BACKGROUND

The chronic kidney disease (CKD)-mineral and bone disorder (MBD) syndrome is an important contributor to the CKD-associated cardiovascular disease and high mortality rates. Sclerostin, a protein synthesized in osteocytes, is a potent downregulator of bone metabolism and a novel candidate for the bone-vascular axis in CKD patients. We tested whether serum sclerostin values are predictive for all-cause mortality and cardiovascular events (CVEs) in a CKD population.

METHODS

Serum sclerostin was obtained from 173 CKD (stage 3-5) and 47 control patients, and its concentration was correlated with estimated glomerular filtration rate and to mineral and vascular abnormalities that are present in the CKD evolution. All-cause mortality and CVEs were also analyzed in relation to serum sclerostin values.

RESULTS

Patients with CKD showed higher sclerostin levels (median 63.5 pmol/L vs 52 pmol/L, P < .001) than controls, with values progressively higher across the CKD stages. In univariate analysis, serum sclerostin concentrations were correlated with gender, estimated glomerular filtration rate, flow-mediated dilatation, and endothelium-independent vasodilatation as markers of endothelial dysfunction and with different serum CKD-MBD-associated parameters. However, in multivariate analysis, only gender, <em>fibroblast</em> <em>growth</em> <em>factor</em>-23, phosphate, flow-mediated dilatation, and cholesterol remained significantly associated with sclerostin levels. During the observational period, there were <em>19</em> deaths and 50 CVEs. In survival analysis, different sclerostin levels were associated with all-cause mortality and CVEs in these patients.

CONCLUSIONS

This is the first study that shows that serum sclerostin values are associated, even after multiple adjustments, with fatal and nonfatal CVEs in a nondialyzed CKD population.

We isolated the cDNA encoding a novel member of the human <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family from the lung. The cDNA encodes a protein of 208 amino acids with high sequence homology (95.6%) to rat FGF-10, indicating that the protein is human FGF-10. Human FGF-10 as well as rat FGF-10 has a hydrophobic amino terminus ( approximately 40 amino acids), which may serve as a signal sequence. The apparent evolutionary relationships of human FGFs indicate that FGF-10 is closest to FGF-7. Chromosomal localization of the human FGF-10 gene was examined by in situ hybridization. The gene was found to map to the 5p12-p13 region. Human FGF-10 (amino acids 40 to 208 with a methionine residue at the amino terminus) was produced in Escherichia coli and purified from the cell lysate. Recombinant human FGF-10 (approximately <em>19</em> kDa) showed mitogenic activity for fetal rat keratinizing epidermal cells, but essentially no activity for NIH/3T3 cells, <em>fibroblasts</em>. The specificity of mitogenic activity of FGF-10 is similar to that of FGF-7 but distinct from that of bFGF. In structure and biological activity, FGF-10 is similar to FGF-7.

Two putative receptors for <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) of approximately 150 and 200 kD were identified in membrane preparations from chick embryos. Specific binding (femtomoles/milligram) of 125I-aFGF to whole chick embryonic membranes was relatively constant from day 2 to 7, then decreased fivefold between days 7 and 13. Day-<em>19</em> chick embryos retained 125I-aFGF binding at low levels to brain, eye, and liver tissues but not to skeletal muscle or cardiac tissues. The 200-kD FGF receptor began to decline between day 4.5 and 7 and was barely detectable by day 9, whereas the 150-kD FGF receptor began to decline by day 7 but was still detectable in day-9 embryonic membranes. It is not known whether the two FGF-binding proteins represent altered forms of one polypeptide, but it is clear that their levels undergo differential changes during development. Because endogenous chick FGF may remain bound to FGF receptor in membrane preparations, membranes were treated with acidic (pH 4.0) buffers to release bound FGF; such treatment did not affect 125I-aFGF binding and moderately increased the number of binding sites in day-7 and -<em>19</em> embryos. Consequently, the observed loss of high affinity 125I-aFGF binding sites and FGF-binding polypeptides most likely represents a loss of FGF receptor protein. These experiments provide in vivo evidence to support the hypothesis that regulation of FGF receptor levels may function as a mechanism for controlling FGF-dependent processes during embryonic development.

