Search PubMed⌕ Search

Biomedical subjects

G Rothe

Publications and source records attributed to G Rothe.

At least 55 records · Page 3Linked to original sources
PubMed1998

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells. European Working Group on Clinical Cell Analysis.

The need for a rapid and reliable marker for the engraftment potential of hematopoietic stem and progenitor cell (HPC) transplants has led to the development of flow cytometric assays to quantitate such cells on the basis of their expression of CD34. The variability associated with enumeration of low-frequency cells (i.e., as low as 0.1% or 5 cells/microl) is exceedingly large, but recent developments have improved the accuracy and precision of the assay. Here, we review and compare the major techniques. Based on the current state of the art, we recommend 1) bright fluorochrome conjugates of class II or III monoclonal antibodies (mAbs) that detect all glycoforms of CD34, 2) use of a vital nucleic acid dye to exclude platelets, unlysed red cells, and debris or use of 7-amino actinomycin D to exclude dead cells during data acquisition, 3) counterstaining with CD45 mAb to be included in the definition of HPC, 4) during list mode data analysis, Boolean gating to resolve the CD34+ HPCs from irrelevant cell populations on the basis of the low levels of CD45 expression and low sideward light-scatter signals of HPCs, 5) inclusion of CD34dim and CD34bright populations in the CD34+ cell count, 6) omission of the negative control staining, and 7) for apheresis products, enumeration of at least 100 CD34+ cells to ensure a 10% precision. Unresolved technical questions are 1) the replacement of conventional dual-platform by single-platform assay formats, i.e., derivation of absolute CD34+ cell counts from a single flow cytometric assessment instead of from combined flow cytometer (percent CD34+) and hematology analyzer (absolute leukocyte count) data, 2) the cross-calibration of the available single-platform assays, and 3) the optimal method for sample preparation. An important clinical question to be addressed is the definition of the precise phenotypes and required numbers of HPCs responsible for short- and long-term recovery to optimize HPC transplant strategies.

Antigens, CD34↗
PubMed1998

T-lymphocytes and monocytes in atherogenesis.

Atherosclerosis is characterized as a chronic inflammatory-fibroproliferative disease of the vessel wall. The attachment of monocytes and T-lymphocytes to the injured endothelium followed by their migration into the intima is one of the first and most crucial steps in lesion development. The co-localization of CD4+ T-cells and macrophages in the lesion, the abundant expression of HLA Class II molecules and the co-stimulatory molecule CD40 and its ligand (CD40L) indicate a contribution of cell-mediated immunity to atherogenesis. Transgenic mouse models revealed that dependent on the model T- and B-cells may promote lesion progression, monocytes and macrophages are in contrast essential for the development of atherosclerotic lesions. Apart from the local process in the vessel wall, systemic signs of an inflammatory reaction are also associated with lesion development. Thus plasma levels of C-reactive protein and fibrinogen and the white blood cell count are positively correlated to the risk of cardiovascular disease. Recently, an inflammatory phenotype of circulating peripheral blood monocytes could be demonstrated as a specific cellular correlate to lipid and lipoprotein risk factors. Thus the pool size of LPS receptor (CD14)dim and Fc gamma IIIa receptor (CD16a)+ monocytes positively correlates to plasma cholesterol levels, to triglycerides levels and to the apolipoprotein E4 (apo E4) phenotype in contrast to a negative correlation to the high density lipoprotein (HDL) cholesterol concentration. This CD14dim CD16a+ monocytes are further characterized by a high expression of beta 1- and beta 2-integrins, suggesting a higher capacity for attachment at sites of inflammation. A proinflammatory cytokine pattern and an expansion of these cells in other inflammatory diseases are indicating that these cells promote the inflammatory process during atherogenesis. Surface expression of the activation antigen CD45RA on monocytes in correlation to plasma LDL cholesterol and Lp(a) levels further indicates an inflammatory reaction. Regarding the potential mechanisms of the phenotypic changes of peripheral blood monocytes, in a serum free in vitro differentiation model supplemented with M-CSF monocytes from probands which are homozygous for apo E4 showed a significantly higher increase of CD16a expression compared to apo E3/E3 cells indicating that a genetic polymorphism of a single apolipoprotein gene locus may affect monocyte differentiation. The further characterization of the cellular immunology of monocytes and T-lymphocytes in lesion development will provide new specific diagnostic and therapeutic targets in atherogenesis.

