In vitro cultures of neural precursor cells are useful experimental tools for studies on the mechanisms of brain development, as well as for generating renewable sources in cell therapy for neurodegenerative disorders. The systematic characterization of cultured neural precursors is a prerequisite for obtaining basic information on brain development. Here, we examine the cell survival, proliferation, and differentiation potential of cultured neural precursors from different embryonic ages and those of the precursors expanded in vitro for different periods of time. Precursor cells were isolated at rat embryonic days 14 (E14) and <em>19</em> (E<em>19</em>) and cultured in the presence of a mitogen basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). The numbers of TUNEL+ and BrdU+ cells in E<em>19</em> cortical precursor cultures were significantly lower than those in E14 cultures, indicating that the programmed cell death and proliferation potential of neural precursors are reduced during the progression of brain development. E14 cells tended to differentiate into neurons, and E<em>19</em> cells into astrocytes. To determine whether the intrinsic properties of neural precursors are similarly altered during in vitro culture, E14 precursor cells were expanded for different periods. Precursor cells expanded for longer periods displayed lower apoptotic and proliferation indices, as well as astrogenic developmental potential. Clonal analysis data confirmed the transition of precursor differentiation potential from neurogenic to astrogenic over the culture period. Our findings collectively suggest that neural precursor cells undergo time-dependent changes in properties via an intrinsic program, both in vivo and in vitro.

Flexor tendon wound healing in zone II is complicated by adhesions to the surrounding fibro-osseous sheath. These adhesions can significantly alter tendon gliding and ultimately hand function. Lactate and transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) are two important mediators of wound healing that have been demonstrated to independently increase collagen production by cells of the tendon sheath, epitenon, and endotenon. This study examined the effects of lactate on TGF-beta peptide and receptor production by flexor tendon cells. Tendon sheath <em>fibroblasts</em>, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendons and cultured separately. Cell cultures were supplemented with 50 mM lactate, and the expression of three TGF-beta peptide isoforms (beta1, beta2, and beta3) and three receptor isoforms (R1, R2, and R3) was quantified with enzyme-linked immunosorbent assays. TGF-beta functional activity was also assessed with the addition of tendon cell conditioned media to mink lung epithelial cells transfected with a luciferase reporter gene expression construct responsive to TGF-beta. Supplementation of the cell culture medium with lactate significantly (p < 0.05) increased the expression of all TGF-beta peptide and receptor isoforms in all three cell lines. Tendon sheath <em>fibroblasts</em> exhibited the greatest increases in beta1 and beta2 peptide isoform expression (30 and 23 percent, respectively), whereas endotenon tenocytes demonstrated the greatest increase in beta3 peptide expression (32 percent). Epitenon tenocytes exhibited the greatest increases in receptor isoform R1 and R2 expression (17 and <em>19</em> percent, respectively). All three tendon cell types demonstrated significant (p < 0.05) increases in TGF-beta functional activity when exposed to lactate. Epitenon tenocytes demonstrated the greatest increase in activity (>4 times control values), whereas tendon sheath <em>fibroblasts</em> demonstrated the highest overall levels of total TGF-beta functional activity. Lactate significantly increased TGF-beta peptide (beta1, beta2, and beta3) expression, receptor (R1, R2, and R3) expression, and functional activity, suggesting a common pathway regulating tendon cell collagen production. Modulation of lactate and TGF-beta levels may provide a means of modulating the effects of TGF-beta on adhesion formation in flexor tendon wound healing.

OBJECTIVE

Recent experimental evidence has suggested that pressure may play an important role in the pathogenesis of arthritic diseases such as temporomandibular disorders (TMDs), rheumatic diseases and osteoarthritis. This study examines the effects of hydrostatic pressure (HP) on cytoskeleton and protein production of bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta (TGF-beta) and the SRY HMG box related gene 9 (SOX-9) in synovial fibroblasts (SFs) of rat temporomandibular joint (TMJ).

METHODS

SFs derived from rat TMJ were grown to confluence in Dulbecco's modified Eagle medium supplemented with 15% fetal calf serum. The monolayer of SFs was subjected to different HPs (0, 30, 60, and 90kPa) by an in-house designed pressure chamber for 12h. Changes of cell morphology were observed by fluorescent microscope. Production of TGF-beta, BMP-2 and SOX-9 was examined by immunocytochemical assay and western blot.

RESULTS

Compared with the untreated control, the cellular actin configuration of SFs became elongated and more intense F-actin stress fiber staining was observed after HP loading. Exposure of SFs to HP for 12h resulted in significant up-regulation of BMP-2 by 46, 54, and 66% at 30, 60, and 90kPa, respectively, whilst TGF-beta increased by 11, 19, and 28% at 30, 60, and 90kPa, respectively. HP also induced the increase of SOX-9 by 72% at 30kPa and 83% at 60kPa, but only 54% at 90kPa.

CONCLUSIONS

The obtained data suggest that HP induced the alteration of cytoskeleton and bone-morphogenetic-related proteins' production of SFs, which may influence the pathological condition of TMDs.

