Biomedical subjects
A Krause
Publications and source records attributed to A Krause.
[Heterogeneity of Borrelia burgdorferi: etiopathogenetic relevance and clinical implications].
Of the ten different species of Borrelia (B.) burgdorferi sensu lato which have been characterized to date, only B. burgdorferi sensu stricto, B. garinii and B. afzelii have been identified as pathogenic in humans. It was suggested that different species possess different organotropisms and may preferentially cause distinct clinical manifestations of Lyme disease. Molecular analyses revealed a strong association of B. afzelii with the late cutaneous manifestation acrodermatitis chronica atrophicans, whereas B. garinii was predominantly identified in clinical samples from patients with neuroborreliosis. PCR-based analyses of samples from European patients with Lyme arthritis had given controversial results, but B. burgdorferi sensu stricto appears to be the major pathogen. The identity of the infecting species seems to be a major determinant in the pathogenesis of Lyme arthritis, although its complex immunopathological background and its clinical heterogeneity clearly indicate concomitant factors. Thus, characterization of the infecting organism at the species level on the one hand and linkage of clinical data with pathogenetically relevant immune parameters on the other, shall lead to a more precise understanding of the pathogenesis and the individual clinical course of Lyme borrelioses.
[Epidemiology and therapy of Lyme arthritis and other manifestations of Lyme borreliosis in Germany: results of a nation-wide survey].
AIM OF THE STUDY: Only little is known about the epidemiology of Lyme borreliosis in Germany. As an example, it is still unclear if there are regional differences in the incidence of Lyme disease in general or of certain clinical manifestations like Lyme arthritis. Moreover, standardization of diagnostic or therapeutic procedures does not exist. Therefore, a Germany-wide questionnaire-based survey was conducted in order to achieve more epidemiological data and to obtain more information about the diagnostic and therapeutic approaches of general practitioners and specialists. METHODS: A self-designed questionnaire was distributed along with two editions of the journal "Deutsches Arzteblatt" (which is delivered to every physician in Germany) and additionally by a pharmaceutical company. During the collection period from March 1, 1998 to February 28, 1999, patients with Lyme disease were reported and information was given about site of infection, diagnostic procedures, clinical symptoms, treatment, and outcome. RESULTS: Altogether 3935 patients were reported. Their mean age was 43.4 years with the peak incidences around the ages of 10 and 60 years. 37.3% of the questionnaires were sent in by general practitioners, 17.6% by dermatologists, 15.7% by pediatricians, 9.7% by internists, and 2.7% by neurologists. 83% of the patients did not have a special infecion risk. The most frequent clinical Lyme manifestation was erythema migrans (EM), which occurred in 50.9% of the patients. 21.3% suffered from general symptoms. Of special interest, 24.5% of the patients had Lyme arthritis (14.7% mon- or oligoarthritis, 9.8% polyarthritis). Therefore, arthritis was more frequently reported than neuroborreliosis (18.4%). Only 16% of the neuroborreliosis patients and 32% of the arthritis patients remembered having had an EM. 189 patients (4.8%) with lymphadenosis cutis benigna and 100 patients (2.5%) with acrodermatitis chronica atrophicans were reported. In 80.4% of the patients, positive Lyme serology was detected. In a few cases, the diagnosis was established by isolation of borreliae, PCR or histology. 3754 patients were treated by antibiotics. The most frequently used compounds were doxycycline (50.4%), followed by ceftriaxone (22.4%), amoxicillin (13.6%), penicillin (7%), and erythromycin (4.2%) with differences depending on clinical manifestations and specialization of the prescribing physician. In less than 10% of the cases, not evaluated or recommended therapeutic procedures were performed. DISCUSSION: Lyme disease is endemic throughuot Germany. The most frequent manifestations are EM, followed by Lyme arthritis and neuroborreliosis. Less than one third of patients suffering from disseminated or chronic Lyme disease remembered an EM. Most of the physicians taking part in this survey follow treatment recommendations concerning choice of antibiotics and treatment durations.
