Phenotyping renal leukocyte subsets by four-color flow cytometry: Characterization of chemokine receptor expression

To investigate mechanisms of cell-mediated injury in renal inflammatory disease it is critical to determine the surface phenotype of infiltrating renal leukocyte subsets. However, the cell-specific expression of many leukocyte receptors is difficult to characterize in vivo. Here, we report a protocol based on flow cytometry that allows simultaneous characterization of surface receptor expression on different subsets of infiltrating renal leukocytes. The described technique combines an adapted method to prepare single cell suspensions from whole kidneys with subsequent four-color flow cytometry. We recently applied this technique to determine the differential expression of murine chemokine receptors CCR2 and CCR5 on infiltrating renal leukocyte subsets. In this article, we summarize our current findings on the validity of the method as compared with immunohistology and in situ hybridization in two murine models of nonimmune ( obstructive nephropathy) and immune-mediated ( lupus nephritis) inflammatory renal disease. Flow cytometry analysis revealed an accumulation of CCR5-, but not CCR2-positive lymphocytes in inflamed kidneys, compared to the peripheral blood. Particularly renal CD8(+) cells expressed CCR5 (79% in obstructed kidneys, 90% in lupus nephritis). In both models, infiltrating renal macrophages were positive for CCR2 and CCR5. These data corresponded to immunohistological and in situ hybridization results. They demonstrate that flow cytometric analysis of single cell suspensions prepared from inflamed kidneys is a rapid and reliable technique to characterize and quantify surface receptor expression on infiltrating renal leukocyte subsets

Keratin 8 expression in head and neck epithelia

<p>Abstract</p> <p>Background</p> <p>The intermediate filament forming protein keratin 8 (K8) is a tumour-associated antigen, which was shown to be over-expressed in a variety of malignancies. Here, we present a study of K8 expression in squamous epithelia of the head and neck area, including normal mucosa, hyperplastic and dysplastic leukoplakia, carcinomas of different sub-localisations, and lymph node metastases.</p> <p>Methods</p> <p>K8 expression was assessed upon immunohistochemistry with specific antibodies in cryosections of primary tumours of the head and neck area.</p> <p>Results</p> <p>K8 expression was characteristic of transformed tissue and marked early stages of disease, <it>i.e. </it>dysplastic oral leukoplakia, but not normal or hyperplastic epithelium. With the exception of carcinomas of the larynx and the tongue, K8 expression also strictly differentiated carcinomas from normal epithelium of the same origin. Furthermore, K8^highwas characteristic of cells, which had detached from the sites of primary tumours and had been invading the surrounding tissue at the time point of surgery.</p> <p>Conclusion</p> <p>K8 is an excellent marker for head and neck malignancies, which allows for early detection as well as for visualisation of potentially disseminating tumour cells <it>in vivo</it>.</p

An empirical analysis on the practice of the leadership in the Brethren Churches in Bund Evangelisch-Freikirchliche Gemeinden in the German Democratic Republic from 1974 until 1989

German textIn dieser empirischen Untersuchung wird die Leitungskultur der Brüder-Gemeinden im Bund Evangelisch Freikirchlicher Gemeinden (BEFG) in der Deutschen Demokratischen Republik (DDR) dargestellt. Dafür wurden Informationen von Brüdern, die damals in Leitungsverantwortung standen, mittels eines schriftlichen Fragebogens eingeholt. Zusätzlich wurde aus diversen schriftlichen Quellen das Leitungsverständnis der Brüderbewegung in Deutschland während verschiedener Epochen eruiert. Anhand der verschiedenen Ergebnisse wird die Leitungskultur in den Brüder-Gemeinden im BEFG in der DDR kritisch bewertet. Diese qualitative und quantitative empirische Forschung will einen Beitrag zur Erforschung der Brüder-Gemeinden in der DDR leisten. Gleichzeitig zeigt sie auf, wie die Leitung einer christlichen Gemeinde in einem antichristlichen Überwachungsstaat funktioniert hat.This empirical study will show the style of leadership in the Brethren church of the “Bund Evangelisch Freikirchlicher Gemeinden” (BEFG) in the former German Democratic Republic (GDR). Information was obtained from brothers, who held a managing position during this time, by answering a written questionnaire. Studies of source material as well as thematic analyses were realized as part of this research to show an overall view of the understanding of leading the Brethren Church of BEFG in Germany in different epochs. The style of leadership will be critically evaluated based on the different results. This quantitative and qualitative study will not only contribute to the research of the BEFG in the former GDR but will also show how the leading of a parish in an antichristian surveillance state worked.Philosophy and Systematic TheologyM. Th. (Theological Ethics

