OBJECTIVE

Cord blood banks provide fully human leukocyte antigen-typed cells, from which a set of standard induced pluripotent stem (iPS) cells for use in allogenic transplantation can be derived. Hence, the ability to generate iPS cells from cord blood cells has the potential to provide a suitable source for clinical transplantation. The aim of this work is to determine the reprogramming methods, culture conditions, and cell fractions that can be used to generate iPS cells from cord blood cells effectively.

METHODS

CD34(+), mononucleated, and derived adherent cells from cord blood were cultured in hematopoietic medium (X-vivo10 containing 50 ng/mL interleukin-6, 50 ng/mL soluble interleukin-6 receptor, 50 ng/mL stem cell <em>factor</em>, 10 ng/mL thrombopoietin, and <em>20</em> ng/mL Flit3/4 ligand) 3 days prior to viral infection. Cells were then infected with retroviral constructs driving the expression of OCT3/4, SOX2, Krüppel-like <em>factor</em> 4, c-MYC, and enhanced green fluorescent protein together with or without the p53 knockdown lentiviral construct Shp53 pLKO.1-puro. Infected cells were then cultured for an additional 4 days in hematopoietic culture medium before being transferred onto mouse embryonic <em>fibroblast</em> (MEF) or SNL76/7 feeder cells in human embryonic stem cell medium (Dulbecco's modified Eagle medium/F-12 containing <em>20</em>% knockout serum replacement, <em>20</em>0 mM L-glutamine, 1% non-essential amino acids (NEAA), 0.1 mM 2-mercaptoethanol, and 4 ng/mL basic <em>fibroblast</em> <em>growth</em> <em>factor</em>). Subsequently, the number of embryonic stem cell-like colonies that emerged in the following 4 weeks was scored. Expression of a number of pluripotency makers were examined by immunochemistry and reverse transcriptase polymerase chain reaction. Finally, the differentiation potential of selected colonies was determined by teratoma formation in severe combined immunodeficient mice and in vitro culture.

RESULTS

Repression of p53 expression by the addition of a lentiviral p53 short-hairpin RNA expression vector increased the frequency of formation of iPS-like colonies from 1 (on average) to around 100 per 2 x 10(4) cells when infected cells were grown on SNL feeder cells.

CONCLUSIONS

iPS cells can be generated easily from CD34(+) cord blood cells through the addition of p53 inhibition to standard reprogramming conditions.

The objective of this study was to analyze the correlation between matrix metalloproteinases (MMPs) and angiogenic genes and survival in advanced-stage ovarian carcinomas. Primary and metastatic ovarian carcinomas from patients diagnosed with FIGO stage III-IV disease and followed up to <em>20</em> years were studied using mRNA in situ hybridization (ISH). Expression of MMP-2, MMP-9, membrane-type 1-MMP (MT1-MMP), the MMP inhibitor TIMP-2, vascular endothelial <em>growth</em> <em>factor</em> (VEGF), interleukin-8 (IL-8) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) was studied. MMP-2, MMP-9 and TIMP-2 mRNA was detected in both tumor and stromal cells, while MT1-MMP was largely confined to tumor cells. In univariate analysis of primary tumors, TIMP-2 and MMP-9 mRNA expression correlated with poor outcome. In metastatic lesions, mRNA expression of TIMP-2, MMP-2, and MT1-MMP correlated with poor survival. In a multivariate analysis of primary tumors, TIMP-2 expression in stromal cells (P=0.006) and MMP-9 expression in tumor cells (P=0.011) retained their predictive value. Intense expression of bFGF mRNA and weak expression of IL-8 mRNA was detected in both stromal and tumor cells in most cases, while VEGF mRNA expression was limited to a few cases. Angiogenic mRNA expression showed no correlation with disease outcome in survival analysis (P>0.05). We conclude that bFGF is the major angiogenic <em>factor</em> expressed in ovarian carcinoma at the mRNA level. MMP-2, MMP-9, MT1-MMP and TIMP-2 are valid markers of poor survival in advanced-stage ovarian carcinoma.

OBJECTIVE

Osteopontin (OPN) is upregulated in left ventricular hypertrophy and is stimulated by angiotensin II (AngII). Our objective was to determine whether mice deficient in OPN would be protected from AngII-induced cardiac fibrosis.

BACKGROUND

Interstitial fibrosis can lead to myocardial dysfunction and ultimately heart failure. Osteopontin activates integrins that regulate cell adhesion, migration, and growth, thus implicating OPN in the process of cardiac fibrosis.

METHODS

Osteopontin null (OPN(-/-)) mice (n = 18) and wild-type controls (n = 20) were infused with AngII (2.5 or 3.0 microg/kg/min) for four days or three weeks via osmotic mini-pumps. Hearts were assessed morphometrically and histologically, including quantitative assessment of fibrosis via optical microscopic imaging analysis. Cardiac fibroblasts derived from these mice were evaluated for adhesion and proliferation. Cardiac transcript expression for cytokines, extracellular matrix (ECM), integrin, and atrial natriuretic peptide were assessed.

RESULTS

Osteopontin(-/-) mice exhibited less cardiac fibrosis (0.7%) than wild-type mice (8.0%) (p < 0.01) and lowered heart/body weight ratios (0.10% vs. 0.23%) (p < 0.01) after three weeks of AngII infusion. Expression of transforming growth factor-beta, fibronectin, and collagen was not different between OPN(-/-) and wild-type mice, despite the decrease in ECM accumulation in the OPN(-/-) mice. Adhesion to ECM substrates decreased by 30% to 50% in cardiac fibroblasts of OPN(-/-) mice but was restored in OPN(-/-) cells by the addition of recombinant osteopontin.

