Hypericum perforatum L. (St John's wort) extract Ze 117 inhibits dopamine re-uptake in rat striatal brain slices. An implication for use in smoking cessation treatment?

Classic synthetic antidepressant drugs, as well as St John's wort extract (SJW), directly inhibit the re-uptake of norepinephrine (NE) and/or serotonin (5-HT) into pre-synaptic axons. With chronic treatment they induce adaptive changes in a number of neurotransmitter receptors in synaptic membranes. The immediate effects of SJW Ze 117, an extract low in hyperforin content, on the specific dopamine (DA) uptake were studied in rat striatal brain slices and compared with the effects on NE and 5-HT uptake in rat cortical brain slices. Specific DA uptake was inhibited in a dose dependent manner. In contrast to the findings in synaptosomal preparations published so far, the extract showed different inhibitory potencies for the respective transporters. The potencies for the uptake inhibition of NA, DA and 5-HT were 30, 7 and 1, respectively. The results indicate that the SJW Ze 117 extract interferes in three ways with the individual uptakes of the relevant neurotransmitters that are considered to be causal in the development of depression. This observation, the concomitant and potent inhibition of DA re-uptake by SJW extract, may additionally provide a rationale for the treatment of nicotine or drug addiction with SJW.

Differential inhibition of inflammatory effector functions by petasin, isopetasin and neopetasin in human eosinophils.

BACKGROUND: Priming of eosinophils with granulocyte-macrophage colony-stimulating factor (GM-CSF) and subsequent stimulation with platelet-activating factor (PAF) or the anaphylatoxin C5a is associated with a rapid production of leukotrienes (LTs) and release of eosinophil cationic protein (ECP). OBJECTIVE: This study was designed to determine the effects of the sesquiterpene esters petasin, isopetasin and neopetasin on LT generation and ECP release in eosinophils in vitro. METHODS: The model of eosinophil activation described above was used to induce LT production and ECP release. Cells were incubated with petasins and control inhibitors prior to priming and stimulation. To analyse intracellular steps of eosinophil activation and determine potential drug targets, some key signalling events were studied. Activity of cytosolic phospholipase A2 (cPLA(2)) was measured by analysing the generation of arachidonic acid (AA). Translocation of 5-lipoxygenase (5-LO) was observed using immunofluorescence microscopy. Intracellular calcium concentrations [Ca2+]i were measured by a bulk spectrofluorometric assay. RESULTS: Whereas all three compounds inhibited LT synthesis, ECP release from eosinophils was blocked by petasin only, but not isopetasin or neopetasin. Similarly, PAF- or C5a-induced increases in [Ca2+]i were completely abrogated by petasin only, whereas isopetasin and neopetasin had significant lower blocking efficacy. Moreover, only petasin, but not isopetasin or neopetasin, prevented increases in cPLA(2) activity and 5-LO translocation from the cytosolic compartment to the nucleus envelope in calcium ionophore-stimulated eosinophils. CONCLUSION: These data suggest that different petasins may at least partially block different intracellular signalling molecules. To reduce LT synthesis, isopetasin and neopetasin may act at the level of or distal to 5-LO. In contrast, petasin may inhibit inflammatory effector functions in human eosinophils by disrupting signalling events at the level of or proximal to phospholipase Cbeta (PLCbeta), besides its potential inhibitory activity within mitogen-activated protein kinase (MAPK) and LT pathways.

Role of petasin in the potential anti-inflammatory activity of a plant extract of petasites hybridus.

A large production of leukotrienes (LTs) can be induced in human eosinophils or neutrophils by priming with granulocyte-macrophage colony-stimulating factor and subsequent stimulation with platelet-activating factor (PAF) or the anaphylatoxin C5a. Here, we investigated the effects of a plant extract of petasites hybridus (Ze339) and its isolated active sesquiterpene ester petasin in these two in vitro cell models. Zileuton, a 5-lipoxygenase inhibitor, was used as a positive control. All compounds inhibited both cysteinyl-LT synthesis in eosinophils and LTB(4) synthesis in neutrophils. In contrast, only Ze339 and petasin, but not zileuton, abrogated PAF- and C5a-induced increases in intracellular calcium concentrations. These data suggest that Ze339 and petasin may block, compared to zileuton, earlier signalling events initiated by G protein-coupled receptors in granulocytes, perhaps at the level of or proximal to phospholipase C(beta). Taken together, petasin appears to be one major active compound of petasites hybridus extract, since it demonstrates the same inhibitory activities on calcium fluxes and subsequent LT generation in both eosinophils and neutrophils as Ze339 does.

