Interleukin-6 is a pleiotropic cytokine with a wide range of effects, including induction of B-cell and cytotoxic T-cell differentiation, and induction of acute phase reactant production by hepatocytes. Interleukin-6 also can act as an autocrine <em>growth</em> <em>factor</em> in malignancy. Various cell types produce interleukin-6, including T and B cells, monocytes, <em>fibroblasts</em>, and some solid tumor cells. In previous work we detected the production of substantial amounts of interleukin-6 by human ovarian cancer cells, including the ovarian cancer cell lines CAOV-3, OVCAR-3, and SKOV-3, and several primary ovarian tumor cultures. In this study we retrospectively examined 90 separate serum specimens for interleukin-6 in 36 patients with epithelial ovarian cancer. The mean serum interleukin-6 concentration of those ovarian cancer patients with macroscopic disease (n = 57) was 0.26 +/- 0.04 U/ml (mean +/- SEM). Healthy adult donors have interleukin-6 serum levels of 0.12 +/- 0.03 U/ml. Sixteen of 21 ovarian cancer patients with macroscopic disease (76%) had elevated (greater than 0.20 U/ml) levels of serum interleukin-6, with levels approaching 1 U/ml in some patients (p less than 0.01). Of those nine patients with bulky tumor (residual greater than 2 cm), eight (89%) had an elevated interleukin-6 level (mean, 0.31 +/- 0.05), while eight of 12 (66%) with minimal residual disease (less than 2 cm) had elevated levels. Only two of 15 (13%) patients who were in clinical remission and who had microscopic disease had elevated values. Of the 36 patients, 22 were CA 125 negative (less than 35 U/ml), and of these, four had elevated interleukin-6 levels. Of the 14 patients with an elevated CA 125 level, 12 (86%) had elevated interleukin-6 levels. In those <em>16</em> patients in whom serial levels of interleukin-6 were measured, rising levels were found over a 3 to 4 month interval in nine (56%); this correlated with tumor progression. Furthermore, the subsequent survival of patients was shown to correlate with the level of interleukin-6, such that patients whose levels were elevated greater than 0.20 U/ml interleukin-6 survived a mean of 12.5 months, compared with 27.2 months for patients with normal levels (p less than 0.001). These data support the concept that interleukin-6 may be a useful tumor marker in some patients with epithelial ovarian cancer, as it correlates with the tumor burden, clinical disease status, and survival.

We have demonstrated the formation of hybrid insulin/insulin-like <em>growth</em> <em>factor</em>-I(IGF-I) receptors in transfected rodent <em>fibroblasts</em>, which overexpress human receptors, by examining reactivity with species- and receptor-specific monoclonal antibodies. In NIH 3T3 and Rat 1 <em>fibroblasts</em>, endogenous IGF-I receptors were unreactive with anti-(human insulin receptor)monoclonal antibodies (47-9, 25-49, 83-14, 83-7, 18-44). However, in transfected cells expressing high levels of insulin receptors, 60-80% of high-affinity IGF-I receptors reacted with these antibodies, as assessed either by inhibition of ligand binding in intact cells or by precipitation of solubilized receptors. Conversely, endogenous insulin receptors in NIH 3T3 cells were unreactive with anti-(IGF-I receptor) antibodies alpha IR-3 and <em>16</em>-13. However, approx. 50% of high-affinity insulin receptors reacted with these antibodies in cells expressing high levels of human IGF-I receptors. The hybrid receptors in transfected cells bound insulin or IGF-I with high affinity. However, responses to these ligands were asymmetrical, in that binding of IGF-I inhibited subsequent binding of insulin, but prior binding of insulin did not affect the affinity for IGF-I. The existence of hybrid receptors in normal tissues could have important implications for metabolic regulation by insulin and IGF-I.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23) plays important roles in the development of hypophosphatemic diseases such as tumor-induced osteomalacia (TIO) and X-linked hypophosphatemic rickets/osteomalacia (XLH). However, clinical usefulness of measurement of FGF23 has not been established. The objective of this study is to examine the importance of FGF23 measurement in the diagnosis of hypophosphatemic diseases. Biochemical parameters concerning phosphate metabolism were analyzed in a cross-sectional study. 32 patients with TIO, 28 patients with XLH and <em>16</em> hypophosphatemic patients with other causes including vitamin D deficiency, Fanconi's syndrome and Cushing's syndrome were studied. In patients with TIO and XLH, FGF23 was above the upper limit of the reference range in most patients irrespective of medical treatment. The lowest FGF23 in these patients was 38.0 pg/ml. FGF23 in hypophosphatemic patients with other causes was undetectable (less than 3 pg/ml) in 12 patients and the highest FGF23 in this group was 23.9 pg/ml. Relationship between phosphate and FGF23 indicated that TIO and XLH are diseases with high FGF23 and hypophosphatemia judged by age-dependent reference ranges for serum phosphate. FGF23 measurement is useful for differential diagnosis of hypophosphatemic diseases caused by excess actions of FGF23 and other etiologies. High FGF23 with low phosphate judged by age-dependent reference ranges for phosphate establishes the diagnosis of diseases caused by excess actions of FGF23.

