document

©2005 FASEB

The FASEB Journal express article 10.1096/fj.05-4650fje. Published online December 13, 2005.

STAT4- and STAT6-signaling molecules in a murine model of multiple sclerosis Moses Rodriguez,*,† Laurie Zoecklein,* Jeffrey D. Gamez,* Kevin D. Pavelko,* Louisa M. Papke,* Shunya Nakane,* Charles Howe,* Suresh Radhakrishnan,† Michael J. Hansen,† Chella S. David,† Arthur E. Warrington,* and Larry R. Pease†

Departments of *Neurology and †Immunology, Mayo Clinic, Rochester, Minnesota

Corresponding author: Moses Rodriguez, Departments of Neurology and Immunology, 200 1st St. SW, Rochester, MN 55905. E-mail: [email protected]

ABSTRACT

Epidemiological studies suggest that an environmental factor (possibly a virus) acquired early in life may trigger multiple sclerosis (MS). The virus may remain dormant in the central nervous system but then becomes activated in adulthood. All existing models of MS are characterized by inflammation or demyelination that follows days after virus infection or antigen inoculation. While investigating the role of CD4+ T cell responses following Theiler’s virus infection in mice deficient in STAT4 or STAT6, we discovered a model in which virus infection was followed by demyelination after a very prolonged incubation period. STAT4−/− mice were resistant to demyelination for 180 days after infection, but developed severe demyelination after this time point. Inflammatory cells and up-regulation of Class I and Class II MHC antigens characterized these lesions. Virus antigen was partially controlled during the early chronic phase of the infection even though viral RNA levels remained high throughout infection. Demyelination correlated with the appearance of virus antigen expression. Bone marrow reconstitution experiments indicated that the mechanism of the late onset demyelination was the result of the STAT4−/− immune system. Thus, virus infection of STAT4−/− mice results in a model that may allow for dissection of the immune events predisposing to late-onset demyelination in MS.

Key words: Theiler’s murine encephalomyelitis virus • virus persistence • transcription factors

lthough the cause of multiple sclerosis (MS) is unknown, there are two major hypotheses regarding the development of demyelination (1). The first hypothesis predicts that the disease begins in peripheral immune sites. Activated autoimmune T cells enter the

central nervous system and injure the myelin sheath. The second hypothesis predicts that virus infection triggers demyelination. The basis of this hypothesis is the existence of viruses that produce demyelination in animals (i.e., picornaviruses, corona viruses, toga viruses, and herpes viruses) (2) and viruses that cause human disease characterized by demyelination (i.e., papovavirus, retroviruses, and paramyxovirus) (3). Epidemiological data indicate that recent virus infection is associated with acute relapses of MS (4). On the basis of “epidemics” of MS in the Faeroe Islands, it has been proposed that MS is triggered by an environmental factor that occurs in early life. However, the disease remains silent and does not manifest until years later

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(5). Whether or not a virus initiates disease or is one of many factors influencing the course and manifestations of MS remains controversial.

We have proposed two mechanisms by which viruses may cause human demyelinating diseases (6). In the “HIT- HIT” hypothesis, virus enters the CNS and injures oligodendrocytes and myelin by direct viral killing or by inducing an immune response designed to limit the spread of virus, but as a consequence injures the nervous system. In the “HIT-RUN” hypothesis, virus infects the CNS or the periphery, but then is cleared rapidly. This virus infection initiates T cell-based autoimmunity that is directed against the CNS through molecular mimicry, molecular identity, or epitope spreading.

There is a need to develop models of various aspects of MS. Intense inflammation, but minimal parenchymal destruction or demyelination manifested within days following immunization characterizes autoimmune models of MS. Viral models are characterized by demyelination within days to weeks following virus infection. One aspect of the pathogenesis of MS that has not been modeled in animals is the concept that a period of “disease silence” would follow the inciting event, with demyelination progressing much later in the lifespan of the animal. This would model a situation in MS in which exposure to the inciting agent occurs around in childhood or young adulthood, but the disease does not manifest until years later (7).

These experiments used intracerebral injection of Theiler’s murine encephalomyelitis virus (TMEV), a picornavirus, that induces a characteristic CNS disease in mice (8). During the first 12 days of infection, virus replicates in neurons of the brain and anterior cells of the spinal cord, but is rapidly cleared from the brain (9) regardless of MHC haplotype. In animals of susceptible MHC haplotype (H-2s,v,r,u,f), the virus persists in glial cells (10) and macrophages (11, 12) in the spinal cord white matter and brain stem.

The family of transcription factors known at STATs (signal transducers and activators of transcription) play a role in activating T cells and microglia (13). Members of the class I and class II cytokine receptor families transduce their signals via the JAK kinases. Activated JAK kinases phosphorylate STATs. After phosphorylation, STAT proteins dimerize in the cytoplasm and translocate to the nucleus where they bind to DNA regulatory elements activating gene transcription. The STAT-signaling pathway is very specific. Each cytokine or group of redundant cytokines induces transcription of a specific subset of genes dependent on STAT proteins. The STAT4 pathway controls differentiation of CD4+ T cells into a Th1 phenotype. The STAT6 pathway controls the differentiation of CD4+ T cells into a Th2 phenotype (14, 15). STAT4 is predominantly activated in response to IL-12 (16, 17). The phenotype of STAT4−/− mice is very similar to that of IL-12−/− mice, resulting in the failure to produce Th1 CD4+ T cells. STAT6 is activated by IL-4, and the related cytokine IL-13 (16). STAT6−/− mice lack most functions associated with IL-4 (18). There is little information regarding the expression of STAT4 and STAT6 outside the immune system; however members of the JAK/STAT family are expressed and regulated during forebrain development (19).

