Human embryonic stem (hES) cells have traditionally been cultured in medium containing fetal calf serum (FCS) and mouse <em>fibroblasts</em> as feeder cells. The use of animal derived materials carries a risk of transmitting animal pathogens, and they are not optimal in cultures aimed at cell transplantation in humans. This technical study aiming at facilitating IVF units to establish new hES cell lines, has systematically compared the non-differentiated <em>growth</em> of the hES cell line HS237, originally derived and thereafter cultured using human foreskin <em>fibroblasts</em> as feeder cells, by culturing it in media containing serum replacement (SR; 10, <em>15</em>, 20%), FCS, and human serum. In addition, optimal concentrations of insulin-transferrin-selenium (ITS) mixture and the effect of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) have also been studied. Cellular <em>growth</em> was monitored daily and maintenance of their non-differentiated character was studied using antibodies against TRA-1-60, TRA-1-81 and SSEA-4 and expression of Oct-4. The hES cells proliferated fastest when 20% of SR was used. In human serum-containing medium, the cells underwent extensive spontaneous differentiation within a few passages. The FCS supported the non-differentiated <em>growth</em> poorly. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> supported non-differentiated <em>growth</em>, the highest concentration (8 ng/ml) giving the best result, while ITS was not beneficial.

In this report, we demonstrate that insulin receptor substrate-2 (IRS-2) is tyrosyl-phosphorylated following stimulation of 3T3-F442A <em>fibroblasts</em> with <em>growth</em> hormone (GH), leukemia inhibitory <em>factor</em> and interferon-gamma. In response to GH and leukemia inhibitory <em>factor</em>, IRS-2 is immediately phosphorylated, with maximal phosphorylation detected at <em>15</em> min; the signal is substantially diminished by 60 min. In response to interferon-gamma, tyrosine phosphorylation of IRS-2 was prolonged, with substantial signal still detected at 60 min. Characterization of the mechanism of signaling utilized by GH indicated that tyrosine residues in GH receptor are not necessary for tyrosyl phosphorylation of IRS-2; however, the regions of GH receptor necessary for IRS-2 tyrosyl phosphorylation are the same as those required for JAK2 association and tyrosyl phosphorylation. The role of IRS-2 as a signaling molecule for GH is further demonstrated by the finding that GH stimulates association of IRS-2 with the 85-kDa regulatory subunit of phosphatidylinositol 3'-kinase and with the protein-tyrosine phosphatase SHP2. These results are consistent with the possibility that IRS-2 is a downstream signaling partner of multiple members of the cytokine family of receptors that activate JAK kinases.

BACKGROUND

Dermal fibroblast is a primary cell type responsible for synthesis and remodeling of extracellular matrix in human skin. Type I collagen and hyaluronan are main components that have roles in skin fibrosis, wound healing, tissue remodeling as well as skin aging. Several studies have reported cytokine-dependent changes in collagen expression or hyaluronan production; however, the cytokines' effect was controversial in human dermal fibroblasts.

OBJECTIVE

To clarify the role of various growth factors, cytokines or chemokines on the production of interstitial type I collagen and hyaluronan in dermal fibroblasts.

METHODS

We confirmed the presence of various corresponding receptors and assessed the effects of 33 human recombinants on the production of type I collagen and hyaluronan using the assay system in dermal fibroblasts.

RESULTS

Platelet-derived growth factor (PDGF)-AA, PDGF-BB, epidermal growth factor (EGF), transforming growth factor (TGF)-β1, MCP-1, IP-10, interleukin (IL)-1α, IL-1β, and IL-15 were effective on both type I collagen and hyaluronan production, as compared with no stimulated control. On the other hand, IL-10 and IFN- α caused a significant decrease in type I collagen production, and IL-8 and GM-CSF caused a decrease in hyaluronan production compared with no cytokine-treated control. Interestingly, some chemokines, such as MCP-1 (CCL2), RANTES (CCL5), eotaxin-2 (CCL24), IP-10 (CXCL10), or fractalkine (CX3CL1) significantly induced the type I collagen or hyaluronan production.

CONCLUSIONS

Various growth factors and cytokines on the regulation of type I collagen and hyaluronan in human dermal skin probably function as key factors in skin remodeling and skin aging. Our profile may help to apply to cosmeceutical area maintaining as young skin through the increase in extracellular matrix.

