Phenotypes influencing low physical activity in maintenance dialysis.

OBJECTIVE: The "Pas à Pas" initiative aimed at evaluating the weekly physical activity (PA) and its determinants in a large cohort of dialysis patients. SETTING: Physical inactivity is a risk factor for mortality in maintenance dialysis patients and is still poorly documented in this population. DESIGN: A prospective national epidemiological study was performed. SUBJECTS: A total of 1,163 patients on maintenance dialysis (hemodialysis and peritoneal dialysis) were included. INTERVENTION AND MAIN OUTCOME MEASURE: PA was recorded during seven consecutive days using a pedometer to measure daily step numbers. RESULTS: Median age was 63 years (Q1 51-Q3 75). Sixty-three percent were sedentary (<5000 steps/day) with a median of 3,688 steps/day (1,866-6,271)]. PA level was similar between hemodialysis patients and those on peritoneal dialysis (3,693 steps [1,896-6,307] vs. 3,320 [1,478-5,926], P = .33). In hemodialysis patients, PA was lower on dialysis days compared with nondialysis days (2,912 [1,439-5,232] vs. 4,054 [2,136-7,108], respectively, P < .01). PA gradually decreased with age, 57% being sedentary between 50 and 65 years and 83% of patients after 80 years. Beyond this age effect, we identified, for the first time, specific phenotypes of patients with lower PA, such as inflammation, cardiovascular disease, protein energy wasting, obesity, and diabetes. By contrast, previous kidney transplantation and a higher muscle mass were associated with higher PA. CONCLUSIONS: Dialysis patients present a very low level of PA with high sedentary. Acting on patient's modifiable phenotypes may help to increase PA to improve morbidity, mortality, and quality of life.

The role of the Estrogen-Responsive B box protein in skin morphogenesis, wound repair and skin disease

SUMMARY: EBBP is a poorly characterized member of the RBCC/TRIM family (RING finger B-box coiled-coilltripartite motif). It is ubiquitously expressed, but particularly high levels are found in keratinocytes. There is evidence that EBBP is involved in inflammatory processes, since it can interact with pro-interleukin-1 ß (prolL-1 ß) in human macrophages and keratinocytes, and its downregulation results in reduced secretion of IL-1 ß. IL-1ß activation and secretion requires the proteolytic cleavage of prolL-1ß by caspase-1, which in turn is actìvated by a protein complex called the inflammasome. As it has been demonstrated that EBBP can bind two different proteins of the inflammasome (NALP-1 and caspase 1), we assumed that EBBP plays a role in the regulation of inflammation and that the inflammasome, which has as yet only been described in ínflammatory cells, may also exist in keratinocytes. Indeed, I could show in my thesis that the inflammasome components are expressed in human keratinócytes at the RNA and protein level and also in vivo in human epidermis. After irradiation with a physiological dose of UVB, keratinocytes activated prolL-1ß and secreted prolL-1 a, IL-1 ß, prolL-18 and inflammasome proteins, although all these proteins lack a classical signal peptide. The secretion was dependent on caspase-1 activity, but not on de novo protein synthesis. Knock-down of NALP1 and -3, caspase-1 and -5, EBBP and Asc strongly reduced the secretion of IL-1 ß, demonstrating that also in keratinocytes inflammasome proteins are directly involved in maturation of this cytokine. These results demonstrate for the first time the presence of an active inflammasome in non-professional immune cells. Moreover, they show that UV irradiation is a stimulus for inflammasome activation in keratinocytes. For the analysis of the ín vivo functions of EBBP, transgenic mice overexpressing EBBP in the epidermis were generated. To examine the influence of EBBP overexpression on inflammatory processes, we subjected the mice to different challenges, which induce inflammation. Wound-healing, UVB irradiation and delayed hypersensitivity were tested, but we did not observe any phenotype in the K14-EBBP mice. Besides, a conditional ebbp knockout mouse has been obtained, which will allow to determine the effects of EBBP gene deletion in different tissues and organs. RESUME: EBBP est un membre encore mal connu de la famille des RBCC/TRIM (RING finger B-box coiled-coil/tripartite motif). Il est exprimé de manière ubiquitaire, et en particulier dans les kératinocytes. EBBP étant capable d'interagir avec la prointerleukine-1 ß (prolL-1 ß) dans les macrophages et les kératinocytes humains et de réguler la sécrétion de l'IL-1 ß, il est très probable que cette protéine est impliquée dans l'inflammation. L'activation et la sécrétion de l'IL-1 ß requièrent le clivage protéolytique de son précurseur prolL-1ß par la caspase-1, qui est elle-même activée par un complexe protéique appelé l'inflammasome. Comme il a été démontré qu'EBBP peut lier deux protéines de l'inflammasome (NALP-1 et caspase-1), nous avons émis l'hypothèse qu'EBBP joue un rôle dans la régulation de l'inflammation et que l'inflammasome, jusqu'ici décrit exclusivement dans des cellules inflammatoires, existe dans les kératinocytes. En effet, j'ai pu montrer dans ma thèse que les composants de l'inflammasome sont exprimés dans les kératinocytes humains ainsi que in vivo dans l'épiderme humain. Après irradiation avec une dose, physiologique d'UVB, les kératinocytes activent la prolL-1 ß et sécrètent la prolL-1a, l'IL-1 ß, la prolL-18 et des protéines de l'inflammasome, bien que toutes ces protéines soient dépourvues de peptide signal. La sécrétion dépend de la caspase-1 mais pas de la synthèse protéique de novo. Le knock-down de NALP-1 et -3, des caspase-1 et -5, d'EBBP et d'Asc réduit de manière marquée la sécrétion d'IL-1 ß, démontrant que dans les kératinocytes également, les protéines de l'inflammasome sont impliquées directement dans la maturation de cette cytokine. Ces résultats démontrent pour la première fois la présence d'un inflammasome actif dans des cellules immunitaires non professionnelles. De plus, ils montrent que l'irradiation aux UV est un stimulus pour l'activation de l'inflammasome dans les kératinocytes. Pour l'analyse des fonctions d'EBBP in vivo, nous avons généré des souris transgéniques qui surexpriment EBBP dans l'épiderme. En vue d'examiner l'influence de la surexpression d'EBBP sur le processus inflammatoire, nous avons soumis ces souris à differents modèles d'inflammation. Nous avons testé cicatrisation, UVB et hypersensibilité retardée, mais n'avons pas observé de phénotype chez les souris transgéniques. En parallèle, nous avons également généré des souris knock-out pour ebbp qui devraient nous permettre de déterminer les effets de la suppression d'EBBP dans différents tissus et organes.

