A histopathology study of 22 pancreatic adenocarcinoma cases revealed that 13 of the patients presented with hyperplastic lesions (atypical and non-atypical hyperplasia, mucous cell hypertrophy, focal epithelial hyperplasia, and ductal papillary hyperplasia), and 9 exhibited fibrosis adjacent to the carcinoma. All lesions expressed high levels of epidermal <em>growth</em> <em>factor</em> receptor (EGF-R) (p<0.0001 and p=0.0008, respectively) as compared with normal ductal epithelium. Non-atypical and atypical hyperplastic lesions also had a higher proliferating cell nuclear antigen (PCNA) labeling index (p<0.001 and p=0.0008, respectively) than normal ductal epithelium. A gradient in PCNA+ nuclei was found in acinar cells adjacent to the tumors. In <em>16</em> cases with marked fibrosis, we observed a significant increase of PCNA+ nuclei in stromal <em>fibroblasts</em> (p=0.0041) and significant upregulation of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) mRNA expression in adjacent tumor cells (p=0.0213). These data suggest that the production of bFGF by pancreatic cancer cells induces ductal and stromal hyperplasia of the pancreas.

Adipocyte browning is a promising strategy for obesity prevention. Using onion-peel-derived extracts and their bioactive compounds, we demonstrate that onion peel, a by-product of onion, can change the characteristics of white adipocytes to those of brown-like adipocytes in the white adipose tissue of mice and 3T3-L1 cells. The expression of the following brown adipose tissue-specific genes was increased in the retroperitoneal and subcutaneous adipose tissues of 0.5% onion-peel-extract-fed mice: PR domain-containing <em>16</em>, peroxisome proliferator-activated receptor gamma coactivator 1α, uncoupling protein 1, <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 and cell death-inducing DFFA-like effector. In 3T3-L1 adipocytes, onion peel extract induced the expression of brown adipose tissue-specific genes and increased the expression of carnitine palmitoyltransferase 1α. This effect was supported by decreased lipid levels and multiple small-sized lipid droplets. The ethyl acetate fraction of the onion peel extract that contained the highest proportion of hydrophobic molecules showed the same browning effect in 3T3-L1 adipocytes. A high-performance liquid chromatography analysis further identified quercetin as a functional compound in the browning effect of onion peel. The quercetin-associated browning effect was mediated in part by the activation of AMP-activated protein kinase. In summary, our study provides the first demonstration of the browning effects of onion peel and quercetin using both animal and cell models. This result indicates that onion peel has the potential to remodel the characteristics of white adipocytes to those of brown-like adipocytes.

BACKGROUND

Up to 10 % of primary gastric cancers are characterized by FGFR2 amplification, and fibroblast growth factor receptor (FGFR) inhibitors may represent therapeutic agents for patients with these malignancies. However, long-term benefits of the treatment might be limited owing to the occurrence of drug resistance.

METHODS

To investigate the mechanisms of resistance to selective FGFR inhibitors, we established three FGFR2-amplified SNU-16 gastric cancer cell lines resistant to AZD4547, BGJ398, and PD173074, respectively.

RESULTS

The resistant cell lines (SNU-16R) demonstrated changes characteristic of epithelial-to-mesenchymal transition (EMT). In addition, they displayed loss of expression of FGFR2 and other tyrosine kinase receptors concurrent with activation of downstream signaling proteins and upregulation of the transforming growth factor β (TGF-β) level. However, treatment of parental SNU-16 cells with TGF-β1 did not evoke EMT, and pharmacological inhibition of TGF-β receptor I was not sufficient to reverse EMT changes in the resistant cells. Finally, we showed that the SNU-16R cell lines were sensitive to the human epidermal growth factor receptor 2 inhibitor mubritinib and the heat shock protein 90 inhibitor AUY922.

CONCLUSIONS

In conclusion, we provide experimental evidence that EMT-mediated resistance might emerge in gastric cancer patients following treatment with FGFR inhibitors, and mubritinib or AUY922 treatment may be an alternative therapeutic strategy for these patients.

OBJECTIVE

To investigate the differentiation of human umbilical cord blood (HUCB)-derived mesenchymal stem cells (MSCs) into hepatocytes by induction of fibroblast growth factor-4 (FGF-4) and hepatocyte growth factor (HGF), and to find a new source of cell types for therapies of hepatic diseases.

METHODS

MSCs were isolated by combining gradient density centrifugation with plastic adherence. When HUCB-derived MSCs reached 70% confluence, they were cultured in Iscove modified Dulbecco medium (IMDM) supplemented with 10 mL/L FBS, 20 ng/mL HGF and 10 ng/mL FGF-4. The medium was changed every 4 d and stored for albumin, alpha-fetoprotein (AFP) and urea assay. Expression of CK-18 was detected by immunocytochemistry. Glycogen storage in hepatocytes was determined by PAS staining.

