PubMed Health⌕ Search

Biomedical subjects

G Migliorati

Publications and source records attributed to G Migliorati.

At least 19 recordsLinked to original sources
PubMed2005

Human exposure to dioxins through diet in Italy.

We have measured the content of polychlorinated dibenzo-p-dioxins, and polychlorinated dibenzofurans (together defined as "dioxins") in 269 samples of food of animal origin collected through the regional veterinary services, covering the national territory. Quantification of the dioxins was accomplished by isotope dilution method, and toxic equivalents (TEQ) were calculated. The average daily food intake was obtained from two main sources: national data collected by the National Institute of Nutrition, and data from an ongoing cohort study on diet and cancer including 40,000 Italian subjects. The mean value of dioxins measured in food of animal origin was 0.144 +/- 0.266 pg-TEQ/g (range: 0.003-1.655 pg-TEQ/g). Fish was the item with the highest content. The estimated intake of dioxins with main food items of animal origin is presented. The major contribution to dioxins intake with food comes from cow milk and fish consumption. These results are in agreement with what observed in studies conducted in other countries, such as Germany, Finland, Japan, Spain, and are below the limits set by the European legislation.

Animals↗
PubMed2002

Induction of apoptosis by 1,4-benzothiazine analogs in mouse thymocytes.

1,4-benzothiazine (1,4-B) derivatives exert numerous effects in vivo and in vitro, including neurotoxicity and antitumor cytotoxicity. To analyze the mechanisms responsible for 1,4-B-induced cytotoxicity, we performed experiments to evaluate the possible apoptotic effect. For that purpose, we used mouse thymocytes, a cell population well sensitive to induction of apoptosis that has been used to assay apoptosis in many experimental systems. Results indicate that a number of 1,4-B analogs are able to induce both thymocyte apoptosis in vitro and thymus cell loss in vivo. Moreover, analysis of the structure-activity relationship indicate that the sulfur (S) oxidation state, the presence of the carbonyl group, and the nature and position of the side chain modulate the apoptotic efficacy. Moreover, results of in vitro experiments show that the 1,4-B-induced apoptosis associates with different biochemical events including phosphatidylcholine-specific phospholipase C activation, acidic sphingomyelinase activation and ceramide generation, loss of mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c release, and caspase-8, -9, and -3 activation. These results indicate that 1,4-B analogs induce apoptosis through a complex of biochemical events.

Animals↗
PubMed2001

Modulation of T-cell activation by the glucocorticoid-induced leucine zipper factor via inhibition of nuclear factor kappaB.

Previously a novel gene was identified that encodes a glucocorticoid-induced leucine zipper (GILZ) whose expression is up-regulated by dexamethasone. This study analyzed the role of GILZ in the control of T-cell activation and its possible interaction with nuclear factor kappaB (NF-kappaB). Results indicate that GILZ inhibits both T-cell receptor (TCR)-induced interleukin-2/interleukin-2 receptor expression and NF-kappaB activity. In particular, GILZ inhibits NF-kappaB nuclear translocation and DNA binding due to a direct protein-to-protein interaction of GILZ with the NF-kappaB subunits. Moreover, GILZ-mediated modulation of TCR-induced responses is part of a circuit because TCR triggering down-regulates GILZ expression. These results identify a new molecular mechanism involved in the dexamethasone-induced regulation of NF-kappaB activity and T-cell activation. (Blood. 2001;98:743-753)

Active Transport, Cell Nucleus↗
PubMed2000

Group B Streptococcus induces apoptosis in macrophages.

