BACKGROUND:Pemphigus is classified as a group of chronic, recurrent, and potentially fatal bullous autoimmune diseases that leads to blisters and skin lesions resulting from IgG antibodies and the loss of cellular connections in the epidermis. Human endogenous retrovirus (HERV) sequences and their products (RNA, cytosolic DNA, and proteins) can modulate the immune system and contribute to autoimmunity. The extent to which, HERV-W env copies may be involved in the pathogenesis of pemphigus remains to be elucidated. AIM:This study aimed to comparatively evaluate the relative levels of HERV-W env DNA copy numbers in the peripheral blood mononuclear cells (PBMCs) of pemphigus vulgaris patients and healthy controls. METHODS:Thirty-one pemphigus patients and the corresponding age- and sex-matched healthy controls were included in the study. The relative levels of HERV-W env DNA copy numbers were then evaluated by qPCR using specific primers, in the PBMCs of the patients and controls. RESULTS:Our results indicated that relative levels of HERV-W env DNA copy numbers in the patients were significantly higher than that in the controls (1.67±0.86 vs. 1.17±0.75; p = 0.02). There was also a significant difference between the HERV-W env copies of male and female patients (p = 0.001). Furthermore, there was no relationship between the HERV-W env copy number and disease onset (p = 0.19) . According to the obtained data, we could not find any relationship between the HERV-W env copy number and serum Dsg1(p=0.86) and Dsg3 (p=0.76) levels. CONCLUSION:Our results indicated a positive link between the HERV-W env copies and pathogenesis of pemphigus. The association between clinical severity score and HERVW env copies in the PBMCs as a biomarker for pemphigus needs further studies.

Supplemental Methods Figure S1: Reports clinical information's about normal pregnant women, patients with complete hydatiform moles or choriocarcinoma. Figure S2: Experimental procedure. Figure S3: Quantification of EG-VEGF PROKR1 and PROKR2 protein expression in CTL, Complete hydatiform mole (CHM) and choriocarcinoma (CC) placental section. Figure S4: EG-VEGF effect on JEG3 migration, in the absence or presence of PROKR1 or PROKR2 antagonists. Figure S5: JEG3-luc injected mice exhibited arrested gestation and disorganized vascularization. Figure S6: Antibody microarray analysis. Figure S7: Characterization of the angiogenic status of sera and placentas collected from CTL, CHM and CC patients. Figure S8: Antibody angiogenic microarray analysis of sera collected from CTL, CHM and CC patients.

Low concentrations of type‐I interferon (IFN) in blood seem to be associated with more severe forms of Coronavirus disease 2019 (COVID‐19). However, following the type‐I interferon response (IR) in early stage disease is a major challenge. We evaluated detection of a molecular interferon signature on a FilmArray® system, which includes PCR assays for four interferon stimulated genes. We analyzed three types of patient populations: (i) children admitted to a pediatric emergency unit for fever and suspected infection, (ii) ICU‐admitted patients with severe COVID‐19, and (iii) healthcare workers with mild COVID‐19. The results were compared to the reference tools, that is, molecular signature assessed with Nanostring® and IFN‐α2 quantification by SIMOA® (Single MOlecule Array). A strong correlation was observed between the IR measured by the FilmArray®, Nanostring®, and SIMOA® platforms (r‐Spearman 0.996 and 0.838, respectively). The FilmArray® panel could be used in the COVID‐19 pandemic to evaluate the IR in 45‐min with 2 min hand‐on‐time at hospitalization and to monitor the IR in future clinical trials.

The human placenta shares properties with solid tumors, such as rapid growth, tissue invasion, cell migration, angiogenesis, and immune evasion. However, the mechanisms that drive the evolution from premalignant proliferative placental diseases—called hydatidiform moles—to their malignant counterparts, gestational choriocarcinoma, as well as the factors underlying the increased aggressiveness of choriocarcinoma arising after term delivery compared to those developing from hydatidiform moles, are unknown. Using a 730-gene panel covering 13 cancer-associated canonical pathways, we compared the transcriptomic profiles of complete moles to those of postmolar choriocarcinoma samples and those of postmolar to post-term delivery choriocarcinoma. We identified 33 genes differentially expressed between complete moles and postmolar choriocarcinoma, which revealed TGF-β pathway dysregulation. We found the strong expression of SALL4, an upstream regulator of TGF-β, in postmolar choriocarcinoma, compared to moles, in which its expression was almost null. Finally, there were no differentially expressed genes between postmolar and post-term delivery choriocarcinoma samples. To conclude, the TGF-β pathway appears to be a crucial step in the progression of placental malignancies. Further studies should investigate the value of TGF- β family members as biomarkers and new therapeutic targets.

IntroductionWe analysed blood DNAemia of TTV and four herpesviruses (CMV, EBV, HHV6, and HSV-1) in the REAnimation Low Immune Status Marker (REALISM) cohort of critically ill patients who had presented with either sepsis, burns, severe trauma, or major surgery. The aim was to identify common features related to virus and injury-associated pathologies and specific features linking one or several viruses to a particular pathological context.MethodsOverall and individual viral DNAemia were measured over a month using quantitative PCR assays from the 377 patients in the REALISM cohort. These patients were characterised by clinical outcomes [severity scores, mortality, Intensive Care Unit (ICU)-acquired infection (IAI)] and 48 parameters defining their host response after injury (cell populations, immune functional assays, and biomarkers). Association between viraemic event and clinical outcomes or immune markers was assessed using χ2-test or exact Fisher’s test for qualitative variables and Wilcoxon test for continuous variables.ResultsThe cumulative incidence of viral DNAemia increased from below 4% at ICU admission to 35% for each herpesvirus during the first month. EBV, HSV1, HHV6, and CMV were detected in 18%, 12%, 10%, and 9% of patients, respectively. The incidence of high TTV viraemia (>10,000 copies/ml) increased from 11% to 15% during the same period. Herpesvirus viraemia was associated with severity at admission; CMV and HHV6 viraemia correlated with mortality during the first week and over the month. The presence of individual herpesvirus during the first month was significantly associated (p < 0.001) with the occurrence of IAI, whilst herpesvirus DNAemia coupled with high TTV viraemia during the very first week was associated with IAI. Herpesvirus viraemia was associated with a lasting exacerbated host immune response, with concurrent profound immune suppression and hyper inflammation, and delayed return to immune homeostasis. The percentage of patients presenting with herpesvirus DNAemia was significantly higher in sepsis than in all other groups. Primary infection in the hospital and high IL10 levels might favour EBV and CMV reactivation.ConclusionIn this cohort of ICU patients, phenotypic differences were observed between TTV and herpesviruses DNAemia. The higher prevalence of herpesvirus DNAemia in sepsis hints at further studies that may enable a better in vivo understanding of host determinants of herpesvirus viral reactivation. Furthermore, our data suggest that EBV and TTV may be useful as additional markers to predict clinical deterioration in ICU patients.

