Presence of ectopic germinal center structures in autoimmune hepatitis.

Autoimmune tissues may contain ectopic germinal centers (EGCs). However, these structures have never been described in the liver of patients suffering from autoimmune hepatitis (AIH). We retrospectively reviewed histological features of 120 definite AIH cases, and found 10 cases harboring markers of EGCs. In these cases, CD21+ follicular dendritic cells were intermixed with CD3+ T and CD20+ B lymphocytes. The latter expressed the GC-specific marker bcl6, and some were proliferative as assessed by Ki67 expression. Antibody-secreting cells (ASCs) defined by expression of the mum-1 transcription factor and presence of cytoplasmic IgMs were usually present in the periphery of these structures, but some were also present within the EGCs. Notably, some ASCs were IgG-switched. Common treatment applied to AIH patients achieved biochemical normalization as efficiently as in patients without EGCs. In the present study, we provide the proof for the occurrence of functional EGCs enabling differentiation of B cells into ASCs and occurrence of immunoglobulin switch in AIH livers.

Moderate alcohol consumption is associated with increased radiological progression in women, but not in men, with early rheumatoid arthritis: results from the ESPOIR cohort (Étude et Suivi des Polyarthrites Indifférenciées Récentes).

OBJECTIVE: We conducted this study to determine whether alcohol consumption influences radiological progression in early rheumatoid arthritis (RA). METHOD: Patients fulfilling the European League Against Rheumatism/American College of Rheumatology 2010 criteria in the early arthritis cohort ESPOIR (Étude et Suivi des Polyarthrites Indifférenciées Récentes) were included in this study. Alcohol consumption was collected at baseline and at each visit. We classified alcohol consumption into three groups: abstinent (0 g/day), moderate (≤ 20 g/day for women, ≤ 30 g/day for men), and abuse (> 20 g/day for women, > 30 g/day for men). The primary outcome was the occurrence of radiological progression, defined as an increase ≥ 5 points in the total Sharp/van der Heijde score. We investigated whether alcohol consumption is predictive of radiological progression at 1, 3, and 5 years by univariate and multivariate analysis adjusted for age, baseline erosion, rheumatoid factor, anti-citrullinated peptide antibody, smoking status, body mass index, and treatment with leflunomide or methotrexate and biologics. RESULTS: The study included 596 patients. When considering the influence of gender on the interaction between alcohol consumption and radiological progression, we showed a deleterious effect of moderate consumption in women [odds ratio (OR) = 1.73, 95% confidence interval (CI) 1.01; 2.96, p = 0.045] and a trend towards a protective effect of moderate consumption in men (OR = 0.50, 95% CI 0.21; 1.16, p = 0.106) in multivariate analysis. CONCLUSION: Our data suggest a deleterious effect of moderate consumption of alcohol on radiological progression in women, but not in men, with early RA.

Bone mineral density in adults with arthrogryposis multiplex congenita: a retrospective cohort analysis.

The primary objective of this study was to evaluate the prevalence of low femoral and lumbar spine bone mineral density (BMD) in adults with arthrogryposis multiplex congenita (AMC). We performed a retrospective cohort analysis of adults with AMC who were enrolled in the French Reference Center for AMC and in the Pediatric and Adult Registry for Arthrogryposis (PARART, NCT05673265). Patients who had undergone dual-energy X-ray absorptiometry (DXA) and/or vitamin D testing were included in the analysis. Fifty-one patients (mean age, 32.9 ± 12.6 years) were included; 46 had undergone DXA. Thirty-two (32/51, 62.7%) patients had Amyoplasia, and 19 (19/51, 37.3%) had other types of AMC (18 distal arthrogryposis, 1 Larsen). Six patients (6/42, 14.3%) had a lumbar BMD Z score less than - 2. The mean lumbar spine Z score (- 0.03 ± 1.6) was not significantly lower than the expected BMD Z score in the general population. Nine (9/40, 22.5%) and 10 (10/40, 25.0%) patients had femoral neck and total hip BMD Z scores less than - 2, respectively. The mean femoral neck (- 1.1 ± 1.1) and total hip (- 1.2 ± 1.2) BMD Z scores in patients with AMC were significantly lower than expected in the general population (p < 0.001). Femoral neck BMD correlated with height (rs = 0.39, p = 0.01), age (rs = - 0.315, p = 0.48); total hip BMD correlated with height (rs = 0.331, p = 0.04) and calcium levels (rs = 0.41, p = 0.04). Twenty-five patients (25/51, 49.0%) reported 39 fractures. Thirty-one (31/36, 86.1%) patients had 25-hydroxyvitamin D levels less than 75 nmol/l, and 6 (6/36, 16.7%) had 25-hydroxyvitamin D levels less than 75 nmol/l. Adults with AMC had lower hip BMD than expected for their age, and they more frequently showed vitamin D insufficiency. Screening for low BMD by DXA and adding vitamin D supplementation when vitamin D status is insufficient should be considered in adults with AMC, especially if there is a history of falls or fractures.

