OBJECTIVE

To improve the prenatal diagnosis of thanatophoric dysplasia by defining the change in fetal size across gestation and the frequency of sonographic features, and developing non-invasive molecular genetic diagnosis based on cell-free fetal DNA (cffDNA) in maternal plasma.

METHODS

Fetuses with a confirmed diagnosis of thanatophoric dysplasia were ascertained, records reviewed, sonographic features and measurements determined. Charts of fetal size were then constructed using the LMS (lambda-mu-sigma) method and compared with charts used in normal pregnancies and those complicated by achondroplasia. Cases in this cohort referred to our Regional Genetics Laboratory for molecular diagnosis using cffDNA were identified and results reviewed.

RESULTS

Forty-two cases were scanned in our units. Commonly reported sonographic features were very short and sometimes bowed femora, frontal bossing, cloverleaf skull, short fingers, a small chest and polyhydramnios. Limb shortening was obvious from as early as 13 weeks' gestation, with minimal <em>growth</em> after <em>20</em> weeks. Analysis of cffDNA in three of these pregnancies confirmed the presence of the c.742C>CT (p.Arg248Cys) or the c.1948A>AG (p.Lys650Glu) mutation in the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 gene.

CONCLUSIONS

These data should improve the accuracy of the sonographic diagnosis of thanatophoric dysplasia and have implications for reliable and safe targeted molecular confirmation using cffDNA.

We have employed differential screening of a rat aortic smooth muscle cell cDNA library to isolate a cDNA clone, SM-<em>20</em>, whose nucleotide and deduced amino acid sequences are distinct from those reported previously. SM-<em>20</em> encodes an mRNA of approximately 2.5-3.0 kilobase pairs which is present at low levels in quiescent vascular smooth muscle cells. SM-<em>20</em> levels increase within 1 h of treatment with <em>growth</em> agonists (serum, platelet-derived <em>growth</em> <em>factor</em>, angiotensin II), isoproterenol, and forskolin. Cycloheximide induces high levels of SM-<em>20</em> mRNA and superinduces it in the presence of serum, suggesting that the increase in SM-<em>20</em> mRNA levels is not dependent on protein synthesis and is part of the primary response to <em>growth</em> agonists. SM-<em>20</em> is expressed at high levels in tissues and cells derived from muscle (smooth, skeletal, and cardiac) and nerve (brain, PC12 cells) and is not expressed in 3T3 <em>fibroblasts</em> or rat 6 <em>fibroblasts</em>. SM-<em>20</em> encodes a new member of the immediate early gene family and is unusual in that it is expressed in vascular smooth muscle, but not in <em>fibroblasts</em>.

We have isolated a strongly mitogenic, type beta transforming <em>growth</em> <em>factor</em> (beta TGF) released by Snyder-Theilen feline sarcoma virus-transformed rat embryo (FeSV-Fre) cells that induces phenotypic transformation of normal NRK cells when they are concomitantly stimulated by analogues of epidermal <em>growth</em> <em>factor</em> (EGF). Molecule filtration chromatography separates beta TGF from an EGF-like TGF (eTGF) which is also present in acid extracts from medium conditioned by FeSV-Fre cells (J. Massagué, (1983) J. Biol. Chem. 258, 13606-13613). Final purification of beta TGF is achieved by reverse phase high pressure liquid chromatography (HPLC) on octadecyl support, molecular filtration HPLC, and nonreducing dodecyl sulfate-polyacrylamide gel electrophoresis steps, yielding a 300,000-fold purified polypeptide with a final recovery of 21%. The purified rat beta TGF consists of two Mr = 11,000-12,000 polypeptide chains disulfide-linked as a Mr = 23,000 dimer. Induction of anchorage-independent proliferation of NRK cells by rat beta TGF depends on the simultaneous presence of eTGF or EGF. In the presence of a saturating (300 pM) concentration of either rat eTGF or mouse EGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with 4-6 pM rat beta TGF. In the presence of a saturating (<em>20</em> pM) concentration of rat beta TGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with either rat eTGF or mouse EGF at a 50-70 pM concentration. Rat beta TGF is also able to induce DNA synthesis and cell proliferation on <em>growth</em>-arrested NRK, human lung, and Swiss mouse 3T3 <em>fibroblast</em> monolayers, this effect being half-maximal at 2-3 pM beta TGF for NRK cells. These results identify eTGF and beta TGF as the two synergistically acting <em>factors</em> responsible for the transforming action of culture fluids from FeSV-Fre cells.

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 15 to <em>20</em> 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.

OBJECTIVE

Green tea catechins are known to have anticarcinogenic effects. Epigallocatechin-3-gallate (EGCG) accounts for almost 50% of the total catechin content in green tea extract and has very potent antioxidant effects. EGCG also inhibits angiogenesis, possibly through the inhibition of proangiogenic factors including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which in turn, inhibits tumor growth and metastasis. However, the exact molecular mechanism by which EGCG suppresses bFGF expression is not known. Our objective was to elucidate the molecular mechanisms by which EGCG inhibits bFGF expression in colorectal cancer.

