The chorioallantoic membrane (CAM) is one of the most vascularized tissues in the chicken embryo. Capillary <em>growth</em> proceeds until day 10 of development and thereafter abruptly regresses. As it is generally accepted that the formation of new blood vessel is regulated by <em>growth</em> <em>factors</em>, we have investigated the presence of angiogenic and mitogenic <em>factors</em> in the chicken chorioallantois. In the present study, we show that chorioallantoic fluid (CAF) contains angiogenic substances that are probably synthesized in the CAM or the embryonic kidney. When applied in the chorioallantoic membrane assay, CAF from 9 day chicken embryos elicits a strong angiogenic response. This angiogenic activity of CAF is associated with pronounced mitogenic effects in vitro. Comparison of different embryonic fluids reveals that mitogenic activity is particularly evident in the CAF but not detectable in embryonic serum and amnion fluid. Expression of mitogenic activity is found to be temporally correlated with vascular <em>growth</em> in the CAM. High activity is detected in CAF prior to day 10 and then sharply decreases, thus preceding termination of capillary <em>growth</em> by one day. Heparin-sepharose affinity chromatography suggests that the biological activities of CAF probably correspond to the presence of acidic and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF and bFGF). In Western blot analyses of CAF, an immunoreactive bFGF-like protein of about <em>17</em> x 10(3) Mr is recognized by a monospecific anti-bFGF antiserum. This protein elutes at 2.4 M NaCl from the heparin-sepharose. The mitogenic activity of the CAF can be specifically blocked by the anti-bFGF antibody indicating bFGF to be the active mitogenic principle of the CAF. These results strongly suggest that basic and probably acidic FGF play an important role in the regulation of chorioallantoic vascular <em>growth</em>.

CTGF (connective-tissue <em>growth</em> <em>factor</em>) has been characterized as an extracellular-matrix-associated protein that modulates basic-<em>fibroblast</em>-<em>growth</em>-<em>factor</em> signalling and angiogenesis. In the present paper, the cloning of the ctgf gene from human umbilical-vein endothelial cells and expression of the protein in Escherichia coli as an N-terminal hexahistidine fusion protein is described. Recombinant human CTGF (rhCTGF) was expressed and purified so that we could investigate its effect on the proliferation of human embryo <em>fibroblast</em> KMB-<em>17</em> and NIH3T3 cells. The results indicated not only that the protein was properly folded, but also that it had the same specific activity and stability as the native protein. Furthermore, we administered this recombinant protein in a non-human primate [rhesus monkey (Macaca mulatta)] burn-wound model and report the clinical findings and structural effects. Epitheliotrophic effects were conspicuous in wounded tissues at 10-100 ng of CTGF/cm(2), suggesting that administered rhCTGF can play a normal physiological role in wound repairing in a non-human primate model.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> receptors (FGFRs) are tyrosine kinase receptors which have been implicated in breast cancer. The aim of this study was to evaluate FGFR-1, -2, -3, and -4 protein expressions in normal murine mammary gland development, and in murine and human breast carcinomas. Using immunohistochemistry and Western blot, we report a hormonal regulation of FGFR during postnatal mammary gland development. Progestin treatment of adult virgin mammary glands resulted in changes in localization of FGFR-3 from the cytoplasm to the nucleus, while treatment with <em>17</em>-β-estradiol induced changes in the expressions and/or localizations of FGFR-2 and -3. In murine mammary carcinomas showing different degrees of hormone dependence, we found progestin-induced increased expressions, mainly of FGFR-2 and -3. These receptors were constitutively activated in hormone-independent variants. We studied three luminal human breast cancer cell lines <em>growing</em> as xenografts, which particularly expressed FGFR-2 and -3, suggesting a correlation between hormonal status and FGFR expression. Most importantly, in breast cancer samples from 58 patients, we found a strong association (P < 0.01; Spearman correlation) between FGFR-2 and -3 expressions and a weaker correlation of each receptor with estrogen receptor expression. FGFR-4 correlated with c-erbB2 over expression. We conclude that FGFR-2 and -3 may be mechanistically linked and can be potential targets for treatment of estrogen receptor-positive breast cancer patients.

