Methods for identification and in vitro expansion of ventral mesencephalic dopaminergic precursor cells are of interest in the search for transplantable neurons for cell therapy in Parkinson's disease (PD). We investigated the potential use of <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2) and <em>fibroblast</em> <em>growth</em> <em>factor</em> 8 (FGF8) for expansion of such dopaminergic precursor cells, and fetal antigen-1 (FA1), a secreted neuronal protein of unknown function, as a non-invasive dopaminergic marker. Tissue from embryonic day (ED) 12 rat ventral mesencephalon was dissociated mechanically and cultured for 4 days in the presence of FGF2, FGF8, or without mitogens (control). After mitogen withdrawal and addition of 0.5% bovine serum, cells were differentiated for 6 days. Before differentiation, significantly more cells incorporated BrdU in cultures exposed to FGF2 (<em>19</em>-fold; P < 0.001) and FGF8 (3-fold; P < 0.05) compared to controls. After differentiation, biochemical analyses showed significantly more dopamine and FA1 in conditioned medium from both FGF2 and FGF8 expanded cultures than in controls. Correspondingly, numbers of tyrosine hydroxylase (TH)- and FA1-immunoreactive cells had increased 16-fold (P < 0.001) and 2.1-fold (P < 0.001), respectively in the FGF2 group and 10-fold (P < 0.001) and 1.8-fold (P < 0.05), respectively in the FGF8 group. In conclusion, the present procedure allows efficient expansion and differentiation of dopaminergic precursor cells and provides novel evidence of FGF8 as a mitogen for these cells. Furthermore, FA1 was identified as a potential supplementary non-invasive marker of cultured dopaminergic neurons.

BACKGROUND

Thyroid nodules are present in <em>19</em>%-67% of the population and carry a 5%-10% risk of malignancy. Unfortunately, fine-needle aspiration biopsies are indeterminate in 20%-30% of patients, often necessitating thyroid surgery for diagnosis. Numerous DNA microarray studies including a recently commercialized molecular classifier have helped to better distinguish benign from malignant thyroid nodules. Unfortunately, these assays often require probes for >100 genes, are expensive, and only available at a few laboratories. We sought to validate these DNA microarray assays at the protein level and determine whether simple and widely available immunohistochemical biomarkers alone could distinguish benign from malignant thyroid nodules.

METHODS

A tissue microarray (TMA) composed of 26 follicular thyroid carcinomas (FTCs) and 53 follicular adenomas (FAs) from patients with indeterminate thyroid nodules was stained with 17 immunohistochemical biomarkers selected based on prior DNA microarray studies. Antibodies used included galectin 3, growth and differentiation factor 15, protein convertase 2, cluster of differentiation 44 (CD44), glutamic oxaloacetic transaminase 1 (GOT1), trefoil factor 3 (TFF3), Friedreich Ataxia gene (X123), fibroblast growth factor 13 (FGF13), carbonic anhydrase 4 (CA4), crystallin alpha-B (CRYAB), peptidylprolyl isomerase F (PPIF), asparagine synthase (ASNS), sodium channel, non-voltage gated, 1 alpha subunit (SCNN1A), frizzled homolog 1 (FZD1), tyrosine related protein 1 (TYRP1), E cadherin, type 1 (ECAD), and thyroid hormone receptor associated protein 220 (TRAP220). Of note, two of these biomarkers (GOT1 and CD44) are now used in the Afirma classifier assay. We chose to compare specifically FTC versus FA rather than include all histologic categories to create a more uniform immunohistochemical comparison. In addition, we have found that most papillary thyroid carcinoma could often be reasonably distinguished from benign disease by morphological cytology findings alone.

RESULTS

Increased immunoreactivity of CRYAB was associated with thyroid malignancy (c-statistic, 0.644; negative predictive value [NPV], 0.90) and loss of immunoreactivity of CA4 was also associated with malignancy (c-statistic, 0.715; NPV, 0.90) in indeterminate thyroid specimens. The combination of CA4 and CRYAB for discriminating FTC from FA resulted in a better c-statistic of 0.75, sensitivity of 0.76, specificity of 0.59, positive predictive value (PPV) of 0.32, and NPV of 0.91. When comparing widely angioinvasive FTC from FA, the resultant c-statistic improved to 0.84, sensitivity of 0.75, specificity of 0.76, PPV of 0.11, and NPV of 0.99.

