Homing of hematopoietic stem cells (HSC) to their bone marrow (BM) niches is crucial to clinical stem cell transplantation. However, the molecular mechanism controlling this process remains not fully understood. In this study, we aimed to explore novel regulators of HSC homing through investigating downstream signals and effector molecules of the stromal cell-derived factor-1 (SDF-1)/CXCR4 axis. We further characterized specific functions of targeted regulators by in vitro and in vivo migration/homing assays on human cord blood (CB) CD34+ hematopoietic stem/progenitor cells. / In summary, we have provided the first transcriptome profile of CB CD34 + cells downstream of the SDF-1/CXCR4 axis. We also reported the first evidence that HSC homing was regulated by the tetraspanin CD9. By comparing the homing-related responses of CD34+ to SDF-1 and CTCE-0214, we identified RGS13 as another potential regulator of HSC homing. It is anticipated that strategies for modulating the expressions and functions of CD9 and RGS13 might improve HSC homing to their hematopoietic niches. / To investigate the transcriptional regulation provided by the SDF-1/CXCR4 axis, we performed the first differential transcriptome profiling of human CB CD34+ cells in response to a short-term exposure of SDF-1, and identified a panel of genes with putative homing functions. We demonstrated that CD9, a member of the tetraspanin family proteins, was expressed in CD34 +CD38-/lo and CD34+CD38+ cells. CD9 levels were enhanced by SDF-1, which simultaneously downregulated CXCR4 membrane expression. Using specific inhibitors and activators, we demonstrated that CD9 expressions were modulated via the CXCR4, G-protein, PKC, PLC, ERK and JAK2 signals. Pretreatment of CD34+ cells with anti-CD9 mAb ALB6 significantly inhibited SDF-1-mediated transendothelial migration and calcium mobilization, whereas adhesion to fibronectin and endothelial cells were enhanced. Infusion of CD34+ cells pretreated with ALB6 significantly impaired their homing to bone marrow and spleen of sublethally irradiated NOD/SCID mice. There also appeared a preferential homing/retaining of untreated CD34+CD9+ cells to these niches. Our results indicate that CD9, as a downstream member of SDF-1/CXCR4 signals might possess specific and important functions in HSC homing. / We first investigated the effects of SDF-1 and its analog, CTCE-0214 (a small cyclized peptide analog of the SDF-1 terminal regions), on homing-related properties (chemotaxis, transwell migration, adhesion and actin polymerization) of CB CD34+ cells. Our results demonstrated that both SDF-1 and CTCE-0214 induced a robust actin polymerization response and improved adhesion of CD34+ cells to fibronectin. Unlike SDF-1, CTCE-0214 did not induce a chemotactic response when added to the lower chamber of the transwell system. Addition of CTCE-0214 to the upper chamber significantly improved migration of CD34+ cells to a SDF-1 gradient, but there was no preferential enhancement in the migration of specific colony-forming unit (CFU) progenitors or the more primitive CD34+CD38 -/lo subpopulation. Pre-exposure of CD34+ cells to CTCE-0214 for 4 hours promoted cell migration, whereas SDF-1 pretreatment retarded migration. To dissect the molecular mechanisms leading to the observed functional differences mediated by SDF-1 and CTCE-0214, we investigated whether the two compounds differentially regulated the expression of several known regulators of HSC migration. Flow cytometric analysis revealed that the cell surface expression of CD26, CD44, CD49d, CD49e and CD164 was not changed by either compounds. Exposure to SDF-1, but not CTCE-0214, decreased membrane expression of CXCR4 on CD34+ cells. Addition of CTCE-0214 to the upper chamber inhibited the SDF-1-induced CXCR4 downregulation in both migrated and non-migrated cell population in the transwell setting. Notably, SDF-1 and CTCE-0214 had an opposite effect on the expression level of regulator of G-protein signaling 13 (RGS13), a negative regulator of chemokine-induced responses. Treatment of CD34+ with SDF-1 for 4 hours resulted in a significant increase in RGS13 expression, whereas CTCE-0214 induced a time-dependent decrease in RGS13 expression. Our results provide the first evidence that SDF-1 and CTCE-0214 differentially regulate migration of CD34 + cells, and we speculate that this might be attributed to their differential regulation of CXCR4 and RGS13 expression. / Leung, Kam Tong. / Adviser: Karen Kwai Har Li. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 146-167). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Orientador: Sandra Helena Penha de Oliveira / Banca: João Eduardo Gomes Filho / Banca: Ana Paula Campanelli / Resumo: O objetivo do presente estudo foi investigar o papel dos Mastócitos (MAST) na resposta tecidual de camundongos com Diabetes Mellitus (DM) submetidos a Doença Periodontal (DP). Os camundongos foram pré-tratados com uma dose única de estreptozotocina (STZ) para indução do DM. Para avaliar o papel dos MAST no controle da DP, os camundongos foram depletados de MAST pelo tratamento com composto 48/80. Subsequentemente foi realizada a indução da DP nos camundongos com DM e controles pela ligadura dos primeiros molares homólogos. Após um período de 7 e 14 dias os animais foram sacrificados para coleta das amostras para ensaios subseqüentes. Os níveis de reabsorção óssea dos camundongos diabéticos e normais foram avaliados radiograficamente para confirmação da presença da DP. O recrutamento de Neutrófilos (NE) foi avaliado pela produção da enzima Mieloperoxidase (MPO) no tecido gengival. Os níveis de IFN-g, IL-4, RANTES/CCL5, KC/CXCL1 e Linfotactina/ XCL1 nos tecidos gengivais e IFN-g e IL-4 no plasma foram avaliados pelo método imunoenzimático (ELISA). Os resultados mostram que animais diabéticos com DP apresentaram após 14 dias da indução da DP uma perda óssea significativa quando comparado ao grupo controle, diferentemente do grupo de 7 dias. Esta perda foi potenciada nos animais diabéticos com DP depletados de MAST. Verificou-se elevados níveis de MPO nos animais normais e com DM após 14 dias da indução da DP. Nos animais com DM e com DP tratados com 48/80 foi observada uma redução parcial dos níveis de MPO. A produção de IFN-g, IL-4, RANTES/CCL5, KC/CXCL1 e Lin-fotactina/XCL1 foi observada nos animais diabéticos independente da indução da DP após o período 7 e 14 dias. Conclui-se então que o DM favoreceu o aumento da perda óssea e o recrutamento de neutrófilos na DP. Como também induziu a produção de altos níveis dos mediadores... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to investigate the role of Mast cells (MAST) on the tissue response in mice with Diabetes Mellitus (DM) submitted to Periodontal Disease (PD). The mice were pretreated with a single dose of streptozotocin (STZ) for the induction of DM. To evaluate the role of MAST in the PD, the mice were depleted of MAST by a pretreatment with compound 48/80. Subsequently, PD was induced in diabetic and normoglycemics mice by using a ligature around the first molars homologous. Seven and fourteen days after the surgery, the animals were sacrificed and the samples were collected for the subsequent experiments. The levels of bone resorption in diabetic and normoglycemic mice with PD were radiographically evaluated to confirm the presence of the PD. Neutrophil migration (NE) was quantified by the presence of the MPO enzyme in the gingival tissue. The levels of IFN-g, IL-4, RANTES/CCL5, KC/CXCL1 and Lymphotactin/XCL1 from gingival tissues and plasma were evaluated by Enzyme- Linked Immunosorbent Assay (ELISA). The results showed that diabetic mice had a significant bone resorption 14 days after the induction of PD when compared to normoglycemics mice. This bone resorption was higher in the diabetic