RESULTS

By combining gradient density centrifugation with plastic adherence, we could isolate MSCs from 25.6% of human umbilical cord blood. When MSCs were cultured with FGF-4 and HGF, approximately 63.6% of cells became small, round and epithelioid on d 28 by morphology. Compared with the control, the level of AFP increased significantly from d 12 to 18.20+/-1.16 microg/L (t = 2.884, P<0.05) in MSCs cultured with FGF-4 and HGF, and was higher (54.28+/-3.11 microg/L) on d 28 (t = 13.493, P<0.01). Albumin increased significantly on d 16 (t = 6.68, P<0.01) to 1.02+/-0.15 microg/mL, and to 3.63+/-0.30 microg/mL on d 28 (t = 11.748, P<0.01). Urea (4.72+/-1.03 micromol/L) was detected on d 20 (t = 4.272, P<0.01), and continued to increase to 10.28+/-1.06 micromol/L on d 28 (t = 9.276, P<0.01). Cells expressed CK-18 on d 16. Glycogen storage was observed on d 24.

CONCLUSIONS

HUCB-derived MSCs can differentiate into hepatocytes by induction of FGF-4 and HGF. HUCB-derived MSCs are a new source of cell types for cell transplantation therapy of hepatic diseases.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) signalling has been implicated in the generation of mesoderm and neural fates in chordate embryos including ascidians and vertebrates. In Ciona, FGF9/<em>16</em>/20 has been implicated in both of these processes. However, in FGF9/<em>16</em>/20 knockdown embryos, notochord fate recovers during later development. It is thus not clear if FGF signalling is an essential requirement for notochord specification in Ciona embryos. We show that FGF-MEK-ERK signals act during two distinct phases to establish notochord fate. During the first phase, FGF signalling is required during an asymmetric cell division to promote notochord at the expense of neural identity. Consistently, ERK1/2 is specifically activated in the notochord precursors following this cell division. Sustained activation of ERK1/2 is then required to maintain notochord fate. We demonstrate that FGF9/<em>16</em>/20 acts solely during the initial induction step and that, subsequently, FGF8/17/18 together with FGF9/<em>16</em>/20 is involved in the following maintenance step. These results together with others' show that the formation of a large part of the mesoderm cell types in ascidian larvae is dependent on signalling events involving FGF ligands.

Gene transfer is a promising approach to the delivery of chondrotrophic <em>growth</em> <em>factors</em> to promote cartilage repair. It is unlikely that a single <em>growth</em> <em>factor</em> transgene will optimally regulate these cells. The aim of this study was to identify those <em>growth</em> <em>factor</em> transgene combinations that optimally regulate aggrecan, collagen type II and collagen type I gene expression by articular chondrocytes. We delivered combinations of the transgenes encoding <em>fibroblast</em> <em>growth</em> <em>factor</em>-2, insulin-like <em>growth</em> <em>factor</em> I, transforming <em>growth</em> <em>factor</em> beta1, bone morphogenetic protein-2, and/or bone morphogenetic protein-7 and assessed chondrocyte responses by measuring changes in the expression of aggrecan, type II collagen and type I collagen genes. These <em>growth</em> <em>factor</em> transgenes differentially regulated the magnitude and time course of all three-matrix protein genes. In concert, the transgenes regulated matrix gene expression in an interactive fashion that ranged from synergistic to inhibitory. Maximum stimulation of aggrecan (<em>16</em>-fold) and type II collagen (35-fold) expression was with the combination of IGF-I, BMP-2, and BMP-7 transgenes. The results indicate that the optimal choice of <em>growth</em> <em>factor</em> genes for cell-based cartilage repair cannot be predicted from observations of individual transgenes. Rather, such gene therapy will require an empirically based selection of <em>growth</em> <em>factor</em> gene combinations.

Chronic skin ulcers such as diabetic ulcers and venous leg ulcers are increasing and are a costly problem in healthcare. We have developed a novel artificial dermis, collagen/gelatin sponge (CGS), which is capable of sustained release of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) for more than 10 days. The objective of this study was to investigate the safety and efficacy of CGS impregnated with bFGF in the treatment of chronic skin ulcers. Patients with chronic skin ulcers that had not healed in at least 4 weeks were treated with CGS impregnated with bFGF at 7 or 14 μg/cm(2) after debridement, and the wound bed improvement was assessed 14 days after application. Wound bed improvement was defined as a granulated and epithelialized area on day 14 with a proportion to the baseline wound area after debridement of 50% or higher. The wound area, the wound area on day 14, and the granulation area on day 14 were independently measured by blinded reviewers in a central review using digital images of wounds taken with a calibrator. Patients were followed up until 28 days after application to observe the adverse reactions related to the application of CGS. From May 2010 to June 2011, 17 patients were enrolled and, in <em>16</em> patients, the wound bed improved. Among the randomized patients in step 2, no significant difference was seen between the low-dose group and the high-dose group. No serious adverse reactions were observed. Adverse reactions with a clear causal relationship to the study treatment were mild and patients quickly recovered from them. This study is the first-in-man clinical trial of CGS and showed the safety and efficacy of CGS impregnated with bFGF in the treatment of chronic skin ulcers. This combination therapy could be a promising therapy for chronic skin ulcers.

