scholarly journals Heterogeneity of acetylcholine receptor autoantibody-mediated complement activity in patients with myasthenia gravis.

2021 ◽  
Author(s):  
Abeer H Obaid ◽  
Chryssa Zografou ◽  
Douangsone D Vadysirisack ◽  
Bailey Munro-Sheldon ◽  
Miriam L Fichtner ◽  
...  
Keyword(s):  
Myasthenia Gravis ◽  
Cell Line ◽  
Acetylcholine Receptor ◽  
Disease Burden ◽  
Hek293t Cell ◽  
Live Cell ◽  
Complement Activity ◽  
Regulator Genes ◽  
Complement Regulator ◽  
Hek293t Cell Line

Background: Autoantibodies targeting the acetylcholine receptor (AChR) in the serum of myasthenia gravis (MG) patients are broadly polyclonal and heterogeneous in their pathogenic capacity. Specifically, AChR autoantibody-mediated pathology occurs through three mechanisms that include complement-directed tissue damage, blocking of the acetylcholine binding site on the AChR, and modulation (internalization) of the AChR. Clinical assays used for diagnosis and prognosis measure only AChR autoantibody binding and they provide weak association with disease burden, thereby limiting understanding of mechanistic heterogeneity, and monitoring therapeutic response. Objective: To develop an in-vitro cell-based assay that measures AChR autoantibody-mediated complement membrane attack complex (MAC) formation. Methods: A HEK293T cell line, which is commonly used for live cell-based AChR autoantibody binding assays, was modified such that the expression of the complement regulator genes (CD46, CD55 and CD59) were disrupted using CRISPR/Cas9 genome editing. This modified cell line was used to measure serum AChR autoantibody-mediated complement MAC formation via flow cytometry. Results: AChR autoantibody-mediated MAC formation required the use of a modified HEK293T cell line in which the surface expression of three complement regulator genes was absent. Serum samples (n=155) from 97 clinically confirmed AChR patients were tested along with 32 healthy donor (HD) samples; the MG cohort included a wide range of disease burden and AChR autoantibody titer. AChR autoantibodies were detected in 139 of the 155 (89.7%) AChR patient samples via a live cell-based assay. Of the 139 AChR positive samples, autoantibody-mediated MAC formation was detected in 83 (59.7%), while no autoantibodies or MAC formation was detected in samples from the HD group. Autoantibody-mediated MAC formation positively associated with autoantibody binding in most MG patient samples. However, a subset displayed a disassociation between binding and MAC formation. Conclusions: We demonstrate the development of a novel assay for evaluating AChR autoantibody-mediated complement activity. It is anticipated that this assay will afford a deeper understanding of the heterogeneous disease pathology and allow for the identification of MG patients who may benefit from complement inhibitor therapy.

Monoclonal antibody to acetylcholine receptor: cell line established from thymus of patient with Myasthenia gravis

Science ◽  
1982 ◽  
Vol 215 (4535) ◽  
pp. 995-997 ◽  
Author(s):  
I Kamo ◽  
S Furukawa ◽  
A Tada ◽  
Y Mano ◽  
Y Iwasaki ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Myasthenia Gravis ◽  
Cell Line ◽  
Acetylcholine Receptor ◽  
Receptor Cell

scholarly journals Cell Banking of HEK293T cell line for clinical-grade lentiviral particles manufacturing

2020 ◽  
Author(s):  
Unai Perpiña ◽  
Cristina Herranz ◽  
Raquel Martin-Ibañez ◽  
Anna Boronat ◽  
Felipe Chiappe ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Hek293t Cell ◽  
Good Manufacturing Practice ◽  
Clinical Grade ◽  
Cell Bank ◽  
Advanced Therapy ◽  
Working Cell ◽  
Cell Banks ◽  
Hek293t Cell Line

