Antibody and T-Cell Subsets Analysis Unveils an Immune Profile Heterogeneity Mediating Long-term Responses in Individuals Vaccinated Against SARS-CoV-2.

BACKGROUND: Based on the fact that coronavirus disease 2019 (COVID-19) is still spreading despite worldwide vaccine administration, there is an imperative need to understand the underlying mechanisms of vaccine-induced interindividual immune response variations. METHODS: We compared humoral and cellular immune responses in 127 individuals vaccinated with either BNT162b2, mRNA-1273, or ChAdOx1-nCoV-19 vaccine. RESULTS: Both mRNA vaccines induced faster and stronger humoral responses as assessed by high spike- and RBD-specific antibody titers and neutralizing efficacy in comparison to ChAdOx1-nCoV-19 vaccine. At 7 months postvaccination, a decreasing trend in humoral responses was observed, irrespective of the vaccine administered. Correlation analysis between anti-S1 IgG and interferon- (IFN-) production unveiled a heterogeneous immune profile among BNT162b2-vaccinated individuals. Specifically, vaccination in the high-responder group induced sizable populations of polyfunctional memory CD4 helper T cells (TH1), follicular helper T cells (TFH), and T cells with features of stemness (TSCM), along with high neutralizing antibody production that persisted up to 7 months. In contrast, low responders were characterized by significantly lower antibody titers and memory T cells and a considerably lower capacity for interleukin-2 and IFN- production. CONCLUSIONS: We identified that long-term humoral responses correlate with the individuals ability to produce antigen-specific persistent memory T-cell populations.

IgG4 autoantibodies against muscle-specific kinase undergo Fab-arm exchange in myasthenia gravis patients.

Autoimmunity mediated by IgG4 subclass autoantibodies is an expanding field of research. Due to their structural characteristics a key feature of IgG4 antibodies is the ability to exchange Fab-arms with other, unrelated, IgG4 molecules, making the IgG4 molecule potentially monovalent for the specific antigen. However, whether those disease-associated antigen-specific IgG4 are mono- or divalent for their antigens is unknown. Myasthenia gravis (MG) with antibodies to muscle specific kinase (MuSK-MG) is a well-recognized disease in which the predominant pathogenic IgG4 antibody binds to extracellular epitopes on MuSK at the neuromuscular junction; this inhibits a pathway that clusters the acetylcholine (neurotransmitter) receptors and leads to failure of neuromuscular transmission. In vitro Fab-arm exchange-inducing conditions were applied to MuSK antibodies in sera, purified IgG4 and IgG1-3 sub-fractions. Solid-phase cross-linking assays were established to determine the extent of pre-existing and inducible Fab-arm exchange. Functional effects of the resulting populations of IgG4 antibodies were determined by measuring inhibition of agrin-induced AChR clustering in C2C12 cells. To confirm the results, κ/κ, λ/λ and hybrid κ/λ IgG4s were isolated and tested for MuSK antibodies. At least fifty percent of patients had IgG4, but not IgG1-3, MuSK antibodies that could undergo Fab-arm exchange in vitro under reducing conditions. Also MuSK antibodies were found in vivo that were divalent (monospecific for MuSK). Fab-arm exchange with normal human IgG4 did not prevent the inhibitory effect of serum derived MuSK antibodies on AChR clustering in C2C12 mouse myotubes. The results suggest that a considerable proportion of MuSK IgG4 could already be Fab-arm exchanged in vivo. This was confirmed by isolating endogenous IgG4 MuSK antibodies containing both κ and λ light chains, i.e. hybrid IgG4 molecules. These new findings demonstrate that Fab-arm exchanged antibodies are pathogenic.

Antibodies to aquaporins are frequent in patients with primary Sjögren's syndrome.

