| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Departments of
*
Bioregulatory Medicine and Rheumatology,
Homeostasis Medicine, and
Pathology and Immunology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. Our results demonstrate that the ectopic expression of
cyclin-dependent kinase inhibitors not only prevents synovial
overgrowth but also ameliorates the proinflammatory milieu in the
affected joints. The induction of p21Cip1 in rheumatoid
synovial tissues by pharmacological agents may also be an effective
strategy to treat rheumatoid arthritis. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, and IL-6, which are produced in situ, the synovial
fibroblasts proliferate and release tissue-degrading enzymes (1, 2). The resulting hyperplastic synovial membrane, termed pannus
tissue, irreversibly destroys the cartilage and bone of the affected
joints. We previously studied the expression of cyclin-dependent kinase inhibitors (CDKIs) in rheumatoid synovial tissues (3). CDKIs are a group of nuclear proteins that inhibit cyclin-dependent kinases (CDKs). CDKs are essential for the progression of the cell cycle, and the up-regulation of CDKI genes generally results in cell cycle arrest. CDKIs discovered to date fall into two groups, the Cip/Kip family with three members (p21Cip1, p27Kip1, and p57Kip2) and the INK4 family with four members (p15INK4b, p16INK4a, p18INK4c, and p19INK4d) (4). The expression of individual CDKIs is independently regulated, and each appears to play a unique role in controlling the cell cycle. In fibroblasts derived from rheumatoid synovial tissues, neither p16INK4a nor p21Cip1 is expressed in vivo, but both are readily induced when the growth of the fibroblasts is inhibited in vitro. The induction of p21Cip1 is also observed in nonrheumatoid fibroblasts, whereas the induction of p16INK4a is characteristic of rheumatoid synovial fibroblasts. The specific induction of p16INK4a in the rheumatoid fibroblasts prompted us to transfer the p16INK4a gene into the joints of rats with adjuvant arthritis (AA). This treatment suppressed the synovial hyperplasia and associated pathology of the arthritis (3). This was the first evidence that arthritis could be treated by inhibiting the cell cycle of the synovial fibroblasts.
The treatment of arthritis by the local transfer of the
p16INK4a gene compares well with treatment by
conventional anti-rheumatic drugs and recently developed biological
reagents. They all aim primarily at the suppression of inflammatory
mediators involved in RA. Most of the biological reagents neutralize
proinflammatory cytokines such as TNF-, IL-1, or IL-6
(5, 6, 7, 8, 9). Our approach was to directly inhibit the
proliferation of the synovial fibroblasts to prevent destructive
synovial hyperplasia without specifically attempting to interfere with
the proinflammatory cytokines. We believe that synovial tissues
overexpressing cell-cycle-suppressing genes would not invade the bone
or cartilage despite the presence of the proinflammatory cytokines.
In this study, we examined the therapeutic effects of the forced
expression of p21Cip1, as well as that of
p16INK4a, in the inflamed synovial tissues of
mice with collagen-induced arthritis (CIA). As stated before, both
genes are inducible in rheumatoid synovial fibroblasts in vitro
(3). p16INK4a binds to cyclin D and
prevents it from forming a catalytically active kinase complex with
CDK4 or CDK6. Thus, it inhibits the cell cycle at the
G1/S transition (10, 11, 12).
p21Cip1 inhibits a wide variety of cyclin/CDK
complexes (13, 14). It also binds to and inactivates
proliferating cell nuclear Ag, which activates DNA polymerase 
(15, 16, 17, 18). Thus, p21Cip1 inactivates
the kinase activity of cyclin/CDK complexes at every stage of the cell
cycle, and it also inhibits DNA replication.
Like rat AA, the murine CIA model used in this study demonstrates remarkable similarities to human RA (19, 20, 21). It was successfully treated by gene transfer of p21Cip1 as well as that of p16INK4a. This murine model allowed us to further investigate the molecular mechanism of the therapeutic effects of CDKI. We found these effects to be not only anti-proliferative but also anti-inflammatory.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cultured fibroblasts were prepared from synovial tissues of patients with RA undergoing total joint replacement surgery or synovectomy at Nippon Medical School Hospital, Tokyo Metropolitan Bokuto Hospital, or Fuchu Hospital. Consent forms were completed by patients before surgery. RA was diagnosed according to the criteria of the American College of Rheumatology (22). The fibroblasts were cultured as described elsewhere (3).
