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Biomedical subjects

R Piva

Publications and source records attributed to R Piva.

At least 37 records · Page 2Linked to original sources
PubMed1999

Stent thrombosis: incidence and related factors in the R.I.S.E. Registry(Registro Impianto Stent Endocoronarico)

Although stent thrombosis has been greatly reduced by adequate stent expansion with high-pressure balloon inflations and by the use of antiplatelet drugs, this event is still frightening, as it may lead to acute myocardial ischemia resulting in acute myocardial infarction or sudden death. Therefore, the definition of factors associated with stent thrombosis may provide a better understanding of the mechanisms underlying this phenomenon and may permit us to define therapeutic strategies to further reduce its occurrence. The purpose of this study was to assess factors responsible for the occurrence of stent thrombosis after coronary stent implantation in 939 consecutive patients enrolled in the Registro Impianto Stent Endocoronarico (R.I.S.E. Study Group). Consecutive patients undergoing coronary stent implantation at 16 medical centers in Italy were prospectively enrolled in the registry. Clinical data, and qualitative and quantitative angiographic findings were obtained from data collected in case report forms at each investigator site. The study group consisted of 781 men and 158 women with a mean age of 59 yr: 1,392 stents were implanted in 1,006 lesions and expanded at a maximal inflation pressure of 14.7 +/- 3 atm. The great majority of patients (92%) received only antiplatelet drugs after coronary stenting. During hospitalization there were 45 major ischemic complications in 39 patients (4.2%): 13 events were related to acute or subacute thrombosis (1.4%). Another stent thrombotic event occurred in the first month of follow-up. On multivariate logistic regression analysis, stent thrombosis was related to the following factors: unplanned stenting (OR 3.46, 95% CI 1.65-7.23), unstable angina (OR 3.37, 95% CI 1.11-10.14) and maximal inflation pressure (OR 0.83, 95% CI 0.75-0.93). In conclusion, this registry shows that in an unselected population of patients undergoing coronary stenting, stent thrombosis occurs in less than 2% of patients and is significantly related to unplanned stent implantation, unstable angina, and maximal inflation pressure. The incidence of this phenomenon is likely to be further reduced by the use of new potent antiplatelet drugs, such as platelet glycoprotein IIb/IIIa antagonists.

Adult↗
PubMed1999

p27/kip1 expression in oligodendrogliomas and its possible prognostic role.

p27/kip1 regulates the G1-S transition of the cell cycle by inhibiting cyclinD-CDK4, cyclinE-CDK2 and cyclinA-CDK2 complexes. Regulation of p27 levels occurs mainly post-translationally by ubiquitin-mediated proteasomal proteolysis. Although genetic changes of p27/kip1 are extremely rare, in many human carcinomas p27 levels are reduced, correlate with histological malignancy, and are associated with poor prognosis. In astrocytic gliomas, p27 decreases with anaplasia and is almost absent in glioblastomas. p27/kip1 was immunohistochemically studied in 37 oligodendrogliomas, categorized according to WHO classification. In this series, the immunohistochemical reaction for p27 was confined to nuclei. p27 score showed a tendency to decrease with malignancy. When the p27 score was considered as high versus low expression (cut-off of p27 labeling index, LI, at 25%), it represented an independent prognostic factor in univariate (P = 0.02) and in multivariate analysis (P = 0.04). The risk ratio suggested that the p27 low expression group had a threefold increased possibility to show a reduced survival. Moreover, p27 levels did not correlate with MIB-1 LI, suggesting that p27 is not merely associated with the control of proliferation.

Brain Neoplasms↗
PubMed1999

A cell cycle alteration precedes apoptosis of granule cell precursors in the weaver mouse cerebellum.

