In this study, we show that removal of a quorum sensing subtype of stromal macrophage expands the support capacity of ex vivo bone marrow culture. Notably, this system maintains much of the remaining paracrine signaling of the organ, unlike traditional macrophage ablation or cytokine supplemented media and does not place undue stress on the HSPC itself. Recent studies have independently identified alternatively activated macrophages that suppress hematopoiesis in in vitro culture. We have identified for the first time, a small molecule capable of preferentially killing those cells, thus providing a method to both culture unaltered HSPC ex vivo for long periods of time and significantly expand transient progenitor cells to assist transplantation efficiency. Our culture system in unique in its ability to maintain cultured HSPC in the physiological micro-environment of the bone marrow We found the small molecule “999” capable of expanding hematopoietic capacity of stroma culture by selectively eliminating an MHCII-Hi subpopulation of stromal macrophages that suppress HSPC growth. Removal of these macrophages enables long-term HSC ex vivo stability and massive expansion of the MPP and its progeny. Cultures expanded in this manner have increased engraftment potential and behave physiologically normal upon transplantation. This investigation has also helped to uncover the role of TGFB in bone marrow quiescence signaling. The MHCII-HI target cells express TGFB and through it, signal quiescence to the HSPC, likely as a form of quorum sensing. Targeted acute elimination of that signal leads to unabashed expansion of MPP. Furthermore, macrophage polarization in the tumor microenvironment has also been show to promote tumor formation and often leads to poor prognosis. Molecular tools such as 999 that have the ability to alter macrophage polarization ratios may prove to be valuable synergistic tools for oncologists in conjunction with current therapies.

Foi estudado experimentalmente o desempenho biológico de um tipo de enxerto ósseo homógeno, processado quimicamente, esterilizado em óxido de etileno e embebido em medula óssea autógena. De cães doadores adulto-jovens foram obtidos blocos cilíndricos de 1,0 x 1,0 cm da epífise distal do fêmur, com auxílio de uma trefina. Os ossos assim obtidos foram clareados, desengordurados, esterilizados em óxido de etileno e mantidos em estoque. Trinta cães adultos jovens foram usados como receptores do enxerto e foram alocados em dois grupos. No grupo I (experimental) os animais foram operados e, criado, transversalmente, com trefina de 1,0 cm de diâmetro externo, um espaço cilíndrico de 1,0 cm de diâmetro por 1,0 cm de altura na epífise distal do fêmur direito, onde foi encaixado o enxerto preparado e que foi previamente embebido em medula óssea do próprio animal retirada por punção óssea na crista ilíaca. Três semanas depois, o mesmo animal foi submetido ao mesmo procedimento cirúrgico no fêmur esquerdo. Os cães deste grupo foram sacrificados seis semanas após a primeira cirurgia. O grupo II constituiu o controle e foi formado por vinte cães adulto-jovens em que, primeiro, foi retirado um cilindro de osso da epífise distal do fêmur esquerdo e, em seguida, no mesmo tempo cirúrgico, foi criado o mesmo espaço no fêmur direito, com uma trefina de um centímetro de diâmetro interno e, então, encaixado o bloco de osso retirado do fêmur do outro lado do mesmo animal. Estes animais foram subdivididos em dois subgrupos de dez cães cada, em relação ao tempo de sacrifício. Em um subgrupo os cães foram sacrificados três semanas após, enquanto que, no outro subgrupo, foram sacrificados seis semanas após a cirurgia de implantação do enxerto. O processo de incorporação do enxerto foi avaliado pela histologia convencional e histologia de fluorescência óssea, pela injeção prévia de tetraciclina. A comparação foi entre os grupos I e II, nos períodos de três e seis semanas pós-implante. As áreas dos enxertos homógenos com 21 dias pós-implante, geralmente mostraram-se visíveis, na maioria das vezes, formadas por trabéculas finas irregulares, sem osteócitos, porém com áreas de neoformação óssea. Já os enxertos autógenos, a área do osso implantado era bem visível, com trabéculas de osso antigo, mais adelgaçadas, em menor número que o osso receptor, porém com intensa deposição de osso neoformado. Para os enxertos homógenos com 42 dias pós-implante, a área do enxerto estava bem definida e integrada ao osso adjacente, composta por trabéculas antigas com predomínio de osso neoformado sobre a superfície. Nos enxertos autógenos com 42 dias pós-implante, as trabéculas tinham orientação comum, com espaço intertrabecular preenchido por tecido conjuntivo denso, em algumas áreas, e por medula óssea madura em outras. O enxerto homógeno processado e esterilizado em óxido de etileno e embebido em medula óssea apresentou boa atividade biológica, embora, com integração mais lenta e menor desempenho em relação ao enxerto autógeno, o que o torna um bom substituto para este último. / In the present investigation the biological performance of the homogenous bone graft chemically processed, sterilized with ethylene oxide and soaked in autogenous bone marrow was investigated. Thirty dogs were assigned into two groups. In group I, ten young adult dogs received a cylindrical block of the above mentioned graft. Previously, the bone graft was immersed in autogenous bone marrow and then inserted