Glutaconate CoA-transferase from Acidaminococcus fermentans: the crystal structure reveals homology with other CoA-transferases

AbstractBackground: Coenzyme A-transferases are a family of enzymes with a diverse substrate specificity and subunit composition. Members of this group of enzymes are found in anaerobic fermenting bacteria, aerobic bacteria and in the mitochondria of humans and other mammals, but so far none have been crystallized. A defect in the human gene encoding succinyl-CoA: 3-oxoacid CoA-transferase causes a metabolic disease which leads to severe ketoacidosis, thus reflecting the importance of this family of enzymes. All CoA-transferases share a common mechanism in which the CoA moiety is transferred from a donor (e.g. acetyl CoA) to an acceptor, (R)-2-hydroxyglutarate, whereby acetate is formed. The transfer has been described by a ping-pong mechanism in which CoA is bound to the active-site residue of the enzyme as a covalent thiol ester intermediate. We describe here the crystal structure of glutaconate CoA-transferase (GCT) from the strictly anaerobic bacterium Acidaminococcus fermentans. This enzyme activates (R)-2-hydroxyglutarate to (R)-2-hydroxyglutaryl-CoA in the pathway of glutamate fermentation. We initiated this project to gain further insight into the function of this enzyme and the structural basis for the characteristics of CoA-transferases.Results: The crystal structure of GCT was solved by multiple isomorphous replacement to 2.55 Å resolution. The enzyme is a heterooctamer and its overall arrangement of subunits can be regarded as an (AB)4tetramer obeying 222 symmetry. Both subunits A and B belong to the open α/β-protein class and can be described as a four-layered α/α/β/α type with a novel composition and connectivity of the secondary structure elements. The core of subunit A consists of seven α/β repeats resulting in an all parallel central β sheet, against which helices pack from both sides. In contrast, the centre of subunit B is formed by a ninefold mixed β sheet. Inboth subunits the helical C terminus is folded back onto the N-terminal domain to form the third layer of helices.Conclusions: The active site of GCT is located at the interface of subunits A and B and is formed by loops of both subunits. The funnel-shaped opening to the active site has a depth and diameter of about 20 Å with the catalytic residue, Glu54 of subunit B, at the bottom. The active-site glutamate residue is stabilized by hydrogen bonds. Despite very low amino acid sequence similarity, subunits A and B reveal a similar overall fold. Large parts of their structures can be spatially superimposed, suggesting that both subunits have evolved from a common ancestor

Inflammatory monocytes require type I interferon receptor signaling to activate NK cells via IL-18 during a mucosal viral infection

The requirement of type I interferon (IFN) for natural killer (NK) cell activation in response to viral infection is known, but the underlying mechanism remains unclear. Here, we demonstrate that type I IFN signaling in inflammatory monocytes, but not in dendritic cells (DCs) or NK cells, is essential for NK cell function in response to a mucosal herpes simplex virus type 2 (HSV-2) infection. Mice deficient in type I IFN signaling, Ifnar(-/-) and Irf9(-/-) mice, had significantly lower levels of inflammatory monocytes, were deficient in IL-18 production, and lacked NK cell-derived IFN-gamma. Depletion of inflammatory monocytes, but not DCs or other myeloid cells, resulted in lower levels of IL-18 and a complete abrogation of NK cell function in HSV-2 infection. Moreover, this resulted in higher susceptibility to HSV-2 infection. Although Il18(-/-) mice had normal levels of inflammatory monocytes, their NK cells were unresponsive to HSV-2 challenge. This study highlights the importance of type I IFN signaling in inflammatory monocytes and the induction of the early innate antiviral response

Enhanced Quantum Estimation via Purification

We analyze the estimation of a finite ensemble of quantum bits which have been sent through a depolarizing channel. Instead of using the depolarized qubits directly, we first apply a purification step and show that this improves the fidelity of subsequent quantum estimation. Even though we lose some qubits of our finite ensemble the information is concentrated in the remaining purified ones.Comment: 6 pages, including 3 figure