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Supplementary Data from Identification of a New Panel of Serum Autoantibodies Associated with the Presence of In situ Carcinoma of the Breast in Younger Women

Purpose: We examined the feasibility of using a panel of autoantibodies to multiple tumor-associated proteins as a method for early detection of breast cancer and, more particularly, carcinoma in situ (CIS).Experimental Design: PPIA, PRDX2, and FKBP52 were identified as early-stage breast cancer autoantigens by proteomic approaches. The seroreactivity of a panel of antibodies consisting of these three antigens and two previously described autoantigens, HSP60 and MUC1, was tested on 235 samples (60 from primary breast cancer patients, 82 from CIS patients, and 93 from healthy controls) with the use of specific ELISAs. FKBP52, PPIA, and PRDX2 mRNA and protein expression levels were evaluated by reverse transcription-PCR and immunohistochemistry in early-stage breast tumors.Results: Three of five autoantibodies, FKBP52, PPIA, and PRDX2, showed significantly increased reactivity in primary breast cancer and CIS compared with healthy controls. When combined, the five markers significantly discriminated primary breast cancer [receiver operating characteristic area under the curve, 0.73; 95% confidence interval (95% CI), 0.60-0.79] and CIS (receiver operating characteristic area under the curve, 0.80; 95% CI, 0.71-0.85) from healthy individuals. Importantly, the receiver operating characteristic–area under the curve value of the autoantibody panel was able to distinguish CIS, including high grades, from healthy controls in women under the age of 50 years (receiver operating characteristic area under the curve, 0.85; 95% CI, 0.61-0.92). Finally, only FKBP52 mRNA and protein levels were found to be increased in CIS and primary breast cancer compared with healthy breast tissue.Conclusions: This autoantibody assay against a panel of five antigens allows for an accurate discrimination between early-stage breast cancer, especially CIS, and healthy individuals. These results could be of interest in detecting early breast cancer as an aid to mammography, especially in women under the age of 50 years with aggressive cancers.

Background West Nile virus (WNV) and Usutu virus (USUV), two closely related flaviviruses, mainly follow an enzootic cycle involving mosquitoes and birds, but also infect humans and other mammals. Since 2010, their epidemiological situation may have shifted from irregular epidemics to endemicity in several European regions; this requires confirmation, as it could have implications for risk assessment and surveillance strategies. Aim To explore the seroprevalence in animals and humans and potential endemicity of WNV and USUV in Southern France, given a long history of WNV outbreaks and the only severe human USUV case in France in this region. Methods We evaluated the prevalence of WNV and USUV in a repeated cross-sectional study by serological and molecular analyses of human, dog, horse, bird and mosquito samples in the Camargue area, including the city of Montpellier, between 2016 and 2020. Results We observed the active transmission of both viruses and higher USUV prevalence in humans, dogs, birds and mosquitoes, while WNV prevalence was higher in horses. In 500 human samples, 15 were positive for USUV and 6 for WNV. Genetic data showed that the same lineages, WNV lineage 1a and USUV lineage Africa 3, were found in mosquitoes in 2015, 2018 and 2020. Conclusion These findings support existing literature suggesting endemisation in the study region and contribute to a better understanding of USUV and WNV circulation in Southern France. Our study underlines the importance of a One Health approach for the surveillance of these viruses.

Constitution of biobank of human tissues requires careful handling and storage of biological material, to guarantee the quality of samples. Tissue preparation is also critical for further applications such as transcriptomic profiling. In this study, our aim was to evaluate the impact of different disruption techniques (FastPrep-24 instrument, GentleMACS dissociator, and syringe/needle) and homogenizing buffers (RLT versus QIAzol) on RNA purity and quality of metabolic tissues (adipose tissues, liver and skeletal muscle) present in the COMET Biobank. For all homogenization methods used and tissue types, the A260/280 ratios reached values ≥ 1.8, which are in the range of what is found in human tissues and cell lines, while the A260/230 ratios were however ≤ 1.8, with the lowest value obtained with GentleMACS Dissociator. In addition, GentleMACS Dissociator combined with QIAzol reagent gave the highest RIN value and 28S/18S ratio for all tissues tested, except for muscle. Performing RT-qPCR, Ct values for different housekeeping genes can be influenced by extraction methods and RNA quality of samples. In conclusion, we have demonstrated that different disruption techniques and homogenizing buffers impact the purity and some quality markers of RNA, and can also impact quantification of mRNAs by RT-qPCR in human metabolic tissues.

In a rat model of metabolic syndrome (MS), our previous studies have shown that dietary sodium restriction prevents both metabolic and cardiac damages associated with MS. Our aim is now to investigate whether the beneficial effects of sodium restriction could be mediated by endothelial to mesenchymal (EndoMT) transition in the myocardium thereby preventing cardiac fibrosis.High fructose (60%) Sprague Dawley rats were divided into 2 groups: low sodium (in vitro model of EndoMT, using primary human aortic endothelial (HAoE) cells that were transdifferentiated with TGF-β2 (10ng/ml). We evidenced in HAoE cells the co-expression of Pecam-1 and collagen-1, as a signature of EndoMT. Especially, fibulin 5, one of the genes identified by transcriptomics was found upregulated (13 folds) in the presence of TGF-β2, confirming its potential role in EndoMT. Our study shows that EndoMT is involved in the prevention of cardiac fibrosis by sodium restriction in our rat model of MS. We also confirmed the involvement of ew genes that could be of interest to improve the management of cardiac fibrosis.

