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J Pfeilschifter

Publications and source records attributed to J Pfeilschifter.

At least 55 records · Page 3Linked to original sources
PubMed2000

Ceramide binds to the CaLB domain of cytosolic phospholipase A2 and facilitates its membrane docking and arachidonic acid release.

Excessive production of eicosanoids is characteristic of many inflammatory diseases. In this study we show that ceramide, which is an early messenger of inflammatory cytokine action, exerts a dual effect on the cytosolic phospholipase A2 (cPLA2), the rate-limiting enzyme in arachidonic acid release and subsequent eicosanoid formation. Stimulation of renal mesangial cells with exogenous short-chain ceramide analogs for 30 and 60 min leads to a concentration-dependent increase in arachidonic acid release that is not blocked by specific inhibitors of mitogen-activated protein kinase pathways. This suggests that these established upstream activators of cPLA2 are not involved in ceramide-induced arachidonic acid release. By use of photoactivatable ceramide analogs, D- and L-[125I]3-trifluoromethyl-3-(m-iodophenyl)diazirine-ceramides (TID-ceramides), we observed a direct interaction of ceramide with cPLA2. This interaction was independent of the absolute configuration as D- and L-TID-ceramide were equally effective in binding to cPLA2. Moreover, recombinant CaLB domain of cPLA2 as well as a mutant deficient in the connecting 'hinge' domain of cPLA2, efficiently bound D- and L-TID-ceramides, whereas the catalytic domain did not interact with TID-ceramides. In vitro binding assays reveal that stearoyl-arachidonyl-phosphatidylcholine (SAPC)-liposomes containing increasing mol% of ceramide lead to an increased association of recombinant cPLA2 to the liposomes. Furthermore, measurement of cPLA2 activity in vitro shows that the presence of SAPC-liposomes resulted in only weak cPLA2 activity. However, the activity dramatically increases by addition of ceramide to the liposomes. Furthermore, liposomes containing SAPC and sphingomyelin resulted in no better substrate than SAPC liposomes, unless bacterial sphingomyelinase was added to generate ceramide, which then causes a marked increase in cPLA2 activity. These results demonstrate that ceramide can interact directly with cPLA2 via the CaLB domain and thereby serves as a membrane-docking device that facilitates cPLA2 action in inflammatory diseases.

Animals↗
PubMed2000

[Incidental findings in endocrinology. When to observe and when intervention is necessary].

Hirsutism, gynecomastia or delayed puberty are usually benign conditions. However, underlying serious disorders must be excluded. Abnormalities in thyroid hormone measurements with contraceptives, glucocorticoids or severe nonthyroid disease must be differentiated from "true" impairments of thyroid function that require specific treatment. Increasingly, abnormalities are detected incidentally (incidentaloma) by diagnostic imaging procedures in organs such as the pituitary or the adrenal gland. Apart from judgements based on the original size of the tumor or the progression in size during observation, functional evaluation is always necessary to decide whether surgery should be recommended.

Diagnosis, Differential↗
PubMed2000

Regulation and immunhistochemical localization of nitric oxide synthases and soluble guanylyl cyclase in mouse spinal cord following nociceptive stimulation.

The localization and regulation of neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS) and soluble guanylyl cyclase (sGC) were assessed in the spinal cord of mice stimulated by an intraplantar injection of zymosan. Both, nNOS and iNOS were upregulated in the dorsal horns of the spinal cord in response to the zymosan challenge. While nNOS was found in neurons of superficial laminae iNOS occurred in astrocytes. Thus, astrocytes might be involved in the processing of nociceptive stimuli. Expression of sGC was not affected by zymosan treatment. It was found exclusively in nerve fibers suggesting that it was predominantly localized to the presynaptic neuron. This supports the hypothesis that nitric oxide (NO) acts as retrograde messenger in spinal nociceptive processing.

Animals↗
PubMed2000

Concentrations of insulin-like growth factor (IGF)-I, -II, and IGF binding protein-4, and -5 in human bone cell conditioned medium do not change with age.