Abstract Background: Cell banks are widely used to preserve cell properties as well as to record and control the use of cell lines in biomedical research. The generation of cell banks for the manufacturing of Advanced Therapy Medicinal Products, such as cell and gene therapy products, must comply with current Good Manufacturing Practice regulations. The quality of the cell lines used as starting materials in viral-vector manufacturing processes must be also assessed.Methods: Three batches of a Master Cell Bank and a Working Cell Bank of the HEK293T cell line were manufactured under current Good Manufacturing Practices regulations. Quality control tests were performed according to product specifications. Process validation includes the training of manufacturing personnel by performing simulation tests, and the continuous measurement of environmental parameters such as air particles and microorganisms. Cell number and viability of cryopreserved cells were periodically measured in order to define the stability of these cellular products.Results: All batches of HEK293T Master and Working Cell Banks met the acceptance criteria of their specifications showing the robustness and homogeneity of the processes. In addition, both Master and Working Cell Banks maintained the defined cell viability and concentration over a 37 month-period after cryopreservation. Conclusions: Manufacturing cell banks under Good Manufacturing Practice regulations for their use as raw materials or final cellular products is feasible. HEK293T cell banks were used to manufacture clinical-grade lentiviral particles for Chimeric Antigen Receptor T-cell based clinical trials.

scholarly journals EFFECT OF EDEM1 OVEREXPRESSION ON THE GENERATION AND ASSEMBLY OF MAJOR HISTOCOMPATIBILITY COMPLEXES

2016 ◽  
Vol 25 (4) ◽  
pp. 181-186
Author(s):  
Alexandra Circiumaru ◽  
◽  
Gabriela Chiritoiu ◽  
Livia Sima ◽  
Mihai Bojinca ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endoplasmic Reticulum ◽  
Western Blot ◽  
Autoimmune Diseases ◽  
Cell Line ◽  
Hek293t Cell ◽  
Western Blot Assay ◽  
Mhc I ◽  
Transfected Cells ◽  
Hek293t Cell Line

Background. A better understanding of the role of endoplasmic reticulum degradation-enhancing alpha-mannosidase – like protein 1 (EDEM1) in endoplasmic reticulum associated degradation (ERAD) may open new therapeutic approaches in autoimmune diseases. Aim. To study ERAD and EDEM1 in the generation and assembly of MHC I and the potential role in the pathophysiology of autoimmune diseases. Materials and methods. HEK293T cell line (human embrionic kidney cells), A375 cell line (amelanotic melanoma cells) and THP-1 cell line (leukemic monocytes used both as undifferentiated and differentiated) underwent transient transfection with EDEM1 and mock transfection with pTriEx. Western blot experiments assessed the total cellular MHC I levels in cell lysates, while expression on the cellular surface was quantified by flow cytometry of fixed cells. Results were analysed using the FACS Calibur and CellQuest Pro dedicated software. Experiments were done twice with duplicate probes for the Western blot assay and triplicate probes were used for flow cytometry. GraphPad Prism was used for data analysis. Results. MHC I plasma membrane routing and expression was similar in HEK293T and A375 both in mock transfected and non-transfected cells. Western blot assay for EDEM1 transfected cells showed bands corresponding to the total MHC I that migrated at 42kDa mass in non-transfected and mock transfected Hek293T, A375 and undifferentiated THP-1 cells. Mock transfected differentiated THP-1 cells showed a reduction of total MHC I. EDEM1 transfected Hek293T, A375 and undifferentiated THP-1 cells displayed higher levels of total MHC I, while differentiated THP-1 cells showed a marked reduction. Flow cytometry assay showed significantly reduced cell surface MHC I levels in Hek293T cell line. We observed a modest reduction of MHC I complexes on the cellular surface in undifferentiated THP-1 EDEM1 transfected cells, while there was no significant change in the A375 EDEM1 transfected cell line, as well as the differentiated THP-1 EDEM1 transfected cells. Conclusion. The impact of ERAD’s EDEM1 in MHC I reduction may have an important role in autoimmune disease, making ERAD an interesting therapeutic target.

scholarly journals No evidence for genome editing in mouse zygotes and HEK293T human cell line using the DNA-guided Natronobacterium gregoryi Argonaute (NgAgo)

