Anti-inflammatory gene therapy can inhibit inflammation driven by TNFalpha in experimental models of rheumatoid arthritis. However, assessment of the therapeutic effect on cartilage and bone quality is either missing or unsatisfactory. A multimodal imaging approach, using confocal laser scanning microscopy (CLSM) and micro-computed tomography (microCT), was used for gathering 3D quantitative image data on diseased and treated murine joints. As proof of concept, the efficacy of anti-TNF-based gene therapy was assessed, comparing imaging techniques with classical investigations. SCID mice knees were injected with human synoviocytes overexpressing TNFalpha. Two days later, electric pulse-mediated DNA transfer was performed after injection of the pGTRTT-plasmid containing a dimeric soluble-TNF receptor (dsTNFR) under the control of a doxycycline-inducible promoter. After 21 days the mice were sacrificed, TNFalpha levels were measured and the joints assessed for cartilage and bone degradation, using CLSM, microCT and histology. TNFalpha levels were decreased in the joints of mice treated with the plasmid in the presence of doxycycline. Concomitantly, histological analysis showed an increase in cartilage thickness and a decrease in specific synovial hyperplasia and cartilage erosion. Bone morphometry revealed that groups with the plasmid in the presence of doxycycline displayed a higher cortical thickness and decreased porosity. Using an anti-TNF gene therapy approach, known to reduce inflammation, as proof of concept, 3D imaging allowed quantitative evaluation of its benefits to joint architecture. It showed that local delivery of a regulated anti-TNF vector allowed decreasing arthritis severity through TNFalpha inhibition. These tools are valuable for understanding the efficacy of gene therapy on whole-joint morphometry. Copyright (c) 2010 John Wiley & Sons, Ltd.

Epidermolysis bullosa acquisita (EBA) is a severe immunobullous disease and is caused by IgG against type VII collagen (Col VII) of anchoring fibrils. In this study, utilizing ELISA and immunoblot, 13/15 EBA sera but 0/20 bullous pemphigoid sera and 0/30 healthy control sera showed IgG reactivity with distinct recombinant subregions of the non-collagenous domain 1 (NC1) of Col VII. In two EBA patients, IgG titers against Col VII-NC1 were grossly correlated to clinical disease activity. Moreover, Col VII-reactive T cells were identified in a representative EBA patient which recognized identical subdomains of Col VII-NC1. These findings strongly suggest that (1) the Col VII-NC1 ELISA is a powerful tool for making the diagnosis of EBA,(2) Col VII-specific IgG grossly relates to disease activity and (3) IgG reactivity is associated with T cell recognition of identical subdomains of Col VII-NC1.

Summary Growth retardation is an important breeding aim and an essential part of horticultural plant production. Here, the potential of transferring the Arabidopsis short internode (shi) mutant phenotype was explored by expressing the AtSHI gene in the popular ornamental plant Kalanchoë. A 35S-AtSHI construct was produced and transferred into eight genetically different cultivars of Kalanchoë by Agrobacterium tumefaciens. The resulting transgenic plants showed dwarfing phenotypes like reduced plant height and diameter, and also more compact inflorescences, as a result of increased vegetative height. The shi phenotype was stable over more than five vegetative subcultivations. Compared with Arabidopsis, the ectopic expression of AtSHI in Kalanchoë showed several differences. None of the Kalanchoë SHI-lines exhibited alterations in leaf colour or morphology, and most lines were not delayed in flowering. Moreover, continuous treatment of lines delayed in flowering with low concentrations of gibberellins completely restored the time of flowering. These features are very important as a delay in flowering would increase plant production costs significantly. The effect of expression controlled by the native Arabidopsis SHI promoter was also investigated in transgenic Kalanchoë and resulted in plants with a longer flowering period. Two AtSHI like genes were identified in Kalanchoë indicating a widespread presence of this transcription factor. These findings are important because they suggest that transformation with the AtSHI gene could be applied to several species as a tool for growth retardation, and that this approach could substitute the use of conventional chemical growth regulation in plant production.

Vitamin D deficiency results in abnormal mineralization of bones and has resulted in prevention programs for children with supplementation when they are breast fed. Further activities of vitamin D relate to defence of microbial infections, e.g. tuberculosis, prevention of cancer, contractility of muscle cells and counteraction of congestive heart failure. Given early reports in the 1960s on deleterious effects of vitamin D supplementation in rodents, that is ectopic media ossification of arterial vessels, a pro-atherogenic function had been anticipated for humans as well. However, cross-sectional studies reveal that vitamin D deficiency in humans is associated with elevated blood pressure and propagation of atherogenesis. These contradictory findings on the progression of atherosclerosis may be reconciled by dissecting the activation mechanism(s) of vitamin D in rodents versus humans. Notably, novel findings convincingly indicate that vitamin D exerts anti-inflammatory effects. In conclusion, vitamin D supplementation in adults may be regarded as simple means with few potential side effects to prevent atherogenesis or halt its progression and combat arterial hypertension. Adjustment of vitamin D dosing regimens is required in patients with chronic kidney disease; however, prospective clinical trials are urgently needed to guide these recommendations with evidence.

