Colloquium
`
`Mechanism of action of glatiramer acetate in multiple
`sclerosis and its potential for the development of
`new applications
`
`Ruth Arnon* and Rina Aharoni
`
`The Weizmann Institute of Science, P.O. Box 26, Rehovot 76100, Israel
`
`Glatiramer acetate (GA, Copaxone, Copolymer 1) is an approved
`drug for the treatment of multiple sclerosis and is highly effective
`in the suppression of experimental autoimmune encephalomyelitis
`in various species. The mode of action of GA is by initial strong
`promiscuous binding to MHC molecules and consequent competi-
`tion with various myelin antigens for their presentation to T cells.
`A further aspect of its action is potent induction of specific
`suppressor cells of the T helper 2 (Th2) type that migrate to the
`brain and lead to in situ bystander suppression. Furthermore, the
`GA-specific cells in the brain express the antiinflammatory cyto-
`kines IL-10 and transforming growth factor ␤,
`in addition to
`brain-derived neurotrophic factor, whereas they do not express
`IFN-␥. Based on this immunomodulatory mode of action, we
`explored the potential of GA for two other applications: preven-
`tion of graft rejection and amelioration of inflammatory bowel
`diseases. GA was effective in amelioration of graft rejection in two
`systems by prolongation of skin graft survival and inhibition of
`functional deterioration of thyroid grafts, across minor and major
`histocompatibility barriers. In all transplantation systems GA treat-
`ment inhibited the detrimental secretion of Th1 inflammatory
`cytokines and induced beneficial Th2兾3 antiinflammatory re-
`sponse. GA was effective also in combination with low-dose
`immunosuppressive drugs. Inflammatory bowel diseases are char-
`acterized by detrimental imbalanced proinflammatory immune
`reactivity in the gut. GA significantly suppressed the various
`manifestations of trinitrobenzene sulfonic acid-induced colitis,
`including mortality, weight loss, and macroscopic and microscopic
`colonic damage. GA suppressed local lymphocyte proliferations
`and tumor necrosis factor ␣ detrimental secretion but induced
`transforming growth factor ␤, thus confirming the involvement of
`Th1 to Th2 shift in GA mode of action.
`
`Glatiramer acetate (GA), known also as Copolymer 1 (Cop
`
`1, Copaxone), is a synthetic amino acid copolymer that was
`developed in our laboratory (1, 2). GA is an approved drug for
`multiple sclerosis (MS) that slows the progression of disability
`and reduces relapse rate, and it exhibits a very high safety profile
`(3). MS and its animal model, experimental autoimmune en-
`cephalitis (EAE), are inflammatory autoimmune diseases of the
`central nervous system (CNS) characterized by myelin destruc-
`tion and axonal damage. GA, composed of the amino acids
`L-alanine, L-lysine, L-glutamic acid, and L-tyrosine in a molar
`ratio of 4.2:3.4:1.4:1.0, was designed to simulate myelin basic
`protein (MBP), one of the major myelin autoantigens involved
`in EAE and MS, and its immunological cross-reactivity with
`MBP is essential for its activity (4). Indeed, GA suppresses very
`efficiently EAE in several species, including primates. Further-
`more, the suppressive activity of GA is not restricted to MBP,
`because it ameliorates EAE induced by other encephalitogens
`such as protelipid protein and myelin oligodendrocyte glyco-
`protein (2).
`
`The mechanism by which GA induces its beneficial effect in
`animals and patients was extensively investigated over the years
`by us and others. These studies demonstrated that GA exerts its
`therapeutic activity by immunomodulating various levels of the
`immune response, which differ in their degree of specificity. The
`prerequisite step is the binding of GA to MHC class II molecules.
`GA exhibited a very rapid, high, and efficient binding to various
`MHC class II molecules on murine and human antigen-
`presenting cells, and even displaced peptides from the MHC-
`binding site (5). This competition for binding to the MHC can
`consequently lead to inhibition of various pathological effector
`functions. Interestingly, it was recently demonstrated that GA
`promotes T helper 2 (Th2) cell development and increased IL-10
`production through modulation of dendritic cells (6). This
`modulation on the level of antigen-presenting cells is the least
`specific step and can be beneficial for the modulation of detri-
`mental immune responses to various antigens. However, in MS
`and EAE, in addition to the MHC blocking, GA was shown to
`inhibit the response to the immunodominant epitope of MBP
`peptide 82–100 in a strictly antigen-specific manner by acting as
`a T cell receptor antagonist (7).