While investigating the CD4+ T cell Th1 and Th2 response to Theiler’s virus infection we found mice with a demyelinating phenotype that satisfies the requirements for a long incubation period before inflammatory demyelination. Infection of STAT4−/− mice resulted in a phenotype in


which prominent inflammatory demyelination did not occur until after 180 days after intracerebral infection.

MATERIALS AND METHODS

Virus

Daniels strain of TMEV was used for all experiments (8).

Mice

Control BALB/cJ mice and SJL/J mice were obtained from the Jackson Laboratories (Bar Harbor, ME). Stat4tm1Gru (STAT4−/−) and Stat6tm1Gru (STAT6−/−) mice (17) with a targeted disruption of the STAT gene were provided by Michael J. Grusby (Harvard University) and bred at Mayo. Mice had been bred for greater than 10 generations to BALB/cJ mice, shown to be resistant to demyelination (20). Parallel experiments were conducted in STAT4−/− mice and STAT6−/− mice obtained from the Jackson Laboratories, which proved to be identical. STAT4−/−, STAT6−/−, and BALB/cJ control mice were of the MHC H-2d haplotype. We generated F1 mice by crossing STAT4−/− mice to STAT6−/− mice. Both strains were used as either parent to exclude the influence of weaning on phenotype. Experiments were approved by Mayo Institutional Animal Care and Use Committee and conformed to the guidelines for care of animals by the National Institutes of Health. A total of 505 mice were used in these experiments.

Infection and CNS histology

At 4–6 weeks of age, mice were intracerebrally infected with 2 × 105 PFU of TMEV in 10 μl. Mice were perfused via intracardiac puncture with 50 ml of Trump’s fixative. Spinal cords and brains were removed and postfixed for 24–48 h in Trump's fixative. Spinal cords were cut into 1-mm coronal blocks. Every third block was fixed with osmium and embedded in glycol methacrylate. Morphological analysis was performed on 12–15 sections per mouse (21). Each quadrant from every coronal section from each mouse was graded for the presence or absence of gray matter disease, meningeal inflammation, and demyelination. The score was expressed as the percentage of spinal cord quadrants examined with the pathologic abnormality. Grading was performed blinded on coded sections.

Brain pathology

Brain pathology was assessed as described previously (9). Two coronal cuts were made in the brain at time of removal one through the optic chiasm, a second through the infundibulum (see Ref. 22, sections #220 and 350, page 6), resulting in three pieces that were embedded in paraffin. Sections were stained with hematoxylin and eosin. Pathologic scores were assigned without knowledge of experimental group to the cortex, corpus callosum, hippocampus, brainstem, striatum, and cerebellum. Each area was graded on a 4-point scale. 0 = no pathology; 1 = minimal inflammation; 2 = mild tissue destruction and moderate inflammation; 3 = moderate tissue destruction; 4 = necrosis. Meningeal inflammation was graded as follows: 0 = no inflammation; 1 = one cell layer; 2 = two cell layers; 3 = three cell layers; 4 = four or more cell layers.


Spontaneous activity monitoring

Both horizontal and vertical spontaneous activity of chronically virus-infected STAT4−/− and STAT6−/− mice (>270 days after infection) was measured using a VersaMax Activity Monitoring System (AccuScan Instruments Inc., Columbus OH) (23). Fifteen mice of each group that were matched for days after infection were randomly distributed into three boxes of five mice each. Activity was measured for 48 h after a 24-h acclimation period to the novel box environment. The numbers of vertical and horizontal beam breaks were recorded hourly for the six groups of mice.

Virus-specific antibody isotype ELISA and neutralizing antibody titers

Sera were collected and stored at –80°C. Total serum IgG, IgM, and isotypes (IgG1, IgG2a, IgG2b, and IgG3) against TMEV were assessed by ELISA. TMEV DAV virus was adsorbed to 96-well plates (Immulon II; Dynatech Laboratories, Chantilly, VA) and then blocked with 1% bovine serum albumin (BSA; Sigma Chemical, St. Louis, MO) in PBS. Serial serum dilutions were made in 0.2% BSA/PBS and were added in triplicate. Immunoreagents used for IgG and IgM were goat anti-mouse IgG-biotin (Jackson Immuno Research #115-007-003), goat anti-mouse IgM-biotin (Jackson #115-007-020), and alkaline phosphatase conjugated to streptavidin (Jackson #016-050-084). Signals were detected using p-nitrophenyl phosphate as the substrate. Absorbance was read at 405 nm. Isotype ELISA was performed on sera from mice 45 days after infection. Serum dilutions of 1:2000 were made in 0.2% BSA/PBS and added in triplicate. For isotype-specific ELISA, we used goat anti-mouse IgG1, goat anti-mouse IgG2a, goat anti-mouse IgG2b, and goat anti-mouse IgG3 (Sigma Chemical, St. Louis, MO #ISO-2). Signal amplification was done with a horseradish peroxidase-conjugated donkey anti-goat antibody (Santa Cruz Biotechnology, Santa Cruz, CA; #SC-2020). The signal was detected using 3, 3′, 5, 5′-tetramethylbenzidine (TMB) Liquid Substrate System (Sigma Chemical; #T8665) and absorbance read at 450 nm. The neutralizing titers were expressed as the log 2 dilution required to neutralize 90% of the viral plaques by standard plaque assay using L2 cells.

