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*Substance via MeSH
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*Genes and Gene Therapy
The Journal of Immunology, 2000, 164: 2776-2781.
Copyright © 2000 by The American Association of Immunologists

Gene Therapy for Chronic Relapsing Experimental Allergic Encephalomyelitis Using Cells Expressing a Novel Soluble p75 Dimeric TNF Receptor1

J. Ludovic Croxford*, Kostas A. Triantaphyllopoulos{dagger}, Richard M. Neve{dagger}, Marc Feldmann{dagger}, Yuti Chernajovsky{dagger} and David Baker2,*

* Neuroinflammation Group, Institute of Neurology, and Department of Clinical Science, Institute of Ophthalmology, University College London, London, United Kingdom; and {dagger} Kennedy Institute of Rheumatology, London, United Kingdom.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In a murine relapsing experimental allergic encephalomyelitis (EAE) model, gene therapy to block TNF was investigated with the use of a retroviral dimeric p75 TNF receptor (dTNFR) construct. To effectively produce these TNF inhibitors in vivo, a conditionally immortalized syngeneic fibroblast line was established, using a temperature-sensitive SV40 large T Ag-expressing retrovirus. These cells were subsequently infected with a retrovirus expressing soluble dTNFR. CNS-injected cells could be detected 3 mo after transplantation and were shown to produce the transgene product by immunocytochemistry and ELISA of tissue fluids. These levels of dTNFR protein were biologically active and could significantly ameliorate both acute and relapsing EAE. This cell-based gene-vector approach is ideal for delivering proteins to the CNS and has particular relevance to the control of inflammatory CNS disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Experimental allergic encephalomyelitis (EAE)3 is a model of inflammation of the CNS, which has many similarities to multiple sclerosis (MS). TNF-{alpha} and TNF-ß are pleiotropic cytokines produced by macrophages and activated T cells and are thought to be important proinflammatory mediators in many immunological disorders such as rheumatoid arthritis, septic shock, and MS (1, 2). There is now strong evidence to support a role of TNF in the pathogenesis of EAE including cytotoxicity of oligodendrocytes (3), up-regulation of adhesion molecules on CNS endothelia (4), and the use of neutralizing TNF antagonists, notably specific anti-TNF Abs and TNFR-Ig fusion proteins (TNFR-Ig), particularly when injected into the CNS (5, 6, 7). However, treatment was not usually curative and on cessation of TNF blockade, disease progression returned (6, 7). Although studies in EAE using Tnf gene knockout mice have yielded conflicting results, these mice may have severe immunological/physiological defects that may affect normal disease progression (8). Theoretically, gene therapy should allow the efficient delivery of TNF antagonists, allowing long term production of the therapeutic protein from a single injection. Previously, we have shown that a DNA-cationic liposome complex coding for soluble dimeric human p75 TNFR (dTNFR) injected directly into the CNS of mice with EAE ameliorated disease and delayed disease onset and that human p75 dTNFR is functional in mice (9). However, gene delivery by DNA plasmid is known to be inefficient, and delivery of product was limited to days (9). In an attempt to deliver long term anti-TNF therapy, a more stable ex vivo approach using retroviruses was investigated. Retroviral vectors integrate only into replicating cells, and evidence of gene expression has been reported for up to 18 mo (10). However, because the CNS is largely postmitotic, these viral vectors have been used almost exclusively ex vivo. Various cell types have been used as "vehicles" for transgenes in the CNS, including fibroblasts and astrocytes (11, 12). With a cell-based gene-vector system, this study demonstrates that TNF neutralization significantly ameliorates the progression and relapse of CNS inflammatory disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and disease induction

Biozzi AB high (ABH) mice were bred at the Institute of Ophthalmology and maintained with U.K. Home Office approval. Mice 6–8 wk old were injected in the flank with 1 mg ABH mouse spinal cord homogenate (SCH) in Freund’s complete adjuvant (Difco, Detroit, MI) on days 0 and 7, as described previously (13). Animals were monitored up to day 24 postinoculation (p.i.), and clinical signs were assessed as follows: 0 = normal, 1 = flaccid tail, 2 = impaired righting reflex, 3 = partial paralysis, 4 = complete paralysis, as described previously (13). Clinical signs of a lower severity than typically observed were scored 0.5 lower than the grade indicated (14).

