Previous animal data showed that platelets contain <em>growth</em> <em>factors</em> that stimulate capillary endothelial migration (angiogenesis), <em>fibroblast</em> proliferation and migration, and collagen synthesis. This study utilized autologous platelet-derived wound healing <em>factors</em> (PDWHF) to treat 49 patients with chronic nonhealing cutaneous ulcers. Patients were classified on the basis of <em>20</em> clinical and wound status parameters to generate a wound severity index. Forty-nine patients--58% diabetic (<em>20</em>% with renal transplants); 16% with trauma, vasculitis, etc.; 14% with decubitus ulcers; and 6% each with venous stasis or arterial insufficiency--with a total of 95 wounds had received conventional wound care for an average of 198 weeks (range: 1-18<em>20</em> weeks). After informed consent was obtained, patients received autologous PDWHF. Mean 100% healing time for all patients was 10.6 weeks. There was no abnormal tissue formation, keloid, or hypertrophic scarring. A multivariant analysis showed a direct correlation to 100% healing with initial wound size and the initiation of PDWHF therapy. This is the first clinical demonstration that locally acting <em>growth</em> <em>factors</em> promote healing of chronic cutaneous ulcers.

Targeted gene studies have demonstrated the importance of insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) for osteoblast (OB) differentiation and the acquisition of peak bone mineral density (BMD). The skeletal response to allelic differences in IGF-I expression can also be measured in vivo, using congenic mice. We created a congenic strain with reduced (approximately <em>20</em>%) circulating IGF-I (C3H.B6-6T [6T]) by backcrossing a small genomic region (30 cM) of Chromosome 6 (Chr6) from C3H/HeJ (C3H) onto a C57Bl/6J (B6) background. 6T female mice have lower serum IGF-I (P<0.001 vs. B6) but similar <em>growth</em> hormone (GH) and serum IGF binding protein (IGFBP) concentrations as B6. At 16 weeks of age, congenics have greater body fat (P<0.02 vs. B6) despite less total body weight, and exhibit smaller femoral cross-sectional size (P=0.001), reduced cortical thickness (P<0.001) and lower trabecular BV/TV (P<0.05) than B6. 6T mice also have suppressed serum leptin (P<0.01), but compared to B6 have similar markers of bone resorption (i.e., urine CTx and serum TRAP 5B). At 8 weeks of age, skeletal IGF-I mRNA from long bones was reduced by 40% (P<0.05) as were liver mRNA transcripts (i.e., 50%, P<0.01). Osteoblast progenitors from the bone marrow of 6T mice formed less colony forming unit <em>fibroblasts</em> by crystal violet staining than B6 (P<0.007) and had significantly reduced alkaline phosphatase-positive colonies than B6(P<0.0001). In addition, staining of bone marrow with oil red O revealed greater numbers of adipocytes in 6T than B6. Several candidate genes in the Chr6 QTL were excluded by lack of strain-related expression differences in bone, but genes positively regulating adipocyte differentiation including Alox 5 and PPAR-gamma require further study as either "pathway" or candidate genes. In summary, allelic differences in a QTL on Chr6 result in altered IGF-I gene expression, changes in OB lineage allocation, and reduced peak bone mass. Congenic mice are useful models not only for mapping genes related to bone mass but also for elucidating the biology underlying various skeletal phenotypes associated with more subtle manipulation of the mouse genome.

Chemically modified hyaluronic acid (HA)-gelatin hydrogels have been documented to support attachment, <em>growth</em>, and proliferation of <em>fibroblasts</em> in vitro and to facilitate repair and engineering of tissues in vivo. The objective of this study was to determine the optimal composition of a synthetic extracellular matrix (sECM) that would promote wound repair and induce tissue regeneration in a rabbit vocal fold wound healing model. The sECM was formed using a thiol-modified semisynthetic glycosaminoglycan (GAG) derived of HA (Carbylan-SX) mixed with a thiolated gelatin derivative, co-cross-linked with poly(ethylene glycol) diacrylate to form Carbylan-GSX. Forty rabbits underwent vocal fold biopsy bilaterally. Rabbits were treated with Carbylan-SX, which lacks gelatin, or with Carbylan-GSX with different gelatin concentrations (2.5%, 5%, 10%, and <em>20</em>%) via unilateral injection of the vocal fold at the time of biopsy. Saline was injected in the contralateral vocal fold as a control. Three weeks after biopsy and injection, animals were euthanized and mRNA levels of procollagen type 1, fibronectin, transforming <em>growth</em> <em>factor</em> beta 1 (TGF-beta1), fibromodulin, HA synthase 2, hyaluronidase 2, and tissue biomechanics were evaluated. Hyaluronidase mRNA levels were found to be significantly elevated in for Carbylan-GSX <em>20</em>% w/w gelatin compared to controls. Both Carbylan-SX and Carbylan-GSX significantly improved tissue elasticity and viscosity. Carbylan-GSX containing 5% w/w gelatin showed the most promise as a scaffold material for vocal fold tissue regeneration.

