Tissue antibodies in primary biliary cirrhosis, active chronic (lupoid) hepatitis, cryptogenic cirrhosis and other liver diseases and their clinical implications

The sera of virtually all patients with primary biliary cirrhosis contained non-organ specific antibodies, probably directed against mitochondria, which give rise to cytoplasmic `M' immunofluorescence in unfixed sections from various organs in high titres. They were also found in 31% of patients with cryptogenic cirrhosis particularly those with obstructive features, and 28% of cases of active chronic (lupoid) hepatitis. In contrast only two patients out of twenty-eight with extrahepatic biliary obstruction and one patient out of twenty-five with infective hepatitis gave positive reactions and then in very low titre. Uniformly negative results for `M' immunofluorescence were obtained in alcoholic cirrhosis, cholestatic drug jaundice and cholestasis associated with ulcerative colitis. These findings suggest that `M' antibodies do not arise merely in response to liver damage and confirm the value of the test for the differential diagnosis between primary biliary cirrhosis and extrahepatic biliary obstruction although care must be taken in interpretation of the results in cases with associated connective tissue disorders where a significant incidence of positive reactions was observed. Seventy-seven per cent of juvenile cirrhosis, 46% of primary biliary cirrhosis and 38% of the cryptogenic cirrhosis patients had anti-nuclear factors. The incidence of ANF in the other liver groups was no higher than in mixed hospital controls. A high incidence of antibodies staining smooth muscle was observed in the juvenile and primary biliary cirrhosis groups. No liver specific antibodies could be detected by precipitation or tanned red cell agglutination although non-organ-specific antibodies were demonstrated in some instances with the latter technique. Thyroid specific autoantibodies were increased four-fold in females with active chronic hepatitis but the incidence of thyroid and gastric autoantibodies was not significantly different from the controls in all other liver conditions studied. Autoimmune reactions are prominent in primary biliary cirrhosis and in active chronic hepatitis but their role in the pathogenesis of these diseases is still undefined.     

High incidence of poor sulfoxidation in patients with primary biliary cirrhosis

An impaired sulfoxidation pathway has been implicated in the pathogenesis of chlorpromazine-induced hepatotoxicity. Since some patients with chronic chlorpromazine-induced cholestasis may have features of primary biliary cirrhosis, we studied the ability to sulfoxidate the amino acid analogue S-carboxymethyl-cysteine in 44 patients with primary biliary cirrhosis and in two control groups--one without liver disease and one with a variety of liver diseases other than primary biliary cirrhosis. Poor sulfoxidation was observed in 84 percent of the patients with primary biliary cirrhosis, as compared with 24 percent of patients with other liver diseases and 22 percent of normal controls (P less than 0.0005 for both comparisons). Poor sulfoxidation did not correlate with the degree of hyperbilirubinemia or histologic severity of liver disease in any of the groups studied. There was an inverse correlation with age only in the patients with primary biliary cirrhosis (r = -0.44, P less than 0.001). Liver transplantation was performed in six of the patients and improved sulfoxidation in five; in the four with primary biliary cirrhosis, sulfoxidation improved from poor to good or intermediate. We conclude that poor sulfoxidation is closely associated with primary biliary cirrhosis but not with the other liver diseases we studied.     

The prevalence of antibodies to reovirus type 3 in adults with idiopathic cholestatic liver disease

Recent evidence suggests that neonatal infection with Reovirus type 3 (Reo-3) may play an important role in the pathogenesis of certain idiopathic cholestatic liver diseases of children. Whether this virus is of pathogenetic importance in similar diseases seen in adults is unclear. In this study, sera from 22 adults with idiopathic cholestatic liver disease (16 primary biliary cirrhosis (PBC) and six primary sclerosing cholangitis (PSC] were tested for antibody to Reo-3 virus by a standard hemagglutination inhibition assay. The results were compared to antibody prevalence in ten adults with various other causes of chronic liver disease and 11 healthy volunteers. Although patients with idiopathic cholestatic liver disease had a high prevalence of anti Reo-3 antibodies (20/22, 91%), similar prevalence rates were found in the two control populations (7/10, 70% in chronic liver disease controls and 11/11, 100% in healthy volunteers). Moreover, the geometric mean titres of antibody to Reo-3 virus were similar in all study groups. While these results do not exclude a pathogenetic role for Reo-3 viral infection, they do imply that some genetic predisposition must exist if Reo-3 infection is truly of pathogenetic importance in idiopathic cholestatic liver disease of adults.     

