Biomedical subjects

D Barbieri

Publications and source records attributed to D Barbieri.

At least 19 recordsLinked to original sources

PubMed2003

Helicobacter pylori and cagA and vacA gene status in children from Brazil with chronic gastritis.

Helicobacter pylori has been shown to be strongly associated with chronic gastritis, gastric and duodenal ulceration, and is a risk factor for gastric carcinoma. Histology, urease, culture, and polymerase chain reaction have been employed as for H. pylori diagnostic methods, pre and post treatment or during follow-up of dyspeptic adult individuals referred for endoscopy. In order to obtain a more-sensitive and specific method for H. pylori detection, we evaluated gastric body and antrum biopsies of 134 consecutive Brazilian consecutive dyspeptic children aged 1-16 years by rapid urease test, histology and polymerase chain reaction using two pairs of oligonucleotides. Our results indicated that polymerase chain reaction with Southern blotting and hybridization with specific chemiluminescent probes increased the number of positive H. pylori patients by 35%. The genotyping of H. pylori strains directly from gastric biopsy using the same nucleic acid methodology revealed that there is no association of chronic gastritis in our infant patients with vacA s1 and the presence of the cagA gene. These data suggest an initial infection of children with normal mucosa and probably others factors than vacA s1 genotype or the presence of the cagA gene are associated with the onset of gastric disease. Altogether, our results reinforce the need for using more sensitive diagnostic methods in order to understand the role of H. pylori in the genesis of gastric disease in children and its progression in adults.

Antigens, Bacterial↗

PubMed2001

Involvement of PI 3-kinase, PKA and PKC in PDGF- and TGF-beta-mediated prevention of 2-deoxy-D-ribose-induced apoptosis in the insect cell line, IPLB-LdFB.

Activation of phosphatidylinositol (PI) 3-kinase, protein kinase A (PKA) and protein kinase C (PKC) is associated with the survival effect elicited by PDGF-AB and TGF-beta1 against the apoptotic inducer 2-deoxy-D-ribose (dRib) in the fat body cell line, IPLB-LdFB, from the insect Lymantria dispar. dRib induces apoptosis and provokes mitochondrial membrane depolarization (MMD). The antioxidant N -acetyl-L-cysteine annuls only the first effect. These findings suggest that apoptosis and MMD are provoked by two different mechanisms, and that dRib induces apoptosis by oxidative stress.

Acetylcysteine↗

PubMed2001

An anti-Bcl-2 antibody prevents 2-deoxy-D-ribose-induced apoptosis in the IPLB-LdFB insect cell line.

Confocal microscopy reveals that the anti-Bcl-2 antibody (pAb) is able to diffuse across the plasma membrane of the fat body cell line IPLB-LdFB from the insect Lymantria dispar, demonstrating the presence of Bcl-2-like molecules in the cytoplasm. Immunoperoxidase procedure confirms the cellular localization. Furthermore, an immunoprecipitation corresponding to a molecular weight of 29 KDa is observed with western blot analysis using the anti-Bcl-2 pAb. Cytofluorimetric experiments show that anti-Bcl-2 pAb counteracts 2-deoxy-D-ribose-induced apoptosis and provokes morphological changes in the insect cell line, i. e. a reduction in cell size, the disappearance of the vacuola and changes in shape. At the same time, the antibody provokes mitochondrial membrane depolarization, and N-acetyl-L-cysteine is unable to reconstitute the physiological conditions. The present findings suggest that Bcl-2-like proteins play a main role in maintaining of the integrity of cellular components, e.g. mitochondria, rather than in controlling programmed cell death.

Animals↗

PubMed2001

Nitric oxide induces apoptosis in the fat body cell line IPLB-LdFB from the insect Lymantria dispar.

The presence of immunoreactive inducible nitric oxide synthase molecules (ir-iNOS) is demonstrated in the Lymantria dispar IPLB-LdFB cell line. The maximum ir-iNOS inducibility is observed 18 h after incubation with sodium nitroprusside (SNP). The increase in NO provoked by SNP in turn induces apoptosis. However, this phenomenon is observed only after 48 h. The NOS-inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME) and N-[3-(aminomethyl)-benzyl]acetamide (1400W) were both unable to block the SNP-induced apoptosis at all the concentrations used. Incubation with SNP plus N-acetyl-L-cysteine (NAC) further augmented the percentage of cell death with respect to SNP used alone, and this process is seen earlier, i.e. after 24 h. Moreover, the induction of apoptosis in the presence of NAC is time- and concentration-dependent. The high percentage of cell death with SNP+NAC suggests that NAC forms S-nitrosothiols with NO, resulting in an increase in the bioavailability of NO. In conclusion, these findings show the existence of a close relationship between mammalian and invertebrate cells with regards to SNP and NAC induction and the related NO response.

