Regulation of cell survival by secreted proneurotrophins.

Neurotrophins are growth factors that promote cell survival, differentiation, and cell death. They are synthesized as proforms that can be cleaved intracellularly to release mature, secreted ligands. Although proneurotrophins have been considered inactive precursors, we show here that the proforms of nerve growth factor (NGF) and the proforms of brain derived neurotrophic factor (BDNF) are secreted and cleaved extracellularly by the serine protease plasmin and by selective matrix metalloproteinases (MMPs). ProNGF is a high-affinity ligand for p75(NTR) with high affinity and induced p75NTR-dependent apoptosis in cultured neurons with minimal activation of TrkA-mediated differentiation or survival. The biological action of neurotrophins is thus regulated by proteolytic cleavage, with proforms preferentially activating p75NTR to mediate apoptosis and mature forms activating Trk receptors to promote survival.

Effect of ionizing radiation on thymidine uptake, differentiation, and VEGFR2 receptor expression in endothelial cells: the role of VEGF(165).

PURPOSE: Late thrombosis of irradiated vascular segments may be the consequence of endothelial cell (EC) dysfunction after radiation therapy. We investigated the effects of beta ionizing radiation on human EC viability, thymidine uptake, and differentiation. METHODS AND MATERIALS: Endothelial cells were exposed to (32)P-labeled DNA oligonucleotides in incremental doses of 2, 6, and 10 Gy. The modulation of the VEGFR2 receptor expression after irradiation and the overall potential radioprotective effect of VEGF(165) on these functions were assayed. RESULTS: A dose-dependent inhibitory effect of beta irradiation on ECs' thymidine uptake and differentiation was observed. EC viability, however, was not affected at levels of radiation up to 10 Gy. VEGF(165) proved to have a radioprotective effect as ECs' thymidine uptake, after radiation doses of 2, 6, and 10 Gy, was increased by 1.5-, 2-, and 4-fold, respectively, in the presence of 10 ng/ml of VEGF(165) (p < 0.05 vs. LacZ). This concentration of VEGF(165) also proved beneficial in maintaining cell differentiation at 16 h postirradiation when compared to controls. These biologic effects were in direct correlation with the upregulation of VEGFR2 receptor expression in irradiated ECs. CONCLUSIONS: beta irradiation interacts directly with EC functions by significantly reducing their ability to differentiate and proliferate, associated with upregulation of VEGFR2. These effects can be prevented in part by pretreating cells with VEGF(165), an effect potentially favored by the upregulation of VEGFR2 receptor expression after irradiation.

IgG subclass responses to Pseudomonas aeruginosa a- and b-type flagellins in patients with cystic fibrosis: a prospective study.

Sera from 20 cystic fibrosis patients, whose lungs were colonised by Pseudomonas aeruginosa, were examined in a 3-5-year prospective study for any relationship between IgG subclass antibody levels to P. aeruginosa a- and b-type flagellins and pulmonary function (FEV1 and radiological score). Patients were divided into two groups according to their pulmonary status: group 1 comprised 11 patients with poor pulmonary status; group 2 comprised nine patients with relatively good pulmonary status. High concentrations of IgG1, IgG2 and IgG3 antibodies to flagellins, particularly to the b-type, were found in most patients. IgG4 reactivity was observed in only a few patients. Comparison of the two groups of patients showed that those with poor pulmonary status (group 1) had a significantly higher concentration (p < 0.05) of IgG3 for two of the three periods studied and of IgG2 for the last period studied. Moreover, IgG3 and IgG1 reactivities to b-type flagellin and IgG3 to a-type flagellin were also increased significantly (p < 0.05) in group 1 patients between the first and the last period studied. These patients also showed a significant (p < 0.05) time-dependent increase in IgG3 and IgG1 antibody concentrations. These data demonstrate that cystic fibrosis patients with poorer pulmonary status have higher IgG3 levels to flagellin than other cystic fibrosis patients. High concentrations of strong opsonic IgG3 and, to a lesser degree, of IgG1 antibodies may increase pulmonary inflammation and induce heightened pulmonary deterioration.

Production of ScFv antibody fragments following immunization with a phage-displayed fusion protein and analysis of reactivity to surface-exposed epitopes of the protein F of Pseudomonas aeruginosa by cytofluorometry.

To increase the possibilities of obtaining antibodies to surface-exposed epitopes of Pseudomonas aeruginosa protein F, we immunized mice with cloned and expressed oprF gene as a gIII-fusion protein displayed on the M13 phage surface. The fusion protein elicited mouse antibodies reacting with the purified protein F at a limit dilution of 1:10,000. Recombinant clones expressing antibody fragments were constructed from the genes of selected B cells of hyperimmunized mouse after a first round of panning against the protein F. Expression of single chain Fv (ScFv) antibody fragments to the protein of P. aeruginosa was detected by ELISA in 20 of 384 clones obtained after the first panning selection. The 20 positive clones recognizing different protein F epitopes as demonstrated by ELISA were assayed by flow cytometry to identify antibody fragments reacting only with surface-exposed epitopes of the protein F on whole bacteria; one of the 20 clones tested showed a level of reactivity compatible with surface-exposed epitope that can lead to ulterior developments in targeting studies.

Use of oligonucleotide probes to analyse the homology of the oprF gene among clinical and heterologous immunotype strains of Pseudomonas aeruginosa.

The conservation of oprF gene among 25 clinical Pseudomonas aeruginosa strains and a set of 17 serotype-specific representative strains of the international antigen typing scheme (IATS) was analysed by dot-blotting using five specific oligonucleotide probes. The oligo 1, 2, 3, 4, 5 correspond to five different regions of the oprF gene and hybridized strongly with respectively 88%, 88%, 76%, 94% and 71% of the IATS strains and 88%, 96%, 92%, 88% and 92% of the clinical strains. A parallel study performed with the whole oprF gene showed a lack of specificity of this probe: indeed, the probe hybridized not only with the 42 Pseudomonas aeruginosa strains but also with Escherichia coli and Salmonella minnesota. This study suggests that the gene sequence encoding the protein F is not totally conserved among Pseudomonas aeruginosa strains.

Cellular effects of beta-particle delivery on vascular smooth muscle cells and endothelial cells: a dose-response study.

BACKGROUND: Although endovascular radiotherapy inhibits neointimal hyperplasia, the exact cellular alterations induced by beta irradiation remain to be elucidated. METHODS AND RESULTS: We investigated in vitro the ability of 32P-labeled oligonucleotides to alter (1) proliferation of human and porcine vascular smooth muscle cells (VSMCs) and human coronary artery endothelial cells (ECs), (2) cell cycle progression, (3) cell viability and apoptosis, (4) cell migration, and (5) cell phenotype and morphological features. beta radiation significantly reduced proliferation of VSMCs (ED50 1.10 Gy) and ECs (ED50 2.15 Gy) in a dose-dependent manner. Exposure to beta emission interfered with cell cycle progression, with induction of G0/G1 arrest in VSMCs, without evidence of cell viability alteration, apoptosis, or ultrastructural changes. This strategy also proved to efficiently inhibit VSMC migration by 80% and induce contractile phenotype appearance, as shown by the predominance of alpha-actin immunostaining in beta-irradiated cells compared with control cells. CONCLUSIONS: 32P-labeled oligonucleotide was highly effective in inhibiting proliferation of both VSMCs and ECs in a dose-dependent fashion, with ECs showing a higher resistance to these effects. beta irradiation-induced G1 arrest was not associated with cytotoxicity and apoptosis, thus demonstrating a potent cytostatic effect of beta-based therapy. This effect, coupled to that on VSMC migration inhibition and the appearance of a contractile phenotype, reinforced the potential of ionizing radiation to prevent neointima formation after angioplasty.