BACKGROUND

Lung hypoplasia in congenital diaphragmatic hernia (CDH) seems to involve impaired alveolar septation. We hypothesized that disturbed deposition of elastin and expression of fibroblast growth factor 18 (FGF18), an elastogenesis stimulus, occurs in CDH.

OBJECTIVE

To document FGF18 and elastin in human CDH and ovine surgical and rat nitrofen models and to use models to evaluate the benefit of treatments.

METHODS

Human CDH and control lungs were collected post mortem. Diaphragmatic hernia was created in sheep at 85 days; fetal lungs were collected at 139 days (term = 145 days). Pregnant rats received nitrofen at 12 days; fetal lungs were collected at 21 days (term = 22 days). Some of the sheep fetuses with hernia underwent tracheal occlusion (TO); some of the nitrofen-treated pregnant rats received vitamin A. Both treatments are known to promote lung growth.

RESULTS

Coincidental with the onset of secondary septation, FGF18 protein increased threefold in control human lungs, which failed to occur in CDH. FGF18 labeling was found in interstitial cells of septa. Elastin staining demonstrated poor septation and markedly decreased elastin density in CDH lungs. Consistently, lung FGF18 transcripts were diminished 60 and 83% by CDH in sheep and rats, respectively, and elastin density and expression were diminished. TO and vitamin A restored FGF18 and elastin expression in sheep and rats, respectively. TO restored elastin density.

CONCLUSIONS

Impaired septation in CDH is associated with decreased FGF18 expression and elastic fiber deposition. Simultaneous correction of FGF18 and elastin defects by TO and vitamin A suggests that defective elastogenesis may result, at least partly, from FGF18 deficiency.

We have previously reported the presence of proteolytic activity in conditioned medium from human <em>fibroblast</em> cultures that cleaves insulin-like <em>growth</em> <em>factor</em>-binding protein-5 (IGFBP-5) into non-IGF-I-binding fragments. Coincubation of IGF-I or IGF-II and IGFBP-5 with <em>fibroblast</em> cultures decreased proteolysis. The protease was purified by heparin-Sepharose affinity chromatography. The purified protease cleaved IGFBP-5 into <em>22</em>-, 20-, and 17-kilodalton non-IGF-I-binding fragments. Protease inhibitor profiles obtained using partially purified enzyme showed that it was a calcium-dependent serine protease. After chelation with EDTA, the activity could only be partially restored with zinc, indicating that it was probably not a metalloprotease. The protease was specific for IGFBP-5 and did not cleave pure IGFBP-1, -2, -3, or -4. IGF-I and IGF-II caused minimal inhibition of proteolysis in vitro. This suggests that the IGF-I-induced increase in IGFBP-5 in <em>fibroblast</em> medium is only partially due to direct protease inhibition. Heparin, antithrombin-III (AT-III), and heparin co<em>factor</em>-II had inhibitory activity, and heparin potentiated the activity of AT-III. Synthetic peptides, that contained the active sites of AT-III and alpha 1-antichymotrypsin, were also inhibitory. Peptides containing sequences found in two basic regions of IGFBP-5 were tested, and one had inhibitory activity. In summary, <em>fibroblasts</em> secrete a serine protease that cleaves IGFBP-5 and is specific for this form of IGFBP. The protease has properties that are similar to kallikreins, a family of serine proteases that is known to cleave epidermal and nerve <em>growth</em> <em>factor</em>-binding proteins.

OBJECTIVE

Rheumatoid arthritis (RA) is characterized by leukocyte recruitment and angiogenesis. We investigated the effects of sulfasalazine (SSZ) and its metabolites, sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA), on components of angiogenesis, namely, endothelial cell (EC) chemotaxis and proliferation, as well as on EC chemokine and soluble adhesion molecule expression.

METHODS

SSZ, SP, and 5-ASA were assayed for their effects on basic fibroblast growth factor (bFGF)-induced human dermal microvascular endothelial cell (HMVEC) chemotaxis and proliferation. EC were plated on Matrigel to assess the effect of SSZ on EC tube formation. Enzyme-linked immunosorbent assays were performed to determine changes in HMVEC production of interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), growth-related oncogene alpha (GROalpha), epithelial neutrophil-activating peptide 78 (ENA-78), soluble E-selectin (sE-selectin), and soluble intercellular adhesion molecule 1 (sICAM-1) upon treatment with SSZ or its metabolites.

