Coagulation defects in cirrhosis--old dogmas not yet ready for burial.
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Blood Coagulation Disorders↗
Biomedical subjects
F Marra
Publications and source records attributed to F Marra.
Overexpression of Bcl-2 by activated human hepatic stellate cells: resistance to apoptosis as a mechanism of progressive hepatic fibrogenesis in humans.
BACKGROUND AND AIMS: Myofibroblast-like cells, originating from activation of hepatic stellate cells (HSC/MFs), play a key role in liver fibrosis, a potentially reversible process that may rely on induction of HSC/MFs apoptosis. While this possibility has been shown in cultured rat HSC, very limited data are currently available for human HSC/MFs. METHODS: Cultured human HSC/MFs were exposed to several proapoptotic stimuli, including those known to induce apoptosis in rat HSC/MFs, and induction of cell death and related mechanisms were investigated using morphology, molecular biology, and biochemical techniques. RESULTS: In this study we report that fully activated human HSC/MFs did not undergo spontaneous apoptosis and survived to prolonged serum deprivation, Fas activation, or exposure to nerve growth factor, tumour necrosis factor alpha (TNF-alpha), oxidative stress mediators, doxorubicin, and etoposide. Induction of caspase dependent, mitochondria driven apoptosis in HSC/MFs was observed only when protein synthesis or transcription were inhibited. Importantly, the process of HSC activation was accompanied by changes in expression of a set of genes involved in apoptosis control. In particular, activated human HSC/MFs in culture overexpressed Bcl-2. The role of Bcl-2 was crucial as Bcl-2 silenced cells became susceptible to TNF-alpha induced apoptosis. Finally, Bcl-2 was markedly expressed in HSC/MFs present in liver tissue obtained from patients with hepatitis C virus related cirrhosis. CONCLUSIONS: Human activated HSC/MFs are resistant to most proapoptotic stimuli due to Bcl-2 overexpression and this feature may play a key role in the progression of fibrosis in chronic liver diseases.
Apoptosis↗
Bioadhesive monolayer film for the in vitro transdermal delivery of sumatriptan.
The work presented here aims to develop a bioadhesive monolayer film containing sumatriptan as adjuvant for the treatment of headache pain in a severe migraine attack. Permeation experiments were performed from the films prepared and from the respective solution, to evaluate the relevant permeation parameters. The effect of the penetration enhancers Transcutol, 2-pyrrolidone, and polyethylene glycol 600 was evaluated. The results obtained show that Transcutol and 2-pyrrolidone decreased sumatriptan permeation from solution, whereas a modest increase was produced by polyethylene glycol 600. The enhancers produced the same effects when they were included in the film. Compared to solution, the film showed a higher sumatriptan flux in the early times of the experiment. When the film was applied in occlusive conditions the profiles were much higher, indicating the importance of patch drying. Concerning skin retention, the bioadhesive film produced a reduction of the amount of sumatriptan remaining in the skin, but this can be advantageous in the control of drug input, since it reduces the reservoir effect in the skin and allows for an immediate interruption of drug input when the patch is removed.
Administration, Cutaneous↗
Thiazolidinediones and hepatic fibrosis: don't wait too long.
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Drug Administration Schedule↗
CXCR3 and alphaEbeta7 integrin identify a subset of CD8+ mature thymocytes that share phenotypic and functional properties with CD8+ gut intraepithelial lymphocytes.
