Interleukin-10 inhibits IgE-mediated nitric oxide synthase induction and cytokine synthesis in normal human keratinocytes

Human keratinocytes (HK) generate nitric oxide (NO) and proinflammatory mediators following activation with either IgE/anti-IgE immune complexes or a combination of lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Recently, interleukin-10 (IL-10) has been shown to down-regulate various inflammatory responses and to be secreted by lymphocytes and dendritic cells during skin inflammatory reactions. We show here that IL-10 down-regulates the production of tumor necrosis factor (TNF)-α and IL-6 by activated HK. Also, induction of inducible nitric oxide synthase (iNOS) expression in HK by IgE/anti-IgE or LPS/IFN-γ is significantly reduced by the addition of IL-10. This effect is dose dependent and correlates with reduction of iNOS mRNA production and enzyme level. Therefore, IL-10 down-regulates NO-mediated HK inflammatory responses and may thus participate in the regulation of the skin immune network.     

Reliability and validity of the Biodex system 3 pro isokinetic dynamometer velocity,torque and position measurements

This study quantitatively assessed the mechanical reliability and validity of position, torque and velocity measurements of the Biodex System 3 isokinetic dynamometer. Trial-to-trial and day-to-day reliability were assessed during three trials on two separate days. To assess instrument validity, measurement of each variable using the Biodex System 3 dynamometer was compared to a criterion measure of position, torque and velocity. Position was assessed at 5° increments across the available range of motion of the dynamometer. Torque measures were assessed isometrically by hanging six different calibrated weights from the lever arm. Velocity was assessed (30°/s to 500°/s) across a 70° arc of motion by manually accelerating the weighted lever arm. With the exception of a systematic decrease in velocity at speeds of 300°/s and higher, the Biodex System 3 performed with acceptable mechanical reliability and validity on all variables tested.DisclosureThe Biodex dynamometer used for this investigation was donated to the laboratory by Biodex Medical Systems. The authors have no commercial or proprietary interest in this device.     

Mutations in VMK1, a mitogen-activated protein kinase gene, affect microsclerotia formation and pathogenicity in Verticillium dahliae

Verticillium dahliae is an important soil-borne fungal pathogen that causes vascular wilt diseases in a large variety of important crop plants. Due to its persistence in the soil, control of Verticillium wilt relies heavily on soil fumigation. The global ban on methyl bromide, a highly effective soil fumigant, poses an urgent need to develop alternative control measures against Verticillium wilt; and these might be more forthcoming with a better understanding of the molecular and cellular mechanisms that underpin the pathogenicity of V. dahliae. In this study, we assessed the role in growth, development, and pathogenicity of VMK1, a gene encoding a mitogen-activated protein (MAP) kinase (hence, Verticillium MAP Kinase 1). Disruption of VMK1 via Agrobacterium tumefaciens-mediated transformation, in two V. dahliae isolates, one from lettuce and the other from tomato, resulted in severely reduced virulence in diverse host plants, suggesting that VMK1 is essential for pathogenicity and that the MAP kinase-mediated signaling pathway has a conserved role in fungal pathogenicity. The vmk1 mutants also exhibited reduced conidiation and microsclerotia formation, suggesting that the gene is important for multiple cellular processes. P.R. and R.G.B. equally contributed to the work     

Chest radiography in general practice: indications, diagnostic yield and consequences for patient management

BACKGROUND: Chest radiography (CXR) is frequently performed in Western societies. There is insufficient knowledge of its diagnostic value in terms of changes in patient management decisions in primary care. AIM: To assess the influence of CXR on patient management in general practice. DESIGN OF STUDY: Prospective cohort study. SETTING: Seventy-eight GPs and three general hospitals in the Netherlands. METHOD: Patients (n = 792) aged > or =18 years referred by their GPs for CXR were included. The main outcome was change in patient management assessed by means of questionnaires filled in by GPs before and after CXR. RESULTS: Mean age of the patients was 57.3+/-16.2 years and 53% were male. Clinically relevant abnormalities were found in 24% of the CXRs. Patient management changed in 60% of the patients following CXR. Main changes included: fewer referrals to a medical specialist (from 26 to 12%); reduction in initiation or change in therapy (from 24 to 15%); and more frequent reassurance (from 25 to 46%). However, this reassurance was not perceived as such in a quarter of these patients. A change in patient management occurred significantly more frequently in patients with complaints of cough (67%), those who exhibited abnormalities during physical examination (69%), or those with a suspected diagnosis of pneumonia (68%). CONCLUSION: Patient management by the GP changed in 60% of patients following CXR. CXR substantially reduced the number of referrals and initiation or change in therapy, and more patients were reassured by their GP. Thus, CXR is an important diagnostic tool for GPs and seems a cost-effective diagnostic test.     

