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*
Department of Neurology, University of California, San Francisco, CA 94143; and
Rocky Mountain Multiple Sclerosis Center, Englewood, CO 80110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Abs produced by naive B cells are known to integrate the circulating pool of Igs and to act as membrane receptors during Ag recognition (18, 19). The way B cells generate the extraordinary large diversity in the Ab they produce was elegantly brought to light some years ago and involves the usage of a few hundred germline segments to potentially produce billions of different proteins (20, 21). Allelic polymorphism, somatic mutation, imprecise joining, nonencoded nucleotide addition, and exonuclease activity also add to Ab diversity. Upon antigenic stimulation, relevant B cell clones are positively selected and undergo gene hypermutation to produce Abs that best fit the Ag in a process called affinity maturation (22).
The complementary-determining region 3 (CDR3) in both heavy and light chains is the most polymorphic fragment of an Ab structure, and it is the region that most closely interacts with the Ag. Conserved amino acids at the VDJ junction are indicative of Ag specificity among a population of receptors. Thus, analysis of the CDR3 from the B cells found at the site of MS brain lesions would provide important insights into the question of how Abs participate in the formation of demyelinating plaques. CDR3 size spectratyping, originally used for the study of TCR transcripts (23), is a straightforward, PCR-based approach to evaluate CDR3 diversity. In this report B cell spectratyping was used to examine the rearranged IgG heavy chain transcripts repertoire of Ab-producing cells in demyelinating plaques of 10 MS patients. Extensive analysis of the brain IgGVH repertoire was performed, and profiles from patients and controls were compared. Two IgVH families, VH1 and VH4, were cloned and sequenced for the detailed analysis of the VDJ junctions. Our results show a significant perturbation of the B cell repertoire in most individuals. The finding of identical or nearly identical CDR3 sequences corroborates the clonality initially observed by spectratyping and suggests a significant role for clonally expanded B lymphocytes in MS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Postmortem brain specimens were obtained from both MS patients and non-MS controls. Frozen sections from 10 individuals with clinical history of MS and from four non-MS controls were divided in two for both molecular and immunohistochemical analyses. MS samples represent different stages of plaque activity. Control samples included four non-MS brain specimens as well as sequential PBL from three healthy individuals. Each brain sample was analyzed with luxol fast blue (LFB) counterstained with Harris hematoxylin for myelin integrity, trichrome (Tri) for astrocytes, and oil red O counterstained with hematoxylin for neutral lipids and EBM 11 for macrophages. These techniques allow for grading MS plaques according to Tourtellotte’s classification (24).
Oligonucleotide primers design
The design of Ig heavy chain VH1 to
VH6 family-specific oligonucleotide primers was
based on sequence alignments of the germline genes taken from the VBASE
directory of human Ig genes (I. M. Tomlinson, S. C. Williams,
O. Ignatovich, S. J. Corbett, and G. Winter, Medical Research
Council Centre for Protein Engineering, Cambridge, U.K.; this
information is also available on the internet:
http://www.mrc-cpe.cam.ac.uk/imt-doc/). Alignments were performed using
the MegAlign program of the LASERGENE package
(DNAstar, Madison, WI). Due to their size, family
VH1 and VH3 required two
and five different sense primers, respectively. The number of
degenerate nucleotides was maintained to a minimum to assure
specificity. The sequences of primers used in this study are as
follows: VH1a, 5'-AGCACAGCCTACATGGAGCTGAGC-3';
VH1b, 5'-AGCACAGCCTACATGGAGCTGAGG-3';
VH2, 5'-STCACCATCWCCAARGACA-3';
VH3a, 3'-CTGTATCTGCAAATGAACAGCCTG-3';
VH3b, 5'-CTGCAAATGAACAGTCTGARARCCG-3';
VH3c, 5'-CTGTATCTGCAAATGAACAG-3';
VH3d, 5'-CCAGAGACAATTCCARGAACA-3';
VH3e, 5'-CAAATGAACAGYCTGAGAG-3';
VH4, 5'-CTCCCTGAAGCTGAGCTCTGTG-3';
VH5. 5'-CTACCTGCAGTGGAGCAGCCTG-3'; and
VH6, 5'-CCAAGAACCAGTTCTCCCTGC-3'. The antisense
primer was the same for all amplifications and was homologous to the 5'
end of the constant 
gene (C 5'-GGCCAGGGGGAAGACCG-3').
