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Biomedical subjects

A Manzin

Publications and source records attributed to A Manzin.

At least 37 records · Page 2Linked to original sources
PubMed1997

Clinical implications of GBV-C/HGV infection in patients with "HCV-related" chronic hepatitis.

BACKGROUND/AIMS: To evaluate the clinical, biochemical and histological implications of a concomitant HGV infection in "HCV-related" chronic liver disease. METHODS: Eighty-three HCV-RNA positive patients with chronic liver disease were tested for GBV-C/HGV coinfection by heminested PCR. RESULTS: Twenty-two (26.5%) patients were found to be positive for GBV-C/HGV RNA. GBV-C/HGV+ patients differed significantly from GBV-C/HGV- ones for younger age, higher frequency of history of drug addiction, which in turn might favor coinfection with interferon-sensitive HCV genotypes (3a), and increased probability of long-term response to interferon. GBV-C/HGV infection appears to have no responsibility for specific aspects of HCV infection such as biochemical or histological cholestatic features, lymphoid follicles, symptomatic cryoglobulinemia or presence of serum autoantibodies, including LKM1. It does not worsen the HCV-related disease (ALT levels and histological activity) and does not significantly interfere with HCV infection, as explored by the number of hepatocytes positive for HCV antigens. The amount of steatosis (mean score) was shown to be higher in GBV-C/HGV+ patients. A virological follow up was performed in 17 interferon-treated GBV-C/HGV+ patients On the whole, GBV-C/HGV seems to be as sensitive to IFN treatment as HCV, but recurrence after withdrawal is more frequent. In spite of this, ALT levels often remain normal after treatment withdrawal. CONCLUSIONS: The present data suggest that GBV-C/HGV infection, apart from more marked liver steatosis, does not modify the overall picture of chronic hepatitis due to HCV infection.

Adolescent↗
PubMed1997

Dynamic features of human immunodeficiency virus type 1 (HIV-1) viremia: kinetics of cell-free HIV-1 RNA after therapeutic plasma exchange.

To gain insight into the variables that influence the dynamics of human immunodeficiency virus type 1 (HIV-1) viremia levels, HIV-1 RNA molecules were quantified in plasma from an infected patient undergoing therapeutic plasma exchange (TPEx). After each TPEx procedure (2000 mL of fluid exchanged per session), HIV-1 genome molecule levels dropped to 58%-63% of the basal level but rapidly reverted to pre-TPEx values (doubling time = 3.50-4.04 h). Of interest, mobilization of extravascular cell-free virions (on average, 5.15 x 10(4) viral genome molecules/h) had already occurred during TPEx. After three daily TPEx procedures, HIV-1 viremia rebounded to basal values, while HIV-1 proviruses and viral transcripts in peripheral blood lymphocytes constantly tested at stable levels. Overall, this study extends previous analyses of the rate of HIV-1 turnover, using an alternative approach to the use of antiretroviral drugs, and it provides, albeit indirectly, insights into the amount and dynamic features of extravascular cell-free virus.

Cell-Free System↗
PubMed1997

Histological changes but not virus eradication reflect biochemical improvement in chronic hepatitis C patients showing a long-term response to interferon alpha therapy.

AIMS: To assess the relationship between changes in liver histology and virological parameters of HCV infection in patients with a sustained biochemical response to alpha-interferon treatment for chronic hepatitis, with the aim of identifying the most reliable indicator of treatment efficacy. METHODS: Quantitative serial testing of HCV-RNA in plasma samples collected on a monthly basis over the last period of post-treatment follow-up were tested in sixteen subjects with > or = 12 months aminotransferase normalisation following discontinuation of interferon therapy. The quantitative HCV-RNA determination in plasma was performed by a competitive reverse-transcription polymerase-chain reaction. Pretreatment and 12-month post-treatment liver histologies were blindly evaluated using a semi-quantitative scoring system. At these times a qualitative HCV-RNA analysis was also carried out. RESULTS: The post-treatment histological grading score was significantly reduced. Nevertheless, all patients except one tested positive for HCV-RNA in plasma (mean 1.7 x 10(6) molecules/ml): nine were positive in all the serial determinations, while the remaining 6 were intermittently positive. No relationship between genotype, viral load, pattern of viremia (intermittent or continuous) and change in histological score was found. CONCLUSIONS: Changes in liver histology are the most reliable indicator of the efficacy of interferon treatment in hepatitis C related liver disease. HCV-RNA clearance based on serial sampling does not appear to be a reliable indicator, even in the presence of a sustained biochemical response and histological improvement.

Adult↗
PubMed1996

Low hepatitis C viremia levels in patients with anti-liver/kidney microsomal antibody type 1 positive chronic hepatitis.