BACKGROUND: We previously demonstrated the existence of two distinct subsets of T cell receptor (TCR)alphabeta+CD8alphabeta+ single positive (SP) cells in human postnatal thymus which express the chemokine receptor CCR7 or CXCR3 and migrate in vitro in response to their specific ligands. AIM: To investigate whether these two CD8+ thymocyte subsets had distinct peripheral colonisation. METHODS: TCRalphabeta+CD8+ SP cells were obtained from normal postnatal thymus, mesenteric lymph node (LNs), small bowel, and peripheral blood (PB) specimens. Cells were then evaluated for expression of surface molecules, cytolytic potential, telomere length, and profile of cytokine production. RESULTS: CD8+CCR7+CXCR3- thymocytes exhibited CD62L, in common with those which localise to LNs. In contrast, CD8+CCR7-CXCR3+ thymocytes lacked CD62L but exhibited CD103, similar to intraepithelial lymphocytes (IELs) present in the gut mucosa where the CXCR3 ligand, CXCL10, and the CD103 ligand, E-cadherin, are highly and consistently expressed. In addition, thymocytes and gut CD8+CXCR3+CD103+ cells showed comparable telomere length, which was higher than that of PB CXCR3+CD8+ T cells. However, both of these populations contained perforin and granzyme A, and displayed the ability to produce interferon gamma and interleukin 2. Of note, CXCR3 deficient, in comparison with wild-type C57Black/6, mice showed decreased proportions of CD3+CD8alphabeta+ and increased proportions of CD3+CD8alphaalpha+ lymphocytes at gut level. Moreover, adoptive transfer of CD3+CD8alphabeta+ thymocytes from wild-type into CXCR3 deficient mice resulted in a significant increase in CD3+CD8alphabeta+ T cells in the gut mucosa but not in other tissues. CONCLUSIONS: The results of this study demonstrate the existence of a previously unrecognised subset of TCRalphabeta+CD8alphabeta+ SP CXCR3+CD103+ thymocytes which share phenotypic and functional features with CD8+ IELs, thus suggesting the possibility of their direct colonisation of the gut mucosa.
Adoptive Transfer↗
Dose dependent and divergent effects of superoxide anion on cell death, proliferation, and migration of activated human hepatic stellate cells.
BACKGROUND AND AIMS: Activated myofibroblast-like cells, originating from hepatic stellate cells (HSC/MFs) or other cellular sources, play a key profibrogenic role in chronic liver diseases (CLDs) that, as suggested by studies in animal models or rat HSC/MFs, may be modulated by reactive oxygen intermediates (ROI). In this study, human HSC/MFs, exposed to different levels of superoxide anion (O(2)(.-)) and, for comparison, hydrogen peroxide (H(2)O(2)), were analysed in terms of cytotoxicity, proliferative response, and migration. METHODS: Cultured human HSC/MFs were exposed to controlled O(2)(.-) generation by hypoxanthine/xanthine oxidase systems or to a range of H(2)O(2) concentrations. Induction of cell death, proliferation, and migration were investigated using morphology, molecular biology, and biochemical techniques. RESULTS: Human HSC/MFs were shown to be extremely resistant to induction of cell death by O(2)(.-) and only high rates of O(2)(.-) generation induced either necrotic or apoptotic cell death. Non-cytotoxic low levels of O(2)(.-), able to upregulate procollagen type I expression (but not tissue inhibitor of metalloproteinase 1 and 2), stimulated migration of human HSC/MFs in a Ras/extracellular regulated kinase (ERK) dependent, antioxidant sensitive way, without affecting basal or platelet derived growth factor (PDGF) stimulated cell proliferation. Non-cytotoxic levels of H(2)O(2) did not affect Ras/ERK or proliferative response. A high rate of O(2)(.-) generation or elevated levels of H(2)O(2 )induced cytoskeletal alterations, block in motility, and inhibition of PDGF dependent DNA synthesis. CONCLUSIONS: Low non-cytotoxic levels of extracellularly generated O(2)(.-) may stimulate selected profibrogenic responses in human HSC/MFs without affecting proliferation.
Apoptosis↗
Renaming cytokines: MCP-1, major chemokine in pancreatitis.
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Animals↗
Review article: the pathogenesis of fibrosis in non-alcoholic steatohepatitis.
Non-alcoholic steatohepatitis has been recognized as a significant cause of end-stage liver disease and hepatic decompensation. Despite the growing interest in this condition, the molecular mechanisms underlying the development of fibrosis in this setting are only partially understood. In this article, the cellular and molecular basis of fibrosis in chronic liver disease are briefly outlined. In addition, mechanisms specifically operating in the context of fatty liver and steatohepatitis are examined, including: insulin resistance, oxidative stress, and inflammation. Finally, recent developments indicating the possible contribution of cytokines derived from adipose tissue (adipokines) to liver fibrosis is discussed.
Adipocytes↗
Alpha-tocopherol pro-vitamins: synthesis, hydrolysis and accumulation in rabbit ear skin.
