A reconstrução de defeitos ósseos é um problema que afeta milhões de pessoas, que a medicina tenta resolver. Uma alternativa para a solução deste problema tem sido o desenvolvimento de biomateriais que atuem no processo de reparação óssea. O colágeno é um polímero de origem natural capaz de promover cicatrização e regeneração óssea e juntamente com a hidroxiapatita são os principais componentes encontrados no tecido ósseo. Vários trabalhos têm sido reportados com matrizes mineralizadas de colágeno tipo I em diferentes formas como em géis, membranas e esponjas, mas a mineralização in vitro de matrizes acelulares obtidas de tecidos biológicos sem a perda da estrutura colagênica não tem sido descrito. Este trabalho teve como objetivo a mineralização in vitro e a caracterização de matrizes de colágeno aniônico obtidas de pele porcina, pericárdio bovino e serosa porcina. Os tecidos foram tratados em temperatura ambiente com solução alcalina por períodos variáveis de 0 à 96h e mineralizados pelo processo de imersão alternada. Os materiais obtidos foram caracterizados pela avaliação preliminar da citotoxicidade in vitro, termogravimetria (TG/DTG), calorimetria exploratória diferencial (DSC), microscopia eletrônica de varredura (MEV), dispersão de raios X (EDS), difração de raios X (DRX) e absorção no infravermelho (FT-IR). Não foi observada citotoxicidade em nenhuma das matrizes avaliadas, contudo foi necessário um pré-tratamento nas matrizes de pele porcina para remoção de gordura. Os resultados de DSC mostraram a integridade da matriz colagênica após o tratamento alcalino. O aumento no tempo desse tratamento diminui a temperatura de desnaturação sendo observado um efeito maior nas matrizes de pele porcina seguidas por pericárdio bovino e serosa porcina. A mineralização induz a um aumento na temperatura de desnaturação em todos os casos. As curvas TG apresentaram perdas de massa relacionadas à água presente no material, decomposição da proteína e carbonização do material orgânico e um resíduo após 750 °C que foi associado ao material inorgânico presente na forma de hidroxiapatita, sendo as matrizes de serosa porcina as de maior teor de mineralização. As matrizes mineralizadas tendem a um aumento na estabilidade térmica do colágeno quando comparadas com as matrizes hidrolisadas. Os espectros FT-IR mostraram a presença de íons fosfatos e a interação de íons cálcio com o colágeno. As relações Ca/P obtidas por EDS foram aquelas esperadas em comparação com o valor teórico para hidroxiapatita (HA) e resultados de DRX confirmaram a obtenção de HA amorfa como principal produto de mineralização. Pelas fotomicrografias obtidas por MEV pôde-se observar que as fibras de colágeno tornam-se mais desestruturadas quando há um aumento no tempo de hidrolise e que a deposição de sais ocorreu de forma heterogênea, disposta em aglomerados esféricos no formato de agulhas por toda a superfície e interior, exceto para matrizes derivadas de pele porcina que não são mineralizadas internamente devido a sua espessura. Os resultados obtidos demonstraram que é possível a mineralização in vitro de matrizes de colágeno tipo I obtidas de diferentes tecidos biológicos em diferentes tempos de hidrólise, produzindo um material com potencial de uso para regeneração óssea. / The reconstruction of osseous defects is still a problem that affects millions of people and medicine tries to solve it. One alternative to solve these problems has been the development of biomaterials that can be used as inductors in the osseous repair process. Collagen is a natural polymer able to promote healing and bone regeneration, and among hydroxyapatite (HA) is the main component found in bone tissue. Several mineralized collagen scaffolds are described in literature, in the form of gel, membranes and films, however, in vitro mineralization of acellular matrices, obtained from biological tissues without the loss of collagenic structure, has not been reported. The objective of this work was the mineralization and characterization of anionic collagen matrices obtained from porcine skin, bovine pericardium and porcine serosa. Biological tissues were treated at room temperature for 0-96h in alkaline solution and mineralized by alternate soaking method. Materials were characterized by preliminary assay of in vitro cytotoxicity, differential scanning calorimetry (DSC), termogravimetric analysis (TG/DTG), scanning electronic microscopy (SEM), energy dispersive x-ray analysis (EDS), x-ray diffraction (XRD), and Fourier Transform Infrared Spectroscopy (FTIR). No cytotoxicity was observed in any of the evaluated matrices; however, a pre-treatment of porcine skin matrices, for fat removal, was necessary. DSC results showed the integrity of collagen matrices after alkaline treatment. Denaturation temperature is dependent of time of alkaline treatment, and this effect is greater for porcine skin matrix, followed by bovine pericardium and porcine serosa. TG/DTG curves showed weight losses associated with release of water, degradation of protein structure and combustion of residual organic components. Residues were obtained at 750°C and associated to hydroxyapatite, being porcine serosa matrix the most mineralized. All mineralized matrices showed an increase in collagen thermal stability when compared to hydrolyzed matrices. FTIR spectra showed the presence of phosphate ions and the interaction of calcium ions with collagen. Ca/P ratios obtained by EDS were as expected when compared with literature values for HA, and RDX results confirmed amorphous HA as the main mineralization product. MEV analysis showed that collagen fibers were more affected for longer hydrolysis times, and that salt deposition was heterogeneous, with crystals grouped in spherical agglomerates in a needle-like shape throughout surface and inner, except for porcine skin derived matrices that were not internally mineralized due their width. Obtained results demonstrated that in vitro mineralization of type I collagen matrices, using different sources of biological tissues and hydrolysis time was possible, producing a material with potential to be used in bone regeneration.

