Low-carbohydrate/high-fat diets (LC-HFDs) in rodent models have been implicated with both weight loss and as a therapeutic approach to treat neurological diseases. LC-HFDs are known to induce ketosis; however, systematic studies analyzing the impact of the macronutrient composition on ketosis induction and weight loss success are lacking. Male Wistar rats were pair-fed for 4 wk either a standard chow diet or one of three different LC-HFDs, which only differed in the relative abundance of fat and protein (percentages of fat/protein in dry matter: LC-75/10; LC-65/<em>20</em>; LC-55/30). We subsequently measured body composition by nuclear magnetic resonance (NMR), analyzed blood chemistry and urine acetone content, evaluated gene expression changes of key ketogenic and gluconeogenic genes, and measured energy expenditure (EE) and locomotor activity (LA) during the first 4 days and after 3 wk on the respective diets. Compared with chow, rats fed with LC-75/10, LC-65/<em>20</em>, and LC-55/30 gained significantly less body weight. Reductions in body weight were mainly due to lower lean body mass and paralleled by significantly increased fat mass. Levels of β-hydroxybutyate were significantly elevated feeding LC-75/10 and LC-65/<em>20</em> but decreased in parallel to reductions in dietary fat. Acetone was about 16-fold higher with LC-75/10 only (P < 0.001). In contrast, rats fed with LC-55/30 were not ketotic. Serum <em>fibroblast</em> <em>growth</em> <em>factor</em>-21, hepatic mRNA expression of hydroxymethylglutaryl-CoA-lyase, peroxisome proliferator-activated receptor-γ coactivator-1α, and peroxisome proliferator-activated receptor-γ coactivator-1β were increased with LC-75/10 only. Expression of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase was downregulated by 50-70% in LC-HF groups. Furthermore, EE and LA were significantly decreased in all groups fed with LC-HFDs after 3 wk on the diets. In rats, the absence of dietary carbohydrates per se does not induce ketosis. LC-HFDs must be high in fat, but also low in protein contents to be clearly ketogenic. Independent of the macronutrient composition, LC-HFD-induced weight loss is not due to increased EE and LA.

Classical radiation pneumonitis has been described after single dose whole lung irradiation in experimental animals where above a threshold dose of irradiation, there is a sigmoid dose response curve with increasing morbidity and mortality. After clinical fractionated irradiation, however, acute radiation pneumonitis consisting of cough shortness of breath and patchy radiological changes, occurs in <10% of patients, has dyspnoea out of proportion to the volume of lung irradiated and usually resolves completely without long-term effects. There is increasing evidence that this represents a bilateral lymphocytic alveolitis or hypersensitivity pneumonitis and has been termed sporadic pneumonitis. Late radiation toxicity results in pulmonary fibrosis. This is a consequence of repair, which is initiated by tissue injury within the radiation portal. It follows release of chemotactic <em>factors</em> for <em>fibroblasts</em> including transforming <em>growth</em> <em>factor</em>-beta, fibronectin and platelet derived <em>growth</em> <em>factor</em>. Radiation fibrosis is the clinically more significant syndrome for patients. It may result in progressive dyspnoea and mortality in patients. The most predictable change in laboratory lung function tests is a decrease in transfer <em>factor</em> due to damage at the capillary-alveolar level. It also results in decreased lung compliance, which will affect the total lung capacity and the forced vital capacity. The forced expiratory volume in 1 s is less affected, although this seems to depend on the volume of lung irradiated. There is also a decrease in perfusion in the irradiated lung. Radiation fibrosis seems to depend, amongst other <em>factors</em>, on the volume of lung, which is irradiated above a threshold of <em>20</em>-30 Gy. The morbidity of radiation fibrosis may therefore be minimized by the use of dose volume histogram to minimize the volume of normal lung irradiated in patients at high risk, e.g., patients with who present with poor lung function. The importance of the baseline perfusion in the irradiated areas continues to be studied.

The aim of the present study was to examine whether hypoxia preconditioning could improve therapeutic effects of adipose derived mesenchymal stem cells (AMSCs) for diabetes induced erectile dysfunction (DED). AMSCs were pretreated with normoxia (<em>20</em>% O2, N-AMSCs) or sub-lethal hypoxia (1% O2, H-AMSCs). The hypoxia exposure up-regulated the expression of several angiogenesis and neuroprotection related cytokines in AMSCs, including vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and its receptor FIK-1, angiotensin (Ang-1), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), brain-derived neurotrophic <em>factor</em> (BDNF), glial cell-derived neurotrophic <em>factor</em> (GDNF), stromal derived <em>factor</em>-1 (SDF-1) and its CXC chemokine receptor 4 (CXCR4). DED rats were induced via intraperitoneal injection of streptozotocin (60 mg/kg) and were randomly divided into three groups-Saline group: intracavernous injection with phosphate buffer saline; N-AMSCs group: N-AMSCs injection; H-AMSCs group: H-AMSCs injection. Ten rats without any treatment were used as normal control. Four weeks after injection, the mean arterial pressure (MAP) and intracavernosal pressure (ICP) were measured. The contents of endothelial, smooth muscle, dorsal nerve in cavernoursal tissue were assessed. Compared with N-AMSCs and saline, intracavernosum injection of H-AMSCs significantly raised ICP and ICP/MAP (p<0.05). Immunofluorescent staining analysis demonstrated that improved erectile function by MSCs was significantly associated with increased expression of endothelial markers (CD31 and vWF) (p<0.01) and smooth muscle markers (α-SMA) (p<0.01). Meanwhile, the expression of nNOS was also significantly higher in rats receiving H-AMSCs injection than those receiving N-AMSCs or saline injection. The results suggested that hypoxic preconditioning of MSCs was an effective approach to enhance their therapeutic effect for DED, which may be due to their augmented angiogenesis and neuroprotection.