Rheumatic heart disease: proinflammatory cytokines play a role in the progression and maintenance of valvular lesions

Heart lesions of rheumatic heart disease (RHD) patients contain T-cell clones that recognize heart proteins and streptococcal M peptides. To functionally characterize heart-infiltrating T lymphocytes, we evaluated their cytokine profile, both directly in situ and in T-cell lines derived from the heart (HIL). Interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-4, and IL-10 expressions were characterized in 20 heart tissue infiltrates from 14 RHD patients by immunohistochemistry. IFN-gamma-, TNF-alpha-, and IL-10-positive cells were consistently predominant, whereas IL-4 was scarce in the valves. In agreement with these data, the in vitro experiments, in which 13 HILs derived from heart samples of eight patients were stimulated with M5 protein and the immunodominant M5 (81-96) peptide, IL-4 was detected in HIL derived from the atrium (three of six) but not from the valve (zero of seven). IFN-gamma and IL-10 production were detected in culture supernatants in 11 of 13 and 6 of 12 HILs, respectively. The predominant IFN-gamma and TNF-alpha expression in the heart suggests that Th1-type cytokines could mediate RHD. Unlike in reversible myocardium inflammation, the significantly lower IL-4 expression in the valvular tissue (P = 0.02) may contribute to the progression of the RHD leading to permanent valvular damage (relative risk, 4.3; odds ratio, 15.8). The lack of IL-4 in vitro production by valve-derived HIL also emphasizes the more severe tissue destruction in valves observed in RHD.     

The Fanconi anemia pathway is required for the DNA replication stress response and for the regulation of common fragile site stability

Fanconi anemia (FA) is a rare multi-genic, autosomal and X-linked recessive disorder characterized by hematological abnormalities, developmental defects and increased cancer susceptibility. Patient-derived FA cells display heightened sensitivity to DNA cross-linking agents such as mitomycin C (MMC). In response to DNA damaging agents, and during S-phase of the cell cycle, the FA pathway is activated via the mono-ubiquitination of FANCD2 (FANCD2-Ub), signaling its translocation to discrete nuclear foci, where it co-localizes with the central DNA repair proteins BRCA1 and RAD51. However, the exact function of activated FANCD2-Ub remains unclear. Here, we have characterized the role of the FA pathway in response to DNA replicative stress by aphidicolin (APH) and hydroxyurea (HU). The FA pathway is strongly activated in response to both agents. In addition, using patient-derived FA cell lines and siRNA targeting FANCD2, we demonstrate a functional requirement for the FA pathway in response to low doses of APH: a replicative stress treatment known to result in chromosome breakage at common fragile sites. Both the total number of chromosome gaps and breaks and breaks at the specific common fragile sites FRA3B and FRA16D were significantly elevated in the absence of an intact FA pathway. Furthermore, we demonstrate that APH activates the mono-ubiquitination of both FANCD2 and PCNA and the phosphorylation of RPA2, signaling processive DNA replication arrest. Following APH treatment, FANCD2-Ub co-localizes with PCNA (early) and RPA2 (late) in discrete nuclear foci. Our results demonstrate an integral role for the FA pathway in the DNA replication stress response.     

Characterization of Mycobacterium montefiorense sp. nov., a novel pathogenic Mycobacterium from moray eels that is related to Mycobacterium triplex

The characterization of a novel Mycobacterium sp. isolated from granulomatous skin lesions of moray eels is reported. Analysis of the hsp65 gene, small-subunit rRNA gene, rRNA spacer region, and phenotypic characteristics demonstrate that this organism is distinct from its closest genetic match, Mycobacterium triplex, and it has been named M. montefiorense sp. nov.