Lipopolysaccharide (LPS) and monophosphoryl lipid A (MPLA) induce, overall, similar transcriptional profiles in healthy individuals, although LPS has been shown to more potently induce pro-inflammatory cytokines. We explore herein whether MPLA could be considered as a synthetic replacement of LPS in immune functional assays to study anergy of immune cells in septic patients. Ex vivo whole blood stimulation with MPLA revealed a lower induction of the TNFα secreted protein in 20 septic patients (SP) compared to 10 healthy volunteers (HV), in agreement with monocyte anergy. Principal component analysis of the 93-gene molecular response to MPLA and LPS stimulation found that the main variability was driven by stimulation in HV and by pathophysiology in SP. MPLA was a stronger inducer of the HLA family genes than LPS in both populations, arguing for divergent signalling pathways downstream of TLR-4. In addition, MPLA appeared to present a more informative stratification potential within the septic population.

Background Critical illness such as sepsis is a life-threatening syndrome defined as a dysregulated host response to infection and is characterized by patients exhibiting various impaired immune profiles. In the field of diagnosis, a gap still remains in identifying the immune profile of critically-ill patients in the ICU. The availability of an immune profiling tool holds a great potential in providing patients at high risk with more accurate and precise management. In this study, a multiplex immune profiling panel prototype was assessed for its ability to semi-quantify immune markers directly from blood, using the FilmArray® System. Results The Immune Profiling Panel (IPP) prototype consists of 16 biomarkers that target both the innate and adaptive immune responses, pro- and anti-inflammatory mediators as well as genes involved in diverse regulatory pathways. The analytical studies carried out on healthy volunteers showed minimal inter- and intra-variability in testing the samples across the tested lots. The majority of the assays were linear with an R 2 higher than 0.8. Results from the IPP pouch were comparable to qPCR and were within the limits of agreement. Finally, quantification cycle values of the target genes were normalized against reference genes to account for the different composition of cells among specimens. The use of the selected panel of markers in IPP demonstrated various gene modulations that could distinctly differentiate three profiles: healthy, borderline mHLA-DR septic shock patients and low mHLA-DR septic shock patients. Conclusion The Immune Profiling Panel allowed host transcriptomic analysis of immune response biomarkers directly from whole blood in less than an hour. The use of IPP showed great potential for the development of a fully automated, rapid and easy-to-use immune profiling tool, enabling the stratification of critically-ill patients at high risk in the ICU.

