Biomedical subjects
Publications and source records attributed to Stefan Borgmann.
In an initial period (< or =4 h) Toll-like receptor 4 (TLR4) signaling is required for Yersinia enterocolitica YopP-induced dendritic cell (DC) death. Later (>4 h), DC die independent of TLR4 signaling. In TLR4-deficient DC caspase 8 cleavage is delayed, indicating that TLR4 signaling accelerates caspase 8 activation, leading to DC death.
Yersinia enterocolitica (Ye) targets mouse dendritic cells (DCs) and inhibits their ability to trigger T cell activation. Here we have investigated whether Ye might interfere with antigen presentation in DCs. Infection of DCs with the Ye wild-type strain reduced OVA uptake by DCs as demonstrated by flow cytometry and confocal laser scan microscopy. In contrast, DCs infected with Yersinia outer protein P (YopP)-deficient mutant strain rapidly internalized OVA. Furthermore, transfection of DCs with YopP, but not with a cysteine protease deficient YopP-C172A mutant, reduced uptake of OVA. This finding suggests that YopP, a virulence factor of Ye, inhibits OVA uptake by DCs. By the use of MAPK inhibitors we provide evidence that YopP mediates reduction of OVA uptake by its ability to block MAPK signalling pathways in host cells. Using transferrin (Tf) as specific marker for clathrin-mediated endocytosis (CME) and lucifer yellow (LY) as specific marker for macropinocytosis (MP) we could show that YopP inhibits CME, whereas other Yops inhibit MP. In keeping with these data, activation and proliferation of OVA-specific T cells was reduced when DCs were treated with MAPK inhibitors. Together, our data demonstrate that (i) MAPK play an important role in antigen uptake by CME in DCs, and (ii) that YopP inhibits this pathway of antigen uptake in DCs, which might contribute to evasion of adaptive immunity.
Yersinia outer protein P (YopP) is a virulence factor of Yersinia enterocolitica that is injected into the cytosol of host cells where it targets MAP kinase kinases (MKKs) and inhibitor of kappaB kinase (IKK)-beta resulting in inhibition of cytokine production as well as induction of apoptosis in murine macrophages and dendritic cells (DC). Here we show that DC death was only partially prevented by the broad spectrum caspase inhibitor zVAD-fmk, indicating simultaneous caspase-dependent and caspase-independent mechanisms of cell death induction by YopP. Microscopic analyses and measurement of cell size demonstrated necrosis-like morphology of caspase-independent cell death. Application of zVAD-fmk prevented cleavage of procaspases and Bid, decrease of the inner transmembrane mitochondrial potential DeltaPsi(m) and mitochondrial release of cytochrome c. From these data we conclude that YopP-induced activation of the mitochondrial death pathway is mediated upstream via caspases. In conclusion, our results suggest that YopP simultaneously induces caspase-dependent apoptotic and caspase-independent necrosis-like death in DC. However, it has to be resolved if necrosis-like DC death occurs independently from apoptotic events or as an apoptotic epiphenomenon.
This report describes the isolation of a linezolid-resistant Enterococcus faecium and a carbapenem-resistant Acinetobacter baumannii from an infected open patella fracture after a car accident in southern Italy.
In 2001 two outbreak episodes (January-March and June-July) caused by vancomycin-resistant E. faecium (VRE) of the VanA-type were observed at a neonatal intensive care unit (NICU) of a university hospital in south-west Germany. To identify the initial source and the route of transmission environmental samples were examined as well as stool samples from patients and the staff. VRE was not found in environmental samples. However, stool samples from 24 hospitalised children tested positive and bacterial clonality was assessed by Sma1-based macro restriction analysis. Furthermore, esp gene and vancomycin resistance gene carriage were examined as well as bacteriocin production. PCR analysis showed that all 24 isolates carried vanA gene cluster, encoding resistance to vancomycin and teicoplanin. However, five of the vanA-positive isolates were resistant to vancomycin but not to teicoplanin. Only these five isolates produced bacteriocin, but in none of the isolates esp gene was detected. PFGE revealed that both outbreaks were caused by two different clones. The patient initiating the first episode, was identified whereas the origin of the second episode remained unknown. From one of the 40 staff stool samples VRE was isolated. This strain was related to the clone of the summer outbreak. In conclusion there were two independent episodes of self limiting VRE outbreaks and transmission on the ward is highly probable.
