In vivo attenuation and genetic evolution of a ST247-SCCmecI MRSA clone after 13 years of pathogenic bronchopulmonary colonization in a patient with cystic fibrosis: implications of the innate immune response.

Methicillin-resistant Staphylococcus aureus (MRSA) causes chronic pulmonary infections in patients with cystic fibrosis (CF). This study tracks the 13-year evolution (1996-2009) of a single MRSA clone in a male patient with CF, evaluating both the host immunogenic response and the microbial variations. Whole-genome sequencing was performed for the initial (CF-96) and evolved (CF-09) isolates. The immunogenicity of CF-96 and CF-09 was evaluated by incubation with innate immune cells from healthy volunteers. We also studied the patient's innate immune response profile, cytokine production, expression of triggering receptor expressed on myeloid cells-1 (TREM-1), and phagocytosis. A total of 30 MRSA ST247-SCCmecI-pvl(-) isolates were collected, which evidenced a genome size reduction from the CF-96 ancestor to the evolved CF-09 strain. Up to six changes in the spa-type were observed over the course of the 13-year evolution. Cytokine production, TREM-1 expression, and phagocytosis were significantly lower for the healthy volunteer monocytes exposed to CF-09, compared with those exposed to CF-96. Patient monocytes exhibited a reduced inflammatory response when challenged with CF-09. Genetic changes in MRSA, leading to reduced immunogenicity and entry into the refractory state, may contribute to the attenuation of virulence and efficient persistence of the bacteria in the CF lung.

Effects of CPAP on oxidative stress and nitrate efficiency in sleep apnoea: a randomised trial.

BACKGROUND: Previous studies have presented contradictory data concerning obstructive sleep apnoea syndrome (OSAS), lipid oxidation and nitric oxide (NO) bioavailability. This study was undertaken to (1) compare the concentration of 8-isoprostane and total nitrate and nitrite (NOx) in plasma of middle-aged men with OSAS and no other known co-morbidity and healthy controls of the same age, gender and body mass index; and (2) test the hypothesis that nasal continuous positive airway pressure (CPAP) therapy attenuates oxidative stress and nitrate deficiency. METHODS: A prospective, randomised, placebo controlled, double-blind, crossover study was performed in 31 consecutive middle-aged men with newly diagnosed OSAS and 15 healthy control subjects. Patients with OSAS were randomised to receive sham CPAP or effective CPAP for 12 weeks. Blood pressure, urinary catecholamine levels and plasma 8-isoprostane and NOx concentrations were obtained before and after both treatment modalities. RESULTS: Patients with OSAS had significantly higher 8-isoprostane levels (median (IQR) 42.5 (29.2-78.2) vs 20.0 (12.5-52.5) pg/ml, p = 0.041, Mann-Whitney test) and lower NOx levels (264 (165-650) vs 590 (251-1465) micromol/l, p = 0.022) than healthy subjects. Body mass index, blood pressure and urinary catecholamines were unchanged by CPAP therapy, but 8-isoprostane concentrations decreased (38.5 (24.2-58.7) pg/ml at baseline vs 22.5 (16.2-35.3) pg/ml on CPAP, p = 0.0001) and NOx levels increased (280 (177-707) vs 1373 (981-1517) micromol/l, p = 0.0001) after CPAP. CONCLUSIONS: OSAS is associated with an increase in oxidative stress and a decrease in NOx that is normalised by CPAP therapy.

Apoptosis induced by Rac GTPase correlates with induction of FasL and ceramides production.

Rho proteins, members of the Ras superfamily of GTPases, are critical elements in signal transduction pathways governing cell proliferation and cell death. Different members of the family of human Rho GTPases, including RhoA, RhoC, and Rac1, participate in the regulation of apoptosis in response to cytokines and serum deprivation in different cell systems. Here, we have characterized the mechanism of apoptosis induced by Rac1 in NIH 3T3 cells. It requires protein synthesis and caspase-3 activity, but it is independent of the release of cytochrome c from mitochondria. Moreover, an increase in mitochondria membrane potential and the production of reactive oxygen species was observed. Rac1-induced apoptosis was related to the simultaneous increase in ceramide production and synthesis of FasL. Generation of FasL may be mediated by transcriptional regulation involving both c-Jun amino terminal kinase as well as nuclear factor-kappa B-dependent signals. None of these signals, ceramides or FasL, was sufficient to induce apoptosis in the parental cell line, NIH 3T3 cells. However, any of them was sufficient to induce apoptosis in the Rac1-expressing cells. Finally, inhibition of FasL signaling drastically reduced apoptosis by Rac1. Thus, Rac1 seems to induce apoptosis by a complex mechanism involving the generation of ceramides and the de novo synthesis of FasL. These results suggest that apoptosis mediated by Rac1 results from a signaling mechanism that involves biochemical and transcriptional events under control of Rac1.

Protective effect of cyclosporin A and FK506 from nitric oxide-dependent apoptosis in activated macrophages.

