Verónica Iglesias

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.

Early levels in blood of immunoglobulin M and natural killer cells predict outcome in nonseptic critically ill patients

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.

Breast feeding and early life immunomodulation

To the Editor, We have read with interest the article from Belderbos et al. (1) suggesting the ability of breastfeeding to down-modulate the secretion of TNF-a and IL-10 by white blood cells in term children during the first month of life. Also very recently, we have reported the existence of an inverse association between duration of breastfeeding and levels of IL-10 and RANTES secreted by the upper respiratory tract 1 year after birth in preterm children (2). A total of 77 children were recruited in our study. Gestational age (weeks) was (mean, SD) 31.9 (2.9). Sex composition was (women/men): 35/38. Weight at birth (kg) was 1.8 (0.5). Length at birth (cm) was 42.5 (4.2). Eight children had broncho-pulmonary dysplasia at birth. The most frequent accompanying condition was the presence of personal (n = 16) or familiar antecedents (n = 32) of atopic dermatitis. The majority of the children received breastfeeding (60%) for an average of 5.4 months after birth. Cytokines were measured using the 27-plex Bio-Rad assay (Hercules, CA, USA) on a Luminex platform (Austin, TX, USA), including IL-1 receptor antagonist (IL-1RA), IL-9, IL-15, Eotaxin, human fibroblast growth factor–basic (FGF-b): IFN-inducible protein 10 (IP-10), macrophage inflammatory protein 1a (MIP1a), platelet-derived growth factor (PDGF-BB), regulated upon activation, normal T cell expressed, and secreted protein (RANTES), vascular endothelial growth factor (VEGF), IL1b, IL-6, IL-8, IL-7, IL-17, granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1b (MIP-1b), IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, granulocyte macrophage colonystimulating factor (GM-CSF), IFN-c and tumor necrosis factor alpha (TNF-a). Interestingly, duration of breastfeeding (in months) inversely correlated with levels in nasopharyngeal aspirates of IL-1b (Spearman correlation coefficient, p): ( 0.254, 0,035), G-CSF ( 0.236, 0.050), MIP-1b ( 0.250, 0.039), IL-10 ( 0.323, 0,007), and TNF-a ( 0.254, 0,035) (Fig. 1), and also with RANTES, IL-6, IL-17 at the level (p < 0.1). Belderbos et al. normalized cytokines by means of logarithmic transformation. Reproducing this approach, we now used logarithmic values for cytokines. Univariate linear regression analysis was performed again to evaluate the relationship between breastfeeding and cytokine levels 1 year after birth. Potential confounding variables were also evaluated in the univariate analysis: gestational age, sex, weight at birth, height at birth, [breastfeeding (duration in months)], bronchopulmonary dysplasia, atopic dermatitis, smoking habit at home, assistance to daycare, siblings at daycare or at school, number of people living at home, sibilances needing of treatment in the first year of life, [prophylaxis with PVZ (three or more doses)], symptoms of respiratory infection in the first year of life observed by the pediatrician, and necessity of hospitalization in the first year of life. Those variables yielding p values <0.2 in the univariate analysis were entered into a further multivariate analysis. After adjusting for potential confounding variables, duration of breastfeeding was inversely associated with levels of RANTES and IL-10 (Table 1) In turn, the variable (siblings at daycare or at school) was directly associated with IL-10 levels and the variable (prophylaxis with PVZ) was inversely associated with levels of this cytokine. Both RANTES and IL-10 are involved in allergy/asthma pathogenesis. In conclusion, these works are pioneer in providing biological evidence on the immuno-modulatory ability of breastfeeding in early life. This activity could be explained by the protective effect of breastfeeding against infections (3, 4). Alternatively, breastfeeding-induced protection might rely on tolerance induction to common environmental and dietary antigens because of antigen transfer across mammary epithelium or to the presence of factors in breast milk influencing neonatal immune system maturation, including immunoglobulins, oligosaccharides, and antimicrobial proteins/peptides (5). These results could explain thus the potential protective effect of breastfeeding against asthma/allergy.

Evidence of Active Pro-Fibrotic Response in Blood of Patients with Cirrhosis

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.

Cytokine profiles linked to fatal outcome in infective prosthetic valve endocarditis.

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.