Brain-reactive antibodies and disease.

Autoimmune diseases currently affect 5-7% of the world's population; in most diseases there are circulating autoantibodies. Brain-reactive antibodies are present in approximately 2-3% of the general population but do not usually contribute to brain pathology. These antibodies penetrate brain tissue only early in development or under pathologic conditions. This restriction on their pathogenicity and the lack of correlation between serum titers and brain pathology have, no doubt, contributed to a delayed appreciation of the contribution of autoantibodies in diseases of the central nervous system. Nonetheless, it is increasingly clear that antibodies can cause damage in the brain and likely initiate or aggravate multiple neurologic conditions; brain-reactive antibodies contribute to symptomatology in autoimmune disease, infectious disease, and malignancy.

Caspr2-reactive antibody cloned from a mother of an ASD child mediates an ASD-like phenotype in mice.

Autism spectrum disorder (ASD) occurs in 1 in 68 births, preferentially affecting males. It encompasses a group of neurodevelopmental abnormalities characterized by impaired social interaction and communication, stereotypic behaviors and motor dysfunction. Although recent advances implicate maternal brain-reactive antibodies in a causative role in ASD, a definitive assessment of their pathogenic potential requires cloning of such antibodies. Here, we describe the isolation and characterization of monoclonal brain-reactive antibodies from blood of women with brain-reactive serology and a child with ASD. We further demonstrate that male but not female mice exposed in utero to the C6 monoclonal antibody, binding to contactin-associated protein-like 2 (Caspr2), display abnormal cortical development, decreased dendritic complexity of excitatory neurons and reduced numbers of inhibitory neurons in the hippocampus, as well as impairments in sociability, flexible learning and repetitive behavior. Anti-Caspr2 antibodies are frequent in women with brain-reactive serology and a child with ASD. Together these studies provide a methodology for obtaining monclonal brain-reactive antibodies from blood B cells, demonstrate that ASD can result from in utero exposure to maternal brain-reactive antibodies of single specificity and point toward the exciting possibility of prognostic and protective strategies.

A role for plasma cell targeting agents in immune tolerance induction in autoimmune disease and antibody responses to therapeutic proteins.

Antibody responses to life saving therapeutic protein products, such as enzyme replacement therapies (ERT) in the setting of lysosomal storage diseases, have nullified product efficacy and caused clinical deterioration and death despite treatment with immune-suppressive therapies. Moreover, in some autoimmune diseases, pathology is mediated by a robust antibody response to endogenous proteins such as is the case in pulmonary alveolar proteinosis, mediated by antibodies to Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF). In this work, we make the case that in such settings, when the antibody response is high titered, sustained, and refractory to immune suppressive treatments, the antibody response is mediated by long-lived plasma cells which are relatively unperturbed by immune suppressants including rituximab. However, long-lived plasma cells can be targeted by proteasome inhibitors such as bortezomib. Recent reports of successful reversal of antibody responses with bortezomib in the settings of ERT and Thrombotic Thrombocytopenic Purpura (TTP) argue that the safety and efficacy of such plasma cell targeting agents should be evaluated in larger scale clinical trials to delineate the risks and benefits of such therapies in the settings of antibody-mediated adverse effects to therapeutic proteins and autoantibody mediated pathology.

Brain-reactive IgG correlates with autoimmunity in mothers of a child with an autism spectrum disorder.

It is believed that in utero environmental factors contribute to autism spectrum disorder (ASD). The goal of this study was to demonstrate, using the largest cohort reported so far, that mothers of an ASD child have an elevated frequency of anti-brain antibodies and to assess whether brain reactivity is associated with an autoimmune diathesis of the mother. We screened plasma of 2431 mothers of an ASD child from Simon Simplex Collection and plasma of 653 unselected women of child-bearing age for anti-brain antibodies using immunohistology on mouse brain. Positive and negative plasma from mothers with an ASD child were analyzed for anti-nuclear antibodies and for autoimmune disorders. Mothers of an ASD child were four times more likely to harbor anti-brain antibodies than unselected women of child-bearing age (10.5 vs 2.6%). A second cohort from The Autism Genetic Resource Exchange with multiplex families displayed an 8.8% prevalence of anti-brain antibodies in the mothers of these families. Fifty-three percent of these mothers with anti-brain antibodies also exhibited anti-nuclear autoantibodies compared with 13.4% of mothers of an ASD child without anti-brain antibodies and 15% of control women of child-bearing age. The analysis of ASD mothers with brain-reactive antibodies also revealed an increased prevalence of autoimmune diseases, especially rheumatoid arthritis and systemic lupus erythematosus. This study provides robust evidence that brain-reactive antibodies are increased in mothers of an ASD child and may be associated with autoimmunity. The current study serves as a benchmark and justification for studying the potential pathogenicity of these antibodies on the developing brain. The detailed characterization of the specificity of these antibodies will provide practical benefits for the management and prevention of this disorder.

