Influence of cell confluence on the cAMP signalling pathway in vascular smooth muscle cells.

The influence of cell confluence on the ß-adrenoceptor (ß-AR)/cAMP/phosphodiesterase (PDE) pathway was investigated in cultured rat aortic smooth muscle cells (RASMCs). Cells were plated either at low density (LD: 3·103cells/cm2) or high density (HD: 3·104cells/cm2) corresponding to non-confluent or confluent cells, respectively, on the day of experiment. ß-AR-stimulated cAMP was monitored in real-time using the fluorescence resonance energy transfer (FRET)-based cAMP sensor, Epac2-camps. A brief application (15s) of the ß-AR agonist isoprenaline (Iso) induced a typical transient FRET signal, reflecting cAMP production followed by its rapid degradation. The amplitude of this response, which increased with the concentration of Iso (10 or 100nM), was higher in HD than in LD cells, whatever the Iso concentration used. However, activation of adenylyl cyclase by L-858051 (100µM) induced a similar saturating response in both LD and HD cells. A ß1-AR antagonist (CGP 20712A, 100nM) reduced the Iso (100nM) response in HD but not LD cells, whereas a ß2-AR antagonist (ICI 118,551, 5nM) reduced this response in HD cells and almost abolished it in LD cells. Competitive [125I]-ICYP binding experiments with betaxolol, a ß-AR ligand, identified two binding sites in HD cells, corresponding to ß1- and ß2-ARs with a proportion of 11% and 89%, respectively, but only one binding site in LD cells, corresponding to ß2-ARs. Total cAMP-PDE activity (assessed by a radioenzymatic assay) was increased in HD cells compared to LD cells. This increase was associated with a rise in mRNA expression of five cAMP-PDEs subtypes (PDE1A, 3A, 4A, 4B and 7B) in HD cells, and a decrease in basal [cAMP]i (assessed by an EIA assay). PDE4 inhibition with Ro-20-1724 (10µM) strongly prolonged the Iso response in LD and HD cells, whereas PDE3 inhibition with cilostamide (1µM) slightly prolonged Iso response only in LD cells. Interestingly, inhibition of PDE4 unmasked an effect of PDE3 in HD cells. Our results show that in cultured RASMCs, the ß-AR/cAMP/PDE signalling pathway is substantially modulated by the cell density. In HD cells, Iso response involves both ß1- and ß2-AR stimulation and is mainly controlled by PDE4, PDE3 being recruited only after PDE4 inhibition. In LD cells, Iso response involves only ß2-AR stimulation and is controlled by PDE4 and to a lower degree by PDE3. This low density state is associated with an absence of membrane expression of the ß1-AR, a lower cAMP-PDE activity and a higher basal [cAMP]i. This study highlights the critical role of the cellular environment in controlling the vascular ß-AR signalling.

Alteration of vascular reactivity in heart failure: role of phosphodiesterases 3 and 4.

BACKGROUND AND PURPOSE: This study examined the role of the main vascular cAMP-hydrolysing phosphodiesterases (cAMP-PDE) in the regulation of basal vascular tone and relaxation of rat aorta mediated by ß-adrenoceptors, following heart failure (HF). EXPERIMENTAL APPROACH: Twenty-two weeks after proximal aortic stenosis, to induce HF, or SHAM surgery in rats, we evaluated the expression, activity and function of cAMP-PDE in the descending thoracic aorta. KEY RESULTS: HF rat aortas exhibited signs of endothelial dysfunction, with alterations of the NO pathway, and alteration of PDE3 and PDE4 subtype expression, without changing total aortic cAMP-hydrolytic activity and PDE1, PDE3 and PDE4 activities. Vascular reactivity experiments using PDE inhibitors showed that PDE3 and PDE4 controlled the level of PGF2α -stimulated contraction in SHAM aorta. PDE3 function was partially inhibited by endothelial NO, whereas PDE4 function required a functional endothelium and was under the negative control of PDE3. In HF, PDE3 function was preserved, but its regulation by endothelial NO was altered. PDE4 function was abolished and restored by PDE3 inhibition. In PGF2α -precontracted arteries, ß-adrenoceptor stimulation-induced relaxation in SHAM aorta, which was abolished in the absence of functional endothelium, as well as in HF aortas, but restored after PDE3 inhibition in all unresponsive arteries. CONCLUSIONS AND IMPLICATIONS: Our study underlines the key role of the endothelium in controlling the contribution of smooth muscle PDE to contractile function. In HF, endothelial dysfunction had a major effect on PDE3 function and PDE3 inhibition restored a functional relaxation to ß-adrenoceptor stimulation.

