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1.
BMC Palliat Care ; 23(1): 72, 2024 Mar 13.
Article in English | MEDLINE | ID: mdl-38481215

ABSTRACT

BACKGROUND: Children with life-threatening and life-limiting conditions can experience high levels of suffering due to multiple distressing symptoms that result in poor quality of life and increase risk of long-term distress in their family members. High quality symptom treatment is needed for all these children and their families, even more so at the end-of-life. In this paper, we provide evidence-based recommendations for symptom treatment in paediatric palliative patients to optimize care. METHODS: A multidisciplinary panel of 56 experts in paediatric palliative care and nine (bereaved) parents was established to develop recommendations on symptom treatment in paediatric palliative care including anxiety and depression, delirium, dyspnoea, haematological symptoms, coughing, skin complaints, nausea and vomiting, neurological symptoms, pain, death rattle, fatigue, paediatric palliative sedation and forgoing hydration and nutrition. Recommendations were based on evidence from a systematic literature search, additional literature sources (such as guidelines), clinical expertise, and patient and family values. We used the GRADE methodology for appraisal of evidence. Parents were included in the guideline panel to ensure the representation of patient and family values. RESULTS: We included a total of 18 studies that reported on the effects of specific (non) pharmacological interventions to treat symptoms in paediatric palliative care. A few of these interventions showed significant improvement in symptom relief. This evidence could only (partly) answer eight out of 27 clinical questions. We included 29 guidelines and two textbooks as additional literature to deal with lack of evidence. In total, we formulated 221 recommendations on symptom treatment in paediatric palliative care based on evidence, additional literature, clinical expertise, and patient and family values. CONCLUSION: Even though available evidence on symptom-related paediatric palliative care interventions has increased, there still is a paucity of evidence in paediatric palliative care. We urge for international multidisciplinary multi-institutional collaboration to perform high-quality research and contribute to the optimization of symptom relief in palliative care for all children worldwide.


Subject(s)
Palliative Care , Terminal Care , Humans , Child , Palliative Care/methods , Quality of Life , Terminal Care/methods , Pain , Family
2.
EBioMedicine ; 45: 303-313, 2019 Jul.
Article in English | MEDLINE | ID: mdl-31262714

ABSTRACT

BACKGROUND: The complement system is a central component of the innate immune system. Constitutive biosynthesis of complement proteins is essential for homeostasis. Dysregulation as a consequence of genetic or environmental cues can lead to inflammatory syndromes or increased susceptibility to infection. However, very little is known about steady state levels in children or its kinetics during infection. METHODS: With a newly developed multiplex mass spectrometry-based method we analyzed the levels of 32 complement proteins in healthy individuals and in a group of pediatric patients infected with bacterial or viral pathogens. FINDINGS: In plasma from young infants we found reduced levels of C4BP, ficolin-3, factor B, classical pathway components C1QA, C1QB, C1QC, C1R, and terminal pathway components C5, C8, C9, as compared to healthy adults; whereas the majority of complement regulating (inhibitory) proteins reach adult levels at very young age. Both viral and bacterial infections in children generally lead to a slight overall increase in complement levels, with some exceptions. The kinetics of complement levels during invasive bacterial infections only showed minor changes, except for a significant increase and decrease of CRP and clusterin, respectively. INTERPRETATION: The combination of lower levels of activating and higher levels of regulating complement proteins, would potentially raise the threshold of activation, which might lead to suppressed complement activation in the first phase of life. There is hardly any measurable complement consumption during bacterial or viral infection. Altogether, expression of the complement proteins appears surprisingly stable, which suggests that the system is continuously replenished. FUND: European Union's Horizon 2020, project PERFORM, grant agreement No. 668303.