METHODS

We examined posttranslational regulation of bFGF by EGCG in human colorectal cancer cells. We also examined bFGF in intestinal tumor formation of APC(Min/+) mice with and without catechin treatment.

RESULTS

The bFGF protein was quickly degraded in the presence of EGCG, but a proteasome inhibitor suppressed this degradation. EGCG was also found to increase ubiquitination of bFGF and trypsin-like activity of the 20S proteasome, thereby resulting in the degradation of bFGF protein. Furthermore, EGCG suppressed tumor formation in APC(Min/+) mice, compared with vehicle-treated mice, in association with reduced bFGF expression.

CONCLUSIONS

The ubiquitin-proteasome degradation pathway contributes significantly to down-regulation of bFGF expression by EGCG. Catechin compounds have fewer adverse effects than chemotherapeutic agents and hence can be used as proof-of-concept in cancer therapeutics to suppress growth and metastasis by targeting proteins such as bFGF.

Recently we demonstrated that endothelins secreted from human keratinocytes act as intrinsic mitogens and melanogens for human melanocytes in UVB-induced melanosis. We show here that UVA-induced melanosis is associated with other keratinocyte-derived <em>growth</em> <em>factors</em>, secretion of which is specifically stimulated after exposure of human keratinocytes to UVA. Medium conditioned by UVA-exposed human keratinocytes elicited a significant increase in DNA synthesis by cultured human melanocytes in a UVA dose-dependent manner. Analysis of endothelin-1 and interleukin (IL)-1 alpha in the conditioned medium by ELISA, both of which are major keratinocyte-derived cytokines involved in UVB-associated melanocyte activation, revealed that UVA exposure did not cause human keratinocytes to stimulate the secretion of the two cytokines. In contrast, the levels of several other cytokines such as IL-6, IL-8 and granulocyte/macrophage colony-stimulating <em>factor</em> (GM-CSF) were significantly increased in the conditioned medium of human keratinocytes after exposure to UVA at a dose of 1.0 J/cm2. The gel chromatographic profile of UVA-exposed keratinocyte-conditioned medium demonstrated that there were two <em>factors</em> (P-1 and P-2) with molecular masses of approx. <em>20</em> and 1 kDa respectively that stimulate DNA synthesis in human melanocytes, and the larger species (P-1) also increased melanization as assessed by [14C]thiouracil incorporation. Quantitative analysis of cytokines in chromatographic fractions by ELISA revealed the P-1 fraction to be consistent with the molecular mass profile of GM-CSF. Furthermore the stimulatory effect of the P-1 fraction on DNA synthesis in human melanocytes was neutralized by antibodies to GM-CSF, but not to basic <em>fibroblast</em> <em>growth</em> <em>factor</em> or stem cell <em>factor</em>. Binding and proliferation assays with recombinant GM-CSF demonstrated that human melanocytes possess specific binding sites for GM-CSF(Kd 2.11 nM; binding sites, 2.5-3.5 x 10(4) per cell), and recombinant GM-CSF at concentrations of more than 10 nM significantly stimulated DNA synthesis and melanization. These findings suggest that GM-CSF secreted by keratinocytes plays an essential role in the maintenance of melanocyte proliferation and UVA-induced pigmentation in the epidermis.

In this report, we describe the isolation from human urine of a predominant 160-kDa epidermal <em>growth</em> <em>factor</em> (EGF)-immunoreactive glycoprotein that exhibits affinity for heparin. The purification procedure involved concentration and dialysis of <em>20</em>-30-liter batches of fresh urine on a high capacity ultrafiltration apparatus followed by chromatography on DEAE-Sephacel, heparin-agarose, and Sephacryl S-300. A nearly homogeneous preparation of 160-kDa protein was obtained with a yield of approximately 1 mg of 160-kDa protein from 25 liters of urine. The amino-terminal sequence of the purified 160-kDa protein, H2N-SAPQHXSXPEGTXA-, matched residues 21-34 of the predicted sequence of human prepro-EGF and established that the 160 kDa protein (pro-EGF) is a product of the prepro-EGF gene. Characterization of the carboxyl terminus of the purified protein by digestion with carboxypeptidase B and by immunoblotting with antisera against synthetic carboxyl-terminal and juxtatransmembrane peptides of prepro-EGF indicated that the carboxyl terminus has been truncated at an arginine residue that corresponds, most likely, to the carboxyl-terminal arginine of the EGF moiety. The intact 160-kDa pro-EGF is biologically active as evidenced by its specific binding to the EGF receptor and activation of the EGF receptor tyrosine kinase in A-431 cell membranes. Purified pro-EGF competitively inhibited the binding of 125I-EGF to human <em>fibroblasts</em>, and it stimulated the proliferation of these cells in culture. When immobilized onto culture dishes, the heparin-binding pro-EGF appeared to function both as an adhesion molecule and as a <em>growth</em> <em>factor</em> for serum-free mouse embryo cells.