RESULTS

HMVEC incubated with SSZ or SP exhibited reduced bFGF-induced chemotaxis (59%, [n = 7] and 22%, [n = 3], respectively) (P<0.05). SSZ and SP decreased basal HMVEC proliferation, while 5-ASA increased proliferation (P<0.05; [n = 5]). SSZ decreased bFGF-induced HMVEC proliferation (P<0.05 [n = 5]). SSZ inhibited phorbol 12-myristate 13-acetate-induced HMVEC tube formation (P<0.05; [minimum n = 5]). Tumor necrosis factor alpha-stimulated HMVEC shedding of sICAM-1 was reduced by incubation with either SSZ (19%) or 5-ASA (23%) (P<0.05; [n = 6]). SP inhibited cytokine-stimulated HMVEC expression of IL-8 and MCP-1 (P<0.05; [n = 4]). Neither SSZ nor its metabolites had any effect on HMVEC production of sE-selectin, GROalpha, or ENA-78.

CONCLUSIONS

These results demonstrate that SSZ and its metabolite SP may affect the pathogenesis of RA by inhibiting EC chemotaxis, proliferation, tube formation, and expression of sICAM-1, IL-8, and MCP-1.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF or FGF-2), vascular endothelial <em>growth</em> <em>factor</em> (VEGF), and endothelin-1 (ET-1) are peptide <em>growth</em> <em>factors</em> (PGF) mediating normal lung development, maturation, injury, and repair. These PGF may therefore be involved in the pathogenesis of bronchopulmonary dysplasia (BPD). We hypothesized that elevated levels of these PGF in tracheal aspirates would be associated with a) BPD and/or death; b) markers of cell injury and apoptosis; and c) chorioamnionitis, a risk <em>factor</em> for BPD. Tracheal aspirates collected in 29 preterm (<34 wk gestation, 500-2000 g birth weight), mechanically ventilated infants on d 1 of life were assayed for PGF and histone-associated DNA fragments by ELISA and for LDH by enzyme assay. Clinical and pathologic examination was performed for chorioamnionitis. BPD was defined as oxygen requirement/mechanical ventilation at 28 d postnatal age. The birth weight (mean +/- SE) was 1009 +/- 85 g and median gestational age was 26 wk (range, <em>22</em>-33). Eighteen infants died or developed BPD. bFGF levels were elevated in infants who died or developed BPD [median (25%,75%) level of 36 (23, 44) pg/mL versus 14 (6, 30) in the survivors without BPD, p = 0.01]. bFGF levels correlated with apoptosis (r = 0.73, p < 0.001) and LDH levels (r = 0.59, p < 0.001). VEGF and ET-1 levels were not associated with apoptosis or with BPD/death. PGF levels were not associated with chorioamnionitis. We conclude that elevated bFGF levels in the preterm trachea correlate with BPD/death and markers of cell injury and apoptosis but not with chorioamnionitis. We speculate that bFGF may play a role in the development of BPD.

Genome-wide association studies (GWAS) and candidate gene analyses have led to the discovery of several dozen genetic polymorphisms associated with breast cancer susceptibility, many of which are considered well-established risk <em>factors</em> for the disease. Despite attempts to replicate these same variant-disease associations in African Americans, the evaluable populations are often too small to produce precise or consistent results. We estimated the associations between 83 previously identified single nucleotide polymorphisms (SNPs) and breast cancer among Carolina Breast Cancer Study (1993-2001) participants using maximum likelihood, Bayesian, and hierarchical methods. The selected SNPs were previous GWAS hits (n = <em>22</em>), near-hits (n = 19), otherwise well-established risk loci (n = 5), or located in the same genes as selected variants (n = 37). We successfully replicated 18 GWAS-identified SNPs in whites (n = 2,352) and 10 in African Americans (n = 1,447). SNPs in the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 gene (FGFR2) and the TOC high mobility group box family member 3 gene (TOX3) were strongly associated with breast cancer in both races. SNPs in the mitochondrial ribosomal protein S30 gene (MRPS30), mitogen-activated protein kinase kinase kinase 1 gene (MAP3K1), zinc finger, MIZ-type containing 1 gene (ZMIZ1), and H19, imprinted maternally expressed transcript gene (H19) were associated with breast cancer in whites, and SNPs in the estrogen receptor 1 gene (ESR1) and H19 gene were associated with breast cancer in African Americans. We provide precise and well-informed race-stratified odds ratios for key breast cancer-related SNPs. Our results demonstrate the utility of Bayesian methods in genetic epidemiology and provide support for their application in small, etiologically driven investigations.

