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To construct and express a humanized M(2) autoantigen trimer designated as BPO and to apply it in the diagnosis of primary biliary cirrhosis (PBC).cDNA fragments encoding M(2)-reactive epitopes of pyruvate dehydrogenase complex E(2) (PDC-E(2)), branched chain 2-oxo-acid dehydrogenase complex E(2) (BCOADC-E(2)) and 2-oxo-glutarate dehydrogenase complex E(2) (OGDC-E(2)) were amplified with PCR using total RNA extracted from human peripheral mononuclear blood cells. The fragments were cloned into the plasmid vector pQE-30 and then transferred into E. coli M15 (pREP4) for expression, which was induced by isopropylthio-beta-D-galactoside. The expressed recombinant BPO protein was demonstrated by SDS-PAGE, Western-blotting and Immunoabsorption test, its antigenic reactivity and specificity were identified with seven M(2)-positive sera confirmed at Euroimmun Research Center (Germany). Using the purified BPO, M(2) antibodies in sera from patients with PBC and other liver related diseases were detected with ELISA.The expressed BPO was observed with both antigenic reactivity and specificity of M(2) autoantigens. The determination of M(2) antibodies by BPO with ELISA was more sensitive than using the Euroimmun's kit with the coefficients of variation less than 10 % in both interassay and intraassay. With the newly established method, M(2) antibodies were found in 100 % (20/20) of patients with PBC. Six cases of liver disease with unknown etiology and 1 patient with drug induced liver injury had detectable levels of serum M(2) antibodies. There were also 2 patients with autoimmune cholangitis and 1 with autoimmune hepatitis showing M(2)-antibody positive.Compared with the routine immunofluorescence assay and commercially available assay kit using porcine heart mitochondrial protein as the antigen, the detection system established in the present study shows higher sensitivity and specificity and may be used as a powerful tool for the diagnosis of PBC.

AIM: To investigate the presence of M2 antibodies specific for primary biliary cirrhosis (PBC) in asymptomatic Chinese and identify patients with early PBC. METHODS: Enzyme-linked immunosorbent assay (ELISA) tests for M2 antibodies to recombinant protein were performed in 5011 subjects (age range, 26-85 years; mean age: 45.81 ± 15.02 years) who took an annual physical examination. M2-positive subjects were further analyzed for immunoglobulin (Ig) classes and subclasses of M2 antibodies. Clinical, biochemical and immunological data were obtained for M2-positive subjects. In addition, ultrasonography (US) or endoscopic retrograde cholangio-pancreatography (ERCP) was performed to exclude any disorders other than PBC. RESULTS: M2 antibodies were detected in 8 (0.16%) of the 5011 subjects studied. Of the 8 subjects, 7 were female and 1 was male (age range: 40-74 years). An unexplained increase of serum alkaline phosphatase (ALP) and gamma glutamyl transpeptidase (γ-GT) values, often to striking levels, was detected in 4 M2-positive subjects, 3 of them accorded with the diagnostic criteria recommended by the American Association for the Study of Liver Diseases, even though they had no symptoms of PBC (such as fatigue, pruritus or jaundice). Liver biopsy was performed in two M2-positive subjects and the histology was compatible with PBC in both cases. CONCLUSION: Our data, while not assessing the true prevalence of asymptomatic PBC in the general population, suggest that asymptomatic PBC is much more common in China than has been supposed.

To study the intestinal permeability (IP) following stress of abdominal operation and the different effects on IP of enteral nutrition (EN) and parenteral nutrition (PN).Forty patients undergoing abdominal surgery were randomized into EN group and PN group. Each group received nutritional support of the same nitrogen and calorie from postoperative day (POD) 3 to POD 11. On the day before operation (POD-1), POD 7 and POD 12, 10 g of lactulose and 5 g of mannitol were given orally, and urine was collected for 6 hours. Urine excretion ratios of lactulose and mannitol (L/M) were measured.L/M ratios of EN group on POD-1, POD 7 and POD 12 were 0.026+/-0.017, 0.059+/-0.026, 0.027+/-0.017, respectively, and those of PN group were 0.025+/-0.013, 0.080+/-0.032, 0.047+/-0.021, respectively. Patients of both groups had elevated L/M ratios on POD 7 vs. POD-1. However the ratio returned toward control level in EN group by POD 12. In contrast, PN group still had elevated L/M ratios on POD 12.L/M ratio increases for a period of time after surgical trauma and the loss of gut mucosal integrity can be reversed by substitution of enteral nutrition.