Sedimentation field-flow fractionation for rapid phenotypic antimicrobial susceptibility testing: a pilot study.

BACKGROUND: The increase in antibiotic resistance is a major public health issue. The development of rapid antimicrobial susceptibility testing (AST) methods is becoming a priority to ensure early and appropriate antibiotic therapy. OBJECTIVES: To evaluate sedimentation field-flow fractionation (SdFFF) as a method for performing AST in less than 3 h. METHODS: SdFFF is based on the detection of early biophysical changes in bacteria, using a chromatographic-type technology. One hundred clinical Escherichia coli strains were studied. A calibrated bacterial suspension was incubated for 2 h at 37°C in the absence (untreated) or presence (treated) of five antibiotics used at EUCAST breakpoint concentrations. Bacterial suspensions were then injected into the SdFFF machine. For each E. coli isolate, retention times and elution profiles of antibiotic-treated bacteria were compared with retention times and elution profiles of untreated bacteria. Algorithms comparing retention times and elution profiles were used to determine if the strain was susceptible or resistant. Performance evaluation was done according to CLSI and the ISO standard 20776-2:2021 with broth microdilution used as the reference method. RESULTS: AST results from SdFFF were obtained in less than 3 h. SdFFF showed high categorical agreement (99.8%), sensitivity (99.5%) and specificity (100.0%) with broth microdilution. Results for each antimicrobial were also in agreement with the ISO 20776-2 recommendations, with sensitivity and specificity of ≥95.0%. CONCLUSIONS: This study showed that SdFFF can be used as a rapid, accurate and reliable phenotypic AST method with a turnaround time of less than 3 h.

Sedimentation Field-Flow Fractionation: A Diagnostic Tool for Rapid Antimicrobial Susceptibility Testing.

Conventional antimicrobial susceptibility testing (AST) methods require 24-48 h to provide results, creating the need for a probabilistic antibiotic therapy that increases the risk of antibiotic resistance emergence. Consequently, the development of rapid AST methods has become a priority. Over the past decades, sedimentation field-flow fractionation (SdFFF) has demonstrated high sensitivity in early monitoring of induced biological events in eukaryotic cell populations. This proof-of-concept study aimed at investigating SdFFF for the rapid assessment of bacterial susceptibility to antibiotics. Three bacterial species were included (Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa) with two panels of antibiotics tailored to each bacterial species. The results demonstrate that SdFFF, when used in "Hyperlayer" elution mode, enables monitoring of antibiotic-induced morphological changes. The percentage variation of the retention factor (PΔR) was used to quantify the biological effect of antibiotics on bacteria with the establishment of a threshold value of 16.8% to differentiate susceptible and resistant strains. The results obtained with SdFFF were compared to that of the AST reference method, and a categorical agreement of 100% was observed. Overall, this study demonstrates the potential of SdFFF as a rapid method for the determination of antibiotic susceptibility or resistance since it is able to provide results within a shorter time frame than that needed for conventional methods (3-4 h vs 16-24 h, respectively), enabling earlier targeted antibiotic therapy. Further research and validation are necessary to establish the effectiveness and reliability of SdFFF in clinical settings.

Overexpression of sortilin is associated with 5-FU resistance and poor prognosis in colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer worldwide. Even if 5-fluorouracil (5-FU) is used as the first-line chemotherapeutic drug, responsiveness is only 20-30%. Acquired resistance to 5-FU contributes to both poor patient prognosis and relapse, emphasizing the need to identify biomarkers. Sortilin, a vacuolar protein sorting 10 protein (Vps10p), implicated in protein trafficking, is over expressed in CRC cell lines cultured 72 hours in presence of 5-FU. This overexpression was also observed in 5-FU-resistant cells derived from these cell lines as well as in CRC primary cultures (or patients derived cell lines). A significantly higher expression of sortilin was observed in vivo, in 5-FU-treated tumours engrafted in Nude mice, as compared with non-treated tumour. A study of transcriptional regulation allowed identifying a decrease in ATF3 expression, as an explanation of sortilin overexpression following 5-FU treatment. In silico analysis revealed SORT1 expression correlation with poor prognosis. Moreover, sortilin expression was found to be positively correlated with CRC tumour grades. Collectively, our findings identify sortilin as a potential biomarker of 5-FU resistance associated with poor clinical outcomes and aggressiveness in CRC. As a new prognostic factor, sortilin expression could be used to fight against CRC.