We synthesized esters of alpha-tocopherol (VE) with the aim to develop new pro-vitamins, easily reconverted by enzymes in the skin and able to release another active moiety such as an amino acid, in order to obtain a synergic effect. In particular, the attention was dedicated to the amino acids glycine and alanine and to pyroglutamic acid. The sensitivity of pro-vitamins to enzymatic hydrolysis was evaluated in vitro using porcine liver esterase. Permeation experiments were performed using rabbit ear skin, for the quantification of pro-vitamins and derived VE in the epidermis and dermis. The new derivatives synthesized, and in particular the glycine and alanine derivatives, accumulated in rabbit skin in a significant extent and originated substantial amounts of alpha-tocopherol. In comparison with the acetate derivative (VEAc), the amounts accumulated are comparable or higher. Moreover, the new derivatives, being more hydrophilic, allow the use of vehicles such as the mixture water/propylene glycol/ethanol widely employed for the preparation of creams and gels. Finally, the enzymatic metabolism of these new derivatives generates not only VE, but also components that can have a further advantageous action on skin.
Alanine↗
Successful treatment of multidrug-resistant tuberculosis following drug-induced hepatic necrosis requiring liver transplant.
A 28-year-old female developed multidrug-resistant (MDR) tuberculous lymphadenitis following a trip to India. She was initially treated with a four-drug regimen of first-line anti-tuberculosis medications, but when sensitivities indicated resistance to isoniazid and rifampin, her regimen was altered to ciprofloxacin (CFX), pyrazinamide (PZA) and ethambutol. She subsequently developed a rash, flu-like symptoms and fever, which progressed to acute hepatic necrosis despite discontinuation of medication. The clinical presentation and subsequent investigations suggested a hypersensitivity reaction, possibly related to the quinolone. The patient subsequently had an orthoptic liver transplant; second-line anti-tuberculosis medications were restarted to which she responded clinically and radiologically. Our findings raise the possibility that the CFX and PZA combination was responsible for the hepatic necrosis. The patient also illustrates that active, even MDR tuberculosis is not a contraindication to hepatic transplant.
Adult↗
Role of hepatic stellate cells in the pathogenesis of portal hypertension.
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Adult↗
Interferon-inducible protein 10, monokine induced by interferon gamma, and interferon-inducible T-cell alpha chemoattractant are produced by thymic epithelial cells and attract T-cell receptor (TCR) alphabeta+ CD8+ single-positive T cells, TCRgammadelta+ T cells, and natural killer-type cells in human thymus.
Strong reactivity for interferon-inducible protein 10 (IP-10), monokine induced by interferon gamma (Mig), and interferon-inducible T-cell alpha chemoattractant (I-TAC) was found in epithelial cells mainly localized to the medulla of postnatal human thymus. The CXC chemokine receptor common to the 3 chemokines (CXCR3) was also preferentially expressed in medullary areas of the same thymuses and appeared to be a property of 4 distinct populations: CD3+ T-cell receptor (TCR) alphabeta+ CD8+ single-positive (SP) T cells, TCRgammadelta+ T cells, natural killer (NK)-type cells, and a small subset of CD3+(low) CD4+ CD8+ TCRalphabeta+ double-positive (DP) T cells. IP-10, Mig, and I-TAC showed chemoattractant activity for TCRalphabeta+ CD8+ SP T cells, TCRgammadelta+ T cells, and NK-type cells, suggesting their role in the migration of different subsets of mature thymocytes during human thymus lymphopoiesis.
CD8-Positive T-Lymphocytes↗
Signal transduction by the chemokine receptor CXCR3: activation of Ras/ERK, Src, and phosphatidylinositol 3-kinase/Akt controls cell migration and proliferation in human vascular pericytes.
Hepatic stellate cells (HSC) and glomerular mesangial cells (MC) are tissue-specific pericytes involved in tissue repair, a process that is regulated by members of the chemokine family. In this study, we explored the signal transduction pathways activated by the chemokine receptor CXCR3 in vascular pericytes. In HSC, interaction of CXCR3 with its ligands resulted in increased chemotaxis and activation of the Ras/ERK cascade. Activation of CXCR3 also stimulated Src phosphorylation and kinase activity and increased the activity of phosphatidylinositol 3-kinase and its downstream pathway, Akt. The increase in ERK activity was inhibited by genistein and PP1, but not by wortmannin, indicating that Src activation is necessary for the activation of the Ras/ERK pathway by CXCR3. Inhibition of ERK activation resulted in a decreased chemotactic and mitogenic effect of CXCR3 ligands. In MC, which respond to CXCR3 ligands with increased DNA synthesis, CXCR3 activation resulted in a biphasic stimulation of ERK activation, a pattern similar to the one observed in HSC exposed to platelet-derived growth factor, indicating that this type of response is related to the stimulation of cell proliferation. These data characterize CXCR3 signaling in pericytes and clarify the relevance of downstream pathways in the modulation of different biologic responses.
