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Data on the vitamin D status of the population of Wallonia (Belgium, 51°30' North) are scarce. This study was carried out to estimate vitamin D deficiency, identify potential determinants, and analyze their relationship with vitamin D supplementation. We tested the hypothesis that vitamin D deficiency is common in the general population, particularly among subjects without supplementation. Vitamin D deficiency was defined as a serum level of 25-hydroxyvitamin D (25(OH)D) concentration less than 50nmol/L. Data were analyzed from 915 participants of the Nutrition, Environment and Cardio-Vascular Health cross-sectional survey. The median (interquartile range) 25(OH)D level was 53.1 (37.8-69.9) nmol/L, and 44.7% of the subjects were vitamin D deficient. Subjects without vitamin D supplementation were more concerned by vitamin D deficiency than those with supplementation (odds ratio [OR], 3.35; P < .0001). From a multivariate standpoint, the potential determinants of vitamin D deficiency among subjects without vitamin D supplementation were season, specifically spring and winter (OR, 7.36 and 6.44, respectively), obesity (OR, 2.19), low household income (OR, 1.73), and lack of solarium use (OR, 1.79). For subjects with supplementation, the only determinant observed for vitamin D deficiency was obesity (OR, 5.00). This work evidenced the high prevalence of 25(OH)D deficiency in the general population, especially among nonsupplemented subjects with obesity, low household income, and/or lack of light. Vitamin D supplementation looks effective in our population, especially via a stabilization of vitamin D coverage throughout the seasons. The best dietary strategy to achieve optimal 25(OH)D concentrations all year round in the general population requires more research., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Osteoarthritis (OA) is associated with a local inflammatory process. Dyslipidemia is known to be an underlying cause for the development of OA. Therefore, lipid and inflammatory levels were quantified ex vivo in blood and synovial fluid of OA patients (n=29) and compared to those of rheumatoid arthritis (RA) patients (n=27) or healthy volunteers (HV) (n=35). The role of apolipoprotein A-I (ApoA1) was investigated in vitro on inflammatory parameters using human joint cells isolated from cartilage and synovial membrane obtained from OA patients after joint replacement. Cells were stimulated with ApoA1 in the presence or not of serum amyloid A (SAA) protein and/or lipoproteins (LDL and HDL) at physiological concentration observed in OA synovial fluid. In our ex vivo study, ApoA1, LDL-C and total cholesterol levels were strongly correlated to each other inside the OA joint cavity whereas same levels were not or weakly correlated to their corresponding serum levels. In OA synovial fluid, ApoA1 was not as strongly correlated to HDL as observed in OA serum or in RA synovial fluid, suggesting a dissociative level between ApoA1 and HDL in OA synovial fluid. In vitro, ApoA1 induced IL-6, MMP-1 and MMP-3 expression by primary chondrocytes and fibroblast-like synoviocytes through TLR4 receptor. HDL and LDL attenuated joint inflammatory response induced by ApoA1 and SAA in a ratio dependent manner. In conclusion, a dysregulated lipidic profile in the synovial fluid of OA patients was observed and was correlated with inflammatory parameters in the OA joint cavity. Pro-inflammatory properties of ApoA1 were confirmed in vitro.

Background. Monitoring the epidemiology of cardiovascular risk factors (CRFs) and their determinants is important to develop appropriate recommendations to prevent cardiovascular diseases in specific risk groups. The NESCaV study was designed to collect standardized data to estimate the prevalence of CRFs in relation to socioeconomic parameters among the general adult population in the province of Liège, Wallonia, Belgium. Methods. A representative stratified random sample of 1017 subjects, aged 20-69 years, participated in the NESCaV study (2010-2012). A self-administered questionnaire, a clinical examination, and laboratory tests were performed on participants. CRFs included hypertension, dyslipidemia, global obesity, abdominal obesity, diabetes, current smoking, and physical inactivity. Covariates were education and subjective and objective socioeconomic levels. Data were analyzed by weighted logistic regression. Results. The prevalence of hypertension, abdominal obesity, global obesity, current smoking, and physical inactivity was higher in subjects with low education and who considered themselves "financially in need." Living below poverty threshold also increased the risk of global and abdominal obesity, current smoking, and physical inactivity. Conclusion. The study shows that socioeconomic factors impact the prevalence of CRFs in the adult population of Wallonia. Current public health policies should be adjusted to reduce health inequalities in specific risk groups.

Angina is chest pain induced by ischemia of the heart muscle, generally due to obstruction or spasm of the coronary arteries. People that suffer from average to severe cases of angina have an increased percentage of death before the age of 55, usually around 60%. Therefore, prevention of major complications, optimizing diagnosis, prognosis and therapeutics are of primary importance. The main objective of this study was to uncover biomarkers by comparing serum protein profiles of patients suffering from stable or unstable angina and controls. We identified by non-targeted proteomic approach and confirmed by the means of independent techniques, the differential expression of several proteins indicating significantly increased vascular inflammation response, disturbance in the lipid metabolism and in atherogenic plaques stability.

The Elongator complex is composed of 6 subunits (Elp1-Elp6) and promotes RNAPII transcript elongation through histone acetylation in the nucleus as well as tRNA modification in the cytoplasm. This acetyltransferase complex directly or indirectly regulates numerous biological processes ranging from exocytosis and resistance to heat shock in yeast to cell migration and neuronal differentiation in higher eukaryotes. The identity of human ELP1 through ELP4 has been reported but human ELP5 and ELP6 have remained uncharacterized. Here, we report that DERP6 (ELP5) and C3ORF75 (ELP6) encode these subunits of human Elongator. We further investigated the importance and function of these two subunits by a combination of biochemical analysis and cellular assays. Our results show that DERP6/ELP5 is required for the integrity of Elongator and directly connects ELP3 to ELP4. Importantly, the migration and tumorigenicity of melanoma-derived cells are significantly decreased upon Elongator depletion through ELP1 or ELP3. Strikingly, DERP6/ELP5 and C3ORF75/ELP6-depleted melanoma cells have similar defects, further supporting the idea that DERP6/ELP5 and C3ORF75/ELP6 are essential for Elongator function. Together, our data identify DERP6/ELP5 and C3ORF75/ELP6 as key players for migration, invasion and tumorigenicity of melanoma cells, as integral subunits of Elongator.