We examined the effects of age on the secretion of insulin-like growth factor (IGF)-I, -II, and IGF binding protein-4, and -5 in long-term bone cell cultures from 71 female donors, aged 28-79 years. Our results suggest that under basal conditions, the intrinsic capacity of human bone cells to produce these IGF components is largely preserved with age.

Adult↗
PubMed2000

Keratinocyte-derived chemotactic cytokines: expressional modulation by nitric oxide in vitro and during cutaneous wound repair in vivo.

Inhibition of inducible nitric oxide-synthase (iNOS) enzymatic activity during cutaneous wound repair leads to severely impaired tissue regeneration. To assess whether disturbed leukocyte infiltration might participate in impaired repair, we determined expressional kinetics of neutrophil-attracting macrophage inflammatory protein-2 (MIP-2), and monocyte-attracting macrophage chemoattractant protein-1 (MCP-1) using an excisional wound healing model in mice. MCP-1 was induced in epithelial keratinocytes upon wounding, and our data indicate that NO serves a negative regulatory role for MCP-1 expression in vivo, as clearly reduced numbers of wound margin keratinocytes associated with NO-deficient repair compensate for high MCP-1 expression levels observed during normal healing. MIP-2 expression was restricted to hair follicles which were not reduced in number during NO-deficient repair. In vitro studies confirmed a regulatory role of NO for keratinocyte-derived chemokine expression, as NO attenuated IL-1beta- and TNF-alpha-induced MCP-1 mRNA expression, whereas NO augmented IL-1beta-induced IL-8 (functional human homolog to murine MIP-2) mRNA expression in the human keratinocyte cell line HaCaT.

Animals↗
PubMed2000

Large induction of the chemotactic cytokine RANTES during cutaneous wound repair: a regulatory role for nitric oxide in keratinocyte-derived RANTES expression.

We investigated the role of NO on expressional regulation of the chemotactic cytokine RANTES (regulated upon activation, normal T-cell expressed and secreted) during tissue regeneration using an excisional wound-healing model in mice. Wound repair was characterized by a large and sustained induction of RANTES expression, and inhibition of inducible nitric oxide synthase (iNOS) during repair only slightly decreased RANTES expression levels. Immunohistochemical analysis revealed keratinocytes of the wound margins and the hyperproliferative epithelium to be the main RANTES-expressing cell type within the wound. Therefore we analysed the regulation of RANTES expression in vitro in cultured human keratinocytes of the cell line HaCaT. Here we demonstrate that NO very efficiently suppressed interleukin-1beta- and tumour-necrosis-factor-alpha-induced RANTES expression in keratinocytes. Furthermore, down-regulation of cytokine-induced RANTES mRNA in keratinocytes was dependent on endogenously produced NO, as inhibition of the co-induced iNOS by L-N(G)-monomethyl-L-arginine increased cytokine-triggered RANTES expression in the cells. Moreover, we observed strongest RANTES-immunopositive labelling in epithelial areas which were characterized by a NO-mediated low cellularity. Thus our data implicate NO as a negative regulator of RANTES expression during wound repair in vivo, as decreased numbers of keratinocytes observed in the absence of wound-derived NO might compensate for the high levels of RANTES expression which are associated with normal repair.

Animals↗
PubMed2000

[Differential diagnosis of hypercalcemia in adults].

BACKGROUND: Increases in serum calcium are often detected by chance. They warrant a prompt diagnostic work-up to identify the underlying cause. DIAGNOSIS: Measurement of intact parathyroid hormone (PTH) is the first step to further evaluate confirmed hypercalcemia. With few exceptions, marked increases in PTH confirm primary hyperparathyroidism. Mildly elevated PTH levels may also be observed in familial hypocalciuric hypercalcemia. Low-normal or decreased PTH levels are mostly associated with clinically evident malignancies or granulomatous diseases. A careful history will often be sufficient to make the diagnosis of drug-related hypercalcemia. Clinical presentation, measurements of 1,25-dihydroxyvitamin D, PTH-related peptide and efforts to localize the underlying disease are successful in over 90% in finding the cause of hypercalcemia with low PTH. CONCLUSION: Hypercalcemia in adults can have many different causes. Identification of the underlying disease is important, since the corresponding treatment is very different for the different causes of hypercalcemia. In most cases, medical history, clinical presentation, and a few carefully directed laboratory measurements are sufficient to differentiate between the most important hypercalcemic disorders.

