Precipitating anti-VIII:C antibodies in two patients with haemophilia A

S ummary . IgG from the plasmas of two haemophilia A patients with anti-VIII:CAg antibodies (1000 and 200 u/ml) was isolated and labelled with ¹²⁵I. The specific labelled anti-VIII:CAg IgG was further purified by binding to and elution from immobilized factor VIII/von Willebrand factor (F.VIII/vWF). When studied by immunodiffusion and autoradiography, both antibodies gave a precipitin line with normal plasma, serum, cryoprecipitate, purified F.VIII/vWF and the plasmas of two patients with haemophilia A⁺. No precipitin line was observed with the plasmas of 11 patients with haemophilia A⁻ or four patients with severe von Willebrand's disease. Levels of VIII:CAg obtained by radioelectroimmunoassay were in agreement with those obtained by immunoradiometric assay. This study demonstrates that, contrary to previous evidence, human anti-VIII:CAg antibodies are precipitating as well as neutralizing when studied by highly sensitive techniques.     

Gamma-globulins prepared from sera of multiparous women bind anti-HLA antibodies and inhibit an established in vivo human alloimmune response

It has previously been shown that sera from multiparous women have increased levels of anti-idiotypic antibodies specific for anti-HLA molecules. gamma-Globulins prepared from these sera may be superior to commercial preparations of intravenous gamma-globulin (IVIg) for inhibiting HLA alloimmunization. To test this, F(ab')2 fragments prepared from either commercial IVIg or from the sera of men or multiparous women were coupled to CNBr-Sepharose and tested for their ability to bind F(ab')2 fragments derived from polyspecific anti-HLA sera. As determined by flow cytometry, compared with columns coated with F(ab')2 derived from commercial IVIg or sera from men, columns coated with F(ab')2 prepared from the sera of multiparous women bound significantly more anti-HLA. In addition, intact IgG molecules prepared from the sera of multiparous women significantly neutralized the reactivity of the anti-HLA F(ab')2 fragments. To determine whether the intact IgG molecules or their corresponding F(ab')2 fragments could affect in vivo alloimmunity, they were tested for their ability to inhibit an established IgG human alloimmune response in humanized severe combined immunodeficient (SCID) mice. Compared with commercial IVIg, when intact IgG or F(ab')2 fragments derived from multiparous women were administered to SCID mice making human anti-HLA antibodies, a significant reduction in anti-HLA reactivity was observed. The findings suggest that IgG molecules prepared from the sera of multiparous women have increased anti-idiotypic reactivity against anti-HLA antibodies, which can significantly inhibit an established human IgG alloimmune response in an Fc-independent manner.     

Serum IgG antibodies to C1q in hypocomplementemic urticarial vasculitis syndrome

Urticaria, angioedema, and arthritis are cardinal features of hypocomplementemic urticarial vasculitis syndrome (HUVS). Considered to be an immune complex-mediated disorder, HUVS has been differentiated from systemic lupus erythematosus (SLE), based on its clinical manifestations and the C1q precipitin (C1q-p) reaction, which is manifested as gel precipitation of C1q by a small percentage of HUVS IgG molecules. This phenomenon has been attributed to an Fc region abnormality, and the responsible IgG molecules are said to possess C1q-p activity. We purified IgG from 4 HUVS patients and confirmed that HUVS IgG contains C1q binding activity. F(ab')2 fragments from these patients also bound to C1q, as measured by 2 different C1q binding methods at physiologic ionic strength; HUVS IgG Fc fragments did not bind to C1q. Preincubation of HUVS F(ab')2 fragments with antibody to human F(ab')2 prevented subsequent binding to C1q. We conclude that IgG antibodies to C1q are present in HUVS serum, and it is likely that these antibodies are C1q-p. Because the clinical manifestations of HUVS and the presence of anti-C1q antibodies have been described in patients with SLE, our findings support the concept that HUVS is an autoimmune syndrome related to SLE.     

