Human Epidermal Growth Factor Receptor 2 (HER-2/neu) is a breast tumor antigen (Ag) commonly overexpressed in 30% of breast cancer cases. Both HER-2/neu-targeted DNA-based and transgene modified dendritic cell (DC)-based vaccines are potent elements in eliciting HER-2/neu specific antitumor immune responses; however, there has been no side-by-side comparison of these two different immunization methods. We utilized an in vivo murine tumor model expressing the rat neu Ag to compare the immunization efficacy between DC transduced with replication-deficient adenovirus containing neu (AdVneu), to form DCneu, and plasmid DNA (pcDNA) vaccine. DCneu displayed an upregulation of immunologically important molecules and inflammatory cytokines expression such as IL-6 that partially mediated conversion of the regulatory T (Tr) cell suppression. Wildtype FVB/N mice immunized with DCneu induced stronger HER-2/neu-specific humoral and cellular immune responses compared to plasmid DNA immunized mice. Furthermore, mice immunized with DCneu remained completely protected from tumor challenge compared to partial or no protection observed in DNA immunized mice in two tumor animal models. In FVBneuN transgenic mice, which develop spontaneous breast tumors at 4-8 months of age, DCneu significantly delayed tumor onset when immunization conducted in mice at a younger age. Taken together, we demonstrated that a HER-2/neu-gene modified DC vaccine is more potent than a plasmid DNA vaccine in inducing neu specific immune responses resulting in greater protective and preventative effects in the tumor models examined. <p>In another study, we examined the use of a DC-based cancer vaccine involving the phagocytosis of apoptotic tumor cells expressing heat shock protein 70 (HSP70). The dual role of HSP70, as an antigenic peptide chaperone and danger signal, makes it especially important in DC-based vaccination. In this study, we investigated the impacts of apoptotic transgenic MCA/HSP tumor cells expressing HSP70 on DC maturation, T cell stimulation and overall vaccine efficacy. We found that DC with phagocytosis of MCA/HSP in the early phase of apoptosis expressed more peptide-major histocompatibility class (pMHC) I complexes, stimulated stronger cytotoxic T lymphocytes (CTL) responses and induced greater immune protection against MCA tumor cell challenge, compared to mice immunized with DC that phagocytosed MCA/HSP cells in the late phase of apoptosis. Taken together, our data demonstrated that HSP70 expression on apoptotic tumor cells stimulated DC maturation and DC with phagocytosis of apoptotic tumor cells expressing HSP70 in early phase of apoptosis more efficiently induced tumor-specific CTL responses and immunity than DC with phagocytosis of apoptotic tumor cells in late phase of apoptosis. <p>Overall, we have examined variations in designing DC-based cancer vaccines in two completely different model systems. Taken together, our results may have an important impact in designing DC-based antitumor vaccines.

<p>Vaccinia virus (VV) is the most thoroughly studied member of the Poxviridae family and the vaccine used to achieve the only successful eradication of a human disease. Over the years, it has proven itself as a useful tool for the study of antiviral immunity, vaccine development, and potentially cancer immunotherapy. VV is capable of eliciting a robust immune response; however the mechanisms by which VV accomplishes this task remain unknown. The overall goal of this thesis project is to determine how VV activates the innate immune system, and how this activation contributes to viral clearance in vivo. We determined that VV or VV-DNA activated the TLR8-MyD88 pathway in plasmacytoid dendritic cells (pDC), resulting in the production of type I interferons (IFN). We also demonstrated that TLR8-mediated production of type I IFN by pDC was crucial to efficient VV control and clearance in vivo. Moreover, we identified the polyA- and polyT-rich sequences in VV-DNA was the possible motif recognize by TLR8. Type I IFN, known for ability to establish the "antiviral state", are also critical mediators of NK cell activation. In the setting of VV infection, we demonstrated that direct action of type I IFN on NK cells, but not accessory cells such as DC, was necessary for NK cell activation in vivo. We further