Epidermal <em>growth</em> <em>factor</em> (EGF) augments late fetal lung maturation by advancing the ontogeny of fetal lung development and by stimulating surfactant synthesis. Previous studies have indicated that fibroblastalveolar epithelial cell communications mediate surfactant synthesis in the fetal lung and EGF acts through such a mechanism. We investigated the hypothesis that is differential activity and expression of the epidermal <em>growth</em> <em>factor</em> receptor (EGF-R) in fetal lung <em>fibroblasts</em> during the canalicular stage of lung development mediates EGF effects. To test this hypothesis, we examined fetal rat lung <em>fibroblasts</em> (FLFs) and type II cells of late gestation (canalicular and saccular stages; <em>17</em>-22 days) by EGF-R binding techniques, SDS-PAGE, and Western blot analysis. Specific EGF binding increased 181% in day 18 female FLFs, with male FLFs exhibiting a similar increase on day 19. In contrast, specific EGF binding was low in type II cells, did not increase during late gestation, and there were no sex-specific differences. SDS-PAGE and Western blot analysis revealed a predominant <em>17</em>0-kDa EGF-R band in <em>fibroblasts</em> that increased with gestation (peak = 19 days), and was stronger in females. Immunoprecipitation of EGF-treated cells demonstrated the tyrosine kinase activity of the identified receptor. In contrast, type II cells showed minimal signal that did not increase until day 21 of gestation. We also examined whole fetal lung sections by immunohistochemistry to determine cell-specific expression of the EGF-R in vivo. Immunohistochemistry revealed specific EGF-R staining in columnar and cuboidal epithelia of small conducting airways and in mesenchyme of epithelial-mesenchymal borders (including subepithelial mesenchyme). In contrast, alveolar epithelia showed minimal staining, while subalveolar mesenchyme EGF-R staining peaked at day 19 of gestation. We conclude that cell-specific and sex-specific differences in EGF-R binding and EGF-R immunolocalization appears in the fetal lung at a developmental stage that is critical for alveolar epithelial cell differentiation. The results suggest a role for EGF-R activation in late fetal alveolar epithelial cell maturation, which is mediated through mesenchymal-epithelial cell communication.

Oligodendrocytes, the myelinating cells of the central nervous system, arise from a profilerating pool of motile progenitor cells. The proliferation and survival of these cells is dependent on signal transduction via several protein tyrosine kinases (PTKs) including receptors for <em>fibroblast</em> <em>growth</em> <em>factor</em> -2, the platelet-derived <em>growth</em> <em>factor</em> receptors and the neurotrophin receptor, trkC. We hypothesized that additional PTKs could also influence oligodendroglial development. Utilizing RTPCR, we amplified from post-natal day 6 rat oligodendroglia <em>17</em> distinct kinase domain sequences, 14 of which were not previously known to be expressed by oligodendroglia. Amongst the sequences identified were the c-met and Fak genes, whose protein products regulate the motility of other epithelial cell types. Utilizing immunohistochemistry, we confirmed that both c-met and Fak are expressed by cultured oligodendroglia, suggesting that these proteins could also be implicated in regulating the motility of these cells.

Primary cultures of rat hippocampal cells have been used to evaluate trophic effects of neurotrophin-3, brain-derived neurotrophic <em>factor</em>, nerve <em>growth</em> <em>factor</em>, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. There was little survival in cultures prepared from embryonic day <em>17</em> embryos and grown in defined medium without <em>growth</em> <em>factors</em>. Addition of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> produced a massive increase in the number of neurons present in the cultures seven days after plating. This action reflected proliferation of neuronal precursor cells rather than increased survival of initially plated neurons. Brain-derived neurotrophic <em>factor</em> was ineffective under these conditions, whereas neurotrophin-3 produced a very small, but statistically significant increase in neuronal survival in the range of 20%. However, hippocampal neurons were responsive to brain-derived neurotrophic <em>factor</em> and neurotrophin-3 as demonstrated under culture conditions, resulting in survival in absence of the neurotrophins. Acute administration of brain-derived neurotrophic <em>factor</em> and neurotrophin-3 to hippocampal cultures grown at high density stimulated the hydrolysis of phosphatidylinositol, a response earlier shown to be mediated by tyrosine receptor kinase neurotrophin receptors. Furthermore, when such cultures were grown in presence of neurotrophin-3 rates of glutamate and GABA uptake were increased. In contrast to the findings obtained in cultures of embryonic day <em>17</em>, cultures prepared from embryonic day 14 or 15 animals were viable in absence of exogenous <em>growth</em> <em>factors</em>. The specific neurotrophin receptor inhibitor, K-252b reduced survival in these cultures and this effect was partly overcome by exogenous neurotrophin-3. Our findings suggest that hippocampal neuron survival at early embryonic stages may involve paracrine neurotrophin mechanisms, whereas the survival of hippocampal neurons of embryonic day <em>17</em> is not markedly enhanced by brain-derived neurotrophic <em>factor</em> or neurotrophin-3. However, at this embryonic stage there is a functional response to both neurotrophins as made evident by the activation of tyrosine kinase receptor-linked signal transduction mechanisms and by the stimulation of transmitter-specific differentiation.

OBJECTIVE

To describe a case of persistent tumor-induced osteomalacia, determine whether serum fibroblast growth factor-23 (FGF-23) levels postoperatively indicate incomplete tumor resection, and report lumbar spine and forearm bone mineral density (BMD) changes during 5 years of follow-up.