Recombinant adenoviruses
Replication-defective adenoviruses containing a human p16INK4a gene (AxCAp16) or a human p21Cip1 gene (AxCAp21) were prepared as described previously (23). A recombinant adenovirus encoding Escherichia coli lac Z gene (AxCALacZ) was generously provided by Dr. Saito (University of Tokyo, Tokyo, Japan) (24). High-titer recombinant adenoviruses were prepared by amplification in 293 cells and purified by cesium chloride density-gradient centrifugation (25).
Cell proliferation assay
In vitro gene transfer of the recombinant adenoviruses and measurement of [3H]thymidine incorporation by adenovirus-infected cells were performed as described elsewhere (23).
Induction of CIA
Male DBA/1J mice were purchased from Japan Charles River Breeding Laboratories (Tokyo, Japan) and housed in the Animal Research Center of Tokyo Medical and Dental University. Bovine type II collagen (Collagen Research Center, Tokyo, Japan) was dissolved at 4 mg/ml in 0.1 M acetic acid, and then emulsified with an equal volume of CFA (Iatron, Tokyo, Japan). For the primary immunization, 100 µl of the immunogen were injected intradermally into 8-wk-old mice at the tail base. After 3 wk, the mice received the same dose of the immunogen s.c. The arthritis developed within 10 days of the second immunization.
In vivo gene transfer
The AxCAp16, AxCAp21, and AxCALacZ adenoviruses were prepared in saline at a concentration of 108 particles/µl. Each mouse received bilateral intraarticular injection at the ankle joints (5 µl per joint) and the knee joints (10 µl per joint), and bilateral periarticular injection at the tarsal joints (5 µl per joint) at the same time. Injection of saline alone served as a control.
Assessment of CIA disease severity
After physical examination, the severity of the arthritis in the hind legs was scored as follows: 0, normal; 1, erythema and mild swelling confined to the ankle joint or toes; 2, erythema and mild swelling extending from the ankle to the midfoot; 3, erythema and severe swelling extending from the ankle to the metatarsal joints; and 4, ankylosing deformation with joint swelling (26). The disease score for each mouse was calculated as the sum of the scores for the two hind legs. The severity of disease was scored by two examiners operating blindly. The ankle and paw widths were measured with a micrometer (Ozaki Manufacturing, Tokyo, Japan). Radiographs of the hind legs were taken by direct exposure on x-ray film (Fuji Photo Film, Tokyo, Japan) at 3.5 mA for 1 min with an SRO-M30 x-ray machine (Sofron, Tokyo, Japan).
RT-PCR analysis
Synovial tissues were isolated from the hind paws by removing
the skin, muscle, fatty tissues, bones, and tendons in the hind legs.
Total RNA was extracted from the synovial tissues with Isogen
(Nippongene, Tokyo, Japan). Complementary DNA was synthesized with
Superscript II reverse transcriptase (Life Technologies, Gaithersburg,
MD) and subjected to 25 cycles of PCR to amplify GAPDH, or 30 cycles of
PCR to amplify murine IL-1ß, IL-6, and TNF- cDNA. The PCR cycle
was 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min. The
products were fractionated by agarose gel electrophoresis and stained
with ethidium bromide. The nucleic acid sequences of the specific PCR
primers were as follows: mouse IL-1ß, 5'-CTG AAA GCT CTC CAC CTC-3'
(sense) and 5'-GGT GCT GAT GTA CCA GTT GG-3' (antisense); mouse IL-6,
5'-GAG ACT TCC ATC CAG TTG CC-3' (sense) and 5'-TTC TGC AAG TGC ATC ATC
G-3' (antisense); mouse TNF-, 5'-GCC ACC ACG CTC TTC TG-3' (sense)
and 5'-ATG GGC TCA TAC CAG GG-3' (antisense); and mouse GAPDH, 5'-AAG
AAG GTG GTG AAG CAG GC-3' (sense) and 5'-TCC ACC ACC CTG TTG CTG TA-3'
(antisense).