TIMP-1 is a key factor of fibrogenic response to bleomycin in mouse lung.

Pulmonary fibrosis is characterized by the excessive deposition of extracellular matrix in the interstitium, resulting in respiratory failure. The role of remodeling mediators such as metalloproteinases (MMPs) and their inhibitors (TIMPs) in the fibrogenic process remains misunderstood. We investigated MMP-9, MMP-2, TIMP-1, TIMP-2 and TIMP-3 in the fibrotic response to bleomycin of fibrosis prone C57BL/6J and fibrosis resistant BALB/c mice. Mice were administered with 0.1 mg bleomycin by intranasal administration. Either 24 h or 14 days after, the mice were anesthetized and underwent either bronchoalveolear lavage (BAL) or lung removal. Collagen deposition in lung tissue was determined by hydroxyproline measurement, MMP activity was analyzed by zymography, and other mediators were analyzed by ELISA. TIMP-1 was localized in lung sections by immunohistochemistry and real time PCR was performed to gene expression in lung. Non parametric Mann-Whitney and Spearman tests were used for statistical analysis. Fourteen days after bleomycin administration, hydroxyproline assay and histological study revealed that BALB/c mice developed significantly less fibrosis compared to C57BL/6J mice. At day 1, bleomycin enhanced TIMP-1, MMP-2 and MMP-9 protein levels in BALF, and induced corresponding genes in lung tissue of both strains. The rise of Timp-1, Mmp-9 and Mmp-2 gene levels were significantly stronger in lungs of C57BL/6J, whereas gelatinase activities of MMP-2 and MMP-9 were similar. Immunohistochemistry revealed that TIMP-1 macrophages and epithelial cells were prominent TIMP-1 producers in both strains. At day 14, neither MMP-2 nor MMP-9 levels exhibited strain-dependent protein level or gene expression, although TIMP-1 was strongly associated with fibrosis. Interestingly, bleomycin induced neither Timp-2 nor Timp-3 in lung tissue at any time of the study. The present study shows that early altered regulation of TIMP-1 following bleomycin administration may be involved in bleomycin-induced pulmonary fibrosis.

Role of matrix metalloproteinases in the development of airway inflammation and remodeling

Matrix metalloproteinases (MMPs) are a major group of proteases known to regulate extracellular matrix (ECM) turnover and so they have been suggested to be important in the process of lung disease associated with tissue remodeling. This has led to the concept that modulation of airway remodeling including excessive proteolysis damage to the tissue may be of interest for future treatment. Within the MMP family, macrophage elastase (MMP-12) is able to degrade ECM components such as elastin and is involved in tissue remodeling processes in chronic obstructive pulmonary disease including emphysema. Pulmonary fibrosis has an aggressive course and is usually fatal within an average of 3 to 6 years after the onset of symptoms. Pulmonary fibrosis is associated with deposition of ECM components in the lung interstitium. The excessive airway remodeling as a result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of ECM components could justify anti-protease treatments. Indeed, the correlation of the differences in hydroxyproline levels in the lungs of bleomycin-treated mice strongly suggests that a reduced molar pro-MMP-9/TIMP-1 ratio in bronchoalveolar lavage fluid is associated with collagen deposition, beginning as early as the inflammatory events at day 1 after bleomycin administration. Finally, these observations emphasize that effective treatment of these disorders must be started early during the natural history of the disease, prior to the development of extensive lung destruction and fibrosis.

Role of matrix metalloproteinases in the development of airway inflammation and remodeling.

Matrix metalloproteinases (MMPs) are a major group of proteases known to regulate extracellular matrix (ECM) turnover and so they have been suggested to be important in the process of lung disease associated with tissue remodeling. This has led to the concept that modulation of airway remodeling including excessive proteolysis damage to the tissue may be of interest for future treatment. Within the MMP family, macrophage elastase (MMP-12) is able to degrade ECM components such as elastin and is involved in tissue remodeling processes in chronic obstructive pulmonary disease including emphysema. Pulmonary fibrosis has an aggressive course and is usually fatal within an average of 3 to 6 years after the onset of symptoms. Pulmonary fibrosis is associated with deposition of ECM components in the lung interstitium. The excessive airway remodeling as a result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of ECM components could justify anti-protease treatments. Indeed, the correlation of the differences in hydroxyproline levels in the lungs of bleomycin-treated mice strongly suggests that a reduced molar pro-MMP-9/TIMP-1 ratio in bronchoalveolar lavage fluid is associated with collagen deposition, beginning as early as the inflammatory events at day 1 after bleomycin administration. Finally, these observations emphasize that effective treatment of these disorders must be started early during the natural history of the disease, prior to the development of extensive lung destruction and fibrosis.