Adult↗
PubMed2000

Identification of copper/zinc superoxide dismutase as a nitric oxide-regulated gene in human (HaCaT) keratinocytes: implications for keratinocyte proliferation.

Recent studies have demonstrated an induction of expression of inducible nitric oxide synthase that is associated with several inflammatory diseases of the skin. To define the mechanisms of action of nitric oxide (NO) in the skin, we attempted to identify genes that are regulated by NO in keratinocytes. Using the human keratinocyte cell line HaCaT as a model system, we identified a Cu/Zn superoxide dismutase (SOD) that was strongly induced by high concentrations (500 microM) of NO-donating agents ¿S-nitrosoglutathione, sodium nitroprusside and (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1,2 -diolate (DETA-NO)¿, but not by serum or by single recombinant growth factors and inflammatory cytokines or by treatment with superoxide anions. Furthermore, endogenously produced NO increased the expression of Cu/Zn SOD mRNA in keratinocytes. Moreover, treatment of HaCaT cells with NO was associated with a biphasic effect on cell proliferation, because low doses (100 microM) of different NO donors (S-nitrosoglutathione and DETA-NO) mediated a proliferative signal to the cells, whereas high concentrations (500 microM) were cytostatic. To determine a possible correlation between the close regulation of Cu/Zn SOD expression and proliferation by NO in keratinocytes, we established a cell line (psp1CZ1N) carrying a human Cu/Zn SOD cDNA under the control of a ponasterone-inducible promoter construct. Ponasterone-induced overexpression of Cu/Zn SOD caused a cytostatic effect in proliferating psp1CZ1N cells. We therefore suggest that the up-regulation of Cu/Zn SOD expression by NO establishes an inhibitory mechanism on keratinocyte proliferation.

Base Sequence↗
PubMed2000

[Adhesion molecules].

Phagocytosis by specialized cells was, expressed in evolutionary terms, one of the first defense strategies of organisms in order to eliminate foreign materials. Migration of professional phagocytic cells to a lesion thus represents one of the most basic aspects of host defense. The article describes different families of adhesion molecules and their contribution to this process.

Animals↗
PubMed2000

Interferon-gamma mediates gene expression of IL-18 binding protein in nonleukocytic cells.

Interleukin-18 (IL-18) binding protein is a soluble decoy receptor for IL-18 which efficiently antagonizes biological functions of IL-18 in vitro and in vivo. Since regulation of IL-18 activity likely contributes to the pathogenesis of inflammatory diseases as well as malignancies, we investigated gene expression of IL-18 binding protein (IL-18BP) in different human cell systems, namely in the keratinocyte cell line HaCaT, in the colon carcinoma cell line DLD-1, and in primary renal mesangial cells. In unstimulated cells only minute amounts of mRNA coding for IL-18 binding protein were detectable. However, in all three cell types gene expression was markedly upregulated by interferon-gamma (IFN-gamma). IL-18 is recognized as a pivotal mediator of IFN-gamma production. Therefore, the present data imply that activity of IL-18 is modulated by a negative feedback mechanism which is mediated by IFN-gamma-induced IL-18 binding protein.

Cell Line↗
PubMed2000

New insights into the mechanism for clearance of apoptotic cells.

