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G Aguiari,
S Savelli,
M Garbo, A Bozza,
G Augello,
L Penolazzi,
E De Paoli Vitali,
C La Torre,
G Cappelli,
R Piva,
L del Senno
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ABSTRACT: Autosomal dominant polycystic kidney disease (ADPKD) is a common disorder mostly characterized by cyst formation in kidney tubules. The majority of ADPKD cases is caused by mutations in the PKD1 gene, but no prevalent mutation has been reported. By heteroduplex analysis of the 3' single-copy region of the gene, we have searched for mutations in subjects from 40 ADPKD families of Northern Italy. Seven novel polymorphisms and three novel disease-associated mutations (R3718Q, L3851P and IVS45+56del25) were identified. Both missense mutations are located in the major extracellular loop of polycystin-1. The 25 bp deletion inside intron 45 did not affect 5' and 3' consensus splicing sites, but caused a 56 nucleotide out of frame-deletion due to activation of a cryptic 3' splice site in exon 46. The mutated RNA should produce a truncated polycystin 1 at the G binding peptide in the intracellular C-terminal end of the protein. RT-PCR analysis showed that the disease-associated mutations were present in transcribed sequences. In particular, RNA analysis of BHK cells transfected with PKD1 genomic DNA, including the deleted intron, showed that no normal transcript is produced by the deleted gene. This intronic mutation, found in a large pedigree, seems to be associated with a prevalence of cerebrovascular disease.
Human Mutation 12/2000; 16(5):444-5. · 5.69 Impact Factor
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G Maio,
P d'Argenio,
T Stroffolini, A Bozza,
L Sacco,
M E Tosti,
M Intorcia,
E Fossi,
G d'Alessio,
L A Kondili,
M Rapicetta,
A Mele
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ABSTRACT: The aim of the study was to estimate the prevalence, risk factors and genotype distribution of hepatitis C virus (HCV) in the general population older than 5 years of age in a southern Italian town. The positive predictive value of alanine transaminase (ALT) screening in identifying HCV positive subjects was also assessed.
Cluster random sampling from the census of the general population was used. ELISA and RIBA tests assessed the presence of anti-HCV; nested reverse transcription polymerase chain reaction (RT-PCR) was used to identify HCV-RNA; genotyping was performed by INNO-LIPA III. The association linking anti-HCV seropositivity with potential risk factors was assessed by multiple logistic regression analysis.
Among the 488 subjects enrolled, 79 (16.2%) were anti-HCV positive. The prevalence increased from 1.2% in subjects 6-29 years of age to 42.1% in those > or = 60 years. Forty percent of these positive subjects also had abnormal ALT level and 54.4% were HCV RNA positive by PCR. The positive predictive value of the ALT test in identifying anti-HCV positive subjects was 65%; however, it was 46.7% in subjects younger than 60 years of age and 90.5% in those 60 or older. Genotype 1b was detected in 74% of subjects, type 2c in 23.3%, and type 1a in 2.3%. The only two variables significantly associated with HCV seropositivity in multivariate analysis were age older than 45 years (O.R. 8.5; CI 95%=3.0-24.1) and past use of glass syringes (O.R. 3.4; CI 95%=1.5-7.6).
These findings confirm that HCV infection is endemic in southern Italy, particularly among the elderly. Percutaneous exposure, such as injections with nondisposable, multiple-use, glass syringes used in the past for medical purposes may have played a major role in the spread of HCV infection. ALT screening is not useful in detecting HCV positive subjects in the general population, particularly among subjects who could benefit from antiviral therapy.
Journal of Hepatology 08/2000; 33(1):116-20. · 9.26 Impact Factor
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G Taliani,
D Celestino,
M C Badolato,
A Pennica, A Bozza,
G Poliandri,
V Riccieri,
G Benfari,
A Sebastiani,
C De Bac,
G Quaranta,
A Aceti
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ABSTRACT: Hepatitis C virus genome (HCV-RNA) has been detected in whole salivary gland tissue of chronically infected patients. However, contamination of the tissue by plasma or blood cells was not excluded by the previous reports.
To assess whether HCV infects the salivary gland epithelial cells in patients with chronic HCV liver disease.