MAST-cell depleted mice with PD. The level of MPO was higher in diabetic and normoglycemic mice 14 days after the induction of PD. Furthermore, it was observed a partial reduction of MPO levels in diabetic mice with PD treated with compound 48/80. The level of IFN-g, IL-4, RANTES/CCL5, KC/CXCL1 and Lymphofotactin/XCL was observed in diabetic mice independently of the induction of PD after 7 or 14 days. In conclusion, DM increased bone resorption and neutrophil recruitment in the PD mice, as well as it induced the production of high levels of IFN-g, IL -4, RANTES/CCL5, KC/CXCL1 and Lymphofotactin/XCL1. The MAST depletion increased bone resorption and reduced NE recruitment... (Complete abstract click electronic access below) / Mestre

Chemokines are responsible for the directed migration of leukocyte chemotactic cytokines, coordinating cell movement during inflammation and the transport of hematopoietic cells. In addition to leukocytes, chemokine receptors are also found in neoplastic cells and tumors associated with stromal cells. Among chemokines, and the CXCR4/CXCL12 CCR7/CCL21 systems have been shown the involvement of lymph node metastases or distant metastases in different cancers. Thus, aim of this study was to evaluate the expression of CXCR4, CXCL12, CCR7, CCL21 and Ki-67 in oral squamous cell carcinoma (SCC) and to correlate these markers with clinicopathological indicators, lymph node metastasis and survival. We conducted a survey of reports and paraffin blocks of excisional biopsies of patients with SCC treated at the Hospital Haroldo JuaÃaba (2001-2009). Data on anatomic location of the lesion, sex, age, patient survival, degree of histological differentiation of the tumor, tumor stage and presence or absence of lymph node metastasis, lymphovascular and perineural invasion, nuclear grade and depth of invasion were collected. For immunohistochemical analysis, followed by the technique of streptavidin-biotin-peroxidase using the anti-CXCR4, anti-CXCL12, anti-CCR7, anti-CCL21 and Ki-67 antibody. Histological sections were photomicrographed in 10 fields chosen randomly and measured for the number of labeled tumor cells and determined the percentage of each labeling antibody. The marking of CXCR4 was detected in the cytoplasm and nucleus, CXCL12, CCR7 and CCL21 were only cytoplasmic, their expression was observed in 18 (60%) 8 (22.66%) 16 (53.3%) and 3 (12%) cases, respectively. We found a significant positive association between lymphovascular invasion and immunostaining of CXCR4 (p = 0.007) and CCR7 (P = 0.01) and among these cases metastasis was present in 62.5% and 37.5%, respectively. When in combination with Ki67, we found a significant positive correlation between CXCR4 (p = 0.0086), CXCL12 (p = 0.036) and CCR7 (p = 0:04). Among patients CXCR4 + over 111 months, only 38.4% were alive (p = 0.845), whereas both patients CCR7 + (p = 0.398) as well as CXCR4 +, and CCR7 + (p = 0.441) after 62 months, everyone had already died. We conclude that these chemokines are associated with lymphovascular invasion and cell proliferation, perhaps favoring the development of metastasis and poor prognosis. / As quimiocinas sÃo citocinas quimiotÃticas responsÃveis pela migraÃÃo direcionada de leucÃcitos, coordenando o movimento celular durante a inflamaÃÃo e o transporte de cÃlulas hematopoiÃticas. AlÃm dos leucÃcitos, os receptores de quimiocinas tambÃm sÃo encontrados em cÃlulas neoplÃsicas e em tumores associados com cÃlulas estromais. Dentre as quimiocinas, os sistemas CXCR4/CXCL12 e CCR7/CCL21 tÃm sido demonstrado no envolvimento de metÃstases linfonodais ou à distÃncia em diferentes tipos de cÃncer. Dessa forma, foi objetivo desse trabalho avaliar a expressÃo de CXCR4, CXCL12, CCR7, CCL21 e Ki-67 em carcinoma de cÃlulas escamosas orais (CEC) e correlacionar estes marcadores com indicadores clÃnicopatolÃgicos, metÃstase linfonodal e sobrevida. Realizou-se um levantamento