Isolation from mouse fibroblasts of a cDNA encoding a new form of the fibroblast growth factor receptor (flg).

Structural definition of the receptors for neurotropic and angiogenic modulators such as fibroblast growth factors and related polypeptides will yield insight into the mechanisms that control early development, embryogenesis, organogenesis, wound repair and neovessel formation. We isolated 3 murine cDNAs encoding different binding domains of these receptors (flg). Comparison of these ectoplasmic portions showed that two of the forms corresponded to previously described murine molecules whereas the third one had a different ectoplasmic portion generated by specific changes in two regions. Interestingly, expression of this third form seems to be restricted in its tissue distribution. Such modifications could influence the ligand specificity of the different receptors and/or their binding affinity.

Altered expression of the transcription factor Mef2c during retinal degeneration in Rpe65-/- mice.

Purpose. To investigate the role of the myocyte enhancer factor 2 (Mef2) transcription factor family in retinal diseases, Mef2c expression was assessed during retinal degeneration in the Rpe65(-/-) mouse model of Leber's congenital amaurosis (LCA). Mef2c-dependent expression of photoreceptor-specific genes was further addressed. Methods. Expression of Mef2 members was analyzed by oligonucleotide microarray, quantitative PCR (qPCR) and in situ hybridization. Mef2c-dependent transcriptional activity was assayed by luciferase assay in HEK293T cells. Results. Mef2c was the only Mef2 member markedly downregulated during retinal degeneration in Rpe65(-/-) mice. Mef2c mRNA level was decreased by more than 2 fold at 2 and 4 months and by 3.5 fold at 6 months in retinas of Rpe65(-/-) mice. Downregulation of Mef2c at the protein level was confirmed in Rpe65(-/-) retinas. The decrease in Mef2c mRNA levels in the developing Rpe65(-/-) retinas, from post-natal day (P)13 onward, was concomitant with the decreased expression of the rod-specific transcription factors Nrl and Nr2e3. Nrl was further shown to drive Mef2c transcriptional activity, supporting a physiological role for Mef2c in the retina. In addition, Mef2c appeared to act as a transcriptional repressor of its own expression, as well as those of the retina-specific retinal G-protein coupled receptor (Rgr), rhodopsin and M-opsin genes. Conclusions. These findings highlight the early altered regulation of the rod-specific transcriptional network in Rpe65-related disease. They further indicate that Mef2c may act as a novel transcription factor involved in the development and the maintenance of photoreceptor cells.

Placental vascular obstructive lesions: risk factor for developing necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a severe neonatal disease affecting particularly preterm infants. Its exact pathogenesis still remains unknown. In this study, we have compared the prevalence of vascular obstructive lesions in placentae of premature newborns which developed NEC and of a control group. We further compared separately the findings of placentae of infants of less than 30 weeks of gestation, the age group in which NEC occurs most frequently. We found signs of fetal vascular obstructive lesions in 65% of the placentae of preterm patients developing NEC, compared to only 17% of the placentae of preterm patients in the control group. In the age groups below 30 weeks of gestation, 58.5% of placentae of later NEC patients presented such lesions compared to 24.5% in the control group. The significant difference between NEC and control group suggests a strong association between fetal vascular obstructive lesions and NEC. Therefore, we propose that fetal vascular obstructive lesions might be considered as a risk factor for the development of NEC in premature infants.

Enhanced visuospatial memory following intracerebroventricular administration of nerve growth factor