Inhibition of the vascular endothelial <em>growth</em> <em>factor</em> VEGF-VEGF receptor (VEGF-R) kinase axes in the tumor angiogenic cascade is a promising therapeutic strategy in oncology. CEP-7055 is the fully synthetic orally active N,N-dimethyl glycine ester of CEP-5214, a C3-(isopropylmethoxy) fused pyrrolocarbazole with potent pan-VEGF-R kinase inhibitory activity. CEP-5214 demonstrates IC(50) values of 18 nM, 12 nM, and 17 nM against human VEGF-R2/KDR kinase, VEGF-R1/FLT-1 kinase, and VEGF-R3/FLT-4 kinase, respectively, in biochemical kinase assays. CEP-5214 inhibited VEGF-stimulated VEGF-R2/KDR autophosphorylation in human umbilical vein endothelial cells (HUVECs) with an IC (50) of approximately 10 nM and demonstrated an equivalent inhibition of murine FLK-1 autophosphorylation in transformed SVR endothelial cells. Evaluation of the antiangiogenic activity of CEP-5214 revealed a dose-related inhibition of microvessel <em>growth</em> ex vivo in rat aortic ring explant cultures and in vitro on HUVEC capillary-tube formation on Matrigel at low nanomolar concentrations. The antiangiogenic activity of CEP-5214 in these bioassays was observed in the absence of apparent cytotoxicity. Single-dose p.o. or s.c. administration of CEP-7055 or CEP-5214 to CD-1 mice at 23.8 mg/kg/dose b.i.d. resulted in a reversible inhibition of VEGF-R2/FLK-1 phosphorylation in murine lung tissues. Administration p.o. of CEP-7055 at 2.57 to 23.8 mg/kg/dose b.i.d. resulted in dose-related reductions in neovascularization in vivo in porcine aortic endothelial cell (PAEC)-VEGF/basic <em>fibroblast</em> <em>growth</em> <em>factor</em>-Matrigel implants in nude mice (maximum, 82% inhibition), significant reductions in granuloma formation (30%) and granuloma vascularity (42%) in a murine chronic inflammation-induced angiogenesis model, and significant and sustained (6 h) inhibition of VEGF-induced plasma extravasation in rats, with an ED(50) of <em>20</em> mg/kg/dose. Chronic p.o. administration of CEP-7055 at doses of 11.9 to 23.8 mg/kg/dose b.i.d. resulted in significant inhibition (50-90% maximum inhibition relative to controls) in the <em>growth</em> of a variety of established murine and human s.c. tumor xenografts in nude mice, including A375 melanomas, U251MG and U87MG glioblastomas, CALU-6 lung carcinoma, ASPC-1 pancreatic carcinoma, HT-29 and HCT-116 colon carcinomas, MCF-7 breast carcinomas, and SVR angiosarcomas. Significant antitumor efficacy was observed similarly against orthotopically implanted LNCaP human prostate carcinomas in male nude mice and orthotopically implanted renal carcinoma (RENCA) tumors in BALB/c mice, in terms of a significant reduction in the metastatic score and the extent of pulmonary metastases. These antitumor responses were associated with marked increases in tumor apoptosis, and significant reductions in intratumoral microvessel density (CD34 and <em>Factor</em> VIII staining) of 22-38% relative to controls depending on the specific tumor xenograft. The antitumor efficacy of chronic CEP-7055 administration was independent of initial tumor volume (in the ASPC-1 pancreatic carcinoma model) and reversible on withdrawal of treatment. Chronic p.o. administration of CEP-7055 in preclinical efficacy studies for periods of up to 65 days was well tolerated with no apparent toxicity or significant morbidity. Orally administered CEP-7055 has entered Phase I clinical trials in cancer patients.

