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A Local Antigen-Driven Humoral Response Is Present in the Inflammatory Myopathies¹

Elizabeth M. Bradshaw^*,, Ana Orihuela, Shannon L. McArdel^*, Mohammad Salajegheh^,,, Anthony A. Amato^,, David A. Hafler^*,, Steven A. Greenberg^2,3,,, and Kevin C. O’Connor^2,3,*,

^* Department of Neurology, Laboratory of Molecular Immunology, Center for Neurologic Diseases and Brigham and Women’s Hospital; Children’s Hospital Informatics Program at the Harvard-Massachusetts Institute of Technology Division of Health Sciences and Technology; and Department of Neurology, Division of Neuromuscular Disease, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115

	Abstract

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The inflammatory myopathies are putative autoimmune disorders characterized by muscle weakness and the presence of intramuscular inflammatory infiltrates. Although inclusion body myositis and polymyositis have been characterized as cytotoxic CD8⁺ T cell-mediated diseases, we recently demonstrated high frequencies of CD138⁺ plasma cells in the inflamed muscle tissue of patients with these diseases. To gain a deeper understanding of the role these B cell family members play in the disease pathology, we examined the molecular characteristics of the H chain portion of the Ag receptor. Biopsies of muscle tissue were sectioned and tissue regions and individual cells were isolated through laser capture microdissection. Ig H chain gene transcripts isolated from the sections, regions, and cells were used to determine the variable region gene sequences. Analysis of these sequences revealed clear evidence of affinity maturation in that significant somatic mutation, isotype switching, receptor revision, codon insertion/deletion, and oligoclonal expansion had occurred within the B and plasma cell populations. Moreover, analysis of tissue regions isolated by laser capture microdissection revealed both clonal expansion and variation, suggesting that local B cell maturation occurs within muscle. In contrast, sequences from control muscle tissues and peripheral blood revealed none of these characteristics found in inflammatory myopathy muscle tissue. Collectively, these data demonstrate that Ag drives a B cell Ag-specific response in muscle in patients with dermatomyositis, inclusion body myositis, and polymyositis. These findings highlight the need for a revision of the current paradigm of exclusively T cell-mediated intramuscular Ag-specific autoimmunity in inclusion body myositis and polymyositis.

	Introduction

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The inflammatory myopathies are a group of autoimmune diseases characterized by progressive skeletal muscle weakness associated with inflammatory cell infiltration within the muscle. The principal subtypes are dermatomyositis, polymyositis, and inclusion body myositis (1). These diseases have distinct clinical and pathological features. Dermatomyositis, a multisystem disease that includes muscle, skin, and sometimes other tissue injury has, for two decades, been viewed as a humorally mediated disorder in which Ab-mediated complement activation results in endothelial cell injury (2). In dermatomyositis, immunohistochemical studies have shown B cells and a CD4⁺ cell infiltrate in muscle, suggesting a class II-restricted immune response. However, a large proportion of these CD4⁺ cells were recently found to be plasmacytoid dendritic cells, not T cells (3). Microarray experiments, confirmed by immunohistochemistry, revealed a signature of type 1 IFN-driven pathology in dermatomyositis muscle and, along with the presence of plasmacytoid dendritic cells, suggest a prominent innate immune response (3). A similar mechanism of tissue pathology has recently been reported in skin in dermatomyositis (4). For inclusion body myositis and polymyositis, self-reactive T cells have been considered to play a central role (2). B cells are sparse among the infiltrating immune cells (5). In these two disorders, CD8⁺ T cells are found near myofibers expressing high levels of MHC class I (6). Evidence that autoreactive T cells contribute to the muscle pathology of polymyositis and inclusion body myositis includes: 1) the presence of a restricted TCR repertoire in a subset of polymyositis and inclusion body myositis muscle biopsies (7, 8, 9); 2) an oligoclonal expansion of muscle-infiltrating T cells in inclusion body myositis (10, 11, 12, 13) and in polymyositis (14, 15); and 3) the long-term presence of clonally expanded T cells in both inclusion body myositis and polymyositis (16, 17, 18). Although these findings suggest that inclusion body myositis and polymyositis are CD8⁺ T cell-mediated autoimmune diseases, whether B cells are also involved in the disease pathogenesis is unknown.