in a cylindrical hole, with the same dimensions, created at the distal femoral epiphysis of the right femur. After three weeks the same animal was submitted to the same surgical procedure, but on the left femur. The animal was killed six weeks later. The group II was made up of twenty dogs that received an autogenous graft obtained from the left distal femur and implanted into the right distal femur (same shape, dimensions and technique). From this group, 10 dogs were killed three weeks later and 10 dogs were killed six weeks later. The graft incorporation was evaluated by light microscopy and fluorescence microscopy. Results showed that for 21 day implant in the homogenous group, there was osteogenesis at the periphery of the bone graft, but most of its trabeculae were still inviable. Conversely, for the autogenous grafts newly-formed bone was found on the trabecular surface, even in the inner parts of the implanted bone. At 42 days there was active osteogenesis in homogenous grafts with new bone deposition on the trabeculae but, in two cases, the graft was not incorporated (reabsorbed in one case and sequestred in the other one). The autogenous graft showed newly formed bone arranged in a well mature fashion with new bone marrow filling the intertrabecular gaps. It was concluded that the homogenous graft as prepared herein is a good alternative for autogenous grafts, although with a slower osteogenesis rate an less biological performance.

Em vista das limitações éticas em torno da obtenção de células-tronco de fetos humanos, o cão é uma alternativa para estes estudos. Além disso, a terapia celular proporciona novas expectativas para o tratamento na espécie. Realizamos o estudo comparativo das células isoladas de saco vitelino, fígado e medula óssea de fetos caninos. As células foram analisadas microscopicamente e ultra estruturalmente. O imunofenótipo das células foi avaliado através de marcadores. Caracterizamos a plasticidade, o cariótipo e o potencial teratogênico destas células. Após expansão as células progenitoras formaram colônias com morfologia fibroblastóide. As células progenitoras do saco vitelino e medula óssea são compostas por: células com alta proporção núcleo-citoplasma e células com citoplasma rico em organelas, enquanto que as células progenitoras do fígado eram semelhantes à célula epitelial e células ricas em organelas. As células-progenitoras dos três tecidos fetais foram positivas para os anticorpos nestina e vimentina, mas negativas para CD45 e CD13. Células progenitoras de medula óssea foram positivas para o marcador CD44. Células progenitoras do fígado e medula óssea expressaram a proteína citoqueratina-18, enquanto as do saco vitelino expressaram ve-caderina. Células positivas para Oct3/4 foram detectadas em todas as células progenitoras. As células-progenitoras do saco vitelino e medula óssea diferenciaram-se em tecidos ósseo, cartilaginoso e muscular; já as do fígado para tecido ósseo e muscular. Nenhum tipo celular diferenciou-se em adipócitos. As células progenitoras da medula óssea diferenciaram em células semelhantes a neurônios. Sugere-se a presença de progenitores semelhantes a células mesenquimais e epiteliais. Todas as células mantiveram o cariótipo estável e não formaram tumores. Células progenitoras de medula óssea apresentaram maior capacidade de proliferação e diversidade de diferenciação. Sugere-se que estas células são possíveis candidatas para a terapia celular. / The use the human fetuses for stem cells isolation have ethical limitations. In this context the dog is an excellent candidate to fetal stem cells. Furthermore, these cells can be used in cell therapy of canine diseases We aimed at isolation and comparative characterization of progenitor cells from yolk sac, liver and bone marrow of canine fetuses. Cells were characterized using stem cells antibodies. Differentiation assays as well as karyotype analysis were performed. Teratogenic properties this cells were evaluated. After establishment of primary culture, best proliferation potential was observed in bone marrow progenitor cells. Bone marrow and liver progenitor cells were more efficient in CFU-F assay, then yolk sac progenitor cells. Evidenced by TEM cells with a high nuclear-to-cytoplasmic ratio and cells with cytoplasm rich in organelles. Cells isolated from liver showed epithelial-like morphology and cytoplasm rich in organelles. The yolk sac, liver bone marrow cells reacted positively with nestin and vimentin, being negative to CD45 and CD13 antibodies. Additionally bone marrow progenitor cells were positive to CD44. Bone marrow and liver progenitor cells reacted positively with cytokeratin 18. Yolk sac progenitor cells were positive to ve-cadherin. A few Oct3/4 positive cells were found in yolk sac, liver and bone marrow. Yolk sac and bone marrow progenitor cells showed successful osteogenic, chondrogenic, myogenic differentiation. Differentiation liver progenitor cells were able to bone and muscle cells. The bone marrow progenitor cells were able to produce neuron-like cells. None of progenitor cells showed adipogenic differentiation. The study suggests the presence of mesenchymal-like and epithelial-like progenitor cells. All the karyotype remained and failed to induce the formation of tumors. Stem cells from bone marrow showed high diversity of differentiation than other cell types. It is suggested that these cells are possible candidates for cell therapy.