Objectives: Several aquaporins (AQPs) are present in the salivary glands, likely contributing to their secretions. AQP dysfunction may contribute to the salivary gland dysfunction in SS. Antibodies to AQP4 and AQP1 are detected in neuromyelitis optica and are believed to play a pathogenic role. We aimed to search for antibodies to several AQPs in the sera from SS patients in an effort to shed light on the pathogenic mechanisms of SS. Methods: We searched for antibodies to six AQPs in the sera of 34 SS patients without neurological findings using ELISAs with synthetic peptides corresponding to the three extracellular domains of each AQP, radioimmunoassays with AQPs, Western blots and competition experiments with cell-embedded AQPs. Results: Thirteen (38.2%) SS patients had antibodies to extracellular domains of AQP1 (two), AQP3 (one), AQP8 (six) or AQP9 (four); none had AQP4 or AQP5 antibodies. Each patient had antibodies to only one extracellular domain. AQP binding was further verified by radioimmunoassay with intact AQPs, western blots and AQP-transfected cells. In contrast, none of the 106 healthy controls or 68 patients with other autoimmune diseases had antibodies to intact AQPs. Expression of AQP8 (the major antibody target) on human salivary glands was shown by immunohistochemistry. Patients with anti-AQP antibodies had more severe xeropthalmia compared with anti-AQP-negative patients, suggesting a potential pathogenic role of these antibodies. Conclusion: Antibodies to AQPs (especially to AQP8 and AQP9) are frequent in SS patients. The likely important role of AQPs in salivary gland secretions justifies further research.

Glial and neuronal antibodies in patients with idiopathic intracranial hypertension.

Headache and visual disturbances are the main presenting symptoms of idiopathic intracranial hypertension (IIH) characterized by increased intracranial pressure (ICP) with an unknown cause. We aimed to investigate the antibodies against optic neuritis-associated glial antigens, aquaporin-4 (AQP4) and myelin oligodendrocyte glycoprotein (MOG) and uncharacterized neuronal membrane antigens in IIH patients. Consecutive patients diagnosed according to Friedman revised diagnostic criteria and control subjects were included after their consent. All serum samples were analyzed for antibodies against AQP4 and MOG using cell-based immunofluorescent assays and for uncharacterized neuronal membrane antigens by indirect immunocytochemistry utilizing live neurons. Sera of 34 patients with IIH and 40 control subjects were investigated but none of the patients showed AQP4 and MOG antibodies. However, serum IgG of five IIH patients showed reactivity against membrane antigens of rat hippocampal and cortical neurons. Interestingly, three out of these five patients had nonspecific white matter lesions on MRI, whereas only four of all other patients had these lesions (p = 0.048). AQP4 and MOG antibodies do not seem to have a role in the pathophysiology of IIH. However, association of immunocytochemistry findings with the presence of white matter lesions may suggest that immunological factors contribute to the pathogenesis of IIH in at least some of the patients.

Low-Density Lipoprotein Receptor-Related Protein 4-Positive Myasthenia Gravis in a Double-Seronegative, Electromyography-Negative Patient.

We describe a patient with ocular myasthenia gravis, where single-fiber electromyography and testing for acetylcholine receptor and muscle-specific kinase antibodies were negative. However, antibodies to low-density lipoprotein receptor-related protein 4 (LRP4) were positive, and this prompted appropriate management. We recommend that testing for LRP4 antibodies be considered when the clinical suspicion for myasthenia gravis is high despite negative conventional diagnostic tests.

Dental follicle mesenchymal stem cell administration ameliorates muscle weakness in MuSK-immunized mice.

BACKGROUND: Myasthenia gravis (MG) is an antibody-mediated autoimmune disease of the neuromuscular junction (NMJ), mostly associated with acetylcholine receptor (AChR) antibodies. Around 5-10 % of MG patients show antibodies to muscle-specific tyrosine kinase (MuSK). Mesenchymal stem cell (MSC) administration has been shown to ameliorate muscle weakness in the experimental autoimmune myasthenia gravis (EAMG) model induced by AChR immunization. METHODS: To investigate the efficacy of stem cell treatment in MuSK-related EAMG, clinical and immunological features of MuSK-immunized mice with or without dental follicle MSC (DFMSC) treatment were compared. RESULTS: MuSK-immunized mice intravenously treated with DFMSC after second and third immunizations showed significantly lower EAMG incidence and severity and reduced serum anti-MuSK antibody, NMJ IgG, and C3 deposit levels and CD11b+ lymph node cell ratios. Moreover, lymph node cells of DFMSC-administered mice showed reduced proliferation and IL-6 and IL-12 production responses to MuSK stimulation. By contrast, proportions of B and T cell populations and production of a wide variety of cytokines were not affected from DFMSC treatment. CONCLUSIONS: Our results suggest that DFMSC treatment shows its beneficial effects mostly through suppression of innate immune system, whereas other immune functions appear to be preserved. Stem cell treatment might thus constitute a specific and effective treatment method in MuSK-associated MG.

