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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.

Producción Científica, The aim of this study was to investigate the relationship between the CD14 rs2569190 polymorphism and death related to septic shock in white European patients who underwent major cardiac or abdominal surgery. We carried out a retrospective study in 205 septic shock patients. The septic shock diagnosis was established by international consensus definitions. The outcome variable was the death within 28, 60 and 90 days after septic shock diagnosis. The CD14 rs2569190 polymorphism was analyzed by Agena Bioscience’s MassARRAY platform. For the genetic association analysis with survival was selected a recessive inheritance model (GG vs. AA/AG). One hundred thirteen out of 205 patients (55.1%) died with a survival median of 39 days (95%CI = 30.6; 47.4). Patients with rs2569190 GG genotype had shorter survival probability than rs2569190 AA/AG genotype at 60 days (62.3% vs 50%; p = 0.035), and 90 days (62.3% vs 52.6%; p = 0.046). The rs2569190 GG genotype was associated with increased risk of septic shock-related death in the first 60 days (adjusted hazard ratio (aHR) = 1.67; p = 0.016) and 90 days (aHR = 1.64; p = 0.020) compared to rs2569190 AA/AG genotype. In conclusion, the presence of CD14 rs2569190 GG genotype was associated with death in shock septic patients who underwent major surgery. Further studies with bigger sample size are required to verify this relationship., Centro Nacional de Genotipado (grant CEGEN-PRB2-ISCIII), Instituto de Salud Carlos III (grants PT13/0001, PI15/01451, CD13/00013, CD14/00002 and CP14CIII/00010), Junta de Castilla y Leon (grants GR463/A/10 and GR773/A/13), PFIZER (grant CT25-ESP01-01)

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.

Infective endocarditis is a disease normally of bacterial cause which affects the endocardic tissue, specifically the valves (native or prosthetic). It is a serious illness and mortality rates remain high, ranging between 20% and 40%. Previous reports have evidenced the potential role of cytokines in the diagnosis of this disease, but no information is available on their relationship with outcome. We recruited 26 consecutive patients with late prosthetic valve endocarditis requiring surgical treatment according to Duke criteria. Eight cytokines were measured in plasma in the first 24 h following diagnosis by using a Bio-Rad multiplex assay. Levels of IL-6, IL-8 and interferon gamma (IFN-γ) were higher in non survivors. Receiver operating characteristic curve analysis evidenced that IL-6, IL-8 and IFN-γ behaved as good diagnostic tests for identifying those patients with fatal outcome (area under the curve, CI 95%, p): IL-6: [0.81 (0.61-1.00) 0.012]; IL-8 [0.76 (0.56-0.96) 0.035]; IFN-γ [0.79 (0.59-0.99) 0.021]. Levels of IL-6, IL-8 and IFN-γ correlated positively between them, indicating that they are produced as consequence of a simultaneous response to the infection. Our findings support the participation of IL-6, IL-8 and IFN-γ in the events linked to fatal outcome in infective prosthetic valve endocarditis.

Background IL-1β is a primary mediator of systemic inflammatory response syndrome (SIRS) and it may lead to shock septic. Our aim was to analyze whether IL-1B rs16944 polymorphism is associated with the onset of septic shock and death after major surgery. Methods We performed a case-control study on 467 patients who underwent major cardiac or abdominal surgery. Out of them, 205 patients developed septic shock (Cases, SS-group) and 262 patients developed SIRS (Controls, SIRS-group). The primary outcome variables were the development of septic shock and death within 90 days after diagnosis of septic shock. The IL-1B rs16944 polymorphism was genotyped by Sequenom's MassARRAY platform. The association analysis was performed under a recessive genetic model (AA vs. GG/GC). Results The frequency of septic shock was higher in patients with IL-1B rs16944 AA genotype than in patients with IL-1B rs16944 GG/AG genotype when all patients were taken into account (63.6% vs 41.8%; p=0.006), cardiac surgery (52.2% vs. 33.3%; p=0.072), and abdominal surgery (76.2% vs. 50.2%; p=0.023). However, the IL-1B rs16944 AA genotype was only associated with higher likelihood of septic shock in the analysis all population [adjusted odds ratio (aOR)=2.26 (95%CI=1.03; 4.97; p=0.042], but not when it was stratified by cardiac surgery (p=0.175) or abdominal surgery (p=0.467). Similarly, IL-1B rs16944 AA genotype was also associated with higher likelihood of septic shock-related death in all population [aOR=2.67 (95%CI=1.07; 4.97); p=0.035]. Conclusions IL-1B rs16944 AA genotype seems to be related to the onset of septic shock and death in patients who underwent major surgery. This article is protected by copyright. All rights reserved.

