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Biomedical subjects

G Valet

Publications and source records attributed to G Valet.

At least 19 recordsLinked to original sources
PubMed2005

Cytomics, the human cytome project and systems biology: top-down resolution of the molecular biocomplexity of organisms by single cell analysis.

A large amount of structural and functional information is obtained by molecular cell phenotype analysis of tissues, organs and organisms at the single cell level by image or flow cytometry in combination with bioinformatic knowledge extraction (cytomics) concerning nuclei acids, proteins and metabolites (cellular genomics, proteomics and metabolomics) as well as cell function parameters like intracellular pH, transmembrane potentials or ion gradients. In addition, differential molecular cell phenotypes between diseased and healthy cells provide molecular data patterns for (i) predictive medicine by cytomics or for (ii) drug discovery purposes using reverse engineering of the data patterns by biomedical cell systems biology. Molecular pathways can be explored in this way including the detection of suitable target molecules, without detailed a priori knowledge of specific disease mechanisms. This is useful during the analysis of complex diseases such as infections, allergies, rheumatoid diseases, diabetes or malignancies. The top-down approach reaching from single cell heterogeneity in cell systems and tissues down to the molecular level seems suitable for a human cytome project to systematically explore the molecular biocomplexity of human organisms. The analysis of already existing data from scientific studies or routine diagnostic procedures will be of immediate value in clinical medicine, for example as personalized therapy by cytomics.

Cell Biology↗
PubMed2004

Cytomics--new technologies: towards a human cytome project.

BACKGROUND: Molecular cell systems research (cytomics) aims at the understanding of the molecular architecture and functionality of cell systems (cytomes) by single-cell analysis in combination with exhaustive bioinformatic knowledge extraction. In this way, loss of information as a consequence of molecular averaging by cell or tissue homogenisation is avoided. PROGRESS: The cytomics concept has been significantly advanced by a multitude of current developments. Amongst them are confocal and laser scanning microscopy, multiphoton fluorescence excitation, spectral imaging, fluorescence resonance energy transfer (FRET), fast imaging in flow, optical stretching in flow, and miniaturised flow and image cytometry within laboratories on a chip or laser microdissection, as well as the use of bead arrays. In addition, biomolecular analysis techniques like tyramide signal amplification, single-cell polymerase chain reaction (PCR), and the labelling of biomolecules by quantum dots, magnetic nanobeads, or aptamers open new horizons of sensitivity and molecular specificity at the single-cell level. Data sieving or data mining of the vast amounts of collected multiparameter data for exhaustive multilevel bioinformatic knowledge extraction avoids the inadvertent loss of information from unknown molecular relations being inaccessible to an a priori hypothesis. CHALLENGE: It seems important to address the challenge of a human cytome project using hypothesis-driven molecular information collection from disease associated cell systems, supplemented by systematic and exhaustive knowledge extraction. This will allow the description of the molecular setup of normal and abnormal cell systems within a relational knowledge system, permitting the standardised discrimination of abnormal cell states in disease. As one of the consequences, individualised predictions of further disease course in patients (predictive medicine by cytomics) by characteristic discriminatory data patterns will permit individualised therapies, identification of new pharmaceutical targets, and establishment of a standardised framework of relevant molecular alterations in disease. This special issue of Cytometry, on new technologies in cytomics, focuses on prominent examples of this presently fast-moving scientific field, and represents one of the preconditions for the formulation of a human cytome project.

