Craniosynostosis is a common disorder with an unknown etiology. Recent genetic mapping studies have demonstrated a strong linkage between several familial craniosynostotic syndromes and mutations in <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGF-R1) and 2 (FGF-R2). The purpose of this experiment was to investigate by immunohistochemistry the protein production of these receptors as well as of their most prevalent ligand, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), before, during, and after sutural fusion in rat cranial sutures. The posterior frontal (normally fuses between postnatal days 12 and 22) and sagittal (remains patent) sutures of embryonic day 20 and neonatal days 6, 12, <em>17</em>, 22, and 62 (n = 3 per group) were harvested, fixed, and decalcified. Five-micrometer sections were stained with polyclonal antibodies against bFGF, FGF-R1, and FGF-R2, and patterns of immunohistochemical staining were assessed by independent reviewers. Our results indicate that increased bFGF production correlates temporally with suture fusion, with increased staining of the dura underneath the fusing suture prior to fusion followed by increased staining within osteoblasts and sutural cells during fusion. FGF-R1 and, to a lesser extent FGF-R2 immunostaining revealed a different pattern of localization with increased immunostaining within the patent sagittal suture at these time points. These results implicate bFGF in the regulation of sutural fusion and may imply autoregulatory mechanisms in <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor expression.

Sl/Sld mutant mice are profoundly deficient in tissue mast cells as a result of a defect in the microenvironment promoting the development of these cells. To facilitate the analysis of the Sl mutation, we attempted to establish an in vitro system in which the in vivo defect of Sl/Sld mice could be reproduced. 3T3 cell lines were established from <em>17</em>-day-old embryos of Sl/Sld and congenic +/+ genotypes and were cocultured with mast cells obtained in vitro from the bone marrow of +/+ mice. All eight 3T3 cell lines derived from +/+ of T-cell-derived <em>growth</em> <em>factors</em>. By contrast, none of eight 3T3 cell lines from Sl/Sld embryos supported mast cells under similar conditions. The defect in Sl/Sld 3T3 cells was further characterized as a failure to induce the G1-to-S transition in synchronized mast cells upon contact, suggesting that the Sl gene product is indispensable for this activity. When 3T3 cells of +/+ genotype, grown on pieces of cellulose acetate membrane, were transplanted into the peritoneal cavity of Sl/Sld mice, mast cells appeared locally in the transplanted 3T3 cell layers. These results suggested an essential role of <em>fibroblasts</em> in vivo as the tissue microenvironment promoting the development of mast cells and that they are defective in Sl/Sld mice. The present coculture system duplicated mast-cell deficiency of Sl/Sld mice in vitro and should prove useful for analysis of the Sl gene product.

BACKGROUND

In adults with heart failure, elevated levels of fibroblast growth factor 23 (FGF23) are associated with mortality. Data on FGF23 levels in pediatric heart failure are lacking.

METHODS

We conducted a cross-sectional study of 17 healthy children (mean age 13 years) and 20 pediatric patients with heart failure (mean age 12 years) who underwent echocardiography and for whom the following measurements were taken: plasma FGF23 and parathyroid hormone (PTH) and serum phosphate, creatinine and N-terminal prohormone brain natriuretic peptide (NT-proBNP). Symptom severity was assessed with the New York Heart Association and the Ross classification systems.

RESULTS

Of the 20 patients, 11 had dilated cardiomyopathy, four had congenital heart disease, three had hypertrophic cardiomyopathy, one had a failing heart transplant and one had pulmonary hypertension. Mean phosphate levels in these patients were within the reported reference range for healthy children. Median PTH levels were in the normal range in patients and controls. The median FGF23 level was higher in patients versus controls (110.9 vs. 66.4 RU/ml; P = 0.03) and higher in patients on diuretics versus other patients (222.4 vs. 82.1 RU/ml; P = 0.01). Levels of FGF23 and NT-proBNP were directly correlated (r = 0.47, P = 0.04), and patients with greater physical functional impairment had higher FGF23 levels (142.5 in those with moderate-severe limitation vs. 92.8 RU/ml in those with no limitation; P = 0.05). Among patients with dilated cardiomyopathy, higher FGF23 levels were associated with a greater left ventricular end-diastolic diameter (r = 0.63, P = 0.04).

CONCLUSIONS

FGF23 levels are elevated in children with heart failure and are associated with diuretic use, severity of heart failure and left ventricular dilation.

