Members of high (22-, 22.5-, 24-, and 34-kDa) and low (18-kDa) molecular mass forms of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) regulate cell proliferation, differentiation, and migration. FGF-2s have been previously shown to accumulate in the nucleus and nucleolus. Although high molecular weight forms of FGF-2 contain at least one nuclear localization signal (NLS) in their N-terminal extension, the 18-kDa FGF-2 does not contain a standard NLS. To determine signals controlling the nuclear and subnuclear localization of the 18-kDa FGF-2, its full-length cDNA was fused to that of green fluorescent protein (GFP). The fusion protein was primarily localized to the nucleus of COS-7 and HeLa cells and accumulated in the nucleolus. The subcellular distribution was confirmed using wild type FGF-2 and FGF-2 tagged with a FLAG epitope. A <em>17</em>-amino acid sequence containing two groups of basic amino acid residues separated by eight amino acid residues directed GFP and a GFP dimer into the nucleus. We systematically mutated the basic amino acid residues in this nonclassical NLS and determined the effect on nuclear and nucleolar accumulation of 18-kDa FGF-2. Lys(119) and Arg(129) are the key amino acid residues in both nuclear and nucleolar localization, whereas Lys(128) regulates only nucleolar localization of 18-kDa FGF-2. Together, these results demonstrate that the 18-kDa FGF-2 harbors a C-terminal nonclassical bipartite NLS, a portion of which also regulates its nucleolar localization.

A receptor for <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF, bFGF) was partially characterized in intact cell cultures, cell plasma membranes, and tissue plasma membrane preparations. Analysis of 24 different cell types from four species identified a 165-kDa FGF receptor present on the cell surface of most mesodermal and neuroectodermal cells. Chemical crosslinking of 125I-aFGF to its cell surface receptor was specifically blocked by a 100-fold molar excess of either aFGF or bFGF. In contrast to the similar molecular weight of FGF receptors, different cell types exhibited significant variations in binding of 125I-aFGF to intact cultures with low values of 8 pM and 700, to high values of 60 pM and 30,000, for the Kd and receptor number per cell, respectively. A binding assay was developed for quantitation of 125I-aFGF binding to cell- and tissue-derived membrane preparations. Membranes prepared from baby hamster kidney cells exhibited a Kd of 55 pM, while a similar Kd of 67 pM was determined for intact baby hamster kidney cells. Although ten different adult bovine tissue membrane preparations and human term placental membranes exhibited no specific binding of 125I-aFGF, FGF receptor was detected in embryonic murine tissues (<em>17</em> days gestation). These results support the existence, in a variety of cells, of either a common FGF receptor that binds both aFGF and bFGF or closely related FGF receptors that cannot be distinguished by molecular weight. The differential binding of FGF to its receptor in embryonic vs. adult tissues suggests a potentially broad role for FGF in embryonic development and a more restrictive role in the adult.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) signalling plays a major role during early vertebrate development. It is involved in the specification of the mesoderm, control of morphogenetic movements, patterning of the anterior-posterior axis, and neural induction. In mammals, 22 FGF ligands have been identified, which can be grouped into seven subfamilies according to their sequence homology and function. We have cloned <em>17</em> fgf genes from Xenopus tropicalis and have analysed their temporal expression by RT-PCR and spatial expression by whole mount in situ hybridisation at key developmental stages. It reveals the diverse expression pattern of fgf genes during early embryonic development. Furthermore, our analysis shows the transient nature of expression of several fgfs in a number of embryonic tissues. This study constitutes the most comprehensive description of the temporal and spatial expression pattern of fgf ligands and receptors during vertebrate development to date. Developmental Dynamics 238:1467-1479, 2009. (c) 2009 Wiley-Liss, Inc.

