This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Davis, J. R. E. Articles by McNeilly, A. S. Search for Related Content PubMed PubMed Citation Articles by Davis, J. R. E. Articles by McNeilly, A. S.

Cell Type-Specific Adenoviral Transgene Expression in the Intact Ovine Pituitary Gland after Stereotaxic Delivery: An in VivoSystem for Long-Term Multiple Parameter Evaluation of Human Pituitary Gene Therapy¹

Endocrine Sciences Research Group and Molecular Medicine and Gene Therapy Unit (S.W., P.R.L., M.G.C.), University of Manchester, Manchester, United Kingdom M13 9PT; and Medical Research Council Human Reproductive Sciences Unit (J.M., G.A.L., A.S.M.), Edinburgh, United Kingdom EH3 9ET

Address all correspondence and requests for reprints to: Prof. J. R. E. Davis, Endocrine Sciences Research Group, University of Manchester, Stopford Building, Oxford Road, Manchester, United Kingdom M13 9PT. E-mail: julian.davis{at}man.ac.uk

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ablative therapies for pituitary tumors commonly cause irreversible damage to normal pituitary cells. Toxin gene therapy should therefore ideally be targeted to specific cell types to avoid collateral cell damage. To evaluate cell-type-specific adenoviral gene transfer in the intact pituitary gland we have used stereotaxic transcranial delivery of recombinant adenoviruses in the sheep with continuous assessment of endocrine function. Adenoviral ß-galactosidase expression was driven either by the human cytomegalovirus (hCMV) promoter or the human PRL gene promoter. The hCMV promoter directed adenoviral ß-galactosidase expression in all pituitary cell types, but the PRL promoter restricted this exclusively to lactotropic cells, indicating that this promoter conferred appropriate cell type specificity in the context of adenoviral transduction in vivo. Serial measurements of plasma hormones showed no disruption of endocrine function over 7 days after intrapituitary injection. In summary, this work shows cell type-specific expression of an adenoviral transgene in the mixed cell population of the intact pituitary gland in vivo in a large animal model and indicates that stereotaxic intrapituitary delivery does not disrupt normal endocrine function.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE ANTERIOR PITUITARY gland contains a mixed cell population of endocrine cells (lactotropic, somatotropic, thyrotropic, corticotropic, and gonadotropic cells, secreting PRL, GH, TSH, corticotropin, and gonadotropins, respectively) together with folliculo-stellate and endothelial cells. Thus, a series of diverse endocrine functions depends on a complex organization of seven different cell types that are closely intermingled. Pituitary adenomas comprise clonal expansions of one of these cell types (1). Thus, any therapy designed to achieve permanent ablation of a pituitary tumor risks collateral damage to the other normal cells, and existing ablative therapies commonly result in a series of irreversible hormonal deficiencies that require life-long treatment (2, 3). Surgical therapy has variable success according to tumor type and size, and the best results are generally disappointing (4, 5).

The development of specific ablation therapy through expression of toxin genes can now be contemplated because of the growth in knowledge of the mechanisms of cell type specificity of hormone gene expression in the pituitary (1, 6). The anterior pituitary gland is an attractive model system in which to study this approach to cell type targeting in vivo, as the cell types can be readily monitored structurally by immunocytochemistry, and functionally by serial measurements of their secreted hormone products in peripheral blood.

Recombinant adenoviruses have become increasingly used as effective tools for gene transfer and are under intensive investigation in human gene therapy protocols. Previous reports have confirmed their efficacy in vitro using cultured pituitary cells (7, 8), in vivo in pituitary tumors propagated in nude mice (9), and in the intact rat pituitary after estrogen/sulpiride administration (10, 11). Further development of such a strategy for potential human therapy requires substantial validation using suitable in vivo systems, in which normal pituitary function should not be disrupted.

