Günter Valet

Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2',7'-dichlorofluorescin.

Hydroethidine (HE) and 2′,7′‐dichlorofluorescin (DCFH) were used for the flow cytometric measurement of reactive oxygen metabolites in leukocytes. Hydroethidine and DCFH were both rapidly oxidized in a cell‐free cuvette assay to ethidium bromide (EB) and 2′,7′‐dichlorofluorescein (DCF) by H2O2 and peroxidase, but not by H2O2 alone, while only HE was oxidized by KO2, a source of O‐ 2. Quiescent lymphocytes, monocytes, and neutrophils spontaneously oxidized HE to EB, while DCFH was only oxidized to a low degree. Neutrophils increased 6.9‐fold in EB red fluorescence and 12.5‐fold in DCF green fluorescence during the respiratory burst induced by phorbol 12‐myristate 13‐acetate or 6.1‐fold and 4.7‐fold, respectively, during the respiratory burst induced by Escherichia coll bacteria. The HE or DCFH oxidation during the respiratory burst, unlike the spontaneous HE oxidation, was not inhibitable by 10 mM NaN. indicating a non‐mitochondrial source of cellular oxidants during the respiratory burst such as NADPH oxidase, which produces O‐ 2. The oxidation of DCFH, but not of HE, was decreased in stimulated neutrophils, which were simultaneously loaded with HE and DCFH. Intracellular DCFH oxidation induced by incubation of resting neutrophils with extracellular H2O2 was not influenced by the presence of HE. This indicates that HE is oxidized at an earlier step in the reactive oxygen metabolism of neutrophils than DCFH, i.e., by early oxygen metabolites like O2 ‐, while DCFH is oxidized in part by H2O2 and phagosomal peroxidases. The differential oxidation of HE and DCFH during simultaneous cellular staining permits the analysis of up to three functionally different neutrophil populations in septic patients. This is of interest for the determination of disease‐related alterations of oxygen metabolism in quiescent and stimulated leukocytes.

Modulation of intracellular formation of reactive oxygen intermediates in peritoneal macrophages and microglia/brain macrophages by propentofylline.

Ischemia-induced nerve cell death can partly be prevented by propentofylline, a pharmacon structurally related to xanthine derivates that interacts with the neuromodulatory function of endogenous adenosine. To evaluate a possible mechanism of neuroprotection by propentofylline, we studied its effect on the cellular production of reactive oxygen intermediates in microglial cells, which under pathological conditions can differentiate into brain macrophages, in comparison to peritoneal macrophages. Using a flow cytometric assay, we determined the intracellular formation of reactive oxygen intermediates by measuring the oxidation of the membrane-permeable and nonfluorescent dihydrorhodamine 123 to the cationic and intracellularly trapped, green fluorescent rhodamine 123 in single viable cells. Propentofylline at the therapeutic concentration of 50 μM completely inhibited the Ca2+-dependent Con A-induced increase in the production of reactive oxygen intermediates in peritoneal macrophages. In isolated and cultured microglial cells, which have a high spontaneous respiratory burst activity, the spontaneous production of reactive oxygen intermediates was reduced by ∼30%. A phorbol 12-myristate 13-acetate-induced rise in the respiratory burst activity could not be inhibited by propentofylline in either cell type. An increased generation of reactive oxygen intermediates is thought to contribute to nerve cell death after brain ischemia, edema, and neurodegenerative diseases like Alzheimers disease. These pathological conditions are all accompanied by an activation of microglial cells. We therefore suggest that the neuroprotective properties of propentofylline might in part be due to a modulation of the microglial production of potentially harmful reactive oxygen intermediates.

