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Biomedical subjects

M Clementi

Publications and source records attributed to M Clementi.

At least 127 records · Page 7Linked to original sources
PubMed1996

Hepatitis C virus infection and mixed cryoglobulinaemia: assessment of HCV RNA copy numbers in supernatant, cryoprecipitate and non-liver cells.

The aim of this study was to characterize hepatitis C virus (HCV) infection in patients with mixed cryoglobulinaemia (MC). The HCV RNA copy number was assayed in clinical specimens from 15 consecutive patients with MC and HCV infection. Absolute quantification of HCV RNA molecules was performed using a competitive reverse transcription-polymerase chain reaction (cRT-PCR). Specific HCV RNA sequences were detected and quantified in plasma samples from all patients (mean HCV RNA copy number 4.9 x 10(6) ml-1 plasma). A high concentration of HCV RNA molecules was detected in the cryoprecipitates of eight of the 15 patients, who had a cryoprecipitate/supernatant ratio higher than 3.0 (range 3.60 to 186.80): in the remaining seven patients this ratio was close to or lower than 1.0 (range 0.13 to 1.60). Quantitative analysis of HCV RNA molecules in cells other than hepatocytes (i.e. peripheral blood mononuclear cells (PBMCs) and bone marrow cells (BMCs), in which the HCV replicative intermediate was detected using strand specific RT-PCR, demonstrated that infection is detectable in nearly 60% of these extrahepatic cells. Quantitative analysis of HCV RNA in PBMCs and BMCs revealed low levels of viral nucleic acids.

Aged↗
PubMed1996

Dynamics and modulation of human immunodeficiency virus type 1 transcripts in vitro and in vivo.

The dynamics of human immunodeficiency virus type 1 (HIV-1) transcription was analyzed in vitro and in vivo by using a specific molecular approach which allows accurate quantitation of the different classes of viral mRNAs. Unspliced (US) and multiply spliced (MS) HIV-1 transcripts were assayed by competitive reverse transcription (cRT)-PCR, using a single competitor RNA bearing in tandem internally deleted sequences of both template species. Acute HIV-1 infection of primary peripheral blood mononuclear cells (PBMCs), monocytes/macrophages cells, and the A3.01 T-lymphocyte-derived cell line was studied; both classes of HIV-1 mRNAs increased exponentially (r2 > 0.98) at days 1 to 3 and 1 to 4 postinfection in HIV(IIIB)-infected A3.01 cells and PBMCs, respectively, whereas monocytes/macrophages infected with monocytotropic HIV(BaL) exhibited a linear (r2 = 0.81 to 0.94) accumulation of US and MS transcripts. Following induction of chronically infected ACH-2 cells, MS transcripts increased 2 h postinduction and peaked at 5 h (doubling time, 58 min), while at 24 h, US mRNAs increased 3,053-fold compared with basal time (doubling time, 137 min). To address the biopathological significance of HIV-1 expression pattern during infection progression, pilot cross-sectional and longitudinal analyses were carried out with samples from untreated and treated HIV-1-infected patients. In almost all untreated (recently infected, long-term nonprogressor, and progressor) patients, MS transcript levels followed the general trend of systemic HIV-1 activity. In patients under treatment with powerful antiretroviral compounds, viral MS transcripts rapidly fell to undetectable levels, indicating that in vivo, levels of MS mRNAs in PBMCs are closely associated with the number of newly infected cells and suggesting a new role for the quantitative analysis of HIV-1 transcription in infected patients.

Cell Line↗
PubMed1996

Clinical application of genetic polymorphism in neurofibromatosis type 1.

The authors report the study of DNA polymorphic sequences, 5 intragenic and 5 flanking the NF1 gene, in 87 Italian NF1 families for a total of 142 affected individuals and 204 non-affected relatives. All PCR-based analyses are easy and simple to perform, and require small amounts of DNA. The non radioactive method used is sensitive, rapid, and has low background. All subjects were informative for at least 2 markers. The use of linkage study to familial cases allowed us to exclude the diagnosis prenatally in two fetuses, and to confirm or exclude diagnosis in those relatives with clinical signs, but not fulfilling the international diagnostic criteria. Furthermore indirect analysis permitted the detection of large gene deletions by loss of heterozygosity of one or more DNA markers in three out of 47 sporadic cases.

Female↗
PubMed1995

Quantitative molecular methods in virology.

During the past few years, significant technical effort was made to develop molecular methods for the absolute quantitation of nucleic acids in biological samples. In virology, semi-quantitative and quantitative techniques of different principle, complexity, and reliability were designed, optimized, and applied in basic and clinical researches. The principal data obtained in successful pilot applications in vivo are reported in this paper and show the real usefulness of these methods to understand more details of the natural history of viral diseases and to monitor specific anti-viral treatments in real time. Theoretical considerations and practical applications indicate that the competitive polymerase chain reaction (cPCR) and competitive reverse-transcription PCR (cRT-PCR) assay systems share several advantages over other quantitative molecular methodologies, thus suggesting that these techniques are the methods of choice for the absolute quantitation of viral nucleic acids present in low amounts in biological samples. Although minor obstacles to a wide use of these quantitative methods in clinical virology still remain, further technical evolution is possible, thus making the quantitative procedures easier and apt to routine applications.