Orientador: Cássio Luís Zanettini Riccetto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T07:18:23Z (GMT). No. of bitstreams: 1 Dias_FernandoGoulartFernandes_M.pdf: 14775900 bytes, checksum: 18591a82df67b2439fbeb82e6cffca13 (MD5) Previous issue date: 2014 / Resumo: INTRODUÇÃO: Telas sintéticas representam, na atualidade, o pilar do tratamento da incontinência urinária e de prolapsos vaginais, sendo o polipropileno monofilamentar o material sintético mais utilizado. Apesar de taxas de cura de até 90%, complicações relacionadas à integração, tais como exposição ou erosão das telas, não podem ser negligenciadas. O colágeno, por ser um material biologicamente compatível, pouco imunogênico e com propriedades moduladoras do processo inflamatório, pode ser utilizado como um importante agente cicatricial melhorando a integração das telas. OBJETIVO: Avaliar, por meio de técnicas imunohistoquímicas, o efeito do recobrimento de tela de polipropileno monofilamentar, implantada no subcutâneo de ratas, com gel purificado de colágeno bovino, do ponto de vista da resposta imuno-inflamatória, do metabolismo do colágeno, angiogênese e citotoxicidade. MATERIAIS E MÉTODOS: Após aprovação no Comite de Ética em Experimentação Animal, foram utilizadas 20 ratas fêmeas da raça Wistar, tendo sido implantadas, em cada animal, de um lado da parede abdominal, uma tela de polipropileno monofilamentar (PP) e, do outro lado, uma tela semelhante recoberta com gel purificado de colágeno (PPC). Os animais foram divididos em quatro sub-grupos contendo 5 animais cada e foram eutanasiados em 7, 14, 21 e 90 dias após o implante. Foram utilizados reagentes específicos para avaliação dos aspectos de interesse: a) Imunológicos (Interleucina 1 ¿ IL-1); b) Metabolismo do colágeno (Metaloproteinases de Matriz 2 e 3 ¿ MMP-2 e 3); c) Angiogênese (Antígeno de Superfície CD-31); d) Citotoxicidade (Receptor do Fator de Necrose Tumoral alfa ¿ TNF ¿?). A análise das imunorreatividades foi realizada com auxílio do software analisador de imagens AxioVisionTM. RESULTADOS: A análise comparativa das variáveis entre os 4 períodos definidos (7,14, 21 e 90 dias) e entre os 2 grupos (PP e PPC) apontou diferença significativa para: CD-31 com maior número de vasos no grupo PPC, no subgrupo 14 dias (p=0.002) em relação ao grupo PP, e diminuição após 90 dias (p=0.002) no grupo PPC; MMP-2 com redução na densidade média no grupo PPC (p=0.046) nos subgrupos 21 e 90 dias em relação ao grupo PP ; MMP-3 com maior estabilidade ao longo do tempo no grupo PPC, de modo que houve queda significativa da área percentual reativa no grupo PP após 14 e 90 dias (p=0.017), bem como redução da densidade média logo após 21 dias, mas apenas após 90 dias no grupo PPC (p<0.001). CONCLUSÃO: O recobrimento com gel purificado de colágeno bovino determinou alterações significantes na resposta tecidual das ratas às telas de polipropileno, do ponto de vista imunohistoquímico, quanto à angiogênese e atividade das metaloproteinases na área do implante, sem influência significativa sobre a resposta imuno-inflamatória local (expressas por meio da IL-1 e TNF¿?) / Abstract: INTRODUCTION: The use of synthetic meshes, specially the polypropylene mesh, has become the standart treatment of urinary incontinence and vaginal prolapses. Even though presenting high cure rates, complications related to integration issues, such as exposure or erosion of the mesh, cannot be neglected. The collagen, well known as an important immunoinflammatory modulator, has been speculated to be a usefull tool in the healing process and possibly improving integration of meshes. OBJECTIVE The aim of this study is to evaluate, using immunohistochemical techniques, the effect of the use of a new purified collagen gel covering the monofilament polypropylene mesh implanted subcutaneously in rats, regarding immune-inflammatory response, collagen metabolism, angiogenesis and cytotoxicity. METHODS: After Ethics Committee on Animal Use¿s approval, in 20 female Wistar rats were implanted, at one side of abdominal wall, a monofilament polypropylene mesh (PP) and on the other side, the same mesh covered with a new developed purified collagen gel (PPC). The animals were divided into four sub-groups containing 5 animals each and were euthanized at 7, 14, 21 and 90 days after implantation. The immunohistochemical assessment of the samples was done by using specific reagents for the evaluation of points of interest: a) Immunologic (Interleukin 1 (IL-1)), b) Collagen metabolism (Metalloproteinases 2 and 3 (MMP-2 and 3)), c) Angiogenesis (surface antigen CD-31), d) Cytotoxicity (Tumor Necrosis Factor-alpha Receptor - TNF- ?). The objective analysis was performed using the image analysis software AxioVision TM. RESULTS: Comparative