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1 From the Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachuestts; and the 2 Laboratory of Retinal and Molecular Biology, National Institutes of Health, Bethesda, Maryland.
Abstract
PURPOSE. To determine whether the inflammation of endotoxin-induced uveitis (EIU) and experimental autoimmune uveoretinitis (EAU) alters key in vivo and in vitro parameters of ocular immune privilege.
METHODS. For EIU induction, C3H/HeN mice received 200 µg lipopolysaccharide (LPS). For EAU induction, B10.A mice were immunized with 50 µg interphotoreceptor retinoid-binding protein (IRBP) mixed with complete Freund’s adjuvant. Aqueous humor (AqH) was collected at periodic intervals and assayed for leukocyte content and the ability to suppress or enhance T-cell proliferation. Eyes with EAU were assessed for the capacity to support anterior chamber (AC)-associated immune deviation (ACAID) induction after injection of ovalbumin (OVA).
RESULTS. Inflammation within the anterior segment in EIU peaked at 12 to 24 hours and was detected from 10 days onward in EAU. In AqH of EIU, protein content rose within 4 hours, followed by infiltrating leukocytes. EIU AqH promptly lost its capacity to suppress T-cell proliferation and became mitogenic for T cells. In AqH of EAU, protein and leukocyte content rose at 11 days and continued to remain elevated thereafter. Whereas 11-day EAU AqH failed to suppress T-cell proliferation, AqH at later time points reacquired immunosuppressive properties. Injection of OVA into the AC of eyes of mice with EAU failed to induce ACAID.
CONCLUSIONS. The intraocular inflammation of EIU and EAU disrupted important parameters of immune privilege, ranging from breakdown of the blood–ocular barrier, to loss of an immunosuppressive microenvironment, to abrogation of ACAID. Because AqH from inflamed EAU reacquired the ability to suppress T-cell proliferation, the authors conclude that the capacity to regulate immune expression and inflammation can be a property even of inflamed eyes.
Immune privilege is a constitutive feature of the anterior chamber (AC) of the normal eye.1 In its strictest definition, immune privilege refers to the fact that foreign tissue grafts placed in the AC survive for prolonged, often indefinite, intervals, whereas placement of such grafts at conventional body sites leads to acute, irreversible immune rejection. As our understanding of the mechanisms responsible for immune privilege have expanded over the past 3 decades, the definition of immune privilege has been relaxed and now embraces, on the one hand, the induction of AC-associated immune deviation (ACAID) after intracameral injection of antigenic materials,2 3 4 and on the other hand, the capacity of the ocular microenvironment (especially aqueous humor [AqH]) to suppress immune effector responses and inflammation.5 6 Existence of ocular immune privilege is believed to serve the purpose of limiting the extent to which innate and adaptive immunity can cause intraocular inflammation. By limiting intraocular inflammation, immune privilege preserves the integrity of the visual axis and thereby prevents blindness.
Ocular inflammation, whether expressed within the cornea or within the uveal tract, is a frequent cause of visual impairment. A variety of experimental models have been developed in laboratory animals as a means of studying the pathogenesis of ocular inflammation.7 8 9 10 Yet, virtually nothing is known about the extent to which ocular inflammation interferes with ocular immune privilege. This may not be an idle concern because inflammation at other body sites can lead, directly or indirectly, to the development of autoimmunity, and as a consequence, local tissue damage is further exaggerated.11 For this reason, and because we wanted to understand the critical factors that contribute to the existence of ocular immune privilege, we examined the so-called immune privileged status of eyes of mice in which intraocular inflammation had been induced experimentally. Using the model systems of endotoxin-induced uveitis (EIU) and experimental autoimmune uveoretinitis (EAU), we found that inflamed eyes displayed weakened or broken blood–ocular barriers and that aqueous humor (AqH) from these eyes either lost or displayed altered immunosuppressive properties. Moreover, EAU-affected eyes no longer supported the induction of ACAID. The relevance of ocular inflammation to retention and maintenance of immune privilege in the AC of the eye is discussed.
Materials and Methods
Animals
C3H/HeN (Taconic Farms, Germantown, NY),C3H/HeJ, and B10.A
(Jackson Laboratory, Bar Harbor, ME) mice were purchased at 6 to 8
weeks of age. Normal BALB/c mice were obtained from our domestic,
inbred mouse-breeding colony. All animals were treated according to the
ARVO Statement for the Use of Animals in Ophthalmic and Vision
Research.