2016 ◽  
Author(s):  
Nay Chi Khin ◽  
Jenna L. Lowe ◽  
Lora M. Jensen ◽  
Gaetan Burgio
Keyword(s):  
Cell Line ◽  
Genome Editing ◽  
Sanger Sequencing ◽  
Hek293t Cell ◽  
Human Cell Line ◽  
Blastocyst Stage ◽  
Editing Efficiency ◽  
Stage Of Development ◽  
Mouse Zygotes ◽  
Hek293t Cell Line

AbstractA recently published research article reported that the extreme halophile archaebacterium Natronobacterium gregoryi Argonaute enzyme (NgAgo) could cleave the cellular DNA under physiological temperature conditions in cell line and be implemented as an alternative to CRISPR/Cas9 genome editing technology. We assessed this claim in mouse zygotes for four loci (Sptb, Tet-1, Tet-2 and Tet-3) and in the human HEK293T cell line for the EMX1 locus. Over 100 zygotes were microinjected with nls-NgAgo-GK plasmid provided from Addgene and various concentrations of 5’- phosphorylated guide DNA (gDNA) from 2.5 ng/μl to 50 ng/μl and cultured to blastocyst stage of development. The presence of indels was verified using T7 endonuclease 1 assay (T7E1) and Sanger sequencing. We reported no evidence of successful editing of the mouse genome. We then assessed the lack of editing efficiency in HEK293T cell line for the EMX1 endogenous locus by monitoring the NgAgo protein expression level and the editing efficiency by T7E1 assay and Sanger sequencing. We reported that the NgAgo protein was expressed from 8 hours to a maximum expression at 48 hours post-transfection, confirming the efficient delivery of the plasmid and the gDNA but no evidence of successful editing of EMX1 target in all transfected samples. Together our findings indicate that we failed to edit using NgAgo.

scholarly journals Cell Banking of HEK293T Cell Line for Clinical-Grade Lentiviral Particles Manufacturing

2020 ◽  
Author(s):  
Unai Perpiña ◽  
Cristina Herranz ◽  
Raquel Martin-Ibañez ◽  
Anna Boronat ◽  
Felipe Chiappe ◽  
...  
Keyword(s):  
Cell Line ◽  
Hek293t Cell ◽  
Cell Number ◽  
Clinical Grade ◽  
Cell Bank ◽  
Master Cell Bank ◽  
Good Manufacturing Practices ◽  
Working Cell ◽  
Cell Banks ◽  
Hek293t Cell Line

Abstract Background: Cell banks have been widely used to preserve cell properties as well as to record and control cell line access in research. However, the generation of cell banks involved in the manufacturing of Advanced therapy medicinal products such as cell or gene therapy products must comply with the current Good Manufacturing Practice regulation. Similarly, the quality of those cell lines used as starting materials in viral-vector manufacturing processes must be also evaluated.Methods: Three batches of both Master Cell Bank and Working Cell Bank of the HEK293T cell line were manufactured under the current Good Manufacturing Practices regulation. Quality control test were performed according to the product specifications. The process validation includes previous qualification of the manufacturing personnel by performing simulation tests as well as the continuous measure of environmental parameters during manufacturing such as air particles and microorganism. Cell number and viability of cryopreserved cells were periodically measured in order to define the stability of these cellular products.Results: All batches of Master Cell Bank and Working Cell Bank fulfilled the acceptance criteria of their specifications showing the robustness and homogeneity of the processes. In addition, both Master and Working cell bank maintain the defined viability and cell number 37 months after cryopreservation. Conclusions: Manufacturing cell banks under Good Manufacturing Practices regulation for its use as raw material or final cellular product is feasible. HEK293T cell banks have been used to manufacture clinical-grade lentiviral particles for Chimeric Antigen Receptor T-cell based clinical trials.

scholarly journals Complement activity in myasthenia gravis is independent of autoantibody titer and disease severity

