Knockdown of Toll-like receptors (TLRs) is a novel therapeutic strategy in treating patients with rheumatoid arthritis (RA). We examined the effects of lentiviral vector-mediated delivery of TLR7 short hairpin RNA gene (Lt.shTLR7) on collagen-induced arthritis (CIA). After being immunized on days 0 and 7, Sprague-Dawley rats received intra-articular (i.a.) injection of Lt.shTLR7 or scramble control vector on days 7 and 10. The therapeutic effects were evaluated by measuring ankle circumferences, articular index, and radiographic and histological scores on killing on day <em>16</em>. Microvessel densities, vascular endothelial <em>growth</em> <em>factor</em> (VEGF) levels, pro-inflammatory cytokine concentrations and T-cell numbers within the synovial tissues were measured. Moreover, VEGF and pro-inflammatory cytokine concentrations in culture supernatants from TLR7-transfected synovial <em>fibroblasts</em> (SFs) stimulated with imiquimod or endogenous ligands were examined. There were significant reduction in ankle circumferences, articular indexes, and radiographic and histological scores. Microvessel densities, VEGF concentrations, interleukin (IL)-1β and IL-6 levels and T-cell densities within synovial tissues were significantly lower. Induction of VEGF, IL-1β and IL-6 production from stimulated SFs was significantly suppressed. Taken together, these data demonstrate the effects of i.a. lentiviral vector-mediated delivery of shTLR7 RNA gene on inhibition of CIA, and implicate the manipulation of TLR7 as a potential therapeutic strategy in RA patients.

This study investigates the feasibility of recombinant spider silk protein as a wound-dressing material for coverage of deep second-degree burn wounds using an animal model. Sixty Sprague-Dawley (SD) rats were randomly divided into four groups (15 rats in each group). Two types of recombinant spider silk proteins, pNSR-<em>16</em> and pNSR-32, as well as collagen (as a control) were applied on the wound; the fourth group was left untreated as a negative control. Each group was evaluated on the 3rd, 5th, 7th, 14th and 21st days for wound-healing rate, histological test, levels of hydroxyproline synthesis and the samples were stained for immunohistochemical detection of the basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). The results of implantation testing showed that wound healing in the treatment groups--recombinant spider silk protein pNSR-<em>16</em> and pNSR-32--was much better than that in the control group (p<0.01). On the 7th, 14th and 21st days, higher expression of bFGF and the increase of hydroxyproline content of the skin indicated good regeneration of wound skin in the treatment groups. Preliminarily, we conclude that the recombinant spider silk protein membrane promotes the recovery of wound skin by increasing the expression and secretion of the <em>growth</em> <em>factor</em> bFGF and hydroxyproline.

We have reported recently that IgG from patients with Graves' disease (GD) can induce the expression of the CD4-specific T lymphocyte chemoattractant, IL-<em>16</em>, and RANTES, a C-C chemokine, in their <em>fibroblasts</em>. This induction is mediated through the insulin-like <em>growth</em> <em>factor</em>-1 receptor (IGF-1R) pathway. We now report that Abs from individuals with active rheumatoid arthritis (RA-IgG) stimulate in their synovial <em>fibroblasts</em> the expression of these same cytokines. IgG from individuals without known autoimmune disease fails to elicit this chemoattractant production. Furthermore, RA-IgG fails to induce IL-<em>16</em> or RANTES expression in synovial <em>fibroblasts</em> from donors with osteoarthritis. RA-IgG-provoked IL-<em>16</em> and RANTES production also appears to involve the IGF-1R because receptor-blocking Abs prevent the response. RA <em>fibroblasts</em> transfected with a dominant-negative mutant IGF-1R fail to respond to RA-IgG. IGF-1 and the IGF-1R-specific analog Des(1-3) also induce cytokine production in RA <em>fibroblasts</em>. RA-IgG-provoked IL-<em>16</em> expression is inhibited by rapamycin, a specific macrolide inhibitor of the Akt/FRAP/mammalian target of rapamycin/p70(s6k) pathway, and by dexamethasone. GD-IgG can also induce IL-<em>16</em> in RA <em>fibroblasts</em>, and RA-IgG shows similar activity in GD <em>fibroblasts</em>. Thus, IgGs from patients with RA, like those associated with GD, activate IGF-1R, and in so doing provoke T cell chemoattraction expression in <em>fibroblasts</em>, suggesting a potential common pathway in the two diseases. Immune-competent cell trafficking to synovial tissue is integral to the pathogenesis of RA. Recognition of this novel RA-IgG/<em>fibroblast</em> interaction and its functional consequences may help identify therapeutic targets.

