胰腺的胰島具有重要的生理功能，表現在系列的荷爾蒙，特別是能夠控制血糖穩態的胰島素的合成和分泌。胰島素的功能受到各種分子信號及環境的調節。在過去的十年裡，腎素血管緊張素系統(RAS)被發現除了調節血壓和體液穩態之外還具有局部性的生理功能。根據我們最近的發現，胰島存在自有的腎素血管緊張素系統並且可能在胰島生理作用和糖尿病方面發揮新穎的作用。同時，越來越多的研究發現一些與臨床相闊的調節因子在胰島的功能和糖尿病中起著關鍵的作用。這些調節因子促進胰島素分泌並且可以調節胰島細胞的生長和凋亡。其中一些調節因子顯示出極大的研究價值。胰高血糖素樣肽-1(GLP-1)能通過它在胰島上的受體改善胰島的功能和血糖的控制;另一方面， 維生素D 也可以通過它在胰島B細胞上的受體來起到調節胰島素分泌及控制糖尿病的作用。像胰島局部RAS一樣， GLP-1 和維生素D 都可以通過它們在同一個靶器官--胰島細胞上的受體來發揮它們的功能。因此，不難想象這三種調節因子之前具有潛在的聯系並且直接或間接地影響胰島功能。此研究可以分為三部分以闡述這三種調節因子在胰島上的新穎作用(1) GLP-l 和RAS 在胰島功能上的潛在協同作用; (2)維生素D 對於胰島RAS 表達的調節作用及對膜島功能的影響；(3) 維生素D 缺乏下的胰島RAS 表達以及胰島功能的改變。 / 在第一部分的研究裡，我們檢測了阻斷血管緊張素一型受體(纈沙坦)和增強GLP-l 作用(DPPIV 抑制劑LAF237) 的復合效應對二型糖尿病小鼠(db/db) 血糖控制和胰島功能方面的影響。我們比較了接受單一給藥和聯合給藥的db/db 小鼠的胰島功能。所有的藥物處理都改善了db/db 小鼠的血糖穩態，而聯合給藥組在增加胰島B細胞面積，減少細胞凋亡，促進增殖以及降低膜島氧化應激和膜島纖維化方面體現出復合效應。另外，短期的聯合給藥顯著促進分離出來的胰島細胞的胰島素分泌。這些結果顯示了血管緊張素型受體阻斷劑和DPPIV 抑制劑在改善胰島的結構與功能以及治療二型糖尿病方面具有復合效應。 / 據研究，維生素D 是種具有抗糖尿病和高血壓作用的荷爾蒙，而不適合的RAS活性能夠減少胰島功能和糖耐量。維生素D 對腎臟腎素的直接抑制作用表明維生素D 可能可以調節胰島得局部RAS 活性進而調節胰島的生理作用。因此第二部分的實驗旨在研究維生素D 是否能夠抑制分離培養的胰島中非正常表達的胰島局部RAS組分並且改善胰島且細胞功能。維生素D 受體存在於胰島且細胞的核與質中，計量依賴性地調節受體對活性維他命D-骨化三醇的反應。骨化三醇的刺激可以通過增加維生素D24羥化黣激發胰島局部維他命D 系統的反饋機制。在分離的胰島中，長期處於高糖的環境，胰島局部RAS 的異常表達可以一定濃度的骨化三醇治療和預防。然而，骨化三醇的送科治療效果，並沒有在生理正常糖濃度的情況下被發現。另外，在高糖環境下，骨化三醇增加胰島素前體合成以及葡萄糖刺激的服島素分泌。這些結果顯示骨化三醇能夠調節以及保護高糖環境引起的異常胰島RAS 組分表達並通過增加胰島素的合成與分泌來改善胰島的功能，為在高血糖和糖尿病情況下的維生素D 與胰島功能關系提供了新的機制。 / 循環中的維生素D 水平與血糖濃度以及糖尿病的患病風險成反比。第二部分的實驗結果現實了維生素D 具有潛在的調節胰島RAS 進而調節胰島功能的作用。因此，在第三部分的實驗裡，我們假設不充足的維生素D 水平可能引起異常的胰島RAS 表達進而引起胰島功能障薇。為了這個目的，我們使用了維生素D 受體缺失的基因敲除小鼠和維生素D 缺乏小鼠來檢測糖代謝，膜島形態以及局部RAS 組分的表達。結果顯示，在缺乏維生素D 以及正常的維生素D 作用的情況下，胰島局部RAS 組分異常表達。而這個維生素D 導致的RAS 異常表達的作用可能發生在高血糖現象之前，從而導致了胰島功能障礙，異常的糖代謝以及減弱的胰島且細胞本身的胰島素作用。這些結果為在生理情況下，維生素D 可以通過調節胰島局部RAS 的表達進而調節胰島功能提供了有力的支持。 / 總括來說，胰島局部RAS 在持續高糖環境下的胰島功能中有著關鍵的作用。GLP-l 和維生素D 都與胰島RAS 具有潛在的生物相關性並可以影響RAS 的表達，進而調節胰島功能和自細胞體積。我們的實驗數據顯示了這三種調節系統共同作用並調節目突島細胞功能以及血糖穩態，進一步提議了它們在二型糖尿病治療中的價值。 / Pancreatic islets perfonn critical biological activities by means of synthesizing and releasing islet peptide honnones, notably insulin that controls our glucose homeostasis. The insulin secretory function is, in turn, governed by various conditions and signaling molecules. In the past decade, it is recognized that the renin-angiotensin system (RAS) has local function rather than the maintenance of blood pressure and fluid homeostasis. With our recent recognition of an islet RAS, it is believed that it has novel roles in islet physiology and diabetes. Meanwhile, more and more clinically relevant regulators that have pivotal roles in islet function and diabetes have been well investigated; such regulators have positive action on insulin secretion, B-cell replication and cell apoptosis/proliferation balance. Of great interest in this context is the glucagon-like peptide I (GLP-I) that improves islet function and glycemic control via its islet specific receptors located on the islets. On the other hand,vitamin D also regulates islet insulin secretion and diabetes via its mediation of receptors on islet B-cells. Like islet RAS, GLPI and vitamin D exert their biological effects via mediation of respective receptors located on the common target, i.e. the islet beta-cells. As such, it is plausible to propose that all these three regulators have potential interactions so as to affect islet functions in a direct or an indirect manner. Accordingly, the primary objective of this study is to examine the potential roles oflocal RAS, GLP-I and vitamin D system in pancreatic islet function. The present study is thus divided into three main parts addressing the issues of these three novel regulators in islet function: (1) the potential synergism of GLP-I and RAS in islet function; (2) the modulatory effects of vitamin D on islet RAS expression and function; (3) The altered islet RAS and islet function under a hypovitaminosis D condition. / In the first part of our study, we examined the combined effect of blocking islet A Tl receptor (ATl receptor blocker: valsartan) and enhancing GLP-l actions (DPP IV inhibitor: LAF237) on islet function and glycemic control in a mouse model with type 2 diabetes, db/db mice. We compared the islet function in db/db mice with either valsartan or LAF237 mono treatment or combined treatment. Consistently, all these treatments improved glucose homeostasis in db/db mice while combined treatment resulted in a significant increase in islet B-cell area by decreasing cell apoptosis and increasing proliferation, together with marked decreases of islet oxidative stress and fibrosis. In addition, a short-term effect on stimulating insulin secretion was also observed in isolated islets with combined treatment. These results indicate that the combination treatments with ATl receptor blocker and DPP IV inhibitor has beneficial additive effects on islet structure and function in type 2 diabetes, compared with their monotherapeutic treatments. / It is reported that vitamin D is a hormone with anti-diabetic and anti-hypertension effects in human while inappropriate RAS activity has been known to reduce islet function and glucose tolerance. The direct suppressive effect of vitamin D on renal renin activity indicates vitamin D may acts as a regulator in RAS activity thus modulate islet physiology. In the second part of our study, it was aimed to study whether vitamin D vitamin D downregulation of abnormal islet RAS activity improves B-cell function using an isolated pancreatic