Acetylcysteine↗

PubMed2000

Apoptosis induced by 2-chloro-adenosine and 2-chloro-2'-deoxy-adenosine in a human astrocytoma cell line: differential mechanisms and possible clinical relevance.

We have previously demonstrated that 2-chloro-adenosine (2-CA) can induce apoptosis of rat astroglial cells (Abbracchio et al. [1995] Biochem. Biophys. Res. Commun. 213:908-915). In the present study, we have characterized, for the first time, the effects induced on a human astrocytoma cell line (ADF cells) by both 2-CA and its related analog 2-chloro-2'-deoxy-adenosine (2-CdA, that is employed as anti-cancer agent in chronic lymphoid malignancies). Exposure of these cells to either adenosine analog resulted in time- and concentration-dependent apoptosis. Experiments with pharmacological agents known to interfere with adenosine receptors, its membrane transporter, and intracellular nucleoside kinases showed that: (i) cell death induced by either adenosine analog did not depend on extracellular adenosine receptors, but on a direct intracellular action; however, only in the case of 2-CA, was entry into cells mediated by the specific nitrobenzyl-tioinosine-sensitive transporter; (ii) for both adenosine analogs, induction of apoptosis required the phosphorylation/activation by specific intracellular nucleoside kinases, i.e., adenosine kinase for 2-CA, and deoxycytidine kinase for 2-CdA. In addition, only in the case of 2-CdA, was induction of apoptosis preceded by a block of cells at the G2/M phase of the cell cycle. Finally, at concentrations of either analog that killed about 80-90% of astrocytoma cells, a significantly lower effect on the viability of primary cortical neurons was observed. In conclusion, both adenosine analogs can trigger apoptosis of human astrocytoma cells, albeit with different mechanisms. This effect together with the relative sparing of neuronal cells, may have potential clinical implications for the therapy of tumors of glial origin.

2-Chloroadenosine↗

PubMed2000

Abnormal phenotype of in vitro dermal fibroblasts from patients with Pseudoxanthoma elasticum (PXE).

Pseudoxanthoma elasticum (PXE) is a genetic connective tissue disease, whose gene and pathogenesis are still unknown. Dermal fibroblasts from patients affected by PXE have been compared in vitro with fibroblasts taken from sex and age-matched normal individuals. Cells were grown and investigated in monolayer, into three-dimensional collagen gels and in suspension. Compared with normal cells, PXE fibroblasts cultured in monolayer entered more rapidly within the S phase and exhibited an increased proliferation index; on the contrary, similarly to normal fibroblasts, PXE cells did not grow in suspension. Furthermore, compared with normal fibroblasts, PXE cells exhibited lower efficiency in retracting collagen type I lattices and lower adhesion properties to collagen type I and to plasma fibronectin. This behavior was associated with higher expression of integrin subunits alpha2, alpha5, alphav, whereas beta1 subunit as well as alpha2beta1 and alpha5beta1 integrin expression was lower than in controls. Compared to controls, PXE fibroblasts had higher CAM protein expression in accordance with their high tendency to form cellular aggregates, when kept in suspension. The demonstration that PXE fibroblasts have altered cell-cell and cell-matrix interactions, associated with modified proliferation capabilities, is consistent with the hypothesis that the gene responsible for PXE might have a broad regulatory role on the cellular machinery.

Adult↗

PubMed2000

Activation of the A3 adenosine receptor affects cell cycle progression and cell growth.