CONCLUSIONS

Osteopontin mediates cardiac fibrosis, probably through the modulation of cellular adhesion and proliferation. Because OPN is increased in cardiac hypertrophy and its lack attenuates fibrosis, understanding of OPN function is essential to extend our knowledge about molecular determinants of cardiac hypertrophy and failure.

Betel quid (BQ) chewing is an etiologic <em>factor</em> of oral cancer and submucus fibrosis (OSF). Keratinocyte inflammation is crucial for the pathogenesis of cancer and tissue fibrosis. We found that areca nut (AN) extract (100-400 micro g/ml) induced PGE2 production by KB cells by 2.34- to 23.1-fold and also TNF-alpha production by gingival keratinocytes (GK). Arecoline (0.2-1.2 mM) elevated PGE2 production by KB cells by 2.5- to 6.1-fold. AN extract (<em>20</em>0-400 micro g/ml) also induced IL-6 production by GK (7.5- to 8.4-fold) and KB cells. In contrast, arecoline (0.1-1.2 mM) suppressed IL-6 production by GK and KB cells, with 42-81 and 41-63% inhibition, respectively. A 48 h exposure of GK to 800-1<em>20</em>0 micro g/ml AN extract led to 37-69% cell death. Arecoline cytotoxicity to GK was noted at concentrations of 0.8-1.2 mM, which led to 28-38% cell death. AN extract (400-800 micro g/ml) induced Cox-2 and IL-6 mRNA expression and also COX-2 protein production by KB cells. IL-6 (5-100 ng/ml) suppressed GK <em>growth</em> by <em>20</em>-33%, but enhanced oral <em>fibroblast</em> (OMF) and KB cell <em>growth</em>. PGE2 (0.05-5 micro g/ml) and anti-IL-6 antibody (ab) (50-1000 ng/ml) showed little effect on GK and KB cell <em>growth</em>. Incubation of GK and KB cells with aspirin, anti-IL-6 ab and anti-TNF-alpha ab showed little effect on arecoline- and AN-induced cytotoxicity, cell cycle arrest and apoptosis. Exposure to anti-TNF-alpha ab slightly affected arecoline- and AN-modulated PGE2 and IL-6 production by GK and KB cells. Arecoline- and AN-conditioned medium decreased phytohemagglutinin-mediated CD4+ and CD8+ T cell activation. These results indicate that BQ chewing contributes to the pathogenesis of cancer and OSF by impairing T cell activation and by induction of PGE2, TNF-alpha and IL-6 production, which affect oral mucosal inflammation and <em>growth</em> of OMF and oral epithelial cells.

The Basidiomycete fungus Agaricus blazei Murill has traditionally been used as a health food for the prevention of cancer, diabetes, hyperlipidemia, arteriosclerosis and chronic hepatitis. In the present study, we examined the antitumor activities of various substances isolated from the lipid fraction of A. blazei. Tumor <em>growth</em> was retarded by the oral administration of the lipid fraction extracted from A. blazei with a chloroform/methanol mixture in sarcoma 180-bearing mice. The substance with the antitumor activity in the lipid fraction was isolated via silica gel column chromatography, eluted with an acetonitrile/methanol (3:2) mixture and identified as ergosterol by direct comparison of the (1)H NMR and mass spectrometry spectral data of an authentic sample. The oral administration of ergosterol to sarcoma 180-bearing mice significantly reduced tumor <em>growth</em> at doses of 400 and 800 mg/kg administered for <em>20</em> d without side effects, such as the decreases in body, epididymal adipose tissue, thymus, and spleen weights and leukocyte numbers induced by cancer chemotherapy drugs. Ergosterol had no cytotoxicity against tumor cells. To clarify the antitumor activity of ergosterol, we examined the effects of ergosterol on tumor-induced angiogenesis using two in vivo models. Intraperitoneal administration of ergosterol at doses of 5, 10 and <em>20</em> mg/kg for 5 consecutive d inhibited the neovascularization induced by Lewis lung carcinoma cell-packed chambers, suggesting that either ergosterol or its metabolites may be involved in the inhibition of tumor-induced neovascularization. Therefore, we further examined the inhibitory effects of ergosterol on Matrigel-induced neovascularization. Female C57BL/6 mice were subcutaneously inoculated with Matrigel containing acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> and heparin with or without ergosterol. Ergosterol inhibited the Matrigel-induced neovascularization, suggesting that ergosterol directly inhibits Matrigel-induced neovascularization. From these results, it seems likely that the antitumor activity of ergosterol might be due to direct inhibition of angiogenesis induced by solid tumors. This is the first report of ergosterol as an antiangiogenic substance.