METHODS

We present clinical, radiologic, histologic, and bone densitometry data as well as serum FGF-23 levels (determined with use of a novel C-terminal enzyme-linked immunosorbent assay) from the study patient and discuss these findings in the context of previous literature.

RESULTS

A 52-year-old man, who presented with muscle weakness and multiple fractures, was found to have low values for serum phosphorus, serum 1,25-dihydroxyvitamin D, and maximal tubular reabsorption of phosphate per glomerular filtration rate, a high level of serum alkaline phosphatase, and a normal serum concentration of parathyroid hormone, characteristic of tumor-induced osteomalacia. Magnetic resonance imaging to evaluate an abnormality of the left foot revealed a soft tissue mass, biopsy of which confirmed the presence of a benign, phosphaturic, mesenchymal tumor. The baseline serum FGF-23 level (2,050 RU/mL) was more than 17 times the upper limit of normal for adults (23 to 118 RU/mL) and decreased substantially within 1 day after partial resection of the tumor but remained above normal postoperatively. BMD changes indicated rapid substantial recovery of vertebral BMD but ongoing loss of forearm bone density.

CONCLUSIONS

The serum FGF-23 level is high in a substantial proportion of patients with tumor-induced osteomalacia. The postoperative above normal levels of serum FGF-23 correlated with known persistence of tumor in our study patient. In a patient with normal renal function, such as our study patient, levels of serum FGF-23 studied with use of the C-terminal enzyme-linked immunosorbent assay reached their nadir within 24 hours postoperatively. This result suggests that this assay can provide clinicians with rapid prognostic information in patients with known or suspected residual tumor. BMD should be assessed at both appendicular and axial sites in patients with persistent tumor-induced osteomalacia.

This study tested the hypothesis that fluid shear stress regulates the release of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-2 from human aortic smooth muscle cells. FGF-2 is a potent mitogen that is involved in the response to vascular injury and is expressed in a wide variety of cell types. FGF-2 is found in the cytoplasm of cells and outside cells, where it associates with extracellular proteoglycans. To test the hypothesis that shear stress regulates FGF-2 release, cells were exposed to flow, and FGF-2 amounts were measured from the conditioned medium, pericellular fraction (extracted by heparin treatment), and cell lysate. Results from the present study show that after 15 minutes of shear stress at 25 dyne/cm(2) in a parallel-plate flow system, a small but significant fraction (<em>17</em>%) of the total FGF-2 was released from human aortic smooth muscle cells. FGF-2 levels in the circulating medium increased 10-fold over medium from static controls (P<0.01). A 50% increase in FGF-2 content versus control (P<0.01) was found in the pericellular fraction (extracted by heparin treatment). Furthermore, a significant decrease in FGF-2 was detected in the cell lysate, indicating that FGF-2 was released from inside the cell. Cell permeability studies with fluorescent dextran were performed to examine whether transient membrane disruption caused FGF-2 release. Flow cytometry detected a 50% increase in mean fluorescence of cells exposed to 25 dyne/cm(2) versus control cells. This indicates that the observed FGF-2 release from human aortic smooth muscle cells is likely due to transient membrane disruption on initiation of flow.

Interleukin-11 (IL-11) is a stromal cell-derived cytokine that belongs to the interleukin-6 family of cytokines. IL-11 has many biological activities and has roles in hematopoiesis, immune responses, the nervous system and bone metabolism. Bone sialoprotein (BSP) is a mineralized tissue-specific protein expressed in differentiated osteoblasts that appears to function in the initial mineralization of bone. IL-11 (20 ng/ml) increased BSP mRNA and protein levels at 12h in osteoblast-like ROS <em>17</em>/2.8 cells. In a transient transfection assay, IL-11 (20 ng/ml) increased luciferase activity of the construct (-116 to +60) in ROS <em>17</em>/2.8 cells and rat bone marrow stromal cells. Introduction of 2 bp mutations to the luciferase constructs showed that the effects of IL-11 were mediated by a cAMP response element (CRE), a <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 response element (FRE) and a homeodomain protein-binding site (HOX). Luciferase activities induced by IL-11 were blocked by protein kinase A inhibitor, tyrosine kinase inhibitor and ERK1/2 inhibitor. Gel shift analyses showed that IL-11 (20 ng/ml) increased nuclear protein binding to CRE, FRE and HOX. CREB1, phospho-CREB1, c-Fos, c-Jun, JunD and Fra2 antibodies disrupted the formation of CRE-protein complexes. Dlx5, Msx2, Runx2 and Smad1 antibodies disrupted FRE- and HOX-protein complex formations. These studies demonstrate that IL-11 stimulates BSP transcription by targeting CRE, FRE and HOX sites in the proximal promoter of the rat BSP gene. Moreover, phospho-CREB1, c-Fos, c-Jun, JunD, Fra2, Dlx5, Msx2, Runx2 and Smadl transcription <em>factors</em> appear to be key regulators of IL-11 effects on BSP transcription.

