Lucia Rico

Immunomodulatory Nanoparticles from Elastin-Like Recombinamers: Single-Molecules for Tuberculosis Vaccine Development

This study investigates both the physicochemical properties and immunogenicity of a genetically engineered elastin-like block corecombinamer (ELbcR) containing a major membrane protein sequence from Mycobacterium tuberculosis. The recombinant production of this ELbcR allows the production of large quantities of safe, antigenic particle-based constructs that directly and reversibly self-assemble into highly biocompatible, multivalent, monodisperse, and stable nanovesicles with a diameter of 55 nm from the same gene product using a highly efficient and cost-effective inverse transition cycling (ITC) procedure. The compositional complexity of these vesicles is retained after secondary processes such as endotoxin removal, sterilization, and lyophilization. An initial pro-chemotactic cytokine response (IL-1β) followed by a pro-Th2/IL-5 response was observed in mice plasma following subcutaneous administration of the antigen-loaded nanovesicles in mice. This biphasic model of cytokine production was coupled with humoral isotype switching from IgM- to IgG-specific antibodies against the antigen, which was only observed in the presence of both the antigen and the polymer in the same construct and in the absence of additional adjuvants.

Host adaptive immunity deficiency in severe pandemic influenza

IntroductionPandemic 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.MethodsWe 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.ResultsThe 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.ConclusionsOur 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.

Early natural killer cell counts in blood predict mortality in severe sepsis

IntroductionHost immunity should play a principal role in determining both the outcome and recovery of patients with sepsis that originated from a microbial infection. Quantification of the levels of key elements of the immune response could have a prognostic value in this disease.MethodsIn 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.ResultsTwenty-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.ConclusionsOur 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.

A new method for detection of pandemic influenza virus using High Resolution Melting analysis of the neuraminidase gene

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.

Host Response Cytokine Signatures in Viral and Nonviral Acute Exacerbations of Chronic Obstructive Pulmonary 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.

Transcriptomic correlates of organ failure extent in sepsis

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.

Viral Infection is Associated with an Increased Proinflammatory Response in Chronic Obstructive Pulmonary Disease

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.

Beneficial role of endogenous immunoglobulin subclasses and isotypes in septic shock

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.

Critical COPD respiratory illness is linked to increased transcriptomic activity of neutrophil proteases genes

BackgroundGene 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.FindingsBy 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.ConclusionStudy 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.

Nasopharyngeal aspirate cytokine levels 1 yr after severe respiratory syncytial virus infection

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.