Proteomic analysis of mononuclear cells of patients with minimal-change nephrotic syndrome of childhood.

Background/Aims. Recently, peripheral blood mononuclear cell transcriptome analysis has identified genes that are upregulated in relapsing minimal-change nephrotic syndrome (MCNS). In order to investigate protein expression in peripheral blood mononuclear cells (PBMC) from relapsing MCNS patients, we performed proteomic comparisons of PBMC from patients with MCNS in relapse and controls. METHODS: PBMC from a total of 20 patients were analysed. PBMC were taken from five patients with relapsing MCNS, four in remission, five patients with other glomerular diseases and six controls. Two dimensional electrophoresis was performed and proteome patterns were compared. RESULTS: Automatic heuristic clustering analysis allowed us to pool correctly the gels from the MCNS patients in the relapse and in the control groups. Using hierarchical population matching, nine spots were found to be increased in PBMC from MCNS patients in relapse. Four spots were identified by mass spectrometry. Three of the four proteins identified (L-plastin, alpha-tropomyosin and annexin III) were cytoskeletal-associated proteins. Using western blot and immunochemistry, L-plastin and alpha-tropomyosin 3 concentrations were found to be enhanced in PBMC from MCNS patients in relapse. Conclusions. These data indicate that a specific proteomic profile characterizes PBMC from MCNS patients in relapse. Proteins involved in PBMC cytoskeletal rearrangement are increased in relapsing MCNS. We hypothesize that T-cell cytoskeletal rearrangement may play a role in the pathogenesis of MCNS by altering the expression of cell surface receptors and by modifying the interaction of these cells with glomerular cells.

Italy's Path to Very Low Fertility : The Adequacy of Economic and Second Demographic Transition Theories

The deep drop of the fertility rate in Italy to among the lowest in the world challenges contemporary theories of childbearing and family building. Among high-income countries, Italy was presumed to have characteristics of family values and female labor force participation that would favor higher fertility than its European neighbors to the north. We test competing economic and cultural explanations, drawing on new nationally representative, longitudinal data to examine first union, first birth, and second birth. Our event history analysis finds some support for economic determinants of family formation and fertility, but the clear importance of regional differences and of secularization suggests that such an explanation is at best incomplete and that cultural and ideational factors must be considered.

Relative and absolute reliability of the clinical version of the Narrow Path Walking Test (NPWT) under single and dual task conditions.

Decline in gait stability has been associated with increased fall risk in older adults. Reliable and clinically feasible methods of gait instability assessment are needed. This study evaluated the relative and absolute reliability and concurrent validity of the testing procedure of the clinical version of the Narrow Path Walking Test (NPWT) under single task (ST) and dual task (DT) conditions. Thirty independent community-dwelling older adults (65-87 years) were tested twice. Participants were instructed to walk within the 6-m narrow path without stepping out. Trial time, number of steps, trial velocity, number of step errors, and number of cognitive task errors were determined. Intraclass correlation coefficients (ICCs) were calculated as indices of agreement, and a graphic approach called "mountain plot" was applied to help interpret the direction and magnitude of disagreements between testing procedures. Smallest detectable change and smallest real difference (SRD) were computed to determine clinically relevant improvement at group and individual levels, respectively. Concurrent validity was assessed using Performance Oriented Mobility Assessment Tool (POMA) and the Short Physical Performance Battery (SPPB). Test-retest agreement (ICC1,2) varied from 0.77 to 0.92 in ST and from 0.78 to 0.92 in DT conditions, with no apparent systematic differences between testing procedures demonstrated by the mountain plot graphs. Smallest detectable change and smallest real change were small for motor task performance and larger for cognitive errors. Significant correlations were observed for trial velocity and trial time with POMA and SPPB. The present results indicate that the NPWT testing procedure is highly reliable and reproducible.

Krypton laser photocoagulation induces retinal vascular remodeling rather than choroidal neovascularization.