A missense mutation in the gene coding for the G-protein-activated inwardly rectifying potassium (GIRK) channel, GIRK2, is responsible for apoptosis in the external germinal layer (EGL) of the cerebellum and a nonapoptotic death of midbrain dopaminergic neurons in the weaver (wv) mouse. Failure of axonogenesis and migration are considered to be the primary consequences of GIRK2 channel malfunction in the cerebellum. We investigated whether a disruption of the cell cycle precedes the failure of migration and axonogenesis and leads to massive apoptosis. To this end, immunohistochemistry and immunoblotting for PCNA, Cdk4, cyclin D, cyclin A, and the Cdk inhibitor p27/kip1, as well as in situ end-labeling for apoptotic DNA fragmentation, were applied to cerebella of P7-P21+/+, wv/+, and wv/wv mice. In +/+ and wv/+ mice, the expression of cell cycle proteins was limited to the outer, premigratory zone of the EGL. Antibodies to p27, a marker of cell differentiation, gave a reverse staining pattern. Due to migration delay, patches of p27-positive cells persisted in the outer EGL in P21 wv/+ mice. On the contrary, marked cell cycle up-regulation and absence of p27 occurred throughout the EGL at all ages in wv/wv mice, indicating an inability to switch off the cell cycle. Mitotic index evaluation showed that cell cycle activation was unrelated to proliferative events. Cell cycle proteins were not expressed in the substantia nigra, suggesting that nonapoptotic death of mature dopaminergic neurons is not preceded by abortive cell cycle re-entry. Our data show that abnormalities of the cell cycle in wv/wv cerebellum represent a major and early consequence of GIRK2 channel malfunction and may strongly influence the susceptibility of EGL cells to apoptosis. These observations may help in understanding the pathogenesis of human neurological channelopathies.

Animals↗
PubMed1999

Mutations in autosomal dominant polycystic kidney disease 2 gene: Reduced expression of PKD2 protein in lymphoblastoid cells.

The polycystic kidney disease 2 (PKD2) gene, encoding a 968-amino acid integral membrane protein with six predicted membrane-spanning domains and intracellular NH2 and COOH termini, is mutated in approximately 15% of the cases of autosomal dominant polycystic kidney disease (ADPKD), a common genetic disease frequently resulting in renal failure. For a better understanding of the cause of this disorder, we searched for mutations in the PKD2 gene in two PKD2-linked families characterized by different clinical phenotypes. A common polymorphism, a nonsense mutation, and a frameshift mutation were found. Both mutations are predicted to produce truncated proteins of 314 and 386 amino acids, arrested at the first extracellular loop of the protein. Restriction enzyme analysis of polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR products, respectively, showed that mutations cosegregated with the disease and mutated alleles were expressed at the messenger RNA level in lymphoblastoid cell lines. However, in these cells, Western blot analysis showed only PKD2 normal protein, and it was expressed at a lower level than that found in cells without the PKD2 mutation. These findings suggest that in lymphoblastoid cells, the truncated protein product of the mutant allele may not be stable.

Aged↗
PubMed1999

Proteasome-dependent degradation of p27/kip1 in gliomas.

p27/kip1 regulates the G1-S transition of the cell cycle by inhibiting cyclin D-CDK4, cyclin E-CDK2, and cyclin A-CDK2. Modulation of p27 cellular abundance occurs mainly at post-translational level by the ubiquitin-proteasome proteolysis. Although rearrangements and mutations of p27/kip1 are extremely rare events, p27 levels are reduced and associated with a poor prognosis in many human carcinomas. In astrocytic tumors, p27 decreases with advancing anaplasia and is almost absent in glioblastomas. To verify whether the degradation of p27 protein was responsible for its reduced levels in malignant gliomas, p27 degradation activity was tested in 22 tissue extracts that represented high, low, and absent p27 protein levels. p27 protein expression was detected by immunohistochemistry and immunoblot analysis and comparable results between the 2 methods were obtained. Low or undetectable p27 degradation activity was found in samples that displayed high levels of p27, i.e. all 4 normal brain biopsies, and 4 out of 6 grade II astrocytomas. Enhanced degradation activity resulted in malignant gliomas with low or absent p27 protein levels. The proteasome inhibitor LLnL abolished p27 degradation, demonstrating that it occurs in a proteasome-dependent manner. These data suggest that proteasome degradation of p27 may be instrumental in the deregulation of the cell cycle and to the malignant transformation of gliomas.

