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Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA 19107
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor knockout mice
immunized with myelin basic protein (MBP) can be curtailed by the
aggressive administration of uric acid (UA) (1, 15), a
known ONOO- scavenger (16, 17). UA
treatment also promotes the recovery of mice with EAE from clinical
signs of the disease (15). In this case, nitrotyrosine, a
marker of ONOO- reactivity, disappears from foci
of inflammation in the CNS within 4 days of UA treatment
(5). Therefore, although it is reasonable to speculate
that the protective effect of UA in EAE is through the inactivation of
ONOO-, or a closely related molecule, produced
at the site of damage in the CNS, there are a number of observations
that confound this interpretation. First, unlike humans, in which UA is
the end product of purine metabolism, mice rapidly metabolize UA
further to allantoin, which does not interact with
ONOO- (16). In our experiments,
after a single 10-mg i.p. dose, elevated UA levels are detectable in
sera for <2 h (15). A minimum of four daily doses is
required to prevent severe EAE in MBP-immunized PLSJL mice, meaning
that UA is available for entry into the CNS from serum for <8 h in a
24-h period. Second, the blood-CNS barrier in healthy animals is
essentially impervious to UA and, therefore, UA does not have access to
the sites where lesions develop until the blood-CNS barrier becomes
compromised as a result of the disease process (18, 19).
Finally, four daily doses of UA begun 5–10 days before the appearance
of clinical signs of EAE in control mice, often prevents both the
development of the disease as well as the associated CNS inflammatory
response (5, 15). Despite the association between high UA
levels and gout in humans, is it possible that UA has
anti-inflammatory properties? Could the administration of a large
quantity of particulate UA to the peritoneum have some deleterious
effect on the development of the immune response to myelin Ags that is
unrelated to its capacity to inactivate ONOO-?
To answer these questions and provide further insight into the
protective role of UA in CNS inflammatory disease we have determined
the effects of UA treatment on the development of MBP-specific
immunity, blood-CNS barrier permeability, and CNS inflammation. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Potential ONOO- scavengers and UA derivatives (all purchased from Sigma, St. Louis, MO, except for 1,3,7,9-tetramethyluric acid, which was obtained from ICN Biomedicals, Costa Mesa, CA and (2-mercaptoethyl)-guanidine, which was the gift or Dr. C. Szabo, Inotek, Beverly, MA) were titrated in the presence of 5 µM dihydrorhodamine 123 (DHR123; Molecular Probes, Eugene, OR) and 100 µM 3-morpholinosydnonimine hydrochloride (SIN-1; Alexis Biochemicals, San Diego, CA) or in the presence of 5 µM DHR123 and 106 activated cells of the mouse monocyte-macrophage cell line RAW. RAW 264.7 cells (American Type Culture Collection, Manassas, VA) were grown to 80% confluence, activated with 1 µg/ml LPS (Escherichia coli serotype 055:B5; Sigma). ONOO--mediated oxidation was measured in vitro by the conversion of DHR123 to fluorescent rhodamine 123 (20, 21). After a 1-h incubation at 37°C, rhodamine 123 fluorescence was measured in a microplate fluorometer (Cytofluor II; PerSeptive Biosystems, Cambridge, MA) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
Western analysis of ONOO--mediated tyrosine nitration
BSA (1 mg/ml) and FBS (1:20 in PBS) were incubated with 1 mM SIN-1 for 2 h at 37°C in the presence or absence of NaHCO3 (10 mM) and UA (200 µM). Immediately after incubation, 5 µl of each sample was separated on a 10% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane (NEN, Boston, MA) (22). Nitrotyrosine-containing proteins were detected with rabbit polyclonal anti-nitrotyrosine Ab (Upstate Biotechnology, Lake Placid, NY) and developed with a diaminobenzidine substrate using the Vectastain detection kit according to the manufacturer’s recommendations (cat. no. PK-6101; Vector Laboratories, Burlingame, CA).