Immune-staining for virus antigen

Immunocytochemistry was performed on paraffin-embedded sections, as described previously (10). Virus antigen staining was performed using polyclonal antisera to TMEV-DA (10) that reacts strongly with the capsid proteins of TMEV. The data were expressed as the percentage of spinal cord quadrants showing virus-antigen-positive cells in either the gray matter or the white matter of the spinal cord.

Immune-staining for CD4, CD8, MHC Class I, MHC Class II, and F4/80

Frozen sections were cut from OCT-embedded spinal cords. Sections were fixed in 95% ethanol at –20°C for 20 min. Sections were blocked with 2% horse serum, 1% BSA, donkey anti-mouse IgG (1:200), and avidin/biotin (Vector) for 10 min each. Antibodies to CD4, CD8, MHC Class I, MHC Class II (clones GK1.5, Cat. # 553728, 53-5.8 Cat. # 553039, MHC H-2Kd, clone SFS-1-1.1Cat. # 550551 and MHC I-Ad clone AMS – 32.1 Cat. # 550554 all from BD PharMingen) and F4/80 (ATTC # HB-198) were used to localize antigens. Appropriate biotin-labeled secondary


antibodies were used to detect primary antibodies. Final detection was performed using avidin/biotin complex methodology (Vector Labs) and 3,3′ diaminobenzidine (Sigma #D-5537).

RT-PCR and real-time analysis for virus RNA

Total RNA was extracted from the brain and spinal cord as described (21). The VP2 fragment of TMEV was amplified by RT-PCR using gene-specific primers. The primer pairs for VP2 of DA virus were forward (5′-TGGTCGACTCTGTGGTTACG-3′) and reverse (5′-GCCGGTCTTGCAAAGATAGT-3′). Gluceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was used as a control for intersample variability. Primer pairs for GAPDH were forward (5′-ACCACCATGGAGAAGGC-3′) and reverse (5′-GGCATGGACTGTGGTCATGA-3′). Sizes of PCR-amplified products were 238 base pairs VP2 and 236 base pairs for GAPDH.

Gene copy standards were generated with each set of samples. Standards were generated by serial 10-fold dilutions of plasmid cDNA. Standards were amplified in parallel with unknown samples by real-time quantitative RT-PCR using the LightCycler (Roche, Indianapolis, IN). Analysis to generate standard curves was performed using LightCycler 3 software. Negative controls (omitting input cDNA) were used in each PCR run. GAPDH mRNA level per sample was log10 7.19+/0.02 (mean ± SEM). The amount of viral RNA was expressed as log10 virus VP2 copy number / 0.5 µg RNA total.

Proliferation of splenocytes to myelin auto-antigens

Six to nine months after infection, splenocytes from five mice per group were harvested and pooled. Cells (3×105 per well) were stimulated against a titrating dose of antigen in triplicate. Antigens tested included UV-irradiated Theiler’s virus, myelin basic protein (Sigma), recombinant MOG (24), and peptides of proteolipid protein (PLP 41-60, PLP 91-100, PLP 139-154, PLP 179-198, and PLP 209-228 synthesized in Mayo Clinic Protein Core Facility). As positive controls, splenocytes from each mouse strain were treated with Con-A (1 μg/ml). After 4 days of stimulation, the cells were pulsed with 0.5 μCi of 3[H] thymidine (Amersham, Piscataway, NJ) for 18 h before harvest and counting (Packard TopCount).

Bone marrow transplant

Bone marrow was obtained from the tibia and femur of donor mice (F1 or STAT4−/− mice) (25). Mature T cells were depleted with CD4+ and CD8+ antibodies bound to paramagnetic beads (MACS magnetic isolation system, Miltenyi Biotec, Auburn, CA). Bone marrow recipients (F1 or STAT4−/−) mice were treated with tetracycline (1 g/ml) in the drinking water for 1 wk before and after bone marrow transplantation. Recipient mice received a lethal dose of irradiation (800 rads) followed by 5 × 106 bone marrow cells intravenously and allowed to recover for 8 wk before infection.

Statistics

Data were analyzed using either the Student's t test (normally distributed) or the Mann-Whitney Rank Sum test (not normally distributed). For comparisons of more than one group, ANOVA was used. The Student-Newman-Keuls test was used for all pair-wise multiple comparisons.


Proportional data were evaluated using the Chi-square-test. The level for significance was set as P < 0.05 for all tests.