Animals↗
PubMed1998

Nitrous oxide impairs the neutrophil oxidative response.

BACKGROUND: Nitrous oxide has been shown inconsistently to impair the oxidative function of neutrophils. The choice of the stimulus, receptor agonists, or stimuli acting independent of receptors seems to determine whether nitrous oxide impairs the oxidative functions, suggesting an interference with the cytosolic signaling of neutrophils. METHODS: Production of hydrogen peroxide by neutrophils was assessed using flow cytometric analysis. N-formyl-methionyl-leucyl-phenylalanine (FMLP), C5a, dioctanylglycerol, and phorbol-12-myristate-13-acetate were used as stimuli. In addition, the expression of receptors for FMLP and the cytosolic-free calcium response of cells were measured. RESULTS: Nitrous oxide depresses C5a- or FMLP-induced generation of reactive oxygen derivatives in a concentration-dependent manner. However, the response with direct activation of protein kinase C was unaffected. Further, the number of FMLP receptors and the cytosolic calcium response were unaffected. Inhibition of the oxidative response was not reversible within the observation period of 4 h. CONCLUSIONS: Nitrous oxide inhibited the intracellular signaling of the investigated G-protein-coupled receptors for chemotactic peptides. No interference of nitrous oxide with reduced nicotinamide adenine dinucleotide phosphate oxidase, the oxidative enzyme system of neutrophils, nor with its activation through protein kinase C was detected.

Adult↗
PubMed1998

Volatile anaesthetics induce changes in the expression of P-selectin and glycoprotein Ib on the surface of platelets in vitro.

Halothane has been shown to inhibit platelet aggregation and may, therefore, prevent intraoperative platelet activation. The aim of this study was to compare the effects of volatile anaesthetics on platelet activation, including the transformation into an adhesive platelet phenotype. After in vitro exposure to volatile anaesthetics, the expression of the adhesion molecule P-selectin and the internalization of the receptor for the von Willebrand factor (GPlb) were analysed by flow cytometry. In contrast to desflurane or N2O, sevoflurane (> or = 0.5 MAC, P < 0.05), halothane (> or = 1.0 MAC, P < 0.01) and isoflurane (> or = 2.0 MAC, P < 0.01) induced a significantly higher expression of P-selectin on the surface of platelets, indicating the degranulation of alpha-granules. In the presence of desflurane (> or = 0.5 MAC, P < 0.05), halothane (> or = 1.0 MAC, P < 0.01), isoflurane and sevoflurane (both > or = 2.0 MAC, P < 0.01), a redistribution of GPlb occurred, indicating platelet activation. N2O had no effect. In conclusion, several of the volatile anaesthetics tested in vitro induced changes in the expression of P-selectin and GPlb, being characteristic of platelet activation. None of the anaesthetics investigated interfered with the platelet response to ADP stimulation.

Adult↗
PubMed1998

Enhanced upregulation of the Fc gamma receptor IIIa (CD16a) during in vitro differentiation of ApoE4/4 monocytes.

