Botanically-Derived Δ9-Tetrahydrocannabinol and Cannabidiol, and Their 1:1 Combination, Modulate Toll-like Receptor 3 and 4 Signalling in Immune Cells from People with Multiple Sclerosis.

The innate immune response to bacterial and viral molecules involves the coordinated production of cytokines, chemokines, and type I interferons (IFNs), which is orchestrated by toll-like receptors (TLRs). TLRs, and their intracellular signalling intermediates, are closely associated with multiple sclerosis (MS) pathogenesis. Recent data from our laboratory reported that the plant-derived cannabinoids, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), regulate viral and bacterial inflammatory signalling pathways controlled by TLR3 and TLR4 in macrophages. The aim of this study was to assess the impact of THC and CBD, when delivered in isolation and in combination (1:1), on TLR3- and TLR4-dependent signalling in peripheral blood mononuclear cells (PBMCs) from people with MS (pwMS; n = 21) and healthy controls (HCs; n = 26). We employed the use of poly(I:C) and lipopolysaccharide (LPS) to induce viral TLR3 and bacterial TLR4 signalling, and PBMCs were pre-exposed to plant-derived highly purified THC (10 µM), CBD (10 µM), or a combination of both phytocannabinoids (1:1 ratio, 10:10 µM), prior to LPS/poly(I:C) exposure. TLR3 stimulation promoted the protein expression of the chemokine CXCL10 and the type I IFN-ß in PBMCs from both cohorts. THC and CBD (delivered in 1:1 combination at 10 µM) attenuated TLR3-induced CXCL10 and IFN-ß protein expression in PBMCs from pwMS and HCs, and this effect was not seen consistently when THC and CBD were delivered alone. In terms of LPS, TLR4 activation promoted TNF-α expression in PBMCs from both cohorts, and, interestingly, CBD when delivered alone at 10 µM, and in combination with THC (in 1:1 combination at 10 µM), exacerbated TLR4-induced TNF-α protein expression in PBMCs from pwMS and HCs. THC and CBD displayed no evidence of toxicity in primary PBMCs. No significant alteration in the relative expression of TLR3 and TLR4 mRNA, or components of the endocannabinoid system, including the cannabinoid receptor CB1 (encoded by CNR1 gene) and CB2 (encoded by CNR2 gene), and endocannabinoid metabolising enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGLL), was determined in PBMCs from pwMS versus HCs. Given their role in inflammation, TLRs are clinical targets, and data herein identify CBD and THC as TLR3 and TLR4 modulating drugs in primary immune cells in vitro. This offers insight on the cellular target(s) of phytocannabinoids in targeting inflammation in the context of MS.

Phytocannabinoid-dependent mTORC1 regulation is dependent upon inositol polyphosphate multikinase activity.

BACKGROUND AND PURPOSE: Cannabidiol (CBD) has been shown to differentially regulate the mechanistic target of rapamycin complex 1 (mTORC1) in preclinical models of disease, where it reduces activity in models of epilepsies and cancer and increases it in models of multiple sclerosis (MS) and psychosis. Here, we investigate the effects of phytocannabinoids on mTORC1 and define a molecular mechanism. EXPERIMENTAL APPROACH: A novel mechanism for phytocannabinoids was identified using the tractable model system, Dictyostelium discoideum. Using mouse embryonic fibroblasts, we further validate this new mechanism of action. We demonstrate clinical relevance using cells derived from healthy individuals and from people with MS (pwMS). KEY RESULTS: Both CBD and the more abundant cannabigerol (CBG) enhance mTORC1 activity in D. discoideum. We identify a mechanism for this effect involving inositol polyphosphate multikinase (IPMK), where elevated IPMK expression reverses the response to phytocannabinoids, decreasing mTORC1 activity upon treatment, providing new insight on phytocannabinoids' actions. We further validated this mechanism using mouse embryonic fibroblasts. Clinical relevance of this effect was shown in primary human peripheral blood mononuclear cells, where CBD and CBG treatment increased mTORC1 activity in cells derived from healthy individuals and decreased mTORC1 activity in cells derived from pwMS. CONCLUSION AND IMPLICATIONS: Our findings suggest that both CBD and the abundant CBG differentially regulate mTORC1 signalling through a mechanism dependent on the activity of the upstream IPMK signalling pathway, with potential relevance to the treatment of mTOR-related disorders, including MS.

Longitudinally extensive transverse myelitis after Campylobacter jejuni enteritis.