BACKGROUND

Fibroblast growth factor 23 (FGF23) is a phosphaturic factor and a suppressor of 1alpha-hydroxylase activity in the kidney. Although its importance in chronic kidney disease (CKD) has been demonstrated in adults, there is little information in pediatric patients.

OBJECTIVE

The aims of this study were: 1) to determine reference values for FGF23 serum levels according to glomerular filtration rate (GFR) (measured by the reference standard, inulin clearance), gender, and age; and 2) to evaluate the effects of different etiologies and treatments on FGF23 serum levels in a prospective single-center cohort of 227 CKD children (119 boys).

RESULTS

Age, body weight, height, and GFR (mean +/- sd) values were: 11.3 +/- 4.1 yr, 37 +/- 16 kg, 140 +/- 20 cm, and 98 +/- 34 ml/min per 1.73 m(2), respectively. Calcium, phosphate, PTH, 25 hydroxyvitamin D, 1,25 dihydroxyvitamin D, C-terminal FGF23, and intact FGF23 (mean +/- sd) levels were: 2.43 +/- 0.11 mmol/liter, 1.41 +/- 0.22 mmol/liter, 41 +/- 23 pg/ml, 24 +/- 10 ng/ml, 152 +/- 72 pmol/liter, 76 +/- 134 relative units/ml, and 44 +/- 37 pg/ml, respectively. There was a wide range of FGF23 serum levels, but FGF23 levels increased when GFR decreased. FGF23 serum levels were not modified by gender, but they increased with age. In univariate analysis, corticosteroid therapy seemed to be associated with increased FGF23 serum levels. A multivariate linear regression analysis found a significant impact of GFR, body mass index, and solid organ transplantation on FGF23 serum levels.

CONCLUSIONS

Age, GFR, body mass index, and solid organ transplantation seem to influence FGF23 serum levels in a pediatric population. The impact of corticosteroids on FGF23 metabolism should be further investigated; further longitudinal studies will also help to better define the prognostic impact of FGF23 serum levels in pediatric CKD in terms of disease progression, cardiovascular morbidities, and bone disabilities.

An improved Ham's F12 nutrient medium supplemented with epidermal <em>growth</em> <em>factor</em> (EGF), insulin (INS), and transferrin (TF) was developed for continuous proliferation and clonal <em>growth</em> of primary rabbit tracheal epithelial (TE) cells in culture. The addition of small quantities of fetal bovine serum (FBS) (0.01 to 0.1%) to cultures had little measureable stimulation on TE cell <em>growth</em> and plating efficiency. However, serum levels higher than 0.1% inhibited cell <em>growth</em> and also masked the <em>growth</em> stimulating activities of EGF and INS despite an increase in cell attachment. Under this defined, hormone-supplemented medium, and in the presence of a trace amount of serum (0.01%), 10 to <em>20</em>% of the protease-dissociated TE cells attached to the culture dish followed by at least four population doublings during 7 to 10 d of culture. Clonal <em>growth</em> occurred at a seeding density of 17 cells/cm2 with a plating efficiency of 6 to 8%. Confluent primary cultures could be passaged two to four times by treatment with a 0.1% trypsin-1 mM EDTA solution and a total of 10 to 30 population doublings of in vitro life span were obtained. The epithelial nature of cultured cells was confirmed by indirect immunofluorescent staining with antikeratin antibody as well as by transmission electron microscopy. This study shows that using this improved hormone-supplemented medium, rabbit TE cells can be maintained in culture for extended periods of time without the aid of a <em>fibroblast</em> feeder layer or explant tissue. This system could be useful for the study of cell differentiation of tracheal epithelium.

BACKGROUND

Congestive heart failure (CHF) is a common cause of atrial fibrillation (AF). Oxidative stress and inflammation (profibrotic) and peroxisome proliferator-activated receptor-alpha (PPAR-alpha, antifibrotic) factors may be involved in CHF-related remodeling. We evaluated the effects of simvastatin (antioxidant, anti-inflammatory) and fenofibrate (PPAR-alpha activator) on CHF-related atrial remodeling.

RESULTS

Dogs were subjected to 2-week ventricular tachypacing (VTP) in the absence and presence of simvastatin (20 or 80 mg/day) or fenofibrate. Induced AF duration (DAF) was increased by VTP from 36+/-14 (non-paced controls) to 1005+/-257 s (p<0.01). Simvastatin prevented VTP-induced DAF increases (147+/-37 and 84+/-37 s at 20 and 80 mg/day, respectively), but fenofibrate did not (1018+/-352 s). Simvastatin also attenuated CHF-induced conduction abnormalities (heterogeneity-index reduced from 1.5+/-0.1 to 1.1+/-0.1 and 1.0+/-0.1 at 20 and 80 mg/day, p<0.01) and atrial fibrosis (from 19.4+/-1.3% to 10.8+/-0.8% and 9.9+/-0.8% at 20 and 80 mg/day, p<0.01), while fenofibrate did not. Simvastatin (but not fenofibrate) also attenuated VTP-induced left-ventricular nitric-oxide synthase and nitrotyrosine increases, along with hemodynamic dysfunction. Atrial fibroblast proliferation increased with 24-h fetal bovine serum (FBS) stimulation from 654+/-153 to 7264+/-1636 DPM (p<0.001). Simvastatin, but not fenofibrate, suppressed fibroblast proliferation (664+/-192 DPM, p<0.001). Simvastatin also significantly attenuated transforming growth factor-beta1-stimulated alpha-smooth muscle actin (alpha-SMA) expression (indicating myofibroblast differentiation) from 1.3+/-0.1 to 1.0+/-0.1 times baseline (p<0.05).

