Immunoglobulin G Autoantibodies Research Articles

Index of Suspicion

Index of Suspicion

Detection of anti-Nogo receptor autoantibody in the serum of multiple sclerosis and controls

A myelin-associated neurite outgrowth inhibitor Nogo-A plays a key role in inhibition of axonal regeneration. Axonal damage beginning at the early stage of multiple sclerosis (MS) is responsible for permanent neurological deficits, although its molecular mechanism remains unknown. The aim was to study the prevalence of autoantibodies against Nogo-A and Nogo receptor (NgR) in the serum of MS. The antibodies were identified in the serum of 30 MS patients, 22 patients with non-MS other neurological diseases (OND), and 22 healthy control (HC) subjects by Western blot using recombinant human Nogo-A-specific segment (NAS), the shared segment of Nogo-A and -B (NAB), Nogo-66 (N66), the non-glycosylated form of NgR, the glycosylated NgR (NgR-Fc), and myelin oligodendrocyte glycoprotein (MOG). None showed immunoglobulin G (IgG) antibodies against NAS or NAB. In contrast, 30% of MS, 23% of OND and 32% of HC subjects exhibited anti-N66 IgG, while 27% of MS, 27% of OND and 18% of HC showed anti-MOG IgG. None of HC but 33% of MS and 14% of OND showed anti-non-glycosylated NgR IgG. Furthermore, 60% of MS, 18% of OND and 14% of HC showed anti-NgR-Fc IgG. Because IgG autoantibodies against N66, NgR and MOG are often detected in the serum of MS and controls, they do not serve as an MS-specific marker.

Prevalence of IgG autoantibody against F‐actin in patients suspected of having autoimmune or acute viral hepatitis

Our objectives in this study were to compare results obtained by an enzyme immunoassay (EIA) for F-actin antibody (FAA) immunoglobulin G (IgG) to those determined by an indirect fluorescent antibody (IFA) assay for smooth muscle antibody (SMA) IgG, and to determine the prevalence of FAA in patient sera having serologic evidence of acute viral hepatitis. Sera from 415 patients suspected of having autoimmune hepatitis (AIH), 208 patients suspected of having acute viral hepatitis A, B, or C, and 100 healthy blood donors (HBD) were included in the study. Only one of 100 HBD showed low levels (20-30 Units) of F-actin IgG. In patients suspected of having AIH, the prevalence of FAA increased as SMA titers increased and all sera with SMA titers of >or=1:160 were FAA-positive. In contrast, there were many sera with negative (<1:20) or low (1:20-1:40) SMA titers that contained moderate to high levels (>30 Units) of FAA; many exceeding 80 Units. Moreover, 51.4% of these sera were also positive for anti-nuclear antibody (ANA), which is also utilized in diagnosing type 1 AIH. FAA was detected in 25% of viral hepatitis antibody-positive sera, with the majority (59.3%) containing low levels, and all were ANA-negative.