analysis of variables between the four periods defined (7, 14, 21 and 90 days) and between the 2 groups (PP and PPC) showed: higher vessel density in PPC group after 14 days (p=0.002) and decrease after 90 days (p=0.002); decrease of MMP-2 average density in PPC group after 21 and 90 days (p=0.046); more stability in MMP-3¿s behavior in PPC group along the periods with MMP-3 percent reactive area showing significant decrease just in PP group after 14 and 90 days (p=0.017) and also for MMP-3 average density, in which reduction was significant after 21 days in PP group, but just after 90 days in PPC (p<0.001).CONCLUSION: Highly purified collagen coating causes significant changes in angiogenesis and in metalloproteinase's immunohystochemical expression in meshes implants in rats, without significant influence on the local immuno-inflammatory response (expressed by IL-1 and TNF-?) / Mestrado / Fisiopatologia Cirúrgica / Mestre em Ciências

Orientadores: Cássio Luís Zanettini Riccetto, Benedicto de Campos Vidal / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T17:40:56Z (GMT). No. of bitstreams: 1 Siniscalchi_RodrigoTeixeira_D.pdf: 4712721 bytes, checksum: 84737665d5451453e82931eab2e748c4 (MD5) Previous issue date: 2012 / Resumo: Introdução: O material sintético (tela) mais utilizado atualmente no tratamento da Incontinência Urinária de Esforço (IUE) e dos prolapsos da parede vaginal, também conhecidos como Prolapsos dos Orgãos Pélvicos (POP) é o polipropileno monofilamentar, com índices de cura de até 90%. Porém, as complicações relacionadas à integração tecidual desses implantes são relativamente prevalentes. O colágeno, por ser um material biologicamente compatível, pouco imunogênico e com propriedades moduladoras do processo inflamatório, pode ser utilizado como um importante agente cicatrizante e, nesse sentido, poderia melhorar a integração das telas. Objetivo: Estudar os efeitos biomecânicos e histológicos do recobrimento de tela de polipropileno monofilamentar com gel purificado de colágeno, implantadas no tecido subcutâneo de ratas. Material e Métodos: Vinte ratas foram utilizadas para o estudo histológico e outras 20 para o estudo biomecânico. De um lado da parede abdominal do animal foi implantado um fragmento tela de polipropileno monofilamentar macroporosa medindo 20 x 10 mm, habitualmente utilizada no tratamento da IUE e dos POP (Grupo I), denominada PLP (tela de polipropileno) e do outro lado foi implantada uma tela com as mesmas dimensões recoberta com gel purificado de colágeno (Grupo II), denominada PLP+C (tela de polipropileno recoberta com o gel purificado de colágeno). De acordo com o tempo de eutanásia (7, 30, 90 ou 180 dias) após o implante os animais de cada grupo foram divididos em quatro subgrupos contendo cinco animais cada. Foi então realizada excisão em bloco da parede abdominal para as análises. O estudo biomecânico foi realizado em um tensiômetro de precisão, no qual a tela era tracionada em sentido uniaxial até que se desprendesse da interface tecidual. Para quantificar a aderência do material, em cada grupo, foi analisada a carga máxima necessária para este desprendimento. No estudo histológico foram analisadas as características relativas à inflamação aguda e crônica além do tecido de granulação, formação de granuloma e reação de corpo estranho em lâminas coradas com Hematoxilina-Eosina (HE), utilizando-se de método semiquantitativo. A organização supramolecular da deposição colágena em torno das telas foi estudada através de microscopia de polarização (birrefringência). Resultados: No estudo biomecânico observou-se que a aderência das telas de polipropileno aos tecidos circunvizinhos aumentou significativamente após o recobrimento com o gel purificado de colágeno, como demonstrado na análise dos valores encontrados para a Carga Máxima no 7º (p=0,0016), 14º (p=0,0039), 90º (p=0,0009) e 180º (p=0,0029) dias após o implante. Considerou-se nessa pesquisa, que o aumento da biocompatibilidade da tela de polipropileno seria alcançado quando, na interface tecidual, houvesse redução da intensidade do processo inflamatório. Verificou-se que a resposta inflamatória crônica e aguda (neutrofílica), assim como a formação de tecido de granulação foi menos intensa, respectivamente p=0,004, p<0,001 e p=0,001 nas telas recobertas com o colágeno na fase inicial (7º e 14º dias) e ausente, assim como nas telas não recobertas na fase tardia (90º e 180º dias). A inflamação granulomatosa foi observada de forma menos significativa aos sete dias após o implante nos animais do grupo II (p=0,029) e em ambos os grupos, de maneira similar, diminuiu ao longo do tempo não mostrando diferença significativa. A reação de corpo estranho foi menos intensa na fase inicial no grupo II (p<0,001) e