of the neocortex. NB-3-deficient mice show abnormal apical dendrite projections of deep layer pyramidal neurons in the visual cortex. Both CHL1 and NB-3 interact with protein tyrosine phosphatase alpha (PTPalpha) and regulate its activity. Moreover, deep layer pyramidal neurons of PTPalpha-deficient mice develop misoriented, even inverted, apical dendrites. We propose a signaling complex in which PTPalpha mediates CHL1 and NB-3-regulated apical dendrite projection in the developing caudal cortex.

The neural recognition molecule NB-3, which belongs to the contactin subgroup of the immunoglobulin superfamily, is expressed exclusively in the nervous system and mainly upregulated at the early postnatal stage during mouse brain development. The expression of NB-3 in the cerebellum increases until adulthood. In contrast, the expression in the cerebrum declines to a low level after postnatal day 7. To characterize the functional roles of NB-3 in vivo, we generated NB-3-deficient mice by substituting a part of the NB-3 gene with the beta-galactosidase (Lac Z) gene. Complete overlap of the Lac Z expression in the heterozygous mouse brain with the NB-3 immunostaining pattern in the rat cerebellum and with the previously reported pattern of in situ hybridization of NB-3 transcripts indicated that Lac Z expression reflects the expression of NB-3 in the mouse brain. NB-3-deficient mice were viable and fertile. The formation and organization of all nuclei and layers throughout the brains of mutant mice appeared normal. Behavioral tests to examine motor function showed that the mice deficient for NB-3 were slow to learn to stay on the rotating rod in the rotorod test during repeated trials, and that they displayed dysfunction of equilibrium and vestibular senses in the wire hang and horizontal rod-walking tests. In contrast, the mutant mice showed no difference of grasp force from the wild-type mice. Thus, NB-3-deficient mice are impaired in motor coordination.

In the era of antiretroviral therapy (ART), liver disease has emerged as an important cause of death among persons with human immunodeficiency virus (HIV)/hepatitis C virus (HCV) coinfection. The objective of this study was to estimate the burden of liver disease and evaluate determinants of liver fibrosis and necroinflammatory activity among HIV/HCV coinfected patients receiving ART. We studied 112 randomly selected and 98 referred HCV-infected patients undergoing care in the Johns Hopkins University HIV clinic. Liver disease was characterized clinically and histologically. Of the 210 individuals studied--64% of whom had received ART within 2 years of liver disease assessment--33% had no fibrosis (F0), and 26% had bridging fibrosis or cirrhosis >> or =F3). The median necroinflammatory activity score was 3 (range, 0-9 of 18). ART was not associated with fibrosis; however, significantly less hepatic necroinflammatory activity was observed among persons who had received highly active antiretroviral therapy longer (P = .02) and more effectively (defined by HIV RNA suppression; P < .01). Twelve percent of individuals had previous ART-associated liver enzyme elevations (grades 3-4), but liver fibrosis was not more severe if the liver enzyme elevation resolved. On the other hand, liver fibrosis was more severe in persons with persistent liver enzyme elevations (grades 1-4). In conclusion, despite widespread exposure to ART and documented instances of ART-related hepatitis, we found no evidence that ART caused serious histological liver disease. Recognition of bridging fibrosis and cirrhosis in some but not most patients underscores the importance of identifying and treating liver disease in HIV/HCV coinfected persons.

The direct-acting antivirals (DAAs) constitute an emerging group of small molecule inhibitors that effectively treat hepatitis C virus (HCV) infection, a common comorbidity in end-stage renal disease patients. To date, there are no data to guide use of these agents in kidney transplant patients. The authors collected data from 20 consecutive kidney recipients treated with interferon-free treatment regimens for HCV at their center: 88% were infected with genotype 1; 50% had biopsy-proved advanced hepatic fibrosis on their most recent liver biopsy preceding treatment (Metavir stage 3 fibrosis [F3] or F4); and 60% had failed treatment pretransplantation with interferon-based therapy. DAA treatment was initiated a median of 888 days after renal transplantation. All patients cleared the virus while on therapy, and 100% have achieved a sustained virologic response at 12 weeks after completion of DAA therapy. The most commonly used regimen was sofosbuvir 400 mg daily in combination with simeprevir 150 mg daily. However, four different treatment approaches were used, with comparable results. The DAAs were well tolerated, and less than half of patients required calcineurin inhibitor dose adjustment during treatment. Eradication of HCV infection with DAAs is feasible after kidney transplantation with few treatment-related side effects.

