Comment on Kopanska et al. Disorders of the Cholinergic System in COVID-19 Era-A Review of the Latest Research. Int. J. Mol. Sci. 2022, 23, 672.

We read the recent review article by Marta Kopanska et al. [...].

Reply to Cafiero et al. Comment on "Kopanska et al. Disorders of the Cholinergic System in COVID-19 Era-A Review of the Latest Research. Int. J. Mol. Sci. 2022, 23, 672".

We have carefully read the Letter to the Editor by Concetta Cafiero, Alessandra Micera, Agnese Re, Beniamino Schiavone, Giulio Benincasa, and Raffaele Palmirotta related to our paper entitled "Disorders of the Cholinergic System in COVID-19 Era-A Review of the Latest Research" [...].

Disorders of the Cholinergic System in COVID-19 Era-A Review of the Latest Research.

The appearance of the SARS-CoV-2 virus initiated many studies on the effects of the virus on the human body. So far, its negative influence on the functioning of many morphological and physiological units, including the nervous system, has been demonstrated. Consequently, research has been conducted on the changes that SARS-CoV-2 may cause in the cholinergic system. The aim of this study is to review the latest research from the years 2020/2021 regarding disorders in the cholinergic system caused by the SARS-CoV-2 virus. As a result of the research, it was found that the presence of the COVID-19 virus disrupts the activity of the cholinergic system, for example, causing the development of myasthenia gravis or a change in acetylcholine activity. The SARS-CoV-2 spike protein has a sequence similar to neurotoxins, capable of binding nicotinic acetylcholine receptors (nAChR). This may be proof that SARS-CoV-2 can bind nAChR. Nicotine and caffeine have similar structures to antiviral drugs, capable of binding angiotensin-converting enzyme 2 (ACE 2) epitopes that are recognized by SARS-CoV-2, with the potential to inhibit the formation of the ACE 2/SARS-CoV-2 complex. The blocking is enhanced when nicotine and caffeine are used together with antiviral drugs. This is proof that nAChR agonists can be used along with antiviral drugs in COVID-19 therapy. As a result, it is possible to develop COVID-19 therapies that use these compounds to reduce cytokine production. Another promising therapy is non-invasive stimulation of the vagus nerve, which soothes the body's cytokine storm. Research on the influence of COVID-19 on the cholinergic system is an area that should continue to be developed as there is a need for further research. It can be firmly stated that COVID-19 causes a dysregulation of the cholinergic system, which leads to a need for further research, because there are many promising therapies that will prevent the SARS-CoV-2 virus from binding to the nicotinic receptor. There is a need for further research, both in vitro and in vivo. It should be noted that in the functioning of the cholinergic system and its connection with the activity of the COVID-19 virus, there might be many promising dependencies and solutions.

Myasthenia gravis associated with novel coronavirus 2019 infection: A report of three cases.

Several neurological presentations have been reported following coronavirus 2019 (COVID-19) infection. This case report describes three myasthenia gravis (MG) patients presented following COVID-19 infection. We report three adult patients with myasthenic Gravis and COVID-19 infection. The patients are between 38 and 61 years old. Case 1 is a 61-year-old woman with progressive dysphagia, nasal speech, ocular ptosis, diplopia, and proximal muscle weakness for 10 days. She had a COVID-19 infection 6 weeks ago. Case 2 is a 57-year-old man with clinical symptoms of muscular fatigability, diplopia, ptosis, and dysphagia for a week and a positive COVID-19 infection 10 days ago. Case 3 is a 38-year-old woman with fatigability, ptosis, dysphagia, and a diagnosis of COVID-19 infection 4 weeks ago. All patients had a positive RT-PCR for COVID-19 infection by nasopharyngeal swab test and a high-level acetylcholine receptor antibody in the serum. All patients were treated with pyridostigmine and prednisolone with a favorable outcome. MG may appear following COVID-19 infection, and the role of molecular mimicry and latent MG activation should be considered the cause of the disease onset.

Emergence of myasthenia gravis in dengue infection-first case report.

The association of dengue infection (DI) with atypical neurological manifestations was first reported in 1976. DENV-2 and DENV-3 serotypes are mostly related to neurological problems. DI has shown an overall risk of 21 autoimmune diseases, and 4% may develop neuromuscular complications. The pathogenetic mechanisms behind myasthenia gravis (MG) occurring during DI is thought to be linked to the neurotrophic effect of the infection. We report a unique case of DENV-1 infection presenting with bilateral ptosis and dysphagia in a previously healthy adult.

Impact of Coronavirus Disease 2019 in a French Cohort of Myasthenia Gravis.