RESULTS

STAT4−/− but not STAT6−/− mice developed spinal cord demyelinating lesions very late after virus infection

We asked whether deletion of transcription factors STAT4 or STAT6 (Fig. 1) would influence demyelinating disease after TMEV infection. In previous studies, the absence or presence of demyelination at 45 days after infection has been an indicator of susceptibility to demyelination (26). At 45 days after infection, demyelination in STAT4−/− mice were limited. Analysis of 13 mice resulted in a white matter inflammation score of 2.0 ± 1.3 and a demyelination score of 1.0 ± 0.7. This was confirmed in a separate group of 10 animals from the Jackson Laboratories. Pathology was similar at 90 days, at which time mice contained small areas of demyelination (inflammation score 0.0±0.0, demyelination score 1.4±1.0). At this point, we would have normally characterized these mice as resistant to TMEV-induced demyelination. For comparison, prototypic susceptible SJL/J mice injected with the same lot of virus resulted in a demyelination scores of 23.5 ± 3.9 (n=18) at 45 days and 47.8 ± 9.0 (n=9) at 90 days. By 180 days after infection, STAT4−/− mice presented with severe demyelination and meningeal inflammation. On average, from day 270 to day 390 after infection, 27 to 29% of spinal cord quadrants contained demyelination (Fig. 1). In STAT4−/− mice with demyelination in the spinal cord infected for 180 days or more, the demyelination score was 22.6 ± 3.3 (n=total 57; 10 at 180 days, 22 at 240 days, 10 at 270 days, 10 at 330 days, 5 at 390 days). For comparison, infected SJL/J mice had a demyelination score of 48.8 ± 3.5 (n=4) at 180 days and 44.2 ± 5.7 (n=9) at 330 days. Lesions were characterized by primary demyelination with relative preservation of axons, intense inflammation consisting of mononuclear cells and absence of remyelination (Fig. 2). The character of spinal cord lesions in STAT4−/− mice at 270 days was similar to what is normally observed in SJL/J mice at 45 and 90 days.

We examined STAT6−/− mice for demyelination from day 45 to 270 after infection (Fig. 1). At 45 days after infection, lesions were small, resulting in a demyelination score of 1.3 ± 0.6 (n=12). The demyelination scores for STAT6−/− mice were 7.5 ± 4.3 (n=11) at 150 days, 5.3 ± 3.0 (n=15) at 180 days and 7.0 ± 4.5 (n=10) at 270 days. STAT6−/− mice had some minimal meningeal inflammation and demyelination by days 150 to 270. At these time points, the STAT4−/− mice had extensive demyelination and meningeal inflammation.

We also examined BALB/cJ mice that were used for generating STAT deficient mice. (Fig. 1 and 2). Spinal cord demyelination after infection ranged from 0 to 2.2% (Fig. 1). Animals infected with TMEV for 180 days or longer presented with demyelination scores of 0.3 ± 0.3 (n=22). We concluded that BALB/cJ mice were resistant to TMEV-induced demyelination. Comparisons of mice that were infected with TMEV for 180 to 270 days showed more demyelination in STAT4−/− mice (20.4±3.4; n=42) compared with BALB/cJ mice (0.3±0.3, n=23; P<0.001 by Rank Sum test). From 180 to 270 days after infection there was no significant difference in demyelination scores in STAT6−/− mice (6.0±2.5, n=25) compared with BALB/cJ mice (P=0.152 by Rank Sum test). Comparing scores from mice infected with TMEV for 180 to


270 days demonstrated more demyelination in STAT4−/− mice (20.4±3.4; n=42) compared with STAT6−/− mice (6.0±2.5, n=25, P=0.007 by Rank Sum Test). We concluded that STAT4 deletion resulted in the development of late demyelination following TMEV infection.

Only a minority of mice (~20%) showed typical clinical signs of neurological deficits chronically during infection in these strains. To more accurately assess neurologic function in chronically infected STAT4−/− and STAT6−/− mice (>270 days after infection), spontaneous activity was monitored continuously for 3 days. We have previously shown that vertical activity, which measures rearing, is related to hind limb paralysis and is the more sensitive indicator of disability (23). A rank sum comparison of the combined hourly nocturnal vertical activity (6:00 a.m. to 5:00 p.m.) between groups demonstrated a significant difference in rearing (P<0.001). STAT4−/− deficient mice broke the vertical beam 181 times/h ± 44 compared with 398 ± 30 vertical beam breaks/h for STAT6−/− mice (median values±SD) during their normally active nighttime period.

(STAT4−/− X STAT6−/−) F1 mice did not develop significant spinal cord demyelination

We crossed STAT4−/− mice to STAT6−/− mice to determine whether STAT mutations, rather than some unknown factor in the BALB/cJ background that resulted in spinal cord demyelination. F1 mice were heterozygous for STAT4 and STAT6 and complemented the STAT4 and STAT6 deficiencies such that at least 50% of the normal expression of the transcription factors was present. F1 mice were generally resistant to chronic demyelinating disease. Demyelinating lesions were very small, resulting in demyelination scores of 0.7 ± 0.7 (n=11) at 90 days, 2.9 ± 2.9 (n=10) at 180 days, and 4.0 ± 3.1 (n=10) at 330 days.

A comparison of animals infected for 180 days or longer demonstrated more demyelination in STAT4−/− mice (22.0±3.2, n=62) than in F1 mice (5.2±2.5, n=30, P<0.001 by Rank Sum test), no difference between the demyelination scores of STAT6−/− (6.0±2.5, n=25) and F1 mice (P=0.524 by Rank Sum test) and no difference between the demyelination scores of BALB/cJ mice (0.3±0.3, n=23) and F1 mice (P=0.422 by Rank Sum test). Because F1 mice did not demyelinate as severely as STAT4−/− mice, we concluded that STAT4 deletion rather than a gene in the BALB/cJ background accounted for the observed phenotype.

