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Biomedical subjects

F Favilli

Publications and source records attributed to F Favilli.

At least 19 recordsLinked to original sources
PubMed2005

Human CD56bright and CD56dim natural killer cell subsets respond differentially to direct stimulation with Mycobacterium bovis bacillus Calmette-Guérin.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is capable of directly stimulating several effector functions of human natural killer (NK) cells in the absence of interleukin-12 and professional antigen presenting cells. To assess the contribution of two main human NK-cell subsets (CD56(dim) and CD56(bright)) to the overall in vitro NK-cell response to BCG, peripheral blood mononuclear cells depleted of nylon wool-adherent cells or purified NK cells were stimulated with live BCG. By combining intranuclear bromodeoxyuridine (BrdU) staining and analysis of CD56 marker intensity, statistically higher percentages of BrdU(+) cells were found among the CD56(bright) subset than the CD56(dim) subset after 6 days of stimulation with BCG. Similarly, evaluation of intracellular interferon-gamma (IFN-gamma) revealed that CD56(bright) cells were those mainly involved in IFN-gamma production in response to BCG. In contrast, the CD56(dim) subset contained higher levels of perforin and granzyme A, two key molecules for exocytosis-mediated cytotoxicity, than the CD56(bright) subset. Although 16-20-h stimulation with BCG did not substantially alter the expression of cytotoxic molecules by the two subsets, a decrease in perforin content was observed in the CD56(dim), but not in the CD56(bright) subset, following 4-h incubation with the NK-sensitive target K562 cell line. This decrease in perforin content correlated with the induction by BCG-stimulated NK cells, of early markers of apoptosis on target cells to a greater extent than unstimulated cells suggesting a major role for the CD56(dim) subset in cytotoxic activity in response to BCG. Taken together, these results demonstrate that CD56(bright) and CD56(dim) human NK-cell subsets exert different functional activities in response to a live bacterial pathogen.

Alleles↗
PubMed2002

The role of H2O2 in the platelet-derived growth factor-induced transcription of the gamma-glutamylcysteine synthetase heavy subunit.

This study demonstrates that platelet-derived growth factor (PDGF) increases transcription of the gamma-glutamylcysteine synthetase (GCS) heavy subunit (GCS-HS) in NIH 3T3 fibroblasts via H2O2 and activation of protein kinase C (PKC). The data obtained using catalase, H2O2, phorbol-12-myristate 13-acetate (PMA) or a specific inhibitor of PKC demonstrate the possibility of a PDGF up-regulation pathway of GCS synthesis. Moreover, since PDGF mitogenic activity takes place through PKC activation and sphingosine-1-phosphate (S1P) production, the involvement of sphingosine kinase activity in the PDGF effect was also investigated. No clear direct relationship emerged between S1P production and any PDGF- or H2O2-induced increase in the GCS-HS mRNA level. However, for the first time, in S1P-stimulated NIH 3T3 cells, increased levels of GCS-HS mRNA were shown to be related to increases in the reduced glutathione synthesis rate similar to those obtained after PMA and PDGF stimulation.

3T3 Cells↗
PubMed2001

GSH role on platelet-derived growth factor receptor tyrosine phosphorylation induced by H2O2.

This study, conducted on NIH3T3 cells, demonstrates that GSH depletion obtained by buthionine sulfoximine (BSO) treatment does not affect platelet-derived growth-factor receptor (PDGFr) autophosphorylation or cell protein phosphorylation induced by exogenous addition of H2O2, while it does decrease tyrosine phosphorylation obtained by PDGF stimulation. This last effect seems due to the lack of H2O2 generation; for the first time a relation between intracellular GSH content and H2O2 production induced by PDGF has been demonstrated. Therefore, changes of GSH levels can affect the early events of the PDGFr signal pathways by redox regulation. It has also demonstrated that in NIH3T3 cells, H2O2 can directly activate tyrosine phosphorylation by a reversible effect with the involvement of SH-group. This H2O2 effect is increased by vanadate and by GSH depleting agent, diethylmaleate, which unlike BSO is able to produce H2O2 as the current study shows.