Antigens
Interphotoreceptor retinoid-binding protein (IRBP) was isolated
from bovine retinas as described previously.12
Ovalbumin
(OVA) was purchased from Sigma (St. Louis, MO).
Induction of Uveitis
To induce endotoxin-induced uveitis (EIU), C3H/HeN mice received a
footpad injection of 200 µg lipopolysaccharide (LPS) from Salmonella
typhimurium (Difco, Detroit, MI) in 100 µl phosphate-buffered
saline (PBS) solution. For induction of experimental autoimmune
uveoretinitis (EAU), B10.A mice were immunized subcutaneously with 50
µg IRBP in 0.2 ml emulsion mixed 1:1 with complete Freund’s adjuvant
(CFA; Difco) that had been supplemented with Mycobacterium
tuberculosis to a final concentration of 2.5 mg/ml.
Simultaneously, the mice were injected intraperitoneally with 500 ng
pertussis toxin (Sigma) as an additional adjuvant.
Assessment of Uveitis
Fundus examination of eyes of mice immunized with IRBP was
performed every other day after immunization. These examinations were
performed in masked fashion in which the observer was unaware of the
nature of the prior experimental manipulations. According to the ocular
findings, an arbitrary 4-point score was devised to provide
semiquantitative evaluation13
of the extent of
inflammation and damage. Histopathologic examination of uveitic eyes
was performed on methacrylate-embedded sections of eyes enucleated at
selected times after immunization with IRBP. These sections were
stained with hematoxylin and eosin and evaluated according to criteria
of ocular inflammation described elsewhere.8
Aqueous Humor Collection and Analysis
LPS-induced EIU generates an acute intraocular inflammation that
reaches peak intensity within 24 hours14
15
16
and is
largely dissipated by 48 hours. By contrast, EAU generates intraocular
inflammation that develops over a more protracted course: The clinical
expression is not uniformly evident until 11 days after immunization,
and the inflammation usually persists beyond 28 days.17
18
Therefore, in our experiments the sampling times for AqH were different
in the two model systems. Aqueous humor was obtained from eyes of
C3H/HeN and B10.A mice for in vitro analysis at 0, 2, 4, 6, 8, 12, 24,
and 48 hours after endotoxin injection in EIU-affected mice, and on
days 0, 11, 17, and 28 after IRBP immunization in EAU-affected mice.
Aqueous humor was obtained immediately after death from both eyes,
using a 30-gauge needle and 10 µl micropipets (Fisher Scientific,
Pittsburgh, PA) by capillary attraction and pooled into a siliconized
microcentrifuge tube (Fisher Scientific). Aqueous humor samples from
panels of at least five mice (10 eyes) were pooled and centrifuged at
3000 rpm for 3 minutes, and the cell-free supernatant was frozen
immediately at -70°C. On average, 6 µl AqH was obtained from the
two eyes of each mouse. Leukocytes that were present in the pellet of
centrifuged AqH were resuspended in medium, stained with 0.4% trypan
blue solution, and counted by phase-contrast microscopy. The total
protein content in AqH samples was measured using a protein assay
reagent kit (BCA; Pierce, Rockford, IL) in reference to a bovine
albumin standard. Endotoxin content of AqH was assessed with a limulus
assay kit (Limulus Amebocyte Lysate [LAL]; Bio-Whittaker,
Walkersville, MD). Briefly, AqH from eyes of mice with EIU was mixed
with LAL and incubated at 37°C for 10 minutes. A substrate solution
was then mixed with the LAL sample and incubated at 37°C for 6
minutes. The reaction was stopped with sodium dodecyl sulfate solution,
and absorbance was determined spectrophotometrically at 405 nm. Each
experiment with pooled AqH was repeated at least twice. In the repeat
experiments, AqH was collected again for new groups of mice.
Assay of T-Cell Proliferation
Spleens were removed from naive BALB/c or LPS-resistant C3H/HeJ
mice and pressed through nylon mesh to produce single-cell suspensions.