We have previously shown that human endogenous retrovirus-W-encoded fusogenic membrane protein syncytin-1 plays a role in the pathogenesis of mycosis fungoides (MF), the most common primary cutaneous T-cell lymphoma (CTCL) (Maliniemi et al., 2013Maliniemi P. Vincendeau M. Mayer J. Frank O. Hahtola S. Karenko L. et al.Expression of human endogenous retrovirus-w including syncytin-1 in cutaneous T-cell lymphoma.PLOS ONE. 2013; 8e76281Crossref PubMed Scopus (35) Google Scholar). The expression of syncytin-1 protein was detected in 15 of 30 MF skin lesions but not in skin-homing nonmalignant lymphocytes. Primary cutaneous CD30-positive lymphoproliferative disorders, including lymphomatoid papulosis and primary cutaneous anaplastic large cell lymphoma, are the second most common form of CTCLs and represent approximately 25% of all CTCLs (Nikolaenko et al., 2019Nikolaenko L. Zain J. Rosen S.T. Querfeld C. CD30-positive lymphoproliferative disorders.Cancer Treat Res. 2019; 176: 249-268Crossref PubMed Scopus (6) Google Scholar, Willemze et al., 2019Willemze R. Cerroni L. Kempf W. Berti E. Facchetti F. Swerdlow S.H. et al.The 2018 update of the WHO-EORTC classification for primary cutaneous lymphomas.Blood. 2019; 133: 1703-1714Crossref PubMed Scopus (311) Google Scholar). This study examined the expression and functional properties of syncytin-1 in verified cell lines of the aforementioned forms of CTCLs and also its presence in the extracellular vesicles (EVs) released by the malignant cells. Human endogenous retrovirus-driven syncytin-1 expression is upregulated in a wide variety of human malignancies, such as rectal and colorectal cancer, prostate cancer, bladder cancer, leukemia, and lymphoma (Bastida-Ruiz et al., 2016Bastida-Ruiz D. Van Hoesen K. Cohen M. The dark side of cell fusion.Int J Mol Sci. 2016; 17: 638Crossref Scopus (49) Google Scholar). Functionally, upregulated syncytin-1 has been shown to promote the cell-to-cell fusion of endometrial carcinoma cells (Strick et al., 2007Strick R. Ackermann S. Langbein M. Swiatek J. Schubert S.W. Hashemolhosseini S. et al.Proliferation and cell-cell fusion of endometrial carcinoma are induced by the human endogenous retroviral syncytin-1 and regulated by TGF-beta.J Mol Med (Berl). 2007; 85: 23-38Crossref PubMed Scopus (110) Google Scholar) and breast cancer or squamous cell carcinoma cells to endothelial cells (Bjerregaard et al., 2006Bjerregaard B. Holck S. Christensen I.J. Larsson L.I. Syncytin is involved in breast cancer-endothelial cell fusions.Cell Mol Life Sci. 2006; 63: 1906-1911Crossref PubMed Scopus (130) Google Scholar). Despite these observations, the role of syncytin-1 in cancer remains poorly understood. EVs contain a variety of proteins, lipids, RNAs, and DNA and have recently emerged as an important form of intercellular communication in cancer (Yáñez-Mó et al., 2015Yáñez-Mó M. Siljander P.R. Andreu Z. Zavec A.B. Borràs F.E. Buzas E.I. et al.Biological properties of extracellular vesicles and their physiological functions.J Extracell Vesicles. 2015; 4: 27066Crossref PubMed Scopus (2194) Google Scholar). Malignant cell-derived EVs can be released into the circulation and transfer their contents to nontumor cells to reprogram target cell function or to prepare metastatic niches (Maas et al., 2017Maas S.L.N. Breakefield X.O. Weaver A.M. Extracellular vesicles: unique intercellular delivery vehicles.Trends Cell Biol. 2017; 27: 172-188Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). The current knowledge on the molecular mechanisms of the fusogenic properties of EVs are not yet fully characterized, but syncytins are anticipated to be involved in the binding of EVs to target cells, a process preceding fusion (Prada and Meldolesi, 2016Prada I. Meldolesi J. Binding and fusion of extracellular vesicles to the plasma membrane of their cell targets.Int J Mol Sci. 2016; 17: 1296Crossref Scopus (94) Google Scholar). Using immunocytochemistry and western blot analyses (Supplementary Materials), we observed that both the syncytin-1 protein (Figure 1) and its receptors ASCT1 and ASCT2 (Supplementary Figure S1) are expressed in CTCL cell lines representing MF (MyLa cell line), lymphomatoid papulosis (Mac-1), primary cutaneous anaplastic large cell lymphoma (Mac-2A), and also in the BeWo cell line, which is known to express syncytin-1 (Cheynet et al., 2005Cheynet V. Ruggieri A. Oriol G. Blond J.L. Boson B. Vachot L. et al.Synthesis, assembly, and processing of the env ERVWE1/syncytin human endogenous retroviral envelope.J Virol. 2005; 79: 5585-5593Crossref PubMed Scopus (64) Google Scholar). Normal T cells showed only very limited syncytin-1 expression (Figure 1a) (Sun et al., 2016Sun Y. Zhu H. Song J. Jiang Y. Ouyang H. Huang R. et al.Upregulation of leukocytic syncytin-1 in acute myeloid leukemia patients.Med Sci Monit. 2016; 22: 2392-2403Crossref PubMed Scopus (6) Google Scholar). To analyze the presence of syncytin-1 as cargo of EVs, we isolated EVs from the cell culture medium of the aforementioned cell lines using ultracentrifugation. Upon isolation, nanoparticle tracking analysis was employed to determine the amount and size distribution of cell-derived EVs (Supplementary Figure S2). The content of the EVs was subsequently analyzed by western blotting with syncytin-1-specific antibody and antibodies against HSP70, RAB5, and TSG101 to confirm the specificity of EVs (Figure 2a). Syncytin-1 protein was detected on the surface of EVs as confirmed by immunoelectron microscopy (Figure 2b).Figure 2Syncytin-1 is detected on EVs derived from CTCL cell lines and BeWo cells. (a) EV-derived syncytin-1 is most prominently detected in the Mac-2A and BeWo cell lines by western blot analysis. The molecular weight of EV-derived syncytin-1 is 63 kDa, while the BeWo cell line lysate includes the full-length gp73 form. HSP70, RAB5, and TSG101 identify EVs. (b) Immunoelectron microscopy confirms syncytin-1 on EVs by 5 nm colloidal gold particles. The 15 nm gold particles (red arrowheads) depict the CD63 marker of EVs. The BeWo-derived EVs are typified as 50 nm vesicles with a phospholipid layer and electron-dense inner layer (black arrow). The bottom panel shows CD30 in CTCL cell line-derived EVs (15 nm gold particles, red arrowheads). The negative controls are immunostained without primary antibodies. Bar = 500 nm. BeWo, chorion carcinoma; CTCL, cutaneous T-cell lymphoma; EV, extracellular vesicle; Mac-1, lymphomatoid papulosis; Mac-2A, primary cutaneous anaplastic large cell lymphoma; MyLa, mycosis fungoides.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The full-length transmembrane 73 kDa syncytin-1 protein (composed of surface and transmembrane subunits) was most prominent in the three CTCL cell lines and in the reference BeWo cell line, whereas an additional 63 kDa form was detected in the CTCL cell lines only (Figure 1b). This 63 kDa variant could represent a partially glycosylated protein or a cell type-specific splicing variant, since the expression of endogenous retroviral glycoproteins is known to be controlled by tissue-specific pre-mRNA splicing (Trejbalová et al., 2011Trejbalová K. Blazková J. Matousková M. Kucerová D. Pecnová L. Vernerová Z. et al.Epigenetic regulation of transcription and splicing of syncytins, fusogenic glycoproteins of retroviral origin.Nucleic Acids Res. 2011; 39: 8728-8739Crossref PubMed Scopus (37) Google Scholar). Moreover, EVs derived from the CTCL and BeWo cell lines harbored only the 63 kDa syncytin-1 form. Several studies have reported changes in the glycosylation patterns of EVs in pathological conditions (Yáñez-Mó et al., 2015Yáñez-Mó M. Siljander P.R. Andreu Z. Zavec A.B. Borràs F.E. Buzas E.I. et al.Biological properties of extracellular vesicles and their physiological functions.J Extracell Vesicles. 2015; 4: 27066Crossref PubMed Scopus (2194) Google Scholar). Further studies are needed to investigate if this size variation of syncytin-1 plays a specific role in the pathomechanism of CTCL. In addition, we observed the most abundant syncytin-1 expression in Mac-2As and their EVs. This possibly reflects an advanced disease stage, since Mac-2A was established from the same patient as Mac-1 after progression to an aggressive primary cutaneous anaplastic large cell lymphoma. A knockdown study of syncytin-1 in the BeWo cell line verified the specificity of syncytin-1 antibody used for immunoblotting (Figure 1b, right panel). In preliminary assays to assess the fusogenic effect of syncytin-1 on T cells, we transiently transfected HEK293T cells with phCMV plasmids encoding either human nonfusogenic (gp60) or fusogenic (env) syncytin-1 proteins. We hypothesized that upon expression in cells, these proteins would be captured in EVs released from the cells. Supernatants collected and cleared of transfected HEK293T cells were then applied onto the Jurkat T-cell leukemia cells, and the cell size was analyzed by flow cytometry 24 hours later. We observed an almost two-fold increase in the amount of large cells upon treatment with the supernatant harvested from fusogenic env-transfected cells compared with the nonfusogenic gp60 (Supplementary Figure S3a). Jurkat cells grown 48 hours in the supernatant from env-transfected cells formed plasma membrane-enveloped giant cells, which were undetectable in the control gp60-treated cells (Supplementary Figure S3b). In addition to syncytin-1, we also studied the eventual release of the tumor-associated antigen CD30 (Figure 1a) in EVs. CD30 has been detected in EVs released by Hodgkin's lymphoma cells. CD30 communicates with bystander cells to allow the indirect binding of brentuximab vedotin, a monoclonal anti–CD30 antibody-drug conjugate also effective in the treatment of MF (Hansen et al., 2016Hansen H.P. Trad A. Dams M. Zigrino P. Moss M. Tator M. et al.CD30 on extracellular vesicles from malignant Hodgkin cells supports damaging of CD30 ligand-expressing bystander cells with brentuximab-vedotin, in vitro.Oncotarget. 2016; 7: 30523-30535Crossref PubMed Scopus (30) Google Scholar, Whittaker et al., 2016Whittaker S. Hoppe R. Prince H.M. How I treat mycosis fungoides and sezary syndrome.Blood. 2016; 127: 3142-3153Crossref PubMed Scopus (68) Google Scholar). Using immunoelectron microscopy, we detected and confirmed CD30 expression in the CTCL cell line-released EVs (Figure 2b). Thus, our observations are analogous to those in Hodgkin's lymphoma and may be relevant for the favorable clinical responses even in immunohistologically CD30-negative MF cases (Welborn and Duvic, 2019Welborn M. Duvic M. Antibody-based therapies for cutaneous T-cell lymphoma.Am J Clin Dermatol. 2019; 20: 115-122Crossref PubMed Scopus (10) Google Scholar). Taken together, the presence of syncytin-1, a fusogen, and its receptors in several CTCL cell lines, refers to its potential role in cancer cell fusion mechanisms, similar to the one used by placental trophoblasts (Bastida-Ruiz et al., 2016Bastida-Ruiz D. Van Hoesen K. Cohen M. The dark side of cell fusion.Int J Mol Sci. 2016; 17: 638Crossref Scopus (49) Google Scholar). We show that tumor cell-secreted EVs carry syncytin-1 protein, which may be transferred to recipient cells by the EVs to mediate membrane fusion and transfer tumor cell signals. Thus, we propose that studies on the content and biological function of EVs in CTCL patients will be of pivotal importance in developing novel therapeutic regimens for the currently incurable CTCL pathologies. The data analyzed during this study are included in this published article and its supplementary information files. Kirsi Laukkanen: https://orcid.org/0000-0001-6243-9850 Mirjam Saarinen: https://orcid.org/0000-0002-6345-3790 Francois Mallet: https://orcid.org/0000-0002-1331-096X Maria Aatonen: https://orcid.org/0000-0002-4819-6722 Annika Hau: https://orcid.org/0000-0002-3388-3394 Annamari Ranki: https://orcid.org/0000-0003-4335-0396 FM is an employee of an in vitro diagnostic company. The remaining authors state no conflict of interest. We thank Mrs. Alli Tallqvist and Ms. Inga Liukko for their skillful technical assistance. We thank Professor Robert Gniadecki, Bispebjerg Hospital, Copenhagen University, for the kind gift of the MyLa, Mac-1, and Mac-2A cell lines. We thank the Extracellular Vesicle Core Facility at the University of Helsinki, Finland, for providing nanoparticle tracking analysis and preparation of immunoelectron microscopy samples. Biomedicum Imaging Unit and Biomedicum Flow Cytometry Core facilities at the University of Helsinki, Finland, were used for imaging and flow cytometry analyses. This work was supported by grants from the Cancer Foundation Finland and the Finnish Dermatological Society . Conceptualization: AR, KL; Data curation: KL, AH; Formal analysis: KL, AH; Funding acquisition: AR; Investigation: KL, AH, MS, MA; Methodology: KL, AH, AR; Project Administration: AR; Resources: AR, FM; Supervision: AR, FM; Validation: KL, AH, MA; Visualization: KL, AH; Writing - Original Draft: AR, KL, AH; Writing - Review & Editing: KL, FM, AH, MS, MA, and AR. We used CTCL cell lines representing MF (MyLa cell line), lymphomatoid papulosis (Mac-1), and primary cutaneous anaplastic large cell lymphoma (Mac-2A) (Netchiporouk et al., 2017Netchiporouk E. Gantchev J. Tsang M. Thibault P. Watters A.K. Hughes J.M. et al.Analysis of CTCL cell lines reveals important differences between mycosis fungoides/sezary syndrome vs. HTLV-1+ leukemic cell lines.Oncotarget. 