Mechanical testing has been regarded as the gold standard to investigate the effects of pathologies on the structure-function properties of the skeleton. With recent advances in computing power of personal computers, virtual alternatives to mechanical testing are gaining acceptance and use. We have previously introduced such a technique called structural rigidity analysis to assess mechanical strength of skeletal tissue with defects. The application of this technique is predicated upon the use of relationships defining the strength of bone as a function of its density for a given loading mode. We are to apply this technique in rat models to assess their compressive skeletal response subjected to a host of biological and pharmaceutical stimulations. Therefore, the aim of this study is to derive a relationship expressing axial compressive mechanical properties of rat cortical and cancellous bone as a function of equivalent bone mineral density, bone volume fraction or apparent density over a range of normal and pathologic bones. We used bones from normal, ovariectomized and partially nephrectomized animals. All specimens underwent micro-computed tomographic imaging to assess bone morphometric and densitometric indices and uniaxial compression to failure. We obtained univariate relationships describing 71-78% of the mechanical properties of rat cortical and cancellous bone based on equivalent mineral density, bone volume fraction or apparent density over a wide range of density and common skeletal pathologies. The relationships reported in this study can be used in the structural rigidity analysis introduced by the authors to provide a non-invasive method to assess the compressive strength of bones affected by pathology and/or treatment options.

The aim of this study was to examine the influence of matrix elasticity on the maintenance of the chondrogenic phenotype of chondrocytes cultured in monolayer. We used a 2D-culturing-system in which polyacrylamide gels with different concentrations of BIS-Acrylamide were coated with collagen type I. Matrices with a Young's Modulus of 4 kPa, 10 kPa, 40 kPa, and 100 kPa were produced, as determined by atomic force microscopy. Porcine chondrocytes were cultivated on these matrices at low density for seven days. The proliferation of cells was analyzed by 5-Bromo-2'-deoxy-uridine (BrdU) incorporation. Maintenance of the chondrogenic phenotype was analyzed by measuring collagen type I, type II and aggrecan gene expression, immunofluorescence staining for collagen type II, and phalloidin staining for actin filaments. Cellular proliferation and actin organisation was decreased on matrices of 4 kPa, in comparison to stiffer substrates. The differentiated phenotype of the chondrocytes grown on matrices of 4 kPa was stabilized, indicated by higher collagen type II and aggrecan, and lower collagen type I expression. These findings indicate that chondrocytes sense the elasticity of the matrix and might be used for the design of scaffolds with mechanical properties, specifically tailored to support the chondrogenic phenotype in tissue engineering applications.

BACKGROUND: International studies in the 1990 s and the HYGEA study from Germany in 2002 revealed prevalences of around 50 % of microbiological contaminations in reprocessed flexible endoscopes. Before introducing the colorectal cancer screening programme by colonoscopy in Germany in 2002, the Kassenärztliche Bundesvereinigung (KBV) and the key stakeholders of the public health insurance system agreed on a quality assessment assurance for reprocessing endoscopes where the qualification for refund for colonoscopies from the public health system was made conditional on adequate qualifications of the gastroenterologist; on a minimum number of performed procedures per year; and on adequate endoscope reprocessing documented by negative surveillance cultures two times per year. This study is an implementation and outcome evaluation of the quality assessment assurance in colonoscopy in Germany. METHODS: The following data - per year and per Kassenärztliche Vereinigung (KV)- were obtained from the KBV: the number of endoscopic units performing therapeutic and/or screening colonoscopies within each KV; the results of all microbiological surveillance tests of reprocessing quality (two per year per unit); the number of failed surveillance tests and re-tests; and the number of qualifications for refund from the public health system cancelled due to repeated failure of microbiological surveillance tests. RESULTS: The percentages of actually performed hygiene control tests (out of those prescribed by the assurance system) reached 95 % already in 2004 and remained above or close to this level thereafter. After the introduction of the quality assessment assurance, the percentage of failed microbiological surveillance tests dropped significantly and steadily from close to 17 % in 2003 to below 4 % in 2007. CONCLUSIONS: This study evidences 1. the successful implementation of the quality assessment assurance in Germany and 2. a substantial improvement in the quality of reprocessing flexible endoscopes achieved by these measures with a drop from 50 % of failed tests observed before the introduction in 2000 - 2001 to below 4 % in 2007.

