Biobanco del Sistema Sanitario de Andalucía

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Core Circadian Clock Proteins as Biomarkers of Progression in Colorectal Cancer.
(2021-08-06) Aroca-Siendones, María I; Moreno-SanJuan, Sara; Puentes-Pardo, Jose D; Verbeni, Michela; Arnedo, Javier; Escudero-Feliu, Julia; García-Costela, María; García-Robles, Adelina; Carazo, Ángel; León, Josefa
Colorectal cancer (CRC) is one of the most common tumours in developed countries. Although its incidence and mortality rates have decreased, its prognosis has not changed, and a high percentage of patients with CRC develop relapse (metachronous metastasis, MM, or local recurrence, LR) during their disease. The identification of these patients is very important for their correct management, but the lack of prognostic markers makes it difficult. Given the connection between circadian disruption and cancer development and progression, we aimed to analyse the prognostic significance of core circadian proteins in CRC. We measured the expression of PER1-3, CRY1-2, BMAL1 and NR1D2 in a cohort of CRC patients by immunohistochemistry (IHC) and analysed their prognostic potential in this disease. A low expression of PER2 and BMAL1 was significantly associated with metastasis at the moment of disease diagnosis, whereas a high expression of CRY1 appeared as an independent prognostic factor of MM development. A high expression of NR1D2 appeared as an independent prognostic factor of LR development after disease diagnosis. Moreover, patients with a low expression of BMAL1 and a high expression of CRY1 showed lower OS and DFS at five years. Although these markers need to be validated in larger and different ethnic cohorts, the simplicity of IHC makes these proteins candidates for personalizing CRC treatment.
Low Prevalence of HER2-Positive Breast Carcinomas among Screening Detected Breast Cancers.
(MDPI AG, 2020-06-15) Lopez-Garcia, M Angeles; Carretero-Barrio, Irene; Perez-Mies, Belen; Chiva, Miguel; Castilla, Carolina; Vieites, Begoña; Palacios, Jose; Instituto de Salud Carlos III (ISCIII); CIBERONC; European Development Regional Fund ‘A way to achieve Europe’ (FEDER)
Conflicting results have been reported regarding the prevalence of screen-detected human epidermal growth factor receptor 2 (HER2)-positive breast carcinomas and non-screen detected HER2-positive breast carcinomas. To address this issue, we evaluated the prevalence of HER2-positive breast carcinomas in two independent regional screening programs in Spain. The clinicopathologic and immunohistochemical characteristics of 479 (306 and 173) screen-detected breast carcinomas and 819 (479 and 340) non-screen-detected breast carcinomas diagnosed in women between 50 and 69-year-olds were compared. The prevalence of HER2-positive breast carcinomas was 8.8% and 6.4% in the two series of screen-detected tumors, compared with 16.4% and 13% in non-screen-detected carcinomas. These differences were statistically significant. This lower prevalence of HER2-positive in-screen-detected breast carcinomas was observed in both hormone receptor positive (luminal HER2) and hormone-receptor-negative (HER2 enriched) tumors. In addition, a lower prevalence of triple-negative and a higher prevalence of luminal-A breast carcinomas was observed in screen-detected tumors. Moreover, a literature review pointed out important differences in subrogate molecular types in screen-detected breast carcinomas among reported series, mainly due to study design, technical issues and racial differences.
MASS CYTOMETRY DATA RECLASSIFY SYSTEMIC AUTOIMMUNE DISEASE PATIENTS IN PHENOTYPICALLY DISTINCTIVE GROUPS
(BMJ Group, 2022-06-01) Rybakowska, P.; van Gassen, S.; Perez-Sanchez, C.; Ibanez-Costa, A.; Varela, N.; Castro, R. Ortega; Fernandez-Roldan, C.; Jimenez-Moleon, I.; Ortego, N.; Raya, E.; Quesada, R. Aguilar; Lopez-Pedrera, C.; Estevez, E. Collantes; Saeys, Y.; Alarcon-Riquelme, M.; Maranon, C.; [Rybakowska, P.] Univ Granada, Andalusian Reg Govt, GENYO, PTS,Genom Med,Ctr Genom & Oncol Res Pfizer, Granada, Spain; [Alarcon-Riquelme, M.] Univ Granada, Andalusian Reg Govt, GENYO, PTS,Genom Med,Ctr Genom & Oncol Res Pfizer, Granada, Spain; [Maranon, C.] Univ Granada, Andalusian Reg Govt, GENYO, PTS,Genom Med,Ctr Genom & Oncol Res Pfizer, Granada, Spain; [van Gassen, S.] Univ Ghent, Dept