The aims of this study were therefore to evaluate cell type-specific adenoviral gene transfer in a large animal pituitary gland as a model of potential human pituitary gene therapy. We used stereotaxic transcranial injection of recombinant adenoviruses into the sheep pituitary gland in vivo and measured effects on anterior pituitary gland function using serial hormone measurements over 7 days. Adenoviral ß-galactosidase expression was driven either by the human cytomegalovirus (hCMV) promoter or the human PRL (hPRL) promoter in an effort to achieve lactotroph-specific expression.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recombinant adenovirus vector construction
The recombinant adenovirus vectors were based on adenovirus type 5, in which the E1 and part of the E3 regions were deleted. In one of these, RAd-CMV-ß-gal (also termed RAd-35) the ß-galactosidase gene is driven by the short immediate-early hCMV (sMIEhCMV) promoter inserted in place of the E1 deletion, as previously described (7). In the other vector (RAd-hPRL-ß-gal) the promoter element comprised a -4429/+14 fragment from the pituitary-specific promoter of the human PRL gene (11, 12, 13, 14), constructed as previously described (11).

Animals and stereotaxic pituitary injection
Anestrous Suffolk ewes (2–3 yr old, 35–45 kg) were anesthetized with thiopentone and maintained with fluothane and nitrous oxide during the surgical procedure. Using a stereotaxic frame (14A ), a burr-hole was made in the skull 2 cm anterior to the bregma. A spinal needle was inserted 2 mm lateral to the midline angled cranio-caudally 30° from the vertical and lowered until its tip reached the lateral ventricle, judged by free flow of cerebrospinal fluid (CSF). One milliliter of radioopaque dye (Omnipaque, Birmingham, UK) was instilled into the CSF, and a lateral radiograph was taken 30 sec later (75 kV, 50 mA, 0.4 sec) to allow visualization of the cerebral ventricles (Fig. 1A). The location of the pituitary gland was deduced from the positions of the infundibular and mammillary recesses of the third ventricle (15). A fine-bore metal cannula was inserted through the spinal needle and lowered to the base of the pituitary fossa. It was withdrawn 2 mm, and 250 µl viral vector were injected into the pituitary gland at each of three levels. The cannula and guide needle were left in place for 1 min, then both were completely withdrawn and reintroduced 2 mm anteriorly, and the procedure was repeated. A third injection procedure was carried out posterior to the initial injection site. A total of nine injection sites were used to inject virus, using a total volume of approximately 2.2 ml, usually taking 50–60 min. The procedure was initially validated using injection of India ink (Fig. 1B), confirming accurate targeting of injectate within the pituitary gland. Virus suspensions of RAd-hPRL-ß-Gal or RAd-hCMV-ß-Gal were prepared at a concentration that delivered approximately 1.5 x 10⁸ plaque-forming units/site, giving a total of approximately 14 x 10⁸ plaque-forming units injected into the gland.

View larger version (50K): [in this window] [in a new window]   Figure 1. Stereotaxic injection of pituitary gland. A, Lateral radiograph and diagram showing radioopaque dye in lateral and third ventricles and the position of the guide needle to direct injection into the pituitary gland. B, Low power photomicrograph of sequential thick sagittal sections (200 µm) through the right lobe of an India ink-injected pituitary gland, with the infundibulum located anteriorly to the upper left, oriented as in the radiograph. Note the distribution of ink along needle tracks and at the base of the gland. C, Distribution of adenoviral transgene expression within the pituitary. Low power photomicrograph of ß-galactosidase immunostaining in sections from one lobe of a sheep anterior pituitary gland injected with RAd-CMV-ßgal. The adenoviral transgene expression (stained brown) in the injected gland is distributed along two visible needle tracks and at the base of the gland (arrowed). Sections were 5 µm thick and taken at 150-µm intervals, visualized with diaminobenzidine and lightly counterstained with hematoxylin (magnification, x4). The infundibulum is to the upper left of each section, oriented as in A.