demonstrated that type I IFN-dependent activation of NK cells was required for optimal VV clearance in vivo. Given the importance of NK cells in anti-VV innate immunity, we next examined what role the TLR2-MyD88 pathway, critical for activation of cDC, played in the activation of NK cells. NK cells from TLR2-/- or MyD88-/- mice displayed a reduction in activation and cytolytic function, and this defect was independent of pro-inflammatory cytokine signaling. We were able to demonstrate that direct TLR2 signaling on NK cells was required for their optimal activation and function in response to VV infection. Moreover, we were able to demonstrate that TLR2-MyD88 signaling resulted in the activation of the PI3K-ERK pathway, which was necessary for NK cell cytotoxicity. In addition, we identified the NKG2D pathway as critical for efficient NK cell activation and function in response to VV infection, independent of the TLR2 pathway. Both the NKG2D and TLR2 pathways were crucial for optimal VV clearance and control in vivo. Collectively, this project illuminates the roles and mechanisms of the innate immune system in the control of VV in vivo.</p> / Dissertation

The goal of the work presented was to further understand how both the body and dendritic cells (DCs) interact and respond to biomaterials through receptor-mediated mechanisms. The role of Toll-like receptor 4 (TLR4) was investigated in the host response to biomaterials, and it was found that TLR4-deficient mice (in comparison to wild-type) had a delayed acute inflammatory response as seen through an altered adherent leukocyte profile on implanted polymer discs. However, following a 2 week implantation, the response was resolved potentially through compensatory receptors. Therefore, TLR4 may aid in the initial response to a biomaterial through recognition of 'danger signal' molecules. An investigation into the role of TLR4 in the response of DCs to biomaterials was investigated using murine bone marrow-derived DCs (BMDC), and PLGA film or microparticle treatment of BMDCs resulted in TLR4-dependent signs of slight maturation in non/loosely adherent BMDCs. However, further investigation into BMDC populations within the culture system revealed that non/loosely adherent BMDCs took on an activated/mature phenotype while adherent BMDCs appeared to be less mature and more responsive to both LPS and biomaterial stimuli. Therefore, it was concluded that investigations into the responsiveness of BMDCs to stimuli in the future analyze both adherent and non/loosely adherent populations. Lastly, the role of integrin-mediated adhesion in biomaterial-induced DC maturation was investigated. Gene expression analysis revealed that PLGA treatment of human DCs increased adhesion molecule expression (including β1 and β2 integrin subunits), LPS treatment reduced adhesion molecule expression and agarose treatment did not alter their expression. Antibody blocking techniques pinpointed the role of β2 integrins (and not β1 integrins) in both the adhesion of DCs to TCPS or PLGA substrates and the regulation of a DC maturation marker (CD86). β2 (and not β1) was found co-localized with F-actin in podosomes of DCs adhering to PLGA, and the direct interaction of β2 (and not β1) to PLGA substrate was confirmed through crosslinking and immunofluorescence studies. Therefore, DCs utilized β2 integrins for both adhesion and maintenance of immunomodulatory status. This aids the field of tissue engineering and vaccine design by further developing the criteria for biomaterial-influenced immunomodulation.

Ongoing advances in tissue engineering with the goal to address the clinical shortage of donor organs have encouraged the design and development of biomaterials to be used in tissue-engineered scaffolds. Furthermore, biomaterials have been used as delivery vehicles for vaccines that aim to enhance the protective immunity against pathogenic agents. These tissue-engineered constructs or vaccines are usually combination products that combine biomaterial and biological (e.g. cells, proteins, and/or DNA) components. Upon introduction into the body, the host response towards these products will be a combination of both a non-specific inflammatory response towards the biomaterial and an antigen-specific immune response towards the biological component(s). Recently, the