Autoantibody-mediated Diseases Research Articles

Abrogation of Anti-HLA Antibodies via Proteasome Inhibition

Current treatments for autoantibody-mediated diseases (i.e., systemic lupus erythematosus) and alloantibodies (in transplant) are minimally effective. Although they deplete naïve B cells, plasmablasts, and transiently reduce antibody concentrations, they are minimally effective against long-lived, antibody-producing plasma cells. In transplantation, plasma cells produce antibodies directed against human leukocyte antigen (HLA) antigens causing poor allograft survival. We report the first clinical experience with a plasma cell depleting therapy, bortezomib, to abrogate anti-HLA antibodies in transplantation (outside of rejection) in an attempt to improve long-term allograft survival. Eleven patients with anti-HLA alloantibodies were treated with bortezomib. All patients underwent plasmapheresis to aid in removal of antibodies and to determine the effect of bortezomib. Serial measurements of anti-HLA antibody levels were conducted weekly by single antigen bead on Luminex platform. Bortezomib treatment elicited substantial reduction in both donor-specific antibody (DSA) and non-DSA levels. Antibodies were directed against DSA in 8 of 11 cases. Mean time to antibody appearance was 2 months posttransplant. Within 22 days (median) from treatment initiation, 9 of 11 patients' antibody levels dropped to less than 1000 mean fluorescence intensity. Of two patients without successful depletion, all had peak mean fluorescence intensity more than 10,000. At a mean follow-up of approximately 4 months posttreatment, all patients have stable graft function. Minimal transient side effects were noticed with bortezomib in the form of gastrointestinal toxicity, thrombocytopenia, and paresthesias. Bortezomib therapy effectively abrogates anti-HLA antibodies. Hence, removal of antibodies, by proteasome inhibition, represents a new treatment strategy for transplantation and may have benefit in autoimmune-related disease.

Insertion of a Targeting Peptide on Capsid Surface Loops of Human Papillomavirus Type-16 Virus-like Particles Mediate Elimination of Anti-dsDNA Abs-producing B Cells With High Efficiency

The purpose of this study was to design chimeric human papillomavirus type-16 L1 virus-like particles (VLPs) and to explore the potential capacity of elimination to anti-dsDNA antibody-producing B cells. To test it, VLPs were achieved by combination of human papillomavirus type-16 L1 proteins inserted into a targeting peptide (DWEYSVWLSN) and plasmids encoding diphtheria toxin A ligand. Additionally, VLPs were cocultured with target cells to assess the killing efficiency by lactate dehydrogenase assay in vitro. Lastly, lupus-prone (BWF1) mice vaccinated with VLPs were used as a model to assess the killing efficiency in vivo. The results showed that the VLPs were constructed successfully, and possessed the potential of killing anti-dsDNA antibody-producing B cells with high efficiency. The findings indicate the possibility that the VLPs ablate autoreactive B cells represents a novel strategy in the immunotherapy of autoantibody-mediated diseases.

Rituximab in the Treatment of Acquired Hemophilia a (AHA): A Novel Eradicating Therapy?