There exists diseases in rheumatology fulfilling classification criteria for either rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). They are called "rhupus". We retrospectively analyzed the data base "GLIMS" of the CHU de Liège from the starting date of november 2005 until april 2011 to identified those patients that were positive for the anti-sDNA antibody marker of SLE and for the anti-CCP antibody, marker of RA. Fourteen patients were identified and two other patients were added, one suffering from SLE, and the other from RA, and likely to be rhupus. Of the 16 patients analyzed, 9 were real RA with anti-dsDNA antibodies induced by anti-TNF-alpha therapies. Seven were candidates to be rhupus and 6 were retained. They were all women, with a median age of 51 years and in addition were all anti-SS-A antibody positive.

Objectives: To test the usefulness of procalcitonin serum level for the reduction of antibiotic consumption in intensive care unit patients., Design: Single-center, prospective, randomized controlled study., Setting: Five intensive care units from a tertiary teaching hospital., Patients: All consecutive adult patients hospitalized for >48 hrs in the intensive care unit during a 9-month period., Interventions: Procalcitonin serum level was obtained for all consecutive patients suspected of developing infection either on admission or during intensive care unit stay. The use of antibiotics was more or less strongly discouraged or recommended according to the Muller classification. Patients were randomized into two groups: one using the procalcitonin results (procalcitonin group) and one being blinded to the procalcitonin results (control group). The primary end point was the reduction of antibiotic use expressed as a proportion of treatment days and of daily defined dose per 100 intensive care unit days using a procalcitonin-guided approach. Secondary end points included: a posteriori assessment of the accuracy of the infectious diagnosis when using procalcitonin in the intensive care unit and of the diagnostic concordance between the intensive care unit physician and the infectious-disease specialist., Measurements and Main Results: There were 258 patients in the procalcitonin group and 251 patients in the control group. A significantly higher amount of withheld treatment was observed in the procalcitonin group of patients classified by the intensive care unit clinicians as having possible infection. This, however, did not result in a reduction of antibiotic consumption. The treatment days represented 62.6±34.4% and 57.7±34.4% of the intensive care unit stays in the procalcitonin and control groups, respectively (p=.11). According to the infectious-disease specialist, 33.8% of the cases in which no infection was confirmed, had a procalcitonin value>1µg/L and 14.9% of the cases with confirmed infection had procalcitonin levels<0.25 µg/L. The ability of procalcitonin to differentiate between certain or probable infection and possible or no infection, upon initiation of antibiotic treatment was low, as confirmed by the receiving operating curve analysis (area under the curve=0.69). Finally, procalcitonin did not help improve concordance between the diagnostic confidence of the infectious-disease specialist and the ICU physician., Conclusions: Procalcitonin measuring for the initiation of antimicrobials did not appear to be helpful in a strategy aiming at decreasing the antibiotic consumption in intensive care unit patients.

Background: The recommended target range for serum parathyroid hormone (PTH) in dialysis patients has changed from 150 to 300 pg/mL in the KDOQI guidelines to two to nine times the upper normal limit in the KDIGO ones. Although inclusion/exclusion criteria for the reference population are highly important, they are usually not mentioned in the commercial kits. In this study, we used the same reference population of vitamin D-replete normal subjects to establish reference values for 10 commercial PTH kits. We evaluated whether this may improve the classification of dialysis patients according to the KDIGO compared to the use of reference values proposed by the manufacturers., Methods: We measured serum PTH with 10 different kits in 149 haemodialysis patients, and 240 25-OH-vitamin D-replete (>75 nmol/L) individuals with an estimated glomerular filtration rate >60 mL/min/1.73 m(2)., Results: For the 10 kits, our upper normal limit was lower than those of the manufacturers. The difference was, however, variable from one kit to another. The two kits that yielded the lowest and the highest absolute concentrations classified differently 84/149 patients (56.4%) according to the KDOQI and 53/149 (36.2%) according to the KDIGO using the manufacturers' normal values. Using our normal values significantly decreased the discrepancies with 24/149 patients (16.1%) being still classified differently. Taking the measurement uncertainty into consideration, 8% of the patients only remained differently classified by these two kits., Conclusions: Using the same vitamin-D-replete population to establish the reference range for 10 commercial PTH kits significantly improved the classification of haemodialysis patients according to the KDIGO target range.

Background: More than 90% of vitamin D synthesis is dependent on UV exposure. Photosensitive disorders such as lupus erythematosus, protoporphyria and xeroderma require strict sun avoidance, and vitamin D deficiency has been demonstrated in these patients. Melanoma patients are also instructed to avoid sun exposure and may hence be expected to be vitamin D deficient., Materials and Methods: Winter and summer vitamin D levels were compared in a group of melanoma patients (n =61) and age- and phototype-matched controls (n = 53) without photosensitive disorders., Results: Oral supplementary vitamin D intake was reported in 32.7% of the melanoma patients and in 15.1% in the control group. Despite oral supplementation, only 25% of the melanoma patients and the controls presented with vitamin D levels of 30 ng/mL or higher. In non-supplemented subjects in the melanoma and control groups, respectively, mean winter vitamin D levels were below the recommended threshold at 12.6 ng/mL vs. 13.2 ng/mL, respectively, but not statistically different. These values increased significantly in both groups during the summer to 24.6 and 23.8 ng/mL respectively., Conclusion: Unexpected, significant increases in vitamin D levels were seen in melanoma patients during summer, suggesting non-adherence with photoprotective measures and reflecting a heliophilic behaviour. Vitamin D supplementation is recommended in melanoma patients during both winter and summer., (© 2011 The Authors. Journal of the European Academy of Dermatology and Venereology © 2011 European Academy of Dermatology and Venereology.)