Viewpoint 1

Viewpoint 1

Commentary 3

Compared with other ‘model’ disorders of organ-specific autoimmunity, such as multiple sclerosis, myasthenia gravis or insulin-dependent diabetes mellitus, the pathogenesis of pemphigus is by far unravelled to the greatest extent (1,2). However, the ambitious endeavour to provide a pathogenetic concept that fits for all the clinical variations of pemphigus demonstrates that, apart from the definition of common major autoantigens, the relative contribution of additional factors may vary interindividually. Several brilliant achievements of the past years have helped to dissect crucial events in the effector phase of the immune pathogenesis of pemphigus, even though several aspects in the aetiology and molecular effects leading to autoimmune loss of epidermal adhesion are not yet fully understood. Immunogenetically, there is a clear-cut association of pemphigus with distinct human leukocyte antigen (HLA) class II alleles that have been shown to be critical for T-cell recognition of autoantigenic peptides of desmoglein (Dsg)3 and Dsg1, an initial event required for T-cell-dependent induction and perpetuation of autoantibody production (2–4). The relevance of this concept is supported by the finding that healthy carriers of pemphigus-associated HLA class II alleles have also autoreactive T cells of identical epitope specificity and similar frequency as patients with pemphigus (4–7). We have shown that Dsg3-specific, T-cell-driven peripheral tolerance is at least one mechanism which regulates the clinical outcome of pemphigus by preventing the activation of Dsg3-specific, autoaggressive B cells (7–9). Several human studies in pemphigus vulgaris (PV) and pemphigus foliaceus (PF) (including the endemic variant, fogo selvagem) have shown that, in general, immunoglobulin G (IgG) autoantibody titres against Dsg3 and Dsg1 relate to the clinical activity and phenotype of PV and PF, respectively (10,11), and that their therapeutic removal is of clinical benefit in PV (12,13). Even though there is a proof that the overall picture of the Dsg compensation theory is valid (11), there are several evidences that autoantibody specificities (i.e. Dsg1 vs Dsg3 reactivity) and titres do not always relate to the clinical phenotype and activity of pemphigus (14,15). With regard to intramolecular epitope spreading, there is strong evidence that polyclonal Dsg-specific IgG autoantibodies in the patients’ sera recognize both pathogenic and non-pathogenic epitopes located in the ectodomains of Dsg3 and Dsg1 (16). Experimental in vitro and in vivo data demonstrate that IgG against the NH2-terminal domain of Dsg3 is pathogenic, while IgG against other epitopes requires the presence of autoantibodies targeting additional epitopes resulting in a pathogenic synergism of ‘semipathogenic’ autoantibodies (17–19). This contention is also supported by serological analyses in PV patients showing (i) that IgG against the NH2-terminal aa1–161 relates to disease activity (20) and (i) that the presence of several domain-specific IgG autoantibodies against the Dsg3 ectodomain relates to disease activity in PV (21). In fogo selvagem, the endemic variant of PF, pathogenic IgG autoantibodies against the putative autoantigen, Dsg1, target its NH2-terminus, while patients’ relatives or individuals who develop disease later on have autoantibodies that target ‘non-pathogenic’ regions in the COOH-terminus of the Dsg1 ectodomain (22). Current evidence favours the concept that Dsg1 and Dsg3 are the principal autoantigens in most clinical variants of pemphigus, even though the relative contribution of additional desmosomal (i.e. desmocollins, plakins) and non-desmosomal autoantigens (pemphaxin, α9-acetylcholine receptor) remains a matter of debate (23,24). The critical role of Dsg3 in epidermal adhesion of mucosal and stratified squamous epithelia has been clearly shown in a Dsg3-deficient animal (25). However, we would like to challenge the concept that Dsg-specific autoantibodies alone are the principal effectors in pemphigus because patients on treatment with the anti-CD20 monoclonal antibody, rituximab, frequently show a clinical response despite the persistence of high autoantibody titres (26). Our group has observed in rituximab-treated PV patients that, upon depletion of B cells, the frequencies of autoreactive T helper 1 and 2 cells were immediately reduced to background levels (27). This was not the case with T helper cells responsive to an unrelated antigen, suggesting that rituximab acted specifically on autoreactive T cells which may also be critically involved in the effector phase of PV, apart from their helper function to autoaggressive B cells (28,29). Along this line, therapeutic blockade with infliximab, a monoclonal antibody against the proinflammatory cytokine, tumor necrosis factor-α, which is mainly produced by effector T cells, leads to an immediate amelioration of PV, despite the persistence of tissue-bound and circulating IgG autoantibodies (30). While this may not suffice to demote Dsg-specific autoantibodies to the inferior role of ‘witnesses of disease’, it clearly questions whether these autoantibodies alone are the principal effectors of PV immunopathogenesis.

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Viewpoint 6

In Vitro Keratinocyte Dissociation Assay for Evaluation of the Pathogenicity of Anti-Desmoglein 3 IgG Autoantibodies in Pemphigus Vulgaris