Immunocytochemistry
Sections of each pituitary gland were cut after preparation exactly as described previously (16). Initially, ß-galactosidase staining was assessed at 50-µm intervals to determine the general distribution of expression within the whole pituitary gland. For this initial single staining screen, sections were treated with hydrogen peroxide in methanol to block endogenous peroxidase activity and microwaved in sodium citrate buffer, pH 6.0, before staining with monoclonal anti-ß-galactosidase antibody (1:200; Promega Corp., Madison, WI) with visualization using diaminobenzidine (DAKO Corp.).

Regions of the pituitary glands identified in this way as containing large numbers of ß-galactosidase-positive cells were then subjected to dual immunofluorescence for ß-galactosidase and pituitary hormones (16) using the following rabbit polyclonal antibodies: 1) PRL, ASMcN-R51, 1:2500; 2) LH, ASMcN-R23, 1:100; 3) FSH, M91, 1:100; 4) GH, 1:500, (NIDDK, NIH); 5) TSH, 1:100, (Dr. J. G. Pierce); and 6) ACTH, 1:300. Sections were treated with H₂O₂ in methanol, microwaved as described above, and incubated with anti-ß-galactosidase antibody (1:50) in blocking buffer (normal goat serum, 10% in Tris-buffered saline) overnight at 4 C in a humidity chamber. Second antibody (goat antimouse Ig biotinylated, DAKO Corp.) was added, sections were washed, and avidin and biotinylated horseradish peroxidase complex (DAKO Corp.) were added for 30 min at room temperature. The ß-galactosidase signal was amplified using a tyramide step, and sections were incubated with avidin-fluorescein isothiocyanate conjugate (Sigma, St. Louis, MO). Anti-hormone antibodies were then added at the dilutions indicated above and incubated overnight at 4 C. The hormone signal was visualized using goat antirabbit tetramethylrhodamine isothiocyanate conjugate (Sigma). Dual immunofluorescence images were obtained using an Olympus Corp. Provis fluorescence microscope (New Hyde Park, NY). For each hormone, 40–300 cells were identified in each of two sections taken from two widely separated regions of each pituitary gland to ensure representative estimates of transgene expression in different cell types. A mean result for each animal was generated from these four sections to provide an overall mean for each group of five animals.

Hormone assays and hematology
Plasma concentrations of PRL (17), LH and FSH (18), TSH (19), and GH and cortisol (20) were measured by RIAs as described previously. All samples were measured in one assay, with sensitivities of 0.5 ng (PRL, LH), 0.1 ng (FSH, GH), 0.2 ng (TSH), and 1 ng (cortisol) per ml plasma and intraassay coefficients of variation less than 8% for all assays. Routine hematology was analyzed by the Diagnostic Services Clinical Laboratories, Royal (Dick) Veterinary School (Edinburgh, UK).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Initial studies in two sheep using India ink confirmed that the stereotaxic approach targeted the pituitary gland accurately, with a satisfactory distribution of dye within the gland after slow injection (Fig. 1B). Ten sheep were studied after injection of RAd-hCMV-ß-gal (n = 5), or RAd-hPRL-ß-gal (n = 5). All animals recovered uneventfully, and showed normal behavior thereafter. During removal of pituitary glands at the end of the experimental period, small blood clots were found in the CSF in relation to the needle track in two animals that had been apparently well, but in all the others the CSF was free of blood-staining.

The overall distribution of adenovirally mediated ß- galactosidase expression in the pituitary gland was assessed in animals injected with RAd-hCMV-ß-gal, and an example is shown in Fig. 1C. Intense staining was seen in relation to the needle tracks and at the base of the pituitary, with stronger expression in the animals injected with RAd-CMV-ßgal than in those injected with RAd-hPRL-ßgal.

