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P Pinzani

Publications and source records attributed to P Pinzani.

At least 19 recordsLinked to original sources
PubMed1999

Real-time quantitative PCR for the measurement of MYCN amplification in human neuroblastoma with the TaqMan detection system.

BACKGROUND: Neuroblastoma is the most common extracranial malignant solid tumor in children under 5 years and is characterized by a wide clinical and biological heterogeneity, from spontaneously regressive forms to cancers with a rapid and fatal progression. MYCN oncogene amplification is considered the most important prognostic factor to evaluate survival and therapeutic choices in these patients. METHODS: Here we present a new assay for rapid and accurate measurement of MYCN amplification, based on real-time quantitative PCR with the TaqMan(TM) reaction. The degree of MYCN amplification was derived from the ratio of the MYCN oncogene and the single-copy reference gene, beta-actin. The absolute abundance of these two genes in tumor sample DNA was obtained by extrapolation on external calibration curves generated with reference DNA. RESULTS: We found a variable degree of MYCN amplification, from 2 to 29, in 26 of 49 (53%) neuroblastomas. These results were well correlated to those obtained with a competitive PCR assay in the same samples (r = 0. 987). MYCN amplification was associated mainly with advanced cancer stages, and the analysis of overall survival confirmed that the measurement of MYCN amplification is a predictor of patient outcome in neuroblastoma. Patients without MYCN amplification had a cumulative survival significantly higher than patients with low (<9; P = 0.02) and high (>/=9; P = 0.03) oncogene amplification. CONCLUSION: The assay is rapid and reproducible and does not require any post-PCR analytical procedure.

Calibration↗
PubMed1998

Developments in quantitative PCR.

In recent years the growing interest in quantitative applications of the polymerase chain reaction (PCR) has favoured the development of a large number of assay procedures suitable for this purpose. In this paper we review some basic principles of quantitative PCR and in particular the role of reference materials and calibrators and the different strategies adopted for nucleic acid quantification. We focus on two methodological approaches for quantitative PCR in this review: competitive PCR and real-time quantitative PCR based on the use of fluorogenic probes. The first is one of the most common methods of quantitative PCR and we discuss the structure of the competitors and the various assay procedures. The second section is dedicated to a recent promising technology for quantitative PCR in which the use of fluorogenic probes and dedicated instrumentation allows the development of homogeneous methods. Assay performance of these methods in terms of practicability and reliability indicates that these kinds of technologies will have a widespread use in the clinical laboratory in the near future.

Animals↗
PubMed1997

Evaluation of methotrexate sensitivity in human leukemia cell lines by an adenosine triphosphate bioluminescence assay.

To verify a recently developed in vitro tumor chemosensitivity assay (TCA) based on adenosine triphosphate (ATP) measurement for detection of methotrexate (MTX) sensitivity or resistance in human leukemias and solid tumors in which the antifol is indicated, we investigated the ability of the assay to discriminate between sensitivity and resistance to this antifolate in human leukemia cell lines sensitive (parental CCRF-CEM/S) and resistant (CCRF-CEM/E, CCRF-CEM/T and CCRF-CEM/P sublines) to MTX by virtue of known biochemical mechanisms. Correlation experiments with a standard cell growth inhibition assay and a radiometric method for measurement of thymidylate (dTMP) synthesis ([5-3H]-2'-deoxyuridine tritium release assay) were performed. No significant differences were observed in the IC50 values for the four cell lines tested as determined by cell growth evaluation (cell number counts) and the ATP-TCA after a 72 h MTX exposure. After short-term (4 h) high-dose MTX exposure, no significant correlation between ATP-TCA and the classic [5-3H]-2'-deoxyuridine tritium release assay was observed in both CCRF-CEM/S and CCRF-CEM/P cells. CCRF-CEM/T and CCRF-CEM/E displayed, instead, complete resistance with both methods. When using conditions proposed for clinical application (long-term exposure, i.e. 144 h) the ATP-TCA permitted the identification of cell lines highly resistant to MTX (CCRF-CEM/F and CCRF-CEM/E), while intermediate MTX resistance due to altered polyglutamylation was not detectable. Detection of this kind of resistance was obtained, as expected, using a short-term exposure (4 h) to MTX followed by a long-term efflux (72 h) in drug-free medium. On the basis of these results, ATP-TCA appears to be a suitable method for the evaluation of cytotoxicity induced by MTX.