Patients with pemphigus vulgaris (PV) have circulating anti-desmoglein (Dsg) 3 immunoglobulin G (IgG) autoantibodies that induce blister formation. We developed an in vitro quantitative assay to evaluate the pathogenic strength of anti-Dsg3 IgG autoantibodies in blister formation. To obtain intercellular adhesion mediated dominantly by Dsg3, we used primary cultured normal human keratinocytes expressing low level of Dsg2 in the presence of exfoliative toxin A that specifically digests Dsg1. After incubation with various antibodies, monolayers released by dispase were subjected to mechanical stress by pipetting, and the number of cell fragments were counted. When anti-Dsg3 monoclonal antibodies (mAb) obtained from pemphigus model mice were tested, pathogenic AK23 mAb yielded significantly higher number of cell fragments than AK7 or AK20 non-pathogenic mAb. Dissociation scores, defined with AK23 mAb as the positive control, were significantly higher with active stage PV sera (n=10, 77.4+/-21.4) than controls (n=11, 16.0+/-9.6; p=0.003). When pair sera obtained from 6 PV patients in active stage and in remission were compared, the dissociation scores reflected well the disease activity as those in active stage were four to 17 times higher than those in remission. When sera from different patients showing similar ELISA scores but different clinical severity were tested (n=6), the dissociation scores with sera from severe disease activity were significantly higher than those with sera in remission. These findings indicate that this dissociation assay will provide a simple and objective biological method to measure the pathogenic strength of pemphigus autoantibodies.

Localized pemphigoid on the soles of both feet

An 80-year-old woman was referred to our hospital with a spontaneously appearing bullous dermatosis limited to the soles of both feet, causing an intense pruritus. She also suffered from Parkinson's disease and depression, and had been treated with levodopa, benserazide, and mirtazapine for several years. On clinical examination, we found several tense and hemorrhagic bullae, with a diameter of up to 3 cm, at both plantar sites and multiple, confluent, dyshidrotic vesicles ( Figs 1 and 2). The rest of the skin, including the mucous membranes and palms, was normal. The first clinical diagnosis was podopompholyx, but histopathologic findings and direct immunofluorescence revealed a diagnosis of bullous pemphigoid, showing subepidermal blisters (Fig. 3) and linear deposits of C3 and immunoglobulin G (IgG). Indirect immunofluorescence was positive, the IgG autoantibodies bound to the epidermal site of salt-split skin, and circulating antibodies against bullous pemphigoid antigens 1 and 2 were found. Because of this typical clinical picture, a diagnosis of dyshidrotic pemphigoid, a localized form of bullous pemphigoid, was made. Under systemic treatment with prednisolone, 40 mg/day, the skin healed completely within 2 weeks. Descriptions of dyshidrotic pemphigoid limited to the soles of both feet are very rare, and the clinical findings might easily lead to a misdiagnosis of podopompholyx.

Free Communication: Immunology

*††YukitoshiTakahashi, *NaomiSakaguchi, ‡MasahikoWatanabe, ‡HisashiMori, ‡MasayoshiMishina, §HideoAiba, ∥TomokoMiyajima, ¶YoshiakiSaitou, **YasushiUchida, †NaokiMatsuo, †SatomiHattori, and *NaomiKondo *Department of Pediatrics, Gifu University School of Medicine, Gifu ; †Department of Anatomy and Embryology, Hokkaido University Graduate School of Medicine, Hokkaido ; ‡Department of Molecular Neurobiology and Pharmacology, School of Medicine, University of Tokyo, Tokyo ; §Shizuoka Children's Hospital, Shizuoka ; ∥Shiga Medical Center for Children, Shiga ; ¶Department of Pediatrics, Tokyo Women's Medical University, Tokyo ; **Department of Pediatrics, Nagahama Municipal Hospital, Nagahama ; and ††Gifu Prefectural Gifu Hospital, Gifu, Japan Purpose: Glutamate receptors (GluRs) in the central nervous system play an important role in the excitatory synapse and have been implicated in neurodegenerative diseases and symptomatic epilepsy. After Rogers et al. reported that autoantibodies against GluR3 were involved in some cases of Rasmussen encephalitis (Science 1994; 265:648–51), studies have focused on other neurologic diseases with a possible causal relation to autoantibodies against GluRs. To reveal the pathophysiologic mechanisms of severe epilepsia partialis continua (Y. Takahashi et al. ActaNeurolScand 1997; 96:345–52), we developed a detection system highly sensitive to autoantibodies against the N‐methyl‐d‐aspartate (NMDA) GluRα2 subunit and found autoantibodies against GluRα2 in patients with chronic progressive epilepsia partialis continua of childhood (Y. Takahashi et al. AnnRepJpnEpilepsyResFound 1999; 11:45–52). Serum autoantibodies against GluRα2 in patients with chronic progressive epilepsia partialis continua of childhood have epitopes on the C‐terminal of GluR2 molecules. In this study, we tried to reveal the site of immunogenesis of autoantibodies by comparing the epitopes in serum and cerebrospinal fluid (CSF). Methods: Six patients with immunoglobulin G (IgG) autoantibodies against the NMDA GluRα2 subunit in serum and CSF were examined. The age at onset of their disease ranged from 2 months to 12.5 years for epilepsy, and 7 months to 12.9 years for epilepsia partialis continua. Probable causative factors were aseptic meningitis, encephalopathy, IgA deficiency, influenza infection, and immunization. The patients had intractable epilepsia partialis continua and complex partial seizures. Neuropsychological outcomes were poor in three patients; quadriplegia developed in four patients, and hemiplegia, in one. Four sequences of GluRα2 cDNA (an N‐terminal fragment, and three C‐terminal fragments) were inserted into bacterial fusion protein vectors (pGEX and pMAL), and the production of those bacterial fusion proteins was induced by isopropyl thiogalactose (IPTG). Three C‐terminal peptides existed after the fourth transmembrane region in the order of C1, C2, and C3. Western blots transferred bacterial fusion proteins composed of GluRα2 fragments to nitrocellulose membranes, and the membranes were incubated with the patients' serum or CSF and stained with IgG‐type second antibodies coupled to alkaline phosphatase. Results: In five of the six patients, IgG autoantibodies against GluRα2 in CSF had all four epitopes, and in a patient with mild disease, without mental retardation or motor dysfunction, IgG autoantibodies against GluR2 in CSF had all epitopes except C1. In the five patients with all epitopes in CSF, one patient had one serum epitope, two patients had two serum epitopes, and two patients had three serum epitopes. Their sera showed epitope N in two patients, epitope C1 in two patients, epitope C2 in five patients, and epitope C3 in two patients. The patient with mild disease and three epitopes in CSF had only one epitope, C2, in serum. Conclusions: The diversity of epitopes of IgG autoantibodies against GluRα2 was broader in the CSF than in the sera of all six patients. It is, therefore, suggested that the immunogenesis of IgG autoantibodies against GluRα2 occurs mainly in the central nervous system of patients with chronic progressive epilepsia partialis continua of childhood.