BACKGROUND/AIMS: The majority of adult patients positive for anti-liver-kidney microsomal antibody are also positive for anti-hepatitis C virus and serum HCV RNA. In these patients the role played by hepatitis C virus infection in the progression of liver damage and its relationship with anti-liver-kidney microsomal antibody are, however, still a matter of debate. METHODS: To clarify this point we have compared hepatitis C viremia in sera from 31 hepatitis C virus-related chronic hepatitis patients positive for anti-liver-kidney microsomal antibody with that of 31 patients with hepatitis C virus-related chronic hepatitis without autoantibodies using a newly developed competitive reverse transcription-polymerase chain reaction technique. Reverse transcription-polymerase chain reaction was performed using a synthetic competitor of a length similar to that of wild template (71 bp vs 86 bp). RESULTS: The results obtained have been related to hepatitis C virus genotypes. Anti-liver-kidney microsomal antibody/anti-HCV positive patients show a median value of hepatitis C virus genome molecules (626829/ml, range 9780-25651424), significantly lower than anti-liver-kidney microsomal antibody negative/anti-HCV positive patients (10158314/ml, range 101822-67429974) (p < 0.001). No hepatitis C virus genotype was significantly associated with anti-liver-kidney microsomal antibody, although a predominance of genotype 1 (subtypes a and b) has been observed in these patients. CONCLUSIONS: Since a low hepatitis C viremia has been observed in anti-liver-kidney microsomal antibody positive patients with disease severity comparable to that of patients without autoantibodies, it is conceivable that in them autoimmune mechanisms may cooperate with viral infection in sustaining disease activity.

Adolescent↗
PubMed1996

Differential humoral immune response against hepatitis C virus antigenic synthetic peptides in infected patients with and without mixed cryoglobulinaemia.

In this study we have evaluated the prevalence of antibodies against core region peptides (residues 1-28, 21-38 and 51-68), the envelope 1, the non-structural (NS) 4 and 5 proteins of hepatitis C virus (HCV) in sera from 65 chronically HCV-infected patients, 47 with mixed cryoglobulinaemia (MC+) and 18 without (MC-). The major binding sites were located within the core region. Regions 1-28 and 51-68 were recognized by a similar proportion of MC+ and MC- patients, while peptide 21-38 was less frequently detected by samples from MC+ patients (65.5% versus 100%, P = 0.011). The patterns of the reactions showed a minimum of three binding sites: one, located within region 51-68, was shared by both groups; a second determinant was identified at residues 1-21 for MC+ patients and at residues 28-38 for MC- patients; a third, not exactly localized, lay between residues 1 and 38. Recognition of NS5 peptides was not significantly different between MC+ and MC- patients, but while the former mostly reacted either with peptide 1 (residues 2294-2309) (five of 15 sera) or with peptide 2 (residues 2304-2319) (nine of 15 sera), the latter group showed a more scattered reaction. Antibodies to HCV peptides prevalently belonged to IgGl subclass. However, whereas IgGl antibodies against peptide 21-38 and peptide 1 of NS5 were more frequently found in MC- rather than in MC+ patients (100% versus 63.8%, P = 0.003, and 22.2% versus 4.2%, P = 0.025, respectively), IgG3 antibodies against region 1-28 were more frequent in MC+ patients (53.19% versus 16.6%, P = 0.0078). Overall, the data suggest that a differential humoral immune response to HCV antigens occurs in patients with and without cryoglobulinaemia.

Adult↗
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↗
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

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↗
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↗
PubMed1994

Active hepatitis C virus infection in bone marrow and peripheral blood mononuclear cells from patients with mixed cryoglobulinaemia.

The presence of hepatitis C virus (HCV) genomic sequences was checked in plasma, liver, peripheral blood mononuclear cells (PBMC) and bone marrow cells from 11 patients with mixed cryoglobulinaemia positive for anti-HCV antibodies, and from 11 patients with chronic HCV hepatitis without serological evidence of cryoglobulinaemia. HCV RNA sequences were demonstrated by reverse transcription polymerase chain reaction in seven plasma samples, in six PBMC samples, and in seven bone marrow cell samples from the 11 cryoglobulinaemic subjects; otherwise, viral specific nucleic acids were detected in 10 plasma samples, in one PBMC sample, and in two bone marrow cell samples from the 11 patients with chronic hepatitis. The HCV replicative intermediate was evidenced in four of the six PBMC and in five of the seven bone marrow aspirate HCV RNA-positive samples. Analysis of subpopulations isolated from bone marrow and peripheral blood samples showed HCV RNA sequences in mononuclear cells belonging either the CD2+ subset or to the CD19+ subpopulation or to the adherent cells. Finally, we compared the nucleotide sequences of a large portion (-270 to -59) of the HCV 5'-untranslated region from five patients with mixed cryoglobulinaemia and from seven patients with chronic hepatitis without cryoglobulinaemia; the degree of heterogeneity, compared with the prototype HCV sequence, was similar in both groups. These findings from two groups of HCV-infected patients indicate that transient or permanent active HCV infection of bone marrow and PBMC is frequent in anti-HCV-positive patients with mixed cryoglobulinaemia, and suggest that extra-hepatic infection may play a major role in influencing the pathophysiology of this infection as well as the viral persistence.

Aged↗
PubMed1994

Presence of hepatitis C virus (HCV) genomic RNA and viral replicative intermediates in bone marrow and peripheral blood mononuclear cells from HCV-infected patients.

