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*
Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA 19107; and
Department of Molecular Neuroimmunology, Institute of Anatomy and Cell Biology, Philipps University, Marburg, Germany
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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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, IL-2,
IL-6, TNF-, and IFN-
as well as other immunologically relevant
factors all become elevated in the brains of rats acutely infected with
BDV (16, 17, 18, 19). Both CNS resident cells and invading
macrophages may participate in the inflammatory response that
contributes to the pathogenesis of acute Borna disease by producing
free radicals and other factors that may interfere with the function
of, if not kill, neurons. It is generally accepted that nervous tissue is shielded from the immune system by the blood-brain barrier (BBB), presumably to prevent inflammatory and cytolytic responses from damaging neurons, which have a limited capacity for repair. Restricted expression of class I MHC Ags by neurons (reviewed in Ref. 20) may offer some protection from Ag-specific cytotoxicity, but non-Ag-specific inflammatory mechanisms alone are most likely sufficient to cause substantial CNS damage. Studies of experimental allergic encephalomyelitis (EAE) have provided considerable information concerning CNS inflammatory disease in the absence of an underlying viral infection. In this model, acute neurological disease is triggered by sensitization of CD4 T cells with myelin Ags, and CNS pathology has been associated with free radical production by inflammatory cells (21, 22, 23). Peroxynitrite (ONOO-), the product of NO· and O2·-, is believed to be an important toxic molecule in this context (24, 25, 26, 27). Whether the reactions attributed to ONOO- in vivo are in fact mediated by radicals formed by the interaction of ONOO- and other molecules found in a biological milieu is currently under investigation (28, 29). Therefore, in the context of our work, reference to ONOO- should be taken as including related intermediates. We have recently provided evidence that, in addition to causing pathological changes in CNS tissues, ONOO- plays an important role in promoting inflammatory cell invasion into CNS tissues in EAE (30). For instance, administration of uric acid (UA), a natural inhibitor of select chemical reactions associated with ONOO-, to animals with EAE protects the blood-CNS barrier from permeability changes normally associated with the disease (30). As the production of the ONOO- precursor NO• is a hallmark of the CNS inflammatory response in both EAE (15, 22) and acute Borna disease (15), we postulate that ONOO- may have a physiological role in neuroimmune responses to enhance BBB permeability and promote cell invasion into the CNS. To investigate whether this may be the case for a neurotropic virus infection, we have assessed the effects of UA treatment on BBB permeability, CNS inflammation, and clinical disease in immunocompetent adult rats acutely infected with BDV.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Twelve- to 14-wk-old female Lewis rats (Harlan Breeders,
Indianapolis, IN) were infected under anesthesia with 30 µl per
nostril of a 4% brain homogenate containing 106
focus-forming units of BDV. Following infection, animals were examined
daily for clinical signs of disease. Scores were assigned on the basis
of the presence of the following symptoms: 0, normal; 1, hyperactivity,
aggressiveness, extremely excitable, and respond vigorously to loud
noises; 2, disturbance of motor functions, ataxia; 3, development of
tremors, spasms, convulsions, and paralysis; 4, moribund, no response
to stimuli. Beginning at 10–14 days postinfection (p.i.), groups of
BDV-infected rats were treated i.p. with two daily doses of 100 mg UA,
or its inert oxidation product allantoin, in 1 ml saline or saline
vehicle alone at an interval of 8 h. With respect to UA treatment,
this dose resulted in an elevation of serum UA levels in the rats from
0.5 ± 0.1 SD to a peak of 3.7 ± 0.7 SD mg/dl in 30 min.
Due to the presence of urate oxidase in rats, levels of UA in serum
dropped rapidly from 30 min postadministration, becoming half-maximal
at 2 h, but remaining significantly elevated for up to 4 h
(1.1 ± 0.3 at 4 h, p = 0.01).