The mitogenic activity in the unfractionated mixture of proteins released from adult bovine bone matrix during demineralization with ethylenediaminetetraacetate (EDTA) has been examined. Bovine bone extract (BBE) from 1 to 25 micrograms protein per ml stimulated proliferation of chick embryo calvaria bone cells, newborn mouse bone cells, and osteoblastlike cell lines MMB-1 and ROS <em>17</em>/2.8. BBE also stimulated DNA synthesis in cells from chick embryo cartilage, skin and skeletal muscle tissues and <em>fibroblast</em>like BALB/c 3T3 and NRK cells. BBE contained beta transforming <em>growth</em> <em>factor</em> (TGF) activity (NRK cell colony formation in soft agar in the presence of epidermal <em>growth</em> <em>factor</em> EGF). The cell specificity results suggest that BBE contains more than one <em>growth</em> <em>factor</em>, including a beta TGF and a <em>factor</em> that is not specific for bone cells, and all of the bone derived <em>growth</em> <em>factor</em> activities that have been described previously, including SGF, are apparently present in BBE. Maximal stimulation of chick embryo calvarial cell DNA synthesis by BBE was equal to or exceeded maximal stimulation by nonosseous <em>growth</em> <em>factors</em> that have been reported to stimulate DNA synthesis in bone organ cultures (EGF, <em>fibroblast</em> <em>growth</em> <em>factor</em>, platelet-derived <em>growth</em> <em>factor</em>, insulinlike <em>growth</em> <em>factor</em> I, and multiplication stimulating activity). Combinations of BBE with maximally stimulatory concentrations of each <em>growth</em> <em>factor</em> stimulated DNA synthesis to a greater magnitude than did each <em>growth</em> <em>factor</em> alone. These results suggest that combinations of bone derived and systemic <em>factors</em> can coordinately stimulate bone cell proliferation.

Direct cell to cell contact has been suggested as the means whereby <em>fibroblast</em> <em>factors</em> influence epithelial cell differentiation in the developing lung. To obtain further evidence for this hypothesis, the role of epithelial-mesenchymal cell contacts is now examined when lung development is accelerated after steroid injection. Male and female rat fetuses were studied from day <em>17</em> to day 22 of gestation. The fetuses were from mothers injected with 2 mg/kg of dexamethasone at 2 days before death and [3H]thymidine at 1 hour before death. Steroid injected during the rapid <em>growth</em> phase reduced total DNA in fetal lung, and from autoradiographs, the effect was most noticeable in epithelial cells. Differentiation began sooner after steroid; more epithelial cells contained lamellar bodies and disaturated phosphatidylcholine levels were higher. From quantitative ultrastructural studies, these changes in phospholipid correlated with an increased frequency of epithelial-interstitial cell contacts. Steroid treatment did not abolish the sex-related difference seen in controls; both males and females showed increases in cell to cell contacts and in lipid synthesis in the lung. The results demonstrate that acceleration of epithelial cell maturation with increased surfactant synthesis is associated with increased epithelial-interstitial cell contacts. This supports the concept that regulatory messages from the fetal <em>fibroblast</em> are passed directly to specific epithelial cells to initiate surfactant synthesis.

Undifferentiated fetal rat lung epithelial cells were isolated on gestational days 15 or <em>17</em> (term 22 days) and cultured in a defined medium. On plastic, most of the cells developed structurally abnormal lamellar bodies. On a basement membrane matrix (BMM), they sequentially accumulated glycogen and formed typical lamellar bodies. Biochemical analysis of the latter indicated that they had a phospholipid composition typical of surfactant for cells on BMM but not on plastic and that surfactant protein A appeared on BMM only. Progressing maturation from day 1 to day 6 in culture was demonstrated for <em>17</em>-day cells on BMM by a sevenfold increase of labeled precursor incorporation into surfactant phospholipids. Exposure to medium conditioned by 21-day fetal <em>fibroblasts</em> enhanced incorporation already after a 1-day culture. The antisteroid RU 486 had no effect on differentiation, whereas transforming <em>growth</em> <em>factor</em>-beta, a <em>factor</em> produced by lung mesenchyme at early fetal stages, inhibited it markedly. Alveolar epithelial type II cells appear to be committed early, but their maturational process would be prevented until a definite gestational stage.