Apoptosis is a physiological mechanism for the removal of unwanted or damaged cells. Apoptotic cells are rarely seen in living tissues, however, because of their rapid and efficient removal by phagocytosis. Phagocytotic cells such as macrophages or dendritic cells recognize apoptotic cells by specific changes of cell surface markers, which usually are not present on normal cells. One such event is the exposure of phosphatidylserine, which moves from the plasma membrane inner leaflet to the outer leaflet in preapoptotic cells. An unresolved problem, however, was the nature of the phosphatidylserine receptor on the phagocytotic cells. In a recent issue of Nature, Fadok et al. have reported the cloning of a phosphatidylserine receptor using an antibody raised against activated macrophages. Antibody treatment of these macrophages blocks this capacity to engulf.

Animals↗
PubMed2000

Extracellular nucleotides activate the p38-stress-activated protein kinase cascade in glomerular mesangial cells.

1. Extracellular ATP and UTP have been reported to activate a nucleotide receptor (P2Y2-receptor) that mediates arachidonic acid release with subsequent prostaglandin formation, a reaction critically depending on the activity of a cytosolic phospholipase A2. In addition, extracellular nucleotides trigger activation of the classical mitogen-activated protein kinase (MAPK) cascade and cell proliferation as well as of the stress-activated protein kinase (SAPK) cascade. 2. In this study, we report that ATP and UTP are also able to activate the p38-MAPK pathway as measured by phosphorylation of the p38-MAPK and its upstream activators MKK3/6, as well as phosphorylation of the transcription factor ATF2 in a immunocomplex-kinase assay. 3. Time courses reveal that ATP and UTP induce a rapid and transient activation of the p38-MAPK activity with a maximal activation after 5 min of stimulation which declined to control levels over the next 20 min. 4. A series of ATP and UPT analogues were tested for their ability to stimulate p38-MAPK activity. UTP and ATP were very effective analogues to activate p38-MAPK, whereas ADP and gamma-thio-ATP had only moderate activating effects. 2-Methyl-thio-ATP, beta gamma-imido-ATP, AMP, adenosine and UDP had no significant effects of p38-MAPK activity. In addition, the extracellular nucleotide-mediated effect on p38-MAPK was almost completely blocked by 1 mM of suramin, a putative P2-purinoceptor antagonist. 5. In summary, these results demonstrate for the first time that extracellular nucleotides are able to activate the MKK3/6- p38-MAPK cascade most likely via the P2Y2-receptor. Moreover, this finding implies that all three MAPK subtypes are signalling candidates for extracellular nucleotide-stimulated cell responses.

Activating Transcription Factor 2↗
PubMed2000

Suppression of apoptosis by glucocorticoids in glomerular endothelial cells: effects on proapoptotic pathways.

Tumour necrosis factor-alpha (TNF-alpha)- and lipopolysaccharide (LPS)-induced apoptosis of bovine glomerular endothelial cells is now recognized as an important part in the pathogenesis of glomerulonephritis characterized by early mitochondrial cytochrome c release, mitochondrial permeability transition, Bak protein upregulation, Bcl-X(L) protein downregulation and caspase-3 activation. Co-treatment of cells with 10 nM dexamethasone and TNF-alpha or LPS blocked roughly 90% of apoptotic cell death in glomerular endothelial cells. The action of glucocorticoids could be documented in that they prevented all apoptotic markers such as DNA laddering, DNA fragmentation measured by the diphenylamine assay as well as morphological alterations. To mechanistically elucidate the action of glucocorticoids we evaluated whether glucocorticoids elicit a time-dependent effect. For dexamethasone, to maximally inhibit DNA fragmentation a preincubation period was not required. Even if dexamethasone was supplemented 6 h following TNF-alpha or LPS we observed a maximal inhibitory effect. Concerning its influence on TNF-alpha and LPS signal transduction, we found that dexamethasone only partially prevented cytochrome-c-release as a first sign of apoptotic cell death but efficiently blocked mitochondrial permeability transition. Moreover, TNF-alpha- and LPS-induced Bak upregulation, Bcl-X(L)-downregulation, and the activation of caspase-3-like proteases, measured fluorometrically using DEVD-AMC and PARP cleavage, were efficiently blocked by dexamethasone. We postulate that glucocorticoids exert their inhibitory action upstream of the terminal death pathways but downstream of primary receptor mediated signals by blocking pro-apoptotic signals pre- and/or post cytochrome c release and mitochondrial signalling.