de laudos e blocos parafinados de biopsias excisionais de pacientes portadores de CEC tratados no Hospital Haroldo JuaÃaba (2001 a 2009). Foram coletados dados sobre localizaÃÃo anatÃmica da lesÃo, sexo, idade, sobrevida do paciente, grau de diferenciaÃÃo histopatolÃgica do tumor, estadiamento tumoral e presenÃa ou ausÃncia de metÃstase linfonodal, invasÃo linfovascular e perineural, grau nuclear e profundidade de invasÃo. Para reaÃÃo de imunohistoquÃmica, seguiu-se a tÃcnica da estreptavidina-biotina-peroxidase, utilizando os anticorpos anti-CXCR4, anti-CXCL12, anti-CCR7, anti-CCL21 e Ki-67. As secÃÃes histolÃgicas foram fotomicrografadas em 10 campos escolhidos aleatoriamente e quantificadas quanto ao nÃmero de cÃlulas tumorais marcadas e determinado o percentual de marcaÃÃo de cada anticorpo. A marcaÃÃo de CXCR4 foi detectada em citoplasma e nÃcleo, CXCL12, CCR7 e CCL21 tiveram marcaÃÃo apenas citoplasmÃtica, sendo observada suas expressÃes em 18 (60%), 8 (22,66%), 16 (53,3%) e 3 (12%) casos, respectivamente. Encontrou-se uma associaÃÃo significativa positiva entre a invasÃo linfovascular e a imunomarcaÃÃo do CXCR4 (p=0.007) e CCR7 (p=0.01) e dentre esses casos a metÃstase esteve presente em 62,5% e 37,5%, respectivamente. Quando em associaÃÃo com o Ki67, encontrou-se uma correlaÃÃo positiva significante entre o CXCR4 (p=0.0086), CXCL12 (p=0.036) e CCR7 (p=0.04). Dentre os pacientes CXCR4+, ao longo de 111 meses, apenas 38,4% estavam vivos (p=0.845), ao passo que tanto para pacientes CCR7+ (p = 0.398), quanto CXCR4+ e CCR7+ (p = 0.441), apÃs 62 meses, todos haviam ido a Ãbito. Conclui-se que essas quimiocinas estÃo associadas com a invasÃo linfovascular e proliferaÃÃo celular, talvez favorecendo o desenvolvimento de metÃstases e um pior prognÃstico.

A resposta inflamatória aguda é uma importante defesa contra o Streptococcus pneumoniae, mas a persistência na inflamação pode causar danos aos tecidos. Duas linhagens de camundongos geneticamente selecionadas para resposta inflamatória aguda mínima (AIRmin) e máxima (AIRmax) foram avaliadas frente a um desafio respiratório invasivo com pneumococo. O desafio induziu a morte de 100% dos camundongos AIRmin, e apenas 36,4% dos camundongos AIRmax. A caracterização da resposta imune inata mostrou que ambas as linhagens de camundongos responderam ao desafio com secreção de citocinas pro-inflamatórias. Entretanto, apenas os camundongos AIRmax controlaram a inflamação. Diferenças significativas quanto à expressão de metaloproteases de matriz sugerem o papel destas proteínas no controle da infecção. Além disso, os camundongos AIRmin apresentaram um aumento no número de macrófagos expressando o receptor de manose CD206, após o desafio. Uma menor resistência de macrófagos e neutrófilos dos camundongos AIRmin à morte celular programada, após o desafio, também foi observada. / Acute inflammatory response is an important defense against Streptococcus pneumoniae, but persistence of inflammation may result in tissue damage. The susceptibility against an invasive respiratory pneumococcal challenge was evaluated in two outbred mice strains, genetically selected for maximum (AIRmax) and minimum (AIRmin) acute inflammatory responses. The challenge induced the death of 100% of the AIRmin mice and only 36.4% of the AIRmax mice. Characterization of the innate immune responses showed that both mice strains responded to the challenge with the secretion of inflammatory cytokines. However, only the AIRmax mice controlled the inflammation. Significant differences on the expression of matrix metalloproteinases suggest a role of these proteins in the control of the infection. Moreover, the AIRmin mice presented an increase in the number of macrophages expressing the CD206 mannose receptor after the challenge. A reduced resistance of macrophages and neutrophils from AIRmin mice to programmed cell death, after the challenge, was also observed.