Antilactoferrin antibodies in autoimmune liver disease

Antilactoferrin antibodies have been reported in patients with several autoimmune disorders, including primary biliary cirrhosis, autoimmune hepatitis and autoimmune cholangitis. We investigated the prevalence and the clinical significance of such autoreactivity in patients with autoimmune and viral chronic liver disease. Sera from 39 patients with autoimmune hepatitis, 51 with primary biliary cirrhosis, 17 with autoimmune cholangitis, 24 with primary sclerosing cholangitis and 28 with HCV-related chronic hepatitis were studied. Positivity for antilactoferrin antibodies was evaluated by Western immunoblotting with purified human lactoferrin. Antilactoferrin antibodies were detected more often in autoimmune liver disorders (25% autoimmune hepatitis, 25% primary biliary cirrhosis, 35% autoimmune cholangitis, 29% primary sclerosing cholangitis) than in HCV-related chronic hepatitis (3.5%, P < 0.02 versus all). Positivity for antilactoferrin antibodies was not associated with a particular clinical or biochemical profile of the underlying liver disease. No correlation was observed between antilactoferrin reactivity and perinuclear antineutrophil cytoplasmic antibodies. Antilactoferrin antibodies are present significantly more often in autoimmune than in viral liver disorders, but they cannot be considered the serological marker of a specific autoimmune liver disease.     

Five novel loci for inherited hearing loss mapped by SNP-based homozygosity profiles in Palestinian families

In communities with high rates of consanguinity and consequently high prevalence of recessive phenotypes, homozygosity mapping with SNP arrays is an effective approach for gene discovery. In 20 Palestinian kindreds with prelingual nonsyndromic hearing loss, we generated homozygosity profiles reflecting linkage to the phenotype. Family sizes ranged from small nuclear families with two affected children, one unaffected sibling, and parents to multigenerational kindreds with 12 affected relatives. By including unaffected parents and siblings and screening 250 K SNP arrays, even small nuclear families yielded informative profiles. In 14 families, we identified the allele responsible for hearing loss by screening a single candidate gene in the longest homozygous region. Novel alleles included missense, nonsense, and splice site mutations of CDH23, MYO7A, MYO15A, OTOF, PJVK, Pendrin/SLC26A4, TECTA, TMHS, and TMPRSS3, and a large genomic deletion of Otoancorin (OTOA). All point mutations were rare in the Palestinian population (zero carriers in 288 unrelated controls); the carrier frequency of the OTOA genomic deletion was 1%. In six families, we identified five genomic regions likely to harbor novel genes for human hearing loss on chromosomes 1p13.3 (DFNB82), 9p23–p21.2/p13.3–q21.13 (DFNB83), 12q14.3–q21.2 (DFNB84; two families), 14q23.1–q31.1, and 17p12–q11.2 (DFNB85).     

Nuclear antigens recognized by antibodies present in liver disease sera.

Nuclear and nuclear matrix proteins of HeLa cells were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and subsequently transferred onto nitrocellulose. Antibodies present in sera of patients with primary biliary cirrhosis and autoimmune chronic active hepatitis reacted with some of the blotted proteins. The antibodies were mainly directed against chromatin-associated proteins and protein constituents of discrete RNP particles. In addition, antibodies found in autoimmune liver disease sera detected a hitherto undescribed nuclear protein of 54 kD, and a nuclear matrix protein of approximately 150 kD. Antibodies recognizing a nuclear 25 kD doublet apparently constituted a marker antibody for autoimmune liver disease. Those directed at the 17 kD centromere protein were associated with the primary biliary cirrhosis-related CREST syndrome, while those recognizing La antigen were related to cases of sicca syndrome associated with autoimmune liver diseases.     

