Although anti-angiogenic agents targeting VEGF have shown affordable beneficial outcomes in several human cancer types, in most pre-clinical and clinical studies, these effects are transient and followed by rapid relapse and tumor re<em>growth</em>. Recently, it has been suggested that recruited bone marrow derived cells (BMDCs) to the tumor-microenvironment together with stromal cells play an important role in development of resistance to anti-VEGF therapies. Additionally, acquired resistance to anti-VEGF therapies has shown to be mediated partly through overexpression of different pro-angiogenic cytokines and <em>growth</em> <em>factors</em> including G-CSF, IL-6, IL-8, VEGF and FGF by these cells. Alongside, IL-<em>17</em>, a pro-inflammatory cytokine, mostly secreted by infiltrated CD4+ T helper cells, has shown to mediate resistance to anti-VEGF therapies, through recruiting BMDCs and modulating stromal cells activities including endothelial cells, tumor associated macrophages and cancer associated <em>fibroblasts</em>. Here, we examined the role of BMDCs, tumor stromal cells, IL-<em>17</em> and their negotiation in development of resistance to anti-VEGF targeted therapies.

Interleukin 1 (IL-1) is an important regulator of immune system function. IL 1 also affects haematopoiesis in vitro: it causes release of colony stimulating <em>factors</em> from <em>fibroblasts</em> and endothelial cells and can directly act on primitive haematopoietic stem cells. We investigated IL 1 production in vitro by stimulated peripheral blood mononuclear cells of patients with aplastic anaemia (N = <em>17</em>), patients with other haematologic diseases (N = 27), and normal individuals (N = 22) using a bioassay for IL 1 activity. Ten aplastic patients showed markedly decreased IL 1 production. IL 1 production by fibronectin-affinity purified monocytes was decreased in six of seven of these patients; in three other cases, in which IL 1 mononuclear cell production was undetectable, sufficient monocytes could not be isolated. IL 1 alpha and IL 1 beta precursor molecules were also absent or much decreased when mononuclear cell lysates from these patients were analysed by immunoblot using specific polyclonal sera. Aplastic patients with low IL 1 production were distinguished by the severity of their disease and the degree of neutropenia. Patients with myelodysplasia with comparable degrees of pancytopenia had normal IL 1 production. This is the first example of deficient haematopoietic <em>growth</em> <em>factor</em> production in a bone marrow failure syndrome. Decreased IL 1 production may contribute to the pathogenesis of some cases of aplastic anaemia and to susceptibility to infection.

T helper <em>17</em> (Th<em>17</em>) cells that produce interleukin (IL)-<em>17</em>A and IL-<em>17</em>F have been found to participate in the development of bronchial asthma and bleomycin-induced pulmonary fibrosis. However, whether they play a causative role in the airway remodeling observed in these respiratory diseases remains unclear. Because fibrocytes are involved in tissue repair and fibrosis and are presumably precursors of lung <em>fibroblasts</em> and myo<em>fibroblasts</em>, we examined the effects of IL-<em>17</em>A/F on fibrocyte functions. Both IL-<em>17</em>A and IL-<em>17</em>F enhanced fibrocytes' α-smooth muscle actin expression. Priming fibrocytes with IL-<em>17</em>A enhanced their CD40-mediated IL-6 production, whereas IL-<em>17</em>F-priming increased the CD40-mediated mRNA expression of collagen I, vascular endothelial <em>growth</em> <em>factor</em>, and angiogenin. CD4(+) T cells co-cultured with fibrocytes produced IL-<em>17</em>A, which was inhibited by blocking CD40 and CD40 ligand interactions. These findings suggest that cooperative interactions between fibrocytes and Th<em>17</em> cells play an important role via CD40- and IL-<em>17</em>A/F-mediated signaling for collagen and proangiogenic <em>factor</em> production, which may lead to the extracellular matrix deposition and neovascularization seen in airway remodeling.

Over the last several years, our in vitro and in vivo studies have focused on optimizing the use of fibrin to deliver cells. We have shown that some three-dimensional (3D) fibrin constructs with specific fibrinogen and thrombin concentration support robust proliferation of normal human dermal <em>fibroblasts</em>, whereas different fibrinogen and thrombin concentrations support high mesenchymal stem cell proliferation in 3D fibrin constructs. In this article, we found that normal human epithelial keratinocytes proliferate well in 3D fibrin constructs consist of fibrinogen concentration ranging from <em>17</em> to 33 mg/mL and thrombin concentration of 1 U/mL. Further, using a new proliferation assay, we studied the proliferation of <em>fibroblasts</em> and keratinocytes cocultured in various 3D fibrin constructs of different fibrinogen and thrombin concentrations. We found that 3D fibrin constructs with a range of fibrinogen concentration (5-34 mg/mL) and a thrombin concentration of 1 U/mL produce an optimal cell proliferation for both cell types when cocultured. This profile of proliferation is different from that seen when keratinocytes or <em>fibroblasts</em> are incorporated separately in 3D fibrin constructs. In conclusion, we found that one needs to choose the fibrinogen and thrombin concentration carefully depending on the cell type to deliver; that is, different fibrin constructs with different fibrinogen and thrombin concentration are required to deliver <em>fibroblasts</em> or keratinocytes alone or to codeliver both cell types. Moreover, there seems to be a cross-talk between keratinocytes and <em>fibroblasts</em> when they are cointroduced in 3D fibrin constructs. This feedback could be due to the effects of <em>growth</em> <em>factors</em> produced by the two cell types in the 3D fibrin constructs.