CONCLUSIONS

CHF-induced atrial structural remodeling and AF promotion are attenuated by simvastatin, but not fenofibrate. Statin-induced inhibition of profibrotic atrial fibroblast responses and attenuation of left-ventricular dysfunction may contribute to preventing the CHF-induced fibrotic AF substrate.

Salmonella enterica serovar Typhimurium proliferates within cultured epithelial and macrophage cells. Intracellular bacterial proliferation is, however, restricted within normal <em>fibroblast</em> cells. To characterize this phenomenon in detail, we investigated the possibility that the pathogen itself might contribute to attenuating the intracellular <em>growth</em> rate. S. enterica serovar Typhimurium mutants were selected in normal rat kidney <em>fibroblasts</em> displaying an increased intracellular proliferation rate. These mutants harbored loss-of-function mutations in the virulence-related regulatory genes phoQ, rpoS, slyA, and spvR. Lack of a functional PhoP-PhoQ system caused the most dramatic change in the intracellular <em>growth</em> rate. phoP- and phoQ-null mutants exhibited an intracellular <em>growth</em> rate <em>20</em>- to 30-fold higher than that of the wild-type strain. This result showed that the PhoP-PhoQ system exerts a master regulatory function for preventing bacterial over<em>growth</em> within <em>fibroblasts</em>. In addition, an overgrowing clone was isolated harboring a mutation in a previously unknown serovar Typhimurium open reading frame, named igaA for intracellular <em>growth</em> attenuator. Mutations in other serovar Typhimurium virulence genes, such as ompR, dam, crp, cya, mviA, spiR (ssrA), spiA, and rpoE, did not result in pathogen intracellular over<em>growth</em>. Nonetheless, lack of either SpiA or the alternate sigma <em>factor</em> RpoE led to a substantial decrease in intracellular bacterial viability. These results prove for the first time that specific serovar Typhimurium virulence regulators are involved in a response designed to attenuate the intracellular <em>growth</em> rate within a nonphagocytic host cell. This <em>growth</em>-attenuating response is accompanied by functions that ensure the viability of intracellular bacteria.

<em>Factors</em> that regulate the <em>growth</em> and development of primitive bone marrow stromal cell precursors are not well defined. We have examined 25 purified recombinant <em>growth</em> <em>factors</em> for their ability to initiate and support clonogenic <em>growth</em> of <em>fibroblast</em> colony-forming cells (CFU-F) from adult human bone marrow. Assays were performed using bone marrow mononuclear cells (BMMNC) enriched in CFU-F by magnetic-activated cell sorting (MACS) using the monoclonal antibody (MoAb) STRO-1. A serum-deprived assay was developed to avoid components of fetal calf serum (FCS) that may mask or otherwise modify the response of CFU-F to exogenously added <em>factors</em>. L-ascorbate and the glucocorticoid dexamethasone were found to be essential for CFU-F colony development under serum-deprived conditions. Importantly, clonogenic <em>growth</em> of CFU-F in this culture system was absolutely dependent on an exogenous source of <em>growth</em> <em>factor</em>. Platelet-derived <em>growth</em> <em>factor</em>-BB (PDGF) and epidermal <em>growth</em> <em>factor</em> (EGF) demonstrated the greatest ability to support colony <em>growth</em>. Colony formation was dose-dependent, with half-maximal colony numbers at approximately 0.2 ng/mL for either <em>factor</em> and plateau numbers at concentrations in excess of 1.0 ng/mL. Simultaneous addition of PDGF and EGF had no effect on the number of colonies initiated but resulted in dose-dependent increases in mean colony diameter that were significant (P < or = .05) when compared with the effect of either <em>factor</em> alone or with the size of colonies elicited in control cultures by <em>20</em>% FCS. Fluorescence-activated cell sorting (FACS) of BMMNC using MoAbs to the alpha chain of the PDGF receptor and to the EGF receptor in combination with the Moab STRO-1 demonstrated constitutive expression of both receptors by greater than 90% on CFU-F. Receptors for insulin-like <em>growth</em> <em>factor</em>-1 (IGF-1) and nerve <em>growth</em> <em>factor</em> (NGF) were also detected on STRO-1+ CFU-F, but in vitro both IGF-1 and NGF did not support colony <em>growth</em>. This report demonstrates the development of a simple, reproducible, and stringent culture system for the <em>growth</em> and assay of stromal precursors under serum-deprived conditions and represents an important prerequisite for future studies of the role of <em>growth</em> <em>factors</em> in the regulation of stromal cell proliferation, differentiation, and development.