Anticardiolipin antibodies are elevated in drug-free,multiply affected families with schizophrenia

The objective of this study was to measure anticardiolipin antibodies in patients and healthy relatives in multicase families with schizophrenia. Twenty-eight (28) multicase families with schizophrenia were examined. One hundred three drug-free patients and 66 first-degree relatives consented to evaluation by DSM-III-R criteria. Criteria for patient definition included the following: age 16, a confirmed hospital diagnosis of schizophrenia, knowledge of biological parents, and consent to participate. Additional data were drawn from family history and medical records. Serum samples were tested separately for IgG and IgM anticardiolipin by enzyme-linked immunosorbent assay (ELISA) and designated positive/negative by comparison to the reactivity of an age-matched control group. IgG anticardiolipin antibodies were significantly more common in both patients and relatives compared to controls. IgM anticardiolipin antibodies were significantly more common in patients. In 75% of families at least one member was anticardiolipin positive and this positivity correlated with patient positivity. The relevance of anticardiolipin antibodies in both patients and healthy relatives of some multicase families to the pathogenesis of schizophrenia is discussed.     

Immunological studies in patients with juvenile-onset myasthenia gravis and in their relatives

Fifty-four patients who developed myasthenia before the age of 20 had an increased incidence of autoantibodies similar to that in adult-onset myasthenics, except that there was no significant increase of striated muscle antibodies. The increase in thyroid antibodies was associated with an increase of thyroid disease.

Their 150 relatives showed a significant increase in thyroid antibodies, in thyroid disease, and a non-significant increase in the five other autoantibodies studied. There was aggregation of thyroid antibodies in some families.

Two patients and one relative were deficient for immunoglobulins (one patient and one father for IgA and one patient for IgM).

These familial immunological abnormalities, which are more marked in the families of patients with juvenile-onset myasthenia than in the families of adult-onset patients, point to fundamental genetic immunopathogenesis of the disease.

     

Clinical significance and prevalence of anti-Saccharomyces cerevisiae antibody in Chinese patients with primary biliary cirrhosis

Clinical significance of anti-Saccharomyces cerevisiae antibody (ASCA) and its prevalence in Chinese primary biliary cirrhosis (PBC) patients have not been characterized and therefore needs to be defined. Enzyme-linked immunosorbent assay was used to test ASCA in sera from 198 PBC patients, 85 patients with other liver diseases (OLD) and 35 health controls (HC). Indirect immunofluorescence was used to detect anti-mitochondrial antibodies (AMA) in PBC. Results showed that the frequency of ASCA in PBC, 29.8 %, was higher than other disease groups. And ASCA occurred more frequently in PBC patients with positive anti-gp210 than the negative ones. Also, ASCA was detected in 7 out of 15 PBC negative for AMA. Some liver-related biochemical indices and inflammatory indices were significantly higher in PBC patients with positive ASCA (p < 0.05). In conclusion, the prevalence of ASCA in Chinese PBC patients is 29.8 %. PBC patients with positive ASCA are associated with more severe liver injury, and ASCA-IgA might be related to disease activity of PBC.     

The primary immune response to haemocyanin in patients with primary biliary cirrhosis

Both the humoral and cellular immune response to haemocyanin was measured in normal subjects and patients with liver disease including primary biliary cirrhosis, other forms of cholestasis, and cryptogenic cirrhosis. Significant differences in antibody titre were found 2 weeks after immunization being highest in normal subjects, less in cryptogenic cirrhosis and least in primary biliary cirrhosis. The incidence of positive skin tests, indicating the development of delayed hypersensitivity to haemocyanin, was significantly less in patients with primary biliary cirrhosis and cryptogenic cirrhosis than in those with cholestasis or the normal subjects. It is suggested that the poor antibody response seen in primary biliary cirrhosis, could be related to impaired T cell function and hence lack of the T and B cell co-operation necessary for antibody formation to some antigens.