The expression of oncofetal H19 RNA and its localization/cellular source was analyzed in synovial tissue (ST) and isolated synovial macrophages (Mphi) or synovial <em>fibroblasts</em> (SFBs) by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. RT-PCR showed significantly higher H19 expression in ST from patients with rheumatoid arthritis (RA) (P = 0.000) and osteoarthritis (OA) (P = 0.009) than in normal/joint trauma controls (N/JT), but comparable levels in reactive arthritis. In situ hybridization demonstrated strong signals in all RA-ST samples (n = 8), with>> or =85% positive cells in the lining layer, diffuse infiltrates, and stroma regions. In lymphoid aggregates and endothelial cells only <em>20</em>% were positive. RA-ST contained a significantly higher percentage of strongly positive lining cells than OA-ST and N/JT-ST. H19 RNA was expressed in both Mphi and SFBs, as confirmed by RT-PCR in isolated RA Mphi and SFBs (n = 3). In RA-SFBs, low constitutive H19 RNA expression in culture (10% fetal calf serum) was strongly increased on starvation (3.5-fold, 1% fetal calf serum), with or without the addition of interleukin-1beta (10 to 100 U/ml), tumor necrosis <em>factor</em>-alpha (1 to 25 ng/ml), or platelet-derived <em>growth</em> <em>factor</em>-BB (2.5 to 10 U/ml). In OA-SFBs, this starvation-induced increase was lower (twofold), reaching significant differences compared with RA-SFBs after stimulation with interleukin-1beta and platelet-derived <em>growth</em> <em>factor</em>-BB. In both RA- and OA-SFBs, the MAP-kinase ERK-1/2 pathway and the phosphatidylinositol-3 kinase pathway influenced H19 RNA expression, as shown by inhibitor studies. Significant overexpression of H19 RNA and its increased sensitivity to starvation/cytokine regulation in RA suggests a pathogenetic role of this oncofetal gene, possibly reflecting embryonal dedifferentiation of the adult ST and/or ongoing inflammatory/oxidative stress.

BACKGROUND

Biliary tract cancers (BTCs) typically present at an advanced stage, and systemic chemotherapy is often of limited benefit.

METHODS

Hybrid capture-based comprehensive genomic profiling (CGP) was performed for 412 intrahepatic cholangiocarcinomas (IHCCAs), 57 extrahepatic cholangiocarcinomas (EHCCAs), and 85 gallbladder carcinomas (GBCAs). The mutational profile was correlated with the clinical outcome of standard and experimental therapies for 321 patients. Clinical variables, detected mutations, and administered therapies were correlated with overall survival (OS) in a Cox regression model.

RESULTS

The most frequent genetic aberrations (GAs) observed were tumor protein 53 (TP53; 27%), cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B; 27%), KRAS (22%), AT-rich interactive domain-containing protein 1A (ARID1A; 18%), and isocitrate dehydrogenase 1 (IDH1; 16%) in IHCCA; KRAS (42%), TP53 (40%), CDKN2A/B (17%), and SMAD4 (21%) in EHCCA; and TP53 (59%), CDKN2A/B (19%), ARID1A (13%), and ERBB2 (16%) in GBCA. <em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR; 11%) and IDH mutations (<em>20</em>%) were mostly limited to IHCCA but appeared to be mutually exclusive. In the IHCCA group, TP53 and KRAS mutations were associated significantly with poor OS, whereas FGFR2 mutations were associated with improved OS (P = .001), a younger age at onset, and female sex. IDH1/2 mutations were not prognostic. In a multivariate model, the effects of TP53 and FGFR GAs remained significant (P < .05). Patients with FGFR GAs had superior OS with FGFR-targeted therapy versus standard regimens (P = .006). Targeted therapy in IHCCA was associated with a numerical OS improvement (P = .07).

CONCLUSIONS

This is the largest clinically annotated data set of BTC cases with CGP and indicates the potential of CGP for improving outcomes. CGP should be strongly considered in the management of BTC patients. Cancer <em>20</em>16;122:3838-3847. © <em>20</em>16 American Cancer Society.

OBJECTIVE

To determine factors that control ocular enlargement in experimental form-deprivation myopia and to clarify the mechanism of form-deprivation myopia.

METHODS

After the left eyes of 20 chicks were monocularly occluded for 2 weeks, protein, basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta 2 contents in samples of constant area (circular button, diameter = 8.5 mm) in the retina-retinal pigment epithelium (RPE)-choroid and the sclera in the posterior region of control and myopic eyes were determined by enzyme-linked immunosorbent assay.

RESULTS

The bFGF content (per circular button) and bFGF concentration (per mg protein) were significantly lower in the sclera in the posterior region of the myopic eyes than in control eyes. The bFGF content and concentration were similar in the retina-RPE-choroid in myopic and control eyes. The TGF-beta 2 content and concentration were significantly higher in myopic eyes in both the retina-RPE-choroid and the sclera (P < 0.05).

CONCLUSIONS

These results are consistent with the possibility that bFGF and TGF-beta 2 regulate ocular enlargement or respond to myopiagenic mechanisms in form-deprivation myopia.

BACKGROUND

Calcification inhibitor deficiencies, mineral imbalance, and phenotypic transformation of vascular cells to osteogenic cells initiate and sustain vascular calcification. Fibroblast growth factor-23 (FGF23) is a key molecule regulating mineral homeostasis.

OBJECTIVE

Our objective was to assess the association of serum FGF23 levels with mineral metabolism parameters and abdominal aortic calcification (AAC) in men.

METHODS

This was a cross-sectional analysis in the STRAMBO cohort.

METHODS

Men holding a private health insurance cover with Mutuelle de Travailleurs de la Région Lyonnaise were included in the study.

METHODS

Participants included male volunteers aged 20-87 (n = 1130).

METHODS

Nonfasting blood collection was done. AAC was semiquantitatively assessed from vertebral fracture assessment scans obtained using dual-energy x-ray absorptiometry.

