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Biomedical subjects

G Hartmann

Publications and source records attributed to G Hartmann.

At least 55 records · Page 3Linked to original sources
PubMed1999

[Prevalence of coronary risk factors in 233 men with coronary heart disease. Role of HDL cholesterol].

In 233 male patients (age 35 to 90 years) with coronary heart disease, chiefly myocardial infarction, the prevalence of the established coronary risk factors was investigated: total cholesterol, HDL- and LDL-cholesterol, triglycerides, hypertension, smoking, overweight, diabetes, heredity, physical inactivity and psychosocial stress. Cigarette smoking shows the highest prevalence (54%), followed by hypertension (39%), overweight (38.6%) and hypercholesterolaemia (34.7%). In patients over 65 the prevalence of these coronary risk factors is lower, whereas physical inactivity and diabetes are more frequent than in the younger group. Combinations of the main coronary risk factors are much more frequent in the younger group. HDL-cholesterol was found to be lower than normal (below 0.91 mmol/l) in 27% irrespective of age. The lower the concentration of HDL-cholesterol the more frequent are the following coronary risk factors: smoking, overweight, diabetes, physical inactivity and hypertriglyceridaemia. We suspect that a low HDL-cholesterol concentration in many individuals may be the result of a clustering of the aforementioned risk factors. For practical purposes it seems important to evaluate all relevant risk factors in an individual and to concentrate intervention on those with a fair chance of success, especially smoking and hypertension in the younger subjects. Normal or elevated HDL-cholesterol in itself is not a reliable cardioprotective factor since it is normal or elevated in two thirds of our coronary heart disease patients.

Adult↗
PubMed1999

Heparan sulfate-modified CD44 promotes hepatocyte growth factor/scatter factor-induced signal transduction through the receptor tyrosine kinase c-Met.

CD44 has been implicated in tumor progression and metastasis, but the mechanism(s) involved is as yet poorly understood. Recent studies have shown that CD44 isoforms containing the alternatively spliced exon v3 carry heparan sulfate side chains and are able to bind heparin-binding growth factors. In the present study, we have explored the possibility of a physical and functional interaction between CD44 and hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the receptor tyrosine kinase c-Met. The HGF/SF-c-Met pathway mediates cell growth and motility and has been implicated in tumor invasion and metastasis. We demonstrate that a CD44v3 splice variant efficiently binds HGF/SF via its heparan sulfate side chain. To address the functional relevance of this interaction, Namalwa Burkitt's lymphoma cells were stably co-transfected with c-Met and either CD44v3 or the isoform CD44s, which lacks heparan sulfate. We show that, as compared with CD44s, CD44v3 promotes: (i) HGF/SF-induced phosphorylation of c-Met, (ii) phosphorylation of several downstream proteins, and (iii) activation of the MAP kinases ERK1 and -2. By heparitinase treatment and the use of a mutant HGF/SF with greatly decreased affinity for heparan sulfate, we show that the enhancement of c-Met signal transduction induced by CD44v3 was critically dependent on heparan sulfate moieties. Our results identify heparan sulfate-modified CD44 (CD44-HS) as a functional co-receptor for HGF/SF which promotes signaling through the receptor tyrosine kinase c-Met, presumably by concentrating and presenting HGF/SF. As both CD44-HS and c-Met are overexpressed on several types of tumors, we propose that the observed functional collaboration might be instrumental in promoting tumor growth and metastasis.

Heparan Sulfate↗
PubMed1999

Quality assurance at the heavy-ion therapy facility at GSI.

In the present stage between the technological realization and the clinical phase, quality management gains a predominant role at the heavy ion therapy facility at GSI. Specific quality inspection procedures had to be developed for the subsequent parts of the beam delivery system, the treatment planning, the raster scanning device, the patient positioning, the dose verification and the safety interlock system. In the meantime the acceptance test of the whole facility has been carried out and an overview about our first experience with quality assurance procedures at the heavy ion irradiation facility is given, with a special emphasis on the medical physics part.

Calibration↗
PubMed1999

Mechanisms and therapeutic applications of immune stimulatory cpG DNA.

Aside from its function as the "blueprint of life" that encodes genetic information, DNA can have direct immune activities. The immune system has evolved a defense mechanism that is able to distinguish microbial DNA from our own because of differences in the frequency and methylation of CpG dinucleotides in particular base contexts. Within minutes of detecting such "CpG-S DNA," cells of the innate immune system become activated and produce cytokines that promote the generation of antigen specific T-helper-1-like immune responses. Animal studies indicate therapeutic utility for CpG-S DNA as a vaccine adjuvant and for the immunotherapy of cancer and infectious and allergic diseases.