The idiotypic characteristics of human antibodies to factor VIII

We have prepared an antiidiotype antibody that specifically reacts with and inactivates a human autoantibody to factor VIII (VIII:C). The antiidiotype specificity of this rabbit antibody was evident in solution, as specific inhibition of anti-VIII:C activity, and in solid- phase studies. The antiidiotype antibody inhibited both plasma anti- VIII:C and Fab' fragments prepared from that plasma's IgG. Although the antibody had a greater inhibitory effect on the anti-VIII:C used for immunization than it did for other anti-VIII:C, partial inhibition was identified with ten of 25 other human anti-VIII:C. The demonstration that antiidiotype antibodies can be prepared to human anti-VIII:C indicates the feasibility of this approach to the immunochemical characterization of human anti-VIII:C inhibitors. Moreover, antiidiotype reagents have a potential role in the treatment of inhibitor patients.     

In vivo efficacy of intravenous gammaglobulins in patients with lupus anticoagulant is not mediated by an anti-idiotypic mechanism.

The authors have investigated the presence in commercially available intravenous gammaglobulins (IVIg) of anti-idiotypic antibodies directed to Lupus Anticoagulant (LA). In vitro incubation of 4 LA plasmas with increasing concentrations of IVIg (from 0 to 39 mg/ml) resulted in a dose-dependent inhibition of LA activity (the highest inhibitions ranged from 14.0 to 53.4%). Similar results were obtained when patients' plasma was substituted with total IgG (the highest inhibitions ranged from 43.0 to 55.0% and were obtained at IgG:IVIg molar ratios ranging from 1:15 to 1:50). Also the incubation of patients' F(ab')2 with F(ab')2 from IVIg produced a similar dose-dependent inhibition of LA activity. These data are suggestive of an in vitro idiotypic-anti-idiotypic interaction between LA and IVIg. However, when injected in patients with LA, IVIg do not seem to operate by this mechanism of action. In fact, reduction or disappearance of LA was only observed in 2 out of 4 patients; also the quick reappearance of LA activity was not consistent with the time course of anti-idiotypic response. Finally, this effect was reached by half the IVIg concentrations necessary to produce an appreciable inhibitory effect on LA activity in vitro. Thus, it is concluded that, even if IVIg contain anti-idiotypic antibodies reacting with LA, the clinical efficacy of IVIg treatment in patients with these autoantibodies should be attributed to other mechanisms.     

Origin of anti-idiotypic activity against anti-factor VIII autoantibodies in pools of normal human immunoglobulin G (IVIg).

Therapeutic preparations of polyspecific IgG obtained from plasma pools of a large number of normal donors (IVIg) express anti-idiotypic activity against a wide spectrum of natural and disease-associated autoantibodies. The present study investigated the origin of anti-idiotypic activity against autoantibodies to factor VIII. The neutralizing activity of pools of IgG against patients' anti-factor VIII autoantibodies was not influenced by the presence of individuals with natural anti-factor VIII antibodies among donors contributing to the pool. A higher frequency of neutralizing antibodies against anti-factor VIII autoantibodies was found in aged donors as compared with young adults and in pools of IgG from multiparous women as compared with IgG from random donors. Pooling IgG from several donors synergistically enhanced the inhibitory activity of the pools. Thus, a neutralizing activity against anti-factor VIII autoantibodies was detected in pools of IgG of as few as two to four donors of whom individually tested IgG did not exhibit inhibitory activity against anti-factor VIII autoantibodies. These observations suggest that aged donors and multiparous women may be privileged sources for the anti-idiotypic activity of IVIg against autoantibodies and emphasize that the expression of anti-idiotypic activity in IVIg results from a synergistic participation of anti-idiotypes from each donor contributing to the pool.     

Natural antibodies to factor VIII (anti-hemophilic factor) in healthy individuals.