METHODS

We evaluated the association between FGF23 concentration and AAC severity in men.

RESULTS

In 350 men aged 60 yr or younger, FGF23 levels decreased with age (r = -0.21; P < 0.001) but were not associated with any other parameter. In 780 men aged over 60 yr, serum FGF23 correlated with age (r = 0.37; P < 0.001) and, after adjustment for confounders, with glomerular filtration rate (r = -0.31; P < 0.001) and PTH levels (r = 0.25; P < 0.001). After adjustment for confounders, self-reported ischemic heart disease, diabetes mellitus as well as higher concentrations of C-reactive protein and osteoprotegerin were all associated with higher FGF23 levels. After adjustment for confounders, subjects in the highest FGF23 quartile had higher prevalence of severe AAC compared with the three lower quartiles combined (odds ratio = 1.88; 95% confidence interval = 1.22-2.85; P < 0.005).

CONCLUSIONS

In healthy older men, circulating FGF23 is associated with parameters of mineral metabolism, including bone metabolism-regulating cytokines, and with severe AAC independent of traditional risk factors.

Mechanisms accounting for the development of proliferative vitreoretinopathy (PVR) in patients with rhegmatogenous retinal detachment remain poorly understood. In a previous study, we found the presence of various <em>growth</em> <em>factors</em> in preretinal membranes that were surgically removed from patients with PVR. The present immunohistological study was undertaken in intravitreal and subretinal fluid cells from patients suffering from PVR in various stages of development, in order to seek the presence of 4 <em>growth</em>-promoting <em>factors</em> for retinal pigment epithelial cells: acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF), epidermal <em>growth</em> <em>factor</em> (EGF), insulin-like <em>growth</em> <em>factor</em> type I (IGF-I) and transforming <em>growth</em> <em>factor</em>-beta (TGF-beta). Results were quite similar in vitreous and subretinal fluid. Acidic FGF was found in all vitreous and subretinal specimens, in 30-100% of the examined cells. Immunoreactivity for EGF could be found in 53% of intravitreal cells and 69% of subretinal fluid cells. Positive cells were seen in all vitreous specimens and in all but 1 of the subretinal fluid specimens. IGF-I-containing cells were present in 13 of 15 vitreous specimens and in 18 of <em>20</em> subretinal fluid samples (mean percentages of reactivity in positive specimens 70% and 78%, respectively). In contrast, TGF-beta 1 reactivity was found in only 8 of 15 vitreous specimens and in 11 of <em>20</em> subretinal samples. Mean percentages of reactive cells were 30% and 50%, respectively. These results suggest that several <em>growth</em> <em>factors</em> could be involved in the proliferation and migration of retinal pigment epithelial cells during the course of PVR.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic kidney disease-mineral bone disorder (CKD-MBD) is a systemic disorder that describes the complex bone and mineral abnormalities that occur in CKD. To understand the pathophysiology of CKD-MBD and determine whether the early use of phosphate binders would alter this physiology, we used a naturally occurring, slowly progressive model of CKD-MBD, the Cy/+ rat. Male Cy/+ rats were compared with their normal littermates at <em>20</em> weeks of age after 1 week of no phosphate binder, calcium carbonate, or sevelamer carbonate. The Cy/+ rat had renal function that was 50% of that of normal littermates, elevated parathyroid hormone (PTH) and <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23), decreased 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] levels, but normal calcium and phosphorus levels. There was a significant positive correlation of blood FGF23 and phosphorus levels and blood FGF23 and urine phosphorus levels. There was an inverse correlation between FGF23 and calcium levels. mRNA from the kidney demonstrated 50% reduction in klotho and Npt2a expression but no difference in CYP27B1. In the intestine, CKD animals had reduced active phosphate absorption in the jejunum using modified Ussing chambers and a reduction in Npt2b expression throughout the small intestine compared with normal littermates. In bone, mRNA expression of FGF23 was reduced (driven by lowering with phosphate binders), and TRAP expression was increased in CKD. By histology, there was increased osteoclast activity and number, and there were reductions in some measures of femoral neck mechanical strength. One week of phosphate binders reduced intestinal phosphate flux, serum phosphorus levels, and urinary phosphate excretion. These results demonstrate marked abnormalities in kidney, intestine, and bone in early CKD-MBD. While phosphate binders were effective in lowering urine phosphorus, they had little effect on end organs after 1 week of administration.

