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BackgroundVaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective. Nevertheless, immunocompromised participants were excluded from randomized controlled clinical trials. This study evaluates the efficacy and safety of the Pfizer/BioNTech BNT162b2 (BNT162b2) vaccine in patients with breast and gynecological cancer treated with active anticancer therapy versus a control cohort of healthy participants.MethodsImmune responses to the BNT162b2 vaccine in patients with breast cancer (n = 44) or a gynecological malignancy (n = 6) on active anticancer therapy (28 on chemotherapy, mostly anthracycline- or taxane-based, and 22 on target therapy) and in a control cohort of participants without cancer (n = 67) were investigated by SARS-CoV-2 neutralizing antibody titers measured by S1-binding immunoglobulin G (IgG) concentrations assessed using the LIAISON XL tools (DiaSorin S.p.A.). Response was assessed after a second dose of the BNT162b2 vaccine administered before and at least 3 weeks after the vaccine dose.ResultsOverall, 43/50 (86%) patients of the cancer cohort (74% in the breast cancer group and 100% in the gynecological malignancy group) developed IgG antibodies after the second dose of the BNT162b2 vaccine. There were no statistically significant differences in responder rates between patients treated with chemotherapy and those on target therapy. The majority of patients who received chemotherapy with or without target therapy, 21/28 (75%), developed a reliable antibody titer after a vaccine. All seven non-responder patients were undergoing an anthracycline-based regimen. Based on IgG levels (0–400 AU/ml), patients were classified as negative (‘non-responders’), weakly positive, or strongly positive (‘responders’). No delay in cancer therapy schedule or reported side effects were recorded after BNT162b2 vaccine administration. All healthy participants were strongly positive. Responder rates differed significantly between the two study cohorts (p < 0.001).ConclusionsMost patients develop antibody titers after the second immunization. However, given the persistence of non-responders or weak responders, additional immunization booster seems to be required, along with proactive planning in the vaccination schedule, with vaccine administration spaced out over time with respect to chemotherapy.

Background: Late HIV diagnosis (i.e CD4≤350 cells/µL) is associated with poorer outcomes. However, determinants of long-term mortality and factors influencing immune recovery within the first years after antiretroviral treatment (ART) initiation are poorly defined. Methods: From PISCIS cohort, we included all HIV-positive adults, two-year survivors after initiating ART between 2005-2019. The primary outcome was all-cause mortality according to the two-year CD4 count. We used Poisson regression. The secondary outcome was incomplete immune recovery (i.e., two-year CD4500 cells/µL, reference population). Overall, 113 patients (4·2%) died. Mortality was higher among LP with two-year CD4 count 200-500 cells/µL (aMRR 1·95[95%CI:1·06-3·61]) or 500 cells/µL, regardless of being initially LP or non-LP (aMRR 1·05[0·50-2·21]). Mortality rates within each two-year CD4 strata were not affected by the initial CD4 count at ART initiation (test-interaction, p = 0·48). The stronger factor influencing immune recovery was the CD4 count at ART initiation. First-line integrase-inhibitor-(INSTI)-based regimens were associated with reduced mortality compared to other regimens (aMRR 0·54[0·31-0·93]) and reduced risk of incomplete immune recovery in LP (aOR 0·70[0·52-0·95]). Interpretation: Two-year immune recovery is a good early predictor of long-term mortality in LP after surviving the first high-risk 2 years. Nearly half experienced a favorable immune recovery with a life expectancy similar to non-LP. INSTI-based regimens were associated with higher rates of successful immune recovery and better survival compared to non-INSTI regimens.

ß-glucans are polysaccharides of D-glucose monomers that come from multiple living beings and are known to have multiple beneficial properties such as being immunostimulant, having antitumoral activity and being antioxidant. This Treball Final de Grau aims to carry out a pilot study with the two control groups regarding a R+D project titled “Innovative Barley-Based Functional Foods” by University of Lleida. The pilot study is done to provide useful information concerning improvements for the large-scale investigation and to try out some of the procedures suggested in the R+D project. For this purpose, 30 Balb/cB&J mice from Charles River Laboratories© (Barcelona, Spain) were randomly distributed into groups of five in a way that we ended up having six groups, three of each sex. Four groups were fed a standard diet supplemented with rice (RC), whereas two groups were fed a maintenance standard chow diet (SC). Every two days for six weeks, animals were weighed and exposed to a health evaluation, as well as their feed intake was monitored. After this period, mice were euthanized and blood, thymus, spleen, intestines, liver, and brain samples were collected for further analysis. Results showed there were no differences between diets regarding the thymus and splenic indexes, as well as the number of Peyer’s patches. The flow cytometry analysis and the histological analysis done on the spleen and the Peyer’s patches, respectively, did not show any conclusive results. Nevertheless, it has been useful as a pilot study to detect deficits in the design, establishment of animals and obtaining and processing samples.

Breast cancer; Predictive markers Cáncer de mama; Marcadores predictivos Càncer de mama; Marcadors predictius Trastuzumab acts in part through the adaptive immune system. Previous studies showed that enrichment of immune-related gene expression was associated with improved outcomes in HER2-positive (HER2+) breast cancer. However, the role of the immune system in response to lapatinib is not fully understood. Gene expression analysis was performed in 1,268 samples from the North Central Cancer Treatment Group (NCCTG) N9831 and 244 samples from the NeoALTTO trial. In N9831, enrichment of CD45 and immune-subset signatures were significantly associated with improved outcomes. We identified a novel 17-gene adaptive immune signature (AIS), which was found to be significantly associated with improved RFS among patients who received adjuvant trastuzumab (HR 0.66, 95% CI 0.49–0.90, Cox regression model p = 0.01) but not in patients who received chemotherapy alone (HR 0.96, 95% CI 0.67–1.40, Cox regression model p = 0.97). This result was validated in NeoALTTO. Overall, AIS-low patients had a significantly lower pathologic complete response (pCR) rate compared with AIS-high patients (χ2 p

Combined kinetic analysis of plasma SARS-CoV-2 RNAemia, Nucleocapsid (N)-antigenemia and virus-specific antibodies may help ascertain the role of antibodies in preventing virus dissemination in COVID-19 patients. We performed this analysis in a cohort of 71 consecutive critically ill COVID-19 patients (49 male; median age, 65 years) using RT-PCR assay, lateral flow immunochromatography method and receptor binding domain (RBD) and N-based immunoassays. A total of 338 plasma specimens collected at a median of 12 days after symptoms onset were available for analyses. SARS-CoV-2 RNAemia and N-antigenemia were detected in 37 and 43 specimens from 26 (36.5%) and 30 (42.2%) patients, respectively. Free RNA was the main biological form of SARS-CoV-2 found in plasma. The detection rate for both viral components was associated with viral load at the upper respiratory tract. Median time to SARS-CoV-2-RBD antibody detection was 14 days (range, 4-38) from onset of symptoms. Decreasing antibody levels were observed in parallel to increasing levels of both RNAemia and N-antigenemia, yet overall a fairly modest inverse correlation (Rho = -0.35; P, This work received no private or public funds.

Members of the regulatory Kvβ family modulate the kinetics and traffic of voltage-dependent K+ (Kv) channels. The crystal structure of Kv channels associated with Kvβ peptides suggests a α4/β4 composition. Although Kvβ2 and Kvβ1 form heteromers, evidence supports that only Kvβ2.1 forms tetramers in the absence of α subunits. Therefore, the stoichiometry of the Kvβ oligomers fine-tunes the activity of hetero-oligomeric Kv channel complexes. We demonstrate that Kvβ subtypes form homo- and heterotetramers with similar affinities. The Kvβ1.1/Kvβ2.1 heteromer showed an altered spatial distribution in lipid rafts, recapitulating the Kvβ1.1 pattern. Because Kvβ2 is an active partner of the Kv1.3-TCR complex at the immunological synapse (IS), an association with Kvβ1 would alter this location, shaping the immune response. Differential regulation of Kvβs influences the traffic and architecture of the Kvβ heterotetramer, modulating Kvβ-dependent physiological responses.

The rapid spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerging variants raises concerns about their capacity to evade immune protection provided by natural infection or vaccination. The receptor-binding domain (RBD) of the viral spike protein is the major target of neutralizing antibodies, and viral variants accumulate mutations in this region. In this study, we determined the antibody neutralization capacity against the RBD of SARS-CoV-2 variants Alpha (B.1.1.7), Gamma (P.1), Epsilon (B.1.427), Kappa (B.1.617.1), and Delta (B.1.617.2) in a cohort of healthcare workers naturally infected or receiving COVID-19 mRNA vaccines from Moderna or Pfizer-BioNTech. We show that the five RBD variants displayed an augmented binding to ACE2 compared to the original Wuhan strain. The most significant increase was observed in variants Epsilon and Delta, containing mutation L452R. Using a flow cytometry cell-based assay, we found that SARS-CoV-2-infected subjects presented low levels of RBD-specific neutralizing antibodies against all variants analyzed, except Alpha. However, the neutralizing activity incremented considerably after a subsequent mRNA-vaccine dose, to levels significantly higher than those in naïve individuals receiving two vaccine doses. Importantly, we observed partially impaired neutralizing responses against most variants in fully vaccinated individuals. Variants Gamma and Kappa encompassing RBD E484K/Q mutations presented the highest neutralizing resistance. Furthermore, a wide heterogeneity in the magnitude of RBD-specific neutralizing responses against all tested SARS-CoV-2 variants following both mRNA vaccines was detected. Altogether, our findings provide important knowledge regarding SARS-CoV-2 vaccine-induced immunity, and should be very useful to guide future vaccination regimens and personalized vaccine approaches.

Background Critically ill patients with coronavirus disease 19 (COVID-19) have a high fatality rate likely due to a dysregulated immune response. Corticosteroids could attenuate this inappropriate response, although there are still some concerns regarding its use, timing, and dose. Methods This is a nationwide, prospective, multicenter, observational, cohort study in critically ill adult patients with COVID-19 admitted into Intensive Care Units (ICU) in Spain from 12th March to 29th June 2020. Using a multivariable Cox model with inverse probability weighting, we compared relevant outcomes between patients treated with early corticosteroids (before or within the first 48 h of ICU admission) with those who did not receive early corticosteroids (delayed group) or any corticosteroids at all (never group). Primary endpoint was ICU mortality. Secondary endpoints included 7-day mortality, ventilator-free days, and complications. Results A total of 691 patients out of 882 (78.3%) received corticosteroid during their hospital stay. Patients treated with early-corticosteroids (n = 485) had lower ICU mortality (30.3% vs. never 36.6% and delayed 44.2%) and lower 7-day mortality (7.2% vs. never 15.2%) compared to non-early treated patients. They also had higher number of ventilator-free days, less length of ICU stay, and less secondary infections than delayed treated patients. There were no differences in medical complications between groups. Of note, early use of moderate-to-high doses was associated with better outcomes than low dose regimens. Conclusion Early use of corticosteroids in critically ill patients with COVID-19 is associated with lower mortality than no or delayed use, and fewer complications than delayed use.

The main goal of this Special Issue was to highlight the recent advances made on the role of mast cells (MCs) in host defense and pathology. MCs are recognized as crucial initiators and regulators of both innate and adaptive immune responses against pathogens. MCs are resident tissue cells found throughout the body, mainly in association with blood vessels and nerves, and are preferentially observed at mucosal surfaces and the skin.

Background: Inflammatory breast cancer (IBC) is a rare and understudied disease, with 40% of cases presenting with human epidermal growth factor receptor 2 (HER2)-positive subtype. The goals of this study were to (i) assess the pathologic complete response (pCR) rate of short-term neoadjuvant dual-HER2-blockade and paclitaxel, (ii) contrast baseline and on-treatment transcriptional profiles of IBC tumor biopsies associated with pCR, and (iii) identify biological pathways that may explain the effect of neoadjuvant therapy on tumor response. Patients and Methods: A single-arm phase II trial of neoadjuvant trastuzumab (H), pertuzumab (P), and paclitaxel for 16 weeks was completed among patients with newly diagnosed HER2-positive IBC. Fresh-frozen tumor biopsies were obtained pretreatment (D1) and 8 days later (D8), following a single dose of HP, prior to adding paclitaxel. We performed RNA-sequencing on D1 and D8 tumor biopsies, identified genes associated with pCR using differential gene expression analysis, identified pathways associated with pCR using gene set enrichment and gene expression deconvolution methods, and compared the pCR predictive value of principal components derived from gene expression profiles by calculating and area under the curve for D1 and D8 subsets. Results: Twenty-three participants were enrolled, of whom 21 completed surgery following neoadjuvant therapy. Paired longitudinal fresh-frozen tumor samples (D1 and D8) were obtained from all patients. Among the 21 patients who underwent surgery, the pCR and the 4-year disease-free survival were 48% (90% CI 0.29–0.67) and 90% (95% CI 66–97%), respectively. The transcriptional profile of D8 biopsies was found to be more predictive of pCR (AUC = 0.91, 95% CI: 0.7993–1) than the D1 biopsies (AUC = 0.79, 95% CI: 0.5905–0.9822). Conclusions: In patients with HER2-positive IBC treated with neoadjuvant HP and paclitaxel for 16 weeks, gene expression patterns of tumor biopsies measured 1 week after treatment initiation not only offered different biological information but importantly served as a better predictor of pCR than baseline transcriptional analysis. Trial Registration: ClinicalTrials.gov identifier: NCT01796197 ( https://clinicaltrials.gov/ct2/show/NCT01796197 ); registered on February 21, 2013.