Group B Streptococcus (GBS) is a pathogen that has developed some strategies to resist host immune defenses. Because phagocytic killing is an important pathogenetic mechanism for bacteria, we investigated whether GBS induces apoptosis in murine macrophages. GBS type III strain COH31 r/s (GBS-III) first causes a defect in cell membrane permeability, then at 24 h, apoptosis. Apoptosis was confirmed by several techniques based on morphological changes and DNA fragmentation. Cytochalasin D does not affect apoptosis, suggesting that GBS-III needs not be within the macrophage cytoplasm to promote apoptosis. Inhibition of host protein synthesis prevents apoptosis, whereas inhibition of caspase-1 or -3, does not. Therefore, GBS can trigger an apoptotic pathway independent of caspase-1 and -3, but dependent on protein synthesis. Inhibition of apoptosis by EGTA and PMA, and enhancement of apoptosis by calphostin C and GF109203X suggests that an increase in the cytosolic calcium level and protein kinase C activity status are important in GBS-induced apoptosis. Neither alteration of plasma membrane permeability nor apoptosis were induced by GBS grown in conditions impeding hemolysin expression or when we used dipalmitoylphosphatidylcholine, which inhibited GBS beta-hemolytic activity, suggesting that GBS beta-hemolysin could be involved in apoptosis. beta-Hemolysin, by causing membrane permeability defects, could allow calcium influx, which initiates macrophage apoptosis. GBS also induces apoptosis in human monocytes but not in tumor lines demonstrating the specificity of its activity. This study suggests that induction of macrophage apoptosis by GBS is a novel strategy to overcome host immune defenses.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2000

Respiratory state and phosphatidylserine import in brain mitochondria in vitro.

The mechanism of phosphatidylserine (PS) movement from donor membranes into rat brain mitochondria was investigated. Mitochondria were incubated with liposomes and subjected to density gradient centrifugation. The energized state was monitored by flow cytometry measuring the fluorescence of membrane-potential-sensitive rhodamine-123 dye. Mitochondria density decreased upon increase of the respiratory rate, as a consequence of their association with liposomes. After interaction of mitochondria with (14)C-PS containing liposomes, (14)C-PS became a substrate of PS decarboxylase, as monitored by the formation of (14)C-phosphatidylethanolamine (PE), indicating translocation of (14)C-PS to the inner membrane. The kinetics of (14)C-PE formation showed a high rate upon addition of ADP, malate and pyruvate (state 3) compared to control (state 1). In state 3, (14)C-PE formation decreased in the presence of NaN(3). Mitochondria-associated membranes (MAM) are the major site of PS synthesis. However, their role in the translocation of PS to mitochondria has not been completely elucidated. A crude mitochondrial fraction (P(2)) containing MAM, synaptosomes and myelin was prelabeled with (14)C-PS and incubated in different respiratory states. At a high respiratory rate, low-density labeled mitochondria, whose band overlaps that of synaptosomes, were obtained by centrifugation. A parallel decrease of both radioactivity and protein in MAM fraction was observed, indicating that the association of MAM and mitochondria had occurred. Synthesis and translocation of (14)C-PS in P(2) membranes were also studied by incubating P(2) with (14)C-serine. In the resting state (14)C-PS accumulated in MAM, indicating that the transfer to mitochondria was a limiting step. In state 3 both the transfer rate of (14)C-PS and its conversion to (14)C-PE increased. Respiratory mitochondrial activity modulated the association of MAM and mitochondria, triggering a mechanism that allowed the transport of PS across the outer mitochondrial membrane.

Adenosine Diphosphate↗
PubMed2000

Dexamethasone increases the incorporation of [3H]serine into phosphatidylserine and the activity of serine base exchange enzyme in mouse thymocytes: a possible relation between serine base exchange enzyme and apoptosis.

The exposure of phosphatidylserine toward the external surface of the membrane is a well-established event of programmed cell death. The possibility that an apoptotic stimulus influences the metabolism of this phospholipid could be relevant not only in relation to the previously mentioned event but also in relation to the capability of membrane phosphatidylserine to influence PKC activity. The present investigation demonstrates that treatment of mouse thymocytes with the apoptotic stimulus dexamethasone, enhances the incorporation of [3H]serine into phosphatidylserine. Cell treatment with dexamethasone also enhanced the activity of serine base exchange enzyme, assayed in thymocyte lysate. Both the effects were observed at periods of treatment preceding DNA fragmentation. The addition of unlabelled ethanolamine, together with [3H]serine to the medium containing dexamethasone-treated thymocytes lowered the radioactivity into phosphatidylserine. Serine base exchange enzyme activity was influenced by the procedure used to prepare thymocyte lysate and was lowered by the addition of fluoroaluminate, that is widely used as a G-protein activator. The increase of serine base exchange enzyme activity induced by dexamethasone treatment was observed independently by the procedure used to prepare cell lysate and by the presence or absence of fluoroaluminate.

Aluminum Compounds↗
PubMed2000

Gene structure and chromosomal assignment of mouse GITR, a member of the tumor necrosis factor/nerve growth factor receptor family.

