Communication with young people in paediatric and adult endocrine consultations: an intervention development and feasibility study.

BACKGROUND: Communication is complex in endocrine care, particularly during transition from paediatric to adult services. The aims of this study were to examine the feasibility of interventions to support young people to interact with clinicians. METHODS: Development and evaluation of a complex intervention in 2 phases: Pre-intervention observational study; Intervention feasibility study. Purposive sample of recordings of 62 consultations with 58 young people aged 11-25 years with long-term endocrine conditions in two paediatric and two adult endocrine clinics. Proportion of time talked during consultations, number and direction of questions asked; Paediatric Consultation Assessment Tool (PCAT); OPTION shared decision making tool; Medical Information Satisfaction Scale (MISS- 21). Young people were invited to use one or more of: a prompt sheet to help them influence consultation agendas and raise questions; a summary sheet to record key information; and the www.explain.me.uk website. RESULTS: Nearly two thirds of young people (63%) chose to use at least one communication intervention. Higher ratings for two PCAT items (95% CI 0.0 to 1.1 and 0.1 to 1.7) suggest interventions can support consultation skills. A higher proportion of accompanying persons (83%) than young people (64%) directed questions to clinicians. The proportion of young people asking questions was higher (84%) in the intervention phase than in the observation phase (71%). CONCLUSIONS: Interventions were acceptable and feasible. The Intervention phase was associated with YP asking more questions, which implies that the availability of interventions could promote interactivity.

Phaeochromocytoma and Acromegaly: a unifying diagnosis.

UNLABELLED: A 52-year-old lady was referred after a 5 cm left adrenal mass was detected on computed tomography (CT) scanning. She was asymptomatic although was noted to have acromegalic facies. Blood pressure (BP) was normal but plasma normetanephrines were raised to 2.81 mmol/l (<1.09) and urinary normetadrenaline excretion 5.3 µmol/24 h (0-4.3). Adrenal biochemistry screen was otherwise normal. Metaiodobenzylguanidine (MIBG) scan demonstrated uptake in the adrenal lesion. Growth hormone (GH) nadir on oral glucose tolerance test (OGTT) was 2.2 ng/ml with an elevated IGF1 level of 435 ng/ml (72-215), confirming acromegaly biochemically. The remainder of the pituitary screen was normal. A magnetic resonance imaging (MRI) scan of the pituitary revealed an enlarged pituitary gland with a microadenoma/cyst of 2-3 mm in diameter. Alpha blockade was achieved with a titrated dose of phenoxybenzamine before a successful laparoscopic hand-assisted left adrenalectomy. Postoperative biochemical testing revealed a normal plasma normetanephrine level of 0.6 nmol/l (<1.09) and a metanephrine level of 0.35 nmol/l (<0.46 nmol/l). Nadir on OGTT was normal at 0.07 ng/ml with an IGF1 level within the reference range at 111 ng/ml (75-215). Histology demonstrated a well-circumscribed and encapsulated oval mass with microscopic features typical for a phaeochromocytoma. The sections stained strongly positive for GHRH in 20% of cells on immunocytochemistry. Genetic analysis showed no pathogenic mutation. This is a report of the rare condition of a phaeochromocytoma co-secreting GHRH resulting in clinical and biochemical acromegaly. Neuroendocrine tumours can stain positive for GHRH without coexisting acromegaly, but the resolution of patient symptoms and normalisation of serum GH and IGF1 levels following surgery imply that this was functional secretion. Pituitary surgery should be avoided in such cases. LEARNING POINTS: Incidental findings on imaging require thorough investigation to determine the presence of serious pathology.Acromegaly and phaeochromocytoma are rarely coincident in the same patient. If this occurs, co-secretion of GHRH from the phaeochromocytoma or the presence of underlying genetic abnormalities must be considered.Acromegaly is due to ectopic GHRH-secreting neuroendocrine tumours in <1% of cases, most commonly pancreatic or bronchial lesions.Co-secretion of GHRH from a phaeochromocytoma is extremely rare.In such cases, the pituitary gland may appear enlarged but pituitary surgery should be avoided and surgical treatment of the neuroendocrine tumour attempted.

