Innate IRE1α-XBP1 activation by viral single-stranded RNA and its influence on lung cytokine production during SARS-CoV-2 pneumonia.

The utilization of host-cell machinery during SARS-CoV-2 infection can overwhelm the protein-folding capacity of the endoplasmic reticulum and activate the unfolded protein response (UPR). The IRE1α-XBP1 arm of the UPR could also be activated by viral RNA via Toll-like receptors. Based on these premises, a study to gain insight into the pathogenesis of COVID-19 disease was conducted using nasopharyngeal exudates and bronchioloalveolar aspirates. The presence of the mRNA of spliced XBP1 and a high expression of cytokine mRNAs were observed during active infection. TLR8 mRNA showed an overwhelming expression in comparison with TLR7 mRNA in bronchioloalveolar aspirates of COVID-19 patients, thus suggesting the presence of monocytes and monocyte-derived dendritic cells (MDDCs). In vitro experiments in MDDCs activated with ssRNA40, a synthetic mimic of SARS-CoV-2 RNA, showed induction of XBP1 splicing and the expression of proinflammatory cytokines. These responses were blunted by the IRE1α inhibitor MKC8866, the TLR8 antagonist CU-CPT9a, and knockdown of TLR8 receptor. In contrast, the IRE1α-XBP1 activator IXA4 enhanced these responses. Based on these findings, the TLR8/IRE1α system seems to play a significant role in the induction of the proinflammatory cytokines associated with severe COVID-19 disease and might be a druggable target to control cytokine storm.

Altered Surface Expression of Insulin-Degrading Enzyme on Monocytes and Lymphocytes from COVID-19 Patients Both at Diagnosis and after Hospital Discharge.

Although the COVID-19 disease has developed into a worldwide pandemic, its pathophysiology remains to be fully understood. Insulin-degrading enzyme (IDE), a zinc-metalloprotease with a high affinity for insulin, has been found in the interactomes of multiple SARS-CoV-2 proteins. However, the relevance of IDE in the innate and adaptative immune responses elicited by circulating peripheral blood mononuclear cells is unknown. Here, we show that IDE is highly expressed on the surface of circulating monocytes, T-cells (both CD4+ and CD4-), and, to a lower extent, in B-cells from healthy controls. Notably, IDE's surface expression was upregulated on monocytes from COVID-19 patients at diagnosis, and it was increased in more severe patients. However, IDE's surface expression was downregulated (relative to healthy controls) 3 months after hospital discharge in all the studied immune subsets, with this effect being more pronounced in males than in females, and thus it was sex-dependent. Additionally, IDE levels in monocytes, CD4+ T-cells, and CD4- T-cells were inversely correlated with circulating insulin levels in COVID-19 patients (both at diagnosis and after hospital discharge). Of note, high glucose and insulin levels downregulated IDE surface expression by ~30% in the monocytes isolated from healthy donors, without affecting its expression in CD4+ T-cells and CD4- T-cells. In conclusion, our studies reveal the sex- and metabolism-dependent regulation of IDE in monocytes, suggesting that its regulation might be important for the recruitment of immune cells to the site of infection, as well as for glucometabolic control, in COVID-19 patients.

The age again in the eye of the COVID-19 storm: evidence-based decision making.

BACKGROUND: One hundred fifty million contagions, more than 3 million deaths and little more than 1 year of COVID-19 have changed our lives and our health management systems forever. Ageing is known to be one of the significant determinants for COVID-19 severity. Two main reasons underlie this: immunosenescence and age correlation with main COVID-19 comorbidities such as hypertension or dyslipidaemia. This study has two aims. The first is to obtain cut-off points for laboratory parameters that can help us in clinical decision-making. The second one is to analyse the effect of pandemic lockdown on epidemiological, clinical, and laboratory parameters concerning the severity of the COVID-19. For these purposes, 257 of SARSCoV2 inpatients during pandemic confinement were included in this study. Moreover, 584 case records from a previously analysed series, were compared with the present study data. RESULTS: Concerning the characteristics of lockdown series, mild cases accounted for 14.4, 54.1% were moderate and 31.5%, severe. There were 32.5% of home contagions, 26.3% community transmissions, 22.5% nursing home contagions, and 8.8% corresponding to frontline worker contagions regarding epidemiological features. Age > 60 and male sex are hereby confirmed as severity determinants. Equally, higher severity was significantly associated with higher IL6, CRP, ferritin, LDH, and leukocyte counts, and a lower percentage of lymphocyte, CD4 and CD8 count. Comparing this cohort with a previous 584-cases series, mild cases were less than those analysed in the first moment of the pandemic and dyslipidaemia became more frequent than before. IL-6, CRP and LDH values above 69 pg/mL, 97 mg/L and 328 U/L respectively, as well as a CD4 T-cell count below 535 cells/µL, were the best cut-offs predicting severity since these parameters offered reliable areas under the curve. CONCLUSION: Age and sex together with selected laboratory parameters on admission can help us predict COVID-19 severity and, therefore, make clinical and resource management decisions. Demographic features associated with lockdown might affect the homogeneity of the data and the robustness of the results.