Background: Acquired inhibitors against factor VIII (FVIII), also termed Acquired Hemophilia A (AHA), occur rarely in the nonhemophilic population. Its incidence is approximately 1 to 4 per million/year. The incidence of AHA increases with age and a major peak is seeing in patients aged 68 to 80 years. These autoantibodies are associated with high rate of morbidity (90% of severe bleeding in affected patients) and mortality (8–22% of the cases). The diagnosis of AHA is based on the demonstration of an isolated prolongation of the activated partial thromboplastin time (APTT), not corrected by incubating the patient's plasma with equal volumes of normal plasma (mixing study), associated with FVIII reduced levels and formal evidence of a FVIII inhibitor in a patient with no previous personal or family history of bleeding. In approximately half of affected patients there is no identification of relevant concomitant diseases. The bleeding pattern in AHA is characterized by hemorrhages into the skin, muscles or soft tissues, and mucous membranes. Rituximab (Mabthera®; Roche; Switzerland) is a chimeric monoclonal antibody against the pan B-cell antigen CD20 that induces a rapid in vivo depletion of normal B lymphocytes. Primarily developed to treat B-cell non Hodgkin Lymphomas, more recently, Rituximab has demonstrated effectiveness in a number of autoantibody-mediated diseases including AHA. Case Report: We treated a 69-year-old diabetic and hypertensive patient with AHA. In March 2007, the diagnosis was made by a prolonged APTT (ratio 2.74 sec), not corrected by the mixing study, and FVIII inhibitor 320 Bethesda units. The patient had no evidence of malignancy, autoimmune disease or use of drugs. His bleeding manifestations were large ecchymosis of upper and lower extremities for almost a year, and at admission to the hospital, a hematoma of left calf and gengival bleeding. First line therapy consisted of steroids and cyclophosphamide (CFM). The steroids had to be withdraw after one week of use, because of very difficult glycemic control. The CFM were use in the dose of 50mg/day adding an total accumulative dose of 3g. This protocol were used considering the incapacity of the patient to tolerate higher dose of CFM due to leukopenia. After treatment, the patient was evaluated and neither normalization of APTT nor improvement of clinical findings happened. Therefore, considering all the above, it was decided to use Rituximab 375mg/m2, weekly, for four weeks. In June 2007, the patient received and tolereted very well all the infusions. In August 2007, he had no bleeding symptoms and his APTT was normal. At his laterest visit to the clinic in July 2008, an year after finishing his treatment, he was still very well and his bleeding tests were normal.Conclusion: Rituximab should be considered a treatment option for AHA.

Effectiveness and side effects of anti-CD20 therapy for autoantibody-mediated blistering skin diseases: A comprehensive survey of 71 consecutive patients from the Initial use to 2007

In order to examine the efficacy and side effects of the monoclonal antibody anti-CD20 (rituximab) on autoimmune blistering skin diseases, we performed a comprehensive survey of 71 consecutive patients from initial use up to 2007, using the PubMed database. A heterogeneous group of patients, including 51 patients with pemphigus vulgaris, one with pemphigus vegetans, nine with pemphigus foliaceus, five with paraneoplastic pemphigus, four with epidermolysis bullosa acquisita, and one with both bullous pemphigoid and graft vs host disease was included in this survey. Overall the monoclonal antibody seems to be effective in that 69% of patients showed complete response, 25% of patients showed partial response, whereas 6% of patients showed progressive disease. Six deaths occurred in association with the treatment, with four of these deaths in patients with paraneoplastic pemphigus, a disease characteristically resistant to conventional medication and with a high mortality rate. Of note, 11 patients who received combined rituximab and intravenous immune globulin treatments had the best outcome: complete response without any serious side effects. Therefore further investigation on rituximab with controlled clinical trial is a worthy pursuit.

The proteasome inhibitor bortezomib depletes plasma cells and protects mice with lupus-like disease from nephritis

Autoantibody-mediated diseases like myasthenia gravis, autoimmune hemolytic anemia and systemic lupus erythematosus represent a therapeutic challenge. In particular, long-lived plasma cells producing autoantibodies resist current therapeutic and experimental approaches. Recently, we showed that the sensitivity of myeloma cells toward proteasome inhibitors directly correlates with their immunoglobulin synthesis rates. Therefore, we hypothesized that normal plasma cells are also hypersensitive to proteasome inhibition owing to their extremely high amount of protein biosynthesis. Here we show that the proteasome inhibitor bortezomib, which is approved for the treatment of multiple myeloma, eliminates both short- and long-lived plasma cells by activation of the terminal unfolded protein response. Treatment with bortezomib depleted plasma cells producing antibodies to double-stranded DNA, eliminated autoantibody production, ameliorated glomerulonephritis and prolonged survival of two mouse strains with lupus-like disease, NZB/W F1 and MRL/lpr mice. Hence, the elimination of autoreactive plasma cells by proteasome inhibitors might represent a new treatment strategy for antibody-mediated diseases.