Dual immunofluorescence staining for ß-galactosidase with each of the six pituitary hormones (Fig. 2) showed that in animals injected with RAd-hCMV-ß-gal, ß-galactosidase staining was colocalized with hormone staining in all six endocrine cell types (Fig. 2, left panels) and also in S-100-staining folliculo-stellate cells (data not shown). Transgene expression was detected in varying proportions of all six endocrine cell types, and this was quantitated in multiple sections of the pituitary glands. As the ß-galactosidase expression was regional, sections were chosen for quantitation from ß-galactosidase-positive areas that also contained sufficient numbers of cells that were positive for each of the six hormones, and percentages of the ß-galactosidase-positive cells that expressed each of the pituitary hormones were calculated. Of the ß-galactosidase-positive cells, 29–92% of cells coexpressed PRL in the five animals studied (mean, 69 ± 28%); of the other hormones, coexpression with ß-galactosidase varied from 8% (ACTH) to 33% (TSH) (Fig. 3A). In contrast, in animals injected with the RAd-hPRL-ß-gal vector, 93.0 ± 3.9% of cells staining for ß-galactosidase coexpressed PRL, 4–5% stained for LH or FSH, and less than 2% stained for GH, TSH, or ACTH (Fig. 2, right panels, and Fig. 3B). It should be noted that because of the regional clustering of different cell types within different areas of the anterior pituitary gland, the apparent prevalence of the different endocrine cell types was overestimated by this method, but the approach was used to avoid selection bias and to ensure that adequate numbers of each endocrine cell type were assessed to allow genuine quantitation.

View larger version (102K): [in this window] [in a new window]   Figure 2. Cell type specificity of adenoviral transgene expression. Left panels, Representative dual immunofluorescence staining of pituitary sections from animals treated with RAd-CMV-ßgal injection. ß-Galactosidase staining is shown in green (left panels), hormone staining in red (center panels), and overlay plots show colocalization of ß-galactosidase with hormone as yellow (right panels). ß-Galactosidase expression colocalized with (top to bottom) PRL, LH, FSH, TSH, GH, and ACTH, indicating expression of the viral transgene in all endocrine cell types. All images were taken using a x40 magnification lens. White bar, 50 µm. Right panels, Representative dual immunofluorescence staining of pituitary sections from animals treated with RAd-hPRL-ßgal injection. Panels are presented as in left-hand series, and overlay plots show colocalization of ß-galactosidase only with PRL (top right panel) and not with LH, FSH, GH, TSH, or ACTH. All images were taken using a x40 magnification lens. White bar, 50 µm.

View larger version (20K): [in this window] [in a new window]   Figure 3. Quantitative analysis of cell type specificity of adenoviral transgene expression. A, Cell type specificity of RAd-CMV-ßgal transgene expression. The histogram shows the percentage of ß-galactosidase-positive cells that costained for each of the six pituitary hormones. Data shown are the mean ± SD (n = 5 animals, derived from four separate pituitary sections in each case, with 40–200 cells counted/section). B, Cell type specificity of RAd-hPRL-ßgal transgene expression. The histogram shows the percentage of ß-galactosidase-positive cells that costained for each of the six pituitary hormones. Data shown are means ± SD (n = 5 animals, derived from four separate pituitary sections, with 40–100 cells counted/section).

View larger version (26K): [in this window] [in a new window]   Figure 4. Pituitary function before and after adenovirus injection. Plasma levels of the pituitary hormones PRL, LH, FSH, TSH, and GH and plasma cortisol plotted before (-28 and -4 h) and then daily for 7 days after RAd injection at 0 h. Four animals injected with RAd-hCMV-ß-gal are shown in the left panels, and five injected with RAd-hPRL-ß-gal are shown in the right panels.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This report demonstrates adenoviral transgene expression in the intact eutopic sheep pituitary gland in vivo for at least 7 days and shows that stereotaxic delivery is reliable, accurate, and safe in a large animal. Furthermore, by quantitation of the six endocrine cell types expressing the transgene, we have shown that the PRL gene promoter confers appropriate cell type specificity to transgene expression in vivo in the context of a recombinant adenoviral vector. Most important, in terms of potential clinical application, we found no deleterious effect of adenoviral injection on endocrine function of the pituitary gland.

