国立情報学研究所で電子化したコンテンツを使用している。

The pro-opiomelanocortin (POMC) prohormone produces several biologically active peptides, including £\-melanocyte-stimulating hormones (£\-MSH, £]-MSH, £^-MSH), corticotrophin (ACTH) and £]-endorphin. POMC-expressing neurons in the brain play a major role in the control of pain, energy homeostasis, pigmentation, adrenocortical function, and sebaceous gland lipid production. Recently, the peripheral POMC system is under active investigation to delineate their pathogenic roles in metabolic diseases such as Cushing¡¦s syndrome and obesity. In the present study, we employed adenovirus gene delivery system to achieve POMC overexpression in the livers of adult C57/BL6 mice. In the endocrine system of adrenal glands, hepatic POMC overexpression mice display hypertrophy the ACTH levels elevated concentrations in the blood, the ACTH receptor, melanocortin type 2 receptor (MC2-R) were decrease. This phenomenon explained the local adrenal gland tissue was inhibiting and feedback from central hypothalamic-pituitary- adrenal axis. Meanwhile, we investigated the islets of Langerhans in hepatic POMC overexpression mice, the insulin were disappear but the glucagon were constant, these reflect the blood sugar were loss of balance, maybe progress to metabolic syndrome. Subsequently, hepatic POMC overexpression resulted in liver injuries that the ALT and AST levels were significantly higher, the fat accumulation in the liver and the glycogen were diminished to nearly 1/4 of basal levels. Evidence the hepatic POMC overexpression induced inflammatory and fatty changes in the livers of mice. In summary, POMC gene delivery induces systemic POMC overexpression and results in fatty liver and adrenal dysfunction, which may facilitates a mice model for Cushing¡¦s-like metabolic syndrome.

OBJECTIVE: Adrenal steroidogenesis is important for maturation of fetal organ systems and plays a role in triggering parturition in ovine pregnancies. Studies have suggested a differential increase in baseline cortisol between twin gestations near term. Our aim was to further delineate the mechanisms responsible for the differences between the hypothalamic- pituitary-adrenal axes of twin fetuses in vivo. STUDY DESIGN: Surgery was performed on pregnant ewes (n = 6) with twin gestations to implant fetal vascular catheters. After recovery but while the subjects were resting, plasma cortisol concentrations were similar in both fetuses. Fetuses received, intravenously, boluses of adrenocorticotropic hormone at 2 doses, and plasma samples were obtained for analysis of the cortisol response. This stimulation by adrenocorticotropic hormone was then repeated in the same fetuses approximately 4 days later, after the increase of resting daily cortisol values in one but not the other fetus. RESULTS: Cortisol responses to adrenocorticotropic hormone before changes in daily resting cortisol concentrations were indistinguishable between twins. However, after separation of daily resting cortisol values, fetuses in group A (elevated resting cortisol concentration) demonstrated a significantly increased response to stimulation by adrenocorticotropic hormone. CONCLUSION: These results suggest a differential development in response to stimulation by adrenocorticotropic hormone between twin fetuses in vivo as the mechanism responsible for the asynchronous elevation of one twin's resting plasma cortisol concentration.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

The aim of this thesis is to in various ways explore protein expression in human normal tissue and in cancer and to apply that knowledge in biomarker discovery. In Paper I the prognostic significance of RNA-binding motif protein 3 (RBM3) is explored in malignant melanoma. To further evaluate the prognostic significance of RBM3 expression was assessed in 226 incident cases of malignant melanoma from the prospective populationbased cohort study Malmö Diet and Cancer Study using tissue microarray technique (TMA). RBM3 was shown to be down regulated in metastatic melanoma and high nuclear expression in the primary tumor was an independent marker of prolonged over all survival. As a tool to facilitate clinical biomarker studies the Human Protein Atlas has created a tissue dictionary as an introduction to human histology and histopathology. In Paper II this work is introduced. A cancer diagnosis can be a complex process with difficulties of establishing tumor type in localized disease or organ of origin in generalized disease. Immunohistochemically assisted diagnosis of cancer is common practice among pathologists where its application combined with known protein expression profiles of different cancer types, can strengthen or help dismiss a suspected diagnosis. In Paper III the diagnostic performance of 27 commonly used antibodies are tested in a predominantly metastatic, multicancer cohort using TMA technique. Overall these 27 diagnostic markers showed a low sensitivity and specificity for its intended use, highlighting the need for novel, more specific markers. Breast, ovarian, endometrial and ovarian cancers affect predominantly women. Differential diagnostics between these cancer types can be challenging. In Paper IV an algorithm, based on six different IHC markers, to differentiate between these cancer types is presented. A new diagnostic marker for breast cancer, namely ZAG is also introduced. In Paper V the transcriptomic landscape of the adrenal gland is explored by combining a transcriptomic approach with a immunohistochemistry based proteomic approach. In the adrenal gland we were able to detect 253 genes with an elevated pattern of expression in the adrenal gland, as compared to 31 other normal human tissue types analyzed. This combination of a transcriptomic and immunohistochemical approach provides a foundation for a deeper understanding of the adrenal glands function and physiology.

