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Biomedical subjects

F B Bianchi

Publications and source records attributed to F B Bianchi.

At least 73 records · Page 4Linked to original sources
PubMed1997

Establishment of a novel radioligand assay using eukaryotically expressed cytochrome P4502D6 for the measurement of liver kidney microsomal type 1 antibody in patients with autoimmune hepatitis and hepatitis C virus infection.

BACKGROUND/AIMS: Liver kidney microsomal type 1 antibody (LKM1) is the diagnostic marker of autoimmune hepatitis (AIH) type 2 and is also found in patients with hepatitis C virus (HCV) infection. Cytochrome P4502D6 (CYP2D6) is the documented target antigen of LKM1 in AIH, but not in HCV infection. To compare the reactivity in the two conditions, we established a radioligand assay using eukaryotically expressed CYP2D6 as target. METHODS: A 1.2-kb human CYP2D6 cDNA was isolated from a human liver cDNA library and subcloned into an in vitro transcription vector pSP64 Poly(A). Recombinant CYP2D6 was then produced by in vitro transcription/translation, metabolically labelled with 35S methionine and used in the immunoprecipitation assay. Antibodies that bound radiolabelled CYP2D6 were immunoprecipitated and their levels assessed as cpm. Sera from 50 LKM1-positive patients (26 with AIH; 24 with HCV infection), 128 LKM1-negative patients and 57 normal controls were tested. RESULTS: Reactivity to 35S labelled CYP2D6 was observed in all LKM1-positive sera from patients with AIH and HCV infection, but in none of the controls. The cpm in both conditions were significantly higher than in normal controls (p<0.0001), and were correlated with the immunofluorescence titres of LKM1 (r 0.87, p<0.001 and r=0.64, p<0.001 for AIH and HCV infection, respectively). Reactivity to 35S labelled CYP2D6 was inhibited by addition of an excess of eukaryotically expressed CYP2D6. CONCLUSIONS: CYP2D6 is a major target antigen of both AIH and HCV infection. The novel radioligand assay is highly sensitive and specific.

Adolescent↗
PubMed1997

Antinuclear antibodies and patterns of nuclear immunofluorescence in type 1 autoimmune hepatitis.

To determine the significance of antinuclear antibodies and their patterns of indirect immunofluorescence in type 1 autoimmune hepatitis, sera from 99 patients were evaluated. Patients with antinuclear antibodies had a lower frequency of liver transplantation (6% vs 22%, P = 0.04) than seronegative patients. They were also more commonly HLA-DR4-positive than seronegative patients (56% vs 30%, P = 0.05) and normal subjects (56% vs 30%, P = 0.004). The 42 patients with antinuclear antibodies and a diffuse pattern of indirect immunofluorescence had higher serum titers of ANA (serum titers > or = 1:500, 71% vs 14%, P < 0.0001) and SMA (serum titers > or = 1:500, 69% vs 27%, P = 0.003) than the 22 patients with antinuclear antibodies and a speckled pattern. These patients, however, were otherwise not distinguished by clinical features and treatment response. Patients with a speckled pattern had A1-B8-DR3 more frequently than patients with a diffuse pattern (65% vs 23%, P = 0.005) and normal subjects (65% vs 13%, P < 0.0001), but they had no other salient features. We conclude that patients with antinuclear antibodies have a better long-term prognosis than seronegative patients, and they have HLA-DR4 more commonly. The patterns of indirect immunofluorescence associated with ANA positivity have no practical clinical implications.

Adult↗
PubMed1997

Organ-specific autoantibodies in coeliac disease: do they represent an epiphenomenon or the expression of associated autoimmune disorders?