[Management of nasolabial clefts: the issue in Dakar]. / Prise en charge des fentes nasolabiales: problématique à Dakar.

The management of patients with a cleft lip in developing countries is often the prerogative of humanitarian missions from developed countries. The goal of our work is to conduct a first epidemiological, clinical and therapeutic assessment of the management of cleft lips by a local team and to evidence the difficulties faced by us in our working conditions. In a retrospective study covering a period of about five years (January 2004 to March 2009), 205 cases of nasolabial clefts are assembled. The mean age at the time of the first visit is 17 months. A slight female predominance is observed. The majority of patients are from the capital city. A close relative with a cleft is found in 6.8% of them. In 44.9% of cases, it is a simple cleft lip. A cleft palate is associated in 47.8% of cases. Associated malformations are observed in 10.5% of cases. We operated on 110 patients. The mean age at the first surgery is two years. Millard's technique is our technique of choice. No operative mortality is observed. In 17.4% of cases, operative morbidity occurred in the form of suppuration with partial or complete early suture release. The esthetic result is satisfactory in 67.7% of cases. This management could be improved by creating a multidisciplinary team including--in addition to surgeons--dentists, speech-language pathologists, psychologists, etc.

Basal endothelial nitric oxide synthase (eNOS) phosphorylation on Ser(1177) occurs in a stable microtubule- and tubulin acetylation-dependent manner.

To better understand the relationship between the subcellular compartmentalization of endothelial nitric oxide synthase (eNOS) and its function in endothelial cells, we addressed the roles of the microtubule network and of its dynamics in organizing Golgi-bound eNOS. We found that part of Golgi-bound eNOS localizes to the trans-Golgi network and/or to trans-Golgi network-derived vesicles and membrane tubules that are organized preferentially by stable microtubules. Also, while most of cellular eNOS was recovered in a detergent-resistant microtubule-enriched subcellular fraction, its recovery was impaired after total microtubule disassembly, but not after selective disassembly of dynamic microtubules or after microtubule stabilization. Basal eNOS phosphorylation on Ser(1177) further required the association of the trans-Golgi network to stable microtubules and was enhanced by microtubule stabilization. We finally show that the involvement of stable microtubules in basal eNOS phosphorylation involved alpha-tubulin acetylation. Microtubule-dependent organization of subcellular eNOS and control over its phosphorylation would thus be essential for endothelial cells to maintain their basal eNOS function.

Apolipoprotein A-I and platelet factor 4 are biomarkers for infliximab response in rheumatoid arthritis.