Histology and immunohistochemistry
The hind paws taken from CIA mice 3 wk after the second
immunization were fixed in 10% phosphate-buffered formalin (pH 7.4),
decalcified in 10% EDTA, and embedded in paraffin. Sections (4 µm)
were stained with hematoxylin and eosin. For immunohistochemical
analyses, synovial tissues from the knee joints were embedded in
Tissue-Tek ornithine carbamyl transferase compound (Miles, Elkhart,
IN), frozen in liquid nitrogen, and stored at -80°C. Serial cryostat
sections (8 µm) were air-dried, fixed with cold 4%
phosphate-buffered paraformaldehyde (pH 7.4), and washed with 10 mM
Tris-HCl (pH 7.5) containing 150 mM NaCl and 0.1% saponin. Saponin
permeabilizes the membranes of cells and intracellular organelles,
thereby allowing the detection of intracellular cytokines. This
technique gives positive nuclear staining in conventional
immunohistochemical analyses, thus showing that cytokines can be
stained mostly around the nucleus (27, 28). The sections
were then incubated with 10% normal goat serum for 1 h at room
temperature and treated with rabbit anti-human IL-1ß Ab (LP-712;
Genzyme, Cambridge, MA), rabbit anti-mouse TNF- Ab (IP-400;
Genzyme), or normal rabbit serum overnight at 4°C. They were
subsequently incubated with biotinylated goat anti-rabbit IgG
(Southern Biotechnology Associates, Birmingham, AL), treated with 0.3%
hydrogen peroxide in methanol, and incubated with HRP-labeled
streptavidin (Vector Laboratories, Burlingame, CA). Bound Abs were
visualized with 0.5 mg/ml 3,3'-diaminobenzidine tetrahydrochloride in
PBS (pH 7.4) and 0.02% hydrogen peroxide, then they were stained with
hematoxylin.
Statistics
Statistical analyses were performed with StatView 4.5J software (Abacus Concepts, Berkeley, CA). Statistical differences of the ankle width and the paw width of the CIA mice were assessed by Student’s t test, and the disease scores by the Mann-Whitney U test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The antiproliferative effects of the forced expression of
p21Cip1 as well as p16INK4a
on synovial cells were examined using human synovial fibroblasts.
Synovial fibroblasts prepared from rheumatoid synovial tissues were
infected with a recombinant adenovirus (AxCAp16, AxCAp21, or AxCALacZ
containing the human p16INK4a, human
p21Cip1, and E. coli lac Z gene,
respectively), and their growth was stimulated with 10% FCS or 10
ng/ml platelet-derived growth factor. When the synovial cells were
infected with AxCAp16 or AxCAp21, their proliferation was suppressed in
a dose-dependent manner (Fig. 1
). No
effect was observed with AxCALacZ. The virus-infected cells were
all viable when examined by trypan blue staining. The cell displayed no
signs of apoptosis when the nuclei were stained with Hoechst 33258 or
when the cellular DNA was analyzed with agarose gel electrophoresis
(data not shown). Similar results were obtained when murine NIH3T3
fibroblastoid cells were infected with the same set of viruses and
stimulated in the same manner (data not shown).
|
The same set of adenoviruses was used to introduce the
p16INK4a, p21Cip1, or
lac Z gene in vivo into the synovial tissues of CIA mice.
The mice were immunized twice with type II collagen to induce
arthritis. Gene transfer into the joints was performed either on the
same day as the second immunization and 10 days later, or only 10 days
after the second immunization. The mRNA of the transferred gene was
expressed specifically in the joints that received the recombinant
adenovirus containing that gene (data not shown). During the course of
the disease, the ankle width, paw width, and disease score were
assessed. Both p16INK4a and
p21Cip1 gene transfer markedly ameliorated the
arthritis, whereas lac Z gene transfer and saline injection
had no effect (Fig. 2, A–C).
Suppression of the knee joint swelling was also significant in the
CDKI-treated mice when the width was measured with a micrometer (data
not shown). The differences in the disease scores reached statistical
significance (p < 0.05) 11 days after the
first gene transfer. Compared with the saline-treated mice, the onset
of the disease was delayed 2.4 days on average in the AxCAp16-treated
mice and 3.4 days on average in the AxCAp21-treated mice.
|
, D–F). The differences in the disease scores reached
statistical significance (p < 0.05) 8 days
after gene transfer. The effects of the two CDKIs were not
significantly different.