An integrated view of aromatase and its inhibition.
Aromatase inhibition has become a major treatment strategy for postmenopausal women with oestrogen-dependent breast cancer. Its optimal application is, however, dependent upon (i) the accurate identification of cancers which are ultimately dependent upon the activity of the aromatase enzyme, (ii) the use of the best method/inhibitor by which to blockade aromatase activity. The single best predictor of response to aromatase inhibitors is the presence of tumour oestrogen receptors; receptor-negative cancers rarely respond whereas those with high levels seem particularly likely to benefit. However, there is a need for additional discriminatory markers. The use of microarray technology coupled with neoadjuvant therapy is likely to yield promising candidate genes. The finding that, amongst peripheral tissues, the tumour itself may have high activity has led to the suggestion that the tumour aromatase measurements may be predictive; however, in situ studies and the lack of robust assays for tumour aromatase suggest that tumour aromatase may not be an influential marker. Whilst drugs such as anastrozole, exemestane, formestane and letrozole are all effective and specific inhibitors of aromatase, they differ in structure, potency and mechanism of action. Thus, differential sensitivity of tissues/tumours and non-cross resistance mean inhibitors are not equivalent and individual agents may have differing roles according to the setting in which they will be used. Aromatase inhibitors have evolved as key endocrine agents in the treatment of breast cancer. They offer the promise of rational treatment management based on the accurate identification of individual cohorts of tumours responsive to specific drugs.
Optimized protocol for linear RNA amplification and application to gene expression profiling of human renal biopsies.
Gene expression analysis using high-density cDNA or oligonucleotide arrays is a rapidly emerging tool for transcriptomics, the analysis of the transcriptional state of a cell or organ. One of the limitations of current methodologies is the requirement of a relatively large amount of total or polyadenylated RNA as starting material. Standard array hybridization protocols require 5-15 micrograms labeled RNA. To obtain these quantities from small amounts of starting RNA material, RNA can be amplified in a linear fashion. Here we introduce an optimized protocol for rapid and easy-to-use amplification of as little as 1 ng total RNA. Our analysis shows that this method is linear and highly reproducible and that it preserves similarities as well as dissimilarities between normal and disease-related samples. We applied this technique to the RNA expression profiling of human renal allograft biopsies with normal histology and compared them to the profiles of renal biopsies with histological evidence of chronic transplant nephropathy or chronic rejection. Among others, complement component C1r was found to be significantly up-regulated in chronic rejection and chronic transplant nephropathy biopsies compared to normal samples, while fructose-1,6-biphosphatase showed lower-than-normal expression.
Treatment of active ankylosing spondylitis with infliximab: a randomised controlled multicentre trial.
UNLABELLED: BACKGROUND Treatment options for patients with ankylosing spondylitis are few. We aimed to assess the effectiveness of infliximab, an antibody to tumour necrosis factor (TNF)-alpha, in treatment of such patients. METHODS: In this 12-week placebo-controlled multicentre study, we randomly assigned 35 patients with active ankylosing spondylitis to intravenous infliximab (5 mg/kg) and 35 to placebo at weeks 0, 2, and 6. One patient in the infliximab group was withdrawn from the study. Our primary outcome was regression of disease activity of at least 50%. To assess response, we used validated clinical criteria from the ankylosing spondylitis assessment working group, including disease activity (BASDAI), functional indices (BASFI), metrology (BASMI), and quality of life (short form 36). Analyses were done by intention to treat. FINDINGS: 18 (53%) of 34 patients on infliximab had a regression of disease activity at week 12 of at least 50% compared with three (9%) of 35 on placebo (difference 44% [95% CI 23-61], p<0.0001). Function and quality of life also improved significantly on infliximab but not on placebo (p<0.0001 and p<0.0001, respectively). Treatment with infliximab was generally well tolerated, but three patients had to stop treatment because of systemic tuberculosis, allergic granulomatosis of the lung, or mild leucopenia. INTERPRETATION: Our results show that treatment with infliximab is effective in patients with active ankylosing spondylitis. Since there are some potentially serious adverse effects, we recommend that this treatment mainly be used in co-operation with rheumatological centres.