GITR is a type I transmembrane protein that belongs to the tumor necrosis factor/nerve growth factor receptor (TNF/NGFR) family. This receptor is preferentially expressed in activated T lymphocytes and may function as signaling molecule during T-cell development. In the present study, we examined the genomic organization of the entire mouse GITR (mGITR) gene. The gene spans a 2543-bp region and consists of five exons (with a length ranging from 88 bp to 395 bp) and four introns (67 bp to 778 bp). In agreement with GITR expression in activated T cells, consensus elements for transcription factors involved in T-cell development and activation were identified in the 5' flanking region, including a consensus element for NF-kappaB. Two highly significant binding sites for MyoD and one binding site for myogenin were also found, suggesting involvement of GITR in muscle development. The mGITR gene contains 17 transcription initiation sites distributed over a 76-bp region, all used with the same frequency. We localized mGITR to the murine chromosome 4 (E region), where other 4 TNF/NGFR members localize, including m4-1BB and mOX40. These results further indicate that GITR shares several features with OX40, 4-1BB, and CD27, suggesting the existence of a new subfamily of the TNFR family, as also confirmed by the similarity of their cytoplasmic domains.

Amino Acid Sequence↗
PubMed2000

Effect of interleukin-2 on generation of natural killer cells: role of major histocompatibility complex class I in B6 and TAP-1-/- mice.

Long-term bone marrow cultures were used to investigate the effect of IL-2, a cytokine widely used in immunotherapy, on natural killer cell differentiation. Specifically, the role of MHC was evaluated by comparing normal B6 and class I-deficient TAP-1-/- mice. The number of cells generated after a 13-day culture was the same in cell cultures from TAP-1-/- or B6 mice but the relative number of natural killer cells, identified as NK-1.1+CD3- cells by flow cytometry analysis, was increased in TAP-1-/- compared to B6 cultures (74.4% and 63.9%, respectively). Addition of an anti-class I mAb determined a strong inhibition of natural killer cell generation in B6 cultures, and its effect was specific since no effect was seen in TAP-1-/- cell cultures. TAP-1-/- natural killer cells or the few natural killer cells escaping the inhibitory effect of anti-class I mAb, were less cytotoxic than total B6 natural killer cells against target cell lines of different haplotype.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Cloning and expression of a short Fas ligand: A new alternatively spliced product of the mouse Fas ligand gene.

The Fas/FasL system mediates apoptosis in several different cell types, including T lymphocytes. Fas ligand (FasL), a 40-kD type II membrane protein also expressed in activated T cells, belongs to the tumor necrosis factor ligand family. We describe a new alternative splicing of mouse FasL, named FasL short (FasLs), cloned by reverse transcriptase-polymerase chain reaction. FasLs is encoded by part of exon 1 and part of exon 4 of FasL gene. The protein encoded by FasLs mRNA has a putative initiation code at position 756 and preserves the same reading frame as FasL, resulting in a short molecule lacking the intracellular, the transmembrane, and part of the extracellular domains. RNase protection and immunoprecipitation analysis showed that FasLs is expressed in nonactivated normal spleen cells and in hybridoma T cells and that it is upregulated upon activation by anti-CD3 monoclonal antibody (MoAb). Moreover, FasLs-transfected cells expressed soluble FasLs in the supernatant and became resistant to apoptosis induced by agonist anti-Fas MoAb. Thus, FasLs, a new alternative splicing of FasL, is involved in the regulation of Fas/FasL-mediated cell death.

Alternative Splicing↗
PubMed1999

Dexamethasone-induced thymocyte apoptosis: apoptotic signal involves the sequential activation of phosphoinositide-specific phospholipase C, acidic sphingomyelinase, and caspases.

