Integrating personalized gene expression profiles into predictive disease-associated gene pools.

Gene expression data are routinely used to identify genes that on average exhibit different expression levels between a case and a control group. Yet, very few of such differentially expressed genes are detectably perturbed in individual patients. Here, we develop a framework to construct personalized perturbation profiles for individual subjects, identifying the set of genes that are significantly perturbed in each individual. This allows us to characterize the heterogeneity of the molecular manifestations of complex diseases by quantifying the expression-level similarities and differences among patients with the same phenotype. We show that despite the high heterogeneity of the individual perturbation profiles, patients with asthma, Parkinson and Huntington's disease share a broadpool of sporadically disease-associated genes, and that individuals with statistically significant overlap with this pool have a 80-100% chance of being diagnosed with the disease. The developed framework opens up the possibility to apply gene expression data in the context of precision medicine, with important implications for biomarker identification, drug development, diagnosis and treatment.

Utility of animal and in vivo experimental infection of humans with rhinoviruses in the development of therapeutic agents for viral exacerbations of asthma and chronic obstructive pulmonary disease.

There is an association with acute viral infection of the respiratory tract and exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Although these exacerbations are associated with several types of viruses, human rhinoviruses (HRVs) are associated with the vast majority of disease exacerbations. Due to the lack of an animal species that is naturally permissive for HRVs to use as a facile model system, and the limitations associated with animal models of asthma and COPD, studies of controlled experimental infection of humans with HRVs have been used and conducted safely for decades. This review discusses how these experimental infection studies with HRVs have provided a means of understanding the pathophysiology underlying virus-induced exacerbations of asthma and COPD with the goal of developing agents for their prevention and treatment.

Local production of inflammatory mediators during childhood parainfluenza virus infection.

OBJECTIVE: To describe the clinical manifestations of parainfluenza virus (PIV) infection and to characterize biochemical markers of PIV disease severity. PATIENTS AND METHODS: We reviewed the medical records of 165 children who had a nasal wash culture positive for PIV at our institution between 1998 and 2008. Nasal wash samples were assayed for 26 inflammatory mediators using Luminex bead proteomics. RESULTS: A total of 153 patients, ages 2 weeks to 12 years, with single virus infection were included in our final analysis. Fifty-two patients were infected with PIV1, 19 with PIV2, 74 with PIV3, and 8 with PIV4. Lower respiratory tract infection (LRTI) was diagnosed in 67 (44%) patients, 21 (14%) had laryngotracheobronchitis, and 49 (32%) had an upper respiratory infection other than laryngotracheobronchitis. LRTI was diagnosed in 54% of patients infected with PIV3, 35% of those infected with PIV1, 26% of those with PIV2, and 50% of those with PIV4. Compared with uninfected control patients, PIV-infected patients had higher nasal wash concentrations of interleukin-6, CX-chemokine ligand 8 (CXCL8 or interleukin-8), CCL3 (macrophage inflammatory protein-1alpha), CCL4 (macrophage inflammatory protein-1beta), CXCL9 (monokine induced by interferon gamma), and CCL5 (regulated upon activation, normal T cell expressed and secreted (RANTES). Patients with LRTI, moderate or severe illness, and PIV 1 or 3 (respirovirus) infection had higher nasal wash concentrations of CXCL8 when compared with patients with upper respiratory infection, mild illness, or PIV 2 and 4 (rubulavirus) infection (P < 0.05). CONCLUSIONS: PIV infection causes a spectrum of illnesses associated with the expression and release of several proinflammatory mediators. Of note, elevated concentrations of CXCL8 in nasal wash samples are associated with more severe forms of PIV disease.

TLR3 activation stimulates cytokine secretion without altering agonist-induced human small airway contraction or relaxation.

Respiratory infections exacerbate chronic lung diseases promoting airway inflammation and hyperreactivity. Toll-like receptor 3 (TLR3) recognizes viral double-stranded (ds) RNA such as polyinosinic-polycytidylic acid [poly(I:C)] and stimulates innate immune responses. The objective of this study was to test the hypothesis that dsRNA promotes lung inflammation and alters airway responsiveness to cholinergic and beta-adrenergic receptor agonists in human lung slices. Human airway smooth muscle (ASM) was incubated for 24 h in poly(I:C) +/- TNFalpha and a TLR3 monoclonal antibody. Precision-cut lung slices (PCLS; 250-microm thickness) from healthy human lungs containing a small airway were incubated in 0, 10, or 100 microg/ml poly(I:C) for 24 h. Intravital microscopy of lung slices was used to quantify contractile and relaxation responsiveness to carbachol and isoproterenol, respectively. Supernatants of ASM and PCLS were analyzed for cytokine secretion using a 25-multiplex bead assay. In human ASM, poly(I:C) (0.5 microg/ml) increased macrophage inflammatory protein-1alpha (MIP-1alpha) and RANTES that was prevented by a TLR3 monoclonal receptor antibody. Incubation of human PCLS with poly(I:C) (10 and 100 microg/ml) had little effect on the log EC(50) or maximum drug effect (E(max)) for contraction and relaxation in response to carbachol and isoproterenol, respectively. The responsiveness of the same human PCLS to poly(I:C) incubation was confirmed by the robust increase in chemokines and cytokines. In separate experiments, incubation of PCLS with IL-13 or TNFalpha (100 ng/ml) increased airway sensitivity to carbachol. Poly(I:C) promotes inflammatory mediator release that was not associated with enhanced bronchoconstriction or attenuated bronchodilation in normal healthy human lung slices. Transduction at the TLR3 initiated by dsRNA stimulates downstream innate immune responses.

