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In previous studies we have demonstrated high levels of rheumatoid factor (RF) and large-size (greater than 22S) circulating immune complexes (CIC) in the serum of rheumatoid arthritis (RA) patients with extra-articular disease. These findings were paralleled by a concurrent increase in the level of RF-associated cross-reactive idiotypes (CRI) and an apparent diversification of the RF repertoire detected in the serum of the same patients. In the present study we examine the ability of CICs to activate the complement system in vivo, and its possible influence on expanding the RF repertoire in RA patients with extra-articular disease. Activation of complement by CICs is the key for germinal centre localization and long-term retention of such complexes on the surface of follicular dendritic cells (FDC), and so provides a source for the selection of cells with high affinity receptors for IgG and leads to the establishment of immunological memory. CICs containing different immunoglobulin isotypes and from different patients localized in mouse spleen germinal centres. However, intense localization was mainly seen for IgG-containing complexes from the serum of patients with large-size (greater than 22S) IgG-IgM RF complexes. The ability of these complexes to localize in mouse spleen germinal centres was related to activation of the complement system via the classical pathway in the patients' sera. Localization of IgG complexes was significantly (P less than 0.05) higher in sera from RA patients with extra-articular disease than those with articular disease alone. This study demonstrates the ability of large-size (greater than 22S) IgG-IgM RF complexes to activate complement, and suggests a possible role for such complexes in modulating the immune response to IgG in RA patients with extra-articular disease.

The size and composition of circulating immune complexes in the sera of patients with rheumatoid arthritis (RA) were studied in relation to different manifestations of the disease. Circulating immune complexes from the sera of 94 patients (50 with extra-articular disease) and 10 matched controls were fractionated by sucrose density gradient ultracentrifugation. The composition, immunoglobulin and rheumatoid factor (RF) concentrations within each of the fractions were determined by a sensitive enzyme linked immunosorbent assay (ELISA). Intermediate size (14S-21S) IgG complexes containing RF activity and 22S IgG-IgM RF complexes were found in the sera of 40 patients with RA, while intermediate size complexes of self associated IgG RF and larger size complexes (greater than 22S) of IgG RF and IgM RF were associated with extra-articular features of RA (50% of extra-articular disease). Complexes containing IgA were found in the sera of many patients with RA, and dimeric IgA RF mainly in patients with extra-articular disease. These results support the view that whereas small size circulating immune complexes are of no primary pathogenic importance in synovitis, large size (greater than 22S) circulating immune complexes may play a role in extra-articular disease in RA. Current understanding of the formation of large complexes provides a biological explanation for their occurrence and effects.

Sera from patients with rheumatoid arthritis (RA), patients with infectious mononucleosis (IM), and blood donors were tested by indirect immunofluorescence for the presence of antikeratin antibody (AKA), antibody to cytoskeletal intermediate filaments of prekeratin or vimentin type (AIFA) and antiperinuclear factor (APF). In 81.9% of the RA sera and 92.5% of the IM sera AIFA of IgM class was found at titres up to and in some cases exceeding 1/160. In blood donors the incidence of AIFA was 26%, at titres not exceeding 1/20. AKA and APF, always of IgG class, were found in 54.2% and 73.6% of rheumatoid sera. A weak correlation was found in RA between the incidence of AIFA and APF. AKA was not present in either IM or blood donor sera, and APF was found in only 2.5% and 3.2% of IM or blood donors respectively.

SUMMARY A previous study using immunofluorescent techniques showed J-chain to be present in the uninvolved skin of patients with papillary IgA dermatitis herpetiformis (DH) in a distribution that was coextensive with the IgA. This implied that the IgA was dimeric and of mucosal origin. In this study, fifteen patients with papillary IgA deposits, fifteen with homogeneous-linear (HL) IgA deposits and four patients with granular-linear (GL) IgA deposits were tested for the presence of in vivo bound J-chain. All fifteen patients with papillary IgA deposits and all four with GL IgA deposits had J-chain staining coextensive with the IgA. However, only one of fifteen patients with HL IgA deposits demonstrated in vivo bound J-chain that could not be accounted for by coexisting IgM deposits. These findings indicate that the IgA in patients with HL deposits is qualitatively different from that in patients with papillary and GL IgA deposits and makes the distinction between the two types of linear fluorescence particularly important.

A simple procedure is described for distinguishing between IgG preparations which on heating form aggregates that are subsequently either stable or unstable in the absence of additional protein. Of 4 commercially available IgG preparations tested in this manner, only one gave IgG aggregates that were unstable.