Radiological examination of the ankle joints 3 wk after the second
immunization revealed that bone erosion was markedly inhibited in the
joints of the CDKI-treated mice (Fig. 3, A–E). The treated joints were examined histologically at
the same time (Fig. 3, F–J). Joints treated with either
AxCAp16 or AxCAp21 exhibited greatly reduced synovial hyperplasia
compared with joints treated with AxCALacZ or saline. The infiltration
of mononuclear cells into the synovial tissues and the formation of
pannus were suppressed, and no destruction of the cartilage or bone was
seen. As with the clinical results, no significant differences were
observed between the joints treated with AxCAp16 and those treated with
AxCAp21.
|
In CIA, as in RA, TNF- and IL-1, which are mainly secreted from
the synovial cells, largely account for the pathology of the arthritis
(29, 30, 31, 32). Thus, their expression level in the synovial
tissues reflects the inflammatory milieu in the affected joints. We
studied the expression of the proinflammatory cytokines IL-1ß, IL-6,
and TNF- in arthritic joints treated with AxCAp16, AxCAp21,
AxCALacZ, or saline. Synovial tissues from the hind paws were
collected on the day of the histopathological examination. From the
CDKI-treated joints, synovial tissues from the joints with the residual
inflammation (disease score 
3) were collected. When the mRNA
expression levels of the cytokines were examined by RT-PCR, all of the
cytokine mRNAs were found abundant in the AxCALacZ- and
saline-treated synovial tissues (Fig. 4).
In contrast, they were undetectable or barely detectable in the
synovial tissues treated with AxCAp16 or AxCAp21. The amount of mRNA
used for each RT-PCR was standardized with respect to the amount of the
extracted RNA. The mRNA of GAPDH was detected at comparable levels in
all of the joints.
|
in the synovial tissues. In the joints
treated with AxCALacZ or saline, the cells expressing these cytokines
were found mainly in the synovial lining layer and in the intimal
synovial tissues (Fig. 5, A–F). In accord with previous observations (27, 28), these cytokines were localized primarily in the
nuclear regions, but cytoplasmic staining was also found. In
contrast, in the synovial tissues from the AxCAp16- and
AxCAp21-treated joints, the staining of IL-1ß and TNF- was
very faint (Fig. 5, G–L). Specimens incubated with normal
rabbit serum and subsequently treated in the same manner yielded no
nonspecific staining (Fig. 5, C, F, I, and L).
Thus, the expression of proinflammatory cytokines in the CDKI-treated
joints was markedly down-regulated.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Although p21Cip1, in contrast to p16INK4a, inhibits the kinase activity of all CDKs as well as DNA replication, the physiological expression of p21Cip1 is usually transient. Because adenoviral gene transfer would be expected to overcome this constraint, we assumed that the overexpression of p21Cip1 would suppress cell proliferation more profoundly than that of p16INK4a. However, we observed that both CDKI genes equally suppressed both the in vitro proliferation of synovial fibroblasts and the pathology of CIA. Presumably, other CDKI genes would also suppress the arthritis if they were transferred into the arthritic synovial tissues. However, in this study, an emphasis was placed on the effects of p16INK4a and p21Cip1 because their endogenous expression can be artificially induced in rheumatoid synovial fibroblasts. This suggests that synthetic compounds that selectively induce the expression of p16INK4a or p21Cip1 could act as therapeutic agents against RA. However, the systemic expression of p16INK4a or p21Cip1 may provoke serious adverse effects by inhibiting cell division essential for natural cell turnover. Because the induction of p16INK4a, unlike that of p21Cip1, under growth inhibitory conditions is characteristic of rheumatoid synovial fibroblasts, the specific induction of p16INK4a in rheumatoid synovial fibroblasts by pharmacological agents might be preferable.
The regulation of p16INK4a gene expression is still too poorly understood to guide us in developing p16INK4a-inducing compounds. p16INK4a is usually expressed in terminally differentiated or replicative senescent cells (33, 34, 35, 36, 37, 38). Although sustained activation of the Ras oncogene leads to the expression of endogenous p16INK4a, the physiological role of this induction seems to be limited to the inhibition of tumor development (39). UV irradiation, bleomycin, or actinomycin D induces transient or delayed expression of p16INK4a in certain cell types (40, 41). In contrast, the expression of p21Cip1 is immediately though transiently induced by a p53 transcription factor in response to DNA damage (42, 43). Many compounds that induce p21Cip1 in various human cell lines have already been identified (44, 45, 46). Therefore, artificial induction of endogenous p21Cip1 might prove easier, although the expression should be made tissue specific.
It should be noted that the dose and timing of gene transfer were not necessarily optimized for practical clinical use as in our previous studies (3). Gene transfer was used to show the effect of CDKI gene expression in the synovial cells. Repeated adenoviral gene transfer could induce tissue injury through an immune reaction against viral gene products. Although the observation period was designed to be short to minimize the effects of secondary immune reactions, the remaining inflammation in the CDKI-treated joints might be related to such effects.
