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Biomedical subjects

A Cerino

Publications and source records attributed to A Cerino.

At least 37 records · Page 2Linked to original sources
PubMed1995

Differential distribution of hepatitis C virus genotypes in patients with and without liver function abnormalities.

Hepatitis C virus (HCV) infection persists for an indefinite length of time in a major proportion of patients, inducing chronic liver lesions that evolve to cirrhosis and hepatocellular carcinoma (HCC) in approximately 20% of cases. We studied HCV viremia and genotypes by reverse transcription-polymerase chain reaction (RT-PCR) in 341 consecutive anti-HCV-positive patients. Of these, 167 patients had persistently normal or near normal alanine aminotransferase (ALT) levels (fluctuations < or = 5 IU above the upper limit of normal); the remaining 174 patients presented with elevated ALT and histological evidence of chronic liver disease. Seventy percent of patients with normal ALT values had circulating HCV RNA despite the absence of biochemical indicators of liver damage and mild histological forms of chronic hepatitis were detected in most patients who underwent liver biopsy. Isolated genotype III infection was significantly more prevalent in this patient group with respect to control patients with abnormal ALT values (70% vs. 39%; P < .001). Conversely, isolated genotype II was more frequently found in patients with elevated ALT values and evidence of chronic liver disease (45% vs. 23%; P < .01) and it was progressively more represented in advanced liver disease, such as cirrhosis and HCC. Virological features of HCV infection might be associated with different clinical manifestations, suggesting a potential prognostic significance on disease outcome.

Adolescent↗
PubMed1994

Hepatitis C virus infection in children treated for acute lymphoblastic leukemia.

We studied 102 consecutive subjects after their completion of acute lymphoblastic leukemia (ALL)-directed chemotherapy, for evidence of hepatitis C virus (HCV) infection by enzyme immunoassay 2 and 3, second generation recombinant immunoblot assay and reverse transcription-polymerase chain reaction (PCR) for detection of circulating HCV-RNA. Forty-four patients (43%) had evidence of exposure to HCV; 30 of these were anti-HCV+. Of the 23 patients who were positive for both anti-HCV and HCV-RNA, 16 (69%) had a moderate increase in serum alanine aminotransferase (ALT) activity without clinical signs of liver disease. Fourteen patients were seronegative despite the presence of HCV-RNA in the serum. The proportion of different HCV genotypes was not significantly different from other anti-HCV+ patient groups. Although half of the patients with genotype III had normal ALT value, patients with normal ALT levels were represented in all genotype groups. Our study documents the prevalence of HCV infection in childhood ALL survivors, which is responsible for the majority of cases of non-B chronic liver disease in these patients. Whereas serologic screening identifies over 70% of patients with ongoing HCV infection, real HCV infection may be present even in the absence of a detectable humoral immune response to the virus. Based on this observation, determination of HCV-RNA by PCR should be recommended in patients in prolonged remission even if they test negative on serological assay. Normal ALT levels do not exclude the presence of HCV infection because the values were repeatedly normal in over half of our viremic patients.

Adolescent↗
PubMed1994

Hepatitis C virus (HCV) core serotypes in chronic HCV infection.