Excess ethanol consumption has serious pathologic consequences. In humans, repeated episodes of binge drinking can lead to liver damage and have adverse effects on other organs such as pancreas and brain. Long term chronic consumption of ethanol can also result in progressive alcoholic liver disease and cirrhosis. Fibroblast growth factor 21 (FGF21) is a metabolic regulator with multiple physiologic functions. FGF21 is a novel biomarker for non-alcoholic fatty liver disease (NAFLD) in humans and limits hepatotoxicity in mice. Therefore, we explored the possibility that FGF21 plays a role in response to ethanol consumption in both humans and mice.

We used a binge drinking paradigm in humans to examine the effect of acute ethanol consumption on circulating FGF21. We adapted this paradigm to evaluate the acute response to ethanol in mice. We then examined the role of FGF21 on liver pathology in two models of chronic ethanol consumption in both wild type (WT) mice and mice lacking FGF21 (FGF21-KO).

Acute ethanol consumption resulted in a robust induction of serum FGF21 after 6 h in both humans and mice. Serum ethanol peaked at 1 h in both species and was cleared by 6 h. Ethanol clearance was the same in WT and FGF21-KO mice, indicating that FGF21 does not play a major role in ethanol metabolism in a binge paradigm. When FGF21-KO mice were fed the Lieber-DeCarli diet, a high fat diet supplemented with ethanol, a higher mortality was observed compared to WT mice after 16 days on the diet. When FGF21-KO mice consumed 30% ethanol in drinking water, along with a normal chow diet, there was no mortality observed even after 16 weeks, but the FGF21-KO mice had significant liver pathology compared to WT mice.

Acute or binge ethanol consumption significantly increases circulating FGF21 levels in both humans and mice. However, FGF21 does not play a role in acute ethanol clearance. In contrast, chronic ethanol consumption in the absence of FGF21 is associated with significant liver pathology alone or in combination with excess mortality, depending on the type of diet consumed with ethanol. This suggests that FGF21 protects against long term ethanol induced hepatic damage and may attenuate progression of alcoholic liver disease. Further study is required to assess the therapeutic potential of FGF21 in the treatment of alcoholic liver disease.

The E5 oncoprotein of the human papillomavirus type <em>16</em> (HPV<em>16</em> E5) deregulates epithelial homeostasis through the modulation of receptor tyrosine kinases and their signaling. Accordingly, the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2b (FGFR2b/KGFR), epithelial splicing transcript variant of the FGFR2, is down-modulated by the viral protein expression, leading to impairment of keratinocyte differentiation. Here, we report that, in cell models of transfected human keratinocytes as well as in cervical epithelial cells containing episomal HPV<em>16</em>, the down-regulation of FGFR2b induced by <em>16</em>E5 is associated with the aberrant expression of the mesenchymal FGFR2c isoform as a consequence of splicing switch: in fact, quantitative RT-PCR analysis showed that this molecular event is transcriptionally regulated by the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) and is able to produce effects synergistic with those caused by TGFβ treatment. Immunofluorescence analysis revealed that this altered FGFR2 splicing leads to changes in the specificity for the ligands FGFs and in the cellular response, triggering epithelial-mesenchymal transition (EMT). Through <em>16</em>E5 or FGFR2 silencing as well as inhibition of FGFR2 activity we demonstrated the direct role of the viral protein in the receptor isoform switching and EMT, suggesting that these early molecular events during HPV infection might represent additional mechanisms driving cervical transformation and tumor progression.

METHODS

We collected the specimens of lumbar intervertebral discs from patients with discogenic low back pain, to study the histopathological features and connective tissue growth factor (CTGF) expressions.

OBJECTIVE

To study the expression and role of CTGF in fibrosis and degeneration of painful disc tissue.

BACKGROUND

Previous studies have demonstrated that degenerative disc commonly showed fibrosis in histology. CTGF, a downstream effector mediated by transforming growth factor-beta1 (TGF-beta1), is commonly related to tissue fibrosis. We do not know whether CTGF is expressed in painful disc, and related to painful disc degeneration and fibrosis.

METHODS

This study included 43 lumbar intervertebral disc specimens from 28 patients with discogenic low back pain obtained during posterior lumbar interbody fusion and 16 asymptomatic degenerative discs from patients without low back pain. Further, 8 normal discs were included as controls. Their histopathological features were studied, and the expression of CTGF was assessed using immunohistochemistry.