Cell Biology↗
PubMed2003

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

BACKGROUND: 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. METHODS: Sixty-seven parameters of AML patients at diagnosis were classified for predictive information by algorithmic data sieving using iteratively self optimizing triple matrix data pattern analysis (http://www.biochem.mpg.de/valet/classif1.html). RESULTS: Pretherapeutic predictive values for nonsurvival within five years and two years were 100.0% and 83.2%, respectively, compared to 13.9% and 47.4% for the prediction of survival at five years and two years, respectively. At diagnosis, five-year nonsurvivors showed increased patient age and higher concentration of cells in the analyzed specimen, as well as increased levels of % CD2, CD4, CD13, CD36, and CD45 positive AML blasts. Two-year nonsurvivors were characterized by a data pattern of increased patient age and levels of % CD4, CD7, CD11b, CD24, CD45, TH126, and HLA-DR positive AML blasts and decreased levels of % CD1, CD65, CD95, and TC25 positive AML blasts. Cytogenetic abnormalities were not selected for the optimized discriminatory data patterns. CONCLUSIONS: The comparatively accurate pretherapeutic identification of high-risk AML patients may prove useful for the development of individualized therapy protocols in stratified clinical patients groups.

Acute Disease↗
PubMed2001

Preoperative prediction of postoperative edema and effusion in pediatric cardiac surgery by altered antigen expression patterns on granulocytes and monocytes.

Postoperative edema and effusion (POEE) following cardiopulmonary bypass (CPB) surgery in children retards recovery and may aggravate postpericardiotomy (PPS), capillary leak syndrome (CLS), or multiorgan failure (MOF). Compared with complication-free children, POEE affected children have different preoperative serum levels of circulating cytokines and adhesion molecules. These levels may be used preoperatively to assess POEE, but their determination is time consuming, costly, and a substantial blood volume is required. Altered serum levels of cytokines and adhesion molecules also may be reflected in altered antigen expression on circulating blood leukocytes. The predictive potential of flow cytometric (FCM) leukocyte immunophenotyping was explored as a sensitive and fast method that required small blood samples. Blood samples taken 24 h preoperatively from 49 patients (3-18 years old) were stained with monoclonal antibodies for adhesion molecules (ICAM-1, LFA-1, Mac-1) or constitutive/activation markers (CD4, CD14, CD16, CD25, CD54, CD69, HLA-DR) and measured on a microbead calibrated FCM. Neutrophils, monocytes, and eosinophils from POEE patients express higher preoperative levels of LFA-1, monocytes, HLA-DR, and other activation markers (all P < 0.03). Over 89% of the patients were classified correctly by using two discriminant analysis methods (sensitivity, >76%; specificity, >86%; positive prediction, >80%; negative prediction, >83%). Granulocytes and monocytes of postoperative POEE patients exhibit significant preoperative immune activation, suggesting an increased risk for patients with atopic/allergic predisposition. Surgical trauma and CPB cause additional immune activation, leading to POEE by a summative response. Most patients at risk for POEE can be identified preoperatively by using data pattern analysis on FCM-derived parameters.

Adolescent↗
PubMed2000

Lymphoma discrimination by computerized triple matrix analysis of list mode data from three-color flow cytometric immunophenotypes of bone marrow aspirates.

BACKGROUND: The goal of this study was to evaluate a self-learning algorithm for the computer classification of information extracted from flow cytometric immunophenotype list mode files from high-grade non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), and multiple myeloma (MM). Materials and Methods Bone marrow aspirates (BMA) were obtained from untreated NHL (n = 51), HD (n = 9), or MM (n = 13) patients. Bone marrow aspirates were not infiltrated in NHL and HD patients as confirmed by thorough histologic and cytologic investigation; however, MM patients showed an infiltration rate >50% by malignant myeloma cells. Peripheral blood leukocyte (PBL) samples were taken from age-matched healthy volunteers (n = 44) as easily available control material. A second control group of 15 healthy volunteers, from whom BMA and PBL samples were available, allowed us to differentiate whether the observed classification results on malignant samples were due to the malignant process or simply to the inherent differences between BMA and PBL. Bone marrow aspirates and PBL were analyzed by the same immunophenotyping antibody panel (CD45/14/20, CD4/8/3, kappa/CD19/5, lambda/CD19/5). The acquired list mode data files were analyzed and classified by the self-learning triple matrix classification algorithms CLASSIF1 following a priori separation of the data into a learning set and unknown test set. After completion of the learning phase, known patient samples were reclassified and unknown samples prospectively classified by the algorithm. RESULTS: Highly discriminatory information was extracted for the various lymphoma entities. The most discriminating information was encountered in antibody binding, antibody binding ratios, and relative antibody surface density parameters of leukocytes rather than in percentage frequencies of discrete leukocyte subpopulations. Samples from healthy controls were classified as normal in 97.2% of the cases, whereas those of NHL, HD, and MM patients were on average correctly classified in 80. 8% of the cases. CONCLUSIONS: Although no detectable lymphoma cells were present in BMA of NHL and HD patients, the CLASSIF1 classification of the immunophenotypes of morphologically normal cells provided a surprisingly good disease discrimination equal or better than that obtained by examining pathological lymph nodes according to the respective literature. The results are suggestive for a lymphoma-related and disease-specific antigen expression shift on normal hematopoietic bone marrow cells that can be used to discriminate the underlying disease (specificity of unspecific changes), i.e., in this case NHL from HD. Multiple myeloma patients were discriminated by changes on malignant as well as on normal bone marrow cells.

