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Biomedical subjects

M Vitale

Publications and source records attributed to M Vitale.

At least 37 records · Page 2Linked to original sources
PubMed2001

The G972R variant of the insulin receptor substrate-1 (IRS-1) gene, body fat distribution and insulin-resistance.

AIMS/HYPOTHESIS: Insulin resistance is recognised as the core factor in the pathogenesis of Type II (non-insulin-dependent) diabetes mellitus, hypertension and atherosclerosis. Several studies indicate the possible role of mutations of the insulin receptor substrate-1 (IRS-1) gene in the pathogenesis of insulin-resistance and suggest a possible interaction between the IRS-1 gene and obesity, either by an effect on the development of obesity or by causing or aggravating the obesity-associated insulin resistance. Therefore, the prevalence of the G972R mutation of the IRS-1 gene was compared in 157 non-diabetic obese subjects (BMI > 30 m/kg2) and in 157 lean subjects (BMI < 28 m/kg2). By investigating the relation between this IRS-1 mutation, measures of obesity and metabolic parameters, we explored the possible influence of this mutation on body fat distribution and insulin resistance. METHODS: The G972R mutation was detected by PCR amplification and BstN-1 restriction enzyme digestion. Data were analysed by univariate and multivariate analysis. RESULTS: The G972R allele was significantly more frequent in obese subjects than in lean subjects (p < 0.002); however, no difference was found between centrally and peripherally obese subjects. Obese G972R carriers had significantly higher BMI (p < 0.001), fasting insulin (p < 0.001), triglycerides (p < 0.03) and HOMAIR (p < 0.001) than obese non-carriers. No differences were observed between G972R carriers and non-carriers among control subjects. Multivariate analysis confirmed that the IRS-1 G972R mutation was significantly and independently associated with reduced insulin sensitivity (p < 0.009) in the obese group. CONCLUSION/INTERPRETATION: The G972R mutation of the IRS-1 gene associates with obesity, but not with fat distribution, in this Italian cohort, and within the obese subjects this IRS-1 variant strongly associates with metabolic parameters suggesting greater insulin-resistance. These findings indicate a possible interaction between the IRS-1 variant and obesity in worsening insulin sensitivity.

Adipose Tissue↗
PubMed2001

Human natural killer cell receptors and co-receptors.

In the absence of sufficient signaling by their HLA class I-specific inhibitory receptors, human natural killer (NK) cells become activated and display potent cytotoxicity against cells that are either HLA class I negative or deficient. This indicates that the NK receptors responsible for the induction of cytotoxicity recognize ligands on target cells different from HLA class I molecules. On this basis, the process of NK-cell triggering can be considered as a mainly non-MHC-restricted mechanism. The recent identification of a group of NK-specific triggering surface molecules has allowed a first series of pioneering studies on the functional/molecular characteristics of such receptors. The first three members of a receptor family that has been termed natural cytotoxicity receptors (NCR) are represented by NKp46, NKp44 and NKp30. These receptors are strictly confined to NK cells, and their engagement induces a strong activation of NK-mediated cytolysis. A direct correlation exists between the surface density of NCR and the ability of NK cells to kill various target cells. Importantly, mAb-mediated blocking of these receptors has been shown to suppress cytotoxicity against most NK-susceptible target cells. However, the process of NK-cell triggering during target cell lysis may also depend on the concerted action of NCR and other triggering receptors, such as NKG2D, or surface molecules, including 2B4 and NKp80, that appear to function as co-receptors rather than as true receptors. Notably, a dysfunction of 2B4 has been associated with a severe form of immunodeficiency termed X-linked lymphoproliferative disease. Future studies will clarify whether also the altered expression and/or function of other NK-triggering molecules may represent a possible cause of immunological disorders.

Cytotoxicity, Immunologic↗
PubMed2001

Interleukin 2 and interleukin 15 differentially predispose natural killer cells to apoptosis mediated by endothelial and tumour cells.

