A novel luminescence-based method for the detection of functionally active antibodies to muscarinic acetylcholine receptors of the M3 type (mAchR3) in patients' sera.

In different bioassays, functional antibodies reacting with the human muscarinic acetylcholine receptor M3(mAchR3) have been detected in sera from patients with Sjögren's syndrome (SS), and there is strong evidence that those antibodies may have pathogenetic relevance. However, depending on the method of detection, their prevalence varied. Furthermore, those bioassays are difficult to standardize. We report on the development and optimization of a novel test system based on a luminometric method to determine downstream signalling of mAchR3 which produces specific and reproducible results. Chinese hamster ovarian (CHO) cells were transfected with plasmids encoding mAchR3 and a green fluorescence protein (GFP)/aequorin fusion protein. Incubation of cells with carbachol resulted in an increase in intracellular [Ca(2+)], which was detected by measuring light emission with a luminometer, and the effect of incubation with patients' immunoglobulins (Ig) was evaluated. Optimal cell density, Ig preparation and time of incubation with patients' sera were determined. Sera from patients with primary Sjögren's syndrome (pSS; n = 40), systemic sclerosis (SSc; n = 47), myasthenia gravis (MG; n = 133) and 50 blood donors were analysed. Optimal assay conditions were obtained with a cell density of 100 000 cells/ml, isolation of Ig by ammonium sulphate precipitation and short-term incubation. Based on this highly reliable assay, 50% of the pSS patients had antibodies which inhibited carbachol-induced activation of mAchR3; none of the SSc patients, 6% of the patients with MG and 12% of the blood donors had antibodies which reacted with the mAchR3. This method facilitates the determination of functional anti-mAchR3 antibodies in patients' sera, confirmed their high prevalence in pSS patients and may, therefore, help to analyse their pathogenetic and clinical relevance in more detail.

Detection of novel non-M2-related antimitochondrial antibodies in patients with anti-M2 negative primary biliary cirrhosis.

OBJECTIVE: In 95% of patients with primary biliary cirrhosis (PBC) antimitochondrial antibodies (AMAs) can be detected reacting with at least one of the five components of the M2 antigen identified as the 2-oxoacid dehydrogenase complex (OADC). However, among our PBC sera 15-20% are anti-M2 negative by ELISA and western blotting but in the immunofluorescence test (IFT) they show the typical AMA staining. The aim of the present study was to characterise the target antigen(s) of these non-M2-related AMAs. PATIENTS AND METHODS: We analysed sera from 27 patients with clinically and histologically proven PBC being AMA positive by the IFT but anti-M2 negative by ELISA and western blotting. They were tested by western blotting against various 100,000 g supernatants obtained after sonication of mitochondria from rat liver, bovine heart and pig kidney. These were further separated by isopycnic sucrose density centrifugation using different sucrose density fractions. RESULTS: Fourteen of the 27 AMA positive/anti-M2 negative sera (52%) reacted in the western blotting with a 60 kDa protein and eight (29%) with an 80 kDa protein, both present in the 100 000 g supernatant from bovine heart mitochondria accumulating at sucrose densities of 1.14-1.16. An identity of these determinants with any of the M2-related antigens could be excluded. In the 60 kDa band components of the mitochondrial enzymes F(1)F(0)-ATPase, ubiquinone cytochrome c reductase and acyl CoA dehydrogenase were detected by MALDI-TOF analysis; the 80 kDa protein could not be further characterised. CONCLUSIONS: AMA positive/anti-M2 negative PBC sera contain antibodies to further mitochondrial antigens at 60 and 80 kDa which do not correspond to any of the M2 determinants. Those antibodies can be detected to a lesser extent in sera from patients with classical anti-M2 positive PBC but not in patients with other hepatic and non-hepatic disorders and may, therefore, represent additional marker antibodies for the serological diagnosis of PBC.

Demonstration of autoantibodies to recombinant human sulphite oxidase in patients with chronic liver disorders and analysis of their clinical relevance.

It has been shown previously that sera from patients with cholestatic liver diseases react with sulphite oxidase (SO) prepared from chicken liver. In order to analyse this reactivity and the clinical relevance of anti-SO antibodies in more detail, we produced human recombinant SO. Human recombinant SO (60 kDa) was expressed in Escherichia coli and applied to enzyme-linked immunosorbent assay and Western blot. Sera from patients with autoimmune liver disorders [primary biliary cirrhosis (PBC) n = 96; autoimmune hepatitis (AIH) n = 77; primary sclerosing cholangitis (PSC) n = 39], and from patients with other hepatic (n = 154) and non-hepatic chronic inflammatory disorders (n = 113) were investigated. Highest incidence and activities of IgG-anti-SO antibodies were observed in PSC patients. Nine of 16 untreated (56%) and four of 23 PSC patients treated with ursodeoxycholic acid (UDCA) (17%) were positive. Antibody activity decreased significantly during UDCA treatment. Five per cent of PBC and 9% of AIH patients, but also 15% of patients with alcoholic liver disease, were IgG anti-SO-positive. In patients with viral hepatitis and non-hepatic disorders they could be hardly detected. Anti-SO antibodies are further anti-mitochondrial antibodies in chronic liver diseases. They occur predominantly in PSC, and UDCA treatment seams to decrease antibody activity. Whether these antibodies are primary or secondary phenomena and whether they are related to the aetiology or pathogenesis, at least in a subgroup of patients with chronic liver diseases, has still to be evaluated.

[Noninvasive diagnosis in the evaluation of shunt size in children with atrial septal defect--an evaluation scheme]. / Nichtinvasive Diagnostik zur Beurteilung der Shuntgrösse bei Kindern mit Vorhofseptumdefekt--ein Beurteilungsschema.

A cumulative score of five non-invasive diagnostic procedures is used to predict the size of the shunt in atrial septal defect (type II) in children. Each of those diagnostic managements being represented by zero, plus one, or two points: a higher score value refers to a more significant shunt volume. Summing up the points you will find 10 to 7 in an atrial septal defect that is worth being operated. In 6 and 5 points the prediction is unsure by scoring. Values of 4 or less points indicate a septal defect with no significant shunting. The validity of this scoring system has been tested in 35 children each examined by heart catheterization.

[Incidence and significance of candidiasis in biopsy material of gastric ulcers (author's transl)]. / Häufigkeit und Bedeutung der Soormykose im Biopsie-Material des Ulcus ventriculi.

121 cases of candidiasis were histologically demonstrated in the course of histological studies of gastric mucosal biopsies in 20 401 patients. Infestation of necrotic tissue with Candida albicans was found exclusively in patients with gastric ulcer, ulcerating carcinoma or lymphoma. Candidiasis was twice as common in carcinoma as in non-carcinomatous gastric ulcer. In the majority of those patients with ulcer who also had Candida albicans mycosis there was was at the same time atrophic or dysplastic gastric mucosa at the edge of the ulcer. Demonstration of candidiasis in biopsy material from gastric ulcer should thus be interpreted as suspicious of carcinoma, until and unless further studies confirm or exclude it.