Defects in multidrug resistance 3 gene (MDR3), which encodes the canalicular phospholipid flippase, cause a wide spectrum of cholangiopathy phenotypes in humans. Mice deficient in Mdr2 (murine ortholog of MDR3) develop liver diseases that closely reproduce the biochemical, histological, and clinical features of human cholangiopathies such as progressive familial intrahepatic cholestasis and primary sclerosing cholangitis. We hypothesized that modulating bile acid metabolism by the gut hormone <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) may represent a novel approach for treating cholangiopathy and comorbidities. We introduced adeno-associated virus carrying the gene for either the endocrine hormone FGF<em>19</em> or engineered FGF<em>19</em> variant M70 to 12-week old Mdr2-deficient mice with fully established disease. Effects on serum levels of liver enzymes, liver histology, and bile acid homeostasis were evaluated. FGF<em>19</em> and M70 rapidly and effectively reversed liver injury, decreased hepatic inflammation, attenuated biliary fibrosis, and reduced cholecystolithiasis in Mdr2-deficient mice. Mechanistically, FGF<em>19</em> and M70 significantly inhibited hepatic expression of Cyp7a1 and Cyp27a1, which encode enzymes responsible for the rate-limiting steps in the classic and alternate bile acid synthetic pathways, thereby reducing the hepatic bile acid pool and blood levels of bile acids. Importantly, prolonged exposure to FGF<em>19</em>, but not M70, led to the formation of hepatocellular carcinomas in the Mdr2-deficient mice. Furthermore, M70 ameliorated the hepatosplenomegaly and ductular proliferation that are associated with cholangiopathy.

CONCLUSIONS

These results demonstrate the potential for treating cholangiopathy by safely harnessing FGF<em>19</em> biology to suppress bile acid synthesis.

Chronic diarrhoea induced by bile acids is common and the underlying mechanisms are linked to homeostatic regulation of hepatic bile acid synthesis by <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>). Increasing evidence, including that from several large case series using SeHCAT (selenium homocholic acid taurine) tests for diagnosis, indicates that bile acid diarrhoea (BAD) accounts for a sizeable proportion of patients who would otherwise be diagnosed with IBS. Studies of other approaches for diagnosis of BAD have shown increased bile acid synthesis, increased faecal levels of primary bile acids, dysbiosis and different urinary volatile organic compounds when compared with healthy controls or with other diseases. The role of the ileal hormone FGF<em>19</em> in BAD has been strengthened: a prospective clinical study has confirmed low FGF<em>19</em> levels in BAD, and so a test to measure these levels could be developed for diagnosis. In animal models, FGF<em>19</em> depletion by antibodies produces severe diarrhoea. Bile acids affect colonic function through farnesoid X receptor (FXR) and TGR5 receptors. As well as these effects in the colon, FXR-dependent stimulation of ileal FGF<em>19</em> production could be a logical mechanism to provide therapeutic benefit in BAD. Further studies of FGF<em>19</em> in humans hold promise in providing novel treatments for this cause of chronic diarrhoea.

The endocrine-derived hormone <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>19</em> has recently emerged as a potential target for treating metabolic disease. Given that skeletal muscle is a key metabolic organ, we explored the role of FGF<em>19</em> in that tissue. Here we report a novel function of FGF<em>19</em> in regulating skeletal muscle mass through enlargement of muscle fiber size, and in protecting muscle from atrophy. Treatment with FGF<em>19</em> causes skeletal muscle hypertrophy in mice, while physiological and pharmacological doses of FGF<em>19</em> substantially increase the size of human myotubes in vitro. These effects were not elicited by FGF21, a closely related endocrine FGF member. Both in vitro and in vivo, FGF<em>19</em> stimulates the phosphorylation of the extracellular-signal-regulated protein kinase 1/2 (ERK1/2) and the ribosomal protein S6 kinase (S6K1), an mTOR-dependent master regulator of muscle cell <em>growth</em>. Moreover, mice with a skeletal-muscle-specific genetic deficiency of β-Klotho (KLB), an obligate co-receptor for FGF15/<em>19</em> (refs. 2,3), were unresponsive to the hypertrophic effect of FGF<em>19</em>. Finally, in mice, FGF<em>19</em> ameliorates skeletal muscle atrophy induced by glucocorticoid treatment or obesity, as well as sarcopenia. Taken together, these findings provide evidence that the enterokine FGF<em>19</em> is a novel <em>factor</em> in the regulation of skeletal muscle mass, and that it has therapeutic potential for the treatment of muscle wasting.

Well-differentiated liposarcoma (WDLS) is one of the most common malignant mesenchymal tumors and dedifferentiated liposarcoma (DDLS) is a malignant tumor consisting of both WDLS and a transformed nonlipogenic sarcomatous component. Cytogenetically, WDLS is characterized by the presence of ring or giant rod chromosomes containing several amplified genes, including MDM2, TSPAN31, CDK4, and others mainly derived from chromosome bands 12q13-15. However, the 12q13-15 amplicon is large and discontinuous. The focus of this study was to identify novel critical genes that are consistently amplified in primary (nonrecurrent) WDLS and with potential relevance for future targeted therapy. Using a high-resolution (5.0 kb) "single nucleotide polymorphism"/copy number variation microarray to screen the whole genome in a series of primary WDLS, two consistently amplified areas were found on chromosome 12: one region containing the MDM2 and CPM genes, and another region containing the FRS2 gene. Based on these findings, we further validated FRS2 amplification in both WDLS and DDLS. Fluorescence in situ hybridization confirmed FRS2 amplification in all WDLS and DDLS tested (n = 57). Real time PCR showed FRS2 mRNA transcriptional upregulation in WDLS (n = <em>19</em>) and DDLS (n = 13) but not in lipoma (n = 5) and normal fat (n = 9). Immunoblotting revealed high expression levels of phospho-FRS2 at Y436 and slightly overexpression of total FRS2 protein in liposarcoma but not in normal fat or preadipocytes. Considering the critical role of FRS2 in mediating <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor signaling, our findings indicate that FRS2 signaling should be further investigated as a potential therapeutic target for liposarcoma.