BACKGROUND: The activation of neutrophils and monocytes by ANCA, resulting in the release of reactive oxygen species and proteases like myeloperoxidase (MPO), is essential to the pathogenesis of ANCA-associated vasculitis. As the A allele of the G-463A MPO gene polymorphism is associated with diminished activity of MPO, it is conceivable that the presence of this allele protects against MPO-ANCA-associated vasculitis. METHODS: Allelic frequencies of the G-463A polymorphism were studied in 119 ANCA-associated vasculitis patients, 48 with MPO-ANCA and 71 with proteinase 3 (PR3)-ANCA. RESULTS: Allelic frequencies of MPO G-463A promoter polymorphism did not differ between MPO-ANCA- and PR3-ANCA-associated vasculitis patients. Moreover, allelic distribution was similar to that of the normal population. CONCLUSIONS: The data suggest that G-463A polymorphism does not seem to contribute to either MPO-ANCA- or PR3-ANCA-associated vasculitis formation.
Yersinia enterocolitica evades innate immunity by expression of a variety of pathogenicity factors. Therefore, adaptive immunity including CD4(+) T cells plays an important role in defense against Y. enterocolitica. We investigated whether Y. enterocolitica might target dendritic cells (DC) involved in adaptive T-cell responses. For this purpose, murine DC were infected with Y. enterocolitica wild-type and mutant strains prior to incubation with ovalbumin (OVA) as antigen and 5-(6)-carboxyfluorescein diacetate N-succinimidyl ester-labeled OVA-specific T cells from DO11.10 mice. While T-cell proliferation was partially affected by infection of DC with plasmid-cured and YopP-deficient Yersinia mutant strains, no T-cell proliferation occurred after infection of DC with wild-type Y. enterocolitica. Infection of DC with Y. enterocolitica wild type resulted in decreased up-regulation of major histocompatibility complex class II, CD54 (intercellular adhesion molecule 1), CD 80, and CD86 expression. Experiments with plasmid-cured Y. enterocolitica or a YopP-deficient mutant strain revealed that YopP accounts for inhibition of surface molecule expression. Wild-type Y. enterocolitica suppressed the release of KC, tumor necrosis factor alpha, interleukin-10 (IL-10), and IL-12 by DC, while infection of DC with plasmid-cured Y. enterocolitica or with the YopP-deficient mutant resulted in the production of these cytokines. Moreover, infection with wild-type Y. enterocolitica induced apoptosis in DC mediated by YopP. Apoptosis occurred despite translocation of NF-kappaB to the nucleus, as demonstrated by electromobility shift assays. Together, these data demonstrate that Y. enterocolitica targets functions of murine DC that are required for T-cell activation. This might contribute to evasion of adaptive immune responses by Y. enterocolitica.
The passage of leukocytes through basement membranes involves proteolytic degradation of extracellular matrix (ECM) components executed by focalized proteolysis. We have investigated whether the migration of leukocytes through 3-dimensional collagenous tissue scaffolds requires similar ECM breakdown. Human T blasts and SupT1 lymphoma cells expressed mRNA of MMP-9, MT1-MMP, MT4-MMP, cathepsin L, uPA, and uPAR as well as ADAM-9, -10, -11, -15, and -17. Upon long-term migration within 3-dimensional collagen matrices, however, no in situ collagenolysis was obtained by sensitive fluorescein isothiocyanate-collagen fragmentation analysis and confocal fluorescence/backscatter microscopy. Consistent with nonproteolytic migration, T-cell crawling and path generation were not impaired by protease inhibitor cocktail targeting MMPs, serine proteases, cysteine proteases, and cathepsins. Dynamic imaging of cell-ECM interactions showed T-cell migration as an amoeba-like process driven by adaptive morphology, crawling along collagen fibrils (contact guidance) and squeezing through pre-existing matrix gaps by vigorous shape change. The concept of nonproteolytic amoeboid migration was confirmed for multicomponent collagen lattices containing hyaluronan and chondroitin sulfate and for other migrating leukocytes including CD8+ T blasts, monocyte-derived dendritic cells, and U937 monocytic cells. Together, amoeboid shape change and contact guidance provide constitutive protease-independent mechanisms for leukocyte trafficking through interstitial tissues that are insensitive toward pharmacologic protease inhibitors.