2017; 8: 95981-95998Crossref PubMed Scopus (25) Google Scholar). The Mac-1 cell line is derived from circulating Sézary-like cells in the peripheral blood of a patient with indolent lymphomatoid papulosis, while Mac-2A was established from skin tumor nodules of the same patient after progression to aggressive primary cutaneous anaplastic large cell lymphoma (Davis et al., 1992Davis T.H. Morton C.C. Miller-Cassman R. Balk S.P. Kadin M.E. Hodgkin's disease, lymphomatoid papulosis, and cutaneous T-cell lymphoma derived from a common T-cell clone.N Engl J Med. 1992; 326: 1115-1122Crossref PubMed Scopus (259) Google Scholar). Although clonally related, Mac-1 and Mac-2A are genomically and transcriptionally distinct (Ehrentraut et al., 2013Ehrentraut S. Nagel S. Scherr M.E. Schneider B. Quentmeier H. Geffers R. et al.t(8;9)(p22;p24)/PCM1-JAK2 activates SOCS2 and SOCS3 via STAT5.PLOS ONE. 2013; 8e53767Crossref PubMed Scopus (31) Google Scholar). The MF-derived cell line MyLa and human embryonic kidney cell line HEK293T were grown in DMEM supplemented with Glutamax, penicillin-streptomycin, and 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Waltham, MA). The Mac-1 and Mac-2A cell lines were grown in RPMI 1640 medium supplemented with Glutamax, penicillin-streptomycin, and 10% FBS. The human BeWo chorioncarcinoma cell line was maintained in Ham's F12 medium supplemented with Glutamax, penicillin-streptomycin, and 10% FBS. Normal T cells were isolated from a buffy coat of a healthy blood donor (Finnish Red Cross Blood Service, Helsinki, Finland) with the Lympholyte-H gradient separation method (CL5015, Cedarlane Corporation, Burlington, ON, Canada) followed by enrichment with a Pan T cell Isolation Kit (130-096-535, Milteney Biotec, Auburn, CA) according to the manufacturer's instructions. The enriched cells were immunostained with CD3 and CD19 antibodies and analyzed by flow cytometry (Biomedicum Flow Cytometry Core, Helsinki, Finland) and immunocytochemistry staining with CD3 antibody (MF7254, Dako, Glostrup, Denmark) to confirm the T cell nature of the enriched cells (data not shown). The cell lines were authenticated at the Finnish Institute for Molecular Medicine, Helsinki, Finland, and tested routinely for mycobacterial contamination using a MycoAlert Mycoplasma Detection Kit (LT07, Lonza, Basel, Switzerland). Immunocytochemistry was used to the detect expression of syncytin-1 (1:25, SC-50369, Santa Cruz Biotechnology, Santa Cruz, CA), ASCT-1 (1:25 or 1:5, SC-134846, Santa Cruz Biotechnology), and ASCT-2 (1:25, 5100, Cell Signaling Technology, Leiden, Netherlands) in cytocentrifuge preparations of Mac-1, Mac-2A, Myla, BeWo, and T cells. Anti–CD30 antibody (1:400, 134080, Abcam, Cambridge, UK) was used to detect CD30 expression on CTCL and BeWo cells. The ImmPRESS Reagent kit (MP-7500, Vector Laboratories, Burlingame, CA) was used according to the manufacturer's instructions. NovaRed (SK-4800, Vector Laboratories) and Mayer's hemalum solution (Merck, Darmstadt, Germany) were used as chromogens for ASCT-1/ASCT-2 receptors and CD30. Horseradish Peroxidase Green substrate buffer (KDB-10049, Nordic BioSite, Täby, Sweden) and Nuclear Fast red (60700 Sigma-Aldrich, Darmstadt, Germany) were used for syncytin-1. Ultracentrifugation was used to isolate the EVs. Cell lines were grown for 72 hours in supplemented media as described above, but 10% EV-depleted FBS was used. FBS was depleted from EV using polyethylene glycol (PEG). Heat-inactivated FBS (Gibco, Thermo Fisher Scientific) was treated with PEG (P6667, Sigma-Aldrich, St. Louis, MO). A sterile-filtered (0.2 μm filter) 50% (w/v) stock solution of PEG prepared in 1× Dulbecco's phosphate buffered saline (PBS) was protected from light and stored at 4 °C. The FBS and PEG stock solutions were mixed in a 5:1 ratio by gently inverting 5 to 10 times and incubated for 2 hours at 4 °C protected from light before centrifugation for 30 minutes at 4 °C at 1,500g in a swinging-bucket rotor. The supernatant was collected leaving a layer of at least 0.5 cm on top of the pellet, and sterile filtered (0.1 μm filter) into aliquots stored at –20 °C until use. For EV purification, cell supernatants were centrifuged at 3,000g at 4 °C for 25 minutes, followed by centrifugation at 100,000g at 4 °C for 2 hours to isolate the microvesicle and exosome fractions. EV pellets were washed with 0.1 μm filtered Dulbecco's PBS, followed by centrifugation at 100,000g at 4 °C for 2 hours. Purified EV samples were analyzed by nanoparticle tracking analysis using Nanosight model LM14 (Malvern Panalytical Ltd, Malvern, United Kingdom) equipped with blue (404 nm, 70 mW) laser and a Scientific Complementary metal-oxide-semiconductor camera. The samples were diluted in Dulbecco's PBS, and three 60-second videos were recorded using camera level 13. The data were analyzed using Nanoparticle Tracking Analysis software 3.0 (Malvern Panalytical Ltd). Mac-1, Mac-2A, MyLa, and BeWo cells were harvested either by centrifugation or mechanical scraping and washed twice with ice-cold PBS. Cell pellets were resuspended in 1× PBS buffer/0.5% Triton X-100 with EDTA-free protease inhibitors (Roche, Basel, Switzerland) and incubated on ice for 60 minutes. The protein concentration was determined using the DC protein assay kit (Bio-Rad, Göteborg, Sweden). A total of 30 μg and 40 μg of protein was used for ASCT-1/ASCT-2 and syncytin-1 western blot analyses, respectively. The samples were boiled at 95 °C for 5 minutes in Laemmli Sample Buffer (Bio-Rad) with 2-mercaptoethanol. Protein lysates were separated by 10% SDS-PAGE electrophoresis and transferred onto a nitrocellulose membrane (Whatman, Boston, MA) at 100 V for 90 minutes. Membranes were incubated with anti–ASCT-1 (1:200, SC-134846, Santa Cruz Biotechnology), anti–ASCT-2 (1:200, 5100, Cell Signaling Technology), and anti–syncytin-1 (1:200, SC-50369, Santa Cruz Biotechnology) antibodies overnight at 4 °C. Incubation with anti–β-actin antibodies (4970L, 1:1,000, Cell Signaling Technology) served as a loading control. After washing, the membranes were incubated with anti–rabbit horseradish peroxidase-conjugated secondary antibody (PO448, Dako) for 1 hour at room temperature. The proteins were detected using Clarity ECL reagent (Bio-Rad), and expression values were normalized against β-actin using the ChemiDoc MP Imaging System (Bio-Rad). Adjusted density values were calculated using Image Lab software (Bio-Rad). Western blot analyses of EVs were performed using 5 μg of protein. The samples were boiled at 95 °C for 5 minutes in Laemmli Sample Buffer (Bio-Rad) with 2-mercaptoethanol and additional SDS (total 8%). Protein lysates were separated as described above. In addition to probing with anti–syncytin-1 antibody, anti–HSP70 (1:500, 610608, BD Biosciences, San Jose, CA), anti–RAB5 (1:100, PA5-29022, ThermoFisher Scientific), and anti–TSG101 (1:100, MA1-23296, ThermoFisher Scientific) antibodies were used as EV-specific markers, followed by incubation with anti–rabbit or anti–mouse horseradish peroxidase-conjugated secondary antibody (PO448 or PO447, Dako). BeWo cells were transduced with pTIP and pTIP-short hairpin RNA knockdown lentiviruses and selected using 3 μg/ml puromycin (Sigma-Aldrich, St. Louis, MO) to stably express either control plasmid (pTIP) or a TetON-inducible short hairpin RNA knockdown construct against syncytin-1 (pTIP-short hairpin RNA knockdown). The vectors were kindly provided by Dr Marcus Peter, Northwestern University, Chicago, IL, and published by Hau et. al. (2012). The sequence of the knockdown oligo was already published by Aagaard et al. (2012). The expression of Syncytin-1 short hairpin RNA was induced using 100 ng/ml doxycycline (Sigma-Aldrich, St. Louis, MO) for 10 days. Cells were then lysed and probed for syncytin-1 using Santa Cruz anti–syncytin-1 antibody (SC-50369, Santa Cruz Biotechnology). EV samples from cell lines were prepared for electron microscopy and imaged as previously described (Puhka et al., 2017Puhka M. Nordberg M.E. Valkonen S. Rannikko A. Kallioniemi O. Siljander P. et al.KeepEX, a simple dilution protocol for improving extracellular vesicle yields from urine.Eur J Pharm Sci. 2017; 98: 30-39Crossref PubMed Scopus (37) Google Scholar) with the addition of an immunostaining step. Briefly, after being loaded onto 200 mesh copper grids, the samples were permeabilized with 0.01% saponin with a prefixation of 2% paraformaldehyde. The samples were then incubated with or without (negative control) anti–syncytin-1 antibody (1:100, SC-50369, Santa Cruz Biotechnology) and anti–CD30 antibody (1:400, 134080, Abcam) followed by 5 nm or 15 nm colloidal gold conjugated goat-anti–rabbit-IgG (1:150, BBI Solutions, Cardiff, UK), respectively. Next, a post-fixation step with 1% glutaraldehyde was performed. In addition, anti–CD63 staining was performed as an EV-specific control. The samples were blocked and incubated with anti–CD63 antibody (1:250, Pelicluster, Sanquin, Amsterdam, The Netherlands) followed by 10-nm colloidal gold conjugated goat-anti–mouse-IgG secondary antibody (1:80, BBI Solutions). All the samples were viewed with transmission electron microscopy using Jeol JEM-1400 (Jeol Ltd, Tokyo, Japan). Images were taken with a Gatan Orius SC 1000B CCD-camera (Gatan Inc, Pleasanton, CA). Human embryonic kidney HEK293T cells were seeded a day before transfection. On the day of transfection, DMEM-containing EV-depleted FBS (centrifuged at 100,000g for 18 hours) was placed onto cells, and the cells were transfected with 1.5 μg of plasmids according to manufacturer's instructions using Lipofectamine 2000 (Thermo LifeSciences, Carlsbad, CA). The nonfusogenic recombinant form of pcDNA–CMV-human gp60 syncytin-1 (hydrophobic tail deleted and RNKR cleavage site mutated) (Antony et al., 2007Antony J.M. Ellestad K.K. Hammond R. Imaizumi K. Mallet F. Warren K.G. et al.The human endogenous retrovirus envelope glycoprotein, syncytin-1, regulates neuroinflammation and its receptor expression in multiple sclerosis: A role for endoplasmic reticulum chaperones in astrocytes.J Immunol. 2007; 179: 1210-1224Crossref PubMed Scopus (96) Google Scholar) and fusogenic pcDNA–CMV-human env syncytin-1 plasmids were used (a kind gift from Dr. Francois Mallet, BioMerieux, France). Approximately 6 hours after the addition of lipofectamine complexes, media were replaced and supplemented with EV-depleted FBS. The following day, media from each transfection were harvested, and cells and debris were removed by centrifugation (860g for 5 minutes). Jurkat cells were counted, and 80,000 to 200,000 cells were resuspended into media harvested from the transfected HEK293T cells. Approximately 24 hours later, the Jurkat cells were harvested, fixed for 30 minutes at room temperature with 2% paraformaldehyde in PBS, and rinsed and stored at 4 °C until FACS analysis. In a parallel experiment, Jurkat cells were incubated with supernatants for 48 hours, and photomicrographs were taken. Cell-size analysis was performed using a BD Accuri C6 (BD Biosciences, San Jose, CA) and FlowJo version 10.5.3. (Treestar Inc, Ashland, OR). The cell size was determined by setting a gate to exclude the normal-sized cells of the control sample and only quantifying the cells that were in this "large" gate, specifically cells that had increased forward scatter or side scatter (Hadji et al., 2014Hadji A. Ceppi P. Murmann A.E. Brockway S. Pattanayak A. Bhinder B. et al.Death induced by CD95 or CD95 ligand elimination.Cell Rep. 2014; 7: 208-222Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar).