BACKGROUND AND AIMS: The occurrence of autoimmune disorders and organ-specific autoantibodies has been reported in coeliac disease. We assessed the prevalence of organ-specific autoantibodies in coeliac patients and evaluated whether their finding is an expression of associated autoimmune diseases. METHODS: Sera from 70 coeliac disease patients were tested for thyroid microsomal, gastric parietal cell, adrenal cortex and pancreatic islet cell antibodies by indirect immunofluorescence on O blood group human tissues. RESULTS: Eighteen coeliacs (26%) were positive for at least one of the autoantibodies studied; thyroid microsomal antibodies showed a higher prevalence (21%) than parietal cell (11%), adrenal cortex (4%) and islet cell antibodies (3%). In 15 (21%) of the 70 coeliacs studied an association with autoimmune diseases was found, including insulin dependent diabetes mellitus (6 cases), autoimmune hepatitis (3 cases), hypothyroidism (4 cases), thyrotoxicosis (1 case) and dermatomyositis (1 case). One or more organ-specific autoantibodies were positive in 12 (80%) of the 15 coeliacs with autoimmune disorders in comparison with their positivity in 6 (11%) of the 55 coeliacs without autoimmune diseases (p < 0.0001). CONCLUSIONS: The finding of organ-specific autoantibodies in coeliac patients discloses the coexistence of a wide spectrum of immunological diseases.

Adolescent↗
PubMed1996

Quantification of hepatitis C virus-infected peripheral blood mononuclear cells by in situ reverse transcriptase-polymerase chain reaction.

Hepatitis C virus (HCV) is known to infect peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis C, but the proportion of HCV-infected circulating cells is not detectable by conventional reverse transcriptase-polymerase chain reaction (RT-PCR) and the pathogenic significance of HCV lymphotropism is still unclear. Therefore, we have devised an in situ RT-PCR technique using fluorescein-labeled HCV-specific primers revealed by flow cytometry. PBMC were isolated from 28 patients with chronic HCV-related liver disease; of these, 6 had previously received an orthotopic liver transplantation (OLT) and were on immuno-suppressive treatment. Fourteen patients (50%) were found positive for HCV genome within PBMC by in situ RT-PCR, the proportion of HCV-infected cells ranging from 0.2% to 8.1%. All 6 OLT patients tested positive. The fluorescent signal, corresponding to the HCV-specific 340-bp amplicon, was confined to part of the cytoplasmic compartment of scattered PBMC. Of these 14 patients, 12 had also negativestrand HCV RNA within PBMC detected by "tagged" RT-PCR. We conclude that HCV may infect a significant proportion of PBMC in chronic hepatitis C patients, especially immunosuppressed OLT cases, and that viral replication within PBMC is a common occurrence. Over time, the persistence of HCV-infected immune system cells might interfere with normal immunologic mechanisms and play a role in the pathogenic processes leading to extrahepatic disorders such as mixed cryoglobulinemia and B-cell malignant lymphoma.

Adult↗
PubMed1996

Frequency and significance of antibodies to actin in type 1 autoimmune hepatitis.

Antibodies to actin have been proposed as diagnostic markers for type 1 autoimmune hepatitis. Our aims were to determine 1) if testing for antibodies to actin is superior to testing for smooth muscle antibodies (SMA); 2) if these antibodies identify patients with distinctive clinical features; and 3) if the production of antibodies to actin is associated with genetic risk factors for autoimmune hepatitis. Sera from 99 patients with type 1 autoimmune hepatitis were tested. The frequencies of HLA B8, DR3, DR4, and A1-B8-DR3 in patient subsets were compared with those in 80 normal subjects. Seventy-three patients (74%) had antibodies to actin. Antibodies to actin were found more commonly in patients with SMA than in patients without them (86% vs. 7%, P < .0001). Screening only for antibodies to actin and antinuclear antibodies (ANA) failed to establish the diagnosis of autoimmune hepatitis in 5 patients. Patients with antibodies to actin were younger than seronegative patients. They were also more commonly DR3-positive than normal subjects and more frequently B8-positive than patients with non-actin-associated SMA (49% vs. 0%, P = .004). Only patients with antibodies to actin died of liver failure (6% vs. 0%), and 10 of 11 patients requiring liver transplantation were seropositive for these antibodies. Indeed, death and liver transplantation occurred more frequently in these patients than in actin-negative patients with ANA (19% vs. 0%, P = .03). We conclude that routine screening for antibodies to actin may miss patients with type 1 autoimmune hepatitis. Antibodies to actin are associated with HLA B8 and DR3, and they identify patients with a poor prognosis.

Actins↗
PubMed1996

Impact of international autoimmune hepatitis group scoring system in definition of autoimmune hepatitis. An Italian experience.