We have previously characterized in Chinese hamster lung <em>fibroblasts</em> a <em>growth</em> <em>factor</em> activatable and amiloride-sensitive Na+/H+ antiport (Pouysségur, J., Chambard, J. C., Franchi, A., Paris, S., and Van Obberghen-Schilling, E. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3935-3939). In this report, we compared the affinity of 28 analogs of amiloride for inhibition of the Na+/H+ antiport and inhibition of <em>growth</em> <em>factor</em>-induced DNA synthesis. We showed that the guanidino moiety of amiloride must be protonated to elicit inhibition of the Na+/H+ exchange. Substitutions within this moiety by methyl, phenyl, or benzyl groups reduced the activity <em>20</em>- to 1000-fold. On the contrary, substitution of the proton(s) of the 5-amino group of amiloride with alkyl or alkenyl groups increases potency up to 100-fold (5-N,N-diethylamiloride has a KI of 4 X 10(-8) M). In HCO-3-free medium and at lower [Na+]0 (25 or 50 mM) to reduce competition with amiloride, we found that <em>growth</em> <em>factor</em>-stimulated DNA synthesis of G0-arrested cells is inhibited by amiloride and its analogs with the same rank order as that for Na+/H+ antiporter inhibition. Over a range of 3 logs of concentration, a tight correlation was established between IC50 for the blockade of both processes, Na+/H+ exchange and percentage of cells entering the S phase upon <em>growth</em> <em>factor</em> action. These findings indicate that, in HCO-3-free medium, the functioning of the Na+/H+ exchange system is required for <em>growth</em> <em>factor</em>-induced DNA synthesis.

Mutation in either TSC1 or TSC2 causes the autosomal dominant disorder tuberous sclerosis, in which widespread hamartomas are seen, some of which have a high level of vascularization. Tuberous sclerosis complex (TSC) gene products negatively regulate mammalian target of rapamycin (mTOR) activity. We found that vascular endothelial <em>growth</em> <em>factor</em> (VEGF) is secreted by Tsc1- or Tsc2-null <em>fibroblasts</em> at high levels compared with wild-type cells. In Tsc1+/- mice, serum levels of VEGF were increased and appeared to be associated with the extent of tumor development. Rapamycin, a mTOR inhibitor, reduced the production of VEGF by Tsc1- and Tsc2-null <em>fibroblasts</em> to normal levels. Moreover, short-term treatment of Tsc1+/- mice with rapamycin at <em>20</em> mg/kg led to some changes in tumor morphology and a reduction in serum VEGF levels. These observations have three implications. First, TSC gene products regulate VEGF production through a mTOR signaling pathway. Second, serum VEGF levels may be a useful clinical biomarker to monitor the progression of TSC-associated lesions. Last, rapamycin or related inhibitors of mTOR may have therapeutic benefit in TSC both by direct tumor cell killing and by inhibiting the development of TSC lesions through impairment of VEGF production.

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.

Levels of mRNA for IFN-beta 2/B cell differentiation <em>factor</em>2/hepatocyte-stimulating <em>factor</em> (IFN-beta 2) in confluent quiescent cultures of human diploid <em>fibroblasts</em> (FS-4 strain) are enhanced by TNF, IL-1 alpha and beta, platelet-derived <em>growth</em> <em>factor</em> (PDGF) and IFN-beta 1. Of these cytokines, IL-1 alpha and beta cause a particularly strong increase in the accumulation of IFN-beta 2 mRNA in <em>fibroblasts</em>. We have evaluated whether the IFN-beta 2 gene is regulated at the transcriptional level by using nuclear run-on transcription assays. We observed that the IFN-beta 2 gene is transcribed at a low level in uninduced FS-4 cells and that this transcriptional activity is increased 2- to 3-fold in cycloheximide-treated cells, <em>20</em>- to 35-fold in IL-1 alpha-treated cells, and 5- to 15-fold in TNF-treated cells. PDGF and IFN-beta 1 enhance transcription across the IFN-beta 2 gene 2- to 3-fold. The enhancing effect of IL-1 alpha on IFN-beta 2 gene transcription, but not that of TNF, PDGF, or IFN-beta 1, is inhibited by cycloheximide, suggesting that newly-synthesized protein is involved in the increase in IFN-beta 2 transcription in response to IL-1 alpha but not in the response to the other stimuli. Furthermore, the enhancement of IFN-beta 2 transcription is sustained for up to 14 h after IL-1 alpha induction but is transient and declines to base line levels within 6 h after TNF addition. These observations suggest that there are important differences in the mechanisms by which IL-1 alpha and TNF increase IFN-beta 2 gene transcription in <em>fibroblasts</em>.