The presence of delayed hypersensitivity to haemocyanin as measured by the skin test was compared to other tests of delayed hypersensitivity including the tuberculin skin test, DNCB sensitization and PHA-stimulated in vitro lymphocyte transformation. Of twenty patients with primary biliary cirrhosis the results of all four tests correlated in nine but did not agree in the remaining eleven patients. These results emphasize the importance of using a number of indices when assessing the degree of anergy of any one patient.

     

Epstein-Barr virus-related antibody patterns in ataxia-telangiectasia.

Epstein-Barr virus-related antibody titres were determined in twenty-seven patients with ataxia-telangiectasia (AT) and twenty-two healthy members of their families, in twenty-two patients with other diseases, among them ten with Behçet''s disease and ten with various primary immune deficiencies, and fifteen healthy members of their families, and in twenty-three unrelated healthy controls. The AT patients showed an increased incidence (55.6%) of high antibody titres (greater than or equal to 1:320) to viral capsid antigen (VCA), and also a high incidence (48.2%) of antibodies to Epstein-Barr virus (EBV) induced early antigens (EA), but low titres (less than 1:10) of antibodies to the EBV-associated nuclear antigen (EBNA) in 44.2% of the cases. The geometric means of anti-VCA were three-to four-fold higher, and of anti-EBNA six-fold lower, than those of the control groups. The patients with the other diseases did not differ significantly from the controls except for a higher incidence of anti-EBNA titres of less than 1:10 (38.1% vs 4--5%). AT patients with low anti-EBNA titres tended to have more advanced T cell deficiencies than AT patients with moderate anti-EBNA titres, as detected by counts of total lymphocytes and E-rosetting cells, and skin test responses. The results support the hypothesis that a functioning T cell system is required to release EBNA from EBV genome-carrying cells for initial and maintained production of anti-EBNA.     

Uptake of carrier testing in families after cystic fibrosis diagnosis through newborn screening

Newborn screening (NBS) for cystic fibrosis (CF) provides the opportunity for cascade carrier testing of relatives. Uptake of testing by adult non-parent relatives of children diagnosed with CF through NBS has not been previously described, and this study describes uptake by both parents and adult non-parent relatives in Victoria, Australia. Pedigrees were taken from parents of children who were born in 2000–2004 and diagnosed with CF. A total of 40 families were eligible for the study and 30 (75%) were recruited. In all, 716 non-parent relatives were identified from the pedigrees as eligible for carrier testing, and 82 (adjusted uptake percentage: 11.8% 95% confidence interval 8.0–15.7) have had carrier testing by March 2009. On average, 2.7 non-parent relatives per family had CF carrier testing after diagnosis through NBS. The odds of being tested were greater for females than males (adjusted odds ratio 1.61; 95% confidence interval 1.11–2.33; P=0.01) and greater for those more closely related to the child with CF (adjusted odds ratio 5.17; 95% confidence interval 2.38–11.24; P<0.001). Most relatives who undergo testing are tested immediately after the baby''s diagnosis; however, some testing is undertaken up to 8 years later. These results indicate that in a clinical setting, the diagnosis of a baby with CF by NBS does not lead to carrier testing for the majority of the baby''s non-parent relatives. We suggest re-contact with parents to offer cascade carrier testing.     

Production of interleukin-1 by mononuclear cells of newborns and their mothers.

We have examined neutrophil phagocytosis and intracellular killing of Staphylococcus aureus in patients with primary biliary cirrhosis, alcoholic liver disease and chronic active hepatitis in comparison with age and sex matched controls. Significant decrease in neutrophil phagocytosis was found in both early and advanced primary biliary cirrhosis while impaired phagocytosis was seen in alcoholic cirrhosis but not in alcoholic fatty liver. In both disorders the effect appeared to be mediated by the patient's serum as there was no difference between patient and control neutrophil function when the test was performed in AB serum. Although total bacterial killing was reduced in chronic active hepatitis, phagocytosis was not impaired. Intracellular killing was not affected in any of the liver disease groups. These results support the view that serum factors exist in patients with liver disease which inhibit neutrophil phagocytosis. While these studies confirm earlier finding in alcoholic liver disease, this is the first demonstration of such a defect in primary biliary cirrhosis.     