Low-grade diffuse astrocytomas are generally slow-<em>growing</em> tumors; however, they may progress to anaplastic astrocytomas or glioblastomas. As existing grading systems fail to distinguish these tumors, ongoing research strives to identify new prognostic markers. In this study, <em>22</em> adult patients with supratentorial diffuse astrocytomas, WHO grade II, were investigated to clarify whether proliferative activity, assessed by different Ki67 antibodies (MIB-1, NC-MM1, NC-Ki67p, rahKi67), expression of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) or microvessel density have prognostic power. The Ki67 antibodies revealed low proliferation indices (PI); however, there was a wide spread of values, ranging from 0.1% to about 10%. In general, a positive correlation between the different Ki67 PIs was found. In 12 of <em>22</em> cases, bFGF immunoreactivity was recorded. Microvessel density was generally low. MIB-1 PI was the only prognostic <em>factor</em> of statistical significance. Ki67 PI, obtained by using the monoclonal antibody MIB-1, can thus serve as a prognostic <em>factor</em> capable of identifying subsets of low-grade diffuse astrocytomas with a potentially more aggressive clinical behavior.

BACKGROUND

Although advanced esophageal squamous-cell carcinoma (ESCC) is treated using a multidisciplinary approach, outcomes remain unsatisfactory. The microenvironment of cancer cells has recently been shown to strongly influence the biologic properties of malignancies. We explored the effect of supernatant from esophageal fibroblasts on the cell growth and chemo-resistance of ESCC cell lines.

METHODS

We used 22 ESCC cell lines, isolated primary human esophageal fibroblasts and immortalized fibroblasts. We first examined cell proliferation induced by fibroblast supernatant. The effect of supernatant was evaluated to determine whether paracrine signaling induced by fibroblasts can influence the proliferation of cancer cells. Next, we examined the effects of adding growth factors HGF, FGF1, FGF7, and FGF10, to the culture medium of cancer cells. These growth factors are assumed to be present in the culture supernatants of fibroblasts and may exert a paracrine effect on the proliferation of cancer cells. We also examined the intrinsic role of HGF/MET and FGFs/FGFR in ESCC proliferation. In addition, we examined the inhibitory effect of lapatinib on ESCC cell lines and studied whether the fibroblast supernatants affect the inhibitory effect of lapatinib on ESCC cell proliferation. Finally, we tested whether the FGFR inhibitor PD-173074 could eliminate the rescue effect against lapatinib that was induced by fibroblast supernatants.

RESULTS

The addition of fibroblast supernatant induces cell proliferation in the majority of cell lines tested. The results of experiments to evaluate the effects of adding growth factors and kinase inhibitors suggests that the stimulating effect of fibroblasts was attributable in part to HGF/MET or FGF/FGFR. The results also indicate diversity in the degree of dependence on HGF/MET and FGF/FGFR among the cell lines. Though lapanitib at 1 μM inhibits cell proliferation by more than 50% in the majority of the ESCC cell lines, fibroblast supernatant can rescue the growth inhibition of ESCC cells. However, the rescue effect is abrogated by co-treatment with FGFR inhibitor.

CONCLUSIONS

These results demonstrate that cell growth of ESCC depends on diverse receptor tyrosine kinase signaling, in both cell-autonomous and cell-non-autonomous manners. The combined inhibition of these signals may hold promise for the treatment of ESCC.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) is a developmentally regulated transmembrane protein. Three other FGFRs (1, 2, and 4) in conjunction with FGFR3 are part of the receptor tyrosine kinase super-family. Mutations in three of these genes (FGFR1, 2, and 3) have been determined to be the cause of human <em>growth</em> and developmental disorders. We have characterized a <em>22</em>-kb DNA fragment containing the human FGFR3 gene and determined 11 kb of its nucleotide sequence. The gene consists of 19 exons and 18 introns spanning 16.5 kb, and the boundaries between exons and introns follow the GT/AG rule. The translation initiation and termination sites are located in exon 2 and exon 19 respectively. The sequence of the 5'-flanking region (1.5 kb) lacks the typical TATA or CAAT boxes. However, several putative binding sites for transcription <em>factors</em> SP1, AP2, Krox 24, IgHC.4, and Zeste are present. The 0.77-kb region from position -889 (5'-flanking region) to -119 (intron 1) contains a CpG island. A comparative sequence analysis of the human and mouse FGFR3 genes indicates that the overall genomic structure and organization of the human gene are nearly identical to those of its mouse counterpart. Furthermore, there is a striking similarity in the promoter regions of both genes, and several of the putative transcription <em>factor</em>-binding sites are conserved across species, suggesting a definitive role of these <em>factors</em> in the transcriptional regulation of these genes.