Genetic predisposition likely plays an important role in the development of intracranial aneurysms. We carried out a genome search meta-analysis to identified loci associated with intracranial aneurysm. We identified previous whole-genome linkage analyses by searching PUBMED. Five studies reported by separate investigators where detailed data could be obtained were included in our analysis. We synthesized the available genome-wide scan data by using a heterogeneity-based genome search meta-analyses. We identified two linkage sites on chromosomes 3 and <em>17</em> which had P-values <0.01 for association with intracranial aneurysm. Our findings confirm the association of a locus on chromosome <em>17</em> and identify a new linkage site on chromosome 3 for intracranial aneurysm. The new locus contains a number of potential gene candidates including kininogen-1 precursor, <em>fibroblast</em> <em>growth</em> <em>factor</em>-12 and endothelin converting enzyme 2.

We examined the localization of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) in the developing embryonic and newborn rat nervous system using 2 anti-bFGF antibodies. Embryonic (E13, E14, E15, E16, E<em>17</em>, and E18) and newborn tissues were examined. Between E16 and E<em>17</em> strong bFGF immunoreactivity (IR) was detectable in the cortex and striatum and, in addition, in almost all neurons of the brainstem, spinal cord, and spinal ganglia. In contrast, in the newborn rat bFGF-IR was found in neuronal subpopulations of brainstem nuclei, ventral spinal cord, and spinal ganglia as it is known for the respective postnatal/adult parts of the nervous system. At E16 7.0 kb and 3.7 kb bFGF mRNA were present. The identification of bFGF-responsive cells was performed using immunocytochemistry (anti-flg antibody) and 125I bFGF for binding studies. The neuronal localization of FGF-receptor suggests that bFGF mediates its effects in an autocrine or paracrine manner. At the time of strongest bFGF-staining (E16/<em>17</em>), proliferation of neurons is almost completed in most of the nervous system areas. Therefore, it could also be suggested from previous biological experiments that the physiological functions of bFGF could include trophic and/or differentiating effects on developing neurons rather than mitogenic effects. The change of the bFGF-staining pattern after birth could indicate a change in the physiological function of bFGF, i.e., different bFGF effects in the immature and mature nervous systems.

A method for the in vitro proliferation of human bone marrow mesenchymal stem cells (MSCs) employing a medium not containing fetal calf serum (FCS) was developed for a regenerative medicine of cartilage using MSCs. Without using density-gradient centrifugation, the bone marrow aspirate was poured into a dish (6.0 \times 10(5) nucleated cells/cm(2)) with DMEM medium containing 10% serum (FCS or donor serum) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and incubated at 37 degrees C under a 5% CO(2) atmosphere. The density of adhesive cells incubated with the medium containing human serum and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (10 ng/ml) almost reached confluence at 19d and was 1.4-2.7 times that in the medium containing only FCS. The density of cells incubated with the medium containing only human serum was 0.1-0.6 times that in the medium containing only FCS. The content of CD45(-) CD105(+) cells among the cells harvested after a 19-d incubation in the medium containing human serum and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> was higher than 90%. This high content and chondrogenic activity, which was confirmed by pellet cultivation and staining with Safranine O, were maintained even after further subcultivation in the medium to <em>17</em> population doubling levels. Consequently, this method might be applicable to in vitro proliferation of MSCs for the regeneration of cartilage.

Primary cultures of bovine microvascular endothelial cells (BME) isolated from the adrenal cortex, are commonly used to study vascular endothelium, but have a limited life span. To circumvent these limitations, we have immortalized BME cells with either simian virus 40 (SV40) or with a retrovirus containing the coding region of human telomerase reverse transcriptase (hTERT), and have investigated whether the clonal populations obtained, maintain differentiated properties characteristic of microvascular endothelium. Immortalized cells were characterized for maintenance of typical endothelial morphology, marker expression, and functional characteristics including uptake of Acetylated low-density lipoprotein (Ac-LDL), capillary-like tube formation in three-dimensional collagen gels, as well as metalloproteinase (MMP) and plasminogen activator (PA)-mediated extracellular proteolysis. Whilst immortalization of BME cells with SV40 was associated with loss of endothelial-specific properties, hTERT-BME exhibited an endothelial phenotype similar to that of wild-type endothelial cells. Specifically, they showed a typical cobblestone morphology, were contact-inhibited, expressed endothelial cell-specific markers (e.g., CD31, vWF) and both <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGFR-1) and vascular endothelial <em>growth</em> <em>factor</em> receptor-2 (VEGFR-2). In addition, they expressed receptors for LDL. Importantly, when grown on collagen gels, hTERT-BME cells underwent MMP-dependent tube-like structure formation in response to VEGFR-2 activation. In a collagen gel sandwich assay, hTERT-BME formed tubular structures in the absence of exogenously added angiogenic cytokines. Sustained tube formation was induced by VEGF-A alone or in combination with FGF-2. From <em>17</em> sub-clones that displayed a non-transformed phenotype, a high proliferative capacity and tubulogenic properties in three-dimensional collagen gels, we isolated two distinct subpopulations that display a highly specific response to VEGF-A or to FGF-2. We have generated hTERT-BME cells that maintain endothelial-specific properties and function and have isolated clones that respond differentially to VEGF-A or FGF-2. These immortalized cell lines will facilitate the study of endothelial cell biology.