Type 2 diabetes is a chronic disease that can be treated with pharmacologic and/or lifestyle interventions, but in most cases it does not get cured. One of the few interventions, however, that can remit diabetes is the Roux-en-Y gastric bypass (RYGB) surgery. Approximately 63 % of patients undergoing RYGB surgery experience diabetes remission, but the underlying mechanisms are poorly understood. Some studies implicate enterohepatic pathways with bile acids, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>), and glucagon-like peptide 1 (GLP-1) being the primary components. Here, we discuss these enterohepatic changes and highlight the roles of bile acids, FGF<em>19</em>, and GLP-1 in diabetes remission. We also describe how we can now actually predict, prior to surgery, the probability for remitting diabetes after RYGB surgery by using the DiaRem score. Deeper understanding of the mechanisms of diabetes remission by RYGB surgery could provide the basis for developing more effective interventions for curing the disease.

Endothelin-1 (ET-1) mRNA is expressed by the human placenta in a developmentally regulated manner and has been shown to stimulate the <em>growth</em> of placental mesenchymal cells. The ability of placental <em>fibroblasts</em> to express preproET-1 mRNA was studied to determine if ET-1 could potentially participate via autocrine mechanisms in the proliferation of placental <em>fibroblasts</em>. <em>Fibroblasts</em> were isolated from normal placentae at various gestational ages (7-<em>19</em> weeks and term) and their abilities to express preproET-1 mRNA in culture evaluated by Northern analysis. Sparse, rapidly <em>growing</em> cultures of placental <em>fibroblasts</em> expressed preproET-1 mRNA at each gestational age in the presence of 10% FBS. The regulation of preproET-1 expression in placental <em>fibroblasts</em> was studied by exposing cells to known mitogenic stimuli. Quiescent, confluent monolayers of placental <em>fibroblasts</em> expressed no detectable levels of preproET-1 mRNA under basal conditions. Epidermal <em>growth</em> <em>factor</em> (EGF, 10 mg/ml), transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta 1, 5 ng/ml), or interleukin 1 beta (IL-1 beta) alone, had no significant effect on steady state preproET-1 mRNA levels. Cycloheximide, an inhibitor of protein synthesis, increased the steady state levels of preproET-1 mRNA at a concentration of 10 micrograms/ml. In the presence cycloheximide, IL-1 beta markedly stimulated preproET-1 mRNA expression, whereas EGF was less effective. TGF-beta 1 had no effect in the presence or absence of cycloheximide. In contrast, 12-O-tetradecanoylphorbol 13-acetate (TPA, 20 nM) exerted a small stimulatory effect on preproET-1 mRNA expression which was not influenced by cycloheximide.(ABSTRACT TRUNCATED AT 250 WORDS)

Normal B lymphopoiesis is dependent on a close relationship between B-cell precursors and the bone marrow (BM) microenvironment. To further understand the mechanisms regulating the proliferation of the malignant counterpart of B-cell precursors, namely precursor-B acute lymphoblastic leukemia (ALL), we examined the adhesion to BM <em>fibroblasts</em> (BMF) of <em>19</em> cases of precursor-B ALL using a chromium labeling assay. Eleven of <em>19</em> cases showed greater than 10% binding to BMF (range 2.3% to 54.8%, mean <em>19</em>.1%). Binding was increased approximately twofold by preincubation of BMF with tumor necrosis <em>factor</em> and interleukin-4, which also resulted in upregulation of expression of vascular cell adhesion molecule-1 (VCAM-1) on BMF. The mechanism of attachment was investigated using murine monoclonal antibodies to leukocyte integrins, principally the beta, integrins VLA-4 and VLA-5, which were demonstrated to be present on most cases by flow cytometry. Statistically significant inhibition of adhesion was observed with antibodies to the beta 1 common subunit, VLA-4, and VLA-5, whereas little effect was seen with antibodies to VLA-6 or the beta 2 integrin subunit. Preincubation of <em>fibroblasts</em> with an antibody to VCAM-1 (a ligand of VLA-4) inhibited leukemic cell binding in the majority of cases, which was an effect also observed on cytokine-stimulated BMF. However, a minority of cases, as well as the pre-B lines NALM-6 and KM-3, showed no evidence of inhibition of adhesion with anti-VCAM-1 antibodies. Treatment of BMF with antifibronectin antibody alone had little effect on ALL adhesion and did not enhance the inhibitory effect of anti-VCAM-1. These data indicate that precursor-B ALL cells bind to BM stroma through the beta 1 integrins VLA-4 and VLA-5 and that this effect is partly mediated by VCAM-1 on stromal cells, although other undefined VLA ligands are also likely to be involved. Attachment of ALL cells to stroma is likely to play a key role in regulating the survival and <em>growth</em> of these cells through exposure to stromal cytokines.

OBJECTIVE

Because the prevalence of obesity in children is increasing, the frequency of pediatric nonalcoholic fatty liver disease (NAFLD) is growing. A reliable noninvasive biomarker for monitoring progression of liver fibrosis would be useful. In cirrhotic persons serum bile acid (BA) levels are significantly elevated. We hypothesized that BA levels and composition in pediatric NAFLD vary depending on the stage of fibrosis.

METHODS

Children with NAFLD were compared with controls and classified by stages of fibrosis (NAFLD-F0, n = 27; NAFLD-F≥1, n = 65) based on liver-biopsy findings. Fasted metabolic and cholestasis status was assessed by several blood tests. BA profiles were measured by tandem mass spectrometry and compared with healthy controls (n = 105).

