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J R Davis

Publications and source records attributed to J R Davis.

At least 19 recordsLinked to original sources
PubMed2001

Characterization of a prolactin gene polymorphism and its associations with systemic lupus erythematosus.

OBJECTIVE: Hyperprolactinemia is associated with systemic lupus erythematosus (SLE), but the mechanism is unknown. Prolactin is expressed in T lymphocytes and is under the control of an alternative promoter region. We characterized a G/T single-nucleotide polymorphism (SNP) at position -1149 of this promoter and assessed its prevalence in patients with SLE. METHODS: Electrophoretic mobility shift assays (EMSAs) were performed to determine DNA protein complex formation in the prolactin promoter. Transient transfection of reporter gene constructs containing the G/T promoter alleles into the Jurkat T cell line were used to determine transcription activity. Peripheral blood lymphocytes (PBLs) were treated in vitro with phytohemagglutinin (PHA) to determine levels of prolactin messenger RNA (mRNA). RESULTS: EMSAs indicated that binding of a GATA-related transcription factor was altered by the G/T SNP at position -1149. Transient transfection studies in Jurkat cells showed that the G allele consistently produced higher promoter activity. PHA treatment of PBLs in vitro induced a greater increment of prolactin mRNA from patients with the GG(-1149) genotype than from those with the TT(-1149) genotype. Disease association studies in a cohort of SLE patients demonstrated an increased frequency of the prolactin -1149 G allele compared with control subjects. CONCLUSION: We found a functionally significant polymorphism that alters prolactin promoter activity and mRNA levels in the lymphocytes. Altered local prolactin production by immune cells may contribute to disease progression by affecting T cell function.

Alleles↗
PubMed2001

Efficient FLPe recombinase enables scalable production of helper-dependent adenoviral vectors with negligible helper-virus contamination.

Helper-dependent (HD), high-capacity adenoviruses are one of the most efficient and safe gene therapy vectors, capable of mediating long-term expression. Currently, the most widely used system for HD vector production avoids significant contamination with helper virus by using producer cells stably expressing a nuclear-targeted Cre recombinase and an engineered first-generation helper virus with parallel loxP sites flanking its packaging signal. The system requires a final, density-based separation of HD and residual helper viruses by ultracentrifugation to reduce contaminating helper virus to low levels. This separation step hinders large-scale production of clinical-grade HD virus. By using a very efficient recombinase, in vitro-evolved FLPe (ref. 14), to excise the helper virus packaging signal in the producer cells, we have developed a scalable HD vector production method. FLP has previously been shown to mediate maximum levels of excision close to 100% compared to 80% for Cre (ref. 15). Utilizing a common HD plasmid backbone, the FLPe-based system reproducibly yielded HD virus with the same low levels of helper virus contamination before any density-based separation by ultracentrifugation. This should allow large-scale production of HD vectors using column chromatography-based virus purification.

Adenoviridae↗
PubMed2001

Is pituitary gene therapy realistic?

Current therapies for pituitary tumours are moderately successful in many cases but still suffer from significant limitations, with relatively poor long-term rates of endocrine cure from surgery, and long-term hypopituitarism after radiotherapy. Even in the case of the most readily treatable tumours, prolactinomas, medical therapy with dopamine agonists is limited by lack of response or side-effects in up to 10% of patients. This has led to increasing interest in the application of our knowledge of pituitary cell and molecular biology to evaluate the potential of gene therapy. Various vectors are available to facilitate gene delivery, and recombinant adenoviruses have been studied in detail because of their ability to transduce the postmitotic, nondividing cells of the pituitary gland. Various studies with reporter genes such as beta-galactosidase have demonstrated high efficiency and long lasting expression of adenoviral transgenes in cultured pituitary cells in vitro. The feasibility of high level transgene expression has also been shown in vivo, but so far this requires stereotaxic intrapituitary injection to achieve adequate transduction. Ablation of pituitary cells has been demonstrated in cultured cell lines and in subcutaneous tumours in nude mice, though alternative animal models will be required to evaluate efficacy in more slowly proliferating tumours as found in man. Inflammatory responses have been documented in the pituitary gland as in other tissues, and this will require the evaluation of modified vectors to avoid significant adverse effects before human applications can be considered. In summary, gene therapy for pituitary disease is likely to be feasible in the future, but will require careful and extensive evaluation of efficacy and safety, using a variety of possible methods of gene delivery.