Objectives: Validation of the Architect 25-OH vitamin D assay., Design and Methods: Determination of repeatability, reproducibility, accuracy profile and 25(OH)-vitamin D2 recovery on native samples. Comparison with DiaSorin Liaison and RIA., Results and Conclusion: Coefficients of variation: <6% (13.6 ng/mL) and 2.2% (78.1 ng/mL). Functional sensitivity: 5 ng/mL. Accuracy profile shows that the method is validated between 13.6 and 78.1 ng/mL. Recovery of 25(OH)D2: 75,8%( 95% CI: 61.9-89.7%). Good correlation with DiaSorin RIA and Liaison <50 ng/mL; above this threshold a systematic positive bias was observed., (Copyright © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: In the second generation of the point-of-care (POC) assay Roche CARDIAC proBNP, the upper limit of the measuring range was extended from 3000 to 9000 ng/L., Methods: A thirteen-site multicentre evaluation was carried out to assess the analytical performance of the POC NT-proBNP assay and to compare it with a laboratory N-terminal pro-brain natriuretic peptide (NT-proBNP) assay., Results: In method comparisons of six lots of POC NT-proBNP with the lab reference method (Elecsys proBNP) mean bias ranged from -10 to +17%. In lot-to-lot comparisons all six investigated lots of POC NT-proBNP showed excellent agreement, with mean bias between -7% and +2%. The majority of all coefficients of variation obtained from ten-fold measurements using 56 native blood samples were below 8%. No interference was observed with hemolytic blood (hemoglobin concentrations up to 0.12 mmol/L), lipemic blood (triglyceride concentrations up to 14.0 mmol/L) nor icteric blood (bilirubin concentrations up to 63 micromol/L). Hematocrit values between 24% and 51% had no influence on the assay result. High NT-proBNP concentrations above the measuring range of POC NT-proBNP did not lead to false low results due to potential high-dose hook effect. Results with POC NT-proBNP were not influenced by different ambient temperatures (18 degrees C to 32 degrees C), the sample material used, nor by over- or underdosing by 15 microL compared to the regular sample volume of 150 microL., Conclusions: The POC NT-proBNP assay showed an excellent analytical performance including a good agreement with the laboratory method. The assay is therefore suitable for its intended use in point-of-care settings.

Natriuretic peptides, particularly BNP and NT-proBNP, are increasingly used as screening test in patients with symptoms suggestive of heart failure (HF). Due to their high negative predictive values, natriuretic peptide determinations allow to exclude chronic HF with great certainty and to identify patients for whom echography is not necessary. These biomarkers are also useful for diagnostic purposes, high plasma levels being related to an increased risk of cardiovascular hospitalisation and death. Risk stratification in patients with HF symptoms is based on "low" and "high" cut-off limits, for which different values have been proposed. The aim of this paper is to discuss the delineation of the decision limits and the intermediate grey zone in comparison to NT-proBNP reference values obtained in a representative group of subjects living in the Liège area (Belgium). Data were analysed in relation to age and gender, two of the main parameters influencing the natriuretic peptide plasma levels.

Background: The ImmunoCAP© ISAC allows for the determination of specific immunoglobulin E (IgE) against 103 recombinant or purified allergen components in a single analytical step. The aim of our study was to perform a comparison of the specific IgE results measured with the microarray method to those obtained using the traditional method of ImmunoCAP©., Methods: We selected 86 clinically relevant patients on the basis of their specific IgE for recombinant allergens (ImmunoCAP© 250, Phadia). Also, we selected two patients with a high total IgE to evaluate the non-specific binding of IgE. All samples were screened with the ImmunoCAP© ISAC. Then, we compared the 555 specific IgE antibodies results provided by ImmunoCAP© ISAC with the specific IgE levels obtained with ImmunoCAP© 250., Results: We observed that 82 of the 384 results found to be positive with ImmunoCAP© were negative with ISAC© (concordance 78.65%). Of 171 negative results obtained with ImmunoCAP©, 11 were positive with ISAC© (concordance 93.57%). No non-specific binding was observed., Conclusions: Our results show that the ImmunoCAP© ISAC has good analytical performance when compared with the ImmunoCAP© 250 method. We did not observe any non-specific binding. However, better sensitivity for some clinically relevant allergen components, such as rPru p 3 is needed.

Background: We validated the DiaSorin Liaison Calcitonin&#95;II-Gen, an improved method for calcitonin (CT) measurements, compared this method with the Cisbio&#95;h-CT kit and established the reference range of CT in a normal adult population., Methods: We determined the precision, functional sensitivity, traceability to the 2nd IS 89/620, linearity and measurement uncertainty, accuracy profile and β-expectation limits. We evaluated the specificity, the susceptibility to human anti-animal antibodies (HAMA), hook-effect and carry over. To establish a reference range, we selected 267 adults without renal insufficiency presenting with normal thyroid stimulating hormone (TSH), free thyroxin (T4) and calcium concentrations and without anti-thyroglobulin antibodies as our "reference" healthy population. We compared the method with Cisbio on 250 consecutive and 45 samples from a post-pentagastrin stimulation test., Results: Precision (expressed as CV) was < 10% for the measurement range, functional sensitivity: 5.3 ng/L and the method was found linear until to a 1/10 dilution. Uncertainty ranged from 25% to 7.2%, and the risk that one result falls out of the ± 20% acceptance limits was < 5% between 2.9 and 1513 ng/L. The Bland and Altman plot showed no systematic bias between the two methods. The test is still prone to HAMA influence, does not present any hook-effect, although carry over was observed. Ninety-five percent of our adult reference population showed CT concentrations < 7.4 ng/L, with an important gender difference: 95% of the men showed CT values < 9.8 ng/L, whereas 95% of women were < 4.0 ng/L., Conclusions: The Liaison Calcitonin&#95;II-Gen is an analytically robust method. The important difference in gender observed in our population might lead to re-evaluation of the generally used "10 ng/L" cut-off in a multicentre prospective study.