B cell spectratyping
Total RNA was isolated from 100–200 mg of frozen brain tissue
homogenized with the Trizol reagent (Life Technologies, Bethesda, MD).
First-strand cDNA was synthesized with the Superscript II kit (Life
Technologies, Bethesda, MD) primed with random hexamers. The common
IgC primer was end labeled with
[-32P]dATP and T4 polynucleotide kinase (New
England Biolabs, Beverly, MA), and PCR was performed in 11 separate
tubes (one per VH family). Reactions were
conducted in 25 µl of PCR buffer (100 mM Tris-HCl (pH 9.0), 500 mM
KCl, 1% Triton X-100, and 0.2% BSA) containing 0.1 µM of each
primer, 0.2 mM dNTPs, 1.5 mM MgCl2, and 2 U of
Taq polymerase (Perkin-Elmer, Norwalk, CT). After 2 min of denaturation
at 94°C, 30 cycles of 94°C for 1 min, 55°C for 45 s, and
72°C for 1 min were performed, followed by a final extension of 7 min
at 72°C. Typically, 3-µl aliquots were electrophoresed on 6%
acrylamide/bisacrylamide (19/1) gels for 3–4 h at 30 W. The gels were
dried and exposed to high sensitivity radiographic film (Hyperfilm,
Amersham, Aylesbury, U.K.) for 1–3 days. Signals were quantified by
digitizing the films with a scanner (UMAX Data Systems, Taiwan), and
m.w. values for bands were assigned by analyzing the digitized images
with the Gelbase/Gelblot Pro3.3 software (UVP, Upland, CA).
Spectratyping and perturbation charts
The intensity of each band is expressed as a percentage relative to the summation of all the bands on a lane. Average spectratyping profiles of normal PBL were included for each VH family in the respective chart to facilitate visual reading of perturbation (white curve in three-dimensional charts). Perturbations were assigned essentially as reported by Gorochov et al. (25) for the TCR repertoire. Patients’ cumulative perturbation landscapes were assembled by plotting the positive and negative relative values from each VH family. This allows an overall representation of the perturbation for each patient for all Ig VH gene families.
Cloning of PCR products and sequencing
IgVH-rearranged PCR were gel purified with
QIAEX II (Qiagen, Valencia, CA) and cloned into PCR-TOPO 2.1 vector
with the TOPO-TA cloning system (Invitrogen, Carlsbad, CA). Twelve to
fifteen white colonies were randomly picked and grown overnight in
LB/Amp, and plasmids were purified with the kit PerfectPrep (5
Prime
3 Prime, Boulder, CO). Samples were sequenced with both forward
and reverse M13 primers using the ABI PRISM Big Dye Terminator cycle
sequencing kit (Perkin-Elmer, Norwalk, CT) in combination with halfTERM
Dye terminator sequencing reagent (Genpak, Stony Brook, NY) and were
run in the ABI PRISM 377 DNA sequencer (Applied Biosystems, Foster
City, CA).
Sequence analyses
Sequencher 3.1 software (Gene Codes Corp., Ann Harbor, MI) was used to construct a customized database that included all the germline DNA sequences and known alleles for VH1, VH4, and JH genes, as well as for D segments, as reported in the VBASE. The best matches with VH and JH germline genes were first assigned for each sequence. D segments were assigned following the criteria of Corbett et al. (26). Accordingly, neither D segment duplications, inversions, minor D segments, nor D segments with irregular spacer signals (DIR) usage were considered, and in general, a minimum of 10 exact matches with germline sequences was set as the cut-off for a valid association. Four sequences from family VH1 and three sequences from family VH4 were excluded from the analysis because they matched the opposite VH family, possibly as the result of a PCR cross-over (27). In addition, one sequence showed partial homology to the VH4 family but no similarity was found in either the D or the JH segments, and it was therefore discarded from the study.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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summarizes the results of the
immunohistochemistry. MS samples showed perivascular inflammation and a
different degree of myelin degradation. In some samples (WM, ME)
macrophages and astrocytosis were also detected in macroscopically
normal appearing white matter (NAWM; nonplaque; Fig. 1). As indicated by the lesion type in
Table I, the samples used in this study represent different stages of
activity.