Influenza A viruses are amongst the most challenging viruses that threaten both human and animal health. Constantly evolving and crossing species barrier, the emergence of novel zoonotic pathogens is one of the greatest challenges to global health security. During the last decade, considerable attention has been paid to influenza virus infections in dogs, as two canine H3N8 and H3N2 subtypes caused several outbreaks through the United States and Southern Asia, becoming endemic. Cats, even though less documented in the literature, still appear to be susceptible to many avian influenza infections. While influenza epidemics pose a threat to canine and feline health, the risks to humans are largely unknown. Here, we review most recent knowledge of the epidemiology of influenza A viruses in dogs and cats, existing evidences for the abilities of these species to host, sustain intraspecific transmission, and generate novel flu A lineages through genomic reassortment. Such enhanced understanding suggests a need to reinforce surveillance of the role played by companion animals-human interface, in light of the “One Health” concept and the potential emergence of novel zoonotic viruses.

Background Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. Numerous studies have explored the complex and dynamic transcriptome modulations observed in sepsis patients, but a large fraction of the transcriptome remains unexplored. This fraction could provide information to better understand sepsis pathophysiology. Multiple levels of interaction between human endogenous retroviruses (HERV) and the immune response have led us to hypothesize that sepsis is associated with HERV transcription and that HERVs may contribute to a signature among septic patients allowing stratification and personalized management. Methods We used a high-density microarray and RT-qPCR to evaluate the HERV and Mammalian Apparent Long Terminal Repeat retrotransposons (MaLR) transcriptome in a pilot study that included 20 selected septic shock patients, stratified on mHLA-DR expression, with samples collected on day 1 and day 3 after inclusion. We validated the results in an unselected, independent cohort that included 100 septic shock patients on day 3 after inclusion. We compared septic shock patients, according to their immune status, to describe the transcriptional HERV/MaLR and conventional gene expression. For differential expression analyses, moderated t tests were performed and Wilcoxon signed-rank tests were used to analyze RT-qPCR results. Results We showed that 6.9% of the HERV/MaLR repertoire was transcribed in the whole blood, and septic shock was associated with an early modulation of a few thousand of these loci, in comparison to healthy volunteers. We provided evidence that a subset of HERV/MaLR and conventional genes were differentially expressed in septic shock patients, according to their immune status, using monocyte HLA-DR (mHLA-DR) expression as a proxy. A group of 193 differentially expressed HERV/MaLR probesets, tested in an independent septic shock cohort, identified two groups of patients with different immune status and severity features. Conclusion We demonstrated that a large, unexplored part of our genome, which codes for HERV/MaLR, may be linked to the host immune response. The identified set of HERV/MaLR probesets should be evaluated on a large scale to assess the relevance of these loci in the stratification of septic shock patients. This may help to address the heterogeneity of these patients.

