Assessing dystrophies and other muscle diseases at the nanometer scale by atomic force microscopy.

AIM: Atomic force microscopy nanoindentation of myofibers was used to assess and quantitatively diagnose muscular dystrophies from human patients. MATERIALS & METHODS: Myofibers were probed from fresh or frozen muscle biopsies from human dystrophic patients and healthy volunteers, as well as mice models, and Young's modulus stiffness values were determined. RESULTS: Fibers displaying abnormally low mechanical stability were detected in biopsies from patients affected by 11 distinct muscle diseases, and Young's modulus values were commensurate to the severity of the disease. Abnormal myofiber resistance was also observed from consulting patients whose muscle condition could not be detected or unambiguously diagnosed otherwise. DISCUSSION & CONCLUSION: This study provides a proof-of-concept that atomic force microscopy yields a quantitative read-out of human muscle function from clinical biopsies, and that it may thereby complement current muscular dystrophy diagnosis.

Adenovirus-mediated gene transfer into selected liver segments using a vascular exclusion technique.

Adenovirus-mediated gene therapy is hampered by severe virus-related toxicity, especially to the liver. The aim of the present study was to test the ability of a vascular exclusion technique to achieve transgene expression within selected liver segments, thus minimizing both viral and transgene product toxicity to the liver. An E1-E3-deleted replication-deficient adenovirus expressing a green fluorescent protein (GFP) reporter gene was injected into the portal vein of BDIX rats, with simultaneous clamping of the portal vein tributaries to liver segments II, III, IV, V, and VIII. GFP expression and inflammatory infiltrate were measured in the different segments of the liver and compared with those of the livers of animals receiving the viral vector in the portal vein without clamping. The GFP expression was significantly higher in the selectively perfused segments of the liver as compared with the non-perfused segments (p < 0.0001) and with the livers of animals that received the vector in the portal vein without clamping (p < 0.0001). Accordingly, the inflammatory infiltrate was more intense in the selectively perfused liver segments as compared with all other groups (p < 0.0001). Fluorescence was absent in lungs and kidneys and minimal in spleen. The clinical usefulness of adenovirus-mediated gene transfer to the liver largely depends on the reduction of its liver toxicity. Clamping of selected portal vein branches during injection allows for delivery of genes of interest to targeted liver segments. Transgene expression confined to selected liver segments may be useful in the treatment of focal liver diseases, including metastases.

Species-Specific Recognition of Aspergillus fumigatus by Toll-like Receptor 1 and Toll-like Receptor 6.

Background. Aspergillus fumigatus causes invasive aspergillosis, a potentially fatal infection in oncohematological patients. Innate immune detection of A. fumigatus involves Toll-like receptor (TLR) 4 and TLR2, which forms a heterodimer with either TLR1 or TLR6. The role of those coreceptors in Aspergillus sensing is unknown. Methods. Cytokine production was measured in bone marrow-derived macrophages (BMDMs) from wild-type (WT) and TLR-deficient mice after incubation with a WT and an immunogenic RodA-deficient (ΔrodA-47) strain of A. fumigatus and in lungs from these mice after intranasal mold inoculation. Aspergillus fumigatus-mediated NF-κB activation was measured in HEK293T cells transfected with plasmids expressing mouse or human TLRs. Results. Bone marrow-derived macrophages from TLR1- and TLR6-deficient mice produced lower amounts of interleukin 12p40, CXCL2, interleukin 6, and tumor necrosis factor α than BMDMs from WT mice after stimulation with A. fumigatus. Lungs from TLR1- and TLR6-deficient mice had diminished CXCL1 and CXCL2 production and increased fungal burden after intranasal inoculation of ΔrodA A. fumigatus compared with lungs from WT mice. ΔrodA strain-mediated NF-κB activation was observed in HEK293T cells expressing mouse TLR2/1, mouse TLR2/6, and human TLR2/1 but not human TLR2/6. Conclusions. Innate immune detection of A. fumigatus is mediated by TLR4 and TLR2 together with TLR1 or TLR6 in mice and TLR1 but not TLR6 in humans.

Induction of innate immune responses through Nalp3 inflammasome sensing of asbestos and silica

The inhalation of airborne pollutants such as asbestos or silica is linked to inflammation of the lung, fibrosis and lung cancer. How the presence of pathogenic dust is recognised, and how chronic inflammatory diseases are triggered are poorly understood. We will se show that asbestos and silica are sensed by the Nalp3 inflammasome, whose subsequent activation leads to IL-1b secretion. Inflammasome activation is triggered by reactive oxygen species, which are generated by a NADPH oxidase upon particle phagocytosis. In a model of asbestos inhalation, Nalp3_/_ mice showed diminished recruitment of inflammatory cells to the lungs, paralleled by lower cytokine production. Our findings implicate the Nalp3 inflammasome in particulate matter-related pulmonary diseases and support its role as a major proinflammatory ''danger" receptor.