OBJECTIVES: The use of biologicals such as infliximab has dramatically improved the treatment of rheumatoid arthritis (RA). However, factors predictive of therapeutic response need to be identified. A proteomic study was performed prior to infliximab therapy to identify a panel of candidate protein biomarkers of RA predictive of treatment response. METHODS: Plasma profiles of 60 patients with RA (28 non-responders (as defined by the American College of Rheumatology 20% improvement criteria (ACR20)) negative and 32 responders (ACR70 positive) to infliximab) were studied by surface enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF MS) technology on two types of arrays, an anion exchange array (SAX2) and a nickel affinity array (IMAC3-Ni). Biomarker characterisation was carried out using classical biochemical methods (purification by ammonium sulfate precipitation or metal affinity chromatography) and identification by matrix assisted laser desorption/ionisation time-of-flight (MALDI-TOF) MS analysis. RESULTS: Two distinct protein profiles were observed on both arrays and several proteins were differentially expressed in both patient populations. Five proteins at 3.86, 7.77, 7.97, 8.14 and 74.07 kDa were overexpressed in the non-responder group, whereas one at 28 kDa was increased in the responder population (sensitivity>56%, specificity>77.5%). Moreover, combination of several biomarkers improved the sensitivity and specificity of the detection of patient response to over 97%. The 28 kDa protein was characterised as apolipoprotein A-I and the 7.77 kDa biomarker was identified as platelet factor 4. CONCLUSIONS: Six plasma biomarkers are characterised, enabling the detection of patient response to infliximab with high sensitivity and specificity. Apolipoprotein A-1 was predictive of a good response to infliximab, whereas platelet factor 4 was associated with non-responders.

Role of ceramide structure and its microenvironment on the conformational order of model stratum corneum lipids mixtures: an approach by FTIR spectroscopy.

The aim of this study is to investigate the influence of ceramide head group architecture and free fatty acid (another main class of stratum corneum lipids) or protein (keratin), on the lamellar organization of the ceramide auto-associated in model films mimicking lipid organization within the stratum corneum. FTIR spectroscopy is a powerful technique for investigating the structure of such systems. This technique has already been used to characterize phase transitions of the SC and of related model systems. As temperature is known to modify the conformational order of lipids, we used it as a variable parameter to monitor the differences in the conformational stability of ceramides. Our study included four ceramides: ceramide 2, 3, 5 and 6 which differ by their head group architecture. Two kinds of lipid-lipid interactions were studied: non-polar and polar. We noted some structural factors which participated to the organizational behavior: insaturation of alkyl chain, alpha-hydroxyl on fatty acid moiety and sphingosine or phytosphingosine head group. There is a direct interaction of palmitic acid on alkyl chains organization and a weak interaction with polar head group in presence of keratin, both provoking a destabilization of the ceramidic orthorhombic organization.

Liquid chromatography on porous graphitic carbon with atmospheric pressure photoionization mass spectrometry and tandem mass spectrometry for the analysis of glycosphingolipids.

The study of several structural variations (the length, the degree of unsaturation and hydroxylation of the alkyl chains, the number and nature of osidic residues) helped understand the behaviour of neutral glycosphingolipids (GSLs) on porous graphitic carbon stationary phase (PGC). Atmospheric pressure photoionization mass spectrometry (APPI) and tandem mass spectrometry were used to perform the detection and the identification of molecular species in positive mode where [M+H](+) and [M-H(2)O+H](+) ions provided structural information on the fatty acid and the sphingoid base. The retention of GSLs increased with the hydrocarboneous volume of their alkyl chains and with the number of osidic residues in agreement with hydrophobic properties and polar retention effect of graphite, respectively. The presence of polar groups, such as OH-group or double bond within alkyl chains, decreased their retention. The coupling of chromatography on PGC with APPI tandem mass spectrometry detection appeared a powerful technique to discriminate isobaric molecules. Isobaric solutes differing by the position of two double bonds or by the repartition of hydrocarboneous skeleton were discriminated according to their chromatographic comportment or their mass spectrum, respectively. Among isobaric molecules, only few structures differing by the nature of osidic residue were not discriminated (i.e. glucosylceramide and galactosylceramide with similar ceramide skeleton were co-eluted and no difference in mass spectra was observed).

Fluorescence probe assisted post-column detection for lipid analysis in microbore-LC.