Preferential production of IgG1, IL-4 and IL-10 in MuSK-immunized mice.

Myasthenia gravis (MG) is an autoimmune disease characterized by muscle weakness associated with acetylcholine receptor (AChR), muscle-specific receptor kinase (MuSK) or low-density lipoprotein receptor-related protein 4 (LRP4)-antibodies. MuSK-antibodies are predominantly of the non-complement fixing IgG4 isotype. The MuSK associated experimental autoimmune myasthenia gravis (EAMG) model was established in mice to investigate immunoglobulin (Ig) and cytokine responses related with MuSK immunity. C57BL/6 (B6) mice immunized with 30µg of recombinant human MuSK in incomplete or complete Freund's adjuvant (CFA) showed significant EAMG susceptibility (>80% incidence). Although mice immunized with 10µg of MuSK had lower EAMG incidence (14.3%), serum MuSK-antibody levels were comparable to mice immunized with 30µg MuSK. While MuSK immunization stimulated production of all antibody isotypes, non-complement fixing IgG1 was the dominant anti-MuSK Ig isotype in both sera and neuromuscular junctions. Moreover, MuSK immunized IgG1 knockout mice showed very low serum MuSK-antibody levels. Sera and MuSK-stimulated lymph node cell supernatants of MuSK immunized mice showed significantly higher levels of IL-4 and IL-10 (but not IFN-γ and IL-12), than those of CFA immunized mice. Our results suggest that through activation of Th2-type cells, anti-MuSK immunity promotes production of IL-4, which in turn activates anti-MuSK IgG1, the mouse analog of human IgG4. These findings might provide clues for the pathogenesis of other IgG4-related diseases as well as development of disease specific treatment methods (e.g. specific IgG4 inhibitors) for MuSK-related MG.

T cell repertoire in DQ5-positive MuSK-positive myasthenia gravis patients.

Myasthenia gravis (MG) is a prototypical antibody-mediated disease characterized by muscle weakness and fatigability. Serum antibodies to the acetylcholine receptor and muscle-specific tyrosine kinase receptor (MuSK) are found in about 85% and 8% of patients respectively. We have previously shown that more than 70% of MG patients with MuSK antibodies share the HLA DQ5 allele. The aim of the present study was to analyze the T cell receptor (TCR) repertoire specific for recombinant human MuSK protein. We used the CDR3 TRBV-TRBJ spectratyping (immunoscope) to analyze the T cell response to MuSK from 13 DQ5+ MuSK-MG patients and from 7 controls (six DQ5+ MuSK negative subjects and one DQ5- DQ3+ MuSK positive patient). DQ5+ MuSK-MG patients but not controls used a restricted set of TCR VJ rearrangements in response to MuSK stimulation. One semiprivate (TRBV29-TRBJ2.5) rearrangement was found in 5/13 patients, while 4 other semiprivate (one in TRBV28-TRBJ2.1 and in TRBV3-TRBJ1.2, and two in TRBV28-TRBJ1.2) rearrangements were differently shared by 4/13 patients each and were absent in controls. When we sequenced the TRBV29-TRBJ2.5 rearrangement, we obtained 26 different sequences of the expected 130 bp length from 117 samples of the 5 positive patients: two common motifs GXGQET/TEHQET were shared in 4 patients as semiprivate motifs. Thus, the MuSK-specific T-cell response appears to be restricted in DQ5+ MuSK-MG patients, with a semiprivate repertoire including a common motif of TRBV29. This oligoclonal restriction of T cells will allow the identification of immunodominant epitopes in the antigen, providing therefore new tools for diagnosis and targeted therapy.