Adenoviridae↗
PubMed2001

Photoperiodic regulation of prolactin gene expression in the Syrian hamster by a pars tuberalis-derived factor.

Syrian hamsters exhibit a marked seasonal variation in prolactin secretion. The aim of this study was to analyse the nature of the photoperiodic regulation of prolactin gene expression, and to define the role of melatonin and the pars tuberalis of the anterior pituitary in this process. Pituitary prolactin gene expression, restricted to the pars distalis, was increased in hamsters maintained in long daylengths (16 h : 8 h, light : dark) compared to hamsters exposed to short daylengths (8 h : 16 h, light : dark) for 8-12 weeks. Analysis of single cells by in situ hybridization showed that photoperiod had no effect on the percentage of pars distalis cells expressing prolactin mRNA, but shifted the frequency distribution of prolactin mRNA expression per cell, such that in long photoperiods a greater proportion of cells were recruited to a higher expressing population. In vitro coculture of hamster pars tuberalis fragments increased prolactin promoter-driven luciferase activity in stably transfected GH3 cells in a dose- and duration-dependent manner. Conditioned medium from hamster and ovine pars tuberalis also activated the prolactin promoter. Furthermore, basal and forskolin-stimulated conditioned medium from hamster pars tuberalis increased prolactin mRNA expression in primary cultures of pars distalis cells. Melatonin attenuated the activity of pars tuberalis-conditioned medium but had no direct effect on either prolactin mRNA expression or secretion in pars distalis cell cultures. Finally, pars tuberalis fragments from long photoperiod hamsters stimulated prolactin gene promoter activity to a greater extent than those from short photoperiod hamsters. In conclusion, this study provides the first evidence in a seasonal mammal that the synthesis of prolactin depends on photoperiodic modulation of a pars tuberalis-derived factor. Our data support further the hypothesis that seasonal modulation of prolactin gene expression depends upon a melatonin-dependent paracrine action of the pars tuberalis on pars distalis lactotrophic cells.

Animals↗
PubMed2001

Telepathology overview: from concept to implementation.

Telepathology is the practice of pathology at a distance by using video imaging and telecommunications. Significant progress has been made in telepathology. To date, 12 classes of telepathology systems have been engineered. Rapid and ultrarapid virtual slide processors may further expand the range of telepathology applications. Next-generation digital imaging light microscopes, such as miniaturized microscope arrays (MMA), may make virtual slide processing a routine laboratory tool. Diagnostic accuracy of telepathology is comparable with that of conventional light microscopy for most diagnoses. Current telepathology applications include intraoperative frozen sections services, routine surgical pathology services, second opinions, and subspecialty consultations. Three telepathology practice models are discussed: the subspecialty practice (SSP) model; the case triage practice (CTP) model; and the virtual group practice (VGP) model. Human factors influence performance with telepathology. Experience with 500 telepathology cases from multiple organs significantly reduces the video viewing time per case (P < .01). Many technology innovations can be represented as S-curves. After long incubation periods, technology use and/or efficiency may accelerate. Telepathology appears to be following an S-curve for a technical innovation.

Diffusion of Innovation↗
PubMed2001

Leptin administration improves skeletal muscle insulin responsiveness in diet-induced insulin-resistant rats.