We recently reported that acidic and basic <em>fibroblast</em> <em>growth</em> <em>factors</em> (aFGF and bFGF) confer a broad-spectrum chemoresistance in solid tumors, and that inhibitors of these proteins enhanced the antitumor activity of several anticancer drugs. The present study investigated the effect of FGF inhibitors on doxorubicin activity in human prostate PC3 tumors. In in vitro studies, conditioned medium (CM) obtained from histocultures of rat MAT-LyLu lung metastases and different combinations of recombinant FGF induced a 7- to <em>15</em>-fold doxorubicin resistance. Suramin had no effect on the doxorubicin activity in the absence of CM or FGF, but reversed the CM- and FGF-induced resistance by>> or =90% at concentrations that had no cytotoxicity (i.e., 1-17 microM suramin). In the in vivo study, immunodeficient mice bearing well established, subcutaneous PC3 tumors (approximately 100 mg in size) were treated intravenously with doxorubicin (5 mg/kg) and suramin (10 mg/kg), administered twice weekly for 3 weeks. The suramin dose, selected to yield plasma concentration of below 50 microM, had neither antitumor activity nor toxicity. Doxorubicin alone reduced tumor <em>growth</em> rate by approximately 60%, reduced the density of nonapoptotic tumor cells by approximately 60%, enhanced the apoptotic cell fraction by 4-fold, and reduced the body weight by approximately <em>15</em>% (p < 0.05 compared with control). Addition of suramin to doxorubicin therapy did not increase weight loss but significantly enhanced the antitumor effect, resulting in complete inhibition of tumor <em>growth</em>, an additional 3-fold reduction in the density of nonapoptotic tumor cells, and an additional 2-fold enhancement of the apoptotic tumor cell fraction (p < 0.05 compared with all other groups). These data indicate significant enhancement of the effectiveness of doxorubicin in prostate tumors by nontoxic and subtherapeutic doses of suramin.

In many mammals, lactation success depends on substantial use of lipid reserves and requires integrated metabolic activities between white adipose tissue (WAT) and liver. Mechanisms responsible for this integration in lactation are poorly understood, but data collected in other conditions of elevated lipid use suggest a role for <em>fibroblast</em> <em>growth</em> <em>factor</em>-21 (FGF21). To address this possibility in the context of lactation, we studied high-yielding dairy cows during the transition from late pregnancy (LP) to early lactation (EL). Plasma FGF21 was nearly undetectable in LP, peaked on the day of parturition, and then stabilized at lower, chronically elevated concentrations during the energy deficit of EL. Plasma FGF21 was similarly increased in the absence of parturition when an energy-deficit state was induced by feed restricting late-lactating dairy cows, implicating energy insufficiency as a cause of chronically elevated FGF21 in EL. Gene expression studies showed that liver was a major source of plasma FGF21 in EL with little or no contribution by WAT, skeletal muscle, and mammary gland. Meaningful expression of the FGF21 coreceptor β-Klotho was restricted to liver and WAT in a survey of <em>15</em> tissues that included the mammary gland. Expression of β-Klotho and its subset of interacting FGF receptors was modestly affected by the transition from LP to EL in liver but not in WAT. Overall, these data suggest a model whereby liver-derived FGF21 regulates the use of lipid reserves during lactation via focal actions on liver and WAT.

Exposure of quiescent cultures of Swiss 3T3-D1 cells to bovine brain acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) enhanced phosphorylation of a 31-kDa protein tentatively identified as 40S ribosomal subunit S6 (S6). Soluble extracts from FGF-treated as compared with quiescent <em>fibroblasts</em> exhibited up to 3-fold higher kinase activity towards S6 in exogenously added rat liver 40S ribosomes and a synthetic peptide, RRLSSLRA. This peptide was patterned after a phosphorylation site sequence in S6 and was phosphorylated with an apparent Km corresponding to 0.18 mM. Optimal activation of the S6 kinase with pure mitogen at 10 ng/ml occurred within <em>15</em> to 20 min exposure to FGF. Half-maximal stimulation of the FGF-induced S6 kinase was attained with FGF at 0.4 ng/ml. The S6 kinase in crude extracts utilized both [gamma-32P]ATP (apparent Km congruent to 6-8 microM) and [gamma-32P]GTP (apparent Km congruent to 3 microM), but the ability to utilize GTP was lost after partial purification of the kinase. The FGF-stimulated kinase had an apparent Mr of about 95,000 as determined by chromatography on Sephacryl S300 but appeared to be retarded on TSK 400 HPLC columns, since it eluted with an apparent Mr of 29,000. Treatment of Swiss 3T3 cells with the tumor promoter phorbol 12-myristate 13-acetate (PMA) activated the FGF-stimulated S6 kinase. However, protein kinase C was not required to mediate the FGF activation of the S6 kinase, as FGF still evoked a two-fold activation of the S6 kinase in phorbol ester-pretreated, protein kinase C-depleted cells.