Are Anti–Oxidized Low-Density Lipoprotein Antibodies Pathogenic or Protective?

Despite a recent decline, atherosclerosis remains the most common cause of death in the Western world. The disease course of atherosclerosis is characterized by its chronicity, with progression in its initial stages being particularly insidious. Chronic inflammation is the pathological hallmark of atherosclerosis,1–4 and inflammatory processes are instrumental at all stages of this disease. Even before the development of detectable intimal lesions, the expression pattern of the endothelium has been shown to be inflammatory in nature, conforming to the response-to-injury hypothesis first postulated by the late Russell Ross.5 Thus, in lesion-prone sites of the arterial tree, the endothelial expression of adhesion molecules is upregulated, reflecting endothelial dysfunction secondary to unfavorable blood rheology6 and/or hypercholesterolemia.7–9 Atherosclerosis is known today to be associated with the burden of infection as well.10 The presence of infectious/inflammatory pathogeneses raises the autoimmune aspect of the condition.11–14 Anti–oxidized LDL (oxLDL) antibodies are exceptional among the autoantibodies prevalent in atherosclerosis: on the one hand, a correlation was found between the existence and titers of anti-oxLDL antibodies and the extent of atherosclerosis and cardiovascular disease (CVD) (Table 1). On the other hand, experimental data indicate that anti-oxLDL antibodies may be protective. View this table: TABLE 1. Correlation Between Titers of Anti-oxLDL Abs and Atherosclerosis and CVD oxLDL frequently presents in the sera of patients with autoimmune conditions, acute coronary syndrome, or stable coronary artery disease (CAD).15–18 OxLDL has been associated with both subclinical atherosclerosis and inflammatory variables.19 A large amount of oxLDL accumulates in atherosclerotic plaques,20–22 and the serum concentrations of circulating oxLDL may correlate with the severity of CAD23 and acute coronary syndrome.24 OxLDL seems to be an immunogenic molecule that stimulates the induction of anti-oxLDL antibodies. Thus, it is not surprising that an association was found between the presence …

Induction of autoantibodies to CCR5 in macaques and subsequent effects upon challenge with an R5-tropic simian/human immunodeficiency virus.

