Biomedical subjects

M Vitale

Publications and source records attributed to M Vitale.

At least 55 records · Page 3Linked to original sources

PubMed2000

Iodide excess induces apoptosis in thyroid cells through a p53-independent mechanism involving oxidative stress.

Thyroid toxicity of iodide excess has been demonstrated in animals fed with an iodide-rich diet; in vitro iodide is cytotoxic, inhibits cell growth, and induces morphological changes in thyroid cells of some species. In this study, we investigated the effect of iodide excess in an immortalized thyroid cell line (TAD-2) in primary cultures of human thyroid cells and in cells of nonthyroid origin. Iodide displayed a dose-dependent cytotoxicity in both TAD-2 and primary thyroid cells, although at different concentrations, whereas it had no effect on cells of nonthyroid origin. Thyroid cells treated with iodide excess underwent apoptosis, as evidenced by morphological changes, plasma membrane phosphatidylserine exposure, and DNA fragmentation. Apoptosis was unaffected by protein synthesis inhibition, whereas inhibition of peroxidase enzymatic activity by propylthiouracil completely blocked iodide cytotoxicity. During KI treatment, reactive oxygen species were produced, and lipid peroxide levels increased markedly. Inhibition of endogenous p53 activity did not affect the sensitivity of TAD-2 cells to iodide, and Western blot analysis demonstrated that p53, Bcl-2, Bcl-XL, and Bax protein expression did not change when cells were treated with iodide. These data indicate that excess molecular iodide, generated by oxidation of ionic iodine by endogenous peroxidases, induces apoptosis in thyroid cells through a mechanism involving generation of free radicals. This type of apoptosis is p53 independent, does not require protein synthesis, and is not induced by modulation of Bcl-2, Bcl-XL, or Bax protein expression.

Annexin A5↗

PubMed2000

Amiodarone induces cytochrome c release and apoptosis through an iodine-independent mechanism.

Amiodarone (AMD) is one of the most effective antiarrhythmic drugs available. However, its use is often limited by side-effects, mainly hypo- or hyperthyroidism. As AMD displays direct toxic effect on different cell types, we investigated the cytotoxic effect of AMD and its main metabolite, desethylamiodarone (DEA), in thyroid (TAD-2) and nonthyroid (HeLa) cell lines. Both AMD and DEA displayed a dose-dependent toxicity in TAD-2 and HeLa cells, although DEA was more effective. Both TAD-2 and HeLa cells underwent apoptosis, as evidenced by plasma membrane phosphatidylserine exposure and DNA fragmentation. Inhibition of protein synthesis with cycloheximide and inhibition of endogenous peroxidase activity with propylthiouracil did not affect this AMD- and DEA-induced apoptosis in TAD-2 cells. Western blot analysis did not display variations in the expression of p53, Bcl-2, Bcl-XL, and Bax proteins during the treatment with AMD and DEA. Generation of reactive oxygen species, investigated by flow cytometry with dichlorofluorescein diacetate, did not show the production of free radicals during drug treatment. Furthermore, Western blot analysis of cytosolic and mitochondrial fractions prepared from AMD-treated cells demonstrated that AMD induces the release of cytochrome c into the cytosol from the mitochondria. These data indicate that AMD induces cytochrome c release from mitochondria, triggering apoptosis through an iodine-independent mechanism, and that this process is not mediated by modulation of p53, Bcl-2, Bcl-XL, or Bax protein expression and does not involve the generation of free radicals.

Amiodarone↗

PubMed2000

Serum withdrawal-induced apoptosis in thyroid cells is caused by loss of fibronectin-integrin interaction.

In some cell types, including a fetal thyroid cell line, denial of adhesion to extracellular matrix induces a type of apoptosis called anoikis. Serum withdrawal in dog and transformed rat thyroid cells also induces programmed cell death. Because serum can stimulate cells to produce some components of the extracellular matrix, it was of interest to determine the role of the matrix in the apoptosis induced by serum withdrawal in normal human thyroid cells in primary culture. The present report demonstrates that thyroid cells selectively produce and deposit insoluble fibronectin (FN) only when stimulated by serum. Adhesion in the presence of serum is dependent upon integrin-FN interaction. Serum withdrawal determines a degradation of the insoluble FN deposited and a detachment of the cells from the plates. In these conditions, cells undergo anoikis, demonstrated by DNA fragmentation and annexin V staining. Apoptosis was prevented by exogenous FN immobilized onto the plates. These results indicate that serum withdrawal induces apoptosis in human thyroid cells, determining FN degradation and loss of cell-matrix adhesion.