The induction of dihydrofolate reductase (DHFR), a key enzyme in DNA biosynthesis that is induced just before the onset of S phase, is markedly attenuated in senescent human <em>fibroblasts</em> (Pang and Chen, 1994, J. Cell. Physiol., 160:531-538). Footprinting analysis of the 365 bp promoter region of the human DHFR gene (-381 to -<em>17</em>) indicated that nuclear proteins bind to a cluster of cis-elements, including two overlapping E2F binding sequences, two Sp1 sites, and one Yi sequence. Gel mobility shift assays were performed to assess the role of each cis-element in the regulation of DHFR gene expression. We found that 1) Sp1 binding activity was constitutively expressed throughout the cell cycle in early passage and senescent cells; 2) Yi binding activity was undetectable in both early passage and senescent cells; and 3) E2F binding activity was serum-inducible, senescence-dependent, and prominent in presenescent cells but strikingly diminished in senescent cells. Northern blot analysis of the expression of E2F and DP family members showed that the E2F-1, E2F-4, and E2F-5 mRNA was <em>growth</em>- and senescence-dependent, whereas E2F-3, DP-1, and DP-2 expression was constitutive and senescence-independent. In contrast, E2F-2 mRNA was not detectable in IMR-90 or WI-38 human <em>fibroblasts</em>. Western blot analysis showed that among the E2F-associated proteins, the expression of E2F-1, cyclin A, and cyclin B but not p107 was cell cycle- and senescence-dependent. A nuclear extract mixing experiment suggested that an inhibitory <em>factor</em> may further reduce E2F binding activity in senescent cells.

Preantral (stages 1-6) and antral (stages 7-10) follicles from proestrous hamsters were exposed for 24 h to 1, 5, 10, 50, and 100 ng/ml of epidermal <em>growth</em> <em>factor</em> (EGF), insulin-like <em>growth</em> <em>factor</em>-I (IGF-I), and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) and 1 microCi [3H]thymidine to determine the rate of DNA replication. FSH (100 ng/ml) was used as a positive control. Synergism was also studied by using suboptimal doses of <em>growth</em> <em>factors</em> (1 ng/ml) and FSH (5 ng/ml). Optimal doses (50 ng/ml) of EGF, IGF-I, and FGF, and 100 ng/ml FSH significantly enhanced follicular DNA replication, whereas suboptimal doses of FSH, EGF, and IGF-I affected only a few preantral stages, and FGF was totally ineffective. FGF significantly inhibited DNA synthesis induced by a suboptimal dose of EGF but did not affect IGF-I-induced DNA replication. Paradoxically, a combination of suboptimal doses of all three <em>growth</em> <em>factors</em> significantly (p less than 0.05) stimulated DNA synthesis for all stages; FSH (5 ng/ml) had no additive effect. FSH significantly stimulated follicular progesterone (P4), androstenedione (A), and estradiol-<em>17</em> beta (E2) accumulation, but significant P4 accumulation from stages 3-10 was observed only after optimal EGF exposure; A and E2 accumulations were unaffected by any of the <em>growth</em> <em>factors</em>. These results indicate that EGF, IGF-I, and FGF stimulate DNA replication to hamster preantral and antral follicles, and may play roles as intraovarian <em>factors</em> regulating folliculogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

To investigate the role of TNF-like weak inducer of apoptosis (TWEAK) in pathological adipose tissue (AT) remodeling and complications of obesity.

METHODS

Wild type (WT) and TWEAK knockout (KO) mice were fed normal diet (ND) or a high fat diet (HFD) for up to <em>17</em> weeks. Adipocyte death was induced using an established transgenic mouse model of inducible adipocyte apoptosis (FAT-ATTAC). Metabolic, biochemical, histologic, and flow cytometric analyses were performed.

RESULTS

TWEAK and its receptor, fibroblast growth factor-inducible molecule 14 (Fn14) were upregulated in gonadal (g)AT of WT mice after HFD week 4 and 24 h after induction of adipocyte apoptosis. Phenotypes of KO and WT mouse were indistinguishable through HFD week 8. However, at week <em>17</em> obese KO mice had ∼30% larger gAT adipocytes and gAT mass than WT mice, coincident with reduced adipocyte death, enhanced insulin signaling, Th2/M2 immune skewing, fewer thick collagen fibers, and altered expression of extracellular matrix constituents and modulators that is consistent with reduced fibrosis and larger adipocytes. KO mice were less steatotic and became more insulin sensitive and glucose tolerant than WT mice after HFD week 12.

CONCLUSIONS

TWEAK constrains "healthy" gAT expansion and promotes metabolic complications in severe obesity.

