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[Criteria defining antimanic drugs (psychopharmacological specificity and/or nonspecificity?)].

The question as to whether specific antimanic drugs differ in their action profile from nonspecific drugs is addressed in regard to symptomatic, nosographic, regulatory and physiopathological issues. Results from clinical studies have shown that mood stabilizers and typical neuroleptics differ as regards improvement of manic symptoms: the former appear to act more evenly on all symptoms of mania, showing a more total normalization of affect, ideation and behaviour whereas the latter tend to sedate patients or to cause a psychomotor retardation, leaving the core manic symptoms unaffected. This has been many times underlined, in particular for lithium, notwithstanding the fact that rating scales employed in clinical trials have often been charged to fall far short of being sensitive enough to pick up the qualitative changes in manic psychopathology. Antimanic drugs may also be more or less specific in their capacities to treat all facets of the manic episode (psychotic, depressive, irritable) whatever the bipolar subtype (bipolar I, II, rapid and non-rapid cycling, secondary bipolar disorder) or the disease stage (early and late episodes). In this respect divalproate seems to have a broader spectrum of efficacy than other available agents. Newer antipsychotics such as olanzapine are promising too. From a regulatory point of view, the current European requirements for a specific antimanic drug are more stringent than the US requirements of the Food and Drug Administration (FDA). Efficacy must be demonstrated in short-term studies showing an effect in acute mania; moreover it has to be shown that efficacy is to be maintained during the episode. So far, three armed randomized controlled trials are required, in which the test product is compared both with placebo and with a standard treatment. A possible design is a comparison of test product, placebo and active control for 3 weeks followed by a two-arm phase for the remaining 9 weeks, comparing only test product and active control. In addition, a specific antimanic has to demonstrate that it does not cause switching to depression. As regards physiopathology, integrative models of bipolar disorder, ie kindling and behavioural sensitization, offer an exciting perspective on the specificity issue; agents active in these models initialize a cascade of intracellular signaling that leads to changes in the expression of immediate early genes as well as late effector genes in corticolimbic structures: the former may contribute to acute symptomatology whereas the latter give rise to neuroanatomical reorganization which could underlie more stable changes in mood and cognition. Due to their action on intracellular messaging systems, dopamine D(1) receptors, serotonin 5HT(1a) or 5HT(2a) receptors, especially in orbitofrontal circuit, antimanic agents may exhibit a more specific activity than other drugs. This specificity could concern a whole spectrum of bipolarity which might be characterized by impulsivity.

Antimanic Agents↗
PubMed1992

Biphasic production of IL-8 in lipopolysaccharide (LPS)-stimulated human whole blood. Separation of LPS- and cytokine-stimulated components using anti-tumor necrosis factor and anti-IL-1 antibodies.

TNF, IL-1, and IL-6 are integral components of the cytokine cascade released in the response to inflammatory stimuli such as LPS. IL-8 is produced both in response to LPS as well as TNF and IL-1. The early, local production of TNF and IL-1 may therefore contribute to the subsequent expression of IL-8. This hypothesis was tested using LPS-stimulated human whole blood as an ex vivo model of local cytokine production. The production of TNF, IL-1 alpha, IL-1 beta, IL-6, and IL-8 was found to be responsive to a wide range of LPS concentrations (0.1 ng/ml-10 micrograms/ml). These cytokines were first detected between 1 to 4 h post-LPS stimulation, and reached plateau levels after 6 to 12 h. IL-8, however, also displayed a secondary wave of production, with the levels again increasing between 12 to 24 h. The IL-8 present in the plasma after LPS stimulation was biologically active, as assessed by neutrophil chemotaxis. In further studies, addition of anti-TNF and anti-IL-1 neutralizing antibodies, alone and in combination, to LPS-stimulated blood resulted in nearly complete ablation of the secondary phase of IL-8 synthesis at both the levels of protein and mRNA, while leaving the first, LPS-mediated phase of IL-8 synthesis unaffected. This model of cytokine production in human whole blood may reflect the sequence of events in a localized environment of inflammation where both a primary stimulus and the induced early cytokine mediators may serve to elicit multiple, temporally distinct phases of IL-8 production.

