M. Fusconi

IgA antiendomysial antibody test. A step forward in celiac disease screening.

Serum IgA antiendomysial antibodies (EmA) were found in 61 (87%) of 70 adults and children with untreated celiac disease, whereas IgA antigliadin antibodies (AGA) and IgA R1-antireticulin antibodies (R1-ARA) were positive in 71% and 47%, respectively, of the same patients. Two of the nine untreated celiacs negative for IgA EmA showed positivity for IgA AGA. While IgA AGA and R1-ARA disappeared in all the celiacs, tested one year after gluten-free diet, IgA EmA persisted at low titer in seven (18%) of these 38 subjects, although the jejunal biopsy showed a complete regrowth of jejunal villi. All the disease control patients as well as the blood donors tested were always negative for the three IgA antibodies. Our results state that the search for both IgA EmA and AGA gives the best results in the screening of celiac disease, since the positivity for at least one of these two antibodies allows identification with a 100% specificity of the 90% of untreated celiac patients.

Anti-actin antibodies: a new test for an old problem.

Smooth muscle antibodies with anti-actin specificity are commonly regarded as markers of autoimmune liver disease. However, there are interpretational problems because different techniques have been used for their identification and therefore the results are difficult to compare. The present paper reports the results of a new method for the identification of anti-actin antibodies (indirect immunofluorescence on cryostat sections of liver from rats chronically injected with phalloidin). The results have been compared with those obtained by four other techniques: demonstration by immunofluorescence of kidney peritubular reactivity (SMAT), of anti-microfilament antibodies (on HEp-2 cells and vinblastine-treated peripheral blood mononuclear cells) and counterimmunoelectrophoresis with purified muscle actin as antigen. The new method proved to be the most sensitive and specific. Furthermore, its reproducibility was found to be high, the interpretation easy and the cost low. The clinical significance of anti-actin antibodies in patients with chronic liver disease is also discussed.

Anti-cyclic citrullinated peptide positivity in non-rheumatoid arthritis disease samples: citrulline-dependent or not?

Background: Antibodies directed against citrullinated proteins (eg anti-cyclic citrullinated peptide (CCP)) have excellent diagnostic and good prognostic potential for rheumatoid arthritis. Type 1 autoimmune hepatitis (AIH-1) is a chronic liver disease characterised by a variety of serum autoantibodies. Recently, in a large group of patients with AIH-1 without clear rheumatoid arthritis overlap, a relatively high percentage (9%) of anti-CCP2 positivity was scored. Objectives: To characterise the citrulline-dependence of the observed anti-CCP2 positivity in AIH-1 sera as well as in other groups of patients without rheumatoid arthritis (mainly rheumatic diseases). Methods: Serum samples of 57 patients with AIH-1 and 66 patients without rheumatoid arthritis, most of them reported as anti-CCP positive, were tested for citrulline-specific reactivity with a second generation anti-CCP kit, with the citrullinated and the corresponding non-citrullinated (arginine-containing) antigen. A subset of AIH-1 sera was also tested with a CCP1 ELISA (and arginine control). Results: The anti-CCP2 reactivity of most non-rheumatoid arthritis rheumatic diseases samples (87–93%) was citrulline-specific, whereas a relatively high percentage of AIH-1 samples (42–50%) turned out to be reactive in a citrulline-independent manner. The use of citrullinated and non-citrullinated CCP1 peptides confirmed a high occurrence of citrulline-independent reactivity in AIH-1 samples. Conclusions: In rheumatoid arthritis and most non-rheumatoid arthritis rheumatologic disease sera, anti-CCP positivity is citrulline-dependent. However in some patients, particularly patients with AIH-1, citrulline-independent reactivity in the anti-CCP2 test can occur. A positive CCP test in a non-rheumatic disease (eg liver disease) should therefore be interpreted with care, and preferably followed by a control ELISA with a non-citrullinated antigen.

ANTI-NUCLEAR ANTIBODIES OF PRIMARY BILIARY CIRRHOSIS RECOGNIZE 78-92-KD AND 96-100-KD PROTEINS OF NUCLEAR BODIES

The specificities of anti‐nuclear antibodies (ANA) reacting with multiple nuclear dots (MND‐ANA) present in about 15% primary biliary cirrhosis sera were studied by Western blot analysis with nuclear fractions from a human cell line. Reactivity with two broad bands of 78–92 kD and 96–00 kD of the insoluble fraction was present exclusively in MND‐ANA‐positive sera. Antibodies eluted from these proteins specifically retained the immunofluorescence reactivity of MND‐ANA. Immunomorphological analysis by a pre‐embedding technique revealed that the antibody specifically binds to nuclear regions resembling in size and number nuclear bodies. Since these structures are absent in immature rate endometrial cell and can be induced by diethylstilbestrol. we tested MND‐ANA by immunofluorescence on cryostat sections of uteri from hormone‐treated and untreated immature rats. A strong reaction of nuclear dots was observed predominantly in endometrial cells of hormone treated rats. We thus conclude that MND‐ANA present in primary biliary cirrhosis sera are directed against 78–92‐kD and 96–100‐kD nuclear proteins located in nuclear bodies.