Aquaporin-1 antibody in neuromyelitis optical patients.

BACKGROUND/METHODS: To find out the prevalence of aquaporin-antibody (Aqp-Ab) and characterize Aqp-Ab associated clinical features in NMO, Aqp-1 and Aqp-4-Abs were examined using radioimmunoprecipitation and cell-based assays, respectively. RESULTS: Aqp-4 and Aqp-1-Abs were detected in 20/30 and 8/30 NMO patients, respectively. One patient was Aqp-1-Ab single-positive, 13 patients were Aqp-4-Ab single-positive, 7 patients were Aqp-4/Aqp-1-Ab double-positive and 9 patients were seronegative. All double-positive patients had optic neuritis during the first attack. Only 2/29 MS patients and none of the control idiopathic intracranial hypertension patients were Aqp-1-Ab positive. CONCLUSION: Aqp-1-Ab is usually detected in Aqp-4-Ab positive NMO patients and might be involved in optic neuritis pathogenesis.

Neuronal nicotinic acetylcholine receptor antibodies in autoimmune central nervous system disorders.

Background: Neuronal nicotinic acetylcholine receptors (nAChRs) are abundant in the central nervous system (CNS), playing critical roles in brain function. Antigenicity of nAChRs has been well demonstrated with antibodies to ganglionic AChR subtypes (i.e., subunit α3 of α3ß4-nAChR) and muscle AChR autoantibodies, thus making nAChRs candidate autoantigens in autoimmune CNS disorders. Antibodies to several membrane receptors, like NMDAR, have been identified in autoimmune encephalitis syndromes (AES), but many AES patients have yet to be unidentified for autoantibodies. This study aimed to develop of a cell-based assay (CBA) that selectively detects potentially pathogenic antibodies to subunits of the major nAChR subtypes (α4ß2- and α7-nAChRs) and its use for the identification of such antibodies in "orphan" AES cases. Methods: The study involved screening of sera derived from 1752 patients from Greece, Turkey and Italy, who requested testing for AES-associated antibodies, and from 1203 "control" patients with other neuropsychiatric diseases, from the same countries or from Germany. A sensitive live-CBA with α4ß2-or α7-nAChR-transfected cells was developed to detect antibodies against extracellular domains of nAChR major subunits. Flow cytometry (FACS) was performed to confirm the CBA findings and indirect immunohistochemistry (IHC) to investigate serum autoantibodies' binding to rat brain tissue. Results: Three patients were found to be positive for serum antibodies against nAChR α4 subunit by CBA and the presence of the specific antibodies was quantitatively confirmed by FACS. We detected specific binding of patient-derived serum anti-nAChR α4 subunit antibodies to rat cerebellum and hippocampus tissue. No serum antibodies bound to the α7-nAChR-transfected or control-transfected cells, and no control serum antibodies bound to the transfected cells. All patients positive for serum anti-nAChRs α4 subunit antibodies were negative for other AES-associated antibodies. All three of the anti-nAChR α4 subunit serum antibody-positive patients fall into the AES spectrum, with one having Rasmussen encephalitis, another autoimmune meningoencephalomyelitis and another being diagnosed with possible autoimmune encephalitis. Conclusion: This study lends credence to the hypothesis that the major nAChR subunits are autoimmune targets in some cases of AES and establishes a sensitive live-CBA for the identification of such patients.