RESULTS

Compared with controls, all of the NAFLD patients were overweight and showed significantly elevated glucose, insulin, aspartate transaminase, and alanine transaminase levels. Total serum BAs were lower in nonfibrotic NAFLD children than in a control cohort (1.73 vs 3.6 μmol/L) because low glycine-conjugated BA levels were incompletely compensated by increases in taurine-conjugated or unconjugated BA. In patients with fibrotic NAFLD, BA levels were lower than in controls (2.45 vs 3.6 μmol/L) but higher than in nonfibrotic patients (2.45 vs 1.73 μmol/L), and the BA pattern resembled that of healthy controls. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> levels were significantly lower in both NAFLD groups than in controls (P ≤ 0.001) and were positively correlated with ursodeoxycholic acid levels.

CONCLUSIONS

Our data indicate that serum BA levels decrease in early NAFLD and increase during progression to fibrosis. Given that BA levels are increased in cirrhotic adults, we postulate a continuous rise as NAFLD advances. BA may have a value as a noninvasive biomarker in pediatric NAFLD progression.

OBJECTIVE

Bile acids (BAs) traversing the enterohepatic circulation (EHC) influence important metabolic pathways. By determining individual serum BAs in relation to markers of metabolic activity, we explored how diurnal variations in their EHC relate to hepatic metabolism in normal humans.

METHODS

Serum BAs, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>), lipoproteins, glucose/insulin and markers of cholesterol and BA syntheses were monitored for 32 h in 8 healthy males. Studies were conducted at basal state and during initiation of cholestyramine treatment, with and without atorvastatin pretreatment. Time series cross-correlation analysis, Bayesian structural model and Granger causality test were applied.

RESULTS

Bile acids synthesis dominated daytime, and cholesterol production at night. Conjugated BAs peaked after food intake, with subsequent FGF<em>19</em> elevations. BA synthesis was reduced following conjugated BA and FGF<em>19</em> peaks. Cholestyramine reduced conjugated BAs and FGF<em>19</em>, and increased BA and cholesterol production; the latter effects attenuated by atorvastatin. The relative importance of FGF<em>19</em> vs. conjugated BAs in this feedback inhibition could not be discriminated. Unconjugated BAs displayed one major peak late at night/early morning that was unrelated to FGF<em>19</em> and BA synthesis, and abolished by cholestyramine. The normal suppression of serum triglycerides, glucose and insulin observed at night was attenuated by cholestyramine.

CONCLUSIONS

Conjugated and unconjugated BAs have asynchronous rhythms of EHC in humans. Postprandial transintestinal flux of conjugated BAs increases circulating FGF<em>19</em> levels and suppresses BA synthesis. Unconjugated BAs peak late at night, indicating a non-postprandial diurnal change in human gut microflora, the physiological implications of which warrants further study.

In a previous report, we described a method to generate cell lines derived from stromal colonies (colony-derived cell lines [CDCL]). In a first step, colonies were obtained from plating cells from adherent layers of human long-term bone marrow cultures (LTBMC) in methyl-cellulose in the presence of interleukin-1 beta (IL-1 beta) (20 U/mL) and tumor necrosis <em>factor</em>-alpha (TNF-alpha) (200 U/mL). In a second step, cell lines were derived from individual colonies cultured in liquid medium with 20 ng/mL basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). In this report, we describe the phenotype of cells from more than 100 cell lines. CDCL did not contain cells of hematopoietic origin, which indicates that the culture system did not allow the <em>growth</em> of hematopoietic precursors. CDCL did not contain endothelial-like cells, similarly to primary adherent layers. CDCL comprised stromal cells, as defined by membrane antigens recognized by monoclonal antibodies 6-<em>19</em>, Stro-1 and 1B10. These cells belonged to the family of connective tissue-forming cells since they synthesized interstitial collagens I, III, and V; nonplasmatic EDa+ and EDb- fibronectin; tenascin; and chondroitin-sulfate. Study of the time course of CDCL showed that the lines differed from one another according to their proliferative capacity: 32% of CDCL grew quickly, yielding about 10,000 cells in 10 days; 36% of CDCL grew slowly, yielding 1000 cells in 10 days; and the remaining 32% had intermediate proliferative capacity. For CDCL with a high proliferative capacity, a distinctive differentiation pattern could be described. At culture, inception cells from the lines were vimentin and laminin. Over time, several cytoskeletal and extracellular matrix (ECM) proteins indicative of vascular smooth muscle differentiation were expressed, including: the alpha-SM-actin isoform; the actin-binding proteins, smooth muscle myosin-heavy chain (SMMHC), SM1; h-caldesmon; calponin; gelsolin; and the ECM proteins, collagen IV and elastin. In full-grown lines, cells were similar to immature, intimal, vascular smooth muscle cells as found beneath the endothelium in adult aortas. Because of the coupling between proliferation and differentiation, the differentiation pattern seems to be under genetic control. However, since the coupling was not stringent during the whole lifespan of the lines, it is possible that cytokines are also involved, ensuring autocrine regulation of CDCL development.