biomaterial component was shown to influence the immune response towards a co-delivered antigen. Specifically, poly(lactic-co-glycolic acid) (PLGA), but not agarose, scaffolds or microparticles (MPs) enhanced the humoral response to a model antigen, ovalbumin. This in vivo result echoed with the in vitro study that PLGA, but not agarose, supported a mature phenotype of dendritic cells (DCs), the most potent antigen-presenting cells. Therefore, it is hypothesized that the effect of biomaterials on DC phenotype may influence the adaptive immunity against a co-delivered antigen. Understanding how biomaterials affect DC response will facilitate the selection and design of biomaterials that direct a desired immune response for tissue engineering or vaccine delivery applications. The objectives of this research were to elucidate the correlations between material properties and DC phenotype, develop predictive models for DC response based on material properties, and uncover the molecular basis for DC response to biomaterials. Well-defined biomaterial systems, including clinical titanium (Ti) substrates and two polymer libraries, were chosen to study induced DC phenotype. Due to the time-consuming nature of conventional methods for assessing DC phenotype, a high-throughput (HTP) method was first developed to screen for DC maturation based on surface marker expression (CHAPTER 4). A 96-well filter plate-based HTP methodology was developed and validated for the assessment of DC response to biomaterials. A "maturation factor", defined as CD86/DC-SIGN and measured by immunostaining, was found to be a cell number-independent metric for DC maturation and could be adapted to screen for DC maturation in a microplate format. This methodology was shown to reproducibly yield similar results of DC maturation in response to biomaterial treatment as compared to the conventional flow cytometric method upon DC treatment in 6-well plates. In addition, the supernatants from each treatment could easily be collected for cytotoxicity assessment using glucose-6-phosphate dehydrogenase (G6PD)-based assay and cytokine profiling using multiplex technology. In other words, the 96-well filter plate-based methodology can generate three outcomes from one single cell culture: 1) maturation marker expression, 2) cytotoxicity, and 3) cytokine profile. To examine which material properties were critical in determining DC phenotype, a set of three clinical titanium (Ti) substrates with well-defined surfaces was used to treat DCs (CHAPTER 5). These Ti substrates included pretreatment (PT), sand-blasted and acid-etched (SLA), and modified SLA (modSLA), with different roughness and surface energy. DCs responded differentially to these substrates. Specifically, PT and SLA induced a mature DC (mDC) phenotype, while modSLA-treated DCs remained immature based on surface marker expression, cytokine production profiles and cell morphology. Both PT and SLA induced higher CD86 expression as compared to iDC control, while modSLA maintained CD86 expression at a level similar to iDC. PT- or SLA-treated DCs exhibited dendritic processes associated with a mDC phenotype, while modSLA-treated DCs were rounded, a morphology associated with an iDC phenotype. Furthermore, PT induced increased secretion of MCP-1 by DCs compared to iDCs, indicating that PT promoted a pro-inflammatory environment. SLA induced higher IL-16 production, which is a pleiotropic cytokine, by DCs, most likely as a pro-inflammatory response due to the enhanced maturation of DCs induced by SLA. In contrast, modSLA did not induced enhanced production of any cytokines examined. Principal component analysis (PCA) were used to reduce the multi-dimensional data space and confirmed these experimental results, and it also indicated that the non-stimulating property of modSLA co-varied with certain surface properties, such as high surface hydrophilicity, % oxygen and % titanium of the substrates. In contrast, high surface % carbon and % nitrogen were more associated with a mDC phenotype. Furthermore, PCA also suggested that surface line roughness (Ra) did not contribute to the expression of CD86, an important maturation marker, suggesting that roughness had little impact on DC response (CHAPTER 5). DC response-material property relationships were also derived using more