OBJECTIVE

To determine the localisation and distribution of connective tissue growth factor (CCN2; CTGF) and transforming growth factor beta type 1 (TGF-beta1) in uterine tissues from cycling and early pregnant pigs.

METHODS

In situ hybridisation and immunohistochemistry were used to localise CCN2 (CTGF) or TGF-beta1 in uteri obtained from gilts on days 0, 5, 10, 12, 15, and 18 of the oestrous cycle or days 10, 12, 14, 16, 17, and 21 of gestation.

RESULTS

In cycling animals, CCN2 (CTGF) mRNA and protein were abundant in luminal epithelial cells (LECs) and glandular epithelial cells (GECs), with lesser amounts in stromal fibroblasts and little or none in endothelial cells. A similar pattern of staining was seen up to day 10 of pregnancy, except that overall staining intensities for CCN2 (CTGF) mRNA or protein were higher and that stromal and endothelial cells were CCN2 (CTGF) positive. However, on days 12-17 there was a striking decrease in the amount of CCN2 (CTGF) in LECs at the utero-conceptus interface, which was associated with maternal stromal matrix reorganisation and the onset of subepithelial neovascularisation. This differential distribution of CCN2 (CTGF) was localised to those LECs that were in close proximity to or in apposition with trophoblast cells. This decrease in CCN2 (CTGF) staining was transient in nature and high amounts of CCN2 (CTGF) were again apparent in LECs on days 17-21, when endometrial neovascularisation and matrix remodelling were complete. The expression of uterine TGF-beta1 was comparable to that of CCN2 (CTGF) at most stages of the oestrous cycle or early pregnancy. Pre-elongation blastocysts recovered on day 10 were positive for both CCN2 (CTGF) and TGF-beta1 in the extra-embryonic trophectoderm, endoderm, and inner cell mass. On day 12, trophectoderm expressed low amounts of TGF-beta1 mRNA and non-detectable amounts of TGF-beta1 protein or CCN2 (CTGF) mRNA or protein. By days 17-21, the expression of both growth factors in the extra-embyronic/placental membranes increased and frequently exceeded that seen in LECs.

CONCLUSIONS

The pattern of CCN2 (CTGF) production during the initial attachment phase supports a role for this factor in stromal remodelling and neovascularisation, although alternative functions at later stages such as epithelial-epithelial interactions are also possible. In most major cell types in the uterus or utero-placental unit, CCN2 (CTGF) expression was highly correlated with that of TGF-beta(1), indicating that CCN2 (CTGF) may mediate some of the functions of TGF-beta in the reproductive tract during the oestrous cycle and pregnancy. The data further highlight epithelium as an important source of CCN2 (CTGF) in the regulation of uterine function.

BACKGROUND

Some chemokines are known to accelerate wound healing. However, there has been no report on the relationship between Thymus and activation-regulated chemokine (TARC)/CC chemokine ligand (CCL) <em>17</em> and wound healing. The purpose of this study was to determine whether CCL<em>17</em> enhances response to cutaneous injury.

METHODS

We made a full-thickness dorsal wound in transgenic (Tg) mice, in which CCL<em>17</em> was overexpressed and in control mice. Wound size was compared over the course of time. We evaluated the effect of CCL<em>17</em> on fibroblast migration by a Boyden chamber assay and a scratch wound assay.

RESULTS

Wound closure in Tg mice was more accelerated than in control mice. CCL<em>17</em> enhanced nerve growth factor (NGF) production by 2B4, which is mouse T cell hybridoma. Further, in the wound area of Tg mice, the number of CCR4(+) fibroblasts, CCR4(+) lymphocytes and mast cells was increased compared to control mice, as was the number of NGF(+) lymphocytes around the wound area. In vitro assay, CCL<em>17</em> was shown to enhance the migration of fibroblasts.

CONCLUSIONS

These results suggest that CCL<em>17</em> accelerates wound healing, mainly by enhancing fibroblast migration, and possibly by increasing NGF(+) lymphocytes and mast cells, which have independently been reported to enhance wound healing.