BACKGROUND: Small-vessel vasculitides are associated with antineutrophil cytoplasmic antibodies (ANCAs). Cytoplasmic ANCAs are targeted mainly against proteinase 3 (PR3), whereas myeloperoxidase (MPO) is the major antigen of perinuclear ANCAs. These relapsing vasculitides show heterogeneous clinical pictures, and disease severity may vary broadly from mild local organ manifestation to acute organ failure (eg, renal failure). We tested whether two cytokine polymorphisms in the interleukin-1beta (IL-1beta) and IL-1 receptor antagonist (IL-1ra) genes, known to determine cytokine secretion, are associated with clinical manifestations and outcome of ANCA-associated vasculitides. METHODS: Polymerase chain reaction and restriction fragment length polymorphism analyses were performed to determine polymorphisms in the IL-1beta and IL-1ra genes in 79 patients with PR3-ANCA, 30 patients with MPO-ANCA vasculitis, and 196 healthy controls. RESULTS: The frequency of the so-called proinflammatory genotype, characterized by high secretion of IL-1beta and low secretion of its antagonist IL-1ra, was increased significantly in patients with PR3-ANCA with end-stage renal disease. CONCLUSION: Patients with a renal manifestation of PR3-ANCA vasculitis have an increased risk for developing end-stage renal disease when carrying the proinflammatory IL-1beta/IL-1ra genotype. Anti-inflammatory therapy specifically antagonizing the proinflammatory effect of IL-1beta may be a promising treatment for patients with Wegener's granulomatosis with renal manifestations.
Increased prevalence of small-sized low-density lipoprotein (LDL) subclass B (diameter < 25.5 nm) possibly is involved in the multifactorial process of cardiovascular disease in patients with end-stage renal disease. Given these epidemiological observations, mechanisms underlying the combined effect of a proinflammatory insult and LDL of different subclasses (subclass A, diameter > 25.5 nm, and subclass B) in a cellular model were investigated. For this, human umbilical vein endothelial cells were preexposed to LDL, then stimulated with tumor necrosis factor-alpha (TNF-alpha). Modulatory effects of LDL phenotypes on the activation of adhesion molecules, monocyte adherence, and transcriptional activity of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) were investigated. Our data show that subclass B LDLs were metabolized through nonspecific scavenger receptors and specific LDL-receptor pathways in endothelial cells. Furthermore, LDL subclass B in comparison to subclass A more effectively enhanced monocyte recruitment and adhesive properties of endothelial cells in response to TNF-alpha. These effects appeared not to be mediated by oxidative stress-responsive NF-kappaB because modulation of this transcription factor by LDL was moderate and similar for both LDL phenotypes. Conversely, effects of LDL subclass B were considered to be caused by augmented AP-1 binding activity. In conclusion, the present model provides new clues in atherogenic mechanisms of small-sized LDLs, which sensitize vascular cells to inflammatory signals more effectively than normal-sized LDLs.
In patients with PR3-ANCA associated vasculitides the carrier frequency of alpha1-antitrypsin (AAT) deficiency allele PI*Z is increased and linkage disequilibrium between polymorphic markers within a cluster of serine protease inhibitor (serpin) genes, including AAT gene, at chromosome 14q32.1 has been described. A1-antichymotrypsin (AACT), part of an extended serpin gene cluster, was discussed to contribute to PR3-ANCA associated vasculitis formation. To analyse, if an AACT gene polymorphism within the signal peptide sequence is associatedwith antineutrophil cytoplasm autoantibodies (ANCA) vasculitis allelic frequencies of AACT polymorphism were analysed in 128 control persons, 79 PR3-ANCA, and 30 MPO-ANCA patients. In MPO-ANCA patients also phenotyping of AAT was performed as well as frequency and linkage analysis of simple tandem repeat polymorphisms in the genes of cortisol-binding globulin, AAT, protein C inhibitor, and three extragenic markers (S48, S55, S51) of the gene cluster. Allelic frequencies of the AACT polymorphism did not differ between controls and vasculitis patients. In the MPO-ANCA group no patient expressed the Pi*Z defective allele of AAT and the allelic frequencies of polymorphic markers within the serpin gene cluster did not differ from those of the controls. Strong linkage disequilibrium was detected in MPO-ANCA patients neither. Therefore, we can not support the hypothesis that AACT polymorphism contributes to the pathogenesis of PR3-ANCA vasculitis. Nor is it probable that any factor, coded by the serpin gene cluster, contributes to MPO-ANCA vasculitis.