Immunization and treatment of mice
EAE was induced in 8- to 10-wk-old female PLSJL mice (The Jackson Laboratory, Bar Harbor, ME) by s.c. immunization with 100 µg MBP in CFA supplemented with 4 mg/ml Mycobacterium tuberculosis H37RA (Difco, Detroit, MI) on day 0, followed by i.p. injection of 400 ng pertussis toxin (List Biological Laboratories, Campbell, CA) on days 0 and 2. MBP was prepared in the laboratory as previously described (23). In this model, EAE can range in onset and severity with 60–100% of the mice developing clinical signs of the disease between 15 and 30 days after immunization. Clinical severity of EAE was assessed a minimum of twice daily by at least two independent investigators. Scores were assigned on the basis of the presence of the following symptoms: 0, normal mouse; 1, piloerection, tail weakness; 2, tail paralysis; 3, tail paralysis plus hindlimb weakness; 4, tail paralysis plus partial hindlimb paralysis; 5, total hindlimb paralysis; 6, hind and forelimb paralysis; 7, moribund/dead. UA (Sigma) was administered i.p. four times daily at intervals of at least 4 h as 10 mg/100 µl suspensions in 0.8% saline. The oxidative product of UA, allantoin, (10 mg/100 µl saline) or 100 µl saline were similarly administered as controls.
Assessment of inhibition of ONOO--mediated oxidation by UA-treated peritoneal cells (PC)
Groups of mice received 10 mg UA in 100 µl saline i.p. or were left untreated. At intervals of 30, 90, and 120 min afterward, the peritoneal cavities of three mice per group were rinsed with 5 ml PBS, and the recovered PC were washed three times by centrifugation in PBS (10 min at 1000 rpm). PC were resuspended in 1 ml PBS and layered on an isotonic 30:65:100% Percoll (Amersham Pharmacia Biotech, Piscataway, NJ) step gradient to isolate them from any residual particulate UA. PC, collected from the 30:65 interface, were resuspended in PBS (2.5 x 106/ml) and plated in seven replicate 100-µl samples in a 96-well flat-bottom plate (Nalge Nunc International, Rochester, NY). To determine whether the UA-treated PC inhibited ONOO--mediated oxidation, they were mixed in culture with DHR123 (5 µM) as an indicator of oxidation and SIN-1 (100 µM) as a source of ONOO-. After a 1-h incubation at 37°C, culture supernatants were analyzed for rhodamine 123 fluorescence using a microplate fluorometer with excitation at 485 nm and emission at 530 nm.
In vitro assays of immune function
Single cell suspensions were prepared from the spleens and
inguinal and axillary lymph nodes of 5–10 MBP-immunized PLSJL mice by
teasing through stainless steel wire mesh in PBS. Red cells were lysed
by hypotonic shock. After a minimum of two washes by centrifugation in
PBS, cells were resuspended in MEM 
modification (Life Technologies,
Grand Island, NY) supplemented with 4 mM L-glutamine (Life
Technologies), 0.05 mM 2-ME (Sigma), 25 mM HEPES (Life Technologies),
10 µg gentamicin (Life Technologies), and 0.6% fresh mouse serum.
Lymph node cells were cultured (2.5 x
106/ml) with spleen cells (1–2.5 x
106/ml) as APCs, and unselected spleen cells were
cultured alone (1 x 106/ml) in either
200-µl volumes in 96-well round-bottom plates (Nalge Nunc
International) or 2-ml volumes in 24-well plates (Costar, Cambridge,
MA) in the absence or presence of MBP (10 µg/ml). Where indicated,
UA, allantoin, or xanthine was added to the cultures at a final
concentration of 5 mg/dl. At 24-h intervals between 48 and 120 h
of culture, four replicate microtiter cultures or three replicate
100-µl samples from 2-ml cultures were pulsed with 1 µCi of
[methyl-3H]thymidine (specific
activity, 65 Ci/mmol; 1 Ci = 37 GBq; DuPont-NEN, Boston, MA) and
incubated at 37°C for 4 h. The cultures were harvested using a
Mach III M harvester 96 (Tomtec, Orange, CT), and the
[3H]thymidine incorporated into newly
synthesized DNA was estimated by liquid scintillation in a 1450
Microbeta Trilux counter (Wallac Oy, Turku, Finland).
