Lack of apolipoprotein-E exacerbates experimental allergic encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) was found to have a chronic and significantly worse course in apolipoprotein-E (apoE) deficient female mice when compared with matched controls. Disease measures compared included incidence of EAE (64% versus 31%, P < 0.05, chi2 test), maximal clinical score (average +/- SD 2.81 +/- 2.5 versus 0.75 +/- 1.1, P < 0.01, Mann-Whitney test) and mortality (27.3% versus 0%, P = 0.02, Mann-Whitney test and chi2 test). ApoE deficient mice had significantly increased lymphocyte proliferation responses to both myelin antigens and mitogens and significantly more infiltrating lesions in the central nervous system (CNS) in histopathology. Defective neuronal repair mechanisms and enhanced immune reactivity in apoE deficient mice may explain our findings. Clinical implications for MS are discussed.

The Ras-pathway inhibitor, S-trans-trans-farnesylthiosalicylic acid, suppresses experimental allergic encephalomyelitis.

AIM: To evaluate the effects of the synthetic Ras-pathway inhibitor, S-trans-trans-farnesylthiosalicylic acid (FTS) on acute and chronic experimental autoimmune encephalomyelitis (EAE and CR-EAE). BACKGROUND: Treatment of EAE and MS is based on immunosuppression aiming at downregulation of the proliferating myelin-reactive lymphocytes. One of the pathways of lymphocyte activation involves the GTP-binding protein Ras. FTS destabilizes the attachment of Ras to the cell membrane, resulting in an inhibition of the Ras-mediated signal transduction pathways. MATERIALS AND METHODS: EAE was induced in SJL/J mice by immunization with spinal cord homogenate (MSCH) in adjuvant and two i.v. boosts of pertussis antigen and CR-EAE with passive transfer of proteolipid protein (PLP)-activated lymphocytes. Animals were treated daily starting either from the day of EAE-induction (or cell transfer) or at a later stage, with i.p. injections of FTS (5 mg/kg/day). The clinical severity of the disease was evaluated daily and scored using a 0-6 scale. RESULTS: In six separate experiments, 27 of the 38 (71.7%) vehicle-treated animals developed clinical signs of EAE compared to 17/38 (44.7%) of the FTS-treated mice (p=0.02, t-test). The maximal average score in the control group was 2.94+/-2.2, whereas in the FTS group it was significantly lower (1.63+/-2.2, p=0.01). Mortality was 26.3% and 10.5% in the two groups, respectively (p=0.03). When treatment was initiated at a later stage, just before the onset of the clinical signs, the protective effect was even more pronounced. A significant suppression of clinical signs was also observed in the CR-EAE model (p=0.02). Lymphocyte proliferation assays demonstrated a more than twofold decrease in the reactivity to myelin antigens (MBP and PLP) and downregulation of the activated lymphocytes (expressing the CD62L, and IA-k-MHC Class I markers and the Vb17 T-cell receptor) in the FTS-treated group; in vitro FTS suppressed the Ras activity of lymphocytes and inhibited the proliferative ability of the lymphocytes in a dose-dependent manner. CONCLUSIONS: FTS suppresses EAE by downregulation of myelin-reactive activated T-lymphocytes. Since FTS did not induce generalized immunosuppressive effects, it may offer significant advantages over the broad immunosuppressive modalities and may be a candidate treatment for autoimmune diseases, such as MS.

Linomide downregulates autoimmunity through induction of TH2 cytokine production by lymphocytes.

Linomide is a synthetic immunomodulator that has been shown to protect animals against a wide range of spontaneously developing or induced autoimmune diseases. We have previously reported that Linomide blocks both the clinical and the histopathological manifestations of experimental autoimmune encephalomyelitis (EAE) in various animal models. In this study, in an effort to elucidate the mechanisms by which Linomide suppresses EAE, and autoimmunity in general, we investigated the in vivo effects of this drug on the TH1/TH2 lymphocyte balance, which is important for the induction or inhibition of autoireactivity. Naive SJL/J mice were treated orally for 15 days with Linomide (80 mg/kg/day). Spleen cells were obtained at various time points during the treatment period and were stimulated in vitro with concanavalin A. Interleukins IL-4, IL-10 and IL-12, transforming growth factor-beta (TGFbeta) and interferon-gamma (IFNgamma) cytokine production was evaluated both by means of detection of the cytokines in the medium (by ELISA technique) and by detection of the cytokine mRNA production, using a semiquantitative reverse transcriptase polymerase chain reaction method. A significant upregulation of IL-4, IL-10 and TGFbeta was observed following treatment with Linomide, which peaked at day 10 (IL-10) or day 15 (IL-4). On the other hand, IL-12 and IFNgamma production were either unchanged or decreased. It seems therefore that Linomide induces in vivo a shift towards TH2 lymphocytes which may be one of the mechanisms of downregulation of the autoimmune reactivity in EAE. Our observations indicate that downregulation of TH1 cytokines (especially IL-12) and enhancement of TH2 cytokine production may play an important role in the control of T-cell-mediated autoimmunity. These data may contribute to the design of new immunomodulating treatments for a group of autoimmune diseases.

