tion in mice of an antiserum against GH was shown to induce thymic atrophy, whereas neonatal thymectomy was associated with degranulation of GH-secreting acidophil cells in the anterior pituitary [14]. The implantation of GH-secreting cells from GH3 pituitary adenoma increases thymic size in aged rats [15], and GH administration increases thymic cellularity and thymic T cell proliferation in GH- and prolactin (PRL)-deficient dwarf DW/J mice [16]. Nevertheless, the thymotropic effects of GH evidenced in hypophysectomised rodents and in genetic models of pituitary hormone deficiency did not gain universal acceptance [17]. Parameters of immunodeficiency reported in these animals may depend upon breeding conditions [18,19]. The thymotrope properties of GH may be partially explained by counteraction of stress-induced immunosuppressive glucocorticoids. GH is able to activate Jak2/Stat5 pathway [20], and activated Stat5 inhibits 75% of the actions promoted by glucocorticoids in lymphoid cells [21], including apoptosis of pre-T cells. The mechanisms underlying GH thymotropic actions in genetic/hypophysecto- mised models remain to be further deciphered using more specific and sensitive methods. Human recombinant GH also promotes human T cell grafting in SCID mice, and this xenograft is associated with a marked colonization of murine thymus by human T cells [22]. As a major mediator of GH actions, IGF-1 closely regulates thymic homing of T-cell precursors, thymopoi- esis, thymocyte traffic within the thymus microenvironment, as well as a number of peripheral immune functions [23–26]. Very interestingly, it was recently reported that infusion in old mice of ghrelin, a GH secretagogue, significantly improves thymopoiesis, increases the number of recent thymic emigrants, and improves TCR diversity of the peripheral T cell repertoire [27]. While the GH/IGF-1 axis, as well as other hormones, is not an essential factor for normal thymus and T-cell function, there is now strong evidence from animal and preclinical studies that they can be effective modulators and used as pharmacological agents in specific situations. This important and well-documented preclinical background has promoted recent clinical studies that are exploring the use of endocrine-based therapies aiming to enhance thymopoiesis in immunodeficient individuals, and in particular to evaluate the potential benefit of GH treatment for immune restoration through stimulation of thymopoiesis in HIV-1 – infected patients. A first pilot study showed that GH treatment reverses thymic atrophy and enhances the number of circulating naive CD4 T cells in HIV-infected adults [28]. This was confirmed by a prospective randomized study, which further showed that GH treatment strongly increases the number of circulating sjTRECs in peripheral blood mononuclear cells [29]. However, the HIV- infected patients from this latter study received very high doses of GH (3 mg daily for 6 months, followed by 1.5 mg daily), and some interference with anti-retroviral therapy could not be excluded, according to the authors themselves. Until now, no data are available with regard to the thymic function once adult GH deficiency (AGHD) has been diagnosed and documented accord- ing to appropriate guidelines. The principal objective of this clinical research study was to directly investigate the impact of GH deficiency and GH supplementation at physiological doses upon thymus function and thymopoiesis in AGHD. Methods The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1. Thymus Function in AGHD Study design and patients Twenty-two patients were enrolled in this study at the Department of Medicine in Liege University Hospital. There were 10 males and 12 females, with an age range between 27 and 69 years. The protocol was approved by the Ethical Committee of Liege Medical School and University Hospital, and each enrolled patient signed an informed consent. All patients had been diagnosed at least two years before with AGHD according to the guidelines of clinical practice established by the Endocrine Society [30]. Causes of AGHD in the patients are listed in Table 1. At the time of their enrolment, all patients were being treated with recombinant human GH; the range of GH dosage was 0.2–0.5 mg daily in one subcutaneous (sc) injection at bedtime and, for each patient, the dosage has been adjusted for at least 6 months to maintain IGF-1 blood concentrations in physiological levels (maintenance dose). Patients also received conventional and adequate replacement therapy in case of associated pituitary hormone deficiencies. The biological parameters of the study were measured at three different time points: first during GH treatment, secondly 1 month after GH withdrawal, and thirdly 1 month after resumption of GH treatment at the dosage used before discontinuation. Given the design of the study, there were neither major inclusion nor exclusion criteria for this study, and the patients did not present any side effects (hyperglycemia or water retention) due to GH treatment. Another major advantage was that each patient constituted his/her own control, and only one parameter (GH administration or not) was modified during the study. Isolation of peripheral blood mononuclear cells (PBMCs) Twenty-four milliliters of blood were collected at each time of the study, and PBMCs were purified by Ficoll-Paque gradient centrifugation (Becton-Dickinson Vacutainer CPT). Cells were washed twice in HBSS, suspended in DPBS, adjusted at 108 cells/ ml, and stored at 280uC. One million cells were used either for sjTREC or DbTREC measurements. TREC analyses Specific primers for the sjTRECs (dRec-yJa), the 13 different DJbTRECs (Db1–Jb1.1–Jb1.6, and Db2–Jb2.1–Jb2.7), and the human CD3c-chain have been previously defined (7,8). In order to amplify the Db2–Jb2.5 to Db2–Jb2.7 TRECs, a set of 3 additional reverse Jb primers were added to the published ones with the following sequences: 2.5-out (59-GCCGGGACCCGGCTCT- CAGT-39), 2.5-in (59-CGGCTCTCAGTGCTGGGTAA-39), 2.6- out (59-TGACCAAGAGACCCAGTA-39), 2.6-in (59-GTCTG- GTTTTTGCGGGGAGT-39), 2.7-out (59-TGACCGTGCTGGG- TGAGTT-39), 2.7-in (59-GGAGCTCGGGGAGCCTTA-39). All primers were purchased from Eurogentec (Seraing, Belgium). Three plasmids were used to generate standard curves for real-time quantitative PCR-based assay, each containing two inserted amplicons (CD3c with either sjTREC, Db1–Jb1.4, or Db2–Jb2.3) amplified in the same run as experimental samples. Parallel quantification of each TREC together with the CD3c amplicon was performed for each sample using LightCycler Technology (Roche Diagnostics, Basel, Switzerland). This protocol precisely measures the input DNA for quantification, thus providing an absolute number of TRECs per 105 cells. Approximately 106 PBMC were lyzed in Tween-20 (0.05%), Nonidet P-40 (NP-40, 0.05%), and proteinase K (100 mg/ml) for 30 min at 56uC, and for 15 min at 98uC. Multiplex PCR amplification was performed for sjTREC, together with the CD3c chain, in 100 ml (10 min for initial denaturation at 95uC, 30 sec at 95uC, 30 sec at 60uC, 2 min at 72uC for 22 cycles) using the ‘outer’ 39/59 primer pairs. These PCR conditions were used PLoS ONE | www.plosone.org 2 May 2009 | Volume 4 | Issue 5 | e5668