3T3 Cells↗
PubMed2001

Adult human heart microvascular endothelial cells are permissive for non-lytic infection by human cytomegalovirus.

OBJECTIVE: Human cytomegalovirus (CMV) infection has been linked to chronic heart disease. The mechanism of CMV dissemination to the heart remains unknown. CMV antigens and nucleic acid sequences have been detected in endothelial cells (ECs) in vivo, and ECs are fully permissive hosts to CMV replication in vitro. This report examines the characteristics of CMV replication in primary cultures of human heart microvascular ECs (HHMECs). METHODS: Capillary ECs were isolated from heart tissue biopsies of six patients at the time of heart surgery. HHMECs were infected with CMV and viral antigens were detected by immunofluorescence assay using monoclonal antibodies as specific reagents. Cytokine and chemokine release in the supernatant of sham- and CMV-infected cells was quantitated by ELISA. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to analyse expression of mRNA for adhesion molecules. RESULTS: CMV was found to productively infect HHMECs without cytolytic effects. Infected cultures released high levels of pro-inflammatory chemokines and enhanced their adhesion molecule expression. CONCLUSIONS: Our data provide new insights into the mechanism of CMV dissemination to the heart, signalling the need for further investigation of the pathogenetic role of this virus in cardiac disorders.

Adult↗
PubMed2001

Rapid changes in hepatitis C virus quasispecies produced by a single dose of IFN-alpha in chronically infected patients.

The effects of a single dose of 3 international megaunits of interferon-alpha2b (IFN-alpha2b) on hepatitis C virus (HCV) load and quasispecies were examined 24 h after administration in 12 previously untreated, chronically infected patients. All patients had viremia loads appreciably reduced relative to baseline values, thus confirming that the viral load is rapidly affected by IFN-alpha2b. Five patients also exhibited changes in plasma HCV quasispecies composition that were clearly evident by single-strand conformation polymorphism, analysis, thus indicating that one dose of IFN-alpha2b may suffice to produce rapid perturbations in the genetic heterogeneity of circulating HCV. Prior to IFN-alpha2b administration, 3 patients exhibited viral quasispecies differences between plasma and peripheral blood mononuclear cells (PBMC). Interestingly, in 2 such patients, the viral quasispecies found in the 24-h plasma resembled that in the pre-IFN PBMC. The latter finding raises the possibility that in these patients, the differences in quasispecies composition between pre-IFN and post-IFN plasma resulted from increased representation of lymphoid tissue-originated variants in the latter sample, possibly because of poor sensitivity to IFN-alpha2b of HCV replication in the lymphoid compartment.

Base Sequence↗
PubMed2000

Liver damage and kinetics of hepatitis C virus and human immunodeficiency virus replication during the early phases of combination antiretroviral treatment.

In order to assess the relationship between human immunodeficiency virus (HIV) RNA, hepatitis C virus (HCV) RNA, CD4, CD8, and liver enzymes during combination antiretroviral therapy, these parameters were measured in 12 HIV-HCV-coinfected patients (who were naive for antiretrovirals) on the day before and 3, 7, 14, 28, 56, and 84 days after initiating the following treatments: stavudine and lamivudine in all patients, indinavir in 6 patients, and nevirapine in 6 patients. HIV RNA declined rapidly, CD4 cells increased slowly, and CD8 cells and liver enzymes were stable. HCV RNA showed a transient significant increase at days 14 and 21 (7.33+/-0.16 [mean +/- SE] and 7.29+/-0.2 log copies/mL vs. 7+/-0.2 log copies/mL at baseline; P<.05). These changes were similar in both treatment groups. A 2-fold alanine aminotransferase increase was observed in 4 of 12 patients; 4 of 4 patients showed increased HCV RNA. The relationship between HCV RNA increase and HIV RNA decrease indicates virus-virus interference. An HCV RNA increase may cause significant liver damage only in a minority of patients.

Adult↗
PubMed2000

Glutathione transport system in NIH3t3 fibroblasts.