Appl Math Comp Sci & Stat, VIB Ctr Inflammat Res Data Min & Modeling Biomed, Ghent, Belgium; [Saeys, Y.] Univ Ghent, Dept Appl Math Comp Sci & Stat, VIB Ctr Inflammat Res Data Min & Modeling Biomed, Ghent, Belgium; [Perez-Sanchez, C.] Univ Cordoba, Reina Sofia Univ Hosp, Maimonides Inst Res Biomed Cordoba IMIBIC, Cordoba, Spain; [Ibanez-Costa, A.] Univ Cordoba, Reina Sofia Univ Hosp, Maimonides Inst Res Biomed Cordoba IMIBIC, Cordoba, Spain; [Castro, R. Ortega] Univ Cordoba, Reina Sofia Univ Hosp, Maimonides Inst Res Biomed Cordoba IMIBIC, Cordoba, Spain; [Lopez-Pedrera, C.] Univ Cordoba, Reina Sofia Univ Hosp, Maimonides Inst Res Biomed Cordoba IMIBIC, Cordoba, Spain; [Estevez, E. Collantes] Univ Cordoba, Reina Sofia Univ Hosp, Maimonides Inst Res Biomed Cordoba IMIBIC, Cordoba, Spain; [Fernandez-Roldan, C.] Univ Granada, Hosp Univ San Cecilio, Dept Med, Serv Med Interna,Unidad Enfermedades Autoinmunes, Granada, Spain; [Ortego, N.] Univ Granada, Hosp Univ San Cecilio, Dept Med, Serv Med Interna,Unidad Enfermedades Autoinmunes, Granada, Spain; [Jimenez-Moleon, I.] Hosp Univ San Cecilio, Serv Reumatol, PTS, Granada, Spain; [Raya, E.] Hosp Univ San Cecilio, Serv Reumatol, PTS, Granada, Spain; [Quesada, R. Aguilar] Andalusian Publ Hlth Syst Biobank, Biobanco Sistema Sanitario Publ Andalucia, Granada, Spain; Innovative Medicines Initiative 2 Joint Undertaking (JU); European Union; EFPIA
Systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSC), Sjögren’s syndrome (SJS), mixed connective tissue disease (MCTD), primary antiphospholipid syndrome (PAPS) and undifferentiated connective tissue disease (UCTD) are classified as systemic autoimmune diseases (SADs). They are diagnosed based on different clinical and laboratory criteria. Due to their high internal heterogeneity and overlapping symptoms, SADs are difficult to diagnose. Therefore, molecular and cellular-based studies need to be undertaken to precisely classify the patients. Mass cytometry is a single-cell proteomics technology that measures approximately 50 markers per cell, thus it is a suitable tool to perform deep-phenotyping studies in SADs.
Identification of the genetic mechanism that associates L3MBTL3 to multiple sclerosis.
(Oxford University Press, 2022-07-07) Alcina, Antonio; Fedetz, Maria; Vidal-Cobo, Isabel; Andres-Leon, Eduardo; Garcia-Sanchez, Maria-Isabel; Barroso-Del-Jesus, Alicia; Eichau, Sara; Gil-Varea, Elia; Luisa-Maria Villar; Saiz, Albert; Leyva, Laura; Vandenbroeck, Koen; Otaegui, David; Izquierdo, Guillermo; Comabella, Manuel; Urcelay, Elena; Matesanz, Fuencisla
Multiple sclerosis (MS) is a complex and demyelinating disease of the central nervous system. One of the challenges of the post-genome-wide association studies (GWAS) era is to understand the molecular basis of statistical associations to reveal gene networks and potential therapeutic targets. The L3MBTL3 locus has been associated with MS risk by GWAS. To identify the causal variant of the locus, we performed fine mapping in a cohort of 3440 MS patients and 1688 healthy controls. The variant that best explained the association was rs6569648 (P = 4.13E-10, odds ratio = 0.71, 95% confidence interval (CI) = 0.64-0.79), which tagged rs7740107, located in intron 7 of L3MBTL3. The rs7740107 (A/T) variant has been reported to be the best expression and splice quantitative trait locus (eQTL and sQTL) of the region in up to 35 human genotype-tissue expression (GTEx) tissues. By sequencing RNA from blood of 17 MS patients and quantification by digital qPCR, we determined that this eQTL/sQTL originated from the expression of a novel short transcript starting in intron 7 near rs7740107. The short transcript was translated into three proteins starting at different translation initiation codons. These N-terminal truncated proteins lacked the region where L3MBTL3 interacts with the transcriptional regulator Recombination Signal Binding Protein for Immunoglobulin Kappa J Region which, in turn, regulates the Notch signalling pathway. Our data and other functional studies suggest that the genetic mechanism underlying the MS association of rs7740107 affects not only the expression of L3MBTL3 isoforms, but might also involve the Notch signalling pathway.
A new molecular classification to drive precision treatment strategies in primary Sjögren's syndrome.