Human <em>fibroblast</em> <em>growth</em> <em>factor</em> (hFGF-1) is a approximately <em>17</em> kDa heparin binding cytokine. It lacks the conventional hydrophobic N-terminal signal sequence and is secreted through non-classical secretion routes. Under stress, hFGF-1 is released as a multiprotein complex consisting of hFGF-1, S100A13 (a calcium binding protein), and p40 synaptotagmin (Syt1). Copper (Cu(2+)) is shown to be required for the formation of the multiprotein hFGF-1 release complex (Landriscina et al. ,2001; Di Serio et al., 2008). Syt1, containing the lipid binding C2B domain, is believed to play an important role in the eventual export of the hFGF-1 across the lipid bilayer. In this study, we characterize Cu(2+) and lipid interactions of the C2B domain of Syt1 using multidimensional NMR spectroscopy. The results highlight how Cu(2+) appears to stabilize the protein bound to pS vesicles. Cu(2+) and lipid binding interface mapped using 2D (1)H-(15)N heteronuclear single quantum coherence experiments reveal that residues in beta-strand I contributes to the unique Cu(2+) binding site in the C2B domain. In the absence of metal ions, residues located in Loop II and beta-strand IV contribute to binding to unilamelar pS vesicles. In the presence of Cu(2+), additional residues located in Loops I and III appear to stabilize the protein-lipid interactions. The results of this study provide valuable information towards understanding the molecular mechanism of the Cu(2+)-induced non-classical secretion of hFGF-1.

Neurite branching is essential for correct assembly of neural circuits, yet it remains a poorly understood process. For example, the neural cell adhesion molecule KAL-1/anosmin-1, which is mutated in Kallmann syndrome, regulates neurite branching through mechanisms largely unknown. Here, we show that KAL-1/anosmin-1 mediates neurite branching as an autocrine co-<em>factor</em> with EGL-<em>17</em>/FGF through a receptor complex consisting of the conserved cell adhesion molecule SAX-7/L1CAM and the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor EGL-15/FGFR. This protein complex, which appears conserved in humans, requires the immunoglobulin (Ig) domains of SAX-7/L1CAM and the FN(III) domains of KAL-1/anosmin-1 for formation in vitro as well as function in vivo. The kinase domain of the EGL-15/FGFR is required for branching, and genetic evidence suggests that ras-mediated signaling downstream of EGL-15/FGFR is necessary to effect branching. Our studies establish a molecular pathway that regulates neurite branching during development of the nervous system.

BACKGROUND

Murine data indicate that angiogenesis is central to the aetiopathogenesis of Kaposi's sarcoma (KS). Therefore, we measured angiogenic cytokines and growth factors in patients with AIDS-related KS during treatment with both antiretrovirals and second-line paclitaxel chemotherapy. Cytokines measured included tumour necrosis factor-alpha (TNF-alpha), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and the interleukins IL-2, -6 and -12.

METHODS

Enzyme-linked immunosorbent assays (ELISAs) were carried out to measure plasma cytokine levels in 17 patients with AIDS-related KS who had progressed within 6 months of receiving liposomal anthracyclines and were treated with paclitaxel 100 mg/m(2) every 2 weeks. Measurements were carried out before progression, at commencement and at the completion of paclitaxel.

RESULTS

The objective response rate to paclitaxel was 71% (95% confidence interval 60% to 81%). In 17 patients with AIDS-related KS, we observed eight partial responses and four complete responses. Patients with AIDS Clinical Trial Group stage T1 disease had higher plasma VEGF (P = 0.05) and lower plasma TNF-alpha levels (P = 0.05) than patients with earlier stage T0 KS. There were no correlations between plasma cytokines (bFGF, VEGF, TNF-alpha, and IL-2,-6 and -12) and the CD4 and CD8 cell counts or HIV-1 RNA viral load. Response to paclitaxel was associated with a fall in plasma IL-6 levels (P = 0.04) but no change in other cytokines. There were no significant changes in CD4, CD8, CD16/56, CD19 cell counts and HIV-1 viral loads during chemotherapy.

CONCLUSIONS

Angiogenic cytokines may correlate with KS disease extent but not with cellular immune function or HIV viraemia. Response to paclitaxel therapy correlates with a fall in plasma IL-6 levels and recent data indicate this may be a surrogate marker of KS-associated herpesvirus viral load. Overall, clinical response in KS correlates poorly with known angiogenic cytokines.

OBJECTIVE

To examine whether taurine (Tau) or its physiologic chlorinated derivative, taurine chloramine (Tau-CI), affects proliferation of, and proinflammatory cytokine (interleukin-6 [IL-6] and IL-8) production by, fibroblast-like synoviocytes (FLS) isolated from rheumatoid arthritis (RA) patients.

METHODS

FLS, isolated from the synovial tissue of 19 RA patients and cultured in vitro for 3-6 passages, were stimulated with the recombinant human cytokines IL-1beta (1 ng/ml), tumor necrosis factor alpha (TNFalpha; 10 ng/ml), or IL-17 (10 ng/ml) in the presence of either Tau or Tau-Cl, which were added at concentrations of 50-500 microM. Tau and Tau-Cl were added simultaneously with, 2 hours before, or 24 hours after the stimuli. The concentrations of IL-6 and IL-8 were determined in culture supernatants using specific enzyme-linked immunosorbent assays. Proliferation of FLS was estimated on the basis of 3H-thymidine incorporation into the cells, which were cultured for 72 hours in the presence of recombinant human basic fibroblast growth factor (bFGF) (1 ng/ml) and Tau or Tau-Cl, which were added simultaneously at the beginning of the culture.

