Cutaneous wound healing is a complex process involving the migration of inflammatory cells to the wound site, deposition of extracellular matrix, and the reestablishment of an intact epithelial barrier. Re-epithelialization depends on the proliferation and directional migration of keratinocytes from the wound edges. Initially, keratinocytes migrate over a provisional wound matrix that is rich in fibronectin, and as the wound heals the provisional matrix becomes replaced by one consisting of collagen and proteoglycans. Re-epithelialization is tightly regulated by a variety of peptides such as growth factors, cytokines and proteases, and abnormalities may result in chronic non-healing wounds or hypertrophic scars. CCN2 (Connective Tissue Growth Factor) is a multifunctional protein with effects on cells and their interactions with the connective tissue. CCN2 is expressed in a variety of cell types and regulates numerous cell functions including proliferation, differentiation, adhesion, migration and stimulation of collagen production. While the importance of CCN2 for the fibrotic response has been well studied, its involvement in keratinocyte function has not yet been fully explored. Using an in vivo wound model, the expression of CCN2 was captured at the leading keratinocyte edge during re-epithelialization. In vitro, exogenous addition of CCN2 to human keratinocyte cultures promoted keratinocyte migration. Subsequently, integrin a5b1 was identified as an important mediator of CCN2 enhancement of keratinocyte adhesion to fibronectin. CCN2 activated the FAK-MAPK signaling pathway, and pretreatment with MEK1 specific inhibitor PD98059 markedly reduced CCN2-promoted keratinocyte migration. In vitro, CCN2 expression was induced by TGF-β1. Compared with inhibiting the SMAD pathway, blocking MAPK was more effective in reducing TGF-β1-induced CCN2 mRNA and protein expression. In addition, CCN2-induced keratinocyte spreading required FAK. Treatment with CCN2 led to actin disassembly and altered the activity of the Rho proteins and p190RhoGAP in keratinocytes. Furthermore, Cdc42 mediated CCN2-induced cell polarity. In conclusion, using in vivo and in vitro models, CCN2 was shown to regulate keratinocyte function by promoting keratinocyte adhesion, spreading and migration. A complete understanding of CCN2 expression in keratinocytes is crucial in order to develop novel therapies for wound healing.

γδ T cells belong to the T cell lineage however they possess some innate like properties. γδ T cells recognize non-peptidic microbial and stress induced self antigens in a non-MHC restricted manner and are proposed to bridge the gap between innate and adaptive immunity. Dendritic epidermal T cells are a prototypic population of intraepithelial γδ T cells in murine skin. Found in the basal layer of epidermis in close contact with Langerhans cells and keratinocytes DETC facilitate vital immunological and physiological processes e.g. wound healing, homeostasis, tumour surveillance and regulation of inflammation. The purpose of this thesis was to elucidate whether γδ T cells and in particular DETC play a role in generation of adaptive immune responses to foreign cutaneous antigen (OVA) in the context of skin grafts. Skin grafting has long been established as a means to test cutaneous and epithelial immunity. To answer this question, γδ T cell knock-out mice (TCRδ-/-), transgenic K5mOva mice and a skin grafting model were used. It is shown in this study that in the absence of γδ in the skin and not in the circulation there is a lower rejection rate of OVA expressing skin grafts. This phenomenon is observed in both freshly placed and well healed grafts. To understand which part of the immune response is affected by the absence of γδ T cells the priming and effector phases of the immune response was examined in TCRδ-/- mice. The priming phase was studied using two approaches: the first approach was to test priming to maximal doses of subcutaneous antigen in conjunction with an adjuvant and the second approach involved testing priming to an antigen in the context of skin grafts (graft priming). Using ELISPOTs and CFSE proliferation assays we found that while administration of OVA in conjunction with an adjuvant (QuilA) via the subcutaneous route results in sufficient priming in γδ T cell knockout mice, cross priming to OVA in the context of - freshly placed and well healed skin grafts is impaired in TCRδ-/- mice. By immunizing TCRδ-/- mice prior to skin grafting or by transferring in vitro primed OT-I cells to RAG-/- mice grafted with K5mOVA or TCRδ-/-OVA skin it was shown that 100% of all OVA grafts are rejected regardless of presence or absence of γδ T cells, concluding that effector phase of the immune response is not affected in this model. The inability of DETC