RESULTS

Histologic examination revealed that the painful discs showed chronic inflammatory reaction with blood vessel infiltration in varying degrees. The anulus fibrosus had lost its normal lamellar architecture, and instead, disorganization, disruption, and crossed fusion were observed. Normal fibroblasts were replaced by chondrocytes in the anulus fibrosus. The nucleus pulposus showed marked fibrosis, blood vessel infiltration, and inflammatory granulation tissue formation. Immunohistochemical staining demonstrated strong CTGF expression in the painful discs, weak expression in the asymptomatic degenerative disc, and no expression in the control discs.

CONCLUSIONS

The painful degenerative disc is significantly different from the asymptomatic degenerative disc with regard to histopathological findings. The strong CTGF expression in the painful disc may be related to disc fibrosis and degeneration.

Fetal cutaneous wounds that occur in early gestation heal without scar formation. Although much work has been done to characterize the role of transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) isoforms in the adult wound repair process, their function in fetal scarless wound repair is not well understood. The authors hypothesized that the pattern of expression for TGF-beta isoforms and their receptors may influence the phenotypic transition from scarless to scar-forming repair observed during fetal gestation. Using time-dated fetal Sprague-Dawley rat <em>fibroblasts</em> and unwounded skin at gestational ages 14, <em>16</em>, 18, and 21 days postcoitum of the scarless (< or =<em>16</em> days) and scar-forming >><em>16</em> days) periods of gestation (term = 21.5 days), the authors analyzed the endogenous messenger RNA (mRNA) levels of TGF-beta 1 and TGF-beta 3 and their signaling receptors TGF-beta-RI and TGF-beta-RII. Northern blot analyses in both <em>fibroblasts</em> and unwounded skin revealed that levels of TGF-beta 1 were not differentially expressed, whereas more TGF-beta 3 mRNA transcript was found in early than in late gestation. <em>Fibroblast</em> expression of TGF-beta-RI showed no substantial differences, whereas expression of TGF-beta-RII increased during gestation. In contrast, expression of both TGF-beta-RI and TGF-beta-RII in unwounded skin showed decreasing levels as a function of gestational age. The differential levels of TGF-beta 1 and TGF-beta 3 suggest that the ratio of these cytokines may provide a predominantly antiscarring or profibrotic signal upon wounding during the scar-free or scar-forming periods of gestation, respectively. Furthermore, lower amounts of the ligand-binding TGF-beta-RII seen in early gestation <em>fibroblasts</em> suggest a decreased ability to perceive ligand during the period of scarless repair.

Neural progenitor cells may provide for cell replacement or gene delivery vehicles in neurodegen-erative disease therapies. The expression of therapeutic proteins by neural progenitors would be enhanced by viral-mediated gene transfer, but the effects of several common recombinant viruses on primary progenitor cell populations have not been tested. To address this issue, we cultured cells from embryonic day <em>16</em>-18 mouse brain in serum-free medium containing epidermal <em>growth</em> <em>factor</em> or basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and investigated how transduction with recombinant viral vectors affected maintenance and differentiation properties of progenitor cells. Neurosphere cultures were incubated with feline immunodeficiency virus (FIV), adeno-associated virus (AAV) or ade-noviral (Ad) constructs expressing either beta-galactosidase or enhanced green fluorescent protein at low multiplicity of infection. Nestin-positive neurospheres were regenerated after incubation of single progenitor cells with FIV, indicating that FIV-mediated gene transfer did not inhibit progenitor cell self-renewal. In contrast, adenovirus induced differentiation into glial fibrillary acidic protein (GFAP)-positive astrocytes. The AAV serotypes tested did not effectively transduce progenitor cells. FIV-transduced progenitors retained the potential for differentiation into neurons and glia in vitro, and when transplanted into the striatum of normal adult C57BL/6 mice differentiated into glia, or remained undifferentiated. In the presence of tumor cells, FIV-transduced progenitors migrated significantly from the injection site. Our results suggest that FIV-based vectors can transduce progenitor cell populations in vitro, with maintenance of their ability to differentiate into multiple cell types or to respond to injury within the central nervous system. These results hold promise for the use of genetically manipulated stem cells for CNS therapies.