Immortalized fibroblast cell lines

Fibroblasts were cultured from day 18 fetal ABH mouse kidneys in DMEM (Life Technologies, Paisley, U.K.) containing 10% FBS, 1 mM sodium pyruvate, 10 mM nonessential amino acid, 1% glutamine, 0.2 µM 2-ME, and gentamicin (1:1000) (Sigma, Poole, U.K.) at 37°C in a humidified atmosphere containing 5% CO2. These were immortalized using supernatant form the SVU 19.5 cell line, secreting retrovirus encoding a temperature-sensitive non-SV40 origin-binding U19 mutant of the SV40 large T Ag and the neomycin resistance gene (15). Cells were selected in medium containing 0.5 mg/ml G418 (Life Technologies) for 1–2 wk. Cells were cloned by limiting dilution and were shown to express SV40-large T Ag by immunocytochemistry on 4% formaldehyde-fixed, 0.2% Triton-X-permeabilized cells using a SV40 large-T Ag-specific mAb (gift from Dr. P. Jat, Ludwig Institute, London, U.K.). These cells were named temperature-sensitive fibroblasts (tsF).

Construction of human dimeric p75 TNFR retroviral gene vector and transfection to tsF

Production of the retroviral dTNFR vector has been described previously (16). Briefly, the human dTNFR coding the extra cellular domain of human CD120b was dimerized by the use of a polyglycine/serine linker and cloned into the MFG retroviral vector under control of mouse Moloney leukemia virus long terminal repeats (LTR) (16). This was packaged using mouse amphotropic packaging cells GPenvAM12, and viral supernatants were collected and used to infect tsF as described previously (16, 17). The expression of human dTNFR in cells was confirmed by immunoperoxidase staining of cytospins using a human CD120b-specific mAb TNFR (clone 4D1B10(MR2–1) (Caltag, South San Francisco, CA). Nontransduced tsF and tsF expressing a mouse IL-12 p40 homodimer were used as controls for immunoperoxidase staining. These were negative. dTNFR-tsF were cloned by limiting dilution, and dTNFR production assessed by ELISA. One clone (oc5) was selected for use in this study. tsF were also infected with a retrovirus coding for IFN-ß (IFN-ß-tsF) as described previously (17).

ELISA and immunocytochemistry

Supernatants collected from cells in culture or serum, urine, and CSF were stored at -20°C until used. Serum, urine, and CSF were collected from individual mice. Blood was collected from terminally anesthetized mice and allowed to clot. After centrifugation, serum was collected and stored at -20°C until use. Urine was collected at the same time each day by gentle massage of the bladder area into an Eppendorf tube. This was then centrifuged and stored at -20°C until further use. CSF (1–15 µl) was withdrawn from the foramen magnum with a sharpened 2-µl pipette tip into a small tube from terminally CO2-anesthetized mice. Care was taken not to use samples contaminated with blood. Samples were centrifuged, and the supernatant was aliquoted in equal volumes and stored at -20°C until use. Human p75-specific ELISA was purchased from Hbt Hycult Biotechnology (Uden, The Netherlands). The IL-4 and IFN-{gamma} ELISA were performed using the Cytoscreen Immunoassay kit (Biosource International, Camarillo, CA). The IL-10 ELISA was purchased from R&D Systems (Minneapolis, MN). The TGF-ß ELISA was performed using a Promega kit (Madison, WI). All TGF-ß samples were acid activated before ELISA according to the instructions. DMEM, 10% FCS was used to measure background concentrations of TGF-ß in growth media. CNS tissue from animals injected i.c. with dTNFR-tsF or tsF were snap frozen, and 8-µm cryostat sections were cut and fixed in acetone. These were stained by indirect immunoperoxidase using biotinylated 4D1B10(MR2–1) mAb as described previously (13). IFN-{alpha} and IFN-ß production from tsF cells was assessed using supernatant in an anti-viral activity assay on LTK(-) fibroblasts (17). Positive controls consisted of tsF cells retrovirally engineered to produce IFN-ß, and {alpha}-BOSC23, NB100, and COS-7 cells were transiently infected with an IFN-ß construct (17). However, IFNß was not detected in nontransduced tsF (17).

In vivo administration of dTNFR-expressing cells

Various numbers of cells were trypsinized (Sigma) from flasks, suspended in sterile PBS, and injected either i.p. or into the right frontal cortex of halothane (May and Baker, Dagenham, U.K.)-anesthetized mice as described previously (9). Control groups consisted of untreated mice or mice injected with similar numbers of nontransduced tsF. In some instances, the cells had been surface labeled with green fluorescent dye PKH2-GL (Sigma) according to manufacturer’s protocol.