to perform the role of cross presentation leads to the hypothesis that DETC indirectly enhance this process by affecting professional antigen presenting cells (APC) of the skin. Based on the contribution of DETC to wound healing it was hypothesized that the migration of dendritic cells (DCs) from the skin grafts to the lymph nodes may be affected. When this hypothesis was tested using hapten sensitization and congenically marked skin grafts it was shown that migration of DCs from skin grafts is not affected by the absence of DETC. In another hypothesis the co-stimulatory markers CD40 and CD86 were examined on migrating DCs found in the skin draining lymph nodes of grafted mice and it was shown that expression levels of those molecules were lower on DCs from TCRδ-/- grafted mice compared to C57BL/6 control mice. In addition using cytometric bead array, we show that the cytokine milieu in TCRδ-/- skin and skin draining lymph nodes is different from that of wildtype C57 skin and this disparate cytokine profile may be contributing to the less efficient cross priming and graft rejection in TCRδ-/- mice.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 104-120).

Neste trabalho foi estudada a formação de membranas para obtenção de compostos dermo-epidérmicos. A porção dérmica foi desenvolvida utilizando-se mistura de polímeros sintéticos, o poli(álcool vinílico) - PVAl ou poli(vinilpirrolidona) – PVP, com polímero natural, a quitosana. As membranas foram reticuladas pela radiação g ou glutaraldeído. A porção epidérmica destas membranas foi formada por queratinócitos cultivados in vitro, os quais foram semeados sobre as membranas correspondentes e verificada sua interação. As membranas que melhor interagiram com os queratinócitos foram aquelas preparadas com quitosana pela reticulação com glutaraldeído, porém não satisfazendo as características mecânicas de manipulação. As membranas que possuíam as melhores características mecânicas, porém com moderada interação com os queratinócitos, foram as compostas de PVAl, liofilizada e intumescida com quitosana. Os componentes foram caracterizados isoladamente, bem como as membranas formadas pelos mesmos. O PVAl foi caracterizado quanto a sua dose gel e a quitosana quanto à determinação das constantes de Mark-Houwink, grau de acetilação e dissolução em diferentes valores de pH. As membranas foram caracterizadas quanto a sua cinética de intumescimento com água. Na membrana de PVAl com quitosana incorporada foi avaliada sua degradação in vitro, determinada sua cinética de intumescimento com a quitosana e estimado o tamanho do poro. As membranas de quitosana reticuladas com glutaraldeído foram caracterizadas quanto à cinética de intumescimento e verificado o possível desprendimento de glutaraldeído. As duas membranas caracterizadas isoladamente foram unidas para formação de uma única membrana, como a parte dérmica do composto, onde a membrana de PVAl incorporada com quitosana foi recoberta com a membrana de quitosana reticulada com glutaraldeído. Quitosanas de outras procedências foram avaliadas na interação com os queratinócitos. / Membrane formations were studied to obtain dermal-epidermal compounds. The dermal portion was developed using synthetic polymers mixture, poly(vinyl alcohol)-PVAl or poly(vinylpyrrolidone)-PVP, with natural polymers, and chitosan. The membranes were crosslinked by gamma irradiation or glutaraldehyde. The epidermal portion of these membranes was formed by keratinocytes cultured in vitro, seeded on these membranes to verify their interaction. The membranes that interacted better with keratinocytes were those prepared with chitosan by glutraldehyde crosslinking, although not satisfying handling mechanical characteristics. The best mechanical characteristic was observed at PVAl membranes frezed dried and chitosan incorporated, but with moderate keratinocytes interaction. The components were characterized separately as well as the membranes formed by both. The PVAl was characterized as to its gel dose and to chitosan were determined Mark-Houwink equation, deacetilation degree and solubility under changes of pH. The membranes were characterized as to their swelling kinetic degree in water. In the membrane of PVAl with chitosan incorporated was evaluated its degradation in vitro, swelling kinetic degree with chitosan solution and the pore size. The chitosan membranes crosslinked by glutaraldehyde were characterized as to their swelling kinetic degree and verified the possibility of deatached glutaraldehyde. Membranes characterized separetelly were joined to perform the ideal dermal component, where the PVAl with chitosan incorporated membrane was covered by chitosan crosslinked by glutaraldehyde membrane. Chitosan from other sources were evaluated in the interaction with keratinocytes.