The A3 adenosine receptor has been implicated in modulation of cell growth. As a first step to the characterization of the underlying mechanisms, we exposed Chinese hamster ovary (CHO) cells transfected with the human A3 receptor (A3R-CHO) to selective A3 receptor ligands. At micromolar concentrations, the A3 agonists N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) and its 2-chloro derivative Cl-IB-MECA reduced cell number, with no effects on either parental CHO cells (not expressing any adenosine receptor), or CHO cells transfected with the human A1 receptor. Cl-IB-MECA also reduced cell number in the human HEK293 cell line transfected with the human A3 receptor cDNA as opposed to the respective untransfected wild-type cells. In A3R-CHO, agonist-induced effects were antagonized by nanomolar concentrations of A3 antagonists, including the triazoloquinazoline derivative MRS 1220, the dihydropyridine derivative MRS 1191, and the triazolonaphthyridine derivative L-249,313. A3 agonist-induced effects were not due to modulation of cell adhesion, nor to necrosis or apoptosis. Growth curves revealed highly impeded growth, and flow-cytometric analysis showed markedly reduced bromodeoxyuridine incorporation into nuclei. The effect on cell cycle was completely antagonized by MRS1191. Hence, activation of the human A3 receptor in A3R-CHO results in markedly impaired cell cycle progression, suggesting an important role for this adenosine receptor subtype in cell cycle regulation and cell growth.

Adenosine↗

PubMed2000

PDGF and TGF-beta partially prevent 2-deoxy-D-ribose-induced apoptosis in the fat body cell line IPLB-LdFB from the insect Lymantria dispar.

The IPLB-LdFB cell line from the fat body of the insect Lymantria dispar shows the presence of immunoreactive, platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 molecules, as well as the corresponding plasma membrane-like receptors, i.e. PDGFR-alpha, PDGFR-beta and TGFR-beta type II. Cytofluorimetric and morphological studies reveal that the reducing sugar 2-deoxy-D-ribose (dRib), an apoptotic agent for human cells, induces apoptosis in a concentration- and time-dependent manner even in IPLB-LdFB cells. PDGF-AB and TGF-beta1 partially counteract the effect of dRib, indicating a survival role of these factors in this apoptotic model of insect cells.

Journal Article↗

PubMed2000

[Inflammatory bowel diseases]

OBJECTIVE: To present a review about inflammatory bowel diseases in childhood so that pediatricians become familiar with two diseases (ulcerative colitis and Crohn s disease) that are increasing in incidence. METHODS: Articles of specific gastroenterological journals were analyzed. Information was also obtained from the author s extensive professional experience in the area. RESULTS: The two diseases are described. Etiopathogenesis is obscure. Clinical presentation, diagnosis, and treatment are discussed, with details and practical aspects to enable the pediatrician to promptly understand the subject. CONCLUSIONS: The inflammatory bowel disease is a condition of high morbidity, and the pediatrician who assists the case must have a very good technical knowledge about the disease, and must be very comprehensive and emotionally serene, in order to afford the young patient a better quality of life throughout the complicated evolution of this disease.

Journal Article↗

PubMed1999

2-deoxy-d-ribose-induced apoptosis in HL-60 cells is associated with the cell cycle progression by spermidine.

The presence of polyamines is required for the apoptotic program triggered by 2-deoxy-D-ribose (dRib) in HL-60 cells, but their oxidative metabolites does not appear to be involved in the oxidative stress caused by the sugar. The present study points to a relationship between spermidine-induced G1 to S phase transition and the onset of dRib-induced apoptosis. Conversely, the G1 block induced by alpha-difluoromethylornithine (DFMO) is associated with a protective effect against dRib-induced cell suicide. Replenishment of the intracellular spermidine pool by exogenous putrescine and spermidine induces cell cycle progression and restores apoptotic levels. The present data indicate that the induction of cell cycle progression by spermidine is a condition facilitating the activation of the apoptotic process by dRib.

Acetylcysteine↗

PubMed1999

Long-term immunologic effects of thymectomy in patients with myasthenia gravis.