islet model. VDR was localized in islet B-cell nuclei and cytoplasm, mediated responses to active form of vitamin D calcitriol in a dose-dependent manner. This islet local vitamin D system may have its own feedback system as a marked increase ofCYP24 transcription was triggered by calcitriol stimulation. In isolated islets exposed to prolonged high glucose environment, abnormal expressed islet RAS components could be reversed or protected by calcitriol at a specific concentration. However, the inhibition effect of calcitriol on islet RAS were not observed at physiological glucose concentrations. In additon, calcitriol increased islet proinsulin synthesis and insulin secretion with hyperglycemia. These results indicated that calcitriol modulate or protect the abnormal isolated islet RAS component expression against hyperglycemia and improve islet function via increasing insulin synthesis and secretion, which might provide an alternative mechanism by which vitamin D availability enhances islet function in hyperglycemia or diabetes. / The circulating vitamin D level is inversely related to blood glucose level and risks of diabetes. Results in the second part of experiments suggested the potential RAS modulatory effect of vitamin D in isolated islets Therefore, in the third part of our study, we hypothesize that the insufficient vitamin D levels may lead to the inappropriate regulation of islet RAS expression and thus result in islet dysfunction. To achieve this, we examined the potential islet RAS-mediated effect of vitamin D on islet function by accessmg glucose homeostasis, islet histomorphology, and local RAS expression and function by means of using a vitamin D receptor knockout and diet-induced vitamin D deficiency mouse models. Results showed that the islet RAS components were abnormally expressed when lacking a sufficient vitamin D level and normal vitamin D action. These observed effects of insufficient vitamin D might occur prior to onset of hyperglycemia thus modulating islet RAS expression, which in turn lead to islet failure and dysfunctional glucose homeostasis, together with decreased insulin actions in islet B-cells. These results provide supports for the view that vitamin D physiologically exerts modulatory effects on islet function by downregulating islet RAS expression and function. / In summary, islet local RAS may have a central role in islet function under prolonged hyperglycemic stress. GLP-l and vitamin D have biological interactions with the islet RAS by downregulation of its expression and function, thereby affecting islet cell function and cell mass. Our data indicate that all three regulators work together in the regulation of pancreatic islet B-cell functions and glucose homeostasis, further suggestive of their potential values in the treatment of type 2 diabetes. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cheng, Qianni. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves [205]-243). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 摘要 --- p.v / Acknowledgements --- p.viii / List of Publications --- p.x / Table of Contents --- p.xii / List of Abbreviations --- p.xvi / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Endocrine Pancreas --- p.2 / Chapter 1.1.1 --- The structure and composition of endocrine pancreas --- p.3 / Chapter 1.1.2 --- Functions of endocrine pancreas --- p.4 / Chapter 1.1.3 --- Insulin structure and insulin receptors --- p.8 / Chapter 1.1.4 --- Mechanisms of insulin secretion --- p.11 / Chapter 1.1.5 --- Mechanisms of insulin actions --- p.18 / Chapter 1.1.6 --- Disorders of the endocrine pancreas --- p.22 / Chapter 1.2 --- Diabetes mellitus --- p.23 / Chapter 1.2.1 --- Type 1 diabetes mellitus (TlDM) --- p.24 / Chapter 1.2.2 --- Type 2 diabetes mellitus (T2DM) --- p.26 / Chapter 1.2.3 --- Other types of diabetes mellitus --- p.29 / Chapter 1.2.4 --- Islet dysfunction and T2DM --- p.30 / Chapter 1.3 --- Renin-angiotensin system (RAS) --- p.33 / Chapter 1.3.1 --- Components ofRAS --- p.33 / Chapter 1.3.2 --- Tissue local RAS --- p.42 / Chapter 1.3.3 --- Pancreatic local RAS --- p.43 / Chapter 1.4 --- Glucagon like peptide-l (GLP-l) and pancreatic islet function --- p.54 / Chapter 1.4.1 --- Gastrointestinal incretin honnones --- p.54 / Chapter 1.4.2 --- GLP-l and pancreatic islet function --- p.56 / Chapter 1.4.3 --- Incretin based therapies for T2DM --- p.59 / Chapter 1.4.4 --- GLP-lIRAS axis and pancreatic islet function --- p.62 / Chapter 1.5 --- Vitamin D and pancreatic islet function --- p.64 / Chapter 1.5.1 --- Vitamin D synthesis and metabolism --- p.65 / Chapter 1.5.2 --- Vitamin D physiological functions and pancreatic islets --- p.67 / Chapter 1.5.3 --- Vitamin D and diabetes mellitus --- p.68 / Chapter 1.5.4 --- Vitamin D and RAS --- p.70 / Chapter 1.6 --- Objectives --- p.71 / Chapter Chapter 2 --- Materials and Methods --- p.73 / Chapter 2.1 --- Experimental animal models --- p.74 / Chapter 2.1.1 --- Animal model ofT2DM --- p.74 / Chapter 2.1.2 --- Animal model for pancreatic islet isolation --- p.75 / Chapter 2.1.3 --- Vitamin D receptor knockout mice (VDRKO mice) --- p.75 / Chapter 2.1.4 --- Animal model for vitamin D deficiency --- p.76 / Chapter 2.2 --- Pancreatic islet isolation and culture --- p.76 / Chapter 2.2.1 --- Mice pancreatic islet and single B-cell isolation --- p.77 / Chapter 2.2.2 --- Primary