OBJECTIVE

Therapeutic angiogenesis with angiogenic growth factors has described as one of the promising methods for collateral formation in the treatment of ischemic heart diseases. The purpose of this study is to assess the value of intramyocardial injection of slow-released basic fibroblast growth factor microspheres on angiogenesis and cardiac function in the early period of acute infarcted myocardium with dobutamine cardiovascular magnetic resonance tagging.

METHODS

Acute myocardial infarction was made by ligation of the left anterior descending coronary artery distal to its first diagonal branch. Immediately after coronary artery occlusion, 1 ml of saline containing 100 microg of basic fibroblast growth factor microspheres was injected into peri-infarct myocardial area in the basic fibroblast growth factor group, whereas only gelatin hydrogel microspheres with 1 ml of saline was given in control dogs. Cardiac function was evaluated by cine magnetic resonance imaging. Dobutamine cardiovascular magnetic resonance was performed at rest and during low doses of dobutamine to assess regional wall motion. Immunohistochemical study with von Willebrand factor was performed to observe angiogenesis.

RESULTS

Left ventricular ejection fraction improved markedly 10 and 17 days after treatment in the basic fibroblast growth factor group. The basic fibroblast growth factor group had more viable myocardium. Microvessel density was higher in the basic fibroblast growth factor group than in the control group except the first day after treatment.

CONCLUSIONS

Intramyocardial administration of basic fibroblast growth factor microspheres can promote the growth of microvessels and improve left ventricular function and myocardial viability in the early period of acute myocardial infarction.

Tumor necrosis <em>factor</em> (TNF)-like weak inducer of apoptosis (TWEAK) is an inflammatory cytokine that modulates several biological responses by inducing chemokines and proinflammatory cytokines. We hypothesized that TWEAK could promote secretion of IL-<em>17</em>, an amplifier of inflammatory arthritis. To test this, we investigated the capacity of TWEAK to induce IL-<em>17</em> production in T cells via the <em>fibroblast</em> <em>growth</em> <em>factor</em>-inducible gene 14 (Fn14, also known as TWEAK receptor) signal pathway in rheumatoid arthritis (RA). Fn14 and IL-<em>17</em> were highly expressed in arthritic tissues of collagen-induced arthritis (CIA) mice. TWEAK induced production of IL-<em>17</em> alone and synergistically with lipopolysaccharide. In naïve murine T cells, TWEAK promoted Th<em>17</em> differentiation. The expression of Fn14 was predominant in Th<em>17</em> cells. TWEAK and IL-<em>17</em> concentrations were significantly higher in synovial fluid and serum in RA patients than OA patients. In addition, we identified CD4(+)IL-<em>17</em>(+)Fn14(+) cells in synovium from RA patients. TWEAK promoted IL-<em>17</em> production synergistically with IL-23 or IL-21 and blockade of Fn14 with Fn14-Fc suppressed Th<em>17</em> differentiation. Conversely, this treatment enhanced Treg differentiation. These results suggest that TWEAK induces IL-<em>17</em> production and may be a therapeutic target in the treatment of RA.

In the present study we have investigated the role of human breast-cancer-derived <em>fibroblasts</em> in the proliferation of primary cultures of epithelial cells derived from the same tumor. For this purpose, a co-culture system, using Transwell tissue-culture inserts with microporous membranes was employed. <em>Fibroblasts</em> and epithelial cells were enriched according to differences in their density on Percoll density gradients. The co-culture system was first established using MCF-7 breast cancer cells and a human <em>fibroblast</em> line (HF cells). Insulin, <em>17</em> beta-estradiol, EGF and HF cells all significantly stimulated the <em>growth</em> of MCF-7 breast cancer cells. The stimulatory effects of insulin, E2 and EGF were additive to the stimulatory effect of HF cells. These data suggest that (unique) <em>factor</em>(s), other than the above-mentioned <em>growth</em>-promoting compounds, are responsible for the <em>growth</em>-promoting effects of <em>fibroblasts</em>. In half of the human breast cancers investigated, tumor-derived <em>fibroblasts</em> stimulated tumor-derived epithelial cell proliferation. EGF significantly stimulated epithelial cell proliferation in 4 out of 6 cultures. The stimulatory effects of <em>fibroblasts</em> and EGF were additive or synergistic, and were observed in the additional presence of FCS, again suggesting production of unique <em>factor</em>(s) by the <em>fibroblasts</em>. In one culture the <em>fibroblasts</em> significantly inhibited epithelial tumor-cell proliferation. Conversely, the epithelial cells significantly stimulated proliferation of <em>fibroblasts</em> in 3 out of 3 cultures. The somatostatin analogue octreotide significantly inhibited epithelial cell proliferation by 46% in one tumor-cell culture in the absence, but not in the presence, of <em>fibroblasts</em>. In one culture, octreotide significantly inhibited the proliferation of <em>fibroblasts</em> co-cultured with epithelial cells.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-9 (FGF-9) is a steroid-regulated mitogen and survival <em>factor</em> for nerve and mesenchymal cells. In the current study, we determined the expression pattern and functional roles of FGF-9 in the ectopic endometriotic lesions. We found that FGF-9 and its receptors were effectively expressed by ectopic endometriotic tissues. The expression of FGF-9 was greater in the early stage of endometriosis, compared with the severe stage, which is consistent with concentration of <em>17</em> beta-estradiol in the peritoneal fluid of women with endometriosis. In addition, expression of FGF-9 in ectopic endometriotic stromal cell was inhibited by treatment with ICI 182,870 indicating it is likely regulated by estrogen in an autocrine manner. Administration of <em>17</em> beta-estradiol induced FGF-9, FGF receptor 2IIIc, and FGF receptor 3IIIc expression in endometriotic stromal cells. Concordant with this result, treatment of endometriotic stromal cells with 4-hydroxyandrostenedione (an aromatase inhibitor) or ICI 182,870 inhibited their proliferation, and that was reversed by coadministration with <em>17</em> beta-estradiol or FGF-9. In conclusion, expression of FGF-9 in endometriotic stromal cells is associated with aberrant production of estrogen. The capability of proliferation possessed by endometriotic stromal cell during menstruation when ovarian <em>17</em> beta-estradiol is in the nadir may be mediated, at least in part, by autocrined estrogen-stimulated expression of FGF-9 and its receptors.

