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Serum IgG-antilipoprotein-autoantibody activity (at 4 degrees C) of a plane xanthoma patient was shown by double-immunodiffusion method. Cryoglobulins in the serum were dissociated to polyclonal IgG and alpha- and beta-lipoproteins by acidification and were reconstructed by neutralization. IgG fraction of the cryoglobulins precipitated with lipoproteins. The cryoglobulins were thus demonstrated to be immune complexes of polyclonal IgG-antilipoprotein-autoantibody and both alpha- and beta-lipoproteins. A part of the lipoprotein-autoantibody immune complexes was not cryoprecipitable. Antigenic determinants for the autoantibody existed in the lipid moieties of lipoproteins, in contrast to the apoproteins which determined the specificity to heteroimmune antilipoprotein antibody. The presence of more than nine different antigenic determinants against the autoantibody indicated that lipoproteins were immunologically heterogeneous depending upon the lipid moieties. Lipoproteins reactive with the autoantibody varied quantitatively in normal individuals and were not detected in a primary hyper-beta-lipoproteinaemia patient and in a primary biliary liver cirrhosis patient with much lipoprotein-X. The absence of antigenicity in the two patients' sera is most likely caused by abnormal lipid moieties of lipoproteins.
Anti-liver microsomes (anti-LM) autoantibodies in patients with dihydralazine-induced hepatitis were found to react specifically with cytochrome P4501A2 (P4501A2) but not with P4501A1 expressed in yeast and bacteria. These results were confirmed by immunoinhibition of methoxyresorufin-O-demethylase activity (supported by the P4501A subfamily); anti-LM antibodies more strongly inhibited this activity in yeast expressing P4501A2 than in yeast expressing P4501A1. Anti-LM were shown to be specific to the disease; in three cases, these autoantibodies were present at high titers during disease, whereas the titers decreased upon recovery and became undetectable a few months after recovery. Thus, there exists a time-dependent relationship between the disease and the autoantibodies, which does not prove that the autoantibodies are causative of the hepatitis; they might only be a marker. The inductive capacity of dihydralazine toward P450 was also studied. In rats treated in vivo and in human hepatocytes treated in vitro with dihydralazine, a 2-fold increase in P4501A2- and P4501A-supported monooxygenase activities was found. The levels of the other P450 isoforms tested were unchanged during treatment, both in vivo in rats and in vitro in cultures of human hepatocytes. In human hepatocytes, dihydralazine produced a dose-dependent increase in the level of P4501A up to 0.1 mM; induction of P4501A was less strong at 0.2 mM and disappeared at 0.5 mM. The same treatment did not change the level of P4503A4, taken as control. The strong heterogeneity in the expression of P4501A enzymes in human liver and the capacity of these enzymes for induction by dihydralazine and by other compounds might be predisposing factors in this autoimmune disease.
Assays were developed to detect and measure autoantibodies (AA) to thyroglobulin (Tg) and to the thyroid hormones, thyroxine (T4) and triiodothyronine (T3). An ELISA to detect AA to Tg was developed, using purified canine Tg as the antigen and goat anti-canine IgG conjugated with alkaline phosphatase as the second antibody. A highly charged agarose electrophoresis assay was used for determination of AA to T4 and T3. Sera from dogs (n = 119) with clinical signs consistent with hypothyroidism were tested for AA to Tg, T4, and T3. Autoantibodies to at least 1 of the 3 thyroid antigens were detected in 58 of the 119 (48.7%) sera tested. Autoantibodies to Tg were detected more frequently in samples with low serum concentrations of thyroid hormones than in samples with normal concentrations. The presence of AA to T4, T3, or both was not significantly associated with low thyroid hormone concentrations, but this lack of association may have been attributable to binding of AA in the measurement of thyroid hormones by radioimmunoassay.