Presence of brown adipose tissue (BAT), characterized by the expression of the thermogenic uncoupling protein 1 (UCP1), has recently been described in adult humans. UCP1 is expressed in classical brown adipocytes, as well as in "beige cells" in white adipose tissue (WAT). The thermogenic activity of BAT is mainly controlled by the sympathetic nervous system. Endocrine <em>factors</em>, such as <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 (FGF21) and bone morphogenic protein <em>factor</em>-9 (BMP-9), predominantly produced in the liver, were shown to lead to activation of BAT thermogenesis, as well as to "browning" of WAT. This was also observed in response to irisin, a hormone secreted by skeletal muscles. Different approaches were used to delineate the impact of UCP1 on insulin sensitivity. When studied under thermoneutral conditions, UCP1 knockout mice exhibited markedly increased metabolic efficiency due to impaired thermogenesis. The impact of UCP1 deletion on insulin sensitivity in these mice was not reported. Conversely, several studies in both rodents and humans have shown that BAT activation (by cold exposure, β3-agonist treatment, transplantation and others) improves glucose tolerance and insulin sensitivity. Interestingly, similar results were obtained by adipose tissue-specific overexpression of PR-domain-containing <em>16</em> (PRDM<em>16</em>) or BMP4 in mice. The mediators of such beneficial effects seem to include FGF21, interleukin-6, BMP8B and prostaglandin D2 synthase. Interestingly, some of these molecules can be secreted by BAT itself, indicating the occurrence of autocrine effects. Stimulation of BAT activity and/or recruitment of UCP1-positive cells are therefore relevant targets for the treatment of obesity/type 2 diabetes in humans.

KRAS activation and PTEN inactivation are frequent events in endometrial tumorigenesis, occurring in 10% to 30% and 26% to 80% of endometrial cancers, respectively. Because we have recently shown activating mutations in <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 (FGFR2) in <em>16</em>% of endometrioid endometrial cancers, we sought to determine the genetic context in which FGFR2 mutations occur. Analysis of 1<em>16</em> primary endometrioid endometrial cancers revealed that FGFR2 and KRAS mutations were mutually exclusive, whereas FGFR2 mutations were seen concomitantly with PTEN mutations. Here, we show that shRNA knockdown of FGFR2 or treatment with a pan-FGFR inhibitor, PD173074, resulted in cell cycle arrest and induction of cell death in endometrial cancer cells with activating mutations in FGFR2. This cell death in response to FGFR2 inhibition occurred within the context of loss-of-function mutations in PTEN and constitutive AKT phosphorylation, and was associated with a marked reduction in extracellular signal-regulated kinase 1/2 activation. Together, these data suggest that inhibition of FGFR2 may be a viable therapeutic option in endometrial tumors possessing activating mutations in FGFR2, despite the frequent abrogation of PTEN in this cancer type.

A number of <em>factors</em> both stimulating and inhibiting angiogenesis have been described. In the current work, we demonstrate that the angiogenic <em>factor</em> vascular endothelial <em>growth</em> <em>factor</em> (VEGF) activates mitogen-activated protein kinase (MAPK) as has been previously shown for basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. The antiagiogenic <em>factor</em> <em>16</em>-kDa N-terminal fragment of human prolactin inhibits activation of MAPK distal to autophosphorylation of the putative VEGF receptor, Flk-1, and phospholipase C-gamma. These data show that activation and inhibition of MAPK may play a central role in the control of angiogenesis.

Collagenase-3 (MMP-13) is a novel matrix metalloproteinase, the expression of which has so far only been documented in human breast carcinomas and osteoarthritic cartilage. In this study we have examined the expression of MMP-13 during human fetal development. Northern blot hybridizations revealed abundant expression of MMP-13 mRNAs in total RNA from fetal cartilage and calvaria at gestational age of 15 weeks. By in situ hybridization MMP-13 transcripts were detected in chondrocytes of hypertrophic cartilage in vertebrae of the spinal column and in the dorsal end of ribs undergoing ossification, as well as in osteoblasts and periosteal cells below the inner periosteal region of ossified ribs. In contrast, no expression of MMP-13 could be detected in osteoclasts. Furthermore, expression of MMP-13 mRNA was detected in osteoblasts and <em>fibroblasts</em> primarily on the inner side of calvarial bone of the skull at <em>16</em> weeks of gestation. Expression of MMP-13 mRNA by primary human fetal chondrocytes in culture was enhanced by transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) and inhibited by bone morphogenetic protein-2 (BMP-2). No expression of MMP-13 mRNA could be noted in other fetal tissues, including the skin, lungs, neural tissue, muscle, and liver. These results suggest that MMP-13 plays an important role in the extracellular matrix remodeling during fetal bone development both via endochondral and intramembranous ossification.