Characterization of Glioblastoma Cancer Stem Cells Sorted by Sedimentation Field-Flow Fractionation Using an Ultrahigh-Frequency Range Dielectrophoresis Biosensor.

Cancer stem cells (CSCs) appear to be an essential target for cancer therapies, in particular, in brain tumors such as Glioblastoma. Nevertheless, their isolation is made difficult by their low content in culture or tumors (<5% of the tumor mass) and is essentially based on the use of fluorescent or magnetic labeling techniques, increasing the risk of differentiation induction. The use of label-free separation methods such as sedimentation field-flow fractionation (SdFFF) is promising, but it becomes necessary to consider a coupling with a detection and characterization method for future identification and purification of CSCs from patient-derived tumors. In this study, we demonstrate for the first time the capability of using an ultrahigh-frequency range dielectrophoresis fluidic biosensor as a detector. This implies an important methodological adaptation of SdFFF cell sorting by the use of a new compatible carrier liquid DEP buffer (DEP-B). After SdFFF sorting, subpopulations derived from U87-MG and LN18 cell lines undergo biological characterization, demonstrating that using DEP-B as a carrier liquid, we sorted by SdFFF subpopulations with specific differentiation characteristics: F1 = differentiated cells/F2 = CSCs. These subpopulations presented high-frequency crossover (HFC) values similar to those measured for standard differentiated (around 110 MHz) and CSC (around 80 MHz) populations. This coupling appeared as a promising solution for the development of an online integration of these two complementary label-free separation/detection technologies.

A New Label-Free Approach to Glioblastoma Cancer Stem Cell Sorting and Detection.

Cancer stem cells (CSCs) play critical roles in cancer, making them important targets for new diagnostic and therapeutic approaches. Since CSCs are heterogeneous and not abundant in tumors, and few specific markers for these cells currently exist, new methods to isolate and characterize them are required. To address this issue, we developed a new label-free methodology to isolate, enrich, and identify CSCs from an heterogeneous tumor cell subpopulation using a cell sorting method (sedimentation field flow fractionation, SdFFF) and a biosensor as a detector. Enrichment was optimized using an original protocol and U87-MG glioblastoma cells cultured in a normal (N) or defined (D) medium (± fetal bovine serum, FBS) under normoxic (N, pO2 = 20%) or hypoxic (H, pO2 < 2%) conditions to obtain four cell populations: NN, NH, DN, and DH. After elution of CSCs via SdFFF using the hyperlayer mode (inertial elution mode for micrometer-sized species), we isolated eight subpopulations with distinct CSC contents based on phenotypical and functional properties, ranging from NN F1 with a lower CSC content to DH F3 with a higher CSC content. Reflecting biological differences, the intrinsic intracellular dielectric permittivity increased from NN to DH conditions. The largest difference in electromagnetic signature was observed between NN F1 and DH F3, in which the CSC content was lowest and highest, respectively. The results demonstrate that microwave dielectric spectroscopy can be used to reliably and efficiently distinguish stem cell characteristics. This new instrumental and methodological approach is an important innovation that allows both enrichment and detection of CSCs, opening the door to novel diagnostic and therapeutic approaches.

Advanced protocol to functionalize CaP bioceramic surface with peptide sequences and effect on murine pre-osteoblast cells proliferation.