OBJECTIVE

To determine the safe dose range and pharmacokinetics of metronomic oral vinorelbine and obtain preliminary data on biomarkers and efficacy in patients with advanced cancer.

METHODS

Successive cohorts of patients received escalated doses of oral vinorelbine given thrice a week until disease progression, unacceptable toxicity (UT), or consent withdrawal. UT was any grade 4 toxicity, or grade 2 or 3 toxicity that would result to longer than 2-week break during the first 2 months of treatment. Blood samples were collected for pharmacokinetics and quantification of angiogenesis regulatory proteins.

RESULTS

Sixty-two patients (median age, 60 years) enrolled at six dose levels from <em>20</em> to 70 mg and received treatment for median 12.25 weeks (range, 2-216+). Unacceptable toxicity occurred in two of six patients treated at 60 mg (leucopenia grade 4 and epistaxis grade 2) and in one at 70 mg (leucopenia grade 2). The upper metronomic dose was 50 mg. Objective antitumor response documented in eight cases and 32% of patients experienced disease stability for minimum 6 months. Three responders (renal cancer, medullary thyroid carcinoma, and Kaposi sarcoma) received nonstop treatment for over 3 years without overt toxicity. Low pretreatment levels of circulating interleukin-8, vascular endothelial <em>growth</em> <em>factor</em>, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> were found predictors of efficacy. Steady-state concentrations of vinorelbine and its active metabolite ranged from 0.5 to 1.5 ng/mL.

CONCLUSIONS

Metronomic administration of oral vinorelbine is feasible at doses up to 50 mg thrice a week and can yield sustainable antitumor activity without overt toxicity, probably through antiangiogenic mechanism. Further clinical investigation is warranted.

Craniosynostosis describes conditions in which one or more sutures of the infant skull are prematurely fused, resulting in facial deformity and delayed brain development. Approximately <em>20</em>% of human craniosynostoses are thought to result from gene mutations altering <em>growth</em> <em>factor</em> signaling; however, the molecular mechanisms by which these mutations cause craniosynostosis are incompletely characterized, and the causative genes for diverse types of syndromic craniosynostosis have yet to be identified. Here, we show that enhanced bone morphogenetic protein (BMP) signaling through the BMP type IA receptor (BMPR1A) in cranial neural crest cells, but not in osteoblasts, causes premature suture fusion in mice. In support of a requirement for precisely regulated BMP signaling, this defect was rescued on a Bmpr1a haploinsufficient background, with corresponding normalization of Smad phosphorylation. Moreover, in vivo treatment with LDN-193189, a selective chemical inhibitor of BMP type I receptor kinases, resulted in partial rescue of craniosynostosis. Enhanced signaling of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) pathway, which has been implicated in craniosynostosis, was observed in both mutant and rescued mice, suggesting that augmentation of FGF signaling is not the sole cause of premature fusion found in this model. The finding that relatively modest augmentation of Smad-dependent BMP signaling leads to premature cranial suture fusion suggests an important contribution of dysregulated BMP signaling to syndromic craniosynostoses and potential strategies for early intervention.

BACKGROUND

Chronic uremia is considered a proinflammatory state associated with high cardiovascular morbidity and mortality. The aim of the present study is to evaluate the potential relationship between the prevalence of coronary artery calcification (CAC) and selected factors that may be involved in the process of atherogenesis (lipid profile, acute-phase reactants, growth factors, and cytokines).

METHODS

The study group consisted of 43 patients (19 women, 24 men) with a mean age of 50.6 +/- 13.4 years treated with peritoneal dialysis (PD) for a median period of 15 months (range, 2 to 96 months). Only patients with sinus rhythm were included. CAC score (CaSc) was measured using multirow spiral computed tomography (MSCT). As parameters of lipid profile, total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides were assayed. C-reactive protein (CRP) and fibrinogen represented the level of acute-phase activation. Proinflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor-alpha [TNF-alpha]), leptin, and basic fibroblast growth factor (bFGF) also were measured.