Achondroplasia is the most common type of genetic dwarfism. It is characterized by disproportionate short stature and other skeletal anomalies resulting from a defect in the maturation of the chondrocytes in the <em>growth</em> plate of the cartilage. Recent studies mapped the achondroplasia gene on chromosome region 4p16.3 and identified a common mutation in the gene encoding the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3). In an analysis of <em>19</em> achondroplasia families from a variety of ethnic backgrounds we confirmed the presence of the G380R mutation in 21 of 23 achondroplasia chromosomes studied. In contrast, the G380R mutation was not found in any of the 8 hypochondroplasia chromosomes studied. Furthermore, linkage studies in a 3-generation family with hypochondroplasia show discordant segregation with markers in the 4p16.3 region suggesting that at least some cases of hypochondroplasia are caused by mutations in a gene other than FGFR3.

We developed a novel wound dressing composed of a hyaluronic acid (HA) and collagen (Col) spongy sheet containing epidermal <em>growth</em> <em>factor</em> (EGF) or basic fibrolast <em>growth</em> <em>factor</em> (bFGF) by freeze-drying method (EGF-wound dressing or bFGF-wound dressing, respectively). A wound dressing without any <em>growth</em> <em>factor</em> was prepared as a control in a similar manner as above (C-wound dressing). Intermolecular cross-linkage between Col molecules was induced by UV irradiation. The release behavior of free HA from the wound dressing was investigated using a C-wound dressing. The weight of C-wound dressing after 1 day, 3, 5, and 7 days of incubation on top of a Col gel sheet at the air-water interface (wound surface model) was 55, 36, 30, and <em>19</em>% of the original weight, respectively. Most free HA and a part of Col was released from the cross-linked Col network in the wound dressing during incubation, as the original Col content in the wound dressing was 33%. Next, <em>fibroblast</em> proliferation was assessed in conventional culture medium preconditioned by immersion of a piece of C-, EGF-, or bFGF-wound dressing, i.e. C-conditioned medium, EGF-conditioned medium, or bFGF-conditioned medium. Cell proliferation in C-conditioned medium increased to approximately the same level as that in conventional medium. Cell proliferation in EGF- and bFGF-conditioned medium was 1.9 times and 2.6 times greater than that in conventional medium after 7 days of cultivation, respectively. Finally, cytokine production of <em>fibroblasts</em> was assessed in a wound surface model using a <em>fibroblast</em>-incorporating Col gel sheet (cultured dermal substitute [CDS]). CDS was elevated to the air-medium interface, on which each wound dressing was placed and cultured for 7 days. <em>Fibroblasts</em> in CDS covered with EGF-wound dressing released 3.6 times more vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and 4.6 times more hepatocyte <em>growth</em> <em>factor</em> (HGF) when compared with the C-wound dressing. <em>Fibroblasts</em> in CDS covered with bFGF-wound dressing released 10.2 times more VEGF and 6.3 times more HGF when compared with the C-wound dressing. This finding indicates that bFGF-wound dressing can facilitate more effectively the VEGF and FGF production compared with EGF-wound dressing.

Apert syndrome is characterized by craniosynostosis, midfacial hypoplasia and bilateral syndactyly. We document in detail the intrauterine natural history of Apert syndrome by serial sonographic examination. Ultrasound examination of a <em>19</em>-week fetus revealed an abnormal appearance of the skull. The subsequent examination including transvaginal brain scanning demonstrated a deformed occipital part of the cerebrum and lateral ventricles, frontal bossing, a low nasal bridge and an abnormal appearance of the fetal hands and feet. The distortion of the fetal profile became progressively worse with advancing gestation. Towards the end of pregnancy, anterior prominence of the cerebrum, ventricles and corpus callosum was demonstrated and mild non-progressive ventriculomegaly was seen. The female 3152-g newborn with the typical facial appearance of Apert syndrome, bilateral syndactyly of the fingers and toes and isolated cleft palate was delivered at 37 weeks. Postnatal three-dimensional computed tomography scan demonstrated the fusion of the coronal suture and a wide mid-line calvarial defect, and cranial magnetic resonance imaging confirmed the prenatal sonographic findings. Although the karyotype was normal, genomic DNA analysis of the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 revealed Ser252Trp, which is specified in the mutational basis of Apert syndrome. The time course of the prenatal findings in this case may help increase understanding of the intrauterine natural history of Apert syndrome.

The characteristics of the human B-type platelet-derived-<em>growth</em>-<em>factor</em> (PDGF) receptor expressed in Chinese hamster ovary (CHO) cells, were compared with those of a mutant receptor lacking all but <em>19</em> amino acids of the intracellular domain. The transfected wild-type receptor was synthesized as a 160-kDa precursor that was processed to <em>19</em>0 kDa. Each CHO cell expressed 30,000-100,000 receptors which bound PDGF-BB with a Kd of about 0.5 nM. Analysis of PDGF-AB binding yielded non-linear Scatchard plots; the major part of the binding sites had a Kd of 6 nM. PDGF-AA was not bound. The receptors expressed in CHO cells were down-regulated after binding of PDGF-BB, and mediated degradation of 125I-PDGF-BB with similar efficiency as PDGF-B-type receptors in human <em>fibroblasts</em>. The transfected receptor also transduced a mitogenic signal. The mutant receptor was synthesized as a 90-kDa precursor and was processed to 120 kDa with a slightly faster rate than the wild-type receptor. Cells expressing the mutant receptor generally had around 10(6) ligand-binding sites/cell, with a Kd for binding of PDGF-BB of 3 nM. The mutant receptor, which did not transduce a mitogenic response, mediated degradation of 125I-PDGF-BB, albeit less efficiently compared to the wild-type receptor. In contrast to the wild-type receptor, it was down-regulated only to a limited extent and not degraded in response to ligand binding. These findings indicate a role for the intracellular part of the receptor, not only in mitogenic signaling, but also in receptor internalization and intracellular routing.