Natural killer cells activate human dermal fibroblasts.

Human dermal fibroblasts (HDF) undergo activation and secrete cytokines when cocultured with T cells. Here, we identify potent activators of HDF among human peripheral CD2(+)-lymphocytes. Populations with strong HDF activating capacity consisted essentially of cells with a natural killer (NK) surface marker phenotype (CD3(-), CD4(-), CD8(-), CD56(+)). Addition of these cells to HDF resulted in rapid increase of intracellular free calcium concentrations as an early rapid cell activation signal. Upregulation of mRNA encoding for the inflammatory cytokines IL-1 beta and IL-6 as well as for chemokines IL-8 and MCP-1 was detected after cells were cocultured. Elevated concentrations of IL-6 and IL-8 were found in coculture supernatants of HDF and NK-cells. Skin-homing NK cells leaving the blood-stream during an inflammatory skin reaction might therefore represent potent activators of local inflammatory cytokine and chemokine production.

Antioxidant and thyroid hormone status in selenium-deficient phenylketonuric and hyperphenylalaninemic patients.

BACKGROUND: Subjects consuming protein-restricted diets, such as patients with phenylketonuria (PKU) or milder hyperphenylalaninemias (HPAs) are at risk of selenium deficiency. Selenium is a cofactor of the antioxidant enzyme glutathione peroxidase and of the thyroid hormone converting enzyme thyroxine deiodinase. OBJECTIVE: Our goal was to investigate the effects of low plasma selenium on antioxidant and thyroid hormone status. DESIGN: We assessed plasma selenium, plasma total antioxidant status and the individual components thereof, erythrocyte antioxidant status, and plasma thyroid hormones in 24 PKU and 10 HPA patients and in 42 age-matched control subjects. RESULTS: Selenium was significantly lower in both PKU and HPA patients than in control subjects and the PKU patients had lower values than did the HPA patients. Total antioxidant status was lower in both patient groups than in the control group, whereas alpha-tocopherol, albumin, and uric acid were not significantly different among groups. Plasma selenium correlated well (r = 0.76) with erythrocyte glutathione peroxidase. PKU patients had lower glutathione peroxidase activity than did HPA patients and control subjects and lower glutathione concentrations than did control subjects. Both patient groups had lower superoxide dismutase activity than did control subjects. Free triiodothyronine was higher in both patient groups than in control subjects, whereas free thyroxine was higher in the PKU patients only. Free thyroxine and reverse triiodothyronine were inversely correlated with selenium. CONCLUSION: Supplementation with selenium seems to be advisable for patients consuming diets low in natural protein.

Differential regulation of bradykinin receptor density, intracellular Ca2+, and prostanoid release in skin and foreskin fibroblasts. Effects of cell density and interleukin-1alpha.

1. Bradykinin (BK) receptors, cytosolic Ca2+, and prostanoids were studied in human skin and foreskin fibroblasts. 2. Bmax values of BK receptors were higher in foreskin than in skin fibroblasts, increasing with cell densities in both cell types. IL-1alpha-dependent receptor induction was blocked by cycloheximide. 3. BK-stimulated cytosolic Ca2+ elevation was higher in confluent than in non-confluent cultures and larger in foreskin than in skin fibroblasts. Responses were not enhanced after IL-1-alpha-induced up-regulation of BK receptors. 4. Intrinsic prostanoid production was higher in foreskin than in skin fibroblasts at comparable cell densities. In foreskin, but not in skin fibroblasts, BK stimulation increased the release of PGE2 10 fold and that of 6-oxo-PGF1alpha 6-7 fold. 5. Preincubation with IL-1alpha had a marked effect on prostanoid release in foreskin fibroblasts only. Subsequent BK stimulation increased the release of PGE2 and 6-oxo-PGF1alpha 7-10 fold in skin fibroblasts while this increase was only 30% in foreskin fibroblasts. Release of TXA2 reached values up to one third of the other prostanoids. The IL-1alpha induced rise in BK-stimulated PGE2 synthesis was fully abolished by specific inhibition of cyclo-oxygenase 2. 6. IL-1alpha sensitized BK-stimulated prostanoid synthesis and modulated prostanoid patterns differently in fibroblasts from skin and foreskin. The IL-1alpha effects on prostanoid release were not related to BK receptor numbers nor to the BK-stimulated Ca2+ signal but appear to be due to induction of prostanoid synthesizing enzymes. Foreskin fibroblasts seem to be unique and significantly different from fibroblasts of other skin locations in respect to their response to inflammation-associated kinins and cytokines.