The Mitochondrial Isoform of FASTK Modulates Nonopsonic Phagocytosis of Bacteria by Macrophages via Regulation of Respiratory Complex I.

Phagocytosis is a pivotal process by which innate immune cells eliminate bacteria. In this study, we explore novel regulatory mechanisms of phagocytosis driven by the mitochondria. Fas-activated serine/threonine kinase (FASTK) is an RNA-binding protein with two isoforms, one localized to the mitochondria (mitoFASTK) and the other isoform to cytosol and nucleus. The mitoFASTK isoform has been reported to be necessary for the biogenesis of the mitochondrial ND6 mRNA, which encodes an essential subunit of mitochondrial respiratory complex I (CI, NADH:ubiquinone oxidoreductase). This study investigates the role and the mechanisms of action of FASTK in phagocytosis. Macrophages from FASTK─/─ mice exhibited a marked increase in nonopsonic phagocytosis of bacteria. As expected, CI activity was specifically reduced by almost 50% in those cells. To explore if decreased CI activity could underlie the phagocytic phenotype, we tested the effect of CI inhibition on phagocytosis. Indeed, treatment with CI inhibitor rotenone or short hairpin RNAs against two CI subunits (NDUFS3 and NDUFS4) resulted in a marked increase in nonopsonic phagocytosis of bacteria. Importantly, re-expression of mitoFASTK in FASTK-depleted macrophages was sufficient to rescue the phagocytic phenotype. In addition, we also report that the decrease in CI activity in FASTK─/─ macrophages is associated with an increase in phosphorylation of the energy sensor AMP-activated protein kinase (AMPK) and that its inhibition using Compound C reverted the phagocytosis phenotype. Taken together, our results clearly demonstrate for the first time, to our knowledge, that mitoFASTK plays a negative regulatory role on nonopsonic phagocytosis of bacteria in macrophages through its action on CI activity.

Viral RNA load in plasma is associated with critical illness and a dysregulated host response in COVID-19.

BACKGROUND: COVID-19 can course with respiratory and extrapulmonary disease. SARS-CoV-2 RNA is detected in respiratory samples but also in blood, stool and urine. Severe COVID-19 is characterized by a dysregulated host response to this virus. We studied whether viral RNAemia or viral RNA load in plasma is associated with severe COVID-19 and also to this dysregulated response. METHODS: A total of 250 patients with COVID-19 were recruited (50 outpatients, 100 hospitalized ward patients and 100 critically ill). Viral RNA detection and quantification in plasma was performed using droplet digital PCR, targeting the N1 and N2 regions of the SARS-CoV-2 nucleoprotein gene. The association between SARS-CoV-2 RNAemia and viral RNA load in plasma with severity was evaluated by multivariate logistic regression. Correlations between viral RNA load and biomarkers evidencing dysregulation of host response were evaluated by calculating the Spearman correlation coefficients. RESULTS: The frequency of viral RNAemia was higher in the critically ill patients (78%) compared to ward patients (27%) and outpatients (2%) (p < 0.001). Critical patients had higher viral RNA loads in plasma than non-critically ill patients, with non-survivors showing the highest values. When outpatients and ward patients were compared, viral RNAemia did not show significant associations in the multivariate analysis. In contrast, when ward patients were compared with ICU patients, both viral RNAemia and viral RNA load in plasma were associated with critical illness (OR [CI 95%], p): RNAemia (3.92 [1.183-12.968], 0.025), viral RNA load (N1) (1.962 [1.244-3.096], 0.004); viral RNA load (N2) (2.229 [1.382-3.595], 0.001). Viral RNA load in plasma correlated with higher levels of chemokines (CXCL10, CCL2), biomarkers indicative of a systemic inflammatory response (IL-6, CRP, ferritin), activation of NK cells (IL-15), endothelial dysfunction (VCAM-1, angiopoietin-2, ICAM-1), coagulation activation (D-Dimer and INR), tissue damage (LDH, GPT), neutrophil response (neutrophils counts, myeloperoxidase, GM-CSF) and immunodepression (PD-L1, IL-10, lymphopenia and monocytopenia). CONCLUSIONS: SARS-CoV-2 RNAemia and viral RNA load in plasma are associated with critical illness in COVID-19. Viral RNA load in plasma correlates with key signatures of dysregulated host responses, suggesting a major role of uncontrolled viral replication in the pathogenesis of this disease.