Algorithms↗
PubMed1999

Flow-cytometric investigation of cellular metabolism during oxidative stress and the effect of tocopherol.

Many studies and scientific publications report on potentially beneficial effects of the lipophilic anti-oxidant vitamin E on cellular metabolic pathways. The present work presents data on the influence of tocopherol on different intracellular parameters of intact and living human skin fibroblasts by flow-cytometric measurements. The parameters analysed were the intracellular pH, representing cell metabolism and cell function, intracellular glutathione, representing one of the cell's own radical scavenger enzyme systems, membrane potential and cell viability. In order to cause large numbers of free radicals cells were UVB-irradiated prior to measurement. The results of the flow-cytometric measurements indicate that vitamin E has significant protecting effects on the measured biochemical parameters during oxidative stress. In the presence of the lipophilic radical scavenger a significant stabilizing effect on pH, intracellular glutathione levels and membrane potential could be observed. Furthermore, vitamin E administration was associated with increased cell viability after UVB irradiation.

Antioxidants↗
PubMed1998

Overtraining and immune system: a prospective longitudinal study in endurance athletes.

A prospective longitudinal study investigated for 19 +/- 3) months whether immunophenotypes of peripheral leukocytes were altered in periods of severe training. Leukocyte membrane antigens (CD3, CD4, CD8, CD14, CD16, CD19, CD45, CD45RO, and CD56) of endurance athletes were immunophenotyped (dual-color flow cytometry) and list mode data analyzed by a self-learning classification system in a state of an overtraining syndrome (OT; N = 15) and several occasions without symptoms of staleness (NS; N = 70). Neither at physical rest nor after a short-term highly intensive cycle ergometer exercise session at 110% of the individual anaerobic threshold did cell counts of neutrophils, T, B, and natural killer cells differ between OT and NS. Eosinophils were lower during OT, activated T cells (CD3+HLA/DR+) showed slight increases (NS: 5.5 +/- 2.7; OT 7.3 +/- 2.4% CD3+ of cells; means +/- SD; P < 0.01) during OT without reaching pathological ranges. The cell-surface expression of CD45RO (P < 0.001) on T cells, but not cell concentrations of CD45RO+ T cells, were higher during OT. OT could be classified with high specificities (92%) and sensitivities (93%). It is concluded that OT does not lead to clinically relevant alterations of immunophenotypes in peripheral blood and especially that an immunosuppressive effect cannot be detected. Immunophenotyping may provide help with the diagnosis of OT in future, but the diagnostic approach presented here requires improvements before use in sports medicine practice is enabled.

Adult↗
PubMed1995

Flow cytometric determination of aminopeptidase activities in viable cells using fluorogenic rhodamine 110 substrates.

Aminopeptidases (AP) are ubiquitously occurring, nonspecific exopeptidases involved in protein degradation. They cleave the N-terminal amino acid of peptides and occur in practically all mammalian cells and tissues. Physiological and pathological processes such as metastasis of tumors and inflammation have been thought to involve changes in AP activities. Determination of AP (EC 3.4.11.X) activity in viable cells by flow cytometry was the subject of this study because of its general biological and clinical interest. Different bis-substituted rhodamine 110 (R110) peptide derivatives were synthesised and used as AP- and exopeptidase (EC 3.4.13.X-EC 3.4.14.X) substrates for flow cytometric measurements. Intracellular AP activities in viable lympho-, mono-, granulo-, and thrombocytes were detected by fluorescence increase from R110 following intracellular substrate cleavage. Eukaryotic-AP do not cleave D-amino acids and hence NH2(D-Leu)2R110 substrate served as negative control. Specific substrate cleavage by AP is shown by complete inhibition of flFLuorescence generation following preincubation of cells with leucine-chloromethylketone inhibitor. R110 AP- and exopeptidase substrates are suitable indicators for coupled endopeptidase reactions due to their rapid cleavage and largely pH independent generation of intracellular fluorescence.

Amino Acids↗
PubMed1995

[Computer-controlled laser irradiation unit for studies of light-induced processes in cell cultures].

For the photodynamic treatment of tumours, synergistic effects of photosensitizing substances and light (today usually laser light) are used. With the aim of optimizing photosensitizing drugs and therapy, the effects of light and drug dose were studied in cell experiments. To automate and standardize such in vitro experiments, a laser irradiation chamber was developed. Cells cultured from tumour cell lines are placed on micro-titre plates or in petri dishes, together with the photosensitizer, and subsequently irradiated in the irradiation chamber with a well-defined dose of laser light of a wavelength corresponding to the absorbance of the photosensitizing agent. The plates or dishes are irradiated from below. In this way, light dose errors due to refraction from the meniscus of the cell suspension as occurs with irradiation from above, are avoided. During irradiation, speckle effects on the underside of the plates or petri dishes lead to variation in irradiation. A vibrator keeps the light transmission fibre and thus speckle pattern in motion, guaranteeing a homogeneous irradiation of the cells.

Computer Graphics↗
PubMed1994

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 microM completely inhibited the Ca(2+)-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 approximately 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 Alzheimer's 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.

Animals↗
PubMed1993

Detection of lysosomal cysteine proteinases in microglia: flow cytometric measurement and histochemical localization of cathepsin B and L.

The activation and differentiation of microglia is a prominent pathophysiological process in numerous inflammatory and demyelinating diseases of the central nervous system, including Alzheimer's disease and the AIDS encephalopathy. The tissue damage during these diseases has partly been attributed to lipid peroxidating reactive oxygen intermediates for which activated microglia are a major source. The destruction of tissue may also involve the release of proteolytic enzymes, such as the lysosomal cysteine proteinases cathepsin B and L, which are present notably in phagocytic cells. The cathepsins B and L are endopeptidases with a substrate specificity including important proteins, like myelin basic protein, extracellular matrix components, or the class II major histocompatibility complex. Because of this pathophysiological relevance the cathepsins B and L were chosen for histochemical demonstration in isolated and cultured rat microglia and measurement by a new flow cytometric method. Cathepsin B/L activity was measured flow cytometrically in single viable cells by the intracellular cleavage of non-fluorescent (Z-Phe-Arg)2-rhodamine 110 to the green fluorescent monoamide Z-Phe-Arg-rhodamine 110 and rhodamine 110. In microglia we measured a cathepsin B/L activity that was 2.5 times higher than in thioglycolate-elicited, i.e., inflammatory peritoneal rat macrophages. In elicited peritoneal macrophages the formation of fluorescent product was 6.2 times higher than in unstimulated resident peritoneal macrophages, demonstrating that the activation and differentiation of mononuclear phagocytes is accompanied by an increased cathepsin B/L enzyme activity. The subcellular localization of cathepsin B/L activity in plated viable microglia was demonstrated histochemically by the use of Z-Ala-Arg-Arg-4-methoxy-2-naphthylamide. Its blue fluorescent cleavage product 4-methoxy-2-naphthylamide was found in lysosomes. Our study shows that activated microglia are an important potential source of cathepsin B/L. This is particularly interesting as enzymatically active cathepsins have recently been found extracellularly at high levels in the senile plaques of Alzheimer's disease, which are known to contain many activated microglia. The release of proteinases by microglia may play a crucial role in the pathomechanism of tissue-destructing diseases in the brain.

