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Departments of
*
Molecular Genetics and Biochemistry and
Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261;
Megabios, Burlingame, CA 94010; and
§
GenVec, Rockville, MD 20852
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, as well as the degree of synovitis, while maintaining
high levels of cartilage matrix synthesis. Interestingly, an
antiarthritic effect was also observed in opposing contralateral
control knee joints that received only a marker gene. An adenoviral
vector carrying the enhanced green fluorescent protein marker gene was
used to demonstrate that a morphologically similar subset of cells
infected in the injected knee joint are able to traffic to the
uninjected contralateral knee joint. Our results suggest that direct,
local intraarticular delivery of the vIL-10 gene may have polyarticular
therapeutic effects. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1 (2, 3, 4, 5), which are thought to be important
mediators that drive the pathophysiology of RA (6, 7).
Most of the recently described biologic agents are proteins, such as
the soluble receptors for TNF- and IL-1, anti-TNF- Abs, and
IL-1 receptor antagonist protein. Gene transfer can potentially
circumvent the inherent delivery problems associated with proteins by
the transfer of genes encoding the therapeutic agent directly to the
synovial lining of the joint (8).
One cytokine of particular interest as a therapeutic for RA is IL-10.
Originally termed cytokine synthesis inhibitory factor, IL-10
(9) is a 35-kDa homodimeric cytokine product of Th2 cells,
B cells, and macrophages. The actions of IL-10 are diverse in that
IL-10 can be antiinflammatory, immunosuppressive, or immunostimulatory,
depending upon the target cell. Primarily, IL-10 can act as an
antiinflammatory by inhibiting synthesis of macrophage-derived
proinflammatory cytokines, such as TNF-, IL-1, IL-1ß, IL-6, and
IL-8 (10, 11, 12, 13). The classification of IL-10 as an
immunosuppressive agent is due to its ability to inhibit the
Ag-presenting functions of macrophages and dendritic cells through the
down-regulation of MHC class II molecules and the costimulatory
molecules ICAM-1 and B7.1 and B7.2 (14, 15, 16, 17). Aside from
its suppressive activities, IL-10 also retains several stimulatory
properties. Most notable are the potent effects that IL-10 has on B
cell proliferation and differentiation (18) and its
ability to act as a chemoattractant for CD8+ T
cells (19). IL-10 is also able to block the apoptosis of
both CD4+ and CD8+ T cells,
as well as germinal center B cells (20, 21). IL-10 is
elevated in the serum of RA patients; the significance of this for
disease pathology, however, is currently under debate (22, 23). While IL-10 may be acting in a suppressive capacity in RA,
high levels of IL-10 also show strong correlation with rheumatoid
factor titers, as well as spontaneous IgM-rheumatoid factor production
(23). Thus, therapy with exogenous IL-10 has the potential
to enhance humoral immunity and undermine its potential effects as a
therapeutic.
Examination of human IL-10 and murine IL-10 cDNA sequences revealed extensive homology to an open reading frame product from the EBV (24). Originally termed BCRF1, this viral protein product is now considered a viral form of IL-10 (vIL-10) (24, 25). The human IL-10 and vIL-10 mature proteins are 84% identical, with most of the divergence found at the amino terminus. vIL-10 shares many of the antiinflammatory and immunosuppressive properties of IL-10, but seems to lack certain immunostimulatory functions (18, 26). Like its human homologue, vIL-10 has demonstrated suppressive functions in several inflammatory settings. It has been shown to increase the survival of mice with experimental endotoxemia (27), prolong the survival of cardiac allografts in mice (28), and suppress the rejection of allogeneic and syngeneic tumors in mice, while, in contrast, cellular IL-10 stimulated tumor rejection (29). The results of the mouse tumor model, in particular, suggest a potential superiority of vIL-10 as an immunosuppressive agent over that of its cellular homologue.