ELISA for BDV-specific Ab
Sera from rats infected with BDV 22–24 days previously and treated with 100 mg UA, allantoin, or saline from day 10 p.i. were assessed for BDV-specific Abs in solid-phase ELISA. Plates (Polysorb; Nalge Nunc International, Rochester, NY) were coated at room temperature with a 2% homogenate of either uninfected or BDV-infected rat brain diluted in PBS and incubated overnight in a humidified chamber. The plates were washed with PBS containing 0.05% Tween 20 (PBS-Tween 20) and blocked with 5% milk in PBS for 1 h before the addition of serum samples. Samples were diluted 1/10 in PBS and titrated 2-fold down the plate. Following a 2-h incubation at room temperature, plates were washed with PBS-Tween 20. Ab was detected using peroxidase-conjugated anti-rat IgG whole molecule (dilution 1/2000; Sigma, St. Louis, MO) with 3,3',5,5'-tetramethylbenzidine (Sigma) in phosphate-citrate buffer (Sigma) as a substrate. Absorbance was read at 450 nm in a microplate spectrophotometer (Biotek, Winooski, VT).
Real-time quantitative PCR
RNA was isolated from brain tissue of rats infected 22–24 days previously with BDV and treated from day 10 p.i. with two daily i.p. doses of 100 mg UA, allantoin, or with saline vehicle. Rats were anesthetized and perfused with PBS containing heparin (1000 U/L), and then brains were removed and snap frozen. RNA was isolated from brain using TRIzol B (Life Technologies, Grand Island, NY), then DNA contamination was removed by treatment with DNA-free reagent (Ambion, Austin, TX). cDNA was synthesized from 5 µg total RNA using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) and dT15 primer. An equivalent of 50 ng total RNA was used for the PCR with Taqman PCR Core Reagent kit (Applied Biosystems, Foster City, CA). Primers and probes were designed using the Web Primer 3 program (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). Double-labeled probes were purchased from Integrated DNA Technologies (Coralville, IA), and primers were synthesized by our in-house Nucleic Acid Facility (Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA). The 5' ends of probes were labeled with reporter dyes Hex or 6-Fam, and 3' ends with the quencher BHQ-1. Probes and primers were as follows: CD8, probe, TTG CTT GGT AGG GCA GCA TGA CTG GC; 5' primer, AGC CTT GCA GCC ACC ATC TTT; 3' primer, TCT CTC CCA TGA GAC GCC AAA. CD4, probe, CCC ACA CAG TGC CTC TGG CCT CCT; 5' primer, GGC CTG TGG ACC AGA TGA ATG; 3' primer, GAG AAA CCG TGG CCA GTT GTG. MHC class II, RT1B, probe, TCA GAT GGC CCC TCC AGA CAC CCA; 5' primer, GGG CAC CAT CTT CAT CAT TCA AG; 3' primer, TTC TTT CCC AGG GTG TGA CTC AA. GAPDH, probe, AGA AGG TGG TGA AGC AGG CGG C; 5' primer, TGG AGA AAC CTG CCA AGT ATG; 3' primer, GTC CTC AGT GTA GCC CAG GAT. Quantitative PCR was performed using a Bio-Rad iCycler iQ Real Time Detection System (Hercules, CA). Data were calculated based on a threshold cycle (Ct) determined as the cycle with a signal higher than that of the background (signal detected in cycles 2–10) plus 10x its SD. Data are expressed as a fold increase in mRNA expression calculated by exponent (Ct lowest expresser (e.g., normal mice) - Ct test value) divided by the same value determined for the housekeeping gene GAPDH.
