Chemokine profiling in children and adults with symptomatic and asymptomatic respiratory viral infections.

Molecular diagnosis; Viral infection; Chemokines; Disease prognosis; CXCL10; CXCL11; CCL3; CCL4; CCL5; Random forest.

The endotoxin-induced pulmonary inflammatory response is enhanced during the acute phase of influenza infection.

BACKGROUND: Influenza infections are often complicated by secondary infections, which are associated with high morbidity and mortality, suggesting that influenza profoundly influences the immune response towards a subsequent pathogenic challenge. However, data on the immunological interplay between influenza and secondary infections are equivocal, with some studies reporting influenza-induced augmentation of the immune response, whereas others demonstrate that influenza suppresses the immune response towards a subsequent challenge. These contrasting results may be due to the use of various types of live bacteria as secondary challenges, which impedes clear interpretation of causal relations, and to differences in timing of the secondary challenge relative to influenza infection. Herein, we investigated whether influenza infection results in an enhanced or suppressed innate immune response upon a secondary challenge with bacterial lipopolysaccharide (LPS) in either the acute or the recovery phase of infection. METHODS: Male C57BL/6J mice were intranasally inoculated with 5 × 103 PFU influenza virus (pH1N1, strain A/Netherlands/602/2009) or mock treated. After 4 (acute phase) or 10 (recovery phase) days, 5 mg/kg LPS or saline was administered intravenously, and mice were sacrificed 90 min later. Cytokine levels in plasma and lung tissue, and lung myeloperoxidase (MPO) content were determined. RESULTS: LPS administration 4 days after influenza infection resulted in a synergistic increase in TNF-α, IL-1ß, and IL-6 concentrations in lung tissue, but not in plasma. This effect was also observed 10 days after influenza infection, albeit to a lesser extent. LPS-induced plasma levels of the anti-inflammatory cytokine IL-10 were enhanced 4 days after influenza infection, whereas a trend towards increased pulmonary IL-10 concentrations was found. LPS-induced increases in pulmonary MPO content tended to be enhanced as well, but only at 4 days post-infection. CONCLUSIONS: An LPS challenge in the acute phase of influenza infection results in an enhanced pulmonary pro-inflammatory innate immune response. These data increase our insight on influenza-bacterial interplay. Combing data of the present study with previous findings, it appears that this enhanced response is not beneficial in terms of protection against secondary infections, but rather damaging by increasing immunopathology.

Assessment of a molecular diagnostic platform for integrated isolation and quantification of mRNA in whole blood.

Implementation of point-of-care tests may facilitate the health management of infectious diseases by reducing the timeframe on pathogen identification and host response measurements, allowing for immediate diagnosis and guided clinical intervention. In this feasibility study, a novel totally integrated and fully automated real-time polymerase chain reaction (PCR) platform (Idylla™, Biocartis) was assessed to determine the mRNA expression levels of multiple genes from 1 mL of whole blood. To this purpose, a sample-in result-out assay, including mRNA extraction and RT-qPCR-based detection, was ported to the platform. The genes used (matrix metallopeptidase 9, olfactomedin 4, NB1 glycoprotein and lipocalin 2) were previously identified as predictive for severity of disease caused by infection with respiratory syncytial virus (RSV). The reproducibility and robustness of the prototype assay was determined using the blood samples of 21 healthy donors. The data showed that the Idylla™ platform allows for a fast and user-friendly determination of the relative expression levels of the four selected mRNA markers.

Olfactomedin 4 Serves as a Marker for Disease Severity in Pediatric Respiratory Syncytial Virus (RSV) Infection.

BACKGROUND: Respiratory viral infections follow an unpredictable clinical course in young children ranging from a common cold to respiratory failure. The transition from mild to severe disease occurs rapidly and is difficult to predict. The pathophysiology underlying disease severity has remained elusive. There is an urgent need to better understand the immune response in this disease to come up with biomarkers that may aid clinical decision making. METHODS: In a prospective study, flow cytometric and genome-wide gene expression analyses were performed on blood samples of 26 children with a diagnosis of severe, moderate or mild Respiratory Syncytial Virus (RSV) infection. Differentially expressed genes were validated using Q-PCR in a second cohort of 80 children during three consecutive winter seasons. FACS analyses were also performed in the second cohort and on recovery samples of severe cases in the first cohort. RESULTS: Severe RSV infection was associated with a transient but marked decrease in CD4+ T, CD8+ T, and NK cells in peripheral blood. Gene expression analyses in both cohorts identified Olfactomedin4 (OLFM4) as a fully discriminative marker between children with mild and severe RSV infection, giving a PAM cross-validation error of 0%. Patients with an OLFM4 gene expression level above -7.5 were 6 times more likely to develop severe disease, after correction for age at hospitalization and gestational age. CONCLUSION: By combining genome-wide expression profiling of blood cell subsets with clinically well-annotated samples, OLFM4 was identified as a biomarker for severity of pediatric RSV infection.

