Synovial sarcoma is a soft tissue tumor defined by the presence of t(X;18)(p11.2;q11.2), fusing the SYT (SS18) gene on chromosome 18 and one of three SSX genes on chromosome X. T(X;18) results in production of a fusion protein (SYT-SSX) that is thought to underlie synovial sarcoma pathogenesis through aberrant targeting of both activating (trithorax, SWI/SNF) and repressing (Polycomb) transcription factors when expressed in a stem or progenitor-like cellular background. Clinically, synovial sarcomas present considerable diagnostic and therapeutic challenges. Whereas the classical biphasic histology is distinctive, the more common monophasic histology can be difficult to differentiate from other spindle cell tumors. In these situations, detection of t(X;18) is the gold standard for diagnosis, but it is a specialized and time-consuming process. Immunohistochemistry can be helpful, but no marker that is both highly sensitive and specific is available. Here I describe a fluorescence in situ hybridization based method employing an SYT break-apart probe set that can expedite detection of t(X;18). I also report that TLE1, which was identified in gene expression studies as a good discriminator of synovial sarcoma from other mesenchymal tumors, is a highly sensitive and specific immunohistochemical marker for synovial sarcoma. Both of these novel diagnostic techniques are applicable to small tissue samples such as core needle biopsies and are now being used clinically. The diagnosis of synovial sarcoma carries a poor prognosis and the 10-year overall survival rate is approximately 50%, most of whom are young adults. The addition of chemotherapy to surgical resection (the mainstay of treatment) does not appear to improve overall survival. Thus, there is a strong need for development of a clinically effective systemic therapy to improve patient outcome. I describe preclinical studies that demonstrate the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) inhibits proliferation of synovial sarcoma by inducing apoptosis and that this is associated with degradation of multiple receptor tyrosine kinases and disruption of the SYT-SSX-β-catenin interaction. I also identify a subset of synovial sarcoma cells, typified by expression of CD133, which exhibit stem-like properties and are relatively resistant to doxorubicin but susceptible to 17-AAG at clinically relevant concentrations.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / Typescript. Vita. Includes bibliographical references. Also issued on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / "May 2004." Typescript. Vita. Includes bibliographical references. Also issued on the Internet.

Synovial sarcoma is a soft tissue tumor defined by the presence of t(X;18)(p11.2;q11.2), fusing the SYT (SS18) gene on chromosome 18 and one of three SSX genes on chromosome X. T(X;18) results in production of a fusion protein (SYT-SSX) that is thought to underlie synovial sarcoma pathogenesis through aberrant targeting of both activating (trithorax, SWI/SNF) and repressing (Polycomb) transcription factors when expressed in a stem or progenitor-like cellular background. Clinically, synovial sarcomas present considerable diagnostic and therapeutic challenges. Whereas the classical biphasic histology is distinctive, the more common monophasic histology can be difficult to differentiate from other spindle cell tumors. In these situations, detection of t(X;18) is the gold standard for diagnosis, but it is a specialized and time-consuming process. Immunohistochemistry can be helpful, but no marker that is both highly sensitive and specific is available. Here I describe a fluorescence in situ hybridization based method employing an SYT break-apart probe set that can expedite detection of t(X;18). I also report that TLE1, which was identified in gene expression studies as a good discriminator of synovial sarcoma from other mesenchymal tumors, is a highly sensitive and specific immunohistochemical marker for synovial sarcoma. Both of these novel diagnostic techniques are applicable to small tissue samples such as core needle biopsies and are now being used clinically. The diagnosis of synovial sarcoma carries a poor prognosis and the 10-year overall survival rate is approximately 50%, most