Like other types of pre-malignant lesions and carcinoma, angiogenesis is associated with high-grade cervical dysplasia and with invasive squamous carcinoma of the cervix. Vascular endothelial cell <em>growth</em> <em>factor</em> (VEGF) is known to be one of the most important inducers of angiogenesis and is upregulated in carcinoma of the cervix. Human Papilloma Virus <em>16</em> (HPV-<em>16</em>) has been etiologically linked to human cervical cancer, and the major oncogenic proteins encoded by the viral genome, E6 and E7, are involved in the immortalization of target cells. Because several oncogenes including mutant ras, EGF receptor, ErbB2/Her2, c-myc and v-src upregulate VEGF expression, we asked whether HVP-<em>16</em> E6 oncoprotein could act in a similar fashion. We found that HPV-<em>16</em> E6-positive cells generally express high levels of VEGF message. Furthermore, co-expression of the VEGF promoter-Luc (luciferase) reporter gene with E6 in both human keratinocytes and mouse <em>fibroblast</em> showed that E6 oncoprotein upregulates VEGF promoter activity, and does so in a p53 independent manner. An E6 responsive region which comprises four Sp-1 sites, between -194 and -50 bp of the VEGF promoter, is also necessary for constitutive VEGF transcription. Taken together, our results suggest the possibility that the HPV oncoprotein E6 may contribute to tumor angiogenesis by direct stimulation of the VEGF gene.

OBJECTIVE

This study aimed to quantitate and compare the concentration of vascular endothelial growth factor (VEGF) in aqueous humor samples from patients with neovascular glaucoma (NVG), primary open-angle glaucoma (POAG), and cataract, as well as in serum samples of healthy human subjects.

METHODS

The authors collected aqueous humor samples by using their previously published technique of limbal paracentesis. The authors determined the concentration of VEGF by using a competitive enzyme immunoassay system and four-parameter logistic curve fitting and performed statistical analysis by using the Mann-Whitney-Wilcoxon test.

RESULTS

The authors detected VEGF in 12 of 12 samples from patients with NVG (mean +/- standard error of the mean, 29.267 +/- 7.350 ng/ml), 15 of 28 samples from patients with POAG (0.726 +/- 0.204 ng/ml), 4 of 20 aqueous humor samples from patients with cataract (0.257 +/- 0.043 ng/ml), and 16 of 16 human serum samples (20.246 +/- 1.568 ng/ml). The mean concentration of VEGF in aqueous humor of patients with NVG was 40- and 113-fold higher than that in patients with POAG and cataract, respectively, and the difference was statistically significant (P < 0.01). The VEGF level in patients with POAG was elevated compared with that in patients with cataract (P < 0.05). Although the mean concentration of VEGF in aqueous humor of patients with NVG was approximately 1.45-fold higher than that in serum, the difference was not significant (P>> 0.05).

CONCLUSIONS

The authors' findings show that patients with NVG had a significantly increased level of VEGF in the aqueous humor and implicate VEGF as an important factor in the pathogenesis of intraocular neovascularization in these patients. The authors discuss the possible role of the ciliary epithelium, in addition to retina, in the production of VEGF and the complementary function of basic fibroblast growth factor and other growth factors.

<AbstractText>Pegbelfermin (BMS-986036), a PEGylated human <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 (FGF21) analogue, has previously been shown to improve markers of metabolism and liver fibrosis in obese patients with type 2 diabetes. In this phase 2a study, we aimed to evaluate the safety and efficacy of pegbelfermin in patients with non-alcoholic steatohepatitis.</AbstractText><p><div><b>METHODS</b></div>In this multicentre, randomised, double-blind, placebo-controlled, parallel-group, phase 2a study, we recruited adults (aged 21-75 years) with a body-mass index of at least 25 kg/m², biopsy-confirmed non-alcoholic steatohepatitis (fibrosis stage 1-3), and a hepatic fat fraction of at least 10% when assessed by magnetic resonance imaging-proton density fat fraction. These patients were enrolled at 17 medical centres in the USA. Eligible patients were stratified by type 2 diabetes status and they were randomly assigned (1:1:1) by a computer-based system to receive subcutaneous injections of placebo once a day, 10 mg pegbelfermin once a day, or 20 mg pegbelfermin once a week, all for <em>16</em> weeks. Participants, the study team administering treatment, and investigators analysing outcomes (who were independent of the study team and had no further involvement) were masked to treatment groups. The primary outcomes were safety and the absolute change in hepatic fat fraction after <em>16</em> weeks of treatment. All patients who were randomly assigned to groups and received the study drug or placebo were included in the primary analyses. This trial was registered with ClinicalTrials.gov, number NCT02413372.</p><AbstractText>Between May 12, 2015, and Aug 4, 20<em>16</em>, 184 overweight or obese patients with non-alcoholic steatohepatitis were screened for study inclusion. Of these, 95 (52%) patients were excluded because they no longer met study criteria and 80 (43%) patients entered the placebo lead-in phase. After further exclusions, 75 (94%) patients were randomly assigned to groups, received at least one dose of treatment (25 patients to receive 10 mg pegbelfermin once a day; 24 patients to receive 20 mg pegbelfermin once a week, and 26 patients to receive placebo), and were included in the primary analysis. A prespecified interim analysis at week 8 showed a greater than expected change in the primary outcome and supported early closing of patient enrolment, since this analysis indicated that the full planned sample size was not needed. We observed a significant decrease in absolute hepatic fat fraction in the group receiving 10 mg pegbelfermin daily (-6·8% vs -1·3%; p=0·0004) and in the group receiving 20 mg pegbelfermin weekly (-5·2% vs -1·3%; p=0·008) compared with the placebo group. Most adverse events were mild; the most common events were diarrhoea in eight (<em>16</em>%) of 49 patients treated with pegbelfermin and two (8%) of 26 patients treated with placebo and nausea in seven (14%) patients treated with pegbelfermin and two (8%) patients treated with placebo. There were no deaths, discontinuations due to adverse events, or treatment-related serious adverse events.</AbstractText><AbstractText>Treatment with subcutaneously administered pegbelfermin for <em>16</em> weeks was generally well tolerated and significantly reduced hepatic fat fraction in patients with non-alcoholic steatohepatitis. Further study of pegbelfermin is warranted in patients with non-alcoholic steatohepatitis. Additional studies that use liver biopsies would allow for the assessment of pegbelfermin's effects on liver histology. Moreover, further studies should allow assessments of the safety and effectiveness of pegbelfermin in a larger number of patients.</AbstractText><AbstractText>Bristol-Myers Squibb.</AbstractText>