Intravenous immunoglobulin selectively decreases circulating autoantibodies in pemphigus

Autoantibody-mediated diseases such as pemphigus are caused by a single or very limited number of pathogenic autoantibodies. A major problem with all current therapies for these diseases is that they target all antibodies rather than selectively targeting only pathogenic antibodies. The following study was conducted to confirm observations made in a limited number of patients that suggest intravenous immunoglobulin (IVIg) may be able to selectively lower serum levels of only abnormal autoantibodies. The study was conducted in 12 patients who received IVIg for the treatment of recalcitrant pemphigus. Serum levels of antibodies to desmoglein 1 (Dsg 1) and desmoglein 3 (Dsg 3) were measured by enzyme-linked immunosorbent assay immediately before IVIg treatment and following a median of 2 cycles (range, 1-3) of treatment. As control, serum levels of several normal antibodies (against herpes simplex virus types 1 and 2, mumps, and varicella) were measured concurrently. Within a median of 2 weeks following the last cycle of IVIg serum, anti-Dsg 3 declined in all patients who tested positive at baseline and in 8 of 10 (80%) patients testing positive for anti-Dsg 1. On average, anti-Dsg 3 decreased by 45% and anti-Dsg 1 by 32%. By contrast, serum levels of the 4 normal antibodies increased in almost all patients, by an average of 408% (P < .001). Correlation of clinical response to treatment with IVIg was not performed. The sample size was limited. These results indicate that IVIg can selectively and markedly decrease serum levels of abnormal antibodies in pemphigus without decreasing the levels of normal antibodies. Thus IVIg appears able to achieve the ideal goal of treatment in autoantibody-mediated diseases--selectively removing from the circulation only those antibodies that cause the disease.

Monoclonal Antibodies: New Therapeutic Agents for Autoimmune Hemolytic Anemia?

Conventional treatment of autoimmune hemolytic anemia (AIHA) comprises corticosteroids and splenectomy and/or other immunosuppressive drugs for refractory/relapsed patients. Monoclonal antibodies (MoAbs) rituximab and alemtuzumab have gained widespread acceptance in the management of B-cell malignancies. More recently, they have been used to treat a number of autoantibody-mediated diseases, such as both idiopathic and secondary AIHA, with encouraging results. Herein we report an overview of the medical literature on the use of MoAbs to treat AIHA. The therapeutic mechanism of action of rituximab in the treatment of autoimmune diseases such as rheumatoid arthritis and lupus is currently a subject of considerable investigation. We have proposed that cell-associated IgG immune complexes, generated by the binding of rituximab to CD20 on B cells, may serve as decoys that attract FcgammaR-expressing effector cells and downregulate effector cell pathogenic action, thus reducing inflammation and tissue destruction in these diseases. We briefly review evidence that suggests that this immune complex decoy hypothesis plays a role in the therapeutic action of rituximab in AIHA, and we propose new measurements that should allow for a more complete evaluation of the importance of this mechanism in AIHA.

An antibody-based construct carrying DNA-mimotope and targeting CR1(CD35) selectively suppresses human autoreactive B-lymphocytes

There is an urgent and unmet need for therapeutic agents targeting selectively disease-associated B-lymphocytes in autoantibody-mediated diseases. We have constructed a chimeric molecule able to cross-link cell surface immunoglobulin with the inhibitory complement receptor type 1 (CD35) on DNA-specific B cells from SLE (systemic lupus erythematosus) patients with the aim of selectively silencing them. This engineered molecule is made of copies of the DNA-mimotope peptide DWEYSVWLSN coupled to a monoclonal anti-CD35 antibody. We found that the DNA-like peptide chimera induced a dose-dependent decrease in the number of IgG anti-dsDNA antibody producing cells when PBMCs of lupus patients were cultured in its presence. Our data present evidence that clustering BCR and the inhibitory CR1 on disease-associated autoreactive B-lymphocytes selectively suppresses autoantibody production.