A large animal model is essential for evaluation of this potential human application of endocrine gene therapy, as multiparameter longitudinal assessment is needed to determine the effects of adenoviral gene transduction on pituitary function. The ability to take serial blood samples to track hormonal changes over days or weeks is severely limited in small animals such as rats or mice. We selected the sheep as a suitable model because the pituitary gland is of similar size, configuration, and accessibility to the human pituitary and has been the subject of careful immunohistochemical evaluation (16). In addition we have previous experience in longitudinal evaluation of pituitary hormone secretion (19). The sheep pituitary is 10–12 mm in antero-posterior diameter, and the results reported here indicate that a local injection of recombinant adenovirus can achieve effective transgene expression across substantial regions of the gland. This suggests that direct adenoviral injection of a pituitary tumor at transsphenoidal surgery may in principle be able to achieve adequate expression of desired suicide genes for future potential tumor therapy applications, as discussed below.

A key point of this study was to employ transcriptional targeting of transgene expression to a single cell type within a mixed cell population by exploiting the known cell type specificity of PRL gene expression. The human PRL gene promoter was used to generate the recombinant adenovirus vector RAd-hPRL-ß-gal in view of potential future therapeutic applications (11, 21). This gene promoter has been extensively studied in vitro, and the promoter fragment used here confers appropriate hormonal regulation onto reporter gene expression in pituitary cells in both transient and stable transfection systems (12, 13, 14, 22). The present study confirms that the PRL promoter in the context of a recombinant adenovirus is activated specifically in lactotropic cells in vivo, with minimal reporter gene transcription in other cell types. In other words, although the adenovirus vectors are capable of infecting all of the cell types found in the pituitary (Refs. 7 and 11 and the present data), the PRL promoter restricts the activation of adenoviral transgene expression effectively to the lactotroph cell population. The RAd-hPRL-ß-gal vector has also been used in primary cultures of rat anterior pituitary cells and in the rat pituitary gland in vivo, with similar restriction of transgene expression mainly to lactotropic cells (11). The slightly higher than expected transgene expression from this vector in gonadotropic cells may be explained by the intimate relation between lactotropic and gonadotropic cells in vivo (16), which could result in overestimation of apparent transgene expression in gonadotrophs. Recent work (23) has used the GH gene promoter to achieve similar restriction of transgene expression to the rat pituitary gland in vivo.

An important goal in this study was to assess the effect of adenoviral injection on the endocrine function of the normal pituitary gland in a large animal model, and the size of the sheep allowed us to track circulating plasma hormone levels. Single time point analysis 10 days after transcranial or transauricular injection of the rat pituitary indicated little or no major change in circulating hormone levels (10, 11). In the present, more extensive, longitudinal evaluation, we were able to monitor pituitary function serially over 7 days. We found a transient rise in plasma cortisol and PRL, a well recognized feature of an anesthetic stress response in sheep (19). In the following 7 days hormone levels and secretory patterns remained normal, however, with no evidence to suggest disruption of pituitary function. It is encouraging that even injection of relatively large volumes of virus into a tightly organized tissue appears to cause no adverse endocrine or other systemic effects.

If adenoviral gene transfer into the normal eutopic pituitary gland can be confirmed to be safe and effective over the longer term, without disrupting pituitary function as our data suggest, it may be possible to consider the potential for ablative therapy for pituitary tumors using suicide or cytotoxic genes. Although pituitary tumors are not usually lethal in themselves, their treatment remains unsatisfactory, and ablative therapy frequently damages residual pituitary function. Pituitary tumors have the advantage of well understood cell biology, and they are a suitable target for further development of adenoviral gene transfer approaches (21, 24). Future developments are likely to include more heavily deleted ("gutless") adenovirus vectors (see Ref. 24 for review), which will enable use of combinations of tissue-specific promoters together with regulatable transcription factors to obtain highly controlled transgene expression (25, 26, 27). Extensive validation will be required to establish their safety and efficacy in different circumstances (28, 29), and the system presented in this report will be valuable in this effort.