The present work assessed the effects of intracerebroventricular injections of rh recombined human nerve growth factor (rh NGF) (5 micrograms/2.5 microl) at postnatal days 12 and 13 upon the development of spatial learning capacities. The treated rats were trained at the age of 22 days to escape onto an invisible platform at a fixed position in space in a Morris navigation task. For half of the subjects, the training position was also cued, a procedure aimed at facilitating escape and at reducing attention to the distant spatial cues. Later, at the age of 6 months, all the rats were trained in a radial-arm maze task. Treatment effects were found in both immature and adult rats. The injection of NGF improved the performance in the Morris navigation task in both training conditions. There was a significant reduction in the escape latency and an increased bias toward the training platform quadrant during probe trials. The most consistent effect was the precocious development of an adult-like spatial memory. In the radial-arm maze, the NGF-treated rats made significantly fewer reentries than vehicle rats and this effect was particularly marked in the treated female rats. Taken together, these experiments reveal that the development and the maintenance of an accurate spatial representation are tightly related to the development of brain structures facilitated by the action of NGF. Moreover, these experiments demonstrate that an acute pharmacological treatment that leads to a transient modification in the choline acetyltransferase activity can induce a behavioral change long after the treatment.

Polymorphisms of the " macrophage migration inhibitory factor " gene in infectious diseases

Summary The proinflammatory cytokine macrophage migration inhibitory factor (MIF) has emerged as a central mediator of inflammation and innate immune defense against infections. MIF has been shown to play an important role in the pathogenesis of infectious diseases like sepsis, tuberculosis and autoimmune inflammatory diseases, such as arthritis, inflammatory bowel disease and asthma. Two functional polymorphisms of the MIF gene promoter, a five to eight CATT repeat microsatellite at position -794 and a G/C SNP at position -173, have been associated with increased susceptibility to or severity of autoimmune inflammatory diseases like arthritis, colitis and atopy. The aim of this thesis was to define whether, and if so by which mechanisms, MIF gene polymorphisms influence the susceptibility to or the outcome of one of the most severe and one of the most prevalent infectious diseases: meningococcal sepsis and tuberculosis, respectively. The results of the comparison between 1106 patients suffering from severe meningococcal infections and 434 healthy volunteers showed that carriers of the CATT5-5 genotype were protected from meningococcemia. A transmission disequilibrium test involving 106 families confirmed this association. At baseline and after stimulation with Neisseria meningitidis, the CATT5 MIF promoter drove lower transcriptional activity than the CATT6 or CATT7 alleles in human monocytic cells and whole blood of CATT5-5 healthy individuals tended to produce less MIF than whole blood of CATT6-6 individuals. Beyond, we describe several new MIF gene polymorphisms in Africans. Genotyping the CATT microsatellite and the -173*G/C SNP revealed great genetic diversity in six African ethnic groups. Comparing 471 African tuberculosis cases and 932 matched healthy controls, we observed ethnicity dependent associations of the -173*G/C and the CATT5-8 with susceptibility to or severity of tuberculosis, but confirmation in larger cohorts ìs needed. In conclusion, we report that homozygous carriage of a low expression allele of the MIF gene protects from meningococcal disease. These results support the concept that analyses of MIF genotypes in patients with sepsis may help to classify patients into risk categories and to identify those patients who may benefit from anti-MIF therapeutic strategies.

Adenosine A2B receptor-mediated leukemia inhibitory factor release from astrocytes protects cortical neurons against excitotoxicity.

ABSTRACT: BACKGROUND: Neuroprotective and neurotrophic properties of leukemia inhibitory factor (LIF) have been widely reported. In the central nervous system (CNS), astrocytes are the major source for LIF, expression of which is enhanced following disturbances leading to neuronal damage. How astrocytic LIF expression is regulated, however, has remained an unanswered question. Since neuronal stress is associated with production of extracellular adenosine, we investigated whether LIF expression in astrocytes was mediated through adenosine receptor signaling. METHODS: Mouse cortical neuronal and astrocyte cultures from wild-type and adenosine A2B receptor knock-out animals, as well as adenosine receptor agonists/antagonists and various enzymatic inhibitors, were used to study LIF expression and release in astrocytes. When needed, a one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test was used for statistical analysis. RESULTS: We show here that glutamate-stressed cortical neurons induce LIF expression through activation of adenosine A2B receptor subtype in cultured astrocytes and require signaling of protein kinase C (PKC), mitogen-activated protein kinases (MAPKs: p38 and ERK1/2), and the nuclear transcription factor (NF)- 54;B. Moreover, LIF concentration in the supernatant in response to 5'-N-ethylcarboxamide (NECA) stimulation was directly correlated to de novo protein synthesis, suggesting that LIF release did not occur through a regulated release pathway. Immunocytochemistry experiments show that LIF-containing vesicles co-localize with clathrin and Rab11, but not with pHogrin, Chromogranin (Cg)A and CgB, suggesting that LIF might be secreted through recycling endosomes. We further show that pre-treatment with supernatants from NECA-treated astrocytes increased survival of cultured cortical neurons against glutamate, which was absent when the supernatants were pre-treated with an anti-LIF neutralizing antibody. CONCLUSIONS: Adenosine from glutamate-stressed neurons induces rapid LIF release in astrocytes. This rapid release of LIF promotes the survival of cortical neurons against excitotoxicity.

