Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.79 (granzyme B)
 3,301 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The serine protease granzyme B (GrB) is a key effector molecule in cell-mediated immunity, released by cytotoxic lymphocytes (CLs) to induce cell death in neoplastic or virus-infected cells. The ability to detect and measure GrB activity is important for understanding CLs. Unfortunately, such analyses are complicated by significant differences in the substrate specificities of human and mouse GrB, which is reflected by their different activities on commonly used peptide substrates. Here, we present methods for the detection of active human and mouse GrB in extracts from primary cells, and evaluate the sensitivity of the various substrates and inhibitors. Mouse splenocytes produce approximately 120-fold more GrB than similarly activated human cells, which allows the use of the hGrB substrate IETD-AFC to follow mouse GrB activity despite its unfavourable kinetic properties.
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PMID:Detection of human and mouse granzyme B activity in cell extracts. 2226 48

Granzyme B (GrB) is an essential cytotoxic effector in cancer immunotherapy as it can be a potential biomarker to predict the efficacy of immunotherapies including checkpoint inhibitors. Monitoring the Granzyme B activity in cells would help determine a patient's clinical response to treatment and lead to better treatment strategies by preventing administration of ineffective therapies and avoid adverse events resulting in a delay in subsequent treatment. Methods: A microfluidic device with hydrodynamic traps and pneumatic valving system was fabricated using photo and soft lithography. Single cell Granzyme B (GrB) activity was detected and measured fluorometrically using a commercial assay kit with a peptide substrate containing GrB recognition sequence (Ac-IEPD-AFC) and AFC (7-Amino-4-trifluoromethylcoumarin) label. Fluorescence was observed and measured using a confocal microscope with CSU-W1 scanner unit and CCD camera as well as an inverted microscope with photodetector. Model cells (NK-92, GrB-transduced Jurkat, and THP1 cells) and human PBMCs from healthy donor and lung cancer patients including an anti-PD-1 antibody treated patient were profiled of its GrB activity as proof of concept. Results: GrB expression from the model cells was found to be markedly different. NK-92 cells were found to have higher GrB activity than the GrB-transduced Jurkat cells. THP-1 was found to have relatively no significant activity. A marked increase in GrB expression was also observed in anti-PD-1 treated lung cancer patient sample in comparison to PBMC from a healthy donor. TCR+ Ig-G4+ PBMC cells were found to have high activity which signifies a clear response to PD-1 blockade. Conclusion: As proof of concept, we have shown the capability of a microfluidic platform to measure GrB production through a single cell enzymatic activity assay. Our platform might be a promising tool for evaluating the sensitivity of immunotherapies and identifying specific T cell subset responsible for the anti-tumor response.
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PMID:A Microfluidic Platform for Single Cell Fluorometric Granzyme B Profiling. 3190 10