HMGB1 mediates splenomegaly and expansion of splenic CD11b+ Ly-6C(high) inflammatory monocytes in murine sepsis survivors.

BACKGROUND: More than 500,000 hospitalized patients survive severe sepsis annually in the USA. Recent epidemiological evidence, however, demonstrated that these survivors have significant morbidity and mortality, with 3-year fatality rates higher than 70%. To investigate the mechanisms underlying persistent functional impairment in sepsis survivors, here we developed a model to study severe sepsis survivors following cecal ligation and puncture (CLP). METHODS: Sepsis was induced in mice by CLP and survivors were followed for twelve weeks. Spleen and blood were collected and analyzed at different time points post-sepsis. RESULTS: We observed that sepsis survivors developed significant splenomegaly. Analysis of the splenic cellular compartments revealed a major expansion of the inflammatory CD11b+ Ly-6CHigh pool. Serum high-mobility group box 1 (HMGB1) levels in the sepsis surviving mice were significantly elevated for 4-6 weeks after post-sepsis, and administration of an anti-HMGB1 monoclonal antibody significantly attenuated splenomegaly as well as splenocyte priming. Administration of recombinant HMGB1 to naive mice induced similar splenomegaly, leukocytosis and splenocyte priming as observed in sepsis survivors. Interestingly analysis of circulating HMGB1 from sepsis survivors by mass spectroscopy demonstrated a stepwise increase of reduced form of HMGB1 (with known chemo-attractant properties) during the first 3 weeks, followed by disulphide form (with known inflammatory properties) 4-8 weeks after CLP. DISCUSSION: Our results indicate that prolonged elevation of HMGB1 is a necessary and sufficient mediator of splenomegaly and splenocyte expansion, as well as splenocyte inflammatory priming in murine severe sepsis survivors.

Living with coeliac disease: survey results from the U.S.A.

BACKGROUND: The only treatment for coeliac disease is lifelong adherence to a rigorous gluten-free diet. The present study aimed to evaluate the influence of coeliac disease on the social aspects of daily life of individuals in the U.S.A. METHODS: The present study used a self-administered survey including the standard Quality of Life questionnaire (12-item short-form) with validated disease-specific questions. sas statistical software 2010 (SAS Institute, Cary, NC, U.S.A.) was used to calculate the mean (SD). RESULTS: Individuals with coeliac disease overall had a low positive health perception. Validated diet and disease-specific questions revealed a significant negative impact on quality of life in social settings. Specifically, the areas of travel, dining out and family life are most affected. The negative impact of diet significantly decreased over time, although it did not resolve for the domains of dining out of the home and travel. Those diagnosed in childhood and maintained on the diet had less of an impact on the quality of life as an adult. CONCLUSIONS: Individuals with coeliac disease in the U.S.A. have a diminished quality of life, especially in the social aspects of life.

Moving towards a cure: blocking pathogenic antibodies in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterized by the presence of autoantibodies that can mediate tissue damage in multiple organs. The underlying aetiology of SLE autoantibodies remains unknown, and treatments aimed at eliminating B cells, or limiting their function, have demonstrated limited therapeutic benefit. Thus, the current therapies for SLE are based on the concept of nonspecific immunosuppression and consist of nonsteroidal anti-inflammatory drugs (NSAIDS), corticosteroids, anti-malarials and cytotoxic drugs, all of which have serious adverse side effects including organ damage. The major auto-specificity in SLE is double-stranded (ds) DNA. Many anti-dsDNA antibodies cross-react with non-DNA antigens that may be the direct targets for their pathogenic activity. Studying anti-dsDNA antibodies present in SLE patients and in animal models of lupus, we have identified a subset of anti-dsDNA antibodies which is pathogenic in the brain as well as in the kidney. We have recently demonstrated that specific peptides, or small molecules, can protect target organs from antibody-mediated damage. Thus, it might be possible to treat the aspects of autoimmune disease without inducing major immunosuppression and ensuing infectious complications.

A subset of lupus anti-DNA antibodies cross-reacts with the NR2 glutamate receptor in systemic lupus erythematosus.

