L’immunothérapie basée dans l’utilisation des cellules NK pour le traitement de différents types de cancers humains est une stratégie très prometteuse. Les cellules dendritiques plasmacytoïdes (pDC) activées permettent de stimuler les cellules NK pour augmenter leurs propriétés anti-tumorales. Les cellules NK activées par les pDC sont capables de développer in vitro et in vivo une forte réponse cytotoxique contre différentes lignées de leucémie lymphoblastiques pre-B. En revanche, les faibles quantités de pDC obtenues à partir du sang périphérique limitent leur l’utilisation en clinique. L’expansion et la différenciation des pDC in vitro à partir des progéniteurs hématopoïétiques CD34, permet d’obtenir des pDC humaines en grande quantité. Récemment il a été démontré que l’utilisation des antagonistes du récepteur de l’aryl hydroxycarbone (AhR) augmente le nombre des pDC générées in vitro. Cependant, la capacité à activer les cellules NK des pDC différenciées in vitro en présence d’antagonistes de l’AhR n’a pas encore été étudiée. Dans cette étude, nous montrons que les pDC obtenues in vitro ont une expression de molécules d’activation et une sécrétion de IFN plus faibles que celles des pDC du sang périphérique, mais que leur capacité à stimuler des cellules NK est similaire. Ces résultats ouvrent donc la voie à l’utilisation des pDC générées in vitro comme agent immuno-therapeutique visant à stimuler les fonctions anti-tumorales des cellules NK. / NK cells immunotherapy is a promising treatment for different human cancers. An effective approach to stimulate NK cells has been the use of activated plasmacytoid dendritic cells (pDC). NK cell activated by pDC develops a strong cytotoxic response against pre-B acute lymphoblastic leukemia (ALL) cell lines in vitro and in vivo. However, the use of pDC in the clinic has limitations because of its low frequency. One suitable strategy is the differentiation of CD34+ progenitors using different cytokines and chemokines. Recently, it has been demonstrated that antagonists of aryl hydroxyl receptor (AhR) increase the number of pDC obtained after culture of CD34+ cells. Nevertheless, the ability of these in vitro differentiated pDC to induce NK cells activation has not been well documented. In this study, it was showed that activated in vitro differentiated pDC present different characteristics than adult pDC, like a lower expression of activation markers and IFNalpha secretion, but their capacity to stimulate NK cells was similar to that observed in adult pDC. In addition, NK cells activated by in vitro differentiated pDC showed a strong cytotoxicity against the pre-B ALL cell line REH suggesting its effectiveness to treat ALL patients.

This study investigates relaxation polarization in some dielectric components of tester UNISPOT S40 developed by company UNITES Systems a.s. Relaxation polarization in the PCB substrates and in tester’s cable insulation causes undesirable deceleration of some test steps. Relaxation polarization in selected PCB substrates and various cables are examined by method of dielectric relaxation spectroscopy in the frequency domain and by measurement of polarization or depolarization current.

The pathogenesis of oral submucous fibrosis (OSMF) is due to a complex interplay between the production and degradation of extracellular matrix (ECM) protein components. In tissue fibrosis, there is a net accumulation of collagen as a result of an imbalance between enhanced production, deposition and impaired degradation of ECM components. OSMF is a chronic inflammatory condition of the oral cavity and regulation of a number of pro-inflammatory and fibrogenic cytokines such as interleukine-1, -6 and -8 isoforms, TGF-β, PDGF, bFGF, IFN-γ and TNF-α has been reported in OSMF tissues. The expression of these growth factors has a bearing on the epithelial changes as well as proliferation and differentiation of oral fibroblasts into ECM protein producing myofibroblast cells. One key modulator of fibrosis in several organs has been TGF-β. Overproduction of TGF-β mRNA and protein has been reported in several fibrotic disorders including that of skin, lungs, liver, kidney and heart. This is mainly due to stimulation of ECM genes by TGF-β. Although there have been few reports suggesting the over production of TGF-β in OSMF tissues, the specific isoforms involved or the mechanisms are poorly understood. Areca nut components, especially arecoline have been implicated in the pathophysiology of OSMF. Few reports indicate the involvement of arecoline in the regulation of collagen production and activity of collagenases and their inhibitors in oral fibroblast cells. Moreover, the alkaloid is involved in initiating epithelial inflammation by inducing COX-2, prostaglandin E2, IL-1α, IL-6 and IL-8 in KB oral carcinoma cells and oral fibroblast cells. These and other reports strongly suggest that changes in gene expression mediated by Arecoline may be central to the progression of OSMF. Not much is known about arecoline-mediated cellular signaling events except for few recent reports that suggest the activation of MAPK pathways. In neuronal and colonic smooth muscle cells of mouse, rat and rabbit, the actions of Arecoline have been reported to be through the activation of muscarinic acetylcholine receptors. Direct binding of arecoline to human M1, 2 and 3 muscarinic receptor isoforms have been shown in brain tissues. Stimulation of these receptors alters the intracellular levels of Ca+2 and cAMP, which are important second messengers. The cholinergic potential of arecoline may be important for their roles in arecoline-mediated signaling events. The expression of muscarinic acetylcholine receptors has been reported in several cell types besides neuronal and excitatory cells. Although several gene expression changes have been reported following Arecoline treatment of a variety of cells, the mechanism of such regulations is not established. Hence in order to understand the role of arecoline in OSMF disease process, we undertook studies that provide insights into arecoline action in epithelial and fibroblast cells and possible molecular mechanisms. The objectives are to study: 1. The role of arecoline in cellular proliferation, cell-cycle regulation and apoptosis in human normal keratinocytes. 