	Acknowledgments

 
We thank Ian Swanston and Fiona Pitt for hormone assays, Joan Docherty and Norah Anderson and other staff at the Marshall Building for expert technical assistance and animal care, and Ted Pinner and Axel Thompson for assistance with preparation of the figures. We acknowledge the excellent technical assistance of Ms. Tricia Maleniak in the Molecular Medicine and Gene Therapy Unit.

	Footnotes

 
¹ This work was supported by the Medical Research Council, the Biotechnology and Biological Sciences Research Council, and the Royal Society.

² Research Fellow of the Lister Institute of Preventive Medicine.

Received August 9, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Asa SL, Ezzat S 1998 The cytogenesis and pathogenesis of pituitary adenomas. Endocr Rev 19:798–827[Abstract/Free Full Text]
2. Ahmed S, Elsheikh M, Stratton IM, Page RCL, Adams CBT, Wass JAH 1999 Outcome of transphenoidal surgery for acromegaly and its relationship to surgical experience. Clin Endocrinol (Oxf) 50:561–567[CrossRef][Medline]
3. Littley MD, Shalet SM, Beardwell CG, Ahmed SR, Applegate G, Sutton ML 1991 Hypopituitarism following external radiotherapy for pituitary tumours in adults. Q J Med 70:145–160
4. Molitch ME, Thorner MO, Wilson C 1997 Management of prolactinomas. J Clin Endocrinol Metab 82:996–997[Free Full Text]
5. Soule SG, Conway GS, Jacobs HS 1996 The outcome of hypophysectomy for prolactinomas in the era of dopamine agonist therapy. Clin Endocrinol (Oxf) 44:711–716[CrossRef][Medline]
6. Treier M, Gleiberman AS, O’Connell SM, Szeto DP, McMahon JA, McMahon AP, Rosenfeld MG 1998 Multistep signaling requirements for pituitary organogenesis in vivo. Genes Dev 12:1691–1704[Abstract/Free Full Text]
7. Castro MG, Goya RG, Sosa YE, Rowe J, Larregina A, Morelli A, Lowenstein PR 1997 Expression of transgenes in normal and neoplastic anterior pituitary cells using recombinant adenoviruses: long term expression, cell cycle dependency, and effects on hormone secretion. Endocrinology 138:2184–2194[Abstract/Free Full Text]
8. Neill JD, Lois C, Musgrove L, Duck W, Sellers JC 1998 High efficiency method for gene transfer in normal pituitary gonadotropes: adenoviral-mediated expression of G protein-coupled receptor kinase 2 supresses luteinizing hormone secretion. Endocrinology 140:2562–2569[Abstract/Free Full Text]
9. Lee EJ, Anderson LM, Thimmapaya B, Jameson JL 1999 Targeted expression of toxic genes directed by pituitary hormone promoters: a potential strategy for adenovirus-mediated gene therapy of pituitary tumors. J Clin Endocrinol Metab 84:786–794[Abstract/Free Full Text]
10. Windeatt S, Southgate TD, Dewey RA, Bolognani F, Perone MJ, Larregina AT, Maleniak TC, Morris ID, Goya RG, Klatzmann, Lowenstein PR, Castro MG 2000 Adenovirus-mediated herpes simplex virus type-1 thymidine kinase gene therapy suppresses oestrogen-induced pituitary prolactinomas. J Clin Endocrinol Metab 85:1296–1305[Abstract/Free Full Text]
11. Southgate TD, Windeatt S, Smith-Arica, Gerdes CA, Perone MJ, Morris ID, Davis JRE, Klatzmann D, Lowenstein PR, Castro MG 2000 Transcriptional targeting to anterior pituitary lactotrophic cells using recombinant adenovirus vectors in vitro and in vivo in normal and estrogen/sulpiride-induced hyperplasic anterior pituitaries. Endocrinology 141:3493–3505[Abstract/Free Full Text]
12. Berwaer M, Monget P, Peers B, Mathy-Hartert M, Bellefroid E, Davis JRE, Belayew A, Martial JA 1991 Multihormonal regulation of the human prolactin gene-expression from 5000bp of its upstream sequence. Mol Cell Endocrinol 80:53–64[CrossRef][Medline]
13. Hoggard N, Davis JRE, Berwaer M, Monget P, Peers B, Belayew A, Martial JA 1991 Pit-1 binding sequences permit calcium regulation of human prolactin gene expression. Mol Endocrinol 5:1748–1754[Abstract/Free Full Text]