West Nile virus (WNV) and Usutu virus (USUV) are zoonotic arboviruses. These flaviviruses are mainly maintained in the environment through an enzootic cycle involving mosquitoes and birds. Horses and humans are incidental, dead-end hosts, but can develop severe neurological disorders. Nevertheless, there is little data regarding the involvement of other mammals in the epidemiology of these arboviruses. In this study, we performed a serosurvey to assess exposure to these viruses in captive birds and mammals in a zoo situated in the south of France, an area described for the circulation of these two viruses. A total of 411 samples comprising of 70 species were collected over 16 years from 2003 to 2019. The samples were first tested by a competitive enzyme-linked immunosorbent assay. The positive sera were then tested using virus-specific microneutralization tests against USUV and WNV. USUV seroprevalence in birds was 10 times higher than that of WNV (14.59% versus 1.46%, respectively). Among birds, greater rhea (Rhea Americana) and common peafowl (Pavo cristatus) exhibited the highest USUV seroprevalence. Infections occurred mainly between 2016&ndash, 2018 corresponding to a period of high circulation of these viruses in Europe. In mammalian species, antibodies against WNV were detected in one dama gazelle (Nanger dama) whereas serological evidence of USUV infection was observed in several Canidae, especially in African wild dogs (Lycaon pictus). Our study helps to better understand the exposure of captive species to WNV and USUV and to identify potential host species to include in surveillance programs in zoos.

Introduction et but de l’etude Dans les pays developpes, le syndrome metabolique (SM), associant une hypertension arterielle et une insulinoresistance, concerne 25 a 45 % des hommes de plus de 40 ans, les predisposant a un risque d’AVC multiplie par 3. L’augmentation de ces troubles metaboliques va de concert avec la consommation de sucres en constante augmentation dans les pays developpes. L’hypertension participe a la fibrose du myocarde, ce qui aboutit a une dysfonction diastolique et au developpement d’une hypertrophie ventriculaire gauche. Dans ce cadre, nous avons montre qu’un regime alimentaire pauvre en sel pouvait prevenir les dommages cardiaques d’une hypertension associee a une insulinoresistance dans un modele de rat. Nous avons recemment identifie par transcriptomique dans ce modele un groupe de genes module par la restriction sodee, et dont certains sont plus particulierement impliques dans un processus appele transition endothelio-mesenchymateuse. Notre objectif a ete de montrer que le regime desode regule negativement la transition endothelio-mesenchymateuse (TEndM) dans le cœur des rats atteints de syndrome metabolique, ce qui previent le phenomene de fibrose. Materiel et methodes Notre modele d’etude est le rat Sprague Dawley nourri avec 60 % de fructose. Les rats sont divises en 2 groupes : groupe normosode fructose (NSF) (0,64 % NaCl), et groupe desode fructose (DSF) avec 0,01 % de NaCl dans la nourriture. Au bout de 4 semaines de regime, l’angiotensine II est perfusee (200 ng/kg/min) durant 4 semaines additionnelles. Nous avons explore l’expression de marqueurs de la TEndM par western blot (WB) et leur colocalisation par immunofluorescence dans les ventricules gauches des rats. La confirmation des variations d’expression en transcriptomique a ete realisee par qPCR et par WB. La quantification est realisee par le logiciel Image J. Les tests statistiques utilises sont le test de Student ou de Mann et Whitney. Resultats et analyse statistique Deux proteines mesenchymateuses, l’actine du muscle lisse (αSMA), et la vimentine, voient leur expression significativement diminuee en regime desode (p Conclusion Nous montrons que la transition endothelio-mesenchymateuse est impliquee dans la prevention du phenomene de fibrose par le regime desode dans notre modele de SM. Nous avons de plus identifie et valide 17 genes modules par la restriction sodee dont deux d’entre eux inconnus jusqu’alors. Nous esperons par cette etude apporter un argument supplementaire de l’interet d’un regime alimentaire appauvri en sel dans la prevention des dommages cardiaques au cours du syndrome metabolique.

Purpose: Among the FKBP family members, FKBP4 has been described to have a potential role in tumorigenesis, and as a putative tissue marker. We previously showed that FKBP4, an HSP90-associated co-chaperone, can elicit immune response as a tumor-specific antigen, and are overexpressed in breast cancer. Experimental design: In this study, we examined how loss of FKBP4 affect breast cancer progression and exploited protein interactomics to gain mechanistic insight into this process. Results: We found that FKBP4 expression is associated with breast cancer progression and prognosis, especially of ER-negative breast cancer. Furthermore, FKBP4 depletion specifically reduces cell growth and proliferation of triple negative breast cancer cell model and xenograft tumor model. Using specific protein interactome strategy by BirA proximity-dependent biotin identification, we demonstrated that FKBP4 is a novel PI3K-Akt-mTOR proximal interacting protein. Conclusion: Our results suggest that FKBP4 interacts with PI3K and can enhance Akt activation through PDK1 and mTORC2.

