Bordetella Pertussis Toxin does not induce the release of pro-inflammatory cytokines in human whole blood

Pertussis Toxin (PTx) is one of the most important virulence factors of Bordetella pertussis, the cause of whooping cough. Therefore, the inactivated toxin is an obligatory constituent of acellular pertussis vaccines. It is described in the literature that both native PTx and recombinant Pertussis Toxin (PTg) activate human monocytes whereas others report an inhibition of mammalian monocytes during pertussis infection. B. pertussis, as a Gram-negative bacterium, harbours naturally lipopolysaccharide (LPS, also known as endotoxin), one of the strongest stimulators of monocytes. The latter is triggered via the interaction of endotoxin with inter alia the surface receptor CD14. Consequently, it is necessary to consider a potential contamination of Pertussis Toxin preparations with LPS. First, we determined the LPS content in different preparations of PTx and PTg. All preparations examined were contaminated with LPS; therefore, possible PTx- and PTg-driven monocyte activation independently of LPS was investigated. To meet these aims, we examined monocyte response to PTx and PTg while blocking the LPS receptor CD14 with a specific monoclonal antibody (anti-CD14 mAb). In addition, all toxin preparations examined underwent an LPS depletion. Our results show that it is contaminating LPS, not Pertussis Toxin, which activates human monocytes. Blocking the CD14 receptor prevents Pertussis Toxin-mediated induction of pro-inflammatory cytokines in human monocytes. The depletion of LPS from Pertussis Toxin leads to the same effect. Additionally, the PTx toxicity after LPS depletion procedure was confirmed by animal tests. In contrast, the original Pertussis Toxin preparations not treated as mentioned above generate strong monocyte activation. The results in this publication allow the conclusion that purified Pertussis Toxin preparations do not induce the release of pro-inflammatory cytokines in human whole blood.     

Combined characterization of microRNA and mRNA profiles delineates early differentiation pathways of CD133+ and CD34+ hematopoietic stem and progenitor cells

MicroRNAs (miRNAs) have been shown to play an important role in hematopoiesis. To elucidate the role of miRNAs in the early steps of hematopoiesis, we directly compared donor-matched CD133(+) cells with the more differentiated CD34(+) CD133(-) and CD34(-) CD133(-) cells from bone marrow on the miRNA and mRNA level. Using quantitative whole genome miRNA microarray and sequencing-based profiling, we found that between 109 (CD133(+) ) and 216 (CD34(-) CD133(-) ) miRNAs were expressed. Quantification revealed that the 25 highest expressed miRNAs accounted for 73% of the total miRNA pool. miR-142-3p was the highest expressed miRNA with up to 2,000 copies per cell in CD34(+) CD133(-) cells. Eighteen miRNAs were significantly differentially expressed between CD133(+) and CD34(+) CD133(-) cells. We analyzed their biological role by examining the coexpression of miRNAs and its bioinformatically predicted mRNA targets and luciferase-based reporter assays. We provide the first evidence for a direct regulation of CD133 by miR-142-3p as well as tropomyosin 1 and frizzled homolog 5 by miR-29a. Overexpression of miRNAs in CD133(+) cells demonstrated that miR-142-3p has a negative influence on the overall colony-forming ability. In conclusion, the miRNAs expressed differentially between the CD133(+) and CD34(+) CD133(-) cells are involved in inhibition of differentiation, prevention of apoptosis, and cytoskeletal remodeling. These results are highly relevant for stem cell-based therapies with CD133(+) cells and delineate for the first time how the stem cell character of CD133(+) cells is defined by the expression of specific miRNAs.     

Peanut-induced anaphylaxis in children and adolescents: Data from the European Anaphylaxis Registry

Background

Peanut allergy has a rising prevalence in high-income countries, affecting 0.5%–1.4% of children. This study aimed to better understand peanut anaphylaxis in comparison to anaphylaxis to other food triggers in European children and adolescents.