Pathological Reorganization of NMDA Receptors Subunits and Postsynaptic Protein PSD-95 Distribution in Alzheimer's Disease.

In Alzheimer's disease (AD), synaptic alterations play a major role and are often correlated with cognitive changes. In order to better understand synaptic modifications, we compared alterations in NMDA receptors and postsynaptic protein PSD-95 expression in the entorhinal cortex (EC) and frontal cortex (FC; area 9) of AD and control brains. We combined immunohistochemical and image analysis methods to quantify on consecutive sections the distribution of PSD-95 and NMDA receptors GluN1, GluN2A and GluN2B in EC and FC from 25 AD and control cases. The density of stained receptors was analyzed using multivariate statistical methods to assess the effect of neurodegeneration. In both regions, the number of neuronal profiles immunostained for GluN1 receptors subunit and PSD-95 protein was significantly increased in AD compared to controls (3-6 fold), while the number of neuronal profiles stained for GluN2A and GluN2B receptors subunits was on the contrary decreased (3-4 fold). The increase in marked neuronal profiles was more prominent in a cortical band corresponding to layers 3 to 5 with large pyramidal cells. Neurons positive for GluN1 or PSD-95 staining were often found in the same localization on consecutive sections and they were also reactive for the anti-tau antibody AD2, indicating a neurodegenerative process. Differences in the density of immunoreactive puncta representing neuropile were not statistically significant. Altogether these data indicate that GluN1 and PSD-95 accumulate in the neuronal perikarya, but this is not the case for GluN2A and GluN2B, while the neuropile compartment is less subject to modifications. Thus, important variations in the pattern of distribution of the NMDA receptors subunits and PSD-95 represent a marker in AD and by impairing the neuronal network, contribute to functional deterioration.

Prevention and treatment of protein energy wasting in chronic kidney disease patients: a consensus statement by the International Society of Renal Nutrition and Metabolism.