<em>Growth</em> <em>factors</em> may play a significant role in regulating the orderly progression of organ <em>growth</em> and differentiation during fetal development. We hypothesized that epidermal <em>growth</em> <em>factor</em> (EGF) would help regulate the development of surfactant synthesis in the fetal lung by influencing <em>fibroblast</em>-epithelial cell interactions. The effect of EGF (10 ng per ml) on the ability of the fetal lung <em>fibroblast</em> to produce <em>fibroblast</em> pneumonocyte <em>factor</em> (FPF) was studied in sex-specific <em>fibroblasts</em> cultured from day 16, day <em>17</em> or day 18 fetal mouse lungs. FPF which is normally not produced by day 16 <em>fibroblasts</em>, is found only in female <em>fibroblasts</em> on day <em>17</em>, and then in both males and females on day 18. EGF advanced this pattern such that female <em>fibroblasts</em> produced activity on day 16 and <em>fibroblasts</em> from both sexes produced FPF activity on day <em>17</em> and day 18. <em>Fibroblasts</em> from an androgen receptor-deficient mouse model confirmed that the effect of EGF was sex-specific and related to the state of development of the fetal lung. We conclude that EGF advances the fetal lung <em>fibroblast</em> through specific stages of development. It appears, therefore, to help control the timing of the clock regulating fetal lung maturation.

Elevated eicosanoid biosynthesis characterizes certain forms of human and experimental glomerular proliferative disease. Thromboxane A2 (TxA2) and other prostaglandins (PG) act through specific receptors and mechanisms of intracellular signal transduction in human mesangial cells. We studied the actions of U-46619, a TxA2 mimetic which stimulates mesangial phospholipase C, and of the PGI2 analogue, Iloprost, a potent activator of adenylate cyclase, on proliferation of cultured human mesangial cells. When applied alone to quiescent cells, U-46619 had only weak mitogenic activity, as assessed by [3H]thymidine [( 3H]-TdR) incorporation and cell counts. On the other hand, addition of U-46619 10 minutes prior to stimulation of the cells with 1 to <em>17</em>% fetal bovine serum (FBS) for 24 hours, potently and dose-dependently inhibited FBS-stimulated [3H]-TdR incorporation. Similarly, U-46619 inhibited the effects of 10 ng/ml platelet-derived <em>growth</em> <em>factor</em> (PDGF), epidermal <em>growth</em> <em>factor</em> or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> on [3H]-TdR incorporation, by 55, 79 and 88%, respectively. The effects of U-46619 were not mimicked by another stimulus of phospholipase C, angiotensin II. Iloprost also inhibited FBS-activated proliferation. Neither eicosanoid inhibited the rise of cytosolic Ca2+ induced by FBS or PDGF. The actions of TxA2 and Iloprost in cultured cells point to multiple functional interactions between eicosanoids and <em>growth</em> <em>factors</em> in the control of mesangial cell proliferation.