Protein kinase C (PKC) is present in high concentrations in neuronal tissues and participates in various neuronal functions. Ten isoforms have so far been identified and each PKC isoform may be activated by a variety of stimuli. By immunoblot analysis the presence of PKC isoforms was examined in dissociated cell cultures of the hippocampus and during the development in vivo. As soon as embryonic day <em>17</em> the hippocampus contains detectable amounts of PKC epsilon and zeta and low levels of PKC alpha. PKC beta and gamma appear during the first and second post-natal week. All isoforms progressively increase until the adult age. Cultures of hippocampal neurons derived from rat embryos express PKC alpha, epsilon and zeta whereas PKC beta and gamma are undetectable; a distinct pattern is observed in cultures of hippocampal glial cells. The neuronal levels of PKC alpha, epsilon and zeta increase during the period in culture and are enhanced when hippocampal neurons are exposed to the continuous presence of basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. Immunofluorescence of PKC epsilon and zeta occupies all the cytoplasmic neuronal compartment. The early expression of some PKC isoforms in cultures of post-mitotic hippocampal neurons suggest their involvement in morphological events that occur during this period; in particular the neuritic out<em>growth</em>.

The <em>growth</em> and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment (1). Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions (1-2). However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor <em>growth</em> that lead to changes in gene expression, cell division, differentiation and invasion(3). Matrix density (4), stiffness (5-6), and structure (6-7), interstitial fluid pressure (8), and interstitial fluid flow (8) are all altered during cancer progression. Interstitial fluid flow in particular is higher in tumors compared to normal tissues (8-10). The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 μm s(-1), depending on tumor size and differentiation (9, 11). This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability (12). Interstitial fluid flow has been shown to increase invasion of cancer cells (13-14), vascular <em>fibroblasts</em> and smooth muscle cells (15). This invasion may be due to autologous chemotactic gradients created around cells in 3-D (16) or increased matrix metalloproteinase (MMP) expression (15), chemokine secretion and cell adhesion molecule expression (<em>17</em>). However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of <em>fibroblasts</em> into tumor-promoting myo<em>fibroblasts</em> (18), (b) transporting of antigens and other soluble <em>factors</em> to lymph nodes (19), and (c) modulating lymphatic endothelial cell morphogenesis (20). The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion (13-15, <em>17</em>). By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Three new macrocyclic diterpenes, kansuinins F (1), G (2), and H (3), together with four known jatrophane diterpenes, kansuinins D (4), E (5), and A (6) and 3beta,5alpha,7beta,15beta-tetraacetoxy-9alpha-nicotinoyloxyjatropha-6(<em>17</em>)-11E-dien-14-one, were isolated from the roots of Euphorbia kansui. Compounds 1 and 2 were assigned as 6(<em>17</em>)-en-11,12-epoxy-14-one-type jatrophane diterpenes, and compound 3 as a 6(<em>17</em>)-en-11,14-epoxy-12-one jatrophane diterpene. The structures of compounds 1-3 and the relative configurations of compounds 4 and 5 were determined by spectral data analysis. Kansuinin E (5) exhibited a specific survival effect on <em>fibroblasts</em> that expressed TrkA, a high-affinity receptor for nerve <em>growth</em> <em>factor</em>.

OBJECTIVE

CXC chemokine 10 (CXCL10) activates CXC chemokine receptor 3 (CXCR3) and attracts activated T-helper 1 cells. In this study we examined the effects of cytokines on CXCL10 production by human gingival fibroblasts.

METHODS

Human gingival fibroblasts were exposed to pro-inflammatory cytokines (interleukin-1beta, tumor necrosis factor-alpha), a T-helper 1 cytokine (interferon-gamma), T-helper 2 cytokines (interleukin-4, interleukin-13), T-helper 17 cytokines (interleukin-17A, interleukin-22) and regulatory T-cell cytokines (interleukin-10, transforming growth factor-beta1) for 24 h. CXCL10 production by human gingival fibroblasts was examined by enzyme-linked immunosorbent assay.

RESULTS

Human gingival fibroblasts produced CXCL10 protein upon stimulation with interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma. Treatment of human gingival fibroblasts with interferon-gamma in combination with tumor necrosis factor-alpha or interleukin-1beta resulted in a synergistic production of CXCL10. However, interleukin-4 and interleukin-13 inhibited CXCL10 production by interferon-gamma-stimulated or tumor necrosis factor-alpha-stimulated-human gingival fibroblasts. On the other hand, interleukin-17A and interleukin-22 enhanced CXCL10 production by human gingival fibroblasts treated with interferon-gamma and inhibited CXCL10 production by tumor necrosis factor-alpha-stimulated human gingival fibroblasts. Furthermore, the anti-inflammatory cytokine, interleukin-10, inhibited CXCL10 production by both interferon-gamma- and tumor necrosis factor-alpha-stimulated human gingival fibroblasts, but transforming growth factor-beta1 enhanced interferon-gamma-mediated CXCL10 production by human gingival fibroblasts.