Adenosine Triphosphate↗
PubMed1997

Quantitative polymerase chain reaction-based homogeneous assay with fluorogenic probes to measure c-erbB-2 oncogene amplification.

We describe a PCR-based assay for determining c-erbB-2 oncogene amplification in breast cancer in which we use the TaqMan system. Two fluorogenic probes anneal to the target between primers for c-erbB-2 and beta-globin genes and contain both a reporter dye (6-carboxy-fluorescein) and a quencher dye (6-carboxy-tetramethyl-rhodamine). During the extension phase of the PCR cycle, the 5'-->3' exonuclease activity of Taq polymerase cleaves the hybridized fluorogenic probe, resulting in an increase of fluorescence emission of the reporter dye that is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. Assay performance showed adequate precision and a lower detection limit and good correlation with the results obtained in the same samples by a competitive PCR assay (n = 25, r = 0.94, P < 0.01). This homogeneous assay is time-saving, avoids usually cumbersome postamplification procedures (that can be additional sources of inaccuracy and contamination), and seems suitable for determination of c-erbB-2 oncogene amplification in tumor specimens.

Base Sequence↗
PubMed1996

Detection of c-erbB-2 amplification in transitional cell bladder carcinoma using competitive PCR technique.

PURPOSE: We used competitive PCR to verify retrospectively the prognostic significance of c-erbB-2 oncogene amplification in transitional cell bladder carcinomas as a predictive index of patient survival with a maximum follow-up of nine years, and to investigate the variations of c-erbB-2 amplification during bladder carcinoma recurrence and/or progression from superficial to more invasive states. MATERIALS AND METHODS: Oncogene amplification was determined by an accurate and sensitive procedure based on competitive PCR. Measurements were performed in DNA extracted from fresh cancers or from formalin-fixed, paraffin-embedded tumor samples. RESULTS: The overall mean incidence of c-erbB-2 oncogene amplification was 26% (24/92), with a significant relationship with tumor grade (p < 0.0001). We did not find any statistical difference in survival probability between subjects with (20%) or without (30%) oncogene amplification. During tumor progression we observed a limited increase of tumors carrying oncogene amplification (2 of 20) whereas the mean degree of amplification was not affected. CONCLUSIONS: c-erbB-2 amplification seems to be a genetic event related to the degree of bladder tumor differentiation. However the presence and/or the degree of this genetic alteration do not seem predictive of tumor progression, recurrence and survival probability, at least in patients with advanced transitional cell bladder carcinoma. These data seem to indicate that the amplification of c-erbB-2 in bladder carcinoma could be considered as an epiphenomenon, present in a subset of tumors but apparently not related to the clinical outcome.

Aged↗
PubMed1995

Methotrexate chemosensitivity by ATP luminescence in human leukemia cell lines and in breast cancer primary cultures: comparison of the TCA-100 assay with a clonogenic assay.

Chemosensitivity assays are widely used to predict the ability of tumor cell lines to respond to potential or existing cytotoxic drugs. In this study we have compared the cell cloning assay first described by Salmon and Hamburger with a recently developed assay which measures viable cell number by ATP luminescence. Methotrexate (MTX) was chosen as the test agent, since cell lines with varying degrees of sensitivity to this agent were readily available. The results shown good correlation between the two assays, both of which are able to discriminate between the various cell lines used. MTX inhibition of primary breast carcinomas and cell lines shows a steep dose-response curve with a threshold concentration above which increasing dose does not increase sensitivity. In solid tumors, the plateau is usually reached at a level well below 100% inhibition. The ATP luminescence assay allows discrimination of MTX sensitivity between breast carcinomas and has considerable technical advantages over the cloning assay.