Preeclampsia is a pregnancy-related disorder associated with increased cardiovascular risk for the offspring. Endothelial colony-forming cells (ECFCs) are a subset of circulating endothelial progenitor cells that participate in the formation of vasculature during development. However, the effect of preeclampsia on fetal levels of ECFCs is largely unknown. In this study, we sought to determine whether cord blood ECFC abundance and function are altered in preeclampsia. We conducted a prospective cohort study that included women with normal (n=35) and preeclamptic (n=<em>15</em>) pregnancies. We measured ECFC levels in the umbilical cord blood of neonates and characterized ECFC phenotype, cloning-forming ability, proliferation, and migration toward vascular endothelial <em>growth</em> <em>factor</em>-A and <em>fibroblast</em> <em>growth</em> <em>factor</em>-2, in vitro formation of capillary-like structures, and in vivo vasculogenic ability in immunodeficient mice. We found that the level of cord blood ECFCs was statistically lower in preeclampsia than in control pregnancies (P=0.04), a reduction that was independent of other obstetric <em>factors</em>. In addition, cord blood ECFCs from preeclamptic pregnancies required more time to emerge in culture than control ECFCs. However, once derived in culture, ECFC function was deemed normal and highly similar between preeclampsia and control, including the ability to form vascular networks in vivo. This study demonstrates that preeclampsia affects ECFC abundance in neonates. A reduced level of ECFCs during preeclamptic pregnancies may contribute to an increased risk of developing future cardiovascular events.

BACKGROUND

Adipose tissue hypoxia and endoplasmic reticulum (ER) stress may link the presence of chronic inflammation and macrophage infiltration in severely obese subjects. We previously reported the up-regulation of TNF-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) axis in adipose tissue of severely obese type 2 diabetic subjects.

OBJECTIVE

The objective of the study was to examine TWEAK and Fn14 adipose tissue expression in obesity, severe obesity, and type 2 diabetes in relation to hypoxia and ER stress.

METHODS

In the obesity study, 19 lean, 28 overweight, and 15 obese nondiabetic subjects were studied. In the severe obesity study, 23 severely obese and 35 control subjects were studied. In the type 2 diabetes study, 11 type 2 diabetic and 36 control subjects were studied. The expression levels of the following genes were analyzed in paired samples of sc and visceral adipose tissue: Fn14, TWEAK, VISFATIN, HYOU1, FIAF, HIF-1a, VEGF, GLUT-1, GRP78, and XBP-1. The effect of hypoxia, inflammation, and ER stress on the expression of TWEAK and Fn14 was examined in human adipocyte and macrophage cell lines.

RESULTS

Up-regulation of TWEAK/Fn14 and hypoxia and ER stress surrogate gene expression was observed in sc and visceral adipose tissue only in our severely obese cohort. Hypoxia modulates TWEAK or Fn14 expression in neither adipocytes nor macrophages. On the contrary, inflammation up-regulated TWEAK in macrophages and Fn14 expression in adipocytes. Moreover, TWEAK had a proinflammatory effect in adipocytes mediated by the nuclear factor-kappaB and ERK but not JNK signaling pathways.

CONCLUSIONS

Our data suggest that TWEAK acts as a pro-inflammatory cytokine in the adipose tissue and that inflammation, but not hypoxia, may be behind its up-regulation in severe obesity.

Mannose 6-phosphate, insulin like <em>growth</em> <em>factors</em> I and II (IGF I, IGF II), insulin and epidermal <em>growth</em> <em>factor</em> (EGF) induce a 1.5- to 2-fold increase of mannose 6-phosphate binding sites at the cell surface of human skin <em>fibroblasts</em>. The increase is completed within 10-<em>15</em> min, is dose and temperature dependent, reversible and transient even in the presence of the effectors. It is due to a redistribution of mannose 6-phosphate/IGF II receptors from internal membranes to the cell surface, while the affinity of the receptors is not affected. Combinations of mannose 6-phosphate with IGF I, IGF II or EGF stimulate the redistribution of the receptor to the cell surface in an additive manner, while combinations of the <em>growth</em> <em>factors</em> result in a non-additive stimulation of redistribution. The redistribution is not dependent on extracellular calcium and appears also to be independent of changes of free intracellular calcium. Pre-treatment of <em>fibroblasts</em> with cholera toxin or pertussis toxin increases the number of cell surface receptors 2- and 1.5-fold, respectively. Neither of the toxins affects the redistribution of mannose 6-phosphate/IGF II receptors induced by the <em>growth</em> <em>factors</em>, while both toxins abolish the receptor redistribution induced by mannose 6-phosphate. These results suggest a multiple regulation of the cell surface expression of mannose 6-phosphate/IGF II receptors by Gs- and Gi-like proteins sensitive to cholera toxin and pertussis toxin and by stimulation of mannose 6-phosphate/IGF II, IGF I and EGF receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

This study investigates the feasibility of recombinant spider silk protein as a wound-dressing material for coverage of deep second-degree burn wounds using an animal model. Sixty Sprague-Dawley (SD) rats were randomly divided into four groups (<em>15</em> rats in each group). Two types of recombinant spider silk proteins, pNSR-16 and pNSR-32, as well as collagen (as a control) were applied on the wound; the fourth group was left untreated as a negative control. Each group was evaluated on the 3rd, 5th, 7th, 14th and 21st days for wound-healing rate, histological test, levels of hydroxyproline synthesis and the samples were stained for immunohistochemical detection of the basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). The results of implantation testing showed that wound healing in the treatment groups--recombinant spider silk protein pNSR-16 and pNSR-32--was much better than that in the control group (p<0.01). On the 7th, 14th and 21st days, higher expression of bFGF and the increase of hydroxyproline content of the skin indicated good regeneration of wound skin in the treatment groups. Preliminarily, we conclude that the recombinant spider silk protein membrane promotes the recovery of wound skin by increasing the expression and secretion of the <em>growth</em> <em>factor</em> bFGF and hydroxyproline.

