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G Schmitz

Publications and source records attributed to G Schmitz.

At least 109 records · Page 6Linked to original sources
PubMed1999

A new antigenic epitope localized within human kappa light chains specific for rheumatoid arthritis and systemic lupus erythematosus.

Human B cell hybridomas were established to define new autoantigens of importance for autoimmune diseases such as rheumatoid arthritis (RA). One lgG1, lambda monoclonal antibody (FKN-E12), was derived from synovial B lymphocytes of a patient with sero-negative RA. The purified lg was used to select specifically binding peptides from a random peptide phage display library. Only one epitope with the heptamer sequence HLTFGPG was detected and named RASFp1. Very similar and partly identical sequences are found in the variable region of lg kappa light chains in position 96-101, at the junction of framework 2 and the J-region. The antibody FKN-E12 was shown to detect the epitope RASFp1 also on human lgG kappa chains, but only in a specific conformation. The aim of the present study was to analyse human sera from patients with autoimmune diseases, non-autoimmune inflammatory diseases and healthy blood donors for the presence of lgG binding to RASFp1. For this purpose a 15-mer-peptide was synthesized containing RASFp1 within Vk-derived flanking regions, and an ELISA assay established. Sera of 142 individuals were studied. Only <5% of the control sera including sera from patients with non-autoimmune inflammations were positive. In contrast, 45% of sera from patients with RA or SLE contained RASFp1-binding antibodies. Within the 40 RA sera analysed so far, rheumatoid factors and RASFp1-binding antibodies have shown no correlation with each other.

Amino Acid Sequence↗
PubMed1999

Isolation and characterization of two human monoclonal anti-phospholipid IgG from patients with autoimmune disease.

The antigenic specificity of anti-phospholipid antibodies (APA) is a matter of intensive investigation. To further characterize these antibodies, we attempted to isolate human monoclonal APA. B-cells of patients with at least one positive test for antibodies against cardiolipin, phosphatidylserine, beta2-glycoprotein I (beta2-GPI) or the lupus anti-coagulant were immortalized by transformation with Epstein-Barr virus and screened for production of specific IgG. Positive pools were fused with a heteromyeloma cell line and APA-secreting clones were isolated by standard procedures. Two monoclonal APA, HL-5B from a 51-year-old man with primary anti-phospholipid syndrome and recurrent cerebral microinfarctions, and RR-7F from a 48-year-old women with systemic lupus erythematosus but no evidence for thrombotic events were obtained. HL-5B is of the IgG2 subtype with lambda light chains, while RR-7F is IgG2 with kappa light chains. Both monoclonals show reactivity against cardiolipin and phosphatidylserine but lack reactivity against beta2-GPI or lupus anti-coagulant activity. To yield the same OD in the cardiolipin and phosphatidylserine ELISAs RR-7F must be used in an approximately 10-fold higher concentration than HL-5B, indicating a lower affinity towards these antigens. Interestingly, both mAPA can bind to cardiolipin in the absence of beta2-GPI. They do not cross-react with dsDNA but show reactivity against oxidized low-density lipoproteins. Analysis of the heavy chain mRNA of HL-5B and RR-7F showed that both are members of the VH3 family. While HL-5B shows extensive somatic mutations in the CDR1 and 2 regions, indicating that it was derived by a T cell-dependent antigen driven process, RR-7F is apparently germline encoded. The two monoclonal APA can be used as tools in further structural and functional analyses.

Amino Acid Sequence↗
PubMed1999

A more mature phenotype of blood mononuclear phagocytes is induced by fluvastatin treatment in hypercholesterolemic patients with coronary heart disease.

