Search

Background Anti‐SARS‐CoV‐2 S antibodies prevent viral replication. Critically ill COVID‐19 patients show viral material in plasma, associated with a dysregulated host response. If these antibodies influence survival and viral dissemination in ICU‐COVID patients is unknown. Patients/Methods We studied the impact of anti‐SARS‐CoV‐2 S antibodies levels on survival, viral RNA‐load in plasma, and N‐antigenaemia in 92 COVID‐19 patients over ICU admission. Results Frequency of N‐antigenaemia was >2.5‐fold higher in absence of antibodies. Antibodies correlated inversely with viral RNA‐load in plasma, representing a protective factor against mortality (Adjusted HR [CI 95%], p): (S IgM [AUC ≥ 60]: 0.44 [0.22; 0.88], 0.020); (S IgG [AUC ≥ 237]: 0.31 [0.16; 0.61]

The role of neutrophil and lymphocyte counts in blood as prognosis predictors in Community Acquired Pneumonia (CAP) has not been adequately studied. This was a derivation-validation retrospective study in hospitalized patients with CAP and no prior immunosuppression. We evaluated by multivariate analysis the association between neutrophil and lymphocyte counts and mortality risk at 30-days post hospital admission in these patients. The derivation cohort (n = 1550 patients) was recruited in a multi-site study. The validation cohort (n = 2846 patients) was recruited in a single-site study. In the derivation cohort, a sub-group of lymphopenic patients, those with

Background While major progress has been made to establish diagnostic tools for the diagnosis of SARS-CoV-2 infection, determining the severity of COVID-19 remains an unmet medical need. With limited hospital resources, gauging severity would allow for some patients to safely recover in home quarantine while ensuring sicker patients get needed care. We discovered a 5 host mRNA-based classifier for the severity of influenza and other acute viral infections and validated the classifier in COVID-19 patients from Greece. Methods We used training data (N=705) from 21 retrospective clinical studies of influenza and other viral illnesses. Five host mRNAs from a preselected panel were applied to train a logistic regression classifier for predicting 30-day mortality in influenza and other viral illnesses. We then applied this classifier, with fixed weights, to an independent cohort of subjects with confirmed COVID-19 from Athens, Greece (N=71) using NanoString nCounter. Finally, we developed a proof-of-concept rapid, isothermal qRT-LAMP assay for the 5-mRNA host signature using the QuantStudio 6 qPCR platform. Results In 71 patients with COVID-19, the 5 mRNA classifier had an AUROC of 0.88 (95% CI 0.80-0.97) for identifying patients with severe respiratory failure and/or 30-day mortality (Figure 1). Applying a preset cutoff based on training data, the 5-mRNA classifier had 100% sensitivity and 46% specificity for identifying mortality, and 88% sensitivity and 68% specificity for identifying severe respiratory failure. Finally, our proof-of-concept qRT-LAMP assay showed high correlation with the reference NanoString 5-mRNA classifier (r=0.95). Figure 1. Validation of the 5-mRNA classifier in the COVID-19 cohort. (A) Expression of the 5 genes used in the logistic regression model in patients with (red) and without (blue) mortality. (B) The 5-mRNA classifier accurately distinguishes non-severe and severe patients with COVID-19 as well as those at risk of death. Conclusion Our 5-mRNA classifier demonstrated very high accuracy for the prediction of COVID-19 severity and could assist in the rapid, point-of-impact assessment of patients with confirmed COVID-19 to determine level of care thereby improving patient management and healthcare burden. Disclosures ljubomir Buturovic, PhD, Inflammatix Inc. (Employee, Shareholder) Purvesh Khatri, PhD, Inflammatix Inc. (Shareholder) Oliver Liesenfeld, MD, Inflammatix Inc. (Employee, Shareholder) James Wacker, n/a, Inflammatix Inc. (Employee, Shareholder) Uros Midic, PhD, Inflammatix Inc. (Employee, Shareholder) Roland Luethy, PhD, Inflammatix Inc. (Employee, Shareholder) David C. Rawling, PhD, Inflammatix Inc. (Employee, Shareholder) Timothy Sweeney, MD, Inflammatix, Inc. (Employee)

Rationale: A depressed expression of antigen presentation is, along with endothelial dysfunction, a recognized signature of severe community-acquired pneumonia (CAP). We aimed to evaluate the expression of a number of genes involved in the immunological synapse in non-critically ill CAP patients with or without organ dysfunction and to profile endothelial biomarkers such as proendothelin-1 (proET1) and proadrenomedullin (proADM). Methods: A nested study in a prospective cohort in CAP patients was performed. Expression levels of major histocompatibility complex class II DR alpha (HLA-DRA), CD40 ligand (CD40LG), CD3E, CD28, and inducible T-cell costimulator (ICOS) were quantified by using droplet digital polymerase chain reaction and endothelial biomarkers by immunofluorescence. Results: Ninety-four patients were included, 44.7% of whom had organ failure in one or more organs. A significant decrease in the expression of the five genes with increased levels of proadrenomedullin (proADM) and proendothelin-1 (proET1) was found in CAP with organ failure. The depressed expression of HLA-DRA (odds ratio (OR), 2.94), CD40LG (OR, 3.90), and CD28 (OR, 3.48) was independently associated with organ failure after adjustment for age, Charlson score, and severity. Conclusions. CAP with organ failure showed depressed expression of immunological synapse genes with increased levels of biomarkers denoting endothelial damage. Simultaneous profiling of immunological and endothelial signatures could help in the early identification of organ failure in CAP and in the implementation of personalized treatment.

Item does not contain fulltext Increasing evidence supports a central role of the immune system in sepsis, but the current view of how sepsis affects immunity, and vice versa, is still rudimentary. The European Group on Immunology of Sepsis has identified major gaps that should be addressed with high priority, such as understanding how immunological alterations predispose to sepsis, key aspects of the immunopathological events during sepsis, and the long-term consequences of sepsis on patient's immunity. We discuss major unmet topics in those three categories, including the role of key immune cells, the cause of lymphopenia, organ-specific immunology, the dynamics of sepsis-associated immunological alterations, the role of the microbiome, the standardisation of immunological tests, the development of better animal models, and the opportunities offered by immunotherapy. Addressing these gaps should help us to better understand sepsis physiopathology, offering translational opportunities to improve its prevention, diagnosis, and care.

Background: Ventilator-associated pneumonia (VAP) is one of the most commonly encountered intensive care unit (ICU) acquired infections worldwide. The objective of the study was to identify the immune alteration occurring in patients suffering from VAP at the transcriptomic level and explore its potential use for clinical diagnoses of this disease. Methods: We performed a prospective observational study in five medical ICUs. Immunological gene expression profiles in the blood of VAP patients were compared with those of controls by using whole transcriptome microarrays and droplet digital polymerase chain reaction (ddPCR) in the first 24 hours following diagnosis. Results: VAP patients showed significantly lower expression levels of HLA-DOA, HLA-DMA, HLA-DMB, ICOS, ICOSLG, IL2RA, CD1, CD3, CD28 and CD40LG. The molecules coded by these genes participate of the immunological synapse. CD1C, CD40LG and ICOS showed the highest values of area under the receiver operating characteristic curve (AUROC) with a good balance between sensibility and specificity. Conclusions: Patients with VAP show a transcriptomic depression of genes participating of the immunological synapse. It takes a commonplace event, namely VAP, and highlights a quite significant underlying immune suppressive state. In effect this small study will change how we regard VAP, and proposes that we regard it as an infection in an immune compromised host, and that immunity has a central role for ICU acquired infections. This may in time change clinical practice, as it has profound implications for the role of protocolised care, or bundles, in the prevention of VAP. Quantifying the expression in blood of this genes using ddPCR could be a useful approach for the diagnosis of VAP.