Orbital changes in thyroid orbitopathy (TO) result from de novo adipogenesis, hyaluronan synthesis, interstitial oedema and enlargement of extraocular muscles. Cellular immunity, with predominantly CD4+ T cells expressing Th1 cytokines, and overexpression of macrophage-derived cytokines, perpetuate orbital inflammation. Orbital <em>fibroblasts</em> appear to be the major effector cells. Orbital <em>fibroblasts</em> express both thyrotropin receptor (TSHR) and insulin-like <em>growth</em> <em>factor</em>-1 receptor (IGF-1R) at higher levels than normal <em>fibroblasts</em>. TSHR expression increases in adipogenesis; TSHR agonism enhances hyaluronan production. IGF-1R stimulation leads to adipogenesis, hyaluronan synthesis and production of the chemokines, interleukin (IL)-<em>16</em> and Regulated on Activation, Normal T Cell Expression and Secreted, which facilitate lymphocyte trafficking into the orbit. Immune activation uses a specific CD40:CD154 molecular bridge to activate orbital <em>fibroblasts</em>, which secrete pro-inflammatory cytokines including IL-1β, IL-1α, IL-6, IL-8, macrophage chemoattractant protein-1 and transforming <em>growth</em> <em>factor</em>-β, to perpetuate orbital inflammation. Molecular pathways including adenylyl cyclase/cyclic adenosine monophosphate, phophoinositide 3 kinase/AKT/mammalian target of rapamycin, mitogen-activated protein kinase are involved in TO. The emergence of a TO animal model and a new generation of TSHR antibody assays increasingly point towards TSHR as the primary autoantigen for extrathyroidal orbital involvement. Oxidative stress in TO resulting from imbalances of the oxidation-reduction state provides a framework of understanding for smoking prevention, achieving euthyroidism and the use of antioxidants such as selenium. Progress has been made in the understanding of the pathogenesis of TO, which should advance development of novel therapies targeting cellular immunity, specifically the CD40:CD40 ligand interaction, antibody-producing B cells, cytokines, TSHR and IGF-1R and its signalling pathways. Further studies in signalling networks and molecular triggers leading to burnout of TO will further our understanding of TO.

BACKGROUND

Venous hypertension is regarded as an important factor in the pathogenesis of dural arteriovenous fistula (DAVF). We investigate histologic reaction of dural sinus under the condition of venous hypertension using a rat venous hypertension model to present hemodynamic and immunohistochemical effect in the development of DAVF.

METHODS

Twenty-four Sprague-Dawley male rats were divided into venous hypertension and control groups. Venous hypertension was induced with a left common carotid artery-external jugular vein anastomosis and an occlusion of a right posterior facial vein. Measurements of systemic mean arterial pressure, draining vein pressure (DVP), and cerebral perfusion pressure (CPP) were conducted on the next day, at 7 days, and at 28 days after surgery, and the rats were killed for histologic examinations.

RESULTS

Postoperative DVP increased significantly in venous hypertension group compared to control group (35 +/- 5 vs 13 +/- 2 mm Hg, P < .05). Increased DVP remained above 30 mm Hg throughout the observation period. Postoperative CPP decreased significantly in venous hypertension group compared to control group (49 +/- 8 vs 86 +/- 9 mm Hg, P < .05). In venous hypertension group, there was a significant difference between days 1 and 28 (49 +/- 8 vs 64 +/- 8 mm Hg, P < .05). Histologic examination revealed thickening of connective tissues, proliferation of fibroblasts, and strong expression of vascular endothelial growth factor (VEGF) in endothelium under venous hypertension condition. Immunostained VEGF cells decreased significantly from day 7 to day 28 (100 +/- 16 vs 72 +/- 19 cells, P < .05). A positive correlation was observed between DVP and VEGF expression (Pearson correlation coefficient; r = 0.671, P = .0017). There was a negative correlation between CPP and immunostained VEGF cells (r = -0.702, P = .0089).

CONCLUSIONS

These results suggest that venous hypertension is associated with increased expression of VEGF, and a decreased CPP may have a potential effect in VEGF expression under venous hypertension condition. These factors are speculated to play an important role in progression of DAVF.

Active angiogenesis, together with an up-regulation of angiogenic <em>factors</em>, is evident in the synovium of both rheumatoid arthritis (RA) and osteoarthritis (OA). The present study assessed, by immunohistochemistry, the microvessel density in the synovium of these arthritides and in normal controls, in relation to the expression of the angiogenic <em>factors</em> vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and platelet-derived endothelial cell <em>growth</em> <em>factor</em> (PD-ECGF) and the apoptosis-related proteins bcl-2 and p53. More importantly, using the novel 11B5 MAb, the activated "VEGF/flk-1(KDR)-receptor" microvessel density was assessed. VEGF expression in <em>fibroblasts</em> was diffuse in both RA and OA. Diffuse PD-ECGF expression of <em>fibroblasts</em> was noted in all cases of RA, while <em>fibroblast</em> reactivity was focal in the OA material. The standard microvessel density (sMVD), as assessed with the anti-CD31 monoclonal antibody (MAb), was higher in RA (64+/-12) and in OA (65+/-<em>16</em>) than in normal tissues (52+/-8; p=0.008 and 0.0004, respectively). The activated microvessel density (aMVD), assessed with the 11B5 MAb, was significantly higher in RA (29+/-10) than in OA (17+/-4; p<0.0001) and than in normal tissues (14+/-2; p<0.0001). The "activation ratio" (aMVD/sMVD) was statistically higher in RA (0.46+/-0.17) than in OA and normal synovial tissues, the latter two having a similar ratio (0.28+/-0.08 and 0.26+/-0.03, respectively). Cytoplasmic bcl-2 expression was frequent in the synovial cells of OA, but rare in RA. Nuclear p53 protein accumulation was never observed. It is suggested that the angiogenic pathway VEGF/flk-1(KDR) may play an important role in the pathogenesis of RA and OA. Thus, failure of VEGF/flk-1(KDR) activation, in the presence of increased VEGF expression, may indicate a synovium with an impaired capacity to establish a viable vasculature, consistent with the degenerative nature of OA. On the other hand, the activated angiogenesis in RA shows a functional, still pathologically up-regulated VEGF/flk-1(KDR) pathway. Whether restoration of an impaired VEGF/flk-1(KDR) pathway in OA, or inhibition of this in RA, would prove of therapeutic importance requires further investigation.