The purpose of this study is to analyze the retina and choroid response following krypton laser photocoagulation. Ninety-two C57BL6/Sev129 and 32 C57BL/6J, 5-6-week-old mice received one single krypton (630 nm) laser lesion: 50 microm, 0.05 s, 400 mW. On the following day, every day thereafter for 1 week and every 2-3 days for the following 3 weeks, serial sections throughout the lesion were systematically collected and studied. Immunohistology using specific markers or antibodies for glial fibrillary acidic protein (GFAP) (astrocytes, glia and Muller's cells), von Willebrand (vW) (vascular endothelial cells), TUNEL (cells undergoing caspase dependent apoptosis), PCNA (proliferating cell nuclear antigen) p36, CD4 and F4/80 (infiltrating inflammatory and T cells), DAPI (cell nuclei) and routine histology were carried out. Laser confocal microscopy was also performed on flat mounts. Temporal and spatial observations of the created photocoagulation lesions demonstrate that, after a few hours, activated glial cells within the retinal path of the laser beam express GFAP. After 48 h, GFAP-positive staining was also detected within the choroid lesion center. "Movement" of this GFAP-positive expression towards the lasered choroid was preceded by a well-demarcated and localized apoptosis of the retina outer nuclear layer cells within the laser beam path. Later, death of retinal outer nuclear cells and layer thinning at this site was followed by evagination of the inner nuclear retinal layer. Funneling of the entire inner nuclear and the thinned outer nuclear layers into the choroid lesion center was accompanied by "dragging" of the retinal capillaries. Thus, from days 10 to 14 after krypton laser photocoagulation onward, well-formed blood capillaries (of retinal origin) were observed within the lesion. Only a few of the vW-positive capillary endothelial cells stained also for PCNA p36. In the choroid, dilatation of the vascular bed occurred at the vicinity of the photocoagulation site and around it. Confocal microscopy demonstrates that the vessels throughout the path lesion are located within the neuroretina while in the choroid (after separation of the neural retina) only GFAP-positive but no lectin-positive cells can be seen. The involvement of infiltrating inflammatory cells in these remodeling and healing processes remained minimal throughout the study period. During the 4 weeks following krypton laser photocoagulation in the mouse eye, processes of wound healing and remodeling appear to be driven by cells (and vessels) originating from the retina.

Minimal data requirements for a continuous monitoring of the quality of care using the DRG classification.

The DRG classification provides a useful tool for the evaluation of hospital care. Indicators such as readmissions and mortality rates adjusted for the hospital Casemix could be adopted in Switzerland at the price of minor additions to the hospital discharge record. The additional information required to build patients histories and to identify the deaths occurring after hospital discharge is detailed.

Heat perception and signalling in plants: a tortuous path to thermotolerance.

An accurate assessment of the rising ambient temperature by plant cells is crucial for the timely activation of various molecular defences before the appearance of heat damage. Recent findings have allowed a better understanding of the early cellular events that take place at the beginning of mild temperature rise, to timely express heat-shock proteins (HSPs), which will, in turn, confer thermotolerance to the plant. Here, we discuss the key components of the heat signalling pathway and suggest a model in which a primary sensory role is carried out by the plasma membrane and various secondary messengers, such as Ca(2+) ions, nitric oxide (NO) and hydrogen peroxide (H(2) O(2) ). We also describe the role of downstream components, such as calmodulins, mitogen-activated protein kinases and Hsp90, in the activation of heat-shock transcription factors (HSFs). The data gathered for land plants suggest that, following temperature elevation, the heat signal is probably transduced by several pathways that will, however, coalesce into the final activation of HSFs, the expression of HSPs and the onset of cellular thermotolerance.

Both the Fas ligand and inducible nitric oxide synthase are needed for control of parasite replication within lesions in mice infected with Leishmania major whereas the contribution of tumor necrosis factor is minimal.

Following infection with the protozoan parasite Leishmania major, C57BL/6 mice develop a small lesion that heals spontaneously. Resistance to infection is associated with the development of CD4(+) Th1 cells producing gamma interferon (IFN-gamma) and tumor necrosis factor (TNF), which synergize in activating macrophages to their microbicidal state. We show here that C57BL/6 mice lacking both TNF and Fas ligand (FasL) (gld TNF(-/-) mice) infected with L. major neither resolved their lesions nor controlled Leishmania replication despite the development of a strong Th1 response. Comparable inducible nitric oxide synthase (iNOS) activities were detected in lesions of TNF(-/-), gld TNF(-/-), and gld mice, but only gld and gld TNF(-/-) mice failed to control parasite replication. Parasite numbers were high in gld mice and even more elevated in gld TNF(-/-) mice, suggesting that, in addition to iNOS, the Fas/FasL pathway is required for successful control of parasite replication and that TNF contributes only a small part to this process. Furthermore, FasL was shown to synergize with IFN-gamma for the induction of leishmanicidal activity within macrophages infected with L. major in vitro. Interestingly, TNF(-/-) mice maintained large lesion size throughout infection, despite being able to largely control parasite numbers. Thus, IFN-gamma, FasL, and iNOS appear to be essential for the complete control of parasite replication, while the contribution of TNF is more important in controlling inflammation at the site of parasite inoculation.

Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria.

