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The Journal of Immunology, 2004, 173: 649-656.
Copyright © 2004 by The American Association of Immunologists

Comparative Analysis of the CD19+ and CD138+ Cell Antibody Repertoires in the Cerebrospinal Fluid of Patients with Multiple Sclerosis1

Alanna M. Ritchie*, Donald H. Gilden*,{dagger}, R. Anthony Williamson§, Mark P. Burgoon*, Xiaoli Yu*, Karen Helm{ddagger}, John R. Corboy* and Gregory P. Owens2,*

Departments of * Neurology and {dagger} Microbiology and {ddagger} University of Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, CO 80262; and § Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Increased amounts of intrathecally synthesized IgG and oligoclonal bands have long been recognized as a hallmark of multiple sclerosis (MS). B cells and plasma cells are components of the inflammatory infiltrates in both active and chronic MS lesions, and increased numbers of these cells are present in MS cerebrospinal fluid (CSF). Single-cell RT-PCR was used to analyze both the CD19+ B cell and CD138+ plasma cell populations in CSF of two patients with clinically definite MS and of one MS patient whose CSF was obtained after a clinically isolated syndrome, but before the second episode. Sequence analysis of amplified IgG V region sequences identified the rearranged germline segments, extent of somatic mutation, and clonal relationships within and between the two cell populations in the three MS patients. Expanded B cell and plasma cell clones were detected in each MS CSF and in all three patients the CD138+ IgG repertoire was more restricted. However, little if any significant sequence overlap was observed between the CD19+ and CD138+ repertoires of each donor. Detection of plasma cell clones by single-cell PCR will facilitate the in vitro production of recombinant Abs useful in identifying disease-relevant Ags.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Multiple sclerosis (MS)3 is a chronic inflammatory demyelinating disease of the CNS. The inflammatory infiltrates in areas of active demyelination consist of T cells, varying levels of B and plasma cells, and activated macrophages or microglia (1). Consistent with the recruitment of B cells into MS plaques, the brain and cerebrospinal fluid (CSF) of nearly all MS patients contain increased levels of intrathecally produced IgG and oligoclonal bands (OGBs) (reviewed in Ref.2). However, OGBs are also present in other inflammatory and infectious CNS diseases, such as subacute sclerosing panencephalitis, neurosyphilis, and cryptococcal and mumps meningitis. In each instance where specificity has been studied, OGBs were shown to be Abs directed against the agent that causes disease (3, 4, 5, 6). In MS, the specificity of OGBs is unknown. It is also unclear whether Ab produced in MS plaques and CSF reflects a host response against an infectious pathogen or whether Ab plays a role in MS pathology.

Molecular studies to characterize the H chain variable regions of IgG expressed in MS plaques and CSF have shown that clonal expansion of B cells is a prominent feature (7, 8, 9, 10, 11, 12, 13). The extensive somatic hypermutation of IgG V region sequences and the restricted Ig populations observed in each repertoire are consistent with a targeted humoral response in the CNS of MS patients. These characteristics of the IgG repertoire have also been found in CSF of some individuals during the initial episode of neurologic disease that ultimately became clinically definite MS, indicating that B cell-mediated immune responses do occur early in disease (10). We previously used single-cell PCR to study CD19+ B cell clonal expansion in CSF of relapsing-remitting MS patients (8). Because the bulk of IgG and OGBs synthesized in MS plaques and CSF are most likely the products of fully differentiated plasma cells, we have extended these studies to the V region sequences of both B cells and plasma cells recovered from the CSF of MS subjects at different stages of disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
MS CSF collection and single-cell sorting