RESULTS

Median CaSc equaled 17.9 Agatston units (range, 0 to 5,502 Agatston units). No calcification was detected in 20 subjects (46.5%; CaSc < 10 Agatston units). CaSc correlated with age (R = 0.57; P < 0.0001), body mass index (R = 0.42; P < 0.005), and serum leptin (R = 0.3; P < 0.05) and CRP levels (R = 0.38; P < 0.05). The correlation with PD therapy duration was borderline statistically significant (P = 0.063). Patients with the greatest values for CaSc >> 400 Agatston units) were characterized by significantly greater levels of IL-6, bFGF, and CRP compared with subjects with a CaSc less than 10 Agatston units (P < 0.05 for all). Patients with history of coronary artery disease (CAD) had significantly greater CaSc values (median, 778.6 versus 3.3 Agatston units; P < 0.001) compared with those without CAD. Serum triglyceride levels were significantly greater and HDL cholesterol levels were significantly lower in patients with CAD. The first group also was characterized by significantly greater serum TNF-alpha (P < 0.01) and CRP levels (P < 0.005). In multiple regression analysis, only age was independently associated with CaSc (beta = 0.45; P = 0.002).

CONCLUSIONS

Our results may suggest an association between CAC and chronic inflammation activity in the mentioned group of patients. To our knowledge, this is the first study reporting the prevalence of CAC in PD patients using the MSCT method. The association between CaSc results and classic, as well as inflammatory, risk factors for CAD found in this study should be interpreted with caution because of its method limitations (cross-sectional design, heterogeneity of study population, and small number of studied patients).

The mechanism of <em>growth</em> <em>factor</em> protection against metabolic/excitotoxic insults was examined. The time course of changes in ATP levels, mitochondrial transmembrane potential, intracellular free calcium levels ([Ca2+]i), and cell survival resulting from glucose deprivation were assessed in cultured hippocampal neurons. ATP levels were significantly reduced within 1 h of the onset of glucose deprivation and reached less than <em>20</em>% of control levels by 12 h. Mitochondrial transmembrane potential (assessed by rhodamine 123 accumulation in mitochondria) declined progressively between 4 and <em>20</em> h following the onset of glucose deprivation. The [Ca2+]i was reduced during the first 1 h of glucose deprivation, gradually rose through 12 h, and then rose rapidly and was elevated five- to sevenfold after 16 h. The [Ca2+]i did not increase, and mitochondrial dysfunction and cell damage were prevented, in hypoglycemic neurons incubated in Ca(2+)-deficient medium. Elevation of [Ca2+]i by exposure of neurons to glutamate caused loss of rhodamine 123 fluorescence and structural damage to mitochondria. Mitochondrial function could be restored and cell survival maintained by addition of glucose prior to the late elevation of [Ca2+]i. Nerve <em>growth</em> <em>factor</em> (NGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and insulin-like <em>growth</em> <em>factor</em> II (IGF-II) prevented loss of both [Ca2+]i homeostasis and mitochondrial transmembrane potential, and protected hippocampal neurons against hypoglycemic injury, but did not prevent the hypoglycemia-induced reduction in ATP levels. NaCN and 2,4-dinitrophenol (DNP) caused a large elevation of [Ca2+]i, mitochondrial dysfunction, and cell death. NGF, bFGF, and IGF-II each significantly reduced the adverse effects of NaCN and DNP on [Ca2+]i, mitochondrial function, and cell survival. Loss of [Ca2+]i homeostasis may be a critical event leading to mitochondrial damage and cell death resulting from energy failure. Preventing loss of [Ca2+]i homeostasis may be a general mechanism for the neuroprotective action of <em>growth</em> <em>factors</em>.

We have shown previously that epidermal <em>growth</em> <em>factor</em> (EGF) plus <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF2) expands the neural precursor cells in the ependyma of the normal adult rat spinal cord in vivo. To investigate the therapeutic effect of these <em>factors</em> on spinal cord injury (SCI), we administered EGF, FGF2, EGF plus FGF2, or artificial cerebrospinal fluid (aCSF) intrathecally (15 ng/h of EGF or FGF2) for 3 or 14 days after mild (2.4-g) or moderate (<em>20</em>-g) clip compression injury at T1 in adult rats. Histological and functional assessments were used to evaluate the therapeutic effects. The EGF plus FGF2 group, which received these agents for 14 days, showed better functional recovery than the aCSF group 42 days after moderate SCI (p < 0.05). At 14 days, the EGF plus FGF2 group showed a much greater expansion of ependymal cells and astrocytes compared to the other groups, and there was evidence for extensive migration of ependymal cells into the surrounding injured cord. These mitogens did not significantly enhance nestin expression in the ependymal layer or alter the expansion of oligodendrocyte precursor cells or microglia/macrophages, and dividing cells did not show the neuron-specific marker NeuN except immediately adjacent to the ependyma. The exact mechanism for improved functional recovery after EGF plus FGF2 is not known.

