FGF19 is a unique member of the fibroblast growth factor (FGF) family of secreted proteins that regulates bile acid homeostasis and metabolic state in an endocrine fashion. Here we investigate the cell surface receptors required for signaling by FGF19. We show that betaKlotho, a single-pass transmembrane protein highly expressed in liver and fat, induced ERK1/2 phosphorylation in response to FGF19 treatment and significantly increased the interactions between FGF19 and FGFR4. Interestingly, our results show that alphaKlotho, another Klotho family protein related to betaKlotho, also induced ERK1/2 phosphorylation in response to FGF19 treatment and increased FGF19-FGFR4 interactions in vitro, similar to the effects of betaKlotho. In addition, heparin further enhanced the effects of both alphaKlotho and betaKlotho in FGF19 signaling and interaction experiments. These results suggest that a functional FGF19 receptor may consist of FGF receptor (FGFR) and heparan sulfate complexed with either alphaKlotho or betaKlotho.

Pruritus is a seriously disabling symptom accompanying many cholestatic liver disorders. Recent experimental evidence implicated the lysophospholipase, autotaxin (ATX), and its product, lysophosphatidic acid (LPA), as potential mediators of cholestatic pruritus. In this study, we highlight that increased serum ATX levels are specific for pruritus of cholestasis, but not pruritus of uremia, Hodgkin's disease, or atopic dermatitis. Treatment of patients with cholestasis with the bile salt sequestrant, colesevelam, but not placebo, effectively reduced total serum bile salts and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> levels, but only marginally altered pruritus intensity and ATX activity. Rifampicin (RMP) significantly reduced itch intensity and ATX activity in patients with pruritus not responding to bile salt sequestrants. In vitro, RMP inhibited ATX expression in human HepG2 hepatoma cells and hepatoma cells overexpressing the pregnane X receptor (PXR), but not in hepatoma cells in which PXR was knocked down. Treatment of severe, refractory pruritus by the molecular adsorbents recirculation system or nasobiliary drainage improved itch intensity, which, again, correlated with the reduction of ATX levels. Upon reoccurrence of pruritus, ATX activity returned to pretreatment values.

CONCLUSIONS

Serum ATX activity is specifically increased in patients with cholestatic, but not other forms of, systemic pruritus and closely correlates with the effectiveness of therapeutic interventions. The beneficial antipruritic action of RMP may be explained, at least partly, by the PXR-dependent transcriptional inhibition of ATX expression. Thus, ATX likely represents a novel therapeutic target for pruritus of cholestasis.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-<em>19</em> (FGF<em>19</em>) and its rodent ortholog, FGF15, are hormones produced in the distal small intestine and secreted into the circulation after a meal. In addition to controlling the enterohepatic circulation of bile acids, FGF15/<em>19</em> also regulates systemic lipid and glucose metabolism. In these experiments we investigated the hypothesis that, like other gut-derived postprandial hormones, FGF15/<em>19</em> can act in the central nervous system to elicit its metabolic effects. We found that FGF-receptors 1 and 4 are present in rat hypothalamus, and that their expression was reduced by up to 60% in high-fat fed rats relative to lean controls. Consistent with a potential role for brain FGF15/<em>19</em> signaling to regulate energy and glucose homeostasis, and with a previous report that intracerebroventricular (i.c.v.) administration of FGF<em>19</em> increases energy expenditure, we report that acute i.c.v. FGF<em>19</em> reduces 24-h food intake and body weight, and acutely improves glucose tolerance. Conversely, i.c.v. administration of an FGF-receptor inhibitor increases food intake and impairs glucose tolerance, suggesting a physiological role for brain FGF receptor signaling. Together, these findings identify the central nervous system as a potentially important target for the beneficial effects of FGF<em>19</em> in the treatment of obesity and diabetes.

Interleukin-6 (IL-6), a multifunctional cytokine produced in monocytes, <em>fibroblasts</em>, endothelial cells, and keratinocytes, is induced by a variety of stimulating signals, including lipopolysaccharide (LPS), poly (I), poly (C), IL-1, tumor necrosis <em>factor</em> (TNF), and platelet-derived <em>growth</em> <em>factor</em>. Some of these signals induce IL-6 effectively only in one cell type, and this selectivity of induction may explain selectivity of biologic effects. In the present study, we show that IL-1 beta, previously known to be a potent inducer of IL-6 in <em>fibroblasts</em>, endothelial cells, and keratinocytes, but not in monocytes, is also a potent inducer of IL-6 in peripheral blood monocytes. High level IL-6 activity that could be neutralized by specific antibodies to IL-6 was detected in supernatants of IL-1-stimulated monocytes. Maximal induction required IL-1 concentrations of 10 ng/mL. As judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, IL-6 species of relative molecular mass of <em>19</em> to 26 Kd could be specifically immunoprecipitated from supernatants of IL-1-induced monocytes. Size heterogeneity is a reported feature of IL-6 produced in a variety of cell types, and monocyte-derived IL-6 induced by either IL-1 or LPS displayed similar size heterogeneity. The highly purified recombinant IL-1 beta preparation used contained little, if any, LPS. In addition, monocyte production of IL-6, induced by IL-1 beta, was specifically neutralized by anti-IL-1 beta antibodies, demonstrating that IL-1, rather than a contaminant in the IL-1 preparation, was responsible for IL-6 induction. A number of biologic activities have been ascribed both to IL-1 and IL-6. The finding that IL-1 induced IL-6 in monocytes may help in defining the spectrum of biologic activities of each of these interactive cytokines.