Density and duration of experimental human pneumococcal carriage.

The density and duration of pneumococcal carriage are considered to affect the likelihood of transmission and invasive disease. Because of its importance in both spreading and causing disease, carriage has been suggested as an endpoint in future vaccine studies. Culture is the current gold standard for detection, but may not be sensitive enough to detect changes at low density. Healthy adult volunteers received an intranasal inoculation of Streptococcus pneumoniae serotype 6B. Pneumococcal density in nasal washes collected at six time-points post-inoculation was determined by culture and quantitative PCR (qPCR). Natural pneumococcal carriers detected at initial screening were followed in parallel. In 331 nasal washes from 79 volunteers, the sensitivity and specificity of pneumococcal detection by qPCR, as compared with culture, were 92.3% and 75.9%. The estimation of pneumococcal density by culture and qPCR was highly correlated (rs  = 0.73, p <0.0001), although qPCR had a lower detection limit. Pneumococcal density fluctuated within a carriage episode, and occasionally fell below the detection limit of both methods. The duration of carriage episodes was underestimated when only one method was used. Similar fluctuations in density were observed in natural carriers. Pneumococcal carriage is a dynamic event. Culture and qPCR are complementary for surveying the density and duration of pneumococcal carriage episodes.

Diagnostic value of serum pneumococcal DNA load during invasive pneumococcal infections.

Detection of pneumococcal DNA in blood could be a fast alternative for blood culture in invasive pneumococcal disease (IPD). In this study we compared the diagnostic value of the serum pneumococcal DNA load between different clinical syndromes in adults with bacteremic pneumococcal infections, also after initiation of antibiotic treatment. Adults hospitalized with a blood culture proven pneumococcal infection between December 2008 and June 2013 were retrospectively included. Pneumococcal DNA loads in corresponding serum samples were determined by qPCR. Data on clinical diagnosis, course of disease and antibiotic treatment were extracted from medical records. For 53 IPD cases eligible stored serum samples were retrieved. The proportion of samples positive in qPCR was lower in uncomplicated pneumonia compared with other clinical syndromes (59.5 % vs. 100 %, p = 0.005). The pneumococcal DNA load was higher in cases other than uncomplicated pneumonia (p = 0.043) as well as in more severe disease (p-values 0.018, 0.029 and 0.003 for PSI Risk Class IV/V, ICU admission and mortality, respectively). Both detection of pneumococcal DNA and distribution of load did not significantly change over the first days of hospitalization despite treatment with appropriate antibiotics. Detection of pneumococcal DNA in serum was more sensitive in clinical syndromes other than uncomplicated pneumonia. Furthermore, the pneumococcal DNA load was associated with the type of IPD and severity of disease. Since the serum pneumococcal DNA load seemed unaffected by antibiotic treatment during the first days of IPD, it may offer an alternative for culture methods after prior antibiotic use.

Severe viral respiratory infections: are bugs bugging?

Viral respiratory tract infections (RTI) pose a high burden on the youngest members of our society. Several risk factors are known for severe viral respiratory disease. However, a large proportion of the severe RTI cannot be explained by these risk factors. A growing body of evidence shows that the composition of the microbiota has a major influence on the training of both the mucosal and the systemic immune response and can thus potentially determine susceptibility for severe viral infections. In this review, we discuss the current evidence regarding the influence of bacterial colonization on the severity of viral respiratory infections.

NOD2 3020insC mutation and the pathogenesis of Crohn's disease: impaired IL-1beta production points to a loss-of-function phenotype.