Explants of epithelial cells from newborn rat lenses undergo changes characteristic of fibre differentiation when cultured with neural retina or retina-conditioned medium. Here we show that similar changes occur when acidic and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) are used instead of retina-conditioned medium. When cultured without FGF, epithelial explants contained negligible amounts of beta-crystallin, a lens protein found only in fibre cells. However, at saturating concentrations of FGF, about <em>20</em> micrograms beta-crystallin was produced per explant in 5 days. The response was dose-dependent, half maximal response requiring 55 and 290 ng/ml of basic and acidic FGF, respectively. FGF also stimulated cell proliferation and cell migration. All three responses to basic FGF were blocked by an antibody specific for basic FGF. The concentration of FGF required to produce a maximal response was lower for cell proliferation and migration than for beta-crystallin accumulation. The results suggest a possible role for FGF in the control of events in lens development.

BACKGROUND

Laboratory and clinical data suggest that the anti-angiogenic agent, thalidomide, if combined with cytotoxic agents, may be effective against recurrent glioblastoma multiforme (GBM).

OBJECTIVE

To determine 6-month progression-free survival (6PFS) and toxicity of temozolomide plus thalidomide in adults with recurrent GBM.

METHODS

Eligible patients had recurrent GBM after surgery, radiotherapy, and/or adjuvant chemotherapy. Temozolomide was given at 150-<em>20</em>0 mg/m(2)/day on days 1-5 of each 28-day cycle. Thalidomide was given orally at 400 mg at bedtime (days 1-28) and increased to 1,<em>20</em>0 mg as tolerated. Patients were evaluated with magnetic resonance imaging scans every 56 days. The study was designed to detect an increase of the historical 6PFS for GBM from 10 to 30%.

RESULTS

Forty-four patients were enrolled, 43 were evaluable for efficacy and safety. The study population included 15 women, 29 men; median age was 53 years (range 32-84); median Karnofsky performance status was 80% (range 60-100%). Thirty-six (82%) patients were chemotherapy-naïve. There were 57 reports of toxicity of grade 3 or greater. Non-fatal grade 3-4 granulocytopenia occurred in 15 patients (34%). The objective response rate was 7%. The estimated probability of being progression-free at 6 months with this therapy is 24% [95% confidence interval (C.I.) 12-38%]. The median time to progression is 15 weeks (95% C.I. 10-<em>20</em> weeks). There was no observed correlation between serum levels of vascular endothelial <em>growth</em> <em>factor</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and IL-8 and the 6PFS outcome.

CONCLUSIONS

This drug combination was reasonably safe, but with little indication of improvement compared to temozolomide alone.

Accumulation of DNA is essential for muscle <em>growth</em>, yet mechanisms of androgen-induced DNA accretion in skeletal muscle are unclear. The purpose of this study was to determine whether androgen receptors (AR) are present in cultured skeletal muscle satellite cells and myotubes and examine the effects of testosterone on satellite cell proliferation and differentiation. Immunoblot analysis using polyclonal AR antibodies (PG-21) revealed an immunoreactive AR protein of approximately 107 kDa in porcine satellite cells and myotubes. Immunocytochemical AR staining was confined to the nuclei of satellite cells, myotubes, and muscle-derived <em>fibroblasts</em>. Administration of 10(-7) M testosterone to satellite cells, myotubes, and muscle-derived <em>fibroblasts</em> increased immunoreactive AR. In satellite cells and myotubes, AR increased incrementally after 6, 12, and 24 h of exposure to testosterone. Testosterone (10(-10) - 10(-6) M), alone or in combination with insulin-like <em>growth</em> <em>factor</em> I, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, or platelet-derived <em>growth</em> <em>factor</em>-BB, had no effect (P>> 0.01) on porcine satellite cell proliferation, and testosterone pretreatment for 24 h did not alter the subsequent responsiveness of cells to these <em>growth</em> <em>factors</em>. Satellite cell differentiation was depressed (<em>20</em>-30%) on days 2-4 of treatment with 10(-7) M testosterone. This effect was not reversible within 48 h after treatment withdrawal and replacement with control medium. These data indicate that satellite cells are direct targets for androgen action, and testosterone administration increases immunoreactive AR protein and reduces differentiation of porcine satellite cells in vitro.

The development of new vessels (angiogenesis) is essential to wound healing. The center of a wound space is hypoxic, a condition that has been shown to stimulate angiogenesis in animal models of coronary artery occlusion. Because the mechanisms involved in this complex process are difficult to study in situ, an in vitro model would provide a useful complement to in vivo studies. This laboratory has developed and characterized calf pulmonary microvessel endothelial cell (PMVEC) cultures and an in vitro model system of angiogenesis using collagen three-dimensional gels that permit migration of cells into vessel networks. This system was used to study the direct effect of normoxia (<em>20</em>% O2) or hypoxia (5% O2) on PMVEC ability to undergo angiogenesis in vitro. Major changes leading to formation of capillary-like networks occurred during the first 3 days of hypoxic exposure only and included restructuring of actin filament networks, focal changes in distribution of basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and orientation and migration of cell tracts into a collagen gel matrix to form vessel networks.