In the present study, we have tested the ability of adenovirus-mediated gene delivery of vIL-10 to protect rabbit knee joints from Ag-induced arthritis (a.i.a). We found that administration of vIL-10 could effectively halt the progression of experimental arthritis by blocking leukocytic infiltration into the joint, while normalizing cartilage metabolism and reducing the degree of synovitis. Most interestingly, therapeutic effects were also observed in opposing, contralateral arthritic knees that had received only the LacZ marker gene. A morphologically similar subset of adenovirally infected cells was shown to traffic from the treated joint to the untreated, inflamed joint. These results suggest that the therapeutic effects of local gene therapy using vIL-10 may not be limited to the treated joint, but may also affect additional joints within the same animal.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female New Zealand White rabbits weighing 
5–6 lb were
obtained from Myrtles Rabbitry (Thompson Station, TN) and housed at the
Central Animal Facility at the University of Pittsburgh (Pittsburgh,
PA). Animals were allowed to acclimate 3 days before experimentation
and were fed water and chow ad libitum.
Adenovirus vectors
The vectors used in this study were E1- and E3-deleted type 5 replication-defective adenoviruses (30). cDNAs encoding vIL-10, ß-galactosidase (LacZ), and enhanced green fluorescent protein (eGFP) were inserted into the E1 region of the vector with gene expression driven by the human CMV early promoter. High titer virus was produced by permissive replication in the 293 human embryonic kidney cell line (American Type Culture Collection, Manassas, VA), as described previously (31). Viral titers were determined by optical density at 260 nm (OD260) where 1 OD unit = 1012 viral particles (32).
Experimental protocol
Rabbits were sensitized to OVA by a series of two intradermal injections of 5 mg OVA emulsified in CFA in the first injection, and in IFA in the second (33). Two weeks following the second injection, an acute articular arthritis was initiated in both hind knees of the rabbits by the intraarticular administration of 5 mg OVA dissolved in 0.5 ml of saline. Twenty-four hours after induction of a.i.a., 5 x 109 particles of replication-defective adenovirus encoding either vIL-10 or LacZ was suspended in 0.2 ml of sterile saline and injected into the joint space via the patellar tendon.
Rabbit knee joints were lavaged on days 3 and 7 post adenovirus administration, by injection of 1 ml Gey’s balanced salt solution (Life Technologies, Rockville, MD) through the patellar tendon. After manipulation of the joint to allow for ample mixing, the needle was reinserted and the fluid aspirated. Leukocytes in recovered lavage fluids were counted with a hemocytometer. Levels of vIL-10 expression in recovered lavage fluids and serum were measured using a cytokine ELISA kit (R&D Systems, Minneapolis, MN).
Evaluation of arthritis
To measure rates of proteoglycan synthesis, articular cartilage was shaved from the femoral chondyles and weighed. Approximately 30 mg of cartilage were then incubated in 1 ml Neuman & Tyell (Life Technologies) serumless medium with 40 µCi of 35SO4-2 for 24 h at 37°C. Subsequently, medium was recovered and stored at -20°C. Proteoglycans were extracted from the cartilage shavings by incubation for 48 h in 1 ml of 0.5 M NaOH at 4°C with gentle agitation. Following chromatographic separation of unincorporated 35SO4-2 using PD-10 columns (Pharmacia, Piscataway, NJ), radiolabeled glycosaminoglycans (GAGs) released into the culture media or recovered by alkaline extraction from the cartilage were quantitated using scintillation counting, as described previously (34).
To quantitate GAGs released into the joint space as a result of cartilage proteoglycan breakdown, recovered lavage fluids were first centrifuged at 12,000 x g for 10 min to remove debris and the supernatants recovered. Aliquots of 100 µl of lavage fluid were treated with papain: 20 µl of papain suspension (type III, 19 U/mg protein; Sigma, St. Louis, MO) was added to 1 ml of buffer containing 10 mM EDTA and 0.4 M sodium acetate (pH 5.2). The papain solution (100 µl) was added to the lavage fluid (100 µl) and incubated overnight at 60°C. Papain was inactivated by the addition of iodoacetic acid to a final concentration of 4 mM. The samples were then centrifuged at 12,000 x g for 10 min. Afterward, 2 U of hyaluronate lyase (Sigma) were added and the samples incubated at 37°C overnight. Sulfated GAG concentrations were measured by a colorimetric dye binding assay using 1,9-dimethylene blue, as previously described (35).