During T cell-dependent immune responses, Ag-activated B cells migrate into the primary follicles of the peripheral lymphatic organs. These cells are then induced to clonally expand, leading to the formation of germinal centers. Within the germinal centers, B cells may undergo class switching and affinity maturation through the process of somatic hypermutation, which diversifies the repertoire of B cells (19, 20). B cells that acquire higher-affinity receptors are selected to differentiate into memory cells and plasma cells (21, 22, 23). The V regions of maturing Abs show a characteristic pattern of somatic mutations. Mutations resulting in amino acid replacement are mainly seen in the CDRs, whereas silent mutations more often accumulate in the framework. Thus, comparison of V_H region sequences to those of the known germline provides an accurate indication of the maturation or experience of a B cell. Examination of constant regions allows identification of B cell lineages that have undergone class switching. Finally, these analyses can be used to identify clonally related cells, B cells derived from a parental cell that arise during both the affinity maturation and differentiation processes.

Recent analysis of muscle mRNA expression data demonstrated a surprising abundance of Ig transcripts in both inclusion body myositis and polymyositis (24, 25). Although sparse numbers of CD19⁺ or CD20⁺ B cells are present in these muscle biopsies, we found large numbers of CD138⁺ plasma cells (25). Further evidence that the humoral immune branch contributes to tissue injury in inclusion body myositis and polymyositis comes from observations that serum-derived Abs for 50% of patients with inclusion body myositis react with myonuclei (26), whereas a subset of patients with polymyositis have autoantibodies against cytoplasmic tRNA synthetases or signal-recognition particles (27). These findings suggest a role for Ab-mediated autoimmunity in these diseases and led us to hypothesize that we would find evidence of an Ag-driven immune response in the tissue of patients with these two inflammatory myopathies. Thus, we investigated the B cell and plasma cell Ig repertoire in muscle biopsies by examining Ig isotype usage, somatic mutations, and clonal expansion.

	Materials and Methods

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Patients

We studied muscle specimens obtained from biopsies from 17 patients as follows: 7 with inclusion body myositis, 3 with dermatomyositis, 2 with polymyositis, and 5 normal patients using standard diagnostic criteria (Table I and Refs. 3 and 25). None of the patients had undergone immunosuppressive treatment before the biopsy. Muscle biopsies were performed for clinical indications, independent of the current study. Control specimens also included four blood samples (two from the same inclusion body myositis patients whose muscle samples were studied, collected at the same time as the biopsy, and two from normal healthy subjects). An institutional review board approved this study.

RNA was extracted from 8- to 12-µm-thick slices or small pieces of muscle tissue using the Absolutely RNA Nanoprep Kit (Stratagene). Human Ig H chain V_H region genes were amplified with a RT-PCR protocol reported by Wang and Stollar (28), with a modification that included the addition of primers to amplify IgA and a different subcloning vector. The sequences of these IgA-specific primers are as follows: RT reaction, 5'-GAG GCT CAG CGG GAA GAC-3'; first PCR, 5'-GCT CAG CGG GAA GAC CTT-3'; and second PCR, 5'-GAC CTT GGG GCT GGT CGG GGA-3'. The RT-PCR products were cloned with the Zero Blunt TOPO PCR Cloning Kit (Invitrogen Life Technologies), and a sample of clones were picked and sequenced with primers complementary to sequences flanking the cloning site.

V_H sequence alignment and constant region identification

Individual V_H sequences were aligned to V_H germline segments with software and the human V_H region database available on the ImMunoGeneTics web site http://imgt.cines.fr (29). A custom software program was used to both submit sequences to the ImMunoGeneTics web site and tabulate the output files from the site. We identified the most homologous V_H germline segments and the extent of sequence homology of the H chain sequences to the germline. Thus, this alignment allowed the determination of the V_H, D_H, and J_H family usage, the extent of somatic mutations, and the identification of the CDR3 amino acid sequence. Because the CDR3 sequence is unique, this region was used as the basis for identifying clones (30). Thus, a group of two or more V_H sequences were considered derived from the same clone if they had identical sequences between each nested primer, which includes the CDR3 region, and were found in different RT-PCR from the same tissue sample. Similarly, two or more sequences were considered to be derived from clonal variants if they had CDR3 amino acid sequences that were identical or differed by one amino acid and at least two different somatic mutations in the V_H region. To confirm the presence of clonal variants in instances in which the CDR3 differed by one or more amino acid, we examined the V_H region for identical mutations and also the length of the D_H and J_H and the number of N nucleotides comprising the CDR3. Finally, the constant region was identified as the IgM, IgG, or IgA subtype.