Spontaneous inhibitors of factor VIII (FVIII) are pathogenic IgG autoantibodies of restricted isotypic heterogeneity found in the plasma of patients presenting with bleeding episodes and low levels of FVIII. We now report the presence of a natural FVIII-neutralizing activity in 85 of 500 plasma samples (17%) from healthy donors. FVIII-inhibitory activity was present in F(ab')2 fragments of purified IgG and was dose-dependent. The titer of anti-FVIII antibodies in normal plasma ranged between 0.4 (threshold of detection) and 2.0 Bethesda units. Anti-FVIII IgG was also detected in normal plasma by using an ELISA. Anti-FVIII antibodies from healthy individuals did not exhibit restricted isotypic heterogeneity. Mean levels of FVIII activity did not differ significantly between individuals with and without detectable anti-FVIII antibodies in plasma. Natural anti-FVIII IgG inhibited FVIII activity in pools of normal plasma and in plasma of certain donors in the pool but did not inhibit FVIII activity in autologous plasma. These observations demonstrate that polyclonal IgG antibodies against procoagulant FVIII are present in healthy individuals. The antibodies are natural IgG autoantibodies and/or antibodies directed against epitopes associated with a so far unidentified allotypic polymorphism of the human FVIII molecule.     

A monoclonal antibody to VIII:C produced by a mouse hybridoma

Spleen cells of a BALB/c mouse immunized with factor VIII procoagulant activity (VIII:C) (isolated by affinity chromatography) were fused with mouse myeloma cells (P3 x 63 Ag8). After the fusion 12/32 wells produced an inhibitor to VIII:C. Cells from one well (1B3) were subcloned four times in order to isolate the hybridoma that produces the anti-VIII:C antibody. Injection of hybridoma cells in pristane pretreated BALB/c mice results in anti-VIII:C titers of 5000-10,000 Bethesda U/ml. Analysis of the produced immunoglobulin demonstrated heavy chains of IgG1 (produced by the myeloma cell line) and IgG2b subclass. The 1B3 antibody neutralizes VIII:C in LMW FVIII, crysosupernatant, cryoprecipitate, and normal plasma. It was found that binding of the IgG to FVIII results in a delay in its activation and not in an inhibition of its cofactor activity. The antibody removes VIII:C from pooled normal plasma when coupled to Sepharose; when coupled to plastic tubes, it binds VIIICAG from isolated VIII:C, purified FVIII, and pooled normal plasma; it does not bind VIIIR:AG, fibrogen, or serum VIIICAG. The 1B3 antibody can be used successfully in an IRMA for VIIICAG.     

Isolation of Human Antibodies to Factor VIII

It has been claimed that human anti-VIII:C antibodies do not form stable complexes with factor VIII and this fact has hampered in the past the isolation of such antibodies. In this study the purification of human anti-VIII:C antibodies appearing in haemophiliac patients following replacment therapy has been achieved using two different systems. In a liquid phase system, purified human factor VIII was mixed with IgG from a haemophilic patient with a high titre antibody. Specific anti-VIII:C antibodies were recovered following filtration of the antigen-antibody complexes on Biogel A-5m, dissociation of complexes at pH 3.5 and final isolation by filtration on Sephadex G-200. In a solid phase system, the same IgG fraction was specifically bound to insolubilized human factor VIII. Purified anti-VIII:C antibodies were subsequently recovered by elution of antigen-antibody complexes with magnesium chloride. The results demonstrated that stable complexes from between anti-VIII:C antibodies and either the whole factor VIII molecule, or VIII:C dissociated by previous interaction with the antibodies. It is postulated that, in vivo, similar antigen-antibody complexes may form following replacement therapy in haemophilic patients with antibody.     

