[Biological markers of inflammation : an update]. / Les marqueurs biologiques de l'inflammation : faisons le point.

Biomarkers of inflammation such as sedimentation rate, C-reactive protein and procalcitonin are used in daily clinical practice for the diagnosis, prognosis and follow-up of patients with fever or inflammatory syndrome. The purpose of this article is to summarize the current knowledge about these main biological tests and to discuss new biomarkers and new assay approaches such as multiplex technology.

: Le dosage des biomarqueurs de l'inflammation, tels que la vitesse de sédimentation, la protéine-C réactive et la procalcitonine, est utilisé quotidiennement dans le cadre du diagnostic, du pronostic et du suivi des patients fébriles ou souffrant de syndrome inflammatoire. Le but de cet article est de résumer les connaissances actuelles quant à ces principaux tests biologiques et d'aborder les nouveaux biomarqueurs ainsi que les nouvelles approches de dosage comme la technologie multiplex.

[Tandem-mess spectrometry coupled with liquid chromatography (LCMS/MS) : a revolution in the clinical chemistry laboratory]. / La chromatographie liquide couplée à un spectromètre de masse en tandem (LC-MS/MS) : une révolution dans le laboratoire de chimie clinique.

Manual or automated immunoassays are largely used in clinical chemistry laboratories for measuring various compounds like steroid or peptide hormones. However, these methods can lack sensibility and specificity. Hence, during this last decade, tandem-mess spectrometry coupled with liquid chromatography (LC-MS/MS) have emerged as a technique of choice to precisely quantify those molecules. However, these instruments remain quite expensive and need highly trained people.

Les laboratoires de biologie clinique réalisent, depuis de très nombreuses années, des dosages de différentes molécules (hormones, peptides, stéroïdes,…) grâce à des méthodes analytiques utilisant des anticorps. Ces méthodes, faciles à utiliser sont, pour la plupart d'entre elles, totalement automatisables. Cependant, ces méthodes dites «immunologiques¼ manquent parfois de sensibilité et surtout de spécificité. Durant cette dernière décennie, la spectrométrie de masse en tandem couplée à la chromatographie liquide (LC-MS/MS) s'est de plus en plus imposée dans les laboratoires de chimie clinique comme une alternative aux immunodosages. En effet, cette technique permet de séparer et de quantifier avec précision des molécules dont la structure est proche et qui ne sont pas bien différenciées avec des immunodosages. Aussi, la sensibilité de la LC-MS/MS peut être également supérieure à celle des immunodosages. Par contre, ce type de technologie demande du matériel de pointe assez coûteux et un personnel hautement qualifié.

The von Hippel-Lindau tumour suppressor gene: uncovering the expression of the pVHL172 isoform.

BACKGROUND: The von Hippel-Lindau (VHL) gene encodes two mRNA variants. Variant 1 encodes two protein isoforms, pVHL213 and pVHL160, that have been extensively documented in the literature. Variant 2 is produced by alternative splicing of exon 2 and encodes a pVHL isoform of 172 amino acids with a theoretical molecular weight of 19 kDa (pVHL172), the expression of which has never been demonstrated so far due to the absence of suitable antibodies. METHODS: We have generated an anti-pVHL monoclonal antibody (JD-1956) using pVHL172 recombinant protein. We tested the antibody against exogenous or endogenous expressed proteins in different cell lines. We identified the pVHL172 using a silencing RNA strategy. The epitope of the antibody was mapped using a peptide array. RESULTS: We efficiently detected the three different isoforms of pVHL in cell lines and tumorigenic tissues by western blotting and immunohistochemistry and confirmed for the first time the endogenous expression of pVHL172. CONCLUSIONS: The endogenous expression of the three isoforms and particularly the pVHL172 has never been shown before due to a lack of a highly specific antibody since none of the available commercial antibodies distinguish the three isoforms of pVHL in cells or in both normal and cancerous human tissues. Evidence of pVHL172 expression emphasises the need to further study its implication in renal tumorigenesis and VHL disease.

Myhre syndrome.