1. Activation of macrophages with lipopolysaccharide (LPS) and low doses of interferon-gamma (IFN-gamma) induced apoptotic death through a nitric oxide-dependent pathway. 2. Treatment of cells with the immunosuppressors cyclosporin A (CsA) or FK506 inhibited the activation-dependent apoptosis. 3. These drugs decreased the up-regulation of p53 and Bax characteristic of activated macrophages. Moreover, incubation of activated macrophages with CsA and FK506 contributed to maintain higher levels of Bcl-2 than in LPS/IFN-gamma treated cells. 4. The inhibition of apoptosis exerted by CsA and FK506 in macrophages was also observed when cell death was induced by treatment with chemical nitric oxide donors. 5. Incubation of macrophages with LPS/IFN-gamma barely affected caspase-1 but promoted an important activation of caspase-3. Both CsA and FK506 inhibited pathways leading to caspase-3 activation. Moreover, the cleavage of poly(ADP-ribose) polymerase, a well established caspase substrate, was reduced by these immunosuppressive drugs. 6. CsA and FK506 reduced the release of cytochrome c to the cytosol and the activation of caspase-3 in cells treated with nitric oxide donors. 7. These results indicate that CsA and FK506 protect macrophages from nitric oxide-dependent apoptosis and suggest a contribution of the macrophage to innate immunity under conditions of immunosuppression of the host.

Interferon-alpha/beta inhibits the apoptosis induced by lipopolysaccharide and interferon-gamma in murine peritoneal macrophages.

Challenge of elicited peritoneal macrophages with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) was followed by an apoptotic response. These cells expressed cytokine-inducible nitric oxide synthase (iNOS) in response to these stimuli, and the NO released contributed markedly to the apoptotic death, as deduced from the increased viability observed when iNOS activity was inhibited. The antiviral type I IFN (IFN-alpha/beta) down-regulated the high levels of NO produced when cells were stimulated with suboptimal doses of LPS and IFN-gamma. Moreover, IFN-alpha/beta also decreased cell death in LPS/IFN-gamma-activated cells, as determined by the reduction in the content of oligonucleosomal DNA fragments, in the binding of annexin V to the plasma membrane, and in the amount of hypodiploid cells when analyzed by flow cytometry after in vivo staining with propidium iodide. Kinetic analysis of the protection exerted by IFN-alpha/beta) against the apoptosis induced by treatment with LPS and IFN-gamma showed that type I IFNs were very effective when added up to 1 h after IFN-gamma/LPS stimulation. Addition of IFN-alpha/beta 4 h after stimulation with IFN-gamma/LPS failed completely to prevent apoptosis. This inhibition of apoptosis elicited by IFN-alpha/beta suggests the existence of a mechanism intended to improve macrophage viability in the course of certain viral infections.

Triggering of peritoneal macrophages with IFN-alpha/beta attenuates the expression of inducible nitric oxide synthase through a decrease in NF-kappaB activation.

Triggering peritoneal macrophages with IFN-gamma and a low concentration of LPS induced the expression of the inducible form of nitric oxide synthase (iNOS). This process was significantly inhibited when IFN-alpha/beta was added during the initial 2 h after the start of IFN-gamma/LPS activation. Evaluation of the transcriptional activity using run-on assays indicated that IFN-alpha/beta inhibited the transcription of iNOS. Transfection experiments using a 1.7-kb promoter sequence corresponding to the 5' flanking region of the murine iNOS gene showed decreased promoter activity in the presence of type I IFNs. Analysis of the transcription factors that participate in iNOS expression revealed a marked decrease of NF-kappaB activation, a nuclear factor required for the transcription of this gene. The degradation of IkappaB alpha and IkappaB beta, which is required for the translocation of NF-kappaB to the nucleus, was inhibited in the presence of IFN-alpha/beta. However, the activity of other transcription factors such as IFN regulatory factor 1, which is involved in the expression of iNOS in response to IFN-gamma, was not affected by IFN-alpha/beta stimulation. These results suggest that in the presence of IFN-alpha/beta, the activity of the iNOS promoter is impaired, and this attenuated nitric oxide synthase expression could be important in pathophysiologic situations in which secretion of type I IFNs occurs.

Requirement of nitric oxide and calcium mobilization for the induction of apoptosis in adrenal vascular endothelial cells.

Exposure of adrenal vascular endothelial cells (AVEC) to pharmacological nitric oxide (NO) donors, proinflammatory cytokines or lipopolysaccharide was unable to induce apoptosis as occurred when macrophages were treated under identical experimental conditions. However, when the intracellular Ca2+ concentration increased, AVEC displayed apoptotic features upon exposure to NO. This apoptosis was confirmed by the release of oligonucleosomes to the cytosol and by the characteristic DNA laddering observed after electrophoresis in agarose gels. Ca2+-mobilizing agents and interleukin-1beta (IL-1beta) also elicited an apoptotic response in these cells through a mechanism that required NO synthesis. The ability of NO and intracellular Ca2+ to promote apoptosis was dependent on the number of passages of the cells in culture, suggesting the loss of protective factors in the course of ex vivo cell culture. Because AVEC exhibit an important capacity to increase the intracellular Ca2+ concentration in response to a wide array of agonists, this condition might affect the integrity of the vascular system under pathological circumstances such as those prevailing in the course of septic shock.