COVID-19: age, Interleukin-6, C-reactive protein, and lymphocytes as key clues from a multicentre retrospective study.

BACKGROUND: The SARS-CoV-2 infection has widely spread to become the greatest public health challenge to date, the COVID-19 pandemic. Different fatality rates among countries are probably due to non-standardized records being carried out by local health authorities. The Spanish case-fatality rate is 11.22%, far higher than those reported in Asia or by other European countries. A multicentre retrospective study of demographic, clinical, laboratory and immunological features of 584 Spanish COVID-19 hospitalized patients and their outcomes was performed. The use of renin-angiotensin system blockers was also analysed as a risk factor. RESULTS: In this study, 27.4% of cases presented a mild course, 42.1% a moderate one and for 30.5% of cases, the course was severe. Ages ranged from 18 to 98 (average 63). Almost 60 % (59.8%) of patients were male. Interleukin 6 was higher as severity increased. On the other hand, CD8 lymphocyte count was significantly lower as severity grew and subpopulations CD4, CD8, CD19, and NK showed concordant lowering trends. Severity-related natural killer percent descents were evidenced just within aged cases. A significant severity-related decrease of CD4 lymphocytes was found in males. The use of angiotensin-converting enzyme inhibitors was associated with a better prognosis. The angiotensin II receptor blocker use was associated with a more severe course. CONCLUSIONS: Age and age-related comorbidities, such as dyslipidaemia, hypertension or diabetes, determined more frequent severe forms of the disease in this study than in previous literature cohorts. Our cases are older than those so far reported and the clinical course of the disease is found to be impaired by age. Immunosenescence might be therefore a suitable explanation for the hampering of immune system effectors. The adaptive immunity would become exhausted and a strong but ineffective and almost deleterious innate response would account for COVID-19 severity. Angiotensin-converting enzyme inhibitors used by hypertensive patients have a protective effect in regards to COVID-19 severity in our series. Conversely, patients on angiotensin II receptor blockers showed a severer disease.

Usefulness of a single-assay chemiluminescence test (Tularaemia VIRCLIA IgG + IgM monotest) for the diagnosis of human tularemia. Comparison of five serological tests.

The aim of this work was to ascertain the usefulness of a new commercially-available single-assay chemiluminescence test (CHT) for the diagnosis of human tularemia (Tularaemia VIRCLIA IgG + IgM monotest, Vircell, Santa Fe, Granada, Spain). A total of 773 sera from 773 patients including 364 initial sera from patients with diagnosed tularemia, patients with suspected tularemia not confirmed (100), healthy people (152), patients with serology positive to Brucella (97), patients diagnosed with other infectious diseases (30), and patients diagnosed with autoimmune diseases (30) were included. All sera were tested by CHT, "in-house" microagglutination test (MAT), immunochromatographic test (ICT) (Virapid Tularaemia, Vircell, Santa Fe Granada, Spain), and "in-house" ELISA IgG, and ELISA IgM. Of the total initial sera, 334 (sensitivity 91.8%) were positive in the CHT, 332 (sensitivity 91.2%) in the MAT, 330 (sensitivity 90.7%) in the ICT, and 328 (sensitivity 90.1%) in the ELISA IgG and ELISA IgM tests. The specificity of the CHT was 96.7%; of the MAT, 100%; of the ICT, 98.7%; and of the ELISA IgG and ELISA IgM, 97.4%. In the group of patients with serology positive to Brucella, at least 12.4% of sera were positive in tularemia tests (12.4% in ELISA IgM, 13.4% in MAT, 14.4% in ICT, and 15.5% in CHT and ELISA IgG). In conclusion, CHT presents a sensitivity and specificity in early diagnosis of human tularemia, similar to MAT, ICT, and ELISA IgG and ELISA IgM. Its single assay design allows lower costs, especially in areas of low endemicity or inter-epidemic periods.