Glucocorticoid hormones (GCH) have been implicated as regulators of T-lymphocyte growth and differentiation. In particular, it has been reported that GCH can induce thymocyte apoptosis. However, the molecular mechanisms responsible for this GCH-induced death have not been clarified. In this work, the biochemical events associated with apoptosis induced by Dexamethasone (Dex), a synthetic GCH, in normal mouse thymocytes, have been analyzed. Results indicate that Dex-induced thymocyte apoptosis is attributable to an early ceramide generation caused by the activation of an acidic sphingomyelinase (aSMase). Caspase activity plays a crucial role in Dex-induced apoptosis and is downstream the aSMase activation in that inhibition of the early ceramide generation inhibits caspase activation and thymocyte death. Moreover, Dex treatment rapidly induces diacylglycerol (DAG) generation, through a protein kinase C (PKC) and G-protein-dependent phosphatidylinositol-specific phospholipase C (PI-PLC), an event which precedes and is required for aSMase activation. Indeed, PI-PLC inhibition by U73122 totally prevents Dex-induced aSMase activity, ceramide generation, and consequently, caspase activation and apoptosis. All these effects require Dex interaction with GCH receptor (GR), are countered by the GR antagonist RU486, and precede the GCH/GR-activated transcription and protein synthesis. These observations indicate that GCH activates thymocyte death through a complex signaling pathway that requires the sequential activation of different biochemical events.

Animals↗
PubMed1999

TCR kappa, a new splicing of the murine TCR zeta gene locus, is modulated by glucocorticoid treatment.

T-cell receptor (TCR) is a multichain receptor in which the TCRzeta subunit is important for membrane assembly and signal transduction. Four alternative splicings of the murine TCRzeta gene locus have been previously described. We here describe a new alternative splicing of murine TCRzeta gene, TCRkappa, cloned by RT-PCR, that is encoded by exons 1-7, a portion of exon 9 and the whole exon 10 of TCRzeta gene. The protein encoded by TCRkappa mRNA is identical to that encoded by TCReta mRNA, because the stop codon is present in the exon 9 before splicing with exon 10. RNAse protection assays carried out on total RNA from thymocytes indicate that TCRkappa mRNA is 1 half with respect to TCReta mRNA, suggesting that TCRkappa mRNA contributes to determine the TCReta protein levels. The 3' untranslated region of TCRkappa mRNA is different from that of TCReta and this might lead to different t(1/2) for each species in vivo. We also show that dexamethasone (DEX), a synthetic glucocorticoid hormone, increases the amount of TCRkappa in the hybridoma T-cell line 3DO (about 5-fold increase), as indicated by reverse transcriptase-polymerase chain reaction (RT-PCR) and RNAse protection assays. This newly described effect of DEX may constitute a further molecular mechanism that contributes to its immunomodulating activity.

Alternative Splicing↗
PubMed1999

Glucocorticoid hormone-induced modulation of gene expression and regulation of T-cell death: role of GITR and GILZ, two dexamethasone-induced genes.

Regulation of T-cell survival is a physiological process involved in determining the immune response development, and also the expansion of T-cell tumours. Glucocorticoid hormones (GCH) have been implicated as regulators of T-lymphocyte growth and differentiation. In particular, GCH which by themselves are apoptosis activators and induce T-cell death, can also counteract apoptosis activated by other stimuli, for example antigen-TCR interaction. A number of biochemical events constitute different GCH-activated death-triggering pathways and transcription activity regulation, either upstream and/or downstream in the pathways, is essential to apoptosis. Similarly, GCH-mediated inhibition of apoptosis also requires gene transcription regulation. In particular, between a number of GCH-induced genes, GITR and GILZ can inhibit apoptosis through interaction with mechanisms involved in T-cell survival regulation including the NF-kappaB transcription activity and the expression of the Fas/FasL system. These observations indicate that this GCH-activated dual effect, induction and/or inhibition of T-cell death, requires transcription regulation.

Apoptosis↗
PubMed1998

TNFalpha processing enzyme inhibitors prevent aspirin-induced TNFalpha release and protect against gastric mucosal injury in rats.