Adenovirus eGFP (Ad.eGFP) marker studies
Bilateral arthritis was induced in rabbit knees as described above. Twenty-four hours after induction of a.i.a., 5 x 1010 particles of adenovirus encoding the cDNA for eGFP (Ad.eGFP) were injected into one knee joint, while the opposing joint received no treatment. Three days postvirus administration, both knee joints were lavaged and the recovered cells divided into two equal portions. One-half of each recovered lavage fluid was cultured in Ham’s F12 media (Life Technologies) supplemented with 10% FCS. After 24 h, cultures derived from both the injected knee and the contralateral joint were examined by fluorescent microscopy (Phase Contrast-2; Nikon, Melville, NY) for the presence of cells that were expressing eGFP. The other half of each recovered lavage fluid was reserved for analysis by FACS. One-color flow cytometry was performed using an EPICS ELITE flow cytometer (Coulter, Hialeah, FL). As a control, the gate for flow cytometry was set using recovered cells aspirated from an inflamed nontransduced control rabbit knee. Logarithmically amplified fluorescent data were collected on 20,000 cells from each recovered lavage fluid, which had been extensively washed and then resuspended in flow cytometry buffer (0.01% NaN3 and 1% BSA in PBS).
Quantitation of rabbit TNF- by ELISA
Endogenous TNF- levels in serum and lavage fluids were
measured using a sandwich ELISA with specific anti-TNF-
polyclonal Abs (PharMingen, San Diego, CA). Briefly, microtiter plates
were coated with 50 µl anti-rabbit TNF- capture Abs (4
µg/ml) overnight at 4°C, then washed twice with PBS containing
0.05% Tween 20 and blocked overnight at 4°C with 10% FCS in PBS.
After washing the plate four times, 100 µl of standards and samples
were incubated in duplicate overnight at 4°C. Plates were again
washed and a biotin-conjugated anti-TNF- secondary Ab (2
µg/ml) was added for 1 h at room temperature. A 30-min
incubation with a 1:400 dilution of avidin-peroxidase (Sigma) followed
an extensive washing of the plate. Finally, TMBlue (Intergen, Milford,
MA) substrate was added (100 µl/well) and incubated at room
temperature for 30 min. The reaction was stopped by the addition of 0.5
N H2SO4 to each well. The
absorbance was read at 450 nm with a UVmax microplate reader (Molecular
Devices, Menlo Park, CA). Standards were plotted to form a linear
standard curve, and the unknown concentrations were determined by
linear regression analysis.
Histology
Rabbit knees were dissected from euthanized animals at day 7, and tissues were fixed in 10% buffered formalin for several days. The fixed tissues were then embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin. Sections were examined by light microscopy at x40 magnification.
Statistical analysis
Data were analyzed using the Macintosh Statworks software program. Group comparisons were performed using both paired and unpaired Student’s t tests where appropriate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test the ability of vIL-10 to inhibit the inflammatory and chondrodestructive effects of a.i.a. in the rabbit knee joint, arthritis was induced in both knees of 16 rabbits. Twenty-four hours post induction, 5 x 109 particles of adenovirus encoding vIL-10 were injected into the right knee of eight rabbits, and 5 x 109 particles of adenovirus encoding LacZ were injected into the left knee of the same eight rabbits. An arthritic control group of eight rabbits received 5 x 109 particles of Ad.LacZ into both knees. A naïve control group of two rabbits was neither induced with arthritis nor injected with virus. Three days after injection of the adenovirus, both knees of each rabbit were lavaged with saline. At 7 days postinfection, the rabbits were sacrificed, the knees lavaged, dissected, and analyzed for effects of transgene expression. It should be noted that we have previously shown through extensive adenovirus titrations that adenoviral doses of 7 x 109 particles or less produce no substantial leukocytic infiltration in synovial fluid for up to 14 days after injection, while maintaining high levels of transgene expression (36).