Several factors, including fruit and vegetables intakes, have been shown to significantly influence the plasma concentrations of the two antioxidants vitamin C and β-carotene. Deficiency levels of 6 mg/L (34.2 μM) for vitamin C and of 0.22 mg/L (0.4 μM) for β-carotene have been suggested below which cardiovascular risk might be increased. The present study performed on 897 presumably healthy subjects aged 40-60 years aimed to examine how modifiable lifestyle factors may be related to vitamin C and/or β-carotene deficiency. Gender, smoking, lack of regular physical activity and of daily fruit consumption (≥2/day), and social status (in particular, unemployment) were found to be significant risk factors for vitamin C deficiency. For β-carotene deficiency, the same factors were identified except social status; moreover, overweight and OC use in women were also found to have a deleterious effect. For non exposed subjects, the probability of developing vitamin C deficiency was 4% in men and 2.4% in women. This probability increased to 66.3% for men and to 44.3% for women (and even to 50.4% under OC use), when all risk factors were present. For β-carotene deficiency, the corresponding probabilities were equal to 29.7% in men and 13.7% in women (no risk factor present), and to 86.1% for men and 69.9% (91.6% for OC use) for women (all factors present), respectively.

Objectives: To evaluate total salt intake in the adult population through an analysis of sodium in 24-h urine samples in two regions of Belgium., Methods: Urine samples were collected over 24 h from participants and they had to complete a specific questionnaire about salt intake afterwards. Sodium and creatinine concentrations were analysed in these samples., Subjects: The target population comprised adults aged 45-65 years in the region of Ghent and Liege. A total of 123 and 157 volunteers from Ghent and Liege, respectively, were included in the study., Results: The mean creatinine level in Flanders (n=114) amounted to 0.173±0.035 mmol/kg/day, whereas in the Walloon region (n=135) it amounted to 0.161±0.036 mmol/kg/day, after the exclusion of subjects with incomplete urine collection. Intake of sodium in Flanders (n=114) was 4.29±1.29 g/day, whereas in the Walloon region (n=135) it was 3.94±1.44 g/day. In both regions, sodium intake in men was higher than in women., Conclusion: Salt intake was more or less twice as high as the recommended intake. Salt intake as estimated from 24-h urine collections is substantially higher than that previously calculated on the basis of food consumption data. A salt reduction programme for Belgium is primordial.

The oncogenic protein BCL-3 activates or represses gene transcription through binding with the NF-kappaB proteins p50 and p52 and is degraded through a phospho- and GSK3-dependent pathway. However, the mechanisms underlying its degradation remain poorly understood. Yeast two-hybrid analysis led to the identification of the proteasome subunit PSMB1 as a BCL-3-associated protein. The binding of BCL-3 to PSMB1 is required for its degradation through the proteasome. Indeed, PSMB1-depleted cells are defective in degrading polyubiquitinated BCL-3. The N-terminal part of BCL-3 includes lysines 13 and 26 required for the Lys(48)-linked polyubiquitination of BCL-3. Moreover, the E3 ligase FBW7, known to polyubiquitinate a variety of substrates phosphorylated by GSK3, is dispensable for BCL-3 degradation. Thus, our data defined a unique motif of BCL-3 that is needed for its recruitment to the proteasome and identified PSMB1 as a key protein required for the proteasome-mediated degradation of a nuclear and oncogenic IkappaB protein.

The nuclear and oncogenic BCL-3 protein activates or represses gene transcription when bound to NF-kappaB proteins p50 and p52, yet the molecules that specifically interact with BCL-3 and drive BCL-3-mediated effects on gene expression remain largely uncharacterized. Moreover, GSK3-mediated phosphorylation of BCL-3 triggers its degradation through the proteasome, but the proteins involved in this degradative pathway are poorly characterized. Biochemical purification of interacting partners of BCL-3 led to the identification of CtBP as a molecule required for the ability of BCL-3 to repress gene transcription. CtBP is also required for the oncogenic potential of BCL-3 and for its ability to inhibit UV-mediated cell apoptosis in keratinocytes. We also defined the E3 ligase TBLR1 as a protein involved in BCL-3 degradation through a GSK3-independent pathway. Thus, our data demonstrate that the LSD1/CtBP complex is required for the repressing abilities of an oncogenic I kappaB protein, and they establish a functional link between the E3 ligase TBLR1 and NF-kappaB.

Background: Parathyroid carcinoma (PCa) is a rare disease that can be difficult to differentiate initially from severe benign parathyroid adenoma. PCa oversecrete the amino form of PTH, which is recognized by third-generation but not by second-generation PTH immunoassays. In normal individuals, the third-generation to second-generation PTH ratio should be less than 1., Objective: Our objective was to study the utility of the third-generation to second-generation PTH ratio as a means of distinguishing PCa patients (n=24) from control groups with and without disorders of calcium secretion, including patients on renal hemodialysis (n=74), postrenal transplantation (n=60), and primary hyperparathyroidism (PHP; n=30)., Setting and Design: We conducted a retrospective, laboratory-based study at tertiary referral academic centers., Results: The mean third-generation to second-generation ratio was 0.58+/-0.10 in the dialysis patients, 0.54+/-0.10 in the renal transplant group, 0.54+/-0.12 in the elderly healthy patients, and 0.68+/-0.11 in the PHP group. All 245 of these patients presented a PTH third-generation to second-generation ratio of less than 1. In contrast, we observed an inverted third-generation to second-generation PTH ratio of more than one in 20 PCa patients, whereas only four PCa patients had a normal ratio of less than 1., Conclusions: An inverted third-generation to second-generation PTH ratio occurred in the majority of patients with advanced PCa and was absent in all 245 relevant controls. A third-generation to second-generation PTH ratio higher than 1 had a sensitivity of 83.3% and a specificity of 100% among PHP patients as a marker for PCa. This ratio may be useful to identify patients with PCa earlier and to detect patients either at risk of developing PCa or those in whom recurrence is taking place.