PPARs in diseases: control mechanisms of inflammation.

The three isotypes of peroxisome proliferator-activated receptors (PPARs), PPARalpha, beta/delta and gamma, are ligand-inducible transcription factors that belong to the nuclear hormone receptor family. PPARs are implicated in the control of inflammatory responses and in energy homeostasis and thus, can be defined as metabolic and anti-inflammatory transcription factors. They exert their anti-inflammatory effects by inhibiting the induction of pro-inflammatory cytokines, adhesion molecules and extracellular matrix proteins or by stimulating the production of anti-inflammatory molecules. Furthermore, PPARs modulate the proliferation, differentiation and survival of immune cells including macrophages, B cells and T cells. This review discusses the molecular mechanisms by which PPARs and their ligands modulate the inflammatory response. In addition, it presents recent developments implicating PPAR specific ligands in potential treatments of inflammation-related diseases, such as atherosclerosis, inflammatory bowel diseases, Parkinson's and Alzheimer's diseases.

Protein delivery for retinal diseases: from basic considerations to clinical applications.

Because the eye is protected by ocular barriers but is also easily accessible, direct intravitreous injections of therapeutic proteins allow for specific and targeted treatment of retinal diseases. Low doses of proteins are required in this confined environment and a long time of residency in the vitreous is expected, making the eye the ideal organ for local proteic therapies. Monthly intravitreous injection of Ranibizumab, an anti-VEGF Fab has become the standard of care for patients presenting wet AMD. It has brought the proof of concept that administering proteins into the physiologically low proteic concentration vitreous can be performed safely. Other antibodies, Fab, peptides and growth factors have been shown to exert beneficial effects on animal models when administered within the therapeutic and safe window. To extend the use of such biomolecules in the ophthalmology practice, optimization of treatment regimens and efficacy is required. Basic knowledge remains to be increased on how different proteins/peptides penetrate into the eye and the ocular tissues, distribute in the vitreous, penetrate into the retinal layers and/or cells, are eliminated from the eye or metabolized. This should serve as a basis for designing novel drug delivery systems. The later should be non-or minimally invasive and should allow for a controlled, scalable and sustained release of the therapeutic proteins in the ocular media. This paper reviews the actual knowledge regarding protein delivery for eye diseases and describes novel non-viral gene therapy technologies particularly adapted for this purpose.

Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica

The inhalation of airborne pollutants, such as asbestos or silica, is linked to inflammation of the lung, fibrosis, and lung cancer. How the presence of pathogenic dust is recognized and how chronic inflammatory diseases are triggered are poorly understood. Here, we show that asbestos and silica are sensed by the Nalp3 inflammasome, whose subsequent activation leads to interleukin-1beta secretion. Inflammasome activation is triggered by reactive oxygen species, which are generated by a NADPH oxidase upon particle phagocytosis. (NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate.) In a model of asbestos inhalation, Nalp3-/- mice showed diminished recruitment of inflammatory cells to the lungs, paralleled by lower cytokine production. Our findings implicate the Nalp3 inflammasome in particulate matter-related pulmonary diseases and support its role as a major proinflammatory "danger" receptor

Maladies infectieuses en pratique ambulatoire (sauf sida) [Infectious diseases (beside AIDS)]

A simplified version of the US guidelines for prophylaxis of infectious endocarditis was published in 2007. Changes are expected in Switzerland as well. Posaconsole is a new antifungal agent available mostly for prophylaxis in immunocompromised patients. Epidemiology of sexually transmitted infections warrants screening in young adults and after one episode. A meta-analysis clarified the impact of antibiotic therapy in patients with Campylobacter spp. infection. In the field of emerging diseases, we discuss Norovirus epidemics, community-acquired bacteria producing extended-spectrum betalactamases, extensively resistant tuberculosis, and new respiratory viruses. Finally, we address a basic research topic that may change practice in the future: the relationship between individual susceptibility to infection and innate immunity.

New insight in aetiopathogenesis of aortic diseases.

BACKGROUND: Knowledge in the aetiopathogeny of aortic disease helps to characterise aortic lesions better and determine the risk of evolution and therapeutic strategies as well. This article focusses on aneurysms and dissections, and excludes causes related to infection, systemic inflammatory diseases and trauma. METHODS AND RESULTS: The biomedical literature of the past 10 years has been reviewed here. Aortic diseases are heterogeneous along the aorta as far as their genetic determinants, contribution of smooth muscle cells, inflammation and thrombus formation are concerned. Degradation of extracellular matrix by proteases causing aortic disease is a 'terminal' event, modulated by genetic background, haemodynamic strain, cellular events and thrombus formation. New genetic determinants of aortic disease have been identified. Proteases degrading the aortic wall are derived from a variety of cell types in addition to macrophages, including neutrophils on the luminal thrombus, mesenchymal and endothelial cells in the wall. Smooth muscle cells contribute to aortic wall homeostasis against inflammation and proteolysis. The degradation of the wall is followed by, or paralleled with, a failure of aortic reconstruction. CONCLUSIONS: Aortic diseases are diverse, and involve a multiplicity of biological systems in the vascular wall and at the interface with blood. Future research needs to unravel distinct cellular and molecular mechanisms causing the clinical events, in particular, dissection, expansion of already formed aneurysms and rupture.