semelhante entre os grupos na fase tardia. Nas análises de birrefringência foi observado no período inicial (sete dias) uma maior densidade média de brilho (transmitância) a favor das telas não recobertas (p=0,000), porém nos outros períodos analisados a densidade média de brilho foi maior nas tela do grupo II (PLP+C), 14 dias (p=0,000), 90 dias (p=0,000) e em 180 dias (p=0,000). Conclusão: O recobrimento das telas de polipropileno com o gel purificado de colágeno aumentou sua aderência aos tecidos, quando implantadas na interface da parede abdominal de ratas adultas, tanto precocemente quanto tardiamente; promoveu uma resposta inflamatória menos intensa e duradoura e na fase tardia do implante desencadeou maior organização e empacotamento das fibras de colágeno. Estes dados inferem que a tela recoberta com o gel purificado de colágeno quando locada em seu leito de implante terá, provavelmente, menor mobilidade e também uma melhor adaptação e integração ao corpo do hospedeiro resultando em menor chance de complicações / Abstract: Introduction: The synthetic material most currently used in the treatment of stress urinary incontinence (SUI) and vaginal wall prolapse, also known as pelvic organ prolapse (POP) is the monofilament polypropylene mesh (PLP). However, complications related to tissue integration of these implants are relatively prevalent. Collagen is the main structural protein of mammals, which modulates inflammatory process, and can be used as an important healing agent and, accordingly, could improve the integration of the meshes. Objetives: To study the biomechanical and histological effects of PLP, coated with purified collagen gel, implanted in the subcutaneous tissue of adult rats. Methods: Twenty rats were used for histological study and other 20 for the biomechanical study. At one side of the abdominal wall, PLP fragment measuring 20x10 mm was implanted (Group I), and in the other side a mesh fragment with the same dimensions coated with purified collagen gel PLP+C) was implanted (Group II). According to the time of euthanasia (7, 30, 90 or 180 days) after implantation the animals from each group were divided into four subgroups of five animals each. The biomechanical study was performed with a precision tension meter with in bloc fresh abdominal wall sample containing the mesh. The mesh was pulled up in uniaxial direction until complete detachment of the tissue interface and the maximum load required for the detachment was analyzed in each group. In the histological study, it was examined characteristics of the acute and chronic inflammatory reaction, granulation tissue, granuloma formation, and foreign body reaction on slides stained with hematoxylin-eosin. The supramolecular organization of the collagen deposition around the meshes was studied with polarizing microscopy (birefringence analysis). Results: In biomechanical study it was observed that the adhesion of PLP to surrounding tissues increased significantly after coating with purified collagen gel, as shown in the analysis of the maximum load at the 7th (p=0.0016), 14th (p=0.0039), 90th (p=0.0009) and 180th (p=0.0029) days after implantation. It was considered in this research, that increased biocompatibility of PLP would be achieved when, in tissue interface, there was a reduction of the intensity of the inflammatory process. It was found that the acute and chronic inflammatory response, as well as the formation of granulation tissue were less intense, respectively p=0.004, p<0.001 and p=0.001 for PLP+C in the initial phase (7th and 14th days) and missing, as well as on the meshes not covered in late phase (90th and 180th days). Granulomatous inflammation was less significant seven days after implantation in animals of Group II (p=0.029) and in both groups, similarly, decreased over time showing no significant difference. The foreign body reaction was less intense in the initial phase in Group II (p<0.001) and similar between the groups in the late phase. In the birefringence analyses, it was noted a greater average density of brightness (transmittance) in PLP (p=0.000) in the initial period (seven days), but in other periods it was greater for PLP+C [14 days (p=0.000); 90 days (p=0.000) and 180 days (p=0.000)]. Conclusions: Coating of polypropylene meshes with purified collagen gel increased their adhesion to the tissues, when implanted in the subcutaneous of the abdominal wall of adult rats and promoted a less intense and lasting inflammatory response and, in the late stage of the implant, triggered greater organization and packaging of collagen fibers. Based on that, one can suppose that mesh coated with purified collagen gel can fix better to the host tissues preventing its displacing, and can elicit fewer integration defects, resulting in less chance of complications / Doutorado / Fisiopatologia Cirúrgica / Doutor em Ciências