Clinical features, adenovirus types, and local production of inflammatory mediators in adenovirus infections.

BACKGROUND: Adenovirus infection manifests in many ways, with respiratory and gastrointestinal symptoms predominating. METHODS: We performed a retrospective chart review on children evaluated at our center who had a nasal wash culture positive for adenovirus. Archived nasal washes were retrieved. Polymerase chain reaction for 15 respiratory viruses was performed on these samples. Patients who were coinfected with another virus were excluded. Adenovirus typing was performed using polymerase chain reaction primers directed at the conserved hexon gene. Bead proteomics was used to measure concentrations of inflammatory mediators. RESULTS: Seventy-eight patients were infected only with adenovirus. The clinical diagnosis was upper respiratory infection in 60%, pneumonia in 18%, febrile seizure in 8%, and bronchiolitis in 6%. Subgroup-C and B1 infections were most common. Seventy percent of patients with upper respiratory infection and all 5 patients with bronchiolitis had a subgroup-C infection; pneumonia was caused by subgroup-B1 and C viruses. Compared with asymptomatic control patients, adenovirus infected patients had higher nasal wash concentrations of interleukin (IL)-1alpha, IL-6, inducible protein-10, macrophage inflammatory protein-1alpha, tumor necrosis factor alpha, monokine induced by gamma interferon, and interferon-alpha (P < 0.05). In addition, we found that IL-8 and IL-1alpha (P < 0.05) were higher in the nasal washes obtained from hospitalized patients than in nonhospitalized patients. CONCLUSIONS: Adenovirus infection causes an array of clinical disease and is associated with local production of several proinflammatory cytokines. The observation that nasal wash IL-8 and IL-1alpha concentrations were higher in patients requiring hospitalization suggests that these mediators contribute to disease severity.

Interferon gamma enhances clonal expansion and survival of CD4+ T cells.

Interferon-gamma (IFN-gamma) serves numerous functions in the regulation of the immune response. During the early phase of the immune response IFN-gamma is produced by natural killer and natural killer T cells. Although the effects of this cytokine on antigen presenting cells and other cell types are known, its direct role on CD4(+) T cells remains unclear. We demonstrate that CD4(+) T cells exposed to IFN-gamma proliferate more vigorously than the controls in response to signals through the antigen receptor. The increased proliferation of IFN-gamma-treated CD4(+) T cells is not due to enhanced signaling through the antigen receptor, but is accounted for by their increased survival. Our data suggest that enhanced survival of IFN-gamma-treated CD4(+)T cells is independent of signal transducer and activator of transcription 1 (STAT 1), a transcription factor that controls the expression of a variety of IFN-gamma-targeted genes. In addition, we demonstrate that independent of STAT 1, IFN-gamma treatment increases the expression of double-stranded RNA-dependent protein kinase, a kinase involved in regulating protein synthesis. Taken together, our findings suggest a direct role of IFN-gamma on unstimulated CD4(+) T cells that is likely to enhance the advent of adaptive immunity by augmenting their survival during the initiation of the immune response.

Generation of a protective T-cell response following coronavirus infection of the central nervous system is not dependent on IL-12/23 signaling.