Anti-CagA Reactivity in Helicobacter pylori-Negative Subjects (A Comparison of Three Different Methods)

Emerging evidence suggests that infection byCagA-positive Helicobacter pylori strains is related tothe development of more serious gastroduodenal diseases,thus conferring to the determination of anti-CagA antibodies a relevant clinical significance inserological screenings. The detection of anti-CagApositivity in sera negative for anti-H. pyloriantibodies raises the question of whether thisapparently nonsense result is merely due to a falsepositive reaction. To address this issue, we comparedthree different methods for the detection of anti-CagAantibodies. In all, 272 selected sera from patients with precisely defined H. pylori status(positive or negative concordance of five tests, ie,histology by Giemsa in both antrum and corpus, rapidurease test, culture, [13C]urea breath test,IgG ELISA) were tested for anti-CagA reactivity by threedifferent techniques (western immunoblotting, ELISA, andrecombinant immunoblotting assay). In order to assessthe sensibility and specificity of each tests, we considered as “true” anti-CagApositive sera those with two out of three positiveresults. Sera from 70% of H. pylori-positive patientsand 10% from H. pylori-negative patients turned out to be “true” positives foranti-CagA antibodies. The three methods showed similarexcellent results, in terms of both sensitivity andspecificity, always over 93%. It is confirmed that aproportion of patients with a negative conventionalserology against H. pylori possess anti-CagA antibodiesin their sera. In this paper we demonstrate that it canhappen even in patients without any biological signs of actual H. pylori infection. The possibilitythat this can be due to a false positive laboratoryresult is very likely ruled out by the accuracy of thethree methods used. The clinical management of these patients needs further study on largerseries.

Heterogeneity of antimitochondrial antibodies with the M2-M4 pattern by immunofluorescence as assessed by Western immunoblotting and enzyme linked immunosorbent assay.

Seventy seven sera with antimitochondrial antibody exhibiting the M2-M4 pattern in immunofluorescence (56 from primary biliary cirrhosis (PBC), 21 from non-primary biliary cirrhosis patients) were studied by the combined use of Western immunoblotting with beef heart mitochondria and an enzyme linked immunosorbent assay (ELISA) with beef heart submitochondrial particles. Forty seven sera (10 without autoantibodies and 37 with different auto-antibodies) were included as controls. By immunoblotting, seven mitochondrial peptides reacting with antimitochondrial antibody positive sera were detected. These were of molecular weight 74 kD, 58 kD, 55 kD, 52 kD, 51 kD, 46 kD, and 43 kD. All primary biliary cirrhosis sera and 71% of antimitochondrial antibody-positive non-primary biliary cirrhosis sera reacted with one or more of these peptides, while none of the 47 antimitochondrial antibody negative sera reacted in immunoblotting. The 74 kD band was the most frequently detected (84% of primary biliary cirrhosis and 57% of non-primary biliary cirrhosis cases). All the primary biliary cirrhosis sera which failed to react with this peptide, showed a positive reaction with that of molecular weight 52 kD. 67/77 (87%) immunofluorescence antimitochondrial antibody positive sera reacted in the ELISA test (93% of primary biliary cirrhosis and 71% of non-primary biliary cirrhosis cases). All the 47 immunofluorescence antimitochondrial antibody negative sera were confirmed negative by ELISA. The ELISA values correlated with the immunofluorescence titres (p less than 0.05). By comparison of the results obtained by these two techniques, it emerged that the ELISA test (using our preparation of submitochondrial particles) was not able to detect the antibody directed against the mitochondrial peptide of 52 kD, which thus seems to be different from the other specificities.