The minimal replicon of the Pseudomonas plasmid pVS1 was genetically defined and combined with the Escherichia coli p15A replicon, to provide a series of new, oligocopy cloning vectors (5.3 to 8.3 kb). Recombinant plasmids derived from these vectors were stable in growing and nongrowing cells of root-colonizing P. fluorescens strains incubated under different environmental conditions for more than 1 month.

Minimal analytical characterisation of engineered nanomaterials need for hazard assessment in biological matrices

The safe and responsible development of engineered nanomaterials (ENM), nanotechnology-based materials and products, together with the definition of regulatory measures and implementation of "nano"-legislation in Europe require a widely supported scientific basis and sufficient high quality data upon which to base decisions. At the very core of such a scientific basis is a general agreement on key issues related to risk assessment of ENMs which encompass the key parameters to characterise ENMs, appropriate methods of analysis and best approach to express the effect of ENMs in widely accepted dose response toxicity tests. The following major conclusions were drawn: Due to high batch variability of ENMs characteristics of commercially available and to a lesser degree laboratory made ENMs it is not possible to make general statements regarding the toxicity resulting from exposure to ENMs. 1) Concomitant with using the OECD priority list of ENMs, other criteria for selection of ENMs like relevance for mechanistic (scientific) studies or risk assessment-based studies, widespread availability (and thus high expected volumes of use) or consumer concern (route of consumer exposure depending on application) could be helpful. The OECD priority list is focussing on validity of OECD tests. Therefore source material will be first in scope for testing. However for risk assessment it is much more relevant to have toxicity data from material as present in products/matrices to which men and environment are be exposed. 2) For most, if not all characteristics of ENMs, standardized methods analytical methods, though not necessarily validated, are available. Generally these methods are only able to determine one single characteristic and some of them can be rather expensive. Practically, it is currently not feasible to fully characterise ENMs. Many techniques that are available to measure the same nanomaterial characteristic produce contrasting results (e.g. reported sizes of ENMs). It was recommended that at least two complementary techniques should be employed to determine a metric of ENMs. The first great challenge is to prioritise metrics which are relevant in the assessment of biological dose response relations and to develop analytical methods for characterising ENMs in biological matrices. It was generally agreed that one metric is not sufficient to describe fully ENMs. 3) Characterisation of ENMs in biological matrices starts with sample preparation. It was concluded that there currently is no standard approach/protocol for sample preparation to control agglomeration/aggregation and (re)dispersion. It was recommended harmonization should be initiated and that exchange of protocols should take place. The precise methods used to disperse ENMs should be specifically, yet succinctly described within the experimental section of a publication. 4) ENMs need to be characterised in the matrix as it is presented to the test system (in vitro/ in vivo). 5) Alternative approaches (e.g. biological or in silico systems) for the characterisation of ENMS are simply not possible with the current knowledge. Contributors: Iseult Lynch, Hans Marvin, Kenneth Dawson, Markus Berges, Diane Braguer, Hugh J. Byrne, Alan Casey, Gordon Chambers, Martin Clift, Giuliano Elia1, Teresa F. Fernandes, Lise Fjellsbø, Peter Hatto, Lucienne Juillerat, Christoph Klein, Wolfgang Kreyling, Carmen Nickel1, and Vicki Stone.

Minimal clinically meaningful differences for the EORTC QLQ-C30 and EORTC QLQ-BN20 scales in brain cancer patients.

BACKGROUND: We aimed to determine the smallest changes in health-related quality of life (HRQoL) scores in the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire core 30 and the Brain Cancer Module (QLQ-BN20), which could be considered as clinically meaningful in brain cancer patients. Materials and methods: World Health Organisation performance status (PS) and mini-mental state examination (MMSE) were used as clinical anchors appropriate to related subscales to determine the minimal clinically important differences (MCIDs) in HRQoL change scores (range 0-100) in the QLQ-C30 and QLQ-BN20. A threshold of 0.2 standard deviation (SD) (small effect) was used to exclude anchor-based MCID estimates considered too small to inform interpretation. RESULTS: Based on PS, our findings support the following integer estimates of the MCID for improvement and deterioration, respectively: physical (6, 9), role (14, 12), and cognitive functioning (8, 8); global health status (7, 4*), fatigue (12, 9), and motor dysfunction (4*, 5). Anchoring with MMSE, cognitive functioning MCID estimates for improvement and deterioration were (11, 2*) and for communication deficit were (9, 7). Estimates with asterisks were <0.2 SD and were excluded from our MCID range of 5-14. CONCLUSION: These estimates can help clinicians evaluate changes in HRQoL over time, assess the value of a health care intervention and can be useful in determining sample sizes in designing future clinical trials.