Acute/relapsing experimental autoimmune encephalomyelitis: induction of long lasting, antigen-specific tolerance by syngeneic bone marrow transplantation.

Experimental autoimmune encephalomyelitis (EAE) is an inducible autoimmune disease widely used as a model of the acute/relapsing stage of multiple sclerosis. We have previously shown that treatment of EAE-mice with high doses of cyclophosphamide (CY) (350 mg kg), followed by syngeneic bone marrow transplantation (SBMT), completely abrogates the clinical paralytic signs and even prevents the appearance of new relapses in the chronic-relapsing model of the disease. In the present study we examined whether this treatment protocol induces long term tolerance and whether this tolerance is antigen-specific. EAE was induced by immunization with spinal cord homogenate (MSCH) in complete Freund's adjuvant (CFA). The treatment with CY and SBMT was performed on day 6 post immunization. Treated and untreated mice were rechallenged with MSCH, or a non-relevant antigen (OVA) in CFA at various stages after the first paralytic attack. In contrast to previous data showing that animals recovering from acute EAE are usually refractory to re-induction of the disease, repeated injections of MSCH at different sites from the initial immunization, followed by i.v. injection of inactivated Bordetella bacteria, 2, 4 and 6 months after the initial EAE-induction, caused a severe and usually lethal relapse in all the untreated, control animals. Mice treated with CY and SBMT were resistant to all rechallenges with the same encephalitogenic inoculum. Following the second rechallenge, peripheral lymph node cells were examined in vitro for their proliferative responses to myelin antigens or to OVA. Lymphocytes obtained from CY+SBMT treated mice did not proliferate in vitro in response to myelin basic protein (MBP), but proliferated against OVA, when immunized with this antigen, after SBMT. Adoptive transfer of lymphocytes from tolerant mice to naive recipients did not transfer resistance to EAE-induction. Our results indicate that high doses of CY, followed by SBMT, induce long term antigen-specific tolerance presumably by a mechanism of clonal deletion or anergy.

Linomide induces apoptotic death of cortical CD4/CD8 double positive thymocytes and thymic atrophy by a corticosteroid-independent pathway.

Linomide is a synthetic immunomodulator which was shown to protect animals against a wide range of experimental autoimmune diseases. In this study we have investigated the effects of Linomide on the thymus in an effort to elucidate the mechanisms by which this immunomodulator suppresses autoimmune reactivity. Normal or adrenalectomized SJL/J mice were treated orally for 10 days with linomide (80 mg/kg/day). Thymocytes were tested by FACS for the analysis of the CD4 and CD8 markers and TCR expression on their surface. Thymuses from these animals were examined for size and cellularity and immunohistopathologically for the detection of apoptosis and for the expression of the markers CD4 and CD8. A significant reduction in the thymus size and cellularity was observed in mice treated with Linomide, starting from day 3 after treatment, accompanied by an enhanced apoptotic death of cortical thymocytes, which was first noted on day 1 of treatment and peaked on day 3. FACS analysis and immunohistochemistry revealed a significant depletion of the CD4(+)/CD8(+) (double positive) cells with a parallel relative increase of the more mature, medullar, single positive, lymphocytes. These effects on the thymus were not mediated through a corticosteroid-dependent pathway, and were also observed in adrenalectomized and Linomide-treated animals. These observations may be of importance for the clarification of the role of thymus in autoimmunity and the possible ways for immune intervention with immunomodulators like Linomide at this level.

Inhibition of the progression of multiple sclerosis by linomide is associated with upregulation of CD4+/CD45RA+ cells and downregulation of CD4+/CD45RO+ cells.