We have previously shown the development in vitro of tryptase+ human mast cells from fetal liver cells cocultured with murine 3T3 <em>fibroblasts</em>. In this study, recombinant human stem cell <em>factor</em> (rhuSCF), the ligand for the c-kit proto-oncogene product called Kit, stimulated the <em>growth</em> and differentiation primarily of mast cells from dispersed fetal liver cells, whereas recombinant human interleukin-3 (rhuIL-3) stimulated the differentiation of basophils along with other cell types. Cultures of fetal liver cells were initiated and maintained in the presence of rhuSCF or rhuIL-3 for up to 6 weeks. Metachromatic cells in cytospins were identified as mast cells primarily on the basis of tryptase expression, and as MCT or MCTC by immunohistochemistry using monoclonal antibodies against tryptase and chymase, whereas basophils were metachromatic, polymorphonuclear, and lacked these proteases. Levels of tryptase and histamine were measured by radioimmunoassay, tryptase and chymase activities by peptide hydrolysis, and cell surface Kit by flow cytometry with the monoclonal antibody YB5.B8. The predominant presence of mast cells occurred only in the cultures supplemented with rhuSCF. The percentage and total number of mast cells increased over time with increasing concentrations of rhuSCF and reached a plateau at 55 ng/mL. At this concentration of rhuSCF, mast cells first appeared by day 7; by day 42, 106% of the starting number of cells were present and 85% of these were tryptase+, 31% being weakly chymase+. These mast cells appeared immature by ultrastructural criteria; most cells were mononuclear, but some had nuclei with deeply divided lobes. DNA synthesis in tryptase+ mast cells at days 21 and 28 of culture with rhuSCF was demonstrated by incorporation of bromodeoxyuridine. Calculated levels of histamine (1.2 pg/mast cell) and tryptase (0.9 pg/mast cell) were similar to those determined previously in coculture experiments with murine 3T3 <em>fibroblasts</em>. Chymase activity was undetectable in most cell extracts. On day 0, 4% to <em>20</em>% of fetal liver cells expressed cell surface Kit. In the presence of rhuSCF, the percentages and total numbers of Kit+ cells and the apparent concentration of Kit per cell increased along with the number of tryptase+ cells. In the presence of rhuIL-3, toluidine blue+, tryptase- cells first and maximally appeared at day 14 (11% +/- 2.5%). The percentage of these toluidine blue+ cells then declined to about 6% by days 21 and 35, while the total number of positive cells declined over 10-fold. Kit+ cells in the presence of rhuIL-3 declined from 9% on day 3 to 2% on day 35.(ABSTRACT TRUNCATED AT 400 WORDS)

alpha-Thrombin (thrombin), a potent mitogen for CCL39 hamster lung <em>fibroblasts</em>, stimulates phosphoinositide-specific phospholipase C (PI-PLC) and inhibits adenylate cyclase via cleavage of a specific G-protein-coupled receptor (TH-R), recently cloned from human and hamster cells. This action can be entirely mimicked by the synthetic peptide SFFLRNP, referred to here as TMP (thrombin-mimicking peptide). TMP corresponds to the first seven amino acids of the new N-terminus generated by thrombin cleavage of the hamster TH-R. Although thrombin and TMP apparently generate identical early transmembrane signals, only thrombin is mitogenic on its own. TMP needs to be associated with <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF), a tyrosine kinase-activating <em>growth</em> <em>factor</em>, to induce cell-cycle re-entry. Here, we have examined the early and late phase of p44 MAP kinase (p44mapk) activation in G0-arrested CCL39 cells after stimulation by thrombin, TMP, FGF or TMP+FGF. We found that: (i) both thrombin and TMP rapidly activate p44mapk in a dose-dependent manner with maximum activation at around 5 min, (ii) after the initial burst of activation, a second and long-lasting wave of activation is observed in response to thrombin (10-100 nM) but not to TMP (up to 300 microM), (iii) FGF alone (25 ng/ml), like thrombin, rapidly and persistently activates p44mapk (<em>20</em>-fold at 5 min and about 3-fold after 2 h), (iv) TMP added together with FGF strongly potentiates the second and sustained phase of p44mapk activation. From these results we propose that: (1) thrombin-induced mitogenesis is mediated only in part by the TH-R recently cloned and (2) activation of p44mapk, in particular the long-lasting phase that correlates with DNA synthesis, is an obligatory event for cell-cycle re-entry.

The aim of this study was to produce dopaminergic neurons in vitro from human embryonic stem (hES) cells following treatment of various neurotrophic <em>factors</em>. MB03 hES cells were induced by retinoic acid (RA) or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), which were further treated with brain derived neurotrophic <em>factor</em> (BDNF) or transforming <em>growth</em> <em>factor</em> (TGF)-alpha in each induction method during neuron differentiation days. At the final differentiation stage (21 days), all treatment groups revealed very similar levels (bFGF, 76-78%; RA, 70-74%) of mature neurons (anti-NF-<em>20</em>0) in two induction methods irrespective of the addition of BDNF or TGF-alpha. In addition, immunostaining and HPLC analyses revealed higher levels of tyrosine hydroxylase (<em>20</em>+/-2.3%) and dopamine (265.5+/-62.8 pg/ml) in the bFGF- and TGF-alpha-treated hES cells than in RA- or BDNF-treated hES cells. These data are one of the first reports on the generation of dopaminergic neurons of hES cells in vitro. Also, our results indicate that TGF-alpha may be successfully used in the bFGF induction protocol and yield higher numbers of dopaminergic neurons from hES cells.

We investigated the influence of organ microenvironment on the angiogenic phenotype in human renal cell carcinoma (HRCC) cells. HRCC line SN12C was established in vitro from a surgical specimen, and metastatic line SN12PM6 was isolated from a lung metastasis produced by parental cells implanted into the kidney of nude mice. SN12C (low metastasis) and SN12PM6 (high metastasis) cells were injected into the kidney or subcutis of nude mice. The kidney tumors were highly vascularized (as revealed by immunohistochemistry using antibodies against <em>factor</em> VIII), and metastatic, whereas the subcutaneous tumors were not. The expression of mRNA for basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) in kidney tumors was 10 to <em>20</em> times that found in subcutaneous tumors. Similar data were obtained at the protein level by using fluorescence activated cell sorting, immunohistochemistry, and enzyme-linked immunosorbent assay. bFGF was detected in the urine of mice with tumors in the kidney but not subcutaneous tumors. The level of bFGF in the serum of mice with kidney tumors was two to three times that in mice with subcutaneous tumors. The changes in bFGF expression in the tumors was transient. Collectively, these data indicate that the organ microenvironment can influence the expression level of bFGF in HRCC.

