Treatment of MRL/lpr mice, a genetic autoimmune model, with the Ras inhibitor, farnesylthiosalicylate (FTS).

Activation and proliferation of lymphocytes requires the active signal transducer Ras. Activation of lymphocytes, associated with autoimmunity, may therefore be modified by S-farnesylthiosalicylic acid (FTS), a synthetic substance that detaches Ras from the inner cell membrane and induces its rapid degradation. The MRL/lpr mouse is a genetic model of a generalized autoimmune disease sharing many features and organ pathology with systemic lupus erythematosus (SLE) and the primary antiphospholipid syndrome (APS). The objective of the present study was to examine the effect of FTS on laboratory and clinical pathology in the MRL/lpr mouse. Female MRL/lpr (n = 50) and MRL/++ control (n = 35) mice were treated intraperitoneally with either FTS (5 mg/kg/day) or saline between 6 and 18 weeks of age. The mice were weighed, tested for proteinuria and lymphadenopathy, lymphocyte proliferation, antibodies, grip strength and behaviour in an open field. FTS treatment resulted in a 50% decrease in splenocyte proliferation to ConA, LPS and a disease specific antigen, beta(2)-glycoprotein-I, and in a significant decrease in serum antibody levels against cardiolipin and dsDNA. Proteinuria and grip strength were normalized and lymphadenopathy and postmortem lymph node and spleen weights were significantly reduced in FTS treated MRL/lpr mice. These findings indicate that modulation of Ras activation has a significant impact on the MRL/lpr model and may represent a new therapeutic approach for the treatment of systemic autoimmune diseases such as SLE and APS.

The effects of APOE genotype on age at onset and progression of neurodegenerative diseases.

APOE genotype influences the age at onset of some neurodegenerative diseases such as AD and the rate of progression in others such as MS. The authors hypothesize that APOE genotype ubiquitously determines the efficacy of neuronal maintenance and repair in these diseases and that the seemingly divergent clinical effects are due to the stage of disease at which the diagnosis is made. Early diagnosis facilitates the measurement of effects on disease progression rate, whereas late diagnosis results in a marked effect of APOE genotype on disease onset.

APOE genotype is a major predictor of long-term progression of disability in MS.

BACKGROUND AND OBJECTIVE: The authors recently reported that the APOE epsilon4 allele is associated with significantly greater progression of disability in a 2-year follow-up of patients with MS. In this study, these findings are substantiated and extended in a much larger group of patients followed for up to 40 years. METHODS: Two hundred five patients with clinically definite MS who were genotyped for the APOE epsilon4 carrier state were included. Groups of patients with (n = 41) and without (n = 164) APOE epsilon4 alleles were compared for latency to expanded disability status scale (EDSS) scores of 4.0 and 6.0 by Kaplan-Meier analysis with the log rank test. The results were adjusted for age at onset and sex by Cox regression analysis. RESULTS: The APOE epsilon4 allele frequency in patients with MS (0.10) was similar to that in the general Israeli population. There was a significant effect of APOE genotype on the latency to reach EDSS 4.0 and 6.0 (p = 0.0002 and p = 0.0006 by two-tailed log rank test). Median latencies were shorter by 12 and 11 years in the APOE epsilon4 group for these outcomes. These results were significant after adjustment for age at onset and sex. CONCLUSIONS: The APOE epsilon4 allele is associated with significantly faster progression of disability in MS. This is the first genetic factor to be identified with a major impact on the progression of disability in this disease.

Immunomodulating therapeutic approaches for multiple sclerosis.

In this review we delineate the rationale for immunotherapy in multiple sclerosis and describe the various levels at which immune intervention, according to a modern model of the immune system organization, is feasible. Current and future immunosuppressive and immunomodulating therapeutic approaches at the level of antigen presentation and at the lymphocyte and cytokine network levels are discussed.

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.

Treatment of secondary progressive multiple sclerosis with the immunomodulator linomide: a double-blind, placebo-controlled pilot study with monthly magnetic resonance imaging evaluation.