(Nature Publishing Group, 2021-06-10) Soret, Perrine; Le-Dantec, Christelle; Desvaux, Emiko; Foulquier, Nathan; Chassagnol, Bastien; Hubert, Sandra; Jamin, Christophe; Barturen, Guillermo; Desachy, Guillaume; Devauchelle-Pensec, Valerie; Boudjeniba, Cheïma; Cornec, Divi; Saraux, Alain; Jousse-Joulin, Sandrine; Barbarroja, Nuria; Rodriguez-Pinto, Ignasi; De Langhe, Ellen; Beretta, Lorenzo; Chizzolini, Carlo; Kovacs, Laszlo; Witte, Torsten; Bettacchioli, Eleonore; Buttgereit, Anne; Makowska, Zuzanna; Lesche, Ralf; Borghi, Maria Orietta; Martin, Javier; Courtade-Gaiani, Sophie; Xuereb, Laura; Guedj, Mickaël; Moingeon, Philippe; Alarcon-Riquelme, Marta E; Laigle, Laurence; Pers, Jacques-Olivier; Innovative Medicines Initiative Joint Undertaking; European Union’s Seventh Framework Program; PRECISESADS Flow Cytometry Consortium
There is currently no approved treatment for primary Sjögren's syndrome, a disease that primarily affects adult women. The difficulty in developing effective therapies is -in part- because of the heterogeneity in the clinical manifestation and pathophysiology of the disease. Finding common molecular signatures among patient subgroups could improve our understanding of disease etiology, and facilitate the development of targeted therapeutics. Here, we report, in a cross-sectional cohort, a molecular classification scheme for Sjögren's syndrome patients based on the multi-omic profiling of whole blood samples from a European cohort of over 300 patients, and a similar number of age and gender-matched healthy volunteers. Using transcriptomic, genomic, epigenetic, cytokine expression and flow cytometry data, combined with clinical parameters, we identify four groups of patients with distinct patterns of immune dysregulation. The biomarkers we identify can be used by machine learning classifiers to sort future patients into subgroups, allowing the re-evaluation of response to treatments in clinical trials.
Porcine Hemagglutinating Encephalomyelitis Virus Infection In Vivo and Ex Vivo.
(2021-05-24) Mora-Díaz, Juan Carlos; Piñeyro, Pablo E; Rauh, Rolf; Nelson, William; Sankoh, Zianab; Gregg, Edward; Carrillo-Ávila, José Antonio; Shen, Huigang; Nelli, Rahul K; Zimmerman, Jeffrey J; Giménez-Lirola, Luis G
Porcine hemagglutinating encephalomyelitis virus (PHEV) is a betacoronavirus that causes vomiting and wasting disease and/or encephalomyelitis in suckling pigs. This study characterized PHEV infection, pathogenesis, and immune response in cesarean-derived, colostrum-deprived (CDCD) neonatal pigs. Infected animals developed mild respiratory, enteric, and neurological clinical signs between 2 to 13 days postoronasal inoculation (dpi). PHEV did not produce viremia, but virus shedding was detected in nasal secretions (1 to 10 dpi) and feces (2 to 7 dpi) by reverse transcriptase quantitative PCR (RT-qPCR). Viral RNA was detected in all tissues except liver, but the detection rate and RT-qPCR threshold cycle (CT ) values decreased over time. The highest concentration of virus was detected in inoculated piglets necropsied at 5 dpi in turbinate and trachea, followed by tonsils, lungs, tracheobronchial lymph nodes, and stomach. The most representative microscopic lesions were gastritis lymphoplasmacytic, moderate, multifocal, with perivasculitis, and neuritis with ganglia degeneration. A moderate inflammatory response, characterized by increased levels of interferon alpha (IFN-α) in plasma (5 dpi) and infiltration of T lymphocytes and macrophages were also observed. Increased plasma levels of interleukin-8 (IL-8) were detected at 10 and 15 dpi, coinciding with the progressive resolution of the infection. Moreover, a robust antibody response was detected by 10 dpi. An ex vivo air-liquid CDCD-derived porcine respiratory cells culture (ALI-PRECs) system showed virus replication in ALI-PRECs and cytopathic changes and disruption of ciliated columnar epithelia, thereby confirming the tracheal epithelia as a primary site of infection for PHEV.IMPORTANCE Among the ∼46 virus species in the family Coronaviridae, many of which are important pathogens of humans and 6 of which are commonly found in pigs, porcine hemagglutinating encephalomyelitis remains one of the least researched. The present study provided a comprehensive characterization of the PHEV infection process and immune responses using CDCD neonatal pigs. Moreover, we used an ex vivo ALI-PRECs system resembling the epithelial lining of the tracheobronchial region of the porcine respiratory tract to demonstrate that the upper respiratory tract is a primary site of PHEV infection. This study provides a platform for further multidisciplinary studies of coronavirus infections.
Integrative Analysis Reveals a Molecular Stratification of Systemic Autoimmune Diseases.