Summary Background. Pemphigus herpetiformis (PH) is a rare dapsone-responsive variant of pemphigus, characterized by annular and vesiculopustular cutaneous lesions. Most PH serum samples contain autoantibodies against desmoglein (Dsg)1, but not Dsg3, and the presence of the latter is almost invariably associated with mucosal involvement, as predicted based on the 'Dsg compensation theory'. Methods. We describe a patient with features characteristic of PH with histologically eosinophilic spongiosis who repeatedly tested positive for anti-Dsg3 but not anti-Dsg1 autoantibodies by ELISA. To investigate whether the peculiar clinical phenotype was due to a distinct immunological profile, the patient's serum was tested by ELISA and immunoblotting using recombinant forms of Dsg3. Results. Serum samples were found to have low and high reactivity against the EC1 and the EC4 domains of Dsg3, respectively, whereas the autoantibodies belonged predominantly to the IgG1 and IgG4 subclasses. The overall immunological profile was typical of pemphigus vulgaris. The patient finally developed isolated oral erosions 22 months after initial presentation, without significant changes in the autoantibody profile and of the targeted antigenic sites. Conclusions. Our patient presented features characteristic of PH. Although circulating anti-Dsg3 antibodies were present, the patient had only cutaneous involvement for a long period. Our findings indicate that the proposed Dsg compensation theory cannot always explain the clinical phenotype, changes in which may occur without apparent modification of the autoantibody profile and antibody specificity. Hence, additional factors, such as Fcgamma-dependent neutrophil activation, may critically affect the clinical presentation of pemphigus.

OBJECTIVE: To evaluate the utility of the duration of morning stiffness (MS), as a patient-reported outcome (PRO), in assessing rheumatoid arthritis (RA) disease activity. METHODS: We acquired information on 5439 patients in QUEST-RA, an international database of patients with RA evaluated by a standard protocol. MS duration was assessed from time of waking to time of maximal improvement. Ability of MS duration to differentiate RA activity states, based on Disease Activity Score (DAS)28, was assessed by analysis of variance; and a receiver-operating characteristic (ROC) curve was plotted for discriminating clinically active (DAS28 > 3.2) from less active (DAS28 </= 3.2) RA. Mixed-effect analysis of covariance (ANCOVA) models were used to assess the utility of adding MS duration to Routine Assessment of Patient Index Data (RAPID) 3, a PRO index based on physical function, pain, and general health (GH), in predicting the 3-variable DAS28 (DAS28v3). RESULTS: MS duration had moderate correlation (r = 0.41-0.48) with pain, Health Assessment Questionnaire, and GH; and weak correlation (r = 0.23-0.39) with joint counts and erythrocyte sedimentation rate. MS duration differed significantly among patients with different RA activity (p < 0.001). The area under the ROC curve of 0.74 (95% CI 0.72-0.75) showed moderate ability of MS duration to differentiate clinically active from less active RA. ANCOVA showed significant interactive effects between RAPID3 and the MS duration categories (p = 0.0005) in predicting DAS28v3. The effect of MS was found to be clinically important in patients with the low RAPID3 scores (< 6) in whom the presence of MS may indicate clinically active disease (DAS28v3 > 3.2). CONCLUSION: MS duration has a moderate correlation with RA disease activity. Assessment of MS duration may be clinically helpful in patients with low RAPID3 scores.

Abstract Support for the theory of ecological speciation requires evidence for ecological divergence between species which directly or indirectly causes reproductive isolation. This study investigates effects of ecological vs. genetic disparity of parental species on the presence of endogenous selection (deformation and mortality rates) and potential sources of exogenous selection (growth rates and hatch timing) on hybrids. Hybrid embryonic development is analysed in a common-garden full-sib cross of three species belonging to two different ecotypes within the Coregonus lavaretus species flock in the central Alpine region of Europe. Although hatch timing was similar across the three species, embryonic growth rates and egg sizes differed between ecotypes. This led to a mismatch between embryonic growth rate and egg size in hybrid crosses that reveals epistasis between the maternal and embryonic genomes and transgressive hatch times that were asynchronous with control crosses. A strong constraint of egg size to embryo size at late development was also evident. We argue that this demonstrates potential for coadaptation of a maternal trait (egg size) with offspring growth rate to be an important source of selection against hybridization between ecotypes with different egg sizes. Implications for the measurement and quantification of early life-history traits affected by this additive relationship, such as hatch day and larval size, are also discussed.