OBJECTIVE

To determine whether primary drug resistance or rapid relapse explains the poor prognosis seen in t(4;14)-positive multiple myeloma (MM).

METHODS

A total of 131 patients treated with high-dose therapy (HDT) were assessed, of whom <em>19</em> were t(4;14) positive. We examined the presentation features, chemotherapy responsiveness at presentation and to salvage therapies at relapse, and overall survival outcomes.

RESULTS

t(4;14)-positive patients had a predominance of the immunoglobulin A isotype (52.6%) but otherwise baseline characteristics were indistinguishable. After treatment with vincristine, doxorubicin, and dexamethasone or dexamethasone alone, 17 (89.7%) of the <em>19</em> patients achieved a partial response and 11 patients (57.9%) demonstrated an additional 50% reduction in paraprotein after HDT. Thus, t(4;14)-positive patients are chemotherapy sensitive; however, early progression was common, with 26% of patients progressing before HDT and a median progression-free survival after HDT of only 14.1 months. At relapse, a resistance to alkylating agents was evident, with no responses (zero of 11 patients) seen with conventional-dose alkylating agents. Salvage regimens using thalidomide and/or dexamethasone achieved at least minimal response in 59% of patients. The duration of response was short, however, with a median of only 4.7 months. The median overall survival after HDT was 24.2 months.

CONCLUSIONS

We conclude that t(4;14)-positive MM is chemotherapy sensitive but rapid relapse occurs. Resistance to alkylating agents is evident at relapse. The development of novel therapeutic agents is required, including the early clinical study of targeted fibroblast growth factor receptor 3 tyrosine kinase inhibitors, which have shown promise in preclinical studies.

This Phase I study of MMI270, an p.o. administered matrix metalloproteinase inhibitor, assessed toxicity, pharmacokinetics, and tumor response data and investigated markers of biological activity to recommend a dose for Phase II studies. MMI270 was administered continuously at seven dose levels (50 mg once daily to 600 mg three times/day). Patients were evaluated for toxicity and tumor response, and blood and urine samples were taken for pharmacokinetics, bone resorption markers, direct targets of the inhibitor [matrix metalloproteinase-2 (MMP-2), MMP-8, and MMP-9], indirect targets [tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, vascular endothelial <em>growth</em> <em>factor</em>, vascular cell adhesion molecule-1, soluble urokinase plasminogen activator receptor, and cathepsins B and H] and for a tumor necrosis <em>factor</em>-alpha cytokine release assay. Ninety-two patients were entered. There was no myelotoxicity. Eighteen patients developed a widespread maculopapular rash, which increased in frequency and severity at doses>> or = 300 mg bid. Thirty nine patients developed musculoskeletal side effects, which were related to duration of treatment, not to dose level. Pharmacokinetics were linear, and MMI270 was rapidly absorbed and eliminated with minimal accumulation on chronic dosing. Sustained plasma concentrations in excess of 4 x mean IC(50) for the target enzymes were observed at dose levels>> or = 150 mg bid. There were no tumor regressions; however, <em>19</em> patients had stable disease for>> or = 90 days. There was a dose-response increase of MMP-2 and TIMP-1 with MMI270. Transient effects on the bone resorption markers were detected. MMI270 was generally well tolerated, with adequate plasma levels for target enzyme inhibition. The two main toxicities were rash, resulting in a maximum tolerated dose of 300 mg bid and musculoskeletal side effects. Biological marker data indicate drug effects. The rise in TIMP-1 suggests that a reflex rise in inhibitors could modify the effects of MMI270. The recommended Phase II dose is 300 mg bid.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) is a hormone released from the small intestine; recently, it has emerged as an endocrine regulator of glucose and lipid metabolism. The aim of this study was to investigate the role of FGF<em>19</em> in the development of nonalcoholic fatty liver disease (NAFLD).

METHODS

This study included 23 (17 boys) obese adolescents (mean age of 14.1 years) with NAFLD. The control group consisted of 34 (13 boys) obese peers with normal ultrasonographic imaging and normal liver function tests.