Twenty unselected patients with chronic active hepatitis (11 cases) or active cirrhosis (nine cases) were examined. Serum and saliva samples were obtained from all patients, 12 of whom (seven, chronic active hepatitis; five, active cirrhosis) underwent salivary gland biopsy. PCR for HCV-RNA was performed on RNA extracted from serum, saliva and salivary gland epithelial cells collected by isokinetic gradient separation after trypsin digestion of whole salivary gland tissue. Saliva samples were also examined for the presence of secretory IgA anti-HCV by gel chromatography and ELISA testing.
HCV-RNA was detected in all sera with titers ranging from 5.42 x 10(5) genome equivalents/ml to 123.2 x 10(5) genome equivalents/ml. Thirteen patients were infected with genotype 1b, four patients had genotype 1a, two patients had genotype 2a and one patient was unclassifiable. Low titer HCV-RNA (<2 x 10(5) genome equivalents/ml) was detected in 3/20 saliva samples (15%) from highly viremic patients infected with 1b genotype. RNA extracted from salivary gland epithelial cells consistently tested negative for HCV-RNA. In addition, all saliva specimens tested negative for secretory-IgA (S-IgA) anti-HCV, even after a 10-fold concentration of the samples.
There was no evidence that HCV infects the salivary gland epithelial cells in our viremic patients with HCV chronic liver disease. Low level HCV-RNA in saliva is most probably due to virus spillover from blood.
Journal of Hepatology 07/1997; 26(6):1200-6. · 9.26 Impact Factor
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Contributions to nephrology 02/1997; 122:49-52. · 1.49 Impact Factor
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ABSTRACT: Serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) have been examined in 38 patients with chronic hepatitis C liver disease treated with interferon. The sICAM-1 values were found to correlate significantly with the ALT values. Pre-treatment sICAM-1 values of responder and nonresponder patients were not significantly different while, by the end of the treatment, the values of responders were significantly lower compared to those of nonresponders. However, no difference could be found between sustained and relapse responders. Of the 21 patients examined for PBMC HCV-RNA, 15 (71.4%) were found to be positive. Neither the rate of responsivity to interferon treatment, nor the mean sICAM-1 values correlated with the positivity of PBMC HCV-RNA. However, the clearance of serum and PBMC HCV-RNA was associated to a significant decrease of sICAM-1 and ALT levels. In conclusion, sICAM-1 values were found to correlate with ongoing viral replication and liver cytonecrosis, but were not influenced by the concomitant HCV infection of PBMC.
Archives of Virology 02/1997; 142(3):557-65. · 2.11 Impact Factor
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ABSTRACT: We report a large three-generation autosomal dominant polycystic kidney disease family from Northern Italy found to be associated with the PKD2 locus. Hepatic involvement (liver cysts, fibrosis, cholelithiasis or jaundice), subarachnoidal hemorrhage (1 case) and esophageal diverticula (1 case) were present in affected individuals. Among the older members, the males (aged 54-61 years) had hepatic cysts or fibrosis and were on chronic hemodialysis, the females (aged 69 and 70 years) had hepatic cysts, hepatomegaly, mild fibrosis and a mild and moderate renal impairment, respectively. In this family, clinical findings do not differ substantially from those reported for PKD1.
American Journal of Nephrology 02/1997; 17(5):458-61. · 2.54 Impact Factor
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ABSTRACT: We have investigated the Ca2+ dependency of DNA degradation into nucleosome-sized fragments in intact chromaffin-like PC12 cells and PC12 nuclear fractions. In intact cells we were unable to trigger DNA fragmentation by inducing either transient or sustained elevations of cytoplasmic Ca2+ ([Ca2+]i) with the Ca2+ ionophore ionomycin. On the contrary, DNA fragmentation was induced in intact cells by the intracellular Zn2+ chelator NNN'N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN). To characterize further PC12 cell endonuclease activity, we then investigated digestion by purified PC12 cell fractions of exogenously added plasmids. In nuclear fractions two endonuclease activities were identified: an acidic (pH 5.0) endonuclease activity that was fully Ca2+- and Mg(2+)-independent; and a neutral (pH 7.6) endonuclease activity that was Ca(2+)-independent but Mg(2+)-dependent. Both endonuclease activities were inhibited by Zn2+. Nuclear membrane permeabilization greatly enhanced plasmid digestion at pH 7.6, but not at pH 5.0. This suggests that neutral endonuclease was located in a membrane-bound compartment, whereas acidic endonuclease was freely accessible to the substrate even in the presence of an intact nuclear membrane. In intact nuclei, digestion of genomic DNA could not be triggered by increasing the bivalent cation composition of the medium. On the contrary, in hypotonic medium we observed a large spontaneous nucleolytic DNA degradation that was increased by Zn2+ chelation. However, an acidic pH shift was a potent stimulus for DNA fragmentation in isotonic as well as hypotonic medium.