Myhre syndrome (MS) is a developmental disorder characterized by typical facial dysmorphism, thickened skin, joint limitation and muscular pseudohypertrophy. Other features include brachydactyly, short stature, intellectual deficiency with behavioral problems and deafness. We identified SMAD4 as the gene responsible for MS. The identification of SMAD4 mutations in Laryngotracheal stenosis, Arthropathy, Prognathism and Short stature (LAPS) cases supports that LAPS and MS are a unique entity. The long-term follow up of patients shows that these conditions are progressive with life threatening complications. All mutations are de novo and changing in the majority of cases Ile500, located in the MH2 domain involved in transcriptional activation. We further showed an impairment of the transcriptional regulation via TGFß target genes in patient fibroblasts. Finally, the absence of SMAD4 mutations in three MS cases may support genetic heterogeneity.

Feedback on the management of the 2011 measles outbreak by French military general practitioners: an evaluation study.

OBJECTIVE: Preventive measures were implemented in the French armed forces to limit the measles outbreak that occurred in 2010 and 2011. This study aimed to obtain feedback concerning the management of this outbreak by the French military general practitioners. METHOD: A cross-sectional study was conducted among the general practitioners (GPs) in military units located in metropolitan France. The 60 military units that reported at least one measles case in 2011 were included. Data were collected using self-administered questionnaires. RESULTS: The acceptance of preventive measures against measles was good (measures "totally justified" for 77.8%) and most of the military GPs considered that the outbreak had no significant impact on their activities. The management of measles cases was perceived as not very problematic but difficulties were encountered in the identification of contacts around cases (48.1% of respondents) and in the identification of vaccine recipients among these contacts (more than 80% of respondents reporting difficulties in the collection of measles and vaccination histories). The organization of vaccination around cases was also perceived as difficult. CONCLUSIONS: Preventive measures around measles cases were well accepted by the military GPs, which could reflect their preparedness in the face of the outbreak. However, vaccination did not seem to be well understood or accepted by military patients, underlining the essential role of military GPs in patient information. Difficulties in the collection of vaccination and measles histories among contacts could be overcome by an early transcription of individual medical records in the military medical files of newly enlisted personnel. A more generalized use of oral fluid testing devices, which can be shipped at ambient temperature, would simplify diagnosis in the armed forces.

Identification of protein biomarkers associated with cardiac ischemia by a proteomic approach.

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.

[Comparative study of five techniques of preparation of platelet-rich plasma]. / Étude comparative de cinq techniques de préparation plaquettaire (platelet-rich plasma).

AIM OF THE STUDY: Injections of platelet-rich plasma (PRP) constitute a new therapeutic for treating chronic tendinopathies. The injection being carried out in the tendon, the volume of PRP should thus be minimal (to decrease the intratendinous pressure and to minimize pain). This PRP should also have a raised platelet count. The quantity of released growth factors could be related to the system of preparation employed. We thus carried out a comparative study of five techniques of preparation of PRP described in the literature. MATERIALS AND METHODS: Samples of venous blood were taken among five patients in order to compare five techniques of preparation of PRP: University Hospital of Liège technique, Curasan(®) PRP Kit, Plateltex(®), GPS(®)II and RegenLab(®). RESULTS: The various techniques make it possible to obtain more important platelet concentration than in blood, with variable volumes (0,3 to 6ml). The number of platelets per microlitre appears higher with Plateltex(®) and obtains smallest volume of PRP. The other techniques also give small volumes except for the GPS(®)II. The number of collected platelets with this technique appears thus higher. The best collect efficiency is obtained with RegenLab(®). CONCLUSION: The technique Plateltex(®) makes it possible to collect the highest concentration of platelets in the smallest volume available.

Comparison of the platelet concentrations obtained in platelet-rich plasma (PRP) between the GPS™ II and GPS™ III systems.

INTRODUCTION: Platelet growth factors are known for their ability to speed up tissue healing (bone, skin, tendons, muscle). Various techniques make it possible to collect this platelet-rich plasma or PRP. METHODS: This study compares the platelet concentrations obtained from five patients using GPS™ III, which has recently come onto the market, with those obtained using GPS™ II. RESULTS AND CONCLUSION: We obtain a platelet concentration that is six to nine times greater with GPS ™ II and GPS™ III, but there is no significant difference between the concentrations of PRP obtained with the two systems.

The impact of an ultra-trail on the dynamic of cardiac, inflammatory, renal and oxidative stress biological markers correlated with electrocardiogram and echocardiogram.