Acute vascular endothelial dysfunction is a central event in the pathogenesis of sepsis,increasing vascular permeability, promoting activation of the coagulation cascade, tissue edema and compromising perfusion of vital organs. Aging and chronic diseases(hypertension,dyslipidaemia,diabetes mellitus,chronic kidney disease,cardiovascular disease,cerebrovascular disease, chronic pulmonary disease,liver disease or cancer)are recognized risk factors for sepsis. In this article we review the features of endothelial dysfunction shared by sepsis,aging and the chronic conditions preceding this disease. Clinical studies and review articles on endothelial dysfunction associated to sepsis,aging and chronic diseases published in PubMed were considered. The main features of endothelial dysfunction shared by sepsis,aging and chronic diseases were 1.increased oxidative stress and systemic inflammation, 2.glycocalyx degradation and shedding, 3.disassembly of intercellular junctions,endothelial cell death,blood tissue barrier disruption, 4.enhanced leukocyte adhesion and extravasation, 5.induction of a pro-coagulant and anti-fibrinolytic state. In addition,chronic diseases impair the mechanisms of endothelial reparation. In conclusion,sepsis,aging and chronic diseases induce similar features of endothelial dysfunction. The potential contribution of the pre-existent degree of endothelial dysfunction to sepsis pathogenesis deserves to be further investigated, Comment: Review article

Summary Objectives Sepsis is characterised by the frequent presence of organ failure and marked immunologic alterations. We studied the association between the extent of organ failure and the transcriptomic response of septic patients. Methods Gene expression profiles in the blood of 74 surgical patients with sepsis were compared with those of 30 surgical patients with no sepsis. Differentially expressed genes were assessed for their correlation with the sequential organ failure (SOFA) score. Results The expression levels of a group of genes participating in the cell cycle (HIST1H1C, CKS2, CCNA2, CDK1, CCNB2, CIT, CCNB1, AURKA, RAD51), neutrophil protease activity (ELANE, ADORA3, MPO, MMP8, CTSG), IL-1R and IL-18R response correlated directly with SOFA and mortality. Genes involved in T cell (LCK, CD3G, CD3D, ZAP70, ICOS, CD3E, CD28, IL2RB, CD8B, CD8A, CD40LG, IL23A, CCL5, SH2D1A, ITK, CD247, TBX21, GATA3, CCR7, LEF1, STAT4) and NK cell immunity (CD244, KLRK1, KLRD1) were inversely associated with SOFA and mortality. Conclusions The extent of organ failure in sepsis correlates directly with the existence of imbalanced innate and adaptive responses at the transcriptomic level. Quantification of the expression levels of the genes identified here could contribute to the simultaneous assessment of disease severity and immunological alterations in sepsis.

Diphtheria antitoxin for therapeutic use is in limited supply. A potential source might be affinity-purified antibodies originally derived from plasma of adults who received a booster dose of a vaccine containing diphtheria toxoid. These antibodies might be useful for treating even severe cases of diphtheria.

To quantify immunological dysfunction in surgical patients with presence/absence of sepsis using a droplet digital polymerase chain reaction (ddPCR) transcriptomic analysis. The study also aims to evaluate this approach for improving identification of sepsis in these patients.Immune dysregulation is a central event in sepsis. Quantification of the expression of immunological genes participating in the pathogenesis of sepsis could represent a new avenue to improve its diagnosis.Expression of 6 neutrophil protease genes (MMP8, OLFM4, LCN2/NGAL, LTF, PRTN3, MPO) and also of 5 genes involved in the immunological synapse (HLA-DRA, CD40LG, CD3E, CD28, ICOS) was quantified in blood from 101 surgical patients with sepsis, 53 uninfected surgical patients, and 16 blood donors by using ddPCR. Areas under receiver operating characteristic curves (AUROC) and multivariate regression analysis were employed to test individual genes and gene ratios to identify sepsis, in comparison with procalcitonin.Sepsis-induced overexpression of neutrophil protease genes and depressed expression of immunological synapse genes. MMP8/HLA-DRA, LCN2/HLA-DRA outperformed procalcitonin in differentiating between patients with sepsis and surgical controls in the AUROC analysis: LCN2/HLA-DRA: 0.90 (0.85-0.96), MMP8/HLA-DRA: 0.89 (0.84-0.95), procalcitonin: 0.80 (0.73-0.88) (AUROC, confidence interval 95%), and also in the multivariate analysis: LCN2/HLA-DRA: 8.57 (2.25-32.62); MMP8/HLA-DRA: 8.03 (2.10-30.76), procalcitonin: 4.20 (1.15-15.43) [odds ratio (confidence interval 95%)]. Gene expression levels of HLA-DRA were an independent marker of hospital mortality.Quantifying the transcriptomic ratios MMP8/HLA-DRA, LCN2/HLA-DRA by ddPCR is a promising approach to improve sepsis diagnosis in surgical patients.

Objective The impact of endogenous immunoglobulin isotypes on the prognosis of patients with severe sepsis has not been sufficiently explored. The aim of this study was to evaluate the association between immunoglobulin levels in plasma and survival in patients with this condition. Design and patients A prospective multicentre cohort study was conducted. A total of 172 adult patients admitted to the intensive care unit (ICU) with severe sepsis or septic shock were recruited. Patients were classified based on deciles of immunoglobulin concentrations at diagnosis of sepsis. Categorical variables were created and tested for their association with survival during hospitalization in the ICU. Results Overall, 42 patients died in the ICU during the study. Kaplan–Meier analysis showed that immunoglobulin concentrations below 300 mg dL−1 for IgG1, 35 mg dL−1 for IgM and 150 mg dL−1 for IgA were associated with shorter survival times. Multivariate regression analysis showed that IgG1 < 300 mg dL−1 was a risk factor for mortality [odds ratio (OR) 2.50, 95% confidence interval (CI) 1.04–6.03; P = 0.042]. The combined presence of IgG1, IgM and IgA levels below the described thresholds had a synergistic impact on mortality risk (OR 5.27, 95% CI 1.41–19.69; P = 0.013). A similar effect was observed for combined low levels of IgG1 and IgA (OR 4.10, 95% CI 1.28–13.12; P = 0.018) and also of IgG1 and IgM (OR 3.10. 95% CI 1.13–8.49; P = 0.028). Conclusions The combined presence of low levels of the endogenous immunoglobulins IgG1, IgM and IgA in plasma is associated with reduced survival in patients with severe sepsis or septic shock. Assessment of the concentrations of these immunoglobulins could improve the results of treatment with exogenous immunoglobulins in patients with sepsis.