A general approach, still few exploited so far and never associated with microbore-LC, consisting of detection of various lipid classes (i.e. phospholipids, triglycerides, ceramides and glycosphingolipids) by non-covalent association with 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence probe is developed. This mode of detection was coupled with non-aqueous reversed-phase microbore-LC (C18) by using classical post-column fluorescence detection. The classical LC system was first adapted to microbore-chromatography (internal diameter 1 mm) without apparatus miniaturization of the solvent delivery system and the detection cell. For this purpose, the detection parameters (probe concentration, post-column flow rate, post-column reactor length and post-column system temperature) were optimized by a central composite design (CCD) using a mixture of phosphatidylcholine (PC) species as a lipid model and DPH (lambda(ex) = 350 nm, lambda(em) = 430 nm) as a fluorescence probe. The optimal conditions of detection for the various molecular species of PC were determined for a DPH concentration of 3.35 micromol/L, a post-column flow rate of 0.5 mL/min, a reactor length of 1.4 m and a temperature of 35 degrees C. The fluorescence response was linear over a wide range of PC species from 5 microg/mL to 100 microg/mL and the lower limit of detection (signal/noise = 3) was about 1 microg/mL, that is equivalent to evaporative light scattering detection (ELSD). Others molecular species of various classes of lipids, i.e. triglycerides, ceramides and glycosphingolipids were also easily detected. Thus, this study demonstrated the versatility of the proposed system of detection which was shown to be sensitive, easy to perform, non-destructive and allowed, in contrast to ELSD, for a linear response with various polarity lipid classes.

ATR-FTIR spectroscopy: a chemometric approach for studying the lipid organisation of the stratum corneum.

The barrier function of skin resides in the lipid components of the stratum corneum, particularly their spatial organisation. FTIR spectroscopy has already been used as a relevant tool to study this lipid organisation: IR vibration band shifts have been attributed to the variations in lipid organisation induced by temperature. Our study included a stratum corneum model, composed of the three main lipids: palmitic acid as an example of fatty acids, cholesterol and ceramide III as an example of ceramide. Different films with various ratios of these lipids were studied. In our analytical strategy, the interest of using a chemometric analysis of global data obtained from ATR-FTIR spectra to highlight the main interactions involved in the molecular organisation of lipids has been demonstrated. Two kinds of interaction between the three main lipids have been shown: a non polar interaction between the long hydrocarbon chains and a polar interaction as the hydrogen bonding between polar functional groups. By varying the lipid ratio, we have shown first that the relative importance of each interaction was modified, second, that the induced modification of organisation can be detected by chemometric analysis of the ATR-FTIR spectra. The role of each kind of lipid in the organisation has been discussed. In conclusion, associating the ATR-FTIR with chemometric treatment is a promising tool: firstly, to understand the consequence of lipid relative compositions on the structural organisation of the stratum corneum, secondly, to show the relationship between lipid organisation and percutaneous penetration data. Indeed, this methodology will be transposed to in vivo studies with IR measurements through a probe.

Modulated release of triterpenic compounds from a O/W/O multiple emulsion formulated with dimethicones: infrared spectrophotometric and differential calorimetric approaches.

From the use of silicones within O/W/O multiple emulsions, we can expect, two principal advantages: (1) the silicones with the lowest molecular weight decrease the oily touch; (2) due to the large range of viscosity, this excipient should influence the skin distribution of actives after topical application. The purpose of our work is to highlight these advantages. Multiple emulsions were formulated with several dimethicones and with drug model. Firstly, the effects of different dimethicones incorporated within multiple emulsions were studied, through in vitro penetration results. Secondly, we investigated the residual film on the skin by Differential Scanning Calorimetry (DSC) and by Fourier Transform Infrared (FTIR) to determine its structure. Correlations were established between the silicone structure and the distribution of drugs in different skin levels or between the silicone structure and the percutaneous penetration. The incorporation of silicones within O/W/O multiple emulsions seems to be an efficient means of modulating the penetration and the distribution of drugs in the skin.

Assessment of the retention properties of poly(vinyl alcohol) stationary phase for lipid class profiling in liquid chromatography.

Potentialities of polymerized vinyl alcohol on silica gel were assessed for class separation of simple lipids, sphingolipids, glyceroglycolipids and phospholipids by high-performance liquid chromatography. A screening of pure solvents in binary gradient elution and a chemometric approach was used to define a rugged two segment linear gradient formed from four solvents for total lipid class separation. Triethylamine and formic acid were added in all mobile phase components for acidic phospholipid separation and evaporative light scattering response enhancement. Simple analytical procedures are described for the analysis of complex lipid materials.

Retention behaviour of ceramides in sub-critical fluid chromatography in comparison with non-aqueous reversed-phase liquid chromatography.