Exposure to pesticides such as rotenone is a risk factor for Parkinson’s disease. Dopaminergic neurons are especially sensitive to the toxicity of compounds that inhibit the mitochondrial respiratory chain such as rotenone and 1-methyl-4-phenylpyridinium (MPP+). However, there is scarce information on their effects on glia. To evaluate whether these neurotoxicants affect the immune response of glia, primary mouse mixed glial and microglial cultures were treated with interleukin (IL) 4 in the absence and presence of MPP+ or rotenone. Using qRTPCR or western blot, we determined the expression of anti-inflammatory markers, the CD200R1 microglial receptor and its ligand CD200, and genes regulating glycolysis and oxidative metabolism. ATP and lactate levels were additionally determined as an index of cell metabolism. Microglial phagocytosis was also evaluated. MPP+ and rotenone clearly abrogated the IL4-induced expression of anti-inflammatory markers in mixed glial cultures. CD200 and CD200R1 expression and microglia phagocytosis were also affected by the neurotoxicants. Changes in the mRNA expression of the molecules regulating glycolysis and oxidative metabolism, as well as in ATP levels and lactate release suggested that metabolic reprogramming in response to MPP+ and rotenone differs between microglial and mixed glial cultures. These findings support the hypothesis that parkinsonian neurotoxicants may impair brain immune response altering glial cell metabolism., This study was supported by grant PI15/00033 from the Instituto de Salud Carlos III (Spain) with joint financing by FEDER funds from the European Union. NR-L was recipient of a predoctoral FPU contract from the Spanish Ministerio de Ciencia, Innovación y Universidades (FPU13/05491).

COVID-19; Booster; Chronic kidney disease COVID-19; Refuerzo; Enfermedad renal crónica COVID-19; Reforç; Malaltia renal crònica Introduction. Patients on peritoneal dialysis (PD) present an impaired humoral response against SARS-CoV-2, at least after the initial vaccination and booster dose. Until now, the effect of a fourth dose has not been established. The aim of the present study is to evaluate the long-term dynamics of the humoral response of PD patients to multiple doses of SARS-CoV-2 vaccines, focusing on the effect of the fourth dose. Methods. This is an analysis of the prospective and multicentric SENCOVAC study. We included patients on PD without additional immunosuppression that had received at least 3 SARS-CoV-2 mRNA vaccine doses. We evaluated anti-spike antibody titers after the initial vaccination, third and fourth doses, using prespecified fixed assessments (i.e., baseline, 28 days, 3, 6, and 12 months after completing the initial vaccine schedule). Breakthrough infections were also collected. Results. We included 164 patients on PD (69% males, 62 ± 13 years old). In patients who had received only two doses, the rates of positive humoral response progressively decreased from 96% at 28 days to 80% at 6 months, as did with anti-spike antibody titers. At 6 months, 102 (62%) patients had received the third vaccine dose. Patients with the third dose had higher rates of positive humoral response (p = 0.01) and higher anti-spike antibody titers (p < 0.001) at 6 months than those with only 2 doses. At 12 months, the whole cohort had received 3 vaccine doses, and 44 (27%) patients had an additional fourth dose. The fourth dose was not associated to higher rates of positive humoral response (100 vs. 97%, p = 0.466) or to statistically significant differences in anti-spike antibody titers as compared to three doses (p = 0.371) at 12 months. Prior antibody titers were the only predictor for subsequent higher anti-spike antibody titer (B 0.53 [95%CI 0.27–0.78], p < 0.001). The 2 (1.2%) patients that developed COVID-19 during follow-up had mild disease. Conclusions. PD presents an acceptable humoral response with three doses of SARS-CoV-2 vaccines that improve the progressive loss of anti-spike antibody titers following two vaccine doses. The present project has been supported by Diaverum, Vifor Pharma, Vircell, Fundación Renal Iñigo Álvarez de Toledo, and ISCIII FEDER funds RICORS2040 (RD21/0005).

Autosomal inborn errors of type I IFN immunity and autoantibodies against these cytokines underlie at least 10% of critical COVID-19 pneumonia cases. We report very rare, biochemically deleterious X-linked TLR7 variants in 16 unrelated male individuals aged 7 to 71 years (mean: 36.7 years) from a cohort of 1,202 male patients aged 0.5 to 99 years (mean: 52.9 years) with unexplained critical COVID-19 pneumonia. None of the 331 asymptomatically or mildly infected male individuals aged 1.3 to 102 years (mean: 38.7 years) tested carry such TLR7 variants (p = 3.5 × 10-5). The phenotypes of five hemizygous relatives of index cases infected with SARS-CoV-2 include asymptomatic or mild infection (n=2, 5 and 38 years), or moderate (n=1, 5 years), severe (n=1, 27 years), or critical (n=1, 29 years) pneumonia. Two boys (aged 7 and 12 years) from a cohort of 262 male patients with severe COVID-19 pneumonia (mean: 51.0 years) are hemizygous for a deleterious TLR7 variant. The cumulative allele frequency for deleterious TLR7 variants in the male general population is < 6.5x10-4 We also show that blood B cell lines and myeloid cell subsets from the patients do not respond to TLR7 stimulation, a phenotype rescued by wild-type TLR7 The patients' blood plasmacytoid dendritic cells (pDCs) produce low levels of type I IFNs in response to SARS-CoV-2. Overall, X-linked recessive TLR7 deficiency is a highly penetrant genetic etiology of critical COVID-19 pneumonia, in about 1.8% of male patients below the age of 60 years. Human TLR7 and pDCs are essential for protective type I IFN immunity against SARS-CoV-2 in the respiratory tract. Funding: The Laboratory of Human Genetics of Infectious Diseases is supported by the Howard Hughes Medical Institute, Rockefeller University; the St. Giles Foundation; the NIH (R01AI088364), the National Center for Advancing Translational Sciences (NCATS); NIH Clinical and Translational Science Award (CTSA) program (UL1TR001866); a Fast Grant from Emergent Ventures; Mercatus Center at the George Mason University; the Yale Center for Mendelian Genomics and the GSP Coordinating Center funded by the National Human Genome Research Institute (NHGRI) (UM1HG006504 and U24HG008956); the Meyer Foundation; the JPB Foundation; the French National Research Agency (ANR) under the “Investments for the Future” program (ANR-10-IAHU-01) and the Integrative Biology of Emerging Infectious Diseases Laboratory of Excellence (ANR-10-LABX-62-IBEID); the French Foundation for Medical Research (FRM) (EQU201903007798); the FRM and ANR GENCOVID project, the ANRS-COV05, ANR GENVIR (ANR-20-CE93-003), and ANR AABIFNCOV (ANR-20-CO11-0001) projects; the European Union’s Horizon 2020 Research and Innovation Program under grant agreement no. 824110 (EASI-genomics). The French COVID Cohort study group was sponsored by INSERM and supported by the REACTing consortium and by a grant from the French Ministry of Health (PHRC 20-0424). The Cov-Contact Cohort was supported by the REACTing consortium, the French Ministry of Health, and the European Commission (RECOVER WP 6). The Neurometabolic Diseases Laboratory received funding from the European Union’s Horizon 2020 Research and Innovation Program (EasiGenomics grant no. 824110 COVID-19/PID12342). A.P., R.P.d.D., C.R.-G., and C.F. were funded by Instituto de Salud Carlos III (COV20_01333 and COV20_01334), the Spanish Ministry of Science and Innovation (RTC-2017-6471-1; AEI/FEDER, UE), Fundación DISA (OA18/017), and Cabildo Insular de Tenerife (CGIEU0000219140 and “Apuestas científicas del ITER para colaborar en la lucha contra la COVID-19”). The laboratories of G.N. and A.N. were supported by a grant awarded to Regione Lazio (PROGETTI DI GRUPPI DI RICERCA 2020) no. A0375-2020-36663, GecoBiomark. A. Amara’s laboratory was supported by ANR under the “Investments for the Future” program (ANR-10-IAHU-01), the Integrative Biology of Emerging Infectious Diseases Laboratory of Excellence (ANR-10-LABX-62-IBEID), the FRM (EQU202003010193), ANR (ANR-20-COVI-000 project IDISCOVR and ANR-20-CO11-0004 project FISHBP), and the University of Paris (Plan de Soutien Covid-19: RACPL20FIR01-COVID-SOUL). This work was supported, in part, by the Division of Intramural Research, NIAID, NIH (grants 1ZIAAI001265 to H.C.S. and ZIA AI001270 to L.D.N.). The G.C. laboratory was supported by the Italian Ministry of Health (grant COVID-2020-12371617) and the intramural COVID Host Genetics program. The J.L.F. laboratory was supported, in part, by the Coopération Scientifique France-Colciencias (ECOS-Nord/COLCIENCIAS/MEN/ICETEX; 806-2018) and Colciencias contract 713-2016 (no. 111574455633). The V.S. laboratory was supported by ANR DENDRISEPSIS (ANR-17-CE15-0003) and ANR APCOD (ANR-17-CE15-0003-01), a Fast Grant from the Mercatus Center, FRM, University of Paris PLAN D’URGENCE COVID19. The N.M. laboratory was supported by Sidra Medicine (SDR400048) and the Qatar National Research Fund (grant No. NPRP9-251-3-045)

This article belongs to the Special Issue Antimicrobial Peptides and Immunology., Antimicrobial peptide (AMP) genes, triggered by Toll and IMD pathways, are essential components of the innate immune system in the German cockroach Blattella germanica. Besides their role in killing pathogenic bacteria, AMPs could be involved in controlling its symbiotic systems (endosymbiont and microbiota). We found that the IMD pathway was active in the adult female transcriptomes of six tissues (salivary glands, foregut, midgut, hindgut, Malpighian tubules and fat body) and hemolymph. Total expression of AMP genes was high in hemolymph and salivary glands and much lower in the other sample types. The expression of specific AMP genes was very heterogeneous among sample types. Two genes, defensin_g10 and drosomycin_g5, displayed relevant expression in the seven sample types, although higher in hemolymph. Other genes only displayed high expression in one tissue. Almost no expression of attacin-like and blattellicin genes was observed in any sample type, although some of them were among the genes with the highest expression in adult female whole bodies. The expression of AMP genes in salivary glands could help control pathogens ingested with food and even determine gut microbiota composition. The low expression levels in midgut and hindgut are probably related to the presence of beneficial microbiota. Furthermore, a reduction in the expression of AMP genes in fat body could be the way to prevent damage to the population of the endosymbiont Blattabacterium cuenoti within bacteriocytes., This work was funded by the European Regional Development Fund (ERDF) and the Ministerio de Ciencia, Innovación y Universidades (Spain) (PGC2018-099344-B-I00) and Generalitat Valenciana (Prometeo/2018/A/133 and CIPROM/2021/042).

Background: It is suggested that neuroinflammation, in which activated microglial cells play a relevant role, contributes to the development of Parkinson’s disease (PD). Consequently, the modulation of microglial activation is a potential therapeutic target to be taken into account to act against the dopaminergic neurodegeneration occurring in this neurological disorder. Several soluble and membrane-associated inhibitory mechanisms contribute to maintaining microglial cells in a quiescent/surveillant phenotype in physiological conditions. However, the presence of activated microglial cells in the brain in PD patients suggests that these mechanisms have been somehow overloaded. We focused our interest on one of the membrane-associated mechanisms, the CD200-CD200R1 ligand-receptor pair. Methods: The acute MPTP experimental mouse model of PD was used to study the temporal pattern of mRNA expression of CD200 and CD200R1 in the context of MPTP-induced dopaminergic neurodegeneration and neuroinflammation. Dopaminergic damage was assessed by tyrosine hydroxylase (TH) immunoreactivity, and neuroinflammation was evaluated by the mRNA expression of inflammatory markers and IBA1 and GFAP immunohistochemistry. The effect of the modulation of the CD200-CD200R1 system on MPTP-induced damage was determined by using a CD200R1 agonist or CD200 KO mice. Results: MPTP administration resulted in a progressive decrease in TH-positive fibres in the striatum and TH-positive neurons in the substantia nigra pars compacta, which were accompanied by transient astrogliosis, microgliosis and expression of pro- and anti-inflammatory markers. CD200 mRNA levels rapidly decreased in the ventral midbrain after MPTP treatment, while a transient decrease of CD200R1 mRNA expression was repeatedly observed in this brain area at earlier and later phases. By contrast, a transient increase in CD200R1 expression was observed in striatum. The administration of a CD200R1 agonist resulted in the inhibition of MPTP-induced dopaminergic neurodegeneration, while microglial cells showed signs of earlier activation in CD200-deficient mice. Conclusions: Collectively, these findings provide evidence for a correlation between CD200-CD200R1 alterations, glial activation and neuronal loss. CD200R1 stimulation reduces MPTP-induced loss of dopaminergic neurons, and CD200 deficiency results in earlier microglial activation, suggesting that the potentiation of CD200R1 signalling is a possible approach to controlling neuroinflammation and neuronal death in PD., This study was supported by grant PI15/00033 from the Instituto de Salud Carlos III (Spain) with joint financing by FEDER funds from the European Union. NRL was recipient of a predoctoral contract from the Spanish Ministerio de Educación. Cultura y Deporte FPU (FPU13/05491).