Statistical analysis

Results were presented as the mean clinical score (assessing all mice within a group), mean EAE score (assessed using only mice which developed EAE within a group), or mean day of onset ± SEM; and the statistical significance was determined by the Mann-Whitney U nonparametric ranking test, using MINITAB V10 software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell vectors

To produce a stable cell line for gene delivery, Biozzi ABH mouse fetal kidney fibroblasts were first immortalized using a temperature-sensitive SV40-large T Ag (15), expanded, and named tsF. These were then infected with retroviral vectors coding dTNFR, before cloning. Clone oc5 infected with dTNFR in the MFG retroviral vector (16) with expression driven by murine Moloney leukemia virus LTR, produced 1.7 ng/ml/1 x 106 cells/24 h TNFR. Although these cells produced undetectable levels of IFN-{alpha} or IFN-ß by antiviral assay (17) or IFN-{gamma}, IL-10, or IL-4 as assessed by ELISA, a TGF-ß ELISA indicated that transduced p75 TNFR and nontransduced tsF cells produced 0.66 and 2.1 ng/ml/1 x 106 cells/24 h TGF-ß, respectively.

Detection of cells and dTNFR production in vivo after CNS transplantation

Fluorescently (PKH2) labeled dTNFR-tsF (2 x 106) when injected into the right cortex were detected in the parenchyma surrounding the injection site and also in the meninges close to the injection site (data not shown). Cells could be detected in this way up to 3 mo post-intracranial (i.c.) injection but due to the half-life of the fluorescent marker could not be positively identified after this time point. SV40 large T Ag was rapidly shut down after in vivo transplantation and consistent with this, cells were not found to be tumorigenic. These cells were, however, shown to produce their TNFR product by immunocytochemistry in situ (Fig. 1Go) and by ELISA in tissue fluids (Fig. 2Go). The use of human p75 dTNFR allowed the TNFR produced by the retrovirally engineered cells to be distinguished from the endogenously produced mouse TNFR. After i.c. injection of 2 x 106 dTNFR-tsF, human dTNFR could be detected (400–700 pg/ml) in the CSF for at least 5 wk (latest time point examined) (Fig. 2Go). Levels within peripheral compartments were significantly less. Although 80–110 pg/ml CD120b were detectable within the serum 24 h after injection, this decreased to 21–30 pg/ml within the first week after injection, to a level similar to that found in the urine (Fig. 2Go). No evidence of inflammation consistent with a rejection episode were seen in any brains examined. After i.p. injection of 5 x 106 dTNFR-tsF, similar serum and urine levels (15–30 pg/ml) were detected (data not shown).



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  		FIGURE 1. CNS localization of dTNFR-producing tsF cells. dTNFR-tsF cells (2 x 106) were injected into the right cerebral cortex, and the brains were removed 2 days postinjection and stained for human CD120b (p75 dTNFR). Positive peroxidase staining could be detected in vivo on a population of cells close to the injection site. Sections were not counterstained. x200.

 


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  		FIGURE 2. Time course of production of human soluble CD120b from dTNFR-tsF in vivo. Time course of in vivo production of human soluble CD120b (p75 TNFR) was measured in urine (•), serum ({circ}), and CSF ({blacktriangledown}) by ELISA from ABH mice injected with 2 x 106 dTNFR-tsF cells i.c. Samples from untreated mice or tsF-treated mice showed no positivity for human p75 TNFR. Sensitivity of the ELISA was <5 pg/ml.

 
Control of acute phase EAE with dTNFR-expressing cells

Having established that dTNFR production was maintained in vivo for >5 wk, the therapeutic potential of cells to control disease was investigated (Table IGo). After disease induction, dTNFR-tsF cells were administered either systemically, to target peripheral events, or locally, to the CNS, to inhibit the effector responses.