Subject(s)
Communicable Diseases/immunology , Complement Activation/immunology , Complement System Proteins/chemistry , Inflammation/immunology , Adolescent , Adult , C-Reactive Protein/genetics , C-Reactive Protein/immunology , Child , Child, Preschool , Clusterin/genetics , Clusterin/immunology , Communicable Diseases/genetics , Complement Activation/genetics , Complement System Proteins/classification , Complement System Proteins/isolation & purification , Female , Homeostasis , Humans , Infant , Infant, Newborn , Inflammation/genetics , Male , Mass Spectrometry , Middle Aged , Young Adult
3.
Pediatr Infect Dis J ; 37(12): e283-e291, 2018 12.
Article in English | MEDLINE | ID: mdl-30169483

ABSTRACT

BACKGROUND: The influenza H1N1 pandemic of 2009-2010, provided a unique opportunity to assess the course of disease, as well as the analysis of risk factors for severe disease in hospitalized children (< 18 years). METHODS: Retrospective national chart study on hospitalized children with H1N1 infection during the 2009-2010 pH1N1 outbreak. RESULTS: Nine hundred forty patients (56% boys), median age 3.0 years, were enrolled; the majority were previously healthy. Treatment consisted of supplemental oxygen (24%), mechanical ventilation (5%) and antiviral therapy (63%). Fifteen patients died (1.6%), 5 of whom were previously healthy. Multivariable analyses confirmed pre-existent heart and lung disease as risk factors for intensive care unit admission. Risk factors for mortality included children with a neurologic or oncologic disease and psychomotor retardation. CONCLUSIONS: This nationwide overview of hospitalized children confirms known risk groups for severe influenza infections. However, most of the acute and severe presentations of influenza occurred in previously healthy children.


Subject(s)
Child, Hospitalized/statistics & numerical data , Influenza A Virus, H1N1 Subtype , Influenza, Human/epidemiology , Adolescent , Child , Child, Preschool , Disease Outbreaks/statistics & numerical data , Female , Hospitalization/statistics & numerical data , Humans , Infant , Influenza, Human/mortality , Length of Stay/statistics & numerical data , Male , Netherlands/epidemiology , Retrospective Studies , Risk Factors
4.
Microbiome ; 6(1): 10, 2018 01 11.
Article in English | MEDLINE | ID: mdl-29325581

ABSTRACT

BACKGROUND: While almost all infants are infected with respiratory syncytial virus (RSV) before the age of 2 years, only a small percentage develops severe disease. Previous studies suggest that the nasopharyngeal microbiome affects disease development. We therefore studied the effect of the nasopharyngeal microbiome on viral load and mucosal cytokine responses, two important factors influencing the pathophysiology of RSV disease. To determine the relation between (i) the microbiome of the upper respiratory tract, (ii) viral load, and (iii) host mucosal inflammation during an RSV infection, nasopharyngeal microbiota profiles of RSV infected infants (< 6 months) with different levels of disease severity and age-matched healthy controls were determined by 16S rRNA marker gene sequencing. The viral load was measured using qPCR. Nasopharyngeal CCL5, CXCL10, MMP9, IL6, and CXCL8 levels were determined with ELISA. RESULTS: Viral load in nasopharyngeal aspirates of patients associates significantly to total nasopharyngeal microbiota composition. Healthy infants (n = 21) and RSV patients (n = 54) display very distinct microbial patterns, primarily characterized by a loss in commensals like Veillonella and overrepresentation of opportunistic organisms like Haemophilus and Achromobacter in RSV-infected individuals. Furthermore, nasopharyngeal microbiota profiles are significantly different based on CXCL8 levels. CXCL8 is a chemokine that was previously found to be indicative for disease severity and for which we find Haemophilus abundance as the strongest predictor for CXCL8 levels. CONCLUSIONS: The nasopharyngeal microbiota in young infants with RSV infection is marked by an overrepresentation of the genus Haemophilus. We present that this bacterium is associated with viral load and mucosal CXCL8 responses, both which are involved in RSV disease pathogenesis.


