Effect of tissue fixatives on telomere length determination by quantitative PCR

Telomere length is a well established marker of cellular senescence and thus biological age. Quantitative PCR allows the determination even from very low amounts of tissue by using telomere specific and single copy gene primers. Comparing a directly processed tissue sample to a 4% formaldehyde fixed one showed a significantly reduced efficiency of PCR reactions (mainly in single copy gene experiments) in a storage time-dependent manner resulting in an artificial increase in reported relative telomere length. This effect was not seen when the tissue was stored in RNA later solution. In summary, telomere length determination from formaldehyde fixed material by quantitative PCR is not a reliable method. Unfortunately therefore, many easily accessible tissue samples from pathology laboratories are unsuitable for this technique.     

Gastrointestinal zygomycosis caused by Mucor indicus in a patient with acute traumatic brain injury.

We report a gastrointestinal infection caused by Mucor indicus in a patient with severe head injuries. Monotherapy with high-dose liposomal amphotericin B successfully eradicated the mucormycosis. Nevertheless, subsequently a hemicolectomy was necessary due to recurrent bleeding from a deep ulcer. Mucor indicus is an uncommon fungal pathogen, typically found in starters used for food fermentation. Reviewing other reports on Mucor indicus infections, primary gastrointestinal manifestations seem to be typical and indicate an oral route of infection.     

Linkage of the MHC to familial multiple sclerosis suggests genetic heterogeneity. The Multiple Sclerosis Genetics Group

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system. While its etiology is not well understood, genetic factors are clearly involved. Until recently, most genetic studies in MS have been association studies using the case-control design testing specific candidate genes and studying only sporadic cases. The only consistently replicated finding has been an association with the HLA-DR2 allele within the major histocompatibility complex (MHC) on chromosome 6. Using the genetic linkage design, however, evidence for and against linkage of the MHC to MS has been found, fostering suggestions that sporadic and familial MS have different etiologies. Most recently, two of four genomic screens demonstrated linkage to the MHC, although specific allelic associations were not tested. Here, a dataset of 98 multiplex families was studied to test for an association to the HLA-DR2 allele in familial MS and to determine if genetic linkage to the MHC was due solely to such an association. Three highly polymorphic markers (HLA-DR, D6S273 and TNFbeta) in the MHC demonstrated strong genetic linkage (parametric lod scores of 4.60, 2.20 and 1.24, respectively) and a specific association with the HLA-DR2 allele was confirmed (TDT; P < 0.001). Stratifying the results by HLA-DR2 status showed that the linkage results were limited to families segregating HLA-DR2 alleles. These results demonstrate that genetic linkage to the MHC can be explained by the HLA-DR2 allelic association. They also indicate that sporadic and familial MS share a common genetic susceptibility. In addition, preliminary calculations suggest that the MHC explains between 17 and 62% of the genetic etiology of MS. This heterogeneity is also supported by the minority of families showing no linkage or association with loci within the MHC.      

The relevance of non-excitable cells for cardiac pacemaker function

Age-dependent changes in the architecture of the sinus node comprise an increasing ratio between fibroblasts and cardiomyocytes. This change is discussed as a potential mechanism for sinus node disease. The goal of this study was to determine the mechanism through which non-excitable cells influence the spontaneous activity of multicellular cardiomyocyte preparations. Cardiomyocyte monolayers (HL-1 cells) or embryonic stem cell-derived cardiomyocytes were used as two- and three-dimensional cardiac pacemaker models. Spontaneous activity and conduction velocity (θ) were monitored by field potential measurements with microelectrode arrays (MEAs). The influence of fibroblasts (WT-fibs) was determined in heterocellular cultures of different cardiomyocyte and fibroblast ratios. The relevance of heterocellular gap junctional coupling was evaluated by the use of fibroblasts deficient for the expression of Cx43 (Cx43^−/−-fibs). The beating frequency and θ of heterocellular cultures depended negatively on the fibroblast concentration. Interspersion of fibroblasts in cardiomyocyte monolayers increased the coefficient of the interbeat interval variability. Whereas Cx43^−/−-fibs decreased θ significantly less than WT-fibs, their effect on the beating frequency and the beat-to-beat variability seemed largely independent of their ability to establish intercellular coupling. These results suggest that electrically integrated, non-excitable cells modulate the excitability of cardiac pacemaker preparations by two distinct mechanisms, one dependent and the other independent of the heterocellular coupling established. Whereas heterocellular coupling enables the fibroblast to depolarize the cardiomyocytes or to act as a current sink, the mere physical separation of the cardiomyocytes by fibroblasts induces bradycardia through a reduction in frequency entrainment.     