OBJECTIVE

Given the heterogeneity of gene expression patterns and cellular distribution between rheumatoid arthritis (RA) synovial tissues, we sought to determine whether this variability was also reflected at the level of the fibroblast-like synoviocyte (FLS) cultured from RA synovial tissues.

METHODS

Gene expression profiles in FLS cultured from synovial tissues obtained from 19 RA patients were analyzed using complementary DNA microarrays and hierarchical cluster analysis. To validate the subclassification, we performed prediction analysis and principal components analysis. Genes that differed significantly in their expression between FLS cultures were selected using Statistical Analysis of Microarrays software. Real-time quantitative polymerase chain reaction was performed to validate the microarray data. Immunocytochemistry was applied to study the expression of the genes of interest in FLS and synovial tissues.

RESULTS

Hierarchical clustering identified 2 main groups of FLS characterized by distinctive gene expression profiles. FLS from high-inflammation synovial tissues revealed increased expression of a transforming growth factor beta/activin A-inducible gene profile that is characteristic of myofibroblasts, a cell type considered to be involved in wound healing, whereas increased production of growth factor (insulin-like growth factor 2/insulin-like growth factor binding protein 5) appeared to constitute a characteristic feature of FLS derived from low-inflammation synovial tissues. The molecular feature that defines the myofibroblast-like phenotype was reflected as an increased proportion of myofibroblast-like cells in the heterogeneous FLS population. Myofibroblast-like cells were also found upon immunohistochemical analysis of synovial tissue.

CONCLUSIONS

Our findings support the notion that heterogeneity between synovial tissues is reflected in FLS as a stable trait, and provide evidence of a possible link between the behavior of FLS and the inflammation status of RA synovium.

OBJECTIVE

The primary aim of this completed multicentre randomised, parallel, double-blind placebo-controlled study was to elucidate the mechanisms of glucose-lowering with colesevelam and secondarily to investigate its effects on lipid metabolism (hepatic de novo lipogenesis, cholesterol and bile acid synthesis).

METHODS

Participants with type 2 diabetes (HbA(1c) 6.7-10.0% [50-86 mmol/mol], fasting glucose <16.7 mmol/l, fasting triacylglycerols <3.9 mmol/l and LDL-cholesterol >1.55 mmol/l) treated with diet and exercise, sulfonylurea, metformin or a combination thereof, were randomised by a central coordinator to either 3.75 g/day colesevelam (n = 30) or placebo (n = 30) for 12 weeks at three clinical sites in the USA. The primary measure was the change from baseline in glucose kinetics with colesevelam compared to placebo treatment. Fasting and postprandial glucose, lipid and bile acid pathways were measured at baseline and post-treatment using stable isotope techniques. Plasma glucose, insulin, total glucagon-like peptide-1 (GLP-1), total glucose-dependent insulinotropic polypeptide (GIP), glucagon and <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>19</em> (FGF-<em>19</em>) concentrations were measured during the fasting state and following a meal tolerance test. Data was collected by people blinded to treatment.

RESULTS

Compared with placebo, colesevelam improved HbA(1c) (mean change from baseline of 0.3 [SD 1.1]% for placebo [n = 28] and -0.3 [1.1]% for colesevelam [n = 26]), glucose concentrations, fasting plasma glucose clearance and glycolytic disposal of oral glucose. Colesevelam did not affect gluconeogenesis or appearance rate (absorption) of oral glucose. Fasting endogenous glucose production and glycogenolysis significantly increased with placebo but were unchanged with colesevelam (treatment effect did not reach statistical significance). Compared with placebo, colesevelam increased total GLP-1 and GIP concentrations and improved HOMA-beta cell function while insulin, glucagon and HOMA-insulin resistance were unchanged. Colesevelam increased cholesterol and bile acid synthesis and decreased FGF-<em>19</em> concentrations. However, no effect was seen on fractional hepatic de novo lipogenesis.

