Philip R. Deitiker

Antigen mimicry in autoimmune disease. Can immune responses to microbial antigens that mimic acetylcholine receptor act as initial triggers of Myasthenia gravis

Myasthenia gravis (MG) is an autoimmune disease caused by autoantibodies against self acetylcholine receptor (AChR). Although a great deal of information is known about the molecular and cellular parameters of the disease, its initial trigger is not known. In order to study the possibility of the involvement of microbial antigens that mimic AChR in triggering MG, we have searched the microbial proteins in the data bank for regions that are similar in structure to the regions of human (h) AChR alpha chain recognized by autoAbs in MG patients. Hundreds of candidate structures on a large number of bacterial and viral proteins were identified. To test the feasibility of the idea, we synthesized four microbial regions similar to each of the major autodeterminants of hAChR (alpha12-27, alpha111-126, alpha122-138, alpha182-200) and investigated their ability to bind autoAbs in MG and normal sera controls. It was found that MG sera recognized a significant number of these microbial regions. The results indicate that in some MG cases immune responses to microbial antigens may cross-react with self antigen (in this case hAChR) and could constitute initial triggers of the disease.

Human T-cell responses to botulinum neurotoxin Proliferative responses in vitro of lymphocytes from botulinum neurotoxin A-treated movement disorder patients

We determined the T-cell responses against botulinum neurotoxin type A (BoNT/A) and tetanus toxin (TeNT) of peripheral blood lymphocytes from 95 BoNT-treated patients and 63 non-treated control subjects. The patient group included 80 cervical dystonia and 15 other movement disorder cases. Positive T-cell responses to BoNT/A were detected in 70% of the treated patients, and in only 3% of controls. T-cell responses of BoNT-treated patients against BoNT/A did not differ between patients who were clinically responsive and those who had become non-responsive to the treatment. BoNT-treated patients gave significantly higher in vitro T-cell responses to TeNT than did the controls.

Association with HLA DQ of early onset myasthenia gravis in Southeast Texas region of the United States.

Forty‐four Caucasian American myasthenia gravis (MG) patients from Southeast Texas underwent high resolution HLA DQ analysis. For the majority of patients who were late onset or male, no significant associations with DQ were observed. However, associations with DQ increased in female patients and early onset patients. At the allele level, DQB1 *0503, *0604, *0502 and *0402 collectively contributed to a positive association of the DQ locus with early onset MG (EOMG), while individually failing to show significant association. At DQ level, the novel haplotype DQA1*0401:DQB1*0201 was the primary factor in the association of combined DQ loci with early onset. In addition, *0104:*0503, *0102:*0604, *0102:*0502 and *0303:*0402 collectively contributed to the positive association of the haplotype loci. DR3‐DQ2.5cis, a well known risk factor for MG in Western Eurasia, was not found associated with disease in any group. For typical EOMG [early onset, no thymoma, anti‐acetylcholine receptor (AChR) antibody (Ab) positive] no association with DQA1 locus was found, however DQB1*0604 demonstrated an ‘uncorrected’ positive association. A few DQ haplotype (DQA1:DQB1) were positively associated with typical EOMG; a positive individual association for *0401:*0201 was complimented by the contributions of *0102:*0604 and *0303:*0402 haplotypes. A small minority of patients that were atypical and EOMG had a strong genetic association with DQA1*0104:DQB1*0503, the group included an anti‐MuSK Ab positive and an anti‐AChR negative patient. This report finds common ground with European studies regarding MuSK association; however similarities in association for typical early onset disease resembled HLA risk factors in East Asia and Southern Europe.

Association of HLA Class II alleles and haplotypes with cervical dystonia: HLA DR13-DQ6 (DQB1*0604) homozygotes are at greatly increased risk of cervical dystonia in Caucasian Americans

An unanticipated discovery was made while examining genetics of the immune response in patients treated with botulinum neurotoxin (BoNT), which included cervical dystonia (CD) patients. Initial examination of HLA DQA1:DQB1 frequencies revealed an unexpectedly high number of DQA1*0102:DQB1*0604 homozygotes (hz) in the CD patients. We typed the BoNT-treated CD Caucasian subset for HLA-DRB1, DQA1, and DQB1 and succeeded in typing HLA-DRB1, -DQA1, and -DQB1 for 75 of the patients. Two statistical methods found the DQB1 locus associated with CD and one method found a probable association of DQB1*0604. Examination of the allele and haplotype pairing indicated that DQB1*0604 hz comprised most to all of the positive association. Other than this genotype, one other allele, DQB1*0504 contributes to the association of the DQB1 locus. These findings indicate a probable infectious and/or autoimmune component in some CD patients. However, longer distance associations within an extended and conserved DQB1*0604 bearing haplotype leave a possibility that a locus proximal to DQB1 might be involved.