complex materials from a combinatorial library of polymethacrylates (pMAs) (CHAPTER 6). Twelve pMAs were selected and were found to induce differential DC response using the HTP method described in CHAPTER 4. These pMAs resulted in a trend of increasing DC maturation represented by the metric CD86/DC-SIGN, which was consistent with the trends of the production of pro-inflammatory cytokine, TNF-α, and chemokine, IL-8. Interestingly, this set of pMAs induced an opposite trend of IL-16 production, which is most likely released as an anti-inflammatory cytokine in this situation. These polymers were characterized extensively for a number of material properties, including surface chemical composition, glass transition temperature (Tg), air-water contact angle, line roughness (Ra), surface roughness (Sa), and surface area. Similar to the results from the Ti study, PCA determined that surface carbon correlated with enhanced DC maturation, while surface oxygen was associated with an iDC phenotype. In addition, Tg, Ra, and surface area were unimportant in determining DC response. Partial square linear regression (PLSR), a multivariate modeling approach, was implemented using the pMAs as the training set and a separate polymer library, which contained methacrylate- and acrylate-based terpolymers, as the prediction set. This model successfully predicted DC phenotype in terms of surface marker expression with R2prediction = 0.76. Furthermore, prediction of DC phenotype was effective based on only theoretical chemical composition of the bulk polymers with R2prediction = 0.80 (CHAPTER 6). Nonetheless, one should note that a predictive model can be only as good as what it is trained on and cannot be used to predict the DC response induced by a type of materials different from the training set. Also, this model might not contain all the important material properties such as polymer swelling and cannot predict specific types of immune responses. However, these results demonstrated that a generalized immune cell response can be predicted from biomaterial properties, and computational models will expedite future biomaterial design and selection (CHAPTER 6). From the pMA library, pMAs that induced the two extremes of DC phenotype (mature or immature) were identified for elucidating the mechanistic basis of biomaterial-induced DC responses (CHAPTER 7). Two pMAs, polyhydroxyethylmethacrylate (pHEMA) and poly(isobutyl-co-benzyl-co-terahydrofurfuryl)methacrylate (pIBTMA), were selected because they induced the least and the most mature DC phenotype, respectively. These pMAs were used to elucidate the activation profiles of transcription factors in DCs after biomaterial treatment and were compared to the iDC and mDC controls. In addition, a combined treatment of pHEMA and LPS was also included to determine if pHEMA could maintain an iDC phenotype in the presence of LPS. Interestingly, pIBTMA induced DC maturation primarily through the activation of NF-κB, while pHEMA mediated suppression of DC maturation through multiple TFs, including the activation of ISRE, E2F-1, GR-PR, NFAT, and HSF. GR-PR and E2F-1 have been shown to be associated with the suppression of DC maturation; ISRE, E2F-1, and NFAT are linked to apoptosis induction; HSF regulates the production of heat shock proteins (HSPs) that induce DC maturation and inhibit apoptosis. The activation of HSF by pHEMA was most likely a natural defensive mechanism against the other apoptotic signals. Therefore, pHEMA suppressed DC maturation through the induction of apoptosis. Surprisingly, in the presence of pHEMA, the effect of LPS was completely eliminated, suggesting that biomaterials can override the effect of soluble factors. The morphology and surface marker expression of DCs treated with these different biomaterials or controls were consistent with TF activation profiles (CHAPTER 7). Overall, this research illustrates that biomaterial properties, within the chosen biomaterial space, can be correlated to DC phenotype and more importantly, can be used as predictors for relative levels of DC phenotype. Furthermore, the differential responses induced by different biomaterials were mediated through the distinct activation profiles of transcription factors. Together, these findings are expected to facilitate the design and selection of biomaterials that direct desired immune responses.