`The above activities, however, do not necessarily play an
`essential role in the modulation of MS and EAE in vivo, because
`GA is degraded in the periphery and not likely to reach the brain
`and compete with the relevant myelin antigens in situ. It is
`therefore likely that, in CNS diseases, additional immunomodu-
`latory mechanisms兾factors that can access the blood–brain
`barrier mediate the therapeutic activity of GA. We have previ-
`ously demonstrated that GA-treated animals (either by s.c.
`injections or oral administration) develop GA-specific T cells in
`the peripheral immune system. These cells can adoptively trans-
`fer protection against EAE (8). Furthermore, T cell lines and
`hybridomas could be isolated from spleens of animals rendered
`unresponsive to EAE by GA (9). Both cell types act as regulatory
`suppressor cells, as they inhibited in vitro the response of
`MBP-specific effector cells and inhibited in vivo EAE induced by
`different CNS antigens. These GA-induced cells were indeed
`characterized as Th2兾3 cells secreting high amounts of antiin-
`flammatory cytokines such as IL-4, IL-10, and transforming
`growth factor (TGF)-␤, but not Th1 cytokines, in response to
`both GA and MBP (10). Other myelin antigens such as prote-
`
`This paper results from the Arthur M. Sackler Colloquium of the National Academy of
`Sciences, ‘‘Therapeutic Vaccines: Realities of Today and Hopes for Tomorrow,’’ held April
`1–3, 2004, at the National Academy of Sciences in Washington, DC.
`
`Abbreviations: GA, glatiramer acetate; MS, multiple sclerosis; EAE, experimental autoim-
`mune encephalitis; MBP, myelin basic protein; Th, T helper; IBD, inflammatory bowel
`disease; BDNF, brain-derived neurotrophic factor; TGF, transforming growth factor; TNBS,
`trinitrobenzene sulfonic acid; CyAm, cyclosporin A.
`
`*To whom correspondence should be addressed. E-mail: ruth.arnon@weizmann.ac.il.
`
`© 2004 by The National Academy of Sciences of the USA
`
`www.pnas.org兾cgi兾doi兾10.1073兾pnas.0404887101
`
`PNAS 兩 October 5, 2004 兩 vol. 101 兩
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`olipid protein (PLP) and myelin oligodendrocyte glycoprotein
`(MOG) could not activate the GA-induced cells to secrete by
`Th2 cytokines. Yet, the disease induced by PLP and MOG can
`be suppressed by GA as well as by GA-induced cells, probably by
`‘‘bystander mechanisms’’ (11). A shift from a Th1-biased cyto-
`kine profile toward a Th2-biased profile was also observed in
`GA-treated MS patients (12–14),
`indicating that such GA-
`specific cells are involved in the therapeutic effect brought about
`by GA in MS. However, until recently, GA-induced Th2 regu-
`latory cells were demonstrated only in the periphery (spleens
`and lymph nodes of experimental animals or peripheral blood
`mononuclear cells in human) and not in the organ in which the
`pathological processes of EAE and MS occur. There was no
`information indicating whether they can reach the CNS and
`whether they actually function as suppressor cells in situ. In this
`article, we review our recent findings on the immunomodulatory
`activity of GA in the target organ of MS兾EAE, namely the CNS.
`In addition, based on the immunomodulatory mode of action
`manifested by GA, two previously undescribed applications of
`GA for the suppression of graft rejection and inflammatory
`bowel diseases (IBDs) are described.