Antibodies against CCR5, the major coreceptor for human immunodeficiency virus type 1 (HIV-1), may have antiviral potential as viral fusion inhibitors. In this study, we generated a virus-like particle (VLP)-based vaccine that effectively breaks B-cell tolerance and elicits autoantibodies against CCR5 in pig-tailed macaques. Initial studies in mice identified a polypeptide comprising the N-terminal domain of pig-tailed macaque CCR5 fused to streptavidin that, when conjugated at high density to bovine papillomavirus major capsid protein L1 VLPs, induced high-titer immunoglobulin G (IgG) that bound to a macaque CCR5-expressing cell line in vitro. In macaques, CCR5 peptide-conjugated VLP preparations induced high-avidity anti-CCR5 IgG autoantibody responses, and all five immunized macaques generated IgG that could block infection of CCR5-tropic simian/human immunodeficiency virus SHIV(SF162P3) in vitro. Although the anti-CCR5 IgG titers declined with time, autoantibody levels were boosted upon revaccination. Vaccinated macaques remained healthy for a period of over 3 years after the initial immunization, and no decline in the number of CCR5-expressing T cells was detected. To test the prophylactic efficacy of CCR5 autoantibodies, immunized macaques were challenged with SHIV(SF162P3). Although the plasma-associated virus in half of six control macaques declined to undetectable levels, viral loads were lower, declined more rapidly, and eventually became undetectable in all five macaques in which CCR5 autoantibodies had been elicited. In addition, in the four vaccinated macaques with higher autoantibody titers, viral loads and time to control of viremia were significantly decreased relative to controls, indicating the possibility that CCR5 autoantibodies contributed to the control of viral replication.

Autoantibodies frequently detected in patients with aplastic anemia

AbstractAlthough accumulating evidence strongly suggests that aplastic anemia (AA) is a T cell-mediated autoimmune disease, no target antigens have yet been described for AA. In autoimmune diseases, target autoantigens frequently induce not only cellular T-cell responses but also humoral B-cell responses. We hypothesized that the presence of antigen-specific autoantibodies could be used as a “surrogate marker” for the identification of target T-cell autoantigens in AA patients. We screened a human fetal liver library for serologic reactivity against hematopoietic stem/progenitor cell antigens and isolated 32 genes. In 7 of 18 AA patients, an immunoglobulin G (IgG) antibody response was detected to one of the genes, kinectin, which is expressed in all hematopoietic cell lineages tested including CD34+cells. No response to kinectin was detected in healthy volunteers, multiply transfused non-AA patients, or patients with other autoimmune diseases. Epitope mapping of IgG autoantibodies against kinectin revealed that the responses to several of the epitopes were shared by different AA patients. Moreover, CD8+cytotoxic T cells raised against kinectin-derived peptides suppressed the colony formation of granulocyte macrophage colony-forming units (CFU-GMs) in an HLA class I-restricted fashion. These results suggest that kinectin may be a candidate autoantigen that is involved in the pathophysiology of AA. (Blood. 2003;102:4567-4575)

Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice.

Dilated cardiomyopathy is a severe pathology of the heart with poorly understood etiology. Disruption of the gene encoding the negative immunoregulatory receptor PD-1 in BALB/c mice, but not in BALB/c RAG-2-/- mice, caused dilated cardiomyopathy with severely impaired contraction and sudden death by congestive heart failure. Affected hearts showed diffuse deposition of immunoglobulin G (IgG) on the surface of cardiomyocytes. All of the affected PD-1-/- mice exhibited high-titer circulating IgG autoantibodies reactive to a 33-kilodalton protein expressed specifically on the surface of cardiomyocytes. These results indicate that PD-1 may be an important factor contributing to the prevention of autoimmune diseases.

Polyreactivity of disease-associated anti-RBC IgG autoantibodies of patients with warm autoimmune haemolytic anaemia and natural anti-RBC IgG autoantibodies of healthy individuals.