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Oligonucleotide primers were designed to selectively amplify all the members of the VH1 (two sets), VH2, VH3 (five sets), VH4, VH5, and VH6 Ig gene families. The initial experimental design included the comparison of repertoires between the demyelinating tissue and the surrounding nonplaque material. However, when analyzed, Ig transcripts were detected in many of the NAWM samples, with a pattern similar to that observed in the pathological sections (data not shown). This finding together with histopathologic analyses of some of the samples suggest that periplaque tissue may contain B cells as found in the demyelinating plaque tissue. In addition, from the four non-MS controls, only one (with medical history of Alzheimer-type senile dementia) showed detectable RT-PCR amplification, indicating no significant B cell infiltration in the remaining three samples. To determine normal Ig profiles and to assess the CDR3 perturbation, PBL RNA from three healthy donors were also spectratyped, and the profiles were compared. To establish whether the PBL B cell repertoire changed over time, we studied the spectratyping profiles at 0, 2, and 4 wk; no significant changes were observed (data not shown).
The data obtained from an arbitrarily selected brain sample (SF; Fig. 2) illustrate the sequential experimental
analysis. Fig. 2A displays the mean spectratyping profile
for the VH3 family (primer set VH3a) in PBL from
three healthy individuals. Similarly shaped curves were obtained when
the rest of the VH families were analyzed (white
curves in Fig. 3). The observed Gaussian
distribution correlates well with the expected allocation of randomly
rearranged VH, D, and JH
gene segments, indicating that no bias in the CDR3 amplification
occurred. This, therefore, confirms the appropriate design of
VH-specific oligonucleotide primers.
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A) are computed and displayed in a two-dimensional graph
(Fig. 2, B and C). The cumulative perturbations
from the same patient are then arranged in a single three-dimensional
chart (Fig. 2D).
Restricted repertoire patterns were observed in most of the
IgVH families when samples from MS patients were
compared with the mean of normal PBL. As shown in Fig. 3, several
samples show an oligoclonal profile, consistent with a restricted B
cell response. Family IgVH1 (primer sets
VH1a and VH1b) was one of
the families with the highest proportion of samples showing oligoclonal
bands (i.e., samples WM, JR, MF, SF, and ME). These findings are in
contrast with the relatively broader CDR3 expression pattern seen in
samples of the whole IgVH3 family (primer sets
3a–3e). This correlates with previously reported data
(28) and could be explained in part by the larger size of
the family VH3, which counts 22 functional
genomic segments compared with only 11 in family
VH1. However, when the profile of family
VH1 was compared with that of the family
VH4, which is identical in size, the former still
displays a more restricted pattern. The detailed representation of the
perturbation within families VH1 (Fig. 4A) and
VH4 (Fig. 4B) shows extensive positive
and negative perturbation in several samples, with those belonging to
family VH1 being the most clearly affected.
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summarizes the perturbation
profiles obtained for all IgVH families in each
of the 10 MS patients studied. A marked perturbation was observed in
most patients. Although the degree of perturbation varies among
patients, positive differences were observed in all cases when compared
with control PBL, implying a preferential expansion of defined clones
rather than selective suppression of others. No obvious correlation was
observed between the histopathologic classification of the plaque and
the degree of perturbation.
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To further analyze the perturbation patterns, new reverse
transcribed PCR products were cloned into a plasmid vector, and
12–15 colonies/sample were sequenced for both
IgVH families. A total of 108 sequences were
analyzed for the family VH1 clones, and 91
sequences were analyzed for the family VH4
clones. Of those 199 sequences, 36 (18 from each family) corresponded
to nonproductive rearrangements (i.e., contained at least one stop
codon in each possible reading frame, or the CDR3 was not long enough
for VH, D, and JH segments
to be identified) and, thus, were not further analyzed.
Tables II and III summarize the
sequencing data of families VH1 and
VH4 for the MS patients and the non-MS sample
(HJ) used as a control. The names of the closest germline genes are
indicated in parentheses next to each segment. D segments are
underlined when a significant match with the germline sequence has been
found. Not a single productive rearrangement of 12 independent colonies
that were scrutinized could be identified on sample RR for family
VH4. Most of the MS samples showed clusters of
sequences (contigs) that were >98% identical (at the nucleotide
level) throughout their CDR3, reflecting clonal expansion.