`
`InSitu Immunomodulation by GA in the CNS
`The ability of GA-specific T cells, induced in the periphery by
`either injection or oral treatment with GA, to pass the blood–
`brain barrier and accumulate in the CNS was demonstrated by
`their isolation from brains of actively sensitized GA-treated
`mice, as well as by localization of GA-specific cells in the brain
`after their passive transfer to the periphery. Thus, a specific ex
`vivo reactivity to GA, manifested by proliferation and by Th2
`cytokine secretion, was found in whole lymphocyte population
`obtained from brains of EAE induced mice treated by GA
`parenterally (15) or orally (16). Moreover, as demonstrated in
`these studies, highly reactive GA-specific T cell lines that secrete
`in vitro IL-4, IL-5, IL-10, and TGF-␤ in response to GA and
`cross-react with MBP at the level of Th2 cytokine secretion, were
`obtained from both brains and spinal cords of GA-treated mice.
`In contrast, no reactivity to the control antigen lysozyme could
`be obtained in lymphocytes isolated from CNS of mice injected
`with lysozyme. Furthermore, an in situ immunomodulatory
`effect induced by GA treatment in the brains of EAE-induced
`mice was manifested by a decrease in the level of the inflam-
`matory cytokine IFN-␥, and by the secretion of the antiinflam-
`matory cytokine IL-10, in response to the encephalitogen MBP.
`Adoptively transferred fluorescently labeled GA-specific cells
`were found in the brain 7 and 10 days after their injection to the
`periphery (15, 16), whereas lysozyme-specific cells were absent
`in the CNS, indicating that the GA cells induced in the periphery
`penetrate and persist in the CNS. It is noteworthy that the ability
`of GA-induced T cells to persist in the CNS, whereas cells with
`different specificities were completely absent, was previously
`shown and attributed to their cross-reactivity with the myelin
`antigen MBP. This enables their in situ activation, whereas T cells
`that are not able to recognize their specific antigen in the CNS
`decline to baseline level.
`Once the presence of GA-specific Th2 cells in the CNS was
`confirmed, their ability to actually function as suppressor cells in
`the diseased organ and secrete antiinflammatory cytokines in
`situ had to be further verified. We therefore attempted to study
`the reactivity of GA-induced T cells in the brain (17). To trace
`the low amounts of cytokines secreted by a specific subset of T
`cells on the level of the whole CNS tissue, we used a double
`labeling approach in which prelabeled specific T cells were
`adoptively transferred and their expression in the brain was
`subsequently immunohistologically detected. Brain sections con-
`taining the penetrating Hoechst-labeled GA-specific T cells
`(stained blue), their immunohistological staining (red), as well as
`the merged images are demonstrated in Fig. 1. As depicted in
`
`Fig. 1 A, GA-specific cells in the brain manifested intensive
`expression of the two antiinflammatory cytokines, IL-10 and
`TGF-␤, but no trace of the inflammatory cytokine, IFN-␥. Of
`interest is the finding that IL-10 and TGF-␤ were
`special
`expressed not only by the GA-labeled cells themselves, but also
`by unlabeled cells within their vicinity (Figs. 1B). These sur-
`rounding bystander cells had elongated astrocyte-like morphol-
`ogy consistent with activated microglia and their astrocyte
`nature was further corroborated by staining with antibodies to
`glial fibrillary acidic protein (GFAP), which bind to astrocyte
`filaments. The glial cells displayed similar or even higher inten-
`sity of IL-10 and TGF-␤expression compared to the staining of
`the GA-specific population. Such spreading of positive staining
`could result either from genuine IL-10 and TGF-␤ secretion by
`the astrocytes that had been activated by the cytokines stemming
`from the GA-specific cells, or from the binding of IL-10 and
`TGF-␤ secreted by the GA-specific cells to their specific recep-
`tors on these astrocytes. In both cases, this Th2兾3 spreading
`suggests a bystander therapeutic effect of GA on the CNS
`resident cells. IL-10 is a potent regulatory cytokine in autoim-
`munity that inhibits Th1 cells and macrophage activation in
`addition to its effector multifunctional therapeutic reactivity (18,
`19). Moreover, IL-10 can modulate glial cell responses by
`inhibiting MHC class II, as well as nitric oxide and chemokine
`expression (20). TGF-␤ suppresses cytotoxic T cell response,
`production of tumor necrosis factor ␣and IFN-␥, as well as other
`factors that contribute to myelin damage such as lysosomal
`enzymes and nitrogen intermediates (21). Hence, the in situ
`ability of GA-specific infiltrating cells to express and induce the
`expression of these potent modulating cytokines and bystander
`CNS cells may contribute to its therapeutic activity.