Human natural killer (NK) cells constitutively express the beta- and gamma-chains of the interleukin 2 (IL-2)/IL-15 receptor, and both IL-2 and IL-15 are able to activate NK cell proliferation and cytotoxicity. When IL-2-primed human NK cells are exposed to sensitive targets (i.e. K562) they undergo apoptosis mediated by the beta(2)-integrin CD18. Here, we demonstrate that: (i) endothelial cells, similar to K562 tumour target cells, induce apoptosis of IL-2-primed NK cells; (ii) endothelial- and K562 cell-induced apoptosis is significantly lower in IL-15 than in IL-2-stimulated NK cells; (iii) a critical role in the apoptosis of IL-2-primed NK cells is played by the alpha-chain of the IL-2 receptor. Our data show for the first time that IL-2-activated NK cells can die by apoptosis upon contact with the vascular endothelium, which is a necessary step for their extravasation, with a direct pathophysiological relevance on the strategy of adoptive immunotherapy of cancer. On the other hand, IL-15, although generating a similar level of activation of NK cells, largely prevents their apoptotic fate. Therefore, IL-15 produced early in the immune response, when T cells are not yet activated, generates lymphokine-activated killer cells that are efficient killers relatively protected from apoptosis. Once activated, T cells produce IL-2 that overcomes the effect of IL-15 on NK cells, paving the way for their death by apoptosis.

Apoptosis↗
PubMed2001

Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis.

Natural killer cells can discriminate between normal cells and cells that do not express adequate amounts of major histocompatibility complex (MHC) class I molecules. The discovery, both in mouse and in human, of MHC-specific inhibitory receptors clarified the molecular basis of this important NK cell function. However, the triggering receptors responsible for positive NK cell stimulation remained elusive until recently. Some of these receptors have now been identified in humans, thus shedding some light on the molecular mechanisms involved in NK cell activation during the process of natural cytotoxicity. Three novel, NK-specific, triggering surface molecules (NKp46, NKp30, and NKp44) have been identified. They represent the first members of a novel emerging group of receptors collectively termed natural cytotoxicity receptors (NCR). Monoclonal antibodies (mAbs) to NCR block to differing extents the NK-mediated lysis of various tumors. Moreover, lysis of certain tumors can be virtually abrogated by the simultaneous masking of the three NCRs. There is a coordinated surface expression of the three NCRs, their surface density varying in different individuals and also in the NK cells isolated from a given individual. A direct correlation exists between the surface density of NCR and the ability of NK cells to kill various tumors. NKp46 is the only NCR involved in human NK-mediated killing of murine target cells. Accordingly, a homologue of NKp46 has been detected in mouse. Molecular cloning of NCR revealed novel members of the Ig superfamily displaying a low degree of similarity to each other and to known human molecules. NCRs are coupled to different signal transducing adaptor proteins, including CD3 zeta, Fc epsilon RI gamma, and KARAP/DAP12. Another triggering NK receptor is NKG2D. It appears to play either a complementary or a synergistic role with NCRs. Thus, the triggering of NK cells in the process of tumor cell lysis may often depend on the concerted action of NCR and NKG2D. In some instances, however, it may uniquely depend upon the activity of NCR or NKG2D only. Strict NKG2D-dependency can be appreciated using clones that, in spite of their NCR(dull) phenotype, efficiently lyse certain epithelial tumors or leukemic cell lines. Other triggering surface molecules including 2B4 and the novel NKp80 appear to function as coreceptors rather than as true receptors. Indeed, they can induce natural cytotoxicity only when co-engaged with a triggering receptor. While an altered expression or function of NCR or NKG2D is being explored as a possible cause of immunological disorders, 2B4 dysfunction has already been associated with a severe form of immunodeficiency. Indeed, in patients with the X-linked lymphoproliferative disease, the inability to control Epstein-Barr virus infections may be consequent to a major dysfunction of 2B4 that exerts inhibitory instead of activating functions.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Desethylamiodarone antagonizes the effect of thyroid hormone at the molecular level.