CONCLUSIONS

Loss of CA4 and increase in CRYAB immunoreactivity distinguish FTC from FA in indeterminate thyroid nodules on a thyroid TMA with an NPV of 91%. Further studies in preoperative patient fine needle aspiration (FNAs) are needed to validate these results.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) is expressed in human fetal tissues and placenta. We, therefore, determined whether FGF-2 appeared in either fetal or maternal circulations during normal pregnancies [fetuses appropriate for gestational age (AGA)] or those complicated by fetal <em>growth</em> restriction (small for gestational age). Cordocentesis was performed, and matched maternal blood was collected between <em>19</em>-39 weeks gestation, whereas maternal and cord blood and amniotic fluid (AF) were collected at term. FGF-2 was extracted from maternal serum (MS), cord serum (CS), and AF by heparin-Sepharose affinity chromatography and subjected to Western blot analysis or quantified by specific RIA. Western blot analysis of MS, CS, and AF revealed, in each case, a single immunoreactive FGF-2 species of 18 kilodaltons (kDa), although this was not present in nonpregnant serum. In AGA pregnancies, immunoreactive FGF-2 was present in MS from at least 18 weeks gestation and rose to maximum values at the end of second trimester (weeks 28-31; mean +/- SEM, 342 +/- 62 pmol/L), but by term had declined (weeks 40-42; 104 +/- 24 pmol/L). In CS, FGF-2 immunoreactivity was highest at weeks 18-20 of gestation (662 +/- 144 pmol/L), but thereafter, slowly declined to term (weeks 40-42; 1<em>19</em> +/- 28 pmol/L). Immunoreactive FGF-2 levels in MS and CS of small for gestational age pregnancies in the second trimester tended to be lower than those in AGA pregnancies, but differences were not statistically significant. AF also contained immunoreactive FGF-2 at term (91 +/- 35 pmol/L). Neutral gel chromatography on Sephadex G-200 revealed that FGF-2 immunoreactivity eluted as a broad peak with an apparent molecular size of 55-160 kDa. These same fractions contained peptides of 55-60, 90-95, and 120-130 kDa, which were recognized by antisera against the extracellular domain of the high affinity FGF receptor, FGFR1, after Western blot. Ligand blot analysis of the same nitrocellulose filters using 125I-labeled FGF-2 revealed that the 55- to 60-kDa species specifically bound FGF-2. This binding species was not recognized during Western blot analysis using an antiserum raised against the intracellular tyrosine kinase domain of FGFR1, suggesting that it represents a truncated receptor form. Similar FGFR1 immunoreactive species were present in nonpregnant female and male sera, but were barely detectable in term CS or AF.(ABSTRACT TRUNCATED AT 400 WORDS)

BACKGROUND

Arterial hypertension and endothelial dysfunction have a role in the development of athero-sclerosis. This study assessed autocrine-paracrine endothelial function in patients with hypertension associated with renal failure.

METHODS

Angiotensin II (Ang II), endothelin-1 ( ET-1), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), transforming <em>growth</em> <em>factor</em>-beta (TGF-beta), soluble forms of adhesion molecules (ICAM-1, VCAM-1), E-selectin, von Willebrand <em>factor</em> (vWf) and nitric oxide (NO) were measured in 26 patients with hypertension and chronic renal failure (CRF), in <em>19</em> essential hypertensives (EH) and in 28 normotensive healthy subjects.

RESULTS

Plasma concentrations of Ang-II, ET-1, ICAM-1, VCAM-1, E-selectin, bFGF and TGF-beta all were significantly higher in patients than in healthy subjects and EH. Furthermore, in CRF, serum creatinine correlated negatively with NO plasma levels (r = - 0.51; p < 0.0) and this relationship held true after adjusting the data for potential confounders. Plasma NO was inversely related with ET-1 and bFGF (P < 0.01).

CONCLUSIONS

Hypertension in CRF is characterized by biochemical evidence of marked endothelial dysfunction, apparently more pronounced than in patients with EH. Amplified endothelial activation in CRF probably contributes to the high rate of atherosclerotic complications in CRF.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-2 is an abundant astroglial cytokine. We have previously shown that FGF-2 downregulates gap junctions in primary astroglial cultures (B. Reuss et al., <em>19</em>98, Glia 22, <em>19</em>-30). We demonstrate now that FGF-2 induces astroglial dopamine (DA) sensitivity and D1 dopamine-receptor (D1DR) antigen and message in cortical and striatal astroglial cultures. On the functional level 10 micromol/L DA triggered transient increases in astroglial [Ca(2+)](i). In gap-junction-coupled cells, no FGF-2-dependent changes in proportions of DA-responsive cells were observable. However, uncoupling with octanol or 18alpha-glycirrhetinic acid isolated the smaller population of astrocytes intrinsically sensitive to DA which was significantly increased by FGF-2 in cortical and striatal cultures. Administration of DR-specific substances revealed that FGF-2 upregulated D1DR. These results indicate that downregulation of astroglial gap junctions by FGF-2 is accompanied by an upregulation of D1DR and DA sensitivity, adding a new aspect to the role of FGF-2 in the regulation of brain functions.