In serum-deprived human <em>fibroblasts</em> IMR-90 and WI-38 cells, the addition of fetal calf serum or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> stimulates DNA synthesis in an extracellular Ca(2+)-dependent manner; the effect of serum on [3H]thymidine incorporation into DNA is 4-<em>16</em>-fold greater at 2.0 mM CaCl2 as compared with that at 0.03 mM CaCl2. By contrast, in SV40 virus-transformed WI-38 (SV-WI-38) cells DNA synthesis is essentially independent of the extracellular calcium concentration ([Ca]out) and serum <em>growth</em> <em>factors</em>. To explore the role of Ca2+ in mitogenic signal transduction through G1 to S phase cell cycle progression, we studied and compared the effect of [Ca]out on phosphorylation of RB protein, the product of a tumor suppressor retinoblastoma gene. In IMR-90 and WI-38 cells, serum or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> induces an increase in the amount of hyperphosphorylated forms of RB protein in a manner strictly dependent on [Ca]out. In sharp contrast, in SV-WI-38 cells, the extent of RB phosphorylation is little affected by [Ca]out or the presence or absence of serum <em>growth</em> <em>factors</em>. In addition, potent calmodulin antagonists W-7 and calmidazolium, but not an inactive analogue W-12 or W-5, strongly inhibit serum-induced increases in DNA synthesis and RB phosphorylation in IMR-90 and WI-38 cells, whereas in SV-WI-38 cells, the inhibitory effect is much more limited. Under the same treatment conditions, we measured histone H1 kinase activity associated with anti-p34cdc2 immunoprecipitate and found that the serum-induced increase in p34cdc2 kinase activity is strongly dependent on [Ca]out and is potently inhibited by the active calmodulin antagonists in IMR-90 and WI-38 cells, but not in SV-WI-38 cells. In IMR-90 cells that have been incubated with serum in 0.03 mM [Ca]out for 24 h, restoration of [Ca]out to 2.0 mM results in initiation of DNA synthesis after 13 h and concomitant increases in RB phosphorylation and p34cdc2 histone H1 kinase activity. These results suggest that in human <em>fibroblasts</em>, Ca2+/calmodulin regulates the signaling cascade leading to cdc2 kinase activation, RB protein phosphorylation, and DNA synthesis and that this Ca(2+)-dependent regulation is abrogated in SV40-transformed cells.

The t(4;14) translocation detected by fluorescence in situ hybridization (FISH) is an independent prognostic <em>factor</em> for an adverse outcome of multiple myeloma (MM). Because t(4;14) uniquely results in <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) expression, decalcified, paraffin-embedded bone marrow biopsies were immunostained for FGFR3, and its expression was correlated with the t(4;14) status. FISH detected t(4;14) in <em>16</em> (19%) of 85 MM patient specimens, and immunocytochemistry detected aberrant FGFR3 expression in 13 (15%). Twelve (75%) t(4;14)-positive cases expressed FGFR3, and 12 (92%) FGFR3-positive cases harbored a t(4;14). FGFR3 expression and t(4;14) were strongly correlated (P < .001). FGFR3 expression by immunohistochemistry was associated with the immunoglobulin A (IgA) isotype (P < .001), a shorter progression-free survival (median, 11.5 versus 25.8 months; P < .001), and a shorter overall survival (median, 19.2 versus 46.3 months; P < .001).

Nerve <em>growth</em> <em>factor</em>-like substance(s) were identified in both conditioned media of a human prostatic tumor epithelial cell line (TSU-pr1) and a human prostatic stromal cell line (HPS) by Western blot analysis and bioassay of neurite out<em>growth</em> of PC12 cells. Nerve <em>growth</em> <em>factor</em>-beta (NGF) immunofluorescence was also localized to secretory vesicles in the cytoplasm of both the TSU-pr1 and HPS cells. Western blot of the TSU-pr1 and HPS cell-secreted protein identified an Mr 65,000 major protein which immunoreacted with murine NGF antibody. NGF Western blot of HPS cell-secreted protein also identified an Mr 42,000 minor band under reduced and nonreduced conditions and an Mr 61,000 minor band under reduced conditions. The secreted protein from the TSU-pr1 cells (50 micrograms/ml) and HPS (50 micrograms/ml), as well as murine NGF (50 ng/ml) or human recombinant NGF (50 ng/ml), stimulated neurite out<em>growth</em> from PC12 cells. This neurite out<em>growth</em> activity was partially inhibited by treatment with NGF antibody. Neither the serum containing <em>growth</em> medium nor bovine serum albumin (50 micrograms/ml) stimulated neurite out<em>growth</em>. The NGF-like secretory protein appeared to play a role in the paracrine regulation of prostatic <em>growth</em> between TSU-pr1 cells and HPS cells. The relative <em>growth</em> of TSU-pr1 cells, as indicated by [3H]thymidine incorporation, in response to HPS secretory protein was stimulated 2.8-fold in a dose-dependent manner. In the converse interaction, the relative <em>growth</em> of HPS cells in response to TSU-pr1 secretory protein was stimulated 1.8-fold in a dose-dependent manner. Immunoneutralization of TSU-pr1 and HPS secretory protein was performed with antibody against NGF, acidic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. Removal of the NGF-like protein from the maximal stimulatory dose of TSU-pr1 secretory protein (100 micrograms/ml) with NGF antibody reduced HPS proliferation to 52% of maximal levels, and immunoneutralization of the NGF-like protein in the maximal stimulatory dose of HPS secretory protein (20 micrograms/ml) also reduced TSU-pr1 proliferation to <em>16</em>% of maximal levels. Addition of normal rabbit serum or prior immunoprecipitation of either TSU-pr1 or HPS secretory protein with antibody against acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> did not inhibit the proliferation of either cell type. These results suggest that TSU-pr1 tumor cells and HPS cells secrete NGF-like protein(s) which modulate their paracrine interactive <em>growth</em> in vitro.

