Injection of inactive Bordetella pertussis and complete Freund's adjuvant with Torpedo californica AChR increases the occurrence of experimental autoimmune myasthenia gravis in C57BL/6 mice.

An animal model of myasthenia gravis (MG), termed experimental autoimmune MG (EAMG), is an important tool for investigations of disease mechanisms and/or methods of treatment for this disease. EAMG can be induced in C57BL/6 (B6, H-2b) mice by 2-3 times injections at 4 weeks intervals with Torpedo californica (t) acetylcholine receptor (AChR) in complete Freund's adjuvant (CFA). However, the protocol especially with a two-injection schedule occasionally produces a poor incidence of EAMG. We have investigated the efficacy of the additional adjuvant, inactive organisms of Bordetella pertussis (iBP), on the induction with a two-injection schedule. In a group immunized with tAChR in CFA + iBP, 76% of mice developed EAMG (average grade in exercise test, 1.02). Whereas, 46% of mice were found EAMG-positive (average grade, 0.73) in a group injected with tAChR/CFA alone. Thus, the combined use of CFA and iBP significantly increased both the occurrence and severity of clinical MG in the immunized mice. This was accompanied by higher antibody (Ab) and T-cell responses to tAChR. The effect on disease occurrence of the iBP use in a three-injection protocol was also described.

Sharpening the tandem walking test for screening peripheral neuropathy.

OBJECTIVE: Few tests of functional motor behavior are useful for rapidly screening people for lower extremity peripheral neuropathy. The goal of this study was to improve the widely used tandem walking (TW) test. METHODS: We tested "normal" (control) adult and ambulatory patients with peripheral neuropathy (PN) with their eyes open and eyes closed while they performed TW on industrial carpeting in sock-covered feet. Each subject wore a torso-mounted inertial motion unit to measure kinematic data. The data of subjects with PN also were compared with historical data on patients with vestibular impairments. RESULTS: The normal and PN groups differed significantly on TW and on the number of steps completed. PN and vestibular impairments data also differed significantly on both visual conditions. Kinematic data showed that patients with PN were more unstable than normal patients in the group. For the number of steps taken during the eyes open condition, receiver operating characteristic (ROC) values were only 0.81 and for the number of steps taken during the eyes closed condition, ROC values were 0.88. Although not optimal, this ROC value is better. Sensitivity and specificity at a cutoff of two steps were 0.81 and 0.92, respectively, and at a cutoff of three steps were 0.86 and 0.75, respectively. ROC values for kinematic data were <0.8, and when combined with the ROC value for the number of steps, the total ROC value did not improve appreciably. CONCLUSIONS: Although not ideal for screening patients who may have PN, counting the number of steps during TW is a quick and useful clinical test. TW is most sensitive to patients with PN when they are tested with eyes closed.

T-cell recognition of acetylcholine receptor provides a reliable means for monitoring autoimmunity to acetylcholine receptor in antibody-negative myasthenia gravis patients.

Myasthenia gravis (MG) is an autoimmune disease usually associated with autoantibodies (auto-Abs) against nicotinic acetylcholine receptor (AChR). Some MG patients appear negative for anti-AChR Abs (seronegative), and a fraction of these have auto-Abs against muscle-specific kinase. The remaining patients, although displaying MG symptoms, show no detectable auto-Abs. We describe here a possible association of a rare human leukocyte antigen (HLA)-DQ type and AChR Ab-negative MG. We also found that the majority of seronegative patients exhibit an anti-AChR autoimmune T lymphocyte response. We investigated the existence of AChR-reactive T cells in peripheral blood lymphocytes from seronegative patients by their proliferative responses against a mixture of 18 overlapping synthetic peptides encompassing the extracellular part of human AChR α-chain. Of the 10 samples, eight exhibited positive T-cell proliferative responses against the peptide mixtures. The proliferative assay was equally efficient using a mixture of eight peptides frequently recognized by MG T cells. This T-cell proliferative assay should provide a reliable method for monitoring seronegative MG patients.

Expanded CTG repeats within the DMPK 3' UTR causes severe skeletal muscle wasting in an inducible mouse model for myotonic dystrophy.