Bile acids, synthesized in the liver and metabolized by microbiota, have emerged as important signaling molecules regulating immune responses and cell proliferation. However, the crosstalk among nutrition, microbiota, and bile acids remains unclear. Our study indicated that undernutrition in weaning piglets led to intestinal atrophy, increased colonic production, and systemic accumulation of lithocholic acid (LCA), deoxycholic acid (DCA), or their conjugated forms, which might be associated with decreased Lactobacillus abundance. Moreover, undernutrition led to increased portal <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> ( FGF<em>19</em>) level, upregulated hepatic heterodimer partner ( SHP), and downregulated cholesterol 7a-hydroxylase ( CYP7A1) expression. The detrimental effects of DCA and LCA on proliferation and barrier function were confirmed in porcine enterocytes, whereas their roles in weaning piglets warrant further research. In summary, undernutrition in weaning piglets led to increased secondary bile acids production, which might be related to altered gut microbiome and enhanced farnesoid X receptor (FXR) signaling while CYP7A1 expression was suppressed.

BACKGROUND

Impaired wound healing in ischemic tissues such as skin flaps resulting from inefficient perfusion is one major cause of complications in plastic surgery. In present experimental study, we investigated the effects of fibroblast growth factor-2 (FGF-2 or bFGF) and erythropoietin (EPO) in prevention of skin flap necrosis in rats.

METHODS

30 adult albino rats were randomized into 3 groups: in control group, normal saline solution; in EPO group, erythropoietin (100U/kg/day); and in FGF-2 group, fibroblast growth factor-2 (2.5µg/day) were injected subcutaneously in 3 daily consecutive doses in the designated flap areas before creating 4:1 random pattern skin flaps on the dorsum of animals. Areas of ischemic (SI) and necrotic (SN) zones were measured and compared in all groups one week after the flap creations.

RESULTS

The necrotic zone (SN), as well as the ratio of the necrotic zone to the total discolored zone (SN/[SI+SN]) were substantially larger in the control group (41%±7%, 90%±6%) compared to the EPO (20%±2%, 42%±4%) and the FGF-2 (8%±2%, 19%±3%) groups (p<0.001). The differences in these values were also meaningful between the EPO and FGF-2 groups (p<0.001).Vascular density in ischemic area of the control group was less than those in the EPO and the FGF-2 groups; however, the differences were not statistically significant between any of the groups (p>0.05).

CONCLUSIONS

Local administration of erythropoietin or fibroblast growth factor-2 in skin flaps could remarkably increase tissue viability and accelerate the wound healing process. However, the therapeutic effect of fibroblast growth factor-2 in preventing the necrotic event in ischemic zones of skin flaps is much more considerable than that of erythropoietin.

Background: Biliary tract cancer (BTC) has a poor prognosis and lacks a standardized second-line therapy. Vascular endothelial growth factor (VEGF), fibroblast growth factor receptor (FGFR) 4, and platelet-derived growth factor receptor (PDGFR) are highly expressed in BTC. Therefore, lenvatinib (a known inhibitor of VEGF receptors 1-3, FGFRs 1-4, and PDGFR-α) was evaluated for second-line treatment of BTC.

Methods: In this single-arm, multicenter, open-label, phase 2 study, patients with BTC received lenvatinib 24 mg orally once daily in 28-day cycles. The primary endpoint was objective response rate (ORR). Secondary endpoints included overall survival (OS), progression-free survival (PFS), PFS rate at 12 weeks, disease control rate, clinical benefit rate, safety and pharmacokinetic profiles.

Results: Twenty-six Japanese patients were enrolled and treated; 3 had a confirmed partial response per investigator assessment and per independent imaging review (IIR); ORR was 11.5% (90% confidence interval [CI]: 3.2-27.2). Median PFS was 3.19 months (95% CI: 2.79-7.23) per investigator assessment and 1.64 months (95% CI: 1.41-3.19) per IIR. Median OS was 7.35 months (95% CI: 4.50-11.27). Grade ≥ 3 treatment-emergent adverse events (TEAEs) occurred in 21 patients (80.8%) and included hypertension (n = 10 [38.5%]), proteinuria (n = 3 [11.5%]), palmar-plantar erythrodysesthesia (n = 3 [11.5%]), decreased appetite (n = 3 [11.5%]), and anemia (n = 3 [11.5%]). Two deaths occurred due to TEAEs between treatment initiation and 30 days after last dose, but neither were considered treatment related.

Conclusions: Lenvatinib demonstrated antitumor activity in BTC, with a tolerable safety profile, and should be further evaluated as potential second-line therapy for this difficult to treat population.

Trial registration: ClinicalTrials.gov NCT02579616 . Date of registration: October 19, 2015.