Multiple genetic changes take place during tumor development and progression. These genetic changes result in inactivation of tumor suppressor genes and activation of proto-oncogenes. Frequent genetic changes observed in gliomas are losses of chromosomal regions on 9p, 10q, 13q, 17p and on <em>22</em>. Loss of 10q is seen in more than 80% of the glioblastoma multiforme (GBM) tumors suggesting the presence of a gene critical for GBM formation on this chromosome. Amplification of epidermal <em>growth</em> <em>factor</em> receptor gene and expression of platelet derived <em>growth</em> <em>factor</em> and <em>fibroblast</em> <em>growth</em> <em>factor</em> genes are also common among gliomas. The most common genetic abnormality found in medulloblastomas is loss of 17p. The C-myc gene is amplified in a few primary tumors, but the incidence of amplification is higher in medulloblastoma derived cell lines. These findings suggest that the same two genetic processes, gene amplification and regional chromosomal loss, which characterize other primitive childhood neuroectodermal tumors such as retinoblastoma and neuroblastoma are also important in medulloblastomas.

Tissue-specific alternative splicing in the second half of Ig-like domain 3 (D3) of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors 1-3 (FGFR1 to -3) generates epithelial FGFR1b-FGFR3b and mesenchymal FGFR1c-FGFR3c splice isoforms. This splicing event establishes a selectivity filter to restrict the ligand binding specificity of FGFRb and FGFRc isoforms to mesenchymally and epithelially derived <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs), respectively. FGF1 is termed the "universal FGFR ligand" because it overrides this specificity barrier. To elucidate the molecular basis for FGF1 cross-reactivity with the "b" and "c" splice isoforms of FGFRs, we determined the first crystal structure of FGF1 in complex with an FGFRb isoform, FGFR2b, at 2.1 Å resolution. Comparison of the FGF1-FGFR2b structure with the three previously published FGF1-FGFRc structures reveals that plasticity in the interactions of the N-terminal region of FGF1 with FGFR D3 is the main determinant of FGF1 cross-reactivity with both isoforms of FGFRs. In support of our structural data, we demonstrate that substitution of three N-terminal residues (Gly-19, His-25, and Phe-26) of FGF2 (a ligand that does not bind FGFR2b) for the corresponding residues of FGF1 (Phe-16, Asn-<em>22</em>, and Tyr-23) enables the FGF2 triple mutant to bind and activate FGFR2b. These findings taken together with our previous structural data on receptor binding specificity of FGF2, FGF8, and FGF10 conclusively show that sequence divergence at the N termini of FGFs is the primary regulator of the receptor binding specificity and promiscuity of FGFs.

OBJECTIVE

Fibroblast growth factor 18 (FGF18) is expressed in rodent brain and is a trophic factor for neuron-derived cells in culture. The purpose of the present study was to evaluate whether FGF18 was neuroprotective in a rat model of cerebral ischemia and to compare the results with those obtained with FGF2.

METHODS

Cerebral ischemia was produced in rats by a transient 2-hour occlusion of the middle cerebral artery (MCAo) with an intraluminal filament followed by 22-hour reperfusion. Starting 15 minutes after MCAo, FGF18 or FGF2 was administered by a 3-hour intravenous infusion. Infarct volumes and behavioral deficits were measured 24 hours after MCAo.

RESULTS

Infusion of FGF18 produced dose-dependent reductions in infarct volumes and improvements in tests of reference and working memory, motor ability, and exploratory behavior. FGF18 was more efficacious than FGF2 on virtually all measures examined. The reductions in infarct volume and behavioral deficit were associated with FGF-mediated increases in regional cerebral blood flow.

CONCLUSIONS

These results demonstrate that FGF18 is an effective neuroprotective agent in a rat model of transient MCAo.

The development of multicellular embryos depends on coordinated cell-to-cell signalling events. Among the numerous cell-signalling pathways, <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) are involved in important processes during embryogenesis, such as mesoderm formation during gastrulation and <em>growth</em>. In vertebrates, the Fgf superfamily consists of <em>22</em> family members, whereas only few FGFs are contained in the less complex genomes of insects and worms. In the recently sequenced genome of the beetle Tribolium, we identified four Fgf family members representing three subfamilies. Tribolium has Fgf1 genes that are absent in Drosophila but known from vertebrates. By phylogenetic analysis and microsynteny to Drosophila, we further classify Tc-fgf 8 as an ancestor of pyramus and thisbe, the fly Fgf8 genes. Tc-fgf8 expression in the <em>growth</em> zone suggests an involvement in mesoderm formation. In the embryonic head, expression of Tc-fgf8 subdivides the brain into a larger anterior and a smaller posterior region. The Fgf Tc-branchless is expressed in the embryonic tracheal placodes and in various gland-like structures. The expression patterns of the only Tribolium Fgf receptor and the adaptor molecule Downstream-of-Fgfr are largely congruent with Tc-Fgf8 and Tc-bnl. Thus, in contrast to Drosophila, only one Fgf receptor canalises Fgf signalling in different tissues in Tribolium. Our findings significantly advance our understanding of the evolution of Fgf signalling in insects.