To bring osteoinductive properties to calcium phosphate (CaP) bioceramics, a silicon-substituted hydroxyapatite was functionalized by integrin-adhesive cyclic-pentapeptides (c-(DfKRG)). A new two-step protocol was set up to immobilize peptides at low and controlled density on the ceramic surface and limit contamination by adsorbed molecules. To this aim, a spacer bearing c-(DfKRG)-S-PEG6-NHS molecule was synthesized and bonded to an organosilane previously covalently bonded to the ceramic surface. The functionalized ceramic was tested in vitro for MC3T3-E1 murine pre-osteoblasts. CaP ceramic surface retained good biological properties thanks to low density of bonded molecules preserving part of the bioactive CaP surface free of bioorganic molecules. The final SiHA-T-PEG6-S-c-(DfKRG) was shown to increase cell density and to improve proliferation. Furthermore, the use of a strong and stable covalent bond between inorganic and organic parts prevented early burst release of the peptide and increased the persistence of its bioactivity over time. So, this CaP ceramic associating c-(DfKRG) by covalent grafting could be considered as promising new biomaterials for bone tissue engineering.

KLRC3, a Natural Killer receptor gene, is a key factor involved in glioblastoma tumourigenesis and aggressiveness.

Glioblastoma is the most lethal brain tumour with a poor prognosis. Cancer stem cells (CSC) were proposed to be the most aggressive cells allowing brain tumour recurrence and aggressiveness. Current challenge is to determine CSC signature to characterize these cells and to develop new therapeutics. In a previous work, we achieved a screening of glycosylation-related genes to characterize specific genes involved in CSC maintenance. Three genes named CHI3L1, KLRC3 and PRUNE2 were found overexpressed in glioblastoma undifferentiated cells (related to CSC) compared to the differentiated ones. The comparison of their roles suggest that KLRC3 gene coding for NKG2E, a protein initially identified in NK cells, is more important than both two other genes in glioblastomas aggressiveness. Indeed, KLRC3 silencing decreased self-renewal capacity, invasion, proliferation, radioresistance and tumourigenicity of U87-MG glioblastoma cell line. For the first time we report that KLRC3 gene expression is linked to glioblastoma aggressiveness and could be a new potential therapeutic target to attenuate glioblastoma.

Sortilin mediates the release and transfer of exosomes in concert with two tyrosine kinase receptors.

The transfer of exosomes containing both genetic and protein materials is necessary for the control of the cancer cell microenvironment to promote tumor angiogenesis. The nature and function of proteins found in the exosomal cargo, and the mechanism of their action in membrane transport and related signaling events are not clearly understood. In this study, we demonstrate, in human lung cancer A549 cells, that the exosome release mechanism is closely linked to the multifaceted receptor sortilin (also called neurotensin receptor 3). Sortilin is already known to be important for cancer cell function. Here, we report for the first time its role in the assembly of a tyrosine kinase complex and subsequent exosome release. This new complex (termed the TES complex) is found in exosomes and results in the linkage of the two tyrosine kinase receptors TrkB (also known as NTRK2) and EGFR with sortilin. Using in vitro models, we demonstrate that this sortilin-containing complex exhibits a control on endothelial cells and angiogenesis activation through exosome transfer.

Anti-apoptotic role and clinical relevance of neurotrophins in diffuse large B-cell lymphomas.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is a fatal malignancy that needs to identify new targets for additional therapeutic options. This study aimed to clarify the clinical and biological significance of endogenous neurotrophin (nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF)) in DLBCL biopsy samples and cell lines. METHODS: We analysed expression of NGF, BDNF, and their receptors (Trk, p75(NTR)) in 51 biopsies and cell lines by immunohistochemistry, immunofluorescence, and western blotting. To investigate the biological role of BDNF/TrkB/p75(NTR) axis, effects of neurotrophin signalling inhibition were determined on tumour cell survival and vascular endothelial growth factor (VEGF) secretion. The pharmacological pan-Trk inhibitor K252a was used for in vitro and in vivo studies. RESULTS: A BDNF/TrkB axis was expressed in all biopsies, which was independent of the germinal centre B-cell (GCB)/non-GCB profile. p75(NTR), TrkB, and BDNF tumour scores were significantly correlated and high NGF expression was significantly associated with MUM1/IRF4, and the non-GCB subtype. Diffuse large B-cell lymphoma cell lines co-expressed neurotrophins and their receptors. The full-length TrkB receptor was found in all cell lines, which was also phosphorylated at Tyr-817. p75(NTR) was associated to Trk and not to its cell death co-receptor sortilin. In vitro, inhibition of neurotrophin signalling induced cell apoptosis. K252a caused cell apoptosis, decreased VEGF secretion, and potentiated rituximab effect, notably in less rituximab-sensitive cells. In vivo, K252a significantly reduced tumour growth and potentiated the effects of rituximab in a GCB-DLBCL xenograft model. CONCLUSIONS: This work argues for a pro-survival role of endogenous neurotrophins in DLBCLs and inhibition of Trk signalling might be a potential treatment strategy for rituximab resistant subgroups.