(Wiley, 2021-05-29) Barturen, Guillermo; Babaei, Sepideh; Catala-Moll, Francesc; Martinez-Bueno, Manuel; Makowska, Zuzanna; Martorell-Marugan, Jordi; Carmona-Saez, Pedro; Toro-Dominguez, Daniel; Carnero-Montoro, Elena; Teruel, Maria; Kerick, Martin; Acosta-Herrera, Marialbert; Le Lann, Lucas; Jamin, Christophe; Rodriguez-Ubreva, Javier; Garcia-Gomez, Antonio; Kageyama, Jorge; Buttgereit, Anne; Hayat, Sikander; Mueller, Joerg; Lesche, Ralf; Hernandez-Fuentes, Maria; Juarez, Maria; Rowley, Tania; White, Ian; Marañon, Concepcion; Gomes-Anjos, Tania; Varela, Nieves; Aguilar-Quesada, Rocio; Garrancho, Francisco Javier; Lopez-Berrio, Antonio; Rodriguez-Maresca, Manuel; Navarro-Linares, Hector; Almeida, Isabel; Azevedo, Nancy; Brandão, Mariana; Campar, Ana; Faria, Raquel; Farinha, Fatima; Marinho, António; Neves, Esmeralda; Tavares, Ana; Vasconcelos, Carlos; Trombetta, Elena; Montanelli, Gaia; Vigone, Barbara; Alvarez-Errico, Damiana; Li, Tianlu; Thiagaran, Divya; Blanco-Alonso, Ricardo; Corrales-Martinez, Alfonso; Genre, Fernanda; Lopez-Mejias, Raquel; Gonzalez-Gay, Miguel A; Remuzgo, Sara; Ubilla-Garcia, Begoña; Cervera, Ricard; Espinosa, Gerard; Rodriguez-Pinto, Ignasi; De-Langhe, Ellen; Cremer, Jonathan; Lories, Rik; Belz, Doreen; Hunzelmann, Nicolas; Baerlecken, Niklas; Kniesch, Katja; Witte, Torsten; Lehner, Michaela; Stummvoll, Georg; Zauner, Michael; Aguirre-Zamorano, Maria Angeles; Barbarroja, Nuria; Castro-Villegas, Maria Carmen; Collantes-Estevez, Eduardo; de-Ramon, Enrique; Diaz-Quintero, Isabel; Escudero-Contreras, Alejandro; Fernandez Roldan, Maria Concepcion; Jimenez-Gomez, Yolanda; Jimenez Moleon, Inmaculada; Lopez-Pedrera, Rosario; Ortega-Castro, Rafaela; Ortego, Norberto; Raya, Enrique; Artusi, Carolina; Gerosa, Maria; Meroni, Pier Luigi; Schioppo, Tommaso; De-Groof, Aurelie; Ducreux, Julie; Lauwerys, Bernard; Maudoux, Anne-Lise; Cornec, Divi; Devauchelle-Pensec, Valerie; Jousse-Joulin, Sandrine; Jouve, Pierre-Emmanuel; Rouviere, Benedicte; Saraux, Alain; Simon, Quentin; Alvarez, Montserrat; Chizzolini, Carlo; Dufour, Aleksandra; Wynar, Donatienne; Balog, Attila; Bocskai, Marta; Deak, Magdolna; Dulic, Sonja; Kadar, Gabriella; Kovacs, Laszlo; Cheng, Qingyu; Gerl, Velia; Hiepe, Falk; Khodadadi, Laleh; Thiel, Silvia; de-Rinaldis, Emanuele; Rao, Sambasiva; Benschop, Robert J; Chamberlain, Chris; Dow, Ernst R; Ioannou, Yiannis; Laigle, Laurence; Marovac, Jacqueline; Wojcik, Jerome; Renaudineau, Yves; Borghi, Maria Orietta; Frostegard, Johan; Martin, Javier; Beretta, Lorenzo; Ballestar, Esteban; McDonald, Fiona; Pers, Jacques-Olivier; Alarcon-Riquelme, Marta E; Innovative Medicines Initiative Joint Undertaking; Instituto de Salud Carlos III
Clinical heterogeneity, a hallmark of systemic autoimmune diseases, impedes early diagnosis and effective treatment, issues that may be addressed if patients could be classified into groups defined by molecular pattern. This study was undertaken to identify molecular clusters for reclassifying systemic autoimmune diseases independently of clinical diagnosis. Unsupervised clustering of integrated whole blood transcriptome and methylome cross-sectional data on 955 patients with 7 systemic autoimmune diseases and 267 healthy controls was undertaken. In addition, an inception cohort was prospectively followed up for 6 or 14 months to validate the results and analyze whether or not cluster assignment changed over time. Four clusters were identified and validated. Three were pathologic, representing "inflammatory," "lymphoid," and "interferon" patterns. Each included all diagnoses and was defined by genetic, clinical, serologic, and cellular features. A fourth cluster with no specific molecular pattern was associated with low disease activity and included healthy controls. A longitudinal and independent inception cohort showed a relapse-remission pattern, where patients remained in their pathologic cluster, moving only to the healthy one, thus showing that the molecular clusters remained stable over time and that single pathogenic molecular signatures characterized each individual patient. Patients with systemic autoimmune diseases can be jointly stratified into 3 stable disease clusters with specific molecular patterns differentiating different molecular disease mechanisms. These results have important implications for future clinical trials and the study of nonresponse to therapy, marking a paradigm shift in our view of systemic autoimmune diseases.
