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#35044824   2022/01/19 To Up

Up-regulation of proBDNF/p75 signaling in antibody-secreting cells drives systemic lupus erythematosus.

Inappropriate expansion of antibody-secreting cells (ASCs) is typical of systemic lupus erythematosus (SLE), but the regulatory signaling of pathogenic ASCs is unclear. The present study shows that brain-derived neurotrophic factor precursor (proBDNF) and its high-affinity pan-75 neurotrophin receptor (p75) are highly expressed in CD19CD27CD38 ASCs in patients with SLE and in CD19CD44CD138 ASCs in lupus-like mice. The increased proBDNF ASCs were positively correlated with clinical symptoms and higher titers of autoantibodies in SLE. Administration of monoclonal antibodies against proBDNF or specific knockout of p75 in CD19 B cells exerted a therapeutic effect on lupus mice by limiting the proportion of ASCs, reducing the production of autoantibodies and attenuating kidney injury. Blocking the biological function of proBDNF or p75 also inhibits ASC differentiation and antibody production in vitro. Together, these findings suggest that proBDNF-p75 signaling plays a critical pathogenic role in SLE through promoting ASC dysfunction.
Wei-Yun Shen, Cong Luo, Plinio Reinaldo Hurtado, Xiao-Jing Liu, Ru-Yi Luo, Hui Li, Zhao-Lan Hu, Jun-Mei Xu, Elizabeth J Coulson, Ming Zhao, Xin-Fu Zhou, Ru-Ping Dai

2734 related Products with: Up-regulation of proBDNF/p75 signaling in antibody-secreting cells drives systemic lupus erythematosus.

96 wells1 Set1 Set1 Set1 Set50ug11 inhibitors100ug Lyophilized100ug100ug Lyophilized1 Set1 Set

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#35044813   2022/01/19 To Up

From structure to sequence: Antibody discovery using cryoEM.

One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and bioinformatic approach to directly assign the heavy and light chains, identify complementarity-determining regions, and discover sequences from cryoEM density maps of serum-derived polyclonal antibodies bound to an antigen. When combined with next-generation sequencing of immune repertoires, we were able to specifically identify clonal family members, synthesize the monoclonal antibodies, and confirm that they interact with the antigen in a manner equivalent to the corresponding polyclonal antibodies. This structure-based approach for identification of monoclonal antibodies from polyclonal sera opens new avenues for analysis of immune responses and iterative vaccine design.
Aleksandar Antanasijevic, Charles A Bowman, Robert N Kirchdoerfer, Christopher A Cottrell, Gabriel Ozorowski, Amit A Upadhyay, Kimberly M Cirelli, Diane G Carnathan, Chiamaka A Enemuo, Leigh M Sewall, Bartek Nogal, Fangzhu Zhao, Bettina Groschel, William R Schief, Devin Sok, Guido Silvestri, Shane Crotty, Steven E Bosinger, Andrew B Ward

2934 related Products with: From structure to sequence: Antibody discovery using cryoEM.

3 modules1 module2 modules1 module1 module1 module1 module1 module200 ug1 module100 ul1 module

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#35044720   2022/01/19 To Up

High throughput glycosylation analysis of intact monoclonal antibodies by mass spectrometry coupled with capillary electrophoresis and liquid chromatography.

The analysis of monoclonal antibodies glycosylation is a crucial quality control attribute of biopharmaceutical drugs. High throughput screening approaches for antibody glycoform analysis are required in various stages of process optimization. Here, we present high throughput screening suitable mass spectrometry-based workflows for the analysis of intact antibody glycosylation out of cell supernatants. Capillary electrophoresis and liquid chromatography were coupled with quadrupole time-of-flight MS or Orbitrap MS. Both separation methods offer fast separation (10-15 min) and the capability to prevent the separated cell supernatant matrix to enter the MS by post-separation valving. Both MS instruments provide comparable results and both are sufficient to determine the glycosylation pattern of the five major glycoforms of the measured antibodies. However, the Orbitrap yields higher sensitivity of 25 μg/mL (CE-nanoCEasy-Orbitrap MS) and 5 μg/mL (LC-Orbitrap MS). Data processing was optimized for a faster processing and easier detection of low abundant glycoforms based on averaged charge-deconvoluted mass spectra. This approach combines a non-target glycoform analysis, while yielding the same glycosylation pattern as the traditional approach based on extracted ion traces. The presented methods enable the high throughput screening of the glycosylation pattern of antibodies down to low μg/mL-range out of cell supernatant without any sample preparation. This article is protected by copyright. All rights reserved.
Lukas Naumann, Patrick Schlossbauer, Florian Klingler, Friedemann Hesse, Kerstin Otte, Christian Neusüß

1425 related Products with: High throughput glycosylation analysis of intact monoclonal antibodies by mass spectrometry coupled with capillary electrophoresis and liquid chromatography.