Methods

Data was sourced from the European Anaphylaxis Registry via an online questionnaire, after in-depth review of food-induced anaphylaxis cases in a tertiary paediatric allergy centre.

Results

3514 cases of food anaphylaxis were reported between July 2007 - March 2018, 56% in patients younger than 18 years. Peanut anaphylaxis was recorded in 459 children and adolescents (85% of all peanut anaphylaxis cases). Previous reactions (42% vs. 38%; p = .001), asthma comorbidity (47% vs. 35%; p < .001), relevant cofactors (29% vs. 22%; p = .004) and biphasic reactions (10% vs. 4%; p = .001) were more commonly reported in peanut anaphylaxis. Most cases were labelled as severe anaphylaxis (Ring&Messmer grade III 65% vs. 56% and grade IV 1.1% vs. 0.9%; p = .001). Self-administration of intramuscular adrenaline was low (17% vs. 15%), professional adrenaline administration was higher in non-peanut food anaphylaxis (34% vs. 26%; p = .003). Hospitalization was higher for peanut anaphylaxis (67% vs. 54%; p = .004).

Conclusions

The European Anaphylaxis Registry data confirmed peanut as one of the major causes of severe, potentially life-threatening allergic reactions in European children, with some characteristic features e.g., presence of asthma comorbidity and increased rate of biphasic reactions. Usage of intramuscular adrenaline as first-line treatment is low and needs to be improved. The Registry, designed as the largest database on anaphylaxis, allows continuous assessment of this condition.

     

ATP/IL‐33‐triggered hyperactivation of mast cells results in an amplified production of pro‐inflammatory cytokines and eicosanoids

IL‐33 and ATP are alarmins, which are released upon damage of cellular barriers or are actively secreted upon cell stress. Due to high‐density expression of the IL‐33 receptor T1/ST2 (IL‐33R), and the ATP receptor P2X7, mast cells (MCs) are one of the first highly sensitive sentinels recognizing released IL‐33 or ATP in damaged peripheral tissues. Whereas IL‐33 induces the MyD88‐dependent activation of the TAK1‐IKK2‐NF‐κB signalling, ATP induces the Ca²⁺‐dependent activation of NFAT. Thereby, each signal alone only induces a moderate production of pro‐inflammatory cytokines and lipid mediators (LMs). However, MCs, which simultaneously sense (co‐sensing) IL‐33 and ATP, display an enhanced and prolonged activation of the TAK1‐IKK2‐NF‐κB signalling pathway. This resulted in a massive production of pro‐inflammatory cytokines such as IL‐2, IL‐4, IL‐6 and GM‐CSF as well as of arachidonic acid‐derived cyclooxygenase (COX)‐mediated pro‐inflammatory prostaglandins (PGs) and thromboxanes (TXs), hallmarks of strong MC activation. Collectively, these data show that co‐sensing of ATP and IL‐33 results in hyperactivation of MCs, which resembles to MC activation induced by IgE‐mediated crosslinking of the FcεRI. Therefore, the IL‐33/IL‐33R and/or the ATP/P2X7 signalling axis are attractive targets for therapeutical intervention of diseases associated with the loss of integrity of cellular barriers such as allergic and infectious respiratory reactions.     

Ahnak1 abnormally localizes in muscular dystrophies and contributes to muscle vesicle release