International audience; As of 2018, Alzheimer's disease (AD) is the most common form of neurodegenerative dementia. It contributes to a progressive neuron loss, deterioration of memory, and cognitive impairment. Current therapies may provide a symptomatic benefit, but do not treat the underlying process. Ongoing researches focus on understanding the causal mechanisms and finding neuropathological hallmarks of AD. Therapeutic approaches targeting senile plaques or neurofibrillary tangles have not yet resulted in a significant cognitive improvement. However, recent data according to the analysis of AD clinical trials (clinicaltrials.gov database) show promising results. This literature review aims at summarizing the recent advances and at highlighting the most promising results of the ongoing researches. It compares the merits of small-molecules, antibodies, cell, and gene-based therapies and emphasizes the need for treatment at earlier stages of the disease.

International audience; In the view of the relationships between excessive sodium intake, immunity and target organ damage, we hypothesized that reduction in dietary sodium would be beneficial in the prevention of cardiac alterations through a restrained local immunity response in a rat model of metabolic syndrome. Sprague-Dawley rats were fed a 60% fructose diet with either a normal sodium (0.64% NaCl) or a low sodium content (b 0.01% NaCl) for 8 weeks. After 4 weeks, rats were infused or not with angiotensin II (200 ng·kg −1 ·min −1 , sc) for 4 weeks. Tail-cuff blood pressure was determined in conscious rats. Heart and left ventricle weight, cardiomyocyte size, and cardiac fibrosis were evaluated. We performed a transcriptomic analysis in order to identify differentially regulated cardiac mRNAs between normal and low sodium diets. We validated those results using qPCR and immunohistochemistry. Angiotensin II-induced blood pressure rise was blunted (~50%) in the low-sodium fed rats while cardiac hyper-trophy and fibrosis were prevented. Transcriptomic analysis revealed 66 differentially regulated genes including 13 downregulated genes under the low sodium diet and implicated in the innate immune response. This was confirmed by reduced cardiac macrophages infiltration under the low sodium diet. Dietary sodium restriction prevents structural alterations of the heart of rats with fructose-induced insulin resistance and angiotensin II-hypertension. The reduction of cardiac inflammation and macrophage infiltration suggests that innate immunity has an important role in the beneficial effect of sodium restriction on cardiac remodeling.

International audience; Atherosclerosis is a chronic inflammatory disease of the arterial wall. It is already well established that several immune cells (macrophages, T lymphocytes, etc.) modulate atherosclerosis progression whereas the role of the different subpopulations of B lymphocytes emerged only recently. B1 lymphocytes secrete protective IgM antibodies that act as scavenger of deleterious molecules whereas B2 lymphocytes probably worsen the disease by activating pro-inflammatory T lymphocytes. The outcome of these opposite functional properties of B lymphocytes on the evolution of arterial lesions may vary depending on their local environment during the different stages of the disease. In this review, we emphasize recent progresses in understanding the specific contribution of B lymphocytes to atherosclerosis and discuss the interest of targeting them to improve therapy.; L’athérosclérose est une maladie inflammatoire chronique de la paroi artérielle. Il est bien établi que certaines cellules immunitaires (macrophages, lymphocytes T, etc.) interviennent dans la progression de l’athérosclérose. Cependant, le rôle des différentes sous-populations de lymphocytes B n’a été évoqué que récemment. Les lymphocytes B1 sécrètent des anticorps IgM protecteurs permettant l’élimination de molécules délétères, alors que les lymphocytes B2 aggravent probablement la maladie en activant des lymphocytes T pro-inflammatoires. La résultante de ces propriétés fonctionnelles opposées des lymphocytes B sur l’évolution des lésions artérielles peut varier selon leur environnement tissulaire au cours des différents stades de la maladie. Dans cette revue, nous mettons l’accent sur les progrès récents dans la compréhension de la contribution spécifique des lymphocytes B au processus athéromateux, permettant de considérer ces derniers comme des cibles potentielles d’une démarche thérapeutique

International audience; B cells regulate immune responses during infectious, inflammatory and autoimmune diseases. Beside their unique and characteristic antibody production, B lymphocytes can modulate physiological and pathological processes by presenting antigens or synthesizing signaling molecules. In human and mouse diseases, immuno-intervention, targeting B cells, has revealed and highlighted their antibody-independent regulatory contribution. In this review, we focus on B cell-cytokine production, which is commonly disturbed in inflammatory disorders, and describe the B cell cytokine profile in different diseases. Finally, we discuss some key issues for future B cell-targeted therapies.

It is now well established that an immune response to cancer is elicited in humans, as demonstrated in part by the identification of autoantibodies against a number of tumor-associated antigens in sera from patients with different types of cancer. During these past few years, proteomic approaches have been developed to identify tumor-associated antigens and their cognate autoantibodies. Detection of a panel of serum autoantibodies has thus been proposed as a new method for early cancer diagnosis. Early detection seems to be particularly adequate in high-risk populations, such as heavy smokers for lung cancer or in women with high mammographic density for breast cancer. In this review, we highlight the features of serum autoantibody biomarkers and outline the proteomic strategies employed to identify and validate their use in clinical practice for cancer screening and diagnosis. We particularly emphasize the clinical utility of autoantibody signatures, using the examples of lung and breast cancer. Finally, we discuss the challenges remaining for clinical validation.