CSF (5–25 ml) was collected from MS patients according to procedures approved by the University of Colorado School of Medicine Institutional Review Board. All MS patients had multiple white matter lesions demonstrated by brain magnetic resonance imaging, and their CSF contained OGBs and elevated levels of IgG (Table I). None of the patients had received steroids or immunomodulatory drugs for at least 1 mo before sampling. CD19+ B cells and CD138+ plasma cells were recovered from CSF by one of two similar protocols. The CSF from MS subjects 02-19 and 02-24 was first divided into equal aliquots, centrifuged at 1500 rpm at room temperature for 10 min, and each cell pellet was suspended in ~200 µl of residual CSF. One tube was incubated at room temperature with 5 µl of PE-anti-CD138 and 5 µl of FITC-anti-CD3 and the second tube was incubated with 5 µl of PE-anti-CD19 and 5 µl of FITC-anti-CD3. After a 20-min incubation at room temperature in reduced light, cells were mixed with 500 µl of sterile PBS, placed on ice, and immediately sorted using a MoFlo flow cytometer (DakoCytomation, Fort Collins, CO). To directly compare and sort the CD19+ and CD138+ cell populations from the same aliquot of CSF, the cell pellet of CSF–MS03-01 was suspended in ~200 µl of CSF and incubated at room temperature with 5 µl of R-PE-conjugated Ab directed against the human cell surface marker CD138 (PE-anti-CD138; plasma cells), 5 µl of allophycocyanin-conjugated Ab directed against CD19 (allophycocyanin-anti-CD19; B cells), and 5 µl of FITC-Ab directed against CD3 (FITC-anti-CD3; T cells) (Caltag Laboratories, Burlingame, CA). After a 20-min incubation at room temperature in reduced light, cells were diluted with 500 µl of sterile PBS and sorted.


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  		Table I. Clinical features of MS patients undergoing single B cell analysis of CSF

 
After gating for cells in the size range of lymphocytes and plasma cells by light scattering, single CD138+ plasma or CD19+ B cells were deposited into wells of a 200-µl, 96-well PCR plate (catalogue no. T-3060; ISC Bioexpress, Kaysville, UT) containing 20 µl of 1x reverse transcriptase buffer (Invitrogen, Carlsbad, CA). After a preliminary pre-sort to gauge the percentage of CD19+ and CD138+ cells in each CSF, samples were sorted until one to three plates of CD138+ and CD19+ cells were obtained. Because MS02-19 CSF contained a lower proportion of plasma cells, only 55 CD138+ cells were sorted and analyzed. Cells sorted from MS02-24 and MS03-1 CSF were analyzed immediately by RT-PCR on one plate each of CD138+ and CD19+ cells before storage at –70°C. All other sorted cells were stored at –70°C until processing for cDNA synthesis.

cDNA synthesis and amplification of VH and VL sequences

First-strand cDNA synthesis and subsequent PCR amplifications of V region sequences were performed using an I-Cycler (Bio-Rad, Hercules, CA) in a 96-well format as described previously (8). VH and {kappa} or {lambda} V regions were amplified by a nested PCR strategy in which pools of conserved family-based leader and framework 1 primers were used in conjunction with H and L chain C region primers. To amplify IgG VH regions, 5 µl of cDNA was used in the primary PCR with a pool of leader primers specific for VH families 1–5 and the conserved C region primer CH{gamma}1. Similarly, a separate primary PCR was performed for MS subject 03-01 with the same VH leader primers and the modified IgM-specific primer CHµ1 (5'-aaggcagcagcacctgtgagg), followed by a nested PCR using 2 µl of primary reaction product and a pool of conserved framework 1 primers for VH families 1–5 with the conserved IgG and IgM C region primers CH{gamma}2 (5'-caccggttcggggaagtagtcc) and CHµ2 (IgM). See Table II of Ref.8 for a complete list of primer sequences. All PCRs were performed in a 50-µl reaction volume containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.01% gelatin, 200 µM dNTPs, 100 pmol of each primer, and 2 U Taq polymerase (Applied Biosystems, Branchburg, NJ). The cycle conditions for the primary PCR were as follows: an initial denaturation cycle at 95°C for 5 min followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min and a final extension cycle at 72°C for 7 min. The nested PCR followed the same protocol except that the annealing step was conducted at 56.5°C for optimum performance of the VH framework 1 primers. Amplification of {kappa}- or {lambda}-chain sequences was performed similarly with family-based pools of leader and framework 1 primers (V{kappa} families 1–5 or V{lambda} families (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) used in combination with L chain-specific C region primers.


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  		Table II. CD19 and CD138 V region sequences analyzed in MS CSF

 
To minimize the potential for PCR cross-contamination between individual wells, all PCR amplifications were performed in a designated PCR room. Separate multichannel pipettes were used for PCR solutions, for cDNA dispensing, and for post-PCR analyses. The latter was also done in a separate designated area. Primers were aliquoted and stored lyophilized at –20°C until needed. Fresh aliquots of primer were used for each CSF analyzed.