The forced expression of the CDKI genes in murine CIA had similar anti-arthritic effects to the forced expression of the p16INK4a gene in rat AA. Pannus formation was significantly suppressed, and no destruction of the cartilage or bone was seen. Moreover, the infiltration of mononuclear cells into the synovial tissues was also suppressed. The p16INK4a gene therapy of rat AA had profound effects even though not all synovial fibroblasts expressed the exogenous gene. We speculated that the expression of p16INK4a made the synovial cells refractory to proliferative stimuli and that they might also exert anti-inflammatory effects. This hypothesis was addressed in this study by examining the expression of proinflammatory cytokines in the CIA joints.
In CIA, as in RA, joint inflammation and cartilage destruction depend
on IL-1 and TNF- in the affected joints (29, 30, 47, 48, 49). Accordingly, RT-PCR and immunohistochemical analyses
revealed that these cytokines were abundant in control joints treated
with AxCALacZ or saline. However, their expression was significantly
suppressed in the residual hyperplastic synovial tissues from the
CDKI-treated joints. Again, both CDKIs had comparable effects. The
sharp contrast in the expression of proinflammatory cytokines between
CDKI-treated and control joints suggested that the difference in
expression did not merely reflect quantitative difference in the
synovial macrophages. Indeed, CD14 mRNA could be readily detected in
RNA extracted from CDKI-treated joints (data not shown).
To test whether gene transfer of the CDKIs suppresses the production of
proinflammatory cytokines directly, murine RAW264.7 macrophage-like
cells were infected with the AxCAp16, AxCAp21, or control
adenoviruses, and stimulated with LPS or IL-1ß for TNF- secretion.
ELISA of the culture supernatant revealed no suppressive effects of the
CDKI expression (data not shown). Thus, the suppression of the cytokine
production did not appear to be an immediate effect; possibly, an
autocrine or paracrine mechanism operated. Because the expression of
endogenous p16INK4a and
p21Cip1 is associated with cellular senescence,
the cells expressing exogenous p16INK4a or
p21Cip1 may have exerted some inhibitory effects
characteristic of senescent cells. This hypothesis agrees with our
previous observation that not all of the synovial cells expressed the
transgene after the gene transfer (3). The immediate
effect alone would have allowed the uninfected synovial cells to
produce the proinflammatory cytokines. Thus, we believe that the
reduction of the cytokine production was not merely secondary to the
suppressed synovial proliferation or due to an immediate effect of the
CDKI gene expression. Underlying mechanisms are now under
investigation.
By adenoviral transfer of a cell cycle regulator gene, we originally intended to suppress the cell cycle of synovial fibroblasts. However, our results demonstrate that the forced expression of CDKI genes in the synovial cells exerted not only an antiproliferative effect but also an anti-inflammatory effect; it suppressed the production of proinflammatory cytokines. In this regard, our short-term observations revealed that the local expression of CDKI genes in joints with established CIA suppressed further synovial thickening but did not diminish it. However, in the long run, the treatment would probably alleviate persistent arthritis such as RA, because it down-regulates the production of proinflammatory cytokines.
It was reported that adenoviral gene transfer into the joints exerted its effects on the contralateral joints (50, 51). Thus, it might be possible that the recombinant CDKI adenoviruses suppressed general immune reactions, which could contribute to suppression of CIA. However, no significant differences were found in the joint swelling of the forelegs, which were not treated with the gene transfer (data not shown). Moreover, in the previous report, we treated rats with AA successfully by injecting the AxCAp16 adenoviruses into the right knees and the control adenoviruses into the left knees of the same animals (3). These results argue that the therapeutic effects observed in this study are also attributable primarily to the local effect of the gene transfer.
We have shown that the forced expression of CDKI exerted both antiproliferative and anti-inflammatory effects, which would serve the ultimate goal of RA treatment, i.e., long-term prevention of joint destruction. Thus, the local induction of p21Cip1, as well as p16INK4a, in the synovial tissues may provide an ideal approach to the effective treatment of RA.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Hitoshi Kohsaka, Department of Bioregulatory Medicine and Rheumatology, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
3 Abbreviations used in this paper: RA, rheumatoid arthritis; CDKI, cyclin-dependent kinase inhibitor; CIA, collagen-induced arthritis; AA, adjuvant arthritis.
