Chemotaxis of bone marrow derived eosinophils in vivo: A novel method to explore receptor‐dependent trafficking in the mouse

Here, we describe a novel method via which ex vivo cultured mouse bone marrow derived eosinophils (bmEos) can be adoptively transferred into recipient mice in order to study receptor‐dependent recruitment to lung tissue in vivo. Intratracheal instillation of recombinant human eotaxin‐2 (hCCL24) prior to introduction of bmEos via tail vein injection resulted in an approximately fourfold increase in Siglec F‐positive/CD11c‐negative eosinophils in the lungs of eosinophil‐deficient ΔdblGATA recipient mice compared with controls. As anticipated, bmEos generated from CCR3‐gene‐deleted mice did not migrate to the lung in response to hCCL24 in this model, indicating specific receptor dependence. BmEos generated from GFP‐positive BALB/c mice responded similarly to hCCL24 in vitro and were detected in lung tissue of BALB/c WT as well as BALB/c ΔdblGATA eosinophil‐deficient recipient mice, at approximately fourfold (at 5 h post‐injection) and approximately threefold (at 24 h postinjection) over baseline, respectively. Comparable results were obtained with GFP‐positive C57BL/6 bmEos responding to intratracheal hCCL24 in C57BL/6 ΔdblGATA recipient mice. The use of ex vivo cultured bmEos via one or more of these methods offers the possibility of manipulating bmEos prior to transfer into a WT or gene‐deleted recipient host. Thus, this chemotaxis model represents a novel and robust tool for pharmacological studies in vivo.     

Sensitive Detection of KRAS Mutations Using Enhanced‐ice‐COLD‐PCR Mutation Enrichment and Direct Sequence Identification

A number of methods allowing the detection of low levels of KRAS mutations have been developed in the last years. However, although these methods have become increasingly sensitive, they can rarely identify the mutated base directly without prior knowledge on the mutated base and are often incompatible with a sequencing‐based read‐out desirable in clinical practice. Here, we present a modified version of the ice‐COLD‐PCR assay called Enhanced‐ice‐COLD‐PCR (E‐ice‐COLD‐PCR) for KRAS mutation detection and identification, which allows the enrichment of the six most frequent KRAS mutations. The method is based on a nonextendable chemically modified blocker sequence, complementary to the wild‐type (WT) sequence leading to the enrichment of mutated sequences. This assay permits the reliable detection of down to 0.1% mutated sequences in a WT background. A single genotyping assay of the amplification product by pyrosequencing directly following the E‐ice‐COLD‐PCR is performed to identify the mutated base. This developed two‐step method is rapid and cost‐effective, and requires only a small amount of starting material permitting the sensitive detection and sequence identification of KRAS mutations within 3 hr. This method is applied in the current study to clinical colorectal cancer samples and enables detection of mutations in samples, which appear as WT using standard detection technologies.     

Combinations of self‐reported rhinitis,conjunctivitis, and asthma predicts IgE sensitization in more than 25,000 Danes

BackgroundAllergic rhinitis (AR), allergic conjunctivitis (AC), and asthma composing multiple phenotypes and improved understanding of these phenotypes and their respective risk factors are needed.ObjectivesThe objective of this study was to define the prevalence of AR, AC, and asthma and their association with allergen‐specific immunoglobulin E (sIgE) sensitization in a large cohort of blood donors and identify risk factors.MethodsFrom the nationwide population‐based Danish Blood Donor Study, 52,976 participants completed an electronic questionnaire including AR, AC, asthma, allergic predisposition, and childhood residence. Of these, 25,257 were additionally tested for sIgE to inhalation allergens (Phadiatop).ResultsThe prevalence of sIgE sensitization, AR, AC, and asthma was 30%, 19%, 15%, and 9%, respectively. The youngest birth cohorts had the highest prevalence of sIgE sensitization and symptoms of asthma, AR, and AC, and for asthma, they apparently experienced symptoms at an earlier age. The sIgE sensitization was positively associated with male sex. The sIgE seroprevalence was higher in participants with both AR and AC (ARC) than in participants with either AR or AC. Allergic predisposition and sIgE sensitization increased the risk of the diseases, while farm upbringing was associated with reduced prevalence of ARC, however, only in sIgE sensitized participants.ConclusionBirth year, childhood residence, sIgE sensitization, and allergic predisposition were associated with asthma, AR, and AC prevalence. Individuals with self‐reported ARC represent a primarily sIgE‐positive phenotype, while those with either AR or AC represent more diverse phenotypes.     

Treatment of temporomandibular joint luxation: a systematic literature review

Clinical Oral Investigations - To evaluate the effectiveness of surgical and nonsurgical treatment of temporomandibular joint (TMJ) luxation. This systematic literature review searched PubMed, the...