The expressions derived in the previous paper for the respective normal, F3, and shear forces, Fshear, acting along and perpendicular to the axis of a doublet of rigid spheres, were used to determine the hydrodynamic forces required to separate two red cell spheres of antigenic type B crosslinked by the corresponding antibody. Cells were sphered and swollen in isotonic buffered glycerol containing 8 X 10(-5) M sodium dodecyl sulfate, fixed in 0.085% glutaraldehyde, and suspended in aqueous glycerol (viscosity: 15-34 mPa s), containing 0.15 M NaCl and anti-B antibody from human hyperimmune antiserum at concentrations from 0.73 to 3.56 vol%. After incubating and mixing for 12 h, doublets were observed through a microscope flowing in a 178-micron tube by gravity feed between two reservoirs. Using a traveling microtube apparatus, the doublets were tracked in a constantly accelerating flow and the translational and rotational motions were recorded on videotape until breakup occurred. From a frame by frame replay of the tape, the radial position, velocity and orientation of the doublet were obtained and the normal and shear forces of separation at breakup computed. Both forces increased significantly with increasing antiserum concentration, the mean values of F3 increasing from 0.060 to 0.197 nN, and Fshear from 0.023 to 0.072 nN. There was no significant effect of glycerol viscosity on the forces of separation. It was not possible to determine whether the shear or normal force was responsible for doublet separation. Measurements of the mean dimensionless period of rotation, TG, of doublets in suspensions containing 0.73 and 2.40% antiserum undergoing steady flow were also made to test whether the spheres were rigidly linked or capable of some independent rotation. A fairly narrow distribution in TG about the value 15.64, predicted for rigidly-linked doublets, was obtained at both antiserum concentrations.

The physical location of histone molecules in a simian virus 40 DNA-histone complex isolated from purified virions was examined using site-specific restriction endonucleases. The complex contains four host histone species but lacks histone F1. Histones prevent complete cleavage of SV40 DNA by two restriction enzymes, HindIII and EcoRI. From the pattern of DNA fragments resulting from cleavage of the histone-DNA complex by the HindIII endonuclease, which makes six breaks on purified SV 40 DNA, we have concluded that histones are randomly arranged on SV40 DNA relative to restriction enzyme cleavage sites. The EcoRI endonuclease, which makes one break in SV40 NDA, was used to determine the degree of physical coverage of the SV 40 DNA molecule by histones. We observed that 80% of the EcoRI sites in the complex are accessible to the enzyme while 20% are "closed." This degree of coverage is consistent with the mass ratio of DNA:histone in the complex as revealed by the buoyant density of the formaldehyde-fixed complex. We conclude that the histones in the complex are located randomly on the SV 40 genome and cover approximatley 20% of the DNA. These results suggest that the histone species F2b, F2al, F2a2, and F3 are bound without regard to nucleotide sequence of SV 40 DNA.

Recent studies indicate that ovarian cancer may be highly responsive to antivascular therapeutics. We have developed an antivascular tumor therapeutic using the F3 peptide to target cisplatin-loaded nanoparticles (F3-Cis-Np) to tumor vessels. We show that although F3-Cis-Np bind with high specificity to both human ovarian tumor cells and tumor endothelial cells in vitro, they only show cytotoxic activity against the tumor endothelial cells. In vivo these nanoparticles bind primarily to tumor endothelial cells. Therapeutic studies in both flank and orthotopic i.p. murine ovarian tumor models, as well as human tumor xenograft models, show rapid tumor regression with treatment. Treatment was associated with significant vascular necrosis consistent with an antivascular effect. Furthermore, treatment was active in both platinum-sensitive and platinum-resistant cell lines. Importantly, we show that F3-Cis-Np bind to human tumor endothelial cells in vitro and to human tumor vessels in vivo. Therapy targeting human vasculature in vivo with F3-Cis-Np led to near complete loss of all human tumor vessels in a murine model of human tumor vasculature. Our studies indicate that F3-targeted vascular therapeutics may be an effective treatment modality in human ovarian cancer.