Immunohistochemistry
Groups of saline- and UA-treated (from day 14 p.i.) rats were anesthetized with ketamine/xylazine 23 days post-BDV infection and transcardially perfused with PBS containing procaine-HCl (5 g/L), followed by Bouin Hollande fixative, as described elsewhere (18). The perfused brains were removed, dissected into anterior, middle, and posterior parts, and postfixed for 24–48 h in Bouin Hollande fixative. After dehydration in a graded series of 2-propanol solutions, tissues were embedded in Paraplast Plus (Merck, Darmstadt, Germany). Deparaffinized serial sections were stained with Giemsa according to standard procedures for the general assessment of histopathology and inflammation, or stained with one of the following primary Abs: the mouse mAb BO18 (dilution 1/1000, a gift from J. Richt, University of Giessen, Giessen, Germany), which recognizes the BDV 38-kDa Ag, a major protein of BDV expressed at all stages of replication; the mouse anti-rat ED1 mAb (dilution 1/200; Camon, Wiesbaden, Germany) as a specific marker for cells of the monocyte/macrophage lineage and activated microglia; the polyclonal rabbit anti-nitrotyrosine (NT) Ab (dilution 1/4000; Chemicon, Temecula, CA), which is a specific marker for highly reactive nitrogen species derived from NO, such as ONOO-, nitrogen dioxide, and nitryl chloride, which lead to the nitration of tyrosine residues in protein chains (31); the polyclonal rabbit anti-inducible NO synthase (iNOS) Ab (dilution 1/400; Santa Cruz Biotechnology, Heidelberg, Germany); the mouse anti-rat CD4 Ab (dilution 1/25; Serotec, Eching, Germany) recognizing rat CD4 cell surface glycoprotein expressed by Th cells; and the mouse anti-rat CD8 Ab (dilution 1/500; Labgen, Frankfurt, Germany) recognizing rat CD8 cell surface glycoprotein expressed by cytotoxic/suppressor T cells. Sections were incubated with the primary Abs overnight at 18°C, followed by an additional incubation for 2 h at 37°C. After several washes in distilled H2O, followed by rinsing in 50 mM PBS, species-specific biotinylated secondary Abs (dilution 1/200; Dianova, Hamburg, Germany) were applied for 45 min at 37°C. After another series of washes, sections were incubated for 2 h at 37°C with the streptavidin-biotin-HRP complex (Amersham/Buchler, Braunschweig, Germany), as described previously (32), or the ABC reagents (Vectastain; Camon, Wiesbaden, Germany). Immunoreactions were visualized with 3'3-diaminobenzidine (Sigma) enhanced by the addition of 0.08% ammonium nickel sulfate (Fluka, Buchs, Switzerland), resulting in a dark blue staining, as described previously (33). Staining controls consisted of sections treated similarly, but without primary Abs. In these controls, no immunoreactions were visible for any used Ab (data not shown). Sections were analyzed and photographed with an Olympus (Melville, NY) AX 70 light microscope.
BBB permeability
BBB permeability was assessed using a modification of a previously described technique in which fluorescein is used as a tracer molecule (30). Rats, infected with BDV 22–24 days previously and treated with UA, allantoin, or saline from day 14 p.i., received 300 µl of 10% sodium fluorescein in PBS i.v. under isoflurane anesthesia. After 5 min, to allow circulation of the sodium fluorescein, cardiac blood was collected, the animals were transcardially perfused with PBS/heparin, and brains were removed. Brains were weighed, homogenized in 1.5 ml of cold 7.5% trichloroacetic acid, and centrifuged for 10 min at 10,000 x g. Following the addition of 0.25 ml of 5 N NaOH, the fluorescence of 100 µl of supernatant was determined using a Cytofluor II fluorometer (PerSeptive Biosystems, Framingham, MA) with 485 nm excitation and 530 nm emission. Serum levels of sodium fluorescein were assessed as previously described (30). Standards ranging from 125 to 4000 µg/µl were used to calculate the sodium fluorescein content of the samples in µg. Sodium fluorescein uptake into the brain tissue is expressed as (micrograms fluorescence in brain/milligram protein)/(µg fluorescence in sera/microliter 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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In our model, clinical signs of Borna disease appear in Lewis rats
20 days after intranasal infection with BDV (17, 18).
The disease is rapidly progressive, with the majority of infected rats
dying within 3–5 days of the onset of symptoms. As can be readily seen
in Fig. 1
, two daily doses of 100 mg UA
i.p. inhibited the onset of clinical signs of Borna disease and
protected against a lethal outcome within this time frame. To determine
whether UA treatment had any general effect on immunity, we assessed
the levels of BDV-specific IgG Abs in the sera of BDV-infected rats
treated with UA, allantoin, or saline. As shown in Fig. 2, all three groups of BDV-infected rats
exhibited strong Ab responses to BDV. Nevertheless, UA-treated rats
made somewhat less Ab (p < 0.04 by the paired
t test) than either saline or allantoin controls (Fig. 2).
