Biomedical subjects

Gregor Rothe

Publications and source records attributed to Gregor Rothe.

9 recordsLinked to original sources

PubMed2006

Coexpression of CD25 and OX40 (CD134) receptors delineates autoreactive T-cells in type 1 diabetes.

T-cell-mediated loss of pancreatic beta-cells is the crucial event in the development of type 1 diabetes. The phenotypic characteristics of disease-associated T-cells in type 1 diabetes have not yet been defined. The negative results from two intervention trials (the Diabetes Prevention Trial-Type 1 Diabetes and the European Nicotinamide Diabetes Intervention Trial) illustrate the need for technologies to specifically monitor ongoing autoimmune reactions. We used fluorescence-activated cell sorter analysis to study surface marker expression on T-cell lines specific for two major type 1 diabetes autoantigens, GAD65 and proinsulin. We then applied this knowledge in a cross-sectional approach to delineate the phenotype of circulating memory T-cells. The autoreactive T-cells of patients could be distinguished from those of control subjects by their coexpression of CD25 and CD134. Autoantigen-specific T-cells that recognized multiple GAD65- and preproinsulin-derived peptides and coexpressed CD25(+)CD134(+) were confined to patients (n = 32) and pre-diabetic probands (n = 5). Autoantigen-reactive T-cells in control subjects (n = 21) were CD25(+)CD134(-) and recognized fewer autoantigen-derived peptides. Insulin therapy did not induce CD25(+)CD134(+) T-cells in type 2 diabetic patients. The coexpression of CD25 and the costimulatory molecule CD134 on memory T-cells provides a novel marker for type 1 diabetes-associated T-cell immunity. The CD134 costimulatory molecule may also provide a novel therapeutic target in type 1 diabetes.

Autoimmunity↗

PubMed2005

Multicentre evaluation of stable reference whole blood for enumeration of lymphocyte subsets by flow cytometry.

BACKGROUND: Clinical indications for lymphocyte subset enumeration by flow cytometry include monitoring of disease progression and timing of therapeutic intervention in infection with human immunodeficiency virus. Until recently international standardisation has not been possible due to a lack of suitable stable reference material. METHODS: This study consisted of two trials of a stabilised whole blood preparation. Eleven participants were sent two standard protocols for staining plus gating strategy and asked to report absolute counts for lymphocyte subsets. RESULTS: No significant difference was detected between the two methods when results from the two assays and all partners were pooled. Significant differences in results from the different partners were observed. However, representative mean counts were obtained for geometric means, geometric coefficient of variation, and 95% confidence interval for CD3 910 cells/mul, 9%, and 888 to 933, respectively), CD4 (495 cells/mul, 12%, and 483 to 507), and CD8 (408 cells/mul, 13%, and 393 to 422). CONCLUSION: We have introduced a stabilised blood preparation and a well-characterized biological standard. The availability of this reference material greatly simplifies the validation of new techniques for CD4(+) T-cell enumeration and the expansion of external quality assurance programmes for clinical laboratories, including those that operate in resource-restricted environments. (c) 2005 Wiley-Liss, Inc.

Journal Article↗

PubMed2004

Enumeration of antigen-specific CD8+ T lymphocytes by single-platform, HLA tetramer-based flow cytometry: a European multicenter evaluation.

BACKGROUND: HLA class I peptide tetramers represent powerful diagnostic tools for detection and monitoring of antigen-specific CD8(+) T cells. The impetus for the current multicenter study is the critical need to standardize tetramer flow cytometry if it is to be implemented as a routine diagnostic assay. Hence, the European Working Group on Clinical Cell Analysis set out to develop and evaluate a single-platform tetramer-based method that used cytomegalovirus (CMV) as the antigenic model. METHODS: Absolute numbers of CMV-specific CD8(+) T cells were obtained by combining the percentage of tetramer-binding cells with the absolute CD8(+) T-cell count. Six send-outs of stabilized blood from healthy individuals or CMV-carrying donors with CMV-specific CD8(+) T-cell counts of 3 to 10 cells/microl were distributed to 7 to 16 clinical sites. These sites were requested to enumerate CD8(+) T cells and, in the case of CMV-positive donors, CMV-specific subsets on three separate occasions using the standard method. RESULTS: Between-site coefficients of variation of less than 10% (absolute CD8(+) T-cell counts) and approximately 30% (percentage and absolute numbers of CMV-specific CD8(+) T cells) were achieved. Within-site coefficients of variation were approximately 5% (absolute CD8(+) T-cell counts), approximately 9% (percentage CMV-specific CD8(+) T cells), and approximately 17% (absolute CMV-specific CD8(+) T-cell counts). The degree of variation tended to correlate inversely with the proportion of CMV-specific CD8(+) T-cell subsets. CONCLUSIONS: The single-platform MHC tetramer-based method for antigen-specific CD8(+) T-cell counting has been evaluated by a European group of laboratories and can be considered a reproducible assay for routine enumeration of antigen-specific CD8(+) T cells.