Transforming <em>growth</em> <em>factor</em> beta (TGF-beta), a potent regulator of bone formation, has bifunctional effects on osteoblast replication and biochemical activity that appear differentiation dependent. We now show that cell surface binding sites for TGF-beta vary markedly among <em>fibroblasts</em>, bone-derived cells, and highly differentiated osteosarcoma cultures from fetal rats. Expression of betaglycan and type II receptors decline relative to type I receptor expression in parallel with an increase in osteoblast-like activity, predicting that the ratio among various TGF-beta binding sites could influence how its signals are perceived. Bone morphogenetic protein 2 (BMP-2), which induces osteoblast function, does not alter TGF-beta binding or biochemical activity in <em>fibroblasts</em> and has only small effects in less differentiated bone cells. In contrast, BMP-2 rapidly reduces TGF-beta binding to betaglycan and type II receptors in osteoblast-enriched primary cell cultures and increases its relative binding to type I receptors in these cells and in ROS <em>17</em>/2.8 cultures. Pretreatment with BMP-2 diminishes TGF-beta-induced DNA synthesis in osteoblast-enriched cultures but synergistically enhances its stimulatory effects on either collagen synthesis or alkaline phosphatase activity, depending on the present state of bone cell differentiation. Therefore, BMP-2 shifts the TGF-beta binding profile on bone cells in ways that are consistent with progressive expression of osteoblast phenotype, and these changes distinguish the biochemical effects mediated by each receptor. Our observations indicate specific stepwise actions by TGF-beta family members during osteoblast differentiation, developing in part from changes imprinted by BMP-2 on TGF-beta receptor stoichiometry.

Signaling by <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) and their receptors has been previously implicated in control of cell proliferation, differentiation and migration. Here we report a novel role for signaling by the EGL-15 FGFR of Caenorhabditis elegans in controlling protein degradation in differentiated muscle. Activation of EGL-15, by means of a reduction of function mutation (clr-1) affecting an inhibitory phosphatase, triggers protein degradation in adult muscle cells using a pre-existing proteolytic system. This activation is not suppressed by mutation in either of the known genes encoding FGF ligands (egl-<em>17</em> or let-756) but is well suppressed when both are mutated, indicating that either ligand is sufficient and at least one is necessary for FGFR activation. Activity of the Ras pathway through mitogen-activated protein kinase (MAPK) is required to trigger protein degradation. This is the first report that degradation of intracellular protein can be triggered by a <em>growth</em> <em>factor</em> receptor using an identified signal transduction pathway. The data raise the possibility that FGF-triggered proteolysis may be relevant to muscle remodeling or dedifferentiation.

We have developed a genetic method to determine the active orientation of dimeric transmembrane protein helices. The bovine papillomavirus E5 protein, a 44-amino acid homodimeric protein that appears to traverse membranes as a left-handed coiled-coil, transforms <em>fibroblasts</em> by binding and activating the platelet-derived <em>growth</em> <em>factor</em> (PDGF) beta receptor. A heterologous dimerization domain was used to force E5 monomers to adopt all seven possible symmetric coiled-coil registries relative to one another within the dimer. Focus formation assays demonstrated that dimerization of the E5 protein is required for transformation and identified a single preferred orientation of the monomers. The essential glutamine residue at position <em>17</em> resided in the dimer interface in this active orientation. The active chimera formed complexes with the PDGF beta receptor and induced receptor tyrosine phosphorylation. We also identified E5-like structures that underwent non-productive interactions with the receptor.

Phosphaturic mesenchymal tumors of the mixed connective tissue type (PMTMCT) are extremely rare, histologically distinctive neoplasms, which cause tumor-induced osteomalacia (TIO) in most cases through the elaboration of a phosphaturic hormone, <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 (FGF23). Rarely, identical tumors without known TIO may be observed. We studied a large group of PMTMCT for expression of FGF23, using a novel reverse transcription polymerase chain reaction (RT-PCR) assay for FGF23 in formalin-fixed, paraffin-embedded tissues. Twenty-nine PMTMCT (<em>17</em> with and 12 without TIO) and 23 non-PMTMCT (16 various mesenchymal tumors, including 5 chondromyxoid fibroma, 8 chondroblastoma, 1 hemangiopericytoma, 1 aneurysmal bone cyst, and 1 high grade sarcoma; 5 carcinomas; and 2 non-neoplastic tissues) were retrieved. Total RNA was extracted from formalin-fixed, paraffin-embedded sections for RT-PCR analysis. FGF23 was amplified using 3 sets of primers that spanned the intron/exon boundaries to amplify the 3 exons of FGF23 gene (140, 125, and <em>17</em>5 bp). The housekeeping gene phosphoglycerokinase (189 bp) was coamplified to check the RNA quality. Sixteen of <em>17</em> (94%) PMTMCT with TIO were FGF23-positive. Nine of 12 (75%) PMTMCT without TIO were FGF23-positive. Two chondromyxoid fibroma and 1 aneurysmal bone cyst were positive; all other non-PMTMCT were negative. We conclude that RT-PCR for FGF23 is a sensitive and specific means of confirming the diagnosis of PMTMCT both in patients with and without TIO. FGF23 gene expression was present in more than 90% of PMTMCT with known TIO, confirming the role of FGF23 in this syndrome. Rare FGF23-negative PMTMCT with known TIO likely express other phosphaturic hormones (eg, frizzled-related protein 4). Our finding of expression of FGF23 in 75% of histologically identical tumors without known TIO confirms the reproducibility of the diagnosis of PMTMCT, even in the absence of known phosphaturia.