Adjuvants, Immunologic↗
PubMed1999

CpG DNA and LPS induce distinct patterns of activation in human monocytes.

A specific set of immune functions is switched on in response to DNA containing unmethylated CpG dinucleotides in particular base contexts ('CpG motifs'). Plasmids, viral vectors and antisense oligodeoxynucleotides used for DNA vaccination, gene replacement or gene blockade contain immunostimulatory CpG motifs which may have independent biological activity. Although the immune stimulatory effects of CpG motifs on murine cells are well established, the evaluation of their possible effects on human cells is complicated by the higher LPS sensitivity of human leukocytes compared with those in mice. To address this issue, we analyzed CpG- and LPS-mediated immune activation of human PBMC. The biologic activity of LPS could be detected within 4 h using intracellular TNF staining of monocytes with flow cytometry at concentrations just one-twentieth (0.0014 Eu/ml) of the lower detection limit for the routinely used LAL assay (0.03 EU/ml). In contrast to the rapid LPS response, CpG DNA-stimulated TNF and IL-6 synthesis in human monocytes was not detectable until 18 h. E. coli DNA induced IL-6 synthesis in a concentration-dependent manner (30 micrograms/ml E. coli DNA; 409 pg/ml +/- 75 pg/ml, n = 7, IL-6 ELISA), but calf thymus DNA did not (< 10 pg/ml). Likewise, the CpG oligodeoxynucleotides 1760 (phosphorothioate) and 2059 (unmodified) induced IL-6 synthesis, but the corresponding control oligonucleotides 1908 and 2077 did not CpG DNA and LPS enhanced IL-6 synthesis synergistically. ICAM-1-expression of monocytes was increased 4.6-fold by E. coli DNA, 3.5-fold by 1760 and three-fold by 2059, compared with 3.6-fold by a maximal LPS stimulus and no change with non-CpG DNA. In conclusion, CpG-motifs induce TNF, IL-6 and ICAM-1 expression in human monocytes, but the kinetics of this differ from that induced by LPS, which makes it possible to distinguish immune activation by these agents. These results have important implications for the clinical development of therapeutic DNA in humans.

Cells, Cultured↗
PubMed1998

Reconstitution of mammary gland development in vitro: requirement of c-met and c-erbB2 signaling for branching and alveolar morphogenesis.

We have established a cell culture system that reproduces morphogenic processes in the developing mammary gland. EpH4 mouse mammary epithelial cells cultured in matrigel form branched tubules in the presence of hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the c-met tyrosine kinase receptor. In contrast, alveolar structures are formed in the presence of neuregulin, a ligand of c-erbB tyrosine kinase receptors. These distinct morphogenic responses can also be observed with selected human mammary carcinoma tissue in explant culture. HGF/SF-induced branching was abrogated by the PI3 kinase inhibitors wortmannin and LY294002. In contrast, neuregulin- induced alveolar morphogenesis was inhibited by the MAPK kinase inhibitor PD98059. The c-met-mediated response could also be evoked by transfection of a c-met specific substrate, Gab1, which can activate the PI3 kinase pathway. An activated hybrid receptor that contained the intracellular domain of c-erbB2 receptor suffices to induce alveolar morphogenesis, and was observed in the presence of tyrosine residues Y1028, Y1144, Y1201, and Y1226/27 in the substrate-binding domain of c-erbB2. Our data demonstrate that c-met and c-erbB2 signaling elicit distinct morphogenic programs in mammary epithelial cells: formation of branched tubules relies on a pathway involving PI3 kinase, whereas alveolar morphogenesis requires MAPK kinase.

Androstadienes↗
PubMed1998

Engineered mutants of HGF/SF with reduced binding to heparan sulphate proteoglycans, decreased clearance and enhanced activity in vivo.