Monocytes are recruited as the principal inflammatory cells into the atherosclerotic lesion. In a previous study we demonstrated that a low HDL-cholesterol and the apo E4 allele are associated with an increased proportion of blood monocytes that are characterized by a high expression of Fcgamma-RIIIa (CD16), a dim expression of the lipopolysaccharide (LPS) receptor (CD14) and a high expression of beta1- and beta2-integrins (Rothe et al. Arterioscler Thromb Vasc Cell Biol 1996;16:1437-1447). In this study, 79 hypercholesterolemic patients were treated either with the HMG CoA reductase inhibitor fluvastatin in combination with diet or with placebo and diet in a double-blind and randomized multicenter study, and monitored for the potential effects on the phenotype of peripheral blood monocytes. At baseline, in the whole group of hypercholesterolemic patients the population size of these more mature monocytes (CD14dimCD16+) was positively correlated to triglyceride (P = 0.003) and total serum cholesterol levels (P = 0.012) confirming our previous study. Fluvastatin treatment for 52 weeks was associated with a 24.2% reduction in LDL-cholesterol (P < 0.001) as well as a 40.7% decrease in the expression density of CD14 on all monocytes (P = 0.027). A 24.5% decrease (P < 0.001) of the population of less differentiated CD14brightCD16- monocytes and an 83.1% increase (P = 0.029) of the population of more differentiated CD14dimCD16+ monocytes further confirmed this modification of the phenotype of peripheral blood monocytes. The positive pre-study correlation of the CD14dimCD16+ monocyte subset to the serum cholesterol concentration, but inverse changes of both parameters under fluvastatin therapy, in conclusion indicate that fluvastatin exerts an as yet uncharacterized immunomodulatory effect on either monocyte maturation and differentiation, or extravasation which may also depend on the endothelial phenotype that is independent of the change in serum lipids.

Adult↗
PubMed1999

Genetic control of branching in Arabidopsis and tomato.

The patterns of axillary bud formation and the growth characteristics of side-shoots determine to a large extent the form of plants. Characterization of mutants in the monopodial plant Arabidopsis thaliana and in the sympodial tomato, as well as cloning of some of the respective genes, contributes to a better understanding of side-shoot development. Genes have been identified that influence the initiation of axillary meristems and the pattern of their subsequent development.

Arabidopsis↗
PubMed1999

The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease.

Tangier disease (TD) is an autosomal recessive disorder of lipid metabolism. It is characterized by absence of plasma high-density lipoprotein (HDL) and deposition of cholesteryl esters in the reticulo-endothelial system with splenomegaly and enlargement of tonsils and lymph nodes. Although low HDL cholesterol is associated with an increased risk for coronary artery disease, this condition is not consistently found in TD pedigrees. Metabolic studies in TD patients have revealed a rapid catabolism of HDL and its precursors. In contrast to normal mononuclear phagocytes (MNP), MNP from TD individuals degrade internalized HDL in unusual lysosomes, indicating a defect in cellular lipid metabolism. HDL-mediated cholesterol efflux and intracellular lipid trafficking and turnover are abnormal in TD fibroblasts, which have a reduced in vitro growth rate. The TD locus has been mapped to chromosome 9q31. Here we present evidence that TD is caused by mutations in ABC1, encoding a member of the ATP-binding cassette (ABC) transporter family, located on chromosome 9q22-31. We have analysed five kindreds with TD and identified seven different mutations, including three that are expected to impair the function of the gene product. The identification of ABC1 as the TD locus has implications for the understanding of cellular HDL metabolism and reverse cholesterol transport, and its association with premature cardiovascular disease.

ATP Binding Cassette Transporter 1↗
PubMed1999

Transplant characteristics: minimal residual disease and impaired megakaryocytic colony growth as sensitive parameters for predicting relapse in acute myeloid leukemia.

