Gary Hamlin

Detection of viral ribonucleic acid and histologic analysis of inflamed synovium in Ross River virus infection

OBJECTIVE To document the histology of Ross River virus (RRV) arthritis and to examine inflamed synovium for viral RNA. METHODS Biopsy tissue from the inflamed knees of 12 patients with RRV infection was studied using conventional and immunostaining techniques. Reverse transcriptase-polymerase chain reaction technology was used to probe for the presence of viral RNA in the synovial biopsy samples and in serum. RESULTS Hyperplasia of the synovial lining layer, vascular proliferation, and mononuclear cell infiltration were the main histologic changes. RRV RNA was found in knee biopsy tissue that was obtained from 2 patients at 5 weeks after the onset of symptoms. CONCLUSION RRV RNA was identified in inflamed synovium more than a month after symptoms began. Inflammation was apparent in the absence of detectable virus in the majority of patients.

Increased expression of neuronal glucose transporter 3 but not glial glucose transporter 1 following severe diffuse traumatic brain injury in rats.

Traumatic brain injury results in an increased brain energy demand that is associated with profound changes in brain glycolysis and energy metabolism. Increased glycolysis must be met by increasing glucose supply that, in brain, is primarily mediated by two members of the facilitative glucose transporter family, Glut1 and Glut3. Glut1 is expressed in endothelial cells of the blood-brain barrier (BBB) and also in glia, while Glut3 is the primary glucose transporter expressed in neurons. However, few studies have investigated the changes in glucose transporter expression following traumatic brain injury, and in particular, the neuronal and glial glucose transporter responses to injury. This study has therefore focussed on investigating the expression of the glial specific 45-kDa isoform of Glut1 and neuronal specific Glut3 following severe diffuse traumatic brain injury in rats. Following impact-acceleration injury, Glut3 expression was found to increase by at least 300% as early as 4 h after induction of injury and remained elevated for at least 48 h postinjury. The increase in Glut3 expression was clearly evident in both the cerebral cortex and cerebellum. In contrast, expression of the glial specific 45-kDa isoform of Glut1 did not significantly change in either the cerebral cortex or cerebellum following traumatic injury. We conclude that increased glucose uptake after traumatic brain injury is primarily accounted for by increased neuronal Glut 3 glucose transporter expression and that this increased expression after trauma is part of a neuronal stress response that may be involved in increasing neuronal glycolysis and associated energy metabolism to fuel repair processes.

Temporal characterisation of pro- and anti-apoptotic mechanisms following diffuse traumatic brain injury in rats.

Few studies have characterised apoptosis in a brain injury model that causes a significant degree of diffuse axonal injury. Such characterisation is essential from a clinical viewpoint since diffuse axonal injury is a major component of human head injury. The present study therefore, examines the expression of active and proactive caspase-3, and the bax, bcl-2 and bcl-x members of the bcl-2 family, to characterise the temporal profile of apoptosis in a model of traumatic brain injury in rats that produces significant diffuse axonal injury. Pentobarbital anaesthetised male Sprague-Dawley rats were injured using the 2m impact-acceleration model of diffuse traumatic brain injury. After injury, diffuse trauma resulted in an increased bax expression followed by induction of caspase-3. The increase in caspase-3 was simultaneous with an increase in anti-apoptotic bcl-2 expression. Bcl-x levels were increased after induction of caspase-3 and the increased levels of bcl-x were sustained to the end of the 5-day observation period. Increased active caspase-3 expression was associated with the appearance of TUNEL positive cells. These cells were detected in different brain regions at different times, with some regions showing no apoptotic cells until 3 days after injury. No TUNEL positive cells were detected at 7 and 14 days after injury. DNA electrophoresis confirmed that DNA fragmentation was maximal at 3 days after injury. Increased active caspase-3 levels were also significantly correlated with increased bcl-2 levels (r=0.80; P<0.001) suggesting that the apoptotic cascade after diffuse traumatic brain injury is a carefully controlled cellular homeostatic response. Pharmacological manipulation of this balance may offer a therapeutic approach for preventing cell death and improving outcome after diffuse traumatic brain injury.