Orientador: Carlos Eduardo Vergani / Resumo: Nos últimos anos, os microcristais de prata têm se tornado foco de estudos. Uma das propriedades evidenciadas destes materiais é a sua atividade antimicrobiana contra diferentes microrganismos, devido a presença da prata na sua composição. Neste estudo, investigou-se a atividade antifúngica de microcristais de alfa vanadato de prata (α-AgVO3) contra Candida albicans (ATCC 90028) e sua citotoxicidade sobre células do tipo queratinócitos orais normais espontaneamente imortalizados (NOK-si). Os microcristias de α-AgVO3 foram sintetizados pelo método da co-precipitação sob três diferentes temperaturas (10, 20 e 30ºC) e caracterizados através de difração de raios-x, microscopia eletrônica de varredura por emissão de campo e espectroscopia Raman. A atividade antifúngica foi avaliada a partir da microdiluição seriada dos microcristais (de acordo com o Clinical & Laboratorial Standards Institute - CLSI), onde foram determinadas as concentrações inibitória (CIM) e fungicida mínimas (CFM). Imagens de microscopia de fluorescência com os microcristais nas concentrações inibitória e fungicida mínimas foram obtidas a fim de confirmar os achados microbiológicos. A viabilidade celular de células NOK-si foi avaliada através do ensaio Alamar Blue, e imagens de microscopia eletrônica de varredura (MEV) de todos os grupos avaliados foram realizadas. Nos ensaios celulares foram utilizadas apenas quatro concentrações dos microcristais (CIM, CFM, CIM diluída 10 vezes e concentrada 10 vezes). Os r... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Cutaneous lupus erythematosus (CLE) is an autoimmune disease that resolves with or without permanent scars depending on the subtype. Interferons (IFNs), including the skin specific IFNλ mainly activate STAT1, which results in inflammation in CLE and may play a significant role in scar formation in chronic discoid CLE. IL-22 activates STAT3 and it is emerging as a mediator with significant impact on normal wound repair, epidermal hyperproliferation and prevention of fibrosis. This work focussed on understanding the regulation and functional impact of IL-22 and IFNλ on skin cells. The counter-regulatory effect of IL-22 on the activities of IFNλ was assessed through downstream interferon stimulated genes (ISGs) expression in healthy and CLE keratinocytes. Cell proliferation and gap closure were investigated in skin resident cells using cell trace dye and scratch assay. Dermal fibroblasts were assessed for the presence of IFNλR1 and IL-22R1, downstream activities of the receptors. Results showed that IL-22 accelerated “scratch” closure in keratinocytes while IFNλ caused a delay in closure. IL-22 significantly downregulated IFNλ-induced chemokines expression in healthy, but not CLE keratinocytes. Reduced IL-22R1 expression and “STAT3 signature genes” was observed in CLE keratinocytes. A key finding of this project is that dermal fibroblasts respond to both IFNλ and IL-22. This work shows that IL-22 can reduce the damaging effect of IFNs in inflamed skin and also identifies dermal fibroblasts as important cells in skin immune responses. In conclusion, IL-10 family members can have both beneficial and destructive effects on the skin organ depending on the micro milieu and cell-type involved. Manipulating the balance of IL-10 family members in the skin may offer new therapeutic approach for both psoriasis and CLE.