Objective. Using a transcriptional approach on tissue samples, we sought to identify predictive biomarkers of post molar malignant transformation, and of choriocarcinoma chemosensitivity to mono(methotrexate or actinomycin D) or polychemotherapy [EMA(Etoposide, Methotrexate, Actinomycin D)-CO(Cyclophosphamide, Vincristine) and EMA-EP(Etoposide, Cisplatine)] regimens. Methods. We studied the expression of a 760-gene panel (PanCancer Pathway) related to oncogenesis and immune tolerance in tissue samples of complete hydatidiform moles and gestational choriocarcinoma. Results. We did not identify any differentially expressed gene between moles with post molar malignant transformation in choriocarcinoma (n = 14) and moles with remission (n = 20). In monochemoresistant choriocarcinoma (n = 34), four genes (HLA-G, COL27A1, IL1R2 and GLI3) had a significantly reduced expression and one (THEM4) had an increased expression [FDR (false discovery rate) adjusted p-value <= 0.05] when compared to monochemosensitive choriocarcinoma (n = 9). The proportion of trophoblast cells and the intensity of immunohistochemical HLA-G expression were reduced in monochemoresistant choriocarcinoma (p < 0.05). In polychemoresistant choriocarcinoma (n = 20) we did not identify differentially expressed genes with an FDR adjusted p-value <= 0.05 when compared to polychemosensitive choriocarcinoma (n = 15). Gene pathway analysis revealed a predicted activation of IFN gamma in monochemoresistant choriocarcinoma and inhibited IL2 and TNF in polychemoresistant choriocarcinoma. The main biological functions predicted to be altered in chemoresistant choriocarcinoma were related to immunological homeostasis and leukopoiesis. Conclusion. HLA-G is a strong candidate gene to predict choriocarcinoma resistance to monochemotherapy and that further studies are required to implement its routine quantification in the decision process for the man-agement of gestational choriocarcinoma. (c) 2020 Elsevier Inc. All rights reserved.