BACKGROUND: Thymectomy (Tx) is a common therapeutic option to treat myasthenia gravis (MG), but its effects on the immune system are still obscure in humans. OBJECTIVE: We sought to evaluate long-term immunologic effects of therapeutic Tx in patients with MG. METHODS: T- and B-cell subsets and T-cell repertoire were analyzed in 35 patients with MG, 16 with previous Tx (at least 8 years before), 6 with recent (<1 year) Tx, and 13 without Tx, as well as in 32 healthy subjects used as normal control subjects. Serum immunoglobulins and a variety of autoantibodies were also measured. A subsequent 3-year clinical follow-up was performed to verify the possible appearance of systemic autoimmune diseases. RESULTS: The long-term thymectomized (Txd) patients had mild T-cell lymphopenia and an expansion of some Vbeta families among circulating CD4+ and CD8+ T cells. They displayed a normal number of total B and CD5+ B-circulating lymphocytes, but they also displayed a polyclonal increase in serum IgM and IgG associated with the presence of high levels of a variety of organ- and nonorgan-specific autoantibodies, including anti-dsDNA and anticardiolipin, without clinical evidence of autoimmune disease. These serologic abnormalities were not detectable in both non-Txd and recently Txd patients. After 3 years, 2 long-term Txd patients had systemic lupus erythematosus and an undifferentiated connective tissue disease. CONCLUSIONS: The association between MG and laboratory findings of systemic autoimmune disease may be in part related to Tx rather than to MG. Tx may represent a risk for the development of systemic autoimmune disorders over years in patients with MG.

Adult↗

PubMed1998

N1,N12-bis(ethyl)spermine effect on growth of cis-diamminedichloroplatinum(II)-sensitive and -resistant human ovarian-carcinoma cell lines.

The results presented here demonstrate that a cis-diamminedichloroplatinum(II) (DDP)-resistant human ovarian-carcinoma cell line is also cross-resistant to the spermine analogue N1,N12-bis(ethyl)spermine (BESPM). We report that C13* cells, which are approximately 20-fold resistant to DDP, similarly showed 7-fold resistance to BESPM by colony-forming assay with an IC50 value of 24.6 +/- 2 microM vs. 3.4 +/- 0.8 microM of 2008 cells. Resistance appears to be the result of many effects, such as different morphological and functional modifications of mitochondria. Furthermore, although BESPM accumulation was almost identical in sensitive and resistant cells, the intracellular polyamine pool of the 2 cell lines was differentially affected by this polyamine analogue. In fact, when spermidine (SPD) was still detectable in C13* cells, in 2008 cells it was not, and the spermine (SPM) content was always more markedly reduced in sensitive cells than in the resistant variant. The lower polyamine content of 2008 cells could be related to a higher degree of induction of spermidine/ spermine N1-acetyltransferase (SSAT) activity by BESPM in sensitive cells than in their resistant counterpart. Despite the observed cross-resistance, the combination of the 2 drugs resulted in supra-additive and synergistic effects in both cell lines, depending on concentration, as assessed by median-effect analysis of the survival data. The effectiveness of this combination was also confirmed by the increased accumulation of cells in the G2/M phase of the cell cycle in both cell lines. Taken together, these data suggest that BESPM effect on cell growth of DDP-sensitive and DDP-resistant cells involves multiple mechanisms that are differently modulated by the DDP-resistant phenotype.

Acetyltransferases↗

PubMed1998

Modulation of apoptosis in human lymphocytes by adenosine analogues.

We previously described that 2-chloroadenosine (2CA) and 2-chloro-2'-deoxyadenosine (2CdA) induced apoptosis in human peripheral blood mononuclear cells (PBMC). In this study we tested different adenosine analogues on PBMC and we found that the modifications introduced in the 2CA structure prevented the molecule from exerting its apoptotic effect. On the other hand, substitutions on 2CdA are tolerated, although with a significant decrease in activity.

2-Chloroadenosine↗

PubMed1998

Neutral endopeptidase-24.11 (NEP) activity in human fibroblasts during development and ageing.

Neutral endopeptidase-24.11 (NEP, EC 3.4.24.11) is a cell surface Zn metallopeptidase that hydrolyzes bioactive regulatory peptides. Using a spectrofluorimetric procedure, we assessed NEP activity in plasma membranes of normal human skin and lung fibroblasts. We found a considerable increase in NEP activity during fetal-to-adult transition. Adult skin fibroblasts from an old donor exhibited significantly higher levels of NEP activity than cells from young donors. Interestingly, however, the NEP activity of fibroblasts from a centenarian donor was similar to that of cells from young donors. Increased levels of NEP activity were also found in in vitro aged lung fibroblasts. Finally, adrenocorticotropin hormone (ACTH (1-24)), a regulatory peptide that can be cleaved by NEP, provoked an increase in enzymic activity in fetal and young adult donor fibroblasts and a decrease in this activity in fibroblasts from adult and old donors. This finding suggests that ageing may affect NEP activity.