Polypeptide <em>growth</em> <em>factors</em> may play an important role in the regulation of human fetal adrenal cortical <em>growth</em> by mediating the tropic actions of ACTH. The abundance of mRNA encoding insulin-like <em>growth</em> <em>factor</em>-II (IGF-II) is high in the human fetal adrenal gland and is stimulated by ACTH in cultured fetal adrenal cortical cells. Therefore, we studied the mitogenic action, regulation, and localization of IGF-II and a closely related peptide, IGF-I, in primary cultures of human fetal adrenal cortical cells and whole human fetal adrenal glands. Recombinant human IGF-I and IGF-II stimulated proliferation of fetal adrenal cortical cells in a dose-dependent fashion (1-1000 ng/mL; 0.133-133 nM). At 1000 ng/mL (133 nM), both peptides increased cell number 1.8- to 2-fold. Combinations of IGF-I or -II (100 ng/mL; 13.3 nM) with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF; 0.1 ng/mL; 6 pM) or epidermal <em>growth</em> <em>factor</em> (EGF; 1.0 ng/mL; 0.17 nM) had a greater effect on proliferation than bFGF, EGF, or either of the IGFs alone, suggesting an additive interaction. IGF-II mRNA was detected in cultured adrenal cortical cells by in situ hybridization analysis, and its abundance was stimulated by ACTH. In contrast, IGF-I mRNA was not detected in cultured fetal zone cells and was not regulated by ACTH. In whole human fetal adrenal glands, IGF-II mRNA was detected in the definitive and fetal zones and in the capsule, whereas IGF-I mRNA was detected only in the capsule and not in the two cortical zones. Using Northern blot analysis, we found that mRNA encoding IGF-II was present in high abundance in fetal adrenal glands (<em>16</em>-22 weeks) and barely detectable in adult adrenals, whereas mRNA encoding IGF-I was present in very low abundance in the fetal adrenal, but was high in adult human adrenals. As IGF-II expression is high in the human fetal adrenal cortex and is regulated by ACTH, we propose that it is the dominant of the two IGFs regulating human fetal adrenal <em>growth</em>. The cooperative mitogenic effect of IGF-II with bFGF and EGF and the regulation of its expression by ACTH support the hypothesis that IGF-II may act as a mediator, in concert with bFGF and possibly EGF, of the tropic action of ACTH in regulating the rapid <em>growth</em> of the human fetal adrenal cortex during midgestation.

BACKGROUND

Chronic allograft nephropathy (CAN) is a multifactorial process, where both immunological and nonimmunological factors play roles. Microarrays detect thousands of genes simultaneously.

METHODS

We have analyzed gene expression profiles of 16 kidney transplant biopsy samples with CAN by high-density oligonucleotide microarrays, comparing to six normal transplant biopsies. Eight CAN biopsies showed nodular arteriolar hyalinization and one was positive for C4d staining.

RESULTS

Hierarchical clustering analysis of the 22 biopsies revealed differential gene expression patterns in CAN versus the control biopsies. However, microarray analysis did not reveal differential gene expression patterns in patients with or without arteriolar hyalinization. Fifty percent of the 100 genes with highest hybridization intensities in a C4d positive sample were related to cellular and humoral immune response. Although 212 genes were upregulated a minimum of 1.5-fold, 112 genes were downregulated in CAN samples. There was differential expression of profibrotic and growth factors that while transforming growth factor-beta induced factor, thrombospondin 1, and platelet derived growth factor-C were up-regulated, vascular endothelial growth factor, epidermal growth factor, and fibroblast growth factors 1 and 9 were downregulated. Selected differentially expressed genes were confirmed in microdissected samples by real-time quantitative PCR. Immunopathologic examination of biopsies revealed strong TGF-beta but decreased glomerular VEGF expression in CAN.

CONCLUSIONS

Microarrays might be an important tool to uncover the mechanisms of multifactorial diseases, such as CAN.