OBJECTIVE: To describe the clinical characteristics and outcomes of coronavirus disease 2019 (COVID-19) among patients with myasthenia gravis (MG) and identify factors associated with COVID-19 severity in patients with MG. METHODS: The CO-MY-COVID registry was a multicenter, retrospective, observational cohort study conducted in neuromuscular referral centers and general hospitals of the FILNEMUS (Filière Neuromusculaire) network (between March 1, 2020, and June 8, 2020), including patients with MG with a confirmed or highly suspected diagnosis of COVID-19. COVID-19 was diagnosed based on a PCR test from a nasopharyngeal swab or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology, thoracic CT scan, or typical symptoms. The main outcome was COVID-19 severity based on location of treatment/management (home, hospitalized in a medical unit, or in an intensive care unit). We collected information on demographic variables, general history, and risk factors for severe COVID-19. Multivariate ordinal regression models were used to identify factors associated with severe COVID-19 outcomes. RESULTS: Among 3,558 patients with MG registered in the French database for rare disorders, 34 (0.96%) had COVID-19. The mean age at COVID-19 onset was 55.0 ± 19.9 years (mean MG duration: 8.5 ± 8.5 years). By the end of the study period, 28 patients recovered from COVID-19, 1 remained affected, and 5 died. Only high Myasthenia Gravis Foundation of America (MGFA) class (≥IV) before COVID-19 was associated with severe COVID-19 (p = 0.004); factors that were not associated included sex, MG duration, and medium MGFA classes (≤IIIb). The type of MG treatment had no independent effect on COVID-19 severity. CONCLUSIONS: This registry-based cohort study shows that COVID-19 had a limited effect on most patients, and immunosuppressive medications and corticosteroids used for MG management are not risk factors for poorer outcomes. However, the risk of severe COVID-19 is elevated in patients with high MGFA classes (odds ratio, 102.6 [4.4-2,371.9]). These results are important for establishing evidence-based guidelines for the management of patients with MG during the COVID-19 pandemic.

[Review of correlation between human parvovirus B19 and autoimmune disease etiology].

Human parvovirus B19 (PVB19) is a small single strand DNA virus distributed throughout the world, with its encoded products being three known proteins. There is conclusive evidence that PVB19 infection is a crucial inducement of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Hashimoto's thyroiditis (HT), myasthenia gravis (MG) and other autoimmune diseases (AIDs). Recent studies have confirmed that anti-B19-VP1u-IgG antibody is able to increase the activity of cytokines such as interleukin 1 (IL-1), tumor necrosis factor α (TNF-α), matrix metalloproteinase-9 (MMP9); PVB19 protein NS 1 and VP1u are capable of inducing the expression of IL-6; PVB19 can induce the production of Th17 cell-related cytokines, resulting in the decrease of IFN-gamma levels and the increase of IL-4 levels in plasma. In this paper, the structure of PVB19, the mechanism of human infection and the relationship between PVB19 and AIDs are summarized.

Detection of human parvovirus B19 infection in the thymus of patients with thymic hyperplasia-associated myasthenia gravis.

OBJECTIVE: To investigate the association between myasthenia gravis (MG) and human parvovirus B19 (B19V) infection in the thymus. METHODS: The presence of human B19V DNA and protein was assessed in 138 samples-including 68 thymic hyperplasias (39 with MG), 58 thymomas (23 with MG), and 12 normal thymus tissues-using a nested polymerase chain reaction, immunohistochemistry, laser capture microdissection, and sequencing in a double-blinded manner. RESULTS: B19V DNA was detected mainly in thymic hyperplasia, and the positivity rate (41.18%, 28/68) was significantly higher than that in thymoma (3.45%, 2/58) (p <0.001) but not that in normal thymic tissues. Correspondingly, the positivity rate in thymic hyperplasia with MG (30.77%, 12/39) was significantly higher than that in thymoma with MG (4.35%, 1/23) (p=0.021). However, it was higher in thymic hyperplasia without MG (55.17%, 16/29) than in thymic hyperplasia with MG (30.77%, 12/39) (p=0.043). Cells in thymic hyperplasia positive for B19V VP1/VP2 protein (63.24%, 43/68) were identified mainly in ectopic germinal centres and thymic corpuscle epithelial cells, but were rare in thymomas (1.72%, 1/58) (p <0.001). Moreover, the positivity rate was significantly higher in thymic hyperplasia with MG (74.36%, 29/39) than in thymic hyperplasia without MG (48.28%, 14/29) (p=0.027). CONCLUSIONS: To our knowledge, the present study is the first to show that human B19V infection is closely associated with thymic hyperplasia and thymic-hyperplasia-associated MG, but is not related to thymoma or thymoma-associated MG. The findings reveal a previously unrecognized aetiopathogenic mechanism of thymic-hyperplasia-associated MG, evoking numerous questions that require further investigation.