Corneal scarring, whether caused by trauma, laser refractive surgery, or infection, remains a significant problem for humans. Certain ligands for peroxisome proliferator-activated receptor gamma (PPARγ) have shown promise as antiscarring agents in a variety of body tissues. In the cornea, their relative effectiveness and mechanisms of action are still poorly understood. Here, we contrasted the antifibrotic effects of three different PPARγ ligands (<em>15</em>-deoxy-Δ12,14-prostaglandin J2, troglitazone, and rosiglitazone) in cat corneal <em>fibroblasts</em>. Western blot analyses revealed that all three compounds reduced transforming <em>growth</em> <em>factor</em> (TGF)-β1-driven myofibroblast differentiation and up-regulation of α-smooth muscle actin, type I collagen, and fibronectin expression. Because these effects were independent of PPARγ, we ascertained whether they occurred by altering phosphorylation of Smads 2/3, p38 mitogen-activated protein kinase, stress-activated protein kinase, protein kinase B, extracellular signal-regulated kinase, and/or myosin light chain 2. Only p38 mitogen-activated protein kinase phosphorylation was significantly inhibited by all three PPARγ ligands. Finally, we tested the antifibrotic potential of troglitazone in a cat model of photorefractive keratectomy-induced corneal injury. Topical application of troglitazone significantly reduced α-smooth muscle actin expression and haze in the stromal ablation zone. Thus, the PPARγ ligands tested here showed great promise as antifibrotics, both in vitro and in vivo. Our results also provided new evidence for the signaling pathways that may underlie these antifibrotic actions in corneal <em>fibroblasts</em>.

BACKGROUND

Stem cell therapy with bone marrow-derived mononuclear cells (BMMCs) is an option for improving joint function in osteonecrosis of the femoral head (ONFH). Bone marrow-derived mesenchymal stromal cell (MSC) numbers and their osteogenic differentiation are decreased in patients with ONFH. However, whether this decrease also extends to the early stages of ONFH in sickle cell disease (SCD) is still unclear.

METHODS

We conducted a phase I/II, non-controlled study to determine efficacy and safety of BMMC implantation using a minimally invasive technique in SCD patients with ONFH. Eighty-nine patients were recruited and followed up for 60 months after surgery. Clinical and radiographic findings were assessed, and data were completed by in vitro analysis.

RESULTS

At the final follow-up (60 months) there was a significant improvement in clinical joint symptoms and pain relief as measured by the Harris Hip Score (P = 0.0005). In addition, after the BMMC implantation procedure, radiographic assessment showed disease stabilization and only 3.7 % of the treated patients did not achieve a satisfactory clinical result. The amount of fibroblast colony-forming units was 28.2 ± 13.9 per 1 million BMMCs after concentration. Flow cytometry analysis showed a significantly higher number of hematopoietic stem/endothelial progenitor cell markers in concentrated BMMCs when compared with bone marrow aspirate, indicating an enrichment of these cell types. Isolated MSCs from SCD patients with pre-collapse ONFH maintained the replicative capacity without significant loss of their specific biomolecular characteristics, multi-differentiation potential, and osteogenic differentiation activities. Cytokines and growth factors (interleukin-8, transforming growth factor-beta, stromal cell-derived factor-1alpha and vascular endothelial growth factor) that mediate endogenous bone regeneration were also produced by expanded MSCs from SCD patients.

CONCLUSIONS

The autologous BMMC implantation with a minimally invasive technique resulted in significant pain relief and halted the progression of early stages of ONFH in SCD patients. MSCs from SCD patients display biological properties that may add to the efficiency of surgical treatment in ONFH. In summary, our results indicate that infusion of BMMCs enriched with stem/progenitor cells is a safe and effective treatment for the early stages of ONFH in SCD patients.

BACKGROUND

ClinicalTrials.gov NCT02448121; registered 15 May 2015.

The anti-angiogenic activity of endostatin (ES) depends on interactions with heparan sulfate (HS). In the present study, intact HS chains of>>/=<em>15</em> kDa bound quantitatively to ES whereas N-sulfated HS decasaccharides, with affinity for several <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) species, failed to bind. Instead, ES-binding oligosaccharides composed of mixed N-sulfated and N-acetylated disaccharide units were isolated from pig intestinal HS. A 10/12mer ES-binding epitope was identified, with two N-sulfated regions separated by at least one N-acetylated glucosamine unit (SAS-domain). Cleavage at the N-acetylation site disrupted ES binding. These findings point to interaction between discontinuous sulfated domains in HS and arginine clusters at the ES surface. The inhibitory effect of ES on vascular endothelial <em>growth</em> <em>factor</em>-induced endothelial cell migration was blocked by the ES-binding SAS-domains and by heparin oligosaccharides (12mers) similar in length to the ES-binding SAS-domains, but not by 6mers capable of FGF binding. We propose that SAS-domains modulate the biological activities of ES and other protein ligands with extended HS-binding sites. The results provide a rational explanation for the preferential interaction of ES with certain HS proteoglycan species.