Significantly, the sample that showed the fewest number of expanded
clones for both IgVH families was HJ (the non-MS
control), suggesting an unrestricted B cell population. The average
numbers of clones per contig (second column in Table II) were 4.2 and
2.8 for the VH1 and VH4
families, respectively. These data are in agreement with our previous
observation that family VH1 showed a more
restricted spectratyping profile compared with that of family
VH4. From our analysis it would appear that there
is no obvious relationship between either the length of the CDR3 or the
number of N nucleotides added and the type of demyelinating lesion.
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Family-specific sense primers were designed to be homologous to part of the IgVH FR3. Accordingly only the last 60 nucleotides from each VH family gene were identified, on the average. Although this was sufficient to identify most of VH1 and VH4 family members, the specific VH gene, usage in some clones could not be definitively ascertained. In those cases, all the matching members are mentioned in Tables II and III, separated by a backslash. IgVH family members VH4–39 (DP79) and VH4–34 (DP63) accounted for 25% of the total rearranged Ig that used the VH4 family. This correlates well with previously reported data in which VH4–39 was the most frequently expressed VH4 gene in an MS brain sample (27). Our findings are also in agreement with those of Suzuki et al. (29), who reported that in normal individuals VH4–34 is the most used IgVH gene. The most used VH1 gene in our study was VH1–69 (DP10), with a frequency of 30%. The fact that nearly all members from the analyzed VH families were found confirms that the primer design was accurate for our purposes.
Usage of D segments
Diversity segments were assigned according to the criteria of
Corbett et al. (26) based on homology with the VBASE. Only
matches to true D segments were considered without taking into account
the DIR genes or minor D segments. This reasoning also
excludes D duplications and inversions as valid rearrangements.
Our data showed a broad D segment utilization with at least one member
of each family. Known D segments were only found in 49% (21 of 43) and
40% (19 of 48) of the different productive rearrangements for families
VH1 and VH4, respectively.
Reading frame (RF) usage was also studied for the identified D segments
(shown in parentheses next to the D gene name in Tables II and III).
Given that the three possible RF in D segments can be classified as
hydrophilic, hydrophobic, or containing a stop codon, we analyzed their
usage distribution. In agreement with a recently published analysis of
893 rearranged human heavy chain sequences (26), we
detected a strong bias toward the hydrophilic RF usage by
VH1 (81% (17 of 21)) and
VH4 (58% (11 of 19)) for the rearrangements
using a known D segment. As shown in Fig. 6, the frequency of D segment utilization
(by family) correlates reasonably well when compared with frequencies
reported from two other independent studies (26, 30) and
to expected frequencies (calculated according to the size of each
family and assuming a random usage of the known functional D segments).
However, a lower frequency was observed here for D5 and a higher one
for D2 family members (p < 0.05). All three
studies showed significant differences when compared with the expected
frequency of D segments, particularly for families D1 and D3. The
over-representation of segments from family D3 and the
under-representation of those from family D1 in these three studies
suggest that the selective usage of particular D family segments is
independent of their family size. When individual D segments were
analyzed in our study, a significant overuse was found for D2–2,
D2–15, D3–10, D3–22, and D6–19 if compared with the expected
frequencies (p = 0.05). The same tendency for
these segments has been reported previously for the general population
by Corbett et al. (26).
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The utilization of JH genes was analyzed by
aligning the obtained sequences to our CDR3 gene database. Because the
reverse primer in all amplifications was homologous to a constant
gene, the entire JH region was sequenced, and
therefore allele assignment was possible. At least one member of each
family was identified. In a few cases, allelic discrimination between
JH4a and JH4d was not
possible because mutations were found at one or both positions that
differentiate them. In those cases, both members are named and
separated by a backslash in Tables II and III. Nevertheless, the
utilization of JH genes could be compared with
that of two other independent studies as well as to their expected
frequencies (Fig. 6). There were no significant differences among the
three studies in terms of JH family gene
utilization, except for the higher frequency in
JH6 reported by Brezinschek et al.
