Peter I. Karachunski

Prevention of experimental myasthenia gravis by nasal administration of synthetic acetylcholine receptor T epitope sequences.

T cell tolerization prevents and improves T cell-mediated experimental autoimmune diseases. We investigated here whether similar approaches could be used for antibody (Ab)-mediated autoimmune diseases. Myasthenia gravis, caused by IgG Ab against muscle acetylcholine receptor (AChR), is perhaps the best characterized of them. We used an animal model, experimental myasthenia gravis induced in C57Bl/6 mice by immunization with Torpedo acetylcholine receptor (TAChR), to demonstrate that nasal administration of synthetic sequences of the TAChR alpha-subunit- forming epitopes recognized by anti-TAChR CD4+ T helper cells (residues alpha150-169, alpha181-200, and alpha360-378), given before and during immunization with TAChR, causes decreased CD4+ responsiveness to those epitopes and to TAChR, reduced synthesis of anti-TAChR Ab, and prevented experimental myasthenia gravis. These effects were not induced by nasal administration of synthetic epitopes of diphtheria toxin. Secretion of IL-2, IL-4, and IL-10 by spleen T cells from TAChR immunized mice, in response to challenge with TAChR in vitro, indicated that in sham-tolerized mice only Th1 cells responded to TAChR, while peptide-treated mice had also an AChR-specific Th2 response. The TAChR peptide treatment induced also in vitro anergy to the TAChR of the spleen T cells, which was reversed by IL-2.

Absence of IFN-γ or IL-12 Has Different Effects on Experimental Myasthenia Gravis in C57BL/6 Mice

Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). Th1 cells facilitate EMG development. IFN-γ and IL-12 induce Th1 responses: we investigated whether these cytokines are necessary for EMG development. We immunized wild-type (WT) C57BL/6 mice and IFN-γ and IL-12 knockout mutants (IFN-γ−/−, IL-12−/−) with Torpedo AChR (TAChR). WT and IFN-γ−/− mice developed EMG with similar frequency, IL-12−/−mice were resistant to EMG. All strains synthesized anti-AChR Ab that were not IgM or IgE. WT mice had anti-AChR IgG1, IgG2b, and IgG2c, IFN-γ−/− mice had significantly less IgG2c, and IL-12−/− mice less IgG2b and IgG2c. All mice had IgG bound to muscle synapses, but only WT and IFN-γ−/− mice had complement; WT mice had both IgG2b and IgG2c, IFN-γ−/− only IgG2b, and IL-12−/− neither IgG2b nor IgG2c. CD4+ cells from all AChR-immunized mice proliferated in response to AChR and recognized similar epitopes. After stimulation with TAChR, CD4+ cells from IFN-γ−/− mice secreted less IL-2 and similar amounts of IL-4 and IL-10 as WT mice. CD4+ cells from IL-12−/− mice secreted less IFN-γ, but more IL-4 and IL-10 than WT mice, suggesting that they developed a stronger Th2 response to TAChR. The EMG resistance of IL-12−/− mice is likely due to both reduction of anti-TAChR Ab that bind complement and sensitization of modulatory Th2 cells. The reduced Th1 function of IFN-γ−/− mice does not suffice to reduce all complement-fixing IgG subclasses, perhaps because as in WT mice a protective Th2 response is missing.

Motor and cognitive assessment of infants and young boys with Duchenne Muscular Dystrophy: results from the Muscular Dystrophy Association DMD Clinical Research Network

Therapeutic trials in Duchenne Muscular Dystrophy (DMD) exclude young boys because traditional outcome measures rely on cooperation. The Bayley III Scales of Infant and Toddler Development (Bayley III) have been validated in developing children and those with developmental disorders but have not been studied in DMD. Expanded Hammersmith Functional Motor Scale (HFMSE) and North Star Ambulatory Assessment (NSAA) may also be useful in this young DMD population. Clinical evaluators from the MDA-DMD Clinical Research Network were trained in these assessment tools. Infants and boys with DMD (n = 24; 1.9 ± 0.7 years) were assessed. The mean Bayley III motor composite score was low (82.8 ± 8; p ≤ .0001) (normal = 100 ± 15). Mean gross motor and fine motor function scaled scores were low (both p ≤ .0001). The mean cognitive comprehensive (p=.0002), receptive language (p ≤ .0001), and expressive language (p = .0001) were also low compared to normal children. Age was negatively associated with Bayley III gross motor (r = -0.44; p = .02) but not with fine motor, cognitive, or language scores. HFMSE (n=23) showed a mean score of 31 ± 13. NSAA (n = 18 boys; 2.2 ± 0.4 years) showed a mean score of 12 ± 5. Outcome assessments of young boys with DMD are feasible and in this multicenter study were best demonstrated using the Bayley III.

