ABSTRACT
The purpose of this study was to compare mRNA levels of the cytotoxic lymphocyte (CL) gene products: granzyme B (GB), perforin (P), and fas ligand (FasL) in patients with long-term type 1 diabetes and healthy controls. The objective was to utilize this information to follow patients as they undergo islet cell transplantation at our center and to determine if changes in CL gene transcript levels correlate with graft status. We have measured mRNA levels for CL genes in peripheral blood samples from 65 long-term (>5 years) type 1 diabetes patients and 29 healthy controls. Total RNA was extracted from EDTA anticoagulated peripheral blood samples and reverse transcribed into first-strand cDNA using SuperScript II reverse Transcriptase. Quantitative, real-time PCR was utilized to determine CL gene transcript levels. mRNA levels of P and FasL genes were found to be significantly lower for patients with type 1 diabetes compared to normal controls (p < 0.05). However, there was no significant difference for GB mRNA levels between patients and controls (p > 0.05). The decreased expression of P and FasL in patients with long-term type 1 diabetes might contribute to the inability to maintain normal levels of peripheral tolerance, which is essential for protection from autoimmune disease.
Subject(s)
Diabetes Mellitus, Type 1/metabolism , Gene Expression Regulation , Membrane Glycoproteins/biosynthesis , Serine Endopeptidases/biosynthesis , T-Lymphocytes, Cytotoxic/metabolism , Tumor Necrosis Factors/biosynthesis , Diabetes Mellitus, Type 1/genetics , Fas Ligand Protein , Female , Granzymes , Humans , Immune Tolerance , Male , Membrane Glycoproteins/genetics , Perforin , Pore Forming Cytotoxic Proteins , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Serine Endopeptidases/genetics , Tumor Necrosis Factors/geneticsABSTRACT
Studies in nonhuman primates have demonstrated that elevation of the cytotoxic lymphocyte (CL) genes granzyme B, perforin, and Fas ligand in peripheral blood precedes islet allograft rejection. The purpose of this study was to determine whether this approach has utility for prediction of human islet allograft loss. We studied 13 patients who had long-term type 1 diabetes and were treated with steroid-free immunosuppression and given sequential islet cell infusions. All recipients became insulin independent, and eight of them experienced deterioration in glycemic control, followed by reinitiation of insulin therapy. Frequent peripheral blood samples were collected to monitor CL gene mRNA levels with real-time PCR. For the eight back-to-insulin patients, there was a clear elevation of CL gene mRNA levels 25-203 days before the onset of frequent hyperglycemia. Granzyme B was the most reliable indicator of ongoing graft loss. Additional correlations with infection were noted; however, evidence of sensitization in antidonor mixed lymphocyte reaction was observed in seven of eight patients who experienced partial graft loss, whereas this was not seen when upregulated CL gene expression was associated with infection. The results suggest that, when taken into consideration with other clinical parameters, elevated CL gene levels may enable prediction of islet allograft loss.