Annexin A5↗

PubMed2000

[Laparoscopic appendectomy. Retrospective analysis of our experience compared with the literature].

BACKGROUND: The benefits of laparoscopic appendectomy (LA) remain controversial. This study reports a critical examination of our experience in a peripheral hospital. METHODS: A total of 128 appendectomies have been performed since January 1996, of which 63 (49%) were laparoscopic. The patients included 52 (82.5%) females and 11 (17.5%) males with a mean age of 20.8 years (range 11-46). Emergency surgery was required in 6 cases (9.5%) and was elective in 57 (90.5%). Appendectomy was performed during another operation in 3 cases (laparoscopic cholecystectomy). RESULTS: Mortality was nil. Morbidity was 1.7% (1 case). The index of conversion was 0%. Only one major complication occurred. This took the form of perforation of an ileal loop following accidental lesion during adhesiolysis. The intraoperative diagnosis was not confirmed in 12 (19%) cases: 9 ovarian cysts, 1 terminal ilieitis and 2 cases of acute salpingitis. In 10 cases (15%) surgery was associated with adhesiolysis, and in 6 cases (9.3%) the appendix was retrocecal. Mean operating time was 42 min (range 18-105 min). The mean hospitalisation was 3.3 days. CONCLUSIONS: The authors emphasise the numerous advantages of laparoscopic techniques in their experience, including excellent cosmetic results, reduced PO pain, rapid functional recovery, lower incidence of adhesion, wound infection and laparocele, and more cost-effective when mechanical staplers are not used. Owing to the ability to explore the entire abdominal cavity, the main advantage of this technique consisted in a correct differential diagnostic balance, especially in young women of child-bearing age, between appendectomy and pathologies of the uterus and adnexa.

Adolescent↗

PubMed2000

[Hyperthyroidism, therapy with erythropoietin, malnutrition and systolic function in hemodialysis: echocardiography study].

Secondary hyperparathyroidism is a frequent condition of dialysis patients. Endocrine derangements, with disturbance of calcium metabolism are complex, involving bone, heart (left ventricular hypertrophy-dilatation), bone marrow (anemia and erythropoietin resistance), muscle (increase of body fat mass) and insulin resistance. Aim of the study was to assess how these conditions are inter-correlated in the same patients. 45 patients (m 20, f 25; years 61.8 +/- 11.6) in maintenance bicarbonate three-weekly hemodialysis since > 3 years were studied. Cardiac function was assessed by echocardiography (EF%: left ventricular ejection fraction), which showed an inverse correlation both with parathormone (iPTH vs EF%: r = -0.64; p < 0.001) and with erythropoietin (rHu-EPO vs EF%: r = -0.62; p < 0.001). This suggests the possibility of a multi-endocrine resistance in dialysis patients with chronic renal failure, secondary to the degree of malnutrition. Lower lean mass is correlated with hyperparathyroidism (iPTH vs fat mass%: r = 0.37; p < 0.01), with lower left ventricular systolic function (EF% vs fat mass%: r = -0.41; p < 0.005) and with rHu-EPO resistance. Moreover, patients with higher iPTH show a hypercatabolic disposition, assessed as protein catabolic rate (PCR/kg vs iPTH r = 0.54; p < 0.001). This pattern can be a consequence of chronic renal failure, but bio-compatibility of materials can be involved as well.

Body Mass Index↗

PubMed2000

The role of flow cytometry in the study of cell growth in the rat anterior pituitary gland.