In den letzten Jahren ergaben sich vermehrt Hinweise, dass dendritische Zellen (DCs) zu einer Stimulation von Natürlichen „Killer“ (NK)-Zellen in der Lage sind, die als zytotoxische Effektorzellen des angeborenen Immunsystems Tumorzellen eliminieren. Aus diesem Grund bestand ein wesentliches Ziel dieser Arbeit in der Analyse der Wechselwirkungen zwischen nativen DCs und NK-Zellen. Dazu wurden slanDCs verwendet, welche die größte DC-Subpopulation des Blutes repräsentieren. Zunächst wurde evaluiert, ob slanDCs eine effiziente Aktivierung von NK-Zellen bewirken. Als ein Ergebnis zeigte sich, dass Lipopolysaccharid (LPS)-stimulierte slanDCs sowohl zu einer verstärkten Expression des Aktivierungsmarkers CD69 auf der Oberfläche von NK-Zellen als auch zur Induktion der NK-Zell-Proliferation führen. Darüber hinaus wurde erstmals die slanDC-abhängige Erhöhung der Expression von aktivierenden Rezeptoren (NKp46, NKp44, NKp30) und Korezeptoren (2B4, DNAM-1) auf NK-Zellen demonstriert, welche essentiell für die NK-Zell-vermittelte Erkennung und Lyse von Tumorzellen sind. In weiteren Untersuchungen induzierten LPS-aktivierte slanDCs eine erhebliche Produktion von Interferon (IFN)-gamma in NK-Zellen, welches proliferationshemmend auf Tumorzellen und aktivierend auf T-Lymphozyten wirkt. Funktionelle Analysen ergaben, dass aktivierte slanDCs das zytotoxische Potential von NK-Zellen gegenüber der Tumorzelllinie K-562 deutlich verstärken. Untersuchungen der zugrunde liegenden Mechanismen zeigten die herausragende Bedeutung von IL-12, das sowohl die Steigerung der IFN-gamma-Sekretion als auch die Zunahme der zytolytischen Aktivität von NK-Zellen induzierte. Darüber hinaus konnte erstmals gezeigt werden, dass LPS-aktivierte slanDCs eine Zytotoxizität von NK-Zellen gegenüber frisch etablierten Blasten von Patienten mit akuter myeloischer Leukämie induzieren. In weiteren Untersuchungen wurde evaluiert, ob NK-Zellen ihrerseits die immunstimulatorischen Eigenschaften von slanDCs beeinflussen. Die Analysen zeigten erstmals, dass unstimulierte NK-Zellen die Expression von MHC-Klasse II-Molekülen, kostimulatorischen Molekülen und Adhäsionsmolekülen auf slanDCs deutlich erhöhen und somit ihre Fähigkeit zur Aktivierung von CD8+ T-Lymphozyten sowie CD4+ T-Helferzellen fördern. NK-Zellen führen ebenfalls zu einer deutlichen Verstärkung der Produktion von IL-12 durch LPS-stimulierte slanDCs. Darüber hinaus zeigte sich, dass NK-Zellen die Sekretion des immunsuppressiven Zytokins IL-10 durch LPS-stimulierte slanDCs reduzieren. In weiteren Analysen wurde demonstriert, dass die Interaktionen mit NK Zellen die Fähigkeit von LPS-aktivierten slanDCs zur Programmierung naiver CD4+ T-Lymphozyten in IFN-gamma-produzierende T-Helfer-1-Zellen deutlich verstärken. Diese Ergebnisse zeigten deutlich, dass stimulierte slanDCs und NK-Zellen in der Lage sind, sich wechselseitig zu aktivieren. NKT-Zellen repräsentieren eine weitere bedeutende Effektorzellpopulation der frühen antitumoralen Immunabwehr, die durch Sekretion von Zytokinen und ein ausgeprägtes zytolytisches Potential zur Elimination von Tumorzellen beiträgt. Deshalb wurden im Rahmen dieser Arbeit erstmals die Wechselwirkungen zwischen slanDCs und NKT-Zellen analysiert. Dabei verstärkten LPS-stimulierte slanDCs die Expression des Aktivierungsmarkers CD69 auf NKT-Zellen. Darüber hinaus induzierten LPS-aktivierte slanDCs eine deutliche IFN-gamma-Produktion in NKT-Zellen, wobei erneut die zentrale Rolle von IL-12 gezeigt wurde. Diese Ergebnisse demonstrierten, dass stimulierte slanDCs zu einer effektiven Aktivierung von NKT Zellen in der Lage sind. In abschließenden Untersuchungen wurde die Wirkung von NKT-Zellen auf slanDCs evaluiert. Dabei verstärkten NKT-Zellen die Maturierung von slanDCs erheblich und führten zu einer signifikanten Steigerung der IL-12-Produktion sowie zu einer Reduktion der IL-10-Freisetzung in Abhängigkeit von IFN-gamma. Die gewonnenen Daten demonstrierten, dass NKT-Zellen und slanDCs zu einer gegenseitigen Aktivierung befähigt sind. Die im Rahmen dieser Dissertation gewonnenen Erkenntnisse zu den Interaktionen von slanDCs und NK- bzw. NKT-Zellen können einen wesentlichen Beitrag zum Verständnis der Immunabwehr von Tumoren leisten und die Konzeption neuer antitumoraler Therapiestrategien unterstützen.