Effect of oestradiol on the secretory immune system in the rat: an increase in biliary IgM antibodies against a T-cell independent antigen.

The effect of sex hormones on the secretory immune system was studied in rats ooforectomized and substituted with oestradiol in permeable capsules deposited subcutaneously. Ooforectomized rats and sham-operated rats without oestradiol substitution served as controls. Two weeks after the ooforectomy the rats were immunized in the Peyer's patches with Escherichia coli O6 carrying type 1 fimbriae. Some rats were given a booster dose with the same antigen at the same site 3 weeks later. Bile and serum were taken 7 days after the last immunization. The oestradiol treatment did not influence the total level of IgA or IgG or the level of specific IgA or IgG antibodies in bile or serum. Instead there was a specific increase in biliary IgM antibodies against lipopolysaccharide (LPS) as well as a rise in the total IgM concentration in the bile in the oestradiol-treated rats. Despite this there was no difference in the biliary IgM anti-fimbrial antibody level between the different groups. The oestradiol treatment did not change the levels of total immunoglobulins or antibodies against fimbriae and LPS in serum. An oestradiol-induced increase similar to the one seen in biliary IgM anti-LPS antibodies in primary immunized animals was not seen during the secondary response in booster immunized rats. Thus it seemed as if the effect of oestradiol on the secretory immune system in the bile was mainly due to an influence on primary stimulated B-cell clones in the liver, producing IgM antibodies against a T-cell-independent antigen. The effect may be mediated through a direct action of oestradiol on the B lymphocytes.     

Serum interferon-gamma-inducing factor/IL-18 levels in primary biliary cirrhosis

Primary biliary cirrhosis is an autoimmune disease of the liver in which T helper 1 cytokines predominate over those of T helper 2 in the pathogenesis. Interleukin- 18 (IL-18), for which the gene was recently cloned, is a novel T helper 1 cytokine, which augments interferon-gamma production. We designed this study to clarify the role of IL-18 in primary biliary cirrhosis and to examine whether serum IL-18 level can be a prognostic indicator for the disease. Serum IL-18 levels were measured using an enzyme linked immuno sorbent assay with mouse monoclonal antibodies. Twenty-two healthy volunteers, 31 patients with primary biliary cirrhosis (Scheuer's stage I, 13; II, 10; and IV, 8), 20 patients with autoimmune hepatitis, 11 patients with virus-related liver cirrhosis and six patients with obstructive jaundice were enrolled. Significant differences of serum IL-18 levels were observed between patients with Scheuer's stage IV and those with stage I, or II, virus-related liver cirrhosis and obstructive jaundice (P < 0.05). The IL-18 levels in primary biliary cirrhosis increased according to the disease progression, and fell promptly after living-related liver transplantation. Moreover, serum IL-18 levels in primary biliary cirrhosis were correlated with serum bilirubin concentrations and the Risk scores of the Mayo Clinic prognostic model for the disease. The IL-18 levels observed in patients with autoimmune hepatitis were also elevated, and correlated with the activity of the disease. These results indicate that serum interleukin-18 levels reflect the severity of primary biliary cirrhosis, the activity of autoimmune hepatitis, and may be an additive prognostic indicator in primary biliary cirrhosis.     

Red cell survival in biliary cirrhosis

Studies were carried out on eight patients with primary biliary cirrhosis. Four patients were found to have a haemolytic anaemia; one had Coombs red cell antibodies. Two patients had evidence of splenic sequestration of red cells using the radio-chromium technique. There was a significant correlation between the red cell survival and the bromsulphthalein retention test and the red cell survival and the serum level of bilirubin. It was concluded that the anaemia of cirrhosis of the liver, including primary biliary cirrhosis, may be due to a number of mechanisms and a unifying hypothesis based on the degree of liver dysfunction is suggested.     