Abstract Collagen prolyl 4-hydroxylases catalyze the hydroxylation of proline residues in collagens. The vertebrate enzymes are α2β2 tetramers in which the β subunit is identical to protein disulphide isomerase (PDI). Two isoforms of the catalytic α subunit have been identified in vertebrates, forming type I [α(I)]2β2 and type II [α(II)]2β2 collagen prolyl 4-hydroxylase tetramers. This thesis reports on the cloning and characterization of a third vertebrate α subunit isoform, α(III). The recombinant human α(III) isoform associates with PDI to form an active type III collagen prolyl 4-hydroxylase tetramer, and its Km values for the cosubstrates are very similar to those of the type I and II enzymes, those for a peptide substrate and an inhibitor being found to lie between the two. The α(III) mRNA is expressed in all tissues studied but at much lower levels than the α(I) mRNA. A novel mixed tetramer PHY-1/PHY-2/(PDI-2)2 was found to be the main collagen prolyl 4-hydroxylase form produced in the nematode Caenorhabditis elegans in vivo and in vitro. However, mutant nematodes can compensate for the lack of the mixed tetramer by increasing the assembly of PHY-1/PDI-2 and PHY-2/PDI-2 dimers, these forms also being unique. The catalytic properties of the recombinant mixed tetramer were characterized, and it was shown by the analysis of mutant worms that PHY-1 and PHY-2 represent the only catalytic subunits needed for the hydroxylation of cuticular collagens. The roles of the two catalytic sites in a collagen prolyl 4-hydroxylase tetramer were studied by using the C. elegans mixed tetramer and a hybrid C. elegans PHY-1/human PDI dimer. An increase in the chain length of the peptide substrate led to an identical decrease in the Km values in both enzyme forms. It is thus clear that two catalytic sites are not required for efficient hydroxylation of long peptides, and their low Km values most probably result from more effective binding to the peptide-substrate-binding domain. Inactivation of one catalytic site in the mixed tetramer reduced the activity by more than 50%, indicating that the remaining wild-type subunit cannot function fully independently.

Abstract Extraintestinal manifestations in inflammatory bowel disease (IBD) have been described with varying frequencies. The aim of this study was to estimate the prevalence of pancreatic duct abnormalities, exocrine and endocrine dysfunction, elevated pancreatic enzymes, hepato-biliary disease, coexisting cholangiographic and pancreatographic duct changes, and elevated serum levels of fibrosis markers in IBD, and to correlate the findings with clinical, endoscopic and histologic variables. From a local patient register, 237 patients were randomly selected and studied. Of these, 170 had ulcerative colitis (UC), 46 had Crohn's disease (CD), and 21 had indeterminate colitis (IC). A detailed history was obtained from medical records and in a face-to-face interview. The patients were screened with a para-aminobenzoic acid (PABA) test and, for pancreatic enzymes, liver function tests, serum aminoterminal propeptide of type III procollagen (PIIINP), and laminin. Further pancreatic evaluation included endoscopic retrograde cholangiopancreato-graphy (ERCP), ultrasound (US), secretin test, and glucagon C-peptide test. Further hepatobiliary evaluation consisted of ERCP, US, and liver biopsy. In IBD, the prevalence rates of pancreatic duct abnormalities and exocrine dysfunction were 8% and 4%, respectively. Parallel impairment of exocrine and endocrine functions was shown. Acute idiopathic pancreatitis may complicate IBD. About 7-17% presented with elevated pancreatic enzymes. Enzyme elevation was associated with extensive and histologically active disease and, in some cases, with primary sclerosing cholangitis (PSC). Abnormal liver test results were commoner in patients with CD than in patients with UC (30% versus 11%). The prevalence of PSC in IBD was 11%, which is higher than previously reported (3.7-7.5%). PSC was commoner in patients with CD than in patients with UC (17.4% versus 7.6%). About half of the PSC patients had concomitant pancreatic duct changes, and the prevalence of concurrent cholangiographic and pancreatographic duct changes in IBD was 4.6%. Both serum PIIINP and laminin were increased in IBD patients. This was not only seen in patients with hepatobiliary disease and PSC, but also in patients with pancreatic disease. In conclusion, pancreatic and hepatobiliary complications in IBD occur with high and similar frequencies in all IBD categories and are associated with each other. They are not clearly associated with the clinical course of IBD.