We have reclassified 110 patients with autoantibody-positive cryptogenic chronic hepatitis according tot he aggregate scoring system proposed by the International Autoimmune Hepatitis Group for signs of hepatitis C virus (HCV) infection and the newly proposed terminology of "unclassified" chronic hepatitis. Anti-HCV and HCV viremia were assessed by second-generation assays and reverse transcription-polymerase chain reaction. Immunomorphological and immunochemical characterizations of antinuclear, smooth, muscle, liver-kidney microsomal type 1, and liver cytosol type 1 autoantibodies were also performed. All 45 anti-HCV negative patients fulfilled the score criteria for the diagnosis of "definite" or "probable" autoimmune hepatitis (AIH). Eight anti-HCV-positive cases reached the score of "probable" AIH, whereas the remaining 57 cases were diagnosed as unclassified chronic hepatitis. The scoring system allows the correct identification of all autoimmune cases without HCV infection. Autoimmune hepatitis runs a more severe disease course than unclassified chronic hepatitis, whose clinical and histological features are similar to those of autoantibody-negative chronic hepatitis C.

Adolescent↗
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

Human umbilical cord as substrate for IgA antiendomysial antibodies allows large scale screening for celiac sprue.

Sera from 800 patients referred to our laboratory for celiac sprue screening were tested for IgA antiendomysial antibodies on human umbilical cord (HUC-EmA) and for IgA and IgG antigliadin antibodies (AGA) on rodent tissue by indirect immunofluorescence. Thirty-three patients (4.1%) were positive for at least one of the two antibodies; IgA HUC-EmA were found in 25 patients, whereas IgA and IgG AGA were detected in 19 and 24 subjects, respectively. Twenty-seven (82%) of the 33 patients with immunological markers showed sub-total or severe partial villous atrophy consistent with celiac sprue, whereas the remaining six cases had normal histological findings. None of 30 antibody-negative biopsied patients showed abnormalities of duodenal mucosa. Of the 27 newly diagnosed celiacs, only 21 (78%) were positive for IgA and/or IgG AGA, whereas IgA HUC-EmA were found in all but two cases (93%). In these two cases, positive only for IgG AGA, celiac sprue was associated with IgA deficiency. Our study proves that the routine use of IgA HUC-EmA increases celiac sprue findings by up to 15% when compared with AGA.

Adult↗
PubMed1995

Detection of anti-liver cytosol antibody type 1 (anti-LC1) by immunodiffusion, counterimmunoelectrophoresis and immunoblotting: comparison of different techniques.

Liver cytosol specific antibody type 1 (anti-LC1) was first described in a proportion of patients with liver/kidney microsomal antibody type 1 (anti-LKM1)-positive autoimmune hepatitis (AIH) and is routinely evaluated by immunodiffusion (ID). Using human liver cytosol as the source of antigen, we have used ID, counterimmunoelectrophoresis (CIE) and immunoblotting (IB), to test sera from 167 patients with documented chronic liver diseases of different etiology. 15 patients had antinuclear antibody (ANA) and/or smooth muscle antibody (SMA)-positive AIH, 13 had anti-LKM1-positive AIH, four had ANA/SMA/anti-LKM1-negative AIH, 76 had anti-LKM1-positive hepatitis C (recently renamed unclassified chronic hepatitis-UCH), 40 had chronic hepatitis C, 15 had chronic hepatitis B, and 4 had chronic hepatitis D. A precipitin line of identity with an anti-LC1 reference serum was detected both by ID and CIE in 16 patients: six with anti-LKM1-positive 'definite' AIH, four with ANA/SMA/anti-LKM1-negative 'definite' AIH, and six with anti-LKM1-positive UCH. By IB, 14 out of the 16 anti-LC1-positive sera (87.5%) reacted with a 58 kDa human liver cytosolic polypeptide, whereas three out of 16 (19%) recognised an additional 60 kDa band. Compared to ID, CIE is more economical in terms of both time and reagents and provides more clear-cut results. The 58 kDa reactivity by IB was detectable in nearly all CIE/ID anti-LC1-positive patients, was not found among CIE/ID anti-LC1-negative patients. In conclusion, CIE is the ideal screening test for the detection of anti-LC1, an autoantibody that can be regarded as an additional serological marker of AIH and is especially useful in ANA/SMA/anti-LKM1 negative cases.