Animals↗
PubMed2000

Inhibition of NFkappaB-mediated pro-inflammatory gene expression in rat mesangial cells by the enolized 1,3-dioxane-4, 6-dione-5-carboxamide, CGP-43182.

1. CGP-43182 has been described as a potent inhibitor of group IIA secreted phospholipase A(2) (group IIA sPLA(2)) activity in vitro. In rat mesangial cells, inhibition of group IIA sPLA(2) activity by CGP-43182 results in a 70% reduction of cytokine-stimulated prostaglandin E(2) biosynthesis, suggesting that group IIA sPLA(2) participates in arachidonic acid release and eicosanoid formation. Under these conditions the cytosolic phospholipase A(2) is not affected. 2. In mesangial cells, in addition to inhibition of catalytic activity, the membrane-permeant CGP-43182 completely blocked interleukin 1beta (IL1beta)-stimulated group IIA sPLA(2) gene expression. 3. A further action of CGP-43182 was a complete inhibition of cyclo-oxygenase-2 gene expression, resulting in a drastic reduction of prostaglandin formation in mesangial cells. 4. Moreover, CGP-43182 completely blocked IL1beta-induced gene expression of the inducible nitric oxide synthase, leading to an inhibition of cytokine-stimulated nitric oxide formation. 5. In contrast, the stimulatory effect of the cell-permeant cyclic AMP-analogue, dibutyryl-cAMP, on the induction of these enzymes was not inhibited by CGP-43182. These data indicate that CGP-43182 interferes with IL1beta- but not cyclic AMP-activated transcriptional regulation. 6. By studying components of the upstream transcription machinery, we observed an inhibition of NFkappaB activation by CGP-43182 in IL1beta-treated cells. Moreover, we observed that CGP-43182 prevented the phosphorylation and proteolytic degradation of the endogenous NFkappaB inhibitor, IkappaB, a process necessary for NFkappaB activation. 7. From our data, we propose that CGP-43182 is a potent anti-inflammatory drug useful for preventing the consequences of a concerted action of cytokine-stimulated pro-inflammatory genes mediated by NFkappaB.

Animals↗
PubMed2000

Large and sustained induction of chemokines during impaired wound healing in the genetically diabetic mouse: prolonged persistence of neutrophils and macrophages during the late phase of repair.

Chemokines are seen as the stimuli that largely control leukocyte migration. To assess whether the severely impaired process of cutaneous repair observed in genetically diabetic db/db mice is associated with a dysregulated infiltration of immune cells, we determined the expressional kinetics for the murine growth-regulated oncogene/melanoma growth stimulatory activity homolog macrophage inflammatory protein-2, and the macrophage chemoattractant protein-1, respectively. Wound repair in db/db mice was characterized by a sustained inflammatory response and a prolonged expression of macrophage inflammatory protein-2 and macrophage chemoattractant protein-1. Immuno-histochemistry revealed that keratinocytes at the wound margins expressed macrophage chemoattractant protein-1, whereas macrophage inflammatory protein-2 immunopositive signals were observed only in keratinocytes of hair follicles located adjacent to the wound site. Inactivation studies using neutralizing antibodies against macrophage chemoattractant protein-1 or macrophage inflammatory protein-2 indicated that sustained expression of these chemokines participated in a prolonged presence of neutrophils and macrophages at the wound site during diabetic repair. Furthermore, our data provide evidence that late infiltration (day 13 after injury) of neutrophils and macrophages into wounds in db/db mice was associated with a simultaneous downregulation of mRNA for receptors specific for macrophage inflammatory protein-2 and macrophage chemoattractant protein-1 in these animals.