The current study characterizes a mediated transport for GSH uptake in murine fibroblasts NIH3T3. The presence of GSH mediated transport is indicated by the behaviour of GSH uptake time-course, as well as by kinetic saturation and the specific inhibition of the initial rate of GSH transport. Moreover, a concentrative GSH uptake has been measured, whose driving force may be due to a change of membrane potential and the direct involvement of ATP. Hyperbolic kinetic saturation shows a single mediated transport with high affinity for GSH (Km = 0.209 +/- 0.03 mM). High specificity of this GSH transporter for the entire structure of GSH is also demonstrated. To summarize, GSH uptake into NIH3T3 cells occurs by an active transport system and shows characteristics similar to ATP-dependent mechanisms previously identified in hepatocyte membranes. Moreover, a possible physiological role of this GSH transporter related to NIH3T3 cell proliferation has been hypothesized.

3T3 Cells↗
PubMed1999

Increased glutathione synthesis associated with platelet-derived growth factor stimulation of NIH3T3 fibroblasts.

Previous data show a relation between GSH content and proliferation of normal and tumour cells. We recently demonstrated a specific involvement of GSH in the autophosphorylation activity of the platelet-derived growth factor (PDGF) receptor in NIH3T3 fibroblasts. In this study we demonstrate that the stimulation by PDGF of serum-starved NIH3T3 cells increases cellular GSH content, while no change in oxidized GSH content was measured. Experiments performed with actinomycin, cycloheximide and buthionine sulfoximide, a specific inhibitor of the rate-limiting enzyme of the de novo synthesis of GSH gamma-glutamylcysteine synthetase (gamma-GCS), confirm PDGF induction of GSH synthesis. These results provide the first demonstration that PDGF mediated transduction signals seem strictly related to mechanisms involved in the increase of gamma-GCS activity associated with increased gamma-GCS heavy subunit mRNA levels. In fact, serum and epidermal growth factor (EGF) stimulation of quiescent NIH3T3 and NIH3T3, which overexpress EGF receptor, does not affect GSH content or its synthesis. These data may be related to a possible GSH role in the redox regulation of cell proliferation mediated by PDGF.

3T3 Cells↗
PubMed1999

Gluthatione level is altered in lymphoblasts from patients with familial Alzheimer's disease.

Intracellular levels of glutathione (GSH), glutathione disulphide (GSSG), glutamic acid and gamma-glutamyl cysteine synthetase (gamma-GCS) were measured in lymphoblast lines from patients with familial and sporadic Alzheimer's disease (AD) and from age-matched controls. Lymphoblasts carrying presenilins (PS) and amyloid precursor protein (APP) genes mutations showed significantly decreased GSH content with respect to controls. Levels of GSSG and glutamic acid, as well as the activity of gamma-GCS were not significantly different in lymphoblasts carrying genes mutations as compared with control cells. These results indicate that even peripheral cells not involved in the neurodegenerative process of AD show altered GSH content when carrying PS and APP genes mutations. The provided data appear to be in accordance with the known alteration of GSH levels in central nervous system and strengthen the hypothesis of oxidative stress as an important, possibly crucial mechanism in the pathogenesis of AD.

Alzheimer Disease↗
PubMed1999

Evidence of glutathione transporter in rat brain synaptosomal membrane vesicles.

Glutathione (GSH) transport was studied in synaptosomal membrane vesicles (SMV) of rat cerebral cortex. The present study shows that GSH uptake into SMV occurs very quickly in a time-dependent manner into an osmotically active intravesicular space. The initial rate of transport followed Michealis-Menten saturation kinetics with a Km 4.5+/-0.8 microM that shows a high affinity of the transporter for GSH. Therefore GSH uptake in SMV occurs by a mediated transport system which can be activated by either an inward gradient of cations, like Na+ or K+, or membrane depolarization. These results, together with those obtained by valinomycin-induced K+ diffusion potential, indicate that GSH synaptosomal transport is electrogenic by a negative charge transfer. The increase of GSH uptake measured by trans-stimulation experiments confirms a GSH bidirectional mediated transport which seems susceptible of modulation by changes in ionic fluxes and in the membrane potential. These results may indicate a possible involvement of this transporter in the role suggested for GSH in synaptic neurotransmission; also considering that GSH precursor of neuroactive aminoacids (glycine, glutamate), may contribute to regulate their level in synapses. Finally, a GSH transporter in synaptosomes may contribute to maintaining the GSH homeostasis in cerebral cortex, where decreases of GSH levels have been related to susceptibility to neuropathologies.