Altres ajuts: the E-Rare (ERA-Net for research programs on rare diseases) and EuroRETT (a European network on Rett syndrome, funded by the European Commission under its 6th Framework Program since 2006) When considering the clinical applications of autologous cell replacement therapy of human induced pluripotent stem cells (iPSC)-derived cells, there is a clear need to better understand what the immune response will be before we embark on extensive clinical trials to treat or model human disease. We performed a detailed assessment comparing human fibroblast cell lines (termed F1) reprogrammed into human iPSC and subsequently differentiated back to fibroblast cells (termed F2) or other human iPSC-derived cells including neural stem cells (NSC) made from either retroviral, episomal, or synthetic mRNA cell reprogramming methods. Global proteomic analysis reveals the main differences in signal transduction and immune cell protein expression between F1 and F2 cells, implicating wild type (WT) toll like receptor protein 3 (TLR3). Furthermore, global methylome analysis identified an isoform of the human TLR3 gene that is not epigenetically reset correctly upon differentiation to F2 cells resulting in a hypomethylated transcription start site in the TLR3 isoform promoter and overexpression in most human iPSC-derived cells not seen in normal human tissue. The human TLR3 isoform in human iPSC-NSC functions to suppress NF-KB p65 signaling pathway in response to virus (Poly IC), suggesting suppressed immunity of iPSC-derived cells to viral infection. The sustained WT TLR3 and TLR3 isoform overexpression is central to understanding the altered immunogenicity of human iPSC-derived cells calling for screening of human iPSC-derived cells for TLR3 expression levels before applications. Stem Cells 2019;37:476-488.

Antiviral responses are activated rapidly after viral infection in order to control and prevent dissemination of the virus. Different pathways are activated in the immune system, including innate and adaptive responses. On the other hand, viruses have evolved specifi c strategies to evade these responses. Due to the high viral evolutionary rates, escape variants can emerge and spread fast in the population. The co-evolution between viruses and their host is a constant arms race, and is of special interest to understand the viral escape mechanisms that may guide the future development of antiviral treatments and vaccines.

Performance of the QuantiFERON-TB Gold Plus (QFT-Plus) assay could be affected by conditions of immune dysregulation. Little is known about the reliability of QTF-Plus in COVID-19 patients. Our aim was to determine the prevalence and the factors related to an indeterminate QFT-Plus test in COVID-19 hospitalized patients, and to analyze its relationship with in-hospital mortality. A retrospective analysis of all hospitalized COVID-19 patients on whom a QTF-Plus assay was performed in a tertiary care public hospital during the first epidemic wave in Spain (March–April 2020). Out of a total of 96 patients included, 34 (35.4%) had an indeterminate result, in all cases due to a lack of response in the mitogen control. Factors related to COVID-19 severity, such as higher lactate dehydrogenase (LDH) (odds ratio [OR] 1.005 [95% confidence interval [CI] 1.002–1.008]) and previous administration of corticosteroids (OR 4.477 [95% CI 1.397–14.345]), were independent predictors for indeterminate QFT-Plus assay. Furthermore, indeterminate results were more frequent among COVID-19 patients who died during hospitalization (29.1% vs. 64.7%, p = 0.005). We conclude that QFT-Plus assay yielded an unexpected, high prevalence of indeterminate results in severe COVID-19 patients. Factors related to worse COVID-19 outcome, such as LDH, as well as corticosteroid use before the QFT-Plus assay, seem to be predictors for an indeterminate result. The role of an indeterminate QFT-Plus result in predicting COVID-19 severity and mortality should be evaluated.

Background & aims HCV CD4+ and CD8+ specific T cells responses are functionally impaired during chronic hepatitis C infection. DAAs therapies eradicate HCV infection in more than 95% of treated patients. However, the impact of HCV elimination on immune responses remain controversial. Here, we aimed to investigate whether HCV cure by DAAs could reverse the impaired immune response to HCV. Methods We analyzed 27 chronic HCV infected patients undergoing DAA treatment in tertiary care hospital, and we determined the phenotypical and functional changes in both HCV CD8+ and CD4+ specific T-cells before and after viral clearance. PD-1, TIM-3 and LAG-3 cell-surface expression was assessed by flow cytometry to determine CD4+ T cell exhaustion. Functional responses to HCV were analyzed by IFN-γ ELISPOT, intracellular cytokine staining (IL-2 and IFN-γ) and CFSE-based proliferation assays. Results We observed a significant decrease in the expression of PD-1 in CD4+ T-cells after 12 weeks of viral clearance in non-cirrhotic patients (p = 0.033) and in treatment-naive patients (p = 0.010), indicating a partial CD4 phenotype restoration. IFN-γ and IL-2 cytokines production by HCV-specific CD4+ and CD8+ T cells remained impaired upon HCV eradication. Finally, a significant increase of the proliferation capacity of both HCV CD4+ and CD8+ specific T-cells was observed after HCV elimination by DAAs therapies. Conclusions Our results show that in chronically infected patients HCV elimination by DAA treatment lead to partial reversion of CD4+ T cell exhaustion. Moreover, proliferative capacity of HCV-specific CD4+ and CD8+ T cells is recovered after DAA’s therapies., Instituto de Salud Carlos III, cofinanced by the European Regional Development Fund (ERDF): grant numbers PI16/00337, PI18/00210 and PI19/00301. C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CP14/00121 and CPII19/00001) cofinanced by the European Regional Development Fund (ERDF)

Neurodegenerative disorders such as Parkinson's (PD) and Huntington's disease (HD) are characterized by a selective detrimental impact on neurons in a specific brain area. Currently, these diseases have no cures, although some promising trials of therapies that may be able to slow the loss of brain cells are underway. Cell therapy is distinguished by its potential to replace cells to compensate for those lost to the degenerative process and has shown a great potential to replace degenerated neurons in animal models and in clinical trials in PD and HD patients. Fetal-derived neural progenitor cells, embryonic stem cells or induced pluripotent stem cells are the main cell sources that have been tested in cell therapy approaches. Furthermore, new strategies are emerging, such as the use of adult stem cells, encapsulated cell lines releasing trophic factors or cell-free products, containing an enriched secretome, which have shown beneficial preclinical outcomes. One of the major challenges for these potential new treatments is to overcome the host immune response to the transplanted cells. Immune rejection can cause significant alterations in transplanted and endogenous tissue and requires immunosuppressive drugs that may produce adverse effects. T-, B-lymphocytes and microglia have been recognized as the main effectors in striatal graft rejection. This review aims to summarize the preclinical and clinical studies of cell therapies in PD and HD. In addition, the precautions and strategies to ensure the highest quality of cell grafts, the lowest risk during transplantation and the reduction of a possible immune rejection will be outlined. Altogether, the wide-ranging possibilities of advanced therapy medicinal products (ATMPs) could make therapeutic treatment of these incurable diseases possible in the near future.

APDS2 is caused by mutations in PIK3R1 gene resulting in constitutive PI3Kδ activation. PI3Kδ is predominantly expressed in leukocytes and plays critical roles in regulating immune responses. Here we first derived fibroblast primary cells from a skin biopsy of a patient carrying a heterozygous single T deletion in intron 11 of the PIK3R1 gene. We next present the derivation of an induced pluripotent stem cell (iPS) line using a non-integrative reprogramming technology. Pluripotent-related hallmarks are further shown, including: iPSCs self-renewal and expression of pluripotent and differentiation markers after in vitro differentiation towards embryonic germ layers, assessed by RT-PCR and immunofluorescence.

Background JIA-associated uveitis (JIAU) is a serious, sight-threatening disease with significant long-term complications and risk of blindness, even with improved contemporary treatments. The MIWGUC was set up in order to propose specific JIAU activity and response items and to validate their applicability for clinical outcome studies. Methods The group consists of 8 paediatric rheumatologists and 7 ophthalmologists. A consensus meeting took place on November 2015 in Barcelona (Spain) with the objective of validating the previously proposed measures. The validation process was based on the results of a prospective open, international, multi-centre, cohort study designed to validate the outcome measures proposed by the initial MIWGUC group meeting in 2012. The meeting used the same Delphi and nominal group technique as previously described in the first paper from the MIWGUC group (Arthritis Care Res 64:1365–72, 2012). Patients were included with a diagnosis of JIA, aged less than 18 years, and with active uveitis or an uveitis flare which required treatment with a disease-modifying anti-rheumatic drug. The proposed outcome measures for uveitis were collected by an ophthalmologist and for arthritis by a paediatric rheumatologist. Patient reported outcome measures were also measured. Results A total of 82 patients were enrolled into the validation cohort. Fifty four percent (n = 44) had persistent oligoarthritis followed by rheumatoid factor negative polyarthritis (n = 15, 18%). The mean uveitis disease duration was 3.3 years (SD 3.0). Bilateral eye involvement was reported in 65 (79.3%) patients. The main findings are that the most significant changes, from baseline to 6 months, are found in the AC activity measures of cells and flare. These measures correlate with the presence of pre-existing structural complications and this has implications for the reporting of trials using a single measure as a primary outcome. We also found that visual analogue scales of disease activity showed significant change when reported by the ophthalmologist, rheumatologist and families. The measures formed three relatively distinct groups. The first group of measures comprised uveitis activity, ocular damage and the ophthalmologists’ VAS. The second comprised patient reported outcomes including disruption to school attendance. The third group consisted of the rheumatologists’ VAS and the joint score. Conclusions We propose distinctive and clinically significant measures of disease activity, severity and damage for JIAU. This effort is the initial step for developing a comprehensive outcome measures for JIAU, which incorporates the perspectives of rheumatologists, ophthalmologists, patients and families.

Trabajo presentado en 3rd International Conference on Fish and Shellfish Immunology (ISFSI), celebrado en Gran Canaria (España), del 16 al 20 de junio de 2019, Endocrine disruptors in general and oestrogenic compounds in particular have been widely investigated in view of their effects on several physiological processes and, therefore, their ecotoxicologic relevance. The modulation of the immune system by oestrogens has increasingly sparked interest in the research community, that previously had been mainly centered on the reproductive effects of these hormones. In fact, since the industrialization an increasing variety of endocrine disruptors, such as oestrogenic endocrine disruptors, are retrieved in the environment. These oestrogenic endocrine disrupting chemicals (EEDCs) have also been suggested to increase the prevalence of autoimmune diseases and cancer. With regard to the high degree of similarities in the immune system of jawed vertebrates as well as the conserved immunomodulatory roles of oestrogen, environmental EEDCs possibly have the capacity to alter immune system functions of teleost fish, which may impair their capacity to fight infectious diseases and eventually may contribute, together with overfishing, to wild stock losses. Importantly, the most deleterious effects of EEDCs, both in mammals and teleosts, appear to arise when the exposure occurs during specific periods of the immune system ontogenesis, commonly referred as critical windows of exposure. However, in mammals and especially in teleost fish, these stages of the immune system development as well as the EEDC-action remain to be fully identified and characterised. The concept and the importance of developmental immunotoxicity is presented by addressing the mechanisms of oestrogenic regulation and the mode of action of EEDCs from an immunological perspective. Emphasis is given to the critical windows of development of the immune system during which EEDCs may alter the immune system development and function with long-term consequences on immunocompetence. Results from different classes of vertebrates are compiled, highlighting studies on teleost fish and their relevance for the human immune system. Additionally, new results on the effects of environmentally relevant concentration of exogenous estradiol exposure during European sea bass (Dicentrarchus labrax) development will be presented with regard to oestrogen's ability to trigger effects on immunocompetence, contributing to fill the gaps on vertebrate immunotoxicology.