Human corneal endothelial cells (HCEC) were isolated by means of enzymatic treatment of excised corneas. The corneas were incubated for 1.5 hr together with a high concentration of collagenase (0.5%), followed by a long-term incubation (up to <em>16</em> hr) using a low concentration of the enzyme (0.04%). Endothelial cells were enriched against contaminating <em>fibroblasts</em> by using a selective L-valine-free medium which inhibited <em>fibroblast</em> <em>growth</em> during the first passages. Subcultures of HCEC were passaged for more than 20 generations without showing signs of senescence. Laminin and chondroitin sulfate functioned as a substrate for HCEC, promoting proliferation and allowing the cells to grow in monolayer formation. The inclusion of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) as well as chondroitin sulfate in the medium led to an additional increase in the rate of proliferation.

Tumour <em>growth</em> is angiogenesis-dependent; brain tumours have more intense neovascularisation than other tumours and produce basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, a potent angiogenic mediator. Because little is known about the release of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> from brain tumours into extracellular fluids, we tested cerebrospinal fluid (CSF) from 26 children and young adults with brain tumours and 18 controls for basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and for proliferative activity on cultured capillary endothelial cells. We also measured the density of microvessels in tumours by immunohistochemical staining. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> was detected in the CSF of 62% (<em>16</em> of 26) patients with brain tumours but in none of the controls. Specimens with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> stimulated DNA synthesis of capillary endothelial cells in vitro. Endothelial proliferative activity was blocked by neutralising antibodies to basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> correlated with mitogenic activity in CSF in vitro (p < or = 0.0001), and with density of microvessels in histological sections (p < or = 0.005). A microvessel count of>> or = 68 per 200 x field was associated with tumour recurrence (p = 0.005) and with mortality (p = 0.02). Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> in brain tumours may mediate angiogenesis as measured by microvessel density in histological sections, so has potential as both a marker for neoplasia and a target for tumour treatments. Furthermore, evaluation of cerebrospinal fluid basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, along with microvessel quantitation in biopsied tumours, may provide improved prognostic information for the management of patients with brain tumours.

The BCR/ABL fusion protein transforms myeloid stem cells. Both chronic myelogenous leukemias (CML) and a subset of acute lymphoblastic leukemias (ALL) are associated with the expression of BCR/ABL proteins. This knowledge has not yet been translated into any specific tool to control ABL driven neoplastic cells <em>growth</em>. CGP57148B is an ATP-competitive inhibitor of the ABL protein kinase; it has been shown to inhibit the kinase activity of ABL both in vitro and in vivo and to inhibit the <em>growth</em> of v-abl and bcr/abl transfectants, as well as the in vitro formation of bone marrow (BM)-derived colonies in the presence of <em>growth</em> <em>factors</em> in some CML patients. These studies were performed to investigate the activity of CGP57148B on the spontaneous proliferation of both fresh and cultured, leukemic and normal, BCR/ABL positive and negative cells, and to study its mechanism of action. Six cell lines derived from BCR/ABL+ leukemias (K562, BV173, KCL22, KU812, MC3, LAMA84), thirteen BCR/ABL negative lines, both neoplastic (KG1, SU-DHL-1, U937, Daudi, NB4, NB4.306) and derived from normal cells (PHA blasts, LAK, <em>fibroblasts</em>, LCL, renal epithelial cells, endothelial cells, CD34(+) cells), and 14 fresh leukemic samples were tested using a tritiated thymidine uptake assay. The in vivo phosphorylation of the BCR/ABL protein was evaluated by western blot, while apoptosis was detected by the annexin V/propidium binding test. The induction of differentiation was assayed by immunofluorescence using multiple antibodies. All six BCR/ABL+ lines showed a dose dependent inhibition of their spontaneous proliferative rate, which was not accompanied by differentiation. The treatment caused, within minutes, dephosphorylation of the BCR/ABL protein, followed in <em>16</em>-24 hours by a decrease in cycling cells and induction of apoptosis. No significant inhibition of DNA synthesis was observed in any BCR/ABL negative normal or neoplastic line at concentrations </=3 microM, with the exception of <em>fibroblasts</em> and CD34 cells. Proliferation inhibition was observed also when using fresh samples obtained from two Ph+ ALL and 12 consecutive CML patients. Induction of apoptosis was observed in these samples too. The activity of CGP57148B can be monitored in ex vivo isolated or cultured cells using a simple and reproducible assay, without the need for exogenously added <em>growth</em> <em>factors</em>. This molecule possibly exerts its effects through the inhibition of the kinase activity of BCR/ABL and the subsequent initiation of apoptosis, without inducing cell differentiation. Some normal cells are also affected. These data support the use of CGP57148B in initial clinical studies; possible toxic effects on BM and <em>fibroblast</em>-derived cells will have to be closely monitored. The in vivo monitoring of patients will have to be focused on the induction of apoptosis in leukemic cells.

