Detection of the Arg702Trp, Gly908Arg and Leu1007fsinsC polymorphisms of the NOD2/CARD15 gene by real-time PCR with melting curve analysis.

Crohn's disease is a complex disorder, with multiple genetic traits. A frameshift mutation (Leu1007fsinsC) and two missense mutations (Gly908Arg and Arg702Trp) in the NOD2/CARD15 gene are strongly associated with susceptibility to Crohn's disease. The presence of one of these risk alleles confers a 2- to 4-fold increase in the risk of developing Crohn's disease, and the presence of two mutant alleles increases the risk over 20-fold. To facilitate the analysis of these polymorphisms, we developed three LightCycler assays to detect the missense mutations Arg702Trp and Gly908Arg and the frameshift mutation Leu100fsinsC in the NOD2/ CARD15 gene. All three assays can be run simultaneously on one LightCycler using identical cycling parameters. Analysis of 53 DNAs from Crohn's patients helped to identify carriers at allele frequencies similar to other Caucasian populations. The sequencing of such DNAs confirmed the accuracy of the assays. In conclusion, we present three rapid and robust assays to detect the Arg702Trp, the Gly908Arg and the Leu1007fsinsC ins mutations in the NOD2/CARD15 gene [corrected]     

Problems and needs for improving primary care of osteoarthritis patients: the views of patients, general practitioners and practice nurses

Background  

Osteoarthritis (OA) is highly prevalent and has substantial impact on quality of life as well as on healthcare costs. The general practitioner (GP) often is the first care provider for patients with this chronic disease. The aim of this study was to identify health care needs of patients with OA and to reveal possible obstacles for improvements in primary care management of OA patients.     

In vivo measurement of the shear modulus of the human vocal fold: interim results from eight patients

The shear modulus of the vocal fold is an essential parameter required to enhance our understanding of how the vocal fold operates, to develop mathematical models of phonatation, and to provide benchmarks to quantify the effectiveness of surgical procedures. The authors announced the successful deployment of an instrument to measure vocal fold elasticity in vivo last year, and now present the data taken from eight patients in vivo. The shear modulus was measured at the mid-membranous point, in a transverse direction with respect to the axis drawn between the anterior commissure and vocal process. The range of mean shear modulus results is 701–2,225 Pa, with a mean value of 1,371 Pa.     

The effect of estrogens and dietary calcium deficiency on the extracellular matrix of articular cartilage in G?ttingen miniature pigs.

Clinical observations have suggested that estrogens are involved in the pathogenesis of postmenopausal osteoarthritis (OA). However, positive and negative associations between the incidence of OA and serum estrogen concentrations have been reported. In contrast to this, osteoporosis is regarded as a disease with a strong estrogen-dependent component. Moreover, there is an interaction between estrogen and calcium deficiency: calcium supplementation potentiates the effect of estrogen therapy. The present study was designed to investigate how estrogen deficiency affects the articular cartilage depending on calcium supply. The distribution of different types of glycosaminoglycans and collagens can be used as an indicator for extracellular matrix changes induced by estrogen deficiency. Different levels of dietary calcium were therefore fed to intact and ovariectomized G?ttingen miniature pigs for one year before articular cartilage was harvested. The histochemical staining for heavy sulfated glycosaminoglycans in the extracellular matrix of ovariectomized miniature pigs, especially of those fed with a low calcium diet, was stronger in comparison to intact animals. In intact animals type II-collagen was immunodetected in all zones of unmineralized and mineralized articular cartilage, while immunostaining for this protein was negative to weak in the deep radiated fiber zone of ovariectomized minipigs. These results suggest that the synthesis of heavy sulfated glycosaminoglycans and immunohistochemically detectable type II-collagen is possibly influenced by estrogen deficiency. In conclusion, under estrogen deficiency, the extracellular matrix of articular cartilage underwent similar changes to those observed in physiologically aging cartilage where keratan sulfate is increased as a heavy sulfated glycosaminoglycan.     

Genetic and phylogeographic structure of populations of<Emphasis Type="Italic"> Pulex simulans</Emphasis> (Siphonaptera) in Peru inferred from two genes (<Emphasis Type="Italic">CytB</Emphasis> and<Emphasis Type="Italic"> CoII</Emphasis>)

In this paper we discuss the potential usefulness of determining the phylogeographic and phylogenetic patterns of a vector for understanding the spread of pathogens or insecticide resistance. We do so using the example of Pulex simulans in Peru. Six populations from six different localities were investigated. Mitochondrial DNA sequences were obtained and branching patterns were inferred using phylogenetic reconstruction methods and nested clade analyses. Ten different haplotypes were discovered. Phylogenetic analysis revealed P. simulans in Peru as a monophyletic group, containing clades that were generally not geographically correlated. The data suggest that P. simulans is not a single genetic entity but rather that this species shows a high degree of intraspecific variation. Restricted gene flow with long distance dispersal coupled with range expansion and long distance colonization are likely to have contributed to the observed patterns of variation.     

Differentiation markers of Sertoli cells and germ cells in fetal and early postnatal human testis

The definition of the temporal sequence of appearance of fetal markers during prenatal and early postnatal development in Sertoli and germ cells may be important for understanding the mechanisms underlying their reexpression in disorders of the adult testis. For this reason, we studied the expression of Sertoli and germ cell markers in 25 human testes spanning a period from 8 gestational weeks to 4 years. Well-characterized antibodies were employed to anti-Müllerian hormone (AMH), cytokeratin 18 (CK18), vimentin (VIM), M2A-antigen (M2A), germ cell alkaline phosphatase (GCAP), and somatic angiotensin-converting enzyme (sACE) on formalin-fixed and microwave-pretreated paraffin sections. In Sertoli cells, AMH and VIM were consistently present. While VIM and CK18 were coexpressed in embryonic testes, CK18 was progressively downregulated and completely absent from the 20th gestational week. M2A was absent or moderately expressed in fetal Sertoli cells but increased during further development. In germ cells, M2A was consistently found in primordial germ cells (PGCs) as well as in M- and T₁-prespermatogonia. In contrast, sACE and GCAP were absent from PGCs but were a distinct feature of late M- and early T₁-prespermatogonia and appeared predominantly between the 18th and the 22nd gestational weeks. Both T₂-prespermatogonia and postnatal prespermatogonia were devoid of any marker. While CK18 represents a differentiation marker for fetal Sertoli cells, M2A, GCAP, and sACE can be used as differentiation markers for the discrimination of different germ cell types during human prespermatogenesis. Because various immunophenotypes reflect distinct differentiation stages, this knowledge may be important for understanding adult testicular pathology.