Laser capture microdissection of muscle sections

Muscle tissue from one patient each with inclusion body myositis, polymyositis, and dermatomyositis were cryosectioned at 12 µm, mounted onto a glass slide, fixed in 75% ethanol for 30 s, and stained with Histogene Staining Solution (Arcturus). The tissue was then dehydrated in consecutive washes of 75, 95, and 100% ethanol and xylene. Sections of tissue were captured with a PixCell IIe laser capture microdissection instrument and CapSure Macro caps (Arcturus) and immediately stored at –80°C. RNA was isolated with the Absolutely RNA Nanoprep Kit (Stratagene) according to the manufacturer’s protocol. The Ig V region genes were identified as outlined above. This methodology does not allow for distinction among various B cell subsets from which the V region genes were derived. For the identification and capture of individual plasma cells, the tissue was stained with an anti-CD138 Ab (Accurate Chemical & Scientific) after fixation, then counterstained with poly-HRP anti-mouse IgG (Ivax Diagnostics), and dehydrated. RNA was isolated as described above.

	Results

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Muscle-infiltrating B cells have undergone isotype switching and have Ig genes that are highly mutated

Both class switching and the accumulation of mutations within the CDRs are features of an Ag-driven B cell response. To examine the nature of the B cell response in the tissue of patients with these inflammatory myopathies, Ig V_H sequence collections were constructed from muscle tissue and blood of patients and control subjects. The evaluation of the sequence collections from the 12 patients with inflammatory myopathies revealed that the majority of B cells had class switched from IgM to either IgG or IgA (Table II). Naive resting B cells do not usually class switch until they have encountered Ag. In five of the muscle samples, there were no IgM sequences amplified at all. Three of the four control blood samples contained sequences primarily derived from B cells producing IgM (Table II). In previous work, we demonstrated the rarity of either CD19⁺, CD20⁺, or CD138⁺ B cells within muscle tissue from non-neuromuscular disease control patients (25). We confirmed that here by analyzing normal muscle tissue and found zero to three Ig sequences present per muscle biopsy (Table II).

View larger version (18K): [in this window] [in a new window]   FIGURE 1. Distribution of VH region mutations in the Ig sequence collections. A, The combined mutation distribution of the inflammatory myopathy sequence collection and, separately, the combined blood sequence collection. B, Mutation distribution of the combined inflammatory myopathy sequence collection separated by Ig isotype.

A limitation of Ig V_H sequence collections created in a single PCR is that B cell clones with identical sequences cannot be differentiated from identical PCR amplicons of the same template cDNA. Thus, the phenomenon of clonal restriction needs to be carefully distinguished from such repeated amplicon identification. The identification of B cell clonal variants that have highly related but nonidentical sequences is one approach to circumvent this constraint. Groups of clonal variants were identified in 10 (85%) of 12 of the inflammatory myopathy samples (Table II). Up to four different groups of clonal variants were found in a muscle section. Most of these consisted of two clonal variants, but in some of the groups there were up to six different but related clones (Table III). Most of the somatic mutations within the clonal variants resulted in amino acid replacements (Table III).

B cells infiltrating inflammatory myopathy tissue accumulate diversity through codon alteration and receptor revision

Single nucleotide point mutations are the most common mutations that occur during the affinity maturation process. However, insertions and deletions of nucleotides have also been reported (31). Analysis of the Ig transcripts isolated from inflammatory myopathy muscle biopsies identified seven clones (Table IV) that contained nucleotide insertions or deletions (3–12 bp). There was one instance of a deletion found in a blood clone. In all instances, the insertions and deletions occurred in multiples of three. As a result, the original reading frame was maintained, allowing productive translation of the Ig. These transcripts were derived from class-switched Ig and also were among those containing the highest amount of somatic mutation. The insertions and deletions were all either within or adjacent to a CDR region.