Effects of K(ATP) channel modulators on acetylcholine release from guinea-pig isolated atria and small intestine.
The effects of K(ATP) channel blockers (glibenclamide, HMR 1883, HMR 1372) and openers (cromakalim, pinacidil, diazoxide) on the electrically-evoked (5 Hz) release of [(3)H]acetylcholine were studied in isolated guinea-pig atria and myenteric plexus-longitudinal muscle preparations which had been preincubated with [(3)H]choline. Atria: Cromakalim (0.3 microM and 1 microM), pinacidil (10 microM) and diazoxide (30 microM) significantly reduced the stimulation-evoked release of [(3)H]acetylcholine. The inhibition produced by cromakalim and pinacidil was prevented by 1 microM of either HMR 1883, HMR 1372 or glibenclamide. The blockers alone significantly increased the release at concentrations of 30 microM, whereas 1 microM and 10 microM had no effect. Myenteric plexus-longitudinal muscle preparation: The electrically-evoked release of [(3)H]acetylcholine was not affected by K(ATP) channel blockers or openers. In contrast, the contractions of the longitudinal muscle caused by electrical stimulation or by carbachol were strongly inhibited by 1 microM cromakalim which suggests that the relaxant effect of the K(ATP) channel openers is exclusively a direct effect on intestinal smooth muscle. The findings suggest that blockade of activated K(ATP) channels in vagal nerves of guinea-pig atria stimulates acetylcholine release, and that this effect may contribute to the antiarrhythmic actions of K(ATP) channel blockers. By contrast, release of acetylcholine from guinea-pig myenteric plexus is not modulated by K(ATP) channels which suggests heterogeneity of K(ATP) channel distribution in peripheral autonomic nerves.
Immunopathogenesis of rheumatic diseases in the context of neuroendocrine interactions.
Growing evidence supports the hypothesis that alterations of the stress response and interactions between the neuroendocrine and immune systems contribute to the pathogenesis of rheumatic diseases such as rheumatoid arthritis (RA). In particular, the hypothalamus-pituitary-adrenal (HPA) axis and the autonomic nervous system (ANS) are of special interest. Polymorphisms of the corticotropin-releasing hormone (CRH)-regulating region have been described recently. These polymorphisms are differentially distributed in RA patients and healthy subjects of various ethnic origin, thus supporting the hypothesis that they represent a new genetic marker for RA susceptibility. The decreased expression of beta(2)-adrenergic receptors (beta(2)-R) on lymphatic cells in rheumatic diseases like RA, together with an impaired influence of catecholamines on immune function in these patients, further underlines the concept of a dysfunction of the ANS in rheumatic diseases. Results from work in this field will provide more insight into the pathogenesis of RA and help to establish novel therapies for this chronic rheumatic disease.
Decreased catecholamine-induced cell death in B lymphocytes from patients with rheumatoid arthritis.
The expression of beta2-adrenergic receptors (beta2-R) on B lymphocytes and agonist-induced cAMP production is reduced in patients with rheumatoid arthritis (RA). To further study functional consequences of the diminished beta2-R density on B lymphocytes in RA patients, agonist-induced cell death was evaluated and compared to healthy controls. B lymphocytes from patients with RA and healthy controls were activated with anti-IgM-antibody. Coincubation was carried out with isoprenaline (iso, 0.001-10 microM). Apoptotic and necrotic cells were determined using Annexin-V and propidium-iodide staining. beta2-R-induced cell death in B cells from healthy volunteers was stimulated after 24 h (medium, 21.2 +/- 1.6%; iso, 34.6 +/- 4.4%; increase 59.3 +/- 10.1%). However, in RA patients the increase in cell death following beta2-R stimulation (21.8 +/- 8.9%) was significantly impaired (p = 0.02). Our data demonstrate that catecholamine-induced cell death after stimulation of beta2-R on B lymphocytes is decreased in RA patients, possibly contributing to the pathogenesis of the disease.