Subtle differences in HLA DQ haplotype-associated presentation of AChR α-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 α-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 Students T-test analyses on DQ isoform bearing groups revealed that high responses to peptide α34–49 were associated with A1*0102:B1*0602/0604, A1*0301:B1*0302 and A1*0401/0303:B1*0301. Peptide α146–162 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.

Assembly-dependent phosphorylation of myosin and paramyosin of native thick filaments in Caenorhabdiris elegans

Phosphorylation of the thick filament proteins myosin and paramyosin was studied in Caenorhabditis elegans. We have incubated partially purified, native thick filaments with [gamma 32P] ATP in the presence of 50-750 mM NaCl, pH 6.5-8.0. Myosin heavy chain and paramyosin were phosphorylatable only upon solubilization at 450 mM and higher NaCl concentrations. Under conditions preserving native structures, no phosphorylation of these proteins occurred. The phosphorylation required Mg2+ but was unaffected by cAMP, cGMP or Ca2+. The specific inhibitor of cAMP and cGMP kinase catalytic subunits, H8, inhibits the activity. Sedimentation experiments show that the kinase may associate with but is not an intrinsic component of thick filaments. In C. elegans, phosphorylation by the thick filament associated activity of myosin and paramyosin is dependent upon the state of their assembly.

Suppression of experimental myasthenia gravis by monoclonal antibodies against MHC peptide region involved in presentation of a pathogenic T-cell epitope

We have prepared monoclonal antibodies (mAbs) against an antigen-binding region of I-A, region 62-76 of I-Abeta(b), which is involved in the T-cell participation in the pathogenesis of EAMG. The mAbs reacted with its parent molecules and inhibited the proliferation of disease-related T-cells. Passive transfer of these mAbs suppressed the occurrence of clinical EAMG, which was accompanied by decreased T-cell and Ab responses to tAChR. The results indicated that blocking the function of disease-related MHC by targeting a disease-associated region on MHC molecules could be an effective, straightforward and feasible strategy for immunointervention in MG.

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.

Human T-cell responses to botulinum neurotoxin. Responses in vitro of lymphocytes from patients with cervical dystonia and/or other movement disorders treated with BoNT/A or BoNT/B

We have previously reported that botulinum neurotoxin type A (BoNT/A)-specific T-cell responses occur in a majority of patients treated with botulinum neurotoxins (BoNT). In this study, we first determined if T-cell responses against BoNT/A and tetanus toxin (TeNT) differ between cervical dystonia (CD) patients and other movement disorder cases. Secondly, we have examined in CD cases the treatment parameters that may have an effect on the T-cell responses against BoNT/A. We found that T-cell responses to BoNT/A were significantly higher in patients with CD than in those with other movement disorders. An increase in TeNT T-cell response in CD was observed when compared to un-treated controls. CD patients who were injected with BoNT/B mounted higher responses to BoNT/A than patients treated with BoNT/A only. Frequent injections (more than 2.1/year) were associated with a significantly higher T-cell response to BoNT/A in CD. T cell responses to BoNT/A did not differ between CD patients who had clinically responsive and non-responsive status at the time of enrollment.

Vaccination with a MHC class II peptide in Alum and inactive pertussis strongly ameliorates clinical MG in C57BL/6 mice

We have investigated the efficacy of immunization against peptides from predisposing MHC class II molecules in human-compatible adjuvants for ameliorating experimental autoimmune myasthenia gravis (EAMG). C57BL/6 mice were immunized three times with the peptide I-Abetab62-76 in Alum+killed pertussis organisms (PT) prior to two injections with tAChR. The treatment greatly reduced the occurrence and severity of clinical MG relative to controls that received saline/Alum+PT or none. It also reduced antibody and T-cell responses against tAChR. The results have important implications for the possible immunotherapy of MG by targeting disease-associated MHC.