The cellular tropism of hepatitis C virus (HCV) was studied in vivo in samples from patients with persistent HCV infection. Plasma, liver, peripheral blood mononuclear cell (PBMC), and bone marrow cell (BMC) samples from 15 subjects positive for anti-HCV antibodies were tested for the presence of HCV RNA sequences by reverse transcription PCR. Virus-specific RNA sequences were found to be present in liver samples from all subjects (100%), in plasma samples from 13 of 15 patients (86.7%), in PBMC samples from 3 patients (20%), and in BMC samples from 9 (60%) of the 15 anti-HCV-positive patients enrolled in this study. The presence of the molecular intermediate of HCV replication (the negative-stranded HCV RNA) was evident in the two of the three PBMC and in five of the nine BMC HCV RNA-positive samples. Finally, we studied the nucleotide sequence of a large portion (-270 to -59) of the 5'untranslated region of HCV amplified from plasma samples of 12 of the 15 patients with and without HCV in BMCs; the degree of heterogeneity compared with the prototype HCV sequence was similar in both groups. The data principally indicate that HCV infection of PBMCs and BMCs is frequent in persistently infected patients, as shown by the occurrence of positive- and negative-stranded HCV RNA, thus suggesting the possibility of extrahepatic replication of HCV.

Adult↗
PubMed1994

Quantitation of hepatitis C virus genome molecules in plasma samples.

A competitive reverse transcription PCR (cRT-PCR)-based assay for the quantitative detection of hepatitis C virus (HCV) viremia was developed, optimized, and applied to the direct molecular analysis of clinical samples from nine patients with persistent HCV infection. As for other competitive PCR-based applications, this method consists of the reverse transcription and subsequent amplification of two RNA species in the same tube: the wild-type template (to be quantified) and a known amount of a modified synthetic template. These templates have identical primer recognition sites and very similar (but not identical) sizes, thus allowing direct detection of both template species after gel electrophoresis and ethidium bromide staining. The results obtained by this cRT-PCR application for testing clinical samples from HCV-infected patients mainly indicate that the competitive approach reaches the degree of sensitivity (fewer than 5 HCV RNA molecules per 100 microliters) necessary to evaluate viral load in all HCV-infected patients, independently of clinical conditions, and that this technique is flexible enough to quantify highly divergent levels of cell-free HCV genome copy numbers in biological samples. Interestingly, we observed a sample-to-sample variation in the loss of detectable HCV genome molecules in serum in comparison with that in plasma from the same patient, thus indicating that serum specimens, although widely used in the past few years for qualitative molecular investigation of HCV-infected patients, cannot be used to obtain reliable quantitative data on HCV viremia from these patients.

Artifacts↗
PubMed1994

Dynamics of molecular parameters of human immunodeficiency virus type 1 activity in vivo.

The dynamics of viral activity during different phases of human immunodeficiency virus type 1 (HIV-1) infection were investigated by competitive PCR methods. In particular, we studied the time course of three quantitative molecular parameters of viral activity (genomic RNA copy number in plasma and provirus and late HIV-1 transcript molecule copy numbers in peripheral blood CD4+ T lymphocytes) in untreated patients and patients treated with specific anti-HIV-1 compounds. The results shown here indicate that direct RNA parameters are quantitative molecular indices sensitive enough to be used for a more accurate evaluation of the natural history of this infection and that an indirect parameter, the mean transcriptional activity for each provirus in CD4+ T lymphocytes, may be important in studying this infection in vivo at the molecular level. A dramatic decrease of the indices was evident at seroconversion, but the quantitative values were virtually stable throughout the time the untreated patients were studied during the clinical latency phase. Furthermore, the results indicate that an early response to antiretroviral compounds is detected in most subjects as a decrease in the viral activity level.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Chronic liver disease and active hepatitis C virus infection in patients with antibodies to this virus.

AIMS: To assess the association between active hepatitis C virus (HCV) infection and liver damage in randomly selected patients with antibodies to the virus. METHODS: Thirty three consecutive subjects with serologically confirmed positivity for antibodies to HCV were studied for the presence of liver and circulating viral sequences by using the reverse transcription polymerase chain reaction (RT-PCR) and specific primers for the 5'-untranslated region (5'-UTR) of the HCV genome. Parallel clinical, biochemical, and histological investigations were carried out in all cases. RESULTS: A comparative virological and histological investigation showed the presence of molecular signs of active viral replication and different degrees of liver damage in all cases. Baseline values of liver and plasma samples from all the patients showed (with one exception) the presence of detectable HCV RNA sequences, despite alanine amino transferase activities being within normal values or within 1.5 times the upper limit of normal in 13 of them. Examination of percutaneous liver biopsy specimens showed the presence of confirmed liver damage (ranging from chronic persistent hepatitis to cirrhosis) in all 33 patients. CONCLUSIONS: Circulating HCV RNA sequences (a direct sign of active HCV infection) are associated with liver damage, even in the absence of clinical or biochemical signs of overt liver disease. Parallel molecular, histological, and clinical follow up of these patients is needed to understand precisely the natural history of HCV infection and for correct clinical management.

Base Sequence↗