Creation versus destruction of T cell epitopes in the class II MHC pathway.

Proteases perform two key roles in the class II MHC antigen processing pathway. They initiate removal of the invariant chain chaperone for class II MHC and they generate peptides from foreign and self proteins for eventual capture and display to T cells. How a balance is achieved between generation of suitable peptides versus their complete destruction in an aggressive proteolytic environment is not known. Nor is it known in most cases which proteases are actually involved in antigen processing. Our recent studies have identified asparagine endopeptidase (AEP or legumain) as an enzyme that contributes to both productive and destructive antigen processing in the class II MHC pathway. The emerging consensus seems to be that individual proteolytic enzymes make clear and non-redundant contributions to antigen processing.

Bm-CPI-2, a cystatin homolog secreted by the filarial parasite Brugia malayi, inhibits class II MHC-restricted antigen processing.

While interference with the class I MHC pathway by pathogen-encoded gene products, especially those of viruses, has been well documented, few examples of specific interference with the MHC class II pathway have been reported. Potential targets for such interference are the proteases that remove the invariant chain chaperone and generate antigenic peptides. Indeed, recent studies indicate that immature dendritic cells express cystatin C to modulate cysteine protease activity and the expression of class II MHC molecules [1]. Here, we show that Bm-CPI-2, a recently discovered cystatin homolog produced by the filarial nematode parasite Brugia malayi (W. F. Gregory et al., submitted), inhibits multiple cysteine protease activities found in the endosomes/lysosomes of human B lymphocyte lines. CPI-2 blocked the hydrolysis of synthetic substrates favored by two different families of lysosomal cysteine proteases and blocked the in vitro processing of the tetanus toxin antigen by purified lysosome fractions. Moreover, CPI-2 substantially inhibited the presentation of selected T cell epitopes from tetanus toxin by living antigen-presenting cells. Our studies provide the first example of a product from a eukaryotic parasite that can directly interfere with antigen presentation, which, in turn, may suggest how filarial parasites might inactivate the host immune response to a helminth invader.

Changing heat and moisture exchangers every 48 hours does not increase the incidence of nosocomial pneumonia.

This prospective study was conducted to evaluate the risk of nosocomial pneumonia when changing heat and moisture exchangers every 48 hours in 1996 instead of every 24 hours in 1995 for patients needing continuous mechanical ventilation. Medical and surgical patients in the two periods did not differ in terms of demographic characteristics and markers of acute or underlying illnesses. The incidence density of nosocomial pneumonia was not different in the two groups. Extended heat and moisture exchanger use reduces circuit manipulation and cost.

Modulation by epitope-specific antibodies of class II MHC-restricted presentation of the tetanus toxin antigen.

Above a certain affinity the dissociation rate of monovalent antigen from antibody becomes slower than the time taken for antigen capture, endocytosis and processing by professional antigen presenting cells. Thus, when high affinity antibodies drive antigen uptake, either directly via B-cell membrane immunoglobulin or indirectly via Fc receptors, the substrate for processing may frequently be an antigen/antibody complex. Here we review studies using the tetanus toxin antigen which show that bound antibodies can dramatically affect proteolytic processing, dependent on the epitope specificity and multiplicity of antibodies bound. Certain antibodies protect or 'footprint' specific domains of the antigen during processing in B-cell clones resulting in modulation of loading of class II MHC-restricted T-cell epitopes. Processing and class II MHC loading of some T-cell epitopes within the footprinted region was hindered, as might be expected, but, surprisingly, presentation of other T-cell epitopes was boosted considerably. These studies show that protein/protein complexes can be processed in an unpredictable fashion by antigen presenting cells and indicate a possible mechanism whereby cryptic T-cell epitopes might be revealed in autoimmune disease.

An asparaginyl endopeptidase processes a microbial antigen for class II MHC presentation.

Foreign protein antigens must be broken down within endosomes or lysosomes to generate suitable peptides that will form complexes with class II major histocompatibility complex molecules for presentation to T cells. However, it is not known which proteases are required for antigen processing. To investigate this, we exposed a domain of the microbial tetanus toxin antigen (TTCF) to disrupted lysosomes that had been purified from a human B-cell line. Here we show that the dominant processing activity is not one of the known lysosomal cathepsins, which are generally believed to be the principal enzymes involved in antigen processing, but is instead an asparagine-specific cysteine endopeptidase. This enzyme seems similar or identical to a mammalian homologue of the legumain/haemoglobinase asparaginyl endopeptidases found originally in plants and parasites. We designed competitive peptide inhibitors of B-cell asparaginyl endopeptidase (AEP) that specifically block its proteolytic activity and inhibit processing of TTCF in vitro. In vivo, these inhibitors slow TTCF presentation to T cells, whereas preprocessing of TTCF with AEP accelerates its presentation, indicating that this enzyme performs a key step in TTCF processing. We also show that N-glycosylation of asparagine residues blocks AEP action in vitro. This indicates that N-glycosylation could eliminate sites of processing by AEP in mammalian proteins, allowing preferential processing of microbial antigens.