Astrocytoma↗
PubMed1998

K562 erythroid and HL60 macrophage differentiation downregulates polycystin, a large membrane-associated protein.

Polycystin, the PKD1 gene product mutated in autosomal dominant polycystic kidney disease, is a large membrane protein which is important in the differentiation of epithelial tubular structure. Furthermore, PKD1 mRNA is expressed in various tissues and in neoplastic cell lines particularly, suggesting that polycystin might be involved in differentiation and/or proliferation of other cell types. Therefore, in order to investigate such a possible role, polyclonal antibodies against a recombinant polycystin peptide were raised and used to study polycystin expression in human leukemia cell lines committed to differentiation. Using Western blot and laser scanning confocal microscopy analyses, we demonstrated expression of polycystin in erythroleukemia K562 cells as a membrane-associated polypeptide of approximately 450 kDa, mainly localized in cell-cell contacts. Protein size and subcellular distribution were similar to those found in the kidney epithelial KJ29 cell line. In addition, K562 cell erythroid differentiation induced by hemin was characterized by a reduction in polycystin expression, as measured by Western blot and Northern blot analyses. Cytofluorimetric analysis indicated that upon hemin treatment there was a progressive reduction in the number of polycystin-expressing cells as well as in proliferation rate. Furthermore, reduction in proliferating and polycystin-expressing cells was also observed in K562 cells after serum starvation. When serum was added to the serum-deprived cells an increase in cell number as well as in number of polycystin-positive cells was observed. In addition, polycystin, also expressed in promyelocytic leukemia HL60 cells, was downregulated when macrophage differentiation in HL60 was induced by TPA. Therefore, in these leukemic cells downregulation of polycystin appeared to be closely related to reduction in cell proliferation and to induction of differentiation. This suggests that polycystin may play a relevant role in these cell processes.

Antibodies↗
PubMed1998

In vitro stability of polymerase chain reaction-generated DNA fragments in serum and cell extracts.

The potential use of polymerase chain reaction (PCR)-generated DNA fragments (PCR-DNAs) as pharmaceutical agents has previously been suggested, with the demonstration of the in vitro cellular internalization and biologic activity of PCR-DNA decoy molecules targeted to human estrogen receptor gene. In order to provide information on the stability of these double-stranded DNA molecules, the nuclease resistance of PCR-DNAs of different sizes was studied in different conditions and experiments. Simulating in vitro and in vivo transfection protocol, we demonstrated that PCR-DNAs exhibited good stability toward fetal bovine serum (FBS) and adult human serum nuclease digestion. In addition, when the protective activity of liposome-based formulations toward nuclease digestion was tested, it was shown that the stability of PCR-DNAs could be further increased (up to 7 days) when a liposome-mediated delivery system was employed.

Adult↗
PubMed1998

Characterization of murine Girk2 transcript isoforms: structure and differential expression.

A mutation in the G-protein-linked inwardly rectifying K+ channel 2 gene (Girk2) is the cause of the weaver mouse phenotype. We determined that the originally published Girk2 transcript is composed of five exons. The primary coding exon (designated exon 4a in our system) encodes over two-thirds of the protein. Five different full-length Girk2 transcript isoforms (designated Girk2-1, Girk2A-1, Girk2A-2, Girk2B, and Girk2C) originating from different transcriptional start sites and/or alternative splicing were isolated by cDNA RACE. Several of the transcripts were predicted to encode truncated proteins that may lack some of the G-proteincoupling sequence. Northern blotting and in situ hybridization studies with transcript-specific probes indicated that the transcripts were differentially expressed in both normal and weaver mice. All transcripts tested were expressed in the three major targets of action of the weaver mutation: cerebellum, substantia nigra, and testis. Two of the transcripts, Girk2A-1 and Girk2A-2, encode identical proteins and have a distinct pattern of expression in testis, which suggests that they are associated with specific stages of spermatogenesis. An additional transcript, Girk2D, appears to be brain-specific, not polyadenylated, and highly expressed in cerebellar granule cells.