Wnt signalling in pituitary development and tumorigenesis.

Wnt signalling is activated in both pituitary organogenesis and its mature function. Wnt ligands and Wnt signalling pathways are critical for the regulation of the formation of the pituitary. In the mature pituitary, Wnt signalling pathways control cell activity and may stimulate cell proliferation in both physiological and pathological processes. This review compares Wnt signalling pathways active in the developing and mature pituitary and explores how this gives us further insight into the development of pituitary adenomas.

Npas4 is activated by melatonin, and drives the clock gene Cry1 in the ovine pars tuberalis.

Seasonal mammals integrate changes in the duration of nocturnal melatonin secretion to drive annual physiologic cycles. Melatonin receptors within the proximal pituitary region, the pars tuberalis (PT), are essential in regulating seasonal neuroendocrine responses. In the ovine PT, melatonin is known to influence acute changes in transcriptional dynamics coupled to the onset (dusk) and offset (dawn) of melatonin secretion, leading to a potential interval-timing mechanism capable of decoding changes in day length (photoperiod). Melatonin offset at dawn is linked to cAMP accumulation, which directly induces transcription of the clock gene Per1. The rise of melatonin at dusk induces a separate and distinct cohort, including the clock-regulated genes Cry1 and Nampt, but little is known of the up-stream mechanisms involved. Here, we used next-generation sequencing of the ovine PT transcriptome at melatonin onset and identified Npas4 as a rapidly induced basic helix-loop-helix Per-Arnt-Sim domain transcription factor. In vivo we show nuclear localization of NPAS4 protein in presumptive melatonin target cells of the PT (α-glycoprotein hormone-expressing cells), whereas in situ hybridization studies identified acute and transient expression in the PT of Npas4 in response to melatonin. In vitro, NPAS4 forms functional dimers with basic helix loop helix-PAS domain cofactors aryl hydrocarbon receptor nuclear translocator (ARNT), ARNT2, and ARNTL, transactivating both Cry1 and Nampt ovine promoter reporters. Using a combination of 5'-deletions and site-directed mutagenesis, we show NPAS4-ARNT transactivation to be codependent upon two conserved central midline elements within the Cry1 promoter. Our data thus reveal NPAS4 as a candidate immediate early-response gene in the ovine PT, driving molecular responses to melatonin.

The prolactin gene: a paradigm of tissue-specific gene regulation with complex temporal transcription dynamics.

Transcription of numerous mammalian genes is highly pulsatile, with bursts of expression occurring with variable duration and frequency. The presence of this stochastic or 'noisy' expression pattern has been relatively unexplored in tissue systems. The prolactin gene provides a model of tissue-specific gene regulation resulting in pulsatile transcription dynamics in both cell lines and endocrine tissues. In most cell culture models, prolactin transcription appears to be highly variable between cells, with differences in transcription pulse duration and frequency. This apparently stochastic transcription is constrained by a transcriptional refractory period, which may be related to cycles of chromatin remodelling. We propose that prolactin transcription dynamics result from the summation of oscillatory cellular inputs and by regulation through chromatin remodelling cycles. Observations of transcription dynamics in cells within pituitary tissue show reduced transcriptional heterogeneity and can be grouped into a small number of distinct patterns. Thus, it appears that the tissue environment is able to reduce transcriptional noise to enable coordinated tissue responses to environmental change. We review the current knowledge on the complex tissue-specific regulation of the prolactin gene in pituitary and extra-pituitary sites, highlighting differences between humans and rodent experimental animal models. Within this context, we describe the transcription dynamics of prolactin gene expression and how this may relate to specific processes occurring within the cell.

Novel approaches to in vitro transgenesis.