Molecular and clinical relationship between live-attenuated Japanese encephalitis vaccination and childhood onset myasthenia gravis.

OBJECTIVE: The incidence of childhood onset myasthenia gravis (CMG) in China is higher than that in other countries; however, the reasons for this are unclear. METHODS: We investigated the clinical and immunological profiles of CMG, and assessed the potential precipitating factors. For the mouse studies, the possible implication of vaccination in the pathogenesis was explored. RESULTS: In our retrospective study, 51.22% of the 4,219 cases of myasthenia gravis (MG) were of the childhood onset type. The cohort study uncovered that the pathophysiology of CMG was mediated by immune deviation, rather than through gene mutations or virus infections. The administration of the live-attenuated Japanese encephalitis vaccine (LA-JEV), but not the inactivated vaccine or other vaccines, in mice induced serum acetylcholine receptor (AChR) antibody production, reduced the AChR density at the endplates, and decreased both muscle strength and response to repetitive nerve stimulation. We found a peptide (containing 7 amino acids) of LA-JEV similar to the AChR-α subunit, and immunization with a synthesized protein containing this peptide reproduced the MG-like phenotype in mice. INTERPRETATION: Our results describe the immunological profile of CMG. Immunization with LA-JEV induced an autoimmune reaction against the AChR through molecular mimicry. These findings might explain the higher occurrence rate of CMG in China, where children are routinely vaccinated with LA-JEV, compared with that in countries, where this vaccination is not as common. Efforts should be made to optimize immunization strategies and reduce the risk for developing autoimmune disorders among children. Ann Neurol 2018;84:386-400.

Acetylcholine Receptor Antibody Titers and Clinical Course after Influenza Vaccination in Patients with Myasthenia Gravis: A Double-Blind Randomized Controlled Trial (ProPATIent-Trial).

BACKGROUND: It is a continuous matter of discussion whether immune activation by vaccination in general and Influenza vaccination in particular increases the risk for clinical deterioration of autoimmune diseases. This prospective study investigated the serological and clinical course of autoimmune Myasthenia gravis (MG) after a seasonal influenza vaccination. METHODS: This randomized, placebo-controlled, double-blind study enrolled MG patients with antibodies against acetylcholine-receptors (AChR-ab). They were allocated to receive seasonal influenza vaccine or placebo. The primary endpoint was the relative change of AChR-ab-titer over 12weeks. A relative increase of 20% was set as non-inferiority margin. Secondary endpoints were clinical changes in the modified Quantitative Myasthenia Gravis Score (QMG), increase of anti-influenza-ELISA-antibodies, and changes of treatment. The study is registered with Clinicaltrialsregister.eu, EudraCT number 2006-004374-27. FINDINGS: 62 patients were included. Mean±standard deviation (median) in the vaccine and placebo group were AChR-ab-titer changes of -6.0%±23.3% (-4.0%) and -2.8%±22.0% (-0.5%) and QMG score changes of -0.08±0.27 (0.17) and 0.11±0.31 (0.00), respectively. The difference between groups (Hodges-Lehmann estimate with 95% CI) was - for the AChR-ab-titer change 4·0% [-13.3%, 4.5%] (p=0.28 for testing a difference, p<0.0001 for testing non-inferiority) and for the QMG change 0·00 [-0.17, 0.00] (p=0.79 for testing a difference). The occurrence of 74 adverse events (AE) was comparable between groups. The most common AE was flu-like symptoms. One serious AE (hospitalisation following gastrointestinal haemorrhage) in the verum group was not related to the vaccine. INTERPRETATION: Influenza vaccination in MG is safe. Uprating the potential risk of a severe course of MG exacerbation during influenza infection compared to the 95% CI differences for the endpoints, vaccination is principally indicated in this patient population.

Is West Nile Virus infection associated with myasthenia gravis?

BACKGROUND: Myasthenia Gravis (MG) is an autoimmune disease which is characterised by disruption of signal transmission at neuromuscular junction. We aimed to search about a newly reported association between MG and West Nile Virus (WNV) infection. METHODS: We searched WNV IgG by ELISA in serum samples of 50 available MG patients and 38 controls. RESULTS: None of the samples gave positive results for past WNV infection. CONCLUSION: No evidence of past WNV infection was found in our study population of MG patients. This may have been because MG has been showed to be related with neuroinvasive WNV, which none of our study subjects seem to have had based on their stories. New multicentre studies focusing on immunological mechanisms and held with larger groups or especially neuroinvasive disease patients can cast light onto the answer of this question.

Increased expression of Toll-like receptors 7 and 9 in myasthenia gravis thymus characterized by active Epstein-Barr virus infection.