BACKGROUND

Pentraxin 3 (PTX3) is a soluble pattern recognition receptor with non-redundant functions in inflammation and innate immunity. PTX3 is produced by immune and structural cells. However, very little is known about the expression of PTX3 and its role in allergic asthma.

OBJECTIVE

We sought to determine the PTX3 expression in asthmatic airways and its function in human airway smooth muscle cells (HASMC). In vivo PTX3 expression in bronchial biopsies of mild, moderate and severe asthmatics was analyzed by immunohistochemistry. PTX3 mRNA and protein were measured by real-time RT-PCR and ELISA, respectively. Proliferation and migration were examined using (3)H-thymidine incorporation, cell count and Boyden chamber assays.

RESULTS

PTX3 immunoreactivity was increased in bronchial tissues of allergic asthmatics compared to healthy controls, and mainly localized in the smooth muscle bundle. PTX3 protein was expressed constitutively by HASMC and was significantly up-regulated by TNF, and IL-1β but not by Th2 (IL-4, IL-9, IL-13), Th1 (IFN-γ), or Th-<em>17</em> (IL-<em>17</em>) cytokines. In vitro, HASMC released significantly higher levels of PTX3 at the baseline and upon TNF stimulation compared to airway epithelial cells (EC). Moreover, PTX3 induced CCL11/eotaxin-1 release whilst inhibited the <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2)-driven HASMC chemotactic activity.

CONCLUSIONS

Our data provide the first evidence that PTX3 expression is increased in asthmatic airways. HASMC can both produce and respond to PTX3. PTX3 is a potent inhibitor of HASMC migration induced by FGF-2 and can upregulate CCL11/eotaxin-1 release. These results raise the possibility that PTX3 may play a dual role in allergic asthma.

BACKGROUND

Diabetic nephropathy imposes a substantial cardiovascular and renal burden contributing to both morbidity and excess mortality. Progression of chronic kidney disease (CKD) in diabetes mellitus is variable, and few biomarkers are available to predict progression accurately. Identification of novel predictive biomarkers may inform clinical care and assist in the design of clinical trials. We hypothesized that urinary and plasma protein biomarkers predict CKD progression independently of the known clinical markers such as albuminuria and estimated glomerular filtration rate (eGFR) in diabetic nephropathy.

METHODS

We studied 67 US veterans with CKD due to type 2 diabetes mellitus and 20 age-matched controls (no CKD, hypertension or cardiovascular disease). After clinical evaluation and the collection of blood and urine specimens for 24 biomarkers, we followed subjects prospectively for the next 2-6 years. CKD progression was defined in three ways: (i) clinically by examining eGFR versus time plots for each individual (slope progression), (ii) progression to end-stage renal disease (ESRD) and (iii) a composite outcome of ESRD or death.

RESULTS

Among <em>17</em> urinary and 7 plasma biomarkers evaluated, the relationship of the biomarkers with outcome was as follows: (i) for progression identified by eGFR plots, urinary C-terminal <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-23 emerged to have the strongest primary association (adjusted odds ratio [aOR] 2.08, P = 0.008); (ii) for ESRD, plasma vascular endothelial <em>growth</em> <em>factor</em> (VEGF) had an association (aOR: 1.44, P = 0.027) and (iii) for the composite outcome of death and ESRD, plasma C-terminal FGF-23 also had a robust direct association (aOR: 3.07, P = 0.008).

CONCLUSIONS

The relationship of biomarkers with future progression of CKD is complex and depends in part on how CKD progression is defined. Biomarkers in the FGF-23 and VEGF-A pathways predicted patient progression independently of albuminuria levels in this patient cohort. Additional studies in other cohorts will help further validate this pilot study.

OBJECTIVE

The cytochrome P450 enzyme CYP7B catalyzes the conversion of dehydroepiandrosterone (DHEA) into 7alpha-hydroxy-DHEA (7alpha-OH-DHEA). This metabolite can stimulate the immune response. We previously reported that the severity of murine collagen-induced arthritis is correlated with CYP7B messenger RNA (mRNA) expression and activity in the arthritic joint. The purpose of this study was to investigate the presence of 7alpha-OH-DHEA in synovial samples and the cytokine regulation of CYP7B activity in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA).