The mechanism of in vivo activation of transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1), which is critical to its role in many physiological and pathological conditions, is not fully understood. To explore the mechanism by which dermal <em>fibroblasts</em> respond to latent TGF-beta1 directly, the efficacy of either latent TGF-beta1 (LTGF-beta1) alone or LTGF-beta1 plus cell membranes isolated from <em>fibroblasts</em>, mink lung, and one skin-related (Sk23) and two skin-unrelated (U251 and D54MG) transformed cell lines was examined using the mink lung epithelial cell (Mv1Lu) inhibition assay. As a source of LTGF-beta1, PA317 cells were transfected with previously constructed pLin-TGF-beta1 or pLin vectors with no TGF-beta1 insert. LTGF-beta1 expressing PA317 cells were then enriched by <em>growth</em> in the presence of 0.5 mg G-418 for 6-10 days. Eight out of 53 colonies of cells expressing high levels of LTGF-beta1 were selected and their conditioned media were removed after 3 days and used to evaluate the latency and bioactivity of TGF-beta1 using ELISA and Mv1Lu <em>growth</em> inhibition assay, respectively. The level of TGF-beta1 was <em>19</em>-fold greater (21.4 +/- 0.4 vs. 1.1 +/- 0.2 ng/ml) in conditioned medium derived from pLin-TGF-beta1 transfected cells than that of control. These conditioned media were then used for the subsequent cell proliferating experiments. The results showed that latent TGF-beta1, which proved to be inactive in an Mv1Lu inhibition assay, significantly stimulates <em>fibroblast</em> cell proliferation compared to that of control in a dose-dependent fashion. In another set of experiments, cells were treated with either active (acidified/neutralized) or latent TGF-beta1 and the results showed a significant increase in cell proliferation in response to low concentrations of active TGF-beta1. However, high concentrations of active TGF-beta1 markedly suppressed <em>fibroblast</em> proliferation. These dual effects were in contrast to a steady increase in <em>fibroblast</em> proliferation found in response to latent TGF-beta1. To explore why LTGF-beta1 has a differential proliferating effect on epithelial and <em>fibroblast</em> cell proliferation, cell membranes from these cells were isolated and incubated with PA317-conditioned medium containing LTGF-beta1 and then added to mink lung cells. Only isolated <em>fibroblast</em> cell membranes incubated with LTGF-beta1 inhibited Mv1Lu cells. To examine whether the LTGF-beta1 cell proliferating activity is unique to dermal <em>fibroblasts</em> or is a general phenomenon, in similar experimental conditions cell membranes from several cell lines, U251, D54MG, and SK23, were isolated, incubated with LTGF-beta1, and then added to an Mv1Lu inhibition assay. The proliferation of Mv1Lu epithelial cells was significantly (1547 +/- 269 vs. 3568 +/- 23) inhibited with SK23, but not U251 cell membranes plus LTGF-beta1 relative to that of control. The inhibitory effect of SK23 plus LTGF-beta1 was cell membrane dose-dependent. In conclusion, the result of this study shows that LTGF-beta1 may directly modulate cell proliferation of those cells that possess a cell membrane associated LTGF-beta1 activation mechanism.

Hepatocyte <em>growth</em> <em>factor</em> (HGF), a cytokine with multiple functions, exhibits cell-type-specific as well as cytokine- and steroid hormone-regulated expression. The HGF gene is known to be expressed predominately in mesenchymal but not in epithelial cells. In this study, we report the identification of a cell-type-specific transcriptional repressor in the promoter region of the mouse HGF gene, which is evidently responsible for the suppression of HGF expression in epithelial cells. Gel mobility shift assays and DNase I footprinting studies revealed that a 27-bp element (-16 to +11) around the transcription initiation site is responsible for the binding of a nuclear protein which is present in epithelial but not in mesenchymally derived cells. Further analysis of the binding activity of the DNA region with nuclear protein revealed that an approximately <em>19</em>-bp sequence containing a unique palindromic structure (5'-AACCGACCGGTT-3') overlapped by a CAP box is essential for binding. Substitution of a single base (the contact site) within this region by site-directed mutagenesis resulted in total abrogation of the binding of the nuclear protein and a concomitant increase in the transcriptional activity of various lengths of HGF-chloramphenicol acetyltransferase fused genes when transfected into the epithelial cell line RL95-2 but not the mesenchymal cell line NIH 3T3. Southwestern (DNA-protein) analyses revealed that the nuclear protein which binds to this repressor element is a single polypeptide of approximately 70 kDa. Analysis of the nuclear extract prepared from regenerating mouse liver at various times after two-thirds partial hepatectomy by gel mobility shift assay revealed a substantial reduction (more than 75% within 3 h) in the binding of the repressor to its cognate binding site. Our results suggest that a cis-acting transcriptional repressor in the promoter region of the mouse HGF gene is involved in cell-type-specific regulation through binding to its cognate trans-acting protein which exists in epithelial cells but is absent in <em>fibroblast</em> cells.