CONCLUSIONS

Colesevelam, a non-absorbed bile acid sequestrant, increased circulating incretins and improved tissue glucose metabolism in both the fasting and postprandial states in a manner different from other approved oral agents.

BACKGROUND

ClinicalTrials.gov NCT00596427

BACKGROUND

The study was funded by Daiichi Sankyo.

Fibroblast growth factors (FGF) play important roles in development, angiogenesis, and cancer. FGF19 uniquely binds to FGF receptor 4 (FGFR4). Our previous study has shown that FGF19 transgenic tumors have an activated Wnt-pathway phenotype. Wnt signaling is implicated in initiating or promoting FGF signaling in various cell types and organs. In this study, we examined whether FGF19 or inhibition of FGF19 affects the beta-catenin signaling pathway using human colon cancer cell lines (HCT116, Colo201). Our results show that FGF19 increases tyrosine phosphorylation of beta-catenin and causes loss of beta-catenin-E-cadherin binding. FGF19 increases p-GSK3beta and active beta-catenin levels and anti-FGF19 antibody (1A6) treatment abrogates this effect of FGF19. Anti-FGF19 antibody treatment increases S33/S37/T41 phosphorylation and ubiquitination of beta-catenin. Ion-trap mass spectrometric analysis confirmed that 1A6 increases phosphorylation of beta-catenin in the NH(2) terminus. Using HCT116-paired beta-catenin knockout cells, we show that FGF19 induces TCF/LEF reporter activity in parental (WT/Delta45) and in WT/--but not in mutant (-/Delta45) cells, and that inhibition of endogenous FGF19 reduces this reporter activity, indicating that wild-type beta-catenin is accessible for modulation. FGFR4 knockdown using inducible short hairpin RNA significantly reduces the colony-forming ability in vitro and tumor growth in vivo. Although cleaved caspase-3 immunoreactivity remains unchanged, the number of ki67-positive nuclei is reduced in FGFR4 knockdown tumor xenograft tissues. Consistent with the reduced beta-catenin activation, Taqman analyses show that FGF19/FGFR4 inhibition reduced beta-catenin target gene (cyclin D1, CD44, c-jun, Cox-2, UPAR) expression. These findings highlight that FGF19/FGFR4 cross-talk with beta-catenin and that pathway intervention reduces tumor growth.

OBJECTIVE

This review focuses on the latest understanding of the molecular mechanisms underlying the complex interactions between intestine and liver bile acid signaling, gut microbiota, and their impact on whole-body lipid, glucose and energy metabolism.

RESULTS

Hepatic bile acid synthesis is tightly regulated by the bile acid negative feedback mechanisms. Modulating the enterohepatic bile acid signaling greatly impacts the whole-body metabolic homeostasis. Recently, a positive feedback mechanism through intestine farnesoid X receptor (FXR) antagonism has been proposed to link gut microbiota to the regulation of bile acid composition and pool size. Two studies identified intestine Diet1 and hepatic SHP-2 as novel regulators of CYP7A1 and bile acid synthesis through the gut-liver FXR-<em>fibroblast</em> <em>growth</em> <em>factor</em> 15/<em>19</em>-FGF receptor four signaling axis. New evidence suggests that enhancing bile acid signaling in the distal ileum and colon contributes to the metabolic benefits of bile acid sequestrants and bariatric surgery.

CONCLUSIONS

Small-molecule ligands that target TGR5 and FXR have shown promise in treating various metabolic and inflammation-related human diseases. New insights into the mechanisms underlying the bariatric surgery and bile acid sequestrant treatment suggest that targeting the enterohepatic circulation to modulate gut-liver bile acid signaling, incretin production and microbiota represents a new strategy to treat obesity and type 2 diabetes.