The study of gene expression is a major focus in biological research and is recognised to be critical for our understanding of physiological and pathophysiological processes. Methods to study gene expression range from in vitro biochemical assays through cultured cells and tissue biopsies to whole organisms. In the early stages of project development, considerations about which model system to use should be addressed and may influence future experimental procedures. The aim of this review is to briefly describe advantages and disadvantages of the existing techniques available to study eukaryote gene expression in vitro, including the mechanism of transgene integration (transient or stable), the different transgenesis systems available, including plasmids, viruses and targeted integration and knockin approaches, and paying particular attention to expression systems such as bacterial artificial chromosomes and episomal vectors that offer a number of advantages and are increasing in popularity. We also discuss novel approaches that combine some of the above techniques, generating increasingly complex but physiologically accurate expression systems.

Failure of antibody response to polysaccharide antigen in treated panhypopituitary adults.

Although pituitary hormones are known to affect immune function, treated hypopituitarism is not a recognized cause of immune deficiency in humans. We set out to assess integrity of baseline and stimulated immune function in severely hypopituitary adults. Twenty-one panhypopituitary adults (group 1), on stable pituitary replacement including growth hormone, and 12 healthy volunteers (group 2) were studied. Lymphocyte subsets, pneumococcal antibody levels pre- and 1 month after polysaccharide vaccination, T cell numbers and in-vitro interferon (IFN)-gamma response were studied. There were no significant differences in T cell numbers or IFN-gamma secretion. B cell numbers were lower in group 1, especially those with low prolactin levels. Independent of this finding, nine of 21 patients in this group had low antibody response to polysaccharide antigen. This was most striking in those with low insulin-like growth factor 1 levels and appeared to be independent of the use of anti-convulsants or corticosteroid replacement. Significant humoral immune deficiency is seen in panhypopituitarism and may contribute to morbidity.

Human prolactin gene promoter regulation by estrogen: convergence with tumor necrosis factor-alpha signaling.

Estrogens have been implicated in the regulation of prolactin gene expression in man, although previous studies have not defined the molecular mechanism whereby estradiol activates the human prolactin gene promoter (hPrl). We found that estradiol induced a reproducible 1.8-fold activation of the hPrl gene promoter, using pituitary GH3 cells stably transfected with a 5000-bp hPrl promoter fragment linked to luciferase reporter gene. This activation was blocked by treatment with estrogen receptor (ER) antagonists 4-hydroxytamoxifen and ICI-182,780. Promoter deletion and mutagenesis experiments identified a functional estrogen response element (ERE) sequence 1189 bp upstream of the transcription start site that was responsible for estrogen-mediated promoter activation. This site differed from the consensus ERE sequence by two base pairs, one in each half-site. This ERE was identified to be functional through binding ERalpha in EMSAs. Chromatin immunoprecipitation assays confirmed ERalpha binding to this sequence in vivo in the absence of ligand, with increased recruitment when cells were cultured in the presence of estradiol. When cells were treated with both estradiol and TNFalpha, we observed synergistic activation of the hPrl promoter, which was mediated by the -1189-bp ERE. Mutagenesis of this ERE abolished the promoter-activating effect not only of estradiol but also of TNFalpha. These data suggest a novel, promoter-specific signaling interaction between estrogen and TNFalpha signaling, which is likely to be important for prolactin regulation in vivo.

Hyperprolactinaemia.

Hyperprolactinaemia is a frequent cause of reproductive problems encountered in clinical practice. A variety of pathophysiological conditions can lead to hyperprolactinaemia; therefore, pregnancy, drug effects, hypothyroidism and polycystic ovary syndrome should be excluded before investigating for prolactin-secreting pituitary tumours. Prolactinomas are mainly diagnosed in women aged 20-40 years. They present with clinical features of hyperprolactinaemia (galactorrhoea, gonadal dysfunction), and more rarely with large tumours, headache and visual field loss due to optic chiasm compression. Medical therapy with dopamine agonists is the treatment of choice for both micro- and macroprolactinomas. Tumour shrinkage and restoration of gonadal function are achieved in the majority of cases with dopamine agonists. A trial of withdrawal of medical therapy may be considered in many patients with close follow-up. Pituitary surgery and radiotherapy currently have very limited indications. Pregnancies in patients with prolactinomas need careful planning and close monitoring.