This study examined the influence of the following <em>growth</em> <em>factors</em> and cytokines on early embryonic development: insulin-like <em>growth</em> <em>factors</em> I and II (IGF-I, IGF-II), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), transforming <em>growth</em> <em>factor</em> (TGF-beta), granulocyte-macrophage colony-stimulating <em>factor</em> (GM-CSF), and leukemia inhibitory <em>factor</em> (LIF). Synthetic oviduct fluid (SOF) was used as the culture medium. We studied the development of bovine embryos produced in vitro and cultured until Day 9 after fertilization. TGF-beta1, bFGF, GM-CSF, and LIF used on their own significantly improved the yield of hatched blastocysts. IGF-I, bFGF, TGF-beta1, GM-CSF, and LIF significantly accelerated embryonic development, especially the change from the expanded blastocyst to hatched blastocyst stages. Use of a combination of these <em>growth</em> <em>factors</em> and cytokines (GF-CYK) in SOF medium produced higher percentages of blastocysts and hatched blastocysts than did use of SOF alone (45% and <em>22</em>% vs. 24% and 12%; P<0.05) on Day 8 after in vitro fertilization and similar results to use of SOF+10% fetal calf serum (38% and 16%, at the same stages, respectively). The averages of total cells, inner cell mass cells, and trophectoderm cells of exclusively in vitro Day-8 blastocysts for pooled GF-CYK treatments were higher than those for SOF and similar to those for fetal calf serum. The presence of these <em>growth</em> <em>factors</em> and cytokines in the embryo culture medium therefore has a combined stimulatory action on embryonic development; in particular through an increase in hatching rate and in the number of cells of both the inner cell mass and trophoblast. These results are the first to demonstrate that use of a combination of recombinant <em>growth</em> <em>factors</em> and cytokine, as IGF-I, IGF-II, bFGF, TGF-beta1, LIF, and GM-CSF, produces similar results to 10% fetal calf serum for the development of in vitro-produced bovine embryos. This entirely synthetic method of embryo culture has undeniable advantages for the biosecurity of embryo transfer.

Eicosapentaenoic acid (EPA; 20:5, n-3) can restrain tumor <em>growth</em> and metastasis in vivo; however, the mechanism of its antitumor effect is still not fully understood. Angiogenesis is a crucial process for tumor <em>growth</em> and metastasis and inhibition of tumor angiogenesis can suppress tumor <em>growth</em> and metastasis in vivo. Vascular endothelial <em>growth</em> <em>factor</em> (VEGF) is an important angiogenic <em>factor</em>. In this study, we investigated the mechanisms of the inhibitory effect of EPA on VEGF-induced proliferation of bovine carotid artery endothelial (BAE) cells. BAE cells, treated with 0-5 microg/ml EPA for 48 h, displayed a dose-dependent suppression to VEGF (0.2 nM)-induced proliferation. Similar inhibitory effect was not found in BAE cells treated with arachidonic acid (AA; 20:4, n-6), or docasahexaenoic acid (DHA; <em>22</em>:5, n-3). In contrast to its effect on VEGF-induced proliferation, EPA had no inhibition to basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF, 0.2 nM)-induced proliferation in BAE cells. Both VEGF and bFGF activated mitogen-activated protein (MAP) kinase in BAE cells; however, EPA selectively inhibited VEGF-induced, but not bFGF-induced activation of MAP kinase. Flk-1 expression was inhibited dose-dependently in EPA-treated cells, whereas Flt-1 expression was increased in EPA treated cells. This in vitro inhibitory effect by EPA on Flk-1 receptor expression provides indirect evidence that one of the mechanisms of EPA for antitumor action in vivo maybe related to its antiangiogenic action.

The 3T3-TNR9 cell line is a variant of Swiss 3T3 cells which does not respond mitogenically to tumor promoters, but does respond mitogenically to epidermal <em>growth</em> <em>factor</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em>, and serum. To elucidate differences between tumor promoters and polypeptide mitogens in the pathway(s) of mitogenesis which might be responsible for the nonresponsiveness of the 3T3-TNR9 cells, we have examined in these cells the early protein phosphorylation events known to be associated with mitogenesis in the parental 3T3 cells. We find that the 3T3-TNR9 cells display levels of tetradecanoyl phorbol acetate binding and of a calcium- and phospholipid-dependent protein kinase activity which are at least the equal of those seen in the parental 3T3 cells, implicating some postreceptor event in the nonmitogenic phenotype. In addition, we find that phosphorylation of the epidermal <em>growth</em> <em>factor</em> receptor and of 80-kDa and <em>22</em>-kDa proteins, as well as the tyrosine phosphorylation of a 42-kDa protein, all proceed normally in the nonmitogenic variant, even though these phosphorylations must depend on the activation of different kinases. Thus, all these early phosphorylation reactions are intact in the 3T3-TNR9 cells. Although these phosphorylations may be necessary, they clearly are insufficient to trigger mitogenesis.