Antibodies, Monoclonal↗
PubMed1998

[Endothelial damage and blood coagulation activation in preeclampsia].

BACKGROUND: The aim of this study was the evaluation of the endothelial cell damage and of the likely activation of coagulative cascade in preeclampsia. METHODS: Forty-seven pregnant women of gestational age from 30 to 34 weeks of gestation were studied: 30 normal pregnancies (N) and 17 women suffering from preeclampsia (P). The plasma factors studied were the following: 1) plasma fibronectin and thrombomodulin as markers of endothelial cell damage; 2) beta-thromboglobulin as platelet activity markers; 3) VIII:C factor and fibrinopeptide A as coagulation activity markers; 4) coagulation inhibitors such as protein C and protein S activity; 5) tissue plasminogen activator (t-PA), plasminogen level and plasminogen activator inhibitors (PAI) as fibrinolytic activity markers. All hypertensive patients didn't use heparin. Data are presented as mean +/- 1SD. Mann-Whitney "U" -test was used for statistical analysis. A p value of < or = 0.05 was regarded as statistically significant. RESULTS: In preeclampsia the plasma fibronectin is increased (P: 115 +/- 64 ng/ml, N: 73 +/- 47 ng/ml; p = 0.023) as well as VIII:C factor activity (P: 151 +/- 13.5%, N: 117.2 +/- 23%; p = 0.0005). CONCLUSIONS: Endothelial cell damage (increase of plasma fibronectin) and a slight thrombin generation (increase of VIII:C factor activity) are to be found in preeclampsia. We can't say which event starts the process. Intravascular coagulative cascade activation with platelet consumption, fibrin production and fibrinolytic activation occurs only in a restricted number of preeclamptic patients, in a late and worsening stage of illness, as a consequence of massive endothelial damage at placental and systemic level.

Adult↗
PubMed2005

Neuronal nitric oxide synthase activity in rat urinary bladder detrusor: participation in M3 and M4 muscarinic receptor function.

1. The aim of this paper was to determine the different signalling cascades involved in contraction of the rat urinary bladder detrusor muscle mediated via muscarinic acetylcholine receptors (muscarinic AChR). Contractile responses, phosphoinositides (IPs) accumulation, nitric oxide synthase (NOS) activity and cyclic GMP (cGMP) production were measured to determine the reactions associated with the effect of cholinergic agonist carbachol. The specific muscarinic AChR subtype antagonists and different inhibitors of the enzymatic pathways involved in muscarinic receptor-dependent activation of NOS and cGMP were tested. 2. Carbachol stimulation of M(3) and M(4) muscarinic AChR increased contractility, IPs accumulation, NOS activity and cGMP production. All of these effects were selectively blunted by 4-DAMP and tropicamide, M(3) and M(4) antagonists respectively. 3. The inhibitors of phospholipase C (PLC), calcium/calmodulin (CaM), neuronal NOS (nNOS) and soluble guanylate cyclase, but not of protein kinase C and endothelial NOS (eNOS), inhibited the carbachol action on detrusor contractility. These inhibitors also attenuated the muscarinic receptor-dependent increase in cGMP and activation of NOS. 4. In addition, sodium nitroprusside and 8-bromo-cGMP, induced negative relaxant effect. 5. The results obtained suggest that carbachol activation of M(3) and M(4) muscarinic AChRs, exerts a contractile effect on rat detrusor that is accompanied by an increased production of cGMP and nNOS activity. The mechanism appears to occur secondarily to stimulation of IPs turnover via PLC activation. This in turn, triggers cascade reactions involving CaM, leading to activation of nNOS and soluble guanylate cyclase. They, in turn, exert a modulator inhibitory cGMP-mediated mechanism limiting the effect of muscarinic AChR stimulation of the bladder.

Animals↗
PubMed2000

Psychosocial stress and HPA functioning: no evidence for a reduced resilience in healthy elderly men.