A Th17- and Th2-skewed Cytokine Profile in Cystic Fibrosis Lungs Represents a Potential Risk Factor for Pseudomonas aeruginosa Infection.

Rationale: Cystic fibrosis (CF) is characterized by progressive pulmonary inflammation that is infection-triggered. Pseudomonas aeruginosa represents a risk factor for deterioration of lung function and reduced life expectancy. Objectives: To assess T-cell cytokine/chemokine production in clinically stable children with CF and evaluate the association between T-cell subtypes and susceptibility for infection with P. aeruginosa. Methods: T-cell cytokine/chemokine profiles were measured in bronchoalveolar lavage fluid (BALF) from children with CF (n = 57; 6.1 ± 5.9 yr) and non-CF control subjects (n = 18; 5.9 ± 4.3 yr). Memory responses to Aspergillus fumigatus and P. aeruginosa were monitored. High-resolution computed tomography-based Helbich score was assessed. In a prospective observational trial the association between BALF cytokine/chemokine profiles and subsequent infection with P. aeruginosa was studied. Measurements and Main Results: Th1- (INF-γ), Th2- (IL-5, IL-13), Th17- (IL-17A), and Th17-related cytokines (IL-1β, IL-6) were significantly up-regulated in airways of patients with CF. IL-17A, IL-13, and IL-5 were significantly higher in BALF of symptomatic as compared with clinically asymptomatic patients with CF. IL-17A and IL-5 correlated with the percentage of neutrophils in BALF (r = 0.41, P < 0.05 and r = 0.46, P < 0.05, respectively). Th17- (IL-17A, IL-6, IL-1β, IL-8) and Th2-associated cytokines and chemokines (IL-5, IL-13, TARC/CCL17), but not IFN-γ levels, significantly correlated with high-resolution computed tomography changes (Helbich score; P < 0.05). P. aeruginosa- and A. fumigatus-specific T cells from patients with CF displayed significantly higher IL-5 and IL-17A mRNA expression. IL-17A and TARC/CCL17 were significantly augmented in patients that developed P. aeruginosa infection within 24 months. Conclusions: We propose a role for Th17 and Th2 T cells in chronic inflammation in lungs of patients with CF. High concentrations of these cytokines/chemokines in CF airways precede infection with P. aeruginosa.

Fourth European Conference on Infections in Leukaemia (ECIL-4): guidelines for diagnosis, prevention, and treatment of invasive fungal diseases in paediatric patients with cancer or allogeneic haemopoietic stem-cell transplantation.

Invasive opportunistic fungal diseases (IFDs) are important causes of morbidity and mortality in paediatric patients with cancer and those who have had an allogeneic haemopoietic stem-cell transplantation (HSCT). Apart from differences in underlying disorders and comorbidities relative to those of adults, IFDs in infants, children, and adolescents are unique with respect to their epidemiology, the usefulness of diagnostic methods, the pharmacology and dosing of antifungal agents, and the absence of interventional phase 3 clinical trials for guidance of evidence-based decisions. To better define the state of knowledge on IFDs in paediatric patients with cancer and allogeneic HSCT and to improve IFD diagnosis, prevention, and management, the Fourth European Conference on Infections in Leukaemia (ECIL-4) in 2011 convened a group that reviewed the scientific literature on IFDs and graded the available quality of evidence according to the Infectious Diseases Society of America grading system. The final considerations and recommendations of the group are summarised in this manuscript.

Tuberculosis comorbidity with communicable and non-communicable diseases: integrating health services and control efforts.

Recent data for the global burden of disease reflect major demographic and lifestyle changes, leading to a rise in non-communicable diseases. Most countries with high levels of tuberculosis face a large comorbidity burden from both non-communicable and communicable diseases. Traditional disease-specific approaches typically fail to recognise common features and potential synergies in integration of care, management, and control of non-communicable and communicable diseases. In resource-limited countries, the need to tackle a broader range of overlapping comorbid diseases is growing. Tuberculosis and HIV/AIDS persist as global emergencies. The lethal interaction between tuberculosis and HIV coinfection in adults, children, and pregnant women in sub-Saharan Africa exemplifies the need for well integrated approaches to disease management and control. Furthermore, links between diabetes mellitus, smoking, alcoholism, chronic lung diseases, cancer, immunosuppressive treatment, malnutrition, and tuberculosis are well recognised. Here, we focus on interactions, synergies, and challenges of integration of tuberculosis care with management strategies for non-communicable and communicable diseases without eroding the functionality of existing national programmes for tuberculosis. The need for sustained and increased funding for these initiatives is greater than ever and requires increased political and funder commitment.