OBJECTIVE: To evaluate the molecular mechanisms of the inhibitory effects of amiodarone and its active metabolite, desethylamiodarone (DEA) on thyroid hormone action. MATERIALS AND METHODS: The reporter construct ME-TRE-TK-CAT or TSHbeta-TRE-TK-CAT, containing the nucleotide sequence of the thyroid hormone response element (TRE) of either malic enzyme (ME) or TSHbeta genes, thymidine kinase (TK) and chloramphenicol acetyltransferase (CAT) was transiently transfected with RSV-TRbeta into NIH3T3 cells. Gel mobility shift assay (EMSA) was performed using labelled synthetic oligonucleotides containing the ME-TRE and in vitro translated thyroid hormone receptor (TR)beta. RESULTS: Addition of 1 micromol/l T4 or T3 to the culture medium increased the basal level of ME-TRE-TK-CAT by 4.5- and 12.5-fold respectively. Amiodarone or DEA (1 micromol/l) increased CAT activity by 1.4- and 3.4-fold respectively. Combination of DEA with T4 or T3 increased CAT activity by 9.4- and 18.9-fold respectively. These data suggested that DEA, but not amiodarone, had a synergistic effect with thyroid hormone on ME-TRE, rather than the postulated inhibitory action; we supposed that this was due to overexpression of the transfected TR into the cells. When the amount of RSV-TRbeta was reduced until it was present in a limited amount, allowing competition between thyroid hormone and the drug, addition of 1 micromol/l DEA decreased the T3-dependent expression of the reporter gene by 50%. The inhibitory effect of DEA was partially due to a reduced binding of TR to ME-TRE, as assessed by EMSA. DEA activated the TR-dependent down-regulation by the negative TSH-TRE, although at low level (35% of the down-regulation produced by T3), whereas amiodarone was ineffective. Addition of 1 micromol/l DEA to T3-containing medium reduced the T3-TR-mediated down-regulation of TSH-TRE to 55%. CONCLUSIONS: Our results demonstrate that DEA, but not amiodarone, exerts a direct, although weak, effect on genes that are regulated by thyroid hormone. High concentrations of DEA antagonize the action of T3 at the molecular level, interacting with TR and reducing its binding to TREs. This effect may contribute to the hypothyroid-like effect observed in peripheral tissues of patients receiving amiodarone treatment.

3T3 Cells↗
PubMed2001

Lovastatin-induced apoptosis in thyroid cells: involvement of cytochrome c and lamin B.

OBJECTIVE: The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, lovastatin, induces apoptosis in the thyroid cell line TAD-2 and in proliferating normal human thyroid cells in culture, through a p53-independent mechanism involving caspase-3-like proteases. The combination of lovastatin with other anti-neoplastic drugs potentiates chemotherapy of tumors. This drug has been suggested for the chemotherapy of tumors and is potentially useful in the treatment of thyroid proliferative diseases. Based on this premise, we analyzed in more detail the role of some molecular effectors and the role of the caspase family proteases in the lovastatin-induced apoptotic pathway in TAD-2 cells. METHODS: TAD-2 cells were treated with lovastatin to induce apoptosis, and expression of p53, Bc1-2, Bcl-XL and Bax was analyzed by Western blot. Caspase activation was evaluated by the assay of enzymatic activity with chromogenic peptides and Western blot. Nuclear, cytosolic and mitochondrial fractions were prepared by differential centrifugation and the presence of cytochrome c and lamin B was evaluated by Western blot. RESULTS: p53, Bc1-2, Bcl-XL and Bax protein expression were unchanged during apoptosis. Cytochrome c was released from mitochondria into the cytosol, a pivotal event in the activation of caspase-3. Caspase-3 and -6 but not caspase-2 were activated, and proteolysis of PARP and lamin B, a caspase-6 substrate located in the inner nuclear membrane, was demonstrated by Western blot. The nuclear localization of lamin B was also inhibited by lovastatin. CONCLUSIONS: These data demonstrate that, in TAD-2 thyroid cells, lovastatin induces lamin B proteolysis and inhibits its nuclear localization and induces cytochrome c release from mitochondria into the cytosol.

Antibodies, Monoclonal↗
PubMed2001

[Heart failure and nutritional status in hemodialysis].