In order to investigate if HPA functioning is altered with age, the present study was conducted. Fifteen healthy elderly men (60-76 years; mean age 66.5 +/- 1.48 yrs.) and 12 younger adults (20-29 years; mean age 25.6 +/- 0.77 yrs.) collected salivary free cortisol profiles after awakening for basal HPA activity. Then, all subjects were exposed to the "Trier Social Stress Test" (TSST). This psychosocial stress protocol consists of a free speech and a mental arithmetic task of 13 minutes duration performed in front of an audience. Beside the assessment of endocrine and cardiovascular responses to the stressful task ratings of depression, mood and perceived stressfulness were obtained. Results show that younger and elderly men had similar morning cortisol profiles after awakening with both groups showing the expected rise after awakening (P=0.004). The TSST induced significant increases in ACTH, total plasma cortisol, saliva free cortisol, and heart rates (all P<0.0001). Regardless of age, both age groups showed comparable endocrine response patterns when confronted with the stressor. However, cardiovascular responses were significantly higher in younger men compared to elderly men (P=0.03). Catecholamine data revealed significant norepinephrine and epinephrine increases due to the stressor (both P<0.0001) with a trend toward elevated norepinephrine levels in elderly men (P=0.058). In sum, the investigated basal and response parameters of HPA functioning neither support the idea of a reduced resilience in healthy aged humans nor do they appear to strengthen assumptions derived from the so called "glucocorticoid cascade hypothesis".

Adaptation, Physiological↗
PubMed2003

Novel diagnostic test for acute stroke.

BACKGROUND AND PURPOSE: The absence of a widely available and sensitive diagnostic test for acute cerebral ischemia remains a significant limitation in the diagnosis and management of stroke. The objective of this study was to examine the feasibility of developing a diagnostic panel of blood-borne biochemical markers of cerebral ischemia. METHODS: Serial blood samples were obtained from patients (n=65 with suspected ischemic stroke, n=157 control subjects) presenting to an academic medical center emergency department. We analyzed 26 blood-borne markers believed to play a role in the ischemic cascade and created a 3-variable logistic regression model to predict the clinical diagnosis of stroke, defined as persistent neurological symptoms of cerebral ischemia lasting >24 hours. RESULTS: Of the 26 blood-borne markers analyzed, univariate logistic analysis revealed that 4 were highly correlated with stroke (P<0.001): a marker of glial activation (S100beta), 2 markers of inflammation (matrix metalloproteinase-9 and vascular cell adhesion molecule), and 1 marker of thrombosis (von Willebrand factor). When the outcome level was set to a cutoff of P=0.1, our logistic model provided a sensitivity and specificity of 90% for predicting stroke. CONCLUSIONS: A panel of blood-borne biochemical markers may be helpful in identifying patients with acute cerebral ischemia who could benefit from urgent care. Such a test may also be helpful in identifying stroke patients in the prehospital setting so that they could be fast-tracked to an institution equipped to care for patients with acute stroke.

Acute Disease↗
PubMed1994

Biocompatibility of potential wound management products: fungal mycelia as a source of chitin/chitosan and their effect on the proliferation of human F1000 fibroblasts in culture.

Aspergillus oryzae, Mucor mucedo, and Phycomyces blakesleeanus cultures were examined as sources of chitin/chitosan. The nitrogen content of the alkali-treated mycelia/sporangiophores of A. oryzae, M. mucedo, and P. blakesleeanus was 2.52, 3.61, and 6.27% w/w, which relates to an estimated chitin content of 37, 52, and 91%, respectively. The effect of these fungal materials on the rate of proliferation of human F1000 fibroblasts in culture was examined. At 0.01% w/v, all three materials exhibited significant (P < .05) proproliferant activity over a period of 13 days. However, at 0.05% w/v, P. blakesleeanus further enhanced cell proliferation, whereas A. oryzae and M. mucedo produced a significant (P < .05) antiproliferant effect. Higher concentrations of P. blakesleeanus (0.1 and 0.5%) caused marked inhibition of F1000 cell proliferation when measured on days 3 and 6. Only the proproliferant effect of these fungal materials appears to correlate to their chitin content. Furthermore, the cytomorphology of the fibroblasts indicated that P. blakesleeanus, and to a lesser extent M. mucedo, possessed cell attractant properties, again correlating with chitin content. If developed for use as wound management materials, the sporangiophores of P. blakesleeanus and the mycelium of M. mucedo could possibly promote the growth of fibroblasts and provide a matrix for their anchorage, thus contributing to the granulation phase of the healing cascade.