2. Mechanism of regulation of gene expression by arecoline in normal keratinocytes. 3. Mechanism of regulation of gene expression by arecoline in human normal oral fibroblasts. In order to achieve the above objectives, a human keratinocyte cell line, HaCaT and an oral periodontal fibroblast cell line (PDC) were utilized. The cells were treated with arecoline and a variety of assays including RT-PCR analysis of mRNA of several genes, phosphorylation status of MAPK pathway intermediates, cell cycle analysis and other cellular and molecular methods have been employed. Following arecoline treatment, there is induction of oxidative stress, growth arrest and epithelial cell death. Since actions of TGF-β are central to most fibrotic disorders and arecoline has been implicated in OSMF, it is hypothesized that arecoline may influence fibrosis via TGF-β pathway. Towards this, several TGF-β target genes that may have a possible role in fibrosis have been studied in arecoline treated epithelial and fibroblast cells. Since arecoline mediated oxidative stress has been reported, the regulation of genes that are involved in stress response pathway have been studied for induction by arecoline in epithelial cells. The results presented in this thesis suggest the up regulation of oxidative stress-responsive genes in HaCaT cells including HOX-1, FTL, G6PD, GCLC and GRD in HaCaT cells. Oxidative stress is a major inducer of inflammatory response in the epithelial tissues. The expression of IL-1α, an important inflammatory cytokine is induced by arecoline in HaCaT cells in response to oxidative stress via the activation of p38 MAPK pathway. Interestingly, activation of MAPK pathways by arecoline is involved in the regulation of common target genes of arecoline and TGF-β and also in the induction of TGF-β−responsive promoter reporter construct, p3TP-lux activity in HaCaT cells. Due to the involvement of TGF-β in fibrosis, regulation of TGF-β pathway genes by arecoline has been studied both in HaCaT and PDC cells. In HaCaT cells, arecoline induces the expression of TGF-β2 mRNA while TβRII expression is down regulated. The expression of the rest of TGF-β/SMAD pathway genes including TGF-β1, β3, TβRI, SMAD1, 2, 3, 4 and 7 are not affected by arecoline in HaCaT cells. Over expression of TGF-β2 is also observed in most of the OSMF tissues compared to normal oral tissues. However, in normal oral fibroblast cells, we observed that the TGF-β/SMAD pathway genes are not regulated by arecoline. These results suggest the possible involvement of arecoline-mediated induction of TGF-β2 in epithelial cells in OSMF disease development. We investigated the signaling pathways involved in the regulation of TGF-β2 and found that stimulation of M3 muscarinic receptor by arecoline leads to the induction of TGF-β2 expression in HaCaT cells via PKC pathway. TGM-2 is an important TGF-β target gene involved in the cross linking of ECM proteins. Arecoline-mediated induction of TGM2 mRNA and transglutaminase activity are observed in oral fibroblast cells, PDC. The induction of TGM-2 was found to be independent of oxidative stress and TGF-β, but dependent on muscarinic acid receptor activation by arecoline and involves cytosolic cAMP. When tested in OSMF tissues, there was an increased expression of TGF-β2, TSP1 and TGM2 as compared to normal tissues suggesting a possible role of these genes in arecoline-mediated progression of OSMF. Interleukin-8 (IL-8), which is involved in inflammation has been reported to be regulated by TGF-β in a cell type specific manner. In several cell types including human endometrial stromal cells, LnCaP (prostate cancer cells), human retinal pigment epithelial cells and rat lung alveolar epithelial (LM5) cells etc., TGF-β up regulates the expression of IL-8 mRNA. Arecoline was found to down regulate IL-8 expression in PDC cells as measured by RT-PCR. Interestingly, the presence of serum along with arecoline induces the expression of IL-8 in PDC cells suggesting the modulation of arecoline-mediated gene regulation by a serum activated signaling pathway. Intriguingly, arecoline treatment led to down regulation of collagens in PDC cells. However, collagen genes are induced in PDC cells in the presence of HaCaT spent medium by arecoline suggesting a role for factor(s) secreted by epithelial cells in the regulation of collagen genes by arecoline. This factor could be an isoform of TGF-β as shown by blocking the induction of collagens by the TGF-β inhibitor, βLAP. Taken together, all these results indicate the ability of arecoline to cause fibrosis in a tissue environment where both epithelial and fibroblasts respond to arecoline and mutually contribute to the disease manifestation. Major conclusions from this study includes, 1] cell death in epithelial cells due to oxidative stress following arecoline treatment, 2] regulation of gene expression by arecoline involves MAPK, PKC pathways, 3] muscarinic acid receptor and oxidative stress are also important for regulation gene expression by arecoline. The most important inference from this study is the possible paracrine influence of TGF-β isoforms secreted by epithelial cells on the oral fibroblasts in determining the progression of OSMF. In summary, in this thesis, an attempt has been made to study the molecular mechanisms and role of arecoline, an alkaloid in conferring gene expression changes that may lead to the initiation and progression of oral sub mucous fibrosis.