The aim of this study was to describe the effects of a 64.2 km ultra-trail on the biomarkers of muscle damage, inflammation and oxidative stress, and compare the results observed with an ECG and an echocardiogram, both performed before and after the race.Thirty-three ultra-trail volunteers (45.8 ± 8.7 years old) were enrolled in our study. Three blood tests were drawn from each runner, one just before (TPRE), one just after (TPOST) and the last 3 h after the end of the race (TPOST3h).All the markers increased. The maximum concentrations observed were at TPOST3h and were significant (p < 0.001) for creatine kinase, creatine kinase isoform MB, high-sensitivity C-reactive protein, uric acid and for the ratio of reduced glutathione to oxidised glutathione. However, in the case of myoglobin, high-sensitive troponin T, N-terminal pro-brain natriuretic peptide, oxidised glutathione, myeloperoxidase, cystatin C and creatinine, the most significant increases were at TPOST (p < 0.001). Modifications were observed in the medical imaging using echocardiography such as reduction of left ventricule end-sytolic and diastolic volumes and left ventricular global longitudinal strain. ECG showed electrical criteria for left ventricular hypertrophy and incomplete right bundle branch block after the race.Endurance races cause significant physiological stress to the body that can be measured by the increase of different biomarkers. From a laboratory perspective, it is important to take into account the possible exercise performed previous to the testing to avoid a misinterpretation of the results. From a training perspective, due to these increases in biomarkers, it is recommended that runners wait at least 72 h after an ultra-trail before subsequent training. In addition a transient impairment of ventricular function due to dehydration were observed.

Intense sport practices and cardiac biomarkers.

Biomarkers are well established for the diagnosis of myocardial infarction, heart failure and cardiac fibrosis. Different papers on cardiac biomarker evolution during exercise have been published in the literature and generally show mild to moderate elevations. However, the mechanism responsible for these elevations, reflecting physiological or even pathophysiological changes, still has to be clearly elucidated. There are also indications of higher cardiac risk in poorly trained athletes than in well-trained athletes. Whether regular repetition of intensive exercise might lead, in the longer term, to fibrosis and heart failure remains to be determined. In this review, we summarized the main research about the effects of intense exercise (in particular, running) on cardiac biomarkers (including troponins, natriuretic peptides, etc.). We found that cardiac fibrosis biomarkers seemed to be the most informative regarding the biological impact of intense physical activity.

[Osteogenesis imperfecta]. / Osteogenesis imperfecta.

We report the case of a young boy who had had multiple bone fractures (more than 10) since the age of 19 months. The father had the same clinical history. The clinical examination was normal for his age except blue sclera. The bone densitometry showed a severe osteoporosis for his age. Biological exam swere correct. The genetic exploration revealed mutation of COL1A2 gene. With this clinical history, the diagnosis of Osteogenesis imperfecta (OI) was retained. OI is a hereditary dystrophy with abnormal synthesis or metabolism of collagen with, often, mutation of COL1A1 or COL1A2 genes. There are 7 different forms. We consider the possible differential diagnoses. The goal of any treatment is to promote bone remineralisation and to decrease the fracture frequency. The treatment includes calcium and vitamin D, and in the presence of some precise criteria, biphosphonate therapy.

Re-certification of hydroxyvitamin D standards by isotope pattern deconvolution.

BACKGROUND: Vitamin D testing in analytical clinical laboratories has been experiencing a rapid increase of demand over the last years, as it plays a key role in several disorders. Due to the narrow ranges of medical significance regarding its concentration levels in human serum, accurate and precise determinations of vitamin D metabolites are required. METHODS: We present an isotope dilution mass spectrometry quantification method for the re-certification of routine commercial standards used in method validation steps, isotope pattern deconvolution (IPD) based on LC-MS/MS. RESULTS: IPD allowed to compensate for the observed biases of +4.7% for 25(OH)D3, -29% for 25(OH)D2 and -30% for 24,25(OH)2D3 standard concentrations, respectively in an easy, cheap and straightforward way. CONCLUSIONS: Is has been observed that, in some cases, discrepancies may exist between stated purity or amount of routinely used commercial standards and actual values, which would lead to unwanted bias in the developed methodologies. The present correction has helped meeting the regulations established by international standardization programs, including Vitamin D Standardization Program (VDSP).