CONCLUSIONS

These results mean that the balance of cytokines in periodontally diseased tissue may be essential for the control of CXCL10 production by human gingival fibroblasts, and the production of CXCL10 might be important for the regulation of T-helper 1 cell infiltration in periodontally diseased tissue.

Patients with traumatic brain injury (TBI) and skeletal injuries have increased rates of excessive bone healing (EH = hypertrophic callus formation and/or heterotopic ossification). Polytrauma patients are often attributed higher rates of delayed fracture union. This study compares 182 total fractures in 29 isolated polytrauma patients (POLY) and 48 patients after TBI and polytrauma (TBI+POLY), examining the clinical parameters of EH versus delay. A subset of 28 patients (13 TBI+POLY, 15 POLY) underwent serological testing for the following bone turnover parameters: carboxy-terminal extension peptide of type 1 procollagen (P1CP), pyridinolene cross-linked carboxy-terminal telopeptide (1CTP), insulin-like <em>growth</em> <em>factor</em>-1 (IGF-1), insulin-like <em>growth</em> <em>factor</em> binding protein-3 (IGFBP-3), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). There were higher rates of delayed union in the POLY patients (45% vs. 23%) and EH in the TBI+POLY patients (33% vs. <em>17</em>%) (not significant = NS). More delayed unions were observed in diaphyseal fractures suffered by POLY (28%) than in TBI+POLY (15%) patients (NS). EH after pelvic fracture was apparent in 52% TBI+POLY and in 21% POLY fractures (NS). P1CP levels did not differ between the groups, but the collagen breakdown parameter 1CTP was significantly higher in the POLY group (p = 0.01-0.04). IGF-1 levels were below normal in both groups, and did not differ. IGFBP-3, an IGF-1-inhibiting and collagenase-3-activating protein, was significantly higher in POLY patients (p = 0.0<em>17</em>-0.037). bFGF levels did not vary between groups. Increased serum levels of 1CTP and IGFBP-3 in POLY patients suggest that EH in TBI patients is secondary to decreased collagen breakdown rather than increased synthesis.

TTSPs [type II TMPRSSs (transmembrane serine proteases)] are a <em>growing</em> family of trypsin-like enzymes with, in some cases, restricted tissue distribution. To investigate the expression of TTSPs in the nervous system, we performed a PCR-based screening approach with P10 (postnatal day 10) mouse spinal cord mRNA. We detected the expression of five known TTSPs and identified a novel TTSP, which we designated neurobin. Neurobin consists of 431 amino acids. In the extracellular part, neurobin contains a single SEA (sea-urchin sperm protein, enterokinase and agrin) domain and a C-terminal serine protease domain. RT-PCR (reverse transcription-PCR) analysis indicated the expression of neurobin in spinal cord and cerebellum. Histochemical analysis of brain sections revealed distinct staining of Purkinje neurons of the cerebellum. Transiently overexpressed neurobin was autocatalytically processed and inserted into the plasma membrane. Autocatalytic activation could be suppressed by mutating Ser(381) in the catalytic pocket to an alanine residue. The protease domain of neurobin, produced in Escherichia coli and refolded from inclusion bodies, cleaved chromogenic peptides with an arginine residue in position P(1). Serine protease inhibitors effectively suppressed the proteolytic activity of recombinant neurobin. Ca2+ or Na+ ions did not significantly modulate the catalytic activity of the protease. Recombinant neurobin processed <em>17</em>-kDa FGF-2 (<em>fibroblast</em> <em>growth</em> <em>factor</em>-2) at several P(1) lysine and arginine positions to distinct fragments, in a heparin-inhibitable manner, but did not cleave FGF-7, laminin or fibronectin. These results indicate that neurobin is an authentic TTSP with trypsin-like activity and is able to process FGF-2 in vitro.

OBJECTIVE

Previously reported from this laboratory are two distinct factors responsible for corneal endothelium modulation: basic fibroblast growth factor (bFGF) and the corneal endothelium modulation factor (CEMF) that is released by inflammatory cells. The altered phenotypes mediated by these two distinct factors--marked increase in cell proliferation, cell shape changes, and synthesis of fibrillar collagens--are identical. The current study sought to determine if bFGF is the direct mediator for corneal endothelium modulation and if CEMF plays a role in inducing bFGF production.

METHODS

bFGF synthesis mediated by CEMF was analyzed by immunoblot assay; cycloheximide was used to block protein synthesis. bFGF-Specific antisense oligonucleotide primer was used to inhibit CEMF-mediated bFGF synthesis and to block further the autocrine activity of bFGF. Cell proliferation was measured by cell counting. The steady-state levels of RNA were determined by Northern blot analysis. CEMF was further purified to homogeneity by sequential electrophoresis, elution, and renaturation of protein.