The chemokine (C-C motif) receptor CCR5 and its ligand CCL5 play key roles in the intra-articular recruitment of peripheral blood mononuclear cells (PBMC) in rheumatoid arthritis (RA). Therefore, using quantitative cytofluorometry, we followed T4 cell surface CCR5 density in 27 subjects with RA before and after treatment with the anti-CD20 monoclonal antibody rituximab. We observed low T4 cell surface CCR5 densities before treatment, which correlated positively with disease activity, as determined using a disease activity score evaluated on 28 joints (DAS 28), and negatively with CCL5 mRNA concentrations in PBMC, contrasting with a high proportion of intracellular CCR5 molecules, a pattern compatible with ligand-induced CCR5 internalization. At 3 months post-treatment, CCL5 mRNA expression in PBMC declined, whereas T4 cell surface CCR5 densities increased proportionally to the decrease in DAS 28. Thus, peripheral blood T4 cell surface CCR5 density is a good surrogate marker of RA activity and of the efficiency of anti-CD20 therapy.

International audience; Discovery of new serum biomarkers exhibiting an increased sensitivity and specificity for cancer are of major importance for diagnosis improvement. There is considerable evidence for an immune response to cancer in humans, as demonstrated in part by the identification of autoantibodies against a number of tumour-associated antigens in sera from patients with different cancer types. Thus, identification of tumour-associated antigens and their associated antibodies is a promising strategy to find relevant biomarkers. Proteomic approaches such as SEREX and SERPA have allowed identification of great numbers of antigens and their cognate autoantibodies during these past few years. They show many advantages, and allowed identification of relevant autoantigens in different types of cancer. However, they are also time consuming, and lack sensibility and specificity. To circumvent these drawbacks, new proteomic techniques, based on protein or antibody arrays, allow high throughput analysis of multiple targets in a single experiment. Specific combinations of markers should thus be identified, theoretically being more efficient to detect a tumor compared to a single marker. In conclusion, these approaches promise great advances in the field of biomarkers for cancer. They also further need to be validated for clinical application on large populations.

Atherosclerosis is a chronic inflammatory disease of the arterial wall. It is already well established that several immune cells (macrophages, T lymphocytes, etc.) modulate atherosclerosis progression whereas the role of the different subpopulations of B lymphocytes emerged only recently. B1 lymphocytes secrete protective IgM antibodies that act as scavenger of deleterious molecules whereas B2 lymphocytes probably worsen the disease by activating pro-inflammatory T lymphocytes. The outcome of these opposite functional properties of B lymphocytes on the evolution of arterial lesions may vary depending on their local environment during the different stages of the disease. In this review, we emphasize recent progresses in understanding the specific contribution of B lymphocytes to atherosclerosis and discuss the interest of targeting them to improve therapy.

Animal models of atherosclerosis suggest that B cells have contradictory protective or proatherogenic effects that are also subset and context dependent. To further understand the pathophysiology of human atheroma, we characterized local Ig production and functional properties of resident B cells in human arterial lesions. Ig repertoires were analyzed by RT-PCR in carotid endarterectomy samples. Cytokine, differentiation marker and transcription factor mRNA expression was studied on arterial wall lymphocytes isolated by laser capture microdissection. Ig sequence analysis revealed that individual samples each contained a limited number of B cell clones. Functional α and γ mRNAs made up the majority of H chain mRNAs in the adventitia. Clonal evolution of Ig V regions, expression of activation-induced cytidine deaminase, clonal H chain switch, and an inverted λ/κ ratio of Ig L chain usage indicated that a local differentiation process was taking place in arterial walls. Clonotypic markers revealed different plaque and adventitia Ig repertoires and a B cell recirculation between adventitia and draining lymph nodes. Microdissected mononuclear cells had an activated phenotype expressing IL-6, GM-CSF, and TNF-α, whereas IL-2, IL-4, IL-10, M-CSF, and IFN-γ were not detected. Adventitial oligoclonal resident B cells of atherosclerotic patients are mainly mature B2 (conventional) CD20− plasmablasts lacking markers of terminal differentiation to plasma cell (CD138 and Blimp-1). They present hallmarks of Ag-driven maturation and could act on inflammation and disease progression directly or by promoting polarization of other immune cells.

International audience; Biomarkers that show high sensitivity and specificity are needed for the early diagnosis and prognosis of cancer. An immune response to cancer is elicited in humans, as demonstrated, in part, by the identification of autoantibodies against a number of tumor-associated antigen (TAAs) in sera from patients with different types of cancer. Identification of TAAs and their cognate autoantibodies is a promising strategy for the discovery of relevant biomarkers. During the past few years, three proteomic approaches, including serological identification of antigens by recombinant expression cloning (SEREX), serological proteome analysis (SERPA) and, more recently, protein microarrays, have been the dominant strategies used to identify TAAs and their cognate autoantibodies. In this review, we aim to describe the advantages, drawbacks and recent improvements of these approaches for the study of humoral responses. Finally, we discuss the definition of autoantibody signatures to improve sensitivity for the development of clinically relevant tests.