Keywords: Ampulla of Vater; Biliary tract cancer; Cholangiocarcinoma; Gallbladder cancer; Lenvatinib.

BACKGROUND

While dysmetabolism is common in patients with chronic kidney disease (CKD) and associated with mortality, the mechanisms mediating these changes are unclear. New data implicate <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-<em>19</em> as a possible entero-hepatic modulator of lipid metabolism.

METHODS

Using samples previously gathered as part of a randomized placebo-controlled study of antioxidative therapy for postprandial dysmetabolism, we investigated short-term (4 h) postprandial changes in circulating FGF-<em>19</em> (ELISA) and the relationship to metabolic markers in six haemodialysis (HD) patients and nine matched healthy subjects (HS), with each participant assessed on four separate occasions.

RESULTS

The postprandial FGF-<em>19</em> response was blunted in patients [maximum change +34.63 (0.24-186) pg/mL] versus controls [maximum change +150.3 (31.2-378.7) pg/mL; P < 0.0001], and the area under the curve (AUC; pg × min × mL(-1)) was also significantly lower 18 0<em>19</em> (12 513-44 387) versus 38 517 (<em>19</em> 775-72 816; P < 0.01). In patients, we found univariate correlations between AUC FGF-<em>19</em> with AUC C-peptide (rho = 0.71; P = 0.001), AUC insulin (rho = 0.63; P = 0.001), but not with AUCs for triglycerides (TG) or glucose. Finally, treatment with the antioxidative compounds N-acetyl cysteine or MP865, but not with placebo, was associated with higher plasma FGF-<em>19</em> (NAC and MP865 coefficients -0.28 and -0.23, P < 0.05, respectively).

CONCLUSIONS

In advanced CKD, the postprandial FGF-<em>19</em> response appears to be blunted, with partial normalization following antioxidative treatments. A blunted FGF-<em>19</em> response was associated with impaired insulin and C-peptide signalling.

The mechanisms underlying retinal cell diversification are crucial to proper neural development. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (Fgf<em>19</em>) is expressed by developing horizontal cells (HCs) in the chicken retina. Although there are two major HC subtypes, axon-bearing and axon-less, the precise subtype expressing Fgf<em>19</em> remains uncertain. Here we characterize Fgf<em>19</em>-expressing cells by co-labeling with antibodies against Lim1 (LIM homeodomain 1, or Lhx1), Islet1, and Prox1 (prospero-related homeobox 1) which are axon-bearing HC, axon-less HC, and pan-HC markers, respectively. We found that a subset of Fgf<em>19</em>-expressing cells was positive for Prox1 and Lim1 in the vitread neuroepithelium at embryonic day 4 (E4). By E9, the majority of Fgf<em>19</em>-expressing cells became positive for Prox1 and Lim1 prior to arrival at the prospective HC layer. In contrast, Fgf<em>19</em>-expressing cells did not overlap with the Islet1-positive population at any stage examined. These results suggest that Fgf<em>19</em> is expressed by the early migratory horizontal precursors, and later by the presumptive axon-bearing HCs.

Increased aortic pulse wave velocity (AoPWV) has been identified as a risk <em>factor</em> for cardiovascular morbidity in the general population and in patients on dialysis. Most of the studies in ESRD patients refer to subjects on hemodialysis. Influence of the inflammatory process on aortic stiffening remains largely unknown. The aim of the present study was to evaluate potential relationships between AoPWV and blood pressure, basic anthropometric parameters, selected <em>growth</em> <em>factors</em> and markers of the inflammatory process in ESRD patients treated with peritoneal dialysis. The study population consisted of 43 patients (<em>19</em> F, 24 M) with a mean age of 50.6 +/- 13.4 years on PD for a mean period of 21.9 +/- 20.7 months. AoPWV was measured using two pressure transducers placed on the carotid and femoral arteries and connected to an automatic processor (Complion Colson AS, Paris, France). Serum levels of Tumor Necrosis <em>Factor</em> alpha (TNFalpha), interleukin 6 (IL-6) and plasma basic <em>Fibroblast</em> <em>Growth</em> <em>Factor</em> (bFGF) were measured with ELISA; C-reactive protein and fibrinogen with nephelometry. Serum lipid profile was also assessed. Blood pressure was measured in an outpatient department under standardized conditions. Mean aortic pulse wave velocity in the study population was 10.7 +/- 2.1 m/s. No difference in AoPWV was found between men and women. AoPWV correlated significantly with age (R = 0.41; p < 0.01) but not with time on dialysis. Positive relationship between AoPWV and body weight and BMI was shown (R = 0.31; p < 0.05 and R = 0.35; p < 0.05, respectively). AoPWV correlated significantly with systolic blood pressure (SBP), mean arterial pressure (MAP) and pulse pressure (PP) (R = 0.46, p < 0.005, R = 0.46, p < 0.005 and R = 0.43, p < 0.01, respectively). AoPWV correlated with serum IL-6 and plasma bFGF (R = 0.32, p < 0.05 and R = 0.4, p < 0.01; respectively). The correlation with serum CRP was borderline significant (p < 0.53). In multiple regression analysis age (beta 0.38; p < 0.005), plasma bFGF level (beta 0.3; p < 0.05), and systolic blood pressure (beta 0.29; p < 0.05) were independently associated with pulse wave velocity. Our results suggest that AoPWV values in patients on PD are associated with <em>factors</em> similar to those encountered in the general population. We suggest that increased aortic stiffening may also be related to the chronic inflammatory process in PD patients.