Aspergillus oryzae↗
PubMed2000

Role of Cdc42p in pheromone-stimulated signal transduction in Saccharomyces cerevisiae.

CDC42 encodes a highly conserved GTPase of the Rho family that is best known for its role in regulating cell polarity and actin organization. In addition, various studies of both yeast and mammalian cells have suggested that Cdc42p, through its interaction with p21-activated kinases (PAKs), plays a role in signaling pathways that regulate target gene transcription. However, recent studies of the yeast pheromone response pathway suggested that prior results with temperature-sensitive cdc42 mutants were misleading and that Cdc42p and the Cdc42p-PAK interaction are not involved in signaling. To clarify this issue, we have identified and characterized novel viable pheromone-resistant cdc42 alleles that retain the ability to perform polarity-related functions. Mutation of the Cdc42p residue Val36 or Tyr40 caused defects in pheromone signaling and in the localization of the Ste20p PAK in vivo and affected binding to the Ste20p Cdc42p-Rac interactive binding (CRIB) domain in vitro. Epistasis analysis suggested that they affect the signaling step at which Ste20p acts, and overproduction of Ste20p rescued the defect. These results suggest that Cdc42p is in fact required for pheromone response and that interaction with the PAK Ste20p is critical for that role. Furthermore, the ste20DeltaCRIB allele, previously used to disrupt the Cdc42p-Ste20p interaction, behaved as an activated allele, largely bypassing the signaling defect of the cdc42 mutants. Additional observations lead us to suggest that Cdc42p collaborates with the SH3-domain protein Bem1p to facilitate signal transduction, possibly by providing a cell surface scaffold that aids in the local concentration of signaling kinases, thus promoting activation of a mitogen-activated protein kinase cascade by Ste20p.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Blood compatibility of titanium oxides with various crystal structure and element doping.

BACKGROUND: Titanium oxides are known to be good hemocompatible, therefore they are suggested as coatings for blood contacting implants. But little is known about the influence of physical characteristics like crystal structure, roughness and electronic state on the activation of blood platelets and the blood clotting cascade. METHODS: Titanium oxide films were produced by metal plasma deposition and implantation in the form of rutile, crystalline and nanocrystalline anatase + brookite and amorphous TiO2. The redox potential was reduced by implantation of chromium ions, the Fermi level of the semiconductive oxide was shifted by ion implantation of the electron donor phosphorous. Hemocompatibility was determined by measuring the adhesion of blood platelets, their P-selectine expression, and of the blood clotting time on these samples. RESULTS: The crystalline titanium oxides had a slightly higher activation of the clotting cascade but lower platelet adhesion than nanocrystalline and amorphous titanium oxides. The surface roughness below 50 nm had no obvious effect. Both, implantation of phosphorous or chromium ions, strongly reduced the activation of the clotting cascade, but only the phosphorous implanted surface also showed a reduced platelet activation, whereas platelet adhesion and activation was strongly increased on the chromium implanted surfaces. CONCLUSION: Phosphorous doping of rutile TiO2 can increase its hemocompatibility, both concerning blood platelets and blood clotting cascade, but the biochemical mechanism has to be worked out.

Biocompatible Materials↗
PubMed2002

[Screening for carriers of the fragile X syndrome; ethical exploration].

Large-scale population screening for carriers of fragile X syndrome is premature. Notably the limited possibilities to detect carriers (of an instable premutation) in the general population with certainty, poses some ethical dilemmas. The possible psychological consequences of this are unknown. A pilot study can only be started if the added value of population screening as opposed to cascade screening is plausible, and the requirement of proportionality is satisfied. If this is the case, preconceptional screening is preferable to prenatal screening. Information should be provided in a process-like manner as this fits in best with the decision-making process that possibly follows participation in a population screening for carriers of fragile X syndrome. Further research into the ethics of cascade screening is desirable. This screening can be carried out on a complementary basis to population screening.