TERE1, a recently discovered gene/protein appears to play a role in bladder tumor <em>growth</em> regulation but to date does not have clear functional correlates. The objective of this study was to gain further insight into the function of the TERE1 protein by identifying potential protein to protein interactions with TERE1 and determining whether these interactions are associated with putative <em>growth</em> regulatory pathways and/or bladder tumor formation. Towards this aim, we have performed a bacterial two hybrid assay and isolated interacting clones, which then were sequenced and further examined by affinity chromatography and immunoprecipitation. From among several positive clones, we isolated a putative interacting plasmid containing the C-terminal portion of preapolipoprotein E starting from amino acid number 124 from the pBT-TERE1/pTarget-cDNA bacterial two hybrid system. The C-terminal portion of apoE interaction with the TERE1 was confirmed using ProBond columns by the expression of 6XHis recombinant and (35)S methionine/cysteine labeled proteins. We found that there was ubiquitous expression of the apoE transcript in normal bladder and in various grades and stages of transitional cell carcinoma (TCC) of the bladder. Likewise, we detected the apoE protein in both normal and malignant bladder tissues by Western blot. There was a significant decrease in the apoE protein in 12 of <em>16</em> muscle invasive TCCs of the bladder compared to normal bladder mucosa samples. Previous studies in rat <em>fibroblasts</em> have found that expression of apoE can decrease the phosphorylation of the <em>growth</em> <em>factor</em>-related p42/44 MAP kinase. A significant decrease in p44/p42 MAPK phophorylation was also apparent using a phosphorylation specific antibody in human 293 kidney cells upon transfection and expression of apoE. In conclusion, the results from this study suggest that the expression and regulation of the apoE pathway may yield clues toward understanding the function of TERE1.

To explore the local mechanisms of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) 23 regulations, we examined the FGF23 expression patterns in an osteoblast culture model. The characteristics of cultured rat calvaria osteoblasts in half-confluence, confluence, osteoid deposition, and osteoid mineralization stages might reflect the proliferation, differentiation, maturation, and matrix mineralization stages, respectively. Compared with proliferating cells in half-confluence, FGF23 expression was upregulated by 7.5-fold at the mRNA level and 126% at the protein level in confluent differentiated cells as determined by real-time RT-PCR and Western blot analysis. Interestingly, mRNA levels of CYP27B1 (the gene coding for 1alpha-hydroxylase enzyme which catalyses the conversion of 1alpha,25-dihydroxyvitamin D, 1alpha,25[OH]2D, from its inactive form, 25-hydroxycholecalciferol, 25[OH]D) and CYP24A (the gene coding for 24-hydroxylase, a target gene of 1alpha,25[OH]2D) were significantly increased by twofold and 34-fold, respectively, in differentiated osteoblasts compared with proliferating cells. We next examined if the local production of 1alpha,25(OH)2D might contribute to the FGF23 upregulation. We cultured osteoblasts in serum-free medium with or without 25-(OH)D (the substrate of 1alpha-hydroxylase). FGF23 mRNA levels were increased in proliferating cells (<em>16</em>-fold) and in differentiated cells (28-fold) by the physiological dose of 25-(OH)D3 treatment. CYP27B1 was slightly but significantly upregulated and CYP24A was increased by 1,700-fold and 800-fold, respectively, in transcriptional levels. Because FGF23 was upregulated in confluent osteoblasts regardless of the presence or absence of 25-(OH)D in serum-free medium, we further examined the possible impact of cell communication on FGF23 expression. We treated osteoblasts with carbenoxolone, a gap junction Cx43 blocker in serum-free medium. The FGF23 mRNA level was reduced by 50% in confluent differentiated cells and slightly but not significantly reduced in half-confluent cells by carbenoxolone treatments. The results suggested that upregulation of FGF23 in differentiated osteoblast appeared to be due to increased autocrine/paracrine action of osteoblast-derived 1alpha,25(OH)2D and increased cell communication, which were identified in cultured rat calvaria osteoblasts. These results indicate that FGF23 expression was stimulated not only by circulating calcitriol but also by locally produced 1alpha,25(OH)2D. The local mechanisms of FGF23 expression remain to be characterized.

Keratinocyte <em>growth</em> <em>factor</em> (KGF) is a member of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) group of heparin-binding polypeptides. In the present study we sought to determine whether KGF is expressed in human pancreatic cancers. Using reverse transcriptase polymerase chain reaction (RT-PCR), a cDNA fragment of KGF was cloned and used to analyze Northern blots of RNA isolated from normal and cancerous human pancreatic tissues. Seven of <em>16</em> (44%) pancreatic cancer samples revealed significant overexpression of the 2.4 kilobase KGF mRNA transcript by comparison with the normal pancreas. Northern blot analysis failed to reveal the KGF transcript in several cultured human pancreatic cancer cell lines. However, by PCR analysis, some of the cell lines expressed KGF mRNA. Furthermore, 5 of 7 tested cell lines expressed the KGF receptor, and the <em>growth</em> of one cell line was enhanced by human recombinant KGF. These results suggest that KGF may participate in aberrant paracrine and autocrine pathways in human pancreatic cancer.

