Early assessment of apoptosis in isolated islets of langerhans

Pierre Cattan, Thierry Berney, Stefano Schena, Ruth Molano, Antonello Pileggi, Caterina Vizzardelli, Camillo Ricordi, Luca A Inverardi

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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 β, interferon γ, and tumor necrosis factor α, 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.

Original languageEnglish
Pages (from-to)857-862
Number of pages6
JournalTransplantation
Volume71
Issue number7
StatePublished - Apr 15 2001

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Islets of Langerhans
Annexin A5
Caspase 3
Apoptosis
Cytokines
DNA Fragmentation
Electrophoresis
Gels
Islets of Langerhans Transplantation
DNA Nucleotidylexotransferase
DNA
Interleukin-1
Interferons
Tumor Necrosis Factor-alpha
Cell Membrane
Transplants
Therapeutics

ASJC Scopus subject areas

  • Transplantation
  • Immunology

Cite this

Cattan, P., Berney, T., Schena, S., Molano, R., Pileggi, A., Vizzardelli, C., ... Inverardi, L. A. (2001). Early assessment of apoptosis in isolated islets of langerhans. Transplantation, 71(7), 857-862.

Early assessment of apoptosis in isolated islets of langerhans. / Cattan, Pierre; Berney, Thierry; Schena, Stefano; Molano, Ruth; Pileggi, Antonello; Vizzardelli, Caterina; Ricordi, Camillo; Inverardi, Luca A.

In: Transplantation, Vol. 71, No. 7, 15.04.2001, p. 857-862.

Research output: Contribution to journalArticle

Cattan, P, Berney, T, Schena, S, Molano, R, Pileggi, A, Vizzardelli, C, Ricordi, C & Inverardi, LA 2001, 'Early assessment of apoptosis in isolated islets of langerhans', Transplantation, vol. 71, no. 7, pp. 857-862.
Cattan P, Berney T, Schena S, Molano R, Pileggi A, Vizzardelli C et al. Early assessment of apoptosis in isolated islets of langerhans. Transplantation. 2001 Apr 15;71(7):857-862.
Cattan, Pierre ; Berney, Thierry ; Schena, Stefano ; Molano, Ruth ; Pileggi, Antonello ; Vizzardelli, Caterina ; Ricordi, Camillo ; Inverardi, Luca A. / Early assessment of apoptosis in isolated islets of langerhans. In: Transplantation. 2001 ; Vol. 71, No. 7. pp. 857-862.
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N2 - 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 β, interferon γ, and tumor necrosis factor α, 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.

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