IgG aggregates produced by heating gamma globulin solutions were freeze-dried, kept at 4 degrees C and reconstituted up to 4 months later. By comparison with frozen (-20 degrees C) preparations, only minimal changes in biological reactivity and in physical integrity occurred during this period. These results demonstrate that freeze-dried preparations of heat-aggregated IgG are potentially useful as a reference reagent for the comparative evaluation and standardisation of immune complex assays.

Leucocyte migration inhibition (LMI) by paired samples of autologous serum and synovial fluid IgG as well as homologous IgG and also heat-aggregated forms of all three IgGs was studied for patients with rheumatoid arthritis (RA) and compared with the response to these antigens by leucocytes from healthy individuals. Autologous and homologous serum IgGs did not markedly affect leucocyte migration, whereas synovial fluid IgGs caused occasional pronounced LMI with autologous (rheumatoid) leucocytes. Although aggregated IgG produced LMI in both RA patients and controls, the means of the migration indexes were significantly different between both groups. Peripheral blood mononuclear cells from RA patients and healthy controls were also cultured with both native rheumatoid and normal IgG; only supernatants from RA cells incubated with rheumatoid IgG showed LMI activity, in contrast to the other culture supernatants. These results indicate that LMI to native IgG in RA may represent a form of specific cell-mediated immunity, although LMI to aggregated IgG might be explained by non-specific factors.

Human smooth muscle autoantibody (SMA) of defined anti-actin specificity was tested by the indirect immunoperoxidase staining method on frozen sections of tissues and on cultured rat lung fibroblasts. The serum stained tissue sections and cultured fibroblasts in a pattern identical with that obtained by indirect immunofluorescence. Ultrastructural studies carried out on the immunoperoxidase stained cells showed that the long parallel filaments spanning the long axis of cultured fibroblasts seen by light microscopy correspond with the thick bundles of microfilaments.

The R1 type anti-reticulin antibody (ARA) is closely associated with gluten-sensitive enteropathy. It disappears from the circulation within a few weeks of starting on a gluten-free diet and often reappears following gluten challenge. It is not clear how gluten ingestion leads to the production of the ARA. We have investigated four possibilities. (1) The ARA is simply a food antibody generated against meats in the diet. (2) The ARA is an anti-gluten antibody which cross-reacts with reticulin. (3) Gluten binds to gut reticulin in vivo rendering reticulin autoimmunogenic. (4) Immune complexes of gluten and anti-gluten antibody bind to reticulin (by virtue of the affinity that gluten has for reticulin) to give the appearance of an ARA in immunofluorescence tests. Our results do not support any of these possible explanations, and the significance of the ARA remains obscure.

A discriminating direct immunofluorescent test has been used to identify immunoglobulin inclusions in polymorphonuclear leucocytes (PMNs) isolated from the blood of patients with rheumatoid arthritis. These inclusions are thought to represent phagocytosed immune complexes, since normal PMNs incubated in RA sera known to contain raised levels of immune complexes developed similar immunoglobulin inclusions. Inclusions did not develop in normal PMNs incubated in normal serum. No correlation was found between the percentage of either RA blood PMNs with immunoglobulin inclusions or normal PMNs developing inclusions after incubation in RA sera, and levels of immune complexes in the corresponding sera. Using heat-aggregated IgG as a laboratory model of immune complexes, a simple relationship has been demonstrated between the uptake of IgG aggregates by normal PMNs and the concentrations of IgG aggregates in the test solutions over a concentration range of 12.5-200 micrograms . ml-1. These results indicate that the C1q- PEG test gives no measure of the actual amounts of immune complexes available in serum for phagocytosis.

A multi-centre study is described in which thirty-five adult patients with papillary IgA dermatitis herpetiformis (DH) were compared with forty-two patients with linear IgA deposits, of whom thirty-four had homogeneous-linear (HL) and eight had granular-linear (GL) IgA deposits. The three groups were similar with regard to age of onset, presence of circulating immune complexes and auto-antibodies, incidence of spontaneous remission, histology of lesional skin and response to dapsone. There was a female predominance in the HL group in contrast to the male predominance in the other two. It was not possible to diagnose the HL group clinically. Some patients had a rash typical of DH whilst others resembled pemphigoid. In the majority, however, no specific diagnosis could be made with confidence. The GL group clinically resembled the DH group. The incidence of positive potassium iodide patch tests was greater in the DH group than in the other two. An associated enteropathy was found in 24% of patients in the HL group, 30% of patients in the GL group and 85% of patients in the DH group. Fifty-six percent of HL patients had HLA-B8 compared with 50% in the GL group and 88% in the DH group. Patients with linear IgA deposits may not be a uniform group, but until they can be divided into specific subgroups (e.g. by ultrastructural localization of the deposit or by response to a gluten-free diet) we propose that the term adult linear IgA diseases should be used to distinguish these patients from those with papillary IgA deposits.