T cell-targeted immunotherapy in murine collagen-induced arthritis.

Counteracting the effect of autoimmunity can be achieved by elimination or inactivation of autoreactive T cells. We have focused on two approaches targeting on autoaggressive T cells in the model of collagen-induced arthritis (CIA) in mice. First, type II collagen (CII) primed DBA/1 mice were treated with various monoclonal antibodies (mAb) specific for the beta chains of the T cell receptor (TCR) using a protocol resulting in a long-term elimination of the target T cells. Indeed, CIA could be suppressed by injection of anti-V beta 8.1, 2 mAb and down-regulated by that of anti-V beta 2 and/or anti-V beta 5, presumably by deleting pathogenic T cell clones. In contrast, treatment with either anti-V beta 6 or anti-V beta 11 mAb did not alter CIA. Second, we generated CII-specific T cell hybrid clones that recognize the antigenic peptides in association with Kq and IA(q) molecules respectively for CD8+ and CD4+ cells. Vaccination with the irradiated hybrid clones, 3 weeks prior to immunization, was effective in preventing the development of arthritis. Furthermore, this suppression was antigen and disease specific. Most importantly, one CD8+ clone could reverse the ongoing disease. These new therapeutic approaches derived from animal models may offer a hope of more selective interventions for the treatment of human autoimmune diseases.

T cell regulation of collagen-induced arthritis in mice. II. Immunomodulation of arthritis by cytotoxic T cell hybridomas specific for type II collagen.

Injection of native type II collagen (CII) to susceptible strains of mice (H-2q) induces a rheumatoid arthritis-like disease. To study the role of CD8+ T cells in the collagen-induced arthritis (CIA), we generated CII-specific T cell hybridomas by fusion of cells from arthritic C3H. Q mice and an AKR thymoma. Two hybrid clones (P3G8 and P2D9) were selected for their ability to lyse syngeneic CII-pulsed macrophages and recognize different antigenic epitopes in association with Kq molecules. When these T cell clones were irradiated and inoculated into (C3H.Q x AKR)F1 mice 21 days prior to priming with native CII/complete Freund's adjuvant, the incidence and the duration of CIA were significantly reduced in comparison to groups receiving saline or control T cell hybridoma. Furthermore, both anti-CII T cell hybridomas were able to attenuate CIA in highly susceptible inbred strains of mice and this suppression was antigen and disease specific. The protective activity seems to require intact cells as neither membrane fractions nor cytosolic preparations of the hybridoma T cells retained the vaccinating activity. Most importantly, one of the hybrid clones (P3G8) had a therapeutic effect on CIA since its administration to arthritic DBA/1 mice on day 30 after priming down-regulated the ongoing disease. Taken together, these findings suggest that anti-CII cytotoxic T cell clones can vaccinate against CIA and even reverse the disease.

[Torulopsis glabrata septicemia: apropos of a case]. / Septicémie à Torulopsis glabrata: à propos d'un cas.

A 74 year's old patient with cancer of oesophagus has been operated. An atelectasis occurred just after surgery, then a septic shock with a septicaemia. Blood cultures were positive with Torulopsis glabrata. Amphotericin B and flucytosine, followed by flucytosine and miconazole cured septicaemia. These septicaemia happen often to diabetics patients, to patients with malignant tumours, or after wide spectrum antibiotics or during prolonged parenteral nutrition or after surgery.

[Laboratory contributions in systemic Candida albicans infections]. / Apport du laboratoire dans les infections systémiques à Candida albicans.

The authors have studied Candida albicans' serologies for seven patients in an intensive care unit. The comparison with the clinical evolution and results' laboratory displays an discordance for two cases. For these patients with many risk's factors, the observation of serology seems interesting on a prolonged period.

[Heart conduction disorders in acute maprotiline poisoning]. / Troubles de la conduction cardiaque lors d'une intoxication aiguë à la maprotiline.

A case is reported of self-poisoning with maprotiline followed by prolonged disorders of intraventricular conduction. The cardiotoxicity of tetracyclic antidepressants was well established, but few cases were reported. Maprotiline pharmacokinetics explained the duration of the cardiovascular side-effects. The severity of maprotiline overdoses should not be underestimated.