Clustering of ANA and SMA was evaluated in patients with various internal diseases as a pattern of autoantibody formation. SMA was found in ANA positive patients with chronic hepatitis, undefined collagenoses and autoallergic thyroid diseases significantly more frequently, than in patients without any autoallergic disorders. The incidence of SMA in ANA positive cases with SLE and RA did not exceed their predictable by chance incidence. It was not found that clustering of autoantibodies is correlated with the E and C lymphocyte receptor abnormality as compared to the control group. The lowest count of E-RFC was found in SLE cases which differed significantly in this respect from the control group and also from chronic vasculitis cases. The clustering of autoantibodies is not correlated with hyperimmunoglobulinemia.
The binding affinity of cryoglobulin IgM rheumatoid factors (RF) for human IgG and of human IgG anti-albumin autoantibodies for HSA was measured by the molecular sieving technique. The binding affinities of the two autoantibodies were consistently low (10(4)-10(5) 1/M) as compared to the affinities of corresponding hyperimmune animal antibodies (10(6)-10(8) 1/M). The findings were discussed in relation to theories on human autoimmunity. The existence of strict autotolerance at the T cell level and of autoreactivity at the low affinity B cell level was considered to be best compatible with the findings of this study and with the major known facts of autoimmunity.
Thirteen smooth muscle antibody (SMA) sera obtained from patients with active chronic hepatitis were examined for immunofluorescence reactivity with gastric mucosal cells. Eight out of 13 sera stained the cytoplasm of gastric parietal cells in a pattern indistinguishable from that obtained with parietal cell autoantibody (PCA). The staining reaction was localised to parietal cells by the demonstration that the same cells stained with both SMA and PCA in double immunofluorescent tests. The SMA staining intensity for parietal cells was weaker than that for smooth muscle. Specificity of the staining reaction for actin was established by the observation that parietal cell staining by SMA was inhibition by serum absorption with skeletal muscle F-actin but not by a microsomal fraction derived from gastric mucosa.
Insulin autoantibodies (IAA) have been identified in newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients and in individuals at high risk of developing the disease. However, the literature is not in agreement regarding the prevalence, significance, and predictive value of IAA. Previous workshops have shown that certain sera give markedly different results depending upon assay methodology and, therefore, have suggested that these discrepancies may be due to variations in the assay methodologies used: either the fluid phase RIA or the solid phase enzyme-linked immunosorbent assay (ELISA). Sera from controls (n = 61), newly diagnosed IDDM patients (n = 30), healthy subjects who later became diabetic (n = 8), and first degree relatives of diabetic probands (n = 22) were randomly numbered and sent without category identification to 19 RIA and 10 ELISA laboratories. Each laboratory's raw data from the control sera were used to determine the cut-off point for positive (3 SD above the mean of control sera) in the disease relevant categories. RIA and ELISA methods were comparable in obtaining a low frequency of IAA in control sera. However, the laboratories using RIA methods found a much higher percentage of sera to be IAA positive among both newly diagnosed patients and healthy individuals who later developed diabetes than laboratories using ELISA methods (P less than 0.005). In contrast, there was considerable overlap in the percentage of sera from first degree relatives found positive by both assays. These data strongly suggest that IAA measured by RIA methodology are more disease related than those measured by ELISA methods. Consequently, RIA assays or assays proven to perform as well should be used to measure IAA associated with IDDM.
Comparisons made between old and young people depicted the increase with aging in ANA and thyroid antibody incidence. In contrast to these antibodies SMA were frequently seen in young women. Also in women serum IgM levels were higher as compared to men. Conversely serum IgD levels were higher in men; both these differences were seen in the young group only. Proportions and numbers of E-RFC were significantly reduced in the older group of people as compared to the values of the younger group. Interestingly, the autoantibodies appearing were correlated in women with a lower percentage of E-RFC. Also in women low IgD levels were associated significantly with higher percentage of E-RFC.