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As UA treatment can prevent the development of CNS inflammation in
a conventional mouse model of EAE (30), it seemed possible
that a similar effect was operative in Borna disease. Nevertheless, it
was also conceivable that the administration of UA had either a direct
or indirect effect on the BDV infection, thereby leading to a reduced
immune and inflammatory stimulus. To test these possibilities, we used
real-time quantitative RT-PCR to assess the levels of mRNAs specific
for CD4, CD8, and MHC class II in brains from UA- and saline
vehicle-treated, BDV-infected rats (Fig. 3). In this experiment, UA treatment
delayed the appearance and severity of clinical signs of BD, but when
the tissues were taken (days 22–24 p.i.), the treated animals showed
signs of disease (average 2 vs 3 in saline controls). Nevertheless,
while the levels of BDV RNA were comparable in both groups of rats
(data not shown), those of mRNA specific for CD4, CD8, and MHC class II
were all significantly reduced by UA treatment
(p < 0.001 by ANOVA) (Fig. 3). Similar results
were obtained for IFN- and iNOS mRNA (data not shown). These results
suggest that UA treatment has no effect on the replication of BDV, but
inhibits the appearance in infected CNS tissue of CD4 and CD8 cells as
well as activated cells of the monocyte lineage.
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To assess the effects of UA treatment on the distribution of BDV
and immune/inflammatory cells in the infected brain, serial sections of
brain tissue from control (saline)- and UA-treated rats infected with
BDV 23 days previously were examined for BDV Ags as well as the
inflammatory changes that normally accompany acute BDV disease. To
assess whether UA treatment either prevented the spread of BDV through
the CNS or promoted the clearance of the virus, sections from the
brains of BDV-infected rats treated with either saline or UA were
assessed for the presence of BDV Ag. High levels of BDV Ags were
detected in brain tissue, regardless of whether UA had been
administered, indicating that UA does not influence virus spread in the
BDV-infected brain (Fig. 4, B
and C). In the normal rat brain (Ctrl), very few
inflammatory cells are seen by Giemsa (Fig. 4D) and ED1
immunostaining (Fig. 4G). Pronounced accumulations of
inflammatory cells, stained with Giemsa (Fig. 4E) and ED1
(Fig. 4H), are found in the subarachnoid space, and in
neocortical parenchyma of the BDV-infected brain (Fig. 4, E
and H). UA treatment restricted Giemsa-stained inflammatory
cells (Fig. 4F) and ED1-positive cells (Fig. 4I)
to the subarachnoid space and blood vessels in the BDV-infected brain.
In contrast, such cells are scarce in the brain parenchyma of
UA-treated, BDV-infected rats. To assess the nature of the infiltrating
cells in the BDV-infected rat brain as well as test for evidence of
ONOO- formation, serial sections from the brains
of control, BDV-infected, and UA-treated BDV-infected rats were stained
with Abs specific for NT, iNOS, CD4, and CD8 (Fig. 5). Although there is little evidence of
these markers in the uninfected control rat brain (Fig. 5, A, D, G, and J), they are
extensively expressed throughout BDV-infected brain tissue (Fig. 5, B, E, H, and K). Cells
staining for NT (Fig. 5B), iNOS (Fig. 5E), CD4
(Fig. 5H), and CD8 (Fig. 5K) are seen in the
subarachnoid space as well as massive infiltration into the neocortical
parenchyma. This pattern was considerably changed by UA treatment
following BDV infection. In this case, moderate numbers of cells
stained for NT (Fig. 5C), iNOS (Fig. 5F), CD4
(Fig. 5I), and CD8 (Fig. 5L) accumulate in the
subarachnoid space and in blood vessels, but infiltration into the
neocortical parenchyma is minimal. In general, CD4 and CD8 cells were
restricted to regions containing NT and some evidence of iNOS
expression (Fig. 5, C, F, I, and
L).
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, UA treatment also
prevented the extensive tissue damage normally associated with acute
Borna disease. In the BDV-infected rat, a marked degeneration and
accumulation of inflammatory cells into the brain parenchyma are seen
(Fig. 6C). The damage is most pronounced in the temporal
lobe and the hippocampus, particularly in the pyramidal cell layer as
well as in the dentate gyrus. The brain from a UA-treated, BDV-infected
rat remained histologically normal with the exception of cells
accumulating in the vasculature (Fig. 6D).