Animals↗
PubMed1998

Cyclin D1 expression in gliomas.

Cyclin D1 (cycD1) expression was defined immunohistochemically using monoclonal antibody DCS-6 and polyclonal antiserum H-295 in 50 glioma biopsies. The number of positive nuclei was higher for H-295 than for DCS-6, with a ratio of 3:1. The labelling index (LI) was compared to the grade of histological malignancy and to Ki-67 MIB-1 LI. The LI for cycD1 increased with histological malignancy, in parallel with the increase in MIB-1 LI. In most tumours, the maximum LI for cycD1 and MIB-1 were found in the same areas. The mean MIB-1 LI: mean cycD1 LI ratio does not vary in the three grades of astrocytic tumours. However, in this study the correlation between the two LIs was not statistically significant. Staining for cycD1 antigen does not necessarily imply that the gene is overexpressed since other molecular mechanisms can also be responsible for cell cycle deregulation. In invasive areas, the cycD1 LI is frequently higher than in solid tumour, either because more tumour cells are positive or because reactive astrocytes and activated microglia express cycD1. The relative contribution of neoplastic and reactive cells remains to be defined.

Astrocytes↗
PubMed1998

Modulation of estrogen receptor gene expression in human breast cancer cells: a decoy strategy with specific PCR-generated DNA fragments.

Transcriptional activity of human estrogen receptor (hER) gene was modulated by competition with double-stranded PCR-generated DNA fragments (decoys) that contain 5' upstream sequences of the hER gene. Two DNA fragments belonging to the P1 canonical promoter and the P3 distal promoter, 120 and 102 bp in size respectively, were produced by PCR and directly transfected in MCF7 breast cancer cells. After 24 hours transfection, RT-PCR analysis revealed that the 120 bp decoy significantly reduced the expression of the ER gene and estrogen responsive genes (PR and c-myc), whereas the 102 bp decoy increased the ER mRNA level. An ER unrelated PCR product, used as control, had no activity. The biological activity of these ds DNAs was related to their high stability, binding affinities, and lack of cytotoxicity. These findings suggest that such PCR product decoys may be a non-antisense tool to analyze putative regulatory sequences and to study the function of DNA-binding transcription factors.

Breast Neoplasms↗
PubMed1998

CDKN2/p16 inactivation and p16 immunohistochemistry in astrocytic gliomas.

CDKN2/p16 inactivation is the most frequent alteration in the molecular regulation of G1-S transition. CDKN2/p16 homozygous deletions was studied in paraffin-embedded sections of 45 astrocytic tumours by multiplex PCR. Immunohistochemistry for p16 and proliferation marker Ki-67 MIB-1 was performed in adjacent sections; their labelling index (LI) have been calculated. CDKN2/p16 gene was not deleted in astrocytomas, while homozygous deletion was found in 26.7% anaplastic astrocytomas, and in 55.0% of glioblastomas. Analysis of CDKN2/p16 homozygous deletion in discrete areas of the same tumour, showed that the deletion occurred independently of the phenotypic aspect of the areas. Nevertheless a genotypic and phenotypic heterogeneity is present in few cases. p16 immunohistochemistry mostly corresponds to the genotypic pattern. No correlation was found between CDKN2/p16 homozygous deletion and MIB-1 LI.

Astrocytoma↗
PubMed1998

Predictors of major in-hospital ischemic complications and length of hospital stay after coronary stenting.