Red blood cells were lysed with Tris-NH4Cl. T
cells were subsequently purified by passage through a T-cell enrichment
column (R&D Systems, Minneapolis, MN) according to the manufacturer’s
directions. The enriched, naive T cells (>95% Thy+ cells, measured by
flow cytometry) were suspended in serum-free medium. Serum-free medium
was composed of RPMI 1640 medium, 10 mM HEPES, 0.1 mM nonessential
amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml
streptomycin (all from Bio-Whittaker) and 1x
10-5 M 2-ME (Sigma) and supplemented with 0.1%
bovine serum albumin (Sigma), ITS+ culture supplement (1 µg/ml
iron-free transferrin, 10 ng/ml linoleic acid, 0.3 ng/ml
Na2Se, and 0.2 µg/ml
Fe[NO3]3; Collaborative
Biomedical Products, Bedford, MA). The proliferation assay used was a
modification of one described previously.19
To individual
wells of a 96-well V-shape bottomed plate (Corning, Corning, NY), we
added 10 µl 2.5 x 104 enriched T cells,
10 µl hamster anti-mouse CD3e IgG (2C11, 2.5 µg/ml; PharMingen, San
Diego, CA), or 10 µl serum-free medium, and 5 µl AqH or PBS. Total
reaction volume was kept constant at 25 µl. The cells were pulsed
with 2.5 µl 20 µCi/ml [3H]thymidine for the
final 8 hours of the 72-hour incubation (37°C; 5%
CO2–95% humidified air mixture). On day 3, the
cells were recovered using a cell harvester (model 96; Tomtec, Orange,
CT), and [3H]thymidine incorporation was
measured in counts per minute, using a liquid scintillation counter
(Betaplate 1205; Wallac, Gaithersburg, MD).
Induction and Assay of Delayed Hypersensitivity and ACAID
To induce delayed hypersensitivity, mice were immunized
subcutaneously with 100 µg OVA emulsified 1:1 in CFA in a total
volume 100 µl. To induce ACAID, OVA was injected (50 µg/3 µl PBS)
into the AC of one eye of recipient mice. One week later these mice
were immunized with OVA-CFA injected into the nape of neck. After seven
days, OVA (200 µg/10 µl) was injected into the right ear pinna, and
ear swelling responses were assessed 24 and 48 hours later using an
engineer’s micrometer (Mitutoyo 227-101; MTI, Paramus, NJ). Ear
swelling was expressed as follows: specific ear swelling =
(24-hour measurement of right ear - 0-hour measurement of right
ear) - (24-hour measurement of left ear - 0-hour
measurement of left ear) x 10-3 mm. Ear
swelling responses of groups of mice are presented as mean ± SEM.
Statistical Evaluations
Significance of differences between mean values of ear swelling
responses was evaluated using Student’s t-test.
P < 0.05 was deemed significant.
Results
Our experimental plan was to induce EIU and EAU in C3H/HeN and B10.A mice, respectively, and to examine the immunosuppressive properties of AqH and the capacity of inflamed eyes to support induction of ACAID. At selected times after the initiating injection, AqH was collected from eyes of panels of mice (at least five animals per time point), pooled, and assayed for protein content, content of leukocytes, and capacity to inhibit anti-CD3–driven T-cell proliferation. Because the ocular inflammation induced in mice by footpad injection of endotoxin and by IRBP immunization varies somewhat from laboratory to laboratory, it was necessary for us to describe the clinical and histologic features of EIU and EAU in the animals from which we collected AqH and in which we attempted to induce ACAID.
Clinical Assessment of Ocular Inflammation in Experimental Mice
Few signs of anterior segment inflammation were observed in eyes
of C3H/HeN mice that received endotoxin by footpad, although the
corneal surface was sometimes covered with a discharge. In B10.A mice
with EAU, the first sign of ocular inflammation was hyperemia of limbal
vessels, which was detected at day 11. Evidence of inflammation in the
anterior ocular segment was minimal. In a few instances, posterior
synechiae were observed between days 14 and 20. However, marked aqueous
flare or definite evidence of leukocytes in the AC were not observed.
EAU is primarily a posterior uveitis. In immunized mice, the frequency
of clinically detectable posterior inflammation approached 80% (Fig. 1)
. After day 11, signs of retinal vasculitis developed in increasing
numbers of eyes. In the more severe cases, retinal or subretinal
exudates, retinal hemorrhages, and/or disc edema were observed. Retinal
detachments were often severe and were detected between days 14 and 22
in severely affected eyes.