Automated extraction of genomic DNA from medically important yeast species and filamentous fungi by using the MagNA Pure LC system

A fully automated assay was established for the extraction of DNA from clinically important fungi by using the MagNA Pure LC instrument. The test was evaluated by DNA isolation from 23 species of yeast and filamentous fungi and by extractions (n = 28) of serially diluted Aspergillus fumigatus conidia (10(5) to 0 CFU/ml). Additionally, DNA from 67 clinical specimens was extracted and compared to the manual protocol. The detection limit of the MagNA Pure LC assay of 10 CFU corresponded to the sensitivity when DNA was extracted manually; in 9 of 28 runs, we could achieve a higher sensitivity of 1 CFU/ml blood, which was found to be significant (p 相似文献   

Pattern of time-dependent reduction of histologically determined infarct volume after focal ischaemia in mice

The mouse model of transcranial permanent occlusion of the middle cerebral artery (tpMCAO) is widely used in stroke research. Here we quantified infarct size using a conventional histological method at several post-ischaemic times, going beyond the commonly analysed period of up to 2 days, following artery occlusion. Two different mouse strains, which are widely used for pharmacological studies of neuroprotection and for genetic engineering, were used. A drill whole was made into the skull of anaesthetised mice and ischaemia was induced by electrocoagulation of the middle cerebral artery. In both mouse strains tested (C57Black/6 and NMRI), the measured infarct volumes decreased significantly during the first days after tpMCAO. Notably, 13 days after surgery, ischaemic and sham-operated animals had indistinguishably small lesions, which where in the range of only 5% of the infarct size on day 2 post-ischaemia. The standard method of calculating oedema and shrinkage correction provided no sufficient explanation for this significant decrease in infarct volume. There was, however, evidence that structural changes in the residual ipsilateral hemisphere may compromise the significance of results arising from the method of calculating oedema and shrinkage correction. In conclusion, our study indicates that the pronounced and fast, time-dependent decrease in histologically defined infarct volume can compromise results when studying the lasting neuroprotective effects of potential drugs.     

Expression of interleukin-8 gene in inflammatory bowel disease is related to the histological grade of active inflammation.

Interleukin-8 (IL-8) is a potent cytokine for recruitment and activation of neutrophils. To visualize its distribution in the intestinal mucosa and to understand better its possible role in the induction and promotion of inflammatory bowel disease, expression of the IL-8 gene was analyzed in resected bowel segments of 14 patients with active Crohn's disease or ulcerative colitis. In situ hybridization with IL-8 anti-sense RNA probes revealed strong and specific signals in the histologically affected mucosa. The number of cells expressing IL-8 gene correlated with the histological grade of active inflammation. In accordance with the characteristic histological signs of active disease, IL-8-expressing cells were diffusely distributed over the entire affected mucosa in patients with ulcerative colitis, whereas in patients with Crohn's disease, IL-8-expressing cells showed a focal distribution pattern. Cells expressing IL-8 were mainly located at the base of ulcers, in inflammatory exudates on mucosal surfaces, in crypt abscesses, and at the border of fistulae. Analysis of semi-serial sections pointed to macrophages, neutrophils, and epithelial cells as possible sources of this cytokine in active inflammatory bowel disease. We consistently failed to detect IL-8 messenger RNA in the mucosa of uninvolved bowel segments and in normal-appearing control mucosa of patients with colon cancer. In contrast, tissue specimens from two patients with acute appendicitis displayed IL-8-expressing cells in the mucosa. These results support the notion that IL-8 plays and important but nonspecific role in the pathogenesis of inflammatory bowel disease and that the production of IL-8 messenger RNA is restricted to areas with histological signs of inflammatory activity and mucosal destruction.     

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.