METHODS

The definition of NAFLD was based on clinical criteria: elevated alanine aminotransferase (>35 U/L) and liver steatosis features on ultrasound imaging. Serum FGF<em>19</em> levels were measured in a fasting blood sample. The definition of insulin resistance was based on the homeostasis model assessment (HOMA) threshold: >2.5.

RESULTS

There was a significant difference between mean FGF<em>19</em> levels in patients with NAFLD and controls (142.2 vs. 206 pg/mL, p=0.04). Mean fasting FGF<em>19</em> levels were decreased in insulin-resistant patients in comparison with the non-insulin-resistant group (155.0 vs. 221.0 pg/mL, p=0.05). There was an inverse correlation between FGF<em>19</em> and alanine aminotransferase levels (R=-0.3, p<0.05) and triglycerides (R=-0.27, p<0.05).

CONCLUSIONS

A decrease in fasting FGF<em>19</em> is associated with the development of NAFLD in obese adolescents. A decrease in fasting FGF<em>19</em> levels may be a new important risk <em>factor</em> for NAFLD and the metabolic syndrome in adolescents. Further studies are needed to explain whether exogenous delivery of FGF<em>19</em> might be therapeutically beneficial.

It has been suggested that hepatocytes have the ability to form bile ductal structures during normal development and in various pathological conditions of the liver. In the present study, we attempted to establish an in vitro model of ductal morphogenesis of hepatocytic cells by combining an aggregate culture and a type I collagen gel culture. When spheroidal aggregates of rat or mouse primary hepatocytes were embedded within the collagen gel matrix and then cultured with a medium containing a <em>fibroblast</em>-conditioned medium, the aggregates extended many dendritic processes composed of a trabecular arrangement of cells. Dendritic morphogenesis was also seen in embedded aggregates of immortal liver epithelia] cell lines, which spontaneously emerged during long-term cultures of mouse primary hepatocytes. A similar morphogenesis was induced by the presence of insulin in the medium. Although epidermal <em>growth</em> <em>factor</em> (EGF) and hepatocyte <em>growth</em> <em>factor</em> (HGF) showed only a small effect on the morphogenesis of most of the hepatocytic cells when used alone, these <em>factors</em>, especially EGF, enhanced the morphogenetic effect of insulin. Electron microscopical observations revealed luminal structures lined by microvilli within these dendritic processes, indicating ductal differentiation. Immunocytochemically, the dendritic processes were positive for cytokeratin <em>19</em>, a marker for bile duct cells. On the other hand, an H-ras-transformed mouse liver epithelial cell line and rat hepatocellular carcinoma cell lines did not demonstrate the organized morphogenesis. Our results indicate that hepatocytic cells can produce bile duct-like structures in the presence of the type I collagenous matrix and soluble morphogenetic <em>factors</em>.

Human embryonic stem cell (hESC) culture is routinely performed using inactivated mouse embryonic <em>fibroblasts</em> (MEFs) as a feeder cell layer (FL). Although these cells maintain pluripotency of hESCs, the molecular basis for this is unknown. Objectives of this study were to determine whether timing between MEF inactivation and their use as a FL influenced hESC <em>growth</em> and differentiation, and to begin defining the mechanism(s) involved. hESCs were plated on MEFs prepared 1 (MEF-1), 4 (MEF-4), and 7 (MEF-7) days earlier. hESC colony morphology and Oct3/4 expression levels were evaluated to determine the influence of different FLs. Significant enhancement of hESC <em>growth</em> (self-renewal) was observed on MEF-1 compared with MEF-4 and/or MEF-7. Conditioned media (CM) collected from MEF-1 supported significantly better hESC <em>growth</em> in a FL-free system compared to MEF-7 CM. Effects of MEFs on hESC <em>growth</em> were not caused by differences in cell density or viability, although indications of apoptosis were observed in MEF-7. Scanning electron microscopy demonstrated that MEF-7 were morphologically distinct from MEF-1 and MEF-4. Microarray analysis identified <em>19</em> genes related to apoptosis with significantly different levels of expression between MEF-1 and MEF-7. Several differentially expressed RNAs had gene ontology classifications associated with extracellular matrix (ECM) structural constituents and <em>growth</em> <em>factors</em>. Because members of Wnt signaling pathway were identified in the array analysis, we examined the ability of the Wnt1 CM and secreted frizzled-related proteins to affect hESC <em>growth</em> and differentiation. The addition of Wnt1 CM to both MEF-1 and MEF-7 significantly increased the number of undifferentiated colonies, while the addition of Sfrps promoted differentiation. Together, these results suggest that microenvironment, ECM, and soluble <em>factors</em> expressed by MEF-1 are significantly better at maintaining self-renewal and pluripotency of hESCs. Our findings have important implications in the optimization of hESC culture when MEFs are used as FL or CM is used in FL-free culture.