Subject(s)
Haemophilus/classification , Interleukin-8/metabolism , Nasopharynx/microbiology , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Viruses/physiology , DNA, Bacterial/genetics , DNA, Ribosomal/genetics , Female , Haemophilus/genetics , Haemophilus/isolation & purification , Hospitalization , Humans , Infant , Infant, Newborn , Male , RNA, Ribosomal, 16S/genetics , Respiratory Syncytial Virus Infections/immunology , Sequence Analysis, DNA/methods , Up-Regulation , Viral Load
5.
Eur J Immunol ; 48(4): 621-631, 2018 04.
Article in English | MEDLINE | ID: mdl-29266251

ABSTRACT

Interferon gamma (IFN-γ) plays an important role in the antiviral immune response during respiratory syncytial virus (RSV) infections. Monocytes and T cells are recruited to the site of RSV infection, but it is unclear whether cell-cell interactions between monocytes and T cells regulate IFN-γ production. In this study, micro-array data identified the upregulation of sialic acid-binding immunoglobulin-type lectin 1 (Siglec-1) in human RSV-infected infants. In vitro, RSV increased expression of Siglec-1 on healthy newborn and adult monocytes. RSV-induced Siglec-1 on monocytes inhibited IFN-γ production by adult CD4+ T cells. In contrast, IFN-γ production by RSV in newborns was not affected by Siglec-1. The ligand for Siglec-1, CD43, is highly expressed on adult CD4+ T cells compared to newborns. Our data show that Siglec-1 reduces IFN-γ release by adult T cells possibly by binding to the highly expressed CD43. The Siglec-1-dependent inhibition of IFN-γ in adults and the low expression of CD43 on newborn T cells provides a better understanding of the immune response against RSV in early life and adulthood.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Interferon-gamma/biosynthesis , Monocytes/immunology , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus, Human/immunology , Sialic Acid Binding Ig-like Lectin 1/immunology , Age Factors , Humans , Infant, Newborn , Interferon-gamma/immunology , Leukosialin/metabolism , Respiratory Syncytial Virus Infections/virology , Up-Regulation/immunology
6.
J Virol ; 91(21)2017 11 01.
Article in English | MEDLINE | ID: mdl-28794038

ABSTRACT

Respiratory syncytial virus (RSV) is the leading cause of severe respiratory illness in infants. At this young age, infants typically depend on maternally transferred antibodies (matAbs) and their innate immune system for protection against infections. RSV-specific matAbs are thought to protect from severe illness, yet severe RSV disease occurs mainly below 6 months of age, when neutralizing matAb levels are present. To investigate this discrepancy, we asked if disease severity is related to antibody properties other than neutralization. Some antibody effector functions are mediated via their Fc binding region. However, it has been shown that this binding may lead to antibody-dependent enhancement (ADE) of infection or reduction of neutralization, both possibly leading to more disease. In this study, we first showed that high levels of ADE of RSV infection occur in monocytic THP-1 cells in the presence of RSV antibodies and that neutralization by these antibodies was reduced in Vero cells when they were transduced with Fc gamma receptors. We then demonstrated that antibodies from cotton rats with formalin-inactivated (FI)-RSV-induced pulmonary pathology were capable of causing ADE. Human matAbs also caused ADE and were less neutralizing in vitro in cells that carry Fc receptors. However, these effects were unrelated to disease severity because they were seen both in uninfected controls and in infants hospitalized with different levels of RSV disease severity. We conclude that ADE and reduction of neutralization are unlikely to be involved in RSV disease in infants with neutralizing matAbs.IMPORTANCE It is unclear why severity of RSV disease peaks at the age when infants have neutralizing levels of maternal antibodies. Additionally, the exact reason for FI-RSV-induced enhanced disease, as seen in the 1960s vaccine trials, is still unclear. We hypothesized that antibodies present under either of these conditions could contribute to disease severity. Antibodies can have effects that may lead to more disease instead of protection. We investigated two of those effects: antibody-dependent enhancement of infection (ADE) and neutralization reduction. We show that ADE occurs in vitro with antibodies from FI-RSV-immunized RSV-infected cotton rats. Moreover, passively acquired maternal antibodies from infants had the capacity to induce ADE and reduction of neutralization. However, no clear association with disease severity was seen, ruling out that these properties explain disease in the presence of maternal antibodies. Our data contribute to a better understanding of the impact of antibodies on RSV disease in infants.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Receptors, IgG/metabolism , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus Vaccines/administration & dosage , Respiratory Syncytial Viruses/immunology , Severity of Illness Index , Animals , Antibodies, Viral/blood , Antibody-Dependent Enhancement , Case-Control Studies , Chlorocebus aethiops , Female , Humans , Infant , Lung/immunology , Lung/pathology , Lung/virology , Monocytes/immunology , Monocytes/pathology , Monocytes/virology , Neutralization Tests , Rats , Receptors, IgG/immunology , Respiratory Syncytial Virus Infections/prevention & control , Sigmodontinae , Vaccination , Vero Cells , Viral Envelope Proteins/immunology , Viral Fusion Proteins/immunology
7.
BMJ Open ; 7(6): e014596, 2017 06 30.
Article in English | MEDLINE | ID: mdl-28667205