Antibodies to actin in autoimmune neutropenia

In an effort to characterize the cellular antigens recognized by anti-neutrophil antibodies in autoimmune neutropenia, we studied sera, purified immunoglobulin G (IgG) and isolated F(ab')2 from 70 neutropenic patients suspected of this diagnosis. Anti-neutrophil antibodies were found in the sera of 36 of these patients by either 125I-staph A binding or immunofluorescence cytometric techniques that detected increased binding of patients' IgG to normal neutrophils. Anti-neutrophil antibody positive sera were then evaluated for specific binding to electrophoretically separated neutrophil membrane-associated proteins by immunoblotting. A 43-Kd protein was consistently identified by eight anti-neutrophil antibody positive sera. The specificity of binding to this protein was confirmed with affinity purified IgG and F(ab')2 fragments prepared from these sera. Sera from 20 healthy normal controls and from 22 non-neutropenic, anti-neutrophil antibody negative rheumatoid arthritis patients failed to bind this protein. Separate studies identified the 43-Kd protein as actin. Purified Acanthamoeba actin comigrated with the protein and was specifically bound by anti-neutrophil antibody positive IgG. Moreover, two actin-specific monoclonal antibodies bound to the 43-Kd membrane-associated protein in immunoblots. In addition, a rabbit anti-actin antiserum not only bound to this same 43-Kd protein but also expressed anti-neutrophil antibody activity against normal human neutrophils, as did purified human anti-actin IgG prepared by affinity chromatography from the serum of one of the index patients. These studies indicate that the anti-neutrophil antibodies of certain patients with autoimmune neutropenia include autoantibodies specific for actin. The molecules on the surface of neutrophils, which have actin-like antigenic epitopes and are recognized by these anti-actin antibodies, remain to be characterized.     

Fractionation of human antibody to factor VIII:C: an IRMA for phospholipid binding sites on factor VIII C: Ag

Labelled Fab' fragments, derived from the plasma of a severe haemophiliac with antibody directed against factor VIII clotting antigen (VIII C:Ag), were fractionated by immunoabsorption with, first, a complex of phospholipid (PL) vesicles and factor VIII and, second, with factor VIII alone. Two pools of labelled anti-VIII C:Ag were obtained and were used in immunoradiometric assays (IRMAs) for VIII C:Ag. With one pool (non-PL-site antibody) VIII C:Ag assays were unaffected by pre-incubation of factor VIII with PL vesicles; however, binding of the second pool of antibody to VIII C:Ag was prevented by PL preincubation, indicating that these antibody molecules bind at or near a phospholipid binding site on VIII C:Ag (PL-site antibody). Assays of VIII C:Ag in an intermediate purity factor VIII concentrate with these two antibody pools indicate that more than one third of the VIII C:Ag may be bound to PL.     

Possible presence of enhancing antibodies in idiopathic thrombocytopenic purpura

It is difficult to detect IgG anti-platelet autoantibodies in idiopathic thrombocytopenic purpura (ITP). Recently, it was reported that reactivity with glycoprotein IIb/IIIa was lost when IgG anti-GPIIb/IIIa antibodies from seven ITP patients were digested with pepsin to yield F(ab')2 fragments. These findings suggested that some IgG antiplatelet autoantibodies in ITP may be of low affinity and thus require the presence of 'enhancing' anti-IgG antibodies (i.e. rheumatoid factors, RFs) for detection. To test this hypothesis, we used a phage display technique to isolate five IgG RFs from an ITP patient (patient 1). Sequence analysis revealed that these RFs consisted of two clones, represented by GG3 and GG48. Both representative RFs bound specifically to IgG Fc fragments with apparent dissociation constants of 8.2 x 10(-8) M and 8.8 x 10(-7) M, respectively. Moreover, IgG RFs were subsequently found in a serum sample from patient 1. Combined, these results suggest that IgG RFs may occur in ITP, and may be required for the detection of some IgG anti-platelet autoantibodies and for the corresponding antibody-mediated platelet destruction in autoimmune ITP.     