Linomide (quinoline-3-carboxamide) is a synthetic immunomodulator that increases the natural killer cell activity. We previously demonstrated that linomide effectively inhibited the clinical and histopathologic signs of acute and chronic relapsing experimental autoimmune encephalomyelitis. We report a double-blind, placebo-controlled study to evaluate tolerability and to obtain preliminary indications of the clinical efficacy of linomide on secondary progressive MS. Thirty patients suffering from clinically definite and laboratory-supported secondary progressive MS, with an expanded disability status scale (EDSS) of 3.0 to 7.0, were included in this study. Patients were treated daily with linomide (2.5 mg) or placebo orally and were followed up for side effects and changes in their neurologic status; monthly MRI scans were taken throughout the treatment period. Twenty-four patients completed at least 6 months of treatment. Mild to moderate side effects, including muscle pains, arthralgia, and edema, were present in 11 of the 15 patients receiving placebo and in 13 of the 15 patients treated with linomide. At 24 weeks, the mean shift in EDSS was +0.272 +/- 0.156 in the placebo group versus -0.166 +/- 0.167 in the linomide group (p = 0.0451). The percentage of patients with evidence of "activity" on their MRI (new, enlarging, or new gadolinium diethylenetriaminepentaacetic acid [Gd-DTPA]-enhancing lesions) throughout the treatment period was 75% in the placebo group and 33% in the linomide group (p = 0.0205). The mean total number of new Gd-DTPA-enhancing lesions per MRI scan for the same period was 0.42 +/- 0.143 in the placebo group and 0.19 +/- 0.114 in the linomide group (p = 0.0387). In this study, linomide proved to be safe and well tolerated in patients with secondary progressive MS. In addition, our results indicate that linomide tends to inhibit the progression of the disease, especially preventing the appearance of new active lesions in the MRI scans. Based on these results, two multicenter phase III trials are currently under way in the United States and in Europe and Australia.

Immunomodulation of autoimmunity in MRL/lpr mice with syngeneic bone marrow transplantation (SBMT).

MRL-lpr/lpr mice spontaneously develop a severe autoimmune syndrome, characterized by massive generalized lymphadenopathy, arthritis, arteritis, dermatitis and immune complex-mediated glomerulonephritis. Bone marrow transplantation (BMT) from MHC-matched systemic lupus erythematosus (SLE)-resistant donors to susceptible recipients has proved effective in correcting autoimmune manifestations in autoimmune-prone mice. We investigated the effect of syngeneic BMT from MRL/lpr (donor) to immunocompromised MRL/lpr (recipient), after purging the bone marrow inoculum with MoAbs against mature T cells (anti-Thy 1.2). All the untreated mice developed lymphadenopathy and by the age of 36 weeks five of the eight were dead; in contrast, all the mice which underwent syngeneic BMT following acute immunosuppression with total body irradiation (900 cGy) (TBI) remained disease-free. In an additional experiment, it was found that conditioning with cyclophosphamide (CY) before BMT was more effective than TBI in inhibiting delayed-onset autoimmune manifestations (mean survival 350 days in the CY group and 305 days in the TBI group, versus 197 days in untreated controls). Under both immunosuppressive regimens T cell-depleted bone marrow grafts produced far better results than did unmanipulated BMT. Following syngeneic BMT the incidence of proteinuria and the level of serum anti-DNA (dd) antibodies were significantly reduced, compared with that of the age-matched untreated controls. CY was more effective than TBI in reducing the anti-DNA titres. Likewise, T depletion of bone marrow inocula before BMT induced a more drastic drop in autoantibodies, following both CY and TBI conditioning protocols. After syngeneic BMT (either CY or TBI) no signs of lymphadenopathy were observed even at an advanced age. Upon histopathological examination, the BMT-treated mice displayed normal glomeruli with occasional minimal signs of glomerulonephritis. Syngeneic T cell-depleted BMT following acute cytoreduction of anti-self immune lymphocytes may represent a new therapeutic approach for drug-resistant autoimmune diseases.

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.

Chronic-relapsing experimental autoimmune encephalomyelitis (CR-EAE): treatment and induction of tolerance, with high dose cyclophosphamide followed by syngeneic bone marrow transplantation.

We examined the effect of acute immunosuppression with high dose cyclophosphamide (CY), followed by syngeneic T-cell-depleted bone marrow transplantation (SBMT) on chronic-relapsing autoimmune encephalomyelitis (CR-EAE) induced in SJL/J mice by immunization with mouse spinal cord homogenate (MSCH) in adjuvant. Treatment of mice on day 9 post immunization, before the appearance of clinical signs of the disease, delayed the onset of paralysis, but did not affect its clinical course. Treatment on day 2-3 after the first clinical signs led to complete regression of the disease. During a period of 3 months, only one of the 15 mice treated after the the onset of CR-EAE relapsed, as compared to a total of 21 relapses in the 15 untreated animals. A rechallenge with MSCH in adjuvant on day 78 after immunization induced a severe relapse in all untreated mice, with 78% mortality; in contrast, only 25% of mice treated with CY and SBMT relapsed when similarly rechallenged. Lymphocytes from mice treated with CY and SBMT showed reduced in vitro proliferative responses to myelin basic protein (GMBP) and PPD, even after the rechallenge with MSCH. Our results show that high dose CY for elimination of immunocompetent lymphocytes, followed by SBMT rescue, suppresses CR-EAE and induces tolerance to the immunizing antigens. These results may encourage attempts to apply a similar therapeutic principle in life-threatening human neurological autoimmune diseases.

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.