La maladie de Parkinson (MP) est marquée par la présence d'une inflammation, pouvant être bénéfique ou délétère à la neurodégénérescence dopaminergique (DAgique). Nous avons adressé la nature des interactions pathologiques entre neurones DAgiques, cellules gliales et leucocytes infiltrant, nécessaire à la mise en place de cette inflammation. Dans un modèle MPTP murin de la MP, les objectifs de ma thèse ont été 1) d'identifier des signaux inflammatoires neuronaux et gliaux, par une approche transcriptomique associée à de la microdissection laser et 2) de déterminer leurs rôles dans la neuroinflammation ainsi que leurs effets sur la perte DAgique. Nous avons retenu parmi les candidats identifiés: les axes CXCL16-CXCR6 et CCL2-CCR2. Nous reportons dans le modèle MPTP, une expression microgliale de CXCL16 ainsi qu'une infiltration de population lymphocytaire CXCR6. Bien que la déplétion de CXCR6 permette de réduire cette infiltration, aucun effet n'est observé sur la perte DAgique. Nous décrivons une infiltration de monocyte CCR2 en concomitance avec une expression astrocytaire précoce de CCL2 dans le modèle MPTP murin, ainsi qu'une expression plus prolongée de CCL2 chez le primate non-humain MPTP, suggérant une relevance de l'axe CCL2-CCR2 dans la MP. En effet nous montrons que des souris surexprimant CCL2, intoxiquées au MPTP, ont non seulement une augmentation accrue de l'infiltrat monocytaire CCR2, mais également de la lésion DAgique. De manière inattendue, nous montrons que la neurotoxicité accrue observée chez des souris CX3CR1-/- MPTP passe indirectement par la voie CCL2-CCR2. Ainsi, nos données supportent l'hypothèse d'une neurotoxicité des monocytes CCR2 dans la MP. / Parkinson's disease (PD) presents signs of neuroinflammation, which can be beneficial or deleterious for dopaminergic (DA) neurodegeneration. We have analyzed the characteristics of such pathological interactions between DA neurons, glial cells and infiltrating immune cells. Using the neurotoxic MPTP mouse model of PD and focusing on chemokines, my thesis objectives were: 1) to identify using laser-microdissection and RNA profiling, the neuronal and glial inflammatory signals in the affected Substantia Nigra (SN) and 2) to assess the role of promising identified chemokine candidates during DA neurodegeneration. We have focused on the lymphocytic CXCL16-CXCR6 and the monocytic CCL2-CCR2 axes. We have found early microglial CXCL16 induction and parallel SN infiltration of CXCR6 lymphocyte subpopulations. CXCR6-deletion reduced infiltration of specific lymphocyte subpopulations, but did not affect the known deleterious infiltration of CD4 T-lymphocytes. For the CCL2-CCR2 axis, we found evidence for limited SN infiltration of CCR2 monocytes, which was preceded by transient astrocytic CCL2 induction in MPTP mice, but a prolonged CCL2 induction in MPTP monkeys, suggesting a potential relevance for human PD. While CCR2-gene deletion did not affect loss of DA neurons, astrocytic CCL2 overexpression increased MPTP induced DA neural loss, revealing the principally neurotoxic nature of infiltrating CCR2 monocytes in a PD-like environment. Unexpectedly, we also found that the known increased DA loss in CX3CR1-KO mice was mediated indirectly via over-induction of the CCL2-CCR2 axis. Combined, our results suggest a potential deleterious role of the CCL2-CCR2 axis in actual human PD.

Fetal alcohol spectrum disorders (FASD) are caused by alcohol exposure during pregnancy and is the leading cause of mental retardation. Alcohol exposure during development results in the loss of neurons in the developing brain. The underlying molecular mechanisms are unclear and there currently is no cure for FASD. Ethanol-induced neuronal death is accompanied by neuroinflammation. Chemokine monocyte chemoattractant protein 1 (MCP-1) and its receptor C-C chemokine receptor type 2 (CCR2) are critical mediators of neuroinflammation and microglial activation. Using a third trimester equivalent mouse model of ethanol exposure, we found that treatment of Bindarit (MCP-1 synthesis inhibitor) and RS504393 (CCR2 antagonist) significantly reduced ethanol-induced microglia activation/neuroinflammation, and neuroapoptosis in the developing brain. Moreover, ethanol plus MCP-1 caused more neuronal death in a neuron/microglia co-culture system than neuronal culture alone, and Bindarit and RS504393 attenuated ethanol-induced neuronal death in the co-culture system. Ethanol activated TLR4 and GSK3β, two key mediators of microglial activation in the brain and cultured microglial cells (SIM-A9). Blocking MCP-1/CCR2 signaling attenuated ethanol-induced activation of TLR4 and GSK3β. Further, we determined whether knocking out of MCP-1/CCR2 ameliorates neonatal alcohol exposure-induced long-lasting behavioral deficits in adolescent and adult mice. C57BL/6 and MCP-1-/-/CCR2-/- mice were exposed to alcohol (5 g/kg) by subcutaneously injection on PD4. A series of behavioral tests including Open Field (PD 35-36 and PD 70-71), Rotor-Rod (PD 38 and PD 73), Balance Beam (PD 40 and PD75) and Morris Water Maze (PD 42 and PD77) were performed in the adolescence and adulthood. We found that MCP-1-/-/CCR2-/- mice were resistant to neonatal alcohol exposure-induced deficits in motor function in the Rotor-Rod and Balance Beam tests; MCP-1 and CCR2 deficiency also protected mice against neonatal ethanol exposure induced long lasting deficits in learning and memory in the Morris Water Maze testing. Collectively, these results suggest that MCP-1/CCR2 signaling plays an important role in ethanol-induced microglial activation/neuroinflammation and neurodegeneration in the developing brain and also plays an important role in developmental alcohol exposure induced long-lasting behavioral deficits in adolescence and adulthood.