We recently reported a positive correlation of the pool size of lipopolysaccharide receptor (CD14)dim and Fc gamma receptor IIIa (CD16a)+ monocytes in peripheral blood to the apolipoprotein E4 (apoE4) phenotype and a negative correlation to high density lipoprotein (HDL) cholesterol levels (Arterioscler Thromb Vasc Biol. 1996;16:1437-1447). In this study, the in vitro differentiation of mononuclear phagocytes derived from healthy blood donors homozygous for the E3/3 or the E4/4 phenotype was analyzed during 7 days of culture in serum-free medium supplemented with macrophage colony-stimulating factor (M-CSF). The CD16a expression, which indicates Fc receptor-dependent phagocytic activity, increased to a significantly higher level in apoE4/4 monocytes than in apoE3/3 cells. The costimulatory molecule CD40, which indicates antigen-presenting capacity, was upregulated more strongly in apoE3/3 monocytes compared with E4/4 cells, but the difference did not reach a significant level. The expression of differentiation-associated surface proteins (CD14, CD33, CD45) and adhesion molecules (CD11a, CD11b, CD11c, CD49d) was not significantly different between apoE3/3 and apoE4/4 monocytes. However, a significantly decreased intracellular apoE concentration and a reduced amount of secreted apoE were found in apoE4/4 monocytes during in vitro differentiation. No differences were found in the surface expression of the low density lipoprotein receptor-related protein (CD91) and the uptake of fluorescence labeled low density lipoprotein between apoE3/3 and apoE4/4 monocytes. These data indicate that the apoE4/4 phenotype significantly influences the M-CSF-dependent differentiation of monocytes toward a more CD16a-positive phagocytic phenotype.

Adult↗
PubMed1998

Serum dehydroepiandrosterone (DHEA) and DHEA sulfate are negatively correlated with serum interleukin-6 (IL-6), and DHEA inhibits IL-6 secretion from mononuclear cells in man in vitro: possible link between endocrinosenescence and immunosenescence.

Interleukin-6 (IL-6) is one of the pathogenetic elements in inflammatory and age-related diseases such as rheumatoid arthritis, osteoporosis, atherosclerosis, and late-onset B cell neoplasia. In these diseases or during aging, the decrease in production of sex hormones such as dehydroepiandrosterone (DHEA) is thought to play an important role in IL-6-mediated pathogenetic effects in mice. In humans, we investigated the correlation of serum levels of DHEA, DHEA sulfate (DHEAS), or androstenedione (ASD) and IL-6, tumor necrosis factor-alpha, or IL-2 with age in 120 female and male healthy subjects (15-75 yr of age). Serum DHEA, DHEAS, and ASD levels significantly decreased with age (all P < 0.001), whereas serum IL-6 levels significantly increased with age (P < 0.001). DHEA/DHEAS and IL-6 (but not tumor necrosis factor-alpha or IL-2) were inversely correlated (all patients: r = -0.242/-0.312; P = 0.010/0.001). In female and male subjects, DHEA and ASD concentration dependently inhibited IL-6 production from peripheral blood mononuclear cells (P = 0.001). The concentration-response curve for DHEA was U shaped (maximal effective concentration, 1-5 x 10(-8) mol/L), which may be the optimal range for immunomodulation. In summary, the data indicate a functional link between DHEA or ASD and IL-6. It is concluded that the increase in IL-6 production during the process of aging might be due to diminished DHEA and ASD secretion. Immunosenescence may be directly related to endocrinosenescence, which, in turn, may be a significant cofactor for the manifestation of inflammatory and age-related diseases.

Adolescent↗
PubMed1998

Inhibition of fibrinogen binding and surface recruitment of GpIIb/IIIa as dose-dependent effects of the RGD-mimetic MK-852.

Conformational activation of platelet GpIIb/IIIa which is coupled to increased affinity for binding of soluble fibrinogen is an important step in platelet aggregation. Antibodies, peptides, and small molecules which antagonise fibrinogen binding, therefore, have been developed as a new class of potent anti-aggregatory compounds. The binding of these artificial ligands, however, similar to fibrinogen binding may also stimulate platelet adhesion and degranulation. Therefore, in this study effects of MK-852, a cyclic hexapeptide based on the RGD-template, on the platelet phenotype were analysed by whole blood flow cytometry. MK-852 already in the nanomolar range completely inhibited the surface binding of fibrinogen to ADP or TRAP-6 stimulated platelets. In the micromolar range, MK-852 induced an up to four-fold increase of GpIIb/IIIa surface expression in the absence of increased P-selectin expression. In conclusion our results suggest that the selective surface recruitment of GpIIb/IIIa may be a side effect of exposure to RGD-analogues which occurs at concentrations well above inhibition of fibrinogen binding.