A new test for the detection of antibodies to gliadin in the sera of children who are gluten sensitive is described. This test is based on the observation that wheat protein binds selectively to connective tissue fibres in cryostat sections of mammalian tissues. Sera containing antibodies to gliadin give a reticulin pattern of staining on section pretreated with wheat gliadin if tested by indirect immunofluorescence. Antibodies to gliadin were detected in this manner in sera fom all of 32 children with coeliac disease, in 16 of whom a provisional diagnosis had been given and in 16 an established diagnosis confirmed by gluten challenge. The incidence of reticulin antibodies detected on untreated sections (in the absence of gliadin) was only 28%. Gliadin antibodies were also present in sera from 15% of 152 children with gastroenterological disorders other than coeliac disease, but most of these non-positive results for coeliac disease were in patients with transient gluten in tolerance, cows' milk-sensitive enteropathy, or Crohn's disease. This new test, although not specific for coeliac disease, promises to be of value as an indicator of gluten sensitivity, and when negative excludes a diagnosis of coeliac disease. It may also be useful in monitoring diet, and in determining when rebiopsy after gluten challenge in appropriate.

Blood lymphocytes from rheumatoid patients and normal subjects were examined for responsiveness in culture to Epstein-Barr virus (EBV) infection by outgrowth assay and 3HTdR uptake. With both unseparated and B-cell-enriched lymphocytes the frequency and rate of outgrowth to form permanent cell lines were significantly higher for rheumatoid than for normal cells. In B-enriched rheumatoid preparations the proportion of responsive cells was also greater, and DNA synthesis was induced by a lower infecting dose of EBV in rheumatoid than in normal cells. The percentage of autologous T cells needed to ensure regression of B-cell proliferation in EBV-infected cultures was considerably higher with rheumatoid than with normal cells. These findings suggest that in rheumatoid arthritis the abnormal lymphocyte responsiveness to EBV has two components, a T-cell immunoregulatory defect, and a separate increased responsiveness of B cells to EBV.

Rheumatoid peripheral blood mononuclear cells show an increased responsiveness to superinfection with Epstein-Barr virus (EBV). We have investigated the role of adherent cells in this hyperresponsiveness using two different methods of adherent cell depletion. Depletion of adherent cells from both rheumatoid and normal mononuclear cells, using either dextran bead columns or plastic petri dishes, produced inconsistent changes in the response of autologous non-adherent cells to EBV. The addition of supernatants of cultured rheumatoid adherent cells also produced an inconsistent change in response although normal adherent cell supernatants increased the responsiveness of autologous non-adherent cells to EBV. The inconsistencies observed are discussed with respect to adherent cell subpopulations present in rheumatoid and normal peripheral blood mononuclear cell preparations when using recognized methods of adherent cell depletion.

Attempts to use the rapid single-step Ficoll-Hypaque centrifugation procedure for the purification of mononuclear and polymorphonuclear leucocytes from the blood of normal individuals and rheumatoid arthritis patients have sometimes been unsuccessful, largely because the erythrocytes would not sediment through the centrifugation medium. Re-evaluation of the factors (e.g. Ficoll concentration, temperature, and ratio of the diatrizoate salts) which affect these separations showed that under our conditions it was advantageous to use a medium with a lower viscosity (Ficoll concentration) and/or a higher osmotic strength (increased sodium diatrizoate: meglumine diatrizoate) than had been recommended previously (Ferrante and Thong, 1978; 1980; Ferrante et al., 1982). Higher osmotic strength media must be used for separating the components of blood from rheumatoid arthritis patients than from normal individuals because rheumatoid arthritis erythrocytes have a lower buoyant density than normal erythrocytes.

Cell electrophoresis was performed on peripheral-blood lymphocytes obtained from 11 patients with rheumatoid arthritis (R.A.) and 16 normal controls. On the basis of cell frequency/mobility histograms each subject's lymphocyte electrophoretic profile was resolved into a bimodal distribution of fast and slow cells. The mean electrophoretic mobility (E.P.M.) of the fast-cell population was similar in controls and in R.A. patients but the mean E.P.M. of the slow population was significantly slower in R.A. than in the controls. In the R.A. patients the E.P.M. of the polymorphonuclear cell population was significantly slower than that of P.M.N. cells in the peripheral blood of controls.