Myasthenia in SCID mice grafted with myasthenic patient lymphocytes Role of CD4 and CD8 cells

Objectives: Acetylcholine receptor (AChR)-specific CD4+ cells are present in MG patients, and synthesis of the high-affinity immunoglobulin G (IgG) autoantibodies (autoAb) against the muscle AChR that causes MG symptoms requires intervention of CD4+ cells. The role of CD4+ cells in MG pathogenesis has been postulated but never proven. MG patients do not have anti-AChR cytotoxic phenomena, and it has been assumed that CD8+ cells do not have a pathogenic role in MG. However, CD8+ cells may facilitate rodent experimental MG, raising the possibility that CD8+ cells might be necessary also in MG. In this study we examined whether CD4+ and CD8+ cells play a role in the pathogenesis of MG and whether CD4+ cells specific for AChR epitope sequences recognized by most MG patients (“universal” epitopes) drive the synthesis of pathogenic antibodies. Methods: First we characterized a chimeric human-mouse model of MG in severe combined immunodeficiency (SCID) mice engrafted with blood lymphocytes (BL) from MG patients. We used that model to determine whether CD4+ and CD8+ cells are necessary for transfer of MG symptoms. We engrafted SCID mice intraperitoneum with BL from 19 MG patients and 5 healthy controls. We engrafted some mice with either BL, BL depleted in CD4+ or CD8+ cells from the same patient, or CD4+ depleted BL reconstituted with CD4+ T cells from the same patient, specific for “universal” AChR epitopes or for two unrelated antigens, tetanus and diphtheria toxoids. We tested the mice for myasthenic symptoms for 7 to 18 weeks. Results: Mice transplanted with BL, or CD8+ depleted BL, or CD4+-depleted BL reconstituted with anti-AChR CD4+ cells from MG patients frequently developed myasthenic weakness. The mice had human anti-AChR Ab in the serum and bound to muscle AChR. Mice transplanted with BL from controls, or CD4+-depleted BL from MG patients, or CD4+-depleted BL from an MG patient reconstituted with CD4+ cells specific for tetanus or diphtheria toxoids did not develop myasthenic weakness or anti-AChR Ab. Conclusions: CD4+ cells are necessary for MG pathogenesis; CD8+ cells may not be. CD4+ cells specific for “universal” AChR epitopes help the synthesis of pathogenic Ab.

Ataluren in patients with nonsense mutation Duchenne muscular dystrophy (ACT DMD): a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial

BACKGROUND Duchenne muscular dystrophy (DMD) is a severe, progressive, and rare neuromuscular, X-linked recessive disease. Dystrophin deficiency is the underlying cause of disease; therefore, mutation-specific therapies aimed at restoring dystrophin protein production are being explored. We aimed to assess the efficacy and safety of ataluren in ambulatory boys with nonsense mutation DMD. METHODS We did this multicentre, randomised, double-blind, placebo-controlled, phase 3 trial at 54 sites in 18 countries located in North America, Europe, the Asia-Pacific region, and Latin America. Boys aged 7-16 years with nonsense mutation DMD and a baseline 6-minute walk distance (6MWD) of 150 m or more and 80% or less of the predicted normal value for age and height were randomly assigned (1:1), via permuted block randomisation (block size of four) using an interactive voice-response or web-response system, to receive ataluren orally three times daily (40 mg/kg per day) or matching placebo. Randomisation was stratified by age (<9 years vs ≥9 years), duration of previous corticosteroid use (6 months to <12 months vs ≥12 months), and baseline 6MWD (<350 m vs ≥350 m). Patients, parents and caregivers, investigational site personnel, PTC Therapeutics employees, and all other study personnel were masked to group allocation until after database lock. The primary endpoint was change in 6MWD from baseline to week 48. We additionally did a prespecified subgroup analysis of the primary endpoint, based on baseline 6MWD, which is reflective of anticipated rates of disease progression over 1 year. The primary analysis was by intention to treat. This study is registered with ClinicalTrials.gov, number NCT01826487. FINDINGS Between March 26, 2013, and Aug 26, 2014, we randomly assigned 230 patients to receive ataluren (n=115) or placebo (n=115); 228 patients comprised the intention-to-treat population. The least-squares mean change in 6MWD from baseline to week 48 was -47·7 m (SE 9·3) for ataluren-treated patients and -60·7 m (9·3) for placebo-treated patients (difference 13·0 m [SE 10·4], 95% CI -7·4 to 33·4; p=0·213). The least-squares mean change for ataluren versus placebo in the prespecified subgroups was -7·7 m (SE 24·1, 95% CI -54·9 to 39·5; p=0·749) in the group with a 6MWD of less than 300 m, 42·9 m (15·9, 11·8-74·0; p=0·007) in the group with a 6MWD of 300 m or more to less than 400 m, and -9·5 m (17·2, -43·2 to 24·2; p=0·580) in the group with a 6MWD of 400 m or more. Ataluren was generally well tolerated and most treatment-emergent adverse events were mild to moderate in severity. Eight (3%) patients (n=4 per group) reported serious adverse events; all except one event in the placebo group (abnormal hepatic function deemed possibly related to treatment) were deemed unrelated to treatment. INTERPRETATION Change in 6MWD did not differ significantly between patients in the ataluren group and those in the placebo group, neither in the intention-to-treat population nor in the prespecified subgroups with a baseline 6MWD of less than 300 m or 400 m or more. However, we recorded a significant effect of ataluren in the prespecified subgroup of patients with a baseline 6MWD of 300 m or more to less than 400 m. Baseline 6MWD values within this range were associated with a more predictable rate of decline over 1 year; this finding has implications for the design of future DMD trials with the 6-minute walk test as the endpoint. FUNDING PTC Therapeutics.

Subcutaneous administration of T-epitope sequences of the acetylcholine receptor prevents experimental myasthenia gravis

Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). The s.c. administration to C57B1/6 mice of synthetic AChR CD4+ epitopes, before and during AChR immunization, reduced the epitope-specific CD4+ responses and the anti-AChR Ab synthesis, and prevented EMG. The s.c. administration of solubilized AChR had effects similar to those of peptide treatment. Sham-tolerized mice had only Th1 anti-AChR cells, whereas peptide-treated mice had also Th2 cells, and Th2-induced anti-peptide Ab. Established EMG was not affected by s.c. peptide treatment, whereas it worsened after s.c. administration of solubilized AChR.

Interleukin-4 deficiency facilitates development of experimental myasthenia gravis and precludes its prevention by nasal administration of CD4+ epitope sequences of the acetylcholine receptor.

Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). We investigated EMG in interleukin (IL)-4 knock out B6 (KO) mice, that lack Th2 cells. EMG was more frequent in KO than in wild type B6 mice. KO and B6 mice developed similar amounts of anti-AChR antibodies. They were IgG2a and IgG2b in KO mice, IgG1 and IgG2b in B6 mice. CD4+ cells from KO and B6 mice recognized the same AChR epitopes. Nasal administration of synthetic AChR CD4+ epitopes reduced antibody synthesis and prevented EMG in B6, not in KO mice. Thus, Th2 cells may have protective functions in EMG.

Transgenic Expression of IL-10 in T Cells Facilitates Development of Experimental Myasthenia Gravis

Ab to the acetylcholine receptor (AChR) cause experimental myasthenia gravis (EMG). Th1 cytokines facilitate EMG, whereas Th2 cytokines might be protective. IL-10 inhibits Th1 responses but facilitates B cell proliferation and Ig production. We examined the role of IL-10 in EMG by using wild-type (WT) C57BL/6 mice and transgenic (TG) C57BL/6 mice that express IL-10 under control of the IL-2 promoter. We immunized the mice with doses of AChR that cause EMG in WT mice or with low doses ineffective at causing EMG in WT mice. After low-dose AChR immunization, WT mice did not develop EMG and had very little anti-AChR serum Ab, which were mainly IgG1, whereas TG mice developed EMG and had higher levels of anti-AChR serum Ab, which were mainly IgG2, in addition to IgG1. At the higher doses, TG mice developed EMG earlier and more frequently than WT mice and had more serum anti-AChR Ab. Both strains had similar relative serum concentrations of anti-AChR IgG subclasses and IgG and complement at the muscle synapses. CD8+-depleted splenocytes from all AChR-immunized mice proliferated in the presence of AChR and recognized a similar epitope repertoire. CD8+-depleted splenocytes from AChR-immunized TG mice stimulated in vitro with AChR secreted significantly more IL-10, but less of the prototypic Th1 cytokine IFN-γ, than those from WT mice. They secreted comparable amounts of IL-4 and slightly but not significantly reduced amounts of IL-2. This suggests that TG mice had reduced activation of anti-Torpedo AChR Th1 cells, but increased anti-AChR Ab synthesis, that likely resulted from IL-10-mediated stimulation of anti-AChR B cells. Thus, EMG development is not strictly dependent on Th1 cell activity.