Post-mitotic cultures of human mesangial cells were maintained in media containing 4-30 mM D-glucose for up to 28 days. Changes in mRNA and protein levels for specific macromolecules occurred between 7 and 14 days after initiating hyperglycaemic conditions. Slot blot analysis showed 2-3-fold increases in mRNAs for collagen type I, fibronectin, versican and perlecan, whereas mRNA for decorin was increased by up to <em>20</em>-fold. Levels of mRNAs for biglycan and syndecan were unaffected by hyperglycaemic culture. Reverse transcriptase PCR (RT-PCR) confirmed that decorin mRNA levels are greatly elevated and also showed increased transcription of the TGF-beta 1 gene in hyperglycaemic cultures. Western analysis and ELISA indicated accumulations of collagen types I and III, laminin and fibronectin in the cell layers and media of hyperglycaemic cultures with increasing time. Type IV collagen did not accumulate in either compartment of hyperglycaemic mesangial cell cultures. Collagen types I, III, and fibronectin did not accumulate in the cell layers of hyperglycaemic human dermal <em>fibroblasts</em>, indicating a cell-specific response in mesangial cultures. Decorin and versican, but not biglycan, were increased in the hyperglycaemic mesangial cell culture media. There were no apparent changes in core proteins for decorin and biglycan in <em>fibroblast</em> media. Transforming <em>growth</em> <em>factor</em> beta 1 (TGF-beta 1) in hyperglycaemic mesangial cell cultures increased 5-fold after 7 days, but decreased thereafter to only approx. 2-fold after 28 days. The changes in TGF-beta 1 mRNA, as detected by RT-PCR, and protein followed one another closely.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> 21 (FGF21) is an important endocrine metabolic regulator expressed in multiple tissues including liver and adipose tissue. Although highest levels of expression are in pancreas, little is known about the function of FGF21 in this tissue. In order to understand the physiology of FGF21 in the pancreas, we analyzed its expression and regulation in both acinar and islet tissues. We found that acinar tissue express <em>20</em>-fold higher levels than that observed in islets. We also observed that pancreatic FGF21 is nutritionally regulated; a marked reduction in FGF21 expression was noted with fasting while obesity is associated with 3-4 fold higher expression. Acinar and islet cells are targets of FGF21, which when systemically administered, leads to phosphorylation of the downstream target ERK 1/2 in about half of acinar cells and a small subset of islet cells. Chronic, systemic FGF21 infusion down-regulates its own expression in the pancreas. Mice lacking FGF21 develop significant islet hyperplasia and periductal lymphocytic inflammation when fed with a high fat obesogenic diet. Inflammatory infiltrates consist of TCRb+ Thy1+ T lymphocytes with increased levels of Foxp3+ regulatory T cells. Increased levels of inflammatory cells were coupled with elevated expression of cytokines such as TNFα, IFNγ and IL1β. We conclude that FGF21 acts to limit islet hyperplasia and may also prevent pancreatic inflammation.

BACKGROUND

Von Hippel-Lindau (VHL) disease induces vascular neoplasms in multiple organs. We evaluated the safety and efficacy of sunitinib in VHL patients and examined the expression of candidate receptors in archived tissue.

METHODS

Patients with VHL were given four cycles of 50 mg sunitinib daily for 28 days, followed by 14 days off. Primary end point was toxicity. Modified RECIST were used for efficacy assessment. We evaluated <em>20</em> archival renal cell carcinomas (RCCs) and <em>20</em> hemangioblastomas (HBs) for biomarker expression levels using laser-scanning cytometry (LSC).

RESULTS

Fifteen patients were treated. Grade 3 toxicity included fatigue in five patients. Dose reductions were needed in 10 patients. Eighteen RCC and 21 HB lesions were evaluable. Six of the RCCs (33%) responded partially, versus none of the HBs (P = 0.014). LSC revealed that mean levels of phosphorylated vascular endothelial growth factor receptor-2 were lower in HB than in RCC endothelium (P = 0.003) and mean phosphorylated fibroblast growth factor receptor substrate-2 (pFRS2) levels were higher in HB (P = 0.003).

CONCLUSIONS

Sunitinib treatment in VHL patients showed acceptable toxicity. Significant response was observed in RCC but not in HB. Greater expression of pFRS2 in HB tissue than in RCC raises the hypothesis that treatment with fibroblast growth factor pathway-blocking agents may benefit patients with HB.

OBJECTIVE

The present study investigated whether the simultaneous application of basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) enhances blood vessel formation in murine ischemic hindlimb compared with bFGF or HGF applied alone.

METHODS

Unilateral hindlimb ischemia was created in C57BL/6 mice. Hindlimb blood flow was evaluated by laser Doppler perfusion image index (LDPII) (ratio (%) of ischemic-to-normal-limb blood flow). The ischemic limbs were treated with bFGF and HGF separately, or bFGF and HGF together, and their therapeutic effects were assessed. Collagen microspheres (CM) were used as a sustained-release carrier for bFGF and HGF.