View larger version (62K): [in this window] [in a new window]   FIGURE 2. Alignment of germline and expressed VH genes from clonal variants, which may represent receptor revision. The clonal variant pairs, 156A and 156B (A) and 179A and 179B (B), have identical CDR3 and JH regions, but appeared to have different VH regions. Common somatic mutations in the VH regions are indicated in bold. With the exception of one, all of the somatic mutations are in the C terminus of the VH region just before the CDR3. The dashes signify that the nucleotide is the same as the top germline VH region. The dots compensate for the different lengths of different VH region genes. The CDR regions are indicated, and a indicates a difference in CDR length.

The identification of clonal variants within a single PCR circumvents the limitation that PCR imposes on the identification of clones. Another strategy that eliminates this constraint is the analysis of sequences from multiple sections of a single tissue sample. Accordingly, to further assess the degree of clonal expansion within inflammatory myopathy tissue, laser capture microdissection was used to divide three tissue specimens (one each with inclusion body myositis, dermatomyositis, and polymyositis) into distinct sections, from which Ig V_H sequence collections were prepared (Fig. 3). In the inclusion body myositis sample, all 9 pieces had Ig sequences, in the dermatomyositis sample 5 pieces of 13, and in the polymyositis 4 of 13 pieces were found to have Ig sequences. Analysis of the sequences revealed evidence of B cell clonal expansion in the inclusion body myositis (P-83) and dermatomyositis (P-258) samples, whereas there was no evidence found in the polymyositis (P-156) specimen (Table V). In the inclusion body myositis sample, five different clones found in this tissue slice were also found in the Ig V_H sequence collection from a different tissue slice from the same patient, P-83 (Fig. 3 and Table V). Clone 1 was also found in three contiguous sections. Clones 4 and 5 were both found in nonadjacent sections, as well as the whole muscle section. Moreover, the sequences comprising clone 5 included variants that were present within unique tissue sections. Variants of clone 3 were also present in the whole muscle sequence collection. The dermatomyositis specimen contained two clones that were found in the whole muscle section sequence collection along with a clonal variant. One of these clones was also found in two separate pieces of the tissue slice. These data clearly demonstrate that clones and clonal variants of B and/or plasma cells are present in muscle tissue. Furthermore, these findings suggest that this expansion has occurred within inflamed muscle tissue.

View larger version (114K): [in this window] [in a new window]   FIGURE 3. Laser capture microdissection of muscle sections. Sample from patient P-83 with inclusion body myositis. A, A cap is placed over the desired section of muscle, and the plastic bottom is melted to the desired tissue. B, Once the cap is removed, most of the desired tissue is removed with it. C, Most of the muscle tissue can be seen on the cap. D, H chain sequence collections were made from the nine sections separated by laser capture microdissection. Five clones were found in both the sections and a distinct piece of whole muscle used to generate a sequence collection. Clone 1 was also found in three contiguous sections. Clones 4 and 5 were found in non-neighboring sections, and clone 5 had two clonal variants in the sections. The H chain CDR3 regions of the clones are listed.

Additional evidence to support an Ag-driven response

The nucleotide mutations within clonal variants demonstrate that affinity maturation is occurring. In addition, common amino acid mutations found in unrelated clones suggest that a common Ag may be driving the response. It has been shown in myasthenia gravis, where the Ag is known, that different clones that use the same V_H region gene have identical amino acid mutations (34). We found a similar pattern in the CDR regions between unrelated clones by studying the entire collection of sequences derived from the inflammatory myopathy patient tissue (Table VI). A number of identical replacement mutations were found in clones of the same patient, whereas others were found in many clones across different patients. The former suggests that an Ag is driving the mutation pattern, the latter that mutational hot spots are present.