BACKGROUND: Although previous studies indicate that prevention of tumour necrosis factor alpha (TNFalpha) release protects against NSAID-induced gastric mucosal injury, intracellular pathways by which aspirin causes TNFalpha release are unknown. TNFalpha is synthesized as a precursor which is proteolytically cleaved by a specific converting enzyme, TACE, to release the mature cytokine. TACE inhibitors prevent TNFalpha release and protect against TNFalpha-mediated disease. AIM: To investigate: (i) molecular events that regulate TNFalpha secretion in response to aspirin in vivo and in vitro; (ii) whether TNFalpha secretion inhibitors prevent aspirin-induced TNFalpha release and protect against gastric mucosal damage; and (iii) whether TNFalpha exerts a direct cytotoxic effect on gastric epithelial cells. METHODS: In vitro studies were carried out on mouse macrophages and rat gastric mucosal cells. Gastric mucosal damage was induced in rats by oral administration of 300 mg/kg aspirin. TNFalpha cytotoxicity on gastric mucosal cells was examined by treating rats with lipopolysaccharide to release TNFalpha or by incubating dispersed gastric mucosal cells with increasing concentrations of TNFalpha. RESULTS: Aspirin increases intracellular calcium (Ca2+) levels and causes a time and concentration dependent increase in macrophage TNFalpha mRNA accumulation and cytokine release. Agents that cause Ca2+ mobilization with a receptor-independent mechanism, such as ionomycin and thapsigargin, stimulate TNFalpha release. Incubating the macrophages in a Ca2+ free medium inhibited TNFalpha secretion. Agents that prevent TNFalpha mRNA transcription, e.g. lisophylline, PGE2, interleukin-10 and 8-BrcAMP, or TACE inhibitors, e.g. EDTA, TAPI-2 and BB-3103, inhibit TNFalpha release and protect rats against gastric mucosal injury induced by oral administration of aspirin. TNFalpha exerts a direct cytotoxic effect on gastric epithelial cells as demonstrated by the reduced viability observed in gastric mucosal cells prepared from rats treated with lipopolysaccharide, or directly incubated with increasing concentrations of TNFalpha. CONCLUSIONS: (i) Aspirin directly stimulates TNFalpha gene transcription; (ii) TACE inhibitors protect against aspirin-induced gastric mucosal injury; and (iii) TNFalpha exerts a direct cytotoxic effect on gastric epithelial cells.

Animals↗
PubMed1998

Suppression of natural killer cell differentiation by activated T lymphocytes in long-term cultures of mouse bone marrow.

The goal of the present work was to study the regulatory role of T lymphocytes on natural killer (NK) cell generation in NK long-term bone marrow cultures (LTBMCs), an established mouse long-term bone marrow (BM) culture system used for the study of NK cell differentiation from precursors. Activation of the few T cells present in NK-LTBMCs by addition of anti-CD3 monoclonal antibody (mAb) together with interleukin (IL)-2 inhibited the generation of NK cells. Coculture with NK-LTBMCs of a pure population of preactivated BM T cells completely inhibited NK cell development even when the T cells were separated from the NK-LTBMCs by transwells. Depletion of IL-2 by activated T cells was not the mechanism of the negative regulation because anti-CD3 mAb added to the cultures inhibited the generation of NK cells even in the presence of 10-fold higher concentrations of exogenous IL-2 than that used in controls. Medium from cultures in which suppression had occurred was also suppressive, suggesting that one or more soluble factors released in the medium was responsible. That this effect was exerted on NK cell development from precursors was indicated by the finding that T cell-conditioned medium stimulated proliferation of mature NK cells. In our experimental conditions, monoclonal antibodies to IL-10, IL-13, transforming growth factor-beta, and tumor necrosis factor receptor failed to reverse the inhibitory effect.

Animals↗
PubMed1998

Functional expression of Fas on mouse bone marrow stromal cells: upregulation by tumor necrosis factor-alpha and interferon-gamma.

In this study we describe the expression and function of Fas in mouse bone marrow (BM) stromal cells (SCs) and cell lines derived from long-term BM cultures. Flow cytometry analysis showed that Fas was expressed on adherent cells from freshly isolated BM and on all cloned SC lines tested. The SC line ME-25 was Fas+ but negative for FasL as detected by reverse transcriptase-polymerase chain reaction. Furthermore, ME-25 was CD44+, VCAM-1+, Mac-3-, Gr-1-, and type IV collagen-. ME-25 treatment with interferon-gamma or tumor necrosis factor-alpha significantly induced upregulation of Fas expression as detected by both flow cytometry and Western blot immunoassay. The same treatment with interleukin (IL)-1, IL-2, or IL-13 had no effect. Functional studies demonstrated that Fas induced a strong increase in apoptosis when engaged with an anti-Fas monoclonal antibody (MoAb). Activated BM T cells induced Fas-dependent cytotoxicity of ME-25 insofar as blocking anti-FasL MoAb inhibited the killing of ME-25 induced by activated BM T cells. These data suggest a possible involvement of Fas-expressing SCs in negative regulatory functions in the BM and provide a starting point for further studies on the role of Fas+ SCs.

Animals↗