Ahnak1 is a giant, ubiquitously expressed, plasma membrane support protein whose function in skeletal muscle is largely unknown. Therefore, we investigated whether ahnak would be influenced by alterations of the sarcolemma exemplified by dysferlin mutations known to render the sarcolemma vulnerable or by mutations in calpain3, a protease known to cleave ahnak. Human muscle biopsy specimens obtained from patients with limb girdle muscular dystrophy (LGMD) caused by mutations in dysferlin (LGMD2B) and calpain3 (LGMD2A) were investigated for ahnak expression and localization. We found that ahnak1 has lost its sarcolemmal localization in LGMD2B but not in LGMD2A. Instead ahnak1 appeared in muscle connective tissue surrounding the extracellular site of the muscle fiber in both muscular dystrophies. The entire giant ahnak1 molecule was present outside the muscle fiber and did only partially colocalize with CD45-positive immune cell infiltration and the extracelluar matrix proteins fibronectin and collagenVI. Further, vesicles shedded in response to Ca(2+) by primary human myotubes were purified and their protein content was analysed. Ahnak1 was prominently present in these vesicles. Electron microscopy revealed a homogenous population of vesicles with a diameter of about 150?nm. This is the first study demonstrating vesicle release from human myotubes that may be one mechanism underlying abnormally localized ahnak1. Taken together, our results define ahnak1 in muscle connective tissue as a novel feature of two genetically distinct muscular dystrophies that might contribute to disease pathology.     

Continuously moving table time‐of‐flight angiography of the peripheral veins

Time‐of‐flight (TOF) MR angiography allows for noninvasive vessel imaging. To overcome the limited volumetric coverage of standard TOF techniques, the aim of this study was to investigate the combination of TOF and continuously moving table (CMT) acquisitions for peripheral vein imaging based on image subtraction. Two acquisition strategies are presented: a simple two‐step method based on 2‐fold CMT acquisition and an advanced one‐step method requiring only one continuous scan. Image quality of both CMT TOF techniques was evaluated by semiquantitative image grading and by signal‐to‐noise ratio and contrast‐to‐noise ratio analysis for veins of the upper and lower leg in 10 healthy volunteers. Results were compared to a standard stationary two‐dimensional (2D) TOF multistation acquisition. Image grading revealed good image quality for both CMT TOF methods, thereby confirming the feasibility of axial 2D CMT TOF to assess the veins of the lower extremities during a single scan. Quantitative evaluation showed no significant difference in signal‐to‐noise ratio and contrast‐to‐noise ratio compared to the stationary experiment. Additional measurements in three patients with postthrombotic changes and varicosities demonstrated the clinical applicability of the presented methods. CMT TOF provides promising results and permits the detection of various pathologic changes of the venous system. Magn Reson Med 63:1219–1229, 2010. © 2010 Wiley‐Liss, Inc.     

Initial characterization of Chlamydophila (Chlamydia) pneumoniae cultured from the late-onset Alzheimer brain

Previous studies from this laboratory provided evidence that the intracellular bacterial pathogen Chlamydophila (Chlamydia) pneumoniae is present in the late-onset Alzheimer's disease (AD) brain. Here we report culture of the organism from two AD brain samples, each of which originated from a different geographic region of North America. Culturable organisms were detectable after one and two passages in HEp-2 cells for the two samples. Both isolates, designated Tor-1 and Phi-1, were demonstrated to be authentic C. pneumoniae using PCR assays targeting the C. pneumoniae-specific genes Cpn0695, Cpn1046, and tyrP. Assessment of inclusion morphology and quantitation of infectious yields in epithelial (HEp-2), astrocytic (U-87 MG), and microglial (CHME-5) cell lines demonstrated an active, rather than a persistent, growth phenotype for both isolates in all host cell types. Sequencing of the omp1 gene from each isolate, and directly from DNA prepared from several additional AD brain tissue samples PCR-positive for C. pneumoniae, revealed genetically diverse chlamydial populations. Both brain isolates carry several copies of the tyrP gene, a triple copy in Tor-1, and predominantly a triple copy in Phi-1 with a minor population component having a double copy. This observation indicated that the brain isolates are more closely related to respiratory than to vascular/atheroma strains of C. pneumoniae.     