Severe skeletal muscle wasting is the most debilitating symptom experienced by individuals with myotonic dystrophy type 1 (DM1). We present a DM1 mouse model with inducible and skeletal muscle-specific expression of large tracts of CTG repeats in the context of DMPK exon 15. These mice recapitulate many findings associated with DM1 skeletal muscle, such as CUG RNA foci with Muscleblind-like 1 (MBNL1) protein colocalization, misregulation of developmentally regulated alternative splicing events, myotonia, characteristic histological abnormalities, and increased CUGBP1 protein levels. Importantly, this DM1 mouse model recapitulates severe muscle wasting, which has not been reported in models in which depletion of MBNL1 is the main feature. Using these mice, we discovered previously undescribed alternative splicing events that are responsive to CUGBP1 and not MBNL, and these events were found to be misregulated in individuals with DM1. Our results indicate that increased CUGBP1 protein levels are associated with DMPK-CUG RNA expression, suggesting a role for CUGBP1-specific splicing or cytoplasmic functions in muscle wasting.

Subtle differences in HLA DQ haplotype-associated presentation of AChR alpha-chain peptides may suffice to mediate myasthenia gravis.

The HLA DQA1 and DQB1 alleles were determined on a set of 24 myasthenia gravis patients that had previously been examined for their T-cell proliferative responses to the 18 overlapping peptides representing the extracellular domain of hAChR alpha-chain. Patient responses according to assumed cis or trans haplotypes were significantly higher in most cases relative to normal controls. Comparisons of in vitro peptide-stimulated T-cell responses of patient pairs which had DQA1:DQB1 in common displayed responses in tighter distribution relative to comparisons in which patient pairs did not share the same DQA1:DQB1 haplotype. Similar haplotypes, such as DQA1*0102:DQB1*0602 and DQA1*0102:DQB1*0604, tended to exhibit similar responses and were grouped according to this similarity. Modified F-test and Student's T-test analyses on DQ isoform bearing groups revealed that high responses to peptide alpha34-49 were associated with A1*0102:B1*0602/0604, A1*0301:B1*0302 and A1*0401/0303:B1*0301. Peptide alpha146-162 showed higher responses in A1*0301:B1*0302 group and moderate responses in A1*0401/0303:B1*0301 groups. Differences in the age of disease onset relative to DQ haplotypes were also observed. Groups of A1*0301:B1*0302, A1*0501:B1*0201 and A1*0102:B1*0604 showed earlier ages of disease onset relative to those of A1*0102:B1*0602 or A1*0505:B1*0301.

Myasthenia gravis induced in mice by immunization with the recombinant extracellular domain of rat muscle-specific kinase (MuSK).

UNLABELLED: Myasthenia gravis (MG) is mostly caused by anti-acetylcholine receptor (AChR) auto-antibodies (Abs). Such Abs are undetectable in 10-15% of MG patients, but many have anti-muscle-specific kinase (MuSK) Abs. We injected recombinant rat-MuSK extracellular domain in H-2(a), H-2(b), H-2(bm12) and H-2(d) mice. Certain strains exhibited exercise-induced fatigue, tremors, weight loss, and some died after 2-3 injections. Compound muscle action potentials showed decrement with low-frequency repetitive nerve stimulation. Miniature endplate potentials decreased, suggesting lower numbers of endplates functional AChRs. Myasthenic sera inhibited agrin-induced AChR aggregation in C2C12 myotubes. CONCLUSION: Anti-MuSK Abs induce MG, which might also result from blocking the agrin-signaling pathway.

Suppression by mAbs against DQB1 peptides of in vitro proliferation of AChR-specific T cells from myasthenia gravis patients.

It has been indicated that multiple genes, including HLA genes, are collectively involved in the susceptibility to myasthenia gravis (MG). DQB1 alleles represent one of those associated with MG. We have prepared B-cell hybridomas that produce mAbs against peptides corresponding to the tip of the MHC antigen-binding cavity (region 70-90) of alleles DQB1*02, *03, *05 and *06. The mAbs bound to DQ molecules isolated from cells. In the assays using peripheral blood lymphocytes (PBL) from patients with MG, the mAbs against peptides of the correlate HLA DQ sequences inhibited the in vitro proliferation of acetylcholine receptor (AChR)-specific T cells. The results indicate that the function of disease-related MHC alleles may be blocked by directly and selectively targeting the antigen-presenting region on these MHC molecules. The results also suggest that DQ molecules are one of those involved in the restriction of autoimmune anti-AChR responses in MG. The strategy could provide an effective means for immunointervention in MG. It may also potentially be adapted for down-regulation of undesirable immune responses such as in other autoimmune diseases, allergic reactions, or clinical conditions where immune responses to a therapeutic protein develop.