Animals↗
PubMed1998

Glutathione involvement on the intestinal Na+-dependent D-glucose active transporter.

Glutathione and its related enzymes are present in intestinal epithelium. Depletion or alteration of glutathione levels have been related to different physiological and pathological conditions. Glutathione also seems to be related to the regulation of some protein activities. The present study, by in vivo experiments, shows a specific relationship between D-glucose Na+-dependent active transporter activity in rat intestine brush-border membranes and reduced glutathione/oxidized glutathione ratio levels. Changes of the kinetic parameters show that an increase of this ratio is related to an increase of the affinity of glucose for its binding sites and a higher transport capacity of the transporter. Neither alteration in the activity of other substrate transport systems nor change in the specific activity of the key enzymes related to glutathione and glucose metabolism are found. These findings suggest the possibility that D-glucose transporter activity is modulated through the change in the redox status of glutathione.

Animals↗
PubMed1997

Glutathione transport system in human small intestine epithelial cells.

The present study characterizes for the first time a GSH specific transporter in a human intestinal epithelial cell line (I407). GSH metabolism is very important for the antioxidant and detoxifying action of intestine and for the maintenance of the luminal thiol-disulfide ratio involved in regulation mechanisms of the protein activity of epithelial cells. GSH level decreases have been related to physio-pathological alterations either of intestine or other organs. GSH specific transport systems have been identified in membranes of various cell types of rat, mice and rabbit. The presence of a Na+-independent transport system of GSH is confirmed by the similar behaviour of GSH uptake time-courses when Na+ in extracellular uptake medium was replaced with choline+ or K+ as well as by kinetic saturation and by the trans-stimulation effect on GSH uptake in GSH preloaded cells. Moreover, this transporter is activated when cations are present in extracellular medium and it is affected by membrane potential changes with an increase in GSH uptake values when membrane depolarization occurs. The present results also show a remarkable affinity and specificity of this transporter for GSH; in fact, Km value is very low (90 +/- 20 microM) and only compounds strictly related to GSH structure, such as GSH S-conjugates and GSH-ethyl ester, inhibit GSH uptake in 1407 cells. Finally, a possible hormonal control and modulation by the thiol-disulfide status of GSH transporter activity is suggested.

Animals↗
PubMed1997

Effect of orally administered glutathione on glutathione levels in some organs of rats: role of specific transporters.

The present study reports data on absorption of orally administered glutathione (GSH) in rat jejunum and in other organs, and the possible role of specific transport systems of GSH and gamma-glutamyltranspeptidase (EC 2.3.2.1; gamma-GT) activity. GSH levels were measured simultaneously in various organs after oral GSH administration to untreated rats and rats treated with L-buthionine sulfoximine (BSO) or acivicin (AT125). BSO selectively inhibits GSH intracellular synthesis and AT125 is a specific inhibitor of gamma-GT activity. GSH levels were also measured after oral administration of an equivalent amount of the constituent amino acids of GSH to untreated and BSO-treated rats. Significant increases in GSH levels were found in jejunum, lung, heart, liver and brain after oral GSH administration to untreated rats. GSH increases were also obtained in all organs, except liver, when GSH was administered to rats previously GHS-depleted by treatment with BSO. The analysis of all results allowed us to distinguish between the increase in GSH intracellular levels due to intact GSH uptake by specific transporters, and that due to GSH degradation by gamma-GT activity and subsequent absorption of degradation products with intracellular resynthesis of GSH; both these mechanisms seemed to be involved in increasing GSH content in heart after oral GSH administration. Jejunum, lung and brain took up GSH mostly intact, by specific transport systems, while in liver GSH uptake occurred only by its breakdown by gamma-GT activity followed by intracellular resynthesis.