OBJECTIVE

Stroke is a cerebrovascular defect that leads to many adverse neurological complications. Current pharmacological treatments for stroke remain unclear in their effectiveness, whereas stem cell transplantation shows considerable promise. Previously, we have shown that human umbilical mesenchymal stem cells (HUMSCs) can differentiate into neurons in neuronal-conditioned medium. Here we evaluate the therapeutic potential of HUMSC transplantation for ischemic stroke in rats.

METHODS

Focal cerebral ischemia was produced by middle cerebral artery occlusion and reperfusion. The HUMSCs treated with neuronal-conditioned medium or not treated were transplanted into the ischemic cortex 24 hours after surgery.

RESULTS

Histology and MRI revealed that rats implanted with HUMSCs treated with neuronal-conditioned medium or not treated exhibited a trend toward less infarct volume and significantly less atrophy compared with the control group, which received no HUMSCs. Moreover, rats receiving HUMSCs showed significant improvements in motor function, greater metabolic activity of cortical neurons, and better revascularization in the infarct cortex. Implanted HUMSCs, treated or not treated, survived in the infarct cortex for at least 36 days and released neuroprotective and <em>growth</em>-associated cytokines, including brain-derived neurotrophic <em>factor</em>, platelet-derived <em>growth</em> <em>factor</em>-AA, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, angiopoietin-2, CXCL-<em>16</em>, neutrophil-activating protein-2, and vascular endothelial <em>growth</em> <em>factor</em> receptor-3.

CONCLUSIONS

Our results demonstrate the therapeutic benefits of HUMSC transplantation for ischemic stroke, likely due to the ability of the cells to produce growth-promoting factors. Thus, HUMSC transplantation may be an effective therapy in the future.

BACKGROUND

Vascular endothelial growth factor (VEGF) is a proangiogenic molecule produced by melanoma cells. We hypothesized that administration of bevacizumab (Bev), a monoclonal antibody that neutralizes VEGF, with low-dose interferon alfa-2b (IFN-alpha2b), an inhibitor of basic fibroblast growth factor (FGF), would lead to the regression of metastatic melanoma.

METHODS

Patients with metastatic melanoma were randomized to receive Bev (15 mg/kg intravenously every 2 weeks) with or without low-dose IFN-alpha2b (1 MU/m2 subcutaneously daily). Patients exhibiting a clinical response or stable disease after 12 weeks were treated until disease progression.

RESULTS

Thirty-two patients (16 per arm) were accrued (18 male, 14 female; mean age 57.5 years). Both regimens were well tolerated. Six patients developed easily managed exacerbations of preexisting hypertension. Two patients developed grade 3 proteinuria that resolved after a treatment break. IFN-alpha2b therapy was associated with grade 1 to 2 constitutional symptoms. Arterial thromboembolic complications were observed in three patients (two mild myocardial infarctions, one transient ischemic attack), all of whom had risk factors. One patient (Bev plus IFN-alpha2b arm) had locally recurrent scalp disease that partially responded to therapy. Eight patients (five Bev, three Bev plus IFN-alpha2b) had prolonged disease stabilization (24 to 146 weeks). Plasma levels of VEGF and FGF did not correlate with any clinical parameter. The patient with the longest period of stable disease had the highest baseline VEGF and FGF.

CONCLUSIONS

Bev was well tolerated at this dose and prolonged disease stabilization was achieved in one-quarter of metastatic melanoma patients. Low-dose IFN-alpha2b did not augment the activity of Bev.