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Regarding EAU, by approximately 11 days after immunization with IRBP, eyes removed from B10.A mice displayed dilatation of the choroidal and retinal vessels. By 17 days after immunization, numerous leukocytes were observed in the vitreous, within the neuronal retina, in the subretinal space, and even in the AC. An intense accumulation of leukocytes was often observed in the angle between the iris and the corneal endothelium. At this time, more than 40% of the eyes displayed moderate to severe retinal detachments. Intraocular evidence of intense inflammation persisted in eyes removed for study 28 days after immunization.
Protein and Leukocyte Content of Aqueous Humor from Eyes with EIU
Aqueous humor was removed from eyes of C3H/HeN mice with EIU at
selected times and separated by centrifugation into a cellular fraction
(pellet) and a soluble fraction (supernatant). The samples were
evaluated for protein concentration and content of leukocytes (Fig. 2)
. As anticipated, no leukocytes were detected in control AqH samples.
Moreover, no leukocytes were found in samples harvested at 2, 4, or 6
hours after LPS injection. At 8 hours and thereafter, leukocytes
(chiefly polymorphonuclear neutrophils) were detected, and peak
concentration of these cells was reached at 12 hours (70.3 ± 5.5
cells/µl). Considerably reduced numbers of these cells were detected
in AqH removed at 24 and 48 hours after endotoxin injection. The
protein concentration in control AqH was low (1.8 ± 0.1 mg/ml).
It remained at this low level in samples of AqH obtained at 2 hours
after LPS footpad injection, but a significant increase in protein
concentration was detected at 4 hours, and the level continued to
increase at 6, 8, 12, and 24 hours. At the latter time point, protein
concentration in AqH was 5.8 mg/ml. Although the peak protein
concentration differed among experiments, it occurred at either 12 or
24 hours. Protein concentration in AqH returned almost to baseline by
48 hours. If elevated protein levels in AqH is taken as evidence of
breakdown of the blood–ocular barrier, these results indicate that
endotoxin injection into the footpad induced a leak through this
barrier within 2 to 4 hours. These results indicate that
endotoxin-dependent breakdown of the blood–ocular barrier, measured by
protein concentration, precedes intrusion of leukocytes into the AC.
This result could mean that leukocyte infiltration into the AC is a
secondary consequence of the action of LPS, and/or it could mean that
LPS (directly or indirectly) alters ocular microvessels, rendering them
permissive for leukocyte immigration.
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Potent immune regulatory forces are operative in the normal eye.5 6 21 22 The existence of immune privilege in the eye offers experimental verification that these forces exist. The ability of the eye to regulate immunity is twofold. On the one hand, the eye creates a microenvironment that insures that antigens that are injected into, or arise within, the AC induce a deviant systemic immune response termed ACAID, in which T cells and antibodies that evoke immunogenic inflammation are suppressed.2 3 4 On the other hand, the ocular microenvironment is inhospitable to the expression of those forms of immunity that use nonspecific inflammation in performing their effector function.5 6 By shaping the immune response to eye-derived antigens at both the induction and expression stages, the eye is relatively protected from the vision-damaging effects of intraocular inflammation. The term "relatively" is important in the preceding sentence because the eye can, and does, experience inflammation. In clinical ophthalmology, acute and chronic uveitis are common afflictions that all too often lead to visual impairment and blindness, and in the laboratory intraocular inflammation can be evoked in informative model systems.9 10 The experiments reported here have taken advantage of two different model systems with which to explore the extent to which intraocular immune regulatory mechanisms are compromised in inflamed eyes.
By harvesting AqH at periodic intervals after induction of EIU and EAU
in mice we have been able to identify the times at which the
blood–ocular barrier is broken and to examine the intervals during
which the ability of this fluid to inhibit T-cell activation is
abrogated. Our results indicate that leakage of plasma proteins into
AqH occurred early in the course of both EIU and EAU—in fact, before
the first clinical evidence of disease was apparent. Moreover, in both
types of ocular inflammation, the AqH promptly lost its capacity to
suppress T-cell proliferation. Four factors could contribute to the
loss of immunosuppressive capacity of AqH from inflamed eyes. First,
plasma proteins display protease activity, and many of the
immunosuppressive factors in AqH (e.g., 
-MSH, VIP,
CGRP)19
23
24
25
are neuropeptides with exquisite
vulnerability to enzymatic degradation. Thus, the mere entry of plasma
proteins into AqH may cause the depletion of factors that suppress
T-cell proliferation. Second, shortly after plasma proteins leaked into
AqH, leukocytes also penetrated into the intraocular microenvironment.