Ethics, Professional↗
PubMed1998

Association of pp60c-src with alpha IIb beta 3 in resting platelets.

To detect whether 125I-alpha IIb beta 3 is associated with tyrosine kinases in platelets, antibodies specific to pp60c-src, pp54/58lyn, and pp62Fyn were used to precipitate their homologous antigens. In contrast to Lyn and Fyn kinases, pp60c-src appears to be complexed with alpha IIb beta 3. Both proteins, pp60c-src and alpha IIb beta 3, coprecipitated when antibodies to pp60c-src were used in the immunoprecipitation experiments. This conclusion was further supported by immunoprecipitation of alpha IIb beta 3 from Triton X-100 extracts of nonlabelled platelets with P2 antibodies. There was no pp60c-src detectable in immunoprecipitates obtained with antibodies specific to alpha 2 beta 1 or GPIb. Since PGE1 was used to prevent platelet activation in buffers throughout all procedures and there was no phosphorylation of pp72syk we assume that the platelets were in the resting state. Therefore, we conclude that alpha IIb beta 3 and pp60c-src can form a complex in resting platelets suggesting that pp60c-src is directly involved in initiating the outside-in signaling cascades in blood platelets.

Adult↗
PubMed2006

On the advantages of multi-input single-output parallel cascade classifiers.

Parallel Cascade Identification (PCI) has been successfully applied to build dynamic nonlinear systems that address diverse challenges in the field of bioinformatics. PCI may be used to identify either single-input single-output (SISO) or multi-input single-output (MISO) models. Although SISO PCI models have typically sufficed, it has been suggested that MISO PCI systems could also be used to form bioinformatics classifiers, and indeed they were successfully applied in one study. This paper reports on the first systematic comparison of MISO and SISO PCI classifiers. Motivation for using the MISO structure is given. The construction of MISO parallel cascade models is also briefly reviewed. In order to compare the accuracy of SISO and MISO PCI classifiers, genetic algorithms are applied to optimize the model architecture on a number of equivalent single-input and multi-input biological training datasets. Through evaluation of both model structures on independent test datasets, we establish that MISO PCI is capable of building classifiers of equal accuracy to those resulting from SISO PCI models. Moreover, we discuss and illustrate the benefits of the MISO approach, including significant reduction in training and testing times, and the ability to adjust automatically the weighting of individual inputs according to information content.

Algorithms↗
PubMed2000

Ischemic preconditioning: from basic mechanisms to clinical applications.

When the heart is subjected to a transient nonlethal period of ischemia, it quickly adapts itself to become resistant to infarction from a subsequent ischemic insult. This adaptation is called preconditioning. This cardioprotection has been shown to be mediated by stimulation of receptors linked to protein kinase C (PKC) (adenosine, bradykinin, opioids, etc.), and these receptors protect by activating PKC. PKC appears to be the first element of a complex kinase cascade that is activated during the prolonged ischemia in the preconditioned heart. Recent studies imply that p38 mitogen-activated protein kinase carries the signal from PKC to the mitochondrial K(ATP) channels, causing them to open and thus protect the heart. The cardioprotection of preconditioning occurs in all species tested to date, and possibly also humans. It is expected that as the mechanism of preconditioning is more thoroughly understood, pharmacological preconditioning will become practical for clinical use.

Animals↗
PubMed1997

Photosensory and thermosensory responses in Dictyostelium slugs are specifically impaired by absence of the F-actin cross-linking gelation factor (ABP-120).

Chemotactic aggregation of starving amoebae of Dictyostelium discoideum leads to formation of a motile, multicellular organism - the slug - whose anterior tip controls its phototactic and thermotactic behaviour. To determine whether proteins that regulate the in vitro assembly of actin are involved in these responses, we tested phototaxis and thermotaxis in mutant slugs in which the gene encoding one of five actin-binding proteins had been disrupted. Of the proteins tested - severin, alpha-actinin, fimbrin, the 34 kD actin-bundling protein and the F-actin cross-linking gelation factor (ABP-120) - only ABP-120 proved essential for normal phototaxis and thermotaxis in the multicellular slugs. The related human protein ABP-280 is required for protein phosphorylation cascades initiated by lysophosphatidic acid and tumor necrosis factor alpha. The repeating segments constituting the rod domains of ABP-120 and ABP-280 may be crucial for the function of both proteins in specific signal transduction pathways by mediating interactions with regulatory proteins.