Previously we reported the purification of the heparin-binding <em>growth</em> <em>factor</em> pleiotrophin (PTN) from supernatants of the human breast cancer cell line MDA-MB-231. To investigate further the biological activities of PTN and its potential role in cancer, we cloned a PTN cDNA and expressed the gene in a human kidney and in a human adrenal carcinoma cell line (SW-13). The supernatants harvested from cells transfected with PTN contained a heparin-binding specific protein of an apparent molecular mass of 18 kDa. These supernatants stimulated the proliferation of endothelial cells as well as the anchorage-independent <em>growth</em> of SW-13 cells and of normal rat kidney <em>fibroblasts</em>. Furthermore, SW-13 cells transfected with PTN acquired autonomous <em>growth</em> in soft agar and were tumorigenic in athymic nude mice. In contrast to these results with PTN from human cells, PTN obtained from insect cells (Sf9) using recombinant baculovirus as a vector was biologically inactive. We detected high levels of PTN mRNA in <em>16</em> of 27 primary human breast cancer samples (62%) as well as in 8 of 8 carcinogen-induced rat mammary tumors. Furthermore, 9 of 34 human tumor cell lines of different origin showed detectable PTN mRNA. We conclude that PTN may function as a tumor <em>growth</em> and angiogenesis <em>factor</em> in addition to its role during embryonic development.

OBJECTIVE

To relate the oxygen environment of the retina to photoreceptor stability, protection, and function in the P23H rat.

METHODS

Heterozygote P23H-3 (Line 3) rats were studied. Photoreceptor death rates were assessed with the TUNEL technique for detection of fragmenting DNA, in a developmental series from postnatal day (P)<em>16</em> to P105 (adult). In adult retinas, trophic <em>factor</em> status was assessed with immunohistochemistry, intraretinal oxygen environment with O(2)-sensing electrodes, and photoreceptor function by the flash-evoked, dark-adapted electroretinogram (ERG), recorded in anesthetized animals.

RESULTS

Photoreceptor death begins by P<em>16</em>; peaks at P25, when the frequency of TUNEL(+) profiles exceeds 70/mm of retina; and then declines to low (<5/mm) adult rates. Compared with that in nondegenerative Sprague-Dawley (SD) rats, the rate of photoreceptor death is abnormally high from P<em>16</em> and remains several-fold higher than normal into young adulthood. In addition, the outer nuclear layer is reduced to approximately half of control thickness, and the levels of ciliary neurotrophic <em>factor</em> (CNTF), glial fibrillary acidic protein (GFAP), fibroblast growth <em>factor</em> (FGF)-2, and FGF-2/FGFR1 colocalization are markedly upregulated. O(2) tension and uptake are relatively normal in the inner retina, but uptake is considerably reduced, and O(2) tension is significantly raised in the outer retina. Surviving photoreceptors generate an a-wave with normal peak latency but sharply reduced amplitude.

CONCLUSIONS

Excess photoreceptor degeneration in the P23H-3 retina begins just after eye opening, peaks in early postnatal life, and then slows, but persists into adulthood. In the adult retina, surviving photoreceptors operate in an environment that is chronically hyperoxic (and therefore toxic) and in which protective factors (CNTF, FGF-2) are chronically upregulated. The net result, slow degeneration and degraded function in an environment that is both toxic and protective, may be representative of adult photoreceptor status in a number of human retinal degenerations. Hyperoxia-induced photoreceptor death may be a self-reinforcing factor that increases oxidative stress in surviving photoreceptors.

We recently reported <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor-type 1 (FGFR1) amplification to be associated with therapeutically tractable FGFR1 dependency in squamous cell lung cancer. This makes FGFR1 a novel target for directed therapy in these tumors. To reproducibly identify patients for clinical studies, we developed a standardized reading and evaluation strategy for FGFR1 fluorescence in-situ hybridization (FISH) and propose evaluation criteria, describe different patterns of low- and high-level amplifications and report on the prevalence of FGFR1 amplifications in pulmonary carcinomas. A total of 420 lung cancer patients including 307 squamous carcinomas, 100 adenocarcinomas of the lung and 13 carcinomas of other types were analyzed for FGFR1 amplification using a dual color FISH. We found heterogeneous and different patterns of gene copy numbers. FGFR1 amplifications were observed in 20% of pulmonary squamous carcinomas but not in adenocarcinomas. High-level amplification (as defined by an FGFR1/centromer 8 (CEN8) ratio ≥2.0, or average number of FGFR1 signals per tumor cell nucleus ≥6, or the percentage of tumor cells containing ≥15 FGFR1 signals or large clusters ≥10%) was detected at a frequency of <em>16</em>% and low-level amplification (as defined by ≥5 FGFR1 signals in ≥50% of tumor cells) at a frequency of 4%. We conclude that FGFR1 amplification is one of the most frequent therapeutically tractable genetic lesions in pulmonary carcinomas. Standardized reporting of FGFR1 amplification in squamous carcinomas of the lung will become increasingly important to correlate therapeutic responses with FGFR1 inhibitors in clinical studies. Thus, our reading and evaluation strategy might serve as a basis for identifying patients for ongoing and upcoming clinical trials.