Two‐Step Mechanism of Virus‐induced Autoimmune Hemolytic Anemia

Viruses are associated with the development of autoantibody-mediated blood autoimmune diseases. A two-step mechanism could explain virus involvement in the development of experimental hemolytic anemia. Immunization of normal mice with rat erythrocytes results in an autoantibody production that could be enhanced by viral infection, without erythrocyte destruction. Inoculation of the same virus when autoantibodies are at high levels triggers clinical anemia. This results from macrophage activation by gamma-interferon, leading to exacerbated erythrophagocytosis. Thus the development of anemia during the course of viral infection may require two independent stimuli, in which the first triggers autoantibody production and the second enhances the pathogenicity of these autoantibodies.

T Helper Type 2-Biased Natural Killer Cell Phenotype in Patients with Pemphigus Vulgaris

Pemphigus vulgaris (PV) is an autoantibody-mediated bullous disease, but the role of natural killer (NK) cells in its pathogenic process has never been examined in detail. Circulating CD56+ CD3- NK cells as well as CD69+-activated NK cells were increased in PV patients compared with healthy controls and patients with other autoantibody-mediated autoimmune diseases, including immune thrombocytopenic purpura and myasthenia gravis. Gene expression analysis of highly purified NK cells demonstrated an increased expression of IL-10 and decreased expression of IL-12Rbeta2, perforin, and granzyme B ex vivo in PV patients versus healthy controls. The NK cells from PV patients also showed impaired signal transducer and activator of transduction4 phosphorylation upon in vitro IL-12 stimulation. Moreover, NK cells from PV patients exhibited reduced IL-10 production in response to in vitro stimulation with IL-2/IL-12. Finally, IL-5 expression in NK cells was exclusively detected ex vivo in PV patients with active disease, and was lost in subsequent analyses performed during disease remission. Together these findings suggest that NK cells contribute to a T helper type 2-biased immune response in PV patients through impaired IL-12 signaling and an upregulation of IL-10 and IL-5.

Toll‐like receptor 9 signaling protects against murine lupus

Hypomethylated CpG-containing DNA, which is recognized by Toll-like receptor 9 (TLR-9), has been strongly implicated in the pathogenesis of autoantibody-mediated diseases such as systemic lupus erythematosus. This study was undertaken to determine the role of TLR-9 in the MRL/+ and MRL/lpr models of murine lupus. TLR-9-deficient MRL mice were generated by backcrossing a TLR-9-deficient allele against the MRL backgrounds by a speed congenic technique. Parameters of murine lupus were examined by routine methods. Regulatory T cell activity was assessed by autologous mixed lymphocyte reaction (AMLR), an in vitro assay for autoreactivity. Surprisingly, TLR-9-deficient animals of both the MRL/+ and the MRL/lpr backgrounds developed more severe lupus, as judged by anti-DNA and rheumatoid factor autoantibodies, total serum Ig isotypes, lymphadenopathy, inflammatory infiltrates in the salivary gland and kidney, proteinuria, and mortality, in comparison with their TLR-9-sufficient littermates. In vitro, regulatory T cells from TLR-9-deficient animals were impaired in their ability to suppress the AMLR. In the MRL model of murine lupus, TLR-9 signaling plays a protective role, perhaps by modulating the activity of regulatory T cells. These results contrast with findings of recent studies that implicate TLR-9 in the pathogenesis of anti-DNA responses, based in part on investigations in incompletely backcrossed TLR-9-deficient MRL/lpr mice in vivo or transgenic B cells in vitro. The present results highlight the need for caution in the assessment of disease paradigms based on the study of isolated cell populations in vitro, as well as in vivo studies of knockout animals involving non-ideal genetic models.