BACKGROUND AND OBJECTIVES: Autoantibodies targeting the acetylcholine receptor (AChR), found in patients with myasthenia gravis (MG), mediate pathology through 3 mechanisms: complement-directed tissue damage, blocking of the acetylcholine binding site, and internalization of the AChR. Clinical assays, used to diagnose and monitor patients, measure only autoantibody binding. Consequently, they are limited in providing association with disease burden, understanding of mechanistic heterogeneity, and monitoring therapeutic response. The objective of this study was to develop a cell-based assay that measures AChR autoantibody-mediated complement membrane attack complex (MAC) formation. METHODS: An HEK293T cell line-modified using CRISPR/Cas9 genome editing to disrupt expression of the complement regulator genes (CD46, CD55, and CD59)-was used to measure AChR autoantibody-mediated MAC formation through flow cytometry. RESULTS: Serum samples (n = 155) from 96 clinically confirmed AChR MG patients, representing a wide range of disease burden and autoantibody titer, were tested along with 32 healthy donor (HD) samples. AChR autoantibodies were detected in 139 of the 155 (89.7%) MG samples through a cell-based assay. Of the 139 AChR-positive samples, autoantibody-mediated MAC formation was detected in 83 (59.7%), whereas MAC formation was undetectable in the HD group or AChR-positive samples with low autoantibody levels. MAC formation was positively associated with autoantibody binding in most patient samples; ratios (mean fluorescence intensity) of MAC formation to AChR autoantibody binding ranged between 0.27 and 48, with a median of 0.79 and an interquartile range of 0.43 (0.58-1.1). However, the distribution of ratios was asymmetric and included extreme values; 16 samples were beyond the 10-90 percentile, with high MAC to low AChR autoantibody binding ratio or the reverse. Correlation between MAC formation and clinical disease scores suggested a modest positive association (rho = 0.34, p = 0.0023), which included a subset of outliers that did not follow this pattern. MAC formation did not associate with exposure to immunotherapy, thymectomy, or MG subtypes defined by age-of-onset. DISCUSSION: A novel assay for evaluating AChR autoantibody-mediated complement activity was developed. A subset of patients that lacks association between MAC formation and autoantibody binding or disease burden was identified. The assay may provide a better understanding of the heterogeneous autoantibody molecular pathology and identify patients expected to benefit from complement inhibitor therapy.
The prevalence of glutamic acid decarboxylase autoantibodies was determined with an immunotrapping enzyme activity assay in newly-diagnosed Type 1 (insulin-dependent) diabetic patients as well as in first-degree relatives using rat brain homogenate as a source of glutamate decarboxylase. Twenty-six out of 86 islet-cell cytoplasmic auto-antibody positive and one out of 24 islet cell autoantibody negative patients of recent onset, had autoantibodies to glutamate decarboxylase above the upper 99% confidence limit obtained from 89 control sera. Among 27 islet cell autoantibody positive relatives including 19 siblings and 8 parents, antibodies to glutamate decarboxylase were found in 8 of 9 (89%) relatives and 7 of 8 (87.5%) siblings with islet cell auto-antibody titres above 20 JDF units, in 1 of 19 (5.2%) relatives with islet cell autoantibody titres between 2 and 5 JDF units, in 2 of 263 (0.7%) siblings and 1 of 139 parents without islet cell autoantibodies. In first-degree relatives, high titre islet cell autoantibodies and autoantibodies to glutamate decarboxylase were tightly associated (X2 = 182, p = 0.0001). None of the relatives with low genetic risk (n = 64), i.e. HLA-different to the diabetic proband, was found to be antibody positive. Antibodies to glutamate decarboxylase were present only in those relatives sharing at least one haplotype with the diabetic proband, including two islet cell autoantibody negative but HLA-identical siblings. Autoantibodies to glutamate decarboxylase were present in 7 of 9 (77%) relatives who developed the disease, including one islet cell autoantibody negative sibling.(ABSTRACT TRUNCATED AT 250 WORDS)
Hypocomplementemic urticarial vasculitis syndrome (HUVS) is an apparent autoimmune disorder that resembles SLE. We previously showed that C1q precipitins in HUVS sera are IgG autoantibody to human C1q. We have compared HUVS anti-C1q autoantibody to a similar autoantibody in the serum of some patients with SLE. As with anti-C1q autoantibody in SLE sera, the HUVS autoantibody binds only to the collagen-like region (CLR) of C1q. In both HUVS and SLE, IgG2 is the predominant subclass of IgG autoantibody and IgM autoantibody to C1q is uncommon. In both diseases, anti-C1q autoantibodies bind preferentially to surface-adsorbed C1q or CLR fragments compared to these antigens in solution. Finally, when HUVS or SLE autoantibodies were added to CLR-coated wells already bound, respectively, by SLE or HUVS autoantibodies, no increases in CLR binding were observed, suggesting that HUVS and SLE autoantibodies to C1q bind to the same CLR epitope(s).