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  		Table I. Therapeutic effects of systemic gene-delivered dTNF during the priming stage of EAE1

 
Systemic administration of 1 x 106 dTNFR-tsF cells administered i.p. had no effect on disease severity (Table IGo). However, increasing the dose to 5 x 106 dTNFR-tsF cells significantly inhibited the development of disease and delayed the onset of clinical signs, compared with tsF-treated mice (Table IGo). There was no significant increase in disease inhibition by increasing the dose of cells to 1 x 107 on day 7 p.i. compared with that seen with 5 x 106 cells, suggesting that the optimal response had been achieved (Table IGo). Comparison of 5 x 106 dTNFR-tsF cells administered systemically at day 0 or day 7 p.i. suggests that systemic TNF neutralization during early or late time points of the priming stage of disease can inhibit EAE to a similar degree (Tables I and II). In groups of mice treated with dTNFR-tsF, the mean percentage incidence of mice exhibiting EAE was significantly reduced (69.6%) compared with tsF (90.5%) and untreated controls (96.8%) (p < 0.03). A further control consisted of tsF retrovirally engineered to produce mouse IFN-ß (IFN-ß-tsF). When 2 x 106 IFNß-tsF cells were injected i.c. on day 12 p.i. there was no effect on disease severity (mean clinical score, 3.5 ± 0.1; n = 6) or mean day of onset (18.2 ± 0.4) compared with tsF-treated mice (mean clinical score, 3.8 ± 0.2; mean day of onset, 17.5 ± 0.5; n = 6). The lack of efficacy seen with the IFNß-tsF may be due to the low level of cytokine produced (17).

The efficacy of dTNFR-tsF cells in EAE inhibition could be further increased when implanted into the CNS. A lower dose of dTNFR-tsF (2 x 106) cells implanted into the CNS on day 7 p.i. or just before disease onset (day 12 p.i.) inhibited disease severity to a equal or greater degree than systemic administration of more than double the number of cells (Table IIGo). In addition, CNS administration of 2 x 106 dTNFR-tsF could inhibit EAE after the onset of clinical disease (Table IIGo).


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  		Table II. Local administration to the CNS increases the efficacy of gene-delivered dTNFR1

 
Mice injected with nontransduced tsF at all time points and by all routes showed no significant difference in mean disease score or onset of disease when compared with the untreated SCH inoculated mice (Tables I–III).

Control of relapse phase EAE with dTNFR-expressing cells.

EAE in ABH mice follows a relapsing-remitting disease profile, which allows investigation of the relapse phase, which may be more clinically relevant to MS than acute disease models of EAE (13).

The dTNFR-tsF cells have been shown to express their transgene product for at least 5 wk in vivo. Therefore, mice injected with dTNFR-tsF cells in the CNS on day 7 p.i. were allowed to pass through the acute phase of disease to study the long term inhibition of TNF on the relapse phase of disease. As seen in previous experiments, dTNFR-tsF-treated mice had a significantly ameliorated mean clinical score compared with tsF-treated mice (p < 0.0005) during the acute phase, with an EAE incidence of 53% compared with 100% for tsF-treated and untreated mice (Table IIIGo). In addition, the dTNFR-tsF-treated group exhibited a lower incidence rate of relapse (41%) than did the tsF-treated group (69%), and the mean clinical score was significantly reduced compared with tsF-treated mice (p < 0.05) (Table IIIGo; Fig. 3Go).


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  		Table III. Therapeutic effect of gene-delivered dTNFR to the CNS in the relapse phase of EAE1

 


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  		FIGURE 3. Anti-TNF therapy of acute and relapse phase of EAE from a single injection of dTNFR-tsF i.c. on day 7 p.i. Data show that the acute phase of EAE can be ameliorated with local delivery to the CNS of human p75 dTNFR from dTNFR-tsF (p < 0.0005) compared with mice injected at the same time point and route with immortalized fibroblasts only (tsF). Mice were injected i.c. with 2 x 106 dTNFR-tsF ({circ}) or tsF (•) day 7 p.i. ({downarrow}). Although the single injection of dTNFR-tsF did not prevent onset of the relapse phase of EAE it could still ameliorate the severity of disease 5 wk later (p < 0.05) to a degree similar to that seen when dTNFR-tsF were administered during remission (day 28 p.i.) (Fig. 4Go). Results represent the mean clinical score of all animals in the group (n = 13–17 mice).