METHODS

The presence of 7alpha-OH-DHEA was examined in synovial biopsy tissues, synovial fluid, and serum by radioimmunoassay. The effect of cytokines on CYP7B mRNA expression and CYP7B activity in FLS was examined by determining the formation of the CYP7B enzyme product 7alpha-OH-DHEA with the use of high-performance liquid chromatography.

RESULTS

The CYP7B enzyme product 7alpha-OH-DHEA was found in synovial biopsy tissues, synovial fluid, and serum from RA patients. The proinflammatory cytokines tumor necrosis factor alpha (TNFalpha), interleukin-1alpha (IL-1alpha), IL-1beta, and IL-17 up-regulated CYP7B activity in an FLS cell line 2-10-fold. Enhanced CYP7B activity was correlated with an increase in CYP7B mRNA. The cytokine transforming growth factor beta inhibited CYP7B activity. Moreover, CYP7B activity was detected in 10 of 13 unstimulated synovial fibroblast cell lines. Stimulation with TNFalpha increased CYP7B activity in all cell lines tested.

CONCLUSIONS

Exposure to the proinflammatory cytokines TNFalpha, IL-1alpha, IL-1beta, and IL-17 increases CYP7B activity in synovial tissue. Increased CYP7B activity leads to higher levels of the DHEA metabolite 7alpha-OH-DHEA in synovial fluid, which may contribute to the maintenance of the chronic inflammation observed in RA patients.

OBJECTIVE

The purpose of this study was to characterize cell cultures and xenografts derived from patients with ovarian cancer.

METHODS

Ninety specimens from 67 patients were plated in RPMI 1640 or inoculated in nude mice. Growth characteristics of cell cultures and xenografts were determined. Expression of receptors for estrogen, progesterone, androgen, epithelial growth factor, fibroblast growth factor, HER-2/erbB-2/c-neu proto-oncogene, and the P53 expression were characterized by immunocytochemistry in 28 cell cultures.

RESULTS

Forty-nine percent of samples were cultured successfully in vitro. Ascitic and pleural effusion specimens were more likely to produce a cell culture or a xenograft than solid tissue specimens (P < 0.005). All of the cell cultures had an epithelial morphology, and 89% were aneuploid with a mean DNA index of 1.6 (range, 0.9-3.0). Of 54 and 61 specimens inoculated into nude mice i.p. and s.c., 15 (28%) and 18 (30%) produced a xenograft, respectively, with two-thirds of these xenografts being reproducibly tumorigenic. The median time to first passage was 21 weeks for cell cultures and 8-12 weeks for xenografts. Expression of epithelial growth factor receptor, HER-2/erbB-2/c-neu proto-oncogene, fibroblast growth factor receptor, estrogen, progesterone, and androgen was seen in 24, 21, 31, 17, 43, and 18%, respectively. P53 was overexpressed in 62% of cell cultures analyzed.

CONCLUSIONS

Ovarian cancer cells collected from effusions are easier to grow in vitro than in vivo. The only characteristic that may be associated with tumorigenicity was abnormal P53 expression. This panel of ovarian cancer materials provides useful models for biological or therapeutical studies.

Acidic (a) and basic (b) <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) are two related mitogenic and angiogenic <em>factors</em>. They are multifunctional in that they can affect proliferation and induce or delay differentiation. Both aFGF and bFGF were shown to stimulate proliferation of calvaria cells in situ as well as osteoblast-enriched calvaria-derived cells. bFGF was also found to suppress the expression of alkaline phosphatase, parathyroid hormone stimulatable adenylate cyclase, osteocalcin, and type I collagen in the osteoblastic ROS <em>17</em>/2.8 cells. To explore a possible role for guanine nucleotide binding proteins we assessed the effects of pertussis toxin (PT) on FGF action. PT had opposite effects to those of bFGF on all parameters examined.