Mice immunized with cross-reacting rat erythrocytes develop autoimmune haemolytic anaemia as indicated by haematological data and erythrocyte autoantibodies. With continued immunization the mice recover haematologically but remain Coombs' positive. Coombs' tests were performed using monospecific antisera to determine whether the recovery from anaemia was associated with a change in the class or subclass of the autoantibodies produced. In both splenectomized mice and in unsplenectomized mice the following subclass of erythrocyte autoantibodies were present: IgG1, IgG2A, IgG2B. IgA autoantibodies were not detected and IgM autoantibodies were only detected in splenectomized mice 1-3 weeks after the IgG autoantibodies had appeared. After six immunizations the frequency of IgG2B autoantibodies decreased and by the tenth immunization and thereon, IgG2B autoantibodies were not detected. It is proposed from these results that the anaemia is caused by IgG2B autoantibodies and the the sudden exacerbation in the anaemia that occurs in splenectomized mice is due to the production of IgM autoantibodies.
BACKGROUND: The determinants of the species barrier preventing human infections with avian influenza A viruses (IAV) are incompletely understood. We previously identified loss-of-function variants of the interferon-regulated antiviral factor MxA as a genetic factor for increased susceptibility to infections with the H7N9 subtype. Given the central role of type I IFNs (IFN-I) in antiviral defence, we hypothesised that IFN-I-neutralising autoantibodies may similarly predispose to zoonotic H7N9 infection. METHODS: In this observational case-control study, serum samples collected between 2013 and 2017 from 199 Chinese patients with laboratory-confirmed H7N9 infection and 531 healthy, uninfected controls (269 poultry workers, 262 close contacts) were screened for IgG autoantibodies binding IFNα2, IFNβ1b, or IFNω using a multiplex bead-based assay. Positive samples were tested for IFN-neutralising activity in a luciferase-based reporter assay. To confirm their ability to block IFNα2-mediated antiviral activity, selected samples (n = 19) were analysed in IAV infection experiments. Associations between age, sex, H7N9 case status, case fatality, and the presence of neutralising autoantibodies were evaluated by logistic regression. Available whole-genome sequencing data from 26 individuals with neutralising autoantibodies were screened for variants in genes linked to IFN-I autoimmunity. FINDINGS: Neutralising autoantibodies against at least one IFN-I were detected in 19.1% (38/199) of patients but in only 1.1% (6/531) of controls, consistent with published general population data. Most patient sera targeted IFNα2 and/or IFNω (35/199), and 18.1% (36/199) neutralised even high IFN-I concentrations of 1-10 ng/ml. The presence of neutralising autoantibodies was associated with 8.2- to 25.3-fold higher odds of H7N9 infection (p < 0.0001), depending on antibody specificity and reference group. Autoantibody prevalence increased significantly with age in patients (44.8% ≥70 years; OR = 1.05; 95% CI 1.02-1.07; p = 0.0001), but was not associated with sex (OR for males vs. females = 0.52; 95% CI 0.23-1.14; p = 0.106). All selected sera containing neutralising autoantibodies blocked IFNα2-induced antiviral activity in cell culture. No known genetic predisposition for IFN-I autoimmunity was identified. INTERPRETATION: Our findings suggest that IFN-I-targeting autoimmunity is associated with susceptibility to zoonotic IAV infection with the H7N9 subtype, and possibly also other subtypes, including panzootic H5N1. Given the ease of implementation, screening for anti-IFN-I autoantibodies could be readily integrated into surveillance or targeted testing. This could be relevant in environments with increased exposure to zoonotic IAVs. FUNDING: Shenzhen Medical Research Fund, National Natural Science Foundation of China, Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences, Guangdong Provincial Science and Technology Program, Program for Youzuzhikeyan of Shenzhen University, German Research Foundation, Swiss National Science Foundation.