A human clonal glioma cell line, U-343 MGa Cl 2, cultured under serum-free conditions, was found to release a <em>factor</em> that competed with 125I-labeled platelet-derived <em>growth</em> <em>factor</em> (125I-PDGF) for binding to human foreskin <em>fibroblasts</em>. The concentration of competing activity in conditioned medium was equal to <em>20</em>-30 ng of PDGF per ml. The PDGF receptor competing activity had an elution position on Sephadex G-<em>20</em>0 close to that of tracer PDGF. The same fractions in the chromatogram also contained <em>growth</em>-promoting activity and material active in a PDGF radioimmunoassay. Incubation of partially purified, 125I-labeled glioma <em>factor</em> with <em>fibroblasts</em>, or rabbit anti-PDGF serum, led to the selective binding of a component with an estimated Mr of 31,000, as shown by NaDodSO4/gel electrophoresis under nonreducing conditions. After reduction this component migrated as a Mr 18,000 protein. Thus, the behavior in NaDodSO4/gel electrophoresis was similar to that of PDGF. Furthermore, incubation of partially purified glioma <em>factor</em> with immobilized PDGF antibodies markedly decreased the amount of PDGF receptor competing activity remaining in the supernatant. These results suggest that the <em>factor</em> produced by glioma cells has structural, immunological, and functional resemblance to PDGF. We previously reported that a human osteosarcoma cell line produces a PDGF-like molecule with <em>growth</em>-promoting activity. Taken together with the recent finding that PDGF is homologous to the transforming gene product of simian sarcoma virus, our present data give additional support for the idea that an autocrine activation of the PDGF receptor may be operational in the <em>growth</em> of human tumors of mesenchymal or glial origin.

Progesterone is the hormone of pregnancy and unequivocally required in all mammals for maternal support of conceptus (embryo/fetus and associated membranes) survival and development. The actions of progesterone are mediated by the progesterone receptor (PR). However, the endometrial lumenal (LE) and glandular epithelia (GE) of a number of species exhibit a loss of PR expression prior to the stages of uterine receptivity and implantation. In sheep, PR expression becomes undetectable in the endometrial LE after Day 11 and then in the GE after Day 13. Loss of PR in the GE appears to be required for onset of differentiated functions in terms of production of secretory proteins, such as uterine milk proteins (UTMP) and osteopontin (OPN). Therefore, the actions of progesterone on endometrial epithelia during most of gestation appear to be mediated by the endometrial stroma that remains PR-positive throughout pregnancy. Stromal cells produce several <em>growth</em> <em>factors</em>, such as hepatocyte <em>growth</em> <em>factor</em> (HGF) and <em>fibroblast</em> <em>growth</em> <em>factors</em>-7 and -10 (FGF-7, FGF-10), that have receptors expressed specifically in the endometrial epithelia. These <em>factors</em> may be progesterone-responsive and mediate epithelial-mesenchymal interactions that are crucial for support of pregnancy. Studies of the uterine gland knockout (UGKO) ewe indicate that uterine glands and, by default, their secretions are required for peri-implantation conceptus survival and <em>growth</em>. A complex servomechanism, involving hormones from the ovary and conceptus as well as endogenous betaretroviruses expressed in the endometrial LE and GE, is proposed to regulate endometrial gland differentiation and function during gestation. At estrus, estrogen increases PR expression in the endometrial epithelia. High levels of endogenous Jaagsiekte sheep retroviruses (enJSRVs) are expressed in the PR-positive endometrial LE and GE in response to increasing progesterone and are hypothesized to stimulate trophoblast proliferation and production of interferon (IFN) tau. IFN tau, the pregnancy recognition hormone produced by the trophoblast from Days 10 to 21, acts in a paracrine manner on the PR-negative endometrial LE and superficial GE to inhibit transcription of estrogen receptor alpha (ER) and oxytocin receptor (OTR) genes. These actions of IFN tau maintain progesterone production from the corpus luteum by abrogating release of luteolytic pulses of prostaglandin F2 alpha (PGF) from the endometrial epithelium. The antiluteolytic effects of IFN tau are dependent on progesterone. Progesterone stimulation over 8-10 days suppresses expression of the PR gene in the LE and then GE. Loss of the PR in the LE is concomitant with decreases in mucin glycoprotein one (MUC-1), an inhibitor of blastocyst implantation. As the conceptus begins implantation on Day 15, the binucleate trophectodermal cells then differentiate and produce placental lactogen (PL), a member of the prolactin (PRL) and <em>growth</em> hormone (GH) family. PL stimulates GE proliferation and production of secretory proteins, such as UTMP and OPN. Interestingly, the effects of PL on the GE appear to require the absence of PR and prior exposure to IFN tau. During mid-pregnancy, the mononuclear trophectodermal cells produce GH that can also act on a progestinized uterus to stimulate GE hypertrophy and secretory function. The actions of this servomechanism are proposed to stimulate GE hyperplasia from Days <em>20</em> to 50 and then GE hypertrophy and maximal differentiated function after Day 50 when the majority of fetal <em>growth</em> and development occurs during gestation.