Angora is an autosomal recessive mouse mutation caused by a deletion of approximately 2 kilobases in the <em>fibroblast</em> <em>growth</em> <em>factor</em> 5 (Fgf5) gene. Phenotypically, homozygous angora (Fgf5go/Fgf5go) mice have excessively long truncal hair and can be differentiated from heterozygous (+/Fgf5go) and wild-type (+/+) littermates by 21 days of age. Abnormal hair length is due to a prolongation of the anagen phase of the hair cycle of approximately 3 days. In addition, widely scattered hair follicles produce structurally defective hair shafts that twist within the follicle, presumably causing secondary hyperplasia of the outer root sheath and epidermis adjacent to the follicle. These follicular abnormalities were accentuated by immunohistochemical detection of mouse specific keratin 6, a nonspecific marker of epidermal hyperplasia. These abnormalities could be identified from birth throughout life in angora mice genotyped by polymerase chain reaction techniques. Moreover, the long truncal hair phenotype was maintained in skin grafted onto C.B-<em>17</em>/Sz-scid/scid mice that had normal pelage hairs and hair cycles, suggesting that circulating or diffusible humoral <em>factors</em> regulating the mouse hair cycle are not involved in this mutation. The angora mutation provides another useful mouse model for studying the hair cycle and its modulation.

Mast cells are tissue-bound cells of the innate immune system which are well known for immunoglobuline (Ig)E-triggered degranulation in allergic reactions. More recently, an important role of mast cells has been described in chronic inflammatory and autoimmune disorders which are often associated with fibrosis or sclerosis. Innate immune receptors such as Fc-, toll-like- or NOD-like receptors stimuli can trigger mast cell degranulation and enhance immunological danger signals. Whereas fulminant degranulation of mast cell vesicles is observed in anaphylaxis, piecemeal degranulation or transgranulation are mechanisms for a slower release of their granula. A cocktail of cytokines, <em>growth</em> <em>factors</em> and proteoglycans is produced and stored in granula of mast cells. Mast cells are a substantial reservoir of both preformed inflammatory <em>factors</em> (i.e., TNF-alpha and IL-<em>17</em>) and <em>factors</em> that can trigger a profibrotic, Th-2-polarised inflammation (i.e., IL-4 and IL-10). In systemic sclerosis, mast cell vesicles are the main source of transforming <em>growth</em> <em>factor</em> (TGF)-beta. Cell-to-cell contact between mast cells and <em>fibroblasts</em> occurs in the affected tissue, supporting the hypothesis that transgranulation might be an important mechanism in fibrosis. The direct release of proteoglycans such as hyaluronic acid into the interstitial space is a further stimulus for matrix remodelling. Mast cell hyperactivity has also been demonstrated in primary fibrotic disorders such as lung, cardiac or renal fibrosis. The exact trigger for mast cell degranulation however is not known. Notwithstanding, at a very early time point of fibrosis, mast cell inhibition by stabilisers or blockage of the tyrosine kinase receptor c-kit by masitinib could be a therapeutic option.

BACKGROUND

We sought to investigate the capacity of interleukin (IL)-7 to enhance collagen-induced arthritis and to study by what mechanisms this is achieved.

METHODS

Mice received multiple injections with IL-7 or phosphate-buffered saline (PBS) as a control. Arthritis severity and incidence were determined by visual examination of the paws. Joint destruction was determined by assessing radiographs and immunohistochemistry of the ankle joints. Total cellularity and numbers of T-cell and B-cell subsets were assessed, as well as ex vivo production of interferon-γ (IFN-γ), IL-<em>17</em>, and IL-4. Proinflammatory mediators were measured in serum with multianalyte profiling.

RESULTS

IL-7 increased arthritis severity and radiology-assessed joint destruction. This was consistent with IL-7-increased intensity of cell infiltrates, bone erosions, and cartilage damage. Splenic CD19+ B cells and CD19+/GL7+ germinal center B cells, as well as CD4 and CD8 numbers, were increased by IL-7. IL-7 expanded memory T cells, associated with increased percentages of IFN-γ-, IL-4-, and IL-<em>17</em>-producing CD4+ T cells. On antigen restimulation of draining lymph node cells in vitro IL-7 treatment was found to increase IFN-γ and IL-<em>17</em> production, whereas IL-4 was reduced. IL-7 also increased concentrations of proinflammatory mediators, indicative of T-cell activation (sCD40L), vascular activation (VCAM-1, VEGF), tissue destruction (fibroblast growth factor-basic (FGF-b), LIF), and chemotaxis (MIP-1γ, MIP-3β, lymphotactin, MDC, and MCP-5).