The functional role of IL-12 and IL-23 in host defense and disease following viral infection of the CNS was determined. Instillation of mouse hepatitis virus (MHV, a positive-strand RNA virus) into the CNS of mice results in acute encephalitis followed by a chronic immune-mediated demyelinating disease. Antibody-mediated blocking of either IL-23 (anti-IL-23p19) or IL-12 and IL-23 (anti-IL-12/23p40) signaling did not mute T-cell trafficking into the CNS or antiviral effector responses and mice were able to control viral replication within the brain. Therapeutic administration of either anti-IL-23p19 or anti-IL-12/23p40 to mice with viral-induced demyelination did not attenuate T-cell or macrophage infiltration into the CNS nor improve clinical disease or diminish white matter damage. In contrast, treatment of mice with anti-IL-12/23p40 or anti-IL-23p19 resulted in inhibition of the autoimmune model of demyelination, experimental autoimmune encephalomyelitis (EAE). These data indicate that (1) IL-12 and IL-23 signaling are dispensable in generating a protective T-cell response following CNS infection with MHV, and (2) IL-12 and IL-23 do not contribute to demyelination in a model independent of autoimmune T-cell-mediated pathology. Therefore, therapeutic targeting of IL-12 and/or IL-23 for the treatment of autoimmune diseases may offer unique advantages by reducing disease severity without muting protective responses following viral infection.

Chemokine expression during mouse-hepatitis-virus-induced encephalitis: contributions of the spike and background genes.

Infection of mice with mouse hepatitis virus (MHV) strain JHM (RJHM) induces lethal encephalitis, with high macrophage and neutrophil, but minimal T-cell, infiltration into the brain when compared to the neuroattenuated strain RA59. To determine if chemokine expression corresponds with the cellular infiltrate, chemokine protein and RNA levels from the brains of infected mice were quantified. RJHM-infected mice had lower T-cell (CXCL9, CXCL10), but higher macrophage-attracting (CCL2), chemokine proteins compared to RA59. RJHM also induced significantly higher CXCL2 (a neutrophil chemoattractant) mRNA compared to RA59. The neurovirulent spike gene chimera SJHM/RA59 induces high levels of T cells and macrophages in the brain compared to the attenuated SA59/RJHM chimera. Accordingly, SJHM/RA59 induced higher levels of CXCL9, CXCL10, and CCL2 protein compared to SA59/RJHM. Chemokine mRNA patterns were in general agreement. Thus, chemokine patterns correspond with the cellular infiltrate, and the spike protein influences levels of macrophage, but not T-cell, chemokines.

Characterization of the epitope for anti-human respiratory syncytial virus F protein monoclonal antibody 101F using synthetic peptides and genetic approaches.

Chimeric 101F (ch101F) is a mouse-human chimeric anti-human respiratory syncytial virus (HRSV) neutralizing antibody that recognizes residues within antigenic site IV, V, VI of the fusion (F) glycoprotein. The binding of ch101F to a series of peptides overlapping aa 422-438 spanning antigenic site IV, V, VI was analysed. Residues 423-436 comprise the minimal peptide sequence for ch101F binding. Substitution analysis revealed that R429 and K433 are critical for ch101F binding, whilst K427 makes a minor contribution. Binding of ch101F to a series of single mutations at positions 427, 429 and 433 in the F protein expressed recombinantly on the cell surface confirmed the peptide results. Sequence analysis of viruses selected for resistance to neutralization by ch101F indicated that a single change (K433T) in the F protein allowed ch101F escape. The results confirm that ch101F and palivizumab have different epitope specificity and define key residues for ch101F recognition.

Relationship between the loss of neutralizing antibody binding and fusion activity of the F protein of human respiratory syncytial virus.

To elucidate the relationship between resistance to HRSV neutralizing antibodies directed against the F protein and the fusion activity of the F protein, a recombinant approach was used to generate a panel of mutations in the major antigenic sites of the F protein. These mutant proteins were assayed for neutralizing mAb binding (ch101F, palivizumab, and MAb19), level of expression, post-translational processing, cell surface expression, and fusion activity. Functional analysis of the fusion activity of the panel of mutations revealed that the fusion activity of the F protein is tolerant to multiple changes in the site II and IV/V/VI region in contrast with the somewhat limited spectrum of changes in the F protein identified from the isolation of HRSV neutralizing antibody virus escape mutants. This finding suggests that aspects other than fusion activity may limit the spectrum of changes tolerated within the F protein that are selected for by neutralizing antibodies.

Diminished inflammatory responses to natural pneumovirus infection among older mice.

Immune responses to virus infection undergo significant change as part of the aging process. Here we examine the inflammatory responses of older, but otherwise immunologically naive mice to infection with pneumonia virus of mice (PVM). Although we see no changes in the extent or kinetics of virus replication, we observe diminished local production of inflammatory mediators, including MIP-1alpha, JE/MCP-1, IFN-gamma and IFN-gamma-induced MIG and IP-10, and interleukins (IL)-6 and IL-17. Levels of KC and IL-1alpha remained unchanged. Age-dependent diminished production of proinflammatory mediators was associated with diminished recruitment of granulocytes and reduced severity of clinical responses, including weight loss and respiratory dysfunction. The differences observed when comparing these results to those reported among elderly human subjects may be related to the specific extent of aging and its impact on biochemical and cellular inflammatory responses and/or the role of lifetime virus re-exposure on the clinical outcome from acute pneumovirus disease.