Using cultures of Schwann cells from neonatal rat sciatic nerves, we examined the mitogenic activity of an axolemmal fraction from adult rat CNS. Axolemmal fraction proved a potent mitogen, stimulating [3H]thymidine incorporation into Schwann cell DNA 13.5-fold over control values when axolemmal fraction equivalent to <em>16</em> micrograms of protein per culture microwell or more was added. Half maximal stimulation was obtained with addition of axolemmal fraction equivalent to 4 micrograms of protein. The concentration-dependence and magnitude of the mitogenic response of the cultured cells were nearly identical whether they were maintained in vitro for 1 day or for 2 weeks prior to addition of the axolemmal fraction. A study of the time-course of the effect of axolemmal fraction on Schwann cell mitosis showed that maximal [3H]thymidine incorporation took place during the fifth day after addition of axolemmal fraction. Axolemmal fraction also produced stimulation of [3H]thymidine incorporation into Schwann cells, seeded and cultured in a serum-free defined medium. Though the concentration-dependence of the mitogenic effect in the absence of serum was similar to that in a serum-containing medium, maximal stimulation in the defined medium was only 2.8-fold. The mitogenic activity of axolemmal fraction was rapidly and almost totally inactivated by sonication or homogenization, and was partially lost after exposure to heat. The mitogenic activities of plasma membrane fragments from rat skeletal muscle or rat erythrocytes, and of mitochondrial fragments (the major contaminant of the axolemmal fraction) were one-tenth that of axolemmal fraction or less. In contrast to glial <em>growth</em> <em>factor</em> prepared from bovine pituitaries (GGF-BP), which stimulates proliferation of both <em>fibroblasts</em> and Schwann cells, axolemmal fraction induced proliferation of Schwann cells but not of endoneurial <em>fibroblasts</em>; cultures treated with axolemmal fraction demonstrated a 3-fold increase in Schwann cell population in 10 days without detectable increase in number of <em>fibroblasts</em>. Also in contrast to GGF-BP, the mitogenic effect of which is considerably enhanced by simultaneous addition of cholera toxin to the medium, cholera toxin had no effect on the Schwann cell proliferative response to axolemmal fraction.

The clonal osteoblast-like cell line MC3T3-E1 undergoes time-dependent morphological changes leading to the formation of bone nodules in vitro. Serial analysis of gene expression was used to identify genes expressed in MC3T3-E1 cells, including known osteoblast markers, structural and matrix proteins, transcription <em>factors</em>, cell-cycle regulators, and housekeeping genes. Relative changes in the expression of 92 representative transcripts were determined by arrayed cDNA hybridization. Complex probes were derived from MC3T3-E1 cells during the proliferation, differentiation, and matrix mineralization stages, and from cells grown with all- trans-retinoic acid (RA), a potent bone morphogen. We found that the relative expression of 68 of these genes was higher during differentiation than in the earlier proliferative phase. In the mineralization phase, all but <em>16</em> cDNAs had lower normalized hybridization intensity ratios as compared with the differentiation phase. cDNAs for vimentin (Vim), presenilin 2 (Psen2), guanine nucleotide binding protein alpha stimulating (Gnas), gap-junction membrane channel protein beta 1 (Gjb1), <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (Fgfr1), eukaryotic translation elongation <em>factor</em> 2 (Eef2), and calponin 2 (Cnn2) had higher normalized hybridization intensities in both differentiation and mineralization than in proliferation. RA treatment during the differentiation phase appeared to reduce the expression of the 92 genes examined, as 62 cDNAs had lower hybridization intensities with complex cDNA probes derived from RA-treated cells than with the probe from untreated cells. Several cDNAs representing genes with previously unrecognized RA responsiveness were identified by this comparison, including the receptor for bone morphogenetic proteins 2 and 4 (Bmp2/Bmp4) and hemoglobin Y.

BACKGROUND

Activating FGFR2 mutations are found in 10-<em>16</em>% of primary endometrial cancers and provide an opportunity for targeted therapy. We assessed the safety and activity of dovitinib, a potent tyrosine-kinase inhibitor of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors, VEGF receptors, PDGFR-β, and c-KIT, as second-line therapy both in patients with FGFR2-mutated (FGFR2(mut)) endometrial cancer and in those with FGFR2-non-mutated (FGFR2(non-mut)) endometrial cancer.

METHODS

In this phase 2, non-randomised, two-group, two-stage study, we enrolled adult women who had progressive disease after first-line chemotherapy for advanced or metastatic endometrial cancer from 46 clinical sites in seven countries. We grouped women according to FGFR2 mutation status and gave all women dovitinib (500 mg per day, orally, on a 5-days-on and 2-days-off schedule) until disease progression, unacceptable toxicity, death, or study discontinuation for any other reason. The primary endpoint was proportion of patients in each group who were progression-free at 18 weeks. For each group, the second stage of the trial (enrolment of 20 additional patients) could proceed if at least eight of the first 20 treated patients were progression free at 18 weeks. Activity was assessed in all enrolled patients and safety was assessed in all patients who received at least one dose of dovitinib. The completed study is registered with ClinicalTrials.gov, number NCT01379534.