RESULTS

The synergistic effect of CEMF and bFGF on corneal endothelial cells was measured by their growth-promoting activity on quiescent corneal endothelial cells. There was a dose-dependent cell proliferation mediated by bFGF at any given CEMF concentration. Thus, bFGF at 10 ng/ml with CEMF at 2.5 micrograms/ml demonstrated saturable synergistic activity on endothelial cell proliferation. When the steady-state levels of collagen RNA were measured under these conditions, the untreated cells showed the doublets of 5.6 and 5.0 kb of alpha 2(I) collagen RNA. The cells treated simultaneously with bFGF and CEMF contained mostly lower transcript, compared to the significant level of upper transcript in control cells. However, there was no significant change in the level of 6.9-kb type IV collagen RNA qualitatively or quantitatively; nonetheless, the level of alpha 2(IV) collagen RNA was lowest in cells treated with bFGF plus CEMF. Neither exogenous bFGF nor CEMF caused induction of bFGF messenger RNA in corneal endothelial cells, whereas simultaneous treatment with bFGF and CEMF selectively enhanced the 4.9-kb transcript. When protein synthesis was inhibited by cycloheximide, bFGF synthesis was blocked in the presence of CEMF, leading to inhibition of corneal endothelium modulation. The effect on endothelial cell growth of bFGF antisense primer was analyzed. Antisense primer blocked by 50% the enhanced growth potential mediated by bFGF induced with CEMF. Finally, CEMF was purified to homogeneity: the purified protein is approximately 17 kD and assumes the modulating activities.

CONCLUSIONS

These findings suggest that de novo synthesis of bFGF induced by CEMF is required for corneal endothelium modulation.

Development of the myotome within somites depends on unknown signals from the neural tube. The present study tested the ability of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1) and dorsalin-1 (dsl-1) to promote myogenesis in stage 10-14 chick paraxial mesoderm utilizing 72 hour explant cultures. Each of these <em>factors</em> alone and the combination of bFGF with dsl-1 had limited to no myogenic-promoting activity, but the combination of bFGF with TGF-beta1 demonstrated a potent dose-dependent effect. In addition, bFGF enhanced the survival/proliferation of somite cells. 98% of stage 10-11 caudal segmental plate explants treated with bFGF plus TGF-beta1, exhibited myosin heavy chain (MHC)-positive cells (avg.=60 per explant), whereas only 15% of similarly treated somites responded with an average of 5 MHC-positive cells. Thus at stage 10-11, there are rostrocaudal differences in myogenic responsiveness with the caudal (more 'immature') paraxial mesoderm being more myogenically responsive to these <em>factors</em> than are somites. It was also discovered that <em>17</em>% of stage 10-11 caudal segmental plate explants exhibited several MHC-positive cells even when cultured without added <em>growth</em> <em>factors</em>, further demonstrating a different myogenic potential of the caudal paraxial mesoderm. Stage 13-14 paraxial mesoderm also exhibited a myogenic response to bFGF/TGF-beta1 but, unlike stage 10-11 embryos, both somites and segmental plate exhibited a strong response. A two-step mechanism for the bFGF/TGF-beta1 effect is suggested by the finding that only TGF-beta1 was required during the first 12 hours of culture, whereas bFGF plus a TGF-beta-like <em>factor</em> were required for the remainder of the culture. The biological relevance of the findings with bFGF is underscored by the observation that a monoclonal antibody to bFGF inhibited myogenic signaling from the dorsal neural tube. However, a monoclonal antibody that can neutralize the three <em>factors</em> TGF-beta1, TGF-beta2 and TGF-beta3 did not block myogenic signals from the neural tube, raising the possibility that another TGF-beta family member may be involved in vivo.

BACKGROUND

Renal cell carcinoma (RCC) is recognized as a neoplasm resistant to chemotherapy. In vitro experiments demonstrated that suramin, at noncytotoxic doses, enhanced the activity of chemotherapy including 5-fluorouracil (5-FU) in xenograft models.

METHODS

A phase I/II trial of noncytotoxic suramin in combination with weekly 5-FU in patients with metastatic RCC was conducted. The treatment consisted of intravenous (i.v.) suramin followed by a 500 mg/m2 i.v. bolus of 5-FU given 4.5 hours after starting suramin. In the phase I portion, a cohort of 6 patients received a suramin dose calculated to achieve a plasma level of 10-50 micromol/L. Therapy was administered once weekly for 6 doses, followed by 2 weeks off. This was followed by a phase II portion in which the primary goal was to determine the objective response rate.

RESULTS

Twenty-three patients were enrolled in the study: 6 in the phase I portion and <em>17</em> in phase II. Seventy-eight percent of patients were men, the mean age was 58.8 years, 96% had previous nephrectomy, and 70% had received previous systemic therapy. Histologic subtype was clear cell in 91%. Dose-limiting toxicity was observed in 1 of 6 patients (grade 3 hypersensitivity related to suramin infusion). The suramin dosing nomogram used in phase I and II portions of the trial yielded the desired plasma level of 10-50 micromol/L from 4.5 hours to 48 hours after infusion in 94 of 115 treatments. No objective responses were noted, and the median time to treatment failure was 2.5 months. The major toxicities (all grades) were fatigue (83%), nausea/vomiting (78%), diarrhea (61%), and chills (61%).