Actins↗
PubMed2002

Protein kinase C (PKC) dependent induction of tissue factor (TF) by mesangial cells in response to inflammatory mediators and release during apoptosis.

1. In inflammatory kidney diseases procoagulatory activity (PCA) becomes evident. Glomerular fibrin deposits and capillary microthrombi are histopathological hallmarks in most forms of glomerulonephritis. 2. Therefore in this study the expression of tissue factor (TF) as the main inducer of thrombogenesis was examined in cultured human mesangial cells (MC) in response to proinflammatory stimuli such as interleukin-1 (IL-1 beta), tumour necrosis factor alpha (TNF-alpha) and lipopolysaccharide (LPS). Also main signalling pathways were investigated. 3. IL-1 beta, TNF-alpha and LPS induced TF in MC in a time and dose dependent manner on mRNA and protein levels. Highest activity was found after 12 h of stimulation. Induction of TF was completely blockable by BAPTA-AM, a chelator of intracellular [Ca(2+)](i) as well as calphostin, a protein kinase C (PKC) inhibitor. Activation of the protein kinase A (PKA) pathway had no influence on basal TF expression, but down-regulated cytokine-induced TF. The PKA blocker, KT5720, increased TF formation significantly. Since TF exerts its activity primarily on the surface of cells and after release of encrypted receptors we further tested TF activity in MC supernatants. IL-1 beta did not significantly increase TF activity in supernatants of intact cells. However, when MC were rendered apoptotic by oxidative metabolites, IL-1 beta treated MC released highly stimulated TF activity into the supernatants, suggesting that a paracrine activation of the coagulatory cascade can take place under such conditions. 4. Inflammatory mediators up-regulate TF expression in MC by a PKC dependent pathway whereas PKA can serve as a negative feed-back link. Apoptosis of inflammatory MC may trigger to spread PCA.

Apoptosis↗
PubMed2000

Inositol 1,4,5-trisphosphate synthesis in mononuclear white blood cells of malignant hyperthermia-susceptible and normal human beings, following in vitro exposure to halothane, caffeine and ryanodine.

Despite a plethora of findings associated with the pathophysiology of malignant hyperthermia (MH), the in vitro contracture test (IVCT) is the only reliable test for diagnosis of this heterogeneous syndrome in man. An increase of 1,4,5-IP3 (inositol 1,4,5-trisphosphate), a second messenger involved in cellular calcium homeostasis, has been observed in muscle tissue of MH susceptible (MHS) patients. The aim of this study was to evaluate if the known differences of 1,4,5-IP3 content in muscle tissue might be reproduced in mononucleated white blood cells (MWBCs). Subsequently, MWBCs of 23 healthy controls and 12 patients with a clinical suspicion for MH disposition were isolated and screened for 1,4,5-IP3 content. An IVCT according to the protocol of the European Malignant Hyperpyrexia Group (EMHG) was performed on muscle specimens of 12 patients. Eight MHN and four MHS individuals were diagnosed. Additionally, 1,4,5-IP3 synthesis in MWBCs was detected following in vitro exposure to IVCT test substances halothane (2%), caffeine (1-30mM), and ryanodine (1-5 microM). A broad inter-individual variability of 1,4,5-IP3 content was observed in MWBCs of all volunteers, but no differences were detected between MHS and MHN individuals. These findings are in strong contrast to those observed in muscle tissue. In vitro exposure of isolated MWBCs to halothane, caffeine and ryanodine yielded no statistically significant differences between groups. A time- and concentration-dependent increase in cellular 1,4,5-IP3 content could be induced in some but not all individuals of both groups. Since no correlation was obtained between induction of 1,4,5-IP3-synthesis following in vitro exposure of MWBCs to MH test substances and MH disposition, this study was terminated. We conclude from our data that the detection of 1,4,5-IP3 synthesis in MWBCs is not suitable for diagnosis of MH disposition. It remains questionable whether an altered 1,4,5-IP3 metabolism in MWBCs is involved in pathologic cascades of MH. Therefore, other cell tissues should be evaluated in further studies to clarify the role of the 1,4,5-IP3 metabolism in MH.

