scholarly journals A Stable Cell Line Expressing Clustered AChR: A Novel Cell-Based Assay for Anti-AChR Antibody Detection in Myasthenia Gravis

2021 ◽  
Vol 12 ◽  
Author(s):  
Yu Cai ◽  
Lu Han ◽  
Desheng Zhu ◽  
Jing Peng ◽  
Jianping Li ◽  
...  

Cell-based assays (CBAs) and radioimmunoprecipitation assay (RIPA) are the most sensitive methods for identifying anti-acetylcholine receptor (AChR) antibody in myasthenia gravis (MG). But CBAs are limited in clinical practice by transient transfection. We established a stable cell line (KL525) expressing clustered AChR by infecting HEK 293T cells with dual lentiviral vectors expressing the genes encoding the human AChR α1, β1, δ, ϵ and the clustering protein rapsyn. We verified the stable expression of human clustered AChR by immunofluorescence, immunoblotting, and real-time PCR. Fluorescence-activated cell sorting (FACS) was used to detect anti-AChR antibodies in 103 MG patients and 58 healthy individuals. The positive results of MG patients reported by the KL525 was 80.6% (83/103), 29.1% higher than the 51.4% (53/103) of RIPA. 58 healthy individuals tested by both the KL525 CBA and RIPA were all negative. In summary, the stable expression of clustered AChR in our cell line makes it highly sensitive and advantageous for broad clinical application in CBAs.

2003 ◽  
Vol 77 (23) ◽  
pp. 12901-12906 ◽  
Author(s):  
Michael K. Lo ◽  
Mark Tilgner ◽  
Pei-Yong Shi

ABSTRACT Prevention and treatment of infection by West Nile virus (WNV) and other flaviviruses are public health priorities. We describe a reporting cell line that can be used for high-throughput screening of inhibitors against all targets involved in WNV replication. Dual reporter genes, encoding Renilla luciferase (Rluc) and neomycin phosphotransferase (Neo), were engineered into a WNV subgenomic replicon, resulting in Rluc/NeoRep. Geneticin selection of BHK-21 cells transfected with Rluc/NeoRep yielded a stable cell line that contains persistently replicating replicons. Incubation of the reporting cells with known WNV inhibitors decreased Rluc activity, as well as the replicon RNA level. The efficacies of the inhibitors, as measured by the depression of Rluc activity in the reporting cells, are comparable to those derived from authentic viral infection assays. Therefore, the WNV reporting cell line can be used as a high-throughput assay for anti-WNV drug discovery. A similar approach should be applicable to development of genetics-based antiviral assays for other flaviviruses.


2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Suleyman Vural ◽  
Alida Palmisano ◽  
William C. Reinhold ◽  
Yves Pommier ◽  
Beverly A. Teicher ◽  
...  

Abstract Background Altered DNA methylation patterns play important roles in cancer development and progression. We examined whether expression levels of genes directly or indirectly involved in DNA methylation and demethylation may be associated with response of cancer cell lines to chemotherapy treatment with a variety of antitumor agents. Results We analyzed 72 genes encoding epigenetic factors directly or indirectly involved in DNA methylation and demethylation processes. We examined association of their pretreatment expression levels with methylation beta-values of individual DNA methylation probes, DNA methylation averaged within gene regions, and average epigenome-wide methylation levels. We analyzed data from 645 cancer cell lines and 23 cancer types from the Cancer Cell Line Encyclopedia and Genomics of Drug Sensitivity in Cancer datasets. We observed numerous correlations between expression of genes encoding epigenetic factors and response to chemotherapeutic agents. Expression of genes encoding a variety of epigenetic factors, including KDM2B, DNMT1, EHMT2, SETDB1, EZH2, APOBEC3G, and other genes, was correlated with response to multiple agents. DNA methylation of numerous target probes and gene regions was associated with expression of multiple genes encoding epigenetic factors, underscoring complex regulation of epigenome methylation by multiple intersecting molecular pathways. The genes whose expression was associated with methylation of multiple epigenome targets encode DNA methyltransferases, TET DNA methylcytosine dioxygenases, the methylated DNA-binding protein ZBTB38, KDM2B, SETDB1, and other molecular factors which are involved in diverse epigenetic processes affecting DNA methylation. While baseline DNA methylation of numerous epigenome targets was correlated with cell line response to antitumor agents, the complex relationships between the overlapping effects of each epigenetic factor on methylation of specific targets and the importance of such influences in tumor response to individual agents require further investigation. Conclusions Expression of multiple genes encoding epigenetic factors is associated with drug response and with DNA methylation of numerous epigenome targets that may affect response to therapeutic agents. Our findings suggest complex and interconnected pathways regulating DNA methylation in the epigenome, which may both directly and indirectly affect response to chemotherapy.


2006 ◽  
Vol 98 (1) ◽  
pp. 134-145 ◽  
Author(s):  
Xavier Z. Khawaja ◽  
Deborah L. Smith ◽  
Stanley P. Nawoschik ◽  
Jean Zhang ◽  
John Dunlop ◽  
...  