Adenocarcinoma↗
PubMed1995

Gene amplification for c-erbB-2, c-myc, epidermal growth factor receptor, int-2, and N-myc measured by quantitative PCR with a multiple competitor template.

We recently proposed a quantitative PCR procedure for the absolute measurement of c-erbB-2 oncogene amplification, based on the simultaneous polymerase chain reaction (PCR) amplification of the target gene and of a competitor DNA molecule acting as internal standard. To increase the number of assayable oncogenes and the accuracy of the quantitative comparison of gene amplification degree within the same tumor, we have now constructed a single synthetic competitor for int-2, c-myc, N-myc epidermal growth factor receptor, and c-erbB-2 genes, and for the reference gene beta-globin. This competitor was constructed by a two-step recombinant PCR procedure and inserted as a 297-bp sequence in a plasmid. The order of primer insertion was designed to obtain competitors of comparable sizes to those of the respective genomic targets, but still easily recognizable from the latter ones by gel electrophoresis. The clone competitor was tested to evaluate the linearity range for each assay. The present application of quantitative PCR based on a multiple competitor represents the first approach for the achievement of a single reagent for the evaluation of a panel of genes potentially amplified in human tumors.

Base Sequence↗
PubMed1994

Adenosine triphosphate release by osmotic shock and hemoglobin A1C in diabetic subjects' erythrocytes.

We investigated the significance of adenosine triphosphate (ATP) release from diabetic subjects' red blood cells (RBCs) following osmotic shock (OS) and its possible relationship with hemoglobin A1C (HbA1C) and with the RBC membrane protein skeleton. RBCs from type I (insulin-dependent [IDDM]) and type II (non-insulin-dependent [NIDDM]) diabetic subjects and age- and sex-matched control subjects were submitted to OS using NaCl solutions (from 0.9% to 0.045% final concentration). ATP release values were determined by the bioluminescent method. For pattern study, they were expressed both as absolute values and as percentages (%) of ATP maximum release (at 0.045% NaCl solution). Twenty-seven IDDM and 25 NIDDM subjects and two control groups were investigated. ATP content in RBCs was 2.08 +/- 0.19 pmol/10(4) RBC in IDDM and 1.23 +/- 0.20 pmol/10(4) RBC in NIDDM subjects. The ATP content of IDDM subjects' RBCs was significantly higher than that of the corresponding control group. ATP release at 0.49% NaCI OS, both as absolute value and as percentage value, was significantly lower in both diabetic groups, and ATP% was inversely correlated with HbA1" (IDDM: r = -.489, P < .01; NIDDM: r = -.654, P < .01), suggesting a possible relationship between Hb glycation, RBC membrane protein skeleton glycation, and its influence on ATP release by OS. In conclusion, the proposed method seems useful for measuring RBC ATP content and, at the same time, for monitoring the leak effect of the RBC membrane before it bursts.

Adenosine Triphosphate↗
PubMed1994

Measuring c-erbB-2 oncogene amplification in fresh and paraffin-embedded tumors by competitive polymerase chain reaction.

We present an original application of competitive polymerase chain reaction (PCR) for measuring oncogene amplification in DNA from human tumors by simultaneous PCR amplification of genomic DNA with fixed amounts of an internal standard (competitor DNA). Competitors share the same sequence as the target genes but contain an additional 15- to 20-base-pair insert, which allows resolution of the amplified products after polyacrylamide gel electrophoresis and ethidium bromide staining. The gene copy number is derived from the ratio between the intensities of the bands corresponding to the amplified products. Using this procedure, we measured c-erbB-2 amplification in breast and bladder carcinomas in both fresh tumor tissues and paraffin-embedded tissue samples and assessed the precision, sensitivity, and accuracy of the assay. Competitive PCR is a simple, reliable, and accurate method for the evaluation of c-erbB-2 amplification and is potentially suitable for use in the clinical laboratory.

Base Sequence↗
PubMed1989

Measurement of estrone-3-glucuronide and pregnanediol-3 alpha-glucuronide in early morning urine samples to monitor ovarian function.