Purpose There is increasing evidence on the relationship between endogenously produced immunoglobulins and the clinical outcome in septic shock (SS). Materials and methods Levels of immunoglobulin G (IgG) subclasses, immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin E were measured in plasma from 42 patients with SS and in 36 patients with systemic inflammatory response syndrome at diagnosis. Association of immunoglobulins levels with disease severity and outcome was evaluated. Results Eighteen patients with SS finally died. Both patients with systemic inflammatory response syndrome and SS showed subnormal levels of total IgG, IgG2, and IgM. Patients with SS who died showed the lowest levels of total IgG and IgG1. Total IgG, IgG1, IgG2, IgG3, IgG4, and IgA correlated inversely with Acute Physiology and Chronic Health Evaluation II score in SS. Univariate Cox regression analysis showed that levels of IgG1, IgG2, IgG3, IgM, IgA, and total IgG were inversely associated to the probability of death at 28 days. Multivariate analysis showed that IgG1, total IgG, IgM, and IgA behaved as independent protective factors against mortality (hazard ratio, P ): 0.23, 0.026; 0.16, 0.028; 0.11, 0.042; 0.05, 0.010, respectively, whereas IgG3 showed a protective trend also. Conclusions Our study evidenced that, in addition to IgG1, other major endogenous immunoglobulins isotypes and subclasses seem to play a beneficial role in SS.

Conventional clinical parameters are not sensitive or specific enough for detecting ongoing disease activity in the Systemic Lupus Erythematosus (SLE). Measurement of cytokines in urine is an encouraging approach to detection of early flares in this disease. Here we have profiled 27 different cytokines, chemokines and celular growth factors in the urine of 48 patients previously diagnosed of SLE as potential biomarkers of disease activity. Correlation analysis with Bonferroni correction showed that MCP-1 was the only immune mediator which levels in urine correlated directly with the SLE Disease Activity Index 2000 (SLEDAI-2K) score (correlation coefficient, p): MCP-1 (0.45,0.003). MCP-1 correlated inversely with levels of C3 complement protein in serum (-0.50,0.001). MCP-1 showed significant higher levels in patients with severe disease activity in comparison with those exhibiting mild activity. Levels of this chemokine were also higher in patients with severe disease activity in comparison with patients with inactive disease and healthy controls. Areas under receiver operating characteristic curves (AUROC) for detection of severe disease (SLEDAI⩾8) was as follows for MCP-1: [AUROC, (IC95%), p]: [0.81 (0.65-0.96) 0.003]. In addition, MCP-1 showed a good result in the AUROC analysis for detecting renal involvement [0.70 (0.52-0.87) 0.050]. When correlation analysis were repeated excluding those patients with active renal disease (n=14), levels of MCP-1 in urine kept on showing a significant positive association with SLEDAI-2K score. In conclusion, multiplex-based cytokine profiling in urine demonstrated the superiority of MCP-1 over a wide range of cytokines as biomarker of disease activity in SLE.

Three years after the pandemic, major advances have been made in our understanding of the innate and adaptive immune responses to the influenza A(H1N1)pdm09 virus and those responses' contribution to the immunopathology associated with this infection. Severe disease is characterized by early secretion of proinflammatory and immunomodulatory cytokines. This cytokine secretion persisted in patients with severe viral pneumonia and was directly associated with the degree of viral replication in the respiratory tract. Cytokines play important roles in the antiviral defense, but persistent hypercytokinemia may cause inflammatory tissue damage and participate in the genesis of the respiratory failure observed in these patients. An absence of pre-existing protective antibodies was the rule for both mild and severe cases. A role for pathogenic immunocomplexes has been proposed for this disease. Defective T cell responses characterize severe cases of infection caused by the influenza A(H1N1)pdm09 virus. Immune alterations associated with accompanying conditions such as obesity, pregnancy or chronic obstructive pulmonary disease may interfere with the normal development of the specific response to the virus. The role of host immunogenetic factors associated with disease severity is also discussed in this review. In conclusion, currently available information suggests a complex immunological dysfunction/alteration that characterizes the severe cases of 2009 pandemic influenza. The potential benefits of prophylactic/therapeutic interventions aimed at preventing/correcting such dysfunction warrant investigation.

The development of new diagnostic methods based on molecular biology has led to evidence of the important role of respiratory viruses in chronic obstructive pulmonary disease (COPD) exacerbations. Cytokines and chemokines are recognized as key actors in the pathogenesis of COPD. The objective of this study was to evaluate the association between viral infection and host cytokine responses in 57 COPD patients hospitalized with an acute exacerbation. Seventeen cytokines were profiled using a Luminex-Biorad multiplex assay in plasma samples collected in the first 24 h following hospital admission. Stepwise linear regression analysis was performed, taking into account the influence of seven potential confounding factors in the results. Twenty-four out of 57 showed radiological signs of community-acquired pneumonia (CAP) at hospital admission, 25 patients required admission to the intensive care unit (ICU), 20 had a bacterial infection, and 20 showed a detectable respiratory virus in pharyngeal swabs. Regression analysis showed that viral infection correlated with higher levels of interleukin-6 (IL-6) (log value of the coefficient of regression B, p=0.47, 0.044), and monocyte chemoattractant protein-1 (MCP-1) (p=0.43, 0.019), and increased admission to the ICU. Viral infection also correlated with higher levels of interferon-γ (IFN-γ) (p=0.70, 0.026), which, in turn, was inversely associated with the severity of illness. Finally, viral infection was independently associated with higher levels of tumor necrosis factor-α (TNF-α) (p=0.40, 0.002). Thus our study demonstrates that in patients with COPD exacerbations, viral infection is directly associated with higher systemic levels of cytokines central to the development of the antiviral response, which are also known to contribute to inflammation-mediated tissue damage. These results reveal a potential specific role of viral infection in the pathogenesis of COPD exacerbations.

Viruses are now thought to play an important role in chronic obstructive pulmonary disease pathogenesis. Viral infection and adaptive immunity show complex interactions in this disease. Tobacco, pollutants, immunosenescence, and chronic intake of steroids alter the adaptive immune response. Impairment of the adaptive response potentially predisposes to infection by respiratory viruses. Viral infection in turn impacts on the immune response, increasing the risk of bacterial infections, inducing disease impairment and chronic illness. The immunologic basis and clinical implications of this complex relationship are discussed in this study.