The concentration of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) was determined in the serum of 90 untreated and 42 treated metastatic cancer patients, including patients with colorectal, breast, ovarian and renal carcinomas, with an enzyme-linked immunosorbent assay (ELISA). Levels higher than the 95th percentile of the concentrations of a control group, i.e. 7.5 pg ml(-1) for bFGF and 500 pg ml(-1) for VEGF, were identified as 'elevated'. One measurement during follow-up was included into the analysis per patient. For <em>19</em> treated patients, consecutive serum samples were analysed. Fifty-seven per cent of all untreated patients had elevated serum levels of one or both angiogenic <em>factors</em>. The fraction of patients with elevated serum levels of bFGF and/or VEGF was similar in the different tumour types. Agreement of bFGF levels and VEGF levels, classified in relation to their respective cut-off values, was present in 67% of all patients. Fifty-eight per cent of the patients with progressive disease during treatment compared with 15% of the patients showing response to treatment (chi-squared test P < 0.05) had elevated bFGF and/or VEGF serum levels. When consecutive serum samples were analysed, two-thirds of the patients showing progressive disease had increasing serum levels of the angiogenic <em>factors</em> compared with less than one-tenth of the patients showing response (chi-squared test P < 0.05). The lack of association between the serum bFGF and VEGF levels and the tumour type may suggest an aspecific host reaction responsible for solid tumour-related angiogenesis. The main determinants of the serum bFGF and VEGF concentration are the progression kinetics of the metastatic carcinomas.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) plays a crucial role in the negative feedback regulation of bile salt synthesis. In the postprandial state, activation of ileal farnesoid X receptor (FXR) by bile salts results in transcriptional induction of FGF<em>19</em> and elevation of circulating FGF<em>19</em> levels. An intestinal-liver axis of FGF<em>19</em> signaling results in down-regulation of bile salt synthesis. The aim of this study was to explore a broader signaling activity of FGF<em>19</em> in organs engaged in the enterohepatic circulation of bile salts. For this aim, FGF<em>19</em> expression and aspects of FGF<em>19</em> signaling were studied in surgical specimens and in cell lines of hepatobiliary and intestinal origin. FGF<em>19</em> messenger RNA was found to be abundantly expressed in the human gallbladder and in the common bile duct, with only minor expression observed in the ileum. Interestingly, human gallbladder bile contains high levels of FGF<em>19</em> (21.9 ± 13.3 versus 0.22 ± 0.14 ng/mL in the systemic circulation). Gallbladder explants secrete 500 times more FGF<em>19</em> than FXR agonist-stimulated ileal explants. <em>Factors</em> required for FGF<em>19</em> signaling (i.e., FGFR4 and βKlotho) are expressed in mucosal epithelial cells of the gallbladder and small intestine. FGF<em>19</em> was found to activate signaling pathways in cell lines of cholangiocytic, enteroendocrine, and enterocytic origin.

CONCLUSIONS

The combined findings raise the intriguing possibility that biliary FGF<em>19</em> has a signaling function in the biliary tract that differs from its established signaling function in the portal circulation. Delineation of the target cells in bile-exposed tissues and the affected cellular pathways, as well as a possible involvement in biliary tract disorders, require further studies.

While it has long been recognized that bile acids are essential for solubilizing lipophilic nutrients in the small intestine, the discovery in <em>19</em>99 that bile acids serve as ligands for the nuclear receptor farnesoid X receptor (FXR) opened the floodgates in terms of characterizing their actions as selective signaling molecules. Bile acids act on FXR in ileal enterocytes to induce the expression of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)15/<em>19</em>, an atypical FGF that functions as a hormone. FGF15/<em>19</em> subsequently acts on a cell surface receptor complex in hepatocytes to repress bile acid synthesis and gluconeogenesis, and to stimulate glycogen and protein synthesis. FGF15/<em>19</em> also stimulates gallbladder filling. Thus, the bile acid-FXR-FGF15/<em>19</em> signaling pathway regulates diverse aspects of the postprandial enterohepatic response. Pharmacologically, this endocrine pathway provides exciting new opportunities for treating metabolic disease and bile acid-related disorders such as primary biliary cirrhosis and bile acid diarrhea. Both FXR agonists and FGF<em>19</em> analogs are currently in clinical trials.

Fibrillin-1 (FBN1) contains 47 epidermal <em>growth</em> <em>factor</em> (EGF)-like domains characterized by six conserved cysteine residues. Cysteine substitutions that disrupt one of the three disulfide bonds are frequent causes of Marfan syndrome (MFS). We identified <em>19</em> new substitutions involving cysteine residues in each of the six positions of EGF-like domains. Allele-specific mRNA assays revealed equal abundance of mutant and normal FBN1 transcripts in all 10 individuals studied. Quantitative pulse-chase analysis of fibrillin protein was performed on 25 mutant <em>fibroblast</em> strains with substitutions of 22 different cysteine residues in 18 different EGF-like domains spanning the entire gene. Normal synthesis and stability of mutant fibrillin molecules was seen in 20/25 individuals, 11 of whom showed delayed intracellular processing and/or secretion. In the remaining five cases, the mutant protein was apparently unstable. In four of these five cases, the second or third disulfide bond of EGF-like domains immediately preceding an 8-cysteine or hybrid domain was affected. All but two mutations caused severe reduction of matrix deposition, which was attributed to a dominant-negative effect of mutant molecules. For genotype/phenotype comparisons, clinical data on 25 probands and <em>19</em> mutation-positive family members were analyzed. Ocular manifestations were among the most consistent features (ectopia lentis in 86%, myopia in 80%). Nine mutations encoded by exons 26-32 resulted in early-onset classic MFS and, in one case, neonatal-lethal MFS. Mutations outside this region were associated with variable clinical phenotypes, including individuals with fibrillinopathies not meeting diagnostic criteria for MFS.

Transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) stimulation of Type I collagen gene (COL1A2) transcription involves the Smad signal transduction pathway, but the mechanisms of Smad-mediated transcriptional activation are not fully understood. We now demonstrate that the ubiquitous transcriptional coactivators p300 and CREB-binding protein (CBP) enhanced basal as well as TGF-beta- or Smad3-induced COL1A2 promoter activity, and stimulated the expression of endogenous Type I collagen. The adenoviral E1A oncoprotein abrogated stimulation of COL1A2 activity in transfected <em>fibroblasts</em>, and reduced the basal level of collagen gene expression. This effect was due to specific interaction of E1A with cellular p300/CBP because (a) a mutant form of E1A defective in p300 binding failed to abrogate stimulation, and (b) forced expression of p300/CBP restored the ability of TGF-beta to stimulate COL1A2 promoter activity in the presence of E1A. The effect of p300 on COL1A2 transcription appeared to be due, in part, to its intrinsic acetyltransferase activity, as stimulation induced by a histone acetyltransferase-deficient mutant p300 was substantially reduced. Transactivation of COL1A2 by p300 involved the Smad signaling pathway, as Smad4-deficient cells failed to respond to p300, and stimulation was rescued by overexpression of Smad4. Furthermore, minimal constructs containing only the Smad-binding CAGACA element of COL1A2 were transactivated by p300 in the presence of TGF-beta. These results indicate, for the first time, that the multifunctional p300/CBP coactivators play a major role in Smad-dependent TGF-beta stimulation of collagen gene expression in <em>fibroblasts</em>. Oncogene (2000) <em>19</em>, 3546 - 3555

FoxG1 is an evolutionarily conserved, winged-helix transcriptional repressor that maintains progenitor cells in the vertebrate forebrain. How the activity of FoxG1 is regulated is not known. Here, we report that in the developing Xenopus and mouse forebrain, FoxG1 is nuclear in progenitor cells but cytoplasmic in differentiating cells. The subcellular localisation of FoxG1 is regulated at the post-translational level by casein kinase I (CKI) and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) signalling. CKI phosphorylation of Ser <em>19</em> of FoxG1 promotes nuclear import, whereas FGF-induced phosphorylation of Thr 226 promotes nuclear export. Interestingly, FGF-induced phosphorylation of FoxG1 is mediated Akt kinase (also known as protein B kinase, PKB) kinase, rather than the MAPK pathway. Phosphorylation of endogenous FoxG1 is blocked by CKI and Akt inhibitors. In the mouse ol<em>factor</em>y placode cell line OP27, and in cortical progenitors, increased FGF signalling causes FoxG1 to exit the nucleus and promotes neuronal differentiation, whereas FGF and Akt inhibitors block this effect. Thus, CKI and FGF signalling converge on an antagonistic regulation of FoxG1, which in turn controls neurogenesis in the forebrain.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> <em>19</em> (FGF<em>19</em>) and 21 (FGF21) have emerged as key regulators of energy metabolism. Several studies have been conducted to understand the mechanism of FGF<em>19</em> and FGF21 action, however, the data presented has often been inconsistent and at times contradictory. Here in a single study we compare the mechanisms mediating FGF<em>19</em>/FGF21 actions, and how similarities/differences in actions at the cellular level between these two <em>factors</em> translate to common/divergent physiological outputs. Firstly, we show that in cell culture FGF<em>19</em>/FGF21 are very similar, however, key differences are still observed differentiating the two. In vitro we found that both FGF's activate FGFRs in the context of βKlotho (KLB) expression. Furthermore, both <em>factors</em> alter ERK phosphorylation and glucose uptake with comparable potency. Combination treatment of cells with both <em>factors</em> did not have additive effects and treatment with a competitive inhibitor, the FGF21 delta N17 mutant, also blocked FGF<em>19</em>'s effects, suggestive of a shared receptor activation mechanism. The key differences between FGF21/FGF<em>19</em> were noted at the receptor interaction level, specifically the unique ability of FGF<em>19</em> to bind/signal directly via FGFR4. To determine if differential effects on energy homeostasis and hepatic mitogenicity exist we treated DIO and ob/ob mice with FGF<em>19</em>/FGF21. We find comparable efficacy of the two proteins to correct body weight and serum glucose in both DIO and ob/ob mice. Nevertheless, FGF21 and FGF<em>19</em> had distinctly different effects on proliferation in the liver. Interestingly, in vivo blockade of FGF21 signaling in mice using ΔN17 caused profound changes in glycemia indicative of the critical role KLB and FGF21 play in the regulation of glucose homeostasis. Overall, our data demonstrate that while subtle differences exist in vitro the metabolic effects in vivo of FGF<em>19</em>/FGF21 are indistinguishable, supporting a shared mechanism of action for these two hormones in the regulation of energy balance.