Flow cytometry is a suitable technique for studying in vivo and in vitro the cell cycle kinetics of different animal and human tissues, both in normal and tumoral conditions. The rat anterior pituitary gland is a model to investigate cell growth and replication of differentiated, neuroendocrine cells, and we report current evidence on its cell cycle kinetics as well as on the role played by flow cytometry in this type of study. The proliferation potential of normal anterior pituitary cells is related to a number of different conditions, including heterogeneity of cell types, age and sex of donors, and circadian influences. In addition, the trend of cell proliferation in both in vivo and in vitro studies is similar, suggesting that cultured anterior pituitary elements may, at least in part, retain growth features analogous to those of the intact gland. Sorting of selective cell types and analysis of the relation between proliferating anterior pituitary cells and the light-dark cycle have shown that flow cytometry may be useful to investigate the replication process of the gland. By using a combination of flow cytometry, light microscopic immunocytochemistry and morphometry, we have reported a peculiar trend of proliferation in primary monolayer cultures of rat anterior pituitary gland, characterized by a non-linear reduction in their proliferation rate with advancing age, primarily dependent on a reduced transition of cells from the G0/G1- to the early S-phase pool. These studies indicate that flow cytometry offers insights into cell cycle check points of anterior pituitary cells, and suggest that it might be applied to the study of growth of selective pituitary elements, both in normal and tumoral conditions.

Animals↗

PubMed1999

Engagement of p75/AIRM1 or CD33 inhibits the proliferation of normal or leukemic myeloid cells.

P75/AIRM1 is a recently identified surface molecule that belongs to the sialoadhesin family and displays homology with the myeloid cell antigen CD33. In lymphoid cells, p75/AIRM1 is confined to natural killer cells and mediates inhibition of their cytolytic activity. In this study, we show that p75/AIRM1 is also expressed by cells of the myelomonocytic cell lineage, in which it appears at a later stage as compared with CD33. In vitro proliferation and differentiation of cord blood-derived CD34(+) cells (induced by stem cell factor and granulocyte-macrophage colony-stimulating factor) were consistently inhibited by the addition of anti-p75/AIRM1 mAb. Engagement of CD33 led to inhibition in some experiments. A sharp decrease of cell proliferation/survival was detected in all three p75/AIRM1+ chronic myeloid leukemias analyzed when cultured in the presence of either anti-p75/AIRM1 or anti-CD33 mAbs. Thus, the present study suggests that p75/AIRM1 and CD33 may play a regulatory role in normal myelopoiesis and may be viewed as suitable target molecules to counteract the proliferation/survival of chronic myeloid leukemias.

Antibodies, Monoclonal↗

PubMed1999

Dietary and hypothyroid hypercholesterolemia induces hepatic apolipoprotein E expression in the rat: direct role of cholesterol.

Apolipoprotein E (apo E) exerts a protective effect against atherosclerosis, related to its role in intracellular cholesterol removal and remnants clearance. In this study we investigated the effect of dietary and hypothyroid hypercholesterolemia, induced respectively by a high cholesterol diet and by propylthiouracil, on hepatic apo E expression in Wistar male rats. The Northern and Western blot analysis of hepatic mRNA and protein levels showed a 2-3-fold increase of apo E in hypercholesterolemic rats compared to controls. The incubation of FAO rat hepatoma cells with 25-OH cholesterol and mevalonate led to a three-fold increase of apo E mRNA, demonstrating a direct role of cholesterol on apo E expression. This effect was completely abolished by elevating intracellular cAMP levels with forskolin. Immunoblot and immunofluorescence analysis revealed that 25-OH cholesterol/mevalonate strongly increased also apo E protein synthesis and secretion in FAO cells. Our data demonstrate that hypercholesterolemia, apart of the cause (diet or hypothyroidism) induces liver apo E expression in the rat and that this effect can be directly related, via cAMP, to cholesterol.

Animals↗

PubMed1999

Apoptosis induced by denied adhesion to extracellular matrix (anoikis) in thyroid epithelial cells is p53 dependent but fails to correlate with modulation of p53 expression.

In normal epithelial cells, impaired cell-matrix contact leads to induction of programmed cell death, a process that has been termed 'anoikis'. We investigated the role of p53 and other apoptotic proteins in anoikis in thyroid epithelial cells. Western blot analysis demonstrated that neither p53 nor Bcl-2, Bcl-XL and Bax protein expression changed during anoikis. However, loss of endogenous p53 activity in cells transfected with a dominant-negative mutated p53 inhibited anoikis demonstrating the involvement of p53-dependent processes. The phosphatase inhibitor sodium orthovanadate opposed anoikis when added to the cells within 6 h, suggesting a role for phosphorylated proteins.