Summary Objective As reported by WHO in its most recent report, H5N1 influenza virus affects mostly children. Differences in morbidity depending on the age could be explained, at least in part, by the differences in the immune response between children and adults. The main objective of the study was to evaluate the effect of H5N1 influenza haemagglutinin on the cytokine secretion profiles of peripheral blood mononuclear cells (PBMCs) from both children and adult healthy donors. Materials and methods We compared the profiles of 19 cytokines and chemokines released after exposure to PBMCs obtained from 30 healthy children and 30 healthy adults to a recombinant glycosylated H5 haemagglutinin after 24 h of culture with this antigen, compared to untreated control cells. Results Donors had not been exposed previously to the virus, as evidenced by the absence of haemagglutination inhibition activity in their plasma samples. Direct contact of PBMCs from both children and adults with the haemagglutinin of H5N1 virus induced increased levels of IL-6, MCP-1 and MIP-1β in the culture supernatants compared to control. Supernatants of adult PBMCs also showed increased levels of IL-8 and IP-10. Conclusions The results of our study supports the idea that, in the absence of protective immunity, the haemagglutinin of the H5N1 virus is able to induce the release of pro-inflammatory mediators by direct contact with PBMCs. Some of these mediators (IL-6, MCP-1 and MIP-1β) are induced regardless of age and have been previously reported to be associated with a poor control of viral replication in severe patients and animal models.

The pathogenesis of systemic lupus erythematosus (SLE) is far from having been elucidated at the molecular level. Using a multiplex system, we profiled 18 immune mediators in the plasma from 57 patients with SLE. Thirteen of them showed mild to moderate disease activity, and 29 showed severe activity, based upon the SLEDAI score. Fifteen patients were in complete clinical remission. Those patients with active disease, and those in clinical remission had been undergoing immunomodulatory treatment for an average of 10.7 months and 19.2 months respectively at the time of the visit. Samples obtained from 10 healthy volunteers were used as control. Patients with active disease and those with inactive disease showed elevated levels of the chemoattractant proteins MCP-1/CCL2, MIP1-beta/CCL4 and IL-8/CXCL8 as compared to the control (p < 0.05). This pattern of increased mediator levels was observed regardless of the immunomodulatory drug regimen received (non-steroidal anti-inflammatory, steroids or immunosuppressants), and of the degree of tissue damage. Patients with anticardiolipin antibodies (ACAs) showed significantly higher levels of IL-8 and MIP-1beta than those with no ACAs. Levels of MCP-1/CCL2, MIP1-beta/CCL4 and IL-8/CXCL8 correlated significantly, indicating a coordinated regulation of their secretion. Conversely, levels of Th1, Th2, Th17 cytokines, IFN-gamma and growth factors did not differ from those found in the healthy controls. IFN-alpha, IL-1beta, IL-6, IL-7 and IL-13 were undetectable. In conclusion, long term treatment of SLE with standard immunomodulatory drug regimens fails to normalize levels of key chemoattractant proteins linked to innate immunity. This might suggest the existence of a basal, pro-inflammatory state in patients with lupus, even in the absence of symptoms, which could serve as a "substratum" or initiator of the immunological events taking place during a flare-up of the disease.

Summary Objectives Immunological dysregulation is now recognised as a major pathogenic event in sepsis. Stimulation of immune response and immuno-modulation are emerging approaches for the treatment of this disease. Defining the underlying immunological alterations in sepsis is important for the design of future therapies with immuno-modulatory drugs. Methods Clinical studies evaluating the immunological response in adult patients with Sepsis and published in PubMed were reviewed to identify features of immunological dysfunction. For this study we used key words related with innate and adaptive immunity. Results Ten major features of immunological dysfunction (FID) were identified involving quantitative and qualitative alterations of [antigen presentation](FID1), [T and B lymphocytes] (FID2), [natural killer cells] (FID3), [relative increase in T regulatory cells] (FID4), [increased expression of PD-1 and PD-ligand1](FID5), [low levels of immunoglobulins](FID6), [low circulating counts of neutrophils and/or increased immature forms in non survivors](FID7), [hyper-cytokinemia] (FID8), [complement consumption] (FID9), [defective bacterial killing by neutrophil extracellular traps](FID10). Conclusions This review article identified ten major features associated with immunosuppression and immunological dysregulation in sepsis. Assessment of these features could help in utilizing precision medicine for the treatment of sepsis with immuno-modulatory drugs.

20 p.- 5 fig.- 1 tab., Background: The interaction between influenza virus and the host response to infection clearly plays an important role in determining the outcome of infection. While much is known on the participation of inflammation on the pathogenesis of severe A (H1N1) pandemic 09-influenza virus, its role in the course of non-fatal pneumonia has not been fully addressed., Methods: A systems biology approach was used to define gene expression profiles, histology and viral dynamics in the lungs of healthy immune-competent mice with pneumonia caused by a human influenza A (H1N1) pdm09 virus, which successfully resolved the infection., Results: Viral infection activated a marked pro-inflammatory response at the lung level paralleling the emergence of histological changes. Cellular immune response and cytokine signaling were the two signaling pathway categories more representative of our analysis. This transcriptome response was associated to viral clearance, and its resolution was accompanied by resolution of histopathology., Discussion: These findings suggest a dual role of pulmonary inflammation in viral clearance and development of pneumonia during non-fatal infection caused by the 2009 pandemic influenza virus. Understanding the dynamics of the host's transcriptomic and virological changes over the course of the infection caused by A (H1N1) pdm09 virus may help identifying the immune response profiles associated with an effective response against influenza virus., This work was supported by Instituto de Salud Carlos III: "Programa de Investigacion Comisionada en Gripe (GR09/0021) and Programa para favorecer la incorporacion de grupos de investigacion en las Instituciones del Sistema Nacional de Salud" (EMER07/050). This work was also supported by Biotechnology and Biological Sciences Research Council (BBSRC) grant (BBS/E/I/00002014). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Mechanisms underlying multiple sclerosis (MS) fatigue and the causes of the beneficial effect of exercise on this symptom are not clarified. Our aim was to evaluate gene expression profiles in MS patients who improved their fatigue status after an exercise program and to compare them with healthy controls (HC). Gene expression in whole blood was profiled at baseline in 7 HC and also in 7 fatigued-MS patients. Patients underwent an exercise program for 6 months, and their fatigue status and gene expression profiles were again analyzed. MS patients showed a significant activation of genes participating in the systemic interferon response in comparison with HC that disappeared at the end of the program. Our results provide a biological basis for the observed benefit of exercise in MS.

Little is known on the participation of immunoglobulin isotypes and subclasses in the pathogenesis of the severe disease caused by the pandemic influenza virus (influenza A(H1N1)pdm09).(1) To evaluate the association between plasma levels of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and outcome in patients with severe pandemic influenza. (2) To evaluate the association between immunoglobulin and cytokine levels in these patients.40 critically ill patients with community acquired pneumonia and influenza A(H1N1)pdm09 infection were recruited from November 2010 to February 2011. Plasma samples were collected during the first 24h following admission to the ICU. Immunoglobulins and 17 major cytokines were profiled in plasma.15 patients died (37.5%). When the association between clinical variables and prognosis was assessed, prior immunosuppression, APACHE II score, levels of IgG2 and levels of IgM were associated with outcome in a univariate Cox regression analysis. Kaplan Meier analysis showed that patients with levels of IgG2 and IgM59 and58 mg/dl respectively died earlier. Multivariate Cox regression analysis showed that APACHE II score and levels of IgM were the best predictors of outcome, being levels of IgM a protective factor against mortality. IgM was the immunoglobulin showing the largest number of negative correlations with cytokine levels.Our results support a central role of IgM in preventing uncontrolled inflammatory response and mortality in severe pandemic influenza. Early assessment of IgM could contribute to guide clinical decisions in these patients.

