Table 1.
Patient demographics and clinical data for the 10 post-liver transplant patients and 9 control patients whose liver sections were analyzed for markers of apoptosis.
Fig 1.
Increased hepatocyte apoptosis is seen after liver transplantation on immunohistochemistry staining of liver tissue.
Photomicrographs show immunohistochemical staining of human liver sections for M30 CytoDEATH and cleaved PARP at 200x magnification, indicating increased levels of these markers of apoptosis in post-liver transplant patients compared to healthy subjects (arrows indicate positive staining cells). Graphs show the relative staining of these apoptotic markers in the liver of each patient assessed by computerized image capture quantification with (A) M30 CytoDEATH and (B) cleaved PARP results expressed as mean proportional area stained within the given area and error bars represent SEM.
Fig 2.
Cyclosporine at therapeutic concentrations does not increase hepatocyte cell death.
(A) Percentage reduction in cell viability from crystal violet assays of PMoH treated with 1 μg/ml of cyclosporine compared to untreated cells. (B) Western blots of cleaved PARP and cleaved caspase 3 levels in PMoH treated with 1 μg/ml of cyclosporine compared to untreated cells. Graphs show fold change in cleaved PARP and cleaved caspase 3 levels relative to untreated cells. Each bar represents the average of 3 experiments and error bar represents SEM. P-values are compared to untreated hepatocytes.
Fig 3.
Mycophenolate mofetil reduces hepatocyte cell death in a dose-dependent manner.
(A) Percentage increase in cell viability from crystal violet assays of PMoH treated with 5 μg/ml of MMF, compared to untreated cells. (B) Western blots of cleaved PARP and cleaved caspase 3 levels in PMoH treated with 5 μg/ml of MMF at 48 hours compared to untreated cells. Graphs show fold change in cleaved PARP and cleaved caspase 3 levels relative to untreated cells. Each bar represents the average of 3 experiments and error bar represents SEM. P-values are compared to untreated hepatocytes.
Fig 4.
The combination of cyclosporine and mycophenolate mofetil enhances cell death in hepatocytes.
(A) Percentage reduction in cell viability from crystal violet assays of PMoH treated with 1 μg/ml of cyclosporine and/or 5 μg/ml of MMF compared to untreated cells. (B) Western blots of cleaved PARP and cleaved caspase 3 levels in PMoH treated with 1 μg/ml of cyclosporine and/or 5 μg/ml of MMF compared to untreated cells. Graphs show fold change in cleaved PARP and cleaved caspase 3 levels in cyclosporine- and/or MMF-treated PMoH at 48 hours relative to untreated cells. Each bar represents the average of 3 experiments and error bar represents SEM. P-values are compared to untreated hepatocytes.
Fig 5.
Tacrolimus at therapeutic concentrations does not affect hepatocyte cell death.
(A) Percentage reduction in cell viability from crystal violet assays of PMoH treated with 0.005 μg/ml of tacrolimus at 24–72 hours compared to untreated cells. (B) Western blots of cleaved PARP and cleaved caspase 3 levels in PMoH treated with 0.005 μg/ml of tacrolimus at 48 hours compared to untreated cells. Graphs show fold change in cleaved PARP and cleaved caspase 3 levels in tacrolimus-treated PMoH relative to untreated cells. Each bar represents the average of 3 experiments and error bar represents SEM. P-values are compared to untreated hepatocytes.
Fig 6.
The combination of tacrolimus and MMF promotes cell death in hepatocytes.
(A) Percentage reduction in cell viability from crystal violet assays of PMoH treated with 0.005 μg/ml of tacrolimus ± 5 μg/ml of MMF or 1 μg/ml of cyclosporine + 5 μg/ml of MMF at 24–72 hours compared to untreated cells. (B) Western blots of cleaved PARP and cleaved caspase 3 levels in PMoH treated with 0.005 μg/ml of tacrolimus ± 5 μg/ml of MMF or 1 μg/ml of cyclosporine + 5 μg/ml of MMF at 48 hours compared to untreated cells. Graphs show fold change in cleaved PARP and cleaved caspase 3 levels in tacrolimus/MMF-treated PMoH and cyclosporine/MMF-treated cells compared to untreated hepatocytes. Each bar represents the average of 3 experiments and error bar represents SEM. P-values are compared to untreated hepatocytes.
Fig 7.
Sirolimus at therapeutic concentrations does not increase hepatocyte cell death.
(A) Percentage reduction in cell viability from crystal violet assays of PMoH treated with 0.01 μg/ml of sirolimus at 24–72 hours compared to untreated cells. (B) Western blots of cleaved PARP and cleaved caspase 3 levels in PMoH treated with 0.01 μg/ml of sirolimus at 48 hours compared to untreated cells. Graphs show fold change in cleaved PARP and cleaved caspase 3 levels in sirolimus-treated PMoH compared to untreated cells. Each bar represents the average of 3 experiments and error bar represents SEM. P-values are compared to untreated hepatocytes.
Fig 8.
The combination of sirolimus and mycophenolate mofetil has no significant effect on cell death in hepatocytes.
(A) Percentage reduction in cell viability from crystal violet assays of PMoH treated with 0.01 μg/ml of sirolimus ± 5 μg/ml of MMF or 0.005 μg/ml of tacrolimus + 5 μg/ml of MMF at 24–72 hours compared to untreated cells. (B) Western blots of cleaved PARP and cleaved caspase 3 levels in PMoH treated with 0.01 μg/ml of sirolimus ± 5 μg/ml of MMF or 0.005 μg/ml of tacrolimus + 5 μg/ml of MMF at 48 hours compared to untreated cells. Graphs show fold change in cleaved PARP and cleaved caspase 3 levels in sirolimus/MMF-treated PMoH and tacrolimus/MMF-treated cells compared to untreated hepatocytes. Each bar represents the average of 3 experiments and error bar represents SEM. P-values are compared to untreated hepatocytes.