The expression of the gene encoding the facilitated glucose transporter (GT) protein was studied in <em>fibroblast</em> cell lines. Addition of 15% calf serum to confluent BALB/c3T3, NIH3T3, or Rat-2 cells rapidly induced a 5-10-fold increase in GT mRNA, as determined by hybridization of size-fractionated total RNA to a rat brain GT cDNA. The rise in GT mRNA was maximal at 3-4 h after stimulation, and then returned to basal values by <em>16</em> h. The serum-stimulated increase in GT mRNA was not blocked by the protein synthesis inhibitors cycloheximide (10 micrograms/ml) or anisomycin (100 microM). In BALB/c3T3 cells, <em>fibroblast</em> <em>growth</em> <em>factor</em> (100 ng/ml), platelet-derived <em>growth</em> <em>factor</em> (5 units/ml), and epidermal <em>growth</em> <em>factor</em> (40 ng/ml) stimulated GT mRNA accumulation, although, when added individually, none of these <em>growth</em> <em>factors</em> increased DNA synthesis. The tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), which activates the enzyme protein kinase C, also caused GT mRNA accumulation in BALB/c3T3 and NIH3T3 cells. Prolonged pretreatment of cells with TPA abolished the response to TPA but not <em>fibroblast</em> <em>growth</em> <em>factor</em>. The involvement of GT gene transcription was assessed by the nuclear run-on technique. Treatment of NIH3T3 cells with serum increased transcription at least 10-20-fold by 30 min and returned to near basal levels by 2 h. This rapid activation paralleled that of the c-fos gene, but preceded the increase in c-myc gene transcription. These data indicate the following: 1) serum <em>growth</em> <em>factors</em> increase glucose transporter mRNA levels by a process not requiring intermediary new protein synthesis and clearly dissociable from mitogenesis, 2) the changes in GT mRNA are preceded by a rapid and transient activation of GT gene transcription, and 3) there exist protein kinase C-dependent and independent pathways for regulation of GT gene expression.

Arterial medial calcification (AMC), a hallmark of vascular disease in uremic patients, is highly correlated with serum phosphate levels and cardiovascular mortality. To determine the mechanisms of AMC, mice were made uremic by partial right-side renal ablation (week 0), followed by left-side nephrectomy at week 2. At 3 weeks, mice were switched to a high-phosphate diet, and various parameters of disease progression were examined over time. Serum phosphate, calcium, and <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF-23) were up-regulated as early as week 4. Whereas serum phosphate and calcium levels declined to normal by 10 weeks, FGF-23 levels remained elevated through <em>16</em> weeks, consistent with an increased phosphate load. Elastin turnover and vascular smooth muscle cell (VSMC) phenotype change were early events, detected by week 4 and before AMC. Both AMC and VSMC loss were significantly elevated by week 8. Matrix metalloprotease 2 (MMP-2) and cathepsin S were present at baseline and were significantly elevated at weeks 8 and 12. In contrast, MMP-9 was not up-regulated until week 12. These findings over time suggest that VSMC phenotype change and VSMC loss (early phosphate-dependent events) may be necessary and sufficient to promote AMC in uremic mice fed a high-phosphate diet, whereas elastin degradation might be necessary but is not sufficient to induce AMC (because elastin degradation occurred also in uremic mice on a normal-phosphate diet, but they did not develop AMC).

Bile acids activate farnesoid X receptor (FXR) and G protein-coupled bile acid receptor-1 (aka Takeda G protein-coupled receptor-5 [TGR5]) to regulate bile acid metabolism and glucose and insulin sensitivity. FXR and TGR5 are coexpressed in the enteroendocrine L cells, but their roles in integrated regulation of metabolism are not completely understood. We reported recently that activation of FXR induces TGR5 to stimulate glucagon-like peptide-1 (GLP-1) secretion to improve insulin sensitivity and hepatic metabolism. In this study, we used the intestine-restricted FXR agonist fexaramine (FEX) to study the effect of activation of intestinal FXR on the gut microbiome, bile acid metabolism, and FXR and TGR5 signaling. The current study revealed that FEX markedly increased taurolithocholic acid, increased secretion of <em>fibroblast</em> <em>growth</em> <em>factors</em> 15 and 21 and GLP-1, improved insulin and glucose tolerance, and promoted white adipose tissue browning in mice. Analysis of <em>16S</em> ribosomal RNA sequences of the gut microbiome identified the FEX-induced and lithocholic acid-producing bacteria Acetatifactor and Bacteroides. Antibiotic treatment completely reversed the FEX-induced metabolic phenotypes and inhibited taurolithocholic acid synthesis, adipose tissue browning, and liver bile acid synthesis gene expression but further increased intestinal FXR target gene expression. FEX treatment effectively improved lipid profiles, increased GLP-1 secretion, improved glucose and insulin tolerance, and promoted adipose tissue browning, while antibiotic treatment reversed the beneficial metabolic effects of FEX in obese and diabetic mice.

CONCLUSIONS

This study uncovered a mechanism in which activation of intestinal FXR shaped the gut microbiota to activate TGR5/GLP-1 signaling to improve hepatic glucose and insulin sensitivity and increase adipose tissue browning; the gut microbiota plays a critical role in bile acid metabolism and signaling to regulate metabolic homeostasis in health and disease. (Hepatology 2018).