Infection and immune response to porcine hemagglutinating encephalomyelitis virus in grower pigs.
(2020-12-16) Mora-Díaz, Juan Carlos; Temeeyasen, Gun; Magtoto, Ronaldo; Rauh, Rolf; Nelson, William; Carrillo-Ávila, José Antonio; Zimmerman, Jeffrey; Piñeyro, Pablo; Giménez-Lirola, Luis
Porcine hemagglutinating encephalomyelitis virus (PHEV) is the cause of acute outbreaks of vomiting and wasting disease and/or encephalomyelitis in neonatal pigs, with naïve herds particularly vulnerable to clinical episodes. PHEV infections in older pigs are generally considered to be subclinical, but are poorly characterized in the refereed literature. In this study, twelve 7-week-old pigs were oronasally inoculated with 0.5 mL (1:128 HA titer) PHEV (Mengeling strain) and then followed through 42 days post inoculation (dpi). Fecal and oral fluid specimens were collected daily to evaluate viral shedding. Serum samples were tested for viremia, isotype-specific antibody responses, cytokine, and chemokine responses. Peripheral blood mononuclear cells were isolated to evaluate phenotype changes in immune cell subpopulations. No clinical signs were observed in PHEV inoculated pigs, but virus was detected in oral fluid (1-28 dpi) and feces (1-10 dpi). No viremia was detected, but a significant IFN-α response was observed in serum at 3 dpi, followed by the detection of IgM (dpi 7), and IgA/IgG (dpi 10). Flow cytometry revealed a one-off increase in cytotoxic T cells at 21 dpi. This study demonstrated that exposure of grower pigs to PHEV results in subclinical infection characterized by active viral replication and shedding followed by an active humoral and cell-mediated immune response that attenuates the course of the infection and results in viral clearance.
Pluripotent stem cell regulation in Spain and the Spanish National Stem Cell Bank.
(2020-08-24) Aran, Begoña; Lukovic, Dunja; Aguilar-Quesada, Rocio; Veiga, Anna
The Spanish National Stem Cell Bank (Banco Nacional de Líneas Celulares, BNLC) was established in 2006 thanks to a change in the legislative framework in Spain. The Law 14/2006 updated the previous Assisted Reproduction Techniques Law (Law 45/2003) allowing the use of the surplus frozen embryos following IVF for research. The BNLC has a network structure with 3 nodes: the Regenerative Medicine Program (IDIBELL), the Principe Felipe Research Center (CIPF) in Valencia and the Andalusian Public Health System Biobank (SSPA Biobank) in Granada. The aim of the BNLC is to guarantee throughout the national territory the availability of human stem cell lines for biomedical research. At present time, there are 40 human embryonic stem cell lines (hESC) and 171 human induced pluripotent stem cell lines (hiPSC) registered in the BNLC. These lines are fully characterized and available in the context of research projects approved by the Technical Committee of the BNLC.
Biobanking Spotlight on Europe, Middle East, and Africa: Presenting the Collective Experience of the ISBER-EMEA Regional Ambassadors.
(2020-08-11) Afifi, Nahla M; Anisimov, Sergey V; Aguilar-Quesada, Rocío; Kinkorova, Judita; Marrs, Stephen; Nassimbwa, Sureyah; Kozlakidis, Zisis; Parry-Jones, Alison
Mutation in ROBO3 Gene in Patients with Horizontal Gaze Palsy with Progressive Scoliosis Syndrome: A Systematic Review.
(2020-06-22) Pinero-Pinto, Elena; Pérez-Cabezas, Verónica; Tous-Rivera, Cristina; Sánchez-González, José-María; Ruiz-Molinero, Carmen; Jiménez-Rejano, José-Jesús; Benítez-Lugo, María-Luisa; Sánchez-González, María Carmen
Horizontal gaze palsy with progressive scoliosis (HGPPS) is a rare, inherited disorder characterized by a congenital absence of conjugate horizontal eye movements with progressive scoliosis developing in childhood and adolescence. Mutations in the Roundabout (ROBO3) gene located on chromosome 11q23-25 are responsible for the development of horizontal gaze palsy and progressive scoliosis. However, some studies redefined the locus responsible for this pathology to a 9-cM region. This study carried out a systematic review in which 25 documents were analyzed, following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) standards. The search was made in the following electronic databases from January 1995 to October 2019: PubMed, Scopus, Web of Science, PEDRO, SPORT Discus, and CINAHL. HGPPS requires a multidisciplinary diagnostic approach, in which magnetic resonance imaging might be the first technique to suggest the diagnosis, which should be verified by an analysis of the ROBO3 gene. This is important to allow for adequate ocular follow up, apply supportive therapies to prevent the rapid progression of scoliosis, and lead to appropriate genetic counseling.