Dose escalation during consolidation therapy of de novo AML, including myeloablative chemotherapy supported with autologous peripheral blood stem cell transplantation (aPBSCT), continuously improved outcome. Therefore, quality control of transplants is getting increasing interest. We studied leukapheresis products (LPs), consecutively collected during postremission treatment of 20 patients with de novo AML for minimal residual disease (MRD) by 5-parametric flow cytometry and for myelodysplasia (MDS)-associated alterations by paired lineage-selected colony assays for colony-forming units-megakaryocytes (CFU-mega) and burst-granulocytes-monocytes colony-forming units (CFU) to evaluate the predictive value of these transplant-associated parameters on outcome. We defined the leukemia-associated immunophenotype at diagnosis and studied the impact of MRD detection in LPs collected after double induction with TAD (thioguanine, daunorubicin, cytarabine) and HAM (mitoxantrone, high-dose cytarabine, n=18 patients) and TAD consolidation treatment (n=20 patients) on outcome after aPBSCT. The level of MRD in the transplants correlated with the relapse-free survival (RFS) using a cut-off level of 1 x 10(-3) residual leukemic cells. The median RFS was 6 months for the group with > or = 1 x 10(-3) residual leukemic cells and has not been reached in the group with low MRD levels (< 1 x 10(-3)). By using the same cut-off level a weak correlation could also be demonstrated between MRD in the pregraft bone marrow and RFS (P = 0.04). Quantitatively abnormal megakaryocytic colony growth in the back-up LPs collected after double induction and in the transplant LPs was characterized by the ratio CFU-mega/CFU. In the group of relapsing patients the ratio CFU-mega/CFU was significantly lower than in the group of patients with CCR (P = 0.004), both in the back-ups and in the transplants. All patients with CFU-mega/CFU ratios < 0.12 relapsed, five of seven patients developed MDS before progressing to full leukemic relapse. Using the optimized cut-off level for the ratio CFU-mega/CFU (< vs > or = 0.12), seven of 10 relapsing patients (70%) could be identified to be at risk of relapse, whereas MRD in the transplants identified only 50% of the relapses and MRD in the pregraft bone marrow 25%. In conclusion, the study could identify two pretransplant risk factors predicting relapse in patients with AML receiving aPBSCT in first CR: MRD in transplants as well as MDS-like alterations within the transplants. These results may have multifold implications on the design of risk-adapted chemotherapy as well as on purging techniques and may contribute to a better understanding of leukemogenesis.

Acute Disease↗
PubMed1999

Frequency and significance of the A-->G (-3826) polymorphism in the promoter of the gene for uncoupling protein-1 with regard to metabolic parameters and adipocyte transcription factor binding in a large population-based Caucasian cohort.

BACKGROUND: The recently described A-->G (-3826) point mutation within the distal region of the UCP-1 promoter is possibly involved in the development of obesity, diabetes and related metabolic disorders. It was the aim of this study to examine the allelic frequency and the prevalence of the three UCP-1 genotypes in a broad caucasian cohort and to investigate the significance of this polymorphism for obesity and diabetes. METHODS: 1020 subjects were randomly chosen from 6450 participants in the Diabetomobile Study. The UCP-1 genotype was determined by genomic PCR and Bcl-I-RFLP analysis in 1020 subjects and tested for association with a variety of metabolic parameters. In addition, the influence of this mutation on adipocyte nuclear factor binding was investigated by electrophoretic mobility shift assays (EMSA). RESULTS: The genotype frequencies in 1020 subjects were: AA genotype, 57.0%; AG genotype, 35.4%; GG genotype, 7.6%; with allelic frequencies of 0.75 for allele A and 0.25 for allele G. No significant differences between the genotypes and age, gender, BMI, leptin, glucose, fasting insulin, C-peptide, HbA1c, diabetes manifestation, total cholesterol, and HDL cholesterol were found. Analysis of the Trp64Arg polymorphism of the beta3-adrenergic receptor in a subgroup of 343 subjects revealed no additive effect to the UCP-1 polymorphism. An yet unknown adipocyte-specific factor of nuclear extracts from 3T3-L1 adipocytes during differentiation is able to bind specifically to the distal UCP-1 promoter region and this binding ability can not be abolished by the mutation. CONCLUSIONS: We determined the genotype and allelic frequency of the UCP-1 promoter polymorphism in the largest known population-based study. The results from genotyping demonstrate clearly that this polymorphism does not play a major role in the pathogenesis obesity and diabetes. A yet unknown adipocyte derived and differentiation-dependent regulated transcription factor is able to bind to the distal UCP-1 promoter surrounding -3826 bp. This binding is not affected by presence of the mutation.