In a recent double-blind, phase II study, conducted in our department, we showed that Linomide-treated MS patients had significantly less active lesions (in serial monthly MRI tests) and a tendency for clinical stabilization. Here we present the immunological evaluation of the patients who participated in this study and propose a novel mechanism by which Linomide downregulates autoreactivity. Peripheral blood leukocytes (PBLs), serum, and CSF samples were obtained at two to four time points over the 6 months of the trial. Flow cytometric analysis (FACS) of the CD5/CD19, CD4/CD8, CD14/CD3, CD16/CD3, CD45RA/CD4, and CD45RO/CD4 surface markers on PBLs was performed and the levels of the IL-1beta, IL-2, IL-4, IL-6, IL-10, TNF-alpha, IFN-gamma, and IL-2R were also examined. White blood counts of Linomide-treated patients were consistently elevated throughout the treatment period (P = 0.002-0.04). Cytokines levels in serum and CSF were highly fluctuating and we could not detect any clear trend as a result of Linomide treatment. FACS analysis showed that Linomide treatment significantly increased the percentage of the CD4+/CD45RA+ cells (from 35.5% at baseline to 42.3% at week 24; P = 0.02), and decreased CD4+/CD45RO+ lymphocytes (62.6% at baseline vs 53.7% at week 24, P = 0.02). Linomide also induced a transient increase in the NK-cells, the NK 1.1 cells, and the CD5 B-cells (P = 0.02). Upregulation of naive CD45RA T-lymphocytes and parallel downregulation of memory CD45RO cells seems to be one of the main mechanisms by which Linomide inhibits MS activity and may represent an alternative immunomodulating approach for the treatment of MS and autoimmunity in general.

Immunomodulation of autoimmunity by linomide: inhibition of antigen presentation through down regulation of macrophage activity in the model of experimental autoimmune encephalomyelitis.

Linomide (quinoline-3-carboxamide, LS-2616), a synthetic immunomodulator, protects animals against a variety of experimental autoimmune diseases. In experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (MS), linomide blocks both the clinical and histological signs of the disease, without inducing generalized immunosuppression. In the first clinical trial in patients with MS, linomide was shown to inhibit the progression of the disease. In the present study we investigated several aspects of the mechanisms of action of this immunomodulator. We found that linomide can inhibit acute EAE even when given as pretreatment, prior to induction of disease (days - 10 to 0). This inhibitory effect was reversed by adoptive transfer of naive spleen cells. A short course (7 days) of linomide treatment also inhibited EAE, especially when administered immediately after disease induction. Spleen cells from linomide-treated mice failed to present myelin antigens to T-cell lines in vitro. The defective antigen presentation was normalized by anti-oxidants such as 2-mercaptoethanol. The proportion of Mac1+ cells in the spleens of linomide-treated mice was significantly reduced and macrophage growth was inhibited in long term cultures of spleen cells derived from linomide-treated animals. Our findings suggest that the effect of linomide on EAE may be attributed, at least in part, to inactivation of antigen presenting cells, possibly following a short period of over-stimulation and increased oxidant production. This mechanism may play a universal role in the regulation of autoimmune reactivity and merits further investigation.

Immunomodulation of experimental autoimmune myasthenia gravis with linomide.

Linomide, a synthetic immunomodulator, increases natural killer (NK) activity and markedly activates several lymphocyte populations in both experimental animals and humans. It has been shown to ameliorate the autoimmune manifestations of lupus-like disease in MRL/lpr mice and the clinical and pathological signs of acute and chronic-relapsing experimental autoimmune encephalomyelitis (EAE) in SJL/J mice. We examined the effect of linomide (100 mg/kg/day; administered in drinking water) on rabbits and rats with experimental autoimmune myasthenia gravis (EAMG). Following immunization with Torpedo acetylcholine receptor (AChR), all control rabbits developed clinical signs of severe weakness and exhibited a decrement of muscle action potential upon repetitive stimulation. In contrast, mild signs of weakness appeared in only two of five linomide-treated rabbits, with EMG borderline positive in one of them. Booster immunization with Torpedo AChR induced severe relapse and death in two EAMG control rabbits, whereas the two linomide-treated animals remained free of myasthenic symptoms. The serum level of antibodies against both Torpedo and rat AChR were markedly suppressed in the linomide-treated animals. Similar inhibition of clinical signs of EAMG was observed in the EAMG rat model. Furthermore, the in vitro proliferative response of lymph node cells to Torpedo AChR and the purified protein derivative of Mycobacterium tuberculosis was significantly lower in the linomide-treated EAMG rats than in the controls. Linomide may constitute a new immunomodulating agent for the treatment of myasthenia gravis.