Natural autoantibodies are primarily immunoglobulin M (IgM) antibodies that bind to a variety of self-antigens, including self-IgG. Accounting for a large proportion of the early B cell repertoire, such polyspecific autoantibodies are speculated to contribute to the homeostasis and/or competence of the primary humoral immune system. Recent studies indicate that the leukemia cells from most patients with chronic lymphocytic leukemia (CLL) also express such IgM autoantibodies. Similarly, the leukemia cells from many CLL patients react with murine monoclonal antibodies (mAbs) specific for crossreactive idiotypes (CRIs) associated with human IgM autoantibodies. In particular, leukemic cells frequently react with G6, a mAb specific for an Ig heavy chain (H chain)-associated CRI, and/or with 17.109, a mAb that defines a kappa light chain (L chain)-associated CRI. Generated against IgM rheumatoid factor (RF) paraproteins, G6 and 17.109 each recognize a major CRI that is present in many IgM RF paraproteins. Furthermore, over 90% of the IgM paraproteins found to bear both H and L chain-associated CRIs also are found to have RF activity. Molecular characterization of these CRIs demonstrates that each is a serologic marker for expression of a highly conserved Ig V gene. As such, the frequent production of IgM polyspecific autoantibodies in CLL simply may reflect the frequent use of such highly conserved autoantibody-encoding Ig V genes with little or no somatic mutation. To test this hypothesis, we generated murine transfectomas to pair the 17.109-reactive kappa L chain of SMI, a 17.109/G6-reactive CLL population, with the Ig H chain of SMI or other G6-reactive leukemia cells or tonsillar lymphocytes. Cotransfection of vectors encoding the Ig H and L chains of SMI generated transfectomas that produce IgM kappa RF autoantibodies reactive with human IgG1 and IgG4. In contrast to G6/17.109-reactive IgM kappa RF Waldenstrom's paraproteins, the SMI IgM kappa also reacts with several other self-antigens, including myoglobin, actin, and ssDNA. However, cotransfection of the SMI L chain with a vector encoding any one of 10 different G6-reactive Ig H chains generated transfectomas that produce IgM kappa antibodies without detectable polyspecific autoantibody activity. These results indicate that polyspecific antiself-reactivity of G6/17.019-reactive Ig is dependent on the somatically generated Ig third complementarity determining region. Collectively, these studies imply that selection may be responsible for the frequent expression of polyspecific autoantibodies in CLL and early B cell ontogeny.