BACKGROUND

We have shown that the angiogenic peptides basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) enhance canine coronary collateral development when administered for>> or = 4 weeks. bFGF, a pluripotent mitogen of mesodermally derived cells, could theoretically exacerbate neointimal smooth muscle cell hyperplasia, a fundamental component of atherosclerosis. VEGF, an endothelial cell-specific mitogen and vascular permeability factor, could have deleterious effects related to vascular hyperpermeability. The present investigation had two aims: (1) to ascertain whether brief (7-day) systemic arterial treatment with bFGF or VEGF would improve myocardial collateral perfusion and (2) to determine whether these peptides induce neointimal accumulation in vivo.

RESULTS

Dogs were subjected to ameroid-induced occlusion of the left circumflex coronary artery and randomized to bFGF 1.74 mg (n = 9), VEGF 0.72 mg (n = 9), or saline (n = 10) as a daily left atrial bolus (days 10 to 16). Additional dogs were randomized to VEGF 0.72 mg (n = 6) or saline (n = 5); however, treatment was delayed by 1 week. Coincident with the institution of treatment, all dogs underwent balloon denudation injury of the iliofemoral artery. bFGF markedly increased maximal collateral flow but did not exacerbate neointimal accumulation. VEGF had no discernible effect on maximal collateral flow, but it exacerbated neointimal thickening after vascular injury.

CONCLUSIONS

Short-term treatment with bFGF enhanced collateral development without increasing neointimal accumulation at sites of vascular injury. Although VEGF did not increase collateral development as administered in this study, it significantly exacerbated neointimal accumulation. These data provide support for the clinical investigation of bFGF in selected patients with ischemic heart disease.

OBJECTIVE

In COPD, the airways are chronically inflamed, and we have now observed fragmentation of the reticular basement membrane (Rbm). This appears to be a hallmark of the process known as epithelial mesenchymal transition (EMT), in which epithelial cells migrate through the Rbm and differentiate into fibroblasts. The aim of this study was to confirm the extent and relevance of Rbm fragmentation in smokers and patients with COPD, and to undertake a preliminary analysis of some classical markers of EMT.

METHODS

Endobronchial biopsies from current smokers (CS; n = 17) and ex-smokers with COPD (ES; n = 15), smokers with normal lung function (NS; n = 16) and never-smoking control subjects (NC; n = 15) were stained for the EMT markers, S100A4, vimentin, epidermal growth factor receptor and matrix metalloproteinase-9.

RESULTS

Compared with NC, there was significant Rbm fragmentation in the CS, ES and NS groups, which was positively associated with smoking history in subjects with COPD. Staining for basal epithelial S100A4, epithelial epidermal growth factor receptor and matrix metalloproteinase-9 in cells within Rbm clefts, and for S100A4 in Rbm cells, was increased in the CS, NS and ES groups compared with the NC group. There was also increased Rbm cell S100A4 staining in the CS group compared with the ES and NS groups. Basal epithelial cell staining for S100A4 was inversely correlated with airflow limitation. Double staining for both S100A4 and vimentin further strengthened the likelihood that these changes represented active EMT.

CONCLUSIONS

This is the first detailed description of fragmentation and cellularity of the Rbm in smokers, which were most marked in subjects with COPD. The data are consistent with active EMT in these subjects.

OBJECTIVE

The fibroblast growth factor receptor (FGFR)-3 fusion genes have been recently demonstrated in a subset of non-small cell lung cancer (NSCLC). To aid in identification and treatment of these patients, we examined the frequency, clinicopathologic characteristics, and treatment outcomes of patients who had NSCLC with or without FGFR fusions.

METHODS

Fourteen known FGFR fusion variants, including FGFR1, FGFR2, and FGFR3, were detected by RT-PCR and verified by direct sequencing in 1,328 patients with NSCLC. All patients were also analyzed for mutations in EGFR, KRAS, HER2, BRAF, ALK, RET, and ROS1. Clinical characteristics, including age, sex, smoking status, stage, subtypes of lung adenocarcinoma, relapse-free survival, and overall survival, were collected.

RESULTS

Of 1,328 tumors screened, two (0.2%) were BAG4-FGFR1 fusion and 15 (1.1%) were FGFR3-TACC3 fusion. Six of 1,016 patients with lung adenocarcinoma were FGFR3-TACC3 fusions and 11 of 312 lung squamous cell carcinoma harbored BAG4-FGFR1 or FGFR3-TACC3 fusions. Compared with the FGFR fusion-negative group, patients with FGFR fusions were more likely to be smokers (94.1%, 16 of 17 patients, P < 0.001), significantly associated with larger tumor (>3 cm; 88.2%, 15 of 17 patients, P < 0.001) and with a tendency to be more poorly differentiated (53.9%, nine of 17 patients, P = 0.095).

CONCLUSIONS

FGFR fusions define a molecular subset of NSCLC with distinct clinical characteristics. FGFR is a druggable target and patients with FGFR fusions may benefit from FGFR-targeted therapy, which needs further clinical investigation.