Producción Científica, Cell counts of leukocytes subpopulations are demonstrating to have an important value in predicting outcome in severe infections. We evaluated here the render of leukogram counts to predict outcome in patients with ventilator-associated pneumonia (VAP) caused by Staphylococcus aureus. Data from patients admitted to the ICU of Hospital Clínico Universitario de Valladolid from 2006 to 2011 with diagnosis of VAP caused by S. aureus were retrospectively collected for the study (n = 44). Leukocyte counts were collected at ICU admission and also at VAP diagnosis. Our results showed that nonsurvivors had significant lower eosinophil counts at VAP diagnosis. Multivariate Cox regression analysis performed by the Wald test for forward selection showed that eosinophil increments from ICU admission to VAP diagnosis and total eosinophil counts at VAP diagnosis were protective factors against mortality in the first 28 days following diagnosis: (HR [CI 95%], P): (0.996 [0.993–0.999], 0.010); (0.370 [0.180–0.750], 0.006). Patients with eosinophil counts

Pandemic H1N1 influenza A (H1N1pdm) is currently a dominant circulating influenza strain worldwide. Severe cases of H1N1pdm infection are characterized by prolonged activation of the immune response, yet the specific role of inflammatory mediators in disease is poorly understood. The inflammatory cytokine IL-6 has been implicated in both seasonal and severe pandemic H1N1 influenza A (H1N1pdm) infection. Here, we investigated the role of IL-6 in severe H1N1pdm infection. We found IL-6 to be an important feature of the host response in both humans and mice infected with H1N1pdm. Elevated levels of IL-6 were associated with severe disease in patients hospitalized with H1N1pdm infection. Notably, serum IL-6 levels associated strongly with the requirement of critical care admission and were predictive of fatal outcome. In C57BL/6J, BALB/cJ, and B6129SF2/J mice, infection with A/Mexico/4108/2009 (H1N1pdm) consistently triggered severe disease and increased IL-6 levels in both lung and serum. Furthermore, in our lethal C57BL/6J mouse model of H1N1pdm infection, global gene expression analysis indicated a pronounced IL-6 associated inflammatory response. Subsequently, we examined disease and outcome in IL-6 deficient mice infected with H1N1pdm. No significant differences in survival, weight loss, viral load, or pathology were observed between IL-6 deficient and wild-type mice following infection. Taken together, our findings suggest IL-6 may be a potential disease severity biomarker, but may not be a suitable therapeutic target in cases of severe H1N1pdm infection due to our mouse data., This work was supported by the Li Ka Shing Foundation and Immune Diagnostics & Research. Dr. Alyson A. Kelvin is the Scientific Director for Immune Diagnostics & Research and had input into experimental design and write-up of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Background Gene expression profiling (GEP) in cells obtained from peripheral blood has shown that this is a very useful approach for biomarker discovery and for studying molecular pathogenesis of prevalent diseases. While there is limited literature available on gene expression markers associated with Chronic Obstructive Pulmonary Disease (COPD), the transcriptomic picture associated with critical respiratory illness in this disease is not known at the present moment. Findings By using Agilent microarray chips, we have profiled gene expression signatures in the whole blood of 28 COPD patients hospitalized with different degrees of respiratory compromise.12 of them needed of admission to the ICU, whilst 16 were admitted to the Respiratory Medicine Service. GeneSpring GX 11.0 software was used for performing statistical comparisons of transcript levels between ICU and non-ICU patients. Ingenuity pathway analysis 8.5 (IPA) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to select, annotate and visualize genes by function and pathway (gene ontology). T-test showed evidence of 1501 genes differentially expressed between ICU and non-ICU patients. IPA and KEGG analysis of the most representative biological functions revealed that ICU patients had increased levels of neutrophil gene transcripts, being [cathepsin G (CTSG)], [elastase, neutrophil expressed (ELANE)], [proteinase 3 (PRTN3)], [myeloperoxidase (MPO)], [cathepsin D (CTSD)], [defensin, alpha 3, neutrophil-specific (DEFA3)], azurocidin 1 (AZU1)], and [bactericidal/permeability-increasing protein (BPI)] the most representative ones. Proteins codified by these genes form part of the azurophilic granules of neutrophils and are involved in both antimicrobial defence and tissue damage. This “neutrophil signature” was paralleled by the necessity of advanced respiratory and vital support, and the presence of bacterial infection. Conclusion Study of transcriptomic signatures in blood suggests an essential role of neutrophil proteases in COPD patients with critical respiratory illness. Measurement and modulation of the expression of these genes could present an option for clinical monitoring and treatment of severe COPD exacerbations.

It remains unknown why some intubated patients remain infection-free while others develop tracheobronchitis (VAT) or pneumonia (VAP).To identify and compare VAP/VAT gene expression "signatures" using genome-wide oligonucleotide microarrays.A prospective translational study of gene expression profiles of VAP and VAT groups was carried out, establishing comparisons in both pre-infection and infection phases. Pathway and functional analyses were performed with Ingenuity Pathway Analysis (IPA). Data analysis and hierarchical clustering of the genes involved in the signalling pathways expressed differentially in the two groups were performed with GeneSpring GX 11.0.Eight patients developing respiratory infections (3 VAP and 5 VAT) after 4 days of mechanical ventilation were assessed. Comparison of gene expression profiles in the pre-infection period revealed 5595 genes expressed differentially between VAP and VAT (p0.01, fold change2). Comparative IPA analysis identified a significant depression of the complement system signalling pathway in the VAP group, affecting the classical pathway along with the final common pathway (p0.05). In addition, the cAMP and calcium signalling pathways were also significantly depressed in the VAP group during the pre-infection phase also.Intubated patients complicated with pneumonia developed immune impairment in the pre-infection period, manifesting as a relatively lower expression of genes involved in the complement system that differed from patients developing tracheobronchitis. These findings suggest that a significant proportion of VAP episodes cannot be prevented, but might be treatable through pre-emptive therapy.

Identification of patients at increased risk of death is dramatically important in severe sepsis. Cytokines have been widely assessed as potential biomarkers in this disease, but none of the cytokines studied has evidenced a sufficient specificity or sensitivity to be routinely employed in clinical practice. In this pilot study, we profiled 17 immune mediators in the plasma of 29 consecutively recruited patients with severe sepsis or septic shock, during the first 24h following admission to the ICU, by using a Bio-Plex Human Cytokine 17-Plex Panel (Bio-Rad). Patients were 66.1year old in average. Twelve patients of our cohort died during hospitalization at the ICU, eight of them in the first 72h due to multiorganic dysfunction syndrom (MODS). Levels in plasma of three pro-inflammatory mediators (IL-6, IL-8, MCP-1) and of an immunosuppressive one (IL-10) were higher in those patients with fatal outcome. We developed a combined score with those cytokines showing to better predict mortality in our cohort based on the results of Cox regression analysis. This way, IL-6, IL-8 and IL-10 were included in the score. Patients were split into two groups based on the percentile 75 (P75) of the plasma levels of these three interleukins. Those patients showing at least one interleukin value higher than P75 were given the value "1". Those patients showing IL-6, IL-8, IL-10 levels below P75 were given the value "0". Hazard ratios for mortality at day 3 and day 28th obtained with the combined score were 2-3-fold higher than those obtained with the individual interleukins values. In conclusion, we have described a combined cytokine score associated with a worse outcome in patients with sepsis, which may represent a new avenue to be explored for guiding treatment decisions in this disease.