RESULTS

A single intramuscular injection of 5 microg or less of bFGF-incorporated CM (bFGF/CM) into the ischemic limb did not significantly increase the LDPII compared with the control (no treatment) 4 weeks after the treatment. Similarly, 20 microg or less of HGF/CM did not increase LDPII. Based on these results, we compared the dual release of CM incorporating 5 microg of bFGF and 20 microg of HGF with either the single release of 5 mug of bFGF/CM alone or 20 microg of HGF/CM alone. The LDPII of the dual release (94.2% +/- 10.9%) was higher than either single release (51.2% +/- 5.8% or 52.5% +/- 8.0%, P < .01). Furthermore, the LDPII in the dual release (94.2% +/- 10.9%) was equivalent to that with 80 microg of bFGF/CM (95.1% +/- 7.6%) alone or 80 microg of HGF/CM (92.8% +/- 7.6%) alone. A histologic evaluation at 4 weeks showed capillary density in the dual release (868 +/- 173 vessels/mm(2)) was higher than that in either single release (204 +/- 68 vessels/mm(2) or 185 +/- 98 vessels/mm(2) , P < .01). The percentage of mature vessels assessed by alpha-smooth muscle actin staining was also higher in the dual release (43.8% +/- 7.8% vs 9.5% +/- 3.0% or 11.7% +/- 3.8%, respectively; P < .01).

CONCLUSIONS

This study demonstrates that the sustained dual release of a lower dose of bFGF and HGF from a carrier matrix can achieve equivalent blood perfusion recovery and more mature vasculature in the ischemic limb than a higher dose of bFGF or HGF alone. This approach may be a highly promising strategy for the future treatment of peripheral vascular disease.

OBJECTIVE

The purpose of this study was to analyze the possible correlation between PEA3 mRNA expression and survival in advanced-stage ovarian carcinomas, studying two patient groups with extremely different disease outcome.

METHODS

Sections from 61 primary ovarian carcinomas and metastatic lesions from 36 patients diagnosed with advanced-stage ovarian carcinoma [International Federation of Gynecologists and Obstetricians (FIGO) stages III-IV] were evaluated for expression of PEA3 using mRNA in situ hybridization. Patients were divided into long-term (n = 16) and short-term (n = <em>20</em>) survivors.

RESULTS

The mean values for disease-free survival and overall survival were 119 and 137 months for long-term survivors, as compared with 4 and 22 months for short-term survivors, respectively. Expression of PEA3 mRNA was detected in carcinoma cells and stromal cells in 56 of 61 lesions (92%) and 54 of 61 lesions (89%), respectively. Intense stromal expression was detected only in the vicinity of grade 2-3 tumors (P = 0.04). PEA3 expression in stromal cells showed a significant association with matrix metalloproteinase 2 mRNA expression in carcinoma cells (P = 0.022). PEA3 expression in carcinoma cells showed an association with mRNA expression of the beta(1) integrin subunit in the same compartment (P = 0.039). It was also associated with mRNA expression of beta(1) integrin subunit (P = 0.012), basic fibroblast growth factor (P = 0.036), and the matrix metalloproteinase inducer EMMPRIN (P = 0.038) in stromal cells. PEA3 mRNA was detected more often in both carcinoma and stromal cells in tumors of short-term survivors (P = 0.021 for stromal cells). In univariate survival analysis, PEA3 expression in stromal cells correlated with both shorter disease-free survival (P = 0.019) and overall survival (P = 0.029), whereas tumor cell expression predicted poor overall survival (P = 0.049). PEA3 mRNA expression in stromal cells emerged as an independent predictor of poor outcome in multivariate survival analysis, in which all molecules previously studied in this patient cohort were included (P = 0.015).

CONCLUSIONS

To the best of our knowledge, this is the first evidence associating PEA3 mRNA expression and poor survival in human epithelial malignancy. PEA3 is thus a novel prognostic marker in advanced-stage ovarian carcinoma. The association between PEA3 mRNA expression and the expression of the beta(1) integrin subunit, basic fibroblast growth factor, and EMMPRIN, first documented in our patient cohort, points to the central role of this transcription factor in tumor progression in ovarian carcinoma.

Obesity is a chronic inflammation with increased serum levels of insulin, insulin-like <em>growth</em> <em>factor</em> 1 (IGF1), and interleukin-17 (IL-17). The objective of this study was to test a hypothesis that insulin and IGF1 enhance IL-17-induced expression of inflammatory chemokines/cytokines through a glycogen synthase kinase 3β (GSK3B)-dependent mechanism, which can be inhibited by melatonin. We found that insulin/IGF1 and lithium chloride enhanced IL-17-induced expression of C-X-C motif ligand 1 (Cxcl1) and C-C motif ligand <em>20</em> (Ccl<em>20</em>) in the Gsk3b(+/+) , but not in Gsk3b(-/-) mouse embryonic <em>fibroblast</em> (MEF) cells. IL-17 induced higher levels of Cxcl1 and Ccl<em>20</em> in the Gsk3b(-/-) MEF cells, compared with the Gsk3b(+/+) MEF cells. Insulin and IGF1 activated Akt to phosphorylate GSK3B at serine 9, thus inhibiting GSK3B activity. Melatonin inhibited Akt activation, thus decreasing P-GSK3B at serine 9 (i.e., increasing GSK3B activity) and subsequently inhibiting expression of Cxcl1 and Ccl<em>20</em> that was induced either by IL-17 alone or by a combination of insulin and IL-17. Melatonin's inhibitory effects were only observed in the Gsk3b(+/+) , but in not Gsk3b(-/-) MEF cells. Melatonin also inhibited expression of Cxcl1, Ccl<em>20</em>, and Il-6 that was induced by a combination of insulin and IL-17 in the mouse prostatic tissues. Further, nighttime human blood, which contained high physiologic levels of melatonin, decreased expression of Cxcl1, Ccl<em>20</em>, and Il-6 in the PC3 human prostate cancer xenograft tumors. Our data support our hypothesis and suggest that melatonin may be used to dampen IL-17-mediated inflammation that is enhanced by the increased levels of insulin and IGF1 in obesity.