In addition to suppressing appetite, leptin may also modulate insulin secretion and action. Leptin was administered here to insulin-resistant rats to determine its effects on secretagogue-stimulated insulin release, whole body glucose disposal, and insulin-stimulated skeletal muscle glucose uptake and transport. Male Wistar rats were fed either a normal (Con) or a high-fat (HF) diet for 3 or 6 mo. HF rats were then treated with either vehicle (HF), leptin (HF-Lep, 10 mg. kg(-1). day(-1) sc), or food restriction (HF-FR) for 12-15 days. Glucose tolerance and skeletal muscle glucose uptake and transport were significantly impaired in HF compared with Con. Whole body glucose tolerance and rates of insulin-stimulated skeletal muscle glucose uptake and transport in HF-Lep were similar to those of Con and greater than those of HF and HF-FR. The insulin secretory response to either glucose or tolbutamide (a pancreatic beta-cell secretagogue) was not significantly diminished in HF-Lep. Total and plasma membrane skeletal muscle GLUT-4 protein concentrations were similar in Con and HF-Lep and greater than those in HF and HF-FR. The findings suggest that chronic leptin administration reversed a high-fat diet-induced insulin-resistant state, without compromising insulin secretion.

3-O-Methylglucose↗
PubMed2001

Polymorphisms of the human prolactin gene--implications for production of lymphocyte prolactin and systemic lupus erythematosus.

Hyperprolactinaemia is associated with systemic lupus erythematosus (SLE) but the mechanism is unknown. Prolactin is expressed not only by pituitary lactotrophic cells but also by T-lymphocytes under the control of an alternative upstream promoter region. T-lymphocytes from SLE patients have been shown to secrete more prolactin than controls, thus implying a possible underlying difference in regulation. This may be due to genetic polymorphism that can be determined by scanning for mutations and using a variety of methods to determine their function. A polymorphism may also be used in disease association studies as it may be in linkage disequilibrium with a disease gene on the same haplotype. Single nucleotide polymorphisms (SNPs) have been found across the prolactin gene region including the extrapituitary and the pituitary promoter regions. These SNPs have been examined for genetic association with SLE and potential effects upon the function of the gene. One SNP in the lymphocyte specific upstream promoter affects prolactin transcription and disease association studies in a cohort of SLE cases demonstrated an increased frequency of the PRL-1149 G allele compared to control subjects. This indicates a possible mechanism for the association of prolactin with SLE. Although prolactin is likely to be one of several predisposing factors in the pathogenesis and progression of SLE, this suggests that manipulation of lymphocyte prolactin production (rather than pituitary production) might be a useful therapeutic approach.

Gene Expression Regulation↗
PubMed2001

Cell type-specific adenoviral transgene expression in the intact ovine pituitary gland after stereotaxic delivery: an in vivo system for long-term multiple parameter evaluation of human pituitary gene therapy.

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.

Adenoviridae↗
PubMed2001

Persistent synchronized oscillations in prolactin gene promoter activity in living pituitary cells.

PRL gene expression in the anterior pituitary gland responds rapidly to different hormonal signals. We have investigated the long-term timing of transcriptional activation from the PRL, GH, and cytomegalovirus promoters in response to different stimulus duration, using real-time imaging of luciferase expression in living stably transfected GH3 cells. Long-term stimulation of serum-starved cells with 50% serum induced a homogeneous rise in PRL promoter activity, with subsequent heterogeneous fluctuations in luciferase activity in individual cells. When cells were subjected to a 2-h pulse of 50% serum, followed by serum-free medium, there were long-term (approximately 50 h) synchronized, homogeneous oscillations in PRL promoter activity. This response was PRL-specific, because in GH3 cells expressing luciferase from the GH or cytomegalovirus promoters, a serum pulse elicited no oscillations in luciferase expression after an initial transient response to serum. The PRL promoter may therefore be a template for an unstable transcription complex subject to stochastic regulation, allowing an oscillatory transcriptional response to physiological signals. This suggests that precise timing and coordination of cell responses to different signal-duration may represent a novel mechanism for coordinating long-term dynamic changes in transcription in cell populations.