Role of FAST Kinase Domains 3 (FASTKD3) in Post-transcriptional Regulation of Mitochondrial Gene Expression.

The Fas-activated serine/threonine kinase (FASTK) family of proteins has recently emerged as a central regulator of mitochondrial gene expression through the function of an unusual RNA-binding domain named RAP (for RNA-binding domain abundant in Apicomplexans), shared by all six members of the family. Here we describe the role of one of the less characterized members, FASTKD3, in mitochondrial RNA metabolism. First, we show that, in contrast to FASTK, FASTKD2, and FASTKD5, FASTKD3 does not localize in mitochondrial RNA granules, which are sites of processing and maturation of mtRNAs and ribosome biogenesis. Second, we generated FASTKD3 homozygous knock-out cell lines by homologous recombination and observed that the absence of FASTKD3 resulted in increased steady-state levels and half-lives of a subset of mature mitochondrial mRNAs: ND2, ND3, CYTB, COX2, and ATP8/6. No aberrant processing of RNA precursors was observed. Rescue experiments demonstrated that RAP domain is required for FASTKD3 function in mRNA stability. Besides, we describe that FASTKD3 is required for efficient COX1 mRNA translation without altering mRNA levels, which results in a decrease in the steady-state levels of COX1 protein. This finding is associated with reduced mitochondrial complex IV assembly and activity. Our observations suggest that the function of this family of proteins goes beyond RNA processing and ribosome assembly and includes RNA stability and translation regulation within mitochondria.

Molecular investigation of tularemia outbreaks, Spain, 1997-2008.

Tularemia outbreaks occurred in northwestern Spain in 1997-1998 and 2007-2008 and affected >1,000 persons. We assessed isolates involved in these outbreaks by using pulsed-field gel electrophoresis with 2 restriction enzymes and multilocus variable number tandem repeat analysis of 16 genomic loci of Francisella tularensis, the cause of this disease. Isolates were divided into 3 pulsotypes by pulsed-field gel electrophoresis and 8 allelic profiles by multilocus variable number tandem repeat analysis. Isolates obtained from the second tularemia outbreak had the same genotypes as isolates obtained from the first outbreak. Both outbreaks were caused by genotypes of genetic subclade B.Br:FTNF002-00, which is widely distributed in countries in central and western Europe. Thus, reemergence of tularemia in Spain was not caused by the reintroduction of exotic strains, but probably by persistence of local reservoirs of infection.

Development of a novel in vitro model to study the modulatory role of the respiratory complex I in macrophage effector functions.

Increasing evidence demonstrate that the electron transfer chain plays a critical role in controlling the effector functions of macrophages. In this work, we have generated a Ndufs4-/- murine macrophage cell lines. The Ndufs4 gene, which encodes a supernumerary subunit of complex I, is a mutational hotspot in Leigh syndrome patients. Ndufs4-/- macrophages showed decreased complex I activity, altered complex I assembly, and lower levels of maximal respiration and ATP production. These mitochondrial respiration alterations were associated with a shift towards a pro-inflammatory cytokine profile after lipopolysaccharide challenge and improved ability to phagocytose Gram-negative bacteria.

The mitochondrial succinate dehydrogenase complex controls the STAT3-IL-10 pathway in inflammatory macrophages.

The functions of macrophages are tightly regulated by their metabolic state. However, the role of the mitochondrial electron transport chain (ETC) in macrophage functions remains understudied. Here, we provide evidence that the succinate dehydrogenase (SDH)/complex II (CII) is required for respiration and plays a role in controlling effector responses in macrophages. We find that the absence of the catalytic subunits Sdha and Sdhb in macrophages impairs their ability to effectively stabilize HIF-1α and produce the pro-inflammatory cytokine IL-1ß in response to LPS stimulation. We also arrive at the novel result that both subunits are essential for the LPS-driven production of IL-10, a potent negative feedback regulator of the macrophage inflammatory response. This phenomenon is explained by the fact that the absence of Sdha and Sdhb leads to the inhibition of Stat3 tyrosine phosphorylation, caused partially by the excessive accumulation of mitochondrial reactive oxygen species (mitoROS) in the knockout cells.