Considerable data implicate the thymus as the main site of autosensitization to the acetylcholine receptor in myasthenia gravis (MG), a B-cell-mediated autoimmune disease affecting the neuromuscular junction. We recently demonstrated an active Epstein-Barr virus (EBV) infection in the thymus of MG patients, suggesting that EBV might contribute to the onset or maintenance of the autoimmune response within MG thymus, because of its ability to activate and immortalize autoreactive B cells. EBV has been reported to elicit and modulate Toll-like receptor (TLR) 7- and TLR9-mediated innate immune responses, which are known to favor B-cell dysfunction and autoimmunity. Aim of this study was to investigate whether EBV infection is associated with altered expression of TLR7 and TLR9 in MG thymus. By real-time PCR, we found that TLR7 and TLR9 mRNA levels were significantly higher in EBV-positive MG compared to EBV-negative normal thymuses. By confocal microscopy, high expression levels of TLR7 and TLR9 proteins were observed in B cells and plasma cells of MG thymic germinal centers (GCs) and lymphoid infiltrates, where the two receptors co-localized with EBV antigens. An increased frequency of Ki67-positive proliferating B cells was found in MG thymuses, where we also detected proliferating cells expressing TLR7, TLR9 and EBV antigens, thus supporting the idea that EBV-associated TLR7/9 signaling may promote abnormal B-cell activation and proliferation. Along with B cells and plasma cells, thymic epithelium, plasmacytoid dendritic cells and macrophages exhibited enhanced TLR7 and TLR9 expression in MG thymus; TLR7 was also increased in thymic myeloid dendritic cells and its transcriptional levels positively correlated with those of interferon (IFN)-ß. We suggested that TLR7/9 signaling may be involved in antiviral type I IFN production and long-term inflammation in EBV-infected MG thymuses. Our overall findings indicate that EBV-driven TLR7- and TLR9-mediated innate immune responses may participate in the intra-thymic pathogenesis of MG.

In silico study of potential autoimmune threats from rotavirus infection.

Rotavirus, the major cause of infantile nonbacterial diarrhea, was found to be associated with development of diabetes-associated auto-antibodies. In our study we tried to find out further potential autoimmune threats of this virus using bioinformatics approach. We took rotaviral proteins to study similarity with Homo sapiens proteome and found most conserved structural protein VP6 matches at two regions with ryanodine receptor, an autoimmune target associated with myasthenia gravis. Myasthenia gravis, a chronic neurodegenerative autoimmune disorder with no typical known reason, is characterized by fluctuating muscle weakness which is typically enhanced during muscular effort. Affected patients generate auto antibodies against mainly acetyl choline receptor and sarcoplasmic reticulum calcium-release channel protein ryanodine receptor. Further, we observed that two regions which matched with ryanodine receptor remain conserved in all circulating rotaviral strains and showed significant antigenecity with respect to myasthenia gravis associated HLA haplotypes. Overall, our study detected rotaviral VP6 as a potential threat for myasthenia gravis and enlighten an area of virus associated autoimmune research.

West nile virus infection and myasthenia gravis.

INTRODUCTION: Viruses are commonly cited as triggers for autoimmune disease. It is unclear if West Nile virus (WNV) initiates autoimmunity. METHODS: We describe 6 cases of myasthenia gravis (MG) that developed several months after WNV infection. All patients had serologically confirmed WNV neuroinvasive disease. None had evidence of MG before WNV. RESULTS: All patients had stable neurological deficits when they developed new symptoms of MG 3 to 7 months after WNV infection. However, residual deficits from WNV confounded or delayed MG diagnosis. All patients had elevated acetylcholine receptor (AChR) antibodies, and 1 had thymoma. Treatment varied, but 4 patients required acetylcholinesterase inhibitors, multiple immunosuppressive drugs, and intravenous immune globulin or plasmapheresis for recurrent MG crises. CONCLUSIONS: The pathogenic mechanism of MG following WNV remains uncertain. We hypothesize that WNV-triggered autoimmunity breaks immunological self-tolerance to initiate MG, possibly through molecular mimicry between virus antigens and AChR subunits or other autoimmune mechanisms.

Lack of evidence for Epstein-Barr virus infection in myasthenia gravis thymus.

A role for Epstein-Barr virus (EBV) in myasthenia gravis pathogenesis has been suggested recently. Using in situ hybridization for the detection of the EBV-encoded RNAs and EBNA1-specific immunohistochemistry, we found no latently infected cells in a series of thymus specimens from patients with myasthenia gravis showing lymphofollicular thymitis. In addition, using immunohistochemistry and an antibody specific for the viral immediate early protein BZLF1, no evidence of lytic EBV infection was seen in these cases. Our results therefore do not support a direct role of thymic EBV infection in the pathogenesis of myasthenia gravis.