Agrodiag PorCoV: A multiplex immunoassay for the differential diagnosis of porcine enteric coronaviruses.
(2020-06-17) Malbec, Rémi; Kimpston-Burkgren, Kay; Vandenkoornhuyse, Elisa; Olivier, Christophe; Souplet, Vianney; Audebert, Christophe; Carrillo-Ávila, Jose Antonio; Baum, David; Giménez-Lirola, Luis
Three different porcine enteric coronaviruses (PECs), i.e., porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine Deltacoronavirus (PDCoV) are currently circulating in U.S. commercial swine herds. Differential diagnosis of PECs relies on laboratory methods. This study describes the development of an ELISA-like multiplex planar immunoassay based on virus-specific recombinant S1 proteins printed in an array of spots at the bottom of a 96-well microplate for simultaneous detection differential serodiagnosis of PEDV, TGEV, PDCoV in a single sample. The technology overall format and working principle is similar to the solid-phase standard ELISA. After the three typical incubation steps, the reaction was visualized as blue spots which intensity correlated with antibody levels to specific viral antigen target in the array. The diagnostic performance of the assay was evaluated on known status serum samples (n = 480) collected over time (day post-inoculation -7, 0, 7, 14, 21, 28, 35, and 42) from pigs inoculated with PEDV, TGEV Purdue, TGEV Miller, PDCoV (USA/IL/2014), or mock inoculated with culture media under experimental conditions. Antigen-specific cut-offs were selected to ensure 100% diagnostic and analytical specificity for each given antigen target. The overall diagnostic sensitivity was 92% (44/48 positives, 95% confidence interval (CI) 98,100) for PEDV S1, 100% (95/95 positives, 95% CI 98, 100) for TGEV S1, and 98% (47/48 positives, 95% CI 97, 100) for PDCoV S1. The results of this study demonstrate that the AgroDiag PEC multiplex immunoassay is an efficient and reliable test for differential detection and serodiagnosis of PEDV, TGEV and PDCoV.
Detecting and Monitoring Porcine Hemagglutinating Encephalomyelitis Virus, an Underresearched Betacoronavirus.
(2020-05-06) Mora-Díaz, Juan Carlos; Magtoto, Ronaldo; Houston, Elizabeth; Baum, David; Carrillo-Ávila, José Antonio; Temeeyasen, Gun; Zimmerman, Jeff; Piñeyro, Pablo; Giménez-Lirola, Luis
Members of family Coronaviridae cause a variety of diseases in birds and mammals. Porcine hemagglutinating encephalomyelitis virus (PHEV), a lesser-researched coronavirus, can infect naive pigs of any age, but clinical disease is observed in pigs ≤4 weeks of age. No commercial PHEV vaccines are available, and neonatal protection from PHEV-associated disease is presumably dependent on lactogenic immunity. Although subclinical PHEV infections are thought to be common, PHEV ecology in commercial swine herds is unknown. To begin to address this gap in knowledge, a serum IgG antibody enzyme-linked immunosorbent assay (ELISA) based on the S1 protein was developed and evaluated on known-status samples and then used to estimate PHEV seroprevalence in U.S. sow herds. Assessment of the diagnostic performance of the PHEV S1 ELISA using serum samples (n = 924) collected from 7-week-old pigs (n = 84; 12 pigs per group) inoculated with PHEV, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine respiratory coronavirus, or porcine deltacoronavirus showed that a sample-to-positive cutoff value of ≥0.6 was both sensitive and specific, i.e., all PHEV-inoculated pigs were seropositive from days postinoculation 10 to 42, and no cross-reactivity was observed in samples from other groups. The PHEV S1 ELISA was then used to estimate PHEV seroprevalence in U.S. sow herds (19 states) using 2,756 serum samples from breeding females (>28 weeks old) on commercial farms (n = 104) with no history of PHEV-associated disease. The overall seroprevalence was 53.35% (confidence interval [CI], ±1.86%) and herd seroprevalence was 96.15% (CI, ±3.70%).IMPORTANCE There is a paucity of information concerning the ecology of porcine hemagglutinating encephalomyelitis virus (PHEV) in commercial swine herds. This study provided evidence that PHEV infection is endemic and highly prevalent in U.S. swine herds. These results raised questions for future studies regarding the impact of endemic PHEV on swine health and the mechanisms by which this virus circulates in endemically infected populations. Regardless, the availability of the validated PHEV S1 enzyme-linked immunosorbent assay (ELISA) provides the means for swine producers to detect and monitor PHEV infections, confirm prior exposure to the virus, and to evaluate the immune status of breeding herds.
Impact of different stabilization methods on RT-qPCR results using human lung tissue samples.
