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G Schmitz

Publications and source records attributed to G Schmitz.

At least 145 records · Page 8Linked to original sources
PubMed1998

Autoantibodies against human calpastatin in rheumatoid arthritis: epitope mapping and analysis of patient sera.

Autoantibodies against calpastatin have recently been described to be highly prevalent in sera of patients with rheumatoid arthritis (RA). When the sera of 45 patients with RA were analysed for autoantibodies against calpastatin by a newly developed enzyme-linked immunosorbent assay (ELISA), only four sera (8.9%) tested positive, which is not significantly different from the frequency observed in healthy controls. Since the ELISA is based on a synthetic peptide containing the C-terminal 27 amino acids of calpastatin bound to the solid phase, this negative result might be the consequence of the small antigen used. Therefore, a systematic analysis of the epitopes for autoantibodies in calpastatin was performed using sera from RA patients and healthy individuals. Recombinant fusion proteins containing fragments of calpastatin or the complete protein were produced and sera analysed by Western blots. In the N-terminal portion (amino acids 1-369), at least two major epitopes exist, against which 65% of normal sera as well as 76% of RA sera show reactivity in Western blot assays. These epitopes are not useful for clinical diagnostics. Only five out of 45 (11.1%) RA sera reacted against the C-terminal portion (amino acids 363-708) of calpastatin, while four out of 52 (7.7%) control sera showed reactivity. Three of the five RA sera and two out of four control sera had autoantibodies against the C-terminal 27 amino acids of calpastatin. These three patient sera had already been tested positive in the ELISA. The fourth patient positive in the ELISA was Western blot negative. The differences between the group of RA patients and controls are not statistically significant. When the clinical characteristics of the four patients with autoantibodies against the carboxyl end of calpastatin were analysed, it became apparent that all four had significantly elevated C-reactive protein (>50 mg/l). This observation might indicate that calpastatin autoantibodies are found in RA patients with more active disease. Thus, while the majority of RA patients do not have an increased prevalence of calpastatin autoantibodies, it cannot be ruled out definitively that a small subgroup may be characterized by autoantibodies to the C-terminus of calpastatin.

Adult↗
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

Anti-phospholipid-antibodies in patients with relapsing polychondritis.

Relapsing polychondritis (RP) is an extremely rare multisystemic disease thought to be of autoimmune origin. In order to assess if RP is associated with anti-phospholipid antibodies (aPL), clinical data and sera of 21 patients with RP were collected in a multicentre study. Concentration of anti-cardiolipin antibodies (aCL) (IgG-, IgM- and IgA-isotypes), anti-phosphatidylserine-antibodies (aPS) (IgG- and IgM-isotypes) and anti-beta-2-glycoprotein I-antibodies (a beta 2 GPI) were measured by ELISA. In eight patients aCL were found to be elevated. One patient had elevated aPS. No patient had elevated a beta 2 GPI. No patient had clinical signs and symptoms of a aPL syndrome. Interestingly, the two RP patients with the highest aPL had concomitant systemic lupus erythematosus (SLE). Therefore the presence of elevated aPL in RP is probably more closely related to an associated SLE than to RP itself. There is no convincing evidence that aPL are associated with RP.

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↗
PubMed1998

Recent advances in molecular genetics of cardiovascular disorders. Implications for atherosclerosis and diseases of cellular lipid metabolism.

Two developments in molecular genetics will profoundly influence our understanding and the diagnosis of cardiovascular disorders. First, the identification of genes responsible for monogenic and polygenic traits by analysis of e.g. large pedigrees and affected sib pairs provides invaluable data regarding the role of specific genes in common diseases like arteriosclerosis, hypertension, diabetes, thrombosis/hemostasis and obesity. Besides the insights into the underlying pathophysiology, this knowledge will permit to identify persons at high risk for disease development. These patients can then obtain a targeted intervention. The second development is related to the availability of new analytical tools for molecular biology. New methods such as sequencing by hybridisation (SBH), DNA-array technology or matrix assisted laser desorption/ionisation-time of flight mass spectroscopy (MALDI-TOF) permit sequence analysis of complete genes within hours. Automated PCR-technologies with homogenous amplicon detection formats simplify PCR and permit its use in the routine laboratory setting. Considering cardiovascular diseases there is a number of genes involved in lipid metabolism (apolipoproteins, lipoprotein receptors, lipolytic enzymes), thrombosis/hemostasis (platelet receptors, pro- and anticoagulant proteins, fibrinogen, PAI's), hypertension (angiotensin converting enzyme, angiotensinogen) glucose metabolism (glucose transporters, enzymes) and obesity (hormones, receptors), that are interesting candidates for sophisticated genetic risk assessment. Furthermore, there are also gene candidates involved in processes of early atherogenesis and chronic inflammation such as complement proteins, cell adhesion molecules, and cellular receptors and enzymes. Most of these gene candidates were derived from pathophysiologic knowledge and subsequent epidemiological studies. However, it is foreseeable that in the coming years genes will be identified which were not known so far to be involved in cardiovascular diseases. Genetic studies will be of prime importance in this area, as is exemplified by animal models. In the long term, analysis of these candidate genes before the implementation of therapy will permit a targeted intervention approach towards high risk patients. This will reduce the overall costs of health care without reducing the quality.