RESULTS

Of 248 patients with FGFR2 prescreening results, 27 (11%) had FGFR2(mut) endometrial cancer. Between Feb 17, 2012, and Dec 13, 2013, we enrolled 22 patients in the FGFR2(mut) group and 31 patients in the FGFR2(non-mut) group. Seven (31·8%, 95% CI 13·9-54·9) patients in the FGFR2(mut) group and nine (29·0%, 14·2-48·0) in the FGFR2(non-mut) group were progression-free at 18 weeks. On the basis of predefined criteria, neither group continued to stage two: seven (35%) of the first 20 patients in the FGFR2(mut) group were progression free at 18 weeks, as were five (25%) of the first 20 in the FGFR2(mut) population. Rates of treatment-emergent adverse events were similar between groups and events were most frequently gastrointestinal. Overall, the most common grade 3 or 4 adverse events suspected to be related to the study drug were hypertension (nine patients; 17%) and diarrhoea (five; 9%). The most frequently reported serious adverse events suspected to be related to study drug were pulmonary embolism (four patients; 8%), vomiting (four; 8%), dehydration (three; 6%), and diarrhoea (three; 6%). Only one death was deemed to be treatment-related: one patient in the FGFR2(non-mut) group died from cardiac arrest with contributing reason of pulmonary embolism (grade 4, suspected to be study drug related) 4 days previously.

CONCLUSIONS

Second-line dovitinib in FGFR2(mut) and FGFR2(non-mut) advanced or metastatic endometrial cancer had single-agent activity, although it did not reach the prespecified study criteria. Observed treatment effects seemed independent of FGFR2 mutation status. These data should be considered exploratory and additional studies are needed.

BACKGROUND

Novartis Pharmaceuticals.

Studies on human papillomavirus type <em>16</em> have demonstrated that the product of the early gene, E7, plays a key role in the immortalization and malignant transformation of the host cell. Several of the biological activities of HPV<em>16</em> E7 are mediated by inactivation of the members of the pocket protein family, pRb, p107 and p130. In this study, we have characterized the in vitro properties of five E7 proteins from benign and malignant HPV types (10, 32, 48, 54, 77). We show that these E7 proteins associate with pRb and p107 with different efficiencies. All E7s increased the proliferative rate of immortalized rodent <em>fibroblasts</em> cultured in 10% calf serum containing medium. This property is completely independent of their ability to associate with the pocket proteins. Furthermore, all E7s, except HPV10 E7, stimulate G1/S progression and activated the cyclin E and cyclin A promoter in the absence of <em>growth</em> <em>factors</em>. This activity also does not correlate with the E7-efficiency of binding the pocket proteins. Together these data provide evidence that different E7s alter the regulation of the cell cycle by diverse mechanism(s). Finally, this comparative analysis of the different E7 proteins demonstrates that the oncogenicity of a HPV type is not determined by the ability of E7 to associate with the pocket proteins.

Human skin equivalents (HSEs) were used as a model to investigate interleukin (IL)-1 alpha and IL-1 beta secretions by keratinocytes stimulated by Sarcoptes scabiei (SS). SS mites burrowed into the stratum corneum when placed on the surface of cultured HSEs. Mites lived for 14 days. Mites and mite products induced cells in the HSEs to secrete IL-1 alpha and IL-1 beta within <em>16</em> hr. Scabies mites induced production of greater amounts of IL-1 alpha than IL-1 beta. Hepatocyte <em>growth</em> <em>factor</em> in the culture medium at 3 and 30 ng/ml upregulated the secretions of both IL-1 alpha and IL-1 beta by mite-infested skin equivalents, whereas 10 ng/ml of IL-6 upregulated production of only IL-1 beta. Therefore, these cytokines were important immunomodulating <em>factors</em> influencing keratinocyte secretion of IL-1 alpha and IL-1 beta in vitro. The results of this study provide the first evidence that keratinocytes (possibly <em>fibroblasts</em>) in the skin produce these cytokines in response to scabies mites or other ectoparasitic arthropods. Because IL-1 alpha and IL-1 beta are potent inducers of inflammation and keratinocytes are among the first effector cells to encounter scabies mites and their products, these cells may be key initiators of the inflammatory/immune reaction to scabies.

OBJECTIVE

Acute gastrointestinal syndrome (AGS) resulting from ionizing radiation causes death within 7 days. Currently, no satisfactory agent exists for mitigation of AGS. A peptide derived from the receptor binding domain of fibroblast growth factor 2 (FGF-P) was synthesized and its mitigation effect on AGS was examined.

METHODS

A subtotal body irradiation (sub-TBI) model was created to induce gastrointestinal (GI) death while avoiding bone marrow death. After 10.5 to 16 Gy sub-TBI, mice received an intramuscular injection of FGF-P (10 mg/kg/day) or saline (0.2 ml/day) for 5 days; survival (frequency and duration) was measured. Crypt cells and their proliferation were assessed by hematoxylin, eosin, and BrdU staining. In addition, GI hemoccult score, stool formation, and plasma levels of endotoxin, insulin, amylase, interleukin (IL)-6, keratinocyte-derived chemokine (KC) monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis factor (TNF)-alpha were evaluated.

RESULTS

Treatment with FGF-P rescued a significant fraction of four strains of mice (33-50%) exposed to a lethal dose of sub-TBI. Use of FGF-P improved crypt survival and repopulation and partially preserved or restored GI function. Furthermore, whereas sub-TBI increased plasma endotoxin levels and several pro-inflammation cytokines (IL-6, KC, MCP-1, and TNF-alpha), FGF-P reduced these adverse responses.