ABSTRACT

INTRODUCTION: Respiratory viruses causing lower respiratory tract infections (LRTIs) are a major cause of hospital admissions in children. Since the course of these infections is unpredictable with potential fast deterioration into respiratory failure, infants are easily admitted to the hospital for observation. The aim of this study was to examine whether systemic inflammatory markers can be used to predict severity of disease in children with respiratory viral infections. METHODS: Blood and nasopharyngeal washings from children <3 years of age with viral LRTI attending a hospital were collected within 24 hours (acute) and after 4-6 weeks (recovery). Patients were assigned to a mild (observation only), moderate (supplemental oxygen and/or nasogastric feeding) or severe (mechanical ventilation) group. Linear regression analysis was used to design a prediction rule using plasma levels of C reactive protein (CRP), serum amyloid A (SAA), pentraxin 3 (PTX3), serum amyloid P component and properdin. This rule was tested in a validation cohort. RESULTS: One hundred and four children (52% male) were included. A combination of CRP, SAA, PTX3 and properdin was a better indicator of severe disease compared with any of the individual makers and age (69% sensitivity (95% CI 50 to 83), 90% specificity (95% CI 80 to 96)). Validation in 141 patients resulted in 71% sensitivity (95% CI 53 to 85), 87% specificity (95% CI 79 to 92), negative predictive value of 64% (95% CI 47 to 78) and positive predictive value of 90% (95% CI 82 to 95). The prediction rule was not able to identify patients with a mild course of disease. CONCLUSION: A combination of CRP, SAA, PTX3 and properdin was able to identify children with a severe course of viral LRTI disease, even in children under 2 months of age. To assess the true impact on clinical management, these results should be validated in a prospective randomised control study.


Subject(s)
Hospitalization/statistics & numerical data , Respiratory Tract Infections/blood , Severity of Illness Index , Virus Diseases/blood , Acute Disease , Biomarkers/blood , Blood Proteins/analysis , Female , Humans , Infant , Male , Netherlands , Predictive Value of Tests , Prospective Studies , Respiratory Tract Infections/diagnosis , Virus Diseases/diagnosis
8.
PLoS One ; 12(1): e0170877, 2017.
Article in English | MEDLINE | ID: mdl-28135305

ABSTRACT

Respiratory syncytial virus (RSV) is the leading cause for respiratory illness that requires hospitalization in infancy. High levels of maternal antibodies can protect against RSV infection. However, RSV-infected infants can suffer from severe disease symptoms even in the presence of high levels of RSV-specific antibodies. This study analyzes several serological characteristics to explore potential deficiencies or surpluses of antibodies that could relate to severe disease symptoms. We compare serum antibodies from hospitalized patients who suffered severe symptoms as well as uninfected infants. Disease severity markers were oxygen therapy, tachypnea, oxygen saturation, admission to the intensive care unit and duration of hospitalization. Antibodies against RSV G protein and a prefusion F epitope correlated with in vitro neutralization. Avidity of RSV-specific IgG antibodies was lower in RSV-infected infants compared to uninfected controls. Severe disease symptoms were unrelated to RSV-specific IgG antibody titers, avidity of RSV-IgG, virus neutralization capacity or titers against pre- and postfusion F or G protein ectodomains and the prefusion F antigenic site Ø. In conclusion, the detailed serological characterization did not indicate dysfunctional or epitope-skewed composition of serum antibodies in hospitalized RSV-infected infants suffering from severe disease symptoms. It remains unclear, whether specific antibody fractions could diminish disease symptoms.