100.00 ug100 ug2 Pieces/Box100.00 ug100 ug100.00 ug1 mg100 ug100.00 ug1 mg100.00 ug1 mg

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#35044456   2022/01/14 To Up

PTPN2 Regulates the Interferon Signaling and Endoplasmic Reticulum Stress Response in Pancreatic β-Cells in Autoimmune Diabetes.

Type 1 diabetes (T1D) results from autoimmune destruction of β-cells in the pancreas. Protein tyrosine phosphatases (PTPs) are candidate genes for T1D and play a key role in autoimmune disease development and β-cell dysfunction. Here, we assessed the global protein and individual PTP profiles in the pancreas from early onset non-obese diabetic (NOD) mice treated with an anti-CD3 monoclonal antibody and interleukin-1 receptor antagonist. The treatment reversed hyperglycemia and we observed enhanced expression of PTPN2, a PTP family member and T1D candidate gene, and endoplasmic reticulum (ER) chaperones in the pancreatic islets. To address the functional role of PTPN2 in β-cells, we generated PTPN2-deficient human stem cell-derived β-like and EndoC-βH1 cells. Mechanistically, we demonstrated that PTPN2 inactivation in β-cells exacerbates type I and type II interferon signaling networks and the potential progression towards autoimmunity. Moreover, we established the capacity of PTPN2 to positively modulate the Ca2+-dependent unfolded protein response and ER stress outcome in β-cells. Adenovirus-induced overexpression of PTPN2 partially protected from ER-stress induced β-cell death. Our results postulate PTPN2 as a key protective factor in β-cells during inflammation and ER stress in autoimmune diabetes.
Bernat Elvira, Valerie Vandenbempt, Julia Bauzá-Martinez, Raphaël Crutzen, Javier Negueruela, Hazem Ibrahim, Matthew L Winder, Manoja K Brahma, Beata Vekeriotaite, Pieter-Jan Martens, Sumeet Pal Singh, Fernando Rossello, Pascale Lybaert, Timo Otonkoski, Conny Gysemans, Wei Wu, Esteban N Gurzov

1446 related Products with: PTPN2 Regulates the Interferon Signaling and Endoplasmic Reticulum Stress Response in Pancreatic β-Cells in Autoimmune Diabetes.

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#35044295   2022/01/19 To Up

Sensitizing to antibacterial agents by decoding and blocking the lipid flippase MprF.

The pandemic of antibiotic resistance represents a major human health threat demanding new antimicrobial strategies. MprF is the synthase and flippase of the phospholipid lysyl-phosphatidylglycerol that increases virulence and resistance of methicillin-resistant (MRSA) and other pathogens to cationic host defense peptides and antibiotics. With the aim to design MprF inhibitors that could sensitize MRSA to antimicrobial agents and support the clearance of staphylococcal infections with minimal selection pressure, we developed MprF-targeting monoclonal antibodies, which bound and blocked the MprF flippase subunit. Antibody M-C7.1 targeted a specific loop in the flippase domain that proved to be exposed at both sides of the bacterial membrane, thereby enhancing the mechanistic understanding of bacterial lipid translocation. M-C7.1 rendered MRSA susceptible to host antimicrobial peptides and antibiotics such as daptomycin, and it impaired MRSA survival in human phagocytes. Thus, MprF inhibitors are recommended for new anti-virulence approaches against MRSA and other bacterial pathogens.
Christoph Josef Slavetinsky, Janna Nadine Hauser, Cordula Gekeler, Jessica Slavetinsky, André Geyer, Alexandra Kraus, Doris Heilingbrunner, Samuel Wagner, Michael Tesar, Bernhard Krismer, Sebastian Kuhn, Christoph M Ernst, Andreas Peschel

1623 related Products with: Sensitizing to antibacterial agents by decoding and blocking the lipid flippase MprF.

480/kit50 50 ug50 ug50.00 ug1 module10 mg50 ug

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#35044182   2022/01/19 To Up

Novel VHH-Based Tracers with Variable Plasma Half-Lives for Imaging of CAIX-Expressing Hypoxic Tumor Cells.