The aim of this study was to investigate the possible participation of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family members: FGF1, FGF2, and FGF7, and their receptor variants: FGFR, FGFR2IIIb, and FGFR2IIIc in theca interna (TI) and granulosa cell (GC) compartments of bovine follicles during final <em>growth</em>. A classification of follicles into five groups (<0.5; >0.5-5; >5-<em>20</em>;>><em>20</em>-180; >180 ng/ml, respectively) was performed according to the follicular fluid (FF) oestradiol-17beta (E) content. The mRNA expression and protein localization was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. FGF1 mRNA expression was relatively high in TI and lower in GC, and without any regulatory change for both tissue compartments during final follicular <em>growth</em>. The FGF1 protein could be predominantly localized in the cytoplasm of GC, in smooth muscle cells of blood vessels, in the rete ovarii, and at a lesser degree in theca cells. FGF2 mRNA in TI increased significantly in large follicles and was low and without any regulatory change in GC. FGF7 mRNA expression was relatively high in TI and very low in GC. For FGF7 in mature follicles a marked staining of the TI and the basal layers of the GC could be demonstrated. The mRNA signal for the FGFR in TI increased significantly with beginning of E production (E>> 0.5-5 ng/ml FF) and was without any regulatory change in GC. The mRNA expression of FGFR2IIIb was relatively high in GC and increased significantly during final <em>growth</em> of follicles in contrast to the TI with very low expression. The FGFR2IIIc mRNA expression in TI and GC was relatively high but without any clear change. Our results suggest that FGF <em>growth</em> <em>factor</em> family members are involved in process of folliculogenesis and especially during final <em>growth</em> of the preovulatory (dominant) follicle by stimulation of angiogenesis and GC survival and proliferation.

BACKGROUND

<em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGFR1) gene amplification was recently reported as a recurrent abnormality in 10% to <em>20</em>% of primary lung squamous cell carcinomas (SqCCs), and has attracted significant interest as a potential therapeutic target. Limited data are available for its prognostic impact in early-stage SqCC.

METHODS

Tissue microarrays containing 135 primary lung SqCCs and 58 matching lymph node metastases were tested by interphase fluorescence in situ hybridization for DNA copy number (CN) abnormalities at the 8p12 region including FGFR1.

RESULTS

FGFR1amplification was found in 18.2% (22 of 121 evaluable) of primary SqCC, using a definition of average copies of FGFR1 per cell of 5.0 or more. Concordance rate between primaries and matching lymph node metastases was 97.7% (43 of 44; 7 amplified and 37 nonamplified), with the only discordant case showing CN at approximately the dichotomous cutoff. Similarly, concordance between two separate lymph node metastases in each of 10 patients was 100% (1 amplified and 9 nonamplified). Using various CN cutoffs, we found no statistically significant association between FGFR1 CN abnormalities and patient age, sex, tumor grade, stage, smoking status, disease-free survival, cause-specific survival, or overall survival.

CONCLUSIONS

FGFR1 amplification is not prognostic in resected lung squamous cell carcinoma patients.

The aim of this study was to gain information relevant to disc repair processes. Limited degradation of the collagen matrix by matrix metalloproteases (MMPs) may facilitate the loosening of cell-cell and cell-matrix interactions within the injured intervertebral disc (IVD) to favour the penetration of blood vessels and migration of <em>fibroblasts</em> into the defect to promote repair processes. Gelatinase A (MMP-2) has a particularly important role to play in angiogenesis, in the present study we investigated the in vitro regulation of MMP-2 by Transforming <em>Growth</em> <em>Factor</em>-beta 1 (TGF-beta 1) and Insulin-like <em>Growth</em> <em>Factor</em>-1 (beta IGF-I) in cells from the nucleus pulposus (NP) of the ovine IVD. Ovine NP cells were grown in alginate bead cultures in complete medium (10% foetal calf serum) for 7 days, established in serum-free conditions for 24 h, then stimulated with TGF-beta 1 (0.1 or 10 ng/ml) or IGF-I (2 or 50 ng/ml) +/-Concanavalin A (<em>20</em> microg/ml) for an additional 48 h. Conditioned medium was examined for matrix metalloproteases using gelatin zymography, Tissue Inhibitor of Metalloproteinase 2 (TIMP-2) and Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) were immunolocalised in beads. Pro (72 kDa) and active (59 kDa) MMP-2 were the major gelatinolytic MMPs detected in control cultures, the TGF-beta 1 and IGF-I treatments significantly decreased levels of the active MMP-2, inclusion of Concanavalin A resulted in a complete reversal of this trend with IGF-I, and to a lesser extent with TGF-beta 1. Cell surface levels of TIMP-2 and MT1-MMP were decreased by the TGF-beta 1 treatment while IGF-I only appeared to decrease TIMP-2 expression. The findings of this study provide some insight as to why dense avascular connective tissues such as the intervertebral disc have such a poor healing potential.