Recently, two distinct hepatitis C virus (HCV) serologic types have been identified on the basis of amino acid variations in the core region. The two serologic types can readily discriminate between genotypes I-II-V (serotype 1) and III-IV (serotype 2), according to the Okamoto classification. We compared HCV core serotyping with genotyping with sera from 363 anti-HCV-positive patients (309 HCV RNA positive by PCR) using a synthetic core peptide-based enzyme immunoassay and PCR amplification of core region sequences with type-specific primers, respectively. Serologic responses to HCV serotypes were successfully identified in 164 (45%) patients, of whom 153 were viremic. Eighty-nine patients had evidence of exposure to serotype 1: 8 of these were infected with genotype I, 50 were infected with genotype II, 2 were infected with genotype III, 7 were infected with genotype V, 13 had infections with mixed genotypes, 3 were infected with an indeterminate genotype, and 6 were nonviremic. Seventy-four patients had been exposed to serotype 2: 64 were infected with genotype III, 3 were infected with mixed genotypes, 2 were infected with an indeterminate genotype, and 5 were nonviremic. The serum of one patient, infected with genotype III, showed reactivity to both serotypes. Comparative evaluation of HCV core region serotyping and genotyping with sera from 294 viremic patients infected with a known HCV genotype showed a remarkable concordance between HCV core region genotyping and serotyping, with only 2 apparently discordant serum samples (both from patients with genotype III infection) of 148 (1.4%) successfully serotyped samples. Serotype 1 infection was more frequently observed in patients with overt chronic liver disease and accounted for all successfully serotyped samples from intravenous drug abusers. In contrast, serotype 2 was more prevalent in subjects with biochemically silent HCV infection (alanine aminotransferase, < 45 U/liter), in agreement with previous findings at the molecular level. HCV core serologic typing is a simple, inexpensive, and highly reproducible assay that can be applied to more than 50% of viremic HCV antibody carriers prior to the use of more sophisticated molecular typing techniques. Moreover, it may be helpful in tracking transmissions routes, particularly for incorrectly stored samples in which the RNA has degraded or for subjects who have cleared the virus and therefore have only antibodies remaining to testify to a remote infection. The lack of recognition of the core sequence from residues 67 to 81, which contains a minor B-cell epitope used to detect type-specific immunoreactivity, may explain the negative serologic findings for half of the patients.

Adolescent↗
PubMed1994

Significance of the immune response to a major, conformational B-cell epitope on the hepatitis C virus NS3 region defined by a human monoclonal antibody.

The nonstructural protein NS3 of hepatitis C virus (HCV) possesses two enzymatic domains which are thought to be essential for the virus life cycle: an N-terminal serine-type proteinase, responsible for the processing of nonstructural polypeptides, and a C-terminal nucleoside triphosphatase/helicase, presumably involved in the unwinding of the viral genome. The human antibody response to NS3 usually appears early in the course of HCV infection and is predominantly directed against the carboxyl-terminal portion; however, its fine specificity and clinical significance are largely unknown. We have generated a human monoclonal antibody (hMAb), designated CM3.B6, from a cloned B-cell line obtained from the peripheral blood of a patient with chronic HCV infection, which selectively recognized the purified NS3 protein expressed in bacteria or in eukaryotic cells transfected with full-length or NS3 cDNA. Fine-specificity studies revealed that CM3.B6 recognized a 92-amino-acid sequence (clone 8, amino acids 1363 to 1454) selected from an NS3 DNase fragment library but failed to bind to 12-mer peptides synthesized from the same region, suggesting recognition of a conformational B-cell epitope. Experiments using deletion mutants of clone 8 and competitive inhibition studies using a panel of NS3 peptide-specific murine MAbs indicated that limited N-terminal and C-terminal deletions resulted in a significant reduction of hMAb binding to clone 8, thus identifying a minimal antibody binding domain within clone 8. Competition experiments showed that binding of CM3.B6 to the NS3 protein was efficiently inhibited by 39 of 44 (89%) sera from HCV-infected patients, suggesting that the hMAb recognized an immunodominant epitope within the NS3 region. More importantly, recognition of the sequence defined by CM3.B6 appeared to accurately discriminate between viremic and nonviremic anti-HCV positive sera, suggesting potentially relevant clinical applications in the diagnosis and treatment of HCV infection.

Animals↗
PubMed1993

A human monoclonal antibody specific for the N terminus of the hepatitis C virus nucleocapsid protein.

