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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
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↗
PubMed1997

Taming TNF: strategies to restrain this proinflammatory cytokine.

Recent studies have demonstrated the essential role of tumor necrosis factor alpha (TNF-alpha) in rheumatoid arthritis and Crohn's disease. This article discusses agents known to suppress the formation or activity of TNF-alpha, and summarizes clinical studies using anti-TNF-alpha antibodies.

Animals↗
PubMed1997

An investigation into the variability of extract viscosity of wheat-relationship with the content of non-starch-polysaccharide fractions and metabolisable energy for broiler chickens.

The in vitro extract-viscosity and the content of non-starch-polysaccharides were investigated in 34 defined wheat varieties grown at 5 locations each. Both, wheat genotype as well as growing location clearly influenced the viscosity of soluble extract from wheat. Furthermore, the content of non-starch-polysaccharides (soluble/total) and pentosans (soluble/total) were determined in 13 wheat varieties each grown at two locations. Soluble pentosan contents were highly positively correlated with extract viscosity of wheat at the locations Hayn (r = 0.86) and Biendorf (r = 0.90). The classical apparent metabolisable energy of 5 wheat samples having different extract viscosities was assessed. The AMEN values ranged from 14.0 to 14.6 MJ/kg DM and were significant negatively correlated to content of soluble arabinoxylans (r = 0.67) and to the extract viscosity (r = 0.83). Furthermore, the viscosity of jejunal (4.0 to 22.8 mPas) and ileal (13.1 to 78.0 mPas) digesta exhibited a clear relationship with soluble pentosan contents and extract viscosity. Under the conditions applied in this study the technique of extract viscosity measurement can predict the AME.

Analysis of Variance↗
PubMed1997

Cytokines and therapeutic oligonucleotides.

Therapeutic oligonucleotides - short strands of synthetic nucleic acids - encompass antisense and aptamer oligonucleotides. Antisense oligonucleotides are designed to bind to target RNA by complementary base pairing and to inhibit translation of the target protein. Antisense oligonucleotides enable specific inhibition of cytokine synthesis. In contrast, aptamer oligonucleotides are able to bind directly to specific proteins. This binding depends on the sequence of the oligonucleotide. Aptamer oligonucleotides with CpG motifs can exert strong immunostimulatory effects. Both kinds of therapeutic oligonucleotides - antisense and aptamer oligonucleotides - provide promising tools to modulate immunological functions. Recently, therapeutic oligonucleotides have moved towards clinical application. An antisense oligonucleotide directed against the proinflammatory intercellular adhesion molecule 1 (ICAM-1) is currently being tested in clinical trials for therapy of inflammatory disease. Immunostimulatory aptamer oligonucleotides are in preclinical development for immunotherapy. In the present review we summarize the application of therapeutic oligonucleotides to modulate immunological functions. We include technological aspects as well as current therapeutic concepts and clinical studies.

Animals↗
PubMed1996

Urinary sodium excretion: association with hyperinsulinaemia, hypertension and sympathetic nervous system activity in obese and control children.

UNLABELLED: The aim of the present study was to evaluate the association between 24 h urinary excretion of sodium and blood pressure, fasting plasma insulin, renin, aldosterone and serum norepinephrine concentrations in 45 obese and 15 control children. Urinary sodium excretion was significantly lower in obese subjects (1.3 +/- 0.6 mmol/kg/24 h, P < 0.01) than in controls (2.8 +/- 1.3 mmol/kg/24 h). Hyperinsulinaemia did not affect sodium excretion of obese children. Plasma renin and aldosterone levels did not correlate with sodium excretion and were significantly higher in overweight children. Serum norepinephrine levels were also significantly higher in the obese group (0.66 +/- 0.89 microgram/100 ml) as compared to the controls (0.11 +/- 0.03 microgram/100 ml, P < 0.01) and showed significant negative correlation with urinary sodium excretion (r = 0.43, P < 0.05). CONCLUSION: Hyperinsulinaemia and the consequently increased sympathetic nervous system activity might be involved in the development of high blood pressure in obese children by decreasing urinary sodium excretion.

Adolescent↗
PubMed1996

Proton dosimetry intercomparison.

BACKGROUND AND PURPOSE: Methods for determining absorbed dose in clinical proton beams are based on dosimetry protocols provided by the AAPM and the ECHED. Both groups recommend the use of air-filled ionization chambers calibrated in terms of exposure or air kerma in a 60Co beam when a calorimeter or Faraday cup dosimeter is not available. The set of input data used in the AAPM and the ECHED protocols, especially proton stopping powers and w-value is different. In order to verify inter-institutional uniformity of proton beam calibration, the AAPM and the ECHED recommend periodic dosimetry intercomparisons. In this paper we report the results of an international proton dosimetry intercomparison which was held at Loma Linda University Medical Center. The goal of the intercomparison was two-fold: first, to estimate the consistency of absorbed dose delivered to patients among the participating facilities, and second, to evaluate the differences in absorbed dose determination due to differences in 60Co-based ionization chamber calibration protocols. MATERIALS AND METHODS: Thirteen institutions participated in an international proton dosimetry intercomparison. The measurements were performed in a 15-cm square field at a depth of 10 cm in both an unmodulated beam (nominal accelerator energy of 250 MeV) and a 6-cm modulated beam (nominal accelerator energy of 155 MeV), and also in a circular field of diameter 2.6 cm at a depth of 1.14 cm in a beam with 2.4 cm modulation (nominal accelerator energy of 100 MeV). RESULTS: The results of the intercomparison have shown that using ionization chambers with 60Co calibration factors traceable to standard laboratories, and institution-specific conversion factors and dose protocols, the absorbed dose specified to the patient would fall within 3% of the mean value. A single measurement using an ionization chamber with a proton chamber factor determined with a Faraday cup calibration differed from the mean by 8%. CONCLUSION: The adoption of a single ionization chamber dosimetry protocol and uniform conversion factors will establish agreement on proton absorbed dose to approximately 1.5%, consistent with that which has been observed in high-energy photon and electron dosimetry.

Calibration↗