OBJECTIVE

Although the slow healing rate of venous ulcers is well known, the underlying defect in the healing process is not well understood. The purpose of this study was to examine the cellular characteristics of fibroblasts taken from venous ulcers (wound-fb) and compare them with the fibroblasts of normal tissue (normal-fb).

METHODS

Biopsy specimens were obtained from wound margins and normal tissue of the upper thigh in each patient. Dermal fibroblasts were isolated from explant cultures in Dulbecco's modified Eagle's medium supplemented with 10% calf serum. These cells were then plated at 1000 cells per plate, and total cells per plate were counted over time so that growth curves could be generated. In further experimentation, media was supplemented with additional calf serum (20%, 30%, 40%, 50%) and growth factors (epidermal growth factor, basic fibroblast growth factor, interleukin-1 beta) in an attempt to stimulate growth.

RESULTS

Two major differences were noted: (1) normal-fb replicated more rapidly than wound-fb; and (2) the morphologic features of wound-fb were different. Normal-fb were compact and tapered, with well-defined nuclear morphologic features. Wound-fb were larger and polygonal in shape, with less-uniform nuclear morphologic features. Additional calf serum in tissue culture media enhanced normal-fb growth but had no effect on wound-fb. Supplementation of media with growth factors stimulated the growth of wound-fb. Statistically significant differences were noted at day 10 and 14 with basic fibroblast growth factor supplementation (p = 0.02 and 0.0001, respectively) and at day 14 with epidermal growth factor (p = 0.008). Although interleukin-1 beta stimulated cell growth in five of six patients, the differences observed were not statistically significant.

CONCLUSIONS

Our data demonstrate that wound-fb proliferate at a slower rate and are morphologically distinct from normal-fb. These characteristics are typical of aged or senescent cells. This decreased growth can be stimulated by growth factors basic fibroblast growth factor, epidermal growth factor, and interleukin-1 beta. Slowed growth may be partially responsible for the defect in healing of venous stasis ulcers. Furthermore, we believe that in some patients ulcer healing may be improved by exogenous provision of specific growth factors.

OBJECTIVE

Hemangioma is a primary tumor of the microvasculature in which angiogenesis is initially excessive, followed by regression of the newly formed vessels. Intervention is necessary in up to <em>20</em>% of cases, high-dose systemic or intralesional steroids being the first-line treatment. As the mechanism of action of steroids is unknown, we undertook an investigation of the cellular and molecular effects of their action.

METHODS

A unique opportunity to study the effect of steroid treatment was presented when biopsy material was obtained from an infant with an ulcerated proliferating hemangioma before and after intralesional triamcinolone injection, which resulted in an accelerated regression of the lesion. Histochemical quantitation of mast cells, molecular analysis by reverse transcriptase-polymerase chain reaction (RT-PCR) for 7 growth factor transcripts and differential display RT-PCR (DD RT-PCR) were conducted.

RESULTS

After steroid therapy, the mast cell number increased (untreated = 2.22 +/-.27 [standard error of the mean ¿SEM¿]; treated = 8.7 +/-.71 [SEM] mast cells per field, respectively; P <.0001; n = 40 fields for each group), and the transcriptional expression of cytokines: platelet-derived growth factor-A and -B; interleukin-6; transforming growth factor-beta1 and -beta3 decreased, while that of basic fibroblast growth factor (bFGF) and vascular endothelial cell growth factor remained unaltered. Elevated urinary bFGF levels noted in cases of proliferating hemangioma, persisted even after steroid treatment. Using DD RT-PCR an amplicon that shared 100% sequence homology with the human mitochondrial cytochrome b gene was detected in the hemangioma biopsy after steroid treatment.

CONCLUSIONS

The regression of this hemangioma subsequent to steroid therapy was accompanied by a significant increase in mast cell density, reduced transcription of several cytokines, and an enhanced expression of the mitochondrial cytochrome b gene.

<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, <em>20</em>13, and July 5, <em>20</em>17, 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, <em>20</em> 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, 15 (15%; 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 15 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 <em>20</em> 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 15% 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.