CONCLUSIONS

The study data support pursuing FGF-P as a mitigator for AGS.

In this study we have compared the intracellular itineraries of insulin and insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) and their receptors subsequent to ligand internalization in rat <em>fibroblasts</em>. We found that the endocytic rate constant is approximately 3 times as high for insulin as for IGF-I. The dissociation of internalized ligand from its receptor was monitored by the ability of ligand-receptor complexes to precipitate in the presence of polyethylene glycol (PEG). Insulin loses its ability to precipitate with PEG more rapidly than IGF-I. After 60 min, less than 10% of the intracellular insulin remains PEG precipitable, whereas 44% of intracellular IGF-I stays PEG precipitable. Ligand degradation was determined by precipitation with trichloroacetic acid (TCA). Insulin degradation after internalization is more rapid compared with IGF-I degradation; after 2 h, 80% of intracellular IGF-I, in contrast to only 30% of intracellular insulin, remains intact. We measured retroendocytosis of insulin and IGF-I by assessing the amount of internalized ligand that was subsequently released from the cells. When analyzing released ligand by TCA precipitation, we found that 25% of insulin and 53% of IGF-I were TCA precipitable. After 120 min, only <em>16</em>% of insulin and 43% of IGF-I remained intracellular. To provide insight into a possible mechanism that prevents IGF-I from being subjected to the same degree of degradation as insulin, we studied the effect of pH on IGF-I and insulin binding. We found IGF-I binding to be less sensitive to decreases in pH compared with insulin binding. Therefore, it is likely that after internalization, IGF-I does not dissociate from its intracellular receptor as easily as insulin in the acidifying endosome and, therefore, can return to the cell surface via a recycling receptor. In summary, we have observed distinct differences in the intracellular itineraries of IGF-I and insulin and their receptors. IGF-I internalization and degradation proceed less efficiently than insulin internalization, and IGF-I is preferentially targeted into a retroendocytotic pathway in contrast to insulin, which primarily undergoes lysosomal degradation. The differential effect of pH on ligand binding to these structurally related hormone receptors may account for the quantitatively distinct ligand trafficking events.

Understanding altered gene expression in osteoarthritic cartilage can lead to new targets for drug intervention. We established a functional assay based on chondrocyte cluster formation, a phenotype associated with osteoarthritis (OA), to screen an OA cartilage gene library. Previous reports have demonstrated that normal chondrocytes grown in suspension culture maintain their chondrocytic phenotype, however, certain <em>growth</em> <em>factors</em> such as basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) will induce the cells to proliferate in tight clusters similar to those seen in osteoarthritic cartilage. In this study we validate that overexpression of bFGF by retrovirally transduced normal chondrocytes would similarly induce the proliferation of tight cell clusters. We then used this approach as a basis to set up a functional screen where an entire OA cartilage cDNA library was tranduced into normal chondrocytes to search for other genes that would also induce cluster formation. Seven potential genes were isolated from the OA gene library, including BPOZ, IL-17 receptor C, NADH ubiquinone oxidoreductase, COMP, Soluble carrier <em>16</em> (MCT 3), C1r, and bFGF itself. None of the identified genes were upregulated by bFGF, however, all of them upregulated the expression of bFGF suggesting a common pathway. Although cluster formation is not considered to be destructive in OA cartilage, it is consistent with the disease and could yield answers to the altered phenotype. Further studies are needed to elucidate how these genes are linked to the disease state.

Crk is a member of a family of adapter proteins predominantly composed of Src homology 2 and 3 domains, whose role in signaling pathways is presently unclear. Using an in situ electroporation system which permits the introduction of glutathione S-transferase (GST) fusion proteins into cells, we found that c-CrkII bound to p130(cas), but not to paxillin in serum-starved rat-1 <em>fibroblasts</em> overexpressing the human insulin receptor (HIRc cells) in vivo. 17 nM insulin stimulation dissociated the binding of c-CrkII to p130(cas), whereas 13 nM insulin-like <em>growth</em> <em>factor</em>-I, <em>16</em> nM epidermal <em>growth</em> <em>factor</em> (EGF), and 10% serum each showed little or no effect. We found that stress fiber formation is consistent with a change in the p130(cas).c-CrkII interactions before and after <em>growth</em> <em>factor</em> stimulation. Microinjection of either GST-Crk-SH2 or -Crk-(N)SH3 domains, or anti-Crk antibody each inhibited stress fiber formation before and after insulin-like <em>growth</em> <em>factor</em>-I, EGF, and serum stimulation. Insulin stimulation by itself caused stress fiber breakdown and there was no additive effect of microinjection. Microinjection of anti-p130(cas) antibody also blocked stress fiber formation in quiescent cells. Microinjection of the Crk-inhibitory reagents also inhibited DNA synthesis after insulin-like <em>growth</em> <em>factor</em>-I, EGF, and serum stimulation, but not after insulin. These data suggest that the complex containing p130(cas).c-CrkII may play a crucial role in actin cytoskeleton organization and in anchorage-dependent DNA synthesis.