International audience; There is an important need to find relevant biomarkers that show high sensitivity and specificity for early diagnosis and prognosis of cancer. An immune response to cancer is elicited in humans, as demonstrated in part by the identification of autoantibodies against a number of tumor-associated antigens in sera from patients with different types of cancer. Identification of tumor-associated antigens and their cognate autoantibodies is a promising strategy for the discovery of relevant biomarkers. During the past few years, proteomic approaches, including SEREX, SERPA and, more recently, protein microarrays, have been the dominant strategies used to identify tumor-associated antigens and their cognate autoantibodies. In this review, we aim to describe advantages, drawbacks, and recent improvements of these approaches for the study of humoral responses.

Purpose: We examined the feasibility of using a panel of autoantibodies to multiple tumor-associated proteins as a method for early detection of breast cancer and, more particularly, carcinoma in situ (CIS). Experimental Design: PPIA, PRDX2, and FKBP52 were identified as early-stage breast cancer autoantigens by proteomic approaches. The seroreactivity of a panel of antibodies consisting of these three antigens and two previously described autoantigens, HSP60 and MUC1, was tested on 235 samples (60 from primary breast cancer patients, 82 from CIS patients, and 93 from healthy controls) with the use of specific ELISAs. FKBP52, PPIA, and PRDX2 mRNA and protein expression levels were evaluated by reverse transcription-PCR and immunohistochemistry in early-stage breast tumors. Results: Three of five autoantibodies, FKBP52, PPIA, and PRDX2, showed significantly increased reactivity in primary breast cancer and CIS compared with healthy controls. When combined, the five markers significantly discriminated primary breast cancer [receiver operating characteristic area under the curve, 0.73; 95% confidence interval (95% CI), 0.60-0.79] and CIS (receiver operating characteristic area under the curve, 0.80; 95% CI, 0.71-0.85) from healthy individuals. Importantly, the receiver operating characteristic–area under the curve value of the autoantibody panel was able to distinguish CIS, including high grades, from healthy controls in women under the age of 50 years (receiver operating characteristic area under the curve, 0.85; 95% CI, 0.61-0.92). Finally, only FKBP52 mRNA and protein levels were found to be increased in CIS and primary breast cancer compared with healthy breast tissue. Conclusions: This autoantibody assay against a panel of five antigens allows for an accurate discrimination between early-stage breast cancer, especially CIS, and healthy individuals. These results could be of interest in detecting early breast cancer as an aid to mammography, especially in women under the age of 50 years with aggressive cancers.

International audience; Discovery of new serum biomarkers exhibiting an increased sensitivity and specificity for cancer are of major importance for diagnosis improvement. There is considerable evidence for an immune response to cancer in humans, as demonstrated in part by the identification of autoantibodies against a number of tumour-associated antigens in sera from patients with different cancer types. Thus, identification of tumour-associated antigens and their associated antibodies is a promising strategy to find relevant biomarkers. Proteomic approaches such as SEREX and SERPA have allowed identification of great numbers of antigens and their cognate autoantibodies during these past few years. They show many advantages, and allowed identification of relevant autoantigens in different types of cancer. However, they are also time consuming, and lack sensibility and specificity. To circumvent these drawbacks, new proteomic techniques, based on protein or antibody arrays, allow high throughput analysis of multiple targets in a single experiment. Specific combinations of markers should thus be identified, theoretically being more efficient to detect a tumor compared to a single marker. In conclusion, these approaches promise great advances in the field of biomarkers for cancer. They also further need to be validated for clinical application on large populations.

International audience; The kallikrein family (KLK) has been implicated in cancer and may be useful as tumor markers. Here, we compared the 15 KLK genes' expression in malignant and normal breast tissues using real-time quantitative PCR. Most KLKs were expressed at lower levels in breast cancer compared to normal breast tissue. The only exception was the eightfold increase level of KLK4 in breast cancer tissues (P=0.008). KLK4 level was strongly associated with tumor grade (P=0.0015). Interestingly, based on laser cell microdissection analysis and immunochemistry, the up-regulation of kallikrein 4 occurred in the surrounding stromal cells. Our findings suggest that KLK4 may be associated with the development and progression of breast cancer and suggest its potential use in breast cancer monitoring.

