J. Ludovic Croxford

Therapeutic potential of cannabinoids in CNS disease.

The major psychoactive constituent of Cannabis sativa, Δ9-tetrahydrocannabinol (Δ9-THC), and endogenous cannabinoid ligands, such as anandamide, signal through G-protein-coupled cannabinoid receptors localised to regions of the brain associated with important neurological processes. Signalling is mostly inhibitory and suggests a role for cannabinoids as therapeutic agents in CNS disease where inhibition of neurotransmitter release would be beneficial.Anecdotal evidence suggests that patients with disorders such as multiple sclerosis smoke cannabis to relieve disease-related symptoms. Cannabinoids can alleviate tremor and spasticity in animal models of multiple sclerosis, and clinical trials of the use of these compounds for these symptoms are in progress. The cannabinoid nabilone is currently licensed for use as an antiemetic agent in chemotherapy-induced emesis. Evidence suggests that cannabinoids may prove useful in Parkinson’s disease by inhibiting the excitotoxic neurotransmitter glutamate and counteracting oxidative damage to dopaminergic neurons. The inhibitory effect of cannabinoids on reactive oxygen species, glutamate and tumour necrosis factor suggests that they may be potent neuroprotective agents. Dexanabinol (HU-211), a synthetic cannabinoid, is currently being assessed in clinical trials for traumatic brain injury and stroke. Animal models of mechanical, thermal and noxious pain suggest that cannabinoids may be effective analgesics. Indeed, in clinical trials of postoperative and cancer pain and pain associated with spinal cord injury, cannabinoids have proven more effective than placebo but may be less effective than existing therapies. Dronabinol, a commercially available form of Δ9-THC, has been used successfully for increasing appetite in patients with HIV wasting disease, and cannabinoid receptor antagonists may reduce obesity.Acute adverse effects following cannabis usage include sedation and anxiety. These effects are usually transient and may be less severe than those that occur with existing therapeutic agents. The use of nonpsychoactive cannabinoids such as cannabidiol and dexanabinol may allow the dissociation of unwanted psychoactive effects from potential therapeutic benefits. The existence of other cannabinoid receptors may provide novel therapeutic targets that are independent of CB1 receptors (at which most currently available cannabinoids act) and the development of compounds that are not associated with CB1 receptor-mediated adverse effects. Further understanding of the most appropriate route of delivery and the pharmacokinetics of agents that act via the endocannabinoid system may also reduce adverse effects and increase the efficacy of cannabinoid treatment.This review highlights recent advances in understanding of the endocannabinoid system and indicates CNS disorders that may benefit from the therapeutic effects of cannabinoid treatment. Where applicable, reference is made to ongoing clinical trials of cannabinoids to alleviate symptoms of these disorders.

A virus-induced molecular mimicry model of multiple sclerosis

Molecular mimicry is the process by which virus infection activates T cells that are cross-reactive with self antigens. Infection of SJL/J mice with the neurotropic picornavirus Theilers murine encephalomyelitis virus (TMEV) leads to a progressive CD4(+) T cell-mediated demyelinating disease similar to multiple sclerosis. To study the potential of virus-induced molecular mimicry to initiate autoimmune demyelination, a nonpathogenic TMEV variant was engineered to encode a 30-mer peptide encompassing the immunodominant encephalitogenic myelin proteolipid protein (PLP139-151) epitope. Infection with the PLP139-151-encoding TMEV led within 10-14 days to a rapid-onset paralytic demyelinating disease characterized by PLP139-151-specific CD4(+) Th1 responses; insertion of a non-self ovalbumin sequence led to restoration of the normal late-onset disease. Early-onset disease was also observed in mice infected with a TMEV encoding PLP139-151 with an amino acid substitution at the secondary T cell receptor (TCR) contact residue (H147A), but not in mice infected with TMEV encoding a PLP139-151 substitution at the primary TCR contact (W144A). Most significantly, mice infected with TMEV encoding a Haemophilus influenzae mimic peptide, sharing only 6 of 13 amino acids with PLP139-151, displayed rapid-onset disease and developed cross-reactive PLP139-151-specific CD4(+) Th1 responses. To our knowledge, this is the first study showing that a naturally infectious virus encoding a myelin epitope mimic can directly initiate organ-specific T cell-mediated autoimmunity.

Epitope spreading and molecular mimicry as triggers of autoimmunity in the Theiler's virus-induced demyelinating disease model of multiple sclerosis.