Growth Hormone Receptors - their Structure, Location and Role

The cellular receptor for growth hormone (GH) was first described in the rabbit liver by Tsushima and Friesen in 1973 (1). Since then, GH receptors have been identified in many vertebrate cell-types including hepatocytes (2, 3), adipocytes (4, 5) . fibroblasts (6), chondrocytes (7), osteoblasts (unpublished results), @-islet cells (8), macrophages (9), lymphocytes (10) and ventral prostatic epithelial cells (1 1). The receptor occurs as disulphide-linked oligomers of a sialoglycoprotein-binding subunit (Mr 1 10,000) which can be rapidly cleaved to lower molecular weight forms. Accordingly, the receptor has an unusually short half-life of around 45 minutes (12, 13). Although the receptor appears to be subject to short-term down-regulation by GH (10, 14), the long-term effect of GH is probably receptor induction (15, 16). Insulin (17) and pregnancy (18) induce the receptor, whereas fasting (19) and renal insufficiency (20) reduce receptor expression (for review see 2 1).

Differentiation-dependent expression of gelatinase B/matrix metalloproteinase-9 in trophoblast cells

Abstract The purpose of this study was to evaluate the Rcho-1 trophoblast culture system as a model for studying trophoblast invasion and to examine stage-specific expression of enzyme(s) potentially participating in rat trophoblast giant cell invasive behavior. The invasive behavior of the differentiating Rcho-1 trophoblast cells was demonstrated using Matrigel invasion chambers. Gelatin zymography and Western blot analysis of conditioned medium from differentiating Rcho-1 trophoblast cell cultures and rat ectoplacental cone outgrowths revealed a differentiation-dependent increase in gelatinase B/matrix metalloproteinase (MMP-9). Nothern blot and reverse transcriptase polymerase chain reaction (RT-PCR) analyses of Rcho-1 trophoblast or ectoplacental cone cells also showed increasing expression of MMP-9 accompanying cell differentiation. Rcho-1 trophoblast cells stably transfected with MMP-9 promoter/luciferase reporter constructs exhibited a differentiation-dependent increase in MMP-9 promoter activation. In conclusion, trophoblast giant cell differentiation is characterized by transcriptional activation of the MMP-9 gene and appearance of the invasive phenotype.

Hormonal Variation of Rat Uterine Contractile Responsiveness to Selective Neurokinin Receptor Agonists

Abstract Regulated uterine contractions are important in many reproductive functions such as sperm transport and embryo positioning during implantation. The role of classical neurotransmitters including acetylcholine and norepinephrine in regulating myometrial contractility has been well studied; however, the peripheral role of sensory neurotransmitters such as the neurokinins is less clear. The major neurokinins are substance P, neurokinin A, and neurokinin B, which predominantly activate neurokinin receptors (NK-Rs) 1, 2, and 3, respectively. This study utilized selective receptor agonists to examine the role of NK-Rs in uterine contractility. Uterine tissues, obtained from the major stages of the rat estrous cycle, were stimulated with selective NK-R agonists. Addition of each agonist resulted in a significant contractile response. However, the magnitude and nature of the response were dependent upon the stage of the estrous cycle, with responses to all agonists being significantly decreased in tissue from proestrus and estrus. Furthermore, the nature of NK3-R-mediated contraction was different in tissue from proestrus and estrus compared to metestrus and diestrus. The hormonal dependence of NK-R-mediated contractility was then examined in the ovariectomized estrogen-supplemented rat model. These studies confirmed that the magnitude and nature of uterine contractility in response to NK-R activation depend upon the hormonal environment.