Sphingoid bases (sphingosine, dihydrosphingosine and phytosphingosine) have been recently found in the oral cavity where they may serve to fortify innate immunity against commensals and periodontal pathogens. In fact, sphingoid bases have potent antimicrobial activity against Gram- positive and Gram- negative bacteria including oral pathogens like Porphyromonas gingivalis. It is not known whether these lipids are cytotoxic or alter the chemokine and cytokine responses of human dendritic cells, a finding important to their future potential as a therapeutic for treatment of periodontal disease. Objectives: The objective of this study was to determine the effects of sphingoid bases on the cytotoxicity and cytokine responses of human myeloid dendritic cells. Methods: Dendritic cells were treated with sphingoid bases (0.2-80.0 μM) for 16 hours in the presence or absence of 0.02 μM hemagglutinin B, a nonfimbrial adhesin of P. gingivalis used as a pro-inflammatory stimulus. The cytotoxicity of the inocula and its ability to induce the production of chemokines and pro-inflammatory cytokines was determined after 16 hours. Results: Higher concentrations of sphingoid bases were cytotoxic (e.g., 40.0-80.0 μM), but physiologic concentrations of sphingoid bases (e.g., 0.2-20.0 μM) were not. At 5, 10, or 20 μM, sphingosine did not enhance or attenuate any HagB-induced IL-8, GM-CSF, MIP-1α, MIP-1β, or TNFα response of human myeloid dendritic cells. At 5 or 10 μM, neither phytosphingosine nor dihydrosphingosine enhanced or attenuated any HagB- induced IL-8, GM-CSF, MIP-1α, MIP-1β, or TNFα response of human myeloid dendritic cells. Conclusion: Sphingoid bases exhibit dose-dependent cytotoxicity and cytokine responses against human myeloid dendritic cells. But at physiologic concentrations sphingoid bases appear to be safe and efficacious at the doses needed to prevent or treat microbial infections in the oral cavity.

<p>Mast cells are very potent multifunctional effector cells of the immune system normally distributed throughout connective tissues. An accumulation of mast cells has been described in several pathological conditions such as allergic- and autoimmune inflammations and in certain tumours. This necessitates two different processes: 1) Recruitment of mast cell progenitors from peripheral blood; 2) Accretion of mature mast cells at sites of inflammation and tumour areas. Both processes are depending on the local production of chemotactic factors. The aim of this study was to investigate the role of chemokines and their corresponding receptors in mast cell chemotaxis. </p><p>By cloning and mRNA-screening of cord blood derived mast cells several chemokine receptors were found to be expressed. Functional expression was confirmed of chemokine receptors CXCR4, CCR1 and CCR4. CXCL12, the only known ligand for CXCR4, acted as a mast cell chemotaxin and induced migration of progenitor cells with capacity to differentiate into mast cells. Of several ligands known to bind CCR1 and CCR4, only CCL5 induced migration of mast cells. The migration to CCL5 was mediated through both CCR1 and CCR4. In contrast, the ligands to CCR4, CCL17 and CCL22, could inhibit CCL5-induced migration. Expression of CCR1 and CCR4 could also be confirmed on mast cells in lung from asthmatic patients. Furthermore, we could demonstrate that mast cells were attracted by CCL5 produced by tumour cells in Hodgkin´s lymphoma.</p><p>In conclusion, the work in this thesis has identified two chemokines that regulates mast cell migration. This knowledge helps us understand the mechanisms behind homing of mast cell progenitors from the blood into the tissue and the accumulation of mature mast cells at sites of inflammation and tumourigenesis.</p>