A fototerapia laser (FTL) tem sido usada clinicamente para auxiliar na cicatrização de inúmeras doenças bucais, especialmente no tratamento de lesões ulceradas. Os mecanismos celulares através dos quais o laser é capaz de promover a bioestimulação não são completamente compreendidos. Dessa forma, o objetivo do presente estudo foi avaliar in vitro o efeito da FTL no comportamento de queratinócitos bucais no processo de cicatrização. Células epiteliais bucais (NOK-SI) foram cultivadas sob duas condições nutricionais: suplementadas com 10% de soro fetal bovino (FBS) e sob déficit nutricional (2% FBS) seguido de irradiação com laser de diodo InGaAlP (660nm, 40mW, 4 e 20J/cm2, 4 e 20s), através da técnica pontual e em contato. Foram realizados ensaio de viabilidade celular (MTT), migração celular (cicatrização) e análise proteica (Western Blotting e Fluorescência). Os resultados obtidos indicaram que a FTL influencia diretamente a migração epitelial evidenciado pelo fechamento acelerado das feridas irradiadas e polarização do citoesqueleto celular (F-actina). Conclui-se que os efeitos clínicos da FTL estão associados, entre outros fatores, ao aumento da migração epitelial. / Laser phototherapy (LPT) has been used clinically to accelerate wound healing in a variety of oral diseases. The cell mechanisms by which LPT can promote biostimulation have not yet been fully elucidated. Epithelial cells play an important role in the reparative process since it proliferation and migration from the wound margin is crucial for restore epithelial continuity. It is unclear whether LPT has an effect on epithelial cell migration. Based on this, the aim of this study was to investigate the effect of LPT in oral wound healing process using oral keratinocytes. Oral keratinocytes were maintained under two nutritional conditions supplemented with 10% of fetal bovine serum (FBS) and in nutritional deficit (2% FBS). Laser irradiation was delivered with InGaAlP laser (660nm, 40mW, 4 e 20J/cm2, 4 e 20s). Irradiations were performed in contact, using the punctual irradiation mode. The following tests were performed cell viability, cell migration and protein analysis. Results obtained suggest that LPT influences epithelial migration and cytoskeleton polarization. Interestingly, LPT effect under epithelial cell migration occurs independently of cell viability. In conclusion, clinical LPT effects are associated with an increase in epithelial cell migration.

Orientador: Carlos Eduardo Vergani / Resumo: Esta presente tese foi dividida em quatro estudos que tiveram como objetivos. 1. Validar um protocolo e comparar o efeito dos tampões RPMI/MOPS e RPMI/HEPES no desenvolvimento de biofilmes e na viabilidade celular de queratinócitos (NOK-si e HaCat); 2. Comparar o dano celular e a resposta inflamatória induzidos pelos metabólitos de biofilmes simples e misto de Staphylococcus aureus e Candida albicans; 3. Avaliar o tipo de morte celular (apoptose vs. necrose) e a ativação de caspases relacionadas aos metabólitos desses biofilmes e 4. Caracterizar um tecido oral reconstituído e analisar o dano tecidual causado pelo sobrenadante e biofilme propriamente dito desses microrganismos. No estudo 1, a viabilidade celular foi avaliada pelo método colorimétrico do MTT e por imagens da cultura após 12 horas em contato com os meios de cultura. Ambos os tampões permitiram similar crescimento do biofilme. Efeito citotóxico do MOPS foi verificado após 6 horas de crescimento de NOK-si e HaCat. Houve preservação da viabilidade e morfologia quando as células foram expostas a RPMI/HEPES. Conclui-se que RPMI/HEPES pode ser utilizado como um meio tamponamente viável para estudos que avaliam o efeito do biofilme em cultura de queratinócitos ao longo do tempo. No estudo 2, o sobrenadante dos biofilmes de 36 h de C. albicans e S. aureus, isolados ou em associação, foi colocado em contato com NOK-si, HaCat e macrófagos (J774A.1). O dano celular foi avaliado por meio de ensaios de viabilidade celular ... (Resumo completo, clicar acesso eletrônico abaixo) / The present thesis was divided into four studies with the following objectives. 