Inhibition of the oxidative burst response of N-formyl peptide-stimulated neutrophils by serum amyloid-A protein

Strong binding of the acute phase protein serum amyloid-A (SAA) to human neutrophils was found using flow cytometry. This binding was shown to be functionally relevant with respect to the oxidative burst reaction assayed on N-formyl peptide-stimulated neutrophils by the intracellular oxidation of non-fluorescent dihydrorhodamine to fluorescent rhodamine 123. The results show reduction of the oxidative burst response by isolated SAA (and recombinant SAA2). Inhibition was also demonstrated by acute phase as compared to normal human serum. This inhibitory effect was abolished by the purified monoclonal anti-amyloid A antibody mc29, strongly suggesting that SAA counteracts neutrophil responses to cytokines or bacterial products. This newly recognized function of SAA may help to prevent oxidative tissue destruction.

Cytomics in predictive medicine

Patient‐specific, disease‐course predictions with >95% or >99% accuracy during therapy would be highly valuable for everyday medicine. If these predictors were available, disease aggravation or progression, frequently accompanied by irreversible tissue damage or therapeutic side effects, could then potentially be avoided by early preventive therapy. The molecular analysis of heterogeneous cellular systems (cytomics) by cytometry in conjunction with pattern‐oriented bioinformatic analysis of the multiparametric cytometric and other data provides a promising approach to individualized or personalized medical treatment or disease management. As a consequence, better patient care and new forms of inductive scientific hypothesis development based on the interpretation of predictive data patterns are at reach. Cytometry Part B (Clin. Cytometry) 53B:1–3, 2003.

Respiratory burst activity in brain macrophages: a flow cytometric study on cultured rat microglia

A new flow cytometric method for the investigation of the respiratory burst of macrophages/ microglia isolated from neonatal rat brain has been established. Respiratory burst activity was measured quantitatively in single viable cells by the intracellular oxidation of non‐fluorescent dihydrorhodamine 123 (DHR) to fluorescent rhodamine 123. Cultured microglia exhibited high spontaneous respiratory burst activity already before stimulation. After maximal stimulation with phorbol myristate acetate, DHR oxidation rose by 40–95%. The respiratory burst activity in resident or inflammatory, i.e. thioglycolate elicited, peritoneal macrophages was significantly lower than in cultured brain macrophages suggesting a high potential of microglia for oxidative tissue destruction.

Cytomics: An entry to biomedical cell systems biology

BACKGROUND The introduction of high throughput and high content screening has induced a prolific growth of -omic terms such as genomics, proteomics, cytomics and many others (1). Cytomics, for example, may be considered a new name for cytometry. It is legitimate to ask why a successful effort should have a new name because previous work may get lost in searches as a counterproductive byproduct. Nevertheless, it seems important to consider potentials and challenges of cytomics in the cytometry field.

Preoperative prediction of pediatric patients with effusions and edema following cardiopulmonary bypass surgery by serological and routine laboratory data

AimPostoperative effusions and edema and capillary leak syndrome in children after cardiac surgery with cardiopulmonary bypass constitute considerable clinical problems. Overshooting immune response is held to be the cause. In a prospective study we investigated whether preoperative immune status differences exist in patients at risk for postsurgical effusions and edema, and to what extent these differences permit prediction of the postoperative outcome.MethodOne-day preoperative serum levels of immunoglobulins, complement, cytokines and chemokines, soluble adhesion molecules and receptors as well as clinical chemistry parameters such as differential counts, creatinine, blood coagulation status (altogether 56 parameters) were analyzed in peripheral blood samples of 75 children (aged 3–18 years) undergoing cardiopulmonary bypass surgery (29 with postoperative effusions and edema within the first postoperative week).ResultsPreoperative elevation of the serum level of C3 and C5 complement components, tumor necrosis factor-α, percentage of leukocytes that are neutrophils, body weight and decreased percentage of lymphocytes (all P < 0.03) occurred in children developing postoperative effusions and edema. While single parameters did not predict individual outcome, >86% of the patients with postoperative effusions and oedema were correctly predicted using two different classification algorithms. Data mining by both methods selected nine partially overlapping parameters. The prediction quality was independent of the congenital heart defect.ConclusionIndicators of inflammation were selected as risk indicators by explorative data analysis. This suggests that preoperative differences in the immune system and capillary permeability status exist in patients at risk for postoperative effusions. These differences are suitable for preoperative risk assessment and may be used for the benefit of the patient and to improve cost effectiveness.