OBJECTIVE

To examine the effects of blocking p38 mitogen-activated protein kinase (MAPK) on post-injury conjunctival scarring in mice. Its effects on the behaviors of cultured subconjunctival fibroblasts were also investigated.

METHODS

An in vivo study was conducted using an adenoviral vector carrying a dominant-negative (DN)-p38MAPK gene. A circumferential incision was made in the equatorial conjunctiva by scissors in the right eye of generally anesthetized adult C57BL/6 mice. DN-p38MAPK-expressing adenoviral vector was topically applied. The left control eye received non-functioning adenoviral vector. At 2, 5, and 7 days (each, n=22) the eyes were processed for histological or immunohistochemical examination to evaluate the tissue scarring. The expressions of type-I collagen and growth factors were evaluated by real time-reverse transcriptase-polymerase chain reaction. The effects of p38MAPK inhibitor on the proliferation, migration, and fibrogenic gene/protein expression of cultured human fibroblasts were also studied.

RESULTS

The in vivo DN-p38MAPK gene introduction blocked the phospho-p38 expression with reduction of myofibroblast generation and suppression of mRNA expression of connective tissue growth factor (CTGF) and monocyte/macrophage chemoattractant protein-1 (MCP-1) in the mouse-injured conjunctiva. Blocking p38MAPK signal in the fibroblasts by a chemical inhibitor counteracted TGFbeta1's enhancement of expressions of type-I collagen, fibronectin, and CTGF. It also retarded cell migration, but cell proliferation was unchanged.

CONCLUSIONS

Inhibiting p38MAPK signal impairs the fibrogenic reaction induced by the subconjunctival fibroblasts in vivo and in vitro, suggesting its potential effectiveness in preventing excessive scarring following glaucoma filtering surgery.

De novo point mutations arise predominantly in the male germline and increase in frequency with age, but it has not previously been possible to locate specific, identifiable mutations directly within the seminiferous tubules of human testes. Using microdissection of tubules exhibiting altered expression of the spermatogonial markers MAGEA4, FGFR3, and phospho-AKT, whole genome amplification, and DNA sequencing, we establish an in situ strategy for discovery and analysis of pathogenic de novo mutations. In 14 testes from men aged 39-90 y, we identified 11 distinct gain-of-function mutations in five genes (<em>fibroblast</em> <em>growth</em> <em>factor</em> receptors FGFR2 and FGFR3, tyrosine phosphatase PTPN11, and RAS oncogene homologs HRAS and KRAS) from 16 of <em>22</em> tubules analyzed; all mutations have known associations with severe diseases, ranging from congenital or perinatal lethal disorders to somatically acquired cancers. These results support proposed selfish selection of spermatogonial mutations affecting <em>growth</em> <em>factor</em> receptor-RAS signaling, highlight its prevalence in older men, and enable direct visualization of the microscopic anatomy of elongated mutant clones.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> receptors (FGFRs) play an important role in development and tumorigenesis. Mutations in FGFR2 cause more than five craniosynostosis syndromes. The FGFR2 genomic structure is the largest of the FGFR family. We have refined and extended the genomic organization of the FGFR2 gene by sequencing more than 119 kb of PACs, cosmids, and PCR products and assembling a region of approximately 175 kb. Although the gene structure has been reported to include only 20 exons, we have verified the presence of at least <em>22</em> exons, some of which are alternatively spliced. The sizes of six exons differed from those reported previously. Comparison of our sequence and those in the NCBI database detected more than 300 potential single nucleotide polymorphisms (SNPs). However, sequencing regions containing 52 of these potential SNPs verified only 14 in PCR products generated from 16 CEPH alleles. In contrast, direct sequencing of the CEPH DNAs revealed 21 other polymorphisms. Only one SNP was found in the 2,926 bp of coding sequence. Twenty-seven SNPs, two insertion polymorphisms and five microsatellite polymorphisms are contained in approximately 16.6 kb of non-coding sequence. These data yield an average of one polymorphism for approximately 488 bp of non-coding sequence examined. This collection of SNP, insertion, and repeat polymorphisms will aid future association studies between the FGFR2 gene and human disease and will enhance mutation detection.

BACKGROUND

Nephrolithiasis affects about 10% of the population in industrialized countries, with calcium salts composing more than 80% of renal stones. A significant percentage of patients with calcium nephrolithiasis and normal parathyroid function show hypophosphatemia and reduced renal phosphate reabsorption (i.e. a renal phosphate leak).