Soluble IL-1 receptor type I binds to human dermal fibroblasts and induces calcium flux.

Soluble cytokine receptors appear to modify ligand concentrations by stabilizing ligands or by specifically inhibiting interactions of ligands with their membrane-bound receptors. Here we describe a new function of the soluble interleukin-1 receptor type I (IL-1sR I). This receptor induced a transient rise of intracellular free calcium concentration in human dermal fibroblasts in a dose-dependent fashion. Mobilization of calcium by IL-1sR I was abolished in the presence of an equimolar concentration of IL-1 receptor antagonist (IL-1ra). Neutralizing antibodies against IL-1beta also abolished calcium mobilization stimulated with IL-1sR I indicating that IL-1beta is involved. IL-1sR I bound with high affinity (Kd 1-2 nM) to the fibroblasts. In addition, IL-1sR I enhanced expression of IL-6 and IL-8 mRNA. The observation that IL-1sR I can act as a ligand and agonist for membrane IL-1 extends the concept of the ligand-receptor functions of both IL-1 and IL-1sR I and adds a new dimension to the cytokine network.

Partial 3-methylcrotonyl-CoA carboxylase deficiency in an infant with fatal outcome due to progressive respiratory failure.

UNLABELLED: Isolated partial 3-methylcrotonyl-CoA carboxylase (MCC) deficiency has been described to be the cause for a distinct relatively mild clinical picture in a single patient. We describe another patient with isolated partial MCC deficiency who suffered from failure to thrive, muscular hypotonia and progressive respiratory insufficiency with fatal outcome at the age of 6.5 months. MCC deficiency was suspected at 3 months of age on the basis of mildly elevated urinary excretion of 3-hydroxyisovaleric acid and 3-methylcrotonylglycine and confirmed by enzyme analysis in lymphocyte and fibroblast homogenates. Residual MCC activity in lymphocytes was 25% of the mean normal value. Residual activity in fibroblasts was lower than in lymphocytes (3.8% of mean normal) and not significantly different from that in patients with complete MCC deficiency. However, the residual incorporation of 14C-isovalerate into macromolecules in intact fibroblasts, was clearly higher (28% of mean normal) than in fibroblasts with complete MCC deficiency (<4%). In both patients with partial deficiency the residual MCC activity was higher in lymphocytes than in fibroblasts. Clinical symptoms and signs in our patient attributable to MCC deficiency include muscular hypotonia, failure to thrive (already present at birth), progressive respiratory failure due to diaphragmatic paresis and a moderate brain atrophy. The clinical presentation was more severe than in many patients with complete MCC deficiency. Dietary therapy was biochemically effective as shown by normalization of organic acid excretion, however, had no effect on the CNS symptoms. CONCLUSION: We speculate that the severity of the disease could be related primarily to deficiency of MCC activity in the brain. Variable MCC activity among various organs may explain the peculiar clinical picture in this patient.

Weekly versus biweekly lipid removal and effect of statins in severe hypercholesterolemia.

Lipid removal using a continuous-flow extracorporeal system is of proven efficacy in severe hypercholesterolemia. Because of the inconveniences and expenses of extracorporeal removal of lipids, the effects of two treatment intervals (weekly versus biweekly) were assessed in two adolescents with circulating cholesterol higher than 20.0 mmol/L. In both patients, circulating levels were largely lower on a weekly lipid removal interval when compared with a biweekly interval. A rapid reaccumulation of cholesterol was noted after lipid removal. Treatment with simvastatin decreased the rapid reappearance of total cholesterol noted during the first 2 days after lipid removal but without any major effect on the subsequent reaccumulation of cholesterol. Aggressive treatment of severe hypercholesterolemia with statins and especially with extracorporeal lipid removal is now possible and of proven efficacy. The minimal practical lipid removal treatment interval should be used.