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The antiinflammatory effect of UA in Borna disease resembles its
effect on cell infiltration in EAE (30). In the latter
case, UA has been demonstrated to protect the blood-CNS barrier from
permeability changes most likely induced by
ONOO- (30). Since, like EAE, the
CNS inflammatory response in acute Borna disease is associated with the
production of NO· (15), it seemed
possible that BDV infection may also promote cell invasion through
ONOO--mediated disruption of the BBB. We
therefore determined whether the integrity of BBB becomes compromised
in acute Borna disease. As shown in Fig. 7, the permeability of the BBB to
Na-fluorescein was significantly enhanced in rats with clinical signs
of Borna disease. UA treatment (100 mg twice daily), beginning 7–9
days before analysis, largely prevented the increased permeability of
the BBB associated with Borna disease in the control animals.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 5) (30). UA
treatment of BDV-infected rats inhibits the accumulation of cells
expressing iNOS, ED-1, CD4, and CD8 in infected brain tissue, as well
as NT formation and CNS tissue damage. UA-mediated suppression of
inflammatory cell invasion into the BDV-infected brain was associated
with maintenance of normal BBB integrity and prevention of the onset of
clinical Borna disease, which is most often lethal in our model.
Together these findings suggest that the action of
ONOO- on the BBB is an important stage in the
invasion of inflammatory cells, including monocytes and T lymphocytes,
into CNS tissues.
Although UA is an efficient inhibitor of certain chemical reactions
associated with ONOO- in vitro
(35), there is some speculation that its molecular targets
in vivo may be radicals, such as
NO2· and
CO3·-, formed by the
rapid reaction of ONOO- with
CO2 (36). Whatever the case, UA
interferes with reactivity associated with
ONOO-. Shortly after mice with preexisting
clinical signs of EAE begin UA treatment, NT residues, generally
accepted as evidence of ONOO- formation (e.g.,
37, 38, 39), are reduced in areas of iNOS-positive
inflammation in the spinal cord (30), indicating that UA
inactivates ONOO-, or its reactive
intermediates, in vivo. This supports the hypothesis that the
inhibitory effect of UA treatment on cell invasion into the CNS is also
a consequence of the inactivation of ONOO-, and
that this molecule or its products are involved in providing
inflammatory cells access to CNS tissues across the BBB. Because of the
variety of chemical reactions that ONOO- can
undergo, this could be through a number of different mechanisms,
including up-regulation of adhesion molecules on the cerebral
vasculature, physiological effects on endothelial cell tight junctions
and other elements of the BBB, or reactive damage to the BBB. There is
evidence that ONOO- may induce the expression of
adhesion molecules on cultured endothelial cells (40), but
we do not believe that this is the sole effect of
ONOO- on the BBB because the permeability to
molecules like UA and fluorescein is also increased (Fig. 7)
(30). Rather, we expect that the enhanced permeability
facilitates the invasion of cells both physically and by promoting
contact with chemoattractive factors, such as chemokines, which are
expressed in the BDV-infected rat brain in the absence of inflammation
(41).
In both BDV-infected and UA-treated BDV-infected rats, the extent of
CD4 and CD8 T cell invasion into brain tissue paralleled the extent of
NT formation. Cells expressing iNOS were also found throughout
BDV-infected, control-treated brain tissue. UA treatment resulted in
substantial inhibition of the accumulation of cells positive for iNOS,
CD4, and CD8 as well as NT staining following BDV infection. For the
most part, staining for these markers was limited to the meningeal
regions of the subarachnoid space with little penetration deeper into
the tissues. However, iNOS-positive cells appeared to be
disproportionately rare, by comparison with those positive for CD4 and
CD8, in BDV-infected brain tissue from UA-treated rats. It is
conceivable that NT formed under these conditions is the result of the
activity of endothelial NOS, possibly stimulated by activated T cells,
rather than iNOS. In this case, UA, which does not penetrate the intact
BBB (30), may have limited access to the
ONOO- produced by the endothelial cells, and
some ONOO--dependent T cell invasion may still
occur. The rats in this study received two daily doses of 100 mg UA,
which caused serum UA levels to be elevated 4-fold, to an average of 2
mg/dl for 4 h at a time. Although peritoneal macrophages may take up
UA at the site of injection and carry it for several hours
(42), the evidence of NT formation in brain tissue from
UA-treated rats makes it seem unlikely that this treatment protocol
provided sufficient UA to inactivate all of the
ONOO- produced at the BBB. Regardless, the
overlapping geographical distributions of CD4, CD8, and NT in the
BDV-infected CNS, whether UA treated or not, suggest the possibility
that ONOO- may make an important contribution to
the invasion of T cells, as well as iNOS-positive cells, into CNS
tissues.