Expression of water-soluble, ligand-binding concatameric extracellular domains of the human neuronal nicotinic receptor alpha4 and beta2 subunits in the yeast Pichia pastoris: glycosylation is not required for ligand binding.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels that are responsible for cell communication via the neurotransmitter acetylcholine. The predominant nAChR subtype in the mammalian brain with a high affinity for nicotine is composed of α4 and ß2 subunits. This nAChR subtype is responsible for addiction to nicotine and is thought to be implicated in Alzheimer and Parkinson diseases and therefore presents an important target for drug design. In an effort to obtain water-soluble, ligand-binding domains of the human α4ß2 nAChR for structural studies, we expressed the extracellular domains (ECDs) of these subunits in the eukaryotic expression system Pichia pastoris. The wild-type ECDs and their mutants containing the more hydrophilic Cys-loop from the snail acetylcholine-binding protein (individually expressed or coexpressed) did not demonstrate any specific interaction with ligands. We then linked the mutated ECDs with the 24-amino acid peptide (AGS)(8) and observed that the ß2-24-α4 ECD concatamer, but not the α4-24-ß2 one, exhibited very satisfactory water solubility and ligand binding properties. The (125)I-epibatidine and [(3)H]nicotine bound to ß2-24-α4 with dissociation constants (K(d)) of 0.38 and 19 nm, respectively, close to the published values for the intact α4ß2 AChR. In addition, (125)I-epibatidine binding was blocked by nicotine, cytisine, acetylcholine, and carbamylcholine with inhibition constants (K(i)) of 20.64, 3.24, 242, and 2,254 nm, respectively. Interestingly, deglycosylation of the concatamer did not affect its ligand binding properties. Furthermore, the deglycosylated ß2-24-α4 ECD existed mainly in monomeric form, thus forming an appropriate material for structural studies and possibly for pharmacological evaluation of novel α4ß2 nAChR-specific agonists.

Characterization of the nicotinic acetylcholine receptor antibodies after an unexpected increase of antibody titer in thymoma associated myasthenia gravis patients.

Two thymoma-associated myasthenia gravis patients with chronic well-controlled disease but an unexpected increase in anti-nAChR autoantibodies titer are reported. The specificity of anti-nAChR autoantibodies directed against extracellular parts of the receptor was studied in order to investigate the discrepancy between clinical and immunological status. Analysis of the anti-nAChR autoantibodies recognizing the extracellular parts of the nAChR revealed that when the concentration of anti-nAChR autoantibodies titer increased both patients had non-anti-α1 autoantibodies. Since the clinical profile of both patients remained unchanged, the increase of non-anti-α1 autoantibodies did not affect the 2 patients' disease progression. Thus, immunotherapy modification due to an increase of anti-nAChR autoantibodies titer could be erroneous and potentially harmful.

Analysis of nAChR Autoantibodies Against Extracellular Epitopes in MG Patients.

Myasthenia gravis (MG) is an autoimmune disorder caused by autoantibodies targeting components of the postsynaptic membrane of the neuromuscular junction (NMJ), leading to neuromuscular transmission deficiency. In the vast majority of patients, these autoantibodies target the nicotinic acetylcholine receptor (nAChR), a heteropentameric ion channel anchored to the postsynaptic membrane of the NMJ. Autoantibodies in patients with MG may target all the subunits of the receptor at both their extracellular and intracellular regions. Here, we combine immunoadsorption with a cell-based assay to examine the specificity of the patients' autoantibodies against the extracellular part of the nAChR. Our results reveal that these autoantibodies can be divided into distinct groups, based on their target, with probably different impacts on disease severity. Although our findings are based on a small sample group of patients, they strongly support that additional analysis of the specificity of the autoantibodies of patients with MG could serve as a valuable tool for the clinicians' decision on the treatment strategy to be followed.

Novel Cell-Based Assay for Alpha-3 Nicotinic Receptor Antibodies Detects Antibodies Exclusively in Autoimmune Autonomic Ganglionopathy.

