Presence Of Acetylcholine Receptor Research Articles

Simultaneous Presence of Acetylcholine Receptor and Muscle Specific Tyrosine Kinase Antibodies in Myasthenia Gravis

Myasthenia gravis (MG) is known as an autoimmune disorder which affects transmission in neuromuscular junction. The serologic tests used for diagnosis include acetylcholine receptor and muscle specific receptor tyrosine kinase antibodies. Studies often have reported that patients with formal antibody are negative for the latter one. However, very limited studies have reported positive anti-muscle specific receptor tyrosine kinase antibody in a small percentage of patients with acetylcholine receptor antibody. Here, we reported a young woman who was diagnosed with MG and had a rapid and progressive course of the disease. She was seropositive for both acetylcholine receptor and muscle-specific receptor tyrosine kinase antibodies simultaneously. However, she discharged from the hospital with good condition after treatment.

Seropositivity in Myasthenia Gravis as a Predictor of Response to Therapeutic Plasma Exchange

Background: In spite of the increasing availability of immunomodulatory treatments for myasthenia gravis (MG), little is known about factors that predict response to the treatment. We aim to study if the presence of acetylcholine receptor (AChR) antibodies may be one of the predictors of the response to the therapeutic plasma exchange (TPE) in patients with MG. Methods: The study was carried out in 78 patients with moderate to severe MG. They were divided into two groups: TPE group (62 patients) that included the patients who received TPE, and control (non-TPE) group who did not receive TPE and was only on medical treatment (16 patients). Patients in TPE group, then, were subdivided into sero-positive and sero-negative subgroups according to the presence or absence of AChR antibodies respectively. Scoring by Quantitative Myasthenia Gravis Scale (QMGS) was done before and 1 month after the TPE. Results: After 1 month of treatment, the change in QMGS was significantly higher in the TPE group than in the Non-TPE one (t = -6.406) (P < 0.0001). In the sero-positive group, the score of QMGS ranged initially from 19 to 34 (mean 26.48 ± 3.75) and after 1 month from 11 to 30 (mean 18.00 ± 4.45) with the median change of -32. The QMGS change was significantly greater in the sero-positive group than in the sero-negative one (t = -3.516) (P < 0.0001). Conclusions: As regards to the presence of AChR antibodies in patients with MG, both sero-positive and sero-negative groups responded to TPE but the sero-positive group had a better response. J Neurol Res. 2019;9(1-2):8-13 doi: https://doi.org/10.14740/jnr510

Seropositivity in Myasthenia Gravis as a Predictor of Response to Therapeutic Plasma Exchange

Background: In spite of the increasing availability of immunomodulatory treatments for myasthenia gravis (MG), little is known about factors that predict response to the treatment. We aim to study if the presence of acetylcholine receptor (AChR) antibodies may be one of the predictors of the response to the therapeutic plasma exchange (TPE) in patients with MG. Methods: The study was carried out in 78 patients with moderate to severe MG. They were divided into two groups: TPE group (62 patients) that included the patients who received TPE, and control (non-TPE) group who did not receive TPE and was only on medical treatment (16 patients). Patients in TPE group, then, were subdivided into sero-positive and sero-negative subgroups according to the presence or absence of AChR antibodies respectively. Scoring by Quantitative Myasthenia Gravis Scale (QMGS) was done before and 1 month after the TPE. Results: After 1 month of treatment, the change in QMGS was significantly higher in the TPE group than in the Non-TPE one (t = -6.406) (P &lt; 0.0001). In the sero-positive group, the score of QMGS ranged initially from 19 to 34 (mean 26.48 ± 3.75) and after 1 month from 11 to 30 (mean 18.00 ± 4.45) with the median change of -32. The QMGS change was significantly greater in the sero-positive group than in the sero-negative one (t = -3.516) (P &lt; 0.0001). Conclusions: As regards to the presence of AChR antibodies in patients with MG, both sero-positive and sero-negative groups responded to TPE but the sero-positive group had a better response. J Neurol Res. 2019;9(1-2):8-13 doi: https://doi.org/10.14740/jnr510

High numbers of autoantigen-reactive mononuclear cells expressing interferon-gamma (IFN-gamma), IL-4 and transforming growth factor-beta (TGF-beta) are present in cord blood.

Umbilical cord blood of neonates and peripheral blood of healthy adults were analysed by in situ hybridization for numbers of mononuclear cells (MNC) expressing the cytokines IFN-gamma, TGF-beta and IL-4 mRNA without culture and after culture in the presence of acetylcholine receptor (AChR), myelin basic protein (MBP) and peripheral myelin protein P2. These antigens were chosen since they represent autoantigens in putatively immune-mediated neurological diseases. The numbers of cells expressing cytokine mRNA after 72 h culture in the presence of AChR, MBP and P2 were higher in cord blood than in peripheral blood of healthy adults. IFN-gamma, TGF-beta and IL-4 were always elevated in parallel. In cord blood there was a pronounced reactivity to several of the tested antigens, while such broad reactivity was not found in peripheral blood of healthy adults. No differences in cytokine mRNA expression were found between cord blood and peripheral blood of adults when cells were analysed without culture. The results show a capacity of cord blood cells to react to several autoantigens by the up-regulation of cytokine mRNA expression.