OBJECTIVE

The objective of the study was to compare serum levels of fibroblast growth factor 23 (FGF23), a regulator of phosphate homeostasis, in 110 recurrent stone formers with or without renal phosphate leak, six patients affected by X-linked hypophosphatemic rickets, five patients affected by oncogenic osteomalacia, and 60 unrelated healthy controls.

METHODS

This was a prospective interventional study.

METHODS

Renal phosphate leak was identified based on the occurrence of idiopathic hypophosphatemia [serum phosphate concentration < 2.50 mg/dl (<0.80 mmol/liter)] and reduced renal threshold phosphate concentration [<2.2 mg/liter (<0.70 mmol/liter)].

RESULTS

In 22 stone formers with renal phosphate leak, serum FGF23 concentration was significantly higher as compared with 88 stone formers without renal phosphate leak and with controls [83.3 (65.6-101.1) vs. 32.1 (26.8-37.4) and 24.5 (19.8-29.1) reference units (RU)/ml, respectively]. Stone formers with renal phosphate leak showed lower FGF23, compared with patients with oncogenic osteomalacia and X-linked hypophosphatemic rickets [572.3 (235.9-908.7) RU/ml]. Among stone formers and controls, serum FGF23 concentration displayed a strong inverse association with serum phosphate (r = -0.784, P = 0.009) and the rate of tubular phosphate reabsorption (r = -0.791, P = 0.008).

CONCLUSIONS

In our study population, renal phosphate leak affected 20% of stone formers and was strongly associated with increased serum FGF23 concentration.

The platyspondylic lethal skeletal dysplasias (PLSDs) are a heterogeneous group of short-limb dwarfing conditions. The most common form of PLSD is thanatophoric dysplasia (TD), which has been divided into two types (TD1 and TD2). Three other types of PLSD, or TD variants (San Diego, Torrance, and Luton), have been distinguished from TD. The most notable difference between TD and the variants is the presence of large rough endoplasmic reticulum (rER) inclusion bodies within chondrocytes of the variants. We examined <em>22</em> cases of TD variants for the presence of missense mutations in the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) gene. All 17 cases of the San Diego type (PLSD-SD) were heterozygous for the same FGFR3 mutations found in TD1. No mutations were identified in the Torrance and Luton types. Large inclusion bodies were found in all 14 cases of PLSD-SD. Similar inclusion bodies were present in two of 72 TD1 cases, but not in 39 controls. The material retained within the rER stained only with antibody to the FGFR3 protein. The radiographic and morphologic differences between TD and PLSD-SD may be a consequence of other genetic <em>factors</em>, perhaps in the processing of mutant FGFR3 molecules within the rER. The presence of rER inclusion bodies cannot reliably discriminate between closely related skeletal dysplasias.

hPDGF is the major <em>growth</em> <em>factor</em> of human blood serum. In vivo, it is apparently synthesized by megakaryocytes and is transported in blood stored in the alpha granules of platelets. hPDGF is a heterodimer of two homologous polypeptide chains (PDGF-1(A) and PDGF-2(B] linked together by disulphide bonds. The PDGF-1(A) chain is encoded by a gene localized in chromosome 7 and the PDGF-2(B) chain is encoded by the c-sis proto-oncogene localized in chromosome <em>22</em>. The hPDGF heterodimer and its two isoforms, the PDGF-1(A) and PDGF-2(B) homodimers, are potent mitogens and chemoattractants for target cells such as diploid <em>fibroblasts</em>, osteoblasts, arterial smooth muscle cells and brain glial cells. The PDGF-1(A) homodimer binds only to its specific receptor alpha, and the hPDGF heterodimer and PDGF-2(B) homodimer bind to both receptors a and b. In addition to their mitogenic action, PDGF stimulates important cellular metabolic activities, including protein, lipid and prostaglandin synthesis. It appears to be an important <em>factor</em> in early development and in vivo appears to modulate tissue regeneration and remodelling during wound healing and osteogenesis. The inappropriate expression of PDGF genes and their mitogenic products has been linked to several proliferative disorders such as fibrosis, atherosclerosis and neoplasia.