In most diseases, the clinical need for serum/plasma markers has never been so crucial, not only for diagnosis, but also for the selection of the most efficient therapies, as well as exclusion of ineffective or toxic treatment. Due to the high sample complexity, prefractionation is essential for exploring the deep proteome and finding specific markers. In this study, three different sample preparation methods (i.e., highly abundant protein precipitation, restricted access materials (RAM) combined with IMAC chromatography and peptide ligand affinity beads) were investigated in order to select the best fractionation step for further differential proteomic experiments focusing on the LMW proteome (MW inferior to 40,000 Da). Indeed, the aim was not to cover the entire plasma/serum proteome, but to enrich potentially interesting tissue leakage proteins. These three methods were evaluated on their reproducibility, on the SELDI-TOF-MS peptide/protein peaks generated after fractionation and on the information supplied. The studied methods appeared to give complementary information and presented good reproducibility (below 20%). Peptide ligand affinity beads were found to provide efficient depletion of HMW proteins and peak enrichment in protein/peptide profiles., (Copyright 2010 Elsevier B.V. All rights reserved.)

Summary: Due to "measurement uncertainty", the "true" 25-OH vitamin D (25(OH)D) of a patient (whatever the commercially available assay tested) will be >80 nmol/L if its measured concentration is >100 nmol/L. Thus, if a physician considers that a normal VTD status is a 25(OH)D level >or=80 nmol/L, he should ensure that the patient's results are >or=100 nmol/L., Introduction: Many experts recommend that serum levels of 25(OH)D should be above a lower normal limit of 75-80 nmol/L. However, the value delivered by laboratories is only an estimation of the "true" value due to "measurement uncertainty." When using a cut off, measurement uncertainty around the cut off is important because therapeutic actions may differ if the measured value is below or above the limit. We aimed to establish the "measurement uncertainty" at different levels of concentration for several commercially available 25(OH)D analytical techniques., Methods: We constituted three pools of serum with different 25(OH)D concentrations. Each pool was assayed in triplicate during 5 days with the DiaSorin RIA, Liaison, Elecsys, and Chromsystems-HPLC assays., Results: We report a relatively high "measurement uncertainty" for the measurement of 25(OH)D for the four different techniques: the mean relative uncertainties, all techniques confounded were 19.4%, 16.0%, and 11.3% for pool 1 (35.3 nmol/L), pool 2 (79.5 nmol/L), and pool 3 (126.1 nmol/L), respectively., Conclusions: Our results show that, whatever the assay, the "true" 25(OH)D of a patient will be >80 nmol/L if its measured concentration is >100 nmol/L. In other words, if a physician considers that a normal VTD status is defined by a 25(OH)D level >or=80 nmol/L, he should ensure that the patients present a 25(OH)D >or=100 nmol/L.

Lysine acetylation is a post-translational modification that critically regulates gene transcription by targeting histones as well as a variety of transcription factors in the nucleus. More recent reports have also demonstrated that numerous proteins located outside the nucleus are also acetylated and that this modification has profound consequences on their functions. This review describes the latest findings on the substrates acetylated outside the nucleus and on the acetylases and deacetylates that catalyse these modifications. Protein acetylation is emerging as a major mechanism by which key proteins are regulated in many physiological processes such as migration, metabolism and aging as well as in pathological circumstances such as cancer and neurodegenerative disorders.

Background: We report a Dutch-Belgian multicentre evaluation of the Tosoh HLC-723G8 glycohaemoglobin analyser, an ion-exchange HPLC instrument for the separation and quantification of haemoglobin A1c (HbA1c) in whole blood., Methods: We evaluated the analytical performances of the Tosoh G8 analyser and compared the results for blood samples with its predecessor, the Tosoh G7, and with two other widely used analysers, the Bio-Rad Variant II and Adams Arkray HA-8160., Results: Within- and between-batch imprecision [coefficient of variation (CVs)] was <0.5% and 2%, respectively, and compared favourably with the G7. The excellent performances in terms of speed (1.6 min/analysis) did not result in increased variability of the results or carry-over between samples. The method shows no interference from carbamylated haemoglobin, and recognises the presence of haemoglobinopathies, which triggers the correction of the HbA1c result. Comparison with established methods showed good correlation, not only with the G7 but also with the Variant II and HA-8160 systems., Conclusions: With respect to reproducibility, chromatographic resolution, speed of analysis and identification of Hb variants, the Tosoh G8 analyser can be considered to be state of the art.

Recently, anti-deamidated gliadin antibodies were proposed for the serological diagnosis of celiac disease. We evaluate the specificity of different anti-deamidated gliadin antibodies ELISA in comparison with conventional anti-native gliadin kits. Serum samples from 46 non celiac patients were analyzed by five different quantitative ELISA for anti-native gliadin, anti-deamidated gliadin and anti-transglutaminase neo-epitope antibodies together with a screening ELISA. Twenty-four percent of the patients demonstrated anti-native gliadin IgA and 63% IgG antibodies. Using anti-deamidated gliadin antibodies, the number of false positive IgA and, particularly, IgG results, markedly decreased in the non celiac patients: 21 and 24% respectively with anti-Gliadin (GAF-3X) Euroimmun kit, 7 and 26% with Bindazyme Human Anti-Gliadin (MGP) The Binding Site kit and 0 and 41% with Celiac G+ Immco kit. The new assay which makes use of the physiological complex of tissue transglutaminase cross-linked with deamidated gliadin peptides, called neo-epitope, did not improve the differential diagnosis of celiac disease with 30% of false positive results in IgG (2% in IgA). Using the Inova screening kit, a positive result for IgA and/or IgG anti-deamidated gliadin and/or anti-tissue transglutaminase antibodies was obtained in 24% of the non celiac patients. In conclusion, our study assessed the superiority, in terms of specificity, of anti-deamidated gliadin antibodies, over the conventional anti-gliadin antibodies for the differential diagnosis of celiac disease.