Analysis of MBP-specific Abs in ELISA
Ab specificity and isotype were assessed in solid phase ELISA. Plates (Polysorb; Nalge Nunc) were coated at room temperature with 2 µg/ml MBP diluted in PBS and incubated overnight in a humidified chamber. The plates were washed with PBS containing 0.05% Tween 20 and blocked with 1% BSA in PBS for 1 h before the addition of serum samples. Samples were diluted 1:100 in PBS and titrated 2-fold down the plate. Following a 2-h incubation at room temperature, plates were washed with PBS containing 0.05% Tween 20 to remove any unbound primary Ab. Bound Ab was detected by the addition of alkaline phosphatase-conjugated rat monoclonal anti-mouse IgG1 (1:2000 in PBS; PharMingen, San Diego, CA) using p-nitrophenyl phosphate (Sigma) in 0.1 M glycine buffer as a substrate. Absorbance was read in a microplate spectrophotometer (Bio-Tek, Winooski, VT) at 405 nm.
Molecular analysis of iNOS mRNA expression
Total mRNA was extracted using TRIzol (Life Technologies) from lymph node, spinal cord, and PBL isolated from blood by centrifugation on Lymphoprep-M (Cedarlane Laboratories, Hornby, Ontario, Canada). RT-PCR detection of iNOS mRNA was performed using the Access RT-PCR System (Promega, Madison, WI) according to the manufacturer’s instructions. RNA was transcribed and amplified, as previously described (24), using mouse iNOS primers: sense TTC CAG AGT TTC TGG CAG CA; antisense TCT TTA CTC AGT GCC AGA AG, which yield a 524-bp product. RT-PCR products were separated on a 1.5% agarose gel containing ethidium bromide (0.5 µg/ml), and the intensity of the amplified 524-bp fragment quantitated under UV illumination using a Fluor-S Multi Imager with Quantity One Software (Bio-Rad, Richmond, CA). Specificity of amplification was routinely controlled by direct sequence of the RT-PCR product.
Assessment of blood-CNS barrier permeability
Blood-CNS barrier permeability was assessed using a modification of a previously described technique in which sodium fluorescein is used as a tracer molecule (25, 26). Mice received 100 µl of 10% sodium fluorescein in PBS i.v. under isoflurane anesthesia. After 10 min to allow circulation of the sodium fluorescein, cardiac blood was collected and the animals were transcardially perfused with PBS/heparin (1000 U/L). Sodium fluorescein uptake into the spinal cord was measured using a modification of the method of Trout et al. (27). In brief, spinal cord tissue was homogenized in 1.5 ml cold 7.5% TCA and centrifuged for 10 min at 10,000 x g to remove insoluble precipitates. Following the addition of 0.25 ml 5 N NaOH, the fluorescence of a 100-µl supernatant sample was determined using a Cytofluor II fluorometer at excitation 485 nm and emission 530 nm. Serum levels of sodium fluorescein were assessed as described previously (27). Standards (0.125–4 µg/ml) were used to calculate the sodium fluorescein content of the samples in micrograms. Sodium fluorescein uptake into spinal cord tissue is expressed as (µg fluorescence spinal cord/mg protein)/(µg fluorescence sera/µl blood) to normalize values for blood levels of the dye at the time of sacrifice.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our approach to examining the contribution of
ONOO- to the pathogenesis of EAE has been to
determine whether reagents that interfere with its chemical reactions
also inhibit the development or progression of the disease (1, 5, 15). Table I
summarizes the
results of a survey of a variety of compounds that have been assessed
for the capacity to inhibit oxidation of DHR123
by ONOO- produced either chemically by SIN-1 or
biologically by LPS-stimulated RAW monocytes. UA, a purine metabolite
and known ONOO- scavenger (16, 17),
proved to be the most effective reagent tested, particularly in the
more biologically relevant RAW cell assay. UA was selected for further
study because of its activity against ONOO- and
the availability of extensive information concerning its toxicity and
metabolism in humans where, unlike mice, it is found at relatively high
levels (28).
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). Taken in context with our previous
finding that UA treatment of mice with EAE reduces tyrosine nitration
in the CNS (5), these results are consistent with the
hypothesis that UA is therapeutic in EAE through inactivating
ONOO-.