Complete FcRn dependence for intravenous Ig therapy in autoimmune skin blistering diseases

Numerous mechanisms of action have been proposed for intravenous Ig (IVIG). In this study, we used IgG passive transfer murine models of bullous pemphigoid (BP), pemphigus foliaceus (PF), and pemphigus vulgaris (PV) to test the hypothesis that the effect of IVIG in autoantibody-mediated cutaneous bullous diseases is to accelerate the degradation of pathogenic IgG by saturation of the MHC-like Fc receptor neonatal Fc receptor (FcRn). BP, PF, and PV are organ-specific antibody-mediated diseases in which autoantibodies target the hemidesmosomal antigen BP180 and desmosomal antigens Dsg1 and Dsg3, respectively. Antibodies against BP180, Dsg1, and Dsg3, when injected into neonatal mice, induce the BP, PF, and PV disease phenotypes, respectively. We found that FcRn-deficient mice were resistant to experimental BP, PF, and PV. Circulating levels of pathogenic IgG in FcRn-deficient mice were significantly reduced compared with those in WT mice. Administration of high-dose human IgG (HDIG) to WT mice also drastically reduced circulating pathogenic IgG levels and prevented blistering. In FcRn-deficient mice, no additional protective effect with HDIG was realized. These data demonstrate that the therapeutic efficacy of HDIG treatment in the pemphigus and pemphigoid models is dependent on FcRn. Thus, FcRn is a promising therapeutic target for treating such IgG-mediated autoimmune diseases.

Prevention and control of reciprocal T–B cell diversification: implications for lupus-like autoimmunity

Autoimmunity is fundamentally a continuously evolving process. The autoimmune responses shift, drift and diversify with time not only to other epitopes in the original antigen but also to other related and sometimes to unrelated antigens. We have described a form of immune diversification—reciprocal T–B epitope spreading—where the activation of first T cells by epitopes from an autoantibody molecule could lead to help provided to a variety of B cells displaying a cross-reactive version of the original epitope. The response spreads in this way until large cohorts of T and B cells have expanded in lupus-prone mice. Such reciprocal T–B cell response can also be induced in normal animals, its extent is limited by the emergence of inhibitory T cells. The induction of such inhibitory T cells is generally impaired in lupus mice. The delivery of T cell epitopes via plasmid DNA vectors, however, can overcome this impairment in lupus mice. The inhibitory T cells thus induced can suppress autoantibody production and lupus disease by ablating or inhibiting autoreactive B cells. Thus, T–B diversification that develops spontaneously in lupus mice could be curtailed in normal animals by inhibitory T cells that emerge whenever there is an impending ‘danger’ of pathologic autoimmunity. We have successfully exploited this regulatory potential of the normal immune response to inhibit clinical autoimmunity. Understanding the mechanisms of autoimmune diversification in lupus mice and of its down-regulation in normal animals may pave the way for developing novel treatments for autoantibody-mediated diseases such as lupus.

B cell receptor-independent stimuli trigger immunoglobulin (Ig) class switch recombination and production of IgG autoantibodies by anergic self-reactive B cells.

In both humans and animals, immunoglobulin (Ig)G autoantibodies are less frequent but more pathogenic than IgM autoantibodies, suggesting that controls over Ig isotype switching are required to reinforce B cell self-tolerance. We have used gene targeting to produce mice in which hen egg lysozyme (HEL)-specific B cells can switch to all Ig isotypes (SWHEL mice). When crossed with soluble HEL transgenic (Tg) mice, self-reactive SWHEL B cells became anergic. However, in contrast to anergic B cells from the original nonswitching anti-HEL × soluble HEL double Tg model, self-reactive SWHEL B cells also displayed an immature phenotype, reduced lifespan, and exclusion from the splenic follicle. These differences were not related to their ability to Ig class switch, but instead to competition with non-HEL–binding B cells generated by VH gene replacement in SWHEL mice. When activated in vitro with B cell receptor (BCR)-independent stimuli such as anti-CD40 monoclonal antibody plus interleukin 4 or lipopolysaccharide (LPS), anergic SWHEL double Tg B cells proliferated and produced IgG anti-HEL antibodies as efficiently as naive HEL-binding B cells from SWHEL Ig Tg mice. These results demonstrate that no intrinsic constraints to isotype switching exist in anergic self-reactive B cells. Instead, production of IgG autoantibodies is prevented by separate controls that reduce the likelihood of anergic B cells encountering BCR-independent stimuli. That bacteria-derived LPS could circumvent these controls may explain the well-known association between autoantibody-mediated diseases and episodes of systemic infection.