The relationships between cytokine responses in septic shock are currently poorly understood. Some studies have pointed to a biphasic model, with an initial proinflammatory phase, followed by a reactive, anti-inflammatory response to explain the pathogenesis of the most severe form of sepsis. However, evidence for the coexistence of both responses has been found. In this study, the plasma levels of 17 cytokines and chemokines, in 20 patients with septic shock, 11 patients with systemic inflammatory response syndrome (SIRS), during the first 24 hours following diagnosis, and 10 healthy controls, were analyzed and compared. Patients with septic shock showed increased levels of IL-6, IL-8, MCP-1, MIP-1β, IFN-γ, GM-CSF and IL-10 compared to healthy controls. Patients with SIRS showed higher levels of IL-6, IL-8, MCP-1, MIP-1β, G-CSF and IL-10 than controls. Patients with septic shock showed higher levels of IL-8, GM-CSF, MIP-1β than those with SIRS. The Spearman test demonstrated a positive association between the pro-inflammatory mediators IL-6, IL-8, MCP-1, MIP-1β, IFN-γ, GM-CSF and the immunomodulatory cytokine IL-10 in septic shock. Consequently, correlation studies supported the notion that secretion of pro- and anti-inflammatory mediators in septic shock occurs as a simultaneous immune response program initiated early in the course of the disease, revealing that both types of cytokine play a role from the very beginning of this life-threatening condition.

Viruses are strongly associated with acute exacerbations of chronic obstructive pulmonary disease (AECOPD). Interferon-inducible protein-10 has been recently described as a biomarker of human rhinovirus infection, but there are no reports on the role of other immune mediators in AECOPD of viral origin. As an attempt to evaluate the differences in the systemic immune mediators profiles between AECOPD patients with presence/absence of viral infection, we measured 27 cytokines, chemokines, and cellular growth factors in the plasma of 40 patients with AECOPD needing of hospitalization by using a Luminex-based assay. These patients were screened for the presence of 16 different respiratory viruses in pharyngeal swabs. Ten healthy controls were recruited for comparison purposes. Both the group of patients with an associated viral infection (n = 11) and those with no viral infection (n = 29) showed high levels of vascular endothelial growth factor, interleukin-13 (IL-13), and IL-2. On the other hand, viral infection in AECOPD induced a coordinated response of innate immunity chemokines (eotaxin, interferon-inducible protein-10, IL-8), Th1 cytokines (IL-12p70, IL-15), and the immunomodulatory IL-10. This profile corresponds to a typical antiviral response signature previously documented for other viral infections. The identification of early cytokine signatures associated with viral infection in AECOPD could contribute to design better treatment strategies for this disease.

Background: Severe disease caused by 2009 pandemic influenza A/H1N1virus is characterized by the presence of hypercytokinemia. The origin of the exacerbated cytokine response is unclear. As observed previously, uncontrolled influenza virus replication could strongly influence cytokine production. The objective of the present study was to evaluate the relationship between host cytokine responses and viral levels in pandemic influenza critically ill patients. Methods: Twenty three patients admitted to the ICU with primary viral pneumonia were included in this study. A quantitative PCR based method targeting the M1 influenza gene was developed to quantify pharyngeal viral load. In addition, by using a multiplex based assay, we systematically evaluated host cytokine responses to the viral infection at admission to the ICU. Correlation studies between cytokine levels and viral load were done by calculating the Spearman correlation coefficient. Results: Fifteen patients needed of intubation and ventilation, while eight did not need of mechanical ventilation during ICU hospitalization. Viral load in pharyngeal swabs was 300 fold higher in the group of patients with the worst respiratory condition at admission to the ICU. Pharyngeal viral load directly correlated with plasma levels of the pro-inflammatory cytokines IL-6, IL-12p70, IFN-gamma, the chemotactic factors MIP-1 beta, GM-CSF, the angiogenic mediator VEGF and also of the immuno-modulatory cytokine IL-1ra ( p < 0.05). Correlation studies demonstrated also the existence of a significant positive association between the levels of these mediators, evidencing that they are simultaneously regulated in response to the virus. Conclusions: Severe respiratory disease caused by the 2009 pandemic influenza virus is characterized by the existence of a direct association between viral replication and host cytokine response, revealing a potential pathogenic link with the severe disease caused by other influenza subtypes such as H5N1., The authors would like to thank also to the Nursery teams who kindly collected the samples and to Lucia Rico and Veronica Iglesias for their precious laboratory assistance. The study was scientifically supported by the Spanish Society for Critical Care Medicine (SEMICYUC). Funding: MICCIN-FIS/JCYL-IECSCYL-SACYL (Spain): Programa de Investigacion Comisionada en Gripe, GR09/0021-EMER07/050-PI081236-RD07/0067. This work was also supported by Grants or Contracts from ATCC/CDC USA, and University Health Network and IDR Canada (DJK).

Introduction Human host immune response following infection with the new variant of A/H1N1 pandemic influenza virus (nvH1N1) is poorly understood. We utilize here systemic cytokine and antibody levels in evaluating differences in early immune response in both mild and severe patients infected with nvH1N1. Methods We profiled 29 cytokines and chemokines and evaluated the haemagglutination inhibition activity as quantitative and qualitative measurements of host immune responses in serum obtained during the first five days after symptoms onset, in two cohorts of nvH1N1 infected patients. Severe patients required hospitalization (n = 20), due to respiratory insufficiency (10 of them were admitted to the intensive care unit), while mild patients had exclusively flu-like symptoms (n = 15). A group of healthy donors was included as control (n = 15). Differences in levels of mediators between groups were assessed by using the non parametric U-Mann Whitney test. Association between variables was determined by calculating the Spearman correlation coefficient. Viral load was performed in serum by using real-time PCR targeting the neuraminidase gene. Results Increased levels of innate-immunity mediators (IP-10, MCP-1, MIP-1 beta), and the absence of anti-nvH1N1 antibodies, characterized the early response to nvH1N1 infection in both hospitalized and mild patients. High systemic levels of type-II interferon (IFN-gamma) and also of a group of mediators involved in the development of T-helper 17 (IL-8, IL-9, IL-17, IL-6) and T-helper 1 (TNF-alpha, IL-15, IL-12p70) responses were exclusively found in hospitalized patients. IL-15, IL-12p70, IL- 6 constituted a hallmark of critical illness in our study. A significant inverse association was found between IL- 6, IL- 8 and PaO2 in critical patients. Conclusions While infection with the nvH1N1 induces a typical innate response in both mild and severe patients, severe disease with respiratory involvement is characterized by early secretion of Th17 and Th1 cytokines usually associated with cell mediated immunity but also commonly linked to the pathogenesis of autoimmune/inflammatory diseases. The exact role of Th1 and Th17 mediators in the evolution of nvH1N1 mild and severe disease merits further investigation as to the detrimental or beneficial role these cytokines play in severe illness., This work has been made by an international team pertaining to the Spanish-Canadian Consortium for the Study of Influenza Immunopathogenesis. The authors would like to thank Lucia Rico and Veronica Iglesias for their assistance in the technical development of the multiplex cytokine assays, to Bego a Nogueira for her technical support, and to Nikki Kelvin for language revision of this article. This work was possible thanks to the financial support obtained from the Ministry of Science of Spain and Consejeria de Sanidad Junta de Castilla y Leon, Programa de investigacion comisionada en gripe, GR09/0021, Programa para favorecer la incorporacion de grupos de investigacion en las Instituciones del Sistema Nacional de Salud, EMER07/050, and Proyectos en Investigacion Sanitaria, PI081236. CIHR, NIH and LKSF-Canada support DJK. This sponsorship made possible reagent acquisition and sample transportation between participant groups.