<p>This thesis deals with the design of ligands for efficientasymmetric catalysis and studies of the conformation of theligands in the catalytically active complexes. All ligandsdeveloped contain chiral oxazoline heterocycles.</p><p>The conformations of hydroxy- and methoxy-substitutedpyridinooxazolines and bis(oxazolines) during Pd-catalysedallylic alkylations were investigated using crystallography,2D-NMR techniques and DFT calculations. A stabilising OH-Pdinteraction was discovered which might explain the differencein reactivity between the hydroxy- and methoxy-containingligands. The conformational change in the ligands due to thisinteraction may explain the different selectivities observed inthe catalytic reaction.</p><p>Polymer-bound pyridinooxazolines and bis(oxazolines) weresynthesised and employed in Pd-catalysed allylic alkylationswith results similar to those of monomeric analogues;enantioselectivities up to 95% were obtained. One polymer-boundligand could be re-used several times after removal of Pd(0).The polymer-bound bis(oxazoline) was also used in Zn-catalysedDiels-Alder reactions, but the heterogenised catalyst gavelower selectivities than a monomeric analogue.</p><p>A series of chiral dendron-containing pyridinooxazolines andbis(oxazolines) were synthesised and evaluated in Pd-catalysedallylic alkylations. The dendrons did not seem to have anyinfluence on the selectivity and little influence on the yieldwhen introduced in the pyridinooxazoline ligands. In thebis(oxazoline) series lower generation dendrimers had a postiveon the selectivity, but the selectivity and the activitydecreased with increasing generation.</p><p>Crown ether-containing ligands were investigated inpalladium-catalysed alkylations. No evidence of a possibleinteraction between the metal in the crown ether and thenucleophile was discovered.</p><p>A new type of catalyst, an oxazoline-containing palladacyclewas found to be very active in oxidations of secondary alcoholsto the corresponding aldehydes or ketones. The reactions wereperformed with air as the re-oxidant. Therefore, this is anenviromentally friendly oxidation method.</p><p><b>Keywords:</b>asymmetric catalysis, chiral ligand,oxazolines, conformational study, allylic substitution,polymer-bound ligands, dendritic ligands, crown ether,oxidations, palladacycle.</p>

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 132-151). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Dengue, a group of four similar viruses transmitted through the bite of a mosquito, is estimated to infect upwards of 100 million annually in over 100 nations throughout the global equatorial belt. Distribution of global dengue is highly skewed as Southeast Asian and Western Pacific regions endure 75% of the global dengue burden. Similar to other regional countries, Malaysia has been rapidly urbanizing, which has supported a hyperendemic dengue state. The biological pathway by which dengue infection causes a wide range of clinical manifestations, spanning asymptomatic to life-threatening severe complications, is not comprehensively understood. Historically, severe dengue complications have primarily occurred in children. Consequentially, the majority of the dengue biological pathway research has been conducted on children; however, extrapolation of research findings to adults may be inappropriate as dengue manifestations have differed between age groups. As developing countries undergo epidemiologic transitions and dengue continues to spread geographically to non-endemic regions, youth and adult populations have been subjected to more of the severe dengue burden. Epidemiology and laboratory-based evidence has supported both memory T-cell and antibody independent enhancement hypotheses to explain the biological pathway of severe dengue. Both hypotheses employ the central idea that a primary infection alters immune components so that during a secondary heterotypic dengue infection, an individual is more at risk for severe complications. Monocytes, immune cells that are pivotal in both hypotheses, have been highly examined through in vivo and in vitro experimentation; however, epidemiological evidence for monocyte involvement is incomplete. The primary objective of the study was to examine if a difference in absolute monocyte count, considering independent risk factors, is present in individuals with primary and secondary dengue infections. A secondary dengue infection was found to raise absolute monocyte count during the defervescence phase of dengue illness in individuals aged 15 years and older 0.71 ± 0.15 (x10^9) compared to those experiencing primary dengue infection. Gender and distance of study participants' residences from Hospital Ampang were found to be risk factors for the relationship of interest; whereas, age and race were not found to be significant risk factors. The study helps expand current knowledge of the severe dengue biological pathway with respect to immunological differences between primary and secondary dengue infections. Further research is needed to confirm and expand the findings of this initial study, specifically to include infecting dengue serotype, education, and socioeconomic status which are known dengue risk factors.