Differential expression of neurotensin and specific receptors, NTSR1 and NTSR2, in normal and malignant human B lymphocytes.

Neurotensin, a neuropeptide growth factor, and its two specific neurotensin receptors, NTSR1 and NTSR2, were shown to be expressed by human B cell lines. Another NTSR, sortilin, which is common to neurotensin and neurotrophins, was also detected as we have previously described. Neurotensin was functional in B cell lines; it induced their proliferation and inhibited apoptosis induced by serum deprivation or Fas activation. Quantitative study of gene expression in two malignant B cell diseases showed that NTSR2 was overexpressed, NTSR1 decreased, and neurotensin was unexpressed in B cell leukemia patient's cells, as compared with healthy B cells. However, these expressions did not significantly change in large diffuse B cell lymphoma lymph nodes compared with benign ones. This study points out that neurotensin and its two specific receptors are expressed in human B lymphocytes. Such expressions were not described, and their relationship in B cell diseases, especially in chronic B cell leukemia, needs to be considered further in regard to these findings.

Clinical management of molecular alterations identified by high throughput sequencing in patients with advanced solid tumors in treatment failure: Real-world data from a French hospital.

Background: In the context of personalized medicine, screening patients to identify targetable molecular alterations is essential for therapeutic decisions such as inclusion in clinical trials, early access to therapies, or compassionate treatment. The objective of this study was to determine the real-world impact of routine incorporation of FoundationOne analysis in cancers with a poor prognosis and limited treatment options, or in those progressing after at least one course of standard therapy. Methods: A FoundationOneCDx panel for solid tumor or liquid biopsy samples was offered to 204 eligible patients. Results: Samples from 150 patients were processed for genomic testing, with a data acquisition success rate of 93%. The analysis identified 2419 gene alterations, with a median of 11 alterations per tumor (range, 0-86). The most common or likely pathogenic variants were on TP53, TERT, PI3KCA, CDKN2A/B, KRAS, CCDN1, FGF19, FGF3, and SMAD4. The median tumor mutation burden was three mutations/Mb (range, 0-117) in 143 patients with available data. Of 150 patients with known or likely pathogenic actionable alterations, 13 (8.6%) received matched targeted therapy. Sixty-nine patients underwent Molecular Tumor Board, which resulted in recommendations in 60 cases. Treatment with genotype-directed therapy had no impact on overall survival (13 months vs. 14 months; p = 0.95; hazard ratio = 1.04 (95% confidence interval, 0.48-2.26)]. Conclusions: This study highlights that an organized center with a Multidisciplinary Molecular Tumor Board and an NGS screening system can obtain satisfactory results comparable with those of large centers for including patients in clinical trials.

Detection of Glycosylated Markers From Cancer Stem Cells With ColoSTEM Dx Kit for Earlier Prediction of Colon Cancer Aggressiveness.

Nowadays, colon cancer prognosis still difficult to predict, especially in the early stages. Recurrences remain elevated, even in the early stages after curative surgery. Carcidiag Biotechnologies has developed an immunohistochemistry (IHC) kit called ColoSTEM Dx, based on a MIX of biotinylated plant lectins that specifically detects colon cancer stem cells (CSCs) through glycan patterns that they specifically (over)express. A retrospective clinical study was carried out on tumor tissues from 208 non-chemotherapeutic-treated and 21 chemotherapeutic-treated patients with colon cancer, which were stained by IHC with the MIX. Clinical performances of the kit were determined, and prognostic and predictive values were evaluated. With 78.3% and 70.6% of diagnostic sensitivity and specificity respectively, our kit shows great clinical performances. Moreover, patient prognosis is significantly poorer when the MIX staining is "High" compared to "Low", especially at 5-years of overall survival and for early stages. The ColoSTEM Dx kit allows an earlier and a more precise determination of patients' outcome. Thus, it affords an innovating clinical tool for predicting tumor aggressiveness earlier and determining prognosis value regarding therapeutic response in colon cancer patients.