culture of isolated pancreatic islets: --- p.78 / Chapter 2.3 --- Physiological assay for pancreatic islet function --- p.78 / Chapter 2.3.1 --- Measurement of blood glucose and glucose tolerance test --- p.78 / Chapter 2.3.2 --- Measurement of glucose-induced insulin secretion --- p.79 / Chapter 2.3.3 --- Measurement of (pro )insulin biosynthesis --- p.80 / Chapter 2.4 --- Detection ofmRNA expression --- p.80 / Chapter 2.4.1 --- Design of primers --- p.81 / Chapter 2.4.2 --- mRNA extraction and cDNA synthesis --- p.82 / Chapter 2.4.3 --- Detection of mRN A expression by conventional peR --- p.83 / Chapter 2.4.4 --- SYBR Green real-time peR --- p.83 / Chapter 2.4.5 --- Real-time peR analysis using the comparative eT method --- p.84 / Chapter 2.5 --- Detection of protein expression --- p.84 / Chapter 2.5.1 --- Western blot analysis --- p.84 / Chapter 2.5.2 --- Immunostaining assessment --- p.85 / Chapter 2.6 --- In situ detection of oxidative stress, proliferation and apoptosis --- p.88 / Chapter 2.6.1 --- Detection of islet reactive oxygen species --- p.88 / Chapter 2.6.2 --- Detection of cell proliferation --- p.89 / Chapter 2.6.3 --- Measurement of cell apoptosis --- p.90 / Chapter 2.7 --- Statistical data analysis --- p.90 / Chapter Chapter 3 --- Combination of DPP-IV Inhibitor LAF237 with ATl Receptor Antagonist Valsartan Enhances Pancreatic Islet Morphology and Function in a Mouse Model of Type 2 Diabetes (This work has been published in J Pharmacal Exp Ther, 327: PI-9) --- p.91 / Chapter 3.1 --- Abstract --- p.92 / Chapter 3.2 --- Introduction --- p.94 / Chapter 3.3 --- Materials and Methods --- p.96 / Chapter 3.4 --- Results --- p.103 / Chapter 3.4.1 --- Effects of acute treatment with GLP-I and valsartan on insulin secretion in isolated islets --- p.103 / Chapter 3.4.2 --- Effects of LAF237 and valsartan on pancreatic --- p.105 / Chapter 3.4.3 --- Effects of LAF237 and valsartan on --- p.107 / Chapter 3.4.4 --- Effects ofLAF237 and valsartan on islet apoptosis --- p.109 / Chapter 3.4.5 --- Effects of LAF237 and valsartan on islet fibrosis --- p.110 / Chapter 3.4.6 --- Effects of LAF237 and valsartan on pancreatic islet superoxide and nitrotyrosine expression --- p.113 / Chapter 3.4.7 --- Effects of LAF237 and valsartan on bood glucose concentration and glucose tolerance in db/db diabetic mice --- p.116 / Chapter 3.5 --- Discussion --- p.119 / Chapter Chapter 4 --- The Role of Calcitriol in Modulating the Expression and Function of Islet Renin-Angiotensin System in Isolated Mouse Pancreatic Islets --- p.124 / Chapter 4.1 --- Abstract --- p.125 / Chapter 4.2 --- Introduction --- p.127 / Chapter 4.3 --- Materials and Methods --- p.130 / Chapter 4.4 --- Results --- p.135 / Chapter 4.4.1 --- The expression of islet VDR under different glucose conditions and the effects of calcitriol --- p.135 / Chapter 4.4.2 --- The effect of calcitriol on high glucose-modulated islet RAS component expression --- p.140 / Chapter 4.4.3 --- The protective effect of calcitriol against high glucose on islet RAS component expression --- p.144 / Chapter 4.4.4 --- The effect of calcitriol on (pro )insulin biosynthesis and insulin release in isolated islets --- p.148 / Chapter 4.5 --- Discussion --- p.151 / Chapter Chapter 5 --- Altered Islet Local Renin-Angiotensin System and Islet Function in Mice with Hypovitaminosis D --- p.158 / Chapter 5.1 --- Abstract --- p.159 / Chapter 5.2 --- Introduction --- p.160 / Chapter 5.3 --- Materials and methods --- p.163 / Chapter 5.4 --- Results --- p.168 / Chapter 5.4.1 --- Glucose homeostasis and islet morphology in VDR KO mice --- p.168 / Chapter 5.4.2 --- Expression of vitamin D receptor and major RAS components in the pancreatic islets of WT and VDR KO mice --- p.170 / Chapter 5.4.3 --- Vitamin D deficiency in mice on a vitamin D deficient diet --- p.172 / Chapter 5.4.4 --- Altered glucose homeostasis in vitamin D deficient mice --- p.174 / Chapter 5.4.5 --- Islet histomorphology in vitamin D deficient mice --- p.176 / Chapter 5.4.6 --- Regulation of islet RAS components expression in vitamin D deficient mice --- p.179 / Chapter 5.4.7 --- Transcriptional regulation of islet insulin receptor and its substrates in vitamin D deficient mice --- p.181 / Chapter 5.4.8 --- Effect of calcitriol treatment on glucose tolerance in vitamin D deficient mice --- p.183 / Chapter 5.5 --- Discussion --- p.185 / Chapter Chapter 6 --- General Discussion --- p.191 / Chapter 6.1 --- Combination effects of blocking islet RAS components and enhancing incretin activity on improving islet function in type 2 diabetes --- p.193 / Chapter 6.2 --- Potential modulatory effect of vitamin D on islet RAS expression and action --- p.196 / Chapter 6.3 --- The role of vitamin D in modulating islet RAS in glucose homeostasis and islet function --- p.199 / Chapter 6.4 --- The significance ofRAS, GLP-l and vitamin D in the management of T2DM --- p.201 / Chapter 6.5 --- Conclusion --- p.202 / Chapter 6.6 --- Future studies --- p.202 / Chapter Chapter 7 --- Bibliography --- p.205