`It is of special importance that, in addition to the secretion of
`Th2兾3 cytokines, GA-specific cells express in situ the potent
`brain-derived neurotrophic factor (BDNF), as demonstrated in
`Fig. 1C. BDNF is a key regulator of neuronal development that
`supports neuronal survival and regulates neurotransmitter re-
`lease and dendritic growth (22, 23). It can also rescue injured or
`degenerating neurons and induce axonal outgrowth, remyelina-
`tion, and regeneration (24). The full-length BDNF receptor,
`tyrosine kinase receptor B, has been found in neurons in the
`vicinity of MS plaques, and in reactive astrocytes in MS lesions
`(25). It can therefore be postulated that BDNF secreted by the
`GA-infiltrating cells can actually function in the MS兾EAE target
`tissue.
`Intense staining with BDNF, IL-10, and TGF-␤antibodies was
`observed only in brains of mice that had been adoptively
`transferred with GA-specific cells. In control mice, only faint
`background staining was noted (Fig. 1C). The expression of
`IL-10, TGF-␤, and BDNF was clearly verified on single cell level
`by cytoplasmatic as well as membranalic staining (Fig. 1 B and
`C). It is noteworthy that GA-specific cells expressing IL-10,
`TGF-␤, and BDNF were visible in various regions all over the
`brain, including the cortex, the thalamus, the basal ganglia, and
`the hippocampus, mainly in the tissue surrounding the ventricles
`and in perivascular locations. They were observed in brains of
`normal as well as EAE-induced mice, after adoptive transfer of
`GA-specific cells. In both cases, staining intensity on a single cell
`level looked similar. However, the total expression on whole
`tissue level was significantly higher in EAE-induced mice, be-
`cause many more GA-specific cells were present in the brains of
`those mice than in brains of normal mice. Hence, under patho-
`logical conditions, treatment with GA leads to a more extensive
`expression of modulator substances by GA-specific cells in the
`CNS. The expression of BDNF, as well as IL-10 and TGF-␤, by
`the GA-specific cells in the CNS, draws a direct linkage between
`the therapeutic activity of GA in MS兾EAE and its in situ
`immunomodulatory effect.
`
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`Immunohistochemical analysis of cytokines and BDNF expression by GA-specific cells in the brain. Activated Hoechst-labeled GA-specific cells were
`Fig. 1.
`injected into the peritoneum of EAE-induced mice. After 7 days, the mice were perfused, and brain sections (20 ␮m) were stained immunocytochemically for
`IL-10, TGF-␤, INF-␥, and BDNF. (A) (Left) Staining for Hoechst labeling (blue). (Center) Immunohistological staining of the specific cytokines (red). (Right) Merged
`images. (B) Merged images of sections depicting perivascular infiltrations in the cortex. Enlarged regions accentuate the complete overlap on a single cell level.
`(C) Staining for BDNF. (Upper) An area surrounding the lateral ventricle. (Lower Left) Merged image of perivascular area. (Center) Enlarged section of a merged
`image of single cell staining. (Right) Brain section from a control EAE-induced mouse that did not receive GA-labeled cells and stained for BDNF. (Scale bar is
`50 ␮m for A and C Lower Left, 20 ␮m for B, and 100 ␮m for Upper and Control.)
`
`Arnon and Aharoni
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`PNAS 兩 October 5, 2004 兩 vol. 101 兩
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`The Effect of GA on Manifestations of Graft Rejection
`The pathological process of graft rejection is mediated by host
`T cells that recognize the graft alloantigens presented on self
`MHC molecules as nonself, proliferate, secrete cytokines, and
`recruit additional inflammatory and cytotoxic cells (26–28). To
`prevent graft rejection, it is therefore essential to inhibit alloan-
`tigen presentation and suppress the inflammatory processes. In
`view of the immunomodulating activities of GA and the immu-
`nopathological nature of immune rejection, the ability of GA to
`alleviate this detrimental reactivity was investigated. Indeed, in
`our previous studies (29), we showed that GA inhibited mixed
`lymphocyte reaction (MLR), an in vitro assay used clinically to
`assess immune rejection between donor and recipient. Subse-
`quently, by using the B10D2 3 BALB兾c model of lethal graft
`versus host disease (GVHD), which is similar to the MHC-
`matched bone marrow transplantation in human, we demon-
`strated that GA was effective in vivo as well and prevented the
`immune rejection of graft against host. Thus, posttransplanta-
`tion administration of GA over a limited time significantly
`reduced the incidence, onset, and severity of GVHD, resulting
`
`in improved long-term survival (29). However, a more prevalent
`obstruction in human transplantation is the response of the host
`against the graft. Immune-rejection is still the major barrier to
`successful transplantations. Current protocols to prevent rejec-
`tion rely on the use of nonspecific immunosuppressive drugs,
`such as cyclosporin A (CyA) and FK506 (tacrolimus) (30, 31).