International audience; The detection of autoantibodies in cancer patients has been shown to constitute an excellent tool for early diagnosis. Because breast cancer still lacks early diagnostic markers, we investigated novel tumor-associated antigens and related autoantibodies in sera from patients with early stage breast cancer compared to autoimmune disease, other cancers, and healthy volunteers, using a proteomics-based approach. Among the 26 protein antigens specifically recognized by early stage breast cancer sera, we focused on Heat Shock Protein 60 (HSP60). Using ELISA, we investigated the frequency of autoantibodies directed against this protein in the sera of 240 individuals, comprising patients with either ductal carcinoma in situ (DCIS) ( n = 49) or early stage breast cancer ( n = 58), other cancers ( n = 20), autoimmune disease ( n = 20), and healthy subjects ( n = 93). Autoantibodies directed against HSP60 were present in 16/49 (31%) early stage breast cancer and 18/58 (32.6%) DCIS patients, compared to 4/93 (4.3%) healthy subjects. In particular, autoantibodies were present in 11/23 patients (47.8%) with high-grade DCIS, compared to 5/26 (19.2%) with low-grade DCIS. HSP60 mRNA levels were significantly higher in primary breast cancer compared to healthy breast tissues. Using immunohistochemistry, we found that HSP60 expression gradually increases from normal through DCIS to invasive tissues. Our results indicate that HSP60 autoantibodies may be of interest in terms of clinical utility for the early diagnosis of breast cancer and more particularly in DCIS. Moreover, HSP60 overexpression during the first steps of breast carcinogenesis may be functionally correlated to tumor growth and/or progression.

Background HIV-1 triggers infection through interaction with the CD4 receptor and the chemokine receptors, CCR5 or CXCR4, on host cells. The involvement of signaling via the chemokine receptors in viral infection remains an issue of debate. We have previously reported that Galphai1 is involved in the signaling triggered by R5 HIV-1 strains through CCR5 binding to facilitate viral replication in unstimulated peripheral blood mononuclear cells. In this study, we pursued the identification of the downstream signaling molecules in CCR5-mediated infection. We also questioned whether CXCR4 using HIV-1 strains induce the same signaling mechanism. Methods We analyzed by western blotting the coreceptor-mediated activation of various mitogen-acitvated protein kinases, including extracellular signal-regulated kinase (ERK)1/2, p38 and c-jun N-terminal kinase in non-stimulated human peripheral blood mononuclear cells. The involvement of Galphai protein in ERK1/2 activation was tested using pertussis toxin. Using real-time PCR, we studied the role of ERK1/2 in the life cycle of HIV-1. Results We found that pertussis toxin inhibited the replication of X4 as well as R5 strains. Furthermore, both strains activated a pertussis toxin-sensitive mitogen-activated protein kinase pathway involving mitogen-activated protein kinase kinases-1/2 and ERK1/2. The inhibition of ERK1/2 activation by U0126 and PD98059 blocked both R5 and X4 HIV-1 replication. Furthermore, ERK1/2 activity was required for the completion of HIV-1 reverse transcription. Conclusion Our results show that R5 and X4 HIV-1 strains induce the same Galphai-dependent ERK pathway that facilitates reverse transcription. The identification of the signaling pathway required for optimal viral replication sheds a new light on HIV physiopathology and opens new therapeutic possibilities.

Background Prenatal diagnosis of chromosomal abnormalities by cytogenetic analysis is time-consuming, expensive, and requires highly qualified technicians. Rapid diagnosis of aneuploidies followed by reassurance of women with normal results can be performed by molecular analysis of uncultured foetal cells. In the present study, we developed a proteomic fingerprinting approach coupled with a statistical classification method to improve diagnosis of aneuploidies, including trisomies 13, 18, and 21, in amniotic fluid samples. Results The proteomic spectra obtained from 52 pregnant women were compiled, normalized, and mass peaks with mass-to-charge ratios between 2.5 and 50 kDa identified. Peak information was combined together and analysed using univariate statistics. Among the 208 expressed protein peaks, 40 differed significantly between aneuploid and non aneuploid samples, with AUC diagnostic values ranging from 0.71 to 0.91. Hierarchical clustering, principal component analysis and support vector machine (SVM) analysis were performed. Two class predictor models were defined from the training set, which resulted in a prediction accuracy of 92.3% and 96.43%, respectively. Using an external and independent validation set, diagnostic accuracies were maintained at 87.5% and 91.67%, respectively. Conclusion This pilot study demonstrates the potential interest of protein expression signature in the identification of new potential biological markers that might be helpful for the rapid clinical management of high-risk pregnancies.

As we have recently shown that the number of CCR5 molecules at the cell surface determines the efficiency of its function as a chemokine receptor, we tested the hypothesis that cell surface CCR5 density could influence the intensity of T lymphocyte recruitment into the rheumatoid joint. For this purpose, we established two Jurkat cell line-derived clones that differed only by their cell surface CCR5 densities. We studied their chemotaxis towards TNF-α-transduced rheumatoid synoviocytes supernatant. The Jurkat cell subline that expressed the higher cell surface CCR5 density migrated more intensively towards the supernatant of TNF-α-transduced synoviocytes than the Jurkat cell subline that expressed a lower surface CCR5 density. Moreover, this migration was blocked by an anti-CCR5 mAb. The CCR5 density on T cell surface, which is constant over time for a given individual, but varies drastically from one individual to another, might thus be a factor determining the intensity of joint inflammation in the course of RA.