RESULTS

Cultured in vitro, RA FLS spontaneously secreted low levels of IL-6 and IL-8, but when RA FLS were stimulated with IL-1beta, TNFalpha, or IL-17, significantly higher amounts of IL-6 and IL-8 were produced. Tau-Cl, but not Tau, inhibited cytokine-triggered synthesis of IL-6 (50% inhibitory concentration [IC50] approximately 225 microM) and IL-8 (IC50 approximately 450 microM) when added simultaneously with the stimuli. However, IL-17-induced production of IL-8 was not affected by Tau-Cl. In the cells prestimulated with IL-1beta for 24 hours, Tau-Cl still inhibited synthesis of IL-6, but did not affect IL-8 production. Moreover, Tau-Cl inhibited spontaneous and bFGF-triggered proliferation of FLS in a dose-dependent manner. Neither Tau nor Tau-Cl affected cell viability.

CONCLUSIONS

The results of these studies demonstrate that Tau-Cl inhibits production of proinflammatory cytokines by RA FLS, as well as proliferation of these cells. Thus, Tau-Cl may act as a physiologic modulator of FLS functions related to their pathogenic role in RA.

Human fetal osteoblast-like cells formed a regular multilayered structure in vitro with an extensive collagen-based extracellular matrix. With colloidal gold immunocytochemistry, labels for alkaline phosphatase and osteocalcin were distributed in a relatively diffuse pattern, in contrast to the bone <em>growth</em> <em>factors</em>, insulin-like <em>growth</em> <em>factors</em> I and II (IGF-I and IGF-II), transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta 1), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, which were colocalized in the collagenous matrix of the multilayer. The inclusion of <em>17</em> beta-estradiol (10(-11) to 10(-9) M) in the culture medium increased multilayer depths, increased labeling for IGF-I, IGF-II, and TGF-beta 1, and resulted in earlier detection of TGF-beta 1 label. In contrast, the increase in multilayer depth resulting from treatment with human platelets, an exogenous source of <em>growth</em> <em>factors</em>, was not accompanied by an increase in matrix IGF-I, IGF-II, or TGF-beta 1 label, suggesting a particular effect of estradiol to facilitate this process. Because <em>growth</em> <em>factors</em> in bone matrix may act as coupling agents when released during resorption, reduced <em>growth</em> <em>factor</em> incorporation in the presence of reduced sex steroid concentrations may lead to uncoupling of resorption and subsequent formation.

OBJECTIVE

We hypothesized that basic fibroblast growth factor (bFGF) may exert a role in carotid plaque instability by regulating the expression of matrix metalloproteinases (MMP).

METHODS

Plaques obtained from 40 consecutive patients undergoing carotid endarterectomy were preoperatively classified as soft or hard. Serum bFGF was pre- and postoperatively measured. The release of MMP-2 and MMP-9 in the blood serum, and the activity, production and expression in the carotid specimens was analyzed. Specific anti-bFGF inhibition tests were performed in vitro on human umbilical artery smooth muscle cells (HUASMC) to evaluate the role of bFGF in the activity, production and expression of MMP-2 and -9.

RESULTS

Twenty-one (53%) patients had a soft carotid plaque and 19 (48%) a hard plaque. Preoperative bFGF serum levels were higher in patients with soft plaques [soft=34 (28-39) pg/mL and hard=20 (17-22) pg/mL-p<0.001] and postoperatively returned to normal values (when compared to 10 healthy volunteers). The serum levels of MMP-2 in patients' with soft plaques were higher than those in patients' with hard plaques [soft=1222 (1190-1252) ng/mL and hard=748 (656-793)ng/mL-p<0.0001]. MMP-9 serum values were 26 (22-29) ng/mL for soft plaques and 18 (15-21) ng/mL for hard plaques (p<0.0001). We found increased activity, production and expression of MMP-2 and -9 in soft plaques compared to hard plaques (p<0.001). In vitro inhibition tests on HUASMC showed the direct influence of bFGF on the activity, production and expression of MMP-2 and -9 (p<0.001).

CONCLUSIONS

bFGF seems to exert a key role in carotid plaque instability regulating the activity, production and expression of MMP thus altering the physiologic homeostasis of the carotid plaque.

BACKGROUND

Children with faciocraniosynostosis present skull base abnormalities and may develop hydrocephalus or cerebellar tonsils ectopia (CTE). Several pathophysiological hypotheses were formulated in the past decades to explain these associations. However, no study has described in a genetically homogeneous population with confirmed fibroblast growth factor receptor type 2 (FGFR2) mutation eventual correlations between skull base abnormalities and hydrocephalus or CTE.

OBJECTIVE

To illustrate these features in children <2 years of age with a genetically confirmed FGFR2-related faciocraniosynostosis.