OBJECTIVE

A major problem in rat liver endothelial cell culture is the rapid loss of cells after 48 h. This study aimed to develop a protocol that allowed easy maintenance and proliferation of sinusoidal endothelial cells in serum-free culture for 5-6 days.

METHODS

Cells isolated from adult rat liver by collagenase digestion were purified by centrifugal elutriation and cultured on glutaraldehyde-crosslinked collagen.

RESULTS

At high plating densities cells could be maintained serum-free for 6 days in the presence of hydrocortisone and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>; at lower plating densities medium had to be supplemented with additional <em>growth</em>-promoting <em>factors</em>. Conditioned medium of adult rat hepatocytes proved to be the most effective <em>growth</em> stimulus; it increased thymidine incorporation, DNA content and cell number per dish with a half-maximal effect at <em>20</em>% (v/v). Cell proliferation was also observed with either vascular endothelial <em>growth</em> <em>factor</em>, phorbol ester or conditioned media from FAO or HEPG2 liver cell lines provided the cultures were additionally supplemented with 1% newborn calf serum. Vascular endothelial <em>growth</em> <em>factor</em> was detected in all conditioned media. In the absence of hepatocyte-conditioned medium, 1% serum helped to maintain cultures; it itself exerted a low proliferative effect. Higher serum concentrations (>5%), however, led to cell loss after 48 h. The numerous sieve plates of 6-h-old cells progressively disappeared during culture and were replaced by randomly distributed pores, which later grouped together at cell-cell borders. More than 90% of the cells endocytosed acetylated low-density lipoprotein.

CONCLUSIONS

The study shows that cultured hepatocytes secrete growth-promoting substances that stimulate in vitro endothelial cell proliferation in the absence of serum; this effect could be mimicked by the combined addition of vascular endothelial growth factor and 1% serum. The new media formulations should facilitate future research on liver endothelial cells in mono- or coculture.

Cultured bovine aortic endothelial cells secrete a potent migration-stimulating <em>factor</em> for vascular smooth muscle cells (SMCs) and adventitial <em>fibroblasts</em>. Vascular pericytes are <em>20</em>-fold less responsive, and endothelial cells themselves do not respond at all. Checkerboard analysis of SMC migration in a micro-chemotaxis chamber assay shows that the <em>factor</em> is chemotactic. Chemotactic activity for SMCs and adventitial <em>fibroblasts</em> is specifically inhibited by antibodies against platelet-derived <em>growth</em> <em>factor</em>. Endothelial cells cultured on nitrocellulose filters secrete the platelet-derived <em>growth</em> <em>factor</em>-like <em>factor</em> almost exclusively into the basal compartment. We suggest that this <em>factor</em> plays an important role in the recruitment of vascular wall cells during the morphogenesis of blood vessels and pathological conditions, such as atherosclerosis.

We evaluated <em>growth</em> <em>factor</em> contents and clinical efficacy of allogeneic platelet gel (PG) prepared with standard blood banking procedures from routine platelet concentrates (PCs) obtained from buffy coats. The PGs were used to treat 11 hypomobile very elderly patients unable to undergo autologous blood processing and previously ineffectively treated with expensive advanced medications for 8-275 weeks. PGs were prepared by platelet activation with human thrombin or commercial batroxobin. Median and range <em>growth</em> <em>factor</em> contents (ng/mL) were: platelet derived <em>growth</em> <em>factor</em> (PDGF-AB/-BB) 112 (31-157) and <em>20</em> (3.8-34); transforming <em>growth</em> <em>factor</em> (TGF-β1/-β2) 214 (48-289) and 0.087 (0.03-0.28); basic-<em>fibroblast</em> <em>growth</em> <em>factor</em> (b-FGF) 0.03 (0.006-0.214); vascular endothelial <em>growth</em> <em>factor</em> (VEGF) 1.15 (0.18-2.46); epidermal <em>growth</em> <em>factor</em> (EGF) 4.50 (0.87-6.64); insulin-like <em>growth</em> <em>factor</em> (IGF-l) 116 (72-156). In the clinical study, 222 PGs were used within 2 h of activation to treat 14 chronic skin ulcers in the 11 patients. No improvement was seen in 3 patients with 24, 27 and 30 cm(3) ulcers who could be treated for no more than 4, 7 and 8 weeks due to progressively worsening clinical conditions, while 11 ulcers with 3.2 cm(3) median size (range 0.2-3.6) in the remaining 8 patients showed 91 ± 14 % reduction after a median of 12 weeks (range 1-<em>20</em>). Cost of PG treatment (19,976 euro) amounted to about 10% of the ineffective advanced medication hospital reimbursement fees (191,236 euro). This study supports efficacy and feasibility of allogeneic PG to treat recalcitrant ulcers in very elderly hypomobile patients for whom autologous blood processing may be difficult.