The expression of and genes was modulated during follow-up; however, this expression was not similar to controls at D+720 post- HDI-AHSCT (data not shown). In relation to anti-apoptotic gene expression, we observed a re-establishment of and gene expression levels, similar to that found in controls, in patients’ PBMCs at 720 post-HDI-AHSCT (Fig. with glutamic acid decarboxylase autoantibodies (GAD65) autoantibody levels. Taken together, the results suggest that apoptosis-related genes deregulation in patients’ PBMCs might be involved in breakdown of immune tolerance and consequently contribute to T1D pathogenesis. Furthermore, HDI-AHSCT modulated the expression of some apoptotic genes towards the levels similar to controls. Possibly, the expression of these apoptotic molecules could be applied as biomarkers of clinical remission of T1D patients treated with HDI-AHSCT therapy. and (Bcl-2 family); and (IAP family); extrinsic pathway gene and pro-apoptotic genes and (Bcl-2 family), and (death receptor family) was performed by SYBR? Green PCR Master Becampanel Mix Kit (Applied Biosystems, Foster City) on a 7500 real-time PCR system (Applied Biosystems, Foster City). The PCR mixture Becampanel consisted of 40 ng of cDNA, 100 M of forward and reverse primers, 75 L of SYBR? Green PCR Master Mix and 45 l of deionized water to a final volume of 15 l. The PCR conditions were: one cycle at 50C for 2 min, 95C for 10 min and 50 cycles at 95C for 15 s, 54C62C for 25 s (annealing temperatures were determined for each gene) and 72C for 34 s. For detection of Becampanel anti-apoptotic and pro-apoptotic gene expression, we used the sequence primers described in Table 2. The -and genes were used as housekeeping genes and the relative expression of the studied target genes were obtained after normalizing using the geometric average of the housekeeping gene mRNA levels. All reactions were duplicated and gene expression was calculated using the relative expression units (REU) method [32]. Table 2 Primer sequences, amplicon size, and annealing temperature of apoptosis-related genes. < 005) in pro-apoptotic (median: 0066), (0298) and (6101) expression in patients' PBMCs when compared to controls (median (0552), (2543) and (9516) gene expression in T1D patients in relation to controls (median < 005) in anti-apoptotic genes (1080), (2529) and (2577) in patients' PBMCs compared to controls (median (7778) gene expression compared to controls (median and gene expression between T1D patients and controls (data not shown). Figure 8a summarizes the results obtained when gene expression data were compared between patients and controls. Open in a separate window Fig. 1 Apoptosis-related pro-apoptotic gene expression profile in type 1 diabetes (T1D) patients; (aCc) and expression was down-regulated in T1D patients' peripheral blood mononuclear cells (PBMCs) (= 14) in comparison to controls (= 14); (dCf) and expression was up-regulated in T1D patients' PBMCs (= 14) in comparison to controls (= 14). Statistical analysis was performed by MannCWhitney and expression was up-regulated in T1D patients' peripheral blood mononuclear cells (PBMCs) (= 14) in comparison to controls (= 14); (d) expression was down-regulated in T1D patients' PBMCs (= 14) in comparison to controls (= 14). Statistical analysis was performed by MannCWhitney (median pre-Tx: 6784; < 005) and (median pre-Tx: 6101; < 00001) gene expression in post-HDI-AHSCT periods, mainly at Becampanel D+180 (median Agt (median pre-Tx:2529; Becampanel = 0001) gene expression at D+540 (median: 5696) and D+720 (1000) post-HDI-AHSCT periods was detected (Figs 3c and 8b). Open in a separate window Fig. 3 Modulation of apoptosis-related gene expression in type 1 diabetes (T1D) patients by high-dose immunosuppression followed by autologous haematopoietic stem cell transplantation (HDI-AHSCT) therapy; (a,b) and expression was up-regulated in T1D patients’ peripheral blood mononuclear cells (PBMCs) at D+360 post-HDI/AHSCT (= 14) in comparison to pre-HDI-AHSCT (= 14); (c) expression was down-regulated in T1D patients’ PBMCs at D+540 post-HDI/AHSCT (= 14) in comparison to pre-HDI-AHSCT (= 14). Statistical analysis was performed by Friedman followed by Dunns’ post-test. The box-plots show the median (horizontal bars), standard deviation, lower and upper quartiles. and pro-apoptotic gene expression similar to controls (Fig. 8b). The expression of and.