Octavio Ramilo

An interferon-inducible neutrophil-driven blood transcriptional signature in human tuberculosis

Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis, is a major cause of morbidity and mortality worldwide. Efforts to control it are hampered by difficulties with diagnosis, prevention and treatment. Most people infected with M. tuberculosis remain asymptomatic, termed latent TB, with a 10% lifetime risk of developing active TB disease. Current tests, however, cannot identify which individuals will develop disease. The immune response to M. tuberculosis is complex and incompletely characterized, hindering development of new diagnostics, therapies and vaccines. Here we identify a whole-blood 393 transcript signature for active TB in intermediate and high-burden settings, correlating with radiological extent of disease and reverting to that of healthy controls after treatment. A subset of patients with latent TB had signatures similar to those in patients with active TB. We also identify a specific 86-transcript signature that discriminates active TB from other inflammatory and infectious diseases. Modular and pathway analysis revealed that the TB signature was dominated by a neutrophil-driven interferon (IFN)-inducible gene profile, consisting of both IFN-γ and type I IFN-αβ signalling. Comparison with transcriptional signatures in purified cells and flow cytometric analysis suggest that this TB signature reflects changes in cellular composition and altered gene expression. Although an IFN-inducible signature was also observed in whole blood of patients with systemic lupus erythematosus (SLE), their complete modular signature differed from TB, with increased abundance of plasma cell transcripts. Our studies demonstrate a hitherto underappreciated role of type I IFN-αβ signalling in the pathogenesis of TB, which has implications for vaccine and therapeutic development. Our study also provides a broad range of transcriptional biomarkers with potential as diagnostic and prognostic tools to combat the TB epidemic.

A Modular Analysis Framework for Blood Genomics Studies: Application to Systemic Lupus Erythematosus

The analysis of patient blood transcriptional profiles offers a means to investigate the immunological mechanisms relevant to human diseases on a genome-wide scale. In addition, such studies provide a basis for the discovery of clinically relevant biomarker signatures. We designed a strategy for microarray analysis that is based on the identification of transcriptional modules formed by genes coordinately expressed in multiple disease data sets. Mapping changes in gene expression at the module level generated disease-specific transcriptional fingerprints that provide a stable framework for the visualization and functional interpretation of microarray data. These transcriptional modules were used as a basis for the selection of biomarkers and the development of a multivariate transcriptional indicator of disease progression in patients with systemic lupus erythematosus. Thus, this work describes the implementation and application of a methodology designed to support systems-scale analysis of the human immune system in translational research settings.

Correlation of interleukin-1β and cachectin concentrations in cerebrospinal fluid and outcome from bacterial meningitis

Because interleukin-1 beta (IL-1 beta) and cachectin (tumor necrosis factor) are thought to mediate the bodys response to microbial invasion, we measured IL-1 beta and tumor necrosis factor concentrations in paired cerebrospinal fluid (CSF) samples (on admission to the hospital, CSF1; 18 to 30 hours later, CSF2) from 106 infants and children with bacterial meningitis. In CSF1, IL-1 beta was detected in 95% of samples; the mean (+/- 1 SD) concentration was 944 +/- 1293 pg/ml. Patients with CSF1 IL-1 beta concentrations greater than or equal to 500 pg/ml were more likely to have neurologic sequelae (p = 0.001). Tumor necrosis factor was present in 75% of CSF1 samples; the mean concentration was 787 +/- 3358 pg/ml. In CSF2 the mean IL-1 beta concentration was 135 +/- 343 pg/ml, and IL-1 beta concentrations correlated significantly with CSF2 leukocyte count, with glucose, lactate, protein, and tumor necrosis factor concentrations, and with neurologic sequelae. Tumor necrosis factor was detected in CSF2 specimens of 53 of 106 patients, with a mean concentration of 21 +/- 65 pg/ml. Of the 106 patients, 47 received dexamethasone therapy at the time of diagnosis. These patients had significantly lower concentrations of IL-1 beta and higher glucose and lower lactate concentrations in CSF2, and they had a significantly shorter duration of fever compared with the values in patients not treated with steroids (p less than or equal to 0.002). Our data suggest a possible role of IL-1 beta and tumor necrosis factor as mediators of meningeal inflammation in patients with bacterial meningitis, and might explain, in part, the beneficial effect of dexamethasone as adjunctive treatment in this disease.

Induction of ICOS+CXCR3+CXCR5+ TH Cells Correlates with Antibody Responses to Influenza Vaccination