BACKGROUND: Although a number of growth factors bind cell-surface heparan sulphate proteoglycans (HSPGs), the role of this interaction is unclear except for fibroblast growth factor which requires HSPG binding for signalling. Hepatocyte growth factor/scatter factor (HGF/SF) plays important roles in mammalian development and tissue regeneration and acts on target cells through a specific receptor tyrosine kinase encoded by the c-met proto-oncogene. This factor also binds HSPGs with high affinity, but conflicting data have been reported on the role of HSPG binding in HGF/SF signalling. RESULTS: To map the binding sites for HSPG and the Met receptor in HGF/SF, we have engineered a number of HGF/SF mutants in which several clusters of solvent-accessible residues in the hairpin structure of the amino-terminal domain or in kringle 2 have been replaced. Two of the mutants (HP1 and HP2) showed greatly decreased (more than 50-fold) affinity for heparin and HSPGs but retained full mitogenic and motogenic activities on target cells in culture. Furthermore, when compared with wild-type HGF/SF, the HP1 mutant exhibited a delayed clearance from the blood, higher tissue levels and a higher induction of DNA synthesis in normal, adult murine liver. CONCLUSIONS: These results establish the following: the binding sites in HGF/SF for Met and for HSPGs can be dissociated by protein engineering; high-affinity binding of HGF/SF to HSPGs is not essential for signalling; one role of HSPG binding in the HGF/SF system appears to be sequestration and degradation of the growth factor; and HGF/SF mutants with decreased affinity for HSPGs exhibit enhanced activity in vivo.

Animals↗
PubMed1998

Anti-inflammatory activities of cAMP-elevating agents: enhancement of IL-10 synthesis and concurrent suppression of TNF production.

Interleukin-10 (IL-10) and tumor necrosis factor (TNF) exert key roles in some acute and chronic inflammatory diseases. In this study we investigated (1) the potency of different cAMP-elevating agents in enhancing IL-10 synthesis, (2) the involvement of protein kinase A in this enhancement, and (3) the mutual dependence of cAMP-enhanced IL-10 formation and cAMP-suppressed TNF synthesis. Rolipram, a specific phosphodiesterase inhibitor and cicaprost, a prostacyclin analogue, were applied as cAMP-elevating agents. The stable cAMP antagonist (Rp)-cAMPS was used to abrogate activation of protein kinase A. Human peripheral blood mononuclear cells were stimulated with lipopolysaccharide (LPS). TNF was quantified by radioimmunoassay, IL-10 by enzyme-linked immunosorbent assay, and mRNA by reverse transcriptase-polymerase chain reaction. After LPS stimulation alone 253+/-45 pg/mL IL-10 was synthesized, which increased to 644+/-117 pg/mL in the presence of 1 microM rolipram. (Rp)-cAMPS reversed this increase of IL-10 formation. In the same samples, the LPS-stimulated production of TNF was markedly attenuated by rolipram or cicaprost. A kinetic analysis revealed a significant increase in TNF production before IL-10 formation was detectable. These results demonstrate that (1) cAMP-elevating agents enhance IL-10 synthesis and suppress TNF production; (2) these regulative functions of cAMP-elevating agents are mediated by activation of protein kinases A; (3) suppression of TNF synthesis by cAMP in the early phase is not mediated by endogenous IL-10. Taken together, rolipram and cicaprost exert a dual regulatory function by enhancing IL-10 formation and attenuating TNF synthesis.

Cyclic AMP↗
PubMed1998

The hairy cell leukemia cell line Eskol spontaneously synthesizes tumor necrosis factor-alpha and nitric oxide.

Tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) exert a wide array of immunoregulatory, partly related effects. We examined the production of these two mediators by the human hairy cell leukemia cell line Eskol. Combined cell lysate and supernatant of Eskol cells (0.5 x 10(6) cells ml(-1)) incubated for 18 h, contained a mean of 1.5 ng ml(-1) TNF-alpha. This spontaneous TNF-alpha synthesis was enhanced by phorbol ester (PMA) and phytohemagglutinin (PHA) and decreased by dexamethasone. Nitrite, the stable product of NO, accumulated in the supernatant of Eskol cells after prolonged incubation. Maximal nitrite concentrations (range: 0.8-3.5 microM at 2 x 10(6) cells ml(-1)) were detected after 7 days of incubation. NO production was augmented by PHA and reduced by PMA. The inhibitors of NO synthase N(G)-monomethyl-L-arginine (L-NMMA) and aminoguanidine decreased NO synthesis. Simultaneous activation with the proinflammatory cytokines, interferon-gamma, interleukin-1beta and TNF-alpha, increased NO synthesis. These results suggest that NO production in Eskol cells results from inducible NO synthase activity. This is the first direct demonstration of NO formation in human lymphoid cells. The cell line, Eskol, may serve as a model to study regulation of TNF-alpha and NO synthesis in human B-cell leukemia.