Received for publication January 3, 2000. Accepted for publication September 13, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in rheumatoid arthritis. Arthritis Rheum. 38:151.[Medline]
(cA2) versus placebo in rheumatoid arthritis. Lancet 344:1105.[Medline]
holoenzyme. Proc. Natl. Acad. Sci. USA 91:8655., anti-IL-1ß, and IL-1Ra. Arthritis Rheum. 39:797.[Medline]
and transforming growth factor ß during induction of collagen type II arthritis in mice. Proc. Natl. Acad. Sci. USA 89:7375. soluble receptors to rabbit knees with experimental arthritis has local and distal anti-arthritic effects. Proc. Natl. Acad. Sci. USA 95:4613.This article has been cited by other articles:
|
|
 |
|
  Y. NONOMURA, F. MIZOGUCHI, A. SUZUKI, T. NANKI, H. KATO, N. MIYASAKA, and H. KOHSAKA Hypoxia-induced Abrogation of Contact-dependent Inhibition of Rheumatoid Arthritis Synovial Fibroblast Proliferation J Rheumatol, April 1, 2009; 36(4): 698 - 705. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Sekine, T. Sugihara, S. Miyake, H. Hirai, M. Yoshida, N. Miyasaka, and H. Kohsaka Successful Treatment of Animal Models of Rheumatoid Arthritis with Small-Molecule Cyclin-Dependent Kinase Inhibitors J. Immunol., February 1, 2008; 180(3): 1954 - 1961. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yamaguchi, K. Fujio, H. Shoda, A. Okamoto, N. H. Tsuno, K. Takahashi, and K. Yamamoto IL-17B and IL-17C Are Associated with TNF-{alpha} Production and Contribute to the Exacerbation of Inflammatory Arthritis J. Immunol., November 15, 2007; 179(10): 7128 - 7136. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Sakuma, K. Hatsushika, K. Koyama, R. Katoh, T. Ando, Y. Watanabe, M. Wako, M. Kanzaki, S. Takano, H. Sugiyama, et al. TGF-{beta} type I receptor kinase inhibitor down-regulates rheumatoid synoviocytes and prevents the arthritis induced by type II collagen antibody Int. Immunol., February 1, 2007; 19(2): 117 - 126. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Scatizzi, J. Hutcheson, E. Bickel, J. M. Woods, K. Klosowska, T. L. Moore, G. K. Haines III, and H. Perlman p21Cip1 Is Required for the Development of Monocytes and Their Response to Serum Transfer-induced Arthritis Am. J. Pathol., May 1, 2006; 168(5): 1531 - 1541. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ray, A. Shakya, D. Kumar, and B. K. Ray Overexpression of Serum Amyloid A-Activating Factor 1 Inhibits Cell Proliferation by the Induction of Cyclin-Dependent Protein Kinase Inhibitor p21WAF-1/Cip-1/Sdi-1 Expression J. Immunol., April 15, 2004; 172(8): 5006 - 5015. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Nonomura, H. Kohsaka, K. Nagasaka, and N. Miyasaka Gene Transfer of a Cell Cycle Modulator Exerts Anti-Inflammatory Effects in the Treatment of Arthritis J. Immunol., November 1, 2003; 171(9): 4913 - 4919. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Perlman, K. Bradley, H. Liu, S. Cole, E. Shamiyeh, R. C. Smith, K. Walsh, S. Fiore, A. E. Koch, G. S. Firestein, et al. IL-6 and Matrix Metalloproteinase-1 Are Regulated by the Cyclin-Dependent Kinase Inhibitor p21 in Synovial Fibroblasts J. Immunol., January 15, 2003; 170(2): 838 - 845. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), but not lac Z (
), inhibited cell growth in a
viral dose-dependent manner. The points and bars in the graph represent
the mean ± SEM.
) either twice (arrows, on
days 21 and 31) (A–C) or once after the onset of
arthritis (arrow, on day 31) (D–F). The ankle width
(A and D), paw width (B
and E), and disease score (C and
F) of the animals were measured on the indicated day.
The points and bars in the graph represent the mean ± SEM of
seven mice. *, p < 0.05, **,
p < 0.01, ***, p <
0.001 (AxCAp16 vs saline). #, p < 0.05;
##,, p < 0.01; ###,
p < 0.001 (AxCAp21 vs saline).