Adult↗
PubMed1997

[Mononuclear phagocytes in peripheral blood as cellular markers of lipid and lipoprotein metabolism].

The functional specialization related to tissue-specific immune surveillance mononuclear phagocyte differentiation is characterized by and increased by and expression of scavenger receptors and synthesis of apo E. Apo E heterogeneity related to different patterns of the cellular uptake and metabolism of lipoproteins represents an interesting genetical model for the analysis of the interaction between cellular lipid metabolism and monocyte differentiation. In a preliminary study of the expression of differentiation dependent antigens on monocytes of hypercholesterolemic patients they observed an altered expression density of maturation associated antigens on monocytes from patients with familial hypercholesterolaemia. The abnormalities of mononuclear phagocytes may correlate with the cardiovascular state within hypercholesterolemic patients. This stimulated the authors to perform a more detailed study of the immunological phenotypes of circulating blood monocytes and their subpopulations as potential diagnostic parameters which may correlate to the atherogenic risk, status of atherosclerosis or therapy response in patients with hypercholesterolemia.

Biomarkers↗
PubMed1997

Altered mononuclear phagocyte differentiation associated with genetic defects of the lysosomal acid lipase.

Multiparameter flow cytometry reveals a complex heterogeneity of mononuclear phagocyte differentiation within the peripheral blood compartment. In this study, the relation of abnormal cellular lipid metabolism to the phenotype of peripheral blood mononuclear phagocytes, which finally may be related to atherogenesis, was analyzed using recently characterized autosomal recessive defects of lysosomal acid lipase (LAL) expression as model system. The reduction of LAL activity in nine heterozygote, disease free carriers of mutations from two cholesteryl ester storage disease (CESD) pedigrees and the family of a patient with Wolman disease was associated with an increased fraction of monocytes which expressed CD56 (N-CAM) (4.1 +/- 2.7% of monocytes, compared to 2.2 +/- 0.5% in ten controls, P < 0.05), an antigen characteristic of immature myeloid cells, suggesting an increased turnover of monocytes. Furthermore, a trend was observed towards an enhanced blood pool of more mature mononuclear phagocytes which show decreased expression of the 55 kD lipopolysaccharide receptor (CD14) together with either expression of the Fc-gamma-receptor III (CD16) or a high expression of CD33. A similar phenotype of peripheral mononuclear phagocytes was observed in the two CESD patients analyzed. In conclusion, our data suggest that these monogenetic defects of lysosomal lipoprotein metabolism are associated with complex alterations of mononuclear phagocyte differentiation and extravasation.

Adolescent↗
PubMed1997

Predictors of blood loss after coronary artery bypass grafting.

OBJECTIVE: To assess the predictive value of variables possibly associated with blood loss after coronary artery bypass grafting (CABG). DESIGN: A prospective study. SETTING: A university hospital. PARTICIPANTS: Eighty-nine patients scheduled for elective CABG. INTERVENTIONS: Blood samples were drawn before and after surgery. Chest tube drainage was measured hourly until removal of drains. MEASUREMENTS AND MAIN RESULTS: Activation of coagulation and fibrinolysis, routine clotting tests, and expression of platelet surface antigens were analyzed using flow cytometry. A significant correlation was found among blood loss and activated partial thromboplastin time, fibrinogen, prothrombin fragment 1 + 2, D-dimers, platelet count, GPIb and P-selectin expression on platelets, use of internal thoracic artery, cross-clamp time, and thrombin-antithrombin III complex. In a multiple regression model, glycoprotein (GP) Ib expression on platelets, platelet count, use of internal thoracic artery, and D-dimers were significantly associated with blood loss. Logistic regression analysis showed that GPIb and D-dimers predicted an increased blood loss with a positive predictive value of 73% and a negative predictive value of 91%. CONCLUSIONS: Postoperative D-dimers and GPIb expression may be useful to exclude nonsurgical causes in bleeding patients after CABG.

Aged↗
PubMed1997

Enhanced expression of HLA-DR, Fc gamma receptor 1 (CD64) and leukocyte common antigen (CD45) indicate activation of monocytes in regenerative training periods of endurance athletes.