The pathogenesis of multiple sclerosis (MS), a human demyelinating disease of the central nervous system (CNS), is currently unknown. It is widely thought that MS is an autoimmune disease which is supported by animal studies showing that myelin-specific CD4+ T cells can induce similar clinical disease in mice as observed in MS. However, the mechanism(s) of activation of these autoreactive CD4+ T cells are unknown. Although genetic susceptibility is important, other factors may be involved. Viral infections have long thought to be involved in the pathogenesis of MS although there exists little or no direct evidence implicating a role for a specific virus in MS pathogenesis. This review will discuss two models of virus-induced CNS autoimmunity, molecular mimicry and epitope spreading. These two mechanisms of activation of autoreactive T cells are presented in the context of MS.

Viral Delivery of an Epitope from Haemophilus influenzae Induces Central Nervous System Autoimmune Disease by Molecular Mimicry

Multiple sclerosis (MS) is an autoimmune CNS demyelinating disease in which infection may be an important initiating factor. Pathogen-induced cross-activation of autoimmune T cells may occur by molecular mimicry. Infection with wild-type Theiler’s murine encephalomyelitis virus induces a late-onset, progressive T cell-mediated demyelinating disease, similar to MS. To determine the potential of virus-induced autoimmunity by molecular mimicry, a nonpathogenic neurotropic Theiler’s murine encephalomyelitis virus variant was engineered to encode a mimic peptide from protease IV of Haemophilus influenzae (HI), sharing 6 of 13 aa with the dominant encephalitogenic proteolipid protein (PLP) epitope PLP139–151. Infection of SJL mice with the HI mimic-expressing virus induced a rapid-onset, nonprogressive paralytic disease characterized by potent activation of self-reactive PLP139–151-specific CD4+ Th1 responses. In contrast, mice immunized with the HI mimic-peptide in CFA did not develop disease, associated with the failure to induce activation of PLP139–151-specific CD4+ Th1 cells. However, preinfection with the mimic-expressing virus before mimic-peptide immunization led to severe disease. Therefore, infection with a mimic-expressing virus directly initiates organ-specific T cell-mediated autoimmunity, suggesting that pathogen-delivered innate immune signals may play a crucial role in triggering differentiation of pathogenic self-reactive responses. These results have important implications for explaining the pathogenesis of MS and other autoimmune diseases.

Initiation and Exacerbation of Autoimmune Demyelination of the Central Nervous System via Virus-Induced Molecular Mimicry: Implications for the Pathogenesis of Multiple Sclerosis

ABSTRACT Epidemiological studies indicate that infectious agents are important in the pathogenesis of multiple sclerosis (MS). Our previous reports showed that the infection of SJL mice with a nonpathogenic variant of Theilers murine encephalomyelitis virus (TMEV) engineered to express a naturally occurring Haemophilus influenzae-encoded molecular mimic (HI574-586) of an immunodominant self-myelin proteolipid protein epitope (PLP139-151) induced a rapid-onset demyelinating disease associated with the activation of PLP139-151-specific Th1 responses. The current results extend our previous findings in four critical respects. We show that disease initiation by the H. influenzae mimic is prevented by tolerance to the self PLP139-151 epitope, definitively proving the occurrence of infection-induced molecular mimicry. We demonstrate that the H. influenzae mimic epitope can be processed from the flanking sequences within the native mimic protein. We show that the H. influenzae mimic epitope only induces an immunopathologic self-reactive Th1 response and subsequent clinical disease in the context of the TMEV infection and not when administered in complete Freunds adjuvant, indicating that molecular mimicry-induced disease initiation requires virus-activated innate immune signals. Lastly, we show that the infection of SJL mice with TMEV expressing the H. influenzae mimic can exacerbate a previously established nonprogressive autoimmune disease of the central nervous system. Collectively, these findings illustrate the evolving mechanisms by which virus infections may contribute to both the initiation and exacerbation of autoimmune diseases, and they have important implications for MS pathogenesis.