Abstract Type XIII collagen belongs to the group of transmembrane collagens. In this thesis the plasma membrane localisation and function of type XIII collagen have been studied using cell biological methods. Type XIII collagen was found to reside in focal adhesions. It appeared in these structures at a very early stage of their assembly and disappeared from them concurrently with focal adhesion proteins talin and vinculin. Insect cells expressing type XIII collagen showed an enhanced adhesion to certain matrix components. These localisation and adhesion data suggested that the function of type XIII collagen is related to cell adhesion. Supporting this, in tissues type XIII collagen was found to localise to cell-matrix and cell-cell adhesion structures. Type XIII collagen was found to be partly present in cholesterol-enriched membrane microdomains. With other membrane proteins this localisation has been shown to be linked to ectodomain shedding. The connection between the membrane microdomain localisation and the ectodomain shedding of type XIII collagen was also characterised, and it was demonstrated that manipulation of the cellular cholesterol level affected the efficiency of the ectodomain shedding. Additionally, insights into intracellular shedding of type XIII collagen in the Golgi apparatus were obtained. The study of type XIII collagen expression in human cancers revealed that it was enhanced especially in the desmoplastic cancer stroma. Since the increased expression of type XIII collagen was detected during the dysplastic stages, type XIII collagen may be involved in the early pathogenesis of cancer. The result indicated that type XIII collagen is involved in the matrix remodelling. In support of this, the cell culture experiments showed that the soluble type XIII collagen ectodomain altered the vitronectin-rich matrix unfavourable for cell adhesion and spreading. This may enhance cancer metastasis. Type XIII collagen expression was also induced in the remodelled stroma of keratoconus and corneal wounds. Data suggested that myofibroblasts were responsible for the increased expression of type XIII collagen in these situations. Therefore both in cancer and in the corneal pathologies studied, type XIII collagen expression was induced by the activated stromal cells.

Abstract Genetic factors have been implicated to play a role in both degenerative lumbar spinal stenosis (LSS) and intervertebral disc disease (IDD). Sequence variations in the genes coding for collagen IX and inflammatory mediators have been indicated as risk factors for IDD. Nine genes coding for intervertebral disc (IVD) collagens I, II, IX and XI and aggrecan (AGC1) were analyzed for sequence variations in 29 Finnish individuals with LSS. In addition, two polymorphisms in the vitamin D receptor gene and one in the matrix metalloproteinase-3 gene were studied. Study subjects were analyzed both clinically and radiologically. Results indicated an association between the COL11A2 IVS6-4 a to t polymorphism and LSS (p = 0.0016). Moreover, the t/t genotype was found more often in the patient group compared to controls (p = 0.0011). A novel splicing mutation, likely resulting in the synthesis of a truncated protein, was identified in COL9A2. Eight hundred four Chinese individuals were screened for the presence of the Trp2 and Trp3 alleles. The Trp2 allele was found in 20% of the individuals compared to the previously reported 5% in Finnish patients with IDD characterized by sciatica. The Trp2 allele was found to predispose to IVD degeneration and end plate herniations, increasing the risk by 2.4-fold from 40 to 49 years of age. In addition, the degeneration was worse in individuals with the Trp2 allele. The risk for annular tears was 4-fold greater in study subjects from 30 to 39 years of age who were Trp2 positive. Surprisingly, the Trp3 allele was absent even though it was found in about 9% of Finnish individuals. One hundred fifty-five Finnish individuals with IDD characterized by sciatica were analyzed for sequence variations in four genes coding for inflammatory mediators IL1A, IL1B, IL6, and TNFA. In addition, sixteen polymorphisms in inflammatory mediator genes were analyzed. The results identified an association between sciatica and the E5+15T>A polymorphism in IL6 (p = 0.007). A significant association was also seen in the IL6 haplotype analysis (-597 g>a, -572 g>c, -174 g>c and E5+15T>A). The association of the GGGA haplotype with the disease was highly significant (p = 0.0033).