Dexamethasone↗
PubMed1998

C-met activation is necessary but not sufficient for liver colonization by B16 murine melanoma cells.

Metastasis to the liver is a frequent event in clinical oncology, the molecular mechanisms of which are not fully understood. We have recently reported a consistent overexpression of c-met in B16 melanoma cells selected in vivo for enhanced liver metastatic ability. In this study we address the question as to whether constitutive activation of c-met is a necessary and sufficient condition for enhanced liver colonization by B16 melanoma cells. Different levels of c-met expression and/or activation in B16 cells were achieved by subcloning, or by c-DNA transfection with either HGF/SF or the oncogenic form of c-met (tpr-met). Metastatic ability of the different populations was then evaluated in vivo by the lung colonization (experimental metastasis) assay. Results indicate that c-met (but not tpr-met) activation in B16 melanoma cells may increase their liver colonizing potential, probably by enhancing motility and invasion in response to paracrine interactions with its ligand. C-met expression per se, however, is not able to change the organ specificity of the cells. C-met activation appears instead to be required at later stages of liver colonization by B16 melanoma cells, in order to enhance their site-specific metastatic ability.

Animals↗
PubMed1998

Inflammatory bowel disease: no association between allele combinations of the interleukin (IL) I beta and IL-I receptor antagonist gene polymorphisms.

BACKGROUND: Interleukin 1 (IL-1) and its physiological antagonist interleukin-1 receptor antagonist (IL-1 ra) play a crucial role in the pathogenesis of inflammatory bowel disease. Polymorphisms in the genes coding for these cytokines, the restriction enzyme TaqI polymorphism for IL-1 beta and the variable number of tandem repeats (VNTR) polymorphism for IL-1 ra, have been shown to influence cytokine synthesis in vitro. Recently, an association has been described for distinct allele combinations of these two polymorphisms in patients with ulcerative colitis and with Crohn's disease but not in healthy control subjects. METHODS: We studied 56 patients with ulcerative colitis, 64 patients with Crohn's disease and 196 healthy control subjects. All were unrelated Caucasians of European ancestry. After polymerase chain reaction (PCR) the amplification products were analysed on agarose gels. For the IL-1 beta polymorphism the PCR product was additionally digested using the restriction enzyme TaqI. RESULTS: The allele and genotype frequencies as well as the carriage rates of the IL-1 beta TaqI polymorphism in healthy control subjects were in agreement with previous findings in other populations. Allele and genotype frequencies of the IL-1 beta polymorphism were not different in inflammatory bowel disease patients compared with healthy control subjects. Comparing allele combinations of both polymorphisms no association could be identified either within healthy control subjects or in the groups of patients with ulcerative colitis or Crohn's disease. CONCLUSION: Thus, we could not confirm the results of a previous study reporting an association between the IL-1ra and IL-1 beta gene polymorphisms in patients with inflammatory bowel disease.

Adult↗
PubMed1998

In vivo synthesis of tumor necrosis factor-alpha in healthy humans after live yellow fever vaccination.

Most knowledge about the synthesis of human tumor necrosis factor (TNF)-alpha and its regulation derives from in vitro studies. The hypothesis that the syndrome of malaise, myalgia, and low fever observed after a vaccination with live attenuated yellow fever virus could be associated with increased synthesis of TNF was investigated. Plasma samples of 15 healthy subjects taken before and until day 2 (until day 11 in 5 of these subjects) after yellow fever vaccination, showed a significant increase of plasma TNF concentration on day 2 after vaccination and a second peak on day 7. Interleukin-1 receptor antagonist (IL-1ra) concentration was significantly elevated in all subjects on day 2 after vaccination. In a control group receiving non-live vaccinations, TNF and IL-1ra concentrations were unchanged. Thus, yellow fever vaccination represents a routine medical intervention that results in a reproducible increase in the plasma concentration of TNF and secondary cytokines. This may serve as a unique model for the study of the regulation of TNF synthesis in vivo.

Adult↗
PubMed1998

Spontaneous and cationic lipid-mediated uptake of antisense oligonucleotides in human monocytes and lymphocytes.