	Discussion

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We found significant oligoclonal expansion of the B cells infiltrating inflammatory myopathy muscle tissue, analogous to oligoclonal expansion that has been demonstrated in several studies of T cells in polymyositis and inclusion body myositis (10, 15, 16, 18). Moreover, we observed topographical clonal expansion representing the presence of identical B cell clones in neighboring regions of tissue in inclusion body myositis and dermatomyositis. We also found topographical clonal variation by identifying related B cell clones in the same section and different sections of tissue. The application of laser capture microdissection to sections of muscle specimens from patients with inclusion body myositis and dermatomyositis demonstrated that both clonal expansion and clonal variation had occurred through the finding of both identical Ig sequences in different regions as well as closely related but not identical Ig sequences in the same and different regions (clonal variants). Misinterpretation of clonal expansion can be due to PCR amplification of a single amplicon. Our finding of clonal variation, in the whole sections and clonal expansion in the sections separated by laser capture microdissection provides substantial evidence of an Ag-driven response. Although we did not confirm the presence of clonal expansion in polymyositis by the use of laser capture microdissection, only 4 of 13 pieces of the muscle slice had any Ig sequences. Perhaps the slice was taken from a section of muscle that had either less B cell infiltrate or low RNA quality. Regardless, the polymyositis sequence collection for this sample (P-156) had three examples of clonal variants.

Our demonstration of topographical clonal variation, the presence of related sequences within submillimeter adjacent regions of muscle, strongly suggests that affinity maturation is occurring within muscle rather than strictly in a local lymph node as is classically held. The likelihood that such highly related B cells migrate from lymph node to nearly identical locations within muscle seems low. The findings of clonal expansion and clonal variation have been identified in other autoimmune diseases: in the cerebrospinal fluid and brain lesions in multiple sclerosis, in the synovial membrane and fluid in rheumatoid arthritis, and in the thymus in myasthenia gravis, (34, 35, 36, 37). In addition, structures resembling germinal centers have been reported in rheumatoid arthritis, myasthenia gravis, and secondary progressive multiple sclerosis (38, 39, 40), supporting the likelihood that affinity maturation is occurring in target tissues. Whether peripherally activated B cells are selectively recruited to inflammatory myopathy muscle tissue where they differentiate into plasma cells or expand and mature locally in an ectopic germinal center remains to be determined.

Our assembly of related sequences into clones is justified by current knowledge of B cell development. Statistically, it is highly unlikely for two B cells to both recombine the same V_H, D_H, and J_H gene segments and incorporate identical N and P nucleotides. Thus, due to this unique characteristic, the CDR3 region, which is defined by the VDJ joint, is an accurate marker for identification of clones. Several sequences in three specimens, namely, P-7, P-156, and P-181, had CDR3 sequences in which only one amino acid differed from those of clones within the specimen’s sequence collection (Table III). The VDJ joining pattern and the number of untemplated nucleotides were identical. On the basis of current knowledge, it is similarly unlikely that distinct clones arrived at nearly identical CDR3 sequences through convergent mutations, and this possibility seems even more unlikely given that the V_H regions of these sequences have common somatic mutations. Thus, we have interpreted these clones with related sequences as having been derived from the same precursor cell.

Receptor revision is a mechanism that provides further diversity to the Ig repertoire of mature B cells. The result can be enhanced Ag affinity or overcoming self-reactivity. In this secondary rearrangement, a hybrid V_H gene is formed containing portions of two different germline V_H region genes. It has been demonstrated that receptor revision can occur during affinity maturation (32). The analysis of several sequences we collected suggests that receptor revision had occurred in our samples. Two pairs of clonal variants (156A and B, 179A and B) had identical nucleotides in their CDR3s; however, they had different V_H region gene segments, no common mutation pattern, and different V_H region lengths (Fig. 2). The difference in the V_H region length of clones 156A and B and clones 179A and B could be accounted for by an insertion of a codon rather than receptor revision. We favor the latter for the following reasons. There is no common somatic hypermutation pattern throughout the V_H region; in fact, there is only one common nucleotide change in clones 156A and B in the 5' end of the V_H region. However, there are common somatic hypermutations in the 3' end of the V_H region, but this region is downstream of the putative receptor revision switch region. Collectively, these data strongly suggest that these Igs had undergone receptor revision. PCR crossover is another explanation for two clonal variants having different V_H gene usage. To minimize this artifact as being misinterpreted as receptor revision, these sequences were compared for identical somatic mutations to every other sequence that used the same V_H gene. If the 5' region was identical for a large portion of the V_H region, then it was assumed to be PCR crossover and not receptor revision.