BACKGROUND AND OBJECTIVES: Autoantibodies against α3-subunit-containing nicotinic acetylcholine receptors (α3-nAChRs), usually measured by radioimmunoprecipitation assay (RIPA), are detected in patients with autoimmune autonomic ganglionopathy (AAG). However, low α3-nAChR antibody levels are frequently detected in other neurologic diseases with questionable significance. Our objective was to develop a method for the selective detection of the potentially pathogenic α3-nAChR antibodies, seemingly present only in patients with AAG. METHODS: The study involved sera from 55 patients from Greece, suspected for autonomic failure, and 13 patients from Italy diagnosed with autonomic failure, positive for α3-nAChR antibodies by RIPA. In addition, sera from 52 patients with Ca2+ channel or Hu antibodies and from 2,628 controls with various neuroimmune diseases were included. A sensitive live cell-based assay (CBA) with α3-nAChR-transfected cells was developed to detect antibodies against the cell-exposed α3-nAChR domain. RESULTS: Twenty-five patients were found α3-nAChR antibody positive by RIPA. Fifteen of 25 patients were also CBA positive. Of interest, all 15 CBA-positive patients had AAG, whereas all 10 CBA-negative patients had other neurologic diseases. RIPA antibody levels of the CBA-negative sera were low, although our CBA could detect dilutions of AAG sera corresponding to equally low RIPA antibody levels. No serum bound to control-transfected cells, and none of the 2,628 controls was α3-CBA positive. DISCUSSION: This study showed that in contrast to the established RIPA for α3-nAChR antibodies, which at low levels is of moderate disease specificity, our CBA seems AAG specific, while at least equally sensitive with the RIPA. This study provides Class II evidence that α3-nAChR CBA is a specific assay for AAG. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that an α3-nAChR cell-based assay is a more specific assay for AAG than the standard RIPA.

Design and expression of human alpha7 nicotinic acetylcholine receptor extracellular domain mutants with enhanced solubility and ligand-binding properties.

In order to facilitate structural studies of the extracellular domain (ECD) of human alpha7 nicotinic acetylcholine receptor (nAChR), we designed several mutants, since the wild-type-ECD forms large oligomers and microaggregates, and expressed them in the yeast Pichia pastoris. Mutant design was based on a 3D model of human alpha7-nAChR-ECD, constructed using as templates the X-ray crystal structure of the homologous acetylcholine-binding protein (AChBP) and the electron microscopy structure of the Torpedo alpha-nAChR-ECD. At least one mutant, mut10, carrying six single-point mutations (Phe3Tyr, Val69Thr, Cys116Ser, Ile165Thr, Val177Thr, Phe187Tyr) and the replacement of its Cys-loop with the corresponding and more hydrophilic AChBP Cys-loop, was expressed with a 4-fold higher expression yield (1.2 mg/L) than the wild-type alpha7-ECD, existing exclusively as a soluble oligomeric, probably pentameric, form, at concentrations up to at least 10 mg/mL, as judged by gel filtration and dynamic light scattering. This mutant displayed a significantly improved (125)I-alpha-bungarotoxin-binding affinity (K(d)=24 nM) compared to the wild-type-ECD (K(d)=70 nM), the binding being inhibited by unlabelled alpha-bungarotoxin, d-tubocurarine or nicotine (K(i) of 21.5 nM, 127 microM and 17.5 mM, respectively). Circular dichroism studies of mut10 revealed (a) a similar secondary structure composition ( approximately 5% alpha-helix, approximately 45% beta-sheet) to that of the AChBP, Torpedo alpha-nAChR-ECD, and mouse alpha1-nAChR-ECD, (b) a well-defined tertiary structure and (c) binding of small cholinergic ligands at micromolar concentrations. Furthermore, electron microscopy showed well-assembled, probably pentameric, particles of mut10. Finally, since deglycosylation did not alter its solubility or ligand-binding properties, mut10, in either its glycosylated or deglycosylated form, is a promising alpha7-ECD mutant for structural studies, useful for the rational drug design to treat alpha7-nAChR-related diseases.

Deciphering anti-MOG IgG antibodies: Clinical and radiological spectrum, and comparison of antibody detection assays.