Fas-activated serine/threonine phosphoprotein promotes immune-mediated pulmonary inflammation.

We generated Fas-activated serine threonine phosphoprotein (FAST)-deficient mice (FAST(-/-)) to study the in vivo role of FAST in immune system function. In a model of house dust mite-induced allergic pulmonary inflammation, wild type mice develop a mixed cellular infiltrate composed of eosinophils, lymphocytes, and neutrophils. FAST(-/-) mice develop airway inflammation that is distinguished by the near absence of neutrophils. Similarly, LPS-induced alveolar neutrophil recruitment is markedly reduced in FAST(-/-) mice compared with wild type controls. This is accompanied by reduced concentrations of cytokines (TNF-alpha and IL-6 and -23) and chemoattractants (MIP-2 and keratinocyte chemoattractant) in bronchoalveolar lavage fluids. Because FAST(-/-) neutrophils exhibit normal chemotaxis and survival, impaired neutrophil recruitment is likely to be due to reduced production of chemoattractants within the pulmonary parenchyma. Studies using bone marrow chimeras implicate lung resident hematopoietic cells (e.g., pulmonary dendritic cells and/or alveolar macrophages) in this process. In conclusion, our results introduce FAST as a proinflammatory factor that modulates the function of lung resident hematopoietic cells to promote neutrophil recruitment and pulmonary inflammation.

Metabolomics study of COVID-19 patients in four different clinical stages.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the coronavirus strain causing the respiratory pandemic COVID-19 (coronavirus disease 2019). To understand the pathobiology of SARS-CoV-2 in humans it is necessary to unravel the metabolic changes that are produced in the individuals once the infection has taken place. The goal of this work is to provide new information about the altered biomolecule profile and with that the altered biological pathways of patients in different clinical situations due to SARS-CoV-2 infection. This is done via metabolomics using HPLC-QTOF-MS analysis of plasma samples at COVID-diagnose from a total of 145 adult patients, divided into different clinical stages based on their subsequent clinical outcome (25 negative controls (non-COVID); 28 positive patients with asymptomatic disease not requiring hospitalization; 27 positive patients with mild disease defined by a total time in hospital lower than 10 days; 36 positive patients with severe disease defined by a total time in hospital over 20 days and/or admission at the ICU; and 29 positive patients with fatal outcome or deceased). Moreover, follow up samples between 2 and 3 months after hospital discharge were also obtained from the hospitalized patients with mild prognosis. The final goal of this work is to provide biomarkers that can help to better understand how the COVID-19 illness evolves and to predict how a patient could progress based on the metabolites profile of plasma obtained at an early stage of the infection. In the present work, several metabolites were found as potential biomarkers to distinguish between the end-stage and the early-stage (or non-COVID) disease groups. These metabolites are mainly involved in the metabolism of carnitines, ketone bodies, fatty acids, lysophosphatidylcholines/phosphatidylcholines, tryptophan, bile acids and purines, but also omeprazole. In addition, the levels of several of these metabolites decreased to "normal" values at hospital discharge, suggesting some of them as early prognosis biomarkers in COVID-19 at diagnose.

Novel fluorescent-based reporter cell line engineered for monitoring homologous recombination events.

Homologous recombination (HR) faithfully restores DNA double-strand breaks. Defects in this HR repair pathway are associated with cancer predisposition. In genetic engineering, HR has been used extensively to study gene function and it represents an ideal method of gene therapy for single gene disorders. Here, we present a novel assay to measure HR in living cells. The HR substrate consisted of a non-fluorescent 3' truncated form of the eGFP gene and was integrated into the AAVS1 locus, known as a safe harbor. The donor DNA template comprised a 5' truncated eGFP copy and was delivered via AAV particles. HR mediated repair restored full-length eGFP coding sequence, resulting in eGFP+ cells. The utility of our assay in quantifying HR events was validated by exploring the impact of the overexpression of HR promoters and the siRNA-mediated silencing of genes known to play a role in DNA repair on the frequency of HR. We conclude that this novel assay represents a useful tool to further investigate the mechanisms that control HR and test continually emerging tools for HR-mediated genome editing.