Mutation of ornithine transcarbamylase (H136R) in a girl with severe intermittent orotic aciduria but normal enzyme activity.

Ornithine transcarbamylase deficiency shows X-linked inheritance with partial dominant expression in carrier females. We studied a girl with intermittent severe orotic aciduria and mild hyperammonaemia despite apparently normal enzyme activity in the liver. Sequence analysis of all 10 exons of the ornithine transcarbamylase gene revealed a novel A-->G exchange (A502G) in exon 5 which changes His-136 to arginine in the ornithine transcarbamylase protein. Km values for carbamyl phosphate and ornithine determined in the patient's liver were comparable to those of wild-type enzyme but, unlike the wild-type enzyme, the mutant enzyme was unstable upon freezing and thawing. Electron microscopy revealed several giant mitochondria with paracrystalline inclusions. The results are compatible with the assumption that the mutant enzyme cannot form a functional complex with carbamyl phosphate synthetase and the ornithine carrier, resulting in decreased availability of substrates and diminished enzyme activity in vivo.

Autologous versus allogeneic T cell-stimulated IL-6 production by dermal fibroblasts.

T cells adhere to human dermal fibroblasts (HDF). This cellular interaction leads to a pronounced secretion of the proinflammatory cytokines IL-6 and IL-8 via a juxtacrine stimulation induced by HDF-associated IL-1. Upon stimulation, fibroblasts express various surface proteins such as MCH-I molecules, which may interact with corresponding receptors on T cells. The present study was conducted to further investigate the mechanism of this complex interaction with regard to the secretion of IL-6 in cocultures of T cells and HDF. IL-6 was time- and dose-dependently upregulated in such cocultures. Spatial separation of the cells by microporous membranes resulted in a 90% reduction of IL-6 secretion, but when cells had limited cell contact IL-6 secretion was increased again. Allogeneic cocultures of T cells and HDF showed increased capacity of IL-6 stimulation as compared to autologous cultures. Our results suggest that MHC-I/T cell receptor interaction modulates IL-6 secretion in allogeneic and autologous cocultures.

Regulatory adaptation of isoprenoid biosynthesis and the LDL receptor pathway in fibroblasts from patients with mevalonate kinase deficiency.

In a search for the pathophysiologic mechanisms, we estimated isoprenoid synthesis and concentration, cellular growth, and the activity of the LDL receptor pathway in fibroblasts from patients with mevalonate kinase deficiency (MKD), a severe multisystemic disorder of cholesterol and non-sterol isoprenoid biosynthesis. In response to different concentrations of LDL and non-lipoprotein-bound cholesterol, MKD cells partially counteracted their enzyme defect by increased activities of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (results from earlier studies) and the LDL receptor pathway, responses similar to the pharmacologic effects seen upon administration of HMG-CoA reductase inhibitors. Rates of N-linked protein glycosylation, estimated as the amount of [14C]galactose-labeled macromolecules secreted into cell culture medium, were significantly decreased in MKD fibroblasts in comparison with control cells which may indicate alterations in the dolichol or dolichol phosphate pool. In response to exogenous cholesterol, the major feedback inhibitor of isoprenoid biosynthesis, growth velocities of MKD fibroblasts declined in comparison with control cells, further suggesting an impairment of non-sterol isoprenoid biosynthesis in MKD. Our results suggest an imbalance in the multilevel regulation of the biosynthesis of cholesterol and non-sterol isoprenoids in MKD, representing an additional causative factor responsible for the pre- and postnatal pathology of MKD.

Uptake and intracellular stability of glycosylphosphatidylinositol-specific phospholipase D in neuroblastoma cells.