The erythropoietin-producing hepatocellular (Eph) family of ligands and receptors has been implicated in the control of axon guidance and the segmental restriction of cells during embryonic development. In this report, we show that ectopic expression of XLerk, a Xenopus homologue of the murine Lerk-2 (ephrin-B1) transmembrane ligand, causes dissociation of Xenopus embryonic blastomeres by the mid-blastula transition. Moreover, a mutant that lacks the extracellular receptor binding domain can induce this phenotype. The carboxyl-terminal <em>19</em> amino acids of the cytoplasmic domain of XLerk are necessary but not sufficient to induce cellular dissociation. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, but not activin, can rescue both the loss of cell adhesion and mesoderm induction in ectodermal explants expressing XLerk. Collectively, these results show that the cytoplasmic domain of XLerk has a signaling function that is important for cell adhesion, and <em>fibroblast</em> <em>growth</em> <em>factor</em> signaling modulates this function.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) is proposed to be a negative feedback regulator of hepatic bile acid (BA) synthesis. We aimed to clarify the distribution of FGF<em>19</em> expression in human intestine and to investigate induction in a novel explant system. Ileal and colonic mucosal biopsies were obtained at endoscopy and analyzed for FGF<em>19</em> transcript expression. Primary explants were incubated with physiological concentrations of various BA for up to 6 h, and expression of FGF<em>19</em> and other genes was determined. FGF<em>19</em> transcripts were detected in ileum but were unquantifiable in colon. No loss of FGF<em>19</em> mRNA occurred as a consequence of the explant system. Ileal FGF<em>19</em> transcript expression was induced 350-fold by 50 μM chenodeoxycholate (CDCA, n = 24, P < 0.0001) and 161-fold by 50 μM glycochenodeoxycholate (GCDCA, n = 12, P = 0.0005). The responses of other genes to CDCA or GCDCA (50 μM) were smaller: median increases of ileal bile acid binding protein, organic solute transporter-α and -β, and short heterodimer partner were 2.4- to 4.0-fold; apical membrane sodium bile acid transporter and farnesoid X receptor (FXR) showed little change. The EC50 for FGF<em>19</em> transcript induction by CDCA was 20 μM. FGF<em>19</em> protein concentrations were significantly higher in the culture fluid from BA-stimulated explants. FGF<em>19</em> induction with cholate was 81% of that found with CDCA, but deoxycholate (40%) and lithocholate (4%) were significantly less potent. The synthetic FXR agonist obeticholic acid was much more potent than CDCA with a 70-fold FGF<em>19</em> stimulation at 1 μM. We concluded that FGF<em>19</em> expression in human ileum is very highly responsive to BA. Changes in FGF<em>19</em> induction are a potential mechanism involved in disorders of BA homeostasis.

Mouse A9 cells, L-cell-derived mutants deficient in hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) were found to be incapable of binding (125)I-labeled epidermal <em>growth</em> <em>factor</em> (EGF) to the cell surface. The A9 cells were fused with human diploid <em>fibroblasts</em> (WI-38) possessing EGF-binding ability, and human-mouse cell hybrids (TA series) were isolated after hypoxanthine/aminopterin/thymidine/ouabain selection. Analyses of isozyme markers and chromosomes of four representative clones of TA hybrids indicated that the expression of EGF-binding ability is correlated with the presence of human chromosome 7 or <em>19</em>. Four subclones were isolated from an EGF-binding-positive line, TA-4, and segregation of EGF-binding was found to be concordant with the expression of human mitochondrial malate dehydrogenase (MDHM; L-malate:NAD(+) oxidoreductase, EC 1.1.1.37), a marker for chromosome 7, but not with glucosephosphate isomerase (GPI; D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9), a marker for chromosome <em>19</em>. Furthermore, evidence from 27 clones of AUG hybrids that were produced between A9 and another human <em>fibroblast</em> line, GM1696, carrying an X/7 chromosome translocation indicated that EGF-binding ability segregates together with human MDHM and two X-linked markers, HPRT and glucose-6-phosphate dehydrogenase (G6PD; D-glucose-6-phosphate:NADP(+) 1-oxidoreductase, EC 1.1.1.49), that are located on the translocation chromosome 7p(+). These results permit assignment of the gene, designated EGFS, which is associated with the expression of EGF-binding ability, to human chromosome 7 and its localization to the p22-qter region. Because the EGF receptor is reported to be a glycoprotein the EGFS could be either a structural gene(s) for receptor protein or a gene(s) for modifying the receptor protein through glycosylation.