Contribution of cysteine residues in the extracellular domain of the F protein of human respiratory syncytial virus to its function.

The mature F protein of all known isolates of human respiratory syncytial virus (HRSV) contains fifteen absolutely conserved cysteine (C) residues that are highly conserved among the F proteins of other pneumoviruses as well as the paramyxoviruses. To explore the contribution of the cysteines in the extracellular domain to the fusion activity of HRSV F protein, each cysteine was changed to serine. Mutation of cysteines 37, 313, 322, 333, 343, 358, 367, 393, 416, and 439 abolished or greatly reduced cell surface expression suggesting these residues are critical for proper protein folding and transport to the cell surface. As expected, the fusion activity of these mutations was greatly reduced or abolished. Mutation of cysteine residues 212, 382, and 422 had little to no effect upon cell surface expression or fusion activity at 32 degrees C, 37 degrees C, or 39.5 degrees C. Mutation of C37 and C69 in the F2 subunit either abolished or reduced cell surface expression by 75% respectively. None of the mutations displayed a temperature sensitive phenotype.

A MUC1/IL-18 DNA vaccine induces anti-tumor immunity and increased survival in MUC1 transgenic mice.

MUC1 (mucin 1) is a tumor-associated antigen that is overexpressed in many adenocarcinomas. Active immunotherapy targeting tumors expressing MUC1 could have great treatment value. MUC1 DNA vaccines were evaluated in MUC1 transgenic (MUC1.Tg) mice challenged with MC38/MUC1+ tumor cells. Vaccination with MUC1 plasmid DNA (pMUC1) alone was insufficient to induce tumor protection. However, co-administration of pMUC1 with a plasmid encoding murine interleukin-18 (pmuIL-18) resulted in significant tumor protection and survival after tumor challenge. Protection was durable in the absence of additional vaccination, as demonstrated by continued protection of vaccinated mice following tumor rechallenge. Mice surviving challenges with MC38/MUC1+ cells showed significant protection after challenge with MUC1(-) MC38 tumor cells, suggesting that these mice had developed immune responses to epitopes shared between the tumor cell lines. Antibody-mediated depletion of lymphocyte subsets demonstrated that protection was due largely to CD4+ T cells. This work demonstrates that a naked DNA vaccine can break tolerance to MUC1 and induce an immune response capable of mediating both significant protection from tumor challenge and increased survival.

The cytoplasmic domain of the F protein of Human respiratory syncytial virus is not required for cell fusion.

The cytoplasmic domains of the fusion proteins encoded by several viruses play a role in cell fusion and contain sites for palmitoylation associated with viral protein trafficking and virus assembly. The fusion (F) protein of Human respiratory syncytial virus (HRSV) has a predicted cytoplasmic domain of 26 residues containing a single palmitoylated cysteine residue that is conserved in bovine RSV F protein, but not in the F proteins of other pneumoviruses such as pneumonia virus of mice, human metapneumovirus and avian pneumovirus. The cytoplasmic domains in other paramyxovirus fusion proteins such as Newcastle disease virus F protein play a role in fusion. In this study, it was shown that deletion of the entire cytoplasmic domain or mutation of the single cysteine residue (C550S) of the HRSV F protein had no effect on protein processing, cell-surface expression or fusion.

Use of a novel cell-based fusion reporter assay to explore the host range of human respiratory syncytial virus F protein.

Human respiratory syncytial virus (HRSV) is an important respiratory pathogen primarily affecting infants, young children, transplant recipients and the elderly. The F protein is the only virion envelope protein necessary and sufficient for virus replication and fusion of the viral envelope membrane with the target host cell. During natural infection, HRSV replication is limited to respiratory epithelial cells with disseminated infection rarely, if ever, occurring even in immunocompromised patients. However, in vitro infection of multiple human and non-human cell types other than those of pulmonary tract origin has been reported. To better define host cell surface molecules that mediate viral entry and dissect the factors controlling permissivity for HRSV, we explored the host range of HRSV F protein mediated fusion. Using a novel recombinant reporter gene based fusion assay, HRSV F protein was shown to mediate fusion with cells derived from a wide range of vertebrate species including human, feline, equine, canine, bat, rodent, avian, porcine and even amphibian (Xenopus). That finding was extended using a recombinant HRSV engineered to express green fluorescent protein (GFP), to confirm that viral mRNA expression is limited in several cell types. These findings suggest that HRSV F protein interacts with either highly conserved host cell surface molecules or can use multiple mechanisms to enter cells, and that the primary determinants of HRSV host range are at steps post-entry.