Although recent data show that coronary stenting reduces procedural complications and late restenosis, major concern has been expressed about the greater hospital cost associated with the use of this device as compared to conventional coronary angioplasty. Since length of hospital stay after surgical procedures is a major determinant of resource use, the identification of variables associated with an excessively long hospital stay after intracoronary stent placement may have important practical consequences. The purpose of this study was to assess factors responsible for the occurrence of in-hospital complications and prolonged hospital stay after coronary stenting in 939 consecutive patients enrolled in the Registro Impianto Stent Endocoronarico (RISE Study Group). Consecutive patients undergoing coronary stent implantation at 16 medical centers in Italy were prospectively enrolled in the Registry. Clinical data, qualitative and quantitative angiographic findings were obtained from data collected in case report forms at each investigator site. Major ischemic complications were considered death, Q-wave myocardial infarction, emergency bypass surgery and emergency repeat angioplasty. The study group consisted of 939 patients (781 men, 158 women with a mean age of 59 years) in whom 1392 stents were implanted in 1006 lesions and expanded at a maximal inflation pressure of 14.7 +/- 3 atmospheres. The great majority of patients (92%) received only antiplatelet drugs after coronary stenting. During hospitalization, there were 45 major ischemic complications in 39 patients (4.2%): 13 events were related to acute or subacute thrombosis (1.4%). On multivariate logistic regression analysis, the following factors were predictive of in-hospital complications: increasing age (OR 2.19, 95% CI 1.18-4.07), unplanned stenting (OR 3.46, 95% CI 1.65-7.23) and maximal inflation pressure (OR 0.83, 95% CI 0.75-0.93). Mean hospital stay after stent implantation was 4.1 +/- 4.4 days and was related, by multivariate regression analysis, to female sex (p = 0.0001), prior bypass surgery (p = 0.03), non-elective stenting (p = 0.0001), use of anticoagulation (p = 0.0001) and development of major ischemic complications (p = 0.0001). This Registry shows that in an unselected population of patients undergoing coronary stenting, major ischemic complications occur at a relatively low rate (4.2%) and thrombotic events can be kept at 1.4%, despite the omission of anticoagulation in the great majority of patients. Length of hospital stay was affected by the occurrence of major ischemic complications, unplanned stenting, use of anticoagulation, female sex and prior bypass surgery. Accumulating experience, further reduction in complications and complete omission of anticoagulation may decrease length of hospital stay, thus reducing the use of resources after coronary stenting.

Adult↗
PubMed1997

p27/kip1 expression in human astrocytic gliomas.

In the cell cycle, p27/kip1 acts as an inhibitory protein of cyclin-cdk complexes. p27/kip1 immunohistochemistry was performed in 50 gliomas (15 astrocytomas, 15 anaplastic astrocytomas and 20 glioblastomas) by a polyclonal antiserum. In the same tumours, proliferation marker Ki-67 was studied by MIB-1 antibody. For both, a labelling index (LI) was calculated after counting at least 1000 cells at x1000 magnification. p27 is diffusely and strongly expressed in astrocytomas (LI mean = 44.4%), whereas positive nuclei decrease in number and in staining intensity in anaplastic astrocytomas (LI mean = 5.86%) and glioblastomas (LI mean = 2.1%). An inverse correlation has been found between MIB-1 LI and p27 LI calculated in the same areas. Interestingly, in malignant gliomas the absence of p27 was independent from any histological feature of differentiation or anaplasia. p27 expression is thus reduced in malignant gliomas as in other malignancies. Since mutations of p27/kip1 are extremely rare, posttranslational changes are hypothesised also in astrocytic gliomas.

Astrocytoma↗
PubMed1997

Muscle apoptosis in humans occurs in normal and denervated muscle, but not in myotonic dystrophy, dystrophinopathies or inflammatory disease.