<AbstractText>The clinical activity of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) inhibitors seems restricted to cancers harbouring rare FGFR genetic aberrations. In preclinical studies, high tumour FGFR mRNA expression predicted response to rogaratinib, an oral pan-FGFR inhibitor. We aimed to assess the safety, maximum tolerated dose, recommended phase 2 dose, pharmacokinetics, and preliminary clinical activity of rogaratinib.</AbstractText><AbstractText>We did a phase 1 dose-escalation and dose-expansion study of rogaratinib in adults with advanced cancers at 22 sites in Germany, Switzerland, South Korea, Singapore, Spain, and France. Eligible patients were aged 18 years or older, and were ineligible for standard therapy, with an Eastern Cooperative Oncology Group performance status of 0-2, a life expectancy of at least 3 months, and at least one measurable or evaluable lesion according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. During dose escalation, rogaratinib was administered orally twice daily at 50-800 mg in continuous 21-day cycles using a model-based dose-response analysis (continuous reassessment method). In the dose-expansion phase, all patients provided an archival formalin-fixed paraffin-embedded (FFPE) tumour biopsy or consented to a new biopsy at screening for the analysis of FGFR1-3 mRNA expression. In the dose-expansion phase, rogaratinib was given at the recommended dose for expansion to patients in four cohorts: urothelial carcinoma, head and neck squamous-cell cancer (HNSCC), non-small-cell lung cancer (NSCLC), and other solid tumour types. Primary endpoints were safety and tolerability, determination of maximum tolerated dose including dose-limiting toxicities and determination of recommended phase 2 dose, and pharmacokinetics of rogaratinib. Safety analyses were reported in all patients who received at least one dose of rogaratinib. Patients who completed cycle 1 or discontinued during cycle 1 due to an adverse event or dose-limiting toxicity were included in the evaluation of recommended phase 2 dose. Efficacy analyses were reported for all patients who received at least one dose of study drug and who had available post-baseline efficacy data. This ongoing study is registered with ClinicalTrials.gov, number NCT01976741, and is fully recruited.</AbstractText><AbstractText>Between Dec 30, 2013, and July 5, 2017, 866 patients were screened for FGFR mRNA expression, of whom 126 patients were treated (23 FGFR mRNA-unselected patients in the dose-escalation phase and 103 patients with FGFR mRNA-overexpressing tumours [52 patients with urothelial carcinoma, eight patients with HNSCC, 20 patients with NSCLC, and 23 patients with other tumour types] in the dose-expansion phase). No dose-limiting toxicities were reported and the maximum tolerated dose was not reached; 800 mg twice daily was established as the recommended phase 2 dose and was selected for the dose-expansion phase. The most common adverse events of any grade were hyperphosphataemia (in 77 [61%] of 126 patients), diarrhoea (in 65 [52%]), and decreased appetite (in 48 [38%]); and the most common grade 3-4 adverse events were fatigue (in 11 [9%] of 126 patients) and asymptomatic increased lipase (in 10 [8%]). Serious treatment-related adverse events were reported in five patients (decreased appetite and diarrhoea in one patient with urothelial carcinoma, and acute kidney injury [NSCLC], hypoglycaemia [other solid tumours], retinopathy [urothelial carcinoma], and vomiting [urothelial carcinoma] in one patient each); no treatment-related deaths occurred. Median follow-up after cessation of treatment was 32 days (IQR 25-36 days). In the expansion cohorts, <em>15</em> (<em>15</em>%; 95% CI 8·6-23·5) out of 100 evaluable patients achieved an objective response, with responses recorded in all four expansion cohorts (12 in the urothelial carcinoma cohort and one in each of the other three cohorts), and in ten (67%) of <em>15</em> FGFR mRNA-overexpressing tumours without apparent FGFR genetic aberration.</AbstractText><AbstractText>Rogaratinib was well tolerated and clinically active against several types of cancer. Selection by FGFR mRNA expression could be a useful additional biomarker to identify a broader patient population who could be eligible for FGFR inhibitor treatment.</AbstractText><AbstractText>Bayer AG.</AbstractText>