The autonomic nervous system serves to maintain a constant inner environment, a process termed homeostasis. Thus, in response to the homeostatic challenge posed by infectious agents, the autonomic nervous system answers to signals from the immune system and elicits adaptive physiological and behavioral reactions. These so called sickness responses include fever, anorexia, hyperalgesia, social avoidance, and the release of stress hormones. Neuropeptides, used in the communication between neurons, are because of their release properties and sustained actions likely mediators of homeostatic responses. The enkephalinergic system constitutes one of the largest neuropeptidergic systems in the brain, but its involvement in inflammatory conditions has been little studied. We first examined the immune-induced activation of the parabrachial nucleus (paper I), an enkephalinergic autonomic relay center in the brain stem. We found that intravenous injection of bacterial endotoxin, lipopolysaccharide (LPS), activated the external lateral parabrachial subnucleus, as measured in terms of Fos expression, but that the enkephalinergic cell population in this subnucleus was largely separated from the LPS-activated neurons. Because Fos may not always be a reliable activity marker, we next examined by in situ hybridization the immune-induced expression of newly transcribed preproenkephalin (ppENK) heteronuclear RNA (hnRNA), which gives a direct indication of the utilization of enkephalin in a particular neuron (paper II). We detected induced expression of ppENK hnRNA in several autonomic structures in the brain, including the paraventricular hypothalamic nucleus (PVH) but not the parabrachial nucleus, indicating increased enkephalinergic signaling activity in the positively labeled structures during inflammatory condition. We then examined the projections of the immune-induced ppENK transcribing PVH neurons by injecting rats intraperitoneally with the retrograde tracer substance Fluoro-Gold, hence labeling neurons with axonal projections outside the blood-brain barrier, followed by systemic injection of LPS (paper III). Dual-labeling histochemical and hybridization techniques showed that the vast majority of the ppENK hnRNA expressing cells were hypophysiotropic cells, hence being involved in neuroendocrine regulation. These findings suggest that centrally produced enkephalin is involved in the coordination of the sickness responses during systemic immune challenge, including the modulation of the release of stress hormones or other hypothalamic hormones during inflammatory conditions. We next turned to the role of prostaglandins in the hypothalamic-pituitary-adrenal (HPA) axis response to inflammation. We injected mice deficient for the terminal prostaglandin (PG) E2 synthesizing enzyme mPGES-1 with LPS and studied their stress hormone release (paper IV). The genetically modified mice displayed attenuated plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone during the later phases of the HPA-axis response compared with wild type mice, and this impairment did not depend on a changed activation pattern in the brain, but instead correlated to an early decrease in corticotropin-releasing hormone mRNA expression in the PVH, hence being the likely cause of the blunted ACTH and corticosterone responses at later time-points. Based on these findings we suggest that a neural, mPGES-1-independent pathway, and a humoral, mPGES-1-dependent pathway act in concert but in distinct temporal patterns to initiate and maintain the HPA-axis response during immune challenge. In addition to activating the central limb of the HPA-axis, inflammatory mediators have been suggested to act directly on the adrenal gland to induce the release of corticosterone, but little is known about the underlying mechanisms. We examined adrenal tissue isolated from rats injected with LPS or interleukin-1β (IL-1β) (paper V), and found that immune stimulation resulted in dynamic changes in the adrenal immune cell population, implying a rapid depletion of dendritic cells in the inner cortical layer and the recruitment of immature cells to the outer layers. These changes were accompanied by an induced production of IL-1β and IL-1 receptor type 1, as well as of cyclo-oxygenase-2 and mPGES-1 in these cells, implying local cytokine-mediated PGE2 production in the adrenals, which also displayed EP1 and EP3 receptors in the cortex and medulla. Additional mechanistic studies using an IL-1 receptor antagonist showed that IL-1β acts locally to affect its own synthesis, as well as that of cyclooxygenase-2. Taken together these data demonstrate a mechanism by which systemic inflammatory agents activate an intrinsically regulated local signaling circuit that may influence the adrenals’ response to immune stress and may help explain the dissociation between plasma levels of ACTH and corticosteroids during chronic immune perturbations.