Trophoblast Cell Differentiation: Expression of the Placental Prolactin Family

Normal growth and maturation of a specialized extraembryonic structure, the placenta, are essential for the establishment and maintenance of pregnancy. The placenta acts as the interface between maternal and embryonic compartments, developing in parallel with the embryo and facilitating the acquisition of maternal resources required for embryonic and fetal development. This essential responsibility of the placenta is accomplished in a number of important ways, including (i) regulation of the transport of nutrients and wastes, (ii) an immunologic role in preventing fetal rejection, and (iii) an endocrine/communication/signaling role between fetal and maternal compartments. Consequently, understanding the control of placental development is fundamental to an appreciation of the initiation and maintenance of pregnancy and various abnormalities associated with altered growth and development. The cells responsible for these diverse functions of the placenta are trophoblast cells.

Estrogen-Dependent Regulation of Neurokinin 3 Receptor-Mediated Uterine Contractility in the Rat

Abstract The receptors for neurokinin 1 (NK1-R), neurokinin 2 (NK2-R), and neurokinin 3 (NK3-R) are expressed and functionally active in the uterus, promoting strong contractions of the myometrium. Previously, we demonstrated that myometrial contractility activated by the NK-Rs is regulated by estrogen. In the current study, we furthered our investigations of the role of estrogen in the regulation of NK3-R-mediated myometrial contractility. Estrogen promotes both heterologous and homologous desensitization of NK3-R-mediated uterine contractility. In tissue obtained from estrogen-dominated rats (ovariectomized estrogen-treated rats and rats in estrus), the magnitude of uterine contractions decreased in response to consecutive additions of the NK3-R-selective agonist senktide. By addition of the fourth dose of agonist, the contractile response was routinely barely above baseline. In contrast, in tissue obtained from non-estrogen-dominated rats consecutive doses of senktide resulted in contractions of identical magnitude. The homologous desensitization was specific to the NK3-R, and the desensitization of the NK3-R-mediated response did not affect the magnitude or nature of uterine contractions in response to NK1-R or NK2-R activation. Furthermore, heterologous and homologous desensitization of NK3-R-mediated contractility is dependent upon the duration of exposure to estrogen. This complex mechanism appears to be important in intact tissue; capsaicin-mediated release of endogenous neuropeptides resulted in a desensitization of response to subsequent stimulation with senktide in estrogen-dominated uterine tissue.

Twelve tips for increasing the defensibility of assessment decisions

Abstract In an era of increasing scrutiny of the performance of graduates in the workplace, there can be frustrations when decisions about learners with borderline or poor performance in formal assessments are over-turned in appeal processes. This article addresses the approach to reducing the uncertainty about assessment decisions and surviving appeals.

Differential responses of phenotypically distinct rat trophoblast cell lines to MHC class I antigen-inducing cytokines

Phenotypically distinct rat trophoblast cell lines, the Rcho-1 and R8RP.3 cells, were compared for their responses to cytokines known to induce major histocompatibility (MHC) class I antigens, tumour necrosis factor (TNF), transforming growth factor-beta (TGF-beta), and interferon-gamma (IFN-gamma). Cell enzyme immunosorbent assays and flow cytometry experiments showed that only IFN-gamma could induce RT1 class I antigens on the Rcho-1 cells. Non-adherent cells were slightly less responsive than adherent, giant cell-like Rcho-1 cells. By contrast, RT1 class I antigens on the R8RP.3 cells were induced by both TGF-beta 1 and IFN-gamma. The cytokines also had different effects on mitochondrial enzyme activity in the two lines. TNF and TGF-beta 1 mRNAs were demonstrated in both lines by using Northern blot hybridization. Rcho-1 but not R8RP.3 cells contained two TNF messages (approximately 2.2, 1.9 kb). Steady state levels of transcripts from the TNF gene, and, to a lesser extent, the TGF-beta 1 gene, were increased in cultures of Rcho-1 cells that contained high proportions of giant cells. Thus, phenotypically distinct rat trophoblast cell lines do not respond identically to TNF, TGF-beta 1 or IFN-gamma, transcription of cytokine genes does not prevent the cells from responding to paracrine cytokine signals, and the cells contain novel TNF transcripts that might be important in cell maturation or differentiation.