MECHANISMS BY WHICH THE I-ABM12 MUTATION INFLUENCES SUSCEPTIBILITY TO EXPERIMENTAL MYASTHENIA GRAVIS : A STUDY IN HOMOZYGOUS AND HETEROZYGOUS MICE

The I‐Abm12 mutation in C57B1/6 (B6) mice yields the B6. C‐H‐2bm12 (bm12) strain, which is resistant to Experimental Myasthenia Gravis (EMG) induced by immunization with Torpedo acetylcholine receptor (TAChR), while the parental B6 strain is highly susceptible to EMG. CD4+ cells from bm12 mice immunized with TAChR do not recognize three sequence regions of the TAChR Q subunit which dominate the CD4+ cell sensitization in B6 mice. We immunized with TAChR bm12, B6 and (bm12B6)Fl mice. B6 and F1 mice developed EMG with comparable frequency. Their CD4+ cells recognized the same TAChR α subunit peptide sequences (Tα150–169, Tα181–200 and Tα360–378). CD4+ cells from TAChR‐sensitized Fl mice were challenged with TAChR and α subunit epitope peptides, using F1, B6 or bml 2 APC. B6 and F1 APC presented all these Ag efficiently, while bm 12 APC presented TAChR and peptide Tα150–169 poorly and erratically. Anti‐TAChR and anti‐α subunit epitope CD4+ lines propagated from Fl and B6 mice had similar TcR Vβ usage. All lines but those specific for the sequence Tα150–169 had unrestricted Vβ usage. Anti‐Tα150–169 lines from both B6 and Fl mice had a strong preferential usage of Vβ6. Anti‐Tα150–169 lines from Fl mice had also a slightly higher Vβ14 usage. B6, bm12 and Fl mice developed similar anti‐TAChR Ab titres, and had Ab bound to muscle AChR in comparable amounts. Therefore EMG resistance of bm12 mice must be due to a subtle shift in the anti‐AChR Ab repertoire, and absence of special Ab able to cause destruction and/or dysfunction of muscle AChR. This is probably related to the absence of CD4+ cells sensitized to epitopes within the sequence Tα 150–160, consequent to the inability of the I‐Abm12 molecule to present this sequence.

Outcome reliability in non-Ambulatory Boys/Men with duchenne muscular dystrophy

Introduction: Therapeutic trials in Duchenne muscular dystrophy (DMD) often exclude non‐ambulatory individuals. Here we establish optimal and reliable assessments in a multicenter trial. Methods: Non‐ambulatory boys/men with DMD (N = 91; 16.7 ± 4.5 years of age) were assessed by trained clinical evaluators. Feasibility (percentage completing task) and reliability [intraclass correlation coefficients (ICCs) between morning and afternoon tests] were measured. Results: Forced vital capacity (FVC), assessed in all subjects, showed a mean of 47.8 ± 22% predicted (ICC 0.98). Brooke Upper Extremity Functional Rating (Brooke) and Egen Klassifikation (EK) scales in 100% of subjects showed ICCs ranging from 0.93 to 0.99. Manual muscle testing, range of motion, 9‐hole peg test, and Jebsen‐Taylor Hand Function Test (JHFT) demonstrated varied feasibility (99% to 70%), with ICCs ranging from 0.99 to 0.64. We found beneficial effects of different forms of corticosteroids for the Brooke scale, percent predicted FVC, and hand and finger strength. Conclusions: Reliable assessment of non‐ambulatory boys/men with DMD is possible. Clinical trials will have to consider corticosteroid use. Muscle Nerve 51: 522–532, 2015