IgG antibodies to myelin oligodendrocyte glycoprotein (MOG) detected by cell based assays (CBA) have been identified in a constantly expanding spectrum of CNS demyelinating disorders. However, a universally accepted CBA has not been adopted yet. We aimed to analyze the clinical and radiological features of patients with anti-MOG IgG1-antibodies detected with a live-cell CBA and to compare the three most popular MOG-CBAs. We screened sera from 1300 Greek patients (including 426 patients referred by our 8 clinics) suspected for anti-MOG syndrome, and 120 controls with the live-cell MOG-CBA for IgG1-antibodies. 41 patients, versus 0 controls were seropositive. Clinical, serological and radiological data were available and analyzed for the 21 seropositive patients out of the 426 patients of our clinics. Their phenotypes were: 8 optic neuritis, 3 myelitis, 3 neuromyelitis optica, 2 encephalomyelitis, 2 autoimmune encephalitis and 3 atypical MS. We then retested all sera of our 426 patients with the other two most popular MOG-CBAs for total IgG (a live-cell and a commercial fixed-cell CBAs). Seven IgG1-seropositive patients were seronegative for one or both IgG-CBAs. Yet, all 21 patients had clinical and radiological findings previously described in MOG-antibody associated demyelination disease supporting the high specificity of the IgG1-CBA. In addition, all IgG1-CBA-negative sera were also negative by the IgG-CBAs. Also, all controls were negative by all three assays, except one serum found positive by the live IgG-CBA. Overall, our findings support the wide spectrum of anti-MOG associated demyelinating disorders and the superiority of the MOG-IgG1 CBA over other MOG-CBAs.

Recent advances in understanding the structure of nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs), members of the Cys-loop ligand-gated ion channels (LGICs) superfamily, are involved in signal transduction upon binding of the neurotransmitter acetylcholine or exogenous ligands, such as nicotine. nAChRs are pentameric assemblies of homologous subunits surrounding a central pore that gates cation flux, and are expressed at the neuromuscular junction and in the nervous system and several nonneuronal cell types. The 17 known nAChR subunits assemble into a variety of pharmacologically distinct receptor subtypes. nAChRs are implicated in a range of physiological functions and pathophysiological conditions related to muscle contraction, learning and memory, reward, motor control, arousal, and analgesia, and therefore present an important target for drug research. Such studies would be greatly facilitated by knowledge of the high-resolution structure of the nAChR. Although this information is far from complete, important progress has been made mainly based on electron microscopy studies of Torpedo nAChR and the high-resolution X-ray crystal structures of the homologous molluscan acetylcholine-binding proteins, the extracellular domain of the mouse nAChR alpha1 subunit, and two prokaryotic pentameric LGICs. Here, we review some of the latest advances in our understanding of nAChR structure and gating.

Retraction Note to: Juvenile-onset myasthenia gravis: autoantibody status, clinical characteristics and genetic polymorphisms.

The Joint Editors-in-Chief have retracted this article [1] at the request of the University of Bergen and the Norwegian Board of Health Supervision.

Characterization of the thymus in Lrp4 myasthenia gravis: Four cases.

Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction. Most patients have pathogenic autoantibodies against the acetylcholine receptor (AChR). In the last years a novel subpopulation of MG patients has been described that harbors antibodies against low-density lipoprotein receptor-related protein 4 (Lrp4), another postsynaptic neuromuscular antigen. In early-onset AChR MG (EOMG), the thymus plays an important role in immunopathogenesis, and early thymectomy is beneficial. It is still unknown if the thymus plays any role in Lrp4-MG. In this pilot study, we compared thymus samples from four patients with Lrp4-MG (one pre-treated with immunosuppressive drugs), four non-MG controls and five EOMG patients (not pretreated with immunosuppressive drugs). Immunohistochemistry of the Lrp4-MG thymi revealed normal architecture, with normal numbers and distribution of B-cells, lymphoid follicles and Hassall's corpuscles. Primary CD23+ lymphoid follicles were similarly infrequent in Lrp4-MG and control thymic sections. In none of the control or Lrp4-MG thymi did we find secondary follicles with CD10+ germinal centers. These were evident in 2 of the 5 EOMG thymi, where primary lymphoid follicles were also more frequent on average, thus showing considerable heterogeneity between patients. Even if characteristic pathological thymic changes were not observed in the Lrp4 subgroup, we cannot exclude a role for the thymus in Lrp4-MG pathogenesis, since one Lrp4-MG patient went into clinical remission after thymectomy alone (at one year follow-up) and one more improved after thymectomy in combination with immunosuppressive therapy.