In systemic lupus erythematosus, antibodies against double-stranded DNA are a major contributor to renal disease. We have previously demonstrated that the pentapeptide Asp/Glu-Trp-Asp/Glu-Tyr-Ser/Gly is a molecular mimic of double-stranded DNA. This sequence is also present in the extracellular domain of murine and human NMDA (N-methyl-D-aspartate) receptor subunits NR2a and NR2b. Here we show that the NR2 receptor is recognized by both murine and human anti-DNA antibodies. Moreover, anti-DNA antibodies with this cross-reactivity mediate apoptotic death of neurons in vivo and in vitro. Finally, we show that the cerebrospinal fluid of a patient with systemic lupus erythematosus contains these antibodies and also mediates neuronal death via an apoptotic pathway. These observations indicate that lupus antibodies cross-react with DNA and NMDA receptors, gain access to cerebrospinal fluid and may mediate non-thrombotic and non-vasculitic abnormalities of the central nervous system.

A new Fc receptor on mouse macrophages binding IgG3.

Monoclonal antibodies to sheep erythrocytes (SRBC) have proved useful in identifying two Fc receptors on mouse macrophages, one for IgG2a, and one for IgG1 and IgG2b. We have used monoclonal IgG3 anti-SRBC to identify a third Fc receptor on mouse macrophages which binds IgG3 uniquely. This receptor is present on primary resident and thioglycolate-induced peritoneal macrophages and on some macrophage cell lines. The binding of IgG3-coated SRBC is inhibited by aggregated byt not monomeric IgG3, and not by IgG1, IgG2a, and IgG2b aggregates. It is unaffected by treating the macrophages with trypsin or cytochalasin B and occurs at both 4 degrees and 37 degrees C. IgG3, like all other IgG subclasses, mediates phagocytosis. We have also generated a variant macrophage line which bears the receptors for IgG1 and IgG2b and for IgG2a, but not for IgG3.

Immunization with a peptide surrogate for double-stranded DNA (dsDNA) induces autoantibody production and renal immunoglobulin deposition.

Anti-double-stranded DNA (dsDNA) antibodies are the serologic abnormality characteristically associated with systemic lupus erythematosus (SLE) and may play an important role in disease pathogenesis. Although the anti-dsDNA antibodies present in SLE are indicative of an antigen-driven response, the antigen has not been conclusively identified. By screening a phage peptide display library, we demonstrated previously that the decapeptide DWEYSVWLSN is specifically bound by the pathogenic murine IgG2b anti-dsDNA antibody R4A. To investigate the possibility that a protein antigen might trigger lupus-like autoimmunity, we immunized BALB/c mice with DWEYSVWLSN in adjuvant. Mice developed significant titers of IgG anti-dsDNA antibodies 2-3 wk after the initial immunization. Immunized mice also developed antibodies against some other lupus autoantigens, and immunoglobulin deposition was present in renal glomeruli at 49 d. Although an immune response to peptide and dsDNA was evident in BALB/c mice, there was little response in other inbred strains. This study demonstrates that lupus-like anti-dsDNA reactivity can be generated in nonautoimmune mice by immunization with a peptide antigen. Peptide-induced autoimmunity may prove useful in understanding the spreading of antigenic specificities targeted in SLE. However, most importantly, the demonstration that a peptide antigen can initiate a SLE-like immune response opens a new chapter on the potential antigenic stimuli that might trigger SLE.

Mutational analysis of an autoantibody: differential binding and pathogenicity.

We have used site-directed mutagenesis to change amino acid residues in the heavy chain of the pathogenic R4A anti-double-stranded DNA (dsDNA) antibody and have looked for resultant alterations in DNA binding and in pathogenicity. The data demonstrate that single amino acid substitutions in both complementarity determining and framework regions alter antigen binding. Changes in only a few amino acids entirely ablate DNA specificity or cause a 10-fold increase in relative binding. In vivo studies in mice of the pathogenicity of the mutated antibodies show that a single amino acid substitution leading to a loss of dsDNA binding leads also to a loss of glomerular sequestration. Amino acid substitutions that increase relative affinity for dsDNA cause a change in localization of immunoglobulin deposition from glomeruli to renal tubules. These studies demonstrate that small numbers of amino acid substitutions can dramatically alter antigen binding and pathogenicity, and that the pathogenicity of anti-DNA antibodies does not strictly correlate with affinity for DNA.

Site of binding of mouse IgG2b to the Fc receptor on mouse macrophages.