Rapid <em>growth</em> of residual tumor after partial hepatectomy has been observed during the period of liver regeneration in children with malignant embryonal hepatoblastoma. The aim of this study was to elucidate the role of hepatocyte <em>growth</em>-<em>factor</em>-scatter <em>factor</em> (HGF-SF) in this phenomenon. Markedly increased serum levels of HGF-SF up to 15 ng/ml were found in 13/18 patients after liver resection and in 6/<em>16</em> patients with regressive tumors after chemotherapy, in comparison with 15 patients with non-pre-treated hepatoblastoma and 20 healthy children of the same age group. In the tumors, epithelial tumor cells highly expressed the HGF-SF receptor c-met, as shown by immunohistochemistry and m-RNA RT-PCR. The hepatoblastoma cell lines HepT1, HepT3 and HUH6 reacted with significantly increased proliferation to rhHGF-SF in these concentrations (1-15 ng/ml). In the tumors, HGF-SF was found to be expressed in the stromal <em>fibroblasts</em>. In culture, hepatoblastoma cells (HepT3, HUH6) stimulated secretion of the <em>factor</em> by human <em>fibroblasts</em>, indicating the paracrine fashion of intratumoral HGF-SF production. Cultured hepatoblastoma cells ceased to proliferate at 20-50 ng/ml HGF-SF, and they underwent cell death at>>/=100 ng/ml. In contrast, the hepatocellular-carcinoma cell line HepG2 decreased <em>growth</em> under HGF-SF in a dose-dependent manner. We conclude that post-operatively secreted and intratumorally produced HGF-SF can function as a <em>growth</em> <em>factor</em> for hepatoblastoma, while the same agent has a cytostatic effect in unphysiologically high concentrations.

Optic nerve axons do not regenerate after lesions in postnatal mammals, except if peripheral nerve transplants are offered as a substrate. In the present study, regeneration was assessed after intracranial freeze-crush lesions of the optic nerve, which completely interrupted all axons. In rats lesioned at the age of 8-9 days and surviving for additional 5-6 days, regenerating retinal fibers were seen to enter and partially cross the lesion site, reaching a maximum distance of 0.8 mm (mean +/- S.E.M. = 0.62 +/- 0.07 mm) in the presence of brain-derived neurotrophic <em>factor</em> (BDNF). Without BDNF, almost all the axons were lost due to axonal die-back. This regeneration distance could be significantly enhanced, up to 1.9 mm, by application of a monoclonal antibody (mAB-IN-1) directed against oligodendrocyte- and myelin-associated neurite <em>growth</em> inhibitory proteins. Similar results were obtained in rats lesioned at <em>16</em>-18 days and surviving for 2 weeks: whereas <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) stimulated sprouting did not exceed distances of 0.5 mm (mean = 0.38 +/- 0.07 mm), FGF and IN-1 antibody treated rats showed regenerations up to 2.3 mm (mean = 1.53 +/- 0.15 mm). The specificity of this effect was confirmed by lesions of myelin- and oligodendrocyte-free optic nerves: optic nerves were locally X-irradiated at birth, day 2, 4 and 6, a procedure which kills the dividing oligodendrocyte precursor cells. When these myelin-free nerves were lesioned at 3 weeks of age, regeneration distances between 2.5 and 3.2 mm were observed 3 weeks later.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin 1-beta (IL-1 beta), IL-2, IL-4, IL-5, IL-6, IL-8, tumour necrosis <em>factor</em>-alpha (TNF-alpha), transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) and granulocyte-macrophage colony-stimulating <em>factor</em> (GM-CSF) gene expression was determined in knee synovium of <em>16</em> patients with rheumatoid arthritis (RA) and <em>16</em> patients with seronegative spondyloarthropathies (SSP), by using polymerase chain reaction (PCR) amplification. The pattern of cytokines observed in RA synovium is of the macrophage-<em>fibroblast</em> type, with the highest expression of IL-1 beta and TGF-beta. GM-CSF and IL-2 bands were visualized in a minority of patients. Neither IL-4 nor IL-5 could be detected. No significant differences were observed in the cytokine profile between patients with early (< 12 months) and more advanced disease. No differences were observed according to gender, age, rheumatoid <em>factor</em> status and the duration of knee synovitis. The pattern of cytokines in the synovium of SSP patients is similar to that observed in RA patients and does not change in relation to disease duration. IL-2 was the only T-cell cytokine observed. These data provide evidence that the macrophage-<em>fibroblast</em> cells have an important role in early and more advanced rheumatoid synovitis, and show that this is also true for SSP peripheral synovitis.