This study was devoted to the development of an analytical method for ceramide analysis in packed subcritical fluid chromatography (pSubFC). Monofunctional grafted silica support was found to be more suitable for ceramide analysis. Five Kromasil columns were coupled and the parameters, temperature, pressure and percentage of organic modifier in CO2 were optimised, considering selectivity and analysis time. The final conditions were 31 degrees C, 6% of methanol (MeOH) and 13 MPa. In these conditions the selectivity for structural differences (methylene group, unsaturation or two different bases) were studied. As classically observed, the methylene selectivity decreased with the increase of the eluotropic strength. Moreover, unlike in non-aqueous reversed-phase liquid chromatography (NARP-LC), adding a further unsaturation and two further methylene groups on ceramide results to an increase of retention in pSubFC. Moreover, this last technique allowed to separate ceramides with the same total number of carbons containing unsaturated fatty acids, when the distribution of carbon number of the two chain is very different. These results had enabled to plot retention chart in order to predict ceramide structure in view to identify additional ceramide. This retention chart was finally compared with the one already obtained in NARP-LC.

Structural analysis of commercial ceramides by gas chromatography-mass spectrometry.

A simple method using gas chromatography-mass spectrometry was applied to analyse structures of ceramides. Identification of trimethylsilylated ceramides were obtained in short analysis times (derivatization of ceramides in 30 min at room temperature and 20 min gas chromatography mass spectrometry run) even for complex mixtures. For example in ceramide Type III, 18 peaks were observed which represent 27 various structures. The coeluted compounds were ceramides containing the same functional groups and the same carbon number but with a different distribution on the two alkyl chains of the molecule. They were accurately differentiated by mass spectrometry. Therefore, 83 structures of trimethylsilylated ceramides were identified in 11 different commercial mixtures. For 52 structures of these, mass spectral data were not described in the literature, neither full mass spectra nor characteristic fragments.

Postcolumn fluorescence as an alternative to evaporative light scattering detection for ceramide analysis with gradient elution in non-aqueous reversed-phase liquid chromatography.

Ceramide analysis was developed with gradient elution in non-aqueous reversed-phase liquid chromatography with evaporative light scattering detection (ELSD) or postcolumn fluorescence detection. Fluorescence detection (excitation, 360 nm; emission, 425 nm) after postcolumn formation of mixed assemblies between eluted ceramides and 1,6-diphenyl-1,3,5-hexatriene was developed. In comparison with ELSD, fluorescence detection allows a better detection of the minor species ceramide from ceramide type III (commercial mixture of non-hydroxy fatty acid-sphingosine) and appears to be more sensitive for quantitation of ceramides at low concentrations. The fluorescence response is linear over a wide range of injected amount of ceramide III (expressed as stearoyl-phytosphingosine): 10 ng to 1000 ng. The response of ELSD is non linear but can be linearized in double logarithmic coordinates for calculations over a narrow range, e.g. between 10 to 350 ng ceramide III injected. The lower quantitation limits of these two detectors are similar: 5 ng ceramide III was injected.

Optimisation of the separation of four major neutral glycosphingolipids: application to a rapid and simple detection of urinary globotriaosylceramide in Fabry disease.

A simple method for the separation of the four major neutral glycosphingolipids, present in all human tissue, was developed. This gradient normal phase-HPLC method utilises a polyvinyl alcohol bonded stationary phase and an evaporative light-scattering detection (ELSD). Screening pure solvents in a binary gradient elution mode allowed, in a first step, to assess the behaviour of the studied solutes and to select the solvents for further mobile phase optimisation. The proportion of the remaining solvents was defined to reach a maximal resolution. The reduction of the analysis time and the enhancement of the signal were obtained by optimising the gradient slope and the flow-rate. Optimal levels of triethylamine and formic acid (TEA-FA) for the enhancement of the evaporative light scattering detector response were established at 0.1% (v/v). Thus, the optimal conditions for the separation of the four glycosphingolipids was obtained with a gradient elution from a 100% chloroform to a 100% acetone:methanol (90:10 (v/v)) mobile phase at 0.2 ml min-1, using a 10% min-1 gradient slope. Finally, this method was applied to detect the excess of one of the neutral sphingolipids, namely globotriaosylceramide (Gb3) in the urine of patients affected with Fabry disease. A liquid-liquid extraction of the sediments obtained from an aliquot of only ten ml of urine proved sufficient to detect the excess of Gb3 present in both hemizygote and heterozygote patients. In all, the ability of our method to detect abnormal amounts of Gb3 in urinary sediments could allow the diagnosis of weakly symptomatic Fabry patients in large screening programs

Quantitative HPLC analysis of sunscreens and caffeine during in vitro percutaneous penetration studies.