of whom are young adults. The addition of chemotherapy to surgical resection (the mainstay of treatment) does not appear to improve overall survival. Thus, there is a strong need for development of a clinically effective systemic therapy to improve patient outcome. I describe preclinical studies that demonstrate the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) inhibits proliferation of synovial sarcoma by inducing apoptosis and that this is associated with degradation of multiple receptor tyrosine kinases and disruption of the SYT-SSX-β-catenin interaction. I also identify a subset of synovial sarcoma cells, typified by expression of CD133, which exhibit stem-like properties and are relatively resistant to doxorubicin but susceptible to 17-AAG at clinically relevant concentrations. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

A cartilagem articular é um tecido avascular, com baixa celularidade, composta principalmente de colágeno extracelular e proteoglicanos, com uma capacidade limitada de regeneração. Nos últimos anos, diversas abordagens clínicas e de pesquisa têm sido adotadas para reparar danos na cartilagem articular, como transplante de condrócitos, enxerto de periósteo, células-tronco mesenquimais e tecidos derivados dessas células. O isolamento de células-tronco mesenquimais foi relatado a partir de diferentes tecidos, incluindo medula óssea, tecido adiposo, sangue do cordão umbilical, sangue periférico e saco vitelino em equinos. As células-tronco mesenquimais derivadas de líquido e membrana sinoviais foram obtidas em humanos, cães, suínos e caprinos e são fontes promissoras para regeneração articular, já que são tecido-específicas e de fácil obtenção e cultivo. O objetivo deste trabalho foi estabelecer e caracterizar a linhagens de células obtidas de membrana e líquido sinoviais equinos. Os fragmentos foram obtidos por meio de artroscopias e cultivados em meios de cultura DMEM-H e MEM para obtenção das linhagens. Para análise da morfologia celular foi realizada a fotodocumentação das garrafas em microscopia invertida. A expressão de marcadores de células-tronco (CD45RO, OCT3/4, NANOG, CD105, CD90, CD34, CD117, CD133, TRA-1-81, VEGF-R1 e Ly6a), marcadores inflamatórios (COX-2, TNF-R1-r, CD11, CD1a e MCP-1) e marcadores envolvidos na checagem e progressão do ciclo celular (Caspase-3 fosforilada, HSP-47, P21, Ki67, Ciclina D1 e P53) mostraram diferencialmente expressos, sugerindo que podemos considerá-las uma possível fonte de células-tronco mesenquimais. Devido ao grande impacto que patologias na articulação têm sobre o desempenho atlético em cavalos, os resultados demonstrados neste trabalho será uma base para conduzir outros experimentos na avaliação das aplicações terapêuticas dessas células. / The articular cartilage is an avascular tissue with low cellularity composed of extracellular collagen and proteoglicans. It has a limited capacity of regeneration. Condrocyte transplantation, mesenchymal stem cells and tissues derived from these cells has been used by several researches to repair damage to the articular cartilage. The isolation of mesenchymal stem cells has been reported from different tissues such as bone marrow, adipose tissue, blood, umbilical cord, and yolk sac. The mesenchymal stem cells from synovial fluid and synovial membrane were obtained from humans, dogs, pigs and goats. These cells are tissue specific and easy to obtain and cultivate. The objective of this research is to obtain and characterize cells from equine synovial fluid and synovial membrane. The samples were obtained by arthroscopy and cultivated in the DMEM-H and MEM media. Cell morphology analyses were made by photodocumentation in inverted microscopy. The expression of stem cell markers (CD45RO, OCT3/4, NANOG, CD105, CD90, CD34, CD117, CD133, TRA-1-81, VEGF-R1 and Ly6a), inflammation markers (COX-2, TNF-R1-r, CD11, CD1a and MCP-1) and markers involved in checking and cell cycle progression (Caspase-3, HSP-47, P21, Ki67, Ciclina D1 and P53) showed differentially expressed. Mesenchymal stem cells from synovial membrane and synovial fluid provide promise for cell-based therapies for articular cartilage repair. These results may lead other experiments to use these cells to new therapeutic applications.