In vivo decomplementation of mice with cobra factor completely prevented trapping of aggregated human IgG within splenic germinal centres. Observations of the intrasplenic position of the aggregated material within 8 hr of its injection into normal and decomplemented mice suggest that the early phase of localisation in germinal centres depends on binding to lymphocyte C3 receptors.

Measurement of the absorbance due to light scattering at 40 nm proved to be a simple and reliable way of assessing the extent of aggregation in heat-treated IgG solutions. Using this technique the rate of aggregation was demonstrated to be markedly temperature dependent with a sharp inflexion in the curve close to 63 degrees C. During heating at 63 degrees C the concentration of unaggregated IgG fell in a manner consistent with a first-order process and the mean size of the IgG aggregates increased with time. IgG aggregates could be selectively removed from heat-treated IgG solutions and concentrated by precipitation with 3.5% polyethylene glycol without altering their size distribution. Furthermore, and in contrast with a previous report, the IgG aggregates examined in this study were remarkably stable in the absence of any other protein such as serum albumin. In consequence, several important practical recommendations concerning the production of heat-aggregated IgG for use in immune complex assays are made.

Sera from forty-six patients with dermatitis herpetiformis (DH) were examined for anti-gliadin antibodies (AGA) by the enzyme linked immunosorbent assay (ELISA) test and by a simple new immunofluorescent (IF) test. AGA were present in fifteen out of thirty-two patients taking a normal diet, but in none of the fourteen taking a gluten-free diet (GFD). The presence of circulating AGA was related to the severity of the enteropathy. AGA were present in all ten patients with a flat mucosa and in four of six with a convoluted mucosa, but in only one out of thirty patients with normal morphology of the small intestine. However, in those patients taking a normal diet and with a normal morphology of the intestine there was evidence of gluten sensitivity compared to those taking a GFD, as the intraepithelial lymphocyte count (IELC) was significantly raised in the peri-nuclear and supra-nuclear positions. The study shows that the presence of AGA in the serum is a good indication of the degree of gluten sensitivity as expressed by severe mucosal damage in patients with DH.

The unaffected skin of eighteen patients with dermatitis herpetiformis (D.H.), twenty-two patients with cœliac disease (C.D.), and eight controls were examined using direct immunofluorescence and class-specific fluorescein-conjugated anti-human IgA, IgM, and IgG antisera. All eighteen patients with D.H. showed IgA deposits in the skin: in seventeen the deposits were only found in the dermal papillae, whilst in one it was found in a continuous line below the basement membrane, confirmed by immuno-electronmicroscopy. IgM deposits were also found in the dermal papillae in three patients with D.H. and IgG deposits below the basement membrane in one patient. In cœliac disease, however, only one of the twenty-two patients showed papillary IgA deposits and one had continuous IgM deposits. These immunoglobulin deposits in D.H. and C.D. seem to be on the reticulin of the dermal papillae. It is suggested that in D.H. there is a fault of the reticulin in the skin and small intestine, whilst in cœliac disease it is present in the small intestine but not in the skin. The reticulin cross-reacts with gluten complexes to give rise to an immunological reaction. In support of this hypothesis we have demonstrated cross-reactivity between gluten and reticulin.

Immunoglobulin levels in cyst-fluids from 19 odontogenic cysts were significantly higher than in the sera of the same patients. Mean cyst/serum ratios for IgA, IgG, and IgM were 3.0, 2.1, and 1.8 respectively. Immunofluorescent staining of sections of cyst walls showed that the characteristic cellular mural aggregates contained many cells forming immunoglobulins; IgA plasma cells were preponderant, but IgG and IgM cells were also seen. In some cases plasma cells were seen penetrating the epithelial lining of the cyst and entering the cavity. It seems that immunoglobulins reaching the cyst cavity are transported by the cells that make them.