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Although the presence of relatively high levels of UA in humans
makes it highly unlikely that UA has deleterious effects on human
lymphocytes or APC, it is within the realm of possibility that UA is
therapeutic in EAE through a direct inhibitory effect on the activity
of these cells in mice. A likely target of any untoward effect of
exogenous UA administration may be peritoneal macrophages as i.p.
injection of a 10-mg suspension of UA was required to sufficiently
raise serum UA levels in mice in the face of rapid metabolism of the
molecule (15). We have previously demonstrated that UA
does not suppress NO· production by LPS-activated RAW cells
(15). However, as suggested by the experiment summarized
in Table II, UA is apparently taken up by
peritoneal macrophages following i.p. administration of a 10-mg
suspension. Macrophages recovered from the peritoneum of mice treated
for 90–120 min with UA inhibit the detection of
ONOO- in the DHR123 assay
(Table II). This is presumably due to the release of sequestered UA
into the medium as after 60 min of culture only the supernatant had the
capacity to inhibit DHR123 oxidation (data not
shown). The fact that macrophages obtained 30 min after UA
administration do not have this effect indicates that UA uptake may be
an active process requiring a greater period of time (Table II). The
reduced inhibition of DHR123 oxidation seen with
peritoneal macrophages recovered 120 min after UA treatment could be
the result of the rapid clearance of UA, which occurs in mice
(15), or due to trafficking of the cells to other sites.
In either case, macrophages potentially involved in the pathogenesis of
EAE appear to retain UA for some time after elevated levels have
disappeared from the serum (15).
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, UA at levels equivalent to those
found in human serum (which average 4–6 mg/dl, depending on age and
sex) and slightly higher than those attained in the sera of PLSJL mice
by the i.p. administration of 10 mg UA (peak level 30 min
postinjection, 3.0 ± 0.4 mg/dl; mean of six mice ± SEM),
does not significantly inhibit MBP-specific T cell proliferation in
vitro. Effects due to the addition of the UA precursor xanthine or
oxidative product allantoin were also minimal (Table III). These
results indicate that both the presentation of MBP and T cell
recognition of the Ag in EAE are unlikely to be negatively influenced
by UA or allantoin.
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Even if UA does not have a direct inhibitory effect on APC or CD4
T cell function, it is possible that its administration may influence
some aspect of immunity in mice. As shown in Fig. 2, there is no statistically significant
difference between the MBP-specific in vitro proliferative responses of
lymph node cells from mice immunized with MBP and treated with saline,
allantoin, or UA for 7 and 10 days following immunization. This implies
that UA has no effect on Ag presentation or T cell priming and
expansion in vivo. The kinetics and magnitude of the MBP-specific
proliferative responses of spleen cells from the MBP-immunized, UA-,
allantoin-, and saline-treated animals are also essentially the same
(Fig. 3), which implies that any effect
of UA on the recirculation of T cells or APC from the lymph nodes
draining the site of immunization to the spleen is minimal. UA
treatment also had no effect on the development of the Ab response to
MBP. MBP-specific IgG1 (Fig. 4), IgG2a,
and IgG2b (data not shown) Abs appeared in equivalent levels between
days 7 and 10 after immunization in both UA- and control-treated
mice.
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). Although cells expressing significant
levels of iNOS mRNA are not found in the peripheral blood of the
majority of nonimmune PLSJL mice, such cells appear in the circulation
of most mice between 12 and 16 days after immunization with MBP
regardless of whether they have been treated with UA (Fig. 6). At 20 days postimmunization, clinical
signs of EAE and the presence of iNOS-mRNA in spinal cord were assessed
in control and UA-treated mice that were positive for iNOS mRNA in
peripheral blood cells 4 days previously (Fig. 7). The development of clinical signs of
EAE in MBP-immunized PLSJL mice was associated with the appearance of
iNOS mRNA in spinal cord tissue. Both the development of clinical signs
and the appearance of iNOS-positive cells in spinal cord tissue were,
in most cases, prevented by UA administration (Fig. 7). Note that the
rare UA-treated animal that expresses iNOS mRNA in the spinal cord does
not necessarily exhibit signs of clinical disease.
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As the blood-CNS barrier normally becomes compromised in EAE and
UA treatment of PLSJL mice with the disease promotes recovery of
blood-CNS barrier integrity (5), we thought that the
failure of iNOS-positive cells to reach spinal cord tissue in the
UA-treated, MBP-immunized animals may be related to a UA-mediated
effect on the blood-CNS barrier. Therefore, we tested whether UA
administration beginning before the onset of EAE protects against the
blood-CNS barrier permeability changes associated with the disease.