CONCLUSIONS

In arthritic mice, IL-7 causes expansion of T and B cells, associated with increased levels of proinflammatory mediators. IL-7 intensifies arthritis severity and joint destruction, accompanied by increased Th1 and Th<em>17</em> activity. These data indicate that IL-7 could be an important mediator in arthritic conditions and that targeting IL-7 or its receptor represent novel therapeutic strategies.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) regulate many important developmental and homeostatic physiological events. The FGF superfamily contains several families. In this review, we present recent findings on the two FGFs of the nematode Caenorhabditis elegans from both functional and phylogenic points of view. C. elegans has a single FGFR (EGL-15) with two functionally exclusive isoforms, and two FGFs (LET-756 and EGL-<em>17</em>), which play distinct roles: an essential function for the former, and guidance of the migrating sex myoblasts for the latter. Regulation of homeostasis by control of the fluid balance could be the basis for the essential function of LET-756. Phylogenetic and functional studies suggest that LET-756, like vertebrate FGF9, -16, and -20, belongs to the FGF9 family, whereas EGL-<em>17</em>, like vertebrate FGF8, -<em>17</em>, and -18, could be included in the FGF8 family.

Acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) is a potent mitogen that can induce angiogenesis in vivo. We have recently reported a marked increase of basic FGF in the pericardial fluid of patients with severe coronary stenosis and an increase in vascular endothelial <em>growth</em> <em>factor</em> (VEGF) in the pericardial fluid of patients with severe myocardial ischemia. The purpose of this study was to evaluate whether acidic FGF levels in the pericardial fluid are associated with severe myocardial ischemia. Immediately after incision of the pericardium in 48 patients during open-heart surgery, 3-5ml of pericardial fluid was obtained. Concentrations of basic FGF and VEGF in the pericardial fluid were measured using an enzyme-linked immunosorbent assay (ELISA). The ELISA system for human acidic FGF was newly developed using a rabbit antibovine acidic FGF antibody. The patients were divided into three groups (group A: 13 patients undergoing emergency coronary artery bypass grafting (CABG) for unstable angina; group B: <em>17</em> patients undergoing elective CABG for stable angina; group C: 18 patients undergoing nonischemic open-heart surgery). The VEGF level in the pericardial fluid in group A was 68 +/- 59pg/ml, which was significantly higher than 33 +/- 9 pg/ml in group B and 31 +/- 20 pg/ml in group C (P < 0.05). The concentrations of basic FGF in the pericardial fluid in groups A and B were 722 +/- 601 and 773 +/- 763pg/ml, respectively, significantly higher than 263 +/- 349pg/ml in group C. The pericardial acidic FGF level in group A was 4,291 +/- 2,336 pg/ml, which was also significantly higher than 2,386 +/- 1,048 pg/ml in group B and 2,589 +/- 990 pg/ml in group C (P < 0.05). The acidic FGF level correlated well with the level of VEGF (r = 0.61, P < 0.0001). It is concluded that the level of acidic FGF in pericardial fluid is associated with severe myocardial ischemia. This result indicates that the release of acidic FGF from the myocardial tissue into pericardial fluid is closely related to severe myocardial ischemia.

BACKGROUND

Chronic allograft nephropathy (CAN) is the most frequent cause of chronic dysfunction and late loss of renal allografts. Epithelial mesenchymal transition (EMT) has been identified as responsible for the presence of activated interstitial fibroblasts (myofibroblasts) and transforming growth factor beta (TGF-beta)/Smad is the key signaling mediator. It has been proposed that the bone morphogenetic protein 7 (BMP-7) antagonist, Gremlin, could participate in EMT, as a downstream mediator of TGF-beta.

METHODS

We evaluated 33 renal allograft biopsies, 16 of which showed CAN, versus 17 controls. By in situ hybridization we studied the expression of TGF-beta and Gremlin mRNA. Gremlin, BMP-7, E-cadherin, and alpha-smooth muscle actin (alpha-SMA) proteins were evaluated by immunohistochemistry and Smad3 activation by Southwestern. In cultured human tubuloepithelial cells (HK2 cell line), Gremlin induction by TGF-beta was studied by confocal microscopy.

RESULTS

Among renal biopsies of transplanted patients with CAN, we detected up-regulation of TGF-beta in colocalization with Gremlin (RNA and protein), mainly in areas of tubulointerstitial fibrosis. In the same tubules, we observed decreased expression of E-cadherin and induction of vimentin and alpha-SMA. BMP-7 was significantly decreased in the CAN biopsies. In addition, HK2 stimulated with TGF-beta (1 ng/mL) induced Gremlin production at 72 hours.