PBMC from a patient with chronic hepatitis C virus (HCV) infection were immortalized with EBV and plated by limiting dilution. Cultures secreting antibodies reactive in a commercial HCV II generation ELISA, which incorporates Ag derived from the nucleocapsid, NS3, and NS4 regions, were repeatedly cloned in the presence of feeder cells and growth factors. Of 23 initially immunoreactive cultures, only one cloned line, designated B12.F8, secreted HCV nucleoprotein-specific IgG1(kappa), whereas no reaction with recombinant polypeptides derived from NS3, NS4, and NS5 regions were documented. Human mAb (hmAb) B12.F8 was shown to recognize the native HCV nucleoprotein expressed in eukaryotic cells transfected with a core cDNA construct by immunofluorescence. The fine specificity of this hmAb was evaluated using synthetic oligopeptides covering the entire HCV nucleocapsid region. A weak but consistent reactivity was observed by PEPSCAN using a 12-mer encompassing residues 34-45 of the HCV-deduced amino acid sequence. Such weak reactivity is indicative for conformational epitopes and, in concurrence with this assumption, we found that longer peptides from the region containing residues 27-59 were more efficiently recognized and effectively inhibited binding of hmAb B12.F8 to recombinant nucleocapsid protein. Several overlapping immunoreactive fragments from the nucleocapsid region were selected from a random cDNA library consisting of DNase I fragments of recombinant core Ag. Best reactive recombinants were identified within residues 1-78 of the HCV sequence, in agreement with the results obtained using synthetic peptides. Comparative experiments on the fine specificity of sera from HCV-infected patients with anticore antibodies invariably showed recognition of peptides 8-40 and 27-59, as well as recombinant fragments spanning from residues 1 to 73, suggesting that hmAb B12.F8 identifies a major B cell epitope within the immunodominant nucleoprotein amino terminal subregion.

Amino Acid Sequence↗
PubMed1993

Properties of a human monoclonal antibody specific for the NS4 region of hepatitis C virus.

A human monoclonal IgG1(kappa) antibody (hmAb) specific for a sequential epitope comprised within the 5-1-1 fragment of the NS4 region of hepatitis C virus (HCV) has been recently generated (1). In this study, B-cell clone supernatant containing the hmAb was purified by passage over a protein A affinity column. Preincubation with synthetic oligopeptides containing the epitope recognized by the hmAb resulted in complete inhibition of binding to the whole recombinant protein, attesting to its specificity. Calculation of the dissociation constant (Kd) using a synthetic 20-mer as antigen gave a value of 3.3372 x 10(-8) M, consistent with that of a human IgG. We predict that this hmAb will be helpful in characterizing the as yet unidentified native NS4 protein of HCV.

Antibodies, Monoclonal↗
PubMed1993

Virological features of hepatitis C virus infection in hemodialysis patients.

The clinical and epidemiological relevance of circulating antibodies to hepatitis C virus (HCV) in hemodialysis patients is uncertain, since clinical signs of infection are often mild or absent, with alanine aminotransferase (ALT) values that are virtually always normal, and liver biopsies are only rarely performed. Determination of HCV RNA in serum is therefore critical for distinguishing chronic HCV infection from previous exposure to the virus. We studied HCV viremia by reverse transcription polymerase chain reaction (RT-PCR) in the 5'-noncoding region of the viral genome in 77 dialysis patients who were screened for anti-HCV by a second-generation enzyme-linked immunosorbent assay (the enzyme immunoassay II; Ortho HCV, 2nd generation, Ortho Diagnostic Systems Raritan, N.J.) and a second-generation recombinant immunoblot assay (Chiron Corporation and Ortho Diagnostic Systems) and prospectively evaluated for ALT elevations over a period of 5 years. Of 77 patients tested, 29 (38%) had active infection as shown by a positive PCR assay result, and of these, 26 were anti-HCV positive. Although a good correlation was found between circulating anti-HCV and HCV RNA in serum, 10 (28%) of 36 anti-HCV-positive patients were HCV RNA negative by PCR, suggesting either low levels of viremia or past exposure to HCV and subsequent recovery. On the other hand, 3 (7.3%) of 41 anti-HCV-negative patients had HCV RNA in their sera, indicating seronegative HCV infection. The ALT level had no predictive value for HCV infection, because it was repeatedly normal in 18 (62%) of 29 viremic patients. HCV genotyping was also performed and indicated that all four known genotypes of HCV were present in our group. In conclusion, serological assays are reliable for detecting exposure to HCV in hemodialysis patients; however, direct identification of the viral genome is required to document current infection.