11. Allergic and immunologic diseases of the skin

Many skin diseases have an inflammatory or immune component, and anti-inflammatory drugs comprise a major portion of a dermatologist's therapeutic armamentarium. Although causes of most of these diseases remain obscure, mechanisms of lesion formation and explanations for symptoms are increasingly well documented. These developments, coupled with the expected availability of novel selective immunomodulatory agents, herald a new era for immunodermatology. Patients with psoriasis, allergic contact dermatitis, atopic dermatitis, urticaria, and autoantibody-mediated blistering diseases are among those who are likely to benefit from advances in the understanding of disease pathogenesis and the emergence of immunotherapeutics. (J Allergy Clin Immunol 2003;111:S560-70.)

Vaccination with Minigenes Encoding VH-derived Major Histocompatibility Complex Class I–binding Epitopes Activates Cytotoxic T Cells that Ablate Autoantibody-producing B Cells and Inhibit Lupus

Current treatments for autoantibody-mediated diseases, such as lupus, can cause nonspecific immune suppression. In this paper, we used a bioinformatic approach to identify major histocompatibility complex class I–binding epitopes in the heavy chain variable region of anti-DNA antibodies from lupus-prone (NZB/NZW F1) mice. Vaccination of such mice with plasmid DNA vectors encoding these epitopes induced CD8+ T cells that killed anti-DNA antibody-producing B cells, reduced serum anti-DNA antibody levels, retarded the development of nephritis, and improved survival. Vaccine-mediated induction of anti-VH cytotoxic T lymphocytes that ablate autoreactive B cells represents a novel approach to treat autoantibody-mediated diseases.

Characterization of autoantibodies from patients with Goodpasture's disease using a resonant mirror biosensor.

Goodpasture's disease is characterized by the binding of IgG autoantibodies to the glomerular basement membrane, leading to glomerular inflammation. The autoantigen has been identified as the noncollagenous domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1). We have used the IAsys resonant mirror biosensor to analyse the extent and affinity of binding of anti-GBM antibodies from sera of patients to purified alpha3(IV) NC1. alpha3(IV) NC1 monomers were immobilized to a carboxylate cuvette, with the simultaneous use of a control well. The binding of serum from patients with Goodpasture's disease (n = 12), normal controls (n = 14) and disease controls with vasculitis (n = 14) was analysed. Antibody binding was detected in sera from all patients with Goodpasture's disease but not from controls. IAsys measurements of binding correlated with antibody levels assessed by the standardized ELISA used for clinical assays. Both ELISA and biosensor measurements showed declining antibody levels in serial serum samples from treated patients; however, the biosensor detected antibody recrudescence when ELISA remained negative. Autoantibodies from patients' serum had average affinity constants (Kd) of 6.5 x 10-11M to 52.07 x 10-10M, as determined by an inhibition assay, indicating high affinity. Sips analysis showed that the antibody response was relatively homogeneous (values of 0.46-1). Biosensor techniques can therefore be used to detect and characterize anti-GBM antibodies in serum from patients, with high sensitivity and without need for antibody purification. This technique may be useful in diagnosis and monitoring of patients with Goodpasture's disease, and may be applicable to other autoantibody mediated diseases.