Home administration of lanreotide Autogel by patients with acromegaly, or their partners, is safe and effective.

OBJECTIVE: The introduction of ready-to-use lanreotide Autogel has presented the possibility of patients receiving their acromegaly treatment at home. The objective of this study was to assess the ability of patients (or their partners) to administer repeat, unsupervised, injections of lanreotide Autogel without compromising efficacy or safety. DESIGN: Multicentre (10 UK regional endocrine centres), open-label, nonrandomised, controlled study. Patients elected either to receive/administer unsupervised home injections after injection technique training (Test group) or continued to receive injections from a healthcare professional (Control group). Patients received monthly injections of lanreotide Autogel at their established dose. Effects were monitored for up to 40 weeks. PATIENTS: Thirty patients (15 per treatment group) with acromegaly treated with a stable dose of lanreotide Autogel (60, 90 or 120 mg) for > or = 4 months before screening. Measurements The main outcome measure was the proportion of patients/partners who successfully administered injections throughout the study. RESULTS: All Test group patients/partners qualified to administer injections. Fourteen of 15 patients fulfilled all criteria for successful administration of unsupervised injections (95% confidence interval, 70%-99%). Fourteen of 15 Test and 14/15 Control patients maintained growth hormone and IGF-1 control. Local injection tolerability was good for both treatment groups, and safety profiles were similar. All Test group patients continued with unsupervised injections after the study. CONCLUSIONS: Patients with acromegaly or their partners were able to administer lanreotide Autogel injections with no detrimental effect on efficacy and safety; therefore, unsupervised home injections are a viable alternative to healthcare professional injections for suitably motivated patients.

Cryptic loxP sites in mammalian genomes: genome-wide distribution and relevance for the efficiency of BAC/PAC recombineering techniques.

Cre is widely used for DNA tailoring and, in combination with recombineering techniques, to modify BAC/PAC sequences for generating transgenic animals. However, mammalian genomes contain recombinase recognition sites (cryptic loxP sites) that can promote illegitimate DNA recombination and damage when cells express the Cre recombinase gene. We have created a new bioinformatic tool, FuzznucComparator, which searches for cryptic loxP sites and we have applied it to the analysis of the whole mouse genome. We found that cryptic loxP sites occur frequently and are homogeneously distributed in the genome. Given the mammalian nature of BAC/PAC genomic inserts, we hypothesised that the presence of cryptic loxP sites may affect the ability to grow and modify BAC and PAC clones in E. coli expressing Cre recombinase. We have observed a defect in bacterial growth when some BACs and PACs were transformed into EL350, a DH10B-derived bacterial strain that expresses Cre recombinase under the control of an arabinose-inducible promoter. In this study, we have demonstrated that Cre recombinase expression is leaky in un-induced EL350 cells and that some BAC/PAC sequences contain cryptic loxP sites, which are active and mediate the introduction of single-strand nicks in BAC/PAC genomic inserts.

Fetal gonadotrope cell origin of FSH-secreting pituitary adenoma - insight into human pituitary tumour pathogenesis.

