Histol Histopathol (2011) 26: 471-479 Histology and http://www.hh.um.es Histopathology Cellular and Molecular Biology Neonatal thymulin gene therapy in nude mice: Effects on the morphology of the pituitary corticotrope population Eliana Martines1,2*, Paula C. Reggiani1,3*, José I. Schwerdt1,3, Rodolfo G. Goya1,3 and Gloria Cónsole1,4 1Department of Cytology, Histology and Embryology, Faculty of Medicine, National University of La Plata, 2Adventist University of La Plata (UAP), Entre Ríos, 3INIBIOLP-UNLP and 4CICPBA, Argentina *These two authors contributed equally to this study Summary. The integrity of the thymus during early life and athymic mice. Athymic mice showed only a is necessary for a proper maturation of the marginal reduction in corticotrope CD, VD and CS. In neuroendocrine system, including the adrenal axis. The these mutants hypothalamic CRH content was slightly thymic metallopeptide thymulin seems to be a increased, whereas adrenal corticosterone tended to be central physiologic mediator of thymus-pituitary lower. Thymulin administration to adult mice tended to communication. Furthermore, neonatal thymulin gene reverse these changes. Our results suggest a possible therapy has been shown to prevent the typical alterations modulating effect of thymulin on the corticotrope of gonadotrophic cell number and morphology and population and the adrenal gland, confirming the serum gonadotropin levels in nude female mice. In the existence of a bidirectional thymus-pituitary-adrenal present study we assessed the impact of athymia and the axis. effect of neonatal thymulin gene therapy on the corticotropic cell population in nude mice. The effect of Key words: Thymus-pituitary axis, Thymulin gene thymulin administration to adult nudes on their therapy, Nude mice, Hypophysiotropic activity, RAd- hypothalamic content of corticotropin-releasing hormone FTS (CRH) and the adrenal content of corticosterone was also determined. We used an adenoviral vector expressing a synthetic gene for the thymic peptide Introduction thymulin (metFTS) termed RAd-FTS. On postnatal day 1 or 2, heterozygous (nu/+) and homozygous (nu/nu) Important contributions to the study of the pups of both sexes received a single bilateral i.m. relationship between the thymus and the adrenal axis injection of RAd-FTS or RAd-GFP, a control vector. On have been made, thus showing that adrenal postnatal day 71, mice were bled and sacrificed, and corticosteroids affect the size and morphology of the their pituitaries were immediately dissected, fixed and thymus gland. In an in vitro study, corticotropin (ACTH) immunostained for corticotropin. Morphometry was increased the release of the thymic metallopeptide performed by means of an image-analysis system. The thymulin in a dose-dependent fashion (Hadley et al., following parameters were calculated: volume density 1997). In vivo ACTH administration or stress conditions (VD: Σ cell area/reference area), cell density (CD: result in a marked decrease of T lymphocytes and number of cells/reference area), and cell surface (CS: thymus size. Moreover, adrenalectomy in animals expressed in µm2). Serum thymulin levels were derived in a thymic hyperplasia (Fisher, 1964). measured by a bioassay, and CRH as well as Conversely, the integrity of the thymus during perinatal corticosterone were determined by IRMA and RIA, life is necessary for a proper maturation of the thymus- respectively. Neonatal thymulin gene therapy in the pituitary axis, as revealed by the endocrine alterations athymic mice restored their serum thymulin levels and caused by neonatal thymectomy (Michael et al., 1980) or increased corticotrope CD, VD and CS in both control congenital absence of the thymus in mice (Rebar et al., 1980). Morover, thymectomy diminished ACTH and Offprint requests to: Gloria M. Console, Department of Cytology, corticosterone levels in newborn rats, whereas Histology and Embryology B - CICBA, Faculty of Medicine, University of replacement therapy with thymosin fraction 5 (a partially La Plata, Argentina, CC 455, La Plata 1900, Argentina (zip 1900) La purified thymus extract) in thymectomized (Tx) animals Plata, Argentina. e-mail: [email protected] not only restored the immune response but also 472 Morphological effects on corticotropes by thymulin normalized ACTH and corticosterone levels (Deschaux described in detail elsewhere (Reggiani et al., 2006). et al., 1979). Briefly, this kit uses a shuttle plasmid (pDC515) and Thymulin is the best characterized thymic hormone. the genomic plasmid pBHGfrt(del)E1,3 FLP. The It is exclusively produced by thymic epithelial cells thymulin synthetic gene (metFTS) was inserted into the (TEC), and consists of a nonapeptide component (facteur shuttle and both plasmids were cotransfected into thymique sérique or FTS) coupled in an equimolecular HEK293 cells. In cotransfected HEK293 cells enzyme- ratio to the ion zinc (Dardenne et al., 1982). A number of directed recombination generated the genome of the secretagogues for thymulin have been reported, desired recombinant adenoviral vector, RAd-FTS. The including dexamethasone, progesterone, testosterone, newly generated RAd was rescued from HEK293 cell estradiol (E2), PRL, GH and thyroid hormones. In TEC lysates and plaque purified. It was further purified by cultures, each of these substances had receptors and ultracentrifugation in CsCl gradient and titrated by a stimulated thymulin release (Savino and Dardenne, serial dilution plaque assay. 