The role of <em>growth</em> <em>factors</em> in a variety of bone and soft tissue healing processes has been studied extensively in numerous recent models, yet little is known about the specific <em>growth</em> <em>factors</em> that may be playing a role in flexor tendon healing. We used a number of established protein purification techniques and bioassays to isolate and partially characterize a heparin-binding <em>growth</em> <em>factor</em> from unoperated canine tendons. Our data provide evidence that basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, a potent angiogenic <em>growth</em> <em>factor</em>, is present in normal canine intrasynovial flexor tendons. We then studied repaired canine flexor tendons to further elucidate the role of <em>growth</em> <em>factors</em> in the tendon healing process. Heparin-sepharose elution profiles from three repair intervals (3, 10, and <em>17</em> days) were graphed and compared to known profiles of isolated <em>growth</em> <em>factors</em>. The three repair intervals demonstrated two elution profile peaks, consistent with varying amounts of platelet-derived <em>growth</em> <em>factor</em> and epidermal <em>growth</em> <em>factor</em>. Although additional experimentation is required to identify definitively the various protein isolates, these data provide compelling evidence that a variety of <em>growth</em> <em>factors</em> are present in uninjured and healing digital flexor tendons.

The E5 oncoprotein of bovine papillomavirus type 1 is a 44-amino-acid, hydrophobic polypeptide which localizes predominantly in Golgi membranes and appears to transform cells through the activation of tyrosine kinase <em>growth</em> <em>factor</em> receptors. In <em>fibroblasts</em>, E5 interacts with both the 16-kilodalton vacuolar ATPase subunit and the platelet-derived <em>growth</em> <em>factor</em> receptor (PDGF-R) via its hydrophobic transmembrane domain and induces autophosphorylation of the receptor. To further analyze the correlation between E5 biological activity and its ability to bind these cellular proteins, a series of nine E5 transmembrane mutants was evaluated. In 32D mouse hematopoietic cells, there was an incomplete correlation between the abilities of the E5 mutant proteins to associate the PDGF-R and to transform cells. However, all transforming E5 mutant proteins induced PDGF-R tyrosine phosphorylation. In NIH 3T3 and C127 mouse <em>fibroblasts</em>, both transforming and nontransforming E5 mutant proteins were defective for PDGF-R binding. In addition, while most of the transforming E5 proteins induced PDGF-R phosphorylation, one hypertransforming mutant (serine <em>17</em>) neither bound nor induced receptor autophosphorylation. These findings support the hypothesis that the transformation of <em>fibroblasts</em> by E5 transmembrane mutants can involve alternative cellular targets or potentially independent activities of the E5 protein. In addition, these results underscore the critical role of the transmembrane domain in mediating E5 biological activities.

We investigated the binding properties of the type I insulin-like <em>growth</em> <em>factor</em> (IGF) receptor expressed in NIH-3T3 <em>fibroblasts</em> transfected with a human type I receptor cDNA. Cell surface receptors bound IGF-I with KD = 1 nM as predicted. Although recent studies have suggested that IGF-I and IGF-II bind to type I receptors with near-equal affinity, the receptors in this system bound IGF-II with much lower affinity (KD = 15-20 nM). When type I receptors from the transfected cells were solubilized and immunopurified, however, both 125I-IGF-I and 125I-IGF-II bound to the purified receptors with extremely high and relatively similar affinities (KD = 8 and <em>17</em> pM respectively). Thus the immunopurified receptors had higher affinity but lower specificity for the two ligands. The monoclonal antibody alpha IR-3 effectively inhibited IGF-I binding to cell surface receptors (75 +/- 10%), but did not inhibit IGF-II binding. In the purified receptor assay, alpha IR-3 also inhibited IGF-I binding more effectively than IGF-II binding (38 +/- 7% versus 10 +/- 4%). We conclude that the products of this cDNA can account for the binding patterns that we previously observed in receptors immunopurified from human placenta. The differential effect of alpha IR-3 on IGF-I versus IGF-II raises the possibility that these homologous <em>growth</em> <em>factors</em> bind to immunologically distinct epitopes on the type I receptor.