Apoptosis↗

PubMed1999

Identification and molecular cloning of p75/AIRM1, a novel member of the sialoadhesin family that functions as an inhibitory receptor in human natural killer cells.

In this study, by the generation of a specific monoclonal antibody, we identified p75/AIRM1 (for adhesion inhibitory receptor molecule 1), a novel inhibitory receptor that is mostly confined to human natural killer cells. p75/AIRM1 is a 75-kD glycoprotein that, upon sodium pervanadate treatment, becomes tyrosine phosphorylated and associates to src homology 2 domain-bearing protein tyrosine phosphatase (SHP)-1. The p75/AIRM1 gene is located on human chromosome 19 and encodes a novel member of the sialoadhesin family characterized by three immunoglobulin-like extracellular domains (one NH(2)-terminal V-type and two C2-type) and a classical immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic portion. The highest amino acid sequence similarity has been found with the myeloid-specific CD33 molecule and the placental CD33L1 protein. Similar to other sialoadhesin molecules, p75/AIRM1 appears to mediate sialic acid-dependent ligand recognition.

Amino Acid Sequence↗

PubMed1999

Inefficient phospholipase C activation and reduced Lck expression characterize the signaling defect of umbilical cord T lymphocytes.

Adult and neonatal immunocompetent cells exhibit important functional distinctions, including differences in cytokine production and susceptibility to tolerance induction. We have investigated the molecular features that characterize the immune response of cord blood-derived T lymphocytes compared with that of adult T lymphocytes. Our findings demonstrate that phospholipase C (PLC) isozymes, which play a pivotal role in the control of protein kinase C activation and Ca2+ mobilization, are differently expressed in cord and adult T lymphocytes. PLCbeta1 and delta1 are expressed at higher levels in cord T cells, while PLCbeta2 and gamma1 expression is higher in adult T lymphocytes. PLCdelta2 and gamma2 appear to be equally expressed in both cell types. In addition, a functional defect in PLC activation via CD3 ligation or pervanadate treatment, stimuli that activate tyrosine kinases, was observed in cord blood T cells, whereas treatment with aluminum tetrafluoride (AlF4-), a G protein activator, demonstrated a similar degree of PLC activation in cord and adult T cells. The impaired PLC activation of cord blood-derived T cells was associated with a a very low expression of the Src kinase, Lck, along with a reduced level of ZAP70. No mitogenic response to CD3 ligation was observed in cord T cells. However, no signaling defect was apparent downstream of PLC activation, as demonstrated by the mitogenic response of cord T cells to the pharmacologic activation of protein kinase C and Ca2+ by treatment with PMA and ionomycin. Thus, neonatal cord blood-derived T cells show a signaling immaturity associated with inadequate PLCgamma activation and decreased Lck expression.

Adult↗

PubMed1999

Selective modulation of specific protein kinase C (PKC) isoforms in primary human megakaryocytic vs. erythroid cells.

We have investigated the pattern of expression of classical (alpha, betaI, betaII, gamma), novel (delta) and atypical (zeta) protein kinase C (PKC) isoforms during the course of human hematopoietic differentiation along the closely related megakaryocytic and erythroid lineages. Using in situ immunofluorescence analysis, freshly isolated human pluripotent CD34+ hematopoietic progenitor cells expressed detectable amounts of all the PKC isoforms investigated. On the other hand, clear-cut differences in terms of PKC staining were noticed between cells belonging to the erythroid and megakaryocytic lineages, obtained after 9 days of serum-free liquid culture in the presence of specific growth factors. Specifically, 1) erythroid cells showed a very weak expression of PKC-alpha, -betaI, -betaII, and -gamma, while megakaryocytes showed an enhanced expression of all classical PKC isoforms, predominantly confined to the cytoplasm; 2) the expression of PKC-delta increased in the cytoplasmic and nuclear compartments of both erythroid and megakaryocytic cells with respect to CD34+ cells; and 3) atypical PKC-zeta isoform showed a striking accumulation in the nucleus during both erythroid and megakaryocytic differentiation.