Our previous studies showed that degradable dextran hydrogels are rapidly formed in situ upon mixing aqueous solutions of dextran vinyl sulfone (dex-VS) conjugates and tetrafunctional mercapto poly(ethylene glycol) (PEG-4-SH) by Michael addition. The hydrogel degradation time and storage modulus could be controlled by the degree of vinyl sulfone substitution (DS) and dextran molecular weight. The degradation time could further be adjusted by the spacer between the thioether and the ester bond of the dex-VS conjugates (ethyl vs. propyl, denoted as dex-Et-VS and dex-Pr-VS, respectively). In this paper, the release of three model proteins, i.e. immunoglobulin G (d(h)=10.7 nm, IgG), bovine serum albumin (BSA, d(h)=7.2 nm) and lysozyme (d(h)=4.1 nm), as well as basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) from these in situ forming dextran hydrogels is studied. Proteins could be easily loaded into the hydrogels by mixing protein containing solutions of dex-VS and PEG-4-SH. The release of IgG from dex-Et-VS hydrogels followed biphasic release kinetics, with a slow, close to first order release for the first 9 days followed by an accelerated release and over 80% of IgG was released in 12 to 25 days. Interestingly, the release of IgG from dex-Pr-VS hydrogels followed close to zero order kinetics, wherein approximately 95% was released in 21 days. The release of BSA from dex-Pr-VS hydrogels followed biphasic kinetics, with almost first order release followed by close to zero order release. Approximately 75% of the entrapped BSA could be released from dex-Pr-VS hydrogels in <em>16</em> days. Dex-Pr-VS hydrogels released 40% of lysozyme in 14 days, with full preservation of the enzymatic activity of the released lysozyme, as determined by bacteria lysis experiments. The release of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) from dex-Pr-VS hydrogels showed first order kinetics, with quantitative release in 28 days. These results show that the in situ forming degradable dextran hydrogels can be used for the controlled release of proteins.

With the aim of interrupting the <em>growth</em> <em>factor</em>-stimulated Ras signaling pathway at the level of the Grb2-Sos interaction, a peptidimer, made of two identical proline-rich sequences from Sos linked by a lysine spacer, was designed using structural data from Grb2 and a proline-rich peptide complexed with its SH3 domains. The peptidimer affinity for Grb2 is 40 nM whereas that of the monomer is <em>16</em> microM, supporting the dual recognition of both Grb2 SH3 domains by the dimer. At 50 nM, the peptidimer blocks selectively Grb2-Sos complexation in ER 22 (CCL 39 <em>fibroblasts</em> overexpressing epidermal <em>growth</em> <em>factor</em> receptor) cellular extracts. The peptidimer specifically recognizes Grb2 and does not interact with PI3K or Nck, two SH3 domain-containing adaptors. The peptidimer was modified to enter cells by coupling to a fragment of Antennapedia homeodomain. At 10 microM, the conjugate inhibits the Grb2-Sos interaction (100%) and MAP kinase (ERK1 and ERK2) phosphorylation (60%) without modifying cellular <em>growth</em> of ER 22 cells. At the same concentration, the conjugate also inhibits both MAP kinase activation induced by nerve <em>growth</em> <em>factor</em> or epidermal <em>growth</em> <em>factor</em> in PC12 cells, and differentiation triggered by nerve <em>growth</em> <em>factor</em>. Finally, when tested for its antiproliferative activity, the conjugate was an efficient inhibitor of the colony formation of transformed NIH3T3/HER2 cells grown in soft agar, with an IC50 of around 1 microM. Thus, the designed peptidimers appear to be interesting leads to investigate signaling and intracellular processes and for designing selective inhibitors of tumorigenic Ras-dependent processes.

The endometrium is the only human tissue to undergo cyclic breakdown and regeneration. This physiological alternation renders it an advantageous system for studying tissue remodelling. Our previous observations indicate that menstrual endometrial breakdown is initiated by matrix metalloproteinases (MMPs), which are controlled overall by ovarian steroids but are also locally regulated by cytokines. We have therefore compared the effect of several endometrial cytokines on the gene expression of eight MMPs and their tissue inhibitors (TIMP)-1, -2 and -3, in primary cultures of human endometrial <em>fibroblasts</em>. Three categories of gene expression were identified: (a) MMP-13, -15 and -<em>16</em> mRNAs were not detected despite stimulation by various cytokines; (b) MMP-2 and -14 as well as TIMP-1, -2 and -3 mRNAs were constitutively expressed but not markedly affected by the six cytokines tested; (c) mRNAs for MMP-1, -9 and -11 were selectively induced by specific cytokines: insulin-like <em>growth</em> <em>factor</em>-II, epidermal <em>growth</em> <em>factor</em> (EGF), platelet derived <em>growth</em> <em>factor</em> (PDGF)-BB and interleukin (IL)-6 stimulated MMP-11 expression; MMP-1 was induced by EGF, PDGF-BB, tumour necrosis <em>factor</em> (TNF)alpha and IL-1alpha, which also exerted additive effects. In contrast with MMP-1 and MMP-11 gene expression, which was sustained for 48 h, MMP-9 mRNA was quickly induced by TNFalpha, but disappeared within 12 h despite continuing stimulation. These results show that several cytokines are able to induce the selective expression of MMPs in cultured human endometrial <em>fibroblasts</em> and are thus good candidates for involvement in local triggering of menstrual tissue breakdown.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) is a potent angiogenic <em>factor</em> and a mitogen for a variety of mesoderm- and neuroectoderm-derived cell types (e.g., <em>fibroblasts</em>, endothelial cells, astrocytes, oligodendrocytes). After application of a monospecific polyclonal antiserum, we localized basic FGF on frozen sections of 73 human brain tumors using immunohistochemistry. FGF was present in a variable number of tumor cells (<em>16</em>/<em>16</em> astrocytomas, 5/5 ependymomas, 0/3 benign and 4/7 anaplastic oligodendrogliomas, 11/12 glioblastomas, 11/11 meningiomas, 6/6 neurilemmomas, 0/3 pituitary adenomas, 2/2 choroid plexus papillomas, 0/1 neurocytoma, 2/2 benign fibrous histiocytomas, 2/5 metastatic carcinomas). FGF was detected in vascular cells of 59 tumors and in <em>fibroblasts</em> of connective tissue stroma from all papillomas and metastases. These results tend to indicate FGF involvement in the malignant progression of gliomas due to an autocrine or paracrine action. Histopathological aspects of malignant gliomas (e.g., pseudopalisading or pathological vessels) could be related to FGF activity.