It is possible that activated neutrophils or macrophages could take up
and/or destroy factors in AqH that confer on the fluid its
immunosuppressive properties. Third, the rapid turnover of AqH in the
eye applies to its content of immunosuppressive factors. It is possible
that endotoxin in EIU, or the autoimmune attack directed at IRBP in
EAU, halts the intraocular production of immunosuppressive factors
(e.g., TGFß-2)26
27
and that the new AqH that
is formed in inflamed eyes is deficient in these immunosuppressive
factors. Fourth, AqH obtained from eyes of mice with EIU and EAU (to a
more limited extent) was found to stimulate T-cell proliferation in
vitro, in the absence of the addition to the culture of a T-cell
mitogen. Limulus assay showed that AqH from eyes of mice with EIU
contained significant amounts of LPS, yet this did not appear to be
responsible for the mitogenic activity. By using T cells from
LPS-resistant C3H/HeJ mice, we determined that AqH from EIU eyes still
induced proliferation. Therefore, the abnormal microenvironment
associated with inflamed eyes may actually promote, rather than
inhibit, T-cell activation. At present, we have no information
concerning the nature of the proliferation-inducing property discovered
in AqH obtained during intraocular inflammation.
In AqH obtained at periodic intervals from eyes with EIU, a relatively good correlation was observed when the amount and timing of protein concentration were compared with the AqH’s loss of immunosuppressive activity. Maximal loss of AqH ability to inhibit T-cell proliferation (4–24 hours, with peak at 6 hours after LPS footpad injection) corresponded reasonably well to maximum protein concentration (4–24 hours, with peak at 12–24 hours). A similarly good correlation was not observed in periodic AqH samples obtained from eyes of mice with EAU. High levels of protein were observed in AqH from EAU-affected eyes at days 11 and 17, but only AqH from day 11 displayed a significant loss of immunosuppressive activity. Aqueous humor removed from eyes with EAU on day 17 inhibited T-cell proliferation in vitro as profoundly as did normal AqH. No better correlation could be made between loss of immunosuppressive capacity and the number of leukocytes found in AqH of mice with EAU. Although AqH in EAU lost its T-cell proliferation–inhibiting capacity coincident with the breakdown of the blood–ocular barrier, continued loss of barrier function did not prevent these eyes from restoring their immunosuppressive microenvironment, at least in the AC. We are eager to identify the mechanism responsible for reestablishment of immunosuppression in these inflamed eyes.
In the aggregate, the in vitro studies of AqH from EIU- and EAU-inflamed eyes confirm that experimentally induced intraocular inflammation abrogates (partially to completely) one important dimension of immune privilege—that is, the ability to suppress T-cell–dependent immunity within the eye. In addition, our experiments attempting to induce ACAID by injecting OVA into the AC of eyes of mice with EAU indicate that intraocular inflammation also interferes with another important dimension of the privileged state—that is, the capacity of the eye to shape the nature of systemic immune responses to eye-derived antigens. B10.A mice that received a uveitogenic IRBP regimen 14 days previously and that showed development of intense posterior uveitis, did not acquire OVA-specific ACAID when OVA was injected into the AC. This finding implies that intraocular inflammation, even if focused on the posterior segment, robs the AC of its capacity to support ACAID induction. More important, the loss of the ability to promote ACAID extends to antigens unrelated to the autoantigens that are the targets of the autoimmune disease. Whereas in our experiments the unrelated exogenous antigen was OVA , a nonpathogenic molecule, the threat posed by intraocular inflammation is that ACAID may not develop when it is needed—for example, when a corneal allograft is placed orthotopically. Experimental evidence in mice indicates that ACAID contributes to the long-term success of keratoplasties.28 29 30 We suspect that if we can learn how to restore the capacity of an inflamed eye to promote ACAID, we may generate therapeutic strategies that will ensure engraftment of allogeneic corneas and perhaps mitigate the destructive potential of intraocular infection with herpes viruses and other pathogens.
Acknowledgements
The authors thank Jacqueline M. Doherty for her valuable help during the preparation of the manuscript.
Footnotes
Reprint requests: J. Wayne Streilein, Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114.
Supported by Grant EY05678 from the National Institutes of Health.
Submitted for publication November 30, 1998; revised February 2, 1999; accepted April 9, 1999.
Proprietary interest category: N.
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