Cell Differentiation↗

PubMed1999

NKp44, a triggering receptor involved in tumor cell lysis by activated human natural killer cells, is a novel member of the immunoglobulin superfamily.

Surface receptors involved in natural killer (NK) cell triggering during the process of tumor cell lysis have recently been identified. Of these receptors, NKp44 is selectively expressed by IL-2- activated NK cells and may contribute to the increased efficiency of activated NK cells to mediate tumor cell lysis. Here we describe the molecular cloning of NKp44. Analysis of the cloned cDNA indicated that NKp44 is a novel transmembrane glycoprotein belonging to the Immunoglobulin superfamily characterized by a single extracellular V-type domain. The charged amino acid lysine in the transmembrane region may be involved in the association of NKp44 with the signal transducing molecule killer activating receptor-associated polypeptide (KARAP)/DAP12. These molecules were found to be crucial for the surface expression of NKp44. In agreement with data of NKp44 surface expression, the NKp44 transcripts were strictly confined to activated NK cells and to a minor subset of TCR-gamma/delta+ T lymphocytes. Unlike genes coding for other receptors involved in NK cell triggering or inhibition, the NKp44 gene is on human chromosome 6.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Lineage-restricted expression of protein kinase C isoforms in hematopoiesis.

The pattern of expression of several protein kinase C (PKC) isoforms (alpha, betaI, delta, epsilon, eta, and zeta) during the course of hematopoietic development was investigated using primary human CD34(+) hematopoietic cells and stable cell lines subcloned from the growth factor-dependent 32D murine hematopoietic cell line. Each 32D cell clone shows the phenotype and growth factor dependence characteristics of the corresponding hematopoietic lineage. Clear-cut differences were noticed between erythroid and nonerythroid lineages. (1) The functional inhibition of PKC-epsilon in primary human CD34(+) hematopoietic cells resulted in a twofold increase in the number of erythroid colonies. (2) Erythroid 32D Epo1 cells showed a lower level of bulk PKC catalytic activity, lacked the expression of epsilon and eta PKC isoforms, and showed a weak or absent upregulation of the remaining isoforms, except betaI, upon readdition of Epo to growth factor-starved cells. (3) 32D, 32D GM1, and 32D G1 cell lines with mast cell, granulo-macrophagic, and granulocytic phenotype, respectively, expressed all the PKC isoforms investigated, but showed distinct responses to growth factor readdition. (4) 32D Epo 1.1, a clone selected for interleukin-3 (IL-3) responsiveness from 32D Epo1, expressed the epsilon isoform only when cultured with IL-3. On the other hand, when cultured in Epo, 32D Epo1.1 cells lacked the expression of both epsilon and eta PKC isoforms, similarly to 32D Epo1. (5) All 32D cell lines expressed the mRNA for PKC-epsilon, indicating that the downmodulation of the epsilon isoform occurred at a posttranscriptional level. In conclusion, the PKC isoform expression during hematopoiesis appears to be lineage-specific and, at least partially, related to the growth factor response.

Animals↗

PubMed1999

Lineage-related susceptibility of human hemopoietic cell lines to apoptosis.

Apoptosis plays a fundamental role in shaping normal hematopoiesis. We have investigated the relationship existing between susceptibility to apoptosis and lineage commitment in hemopoietic cells. The presence and degree of apoptosis were investigated in myeloid (HL-60 and K562), T (Jurkat and MOLT-4), and B (CESS and Raji) lymphoid cell lines by using a variety of techniques-transmission electron and light microscopy, flow cytometry and DNA gel electrophoresis. The major achievement of this study is that hematopoietic cells respond to different chemical (staurosporin, tiazofurin, camptothecin) and physical (hyperthermia or hypothermia) stimuli by apoptosis in a lineage-related way. Moreover, with respect to the methods used to detect apoptosis, a strong correlation was observed between the presence of the hypodiploid peak determined by flow cytometry and the DNA laddering evaluated by gel electrophoresis, but both techniques failed to demonstrate the presence of apoptosis in some cases. We conclude that cells of different hematopoietic lineages mostly show a lineage-related behaviour in their apoptotic response to different stimuli, suggesting that the lineage commitment and the stage of differentiation can confer different sensitivities to specific apoptotic stimuli. Moreover, morphological techniques still represent the most reliable approach to detect apoptosis in hemopoietic cells.