Biochemical Journal 12/1995; 311 ( Pt 3):1033-8. · 4.90 Impact Factor
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ABSTRACT: The polymerase chain reaction (PCR) was used to investigate the presence of positive and negative hepatitis C virus (HCV) RNA strands in serum and peripheral blood mononuclear cells (PBMC) of 20 patients with histologically proven HCV-related chronic liver disease. All patients completed a course of interferon (IFN) treatment (6 MU of IFN-alpha 2b three times a week for 24 weeks) and were followed-up for 12 months after treatment was discontinued. Pre-treatment, end-treatment and 6-month follow-up serum and PBMC samples were examined. At enrollment, the positive strand of HCV-RNA was detected in serum of 18 patients (90%), the negative strand in none. Positive-stranded HCV-RNA was detected in PBMC of 15 patients (75%), 13 of whom also had detectable levels of negative-stranded HCV-RNA in PBMC. By the end of the treatment, 12 patients (60%) were responders. The pre-treatment HCV infection of PBMC, indicated by the presence of both RNA strands, was found in 8 (66.7%) responders compared to 5 (62.5%) non-responders (P = n.s.). End-treatment loss of PBMC HCV-RNA correlated significantly with the response since it occurred in all responders compared to 2 non-responders (P = 0.02). However, end-treatment-negative serum and PBMC HCV-RNA did not predict the occurrence of a sustained response, which was observed at month 12 in 5 of 12 responders (P = n.s.). On the other hand, the persistent absence of HCV RNA in serum and PBMC at the end of the 6-month follow-up was significantly associated with the occurrence of a sustained response (P < 0.0001).
Journal of Medical Virology 10/1995; 47(1):16-22. · 2.82 Impact Factor
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ABSTRACT: The genetic mutation underlying Huntington's disease (HD) has been identified as an expansion and instability of a specific CAG repeat sequence in a gene on chromosome 4. A simple polymerase chain reaction assay has been used for the assessment of the (CAG)n expansion in a 72-year-old woman with typical HD symptoms, but no family history of the disorder. The DNA analysis showed that the patient had an allele with 41 repeat units, in the size range seen in HD chromosomes. Therefore, HD diagnosis is confirmed in this seemingly sporadic case and the disease is newly diagnosed in a large family. The risk of inheriting this unstable expanded allele is discussed. INTRODUCTION--The discovery of an expansion of a trinucleotide (CAG) repeat region in the IT15 gene on the short arm of chromosome 4 has identified the mutational mechanism causing Huntington's disease (HD) and enables the direct diagnosis of affected subjects based on DNA analysis alone. Here a 72-year-old woman with typical HD symptoms, but no family history of the disorder, has been unambiguously diagnosed by using a quick DNA analysis. This is relevant because the disease is newly diagnosed in a large family. MATERIAL AND METHODS--A labelled polymerase chain reaction (PCR) test has been used to amplify the repeat region of the IT15 gene and DNA fragments were analyzed by Polyacrylamide gel electrophoresis. RESULTS--The number the CAG repeats in the proband displayed two alleles of 23 and 41 repeats, respectively. Since normal chromosomes are reported to contain 11-34 repeats, the clinical appearance of HD in the proband is explained by the presence of the repeat expansion. DISCUSSION--The parents of the proposita both died aged over 80 y apparently without neurological signs referable to HD. Hence, this is presumably a sporadic case of the disease. Because of the length of 41 repeats of this HD chromosome, offspring of this proband could inherit the expanded allele with 37 repeats, as expected for the reversal of the trinucleotide expansion. A subject with this intermediate allele could be affected, but would not be affected if the HD IT gene with reduced triplets had recovered its normal function. Thus, in a seemingly sporadic case like the one reported here, despite the PCR analysis, the risk of transmission of HD to her offspring may remain uncertain.
