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1.  Autoantibodies Produced at the Site of Tissue Damage Provide Evidence of Humoral Autoimmunity in Inclusion Body Myositis 
PLoS ONE  2012;7(10):e46709.
Inclusion body myositis (IBM) belongs to a group of muscle diseases known as the inflammatory myopathies. The presence of antibody-secreting plasma cells in IBM muscle implicates the humoral immune response in this disease. However, whether the humoral immune response actively contributes to IBM pathology has not been established. We sought to investigate whether the humoral immune response in IBM both in the periphery and at the site of tissue damage was directed towards self-antigens. Peripheral autoantibodies present in IBM serum but not control serum recognized self-antigens in both muscle tissue and human-derived cell lines. To study the humoral immune response at the site of tissue damage in IBM patients, we isolated single plasma cells directly from IBM-derived muscle tissue sections and from these cells, reconstructed a series of recombinant immunoglobulins (rIgG). These rIgG, each representing a single muscle-associated plasma cell, were examined for reactivity to self-antigens. Both, flow cytometry and immunoblotting revealed that these rIgG recognized antigens expressed by cell lines and in muscle tissue homogenates. Using a mass spectrometry-based approach, Desmin, a major intermediate filament protein, expressed abundantly in muscle tissue, was identified as the target of one IBM muscle-derived rIgG. Collectively, these data support the view that IBM includes a humoral immune response in both the periphery and at the site of tissue damage that is directed towards self-antigens.
PMCID: PMC3465259  PMID: 23071619
2.  Related B cell clones that populate the CSF and CNS of patients with multiple sclerosis produce CSF immunoglobulin 
Journal of neuroimmunology  2011;233(1-2):245-248.
We investigated the overlap shared between the immunoglobulin (Ig) proteome of the cerebrospinal fluid (CSF) and the B cell Ig-transcriptome of CSF and the central nervous system (CNS) tissue of three patients with multiple sclerosis. We determined the IgG-proteomes of CSF by mass spectrometry, and compared them to the IgG-transcriptomes from CSF and brain lesions, which were analyzed by cDNA cloning. Characteristic peptides that were identified in the CSF-proteome could also be detected in the transcriptomes of both, brain lesions and CSF, providing evidence for a strong overlap of the IgG repertoires in brain lesions and in the CSF.
PMCID: PMC3090654  PMID: 21353315
multiple sclerosis; B cells; cerebrospinal fluid; central nervous system; oligoclonal bands
3.  Related B cell clones populate the meninges and parenchyma of patients with multiple sclerosis 
Brain  2011;134(2):534-541.
In the central nervous system of patients with multiple sclerosis, B cell aggregates populate the meninges, raising the central question as to whether these structures relate to the B cell infiltrates found in parenchymal lesions or instead, represent a separate central nervous system immune compartment. We characterized the repertoires derived from meningeal B cell aggregates and the corresponding parenchymal infiltrates from brain tissue derived primarily from patients with progressive multiple sclerosis. The majority of expanded antigen-experienced B cell clones derived from meningeal aggregates were also present in the parenchyma. We extended this investigation to include 20 grey matter specimens containing meninges, 26 inflammatory plaques, 19 areas of normal appearing white matter and cerebral spinal fluid. Analysis of 1833 B cell receptor heavy chain variable region sequences demonstrated that antigen-experienced clones were consistently shared among these distinct compartments. This study establishes a relationship between extraparenchymal lymphoid tissue and parenchymal infiltrates and defines the arrangement of B cell clones that populate the central nervous system of patients with multiple sclerosis.
PMCID: PMC3030766  PMID: 21216828
multiple sclerosis; B cells; clonal expansion; antigen experience; central nervous system
5.  Structural Basis for Apoptosis Inhibition by Epstein-Barr Virus BHRF1 
PLoS Pathogens  2010;6(12):e1001236.
Epstein-Barr virus (EBV) is associated with human malignancies, especially those affecting the B cell compartment such as Burkitt lymphoma. The virally encoded homolog of the mammalian pro-survival protein Bcl-2, BHRF1 contributes to viral infectivity and lymphomagenesis. In addition to the pro-apoptotic BH3-only protein Bim, its key target in lymphoid cells, BHRF1 also binds a selective sub-set of pro-apoptotic proteins (Bid, Puma, Bak) expressed by host cells. A consequence of BHRF1 expression is marked resistance to a range of cytotoxic agents and in particular, we show that its expression renders a mouse model of Burkitt lymphoma untreatable. As current small organic antagonists of Bcl-2 do not target BHRF1, the structures of it in complex with Bim or Bak shown here will be useful to guide efforts to target BHRF1 in EBV-associated malignancies, which are usually associated with poor clinical outcomes.