Pancreatic cancer is one of the most lethal cancers and despite all research efforts the last 50 years, there are still no effective therapy for this terrible disease. Until quite recently most research in the field of pancreatic duct adenocarcinoma (PDAC) was focused on the tumor cells and mechanisms essential for their proliferation and survival. However, the tumor does not only consist of tumor cells, rather a combination of tumor cells and numerous stroma cell types, i.e. the tumor microenvironment. The tumor cells have developed the ability to activate the surrounding cells to produce factors important for the progression of the tumor. Cancer associated fibroblasts (CAFs) are the major stroma component and as much as 70% of the total PDAC tumor mass consists of these cells. In this thesis I have investigated the mechanisms involved in the cross-talk between tumor cells and CAFs and distinguished the local and systemic effects of this communication. Tumor derived IL-1α was identified as an important factor creating the inflammatory profile seen in CAFs. In PDAC patients, IL-1α was detected in 90% of the tumors and high expression was associated with poor clinical outcome. Moreover, the PDAC tumors had elevated expression levels of many inflammatory factors that were induced in CAFs by the tumor derived IL-1α in vitro. Consequently, this high expression of inflammatory factors in CAFs will attract immune cells including tumor associated macrophages (TAMs), dendritic cells (DCs), and CD8+ T cells. This indicates an immune suppressive role of CAFs, protecting the tumor cells by acting as decoy targets for immune cells homing into the tumor. The inflammatory factors produced in the PDAC microenvironment did not only affect the infiltrating immune cells, but had also systemic effects that included decreased levels of blood DCs in PDAC patients. Furthermore, these myeloid and plasmacytoid DCs were partly activated and had a semi mature phenotype and impaired immunostimulatory function. Low levels of blood DCs were direct associated with poor patient prognosis and the same was seen for low expression of ICOSL by the DCs. The findings presented in this thesis indicate an essential role for the cross-talk between tumor cells and stroma in the production of tumor  promoting factors. Treatment of PDAC patients with drugs that target the IL-1α signaling pathway could prevent the communication between these cells, thus reduce the amount of inflammatory factors both locally and systemically. Altogether, our findings support the idea that neutralization of the IL-1α signaling molecule could be a promising therapy for pancreatic cancer. The findings presented in this thesis indicate an essential role for the cross-talk between tumor cells and stroma in the production of tumor promoting factors. Treatment of PDAC patients with drugs that target the IL-1α signaling pathway could prevent the communication between these cells, thus reduce the amount of inflammatory factors both locally and systemically. Altogether, our findings support the idea that neutralization of the IL-1α signaling molecule could be a promising therapy for pancreatic cancer. / Mindre än 5% av patienterna som drabbas av cancer i bukspottkörteln förväntas överleva i mer än fem år. De typiska symtomen kommer sent och sjukdomen framskrider snabbt. Några av de riskfaktorer som identifierats är tobaksrökning, fetma och typ 2 diabetes. Forskningen har hittills siktat in sig på tumörcellerna och de mekanismer de använder för att överleva och föröka sig. Men en tumör innehåller också normala kroppsceller och vid bukspottkörtelcancer kan så mycket som 70 procent bestå av i sig ofarliga bindvävsceller. Miljön i tumören skapas av samspelet mellan dessa celltyper. De cancerceller som är bäst på att utnyttja omgivningen för sin tillväxt fortlever och för sina egenskaper vidare. En sådan egenskap är att kunna manipulera bindvävsceller till att producera signalsubstanser och tillväxtfaktorer som gynnar tumören. Mekanismerna bakom denna kommunikation har studerats och ett viktigt fynd var att tumörcellerna producerar signalämnet interleukin 1-alpha (IL-1a). Detta protein upptäcktes i 90 procent av de undersökta tumörerna, och var kopplat till dålig prognos hos patienterna. Signalen via IL-1a sätter igång tillverkningen av substanser som behövs för nybildning och tillväxt av blodkärl, i sin tur en förutsättning för att tumören ska leva vidare och växa. Proteinet stimulerar också celldelning i tumören, bidrar till att lura kroppens immunförsvar och underlättar spridning av dottertumörer till andra delar av kroppen. När vi slår ut signaleringen kan tumörcellerna inte längre påverka bindvävscellerna lika effektivt, och således minskar förekomsten av flera faktorer som gynnar tumörtillväxten. IL-1a kan därför vara en lovande kandidat att utforska vidare för framtida som ett läkemedel mot bukspottkörtelcancer.

Glial cells represent the most abundant cell population in the central nervous system (CNS), and yet, have historically been thought of as merely support cells for neurons. Over the past few decades, however, the number of identified roles that glial cells play in the CNS has expanded at an exponential rate, revealing new and exciting functions in neuron-glial communication. At synapses, astrocytes are now recognized as part of a "tripartite" complex with pre- and postsynaptic structures and can modulate synaptic transmission and plasticity. Accumulating evidence has also revealed new roles for oligodendrocytes in regulating axon diameter and integrity, and ion channel clustering. Despite our knowledge of the physiological connections between neurons and glia, relatively little is known about the morphological interplay of these cells during development and in the mature brain. The results presented in this thesis reveal the extent and time-course of rapid remodelling of astrocytes and oligodendrocytes in close proximity to dendritic spines and axons respectively. These findings provide further evidence that glia play an important role in regulating the structural plasticity of the brain. The methodology developed also provides a powerful system for the study of neuron-glial structural dynamics and may contribute to the development of novel therapeutic strategies for diseases affecting the central nervous system.