Purification and analyses

All PCR products were verified by agarose gel electrophoresis and purified with the Qiaquick PCR Purification kit (Qiagen, Valencia, CA) or the Montage PCR purification kit (Millipore, Bedford, MA). PCR products were sequenced by the University of Colorado-Cancer Center DNA Sequencing & Analysis Core (University of Colorado Health Sciences Center, Denver, CO) with conserved C region primers as described elsewhere (8). Individual sequences were analyzed with DNASIS Max software and aligned with the database V Base using the online software program DNAPlot from the Cambridge Center for Protein Engineering (http:/www.mrc-cpe.cam.ac.uk). This online service was used to identify the closest V region germline segments and to determine the extent of sequence homology for each of the Ab H and L chain sequences analyzed. For consistency, homologies to germline segments of donor DP (14) were used when applicable. Otherwise, the gene locus was used to identify the most homologous germline segment.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sorting of CD19+ and CD138+ cells and amplification of V region sequences

In CSF of the three MS subjects, CD19+ cells comprised ~4% (3.7–4.4%) of the cell population compared with CD138+ cells which varied from 0.25 to 3.5% of cells. An analysis of cells sorted from MS02-24 CSF is shown in Fig. 1, A–C. Fig. 1B shows that from the 32,508 cells selected by light scattering (Fig. 1A), 1151 were CD138+ cells. Fig. 1C shows the sorting of CD19+ cells from a separate aliquot of CSF from the same patient. From the 3,744 cells analyzed in Fig. 1C, 137 CD19+ cells were obtained. In MS03-01 CSF, in which the CD19+ and CD138+ cell populations could be viewed simultaneously, 634 CD19+ (Fig. 1D) and 109 CD138+ cells (data not shown) were obtained from the 16,166 cells sorted. After depositing single cells from each population into 96-well plates, cDNA was synthesized and nested PCR was used to amplify the H and L chain V region sequences. Positive PCR products were analyzed and their sequences were aligned to those in the V Base database containing all known H and L chain germline segments. The germline family was identified along with the most homologous germline segment and extent of homology for each H and L chain V region sequence. The Ig H and L chain CDR3 amino acid sequence expressed by each cell was used as a unique clonal identifier to categorize B and plasma cell populations.



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  		FIGURE 1. FACS purification of CD138+ plasma cells and CD19+ B cells from MS02-24 CSF (A–C) and CD19+ cells from MS03-01 CSF (D). CSF from MS02-24 was divided into two aliquots and cells were collected by centrifugation. Cells were suspended in residual CSF (200 µl) and incubated with 5 µl each of fluorescent-conjugated Abs to human CD3 and CD138 or with Abs to human CD3 and CD19 (see Materials and Methods). Cells in the approximate size range of lymphocytes and plasma cells (red) were selected first by light scattering (forward scatter, FSC; side scatter, SSC; A), then by either CD138 or CD19 expression (R3 in B and C, respectively), and single cells were expelled into separate wells of a 96-well PCR plate. Note that the sorted CD138+ cells exhibited increased light scattering compared with the lymphocyte population, as expected for the larger plasma cells. Cells in MS03-1 CSF were collected by centrifugation, suspended in 200 µl of residual CSF, incubated with labeled Abs to CD3, CD138, and CD19, and sorted as described in Materials and Methods. As shown in D for the sorting of the CD19+ cell population (R7), some CD138+ cells (red) that are weak expressers of CD19 are included in this gate and account for ~13% of the CD19+ cell population.

 
Table II lists the number of CD19+ and CD138+ V region sequences analyzed from each MS CSF. cDNA synthesis immediately after cell sorting enhanced PCR amplification efficiency of VH sequences. When only IgG-expressing cells were examined, the efficiency of V region amplification approached 50% for CD19+ cells in MS02-24 and >70% for MS03-01 when both IgG and IgM repertoires were analyzed. In contrast, the efficiency of IgG amplification in MS02-19 CD19+ CSF cells, which had been stored frozen at –80°C before cDNA synthesis, was only ~10%. The efficiency of IgG VH amplification in CD138+ plasma cells from MS02-24 and MS03-01 CSF was ~80% compared with 45% for CD138+ cells from MS02-19. The increased efficiency of V region amplification in CD138+ cells may reflect the increased levels of IgG mRNA in plasma cells.