Estudos anteriores demonstraram que a crotoxina (CTX), o principal componente do veneno de Crotalus durissus terrifucus, apresenta ação anti-inflamatória, inibindo a migração celular e a atividade fagocítica de neutrófilos peritoneais. Esses efeitos inibitórios são prolongados, uma vez que podem ser observados até 14 dias após a administração de uma única dose dessa toxina. Considerando-se a vida média curta dos neutrófilos, é difícil explicar como a ação inibitória da CTX sobre os neutrófilos circulantes e peritoneais persiste por períodos prolongados após a administração de uma única dose da toxina. Dessa forma, o objetivo desse estudo foi avaliar o efeito in vitro e in vivo da CTX sobre a atividade funcional dos neutrófilos da medula óssea de camundongos e alguns dos mecanismos moleculares envolvidos na ação inibitória da CTX sobre as funções avaliadas. Para os ensaios in vitro, os neutrófilos foram incubados com a CTX (0,08 μg/mL), por 1 ou 24 horas. Para os ensaios in vivo, os animais foram pré-tratados com uma única administração de CTX (44 mg/kg), 1 dia antes do isolamento das células. Uma vez obtidos os neutrófilos, os seguintes parâmetros funcionais foram avaliados: quimiotaxia, adesão à fibronectina, fagocitose, produção de espécies reativas do oxigênio e desgranulação. Os resultados obtidos demonstraram que a CTX, in vitro e in vivo, inibiu os processos de quimiotaxia, adesão à fibronectina e fagocitose de partículas opsonizadas, entretanto não alterou a produção de espécies reativas do oxigênio ou a desgranulação em neutrófilos da medula óssea. Esses resultados demonstram que a CTX induz efeito inibitório sobre a funcionalidade dos neutrófilos da medula óssea, particularmente sobre funções associadas à polimerização de actina e consequente reorganização do citoesqueleto. Ainda, com o objetivo de elucidar os possíveis mecanismos envolvidos neste efeito inibitório, foram realizados ensaios para a análise da expressão do receptor CR3, bem como para a avaliação da expressão total e da atividade de proteínas de sinalização intracelular envolvidas na polimerização de actina nos neutrófilos. Os resultados obtidos mostraram que a CTX inibiu a expressão de ambas as subunidades (CD11b e CD18) do receptor CR3, bem como inibiu a atividade de Syk, Vav1, Cdc42, Rac1 e RhoA e a expressão da subunidade 1B do complexo Arp2/3. Em conjunto, os resultados desse estudo mostraram que a CTX inibe a funcionalidade dos neutrófilos da medula óssea e que essa ação está associada à inibição do receptor CR3, bem como à inibição da atividade de Syk e de suas proteínas downstream, o que resulta na redução da formação de filamentos de F-actina. Os resultados desse estudo comprovam a hipótese de que ação inibitória prolongada da CTX sobre a atividade dos neutrófilos circulantes e peritoneais está associada a alterações funcionais dos neutrófilos da medula óssea. Ainda, considerando-se a participação central dessas células na resposta inflamatória aguda, esse estudo contribui para a elucidação do efeito anti-inflamatório prolongado da CTX / Previous studies demonstrated that crotoxin (CTX), the main component of Crotalus durissus terrificus venom, presents anti-inflammatory properties, inhibiting cell migration and the phagocytic activity of peritoneal neutrophils. These inhibitory effects are long-lasting, since it can be observed up to 14 days after a single administration of this toxin. