Cytotoxic and genotoxic evaluation of different synthetic amorphous silica nanomaterials in the V79 cell line.

The nature of occupational risks and hazards in industries that produce or use synthetic amorphous silica (SAS) nanoparticles is still under discussion. Manufactured SAS occur in amorphous form and can be divided into two main types according to the production process, namely, pyrogenic silica (powder) and precipitated silica (powder, gel or colloid). The physical and chemical properties of SAS may vary in terms of particle size, surface area, agglomeration state or purity, and differences in their toxicity potential might therefore be expected. The aim of this study was to compare the cytotoxicity and genotoxicity of representative manufactured SAS samples in Chinese hamster lung fibroblasts (V79 cells). Five samples from industrial SAS producers were evaluated, that is, two pyrogenic SAS powders (with primary particle sizes of 20 nm and 25/70 nm), one precipitated SAS powder (20 nm) and two precipitated SAS colloids (15 and 40/80 nm). V79 cell cultures were treated with different concentrations of SAS pre-dispersed in bovine serum albumin -water medium. Pyr (pyrogenic) 20, Pre (precipitated) 20 and Col (colloid) 15 significantly decreased the cell viability after 24 h of exposure, whilst Pyr 25/70 and Col 40/80 had negligible effects. The cytotoxicity of Pyr 20, Pre 20 and Col 15 was revealed by the induction of apoptosis, and Pyr 20 and Col 15 also produced DNA damage. However, none of the SAS samples generated intracellular reactive oxidative species, micronuclei or genomic mutations in V79 cells after 24 h of exposure. Overall, the results of this study show that pyrogenic, precipitated and colloidal manufactured SAS of around 20 nm primary particle size can produce significant cytotoxic and genotoxic effects in V79 cells. In contrast, the coarser-grained pyrogenic and colloid SAS (approximately 50 nm) yielded negligible toxicity, despite having been manufactured by same processes as their finer-grained equivalents. To explain these differences, the influence of particle agglomeration and oxidative species formation is discussed.

Cytotoxicity and genotoxicity of panel of single- and multiwalled carbon nanotubes: in vitro effects on normal Syrian hamster embryo and immortalized v79 hamster lung cells.

Carbon nanotubes (CNTs) belong to a specific class of nanomaterials with unique properties. Because of their anticipated use in a wide range of industrial applications, their toxicity is of increasing concern. In order to determine whether specific physicochemical characteristics of CNTs are responsible for their toxicological effects, we investigated the cytotoxic and genotoxic effects of eight CNTs representative of each of the commonly encountered classes: single- SW-, double- DW-, and multiwalled (MW) CNTs, purified and raw. In addition, because most previous studies of CNT toxicity were conducted on immortalized cell lines, we decided to compare results obtained from V79 cells, an established cell line, with results from SHE (Syrian hamster embryo) cells, an easy-to-handle normal cell model. After 24 hours of treatment, MWCNTs were generally found to be more cytotoxic than SW- or DWCNTs. MWCNTs also provoked more genotoxic effects. No correlation could be found between CNT genotoxicity and metal impurities, length, surface area, or induction of cellular oxidative stress, but genotoxicity was seen to increase with CNT width. The toxicity observed for some CNTs leads us to suggest that they might also act by interfering with the cell cycle, but no significant differences were observed between normal and immortalized cells.

In vitro cytotoxicity and transforming potential of industrial carbon dust (fibers and particles) in syrian hamster embryo (SHE) cells.

Carbon fibers have many applications, mainly in high-tech industries such as the aviation industry. Eleven carbon samples (fibers and particles) coming from an aeronautic group were tested for their cytotoxicity and carcinogenic potential using in vitro short-term assays in Syrian hamster embryo cells. These samples were taken during each important step of the process, i.e. from the initial heating of polyacrylonitrile fibers to pure carbon fibers. They were compared to an asbestos fiber, an amorphous silica, and two commercial graphite powders. Their physical-chemical characteristics and their capacity to release reactive oxygen species (ROS) were determined. This study showed that none of the carbon samples was able to generate ROS as measured by Electron Paramagnetic Resonance analysis, and in our biological assays, they demonstrated no morphological transformation potential and low cytotoxicity compared to positive control (chrysotile asbestos).

Microstructure and electrical properties of diborides modified by rapid thermal annealing.

Diborides of Ti, Hf and Zr are thermally, mechanically and chemically stable with good thermal and electrical conductivity. We tested their properties in front-end processes used in Si integrated circuits (IC). Films were deposited by e-beam evaporation either on Si, for the formation of contacts to the source/drain (S/D) regions, or on Si oxides, for the formation of metal gates in p-type metal-oxide-semiconductor (PMOS) transistors. We focused on their crystallization caused by rapid thermal processing (RTP) at temperatures up to 1100 degrees C. Transmission electron microscopy was used for identification of nanocrystallites of TiB(2), ZrB(2), and HfB(2). The grain growth was correlated with temperature and time of RTP. Of all borides, HfB(2) resulted in the most complete crystallization with little amorphous phase left. There was no crystallographic degradation of the interface with Si or dielectrics, except for extreme thermal budgets. Complementary techniques were used for monitoring chemical stability and electrical parameters of test structures to assess the role of recrystallization in device behaviour.

SF-1 (steroidogenic factor-1), C/EBPbeta (CCAAT/enhancer binding protein), and ubiquitous transcription factors NF1 (nuclear factor 1) and Sp1 (selective promoter factor 1) are required for regulation of the mouse aldose reductase-like gene (AKR1B7) expression in adrenocortical cells.

The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) that is responsible for detoxifying isocaproaldehyde generated by steroidogenesis. In adrenocortical cell cultures, hormonal regulation of MVDP gene occurs through the cAMP pathway. We show that in adrenals, the pituitary hormone ACTH regulates MVDP gene expression in a coordinate fashion with steroidogenic genes. Cell transfection and DNA-binding studies were used to investigate the molecular mechanisms underlying MVDP gene regulation in Y1 adrenocortical cells. Progressive deletions of upstream regulatory regions identified a -121/+41 fragment that was sufficient for basal and cAMP-mediated transcriptional activities. Gel shift assays showed that CTF1/nuclear factor 1 (NF1), CCAAT enhancer binding protein-ss (C/EBPss), and selective promoter factor 1 (Sp1) factors bound to cis-acting elements at positions -76, -61, and -52, respectively. We report that the cell-specific steroidogenic factor-1 (SF-1) interacts specifically with a novel regulatory element located in the downstream half-site of the proximal androgen response element (AREp) at position -102. Functional analysis of SF-1 and NF1 sites in the -121/+41 promoter showed that mutation of one of them decreases both constitutive and forskolin-stimulated promoter activity without affecting the fold induction (forskolin stimulated/basal). Individual mutations of C/EBP and Sp1 sites resulted in a loss of more than 50% of the cAMP-dependent induction. When both sites were mutated simultaneously, cAMP responsiveness was nearly abolished. Thus, in adrenocortical cells, both SF-1 and NF1 are required for high expression of the MVDP promoter while Sp1 and C/EBPss functionally interact in an additive manner to mediate cAMP-dependent regulation. Furthermore, we report that MVDP gene regulation is impaired in stably transfected Y1 clones expressing DAX-1. Taken together, our findings suggest that detoxifying enzymes of the aldose reductase family may constitute new potential targets for regulators of adrenal and gonadal differentiation and function, e.g. SF-1 and DAX-1.

Down-regulation of AP1 activities after polarization of vas deferens epithelial cells correlates with androgen-induced gene expression.

Vas deferens epithelial cell subcultures were used to study the sequential regulation of jun/fos proto-oncogene expression and AP1 activities during cell proliferation, polarization and androgen-induced expression of a terminal differentiation marker, i. e. the mvdp gene. Proliferation of epithelial cells is associated with a high expression in the nucleus of most Jun and Fos oncoproteins. After cell seeding on an extracellular matrix which allows polarization and expression of the mvdp gene in response to androgens, AP1 protein accumulation is greatly altered and consists in a loss of JunB, Fra1, FosB and a decrease in c-Fos, c-Jun and Fra2, while JunD remained at the same level. This was correlated with a drop in AP1 binding activity as evaluated by gel shift assay using either AP1 consensus sequence or AP1 binding sites of the mvdp gene promoter region, and in AP1 transactivating activity, as estimated by stable transfection experiments using an AP1 responsive promoter (TRE-TK-luc). Androgens did not significantly influence AP1 activities. On the contrary, stimulation of AP1 proteins by the tumor-promoting phorbol ester caused a decrease in androgen-induced mvdp mRNA accumulation, and this effect was reversed by staurosporine, a potent inhibitor of PKC. Our data suggest that a down-regulation of AP1 activities induced by epithelial cell differentiation is a prerequisite to androgen-induced mvdp gene expression. The high AP1 activities observed during proliferative state or induced in TPA-treated polarized cells, exert a repressive effect on androgen action.

Phorbol ester causes ligand-independent activation of the androgen receptor.

We have examined the human androgen receptor (hAR) for its ability to activate AR-dependent transcription of a transgene in a ligand-independent manner. The transcriptional activity was determined by analysis of chloramphenicol acetyltransferase (CAT) activity in T47D cells cotransfected with a plasmid expressing the hAR and a natural AR-regulated promoter (the MVDP androgen-dependent enhancer) ligated to the reporter CAT gene. In this study, the effects of the protein kinase C (PKC) activator 12-O-tetradecanoyphorbol-13 acetate (TPA) on AR activity were tested. We demonstrated that in the absence of androgen, TPA enhanced AR-mediated transactivation by 10-12-fold. This effect was specific of the PKC pathway since stimulation to the PKA pathway did not activate the unliganded AR. This ligand-independent pathway can function through another androgen-regulated promoter as shown by the use of the mouse mammary tumor virus MMTV-CAT reporter. The human glucocorticoid receptor (hGR) and the rabbit progesterone receptor (rPR) could not be activated by TPA, indicating that the effects are not universal for steroid receptors. A reporter plasmid containing the MVDP androgen response element (ARE) in front of the thymidine kinase promoter ligated to the CAT gene was activated by DHT but not by TPA, indicating that the context of the natural promoter is critical for ligand-independent activation of the AR. Exogenous c-jun enhanced transcriptional activation by the AR in a ligand-dependent manner, but had no effect in the absence of DHT. Base pair substitutions in both AR-binding (5'-TGTTCT-3' to 5'-TTTTTT-3') and NF1-binding (5'-GTGGCTG-3' to 5'-GTTTTTG-3') sites resulted in a loss of TPA responsiveness. Our results suggest that ligand-independent activation of the AR by TPA results from interaction of unliganded AR with other proteins in the transcription machinery.

Ubiquitous transcription factors NF1 and Sp1 are involved in the androgen activation of the mouse vas deferens protein promoter.

Transcription of the mouse vas deferens protein (MVDP) gene is stimulated by androgens and we have previously shown that in a 162 bp fragment, located at position -121 to +41, a TGAAGTtccTGTTCT sequence functions as an androgen-dependent enhancer. To determine which factors are involved in the hormonally regulated MVDP gene transcription, we have used DNase I footprinting and band-shift assays to examine in vitro binding of proteins to the enhancer and promoter sequences and have determined the functional significance of the recognized DNA sequences in transient transfection assays. Studies using recombinant proteins such as the DNA binding domain of the androgen receptor (AR-DBD) and Sp1, and crude cellular extracts from T47D and vas deferens epithelial cells (VDEC) showed that in addition to AR-DBD, the transcriptional activators NF1 and Sp1 interact with the -121/+41 fragment in a specific manner. Transient transfection assays revealed that site-directed mutations in the NF1 and Sp1 binding elements strongly reduced (NF1) or abolished (Sp1) androgen induced expression. The results demonstrate that the -121/+41 sequence is a composite site for the androgen receptor mediated transactivation of the MVDP gene.

Treatment of respiratory failure using minitracheotomy and intratracheal oxygenation in selected patients with chronic lung disease.

OBJECTIVE: To evaluate the efficacy of minitracheotomy (MT) insertion for intratracheal oxygen insufflation (ITO2) on arterial blood gases and survival in patients with respiratory failure from chronic lung disease. DESIGN: Open, prospective clinical study. SETTING: A 12-bed medical intensive care unit in a non-university hospital. PATIENTS: 20 patients (14 males and 6 females, mean age 74.8 +/- 2.6 years), admitted for respiratory failure and denied mechanical ventilation. INTERVENTION: Percutaneous insertion of an MT for ITO2. Arterial blood gases were drawn just prior to, then 3, 24, 48 h and 1 week after MT insertion. Data are evaluated with a two-way analysis of variance for distribution-free data (Friedman's rank sums test). MEASUREMENTS AND RESULTS: Three hours after starting ITO2, the partial pressure of oxygen in arterial blood (PaO2) and the arterial oxygen saturation (SaO2) both increased from 51.7 +/- 2.8 to 85.4 +/- 5.6 mmHg and from 79.7 +/- 3.1 to 93.7 +/- 0.9%, respectively (p < 0.001 for both), along with a slight worsening in the partial pressure of carbon dioxide in arterial blood (PaCO2), from 59.6 +/- 2.5 to 63.5 +/- 3.0 mmHg (p < 0.05). At 1 week, improvements in PaO2 and SaO2 were maintained in all patients, while PaCO2 decreased in 14 patients (mean decrease 8.3 mmHg) and increased in the remaining patients (mean 12.5 mmHg), when compared to pre-ITO2 values. Seven patients died during follow-up, leading to a success rate of 65%. Eight and 4 patients were discharged home and to a nursing home, respectively, 9 still receiving ITO2 via MT as chronic oxygen therapy. CONCLUSION: Our results suggest that MT insertion for ITO2 may be a therapeutic option in selected patients with respiratory failure from CLD.

Protein kinase C pathway potentiates androgen-mediated gene expression of the mouse vas deferens specific aldose reductase-like protein (MVDP).

Transcription of the mouse vas deferens protein (MVDP) gene, a member of the aldo-keto reductase superfamily, is stimulated by androgens via the androgen responsive element (ARE) located in the proximal promoter (-111 to -97). We investigated interaction between androgens and the protein kinase C (PKC) signalling pathway. Transcriptional regulation was determined by analysis of chloramphenicol acetyltransferase (CAT). T47D cells were transiently transfected with 5' flanking MVDP DNA promoter sequences (-1804 to +41; -510 to +41 and -121 to +41) fused to the reporter (CAT) gene. Androgen-induced transcriptional activity can be enhanced from 6 (1.8 and 0.5 kb MVDP-CAT constructs) to 18 fold (0.16 kb MVDP-CAT construct), in a time and dose-dependent manner, by the PKC activator 12-o-tetradecanoylphorbol-13 acetate (TPA). A mutation in the proximal ARE abolished both androgen and TPA-dependent gene enhancement. TPA influenced minimally MMTV promoter in T47D cells and MVDP promoter in CV1 cells suggesting that the effects of the PKC activator are probably promoter and cell-specific. In contrast, activation of protein kinase A (PKA) via addition of dibutyryl-cAMP (db-cAMP) reduced androgen induction of the MVDP gene.

Characterization of the promoter of the gene for a mouse vas deferens protein related to the aldo-keto reductase superfamily: effect of steroid hormones and phorbol esters.

Mouse vas deferens protein (MVDP) is a member of the aldo-keto reductase family regulated by androgens. The expression of a hybrid gene containing the promoter of the MVDP gene and the chloramphenicol acetyl transferase (CAT) gene coding region was analyzed in two cell lines that do not normally express the MVDP gene: T47D and CV1 cells. A small region of the promoter (-121 to +41) was able to direct significant expression of the reporter gene in both cell lines. Additional elements, between -510 and -121 modulate basal expression in a cell-dependent manner. Interestingly, the 162 bp fragment serves as an androgen-dependent enhancer, and mutation of the consensus ARE sequence located between positions -111 and -97 resulted in a loss of androgen response in both cell lines. Additional elements, upstream of the enhancer, modulate induction positively or negatively in relation to the cell line used. The expression of different MVDP-CAT constructs was more effectively induced by androgens than by glucocorticoids at physiological hormonal concentrations. In addition to the 162 bp enhancer, sequences upstream of -510 were also required for specific androgen regulation. Phorbol 12-myristate 13-acetate (TPA) had no effect on basal activity of the 1.8 kb MVDP-CAT construct but strongly enhanced the induction by androgens.

Vas deferens epithelial cells in subculture: a model to study androgen regulation of gene expression.

The understanding of androgen-regulated gene expression requires a cell cultures system that mimics the functions of cells in vivo. In the present paper we have examined a vas deferens epithelial cell subculture system. Cultured vas deferens epithelial cells have been shown to exhibit polarized properties characteristic of functioning epithelia and to display a high level of androgen receptors. Incubation of cells with androgen caused a decrease in cellular androgen receptor mRNA that was time-dependent. Total suppression was observed after 24h of exposure to androgen. By contrast, incubation of vas deferens epithelial cells with androgen resulted in a threefold increase in the cellular content of androgen receptor protein, as assayed by ligand binding. In response to androgens, vas deferens epithelial cells expressed mouse vas deferens protein mRNA (MVDP mRNA). Maximum expression of the MVDP gene, at both mRNA and protein levels, was observed after 24h of androgen induction. DEAE-dextran transfection conditions were defined using the MMTV-CAT gene in vas deferens epithelial cells in a dose- and time-dependent manner. No induction was seen when fragments of the MVDP promoter region were cloned directly in front of the CAT gene and transiently transfected into vas deferens epithelial cells. It was found that cotransfection of cells with MVDP-CAT gene and transfection of cells with MVDP-CAT constructs and with an androgen receptor expression vector resulted in a small but consistent androgen-dependent increase in reporter gene activity. Transiently transfected vas deferens epithelial cells are a suitable model with which to study the effect of androgen on gene regulatory elements.

Functional results of single-lung transplantation for chronic obstructive lung disease.

The feasibility and immediate tolerance of single-lung transplantation were recently demonstrated in patients with severe obstructive lung disease. Since initial reports, hundreds of procedures have been performed worldwide in such patients, but views regarding the results are still controversial. Since few data concerning medium-term functional results are available, we report here our series of 20 patients with chronic obstructive pulmonary disease who received a single-lung transplant. A group of 16 patients who survived for 6 mo or more form the basis of this report. Current 1- and 2-yr actuarial survival are 75 and 70%, respectively, with 4 perioperative deaths and 2 deaths at 9 and 15 mo after transplantation. Before transplantation the patients were severely obstructive, with a FEV1 of 17 +/- 6% of predicted, a PaO2 of 51 +/- 10 mm Hg, a PaCO2 of 49 +/- 11 mm Hg, and a 6 min walk test of 99 +/- 84 m. A significant functional improvement was observed postoperatively, the patients' FEV1 at 3 mo reached 53 +/- 13%, PaO2 81 +/- 3 mm Hg, and PaCO2 39 +/- 3 mm Hg. The distance covered during 6 min was 587 +/- 147 m at 6 mo. Throughout postoperative follow-up, lung function remained stable in some patients but decreased in others after several mo, this decline related to the occurrence of bronchiolitis obliterans, except in two patients who had airway complications. Impairment in lung function led to retransplantation in four patients, with good clinical results in three patients, one patient dying postoperatively.(ABSTRACT TRUNCATED AT 250 WORDS)

[Bronchiolitis obliterans after lung transplantation]. / Bronchiolite oblitérante après transplantation pulmonaire.

Lung transplantation has become a realistic treatment in some patients with severe respiratory impairment or severe pulmonary arterial hypertension. Immunosuppression therapy is about the same in all transplantation units and includes cyclosporin, corticosteroids and azathioprine. The diagnosis of acute rejection episodes has been greatly facilitated by the histological study of transbronchial biopsies obtained by endoscopy. Improvements in the short-term prognosis of these patients have made it possible to individualize an unusual and delayed complication: bronchiolitis obliterans. This progressive and diffuse obstruction followed by destruction of the transplant's bronchioles is interpreted as a consequence of chronic rejection. The diagnosis of bronchiolitis obliterans is difficult and rests essentially on degradation of the respiratory function resisting to increased immunosuppression. Some viral infections perhaps contribute to its development, and it may be so severe as to require another lung transplantation.

Pulmonary lymphangiomyomatosis treated by single lung transplantation.

Pulmonary lymphangiomyomatosis is a rare disease resistant to almost all medical treatments to date. We describe the case of a 44-yr-old woman with end-stage pulmonary lymphangiomyomatosis who was treated by single-lung transplantation. The patient is doing well in her sixteenth post-transplantation month and has a marked improvement in her pulmonary function tests and walking distance as compared with preoperative values, and she is enjoying an unrestricted life-style.

[3 cases of Kituchi's lymphadenitis in systemic lupus erythematosus. Role of the parvovirus B19]. / Trois cas de lymphadénite de Kikuchi au cours du lupus érythémateux systémique. Rôle du parvovirus B19.

Florid necrotizing lymphadenitis, characterized by segmental infarction and lymphoid hyperplasia, is an uncommon feature of systemic lupus erythematosus (SLE). Kikuchi's disease is a well-defined clinicopathological entity, with a strong preference for the cervical lymph nodes of young women. The etiology of histiocytic necrotizing lymphadenitis (HNL) remains unknown, although viral agents have been proposed. HNL may reflect a self-limited SLE-like autoimmune disease but full-blown SLE associated with this condition has not, to the best of our knowledge, been reported. Thus, ours is the first description of the coexistence of SLE and HNL in 3 patients with immunologically proven parvovirus B19 infections. SLE and HNL were diagnosed simultaneously in 2 patients, but was retrospective in the third, in whom anti-tuberculous therapy was ineffective. Patients 1 and 2 were treated with prednisone (1 mg/kg/d) and responded rapidly. These data suggest that both HNL and SLE flares can be caused by parvovirus B19 infection.

Nosocomial pneumonia in patients receiving continuous mechanical ventilation. Prospective analysis of 52 episodes with use of a protected specimen brush and quantitative culture techniques.

Epidemiologic studies of nosocomial bacterial pneumonia in patients requiring mechanical ventilation have been limited because of the poor reliability of diagnosis procedures in this setting. To determine prognostic and descriptive factors of ventilator-associated (V-A) pneumonia, we prospectively studied 567 patients who had been receiving mechanical ventilation for more than 3 days in our unit. Fiberoptic bronchoscopy using a protected specimen brush (PSB) was performed on each patient suspected of having pneumonia because of the presence of a new pulmonary infiltrate and purulent tracheal secretions. The diagnosis of V-A pneumonia was retained only if PSB specimens yielded greater than 10(3) cfu/ml of at least one microorganism, unless this result was established to be a false positive result on follow-up. V-A pneumonia developed in 49 patients for a total of 52 episodes (9%). The actuarial risk of V-A pneumonia was 6.5% at 10 days, 19% at 20 days, and 28% at 30 days of ventilation. Patients with pneumonia were significantly older (65 versus 57 yr of age, p less than 0.01) and more frequently had severe underlying illnesses (24 versus 10%, p less than 0.01) than did patients without pneumonia. A total of 84 microorganisms (51 gram-negative and 33 gram-positive) were isolated in significant concentrations from PSB specimens. Pseudomonas aeruginosa and Staphylococcus aureus were involved in 31 and 33% of these pneumonias, respectively. Forty percent of all specimens yielded a polymicrobial flora with more than one potential pathogen.(ABSTRACT TRUNCATED AT 250 WORDS)