Antibodies to nuclear lamin proteins in liver disease

Sera of patients with autoimmune liver disease contained antibodies reactive with nuclear lamins. These antigens were identified in immunoblotting experiments, using isolated nuclei, nuclear matrices, nuclear lamina-pore complexes and purified lamins as antigen source. The lamins were, furthermore, characterized by 2-dimensional gel electrophoresis. Antibodies to nuclear lamins were found in 75 per cent of the active lupoid hepatitis cases, but not in patients with inactive disease. Anti-lamin antibodies were detected in 8 per cent of primary biliary cirrhosis sera. The autoimmune liver disease sera recognized predominantly the nuclear lamins A/C, and less frequently the lamins A/B/C or lamin B.     

Antibodies to nuclear Lamin proteins in liver Disease

Sera of patients with autoimmune liver disease contained antibodies reactive with nuclear lamins. These antigens were identified in immunoblotting experiments, using isolated nuclei, nuclear matrices, nuclear lamina-pore complexes and purified lamins as antigen source. The lamins were, furthermore, characterized by 2-dimensional gel electrophoresis. Antibodies to nuclear lamins were found in 75 per cent of the active lupoid hepatitis cases, but not in patients with inactive disease. Anti-lamin antibodies were detected in 8 per cent of primary biliary cirrhosis sera. The autoimmune liver disease sera recognized predominantly the nuclear lamins A/C, and less frequently the lamins A/B/C or lamin B.     

Cryoglobulins in acute and chronic liver diseases

Cryoglobulins were detected in the sera of thirteen patients with acute viral hepatitis and of twelve with chronic hepatic diseases (active chronic hepatitis, primary biliary cirrhosis and cryptogenic cirrhosis). Their nature and antibody activity was studied. In both groups, most of them consisted of mixed cryoimmunoglobulins (IgM, IgG and/or IgA), but some were single-class immunoglobulins with one or both types of light chains. Unusual components were also found.α₁-fetoprotein was present in four cryoprecipitates: in two as the single constituent and in two associated to immunoglobulins; hepatitis-associated antigen co-existed in one of the latter. Some cryoglobulins showed antibody activity against human IgG, smooth muscle and mitochondrial antigens. In one case, the IgM-kappa of the cryoprecipitate had antibody activity against α₁-fetoprotein; this antigen was also present in the cryoprecipitate, suggesting immune-complex formation.Autoantibodies were also looked for in the sera of the twenty-five patients; apart from the most common ones, antibodies to α₁-fetoprotein were found in two patients.     

HLA DQA1*0501 and DQB1*02 in Cuban celiac patients

Celiac disease (CD) susceptibility has been strongly associated with HLA-DQ2 and HLA-DQ8. The main objective of this study was to assess the distribution of HLA DQA1*0501 and DQB1*02 alleles (DQ2) for the first time in a group of Cuban celiac patients. We evaluated 22 patients, 54 first-degree relatives, and 60 controls for detection of antitissue transglutaminase (tTG)-specific antibodies in serum. We found that 100% of the probands and 19% of the first-degree relatives were positive for the antibodies in serum. We did not detect any specific response for the healthy control individuals. We observed a significant over-representation of DQ2 heterodimer, both in patients and relatives. In the group of patients, 86.3% were positive for DQA1*0501, 90.2% were positive for DQB1*02, and 86.3% were positive for both alleles. The frequencies in relatives and controls were as follows: 70%, 90%, and 70%; and 56.6%, 45%, and 20%, respectively. In conclusion, we found that the proportion of our celiac patients carrying DQ2 was similar to the proportion of CD patients reported in populations with different genetic backgrounds. These results underline the primary importance of HLA-DQ alleles in susceptibility to celiac disease.