Blood Physiological Phenomena↗
PubMed2001

Identification of caveolae and detection of caveolin in normal human osteoblasts.

Caveolae, specialised regions of the cell membrane which have been detected in a wide range of mammalian cells, have not been described in bone cells. They are plasmalemmal invaginations, 50 to 100 nm in size, characterised by the presence of the structural protein, caveolin, which exists as three subtypes. Caveolin-1 and caveolin-2 are expressed in a wide range of cell types whereas caveolin-3 is thought to be a muscle-specific subtype. There is little information on the precise function of caveolae, but it has been proposed that they play an important role in signal transduction. As the principal bone-producing cell, the osteoblast has been widely studied in an effort to understand the signalling pathways by which it responds to extracellular stimuli. Our aim in this study was to identify caveolae and their structural protein caveolin in normal human osteoblasts, and to determine which subtypes of caveolin were present. Confocal microscopy showed staining which was associated with the plasma membrane. Transmission electron microscopy revealed the presence of membrane invaginations of 50 to 100 nm, consistent with the appearance of caveolae. Finally, we isolated protein from these osteoblasts, and performed Western blotting using anti-caveolin primary antibodies. This revealed the presence of caveolin-1 and -2, while caveolin-3 was absent. The identification of these structures and their associated protein may provide a significant contribution to our further understanding of signal transduction pathways in osteoblasts.

Blotting, Western↗
PubMed2001

Pituitary tumours.

Pituitary tumours are a common type of intracranial neoplasm and, depending on the cell type of origin, have diverse endocrine and reproductive effects. The developmental biology of the different cell types is understood to result from a sequential activation of a cascade of transcription factors, and mutations in these factors result in various forms of hypopituitarism. Tumours in the pituitary gland arise from activation of dominantly acting oncogenes such as gsp, or from loss of function of a series of tumour suppressor genes such as MEN1. Abnormal patterns of DNA methylation may be implicated in the allelic losses that cause tumour suppressor gene silencing. The different clinically recognized types of pituitary tumour are currently treated by medical therapies such as dopamine and somatostatin agonists, surgery or radiotherapy. However, these treatments are not entirely satisfactory and recent advances in gene therapy may offer valuable new therapeutic opportunities for patients with aggressive tumours that fail to respond to traditional approaches.

Adenoma↗
PubMed2001

Nuclear chromatin patterns in normal, hyperplastic and atypical endometrium.

OBJECTIVE: To characterize the nuclei of endometrial lesions for the diagnostic categories of normal glandular tissue, simple hyperplasia, atypical hyperplasia and adenocarcinoma of the endometrium, with the specific goal of probing for heterogeneity. STUDY DESIGN: For each diagnostic category the images of 360 nuclei were recorded on a high-resolution video microphotometer. Features descriptive of the statistical and spatial distribution of nuclear chromatin were computed for each nucleus. A nonsupervised learning algorithm, P-index, was employed to establish subsets of nuclei within each diagnostic category and to determine whether these subsets were statistically significantly different in the nuclear chromatin pattern. RESULTS: Lesions from cases of hyperplasia, atypical hyperplasia and adenocarcinoma of the endometrium each contained several subsets of nuclei with statistically significantly different chromatin patterns. For one such subset from each diagnostic category, a clear trend of progression toward adenocarcinoma could be demonstrated. CONCLUSION: The nuclei in endometrial lesions represent a highly heterogeneous set. Any measure of lesion progression or regression due to chemopreventive intervention, in an individual case, will have to examine the proportion of nuclei in each of these subsets as well as measures of deviation from normal for each subset.

Adenocarcinoma↗
PubMed2001

Nuclear chromatin characteristics of breast solid pattern ductal carcinoma in situ.