(28). However, all three studies showed a significant
over-representation of JH4 family members
(p = 0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been previously shown that oligoclonal Igs are produced within the CNS in MS (2, 32). In a series of elegant experiments, Knopf and colleagues (32) demonstrated in a rat model with an intact blood-brain barrier the trafficking of activated Ag-specific B cells into the brain, retention, and Ab production. Their data demonstrated that the brain microenvironment supports the development of Ag-directed humoral immunity. Following activation and somatic hypermutation in peripheral lymphoid organs, B cells home to the CNS where they mature to plasma cells (33). The high level of rearranged transcripts we observed in MS brains, but not in non-MS noninflammatory brain tissues suggests that the oligoclonal bands found by spectratyping are likely to be the result of Ab synthesis within the plaques. Furthermore, the morphological analysis of early MS plaques revealed that most of the infiltrating cells positive for intracellular Ig staining were plasma cells (1).
Surprisingly, similar patterns of amplification were observed in some
nonplaque tissue samples, indicating a specific, but disseminated,
humoral immune response in the MS CNS. Histopathologic analysis of MS
brain tissue showed clear evidence of local inflammation in the
demyelinating plaque and periplaque tissue as well as in NAWM
demonstrated by the presence of perivascular hypercellularity, mainly
mononuclear cells, macrophages, and astrocytes (Fig. 1). In a recent
report, Goodkin and colleagues (34) detected significant
pathological characteristics in the NAWM of MS brains when analyzed by
sophisticated imaging techniques such as gadolinium enhancement, water
proton density, water proton T2 relaxation time constants,
magnetization transfer ratio, and T1-weighted signal intensity.
Abnormalities in these NAWM regions generally preceded the appearance
of the macroscopically visible lesion. With one notable exception (a
specimen from a patient with Alzheimer-type senile dementia; HJ), our
attempts to amplify IgVH rearranged genes from
brain samples with no evidence of inflammatory disease failed
consistently, indicating that few or no plasma cells were present in
such tissue. On the other hand, MS samples showed robust and
reproducible CDR3 amplifications, but significantly different from the
normal distribution observed in PBL, suggesting specific B cell
expansions.
Spectratyping of IgVH rearrangements showed a relatively restricted VH1 repertoire. A recent study provides evidence that the CDR3 structure, independent from the VH framework, is sufficient to define the specificity of an Ab (35). When a more detailed anal-ysis of the CDR3 region in VH1 and VH4 gene families (both account for 11 genes) was performed, the former still showed a more restricted pattern in most samples. Rearranged transcripts from these two families were cloned and sequenced, and a direct correlation between the restricted patterns in the spectratyping analysis and the number of identical clones was observed. In most cases where an oligoclonal pattern was found, a homogeneous population was identified by sequencing, and generally, the more restricted the pattern, the larger the resulting contig. In this context, Hauser and colleagues demonstrated that clonally restricted B cell populations are also present in peripheral blood from MS patients (36). More recently, Qin et al. (4) and Owens et al. (27) have shown that restricted B cell populations can be found in the brain and CSF of such patients.
Family VH4 has been reported to be the most frequently used VH family in the blood from rheumatoid arthritis patients (37) and in MS brains (27), occurring well above the expected frequency. In those and other quantitative studies, family VH1 was markedly under-represented (28, 38, 39). In our study we found a higher clonal distribution for members of the family VH1 compared with those of VH4, suggesting the relevance of this IgVH gene family in MS. However, given that our study was not intended to be quantitative, we could not compare frequencies, and the relative role and involvement of VH1 vs VH4 families remain to be determined. The frequency of D segment usage reported here correlates well with the findings of other studies involving both autoimmune diseases and normal individuals. This suggests that the bias found toward the usage of particular D segments may be due to molecular, rather than immune, selection mechanisms.
In a recent study Corbett et al. (26), using 10 nucleotides as the minimum cut-off for aligning a D segment, were unable to find any evidence for the use of DIR segments and minor segments or for any other mechanisms such as D duplication or inversion as previously suggested (28, 40). Using similar stringent criteria, we found that nearly half the MS brain sequences we analyzed did not match a germline D segment, a finding in complete agreement with the analysis of 893 rearranged transcripts reported by the same investigators (26). The reason for this is not yet clear, but it is possible that exonuclease activity, somatic mutation, and addition of N nucleotides have modified the sequence of a germline D segment to the point of making it different enough to be recognized by the alignment algorithm. As far as the usage of JH genes is concerned, family JH4 was the most frequently used. However, this over-representation has no direct relationship to the type of lesion in our cohort of MS patients. In studies of other autoimmune diseases and normal individuals, this gene family has been found to be over-represented as well (28, 29, 41). This suggests that the preferential usage of this gene family is not related to an Ag-mediated immune response but, rather, would correspond to molecular mechanisms. In this regard, it has been proposed that because family JH4 is the only one with a conventional 23-bp spacer between the 5' heptamer and nonamer coding sequences, the JH-D recombination would be facilitated (28).