Bone loss and increased fractures are common complications in chronic kidney disease. Because Wnt pathway activation is essential for normal bone mineralization, we assessed whether Wnt inhibition contributes to high-phosphorus-induced mineralization defects in uremic rats. By week 20 after 7/8 nephrectomy, rats fed a high-phosphorus diet had the expected high serum creatinine, phosphorus, parathyroid hormone, and <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23) levels and low serum calcium. There was a <em>15</em>% reduction in tibial mineral density and a doubling of bone cortical porosity compared to uremic rats fed a normal-phosphorus diet. The decreases in tibial mineral density were preceded by time-dependent increments in gene expression of bone formation (Osteocalcin and Runx2) and resorption (Cathepsin K) markers, which paralleled elevations in gene expression of the Wnt inhibitors Sfrp1 and Dkk1 in bone. Similar elevations of Wnt inhibitors plus an increased phospho-β-catenin/β-catenin ratio occurred upon exposure of the osteoblast cell line UMR106-01 either to uremic serum or to the combination of parathyroid hormone, FGF23, and soluble Klotho, at levels present in uremic serum. Strikingly, while osteoblast exposure to parathyroid hormone suppressed the expression of Wnt inhibitors, FGF23 directly inhibited the osteoblastic Wnt pathway through a soluble Klotho/MAPK-mediated process that required Dkk1 induction. Thus, the induction of Dkk1 by FGF23/soluble Klotho in osteoblasts inactivates Wnt/β-catenin signaling. This provides a novel autocrine/paracrine mechanism for the adverse impact of high FGF23 levels on bone in chronic kidney disease.

The t(4;14) translocation detected by fluorescence in situ hybridization (FISH) is an independent prognostic <em>factor</em> for an adverse outcome of multiple myeloma (MM). Because t(4;14) uniquely results in <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) expression, decalcified, paraffin-embedded bone marrow biopsies were immunostained for FGFR3, and its expression was correlated with the t(4;14) status. FISH detected t(4;14) in 16 (19%) of 85 MM patient specimens, and immunocytochemistry detected aberrant FGFR3 expression in 13 (<em>15</em>%). Twelve (75%) t(4;14)-positive cases expressed FGFR3, and 12 (92%) FGFR3-positive cases harbored a t(4;14). FGFR3 expression and t(4;14) were strongly correlated (P < .001). FGFR3 expression by immunohistochemistry was associated with the immunoglobulin A (IgA) isotype (P < .001), a shorter progression-free survival (median, 11.5 versus 25.8 months; P < .001), and a shorter overall survival (median, 19.2 versus 46.3 months; P < .001).

Insulin, whole serum, phorbol esters and epidermal <em>growth</em> <em>factor</em> each rapidly stimulate protein synthesis in serum-depleted Swiss 3T3 <em>fibroblasts</em>. The activation of protein synthesis by each of these agents is associated with stimulation of the activity of the guanine-nucleotide-exchange <em>factor</em> (GEF). This protein recycles the initiation <em>factor</em> eIF-2 by promoting exchange of GDP bound to eIF-2 for GTP. Activation of GEF is rapid, becoming maximal within <em>15</em> min. The degree of activation of GEF by these stimuli (to greater than 170% of control for insulin, serum or epidermal <em>growth</em> <em>factor</em>; 120% for phorbol dibutyrate) is more than enough to account for their effects on the overall rate of translation. Stimulation of protein synthesis and GEF activity occurs at low nanomolar insulin concentrations, indicating they are mediated through the insulin receptor. The best-characterized mechanism for regulating GEF activity is through changes in the phosphorylation of the smallest subunit of eIF-2 (eIF-2 alpha); however, none of the stimuli studied altered the level of phosphorylation of eIF-2 alpha in Swiss <em>fibroblasts</em>. It seems that direct regulation of GEF activity may be occurring here, and possible mechanisms for this are discussed.

Translocations involving the immunoglobulin heavy-chain switch region and <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) are identified in 10% to <em>15</em>% of patients with myeloma. In previous research we overexpressed FGFR3 or the constitutively active FGFR3-TD mutant in an interleukin-6 (IL-6)-dependent murine myeloma cell line, B9. FGFR3-enhanced IL-6 responsiveness increased phosphorylation of STAT3 and up-regulated Bcl-x(L). Since Bcl-x(L) was up-regulated, we have tested FGFR3-expressing B9 cells for chemotherapy sensitivity. FGFR3 expression did not alter sensitivity to melphalan or doxorubicin. In contrast, B9 cells overexpressing FGFR3 were resistant to treatment with dexamethasone, a phenomenon successfully reversed using a Bcl-x(L) antisense oligonucleotide. These data demonstrate that the overexpression of FGFR3 in B9 cells confers resistance to dexamethasone but not to anthracyclines or alkylating agents, at least in part through the up-regulation of Bcl-x(L). This finding has potential implications for the use of chemotherapy in t(4;14)-positive myeloma.