Comparison of isotope pattern deconvolution and calibration curve quantification methods for the determination of estrone and 17ß-estradiol in human serum.

A Liquid Chromatography coupled to tandem mass spectrometry (LC-MS/MS) based method have been developed for the determination of the main estrogen compounds -estrone (E1) and 17ß-estradiol (E2)- in human serum. Two isotope dilution mass spectrometry (IDMS) quantification procedures have been used: a classical calibration curve-based method were compared to a recently developed isotope pattern deconvolution (IPD) method. IPD is based on isotopic abundance measurements and multiple linear regression. Validation was performed in terms of intra-assay repeatability (n = 5), inter-assay reproducibility (n = 9) and accuracy using spiked steroid-free serum at 5 concentration levels and 3 certified reference materials. Both methodologies meet EMEA requirements yielding recoveries between 79-106% and coefficient of variations of 1.7-8.3% along all experiments. Limits of quantification as low as 5 ng/L were achieved. 40 real samples were analysed for comparison purposes showing a great correlation between calibration and IPD concentration values. Real samples were also quantified by routine immunoassay analysis, which showed a significant proportional bias of 2.55 for E1 and good correlation for E2. While methods were considered suitable for routine or countercheck analysis within the context of hospital's needs, IPD has demonstrated to be faster and cost saving.

[How I explore ... review of the principals of antibodies]. / Comment j'explore ... revue des principaux auto-anticorps.

Auto-immune diseases represent the 3rd cause of morbidity after cardiovascular and oncologic diseases. They often occur in young subjects. Their presence is not synonymous of disease and must be associated to clinical signs to be pathological. However, their discovery can require a complement of investigations and the possibility of a follow-up because some auto-antibodies are predictive of disease. This paper is concerned with the main autoantibodies that can be picked out at the laboratory of immunology. Some technical explanations and INAMI rules are explained too.

[Comparison of five techniques to detect anti-Saccharomyces cerevisiae antibodies (ASCA) in serum for diagnosing Crohn's disease]. / Comparaison de cinq techniques de détection des anticorps anti-Saccharomyces cerevisiae (ASCA) dans le sérum pour le diagnostic de la maladie de Crohn.

OBJECTIVE: the anti-Saccharomyces cerevisiae antibodies (ASCA) are diagnostic markers found in Crohn's disease patients. The aim of this study was to compare three Elisa (enzyme linked immunosorbent assay) kits with the indirect immunofluorescence (IFI) technique and an immunodot for ASCA detection. MATERIALS AND METHODS: we compared the results obtained using IFI (IgA and IgG) and Elisa (IgA and IgG) in 139 patients (37 Crohn's disease). An immunodot (IgA+IgG) was tested in a sub-group of 24 patients (18 Crohn's disease). RESULTS AND DISCUSSION: for the different techniques by Elisa (IgA or IgG), the sensitivity ranged from 65% to 76%, the specificity from 88% to 98%, the positive predictive value (PPV) from 84% to 94% and the negative predictive value (NPV) from 88% to 93%. For IFI, the sensitivity was 81%, the specificity 100%, the PPV 100% and the NPV 93%. The immunodot showed a specificity and PPV of 100% and NPV of 33%. CONCLUSION: the detection of the ASCA is useful in the diagnosis of Crohn's disease. IFI appears as the method of choice for its excellent sensitivity and specificity, and affordable costs.

[Conservation of the MC domains in eukaryotic termination factor eRF3].

Eukaryotic translation termination employs two protein factors, eRF1 and eRF3. Proteins of the eRF3 family each consist of three domains. The N and M domains vary in different species, while the C domains are highly homologous. The MC domains of Homo sapiens eRF3a (hGSPT I), Xenopus laevis eRF3 (XSup35), and Mus musculus eRF3a (mGSPTI) and eRF3b (mGSPT2) were found to compensate for the sup35-21(ts) temperature-sensitive mutation and lethal disruption of the SUP35 gene in yeast Saccharomyces cerevisiae. At the same time, strains containing the MC domains of the eRF3 proteins from different species differed in growth rate and the efficiency of translation termination.