CONCLUSIONS

Suramin levels expected to reverse fibroblast growth factor-induced resistance can be achieved with the dosing regimen used in this study. The toxicity observed with suramin and 5-FU was acceptable. The combination does not have clinical activity in patients with metastatic RCC.

Osteoblast-like cells, such as UMR 106 osteosarcoma cells, are known to be <em>growth</em> stimulated by <em>growth</em> <em>factors</em> such as EGF. In contrast, <em>factors</em> such as PTH and prostaglandin E2 inhibit their <em>growth</em>. The exact signal transduction mechanisms by which these latter <em>factors</em> act remain to be elucidated. Here we show that simultaneous treatment of UMR 106 cells with EGF and PTH-(1-34) resulted in a level of DNA synthesis intermediate between the levels of treatment with epidermal <em>growth</em> <em>factor</em> (EGF) and PTH alone. This correlated with the interference of PTH-(1-34) early in an EGF receptor-linked signal transduction pathway, i.e. the EGF-induced activation of p42 mitogen-activated protein (MAP) kinase. This effect was also found for prostaglandin E2, and could be potentiated by the phosphodiesterase inhibitor isobutyl-methylxanthine and mimicked by forskolin and 8-bromo-cAMP. There was a strict correlation between the lowest concentration of PTH-(1-34) required to enhance protein kinase A (PKA) activity and that required to inhibit MAP kinase activation, whereas saturating amounts of PTH-(3-34), a PTH analog unable to elevate PKA activity, had no effect. Lysophosphatidic acid- and 12-O-tetracanoylphorbol-13-acetate-induced MAP kinase activation were also inhibited by PTH-(1-34) and forskolin in these cells. Similar effects were seen on basic <em>fibroblast</em> <em>growth</em> <em>factor</em>-mediated MAP kinase activation in ROS <em>17</em>/2.8 cells, indicating that this mechanism is a general feature of PTH in osteosarcoma cells. The inhibition of this mitogenic pathway through activation of PKA might play an important role in PTH-induced changes in proliferation and differentiation of osteoblasts.

Human interferon beta (IFN-beta) has been developed as a major biotherapeutic agent for the treatment of multiple sclerosis. Since World Health Organization (WHO) international standards (IS) for IFN-beta were established several years prior to the development of clinical grade IFN-beta products, a number of scientific issues with regard to the biological standardisation of natural and recombinant IFN-beta products have emerged. In order to address these issues, an international collaborative study to evaluate WHO IS and candidate international standards (CIS) of IFN-beta was instigated by the National Institute for Biological Standards and Control (NIBSC) in 2000 and was carried out in the succeeding year. Sixteen expert laboratories from 8 countries worldwide participated in the study. They performed titrations on 8 different IFN-beta preparations, including IS and new CIS, in a variety of mainly antiviral- but also including some antiproliferative- and reporter gene-assays, and contributed raw data from these assays to NIBSC for statistical analysis and calculation of potencies. While both intra- and inter-laboratory variation of potency estimates was evident, overall validity of the study as a whole was clearly shown by comparison of two pairs of internal coded duplicates, which gave the expected relative potency of 1 and the lowest inter-laboratory variability of potency estimates in all assay types. The CIS containing Chinese hamster ovary (CHO) cell- or human <em>fibroblast</em>-derived, glycosylated, IFN-beta gave similar low inter-laboratory variation in potency estimates one to another as the coded duplicates, which was significantly less than to the 2nd WHO IS of IFN-beta, human <em>fibroblast</em>-derived, Gb23-902-531. One of these CIS, designated 00/572, containing CHO cell-derived IFN-beta and formulated with both bovine casein and human serum albumin, could be assigned a potency, consistent for all assay types, of 40,000 international units (IU) per ampoule relative to the IU of the 2nd IS of IFN-beta, Gb23-902-531. Other CIS containing glycosylated IFN-beta, either CHO cell- or human-<em>fibroblast</em>-derived, could also be assigned potency values that were continuous with the IU of Gb23-902-531 and 00/572. However, greater inter-laboratory variations in estimates were evident from comparisons of Gb23-902-531 or 00/572 with either the 1st IS for E. coli-derived, non-glycosylated, IFN-beta with serine substitution at position <em>17</em> (IFN-beta Ser <em>17</em> mutein), Gxb02-901-535, or with a CIS (00/574) containing IFN-beta Ser <em>17</em> mutein. Indeed, variations in potency estimates for preparations containing IFN-beta Ser <em>17</em> mutein were sufficiently large to indicate that assays could distinguish preparations of IFN-beta Ser <em>17</em> mutein from preparations of glycosylated IFN-beta. Thus, neither the 2nd IS of IFN-beta, Gb23-902-531, containing <em>fibroblast</em>-derived IFN-beta, nor CIS, 00/572, containing CHO cell-derived IFN-beta, was appropriate for standardisation of preparations of IFN-beta Ser <em>17</em> mutein. Conversely, neither the IS of IFN-beta Ser <em>17</em> mutein, Gxb02-901-535, or a CIS of IFN-beta Ser <em>17</em> mutein, 00/574, was appropriate for the standardisation of preparations of glycosylated IFN-beta. CIS 00/572, containing CHO cell-derived, glycosylated IFN-beta, was clearly shown to be suitable to serve as a primary standard for glycosylated forms of IFN-beta, especially clinical grade IFN-beta-1a products. It was further shown to exhibit high thermal and long-term stability. Since the CHO cell-derived IFN-beta used for preparation of 00/572 was of a greater purity than the IFN-beta used for the 2nd IS of IFN-beta, Gb23-902-531, it was recommended by the WHO Informal Consultation on the Standardisation of Cytokines, <em>Growth</em> <em>Factors</em> and Other Endocrinological Substances, which met in October 2003, that 00/572 should replace Gb23-902-531 as the IS for glycosylated IFN-beta. This recommendation was accepted by the WHO Expert Committee on Biological Standardization (ECBS) at its annual meeting in November 2003 and 00/572 was established as the 3rd IS for human glycosylated IFN-beta with an assigned potency of 40,000 IU. As this study identified no advantage to replacing the existing 1st IS for IFN-beta Ser <em>17</em> mutein, Gxb02-901-535, WHO ECBS accepted that this should continue to serve as the IS for this material.