Dendritic cells (DCs), a hematopoietic cell type belonging to the sub-group of cells called antigen presenting cells (APCs), inhabit a central role in innate and adaptive immunity. Although the DC family is very heterogeneous, all members share unique features. Most importantly, DCs can stimulate an immune response. This is due to the cells’ ability to capture and process antigens and to maturate in the presence of danger signals presented by pathogens. Maturation in turn results in the migration of DCs from the tissue they reside in to the draining lymph nodes, as well as in the subsequent presentation of the acquired antigens to T cells. In the skin, which is one of the most immunogeneic organs, DCs are present in sizable numbers in both the epidermis and the dermis. This study focused on two types of DCs: epidermal Langerhans cells (LCs) and dermal DCs (DDCs). While much is understood about LCs, far less is known about the role that DDCs play in skin immunity. Therefore one purpose of this study was to characterize DDCs and to compare their phenotype and functions to that of LCs. This study used two different methods to characterize human skin resident immune cells with regard to their number and distribution. First, a stable analytical immunohistochemistry-based method was developed and applied to a substantial number of healthy skin donors. This enabled a quantitative analysis of skin DC types and skin resident T cells at different anatomical locations in situ. A novel method to count dermal cell populations in situ was developed that resulted in the first published quantification of APCs, DDCs, as well as T cells in human dermis. Second, the traditional form of the emigration assay, which selectively enriches vital cells capable of ex vivo emigration from the skin, was upgraded toward a stable analytical method to separate epidermal LCs from DDCs. In this way, both skin DC types became accessible in sufficient numbers to allow for a comparison of phenotypes and functions in vitro. The resulting phenotypic observations clearly showed that both, LCs and DDCs are not fully mature after their emigration ex vivo and that both can be transformed into a phenotypically more mature state by treating them with inflammatory cytokines. What’s more, LCs are also functionally in an immature state after their emigration. They efficiently took up antigen, showed a low capacity to trans-migrate in response to chemokines, and demonstrated a low capacity to stimulate allogeneic T cells in a mixed leukocyte reaction (MLR). For the first time this study observed all these main APC functions not only for LCs but additionally for DDCs. As these observations were made in relation to LCs of the same donor, it could be concluded that DDCs are functionally more mature than LCs after emigration. DDCs showed a lower antigen uptake capacity than LCs but were superior in terms of their migratory and stimulatory capacity. However, treatment with cytokines could skew LC functions toward functional capacities observed for DDCs, i.e., it decreased LCs’ Ag uptake and increased their migratory and stimulatory capacity, whereas the cytokine treatment did not alter DDCs’ functional capacities. After improving immuno-histochemistry and the emigration assay using healthy skin samples, these newly developed techniques were implemented in clinical trials to observe the number, distribution and migratory capacity of skin DCs and T cells in patients undergoing allogeneic hematopoietic cell transplantation (aHSCT). Such a study is of importance because the turnover of DCs and T cells is closely associated with the occurrence of acute graft-versus-host disease (aGvHD), the major cause of morbidity and mortality after aHSCT. Due to the study design used, this study concisely demonstrate that at the onset of aGvHD, different DC types accumulate along with effector T cells in skin lesions of aGvHD but not in uninvolved skin of the same patient. These results suggest that in addition to donor T cells LCs and DDCs play a role during the early phase of cutaneous aGvHD directly within the site of inflammation. The view of many authors that DC depletion in the transplant recipient, especially in target organs, is a promising approach for aGvHD prophylaxis and therapy is further underscored by these results. One targeting strategy to inhibit GvHD by eliminating recipient DCs may be the use of DC specific monoclonal antibodies. Alemtuzumab (anti-CD52) is a monoclonal antibody and has proven effective in preventing aGvHD after aHSCT. It may, despite depleting donor T cells, also work by targeting recipient DCs. To determine whether the last mechanism of action is significant, a second clinical study investigated the effects of intravenous alemtuzumab on DCs by comparing the number of these cells in skin and blood of patients before and after a 4-week course of alemtuzumab treatment. The result was that although skin DCs weakly express the target antigen CD52 the number of these cells was not consistently reduced by alemtuzumab. In contrast, circulating blood DCs have a stronger CD52 expression and were significantly reduced by the treatment. In conclusion, this work provides new insights into the phenotypical and functional characteristics of human skin DCs, as well as into the fate of these cell types during aHSCT. The investigation of the APC system during aGvHD as carried out here will help to understand the process of aGvHD in more detail. All these efforts may hopefully support the development of new approaches for therapy and prevention of this major limitation of aHSCT and may help to improve this only curative therapy for several life-threatening diseases.