Adipocytes↗
PubMed1999

Flow cytometric immunophenotyping of mature lymphatic neoplasias using knowledge guided cluster analysis.

Flow cytometry is widely used for the immunological characterization of hematopoietic malignancies. Discrimination of normal and malignant cellular immunophenotypes is the most critical step in data analysis, especially if multi-color analysis is performed on highly heterogenous cell suspensions. We therefore investigated, whether adaptive, simultaneous multiparameter gating allowed automated, operator independent analysis of data obtained from the immunophenotyping of blood or bone marrow samples with regard to the presence of non-Hodgkin lymphoma cells. The identification of physiological and malignant cells was achieved by predefining population boundaries, based on the expectations of the population's location in two-dimensional dot plots. The prospective application of these predefined region boundaries in 52 blood and bone marrow samples enabled identification of lymphoma cells with regard to their presence and immunophenotype, based on the correlation of markers as defined in multiple tubes. Our data confirm that highly standardized data analysis methods can reduce the variability of analysis and support the expert in establishing a rapid classification of the sample.

B-Lymphocytes↗
PubMed1999

ATP-binding cassette transporter A1 (ABCA1) in macrophages: a dual function in inflammation and lipid metabolism?

Activated lipid-laden macrophages in the vascular wall are key modulators of the inflammatory processes underlying atherosclerosis. We demonstrate here that the ATP-binding cassette (ABC) transporter ABCA1 is induced during differentiation of human monocytes into macrophages. ABCA1 has been implicated in macrophage interleukin-1beta secretion and apoptosis. Moreover, ABCA1 mRNA and protein levels are strongly upregulated by uptake of modified LDL and downregulated by HDL(3)-mediated lipid efflux in macrophages. Mutation analysis in patients with the classical Tangier disease (TD), a monogenetic disorder characterized by hypersplenism, macrophage accumulation and deposition of cholesteryl esters in the reticuloendothelial system, low plasma HDL and premature atherosclerosis, revealed deleterious mutations in their ABCA1 gene. The localization pattern of the mutations within the ABCA1 protein appears to determine the tropism for either the reticuloendothelial system, as seen in the classical TD phenotype, or the artery wall, as in the case of HDL deficiency in the absence of splenomegaly. In a comprehensive analysis of the expression and regulation of all currently known human ABC transporters, we identified additional cholesterol-responsive genes that are induced during monocyte differentiation into macrophages. Our results indicate a dual regulatory function for ABCA1 in macrophage lipid metabolism and inflammation.

ATP Binding Cassette Transporter 1↗
PubMed1999

The scavenger receptor CD163: regulation, promoter structure and genomic organization.

CD163 is a recently identified member of the scavenger receptor cysteine-rich superfamily expressed on peripheral blood monocytes and most tissue macrophages. We demonstrate that in vitro culture of human blood monocytes with recombinant M-CSF induces CD163 transcription. In contrast, dendritic differentiation in the presence of GM-CSF and IL-4 suppresses CD163 mRNA and protein levels. Because an important function of CD163 in inflammation has been suggested, we investigated the influence of pro- and anti-inflammatory stimuli on CD163 expression and found a significant suppression by lipoposaccharide and IFN-gamma, whereas IL-10 or dexamethasone strongly induced the expression of CD163. The induction of CD163 mRNA by dexamethasone is suggested to be mediated by several glucocorticoid receptor binding sites located in the proximal promoter region. In addition, this sequence contains potential binding sites for the transcription factors Sp1, C/EBPalpha, Ets-2, PU.1 and AP-1, which have been shown to play an important role in myeloid-specific gene expression. We also identified an L1-transposable element 1.4 kb upstream of the transcription start site that might influence the promoter activity. The function of CD163 may also depend on the use of different isoforms. Several variants of CD163 mRNA have been described that encode proteins with altered cytoplasmic or extracellular domains and thus may differ in their functional properties. We analyzed the genomic organization of the CD163 gene and could demonstrate that these isoforms result from alternative splicing. Further characterization of the isoforms may help to understand the complete function of CD163.