Maternal Immunologic Diseases and Neonatal Disorders

After completing this article, readers should be able to: 1. Explain why only certain maternal autoimmune diseases result in perinatal and neonatal disorders. 2. Delineate the criteria for diagnosis, the perinatal consequences, and the approaches to treatment of maternal antiphospholipid syndrome. 3. Differentiate between benign gestational thrombocytopenia and maternal immune thrombocytopenia. 4. Outline management considerations for the mother and baby in maternal immune thrombocytopenia. 5. Describe the clinical manifestations, pathophysiology, and guidelines for treatment of the neonatal lupus syndrome. Autoimmune diseases are characterized by an immune attack on tissues of the body in the apparent absence of active infection. Symptoms arise from the resulting impairment of cell or organ function. Women are disproportionately affected by many autoimmune diseases, especially those conditions whose typical age of onset is in the early childbearing years. (1) For women who have autoimmune diseases, the physiologic changes of pregnancy often have consequences for the course of the autoimmune disease. (2) In turn, autoimmune disease or its treatment may affect the developing fetus or the newborn. Autoimmune diseases can be classified into two broad categories, based on the primary immunologic effector mechanism that causes disease. In one class of autoimmune disease, CD4+ T cells are believed to be the major effector cells, although they enlist other arms of the immune response, such as CD8+ T cells or macrophages, during the disease course. Examples of this class of autoimmune disease include insulin-dependent diabetes mellitus, rheumatoid arthritis, and multiple sclerosis. The other class of autoimmune diseases is caused by antibodies that are directed against self-antigens. (It should be noted that production of these antibodies is generally T-cell-dependent). Diseases in this class include myasthenia gravis, Goodpasture syndrome, Graves’ disease, antiphospholipid antibody syndrome (APS), immune thrombocytopenic purpura (ITP), and systemic lupus erythematosus (SLE). The autoantibody-mediated diseases can have direct consequences on the fetus …

Granzyme B proteolysis of a neuronal glutamate receptor generates an autoantigen and is modulated by glycosylation.

Autoimmune processes are initiated when tolerance to self-proteins fails to be established or maintained and immune cells are stimulated by self-Ags. Although intracellular autoantigens are common, the origin of extracellular autoantigens is poorly defined and possibly more dangerous. In this study, we considered a mechanism for the origin of an extracellular autoantigen from the neuronal glutamate receptor subunit 3 (GluR3) in Rasmussen's encephalitis, a severe form of pediatric epilepsy. We demonstrate that specific cleavage of GluR3 by granzyme B (GB), a serine protease released by activated immune cells, can generate the GluR3B autoantigenic peptide, but only if an internal N:-linked glycosylation sequon within the GluR3-GB recognition sequence (ISND*S) is not glycosylated. However, this N:-glycon sequon while glycosylated normally is inefficiently used and glycosylation can fail. These results suggest that GB/N:-glycon sites may escape normal tolerance mechanisms and contribute to autoantibody-mediated immune diseases.

Chemically modified ribozyme to V gene inhibits anti-DNA production and the formation of immune deposits caused by lupus lymphocytes.

A variety of autoantibodies is responsible for the tissue injury in autoimmune diseases. We have demonstrated that the human anti-DNA Ab O-81, of which Ids are commonly detected in renal glomeruli of active lupus nephritis, uses the V3-7 gene. We tried to develop a new therapy for lupus nephritis by using chemically modified ribozymes to specifically inhibit the expression of the mRNA of Ig V gene. The transfection of hammerhead ribozyme or the addition of chemically modified ribozyme against the flanking region of V3-7 caused a potent and selective inhibition of anti-DNA production in V3-7-using B cell clones, but not in irrelevant V gene-using clones in vitro. Chemically modified ribozyme was long-acting and resistant to RNase, and nonspecific cytotoxicity of the ribozyme was negligible. To know the efficacy of the ribozyme in vivo, we used a model of immune complex nephritis in SCID mice in which 5 x 10(6) PBLs from patients with active lupus nephritis (lupus PBL) were transferred twice. The injection of lupus PBL in combination with chemically modified ribozyme to increase resistance to RNase significantly reduced anti-DNA Ab levels in blood and decreased levels of urinary protein in the immune deposit models. Immunofluorescence study also revealed a marked decrease in IgG deposits at renal glomeruli in the ribozyme-treated group. These results indicate an efficacy of chemically modified ribozyme therapy for autoantibody-mediated immune diseases.