Such experiments are made difficult to interpret because of several
factors including 1) failure of up to 40% of MBP-immunized PLSJL mice
to develop EAE within a reasonable period of time; 2) variability in
onset of clinical signs of EAE between individual mice; and 3) absence
of disease in the majority of UA-treated mice. Because of the latter,
both treated and control mice must be sampled based on the predicted
onset of disease in the control group. In the experiment shown in Fig. 8, the permeability of the blood-CNS
barrier was assessed 18 days postimmunization with MBP when only 6 of
the 40 mice had shown mild signs of clinical EAE (score 
3). Based on
blood-CNS barrier permeability, control- and UA-treated animals each
segregate into two statistically different groups
(p < 0.001 for the control groups, and
p < 0.02 for the UA-treated groups; Mann-Whitney
U test). However, although blood-CNS barrier permeability
was clearly elevated in 13 of 27 control-treated mice, only 2 of 13
UA-treated mice showed a similar change. UA treatment significantly
reduced the incidence of mice with evidence of compromised blood-CNS
barriers (
2 test, p < 0.023).
A similar relationship was seen in the expression of iNOS mRNA in the
spinal cords of control- vs UA-treated mice 20–21 days after MBP
immunization (Fig. 9). Control mice
segregated into two statistically distinct groups
(p < 0.001 by the Mann-Whitney U
test) with 12 of 25 mice expressing high levels of iNOS mRNA. Eleven of
the 12 mice exhibited clinical scores higher than 2, whereas only one
was not sick and another had clinical signs of EAE and mRNA levels
within the lower range. In contrast, only one of nine UA-treated
animals expressed high levels of iNOS mRNA in the spinal cord. None of
these mice showed clinical signs of EAE.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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15-fold less effective at inhibiting
DHR123 oxidation mediated by
ONOO- elaborated by activated macrophages. More
important in the context of nitrotyrosine formation in MS and EAE
(1, 2, 3, 4, 5), UA efficiently inhibits
ONOO--mediated tyrosine nitration in the
presence and absence of a source of carbonyl anion. Apart from
inactivating ONOO-, UA does not appear to have
any significant effect on in vitro measures of immune and inflammatory
cell function relevant to EAE. Although peritoneal macrophages appear
to take up UA, the expression of iNOS and production of NO· by
monocytes in MBP-immunized mice are not diminished by UA treatment. The
MBP-specific T cell proliferative response is also unchanged in the
presence of UA. Thus it is not surprising that the in vivo immune
response to MBP appears to be largely unaffected by the administration
of UA even if begun on the day of immunization as opposed to 5–10 days
later, which still successfully interferes with the development of
clinical EAE (15). The levels of MBP-specific IgG Abs
produced by MBP-immunized mice, whether treated with UA or not, are
equivalent, as is the capacity of T cells recovered from such animals
to proliferate in response to MBP in vitro. More importantly, with
respect to the putative association between iNOS,
ONOO-, and the pathogenesis of EAE, it is
readily apparent that UA treatment does not interfere with iNOS
expression in the lymph nodes or peripheral blood of mice subjected to
the stimulus that triggers EAE. Therefore, we conclude that UA
treatment does not affect the MBP-induced activation of monocytes to
express iNOS or the circulation of these cells through peripheral
blood, a necessary step toward the invasion of CNS tissue. However, UA
treatment evidently prevents iNOS-positive cells from entering CNS
tissue. A variety of observations have provided evidence that ONOO- may have deleterious effects on the integrity of the blood-CNS barrier (5, 31, 32, 33). The current findings support the hypothesis that one therapeutic mode of action of UA in EAE is through interfering with inflammatory cell invasion into the CNS by blocking ONOO--mediated permeability changes in the neurovasculature. Presumably, for this to be the case UA would have to be available at the blood-CNS barrier consistently, yet we can only detect it in serum for <2 h following each dose (15). Although it is possible that even the transient presence of UA could sufficiently reduce the effect of ONOO- on the blood-CNS barrier to help maintain its integrity, the fact that peritoneal macrophages are probably taking up UA provides another possible explanation. Peritoneal macrophages from UA-treated mice show peak antioxidant capacity when serum UA levels have already deteriorated. Therefore, monocytes may serve as a short-term reservoir of UA that is not subject to rapid breakdown by urate oxidase in the liver (34) and can be targeted to areas of inflammatory cell accumulation.