The sigma factor AlgU (AlgT) controls exopolysaccharide production and tolerance towards desiccation and osmotic stress in the biocontrol agent Pseudomonas fluorescens CHA0.

A variety of stress situations may affect the activity and survival of plant-beneficial pseudomonads added to soil to control root diseases. This study focused on the roles of the sigma factor AlgU (synonyms, AlgT, RpoE, and sigma(22)) and the anti-sigma factor MucA in stress adaptation of the biocontrol agent Pseudomonas fluorescens CHA0. The algU-mucA-mucB gene cluster of strain CHA0 was similar to that of the pathogens Pseudomonas aeruginosa and Pseudomonas syringae. Strain CHA0 is naturally nonmucoid, whereas a mucA deletion mutant or algU-overexpressing strains were highly mucoid due to exopolysaccharide overproduction. Mucoidy strictly depended on the global regulator GacA. An algU deletion mutant was significantly more sensitive to osmotic stress than the wild-type CHA0 strain and the mucA mutant were. Expression of an algU'-'lacZ reporter fusion was induced severalfold in the wild type and in the mucA mutant upon exposure to osmotic stress, whereas a lower, noninducible level of expression was observed in the algU mutant. Overexpression of algU did not enhance tolerance towards osmotic stress. AlgU was found to be essential for tolerance of P. fluorescens towards desiccation stress in a sterile vermiculite-sand mixture and in a natural sandy loam soil. The size of the population of the algU mutant declined much more rapidly than the size of the wild-type population at soil water contents below 5%. In contrast to its role in pathogenic pseudomonads, AlgU did not contribute to tolerance of P. fluorescens towards oxidative and heat stress. In conclusion, AlgU is a crucial determinant in the adaptation of P. fluorescens to dry conditions and hyperosmolarity, two major stress factors that limit bacterial survival in the environment.

Controlled nerve growth factor release from multi-ply alginate/chitosan-based nerve conduits.

The delivery kinetics of growth factors has been suggested to play an important role in the regeneration of peripheral nerves following axotomy. In this context, we designed a nerve conduit (NC) with adjustable release kinetics of nerve growth factor (NGF). A multi-ply system was designed where NC consisting of a polyelectrolyte alginate/chitosan complex was coated with layers of poly(lactide-co-glycolide) (PLGA) to control the release of embedded NGF. Prior to assessing the in vitro NGF release from NC, various release test media, with and without stabilizers for NGF, were evaluated to ensure adequate quantification of NGF by ELISA. Citrate (pH 5.0) and acetate (pH 5.5) buffered saline solutions containing 0.05% Tween 20 yielded the most reliable results for ELISA active NGF. The in vitro release experiments revealed that the best results in terms of reproducibility and release control were achieved when the NGF was embedded between two PLGA layers and the ends of the NC tightly sealed by the PLGA coatings. The release kinetics could be efficiently adjusted by accommodating NGF at different radial locations within the NC. A sustained release of bioactive NGF in the low nanogram per day range was obtained for at least 15days. In conclusion, the developed multi-ply NGF loaded NC is considered a suitable candidate for future implantation studies to gain insight into the relationship between local growth factor availability and nerve regeneration.

An European Organisation for Research and Treatment of Cancer phase I study of lapatinib and docetaxel as neoadjuvant treatment for Human Epidermal Growth Factor Receptor 2 (HER2) positive locally-advanced/inflammatory or large operable breast cancer.

BACKGROUND: Lapatinib is an effective anti-HER2 therapy in advanced breast cancer and docetaxel is one of the most active agents in breast cancer. Combining these agents in pre-treated patients with metastatic disease had previously proved challenging, so the primary objective of this study aimed to determine the maximum tolerated dose (MTD) in treatment-naive patients, by identifying acute dose-limiting toxicities (DLT) during cycle 1 in the first part of a phases 1-2 neoadjuvant European Organisation for Research and Treatment of Cancer (EORTC) trial. PATIENTS AND METHODS: Patients with large operable or locally-advanced HER2 positive breast cancer were treated with continuous lapatinib, and docetaxel every 21days for 4 cycles. Dose levels (DLs) were: 1000/75, 1250/75, 1000/85, 1250/85, 1000/100 and 1250/100 (mg/day)/(mg/m(2)). RESULTS: Twenty-one patients were included. Two DLTs occurred at dose level 5 (1000/100); one grade 4 neutropenia ⩾7days and one febrile neutropenia. A further 3 patients were therefore treated at the same dose with prophylactic granulocyte-colony stimulating factor (G-CSF), and 3 patients at dose level 6. No further DLTs were observed. CONCLUSIONS: Our recommended dose for phase II is lapatinib 1000mg/day and docetaxel 100mg/m(2) with G-CSF in HER2 positive non-metastatic breast cancer. The dose of lapatinib should have been 1250mg/day but we were mindful of the high rate of treatment discontinuation in GeparQuinto with lapatinib 1250mg/day combined with docetaxel. No grade 3-4 diarrhoea was observed. Pharmacodynamics analysis suggests that concomitant medications altering P-glycoprotein activity (in addition to lapatinib) can modify toxicity, including non-haematological toxicities. This needs verification in larger trials, where it may contribute to understanding the sources of variability in clinical toxicity and treatment discontinuation.

Further insights on the French WISC-IV factor structure through Bayesian structural equation modeling

The interpretation of the Wechsler Intelligence Scale for Children-Fourth Edition (WISC-IV) is based on a 4-factor model, which is only partially compatible with the mainstream Cattell-Horn-Carroll (CHC) model of intelligence measurement. The structure of cognitive batteries is frequently analyzed via exploratory factor analysis and/or confirmatory factor analysis. With classical confirmatory factor analysis, almost all crossloadings between latent variables and measures are fixed to zero in order to allow the model to be identified. However, inappropriate zero cross-loadings can contribute to poor model fit, distorted factors, and biased factor correlations; most important, they do not necessarily faithfully reflect theory. To deal with these methodological and theoretical limitations, we used a new statistical approach, Bayesian structural equation modeling (BSEM), among a sample of 249 French-speaking Swiss children (8-12 years). With BSEM, zero-fixed cross-loadings between latent variables and measures are replaced by approximate zeros, based on informative, small-variance priors. Results indicated that a direct hierarchical CHC-based model with 5 factors plus a general intelligence factor better represented the structure of the WISC-IV than did the 4-factor structure and the higher order models. Because a direct hierarchical CHC model was more adequate, it was concluded that the general factor should be considered as a breadth rather than a superordinate factor. Because it was possible for us to estimate the influence of each of the latent variables on the 15 subtest scores, BSEM allowed improvement of the understanding of the structure of intelligence tests and the clinical interpretation of the subtest scores.

Identification and functional characterization of Rca1, a transcription factor involved in both antifungal susceptibility and host response in Candida albicans.