A T cell subset that emerges in blood after seasonal influenza vaccinations correlates with the development of protective antibody responses. What Lies Beneath Although the seasonal flu vaccine, which can protect 60 to 90% of young healthy adults, has been in use for decades, we still know surprisingly little about how it actually induces protective antibody responses. This information is especially important to improve vaccination efficacy in populations that are more susceptible to infection such as the very young and the elderly. Now, Bentebibel et al. take us a step further into understanding what is required for protective antibody responses in humans. The authors identified a subset of CD4+ T cells that were associated with protective antibody responses after seasonal flu vaccination in humans. These cells expressed the costimulatory molecules ICOS as well as two chemokine receptors, CXCR3 and CXCR5, which identify these cells as circulating memory T follicular helper (TFH) cells. TFH cells traditionally are thought to reside in the B cell follicles and be instrumental for germinal center formation and subsequent memory antibody response. Indeed, these circulating cells were influenza antigen–specific, could induce memory B cells to differentiate into plasma cells, and correlated with specific antibody titer. Further studies that find ways to harness these cells could thus improve vaccine design. Seasonal influenza vaccine protects 60 to 90% of healthy young adults from influenza infection. The immunological events that lead to the induction of protective antibody responses remain poorly understood in humans. We identified the type of CD4+ T cells associated with protective antibody responses after seasonal influenza vaccinations. The administration of trivalent split-virus influenza vaccines induced a temporary increase of CD4+ T cells expressing ICOS, which peaked at day 7, as did plasmablasts. The induction of ICOS was largely restricted to CD4+ T cells coexpressing the chemokine receptors CXCR3 and CXCR5, a subpopulation of circulating memory T follicular helper cells. Up to 60% of these ICOS+CXCR3+CXCR5+CD4+ T cells were specific for influenza antigens and expressed interleukin-2 (IL-2), IL-10, IL-21, and interferon-γ upon antigen stimulation. The increase of ICOS+CXCR3+CXCR5+CD4+ T cells in blood correlated with the increase of preexisting antibody titers, but not with the induction of primary antibody responses. Consistently, purified ICOS+CXCR3+CXCR5+CD4+ T cells efficiently induced memory B cells, but not naïve B cells, to differentiate into plasma cells that produce influenza-specific antibodies ex vivo. Thus, the emergence of blood ICOS+CXCR3+CXCR5+CD4+ T cells correlates with the development of protective antibody responses generated by memory B cells upon seasonal influenza vaccination.

Challenges in infant immunity: Implications for responses to infection and vaccines

Infections in infants continue to be an important cause of morbidity and mortality worldwide. Understanding the immune mechanisms that operate in infants is necessary for the development of new approaches to improve the health of infants around the world.

Elevated cytokine concentrations in the nasopharyngeal and tracheal secretions of children with respiratory syncytial virus disease.

BACKGROUND Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract disease in infants. The role of inflammatory mediators in the pathogenesis of RSV disease is not well-understood. The present study was designed (1) to determine whether RANTES (regulated on activation, normal T cell expressed and presumably secreted), macrophage-inflammatory protein-1-alpha (MIP-1-alpha), interleukin (IL)-6, IL-8 and IL-10 can be detected in respiratory secretions of children with RSV infection and (2) to assess whether the concentrations of these cytokines in respiratory secretions correlate with white blood cell (WBC) counts and RSV concentrations and with disease severity. METHODS During the 1996 to 1997 RSV season, we studied prospectively 14 intubated and 14 nonintubated children hospitalized with RSV disease. Nasal wash (NW) and tracheal aspirate (TA) samples were obtained from intubated patients on Hospital Days 1, 3 and 5. NW samples were obtained from nonintubated patients on hospital days 1 and 3. Seven healthy children undergoing elective surgery served as controls. All samples were analyzed for: (1) WBC and differential counts; (2) concentrations of RANTES, MIP-1-alpha, IL-6, IL-8 and IL-10; and (3) quantitative RSV cultures, except in control patients. RESULTS RANTES, MIP-1-alpha, IL-6, IL-8 and IL-10 were detected in NW and TA samples from all children with RSV infection. The concentrations of these cytokines in samples obtained from children with RSV infection were significantly greater than those in samples obtained from control children. NW WBC counts significantly correlated with NW RANTES, IL-6, IL-8 and IL-10 concentrations, whereas TA WBC counts significantly correlated with TA IL-6, IL-8, IL-10 and MIP-1-alpha concentrations. NW RSV concentrations correlated with NW WBC counts and with NW cytokine concentrations. Among children with RSV infection nonintubated patients had greater NW WBC counts and NW RANTES concentrations than intubated patients. TA RANTES, IL-8 and IL-10 concentrations inversely correlated with clinical markers of RSV disease severity. CONCLUSION The presence of cytokines in NW and TA samples of children with RSV infection suggests that they have a role in mediating the respiratory tract inflammation induced by RSV. These observations could have implications for designing new therapeutic strategies directed at immunomodulation of RSV disease.

Blood leukocyte microarrays to diagnose systemic onset juvenile idiopathic arthritis and follow the response to IL-1 blockade

Systemic onset juvenile idiopathic arthritis (SoJIA) represents up to 20% of juvenile idiopathic arthritis. We recently reported that interleukin (IL) 1 is an important mediator of this disease and that IL-1 blockade induces clinical remission. However, lack of specificity of the initial systemic manifestations leads to delays in diagnosis and initiation of therapy. To develop a specific diagnostic test, we analyzed leukocyte gene expression profiles of 44 pediatric SoJIA patients, 94 pediatric patients with acute viral and bacterial infections, 38 pediatric patients with systemic lupus erythematosus (SLE), 6 patients with PAPA syndrome, and 39 healthy children. Statistical group comparison and class prediction identified genes differentially expressed in SoJIA patients compared with healthy children. These genes, however, were also changed in patients with acute infections and SLE. An analysis of significance across all diagnostic groups identified 88 SoJIA-specific genes, 12 of which accurately classified an independent set of SoJIA patients with systemic disease. Transcripts that changed significantly in patients undergoing IL-1 blockade were also identified. Thus, leukocyte transcriptional signatures can be used to distinguish SoJIA from other febrile illnesses and to assess response to therapy. Availability of early diagnostic markers may allow prompt initiation of therapy and prevention of disabilities.