In the case of Parkinson’s disease (PD), epidemiological studies have reported that pesticide exposure is a risk factor for its pathology. It has been suggested that some chemical agents, such as rotenone and paraquat, that inhibit the mitochondrial respiratory chain (in the same way as the PD mimetic toxin 1-methyl-4-phenylpyridinium, MPP+) are involved in the development of PD. However, although the neurotoxic effect of such compounds has been widely reported using in vivo and in vitro experimental approaches, their direct effect on the glial cells remains poorly characterized. In addition, the extent to which these toxins interfere with the immune response of the glial cells, is also underexplored. We used mouse primary mixed glial and microglial cultures to study the effect of MPP+ and rotenone on glial activation, in the absence and the presence of a pro-inflammatory stimulus (lipopolysaccharide plus interferon-γ, LPS+IFN-γ). We determined the mRNA expression of the effector molecules that participate in the inflammatory response (pro-inflammatory cytokines and enzymes), as well as the nitric oxide (NO) and cytokine production. We also studied the phagocytic activity of the microglial cells. In addition, we evaluated the metabolic changes associated with the observed effects, through the measurement of adenosine triphosphate (ATP) production and the expression of genes involved in the control of metabolic pathways. We observed that exposure of the glial cultures to the neurotoxins, especially rotenone, impaired the pro-inflammatory response induced by LPS/IFN-γ. MPP+ and rotenone also impaired the phagocytic activity of the microglial cells, and this effect was potentiated in the presence of LPS/IFN-γ. The deficit in ATP production that was detected, mainly in MPP+ and rotenone-treated mixed glial cultures, may be responsible for the effects observed. These results show that the response of glial cells to a pro-inflammatory challenge is altered in the presence of toxins inhibiting mitochondrial respiratory chain activity, suggesting that the glial immune response is impaired by such agents. This may have relevant consequences for brain function and the central nervous system’s (CNS’s) response to insults., NR-L was recipient of an FPU grant (FPU13/05491) from the Spanish Ministerio de Educación, Cultura y Deporte. This study was supported by grants PI14/00302 and PI15/00033 from the Instituto de Salud Carlos III (Spain) with joint financing by FEDER funds from the European Union. We acknowledge support for the publication fee, by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Altres ajuts: This work was supported by grants from: (i) the Isolana Foundation, (ii) the Maria Francisca de Roviralta Foundation expedient AT/MA 1-19/07/2017, AT/MA 70-27/04/2016, and AT/MA 3-22/10/2014 and (vi) Fundació Recerca i Docència Mútua Terrassa. Altres ajuts: PERIS/SLT006-17-144 In recent years, pediatric research on tuberculosis (TB) has focused on addressing new biomarkers with the potential to be used as immunological non-sputum-based methods for the diagnosis of TB in children. The aim of this study was to characterize a set of cytokines and a series of individual factors (ferritin, 25-hydroxyvitamin D [25(OH)D], parasite infections, and nutritional status) to assess different patterns for discriminating between active TB and latent TB infection (LTBI) in children. The levels of 13 cytokines in QuantiFERON-TB Gold In-Tube (QFT-GIT) supernatants were analyzed in 166 children: 74 with active TB, 37 with LTBI, and 55 uninfected controls. All cytokines were quantified using Luminex or ELISA. Ferritin and 25(OH)D were also evaluated using CLIA, and Toxocara canis Ig-G antibodies were detected with a commercial ELISA kit. The combination of IP-10, IFN-γ, ferritin, and 25(OH)D achieved the best diagnostic performance to discriminate between active TB and LTBI cases in children in relation to the area under receiver operating characteristic (ROC) curve 0.955 (confidence interval 95%: 0.91-1.00), achieving optimal sensitivity and specificity for the development of a new test (93.2 and 90.0%, respectively). Children with TB showed higher ferritin levels and an inverse correlation between 25(OH)D and IFN-γ levels. The model proposed includes a combination of biomarkers for discriminating between active TB and LTBI in children to improve the accuracy of TB diagnosis in children. This combination of biomarkers might have potential for identifying the onset of primary TB in children.

Primary hypercholesterolemia (PH) is associated with a low grade systemic inflammation that is likely the main driver of premature atherosclerosis. Accordingly, we characterized the immune cell behaviour in PH and its potential consequences. Whole blood from 22 PH patients and 21 age-matched controls was analysed by flow cytometry to determine the percentage of leukocyte immunophenotypes, activation, and platelet-leukocyte aggregates. Plasma markers were determined by Enzyme-Linked ImmunoSorbent Assay (ELISA). The adhesion of platelet-leukocyte aggregates to tumor necrosis factor-&alpha, (TNF&alpha, )-stimulated arterial endothelium was investigated using the dynamic model of the parallel-plate flow chamber. PH patients presented greater percentage of Mon 3 monocytes, Th2 and Th17 lymphocytes, activated platelets, and leukocytes than controls. The higher percentages of circulating platelet-neutrophil, monocyte and lymphocyte aggregates in patients caused increased platelet-leukocyte adhesion to dysfunctional arterial endothelium. Circulating CXCL8, CCL2, CX3CL1, and IL-6 levels positively correlated with key lipid features of PH, whereas negative correlations were found for IL-4 and IL-10. We provide the first evidence that increased platelet and leukocyte activation leads to elevated platelet-leukocyte aggregates in PH and augmented arterial leukocyte adhesiveness, a key event in atherogenesis. Accordingly, modulation of immune system behavior might be a powerful target in the control of further cardiovascular disease in PH.

Our goal in this doctoral thesis was to study the immunomodulatory effects of CD5 and CD6, two proteins expressed on the lymphocytes membrane. These two proteins belong to the Scavenger Receptors Cystein- Rich superfamily, characterized by the presence of one or more cysteine rich domains. The interaction between CD6 and ALCAM, its principal ligand, is well established but recently more ligands of CD6 have been described. On the other hand, to date there is no consensus about the CD5 ligand/s, although several candidates have been proposed. Previous work carried out with CD5 and CD6-deficient mice revealed the role as negative modulators of the T/B cell receptor signaling. It was observed how its blockade/absence led to a hyper-activation post T cell activation by increment of cell proliferation and calcium mobilization. We decided to generate a transgenic mouse that would expresses high levels of the extracellular region of human CD5 constitutively, in order to block the interactions mediated by membrane-bound CD5 with its ligand/s. This “functional knockout" would resemble what would happen in a clinical application, by injecting the soluble recombinant protein to humans. These mice presented indeed soluble human CD5 in serum, which resulted in immunophenotypical changes that were reproduced in wild-type mice after repeated administration of exogenous rshCD5 protein. The transgenic mice did not reveal any gross alterations in major lymphocyte subpopulations. However, a decrease in T and B cell subsets with regulatory function, as well as an increment in NKT cells were observed. They also presented more severe disease outcome to two different induced experimental autoimmune disease models and a better anti- tumor response against murine melanoma cells (B16-F0) and a genetically modified line of thymoma tumor cells (EG7-OVA). We have observed that this effect is due to the increase in number and activity of the effector cells of the immune system as well as the lowering of the regulatory T cells in the tumor draining lymph nodes. On the other hand, wild-type mice challenged with melanoma showed an improved anti-tumor response as well as changes in the tumor draining lymph node after therapeutic administration of the protein. In addition, in this model, a decrease in IL-6 expression levels was observed, and the loss of efficacy obtained with CD5 administration by depleting NK cells. We also generated another murine model expressing elevated levels of human soluble CD6 in a constitutive way, with the objective of blocking the heterophillic and homophillic interactions of CD6 and its ligands. These mice had a decrease in the spleen and lymph nodes total cell number. Their B cells have reduced proliferative capacity and the regulatory T cell less suppressive activity aside to a reduction of their number. We observed an increment of the anti-tumor response to different tumor cell lines, but their autoimmune response was not exacerbated, as it was expected. These results were reproduced when recombinant soluble human CD6 was injected repeatedly into wild-type mice. The results obtained demonstrate that counteracting the function of membrane-bound CD5/CD6 with sCD5/CD6 may enhance current immunotherapies for cancer, as well as be used in other possible applications such as in acute or chronic infections (CD5 recognizes fungal and viral components, and CD6 bacterial components) and vaccines., El objetivo de esta tesis doctoral ha sido el estudio de los efectos inmunomoduladores de CD5 y CD6, dos proteínas expresadas en la membrana de los linfocitos. Estas dos proteínas pertenecen a la superfamilia de receptores "Scavenger Receptor Cystein-Rich", caracterizada por la presencia de uno o varios dominios ricos en cisteínas. En cuanto a CD6, están descritos varios ligandos, sin embargo, a día de hoy no existe un consenso acerca del ligando/s de CD5.Trabajos previos llevados a cabo con ratones deficientes para CD5/CD6, pusieron de manifiesto sus papeles como moduladores negativos de la señal del receptor de células T/B. Decidimos generar un ratón que expresara niveles elevados de la región extracelular de CD5 humano constitutivamente, con el objetivo de bloquear las interacciones mediadas por el CD5 de membrana con su ligando/s. Observamos que estos ratones presentaron una respuesta autoinmune exacerbada pero mejor respuesta anti-tumoral frente a células murinas de melanoma y linfoma. Este efecto se debió al aumento de número y actividad de las células efectoras del sistema inmune, así como a la bajada de las células T reguladoras en los ganglios drenantes del tumor. Por otro lado, ratones silvestres a los que se les indujo melanoma mostraron una respuesta anti-tumoral mejorada, así como cambios en el ganglio drenante, tras la administración de la proteína de forma terapéutica. Además, la eficacia obtenida con la administración de CD5 se perdió al deplecionar las células NK. Además, se generó otro modelo murino que expresara niveles elevados de CD6 soluble humano de manera constitutiva, con el mismo objetivo. Estos ratones presentaron una bajada en cuanto al número de células en bazo y nódulos linfáticos debido a la capacidad proliferativa reducida de los linfocitos B. Además de una disminución en número, las células T reguladoras presentaron una menor actividad supresora. Así, observamos un aumento de la respuesta anti-tumoral frente a diferentes líneas tumorales, pero su respuesta autoinmune no se encontraba exacerbada. Estos resultados se reprodujeron cuando se inyectó CD6 soluble a ratones silvestres. Los resultados obtenidos denotan la importancia de CD5 y CD6 en la respuesta inmune, y su posible aplicación en combinación con las actuales terapias inmunomoduladoras.

[eng] Induced pluripotent stem cells (iPSC) can be made from adult somatic cells by reprogramming them with Oct4, Sox2, Klf4 and c-Myc. IPSC have given rise to a new technology to study and treat human disease (Takahashi et al., 2007). However, before iPSC clinical application, we need to step back and address two main challenges: (i) Genetic stability of iPSC. (ii) Immune response of iPSC-derived cells. To address these key issues, the overall mission of this PhD thesis is to advance iPSC technology by addressing two objectives. First, is to replace c-Myc with Cyclin D1 in the reprogramming cocktail (Oct4, Sox2, Klf4 and c-Myc or Cyclin D1) and second, to study the immune response of iPSC-derived cells. The quality of the starting iPSC determines the quality of the differentiated cells to be transplanted for clinical applications. In terms of genetic stability, aberrant cell reprogramming leads to genetic and epigenetic modifications that are the most significant barriers to clinical applications of patient iPSC derivatives (Gore et al., 2011). Such aberrations can result from the cellular stress that accompanies reprogramming or from the reprogramming factors themselves (Lee et al., 2012a). IPSC made with c-Myc are neoplastic in mouse models and have a higher tumorigenic potential than embryonic stem cells, prompting a search for new pluripotency factors that can replace the oncogenic factors Klf4 and c-Myc (Huangfu et al., 2008; Miura et al., 2009; Okita et al., 2007). We chose Cyclin D1 to replace c-Myc because of previous observation it can be used to reprogram cells to iPSC (Edel et al., 2010) and because of its DNA repair function (Chalermrujinanant et al., 2016). In this thesis we adopt a synthetic mRNA method to demonstrate that Cyclin D1 and c-Myc made iPSC have equal pluripotency using standard methods of characterisation. Moreover, no significant changes in copy number variation were found between starting skin cells and iPSC highlighting it is the method of choice for generating high quality iPSC. Further in- depth analysis revealed that Cyclin D1 made iPSC have reduced genetic instability assessed by: (i) reduced DNA double strand breaks (DSB), (ii) higher nuclear amount of the homologous recombination key protein Rad51, (iii) reduced multitelomeric signals (MTS) and (iv) reduced teratoma growth kinetics in vivo, compared to c-Myc made iPSC. Moreover, we demonstrate that Cyclin D1 iPSC derived neural stem cells engraft successfully, survive long term and differentiate into mature neuron cell types with high efficiency, with no evidence of pathology in a spinal cord injury rat model. As we move towards the clinic with iPSC-derived cells for cell transplantation, the immunogenic response is thought to be one of the main advantages of iPSC technology for clinical application, because of its perceived lack of immune rejection of autologous cell therapy. We hypothesize that iPSC derived cells are unlikely to provoke an immune response. Here we have performed an analysis of the innate and adaptive immune response of human skin cells (termed F1) reprogramed to iPSC and then compared to iPSC-derived cells (termed F2) using proteomic and methylome arrays. We found little differences between MHCI expression and function; however, we discovered a short isoform of the Toll-like receptor 3 (TLR3), essential for viral dsRNA innate immune recognition, which is predominantly upregulated in all iPSC derived cells analysed and not seen in normal endogenous cells. High levels of the TLR3 isoform is associated with unresponsiveness to viral stimulation measured by lack of IL6 secretion in iPSC derived neural stem cells. We propose a new model that TLR3 short isoform competes with the full length wild type isoform destabilizing the essentially required TLR3 dimerization process. These differences could result in supressed inflammatory effects for transplanted human iPSC-derived cells in response to viral or bacterial insult. Further work to determine the in vivo effects is warranted and calls for screening of iPSC lines for TLR3 isoform expression levels before clinical use. In conclusion, this thesis has advanced iPSC technology by defining a new method that is a significant advance with novel insights that has immediate impact on current methods to generate iPSC for clinical application and more accurate disease modelling., [cat] Les cèl·lules mare pluripotents induïdes (iPSC) es poden derivar de cèl·lules somàtiques adultes mitjançant la reprogramació amb Oct4, Sox2, Klf4 i c-Myc. Les iPSC han donat lloc a una nova tecnologia per estudiar i tractar malalties humanes (Takahashi et al., 2007). No obstant, abans de la aplicació clínica de les iPSC, dos problemes principals han de ser adreçats: (i) Estabilitat genètica de les iPSC. (ii) Resposta immune de les cèl·lules derivades de iPSC. Per adreçar aquests dos qüestions cabdals, la missió principal d’aquest doctorat és avançar la tecnologia de les iPSC adreçant dos objectius. El primer, és la substitució de c-Myc per Ciclina D1 al còctel de reprogramació (Oct4, Sox2, Klf4 and c-Myc o Ciclina D1) i segon, estudiar la resposta immune de les cèl·lules derivades de iPSC. Hem escollit Ciclina D1 per substituir c-Myc atès a observacions prèvies que pot ser emprat per reprogramar (Edel et al., 2010) i donada la seva funció en reparació de l’ADN (Chalermrujinanant et al., 2016). Les iPSC reprogramades amb Ciclina D1 presenten una pluripotència similar a les reprogramades amb c-Myc, l’anàlisi en profunditat mostra però, que les iPSC reprogramades amb Cyclin D1 tenen una reduïda inestabilitat genètica adreçada per: (i) reducció en ruptures de doble cadena de DNA, (ii) major quantitat nuclear de la proteïna clau en la recombinació homòloga Rad51, (iii) reducció en senyals multitelomèriques (MTS) i (iv) reducció en la cinètica de creixement de teratomas in vivo, en comparació amb iPSC reprogramades amb c-Myc. A més a més, demostrem que les cèl·lules mare neuronals derivades d’aquestes iPSC son capaces de implantar-se exitosament, sobreviure a llarg termini i diferenciar a neurones madures sense evidències de patologia en un model de dany medul·lar. També hem realitzat un anàlisi del sistema immune innat i adaptatiu de cèl·lules humanes de la pell (nomenades F1) reprogramades a iPSC i comparades amb cèl·lules derivades de iPSC (nomenades F2). Hem descobert una isoforma curta del Toll-Like Receptor 3 (TLR3), essencial en el reconeixement de RNA de doble cadena d’origen víric, que està predominantment sobreexpresada en totes les cèl·lules derivades de iPSC analitzades i no trobat en cèl·lules endògenes. Nosaltres proposem un nou model per el qual la isoforma curta del TLR3 competeix amb la isoforma llarga wild type desestabilitzant el procés essencial de dimerització del TLR3.