Heparin-binding protein <em>17</em>/<em>fibroblast</em> <em>growth</em> <em>factor</em>-binding protein-1 (HBp<em>17</em>/FGFBP-1) is a secreted protein that releases immobilized <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) from the extracellular matrix and plays a critical role in FGF bioactivation. In the present study co-localization of FGF-2 and HBp<em>17</em>/FGFBP-1 was observed in oral tissues including normal mucosa, hyperplasia, dysplasia of different degrees and oral squamous cell carcinoma (OSCC). The expression score for HBp<em>17</em>/FGFBP-1, FGF-2 as well as vascular endothelial <em>growth</em> <em>factor</em> A (VEGF-A) became higher with the severity of epithelial dysplasia and was highest in severe dysplasia. The expression of HBp<em>17</em>/FGFBP-1, FGF-2 and VEGF-A showed significant association with microvessel density, but no correlation with TNM stages or OSCC recurrence interval. Our results demonstrated that HBp<em>17</em>/FGFBP-1, like VEGF-A and FGF-2, might also promote the induction of tumor angiogenesis. The strongest expression of angiogenic <em>factors</em> in severe dysplasia suggests a potential point for targeting novel anti-angiogenic therapeutic strategies.

The purpose of this study was to elucidate the relationship between pituitary tumor transforming gene (PTTG) and invasiveness in pituitary macroadenomas and to determine the association between PTTG and both the tumor proliferative activity marker proliferation cell nuclear antigen (PCNA) and the angiogenic <em>factor</em> basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. A total of 70 patients with pituitary adenomas who underwent transsphenoidal or craniotomy surgical resection were enrolled. The average age were 42.5 ± 13.7 years (<em>17</em>-64 years) for the invasive group and 46.8 ± 12.1 years (16-71 years) for the non-invasive group, with no significant difference (P=0.<em>17</em>9) between the two groups. RT-PCR analysis of a group of pituitary macroadenomas demonstrated that the expression levels of PTTG and PCNA in invasive pituitary adenomas were significantly higher than in non-invasive pituitary adenomas. Both <em>factors</em> are both closely related to the invasive <em>growth</em> of pituitary adenomas and may possibly serve as important markers of this <em>growth</em>. In conclusion, PTTG may promote invasive tumor <em>growth</em> by stimulating pituitary adenomas proliferation. The mechanisms of tumor <em>growth</em> promotion and invasion of the surrounding structures by PTTG need to be further explored.

A radioimmunoassay (RIA) was developed to measure <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) using antiserum generated against a synthetic replicate of [Tyr10]FGF(1-10). The antisera, previously shown to be capable of inhibiting the biological action of FGF on bovine aortic arch endothelial cells in vitro, are highly specific for the amino-terminus of FGF. In the RIA, the antisera recognize the decapeptide antigen [Tyr10]FGF(1-10) and the intact mitogen on an equimolar basis and show less than 0.01% cross-reactivity with N-acetyl-[Tyr10]FGF(1-10). Bovine adenohypophysial cells maintained in primary monolayer culture release and ir-FGF which is indistinguishable from the intact mitogen in as much as it is retained on heparin-Sepharose affinity columns and shows a dose-dependent and parallel displacement in RIA. The release of ir-FGF by the bovine adenohypophysis can be increased with forskolin (10(-5) M) or KCl (50 mM). Preincubation of pituitary cells with <em>17</em> beta-estradiol has no measurable effects on basal ir-FGF, but increases the release after KCl treatment 2-3-fold. These results show that ir-FGF can be released by the bovine adenohypophysis in vitro and lend credence to the hypothesis that FGF plays a physiological role in the homeostatic mechanisms regulating mesoderm-derived cell <em>growth</em>.