Protein energy wasting (PEW) is common in patients with chronic kidney disease (CKD) and is associated with adverse clinical outcomes, especially in individuals receiving maintenance dialysis therapy. A multitude of factors can affect the nutritional and metabolic status of CKD patients requiring a combination of therapeutic maneuvers to prevent or reverse protein and energy depletion. These include optimizing dietary nutrient intake, appropriate treatment of metabolic disturbances such as metabolic acidosis, systemic inflammation, and hormonal deficiencies, and prescribing optimized dialytic regimens. In patients where oral dietary intake from regular meals cannot maintain adequate nutritional status, nutritional supplementation, administered orally, enterally, or parenterally, is shown to be effective in replenishing protein and energy stores. In clinical practice, the advantages of oral nutritional supplements include proven efficacy, safety, and compliance. Anabolic strategies such as anabolic steroids, growth hormone, and exercise, in combination with nutritional supplementation or alone, have been shown to improve protein stores and represent potential additional approaches for the treatment of PEW. Appetite stimulants, anti-inflammatory interventions, and newer anabolic agents are emerging as novel therapies. While numerous epidemiological data suggest that an improvement in biomarkers of nutritional status is associated with improved survival, there are no large randomized clinical trials that have tested the effectiveness of nutritional interventions on mortality and morbidity.

Islet-brain1/C-Jun N-terminal kinase interacting protein-1 (IB1/JIP-1) promoter variant is associated with Alzheimer's disease.

Islet-brain1 (IB1) or c-Jun NH2 terminal kinase interacting protein-1 (JIP-1), the product of the MAPK8IP1 gene, functions as a neuronal scaffold protein to allow signalling specificity. IB1/JIP-1 interacts with many cellular components including the reelin receptor ApoER2, the low-density lipoprotein receptor-related protein (LRP), kinesin and the Alzheimer's amyloid precursor protein. Coexpression of IB1/JIP-1 with other components of the c-Jun NH2 terminal-kinase (JNK) pathway activates the JNK activity; conversely, selective disruption of IB1/JIP-1 in mice reduces the stress-induced apoptosis of neuronal cells. We therefore hypothesized that IB1/JIP-1 is a risk factor for Alzheimer's disease (AD). By immunocytochemistry, we first colocalized the presence of IB1/JIP-1 with JNK and phosphorylated tau in neurofibrillary tangles. We next identified a -499A>G polymorphism in the 5' regulatory region of the MAPK8IP1 gene. In two separate French populations the -499A>G polymorphism of MAPK8IP1 was not associated with an increased risk to AD. However, when stratified on the +766C>T polymorphism of exon 3 of the LRP gene, the IB1/JIP-1 polymorphism was strongly associated with AD in subjects bearing the CC genotype in the LRP gene. The functional consequences of the -499A>G polymorphism of MAPK8IP1 was investigated in vitro. In neuronal cells, the G allele increased transcriptional activity and was associated with an enhanced binding activity. Taken together, these data indicate that the increased transcriptional activity in the presence of the G allele of MAPK8IP1 is a risk factor to the onset of in patients bearing the CC genotype of the LRP gene.

Molecular chaperones and associated cellular clearance mechanisms against toxic protein conformers in Parkinson's disease.

Parkinson's disease (PD) is a slowly progressive neurodegenerative disorder marked by the loss of dopaminergic neurons (in particular in the substantia nigra) causing severe impairment of movement coordination and locomotion, associated with the accumulation of aggregated α-synuclein (α-Syn) into proteinaceous inclusions named Lewy bodies. Various early forms of misfolded α-Syn oligomers are cytotoxic. Their formation is favored by mutations and external factors, such as heavy metals, pesticides, trauma-related oxidative stress and heat shock. Here, we discuss the role of several complementing cellular defense mechanisms that may counteract PD pathogenesis, especially in youth, and whose effectiveness decreases with age. Particular emphasis is given to the 'holdase' and 'unfoldase' molecular chaperones that provide cells with potent means to neutralize and scavenge toxic protein conformers. Because chaperones can specifically recognize misfolded proteins, they are key specificity factors for other cellular defenses, such as proteolysis by the proteasome and autophagy. The efficiency of the cellular defenses decreases in stressed or aging neurons, leading to neuroinflammation, apoptosis and tissue loss. Thus, drugs that can upregulate the molecular chaperones, the ubiquitin-proteasome system and autophagy in brain tissues are promising avenues for therapies against PD and other mutation-, stress- or age-dependent protein-misfolding diseases.

Association of a common vitamin D-binding protein polymorphism with inflammatory bowel disease.