STAT4−/− mice had normal humoral immune responses to TMEV infection

Deficiency of transcription factors from birth could have affected protective humoral response directed against virus and predisposition to the late demyelinating disease. We assessed antibody responses in serum directed against purified virus antigens by ELISA (Fig. 3). STAT4−/− mice had similar IgM responses after infection compared with the other two strains (Fig. 3) and in IgG responses at 45 days. At 270 days, there was a statistically significant increased IgG response in STAT4−/− mice. We analyzed isotype-specific responses by ELISA since deletion of Th1 or Th2 responses may have altered IgG1, IgG2a, IgG2b, or IgG3 to virus antigens. Statistical comparisons (ANOVA or ranks) of isotype responses demonstrated more IgG1 in STAT4−/− mice compared with uninfected mice (P<0.001). There was a statistical increase in IgG2a in STAT4−/−, STAT6−/−, and F1 mice as compared with uninfected mice. There was a statistical increase in IgG2b and IgG3 in STAT4−/− mice as compared with STAT6−/− mice, but the


highest response to these isotypes occurred in the F1 mice. To determine whether these changes in antibody were playing a role in neutralizing virus, we analyzed virus-specific neutralization using antiserum from STAT4−/−, STAT6−/−, and F1 mice. The neutralizing titers, expressed as the log 2 dilution required to neutralize 90% of the viral plaques, were 11.4 ± 0.4 for STAT4−/− mice, 11.6 ± 0.3 for STAT6−/− mice and 11.5 ± 0.6 for F1 mice at 45 days and 12.4 ± 0.6 for STAT4−/− mice, 12.0 ± 0.6 for STAT6 mice and 10.4 ± 0.2 for F1 mice at 270 days. There was no statistical difference in virus neutralization between the strains (ANOVA).

Lesions in STAT4−/− mice showed CD4+ T cells, CD8+ T cells, macrophages and expression of MHC Class I and Class II antigens

We asked whether STAT4 or STAT6 deficiency altered the distribution of inflammatory cells or expression of MHC in the CNS following virus infection (Fig. 4). We chose the in situ technique (rather than FACS) because we wanted to precisely correlate immune factors with demyelination. Immunostaining was performed on spinal cord sections from STAT4−/−, STAT6−/−, and F1 mice at 270 days after infection. F1 immunostaining (not shown) was similar to that in STAT6−/− mice. CD4+ (Fig. 4A and B) and CD8+ T cells (Fig. 4C and D) were observed in the spinal cords of STAT4−/− mice, but not in STAT6−/− or F1 mice. In STAT4−/− mice, Class I was distributed in the CNS, but only minimally in STAT6−/− mice or F1 mice (Fig. 4E and F). In STAT4−/− mice Class II (Fig. 4G and H) was expressed in the CNS but less so in STAT6−/− mice or F1 mice. There was greater staining for F4/80, which marks microglia and a subset of macrophages, in STAT4−/− mice than STAT6−/− mice or F1 mice (Fig. 4I and J). Thus virus infection of STAT4−/− mice resulted in a pathologic immune profile in chronic lesions similar to that observed in MS.

Deletion of STAT4 or STAT6 did not alter brain neuronal damage following virus infection

Having established that deficiency of STAT4 predisposed mice to late demyelination, we asked whether deficiency in STAT4 or STAT6 would predispose specific populations of brain neurons to late virus-induced injury (Fig. 5). We analyzed brain neuronal pathology at 7 days to establish whether a similar extent of brain disease was present. All strains demonstrated a similar extent of pathology in cortex, hippocampus, and striatum, with minimal disease in the cerebellum. By day 90, most neuronal brain disease had subsided in all strains with only occasional mice showing persistent neuronal pathology. On day 270, when STAT4−/− mice showed late demyelination in the spinal cord, there was minimal neuronal disease in the brain (Fig. 5). We concluded that the STAT4 deletion exclusively altered the late demyelinating disease.

STAT4−/− mice propagated viral RNA in the spinal cord during all stages of infection

Reports have indicated that viral RNA persists following TMEV infection during chronic disease even though it is difficult to detect infectious virus by plaque assay (27). We developed a sensitive and quantitative RT-PCR assay to measure copy number of VP2-specific RNA in brain and spinal cord of infected mice (Fig. 6). All experiments were controlled to GAPDH RNA that was consistent among strains and time points after virus infection.


High expression of virus RNA was observed during acute and early chronic infection (7, 21, and 45 days) in the brain and spinal cord of all three strains. Because we were analyzing three strains, all statistical comparisons were made by one-way ANOVA with pair-wise comparisons using the Student-Newman-Keuls method. At 7 days, there was an increased level of virus RNA expression in the brain (P=0.001) and in the spinal cord (P<0.001) in STAT4−/− mice compared with STAT6−/− or F1 mice. No difference in virus RNA in the brain was measured at 21 and 45 days. In the spinal cord of STAT4−/− mice, more viral RNA was measured as compared with F1 mice (21 days, P=0.04) and STAT6−/− mice (45 days, P=0.002). There was more viral RNA in the brain and spinal cord of STAT4−/− mice at 90, 180, and 360 days compared with STAT6−/− or to F1 mice (P<0.001).