Akademisk avhandling--Lund. / Extra t.p., with thesis statement, inserted. Errata slip inserted. Bibliography: p. [117]-125.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

A cartilagem articular é um tecido avascular, com baixa celularidade, composta principalmente de colágeno extracelular e proteoglicanos, com uma capacidade limitada de regeneração. Nos últimos anos, diversas abordagens clínicas e de pesquisa têm sido adotadas para reparar danos na cartilagem articular, como transplante de condrócitos, enxerto de periósteo, células-tronco mesenquimais e tecidos derivados dessas células. O isolamento de células-tronco mesenquimais foi relatado a partir de diferentes tecidos, incluindo medula óssea, tecido adiposo, sangue do cordão umbilical, sangue periférico e saco vitelino em equinos. As células-tronco mesenquimais derivadas de líquido e membrana sinoviais foram obtidas em humanos, cães, suínos e caprinos e são fontes promissoras para regeneração articular, já que são tecido-específicas e de fácil obtenção e cultivo. O objetivo deste trabalho foi estabelecer e caracterizar a linhagens de células obtidas de membrana e líquido sinoviais equinos. Os fragmentos foram obtidos por meio de artroscopias e cultivados em meios de cultura DMEM-H e MEM para obtenção das linhagens. Para análise da morfologia celular foi realizada a fotodocumentação das garrafas em microscopia invertida. A expressão de marcadores de células-tronco (CD45RO, OCT3/4, NANOG, CD105, CD90, CD34, CD117, CD133, TRA-1-81, VEGF-R1 e Ly6a), marcadores inflamatórios (COX-2, TNF-R1-r, CD11, CD1a e MCP-1) e marcadores envolvidos na checagem e progressão do ciclo celular (Caspase-3 fosforilada, HSP-47, P21, Ki67, Ciclina D1 e P53) mostraram diferencialmente expressos, sugerindo que podemos considerá-las uma possível fonte de células-tronco mesenquimais. Devido ao grande impacto que patologias na articulação têm sobre o desempenho atlético em cavalos, os resultados demonstrados neste trabalho será uma base para conduzir outros experimentos na avaliação das aplicações terapêuticas dessas células. / The articular cartilage is an avascular tissue with low cellularity composed of extracellular collagen and proteoglicans. It has a limited capacity of regeneration. Condrocyte transplantation, mesenchymal stem cells and tissues derived from these cells has been used by several researches to repair damage to the articular cartilage. The isolation of mesenchymal stem cells has been reported from different tissues such as bone marrow, adipose tissue, blood, umbilical cord, and yolk sac. The mesenchymal stem cells from synovial fluid and synovial membrane were obtained from humans, dogs, pigs and goats. These cells are tissue specific and easy to obtain and cultivate. The objective of this research is to obtain and characterize cells from equine synovial fluid and synovial membrane. The samples were obtained by arthroscopy and cultivated in the DMEM-H and MEM media. Cell morphology analyses were made by photodocumentation in inverted microscopy. The expression of stem cell markers (CD45RO, OCT3/4, NANOG, CD105, CD90, CD34, CD117, CD133, TRA-1-81, VEGF-R1 and Ly6a), inflammation markers (COX-2, TNF-R1-r, CD11, CD1a and MCP-1) and markers involved in checking and cell cycle progression (Caspase-3, HSP-47, P21, Ki67, Ciclina D1 and P53) showed differentially expressed. Mesenchymal stem cells from synovial membrane and synovial fluid provide promise for cell-based therapies for articular cartilage repair. These results may lead other experiments to use these cells to new therapeutic applications.

A atividade física, dependendo da intensidade e duração, provoca uma resposta inflamatória nos tecidos articulares dos eqüinos. Como ocorre nos cavalos de pólo que percorrem grandes distâncias em alta velocidade com paradas bruscas do movimento, submetendo suas articulações a esforços intensos e constantes. A finalidade deste trabalho foi avaliar os efeitos da atividade esportiva no metabolismo articular, por meio de exame físico, das dosagens de mediador inflamatório (PGE2) e citocinas (IL-1 e TNF-&alpha;), da concentração de glicose, proteína total e da análise quantitativa e qualitativa das células presentes no líquido sinovial de 20 eqüinos atletas em três estágios diferentes de treinamento, a cada 30 dias, durante o período de 360 dias: animais sem treinamento (grupo controle), animais em início de treinamento (G1) e animais com mais de 5 anos de treinamento (G2). Além disso, estabelecer uma relação entre os resultados laboratoriais com os exames radiográficos e ultra-sonográficos articulares. Numa segunda fase do experimento foi avaliado o líquido sinovial de oito eqüinos atletas antes do exercício e 3, 6 e 24 horas após o término deste. As principais alterações foram encontradas nos animais do G2, ou seja, aumento da circunferência articular (P<0,05), mudança da coloração do líquido sinovial (P<0,05), maior volume de líquido sinovial obtido das articulações metacarpofalangeanas (P<0,05), aumento nas concentrações de proteína (P<0,05), glicose (P<0,05) e PGE2 (P<0,05). Concomitantemente, estes animais apresentaram aumento de volume dos tecidos moles periarticulares, osteófitos, esclerose óssea e diminuição da flexão articular (P<0,05) nos exames radiográficos; além de maior espessura e menor homogeneidade da cápsula articular fibrosa, e maior quantidade de líquido articular visualizado nos exames ultra-sonográficos (P<0,05). Já a expressão dos receptores de TNF do tipo II foi maior nas células do líquido sinovial dos animais do G1. Os animais dos grupos 1 e 2 apresentaram maior porcentagem de macrófagos e sinoviócitos (P<0,05), do que os animais do grupo controle. Na segunda fase do experimento, foram observadas às 3 e 6 horas mudanças na coloração do líquido sinovial (P<0,05), diminuição no volume obtido (P<0,05), diminuição da concentração de glicose (P<0,05), aumento na concentração de proteína total (P<0,05), aumento na concentração de PGE2 (P<0,05). As células do líquido sinovial apresentaram intensidade acentuada de marcação para os receptores de TNF do tipo II também após 3 horas do término do exercício. Conclui-se que um processo inflamatório articular é desencadeado após o exercício intenso, e a resposta dos tecidos articulares frente a este insulto mecânico, com maior intensidade 3 horas após o término da atividade esportiva, e retornando aos valores basais 24 horas após, revela a excelente adaptação articular ao estresse físico. Esta adaptação também foi observada a longo prazo, em cavalos com maior tempo de carreira esportiva, ou seja, estes animais podem apresentar alterações articulares sem sinais de dor e claudicação. Contudo, isto não impede que cavalos com carreira atlética longa desenvolvam osteoartrite. / Physical activity causes an inflammatory response in joints depending on intensity and duration of the exercise. Polo ponies, for example, cross large distances at high speed, with abrupt stops of movement, subjecting the joints to intense and constant effort. The purpose of this study was to evaluate the effects of the sporting activity on joint metabolism, by means of physical examination, measurements of inflammatory mediator (PGE2) and cytokines (IL-1 and TNF-&alpha;), glucose and total protein concentrations, and quantitative and qualitative analyses of synovial fluid cells of 20 athletic horses on 3 different stages of training, every 30 days, during a period of 360 days: animals without training (control group), animals in the beginning to train (G1), and animals with more than five years of training (G2). Moreover, the study aimed to establish a relationship between laboratorial results and radiographic and ultrasonographic exams. In a second phase of the trial, synovial fluid analysis was done from samples of eight athletic horses before, and 3, 6 and 24 hours after exercise. Main alterations were found in horses of group 2, which were increase in joint circumference (P<0.05), alteration of the synovial fluid color (P<0.05), greater synovial fluid volume, obtained from metacarpophalangeal joints (P<0.05), greater protein (P<0.05), glucose (P<0.05), and PGE2 (P<0.05) concentrations. Concomitantly, these animals showed an increase of the periarticular soft tissues, ostephytes, subchondral bone sclerosis, and a diminished joint flexion in radiographic exams (P<0.05), and also a thick and less homogeneous articular capsule, and an increase of the joint fluid quantity in ultrasonographic exams (P<0.05). The TNF type II receptor expression was greater in synovial fluid cells of group 1 horses. Horses of the groups 1 and 2 showed greater percentage of macrophages and synoviocytes (P<0.05) than the animals of the control group. In the second phase of the study, it was observed a change of the synovial fluid color at 3 and 6 hours post-exercise (P<0.05), reduction of the obtained volume (P<0.05) and glucose concentration (P<0.05), and an increase of total protein (P<0.05) and PGE2 (P<0.05) concentrations. Also, the synovial fluid cells showed an accentuated intensity of markers for TNF type II receptor 3 hours after sports activity was finished. An inflammatory process of the joint is triggered after intense exercise, but the response of the articular tissue facing this mechanical insult, more intense within 3 hours after the end of the sports activity, and the return to basal values after 24 hours, reveal the excellent adaptation of the articular tissue to physical stress. This adaptation can also be observed in animals with more time of sports career, which can have articular changes without signs of pain or lameness. Nevertheless, this doesn\'t prevent horses with longer sports careers to develop osteoarthritis.