DNA, Viral↗
PubMed1995

Genetic control of the CD4/CD8 T-cell ratio in humans.

We studied the genetic pattern of inheritance of the ratio between circulating CD4+ and CD8+ T lymphocytes in a population of healthy donors. The distribution of the CD4/CD8 ratio in males and females was significantly different and was significantly affected by age. In 46 randomly selected families, the parental CD4/CD8 ratio significantly influenced the ratio in offspring. Complex segregation analysis of the data rejected the non-genetic hypothesis; among the genetic models tested, a major recessive gene with a polygenic component and random environmental effects was the most parsimonious model. These findings indicate that the ratio of CD4+ and CD8+ T cells is genetically controlled in humans.

Adult↗
PubMed1995

Hepatitis B virus precore mutants in HBeAg carriers with chronic hepatitis treated with interferon.

Precore mutants of hepatitis B virus (HBV) were looked for in 18 hepatitis B e antigen (HBeAg) carriers who were treated with recombinant interferon-alpha (rIFN) and the results were compared with those obtained in 12 untreated carriers who underwent spontaneous HBeAb seroconversion. Molecular analysis of the HBV precore region was carried out by polymerase chain reaction (PCR) amplification and direct sequencing. Precore mutants with a stop codon at codon 28 were detectable at baseline in 19/30 carriers. However, wild-type strains predominated in the baseline sera of both treated (n = 16) and untreated (n = 10) patients. Sera from the remaining four patients contained predominantly or exclusively mutant virions. Following IFN treatment, there was a shift from the wild-type pattern to the mutant pattern in all patients, with the precore pattern prevailing in long-term responders (six out of nine) compared with the non-responders (none of nine). The wild-type pattern predominated among the non-responders (eight vs three), suggesting that the long-term response to IFN was associated with take-over of precore mutants. There were no relationships between any pretreatment precore molecular pattern and disease severity or outcome of treatment. Precore mutants also took over in 10 of the 12 untreated patients (83%), who underwent spontaneous HBeAb seroconversion. Thus, a shift from wild-type to precore mutant pattern occurs in most Italian patients undergoing IFN-induced or spontaneous HBeAb seroconversion.

Adolescent↗
PubMed1995

Quantitative molecular monitoring of human immunodeficiency virus type 1 activity during therapy with specific antiretroviral compounds.

Methods for the absolute quantitation of nucleic acids present in small amounts in biological samples (competitive PCR and competitive reverse transcription PCR) were applied to the direct monitoring of specific anti-human immunodeficiency virus type 1 (HIV-1) therapy. With these techniques, different parameters of HIV-1 activity (including genomic RNA copy numbers in plasma, proviral and late transcript copy numbers in peripheral blood lymphocytes, and mean transcriptional activity per each HIV-1 provirus) were monitored during therapy with azidothymidine or ddI. In most of these treated patients, a direct response to the antiretroviral compounds employed was detected during the first few weeks of treatment, as documented by a fast decrease of all molecular indexes of HIV-1 activity. However, residual viral replication (albeit at minimal levels) was documented during therapy in all subjects monitored in this study. In a minority of the patients under study (3 of 12), the drug-dependent viral inhibition was maintained throughout the observation time (213 to 791 days), but in 9 patients a rebound in viremia level was detected during therapy with competitive reverse transcription PCR. Sequencing analysis of a portion of the HIV-1 gene pol from cell-free virions showed that circulating viral variants bearing at least two mutations compatible with azidothymidine or ddI resistance were detectable in the patients who exhibited a rebound in cell-free HIV-1 genomic RNA copy numbers in plasma but not in one patient who maintained (for 455 days) lowered levels of viral load during ddI treatment.

Amino Acid Sequence↗
PubMed1995

Adenoma-carcinoma sequence of colorectum. Prevalence of K-ras gene mutation in adenomas with increasing degree of dysplasia and aneuploidy.

One hundred and fifty colorectal adenomas were investigated in order to detect the presence of K-ras gene mutation. The adenomas were classified according to the severity of the histological lesion (mild, moderate, or severe dysplasia and carcinomatous transformation) and to the degree of aneuploidy. K-ras mutation was found in 30.8% of cases, mostly consisting of a point mutation of codon 12. K-ras mutation was more frequently found in adenomas > 1 cm and in the villous type. No correlation was otherwise demonstrable with the ploidy pattern of the lesion.

Adenoma↗
PubMed1995

[Pancreatic assessment by magnetic resonance imaging: an improvement in the diagnostic results with new study technics].