Adolescent↗
PubMed1992

Monoclonal antibodies to human DNA topoisomerase I and the two isoforms of DNA topoisomerase II: 170- and 180-kDa isozymes.

Several monoclonal antibodies of different isotypes specific to human DNA topoisomerase I, to 170- and 180-kDa DNA topoisomerase II isozymes, were produced and characterized. The specificity of monoclonal antibodies was confirmed by comparison with polyclonal antibodies by Western blot, by immunoprecipitation of enzyme activity, and by immunoprecipitation of DNA topoisomerases with characterized polyclonal antisera. Morphological studies performed by immunofluorescence indicate that the three groups of monoclonal antibodies (MoAbs) stain the nucleus with characteristic patterns, which can be compared with those obtained with polyclonal antibodies. In particular the MoAbs to the 100-kDa DNA topoisomerase I stain the nucleolus and the nucleoplasm; the MoAbs to 170- and 180-kDa DNA topoisomerase II give completely distinct intranuclear patterns: those to the 170-kDa protein stain mainly the nucleoplasm, whereas those to the 180-kDa protein stain only the nucleolus. The two DNA topoisomerase II isozymes clearly exhibit fluctuations in their expression during cell growth: the 170-kDa isozyme is more abundant during the logarithmic phase of growth, while the 180-kDa isozyme is mainly present during the plateau phase of growth.

Antibodies, Monoclonal↗
PubMed1992

High prevalence of antibodies to hepatitis C virus in hemodialysis units using a second generation assay.

We studied the prevalence and significance of antibodies to hepatitis C virus (HCV) in patients and staff from 3 dialysis units, using a 2nd generation assay (2nd g.a.; Ortho HCV). Of 277 patients, 151 (55%) were positive by 2nd and 85 (31%) by 1st g.a. Significant associations with the anti-HCV carrier status were: blood transfusions, retrospective finding of elevated ALT and duration of dialysis treatment, independently of transfusions. Of the 74 staff members, 5 were positive by 2nd and 3 by 1st g.a. Our data suggest that the 2nd g.a. is more sensitive in detecting HCV exposure in dialysis units and that duration of dialysis is a significant factor in acquiring HCV infection.

Ambulatory Care Facilities↗
PubMed1991

Identification of an immunodominant B cell epitope on the hepatitis C virus nonstructural region defined by human monoclonal antibodies.

Several EBV-transformed B cell lines (BCL) were obtained from two patients with chronic hepatitis C virus (HCV) infection that secreted IgG class antibodies to the HCV nonstructural Ag c100-3. Two cloned BCL, derived from the same parental line, generated stable cloned lines that secreted up to 20 mg/liter of specific IgG1(kappa). Supernatants from oligoclonal and cloned BCL were also analyzed by immunoblot and all strongly reacted with recombinant polypeptides derived from the putative NS4 region of HCV, c100-3 and 5-1-1 (a 42-amino acid fragment of c100-3), whereas no reaction with the viral nucleoprotein, the NS3 nonstructural protein or the superoxide dismutase moiety of the c100-3 fusion protein could be documented. The fine specificity of these antibodies was also evaluated using overlapping synthetic peptides (20-mers) covering the 5-1-1 sequence. All oligoclonal and clonal IgG displayed high affinity binding to peptides covering residues 120-137 of Chiron's c100-3 sequence at the aminoterminus of 5-1-1. In addition, a minimal B cell epitope, N-VLYREF-C, was defined by human oligoclonal and monoclonal antibodies corresponding to residues 132-137. Interestingly, predominant recognition of the N-terminus of 5-1-1 was also observed in more than 80% of sera from patients with HCV infection. In conclusion, we have successfully produced human B cell cloned lines that secrete abundant quantities of IgG1(kappa)-specific for a polypeptide encoded by the NS4 region of HCV. Such antibodies recognize an immunodominant epitope, relative to this region, located at the N-terminus of the 5-1-1 fragment.