Endostatin is a Mr <em>20</em>,000 COOH-terminal fragment of collagen XVIII that inhibits the <em>growth</em> of several primary tumors. We report here the cloning and expression of mouse endostatin in both prokaryotic and eukaryotic expression systems. Soluble recombinant protein expressed in yeast (15-<em>20</em> mg/L) inhibited the proliferation and migration of endothelial cells in response to stimulation by basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. A rabbit polyclonal antibody was raised that showed positive immunoreactivity to the recombinant protein expressed from both systems. Importantly, the biological activity of the mouse recombinant protein could be neutralized by this antiserum in both endothelial proliferation and chorioallantoic membrane assays. Systemic administration of endostatin at 10 mg/kg suppressed the <em>growth</em> of renal cell cancer in a nude mouse model. The inhibition of tumor <em>growth</em> with soluble yeast-produced protein was comparable to that obtained with non-refolded precipitated protein expressed from bacteria. In addition, two closely related COOH-terminal deletion mutants of endostatin were also tested and showed strikingly differing activity. Collectively, these findings demonstrate the expression of a biologically active form of mouse endostatin in yeast, define a role for the molecule in inhibiting endothelial cell migration, extend its antitumor effects to renal cell carcinoma, and provide a formal proof (via the neutralizing antiserum experiments and the mutant data) that endostatin (and not a possible contaminant) acts as an antiangiogenic agent. Finally, the high level expression of mouse endostatin in yeast serves as an endotoxin free, soluble source of protein for fundamental studies on the mechanisms of tumor <em>growth</em> suppression by angiogenesis inhibitors.

BACKGROUND

Basic fibroblast growth factor (bFGF) is an important positive regulator of tumor angiogenesis. This study evaluated the role of serum bFGF as a biological marker of tumor invasiveness and postresection recurrence in hepatocellular carcinoma (HCC).

METHODS

Concentrations of bFGF in preoperative serum samples in 88 patients undergoing resection of HCC were measured by a quantitative enzyme-linked immunosorbent assay. A single pathologist performed histopathologic examination of all tumor specimens. All patients were prospectively monitored for tumor recurrence.

RESULTS

The preoperative serum bFGF levels ranged from <0.22 to 71.2 pg/mL (median 10.8 pg/mL). There was significant correlation between high serum bFGF levels and large tumor >5 cm, presence of venous invasion or advanced pTNM stage. Patients with a serum bFGF level >10.8 pg/mL had worse disease-free survival than those with a level <10.8 pg/mL (median disease-free survival 11.2 versus 20 months, P = 0.044). Serum bFGF level >10.8 pg/mL (P = 0.035) and tumor size >5 cm (P = 0.004) were independent preoperative factors that predicted early recurrence after resection of HCC.

CONCLUSIONS

This study supports a role of bFGF in tumor growth and invasion in HCC. A high preoperative serum bFGF level appears to be predictive of invasive tumor and early postoperative recurrence. The clinical implications of serum bFGF level in HCC warrant further investigation.

Recent investigations have suggested an active role for endothelial cells in organ development, including the lung. Herein, we investigated some of the molecular mechanisms underlying normal pulmonary vascular development and their influence on epithelial branching morphogenesis. Because the lung in utero develops in a relative hypoxic environment, we first investigated the influence of low oxygen on epithelial and vascular branching morphogenesis. Two transgenic mouse models, the C101-LacZ (epithelial-LacZ marker) and the Tie2-LacZ (endothelial-LacZ marker), were used. At embryonic day 11.5, primitive lung buds were dissected and cultured at either <em>20</em> or 3% oxygen. At 24-h intervals, epithelial and endothelial LacZ gene expression was visualized by X-galactosidase staining. The rate of branching of both tissue elements was increased in explants cultured at 3% oxygen compared with <em>20</em>% oxygen. Low oxygen increased expression of VEGF, but not that of the VEGF receptor (Flk-1). Expression of two crucial epithelial branching <em>factors</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em>-10 and bone morphogenetic protein-4, were not affected by low oxygen. Epithelial differentiation was maintained at low oxygen as shown by surfactant protein C in situ hybridization. To explore epithelial-vascular interactions, we inhibited vascular development with antisense oligonucleotides targeted against either hypoxia inducible <em>factor</em>-1 alpha or VEGF. Epithelial branching morphogenesis in vitro was dramatically abrogated when pulmonary vascular development was inhibited. Collectively, the in vitro data show that a low-oxygen environment enhances branching of both distal lung epithelium and vascular tissue and that pulmonary vascular development appears to be rate limiting for epithelial branching morphogenesis.