Objective- In patients with stable coronary artery disease, conventional risk <em>factors</em> provide limited incremental predictive value for cardiovascular events. We sought to investigate whether a panel of cardiometabolic biomarkers alone or combined with conventional risk <em>factors</em> would exhibit incremental value in the prediction of cardiovascular events. Approach and Results- In the discovery cohort, we measured serum adiponectin, A-FABP (adipocyte fatty acid-binding protein), lipocalin-2, FGF (<em>fibroblast</em> <em>growth</em> <em>factor</em>)-<em>19</em> and 21, plasminogen activator inhibitor-1, and retinol-binding protein-4 in 1166 Chinese coronary artery disease patients. After a median follow-up of 35 months, 170 patients developed new-onset major adverse cardiovascular events (MACE). In the model with age ≥65 years and conventional risk <em>factors</em>, area under the curve for predicting MACE was 0.68. Addition of lipocalin-2 to the age-clinical risk <em>factor</em> model improved predictive accuracy (area under the curve=0.73). Area under the curve further increased to 0.75 when a combination of lipocalin-2, A-FABP, and FGF-<em>19</em> was added to yield age-biomarkers-clinical risk <em>factor</em> model. The adjusted hazard ratio on MACEs for lipocalin-2, A-FABP, and FGF-<em>19</em> levels above optimal cutoffs were 2.23 (95% CI, 1.62-3.08), 1.99 (95% CI, 1.43-2.76), and 1.65 (95% CI, 1.15-2.35), respectively. In the validation cohort of 1262 coronary artery disease patients with type 2 diabetes mellitus, the age-biomarkers-clinical risk <em>factor</em> model was confirmed to provide good discrimination and calibration over the conventional risk <em>factor</em> alone for prediction of MACE. Conclusions- A combination of the 3 biomarkers, lipocalin-2, A-FABP, and FGF-<em>19</em>, with clinical risk <em>factors</em> to yield the age-biomarkers-clinical risk <em>factor</em> model provides an optimal and validated prediction of new-onset MACE in patients with stable coronary artery disease.

Treatment of MSU-1.1 cells, a near-diploid, karyotypically stable, infinite life-span human <em>fibroblast</em> strain, with (+-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene induced focus formation. Eight independent foci were isolated and the cell strains developed from them were examined for characteristics of malignant cells. Each grew to a higher density in medium containing 1% serum than did the MSU-1.1 cells. Three of the eight grew rapidly in serum-free medium without added <em>growth</em> <em>factors</em>, formed colonies in agarose with diameters of greater than or equal to 120 microns at a frequency of 5-<em>19</em>%, exhibited loss of genetic material, and, when injected into athymic mice, formed sarcomas that reached 6 mm in diameter within 2-3 wk. One produced high-grade sarcomas (progressively <em>growing</em>, invasive tumors exhibiting high mitotic activity); the other two produced low-grade sarcomas (tumors with a lower degree of mitotic activity) that developed focal areas of high-grade malignant cells if left in the animals for greater than 4 wk. A fourth cell strain formed high-grade sarcomas only after 2.5-3 mo, but the tumor-derived cells analyzed showed the same <em>growth</em> properties as the three malignant cell strains described above, exhibited loss of genetic material, and, when reinjected into athymic mice, produced high-grade sarcomas with a short latency period.