Sera from 101 patients with adult coeliac disease, 46 patients with childhood coeliac disease, 50 patients with dermatitis herpetiformis, and 479 patients with various other diseases, including skin, gastrointestinal, haematological, and immunological disorders, have been tested for the presence of the antireticulin antibody. Positive sera were retested at higher dilutions. Antireticulin antibody was only found in a significant proportion of patients with three diseases, ie, coeliac disease, dermatitis herpetiformis, and Crohn's disease. Antireticulin antibody was present in 38 out of 101 patients (38%) with adult coeliac disease, 27 out of 46 patients (59%) with childhood coeliac disease, 11 out of 50 patients (22%) with dermatitis herpetiformis, and nine out of 38 patients (24%) with Crohn's disease. In the 434 other patients with various disorders the antireticulin antibody was present in only six 1.4%) (two patients were pregnant, one had vitiligo, one had tropical sprue, one had reticulum cell sarcoma, and one had pernicious anaemia). In patients with gluten-sensitive enteropathy, ie, coeliac disease and dermatitis herpetiformis, there was a significantly higher incidence in patients taking a normal diet compared with those on a gluten-free diet. The presence of antireticulin antibody would appear to be particularly helpful in diagnosing childhood coeliac disease as it was found in 22 out of 26 patients (85%) taking a normal diet.

Without prior knowledge of the diagnosis or clinical status, sera from patients with various arthritides were screened for antibody against rheumatoid-arthritis-associated nuclear antigen (RANA) with indirect immunofluorescence on cytocentrifuged Raji cell preparations. 93% of 103 seropositive rheumatoid patients had anti-RANA antibody, in contrast to 16% of 50 normal controls and a mean of 19% (10-29%) of 122 patients with other arthritides. Anti-RANA antibody was also demonstrated in 95% of 21 patients with seronegative rheumatoid arthritis characterised by symmetrical erosive peripheral polyarthritis. No correlation was found in rheumatoid arthritis between anti-RANA antibody and disease activity, duration of disease, drug therapy, or extra-articular manifestations. RANA staining was absent in a non-Epstein-Barr-virus (EBV) cell line (Ramos). In 10 rheumatoid patients, discordance between the presence of antibodies to EBV antigens (viral capsid antigen [VCA] and EBNA) ANd RANA antibody was demonstrated. Anti-RANA antibody could be a useful marker for rheumatoid arthritis.

Serum antibodies reactive with intermediate filaments of the cytoskeleton (anti-IF antibodies) are often present in infectious mononucleosis, some other viral diseases, and rheumatoid arthritis. The mechanism of their production is not known, but it is possible that the formation of this and other autoantibodies result from polyclonal activation of B-cells. Peripheral blood mononuclear cells from subjects with or without serum anti-IF antibody were therefore cultured in the presence or absence of Epstein-Barr virus (EBV). IgM anti-IF antibody was produced in both unfractionated and T-cell-depleted cultures, but not in the supernatants of the same cells cultured without added EBV.

Smooth muscle antibody at titres of 1/10 or more was found in 54 (67·5%) out of 80 patients with malignant disease and in 9 (20%) out of 46 controls. The incidence of S.M. antibody ranged from 18/30 (60%) in malignant melanoma to 7/8 (83%) in carcinoma of the ovary. The presence of this antibody is possibly related to changes in the malignant cell membrane. A new antibody directed at an antigen presumed to be located in bile canaliculi among other sites is described.

The peripheral-blood lymphocytes from eight patients with infectious mononucleosis (I.M.) included an abnormally high proportion (45·4%) of cells forming spontaneous rosettes with sheep red blood-cells (mean normal 30·3%). The rosette-forming cells were preponderantly the atypical lymphocytes characteristic of the acute stage. In one patient with numerous circulating atypical cells (84%) the rosette-forming cells were equally numerous, and phytohaemagglutinin (P.H.A.) responsiveness was much depressed, on serial study a fall in rosetted cells paralleled the loss of atypical cells, while P.H.A. responsiveness returned. It is concluded that the atypical cells of I.M. are derived from the T lymphocyte population.

A new antibody of IgG class reacting with connective tissue in rat and human organs was found in 17% of 29 patients with dermatitis herpetiformis and in 36% of 31 patients with adult coeliac disease. The antibody appeared to be directed against reticulin rather than basement membrane. In 34% of the dermatitis-herpetiformis patients antinuclear factors of IgG or IgM class were present.

A method for the detection in human sera of precipitating antibody specific for D.N.A. by two-stage electroimmunodiffusion is described. The results obtained in a pilot study on 351 sera indicate that the significance of this antibody may be different from that attributed to D.N.A. antibodies detected by other procedures, since development of nephropathy in cases of systemic lupus erythematosus was associated with absence of the antibody, while its presence correlated with the occurrence of cutaneous vasculitic lesions.