OBJECTIVE: Inflammatory bowel diseases (IBDs), Crohn's disease, and ulcerative colitis (UC), are multifactorial disorders, characterized by chronic inflammation of the intestine. A number of genetic components have been proposed to contribute to IBD pathogenesis. In this case-control study, we investigated the association between two common vitamin D-binding protein (DBP) genetic variants and IBD susceptibility. These two single nucleotide polymorphisms (SNPs) in exon 11 of the DBP gene, at codons 416 (GAT>GAG; Asp>Glu) and 420 (ACG>AAG; Thr>Lys), have been previously suggested to play roles in the etiology of other autoimmune diseases. METHODS: Using TaqMan SNP technology, we have genotyped 884 individuals (636 IBD cases and 248 non-IBD controls) for the two DBP variants. RESULTS: On statistical analysis, we observed that the DBP 420 variant Lys is less frequent in IBD cases than in non-IBD controls (allele frequencies, P=0.034; homozygous carrier genotype frequencies, P=0.006). This inverse association between the DBP 420 Lys and the disease remained significant, when non-IBD participants were compared with UC (homozygous carrier genotype frequencies, P=0.022) or Crohn's disease (homozygous carrier genotype frequencies, P=0.016) patients separately. Although the DBP position 416 alone was not found to be significantly associated with IBD, the haplotype DBP_2, consisting of 416 Asp and 420 Lys, was more frequent in the non-IBD population, particularly notably when compared with the UC group (Odds ratio, 4.390). CONCLUSION: Our study adds DBP to the list of potential genes that contribute to the complex genetic etiology of IBD, and further emphasizes the association between vitamin D homeostasis and intestinal inflammation.

Genetic Loci Associated with C-reactive Protein Levels and Risk of Coronary Heart Disease.

CONTEXT: Plasma levels of C-reactive protein (CRP) are independently associated with risk of coronary heart disease, but whether CRP is causally associated with coronary heart disease or merely a marker of underlying atherosclerosis is uncertain. OBJECTIVE: To investigate association of genetic loci with CRP levels and risk of coronary heart disease. DESIGN, SETTING, AND PARTICIPANTS: We first carried out a genome-wide association (n = 17,967) and replication study (n = 13,615) to identify genetic loci associated with plasma CRP concentrations. Data collection took place between 1989 and 2008 and genotyping between 2003 and 2008. We carried out a mendelian randomization study of the most closely associated single-nucleotide polymorphism (SNP) in the CRP locus and published data on other CRP variants involving a total of 28,112 cases and 100,823 controls, to investigate the association of CRP variants with coronary heart disease. We compared our finding with that predicted from meta-analysis of observational studies of CRP levels and risk of coronary heart disease. For the other loci associated with CRP levels, we selected the most closely associated SNP for testing against coronary heart disease among 14,365 cases and 32,069 controls. MAIN OUTCOME MEASURE: Risk of coronary heart disease. RESULTS: Polymorphisms in 5 genetic loci were strongly associated with CRP levels (% difference per minor allele): SNP rs6700896 in LEPR (-14.8%; 95% confidence interval [CI], -17.6% to -12.0%; P = 6.2 x 10(-22)), rs4537545 in IL6R (-11.5%; 95% CI, -14.4% to -8.5%; P = 1.3 x 10(-12)), rs7553007 in the CRP locus (-20.7%; 95% CI, -23.4% to -17.9%; P = 1.3 x 10(-38)), rs1183910 in HNF1A (-13.8%; 95% CI, -16.6% to -10.9%; P = 1.9 x 10(-18)), and rs4420638 in APOE-CI-CII (-21.8%; 95% CI, -25.3% to -18.1%; P = 8.1 x 10(-26)). Association of SNP rs7553007 in the CRP locus with coronary heart disease gave an odds ratio (OR) of 0.98 (95% CI, 0.94 to 1.01) per 20% lower CRP level. Our mendelian randomization study of variants in the CRP locus showed no association with coronary heart disease: OR, 1.00; 95% CI, 0.97 to 1.02; per 20% lower CRP level, compared with OR, 0.94; 95% CI, 0.94 to 0.95; predicted from meta-analysis of the observational studies of CRP levels and coronary heart disease (z score, -3.45; P < .001). SNPs rs6700896 in LEPR (OR, 1.06; 95% CI, 1.02 to 1.09; per minor allele), rs4537545 in IL6R (OR, 0.94; 95% CI, 0.91 to 0.97), and rs4420638 in the APOE-CI-CII cluster (OR, 1.16; 95% CI, 1.12 to 1.21) were all associated with risk of coronary heart disease. CONCLUSION: The lack of concordance between the effect on coronary heart disease risk of CRP genotypes and CRP levels argues against a causal association of CRP with coronary heart disease.