Science ◽  
1982 ◽  
Vol 215 (4535) ◽  
pp. 995-997 ◽  
Author(s):  
I Kamo ◽  
S Furukawa ◽  
A Tada ◽  
Y Mano ◽  
Y Iwasaki ◽  
...  

2021 ◽  
Vol 13 ◽  
pp. 117957352110160
Author(s):  
Thomas Mathew ◽  
Kurian Thomas ◽  
Saji K John ◽  
Shruthi Venkatesh ◽  
Raghunandan Nadig ◽  
...  

Background: Rituximab is reserved for treating refractory myasthenia gravis (MG) patients. Here we report our experience with rituximab in AChR antibody positive generalized MG (gMG) and impending myasthenic crisis (IMC). Methods: This retrospective, observational study, conducted at a tertiary care, neuroimmunology clinic, analyzed the data of patients with AChR antibody positive gMG, treated with rituximab between 1st January 2016 and 30th October 2018. Results: Eleven patients with AChR antibody positive gMG received rituximab. Mean age of the cohort was 50.54 ± 18.71 years with 9 males. Seven out of 11 patients received rituximab in the early stage (<2 years from onset) and had good response to treatment. Four of the 5 patients with IMC improved with rituximab alone. In the 10 patients who regularly followed up, there was a significant difference between the QMG scores at baseline and at 1, 2, 6, 12, and 18 months ( P < .0001). Conclusion: Rituximab appears to be a potentially effective early treatment option for AChR antibody positive generalized MG and impending myasthenic crisis.


Development ◽  
2002 ◽  
Vol 129 (10) ◽  
pp. 2419-2433 ◽  
Author(s):  
Jean-Louis Couderc ◽  
Dorothea Godt ◽  
Susan Zollman ◽  
Jiong Chen ◽  
Michelle Li ◽  
...  

The bric à brac (bab) locus acts as a homeotic and morphogenetic regulator in the development of ovaries, appendages and the abdomen. It consists of two structurally and functionally related genes, bab1 and bab2, each of which encodes a single nuclear protein. Bab1 and Bab2 have two conserved domains in common, a BTB/POZ domain and a Psq domain, a motif that characterizes a subfamily of BTB/POZ domain proteins in Drosophila. The tissue distribution of Bab1 and Bab2 overlaps, with Bab1 being expressed in a subpattern of Bab2. Analysis of a series of mutations indicates that the two bab genes have synergistic, distinct and redundant functions during imaginal development. Interestingly, several reproduction-related traits that are sexually dimorphic or show diversity among Drosophila species are highly sensitive to changes in the bab gene dose, suggesting that alterations in bab activity may contribute to evolutionary modification of sex-related morphology.


2020 ◽  
Vol 11 ◽  
Author(s):  
Konstantinos Lazaridis ◽  
Socrates J. Tzartos

Myasthenia gravis (MG) is the most common autoimmune disorder affecting the neuromuscular junction, characterized by skeletal muscle weakness and fatigability. It is caused by autoantibodies targeting proteins of the neuromuscular junction; ~85% of MG patients have autoantibodies against the muscle acetylcholine receptor (AChR-MG), whereas about 5% of MG patients have autoantibodies against the muscle specific kinase (MuSK-MG). In the remaining about 10% of patients no autoantibodies can be found with the classical diagnostics for AChR and MuSK antibodies (seronegative MG, SN-MG). Since serological tests are relatively easy and non-invasive for disease diagnosis, the improvement of methods for the detection of known autoantibodies or the discovery of novel autoantibody specificities to diminish SN-MG and to facilitate differential diagnosis of similar diseases, is crucial. Radioimmunoprecipitation assays (RIPA) are the staple for MG antibody detection, but over the past years, using cell-based assays (CBAs) or improved highly sensitive RIPAs, it has been possible to detect autoantibodies in previously SN-MG patients. This led to the identification of more patients with antibodies to the classical antigens AChR and MuSK and to the third MG autoantigen, the low-density lipoprotein receptor-related protein 4 (LRP4), while antibodies against other extracellular or intracellular targets, such as agrin, Kv1.4 potassium channels, collagen Q, titin, the ryanodine receptor and cortactin have been found in some MG patients. Since the autoantigen targeted determines in part the clinical manifestations, prognosis and response to treatment, serological tests are not only indispensable for initial diagnosis, but also for monitoring treatment efficacy. Importantly, knowing the autoantibody profile of MG patients could allow for more efficient personalized therapeutic approaches. Significant progress has been made over the past years toward the development of antigen-specific therapies, targeting only the specific immune cells or autoantibodies involved in the autoimmune response. In this review, we will present the progress made toward the development of novel sensitive autoantibody detection assays, the identification of new MG autoantigens, and the implications for improved antigen-specific therapeutics. These advancements increase our understanding of MG pathology and improve patient quality of life by providing faster, more accurate diagnosis and better disease management.


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