Author Summary
Altruistic suicide of infected host cells is a key defense mechanism to combat viral infection. To ensure their own survival and proliferation, certain viruses, including Epstein-Barr virus (EBV), have mechanisms to subvert apoptosis, including the expression of homologs of the mammalian pro-survival protein Bcl-2. EBV was first identified in association with Burkitt lymphoma and it is also linked to certain Hodgkin's lymphomas and nasopharyngeal carcinoma. Whereas increased expression of Bcl-2 promotes malignancies such as human follicular lymphoma, the precise role of the EBV encoded Bcl-2 homolog BHRF1 in EBV-associated malignancies is less well defined. BHRF1 is known to bind the pro-apoptotic BH3-only protein Bim, and here we demonstrate that it also binds other pro-apoptotic proteins (Bid, Puma, Bak) expressed by host cells. Crystal structures of BHRF1 with the BH3 regions of Bim and Bak illustrate these interactions in atomic detail. A consequence of BHRF1 expression is marked resistance to a range of cytotoxic agents, and we show that its expression renders a mouse model of Burkitt lymphoma untreatable. As current antagonists of Bcl-2 do not target BHRF1, our crystal structures will be useful to guide efforts to target BHRF1 in EBV-associated malignancies, which are usually associated with poor clinical outcomes.
PMCID: PMC3009601  PMID: 21203485
6.  Epstein–Barr virus infection is not a characteristic feature of multiple sclerosis brain 
Brain  2009;132(12):3318-3328.
Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system (CNS) that is thought to be caused by a combination of genetic and environmental factors. To date, considerable evidence has associated Epstein–Barr virus (EBV) infection with disease development. However, it remains controversial whether EBV infects multiple sclerosis brain and contributes directly to CNS immunopathology. To assess whether EBV infection is a characteristic feature of multiple sclerosis brain, a large cohort of multiple sclerosis specimens containing white matter lesions (nine adult and three paediatric cases) with a heterogeneous B cell infiltrate and a second cohort of multiple sclerosis specimens (12 cases) that included B cell infiltration within the meninges and parenchymal B cell aggregates, were examined for EBV infection using multiple methodologies including in situ hybridization, immunohistochemistry and two independent real-time polymerase chain reaction (PCR) methodologies that detect genomic EBV or the abundant EBV encoded RNA (EBER) 1, respectively. We report that EBV could not be detected in any of the multiple sclerosis specimens containing white matter lesions by any of the methods employed, yet EBV was readily detectable in multiple Epstein–Barr virus-positive control tissues including several CNS lymphomas. Furthermore, EBV was not detected in our second cohort of multiple sclerosis specimens by in situ hybridization. However, our real-time PCR methodologies, which were capable of detecting very few EBV infected cells, detected EBV at low levels in only 2 of the 12 multiple sclerosis meningeal specimens examined. Our finding that CNS EBV infection was rare in multiple sclerosis brain indicates that EBV infection is unlikely to contribute directly to multiple sclerosis brain pathology in the vast majority of cases.
PMCID: PMC2792367  PMID: 19638446
B cells; Epstein–Barr virus; multiple sclerosis brain
7.  The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized 
Cancer cell  2006;10(5):389-399.
Since apoptosis is impaired in malignant cells overexpressing pro-survival Bcl-2 proteins, drugs mimicking their natural antagonists, BH3-only proteins, might overcome chemoresistance. Of seven putative BH3 mimetics tested, only ABT-737 triggered Bax/Bak-mediated apoptosis. Despite its high affinity for Bcl-2, Bcl-xL and Bcl-w, many cell types proved refractory to ABT-737. We show that this resistance reflects its inability to target another pro-survival relative, Mcl-1. Down-regulation of Mcl-1 by several strategies conferred sensitivity to ABT-737. Furthermore, enforced Mcl-1 expression in a mouse lymphoma model conferred resistance. In contrast, cells overexpressing Bcl-2 remained highly sensitive to ABT-737. Hence, ABT-737 should prove efficacious in tumors with low Mcl-1 levels, or when combined with agents that inactivate Mcl-1, even to treat those tumors that overexpress Bcl-2.
Targeting the pro-survival Bcl-2-like proteins for cancer therapy is attractive because their overactivity promotes tumor formation and often limits responses to cytotoxic agents. Hence, drugs mimicking their antagonists, BH3-only proteins, offer promise as anti-cancer agents. Unlike other putative BH3 mimetics tested, ABT-737 induced apoptosis by the expected mechanism. Because it targets only certain pro-survival proteins (Bcl-2, Bcl-xL, Bcl-w), the efficacy of ABT-737 as a single agent is restricted to tumors where pro-survival Mcl-1 is low. We show that resistant cells can be sensitized to ABT-737 by approaches that down-regulate, destabilize or inactivate Mcl-1. Our studies provide a rational basis for designing clinical trials of this highly promising agent and a benchmark for systematically evaluating BH3 mimetic compounds.
PMCID: PMC2953559  PMID: 17097561
8.  Interleukin 15–mediated survival of natural killer cells is determined by interactions among Bim, Noxa and Mcl-1 
Nature immunology  2007;8(8):856-863.