Craniosynostosis is one of the most common craniofacial disorders encountered in clinical genetics practice, with an overall incidence of 1 in 2,500. Between 30% and 70% of syndromic craniosynostoses are caused by mutations in hotspots in the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) genes or in the TWIST1 gene with the difference in detection rates likely to be related to different study populations within craniofacial centers. Here we present results from molecular testing of an Australia and New Zealand cohort of 630 individuals with a diagnosis of craniosynostosis. Data were obtained by Sanger sequencing of FGFR1, FGFR2, and FGFR3 hotspot exons and the TWIST1 gene, as well as copy number detection of TWIST1. Of the 630 probands, there were 231 who had one of 80 distinct mutations (36%). Among the 80 mutations, <em>17</em> novel sequence variants were detected in three of the four genes screened. In addition to the proband cohort there were 96 individuals who underwent predictive or prenatal testing as part of family studies. Dysmorphic features consistent with the known FGFR1-3/TWIST1-associated syndromes were predictive for mutation detection. We also show a statistically significant association between splice site mutations in FGFR2 and a clinical diagnosis of Pfeiffer syndrome, more severe clinical phenotypes associated with FGFR2 exon 10 versus exon 8 mutations, and more frequent surgical procedures in the presence of a pathogenic mutation. Targeting gene hot spot areas for mutation analysis is a useful strategy to maximize the success of molecular diagnosis for individuals with craniosynostosis.

The assignments of the 1H, 15N, 13CO and 13C resonances of recombinant human basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2), a protein comprising of 154 residues and with a molecular mass of <em>17</em>.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N-/13C-labeled FGF-2 with an isotope incorporation>> 95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of C alpha, C beta and H alpha to the backbone amide 1H and 15N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of C alpha, C beta and H alpha to the backbone amide 1H and 15N in the CBCANH and HNHA experiments. In addition, C alpha and C beta chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and C alpha correlations from the HNCA experiment. Aliphatic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic 1H and 13C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, H alpha and H beta protons as well as 3JHNH alpha coupling constants, amide exchange and 13C alpha and 13C beta secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel beta-sheets (residues 30-34, 39-44, 48-53, 62-67, 71-76, 81-85, 91-94, 103-108, 113-118, 123-125 and 148-152) and a helix-like structure (residues 131-136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131-136 were defined as beta-strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128-138) instead of the beta-strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9-28. This is consistent with the X-ray structures of FGF-2, where the first <em>17</em>-19 residues were ill defined.

The chromosomal breakage syndromes--ataxia-telangiectasia, Fanconi's anemia, and Bloom's syndrome--are associated with <em>growth</em> failure, neurologic abnormalities, immunodeficiency, and an increased incidence of malignancy. The relationship between these features is unknown. We recently evaluated a 21-year-old female with more severe chromosomal breakage, immunodeficiency, and <em>growth</em> failure than in any of the mentioned disorders. As of November 1985, the patient remains clinically free of malignancy. At age 18, the patient's weight was 22.6 kg (50th percentile for seven years), height was 129 cm (50th percentile for eight years), and head circumference was 42 cm (50th percentile for six months). Laboratory studies demonstrated a marked decrease in both B and T cell number and function. The peripheral blood contained 400 to 900 lymphocytes/microL with 32% T11 cells, <em>17</em>% T4 cells, and 21% T8 cells. The proliferative responses to phytohemagglutinin (PHA), pokeweed mitogen, and concanavalin A were less than 10% of control. There were 1% surface IgM positive cells, and serum IgG was 185 mg/dL, IgM 7 mg/dL, IgA 5 mg/dL. In lymphocyte cultures stimulated with the T cell mitogens PHA, phorbol ester, and interleukin 2, 55% of the banded metaphases demonstrated breaks or rearrangements. The majority of the breaks involved four fragile sites on chromosomes 7 and 14, 7p13, 7q35, 14q11, and 14q32. These are the sites of the genes for the T cell-antigen receptor and the immunoglobulin heavy chain and are sites of gene rearrangement in lymphocyte differentiation. Epstein-Barr virus stimulated B cells and <em>fibroblast</em> cultures also demonstrated a high incidence of breaks, but the sites were less selective. These findings suggest that the sites of chromosomal fragility in the chromosomal breakage syndromes may be informative and that <em>factors</em> other than the severity of the immunodeficiency or the high incidence of chromosomal damage may contribute to the occurrence of malignancy in the chromosomal breakage syndromes.