Administration, Oral↗
PubMed1994

Glutathione metabolism in Crohn's disease.

A statistically significant decrease of glutathione (GSH) and an increase of GSH disulfide (GSSG) both in healthy and ill ileum of patients with Crohn's disease in comparison with the controls (without this pathology) is demonstrated. However, the lowering of these levels was more remarkable in ill ileum in which high levels of GSSG were detected, too. These alterations may be in part explained by the changes obtained in GSH-related enzyme levels. Finally, considering the results that others and we obtained by studies on GSH oral absorption in rat intestine, an oral therapy of GSH in Crohn's disease is suggested.

Administration, Oral↗
PubMed1993

Age and GSH metabolism in rat cerebral cortex, as related to oxidative and energy parameters.

A comprehensive study on GSH metabolism in relation to some markers of oxidative and energy status in rat cerebral cortex as a function of age was performed. Reduced GSH, total GSH and the GSH Redox Index decreased both during growth (defined as the period between 1 and 5 months) and during aging (defined as the period between 5 and 27 months) while GSSG levels increased during the two periods, but most significantly during aging. Also GSH-associated enzymes and adenine-pyridine nucleotide levels show age characteristic changes. The obtained results suggest that decreases in oxidative and energy metabolism occur during aging. They probably contribute to decreases in the activity of the biosynthetic processes (i.e., NADP+(H) and GSH synthesis) and in the antioxidant capacity of the GSH system. However, the oxidative stress does not seem to be a typical characteristic of the aging period; as an oxidative status is present during the growth period too. Typical parameters of aging process are mainly the low levels of reduced GSH, total GSH and GSH Redox Index and the high levels of GSSG as well as the high levels of GSH peroxidase and GSH transferase and the low levels of gamma-glutamylcysteine synthetase.

Adenosine Diphosphate↗
PubMed1993

Altered metabolism of glutathione in cells transformed by oncogenes which cause resistance to ionizing radiations.

We measured glutathione (GSH) metabolism in normal NIH/3T3 fibroblasts, and in cells transformed by the oncogenes sis, erbB, src, ras, dbl, and raf.erbB,src,ras and raf, but not sis and dbl transformants, showed increased level of total and reduced GSH as compared with normal NIH/3T3 fibroblasts; oxidized GSH was elevated only in src- and ras-transformed cells. Increased total GSH content was associated with decreased activity of the synthetic enzyme gamma-glutamylcysteine synthetase, and oxidized GSH level with increased activity of GSH reductase. These data suggest that GSH synthesis was selectively enhanced in cells transformed by specific oncogenes, with resulting down-regulation of its synthetic enzyme; alterations of GSH metabolism appeared to be peculiar of transformation by specific oncogenes, and not trivial epiphenomena of neoplastic transformation. Oncogenic transformants that presented elevated level of GSH were also those reported to be resistant to antineoplastic drugs and ionizing radiations, thus confirming a possible link between altered GSH metabolism and resistance to antineoplastic treatment.

3T3 Cells↗
PubMed1991

Intestinal glutathione transport system: a possible detoxication role.

The epithelium of the small intestine act by the formation of GSH-S-conjugation, as a first line of defence against various ingested toxic chemicals. GSH and GSH-dependent enzymes are present in the gastrointestinal wall. We and others have characterized the GSH-specific transport systems in intestinal brush-border and in basolateral membrane vesicles, in which gamma-glutamyltranspeptidase (gamma-GT) activity was inactivated by AT-125. In the present study we use inhibition experiments, kinetic studies, trans-stimulation of GSH uptake and HPLC determination to demonstrate (for the first time) that GSH and two GSH-S-conjugates (chosen as model compounds) share a common transport system. Plasma GSH-S-conjugates that may enter the intestinal cells via basolateral membrane, and GSH-S-conjugates that form in intestinal cells, may be eliminated directly by this GSH transporter across brush-border membranes or transported into lumen to the active site of gamma-GT; they are then further metabolized and excreted by various routes. This transport system may thus contribute to the intestinal detoxication role.

Amino Acid Sequence↗