OBJECTIVE

Vocal fold scarring disrupts the layer structure of the vocal fold lamina propria that is essential for optimal mucosal vibration. Prevention of vocal fold scarring remains challenging. Hepatocyte growth factor (HGF) has strong antifibrotic activity. The authors' previous studies have found that HGF stimulates hyaluronic acid production and suppresses collagen production from vocal fold fibroblasts, suggesting that HGF has therapeutic potential in prevention of vocal fold scarring. The present study aimed to demonstrate the effects of HGF on vocal fold scarring in an in vivo rabbit model.

METHODS

Animal experiment.

METHODS

The vocal fold mucosa was stripped unilaterally in 20 rabbits, then HGF or saline (sham-treated group) was immediately injected into the injured site. At 6 months after the procedure, histological, rheological, and physiological examinations of vibratory behavior were completed.

RESULTS

Histological examination revealed excessive collagen deposition and disorganized elastin in the sham-treated group, whereas the HGF-treated group presented with better wound healing exhibiting less collagen deposition. Contraction of the injured vocal folds observed in the sham-treated group did not occur in the HGF-treated group. Rheological data indicated that the HGF-treated vocal folds were less stiff and viscous compared with the sham-treated group. Mucosal vibration of HGF-treated vocal folds appeared much better than the sham-treated group in terms of phonation threshold pressure, vocal efficiency, mucosal wave amplitude, and glottal closure.

CONCLUSIONS

Hepatocyte growth factor proved to be useful in preventing vocal fold scarring and maintaining viscoelastic shear properties of the vocal fold.

Connective tissue <em>growth</em> <em>factor</em> (CTGF) is a <em>growth</em> and chemotactic <em>factor</em> for <em>fibroblasts</em> encoded by an immediate early gene that is transcriptionally activated by transforming <em>growth</em> <em>factor</em> ss. Although the primary translational product of the pig CTGF gene is predicted to be of approximate Mr 38 000, pig uterine luminal flushings (ULF) contained 10- to <em>20</em>-kDa CTGF proteins that were heparin-binding and mitogenic, whereas 38-kDa CTGF was not apparent. The N-termini of two microheterogeneous forms of 16-kDa CTGF, as well as 18-kDa and <em>20</em>-kDa forms of CTGF, commenced at, respectively, Cys199, Ala197, Asp186, and Asp186 and did not correspond to intron-exon boundaries in the CTGF gene. Northern blotting revealed a single porcine (p) CTGF transcript of 2.4 kilobases in endometrium from Day 10 to 16 cycling or pregnant pigs. Ten- to twenty-kilodalton pCTGF proteins in ULF were stable for 48 h at 37 degreesC whereas native 38-kDa pCTGF was degraded within 10 min under the same conditions. CTGF-degrading activity in pig ULF was heat-sensitive and concentration- and time-dependent. Ten- to twenty-kilodalton CTGF levels in ULF peaked on Day 16 of the cycle and on Day 12 of pregnancy and were highly correlated with the levels of proteolytic activity for 38-kDa CTGF. Collectively these data suggest that bioactive 10- to <em>20</em>-kDa CTGF proteins are generated in utero through limited proteolysis of the 38-kDa CTGF primary translational product.

Previous studies have indicated that the Ca(2+)-dependent protease, calpain, is activated in platelets within 30-60 s of thrombin stimulation, but specific roles of calpain in platelets remain to be identified. To directly test the functions of calpain during platelet activation, a novel strategy was developed for introducing calpain's specific biological inhibitor, calpastatin, into platelets prior to activation. This method involves treatment of platelets with a fusion peptide, calpastat, consisting of the cell-penetrating signal sequence from Kaposi's <em>fibroblast</em> <em>growth</em> <em>factor</em> connected to a calpain-inhibiting consensus sequence derived from calpastatin. Calpastat specifically inhibits thrombin peptide (SFLLR)-induced alpha-granule secretion (IC(50) = <em>20</em> microM) during the first 30 s of activation, thrombin-induced platelet aggregation (IC(50) = 50 microM), and platelet spreading on glass surfaces (IC(50) = 34 microM). Calpastat-Ala, a mutant peptide in which alanine is substituted at conserved calpastatin residues, lacks calpain inhibitory activity and fails to inhibit secretion, aggregation, or spreading. The peptidyl calpain inhibitors calpeptin, MDL 28,170 (MDL) and E64d also inhibit secretion, aggregation and spreading, but require 3-10-fold higher concentrations than calpastat for biological activity. Together, these findings demonstrate that calpain regulates platelet secretion, aggregation, and spreading and indicate that calpain plays an earlier role in platelet activation following thrombin receptor stimulation than had been previously detected.