Human embryonic stem cells (hESCs) can be maintained undifferentiated (pluripotent) or differentiated to basically all functional cell types, depending on the culture conditions used. Culture of hESCs in the presence of medium conditioned by mouse embryonic <em>fibroblasts</em> (MEFs) can be used to keep hESCs undifferentiated. This observation suggests that MEFs produce <em>factors</em> required for the pluripotency of hESCs. The data presented here show that <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2) treatment of MEFs is crucial for the production of these <em>factors</em>. To identify the potential <em>factors</em> that are expressed in the presence of FGF2 in MEFs, a global expression profile analysis using microarrays was performed. This analysis indicated that <em>17</em> secreted <em>factors</em> are downregulated in the absence of FGF2. These <em>factors</em> include several ligands for known signaling receptors, extracellular proteases and components of the extracellular matrix, that may all be involved in signaling events. Surprisingly, we found that selective blocking of extracellular signal-regulated kinase (ERK) signaling by the MAPK/ERK kinase (MEK) inhibitor U0126 affected the expression of only some of the FGF2-regulated genes, suggesting FGF2-induced pathways that are independent of ERK signaling. It has been shown recently that activation of Activin/Nodal signaling and inhibition of bone morphogenetic protein signaling are required for the maintenance of pluripotency. Accordingly, among the <em>17</em> FGF2-regulated genes we found inhibin beta B that can lead to the assembly of Activin B and gremlin 1 that codes for an antagonist of bone morphogenetic proteins. This study identifies potentially important <em>factors</em> involved in the maintenance of pluripotency in hESCs and may allow the development of defined culture conditions without contaminating material from animal cells.

We previously reported that overexpression of full-length periostin, Pn-1, resulted in ventricular dilation with enhanced interstitial collagen deposition in a rat model. However, other reports have documented that the short-form splice variants Pn-2 (lacking exon <em>17</em>) and Pn-4 (lacking exons <em>17</em> and 21) promoted cardiac repair by angiogenesis and prevented cardiac rupture after acute myocardial infarction. The apparently differing findings from those reports prompted us to use a neutralizing antibody to selectively inhibit Pn-1 by blockade of exon <em>17</em> in a rat acute myocardial infarction model. Administration of Pn neutralizing antibody resulted in a significant decrease in the infarcted and fibrotic areas of the myocardium, which prevented ventricular wall thinning and dilatation. The inhibition of fibrosis by Pn neutralizing antibody was associated with a significant decrease in gene expression of fibrotic markers, including collagen I, collagen III, and transforming <em>growth</em> <em>factor</em>-β1. Importantly, the number of α-smooth muscle actin-positive myo<em>fibroblast</em>s was significantly reduced in the hearts of animals treated with Pn neutralizing antibody, whereas cardiomyocyte proliferation and angiogenesis were comparable in the IgG and neutralizing antibody groups. Moreover, the level of Pn-1 expression was significantly correlated with the severity of myocardial infarction. In addition, Pn-1, but not Pn-2 or Pn-4, inhibited <em>fibroblast</em> and myocyte attachment, which might account for the cell slippage observed during cardiac remodeling. Collectively, these results indicate that therapeutics that specifically inhibit Pn exon-<em>17</em>, via a neutralizing antibody or drug, without suppressing other periostin variants might offer a new class of medication for the treatment of acute myocardial infarction patients.

Based on the K8/JTS-1-mediated transfection technique, we developed an in vivo protocol for an efficient transfer of plasmid DNA to ocular cells. As determined with condensed plasmids containing reporter genes for either beta-galactosidase (pcDNA-lacZ) or enhanced green fluorescent protein (pREP-EGFP), the immortalized human retinal epithelial cells RPE D407 and human embryonic kidney 293 cells can be transfected with typical efficiencies of 11 and 19%, respectively. Unlike 293 cells, RPE D407 cells had a reduced viability on transfection with both plasmids. In vivo, subretinal injections of DNA-K8/JTS-1 complexes revealed reporter gene expression in choroidal and RPE cells of normal pink-eyed Royal College of Surgeons (RCS) rats. The validity of this transfection technique in terms of retinal cell survival in RCS rats was then examined by using pREP-hFGF2 plasmid, which encodes the human basic <em>fibroblast</em> <em>growth</em> <em>factor</em> isoforms (hFGF2). Subretinal injection of pREP-hFGF2-K8/JTS-1 complexes into 3-week-old dystrophic RCS rat eyes reveals a delayed photoreceptor cell degeneration 60 days postinjection. In this case, the average analyzed field points with delayed cell dystrophy represent 14 to <em>17</em>% of the retinal surface as compared with 2.6 and 4% in pREP5beta and vehicle-injected eyes, respectively. Peptide-mediated in oculo transfection thus appears to be a promising technique for the treatment of retinal cell and photoreceptor degenerations.