 
This study also investigated the inhibition of the relapse phase in mice that had been allowed to pass through the acute phase of disease without treatment. After acute phase disease (day 15–25 p.i.), mice were injected i.c. with 2 x 106 dTNFR-tsF or nontransduced tsF cells during the remission phase (day 28 p.i.). After transplantation, mice injected with dTNFR-tsF cells showed a significantly lower clinical score during the relapse phase (day 30 onward) compared with control tsF-treated mice (p < 0.05) (Table IIIGo; Fig. 4Go). The incidence of relapse was reduced (33%) compared with either tsF-treated group (100%) or untreated group (78%) (Table IIGo).



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  		FIGURE 4. Anti-TNF therapy of the relapse phase of EAE with a single injection of dTNFR-tsF i.c. on day 28 p.i. Mice were injected i.c. with 2 x 106 dTNFR-tsF ({circ}) or tsF (•) during remission of EAE day 28 p.i. ({downarrow}). Data show that although the single injection of dTNFR-tsF expressing human p75 dTNFR did not prevent onset of the relapse phase of EAE, it could still ameliorate the severity of relapse (p < 0.05) compared with mice treated with the same quantity of tsF at the same time point. The results represent the mean clinical score of all animals in the group (n = 9–12 mice).

 
It appeared that the reduction of clinical disease score in affected mice treated with dTNFR-tsF cells was largely due to animals that were completely protected from clinical disease, although after i.c. administration the severity of clinical signs were significantly reduced (Table IIGo). Lumbar regions of spinal cord were taken from mice on day 18–19 p.i. when there was a maximal difference in clinical disease between dTNFR-tsF-treated and other groups. Spinal cords from dTNFR-tsF-treated mice (n = 5) that had no clinical disease and no weight loss showed no perivascular infiltrate, or lesions in the parenchyma, although there was slight infiltration in the meninges of one animal. This contrasts to mice from nontransduced tsF-treated (n = 3) or control SCH (n = 2) mice that were at maximum clinical disease (grade 4) and showed perivascular lesions and substantial infiltrate in the parenchyma and meninges (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that genetically engineered cell vectors can survive long term and produce a therapeutic concentration of protein capable of inhibiting CNS-inflammatory disease.

Previously, it has been reported that DNA constructs coding for dTNFR complexed with cationic liposome can ameliorate acute EAE when injected into the CNS (9). However, production was limited to days, in contrast to using a retroviral/cell vector system as described here where transgene production could be detected for weeks. After CNS delivery, high levels of human dTNFR could be detected in the CSF compared with that detected in peripheral compartments. The capacity to maintain a concentration gradient of immunosuppressive agents within the CNS may be important to limit nonspecific effects in the periphery and will be aided by the relatively short half-life of dTNFR (18). However, this study suggests that dTNFR is active when administered both centrally and systemically. The dTNFR-tsF cells were at least as effective at inhibiting EAE as previous reports using multiple dose protein therapy, despite producing 1000-fold lower protein concentrations than bolus protein injections (5, 6, 7). Furthermore, the neutralization of TNF and lymphotoxin in EAE by dTNFR-expressing cells was comparable with a delay in onset of EAE seen in Tnf gene knockout mice (19) and supports studies using anti-TNF mAb or TNF fusion proteins to inhibit TNF-mediated pathology (7, 9).

The p75 (CD120b) TNFR gene product used here inhibited disease development, particularly when administered into the CNS. This would be consistent with an important TNF-dependent mechanism in the CNS, which is involved in the pathogenesis of EAE.

TNF is a pleiotropic cytokine and is thought to have a major role in the recruitment of inflammatory cells, possibly through the regulation of vascular adhesion molecules or the induction of other proinflammatory cytokines (20, 21, 22). However, it has also been suggested that TNF-specific (p55 TNFR-Ig) treatment may decrease the activation state of encephalitogenic T cells rather than inhibiting T cell infiltration into the CNS (23). The onset of weight loss correlates with blood-brain barrier dysfunction and cellular recruitment (24); and in some cases of TNF-specific protein therapy, animals showed weight loss even though disease was reduced (23). Although some dTNFR-tsF-treated mice in this study did develop EAE, there were a number of animals that were completely protected from EAE, showing stable weight progression and no CNS infiltrate. This may indicate a higher degree of TNF neutralization by the dTNFR-tsF cells, inhibiting TNF-induced cachexia, compared with the bolus protein administration in some studies (23). This study also demonstrates that both the development of acute phase EAE and the development of relapse may also be inhibited by dTNFR.