OBJECTIVE

The epidermal growth factor receptor (EGFR) mutation status has emerged as a validated biomarker for predicting the response to treatment with EGFR-tyrosine kinase inhibitors (EGFR-TKI) in patients with non-small cell lung cancer. However, the responses to EGFR-TKIs vary even among patients with EGFR mutations. We studied several other independently active biomarkers for EGFR-TKI treatment.

METHODS

We retrospectively analyzed the serum concentrations of 13 molecules in a cohort of 95 patients with non-small cell lung adenocarcinoma who received EGFR-TKI treatment at three centers. The pretreatment serum concentrations of amphiregulin, β-cellulin, EGF, EGFR, epiregulin, fibroblast growth factor-basic, heparin-binding EGF-like growth factor, hepatocyte growth factor (HGF), platelet-derived growth factor β polypeptide, placental growth factor, tenascin C, transforming growth factor-α, and vascular endothelial growth factor (VEGF) were measured using enzyme-linked immunosorbent assay and a multiplex immunoassay system. The associations between clinical outcomes and these molecules were evaluated.

RESULTS

The concentrations of HGF and VEGF were significantly higher among patients with progressive disease than among those without progressive disease (P < 0.0001). HGF and VEGF were strongly associated with progression-free survival (PFS) and overall survival (OS) in a univariate Cox analysis (all tests for hazard ratio showed P < 0.0001). A stratified multivariate Cox model according to EGFR mutation status (mutant, n = 20; wild-type, n = 23; unknown, n = 52) showed that higher HGF levels were significantly associated with a shorter PFS and OS (P < 0.0001 for both PFS and OS). These observations were also consistent in the subset analyses.

CONCLUSIONS

Serum HGF was strongly related to the outcome of EGFR-TKI treatment. Our results suggest that the serum HGF level could be used to refine the selection of patients expected to respond to EGFR-TKI treatment, warranting further prospective study.

Multiwall carbon nanotubes (MWCNTs) have been widely used in many disciplines due to their unique physical and chemical properties, but have also raised great concerns about their possible negative health impacts, especially through occupational exposure. Although recent studies have demonstrated that MWCNTs induce granuloma formation and/or fibrotic responses in the lungs of rats or mice, their cellular and molecular mechanisms remain largely unaddressed. Here, it is reported that the TGF-β/Smad signaling pathway can be activated by MWCNTs and play a critical role in MWCNT-induced pulmonary fibrosis. Firstly, in vivo data show that spontaneously hypertensive (SH) rats administered long MWCNTs (<em>20</em>-50 μm) but not short MWCNTs (0.5-2 μm) exhibit increased <em>fibroblast</em> proliferation, collagen deposition and granuloma formation in lung tissue. Secondly, the in vivo experiments also indicate that only long MWCNTs can significantly activate macrophages and increase the production of transforming <em>growth</em> <em>factor</em> (TGF)-β1, which induces the phosphorylation of Smad2 and then the expression of collagen I/III and extracellular matrix (ECM) protease inhibitors in lung tissues. Finally, the present in vitro studies further demonstrate that the TGF-β/Smad signaling pathway is indeed necessary for the expression of collagen III in <em>fibroblast</em> cells. Together, these data demonstrate that MWCNTs stimulate pulmonary fibrotic responses such as <em>fibroblast</em> proliferation and collagen deposition in a TGF-β/Smad-dependent manner. These observations also suggest that tube length acts as an important <em>factor</em> in MWCNT-induced macrophage activation and subsequent TGF-β1 secretion. These in vivo and in vitro studies further highlight the potential adverse health effects that may occur following MWCNT exposure and provide a better understanding of the cellular and molecular mechanisms by which MWCNTs induce pulmonary fibrotic reactions.

1. We examined the effects of endogenous prostaglandin E(2) (PGE(2)) on the production of interleukin-6 (IL-6), macrophage colony stimulating <em>factor</em> (M-CSF), and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) by interleukin-1beta (IL-1beta)-stimulated human synovial <em>fibroblasts</em>. 2. NS-398 (1 microM), a cyclo-oxygenase-2 (COX-2) inhibitor, inhibited IL-6 and VEGF production (35+/-4% and 26+/-2%, respectively) but enhanced M-CSF production (38+/-4%) by IL-1beta (1 ng ml(-1)) in synovial <em>fibroblasts</em> isolated from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Exogenous PGE(2) completely abolished the effects of NS-398 on the production of each mediator by OA <em>fibroblasts</em> stimulated with IL-1beta. 3. 8-Bromo cyclic AMP and dibutyryl cyclic AMP, cyclic AMP analogues, mimicked the effects of PGE(2) on IL-6, M-CSF, and VEGF production by OA <em>fibroblasts</em>. 4. The EP(2) selective receptor agonist ONO-AE1-259 (2 nM) and the EP(4) selective receptor agonist ONO-AE1-329 (2 or <em>20</em> nM), but not the EP(1) selective receptor agonist ONO-DI-004 (1 microM) and the EP(3) selective receptor agonist ONO-AE-248 (1 microM), replaced the effects of PGE(2) on IL-6, M-CSF, and VEGF production by OA and RA <em>fibroblasts</em> stimulated with IL-1beta in the presence of NS-398. 5. Both OA and RA <em>fibroblasts</em> expressed mRNA encoding EP(2) and EP(4) but not EP(1) receptors. In addition, up-regulation of EP(2) and EP(4) receptor mRNAs was observed at 3 h after IL-1beta treatment. 6. These results suggest that endogenous PGE(2) regulates the production of IL-6, M-CSF, and VEGF by IL-1beta-stimulated human synovial <em>fibroblasts</em> through the activation of EP(2) and EP(4) receptors with increase in cyclic AMP.