Subject(s)
Antibodies, Viral/blood , Antibodies, Viral/immunology , Antibody Specificity/immunology , Hospitalization , Respiratory Syncytial Virus Infections/blood , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus, Human/immunology , Acute Disease , Antibody Affinity/immunology , Antigens, Viral/immunology , Epitopes/immunology , Female , Glycoproteins/immunology , Humans , Immunoglobulin G/blood , Infant , Male , Neutralization Tests , Severity of Illness Index
9.
Sci Rep ; 6: 36603, 2016 11 11.
Article in English | MEDLINE | ID: mdl-27833115

ABSTRACT

Respiratory syncytial virus (RSV) causes infections that range from common cold to severe lower respiratory tract infection requiring high-level medical care. Prediction of the course of disease in individual patients remains challenging at the first visit to the pediatric wards and RSV infections may rapidly progress to severe disease. In this study we investigate whether there exists a genomic signature that can accurately predict the course of RSV. We used early blood microarray transcriptome profiles from 39 hospitalized infants that were followed until recovery and of which the level of disease severity was determined retrospectively. Applying support vector machine learning on age by sex standardized transcriptomic data, an 84 gene signature was identified that discriminated hospitalized infants with eventually less severe RSV infection from infants that suffered from most severe RSV disease. This signature yielded an area under the receiver operating characteristic curve (AUC) of 0.966 using leave-one-out cross-validation on the experimental data and an AUC of 0.858 on an independent validation cohort consisting of 53 infants. A combination of the gene signature with age and sex yielded an AUC of 0.971. Thus, the presented signature may serve as the basis to develop a prognostic test to support clinical management of RSV patients.


Subject(s)
Bronchiolitis, Viral , Gene Expression Profiling , Respiratory Syncytial Virus Infections , Respiratory Syncytial Viruses/metabolism , Severity of Illness Index , Support Vector Machine , Transcriptome , Bronchiolitis, Viral/diagnosis , Bronchiolitis, Viral/metabolism , Female , Humans , Infant , Infant, Newborn , Male , Prognosis , Respiratory Syncytial Virus Infections/diagnosis , Respiratory Syncytial Virus Infections/metabolism
10.
BMC Infect Dis ; 16: 129, 2016 Mar 17.
Article in English | MEDLINE | ID: mdl-26983753

ABSTRACT

BACKGROUND: Respiratory syncytial virus (RSV) is an important cause of lower respiratory tract infections in infants. A small percentage of the infected infants develops a severe infection, while most of these severely ill patients were previously healthy. It remains unclear why these children develop severe RSV infections. In this study, we investigate whether pneumococcal nasopharyngeal carriage patterns correlate with mucosal inflammation and severity of disease. METHODS: In total, 105 infants hospitalized with RSV infection were included and recovery samples were taken from 42 patients. The presence and density of Streptococcus pneumoniae was determined by RT qPCR to study its relation to viral load, inflammation (MMP-9 and IL-6) and severity of RSV disease. RESULTS: We show that pneumococcal presence or absence in the nasopharynx does not correlate with viral load, inflammation or severity of disease. However, when pneumococcus is present in patients, a higher nasopharyngeal pneumococcal density was correlated with a higher RSV load, higher MMP-9 levels and a less severe course of disease. CONCLUSIONS: Our results show correlations between S. pneumoniae density and viral load, inflammation and disease severity, suggesting that pneumococcal density may be an indicator for severity in paediatric RSV disease.