In vivo pH measurement at the site of calcification in an octocoral.

Calcareous octocorals are ecologically important calcifiers, but little is known about their biomineralization physiology, relative to scleractinian corals. Many marine calcifiers promote calcification by up-regulating pH at calcification sites against the surrounding seawater. Here, we investigated pH in the red octocoral Corallium rubrum which forms sclerites and an axial skeleton. To achieve this, we cultured microcolonies on coverslips facilitating microscopy of calcification sites of sclerites and axial skeleton. Initially we conducted extensive characterisation of the structural arrangement of biominerals and calcifying cells in context with other tissues, and then measured pH by live tissue imaging. Our results reveal that developing sclerites are enveloped by two scleroblasts and an extracellular calcifying medium of pH 7.97 ± 0.15. Similarly, axial skeleton crystals are surrounded by cells and a calcifying medium of pH 7.89 ± 0.09. In both cases, calcifying media are more alkaline compared to calcifying cells and fluids in gastrovascular canals, but importantly they are not pH up-regulated with respect to the surrounding seawater, contrary to what is observed in scleractinians. This points to a potential vulnerability of this species to decrease in seawater pH and is consistent with reports that red coral calcification is sensitive to ocean acidification.

Myoferlin regulates cellular lipid metabolism and promotes metastases in triple-negative breast cancer.

Myoferlin is a multiple C2-domain-containing protein that regulates membrane repair, tyrosine kinase receptor function and endocytosis in myoblasts and endothelial cells. Recently it has been reported as overexpressed in several cancers and shown to contribute to proliferation, migration and invasion of cancer cells. We have previously demonstrated that myoferlin regulates epidermal growth factor receptor activity in breast cancer. In the current study, we report a consistent overexpression of myoferlin in triple-negative breast cancer cells (TNBC) over cells originating from other breast cancer subtypes. Using a combination of proteomics, metabolomics and electron microscopy, we demonstrate that myoferlin depletion results in marked alteration of endosomal system and metabolism. Mechanistically, myoferlin depletion caused impaired vesicle traffic that led to a misbalance of saturated/unsaturated fatty acids. This provoked mitochondrial dysfunction in TNBC cells. As a consequence of the major metabolic stress, TNBC cells rapidly triggered AMP activated protein kinase-mediated metabolic reprogramming to glycolysis. This reduced their ability to balance between oxidative phosphorylation and glycolysis, rendering TNBC cells metabolically inflexible, and more sensitive to metabolic drug targeting in vitro. In line with this, our in vivo findings demonstrated a significantly reduced capacity of myoferlin-deficient TNBC cells to metastasise to lungs. The significance of this observation was further supported by clinical data, showing that TNBC patients whose tumors overexpress myoferlin have worst distant metastasis-free and overall survivals. This novel insight into myoferlin function establishes an important link between vesicle traffic, cancer metabolism and progression, offering new diagnostic and therapeutic concepts to develop treatments for TNBC patients.

Using platelet-rich plasma to treat jumper's knees: Exploring the effect of a second closely-timed infiltration.

OBJECTIVES: Some clinical series have evaluated the effect of platelet-rich plasma (PRP) in the treatment of proximal patellar tendinopathy. Although it is possible that a single infiltrative administration may prove to be an effective treatment for this indication, most of the existing studies evaluated the effects of two or three successive infiltrations. The aim of this study was to evaluate whether two infiltrations of PRP proves more effective than a single treatment. DESIGN: Prospective, randomized and comparative study of level 2. METHODS: Twenty patients suffering from chronic proximal patellar tendinopathy were enrolled into the study and split into two randomized groups (one or two infiltrations of PRP, respectively). The 3-month follow-up evaluation consisted of VAS, IKDC and VISA-P scores, along with algometer, isokinetic and ultrasounds evaluations. After 1 year, subjects were contacted to define their functional evolution. RESULTS: The concentration of the PRP used for each infiltration was similar in both groups, and contained no red or white cells. Results revealed no difference in treatment efficacy between the groups. CONCLUSIONS: The comparison between one or two infiltrations of PRP did not reveal any difference between the two groups at short to mid term. A second closely-timed infiltration of PRP to treat proximal patellar tendinopathies is not necessary to improve the efficacy of this treatment in the short term.