Three mouse immunoglobulins with altered heavy chains have been used to study the specificity of the mouse IgG2b Fc receptor on mouse macrophages. These immunoglobulins were synthesized by variant clones derived from the MPC 11, IgG2b-producing mouse myeloma cell line. One variant, whose Fc receptor. A second variant, which makes a short heavy chain lacking the CH3 domain, binds specifically to the IgG2b Fc receptor. The third variant makes a hybrid IgG2b-IgG2a heavy chain whose CH3 domain is enterely IgG2a-like and binds to both IgG2a and IgG2b Fc receptors. These data suggest that the binding of mouse IgG2b immunoglobulins to the mouse macrophage Fc receptor involves a site within the CH2 domain and indicate that immunoglobulins with altered heavy chains are a useful tool to probe Fc receptors.

A novel class of anti-DNA antibodies identified in BALB/c mice.

We have characterized four IgG monoclonal antibodies (mAbs) derived from BALB/c mice that bind double-stranded DNA (dsDNA) with high affinity. The hydridomas were selected for expression of a member of the VHS107 family. Three of the four cell lines use the VH11 gene and one uses the VH1 gene. These antibodies exhibit many characteristics of pathogenic anti-DNA antibodies. They are high affinity and not broadly crossreactive. Unlike the anti-DNA antibodies in autoimmune mice, they exhibit no somatic mutation in their VH genes. These results demonstrate that somatic mutation of VHS107 genes is not necessary for generating high affinity dsDNA binding. The fact that such antibodies have not previously been reported suggests that they are rare and that their expression may be downregulated in both nonautoimmune and autoimmune individuals.

Molecular characteristics of antibodies bearing an anti-DNA-associated idiotype.

Anti-double-stranded DNA antibodies are the hallmark of the disease systemic lupus erythematosus and are believed to contribute to pathogenesis. While a large number of anti-DNA antibodies from mice with lupus-like syndromes have been characterized and their variable region genes sequenced, few human anti-DNA antibodies have been reported. We describe here the variable region gene sequences of eight antibodies produced by Epstein-Barr virus (EBV)-transformed B cells that bear the 3I idiotype, an idiotype expressed on anti-DNA antibodies and present in high titer in patients with systemic lupus. The comparison of these antibodies to the light chains of 3I+ myeloma proteins and serum antibodies reveals that EBV transformation yields B cells producing antibodies representative of the expressed antibody repertoire. The analysis of nucleotide and amino acid sequences of these antibodies suggests the first complementarity determining region of the light chain may be important in DNA binding and that paradigms previously generated to account for DNA binding require modification. The understanding of the molecular genetics of the anti-DNA response requires a more complete description of the immunoglobulin germ line repertoire, but data reported here suggest that somatic diversification is a characteristic of the anti-DNA response.

Antibody-mediated autoimmune myocarditis depends on genetically determined target organ sensitivity.

Injury to cardiac myocytes often leads to the production of anti-myosin antibodies. While these antibodies are a marker of myocardial injury, their contribution to pathogenesis in diseases such as autoimmune myocarditis or rheumatic fever is much less clear. We demonstrate in this report that monoclonal anti-myosin antibodies can mediate myocarditis in a susceptible mouse strain. Additionally, we show disease susceptibility depends on the presence of myosin or a myosin-like molecule in cardiac extracellular matrix. This study demonstrates that susceptibility to autoimmune heart disease depends not only on the activation of self-reactive lymphocytes but also on genetically determined target organ sensitivity to autoantibodies.

Light chain usage in anti-double-stranded DNA B cell subsets: role in cell fate determination.

Two major mechanisms for the regulation of autoreactive B cells that arise in the bone marrow are functional silencing (anergy) and deletion. Studies to date suggest that low avidity interactions between B cells and autoantigen lead to B cell silencing, whereas high avidity interactions lead to deletion. Anti-double stranded (ds) DNA antibodies represent a pathogenic autospecificity in Systemic Lupus Erythematosus (SLE). An understanding of their regulation is critical to an understanding of SLE. We now demonstrate in a transgenic model in which mice express the heavy chain of a potentially pathogenic anti-DNA antibody that antibody affinity for dsDNA does not alone determine the fate of anti-dsDNA B cells. B cells making antibodies with similar affinities for dsDNA are regulated differently, depending on light chain usage. A major implication of this observation is that dsDNA may not be the self antigen responsible for cell fate determinations of anti-dsDNA B cells. Light chain usage may determine antigenic cross-reactivity, and cross-reactive antigens may regulate B cells that also bind dsDNA.

Alterations in Blood-Brain Barrier Permeability in Patients with Systemic Lupus Erythematosus.