Enzyme linked immunosorbent assay (ELISA) for the measurement of factor VIII coagulant antigen (CAg) using haemophilic antibodies

A method for the quantitation of factor VIII clotting antigen (VIII:CAg) has been developed based on a micro enzyme linked immunosorbent assay (ELISA) principle employing antibodies from two polytransfused haemophilia A patients. Solid polystyrene support bound IgG fraction of inhibitor plasma extracted VIII:CAg from normal plasma and samples. Bound VIII:CAg was detected by peroxidase labelled F(ab')2 fragment of the IgG used for solid phase. Two assays, each based on its particular inhibitor antibody, were set up. The F VIII clotting antigen in plasma of 30 healthy persons was found identical with the two VIII:CAg assays (r=0.97) and closely correlating with clotting activity (VIII:C) (r=0.84). Serum VIII:CAg was 67% (+/-14.5%) of the corresponding plasma value. In severe haemophilia A, 17 out of 19 had VIII:CAg values less than 1 U/dl. Two patients with cross-reactive material (CRM+) were found. In some milder cases of haemophilia A, higher values of VIII:CAg than VIII:C was recorded. The sensitivity of the method was 0.08 U/dl. Inter assay coefficient of variation at the 100 U/dl level was 9.5% (CV%), at the 2 U/dl level 16.4% (CV%). Mainly due to the great stability of enzyme conjugated antibody compared to the natural decay of radioiodinated material and subsequent loss of detecting material, ELISA was found superior to immunoradiometric assay (IRMA).     

IVIg inhibits reticuloendothelial system function and ameliorates murine passive-immune thrombocytopenia independent of anti-idiotype reactivity

Although the mechanism of action of intravenous immunoglobulin (IVIg) in treating antibody-dependent thrombocytopenia remains unclear, most studies have suggested that IVIg blocks the function of Fc receptors in the reticuloendothelial system (RES) and/or the protective effect may be due to the presence of variable region-reactive (anti-idiotype) antibodies within IVIg. We evaluated the effect of IVIg on platelet counts in a murine model of passively induced immune thrombocytopenia (PIT). Although IVIg was unable to neutralize the binding of two platelet-specific monoclonal antibodies to their target antigens either in vivo or in vitro, it was able to prevent PIT as well as ameliorate pre-established PIT mediated by these antibodies. IVIg adsorbed against the antibody used to induce thrombocytopenia or endogenous murine immunoglobulin also protected against PIT, indicating that antibodies with anti-idiotype activity present in IVIg are not necessary for its effective treatment of PIT. IVIg significantly blocked the ability of the RES to clear antibody-sensitized red blood cells. F(ab')2 fragments of IVIg, which are unable to block the RES but retain the idiotypic regions, were ineffective at protecting mice from PIT. Our data suggest that IVIg exerts its rapid effect by inhibiting RES function and that anti-idiotype interactions are not required.     

Identification of class II HLA alloantibodies in placenta-eluted gamma globulins used for treating rheumatoid arthritis

Placenta-eluted gamma globulins (PEGG) have been recently and successfully used in the treatment of patients with rheumatoid arthritis. PEGG, eluted at acid pH from large pools of human placentas, contained 99% IgG material. Sephacryl S300 gel filtration revealed a main fraction (76%) of native IgG accompanied by 10% aggregates and 14% digested fragments (as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoelectrophoresis with specific antisera). Previous in vitro data had suggested that alloantibodies to class II HLA antigens were present in this preparation. This study confirms that PEGG and F(ab')2 fragments were able to inhibit stimulating cells in mixed lymphocyte reactions. Additional findings showed that: IgG from PEGG were cytotoxic for the non-T cell population; IgG or F(ab')2 from PEGG bound only to class II HLA-bearing cells; F(ab')2 from PEGG were able to block the complement-mediated cytotoxicity of anti-HLA-DR and anti-DQw1 alloantibodies. These data confirm the presence of class II HLA alloantibodies in PEGG. These antibodies may account for the clinical improvement reported in patients with rheumatoid arthritis. Our findings are similar to recent data showing that the injection of anti-Ia antibodies in experimental animal models decreases the autoimmune process.     

Felty syndrome: autoimmune neutropenia or immune-complex-mediated disease?