1. Validate a protocol and compare the effect of RPMI/MOPS and RPMI/HEPES buffers on the development of biofilms and keratinocyte cell viability (NOK-si and HaCat); 2. Compare the cellular damage and the inflammatory response induced by the metabolites of simple and mixed biofilms of Staphylococcus aureus and Candida albicans; 3. Evaluate the type of cell death (apoptosis vs. necrosis) and the activation of caspases related to the metabolites of these biofilms and 4. Characterize the reconstituted oral tissue and analyze the tissue damage caused by the supernatant and biofilm of these microorganisms. In study 1, cell viability was evaluated by the MTT colorimetric method and by culture images after 12 hours in contact with the culture media. Both buffers permitted similar biofilm growth. The cytotoxic effect of MOPS was observed after six hours of NOK-si and HaCat growth. There was preservation of viability and morphology when cells were exposed to RPMI/HEPES. It was concluded that RPMI/HEPES can be used as a buffering medium for studies evaluating the effect of biofilm on keratinocyte culture over time. In study 2, the supernatant of the 36-hour biofilms of C. albicans and S. aureus, isolated or in combination, was placed in contact with NOK-si, HaCat and macrophages (J774A.1). Cell damage was assessed by cell viability assays (MTT) and LDH enzyme release. Cytokine production was analyzed by the ELISA method and evaluation of the type of cell death by the staining of the apoptotic cells with annexin V and the necrotic cells with propidium iodide. The mixed biofilm and biofilm of C. albicans were more cytotoxic, and the mixed biofilm caused greater cellular damage through the release of the LDH enzyme. S. aureus biofilm metabolites stimulated greater production of NO, IL-6 and TNF-α... (Complete abstract electronic access below) / Doutor

A fototerapia laser (FTL) tem sido usada clinicamente para auxiliar na cicatrização de inúmeras doenças bucais, especialmente no tratamento de lesões ulceradas. Os mecanismos celulares através dos quais o laser é capaz de promover a bioestimulação não são completamente compreendidos. Dessa forma, o objetivo do presente estudo foi avaliar in vitro o efeito da FTL no comportamento de queratinócitos bucais no processo de cicatrização. Células epiteliais bucais (NOK-SI) foram cultivadas sob duas condições nutricionais: suplementadas com 10% de soro fetal bovino (FBS) e sob déficit nutricional (2% FBS) seguido de irradiação com laser de diodo InGaAlP (660nm, 40mW, 4 e 20J/cm2, 4 e 20s), através da técnica pontual e em contato. Foram realizados ensaio de viabilidade celular (MTT), migração celular (cicatrização) e análise proteica (Western Blotting e Fluorescência). Os resultados obtidos indicaram que a FTL influencia diretamente a migração epitelial evidenciado pelo fechamento acelerado das feridas irradiadas e polarização do citoesqueleto celular (F-actina). Conclui-se que os efeitos clínicos da FTL estão associados, entre outros fatores, ao aumento da migração epitelial. / Laser phototherapy (LPT) has been used clinically to accelerate wound healing in a variety of oral diseases. The cell mechanisms by which LPT can promote biostimulation have not yet been fully elucidated. Epithelial cells play an important role in the reparative process since it proliferation and migration from the wound margin is crucial for restore epithelial continuity. It is unclear whether LPT has an effect on epithelial cell migration. Based on this, the aim of this study was to investigate the effect of LPT in oral wound healing process using oral keratinocytes. Oral keratinocytes were maintained under two nutritional conditions supplemented with 10% of fetal bovine serum (FBS) and in nutritional deficit (2% FBS). Laser irradiation was delivered with InGaAlP laser (660nm, 40mW, 4 e 20J/cm2, 4 e 20s). Irradiations were performed in contact, using the punctual irradiation mode. The following tests were performed cell viability, cell migration and protein analysis. Results obtained suggest that LPT influences epithelial migration and cytoskeleton polarization. Interestingly, LPT effect under epithelial cell migration occurs independently of cell viability. In conclusion, clinical LPT effects are associated with an increase in epithelial cell migration.