BACKGROUND AND PURPOSE: Neuropsychiatric systemic lupus erythematosus refers to central and peripheral nervous system involvement, which may occur secondary to antineuronal antibodies crossing the blood-brain barrier that preferentially target cells in the hippocampus leading to abnormal hypermetabolism and atrophy. Thus, we hypothesized that alterations in BBB permeability, detected on dynamic contrast-enhanced MR imaging, occur in the hippocampus in patients with systemic lupus erythematosus before development of neuropsychiatric systemic lupus erythematosus. MATERIALS AND METHODS: Six patients with systemic lupus erythematosus without neuropsychiatric systemic lupus erythematosus and 5 healthy controls underwent dynamic contrast-enhanced MR imaging with postprocessing into BBB permeability parameters (K trans and Ve) and CBF. Standardized methods selected ROI sampling of the abnormal brain regions detected on FDG-PET. The mean and SD of K trans, Ve, and CBF were calculated. Linear regression and nonparametric Spearman rank correlation analyses of K trans and Ve with CBF were performed. Dynamic contrast-enhanced curves and the area under the curve were generated for each brain region. Student t test comparisons were performed. RESULTS: Quantitative data revealed that patients with systemic lupus erythematosus have statistically increased K trans (P < .001) and Ve (P < .001) compared with controls. In patients with systemic lupus erythematosus, statistically significant positive correlations were seen between K trans (P < .001) and Ve (P < .001) with CBF. Furthermore, the mean area under the curve revealed statistically increased BBB permeability in the hippocampus (P = .02) compared with other brain regions in patients with systemic lupus erythematosus compared with controls. CONCLUSIONS: These initial findings are proof-of-concept to support the hypothesis that patients with systemic lupus erythematosus have increased BBB permeability, specifically in the hippocampus, compared with other brain regions. These findings may advance our understanding of the underlying pathophysiology affecting the brain in autoimmune diseases.

Dynamic Contrast-Enhanced MRI Reveals Unique Blood-Brain Barrier Permeability Characteristics in the Hippocampus in the Normal Brain.

We report a prospective dynamic contrast-enhanced MR imaging analysis of region-specific blood-brain barrier permeability in 5 healthy subjects. By means of standardized postprocessing and ROI sampling methods, the hippocampi revealed significantly elevated area under the dynamic contrast-enhanced curve and significantly increased blood-brain barrier permeability metrics (volume transfer constant and volume in the extravascular extracellular space) from model-based quantitation. These findings suggest unique blood-brain barrier permeability characteristics in the hippocampus, which are concordant with previous animal studies, potentially laying the groundwork for future studies assessing patient populations in which hippocampal pathology plays a role.

Suppression of anti-DNA antibody synthesis in vitro by a cross-reactive antiidiotypic antibody.

An understanding of the regulatory mechanisms that govern the humoral immune response will be facilitated by the availability of techniques for the measurement of specific antibody production in vitro. We have developed an enzyme-linked immunosorbent assay system for the measurement of in vitro anti-DNA antibody production by peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus. Using this technique, the PBMC from 74% of serologically active SLE patients produced levels of anti-DNA antibodies that were greater than 2 SD above the mean of 18 +/- 9 IU/ml for normal subjects. Furthermore, the addition of 3I, a monoclonal anti-idiotypic antibody that recognizes a cross-reactive determinant on anti-DNA antibodies, was shown to specifically inhibit anti-DNA production in vitro.

Molecular analysis of the human immunoglobulin V lambda II gene family.

The 8.12 idiotype characterizes a subpopulation of anti-DNA antibodies in patients with systemic lupus erythematosus (SLE). The idiotype is present on lambda light chains and has previously been shown to be exclusively encoded by V lambda II light chains. RFLP analysis of the V lambda II gene family has shown the family to consist of 10 to 15 members. Thus far, the sequences of seven V lambda II germline genes are reported in the literature with one of these a pseudogene. To identify the V lambda II genes that encode 8.12 positive antibodies and to further characterize the V lambda II family, germline V lambda II clones were derived from a patient with SLE. Two libraries were constructed: a genomic DNA library and a library of PCR-derived V lambda II gene products obtained using a conserved V lambda II leader region primer and a primer for the nonamer region 3' of the coding sequence. We now describe seven new germline genes, two of which are pseudogenes. Comparison of V lambda II germline genes to sequences of 8.12 positive light chains produced by EBV-transformed B cell lines show that all 8.12 positive light chains are encoded by a limited number of highly homologous members of the V lambda II family. 8.12 negative V lambda II encoded light chains also derive from a limited number of V lambda II genes, suggesting that only a subset of the apparently available V lambda II genes are commonly expressed.