An autoantibody to liver cytosol was previously described in childhood autoimmune chronic active hepatitis type 2. The antigen, liver cytosol antigen type 1, was for the first time partially purified using gel filtration and ion exchange chromatography, and it was characterized using immunodiffusion, immunoblot and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunoprecipitate. Immunoblot detected a unique antigenic peptide at 62 kD from human cytosol and at 58 kD from rat cytosol. The same peptides were also detected when immunoprecipitates of liver cytosol antigen type 1 and autoantibodies to liver cytosol antigen were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polymeric structure, probably a tetramer, is suggested for native liver cytosol antigen type 1 because in gel filtration chromatography liver cytosol antigen type 1 was eluted as a protein of a molecular weight between 240 and 290 kD when human liver cytosol was fractionated and between 220 and 270 kD from rat liver cytosol. Liver cytosol antigen type 1 is probably poor in carbohydrates because it was not stained by periodic acid-Schiff stain. The autoantibodies to liver cytosol were frequently found in association with antiliver kidney microsomal autoantibodies type 1, which are directed against the cytochrome P-450 of the IID6 subfamily. Antiliver kidney microsomal autoantibodies type 1 but not antiliver cytosol autoantibodies were found in association with antibodies to hepatitis C virus. Autoantibodies to liver cytosol antigen type 1 seem to be a more specific marker for autoimmune hepatitis type 2 than antiliver kidney microsomal antibodies type 1 autoantibodies.
A radiobinding assay for the detection of autoantibodies against islet amyloid polypeptide was developed, analytically validated, and--in parallel with a similar assay for the detection of autoantibodies against insulin--applied to sera from recent-onset Type 1 (insulin-dependent) diabetic patients and from age- and sex-matched control subjects. There was no difference in islet amyloid polypeptide autoantibody titres between patient groups and matched control subjects, nor within subject groups according to age. At onset of Type 1 diabetes, elevated islet amyloid polypeptide-autoantibody levels (> 97th percentile of control subjects) were only detected in 1 of 30 patients aged 0-19 years and in 2 of 35 patients aged 20-39 years. By contrast, insulin autoantibodies were frequently demonstrated, in particular at onset of diabetes under age 20 (0-19 years: 18 of 30 patients; 20-39 years: 10 of 35 patients; p < 0.01 vs matched control subjects). Islet amyloid polypeptide autoantibodies were not detectable in 3 insulinoma patients nor in 37 patients (aged 33-70 years) with Type 2 diabetes (vs 1 of 40 in matched control subjects). In positive serum, adsorption onto protein A-Sepharose removed islet amyloid polypeptide binding activity, hereby confirming its antibody nature. In conclusion, Type 1 diabetes is associated with an age-dependent autoantibody reaction against insulin but not against islet amyloid polypeptide. Conditions associated with amyloid deposition in islets (Type 2 diabetes, insulinoma and ageing) do not favour the formation of autoantibodies against islet amyloid polypeptide.
Mice with SCID disease have previously been successfully engrafted with human peripheral blood mononuclear cells (PBMC) obtained from normal individuals and from patients with various diseases. To determine whether SCID mice engrafted with SLE PBMC produced autoantibodies with specificities similar to those in the SLE donor, and to investigate which variables influence autoantibody production in the SCID recipients, we injected PBMC from 16 SLE patients into SCID mice and tested the recipients for autoantibodies to DNA and to five recombinant autoantigens. Ten out of 16 (68%) lupus and six out of nine (67%) normal grafts were successful as determined by the presence of human IgG greater than or equal to 5 micrograms/ml of SCID serum post-transfer. Autoantibodies to La/SSB, Ro/SSA, and RNP were detected in five out of 10 SCID-SLE recipients by ELISA and immunoblotting up to 22 weeks post-engraftment. The detection of autoantibodies in SCID-SLE mice was more closely related to autoantibody levels in donor sera than to total IgG concentrations in the SCID recipients. Autoantibody activity/mg IgG was similar in the donor and recipient sera. Histological evaluation of eight SCID-SLE mice killed 4-22 weeks post-transfer revealed population of the SCID thymus and spleen with mononuclear cells, but no evidence of lupus nephritis or dermatitis. These findings indicate that SCID mice can be engrafted with PBMC from patients with lupus and that specific autoantibodies are produced up to 5 months post-transfer. Failure to develop glomerulonephritis may be explained by low or absent anti-DNA antibodies or by changes in the cellular composition of the PBMC grafts.