The identification of novel transcription factors associated with antifungal response may allow the discovery of fungus-specific targets for new therapeutic strategies. A collection of 241 Candida albicans transcriptional regulator mutants was screened for altered susceptibility to fluconazole, caspofungin, amphotericin B, and 5-fluorocytosine. Thirteen of these mutants not yet identified in terms of their role in antifungal response were further investigated, and the function of one of them, a mutant of orf19.6102 (RCA1), was characterized by transcriptome analysis. Strand-specific RNA sequencing and phenotypic tests assigned Rca1 as the regulator of hyphal formation through the cyclic AMP/protein kinase A (cAMP/PKA) signaling pathway and the transcription factor Efg1, but also probably through its interaction with a transcriptional repressor, most likely Tup1. The mechanisms responsible for the high level of resistance to caspofungin and fluconazole observed resulting from RCA1 deletion were investigated. From our observations, we propose that caspofungin resistance was the consequence of the deregulation of cell wall gene expression and that fluconazole resistance was linked to the modulation of the cAMP/PKA signaling pathway activity. In conclusion, our large-scale screening of a C. albicans transcription factor mutant collection allowed the identification of new effectors of the response to antifungals. The functional characterization of Rca1 assigned this transcription factor and its downstream targets as promising candidates for the development of new therapeutic strategies, as Rca1 influences host sensing, hyphal development, and antifungal response.

Responsiveness of astrocytes in serum-free aggregate cultures to epidermal growth factor: dependence on the cell cycle and the epidermal growth factor concentration.

Serum-free aggregating cell cultures of fetal rat telencephalon treated with low doses (0.5 nM) of epidermal growth factor (EGF) showed a small, transient increase in DNA synthesis but no significant changes in total DNA and protein content. By contrast, treatment with high doses (13 nM) of EGF caused a marked stimulation of DNA synthesis as well as a net increase in DNA and protein content. The expression of the astrocyte-specific enzyme, glutamine synthetase, was greatly enhanced both at low and at high EGF concentrations. These results suggest that at low concentration EGF stimulates exclusively the differentiation of astrocytes, whereas at high concentration, EGF has also a mitogenic effect. Nonproliferating astrocytes in cultures treated with 0.4 microM 1-beta-D-arabinofuranosyl-cytosine were refractory to EGF treatment, indicating that their responsiveness to EGF is cell cycle-dependent. Binding studies using a crude membrane fraction of 5-day cultures showed a homogeneous population of EGF binding sites (Kd approximately equal to 2.6 nM). Specific EGF binding sites were found also in non-proliferating (and nonresponsive) cultures, although they showed slightly reduced affinity and binding capacity. This finding suggests that the cell cycle-dependent control of astroglial responsiveness to EGF does not occur at the receptor level. However, it was found that the specific EGF binding sites disappear with progressive cellular differentiation.

Hypotensive effect of the active fragment derived from factor XII is mediated by an activation of the plasma kallikrein-kinin system.

Plasma protein fraction (PPF) contaminated by factor XII active fragment (XIIf) may cause hypotensive reactions when infused to patients. This study was planned to assess in conscious normotensive rats whether the blood pressure response to the factor XIIf is mediated by an activation of the plasma kallikrein-kinin system or by stimulation of prostaglandin synthesis. To test whether the factor XIIf-induced blood pressure fall is due partially to an enhanced generation of vasodilating prostaglandins, the blood pressure effect of XIIf (1 microgram i.v.) was investigated 15 min after treatment with indomethacin (5 mg i.v.), an inhibitor of cyclo-oxygenase. Factor XIIf reduced mean blood pressure similarly in indomethacin- and vehicle-treated rats (-23 +/- 4 mmHg, n = 5, and -23 +/- 5 mmHg, n = 4, respectively). Other rats received factor XIIf 15 min after depletion of circulating prekallikrein by the administration of dextran sulfate. Thirty minutes after a 0.25 mg i.v. dose of this agent, plasma prekallikrein activity averaged 0.12 +/- 0.015 mumol/min/ml (n = 6) as compared to 2.48 +/- 0.31 mumol/min/ml in control rats (n = 4, P less than .001). Factor XIIf decreased mean blood pressure by only 4 +/- 2 mm Hg in rats pretreated with dextran sulfate. Thus, it was possible to blunt the acute hypotensive effect of factor XIIf by depleting circulating prekallikrein, but not by inhibiting prostaglandin production. This strongly suggests that the blood pressure effects of factor XIIf is mediated by a stimulation of the plasma kallikrein-kinin system.