BACKGROUND

The Japan-Multinational Trial Organization (JMTO) lung cancer (LC) 0003 was a prospective randomized phase III trial investigating advanced non-small cell lung cancer comparing paclitaxel (P) plus carboplatin (C) versus vinorelbine (V), gemcitabine (G) followed by docetaxel (D). This trial was conducted with Southwest Oncology Group (SWOG) 0003 using a common arm of PC. An analysis of SWOG 0003 samples showed that low osteopontin (OPN) plasma levels were highly prognostic for a better outcome. We performed an independent investigation to validate these results using samples from Japanese patients enrolled in the JMTO LC 0004, a correlative study associated with JMTO LC 0003.

METHODS

A total of <em>20</em> ml of blood was collected before treatment from patients enrolled in JMTO LC 0003. Serum concentrations of OPN and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) were measured by enzyme-linked immunosorbent assay. Effects of OPN and bFGF levels on tumor response, progression-free survival (PFS), and overall survival (OS) were examined.

RESULTS

Seventy-one samples were obtained, including 32 specimens from the PC arm and 39 from the VGD arm. There were no significant relationships between either OPN or bFGF levels with patient characteristics. In an analysis of clinical outcome, low OPN levels correlated with better OS and progression-free survival (hazard ratio [HR] = 0.57; 95% confidence interval [CI], 0.33-0.97; p = 0.037, HR = 0.42; 95% CI, 0.25-0.70; p = 0.001, respectively) and high bFGF levels correlated with better OS (HR = 0.53; 95% CI, 0.31-0.90; p = 0.018).

CONCLUSIONS

Consistent with the findings from SWOG 0003, low OPN serum levels were significantly associated with a favorable prognosis in the JMTO LC 0004. Additionally, high bFGF levels were associated with improved survival.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF or FGF-2) is produced by nearly all melanomas in vitro and in vivo but not by normal melanocytes, which require exogenous bFGF for <em>growth</em>. In this study, we transduced normal human melanocytes to overexpress two forms of bFGF: (bFGF-Long and bFGF-Short) using replication-deficient adenovirus 5 vectors. bFGF-Long induced the 17.8, 22.5, 23.1 and 24.2 kDa forms of bFGF, whereas bFGF-Short induced only the 17.8 kDa mature form. <em>Growth</em> of cultured melanocytes transduced with either vector was similar to that of nevus and melanoma cells and was independent of exogenous bFGF and of insulin/insulin-like <em>growth</em> <em>factor</em> 1, and cyclic AMP enhancers, requiring only phorbol ester as an exogenous mitogen. Like primary melanoma cells, transduced normal melanocytes grew anchorage independently in soft agar. When injected into the dermis of human skin grafted to mice, bFGF-transduced melanocytes proliferated for at least <em>20</em> days, whereas cells from control cultures showed poor survival and no proliferation. These results demonstrate that bFGF upregulation is a critical component in melanoma progression.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) influences the differentiation and survival of retinal photoreceptors in vivo and in vitro, but it is not known whether it acts directly on photoreceptor FGF receptors or indirectly through activation of surrounding cells. To clarify the effects of FGF-2 on photoreceptor survival, we developed a purified photoreceptor culture system. The outer nuclear layers of postnatal day 5-15 rat retinas were isolated by vibratome sectioning, and the photoreceptor fractions obtained were enzymatically dissociated. Photoreceptors were maintained in monolayer culture for 1 week in a chemically defined medium. Immunocytochemical labeling showed that >99.5% of cells were photoreceptors, and glial contamination represented approximately 0. 2%. Photoreceptors from postnatal day 5-9 retinas survived for at least 24 hr in vitro, whereas cells from postnatal day 10-15 retinas died rapidly. Subsequent studies performed with postnatal day 5 photoreceptors showed that their survival was increased in a dose-dependent manner after the addition of FGF-2. In control cultures, 36% of originally seeded photoreceptors were alive after 5 d in vitro, and in the presence of <em>20</em> ng/ml FGF-2 this number was doubled to 62%. This increase was not caused by proliferation of photoreceptor precursors. Denaturing or blocking FGF-2 prevented enhancement of survival. Conversely, only 25.5% of photoreceptors survived in the presence of epidermal <em>growth</em> <em>factor</em> (EGF). FGF- and EGF-receptor mRNA and proteins were detected in purified photoreceptors in vitro, and addition of FGF-2 or EGF led to tyrosine phosphorylation of photoreceptor proteins. These data support a direct mechanism of action for FGF-2 stimulation of photoreceptor survival.