Anesthetics, Inhalation↗
PubMed2001

Coagulation process proceeds on cultured human mesangial cells via expression of factor V.

BACKGROUND: In a previous clinicopathological study, we observed mesangial factor V expression accompanied by the intact form of cross-linked fibrin deposition in the active type of IgA nephropathy. The conversion of prothrombin to thrombin by factor Xa is potently accelerated more than 104-fold by the presence of factor V, which is a membrane-bound cofactor. Another membrane-bound cofactor, tissue factor, is known to play an initiating role in the coagulation cascade and to be synthesized in mesangial cells (MCs) by the stimulation of tumor necrosis factor-alpha (TNF-alpha). However, the synthesis of factor V, which plays on the terminating stage of prothrombin activation, has not been reported previously in MCs by in vitro study. Our current study tested the coagulation process via expression of factor V by the stimulation of proinflammatory cytokine, TNF-alpha, in cultured human MCs. METHODS: To evaluate factor V protein expression, immunoperoxidase staining with densitometric evaluation and Western blot analysis were conducted after stimulation of TNF-alpha. To test factor V activity, stimulated MCs were incubated in combination with factor Xa, prothrombin, fibrinogen and factor XIII, and fibrin production on MCs was assessed after immunoperoxidase staining on the cell surface. In a blocking test using an antibody against factor V, suppression of fibrin production was evaluated to clarify the role of factor V activity. For the evaluation of factor V mRNA expression in cultured human MCs, in situ hybridization and Northern blot analysis were performed. RESULTS: Factor V protein expression in MCs after TNF-alpha stimulation increased both time- and dose-dependently. As a marker of factor V activity with exogenous factor Xa, fibrin production on TNF-alpha-stimulated MCs was increased in a time-dependent manner and was inhibited by the addition of anti-factor V antibody. Factor V mRNA was identified in MCs by in situ hybridization and showed an increase after stimulation with TNF-alpha on Northern blot analysis. CONCLUSIONS: Our data suggest that the coagulation process proceeds on MCs as the result of increased expression of endogenous factor V activity on its cell surface in cooperation with exogenous factor Xa.

Antibodies↗
PubMed2006

Endogenous opioids modulate the growth of the biliary tree in the course of cholestasis.

BACKGROUND & AIMS: There is poor knowledge on the factors that modulate the growth of cholangiocytes, the epithelial cell target of cholangiopathies, which are diseases leading to progressive loss of bile ducts and liver failure. Endogenous opioids are known to modulate cell growth. In the course of cholestasis, the opioidergic system is hyperactive, and in cholangiocytes a higher expression of opioid peptide messenger RNA has been described. This study aimed to verify if such events affect the cholangiocyte proliferative response to cholestasis. METHODS: The presence of the delta opioid receptor (OR), muOR, and kappaOR was evaluated. The effects on cholangiocyte proliferation of the in vitro and in vivo exposure to their selective agonists, together with the intracellular signals, were then studied. The effects of the OR antagonist naloxone on cell growth were also tested both in vivo and in vitro. RESULTS: Cholangiocytes express all 3 receptors studied. deltaOR activation strongly diminished the proliferative and functional response of cholangiocytes to cholestasis, whereas muOR resulted in a slight increase in cell growth. The deltaOR signal is mediated by the IP3/CamKIIalpha/PKCalpha pathway, which inhibits the cAMP/PKA/ERK1/2/AKT cascade. In contrast, muOR activation stimulates the cAMP/PKA/ERK1/2/AKT cascade but does not affect the IP3/CamKIIalpha/PKCalpha pathway. The blockage of endogenous opioid peptides by naloxone further enhanced cholangiocyte growth both in vivo and in vitro. CONCLUSIONS: The increase in opioid peptide synthesis in the course of cholestasis aims to limit the excessive growth of the biliary tree in the course of cholestasis by the interaction with the deltaOR expressed by cholangiocytes.

Animals↗