Chemoresistance is one of the causes of poor prognosis in pancreatic cancer patients. However, the mechanisms of resistance remain unclear. Here we screened miRNAs associated with drug resistance in pancreatic cancer, and identified a panel of miRNAs dysregulated in gemcitabine-resistance pancreatic cancer cells, including 13 downregulated miRNAs and <em>20</em> upregulated miRNAs. Further studies focusing on miR-497 demonstrated that miR-497 suppressed cells proliferation, decreased the percentage of S phase cells, re-sensitized cells to gemcitabine and erlotinib, and attenuated migration and invasion capacities. Furthermore, <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 and <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 were confirmed as miR-497 targets. Overexpression of miR-497 inhibited tumor <em>growth</em> in vivo. Additionally, miR-497 expression was significantly downregulated in pancreatic cancer tissues compared with tumor-adjacent samples (P=0.000). Low expression of miR-497 was an independent adverse prognostic <em>factor</em> of pancreatic cancer (P=0.01, hazard ratio=2.762, 95% confidence interval: 1.159-6.579). Together these results indicate that miR-497 could be a new therapeutic target and prognostic marker of pancreatic cancer.

UNASSIGNED

Coronary artery calcification (CAC) is highly prevalent in dialysis-naive patients with chronic kidney disease (CKD). However, there are sparse data on the association of CAC with subsequent risk of cardiovascular disease and all-cause mortality in this population.

UNASSIGNED

To study the prospective association of CAC with risk of cardiovascular disease and all-cause mortality among dialysis-naive patients with CKD.

UNASSIGNED

The prospective Chronic Renal Insufficiency Cohort study recruited adults with an estimated glomerular filtration rate of <em>20</em> to 70 mL/min/1.73 m2 from 7 clinical centers in the United States. There were 1541 participants without cardiovascular disease at baseline who had CAC scores.

UNASSIGNED

Coronary artery calcification was assessed using electron-beam or multidetector computed tomography.

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Incidence of cardiovascular disease (including myocardial infarction, heart failure, and stroke) and all-cause mortality were reported every 6 months and confirmed by medical record adjudication.

UNASSIGNED

During an average follow-up of 5.9 years in 1541 participants aged 21 to 74 years, there were 188 cardiovascular disease events (60 cases of myocardial infarction, 1<em>20</em> heart failures, and 27 strokes; patients may have had >1 event) and 137 all-cause deaths. In Cox proportional hazards models adjusted for age, sex, race, clinical site, education level, physical activity, total cholesterol level, high-density lipoprotein cholesterol level, systolic blood pressure, use of antihypertensive treatment, current cigarette smoking, diabetes status, body mass index, C-reactive protein level, hemoglobin A1c level, phosphorus level, troponin T level, log N-terminal pro-B-type natriuretic peptide level, fibroblast growth factor 23 level, estimated glomerular filtration rate, and proteinuria, the hazard ratios associated with per 1 SD log of CAC were 1.40 (95% CI, 1.16-1.69; P < .001) for cardiovascular disease, 1.44 (95% CI, 1.02-2.02; P = .04) for myocardial infarction, 1.39 (95% CI, 1.10-1.76; P = .006) for heart failure, and 1.19 (95% CI, 0.94-1.51; P = .15) for all-cause mortality. In addition, inclusion of CAC score led to an increase in the C statistic of 0.02 (95% CI, 0-0.09; P < .001) for predicting cardiovascular disease over use of all the above-mentioned established and novel cardiovascular disease risk factors.

UNASSIGNED

Coronary artery calcification is independently and significantly related to the risks of cardiovascular disease, myocardial infarction, and heart failure in patients with CKD. In addition, CAC improves risk prediction for cardiovascular disease, myocardial infarction, and heart failure over use of established and novel cardiovascular disease risk factors among patients with CKD; however, the changes in the C statistic are small.