This report describes rapid analytical HPLC for the quantification of five UV filters (octyl methoxycinnamate, benzophenone-3, benzophenone-4, octyl triazone and octocrylene) and of caffeine in various skin layers (stratum corneum, dermis, epidermis and receptor fluid) and in cosmetic preparations. The predominant purpose of the study was to establish standard operating procedures for rapid analysis of the compounds in various skin samples. Particular attention was paid to the preparation of biological samples whose natural constitution could interfere with the quantitative analysis. Our methods used the isocratic chromatographic mode in an RP-HPLC with UV detection and did not involve centrifugation or evaporation. Our results were validated in terms of specificity, linearity, precision, accuracy and limits of detection and quantification. The first results, obtained after in vitro experiments, are presented in this report.

Liquid chromatographic analysis of monosaccharides with phenylisocyanate derivatization.

Because of the lack of sensitivity in carbohydrate analysis, HPLC pre-column derivatization techniques which give strongly UV absorbing compounds have been reported. These techniques have the disadvantage of leading to several chromatographic peaks from each reducing sugar. To enhance both the sensitivity and the selectivity of such specific separation problems, a simplex procedure was applied to optimize the phenylisocyanate derivatization of monosaccharides: a major and stable derivative was formed under the optimal conditions established. The method was extended to deoxysugars and methylglycosides. The limit of detection was 0.2-1 ng for all sugars tested.

[Computerized tomography-guided needle biopsy for the diagnosis of pulmonary nodules: analysis of a series of 41 patients]. / Ponction à visée diagnostique de nodules pulmonaires sous repérage scanographique: analyse d'une série de 41 patients.

This study was aimed at assessing percutaneous biopsy under computerized tomography (CT) guidance, its results in terms of diagnostic value, factors contributing to its complications or success, and its indications. Results obtained from a series of 41 patients and data from previous clinical trials were compared. Patients who were not eligible due to either poor physical condition or a tumor that could not be reached were not included into the study. Various factors, ie, the tumor location, the size of the lesion, the distance between the lesion and the thoracic wall, and the type (histological and/or cytological) of samples, were analyzed. Results were evaluated according to the disease evolution or pathological findings. Tumoral cells were found in 23 samples. Eighteen samples were negative for malignancy, with eight benign lesions and ten false negative. Complications that occurred just after the biopsy were recorded. Failures and complication factors were analyzed and compared to those already described. CT-guided needle biopsy is an admitted alternative to thoracotomy, even for small nodules deeply located. The use of this technique should be discussed when fiberoptic endoscopy leads to negative results. Various tumors cannot be reached using CT-guided needle biopsy and this technique is more efficacious for bulky nodules. The thickness of lung parenchyma passed through does not seem to change the risk of pneumothorax. The use of laser guidance might help locate the tumor with more accuracy.

[Epidemiology of suicide attempts in Reunion Island]. / Epidémiologie des tentatives de suicide à l'Ile de la Réunion.

In order to specify the epidemiological characteristics of attempted suicides on Reunion island, a quantitative study was carried out, using Emergency Room registers of all the hospitals on the island. This enabled collection of 9526 files. The results revealed a high proportion of suicide attempts (335/100,000/year), particularly prevalent in rural areas to the south of the island. There was a tendency towards over representation of women, though less distinct than in Europe, as well as an abnormally frequent use of pesticides or herbicides. Medication and violent means were not used as often as in France. These outcomes, in particular the very high frequency (especially in the rural areas of the south), along with the significance of medico-psychological factors, should be taken into account by anthropologists, linguists, clinical specialists, in order to help determine preventive measures.