Summary Immunofluorescence on polymorphonuclear cells (PMN) of patients with Felty syndrome (FS) revealed increased amounts of IgG, IgA, and IgM bound to the PMN surface compared with PMN of patients with rheumatoid arthritis alone. A positve correlation was found between the score for surface-bound immunoglobulins on FS-PMN and the results of the C1q binding assay in FS sera. After preincubation with sera from 20 patients with FS, immunofluorescence on PMN from healthy controls (HC) showed that these cells had bound IgG, IgA, and IgM. However F(ab')₂ fragments of IgG from FS sera did not bind to PMN, although the antigen-binding reactivity of the F(ab')₂ fragments was maintained as shown by control experiments. Immunoglobulins eluted from FS-PMN failed to bind to HC-PMN, whereas the corresponding IgG of patients with autoimmune neutropenia was bound. Gel filtration of FS sera on Sepharose 4B showed that the binding of IgG in FS sera to PMN did not coincide with the 7S peak but occurred mainly in fractions containing larger material. No binding of IgA and IgM to HC-PMN was found after incubation with FS sera pretreated with polyethylene glycol (PEG) to precipitate immune complexes. These results indicate that in sera of patients with FS the PMN-binding reactivity of IgG, IgA, and IgM is due to the binding of immune complexes containing these immunoglobulins and not to presence of autoantibodies directed to antigens on the neutrophil surface.     

Fc(gamma) receptors IIa on cardiomyocytes and their potential functional relevance in dilated cardiomyopathy.

OBJECTIVES: The purpose of this study was to investigate how cardiac autoantibodies might contribute to cardiac dysfunction in patients suffering from dilated cardiomyopathy (DCM). BACKGROUND: In the majority of DCM patients, it is possible to detect antibodies with negative inotropic effect on cardiomyocytes. The manner in which these antibodies impair cardiac function is poorly understood. METHODS: Immunoglobulin (Ig)G was prepared from plasma of 11 DCM patients containing antibodies that induced a negative inotropic effect on cardiomyocytes. We analyzed the effects of antibodies/IgG fragments on calcium transients and on systolic cell shortening of adult rat cardiomyocytes and investigated the dependency of these effects on potential cardiomyocyte Fc receptors. RESULTS: In contrast to control subjects, intact IgG from DCM patients reduced calcium transients and cell shortening of cardiomyocytes. The F(ab')2 fragments of these antibodies did not induce these effects but inhibited the functional effects of DCM-IgG of the respective patients' IgG. These effects were also inhibited by Fc fragments of normal IgG. Reconstitution of the Fc part by incubation of cardiomyocytes with DCM-F(ab')2 fragments followed by goat-anti-human-F(ab')-IgG again induced reduction of cell shortening and of calcium transients. In rat and human ventricular cardiomyocytes, Fc(gamma) receptors IIa (CD32) were demonstrated by immunofluorescence. CONCLUSIONS: Our findings indicate that DCM-IgG-F(ab')2 bind to their cardiac antigen(s), but the Fc part might trigger the negative inotropic effects via the newly detected Fc(gamma) receptor on cardiomyocytes. These results point to a novel potential mechanism for antibody-induced impairment of cardiac function in DCM patients.     

Inhibition of cell adhesion by antibodies to Arg-Gly-Asp (RGD) in normal immunoglobulin for therapeutic use (intravenous immunoglobulin, IVIg).

Intravenous immunoglobulin (IVIg) therapy is associated with a broad range of immunomodulatory activities. Several of the postulated mechanisms of IVIg action relate to the presence of antibodies to molecules relevant for regulation of the immune response. This article reports that IVIg contains antibodies to the Arg-Gly-Asp (RGD) sequence, and the attachment site of a number of adhesive extracellular matrix proteins, including ligands for beta1, beta3, and beta5 integrins. Anti-RGD antibodies were identified in IVIg by enzyme-linked immunosorbent assay and by using the BIAcore (BIAcore, Uppsala, Sweden) technology. The affinity of anti-RGD antibodies to a synthetic RGD-containing peptide and to fibronectin (Fn) was found to be in the micromolar range. F(ab')2 fragments specific for RGD were purified from IVIg by affinity chromatography. Anti-RGD F(ab')2 antibodies inhibited adenosine diphosphate induced alphaIIb/beta3 integrin-mediated platelet aggregation and the adhesion of activated alpha4beta1 integrin-expressing B cells to Fn. Adhesion of unstimulated platelets to fibrinogen (Fg) involving both the gamma-chain dodecapeptide sequence and the RGD sequence was inhibited by anti-RGD antibodies. In addition, adhesion of thrombin-stimulated platelets to von Willebrand factor or Fg was completely inhibited by affinity-purified anti-RGD antibodies. Our results suggest that the presence of natural IgG antibodies to the RGD motif may contribute to the immunomodulatory and anti-inflammatory effects of therapeutic preparations of normal IgG.     