A new MALDI-TOF approach for the quick sequence type identification of Legionella pneumophila.

BACKGROUND: Recently, MALDI-TOF has emerged as a quick tool for bacterial typing. The aim was to evaluate if MALDI-TOF based typing of Legionella pneumophila can achieve the same discriminatory power as that of the Sequence Based Typing (SBT) method. METHODS: The Sequence Type (ST) was obtained from the 90 strains included in the training set and an in-house MALDI-TOF library based on the Main Spectra Profile (MSP) was generated for the identification of such ST. Then, our library was validated by three procedures: a) creating a dendrogram, b) searching for specific peaks present exclusively in each MSP entry, and c) analysing a validation set composed of 14 strains with known ST. Fully characterized L. pneumophila ATCC 33152, which belongs to ST 36, was used as a control strain. RESULTS: In the training set, 17 strains belonged to ST 1, 1 to ST 20, 63 to ST 22, 1 to ST 146, 6 to ST 578, and 2 to ST 1086. Specific peaks present in each MSPs spectrum, which are considered type-specific biomarkers, ranged from 2 to 11; more concretely, MSP for ST 1 identification shows 2 specific peaks; MSP for ST 20 identification: 9 specific peaks; MSP for ST 22 and ST 36 identification: 11 specific peaks; MSP for ST 146 identification: 5 specific peaks; and MSP for ST 578 and ST 1086 identification: 3 specific peaks. Using the validation set (nine strains belonging to ST 22 and five to ST 1), MALDI-TOF assigned accurately the ST in 30 min per tested strain with a full match. CONCLUSIONS: The ST of L. pneumophila can be identified and reported in few minutes directly from colonies grown on BCYE agar using MALDI-TOF.

Quick identification and epidemiological characterization of Francisella tularensis by MALDI-TOF mass spectrometry.

INTRODUCTION: Currently, Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is being evaluated for its efficacy as a fast bacterial typing tool due to its great speed compared to other molecular methods. In this study, we evaluated MALDI-TOF as a tool for quick identification and typing of Francisella tularensis. MATERIALS AND METHODS: This study encompassed 86 strains from two different geographical origins (Spain and the Czech Republic), which were previously characterised by Pulsed-Field Gel Electrophoresis (PFGE) and Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA). The direct colony method was used for microbial identification. High-quality spectra of the 86 strains were obtained and their main spectra profiles (MSPs) were created for epidemiological typing using MALDI-TOF. Based on the MSPs, principal components were generated and a dendrogram was constructed. An in-house MALDI-TOF library entry was created for each group of PFGE and MLVA strains based on their high-quality spectra. Two dendrograms were obtained using these entries and the unique peaks in each entry were searched. RESULTS: All strains were correctly identified to the species level. No clear divisions were found in the 86-strain dendrogram; however, Spanish and Czech strains appeared separately in dendrograms created using MLVA and PFGE entries. Entries from our in-house MALDI-TOF library revealed 2-4 biomarker peaks for the detection of the five PFGE groups and 1-12 biomarker peaks for the detection of the seven MLVA groups. Finally, two and one specific biomarkers were found in the Czech and Spanish strains, respectively. CONCLUSION: MALDI-TOF can be used to accurately identify F. tularensis strains in less than 15 min. Moreover, data on geographical origin and PFGE and MLVA groups could be obtained in less than one hour after colony growing.

GEHEP 010 study: Prevalence and distribution of hepatitis B virus genotypes in Spain (2000-2016).

OBJECTIVE: To study the prevalence and distribution of HBV genotypes in Spain for the period 2000-2016. METHODS: Retrospective study recruiting 2559 patients from 17 hospitals. Distribution of HBV genotypes, as well as sex, age, geographical origin, mode of transmission, HDV-, HIV- and/or HCV-coinfection, and treatment were recorded. RESULTS: 1924 chronically HBV native Spanish patients have been recruited. Median age was 54 years (IQR: 41-62), 69.6% male, 6.3% HIV-coinfected, 3.1% were HCV-coinfected, 1.7% HDV-co/superinfected. Genotype distribution was: 55.9% D, 33.5% A, 5.6% F, 0.8% G, and 1.9% other genotypes (E, B, H and C). HBV genotype A was closely associated with male sex, sexual transmission, and HIV-coinfection. In contrast, HBV genotype D was associated with female sex and vertical transmission. Different patterns of genotype distribution and diversity were found between different geographical regions. In addition, HBV epidemiological patterns are evolving in Spain, mainly because of immigration. Finally, similar overall rates of treatment success across all HBV genotypes were found. CONCLUSIONS: We present here the most recent data on molecular epidemiology of HBV in Spain (GEHEP010 Study). This study confirms that the HBV genotype distribution in Spain varies based on age, sex, origin, HIV-coinfection, geographical regions and epidemiological groups.