Amino Acid Sequence↗
PubMed1991

Synthetic and natural factors promoting production of murine and human monoclonal antibodies.

Human endothelial culture supernatant (HECS) contains factor(s) which promote the growth and the stability of hybridoma cells, and in these respects is superior to the commonly used feeder cells. In particular: HECS increases the recovery of hybridomas after fusion as compared to that in the presence of feeder cells; HECS can substitute feeder cells supporting the growth of hybridomas at the single-cell level under limiting dilution conditions; HECS prolongs the stability of human-mouse hybridomas producing human immunoglobulins (Ig). The active principle which is present in HECS can be different from IL-6. Furthermore the effects of a polyamine derivative, carboxyethyl gamma-aminobutyric acid (CEGABA), were studied on the growth of hybridomas and Epstein-Barr Virus-transformed cell lines. CEGABA enhances the growth of hybridomas through all the steps of hybridoma technology and induces the formation of a large number of EBV-transformed lymphocytes, when added to the culture medium immediately after EBV infection. However, CEGABA shows only a moderate effect on the stability of EBV-transformed cell lines over time, and does not effect the growth of stabilized cell lines. It is possible that CEGABA acts on cells other than EBV-transformed lymphocytes (in fact, after EBV infection all types of mononuclear cells from the blood are present in the culture) and indirectly promotes the growth of EBV-transformed lymphocytes.

Animals↗
PubMed1990

Antibodies from patients with rheumatoid arthritis and systemic lupus erythematosus recognize different epitopes of a single heterogeneous nuclear RNP core protein. Possible role of cross-reacting antikeratin antibodies.

Antibodies to recombinant heterogeneous nuclear RNP core protein A1 were detected in sera from 27 of 58 patients with rheumatoid arthritis (RA) and from 7 of 31 patients with systemic lupus erythematosus, by immunoblotting and enzyme-linked immunosorbent assay. Protein A1 consists of 2 distinct domains: The N-terminal sequence is identical to a single-stranded DNA binding protein termed UP1, and the C-terminal domain shows a partial homology with keratin. All 7 A1-positive systemic lupus erythematosus sera reacted with UP1, whereas 9 of the 27 A1-positive RA sera did not. In RA, anti-A1 activity was significantly associated with antikeratin antibodies (AKA); these antibodies were present in 23 of 27 A1-positive sera and 10 of 31 A1-negative sera (P less than 0.01). Immunoabsorption with recombinant protein A1 resulted in a significant reduction of AKA titers in 6 of 10 RA sera tested, suggesting that AKA from RA patients may cross-react with the C-terminal portion of the heterogeneous nuclear RNP protein A1.

Adolescent↗
PubMed1990

Autoantibodies to poly(ADP-ribose)polymerase in autoimmune diseases.

Antibodies to various nuclear proteins are frequently found in sera of patients affected by connective tissue diseases and other autoimmune diseases. We investigated the specificity of circulating autoantibodies to poly(ADP-ribose)polymerase (pADPRP) in different autoimmune and connective tissue diseases: Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), Progressive Systemic Sclerosis (PSS), Sjogren's Syndrome (SS), Undifferentiated Connective Tissue Disease (UCTD), Cryptogenic Fibrosing Alveolitis (CFA) and Sarcoidosis. pADPRP was purified from calf thymus. Antibody specificity was detected by ELISA, western blot and enzyme activity precipitation. Positive values (mean O.D. values + 3 S.D. of 36 normal controls) were obtained in 7/15 SLE patients, 1/18 RA patients, 1/30 PSS, 3/14 SS, 0/5 UCTD, 5/21 CFA and 4/25 Sarcoidosis. The positive sera also recognized the pADPRP protein when tested by western blot. Furthermore the enzyme activity was inhibited after immunoprecipitation by some highly positive sera.