ELISA measurements of vIL-10 levels in recovered lavage fluids detected
17 ng/ml and 22 ng/ml at days 3 and 7, respectively, in knees that
received the adenovirus encoding vIL-10 (Fig. 1
). vIL-10 was not detectable in sera
(data not shown) or lavage fluid recovered from knees contralateral to
those knees receiving the adenovirus encoding vIL-10.
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Leukocytes in the recovered lavage fluids from each group of
rabbits were counted and compared as a quantitative measure of
inflammation. As shown in Fig. 2, at day
3, the control group of rabbits, which were injected with Ad.LacZ into
both knees, exhibited severe joint inflammation with a mean level of
infiltrating leukocytes exceeding 7.7 x 106
per ml of recovered lavage fluid. By day 7, this level had risen to
9.3 x 106 leukocytes per ml of recovered
lavage fluid. In comparison, the group of rabbits that was injected
with Ad.vIL-10 in the right knee and Ad.LacZ in the left knee had
significantly lower white blood cell infiltrates in the right knees
than in control knees. Levels in the vIL-10 knees at day 3 averaged
3.2 x 106 infiltrating cells, a 72%
reduction. By day 7, this reduction further increased to 81%.
Interestingly, the knees receiving Ad.LacZ, contralateral to the
Ad.vIL-10 treated knees, also displayed a reduction in the amount of
cellular infiltration into the joint space. Rabbits injected with
Ad.vIL-10 into the right knees and Ad.LacZ into the left, displayed a
23% reduction in the mean infiltration into the contralateral joint,
and, by day 7, this difference increased to 60%, compared with the
knees of rabbits receiving only Ad.LacZ.
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To examine the effect of intraarticular expression of vIL-10 on
cartilage matrix degradation, the levels of GAGs released into synovial
fluid as a result of proteoglycan breakdown were measured in recovered
lavage fluids. As shown in Fig. 3A, expression of vIL-10
inhibited cartilage breakdown. The control group of rabbits receiving
injections of Ad.LacZ in both knees had very high levels of GAGs in the
lavage fluids of both knees at both days 3 and 7. Rabbits injected with
Ad.vIL-10 in the right knee and Ad.LacZ in the opposite joint showed
reductions in the levels of released GAGs. By day 3, knees receiving
Ad.vIL-10 displayed a 56% reduction in the amount of GAGs released
into the joint space. This difference increased to 72% by day 7. Once
again, the joints contralateral to those that received vIL-10 displayed
a moderate reduction in GAG release at day 3 (38%), with a more
significant reduction at day 7 (53%).
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B). Compared with
naïve controls, the rates of GAG synthesis were depressed by
60% in a.i.a. knees of arthritic control rabbits that received only
the Ad.LacZ virus into both joints. In contrast, GAG synthesis rates in
knees that received Ad.vIL-10 were moderately protected with synthesis
levels only 30% lower than those of naïve joints. Joints
receiving Ad.LacZ contralateral to those that received Ad.vIL-10
exhibited only a 40% decline in synthesis rates, compared with
naïve knee joints. However, due to interrabbit variability, the
effect of vIL-10 on matrix synthesis in the injected and contralateral
joints was not statistically significant. Histological analysis
The histological analysis of tissue recovered from the knees of
each group of rabbits is shown in Fig. 4.
Compared with tissue recovered from normal naïve rabbits (Fig. 4A), sections from the Ad.LacZ control group displayed the
severe synovitis typically observed with a.i.a. (Fig. 4B).
The synovium was thickened, fibrous, hyperplastic, and hypertrophic due
to synovial cell proliferation and infiltration by mononuclear
leukocytes. Treatment of a.i.a. knees with Ad.vIL-10 (Fig. 4C) blocked disease to such a degree that these knees were
largely indistinguishable from naïve control knees (Fig. 4A). Opposing contralateral joints (Fig. 4D) also
displayed a large reduction in the amount of synovitis.