The aim of this study was to test the hypothesis whether endurance athletes have distinct expressions of functionally relevant surface antigens of monocytes. Under standardized experimental and laboratory conditions CD 14+ blood monocytes of 55 males (22 sedentary controls [CO], individual anaerobic threshold [IAT]: 2.2 +/- 1.2 W.kg-1, VO2 max: 47 +/- 12 ml.min-1.kg-1; 13 endurance athletes, training: 3-10 hours weekly [E1], IAT: 3.0 +/- 1.1 W.kg-1, VO2max 57 +/- 5 ml.min-1.kg-1; 20 endurance athletes training: > 10 hours weekly [E2], IAT: 3.7 +/- 1.3 W.kg-1, VO2max: 64 +/- 6 ml.min-1.kg-1) were investigated for relative surface receptor expression of CD45, CD64 and HLA-DR (direct immunofluorescence, flow cytometry). E1 and E2 showed significantly (ANOVA, Tukey's HSD test; each p < 0.01) higher relative receptor expressions for CD64 in comparison to CO (E1: +62/E2: +71%; percentage of increase compared to CO value), CD45 (+44/+32%) and HLA-DR (+22/+35%). Cell counts of four monocyte subpopulations (CD56+, CD14bright+CD16-, CD14bright+CD16dim+, CD14dim(+)-CD16bright+) did not show significant differences between the groups. These data suggest that in endurance athletes during regenerative training period circulating monocytes show an enhancement of functionally relevant surface receptors indicating an activation monocytes.

Adult↗
PubMed1997

Scavenging, signalling and adhesion coupling in macrophages: implications for atherogenesis.

Integrins are in physical association and functional cooperation with other membrane proteins that include receptors with scavenger functions, glycosyl-phosphatidylinositol-linked receptors, the family of integrin associated multimembrane spanning signalling proteins and possibly other less characterized proteins with a coupling or signalling function. Monocyte adhesion, migration and differentiation to phagocytically active scavenger cells are directly coupled processes, involving integrins as common transducers for a panel of integrin-linked specific receptors, which assemble a master cluster to coordinate adhesion, migration, scavenging and associated metabolic pathways of the lysosomal and secretory route, and also processes involved in host response. As macrophages represent highly heterogeneous cells that have major phenotypical and functional differences associated with specific patterns of integrin expression, the functional cooperation of integrins with scavenger receptors has to be related to specialized subsets of monocytes and macrophages as well as to ligand specific effects which mediate receptor coupling.

Animals↗
PubMed1997

[Effect of processed blood volume, leukocyte count and concentration of CD34-positive cells in peripheral blood on efficiency of stem cell apheresis].

Despite many published studies no parameter could be identified yet to acceptably and individually predict collection results in stem cell apheresis. We analyzed leukocyte counts and processed blood volume, absolute and relative CD34+ cell counts, and overall collection efficiency in 120 patients with hematological and solid malignancies (354 leukaphereses using the Cobe Spectra cell separator, a median of 3 per patient, span 1-9). Stem cells were mobilized into peripheral blood by conventional chemotherapy followed by daily doses of G-CSF. CD34+ progenitor cell counts were monitored through multiparametric flow cytometry. Blood and collection flows varied in the range of 45-90 ml/min and 0.7-1.5 ml/min, respectively. CD34+ progenitor cells were enriched 38-fold in the apheresis product as compared to peripheral blood at a processed blood volume lower than one total blood volume. Efficiency continuously declined, on to a 25-fold concentration at a processed blood volume above the 3-fold total blood volume. Total collection efficiency, calculated from the absolute content of CD34+ progenitor cells in peripheral blood and apheresis concentrate (a parameter for progenitor cell mobilization during the apheresis), reached a plateau at a processed blood volume above the 3-fold total blood volume. However, variation among individual patients was high. The concentration rate of CD34+ cells at a leukocyte count below 5,000/microliter averaged 50 and declined continuously to 8 at leukocyte counts between 45,000 and 50,000/microliter. To summarize, in 70% of patients with leukocyte counts below 5,000/microliter and CD34+ progenitor cell counts above 10,000/ml, more than 1.5 x 10(6) progenitors per kg body weight could be collected in a single leukapheresis. According to the presented data, the variation in overall collection efficiency is mainly due to: 1) varying mobilization of progenitors during the apheresis procedures itself and 2) dependence on peripheral leukocyte counts.