Selective attenuation of Toll-like receptor 2 signalling may explain the atheroprotective effect of sphingosine 1-phosphate.

AIMS: Vascular inflammation is a major atherogenic factor and Toll-like receptor (TLR) 2 ligands, including bacterial and serum lipoproteins, seem to be involved in atherogenesis. On this basis, we analysed the effect of lipoproteins and different lipid components on TLR2-dependent signalling. METHODS AND RESULTS: In TLR2-transfected human embryonic kidney 293 cells and human monocytes, oxidized low-density lipoproteins inhibited nuclear factor (NF)-kappaB-driven transcriptional activity and chemokine gene expression in response to TLR2 ligands. Sphingosine 1-phosphate (S1P) and oxidized palmitoyl-arachidonoyl-phosphatidylcholine, but not lipoprotein-carried lysophospholipids, inhibited TLR2 activation. Silencing experiments in TLR2-transfected 293 cells showed that the S1P-mediated attenuation effect is mediated by S1P receptors type 1 and type 2. To address the physiological significance of these findings, additional experiments were performed in human peripheral blood monocytes and monocyte-derived macrophages. In both cell types, S1P selectively attenuated TLR2 signalling, as NF-kappaB and extracellular signal-regulated kinase activation, but not c-Jun amino terminal kinase phosphorylation, were inhibited by physiologically relevant concentrations of S1P. Moreover, the attenuation of TLR2 signalling was partially reverted by pharmacological inhibition of phosphoinositide 3-kinase (PI3K) and Ras pathways. In addition, S1P inhibited the chemokine gene expression elicited by TLR2, but not by TLR4 ligands. CONCLUSION: These findings disclose a cross-talk mechanism between lipoprotein components and TLR in which engagement of S1P receptors exert selective attenuation of TLR2-dependent activation via PI3K and Ras signalling. A corollary to these data is that the negative cross-talk of S1P receptors and TLR2 signalling might be involved in the atheroprotective effects of S1P.

Epidemiological surveillance and wild-type MIC distribution of Legionella pneumophila in north-western Spain. 2003-2016.

PURPOSE: To perform epidemiological surveillance of Legionella pneumophila in recreational swimming pools in the city of Valladolid (Spain), an area with a continental climate and low incidence of legionella-associated infections. Additionally, wild-type minimum inhibitory concentration (MIC) distributions for eight antibiotics commonly used for the treatment of legionellosis were calculated from the isolates obtained. METHODS: Twelve recreational pools were enrolled between June 2003 and December 2016 and 7221 water samples were taken from three different points of the water network (tank, tap and shower). Legionella culture was performed according to ISO 11731 and 11731-2 standards. MICs of antibiotics were obtained by a gradient test. RESULTS: 1.44% of the water samples were positive for L. pneumophila. 60 strains (57.69%) were isolated from showers, 26 (25.00%) from tanks and 18 (17.31%) from taps. L. pneumophila counts were <100CFU/L in 75 samples (72.12%), 100-1000CFU/L in 17 (16.35%) and >1000CFU/L in 12 (11.54%). The MIC90 values obtained were for Rifampicin 0.125mg/L; Trimethoprim-Sulfamethoxazole 0.25mg/L; Azithromycin and Levofloxacin 0.5mg/L; Clarithromycin and Ciprofloxacin 1.0mg/L; Doxycycline and Tigecycline 4.0mg/L. CONCLUSIONS: The use of showers in recreational pools can become a potential pathway for exposure to L. pneumophila, even in cold climates. The wild-type MIC distributions presented in this article may be useful for a better detection of antibiotic resistance and can contribute to improvements in the choice of the antibiotic treatment of legionellosis.