The determination of the concentration of estrone-3-glucuronide and pregnanediol-3 alpha-glucuronide has been performed by a chemiluminescent immunoassay in early morning urine samples of 14 normal menstruating women and 11 women affected by luteal phase defect. The early morning urine samples were daily collected for an entire menstrual cycle. We have employed a timed and measured volume collection procedure as correction factor. The integrated values of the hormonal data in definite time intervals were used to create a nomogram. By means of this method, it was possible to completely separate normal from luteal insufficiency subjects and to distinguish two different types of luteal phase defects. Moreover, the same approach was applied to the study of the role and the frequency of luteal phase defect in 15 patients affected by habitual abortion and in 17 premenopausal women who had undergone quadrantectomy for T1a No Mo breast cancer. A luteal phase defect was detected in nine of the aborting patients (60%) and in eight women affected by breast cancer (47%). Finally estrone-3-glucuronide was measured in early morning urine samples of 96 prepubertal and pubertal girls in different pubertal stages and in one patient affected by precocious puberty, before and during an agonist GnRH treatment. The urinary test of ovarian function seems to be suitable for diagnostic purposes and for clinical studies.

Abortion, Habitual↗
PubMed1989

A chemiluminescent immunoassay for the direct measurement of urinary 5 alpha-androstane-3 alpha, 17 beta-diol-glucuronide in urine.

We described a chemiluminescent immunoassay (CIA) for 5 alpha-androstane-3 alpha, 17 beta-diol-glucuronide (3 alpha-diol-G) in human diluted urine. This method allowed the direct measurement in 1 microliter of urine avoiding the hydrolysis and extraction steps for sample pretreatment commonly used in routine methods. The hapten 3 alpha-diol-G was synthesized by a Koenigs-Knorr reaction. The immunogenic complex, 3 alpha-diol-G conjugated to bovine serum albumin (BSA), was employed to induce the formation of specific antibodies in New Zealand rabbits. In addition, the required chemiluminescent (CL) tracer was prepared. The characteristics of the antibody was determined as regard to specificity and sensitivity and the precision of the assay methods established. In 22 hirsute women affected by polycystic ovarian syndrome we found 3 alpha-diol-G values significantly (p less than 0.01) higher (146.28 +/- 73.77 micrograms/g of creatinine; mean +/- SD) than those observed in normal women (72.1 +/- 32.58 micrograms/g of creatinine; mean +/- SD).

Adult↗
PubMed1989

A chemiluminescent method for the measurement of pregnanetriol-3 alpha-glucuronide in human diluted urine.

Pregnanetriol-3 alpha-glucuronide (PTG) is the majority urinary metabolite of 17-hydroxyprogesterone (17OHP) and it typically increases in the commonest form of congenital adrenal hyperplasia (CAH), due to 21 hydroxylase deficiency. We developed a simple chemiluminescent immunoassay for the direct measurement of PTG in diluted urine in order to avoid the preliminary hydrolysis and extraction steps that are usually employed in gas-liquid chromatographic methods. The immunogenic complex PTG-bovine-serum-albumin was used to induce the formation of specific antibodies in New Zealand rabbits. In addition, PTG was conjugated to aminoethylethylisoluminol and the resulting tracer was characterized by mass spectrometry and used to monitor the immunological reaction. The characteristics of the antibody were determined with regard to specificity and sensitivity. The precision of the assay method was also established. PTG excretion was studied before and after the ACTH stimulation test (1 mg synthetic ACTH i.m.) in 11 normal women and in one subject affected by CAH due to 21-hydroxylase deficiency. PTG levels well correlated with 17OHP plasma concentrations both under basal and stimulated conditions, in normal women as well as in the patient affected by CAH.

17-alpha-Hydroxyprogesterone↗
PubMedDate not supplied

Total antioxidant capacity (TAC): is it an effective method to evaluate the oxidative stress in uraemia?