OBJECTIVE: The pathogenesis of human pituitary adenomas remains unclear, but we report a case of FSH-secreting pituitary adenoma whose monohormonal phenotype suggests it was of fetal origin. PATIENTS: A 28-year-old woman presented with abdominal discomfort and irregular menses, enlarged multicystic ovaries and elevated serum oestradiol, with sustained high-normal FSH and low LH levels. MEASUREMENTS: Endocrine studies were performed before and after curative surgery, with assessment of tumour hormone secretion in vitro, and immunostaining of tumour tissue for a series of gonadotrope proteins. RESULTS: Immunocytochemistry showed that tumour cells were monohormonal for FSH. Normal components of gonadotrope signalling pathways were expressed, including oestrogen receptor-alpha, activin receptors, secretogranin-II and chromogranin-A. beta-glycan, the putative inhibin coreceptor, was absent. Tumour culture in vitro confirmed secretion of FSH with minimal LH, that was unsuppressed by oestradiol or inhibin-A. Human fetal pituitary tissue contained FSH-only cells at 18 weeks gestation, whereas normal adult pituitary tissue contained only bihormonal gonadotropes. CONCLUSIONS: We propose that this pituitary adenoma represents an indolent tumour of monohormonal fetal gonadotrope cells that originated early in gestation. Pituitary tumours may therefore arise from abnormal persistence of fetal cell types, with extremely slow growth over many years until reaching a size threshold to generate an endocrine syndrome. Understanding fetal pituitary architecture and function may be more informative for new insights into pituitary tumour pathogenesis than classical theories of cancer biology that invoke unrestrained cell proliferation.

Steroid withdrawal syndrome after successful treatment of Cushing's syndrome: a reminder.

Steroid withdrawal syndrome (SWS) usually refers to relapse of the disease being treated after withdrawal of glucocorticoid therapy, or the symptoms of adrenal insufficiency which occur when glucocorticoids are rapidly reduced or stopped. A less well-recognised form of SWS is that which develops when patients experience a symptom complex similar to that of adrenal insufficiency despite acceptable cortisol levels. We describe three patients who presented with this form of SWS following surgical treatment for endogenous Cushing's syndrome. All responded well to a short-term increase in the dose of glucocorticoid replacement therapy, with the median duration of the syndrome being 10 months (range 6-10 months). Trough serum cortisol levels above 100 nmol/l, with peaks between 460 and 750 nmol/l were documented in the first two patients at presentation with SWS. It is thought that the syndrome may result from development of tolerance to glucocorticoids, and mediators considered to be important in its development include interleukin-6, corticotrophin-releasing hormone, vasopressin, and central noradrenergic and dopaminergic systems. The exact underlying mechanism for SWS remains unclear. However, with increasing recommendations for use of lower doses of replacement glucocorticoids, its incidence may increase. Physicians need to be aware of this condition, which is self-limiting and easily treated by a temporary increase in the dose of glucocorticoid replacement therapy. It is possible that a slower glucocorticoid tapering regimen than that used in the standard postoperative management of patients undergoing pituitary surgery may reduce the risk of development of SWS.

Real-time imaging of gene promoter activity using an adenoviral reporter construct demonstrates transcriptional dynamics in normal anterior pituitary cells.

Although analysis of luciferase activity using luminescence imaging has provided new insights into the dynamic regulation of gene expression in living tIssues, studies in vitro have relied on stably transfected clonal cell lines, limiting the choice of cell type and species, or DNA microinjection, which is arduous and highly selective. We report here the first use of a recombinant adenovirus in which the firefly luciferase reporter gene was regulated by the prolactin gene promoter, to study temporal dynamics of promoter activity. This vector was used to infect the pituitary GH3 cell line, and also primary cultures of Syrian hamster pituitary cells. We show that adenovirally transduced cells retained normal regulation of the promoter-reporter transgene by appropriate signals. Furthermore, microscopic imaging studies indicated that both clonal and primary pituitary cells were transduced efficiently, giving readily detectable luminescence signals in real-time over long periods. Finally, analysis of single-cell expression patterns indicated that prolactin promoter activity was highly dynamic with pulses in gene expression, revealing that the transcriptional instability seen in clonal cells is a feature of normal pituitary cells. Adenoviral vectors offer a valuable tool for studies of gene regulation where conventional transgenesis and clonal cell lines are not available.

Dynamic patterns of growth hormone gene transcription in individual living pituitary cells.