Achievement of at least very good partial response is a simple and robust prognostic factor in patients with multiple myeloma treated with high-dose therapy: long-term analysis of the IFM 99-02 and 99-04 Trials.

PURPOSE: The prognostic impact of complete response (CR) achievement in multiple myeloma (MM) has been shown mostly in the context of autologous stem-cell transplantation. Other levels of response have been defined because, even with high-dose therapy, CR is a relatively rare event. The purpose of this study was to analyze the prognostic impact of very good partial response (VGPR) in patients treated with high-dose therapy. PATIENTS AND METHODS: All patients were included in the Intergroupe Francophone du Myelome 99-02 and 99-04 trials and treated with vincristine, doxorubicin, and dexamethasone (VAD) induction therapy followed by double autologous stem-cell transplantation (ASCT). Best post-ASCT response assessment was available for 802 patients. RESULTS: With a median follow-up of 67 months, median event-free survival (EFS) and 5-year EFS were 42 months and 34%, respectively, for 405 patients who achieved at least VGPR after ASCT versus 32 months and 26% in 288 patients who achieved only partial remission (P = .005). Five-year overall survival (OS) was significantly superior in patients achieving at least VGPR (74% v 61% P = .0017). In multivariate analysis, achievement of less than VGPR was an independent factor predicting shorter EFS and OS. Response to VAD had no impact on EFS and OS. The impact of VGPR achievement on EFS and OS was significant in patients with International Staging System stages 2 to 3 and for patients with poor-risk cytogenetics t(4;14) or del(17p). CONCLUSION: In the context of ASCT, achievement of at least VGPR is a simple prognostic factor that has importance in intermediate and high-risk MM and can be informative in more patients than CR.

Transcriptional activation of the GLUT2 gene by the IPF-1/STF-1/IDX-1 homeobox factor.

The homeodomain protein PDX-1, referred as IPF-1/STF-1/IDX-1, is a transcriptional factor that plays a critical role in the control of several genes expressed in the pancreatic islet. PDX-1 gene expression has been previously shown to be reduced in cultured beta-cell lines chronically exposed to high glucose concentrations. As the glucose transporter type 2 (GLUT2) gene expression is selectively decreased in the beta-pancreatic cells of experimental models of diabetes, we postulated that the loss of GLUT2 gene expression in the pancreatic islets of diabetic animals may be due to the loss of PDX-1 transacting function on the GLUT2 gene. We, therefore, investigated the potential role of PDX-1 in the transcriptional control of GLUT2. We have identified a repeat of a TAAT motif (5'-TAATA-ATAACA-3') conserved in the sequence of the human and murine GLUT2 promoters. Recombinant PDX-1 binds to this GLUT2TAAT motif in electrophoretic mobility shift experiments. PDX-1 antiserum detects the formation of the complex of PDX-1 with the GLUT2TAAT motif in nuclear extracts from the pancreatic insulin-secreting cell line, beta TC3. The GLUT2TAAT motif was mutated in the murine GLUT2 promoter (-1308/+49 bp) linked to a luciferase reporter gene and transfected into beta TC3 cells. Compared with the transcriptional activity of the wild type promoter, that of the mutated promoter decreases by 41%. Multiple copies of the GLUT2TAAT motif were ligated 5' to a heterologous promoter and transfected into a PDX-1-expressing cell line (beta TC3) and into cell lines lacking the homeobox factor (InR1-G9 and JEG-3). The GLUT2TAAT motif mediates the activation of the heterologous promoter in the PDX-1-expressing cell line but not in InR1-G9 or JEG-3 cell lines. Furthermore, cotransfection in a PDX-1-deficient cell line with the expression vector encoding PDX-1 transactivates specifically the heterologous promoter containing the multimerized GLUT2TAAT motif. These data demonstrate that the murine GLUT2 promoter is controlled by the PDX-1 homeobox factor through the identified GLUT2TAAT motif.