Apoptosis↗

PubMed1999

Molecular and functional characterization of IRp60, a member of the immunoglobulin superfamily that functions as an inhibitory receptor in human NK cells.

In this study we describe the functional and molecular characterization of IRp60 (inhibitory receptor protein 60), an inhibitory receptor expressed on all human NK cells. The IRp60 molecule has been identified by the generation of three novel monoclonal antibodies (mAb). Cross-linking of IRp60 by specific mAb strongly inhibits the spontaneous cytotoxicity of NK cells as well as the NK-mediated cytolytic activity induced via different non-HLA-specific or HLA-specific activating receptors. IRp60 is a 60-kDa glycoprotein that, upon sodium pervanadate treatment, becomes tyrosine phosphorylated and associates with the SH2-containing phosphatases SHP-1 and SHP-2. The IRp60 gene is located on human chromosome 17 and encodes a molecule belonging to the immunoglobulin (Ig) superfamily characterized by a single V-type Ig-like domain in the extracellular portion. The cytoplasmic tail contains three classical immunoreceptor tyrosine-based inhibitory motifs. Southern blot analysis revealed cross-hybridization with monkey and mouse genomic DNA, thus suggesting that IRp60 may be conserved among different species. Moreover, based on the use of different anti-IRp60 mAb, we could identify two IRp60 allelic variants. Since IRp60 is also expressed by other cell types, including T cell subsets, monocytes and granulocytes, it may play a more general role in the negative regulation of different leukocyte populations.

Amino Acid Sequence↗

PubMed1999

Evaluation of disease activity by IGF-I and IGF binding protein-3 (IGFBP3) in acromegaly patients distributed according to a clinical score.

UNLABELLED: To facilitate the estimation of acromegalic activity a prospective study was done comparing, against a clinical score, the effectiveness of serum IGF-I, IGFBP3 and the IGF-I/IGFBP3 molar ratio. Sixty nine observations were distributed in three groups: Group I=patients before surgery; group II=patients improved but still clinically active; group III=patients clinically inactive. Suppression of serum GH levels one hour after an oral glucose load was in agreement with the clinical score in 21/22 observations. Increases in serum IGF-I and IGFBP3 levels were similarly frequent: both 100% in group I, 80% and 95% in group II, 9% and 36% in group III, respectively. The frequency of abnormal molar ratios was 95%, 40% and 0% in the same groups. Log IGF-I, log IGFBP3, and log molar ratio correlated significantly with the clinical scores (r=0.873, r=0.692, and r=0.829, respectively). CONCLUSIONS: The IGF-I/IGFBP3 molar ratio was not better than either IGF-I or IGFBP3 in detecting activity in the three groups of patients studied. Both IGF-I and IGFBP3 appear comparably useful for the diagnosis and follow-up of acromegalic patients. Since IGF-I is a more biologically meaningful parameter it might be preferable.

Acromegaly↗

PubMed1999

Expression of integrins of the beta1 family in thyroid cells from patients with Graves' disease in vivo and in vitro.

The expression of the beta1 family of integrins was determined in thyroid follicular cells from patients with Graves' disease (GD). Integrin expression was quantitated by flow fluorocytometry of single cell suspensions with antibodies against the common beta1 chain and the alpha1-alpha6 subunits. Results indicated that also in thyroid glands of GD, as previously observed in nodular goiters, two follicular cell populations with different patterns of beta1 integrin expression coexist (VLAalpha3beta1 and VLAalpha1,3,5,6beta1). The VLAalpha1,3,5,6beta1 thyrocyte population in GD was more abundant than in nodular goiters, ranging from 40 to 70% of the total follicular cells and the overall expression of the beta1 integrins was a two-fold higher. In thyrocytes from patients with GD cultured in vitro, alpha3 and alpha2 expression was regulated by cell-to-cell contact as previously described in normal thyroid cells, while the expression of alpha1, alpha5 and alpha6 was quickly lost during the culture. Our data suggest that the integrin profile of the VLAalpha1,3,5,6beta1 thyrocyte population in GD is induced by micro-environmental conditions rather than being the expression of a constitutive phenotype.

Cells, Cultured↗