Inoculation of BCL1 lymphoma cells into CSJL/J F1 mice inhibits acute experimental autoimmune encephalomyelitis.

T cell vaccination, which protects rodents against experimental autoimmune encephalomyelitis (EAE), has been shown to induce anti-idiotypic response in the T cell compartment. CD5 B cells (B1 cells) are the main source of natural autoantibodies, and are often characterized by high idiotypic connectivity. In this study we examined the possibility that idiotypic connectivity in the B cell compartment may also play a role in the regulation of EAE. We inoculated CSJLF1 mice (H-2d,s) with a CD5 B cell line, the BCL1 lymphoma cells (H-2d), and subsequently induced EAE. The injection of as few as 1,000 BCL1 lymphoma cells significantly blocked the development of EAE. Injection of CD5-negative myeloma cells (SP2) had no effect on the pathogenesis of the disease. Unlike control animals, lymphocytes from BCL1 lymphoma-injected mice significantly proliferate in response to interleukin-5, a growth factor to CD5 B cells. The proliferative response of lymphocytes from BCL1 inoculated mice to mitogenic stimulation was rather unchanged, indicating that no general immunosuppression has been induced by inoculating BCLJ lymphoma. These experiments suggest that CD5 B cells may be involved in the regulation of EAE.

Inhibition of acute, experimental autoimmune encephalomyelitis by the synthetic immunomodulator linomide.

Linomide (LS-2616, quinoline-3-carboxamide) is a synthetic immunomodulator that stimulates natural killer cell activity and activates several lymphocytic subpopulations in experimental animals and humans. In this study we determined the effect of oral treatment with linomide on the development of experimental autoimmune encephalomyelitis, an animal model for immune-mediated human demyelinating disorders. Experimental autoimmune encephalomyelitis was induced in SJL/J mice and in an outbred strain of rats (Sabra) by subcutaneous injection of spinal cord homogenate in adjuvant followed by inoculation with Bordetella pertussis. Linomide was administered in drinking water, at an estimated dose of 50 to 100 mg/kg/day. None of the linomide-treated mice (0/41) and Sabra rats (0/15) developed any clinical or pathological signs of experimental autoimmune encephalomyelitis, whereas almost all control animals (48/53 and 18/19, respectively) were severely paralyzed and 64.5% died from the disease. Lymphocytes obtained from linomide-treated animals had reduced in vitro proliferative responses to guinea pig myelin basic protein, proteolipid protein of the myelin, and tuberculin-purified protein derivative, unlike antigen-independent proliferation which was rather unaffected. Natural killer cell activity (tested by a cytotoxic assay on radiolabeled YAC-1 target cells) was significantly enhanced in mice treated with linomide. Our results indicate that modulation of the immune system with linomide leads to complete inhibition of experimental autoimmune encephalomyelitis in the absence of systemic immunosuppression. Linomide could therefore be of use in future clinical trials for the treatment of human autoimmune demyelinating disorders.

Prevention and reversal of adoptively transferred, chronic relapsing experimental autoimmune encephalomyelitis with a single high dose cytoreductive treatment followed by syngeneic bone marrow transplantation.

A chronic relapsing form of experimental autoimmune encephalomyelitis (CR-EAE) was induced in SJL/J mice by adoptive transfer of lymph node cells (LNC) sensitized to guinea pig myelin basic protein (GMBP). We examined the efficacy of high dose immunosuppressive regimens (cyclophosphamide [CY] 300 mg/kg or total body irradiation [TBI] 900 cGy) followed by syngeneic bone marrow transplantation (SBMT) in prevention and treatment of already established CR-EAE. Treatment with TBI and SBMT on day 5 after the induction of CR-EAE, just before the onset of clinical signs, completely inhibited the appearance of the paralytic signs. The same treatment, applied 4 d after the clinical onset of the disease, led to a significant regression of the paralytic signs and to a total inhibition of spontaneous relapses during a follow-up period of 2 mo. Challenge of mice with GMBP+CFA 78 d after the passive induction of CR-EAE induced a relapse of the disease 7 d later in almost all of the untreated mice; in contrast, the same challenge given to TBI+SBMT-treated mice caused a delayed relapse (30 d later) in only a minority (3/7) of the challenged mice. In vitro lymphocytic proliferative responses to GMBP and purified protein derivative were significantly lower in TBI/SBMT-treated mice before and after the GMBP challenge, although these mice were fully immunocompetent, as evidenced by their normal lymphocytic proliferation to concanavalin A (ConA) and the FACS analysis of their lymphocytic subpopulations. A similar beneficial therapeutic effect was observed in mice treated with CY followed by SBMT, after the onset of CR-EAE. Our results could support possible clinical applications of similar therapeutic strategies, involving acute immunosuppression followed by stem cell transplantation and retolerization of the reconstituting immune cells in life-threatening neurological and multisystemic autoimmune diseases.