The D-type cyclins (D1, D2, and D3) are involved in progression through the G1 phase of the cell cycle and are induced as part of the delayed early response to <em>growth</em> <em>factor</em> stimulation. To better understand the role of D-type cyclins in pituitary cell function and the regulatory role of <em>growth</em> <em>factors</em> in the cell cycle, we analyzed the expression and regulation of D-type cyclins in normal and neoplastic rat pituitary cells. Immunocytochemical and RT-PCR analyses showed expression of all three D-type cyclins in the normal pituitary, with higher percentages of positive cells by immunocytochemistry in the nuclei of normal pituitaries (D1, 20-30%; D2, 50-60%; D3, 70-80%), compared with GH3 cells. In the normal pituitary, there were significantly higher levels of cyclins D2 and D3 in PRL, GH, LH, and TSH cells, compared with ACTH cells. Cyclin D1 protein was not detected in GH3 cells, while D2 was present in less than 1 percent and D3 in 10-<em>15</em> percent of GH3 cells. There were low levels of cyclin D1 and D2 messenger RNA expression in GH3 cells, by RT-PCR. When dissociated rat pituitary cells were cultured in the presence of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (5.6 nM) for 3 days, cyclin D2 was up-regulated 2-fold in normal PRL cells (control, 33 +/- 1%; treated, 68 +/- 2%). Similarly, bFGF treatment stimulated cyclin D3 expression 3-fold in GH3 cells (control, <em>15</em> +/- 1%; treated, 44 +/- 1%). Treatment of GH3 cells with 5-aza-2'-deoxycytidine, which induces gene demethylation, produced marked increases in cyclin D2 and D3 expression. Transfection of mouse cyclin D1 complementary DNA, driven by a cytomegalovirus promoter into GH3 cells, led to ectopic cyclin D1 expression; and there was a slight stimulation of cell proliferation and increased apoptosis in GH3 cells. These results indicate that there is a differential expression of various D-type cyclins in different types of normal pituitary cells and between normal pituitary and GH3 cells. <em>Growth</em> <em>factors</em>, such as bFGF and demethylation increased D-type cyclin expression, whereas ectopic overexpression of cyclin D1 stimulates cell proliferation and increases apoptosis in GH3 pituitary tumor cells.

Short-term cultures from 1<em>15</em> squamous cell carcinomas (SCC) of the head and neck were cytogenetically investigated. Thirty-six of the tumors have been reported previously, whereas 79 are new cases. The material was divided into two series based on the medium used. The 80 tumors of series I were cultured in RPMI 1640 supplemented with fetal calf serum, glutamine, antibiotics, insulin, cholera toxin, and epidermal <em>growth</em> <em>factor</em>. The 35 tumors of series II were cultured in a chemically defined, serum-free medium with a low calcium concentration, MCDB <em>15</em>3, which stimulates epithelial <em>growth</em> while inhibiting <em>fibroblasts</em>. A total of 83 tumors with clonal karyotypic abnormalities were detected in the two series. Series II had a higher proportion of tumors with complex karyotypic changes than series I (43% versus <em>15</em>%), a lower proportion of tumors with pseudo- or neardiploid clones characterized by simple rearrangements (3% versus 34%), and a lower frequency of unrelated clones (3% versus 24%), indicating that the different culture conditions favored <em>growth</em> of different cell populations. Except for rearrangements of 1p22, which were mainly found in series I, the distribution of breakpoints in structural aberrations was similar in the two series and clustered to several chromosomal bands or regions, in particular 11q13, 1p22, 1p11-12, 3p11-q11, 5q13, 1q25, <em>15</em>q10, and 8q10. Unbalanced structural aberrations were more common in series II, frequently leading to loss of segments from chromosome arms 3p, 7q, 8p, 11q, 13p, 14p, and <em>15</em>p, whereas gain of genetic material often involved chromosome arms 1q, 3q, 8q, and <em>15</em>q.

OBJECTIVE

Fibroblast growth factor receptor 3 (FGFR3) mutations were reported recently at a high frequency in low-grade urothelial cell carcinoma (UCC). We investigated the feasibility of combining microsatellite analysis (MA) and the FGFR3 status for the detection of UCC in voided urine.

METHODS

In a prospective setting, 59 UCC tissues and matched urine samples were obtained, and subjected to MA (23 markers) and FGFR3 mutation analysis (exons 7, 10, and 15). In each case, a clinical record with tumor and urine features was provided. Fifteen patients with a negative cystoscopy during follow-up served as controls.

RESULTS

A mutation in the FGFR3 gene was found in 26 (44%) UCCs of which 22 concerned solitary pTaG1/2 lesions. These mutations were absent in the 15 G3 tumors. For the 6 cases with leukocyturia, 46 microsatellite alterations were found in the tumor. Only 1 of these was also detected in the urine. This was 125 of 357 for the 53 cases without leukocyte contamination. The sensitivity of MA on voided urine was lower for FGFR3-positive UCC (15 of 21; 71%) as compared with FGFR3 wild-type UCC (29 of 32; 91%). By including the FGFR3 mutation, the sensitivity of molecular cytology increased to 89% and was superior to the sensitivity of morphological cytology (25%) for every clinical subdivision. The specificity was 14 of 15 (93%) for the two (molecular and morphological) cytological approaches.

CONCLUSIONS

Molecular urine cytology by MA and FGFR3 mutation analysis enables a highly sensitive and specific detection of UCC. The similarity of molecular profiles in tumor and urine corroborate their clonal relation.