Magnetic resonance imaging of the pancreas has been, limited by a series of artifacts with a resulting poor contrast to noise ratio. Nevertheless, technological progress has allowed to reduce not only scanning time but also the number of artifacts, by increasing the number of excitations and matrix size. Moreover, tissue contrast can now be modified. In our study performed on 5 normal volunteers and 20 patients with different pancreatic diseases conditions, fast SE sequences with and without fat suppression were used, with an overall increase in contrast to noise ratio. In all patients MR images allowed the accurate definition of the lesions: in 8 adenocarcinoma patients the lesion could be depicted, including the 2 lesions CT had poorly demonstrated. Also in the 3 patients with endocrine tumors, MRI depicted small tumors which were later confirmed at surgery. This new protocol makes MRI a very sensitive and accurate tool in the study of neoplastic and inflammatory pancreatic diseases thus, MRI is not only a complementary tool to CT but an even better study technique. In particular, its higher contrast to noise ratio allows a dramatic improvement in the depiction of small solid lesions, especially those not altering pancreatic outline. This study proves that MRI, when adequately performed, is a very accurate tool to study, in a very short time, the pancreas and peripancreatic region, yielding much better results than all the other diagnostic imaging modalities.

Acute Disease↗
PubMed1995

[Magnetic resonance angiography of the intracranial circle using magnetization transfer contrast (MTC)].

Magnetic Resonance Angiography (MRA) is becoming an accurate technique to study vascular conditions, especially when the intracranial circulation is involved. Nevertheless, the contrast-to-noise (C/N) ratio in MRA images is often inadequate to obtain diagnostic images. C/N ratio can be increased by using magnetization transfer contrast (MTC), the technique which allows to reduce the signal from the stationary tissues surrounding vascular structures. To demonstrate the value of MTC, 10 healthy volunteers and 10 patients with different cerebral vascular conditions were examined. All exams were performed using 3D MOTSA (Multiple Overlapping Thin Slab Acquisition) Time of Flight sequences, with and without MTC. A quantitative analysis was carried out in the images with and without MTC, measuring the signal intensity value in the regions of interest at the vascular and stationary structures. A qualitative analysis was also carried out with a double blind study by two independent radiologists. Our results showed that MTC allows to reduce stationary tissues signal intensity, without affecting flowing blood signal intensity. Therefore, the C/N ratio is increased, thus allowing better depiction of smaller peripheral branches. In conclusion, MTC applied to intracranial MRA allows image quality to be markedly improved, with an overall increase in the diagnostic accuracy of this technique.

Cerebrovascular Circulation↗
PubMed1994

Competitive polymerase chain reaction for quantitating feline immunodeficiency virus load in infected cat tissues.

To quantitate FIV provirus copy numbers present in tissue of infected cats, we have applied a competitive polymerase chain reaction (cPCR) recently described for HIV. The method consists in coamplifying a fixed amount of the DNA to be examined with graded copy numbers of a DNA competitor incorporating a short deletion and bearing the same primer recognition sequences. These conditions ensure almost identical thermodynamic and amplification efficiency for both template species but permit a prompt recognition of the two amplification products by gel electrophoresis. Since the amounts of the two amplicons are dependent on relative initial template concentrations, the number of FIV genomes in the sample can be calculated by densitometric analysis of the electrophoretic bands. After validation, the method has been applied to study the provirus loads in the tissues of cats infected with the Pisa-M2 isolate of FIV.

Animals↗
PubMed1994

766 cases of oral cleft in Italy. Data from Emilia Romagna (IMER) and northeast Italy (NEI) registers.

Epidemiological and genetic variables for oral clefts were analysed for the years 1981-1989 in a case-control study of congenital malformations in the Emilia Romagna, Veneto, and Friuli regions, and in the Trento and Bolzano hospitals. Birth prevalence for all cases of cleft lip with or without cleft palate (CL(P)) was 8.2 per 10,000 births, and that for cleft palate only (CP) was 6.1 per 10,000. Coexisting abnormalities were found in 23% of CL(P) cases and in 43% of CP. No clusters in time or space were detected. For isolated clefts, a predominance of males among CL(P) and of females among CP was found; epilepsy was the only maternal risk factor correlated with clefts, and an association between clefting and consanguinity was found. Empirical recurrence risks were calculated in both isolated CL(P) and CP.

Abnormalities, Multiple↗
PubMed1994

Competitive polymerase chain reaction and analysis of viral activity at the molecular level.

Due to the high sensitivity level (which can be pushed to the limit of one molecule) and its extraordinary flexibility, the polymerase chain reaction (PCR) is the method of choice for the detection of nucleic acids present in very low concentration in biological samples. Since the qualitative features of PCR amplification have limited its use, several PCR-based approaches for the quantitation of low-abundance nucleic acid species have been planned and proposed in the last few years. Recently, different lines of evidence have indicated that competitive PCR and competitive reverse-transcription-PCR share several advantages over other quantitative approaches. This evidence opens up unexpected possibilities in many biological fields, including virology; in fact, availability of reliable techniques for the absolute quantitation of DNA and RNA species may be the key to a better understanding of the pathogenic steps of most viral diseases and for a more precise monitoring of patients treated with specific antiviral compounds. In this review article, we summarize the procedures adopted for this quantitative molecular approach; additionally, several important technical aspects to plan novel competitive PCR-based applications are analyzed, and early results obtained using cPCR for the direct evaluation of viral activity in vivo are discussed.

DNA, Viral↗