Human cytome project, cytomics, and systems biology: The incentive for new horizons in cytometry

SYSTEMS BIOLOGY Systems biology aims at the quantification of molecular elements and their interrelations in biological systems in response to perturbation to integrate the variety of information into mathematical network models and to correctly predict biological system response to stimuli (1–4). Although promising for single-cell systems such as bacteria, yeast, or cell cultures, difficulties arise for complex mammalian organisms with numerous genes, diversity of genotypes among individuals, and variable exposure histories to environmental influences. Additional complexity derives from the diversity of organs and specialized tissues, each consisting of multiple cell types of significant internal heterogeneity, e.g, of cell cycle, functional status, size, and molecule content. Cell cultures or diseased and genetically modified animals may in certain circumstances serve as model systems for human disease. However, significant concerns as to the ultimate validity of the conclusions from such model systems for the human in vivo situation, especially in disease, or in the search for new drug targets have been raised (5). Cell or animal model systems may ultimately not permit one to generate valid mathematical models of the human organism by systems biology, permitting, e.g., the accurate prediction of a particular patient’s reaction to stimuli such as microbial infection, interaction with toxic substances, or adverse drug reactions from mathematical network models. It is also questionable whether a preexisting mathematical model for an entire biological system such as the human organism represents a prerequisite for the understanding and mathematical modeling of specific molecular disease pathways in complex diseases such as cancer, allergies, rheumatoid arthritis, asthma, diabetes, or infections. MOLECULAR CELL PHENOTYPING BY CYTOMICS The analysis of molecular cell phenotypes by cytomics (6,7) represents a simplifying strategy for the dissection of molecular disease networks. Differential molecular screens of single cells from diseased versus reference persons in disease-associated cell systems provide discriminatory data patterns or biomarker patterns for predictive medicine by cytomics and for the detection of new drug targets (8,9) by using hypothesis-driven selection of screening parameters. Reference persons can be healthy individuals or stationary status or survivor patients. The differentially obtained discriminatory data patterns from cells represent important entry points for molecular reverse engineering by biomedical cell systems biology (7). The reasons for this are that diseases are caused by molecular perturbations in cells and cells represent the elementary construction and function units of cellular systems, organs, and organisms. Reverse engineering of biological complexity (10) using discriminatory data patterns from patients (8) has the potential to directly elaborate mathematically modeled molecular disease pathways without exact knowledge about the normal condition or the disease-causing mechanisms. Therefore, analysis of molecular differentials in disease circumvents a priori mathematical modeling of systematically perturbed biological systems. An important advantage for the molecular cell phenotype screening concept consists of the plethora of avail-

Pretherapeutic identification of high-risk acute myeloid leukemia (AML) patients from immunophenotypic, cytogenetic, and clinical parameters.

The goal of this study concerned the pretherapeutic identification of high‐risk acute myeloid leukemia (AML) patients by data pattern analysis from flow cytometric immunophenotype, cytogenetic, and clinical data.

A New Method for Fast Blood Cell Counting and Partial Differentiation by Flow Cytometry

SummaryA new blood counting method by flow cytometry is described which determines absolute counts and relative proportions of erythrocytes, reticulocytes, thrombocytes, lymphocytes and granulocytes from one sample of saline diluted human or animal blood. Staining time is 2 to 5 min and measuring time between 1 and 2 additional minutes. Measured simultaneously are the electrical cell volume, the green and optionally also the red fluorescence of the transmembrane potential sensitive dye 3,3-dihexyloxacarbocyanine DiOC6(3) and the RNA/DNA stain acridine orange (AO). Work is under way to fully automate staining, measurement and data evaluation. The use of stains by which blood cell counting and biochemical analysis can be combined offers new possibilities for routine blood cell counting without requirement for additional time. The potential of such stains is that pathologic cell conditions which are not, or not yet reflected in the cell count may be earlier detectable by biochemical stains.