A number of angiogenic <em>growth</em> <em>factors</em> have been shown to accelerate wound healing. Previous work has demonstrated that topical application of epidermal <em>growth</em> <em>factor</em> is effective in healing chronic tympanic membrane perforations in an animal model. Theoretically, <em>fibroblast</em> <em>growth</em> <em>factor</em> may result in a superior healed membrane through preferential stimulation of the <em>fibroblasts</em> within the middle layer of the tympanic membrane. To test this hypothesis, the effects of exogenously applied <em>fibroblast</em> <em>growth</em> <em>factor</em> on the chronically perforated tympanic membrane were evaluated. A buffered solution of <em>fibroblast</em> <em>growth</em> <em>factor</em> (25 microliters of <em>fibroblast</em> <em>growth</em> <em>factor</em>, 0.2 mg/ml) was administered to a Gelfoam pledget placed over chronic tympanic membrane perforations in chinchillas. Control ears were treated with Gelfoam and the buffer solution only. Complete closure of the tympanic membrane perforation was observed in 81% (13 of 16) of the <em>fibroblast</em> <em>growth</em> <em>factor</em>-treated ears, but in only 41% (7 of <em>17</em>) of the controls (p = 0.05). Heading took place gradually, requiring an average of 4 weeks for the <em>fibroblast</em> <em>growth</em> <em>factor</em>-treated and 6.5 weeks for the control ears that healed. The relatively high healing rate for the control group does not imply that the pretreatment perforations were not chronic, rather there appears to be some efficacy to the control protocol of repeated applications of Gelfoam and buffer. A histologic analysis of the <em>fibroblast</em> <em>growth</em> <em>factor</em>-healed eardrums immediately after closure demonstrated hypertrophy of the squamous and fibrous layers of the tympanic membrane. Over time, the eardrum thinned to reach proportions similar to those of the normal tympanic membrane, including the presence of a substantial middle fibrous layer. A screening ototoxicity study revealed no structural damage to the organ of Corti after <em>growth</em> <em>factor</em> treatment. To assess the potential for systemic toxicity, blood and peripheral tissues were analyzed for radioactivity at time points during a 48-hour period after application of 25 microliters of 125I-<em>fibroblast</em> <em>growth</em> <em>factor</em> to the perforated tympanic membrane. More than 78% of the radioactivity remained at the application site. Given the tiny original dosage, the small fraction absorbed systemically is minuscule and highly unlikely to induce adverse effects in light of published toxicity data. On the basis of these promising safety and efficacy data in the chinchilla model, clinical trials of <em>fibroblast</em> <em>growth</em> <em>factor</em> in repair of chronic tympanic membrane perforations in human beings are being initiated.

The vertebrate brain is regionalized during development into forebrain, midbrain and hindbrain. <em>Fibroblast</em> <em>growth</em> <em>factor</em> 8 (FGF8) is expressed in the midbrain/hindbrain boundary (MHB) and functions as an organizer molecule. Previous studies demonstrated that the brain of basal chordates or ascidians is also regionalized at least into fore/midbrain and hindbrain. To better understand the ascidian brain regionalization, the expression of the Ciona Fgf8/<em>17</em>/18 gene was compared with the expression of Otx, En and Pax2/5/8 genes. The expression pattern of these genes resembled that of the genes in the vertebrate forebrain, midbrain, MHB and hindbrain, each of those domains being characterized by sole or combined expression of Otx, Pax2/5/8, En and Fgf8/<em>17</em>/18. In addition, the putative forebrain and midbrain expressed Ci-FgfL and Ci-Fgf9/16/20, respectively. Therefore, the regionalization of the ascidian larval central nervous system was also marked by the expression of Fgf genes.

BACKGROUND

Until recently thought to be of little significance unless occurring during pregnancy, Toxoplasma gondii infection of human hosts is now known to play a larger role in mental health and is a growing concern in the health care community. We sought to elucidate a possible mechanism by which Toxoplasma infection may cause some of the behavioural pathology now associated with infection. We hypothesized that exosomes may be playing a role.

METHODS

We utilized electron microscopy to detect the presence and size of extracellular vesicles in the supernatants of Toxoplasma-infected human foreskin fibroblasts (HFF). We then utilized microarray analysis to discern mRNA and miRNA content of the vesicles isolated from supernatants of Toxoplasma-infected (Toxo) and serum-starved (SS) HFF.

RESULTS

We recovered extracellular vesicles with a size consistent with exosomes that we called exosome-like vesicles (ELVs) from the supernatants of SS and Toxo cultures. The mRNA and miRNA content of these ELVs was highly regulated creating specific and unique expression profiles comparing Toxo ELVs, SS ELVs and RNA isolated from whole cell homogenates. Interestingly, among the most enriched mRNA isolated from ELVs of Toxo cells are 4 specific mRNA species that have been described in the literature as having neurologic activity: Rab-13, eukaryotic translation elongation factor 1 alpha 1, thymosin beta 4 and LLP homolog. In addition, miRNA species uniquely expressed in Toxo ELVs include miR-23b, a well-known regulator of IL-17.