To determine whether the human c-fes gene, a homologue of the feline v-fes oncogene, can play a role in the malignant transformation of human <em>fibroblasts</em>, we transfected a near-diploid, infinite life span, <em>growth</em> <em>factor</em>-independent, human <em>fibroblast</em> cell strain, MSU-1.2, with plasmids carrying a fes gene along with a selectable marker. The fes gene was either the v-fes oncogene from the Gardner-Arnstein strain of feline sarcoma virus or a chimeric construct in which 835 base pairs representing exons 10-<em>19</em> from the human c-fes proto-oncogene had been substituted for the corresponding feline sequence in the v-fes oncogene. The transfected cells were selected in appropriate medium, and a number of drug-resistant clones were isolated, and the progeny cells were assayed for fes expression by immunoprecipitation analysis. Six independent clones that expressed the v-fes protein and four that expressed the gag-c-fes protein were further characterized. The former exhibited anchorage independence and formed progressively <em>growing</em>, invasive, spindle cell sarcomas in athymic mice after only a short latency period. The latter strains were not anchorage independent and did not form tumors in athymic mice. These results show that the v-fes oncogene can malignantly transform an infinite life span human <em>fibroblast</em> cell strain, but the human c-fes gene cannot.

The aim of this study was to establish a long-term culture system for rat colon epithelial cells. Colonic crypts were isolated by incubating a 4-cm-long rat colon segment cut longitudinally with an ethylenediaminetetraacetic acid [disodium salt]-containing buffer, taken up in conditioned medium from the normal rat kidney <em>fibroblast</em> cell line NRK (i.e., the supernatant of pure NRK cultures), directly plated on mitomycin C-treated NRK cells and subcultured with conditioned medium from NRK cells. Cells started to migrate out of the crypts shortly after plating them on NRK feeder layers. Some of the crypts fell apart during the isolation procedure, whereas the vast majority of them did it within 1 to 2 h after plating. The cells proliferated extremely slowly but continuously over a period of 4 mo and were epithelial because they expressed cytokeratin <em>19</em> and were stained by crystal violet at pH 2.8. In conclusion, the experimental system described in this study allows to maintain rat colon epithelial cells for up to 4 mo in culture and can be used to study the effects of a variety of tumor-modulating <em>factors</em> on <em>growth</em> and gene expression of normal colon epithelial cells in vitro.

BACKGROUND

The "seed and soil" hypothesis emphasizes the importance of interactions between tumor cells and their microenvironment. CAFs (Cancer associated fibroblasts) are important components of the tumor microenvironment. They were widely involved in cancer cells growth and metastasis. Fibroblasts may also play a role in inflammatory disease. The phenotype conversion of fibroblasts in lung diseases has not been investigated previously. We hypothesized that fibroblasts phenotypes may vary among different types of lung disease.

METHODS

The study included six types of lung tissues, ranging from normal lung to lung adenocarcinoma with lymphatic metastasis. Para-carcinoma tissues which were 2-cm-away from the tumor focus were also included in the analysis. The expression of target proteins including alpha-SMA (smooth muscle actin), FAP (fibroblast activation protein), vimentin, E-cadherin, and CK-19 (cytokeratin-19) were examined by immunohistochemistry. TGF-beta(transforming growth factor) and Twist were detected simultaneously in all samples.

RESULTS

A progressive increase in the levels of alpha-SMA, vimentin and CK-19 was observed in correlation to the degree of malignancy from normal lung tissue to lung adenocarcinoma with lymphatic metastasis, whereas E-cadherin expression showed the opposite trend. TGF-beta and Twist were detected in cancer tissues and inflammatory pseudotumors. None of the proteins were detected in para-carcinoma tissues.

CONCLUSIONS

Fibroblast phenotypes varied according to the type and degree of lung malignancy and fibroblasts phenotypic conversion occurs as a gradual process with specific spatiotemporal characteristics. Similar fibroblast phenotypes in inflammatory diseases and cancer tissues suggested a correlation between inflammation and cancer and implied a common mechanism underlying the formation of fibroblasts in inflammatory diseases and lung cancer.

Since early diagnosis is very important for treating CRC, we decided to detect peripheral serum canopy <em>fibroblast</em> <em>growth</em> <em>factor</em> signaling regulator 2 (CNPY2) isoform 2 to verify its diagnostic value for CRC patients. Serum samples were collected from 430 CRC patients and 201 healthy controls. Enzyme-linked immunosorbent assay (ELISA) detection kits for CNPY2 isoform 2 were generated and then applied to measure serum CNPY2 isoform 2 concentrations. Serum carcinoembryonic antigen (CEA) and carbohydrate antigen <em>19</em>-9 (CA<em>19</em>-9) were also measured. The median serum CNPY2 isoform 2 concentrations in all CRC patients were significantly higher than those in the healthy control group (all P<0.001). Those with stage I CRC presented the highest area under the receiver operating characteristic curve (AUC) for CNPY2 isoform 2 [0.707, 95% confidence interval (CI): 0.649-0.765, P<0.001]. The diagnostic efficiency of the combination of CNPY2 isoform 2, CEA and CA<em>19</em>-9 was significantly higher than that of each biomarker detected separately (all P<0.0167). Serum CNPY2 isoform 2 may be a valuable biomarker for the early detection of CRC and presents an improvement in the diagnostic efficiency by combination of CEA and CA<em>19</em>-9.