Analysis of glial acidic fibrillary protein in the human entorhinal cortex during aging and in Alzheimer's disease.

Glial fibrillary acidic protein, GFAP, is a major intermediate filament protein of glial cells and major cytoskeletal structure in astrocytes. The entorhinal cortex has a key role in memory function and is one of the first brain areas to reveal hallmark structures of Alzheimer's disease and therefore provides an ideal tissue to investigate incipient neurodegenerative changes. Here we have analyzed age- and disease-related occurrence and composition of GFAP in the human entorhinal cortex by using one- and two-dimensional electrophoresis, Western blots and immunocytochemistry combined with confocal microscopy. A novel monoclonal antibody, GF-02, was characterized that mainly reacted with intact GFAP molecules and indicated that more acidic and soluble GFAP forms were also more susceptible to degradation. GFAP and vimentin increased with aging and in Alzheimer's disease (AD). Two-dimensional electrophoresis and Western blots revealed a complex GFAP pattern, both in aging and AD with different modification and degradation forms. Immunohistochemistry indicated that reactive astrocytes mainly accumulated in relation to neurofibrillary tangles and senile plaques in deeper entorhinal cortex layers. GFAP may be used as an additional but not exclusive diagnostic tool in the evaluation of neurodegenerative diseases because its levels change with age and respond to senile plaque and tangle formation.

Loss of the thyroid hormone-binding protein Crym renders striatal neurons more vulnerable to mutant huntingtin in Huntington's disease.

The mechanisms underlying preferential atrophy of the striatum in Huntington's disease (HD) are unknown. One hypothesis is that a set of gene products preferentially expressed in the striatum could determine the particular vulnerability of this brain region to mutant huntingtin (mHtt). Here, we studied the striatal protein µ-crystallin (Crym). Crym is the NADPH-dependent p38 cytosolic T3-binding protein (p38CTBP), a key regulator of thyroid hormone (TH) T3 (3,5,3'-triiodo-l-thyronine) transportation. It has been also recently identified as the enzyme that reduces the sulfur-containing cyclic ketimines, which are potential neurotransmitters. Here, we confirm the preferential expression of the Crym protein in the rodent and macaque striatum. Crym expression was found to be higher in the macaque caudate than in the putamen. Expression of Crym was reduced in the BACHD and Knock-in 140CAG mouse models of HD before onset of striatal atrophy. We show that overexpression of Crym in striatal medium-size spiny neurons using a lentiviral-based strategy in mice is neuroprotective against the neurotoxicity of an N-terminal fragment of mHtt in vivo. Thus, reduction of Crym expression in HD could render striatal neurons more susceptible to mHtt suggesting that Crym may be a key determinant of the vulnerability of the striatum. In addition our work points to Crym as a potential molecular link between striatal degeneration and the THs deregulation reported in HD patients.

Protein-binding microarrays: probing disease markers at the interface of proteomics and genomics.

DNA-binding proteins mediate a variety of crucial molecular functions, such as transcriptional regulation and chromosome maintenance, replication and repair, which in turn control cell division and differentiation. The roles of these proteins in disease are currently being investigated using microarray-based approaches. However, these assays can be difficult to adapt to routine diagnosis of complex diseases such as cancer. Here, we review promising alternative approaches involving protein-binding microarrays (PBMs) that probe the interaction of proteins from crude cell or tissue extracts with large collections of synthetic or natural DNA sequences. Recent studies have demonstrated the use of these novel PBM approaches to provide rapid and unbiased characterization of DNA-binding proteins as molecular markers of disease, for example cancer progression or infectious diseases.

Protein level expression of Toll-like receptors 2, 4 and 9 in renal disease.

Background. Toll-like receptors (TLR) recognize a variety of ligands, including pathogen-associated molecular patterns and link innate and adaptive immunity. Individual receptors can be up-regulated during infection and inflammation. We examined the expression of selected TLRs at the protein level in various types of renal disease.Methods. Frozen sections of renal biopsies were stained with monoclonal antibodies to TLR-2, -4 and -9.Results. Up-regulation of the three TLRs studied was seen, although the extent was modest. TLR-2- and -4-positive cells belonged to the population of infiltrating inflammatory cells; only in the case of TLR-9 were intrinsic glomerular cells positive in polyoma virus infection and haemolytic uraemic syndrome (HUS).Conclusions. Evidence for the involvement of the three TLRs tested in a variety of human renal diseases was found. These findings add to our understanding of the role of the innate immune system in kidney disease.