The c-Myc protein is a helix-loop-helix leucine zipper oncogenic transcription <em>factor</em> that participates in the regulation of cell proliferation, differentiation, and apoptosis. The biochemical function of c-Myc has been well described, yet the identities of downstream effectors are just beginning to emerge. We describe the identification of a set of c-Myc-responsive genes in the Rat1a <em>fibroblast</em> through the application of cDNA representational difference analysis (RDA) to cDNAs isolated from nonadherent Rat1a and Rat1a-myc cells. In this system, c-Myc overexpression is sufficient to induce the transformed phenotype of anchorage-independent <em>growth</em>. We identified <em>20</em> differentially expressed cDNAs, several of which represent novel cDNA sequences. We further characterized one of the novel cDNAs identified in this screen, termed rcl. rcl expression is (i) directly stimulated by c-Myc; (ii) stimulated in the in vivo <em>growth</em> system of regenerating rat liver, as is c-myc; and (iii) elevated in human lymphoid cells that overexpress c-myc. By using an anti-Rcl antibody, immunoblot analysis, and immunofluorescence microscopy, the Rcl protein was found to be a 23-kDa nuclear protein. Ectopic expression of the protein encoded by the rcl cDNA induces anchorage-independent <em>growth</em> in Rat1a <em>fibroblasts</em>, albeit to a diminished extent compared to ectopic c-Myc expression. These data suggest a role for rcl during cellular proliferation and c-Myc-mediated transformation.

The K-sam gene, originally isolated as an amplified gene from the stomach cancer cell line KATO-III, is characterized by its preferential amplification in the undifferentiated type (diffuse type) of stomach cancer and encodes one of the receptors for heparin-binding <em>growth</em> <em>factors</em> or <em>fibroblast</em> <em>growth</em> <em>factors</em>. The K-sam gene has been isolated by different methods and has been designated BEK, TK14, and Cek2. The receptor for keratinocyte <em>growth</em> <em>factor</em> was also found to be encoded by the same gene. To examine the expression of the K-sam protein in stomach cancer, polyclonal antibody pK1-2 was raised against the extracellular domain of the gene product. This antibody detected K-sam proteins by Western blot and flow cytometry analyses in stomach cancer cell lines KATO-III and HSC39, in which the K-sam gene is amplified and overexpressed. By immunohistochemical analysis, <em>20</em> of 38 cases of the undifferentiated type of advanced stomach cancer were K-sam positive, whereas none of 11 cases of the differentiated or intestinal type revealed K-sam staining. The K-sam product was observed predominantly in diffusely infiltrative lesions. In one autopsy case, the K-sam protein was detected only focally in the primary tumor, whereas markedly increased staining for the K-sam product was detected diffusely in the metastasized tumor in the lymph node and liver. These results suggest that K-sam overexpression is associated with the malignant phenotype of the undifferentiated type of stomach cancer, such as infiltrative <em>growth</em> and metastasis.

BACKGROUND

Obesity is associated with hyperparathyroidism and increased bone mass and turnover, but their pathogeneses are unclear.

OBJECTIVE

Our aim was to determine in obesity interrelationships among serum levels of leptin, the mineral-regulating hormones, bone turnover markers, and sclerostin.

METHODS

This case-control study was performed in <em>20</em> women having bariatric surgery and <em>20</em> control women matched for race and age. Anthropometrics and fasting serum biochemistries were measured in controls and in bariatric patients the morning of surgery.

RESULTS

Body mass index (48.9 vs. 25.4 kg/m(2)), weight (128.6 vs. 71.9 kg), serum leptin (74.6 vs. 25.2 ng/ml), PTH (44.5 vs. 28.8 pg/ml), fibroblast growth factor 23 (FGF23) (42.4 vs. 25.9 pg/ml), and bone alkaline phosphatase (BAP) (25.8 vs. 17.5 U/liter) were higher, but height (162.3 vs. 167.7 cm) and 1,25-dihydroxyvitamin D (1,25D) (39.2 vs. 48.7 pg/ml) were lower in bariatric surgery patients than controls. There was no difference in serum sclerostin, amino-terminal collagen cross-links, 25-hydroxyvitamin D (25D), calcium, phosphate, and creatinine between groups. In the combined sample, leptin was positively related to PTH, FGF23, and BAP but not to 1,25D or sclerostin. Multiple regression analysis demonstrated that PTH was predicted by leptin and Ca (R(2) = 0.39); 1,25D by 25D, FGF23, and phosphate (R(2) = 0.43); FGF23 by leptin and 1,25D (R(2) = 0.27); BAP by leptin, PTH, and Ca (R(2) = 0.39); and sclerostin by leptin and PTH (R(2) = 0.<em>20</em>).

CONCLUSIONS

Women having bariatric surgery had higher leptin, PTH, FGF23, and BAP and lower 1,25D than controls. Leptin predicted the serum levels of PTH, 1,25D, and FGF23, the mineral-regulating hormones, and BAP, a bone formation marker, in women with body mass index ranging from 13.9-65.8 kg/m(2). The results suggest that leptin has an endocrine or paracrine effect on PTH and FGF23 production and that PTH may be one of the signals in obesity that leads to increased bone mass.