Codon insertions and deletions have been observed in Ig V_H region gene segments expressed by B cells in the synovial tissue of patients with rheumatoid arthritis, but not at a frequency that varied from normal B cells (41). Having codon insertions/deletions in the CDR regions as opposed to the framework regions creates more diversity, but also does not interfere with the proper folding of the Ig protein. We observed that these forms of diversification are features of the somatic mutation pattern of B cells in inflammatory myopathy tissue samples. In seven Ig transcripts, we observed codon insertions or deletions when compared with the best-matching V_H region gene segment.

Our data also suggest that IgA class-switched Igs play a broader role in autoimmunity than currently appreciated. Immunohistochemistry of inclusion body myositis samples previously showed IgG-, IgM-, and IgA-, but not IgD-positive cells within the muscle tissue (25). IgG and IgA are generally believed to have different roles in the immune system. IgA is the predominate isotype at mucosal surfaces. However, IgA (along with IgG- and IgM-)- secreting plasma cells have been identified in the cerebrospinal fluid and lesions of patients with multiple sclerosis (42, 43). In the inflammatory myopathies, there were a significant number of Ig sequences of the IgA isotype. Among the clonal variant groups identified, one group from a dermatomyositis patient had clonal variants that were from different isotypes (IgG/IgA), which is evidence that Ig class switching may be occurring in muscle from subjects with dermatomyositis. Although we found this switching to occur only in dermatomyositis, larger studies will be needed to confirm that this phenomenon is restricted to this disease. Microarray data from inclusion body myositis and polymyositis muscle further corroborate an abundance of IgA transcripts in muscle of some patients. In our previous microarray studies, the IgA H chain transcript was the highest differentially up-regulated transcript, with a fold ratio of 142, among 13,000 transcripts assayed in inclusion body myositis tissue compared with control tissue (25). The IgA isotype, whose role is not often considered in autoimmunity, may play an important role in the pathogenesis of these inflammatory myopathies.

In conclusion, Ig sequences from inflammatory myopathies muscle tissue have class switched, and the V_H region genes are extensively mutated. Clonal sequences with identical or nearly identical CDR3 VDJ rearrangements and varying patterns of somatic mutations in CDR1 and CDR2 show that intraclonal diversification has occurred. Collectively, these data suggest that, in the major subgroups of the inflammatory myopathies, Ags in muscle drive a B cell Ag-specific response and that B cells mature in part within muscle itself. These findings highlight the need for a revision of the current paradigm of exclusively T cell-mediated i.m. Ag-specific autoimmunity in inclusion body myositis and polymyositis in particular. Efforts to elucidate the nature of the Ags are of paramount importance to understand the immunopathology of these myopathies.

	Acknowledgments

 
We are grateful for the technical assistance of Dr. C. Vanderburg of the Harvard Center for Neurodegeneration and Repair and Julia S. Kingsdale. We thank Brigham and Women’s Hospital Editorial Service for careful review of this manuscript.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These studies were supported by grants to S.A.G. from the National Institutes of Health (R01 NS043471), the Muscular Dystrophy Association, and the Sporadic Inclusion Body Myositis Research Foundation and to D.A.H. from the National Institutes of Health (RO1 NS24247). These studies were also supported, in part, by a Career Transition Fellowship awarded to K.C.O. from the National Multiple Sclerosis Society (TA 3000A2/1). E.M.B. is a National Institute of Allergy and Infectious Diseases, Kirschstein-National Research Service Award Fellowship Recipient (F32 AI065100-01 A1).

² S.A.G. and K.C.O. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Kevin C. O’Connor, Harvard Medical School, Center for Neurologic Diseases, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: koconnor{at}rics.bwh.harvard.edu and Dr. Steven A. Greenberg, Department of Neurology, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115. E-mail address: sagreenberg{at}partners.org

Received for publication March 9, 2006. Accepted for publication October 10, 2006.

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