We have shown that the intensity of expression of the C-C chemokine receptor CCR5 at the single CD4(+) cell level strongly determines the efficiency of its function as a coreceptor for human immunodeficiency virus type 1. By analogy, we examined if the number of CCR5 molecules at the cell surface might determine its chemotactic response to CCR5 ligands. To test this hypothesis, we measured by flow cytometry the migration of primary human T cells towards the CCR5-binding chemokine CCL5 in vitro. First, we observed a dose-dependent blockage of this migration exerted by an anti-CCR5 monoclonal antibody. Second, we sorted peripheral blood mononuclear cells into five subpopulations expressing various cell surface CCR5 densities, and observed a correlation between the intensity of migration towards CCL5 and the level of CCR5 expression on these subpopulations. Third, we transduced CCR5(+) peripheral blood mononuclear cells with the CCR5 gene, and observed that the CCR5 over-expression induced an over-migration towards CCL5. Finally, we observed in healthy donors a correlation between the chemotactic response of peripheral blood CD8(+) T cell to CCL5 and their level of surface CCR5 expression. T-cell surface CCR5 density, which is constant over time for a given individual, but varies drastically among individuals, might therefore be an important personal determinant of T-cell migration in many biological situations where CCR5-binding chemokines play a role, such as graft rejection, T helper 1-mediated auto-immune diseases, and infectious diseases involving CCR5. Moreover, our data highlight the therapeutic potential of CCR5 antagonists in these situations.

Objectif L’objectif est de comparer l’effet d’un regime desode (DS, Materiels et methodes La pression arterielle (TCP) est mesuree avant et apres AngII. L’index de masse cardiaque (IMCard, poids du cœur/poids du corps) et la taille des cardiomyocytes sont mesures en fin d’etude. L’expression cardiaque de divers genes est determinee par PCR en temps reel (qPCR). La presence de monocytes/macrophages est determinee par immunomarquage et l’expression du CD68. Le facteur de transcription NF-kBp65 et la presence de lymphocytes T CD4 + et CD8 + ainsi que de lymphocytes B (CD20) est mesuree. Resultats L’AngII induit une augmentation de la TCP de 40 ± 5 % en regime NSF et de 20 ± 4 % en regime DSF. L’IMCard est augmente par l’AngII dans les deux regimes NSF et DSF (20 et 10 %, respectivement). L’augmentation de l’aire des cardiomyocytes induite par l’AngII en regime NSF n’est pas observee en regime DSF. L’infiltration macrophagique cardiaque (marquage CD68) et l’expression de CD68 est significativement plus basse en regime desode en absence (9,5 ± 0,8.10−2 vs 7,1 ± 0,5.10−2) et en presence d’AngII (10,6 ± 1,5.10−2 vs 5,6 ± 0,4.10−2). De meme, l’expression de CD4 et de CD8 est reduite en regime desode en absence ou en presence d’AngII. Aucune difference significative n’est retrouvee pour CD20. L’expression de NF-kBp65 est reduite significativement par le regime desode en presence ou absence d’AngII. Conclusion L’effet benefique cardiaque de la restriction sodee est conserve quand la dysfonction metabolique est associee a une hypertension arterielle. L’effet benefique est vraisemblablement lie a la baisse de pression arterielle ainsi qu’a la diminution de l’inflammation et de l’infiltration des cellules de l’immunite (macrophages, cellules T mais pas B) au niveau cardiaque.

Objectif Nous avons montre que la restriction sodee prevenait les lesions renales associees a l’insulinoresistance en partie par un effet anti-inflammatoire. L’objectif de cette etude est d’evaluer l’effet du regime desode sur les atteintes cardiovasculaires induites par un regime riche en fructose, modele d’insulinoresistance chez le rat. Materiels et methodes Les rats recoivent un regime normosode standard (NSS), normosode-60% fructose (NSF), desode-60% fructose (DSF) pendant 12 semaines. La pression arterielle moyenne (PAM), la morphologie et la fonction cardiaques sont evaluees par echocardiographie. L’epaisseur de la paroi carotidienne, la taille des cardiomyocytes et la fibrose sont mesurees histologiquement. La vasorelaxation a l’acethylcholine est evaluee sur des anneaux aortiques. L’inflammation et le stress oxydant cardiaques sont estimes par immunomarquage (ED1 et nitrotyrosine, respectivement). Resultats Le fructose induit une hypertrophie ventriculaire gauche (1,74 ± 0,11 vs 1,63 ± 0,06 mg/g) de type concentrique (RWT : 0,35 ± 0,01 vs 0,29 ± 0,02) avec alteration de la fraction de raccourcissement (FR : 44 ± 2 vs 52 ± 3) sans modifier la PAM (122 ± 4 vs 121 ± 4 mmHg). L’aire de section de la carotide (ASC) est plus grande chez les rats NSF compares aux rats NSS et s’accompagne d’une alteration de la vasorelaxation. Les alterations cardiaques sont associees a une augmentation du collagene (3,2 ± 0,8 vs 1,5 ± 0,2%), a un doublement de l’infiltration macrophagique et a une augmentation du stress oxydant myocardique (nitrotyrosine, 2,3 ± 0,3% vs 1,1 ± 0,3%). Le regime desode previent l’hypertrophie VG (IMVG : 1,41 ± 0,01 mg/g ; RWT : 0,30 ± 0,02) et carotidienne, l’alteration de la fonction systolique (FR : 48 ± 1%) et la fibrose myocardique (Rouge Sirius : 1,40 ± 0,04%) sans influencer la pression arterielle ni la dysfonction endotheliale. Ces effets benefiques de la restriction sodee s’accompagnent d’une diminution de l’infiltration macrophagique (cellules ED1 : 0,4 ± 0,0 par mm2) et du score nitrotyrosine (1,4 ± 0,3%) myocardiques. Conclusion La restriction sodee previent l’hypertrophie cardiovasculaire et la fibrose cardiaque associees a l’insulinoresistance par un effet anti-inflammatoire et anti-oxydant tissulaires.