Lipoprotein abnormalities are common in uraemia and are considered important factors for development of atherosclerosis and progression of renal disease. Reduction of total antioxidant capacity (TAC) and lipid peroxidation (LP) probably play a major role in both processes. The aim of this study was to assess the effect of renal function, dietary manipulation and lipids on TAC of uraemic patients with different chronic renal failure (CRF). Sixty patients (36M, 24F), aged 60 +/- 12 years were divided into five groups according to serum creatinine levels (sCr,mg/dl)--CRFI, 1.5-3; CRFII, > 3-5.5; CRFIII, > 5.5; CRFIV, > 3 on vegetarian supplemented diet (SD); CRFV haemodialysis patients (HD)- and investigated for TAC by enhanced chemiluminescent assay, autoantibodies against oxidized LDL (oxLDLAb), lipids, apolipoprotein AI, B, Lp(a) and uric acid (UA). The results were compared to a control group of 19 people (8M, 11F), aged 52 +/- 11 years with sCr < 1.5. TAC increased significantly with the progression of CRF and was strongly related to both sCr and UA. Lipids and SD did not show any influence on TAC. Unexpectedly, lipid peroxidation did not correlate to TAC, neither to sCr or UA. HD accounted for a mild reduction of both TAC and LP. Patients on SD showed a marked reduction of LP as compared to patients with a similar degree of renal failure (CRF-III) but on conventional diet. Our results suggest that elevated TAC in uraemia is likely to be dependent on increased UA levels and does not seem to induce an effective protection in vivo from oxidative stress. In conclusion, TAC does not appear to be a reliable method for assessing the oxidative susceptibility of CRF patients.

Adult↗
PubMedDate not supplied

Serum antioxidant capacity in healthy and diabetic subjects as determined by enhanced chemiluminescence.

Free radicals are considered to be important factors involved in many physiopathological processes. Several methods have been proposed for studying the mechanisms of antioxidant protection against free radical-induced injury, including the measurement of the total antioxidant capacity (TAC) in body fluids, based on enhanced chemiluminescence. This technique is calibrated against Trolox and assay results are expressed as mumol/L of Trolox equivalents. Since many of the complications induced by diabetes appear to be mediated by oxygen free radical generation, we have investigated serum antioxidant capacity in a group of healthy subjects and in insulin-dependent diabetic (IDDM) subjects. A statistically significant difference was noticed in TAC values between the IDDM group and the young control group. Even if the biological meaning of this significant reduction in TAC remains to be explained, an overproduction of precursors of reactive oxygen free radicals and/or a decreased scavenger systems efficiency can be associated with the increased risk of atherosclerotic cardiovascular disease in diabetic patients.

Adult↗
PubMedDate not supplied

Measurement of urinary somatomedin C by a chemiluminescent method.

We present a method for the measurement of the total Somatomedin C (SmC) content in human early morning urine samples after dialysis, extraction, and concentration. We modified a chemiluminescence immunoassay, previously developed for SmC determination in serum, for analysis of SmC in urine. Appropriate sensitivity was obtained by the preparation of a new chemiluminescent tracer (AEEI-COOH-SmC) and the optimization of a competitive non-equilibrium immunoassay system which had a detection limit of 0.24 fmol SmC per tube.

Carrier Proteins↗
PubMedDate not supplied

Image analysis in quantitative PCR. An application for the measurement of c-erbB-2 oncogene amplification in DNA from human tumours.

We present an application of image analysis for the direct quantification of PCR products after gel electrophoresis and ethidium bromide staining of DNA. This procedure has been applied to the development of an assay based on competitive PCR for the measurement of the degree of amplification of c-erbB-2 oncogene in DNA from human tumours. In this method two DNA species (genomic and competitor) compete for PCR amplification. Since results are calculated from the final competitor/genomic ratio any variable affecting the rate of PCR amplification has no effect on the accuracy of the ratio measurement. Results are reported which show that even large variations in the experimental conditions (number of PCR cycles, sample volumes and extracted DNA quality) did not interfere with the precision of the measurement of the competitor/genomic ratio.

Binding, Competitive↗