`These drugs, however, induce severe side effects and render
`patients vulnerable to infections. Consequently, they have a
`narrow therapeutic window, and any deviation from this range
`results in rejection of the transplanted organ or in intolerable
`toxicity, infections, and malignancy. It was therefore of interest
`to examine whether GA was also effective in preventing the
`response of host against graft. We investigated the ability of GA
`by itself as well as in combination with two immunosuppressive
`drugs, CyA and FK506, to suppress the rejection of strongly
`mismatched allografts. This was tested in three experimental
`systems: skin-graft transplantation and thyroid-graft assay in
`mice and heterotropic heart transplantation in rats.
`In the skin transplantation system, GA significantly prevented
`skin graft rejection, as demonstrated in four strain combinations
`
`The effect of GA, CyA, and FK506 on the survival of strongly mismatched skin grafts. BALB兾c mice were transplanted with skin grafts originated in
`Fig. 2.
`C57BL兾6 donors. (A) The effect of GA in comparison to various doses of immunosuppressive drugs: 100 mg兾kg GA starting 2 weeks before transplantation; 5,
`10, or 15 mg兾kg CyA starting 6 days before transplantation; and 5 or 10 mg兾kg FK506 starting 6 days before transplantation. (B) The effect of GA in combination
`with immunosuppressive drugs: 100 mg兾kg GA was administered with either 7.5 mg兾kg CyA or 5 mg兾kg FK506. The effect of the combined treatment in
`comparison to the immunosuppressive drugs alone is demonstrated. Grafts were considered rejected when no viable donor epidermis remained. At least seven
`mice were tested in each group. Statistical significance for graft survival over untreated control (P ⬍ 0.05 by Kaplan–Meier test) was obtained for GA, CyA (15
`mg兾kg), and FK506 (10 mg兾kg) (A), and for the two combination treatments (B). (Inset) One mouse treated by combination of GA and FK506 in which engraftment
`was sustained for 45 days even though treatment was discontinued 20 days after transplantation.
`
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`across minor and major MHC barriers (32). The ability of GA,
`in comparison to CyA and FK506, to suppress the rejection of
`strongly mismatched allografts across MHC barriers (C57BL兾6
`3 BALB兾c) is demonstrated in Fig. 2A. The prolongation of
`skin graft survival by 100 mg兾kg GA (27% increase in survival
`compared with untreated control) was longer than that obtained
`by 5 mg兾kg CyA or FK506 (14% and 12% respectively), and
`similar to that of 10 mg兾kg CyA. GA was less effective than the
`highest doses of these immunosuppressive drugs, 15 mg兾kg CyA
`or 10 mg兾kg FK506 (62% and 88% prolongation). However,
`these effective doses of CyA and FK506 had a toxic effect on the
`mice, inducing considerable weight loss, weakness, and mortality
`in 20% of the mice, whereas mice injected daily with a much
`larger dose (100 mg兾kg) of GA looked completely healthy.
`In an attempt to reduce the dosage and toxicity of the current
`immunosuppressive regimens and to improve their efficacy, we
`investigated the ability of GA to inhibit graft rejection in a
`combined treatment with suboptimal and less toxic doses of
`these suppressants. We found that combined treatments of GA
`with various doses of CyA or FK506 significantly extended skin
`graft survival compared with the effect of each drug alone.