<p>Hyaluronan (HA), a structural component in the extracellular matrix (ECM), has been recognized as a signaling molecule. It is important during various biological activities such as embryogenesis, angiogenesis, wound healing and tumor progression. Increased amount of hyaluronan during embryonic development is necessary for cell migration and differentiation, but the increased production of hyaluronan by tumor cells or tissue fibroblasts is correlated to poor prognosis for tumor progression and chronic inflammation, respectively. Therefore, understanding the mechanisms regulating HA-enriched matrices and the roles of HA in the biological functions is of fundamental biological importance.</p><p>Four novel findings are described in this thesis: (1) HA fragments (HA12) and the known angiogenic factor FGF-2 promote endothelial cell differentiation by induction of common but also distinct sets of genes, particularly, upregulation of the chemokine <i>CXCL1/GRO1</i> gene is necessary for HA12-induced angiogenesis and this effect is dependent on CD44 activation. (2) High concentrations of hyaluronan suppress PDGF-BB-induced fibroblasts migration and PDGFRβ tyrosine phosphorylation upon activation of hyaluronan receptor CD44, probably by recruiting a CD44-associated phosphatase to the PDGFRβ. (3) PDGF-BB stimulates <i>HAS2</i> transcriptional activity and HA synthesis through upregulation of MAP kinase and PI3 kinase signaling pathways in human dermal fibroblasts. (4) Specific suppression of <i>HAS2</i> gene in the invasive breast cancer cell line Hs578T by RNA interference (RNAi) leads to a less aggressive phenotype of breast tumor cells. This suppressive effect can be reversed by exogenously added hyaluronan.</p><p>In conclusion, binding of hyaluronan to CD44 plays an important role in cell signaling, inflammation and tumor progression. Further studies are required to elucidate the molecular mechanisms through which hyaluronan levels are regulated under physiological or pathological conditions, and to explore compounds involved in hyaluronan accumulation and activity as targets for therapies of chronic inflammation and tumors.</p>

<p>Enterovirus (EV) infections have been associated with the pathogenesis of Type 1 Diabetes (T1D). However, the pathway(s) by which EV may induce or accelerate diabetes is not well understood. The purpose of this thesis was to obtain new information on the mechanism by which EV infections, with different strains of EV, could cause damage to the insulin-producing β-cells in isolated human islets and in a rat insulin-producing cell line (RINm5F). </p><p>Infection with EV strains isolated from T1D patients revealed replication/cell destruction in human islets and EV-like particles in the cytoplasm of the β-cell and infection with the isolates affected the release of insulin in response to glucose stimulation as early as three days post infection, before any decrease in cell viability was observed. A decrease in the induction/secretion of the chemokine RANTES in human islets during EV infection was also detected. When islets were cultured with nicotinamide (NA) the secretion of RANTES was increased irrespectively if the islets were infected or not. In addition, the degree of virus-induced cytolysis of human islets was reduced by NA, suggesting an antiviral effect of NA. Infection with EV strains revealed permissiveness to islet-derived cells. </p><p>All EV strains used for infection were able to replicate in the RIN cell clusters (RCC) but not in the RIN cells that were cultured as a monolayer. This might be due to the differences in expression of the Coxsackie-adenovirus receptor (CAR), which only could be detected on the RCC. Infection of RCC with a CBV-4 strain did not affect cell viability and did not induce nitric oxide (NO) production alone or with the addition of IFN-γ. This was in contrast to the results obtained with synthetic dsRNA, poly(IC), which induced NO, suggesting that synthetic dsRNA does not mimic enteroviral intermediate dsRNA.</p><p>During analyses performed with the samples from a family where the mother and one son where diagnosed with T1D on the same day, the results showed that the whole family had a proven EV infection at the time diagnosis.</p><p>To conclude, the ability of EV strains to replicate in RIN cells is dependent on the growth pattern of the cells and this may be due to the upregulation and/or changed expression pattern of CAR in these cells. In the RIN cells, contrary to artificial dsRNA, viral dsRNA does not induce NO. The isolated EV virus strains used were able to infect and affect human pancreatic islets in vitro. The chemokine RANTES is reduced during an EV infection of human pancreatic islets and NA causes upregulation of RANTES in infected and uninfected islets. </p>