Recent data suggest that death of muscle cells during development and in selected pathological conditions occurs via apoptosis. We investigated the occurrence of apoptosis in normal and pathological human skeletal muscle, using in situ end-labeling (ISEL) to detect DNA fragmentation, and immunohistochemistry for the expression of tissue transglutaminase and interleukin-1beta-converting enzyme (ICE)-like proteases. In normal subjects, apoptotic myonuclei were occasionally observed as evidence of normal tissue turnover. Myonuclear apoptosis due to a deficit of trophic support from nerve cells also occurred in spinal muscular atrophies. No apoptosis of muscle cells was found in dystrophinopathies, myotonic dystrophy and inflammatory myopathies, suggesting that death of myofibers in those conditions is not due to activation of a gene-directed program of death. In dystrophinopathies and inflammatory myopathies, apoptosis was found in interstitial mononuclear cells, as a likely mechanism of clearance of the inflammatory infiltrates.

Adolescent↗
PubMed1997

Role of apoptosis in the prognosis of oligodendrogliomas.

Prognostic factors in oligodendrogliomas are not well defined, even considering the labeling index of proliferation markers. As in other neuroepithelial tumors, the difficulty in calculating cell loss may contribute to this uncertainty. Proliferation markers Ki-67/MIB.1 and PCNA, mitoses, apoptotic nuclei, p53 and bcl-2 expression were investigated in 98 oligodendrogliomas. Apoptosis was assessed by the aspect of nuclei, by in situ end-labeling (ISEL) technique and by c-Jun immunohistochemical demonstration. The Bcl-2 also was immunohistochemically studied for its anti-apoptotic role. Mitotic index (MI), labeling index (LI) for MIB.1 and PCNA and apoptotic index (AI) were calculated and compared among themselves and with histology and survival. It was found that AI correlated with MI (p = 0.001) and was significantly higher in anaplastic than in classic oligodendrogliomas (p = 0.001). Apoptosis occurred only slightly more frequently in cases with high LIs for proliferation markers (MIB.1 and PCNA) (p = non-significant) and it was definitely higher in p53-positive cases (p = 0.008). It did not correlate with bcl-2 which was poorly expressed in oligodendrogliomas, with the exception of cells with astrocytic features. Apoptotic index correlated very weakly with survival (p = 0.05); therefore, it cannot be considered a highly reliable prognostic factor in oligodendrogliomas.

Adult↗
PubMed1997

c-Jun, JNK/SAPK kinases and transcription factor NF-kappa B are selectively activated in astrocytes, but not motor neurons, in amyotrophic lateral sclerosis.

There is increasing evidence that oxidative damage plays a major role in amyotrophic lateral sclerosis (ALS), but how it contributes to motor neuron degeneration and astrocytic gliosis, two pathologic hallmarks of the disease, is unknown. A few studies have suggested that ALS motor neurons die via apoptosis and show upregulation of c-jun, an immediate early gene that is necessary for neuronal apoptosis. In order to elucidate the mechanisms of cell damage induced by oxidant stress, we have studied in ALS and control spinal cord the immunohistochemical expression of c-Jun, of JNK/SAPK, a kinase that activates c-Jun following various types of stress, and of NF-kappa B, a transcription factor that is induced by oxidant stress and has prominent neuroprotective functions. An in situ end-labeling assay was performed for detecting apoptotic cells. We show that (a) the JNK/SAPK-c-Jun pathway is dramatically overexpressed in ALS spinal cord; (b) the strongest activation occurs in astrocytes, while motor neurons show unusually low expression of the pathway; (c) increased JNK/SAPK expression in glial cells is accompanied by NF-kappa B activation, indicating the presence of a protective response to oxidant sress, which is deficient in motor neurons; (d) activation of JNK/SAPK, c-Jun and NF-kappa B is unrelated to apoptotic cell death. These results support the view that astrocytes are directly involved in the pathologic process of ALS, and might explain the selective vulnerability of motor neurons by their relative lack of antioxidant defenses.

Amyotrophic Lateral Sclerosis↗