Although our studies of ONOO- in EAE and Borna
disease have largely focused on the contribution of this molecule to
the pathogenic aspects of CNS inflammation, it is clear that, depending
on the circumstances, ONOO--mediated processes
may be protective. In situations in which a CNS inflammatory response
contributes to recovery from a neurotropic infection, with BDV or other
pathogens, ONOO- may participate in providing
immune cells and Ab access to the focus of infection as well as in the
destruction of the infecting agent and infected cells. In this regard,
we speculate that the somewhat lowered Ab response to BDV seen in
UA-treated rats (Fig. 2) may be due to reduced contact between the
immune system and viral Ags as a consequence of maintained BBB
integrity, lessened CNS inflammation, and diminished damage of infected
neurons. It is conceivable that ONOO- makes
contributions to protective immune responses against infected or
transformed tissue in the periphery, similar to those seen in the CNS.
Nevertheless, UA administration had no inhibitory effect on various
parameters of Ag-specific immunity in mice immunized s.c. with myelin
basic protein, which includes the appearance of iNOS-positive cells in
the circulation (42, 43).
The demonstration that ONOO- participates in opening the BBB and providing inflammatory cells access to the CNS during a neurotropic infection in rodents has implications for immune responses to similar infections in humans. Most mammals, including mice and rats, possess urate oxidase, an enzyme that metabolizes UA, a product of purine metabolism, to allantoin, which does not inactivate ONOO- (35). Thus, serum levels of UA are low in most mammals, and can only be raised by repeated administration of relatively large amounts of UA (26, 27). However, the urate oxidase gene has been inactivated in higher order primates, and their serum UA levels are substantially higher (44, 45). This has been the stimulus of considerable speculation as to the importance of UA to human evolution (e.g., 46, 47, 48). The fact that two daily doses of UA in rats are sufficient to protect CNS tissue from a robust immune response to BDV infection supports the notion that UA may play an important role in protecting the CNS, and possibly other tissues, from ONOO--dependent inflammatory cell invasion and damage. These data also imply that certain immune as well as pathogenic processes that involve ONOO- may be somewhat different in humans by comparison with lower animals. UA evidently does not directly interfere with the development of Ag-specific elements of the immune response to accessible Ags (42). Nevertheless, the relatively high levels of serum UA found in humans and higher order primates should limit ONOO--dependent BBB permeability changes as well as the invasion of immune and inflammatory cells into the CNS. Like BDV-infected rats treated with UA, high UA levels in humans may be expected to inhibit the invasion of monocytes and lymphocytes into the CNS in response to infection. Conceivably, UA may thereby contribute to the persistence of CNS infection with a virus of limited neuropathogenicity. An important example of this in humans may be HIV infection of the CNS. It is conceivable that serum UA levels may be relevant to the establishment of HIV infection in the brain and to the progression of the AIDS-dementia complex.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. D. Craig Hooper, Department of Microbiology and Immunology, Thomas Jefferson University, 1020 Locust Street, JAH Room 454, Philadelphia, PA 19107-6799. E-mail address: douglas.c.hooper{at}mail.tju.edu
3 Abbreviations used in this paper: BDV, Borna disease virus; BBB, blood-brain barrier; EAE, experimental allergic encephalomyelitis; iNOS, inducible NO synthase; NT, nitrotyrosine; ONOO-, peroxynitrite; p.i., postinfection; UA, uric acid.
Received for publication November 14, 2000. Accepted for publication July 9, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-antiproteinase inactivation by ascorbic acid: a comparison with other biological antioxidants. Free Radical Res. 25:275.[Medline]
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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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