Regulation of beta2-adrenergic receptors on CD4 and CD8 positive lymphocytes by cytokines in vitro.
Increasing evidence points to a close relationship between the autonomic nervous system and the immune system. To further investigate mechanisms regulating beta2-adrenergic receptor (beta2R) expression in lymphocytes, the influence of cytokines on the density of beta2R on purified CD4+ and CD8+ lymphocytes was determined in vitro. beta2R were determined by means of a radioligand binding assay with (125I)iodocyanopindolol. CD4+ and CD8+ lymphocytes were incubated with catecholamines, interleukin 1beta (IL-1beta) and interleukin 2 (IL-2) for 6-72 h. The results demonstrate declining beta2R numbers on CD4+ and CD8+ lymphocytes in vitro augmented by epinephrine. IL-1beta has no effect on beta2R expression compared to medium. However, incubation with IL-2 resulted in an up-regulation of beta2R on CD8+ lymphocytes. Thus, the study demonstrates a differential regulation of beta2R on T-lymphocyte subpopulations with CD8+ lymphocytes being more susceptible to mechanisms of beta2R modulation than CD4+ lymphocytes. The findings further strengthen the concept of a close interplay between the autonomic nervous system and the immune system.
Cross-talk between IL-1 and IL-6 signaling pathways in rheumatoid arthritis synovial fibroblasts.
The balance between pro- and anti-inflammatory cytokines plays an important role in determining the severity of inflammation in rheumatoid arthritis (RA). Antagonism between opposing cytokines at the level of signal transduction plays an important role in many other systems. We have begun to explore the possible contribution of signal transduction cross-talk to cytokine balance in RA by examining the effects of IL-1, a proinflammatory cytokine, on the signaling and action of IL-6, a pleiotropic cytokine that has both pro- and anti-inflammatory actions, in RA synovial fibroblasts. Pretreatment with IL-1 suppressed Janus kinase-STAT signaling by IL-6, modified patterns of gene activation, and blocked IL-6 induction of tissue inhibitor of metalloproteases 1 expression. These results suggest that proinflammatory cytokines may contribute to pathogenesis by modulating or blocking signal transduction by pleiotropic or anti-inflammatory cytokines. The mechanism of inhibition did not require de novo gene activation and did not depend upon tyrosine phosphatase activity, but, instead, was dependent on the p38 stress kinase. These results identify a molecular basis for IL-1 and IL-6 cross-talk in RA synoviocytes and suggest that, in addition to levels of cytokine expression, modulation of signal transduction also plays a role in regulating cytokine balance in RA.
Species-specific and conserved epitopes on mouse and human E-selectin important for leukocyte adhesion.
Selectins are C-type, cell surface lectins that are key players in leukocyte adhesion to the blood vessel wall endothelium. We describe here epitopes for a series of novel monoclonal antibodies (moAbs), UZ4-UZ7, directed against mouse E-selectin. All four antibodies specifically bind to mouse E-selectin, but not to P- or L-selectin, and all inhibit the adhesion of granulocytes, peripheral blood lymphocytes, and promyelocytic HL-60 cells to cytokine-activated mouse endothelium. Three moAbs, UZ5, UZ7, and UZ6, specifically inhibit mouse E-selectin-mediated adhesion by binding to epitopes in domains CR1 or CR2. moAb UZ4 inhibits leukocyte adhesion to both human and murine endothelium activated with IL-1 or other proinflammatory stimuli. UZ4 is the first described moAb that detects an epitope in the lectin domain which is conserved in both murine and human E-selectin (CXKKKL), but is not present in the other members of the selectin family, P- and L-selectin. Interestingly, UZ5, UZ6, and UZ7 more efficiently interfere with lymphocyte than with granulocyte adhesion to cytokine-activated endothelium, while UZ4 completely blocks adhesion of PMN, lymphocytes, and HL-60 and U937 cell lines. The data suggest that E-selectin-ligand engagement differs between lymphocytes and PMN, and that these differences may be accentuated by the CR1 and CR2 domains in the E-selectin cell adhesion molecule.