Direct Susceptibility Testing of Mycobacterium tuberculosis for Pyrazinamide by Use of the Bactec MGIT 960 System

Pyrazinamide (PZA) is a key antituberculosis drug, yet no rapid susceptibility test is commercially available. PZA drug susceptibility testing (DST) was performed directly on sputum samples from 327 patients and compared with the indirect method by using the Bactec MGIT 960 system in the context of patient screening for participation in a drug trial. Compared to standard indirect PZA DST, direct DST was successful in only 59% of cases, but results obtained were highly accurate and available faster. Agreement between the direct and indirect methods varied from 90 to 100% in each laboratory. The median times for obtaining PZA results from the time when the specimen was collected ranged from 11 to 16 days for the direct test and 18 to 95 days for the indirect test across laboratories. The direct method is accurate and reproducible across laboratories. It can be expected to accelerate results in >50% of cases, but it cannot replace indirect DST for PZA. Phenotypic methods remain the gold standard for DST in drug trials. If future studies can optimize the method to decrease the number of uninterpretable results, direct MGIT DST could be the new phenotypic DST standard for clinical trials, providing more rapid detection of resistance to new drugs in experimental regimens.     

High Frequency of Pathogenic Rearrangements in SPG11 and Extensive Contribution of Mutational Hotspots and Founder Alleles

Biallelic loss‐of‐function mutations in SPG11 cause a wide spectrum of recessively inherited, neurodegenerative disorders including hereditary spastic paraplegia (HSP), amyotrophic lateral sclerosis, and Charcot‐Marie‐Tooth disease. By comprehensive screening of three large cohorts of HSP index patients, we identified 83 alleles with “small” mutations and 13 alleles that carry large genomic rearrangements. Including relevant data from previous studies, we estimate that copy number variants (CNVs) account for ～19% of pathogenic SPG11 alleles. The breakpoints for all novel and some previously reported CNVs were determined by long‐range PCR and sequencing. This revealed several Alu‐associated recombination hotspots. We also found evidence for additional mutational mechanisms, including for a two‐step event in which an Alu retrotransposition preceded the actual rearrangement. Apparently independent samples with identical breakpoints were analyzed by microsatellite PCRs. The resulting haplotypes suggested the existence of two rearrangement founder alleles. Our findings widen the spectra of mutations and mutational mechanisms in SPG11, underscore the pivotal role played by Alus, and are of high diagnostic relevance for a wide spectrum of clinical phenotypes including the most frequent form of recessive HSP.     

A comprehensive survey for polymorphisms in the bovine IFN-gamma gene reveals a highly polymorphic intronic DNA sequence allowing improved genotyping of Bovinae.

This study aimed to identify interferon-gamma (IFN-gamma) gene variants in cattle for diagnostic purposes. Therefore, the entire bovine IFN-gamma gene (BoIFNG) and 2605 bp of its promoter DNA were sequenced. The BoIFNG DNA sequence conforms to the published part of Bo-IFN-gamma cDNA. Primer extension experiments show the presence of a 5' extension of exon 1 by 42 nucleotides (nt). One SINE element (Bov-A2) is located in the 5'-region, and two SINE elements (Bov-tA, Bov-B) are contained in the 3'-region of BoIFNG. The variants were detected by comparative sequence analysis of PCR amplicons from different bovine species. Four polymorphic mononucleotide repeats are situated in the promoter and in intron 1. Four distinct series of single nucleotide polymorphisms (SNP) were found in functionally important regions of BoIFNG. The region between the two intron 1 microsatellites contains the highest density of SNPs in Bos taurus breeds. One G-T transversion in the coding region of exon 1 causes a Gly(14) to Val(14) exchange in the BoIFNG signal peptide of different bovine species. A G-A transition in exon 2 encodes a Ser(19) to Asn(19) change in the mature protein of the Tibetan yak. Genotyping of randomly sampled Holstein Friesian cows at selected SNPs and of both intron 1 microsatellites revealed two dominant BoIFNG microhaplotypes. The detected SNPs improve the recently reported genotyping system of cattle.