The FIP1L1-PDGFRA oncogene is a common cause of chronic eosinophilic leukemia (CEL), and encodes an activated tyrosine kinase that is inhibited by imatinib. FIP1L1-PDGFRA-positive patients with CEL respond to low-dose imatinib therapy, but resistance due to acquired T674I mutation has been observed. We report here the identification of sorafenib as a potent inhibitor of the FIP1 like 1-platelet-derived growth factor receptor alpha (FIP1L1-PDGFRalpha) (T674I) mutant. Sorafenib inhibited the proliferation of FIP1L1-PDGFRalpha and FIP1L1-PDGFRalpha(T674I)-transformed Ba/F3 cells and induced apoptosis of the EOL-1 cell line at a low nanomolar concentration. Western blot analysis confirmed that these effects were due to a direct effect on FIP1L1-PDGFRalpha and FIP1L1-PDGFRalpha(T674I). Sorafenib was recently approved for the treatment of renal cell carcinoma. Our data suggest that low doses of sorafenib could be efficient for the treatment of FIP1L1-PDGFRA-positive CEL and could be used to overcome resistance to imatinib associated with the T674I mutation.

The Cre-loxP recombination system has been used to great advantage in vivo for conditional gene targeting, lineage tracing, and other applications. To express cre in skeletal myoblasts and muscle fibers, we utilized the well-characterized transcriptional regulatory regions of the muscle determination gene, MyoD. Transgenic mouse lines were produced (F3/-2.5cre) in which the cre gene is driven by the MyoD promoter and core enhancer, which directs the early activation of MyoD. Specificity of cre expression and efficiency of recombination was determined by monitoring reporter gene expression after crossing to the Cre-dependent reporter lines, R26R and Z/AP. Efficient labeling of embryonic and fetal myoblasts and muscle fibers was observed, with timing that was similar (branchial arches and limb buds) or slightly delayed (myotomes) relative to the endogenous MyoD gene. In satellite cell cultures, a strict concordance between MyoD protein and reporter gene expression was observed, demonstrating the muscle specificity and efficiency of Cre-mediated recombination. Nascent muscle fibers were labeled following injury of adult muscle, indicating recombination in satellite cells or their daughters in vivo.

Neural stem cells are mobile, are attracted to regions of brain damage, and can migrate a considerable distance to reach a glioma site. However, the molecular basis of the progression of gliotropism to malignant gliomas remains poorly understood. With the use of clinically and histologically assessed glioma cells, we have assessed their protein and gene profiles via proteomics and microarray approaches, and have identified candidate genes from human glioma tissues. This research is expected to provide clues to the molecular mechanisms underlying the migration of neural stem cells (F3 cell) to glioma sites. The expression of 16 proteins was shown to have increased commonly in human glioma tissues. Among them, the expression of annexin A2, TIMP-1, COL11A1, bax, CD74, TNFSF8, and SPTLC2 were all increased in human glioma cells, as confirmed by Western blotting and immunohistochemical staining. In particular, annexin A2 effects an increase in migration toward F3 and glioblastoma cells (U87 cell) in a Boyden chamber migration assay. An ERK inhibitor (PD98057) and a CDK5 inhibitor (rescovitine) inhibited 50% and 90% of annexin A2-induced migration in F3 cells, respectively. A similar chemotactic migration was noted in F3 and U87 cells. These results demonstrated that 7 candidate proteins may harbor a potential glioma tropism factor relevant to the pathology of malignant glioma. These results reveal that this novel molecular approach to the monitoring of glioma may provide clinically relevant information regarding tumor malignancy, and should also prove appropriate for high-throughput clinical screening applications.

BACKGROUND

The area under the receiver operating characteristic (ROC) curve is widely used as an estimate of the diagnostic value for fibrosis markers. Biopsy length and fragmentation are known as risk factors of false positive or false negative of biopsy but their quantitative impact on area under the receiver operating characteristic curve variability has not been assessed.

OBJECTIVE

To assess these relationships to better compare the fibrosis markers.

METHODS

The area under the ROC curves of FibroTest for the diagnosis of fibrosis was estimated in patients with chronic hepatitis C using an integrated database including 1312 patients with FibroTest and biopsy. To take into account the biopsy length, we used two adjustment factors: one in which an observed area under the ROC curve could be adjusted according to the relative area under the receiver operating characteristic curve of a biopsy of a given length vs. the entire liver and one taking into account the prevalence of each fibrosis stage defining advanced and non-advanced fibrosis.