Seasonal influenza viruses are typically restricted to the human upper respiratory tract whereas influenza viruses with greater pathogenic potential often also target extra-pulmonary organs. Infants, pregnant women, and breastfeeding mothers are highly susceptible to severe respiratory disease following influenza virus infection but the mechanisms of disease severity in the mother-infant dyad are poorly understood. Here we investigated 2009 H1N1 influenza virus infection and transmission in breastfeeding mothers and infants utilizing our developed infant-mother ferret influenza model. Infants acquired severe disease and mortality following infection. Transmission of the virus from infants to mother ferrets led to infection in the lungs and mother mortality. Live virus was also found in mammary gland tissue and expressed milk of the mothers which eventually led to milk cessation. Histopathology showed destruction of acini glandular architecture with the absence of milk. The virus was localized in mammary epithelial cells of positive glands. To understand the molecular mechanisms of mammary gland infection, we performed global transcript analysis which showed downregulation of milk production genes such as Prolactin and increased breast involution pathways indicated by a STAT5 to STAT3 signaling shift. Genes associated with cancer development were also significantly increased including JUN, FOS and M2 macrophage markers. Immune responses within the mammary gland were characterized by decreased lymphocyte-associated genes CD3e, IL2Ra, CD4 with IL1β upregulation. Direct inoculation of H1N1 into the mammary gland led to infant respiratory infection and infant mortality suggesting the influenza virus was able to replicate in mammary tissue and transmission is possible through breastfeeding. In vitro infection studies with human breast cells showed susceptibility to H1N1 virus infection. Together, we have shown that the host-pathogen interactions of influenza virus infection in the mother-infant dyad initiate immunological and oncogenic signaling cascades within the mammary gland. These findings suggest the mammary gland may have a greater role in infection and immunity than previously thought., Author Summary Influenza is known as a respiratory infectious disease. Breastfeeding allows for frequent microbial exchange between infant and mother. Although infants, pregnant women and breastfeeding mothers are more susceptible to severe respiratory disease following influenza virus infection, the mechanisms of disease severity in infants and mothers is poorly understood. We were interested in understanding the immune responses, pathogenicity, and transmission dynamics in the infant-mother system. With this aim we developed an infant-mother ferret influenza model. Influenza infection in infants led to virus transmission to mothers causing severe disease and mortality. Unexpectedly, influenza-infected infant ferrets transmitted the virus to mother mammary glands leading to live influenza virus in expressed breast milk. Gene regulation analysis in the mammary gland showed reduction of milk production genes such as Prolactin and increased genes involved with breast shutdown. Genes associated with cancer development were significantly increased including JUN, FOS and BRCA2. We found human breast cells were able to be infected with the influenza virus suggesting the importance of our results to human health. Our data suggests influenza transmission can occur through the mammary glands initiating immunological and pathological events contributing to influenza disease severity impacting infant and maternal health.

The role of systemic immunity in the pathogenesis of cirrhosis is not fully understood. Analysis of transcriptomic profiles in blood is an easy approach to obtain a wide picture of immune response at the systemic level. We studied gene expression profiles in blood from thirty cirrhotic patients and compared them against those of eight healthy volunteers. Most of our patients were male [n = 21, 70%] in their middle ages [57.4 ± 6.8 yr]. Alcohol abuse was the most frequent cause of cirrhosis (n = 22, 73%). Eleven patients had hepatocellular carcinoma (36.7%). Eight patients suffered from hepatitis C virus infection (26.7%). We found a signature constituted by 3402 genes which were differentially expressed in patients compared to controls (2802 over-expressed and 600 under-expressed). Evaluation of this signature evidenced the existence of an active pro-fibrotic transcriptomic program in the cirrhotic patients, involving the [extra-cellular matrix (ECM)-receptor interaction] & [TGF-beta signaling] pathways along with the [Cell adhesion molecules] pathway. This program coexists with alterations in pathways participating in [Glycine, serine and threonine metabolism], [Phenylalanine metabolism], [Tyrosine metabolism], [ABC transporters], [Purine metabolism], [Arachidonic acid metabolism]. In consequence, our results evidence the co-existence in blood of a genomic program mediating pro-fibrotic mechanisms and metabolic alterations in advanced cirrhosis. Monitoring expression levels of the genes involved in these programs could be of interest for predicting / monitoring cirrhosis evolution. These genes could constitute therapeutic targets in this disease.

This item has no abstract. Use the links below to access the full text.

Producción Científica, Evaluation of host immune response to infection at the molecular level is a promising avenue to obtain diagnostic and prognostic tools for the clinical management of patients with sepsis. A recent report from Cajander and colleagues [1] has shown the potential of HLA-DR mRNA quantification by real-time PCR as a biomarker of immunosuppression in these patients. IgM is the first immunoglobulin produced in response to infection. In a pilot study, we have employed a next generation quantitative PCR method (nanoliter-sized droplet technology paired with digital PCR (ddPCR)) for detecting the early transcriptomic response of IgM in blood from patients with sepsis. Approval for the study protocol for both scientific and ethical aspects was obtained from the Committee for Clinical Research of Hospital Clínico Universitario, Valladolid, Spain. Written informed consent was obtained directly from each patient or a legal surrogate. The target gene transcript was IGHM, which encodes the constant region of the mu heavy chain, which defines the IgM isotype [2]. In blood, the cells producing IgM transcripts are B lymphocytes expressing CD20 [3], which was employed as housekeeping gene., Instituto de Salud Carlos III (grant PI13/02110)