2000). Moreover, it has been observed that the i.p. treatment of senescent mice with hypothalamic extracts RAd-GFP from young mice corrected the deficit of serum thymulin levels (Folch et al., 1986). Exposure to hypothalamic or An adenoviral vector termed RAd-GFP was pituitary extracts derived from young mice in TEC constructed in our laboratory following the above cultures induced an increase in thymulin concentration general procedures and was used as a control vector in in the cell supernatants, and this effect was lower when the gene therapy studies. The vector harbours a hybrid the extracts were obtained from old mice (Goya et al., gene encoding the Aequorea victoria enhanced green 1995). fluorescent protein (GFP) fused to herpes simplex virus Shortly after birth congenitally athymic (nude) mice, type 1 thymidine kinase (a kind gift from Dr. Jacques as well as normal mice neonatally thymectomized, Galipeau, McGill University, Montreal, Canada). The develop not only a severe immunodeficiency but also vector was expanded in HEK293 cells and purified and degenerative alterations in the adrenal glands that can be titrated as indicated above. prevented by thymus neonatal grafting (Pierpaoli and Sorkin, 1972). The removal of the adrenal glands in Animals and experimental procedures male and female mice produced a temporary decrease (for a month) of serum thymulin, with the consequent Gene therapy experiments increment in the intrathymic content of thymulin (Dardenne et al., 1986). The offspring of NIH homozygous (nu/nu) nude Recently, a synthetic DNA sequence coding for male and heterozygous (nu/+) female mice were used. thymulin has been constructed and used to design a The parent mice were purchased from the Animal Core recombinant adenoviral vector termed RAd-FTS. When Facility of the National University of La Plata, intramuscularly (i.m.) administered to adult Argentina. All mice were maintained on a γ-irradiated thymectomized (Tx) mice and rats, the vector RAd-FTS chow diet and sterilized water. Animals had free access induced sustained supraphysiological serum levels of to food and water and were kept at 22°C with a biologically active thymulin which remained high for at light/dark cycle of 12/12 h. All experiments in nude least 112 days in mice and for over 320 days in rats animals were done following the Animal Welfare (Reggiani et al., 2006; Morel et al., 2008). Guidelines of the NIH. In the present study, we assessed the effect of On postnatal day 1 or 2, each experimental neonatal thymulin gene therapy on the morphology of heterozygous (nu/+) and homozygous (nu/nu) pup of the corticotropic cell population at puberty in nude mice, both sexes received a single bilateral i.m. (hindlegs) as well as the effect of thymulin peptide replacement injection of 108 plaque forming units (pfu) with RAd- therapy on hypothalamic CRH content and FTS or RAd-GFP used as a control vector, in 10 µl corticosterone adrenal content in adult nude mice. vehicle (5 µl per side). On postnatal days 71-72, mice were bled and immediately sacrificed by cervical Materials and methods dislocation, their pituitaries were immediately dissected, fixed and immunostained. The experimental groups Adenoviral vectors used analyzed were: nu/nu mice injected with RAd-GFP or RAd-FTS and nu/+ mice injected with RAd-GFP, both RAd-FTS females and males. Thus, six groups of mice were studied. A DNA sequence coding for the biologically active thymulin analog termed methionine-FTS or metFTS, Thymulin replacement therapy experiments was constructed and cloned in a recombinant adenoviral (RAd) vector generated by a variant of the two plasmid Adult mice received 1 ng synthetic thymulin (FTS) ® method (Hitt et al., 1998) employing the AdMax (in 0.1 ml saline containing 1 ng ZnCl2) per day, plasmid kit (Microbix, Canada). This procedure has been administered sucbcutaneously (s.c.) for 10 days. 473 Morphological effects on corticotropes by thymulin Hypothalamic and adrenal extract preparation overnight at room temperature with 200:l of the CRH- IRMA reagent mixture. This consisted of 125I-labelled In some adult mice the medial basal hypothalamus rabbit anti-CRH (36-41)-NH2 IgG (100,000 cpm/tube) (MBH) was immediately dissected after sacrifice and and 1/5,000 guinea pig anti-CRH-(1-20) serum (referred homogenized in 1 ml 0.05M HCl + 1% w/v ascorbic to as "CRH linker") in 0.05 M sodium phosphate buffer acid per each MBH. After thorough homogenization the (pH 7.4) containing 0.5% (w/v) human serum albumin, extract was frozen and thawed three times and then 1% (v/v) normal rabbit serum and 0.01% (w/v) sodium submitted to three 20-s sonication bursts, leaving 10-s azide. Separation of CRH-bound from unbound labelled intervals between bursts.