RESULTS

The mean biopsy length was smaller for cirrhosis (F4, 16 mm) vs. F3, (18 mm, P=0.01) and F0 (19 mm, P=0.01). The mean number of fragments was higher for cirrhosis (F4=4.1 fragments) vs. all the other stages (F0=1.9, F1=1.9, F2=1.9, F3=2.3; P<0.001 vs. F4). The FibroTest area under the ROC curves for the diagnosis of advanced fibrosis, adjusted for stages' prevalence, ranged from 0.80 to 0.98 depending on biopsy length and fragmentation, respectively.

CONCLUSIONS

The comparison of the area under the ROC curves of fibrosis markers should take into account the biopsy length and fragmentation.

Up to 50% of patients with chronic hepatitis C fail to respond to initial therapy with pegylated interferon (PEG-IFN) and ribavirin (RBV). With unsuccessful viral eradication, these patients remain at risk for developing progression of their liver disease. Retreatment with PEG-IFN/RBV yields sustained virologic response (SVR) rates that are under 10%. A wholly synthetic interferon, interferon alfacon-1 or consensus interferon (CIFN) given with RBV, was evaluated in patients who failed initial PEG-IFN/RBV therapy. The intent-to-treat analysis included 487 patients; 245 received CIFN 9 microg/day and RBV, and 242 received CIFN 15 microg/day and RBV. Within this group of patients, 59.3% had documented advanced fibrosis at baseline liver biopsy (stage F3 or F4). SVR rates were 6.9% (17/245 patients) in the 9 microg group and 10.7% (26/242) in the 15 microg group. In the intent-to-treat analysis, SVR rates were higher among patients with a >2-log(10) decrease in hepatitis C virus RNA during prior PEG-IFN/RBV therapy: 11% (4/38) in the 9 mug group and 23% (7/31) in the 15 microg group. Among patients with lower baseline fibrosis scores (F0-F3), SVR rates were 7.8% (15/192) in the 9 microg group and 13.1% (23/175) in the 15 microg group. In this same group of patients (F0-F3), if a >2-log(10) decrease in hepatitis C virus RNA with previous PEG-IFN/RBV treatment was achieved, SVR rates improved to 10.7% and 31.6% in the 9 microg and 15 microg groups, respectively. CIFN/RBV combination retreatment was safe and well tolerated.

CONCLUSIONS

Retreatment of PEG-IFN and RBV nonresponders with CIFN and RBV is safe and efficacious and can be considered a retreatment strategy for patients failing previous therapy with PEG-IFN/RBV, especially in interferon-sensitive patients with lower baseline fibrosis scores.

Circular dichroism spectra have been obtained for albumin, alpha-chymotrypsinogen, collagen, concanavalin A, elastase, hemoglobin, histone f2b, alpha-lactalbumin, lactate dehydrogenase, beta-lactoglobulin, lysozyme, myoglobin, papain, ribonuclease A, and thermolysin in the presence of sodium dodecyl sulfate and dithiothreitol. While all spectra have the shape anticipated for a mixture of random coil and alpha helix, the intensities differ markedly ([theta]222 ranges from --1400 to --15 000 deg cm2/dmol). The variation in the circular dichroism can be quantitatively explained by a model which assumes that the arginyl, histidyl, and lysyl residues have an enhanced probability of propagating a helical segment in the presence of the detergent. The model also permits the computation of dimensional properties (unperturbed end-to-end distance and radius of gyration) for polypeptides of known amino acid sequence. Such computations have been performed for 67 proteins. The computed dimensions are compatible with experimental values and with the molecular weight dependence of the transport properties of the complexes. Furthermore, the model can account for the abnormal transport properties of the sodium dodecyl sulfate complexes formed by ribonuclease A, collagen fragments, and histones f2a1, f2a2, f2b, and f3. Even though some of the protein--sodium dodecyl sulfate complexes have helical contents as high as 50%, their overall conformation more closely approximates that of a random coil than a rod.

