Nuove specificit\ue0 nell'ambito degli Autoanticorpi Antinucleo: anticorpi anti-DFS70

In recent years, a new specificity of ANA has been described on HEp2 cells, with a fluoroscopic pattern called "Dense fine speckled" (DFS). Since the antigen associated with this fluoroscopic pattern is a protein of 70 kDa this antigen has been referred to as DFS70 and, among the 28 fluoroscopic pattern is identified by ICAP, AC-02 is the frame of reference. Recently it has been suggested a role of DFS70 in association with conditions of Thrombophilia. In a French multicentric study Marler and collaborators highlighted in IFI a frequency of anti-DFS70 equal to 11% in subjects affected by thrombotic events or obstetric complications, towards 3% found in the control population. The aim of the study was to verify, in a wide case, the association between anti-DFS70 and thrombotic events. A total of 633 sera of which 413 of patients affected by thrombotic events and 220 control sera were analysed. 116 sera of PCs with antiphosphopid antibody (APS) syndrome have been investigated; 77 sera of PZ with obstetric complications (ABO); 130 sera of pz with deep venous thrombosis; 68 sera of PZ with arterial thrombosis; 22 sera of PZ with arterial or venous thrombosis in heiric therapy. For control we have been studied: 92 sera of PZ in TAO/NAO for atrial fibrillation; 28 Sera of PZ in heiric therapy but without thrombosis; And 100 sera of donors. 12 centers participated in the collection of sera, of which 2 were involved in dosages and 1 center for data collection. The study was conducted in double blind in the two different dosing centers. Center number 1 has performed the IFI on the collected sera (HEp-2 Euroimmun). Center number 2 has performed, on the same sera, the specific research of anti-DFS70 with CLIA (Inova) method on Bioflash. The results show that: IFI DFS70 positive patients with thrombosis are only 5.5% compared to 11% indicated in the French study. In Our study, patients with thrombosis however have a double prevalence compared to controls when analyzed in IFI and triple if analyzed in CLIA. IFI has shown many unconfirmed false positives in CLIA and not even with the Immunoblot method subsequently executed and therefore we confirm that the data provided in the French study cannot be considered reliable. Only sera with double positivity for DFS70 in IFI and CLIA can be considered true positive and, despite the differences observed between the two samples (PZ with thrombosis/Controli 6/1), no statistical significance is reached

Anti-DFS70 antibodies detected by specific methods in patients with thrombosis or recurrent pregnancy loss: no evidence of an association

A dense fine speckled pattern (DFS) caused by antibodies to the DFS70 kDa nuclear protein is a relatively common finding while testing for anti-nuclear antibodies (ANA) by indirect immunofluorescence (IIF) on HEp-2 cells. However, despite many efforts and numerous studies, the clinical significance of anti-DFS70 antibodies is still unknown as they can be found in patients with various disorders and even in healthy subjects. In this study we aimed at verifying whether these antibodies are associated with thrombotic events or with unexplained recurrent pregnancy loss (RPL). We studied 443 patients with venous or arterial thrombosis or RPL and 244 controls by IIF on HEp-2 cells and by a DFS70-specific chemiluminescent immunoassay (CIA). The DFS pattern was observed in IIF in 31/443 (7.0%) patients and in 6/244 (2.5%) controls (p\u2009=\u20090.01) while anti-DFS70 specific antibodies were detected by CIA in 11 (2.5%) patients and in one (0.4%) control (p\u2009=\u20090.06). Positive samples, either by IIF or by CIA, were then assayed by a second DFS70-specific line-immunoassay (LIA) method: 83.3% of the CIA positive samples were confirmed DFS70 positive versus only 29.7% of the IIF positive samples. These findings show that IIF overestimates anti-DFS70 antibody frequency and that results obtained by specific CIA and LIA assays do not indicate that venous or arterial thrombosis or RPL are linked to a higher prevalence of anti-DFS70 antibodies

AnnoMiner is a new web-tool to integrate epigenetics, transcription factor occupancy and transcriptomics data to predict transcriptional regulators

International audienceGene expression regulation requires precise transcriptional programs, led by transcription factors in combination with epigenetic events. Recent advances in epigenomic and transcriptomic techniques provided insight into different gene regulation mechanisms. However, to date it remains challenging to understand how combinations of transcription factors together with epigenetic events control cell-type specific gene expression. We have developed the AnnoMiner web-server, an innovative and flexible tool to annotate and integrate epigenetic, and transcription factor occupancy data. First, AnnoMiner annotates user-provided peaks with gene features. Second, AnnoMiner can integrate genome binding data from two different transcriptional regulators together with gene features. Third, AnnoMiner offers to explore the transcriptional deregulation of genes nearby, or within a specified genomic region surrounding a user-provided peak. AnnoMiner’s fourth function performs transcription factor or histone modification enrichment analysis for user-provided gene lists by utilizing hundreds of public, high-quality datasets from ENCODE for the model organisms human, mouse, Drosophila and C. elegans . Thus, AnnoMiner can predict transcriptional regulators for a studied process without the strict need for chromatin data from the same process. We compared AnnoMiner to existing tools and experimentally validated several transcriptional regulators predicted by AnnoMiner to indeed contribute to muscle morphogenesis in Drosophila . AnnoMiner is freely available at http://chimborazo.ibdm.univ-mrs.fr/AnnoMiner/