Validity of internal expression of the major histocompatibility complex class I in the diagnosis of inflammatory myopathies

Objective The inflammatory myopathies (IMs) are a group of disorders characterised by weakness and inflammation of the skeletal muscles. Muscle biopsy is the most crucial test to confirm the clinical diagnosis, but also the most common cause of misdiagnosis. There are currently no markers specific or sensitive enough to distinguish IMs from other diseases with similar clinical and morphological features, and an international multidisciplinary effort is under way to develop new classification criteria for IMs. Methods Standards for Reporting of Diagnostic Accuracy recommendations to validate a diagnostic test based on the quantification of internal major histocompatibility complex class I (MHC-I) positive fibres were adopted. MHC-I immunostained specimens from 64 patients were scored by two independent blinded investigators, and the percentage of positive fibres was determined. Agreement between investigators was evaluated with the k-weighted statistic. The receiver operating characteristic curve, area under the curve, sensitivity, specificity, and positive and negative predictive values of each percentage range of positive fibres versus the diagnosis of IM were calculated. Results The main difference between IM and non-inflammatory samples was the number of internal MHC-I positive fibres. The k-weighted value was 0.89 for a percentage of MHC-I positive fibres above 50%; the positive predictive value was 100%, and the negative predictive value was 94%. Conclusions This is the first study on the validity of a quantitative analysis of internal MHC-I positive fibres for an IM diagnosis performed according to Standards for Reporting of Diagnostic Accuracy recommendations. The interobserver agreement was almost perfect, thus making the method reproducible. Applying an MHC-I cut-off above 50% is an optimal marker for polymyositis (PM) and dermatomyositis (DM) diagnosis.

Bile duct cell apoptosis is a rare event in primary biliary cirrhosis

BACKGROUND The frequency of apoptosis in bile duct cells of primary biliary cirrhosis is still unclear spanning from rare to 50% in the various reports. AIM To study bile duct cell apoptosis in stage I primary biliary cirrhosis lesions. PATIENTS Nine stage I-II biopsies with a total number of 26 bile ducts of different sizes, selected from a larger series on the basis of the expression on serial frozen sections of HLA-DR and Fas antigens. METHODS Apoptosis was evaluated by a DNA fragmentation assay on frozen sections, according to the manufacturers protocol and by expression of apoptosis related cytokeratin neoepitopes. Bile duct cell proliferation was assessed by MIB1 (Ki-67) expression. RESULTS Apoptosis was frequently found in inflammatory cells of portal tracts and sinusoids. Apoptosis of hepatocytes was also systematically observed. Only 4 positive bile duct cells were found in 3 bile ducts from 3 biopsies. Quantitative evaluation was not attempted. Cholangiocyte proliferation was observed in the same ducts and occasionally in other biopsies. CONCLUSIONS These data suggest that cholangiocyte death by apoptosis at the level of typical primary biliary cirrhosis lesions is a rare event, at least in early stages of the disease. The observed rate of proliferation was consistent with the rate of apoptosis.

Etanercept and infliximab induce the same serological autoimmune modifications in patients with rheumatoid arthritis

Etanercept and infliximab treatments are often associated with autoantibodies induction. Their reported prevalences vary among different studies and the conclusions are somehow conflicting, mainly regarding whether the two drugs induce the same modifications. In this small prospective study, specifically designed to identify transient phenomena, we assess the prevalence of different relevant rheumatologic autoantibodies during anti-TNF-α courses in patients with rheumatoid arthritis. We report that both etanercept and infliximab transiently induce anti-DNA antibodies in 50–78% of patients, respectively, and these antibodies seem to be different from the typical lupus associated ones. Antinuclear antibodies (ANA) increased their titres and were newly produced up to 100% of patients. No other relevant antibodies are affected. Finally, as also confirmed for the first time by the patients switched from one drug to the other, the two TNF-α blockers behave similarly.

Demonstration of peptide-specific and cross-reactive epitopes in proteins reacting with antimitochondrial antibodies of primary biliary cirrhosis

Recently the main targets of antimitochondrial antibodies (AMA) of primary biliary cirrhosis have been identified as parts of three related mitochondrial multienzyme complexes, namely pyruvate dehydrogenase (PDH), branched chain alpha-ketoacid dehydrogenase (BKDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH). Usually AMA-positive PBC serum samples show reactivity to more than one of these, raising the question whether they are exclusively different antibodies or are, at least in part, the result of cross-reactive specificities. With Western immunoblotting, four antigens with molecular masses of 74, 52, 51 and 43 kDa, are recognized by PBC sera. In this study, using affinity purified antibodies from mitochondrial proteins immobilized on nitrocellulose blots, we demonstrate the presence of peptide-specific and cross-reactive epitopes in some targets. In particular, at least three different epitopes present in the 74-kDa protein (presumed to by PDH-E2) are also present in the 51-kDa protein (probably PDH-X), and two in the 52-kDa peptide (possibly BCKDH-E2). Moreover, the 43-kDa mitochondrial protein (the identity of which is more problematic) has three epitopes. One of these is also present in the 74-, 52- and 51-kDa proteins, a second in the 74- and 51-kDa, and a third seems to be peptide-specific. These results show that different sera with the same immunoblotting pattern of reactivity can have antibodies with different antigenic specificities and, conversely, that the same specificity can be responsible for more than one band.