Campylobacter jejuni infection is well-known to precipitate Guillain-Barré syndrome through an immune-mediated attack on the peripheral nervous system. Molecular mimicry between C. jejuni lipo-oligosaccharides on the surface of infectious agents and human gangliosides in the peripheral nerves induces cross-reactive immune responses. Although gangliosides also occur in the central nervous system (CNS), autoimmune CNS disorders rarely follow Cjejuni infections. However, longitudinally extensive transverse myelitis commonly has a parainfectious cause, triggered by a wide range of micro-organisms including viruses and bacteria. We report a patient who developed longitudinally extensive transverse myelitis after Cjejuni enteritis associated with antiganglioside antibodies.

Alemtuzumab treatment of multiple sclerosis: long-term safety and efficacy.

OBJECTIVES: Alemtuzumab is a newly licensed treatment of active relapsing-remitting multiple sclerosis (RRMS) in Europe, which in phase II and III studies demonstrated superior efficacy over ß-interferon in reducing disability progression over 2-3 years. In this observational cohort study, we sought to describe our longer-term experience of the efficacy and safety of alemtuzumab in active RRMS. METHODS: Clinical and laboratory data including serial Expanded Disability Status Scale (EDSS) assessments, from all 87 patients treated with alemtuzumab on investigator-led studies in Cambridge, UK, from 1999 to 2012, were collected. The occurrence of adverse events including secondary autoimmunity, malignancy and death, and pregnancy outcomes was recorded. Baseline variables including age, disease duration and relapse rate were compared in univariate and logistic regression analyses between groups with different disability outcomes. RESULTS: Over a median 7-year follow-up (range 33-144 months), most patients (52%) required just two cycles of alemtuzumab. In the remaining patients, relapses triggered re-treatment to a total of three cycles (36%), four cycles (8%) or five cycles (1%). Using a 6-month sustained accumulation of disability definition, 59/87 (67.8%) of patients had an improved or unchanged disability compared with baseline. By an area under the curve analysis, 52/87 (59.8%) patients had an overall improvement or stabilisation of disability. Higher baseline relapse rate was associated with worse long-term disability outcomes, with trends for longer disease duration and older age at first treatment. Secondary autoimmunity was the most frequent adverse event occurring in 41/86 (47.7%) patients, most commonly involving the thyroid gland. CONCLUSIONS: Alemtuzumab is associated with disease stabilisation in the majority of patients with highly active RRMS over an average seven-year follow-up. No new safety concerns arose over this extended follow-up.

Secondary autoimmune diseases following alemtuzumab therapy for multiple sclerosis.

Alemtuzumab is a humanized monoclonal antibody that causes prolonged lymphopenia. In a recent Phase III trial, alemtuzumab was shown to reduce both the annualized relapse rate and the rate of sustained accumulation of disability by over 70% when compared with IFN-ß1a. However, the drug is associated with thyroid autoimmunity in approximately a third of treated patients, as well as other secondary autoimmune conditions in smaller numbers. Secondary autoimmunity typically arises during reconstitution of the lymphocyte repertoire. Individuals with high baseline circulating levels of IL-21 are at highest risk of developing autoimmunity following treatment with alemtuzumab.

IL-27 mediates the response to IFN-ß therapy in multiple sclerosis patients by inhibiting Th17 cells.

Interferon (IFN)-ß is a commonly used therapy for relapsing remitting multiple sclerosis (RRMS). However its protective mechanism is still unclear and the failure of many patients to respond has not been explained. We have found that IFN-ß suppressed IL-23 and IL-1ß production and increased IL-10 production by human dendritic cells (DC) activated with the TLR2 and dectin-1 agonist zymosan. Furthermore, IFN-ß impaired the ability of DC to promote IL-17 production by CD4(+) T cells, but did not affect IFN-γ production. IFN-ß induced IL-27 expression by DC, and neutralisation of IL-27 abrogated the suppressive effects of IFN-ß on zymosan-induced IL-1 and IL-23 production and the generation of Th17 cells in vitro. Complementary in vivo studies in a mouse model showed that treatment with IFN-ß enhanced expression of IL-27, and reduced IL-17 in the CNS and periphery and attenuated the clinical signs of experimental autoimmune encephalomyelitis (EAE). In addition, the significant suppressive effect of IFN-ß on the ability of DC to promote Th17 cells was lost in cells from IL-27 receptor deficient mice. Finally, we showed that PBMC from non-responder RRMS patients produced significantly less IL-27 in response to IFN-ß than patients who responded to IFN-ß therapy. Our findings suggest that IFN-ß mediates its therapeutic effects in MS at least in part via the induction of IL-27, and that IL-27 may represent an alternative therapy for MS patients that do not respond to IFN-ß.

CD39+Foxp3+ regulatory T Cells suppress pathogenic Th17 cells and are impaired in multiple sclerosis.