Animals↗
PubMed1992

Membrane permeable fluorogenic rhodamine substrates for selective determination of cathepsin L.

The dipeptidyl rhodamine diamide substrates (Z-Phe-Arg)2-R110 and (Z-Arg-Arg)2-R110 are 820- and 360-fold more selective for cathepsin L than for cathepsin B allowing a sensitive determination of cathepsin L activity in the presence of high activity of cathepsin B. The results obtained with cell lysates suggest that the cysteine proteinase activity of vital macrophages detected by flow cytometry with these substrates is mainly due to cathepsin L.

Animals↗
PubMed1992

Flow cytometric analysis of protease activities in vital cells.

The analysis of lysosomal proteases in cell lysates is complicated by pH-dependent and oxidative changes of their activity and complex formation with cytosolic inhibitors. Therefore, new flow cytometric methods were developed for the intracellular measurement of protease activities in viable cells. Intracellular cleavage of substrates such as Z-Arg-Arg-4-trifluoromethylcoumarinyl-7-amide to green fluorescent 7-amino-4-trifluoromethylcoumarin (AFC) in viable neutrophils and monocytes was only detected following phagocytosis of Escherichia coli. A measurement of the cysteine or serine proteinase activities in resting human leukocytes was, however, not possible with AFC derivatives as the overlapping blue fluorescence of the substrates reduces sensitivity. Nonfluorescent bis-substituted peptide derivatives of rhodamine 110 (R110), which are intracellularly cleaved to green fluorescent mono-substituted R110 and free R110 proved to be more sensitive substrates. The activity of the lysosomal cysteine proteinases of human monocytes or rat macrophages, i.e. cathepsin B and L, was specifically measured with (Z-Arg-Arg)2-R110, (Z-Phe-Arg)2-R110, or (Z-Ala-Arg-Arg)2-R110. Fluorescence generation was completely inhibited by Z-Phe-Ala-diazomethane or E-64. The serine proteinases of human neutrophils were analyzed with Elastase-substrates such as (Z-Ala-Ala)2-R110 or (Z-Ala-Ala-Ala)2-R110. Specificity was shown by inhibition with diisopropylfluorophosphate.

Amino Acid Sequence↗
PubMed1992

Prognostic value of flow cytometrically determined DNA-ploidy, intracellular pH and esterase activity of non-small cell lung carcinomas.

30 surgical specimens of patients with non-small cell lung carcinomas (NSCLC) were investigated. Significant increases of intracellular pH values in epithelial and inflammatory cells, in the percentage of dead epithelial and inflammatory cells and in the cell volume of vital inflammatory cells in cancerous lung tissue were encountered. Furthermore, decreases of the esterase activity of vital epithelial cells and of the percentage of free cell nuclei were observed. The DNA aneuploidy in 36.6% of the tumours was frequently associated with non-squamous cell carcinomas and stage II, III, IV tumours. Patients with DNA aneuploid tumours had a significantly shorter survival rate than those with DNA euploid tumours. Within the different tumour stages a similar tendency was observed which was, however, only significant in stage III tumour patients. Stage III tumours constitute therefore a heterogeneous entity with a worse prognosis for DNA aneuploid tumour patients. The intracellular pH values and esterase activity as well as the cell volume, the percentage of free cell nuclei and dead inflammatory or epithelial cells contained no significant prognostic information.

Adult↗