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levels
Since TNF- is thought to be a major proinflammatory contributor
to the pathophysiology of RA and is the target of several novel
therapies, the effect of Ad.vIL-10 therapy on endogenous levels of
rabbit TNF- in lavage fluids was determined (Fig. 5). At days 3 and 7 in naïve
knees, levels of TNF- averaged 30 pg/ml and 15 pg/ml, respectively.
Interestingly, 3 days postadenovirus injection, the arthritic knees
that received Ad.vIL-10 exhibited the highest average TNF- levels,
175 pg/ml, followed by the opposing contralateral knees that received
Ad.LacZ, which averaged 140 pg/ml. Control rabbits that received
Ad.LacZ in both joints had the lowest levels of TNF-, which averaged
100 pg/ml. By day 7, a dramatically different pattern of expression was
observed. TNF- levels in control animals that received Ad.LacZ in
both joints remained high, averaging 160 pg/ml. In stark contrast,
knees that received Ad.vIL-10, as well as the opposing contralateral
Ad.LacZ knees, saw a significant reduction in TNF- levels to near
normal levels of 50 pg/ml and 40 pg/ml, respectively.
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The results of the above experiments suggested that intraarticular
injection of a replication-defective adenovirus vector encoding vIL-10
could have antiarthritic effects at sites distal to the site of
injection. Cell trafficking provides a possible mechanism by which
adenovirally transduced cells may be involved in conferring the
therapeutic effect seen in the contralateral joint. Previous
experiments with an adenoviral vector encoding the luciferase marker
gene suggested that cells from the injected joint were able to traffic
to the contralateral joint and its draining lymph node
(36). In an attempt to visualize the type(s) of cells
trafficking to the contralateral joint, three rabbits were induced
bilaterally with a.i.a., as described in the above experiments.
Twenty-four hours postinduction, 5 x 1010
particles of Ad.eGFP were injected into one knee of each rabbit. Three
days post injection, each joint was lavaged and the recovered fluids
divided for either tissue culture or FACS analysis. FACS analysis of
the lavage fluid recovered from the injected joint showed that 20%
of the cells infiltrating the joint space were GFP-positive, whereas
3% of infiltrating cells from the contralateral joint were positive
for GFP (Fig. 6, A and
B). Fluorescent microscopy detected GFP-positive cells of
various morphologies in cultures established from the ipsilateral joint
(Fig. 6C). In contrast, GFP-expressing cells cultured from
the contralateral joint were of only the single morphology shown in
Fig. 6D. These results suggest that a certain subset of
infected cells is able to traffic from the injected knee joint to the
contralateral joint.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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levels, and the degree
of synovitis in each joint. Furthermore, vIL-10 displayed a strong
chondroprotective effect in blocking cartilage degradation while
maintaining new matrix synthesis. This antiarthritic effect was not
only apparent in treated joints, but also in contralateral joints that
received only the LacZ marker gene, suggesting that local
intraarticular gene therapy may confer polyarticular therapeutic
effects.
The potent antiinflammatory effects of vIL-10 observed in the rabbit
a.i.a. model are consistent with the in vitro actions of vIL-10 on T
cells and macrophages. vIL-10 has been shown to block the expression of
certain cytokines, such as IL-2, IL-12, IFN-
, and GM-CSF by T cells
(9), as well as proinflammatory cytokines, such as TNF-
and IL-1ß by macrophages (10, 11, 12), that are thought to
be important mediators in the pathogenesis of RA (6, 7).