Defective neuromuscular synapses in mice lacking amyloid precursor protein (APP) and APP-Like protein 2.

Biochemical and genetic studies place the amyloid precursor protein (APP) at the center stage of Alzheimer's disease (AD) pathogenesis. Although mutations in the APP gene lead to dominant inheritance of familial AD, the normal function of APP remains elusive. Here, we report that the APP family of proteins plays an essential role in the development of neuromuscular synapses. Mice deficient in APP and its homolog APP-like protein 2 (APLP2) exhibit aberrant apposition of presynaptic marker proteins with postsynaptic acetylcholine receptors and excessive nerve terminal sprouting. The number of synaptic vesicles at presynaptic terminals is dramatically reduced. These structural abnormalities are accompanied by defective neurotransmitter release and a high incidence of synaptic failure. Our results identify APP/APLP2 as key regulators of structure and function of developing neuromuscular synapses.

Responses in vitro of peripheral blood lymphocytes from patients with myasthenia gravis to stimulation with human acetylcholine receptor alpha-chain peptides: analysis in relation to age, thymic abnormality, and ethnicity.

Peripheral blood lymphocytes (PBLs) were isolated from 24 patients with myasthenia gravis of three ethnic groups (Caucasian, African American, and Hispanic) and ten healthy individuals. We determined the in vitro proliferative responses of the PBL samples to each of 18 overlapping synthetic peptides corresponding to the entire main extracellular domain (residues 1-210) of the alpha-subunit of human acetylcholine receptor. The profiles of the T-cell responses (expressed in stimulation index [SI]) to the peptides varied among the 24 patient samples. There was a significant difference in the overall patient responses relative to controls toward 17 of 18 peptides. T cells from the patients gave responses greater than control mean SI + 4 standard deviation (Z(SI) > 4) to 2 approximately 9 peptides/sample. Six peptides, alpha 23-38, alpha 34-49, alpha 78-93, alpha 122-138, alpha 146-162, and alpha 182-198, were recognized with Z > 4 level by 42% to 58% of the patients' PBLs. The grouped patient responses, divided according to age, thymic diagnosis, or ethnicity, were compared with controls and with each other. Significant differences were observed between early- and late-onset cases in recognition of residues alpha 34-49 (p = 0.015) and alpha 78-93 (p = 0.053), and in recognition of residues alpha 12-27, alpha 56-71, alpha 134-150, and alpha 146-162 (0.0072 < p < 0.064) when two ethnic groups were compared with each other.

Reelin promotes peripheral synapse elimination and maturation.

Reelin is an extracellular protein that is crucial for layer formation in the embryonic brain. Here, we demonstrate that Reelin functions postnatally to regulate the development of the neuromuscular junction. Reelin is required for motor end-plate maturation and proper nerve-muscle connectivity, and it directly promotes synapse elimination. Unlike layer formation, neuromuscular junction development requires a function of Reelin that is not mediated by Disabled1 or very-low-density lipoprotein receptors and apolipoprotein E receptor 2 receptors but by a distinct mechanism involving its protease activity.

Immunoglobulin Fc gamma receptor promotes immunoglobulin uptake, immunoglobulin-mediated calcium increase, and neurotransmitter release in motor neurons.

Receptors for the Fc portion of immunoglobulin G (IgG; FcgammaRs) facilitate IgG uptake by effector cells as well as cellular responses initiated by IgG binding. In earlier studies, we demonstrated that amyotrophic lateral sclerosis (ALS) patient IgG can be taken up by motor neuron terminals and transported retrogradely to the cell body and can alter the function of neuromuscular synapses, such as increasing intracellular calcium and spontaneous transmitter release from motor axon terminals after passive transfer. In the present study, we examined whether FcgammaR-mediated processes can contribute to these effects of ALS patient immunoglobulins. F(ab')(2) fragments (which lack the Fc portion) of ALS patient IgG were not taken up by motor axon terminals and were not retrogradely transported. Furthermore, in a genetically modified mouse lacking the gamma subunit of the FcR, the uptake of whole ALS IgG and its ability to enhance intracellular calcium and acetylcholine release were markedly attenuated. These data suggest that FcgammaRs appear to participate in IgG uptake into motor neurons as well as IgG-mediated increases in intracellular calcium and acetylcholine release from motor axon terminals.