Arteriosclerosis↗
PubMed1998

Transcriptional regulation of lysosomal acid lipase in differentiating monocytes is mediated by transcription factors Sp1 and AP-2.

Human lysosomal acid lipase (LAL) is a hydrolase required for the cleavage of cholesteryl esters and triglycerides derived from plasma lipoproteins. It is shown here that during monocyte to macrophage differentiation, the expression of LAL-mRNA is induced. This induction is dependent on protein kinase C activity and protein synthesis. The cell type-specific increase in LAL expression is further investigated in the THP-1 cell line with respect to transcriptional regulation. The human monocytic leukemia cell line THP-1 differentiates into macrophage-like cells when treated with phorbol esters. In order to determine the cis-acting elements necessary for both basal and phorbol 12-myristate-13 acetate (PMA)-enhanced promoter activity, we performed deletion analysis and reporter gene assays. A PMA responsive element has been identified between -182 bp and -107 bp upstream of the major transcription start site. Gel mobility shift assays demonstrated that binding of Sp1 and AP-2 to the LAL promoter is increased by PMA in THP-1 cells. Co-transfections with expression plasmids for Sp1 and AP-2 further emphasized the important role of these transcription factors in both basal and PMA-enhanced LAL expression. Our data suggest that differentiation dependent increase of lysosomal acid lipase (LAL) expression in THP-1 cells is mediated by a concerted action of Sp1 and AP-2.

Cell Differentiation↗
PubMed1997

A bidirectional promoter connects the p14.5 gene to the gene for RNase P and RNase MRP protein subunit hPOP1.

We have identified the functional promoter of the translational inhibitor p14.5, the human homologue to a rat perchloric acid-soluble protein (PSP), a mouse heat-responsive protein (Hrp12) and a goat tumor antigen (UK114). Sequence analysis revealed a GC-rich promoter with several consensus sequences for transcription factors, but no TATA- and CAAT-box. To confirm promoter activity, DNA fragments of the p14.5 5'-flanking region were ligated in front of the luciferase gene and were transfected into HeLa and HepG2 cells. A minimal promoter between nt -104 and nt +88 relative to the transcription start site was responsible for basal activity. Furthermore, we observed a head-to-head orientation of p14.5 to the gene for the protein subunit of RNase P and MRP ribonucleoproteins (hPOP1). Luciferase assays with fragments of the hPOP1 5'-flanking region revealed a minimal promoter between nt -20 and nt +98 relative to the start of transcription. These data indicate that the 102 bp region between p14.5 and hPOP1 can act as a bidirectional promoter. The p14.5-hPOP1-cluster was mapped to chromosome 8q22 using in situ hybridization technique.

Animals↗
PubMed1997

Lipopolysaccharide-binding protein is required to combat a murine gram-negative bacterial infection.

An invading pathogen must be held in check by the innate immune system until a specific immune response can be mounted. In the case of Gram-negative bacteria, the principal stimulator of the innate immune system is lipopolysaccharide (LPS), a component of the bacterial outer membrane. In vitro, LPS is bound by lipopolysaccharide-binding protein (LBP) and transferred to CD14--the LPS receptor on the macrophage surface--or to high-density lipoprotein (HDL) particles. Transfer to CD14 triggers an inflammatory response which is crucial for keeping an infection under control. Here we investigate how LBP functions in vivo by using LBP-deficient mice. Surprisingly, we find that LBP is not required in vivo for the clearance of LPS from the circulation, but is essential for the rapid induction of an inflammatory response by small amounts of LPS or Gram-negative bacteria and for survival of an intraperitoneal Salmonella infection.

Acute-Phase Proteins↗
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

The genomic organization of the genes for human lipopolysaccharide binding protein (LBP) and bactericidal permeability increasing protein (BPI) is highly conserved.