An angiogenic <em>growth</em> <em>factor</em> present in bovine corpus luteum (CL) has been purified to apparent homogeneity by a combination of differential salt precipitation, ion exchange chromatography, and heparin-Sepharose chromatography. It is a single chain polypeptide with an apparent mol wt of 15,000 and an amino acid composition similar to that previously reported for pituitary and brain <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF). Sequence analysis of the first 17 residues of the CL-derived <em>growth</em> <em>factor</em> identified the sequence; His-Phe-Lys-Asp-Pro-Lys-Arg-Leu-Tyr-X-Lys-Asn-Gly-Gly-X-Phe-Leu. This sequence is identical to residues 16-33 of bovine pituitary and brain FGF, indicating that the CL-derived <em>growth</em> <em>factor</em> is an amino-terminally truncated form of FGF and is otherwise similar, if not identical, to FGF. The biological activity of CL FGF is indistinguishable from that of pituitary or brain FGF. It is highly active in triggering the proliferation of cultured bovine vascular endothelial cells derived either from large vessels (aortic arch) or from corpus luteum and adrenal cortex capillaries (half-maximal stimulation at <em>20</em>-40 pg/ml and saturation at 400-600 pg/ml). In vivo implants containing 50 ng to 1 microgram CL-derived <em>growth</em> <em>factor</em> stimulate neovascularization in the chorioallantoic membrane of the chick embryo. In addition to being mitogenic for vascular endothelial cells, CL FGF also stimulates the proliferation of a wide variety of mesoderm- and neuroectoderm-derived cells, including vascular smooth muscle cells, granulosa and adrenal cortex cells, rabbit costal chondrocytes, and corneal endothelial cells.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF), a polypeptide that has been shown to stimulate division in 3T3 cells, was tested for mitogenic effects on diploid, early-passage cells from human and murine sources. The quantitative assay of [3H]thymidine incorporation into acid-insoluble material showed that FGF at low concentrations (10 minus 9 M) was more effective than additional serum for provoking the initiation of DNA synthesis in human foreskin <em>fibroblasts</em> or mouse <em>fibroblasts</em> maintained in 5 or 10% serum, respectively. The <em>growth</em> of the human <em>fibroblasts</em> was twice as fast in the presence of FGF plus 10% calf serum as it was in the presence of 10% calf serum or <em>20</em>% fetal calf serum alone. The addition of FGF to primary cultures of mouse <em>fibroblasts</em> in 0.4% serum resulted in a twofold increase in cell number compared to controls. In contrast to results obtained with 3T3 cells, neither insulin nor a glucocorticoid potentiated the effects of FGF on either human or mouse cells.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) is known for its mitogenic and motogenic effects on breast cancer cells. Here, we demonstrate that FGF-2 is also a potent stimulator of breast cancer cell survival, as it counteracts the apoptotic activity of the C2 ceramide analogue and various chemotherapeutic agents (5-fluorouracil, camptothecin, etoposide) in MCF-7, T47-D and BT-<em>20</em> cells. The use of pharmacological inhibitors (PD98059, wortmannin, LY294002, SN50) and transfection with negative dominants (IkappaBm, p110(PI3K (phosphoinositide 3-kinase))*DeltaK, AktND) or small interfering RNA targeted against Akt indicated that PI3K/Akt and nuclear <em>factor</em>-kappaB (NF-kappaB), but not p42/p44 MAP-kinases, were required to stimulate FGF-2 antiapoptotic activity. The activation of NF-kappaB was dependent on PI3K/Akt, and using a combination of approaches based on immunoprecipitation, Western blotting and proteomics (two-dimensional electrophoresis and mass spectrometry), we identified the beta form of IkappaB kinase (IKKbeta) as a target of Akt signaling. The selective disruption of IKKbeta using small interfering RNA induced a potent inhibition of Akt-mediated activation of NF-kappaB and cell survival, indicating the functional involvement of IKKbeta in FGF-2 antiapoptotic signaling. Together, these results demonstrate Akt/IKKbeta interaction in NF-kappaB pathways, thereby emphasizing the potential of these proteins as therapeutic targets in breast cancer.

Tumor-induced osteomalacia (TIO) is a rare disorder of phosphate wasting due to <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 (FGF23)-secreting tumors that are often difficult to locate. We present a systematic approach to tumor localization and postoperative biochemical changes in 31 subjects with TIO. All had failed either initial localization, or relocalization (in case of recurrence or metastases) at outside institutions. Functional imaging with ¹¹¹Indium-octreotide with single photon emission computed tomography (octreo-SPECT or SPECT/CT), and ¹⁸fluorodeoxyglucose positron emission tomography/CT (FDG-PET/CT) were performed, followed by anatomic imaging (CT, MRI). Selective venous sampling (VS) was performed when multiple suspicious lesions were identified or high surgical risk was a concern. Tumors were localized in <em>20</em> of 31 subjects (64.5%). Nineteen of <em>20</em> subjects underwent octreo-SPECT imaging, and 16 of <em>20</em> FDG-PET/CT imaging. Eighteen of 19 (95%) were positive on octreo-SPECT, and 14 of 16 (88%) on FDG-PET/CT. Twelve of <em>20</em> subjects underwent VS; 10 of 12 (83%) were positive. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were as follows: sensitivity = 0.95, specificity = 0.64, PPV = 0.82, and NPV = 0.88 for octreo-SPECT; sensitivity = 0.88, specificity = 0.36, PPV = 0.62, and NPV = 0.50 for FDG-PET/CT. Fifteen subjects had their tumor resected at our institution, and were disease-free at last follow-up. Serum phosphorus returned to normal in all subjects within 1 to 5 days. In 10 subjects who were followed for at least 7 days postoperatively, intact FGF23 (iFGF23) decreased to near undetectable within hours and returned to the normal range within 5 days. C-terminal FGF23 (cFGF23) decreased immediately but remained elevated, yielding a markedly elevated cFGF23/iFGF23 ratio. Serum 1,25-dihydroxyvitamin D₃ (1,25D) rose and exceeded the normal range. In this systematic approach to tumor localization in TIO, octreo-SPECT was more sensitive and specific, but in many cases FDG-PET/CT was complementary. VS can discriminate between multiple suspicious lesions and increase certainty prior to surgery. Sustained elevations in cFGF23 and 1,25D were observed, suggesting novel regulation of FGF23 processing and 1,25D generation.