Although many studies have demonstrated that TNF and soluble TNFR levels in serum and CSF correlate to severity of MS, there remains much controversy over the results (25, 26, 27, 28). However, in this study, analysis of serum samples for the presence of mouse CD120a and CD120b throughout the relapsing course described by Allen (24), using specific ELISA reagents provided by Dr. W. Buurman, failed to demonstrate any correlation with disease activity (our unpublished observations). The effect of TNF neutralization in vivo in this study, however, further indicates the importance of TNF in the pathogenesis of chronic relapsing EAE and may possibly suggest that this may be a useful target in the control of MS in humans.

However, although TNF-specific mAb have reduced the severity of rheumatoid arthritis and Crohn’s disease (29, 30), the same Ab and a p55 TNFR-IgG1 fusion protein administered to septic shock patients had no survival benefit, and where a high dose of TNFR-IgG1 was administered it was associated with a higher mortality rate among the patients (31, 32). Similarly, it has been reported that a p55 TNFR-Ig fusion protein increased exacerbations in two MS patients (33). Thus, it appears that different TNF antagonists have different immunogenic and pharmacological properties. Indeed, cellular feedback mechanisms tightly control endogenous TNF production (34, 35), making it likely that the degree and timing of TNF neutralization may have important consequences on the development of either pro- or antiinflammatory events. Therefore, care must be taken in moving animal data into the clinic.

Although many cell types are currently being studied for vehicles in gene therapy, fibroblasts are an attractive vector for human therapy because they can be easily cultured from skin biopsies of any individual and thus eliminate any potential allogeneic immune response. These can readily be stably infected by retrovirus ex vivo. At present, fibroblasts are being used as vehicles to deliver therapeutic products in a variety of disease models, such as Parkinson’s disease, leukemia, Alzheimer’s disease, CNS glioma and melanoma, and arthritis (11, 18, 36, 37, 38, 39, 40, 41). Because these cells are viable and are capable of producing endogenous growth factors, added benefit in immunosuppression/neuroprotection and graft survival may come from the use of CNS-derived cells. For example, oligodendrocytes that could be used to promote remyelination in damaged tissue (42). Recent reports of gene therapy in EAE have studied the use of hybridoma cells and CNS Ag-specific T cells to deliver IL-10, IL-4, or TGF-ß (43, 44, 45). This method has the potential to target delivery of cytokines to the CNS by less invasive systemic administration. However, the number of circulating cells entering the CNS is low, and hybridoma cells will continue to proliferate in vivo. Therefore, the concentration of cytokine delivered to the CNS cannot be accurately assessed. Retrovirally engineered cells injected into the CNS, however, although invasive, allow the accurate and reproducible delivery of transgene product directly into the CNS.

This ex vivo gene therapy procedure administered locally could also be used to deliver a variety of other immunosuppressive products or neuroprotection/regeneration agents, either alone or in combination. Although efficacy has been shown here in an inflammatory CNS model, this approach will be of relevance to a variety of other conditions where proteins need to be delivered long term to the CNS.

Recent advances in gene therapy allow the encapsulation of genetically engineered cells in a semipermeable membrane which can be inserted, and importantly removed if necessary, into the lumbar region of the spinal cord. This could allow complete control over the location of engineered cells, time points and combination of product administration, and in limiting potential immune responses to implanted cells (46). These are important safety factors that must be considered when developing their potential use in chronic diseases. This strategy provides the opportunity to deliver proteins long term, without frequent repeat administration unlike Ab or fusion protein therapy. This offers a useful tool to dissect neurological disease pathways experimentally and could be developed toward the control of a number of neurological disorders including MS.


   Acknowledgments
 
We thank the people cited in the article for providing access to and donation of reagents.


   Footnotes
 
1 This work was supported by the Multiple Sclerosis Society of Great Britain and Northern Ireland and The Arthritis and Rheumatism Campaign, U.K. Back

2 Address correspondence and reprint requests to Dr. D. Baker, Neuroinflammation Group, Institute of Neurology, University College London, 1, Wakefield Street, London, WC1N 3BG U.K. E-mail address: Back

3 Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; i.c., intracranial; p.i., postinoculation; MS, multiple sclerosis; ABH, Ab high; TNFR-Ig, TNFR-Ig fusion protein; dTNFR, dimeric human p75 TNFR; SCH, spinal cord homogenate; tsF, temperature-sensitive fibroblasts; LTR, long terminal repeat. Back

Received for publication September 27, 1999. Accepted for publication December 10, 1999.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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