Chronic wounds represent a severe socio-economic burden and a key area of unmet clinical need. PKCα is ubiquitous in the skin, particularly the epidermis and functions in numerous pathways that are fundamental to wound repair. By utilising a global PKCα-/- mouse we have identified PKCα-regulated processes both in unwounded skin and during wound healing. PKCα-/- mice display considerably delayed wound healing with a dramatic reduction in re-epithelialisation. By analysing the ultrastructure of the epidermis, I have shown that this delay directly correlates with a failure of wound edge desmosomes to switch to a their adhesive properties. A major risk factor for the development of chronic wounds is age. Crucially, this delay in modulating cell adhesion is conserved in human chronic wounds and aged murine skin. Furthermore, manipulation of PKCα using an inducible bitransgenic mouse containing epidermal specific constitutively active PKCα can accelerate the modulation of desmosome adhesion and subsequently improve re-epithelialisation. Global gene expression analysis of PKCα-/- skin and wounds revealed further defects. Upon wounding, we observed a failure to correctly regulate expression of key collagen and Wnt signalling genes that are essential for correct and timely wound healing. Finally, intrinsic gene expression changes were identified in the skin of PKCα-/- mice, specifically a downregulation of multiple extracellular matrix genes. Of note was the downregulation of small leucine-rich proteoglycans which led to alterations to dermal collagen structure and skin tensile strength. These changes render the PKCα-/- skin susceptible to breaking and wound development. To conclude, we have identified multiple roles for PKCα intrinsically in the skin and also during cutaneous wound healing. Importantly, these intrinsic changes appear to predispose PKCα-/- skin to the development of cutaneous wounds and altered wound-specific processes that manifest in a delayed healing phenotype.

Abstract Type XIII collagen is an integral membrane protein in type II orientation. In cells and tissues type XIII collagen has been located in various adhesive structures, like focal adhesions. Due to this, its biological role has been implicated in cell adhesion. This collagen also exists as a soluble protein due to the release of the ectodomain from the plasma membrane. In this thesis, ectodomain shedding, i.e. enzymatic release of the extracellular domain, was studied in detail, focusing on the phenomenon as it occurs in mammalian cells. It was found that the ectodomain is released by members of the mammalian proprotein convertase family, e.g. furin. Shedding was shown to take place at the cell surface, but based on additional observations, this cleavage may also take place intracellularly in the Golgi apparatus. Various intracellular mechanisms, depending on cell type, were found to be involved in the regulation of ectodomain shedding. Apparently, due to the liberation of the ectodomain, the level of type XIII collagen on the plasma membrane is maintained at a relatively even amount. The released ectodomain was shown to retain biological activity. It showed distinct matrix-specificity so that on vitronectin its influence on cell functions was anti-adhesive, anti-migratory, anti-proliferative and non-supportive of cell spreading. It was also demonstrated to affect the fibronectin matrix assembly in a manner that resulted in reduced amounts of the fibrillar fibronectin matrix. A large collection of human epithelial and mesenchymal cancer samples were screened for type XIII collagen mRNA expression and compared to the expression levels of pre-malignant and normal samples. It was discovered that malignant transformation upregulates the expression of type XIII collagen in mesenchymal cancers and particularly in the stroma of epithelial cancers, more so than in cancer epithelia. TGF-β1 was demonstrated as one factor contributing to the stimulation of expression. Based on cell culture experiments in this study, it was also deduced that the upregulated expression of type XIII collagen and the concomitant shedding of the ectodomain can remodel the tumour stroma, making it inauspicious for adhesion-dependent cell functions, particularly in vitronectin-rich milieu.