Active immunological profile is associated with systemic Sjögren's syndrome.

BACKGROUND: The aim of this paper was to study the evolution of primary Sjögren's syndrome (pSS) immunological profile, its impact on pSS activity and long-term evolution in a bicentric cohort of French patients with pSS (n = 445, mean age 53.6 ± 14 years, mean follow-up 76.1 ± 51 months). METHODS: This is a retrospective cohort study. RESULTS: Two hundred twelve patients were Sjögren's syndrome A (SSA) positive, and 131 were both SSA and Sjögren's syndrome B (SSB) positive. Sixty-eight patients (15%) had cryoglobulinemia. Active systemic profile (i.e., hypergammaglobulinemia, rheumatoid factor (RF), and anti-Sjögren's syndrome A (anti-SSA), anti-Sjögren's syndrome B (anti-SSB) positivity), associated with multisystemic involvement, leads to an increased utilization of corticosteroid and hydroxychloroquine. Multivariate analysis pointed out independent statistical association between hypergammaglobulinemia, anti-SSA, anti-SSB, and RF. Cryoglobulinemia is associated with multi-systemic involvement, lymphoma, and pSS-related death. CONCLUSION: The subset of patients with active immunological profile is characterized by systemic complications leading to immunosuppressive drug utilization and polyclonal B-cell activation profile.

Intraperitoneal Chemotherapy for Peritoneal Metastases: Technical Innovations, Preclinical and Clinical Advances and Future Perspectives.

(1) Background: Tumors of the peritoneal serosa are called peritoneal carcinosis. Their origin may be primary by primitive involvement of the peritoneum (peritoneal pseudomyxoma, peritoneal mesothelioma, etc.). This damage to the peritoneum can also be a consequence of the dissipation of cancers-in particular, digestive (stomach, pancreas, colorectal, appendix) and gynecological (ovaries) ones in the form of metastases. The aim of the treatment is a maximal reduction of the macroscopic disease called "cytoreduction" in combination with hyperthermic intra-abdominal chemotherapy to treat residual microscopic lesions. (2) Methods: In this narrative review, we fundamentally synthetize the evolution of this process over time and its impact on clinical applications. (3) Results: Over the last past decade, different evolutions concerning both delivery modes and conditions concerning hyperthermic intra-abdominal chemotherapy have been realized. (4) Conclusion: The final objective of these evolutions is the improvement of the global and recurrence-free survival of primary and secondary malignant peritoneal pathologies. However, more large randomized controlled trials are needed to demonstrate the efficacy of such treatments with the help of molecular biology and genetics.

BDNF and pro-BDNF in serum and exosomes in major depression: Evolution after antidepressant treatment.

BACKGROUND: The study of clinically related biological indicators in Major Depression (MD) is important. The Brain Derived Neurotrophic Factor (BDNF) appears to play an important role in MD, through its neurotrophic effect, and its levels are significantly decreased. The variation in the serum levels of its precursor proBDNF, which has opposite effects, is not known. Their distribution between serum and exosomes and their evolution during antidepressant treatment is also not known, and may be important in modulating their effects. The aim of this study is to evaluate whether serum and exosome mBDNF and proBDNF levels are altered in patients with MD during antidepressant treatment compared to controls, and their association with clinical improvement and clinical variables. MATERIALS AND METHODS: 42 MD subjects and 40 controls were included. Questionnaires to assess the severity of depression and cognitive impairment and blood samples were collected during the three visits at D0 (inclusion) and 3 and 7 weeks after the start of antidepressant treatment. Assays for mBDNF and proBDNF levels were performed in serum and exosomes by ELISA. RESULTS: MD subjects had decreased serum and exosomal BDNF levels and increased proBDNF levels at D0 compared to controls. BDNF and pro-BDNF vary in an inverse manner in both serum and exosomes during antidepressant treatment. No relationship of BDNF and proBDNF levels to clinical improvement and depression scales was found. CONCLUSION: We demonstrated an evolution of those molecules either in serum or in exosomes after MD treatment. These transport vesicles could have a role in the regulation of BDNF.