Resistance toward imatinib and other BCR/ABL tyrosine kinase inhibitors remains an increasing clinical problem in the treatment of advanced stages of chronic myeloid leukemia (CML). We recently have identified the heat shock protein 32 (Hsp32)/heme oxygenase-1 (HO-1) as a BCR/ABL-dependent survival molecule in CML cells. We here show that silencing Hsp32/HO-1 in CML cells by an siRNA approach results in induction of apoptosis. Moreover, targeting Hsp32/HO-1 by either pegylated zinc protoporphyrine (PEG-ZnPP) or styrene maleic acid-micelle-encapsulated ZnPP (SMA-ZnPP) resulted in growth inhibition of BCR/ABL-transformed cells. The effects of PEG-ZnPP and SMA-ZnPP were demonstrable in Ba/F3 cells carrying various imatinib-resistant mutants of BCR/ABL, including the T315I mutant, which exhibits resistance against all clinically available BCR/ABL tyrosine kinase inhibitors. Growth-inhibitory effects of PEG-ZnPP and SMA-ZnPP also were observed in the CML-derived human cell lines K562 and KU812 as well as in primary leukemic cells obtained from patients with freshly diagnosed CML or imatinib-resistant CML. Finally, Hsp32/HO-1-targeting compounds were found to synergize with either imatinib or nilotinib in producing growth inhibition in imatinib-resistant K562 cells and in Ba/F3 cells harboring the T315I mutant of BCR/ABL. In summary, these data show that HO-1 is a promising novel target in imatinib-resistant CML.

Insulin-like growth factor-binding proteins (IGFBPs) are a family of secreted proteins that bind insulin-like growth factors I and II (IGFs I and II) and are capable of modulating IGF actions on target cells. We have shown previously that C2 myoblasts secrete a single approximately 29-kDa IGFBP during their terminal differentiation (Tollefsen, S. E., Lajara, R., McCusker, R. H., Clemmons, D. R., and Rotwein, P. (1989) J. Biol. Chem. 264, 13810-13817). In this study, we have purified the protein from C2 cell-conditioned media by conventional and IGF-affinity chromatography, cloned its cDNA by PCR-based and traditional library screening, and identified it as mouse IGFBP-5. The resultant 5561 nucleotide cDNA encodes a 252-amino acid mature protein (predicted M(r) approximately 28,400) that is 97% identical to rat and human IGFBP-5. In differentiating C2 myoblasts and in F3 azamyoblasts the>> 6-kilobase IGFBP-5 mRNA accumulates concomitantly with induction of myogen mRNA, an early marker of muscle differentiation. Ligand blot analysis shows that IGFBP-5 protein is secreted within 12 h of the onset of differentiation in these cells and that it is the only IGFBP produced in several fusing skeletal muscle cell lines. In vivo, IGFBP-5 transcripts are expressed in a variety of mouse tissues including striated muscle, but, unlike other IGFBPs, it is barely detectable in liver. IGFBP-5 is more conserved than other IGFBPs in mammals; its conserved structure and sequence also extends to non-mammalian vertebrates. Hybridization of a mouse BP5 coding region probe to RNA from several chicken and Xenopus tissues demonstrated similarly sized transcripts in these species. A partial Xenopus cDNA is identical in 38/45 deduced amino acids to the mammalian proteins. Identification of an IGF-binding protein that is produced during myoblast differentiation provides a model system in which to study the potential modulatory role of IGFBPs in development.

The suppressor of cytokine signaling-3 (SOCS3/CIS-33/SSI-3) is an important negative regulator of cytokine signaling. Here, we show that an N-terminal truncated isoform (DeltaN-SOCS3) translated from the internal AUG codon 12 was profoundly induced by endoplasmic reticulum (ER) stress- or active double-stranded RNA-activated protein kinase PKR, as a result of induction of eukaryotic initiation factor 2alpha phosphorylation. DeltaN-SOCS3 exhibited a stronger cytokine-inhibitory activity and a higher stability than WT-SOCS3 in Ba/F3 hematopoietic cells. A potential ubiquitination residue, Lys-6, at the N terminus is evolutionary conserved among SOCS3 species. The K6Q-SOCS3 mutant showed a much longer half-life than WT-SOCS3 in Ba/F3 cells. Furthermore, inhibition of the 26 S proteasome pathway increased both ubiquitination and protein levels of WT-SOCS3 but had no effect on K6Q-SOCS3. SOCS3 mutant lacking the carboxyl-terminal SOCS-box exhibited the same stability as K6Q-SOCS3. These observations suggest that the short form of SOCS3 is a naturally occurring stabilized inhibitory protein, whereas WT-SOCS3 is a short-lived protein modulated by Lys-6 ubiquitination and proteasome-dependent degradation. Our findings provide strong evidence for the first time that translational control plays an important role in stabilization and function of SOCS3.