Subject(s)
Respiratory Syncytial Virus Infections/epidemiology , Respiratory Syncytial Viruses/isolation & purification , Respiratory Tract Infections/epidemiology , Streptococcus pneumoniae/isolation & purification , Child Health Services , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Male , Nasopharynx/microbiology , Nasopharynx/virology , Netherlands/epidemiology , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Viruses/pathogenicity , Respiratory Tract Infections/virology , Severity of Illness Index , Streptococcus pneumoniae/pathogenicity , Viral Load
11.
Pediatr Infect Dis J ; 35(3): e89-96, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26658377

ABSTRACT

BACKGROUND: Respiratory syncytial virus (RSV) is a common cause of bronchiolitis in infants with a wide spectrum of disease severity. Besides environmental and genetic factors, it is thought that the innate immune system plays a pivotal role. The aim of this study was to investigate the expression of immune receptors on monocytes and the in vitro responsiveness from infants with severe RSV infections. METHODS: Peripheral blood mononuclear cells (PBMCs) from infants with RSV infections were isolated. Classical, intermediate and nonclassical monocytes were immunophenotyped for the expression of CD14, CD16, human leukocyte antigen (HLA)-ABC and HLA-DR. PBMCs were stimulated with lipopolysaccharide to determine the secretion of tumor necrosis factor and interleukin (IL)-10 with enzyme-linked immunosorbent assay. RESULTS: During RSV infection, intermediate monocytes are increased in the peripheral blood, whereas classical and nonclassical monocytes are reduced. The expression of CD14 and HLA-ABC is increased on monocytes, whereas the expression of HLA-DR is suppressed. Low HLA-DR expression is correlated with increased disease severity. PBMCs from infants with severe RSV infections show an impaired IL-10 response in vitro. CONCLUSIONS: Phenotyping subpopulations of monocytes combined with in vitro responsiveness reveals significant differences between nonsevere and severe RSV infections. Reduced HLA-DR expression and impaired IL-10 production in vitro during severe RSV infections indicate that an imbalanced innate immune response may play an important role in disease severity.


Subject(s)
Gene Expression , HLA-DR Antigens/genetics , HLA-DR Antigens/immunology , Monocytes/immunology , Monocytes/metabolism , Respiratory Syncytial Virus Infections/genetics , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus, Human/immunology , Biomarkers , Case-Control Studies , Cohort Studies , Female , Humans , Immunophenotyping , Infant , Interleukin-10/biosynthesis , Leukocyte Count , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Male , Phenotype , Respiratory Syncytial Virus Infections/diagnosis , Respiratory Syncytial Virus Infections/virology , Risk Factors , Severity of Illness Index
12.
Clin Vaccine Immunol ; 23(3): 243-5, 2015 Dec 09.
Article in English | MEDLINE | ID: mdl-26656116

ABSTRACT

Maternal vaccination is currently considered a strategy against respiratory syncytial virus (RSV) infections. In RSV-infected infants, high mucosal IgG levels correlated better with reduced RSV load and lower mucosal CXCL10 levels than plasma IgG levels. For future vaccination strategies against RSV, more focus should be on the mucosal humoral immune response.


Subject(s)
Antibodies, Viral/blood , Chemokine CXCL10/blood , Immunity, Maternally-Acquired/immunology , Immunity, Mucosal/immunology , Immunoglobulin G/blood , Respiratory Syncytial Virus Infections/prevention & control , Respiratory Syncytial Virus, Human/immunology , Antibodies, Viral/immunology , Female , Humans , Immunoglobulin A/blood , Infant , Male , Respiratory Syncytial Virus Infections/immunology , Viral Vaccines/immunology
13.
Eur Respir J ; 45(3): 718-25, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25261323

ABSTRACT

Respiratory syncytial virus (RSV) causes mild infections in the vast majority of children. However, in some cases, it causes severe disease, such as bronchiolitis and pneumonia. Development of severe RSV infection is determined by the host response. Therefore, the main aim of this study was to identify biomarkers associated with severe RSV infection. To identify biomarkers, nasopharyngeal gene expression was profiled by microarray studies, resulting in the selection of five genes: ubiquitin D, tetraspanin 8, mucin 13, ß-microseminoprotein and chemokine ligand 7. These genes were validated by real-time quantitative PCR in an independent validation cohort, which confirmed significant differences in gene expression between mildly and severely infected and between recovery and acute patients. Nasopharyngeal aspirate samples are regularly taken when a viral respiratory tract infection is suspected. In this article, we describe a method to discriminate between mild and severe RSV infection based on differential host gene expression. The combination of pathogen detection and host gene expression analysis in nasopharyngeal aspirates will significantly improve the diagnosis and prognosis of respiratory tract infections.