2021 ◽  
Author(s):  
Miriam L. Fichtner ◽  
Michelle D. Hoarty ◽  
Douangsone D. Vadysirisack ◽  
Richard J. Nowak ◽  
Kevin C. O’Connor
Keyword(s):  
Myasthenia Gravis ◽  
Disease Burden ◽  
Activity Levels ◽  
Variable Response ◽  
Complement Activity ◽  
Classical Complement Pathway ◽  
Complement Inhibitors ◽  
Autoantibody Titer ◽  
Autoimmune Myasthenia ◽  
Classical Complement

AbstractAcetylcholine receptor (AChR) autoantibodies, found in patients with autoimmune myasthenia gravis (MG), can directly contribute to disease pathology through activation of the classical complement pathway. Accordingly, complement inhibitors are used as a therapeutic strategy, but the response can be heterogeneous even though AChR autoantibodies are present. The mechanisms underlying the variable response are not defined. Yet there is a need for further understanding so that responses can be better predicted. There is a broad spectrum of circulating complement activity levels activity among MG patients. It is not clear whether this activity associates with disease burden or the circulating levels of autoantibodies. We measured complement activity and investigated these associations in MG patients as a means to explore candidate biomarkers. Most study subjects had complement activity within the range defined by healthy controls and no association between this activity and disease burden or AChR autoantibody titer was observed. Assays measuring the complement activating properties of AChR autoantibodies are needed to identifying patients expected to respond to complement inhibitor-based treatments.

scholarly journals The biological properties of HEK293T cell line transfected with mCD150 and nCD150 isoforms of CD150/SLAMF1 receptor

2020 ◽  
Vol 36 (2) ◽  
pp. 99-109
Author(s):  
L. M. Shlapatska ◽  
I. M. Gordiienko ◽  
L. M. Kovalevska ◽  
S. P. Sidorenko
Keyword(s):  
Cell Line ◽  
Hek293t Cell ◽  
Biological Properties ◽  
Hek293t Cell Line

Polycistronic expression of the mitochondrial steroidogenic P450scc system in the HEK293T cell line

2018 ◽  
Vol 120 (3) ◽  
pp. 3124-3136 ◽  
Author(s):  
Vera S. Efimova ◽  
Ludmila V. Isaeva ◽  
Anastasia A. Labudina ◽  
Vadim N. Tashlitsky ◽  
Mikhail A. Rubtsov ◽  
...  
Keyword(s):  
Cell Line ◽  
Hek293t Cell ◽  
Hek293t Cell Line

scholarly journals Generation and utilization of a HEK-293T murine GM-CSF expressing cell line

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0249117
Author(s):  
Elektra Kantzari Robinson ◽  
Sergio Covarrubias ◽  
Simon Zhou ◽  
Susan Carpenter
Keyword(s):  
Dendritic Cells ◽  
Cell Line ◽  
Alveolar Macrophages ◽  
Hek293t Cell ◽  
293T Cell ◽  
Gm Csf ◽  
Viable Cells ◽  
Hek293t Cell Line ◽  
Economical Alternative

Macrophages and dendritic cells (DCs) are innate immune cells that play a key role in defense against pathogens. In vitro cultures of bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) are well-established and valuable methods for immunological studies. Typically, commercially available recombinant GM-CSF is utilized to generate BMDCs and is also used to culture alveolar macrophages. We have generated a new HEK-293T cell line expressing murine GM-CSF that secretes high levels of GM-CSF (~180 ng/ml) into complete media as an alternative to commercial GM-CSF. Differentiation of dendritic cells and expression of various markers were kinetically assessed using the GM-CSF HEK293T cell line, termed supGM-CSF and compared directly to purified commercial GMCSF. After 7–9 days of cell culture the supGM-CSF yielded twice as many viable cells compared to the commercial purified GM-CSF. In addition to differentiating BMDCs, the supGM-CSF can be utilized to culture functionally active alveolar macrophages. Collectively, our results show that supernatant from our GM-CSF HEK293T cell line supports the differentiation of mouse BMDCs or alveolar macrophage culturing, providing an economical alternative to purified GM-CSF.

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