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O uso de dieta hiperlipídica com banha de porco em roedores induz obesidade, resistência à insulina e disfunção das células beta do pâncreas. Em diversos tecidos de animais tratados com dieta hiperlipídica já se observou aumento de expressão e/ou atividade da NAD(P)H oxidase, que pode estar envolvida em processos fisiopatológicos. O objetivo deste trabalho foi avaliar se a dieta hiperlipídica altera a expressão e/ou a atividade da NAD(P)H oxidase em ilhotas pancreáticas e se este fato pode estar associado às disfunções das células beta relatadas na literatura para este modelo animal. As ilhotas pancreáticas dos animais tratados com dieta apresentam maior secreção de insulina em alta glicose, maior metabolização da glicose, menos apoptose, menor expressão protéica de subunidades da enzima, menor produção de superóxido e não apresentam estresse oxidativo. O papel da enzima provavelmente se relaciona ao processo de secreção de insulina. A regulação de sua expressão e atividade deve estar relacionada à adaptação das ilhotas aos efeitos deletérios da dieta. / Feeding animals with high fat diet containing lard causes obesity, insulin resistance and dysfunction of pancreatic beta cells. High fat diet induces oxidative stress and modulates NAD(P)H oxidase expression and activity in many tissues. This enzyme may be involved in many pathophysiological processes. The objective of this study was to evaluate the action of high fat diet on NAD(P)H oxidase activity and expression and if this fact can be connected to the beta cell dysfunction reported in the literature on this animal model. In pancreatic islets of rats fed the high fat diet apoptosis was reduced, glucose metabolism increased, insulin secretion elevated at high glucose, protein expression of NAD(P)H oxidase subunits reduced and the superoxide production was diminished. There was no difference between the groups for oxidative stress markers. It is possible that the enzyme has a role in the process of insulin secretion. Probably the islets are regulating their activity and function to compensate the deleterious effect of lard.

Made available in DSpace on 2014-12-17T15:32:22Z (GMT). No. of bitstreams: 1 KaruzaMAP.pdf: 544780 bytes, checksum: e0fadeed7d2d1ec7568970935306d05c (MD5) Previous issue date: 2006-02-22 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The Human Papillomavirus (HPV) has been strongly implicated on development of some cases of oral squamous cell carcinoma (OSCC). However, the immunological system somehow reacts against the presence of this virus. Among the cells involved on such mechanism of defense detaches the Langerhans cells (LC), which are responsible for processing and presenting antigens. The purpose of this study was to evaluate the immunohistochemical reactivity for Langerhans cells between HPV positive and HPV negative OSCC, as well as, the relation of the immunoreactivity for this cells and the histological grading of malignancy proposed by Bryne (1998) and modified by Miranda (2002). Additionally, HPV infection was evaluated in relation to sex, age, lesion localization and histological grading of malignancy. In the total, 27 cases of OSSC were evaluated, 09 of them HPV positive and 18 HPV negative. Anti S-100 antibody was utilized for the immunohistochemical labelling, followed by the counting of LCs in 5 highpower fields (400x). No statistically significant difference was verified between the variables sex, age, lesion localization, histological grading of malignancy and HPV presence in OSSC. There was neither association between the immunohistochemical labeling for LCs (S-100+) and HPV infection nor correlation between the quantity of LCs labeled and the histological grading of malignancy of OSSC. The results suggest that despite the absence of statistically significant difference, the presence of HPV in such cases of OSCC can alter the immunological system, particularly the Langerhans cells / O Papilomav?rus Humano (HPV) tem sido implicado fortemente no desenvolvimento de alguns carcinomas epiderm?ides orais (CEOs). Contudo, o sistema imunol?gico reage de alguma forma ? presen?a desse v?rus. Dentre as c?lulas envolvidas nesse mecanismo de defesa, destaca-se a c?lula de Langerhans (CL), por serem c?lulas processadoras e apresentadoras de ant?genos. O objetivo desse estudo foi avaliar a marca??o imuno-histoqu?mica das c?lulas de Langerhans entre os casos de CEOs HPV positivos e negativos, bem como a rela??o da imunomarca??o dessas c?lulas e a grada??o histol?gica de malignidade proposta por Bryne (1998) e modificada por Miranda (2002). Adicionalmente, a infec??o pelo HPV foi estudada com rela??o ao sexo, idade, localiza??o da les?o e a grada??o histol?gica de malignidade. Foram analisados 27 casos de CEOs, sendo 09 destes HPV positivos e 18 casos negativos. Para a marca??o imuno-histoqu?mica utilizou-se o anticorpo anti S-100, sendo as CLs quantificadas em 5 campos de maior aumento (400x). A an?lise estat?stica revelou n?o existir rela??o das vari?veis, sexo, idade, localiza??o da les?o e grada??o histol?gica, com a presen?a do HPV nos CEOs estudados. N?o existiu associa??o entre a marca??o imuno-histoqu?mica das CLs(S-100+) e a infec??o pelo HPV, e tamb?m n?o houve correla??o entre as CLs imunomarcadas e a grada??o histol?gica nos casos de CEOs analisados. Diante desses resultados, pode-se sugerir que mesmo n?o havendo diferen?a significativa, a presen?a do HPV nos casos de carcinoma epiderm?ide oral pode alterar o sistema imune, particularmente as c?lulas de Langerhans

Diabetes mellitus tipo 1 resulta da produção insuficiente ou da ausência de insulina, decorrente da destruição de células β, por mecanismo auto-imune. O tratamento deste tipo de diabetes consiste na administração subcutânea de insulina exógena. Recentemente, foi demonstrado que o transplante de ilhotas pancreáticas é capaz de tornar o portador de diabetes tipo 1 independente de insulina exógena. Apesar do sucesso alcançado, a necessidade permanente de imunossupressão é uma das principais barreiras para que o transplante de ilhotas possa ser realizado em número maior de pacientes. Assim, o desenvolvimento de novas metodologias que evitem a rejeição do enxerto, como o macro e o microencapsulamento de ilhotas, continua sendo crucial para o estabelecimento definitivo do transplante de ilhotas como opção terapêutica no tratamento de diabetes tipo 1. Neste trabalho, foi padronizado um modelo animal para avaliar, in vivo, a funcionalidade das ilhotas pancreáticas humanas isoladas e purificadas na Unidade de Ilhotas Pancreáticas Humanas do IQUSP. Ratos NIH nude foram