Adolescent↗
PubMed1995

IgA anti-endomysial antibodies on human umbilical cord tissue for celiac disease screening. Save both money and monkeys.

Since celiac disease screening by traditional IgA anti-endomysial antibody test is limited by high costs of monkey esophagus commercial kits as well as by rising ethical problems related to the endangered species, the identification of an inexpensive and commonly available substrate for this antibody determination is urgently required. To achieve this goal, we compared the prevalence of IgA anti-endomysial antibodies detected on monkey esophagus with that on human umbilical cord. Fifty-seven (95%) of 60 untreated adult celiacs were positive for these antibodies on monkey esophagus as well as on human umbilical cord. IgA anti-endomysial antibodies, detected on both tissues, were negative in all 200 disease and healthy controls tested, displaying a 100% specificity for gluten-sensitive enteropathy. These data suggest that human umbilical cord can replace monkey esophagus for IgA anti-endomysial antibodies test. Human umbilical cord allows unlimited testing for celiac disease screening on wide series of high-risk subjects, permitting identification of greater numbers of asymptomatic celiac patients with a remarkable saving of money and bypassing the ethical problems related to killing monkeys.

Adult↗
PubMed1995

Specific reactivity of fluorescein isothiocyanate-conjugated separated IgG and IgA from celiac disease sera on human tissues.

Study of the reaction of celiac disease-related antibodies with human substrates has been hampered by the immunological cross-reactivity between fluorescein isothiocyanate-conjugated anti-human immunoglobulins and immunoglobulins normally present in human tissues. In order to overcome this problem we extracted IgG from 2 celiac disease sera (positive for specific IgG and IgA antibodies) using a genetically engineered recombinant form of streptococcal protein G covalently immobilized on 4% agarose beads. The separated IgG and IgA was conjugated with fluorescein isothiocyanate and used in direct immunofluorescence on 0 blood group human duodenum, liver, and myocardium. Staining of the lamina propria beneath the villous epithelium, the endomysium of smooth muscle layers in the duodenum, and the liver sinusoidal and pericellular myocardial matrix was observed. The reactivity of celiac disease-related antibodies with various human tissues indicates that these antibodies are the result of a systemic immune response directed against all tissues containing matrix proteins.

Animals↗
PubMed1995

Liver cytosolic 1 antigen-antibody system in type 2 autoimmune hepatitis and hepatitis C virus infection.

Within the multiform liver/kidney microsomal (LKM) family, a subgroup of sera that reacts with a liver cytosolic (LC) protein has been isolated and the new antigen-antibody system is called LC1. Unlike LKM antibody type 1 (anti-LKM1), anti-LC1 is said to be unrelated to hepatitis C virus (HCV) infection and has therefore been proposed as a marker of 'true' autoimmune hepatitis type 2. Altogether 100 LKM1 positive sera were tested by immunodiffusion (ID). Twenty five gave a precipitation line with human liver cytosol; 17 of the 25 also reacted with rat liver cytosol. Thirteen of the 25 sera were anti-HCV positive by second generation ELISA: anti-HCV positive patients were significantly older (p < 0.001) and tended to have less active disease. No difference in anti-LC1 titre or ID immunoreactivity was found between anti-LC1/anti-HCV positive and anti-LC1/anti-HCV negative cases. In Western blotting experiments, 14 of 24 ID positive sera recognised a 58 kD protein of the human cytosolic fraction and 11 gave a similar reactivity when tested with human microsomes, suggesting the presence of the LC1 target antigen also in the microsomal preparation. Western blotting reactivity was similar for both anti-HCV positive and negative sera. These data confirm the existence of the LC1 antigen-antibody system that partially overlaps with LKM1, and that it is an additional marker of juvenile autoimmune hepatitis type 2. It does not, however, discriminate between patients with and without HCV infection.

Adolescent↗
PubMed1995

Heterogeneity of liver/kidney microsomal antibody type 1 in autoimmune hepatitis and hepatitis C virus related liver disease.