`Representative results (Fig. 2B) demonstrate the synergistic
`effect induced by the adding of GA to either 7.5 mg兾kg CyA or
`5 mg兾kg FK506, resulting in a combination effect (the ratio
`between the effect obtained with and without GA) of 5.4 and 3.1,
`respectively. Moreover, in each of these combination groups, one
`mouse survived for a prolonged period (25 and 45 days for GA
`with CyA and FK506, respectively), even though treatment was
`discontinued 20 days after transplantation. These skin grafts
`showed hair growth (Fig. 2B), suggesting that blood supply and
`engraftment have taken place in this fully disparate system by the
`combination treatment with GA. These results are of signifi-
`cance, because skin is one of the most stubborn tissues in
`transplantation, and even a modest prolongation in survival is
`considered meaningful.
`In the thyroid transplantation system, donor’s thyroid glands
`were transplanted in the kidney capsule of recipient mice, and
`the iodine absorbency of the transplanted kidney served as a
`quantitative measure not only for the graft survival but also for
`the function of the transplanted thyroid. The effect of the
`treatment was expressed numerically as the mean function index
`(MFI), which is the ratio between the net iodine absorbance in
`treated versus untreated mice. In this system as well, GA induced
`a significant beneficial effect and consistently improved the
`function of grafted thyroid from various origins, in different
`in the C57BL兾6 3
`strain combinations (32). For example,
`
`BALB兾c strain combination GA treatment resulted in functional
`increase of 3.5- and 2.2-fold over control untreated transplanted
`mice (P ⬍ 0.05). Likewise, the various doses of the two immu-
`nosuppressive drugs induced beneficial effects: the MFI ranged
`between 1.2–6.5 and 3.1–4.1 with increased doses of CyA and
`FK506, respectively. However, the combined treatments of GA
`with either FK506 or CyA improved the grafted thyroid function
`to a greater extent, and resulted in much higher functionality
`values of 12.5–24.1, whereas the MFI of either immunosuppres-
`sant alone never exceeded the value of 10. Thus, significant net
`effects of the combination treatment with GA, ranging between
`2.8- and 20.1-fold for CyA and 2.2- 3.9-fold for FK506, were
`obtained. Furthermore, in both skin and thyroid transplantation,
`the beneficial effect in the mice treated by combination of each
`drug, CyA or FK506, with GA was higher than that obtained with
`double dose of the immunosuppressive drug alone. An even
`more prominent effect was achieved in a heterotropic heart
`transplantation model in rats that allowed the study of vascu-
`larized and perfused organs, which is relevant to human trans-
`plantation (33). In this system, the combination of GA with 2.5
`mg兾kg CyA resulted in 28.7 days of survival, longer than that
`obtained by a 4-fold higher dose of CyA alone.
`Concerning the mechanism by which GA alleviates the im-
`mune rejection, our results indicate that, in the case of bone
`marrow transplantation, GA drastically reduced the cytotoxic
`activity toward host targets (34). In the graft rejection study, GA
`inhibited the responses of spleen and lymph node cells, as well
`as those of the T cell lines originated from them, against the graft
`(32). In both graft-versus-host and host-against-graft systems,
`GA treatment inhibited the secretion of Th1 inflammatory
`cytokines, IL-2 and IFN-␥ and induced the secretion of bene-
`ficial Th2兾3 antiinflammatory cytokines, IL-4, IL-5, IL-10, and
`TGF-␤. This finding is in accord with the mechanism prevailing
`in the GA-induced suppression of EAE兾MS. These data indicate
`that GA acts by immunomodulating the detrimental cellular
`response against the host兾graft, suggesting that application of
`this well tolerated drug may result not only in reduction in the
`adverse effects but also in improvement of efficacy in the current
`immunosuppressive regimens in organ transplantation.