Real-time imaging of the GH gene promoter linked to luciferase in living pituitary cells has revealed surprising heterogeneity and variety of dynamic patterns of gene expression. Cells treated with either forskolin or thyroid hormone generated a consistent and characteristic temporal response from cell populations, but detailed analysis of individual cells revealed different patterns. Approximately 25-26% of cells displayed no response, 25-33% of cells exhibited a sustained progressive rise in luciferase activity, and 41-50% showed a transient phasic, or oscillatory response, after given stimuli. In cells treated consecutively with the two stimuli, the population response to the second stimulus was augmented. Single-cell analysis revealed that this was partly due to an increased number of cells responding, but also that the prevalence of response patterns changed: cells that responded to an initial stimulus were more likely to respond subsequently in a progressive sustained manner. In conclusion, these studies have indicated that GH promoter activity in individual living pituitary cells is unstable and possibly stochastic, with dynamic variations from hour to hour. The prevalence of different temporal patterns of response to hormonal stimulation among a population of cells is altered by the endocrine history of those cells.

Adenovirus-mediated gene transfer in the ovine pituitary gland is associated with hypophysitis.

Gene therapy for pituitary disease requires evaluation for safety as well as efficacy. We have reported results of adenovirus-mediated gene transfer using the sheep as a large animal model that allows longitudinal evaluation of hormone secretion and have confirmed high levels of transgene expression up to 7 days after direct stereotaxic injection into the pituitary gland. Here we report the results of detailed histological examination of the pituitary glands from animals injected with two recombinant adenoviruses expressing the beta-galactosidase marker gene, or with saline vehicle to control for the potential tissue-disruptive effect of the injection volume itself. Pituitaries injected with saline showed no evidence of inflammatory response apart from occasional minor foci of apoptosis. In all other respects they were indistinguishable from normal uninjected control pituitary glands. Glands injected with recombinant adenoviruses containing either the hCMV-beta-gal or the hPRL-beta-gal transgene, on the other hand, displayed variable degrees of inflammatory response, with periglandular fibrosis, lymphocytic infiltrate and venulitis in almost all cases. Focal necrosis and/or apoptosis was noted in six of nine cases. In summary, we have found evidence of severe inflammatory reaction within the first seven days of adenovirus injection, amounting to significant hypophysitis. The histological extent of this reaction has not previously been recognised by studies of the efficacy of gene transfer in rodents, and was underestimated by immunocytochemical studies of hormone and transgene expression. The findings emphasise the need for careful evaluation of the safety of endocrine gene therapy, and for caution with the dose of vector used.

Failure of steroid regulation of the MMTV promoter in a small cell lung cancer cell line is caused by a DNA sequence flanking the glucocorticoid response element.

We have previously described a panel of human small cell lung carcinoma (SCLC) cell lines that have profound glucocorticoid resistance, resulting from various molecular defects in glucocorticoid signalling. However, in one SCLC cell line, CORL103, the cause of the resistance is unknown. These cells are refractory to dexamethasone stimulation of MMTV even when exogenous wild-type glucocorticoid receptor (GR) is co-transfected. This is in contrast to cell lines DMS79 and CORL24 where resistance is overcome by transfection of the wild-type receptor. Sequencing of the GR from CORL103 cells revealed two point mutations, but neither of these induced dominant negative activity. Steroid hormone resistance extended to mineralocorticoid and progesterone receptor (MR, PR) activation of MMTV-luc, whereas oestrogen and thyroid hormone receptor transactivation were normal. A simpler reporter, TAT3-luc, containing three copies of the tyrosine aminotransferase glucocorticoid response element (GRE), was responsive when transfected into CORL103 cells with GR, MR and PR expression vectors and activated with their respective ligands. Similarly, pHH-luc and pAH-luc (truncated MMTV variants containing the GRE region, both derived from a different strain of MMTV), were effectively transactivated with dexamethasone. This suggests that the minor changes in the flanking sequence of the MMTV promoter are critically important in determining steroid responsiveness in CORL103 cells. We propose that minor differences in MMTV may determine recruitment of co-factors, which destabilise GR binding to the MMTV GREs. These findings represent a new, selective, model of glucocorticoid resistance that may explain specific cell and target gene differences in glucocorticoid sensitivity.