In mouse Balb/c3T3 <em>fibroblasts</em>, insulin-like <em>growth</em> <em>factor</em> (IGF)-II activates a calcium-permeable cation channel through a cell surface IGF-II receptor (Kojima, I., Nishimoto, I., Iiri, T., Ogata, E., and Rosenfeld, R. G. (<em>19</em>88) Biochem. Biophys. Res. Commun. 154, 9-<em>19</em>; Matsunaga, H., Nishimoto, I., Kojima, I., Yamashita, N., Kurokawa, K., and Ogata, E. (<em>19</em>88) Am. J. Physiol. 255, C442-C446). In the action of IGF-II, a pertussis toxin (or islet-activating protein; IAP)-sensitive GTP-binding protein (G protein) is inferred to be involved (Nishimoto, I., Hata, Y., Ogata, E., and Kojima, I. (<em>19</em>87) J. Biol. Chem. 262, 12120-12126). In the present study, we examined the direct coupling of the IGF-II receptor with G proteins. In broken Balb/c3T3 cell membranes, 10 nM IGF-II rapidly attenuated the IAP-catalyzed ADP-ribosylation of a 40-kDa protein in a manner requiring magnesium ion. The IGF-II-mediated attenuation in the IAP substrate activity was 80% recovered after washing off IGF-II and inhibited by coexisting guanosine 5'-O-(2-thiodiphosphate), while either aluminum fluoride solution (10 mM NaF plus 100 microM AlCl3) or 100 microM guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) reproduced the action of IGF-II. When purified IAP substrate G proteins (Gi1, Gi2, G0) were incubated with IGF-II in the presence of membranes from IAP-treated Balb/c3T3 cells, the attenuation in the IAP substrate activity was evident in Gi2, but not in Gi1 or G0. On the other hand, 10 nM insulin had no effect on the modification of the 40-kDa IAP substrate in Balb/c3T3 cell membranes, whereas 10 nM IGF-I elicited a slow onset of the IAP sensitivity attenuation from the 40-kDa protein. However, the specific involvement of the IGF-II receptor in the modification of the IAP substrate induced by low concentrations of IGF-II was suggested by the observations that (i) IGF-I receptor-lacking cell membranes were effective for the Gi2 modification by IGF-II, (ii) the ability of membranes to mediate the action of IGF-II was markedly attenuated in IGF-II receptor-lacking cell membranes, and (iii) agonistic anti-IGF-II receptor antibody mimicked the action of IGF-II on the 40-kDa protein in Balb/c3T3 cell membranes in a dose-dependent manner similar to that observed in the antibody-induced blocking of membrane IGF-II binding.(ABSTRACT TRUNCATED AT 400 WORDS)

BACKGROUND

Increased serum phosphate is associated with cardiovascular disease. Compared with whites, blacks have significantly higher serum phosphate and increased risk of hyperphosphataemia in health and chronic kidney disease (CKD). While population-based studies suggest that diminished urinary phosphorus excretion in blacks may explain these differences, few physiological studies explored the potential mechanisms. The aim of this study was to examine racial differences in postprandial urinary mineral ion excretion in health and in CKD.

METHODS

Twenty-eight healthy (18 white and 10 black) and <em>19</em> CKD (9 white and 10 black) subjects consumed a standardized meal; after which, blood and urine samples were collected for 4 h for measurement of phosphate, calcium, parathyroid hormone (PTH) and <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23).

RESULTS

Although serum phosphate did not differ by race, blacks had ∼30% lower postprandial fractional excretion of phosphate than whites in health (P < 0.001) and CKD (P = 0.02). Similarly, blacks had ∼35% lower fractional excretion of calcium in health (P = 0.02) and CKD (P = 0.3). Moreover, the postprandial response in serum calcium among CKD subjects differed by race (P = 0.03), with serum calcium significantly decreasing in whites but not blacks.

CONCLUSIONS

Blacks had lower fractional excretion of phosphate than whites despite similar levels of PTH and FGF23 in health and in CKD, suggesting racial variability in renal sensitivity to phosphaturic hormones. Furthermore, blacks defend postprandial serum calcium more effectively than whites in CKD. Further studies are needed to define the mechanisms underlying these observations and evaluate whether racial differences in mineral ion handling may contribute to disparities in CKD outcomes.

OBJECTIVE

To develop, by specific genetic modification, a differentiated human retinal pigment epithelial (RPE) cell line with an extended life span that can be used for investigating their function in vitro and for in vivo transplantation studies.

METHODS

Primary human RPE cells were genetically modified by transfecting with a plasmid encoding the simian virus (SV)40 large T antigen. After characterization, two cell lines, designated h1RPE-7 and h1RPE-116, were chosen for further investigation, along with the spontaneously derived RPE cell line ARPE-<em>19</em>. <em>Factors</em> reported to be important in RPE and photoreceptor cell function and survival in vivo were examined.