Anti-red blood cell (RBC) immunoglobulin G (IgG) autoantibodies are present in patients with warm autoimmune haemolytic anaemia (WAIHA), and, as natural autoantibodies, in healthy individuals. This study investigated whether the feature of polyreactivity discriminates disease-associated from natural anti-RBC IgG autoantibodies. The patterns of reactivity of purified anti-RBC IgG eluted from the RBC of WAIHA patients and from the RBC of healthy individuals were analysed using quantitative immunoblotting on a panel of whole human tissue or bacterial cell extracts as antigen sources. In parallel, the reactivity patterns of IgG purified from plasma were analysed. Anti-RBC IgG of WAIHA patients and of healthy individuals recognized a wide range of self- and nonself-antigens. The reactivity patterns of anti-RBC IgG were homogeneous among patients and controls, did not differ between patients and controls, and were similar to those of IgG purified from plasma in the case of both patients and healthy individuals. The data demonstrate that the anti-RBC IgG autoantibodies of WAIHA patients share extensive similarity with those of healthy individuals. Polyreactivity is a common feature of both disease-associated and natural anti-RBC IgG autoantibodies.

Effect of low-density lipoprotein (LDL) antigen source on an enzyme-linked immunosorbent assay for autoantibodies against oxidized LDL.

We examined the effect of antigen source on an enzyme-linked immunosorbent assay (ELISA) for autoantibodies against oxidized low-density lipoprotein (LDL). Serum samples from 20 subjects with systemic lupus erythematosus (SLE) and from 20 controls were assayed for immunoglobulin G (IgG) and immunoglobulin M (IgM) autoantibodies against oxidized LDL, using either a pooled or individual (n = 3) LDL preparation as antigen. For IgG autoantibodies against oxidized LDL there was a relationship (r approximately 0.5, P < 0.01) between data obtained using individual versus pooled antigen preparations. Bias plots demonstrated consistent inverse, concentration-dependent relationships (r approximately -0.6, P < 0.001). The difference in IgG autoantibodies against oxidized LDL levels between SLE patients and controls was underestimated (39-58%) when assays used individual rather than pooled LDL antigen. For IgM autoantibodies against oxidized LDL the direct relationships were stronger (r approximately 0.8, P < 0.001) and the concentration-dependent relationships weaker (r approximately -0.3, significance variable) than for IgG autoantibodies against oxidized LDL. Variations between LDL preparations suggested that a pooled antigen would give a more stable assay. Thus, LDL antigen source is important in assays for both IgG and IgM autoantibodies against oxidized LDL.

Beta2-microglobulin-deficient mice are resistant to bullous pemphigoid.

Recent understanding of the mechanism of immunoglobulin G (IgG) catabolism has yielded new insight into antibody-mediated diseases. We proposed that beta2-microglobulin (beta2m)-deficient mice have been protected from systemic lupus erythematosis (SLE)-like syndromes because they lack the beta2m-associated IgG protection receptor (FcRn) and therefore catabolize IgG, including pathogenic IgG autoantibodies, considerably more rapidly than normal mice. Such an hypothesis would predict that beta2m-deficient mice would also be resistant to experimental bullous pemphigoid, a disease with a pathogenesis thought to be much simpler than SLE, being the result of antibody directed toward a pathogenic epitope on the epidermal hemidesmosome that anchors basal keratinocytes to the basement membrane. To test this hypothesis, we administered pathogenic rabbit antibody directed toward the hemidesmosome to beta2m-deficient mice and to normal control mice, both intraperitoneally and intradermally, and assessed the mice clinically, histologically, and immunologically for manifestations of skin disease. We found that the beta2m-deficient mice were protected when the antibody was given intraperitoneally whereas intradermal administration resulted in blisters only slightly less severe than those seen in normal mice. These data would indicate that autoantibody-mediated inflammation might be prevented or controlled by appropriate modulation of FcRn function.

Effect of MHC Transgene Expression on Spontaneous Insulin Autoantibody Class Switch in Nonobese Diabetic Mice

A study of spontaneous anti-insulin autoantibodies in nonobese diabetic (NOD) mice revealed that when first detected, the antibodies are immunoglobulin M (IgM), but by age 10 weeks, immunoglobulin G (IgG) autoantibodies have appeared in many of these animals. When NOD strains, partially or completely protected from IDDM by the insertion of transgenes in the class II region, were compared, it was found that the switch to IgG autoantibodies was inhibited and the autoantibodies remained IgM indefinitely. We speculate that the switch to IgG may be a marker of events leading to IDDM in NOD mice and an indication that T-cell help has been generated for responses to beta-cell antigens. Such help not only directs the development of IgG autoantibodies, but more importantly, allows the emergence of potentially pathogenic T-cell clones that are capable of infiltrating the pancreas and mediating beta-cell damage.