Animals↗
PubMed2000

Nitric oxide modulates expression of matrix metalloproteinase-9 in rat mesangial cells.

UNLABELLED: Nitric oxide modulates expression of matrix metalloproteinase-9 in rat mesangial cells. BACKGROUND: High-output levels of nitric oxide (NO) are produced by rat mesangial cells (MCs) in response to proinflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) by the inducible isoform of NO synthase (iNOS). We tested modulatory effects of NO on the expression and activities of matrix metalloproteinases-9 and -2 (MMP-9 and MMP-2), respectively. Temporal and spatial expression of these MMPs and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), seems to be critical in the extensive extracellular matrix (ECM) remodeling that accompanies sclerotic processes of the mesangium. Methods and Results. Using the NO donors S-Nitroso-N-acetyl-D,L-penicillamine (SNAP) and DETA-NONOate, we found strong inhibitory effects of NO mainly on the IL-1beta-induced MMP-9 mRNA levels. NO on its own had only weak effects on the expression of MMP-9 and MMP-2. The addition of the NOS inhibitor NG-monomethyl L-arginine (L-NMMA) dose dependently increased steady-state mRNA levels of cytokine-induced MMP-9, suggesting that endogenously produced NO exerts tonic inhibition of MMP-9 expression. MMP-9 activity in conditioned media from MCs costimulated with IL-1beta and NO donor contained less gelatinolytic activity than media of cells treated with IL-1beta alone. Exogenously added NO did not alter gelatinolytic activity of MMP-9 in cell-free zymographs. The expression levels of TIMP-1 were affected by NO similarly to the expression of MMP-9. CONCLUSION: We conclude that NO modulates cytokine-mediated expression of MMP-9 and TIMP-1 in rat MCs in culture. Our results provide evidence that NO-mediated attenuation of MMP-9 gelatinolytic activity is primarily due to a reduced expression of MMP-9 mRNA, and not the result of direct inhibition of enzymatic activity.

Animals↗
PubMed2000

Assessment of sex hormone-binding globulin and osteocalcin in patients undergoing coronary artery bypass graft surgery.

OBJECTIVE: To determine sex hormone-binding globulin (SHBG) and osteocalcin (OC) levels in patients undergoing coronary artery bypass graft surgery to clarify the status of peripheral thyroid metabolism and to correlate SHBG and OC with thyroid hormones and adverse postoperative events. DESIGN: Prospective study. SETTING: University medical center. PARTICIPANTS: Fifty randomly selected patients undergoing coronary artery bypass graft surgery. INTERVENTIONS: On the morning of surgery before induction of anesthesia; 30 minutes after cross-clamping of the aorta; 2 hours and 6 hours after aortic cross-clamp removal; and on the first, second, third, and seventh postoperative mornings, blood samples were drawn and analyzed for OC, SHBG, triiodothyronine (tT3), free T3 (fT3), thyroxine (tT4), free T4 (fT4), thyroid-stimulating hormone, and thyroid-binding globulin. Adverse postoperative events were recorded. MEASUREMENTS AND MAIN RESULTS: Mean tT3 and fT3 decreased on average by 35% and 18% but remained within the normal range perioperatively. Similarly, mean SHBG and OC remained within the normal range. More than half of the patients investigated (60%) had OC concentrations below the normal range. Patients with pathologically decreased tT3 (n = 6) and tT4 (n = 16) intraoperatively and postoperatively had SHBG and OC concentrations similar to those in patients with normal tT3 and tT4 levels. Patients with postoperative complications had significantly lower OC levels preoperatively and on the first postoperative morning than those with an uneventful postoperative recovery. CONCLUSION: Despite significant intraoperative and postoperative decreases in levels of thyroid hormones, low T3 syndrome was rare in this patient population. Unchanged concentrations of SHBG and OC in patients with pathologically decreased tT3 or tT4 suggest normal local thyroid exposure at the tissue sites in these patients. OC may act as a predictor for postoperative outcome.

Adult↗