The recent Zika virus (ZIKV) epidemic has highlighted the poor knowledge on its physiopathology. Recent studies showed that ZIKV of the Asian lineage, responsible for this international outbreak, causes neuropathology in vitro and in vivo. However, two African lineages exist and the virus is currently found circulating in Africa. The original African strain was also suggested to be neurovirulent but its laboratory usage has been criticized due to its multiple passages. In this study, we compared the French Polynesian (Asian) ZIKV strain to an African strain isolated in Central African Republic and show a difference in infectivity and cellular response between both strains in human neural stem cells and astrocytes. Consistently, this African strain led to a higher infection rate and viral production, as well as stronger cell death and anti-viral response. Our results highlight the need to better characterize the physiopathology and predict neurological impairment associated with African ZIKV., Highlights • ZIKV from two Asian and African strains infect differentially IPSc-derived NSCs • African ZIKV strain displays more infectivity and anti-viral response • African and Asian ZIKV strains infect and replicate efficiently in human astrocytes Zika virus (ZIKV) is involved in a major epidemic in Latin America. Some neurological complications are reported following infection, in particular microcephaly and Guillain-Barré syndromes. Analyses show that one Asian and two Africans lineages exist. Although originally discovered in Africa in 1947, the actual epidemic is due to Asian ZIKV. Here, we compared the virulence of two African and Asian ZIKV strains in neural cells. We showed that in human neural stem cells, African ZIKV strain had a higher infectivity and triggered stronger cellular response. Altogether, our results highlight the need to better characterize ZIKV lineages to anticipate further epidemics.

International audience; Histopathological diagnosis in most of the world's hospitals is based upon formalin-fixed and paraffin-embedded (FFPE) tissues. Although this standard fixation and embedding procedure keeps the tissue in excellent form for morphological and immunohistological analysis, FFPE is inappropriate for nucleic acids and protein studies. We investigated the potential value of RCL2, a new non-toxic fixative, for sparing proteins preserved in paraffin-embedded tissues. Normal colonic mucosa tissue was fixed in RCL2 prior to paraffin embedding (RCL2P), and then processed for quality and quantity of protein conservation, as compared to frozen and FFPE tissues using complementary proteomic analysis approaches. Using 4 different protein extraction protocols, RCL2P tissue consistently showed the highest protein yield. Similar protein patterns were observed with RCL2P and frozen tissues using mono and bi-dimensional electrophoresis. Moreover, membrane, cytoplasmic and nuclear proteins, as well as phosphorylated proteins, were successfully detected using western-blot. Furthermore, protein patterns observed by mass spectrometry analysis after laser-captured microdissection were found to be identical for frozen and RCL2-fixed tissues. At last, immunohistochemistry using various antibodies showed comparable results between both tissue storage methods. We concluded that RCL2 has great potential for performing both morphological and molecular analyses on the same archival paraffin-embedded tissue sample, and can be a new method for investigating protein biomarkers.

International audience; Histone lysine acetylation is an epigenetic mark regulated by histone acetyltransferases and histone deacetylases (HDAC) which plays an important role in tumorigenesis. In this study, we observed a strong overexpression of class IIa HDAC9, at the mRNA and protein levels, in the most aggressive human breast cancer cell lines (i.e. in basal breast cancer cells vs luminal ones or in malignant vs begnin MCF10A breast epithelial cell lines). HDAC9 overexpression was associated with higher rates of gene transcription and increased epigenetic marks on the HDAC9 promoter. Ectopic expression of HDAC9 in MCF7 luminal breast cancer cells led to an increase in cell proliferation and to a decrease in apoptosis. These effects were associated with a deregulated expression of several genes controlled by HDAC inhibitors such as CDKN1A, BAX and TNFRSF10A. Inversely, knock-down of HDAC9 expression in MDA-MB436 basal breast cancer cells reduced cell proliferation. Moreover, high HDAC9 expression decreased the efficacy of HDAC inhibitors to reduce cell proliferation and to regulate CDKN1A gene expression. Interestingly, the gene encoding the transcription factor SOX9 was identified by a global transcriptomic approach as an HDAC9 target gene. In stably transfected MCF7 cells, SOX9 silencing significantly decreased HDAC9 mitogenic activity. Finally, in a large panel of breast cancer biopsies, HDAC9 expression was significantly increased in tumors of the basal subtype, correlated with SOX9 expression and associated with poor prognosis. Altogether, these results indicate that HDAC9 is a key factor involved in mammary carcinogenesis and in the response to HDAC inhibitors.