We have demonstrated that levels of TNF- in the arthritic knees were
reduced at day 7 following vIL-10 treatment. Thus, it is likely that
the therapeutic effects of vIL-10 seen in this system are due, in part,
to the suppression of TNF- by vIL-10. However, why TNF- is
elevated in lavage fluids from day 3 knees is unclear. In addition to
blocking the production of proinflammatory cytokines and T cell growth
factors, vIL-10 is able to block Ag presentation through the
down-regulation of MHC class II, ICAM-1, and B7 expression on
macrophages and dendritic cells (14, 15, 16, 17).
We observed significant effects of intraarticular vIL-10 expression on cartilage metabolism. The chondroprotective effects demonstrated in this model are unlikely to reflect a direct effect on articular chondrocytes. In vitro transduction of chondrocytes with Ad.vIL-10 shows no effect on normal cartilage metabolism, nor can vIL-10 protect against IL-1ß-induced suppression of matrix synthesis (E. Lechman and P. Robbins unpublished observations). Instead, it is quite possible that the chondroprotective effects observed with vIL-10 treatment are a consequence of the powerful antiinflammatory actions of vIL-10. In agreement with this conclusion, vIL-10 expression has been shown to inhibit synovial cell invasion of articular cartilage in a human SCID model of arthritis, but did not influence chondrocytic chondrolysis (37).
Recently, several groups have demonstrated that adenoviral administration of vIL-10 is efficacious for the treatment of murine collagen-induced arthritis (CIA) (38, 39, 40). However, only a mild therapeutic effect was seen when vIL-10 was introduced in established CIA (38). Nevertheless, in this report, we have demonstrated a more potent therapeutic effect following intraarticular expression of vIL-10 in established rabbit a.i.a. This discrepancy may be due to the inherent differences in the models of disease. In addition, we were able to deliver the genes directly to the joint, whereas others have delivered the adenovirus systemically (38, 39). Direct intraarticular administration and concentrated expression of vIL-10 at the site of Ag presentation may be critical to its ability to suppress disease. It is important to note that other groups have reported an Ad.vIL-10-associated inflammation upon local vector administration to mouse knee joints (38). However, we have not observed this effect in naïve rabbit joints with the doses of virus used in the vIL-10 studies presented here. In fact, we have demonstrated, via extensive titrations of adenovirus in naïve rabbit knees, that adenoviral doses of less than 7 x 109 particles generate little or no measurable inflammation within the synovial fluid (36). This apparent disparity in observations may be due to titers of virus, site of injection, purity of virus preparation, and virus storage buffer conditions. Furthermore, we saw no increase in the persistence of gene expression due to expression of vIL-10 (data not shown) as other groups have reported (41).
The experiments described here suggest that intraarticular delivery of
the vIL-10 gene to a single joint has polyarticular antiinflammatory
effects. This phenomenon was previously reported as a "contralateral
effect" following the administration of soluble receptors for TNF-
and IL-1 by replication-defective adenovirus (36). Levels
of these gene products in the serum and contralateral joint in treated
animals were too low to confer a therapeutic effect. Marker gene
trafficking studies indicated that virus was retained within the
injected joint space. It was also determined that cells that had been
transduced in one joint had migrated to the lymph nodes and synovial
space of the contralateral joint. In this report, we have further
examined the apparent trafficking of cells to the contralateral joint.
Although we cannot completely discount that the presence of
eGFP-positive cells in the contralateral joint may be due to the
leakage of virus into the systemic circulation, the combined results of
our trafficking studies with Ad.eGFP and Ad.Luc suggest that a
population(s) of leukocytes exists that does indeed migrate from the
treated joint to the contralateral joint. These cells may be capable of
carrying therapeutic genes to other sites of inflammation through
either the systemic circulation or the lymphatic system. Interestingly,
it seems that these cells represent a single morphological cell type.
From preliminary morphologic inspection, it appears that the cells
resemble either macrophages or immature dendritic cells. However,
further characterization of the trafficking cells has been hindered by
the lack of appropriate immunological reagents with which to identify
cells of the rabbit immune system. It is interesting to note that cell
trafficking appeared to depend on several parameters, including the
initial amount of inflammation in the Ad.eGFP-infected joint, the
numbers of adenovirally transduced cells within the injected joint, and
the amount of inflammation in the contralateral joint (data not shown).