RESULTS

Both h1RPE-7 and h1RPE-116 cells exhibited epithelial morphology, expressed cytokeratins, and displayed junctional distribution of ZO-1, p100-p120 and beta-catenin. The cells expressed mRNA for RPE65 and cellular retinaldehyde-binding protein (CRALBP) and the trophic and growth factors brain-derived neurotropic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), pigment epithelium-derived factor (PEDF), nerve growth factor (NGF), platelet-derived growth factor (PDGF)-alpha, insulin-like growth factor (IGF)-1, and vascular endothelial growth factor (VEGF). Secreted BDNF, bFGF, and VEGF, but not CNTF, were identified in cell supernatants. The cell lines constitutively expressed HLA-ABC, CD54, CD58, and CD59. After activation with IFN-gamma both HLA-ABC and CD54 were upregulated, and the expression of HLA-DR was induced. Both cell lines failed to express CD80, CD86, CD40, or CD48 in vitro and in a mixed lymphocyte reaction were unable to induce T-cell proliferation. Fas ligand (CD95L) was not detected in vitro by RT-PCR. Similar results were obtained with the ARPE-<em>19</em> cell line.

CONCLUSIONS

RPE lines h1RPE-7 and h1RPE-116 retain many of the morphologic and biochemical characteristics of RPE cells in vivo and may serve as a source of cells for in vitro analysis of RPE cell function, as well as for orthotopic transplantation studies.

The normal development of eyes relies on proper signaling through Fibroblast growth factor (FGF) receptors, but the source and identity of cognate ligands have remained largely unknown. We have found that Fgf19 is expressed in the developing chicken retina. In situ hybridization discloses dynamic expression patterns for Fgf19 in the optic vesicle, lens primordia and retinal horizontal cells. Overall expression pattern of Fgf19 during chicken embryogenesis was also examined: Fgf19 is expressed in the regions associated with cranial placodes induction, boundary regions of rhombomeres, somites, specific groups of neural cells in midbrain, hindbrain, and those derived from epibranchial placodes, and the apical ectodermal ridge of limb buds. Expression pattern of the Fgf19-orthologous gene Fgf15 was further examined in the mouse developing eye. Fgf15 is expressed in the optic vesicle, a subset of progenitor cells of neural retina, and emerging ganglion and amacrine cells during retinogenesis.

The transcription <em>factor</em>, Hoxc8, is a member of the homeobox gene family that is vital for <em>growth</em> and differentiation. Previously, we identified 34 genes whose expression levels were changed at least 2-fold by forced expression of Hoxc8 in C57BL/6J mouse embryo <em>fibroblast</em> cells using a mouse 16,463-gene oligonucleotide microarray. In the present study, we used the combined power of microarray profiling, global Hoxc8 DNA-binding site analysis, and high-throughput chromatin immunoprecipitation assays to identify direct and biologically relevant targets of Hoxc8 in vivo. Here we show that <em>19</em> of the 34 responsive genes contain Hoxc8 consensus DNA-binding sequence(s) in their regulatory regions. Chromatin immunoprecipitation analysis indicated that Hoxc8-DNA interaction was detected in five of the <em>19</em> candidate genes. All of these five target genes have been implicated in oncogenesis, cell adhesion, proliferation, and apoptosis. Overall, the genes described here should aid in the understanding of global regulatory networks of Hox genes and to provide valuable insight into the molecular basis of Hoxc8 in development and carcinogenesis.

OBJECTIVE

To investigate the effects of hyaluronic acid (HA) on the release of proteoglycan by cultured rabbit chondrocytes.

METHODS

Articular cartilage chondrocytes were isolated from the knee joints of New Zealand white rabbits. Proteoglycan synthesis after incubation with HA was determined by measuring 35S-sulfate incorporation. Cells incubated with HA were labeled with 3H-glucosamine and applied to a Sepharose CL-2B column. After incubation of confluent cells with 35S-sulfate and then with HA in various concentrations in the presence or absence of cytokines, proteoglycan release from the cell matrix layer was measured.

RESULTS

HA (M(r) 3 x 10(5) to <em>19</em> x 10(5)), at 10 micrograms/ml to 1 mg/ml, had little effect on the incorporation of 35S-sulfate or 3H-glucosamine into cartilage matrix proteoglycans, or on the hydrodynamic size of proteoglycan monomers, in rabbit chondrocyte cultures. However, at 10-1,000 micrograms/ml, HA suppressed the release of 35S-proteoglycans from the cell matrix layer into the medium in the presence and absence of interleukin-1, tumor necrosis <em>factor</em> alpha, or basic <em>fibroblast</em> <em>growth</em> <em>factor</em>.

CONCLUSIONS

These results suggest that HA is a potent inhibitor of the displacement of matrix proteoglycan into culture medium.

BACKGROUND

We have recently shown, in an animal model, that amniotic fluid can be a source of cells for fetal tissue engineering. This study was aimed at determining whether fetal tissue constructs could also be engineered from cells normally found in human amniotic fluid.