Estimate of the Prevalence of Anti-Gastric Parietal Cell Autoantibodies in Healthy Individuals Is Method Dependent

Objectives Anti-parietal cell antibodies (APCA) are a serologic marker of autoimmune gastritis. Their prevalence in healthy individuals is not well defined. Methods We evaluated APCA prevalence in 515 healthy blood-donors by rat/primate tissue indirect immunofluorescence (IIF), enzyme-linked immunosorbent assay (ELISA), and immunoblot. Results Fifty-three of 515 (10.3%) subjects were positive for APCA by at least one method: 18 only by ELISA, 10 by rodent tissue IIF, and one by primate tissue IIF; 18 were positive by ELISA and primate tissue IIF, and one by ELISA and rodent tissue IIF. Two were positive by both IIF methods, and three were triple positive. APCA positivity was confirmed by immunoblot in 100% of ELISA positive, in 95.8% of positive primate tissue IIF, and in 50% of positive rat tissue IIF. Conclusions A great discrepancy in APCA prevalence detected by different methods in this cohort was apparent. Thus, the results on APCA prevalence in healthy individuals are likely method-dependent

Sphere-forming cell subsets with cancer stem cell properties in human musculoskeletal sarcomas

Musculoskeletal sarcomas are aggressive malignancies often characterized by an adverse prognosis despite the use of intense multiagent chemotherapy or molecular targeted therapy in combination to surgery and radiotherapy. Stem-like cells identified within solid tumors have been recently implicated in drug resistance, metastasis and local relapse. Here, we report the identification of putative cancer stem cells (CSCs) in sarcomas using a sphere culture system. These sarcospheres, able to grow in anchorage-independent and serum-starved conditions, express the pluripotent embryonic stem cell marker genes OCT3/4, Nanog and SOX2. Expression levels of these genes were greater in sarcospheres than in the parental tumor cultures. Importantly, the isolated tumor spheres transplanted into mice were tumorigenic and capable of recapitulating the human disease. Finally, we demonstrated that low (1%) O2 conditions, reproducing those found within the tumor microenvironment, significantly increase the number and the size of sarcospheres. The sphere formation assay is, therefore, a valuable method for the isolation of putative CSCs from human sarcomas and its efficiency is improved by controlling oxygen availability. This method provides a reliable preclinical model that can be used for future studies aimed at investigating crucial aspects of sarcoma biology, such as resistance to treatments and relapse

Organoids as a new model for improving regenerative medicine and cancer personalized therapy in renal diseases

The pressure towards innovation and creation of new model systems in regenerative medicine and cancer research has fostered the development of novel potential therapeutic applications. Kidney injuries provoke a high request of organ transplants making it the most demanding system in the field of regenerative medicine. Furthermore, renal cancer frequently threaten patients\u2019 life and aggressive forms still remain difficult to treat. Ethical issues related to the use of embryonic stem cells, has fueled research on adult, patient-specific pluripotent stem cells as a model for discovery and therapeutic development, but to date, normal and cancerous renal experimental models are lacking. Several research groups are focusing on the development of organoid cultures. Since organoids mimic the original tissue architecture in vitro, they represent an excellent model for tissue engineering studies and cancer therapy testing. We established normal and tumor renal cell carcinoma organoids previously maintained in a heterogeneous multi-clone stem cell-like enriching medium. Starting from adult normal kidney specimens, we were able to isolate and propagate organoid 3D-structures composed of both differentiated and undifferentiated cells while expressing nephron specific markers. Furthermore, we were capable to establish organoids derived from cancer tissues although with a success rate inferior to that of their normal counterpart. Cancer cultures displayed epithelial and mesenchymal phenotype while retaining tumor specific markers. Of note, tumor organoids recapitulated neoplastic masses when orthotopically injected into immunocompromised mice. Our data suggest an innovative approach of long-term establishment of normal- and cancer-derived renal organoids obtained from cultures of fleshly dissociated adult tissues. Our results pave the way to organ replacement pioneering strategies as well as to new models for studying drug-induced nephrotoxicity and renal diseases. Along similar lines, deriving organoids from renal cancer patients opens unprecedented opportunities for generation of preclinical models aimed at improving therapeutic treatments