Angiogenesis plays an important role in the <em>growth</em>, progression, and metastasis of solid tumors. Among angiogenic <em>factors</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) appear to be useful markers in adults with cancer. The aim of this pilot study was to determine the levels of VEGF in serum and bFGF in serum and urine of children with solid tumor at diagnosis (as measured by ELISA), and to investigate whether these parameters provide prognostic information. Forty consecutive patients with different types of cancer were prospectively included in this study. Median values of all studied angiogenic <em>factors</em> were higher in patients than in controls (n = 40), and the differences were statistically significant for bFGF in serum and urine: 10 versus 3 pg/ml (P = 0.0004) and 6406 versus 0 pg/g of creatinine (P < 0.0001), respectively. Among patients, median serum values of bFGF and VEGF were higher in children with metastatic disease (n = 14) than in those with localized disease (n = 26). The difference was statistically significant for serum bFGF: 17.5 versus 6 pg/ml (P = 0.02). Serum angiogenic <em>factor</em> levels correlated with outcome. The estimated event-free survival at 3 years was 79% for patients with normal bFGF values (n = 13) versus 42% (n = 26; P = 0.02) for those with high levels, and 71% in case of normal VEGF values (n = 20) versus 38% (n = <em>19</em>; P = 0.04) for those with high levels. No benefit of normal urinary bFGF values was observed. Our results provide a rationale for exploring the clinical interest of bFGF and VEGF measurements in body fluids of a larger group of children with cancer.

BACKGROUND

Angiogenesis plays a significant role in the growth and progress of cancer, thus we evaluated the levels of urinary basic fibroblast growth factor (bFGF) in bladder cancer (Ca) patients and investigated any possible correlation between this angiogenic factor with tumor stage and grade.

METHODS

Urine samples from 41 patients with bladder Ca, 11 patients with history of bladder Ca but negative follow-up cystoscopy, 18 patients with benign prostate hyperplasia (BPH) and 15 normal healthy volunteers were assayed using an enzyme-linked immunosorbent assay for bFGF. Resulting values were normalized against urine creatinine and expressed as pg/g.

RESULTS

The median urinary bFGF level of patients with active disease, history of bladder carcinoma and negative follow-up cystoscopy, BPH, and healthy volunteers were 2,717, 1,009, 1,414 and 1,100 pg/g, respectively. There was a statistically significant difference between median bFGF levels of patients with active bladder Ca and those of the other groups (p = 0.000). Eleven patients with invasive bladder Ca had a median bFGF value of 6,880 pg/g that was significantly increased (p = 0.002) compared to the median of 2,312 pg/g of those with superficial tumors (Ta 14, T1 16). Grades 1, 2 and 3 carcinoma were found in 5, 19 and 17 patients which had a median bFGF of 2,717, 1,762 and 3,617 pg/g, respectively, but the difference was not statistically significant (p = 0.13).

CONCLUSIONS

Our results confirm the implication of bFGF in the biology of bladder cancer, and demonstrate that urinary bFGF concentration seems to be significantly related to the stage but not to the grade of the disease supporting the proposed mechanisms of release of bFGF. Further studies are required in order to validate the potential clinical applications of bFGF for specific groups of bladder cancer patients.

Our group has recently developed 1-(t)butyl carbamoyl, 7-methyl-indole-3-ethyl isothiocyanate (NB7M), a novel indole ethyl isothiocyanate analog. We now describe its selective cytotoxicity in both central nervous system (CNS) and neuroblastoma (NB) cancer cells. In an effort to understand its mechanism of action we examined the effects of NB7M on apoptosis, cell cycle arrest, and pro-survival/mitogen-activated protein kinase (MAPK) signaling in neuroblastoma cells. NB7M proved highly cytotoxic to NB cell lines (SMS-KCNR, SK-N- SH, SH-SY5Y, IMR-32) with IC(50) values ranging from 1.0-2.0 microM, whereas lung <em>fibroblasts</em> were less affected (IC(50)>> or =10 microM). In the NCI 60 cell screen 1-dose assay, NB7M (10 microM) reduced the <em>growth</em> (-89 to -27 % <em>growth</em>) of CNS cancer cell lines SF-268, SF-295, SNB-75 (glioblastoma), SF-539 (gliosarcoma), and U251 (astroglioma) while SNB-<em>19</em> glioblastoma cells were relatively resistant (<em>19</em>% <em>growth</em>). Hoechst staining of SMS-KCNR cells treated with NB7M (3 microM) for 24 hrs exhibited significant chromatin condensation and DNA fragmentation, whereas Annexin-v/7AAD staining revealed that the majority of cells accumulated in the early-apoptotic and late-apoptotic/necrotic stages. NB7M treatment of SMS-KCNR and SH-SY5Y cells also led to the cleavage of procaspases-3, and PARP-1 while causing activation of pro-apoptotic MAPKs and down-regulation of pro-survival <em>factors</em> AKT and PI-3K. Furthermore, NB7M treatment caused S-phase arrest in SMSKCNR and G1-phase arrest in SH-SY5Y cells. NB7M is active against CNS cancers and NB.