Whether these cells actually contribute to the contralateral effect, or
are mere spectators, remains unclear.
We also have observed a similar "contralateral" joint effect with vIL-10 in the murine CIA model, in that periarticular injection of Ad.vIL-10 into an ankle joint at the onset of disease protects the remaining three uninjected paws (39). Expression of vIL-10 was shown in the injected paw as well as the draining lymph node (39). In a delayed-type hypersensitivity (DTH) model, where the mouse is first immunized to Ag and then challenged by injection of Ag into the footpad, prior injection of Ad.vIL-10 into that footpad blocks the DTH response. Interestingly, local footpad injection of Ad.vIL-10 also reduces the DTH response in the contralateral footpad injected with Ag (J. Whaler and C. Evans, unpublished observations). Taken together with our data in the rabbit model of a.i.a., these results are consistent with a model in which local injection of Ad.vIL-10 results in the genetic modification of cells, possibly APCs, that are able to traffic to spleen, lymph nodes, and sites of inflammation. Exactly how and where the therapeutic effect is conferred is still unclear. However, given that a similar effect has been observed in the rabbit a.i.a. model following injection of adenoviral vectors expressing inhibitors of TNF and IL-1, vIL-10 might be working in part through the down-regulation of proinflammatory cytokines by APCs. Clearly, a better understanding of the cell types important for conferring the therapeutic effects following local intraarticular injection of Ad.vIL-10 could provide insights into how to more effectively treat autoimmune and inflammatory diseases by gene therapy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paul D. Robbins, Department of Molecular Genetics and Biochemistry, W1246 Biomedical Science Tower, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261. E-mail address:
3 Abbreviations used in this paper: RA, rheumatoid arthritis; Ad., adenovirus; a.i.a., Ag-induced arthritis; vIL-10, viral IL-10; CIA, collagen-induced arthritis; GAG, glycosaminoglycan; LacZ, ß-galactosidase gene; eGFP, enhanced green fluorescent protein.
Received for publication February 12, 1999. Accepted for publication June 3, 1999.
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E. Crawley, S. Kon, and P. Woo Hereditary predisposition to low interleukin-10 production in children with extended oligoarticular juvenile idiopathic arthritis Rheumatology, May 1, 2001; 40(5): 574 - 578. [Abstract] [Full Text] [PDF]
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S. H. Kim, S. Kim, C. H. Evans, S. C. Ghivizzani, T. Oligino, and P. D. Robbins Effective Treatment of Established Murine Collagen-Induced Arthritis by Systemic Administration of Dendritic Cells Genetically Modified to Express IL-4 J. Immunol., March 1, 2001; 166(5): 3499 - 3505. [Abstract] [Full Text] [PDF]
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K. Nasu, H. Kohsaka, Y. Nonomura, Y. Terada, H. Ito, K. Hirokawa, and N. Miyasaka Adenoviral Transfer of Cyclin-Dependent Kinase Inhibitor Genes Suppresses Collagen-Induced Arthritis in Mice J. Immunol., December 15, 2000; 165(12): 7246 - 7252. [Abstract] [Full Text] [PDF]
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K. Setoguchi, Y. Misaki, Y. Araki, K. Fujio, K. Kawahata, T. Kitamura, and K. Yamamoto Antigen-Specific T Cells Transduced with IL-10 Ameliorate Experimentally Induced Arthritis Without Impairing the Systemic Immune Response to the Antigen J. Immunol., November 15, 2000; 165(10): 5980 - 5986. [Abstract] [Full Text] [PDF]
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 W. C. Russell Update on adenovirus and its vectors J. Gen. Virol., November 1, 2000; 81(11): 2573 - 2604. [Full Text]
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 W OLLIER Rheumatoid arthritis and Epstein-Barr virus: a case of living with the enemy? Ann Rheum Dis, July 1, 2000; 59(7): 497 - 499. [Full Text]
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