Alterations in the expression of integrin receptors for extracellular matrix (ECM) proteins are strongly associated with the acquisition of invasive and/or metastatic properties by human cancer cells. Despite this, comparatively little is known of the biochemical mechanisms that regulate the expression of integrin genes in cells. Here we demonstrate that the Ras-activated Raf-MEK-extracellular signal-regulated kinase (ERK) signaling pathway can specifically control the expression of individual integrin subunits in a variety of human and mouse cell lines. Pharmacological inhibition of MEK1 in a number of human melanoma and pancreatic carcinoma cell lines led to reduced cell surface expression of alpha6- and beta3-integrin. Consistent with this, conditional activation of the Raf-MEK-ERK pathway in NIH 3T3 cells led to a 5 to <em>20</em>-fold induction of cell surface alpha6- and beta3-integrin expression. Induced beta3-integrin was expressed on the cell surface as a heterodimer with alphav-integrin; however, the overall level of alphav-integrin expression was not altered by Ras or Raf. Raf-induced beta3-integrin was observed in primary and established mouse <em>fibroblast</em> lines and in mouse and human endothelial cells. Consistent with previous reports of the ability of the Raf-MEK-ERK signaling pathway to induce beta3-integrin gene transcription in human K-562 erythroleukemia cells, Raf activation in NIH 3T3 cells led to elevated beta3-integrin mRNA. However, unlike immediate-early Raf targets such as heparin binding epidermal <em>growth</em> <em>factor</em> and Mdm2, beta3-integrin mRNA was induced by Raf in a manner that was cycloheximide sensitive. Surprisingly, activation of the Raf-MEK-ERK signaling pathway by <em>growth</em> <em>factors</em> and mitogens had little or no effect on beta3-integrin expression, suggesting that the expression of this gene requires sustained activation of this signaling pathway. In addition, despite the robust induction of cell surface alphavbeta3-integrin expression by Raf in NIH 3T3 cells, such cells display decreased spreading and adhesion, with a loss of focal adhesions and actin stress fibers. These data suggest that oncogene-induced alterations in integrin gene expression may participate in the changes in cell adhesion and migration that accompany the process of oncogenic transformation.

We have examined the effect of peptide <em>growth</em> <em>factors</em> on DNA and proteoglycan synthesis by adult bovine articular cartilage in organ culture. The actions of somatomedin-C/insulin-like <em>growth</em> <em>factor</em> I (Sm-C/IGF-I), insulin, epidermal <em>growth</em> <em>factor</em> (EGF), and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) from bovine pituitary were investigated individually and in combination. FGF stimulated a 10-fold increase in tritiated thymidine incorporation while other <em>factors</em> used individually did not influence mitotic activity. Used in concert, insulin with EGF and insulin with FGF acted synergistically in stimulating DNA synthesis <em>20</em>-fold and 40-fold, respectively. All of these <em>growth</em> <em>factors</em>, acting individually, significantly enhanced radiosulfate incorporation. This stimulation was additive for Sm-C/IGF-I in combination with EGF or FGF, but not with insulin. These data indicate that adult bovine articular chondrocytes possess the capacity to augment both mitotic and differentiated cell functions in response to <em>growth</em> <em>factors</em>. The data further suggest that, with the exception of insulin and Sm-C/IGF-I, which appear to share a common mechanism of action, these <em>factors</em> produce their cellular effects via different receptor or postreceptor pathways.

Osteoblast-like cells have been shown to be sensitive to the proliferative action of a wide variety of <em>growth</em> <em>factors</em>. Many of these <em>growth</em> <em>factors</em> have been isolated from platelets and are thought to be released at local sites in response to injury. In this study, we tested whether human platelet concentrate, as a supplement to basic medium, would support the proliferative and functional activity of human fetal osteoblast-like cells in both short-term and long-term culture. In short-term studies, uptake of [3H]thymidine was increased in platelet-treated cultures by more than 4-fold compared with 10% serum-supplemented controls. When cultured for prolonged periods on coverslips, the cells formed multilayers, with a collagen-based matrix separating the layers. Long-term cultures that were treated with 1.5% (vol/vol) platelets in serum-supplemented medium showed increases in the depth of the multilayers of as much as 36-fold at 30 days after confluence, compared with the 10% serum-supplemented controls; this difference persisted until day 50. Incorporation of <em>growth</em> <em>factor</em> in the matrix was examined with the use of colloidal gold immunoelectron microscopy. Immunogold labeling intensities for transforming <em>growth</em> <em>factor</em>-beta 1 were significantly lower in the platelet-treated cultures at <em>20</em> days and then increased to a maximum level of 2.1-fold more than in the controls at 40 days. Labeling intensities for insulin-like <em>growth</em> <em>factor</em>-I and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> were significantly lower in the platelet-treated cultures than in the controls at all stages of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) was found to protect bovine aortic endothelial cells against the lethal effects of ionizing radiation by inhibiting the programmed cell death (apoptosis) induced in these cells by radiation exposure. The involvement of the bFGF receptor tyrosine kinase in this function was demonstrated by abrogation of the radioprotective effect of bFGF by a specific inhibitor of the bFGF receptor tyrosine kinase, the tyrphostin AG213. The downstream signaling after stimulation of the bFGF receptor tyrosine kinase in bovine aortic endothelial cells involved translocation of the alpha isotype of cytoplasmic protein kinase C (PKC) into the membrane and its activation within 30 s after bFGF stimulation. The involvement of PKC in the radioprotective effect conferred by bFGF was suggested by the demonstration that nonspecific PKC activation by short-term exposure (30 min) to the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA; 30 ng/ml) mimicked the radioprotective effect of bFGF. Furthermore, treatment of the cells with the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (<em>20</em> microM) abrogated the radioprotective effect of bFGF, as was observed after the depletion of cellular PKC by overnight preincubation with high-dose TPA (<em>20</em>0 nM). Agarose gel electrophoresis of DNA extracted from irradiated bovine aortic endothelial cells showed that both TPA (30 ng/ml; 30 min) and bFGF (1 ng/ml) inhibited the apoptotic degradation of DNA induced in these cells by radiation exposure (500 cGy). Both the bFGF- and the TPA-mediated inhibition of apoptosis could be reversed by the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (<em>20</em> microM). These data demonstrate the involvement of PKC in the inhibition of radiation-induced apoptosis by bFGF and the rescue of endothelial cells from this mode of radiation-induced cell death.