To grow and metastasize, solid tumours must develop their own blood supply by neo-angiogenesis. Thalidomide inhibits the processing of mRNA encoding peptide molecules including tumour necrosis <em>factor</em>-alpha (TNF-alpha) and the angiogenic <em>factor</em> vascular endothelial <em>growth</em> <em>factor</em> (VEGF). This study investigated the use of continuous low dose Thalidomide in patients with a variety of advanced malignancies. Sixty-six patients (37 women and 29 men; median age, 48 years; range 33-62 years) with advanced measurable cancer (<em>19</em> ovarian, 18 renal, 17 melanoma, 12 breast cancer) received Thalidomide 100 mg orally every night until disease progression or unacceptable toxicity was encountered. Three of 18 patients with renal cancer showed partial responses and a further three patients experienced stabilization of their disease for up to 6 months. Although no objective responses were seen in the other tumour types, there were significant improvements in patients' sleeping (P < 0.05) and maintained appetite (P < 0.05). Serum and urine concentrations of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), TNF-alpha and VEGF were measured during treatment and higher levels were associated with progressive disease. Thalidomide was well tolerated: Two patients developed WHO Grade 2 peripheral neuropathy and eight patients developed WHO grade 2 lethargy. No patients developed WHO grade 3 or 4 toxicity. Further studies evaluating the use of Thalidomide at higher doses as a single agent for advanced renal cancer and in combination with biochemotherapy regimens are warranted.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGF) are a family of ligands that bind to four different types of cell surface receptor entitled, FGFR1, FGFR2, FGFR3 and FGFR4. These receptors differ in their ligand binding affinity and tissue distribution. The prototypical receptor structure is that of an extracellular region comprising three immunoglobulin (Ig)-like domains, a hydrophobic transmembrane segment and a split intracellular tyrosine kinase domain. Alternative gene splicing affecting the extracellular third Ig loop also creates different receptor isoforms entitled FGFRIIIb and FGFRIIIc. Somatic <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) mutations are implicated in different types of cancer and germline FGFR mutations occur in developmental syndromes particularly those in which craniosynostosis is a feature. The mutations found in both conditions are often identical. Many somatic FGFR mutations in cancer are gain-of-function mutations of established preclinical oncogenic potential. Gene amplification can also occur with <em>19</em>-22% of squamous cell lung cancers for example having amplification of FGFR1. Ontologic comparators can be informative such as aberrant spermatogenesis being implicated in both spermatocytic seminomas and Apert syndrome. The former arises from somatic FGFR3 mutations and Apert syndrome arises from germline FGFR2 mutations. Finally, therapeutics directed at inhibiting the FGF/FGFR interaction are a promising subject for clinical trials.

Thirty-five morbidly obese patients underwent Roux-en-Y gastric bypass surgery (RYGB). In addition to weight loss, these patients showed significant improvement of insulin resistance and a reduction of hepatic fat content. Three months after surgery, the serum bile salts were slightly but significantly elevated, and the levels of the endocrine-acting <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) and FGF21 were increased. FGF<em>19</em> and FGF21 play a role as regulators of hepatic lipid and glucose metabolism. These results show that RYGB surgery improves metabolism and that this improvement is still apparent 3 months after surgery. Bile salts may play a key role in the improvement of metabolism after RYGB. Why serum bile salt concentrations are elevated after RYGB needs to be investigated.

BACKGROUND

Cold exposure stimulates fibroblast growth factor 21 (FGF21) secretion in animals, enhancing the cold-induced thermogenesis (CIT) response through browning of white adipose tissue. In humans, the effects of cold exposure on circulating FGF21 levels are unknown.

OBJECTIVE

Our objective was to evaluate the effects of mild cold exposure on circulating FGF21 and its relationship with CIT and lipolysis in humans.

METHODS

We conducted a randomized, single-blind, crossover intervention study at the National Institutes of Health Clinical Center.

METHODS

Participants were healthy adults.

METHODS

Subjects were exposed to a 12-h exposure to 24 or 19 C in a whole-room indirect calorimeter.

METHODS

Energy expenditure, plasma FGF 21, nonesterified fatty acid, and adipose tissue microdialysis glycerol concentrations were evaluated.