Hypoxic areas are present in the majority of solid tumors, and hypoxia is associated with resistance to therapies and poor outcomes. A transmembrane protein that is upregulated by tumor cells that have adapted to hypoxic conditions is carbonic anhydrase IX (CAIX). Therefore, noninvasive imaging of CAIX could be of prognostic value, and it could steer treatment strategies. The aim of this study was to compare variants of CAIX-binding VHH B9, with and without a C-terminal albumin-binding domain with varying affinity (ABD and ABD), for SPECT imaging of CAIX expression. The binding affinity and internalization of the various B9-variants were analyzed using SK-RC-52 cells. Biodistribution studies were performed in mice with subcutaneous SCCNij153 human head and neck cancer xenografts. Tracer uptake was determined by radioactivity counting and visualized by SPECT/CT imaging. Furthermore, autoradiography images of tumor sections were spatially correlated with CAIX immunohistochemistry. B9-variants demonstrated a similar moderate affinity for CAIX . Maximal tumor uptake and acceptable tumor-to-blood ratios were found in the SCCNij153 model at 4 h post injection for [In]In-DTPA-B9 (0.51 ± 0.08%ID/g and 8.1 ± 0.85, respectively), 24 h post injection for [In]In-DTPA-B9-ABD (2.39 ± 0.44%ID/g and 3.66 ± 0.81, respectively) and at 72 h post injection for [In]In-DTPA-B9-ABD (8.7 ± 1.34%ID/g and 2.43 ± 0.15, respectively) An excess of unlabeled monoclonal anti-CAIX antibody efficiently inhibited tumor uptake of [In]In-DTPA-B9, while only a partial reduction of [In]In-DTPA-B9-ABD and [In]In-DTPA-B9-ABD uptake was found. Immunohistochemistry and autoradiography images showed colocalization of all B9-variants with CAIX expression; however, [In]In-DTPA-B9-ABD and [In]In-DTPA-B9-ABD also accumulated in non-CAIX expressing regions. Tumor uptake of [In]In-DTPA-B9-ABD and [In]In-DTPA-B9-ABD, but not of [In]In-DTPA-B9, could be visualized with SPECT/CT imaging. In conclusion, [In]In-DTPA-B9 has a high affinity to CAIX and shows specific targeting to CAIX in head and neck cancer xenografts. The addition of ABD prolonged plasma half-life, increased tumor uptake, and enabled SPECT/CT imaging. This uptake was, however, partly CAIX- independent, precluding the ABD-tracers for use in hypoxia quantification in this tumor type.
Sanne A M van Lith, Fokko J Huizing, Gerben M Franssen, Bianca A W Hoeben, Jasper Lok, Sofia Doulkeridou, Otto C Boerman, Martin Gotthardt, Paul M P van Bergen En Henegouwen, Johan Bussink, Sandra Heskamp

1961 related Products with: Novel VHH-Based Tracers with Variable Plasma Half-Lives for Imaging of CAIX-Expressing Hypoxic Tumor Cells.

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#35043970   2022/01/19 To Up

History and development of staining methods for skeletal muscle fiber types.

The contractile and metabolic properties of skeletal muscles depend on the composition of muscle fibers. There are two major fiber types: type 1 and type 2. Type 2 fibers are further subdivided into type 2A, 2X, and 2B fibers. Muscle fiber type composition is an important property that affects sports performance and metabolic ability in humans, and meat quality in domestic animals. In this review, we summarize the history of muscle fiber type classification based on various staining methods for skeletal muscle sections. The history illustrates the development of an experimental method to detect myosin heavy chain (MyHC) proteins, which are the most common marker molecules for muscle fiber type. Metabolic enzymes, such as nicotinamide adenine dinucleotide-tetrazolium reductase and succinate dehydrogenase are also described for histochemical staining combined with myosin ATPase staining. We found an improvement in the quality of antibodies used for immunostaining of MyHC, from polyclonal antibodies to monoclonal antibodies (mAbs) and then to mAbs produced by synthetic peptides as antigens. We believe that the information presented herein will assist researchers in selecting optimal staining methods, dependent on the experimental conditions and purposes.
Shoko Sawano, Wataru Mizunoya

1902 related Products with: History and development of staining methods for skeletal muscle fiber types.

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#35043561   2022/01/19 To Up

Rapid monitoring of high-mannose glycans during cell culture process of therapeutic monoclonal antibodies using lectin affinity chromatography.

We developed a simple HPLC assay to monitor high-mannose glycans in monoclonal antibodies by monitoring terminal alpha-mannose as a surrogate marker. Analysis of glycan data of therapeutic monoclonal antibodies by 2-aminobenzamide assay showed a linear relationship between high mannose and terminal mannose of Fc glycans. Concanavalin A has strong affinity to alpha-mannose in glycans of typical therapeutic monoclonal antibodies. To show that terminal mannose binds specifically to Concanavalin A column, exoglycosidase-treated monoclonal antibodies were serially blended with untreated monoclonal antibodies. Linear responses of terminal-mannose binding to the column and comparable data trending with high mannose levels by 2-aminobenzamide assay confirmed that terminal-mannose levels measured by the Concanavalin A column can be used as surrogate for the prediction of high-mannose levels in monoclonal antibodies. The assay offers a simple, fast, and specific capability for prediction of high-mannose content in samples compared with traditional glycan profiling by 2-aminobenzamide, or mass spectrometry-based methods. When the Concanavalin A column was coupled with protein A column for purification of antibodies from cell culture samples in a fully automated two-dimensional analysis, high-mannose data could be relayed to the manufacturing team in less than 30 minutes, allowing near-real-time monitoring of high-mannose levels in cell culture process. This article is protected by copyright. All rights reserved.
Jun Kim, Methal Albarghouthi

2267 related Products with: Rapid monitoring of high-mannose glycans during cell culture process of therapeutic monoclonal antibodies using lectin affinity chromatography.

100.00 ug100.00 ug100.00 ug100.00 ug100.00 ug200 ug50 g5 mg100.00 ug100.00 ug1100ul

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