The clinical, human and economic burden associated with sepsis is huge. Initiatives such as the Surviving Sepsis Campaign aim to effectively reduce risk of death from severe sepsis and septic shock. Nonetheless, although substantial benefits raised from the implementation of this campaign have been obtained, much work remains if we are to realise the full potential promised by this strategy. A deeper understanding of the processes leading to sepsis is necessary before we can design an effective suite of interventions. Dysregulation of the immune response to infection is acknowledged to contribute to the pathogenesis of the disease. Production of both proinflammatory and immunosuppressive cytokines is observed from the very first hours following diagnosis. In addition, hypogammaglobulinemia is often present in patients with septic shock. Moreover, levels of IgG, IgM and IgA at diagnosis correlate directly with survival. In turn, nonsurvivors have lower levels of C4 (a protein of the complement system) than the survivors. Natural killer cell counts and function also seem to have an important role in this disease. HLA-DR in the surface of monocytes and counts of CD4+CD25+ T-regulatory cells in blood could also be useful biomarkers for sepsis. At the genomic level, repression of networks corresponding to major histocompatibility complex antigen presentation is observed in septic shock. In consequence, cumulative evidence supports the potential role of immunological monitoring to guide measures to prevent or treat sepsis in a personalised and timely manner (early antibiotic administration, immunoglobulin replacement, immunomodulation). In conclusion, although diffuse and limited, current available information supports the development of large comprehensive studies aimed to urgently evaluate immunological monitoring as a tool to prevent sepsis, guide its treatment and, as a consequence, diminish the morbidity and mortality associated with this severe condition.

Several clinical studies have confi rmed an association between persistent hypercytokinemia and severe 2009 pandemic infl uenza, but none of these have used predictive models to analyze the relationship between the cytokines involved and disease outcome [1-4]. In the present work, we re-analyzed the results from two cohorts of critically ill patients suff ering from pandemic infl uenza infection in 2009 [1,2]. Th irty-fi ve critically ill patients hospitalized with primary viral pneumonia were included in the analysis. Th e levels of 27 cytokines in peripheral blood measured during the fi rst 24 hours following admission to the hospital were included in a Cox regression analysis to evaluate their association with mortality at 28 days. Th is analysis was adjusted by APACHE II score and the presence/absence of mechanical ventilation in order to preclude their potential infl uence on the results. IL-6, IL-8, IL-7, IL-17, and granulocyte colony-stimulating factor (G-CSF) yielded P-values

Producción Científica, In an attempt to evaluate the quantitative changes in the status of immunocompetence in severe sepsis over time and its potential influence on clinical outcome, we monitored the evolution of immunoglobulins (Igs) (IgG, IgA and IgM), complement factors (C3 and C4) and lymphocyte subsets (CD4+ T cells, CD8+ T cells, B cells (CD19+) and natural killer (NK) cells (CD3-CD16+CD56+)) in the blood of 50 patients with severe sepsis or septic shock at day 1, day 3 and day 10 following admission to the ICU. Twenty-one patients died, ten of whom died within the 72 hours following admission to the ICU. The most frequent cause of death (n = 12) was multiorgan dysfunction syndrome. At day 1, survivors showed significantly higher levels of IgG and C4 than those who ultimately died. On the contrary, NK cell levels were significantly higher in the patients who died. Survivors exhibited a progressive increase from day 1 to day 10 on most of the immunological parameters evaluated (IgG, IgA, IgM, C3, CD4+, CD8+ T cells and NK cells). Multivariate Cox regression analysis, including age, sex, APACHE II score, severe sepsis or septic shock status and each one of the immunological parameters showed that NK cell counts at day 1 were independently associated with increased risk of death at 28 days (hazard ratio = 3.34, 95% CI = 1.29 to 8.64; P = 0.013). Analysis of survival curves provided evidence that levels of NK cells at day 1 (> 83 cells/mm3) were associated with early mortality. Our results demonstrate the prognostic role of NK cells in severe sepsis and provide evidence for a direct association of early counts of these cells in blood with mortality.

Introduction: Pandemic A/H1N1/2009 influenza causes severe lower respiratory complications in rare cases. The association between host immune responses and clinical outcome in severe cases is unknown. Methods: We utilized gene expression, cytokine profiles and generation of antibody responses following hospitalization in 19 critically ill patients with primary pandemic A/H1N1/2009 influenza pneumonia for identifying host immune responses associated with clinical outcome. Ingenuity pathway analysis 8.5 (IPA) (Ingenuity Systems, Redwood City, CA) was used to select, annotate and visualize genes by function and pathway (gene ontology). IPA analysis identified those canonical pathways differentially expressed (P < 0.05) between comparison groups. Hierarchical clustering of those genes differentially expressed between groups by IPA analysis was performed using BRB-Array Tools v.3.8.1. Results: The majority of patients were characterized by the presence of comorbidities and the absence of immunosuppressive conditions. pH1N1 specific antibody production was observed around day 9 from disease onset and defined an early period of innate immune response and a late period of adaptive immune response to the virus. The most severe patients (n = 12) showed persistence of viral secretion. Seven of the most severe patients died. During the late phase, the most severe patient group had impaired expression of a number of genes participating in adaptive immune responses when compared to less severe patients. These genes were involved in antigen presentation, B-cell development, T-helper cell differentiation, CD28, granzyme B signaling, apoptosis and protein ubiquitination. Patients with the poorest outcomes were characterized by proinflammatory hypercytokinemia, along with elevated levels of immunosuppressory cytokines (interleukin (IL)-10 and IL-1ra) in serum. Conclusions: Our findings suggest an impaired development of adaptive immunity in the most severe cases of pandemic influenza, leading to an unremitting cycle of viral replication and innate cytokine-chemokine release. Interruption of this deleterious cycle may improve disease outcome., This work has been developed by an international team pertaining to the Spanish-Canadian Consortium for the Study of Influenza Immunopathogenesis. The authors would like to thank also to the Nursery teams who kindly collected the samples. The authors would like to thank Nikki Kelvin for language revision of this article. The study was scientifically sponsored by the Spanish Society for Critical Care Medicine (SEMICYUC). Funding: MICCIN-FIS/JCYL-IECSCYL-SACYL (Spain): Programa de Investigacion Comisionada en Gripe, GR09/0021 EMER07/050 PI081236 RD07/0067. CIHR NIH-Sardinia Recherche-LKSF Canada support DJK.

As highlighted by Rajesh Gupta (October issue), strategies for developing treatments for Ebola virus disease (EVD) need to be rethought. New treatments have to be safe and effective, and in this sense testing already-approved drugs for a new indication is a good approach. With the same philosophy in mind, there are old approaches that could be saving lives in the current outbreak of EVD. Hyperimmune serum from convalescent patients is regarded by WHO as a potential treatment for EVD. The rationale for convalescent serum treatment is provided by limited experiences during the Ebola outbreak in the Democratic Republic of Congo in 1995 and also from tests in non-human primates. Additionally, con valescent serum has been used in Spain and other countries to treat patients with EVD during the current outbreak. Nonetheless, obtaining convalescent serum is a diffi cult task and not free of risk. The logistics of identifying survivors is not easy. Also, blood from survivors needs to be tested to rule out presence of virus. Vaccinated individuals might represent a safer source of hyperimmune serum. Two vaccine candidates will be tested soon in west Africa. If serum from vaccinated individuals has neutralising activity against the virus in cell models, this serum could be used as adjuvant therapy for EVD. In parallel, animal models could be developed to compare hyperimmune serum against a placebo. In the current scenario, this strategy could offer some hope to patients with this devastating disease.