Clonal expansion of B and plasma cells in MS CSF at all stages of disease

Expanded B cell and plasma cell clones were detected in the CSF of each MS subject. CSF IgG-positive B cells in clonal populations (Table II) varied from 12% (MS02-24) to 39.5% (MS03-01), whereas the IgM repertoire of CD19+ cells recovered from the CSF of MS03-01 was polyclonal. The fraction of CSF plasma cells within clonal groups was even more pronounced, ranging from 26% (MS02-24) to >75% (MS02-19 and MS03-01).

Table III lists the CDR3 amino acid sequences and features of all of the VH and matching VL sequences amplified from CD138+ and CD19+ cells of MS subject 02-19 CSF, while Tables IV and V list those of CD138+ and CD19+ clones detected in MS02-24 and MS03-01 CSF. The majority of V region sequences analyzed represented functional rearrangements, although occasional nonproductive rearrangements, usually L chains, were sometimes detected in cells that also expressed a functional L chain rearrangement (clone 2 in Tables IV and V). In the MS02-19 plasma cell population, eight distinct clones were detected, and cells of each clone expressed an identical V(D)J (H chain) and/or VJ (L chain) recombination. Alignment of H and L chain sequences within a group revealed common sets of somatic mutations that confirmed their clonal relationships, e.g., all VH sequences from clone 1 of MS02-19 shared 17 identical somatic mutations and an additional 2 somatic mutations in one plasma cell from clone 1 indicated a degree of clonal variation within this population. Intraclonal variants were also seen in plasma cell populations from the other MS CSFs (Tables IV and V). CSF of MS02-24 CSF contained 8 unique clones (Table IV) and MS03-01 contained 13 (Table V), whereas relatively fewer clones were present in each of the CD19+ populations. Four B cell clones were detected in CSF of MS subjects 02-19 and 03-01 and two B cell clones were found in CSF of MS02-24. Most IgG sequences from both B and plasma cell populations were mutated, some quite extensively, with homologies ranging from 85 to 99% to their closest germline segment.


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  		Table III. Features of V region sequences amplified from MS02–19 CSF CD138+ and CD19+ populationsa

 

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  		Table IV. Features of clonal V region sequences amplified from MS02–24 CD138+ and CD19+ cellsa

 

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  		Table V. Features of clonal V region sequences amplified from MS03–1 CD138+ and CD19+ cell populationsa

 
The clonal relationships between the CD19+ and CD138+ cells in each CSF were minimal. For example, of the eight plasma cell clones identified in MS02-19 (Table III), only clone 5 (CDR3 amino acid sequence IRSMHYLDH) was detected as a clonal population in the CD19+ repertoire (clone 1). Of the 13 CD138+ clones recovered from the CSF of MS03-01, only clones 1 and 5 were also detected as CD19+ cell clones (Table V) and sequences related to plasma cell clones 6, 9, and 11 were detected once in the CD19+ repertoire. Because some CD138+ cells in the CD19+ cell population express low levels of CD19 as shown in Fig. 1D for MS03-1 CSF (CD138+ cells indicated in red), much of the overlap between the CD19+ and CD138+ repertoires of each CSF was probably due to the presence of some CD138+ cells in the CD19+ pool. In MS03-1, where the sorting of CD19+ and CD138+ cells was combined, 13% of the sorted CD19+ cell population (Fig. 1D, R7) was also CD138+. In each CSF, there were also CD19+ clones that did not appear in the CD138+ repertoire. Although clonally related, the few cells appearing as single sequences in both the CD19+ and CD138+ cell populations of each CSF (boldface sequences in Tables III–V) were not included in the clonal populations counted in Table II.

