Thymus Function in AGHD 47.2 221.4 4.6 106.3 60.3 2.4 0.1 29.7 8.1 8.9 21.0 73.8 44.0 42.0 24.7 n.d. n.d. n.d. 54.2 14.5 2.2 118.2 Nu 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Cause of AGHD Craniopharyngioma Traumatic brain injury Secreting pituitary adenoma HIV infection and therapy Secreting pituitary adenoma Secreting pituitary adenoma Secreting pituitary adenoma Craniopharyngioma Craniopharyngioma Meningioma Radiotherapy Congenital GH deficiency Nonsecreting pituitary adenoma Sheehan’s syndrome Nonsecreting pituitary adenoma Craniopharyngioma Traumatic brain injury Traumatic brain injury Pinealoma Nonsecreting pituitary adenoma Isolated/idiopathic GH deficiency Secreting pituitary adenoma 67 37 52 54 57 51 65 69 38 64 48 27 62 61 43 51 36 57 46 69 28 49 F M F M F F F M M F M M M F M F M F F F F M 172 330 280 318 232 304 87 123 126 323 136 248 263 154 202 82 365 233 430 178 116 134 119.4 167.9 88.2 778.3 1452.9 361.7 21.9 1.2 30.7 68.4 107.9 216.2 39.3 970.3 650.6 1135.5 656.7 103.4 2833.3 62.1 68.5 3405.9 Table 1. Clinical and baseline characteristics of patients with AGHD before GH withdrawal. Age Sex IGF-1 (ng/ml) sj/ß TREC ratio sjTREC frequency (n/105 cells) n.d.:not determined. doi:10.1371/journal.pone.0005668.t001 for all subsequent experiments. Following the first round of amplification, PCR products were diluted 10-fold prior to online real-time amplification using LightCycler Technology. PCR conditions in LightCycler experiments were as follows: 1 min for initial denaturation at 95uC, 1 sec at 95uC, 15 sec at 60uC, 15 sec at 72uC for 40 cycles. Fluorescence measurements were performed at the end of the elongation steps. For each PCR product, the TREC and CD3c second-round PCR quantifications were performed in separate capillaries and in independent LightCycler experiments, but quantified on the same first-round serially diluted standard curve. This highly sensitive nested quantitative PCR assay allows the detection of one copy per PCR reaction for each DNA circle. The results are expressed as absolute number of TRECs per 105 cells. The quantification of sjTREC frequencies was performed in duplicate. For DbTREC amplification, the first round was performed with 9 primers for different DbTREC amplification. The temperature and cycles were identical to sjTREC amplification. Light Cycler quantification of Db1-Jb1TRECs and Db2-Jb2TRECs were performed independently as described. IGF-1 measurement Serum IGF-1 concentrations were determined using a specific radioimmunoassay (BioSource, Nivelles, Belgium). Statistical analyses Differences between paired groups were assessed by Wilcoxon test. Spearman’s test was used to assess correlation between two distinct parameters. Age- and IGF-1–dependency of sjTREC were fitted to an exponential regression model [6]. All statistical analyses were performed on GraphPad Prism4. Results Plasma IGF-1 concentrations As shown in Fig. 1A and as expected, plasma IGF-1 concentrations significantly decreased one month after interrup- tion of GH treatment in 21 out of 22 patients with GHDA. One month after reintroduction of GH supplementation, plasma IGF-1 concentrations significantly increased in 21 out of 22 patients to reach a level, which was similar to the one measured before stopping GH treatment. The most important decrease in plasma IGF-1 was observed in patient #19, a female (46 yr-old) with a surgically treated pinealoma. sjTREC quantification In 19 out of 22 patients with GHDA, the arrest of treatment induced a significant decrease in sjTREC frequency, while GH resumption was followed by a significant rebound in the same patients (Fig. 1B). Two dramatic decreases were observed: the first patient (from 900 to 30 sjTREC/105 cells) was patient #4, a male (44 yr-old) with AGHD in the context of a treated HIV infection, the second patient (from 1000 to 2 sjTREC/105 cells) was patient #5, a female (57 yr-old) with a PRL-secreting adenoma that had been surgically treated. A significant positive correlation (P,0.01) was observed between sjTREC levels and plasma IGF-1 concentrations (Fig. 1C). PLoS ONE | www.plosone.org 3 May 2009 | Volume 4 | Issue 5 | e5668