Background: The goal of this study was to validate the DiaSorin Liaison BAP OSTASE, a new method for measurement of bone alkaline phosphatase (BAP), and to compare this method with the Beckman-Coulter Access Ostase. We also wanted to establish the reference range for BAP in adults and children., Methods: We determined the precision, functional sensitivity, recovery, linearity and measurement uncertainty, accuracy profile and beta-expectation limits. We defined an adult reference interval using individuals with 25-OH vitamin D >80 nmol/L, parathormone <58 ng/L, and normal calcium, phosphorous and estimated glomerular filtration rate. Each adult subclass (men/non-menopausal women/menopause women) contained 120 individuals. We also determined the 2.5th and 97.5th percentiles from a population of 450 children, stratified according to age and gender., Results: The results of the validation showed: precision <6%, functional sensitivity <0.74 microg/L, mean recovery 98.8+/-4.2% and good linearity. Relative uncertainty ranged from 9.0% to 12.9%, and the risk of one result falling out of the +/-15% acceptance limits was <5% for concentrations between 7 and 94 microg/L. The Bland-Altman plot showed no systematic bias between the two methods. In adults, we did not find any statistical difference between the different subclasses. The upper limit of normality observed in the entire population (n=360) was 21.3 microg/L (90% CI: 18.3-24.2 microg/L)., Conclusions: The Liaison BAP OSTASE is a robust method, and is completely validated between 7 and 93 microg/L: in this range, 95% of the values obtained will be within +/-15% of the true value.

Protein profiling using SELDI-TOF-MS has gained over the past few years an increasing interest in the field of biomarker discovery. The technology presents great potential if some parameters, such as sample handling, SELDI settings, and data analysis, are strictly controlled. Practical considerations to set up a robust and sensitive strategy for biomarker discovery are presented. This paper also reviews biological fluids generally available including a description of their peculiar properties and the preanalytical challenges inherent to sample collection and storage. Finally, some new insights for biomarker identification and validation challenges are provided.

Background: The cobas h 232 point-of-care analyzer by Roche is the instrument successor of the Cardiac reader allowing the quantitative determination of troponin T, creatine kinase MB, myoglobin, NT-proBNP and D-dimer., Methods: In this study 1329 patients with acute coronary syndromes, heart failure, thromboembolic or other diseases and 945 healthy donors were assessed. Comparisons versus central laboratory methods were carried out with 2379 samples from these individuals; out of these, 1591 samples gave quantitative results within the measuring range and were included in the evaluation., Results: The point-of-care assays for creatine kinase MB, myoglobin, NT-proBNP and D-dimer were within a relative bias range of -5.9 to +6.9% compared to the laboratory assay. The troponin T assay showed a bias of -11.0% and after change of the calibration procedure of +1.9%. None of the five point-of-care assays had a relative difference between the new system and the precursor device that was higher than +5.0%. Within-series coefficients of variation of patient samples were found in a range from 4.8 to 14.8%. No significant interference was observed with lipemic, hemolytic and icteric blood or at different hematocrit values., Conclusions: Due to its good analytical agreement with the laboratory methods and with its precursor device, the cobas h 232 system can be reliably used to support on-site decision making for cardiovascular patients in acute and non-acute settings.

Background and Objectives: Stability of parathyroid hormone (PTH) at -80 degrees C for long storage periods has never been studied. This can be of importance for the conclusions of studies where blood banks have been constituted. The study's aim was to evaluate stability of PTH when stored as serum or plasma EDTA samples at -80 degrees C., Design, Setting, Participants & Measurements: Samples were collected from 16 chronic hemodialysis patients using EDTA and gel-separator tubes. Plasma and serum were aliquoted; one aliquot was assayed with Elecsys and Liaison methods to determine the "baseline" values and another aliquot after 1, 3, 6, and 12 mo. The factors "method," "tubes," "subjects," and "time" were included in a mixed linear model to evaluate their effects on measured PTH values. The prediction interval methodology was used to assess where a future result could be obtained with a defined probability., Results: With the Liaison method, the maximum storage times with either dry or EDTA tubes were estimated to be 9 and 2 mo, respectively. With the Elecsys method, samples could be stored at least 2 yr with acceptable level of degradation., Conclusion: PTH stability at -80 degrees C is not infinite. Maximum storage time and acceptance limits (30%) were defined, showing that with one method, samples should be stored for not more than 2 mo, whereas the other could be stored for up to 2 yr. With any PTH assay, the maximum storage time should be evaluated to ascertain that samples will keep their initial reactive profile after prolonged storage periods.

The consequences of Hymenoptera venom anaphylaxis are very severe but it is not obvious to predict which reactions will occur in one single patient when he is stung for the second time. For a couple of years, many new laboratory tests have been experimented and many studies published. CAST, BAT, WB, tryptase, sIgE and sIgG4 are the new valuable additional diagnostic tools that can help the decision to perform an immunotherapy or to discontinue this therapy after 3 years. The aim of our study was to determine which could be the profile of a desensitized patient and to screen for good candidates for venom immunotherapy.

The 2007 international consensus about the standardization of HbA(1c) determination and expression of results is progressively implemented in most countries. In France, a common working group of the Société française de biologie clinique (SFBC) and the Société francophone de diabétologie (SFD) has expressed the following recommendations. HbA(1c) results are expressed in percentage of total hemoglobin and in mmol HbA(1c)/mol Hb, but are not converted into estimated average glucose. A table indicating the correspondence between HbA(1c) and estimated average glucose may be given with the results, subject to precautions of interpretation at the individual level.

Ceramides are central molecules in sphingolipid metabolism. They are involved in the regulation of cancer-cell growth, differentiation, senescence and apoptosis. To better understand how these secondary messengers induce their biological effects, adenocarcinoma cells (HCT116) were treated with exogenous long-chain ceramides (C16-ceramide) in order to mimic endogenous sphingolipids. This treatment induced a decrease of cell viability partly due to apoptosis as shown by PARP cleavage and a decrease of pro-caspase 3. Two-dimensional differential in-gel electrophoresis (2D-DIGE) revealed the differential expression of 51 proteins in response to C16-ceramide. These proteins are notably involved in cell proliferation, apoptosis, protein transport and transcriptional regulation. Among them, the cell death-promoting factor Btf was found to be implicated in the apoptotic signal triggered by ceramide. In adenocarcinoma cells, Btf regulates apoptosis related proteins such as Mdm2, p53, BAX and pBcl-2 and thus plays an important role in the ceramide mediated cell death. These findings bring new insight into the proapoptotic ceramide-dependent signaling pathway.