It is now well established that a higher than expected replacement/silent (R/S) ratio in CDR and a lower than expected R/S ratio in FR regions usually reflect positive and negative selection pressures, respectively; this is consistent with Ag-driven affinity maturation. Given our experimental design, only CDR3 and part of FR3 were sequenced; hence, CDR1, CDR2, and FR could not be analyzed for R/S ratios. However, the fact that only a single two-sequence contig was found in the control sample (HJ) is consistent with Ag-specific activation of the expanded clones through positive selection. Dominant CDR3 motifs among different patients have not been detected. Genetic heterogeneity among the studied individuals and/or determinant spreading (42) may explain different epitope selection. Our experimental approach requires considering the magnitude of the polymerase-induced error rate. Previous studies studying PCR fidelity showed that the error rate could be as low as 8 x 10-6 (43) and as high as 2.1 x 10-4 (44) depending on the templates and PCR conditions. Breszinchek et al. (28) reported an error rate of 1–2 x 10-3 for single-cell PCR of rearranged IgM genes on CD5+ B cells, corresponding to 0.3–0.5 mutations per VH gene. These data indicate that PCR-generated mutations do not interfere significantly with our results.
In conclusion, our findings are consistent with the hypothesis that an Ag-driven process may be responsible for the accumulation of particular subsets of B cells in the MS brain rather than this being the result of random B cell activation. Moreover, it is conceivable that such B cells underwent clonal expansion in response to chronic stimulation by pathogens or autoantigens. The identification of the Ag or Ags against which this selective B cell response occurs may provide important insights into the question of blood-brain barrier disruption, immune-mediated demyelination, and atrophy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jorge Oksenberg, Department of Neurology, University of California, 513 Parnassus Avenue, Medical Science Building, Room S-256, San Francisco, CA 94143-0435. E-mail address:
3 Abbreviations used in this paper: MS, multiple sclerosis; CSF, cerebrospinal fluid; CDR3, complementary-determining region 3; D, diversity genes; JH, joining segments; MBP, myelin basic protein; LFB, luxol fast blue; Tri, trichrome; DIR, D segments with irregular spacer signals; NAWM, normal appearing white matter; FR, framework region; RF, reading frame; R/S, replacement to silent ratios.
Received for publication June 8, 1999. Accepted for publication August 24, 2999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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staining patterns. Ann. Neurol. 11:408.[Medline]
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R. Hohlfeld and H. Wekerle Autoimmune concepts of multiple sclerosis as a basis for selective immunotherapy: From pipe dreams to (therapeutic) pipelines PNAS, October 5, 2004; 101(suppl_2): 14599 - 14606. [Abstract] [Full Text] [PDF]
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A. Corcione, S. Casazza, E. Ferretti, D. Giunti, E. Zappia, A. Pistorio, C. Gambini, G. L. Mancardi, A. Uccelli, and V. Pistoia Recapitulation of B cell differentiation in the central nervous system of patients with multiple sclerosis PNAS, July 27, 2004; 101(30): 11064 - 11069. [Abstract] [Full Text] [PDF]
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A. M. Ritchie, D. H. Gilden, R. A. Williamson, M. P. Burgoon, X. Yu, K. Helm, J. R. Corboy, and G. P. Owens Comparative Analysis of the CD19+ and CD138+ Cell Antibody Repertoires in the Cerebrospinal Fluid of Patients with Multiple Sclerosis J. Immunol., July 1, 2004; 173(1): 649 - 656. [Abstract] [Full Text] [PDF]
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G. P. Owens, A. M. Ritchie, M. P. Burgoon, R. A. Williamson, J. R. Corboy, and D. H. Gilden Single-Cell Repertoire Analysis Demonstrates that Clonal Expansion Is a Prominent Feature of the B Cell Response in Multiple Sclerosis Cerebrospinal Fluid J. Immunol., September 1, 2003; 171(5): 2725 - 2733. [Abstract] [Full Text] [PDF]