(Nature Publishing Group, 2020-01-20) Esteva-Socias, Margalida; Gomez-Romano, Fernando; Carrillo-Avila, Jose Antonio; Sanchez-Navarro, Alicia Loreto; Villena, Cristina; Ministerio de Ciencia, Innovación y Universidades of Spain and Instituto de Salud Carlos III; Spanish Biobank Network; Conselleria d’Innovació, Recerca i Turisme del Govern de les Illes Balears
Aiming to increase the reproducibility of biomedical research results, biobanks obtain human tissues of the highest quality and carry out different storage methods adapted to the needs of analytical technique to be performed by the biomedical researchers. However, there is much controversy and little data concerning the real impact of different stabilization methods on tissue quality, integrity and functionality of derived biomolecules. The influence of four stabilization methods [RNAlater (RNL), snap freezing (SF), snap freezing using Optimal Cutting Tissue compound (SF-OCT) and formalin-fixed paraffin-embedded (FFPE)] on RNA quality and integrity was evaluated in paired samples of lung tissue. RNA integrity was evaluated through PCR-endpoint assays amplifying six fragments of different length of the HPRT1 gene and RNA Integrity Number (RIN). To evaluate the difference of tissue functionality among the stabilization methods tested, RT-qPCRs were performed focusing on the differential expression of the HPRT1, SNRPD3 and Jun genes. RNA from the samples preserved with the RNL or SF-OCT method showed better integrity compared to SF and FFPE, measured by PCR-endpoint and RT-qPCR assays. However, only statistically significant differences were observed between the RNA from FFPE and other stabilization methods when gene expression of HPRT1, SNRPD3 and Jun housekeeping genes were determined by RT-qPCR. For the three mentioned genes, Cq and RIN values were highly correlated. The present work describes the fragility of SF samples, being critical the moment just before RNA extraction, although further experiments of tissue RNA are needed. Standardization pre-analytic workflow can lead to improved reproducibility between biomedical research studies. The present study demonstrated clear evidences about the impact of the stabilization method on RNA derived from lung human tissue samples.
IL8 and EDEM3 gene expression ratio indicates peripheral blood mononuclear cell (PBMC) quality.
(2018-11-26) Kofanova, Olga; Bellora, Camille; Quesada, Rocio Aguilar; Bulla, Alexandre; Panadero-Fajardo, Sonia; Keipes, Marc; Shea, Kathi; Stone, Mars; Lescuyer, Pierre; Betsou, Fay
Uncontrolled preanalytical variables can reduce the accuracy and reproducibility of downstream analytical results from peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from EDTA and citrate-anticoagulated blood samples, obtained from healthy subjects and patients with inflammatory and infectious conditions. PBMC-derived RNA samples were examined for gene expression changes induced by extended blood pre-centrifugation delays at 4 °C and RT. We used Taqman RTqPCR to evaluate the combination of two target genes for their "diagnostic performance" in identifying EDTA and citrate-anticoagulated PBMC samples with extended pre-centrifugation times. We established the PBMC preanalytical score, a gene expression metric to asses the PBMC quality related to the pre-centrifugation delay at room temperature for different anticoagulants. The PBMC preanalytical score measurement can identify: CONCLUSION: The proposed PBMC preanalytical score may enable objective PBMC sample qualification for downstream applications, which may be influenced by blood precentrifugation delays.
Identification of the Missing Protein Hyaluronan Synthase 1 in Human Mesenchymal Stem Cells Derived from Adipose Tissue or Umbilical Cord.
(2018-07-06) Clemente, Luis Felipe; Hernáez, María Luisa; Ramos-Fernández, Antonio; Ligero, Gertrudis; Gil, Concha; Corrales, Fernando José; Marcilla, Miguel
Currently, 14% of the human proteome is made up of proteins whose existence is not confirmed by mass spectrometry. We performed a proteomic profiling of human mesenchymal stem cells derived from adipose tissue or umbilical cord (PRIDE accession number: PXD009893) and identified peptides derived from 13 of such missing proteins. Remarkably, we found compelling evidence of the expression of hyaluronan synthase 1 (NX_Q92839-1) and confirmed its identification by the fragmentation of four heavy-labeled peptides that coeluted with their endogenous light counterparts. Our data also suggest that mesenchymal stem cells constitute a promising source for the detection of missing proteins.
West Nile virus outbreak in humans and epidemiological surveillance, west Andalusia, Spain, 2016.
(2018) López-Ruiz, Nuria; Montaño-Remacha, María Del Carmen; Durán-Pla, Enric; Pérez-Ruiz, Mercedes; Navarro-Marí, Jose María; Salamanca-Rivera, Celia; Miranda, Blanca; Oyonarte-Gómez, Salvador; Ruiz-Fernández, Josefa
In Andalusia, Spain, West Nile virus (WNV) surveillance takes place from April to November, during the active vector period. Within this area seroconversion to this virus was evidenced in wild birds in 2004, affecting horses and two humans for the first time in 2010. Since 2010, the virus has been isolated every year in horses, and national and regional surveillance plans have been updated with the epidemiological changes found. WNV is spreading rapidly throughout southern Europe and has caused outbreaks in humans. Here we describe the second WNV outbreak in humans in Andalusia, with three confirmed cases, which occurred between August and September 2016, and the measures carried out to control it. Surveillance during the transmission season is essential to monitor and ensure prompt identification of any outbreaks.