Serial monitoring of clinical, haematological and immunological parameters in canine autoimmune haemolytic anaemia.

Clinical, haematological and immunological data are presented from 14 dogs with autoimmune haemolytic anaemia (AIHA) serially monitored for up to 945 days after initial presentation. At the time of diagnosis, all dogs had severe anaemia (mean packed cell volume [PCV] 17.6 +/- 7.1 per cent) with leucocytosis in seven cases and thrombocytopenia in four dogs. The Coombs' test was positive in all cases. Immunoglobulin G (IgG) autoantibody alone was identified in eight cases, a combination of IgG and IgM autoantibodies was recognised in three cases, and in two dogs only IgM autoantibody was recorded (complement fixing in one of these dogs). All dogs were treated with immunosuppressive doses of corticosteroids and some animals also received cyclophosphamide (four cases), azathioprine (two cases), blood transfusion (four cases) or underwent splenectomy (two cases). Two dogs died during the initial episode of AIHA. In 12 dogs, the anaemia was resolved by an average of 36.3 +/- 16.0 days after initial presentation, but autoantibody titre often persisted after clinical improvement and normalisation of PCV. Four dogs had a clinical relapse 67 to 170 days after initial presentation and one of these dogs subsequently died from thromboembolic disease. One dog developed lymphocytic thyroiditis and serum antinuclear antibody at day 691 after initial presentation, and two cases developed disease consistent with autoimmune thrombocytopenia (AITP) at 365 and 618 days post initial presentation. In one of these dogs, AITP was concurrent with multicentric lymphoma. No correlation was recorded between haematological and immunological parameters at presentation and subsequent response to therapy or long-term clinical behaviour.

Detection of Antiendometrial Antibodies in Sera of Patients With Endometriosis by Dual‐Colored, Double‐Labeling Immunohistochemical Method and Western Blot

This study was undertaken to determine whether specific binding activities against endometrial proteins in sera of patients with endometriosis are detectable and, if so, to identify endometrial antigens involved in autoimmunity in endometriosis. Sera from 33 patients with endometriosis and 20 cord sera (controls) were tested against endometria of patients and their protein extracts by dual-colored, double-labeling immunohistochemical method, and Western blotting. Antiendometrial binding activities were detected in sera of 2 (10.0%) control patients and 13 (48.2%) patients with endometriosis by the immunohistochemical method. Endogenous immunoglobulin G (IgG) binding to endometrial proteins had molecular weights (MW) of 26, 28, 54, 85, 107 and 116 kDa. Most sera of both control and patients showed reactivity against endometrial proteins with MW of 34, 36, 56 and 77 kDa. However, there were specific IgG autoantibodies reactive against the endometrial proteins of 71, 92, and 103 kDa in sera of 55.2% (16/29) of patients but not in the control sera. Over 80% (10/12) of patients' sera with binding activities detectable by the immunohistochemical method also tested positive by Western blot analysis. These data show that specific IgG antibodies reactive against endometrial antigens are detectable in sera from some patients with endometriosis.

Effects of human anti-IL-1α autoantibodies on receptor binding and biological activities of IL-1

Immunoglobulin G (IgG) antibodies to interleukin 1α (IL-1α) are frequently found in the sera of healthy human individuals. The effects of these autoantibodies on receptor binding and biological activities of human IL-1 were tested. Using the murine T-lymphocyte line NOB-1, human thyrocytes and human foreskin fibroblasts, the antibodies competitively inhibited the biological activity of human recombinant IL-1α (rIL-1α). The degree of inhibition correlated with 125I-rIL-1α binding to IgG in different immunoglobulin preparations and in individual sera. These antibodies also neutralized the IL-1 activity of isolated membrane fragments and lysates of human blood monocytes activated by lipopolysaccharide. In contrast, the supernatant IL-1 activity was not affected. Stronger inhibition of biological activity and cell binding of 125I-rIL-1α was obtained with NOB-1 cells than with human thyrocytes. The antibodies failed to interfere with the biological activity of rIL-1β. It is concluded that IgG autoantibodies to IL-1α in the sera of healthy humans selectively inhibit the biological activity of the soluble and membrane-associated forms of IL-1α in vitro, and that the degree of biological inhibition afforded by these antibodies depends upon the target cell.