`
`The Effect of GA on IBDs
`IBDs are severe gastrointestinal disorders, characterized by
`detrimental immune reactivity in the gut that also involve mainly
`CD4⫹ Th1 cells, and an imbalance between pro- and antiinflam-
`matory reactivity (35). Current medical treatments for IBD rely
`on the use of nonspecific antiinflammatory, as well as immuno-
`
`Table 1. The effect of GA treatment on the macroscopic manifestations of TNBS colitis in
`different mouse strains
`
`Mouse strain
`BALB兾c
`
`SJL兾JxBALB兾c
`
`SJL兾J
`
`Treatment
`
`Control
`GA oral
`Control
`GA oral
`GA injected
`Control
`GA oral
`GA injected
`
`n
`
`16
`13
`8
`8
`7
`10
`11
`11
`
`Macroscopic scoring of colon damage
`
`Ulceration
`(max 10)
`
`Adhesion
`(max 2)
`
`Diarrhea
`(max 1)
`
`Thickness
`(max 1)
`
`Total score
`(max 14)
`
`7.5
`2.1
`6.8
`3.8
`1.4
`8.4
`7.1
`3.5
`
`1.6
`0.4
`1.6
`1.0
`0.8
`2.0
`1.8
`1.2
`
`0.7
`0.2
`0.7
`0.3
`0.4
`1.0
`0.9
`0.5
`
`0.4
`0.0
`0.5
`0.3
`0.1
`1.0
`0.9
`0.5
`
`10.2
`2.7*
`9.6
`5.4
`2.7*
`12.4
`10.7
`5.7*
`
`Colitis was induced by TNBS in 50% ethanol. GA was administered either orally every other day (0.25 mg, eight
`feedings starting 7 days before disease induction) or by daily injections (2.5 mg per mouse s.c. starting 7 days
`before induction). Colonic damage was examined 5–7 days after disease induction. Asterisks indicate statistically
`significant decrease in macroscopic manifestation in GA-treated versus untreated mice (P ⬍ 0.05). max, maximal
`score.
`
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`suppressive, drugs (36, 37). However, these treatments do not
`modify the disease course, but only alleviate the symptoms while
`inducing severe side effects that limit their use. Moreover, a
`significant percentage of the patients is steroid-resistant. In view
`of the immunopathological ‘‘autoimmune-like’’ nature of IBD
`and the proposed mechanism for GA activity, it was of interest
`to test whether GA can also immunomodulate IBD. Toward this
`end, we tested the effect of GA on trinitrobenzene sulfonic acid
`(TNBS)-induced colitis, a murine model that resembles human
`Crohn’s disease in terms of its histopathological features and
`cytokine reactivity (38).
`The results obtained in three strains of mice clearly indicate
`that GA significantly ameliorates the various pathological man-
`ifestations of TNBS-induced colitis (R. Aharoni, B. Kayhan, and
`R. Arnon, unpublished data). The macroscopic colonic damage
`characteristic to the disease, severe ulceration and兾or inflam-
`mation, adhesion to adjacent organs, diarrhea, and bowel wall
`thickening were all drastically reduced by GA treatment (Table
`1). The prevention of colonic damage by GA treatment was also
`confirmed histologically. Whereas colonic specimens from the
`mice with TNBS-induced colitis revealed transmural inflamma-
`tion, extensive ulceration, and severe disruption of the normal
`architecture, colons of TNBS-induced mice treated with GA
`demonstrated significantly less damaged intestine architecture
`and well conserved glandular structure. GA abrogated the
`weight loss characteristic to this disease, as manifested also in a
`faster subsequent weight regain. The beneficial suppression of all
`of the disease manifestations by GA treatment resulted in
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`improved long-term survival (0–20% mortality in the different
`strains) in comparison to the untreated mice with TNBS-induced
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`even several days after, disease induction. Daily injection of GA
`was the most effective treatment, but oral treatment also resulted
`in disease suppression. The effect of GA was corroborated in
`three highly susceptible strains of mice, including the SJL兾J
`strain in which an exceptional aggressive disease was manifested,
`indicating that the ability of GA to ameliorate TNBS-induced
`colitis represents a general phenomenon.
`In accord with the mechanism demonstrated in EAE兾MS and
`in the transplantation systems, GA treatment resulted in signif-
`icant reduction in the overall secretion of the proinflammatory
`cytokine tumor necrosis factor ␣ and in beneficial elevation in
`the secretion of the Th3 essential cytokine TGF-␤. These results
`suggest that GA can modulate the detrimental immune response
`involved in the pathogenesis of experimental colitis, which
`resembles Crohn’s disease.
`
`Conclusions
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`of GA, support its application for various pathological autoim-
`mune disorders such as MS, immune rejection, and IBD.
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