CD8-Positive T-Lymphocytes↗

PubMed2004

Differential effects of clindamycin on neutrophils of healthy donors and septic patients.

Antibiotics are frequently administered to ICU patients in case of bacterial infections. Little is known, however, about the interference of antibiotics with neutrophil host defence mechanisms in patients with sepsis and multiple organ dysfunction syndrome (MODS). With our study, evidence for differential clindamycin effects on neutrophils in healthy donors and septic patients without or with MODS was sought. Functional parameters (oxidative response and phagocytosis) and fMLP receptor expression were analysed. The study was approved by the local ethical board. Venous blood was drawn from healthy donors and septic patients. Neutrophils in PBS were incubated with 0, 5, 25 or 125 microg/ml clindamycin and analysed flow cytometrically. Neutrophils of patients with sepsis and MODS showed a significantly higher basal activation compared to healthy donors. Clindamycin application led to a dose-dependent significant suppression of the fMLP-induced oxidative response in patients with sepsis and MODS, but not in healthy donors or septic patients in the absence of MODS. In patients with sepsis and MODS, phagocytosis of Escherichia coli and Staphylococcus aureus was significantly suppressed by clindamycin 125 microg/ml. In both other treatment groups, clindamycin did not affect phagocytosis. fMLP receptor expression was not altered by clindamycin. High-dose clindamycin selectively suppresses functional responses of neutrophils in septic patients with MODS. Simultaneously applied drugs, such as general anaesthetics, may potentiate this modulation of antibacterial defence and inflammation.

Anti-Bacterial Agents↗

PubMed2004

Phagocyte function.

Explore the source record for details and available documents.

Calcium Signaling↗

PubMed2004

Mild hyperthermia down-regulates receptor-dependent neutrophil function.

Mild hypothermia impairs resistance to infection and, reportedly, impairs phagocytosis and oxidative killing of unopsonized bacteria. We evaluated various functions at 33 degrees-41 degrees C in neutrophils taken from volunteers. Adhesion on endothelial cells was determined using light microscopy. Adhesion molecule expression and receptors, phagocytosis, and release of reactive oxidants were assessed using flow cytometric assays. Adhesion protein CD11b expression on resting neutrophils was temperature-independent. However, up-regulation of CD11b with tumor necrosis factor (TNF)-alpha was increased by hypothermia and decreased with hyperthermia. Neutrophil adhesion to either resting or activated endothelial cells was not temperature-dependent. Bacterial uptake was inversely related to temperature, more so with Escherichia coli than Staphylococcus aureus. Temperature dependence of phagocytosis occurred only wi thopsonized bacteria. Hypothermia slightly increased N-formyl-L-methionyl-L-leucyl-phenylalanine receptors on neutrophils: hyperthermia decreased expression, especially with TNF-alpha. N-formyl-L-methionyl-L-leucyl-phenylalanine-induced H2O2 production was inversely related to temperature, especially in the presence of TNF-alpha. Conversely, phorbol-13-myristate-12-acetate, an activator of protein kinase C, induced an extreme and homogenous release of reactive oxidants that increased with temperature. In contrast to nonreceptor-dependent phagocytosis and oxidative killing, several crucial receptor-dependent neutrophil activities show temperature-dependent regulation, with hypothermia increasing function. The temperature dependence of neutrophil function is thus more complicated than previously appreciated.

Adult↗

PubMed2003

Plasma ceramide and lysophosphatidylcholine inversely correlate with mortality in sepsis patients.