Adolescent↗
PubMed1990

Friedreich ataxia in Italian families: genetic homogeneity and linkage disequilibrium with the marker loci D9S5 and D9S15.

Friedreich ataxia (FA) is an autosomal recessive degenerative disease of the nervous system of unknown biochemical cause. The FA gene has been shown to be in close linkage with the two chromosome 9 markers D9S5 and D9S15, and linkage disequilibrium between FA and D9S15 has been detected in French families by Hanauer et al. We used new highly informative markers at the above loci to analyze Italian FA families for linkage and linkage disequilibrium. The new markers were a three-allele BstXI RFLP at D9S5 (PIC = .55) and a six-allele microsatellite, typed by polymerase chain reaction, at D9S15 (PIC = .75). We obtained maximum lod scores of 8.25 between FA and D9S5, 10.55 between FA and D9S15, and 9.52 between D9S5 and D9S15, all at zero recombination. Our results, combined with those reported by other authors, reduce maxlod-1 (maximum lod score minus 1) confidence limits to less than 1.1 cM between FA and D9S5, 1.2 cM between FA and D9S15, and 1.4 cM between D9S5 and D9S15. Linkage disequilibrium with FA was found only for D9S15 when all families were evaluated but was also found for a D9S5/D9S15 haplotype in a subgroup of southern Italian families. We conclude that FA, D9S5, and D9S15 are tightly clustered and that studies of geographically restricted groups may reveal a limited number of mutations responsible for the disease in the Italian population. We present preliminary evidence from pulsed-field gel electrophoresis that D9S5 and D9S15 may be less than 450 kb apart. Linkage disequilibrium between FA and D9S15 suggests that the disease gene may be at an even shorter distance from this marker locus, which therefore represents a very good starting point for cloning attempts.

Alleles↗
PubMed1989

Antibodies to hnRNP core protein A1 in connective tissue diseases.

We investigated the specificity of circulating autoantibodies to a heterogeneous nuclear ribonuclear protein A1 (hnRNP A1), obtained by recombinant DNA technique, in different rheumatic diseases: systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), scleroderma, primary Sjogren's syndrome (SS), idiopathic Raynaud (IR), mixed connective tissue disease (MCTD), and healthy donors. All sera were tested by ELISA on hnRNP A1 protein. Positive values were obtained in 22% SLE, 19% scleroderma, 10% IR, 40% (2/5) MCTD, 5% SS, and 50% RA patients. The majority of patients reacted with the aminoterminal part (UP1) of hnRNP A1; however, some RA patients reacted also with the carboxy-terminal part that shows partial homology with keratin. Therefore, hnRNP A1 (UP1) can be considered a target of antinuclear autoimmunity in various rheumatic disorders.

Autoantibodies↗
PubMed1988

Carboxyethyl gamma-aminobutyric acid, a polyamine derivative, improves the recovery of EBV-transformed lymphocytes.

A polyamine derivative, carboxyethyl - Aminobutyric Acid (CEGABA), induces the formation of a large number of Epstein-Barr Virus (EBV) transformed lymphocytes, when added to the culture medium immediately after EBV infection. However, CEGABA shows only a moderate effect on the stability of EBV-transformed cell lines over time, and does not affect the growth of stabilized cell lines. It is possible that CEGABA acts on cells other than EBV transformed lymphocytes (in fact, after EBV infection all types of mononuclear cells from the blood are present in the culture) and indirectly promotes the growth of EBV transformed lymphocytes.

Antibodies, Monoclonal↗