Left ventricular function is impaired in long-term maintenance dialysis as a consequence of several inter-related conditions. Aim of the study was to assess if different degree of malnutrition can affect diastolic and systolic function of dialysis patients. Bio-electrical whole-body multifrequency impedance analysis (BIA) allows a dynamic assessment of body composition according to a multi-compartment model. This allows an assessment of lean (FFM) and fat mass (FAT), metabolically active cell mass (BCM) and a BIA Nutritional Index (NI). We studied 73 patients (53 M, 20 F), aged 54.41 +/- 3.1 yrs, on maintenance bicarbonate dialysis since 57.3 +/- 61.8 mths, by BIA, performed before and after a dialysis. I-PTH and serum albumin are correlated (r = -0.55, p < 0.001); the same trend is shown by I-PTH vs fat free mass (r = -0.47, p < 0.001) and vs NI (r = -0.51, p < 0.001). Ejection fraction (EF) is correlated with age (r = -0.50, p < 0.001) and with I-PTH (r = -0.59, p < 0.001). Moreover, albumin and EF are closely related (r = 0.36, p < 0.01), as well as EF vs FFM (r = 0.47, p < 0.001), and EF vs NI (r = 0.37, p < 0.01). A/E ratio, assumed as index of left ventricular diastolic function, shows a correlation vs albumin (r = -0.33, p < 0.01), BCM (r = -0.34, p < 0.01) and NI (r = -0.40, p < 0.01), but not with I-PTH. Malnutrition, defined both by BIA measurements and lower serum albumin, could exert unfavourable effects on left ventricular systolic and diastolic function of patients on long-term haemodialysis, even independently by hyperparathyroidism.

Female↗
PubMed2000

A role for nuclear phospholipase Cbeta 1 in cell cycle control.

Phosphoinositide signaling resides in the nucleus, and among the enzymes of the cycle, phospholipase C (PLC) appears as the key element both in Saccharomyces cerevisiae and in mammalian cells. The yeast PLC pathway produces multiple inositol polyphosphates that modulate distinct nuclear processes. The mammalian PLCbeta(1), which localizes in the nucleus, is activated in insulin-like growth factor 1-mediated mitogenesis and undergoes down-regulation during murine erythroleukemia differentiation. PLCbeta(1) exists as two polypeptides of 150 and 140 kDa generated from a single gene by alternative RNA splicing, both of them containing in the COOH-terminal tail a cluster of lysine residues responsible for nuclear localization. These clues prompted us to try to establish the critical nuclear target(s) of PLCbeta(1) subtypes in the control of cell cycle progression. The results reveal that the two subtypes of PLCbeta(1) that localize in the nucleus induce cell cycle progression in Friend erythroleukemia cells. In fact when they are overexpressed in the nucleus, cyclin D3, along with its kinase (cdk4) but not cyclin E is overexpressed even though cells are serum-starved. As a consequence of this enforced expression, retinoblastoma protein is phosphorylated and E2F-1 transcription factor is activated as well. On the whole the results reveal a direct effect of nuclear PLCbeta(1) signaling in G(1) progression by means of a specific target, i.e. cyclin D3/cdk4.

Animals↗
PubMed2000

Infection of CD34(+) hematopoietic progenitor cells by human herpesvirus 7 (HHV-7).

To investigate the tropism of the T-lymphotropic human herpesvirus 7 (HHV-7) for hematopoietic progenitors, cord blood CD34(+) cells were inoculated in vitro with HHV-7 and then induced to differentiate along the granulocytic and erythroid lineages by the addition of appropriate cytokine cocktails. In semisolid assays, HHV-7 modestly affected the growth of committed (granulocytic/macrophagic and erythroid) progenitors, whereas it significantly decreased the number of pluripotent (granulocytic/erythroid/ monocytic/megakaryocytic) progenitors. Such inhibitory effect was completely abrogated by incubating HHV-7 inoculum with anti-HHV-7 neutralizing serum. In liquid cultures, HHV-7 hastened maturation along the myeloid but not the erythroid lineage, as demonstrated by the up-regulation of CD33 early myeloid antigen at day 7 of culture, and of CD15 and CD14 antigens at day 15. Moreover, HHV-7 messenger RNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in cells maturating along both the myeloid and the erythroid lineages. To evaluate the relevance of these in vitro findings, the presence of HHV-7 was investigated in bone marrow (BM) unfractionated mononuclear cells (MCs) as well as in purified CD34(+) and CD34(-) cell subsets, obtained from 14 normal adult donors. HHV-7 DNA was detected by DNA-PCR in 4 of 7 BMMC samples, and it was found to be associated with both the CD34(-) (2 of 7) and the CD34(+ )(1 of 7) fractions. These data indicate that HHV-7 infects BM cells in vivo and shows the ability to affect the survival/differentiation of CD34(+) hematopoietic progenitors in vitro by inhibiting more ancestral progenitors and perturbing the maturation of myeloid cells.