Interleukin 15 (IL-15) promotes the survival of natural killer (NK) cells by preventing apoptosis through mechanisms unknown at present. Here we identify Bim, Noxa and Mcl-1 as key regulators of IL-15-dependent survival of NK cells. IL-15 suppressed apoptosis by limiting Bim expression through the kinases Erk1 and Erk2 and mechanisms dependent on the transcription factor Foxo3a, while promoting expression of Mcl-1, which was necessary and sufficient for the survival of NK cells. Withdrawal of IL-15 led to upregulation of Bim and, accordingly, both Bim-deficient and Foxo3a−/− NK cells were resistant to cytokine deprivation. Finally, IL-15-mediated inactivation of Foxo3a and cell survival were dependent on phosphotidylinositol-3-OH kinase. Thus, IL-15 regulates the survival of NK cells at multiple steps, with Bim and Noxa being key antagonists of Mcl-1, the critical survivor factor in this process.
PMCID: PMC2951739  PMID: 17618288
9.  Life in the balance: how BH3-only proteins induce apoptosis 
Current opinion in cell biology  2005;17(6):617-625.
BH3-only members of the Bcl-2 intracellular protein family, which include Bim, Bmf, Bik, Bad, Bid, Puma, Noxa and Hrk, mediate many developmentally programmed and induced cytotoxic signals. They have key roles in development, tissue homeostasis, immunity and tumor suppression, and compounds mimicking them are promising anti-cancer agents. Their activity is normally constrained by transcriptional and/or diverse post-transcriptional controls. When activated, these death ligands engage pro-survival Bcl-2-like proteins via the BH3 domain, inactivating their function. Bim and Puma bind all the pro-survival proteins, whereas others, such as Noxa and Bad, engage distinct subsets and exhibit complementary killing. Hence, multiple pro-survival proteins must be inactivated to unleash Bax and Bak, which drive apoptosis. Whether certain BH3-only proteins also directly activate Bax/Bak remains controversial.
PMCID: PMC2930980  PMID: 16243507
10.  A novel BH3 ligand that selectively targets Mcl-1 reveals that apoptosis can proceed without Mcl-1 degradation 
The Journal of Cell Biology  2008;180(2):341-355.
Like Bcl-2, Mcl-1 is an important survival factor for many cancers, its expression contributing to chemoresistance and disease relapse. However, unlike other prosurvival Bcl-2–like proteins, Mcl-1 stability is acutely regulated. For example, the Bcl-2 homology 3 (BH3)–only protein Noxa, which preferentially binds to Mcl-1, also targets it for proteasomal degradation. In this paper, we describe the discovery and characterization of a novel BH3-like ligand derived from Bim, BimS2A, which is highly selective for Mcl-1. Unlike Noxa, BimS2A is unable to trigger Mcl-1 degradation, yet, like Noxa, BimS2A promotes cell killing only when Bcl-xL is absent or neutralized. Furthermore, killing by endogenous Bim is not associated with Mcl-1 degradation. Thus, functional inactivation of Mcl-1 does not always require its elimination. Rather, it can be efficiently antagonized by a BH3-like ligand tightly engaging its binding groove, which is confirmed here with a structural study. Our data have important implications for the discovery of compounds that might kill cells whose survival depends on Mcl-1.
PMCID: PMC2213596  PMID: 18209102
11.  Protein array–based profiling of CSF identifies RBPJ as an autoantigen in multiple sclerosis 
Neurology  2013;81(11):956-963.
To profile the reactivity of CSF-derived immunoglobulin from patients with multiple sclerosis (MS) against a large panel of antigens, to identify disease-specific reactivities.
CSF from subjects with MS with elevated immunoglobulin G and CSF from control subjects presenting with other inflammatory neurologic disease were screened against a protein array consisting of 9,393 proteins. Reactivity to a candidate protein identified using these arrays was confirmed with ELISA and immunocytochemistry.
Autoantibodies against one protein on the array, recombination signal binding protein for immunoglobulin kappa J region (RBPJ), discriminated between patients with MS and controls (p = 0.0052). Using a large validation cohort, we found a higher prevalence of autoantibodies against RBPJ in the CSF of patients with MS (12.5%) compared with the CSF of patients with other neurologic diseases (1.6%; p = 0.02) by ELISA. This difference in reactivity was restricted to the CSF as serum reactivity against RBPJ did not differ between patients and controls. The presence of CSF autoantibodies against RBPJ was further confirmed by immunocytochemistry.
These data indicate that RBPJ, a ubiquitous protein of the Notch signaling pathway that plays an important role in Epstein-Barr virus infection, is a novel MS autoantigen candidate that is recognized by CSF-derived immunoglobulin G in a subset of patients with MS.
PMCID: PMC3888197  PMID: 23921886

Results 1-11 (11)