Reduction of Respiratory Syncytial Virus (RSV) in Tracheal Aspirates in Intubated Infants by Use of Humanized Monoclonal Antibody to RSV F Protein

Thirty-five children <2 years of age mechanically ventilated for respiratory syncytial virus (RSV) infection were randomized to receive an intravenous infusion of 15 mg/kg MEDI-493 or placebo. RSV concentration was measured in tracheal secretions by plaque assay before and at 24-h intervals after treatment. The reduction in tracheal RSV concentration from day 0 to day 1 (-1.7+/-0.28 vs. -0. 6+/-0.21 log10 pfu/mL; P=.004) and from day 0 to day 2 (-2.5+/-0.26 vs. -1.0+/-0.41 log10 pfu/mL; P=.012) was significantly greater in the MEDI-493 group than in the placebo group. RSV concentration in nasal aspirates did not differ significantly between the groups. No significant differences were observed in the tracheal aspirate white blood cell count, or myeloperoxidase or eosinophilic cationic protein concentration, or in measures of disease severity between the groups. Thus, treatment with 15 mg/kg MEDI-493 intravenously was well-tolerated and significantly reduced RSV concentration in tracheal aspirates of children with respiratory failure due to RSV.

Respiratory Syncytial Virus Induces Pneumonia, Cytokine Response, Airway Obstruction, and Chronic Inflammatory Infiltrates Associated with Long-Term Airway Hyperresponsiveness in Mice

BACKGROUND Respiratory syncytial virus (RSV) infection is associated with acute morbidity (e.g., pneumonia and airway obstruction [AO]) and long-term complications (e.g., airway hyperresponsiveness [AHR]). We present a comprehensive evaluation of the acute and chronic phases of RSV respiratory tract infection, using a mouse model. METHODS BALB/c mice were inoculated with RSV and monitored for 154 days. RSV loads and cytokines were measured in bronchoalveolar lavage (BAL) samples. Pneumonia severity was assessed using a standard histopathologic score, and pulmonary function was determined by plethysmography. RESULTS RSV-infected mice exhibited viral replication that peaked on day 4-5 and became undetectable by day 7. These mice developed acute pneumonia (peak days, 4-5) and chronic pulmonary inflammatory infiltrates that lasted up to 154 days after inoculation. BAL concentrations of tumor necrosis factor- alpha, interleukin (IL)-6, interferon- gamma, IL-4, IL-10, KC (an IL-8 homologue), MIG (CXCL9), RANTES, macrophage inflammatory protein-1 alpha, and eotaxin were significantly higher in RSV-infected mice than in control mice. RSV-infected mice developed acute AO during the first week of infection that persisted for 42 days. RSV-infected mice also showed significant AHR in response to methacholine up to 154 days. CONCLUSION This model provides a means to investigate the immunopathogenesis of RSV infection and its association with reactive airway disease.

Risk factors in children hospitalized with RSV bronchiolitis versus non-RSV bronchiolitis.

BACKGROUND: The trends in hospitalization rates and risk factors for severe bronchiolitis have not been recently described, especially after the routine implementation of prophylaxis for respiratory syncytial virus (RSV) infections. OBJECTIVES: To define the burden of hospitalizations related to RSV and non-RSV bronchiolitis in a tertiary-care childrens hospital from 2002 to 2007 and to identify the risk factors associated with severe disease. METHODS: Medical records of patients hospitalized for bronchiolitis were reviewed for demographic, clinical, microbiologic, and radiologic characteristics as well as the presence of underlying medical conditions. Differences were evaluated between children with RSV and non-RSV bronchiolitis, and multivariable logistic regression analyses were performed to identify independent risk factors for severe disease. RESULTS: Bronchiolitis hospitalizations in children younger than 2 years old (n = 4800) significantly increased from 536 (3.3%) in 2002 to 1241 (5.5%) in 2007, mainly because of RSV infections. Patients with RSV bronchiolitis (n = 2840 [66%]) were younger at hospitalization and had a lower percentage of underlying medical conditions than children hospitalized with non-RSV bronchiolitis (27 vs 37.5%; P < .001). However, disease severity defined by length of hospitalization and requirement of supplemental oxygen, intensive care, and mechanical ventilation was significantly worse in children with RSV bronchiolitis. RSV infection and prematurity, regardless of the etiology, were identified as independent risk factors for severe bronchiolitis. CONCLUSIONS: There was a significant increase in hospitalizations for RSV bronchiolitis from 2002 to 2007. A majority of the children with RSV bronchiolitis were previously healthy, but their disease severity was worse compared with those hospitalized with non-RSV bronchiolitis.