Androstadienes↗
Guanosine 3': 5'-cyclic monophosphate-dependent pathway alterations in ventricular cardiomyocytes of spontaneously hypertensive rats.
1. We investigated the effect of the NO-donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) on cardiomyocytes isolated from control normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. 2. Ventricular cardiomyocytes were isolated from SHR and WKY hearts and imaging analysis of fura-2-loaded cells was performed in order to evaluate calcium transient in electrical field paced (0.5 Hz) cells. 3. In WKY cardiomyocytes, 1 - 200 microM SNAP dose-dependently increased cyclic GMP content. In basal conditions, cyclic GMP content of SHR cardiomyocytes was significantly higher than in WKY, but SNAP failed to further increase cyclic GMP over the basal level. 4. In control conditions, the Delta F/F and decay time of the calcium transient were similar in both strains. In WKY cardiomyocytes, SNAP (1 - 100 microM) reduced the decay time. In SHR cardiomyocytes, SNAP was ineffective. Dibutyryl cyclic GMP (10(-6) - 10(-8) M), a membrane permeable cyclic GMP analogue, behaved similarly to SNAP. 5. In WKY and SHR cardiomyocytes, 10(-8) M isoprenaline similarly increased Delta F/F and decreased the decay time. SNAP and dibutyryl cyclic GMP prevented the effect of isoprenaline in WKY, whereas both molecules were ineffective in SHR cardiomyocytes. In WKY, SNAP effects were blocked by pretreating cells with the cGK inhibitor KT-5823. 6. Western blotting analysis of cGK type I showed that the enzyme was expressed in WKY isolated cardiomyocytes, but absent in four out of five SHR preparations. 7. We concluded that the low expression of cGKI may determine the lack of NO/cyclic GMP-dependent regulation on calcium transient in SHR cardiomyocytes. This alteration may contribute to the development of heart hypertrophy in hypertensive status.
Animals↗
Agonist-specific regulation of monocyte chemoattractant protein-1 expression by cyclooxygenase metabolites in hepatic stellate cells.
Activated hepatic stellate cells (HSC) regulate the liver "wound-healing" response through expression of chemokines, including monocyte chemoattractant protein-1 (MCP-1), which participate in the formation of the inflammatory infiltrate during liver injury. Cyclooxygenase (COX) catalyzes the conversion of arachidonic acid into prostaglandins, which may contribute to the inflammatory response. In this study, we investigated the effects of COX inhibitors on the expression of MCP-1 in cultured HSC. Pretreatment of HSC with nonspecific COX inhibitors such as indomethacin or ibuprofen markedly reduced the expression of MCP-1 caused by exposure to tumor necrosis factor alpha (TNF-alpha) or interleukin-1alpha (IL-1alpha). NS-398, a specific COX-2 inhibitor, also resulted in a dose-dependent inhibition of MCP-1 gene and protein expression. These effects were dependent on reduced MCP-1 transcription, as established using a reporter plasmid. In contrast, the up-regulation of MCP-1 expression caused by interferon gamma (IFN-gamma) was not sensitive to COX inhibitors. Quiescent HSC did not show detectable expression of COX-2, which became evident after activation in culture, and while TNF-alpha and IL-1alpha markedly increased the expression of COX-2, IFN-gamma did not have any effects. Pretreatment of HSC with the stable cyclic adenosine monophosphate (cAMP) analog, 8-bromo cAMP, reverted the effects of the COX-2 inhibitor, but not of a nuclear factor-kappaB (NF-kappaB) inhibitor, demonstrating that prostaglandins modulate MCP-1 expression via production of cAMP. On the other hand, the action of NF-kappaB inhibitors was negligible in IFN-gamma-stimulated cells. These findings indicate that cross-talk between cytokines and a prostaglandin-cAMP pathway differentially regulates the proinflammatory potential of HSC, contributing to the modulation of liver tissue inflammation.
8-Bromo Cyclic Adenosine Monophosphate↗
Cytokine receptors and signaling in hepatic stellate cells.