IL8 and IL16 levels indicate serum and plasma quality.
(2018) Kofanova, Olga; Henry, Estelle; Aguilar Quesada, Rocio; Bulla, Alexandre; Navarro Linares, Hector; Lescuyer, Pierre; Shea, Kathi; Stone, Mars; Tybring, Gunnel; Bellora, Camille; Betsou, Fay
Longer pre-centrifugation times alter the quality of serum and plasma samples. Markers for such delays in sample processing and hence for the sample quality, have been identified. Twenty cytokines in serum, EDTA plasma and citrate plasma samples were screened for changes in concentration induced by extended blood pre-centrifugation delays at room temperature. The two cytokines that showed the largest changes were further validated for their "diagnostic performance" in identifying serum or plasma samples with extended pre-centrifugation times. In this study, using R&D Systems ELISA kits, EDTA plasma samples and serum samples with a pre-centrifugation delay longer than 24 h had an IL16 concentration higher than 313 pg/mL, and an IL8 concentration higher than 125 pg/mL, respectively. EDTA plasma samples with a pre-centrifugation delay longer than 48 h had an IL16 concentration higher than 897 pg/mL, citrate plasma samples had an IL8 concentration higher than 21.5 pg/mL and serum samples had an IL8 concentration higher than 528 pg/mL. These robust and accurate tools, based on simple and commercially available ELISA assays can greatly facilitate qualification of serum and plasma legacy collections with undocumented pre-analytics.
Prevalence and genetic diversity of Trichomonas vaginalis in the general population of Granada and co-infections with Gardnerella vaginalis and Candida species.
(2017-10-04) Carrillo-Ávila, José Antonio; Serrano-García, María Luisa; Fernández-Parra, Jorge; Sorlózano-Puerto, Antonio; Navarro-Marí, José María; Stensvold, C Rune; Gutiérrez-Fernández, Jose
Purulent or exudative genitourinary infections are a frequent cause of consultation in primary and specialized healthcare. The objectives of this study were: to determine the prevalence of Trichomonas vaginalis and co-infections with Candida spp. and Gardnerella vaginalis in vaginal secretion; and to use multilocus sequence typing (MLST) to analyse the genetic diversity of T. vaginalis strains. The samples were submitted for analysis (n=5230) to a third-level hospital in Granada (Southern Spain) between 2011 and 2014; eight T. vaginalis strains isolated during 2015 were randomly selected for MLST analysis. Culture and nucleic acid hybridization techniques were used to detect microorganisms in the samples. The prevalence of T. vaginalis was 2.4 % between 2011 and 2014, being higher during the first few months of both 2011 and 2012. Among samples positive for T. vaginalis, co-infection with G. vaginalis was detected in 29 samples and co-infection with Candida spp. in 6, while co-infection with all three pathogens was observed in 3 samples. The only statistically significant between-year difference in co-infection rates was observed for T. vaginalis with G. vaginalis due to an elevated rate in 2011. MLST analysis results demonstrated a high genetic variability among strains circulating in our setting. These findings emphasize the need for the routine application of diagnostic procedures to avoid the spread of this sexually transmitted infection.
Cell lines authentication and mycoplasma detection as minimun quality control of cell lines in biobanking.
(2017-03-02) Corral-Vázquez, C; Aguilar-Quesada, R; Catalina, P; Lucena-Aguilar, G; Ligero, G; Miranda, B; Carrillo-Ávila, J A
Establishment of continuous cell lines from human normal and tumor tissues is an extended and useful methodology for molecular characterization of cancer pathophysiology and drug development in research laboratories. The exchange of these cell lines between different labs is a common practice that can compromise assays reliability due to contamination with microorganism such as mycoplasma or cells from different flasks that compromise experiment reproducibility and reliability. Great proportions of cell lines are contaminated with mycoplasma and/or are replaced by cells derived for a different origin during processing or distribution process. The scientific community has underestimated this problem and thousand of research experiment has been done with cell lines that are incorrectly identified and wrong scientific conclusions have been published. Regular contamination and authentication tests are necessary in order to avoid negative consequences of widespread misidentified and contaminated cell lines. Cell banks generate, store and distribute cell lines for research, being mandatory a consistent and continuous quality program. Methods implementation for guaranteeing both, the absence of mycoplasma and authentication in the supplied cell lines, has been performed in the Andalusian Health System Biobank. Specifically, precise results were obtained using real time PCR detection for mycoplasma and 10 STRs identification by capillary electrophoresis for cell line authentication. Advantages and disadvantages of these protocols are discussed.

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