Optimization of Standard In-House 24-Locus Variable-Number Tandem-Repeat Typing for Mycobacterium tuberculosis and Its Direct Application to Clinical Material

Variable-number tandem-repeat (VNTR) typing with a panel of 24 loci is the current gold standard in the molecular typing of Mycobacterium tuberculosis complex isolates. However, because of technical problems, a part of the loci often cannot be amplified by multiplex PCRs. Therefore, a considerable number of single-locus PCRs have to be performed for the loci with missing results, which impairs the laboratory work flow. Therefore, the original in-house method described by Supply et al. in 2006 was reevaluated. We modified seven primers and the PCR master mixture and obtained a strongly optimized in-house 24-locus VNTR typing method. The percentage of instantly complete 24-locus VNTR patterns detected in the routine flow of typing activities increased to 84.7% from the 72.3% obtained with the typing conducted with the commercially available Genoscreen MIRU-VNTR typing kit. The analytical sensitivity of the optimized in-house method was assessed by serial dilutions of M. tuberculosis in bronchoalveolar lavage fluid. A 1:10 dilution of the different strains tested was the lowest dilution for the detection of a complete 24-locus VNTR pattern. The optimized in-house 24-locus VNTR typing method will reduce the turnaround time of typing significantly and also the financial burden of these activities.     

The effectiveness of a treatment protocol for male lower urinary tract symptoms in general practice: a practical randomised controlled trial

BACKGROUND: Randomised controlled trials have shown the efficacy of several treatment modalities for lower urinary tract symptoms (LUTS) in selected populations. The effectiveness in daily practice has hardly been investigated, especially in primary care and is dependent on choices between all possible treatment options and best investigated in a comprehensive study, including all treatment modalities (watchful waiting, alpha-blockers, 5-alpha-reductase inhibitors, and surgery). AIM: Assessment of the effectiveness of a comprehensive treatment protocol for LUTS in primary care. DESIGN OF STUDY: Randomised controlled trial. SETTING: Fourteen general practices in the Netherlands. METHOD: Intervention: treatment protocol based on a formalised expert opinion. Control condition: usual care. Study population: 208 subjects with moderate to severe LUTS (IPSS > or =8, median = 13). OUTCOME MEASURES: symptom severity (IPSS [International Prostate Symptom Score]), bother score (Dan-PSS [Danish Prostate Symptom Score]), and maximum urinary flow (Q(max)); incidence of acute urinary retention and urinary tract infections. RESULTS: In the intervention group markedly more subjects used an alpha-blocker at end of follow-up than in the usual care group (24% versus 6%). No significant differences were found between intervention and control group in IPSS, Q(max) or Dan-PSS. CONCLUSION: alpha-blockers and watchful waiting are the most frequent treatment modalities for LUTS in primary care. Our study showed no evidence that a protocol using well-defined indications for all possible treatment modalities based on a formalised expert opinion procedure has added value. Based on our results, we cannot recommend a broadening of the indication for alpha-blockers, which, however, seems to be the current trend.     

Chronic obstructive pulmonary disease is associated with enhanced bronchial expression of FGF-1, FGF-2, and FGFR-1

An important feature of chronic obstructive pulmonary disease (COPD) is airway remodelling, the molecular mechanisms of which are poorly understood. In this study, the role of fibroblast growth factors (FGF-1 and FGF-2) and their receptor, FGFR-1, was assessed in bronchial airway wall remodelling in patients with COPD (FEV1 < 75%; n = 15) and without COPD (FEV1 > 85%; n = 16). FGF-1 and FGFR-1 were immunolocalized in bronchial epithelium, airway smooth muscle (ASM), submucosal glandular epithelium, and vascular smooth muscle. Quantitative digital image analysis revealed increased cytoplasmic expression of FGF-2 in bronchial epithelium (0.35 +/- 0.03 vs 0.20 +/- 0.04, p < 0.008) and nuclear localization in ASM (p < 0.0001) in COPD patients compared with controls. Elevated levels of FGFR-1 in ASM (p < 0.005) and of FGF-1 (p < 0.04) and FGFR-1 (p < 0.001) in bronchial epithelium were observed. In cultured human ASM cells, FGF-1 and/or FGF-2 (10 ng/ml) induced cellular proliferation, as shown by [3H]thymidine incorporation and by cell number counts. Steady-state mRNA levels of FGFR-1 were elevated in human ASM cells treated with either FGF-1 or FGF-2. The increased bronchial expression of fibroblast growth factors and their receptor in patients with COPD, and the mitogenic response of human ASM cells to FGFs in vitro suggest a potential role for the FGF/FGFR-1 system in the remodelling of bronchial airways in COPD.     

Smooth muscle homeostasis in human atherosclerotic plaques through interleukin 15 signalling

Interleukin (IL)-15 is a cytokine that has a broad tissue distribution and is important in maintaining homeostasis of cells and stability of tissues. When II-15 is also expressed by vascular smooth muscle cells (SMC), which are the dominant type of cells in most atherosclerotic plaques, it could be important in maintaining plaque tissue integrity and hence resistance of plaques towards development of clinically relevant complications such as plaque rupture and thrombosis. In this study, IL-15 and IL-15Rα in vitro expression by coronary artery SMC was investigated using RT -PCR and FACS analysis. Immunohistochemistry was used to study in situ expression of IL-15 and IL-15R by SMC of human carotid artery atherosclerotic plaques. Multiplex ligand-dependent probe amplification (MLPA) was used to investigate the mRNA expression of 40 pro- and anti inflammatory genes after stimulating coronary SMC with IL-15. We found that atherosclerotic SMC express both IL-15 and its receptor IL-15R, and TNF-γ and TNF-α enhance IL-15R expression in cultured SMC. MLPA studies on SMC revealed enhanced expression of PDGF beta mRNA after IL15 stimulation. In conclusion, our data suggest that IL-15 may contribute to atherosclerotic plaque integrity by stimulation of smooth muscle cells, probably in a PDGF dependent fashion.     