Adult↗
PubMed2000

TNF-related apoptosis-inducing ligand (TRAIL) as a negative regulator of normal human erythropoiesis.

The impact of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on normal hematopoietic development was investigated using adult peripheral blood CD34(+) hematopoietic progenitor cells, induced to differentiate along the erythroid, megakaryocytic, granulocytic, and monocytic lineages by the addition of specific cytokine cocktails. TRAIL selectively reduced the number of erythroblasts, showing intermediate levels of glycophorin A (glycophorin A(interm)) surface expression, which appeared in liquid cultures supplemented with stem cell factor + interleukin 3 + erythropoietin at days 7-10. However, neither immature (day 4) glycophorin A(dim) erythroid cells nor mature (day 14) glycophorin A(bright) erythroblasts were sensitive to TRAIL-mediated apoptosis. Moreover, pre-exposure to TRAIL significantly decreased the number and size of erythroid colonies in semisolid assays. These adverse effects of TRAIL were selective for erythropoiesis, as TRAIL did not significantly influence the survival of cells differentiating along the megakaryocytic, granulocytic, or monocytic lineages. Furthermore, TRAIL was detected by Western blot analysis in lysates obtained from normal bone marrow mononuclear cells. These findings indicate that TRAIL acts in a lineage- and stage of differentiation-specific manner, as a negative regulator of normal erythropoiesis. (Blood. 2000;95:3716-3724)

Adult↗
PubMed2000

A simple method for non-radioactive PCR-SSCP using MDE gel solution and a midi gel format: application for the detection of variants in the GLUT1 and CTLA-4 genes.

The need to identify disease-causing mutations and DNA polymorphisms has increased with the continuing identification of new candidate genes. PCR single-strand conformation polymorphism (PCR-SSCP) is one of the techniques most widely used to identify a mutant sequence or a polymorphism in a known gene. However, the original SSCP protocols using the incorporation of radioactive label and polyacrylamide gel electrophoresis on sequencing gels for detection were labour intensive and time-consuming. Here we describe a simple SSCP protocol using MDE gel solution and a midi gel format to detect SSCP variations in the glucose transporter gene GLUT1, that we have previously analysed with the standard radioactive SSCP protocol, and we have also tested this method on the previously described point mutation (A/G transition in exon 1) of the CTLA-4 (cytotoxic T lymphocyte associated-4) gene. All known variants were detected. Based on the results, this technique appears to be simple, with no use of radioactive labels and with easy handling of the gel. Furthermore, it needs little optimisation, is relatively rapid and highly sensitive. We propose this method for the first screening for candidate gene variants.

Abatacept↗
PubMed2000

Differential production of IFN-gamma, analyzed at the single-cell level, by specific subsets of human NK and T cells from healthy and HIV(+) subjects.

BACKGROUND: Interferon gamma is a cytokine that plays a central role in immunity, and is physiologically secreted by T and NK cells under appropriate stimuli during the immune response. By means of flow cytometry, we performed a single cell analysis of interferon gamma producing NK cells and their surface phenotype in normal and HIV(+) individuals that show several defects of cytokine production and cellular immunity. METHODS: PBMC or purified NK cells were stimulated for 1-12 h with PMA/ionomycin in the presence of monensin, subsequently stained for surface CD56 and CD3 or CD8, and for intracytoplasmic IFN-gamma, and analysed by flow cytometry. RESULTS: Our results show that CD56(+) NK cells are more efficient interferon gamma producers than T cells. Moreover, within the CD56(+) NK cell population, those that co-express low density CD8 are the best producers. Finally, we show that NK cells during HIV infection are more massively recruited to interferon gamma production than those from normal subjects. CONCLUSIONS: Both in the normal and HIV(+) subjects, a higher percentage of NK cells than T cells can produce IFN-gamma although differences can be identified within the NK cells subset in terms of IFN-gamma production. The production of IFN-gamma is fully achievable in the HIV(+) subjects, which is consistent with their elevated plasmatic levels of the cytokine. The possibility that NK cells that produce interferon gamma could represent a functionally distinct population committed to the production of this cytokine, is discussed.