Glycosylphosphatidylinositol-specific phospholipase D from mammalian serum has been described to be relatively stable towards the action of proteases in vitro, and it has been speculated that the enzyme may only be active on glycosylphosphatidylinositol-anchored substrates after its proteolytic processing in an intracellular compartment following uptake from body fluids. To test this hypothesis, we studied the possible uptake and intracellular processing of purified glycosylphosphatidylinositol-specific phospholipase D into the mouse neuroblastoma cell line N2A. We found that after incubation of neuroblastoma cells with glycosylphosphatidylinositol-specific phospholipase D at 37 degrees C the amount of cell-associated glycosylphosphatidylinositol-specific phospholipase D activity increased in a concentration- and time-dependent way. A similar uptake was also observed with 125I-labeled intact and trypsin-treated form of glycosylphosphatidylinositol-specific phospholipase D. We found that the incorporated radiolabeled proteins were processed intracellularly to distinct low molecular mass products, and that this process was in part inhibited by the presence of chloroquine during incubation.

Treatment with low-dose diazoxide in two growth-retarded prepubertal girls with glycogen storage disease type Ia resulted in catch-up growth.

Two originally prepubertal girls suffering from glycogen storage disease type Ia and short stature were treated with low-dose diazoxide (3-4.8 mg/kg per day) for 7 and 4 years, respectively. Both showed an impressive catch-up growth following this treatment. This appeared to be due to prolongation of normoglycaemia after meals and reduction of fasting lactic acidosis by diazoxide.

Inhibition by vitamin E of drug accumulation and of phospholipidosis induced by desipramine and other cationic amphiphilic drugs in human cultured cells.

1. Cationic amphiphilic drugs (CADs) are widely used in chronic pharmacotherapies in spite of frequently observed side effects connected with lysosomal phospholipid (PL) storage. 2. It has recently been shown that alpha-tocopherol (alpha-Toc) inhibits drug- and PL accumulation in cell cultures chronically exposed to the CAD, amiodarone. 3. The mechanisms of alpha-Toc action on drug kinetics and PL storage were studied in human cultured fibroblasts exposed to single and repetitive doses of desipramine and other CADs. 4. alpha-Toc did not influence the initial, pH-dependent rapid phase of drug uptake. It inhibited, in a dose-dependent manner, the slow and the cumulative phases of drug uptake and coincidently the accumulation of cellular PLs. 5. The inhibitory effects of alpha-Toc on CAD and PL accumulations depends on the ratio between CAD and alpha-Toc concentrations in the medium. This points to competition between alpha-Toc and CADs for PL complex formation. 6. Effectiveness of alpha-Toc on drug uptake varies among different CADs. It depends on its structural integrity but is independent of stereoisomerism. The inhibitory action is restricted to the piggyback slow drug uptake and therefore related to the proportion of membrane-mediated transport to permeation into lysosomes (rapid uptake). This proportion differs among CADs. 7. alpha-Toc prevents lysosomal membrane-PL storage, accelerates disintegration of PL-stores and normalizes drug-related increased membrane fluidity. This strongly suggests that alpha-Toc restores membrane recycling, impaired by CAD exposure. 8. It remains to be tested in vivo whether alpha-Toc reduces CAD side effects without interfering with drug effectiveness.

Juxtacrine stimulation of cytokine production in cocultures of human dermal fibroblasts and T cells.

Adhesion of T cells to fibroblasts activates cells to produce cytokines, either by direct cell contact and/or soluble factors. A cell-associated form of IL-1 beta on fibroblasts might act through a cell contact mediated fashion. To test this hypothesis we analysed the activation of T cells and human dermal fibroblasts (HDF) in coculture experiments. Elevated levels of IL-1 beta, secreted by T cells as well as IL-6 and IL-8, mainly produced by HDF, were found in supernatant fluids of cocultured cells. IL-1 beta mRNA expression was induced in T cells as well as in HDF. While in HDF IL-1 beta remained cell-associated, T cells were activated to produce and secrete soluble IL-1 beta and IL-6. IL-1 beta and possibly other soluble factors increased IL-6 production by fibroblasts. These effects could be mainly attributed to CD8+ T cells. Our results suggest, that IL-1 beta, produced as a cell-associated cytokine by human dermal fibroblasts, acts as a juxtacrine molecule to stimulate T cells. Such a cellular cooperation, could be a powerful mediator in inflammatory response and possibly in wound healing.

Combined 3-methylglutaconic and 3-hydroxy-3-methylglutaric aciduria with endocardial fibroelastosis and dilatative cardiomyopathy in male and female siblings with partial deficiency of complex II/III in fibroblasts.