Neutrophil extracellular traps cause airway obstruction during respiratory syncytial virus disease

Human respiratory syncytial virus (RSV) is the most important cause of severe lower respiratory tract disease (LRTD) in young children worldwide. Extensive neutrophil accumulation in the lungs and occlusion of small airways by DNA‐rich mucus plugs are characteristic features of severe RSV–LRTD. Activated neutrophils can release neutrophil extracellular traps (NETs), extracellular networks of DNA covered with antimicrobial proteins, as part of the first‐line defence against pathogens. NETs can trap and eliminate microbes; however, abundant NET formation may also contribute to airway occlusion. In this study, we investigated whether NETs are induced by RSV and explored their potential anti‐viral effect in vitro. Second, we studied NET formation in vivo during severe RSV–LRTD in infants and bovine RSV–LRTD in calves, by examining bronchoalveolar lavage fluid and lung tissue sections, respectively. NETs were visualized in lung cytology and tissue samples by DNA and immunostaining, using antibodies against citrullinated histone H3, elastase and myeloperoxidase. RSV was able to induce NET formation by human neutrophils in vitro. Furthermore, NETs were able to capture RSV, thereby precluding binding of viral particles to target cells and preventing infection. Evidence for the formation of NETs in the airways and lungs was confirmed in children with severe RSV–LRTD. Detailed histopathological examination of calves with RSV–LRTD showed extensive NET formation in dense plugs occluding the airways, either with or without captured viral antigen. Together, these results suggest that, although NETs trap viral particles, their exaggerated formation during severe RSV–LRTD contributes to airway obstruction. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Structural genomic variation in childhood epilepsies with complex phenotypes

A genetic contribution to a broad range of epilepsies has been postulated, and particularly copy number variations (CNVs) have emerged as significant genetic risk factors. However, the role of CNVs in patients with epilepsies with complex phenotypes is not known. Therefore, we investigated the role of CNVs in patients with unclassified epilepsies and complex phenotypes. A total of 222 patients from three European countries, including patients with structural lesions on magnetic resonance imaging (MRI), dysmorphic features, and multiple congenital anomalies, were clinically evaluated and screened for CNVs. MRI findings including acquired or developmental lesions and patient characteristics were subdivided and analyzed in subgroups. MRI data were available for 88.3% of patients, of whom 41.6% had abnormal MRI findings. Eighty-eight rare CNVs were discovered in 71 out of 222 patients (31.9%). Segregation of all identified variants could be assessed in 42 patients, 11 of which were de novo. The frequency of all structural variants and de novo variants was not statistically different between patients with or without MRI abnormalities or MRI subcategories. Patients with dysmorphic features were more likely to carry a rare CNV. Genome-wide screening methods for rare CNVs may provide clues for the genetic etiology in patients with a broader range of epilepsies than previously anticipated, including in patients with various brain anomalies detectable by MRI. Performing genome-wide screens for rare CNVs can be a valuable contribution to the routine diagnostic workup in patients with a broad range of childhood epilepsies.     

Prekallikrein inhibits innate immune signaling in the lung and impairs host defense during pneumosepsis in mice

Prekallikrein (PKK, also known as Fletcher factor and encoded by the gene KLKB1 in humans) is a component of the contact system. Activation of the contact system has been implicated in lethality in fulminant sepsis models. Pneumonia is the most frequent cause of sepsis. We sought to determine the role of PKK in host defense during pneumosepsis. To this end, mice were infected with the common human pathogen Klebsiella pneumoniae via the airways, causing an initially localized infection of the lungs with subsequent bacterial dissemination and sepsis. Mice were treated with a selective PKK-directed antisense oligonucleotide (ASO) or a scrambled control ASO for 3 weeks prior to infection. Host response readouts were determined at 12 or 36 h post-infection, including genome-wide messenger RNA profiling of lungs, or mice were followed for survival. PKK ASO treatment inhibited constitutive hepatic Klkb1 mRNA expression by >80% and almost completely abolished plasma PKK activity. Klkb1 mRNA could not be detected in lungs. Pneumonia was associated with a progressive decline in PKK expression in mice treated with control ASO. PKK ASO administration was associated with a delayed mortality, reduced bacterial burdens, and diminished distant organ injury. While PKK depletion did not influence lung pathology or neutrophil recruitment, it was associated with an upregulation of multiple innate immune signaling pathways in the lungs already prior to infection. Activation of the contact system could not be detected, either during infection in vivo or at the surface of Klebsiella in vitro. These data suggest that circulating PKK confines pro-inflammatory signaling in the lung by a mechanism that does not involve contact system activation, which in the case of respiratory tract infection may impede early protective innate immunity. © 2019 Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.