This data demonstrate that while all strains replicated TMEV during the early stages of infection STAT4−/− and STAT6−/− mice maintained a high level of viral RNA throughout the course of infection, whereas F1 mice controlled viral RNA in the spinal cord and brain at 90,180, and 360 days after infection. There was a highly significant 105- fold decrease of viral RNA in F1 mice brain (P=0.009) and spinal cord (P=0.005) when comparing the 45- to 90-day time points.

F1 mice that remained resistant to demyelination appeared to control virus RNA after 45 days of infection. To test whether virus RNA was truly “cleared” in F1 mice or was under “control” by the immune system, we treated F1 mice that were infected for 270 days with cyclophosphamide (50 mg/kg), which is immune-suppressive. All 3 mice treated with cyclophosphamide had detectable virus RNA (3 to 4 log10 of virus RNA), whereas none of the PBS-treated mice did (P<0.001, Chi square test). In mice that had controlled virus infection, persistent virus RNA could be detected following immune suppression.

STAT4−/− mice expressed virus antigen in spinal cord during the early chronic and the late chronic demyelinating stages of disease

The distribution of virus antigen in the spinal cords of STAT4−/−, STAT6−/−F1, and Balb/c mice was examined at 45, 90, and 270 days postinfection (Fig. 7). Spinal cord quadrants were scored blindly for the presence or absence of virus-antigen positive cells in gray or white matter. Five to fifteen mice were examined in each group with an average of 8 spinal cord sections representing the cervical, thoracic, and lumbar cord. ANOVA on ranks was used to compare the percentage of virus-antigen positive spinal cord quadrants between mouse strains and time points after infection. At 45 days, there was no difference in virus antigen in white matter between strains. Virus antigen was expressed to a limited extent in all strains at 45 days despite the absence of demyelination in any strain. At both 90 (data not shown) and 270 days after infection (Fig. 7), there was a significant difference (P=0.006) in the percent of white matter quadrants with virus between strains. Multiple pair-wise comparisons by ANOVA on ranks demonstrated an increase in the percentage of white matter quadrants with virus in STAT4−/− compared with F1 and STAT6−/− mice. The majority of the cells were in the white matter. The few cells observed in the gray matter were not neurons but morphologically appeared to be glia or macrophages. An additional group of STAT4−/− mice was examined at 330 days following infection. The percentage of virus antigen positive white matter quadrants in the STAT4−/− mice (n=10) at 330 days was 32.1 +/− 10.0. The corresponding white matter pathologic score for these 10 mice was 27.9 +/− 9.3. Thus, virus antigen in spinal cord white matter of STAT4−/− mice


correlated (linear regression, R=0.850, P=0.002) with demyelination at chronic time points after infection.

Epitope spreading to myelin antigens did not account for the late demyelination in STAT4−/− mice

Epitope spreading may explain the chronic demyelination observed in the prototypic SJL/J strain (28). Even though Balb/cJ mice normally are considered to be resistant to EAE, they have been found to be susceptible to proteolipid protein-induced EAE (29). We determined the proliferative responses of T cells obtained from spleens of chronically TMEV-infected STAT4−/−, STAT6−/−, or F1 mice in response to various CNS specific myelin antigens (Fig. 8). Antigens were chosen because they have been shown to induce EAE in mice (24, 30). The response of splenocytes to UV-inactivated TMEV was used as a control. In response to TMEV, F1 and STAT4−/− mice T cell responded at concentrations from 0.01 to 1 μg. STAT6−/− mice did not respond to TMEV or other antigens. Unexpectedly, T cells from nondemyelinating F1 mice showed the greatest proliferative response to several antigens, including bovine MBP from 10 µg to 0.01 µg of protein. T cells from F1 mice and STAT4−/− mice responded to MOG and PLP139-154 and PLP179-198. Cells from F1 mice responded strongly to PLP41-60 and PLP91-100, whereas a minimal response was measured in STAT4−/− mice. F1 mice responded to PLP209-228 only at high concentrations, whereas STAT4−/− did not respond. Splenocytes from all strains proliferated to Con A (>40,000 cpm) in contrast to splenocytes cultured in media alone (500 cpm).

The fact that F1 mice responded to many antigens, but STAT4−/− mice did not, argues against the possibility that the absence of responses in STAT4−/− mice was a result of failure to recognize the I-Ad haplotype on the BALB/cJ background. Both strains shared the same MHC. These data do not support the hypothesis that mice with chronic demyelination (STAT4−/−) had epitope spreading to myelin antigens, whereas mice that did not demyelinate (F1) had no epitope spreading. It is possible that other autoantigens may have distinguished the demyelinating strain.

The mechanism of the late demyelinating model of the late demyelinating phenotype is dependent on immune cells of the STAT4−/− strain

We explored the mechanism for STAT4−/− mice developing demyelination using bone marrow chimeric adoptive transfer. Mature T cells were deleted from donor bone marrow and transplanted into lethally irradiated recipient mice. Recipient mice developed an immune system composed of donor cells, but maintained radiation-resistant cells of the host CNS. F1 mice were reconstituted with STAT4−/− bone marrow and allowed 8 wk to recover and develop a new immune repertoire. Mice were then infected with TMEV and killed after chronic infection. All 17 F1 mice reconstituted with STAT4−/− bone marrow developed severe demyelination with a score of 35.1 ± 3.9. The extent of demyelination was the same as a parallel group of STAT4−/− mice, which had a score of 36.8 ± 8.4 (n=5) (Student’s t test). Reciprocal STAT4−/− mice reconstituted with F1 bone marrow had significantly less demyelination (13.7±4.6, n=15) compared with F1 mice reconstituted with STAT4−/− bone marrow (P=0.001, Student’s t test). We concluded that the mechanism of the late demyelinating phenotype of STAT4−/− mice was due to immune dysregulation rather than an intrinsic abnormality of the CNS.