Investigation of the cellular uptake of E-Selectin-targeted immunoliposomes by activated human endothelial cells.
In the present study the cellular uptake of targeted immunoliposomes by interleukin-1 activated human endothelial cells has been analysed by several spectroscopical and microscopical fluorescence techniques. Previous in vitro experiments demonstrated that the targeting of immunoliposomes to vascular selectins is a potential way for a selective drug delivery at inflammatory sites. In attempts to further adapt the targeting experiments to physiological conditions, we demonstrate that E-Selectin-directed immunoliposomes are able to bind their target cells under the simulated shear force conditions of capillary blood flow cumulatively for up to 18 h. In order to consequently follow the fate of liposomes after target binding, we analysed the route and degree of liposome internalization of the cells concentrating on cell activation state or various liposomal parameters, e.g., sterical stabilization, type of antibody or antibody coupling strategy. The use of NBD-labelled liposomes and subsequent fluorescence quenching outside the cells with dithionite show that circa 25% of the targeted immunoliposomes were internalized. According to inhibition experiments with agents that interfered with the endocytotic pathway, we found out that the major mechanism of liposome entry is endocytic. The entry involves, at least in part, receptor-mediated endocytosis via E-Selectin, a liposome accumulation in the endosomes and their acidification was proved by pyranine spectroscopic results. Furthermore, microscopical investigations demonstrate that also a fusion of liposomes with the cell membrane occurs followed by a release of entrapped calcein into the cytoplasm. These observations gain insight into the behaviour of E-Selectin-targeted immunoliposomes and indicate that these immunoliposomes have great potential to be used as drug carriers for intracellular drug delivery at inflammatory sites.
Human natriuretic peptides exhibit antimicrobial activity.
Here we describe a novel function for members of the well-characterized human natriuretic peptide family. Human "brain-type natriuretic peptide" (hBNP-32) as well as other members of this peptide class are antimicrobially active against Gram-positive and Gram-negative bacteria and yeast in a dose-dependent manner. This activity of natriuretic peptides is comparable to that of known antimicrobial peptides such as casocidins or magainins.
Levonorgestrel-releasing intrauterine devices.
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Simultaneous immunohistochemical detection of tumor cells in lymph nodes and bone marrow aspirates in breast cancer and its correlation with other prognostic factors.
PURPOSE: We studied the prognostic and predictive value of immunohistochemically detected occult tumor cells (OTCs) in lymph nodes and bone marrow aspirates obtained from node-negative breast cancer patients. All were classified as distant metastases-free using conventional staging methods. PATIENTS AND METHODS: A total of 484 patients with pT1-2N0M0 breast cancer and 70 with pT1-2N1M0 breast cancer and a single affected lymph node participated in our trial. Ipsilateral axillary lymph nodes and intraoperatively aspirated bone marrow were examined. All samples were examined for OTCs using monoclonal antibodies to cytokeratins 8, 18, 19. Immunohistological findings were correlated with other prognostic factors. The mean follow-up was 54 +/- 24 months. RESULTS: OTCs were detected in 180 (37.2%) of 484 pT1-2N0M0 patients: in the bone marrow of 126 patients (26.0%), in the lymph nodes of 31 patients (6.4%), and in bone marrow and lymph nodes of 23 (4.8%) patients. Of the 70 patients with pT1-2N1MO breast cancer and a single involved lymph node, OTCs were identified in the bone marrow of 26 (37.1%). The ability to detect tumor cells increased with the following tumor features: larger size, poor differentiation, and higher proliferation. Tumors of patients with OTCs more frequently demonstrated lymph node invasion, blood vessel invasion, higher urokinase-type plasminogen activator levels, and increased PAI-1 concentrations. Patients with detected OTCs showed reduced disease-free survival (DFS) and overall survival (OAS) rates that were comparable to those observed in patients who had one positive lymph node. Multivariate analysis of prognostic factors revealed that OTCs, histological grading, and tumor size are significant predictors of DFS; OTCs and grading of OAS. CONCLUSION: OTCs detected by simultaneous immunohistochemical analysis of axillary lymph nodes and bone marrow demonstrate independent metastatic pathways. Although OTCs were significantly more frequent in patients with other unfavorable prognostic factors, they were confirmed as an independent prognostic factor for pT1-2N0M0, R0 breast cancer patients.