Human endogenous retroviruses (HERVs) and mammalian apparent long terminal repeat (LTR) retrotransposons (MaLRs) are retroviral sequences that integrated into germ line cells millions of years ago. Transcripts of these LTR retrotransposons are present in several tissues, and their expression is modulated in pathological conditions, although their function remains often far from being understood. Here, we focused on the HERV/MaLR expression and modulation in a scenario of immune system activation. We used a public data set of human peripheral blood mononuclear cells (PBMCs) RNA-Seq from 15 healthy participants to a clinical trial before and after exposure to lipopolysaccharide (LPS), for which we established an RNA-Seq workflow for the identification of expressed and modulated cellular genes and LTR retrotransposon elements. IMPORTANCE We described the HERV and MaLR transcriptome in PBMCs, finding that about 8.4% of the LTR retrotransposon loci were expressed and identifying the betaretrovirus-like HERVs as those with the highest percentage of expressed loci. We found 4,607 HERV and MaLR loci that were modulated as a result of in vivo stimulation with LPS. The HERV-H group showed the highest number of differentially expressed most intact proviruses. We characterized the HERV and MaLR loci as differentially expressed, checking their genomic context of insertion and observing a general colocalization with genes that are involved and modulated in the immune response, as a consequence of LPS stimulation. The analyses of HERV and MaLR expression and modulation show that these LTR retrotransposons are expressed in PBMCs and regulated in inflammatory settings. The similar regulation of HERVs/MaLRs and genes after LPS stimulation suggests possible interactions of LTR retrotransposons and the immune host response.

The complexity of sepsis pathophysiology hinders patient management and therapeutic decisions. In this proof-of-concept study we characterised the underlying host immune response alterations using a standardised immune functional assay (IFA) in order to stratify a sepsis population. In septic shock patients, ex vivo LPS and SEB stimulations modulated, respectively, 5.3% (1/19) and 57.1% (12/21) of the pathways modulated in healthy volunteers (HV), highlighting deeper alterations induced by LPS than by SEB. SEB-based clustering, identified 3 severity-based groups of septic patients significantly different regarding mHLA-DR expression and TNFα level post-LPS, as well as 28-day mortality, and nosocomial infections. Combining the results from two independent cohorts gathering 20 HV and 60 patients, 1 cluster grouped all HV with 12% of patients. The second cluster grouped 42% of patients and contained all non-survivors. The third cluster grouped 46% of patients, including 78% of those with nosocomial infections. The molecular features of these clusters indicated a distinctive contribution of previously described genes defining a "healthy-immune response" and a "sepsis-related host response". The third cluster was characterised by potential immune recovery that underlines the possible added value of SEB-based IFA to capture the sepsis immune response and contribute to personalised management.

A new member of Anelloviridae, named torque teno mini virus (TTMV)-SH, was recently identified in the serum of three Hodgkin's lymphoma patients suggesting that TTMV-SH may be associated with this type of hematological malignancy. We investigated by metagenomic analysis the presence of TTMV-SH-related viruses in plasma samples (n = 323) collected from patients with various hematological malignancies (multiple myeloma (MM, n = 256), non-Hodgkin's lymphoma (NHL, n = 20), acute myeloid leukemia (n = 10)) and from healthy donors (n = 37). TTMV-SH-related strains were identified in 24 samples corresponding to four MM and one NHL patients. Phylogenic analysis revealed that the 24 isolates were close to the TTMV-SH strains previously identified, sharing 79.6-86.7% ORF1 nucleotide sequence identity. These results suggest that TTMV-SH-related viruses might be found in hematological diseases other than Hodgkin's lymphoma. Due to the high genetic variability within Anelloviridae species, the association between a particular medical condition and a new genotype should be interpreted with caution.