<AbstractText>Abnormal autocrine <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) production has been observed in several types of cancers, including hepatocellular carcinoma (HCC). In this study, we investigated the potential of serum FGF<em>19</em> as a novel tumor marker of HCC based on a sandwich enzyme-linked immunosorbent assay (ELISA).</AbstractText><AbstractText>The serum FGF<em>19</em> levels of 304 patients with HCC was measured by ELISA. The serum levels of existing markers, including alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) were determined by chemiluminescence enzyme immunoassay. Both diagnostic value of FGF<em>19</em> and its changes after curative ablation therapy was further examined.</AbstractText><AbstractText>The median FGF<em>19</em> levels in controls, chronic liver disease patients, and primary HCC patients, were 78.8 pg/mL, 100.1 pg/mL, and 214.5 pg/mL, respectively. The subsequent receiver operating characteristic curves (ROC) successfully determined an optimal cut-off value of 200.0 pg/mL. The area under the ROC curve (AUC) of FGF<em>19</em> for HCC detection was comparable to those of AFP and DCP. Of importance, FGF<em>19</em> showed higher sensitivity for the detection of small HCC (solitary cancer with diameter < 20 mm) than those of existing markers. In addition, 43 out of 79 cases (54.4%) with normal AFP and DCP (so-called "double negative HCC") exhibited serum FGF<em>19</em> level ≥ 200 pg/mL. In 45 HCC patients treated with curative ablation therapy, serum FGF<em>19</em> levels changed from 257.4 pg/mL to 112.0 pg/mL after the treatment.</AbstractText><AbstractText>Our findings reveal that FGF<em>19</em> can be a potential novel biomarker for HCC. Although FGF<em>19</em> is not necessarily a substitute for existing markers, it may help improve the prognosis in HCC patients owing to its resourceful use in various aspects of HCC management and treatment.</AbstractText>

BACKGROUND

Micro-ribonucleic acids (miR) are small, noncoding RNA molecules <em>19</em> to 25 nucleotides in length that typically function as negative regulators of expression for many target genes involved in cell proliferation, differentiation, and apoptosis. However, the effects of miR-21 on keloid <em>fibroblasts</em> are currently unknown.

OBJECTIVE

The authors investigate whether miR-21, a specific miR implicated in multiple aspects of keloid fibroblasts, affects the expression of Fas ligand (FasL) in the presence of transforming growth factor (TGF)-β1.

METHODS

The relationship between TGF-β1 and miR-21 expression was investigated by TaqMan quantitative real-time polymerase chain reaction (Life Technologies, Grand Island, New York). FasL protein was determined by Western blotting, and regulation of cell proliferation/migration/apoptosis ability by TGF-β1 inhibitor or plasmid was evaluated respectively by EdU incorporation, Transwell assay, and flow cytometry analysis.

RESULTS

Fibroblasts from keloid tissue were confirmed to express high levels of TGF-β1 and miR-21 compared with normal skin fibroblasts. Expression of TGF-β1 and miR-21 was positively correlated in fibroblasts. In addition, cells transfected with TGF-β1 inhibitor or miR-21 inhibitor showed significant increases in FasL protein levels and number of apoptotic cells compared with control cells, whereas cell growth and migration significantly decreased. The opposite results could also be confirmed when TGF-β1 was upregulated in normal skin fibroblasts.

CONCLUSIONS

TGF-β1 could effectively influence cell proliferation, apoptosis, and migration via its control of miR-21. These findings also identify a novel mechanism of interaction between TGF-β1 and miR-21 in the regulation of FasL protein, which is involved in keloid formation.

Lung epithelial cell differentiation is predominantly regulated by mesenchymal-epithelial cell communication. We have previously shown that epidermal <em>growth</em> <em>factor</em> (EGF) positively influences this process, and that EGF receptor (EGF-R) binding in fetal rat lung <em>fibroblasts</em> peaks on d18-<em>19</em> of gestation, just before the onset of augmented surfactant synthesis. This regulation of EGF-R in late gestation fetal lung <em>fibroblasts</em> may control the timing of mesenchymal-epithelial cell communication leading to surfactant synthesis. Hormones and <em>growth</em> <em>factors</em> exert positive and negative influences on lung development, but whether they regulate the EGF-R is unknown. We hypothesized that positive [EGF, cortisol, retinoic acid (RA)] and negative [transforming <em>growth</em>-<em>factor</em>-beta1 (TGF-beta1), dihydrotestosterone (DHT)] regulators of lung cell development regulate the EGF-R in the fetal lung. We studied EGF-R binding and protein abundance in sex-specific fetal rat lung <em>fibroblasts</em> cultured at d17, d<em>19</em>, and d21. EGF-R binding was significantly elevated after RA (both sexes d17 and d<em>19</em>, females d21) and after DHT (females d<em>19</em>) treatment. EGF and cortisol had minimal or inhibitory effects on EGF-R binding. Western blot analysis showed that the observed changes in EGF-R binding were associated with similar changes in EGF-R protein. We conclude that <em>factors</em> that affect lung maturation continue to regulate EGF-R in a developmental, sex-specific manner during late gestation.