Negative-feedback regulation of CD28 costimulation by a novel mitogen-activated protein kinase phosphatase, MKP6.
TCR and CD28 costimulatory receptor-cooperative induction of T cell IL-2 secretion is dependent upon activation of mitogen-activated protein (MAP) kinases. Using yeast-hybrid technology, we cloned a novel CD28 cytoplasmic tail (CD28 CYT) interacting protein, MAP kinase phosphatase-6 (MKP6), which we demonstrate inactivates MAP kinases. Several lines of evidence indicate that MKP6 plays an important functional role in CD28 costimulatory signaling. First, in human peripheral blood T cells (PBT), expression of MKP6 is strongly up-regulated by CD28 costimulation. Second, transfer of dominant-negative MKP6 to PBT with the use of retroviruses primes PBT for the secretion of substantially larger quantities of IL-2, specifically in response to CD28 costimulation. A similar enhancement of IL-2 secretion is observed neither in response to TCR plus CD2 costimulatory receptor engagement nor in response to other mitogenic stimuli such as phorbol ester and ionomycin. Furthermore, this hypersensitivity to CD28 costimulation is associated with CD28-mediated hyperactivation of MAP kinases. Third, a retroviral transduced chimeric receptor with a CD28 CYT that is specifically unable to bind MKP6 costimulates considerably larger quantities of IL-2 from PBT than a similar transduced chimeric receptor that contains a wild-type CD28 CYT. Taken together, these results suggest that MKP6 functions as a novel negative-feedback regulator of CD28 costimulatory signaling that controls the activation of MAP kinases.
Insights from a novel three-dimensional in vitro model of lyme arthritis: standardized analysis of cellular and molecular interactions between Borrelia burgdorferi and synovial explants and fibroblasts.
OBJECTIVE: To develop a novel 3-dimensional (3-D) in vitro model of Lyme arthritis to use in the study of the interactions between Borrelia burgdorferi (Bb) and human synovial host cells with respect to phagocytosis and potential persistence of Bb as well as the induction of proinflammatory cytokines and chemokines. METHODS: Two distinct culture systems, consisting of synovial membrane explants or interactive synovial cells embedded in 3-D fibrin matrices, were chosen. Both systems were artificially infected with Bb, and the interactions between Bb and synovial tissue/cells were studied by histology, immunohistochemistry, and electron microscopy. Functional analyses included the induction/secretion of cytokines by Bb in the model system. RESULTS: Both culture systems proved to be stable and reproducible. The host cells and spirochetes showed high levels of viability and maintained their physiologic shape for >3 weeks. Bb invaded the synovial tissue and the artifical matrix in a time-dependent manner. Host cells were activated by Bb, as indicated by the induction of interleukin-1beta and tumor necrosis factor alpha. Electron microscopic analysis revealed Bb intracellularly within macrophages as well as synovial fibroblasts, suggesting that not only professional phagocytes, but also resident synovial cells are capable of phagocytosing Bb. Most interestingly, the uptake of the spirochetes appeared to cause severe damage of the synovial fibroblasts, since the majority of these cells displayed ultrastructural features of disintegration. CONCLUSION: A novel 3-D in vitro model has been established that allows the study of distinct aspects of Lyme arthritis under conditions that resemble the pathologic condition in humans. This reproducible, standardized model supplements animal studies and conventional 2-D cultures. The disintegration of synovial fibroblasts containing Bb or Bb fragments challenges the concept of an intracellular persistence of Bb and may instead reflect a mechanism that contributes to the inflammatory processes characteristic of Lyme arthritis.