We report on 2 children, brother and sister, who presented with cardiomyopathy and muscular hypotonia at the age of B months. They both excreted significant amounts of 3-hydroxy-3-methylglutaric acid (3-HMG) and 3-methylglutaconic acid (3-MGC) but no 3-methylglutaric acid (3-MG). Enzyme analysis in fibroblasts revealed normal activities of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase and of 3-methylglutaconyl hydratase and other enzymes of 3-HMG metabolism. Loading tests with leucine did not affect the excretion of 3-HMG and 3-MGC. The girl died as a result of her cardiomyopathy, while the boy recovered and was treated with cardiac supportive therapy. He showed a steady improvement during his clinical course with biochemical normalization of the urinary excretion of 3-HMG, concomitant with marked improvement in the hypertrophic cardiomyopathy. In cultured fibroblasts from both patients a reduced activity of complex II/III of the respiratory chain was measured which may be the cause of this new type of 3-HMG uria. Analysis of mitochondrial DNA heart muscle, liver and fibroblast culture of the patient did not reveal any major mitochondrial DNA rearrangements (deletion, duplication) or any point mutation that had been described in association with mitochondrial cardiomyopathy.

Vitamin E reduces accumulation of amiodarone and desethylamiodarone and inhibits phospholipidosis in cultured human cells.

Chronic administration of amiodarone (AMIO), widely used by clinicians for the treatment of therapy-resistant cardiac arrhythmias, is frequently associated with serious side-effects. AMIO and its main metabolite desethylamiodarone (DEA) are known to induce phospholipidosis in vivo and in cultured cells presumably by inhibition of lysosomal phospholipid degradation. D-alpha-Tocopherol = vitamin E (alpha-TOC) was able to reduce AMIO and DEA toxicity in cell cultures. Results from the present study showed that alpha-TOC reduced phospholipidosis in cultured human skin fibroblasts chronically exposed to micromolar concentrations of AMIO and DEA and inhibited cumulative uptake of the drugs in a dose-dependent manner. A linear correlation was observed between cellular AMIO levels and phospholipid accumulation suggesting a stoichiometric relationship. alpha-TOC was also effective in clearing previously accumulated phospholipids after discontinuation of the drug treatment. The results can best be explained by an interference of alpha-TOC (a) with drug-phospholipid complex formation responsible for both phospholipid storage and drug accumulation, and (b) with pre-existing drug-phospholipid complexes, accelerating their dissociation and rendering phospholipids to substrates for lysosomal phospholipases. The finding raises hope that side-effects of AMIO and DEA can be prevented or made reversible by the administration of alpha-TOC. It must, however, be proven that the antiarrhythmic drug will still be effective.

Leguminosae in the diet: the raffinose-stachyose question.

Adhering to a galactose-free diet by strictly avoiding dairy products and known hidden sources of galac-tose does not completely normalize galactose-1-phosphate (gal-1-P) in erythrocytes from patients with galactosemia. Major neurological complications, even in the best treated patients, are threatening a good clinical outcome and dictate a continuous search for leaks in the dietary regimen. Raffinose and stachyose, present in important amounts in various vegetables, contain alpha-1,4 linked galactose which is cleaved only by bacterial alpha-galactosidases, presumably in the lower part of the gut. In order to test the hypothesis whether galactose released from raffinose and stachyose could be a source of absorbed galactose and a cause of elevated gal-1-P six patients with galactosemia (aged 6-24 years), underwent a raffinose- and stachyose-poor dietary regimen for 2 weeks. Before, after, and during the test period, the daily intake of stachyose and raffinose as well of protein, carbohydrate, fat and minerals was calculated from food protocols obtained from the patients. Plasma galactose and erythrocyte gal-1-P were measured at the end of the three test phases. Stachyose and raffinose intake was reduced to 5%-10% during the experimental diet, which was well tolerated, except for constipation in some patients. In five of the six patients gal-1-P in erythrocytes was somewhat lower (statistically not significant) during the test phase than during regular diet while plasma galactose remained unchanged. Galactose released from raffinose and stachyose may be absorbed and contribute to elevated gal-1-P values in erythrocytes of galactosemic patients.