BACKGROUND

Angiostatic drugs might provide desirable modulation of choroidal angiogenesis-related diseases, including histoplasmosis and the exudative form of age-related macular degeneration. However, the precise effects of this class of compounds in the choroidal neovascularization are still unclear. In the present study, we investigated the effects of triamcinolone acetonide (TA), an angiostatic steroid, on choroidal angiogenesis in vitro.

METHODS

Bovine choroidal endothelial cells (CEC), which are the critical cellular component of choroidal angiogenesis in vivo, were isolated with Lycopersicon esculentum agglutinin-coated Dynabeads and cultured in EGM medium. CEC were treated with basic fibroblast growth factor (bFGF) and TA at various concentrations ranging from 50 to 300 mg/l. The capacities for CEC migration and tube formation were evaluated with the modified Boyden chamber and the Vitrogen collagen assay, respectively. The activities of matrix metalloproteinases (MMP)-2 and -9 were examined using gelatin zymography.

RESULTS

The stimulation of CEC with 50 ng/ml bFGF resulted in an increase of about 100% in migration activity (P<0.01). Preincubation of CEC with TA at the indicated concentrations for 20 min inhibited the bFGF-stimulated migration in a dose-dependent manner (P<0.01). After 5 days, the bFGF-stimulated tube formation in CEC was inhibited by TA at the concentrations 100, 150 and 300 mg/l (P<0.01). Gelatin zymography of the culture media of CEC showed that the bFGF-induced activation of MMP-2 was attenuated by 300 mg/l TA (P<0.05).

CONCLUSIONS

Downregulation of the activation of MMPs in CEC could be one of the mechanisms by which angiostatic steroids inhibit choroidal angiogenesis.

The role of angiogenesis and lymphangiogenesis in thyroid cancer pathogenesis has not been elucidated. Patterns for tumour behaviour and metastasic spread vary according to tumour type and whether differences in the angiogenic or lymphangiogenic phenotype influence the route for tumour metastases or determine a more aggressive behaviour has not been fully explored. The angiogenic and lymphangiogenic phenotypes of a large cohort of thyroid proliferative lesions (n=191) were studied. Using immunohistochemistry for CD34, lymphatic vessel endothelial receptor-1 (LYVE-1) (specific markers for vascular and lymphatic endothelium respectively), vascular endothelial <em>growth</em> <em>factor</em> (VEGF-A), VEGF-C and <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2), this study analyses microvascular density (MVD), lymphatic vascular density (LVD), and expression of angiogenic and lymphangiogenic <em>factors</em> in normal thyroid (NT; n=19), multinodular goitre (n=25), toxic multinodular goitre (n=8), Graves' hyperplasia (n=22), follicular adenoma (n=54), papillary carcinoma (PC; n=27), incidental papillary microcarcinoma (PMC; n=8), follicular carcinoma (FC; n=<em>20</em>) and medullary carcinoma (MC; n=8). MVD was decreased in proliferative lesions, benign and malignant, compared with NT (P<0.0001). In contrast, VEGF-A expression was increased in thyroid carcinomas (PC, FC and MC) when compared with PMC, benign lesions and NT (P<0.0001). LVD was higher in PC and PMC (P=0.001), and VEGF-C expression was increased in PC (P<0.0001). Despite higher LVD and increased expression of VEGF-A and VEGF-C in thyroid cancers, these markers were not related to poor prognosis in terms of tumour size, multifocality and/or presence of lymphatic or distant metastases. In conclusion, angiogenesis is reduced in thyroid proliferative lesions compared with NT tissue. However, VEGF-A expression is upregulated in thyroid cancers. Lymphangiogenesis and VEGF-C expression are increased in thyroid tumours prone to lymphatic metastases. This may be an important mechanism underlying the differences in metastatic behaviour between papillary and follicular thyroid cancer.