Following acute or chronic liver tissue damage, hepatic stellate cells (HSCs) undergo a process of activation toward a phenotype characterized by increased proliferation, motility, contractility, and synthesis of extracellular matrix components. Activation of HSCs is regulated by several soluble factors, including growth factors, cytokines, chemokines, and products of oxidative stress, as well as by extensive changes in the composition and organization of the ECM. Different groups of soluble factors may be classified according to their prevalent biological effect: (a) factors promoting HSC proliferation and/or migration (i.e., platelet-derived growth factor, basic fibroblast growth factor, insulin-like growth factor-1); (b) factors promoting fibrillar ECM accumulation, particularly transforming growth factor-beta1; (c) factors with a prevalent contractile effect on HSCs, such as endothelin-1, thrombin, angiotensin-II and vasopressin, although all these agents also may promote HSC proliferation; (d) proinflammatory cytokines and chemokines; and (e) cytokines with a prominent antiinflammatory/antifibrogenic activity, such as interleukin-10 and interferon-gamma. Additional important issues are represented by the relationship between cytokine and integrin signaling, and by the effects of oxidative stress-related molecules on cytokine signaling. In the past decade the major intracellular signaling pathways elicited by these factors in HSCs have been greatly elucidated.
Cytokines↗
Phosphatidylinositol 3-kinase inhibition enhances human sperm motility.
BACKGROUND: The number of spermatozoa with forward motility after capacitation procedures represents the limiting factor for application of IVF versus intracytoplasmatic sperm injection (ICSI) procedure in cases of oligoasthenozoospermia. The possibility of increasing this number may thus be of help to the patient. A complex array of signalling pathways is involved in the regulation of sperm motility and recent data pointed out an important role for kinase/phosphatase-regulated phosphorylation of proteins. Here, we investigated the role of phosphatidylinositol 3-kinase (PI3K), a lipid and protein kinase involved in the regulation of several biological aspects of somatic cells, on human sperm motility by using the specific PI3K inhibitor LY294002. METHODS AND RESULTS: We demonstrated that in-vitro incubation of swim-up selected or unselected human spermatozoa with LY294002 determined an increase of percentage forward motility in all the treated samples. The effect was dose-dependent with an EC(50) of 1.09 +/- 0.54 micromol/l. LY294002 also increased sperm movement characteristics and hyperactivation as evaluated by computer-assisted motion analyser. The compound was also able to overcome the detrimental effect of hydrogen peroxide and lithium chloride on sperm motility. CONCLUSIONS: Our results suggest a negative role for PI3K in the development and maintenance of sperm motility and suggest a possible use of PI3K inhibitors to enhance motility in cases of asthenozoospermia.
Chromones↗
Role of therapeutic drug monitoring for protease inhibitors.
OBJECTIVE: To review the role of therapeutic drug monitoring (TDM) for protease inhibitors in the treatment of HIV infection. DATA SOURCES: Primary articles were identified using MEDLINE (1966-October 2000), EMBASE (1987-October 2000), AIDSLINE (1980-October 2000), Current Contents, PubMed, and Medscape. Further articles were identified from bibliographic review of primary articles and review papers. Abstracts presented at the World AIDS Conference, Interscience Conference on Antimicrobial Agents and Chemotherapy, and Conference on Retroviruses and Opportunistic Infections were also identified from 1997 to 2000. STUDY SELECTION AND DATA EXTRACTION: All English-language, prospective clinical trials, as well as selected retrospective studies and case series, pertaining to therapeutic drug monitoring of protease inhibitors were included. DATA SYNTHESIS: A number of clinical studies have found a good relationship between concentration and pharmacologic response and/or toxicity as well as wide interpatient variability in the pharmacokinetics of protease inhibitors. There also is some preliminary evidence of the usefulness of plasma drug concentrations to guide dosage adjustments of protease inhibitors in patients with liver dysfunction. Furthermore, there is preliminary evidence of a relationship between drug concentrations and resistance. CONCLUSIONS: A number of clinical studies support the usefulness of TDM of protease inhibitors. However, before TDM can be of the most value, further evaluation requires simplified and standardized assays to be performed routinely by clinical laboratories; determination of the appropriate target concentration and therapeutic range, as well as the best predictor of pharmacologic response; and refined interpretation of plasma drug concentrations. Randomized, controlled clinical trials of patient outcomes are needed to assess the clinical utility of TDM for protease inhibitors.
Clinical Trials as Topic↗