tornados diabéticos através de injeção de estreptozotocina para o implante de ilhotas pancreáticas humanas nuas e microencapsuladas. As ilhotas foram microencapsuladas em Biodritina, um novo heteropolissacarídeo patenteado e cedido ao nosso laboratório, tendo sido possível padronizar a produção de microcápsulas uniformes e homogêneas, com tamanho médio entre 400µm e 600 µm. A reversão do diabetes ocorreu em 24% dos ratos nude transplantados com ilhotas pancreáticas humanas nuas. Por outro lado, não observamos reversão do diabetes quando ilhotas encapsuladas foram implantadas, apesar do teste de atividade funcional realizado in vitro ter demonstrado que elas continuam a secretar insulina e a responder ao estímulo com glicose após o encapsulamento. Para elucidar este efeito, cápsulas vazias foram implantadas em ratos nude e em ratos imunocompetentes, os quais desenvolveram processo inflamatório acompanhado de processo fibrótico no local do implante. Estudo imuno-histoquímico está sendo realizado para esclarecer a natureza e a intensidade destes processos. / Type 1 diabetes mellitus results from insufficient or absence of insulin production, as a consequence of destruction of pancreatic β cells, by an auto-imune mechanism. Treatment for this type of diabetes consists of subcutaneous administration of exogenous insulin. Recently, it has been demonstrated that pancreatic islet cell transplantation is capable of rendering type I diabetic patients independent of exogenous insulin. However, in spite of the success achieved, permanent immunosuppression is still required, being the main barrier to expand this treatment to a large number of patients. Therefore, development of new technologies, such as islet macro and microencapsulation to avoid rejection of the tissue implanted, is still crucial for definitive establishment of islet transplantation as a therapeutic alternative for type I diabetes. In the present work, an animal model was established for in vivo evaluation of the functional ability of human pancreatic islets, which were isolated and purified at the Human Pancreatic Islet Unit of the University of São Paulo Chemistry Institute. Diabetes was induced in NIH nude rats through streptozotocin injection followed by implantation of naked or microencapsulated human pancreatic islets. Biodritin, a new and patented heteropolyssaccaride was used to microencapsulate the islets. The production of uniform and homogeneous microcapsules with diameters in the range of 400µm e 600 µm was successfully established. Reversion of diabetes occurred in 24% of the nude rats transplanted with human pancreatic islets. On the other hand, no reversion of diabetes was observed when encapsulated islets were implanted, although their functional activity in vitro indicated that they secreted insulin and responded to glucose stimulation upon encapsulation. In order to elucidate this effect, empty capsules were implanted in nude rat and in immunocompetent rats, both of which developed an inflammatory process accompanied by a fibrotic process in the site of the implant. Immunohistochemical studies are underway to address the nature and the intensity of these inflammatory processes.

Les Cellules Dendritiques sont un groupe hétérogène de cellules présentatrices d’antigènes, importantes pour le contrôle des réponses innées et adaptatives. Les Cellules Dendritiques Plasmacytoïdes (pCD) en représentent une population unique, aux caractéristiques phénotypiques et fonctionnelles particulières, notamment par leur capacité à produire de grande quantité d’Interféron de type I (IFN). Cette signature IFN marque la physiopathologie du Lupus Erythémateux Systémique (LES), maladie auto-immune systémique. Les mécanismes à l’origine de cette production excessive d’IFN par les pCD restent incomplètement élucidés. Nous montrons, dans notre étude, chez l’homme comme dans un modèle murin que les plaquettes, activées dans le LES, participent à la production d’IFN via le CD40L. Cette production en excès d’IFN, a pour conséquence une maturation et activation d’autres Cellules Dendritiques (CD) entrainant l’activation inappropriée des lymphocytes T. Chez le sujet sain, cette activation inappropriée du système immunitaire adaptatif doit être strictement contrôlée afin d’assurer l’homéostasie du système immunitaire. Il a été montré précédemment que de nombreux lymphocytes aux caractéristiques phénotypiques de type mémoire-effecteur (TEM) peuplent les tissus périphériques, notamment le tissu cutané. Ces TEM sont capables de s’activer et proliférer localement en réponse à un stimulus. Les Cellules de Langerhans (LC) sont des cellules dendritiques résidant au niveau cutané dans l’épiderme. Leur fonction est à ce jour l’objet d’une controverse entre une fonction immuno-stimulante (modèle humain) et une fonction immuno-régulatrice (modèle murin). Nous démontrons dans cette étude que les LC, à l’état basal, chez l’homme, induisent la prolifération de Lymphocytes T régulateurs (Treg) au niveau cutané, capables de bloquer la stimulation inappropriée des TEM cutanés. Cependant en présence d’un stimulus infectieux, les LC induisent préférentiellement la prolifération des TEM en limitant celle des Treg. Les LC semblent être à la fois immuno-régulatrices ou stimulantes en fonction du contexte biologique auquel elles sont confrontées. / Dendritic Cell (DC) are a heterogeneous group of antigen-presenting leukocytes that are important in activation of both the innate and adaptative arms of the immune system. Plasmacytoid Dendritic Cells (pDC) represent a unique population, characterized by their ability to produce large amounts of type I Interferon (IFN). This « IFN signature » is a prominent feature of Systemic Lupus disease (SLE). Mechanisms leading to the excessive production of type I IFN remain largely unknown. Here, in our present study, we demonstrate that platelets are activated in SLE patients by circulating immune complexes and represent a major reservoir of CD40L. Activated platelets potentiate the production of type I IFN by pCD through a CD40L/CD40 interaction. Excessive production of type I IFN by pCD leads to DC activation and maturation and inappropriate activation of auto-reactive T cells.Under steady state condition, inappropriate activation of the immune system must be tightly controlled. It has been previously shown that normal adult human skin contains a large number of resident T cells (TRM) expressing the phenotype of Effector Memory T cells (TEM). These TEMTRM are specific for antigens previously encountered through skin and can be activated and proliferate under specific stimulation. Langerhans Cells (LC) are a group of skin resident DC living in epidermis. There is currently substantial controversy regarding the physiologic role of LC with regard to immunoregulation versus immunostimulation. Here we show that under steady state condition, LC induce the proliferation of a small subset of TRM. These proliferating TRM express the phenotype of TREG and are functional. However this stimulation of TREG could be reversed in the presence of foreign antigen in a dose-dependant fashion, as the addition of a pathogen to LC and TRM led to diminished TREG proliferation and increased TEM proliferation. These findings establish a novel immunological role for LC in human skin, allowing for the constitutive maintenance of tolerance, while also permitting the stimulation of resident immune memory in response to infectious challenge