A combination of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) or <em>16</em> nM 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum induces human SH-SY5Y neuroblastoma cells to undergo differentiation and acquire a neuronal phenotype. Nerve <em>growth</em> <em>factor</em> (NGF) added to SH-SY5Y cells stably transfected with the NGF-receptor TRK-A (SH-SY5Y/trk) induces a similar differentiated phenotype. SH-SY5Y cells express protein kinase C (PKC)-alpha, PKC-beta I, PKC-epsilon, and PKC-zeta protein, and phorbol ester- or <em>growth</em> <em>factor</em>-induced differentiation results in a sustained activation of PKC. The specific PKC inhibitor GF 109203X blocked TPA- and bFGF-IGF-I-induced neurite out<em>growth</em> in wild-type SH-SY5Y cells and NGF-induced neurite out<em>growth</em> in SH-SY5Y/trk cells. When added to differentiated cells, GF 109203X caused rapid retraction of <em>growth</em> cone filopodia. In TPA- and bFGF-IGF-I-treated cells, addition of GF 109203X also blocked induced expression of <em>growth</em> associated protein-43 and neuropeptide tyrosine while the increase in expression of these two genes was only slightly affected by the inhibitor in NGF-treated SH-SY5Y/trk cells. Thus, a portion of the NGF-induced phenotypic changes appears not to be mediated via PKC-dependent signaling. A high concentration of TPA (1.6 microM) down regulated PKC-alpha and PKC-beta I almost completely and PKC-epsilon partially in wild-type SH-SY5Y and SH-SY5Y/trk cells. Cells with down-regulated PKC-alpha and PKC-beta I after 1.6 microM TPA treatment still differentiated with <em>growth</em> <em>factors</em>. In these cells, the PKC-epsilon level was restored, and the PKC-epsilon protein was enriched in the <em>growth</em> cones. The 1.6 microM TPA-induced down-regulation of PKC-epsilon was counteracted by bFGF and NGF but not by platelet-derived <em>growth</em> <em>factor</em> or IGF-I. These data indicate that PKC activity is vital for neurite formation, and that the cells can differentiate under conditions when PKC-alpha and PKC-beta I are extensively down regulated. The close correlation between differentiation and presence of PKC-epsilon protein suggests an important function for this isoform during this process.

Patients with end-stage renal disease (ESRD) suffer exceptionally high mortality rates in their first year of chronic hemodialysis. Both vitamin D and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-23 levels correlate with survival in these patients. Klotho is a protein in the vitamin D/FGF-23 signaling pathway that has been linked with accelerated aging and early mortality in animal models. We therefore hypothesized that genetic variation in the Klotho gene might be associated with survival in subjects with ESRD. We tested the association between 12 single nucleotide polymorphisms (SNPs) in the Klotho gene and mortality in a cohort of ESRD patients during their first year on hemodialysis (n = 1307 white and Asian). We found a significant association between the CC genotype of one tag SNP, rs577912, and increased risk for 1-yr mortality (RR, 1.76; 95% CI, 1.19-2.59; p = 0.003). This effect was even more marked among patients who were not treated with activated vitamin D supplementation (HR, 2.51; 95% CI, 1.18-5.34; p = 0.005). In lymphoblastoid cell lines derived from HapMap subjects, the CC genotype was associated with a <em>16</em>-21% lower Klotho expression compared with the AA/AC genotype. Our data suggest that a specific Klotho variant (rs577912) is linked to survival in ESRD patients initiating chronic hemodialysis and that therapy with activated vitamin D may modify this risk.

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 (17 with and 12 without TIO) and 23 non-PMTMCT (<em>16</em> 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 175 bp). The housekeeping gene phosphoglycerokinase (189 bp) was coamplified to check the RNA quality. Sixteen of 17 (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.

Low-energy helium-neon lasers (632.8 nm) have been employed in a variety of clinical treatments including vitiligo management. Light-mediated reaction to low-energy laser irradiation is referred to as biostimulation rather than a thermal effect. This study sought to determine the theoretical basis and clinical evidence for the effectiveness of helium-neon lasers in treating vitiligo. Cultured keratinocytes and <em>fibroblasts</em> were irradiated with 0.5-1.5 J per cm2 helium-neon laser radiation. The effects of the helium-neon laser on melanocyte <em>growth</em> and proliferation were investigated. The results of this in vitro study revealed a significant increase in basic <em>fibroblast</em> <em>growth</em> <em>factor</em> release from both keratinocytes and <em>fibroblasts</em> and a significant increase in nerve <em>growth</em> <em>factor</em> release from keratinocytes. Medium from helium-neon laser irradiated keratinocytes stimulated [3H]thymidine uptake and proliferation of cultured melanocytes. Furthermore, melanocyte migration was enhanced either directly by helium-neon laser irradiation or indirectly by the medium derived from helium-neon laser treated keratinocytes. Thirty patients with segmental-type vitiligo on the head and/or neck were enrolled in this study. Helium-neon laser light was administered locally at 3.0 J per cm2 with point stimulation once or twice weekly. The percentage of repigmented area was used for clinical evaluation of effectiveness. After an average of <em>16</em> treatment sessions, initial repigmentation was noticed. Marked repigmentation (>50%) was observed in 60% of patients with successive treatments. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> is a putative melanocyte <em>growth</em> <em>factor</em>, whereas nerve <em>growth</em> <em>factor</em> is a paracrine <em>factor</em> for melanocyte survival in the skin. Both nerve <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> stimulate melanocyte migration. It is reasonable to propose that helium-neon laser irradiation clearly stimulates melanocyte migration and proliferation and mitogen release for melanocyte <em>growth</em> and may also rescue damaged melanocytes, therefore providing a microenvironment for inducing repigmentation in vitiligo.