Treatment of chronic-relapsing experimental autoimmune encephalomyelitis with the synthetic immunomodulator linomide (quinoline-3-carboxamide).

Linomide is a synthetic immunomodulator that enhances natural killer cell activity and significantly activates several lymphocytic cell subpopulations in both experimental animals and humans. In this study we examined the effect of linomide (80 mg per kg per day in drinking water) on mice with chronic-relapsing experimental autoimmune encephalomyelitis (CR-EAE), a T-cell-mediated organ-specific autoimmune disease that resembles human multiple sclerosis. None of the mice (n = 17) that were treated with linomide from day 7 after disease induction developed any clinical or histopathological signs of CR-EAE, as compared to 19 of 20 untreated controls that were severely paralyzed and had extensive demyelinating lesions in the central nervous system. Linomide-treated animals were also resistant to an induced attack by a booster injection with a murine spinal cord homogenate. When administered to mice exhibiting severe clinical signs of paralysis, linomide inhibited both spontaneous and induced relapses. Linomide treatment protected mice from passively induced CR-EAE as well, when given from the day of injection with myelin-basic-protein-specific lymphocytes. Lymphocytes obtained from linomide-treated mice had a reduced in vitro proliferative response to the myelin basic protein and to the tuberculin purified protein derivative, whereas the mitogenic response to concanavalin A was not affected. Natural killer cell and lymphokine-activated killer cell activities were enhanced. These results suggest that linomide regulates autoimmunity in the absence of systemic immunosuppression. Since linomide is very well tolerated in experimental animals and humans, it might be used in the treatment of multiple sclerosis.

Prevention of experimental autoimmune encephalomyelitis and induction of tolerance with acute immunosuppression followed by syngeneic bone marrow transplantation.

Experimental autoimmune encephalomyelitis (EAE) is an inducible autoimmune disease widely used as a model of the acute/relapsing stage of multiple sclerosis. In the present study we examined the effect of acute immunosuppression induced by total body irradiation (TBI) (900 to 1100 centigray (cGy)) or by a single high dose of cyclophosphamide (CY) (300 mg/kg), followed by syngeneic bone marrow transplantation (SBMT), on the development of EAE in SJL/J mice. EAE was induced in SJL/J mice by immunization with spinal cord homogenate in adjuvant. Treatment with TBI (900 cGy) and SBMT on day 6 postimmunization caused a delayed onset and a marked reduction in the incidence and severity of EAE. A higher dose of irradiation (1100 cGy) or the administration of CY followed by SBMT completely abrogated the development of paralysis. None of the 21 mice treated with CY and SBMT, and only 1 of 7 mice treated with TBI (1100 cGy) and SBMT developed clinical signs of EAE during a period of 3 months. Furthermore, mice treated with CY and SBMT became resistant to rechallenge with the same encephalitogenic inoculum. In addition, the lymphocytes obtained from these mice did not proliferate in vitro in response to myelin basic protein or tuberculin-purified protein derivative, unlike lymphocytes from immunized but untreated animals. This absence of reactivity was not associated with alterations in the proportion of the L3T4 and Lyt-2 T-cell subsets nor with a loss in T cell competence as evidenced by the full response of lymphocytes to the T cell mitogen Con A and to a nonrelevant Ag (OVA). Our results indicate that the elimination of effector lymphocytes either by myeloablative doses of CY or ionizing irradiation followed by rescue with SBMT inhibits the development of the autoimmune process in EAE and leads to induction of tolerance to the immunizing Ag by newly developing lymphocytes. This approach of combining immunoablation and reconstitution with autologous bone marrow transplantation may be applicable in the treatment of life-threatening neurologic autoimmune diseases.