UNASSIGNED

Bile acid diarrhoea (BAD) is a common cause of chronic diarrhoea with a population prevalence of primary BAD around 1%. Previous studies have identified associations with low levels of the ileal hormone <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>), obesity and hypertriglyceridaemia. The aim of this study was to identify further associations of BAD.

UNASSIGNED

A cohort of patients with chronic diarrhoea who underwent 75selenohomocholic acid taurate (SeHCAT) testing for BAD was further analysed retrospectively. Additional clinical details available from the electronic patient record, including imaging, colonoscopy, chemistry and histopathology reports were used to calculate the prevalence of fatty liver disease, gallstones, colonic neoplasia and microscopic colitis, which was compared for BAD, the primary BAD subset and control patients with diarrhoea.

UNASSIGNED

Of 578 patients, 303 (52%) had BAD, defined as a SeHCAT 7d retention value <15%, with 179 (31%) having primary BAD. 425 had an alanine aminotransferase (ALT) recorded, 184 had liver imaging and 176 had both. Overall, SeHCAT values were negatively associated with ALT (rs=-0.<em>19</em>, p<0.0001). Patients with BAD had an OR of 3.1 for an ALT >31 ng/mL with imaging showing fatty liver (p<0.001); similar figures occurred in the primary BAD group. FGF<em>19</em> was not significantly related to fatty liver but low levels were predictive of ALT >40 IU/L. In 176 subjects with gallbladder imaging, 27% had gallstones, 7% had a prior cholecystectomy and 34% either of these. The median SeHCAT values were lower in those with gallstones (3.8%, p<0.0001), or gallstones/cholecystectomy (7.2%, p<0.001), compared with normal gallbladder imaging (14%). Overall, BAD had an OR of 2.0 for gallstones/cholecystectomy (p<0.05). BAD was not significantly associated with colonic adenoma/carcinoma or with microscopic colitis.

UNASSIGNED

The diagnosis of BAD is associated with fatty liver disease and with gallstones. The reasons for these associations require further investigation into potential metabolic causes.

Severe acute malnutrition (SAM) is a major cause of mortality in children under 5 years and is associated with hepatic steatosis. Bile acids are synthesized in the liver and participate in dietary fat digestion, regulation of energy expenditure, and immune responses. The aim of this work was to investigate whether SAM is associated with clinically relevant changes in bile acid homeostasis.

An initial discovery cohort with 5 healthy controls and 22 SAM-patients was used to identify altered bile acid homeostasis. A follow up cohort of 40 SAM-patients were then studied on admission and 3 days after clinical stabilization to assess recovery in bile acid metabolism. Recruited children were 6-60 months old and admitted for SAM in Malawi. Clinical characteristics, feces and blood were collected on admission and prior to discharge. Bile acids, 7α-hydroxy-4-cholesten-3-one (C4) and FGF-<em>19</em> were quantified.

On admission, total serum bile acids were higher in children with SAM than in healthy controls and glycine-conjugates accounted for most of this accumulation with median and interquartile range (IQR) of 24.6 μmol/L [8.6-47.7] compared to 1.9 μmol/L [1.7-3.3] (p = 0.01) in controls. Total serum bile acid concentrations did not decrease prior to discharge. On admission, fecal conjugated bile acids were lower and secondary bile acids higher at admission compared to pre- discharge, suggesting increased bacterial conversion. FGF<em>19</em> (<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em>), a marker of intestinal bile acid signaling, was higher on admission and was associated with decreased C4 concentrations as a marker of bile acid synthesis. Upon recovery, fecal calprotectin, a marker of intestinal inflammation, was lower.

SAM is associated with increased serum bile acid levels despite reduced synthesis rates. In SAM, there tends to be increased deconjugation of bile acids and conversion from primary to secondary bile acids, which may contribute to the development of liver disease.

BACKGROUND

Hypophosphatemic rickets/osteomalacia is a group of diseases characterised by defective mineralization of bone due to hypophosphatemia and low 1,25-dihydroxy vitamin D. To explore the role of fibroblast growth factor 23 (FGF-23) in the regulation of phosphate homeostasis, we measured the circulating concentrations of this growth factor in healthy individuals and in patients with hypophosphatemic rickets/osteomalacia.

METHODS

Nineteen patients with hypophosphatemic rickets/osteomalacia were included in hypophosphatemic group (HP, 12 female and 7 male, mean age was 30 years), and 19 healthy age-matched individuals served as the control group. Full length FGF-23 fragments were measured by two-site enzyme-linked immunosorbent assay.

RESULTS

Mean FGF-23 concentrations were significantly higher in the HP group ((87.4 +/- 43.6) pg/ml) compared with the control group ((19.2 +/- 6.16) pg/ml; P < 0.001). In 1 patient with tumour-induced osteomalacia, serum FGF-23 concentrations were 84.1 pg/ml; these concentrations were normalized 2 hours after a hemangiopericytoma resection (7.8 pg/ml). Subsequently, serum 1,25(OH)(2) vitamin D3 concentrations significantly increased from 21.3 pg/ml to 89.3 pg/ml, and serum phosphorus levels were normalized.