Recent data indicate that ceramide (Cer) and lysophosphatidylcholine (LPC) regulate immune cell functions. Since these bioactive lipids are generated in blood plasma by inflammatory lipases, we hypothesized that they may be involved in the process of acute systemic sepsis. In order to provide support for this hypothesis, we analyzed the plasma levels of Cer and LPC by quantitative tandem mass spectrometry in 102 sepsis patients starting with the day at which the sepsis criteria were fulfilled for the first time, as well as on day 4 and day 11. The values were compared with 56 healthy controls and correlated with sepsis-related mortality within 30 days of study entry. Most Cer species were increased in sepsis patients, while all LPC species were markedly decreased. In addition, we determined the molar ratios with their precursor molecules sphingomyelin (SPM) and phosphatidylcholine (PC), which reflect the enzymatic reactions responsible for their formation. Species-specific as well as total Cer-SPM ratios were increased, whereas LPC-PC ratios were decreased in sepsis patients. The increased Cer-SPM ratios as well as the decreased LPC-PC ratios showed a strong predictive power for sepsis-related mortality. Together with existing data from in vitro experiments and animal models, the results provide the first ex vivo indication for the role of Cer and lysophospholipids in systemic inflammation in humans.

Adolescent↗

PubMed2002

Leukocyte ABCA1 controls susceptibility to atherosclerosis and macrophage recruitment into tissues.

The ATP-binding cassette transporter 1 (ABCA1) has recently been identified as a key regulator of high-density lipoprotein (HDL) metabolism, which is defective in familial HDL-deficiency syndromes such as Tangier disease. ABCA1 functions as a facilitator of cellular cholesterol and phospholipid efflux, and its expression is induced during cholesterol uptake in macrophages. To assess the role of macrophage ABCA1 in atherosclerosis, we generated low-density lipoprotein (LDL) receptor knockout (LDLr(-/-)) mice that are selectively deficient in leukocyte ABCA1 (ABCA1(-/-)) by using bone marrow transfer (ABCA1(-/-) --> LDLr(-/-)). Here we demonstrate that ABCA1(-/-) --> LDLr(-/-) chimeras develop significantly larger and more advanced atherosclerotic lesions compared with chimeric LDLr(-/-) mice with functional ABCA1 in hematopoietic cells. Targeted disruption of leukocyte ABCA1 function did not affect plasma HDL cholesterol levels. The amount of macrophages in liver and spleen and peripheral blood leukocyte counts is increased in the ABCA1(-/-) --> LDLr(-/-) chimeras. Our results provide evidence that leukocyte ABCA1 plays a critical role in the protection against atherosclerosis, and we identify ABCA1 as a leukocyte factor that controls the recruitment of inflammatory cells.

ATP Binding Cassette Transporter 1↗

PubMed2002

MK-383 (tirofiban) induces a GPIIb/IIIa receptor conformation which differs from the resting and activated receptor.

The platelet integrin alphaIIb beta3 (GPIIb/IIIa) acts as a receptor for fibrinogen, playing a critical role in platelet aggregation. GPIIb/IIIa antagonists, which block the receptor-ligand interaction, have been accused of causing occasional thrombocytopenia, probably via drug-induced platelet activation or immunogenic neoepitopes. We, therefore, analyzed the effects of the GPIIb/IIIa antagonist MK-383 (tirofiban) on platelet activation and GpIIb/IIIa conformation. At a concentration of 10(-7) mol/l, MK-383 completely inhibited fibrinogen binding to in vitro stimulated platelets. Simultaneously, the GPIIb/IIIa expression density increased, similar to that on activated platelets, but no effect on P-selectin expression or the formation of platelet-leukocyte aggregates could be observed, indicating that MK-383 binding did not induce general platelet activation. The GPIIb/IIIa receptor conformation was further analyzed by fluorescence resonance energy transfer analysis between fluorochrome-labeled antibodies against different GpIIb/IIIa epitopes. As a result, MK-383 induced a receptor conformation that differed from the resting as well as the activated receptor as induced by ADP or TRAP-6. This conformational modulation of GPIIb/IIIa presents an interesting mechanism which may be linked to receptor recruitment without inducing general platelet activation.

Flow Cytometry↗