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 M. Zocher, P. A. Baeuerle, T. Dreier, and A. Iglesias Specific depletion of autoreactive B lymphocytes by a recombinant fusion protein in vitro and in vivo Int. Immunol., July 1, 2003; 15(7): 789 - 796. [Abstract] [Full Text] [PDF]
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J. A. Coronella, C. Spier, M. Welch, K. T. Trevor, A. T. Stopeck, H. Villar, and E. M. Hersh Antigen-Driven Oligoclonal Expansion of Tumor-Infiltrating B Cells in Infiltrating Ductal Carcinoma of the Breast J. Immunol., August 15, 2002; 169(4): 1829 - 1836. [Abstract] [Full Text] [PDF]
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H.-C. von Budingen, S. L. Hauser, A. Fuhrmann, C. B. Nabavi, J. I. Lee, and C. P. Genain Molecular characterization of antibody specificities against myelin/oligodendrocyte glycoprotein in autoimmune demyelination PNAS, June 11, 2002; 99(12): 8207 - 8212. [Abstract] [Full Text] [PDF]
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 S.E. Baranzini, K. Laxer, R. Saketkhoo, M.K. Elkins, J.M. Parent, R. Mantegazza, and J.R. Oksenberg Analysis of antibody gene rearrangement, usage, and specificity in chronic focal encephalitis Neurology, March 12, 2002; 58(5): 709 - 716. [Abstract] [Full Text] [PDF]
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M. Jacobsen, S. Cepok, E. Quak, M. Happel, R. Gaber, A. Ziegler, S. Schock, W. H. Oertel, N. Sommer, and B. Hemmer Oligoclonal expansion of memory CD8+ T cells in cerebrospinal fluid from multiple sclerosis patients Brain, March 1, 2002; 125(3): 538 - 550. [Abstract] [Full Text] [PDF]
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R. A. Williamson, M. P. Burgoon, G. P. Owens, O. Ghausi, E. Leclerc, L. Firme, S. Carlson, J. Corboy, P. W. H. I. Parren, P. P. Sanna, et al. Anti-DNA antibodies are a major component of the intrathecal B cell response in multiple sclerosis PNAS, February 1, 2001; (2001) 31567598. [Abstract] [Full Text]
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 D. H. Gilden, M. P. Burgoon, B. K. Kleinschmidt-DeMasters, R. A. Williamson, O. Ghausi, D. R. Burton, and G. P. Owens Molecular Immunologic Strategies to Identify Antigens and B-Cell Responses Unique to Multiple Sclerosis Arch Neurol, January 1, 2001; 58(1): 43 - 48. [Abstract] [Full Text] [PDF]
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S. E. Baranzini, C. Elfstrom, S.-Y. Chang, C. Butunoi, R. Murray, R. Higuchi, and J. R. Oksenberg Transcriptional Analysis of Multiple Sclerosis Brain Lesions Reveals a Complex Pattern of Cytokine Expression J. Immunol., December 1, 2000; 165(11): 6576 - 6582. [Abstract] [Full Text] [PDF]
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 S. A. Riemersma, E. S. Jordanova, R. F. J. Schop, K. Philippo, L. H. J. Looijenga, E. Schuuring, and P. M. Kluin Extensive genetic alterations of the HLA region, including homozygous deletions of HLA class II genes in B-cell lymphomas arising in immune-privileged sites Blood, November 15, 2000; 96(10): 3569 - 3577. [Abstract] [Full Text] [PDF]
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M. Colombo, M. Dono, P. Gazzola, S. Roncella, A. Valetto, N. Chiorazzi, G. L. Mancardi, and M. Ferrarini Accumulation of Clonally Related B Lymphocytes in the Cerebrospinal Fluid of Multiple Sclerosis Patients J. Immunol., March 1, 2000; 164(5): 2782 - 2789. [Abstract] [Full Text] [PDF]
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R. A. Williamson, M. P. Burgoon, G. P. Owens, O. Ghausi, E. Leclerc, L. Firme, S. Carlson, J. Corboy, P. W. H. I. Parren, P. P. Sanna, et al. Anti-DNA antibodies are a major component of the intrathecal B cell response in multiple sclerosis PNAS, February 13, 2001; 98(4): 1793 - 1798. [Abstract] [Full Text] [PDF]
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