14. Takasuka N, White MRH, Wood CD, Robertson WR, Davis JRE 1998 Dynamic changes in prlactin promoter activation in individual living lactotrophic cells. Endocrinology 139:1361–1368[Abstract/Free Full Text]
14. Mori Y, Takeuchi Y, Shimada M, Hayashi S, Hoshino K 1990 Stereotaxic approach to hypothalamic nuclei of the Shiba goat with radiographic monitoring. Jpn J Vet Sci 52:339–349
15. Lignereux Y, Regodon S, Marty M-H, Franco A, Bubien A 1991 Encephalic ventricles of the ewe (Ovis aries L.): conformation, relations and stereotaxic topography. Acta Anat 141:82–84[Medline]
16. Tortonese DJ, Brooks J, Ingleton PM, McNeilly AS 1998 Detection of prolactin gene expression in the sheep pituitary gland and visualization of the specific translation of the signal in gonadotrophs. Endocrinology 139:5215–5223[Abstract/Free Full Text]
17. McNeilly AS, Land RB 1979 Effect of suppression of plasma prolactin on ovulation, plasma gonadotrophins and corpus luteum function in LH-RH-treated anoestrous ewes. J Reprod Fertil 56:601–609[Abstract/Free Full Text]
18. McNeilly AS, Crow WJ, Fraser HM 1992 Suppression of pulsatile luteinizing hormone secretion by gonadotrophin-releasing hormone antagonist does not affect episodic progesterone secretion or corpus luteum function in ewes. J Reprod Fertil 96:865–874[Abstract/Free Full Text]
19. Fraser HM, McNeilly AS 1982 Effect of chronic immunoneutralization of thyrotropin-releasing hormone. Endocrinology 111:1964–1973[Abstract/Free Full Text]
20. McNeilly AS, Brooks AN, Baxter G, Webb R 1994 Sheep adrenal inhibin. In: Burger HG, Findlay JK, deKretser D, Petraglia F (eds) Inhibin and Inhibin-Related Proteins. Frontiers in Endocrinology. Ares-Serono Symposia, Rome, vol 3:261–269
21. Davis JRE, Lowenstein PR, McNeilly AS, Castro MG 1999 Gene therapy for pituitary tumours. Endocr Relat Cancer 6:475–483[CrossRef][Medline]
22. Peers B, Voz ML, Monget P, Mathy-Hartert M, Berwaer M, Belayew A, Martial JA 1990 Regulatory elements controlling the expression of the human prolactin gene. Mol Cell Biol 10:4690–4700[Abstract/Free Full Text]
23. Lee EJ, Thimmapaya B, Jameson JL 2000 Stereotactic injection of adenoviral vectors that target gene expression to specific pituitary cell types: implication for gene therapy. Neurosurgery 46:1461–1468[CrossRef][Medline]
24. Castro MG 1999 Gene therapy strategies for the treatment of pituitary tumours. J Mol Endocrinol 22:9–18[CrossRef][Medline]
25. Yoshida Y, Hamada H 1997 Adenovirus-mediated inducible gene expression through tetracycline-controllable transactivator with nuclear localization signal. Biochem Biophys Res Commun 230:426–430[CrossRef][Medline]
26. Harding TC, Geddes BJ, Murphy D, Knight D, Uney JB 1998 Switching transgene expression in the brain using an adenoviral tetracycline-regulatable system. Nat Biotechnol 16:553–555[CrossRef][Medline]
27. Castro MG, Windeatt S, Smith-Arica J, Lowenstein PR 1999 Cell-type specific expression in the pituitary: physiology and gene therapy. Biochem Soc Trans 27:858–863[Medline]
28. Dewey RA, Morrissey G, Cowsill CM, Stone D, Bolognani F, Dodd NJF, Southgate TD, Klatzmann D, Lassmann H, Castro MG, Lowenstein PR 1999 Chronic brain inflammation and persistent herpes simplex virus 1 thymidine kinase expression in survivors of syngeneic glioma treated by adenovirus-mediated gene therapy: implications for clinical trials. Nat Med 5:1256–1263[CrossRef][Medline]
29. Cowsill C, Southgate TD, Morrissey G, Dewey RA, Morrelli AE, Maleniak TC, Forrest Z, Klatzmann D, Wilkinson GWG, Lowenstein PR, Castro MG 2000 Central nervous toxicity of two adenoviral vectors encoding variants of the herpes simplex virus type 1 thymidine kinase: reduced cytotoxicity of a truncated HSV-1 TK. Gen Ther 7:679–685[CrossRef][Medline]