BACKGROUND: Mutations of the NOD2 gene increase the susceptibility of humans to Crohn's disease. NOD2 is a cytoplasmic receptor for the bacterial product peptidoglycan. There is considerable controversy in the literature whether the most common mutation in Crohn's disease, the 3020insC NOD2, leads to a loss of function, i.e. decreased cytokine production, or to the reverse, i.e. a gain of function. In previous papers we proposed the former, since we could show decreased cytokine production with a net proinflammatory status after exposure to muramyl dipeptide (MDP). METHODS: Because of recent data in the literature showing increased interleukin-beta (IL-1beta) production in mice with the corresponding NOD2 mutation, we investigated the production of this cytokine by cells of patients with Crohn's disease, either homozygous or heterozygous for the 3020insC mutation, and compared it with that of patients with Crohn's disease bearing the wild-type allele. RESULTS: A strongly decreased production of IL-1beta by peripheral mononuclear cells was found upon exposure to either peptidoglycan or peptidoglycan-derived MDP in homozygous patients bearing the 3020insC NOD2mutation. CONCLUSION: This sustains the hypothesis that the 3020insC mutation in the human NOD2 gene leads to a loss-of-function phenotype.

Long term results of pneumatic dilation in achalasia followed for more than 5 years.

OBJECTIVE: We aimed to evaluate the long term therapeutic outcome in achalasia patients treated with pneumatic dilation, specifically focusing on those patients treated more than 15 yr ago. METHODS: All patients treated in our center whose records were available for review were asked to fill out a questionnaire assessing their degree of dysphagia, retrosternal pain, regurgitation, weight loss, and coughing during the night. The number of dilations was collected from the clinical records. The results of the treatment were classified into four different classes (excellent, good, moderate, poor). For those patients who had died, the cause of death was ascertained from the medical records or from the general practitioner. RESULTS: The questionnaires were distributed to 249 patients, 32 of whom had died. Of the 125 patients who completed the questionnaire, 81 (45 male and 36 female) were treated more than 5 yr ago. The mean follow-up was 12+/-1 yr. The therapeutic success rate was 50%, obtained after a median of four dilations (interquartile range = 3-6). Of this cohort, 25 patients (18 male and seven female, aged 35-84 yr) were treated more than 15 yr ago (mean follow-up = 20.5+/-0.5 yr). The median number of dilations was four (interquartile range = 3-7), with a therapeutic success rate of 40%. Two patients experienced a perforation, and seven were referred for surgery. Six patients out of 32 (19%) died of esophageal cancer. CONCLUSIONS: The long term success rate of pneumatic dilation is rather low, resulting in permanent successful treatment of achalasia in only 40-50% of patients. Achalasia is a risk factor for esophageal cancer.

Nicotinic acetylcholine receptor blocking effect of guanethidine in the rat gastric fundus.

1 Guanethidine is commonly used as a drug to investigate adrenergic neurotransmission and, in combination with atropine, to realize non-adrenergic non-cholinergic (NANC) conditions. Previous studies suggested a nicotinic acetylcholine receptor blocking effect of guanethidine. Therefore, we investigated the effect of increasing concentrations of guanethidine (0.1-100 microM) on nicotine-induced relaxations of longitudinal muscle strips of rat gastric fundus. 2 In the presence of 1 microM atropine and 3 microM guanethidine, nicotine (30 microM) induces a fast and sustained relaxation which is partly inhibited by the nitric oxide synthase inhibitors Nomega-nitro-L-arginine (L-NOARG) and Nomega-nitro-L-arginine methyl ester (L-NAME) (both 30 and 100 microM). One microM tetrodotoxin (TTX) completely blocks this nicotine-induced relaxation. 3 High concentrations of guanethidine (> or =10 microM), but not adrenoceptor blockade by the alpha-adrenoceptor antagonist phentolamine in combination with the beta-adrenoceptor antagonist nadolol (both 3 microM), inhibit the nicotine-induced relaxation. 4 Guanethidine (0.1-100 microM) has no effect on relaxations induced by electrical field stimulation (EFS; 1-8 Hz), nitric oxide (NO; 0.01-1 microM), vasoactive intestinal polypeptide (VIP; 0.1-10 nM) or isoprenaline (1-10 nM). 5 We conclude that high concentrations of guanethidine (> or =10 microM) block nicotine-induced NANC relaxations of longitudinal muscle strips of the rat gastric fundus most likely at the level of the nicotinic acetylcholine receptor.