Connective tissue <em>growth</em> <em>factor</em> (CTGF) is a member of the CCN family of immediate early genes, which are involved in cell proliferation, migration, and matrix production. Recently, CTGF was observed to be strongly upregulated in human proliferative and fibrogenic renal disease. By in situ hybridization and reverse transcriptase-PCR, the expression of CTGF was investigated in experimental proliferative glomerulonephritis induced by injection of anti-Thy-1.1 antibody in the rat. CTGF expression in cultured rat mesangial cells and glomerular visceral epithelial cells (GVEC) was studied in response to transforming <em>growth</em> <em>factor</em> beta (TGF-beta), an essential pathogenetic <em>factor</em> in this model. In normal rat kidneys, only some GVEC expressed CTGF mRNA. In anti-Thy-1.1 nephritis, CTGF mRNA expression was strongly increased in extracapillary and mesangial proliferative lesions and in areas of periglomerular fibrosis. Early glomerular CTGF overexpression in GVEC coincided with a striking upregulation of TGF-beta2 and to a lesser extent of TGF-beta3. Glomerular CTGF mRNA expression was maximal at day 7, in association with increased TGF-beta1 mRNA and protein expression. CTGF mRNA overexpression by parietal epithelial cells preceded the periglomerular appearance of alpha-smooth muscle actin-positive <em>fibroblasts</em>. In cultured mesangial cells, TGF-beta1, -beta2, and -beta3 transiently increased the CTGF/glyceraldehyde phosphate dehydrogenase mRNA ratio up to threefold versus control at 4 h. In GVEC, upregulation of CTGF mRNA by these TGF-beta isoforms was more sustained, being 8- to <em>16</em>-fold versus control at 24 h. The kinetics of CTGF expression strongly suggest a role in glomerular repair, possibly downstream of TGF-beta, in this model of transient renal injury.

MicroRNAs (miRNAs) are genomically encoded non-protein-encoding small RNAs, which negatively regulate target gene expression at post-transcriptional level. The present study aimed to investigate whether disorders of miRNAs system were involved in the pathogenesis of hypertension in spontaneously hypertensive rats (SHR). MiRanda, Target Scan and PicTar were utilized for predictive analysis of miRNAs and target genes. MiR-1, miR-133a, miR-155 and miR-208 were selected as the candidate miRNAs potentially related to blood pressure. The expression levels of miR-1, miR-133a, miR-155 and miR-208 in the aorta of 4-, 8-, <em>16</em>- and 24-week-old SHR and age-matched Wistar-Kyoto (WKY) rats were detected by real-time RT-PCR. The mRNA levels of angiotensin II receptor type 1 (AGTR1a), angiotensin II receptor associated protein (AGTRAP), divalent metal transporter 1 (DMT1), low-density lipoprotein-related protein 1B (LRP1B), <em>fibroblast</em> <em>growth</em> <em>factor</em>-7 (FGF-7), protocadherin 9 precursor (PCDH9), chloride channel protein 5 (CLCN-5), small conductance calcium activated potassium channel protein 3 (KCNN3) and thyroid hormone receptor associated protein 1 (THRAP1), which were predicted to be target genes of differentially expressed miRNAs, were further detected by real-time RT-PCR. The results obtained showed that the expression levels of miR-1, miR-155 and miR-208 in the aorta were significantly different from those in the heart of WKY rats. The miR-155 level was significantly lower in aorta of <em>16</em>-week-old SHR than that of age-matched WKY rats (P<0.05), but there was no difference between SHR and WKY rats in other age groups. In addition, miR-155 level was negatively correlated to blood pressure (r=-0.525, P<0.05). Both in WKY rats and SHR, miR-208 was most abundantly expressed in 4-week-old rats, but declined significantly in 8-, <em>16</em>- and 24-week-old rats (P<0.05). No difference in miR-208 levels was observed between age-matched SHR and WKY rats. Moreover, miR-208 expression in aorta was negatively correlated with blood pressure (r=-0.400, P<0.05) and age (r=-0.684, P<0.0001). Neither miR-1 nor miR-133a was differentially expressed in SHR and WKY rats in different age groups. The mRNA levels of predicted target genes were not correlated to miR-155 or miR-208 levels. These results indicate that miR-155 is less expressed in the aorta of adult SHR compared with that of WKY rats and is negatively correlated with blood pressure, suggesting it is possibly involved in the development and pathologic progress of hypertension. The miR-208 expression in rat aorta declines with aging and it may play a role in the blood vessel development.