The VH4 germline family segments dominated the plasma cell repertoire in each MS CSF (Tables III–V). In CSF of MS02-19, 10 (77%) of the 13 unique VH sequences detected in the CD138+ cell population used VH4 germline segments; in MS02-24, 40 (66%) of 61 unique VH sequences utilized VH4 germline sequences, and in MS03-01, VH4 germlines comprised 18 (58%) of 31 unique VH sequences. In each of the CD138+ repertoires, the VH3 germline made up the bulk of the remaining V region segments used. For MS03-01, where both the IgM- and IgG-expressing CD19+ repertoires were analyzed, there was a striking shift to VH4 germline usage in the IgG-expressing CD19+ and CD138+ cells. VH4 germline segments accounted for only 5 (19%) of 26 unique sequences in the polyclonal IgM repertoire (data not shown), but for 12 (44%) of 27 unique sequences in the CD19+ IgG repertoire. Surprisingly, VH1 germline segments were almost completely absent in the CD138+ repertoires; only 2 of 105 (1.9%) unique VH sequences recovered from plasma cells in MS CSF were VH1 germline segments. VH1 genes comprise ~20% of the functional germline segments in the human chromosome, and their appearance in the normal peripheral blood repertoire usually approximates this value (15).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Since brain interstitial fluid extravasates into the subarachnoid and ventricular spaces (16), the cellular and protein composition of human CSF can be a useful surrogate to monitor the microenvironment of the brain. In MS, the CSF is characterized by increased numbers of both B and plasma cells compared with CSF of patients with noninflammatory neurologic disease (17). We have analyzed the humoral immune response in MS CSF of three MS patients using fluorescence-activated cell sorting and single-cell RT-PCR. Both B cell and plasma cell populations, sorted based on the expression of CD19 and CD138, were recovered from CSF of all three subjects, even when, as for subject MS03-01, the cell count was relatively low at less than a single cell per microliter of CSF (Table II). The rearranged V regions expressed by these cells were then amplified and sequenced. The efficiency of V region amplification from single B and plasma cells was significantly improved by synthesizing cDNA immediately after sorting rather than after storage at –70°C. This increased efficiency allowed a meaningful representation of the repertoires for each CSF from a single plate of sorted cells.

Consistent with our previous analyses of the CD19+ B cell repertoire in CSF of patients with relapsing-remitting MS (8), clonal expansion was also a prominent feature of the CD138+ plasma cell repertoire of MS CSF. At least 8 distinct CD138+ clones were detected in the CSF of MS02-19 and MS02-24 and 13 distinct clones were found in the CSF of MS03-01. By comparison, the CD19+ IgG-expressing B cell pool of each CSF was more diverse, contained fewer clones, and had a smaller fraction of CD19+ cells in clonal populations than did the corresponding CD138+ pool. IgM-expressing CD19+ cells have also been found in MS CSF and constitute a significant portion of the B cell repertoire; in MS03-01, the CD19+ repertoire was polyclonal, as previously found in the CSF of other MS patients (8). Overall, increasingly restricted Ab repertoires containing clonal families of closely related sequences were associated with the more mature plasma cell populations. This pattern likely emerges with dominant clonotypes produced by selective pressure during maturation. Coinciding with this selection, the percentage of unique V(D)J recombinations utilizing VH4 germline segments was also increased markedly in MS03-1 CSF when their prevalence in the polyclonal IgM repertoire of CD19+ B cells (18%) was compared with the more mature IgG repertoire of plasma cells (58%).

There was very little sequence overlap between cells populating the CD19+ and CD138+ pools of all three MS subjects. Whether those cells that were identified as clones in the CD138+ and also in the CD19+ populations of each CSF, such as clone 5 in MS02-19 CSF (Table III) and clones 1 and 5 in MS03-1 CSF (Table V), represent a CD19+ pool from which plasma cells differentiate within the CNS is uncertain. As shown in Fig. 1D for MS03-1 CSF, ~13% of the CD19+ cell population were also CD138+ and could account for the appearance of some rearrangements in both populations. In animal models of CNS-targeted humoral responses, activated postgerminal B cells enter the CNS and those cells specific for Ag are retained and differentiate into plasma cells at the site of Ag deposition (18). Although no organized germinal centers are apparent in MS lesions, it is yet to be determined whether germinal center-like reactions occur within the CNS from which plasma cells differentiate or whether these reactions occur primarily in peripheral germinal centers. More stringent sorting of the CD19+ and CD138+ cell populations and/or serial analyses of MS CSF may help to elucidate the dynamic nature of B and plasma cell populations in the CNS of MS patients and the clonal relationships between these cells in MS lesions. Interestingly, germinal center-like structures have been identified in synovial tissue of patients with rheumatoid arthritis; a comparison of the B cell and plasma cell repertoires showed clonal expansion within each pool, but no clear clonal relationships between B cells and plasma cells (19). A dynamic B cell response in which the CD19+ compartment turns over rapidly compared with longer-lived plasma cells that slowly accumulate in synovial tissue might explain those findings. Thus, many of the CD138+ clones may also be long-lived plasma cells that are maintained in the CSF of MS patients. This would account for the continued persistence of the same OGBs in MS patients (20).