CONCLUSIONS

We postulated that Gremlin is a downstream mediator of TGF-beta, suggesting a role for Gremlin in EMT observed in CAN.

OBJECTIVE

Fibroblast growth factor 23 is a phosphate- and vitamin D-regulating hormone. The objective of this study was to determine the effect of ergocalciferol administration on fibroblast growth factor 23 levels in healthy vitamin D-deficient subjects.

METHODS

In this 12-week trial conducted in a clinical research center, 18- to 45-year-old subjects (n=90) with 25-hydroxyvitamin D levels ≤20 ng/ml (by chemiluminescent immunoassay) were randomized to weekly ergocalciferol treatment of 50,000 international units or placebo, while consuming a self-selected diet. Changes in fibroblast growth factor 23, 25-hydroxyvitamin D (by liquid chromatography/tandem mass spectroscopy), 1,25-dihydroxyvitamin D, parathyroid hormone, and serum phosphate were measured.

RESULTS

Mean 25-hydroxyvitamin D (P<0.0001), 1,25-dihydroxyvitamin D (P=0.01), and fibroblast growth factor 23 (P=0.003) increased in the treatment versus placebo group. In the treatment group, 25-hydroxyvitamin D increased from 18 ± 7 to 40 ± 12 ng/ml at week 4 (P<0.0001) and remained stable at 43 ± 12 ng/ml at week 12 (P<0.0001); 1,25-dihydroxyvitamin D increased from 42 ± 17 to 52 ± 18 pg/ml at week 4 (P<0.001) and then remained stable, and fibroblast growth factor 23 increased from 43 ± 17 to 60 ± 33 pg/ml at week 8 (P=0.001) and 74 ± 42 pg/ml at week 12 (P<0.0001). Urinary phosphate excretion increased within the treatment group, but parathyroid hormone and serum phosphate were unchanged.

CONCLUSIONS

Ergocalciferol administration increases circulating fibroblast growth factor 23. When measuring fibroblast growth factor 23, concurrent 25-hydroxyvitamin D measurements should be obtained, because vitamin D deficiency may lower circulating fibroblast growth factor 23 levels.

Neuropathic bladder dysfunction results from abnormal development of the spine, spinal cord injuries, or diseases such as diabetics. Patients with neuropathic bladders often require surgical intervention such as bladder reconstruction to improve incontinence and prevent renal damage. Tissue engineering with ex-vivo cultured bladder cells has been suggested as one means for improving bladder function. However, we previously demonstrated that cultured bladder smooth muscle cells (SMCs) derived from neuropathic bladder exhibit and maintain altered pathologic phenotypes in culture. To identify genes that are responsible for the abnormal neuropathic phenotypes specifically elevated cell proliferation, the expression levels of 1,185 genes were compared between cultured SMCs derived from normal and neuropathic bladders using a cDNA array consisting of well-annotated genes. The expression data were analyzed using several methods to identify differentially expressed genes. The resulting sets of differentially expressed genes were examined by pathway analysis to identify the networks that remain abnormal in the culture-stable phenotype of neuropathic SMCs. A total of 18 genes that are differentially expressed between cultured normal and neuropathic bladder SMCs were identified. Of these <em>17</em> were up-regulated greater than 2-fold in neuropathic bladder SMCs, six of them along with one gene that was not up-regulated greater than 2-fold in cultured neuropathic bladder SMCs were confirmed and identified by more stringent analysis methods including significance analysis of microarrays, class comparison, and class prediction analyses. The major dysregulated pathways include <em>fibroblast</em> <em>growth</em> <em>factor</em> signaling, PTEN signaling, and integrin signaling. Our results further suggest that altered neuropathic bladder SMC phenotypes is stable in the culture environments and that SMCs derived from diseased bladders may not be appropriate for tissue engineering purpose without modification of pathologically altered genes expression.

A recurrent point mutation in the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) gene that converts proline 250 into arginine is commonly associated with coronal craniosynostosis and has allowed definition of a new syndrome on a molecular basis. Sixty-two patients with sporadic or familial forms of coronal craniosynostosis were investigated for the P250R FGFR3 mutation. It was identified in 20 probands originating from 27 unrelated families (74%), while only 6/35 sporadic cases (<em>17</em>%) harboured the mutation. In both familial and sporadic cases, females were significantly more severely affected than males. Hence, while 68% of females carrying the P250R mutation showed brachycephaly, only 35% of males had the same phenotype. In the most severe forms of the disease, the association of bicoronal craniosynostosis with hypertelorism and marked bulging of the temporal fossae were common hallmarks that might be helpful for clinical diagnosis. Taken together, these results indicate that the P250R FGFR3 mutation is mostly familial and is associated with a more severe phenotype in females than in males. The sex related severity of the condition points to the possible implication of modifier genes in this syndrome.