Alternative Splicing↗
PubMed1999

Growth and cell cycle abnormalities of fibroblasts from Tangier disease patients.

We have investigated the abnormal proliferation and morphology of fibroblasts from patients with Tangier disease (TD), a high density lipoprotein (HDL) deficiency syndrome that is characterized by impairment of HDL3-mediated lipid efflux and Gi-protein-mediated signaling via phosphatidylinositol-specific phospholipase C (PI-PLC) and phospholipase D (PLD). TD fibroblasts displayed a 30% to 50% reduced in vitro growth rate and a 1.6-fold increased cell surface area. The response to different mitogens was diminished, and asynchronously growing TD fibroblasts showed 4.4+/-0.3% S-phase and 19.1+/-0.5% G2/M-phase cells compared with 9.7+/-0.6% and 7.8+/-0.5%, respectively, in controls. Monensin, but not brefeldin A, induced an S- and G2/M-phase distribution in control cells similar to that found in TD fibroblasts. This effect of monensin was accompanied by an increase of ceramide levels in controls, whereas TD fibroblasts already had a 2.5-fold increased basal ceramide concentration. Incubation of control cells with C2 ceramide and threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) mimicked the effect of monensin on the cell cycle. The inhibition of neither Gi protein function by pertussis toxin nor PLD by butanol resulted in a G2/M-phase arrest. Propranolol, known to increase phosphatidic acid levels, was ineffective in reversing the G2/M-phase arrest in TD fibroblasts. In addition, cDNA sequences and mRNA expression of the participants of PI-PLC or PLD signaling, ie, G-protein subunits alphai1, alphai2, and alphai3; phosphatidylinositol transfer proteins-alpha and -beta; and ADP ribosylation factors 1 and 3 were found to be normal. Thus, growth and cell cycle abnormalities in TD fibroblasts are likely to be related to impaired Golgi function and sphingolipid signaling rather than inoperative G-protein signal transduction. Because PDMP was also found to decrease HDL3-mediated lipid efflux in control but not TD fibroblasts, similar pathways seem to be involved in the disturbances of lipid transport and growth retardation.

ADP-Ribosylation Factors↗
PubMed1999

Transcription factors Sp1 and AP-2 mediate induction of acid sphingomyelinase during monocytic differentiation.

Cells from the human monocytic leukemia cell line THP-1 differentiate towards a macrophage-like phenotype when stimulated with phorbol 12-myristate -13- acetate (PMA), 1,25-dihydroxy-vitamin D3, and various other agents. We demonstrate here that the expression of the lysosomal enzyme acid sphingomyelinase (ASM; E.C. 3.1.4.12) is induced during this process and is strongly elevated in differentiated THP-1 cells, as well as in differentiated human mononuclear phagocytes. Using Northern blotting, RNase protection assay, and nuclear run-on analyses, we show that the up-regulation of ASM expression is regulated mainly at the level of transcription and that new protein synthesis is required for enhanced ASM activity. This cell-type specific induction by PMA treatment was further investigated with respect to transcriptional control. A series of 5' deletion derivatives of the upstream regulatory region were used in transient transfection assays to identify promoter elements required for basal and inducible gene expression. A PMA responsive element was localized to a region between -319 and -219 bp upstream of the initiation codon and co-transfections with transcription factor expression plasmids for AP-2 and Sp1 resulted in augmented ASM promoter activity, which was abolished when the binding sites for these two factors were deleted. Using electrophoretic mobility shift assays and supershift assays we demonstrate that this region is specifically bound by Sp1 and AP-2. These factors are present in nuclear extracts prepared from both induced and uninduced THP-1 cells. However, the intensity of the complex formed appeared to increase when nuclear extracts from PMA-treated cells were used. From these studies, we conclude that a concerted action of the transcription factors AP-2 and Sp1 is essential for the up -regulation of ASM expression during the process of macrophage differentiation.