OBJECTIVE: To characterize nuclei from breast solid pattern ductal carcinoma in situ (DCIS) by their karyometric features and to search for the presence of statistically significantly different subsets of nuclei. STUDY DESIGN: One hundred nuclei from each of 6 normal, 13 solid DCIS, (9 low and intermediate grade and 4 high grade DCIS) histopathologic samples of breast tissue were digitally recorded. Karyometric features were computed and subjected to a nonsupervised learning algorithm (P-index) to identify significantly different subgroups. RESULTS: Nuclei in low grade lesions displayed a diploid/near diploid pattern, while the majority of intermediate grade lesions fell into a range beyond 5N. The high grade lesions showed substantial genomic instability and represented three statistically different subsets or phenotypes. CONCLUSION: There is a progression of nuclear abnormality from low grade to high grade DCIS. The nuclei from high grade DCIS form a heterogeneous set that represents three phenotypes. One of these phenotypes shows a nuclear chromatin pattern that more closely resembles poorly differentiated, infiltrating disease. The observation of such a phenotype may have prognostic implications.

Breast Neoplasms↗
PubMed2000

Transverse-contour modeling of trunk muscle-distributed forces and spinal loads during lifting and twisting.

STUDY DESIGN: An electromyography-assisted biomechanical model was developed using electromyographic (surface and in-dwelling) data collected during asymmetric lifting and twisting activities. OBJECTIVES: To develop a biomechanical model of the lumbar region that considers the ability of the broad, flat muscles of the trunk (external obliques, internal obliques and latissimus dorsi) to activate different anatomic regions at different intensity levels and then uses this information to describe the spine reaction forces that result during lifting and twisting tasks. SUMMARY OF BACKGROUND DATA: Many biomechanical models of the lumbar region use single-vector representations for the external oblique, internal oblique, and latissimus dorsi muscles. This simplification limits the description of the complexity of the resultant forces produced by these muscles and does not consider their differential activation capacity. METHODS: Human subjects performed lifting and twisting exertions while muscle electromyographic activities were sampled at one location on the rectus abdominis and erector spinae muscles and at multiple locations on the latissimus dorsi, external oblique, and internal oblique muscles. These data were used in conjunction with in vivo digitized muscle origin and insertion points to predict muscle forces and spine loads through the use of the electromyography-assisted modeling method. RESULTS: The measures of model performance such as percentage of error (6-21%) in the prediction of the external torques, correlations (0.83-0.98) between internal and external torques and the values of predicted muscle force capacity were all similar to data collected in previous electromyography-assisted models, but the predictions of spinal loading, particularly shear forces, were quite different. The results have shown that by modeling the broad, flat muscles of the torso using multiple-force vectors, the calculated shear forces in the spine were reduced. CONCLUSIONS: The multivector, transverse-contour model developed in this research illustrates the importance of realistic multiple-vector modeling and the importance of considering the selective-activation capacity of the abdominal oblique musculature.

Abdominal Muscles↗
PubMed2000

Wilms' tumor of the cervix: a case report and review of the literature.

INTRODUCTION: Extrarenal Wilms' tumors are rare, with only 55 well-documented cases in the literature and only 4 confined to the uterus. A fifth case along with review of the current literature is presented. CASE: A 13-year-old girl presented with a 2-month history of irregular vaginal spotting. A 6.5 x 4.5 x 2.5-cm polypoid mass meeting criteria for an extrarenal Wilms tumor was removed from the superior aspect of the cervix, but recurred 8 months later on the posterior lip. The patient underwent a Schauta procedure, lymph node dissection, and bilateral oophoropexy. The surgery was followed by typical renal Wilms' tumor chemotherapy. The patient is without evidence of disease 5 years after surgery. DISCUSSION: This case uniquely suggests that limited excision of the original tumor has inherent risks for relapse. Oophoropexy appears to be reasonable when fertility preservation is desired and reconfirms that treatment mimicking renal nephroblastomas offers good long-term survival.