Humoral immunity appears to be an important component of MS pathogenesis (21), and a high predominance of B cells in MS CSF is associated with more rapid disease progression (17). The features of the humoral response in MS as determined by single-cell analyses, however, appear to be largely independent of disease state. Expanded CD19+ and CD138+ clones were present in MS patients diagnosed with either primary-progressive (MS02-19) or secondary-progressive disease (MS02-24). Our previous analyses of the CD19+ cell repertoire recovered from the CSF of patients with relapsing-remitting disease also revealed B cell clonal expansion in three of four patients studied (8), and it seems reasonable to assume that plasma cell clones are present in those individuals as well. Clonal expansion was also prominent early in disease; the humoral response in the CSF of MS03-01, which was examined during the first clinical episode of disease, was highly restricted and contained as many or more B cell and plasma cell clones than did CSF of MS02-19 and MS02-24, individuals with disease duration of 3 and 20 years, respectively. Qin et al. (10) also identified at least one clonal B cell population in the CSF of 13 of 16 patients classified as clinically isolated syndrome. Careful follow-up revealed a high conversion rate to definite MS for those positive patients, supporting the notion that a targeted humoral response occurs early and may predict disease progression. Analysis of our repertoires revealed no obvious differences in other parameters of the humoral response, such as degree of somatic mutation or V region germline usage that distinguished patients based on disease state or duration.

Single-cell repertoire analyses provides not only compelling evidence for B and plasma cell clonal expansion within the CNS of MS patients, regardless of duration or classification of disease, but also offers the unique opportunity to accurately duplicate the H and L chain pairings of these cells in vitro as recombinant IgG. The specificity of the OGBs found in MS plaques and CSF and their relevance to the pathogenesis of MS remains unknown. The recombinant Abs that can be prepared from single B and plasma cell Ig sequences can be useful tools to determine the specificity of the oligoclonal IgG in MS.


   Acknowledgments
 
We thank Marina Hoffman for editorial review and Cathy Allen for preparing this manuscript.


   Footnotes
 
1 This study was supported in part by Public Health Service Grant NS 32623. Back

2 Address correspondence and reprint requests to Dr. Gregory P. Owens, Department of Neurology, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Mail Stop B182, Denver, CO 80262. E-mail address: greg.owens{at}uchsc.edu Back

3 Abbreviations used in this paper: MS, multiple sclerosis; CSF, cerebrospinal fluid; OGB, oligoclonal band. Back

Received for publication January 16, 2004. Accepted for publication April 26, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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G. P. Owens, A. J. Shearer, X. Yu, A. M. Ritchie, K. M. Keays, J. L. Bennett, D. H. Gilden, and M. P. Burgoon
Screening Random Peptide Libraries with Subacute Sclerosing Panencephalitis Brain-Derived Recombinant Antibodies Identifies Multiple Epitopes in the C-Terminal Region of the Measles Virus Nucleocapsid Protein
J. Virol., December 15, 2006; 80(24): 12121 - 12130.
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 BrainHome page
M. Krumbholz, D. Theil, S. Cepok, B. Hemmer, P. Kivisakk, R. M. Ransohoff, M. Hofbauer, C. Farina, T. Derfuss, C. Hartle, et al.
Chemokines in multiple sclerosis: CXCL12 and CXCL13 up-regulation is differentially linked to CNS immune cell recruitment
Brain, January 1, 2006; 129(1): 200 - 211.
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 Proc. Natl. Acad. Sci. USAHome page
M. P. Burgoon, K. M. Keays, G. P. Owens, A. M. Ritchie, P. R. Rai, C. D. Cool, and D. H. Gilden
Laser-capture microdissection of plasma cells from subacute sclerosing panencephalitis brain reveals intrathecal disease-relevant antibodies
PNAS, May 17, 2005; 102(20): 7245 - 7250.
[Abstract] [Full Text] [PDF]

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