High-sensitivity C-reactive protein predicts future cardiovascular events in both healthy individuals and patients with unstable and stable coronary syndromes. Few data are available about the incidence and the relation to inflammation of troponin elevation following percutaneous coronary intervention (PCI), a potential predictor of longterm outcome. We sought to confirm the impact of embolization on long-term outcome and evaluate the ability of baseline inflammation to predict troponin elevation induced by PCI. We prospectively analyzed 200 patients treated by PCI for stable or Braunwald IIA class unstable angina. The patients were recruited between January 1997 and May 1999, and the population was followed during a mean follow-up of 32 months. Major adverse cardiac events (MACEs) were defined as the occurrence of death, myocardial infarction or recurrent angina requiring repeat PCI, or coronary artery bypass grafting. During the follow-up period, 58 MACEs were observed. By multivariate analysis, independent predictors for the occurrence of MACEs were unstable angina and troponin I level after PCI (P < 0.0001 for both). No correlation was found between baseline inflammation and significant troponin I elevation post PCI and by multivariate analysis, no biological variable was a predictor of troponin I elevation post PCI. Baseline inflammation cannot predict onset of minor myonecrosis damage (expressed by troponin elevation) induced by PCI, a significant predictor of long-term outcome in this setting.

Immunoassays, or assays with antibodies as reagents, are widely used in medical laboratories. These assays are used to identify and quantify various substances in biological fluids, such as specific proteins (various tissue markers, markers of inflammation, hormones, coagulation factors...) or immunoglobulins (viral or bacterial antibodies, auto-antibodies...) and even both viral antigens and antibodies (HIV virology). The use of monoclonal antibodies allowed, through their specificity for a single epitope of the target molecule, the development of increasingly sophisticated immunoassays. In particular, the use of monoclonal antibodies with microarrays permits the simultaneous determination of various proteins (inflammatory profile, cardiac profile, specifics IgE...) quickly and accurately. Very important tools in the clinical laboratory, immunoassays techniques are, however, subject to various analytical interferences which may be responsible for significant changes in the test results.

In contrast to a polyclonal antiserum, a monoclonal antibody is specific to a single epitope on the surface of a complex antigen. In 1975, Kohler and Milstein produced the first monoclonal antibodies by using a method which rapidly became a key technology in immunology. By fusing activated antibody-forming cells (B cells) with myeloma cells, they obtained hybrid cells--the so-called hydridomas--which combine the ability of the activated B cells to secrete a single species of antibody and the immortality of the myeloma cell. The selected hybridomas proliferate continuously, their clonal progeny providing an unending supply of antibody with a single specificity. These antibodies have found many applications in basic research and in vitro diagnosis. In the clinical laboratory, monoclonal antibodies are used as reagents in immunoassays, often replacing traditional antisera. Many years of development and innovation were needed to humanize monoclonal antibodies in order to make them usable in human therapy.

Background: Patients with anorexia nervosa (AN) are at high risk of renal failure. Glomerular filtration rate (GFR) is overestimated when estimated by the creatinine-based equations. We have studied the accuracy and precision of cystatin C-based equations., Method: 27 AN patients were included. GFR was measured with the chromium-51-ethylenediaminetetraacetate (51Cr-EDTA) method. We have compared the accuracy and precision of creatinine-based equations (MDRD and Cockcroft) with those of different new cystatin C-based equations., Results: The creatinine-based equations overestimate measured GFR, especially the MDRD study equation. All the cystatin C-based equations also overestimate measured GFR. The Cockcroft and Gault formula and the cystatin C-based equation published by Rule have the best accuracy and precision, but these last performances remain unsatisfactory., Conclusion: Both creatinine and cystatin C-based equations strongly overestimate measured in patients with AN.

Constitutive nuclear factor (NF)-kappaB activation in haematological malignancies is caused in several cases by loss of function mutations within the coding sequence of NF-kappaB inhibitory molecules such as IkappaBalpha or p100. Hut-78, a truncated form of p100, constitutively generates p52 and contributes to the development of T-cell lymphomas but the molecular mechanism underlying this oncogenic potential remains unclear. We show here that MMP9 gene expression is induced through the alternative NF-kappaB-activating pathway in fibroblasts and also on Hut-78 or p52 overexpression in fibroblasts as well as in lymphoma cells. p52 is critical for Hut-78-mediated MMP9 gene induction as a Hut-78 mutant as well as other truncated NF-kappaB2 proteins that are not processed into p52 failed to induce the expression of this metalloproteinase. Conversely, MMP9 gene expression is impaired in p52-depleted HUT-78 cells. Interestingly, MLL1 and MLL2 H3K4 methyltransferase complexes are tethered by p52 on the MMP9 but not on the IkappaBalpha promoter, and the H3K4 trimethyltransferase activity recruited on the MMP9 promoter is impaired in p52-depleted HUT-78 cells. Moreover, MLL1 and MLL2 are associated with Hut-78 in a native chromatin-enriched extract. Thus, we identified a molecular mechanism by which the recruitment of a H3K4 histone methyltransferase complex on the promoter of a NF-kappaB-dependent gene induces its expression and potentially the invasive potential of lymphoma cells harbouring constitutive activity of the alternative NF-kappaB-activating pathway.

The generation of cortical projection neurons relies on the coordination of radial migration with branching. Here, we report that the multisubunit histone acetyltransferase Elongator complex, which contributes to transcript elongation, also regulates the maturation of projection neurons. Indeed, silencing of its scaffold (Elp1) or catalytic subunit (Elp3) cell-autonomously delays the migration and impairs the branching of projection neurons. Strikingly, neurons defective in Elongator show reduced levels of acetylated alpha-tubulin. Reduction of alpha-tubulin acetylation via expression of a nonacetylatable alpha-tubulin mutant leads to comparable defects in cortical neurons and suggests that alpha-tubulin is a target of Elp3. This is further supported by the demonstration that Elp3 promotes acetylation and counteracts HDAC6-mediated deacetylation of this substrate in vitro. Our results uncover alpha-tubulin as a target of the Elongator complex and suggest that a tight regulation of its acetylation underlies the maturation of cortical projection neurons.