RESULTS

At 24 C, plasma FGF21 exhibited a diurnal rhythm, peaking at 0800 h [110 (59-178) pg/ml], and progressively dropped to a nadir at 1700 h [41 (21-71) pg/ml, P < 0.0001] before rising at 1900 h [60 (11-81) pg/ml, P < 0.0001]. Exposure at 19 C lessened the diurnal reduction of FGF21 observed at 24 C from 0800-1700 h and augmented overall FGF21 levels by 37 ± 45% (P = 0.01). The change in area under the curve plasma FGF21 between 19 and 24 C correlated positively with the change in area under the curve adipose microdialysate glycerol (R(2) = 0.35, P = 0.04) but not with nonesterified fatty acid. Cold-induced increase in FGF21 predicted greater rise in energy expenditure during cold exposure (β = 0.66, P = 0.027), independent of age, gender, fat mass, and lean mass.

CONCLUSIONS

Mild cold exposure increased circulating FGF21 levels, predicting greater lipolysis and CIT. A small reduction in environmental temperature is sufficient to modulate FGF21 diurnal rhythm in humans, which may mediate cold-induced metabolic changes similar to those in animals.

Tubulointerstitial fibrosis correlates closely with renal function, although the mechanism regulating tubulointerstitial fibrogenesis remains poorly understood. Since platelet-derived <em>growth</em> <em>factor</em> (PDGF) is a <em>growth</em> <em>factor</em> for <em>fibroblasts</em>, we examined the effect of daily (for 7 days) PDGF-AA or PDGF-BB administration on renal tubulointerstitial architecture in rats. PDGF-AA administration at a dose of 5 mg/kg did not affect the renal tubulointerstitium. By comparison, PDGF-BB induced dose-dependent renal tubulointerstitial cell proliferation and fibrosis. At 5 mg/kg, PDGF-BB increased BrdU labeling in tubulointerstitial <em>fibroblasts</em> at 24 hours (<em>19</em>-fold), which peaked at 72 hours (23-fold) with bromodeoxyuridine uptake returning to control values by 7 days. Tubulointerstitial proliferation was associated with the differentiation of these cells into myo<em>fibroblasts</em> as evidenced by alpha-smooth muscle actin expression beginning on day 3. The expression of alpha-smooth muscle actin peaked on day 5 and had markedly declined by day 21. In addition, apoptotic cells were identified within the tubulointerstitium on day 3 and progressively increased through day 7, suggesting that the disappearance of myo<em>fibroblasts</em> may have occurred through apoptosis. These changes were accompanied by increased expression of alpha 1 (III) collagen mRNA and interstitial accumulation of type III collagen within the renal cortex. By morphometric analysis, an approximately twofold increase in collagen III immunolabeling within the interstitial compartment was evident at 24 hours and peaked on days 5 to 7 (approximately fourfold). These data suggest that PDGF-BB may be an important mediator of tubulointerstitial hyperplasia and fibrosis.

BACKGROUND

YKL-40 is secreted by macrophages and neutrophils and is a growth factor for vascular endothelial cells and fibroblasts. Elevated serum concentrations of YKL-40 are found in patients with diseases characterized by inflammation or ongoing fibrosis. The aim of this study was to seek association between serum YKL-40 in patients with ulcerative colitis (UC) and Crohn disease (CD) and clinical disease activity.

METHODS

One-hundred-and-sixty-four patients with UC and 173 patients with CD were studied. The Simple Clinical Colitis Activity Index (SCCAI) and the Harvey-Bradshaw (H-B) score were used to assess disease activity. Serum YKL-40 (determined by ELISA) was related to C-reactive protein (CRP) and disease activity.

RESULTS

In patients with UC, the median serum YKL-40 rose with increasing disease activity, and patients with severe active disease had higher serum YKL-40 (median 59 microg/L (95% CI: 26-258 microg/L), P < 0.001) than patients with inactive UC (33 microg/L (19-163)) and age-matched controls (43 microg/L (20-124)). Patients with severe active CD had higher serum YKL-40 (59 microg/L (21-654), P < 0.001) than age-matched controls, but not higher than inactive CD patients (43 microg/L (17-306)). Serum YKL-40 was elevated in 41% of the patients with severe UC, in 10% with inactive UC, in 46% with severe CD and in 30% with inactive CD. Serum YKL-40 correlated with SCCAI in UC patients but not with H-B score in CD patients. In both patient groups, low correlations were found between serum YKL-40 and CRP, albumin and leucocytes.

CONCLUSIONS

Serum YKL-40 is elevated in patients with active IBD and may be complementary to inflammatory markers and clinical characteristics in the assessment of disease activity.

BACKGROUND

Lenvatinib is an oral inhibitor of vascular endothelial growth factor receptor 1-3, fibroblast growth factor receptor 1-4, platelet-derived growth factor receptor alpha, RET, and KIT. This phase 2, single-arm, open-label multicenter study evaluated lenvatinib in advanced hepatocellular carcinoma (HCC).