METHODS

We measured the foramen magnum area (FMA) and its sagittal and transversal components: the right, left, and mean area of the jugular foramen; the posterior fossa volume; and the cerebellar volume on preoperative millimetric computed tomography scan slices in 31 children with an FGFR2 mutation (14 with Crouzon syndrome, 11 with Apert syndrome, and 6 with Pfeiffer syndrome). They were compared with 17 children without synostosis. All children were <24 months of age. We correlated all these measures with the presence of hydrocephalus or CTE.

RESULTS

We observed a significantly small FMA in children with Crouzon (P = .03) and in children with Pfeiffer (P = .05) resulting from a reduced sagittal diameter (P = .02 for Crouzon and P = .002 for Pfeiffer). Hydrocephalus was associated with small FMA (P = .02). The jugular foramen area, posterior fossa volume, and cerebellar volume were not associated with hydrocephalus or CTE. Hydrocephalus and CTE were statistically associated (P = .002).

CONCLUSIONS

Hydrocephalus in FGFR2-related Crouzon and Pfeiffer syndromes is statistically associated with a small FMA. Hydrocephalus is statistically associated with CTE.

Cancer-associated <em>fibroblasts</em> (CAFs) are the main stromal cells in the tumour microenvironment (TME). We found that the distribution of CAFs was significantly increased with tumour progression and led to a poor prognosis. In vitro and in vivo assays revealed that CAFs enhanced colorectal cancer (CRC) metastasis. Based on extraction and identification of exosomes of CAFs and normal <em>fibroblasts</em> (NFs), CAFs-exo showed higher expression of miR-<em>17</em>-5p than NFs-exo and could deliver exosomal miR-<em>17</em>-5p from parental CAFs to CRC cells. Further exploration verified that miR-<em>17</em>-5p influenced CRC metastasis capacity and directly targeted 3'-untranslated regions (UTRs) of RUNX family transcription <em>factor</em> 3(RUNX3). Our findings further revealed that RUNX3 interacted with MYC proto-oncogene(MYC) and that both RUNX3 and MYC bound to the promoter of transforming <em>growth</em> <em>factor</em> beta1(TGF-β1) at base pairs 1005-1296, thereby activating the TGF-β signalling pathway and contributing to tumour progression. In addition, RUNX3/MYC/TGF-β1 signalling sustained autocrine TGF-β1 to activate CAFs, and activated CAFs released more exosomal miR-<em>17</em>-5p to CRC cells, forming a positive feedback loop for CRC progression. Taken together, these data provide a new understanding of the potential diagnostic value of exosomal miR-<em>17</em>-5p in CRC.

<strong class="sub-title"> Keywords: </strong> Cancer-associated <em>fibroblasts</em>; Colorectal cancer; Exosomes; miR-<em>17</em>-5p.

We studied three FLT3 ITD acute myeloid leukemia (AML) patients who relapsed after allogeneic haematopoietic stem cell transplantation (alloHSCT) and received multikinase inhibitor (MKI) sorafenib as part of salvage therapy. MKI was given to block the effect of FLT3 ITD mutation which powers proliferation of blast cells. However, the known facts that sorafenib is more effective in patents post alloHSCT suggested that this MKI can augment the immune system surveillance on leukaemia. In the present study, we investigated in depth the effect of sorafenib on the alloreactivity seen post-transplant including that on leukaemia. The patients (i) responded to the treatment with cessation of blasts which lasted 1, <em>17</em> and 42+ months, (ii) developed skin lesions with CD3+ cell invasion of the epidermis, (iii) had marrow infiltrated with CD8+ lymphocytes which co-expressed PD-1 (programmed cell death protein 1 receptor, CD279) in higher proportions than those in the blood (163±32 x103 cells/μl vs 38±8 x103 cells/μl, p<0.001). The Lymphoprep fraction of marrow cells investigated for the expression of genes involved in lymphocyte activation showed in the patients with long lasting complete remission (CR) a similar pattern characterized by (i) a low expression of nitric oxide synthase 2 (NOS2) and colony stimulating <em>factor</em> 2 (CSF2) as well as that of angiopoietin-like 4 (ANGPTL4) (supporting the immune response and anti-angiogenic) genes, and (ii) higher expression of <em>fibroblast</em> <em>growth</em> <em>factor</em> 1 (FGF1) and collagen type IV alpha 3 chain (COL4A3) as well as toll like receptor 9 (TLR9) and interleukin-12 (IL-12) (pro-inflammatory expression profile) genes as compared with the normal individual. The positive effect in one patient hardly justified the presence of unwanted effects (progressive chronic graft-versus-host disease (cGvHD) and avascular necrosis of the femur), which were in contrast negligible in the other patient. The anti-leukemic and unwanted effects of sorafenib do not rely on each other.