La tolérance périphérique vis-à-vis de molécules potentiellement allergéniques en contact avec la peau joue un rôle essentiel pour prévenir le développement de l’eczéma allergique de contact (EAC). Au cours de ma thèse, j'ai contribué à l'identification des mécanismes et des acteurs responsables de l'induction de la tolérance par voie cutanée et à préciser le rôle respectif des sous-populations de cellules dendritiques (DC) cutanées dans la rupture de la tolérance et l'induction de lymphocytes T (LT) CD8+ initiant l'EAC. A l'aide d'un modèle murin d'induction de tolérance aux haptènes, j’ai pu montrer que les cellules de Langerhans (LC) épidermiques sont les cellules clés pour induire la tolérance cutanée et empêcher le développement d'un EAC médié par les LT CD8+. En effet, suite à l’application épicutanée d’un allergène/haptène faible, le DNTB, les LC migrent de la peau aux ganglions lymphatiques pour présenter l’antigène aux LT CD8+. Des expériences de déplétion in vivo et de transfert adoptif montrent que les LC sont responsables de la suppression de l’EAC en prévenant la différentiation des LT CD8+ spécifiques de l'allergène en cellules T cytotoxiques via deux mécanismes complémentaires: i) l’anergie/délétion des LT CD8+ et ii) l'activation de LT régulateurs Foxp3+ exprimant ICOS. Après avoir identifié des conditions d'immunisation conduisant au développement d'un EAC au DNTB, j'ai montré que la rupture de tolérance à ce type d'allergène est associée à i) à des modifications phénotypiques des LC épidermiques, ii) au recrutement rapide de monocytes inflammatoires Gr1+ dans la peau et iii) à une capacité équivalente des LC et des DC dermiques Langerin- à présenter l'allergène aux LT CD8+ dans les ganglions. Dans cette situation, les LC jouent un rôle pro-inflammatoire puisque leur déplétion réduit de manière dramatique l'induction de LT CD8+ effecteurs et l'EAC. Ces résultats indiquent que les LC jouent un rôle essentiel à la fois dans la prévention et dans l’induction de l’EAC, et que leur fonction tolérogène ou stimulatrice est vraisemblablement conditionnée par le microenvironnement cutané lors de la pénétration de l’allergène / Induction of peripheral tolerance to potentially allergenic molecules in contact with the skin is essential to prevent the development of allergic contact dermatitis (ACD). During my PhD, I contributed to the identification of the mechanisms and actors responsible for the induction of skin tolerance and clarified the respective roles of dendritic cell (DC) subsets in the breakdown of skin tolerance leading to the priming of cytotoxic CD8+ T cells and developpement of ACD. Using a mouse model of cutaneous tolerance to a model weak allergen, we show that epidermal Langerhans cells (LC) are essential to induce CD8+ T cell tolerance and prevent the development of ACD. Indeed, following the epicutaneous delivery of the weak allergen/hapten DNTB, LC were found to migrate from skin to draining lymph nodes to present the allergen to CD8+ T cells. Depletion and adoptive transfer experiments revealed that LC protect from development of ACD by preventing the priming of allergenspecific cytotoxic CD8+ T cells via two complementary mechanisms: i) anergy/deletion of allergen-specific CD8+ T cells and ii) activation of highly suppressive Foxp3+ regulatory T cells expressing ICOS. We identified DNTB skin delivery conditions that allow for CD8+ T cell priming and initiation of ACD. Breakdown of tolerance to this weak allergen was associated with i) phenotypic modifications of epidermal LC, ii) recruitment of inflammatory monocytes to the skin and iii) allergen presentation to CD8+ T cells by both LC and dermal Langerin- DC. In addition, LC are involved in tolerance breakdown as their depletion prior to skin immunization abrogated induction of CD8+ effector cells and ACD. These results demonstrate that LC are essential for both the induction of skin tolerance to weak skin allergens and for the induction of ACD, and suggest that their tolerogenic versus immuno-stimulatory function is likely dictated by signals from the skin microenvironment after penetration of the allergen

L’électroporation (EP) in vivo est utilisée comme stratégie d’amélioration de la réponse immune induite par les vaccins ADN. Cependant son effet sur les acteurs du système immunitaire inné reste méconnu. Dans l’objectif de mettre en évidence le comportement cellulaire sur le site de la vaccination, nous avons développé des approches d’imagerie par fluorescence in vivo chez le macaque. Nos résultats montrent que l’EP locale, augmente non seulement la quantité et la distribution de l’antigène vaccinal, mais induit également la mobilisation et la migration des cellules de Langerhans. De plus, l’EP cause un recrutement de leucocytes dans la peau et le tissu sous-cutané et favorise la production de cytokines pro-inflammatoires dans la peau. Ces évènements précoces, qui résultent de l’utilisation de l’EP en tant que système de délivrance des vaccins ADN, mettent en évidence le potentiel de l’EP en tant qu’adjuvant vaccinal. / In vivo electroporation (EP) is used as a strategy to improve the immune response induced by DNA vaccines. However, its local effect on the innate immune cells has not been fully described. We developed in vivo fluorescence imaging approaches to highlight the cell behavior in the site of vaccination in macaques. Our results show that the local EP not only increases the amount and the distribution of the vaccine antigen, but also induces the mobilization and migration of Langerhans cells. Furthermore, EP causes the recruitment of leukocytes into the skin and subcutaneous tissue and promotes the production of pro-inflammatory cytokines. These early events that result from the use of the EP as a delivery system for DNA vaccines, highlight its potential as a vaccine adjuvant.