OBJECTIVE

Adenoid cystic carcinoma (ACC) is a malignant salivary gland tumor with a high mortality rate due to late, distant metastases. This study aimed at unraveling common genetic abnormalities associated with ACC. Additionally, chromosomal changes were correlated with patient characteristics and survival.

METHODS

Microarray-based comparative genomic hybridization was done to a series of 18 paraffin-embedded primary ACCs using a genome-wide scanning BAC array.

RESULTS

A total of 238 aberrations were detected, representing more gains than losses (205 versus 33, respectively). Most frequent gains (>60%) were observed at 9q33.3-q34.3, 11q13.3, 11q23.3, 19p13.3-p13.11, 19q12-q13.43, 21q22.3, and 22q13.33. These loci harbor numerous <em>growth</em> <em>factor</em> [<em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) and platelet-derived <em>growth</em> <em>factor</em> (PDGF)] and <em>growth</em> <em>factors</em> receptor (FGFR3 and PDGFRbeta) genes. Gains at the FGF(R) regions occurred significantly more frequently in the recurred/metastasized ACCs compared with indolent ACCs. Furthermore, patients with <em>17</em> or more chromosomal aberrations had a significantly less favorable outcome than patients with fewer chromosomal aberrations (log-rank = 5.2; P = 0.02).

CONCLUSIONS

Frequent DNA copy number gains at loci of growth factors and their receptors suggest their involvement in ACC initiation and progression. Additionally, the presence of FGFR3 and PDGFRbeta in increased chromosomal regions suggests a possible role for autocrine stimulation in ACC tumorigenesis.

The new steroidal 5,7-diene, 3beta-hydroxyandrosta-5,7-diene-<em>17</em>beta-carboxylic acid (<em>17</em>-COOH-7DA), was synthesized from 21-acetoxypregnenolone, with the oxidative cleavage of the side chain being dependent on the presence of oxygen. In human epidermal (HaCaT) keratinocytes, <em>17</em>-COOH-7DA inhibited proliferation in a dose-dependent manner, starting at a dose as low as 10(-11) M. This inhibition was accompanied by decreased expression of epidermal <em>growth</em> <em>factor</em> receptor, bcl2 and cyclin E2 mRNAs and by increased expression of involucrin mRNA. Inhibition of proliferation was associated with slowing of the cell cycle in G1/G0 phases but not with cell death. <em>17</em>-COOH-7DA was significantly more potent than pregnenolone, <em>17</em>-COOH-pregnenolone, <em>17</em>-COOCH(3)-7DA and calcitriol. <em>17</em>-COOH-7DA also inhibited proliferation of normal human epidermal melanocytes and human and hamster melanoma lines, however, with lower potency than for keratinocytes. In normal human dermal <em>fibroblasts</em> <em>17</em>-COOH-7DA stimulated proliferation in serum-free media but inhibited it in the presence of 5% serum. <em>17</em>-COOH-7DA inhibited cell colony formation of human and hamster melanoma cells, and induced monocyte-like differentiation of human HL60 leukemia cells. Thus, the new steroidal 5,7-diene, <em>17</em>-COOH-7DA, can serve as an inhibitor of proliferation of normal keratinocytes and normal and malignant melanocytes, as a condition-dependent regulator of <em>fibroblast</em> proliferation and a stimulator of leukemia cell differentiation.

Oncogenes in DNAs from human tumor cell lines have been detected by a new transformation assay. Cellular DNAs are transfected into NIH3T3 murine <em>fibroblasts</em>, and transformed cells are selected by maintaining cell cultures in a defined medium lacking platelet-derived or <em>fibroblast</em> <em>growth</em> <em>factors</em>. DNAs from eight of <em>17</em> human tumor cell lines have yielded transformants by this method. Activated cellular ras genes account for three of the transforming activities. The SAOS2 osteosarcoma cell line contains an activated oncogene distinct from 18 known oncogenes. Another cellular oncogene was detected as the consequence of a fortuitous transfection-mediated DNA rearrangement.