This article has been cited by other articles:

				S. Semprini, S. Friedrichsen, C. V. Harper, J. R. McNeilly, A. D. Adamson, D. G. Spiller, N. Kotelevtseva, G. Brooker, D. G. Brownstein, A. S. McNeilly, et al. Real-Time Visualization of Human Prolactin Alternate Promoter Usage in Vivo Using a Double-Transgenic Rat Model Mol. Endocrinol., April 1, 2009; 23(4): 529 - 538. [Abstract] [Full Text] [PDF]

				J. Acunzo, S. Thirion, C. Roche, A. Saveanu, G. Gunz, A. L. Germanetti, B. Couderc, R. Cohen, D. Figarella-Branger, H. Dufour, et al. Somatostatin Receptor sst2 Decreases Cell Viability and Hormonal Hypersecretion and Reverses Octreotide Resistance of Human Pituitary Adenomas Cancer Res., December 15, 2008; 68(24): 10163 - 10170. [Abstract] [Full Text] [PDF]

				M. P. Gillam, M. E. Molitch, G. Lombardi, and A. Colao Advances in the Treatment of Prolactinomas Endocr. Rev., August 1, 2006; 27(5): 485 - 534. [Abstract] [Full Text] [PDF]

				M Candolfi, G Jaita, D Pisera, L Ferrari, C Barcia, C Liu, J Yu, G Liu, M G Castro, and A Seilicovich Adenoviral vectors encoding tumor necrosis factor-{alpha} and FasL induce apoptosis of normal and tumoral anterior pituitary cells. J. Endocrinol., June 1, 2006; 189(3): 681 - 690. [Abstract] [Full Text] [PDF]

				E. J. Lee and J L. Jameson Gene therapy of pituitary diseases J. Endocrinol., June 1, 2005; 185(3): 353 - 362. [Abstract] [Full Text] [PDF]

				C. Roche, A. J Zamora, D. Taieb, E. Lavaque, R. Rasolonjanahary, H. Dufour, C. Bagnis, A. Enjalbert, and A. Barlier Lentiviral vectors efficiently transduce human gonadotroph and somatotroph adenomas in vitro. Targeted expression of transgene by pituitary hormone promoters J. Endocrinol., October 1, 2004; 183(1): 217 - 233. [Abstract] [Full Text] [PDF]

				U. B. Kaiser Editorial: Pituitary Gene Therapy--Hypotheses on the Hypophysis Endocrinology, February 1, 2001; 142(2): 528 - 531. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Davis, J. R. E. Articles by McNeilly, A. S. Search for Related Content PubMed PubMed Citation Articles by Davis, J. R. E. Articles by McNeilly, A. S.