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.

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.

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.

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.

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.

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.

Diagnostic methods based upon exclusive detection of haemagglutinin do not detect sequence variation in other gene segments of the Influenza A virus. A complementary approach is described based upon high-resolution melting curve analysis of the neuraminidase gene, an approach with the potential ability to detect small changes in the neuraminidase sequence without the need for specific probes.

et al., Respiratory syncytial virus (RSV) infection is an important cause of recurrent wheezing in infants. Nevertheless, the link between RSV infection and wheezing has yet to be elucidated at the molecular level. Here, we present a preliminary study on the evolution of the immune response in the respiratory tract at long-term after RSV infection. Twenty-seven immune mediators were profiled in nasopharyngeal aspirates (NPAs) obtained from 20 children hospitalized due to a severe infection by RSV at discharge from hospital and again 1 yr later. The same mediators were profiled in parallel in NPAs from 12 healthy controls. In the year following discharge, 85% (17/20) of children of the RSV group suffered at least one episode of wheezing documented by the pediatrician. On the contrary, wheezing episodes were observed only in 25% (3/12) of children in the control group. While most of the mediators profiled returned to normal levels by 1 yr after discharge from hospital, RSV children showed a persistent nasal hyper-secretion of VEGF, G-CSF, IL-10, IL-6, IFN-γ, IL-7 and IL-13. In previous works VEGF, IL-10 and IFN-γ have been put in relation with the pathogenesis of post-virus induced asthma. G-CSF, IL-6, IL-7 and IL-13 are increased in respiratory and plasma samples of asthmatic patients. Here, we evidence for the first time a persistent elevation of these mediators as late as 1 yr after severe RSV disease resolution, reinforcing their possible implication in the pathogenesis of wheezing. © 2009 Blackwell Munksgaard., This work has been possible thanks to the financial support from “Fondo de Investigaciones Sanitarias, FIS, Programa para favorecer la incorporación de grupos de investigación en las Instituciones del Sistema Nacional de Salud, EMER07/050”.

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.

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)

Purpose Critical illness results in derangements of all components of the immune response. Nonetheless, most of the efforts evaluating immune status in critically ill patients have been done in the field of sepsis. Here we have evaluated the immunity status at intensive care unit (ICU) admission in a cohort of nonseptic critically ill patients and its influence on their outcome. Material and methods Ninety patients 18 years and older admitted to our ICU were studied for levels of immunoglobulin (Ig) G, IgM, IgA, CD3 + CD4 + T cells, CD3 + CD8 + T cells, B cells, natural killer (NK) cells, and C3 and C4 complement factors in peripheral blood in the next 24 hours after admission to the ICU. Patients with infection, sepsis, immunodeficiency, or concomitant immunosuppressive therapy were excluded. Results Levels of IgM, CD3 + T cells, CD4 + T cells, CD8 + T cells, and B lymphocytes correlated inversely with age. In turn, levels of CD3 + T cells, CD4 + T cells, CD8 + T cells, and C3 factor of the complement system correlated inversely with Acute Physiology and Chronic Health Evaluation II score. Multivariate Cox regression analysis censored at 28 days evidenced that levels of IgM played a protective role, whereas levels of NK cells behaved as a risk factor for mortality. Kaplan-Meier curves showed a cutoff of 58 mg/dL for IgM and 140 cells/mm 3 for NK cells. Conclusions In conclusion, our results demonstrate that IgM plays a protective role in critically ill patients with no sepsis, whereas NK cell counts seem to play a deleterious one. Aging and severity at admission affect levels of key factors of the immune system in the blood of these patients.

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.