METHODS

Cells obtained from the amniotic fluid of pregnant women at 15 to <em>19</em> weeks of gestation (n=6) were cultured in Dulbecco's Modified Eagle's medium (Sigma Chemical, St Louis, MO) containing 20% fetal bovine serum and 5 ng/mL basic <em>fibroblast</em> <em>growth</em> <em>factor</em> in a 95% humidified, 5% CO(2) chamber at 37 degrees C. A subpopulation of morphologically distinct cells was then mechanically isolated from the rest and selectively expanded. The lineage of this subpopulation of amniocytes was determined by immunofluorescent staining with antibodies against standard intermediate filaments and surface antigens. Cell proliferation rates were determined by oxidation assay. After cell expansion, colonies of amniocytes were statically and dynamically seeded onto both unwoven, 1-mm-thick polyglycolic acid polymer scaffold and acellular human dermis for 72 hours. The resulting constructs were analyzed by scanning electron microscopy.

RESULTS

Amniocytes stained positively for smooth muscle actin, vimentin, cytokeratin 18, and fibroblast surface protein, and negatively for desmin, cluster of differentiation 31, and von Willebrand's factor (Dako, Carpenteria, CA). These findings are consistent with a mesenchymal, fibroblast-myofibroblast cell lineage. Mesenchymal amniocytes could be rapidly expanded in culture, based on results of the proliferation assay. Scanning electron microscopy of amniocyte constructs revealed dense, confluent layers of cells surrounding the polymer matrices and firm cell adhesion to both PGA and Alloderm (Lifecell Corp, Branchburg, NJ) scaffolds. No evidence of cell death was observed.

CONCLUSIONS

Subpopulations of fetal mesenchymal cells can be consistently isolated from human amniotic fluid and rapidly expanded in vitro. Human mesenchymal amniocytes attach firmly to both polyglycolic acid polymer and acellular human dermis. The amniotic fluid can be a valuable and practical cell source for fetal tissue engineering.

BACKGROUND

Arresting or regressing kidney scarring is of major clinical relevance. Platelet-derived growth factor D (PDGF-D) is widely expressed in fibrotic kidneys. Administration of the PDGF-D neutralizing fully human monoclonal antibody CR002 in the acute phase of progressive anti-Thy 1.1 glomerulonephritis reduced glomerular and secondary tubulointerstitial damage.

METHODS

Using this model, we now assessed the effects of CR002 (n=15) vs irrelevant control IgG (n=17) administered on days 17, 28 and 35 after disease induction, i.e. after acute glomerular damage had subsided.

RESULTS

In vitro, CR002 inhibited the PDGF-D- but not the PDGF-B-induced proliferation of rat renal fibroblasts. Following the first CR002 injection on day 17, exposure to therapeutic levels was maintained until day 49. Proteinuria in the CR002-treated group was transiently reduced between days 49 and 77 (-19 to -23% in comparison with the controls; P<0.05). On day 100, CR002 treatment reduced the number of rats that had doubled their serum creatinine (CR002: 40 vs controls: 71%; P<0.05). Compared with controls, the CR002 animals, on day 100, significantly lowered glomerular expression of vimentin and collagens as well as tubulointerstitial damage scores, interstitial fibrosis, vimentin and cortical PDGF-D mRNA levels.

CONCLUSIONS

PDGF-D antagonism, even after the phase of acute glomerular damage, exerts beneficial effects on the course of tubulointerstitial damage, i.e. the final common pathway of most renal diseases.

There is no universally available laboratory test to diagnose bile acid diarrhoea (BAD).

To conduct a systematic review and meta-analysis to identify a biomarker for idiopathic BAD in patients with functional bowel disorder (FBD) with diarrhoea.

We searched multiple databases through 15 May 2015. Data were only available to estimate the diagnostic yield of each test (the prevalence of a positive test). Estimates were pooled across studies using the random effects model.

We included 36 studies, enrolling 5028 patients (24 using 75selenium homotaurocholic acid test (75SeHCAT) retention of <10%, 6 using fasting serum C4, 3 using fasting serum <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) and 2 based on total faecal bile acid (BA) excretion over 48 h). The diagnostic yields (and 95% CI) of abnormal tests were: 0.308 (0.247 to 0.377) for 75SeHCAT retention (<10%), 0.171 (0.134 to 0.217) for serum C4, 0.248 (0.147 to 0.385) for serum FGF<em>19</em> and 0.255 (0.071 to 0.606) for total faecal BA excretion over 48 h. The majority of the analyses were associated with substantial heterogeneity. Performance characteristics relative to a gold standard test could not be estimated.

Overall, the test with the highest diagnostic yield conducted in the largest number of studies was 75SeHCAT retention, which is not widely available in many countries outside Europe and Canada. Using different diagnostic tests, 25% (average) of patients with lower FBD with diarrhoea has evidence of idiopathic BAD. These tests serve to identify idiopathic BAD among patients with FBD with diarrhoea. Further studies are required to appraise the performance characteristics of tests for idiopathic BAD.