METHODS

Patients with histologically/clinically confirmed advanced HCC who did not qualify for surgical resection or local therapies received lenvatinib at a dosage of 12 mg once daily (QD) in 28-day cycles. The primary efficacy endpoint was time to progression (TTP) per modified Response Evaluation Criteria in Solid Tumors v1.1; secondary efficacy endpoints included objective response rate (ORR), disease control rate (DCR), and overall survival (OS).

RESULTS

Between July 2010 and June 2011, 46 patients received lenvatinib at sites across Japan and Korea. The median TTP, as determined by independent radiological review, was 7.4 months [95 % confidence interval (CI): 5.5-9.4]. Seventeen patients (37 %) had partial response and 19 patients (41 %) had stable disease (ORR: 37 %; DCR: 78 %). Median OS was 18.7 months (95 % CI: 12.7-25.1). The most common any-grade adverse events (AEs) were hypertension (76 %), palmar-plantar erythrodysesthesia syndrome (65 %), decreased appetite (61 %), and proteinuria (61 %). Dose reductions and discontinuations due to AEs occurred in 34 (74 %) and 10 patients (22 %), respectively. Median body weight was lower in patients with an early (<30 days) dose withdrawal or reduction than in those without.

CONCLUSIONS

Lenvatinib 12-mg QD showed clinical activity and acceptable toxicity profiles in patients with advanced HCC, but early dose modification was necessary in patients with lower body weight. Further development of lenvatinib in HCC should consider dose modification by body weight. TRIAL REGISTRATION ID: www.ClinicalTrials.gov NCT00946153.

Treatment of mild-moderate Alzheimer's disease (AD) subjects (N = 1<em>19</em>) for 52 weeks with the SIRT1 activator resveratrol (up to 1 g by mouth twice daily) attenuates progressive declines in CSF Aβ40 levels and activities of daily living (ADL) scores.

For this retrospective study, we examined banked CSF and plasma samples from a subset of AD subjects with CSF Aβ42 <600 ng/ml (biomarker-confirmed AD) at baseline (N = <em>19</em> resveratrol-treated and N = <em>19</em> placebo-treated). We utilized multiplex Xmap technology to measure markers of neurodegenerative disease and metalloproteinases (MMPs) in parallel in CSF and plasma samples.

Compared to the placebo-treated group, at 52 weeks, resveratrol markedly reduced CSF MMP9 and increased macrophage-derived chemokine (MDC), interleukin (IL)-4, and fibroblast growth factor (FGF)-2. Compared to baseline, resveratrol increased plasma MMP10 and decreased IL-12P40, IL12P70, and RANTES. In this subset analysis, resveratrol treatment attenuated declines in mini-mental status examination (MMSE) scores, change in ADL (ADCS-ADL) scores, and CSF Aβ42 levels during the 52-week trial, but did not alter tau levels.

Collectively, these data suggest that resveratrol decreases CSF MMP9, modulates neuro-inflammation, and induces adaptive immunity. SIRT1 activation may be a viable target for treatment or prevention of neurodegenerative disorders.

ClinicalTrials.gov NCT01504854.

The production of transgenic and null mice with skin abnormalities makes it increasingly important to establish cultures of mouse epidermal keratinocytes for in vitro studies. This requires that each cell line be derived from a single mouse and that the cells be carried for multiple passages. Freezing the cells would also be advantageous by allowing comparison of keratinocytes from several mouse lines at the same time. Mouse keratinocytes, however, have been exceedingly difficult to grow as primary cultures, and subculturing these cells has been virtually impossible until now. We describe a gentle dissociation method and a highly supplemented <em>fibroblast</em> conditioned medium that allows us to grow and subculture total mouse keratinocytes for up to <em>19</em> subcultures, allowing an increase in cell number of greater than 10 logs. Epidermal keratinocytes from newborn mice were grown on collagen IV coated dishes in murine <em>fibroblast</em> conditioned medium with 0.06 mM calcium and added <em>growth</em> <em>factors</em>. The cells could be passaged, frozen as viable stocks, and induced to differentiate. Morphologically the cultured keratinocytes demonstrated a pattern characteristic of basal cells. Stratified cultures which made mouse keratin 1 and profilaggrin through passage 10 were induced by purging the monolayer cultures of <em>growth</em> <em>factors</em>, then adding medium with 0.15 mM calcium; expression of mouse keratin 1 and profilaggrin was lost by passage 15. The methods explained in detail here should be of great interest to investigators who are now trying to analyze skin phenotypes and expression of markers of epidermal differentiation of their transgenic or knockout mice.