DISCUSSION

We describe a phenotype in which virus infection is followed by demyelination in spinal cord after a very prolonged incubation period. This paper models one aspect of MS pathogenesis in which a proposed environmental event (possibly a virus infection) is followed by a long incubation period, during which the individual remains nonsymptomatic. The present studies using infected STAT4−/− mice on a BALB/cJ background mirrors this proposed scenario. Even though the pathology of MS is heterogeneous, this model fulfills many of the criteria for an excellent model of MS. The virus-infected STAT4−/− mice had the following phenotype: 1) spinal cord lesions exhibited extensive primary demyelination with minimal remyelination, 2) lesions consisted of CD4+ and CD8+ T cells and macrophages, 3) MHC class I and II antigens were up-regulated in the CNS, 4) high virus RNA expression was present during the asymptomatic and the symptomatic stages of disease, and 5) virus antigen was expressed during the demyelinating stage of disease. The mechanism of the phenotype was dependent on the reactivation of virus in the CNS due to a genetic deletion resulting in immune dysregulation and thus differed from postinfectious encephalomyelitis where virus does not re-emerge.

In all mouse strains studied to date, infection with Theiler’s virus results in two distinct phenotypes. In animals that are resistant to demyelination, virus replicates in the brain during the first 7 to 10 days of infection resulting in encephalitis. Virus is rapidly controlled such that virus persistence and subsequent spinal cord demyelination does not occur. The CD8+ T cell response is directed against an immunodominant viral peptide that participates in control of virus replication (31). CD8+ T cells may play a protective role as suppressor cells in BALB/c mice (32, 33). In susceptible mice (SJL/J), virus is never fully controlled in the CNS, but persists in glial cells and macrophages of the spinal cord. Demyelination, in association with an intense inflammatory response, is established by day 45. Demyelination worsens until day 100, and then plateaus. Animals continue to worsen functionally as a result of the loss of large diameter axons, progressive spinal cord atrophy, and limited remyelination.

The present model differs from what has been observed previously using Theiler’s virus infection. STAT4−/− mice remained relatively resistant to demyelination for 180 days; after this time point, demyelination became severe. STAT6−/− and F1 mice demonstrated minimal demyelination at this time point. Bone marrow chimeric adoptive transfer experiments established the mechanism of the phenotype because reconstitution of lethally radiated F1 mice with STAT4−/− bone marrow resulted in demyelination following virus infection identical to that of STAT4−/− mice. The difference in response to virus infection in STAT4−/− mice compared with STAT6−/− or F1 mice may provide clues to the immune deficit required to produce the late disease.

The mechanism by which deletion of STAT4 predisposes mice to late demyelination depends on immune dysregulation in STAT4−/− mice rather than on an intrinsic characteristic of the CNS of BALB/cJ mice. Our hypothesis is that, as a consequence of a focused immune dysregulation, more virus replicates later in life. We propose that the virus is partially controlled such that the virus antigen is not expressed in the spinal cord of mice that do not demyelinate. However, as a consequence of the STAT4 deletion and importantly as a function of age, virus antigen becomes expressed in the spinal cord white matter triggering demyelination.


The most unique aspect of this model was that STAT4−/− mice required considerable time for the development of the demyelinating phenotype. This time, from the inciting event to the demyelinating phenotype, may have been the consequence of aging of the nervous system or the immune system. Bone marrow chimeric adoptive transfer experiments supported the hypothesis that STAT4−/− mice develop demyelination due to an immune defect. The STAT4 defect altered the Th1 arm of the immune response, an insight that may be relevant to human disease. With time, the protective MHC Class II Th1 responses (for example, interferon gamma, IL12, or TNF) (34–36) that control of virus infection may have been exhausted. This may have also altered the protective CD8+ T cell response. Possibly as result of STAT4 deficiency from development, there was an age-dependent immune defect that favored virus antigen emergence. STAT4 deletion may have altered memory T cell function for control of virus infection or altered the longevity of auto-specific T cells. By understanding how the virus antigen was partially controlled in STAT4−/− mice during the first 180 days and then understanding why virus antigen increased may provide insight into the immune dysregulation that has been proposed for MS.

Unfortunately, there are only a few studies addressing the role of STAT4 or STAT6 in the CNS or in demyelinating diseases. Not only T cells, but also resident CNS microglia express Jak/STAT proteins (13). These factors control cellular proliferation of resident cells or may function to limit inflammation in the CNS. STAT6 protein is expressed in the brain and is maximal at embryonic stages of the rat (E14 to E18), when there is limited gliogenesis (19). In contrast, STAT4 proteins are not detected in the brain cultured neural cells, or brain-derived neural lines.