<AbstractText>Liver regeneration following partial hepatectomy (PHx) is a complicated process involving multiple organs and several types of signaling networks. The bile acid-activated metabolic pathways occupy an auxiliary yet important chapter in the entire biochemical story. PHx is characterized by rapid but transient bile acid overload in the liver, which constitutes the first wave of proliferative signaling in the remnant hepatocytes. Bile acids trigger hepatocyte proliferation through activation of several nuclear receptors. Following biliary passage into the intestines, enterocytes reabsorb the bile acids, which results in the activation of farnesoid X receptor (FXR), the consequent excretion of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)<em>19</em>/FGF15, and its release into the enterohepatic circulation. FGF<em>19</em>/FGF15 subsequently binds to its cognate receptor, <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 4 (FGFR4) complexed with β-klotho, on the hepatocyte membrane, which initiates the second wave of proliferative signaling. Because some bile acids are toxic, the remnant hepatocytes must resolve the potentially detrimental state of bile acid excess. Therefore, the hepatocytes orchestrate a bile acid detoxification and elimination response as a protective mechanism in concurrence with the proliferative signaling. The response in part results in the excretion of (biotransformed) bile acids into the canalicular system, causing the bile acids to end up in the intestines.</AbstractText><AbstractText>Recently, FXR agonists have been shown to promote regeneration via the gut-liver axis. This type of pharmacological intervention may prove beneficial for patients with hepatobiliary tumors undergoing PHx. In light of these developments, the review provides an in-depth account of the pathways that underlie post-PHx liver regeneration in the context of bile acid homeostasis in the liver and the gut-liver signaling axis.</AbstractText>

Bariatric surgery has proven to be the most effective treatment for controlling hyperglycemia in severely obese patients with diabetes. We show that <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>), a gut hormone, is rapidly induced by bariatric surgery in rodents and humans. Administration of FGF<em>19</em> achieves diabetes remission independent of weight loss in animal models of diabetes, supporting a role for FGF<em>19</em> in the hormonal remodeling that restores metabolic function after the surgery. Through an unbiased, systematic screen in diabetic mice, we identified selective, safe, and effective FGF<em>19</em> analogs. Unexpectedly, a lead FGF<em>19</em> analog, NGM282, did not correct hyperglycemia in patients with type 2 diabetes. In contrast, administration of NGM282 resulted in a rapid, robust, and sustained reduction in liver fat content and an improvement in liver histology in patients with nonalcoholic steatohepatitis, faithfully replicating another key benefit of bariatric surgery. Our work identifies a strategy for replacing the surgery with an equally effective, but less invasive, treatment for nonalcoholic steatohepatitis.

OBJECTIVE

Disruption of the enterohepatic circulation of bile acids (BAs) is part of the gastrointestinal phenotype of cystic fibrosis (CF). Ivacaftor (VX-770), a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator, improves pulmonary function in CF patients with class III gating mutations. We studied the effect of ivacaftor on the enterohepatic circulation by assessing markers of BA homeostasis and their changes in CF patients.

METHODS

In CF patients with an S1251N mutation (N = 16; age 9-35 years S125N study/NTR4873) or a G551D mutation (N = 101; age 10-24 years; GOAL study/ NCT01521338) we analyzed plasma <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) and 7α-hydroxy-4-cholesten-3-one (C4) levels, surrogate markers for intestinal BA absorption and hepatic synthesis, respectively, before and after treatment with ivacaftor.

RESULTS

At baseline, median FGF<em>19</em> was lower (52% and 53%, P < .001) and median C4 higher (350% and 364%, P < .001), respectively, for the S1251 N and G551D mutation patient groups compared to healthy controls. Treatment with ivacaftor significantly increased FGF<em>19</em> and reduced C4 levels towards normalization in both cohorts but this did not correlate with CFTR function in other organs, as measured by sweat chloride levels or pulmonary function.

CONCLUSIONS

We demonstrate that patients with CFTR gating mutations display interruption of the enterohepatic circulation of BAs reflected by lower FGF<em>19</em> and elevated C4 levels. Treatment with ivacaftor partially restored this disruption of BA homeostasis. The improvement did not correlate with established outcome measures of CF, suggesting involvement of modulating <em>factor</em>s of CFTR correction in different organs.