Adult↗

PubMed1998

Growth properties and growth factor responsiveness in skin fibroblasts from centenarians.

Human fibroblast cultures, which have a finite replicative lifespan in vitro, are the most widely used model for the study of senescence at the cellular level. An inverse relationship between replicative capability and donor age has been reported in human fibroblast strains. We studied the growth capacity of fibroblast primary cultures derived from people whose lifespan was as closer as possible to the expected maximum human lifespan, i.e. people over one hundred. Our data suggest that outgrowth of fibroblasts from biopsies, growth kinetics at different population doubling levels, capability to respond to a classical mitogenic stimulus (such as 20% serum) and a variety of growth factors, were remarkably similar in fibroblasts from centenarians and young controls. On the whole, our data challenge the tenet of a simple and strict relationship between in vivo aging and in vitro proliferative capability of human fibroblasts, at least at the individual level.

Adolescent↗

PubMed1998

The highly reducing sugar 2-deoxy-D-ribose induces apoptosis in human fibroblasts by reduced glutathione depletion and cytoskeletal disruption.

2-deoxy-D-Ribose (dRib), the most reducing sugar, induces apoptosis in normal human fibroblasts, as judged by cytoplasmic shrinkage, chromatin condensation, DNA fragmentation and mitochondrial depolarization. This effect is independent from culture conditions, such as cell density and the presence or absence of serum in the culture milieu, suggesting that dRib-induced apoptosis is cell cycle-independent. dRib was found also to provoke disruption of the actin filament network and detachment from the substratum, while at the same time, interestingly, it increases the expression of several integrins and cell adhesion molecules. Furthermore, dRib was found to reduce the intracellular levels of reduced glutathione (GSH). The apoptotic process was not affected by the macromolecular-synthesis inhibitors cycloheximide and actinomycin D. On the contrary, the antioxidant N-acetyl-L-cysteine (NAC) fully blocks the dRib-induced apoptosis by preventing GSH depletion, while it also inhibits actin-filament-network disruption and mitochondrial depolarization. The above indicate that dRib induces apoptosis in human fibroblasts by a mechanism involving glutathione metabolism and oxidative stress, as well as disturbance of cytoskeletal integrity and cell adhesion.

Acetylcysteine↗

PubMed1998

Polyamine depletion protects HL-60 cells from 2-deoxy-D-ribose-induced apoptosis.

We investigated the involvement of natural polyamines in HL-60 cell death triggered by exposure to 2-deoxy-D-ribose (dRib). In contrast to previous studies, exogenous polyamines failed to protect HL-60 cells against apoptosis caused by dRib. Moreover, in our experimental conditions, depletion of intracellular levels of putrescine and spermidine by alpha-difluoromethylornithine (DFMO) delayed the onset of apoptosis by at least a day or so. Exogenous polyamines reversed the beneficial effect of DFMO and restored the apoptotic levels observed in dRib-treated cells. We suggested that polyamines, especially putrescine and spermidine, act as facilitating factors in the induction of apoptosis triggered by dRib in HL-60 cells.

Apoptosis↗

PubMed1998

Antisense inhibition of Bax mRNA increases survival of terminally differentiated HL60 cells.

Cell sensitivity to programmed cell death is primarily modulated by members of the Bcl-2 family, as the balance of homodimer or heterodimer formation between proapoptotic and antiapoptotic members defines apoptosis susceptibility in the great majority of cellular contexts. It is, therefore, important to clarify if the Bax protein is limiting for activation of the genetic program of programmed cell death or can be complemented by different Bcl-2 family members, such as Bak or Bad. To gain some insight into the role of Bax in the molecular mechanisms of apoptosis of myeloid cells, we inhibited this gene in all-trans-retinoic acid (ATRA)-treated HL60 cells using the methodology of antisense oligodeoxynucleotides (AS-ODN). Our results indicate that Bax inhibition has no effect on the proliferation and differentiation capacity of HL60 cells. Instead, the survival rate of terminally differentiated Bax-inactivated HL60 (Bax(-) HL60) cells is almost three times higher in respect to control cultures, indicating that in mature granulocytes Bax is not efficiently complemented by others members of the Bcl-2 family proteins.

Apoptosis↗