Monocytes are important target cells for anti-inflammatory antisense strategies. However, monocytes are characterized by strong phagocytic and catalytic activity which may limit the effect of antisense oligonucleotides. Intracellular distribution of oligonucleotides in monocytes and the effect of cationic lipids on oligonucleotide uptake in monocytes and other leukocytes have not been evaluated. We investigated cationic lipid-mediated uptake of oligonucleotides in human monocytes and lymphocyte subpopulations. Incorporation of oligonucleotides was quantified by flow cytometry and by confocal microscopy. In the absence of cationic lipids, nearly 100% of monocytes and of B lymphocytes incorporated oligonucleotides compared with only 12% of natural killer cells and 1% of T lymphocytes. The amount of oligonucleotide uptake per cell, as determined by mean fluoresence intensity of positive cells, was four times higher in monocytes than in B lymphocytes. Cationic lipids, which form complexes with oligonucleotides, markedly enhanced the amount of oligonucleotide uptake in all cell types and were most effective at a ratio of 1.1 of positive-to-negative molar charges. In monocytes, oligonucleotides incorporated spontaneously (without a lipid carrier) were trapped in cytoplasmic vesicles. In contrast, cationic lipid-mediated uptake of fluorescence-labeled oligonucleotides resulted in cytoplasmic and nuclear staining. We conclude that 1) monocyte and lymphocyte subpopulations differ in the degree of spontaneous oligonucleotide uptake, and 2) lipofectin both quantitatively and qualitatively affects this uptake. Our results may explain the necessary role of cationic lipids in most antisense models with leukocytes as target cells.

Flow Cytometry↗
PubMed1997

Suppression of tumor necrosis factor-alpha production by interleukin-10 is enhanced by cAMP-elevating agents.

The pro-inflammatory peptide tumor necrosis factor-alpha (TNF) stimulates production of the anti-inflammatory cytokine-interleukin-10 by monocytes which in turn inhibits the synthesis of TNF. This inhibitory effect of interleukin-10 may contribute to the balance of pro- and anti-inflammatory cytokines in several diseases, e.g., chronic inflammatory bowel disease. In the present study we addressed the question whether interleukin-10 in combination with other TNF-suppressing agents leads to enhanced suppression of TNF synthesis. We investigated the inhibitory potency of interleukin-10 in combination with rolipram, a specific type IV phosphodiesterase inhibitor, or with cicaprost, a stable prostacyclin analogue in lipopolysaccharide-stimulated human peripheral blood mononuclear cells. Peripheral blood mononuclear cells were stimulated with 10 ng/ml lipopolysaccharide in the absence or presence of interleukin-10 or one of the cAMP-elevating agents. First, we confirmed the TNF-suppressing effect of interleukin-10, rolipram and cicaprost alone and determined the IC50 for these substances. Second, for the combination of interleukin-10 with one of the cAMP-elevating substances we were able to demonstrate enhanced TNF inhibition. Of these, the combination of interleukin-10 and rolipram revealed an additive effect. The maximal TNF synthesis of 5.5 +/- 1.1 ng/ml after lipopolysaccharide stimulation alone was inhibited by 0.1 ng/ml interleukin-10 to 2.7 +/- 0.6 ng/ml TNF and by 100 nM rolipram to 3.1 +/- 0.6 ng/ml TNF. Both substances combined suppressed TNF synthesis to 1.5 +/- 0.3 ng/ml. After stimulation with Staphylococcus epidermidis we could demonstrate a more pronounced inhibition of TNF synthesis by interleukin-10 compared to rolipram which was more effective after stimulation with lipopolysaccharide. Finally, the additive inhibitory effect of interleukin-10 and rolipram could be confirmed on the level of TNF mRNA. The results obtained in the present investigation could form a prerequisite to study the combination of interleukin-10 and cAMP-elevating agents in in vivo models of acute or chronic inflammatory diseases.

Antineoplastic Agents↗
PubMed1997

Domain structure of hepatocyte growth factor/scatter factor (HGF/SF).

The modular structure of hepatocyte growth factor/scatter factor (HGF/SF) has facilitated structure-function analysis. Domain deletion experiments have established that the N-domain, kringle 1 and kringle 2 are essential for HGF/SF activity on target cells and that, conversely, truncated variants containing the N-domain and kringle 1 (NK1) or kringles 1 and 2 (NK2) exhibit partial agonistic or antagonistic activity depending on target cells and the presence of full length HGF/SF. The 3D structures of the six domains of HGF/SF have been modelled on the structure of homologues, offering interesting insights into putative mechanisms of domain interactions, receptor binding and activation. The predictions offered by such models are currently assessed by protein engineering techniques and will ultimately be measured against experimental structures.

Gene Deletion↗