CONCLUSIONS

Serum FGF-23 concentrations were markedly elevated in patients with hypophosphatemic rickets. FGF-23 plays an important role in the pathogenesis of hypophosphatemic rickets/osteomalacia.

We assessed (1) angiogenic <em>factors</em> in patients with stable angina and longstanding >> or =24 months) total occlusion of a single coronary artery and (2) the relation between plasma levels of angiogenic <em>factors</em> and the development of collateral vessels as evaluated by coronary angiography. Plasma concentrations of vascular endothelial <em>growth</em> <em>factor</em> (VEGF(165)), <em>fibroblast</em> <em>growth</em> <em>factor</em>, placenta-derived <em>growth</em> <em>factors</em> (PlGFs), and hepatocyte <em>growth</em> <em>factor</em> were assessed in 96 patients with stable angina and longstanding >> or =24 months) total occlusion of a single coronary artery. According to coronary angiographic results, 18 patients had no visible collaterals (group 0), 21 patients had visible collaterals but no filling of the recipient epicardial vessel (group 1), and 57 patients showed filling (partial or complete) of the recipient epicardial vessel by collaterals (group 2). Plasma VEGF(165) and PlGF concentrations were higher in group 1 than in groups 0 and 2 (VEGF(165) 75 pg/ml, range 24 to 105, vs 23 pg/ml, range 15 to 29, and <em>19</em> pg/ml, range 10 to 41, respectively, F = 5.53, p = 0.006; PlGF 35 pg/ml, range 3.5 to 105, vs 1 pg/ml, range 1 to 38, and 1 pg/ml, range 1 to 5, respectively, F = 7.09, p = 0.008). Plasma VEGF(165) and PlGF levels were similar in groups 0 and 2. There was no significant difference in plasma levels of <em>fibroblast</em> and hepatocyte <em>growth</em> <em>factor</em> concentrations across the 3 groups. In conclusion, plasma levels of angiogenic <em>growth</em> <em>factors</em> differ among patients with stable angina pectoris and longstanding total coronary occlusion.

The tumor inhibitory <em>factor</em>-2 from the conditioned medium of the human rhabdomyosarcoma cell line A673 was purified and sequenced. The <em>19</em> N-terminal amino acid residues were identical to those of human interleukin 1 (IL-1), corresponding to the residues 1<em>19</em>-137 of the IL-1 alpha precursor. The purified material had an apparent molecular weight similar to that of the mature secreted form of IL-1 alpha (Mr 17,400). In addition, similarly to IL-1, it induced the production of IL-2 by T-cells. The purified protein inhibited the <em>growth</em> of the A673 cells from which it was derived, suggesting that it may act as an autocrine <em>growth</em> inhibitor. It also inhibited the <em>growth</em> of a human adenocarcinoma of the lung and three human mammary carcinomas, but not of two human melanoma cell lines. In contrast, it stimulated the proliferation of normal human <em>fibroblasts</em>. These biological activities, previously assigned to a putative tumor inhibitory <em>factor</em> molecule, are apparently due to the production by the tumor cells of IL-1 alpha.

BACKGROUND

Inflammation is an acute, early process during normal wound healing. Talactoferrin, a recombinant human lactoferrin, can induce the secretion of inflammatory mediators.

METHODS

We measured wound healing activity of topical talactoferrin in full-thickness wounds of normal mice and diabetic (db(-)/db(-)) mice, systemic bioavailability, and the potential to modulate inflammation through in vitro and in vivo binding assays and inflammatory mediator measurements.

RESULTS

Talactoferrin significantly increased the closure rate during 12 to <em>19</em> d (maximally on d 3 to 6), the 75% closure incidence, and the time to 50% closure versus vehicle or becaplermin (recombinant human platelet-derived <em>growth</em> <em>factor</em>). Systemic bioavailability was less than 0.5% following administration to open wounds. Talactoferrin bound local dermal cells in vivo and human dermal <em>fibroblasts</em> in vitro, and it induced the migration of dermal <em>fibroblasts</em>, THP-1 macrophages, Jurkat T cells, and mouse granulocytes in vitro. Competition binding assays suggested the involvement of IL-8RB and CCR2 chemokine receptors in binding and/or cell migration. Consistently, the induction of migration was partially inhibited in interleukin (IL)-8RB deficient granulocytes. Talactoferrin also enhanced the production of key repair inflammatory mediators IL-8, IL-6, macrophage inflammatory protein-1 alpha, and tumor necrosis <em>factor</em> alpha in d 3 wounds, and IL-8, IL-6, and monocyte chemotactic protein-1 in cultured dermal <em>fibroblasts</em>.

CONCLUSIONS

Talactoferrin promotes wound repair in vivo, correlating with a modulated enhancement of the early inflammatory phase of wound healing. Based on this data, talactoferrin was subsequently tested clinically in a Phase II trial in patients with diabetic ulcers and was found to be effective and safe. Talactoferrin should be further evaluated in patients with diabetic and other types of ulcers.