Although human endogenous retroviruses (HERVs) expression is a growing subject of interest, no study focused before on specific endogenous retroviruses loci activation in severely injured patients. Yet, HERV reactivation is observed in immunity compromised settings like some cancers and auto-immune diseases. Our objective was to assess the transcriptional modulation of HERVs in burn, trauma and septic shock patients. We analyzed HERV transcriptome with microarray data from whole blood samples of a burn cohort (n = 30), a trauma cohort (n = 105) and 2 septic shock cohorts (n = 28, n = 51), and healthy volunteers (HV, n = 60). We described expression of the 337 probesets targeting HERV from U133 plus 2.0 microarray in each dataset and then we compared HERVs transcriptional modulation of patients compared to healthy volunteers. Although all 4 cohorts contained critically ill patients, the majority of the 337 HERVs was not expressed (around 74% in mean). Each cohort had differentially expressed probesets in patients compared to HV (from 19 to 46). Strikingly, 5 HERVs were in common in all types of severely injured patients, with 4 being up-modulated in patients. We highlighted co-expressed profiles between HERV and nearby CD55 and CD300LF genes as well as autonomous HERV expression. We suggest an inflammatory-specific HERV transcriptional response, and importantly, we introduce that the HERVs close to immunity-related genes might have a role on its expression.

Human Endogenous Retroviruses (HERVs) and Mammalian apparent LTR-retrotransposons (MaLRs) are retroviral sequences that integrated into the germline cells millions year ago. Transcripts of these LTR-retrotransposons are present in several tissues, and their expression is modulated in pathological conditions, although their function remains often far from being understood. In this work, we focused on the HERVs/MaLRs expression and modulation in a scenario of immune system activation. We used a public dataset of Human Peripheral Blood Mononuclear Cells (PBMCs) RNA-seq from 15 healthy participants to a clinical trial before and after the exposure to Lipopolysaccharide (LPS), for which we established an RNA-seq workflow for the identification of expressed and modulated cellular genes and LTR-retrotransposon elements. Importance We described the HERV and MaLR transcriptome in PBMCs, finding that about 8.4 % of the LTR-retrotransposons loci were expressed, and identifying the betaretrovirus-like HERVs as those with the highest percentage of expressed loci. We found 4,607 HERVs and MaLRs loci that were modulated as a result of in vivo stimulation with LPS. The HERV-H group showed the highest number of differentially expressed most intact proviruses. We characterized the HERV and MaLR loci differentially expressed checking their genomic context of insertion and, interestingly, we found a general co-localization with genes that are involved and modulated in the immune response, as consequence of LPS stimulation. The analyses of HERVs and MaLRs expression and modulation show that this LTR-retrotransposons are expressed in PBMCs and regulated in inflammatory settings. The similar regulation of HERVs/MaLRs and genes after LPS stimulation suggests possible interactions of LTR-retrotransposons and the immune host response.

Recent advances in the immunotherapy field require evaluation of the immune function to adapt therapeutic decisions. Immune functional assays (IFA) are able to reveal the immune status and would be useful to further adapt and/or improve patient's care. However, standardized methods are needed to implement IFA in clinical settings. We carried out an independent validation of a published method used to characterize the underlying host response to infectious conditions using an IFA. We evaluated the reproducibility and robustness of this IFA and the associated readout using an independent healthy volunteers (HV) cohort. Expression of a 44-gene signature and IFNγ protein secretion was assessed after stimulation. We observed a strong host-response correlation between the two cohorts. We also highlight that standardized methods for immune function evaluation exist and could be implemented in larger-scale studies. This IFA could be a relevant tool to reveal innate and adaptive immune dysfunction in immune-related disorders patients.

Sepsis, which is the leading cause of death in intensive care units (ICU), has been acknowledged as a global health priority by the WHO in 2017. Identification of biomarkers allowing early stratification and recognition of patients at higher risk of death is crucial. One promising biomarker candidate is pentraxin-3 (PTX3); initially elevated and persistently increased plasma concentration in septic patients has been associated with increased mortality. PTX3 is an acute phase protein mainly stored in neutrophil granules. These cells are responsible for rapid and prompt release of PTX3 in inflammatory context, but the cellular origin responsible for successive days' elevation in sepsis remains unknown. Upon inflammatory stimulation, PTX3 can also be produced by other cell types, including endothelial and immune cells. As in septic patients immune alterations have been described, we therefore sought to investigate whether such cells participated in the elevation of PTX3 over the first days after septic shock onset. To address this point, PTX3 was measured in plasma from septic shock patients at day 3 after ICU admission as well as in healthy volunteers (HV), and the capacity of whole blood cells to secrete PTX3 after inflammatory stimulation was evaluated ex vivo. A significantly mean higher (100-fold) concentration of plasma PTX3 was found in patients compared to HV, which was likely due to the inflammation-induced initial release of the pre-existing PTX3 reservoir contained in neutrophils. Strikingly, when whole blood was stimulated ex vivo with LPS no significant difference between patients and HV in PTX3 release was found. This was in contrast with TNFα which decreased production was illustrative of the endotoxin tolerance phenomenon occurring in septic patients. Then, the release of PTX3 protein from a HV neutrophil-free PBMC endotoxin tolerance model was investigated. At the transcriptional level, PTX3 seems to be a weakly tolerizable gene similar to TNFα. Conversely, increased protein levels observed in anergy condition reflects a non-tolerizable phenotype, more likely to an anti-inflammatory marker. Hence, altered immune cells still have the ability to produce PTX3 in response to an inflammatory trigger, and therefore circulating white blood cell subset could be responsible of the sustained PTX3 plasma levels over the first days of sepsis setting.

Aims: The objectives of this study were to explore circulating tumor cell-secreted extracellular vesicles (EVs) and the presence of the HERV-W-encoded syncytin-1 as an EV cargo protein in primary cutaneous T-cell lymphoma (CTCL) cell lines. EVs exert pleiotropic effects, both in the tumor microenvironment and at long distance.

Human Endogenous Retroviruses (HERVs) and Mammalian apparent LTR-retrotransposons (MaLRs) represent the 8% of our genome and are distributed among our 46 chromosomes. These LTR-retrotransposons are thought to be essentially silent except in cancer, autoimmunity and placental development. Their Long Terminal Repeats (LTRs) constitute putative promoter or polyA regulatory sequences. In this study, we used a recently described high-density microarray which can be used to study HERV/MaLR transcriptome including 353,994 HERV/MaLR loci and 1559 immunity-related genes.