[eng] RATIONALE: Chronic obstructive pulmonary disease (COPD) is characterized by chronic airflow limitation caused by a combination of airways disease (bronchiolitis) and parenchymal destruction (emphysema), whose relative proportion varies from patient to patient. An abnormal immune response to tobacco smoke is one of the main factors, both in the lungs and circulating blood. Both the relation between the pulmonary and systemic inflammation and the regenerative capacity of the lung are also unclear. OBJECTIVES: Divided into 3 specific objectives: 1. To explore and contrast the molecular pathogenesis of emphysema and bronchiolitis in COPD. 2. To characterize and contrast the cellular pulmonary and systemic immune response in COPD patients and healthy controls smokers and non-smokers. 3. To identify, characterize and compare the immunomodulatory capacity of a putative resident stem cell population in the lung. METHODS: For the first objective differential expression and gene co-expression in bronchiolitis and emphysema were analyzed by lung transcriptomics. For the second, flow cytometry from lung tissue and blood was performed to evaluate the cellular immune response. For the third objective, a new cell culture methodology was developed. RESULTS: Specific results for each objective 1. Emphysema signature is different from bronchiolitis with an up-regulated expression of ontologies related with B-cell homing and activation. 2. The lung of mild COPD patients has increase macrophages and decrease T lymphocytes associated to both COPD and smoking status, that is not co-related with systemic immune response. 3. A putative lung resident stem cell population was identify with transcriptional signature of mesenchymal origin and immunomodulatory properties. CONCLUSIONS: The lung immune response is heterogeneous and associated with both the lung regenerative capacity and the clinical heterogeneity/complexity of COPD., [spa] INTRODUCCIÓN: La enfermedad pulmonar obstructiva crónica (EPOC) se caracteriza por una limitación crónica del flujo aéreo causada por una combinación de enfermedad de las vías aéreas (bronquiolitis) y destrucción del parénquima (enfisema). La contribución relativa de cada patología varia entre pacientes. Un respuesta anormal y exagerada frente al humo del tabaco es uno de los principales factores de riesgo. La relación entre la respuesta inmune pulmonar y sistémica y la capacidad regenerativa del pulmón no se encuentran determinadas. OBJETIVOS: Hay 3 objetivos específicos: 1. Comparar las diferencias moleculares entre la patogénesis del enfisema y la bronquiolitis en la EPOC. 2. Caracterizar y comparar la respuesta inmune celular a nivel pulmonar y en sangre periférica en pacientes con EPOC y controles sanos fumadores y no fumadores. 3. Identificar, caracterizar y comparar la actividad inmunomoduladora de una población de células madre residentes en pulmón. MÉTODOS: Para el primer objetivo la expresión diferencial y la co-expresión de los genes se realizó mediante análisis del transcriptoma pulmonar. Para el segundo, se utilizó citometría de flujo para la determinación celular de las poblaciones de la respuesta inmune en pulmón y sangre. Para el tercer objetivo, se ha desarrollado una nueva metodología de cultivo celular. RESULTADOS: Resultados específicos por objetivos 1. La caracterización molecular es diferentes entre la bronquiolitis y el enfisema con un incremento en la expresión de genes relacionados con la activación y movilidad de los linfocitos B. 2. El pulmón de los pacientes con EPOC leve contiene un incremento de macrófagos y una reducción de linfocitos T asociada tanto a la EPOC como al consumo de tabaco. 3. Se ha identificado una población de células madre residentes en pulmón con una transcriptómica de origen mesenquimal y propiedades inmunomoduladoras. CONCLUSIONES: la respuesta inmune pulmonar es heterogénea y está asociada tanto con la capacidad regenerativa como con la complexidad de la EPOC.

[Abstract] Xenogeneic chondrocytes and allogeneic mesenchymal stem cells (MSC) are considered a potential source of cells for articular cartilage repair. We here assessed the immune response triggered by xenogeneic chondrocytes when injected intraarticularly, as well as the immunoregulatory effect of allogeneic bone marrow-derived MSC after systemic administration. To this end, a discordant xenotransplantation model was established by injecting three million porcine articular chondrocytes (PAC) into the femorotibial joint of Lewis rats and monitoring the immune response. First, the fate of MSC injected using various routes was monitored in an in vivo imaging system. The biodistribution revealed a dependency on the injection route with MSC injected intravenously (i.v.) succumbing early after 24 h and MSC injected intraperitoneally (i.p.) lasting locally for at least 5 days. Importantly, no migration of MSC to the joint was detected in rats previously injected with PAC. MSC were then administered either i.v. 1 week before PAC injection or i.p. 3 weeks after to assess their immunomodulatory function on humoral and adaptive immune parameters. Anti-PAC IgM and IgG responses were detected in all PAC-injected rats with a peak at week 2 postinjection and reactivity remaining above baseline levels by week 18. IgG2a and IgG2b were the predominant and long-lasting IgG subtypes. By contrast, no anti-MSC antibody response was detected in the cohort injected with MSC only, but infusion of MSC before PAC injection temporarily augmented the anti-PAC antibody response. Consistent with a cellular immune response to PAC in PAC-injected rats, cytokine/chemokine profiling in serum by antibody array revealed a distinct pattern relative to controls characterized by elevation of multiple markers at week 2, as well as increases in proliferation in draining lymph nodes. Notably, systemic administration of allogeneic MSC under the described conditions did not diminish the immune response. IL-2 measurements in cocultures of rat peripheral blood lymphocytes with PAC indicated that PAC injection induced some T-cell hyporesponsiveness that was not enhanced in the cohorts additionally receiving MSC. Thus, PAC injected intraarticularly in Lewis rats induced a cellular and humoral immune response that was not counteracted by the systemic administration of allogeneic MSC under the described conditions. Instituto de Salud Carlos III; PI11/02799 Instituto de Salud Carlos III; PI11/02799

Protein alpha-helical coiled coil structures are known to induce antibodies able to block critical functions in different pathogens. In a previous study, a total of 50 proteins of Plasmodium vivax erythrocytic asexual stages containing alpha-helical coiled coil structural motifs were identified in silico, and the corresponding peptides were chemically synthesized. A total of 43 peptides were recognized by naturally acquired antibodies in plasma samples from both Papua New Guinea (PNG) and Colombian adult donors. In this study, the association between IgG antibodies to these peptides and clinical immunity was further explored by measuring total IgG antibody levels to 24 peptides in baseline samples from a longitudinal study of children aged 1-3 years (n = 164) followed for 16 months. Samples were reactive to all peptides tested. Eight peptides were recognized by >50% of individuals, whereas only one peptide had < 20% reactivity. Children infected at baseline were seropositive to 23/24 peptides. No significant association was observed between antibody titers and age or molecular force of infection, suggesting that antibody levels had already reached an equilibrium. There was a strong association between antibody levels to all peptides and protection against P. vivax clinical episodes during the 16 months follow-up. These results suggest that the selected coiled coil antigens might be good markers of both exposure and acquired immunity to P. vivax malaria, and further preclinical investigation should be performed to determine their potential as P. vivax vaccine antigens.

[eng] Our goal in this doctoral thesis was to study the immunomodulatory effects of CD5 and CD6, two proteins expressed on the lymphocytes membrane. These two proteins belong to the Scavenger Receptors Cystein- Rich superfamily, characterized by the presence of one or more cysteine rich domains. The interaction between CD6 and ALCAM, its principal ligand, is well established but recently more ligands of CD6 have been described. On the other hand, to date there is no consensus about the CD5 ligand/s, although several candidates have been proposed. Previous work carried out with CD5 and CD6-deficient mice revealed the role as negative modulators of the T/B cell receptor signaling. It was observed how its blockade/absence led to a hyper-activation post T cell activation by increment of cell proliferation and calcium mobilization. We decided to generate a transgenic mouse that would expresses high levels of the extracellular region of human CD5 constitutively, in order to block the interactions mediated by membrane-bound CD5 with its ligand/s. This “functional knockout" would resemble what would happen in a clinical application, by injecting the soluble recombinant protein to humans. These mice presented indeed soluble human CD5 in serum, which resulted in immunophenotypical changes that were reproduced in wild-type mice after repeated administration of exogenous rshCD5 protein. The transgenic mice did not reveal any gross alterations in major lymphocyte subpopulations. However, a decrease in T and B cell subsets with regulatory function, as well as an increment in NKT cells were observed. They also presented more severe disease outcome to two different induced experimental autoimmune disease models and a better anti- tumor response against murine melanoma cells (B16-F0) and a genetically modified line of thymoma tumor cells (EG7-OVA). We have observed that this effect is due to the increase in number and activity of the effector cells of the immune system as well as the lowering of the regulatory T cells in the tumor draining lymph nodes. On the other hand, wild-type mice challenged with melanoma showed an improved anti-tumor response as well as changes in the tumor draining lymph node after therapeutic administration of the protein. In addition, in this model, a decrease in IL-6 expression levels was observed, and the loss of efficacy obtained with CD5 administration by depleting NK cells. We also generated another murine model expressing elevated levels of human soluble CD6 in a constitutive way, with the objective of blocking the heterophillic and homophillic interactions of CD6 and its ligands. These mice had a decrease in the spleen and lymph nodes total cell number. Their B cells have reduced proliferative capacity and the regulatory T cell less suppressive activity aside to a reduction of their number. We observed an increment of the anti-tumor response to different tumor cell lines, but their autoimmune response was not exacerbated, as it was expected. These results were reproduced when recombinant soluble human CD6 was injected repeatedly into wild-type mice. The results obtained demonstrate that counteracting the function of membrane-bound CD5/CD6 with sCD5/CD6 may enhance current immunotherapies for cancer, as well as be used in other possible applications such as in acute or chronic infections (CD5 recognizes fungal and viral components, and CD6 bacterial components) and vaccines., [spa] El objetivo de esta tesis doctoral ha sido el estudio de los efectos inmunomoduladores de CD5 y CD6, dos proteínas expresadas en la membrana de los linfocitos. Estas dos proteínas pertenecen a la superfamilia de receptores "Scavenger Receptor Cystein-Rich", caracterizada por la presencia de uno o varios dominios ricos en cisteínas. En cuanto a CD6, están descritos varios ligandos, sin embargo, a día de hoy no existe un consenso acerca del ligando/s de CD5.Trabajos previos llevados a cabo con ratones deficientes para CD5/CD6, pusieron de manifiesto sus papeles como moduladores negativos de la señal del receptor de células T/B. Decidimos generar un ratón que expresara niveles elevados de la región extracelular de CD5 humano constitutivamente, con el objetivo de bloquear las interacciones mediadas por el CD5 de membrana con su ligando/s. Observamos que estos ratones presentaron una respuesta autoinmune exacerbada pero mejor respuesta anti-tumoral frente a células murinas de melanoma y linfoma. Este efecto se debió al aumento de número y actividad de las células efectoras del sistema inmune, así como a la bajada de las células T reguladoras en los ganglios drenantes del tumor. Por otro lado, ratones silvestres a los que se les indujo melanoma mostraron una respuesta anti-tumoral mejorada, así como cambios en el ganglio drenante, tras la administración de la proteína de forma terapéutica. Además, la eficacia obtenida con la administración de CD5 se perdió al deplecionar las células NK. Además, se generó otro modelo murino que expresara niveles elevados de CD6 soluble humano de manera constitutiva, con el mismo objetivo. Estos ratones presentaron una bajada en cuanto al número de células en bazo y nódulos linfáticos debido a la capacidad proliferativa reducida de los linfocitos B. Además de una disminución en número, las células T reguladoras presentaron una menor actividad supresora. Así, observamos un aumento de la respuesta anti-tumoral frente a diferentes líneas tumorales, pero su respuesta autoinmune no se encontraba exacerbada. Estos resultados se reprodujeron cuando se inyectó CD6 soluble a ratones silvestres. Los resultados obtenidos denotan la importancia de CD5 y CD6 en la respuesta inmune, y su posible aplicación en combinación con las actuales terapias inmunomoduladoras.