We have determined the exon/intron organization of the human lipopolysaccharide binding protein (LBP) and bactericidal permeability increasing protein (BPI) genes. The LBP gene spans approximately 28.5 kb and is composed of 14 exons while the 31.5-kb-long BPI gene is composed of 15 exons. Comparison of the genomic organization of the LBP and BPI genes together with the genomic structures of the PLTP (phospholipid transfer protein) and CETP (cholesteryl ester transfer protein) genes, which all together constitute a gene family of functionally related proteins, revealed high homology with a remarkable conservation of exon/intron transitions. The exon/intron junctions of the LBP, BPI, and PLTP genes are almost identical, with most of the exons being of the same size. In addition, functional domains are conserved in these proteins. The C-terminal octapeptide important for CETP anchoring in lipoprotein particles is also present in LBP, BPI, and PLTP. The LPS binding motif in exons 3 and 4 has been retained in LBP and BPI. Our results indicate that the LBP, BPI, and PLTP genes, and probably the CETP gene, may have evolved from a common primordial gene and may share similar functional properties.

Acute-Phase Proteins↗
PubMed1997

Identification and characterization of a novel monocyte/macrophage differentiation-dependent gene that is responsive to lipopolysaccharide, ceramide, and lysophosphatidylcholine.

A novel differentiation-dependent cDNA (DIF-2) has been isolated from human mononuclear phagocytes by differential display. The full-length cDNA was cloned and sequenced. DIF-2 consists of 156 amino acids and has a predicted isoelectric point of 8.84. The mRNA is expressed in freshly isolated monocytes and is downregulated significantly when monocytes are subjected to differentiation. A similar differentiation-dependent downregulation is observed in normal hepatocytes compared to undifferentiated HepG2 cells. The mRNA expression in monocytes is sensitive to lipopolysaccharide and ceramide which both strongly increase DIF-2 transcription, while lysophosphatidylcholine results in a weaker upregulation of DIF-2 expression. A DIF-2 homologous gene has been previously isolated from mouse fibroblasts and was shown to be a serum growth factor-inducible immediate early gene. Our results indicate that DIF-2 represents a gene which is regulated in differentiation processes and strongly responsive to lipopolysaccharide, ceramide and lysophosphatidylcholine.

Amino Acid Sequence↗
PubMed1997

Acyl-CoA binding protein (ACBP) regulates acyl-CoA:cholesterol acyltransferase (ACAT) in human mononuclear phagocytes.

It is demonstrated that the acyl-CoA:cholesterol acyltransferase (ACAT) enzyme activity in rough endoplasmatic reticulum membranes is regulated by the acyl-CoA binding protein (ACBP). The ACAT activity is strongly inhibited by different ACBP/oleoyl-CoA complexes depending from the molar ratio of protein and fatty acid-CoA. Other lipid binding proteins such as bovine serum albumin and the liver fatty acid binding protein do not show any effects on ACAT activity. In addition, we can show that cholesterol loading with acetylated low density lipoproteins does not lead to an increase of the ACBP mRNA level. Consequently, the increase of the intracellular concentration of fatty acids because of the cholesteryl ester accumulation renders ACAT more active for cholesterol esterification. In binding studies we have characterized binding sites on microsomal membranes for the ACAT substrate oleoyl-CoA and the ACAT inhibitor diazepam. Diazepam competes with oleoyl-CoA and vice versa for its binding to microsomal membranes. This common binding site is suggested to be responsible for the transfer from ACBP-bound oleoyl-CoA to ACAT and, therefore, to be essential for the microsomal cholesterol esterification.

Acyl Coenzyme A↗
PubMed1997

Identification of naturally processed T cell epitopes from glutamic acid decarboxylase presented in the context of HLA-DR alleles by T lymphocytes of recent onset IDDM patients.

Glutamic acid decarboxylase (GAD) has been defined as a major target antigen in insulin-dependent diabetes mellitus (IDDM). To identify the molecular ligands triggering a T cell response to GAD, a panel of human GAD65-specific T lymphocyte lines was generated from peripheral blood of three recent onset IDDM patients. All lines derived from a patient expressing the high-risk-conferring HLA-DR*0301/ *0401 haplotypes recognized a single epitope localized between amino acid positions 270 and 283 of GAD65, a stretch that is located in close proximity to the homology region shared with Coxsackie virus P2-C protein. All lines with this specificity were restricted to the DRA, B1*0401 product of the DR4 haplotype. Analysis of the GAD-specific T cell response in a second patient homozygous for DR4 haplotypes demonstrated that the same DRA, B1*0401 allele selected T cells specific for a different determinant. The T cell response profile in a third patient showed that DR*1501/ *1601-encoding haplotypes could present at least three different epitopes to GAD65-specific T lymphocytes. One of these epitopes was presented by a DR allele associated with the resistance-conferring DRB1*1501 haplotype. GAD-specific T cell lines could not be isolated from HLA class II-matched normal individuals. Our data reveal that (a) the T cell response to GAD65 is quite heterogenous in recent onset IDDM patients; (b) HLA-DR, not DQ, seems to be the principal restriction element used by T cells present at the onset of the disease; and (c) T cells responding to epitopes containing identical sequences to Coxsackie virus P2-C protein were not detected.

Alleles↗