Dendritic cells (DCs), a hematopoietic cell type belonging to the sub-group of cells called antigen presenting cells (APCs), inhabit a central role in innate and adaptive immunity. Although the DC family is very heterogeneous, all members share unique features. Most importantly, DCs can stimulate an immune response. This is due to the cells’ ability to capture and process antigens and to maturate in the presence of danger signals presented by pathogens. Maturation in turn results in the migration of DCs from the tissue they reside in to the draining lymph nodes, as well as in the subsequent presentation of the acquired antigens to T cells. In the skin, which is one of the most immunogeneic organs, DCs are present in sizable numbers in both the epidermis and the dermis. This study focused on two types of DCs: epidermal Langerhans cells (LCs) and dermal DCs (DDCs). While much is understood about LCs, far less is known about the role that DDCs play in skin immunity. Therefore one purpose of this study was to characterize DDCs and to compare their phenotype and functions to that of LCs. This study used two different methods to characterize human skin resident immune cells with regard to their number and distribution. First, a stable analytical immunohistochemistry-based method was developed and applied to a substantial number of healthy skin donors. This enabled a quantitative analysis of skin DC types and skin resident T cells at different anatomical locations in situ. A novel method to count dermal cell populations in situ was developed that resulted in the first published quantification of APCs, DDCs, as well as T cells in human dermis. Second, the traditional form of the emigration assay, which selectively enriches vital cells capable of ex vivo emigration from the skin, was upgraded toward a stable analytical method to separate epidermal LCs from DDCs. In this way, both skin DC types became accessible in sufficient numbers to allow for a comparison of phenotypes and functions in vitro. The resulting phenotypic observations clearly showed that both, LCs and DDCs are not fully mature after their emigration ex vivo and that both can be transformed into a phenotypically more mature state by treating them with inflammatory cytokines. What’s more, LCs are also functionally in an immature state after their emigration. They efficiently took up antigen, showed a low capacity to trans-migrate in response to chemokines, and demonstrated a low capacity to stimulate allogeneic T cells in a mixed leukocyte reaction (MLR). For the first time this study observed all these main APC functions not only for LCs but additionally for DDCs. As these observations were made in relation to LCs of the same donor, it could be concluded that DDCs are functionally more mature than LCs after emigration. DDCs showed a lower antigen uptake capacity than LCs but were superior in terms of their migratory and stimulatory capacity. However, treatment with cytokines could skew LC functions toward functional capacities observed for DDCs, i.e., it decreased LCs’ Ag uptake and increased their migratory and stimulatory capacity, whereas the cytokine treatment did not alter DDCs’ functional capacities. After improving immuno-histochemistry and the emigration assay using healthy skin samples, these newly developed techniques were implemented in clinical trials to observe the number, distribution and migratory capacity of skin DCs and T cells in patients undergoing allogeneic hematopoietic cell transplantation (aHSCT). Such a study is of importance because the turnover of DCs and T cells is closely associated with the occurrence of acute graft-versus-host disease (aGvHD), the major cause of morbidity and mortality after aHSCT. Due to the study design used, this study concisely demonstrate that at the onset of aGvHD, different DC types accumulate along with effector T cells in skin lesions of aGvHD but not in uninvolved skin of the same patient. These results suggest that in addition to donor T cells LCs and DDCs play a role during the early phase of cutaneous aGvHD directly within the site of inflammation. The view of many authors that DC depletion in the transplant recipient, especially in target organs, is a promising approach for aGvHD prophylaxis and therapy is further underscored by these results. One targeting strategy to inhibit GvHD by eliminating recipient DCs may be the use of DC specific monoclonal antibodies. Alemtuzumab (anti-CD52) is a monoclonal antibody and has proven effective in preventing aGvHD after aHSCT. It may, despite depleting donor T cells, also work by targeting recipient DCs. To determine whether the last mechanism of action is significant, a second clinical study investigated the effects of intravenous alemtuzumab on DCs by comparing the number of these cells in skin and blood of patients before and after a 4-week course of alemtuzumab treatment. The result was that although skin DCs weakly express the target antigen CD52 the number of these cells was not consistently reduced by alemtuzumab. In contrast, circulating blood DCs have a stronger CD52 expression and were significantly reduced by the treatment. In conclusion, this work provides new insights into the phenotypical and functional characteristics of human skin DCs, as well as into the fate of these cell types during aHSCT. The investigation of the APC system during aGvHD as carried out here will help to understand the process of aGvHD in more detail. All these efforts may hopefully support the development of new approaches for therapy and prevention of this major limitation of aHSCT and may help to improve this only curative therapy for several life-threatening diseases.