Acta Neurologica Scandinavica 09/1995; 92(2):132-4. · 2.47 Impact Factor
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ABSTRACT: In the spring of 1994, anti-HCV prevalence and associated risk factors were evaluated in 681 subjects representing all age-groups in the general population of a small central Italian town. The overall anti-HCV prevalence was 8.4%, ranging from 3.7% in the 30-39 age-group to 18.2% (p < 0.01) in the 60-70 age-group; no subject below 30 years of age was positive. Multiple logistic regression analysis showed that the only variables independently associated with anti-HCV positivity were awareness of unspecified liver disease (O.R. 3.58), age > 45 years (O.R. 2.72), and lowest number of years of schooling (O.R. 11.0) while no association was found with any parenteral exposure such as blood transfusion, intravenous drug use, major or minor surgical intervention, use of glass syringes or dental therapy. The HBsAg prevalence in this population was 1.3%, which corresponds to the rate reported in central Italy. These findings show a high level of HCV endemicity, with no evidence of parenteral exposure.
The Italian journal of gastroenterology 07/1995; 27(5):235-8.
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ABSTRACT: Two sources of variation in the huntingtin gene, the length of the CCG-rich segment downstream to the (CAG)n stretch undergoing expansion in Huntington disease (HD) and the deletion of 3 bp at codon positions 2642-2645 (delta 2642), were analysed on the normal and HD chromosomes of 80 Italian families affected with HD. No instances of meiotic instability of the CCG-rich segment were detected. A strong linkage disequilibrium was found between the HD mutation and alleles at both polymorphic regions: CCG-rich length alleles different from 176 bp are underrepresented while delta 2642 is overrepresented on HD chromosomes. The presence of such alleles on HD chromosomes does not affect age at onset of the disease. Normal chromosomes displayed a non-random association, shorter (CAG)n segments being preferentially followed by longer CCG-rich segments. Finally, the finding, among normal subjects, of carriers of variants on both chromosomes denotes that variation at either of the two polymorphisms does not impair the function of the huntingtin gene product.
Human Molecular Genetics 08/1994; 3(7):1129-32. · 7.64 Impact Factor
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Journal of Hepatology 07/1994; 20(6):845. · 9.26 Impact Factor
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ABSTRACT: Two (CA) microsatellite polymorphisms have been studied in 209 subjects from Northern Italy, members of 27 Polycystic Kidney Disease families. Polymorphic alleles were analyzed by using a labelled PCR. Results obtained show that these markers highly improve the presymptomatic diagnosis of the disease in subjects at risk. In addition, our findings suggest that a region with a high recombination frequency should exist between PKD1 and SM7.
Bollettino della Società italiana di biologia sperimentale 05/1994; 70(4):129-33.
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ABSTRACT: The effects of androgen and estrogen on cell growth and gene expression were investigated in KJ29 kidney epithelial cells. Incorporation of 3H leucine and 3H thymidine was increased by androgen at 10nM, but not by estrogen. Estrogen however, inhibited the effects induced by androgen. In addition, cell number and the proliferation marker Ki67 were increased by androgen, but not by estrogen. Levels of androgen receptor RNA, as detected by RT-PCR and Northern blot analysis, were not affected by either androgen or estrogen. Levels of estrogen receptor RNA could be detected only by RT-PCR, and disappeared after estrogen treatment. These studies show that sex steroid receptors are differently expressed in KJ29 cells, and suggest that androgen, via its canonic receptor, acts as a mitogenic factor in human kidney cells, whereas estrogen has an antiandrogenic action.
Biochemistry and molecular biology international 04/1994; 32(4):597-604.
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ABSTRACT: The presence of circulating hepatitis C virus genome (HCV-RNA), elevated ALT levels and antibodies to an NS5-derived synthetic peptide have been examined in 13 subjects with isolate positivity for antibodies to the HCV core antigen (C22) on RIBA-2 testing. All subjects were followed up for 8-18 months (mean 12.4 months). In seven subjects (54%), intermittent or persistent viremia was associated with abnormal ALT levels (6 subjects) and with positivity for antibodies to NS5-peptide (6 subjects). On the other hand, in 6 out of 13 subjects (46%) no viral replication, no liver cytonecrosis and no antibodies to NS5 were found. It is concluded that isolate reactivity to C22 by RIBA-2 is a heterogeneous condition that corresponds to two distinct categories of subjects: those with active HCV infection and those without evidence of virus replication. Although HCV-RNA determination is the most reliable means of identifying HCV carriers, antibodies to NS5 can be a useful marker of virus activity. In fact, antibodies to NS5 were detected in 6 out of 7 viremic patients, compared to 0 out of 6 non-viremic patients (P = 0.004). It remains to be elucidated whether the isolate reactivity to core antigen found in non-viremic subjects represents a specific, HCV-induced antibody response, or is an unrelated crossreactivity.