Structural requirements for initiation of cross‐reactivity and CNS autoimmunity with a PLP139–151 mimic peptide derived from murine hepatitis virus

MS is an autoimmune CNS demyelinating disease in which infection appears to be an important pathogenic factor. Molecular mimicry, the cross‐activation of autoreactive T cells by mimic peptides from infectious agents, is a possible explanation for infection‐induced autoimmunity. Infection of mice with a non‐pathogenic strain of Theilers murine encephalomyelitis virus (TMEV) engineered to express an epitope from Haemophilus influenzae (HI) sharing 6/13 amino acids with the dominant proteolipid protein (PLP) epitope, PLP139–151, can induce CNS autoimmune disease. Here we demonstrate that another PLP139–151 mimic sequence derived from murine hepatitis virus (MHV) which shares only 3/13 amino acids with PLP139–151 can also induce CNS autoimmune disease, but only when delivered by genetically engineered TMEV, not by immunization with the MHV peptide. Further, we demonstrate the importance of proline at the secondary MHC class II contact residue for effective cross‐reactivity, as addition of this amino acid to the native MHV sequence increases its ability to cross‐activate PLP139–151‐specific autoreactive T cells, while substitution of proline in the HI mimic peptide has the opposite effect. This study describes a structural requirement for potential PLP139–151 mimic peptides, and provides further evidence for infection‐induced molecular mimicry in the pathogenesis of autoimmune disease.

Effects of cannabinoid treatment on Chagas disease pathogenesis: balancing inhibition of parasite invasion and immunosuppression

Trypanosoma cruzi invades heart cells via a calcium‐dependent, G protein‐mediated mechanism, leading to severe cardiac inflammation considered by some to be autoimmune in nature. Cannabinoids inhibit calcium flux and G protein signalling; as potent immunosuppressive agents, they are effective in the treatment of autoimmune disease but contraindicated for the treatment of infections. We compared the action of the synthetic cannabinoid R(+)WIN55,212 and its inactive isomer S(–)WIN55,212 on cardiac myoblast invasion: R(+)WIN55,212 inhibited invasion by over 85%. We then tested for efficacy in modulating pathogenesis in mice by assaying parasite burden in heart and blood, cellular and humoral immunity to parasite and self antigens, and mortality. R(+)WIN55,212 significantly reduced cardiac inflammation but led to considerably increased parasitaemia. Cardiac parasitosis and mortality were not significantly different in treatment and control groups. We conclude that cannabinoids can block cardiac cell puncture repair mechanisms, thereby inhibiting trypanosome invasion as predicted by the mode of drug action, but, also inhibit immune cell effector functions, offsetting the benefit of inhibition parasite cell invasion. Refined use of cannabinoids may prove therapeutic in the future, but our results raise concern about the effect of cannabis use on those chronically infected by T. cruzi and on heart cell homeostasis generally.

Cross-reactivity between peptide mimics of the immunodominant myelin proteolipid protein epitope PLP139-151: Comparison of peptide priming in CFA vs. viral delivery

Epidemiological evidence suggests that pathogens may trigger the development of autoimmune diseases such as multiple sclerosis (MS). Pathogens may trigger disease via molecular mimicry, wherein T cells generated against foreign epitopes are also cross-reactive with self-epitopes. Five pathogen-derived molecular mimics of PLP(139-151) (the immunodominant CD4(+) T cell myelin epitope in SJL mice) were previously identified. This study examines the degree of cross-reactivity between the different mimics, comparing mice primed with mimic peptide in CFA with mice infected with recombinant mimic-expressing viruses. The pattern of in vitro reactivity and ability to induce CNS disease differs between peptide priming and virus infection.

Mouse Models of Multiple Sclerosis

Experimental autoimmune encephalomyelitis (EAE) and Theilers murine encephalitis virus-induced demyelinating disease (TMEV-IDD) are two clinically relevant murine models of multiple sclerosis (MS). Like MS, both are characterized by mononuclear cell infiltrate into the central nervous system and demyelination. EAE is induced by either the administration of protein or peptide in adjuvant or by the adoptive transfer of encephalitogenic T-cell blasts into naive recipients. The relative merits of each of these protocols are compared. Depending on the type of question asked, different mouse strains and peptides are used. Different disease courses are observed with different strains and different peptides in active EAE. These variations are addressed, and grading of mice in EAE is discussed. In addition to EAE induction, useful references for other disease indicators, such as delayed-type hypersensitivity, in vitro proliferation, and immunohistochemistry, are provided. TMEV-IDD is a useful model for understanding the potential viral etiology of MS. This chapter provides detailed information on the preparation of viral stocks and subsequent intracerebral infection of mice. In addition, virus plaque assay and disease assessment are discussed. Recombinant TMEV strains have been created for the study of molecular mimicry; these strains incorporate 30 various amino acid myelin epitopes within the leader region of TMEV.