Liver/kidney microsomal antibody type 1 (LKM-1), the serological marker of a subset of autoimmune hepatitis, is also present in a proportion of patients with hepatitis C virus (HCV) related chronic liver disease. To characterise further this autoreactivity and to evaluate whether an autoantibody giving an identical immunofluorescence staining, and detected in two different clinical conditions, involves the same antigenic target(s), sera from autoimmune and HCV infected patients were tested with native, recombinant, and synthetic antigens. Sixty five sera were selected on the basis of the typical immunofluorescence pattern: 50 patients had serological markers of HCV infection, the remaining 15 suffered from autoimmune hepatitis. The reactivity of each serum with rat and human microsomal fractions, full length human recombinant CYP2D6, and two synthetic peptides spanning the amino acid regions 257-269 and 373-398 of CYP2D6 was systematically investigated by immunoblotting. Fourteen (93%) sera from autoimmune hepatitis patients and 39 (78%) from HCV infected patients reacted with rat and/or human microsomal polypeptides of 39 kD, 50 kD, 58 kD, and 66 kD in different associations, the 50 kD band being the most frequently observed. Reactivity to CYP2D6 and its amino acid sequence 257-269 was significantly more common in autoimmune hepatitis than in HCV infected patients (p < 0.001 and p < 0.0003, respectively). LKM-1 reactivity is directed against heterogeneous and not entirely defined autoantigens. The main target in autoimmune sera is CYP2D6 and its 257-269 amino acid region, while sera from patients with HCV infection are more likely to recognise other microsomal targets, the molecular identity of which is currently unknown.

Adolescent↗
PubMed1995

Hepatitis C virus (HCV) genotype, tissue HCV antigens, hepatocellular expression of HLA-A,B,C, and intercellular adhesion-1 molecules. Clues to pathogenesis of hepatocellular damage and response to interferon treatment in patients with chronic hepatitis C.

To obtain information on the mechanisms of hepatocellular damage and the determinants of response to interferon, hepatitis C virus (HCV) genotype, tissue HCV antigens, hepatocellular expression of HLA-A,B,C and intercellular adhesion-1 molecules, and the number of lobular T lymphocytes were studied in 38 anti-HCV-positive patients. 14 patients did not show a primary response to interferon treatment. HCV genotype 1b was detected in 11 of them. They displayed higher scores of HCV-positive hepatocytes, HLA-A,B,C, and ICAM-1 molecules expression than with the responders. HCV-infected hepatocytes maintained the capacity to express HLA-A,B,C and ICAM-1 molecules. CD8-positive T cells in contact with infected hepatocytes and Councilman-like bodies were observed. A significant correlation was found between the number of lobular CD8-positive T cells and alanine amino transferase levels. No differences were observed in clinical, biochemical, and histological features between patients with high and low number of hepatocytes containing HCV antigens. These data suggest a prominent role of T cell-mediated cytotoxicity in the genesis of hepatocellular damage. The high expression of interferon-inducible antigens like HLA-A,B,C molecules suggests the presence of strong activation of the interferon system possibly related to high HCV replication in nonresponder patients infected with genotype 1b.

Antigens, Viral↗
PubMed1995

Hepatocellular codistribution of c100, c33, c22, and NS5 hepatitis C virus antigens detected by using immunopurified polyclonal spontaneous human antibodies.

Hepatitis C virus (HCV) antigens in liver biopsy have been detected by immunohistochemistry using both spontaneous human IgG and murine monoclonal or rabbit polyclonal monospecific reagents. Conflicting results have been obtained in different studies. This was probably because of the incapacity of single experimental antibodies, raised against synthetic or recombinant peptides, to recognize native tissue antigens. To overcome this possibility, we immunopurified monospecific spontaneous polyclonal human Ig, therefore induced by native antigens, from the single antigen-containing bands of RIBA 3 strips. Antibodies to c100, c33, c22, and NS5 antigens were obtained from the serum of a patient affected by chronic hepatitis C. The IgG fraction of this serum had proved to stain tissue HCV antigens. Eight biopsies were selected on the basis of strong hepatocellular reactivity with the whole IgG fraction in a variable number (from 5% to 75%) of cells. The four antigens were detected in all biopsies; a clear cellular codistribution was observed on serial sections. These data demonstrate that the possibility to identify HCV antigens in liver biopsies is higher when using human antibodies induced by native antigens rather than experimental antibodies. The approach of immunopurification of human antibodies can be extended to other HCV-related epitopes to obtain reagents useful for the selection and optimization of monoclonal or polyclonal antibodies.

Antigens, Viral↗