Archives of virology. Supplementum 02/1993; 8:219-28.
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ABSTRACT: Ristocetin induces a conformational change on von Willebrand Factor (vWF) similar to that due to the interaction with the subendothelium, by which the former can interact with the Glycoprotein-1 B (GPIB) of the platelet membrane and trigger aggregation and granule content secretion. Platelet Rich Plasma (PRP) treated with Acetyl Salicylic Acid (ASA) loses completely the aggregability induced by addition of Ristocetin whereas ASA-treated and successively Washed Platelets (AWP) supplemented with normal plasma (PPP) give an aggregation and a secretory response to Ristocetin similar to that given by PRP; similarly normal Washed Platelets (WP) supplemented with ASA-treated plasma (APPP) give identical aggregation, and secretion by Ristocetin addition. Ours results indicate that the Ristocetin-vWF complex can trigger two distinct intraplatelet metabolic pathways. A first well known way starts from the activation of Phospholipase A-2 (PL-A2), by which arachidonic acid is produced, that, in turn, undergoes the metabolic pathway leading to Thromboxane A-2; this pathway can be blocked by the intraplatelet ASA by irreversible inactivation of Cyclooxygenase, but it is insensitive to the extra-platelet ASA. A second, independent metabolic pathway, can be triggered by intact vWF, but not by the ASA treated one. It is insensitive to intraplatelet ASA and therefore unrelated to the arachidonic acid metabolism. This pathway could start from the activation of Phospholipase C (PL-C).
Bollettino della Società italiana di biologia sperimentale 11/1992; 68(10):607-12.
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ABSTRACT: Three yeast artificial chromosome (YAC) libraries were constructed using two human cell lines and the pYAC-RC vector. The main differences from the previously described methods were: i) genomic DNA was digested in low melting point (LMP) agarose blocks with the rare cutting enzyme ClaI; ii) DNA was ligated in melted LMP agarose after agarase treatment; iii) spheroplast regeneration plating was done in calcium alginate thin layer. In addition, a panel of PCR primers was used to identify quickly the presence in the libraries of repetitive and single copy human DNA sequences.
Biochemistry international 07/1992; 27(1):45-53.
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ABSTRACT: A simple HPLC assay for serum guanase based on the direct determination of enzymatically formed xanthine was applied to normal and pathological sera. The procedure is sensitive, precise (CV below 5%) and suitable for routine purposes, and the method requires only 50 microL of sample. Using this method the reference range as determined from the sera of 40 healthy adult controls is 0-1.1 U/L. In patients with various liver diseases serum guanase activities were found to be increased 5- to 50-fold compared with the normal mean value.
Biomedical Chromatography 9(3):130-4. · 1.97 Impact Factor
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C De Bac,
C Clementi,
F Duca,
D Livoli,
G Poliandri, A Bozza,
A Osso,
S Martuscelli,
C Pasquazzi,
P Petrucci,
C Furlan,
G Taliani
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ABSTRACT: In order to improve our knowledge of the incidence of liver cirrhosis in Italy, we conducted two epidemiological studies. The first study showed that about 15% of asymptomatic subjects with persistent increase in alanine aminotransferase had histological evidence of cirrhosis. In this setting, cirrhosis was associated with viral aetiology in 91.4% of cases. In the second study, which enrolled cirrhosis patients from 13 centres from all regions of the country, viral infections were detected in 82.6% of patients, the large majority of whom, 71.2%, were positive for hepatitis C virus (HCV). Alcohol abuse was present in 8.7% of cases as exclusive aetiological factor. All the patients were classified according to Child-Pugh and were scored as class A in 62.4%, as class B in 23.8% and as class C in 13.8% of cases. The age distribution showed that about 55% of cirrhosis patients were under 60 years of age; 34.3% of them had a Child-Pugh score of class B or C. These data show that HCV infection represents the predominant aetiological factor of cirrhosis in Italy and that cirrhosis can be found frequently in asymptomatic subjects.
Research in Virology 148(2):139-42.
