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Early assessment of apoptosis in isolated islets of Langerhans. Transplantation 2001 Apr 15;71(7):857-62

Date

05/15/2001

Pubmed ID

11349716

DOI

10.1097/00007890-200104150-00006

Scopus ID

2-s2.0-0035870568 (requires institutional sign-in at Scopus site)   60 Citations

Abstract

BACKGROUND: There is substantial evidence to link early graft loss after islet transplantation to isolation-induced islet cell apoptosis. Measurement of caspase 3 activity and detection of the lost cell membrane asymmetry, revealed by annexin V binding, are newly available assays that allow the analysis of early events of apoptosis.

METHODS: In this study, we compared these tests with the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and analysis of DNA fragmentation after gel electrophoresis in freshly isolated islets obtained from rats, before and after treatment with interleukin-1 beta, interferon gamma, and tumor necrosis factor a, cytokines known to induce islet cell damage.

RESULTS: A measurable level of apoptosis was observed the day after isolation when caspase 3 activity and annexin V binding were used as assays, although no substantial DNA fragmentation was detected with TUNEL assay and DNA gel electrophoresis. Baseline caspase 3 activity was 0.8+/-0.3 U/100 islet equivalents and it increased to 1.4+/-0.45 U/100 islet equivalents 3 hr after cytokine stimulation (P<0.05 vs. unstimulated islets). The baseline level of apoptosis, as detected by annexin V binding, was 21.1%+/-5.8%, and it increased to 27.5%+/-8.1% 6 hr after addition of the cytokine cocktail (P<0.01 vs. unstimulated islets). An increase in the number of TUNEL-positive nuclei was detected 24 hr after stimulation and peaked at 48 hr. DNA laddering was also evident 24 hr after cytokine treatment.

CONCLUSION: These data suggest that measurement of caspase 3 activity and annexin V binding analysis might represent reliable markers of early events of islet cell apoptosis.

Author List

Cattan P, Berney T, Schena S, Molano RD, Pileggi A, Vizzardelli C, Ricordi C, Inverardi L

Author

Stefano Schena MD, PhD Associate Professor in the Surgery department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Animals
Annexin A5
Apoptosis
Biomarkers
Caspase 3
Caspases
DNA Fragmentation
Electrophoresis, Polyacrylamide Gel
In Situ Nick-End Labeling
In Vitro Techniques
Islets of Langerhans
Rats
Rats, Inbred Lew