Microfluidic Lab-on-a-Chip Based on UHF-Dielectrophoresis for Stemness Phenotype Characterization and Discrimination among Glioblastoma Cells.

Glioblastoma (GBM) is one of the most aggressive solid tumors, particularly due to the presence of cancer stem cells (CSCs). Nowadays, the characterization of this cell type with an efficient, fast and low-cost method remains an issue. Hence, we have developed a microfluidic lab-on-a-chip based on dielectrophoresis (DEP) single cell electro-manipulation to measure the two crossover frequencies: fx01 in the low-frequency range (below 500 kHz) and fx02 in the ultra-high-frequency range (UHF, above 50 MHz). First, in vitro conditions were investigated. An U87-MG cell line was cultured in different conditions in order to induce an undifferentiated phenotype. Then, ex vivo GBM cells from patients' primary cell culture were passed through the developed microfluidic system and characterized in order to reflect clinical conditions. This article demonstrates that the usual exploitation of low-frequency range DEP does not allow the discrimination of the undifferentiated GBM cells from the differentiated one. However, the presented study highlights the use of UHF-DEP as a relevant discriminant parameter. The proposed microfluidic lab-on-a-chip is able to follow the kinetics of U87-MG phenotype transformation in a CSC enrichment medium and the cancer stem cells phenotype acquirement.

Targeted Sub-Attomole Cancer Biomarker Detection Based on Phase Singularity 2D Nanomaterial-Enhanced Plasmonic Biosensor.

HIGHLIGHTS: A zero-reflection-induced phase singularity is achieved through precisely controlling the resonance characteristics using two-dimensional nanomaterials. An atomically thin nano-layer having a high absorption coefficient is exploited to enhance the zero-reflection dip, which has led to the subsequent phase singularity and thus a giant lateral position shift. We have improved the detection limit of low molecular weight molecules by more than three orders of magnitude compared to current state-of-art nanomaterial-enhanced plasmonic sensors. Detection of small cancer biomarkers with low molecular weight and a low concentration range has always been challenging yet urgent in many clinical applications such as diagnosing early-stage cancer, monitoring treatment and detecting relapse. Here, a highly enhanced plasmonic biosensor that can overcome this challenge is developed using atomically thin two-dimensional phase change nanomaterial. By precisely engineering the configuration with atomically thin materials, the phase singularity has been successfully achieved with a significantly enhanced lateral position shift effect. Based on our knowledge, it is the first experimental demonstration of a lateral position signal change > 340 µm at a sensing interface from all optical techniques. With this enhanced plasmonic effect, the detection limit has been experimentally demonstrated to be 10-15 mol L-1 for TNF-α cancer marker, which has been found in various human diseases including inflammatory diseases and different kinds of cancer. The as-reported novel integration of atomically thin Ge2Sb2Te5 with plasmonic substrate, which results in a phase singularity and thus a giant lateral position shift, enables the detection of cancer markers with low molecular weight at femtomolar level. These results will definitely hold promising potential in biomedical application and clinical diagnostics.

Autophagy inhibition reinforces stemness together with exit from dormancy of polydisperse glioblastoma stem cells.