Abstract Rheumatoid arthritis (RA) is a systemic autoimmune disease with symmetrical articular manifestations. The etiology of the disease is unknown. The prevalence of RA is approximately 0.5–1.0% in adults. In Finland, the annual incidence is 39/100 000. RA is about three times more common in females than in males. Most commonly the disease affects first the joints of feet and fingers. Chronic inflammation leads to erosions of cartilage, bone and tendons and may destroy the whole joint. The diagnosis of RA is mainly based on the clinical features of the disease. The American College of Rheumatology (ACR) 1987 revised classification criteria of RA have commonly been used for diagnosis. No specific diagnostic test is available. Rheumatoid factor (RF) has traditionally been used in the diagnosis, but only 70 to 80% of RA patients have RF in their serum. Other antibodies found in RA are the antiperinuclear factor (APF), the anti-keratin antibody (AKA) and the antibodies to cyclic citrullinated peptide (CCP), which recognize the citrulline-containing antigenic filaggrin protein. Citrulline is an amino acid that is post-translationally formed from arginine by peptidylarginine deiminase enzymes (PADs). Autoantibodies to citrullinated proteins are more specific for RA than RF. There is no filaggrin in joints, which indicates that the autoantibodies reacting with this protein most probably only reflect immunological cross-reaction. It has been postulated that autoimmunity against collagens might be involved in the pathogenesis of RA. There are antibodies binding to collagen in cartilage (type II collagen) and in bone (type I collagen). They have been tested by using collagen preparations rendered soluble by pepsin digestion. This digestion removes the carboxyterminal (C-terminal) telopeptides of collagen. Autoantibodies to the C-telopeptides of type I and II collagens were studied in this doctoral research. Autoantibodies to the citrullinated C-telopeptides of type I and II collagens were found in the serum of patients with RA. ELISA, CLIA and inhibition ELISA were used to detect these autoantibodies. Automatic CLIA gives a more than twofold number of positive findings compared to previous ELISA. Currently the best method for the detection of these autoantibodies is inhibition ELISA. These autoantibodies are specific for citrulline in the peptide sequence. Autoantibodies that bind the normal C-telopeptides of type I and II collagens were not inhibited by soluble normal or citrullinated telopeptides. However, the antibodies that bind only citrullinated telopeptides could be inhibited by corresponding citrullinated telopeptides. Autoantibodies binding the citrullinated telopeptides of type II collagen and anti-CCP predict synergistically the development of seropositive RA. / Tiivistelmä Nivelreuma (arthritis rheumatoides) on krooninen autoimmuunisairaus, jonka aiheuttajaa ei tunneta. Nivelreuman esiintyvyys aikuisilla on 0.5–1.0 prosenttia. Siihen sairastuu vuosittain 39/100 000 suomalaista aikuista. Naiset sairastavat nivelreumaa kolme kertaa yleisemmin kuin miehet. Sairaus alkaa tavallisesti päkiöistä ja sormien nivelistä. Nivelreuma aiheuttaa ruston, luun ja nivelsiteiden syöpymistä ja voi lopulta tuhota koko nivelen. Nivelreuman diagnoosi perustuu pääasiassa taudin kliinisiin piirteisiin. Yhdysvaltain reumajärjestön (American College of Rheumatology, ACR) vuonna 1987 esittämät luokittelukriteerit ovat yleisesti käytössä. Taudin toteamiseen ei ole spesifistä laboratoriotestiä. Perinteisesti käytettyä reumatekijää esiintyy 70–80 prosentilla potilaista. Muita nivelreumapotilaan seerumista esiintyviä vasta-aineita ovat antiperinukleriaaritekijä (APF), keratiinivasta-aineet (AKA) ja vasta-aineet sykliseen sitrullinisoituneeseen peptidiin (CCP). Sitrulliini on aminohappo, jonka peptidyyliarginiini deiminaasi -entsyymit (PAD) ovat muokanneet arginiinista posttranslationaalisesti. Sitrullinisoituneiden proteiinien autovasta-aineet ovat spesifisempiä nivelreumassa kuin reumatekijä. Nivelissä ei ole filaggriinia, mikä viittaa siihen, että todetut vasta-aineet perustuvat immunologiseen ristireaktioon. On epäilty, että nivelreuman patogeneesiin voisi liittyä autoimmuniteettia kollageeniin. Aiemmin tutkitut luun ja ruston (tyypin I ja II) kollageenivasta-aineet eivät ole olleet sitrullinisoituneita. Autovasta-ainetesteissä on käytetty pepsiinidigestiota, jolla kollageeni on saatu liukoiseksi. Pepsiinidigestio tuhoaa kuitenkin kollageenin karboksyyliterminaaliset (C-terminal) telopeptidit. Tässä väitöskirjatutkimuksessa tutkittiin tyypin I ja II kollageenien C-telopeptidejä. Nivelreumapotilaiden seerumissa todettiin autovasta-aineita, jotka sitoutuvat sitrullinisoituneisiin tyypin I ja II kollageeneihin. Todettuja vasta-aineita voidaan osoittaa ELISA-, CLIA- ja inhibitio-ELISA-menetelmillä. Automaattisella CLIA:lla saadaan kaksi kertaa enemmän positiivisia löydöksiä kuin aiemmin kehitetyllä ELISA:lla. Tällä hetkellä paras menetelmä näiden autovasta-aineiden osoittamiseen on inhibitio-ELISA. Todetut vasta-aineet ovat spesifisiä peptidin sekvenssissä olevaan sitrulliiniin. Vasta-aineita, jotka sitoutuvat normaaliin tyypin I ja II kollageenien C-telopeptidiin, ei voida inhiboida liukoisella normaalilla eikä sitrullinoidulla telopeptidillä. Kuitenkin vasta-aineita, jotka sitoutuvat vain sitrullinisoituihin telopeptideihin, voidaan inhiboida vastaavalla liukoisella sitrullinisoidulla telopeptidillä. Autovasta-aineet, jotka sitoutuvat samanaikaisesti sitrullinisoituneeseen tyypin II kollageenin C-telopeptidiin ja anti-CCP:hen ennustavat seropositiivista nivelreumaa.