We explored if baseline CD4/CD8 T-cell ratio is associated with immunodiscordant response to antiretroviral therapy in HIV-infected subjects. Comparing immunodiscordant and immunoconcordant subjects matched by pretreatment CD4 counts, we observed a lower pretreatment CD4/CD8 T-cell ratio in immunodiscordant subjects. Furthermore, pretreatment CD4/CD8 T-cell ratio, but not CD4 counts, correlated with the main immunological alterations observed in immunodiscordants, including increased regulatory T-cell (Treg) frequency and T-cell turnover-related markers. Then, in a larger cohort, only baseline CD4/CD8 T-cell ratio was independently associated with immunodiscordance, after adjusting by the viral CXCR4-tropic HIV variants. Our results suggest that the CD4/CD8 T-cell ratio could be an accurate biomarker of the subjacent immunological damage triggering immunodiscordance.

[eng] A major challenge of modern Biology is elucidating the genetic basis of adaptation. While there are many SNP-based studies trying to elucidate the genetic basis of genotype-phenotype relationships, the role of transposable element (TE)-induced mutations is understudied. Recent evidences demonstrate that TEs are a powerful tool to identify the genetic basis of adaptive phenotypic traits. Drosophila melanogaster is a good model to study adaptation because it is original from subtropical Africa and only recently colonized out-of-Africa environments. To identify and characterize the role of several candidate TEs in D. melanogaster adaptation, we have followed two different strategies: locus-specific and trait-specific. In the first chapter, we have characterized both at the molecular and phenotypic level FBti0019386, a previously identified candidate adaptive TE. We first elucidated the evolutionary history of this natural insertion and provided evidences of genomic signatures of positive selection. We then explored several phenotypes related to known phenotypic effects of nearby genes, and having plausible connections to fitness variation in nature. We found that flies with FBti0019386 insertion had a shorter developmental time and were more sensitive to stress, which are likely to be the adaptive effect and the cost of selection of this mutation, respectively. Interestingly, these phenotypic effects are not consistent with a role of FBti0019386 in temperate adaptation as has been previously suggested. Indeed, a global analysis of the population frequency of FBti0019386 showed that climatic variables explain well the FBti0019386 frequency patterns only in Australia. These results suggest that further functional validation should be gathered before concluding that a candidate loci is under spatially varying selection. Finally, although FBti0019386 insertion could be inducing the formation of heterochromatin by recruiting HP1a (Heterochromatin Protein 1a) protein, the insertion is associated with up-regulation of sra in adult females. In the second chapter of this thesis, we have studied the impact of several TE insertions in a highly conserved and ecologically relevant trait: the immune response. To do that, we first performed a new genome-wide screening in order to identify a dataset of candidate TEs involved in adaptation. By increasing the number of populations and the number of TEs analyzed compared to similar studies, we were able to increase the number of identified candidate TEs: a total of 121 TEs. Interestingly, we found that genes associated with those TEs are enriched for stress-related functions, specifically we detected a significant enrichment for immune response functions. We combined allele-specific expression (ASE), enhancer assays, and TSS detection experiments to characterize the impact of these TEs in oral immune response to the gram-negative bacteria Pseudomonas entomophila. We were also able to associate the 12 candidate TEs with gene expression changes, and determine some of the molecular mechanisms behind these expression changes. We showed that the allele with the TE was differently expressed in 13 out of the 16 analyzed genes under non-infected and/or infected conditions in at least one of the two genetic backgrounds analyzed. We also show that different TEs alter gene expression by adding promoters and enhancer regulatory sequences to their nearby genes. Although we found evidences pointing to a possible role of TEs in immune response regulation, more experiments should be performed in order to link the identified TEs with a fitness effect in this trait. Overall, our two integrative approaches allowed us to shed light on the role of TEs in generating genomic natural variation potentially underlying adaptation. The results obtained in this work illustrate that TEs are a good tool to bridge the gap between genotypic and phenotypic evolution, [cat] Un dels reptes actuals en biologia és explicar la base genètica de l’adaptació. Mentre que hi ha molts estudis basats en SNPs intentant trobar les bases genètiques de les relacions genotip-fenotip, no s’ha estudiat tant bé el paper de les mutacions generades pels elements mòbils (TEs, de l’anglès Transposable Elements). Evidències recents demostren que els TEs són una eina potent per identificar les bases genètiques dels fenotips adaptatius. Drosophila melanogaster és un bon model per estudiar l’adaptació, ja que és originària de l’Àfrica subtropical i recentment ha colonitzat altres ambients. Per identificar i caracteritzar el paper de diversos TEs candidats per l’adaptació a D. melanogaster, hem seguit dues estratègies diferents: locus-específica i tret-específica. En el primer capítol hem caracteritzat a nivell molecular i fenotípic el TE FBti0019386, identificat prèviament com a candidat per l’adaptació. Primer hem estudiat la història evolutiva d’aquesta inserció i hem demostrat que està associada a senyals genòmiques de selecció positiva. Després hem explorat diferents fenotips relacionats amb els efectes fenotípics coneguts dels gens del costat, que poguessin tenir connexions plausibles amb la variació de la fitness a la natura. En el segon capítol, hem estudiat l’impacte de diferents TEs en la resposta immune. Per això, hem mostrejat el genoma buscant TEs candidats per l’adaptació en quatre poblacions naturals. Després, hem combinat anàlisis d’expressió específica d’al·lel, assajos d’enhancer i detecció de TSS per caracteritzar l’impacte d’aquests TEs durant la resposta a infecció amb el bacteri gram-negatiu Pseudomonas entomophila. Hem trobat que l’al·lel amb el TE s’expressa de manera diferent en 13 dels 16 gens analitzats en condicions control i/o d’infecció en almenys un dels dos fons genètics analitzats. Hem demostrat que alguns d’aquests TEs alteren l’expressió afegint promotors i enhancers als gens del costat. Tot i que les evidències assenyalen cap a un possible paper dels TEs en la regulació de la resposta immune, es requereixen més experiments per associar els TEs identificats amb un efecte en la fitness. En resum, les dues estratègies integratives seguides ens han permès mostrar el paper dels TEs en la generació de variació natural genòmica potencialment implicada en l’adaptació

T-cell homeostasis must be tightly regulated and maintained in order to guarantee appropriate immune responses and prevent immunopathology. This maintenance depends on MHC-TCR interaction and cytokine-mediated signals among others. However, cell intrinsic factors that modulate essential functions in T-cells must be also integrated to support genomic stability and contribute to the control of T-cell homeostasis. The present work establishes a coordinated role of poly (ADP-ribose) polymerase-1 (PARP-1) and PARP-2 in maintaining T-lymphocyte number and function, demonstrated by the defective thymocyte maturation and diminished numbers of peripheral CD4+ and CD8+ T cells in mice bearing a T-cell specific deletion of PARP-2 in a PARP-1-deficient background. Moreover, this T-cell lymphopenia is associated with an increased DNA-damage and concomitant cell death, leading to highly aggressive spontaneous T-cell lymphomas in PARP-1/PARP-2 double-deficient mice. Our findings highlight the importance of understanding the specific involvement of both proteins in key biological processes that could have an impact on the development and exploitation of PARP-inhibitors., L’homeòstasi de la cèl·lula T ha d’estar estrictament regulada per tal de garantir una correcta resposta immunitària i prevenir alhora qualsevol problema immunopatològic. Aquest correcte manteniment depèn, entre d’altres, de la interacció amb el complex MHC-TCR i de les senyals de diferents interleuquines. No obstant, hi ha altres factors intrínsecs que intervenen en la modulació de les funcions vitals de la cèl·lula T i que han d’estar també correctament integrats en tot el sistema per tal de garantir una correcta estabilitat genòmica i contribuir en el control de l’homeòstasi de la cèl·lula T. El present treball estableix el paper coordinat entre els enzims poli (ADP-ribosa) polimerasa-1 (PARP-1) i PARP-2 en el manteniment del nombre i la funció dels limfòcits T, tal i com es demostra amb el defecte en maduració i el descens en el número de cèl·lules CD4+ i CD8+ perifèriques que tenen els ratolins amb deleció de PARP-2 en un background PARP-1 deficient. A més a més, aquesta limfopènia està associada amb un increment del dany en el ADN i una concomitant mort cel·lular, que condueix al desenvolupament espontani de limfomes T molt agressius en els ratolins dobles deficients per PARP-1 i PARP-2. Els nostres resultats posen de manifest la importància de conèixer correctament el paper específic de les dues proteïnes en processos biològics rellevants, ja que podria tenir especial impacte en el desenvolupament i l’explotació dels inhibidors PARP.

Supplemental Digital Content is available in the text, Background: The failure to increase CD4+ T-cell counts in some antiretroviral therapy suppressed participants (immunodiscordance) has been related to perturbed CD4+ T-cell homeostasis and impacts clinical evolution. Methods: We evaluated different definitions of immunodiscordance based on CD4+ T-cell counts (cutoff) or CD4+ T-cell increases from nadir value (ΔCD4) using supervised random forest classification of 74 immunological and clinical variables from 196 antiretroviral therapy suppressed individuals. Unsupervised clustering was performed using relevant variables identified in the supervised approach from 191 individuals. Results: Cutoff definition of CD4+ cell count 400 cells/μl performed better than any other definition in segregating immunoconcordant and immunodiscordant individuals (85% accuracy), using markers of activation, nadir and death of CD4+ T cells. Unsupervised clustering of relevant variables using this definition revealed large heterogeneity between immunodiscordant individuals and segregated participants into three distinct subgroups with distinct production, programmed cell-death protein-1 (PD-1) expression, activation and death of T cells. Surprisingly, a nonnegligible number of immunodiscordant participants (22%) showed high frequency of recent thymic emigrants and low CD4+ T-cell activation and death, very similar to immunoconcordant participants. Notably, human leukocyte antigen - antigen D related (HLA-DR) PD-1 and CD45RA expression in CD4+ T cells allowed reproducing subgroup segregation (81.4% accuracy). Despite sharp immunological differences, similar and persistently low CD4+ values were maintained in these participants over time. Conclusion: A cutoff value of CD4+ T-cell count 400 cells/μl classified better immunodiscordant and immunoconcordant individuals than any ΔCD4 classification. Immunodiscordance may present several, even opposite, immunological patterns that are identified by a simple immunological follow-up. Subgroup classification may help clinicians to delineate diverse approaches that may be needed to boost CD4+ T-cell recovery.

Background: to report an uncommon case of hand, foot and mouth disease, (HFMD) in an immunocompetent adult; a highly infectious disease, characterized by the appearance of vesicles on the mouth, hands and feet, associated with coxsackieviruses and enteroviruses; including a literature review. Case report: a 23 year Caucasian male with no medical or surgical history, no allergies, was not taking any medication and smoked ten cigarettes a day, suffering from discomfort in the oral cavity; itching, burning and pain when swallowing associated with small erythematous lesions located on the hard palate, and small ulcers in tonsillar pillars and right buccal mucosa. Mild fever of 37.8 °C and general malaise. The patient reported he had had contact with a child diagnosed with HFMD. From his background and symptoms, the patient was diagnosed with HFMD. Following symptomatic treatment, the symptoms remitted in 7 days. Methods: a literature review in MEDLINE (PubMed). The inclusion criteria were for studies on humans over the last 5 years, using the keywords HFMD. Results: we found 925 articles, which were subsequently reduced to 52 documents after applying the inclusion criteria. Maculopapular lesions were found on hands and feet. Conclusions: dentists may have a key role diagnosing the disease. A surveillance system to predict future outbreaks, encourage early diagnosis, put appropriate public health measures in place and research vaccine development is vitally important in order to control the disease.

Background The relationship between host microRNAs (miRNA), viral control and immune response has not yet been elucidated in the field of HIV. The aim of this study was to assess the differential miRNA profile in CD8+ T-cells between HIV-infected individuals who differ in terms of viral replication control and immune response. Methods miRNA profile from resting and CD3/CD28-stimulated CD8+ T-cells from uninfected individuals (HIV-, n = 11), Elite Controllers (EC, n = 15), Viremic Controllers (VC, n = 15), Viremic Progressors (VP, n = 13) and HIV-infected patients on therapy (ART, n = 14) was assessed using Affymetrix miRNA 3.1 arrays. After background correction, quantile normalization and median polish summarization, normalized data were fit to a linear model. The analysis comprised: resting samples between groups; stimulated samples between groups; and stimulated versus resting samples within each group. Enrichment analyses of the putative target genes were perfomed using bioinformatic algorithms. Results A downregulated miRNA pattern was observed when resting samples from all infected groups were compared to HIV-. A miRNA downregulation was also observed when stimulated samples from EC, ART and HIV- groups were compared to VP, being hsa-miR-4492 the most downregulated. Although a preferential miRNA downregulation was observed when stimulated samples were compared to the respective resting samples, VP presented a differential miRNA expression pattern. In fact, hsa-miR-155 and hsa-miR-181a were downregulated in VP whereas in the other groups, either an upregulation or no differences were observed after stimulation, respectively. Overall, functional enrichment analysis revealed that the predicted target genes were involved in signal transduction pathways, metabolic regulation, apoptosis, and immune response. Conclusions Resting CD8+ T-cells do not exhibit a differential miRNA expression between HIV-infected individuals but they do differ from non-infected individuals. Moreover, a specific miRNA pattern is present in stimulated CD8+ T-cells from VP which could reflect a detrimental pattern in terms of CD8+ T-cell immune response.