CONCLUSIONS

While the production of ELVs containing mRNAs that modify behaviour are consistent with reported Toxoplasma pathology, the mechanism of enrichment and ultimate in vivo effect of these mRNA and miRNA containing ELVs remains to be investigated.

Vascular endothelial <em>growth</em> <em>factor</em> (VEGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and soluble interleukin-2 receptor (sIL-2R) are important cytokines. They are secreted by normal pituitary glands and those with all types of adenomas and may be involved in pituitary tissue <em>growth</em>. The peripheral blood concentrations of VEGF, bFGF and sIL-2R in nineteen patients (<em>17</em>-70 years) with pituitary tumours and ten healthy subjects (23-34 years) were studied. Hypersecretion of prolactin (five cases), human <em>growth</em> hormone (four cases), and thyroid stimulating hormone (one case) was recorded in some patients, and the remaining subjects were diagnosed as having nonfunctional pituitary tumours. Increased peripheral blood plasma levels of VEGF (310.82 +/- 59.<em>17</em> pg/ml) compared with controls (40.32 +/- 11.80 pg/ml; p < 0.01), as well as bFGF (87.27 +/- 7.58 pg/ml) versus controls (11.14 +/- 2.43 pg/ml; p < 0.001) were recorded. The levels of sIL-2R did not differ between the pituitary tumour patients (4,490.58 +/- 581.50 pg/ml) and control subjects (3,6<em>17</em>.01 +/- 1,397.18 pg/ml; p>> 0.05). The concentrations of VEGF and bFGF in the peripheral blood are useful additional markers of the presence of tumours.

Sternal chondrocytes of <em>17</em>-day-old chick embryos in serum-free agarose culture secrete transforming <em>growth</em> <em>factor</em>-beta. Media conditioned by such cells prevent serum-induced chondrocyte hypertrophy and cause a phenotypic modulation in serum-free culture which is similar to that observed for chondrocytes in monolayer culture. The modulated cells lose the round shape of differentiated chondrocytes and increasingly with time resemble tendon <em>fibroblasts</em> embedded into agarose. In addition, they produce less matrix macromolecules which include collagen I rather than cartilage collagens II, IX, X, and XI. All of these effects are abolished upon addition to the conditioned media of a monoclonal antibody against recombinant human transforming <em>growth</em> <em>factor</em>-beta 2. The same <em>factor</em> caused effects closely similar to those elicited by conditioned media. Therefore, the phenotypic modulation in adhesion-dependent cultures of chondrocytes in vitro does not directly result from cell-matrix interactions but can be produced also in suspension culture under the direction of appropriate diffusible stimuli that include transforming <em>growth</em> <em>factor</em>-beta. In addition, the results support the concept of transforming <em>growth</em> <em>factor</em>-beta as a multifunctional cytokine acting differently on cells of the same developmental origin depending on their stage of differentiation.

OBJECTIVE

Allograft inflammatory factor 1 (AIF-1) was first identified in rat cardiac allografts undergoing chronic rejection. The vasculopathy of chronic allograft rejection is strikingly similar to that seen in patients with systemic sclerosis (SSc). We previously demonstrated AIF-1 expression in inflammatory cells infiltrating skin and lungs from SSc patients, but its role in SSc pathogenesis is unknown. The present study was undertaken to investigate the effects of AIF-1 on T cell migration and production of cytokines capable of modulating normal dermal fibroblast functions.

METHODS

Stably transfected Jurkat T cells expressing 2 AIF-1 splicing variants were prepared, and their migration toward fibroblast monolayers assayed in Transwell cultures. Cytokine production was assessed by real-time polymerase chain reaction (PCR) and multiplex enzyme-linked immunosorbent assay. Fibroblast gene expression was quantified by real-time PCR, and collagen production by Western blot analysis of culture media.

RESULTS

AIF-1 significantly increased Jurkat T cell migration toward fibroblast monolayers. Expression of AIF-1 isoform 2 in Jurkat T cells up-regulated their production of interleukin-4 (IL-4) and IL-17. Conditioned media from AIF-1-expressing clones stimulated synthesis of types I and III collagen and expression of IL-6, transforming growth factor beta, endothelin receptor, and alpha-smooth muscle actin by normal dermal fibroblasts.

CONCLUSIONS

These results suggest that AIF-1 may participate in the early pathogenesis of SSc by promoting tissue T cell infiltration and production of cytokines capable of inducing the expression of a fibrotic phenotype in normal fibroblasts.