Antigens, CD↗
PubMed2000

HIV-1 Tat-mediated inhibition of the tyrosine hydroxylase gene expression in dopaminergic neuronal cells.

Treatment of dopaminergic rat PC12 cells with human immunodeficiency virus, type 1 (HIV-1) Tat protein or tat cDNA inhibited the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme for the dopamine biosynthetic pathway, as well as the production and release of dopamine into the culture medium. Moreover, the Tat addition to PC12 cells up-regulated the expression of the inducible cAMP early repressor (ICER), a specific member of the cAMP-responsive element modulator transcription factor family, in a cAMP-dependent manner. In turn, ICER overexpression abrogated the transcription activity of the TH promoter in PC12 cells, strongly suggesting ICER involvement in Tat-mediated inhibition of TH gene expression. In vivo injection of synthetic HIV-1 Tat protein into the striatum of healthy rats induced a subclinical Parkinson's-like disease that became manifested only when the animals were treated with amphetamine. As early as one week postinjection, the histochemical examination of the rat substantia nigra showed a reduced staining of neurons expressing TH followed by a loss of TH(+) neurons at later time points. As Tat protein can be locally released into the central nervous system by HIV-1-infected microglial cells, our findings may contribute to the explanation of the pathogenesis of the motorial abnormalities often reported in HIV-1 seropositive individuals.

Animals↗
PubMed2000

Ultrastructural characterization of maturation, platelet release, and senescence of human cultured megakaryocytes.

The aim of this study was to evaluate the ultrastructural features of human megakaryocytes cultured in vitro. For this purpose, pluripotent CD34(+) (cluster of differentiation 34) hematopoietic progenitor cells, obtained from the peripheral blood of healthy adult donors, were differentiated along the megakaryocytic lineage in liquid cultures by the addition of the megakaryocyte-specific growth factor thrombopoietin (TPO, 100 ng/ml). After only 6-8 days, virtually all of the CD34-derived cells expressed the early megakaryocytic CD61 antigen, while, after 15-16 days, most cells also expressed the late megakaryocytic CD42a antigen. Ultrastructural analysis of cells obtained after 7 days of culture showed aspects typical of developing megakaryocytes (MK), such as formation of platelet territories and cytoplasmic fragmentation. At later (15-16 day) culture times, two distinct cell populations were observed: fully developed megakaryocytes releasing platelets into the culture medium and senescent megakaryocytes, characterized by morphological features of apoptosis. Analysis of DNA fragmentation in these cells revealed that apoptosis in megakaryocytes occurred in the absence of the internucleosomic cleavage, which is characteristic of most, but not all, types of apoptosis in cells of hematopoietic origin. On the other hand, flow cytometry of the DNA content of senescent megakaryocytes showed a subdiploid peak that was likely due to a loss of micronuclei during processing.

Aging↗
PubMed2000

A stability-indicating HPLC method to determine cyproterone acetate in tablet formulations.

A simple and accurate liquid chromatographic method was developed to estimate cyproterone acetate (CA) in pharmaceuticals. The drug was chromatographed on a reversed-phase C18 column. Eluents were monitored at a wavelength of 254 nm utilizing a mixture (60:40) of acetonitrile and water. Solution concentrations were measured on a weight basis to avoid the use of an internal standard. The method was statistically validated for linearity, accuracy, precision, and selectivity. Due to its simplicity and accuracy, we believe that the method can be used for routine quality control analysis. No specific sample preparation is required except for the use of a column guard and a suitable prefilter attached to the syringe.

Androgen Antagonists↗