[spa] El mantenimiento de la homeostasis del sistema inmune innato es fundamental, ya que éste debe mantener una respuesta equilibrada frente a los patógenos. En mamíferos, uno de los mecanismos por el cual se mantiene la homeostasis es el reflejo inflamatorio, un circuito neural que modula la respuesta inflamatoria previniendo la posibilidad de una sobreactivación. El primer objetivo de esta tesis fue establecer si el reflejo inflamatorio descrito en mamíferos también estaba presente en otros vertebrados, y para ello nos centramos en su estudio en teleósteos, más específicamente en trucha (Oncorhynchus mykiss). Una pieza clave del sistema del reflejo inflamatorio es el receptor nicotínico de acetilcolina alfa 7 (α7nAChR). La secuencia del receptor α7nACh de trucha está altamente conservada en comparación con otras especies y el análisis filogenético lo agrupa con otras especies de peces. En estudios in vivo, la estimulación intraperitoneal con lipopolisacárido (LPS) y un análogo del ARN de doble cadena (poly (I:C)), aumentan la expresión del receptor α7nACh en diferentes tejidos y, además, modulan los niveles de acetilcolina en plasma y en bazo. Adicionalmente, el receptor se expresa en macrófagos aislados de bazo y de riñón anterior, en ambos casos siendo capaz de unir α-bungarotoxina, un antagonista específico del receptor α7nACh. Nuestros resultados muestran que existe una modulación de la nicotina (un agonista del receptor) en la expresión génica de moléculas inflamatorias en macrófagos frente a una estimulación con poly (I:C) y no con LPS, a diferencia de estudios realizados en mamíferos. Esta modulación se realiza a través del receptor α7nACh, ya que la adición de un antagonista específico, produce una disminución del efecto de la nicotina. En conclusión, los resultados presentados otorgan indicios de la existencia de un sistema de reflejo inflamatorio en peces, con similitudes y diferencias con el sistema descrito en mamíferos. La mayoría de los teleósteos responden a los patógenos con un potente sistema inmune innato, lo que convierte la modulación de éste, en una buena herramienta para mejorar la capacidad de respuesta de los peces ante una infección. El sistema inmune de los teleósteos puede ser estimulado a través de patrones moleculares asociados a patógenos (PAMPs), y a su vez, por citoquinas, como potenciadores no específicos. La segunda hipótesis de esta tesis es que nanopartículas proteicas, conocidas como cuerpos de inclusión (CI), pueden ser utilizados como plataformas de entrega de inmunoestimulantes. En este contexto, fueron desarrollados CI con (citoquinas) y sin (proteínas fluorescentes) una función inmunológica en peces. Los CI son internalizados in vitro por hepatocitos del pez cebra (Danio rerio) y por macrófagos de trucha, modulando la expresión génica de genes relacionados con la respuesta inflamatoria. Los resultados de la biodistribución in vivo en trucha mostraron que los CI son acumulados en bazo y en riñón anterior, y son endocitados por células inmunes relevantes como, los macrófagos. Los diferentes CI producidos y administrados intraperitonealmente, protegen al pez cebra frente a una infección bacteriana letal (Pseudomonas aeruginosa), siendo mayor la protección otorgada por los CI formados por citoquinas. Adicionalmente, los CI mantienen su morfología y función frente a condiciones extremas de pH y temperatura, así como durante la liofilización. Al ser administrados oralmente en truchas, los CI llegan a los dos primeros tramos del intestino y son absorbidos por células presentes en la mucosa intestinal, las cuales, por su localización en la mucosa podrían corresponderse con linfocitos intraepiteliales (IELs). Nuestros resultados sugieren que los CI pueden ser una alternativa a los métodos actuales como plataforma de entrega de inmunoestimulantes y proteínas por vía oral sin necesidad de encapsulación adicional, siendo éste uno de los principales objetivos de la industria de cultivo de peces., [eng] The inflammatory reflex is a neural circuit that modulates the innate immune system response preventing an overactivation. The first hypothesis of this thesis was to establish whether the inflammatory reflex described in mammals was also present in other vertebrates, and we focused on its study in Oncorhynchus mykiss. We cloned and characterised one of the main components of the inflammatory reflex: the alpha 7 nicotinic acetylcholine receptor (α7nAChR). The in vivo stimulation with LPS and poly (I:C) increased the receptor gene expression in different tissues and, also, modulated acetylcholine levels in plasma and spleen. The receptor was expressed in trout macrophages and was able to bind bungarotoxin, a specific receptor antagonist. Our results showed that nicotine modulates the gene expression of inflammatory genes in macrophages through α7nAChR after the stimulation with poly (I:C). In conclusion, the results provide evidences of evolutionary conservation of the inflammatory reflex in vertebrates. Teleost respond to an infections with the activation of a potent innate immune system aiming to eliminate pathogens. Thus, the modulation of the innate system would be an excellent target to improve the responsiveness of the fish to an infection. We have been used protein nanoparticles, known as inclusion bodies (IBs) as immunostimulants delivery platform. The IBs were efficiently internalized in vitro by both zebrafish hepatocytes (Danio rerio) and trout macrophages, and when IBs were administered in vivo they accumulated in the spleen and the head kidney of trout. Importantly, the IBs administered by intraperitoneal injection protect zebrafish against a lethal bacterial infection (Pseudomonas aeruginosa). The orally administered IBs were uptaken by cells of the intestinal mucosa. Our results suggest that the IBs could be an attractive alternative to stimulation of the innate immune system and can be administered orally overcoming the harsh conditions of the gastrointestinal tract.

[spa] La sepsis es la culminación de interacciones complejas entre el microorganismo infeccioso y la respuesta inmune, inflamatoria y de la coagulación del huésped. Puede evolucionar a sepsis grave y shock séptico, sus formas graves, que son la causa más frecuente de muerte en las Unidades de Cuidados Intensivos (UCI) no coronarias. En los últimos años se han conseguido avances importantes en el conocimiento de la epidemiología, fisiopatología y tratamiento de la sepsis, habiéndose reducido la mortalidad de forma significativa, sin embargo ésta se sitúa todavía entre el 20-30% en muchas series, fundamentalmente debido al síndrome de disfunción multiorgánica (SDOM). Es por ello necesario mejorar el conocimiento de la fisiopatología de los síndromes sépticos y el SDOM asociado a los mismos, para así encontrar nuevas potenciales dianas terapéuticas que permitan reducir la mortalidad. Los factores de riesgo para fallecer por sepsis se asocian tanto con la predisposición del paciente para presentar una infección como con la probabilidad de progresar a sepsis grave/shock séptico y desarrollar SDOM una vez instaurada la infección. Entre los factores de riesgo conocidos para presentar infecciones que más susceptiblemente desembocarán en sepsis grave o shock séptico se incluyen las enfermedades crónicas (síndrome de inmunodeficiencia adquirida, enfermedad pulmonar obstructiva crónica, neoplasias.) y el uso de fármacos inmunosupresores. La edad, el sexo y la raza también influyen en la incidencia de la sepsis grave. Sin embargo, los factores de riesgo para desarrollar el SDOM en los pacientes con este tipo de infecciones que presentarán sepsis grave o shock séptico no están bien definidos. Probablemente influyen el microorganismo causal, los factores genéticos del huésped, la función previa del órgano alterado y la precocidad en la instauración del tratamiento óptimo para la infección. Por este motivo se planteó la realización de la presente Tesis Doctoral. En ella se incluyen cuatro trabajos que tienen como principales objetivos evaluar y caracterizar algunos de los factores implicados en la fisiopatología de la sepsis y su relación con el SDOM y el pronóstico. Así, se evaluaron la expresión y función las moléculas de adhesión en células mononucleares de sangre periférica y los niveles séricos de moléculas solubles de adhesión endotelial, implicadas en la adhesión endotelio- neutrófilo, en pacientes con sepsis grave o shock séptico secundario a neumonía adquirida en la comunidad (Artículo 1); la existencia y relevancia funcional de los polimorfismos genéticos en el gen de la Lectina fijadora de Manosa (MBL2) y de la MASP2 (por sus siglas en inglés, de MBL-Associated Serine Proteases), asociados con la predisposición a presentar infecciones graves (Artículo 2); los niveles de CD5 y CD6 soluble, moléculas que actúan como receptores de reconocimiento de patrones y juegan un importante papel en la respuesta inmune innata (Artículo 3); y los niveles de ADAMTS-13, proteasa que escinde los multímeros inusualmente grandes del factor de von Willebrand, y que se ha hallado deficitario en pacientes sépticos (Artículo 4). Los principales resultados de los estudios demuestran que los niveles de CD5 y CD6 soluble y de ADAMTS-13 de los pacientes críticamente enfermos con Síndromes sépticos se relacionan con la gravedad de la enfermedad y la progresión a SDOM y la mortalidad. Por otro lado, aunque los genotipos de baja expresión de MBL y de la MASP-2 no se asocian con la gravedad ni con la mortalidad de estos pacientes, el genotipo de alta expresión de MBL parece ser un factor de riesgo para desarrollar SRIS no infeccioso. Finalmente, los pacientes críticamente enfermos con síndromes sépticos secundarios a neumonía adquirida en la comunidad presentan alteraciones en los niveles y en la función de las moléculas de adhesión que parecen estar asociadas con el pronóstico., [eng] Sepsis is the culmination of complex interactions between the infecting organism and the immune, inflammatory and coagulation response of the host. It can lead to severe sepsis and septic shock which are the most common cause of death in non-coronary Intensive Care Units. It is therefore necessary to improve the understanding of the pathophysiology of septic syndromes and multiple organ dysfunction syndrome (MODS), in order to find new potential therapeutic targets to reduce mortality. However, risk factors for developing MODS in patients with sepsis are not well defined. This thesis includes four works whose objectives were to evaluate and characterize some of the factors involved in the pathophysiology of sepsis and its association with MODS and prognosis. We evaluated the expression and function of adhesion molecules and serum levels of soluble endothelial adhesion molecules in patients with severe sepsis or septic shock secondary to community acquired pneumonia (Article 1), the existence and functional significance of genetic polymorphisms in Mannose-binding lectin (MBL 2) and MASP2 (MBL-Associated Serine Proteases) genes regarding their predisposition to present severe infections (Article 2), the levels of soluble CD5 and CD6 molecules, which act as pattern recognition receptors and play an important role in the innate immune response (Article 3), and the levels of ADAMTS-13, protease deficient in septic patients that cleaves the unusually large multimers of von Willebrand factor (Article 4). The main results of these studies show that levels of soluble CD5 and CD6 and ADAMTS-13 in critically ill patients with septic syndromes are related to disease severity and progression to MODS and mortality. Furthermore, although genotypes of low expression of MBL and MASP-2 are not associated with the severity or mortality of these patients, the genotype of high expression of MBL appears to be a risk factor for developing non-infectious SIRS. Finally, critically ill patients with septic syndromes secondary to community acquired pneumonia have alterations in the levels and function of adhesion molecules that appear to be associated with their outcome.

C57Bl/6J mice displayed considerable inter-mice variation in some behaviors in the open-field and the elevated plus-maze (coefficients of variation from 30% to 200%). Those individual differences were not entirely random, because four successive measurements of each behavior were positively correlated. Application of the aggregation principle revealed that (i) in males and females, odd-even correlation coefficients for ambulation (in the open-fieldand in the plus-maze) were of the order of 0.60, (ii) the odd-even correlation coefficient for defecation in the open-field was 0.74 for males and 0.23 forfemales (not significant), (iii) the odd-even correlation coefficient for the antibody response (to immunization with aggregated bovine serum albumin) was 0.43 for males and 0.60 for females. In males, but not in females, these variables were intercorrelated: ambulation in the open-field, ambulation in the enclosed arm of the plus-maze, and defecation in the open-field; the antibody response was uncorrelated with behaviors.