Nicotinic Acetylcholine Receptor Induces Lateral Segregation of Phosphatidic Acid and Phosphatidylcholine in Reconstituted Membranes

Purified nicotinic acetylcholine receptor (AChR) protein was reconstituted into synthetic lipid membranes having known effects on receptor function in the presence and absence of cholesterol (Chol). The phase behavior of a lipid system (DPPC/DOPC) possessing a known lipid phase profile and favoring nonfunctional, desensitized AChR was compared with that of a lipid system (POPA/POPC) containing the anionic phospholipid phosphatidic acid (PA), which stabilizes the functional resting form of the AChR. Fluorescence quenching of diphenylhexatriene (DPH) extrinsic fluorescence and AChR intrinsic fluorescence by a nitroxide spin-labeled phospholipid showed that the AChR diminishes the degree of DPH quenching and promotes DPPC lateral segregation into an ordered lipid domain, an effect that was potentiated by Chol. Fluorescence anisotropy of the probe DPH increased in the presence of AChR or Chol and also made apparent shifts to higher values in the transition temperature of the lipid system in the presence of Chol and/or AChR. The values were highest when both Chol and AChR were present, further reinforcing the view that their effect on lipid segregation is additive. These results can be accounted for by the increase in the size of quencher-free, ordered lipid domains induced by AChR and/or Chol. Pyrene phosphatidylcholine (PyPC) excimer (E) formation was strongly reduced owing to the restricted diffusion of the probe induced by the AChR protein. The analysis of Forster energy transfer (FRET) from the protein to DPH further indicates that AChR partitions preferentially into these ordered lipid microdomains, enriched in saturated lipid (DPPC or POPA), which segregate from liquid phase-enriched DOPC or POPC domains. Taken together, the results suggest that the AChR organizes its immediate microenvironment in the form of microdomains with higher lateral packing density and rigidity. The relative size of such microdomains depends not only on the phospholipid polar headgroup and fatty acyl chain saturation but also on AChR protein-lipid interactions. Additional evidence suggests a possible competition between Chol and POPA for the same binding sites on the AChR protein.

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-gamma, 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.

Organ-specific autoantigens induce interferon-gamma and interleukin-4 mRNA expression in mononuclear cells in multiple sclerosis and myasthenia gravis.

T cells recognizing the myelin components myelin basic protein (MBP) and proteolipid protein (PLP) are increased in multiple sclerosis (MS), and there are elevated numbers of T cells recognizing the nicotinic acetylcholine receptor (AChR) in myasthenia gravis (MG). However, the cytokine repertoires in these diseases are largely unknown. We adopted in situ hybridization with radiolabeled complementary DNA oligonucleotide probes to enumerate mononuclear cells that expressed the T-helper type 1 (Th1) cell-related interferon-gamma (IFN-gamma) and Th2-associated interleukin-4 (IL-4) after short-term culture in the presence of autoantigen. High numbers of IFN-gamma and IL-4 mRNA-expressing cells in response to MBP and PLP were detected in patients with untreated MS, and to AChR in MG. The levels of IFN-gamma and IL-4 mRNA-positive cells in MS after culture in the presence of AChR, and in MG after culture in the presence of MBP or PLP, did not differ from those detected after culture without antigen. The CSF of MS patients contained four- to eightfold more myelin protein-reactive IFN-gamma and IL-4 expressing cells. The findings imply that MS and MG are associated with mixed Th1- and Th2-like cell responses directed to organ-specific target antigens.

Differential scanning calorimetry and Fourier transform infrared analysis of lipid-protein interactions involving the nicotinic acetylcholine receptor

Lipid-protein interactions were studied using Torpedo californica acetylcholine receptor (AChR) as a model system by reconstituting purified AChR into dielaidoylphosphatidylcholine (DEPC, 18:1 trans-9,10) membranes. The structural and thermodynamic behavior of lipids in the vicinity of the protein were studied by differential scanning calorimetry and Fourier transform infrared spectroscopy. The effects of AChR on the thermodynamic parameters associated with lipid phase transitions were to reduce the enthalpy change, lower the transition temperature and reduce the cooperative behavior of the lipid molecules. A stoichiometry of approx. 95 lipids per AChR molecule was found by simulating the decrease in enthalpy in terms of a simple model in which a fixed number of lipid molecules are prevented from undergoing a cooperative phase transition. In parallel, the vibrational spectra of pure DEPC and AChR reconstituted in DEPC membranes at various lipid to protein ratios were examined. Profiles of the 3000-2800 cm −1 C-H stretching region and 1350-950 cm −1 characteristic of the headgroup region of the lipid exhibit little sensitivity to protein/lipid ratio reflecting weak interaction of AChR with DEPC. The lipid carbonyl on the other hand appear to be increasingly hydrogen bonded in the presence of AChR. The results provide new information about the size and physical state of the motionally restricted lipid environment that surrounds the acetylcholine receptor. The results are discussed in the context of lipid-mediated alterations in acetylcholine receptor function.

Propidium as a probe of acetylcholine receptor binding sites

The fluorescence of propidium was enhanced in the presence of acetylcholine receptor (AcChR) purified from Torpedo californica electroplax membranes. This fluorescence enhancement has been exploited to titrate the AcChR and to search for the effects of other cholinergic ligands on propidium binding by equilibrium and stopped-flow kinetic techniques. Propidium bound to half of the α-bungarotoxin binding sites on the AcChR with a K d = 1.8 × 10 −6, M and various bis-quaternary cholinergic analogs acted as competitors of propidium binding, the dissociation constant for these cholinergic ligands being obtained by competitive binding treatment of the data. Carbamylcholine did not displace propidium at concentrations corresponding to its known dissociation constant, but it affected the fluorescence yield of bound propidium in the proposed ternary complex. The site for propidium binding is likely to be a cationic binding site near one of the specific cholinergic neurotransmitter binding sites. The stopped-flow results allow calculation of the rates of dissociation ( k −1 = 0.2 s −1) of propidium from AcChR-propidium complexes and estimation, to a first approximation, of the association rate constant value ( k 1 = 1 × 10 5, M −1 S −1).