Noncoagulation inhibitory factor VIII antibodies after induction of tolerance to factor VIII in hemophilia A patients

We recently described tolerance induction with factor VIII/IX, cyclophosphamide, and high-dose intravenous IgG in hemophilia A or B patients with coagulation inhibitory antibodies. Circulating noninhibitory antibodies complexed with factor IX have been demonstrated in tolerant hemophilia B patients. Similar findings are now described in six tolerant hemophilia A patients. Complexes between factor VIII and the 'tolerant' antibody were demonstrated by subjecting plasma to gel filtration chromatography, void fractions containing factor VIII/vWF complexes being collected and adsorbed to protein A. Using 125I-labeled F(ab')2 fragments against IgG subclass and factor VIII antigen, complexes between an IgG4 antibody and factor VIII were found to adsorb to protein A. After infusion of factor VIII to tolerant patients, all factor VIII circulated in complex with IgG4 antibody. In three of the patients, the 'tolerant' antibodies inhibited an ELISA specific for factor VIII light chain but, unlike the pretolerant antibodies, did not bind radiolabeled factor VIII heavy chain. Although after induction of tolerance the patients still have circulating IgG4 antibodies against factor VIII, the antibodies differ in specificity, lack coagulation inhibitory activity, and do not enhance the rate of elimination of factor VIII.     

Antiidiotypic antibodies neutralize autoantibodies that inhibit cholinergic neurotransmission

OBJECTIVE: Functional autoantibodies that inhibit M(3) muscarinic receptor (M3R)-mediated neurotransmission have been reported in patients with Sj?gren's syndrome (SS) and in patients with scleroderma. Because of limited reports that intravenous immunoglobulin (IVIG) improves dysautonomia in primary SS, we investigated whether IVIG neutralizes the effect of anti-M3R antibodies on colon smooth muscle contractions, in an in vitro functional assay. METHODS: IgG obtained from patients with primary SS, patients with rheumatoid arthritis and secondary SS, and patients with scleroderma was tested, before and after coincubation with equimolar amounts of IVIG or its F(ab')(2) and Fc fractions, for the ability to inhibit carbachol-evoked colon smooth muscle contractions. In addition, patient IgG was passed through an IVIG F(ab')(2) column, and unretained IgG was tested for functional activity on colon smooth muscle strips. Purified IgG obtained from healthy adults was also examined for a neutralizing effect on anti-M3R antibody activity. RESULTS: Inhibition of colon contractions was mediated by the Fab fraction of patient IgG. Coincubation of IgG from the 3 patient groups with IVIG or its F(ab')(2) fragment neutralized anti-M3R antibody-mediated inhibition of cholinergic smooth muscle contractions. Preabsorption of patient IgG with Sepharose-bound IVIG F(ab')(2) removed the anti-M3R inhibitory activity. In addition, purified IgG from each of 4 healthy adults neutralized the functional autoantibodies. CONCLUSION: Anti-M3R antibody activity does not require receptor crosslinking. Antiidiotypic antibodies present in pooled IgG neutralize patient IgG-mediated inhibition of M3R cholinergic neurotransmission, providing a rationale for IVIG as a treatment of autonomic dysfunction in patients with SS and patients with scleroderma. Furthermore, antiidiotypic antibodies in healthy individuals may prevent the emergence of pathogenic anti-M3R autoantibodies.