Subject(s)
Chemokine CCL7/genetics , Host-Pathogen Interactions/genetics , Mucins/genetics , Prostatic Secretory Proteins/genetics , Respiratory Syncytial Virus Infections , Tetraspanins/genetics , Child, Preschool , Female , Gene Expression Profiling , Genetic Markers , Humans , Infant , Male , Microarray Analysis , Nasopharynx/virology , Patient Acuity , Predictive Value of Tests , Prognosis , Recovery of Function/genetics , Respiratory Syncytial Virus Infections/diagnosis , Respiratory Syncytial Virus Infections/genetics , Respiratory Syncytial Virus Infections/physiopathology , Respiratory Syncytial Virus Infections/virology
14.
J Vis Exp ; (82): e50766, 2013 Dec 10.
Article in English | MEDLINE | ID: mdl-24379004

ABSTRACT

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology. Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.


Subject(s)
Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/virology , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Viruses/immunology , HeLa Cells , Humans , Respiratory Syncytial Virus Infections/blood
15.
J Med Virol ; 84(9): 1471-80, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22825827

ABSTRACT

Matrix metalloproteinases (MMPs) play an important role in respiratory inflammatory diseases, such as asthma and chronic obstructive pulmonary disease. It was hypothesized that MMP-8 and MMP-9 may function as biological markers to assess disease severity in viral lower respiratory tract infections in children. MMP-8 and MMP-9 mRNA expression levels in peripheral blood mononuclear cells (PBMCs) and granulocytes obtained in both the acute and recovery phase from 153 children with mild, moderate, and severe viral lower respiratory tract infections were determined using real-time PCR. In addition, MMP-8 and MMP-9 concentrations in blood and nasopharyngeal specimens were determined during acute mild, moderate, and severe infection, and after recovery using ELISA. Furthermore, PBMCs and neutrophils obtained from healthy volunteers were stimulated with RSV, LPS (TLR4 agonist), and Pam3Cys (TLR2 agonist) in vitro. Disease severity of viral lower respiratory tract infections in children is associated with increased expression levels of the MMP-8 and MMP-9 genes in both PBMCs and granulocytes. On the contrary, in vitro experiments showed that MMP-8 and MMP-9 mRNA and protein expression in PBMCs and granulocytes is not induced by stimulation with RSV, the most frequent detected virus in young children with viral lower respiratory tract infections. These data indicate that expression levels of the MMP-8 and MMP-9 genes in both PBMCs and neutrophils are associated with viral lower respiratory tract infections disease severity. These observations justify future validation in independent prospective study cohorts of the usefulness of MMP-8 and MMP-9 as potential markers for disease severity in viral respiratory infections.


Subject(s)
Matrix Metalloproteinase 8/metabolism , Matrix Metalloproteinase 9/metabolism , Respiratory Syncytial Virus Infections/enzymology , Respiratory Syncytial Viruses , Respiratory Tract Infections/enzymology , Female , Gene Expression , Granulocytes/enzymology , HeLa Cells , Humans , Infant , Leukocytes, Mononuclear/enzymology , Male , Matrix Metalloproteinase 8/blood , Matrix Metalloproteinase 8/genetics , Matrix Metalloproteinase 9/blood , Matrix Metalloproteinase 9/genetics , Nasal Mucosa/enzymology , Nasal Mucosa/virology , Neutrophils/enzymology , Neutrophils/virology , Pharynx/enzymology , Pharynx/virology , Prospective Studies , Respiratory Syncytial Virus Infections/pathology , Respiratory Syncytial Virus Infections/virology , Respiratory Tract Infections/pathology , Respiratory Tract Infections/virology , Severity of Illness Index , Statistics, Nonparametric
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