STAT4 and STAT6 have been studied in the induction of EAE (14). EAE is a CD4+ T cell Th1-mediated disease. As expected, and in contrast to our model, STAT4−/− mice were resistant to induction of MOG-induced EAE. In contrast, STAT6−/− mice developed a severe phenotype following EAE induction. Adoptive transfer experiments in EAE demonstrated that primed splenocytes from STAT6−/− mice induced a milder disease. Adoptive transfer of primed splenocytes from STAT4−/− mice failed to induce EAE. In normal mice, expression of STAT4 and STAT6 were undetectable in CNS (37). However, after induction of MOG-induced EAE, STAT4 was expressed in CD3+ T cells in the brain, but not in neurons, astrocytes, or microglia.

The involvement of the STAT4 pathway in demyelinating disease may have implications for MS therapy. Curcumin (1,7-Bis (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3, 5 dione) is a naturally occurring phytochemical isolated from Curcuma longa. Treatment with this compound inhibited EAE and worked by inhibiting IL-12- induced STAT4 transcription factor (38). Statins (3-hydroxy-3-methylglutaryl CoA reductase inhibitors), which are approved for cholesterol reduction, may benefit inflammatory diseases (15). Atorvastatin inhibited EAE by inducing STAT6 phosphorylation and conversely inhibiting STAT4 phosphorylation. The finding that virus infection of STAT4-deficient mice resulted in late demyelination raises concerns as to the use of statins in MS and may provide insight into the polarity of the immune response in the human disease.

ACKNOWLEDGMENTS

Grants from the National Institutes of Health (P01-NS-38468, R01-NS-32129, R01-NS-24180) and the National MS Society (CA-1011A8) supported this work.


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Received July 8, 2005; accepted October 13, 2005


Fig. 1

Figure 1. Percent of spinal cord quadrants with gray matter inflammation, white matter inflammation, or demyelination. Plastic-embedded sections were scored for presence of a particular pathologic abnormality in every spinal quadrant. All strains were tested up to 270 days after infection. To determine whether STAT4−/− mice continued to demyelinate past this time point STAT4−/− and specificity control (STAT4−/− X STAT6−/−), F1 mice were tested up to day 390.


Fig. 2

Figure 2. Spinal cord pathology after TMEV infection. Representative examples of spinal cord white matter pathology are shown in STAT4−/−, STAT6−/−, and F1 (STAT4−/− × STAT6−/−) mice. A) Absence of demyelination in a STAT4−/− mouse (45 days). B) Absence of demyelination in a STAT6−/− mouse (45 days). C) Absence of demyelination in an F1 mouse (45 days). D) Absence of demyelination in a BALB/cJ mouse (45 days). E) Presence of white matter inflammation and demyelination in a STAT4−/− mouse (270 days). F) Presence of white matter inflammation and demyelination in a STAT6−/− mouse (270 days). G) Absence of demyelination in an F1 mouse (270 days). H) Absence of demyelination in a BALB/cJ mouse (270 days). I) Higher magnification (Scale bar: 100 µm) of demyelinated axons (arrow) without remyelination in the spinal cord of a 270-day infected STAT4−/− mouse. Arrowheads point to macrophages with myelin debris. Figures A to H were taken at the same magnification (Scale bar: 100 µm).


Fig. 3

Figure 3. Virus-specific humoral immune responses. ELISA for serum IgM and IgG antibodies directed against TMEV antigens in STAT4−/− (n=8 at 45 days, n=22 at 270 days), STAT6−/− (n=12 at 45 days and n=10 at 270 days) and F1 (n=7 at 45 days, n=10 at 270 days) mice. Negative controls are from mice not infected with TMEV. Isotype-specific ELISA was performed from mice infected for 45 days.


Fig. 4

Figure 4. Immune staining for T cells, macrophages, and MHC. Immunoperoxidase staining was performed for CD4+ T cells (A, B), CD8+ T cells (C, D), Class I MHC (E, F), Class II MHC (G, H), and F4/80 (I, J) in the spinal cords of STAT6−/− (A, C, E, G, and I) and STAT4−/− mice (B, D, F, H, and J). Note general absence of staining for any marker in infected STAT6−/− mice. In contrast, there was staining for all of the markers in the spinal cords of infected STAT4−/− mice (Scale bar: 100 µm). Frozen spinal cord sections were from mice infected with virus for 270 days. Samples were counterstained with hematoxylin.


Fig. 5

Figure 5. Pathology in the brain. Pathologic analysis of brain areas after TMEV infection. Pathologic qualitative scores from 0 to 4 are described in Materials and Methods. Each symbol represents one mouse.


Fig. 6

Figure 6. Virus RNA expression. Virus RNA expression in STAT4−/−, STAT6−/−, and F1 mice after TMEV infection was analyzed independently in the brain and spinal cord. Levels of VP2 RNA message was quantified by light cycler PCR. Data are expressed as mean ± SD in 5 mice per group.


Fig. 7

Figure 7. Virus antigen positive cells. Immunoperoxidase staining was used to identify virus- antigen positive cells. Data were expressed as the percent of spinal quadrants showing virus-antigen positive cells in either the gray matter or the white matter. Each bar represents one animal.


Fig. 8

Figure 8. Proliferation of splenocytes to antigens. Six to nine months after infection, splenocytes from chronically infected mice (five per group) were harvested and pooled. Cells were stimulated against titrating dose of antigen in triplicate. Data are expressed as counts per minute (CPM).


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