Background: Management of a transplant recipient involves the use of multiple immunosuppressant drugs. Currently there is no test that reflects the overall immune status of the patient. This results in under or over suppression of the immune system and consequently increases in morbidity and mortality rates. Evaluation of the proliferative response of PBMC's to a mitogen PHA by measurement of intracellular ATP was evaluated as a tool to assess the immune response in kidney transplant patients. Method: PBMC's were separated from the blood samples of healthy controls and kidney transplant patients on cyclosporine, sirolimus, and tacrolimus based regimens by density gradient centrifugation, cells were counted and incubated overnight with and without PHA. The luciferin-Iuciferase enzyme reaction which induces bioluminescence and the Turner Biosystem luminometer were used to measure intracellular ATP levels in relative light units (RLU). An A TP standard curve was generated for each test. Results: The ATP (nglml) levels measured in the transplant recipients were lower and statistically significantly different (p< 0.0001) than the healthy controls. No statistically significant difference was measured between the cycIosporine and sirolimus drug groups. Patients on tacrolimus gave a statistically significant (p<O.0001) stronger immune response than those receiving cyclosporine and sirolimus. Overall, the immune response results of kidney transplant patients were statistically significantly lower than the healthy control by 981 nglml. Linear regression analysis revealed no correlation between patient A TP (nglml) levels and therapeutic drug blood levels, immunosuppressant drug dosages, creatinine levels and white cell counts. The immune responses of patients who were diagnosed with infection or were clinically stable were characterised as low or moderate, of interest, one patient who was diagnosed with rejection was found to have a strong immune response (>501 nglml ATP). Conclusion: Future studies to determine the predictive value of the A TP assay in directing immunosuppressive therapy are required. The assay described in this study is simple, sensitive and rapid and has possible application in immunological monitoring in a variety of conditions that affects the immune system. Keywords: kidney transplantation, immunosuppression, bioluminescence, lymphocyte, Adenosine Triphosphate (A TP), Phytohemmagglutinin (PHA) / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2008.

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Existem evidências na literatura que demonstram a existência de células indiferenciadas na capsula adrenal, e que o ACTH poderia estimular estas células. Porém não se sabe quais os genes e vias que desencadeiam esta resposta. Através de animais Cre-Lox induzível por Tamoxifeno, silenciamos o gene Pomc em camundongos adultos e avaliamos o efeito do ACTH nessas células. Foram utilizadas placas de PCR array para análise de genes relacionados com células progenitoras em amostras obtidas pela técnica de rolamento, e validação por PCRq com amostras microdissecadas da zona capsular/subcapsular da adrenal. Após caracterização dos animais com o gene Pomc silenciado e tratamentos com ACTH observamos o aumento da expressão de genes relacionados com as vias Wnt, Igf1 e Notch. Esses dados corroboram evidencias descritas na literatura que mostram a importância dessas vias no desenvolvimento e manutenção do córtex adrenal, e sugerem o envolvimento do ACTH nesses processos que envolvem as células progenitoras do córtex adrenal. / There is evidence in the literature demonstrating the existence of stem cells in the adrenal capsule, and that ACTH could stimulate these cells. However, it remains unknown which genes and pathways that trigger this response. By using a tamoxifen-inducible Cre-Lox mice strain, we knocked-out Pomc gene in adult mice and evaluated the effect of ACTH in these cells. PCR array technique was used to determine the expression level of key genes related to progenitor cells in samples obtained by the technique of \"rolling bearing\". Also, we validated the data by qPCR using samples from microdissected capsular areas of the adrenal gland. After characterization of animal model, the results show that treatment with ACTH increase the expression of genes related to Wnt, Igf1 and Notch pathways. These data corroborate with the literature, reinforcing the importance of these pathways in the development and maintenance of the adrenal cortex, and also suggesting the involvement of ACTH in these processes involving the progenitor cells of the adrenal cortex.