Adolescent↗
PubMed2000

Transcriptional targeting to anterior pituitary lactotrophic cells using recombinant adenovirus vectors in vitro and in vivo in normal and estrogen/sulpiride-induced hyperplastic anterior pituitaries.

The use of pituitary cell type-specific promoters is a powerful molecular tool to achieve pituitary cell type-specific transcriptional targeting of transgenes encoded by viral vectors. It has recently been proposed that transcriptional targeting of therapeutic genes could be harnessed as a gene therapy strategy for the treatment of pituitary disease. We describe the successful use of the human PRL promoter (hPrl) encoded within recombinant adenovirus vectors to target transgene expression of Herpes Simplex Virus Type 1-Thymidine Kinase (HSV1-TK) or beta-galactosidase to lactotrophic cells in vitro and in vivo. Functionally, the restriction of expression of HSV1-TK to lactotrophic tumor cells, using the hPrl promoter, resulted in the cell type-specific induction of apoptosis in the lactotrophic GH3 tumor cell line, in the presence of ganciclovir (GCV). In the corticotrophic AtT20 cell line, we detected neither HSV1-TK expression, nor apoptosis in the presence of GCV. The hPrl promoter encoded within a recombinant adenoviral vector also restricted transgene expression to lactotrophic cells in primary anterior pituitary (AP) cultures, and importantly, within the anterior pituitary gland in vivo. When the HSV1-TK driven by hPrl promoter was used in an in vivo model ofestrogen/sulpiride lactotroph induced hyperplasia within the AP in situ, the treatment was not effective in either reducing the weight of the gland, the number of lactotrophic cells within the transduced area in vivo, or the circulating PRL levels. This is in contrast to the human cytomegalovirus promoter (hCMV) driving expression of HSV1-TK in the same experimental paradigm, which was effective in reducing pituitary weight and circulating PRL levels. Our results have important implications in the design of gene therapy strategies for pituitary tumors. We demonstrate that both the choice of the in vivo animal model, i.e. adenoma in the AP gland in situ, and the particular gene therapy strategy chosen, i.e. use of strong ubiquitous promoters vs. weaker but cell type-specific promoters, determine the experimental therapeutic outcome.

Adenoviridae↗
PubMed1999

Interaction of glucocorticoid receptor isoforms with transcription factors AP-1 and NF-kappaB: lack of effect of glucocorticoid receptor beta.

Glucocorticoids act through the glucocorticoid receptor (GR) to enhance or repress transcription of glucocorticoid responsive genes depending on the promoter context and cellular background. The human GR primary transcript is alternatively spliced resulting in hGR alpha and hGR beta isoforms. Transactivation and transrepression are mediated by hGR alpha and while it has been demonstrated that hGR beta, can act as a dominant negative inhibitor of hGR alpha mediated transactivation, its effects on transrepression are not known. To investigate hGR beta actions, we used GR-deficient COS-7 and HEK-293 cells. When hGR alpha (0.5 microg 10(6) cells(-1)) was transfected into COS-7 cells dexamethasone (150 nM) inhibited TNF alpha (80 U ml(-1)) effects on a NF-kappaB responsive reporter gene by 40%. There was no evidence of a dominant negative effect when hGR beta (1-10 microg) was co-transfected with hGR alpha up to ratios of 10:1. Similarly hGR beta had no effect on hGR alpha inhibition of a phorbol ester stimulated Ap-1-responsive reporter gene in COS-7 or HEK-293 cells. In comparison, an apparent dominant negative effect of hGR beta on hGR alpha-mediated transactivation was found to be attributable to non-specific transcriptional squelching in COS-7 cells. In summary, the potential for hGR beta, to act as a dominant negative inhibitor of hGR alpha-mediated transactivation remains controversial, but our data suggest that hGR beta, was unable to act as a dominant negative inhibitor of either hGR alpha-mediated transrepression or transactivation in these promoter and cell contexts.

Animals↗