The role of vitamin D in maintaining bone health has been known for decades. Recently, however, the discovery that many tissues expressed the vitamin D receptor and were able to transform the 25-OH vitamin D into its most active metabolite, 1,25-(OH)(2) vitamin D, has led to a very promising future for this "old" molecule. Indeed, observational studies, and more and more interventional studies, are raising the importance of a significant vitamin D supplementation for not-only skeletal benefits. Among them, 25-OH vitamin D has been found to play an important role in prevention of cancers, auto-immune diseases, cardiovascular diseases, diabetes, and infections. Vitamin D deficiency, defined as serum 25-OH vitamin D levels <30 ng/mL, is very common in our population. The cost/benefit ratio and some recently published studies are clearly now in favor of a controlled and efficient vitamin D supplementation in these patients presenting a 25-OH vitamin D level <30 ng/mL. More attention should also be focused on pregnant and lactating women, as well as children and adolescents.

Background: Cystatin C-based equations are used to estimate GFR. However, three cystatin C immunoassays are on the market. Difference in cystatin C assays could have strong consequences on the accuracy and precision of cystatin C-based equations. We have performed an analytical study of these three assays and studied potential differences between assays on the precision of cystatin C-based equations., Methods: We have studied imprecision, recovery, linearity and interferences of the three immunoassays (nephelometric assay from Siemens and turbidimetric assays from Dako and Gentian). The impact of differences in cystatin C assays has been studied for the equations published by Levey (Siemens assay) and Grubb (Dako assay)., Results: Analytical performance of the Dako assay is slightly less high. For cystatin C values below 2.5 mg/L, no statistical difference is found between results given by the Dako and the Gentian assays. So, both assays can be used in the Grubb equation. Cystatin C results are different with the Siemens assay. The Levey equation, built with the Siemens assay, can only be used with cystatin C values measured with this assay. Using the Dako or Gentian assay results in the Levey equation can lead to differences in estimating GFR up to 6 mL/min/1.73 m2. Differences can reach 9.5 mL/min/1.73 m2 if the Siemens assay is used in the Grubb equation., Conclusion: The Siemens and Gentian assays seem analytically more valid than the Dako assay for cystatin C determination. Differences in cystatin C assays can lead to significant differences in cystatin C-based equations. However, these differences seem less important than the differences observed with creatinine and creatinine-based equations.

Purpose: Nearly one billion people around the world are deficient in vitamin D and need to be supplemented. Vitamin D is available in medicines and fortified foods. It is available in two forms: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol)., Key Points: The pharmacopeiae consider these steroid hormones as equivalent and interchangeable. However, several studies have showed that serum level of 25(OH)D is increased more effectively with vitamin D3 than vitamin D2. Vitamin D2 has shorter plasma half-life and a lower affinity for the vitamin D binding protein, the hepatic vitamin D hydroxylase and the vitamin D receptor., Conclusion: Vitamin D2 should not be regarded anymore as suitable for supplementation or fortification. Currently though, it is still the most used in some countries such as Portugal and Australia.

Background: Determination of glomerular filtration rate plays an important role in nephrological practice. Iohexol is a reference marker for glomerular filtration rate determination. It is available and safe. The aim of this study was to develop a simple, efficient and easy to use analytical method for the quantification of iohexol in serum and urine by high performance liquid chromatography and to thoroughly validate this method., Methods: The HPLC method was inspired from the method published by Krutzen. The e.noval software V2.0 (Arlenda, Liège, Belgium) was used to compute all validation results., Results: The validation results indicate that the method will give accurate and reliable results for serum values ranging from 12.95 to 1295 microg/ml and for urine values ranging from 86.0 to 4144 microg/ml. In routine practice, iohexol concentrations found in plasma after injection range from 40 to 600 microg/ml. The expected urinary values are much wider. One should not hesitate to dilute urine samples to fit with the validated range over 5000 microg/ml., Conclusion: This is the first time that a reference method for the determination of GFR is validated with such a rigorous and thorough protocol. Contrary to other GFR markers, iohexol is now strongly validated from an analytical point of view.

Ionized calcium is the only physiologically active form of calcium. Because of the variation of albumin, pH and haemoconcentration observed during haemodialysis session in patients with chronic renal failure, measure of total calcium does not reflect the real variation of ionized calcium. However, many formulae to correct total calcium by albumin have been proposed but none of them has been validated in dialysis patients. At present time, computing progress permit laboratory to systematically provide a value of corrected total calcium on protocols but is it really indicated? Our results showed that any of those formulae allows obtaining a value of total calcium that possesses a significant critical difference in relation to total calcium. Thus, correction formulae must be abandoned in aid of ionized calcium in haemodialysis patients.

Objectives: Infliximab is the first anti-TNFalpha accepted by the Food and Drug Administration for use in inflammatory bowel disease treatment. Few clinical, biological and genetic factors tend to predict response in Crohn's disease (CD) patient subcategories, none widely predicting response to infliximab., Design and Methods: Twenty CD patients showing clinical response or non response to infliximab were used for serum proteomic profiling on Surface Enhanced Lazer Desorption Ionisation-Time of Flight-Mass Spectrometry (SELDI-TOF-MS), each before and after treatment. Univariate and multivariate data analysis were performed for prediction and characterization of response to infliximab., Results: We obtained a model of classification predicting response to treatment and selected relevant potential biomarkers, among which platelet aggregation factor 4 (PF4). We quantified PF4, sCD40L and IL-6 by ELISA for correlation studies., Conclusions: This first proteomic pilot study on response to infliximab in CD suggests association between platelet metabolism and response to infliximab and requires validation studies on a larger cohort of patients.