We describe 2 cases of angioblastoma, a rare, destructive pediatric tumor, treated with interferon alfa 2b (IFNalpha2b). The first patient is a 10-month-old male who presented with an ulcerated palatal neoplasm that could not be completely resected. The second is a male neonate with a congenital tumor of the right hand that invaded the hypothenar eminence, destroying the fourth and fifth metacarpals. Biopsy in both patients was interpreted as giant cell angioblastoma. Angioblastoma is rare; there is only 1 reported case that necessitated amputation of an upper extremity, also initially recommended for our patient. Because there is little experience with chemotherapy, permission was granted to employ an antiangiogenic regimen of IFNalpha2b. The angiogenic protein, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), was abnormally elevated in both patients. Both patients received IFNalpha2b. In the first child, it was used after incomplete resection, because biopsy-proven tumor was present at the margin and in the nasopharynx. Biopsies <em>15</em> months after initiation of IFN2alphab were negative for tumor. Therapy was stopped after 3 years. Eighteen months later, the patient remains disease-free. In the second child, IFNalpha2b was started after debridement of the ulcerated tumor. Over 11 months, the tumor completely regressed and there was bony regeneration of the metacarpals. The fifth digit was amputated because of damage to the metacarpophalangeal joint by the tumor. IFNalpha2b therapy was discontinued after 1 year of treatment, and the child remains disease-free 2 years and 8 months later. In conclusion, this report demonstrates that: 1) a bFGF-overexpressing low-grade tumor can respond to IFNalpha2b in a manner similar to life-threatening infantile hemangiomas, 2) urinary bFGF levels can help guide IFNalpha dosage in such patients, and 3) although bFGF-mediated tumor angiogenesis is inhibited by IFNalpha, physiologic angiogenesis seems to be unaffected.

BRAF (7q24) encodes a serine/threonine protein kinase, and its expression level varies in different tissues. Although a high prevalence of BRAF mutation has been suggested as an important event in thyroid tumorigenesis, little is known about the expression pattern of B-Raf in the thyroid. Thus, we examined the expression of B-Raf in various neoplastic and nonneoplastic thyroid tissues and compared it with BRAF mutational status. Normal and hyperplastic thyroid tissues showed focal and faint immunoreactivity for B-Raf, especially in cuboidal follicular cells of small follicles. In contrast, diffuse expression of B-Raf was observed in follicular adenomas and well-differentiated carcinomas. The missense point mutation BRAF(V600E) was identified in 42% (13/31 cases) of papillary carcinomas and 33% (5/<em>15</em> cases) of undifferentiated carcinomas but not in normal thyroid tissues, nodular hyperplasia, follicular adenomas, or follicular carcinomas. The immunohistochemical expression of B-Raf did not correlate with BRAF mutational status. Moreover, Western blotting revealed that B-Raf expression in thyroid carcinoma cell lines was also independent of BRAF mutation. Serum or <em>fibroblast</em> <em>growth</em> <em>factor</em>-1 stimulation further activates ERK1/2 in heterozygous BRAF(V600E)-positive carcinoma cells as well as BRAF(V600E) mutation-negative carcinoma cells. In conclusion, heterogeneous focal expression of wild-type B-Raf in nonneoplastic tissues may play a role in the <em>growth</em> or functional activity of thyroid follicular cells. In contrast, diffuse expression of wild-type and/or mutant B-Raf may be involved in the tumorigenic process resulting in activation of the mitogen-activated protein kinase signaling pathway in cooperation with other genetic abnormalities and activation of ligand-receptor signaling pathways.

Subconfluent bovine pulmonary artery endothelial cells on rigid substrates were exposed to 1.5-<em>15</em> cm H2O sustained hydrostatic pressure for up to 7 days and exhibited elongation, cytoskeletal rearrangement, increased cell proliferation, and bilayering. The role of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) in the mechanism(s) of these endothelial cell responses to sustained hydrostatic pressure was investigated. Evidence that bFGF was released from endothelial cells exposed to sustained hydrostatic pressure or compression was provided by the following experimental results: 1) Cells exposed to control (3 mm H2O) pressure displayed intense nuclear and cytoplasmic bFGF staining by immunocytochemical techniques; this staining was absent in cells exposed to 10 cm H2O for 7 days. 2) Conditioned medium from endothelial cells exposed to 10 cm H2O for 7 days contained a transferable, <em>growth</em>-promoting activity exhibiting heparin-Sepharose affinity, lability to both heat and freeze/thawing, and neutralization by anti-bovine bFGF. 3) Suramin (0.1 mM), a <em>growth</em>-<em>factor</em> receptor inhibitor, abrogated the proliferative and morphological responses of endothelial cells exposed to sustained hydrostatic pressure. Endothelial cells exposed to elevated hydrostatic pressure demonstrated no detectable decrement in cell viability as assessed by Trypan blue exclusion. The results of the present study indicate that hydrostatic pressure or compression can induce bFGF release from endothelial cells independent of cell injury or death; bFGF is subsequently responsible for the morphological, proliferative, and bilayering responses of endothelial cells to hydrostatic pressure.