OBJECTIVE

Transient elastography (TE) can non-invasively diagnose cirrhosis and portal hypertension (PHT). New TE reliability criteria suggest classifying measurements as very reliable (IQR/M < 0.1), reliable (IQR<0.3 or >0.3, if TE < 7.1 kPa) and poorly reliable (IQR/M>> 0.3, if TE>> 7.1 kPa). Compare traditional (reliable: success rate >60% + IQR/M ≤ 0.30) and new TE quality criteria (accurate: very reliable + reliable) regarding their diagnostic accuracy for cirrhosis and PHT and to identify potential confounders (age, aetiology, necroinflammatory activity, steatosis, siderosis, cholestasis, aminotransferases) of TE performance.

METHODS

Patients undergoing simultaneous measurements of TE, portal pressure (hepatic venous pressure gradient, HVPG) and liver biopsy were analysed.

RESULTS

Among 226 patients (48.7 ± 13.1 years, 74.7% male, 75.7% viral aetiology, 57% F3/F4), traditional TE quality criteria identified 71.6% reliable measurements, while new criteria yielded in 83.2% accurate results. Reliable TE values according to both criteria significantly correlated with fibrosis stage (r = 0.648 vs. r = 0.636) and HVPG (r = 0.836 vs. r = 0.846). Diagnostic accuracy for cirrhosis (cut-off >14.5 kPa) was 76.5% (AUC: 0.863) and 75.0% (AUC: 0.852) for traditional and new TE criteria, respectively, while for predicting HVPG ≥ 10 mmHg (>16.1 kPa), the accuracies were 88.9% (AUC: 0.957) and 89.8% (AUC: 0.962). New TE criteria allowed a better discrimination of reliable and non-reliable results for prediction of fibrosis and CSPH. Only aetiology and aminotransferases were independent confounders of the correlation of TE and fibrosis stage, while no confounder affected the correlation of TE and HVPG.

CONCLUSIONS

New reliability criteria for TE measurements increase the number of patients with accurate measurements without affecting diagnostic performance for detecting cirrhosis and portal hypertension. Aetiology of liver disease and aminotransferases should be considered when assessing liver fibrosis by TE.

A sulfated fucan containing fraction (SmWE) was isolated from water extract of the brown seaweed Stoechospermum marginatum collected from the Arabian Sea. Anion exchange chromatography of the crude fraction results in the production of a sulfated fucan (F3) having a molecular mass of 40 kDa and specific rotation [alpha]D(30) - 124 degrees (c 0.5, H2O). NMR spectroscopic studies and methylation analysis suggested that the polymer consists of a backbone of (1-->4)- and (1-->3)-linked-alpha-L-fucopyranosyl residues that are substituted at C-2 and C-3, and that fucosyl residues are sulfated mostly at C-2 and/or C-4. SmWE and F3 were selective inhibitors of herpes simplex virus type 1 (strain F, thymidine kinase-deficient strains field and B2006 and syncytial variants arising after selection with a natural carrageenan syn 13-8 and 14-1) and type 2 (strain MS) in Vero cells, with antiviral effective concentration 50% (EC50) values in the range 0.63-10.0 microg/ml. The compounds were highly selective due to the lack of cytotoxicity. The antiviral activity was dependent on the presence of the sulfated fucans during the adsorption period. No direct inactivating effect on virions was observed in a virucidal assay. The absence of anticoagulant activity at concentrations near EC50 confirmed that there was no correlation between the antiviral and anticoagulant properties.

Chemokines are chemotactic cytokines that direct the traffic of leukocytes and other cells in the body. Chemokines bind to G protein-coupled receptors expressed on target cells to initiate signaling cascades and induce chemotaxis. Although the cognate receptors of most chemokines have been identified, the receptor for the mucosal chemokine CXCL17 is undefined. In this article, we show that GPR35 is the receptor of CXCL17. GPR35 is expressed in mucosal tissues, in CXCL17-responsive monocytes, and in the THP-1 monocytoid cell line. Transfection of GPR35 into Ba/F3 cells rendered them responsive to CXCL17, as measured by calcium-mobilization assays. Furthermore, GPR35 expression is downregulated in the lungs of Cxcl17(-/-) mice, which exhibit defects in macrophage recruitment to the lungs. We conclude that GPR35 is a novel chemokine receptor and suggest that it should be named CXCR8.