<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 <em>20</em> mg pegbelfermin once a week, all for 16 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 16 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, <em>20</em>15, and Aug 4, <em>20</em>16, 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 <em>20</em> 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 <em>20</em> 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 (16%) 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 16 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>

This study determined the presence of interleukin 1 (IL-1), interleukin 6 (IL-6), tumour necrosis <em>factor</em> alpha (TNF alpha), tumour necrosis <em>factor</em> beta (TNF beta), interferon gamma (IFN gamma), transforming <em>growth</em> <em>factor</em> beta 2 (TGF beta 2) and <em>fibroblast</em> proliferation activity (FPA) in vitreous aspirates from eyes undergoing vitrectomy for the treatment of retinal detachment complicated by proliferative vitreoretinopathy (PVR) or uncomplicated retinal detachment (RD). Cadaveric vitreous from normal subjects were used as controls. The results showed that IL-1 and IL-6 predominated in vitreous from eyes with PVR or RD, and that concentrations of IL-6 greater than <em>20</em> pg/ml were more frequently found in PVR than in RD (p = 0.031) or control specimens (p = 0.006). Low levels of TNF alpha were observed in 4/18 eyes with PVR, 1/15 eyes with RD and 1/15 control vitreous, and small concentrations of TNF alpha were seen in 3/18 eyes with PVR, 1/15 eyes with RD and 2/15 control vitreous. IFN gamma was detected in 12/18 eyes with PVR, but only in 5/15 eyes with RD (p = 0.048) and 6/15 control specimens. TGF beta 2 was present in all vitreous samples at concentrations ranging from 100 to 4,500 pg/ml with no significant differences among the three groups. Control vitreous possessed the greatest FPA when compared with vitreous from eyes with PVR (p = 0.031) or RD (p = 0.048). These observations provide further evidence that cytokine-mediated pathways of inflammation are involved in the pathogenesis of PVR and point to the possible involvement of IL-1, IL-6 and IFN gamma in cellular interactions leading to chronicity.

OBJECTIVE

Mutations in the fibroblast growth factor receptor 3 (FGFR3) occur in 50% of primary bladder tumors. An FGFR3 mutation is associated with good prognosis, illustrated by significantly lower percentage of patients with progression and disease-specific mortality. FGFR3 mutations are especially prevalent in low grade/stage tumors, with pTa tumors harboring mutations in 85% of the cases. These tumors recur in 70% of patients. Efficient FGFR3 mutation detection for prognostic purposes and for detection of recurrences in urine is an important clinical issue. In this paper, we describe a simple assay for the simultaneous detection of nine different FGFR3 mutations.

METHODS

The assay consists of one multiplex PCR, followed by extension of primers for each mutation with a labeled dideoxynucleotide. The extended primers are separated by capillary electrophoresis, and the identity of the incorporated nucleotide indicates the presence or absence of a mutation.

RESULTS

The assay was found to be more sensitive than single-strand conformation polymorphism analysis. Mutations could still be detected with an input of only 1 ng of genomic DNA and in a 20-fold excess of wild-type DNA. Moreover, in urine samples from patients with a mutant tumor, the sensitivity of mutation detection was 62%.

CONCLUSIONS

We have developed a fast, easy to use assay for the simultaneous detection of FGFR3 mutations, which can be of assistance in clinical decision-making and as an alternative for the follow-up of patients by invasive cystoscopy for the detection of recurrences in urine.

OBJECTIVE

To perform a phase I trial of recombinant human endostatin (rhEndostatin; EntreMed, Rockville, MD) given as a daily <em>20</em>-minute intravenous (IV) injection in adult patients with refractory solid tumors.

METHODS

The daily dose was increased from 15 to 240 mg/m(2) by a factor of 100% in cohorts of three patients. In the absence of dose-limiting toxicity, uninterrupted treatment was continued until the tumor burden increased by more than 50% from baseline. Correlative studies included dynamic contrast-enhanced magnetic resonance imaging of tumor blood flow, urinary vascular endothelial growth factor and basic fibroblast growth factor levels, rhEndostatin serum pharmacokinetics, and monitoring of circulating antibodies to rhEndostatin.

RESULTS

There were no notable treatment related toxicities among 15 patients receiving a total of 50 monthly cycles of rhEndostatin. One patient with a pancreatic neuroendocrine tumor had a minor response and two patients showed disease stabilization. Linearity in the pharmacokinetics of rhEndostatin was indicated by dose-proportionate increases in the area under the curve for the first dose and the peak serum concentration at steady state. Daily systemic exposure to rhEndostatin in patients receiving 240 mg/m(2)/d was approximately 50% lower than that provided by the therapeutically optimal dose in preclinical studies.

CONCLUSIONS

rhEndostatin administered as a <em>20</em>-minute daily IV injection at doses up to 240 mg/m(2) showed no significant toxicities. Evidence of clinical benefit was observed in three patients. Due to high variability between the peak and trough serum concentrations associated with the repeated short IV infusion schedule, daily serum drug levels only briefly exceeded concentrations necessary for in vitro antiangiogenic effects.