Therapeutic resistance and infiltrative capacities justify the aggressiveness of glioblastoma. This is due to cellular heterogeneity, especially the presence of stemness-related cells, i.e. Cancer Stem Cells (CSC). Previous studies focused on autophagy and its role in CSCs maintenance; these studies gave conflicting results as they reported either sustaining or disruptive effects. In the present work, we silenced two autophagy related genes -either Beclin1 or ATG5- by shRNA and we explored the ensuing consequences on CSCs markers' expression and functionalities. Our results showed that the down regulation of autophagy led to enhancement in expression of CSCs markers, while proliferation and clonogenicity were boosted. Temozolomide (TMZ) treatment failed to induce apoptotic death in shBeclin1-transfected cells, contrary to control. We optimized the cellular subset analysis with the use of Sedimentation Field Flow Fractionation, a biological event monitoring- and cell sorting-dedicated technique. Fractograms of both shBeclin1 and shATG5 cells exhibited a shift of elution peak as compared with control cells, showing cellular dispersion and intrinsic sub-fraction modifications. The classical stemness fraction (i.e. F3) highlighted data obtained with the overall cellular population, exhibiting enhancement of stemness markers and escape from dormancy. Our results contributed to illustrate CSCs polydispersity and to show how these cells develop capacity to bypass autophagy inhibition, thanks to their acute adaptability and plasticity.

Articular manifestations in primary Sjögren's syndrome: clinical significance and prognosis of 188 patients.

OBJECTIVES: Articular manifestations (AMs) occurred in approximately 30-60% of patients with primary SS (pSS). We conducted the current study to describe clinical presentation, specific treatment and to report clinical outcome of pSS patients with AM in a large bicentric French cohort. METHODS: Clinical, biological and immunological features of 419 consecutive patients with pSS were recorded in order to describe the clinical and immunological course of pSS AM and to point out the impact of those rheumatological features on pSS evolution. RESULTS: A total of 188 patients with pSS (172 women, 16 men) exhibited AM. They preceded sicca symptoms in 32, were simultaneous to pSS diagnosis in 98 and followed diagnosis in 59 patients. Clinical presentation was polyarticular and concerned mostly peripheral joints (synovitis, n = 66). Symptoms responded readily to symptomatic treatment in 45 cases (24%). DMARDs or immunosuppressive treatments were introduced in 133 patients: HCQ (n = 111), corticosteroid (n = 53), MTX (n = 12), SSZ (n = 6), AZA (n = 3), LEF (n = 1), etanercept (n = 1) and allochrysine (n = 1). Only one case of RA was diagnosed during the evolution. Statistical analysis identified clinical and biological factors associated with AM (P < or = 0.05): RP, muscular manifestations, renal involvement, peripheral neuropathy, cutaneous vasculitis, and positivity of RF, anti-SSB antibodies and cryoglobulinaemia. Patients with AM at diagnosis were characterized by a multisystemic involvement at the end of the follow-up period (P < 0.001). CONCLUSION: Although AMs are frequent and usually mild in pSS, these manifestations are associated with a pluri-systemic involvement of pSS.

Role of endogenous brain-derived neurotrophic factor and sortilin in B cell survival.

Brain-derived neurotrophic factor (BDNF), a major neuronal growth factor, is also known to exert an antiapoptotic effect in myeloma cells. Whereas BDNF secretion was described in B lymphocytes, the ability of B cells to produce sortilin, its transport protein, was not previously reported. We studied BDNF production and the expression of its receptors, tyrosine protein kinase receptor B and p75 neurotrophin receptor in the human pre-B, mature, and plasmacytic malignant B cell lines under normal and stress culture conditions (serum deprivation, Fas activation, or their combination). BDNF secretion was enhanced by serum deprivation and exerted an antiapoptotic effect, as demonstrated by neutralization experiments with antagonistic Ab. The precursor form, pro-BDNF, also secreted by B cells, decreases under stress conditions in contrast to BDNF production. Stress conditions induced the membranous expression of p75 neurotrophin receptor and tyrosine protein kinase receptor B, maximal in mature B cells, contrasting with the sequestration of both receptors in normal culture. By blocking Ab and small interfering RNA, we evidenced that BDNF production and its survival function are depending on sortilin, a protein regulating neurotrophin transport in neurons, which was not previously described in B cells. Therefore, in mature B cell lines, an autocrine BDNF production is up-regulated by stress culture conditions and exerts a modulation of apoptosis through the sortilin pathway. This could be of importance to elucidate certain drug resistances of malignant B cells. In addition, primary B lymphocytes contained sortilin and produced BDNF after mitogenic activation, which suggests that sortilin and BDNF might be implicated in the survival and activation of normal B cells also.