[cat] TREX2 (three-prime repair exonuclease 2) és una exonucleasa no processiva 3'-5', expressada específicament en la pell que pot estar implicada en el manteniment de la integritat del genoma editant els extrems 3' del DNA en diversos processos, des de la replicació a la reparació del dany passant per la degradació en l'apoptosi. L'estudi de TREX2 mitjançant immunohistofluorescència de mostres humanes de lesions precanceroses (queratosis actínica) i tumors derivats de queratinòcits, incloent cSCC (cutaneous squamous cell carcinomas) i HNSCCs (head and neck SCC), revela que en les carcinogènesis escamoses l'expressió de TREX2 pateix es troba alterada. La deleció de Trex2 en ratolins provoca un augment de la susceptibilitat de tumors induïts en pell provocats per radiació ultraviolada B (UVB). TREX2 promou l'eliminació del DNA danyat i l'apoptosi dels queratinòcits en les pells de ratolí irradiades amb UVB. Les lesions al DNA induïdes per UVB (CPD: cyclobutane pyrimidine_dimers), són més persistents en les pells dels ratolins Trex21- respecte els wt, paral• lelament també es dóna una menor activació de la caspasa 3, indicador d'apoptosi, en les pells Trex21-. Els resultat obtinguts en queratinòcits wt i deficients en TREX2 in vitxo reflexen una multifuncionalitat de la proteïna TREX2 intervenint en la reparació del dany al DNA en resposta a la radiació UV i promovent l'apoptosi de forma irreversible, essent aquesta última funció la que preval. TREX2 pot ser reclutada diferencialment a diferents localitzacions subnuclears en funció de l'estat de la cromatina, com per exemple els micronuclis i les regions de cromatina poc condensada. Els estudis de co-immunoprecipitació demostren que TREX2 interacciona amb yH2AX, així TREX2 pot accedir als extrems lliures del DNA interaccionant amb la proteïna H2AX fosforilada, preferentment en la cromatina destinada a ser degradada. L'estudi histopatològic de les pells de ratolins irradiats amb UVB, revela que els ratolins Trex21- són més resistents que els wt a la inflamació de la pell induïda per UVB. Addicionalment, en les pells irradiades repetidament del ratolí Trex2-/, l'augment en el nombre de capes cel• lulars en l'epidermis com a mesura de protecció de la pell contra la irradiació, és menor que en el wt. En resum, les nostres dades subratllen la rellevància de TREX2 a l'hora de promoure la resposta inflamatòria contra la UVB. Per altra part, a lesions psoriàsiques es dóna un fort increment i canvis en el patró d'expressió de TREX2 respecte la pell sana. La deficiència de TREX2 a ratolins disminueix el fenotip psoriàsic induït per l'Imiquimod. Concretament, TREX2 afavoreix la mort cel• lular i la resposta inflamatòria inicial. A més a més, TREX2 està implicada en la degradació del DNA durant la diferenciació terminal dels corneòcits, disminuint la paraqueratosi. Així, TREX2 té un paper actiu i rellevant en el desenvolupament de la patogènesi associada a la psoriasi., [eng] TREX2 is a 3 '-DNA exonuclease specifically expressed in keratinocytes that acts as tumor suppressor in skin carcinogenesis, but the importance in human pathologies like carcinogenesis and psoriasis and the underlying mechanisms remained unknown. Here, we investigated the relevance and molecular events of TREX2 in the DNA damage skin response. TREX2 expression was up-regulated by chronic exposure to ultraviolet (UV) radiation, whereas was de-regulated or lost in human squamous cell carcinomas (SCCs). TREX2 deficiency in mice enhanced the susceptibility to UVB-induced skin carcinogenesis, which was associated with alterations in DNA damage removal and degradation, cell death and inflammation, indicating multifaceted functions of TREX2. Notably, TREX2 deficiency diminished the up-regulation of key cytokines related to DNA repair and antitumor immunity. Interestingly, TREX2 was recruited to low-density chromatin regions in nuclei that were beginning to condensate chromatin and to micronuclei, interacting with phosphorylated H2AX histone (yH2AX). Altogether, our data establish cell and non-cell autonomous functions of TREX2 in the defense against UV-induced skin carcinogenesis. Moreover TREX2 is overexpressed in psoriasic lesions. To elucidate the importance of this exonuclease in the development of the disease, we induced a skin inflammation in mice by topical application of Imiquimod. The Trex24- mice were more resistant to the Imiquimod-induced skin inflammation than wt. Thus, clinical parameters like skin thickness, erythema and scaling were lower in the Trex24- mice compared to the wt, whereas parakeratosis was more abundant in the Trex24- skins. Altogether, our results show thatTREX2 contribute to the pathologic features of psoriasis by enhancing the keratinocyte apoptosis and the inflammation in response to Imiquimod application.

Actualment, l’estudi de la relació entre nutrició i salut ha comportat que la immunonutrició esdevingui una ciència d’interès creixent. El cacau és un aliment ric en macronutrients (fibra, proteïnes, carbohidrats i lípids), micronutrients (minerals i vitamines) i conté una quantitat relativament elevada de flavonoides. Aquests inclouen majoritàriament els flavan-3-ols, com (-)-epicatequina, (+)-catequina i procianidines, compostos amb capacitat antioxidant i amb efectes antitumorals, en processos neurodegeneratius i sobre el sistema cardiovascular i el sistema immunitari. És per aquest motiu que el cacau s’ha convertit en un aliment amb gran interès com a potencial nutracèutic. L’al·lèrgia alimentària representa un problema de salut important en els països industrialitzats amb elevada prevalença en la població adulta i sobre tot en infants. Els individus afectats presenten una resposta immunitària anòmala quan ingereixen certs aliments o components alimentaris. La seva patogènia inclou la pèrdua de tolerància oral a proteïnes dels aliments i, en la majoria de casos, s’acompanya de la producció d’IgE. Entre les diferents manifestacions clíniques destaquen les alteracions gastrointestinals, les afeccions cutànies i l’anafilaxi, que pot arribar a xoc anafilàctic i comprometre la vida del pacient. En base a aquests antecedents, l’objectiu principal de la tesi ha estat determinar si la capacitat immunomoduladora del cacau podia ser útil per controlar la patogènia de l’al·lèrgia alimentària i establir el paper dels flavonoides en aquest efecte. De forma preliminar, es va desenvolupar un model d’al·lèrgia no alimentària, que va permetre estudiar els efectes d’una dieta enriquida amb cacau convencional sobre la resposta immunitària i establir variables objectives per avaluar la resposta anafilàctica. L’efecte del cacau en el model d’al·lèrgia desenvolupat mitjançant una única immunització intraperitoneal d’ovoalbúmina (al·lergogen), hidròxid d’alumini i toxina de Bordetella pertussis (adjuvants) va revelar, principalment, que el cacau atenuava la síntesi d’anticossos específics relacionats amb la resposta immunitària Th2. D’altra banda, en aquest mateix model, es van quantificar els canvis produïts en l’activitat motora immediatament després de realitzar una provocació oral. D’aquesta manera es va poder establir una variable objectiva per avaluar l’anafilaxi que es correlacionava inversament amb la concentració de proteasa II mastocitària sèrica. Posteriorment es va desenvolupar un model d’al·lèrgia alimentària en rata mitjançant la combinació d’una única immunització intraperitoneal, com la descrita anteriorment, amb l’administració oral de l’al·lergogen catorze dies més tard i durant tres setmanes. Aquest model es caracteritza principalment per l’exacerbació en la síntesi d’anticossos relacionats amb la resposta Th2 (IgE, IgG1, IgG2a) conseqüent a l’inici de l’administració oral i que ja és patent a la primera setmana. A més, la provocació oral dels animals amb al·lèrgia alimentària va provocar un increment de fins a sis vegades de la concentració de proteasa mastocitària II i de la seva expressió gènica a nivell intestinal. Un cop desenvolupat i caracteritzat el model d’al·lèrgia alimentària, es va determinar l’efecte d’una dieta enriquida amb cacau en aquest model. Els animals van rebre una dieta rica en cacau convencional des del moment de la immunització intraperitoneal i fins al final de l’estudi. El cacau va ser capaç de reduir la síntesi d’anticossos específics associats a la resposta tipus Th2, especialment d’isotip IgE. També va inhibir la secreció de citocines de tipus Th2 i, a més, en realitzar una provocació oral, va protegir parcialment de la resposta anafilàctica ja que va disminuir la síntesi i alliberament de la proteasa mastocitària II. Per identificar si els responsables d’aquest efecte eren els flavonoides, es va determinar sobre aquest mateix model d’al·lèrgia alimentària, l’efecte d’una dieta enriquida amb cacau no fermentat, el qual proporcionava principalment flavonoides i menys proporció d’altres components. Els cacau no fermentat va tenir un cert impacte en l’al·lèrgia alimentària però els seus efectes van ser menys potents que els observats amb la dieta enriquida amb cacau convencional. Per tant, a la vista dels resultats obtinguts, es pot concloure que els flavonoides del cacau no són els únics responsables del seu poder modulador en la resposta Th2. En resum, la intervenció nutricional amb una dieta enriquida en cacau en el model d’al·lèrgia alimentària desenvolupat, demostra l’efecte beneficiós d’aquest aliment i li confereix un interès especial en el camp de la immunonutrició com a potencial nutracèutic., Nowadays, immunonutrition has become a very interesting issue of study. Due to cocoa has a relatively high content of flavonoids (mainly flavanols such as epicatechin, catechin and procyanidins), and it has a beneficial role in cardiovascular, chronic inflammation and cancer, cocoa has a potential role in immunonutrition as a nutraceutical. On the other hand, food allergy is an immune disease which is caused by food allergens, and is mainly mediated by IgE. During the past decades has become a major public health in westernized countries with an increasing prevalence. Based on these antecedents, the aim of the present study was to ascertain the effect of a cocoa-enriched diet on a food allergy rat model and also to establish the role of cocoa flavonoids in these effects. As a preliminary study, an allergy model by means of an intraperitoneal immunization with ovalbumin (allergen), aluminum hydroxide and toxin from Bordetella pertussis (adjuvants), was achieved. Using this rat model, a diet with conventional cocoa was tested and animals were orally provoked with the allergen to establish unbiased variables to study an anaphylactic response. The conventional cocoa diet was able to reduce Th2-associated antibodies, mainly specific IgE, and cytokines. The anaphylactic response produced a decrease in the number of animal movements which was inversely correlated with serum mast cell protease release. Then, a food allergy model was set up by means of the combination of only one intraperitoneal immunization (as above) with an oral administration of the allergen, fourteen days later and for three weeks. The model was achieved after the first week of oral gavage as demonstrated by a rise in the synthesis of specific antibodies related with Th2 immune response. Finally, using the food allergy model, a nutritional intervention with cocoa was assay. The animals received a conventional cocoa-enriched diet which was able to reduce the synthesis of Th2-specific antibodies and also partially inhibit the anaphylactic response because the diet produced a reduction in the serum concentration of mast cell protease and a down-regulation of its intestinal gene expression. To establish the role of cocoa flavonoids in this immunomodulation, food allergic animals received a diet containing non fermented cocoa which provided mainly flavonoids and no other cocoa components. Although a certain tendency of protection was detected, the conventional cocoa effect was stronger demonstrating that not only flavonoids are responsible for the protection in front of food allergy, and suggesting that cocoa has a high interest in the field of health and immunonutrition.

The goal of this report is to find out whether early exposure of mice to the open-field results in altered behavior of the same adult mice in the same open-field. Early exposure to the open-field was carried out between birth and weaning; two control groups were included: control 2 (mice exposed to a reduced dark space) and control 1 (mice left undisturbed). The (male and female) mice were of the Balb/c and C57Bl/6 strains. Adult C57Bl/6 female mice of the open-field and control 2 groups ambulated to the same extent in the periphery of the open-field, and mice of both groups ambulated more than mice of the control 1 group; no consistent difference between the three groups was noticed on defecation, or ambulation in the center of the field. No effects of early exposure to the open-field were observed in mice of the Balb/c strain. The effect of early exposure to the open-field, or to a dark space, on adult behavior depends on the murine strain, on sex, and on the behavior measured., El objetivo de este artículo es averiguar si la exposición temprana de los ratones al campo abierto se refleja en conducta alterada de los mismos ratones adultos en el mismo campo abierto. La exposición temprana al campo abierto se llevó a cabo entre el nacimiento y el destete; se incluyeron dos grupos control: control 2 (ratones expuestos a un espacio reducido y obscuro) y control 1 (ratones no manipulados). Los ratones, machos y hembras, pertenecían a las cepas Balb/c y C57Bl/6. Las hembras adultas de la cepa C57Bl/6 pertenecientes a los grupos campo abierto y control 2 deambularon lo mismo en la periferia del campo abierto, y los ratones de ambos grupos deambularon más que los ratones del grupo control 1; no se observó ninguna diferencia consistente entre los ratones de los tres grupos en la defecación, o en la deambulación en el centro del campo. No se observaron efectos consistentes de la exposición temprana al campo abierto sobre la conducta adulta en los ratones de la cepa Balb/c. El efecto de la exposición temprana al campo abierto, o a un espacio obscuro, depende de la cepa de ratón, del sexo, y de la conducta medida.

Background The effect of maraviroc on the maintenance and the function of HIV-1-specific T cell responses remains unknown. Methods Subjects recently infected with HIV-1 were randomized to receive anti-retroviral treatment with or without maraviroc intensification for 48 weeks, and were monitored up to week 60. PBMC and in vitro-expanded T cells were tested for responses to the entire HIV proteome by ELISpot analyses. Intracellular cytokine staining assays were conducted to monitor the (poly)-functionality of HIV-1-specific T cells. Analyses were performed at baseline and week 24 after treatment start, and at week 60 (3 months after maraviroc discontinuation). Results Maraviroc intensification was associated with a slower decay of virus-specific T cell responses over time compared to the non-intensified regimen in both direct ex-vivo as well as in in-vitro expanded cells. The effector function profiles of virus-specific CD8+ T cells were indistinguishable between the two arms and did not change over time between the groups. Conclusions Maraviroc did not negatively impact any of the measured parameters, but was rather associated with a prolonged maintenance of HIV-1-specific T cell responses. Maraviroc, in addition to its original effect as viral entry inhibitor, may provide an additional benefit on the maintenance of virus-specific T cells which may be especially important for future viral eradication strategies.