The fact that UA treatment is therapeutic against the development of EAE in the rare instance where iNOS mRNA-positive cells appear in the spinal cord despite UA administration is consistent with our previous observation that UA can reverse the progression of clinical disease in mice (15). The mode of action in this scenario is presumably that UA penetrates the compromised blood-CNS barrier and inactivates ONOO- produced at the site of damage. The best evidence supporting this possibility is that UA treatment is associated with inhibition of tyrosine nitration but not iNOS expression in the lesion (5). However, concomitant with recovery from EAE, UA-treated mice also show a return of blood-CNS barrier permeability to normal (5). Our current findings support the concept that a major protective effect of UA in EAE is through the maintenance of blood-CNS barrier integrity, which prevents iNOS-positive cells and, likely, other pathogenic cells and factors from reaching spinal cord tissue. In this case, ONOO-, or a closely related product, must play a principal role in providing activated monocytes access to CNS tissues. Further studies are necessary to determine whether this is through damage or a signaling process and whether there is a contribution from up-regulation of adhesion molecules on vascular endothelial cells in addition to the enhanced physical permeability of the blood-CNS barrier.
We have previously observed significantly lower serum UA levels in a group of MS patients by comparison with age- and sex-matched controls (15). The current findings suggest that UA does not directly inhibit the immune mechanisms responsible for the pathogenesis of EAE, an animal correlate of MS, but instead protects against the development of the disease by preventing blood-CNS barrier breakdown and the associated inflammatory cell invasion into CNS tissues. However, UA treatment also promotes the recovery of mice with pre-existing EAE (5, 15). We theorize that the role of UA in human physiology is to maintain blood-CNS barrier integrity as well as to prevent tissue damage due to ONOO- or associated radicals (35). In this case, low serum UA levels in humans may predispose toward the development of MS and other CNS inflammatory diseases.
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Acknowledgments |
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Footnotes |
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2 R.B.K. and S.V.S. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. D. C. Hooper, Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, JAH Room 454, Philadelphia, PA 19107-6799.
4 Abbreviations used in this paper: ONOO-, peroxynitrite; MS, multiple sclerosis; EAE, experimental allergic encephalomyelitis; UA, uric acid; MBP, myelin basic protein; iNOS, inducible NO synthase; PC, peritoneal cells; DHR123, dihydrorhodamine 123; SIN-1, 3-morpholinosydnonimine hydrochloride.
Received for publication March 10, 2000. Accepted for publication August 30, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-antiproteinase inactivation by ascorbic acid: a comparison with other biological antioxidants. Free Radic. Res. 25:275.[Medline]
-l-antiproteinase by hydroxyl radicals: the effect of uric acid. FEBS Lett. 244:76.[Medline]
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G. S. Scott, R. B. Kean, T. Mikheeva, M. J. Fabis, J. G. Mabley, C. Szabo, and D. C. Hooper The Therapeutic Effects of PJ34 [N-(6-Oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide.HCl], a Selective Inhibitor of Poly(ADP-Ribose) Polymerase, in Experimental Allergic Encephalomyelitis Are Associated with Immunomodulation J. Pharmacol. Exp. Ther., September 1, 2004; 310(3): 1053 - 1061. [Abstract] [Full Text] [PDF]
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G. S. Scott, S. V. Spitsin, R. B. Kean, T. Mikheeva, H. Koprowski, and D. C. Hooper Therapeutic intervention in experimental allergic encephalomyelitis by administration of uric acid precursors PNAS, December 10, 2002; 99(25): 16303 - 16308. [Abstract] [Full Text] [PDF]
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 M. L. PALL NMDA sensitization and stimulation by peroxynitrite, nitric oxide, and organic solvents as the mechanism of chemical sensitivity in multiple chemical sensitivity FASEB J, September 1, 2002; 16(11): 1407 - 1417. [Abstract] [Full Text] [PDF]
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D. C. Hooper, R. B. Kean, G. S. Scott, S. V. Spitsin, T. Mikheeva, K. Morimoto, M. Bette, A. M. Rohrenbeck, B. Dietzschold, and E. Weihe The Central Nervous System Inflammatory Response to Neurotropic Virus Infection Is Peroxynitrite Dependent J. Immunol., September 15, 2001; 167(6): 3470 - 3477. [Abstract] [Full Text] [PDF]
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0.55 by Student’s t
test).
, dashed line). Data is represented as mean ± SD. Serum Ab
responses were negligible at 7 days postimmunization and are
indistinguishable on the graph.