The stroma of the human postmenopausal ovary is postulated to produce androgens, but evidence for and against this idea exits in the literature. The purpose of this study was to determine whether key steroidogenic enzymes involved in androgen synthesis are expressed in the postmenopausal ovarian stroma. Stromal cells were isolated from postmenopausal ovaries and expression for genes involved in steroidogenesis [steroidogenic acute regulatory protein (StAR), P450scc, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) P450c17, and P450c27] as well as for several <em>growth</em> <em>factor</em> binding proteins [gremlin, IGF binding protein-4, follistatin, and secreted frizzled-related protein (sFRP)-1 and -4], were compared with cultured human theca cells and dermal <em>fibroblasts</em>. Production of steroids (pregnenolone, progesterone, and hydroxysterol metabolites) and the metabolism of [3H] pregnenolone by ovarian stromal cells were also assessed. Isolated ovarian stromal cells from different subjects had a uniform morphology within and across cultures. Quantitative real time RT-PCR revealed that StAR, P450scc, and 3 beta-HSD transcripts were, respectively 30, 25, and 45 times more abundant in theca cells than in stromal cells. Mean levels of P450scc and 3 beta-HSD transcripts in stromal cells were similar to those found in dermal <em>fibroblasts</em>, whereas StAR transcripts in stromal cells were 285-fold more abundant than in <em>fibroblasts</em>. There was no significant expression of P450c17 in ovarian stromal cells or <em>fibroblasts</em> ( approximately 2000-fold less than in theca cells). Western analysis demonstrated the presence of the 30-kDa StAR mature protein in the cultured stromal cells, whereas P450c17 protein was not detectable. Ovarian stromal cells did not metabolize [3H] pregnenolone in the presence or absence of 8-Br-cAMP. Furthermore, pregnenolone and progesterone secretion by stromal cells was also undetectable, even in the presence of <em>22</em>-hydroxycholesterol. P450c27 protein was detected in ovarian stromal cells and its metabolic products (i.e. 27-hydroxycholesterol and cholestenoic acid) were found in the culture media, reflecting functional cholesterol 27-hydroxylase activity. Follistatin, gremlin, IGF binding protein-4, and sFRP-1 and -4 transcripts were detected in the stromal cells in relative amounts significantly higher than theca cells, but not significantly different from <em>fibroblasts</em>, except for sFRP-1, which was significantly higher in stromal cells. Our observations demonstrate that stromal cells of the postmenopausal ovary have a signature biochemical and molecular phenotype that can be distinguished from <em>fibroblasts</em>. These cells do not appear to have significant steroidogenic potential in vitro, but they do metabolize cholesterol into hydroxysterols. We conclude that the predominant stromal cells of the postmenopausal ovary are not a significant site of androgen biosynthesis.

We have conjugated a murine monoclonal antibody (B4G7) against the human epidermal <em>growth</em> <em>factor</em> (EGF) receptor to gelonin, a 60S ribosome inactivating protein, via N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and 2-iminothiolane. The B4G7-gelonin conjugate bound to the cell surface in proportion to the number of EGF receptors and competed with B4G7 antibody for binding to EGF receptors. The conjugate killed EGF receptor-hyperproducing squamous carcinoma cells (A431, NA, Ca9-<em>22</em>, TE5), and to some extent, human <em>fibroblasts</em> (HFO). It did not kill EGF receptor-deficient small-cell lung cancer cells (H69) and mouse <em>fibroblasts</em> (Swiss/3T3). Free B4G7, gelonin or a mixture of B4G7 and gelonin did not kill A431 cells. The number of EGF receptors was correlated to cytotoxicity at 10(-8) M of the conjugate, and the data were fitted to the regression equation: y = -35.83 log x +233.4 (correlation coefficient = -0.9995). These results suggest that the B4G7-gelonin conjugate may be a useful weapon for targeting therapy to squamous-cell carcinomas.

Thalidomide is reported to suppress levels of several cytokines, angiogenic and <em>growth</em> <em>factors</em> including TNF-alpha, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and interleukin-6 (IL-6). The resulting anti-angiogenic, immunomodulatory and <em>growth</em> suppressive effects form the rationale for investigating thalidomide in the treatment of malignancies. We have evaluated the use of high-dose oral thalidomide (600 mg daily) in patients with renal carcinoma. 25 patients (all men; median age, 51 years; range 34-76 years) with advanced measurable renal carcinoma, who had either progressed on or were not suitable for immunotherapy, received thalidomide in an escalating schedule up to a maximum dose of 600 mg daily. Treatment continued until disease progression or unacceptable toxicity were encountered. <em>22</em> patients were assessable for response. 2 patients showed partial responses (9%; 95% CI: 1-29), 7 (32%; 95% CI: 14-55) had stable disease for more than 6 months and a further 5 (23%; 95% CI: 8-45) had stable disease for between 3 and 6 months. We also measured levels of TNF-alpha, bFGF, VEGF, IL-6 and IL-12 before and during treatment. In patients with SD>> or = 3 months or an objective response, a statistically significant decrease in serum TNF-alpha levels was demonstrated (P = 0.05). The commonest toxicities were lethargy >> or = grade II, 10 patients), constipation >> or = grade II, 11 patients) and neuropathy >> or = grade II, 5 patients). Toxicities were of sufficient clinical significance for use of a lower and well tolerated dose of 400 mg in currently accruing studies.