Base Sequence↗
PubMed1999

Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS).

Ceramide (CER) is an important signaling molecule involved in a variety of cellular processes, including differentiation, cell growth, and apoptosis. Currently, different techniques are applied for CER quantitation, some of which are relatively insensitive and/or time consuming. Tandem mass spectrometry with its high selectivity and sensitivity is a very useful technique for detection of low abundant metabolites without prior purification or derivatization. In contrast to existing mass spectrometry methods, the developed electrospray tandem mass spectrometry (ESI-MS/MS) technique is capable of quantifying different CER species from crude cellular lipid extracts. The ESI-MS/MS is performed with a continuous flow injection and the use of an autosampler, resulting in a high throughput capability. The collision-induced fragmentation of CER produced, in addition to others, a characteristic fragment of m/z 264, making a precursor ion scan of 264 well suited for CER quantitation. Quantitation is achieved by use of a constant concentration of a non-naturally occurring internal standard C8-CER, together with a calibration curve established by spiking different concentrations of naturally occurring CER. The calibration curves showed linearity over a wide concentration range and sample volumes equivalent to 10 microg of cell protein corresponding to about 20, 000 fibroblasts were sufficient for CER analysis. Moreover this assay showed a detection limit at the subpicomole level. In summary, this methodology enables accurate and rapid analysis of CER from small samples without prior separation steps, thus providing a useful tool for signal transduction research.

Ceramides↗
PubMed1999

Influence of crystal orientation on oxygen analysis in NiO with energy dispersive X-ray analysis

Characteristic X-ray spectra of stoichiometric NiO were investigated by EDX. Peak intensities of Ni-Kalpha and O-Kalpha are determined for several zone axes and also off-axis specimen orientations. The intensity ratios depend strongly on specimen orientation, resulting in errors of up to 30% in concentration analysis. Based on Bloch wave calculations, the dependence on the zone axis is explained qualitatively by the ordered structure of NiO, assuming the atoms to be ideal point scatterers. However, in order to get a quantitative description this simple model is not sufficient: the spatial structure of the electron shells must also be considered.

Journal Article↗
PubMed1999

Clinically useful information provided by the flow cytometric immunophenotyping of hematological malignancies: current status and future directions.

BACKGROUND: At present, immunophenotyping of hematological malignancies represents one of the most relevant clinical applications of flow cytometry. In recent years, its use has extended from clinical research to diagnostic laboratories. The aim of this report is to critically review the type of information provided by the flow cytometric immunophenotyping of hematological malignancies and its clinical impact as well as to highlight its potential future applications. METHODS: The currently available information, including that provided by different international consensus groups on the phenotypic characterization of hematologic malignancies, was reviewed. Additionally, recent reports on the immunophenotypic analysis of hematological malignancies published in hematology, oncology, pathology, immunology, and cell biology journals were also analyzed. RESULTS: A careful review of the literature showed that in spite of the well-established utility of immunophenotyping for the diagnosis, classification, prognostic stratification, and monitoring of hematological malignancies, only a small part of the information on the immunophenotypic characteristics of pathological hemopoietic cells has been used routinely. Specific and sensitive identification of neoplastic cells and their accurate enumeration and phenotypic characterization represent the major aims of these procedures. Similarities between leukemic and healthy cells allow the establishment of the lineage and maturation stage of the pathologic cells, this information being of great utility for the diagnosis, classification, and prognostic evaluation of different subtypes of hematological malignancies. On the other hand, the phenotypic aberrations displayed by leukemic cells could allow the selection of cases carrying specific genetic abnormalities in which further confirmatory molecular studies will be performed. CONCLUSIONS: The information provided by the flow cytometric immunophenotyping of hematological malignancies is of great clinical utility, with a major challenge for the near future being the standardization of technical procedures, data interpretation, and reporting.

Flow Cytometry↗