Antigens, CD34↗
PubMed1997

[Specific Lp(a) apheresis for secondary prevention of arteriosclerosis].

Lipoprotein(a) (Lp(a)) is an independent risk factor for arteriosclerosis. It consists of the Lp(a)-specific apo(a) which is bound to the apo-B of an LDL particle by a disulfide bridge. Apo(a) is homologous to parts of the plasminogen molecule: It consists of one kringle 5 and 10-40 kringles 4 of the plasminogen molecule. Due to the lack of alternative drug treatment, 3 patients with early onset of arteriosclerosis, rapid progression, and elevated Lp(a) as their dominating risk factor were treated weekly with specific Lp(a)-aphereses. Since October 1992, we carried out 229 immunoadsorptions (IA) with specific columns containing anti-Lp(a) antibodies covalently bound to sepharose. To reduce Lp(a) from preapheresis values of 142 +/- 53 mg/dl to 25 +/- 11 mg/dl immediately after apheresis, we had to adsorb 1.4-3 patient's plasma volumes. Lp(a) rise to preapheresis values took 3-4 days. Protein reduction caused by loss of plasma during column changes remained tolerable (total protein before IA: 71 +/- 4 g/l, after IA: 56 +/- 4 g/l. Immediately after IA, these values were measured after the application of 991 +/- 207 ml of ACDB with 5,000 IU of heparin as anticoagulant. Hemoglobin remained unchanged (before IA: 13.4 +/- 1.4 g/dl, after IA: 13.6 +/- 1.5 g/dl). Side effects were mainly flush and tachycardia. They were seen especially when using new columns and plasma flow rates above 55 ml/min and were immediately reverted by interrupting the IA. Control angiography performed after 2 years in 2 patients showed no progression of disease, in the 3rd patient, the stress test showed a significant improvement as did clinical parameters. In our hands, IAs are a safe and efficient method for Lp(a) reduction and secondary prevention of myocardial infarction. Therapeutic efficiency should further be proven by a controlled trial.

Adult↗
PubMed1997

[Clinical and flow cytometry outcome of improved photopheresis methods in dermatologic patients].

In photopheresis, 8-methoxypsoralen (8-MOP) is added to a mononuclear cell concentrate and then activated by UVA light, thus forming covalent bonds between DNA strands. Infusion of these modified cells that are not able to replicate any more seems to lead to the elimination of pathogenic T-cell clones and clinical improvement in patients suffering from cutaneous T-cell lymphoma, graft versus host reactions, and various autoimmune diseases. The original method described by Edelson et al. (1987) was improved by the following modifications proposed by Andreu et al. (1994): i) by using a cell separator (COBE Spectra) that produced purer concentrate with less red blood cells absorbing UVA light. ii) By applying 8-MOP directly into the collection bag, the drug side effects due to the oral application of the thousandfold dose and varying serum levels were avoided. iii) By irradiating the concentrate after collection, all cells received the same irradiation dosage. We treated 2 men with cutaneous T-cell lymphoma, 2 women with atopic eczema and 1 man with severe pustular psoriasis with overall 123 extracorporeal photochemotherapy (ECPC) sessions. In addition to routine laboratory analysis, detailed characterization of lymphocyte subpopulations was carried out by flow cytometry to differentiate T-helper and T-suppressor cells and their activation (HLA-DR, CD 25), B, NK cells, and monocyte subpopulations. The following mediators soluble were analyzed as well: Il-2-receptor and as indicators of acute phase reaction Il-8, neopterin and Il-1 beta. We observed a good clinical improvement independent of no significant trend in the immunological phenotype of circulating blood leukocytes. Our results suggest that ECPC effects are not mediated by a systemic immune response or alternatively are not measured in the blood compartment.

Dermatitis, Atopic↗