Despite the fact that CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg cells) play a central role in maintaining self-tolerance and that IL-17-producing CD4(+) T cells (Th17 cells) are pathogenic in many autoimmune diseases, evidence to date has indicated that Th17 cells are resistant to suppression by human Foxp3(+) Treg cells. It was recently demonstrated that CD39, an ectonucleotidase which hydrolyzes ATP, is expressed on a subset of human natural Treg cells. We found that although both CD4(+)CD25(high)CD39(+) and CD4(+)CD25(high)CD39(-) T cells suppressed proliferation and IFN-gamma production by responder T cells, only the CD4(+)CD25(high)CD39(+), which were predominantly FoxP3(+), suppressed IL-17 production, whereas CD4(+)CD25(high)CD39(-) T cells produced IL-17. An examination of T cells from multiple sclerosis patients revealed a normal frequency of CD4(+)CD25(+)CD127(low)FoxP3(+), but interestingly a deficit in the relative frequency and the suppressive function of CD4(+)CD25(+)CD127(low)FoxP3(+)CD39(+) Treg cells. The mechanism of suppression by CD39(+) Treg cells appears to require cell contact and can be duplicated by adenosine, which is produced from ATP by the ectonucleotidases CD39 and CD73. Our findings suggest that CD4(+)CD25(+)Foxp3(+)CD39(+) Treg cells play an important role in constraining pathogenic Th17 cells and their reduction in multiple sclerosis patients might lead to an inability to control IL-17 mediated autoimmune inflammation.

Reactivation of BK polyomavirus in patients with multiple sclerosis receiving natalizumab therapy.

Natalizumab therapy in multiple sclerosis has been associated with JC polyomavirus-induced progressive multifocal leucoencephalopathy. We hypothesized that natalizumab may also lead to reactivation of BK, a related human polyomavirus capable of causing morbidity in immunosuppressed groups. Patients with relapsing remitting multiple sclerosis treated with natalizumab were prospectively monitored for reactivation of BK virus in blood and urine samples, and for evidence of associated renal dysfunction. In this cohort, JC and BK DNA in blood and urine; cytomegalovirus (CMV) DNA in blood and urine; CD4 and CD8 T-lymphocyte counts and ratios in peripheral blood; and renal function were monitored at regular intervals. BK subtyping and noncoding control region sequencing was performed on samples demonstrating reactivation. Prior to commencement of natalizumab therapy, 3 of 36 patients with multiple sclerosis (8.3%) had BK viruria and BK reactivation occurred in 12 of 54 patients (22.2%). BK viruria was transient in 7, continuous in 2 patients, and persistent viruria was associated with transient viremia. Concomitant JC and CMV viral loads were undetectable. CD4:CD8 ratios fluctuated, but absolute CD4 counts did not fall below normal limits. In four of seven patients with BK virus reactivation, transient reductions in CD4 counts were observed at onset of BK viruria: these resolved in three of four patients on resuppression of BK replication. No renal dysfunction was observed in the cohort. BK virus reactivation can occur during natalizumab therapy; however, the significance in the absence of renal dysfunction is unclear. We propose regular monitoring for BK reactivation or at least for evidence of renal dysfunction in patients receiving natalizumab.

The patient knows best: significant change in the physical component of the Multiple Sclerosis Impact Scale (MSIS-29 physical).

AIM: The aims of this study were to determine the reliability, responsiveness and minimally important change score of the Multiple Sclerosis Impact Scale (MSIS)-29 physical using the Expanded Disability Status Scale (EDSS) as an anchor measure. METHODS: 214 patients with multiple sclerosis (MS) (EDSS 0-8.5) had concurrent MSIS-29 and EDSS assessments at baseline and at up to 4 years of follow-up. RESULTS: 116 patients had unchanged EDSS scores. Stability of the MSIS-29 physical (mean change 0.1 points) was better in the 85 patients with EDSS 0-5.0 than in the 31 patients with EDSS 5.5-8.5 in whom the MSIS-29 physical score fell by 8 points, a response shift phenomenon. A floor effect for the MSIS-29 was observed in 5% of stable patients at both time points. 98 patients experienced EDSS change with moderately strong statistically significant correlations between change scores in the EDSS and the MSIS-29 physical (r = 0.523, p<0.0001). Effect sizes for MSIS-29 physical change were moderate to large. Using receiver operating characteristic curves, the MSIS-29 change score which produced a combination of optimal sensitivity and specificity was chosen for both EDSS ranges. For EDSS range 5.5-8, a change score of 8 had a sensitivity of 87% and specificity of 67%. For EDSS 0-5.0, a change score of 7 had a sensitivity of 78% and a specificity of 51%. CONCLUSIONS: The MSIS-29 physical performs well over time, and is suitable for use in trials; a minimal change score of 8 points in the MSIS-29 is clinically significant.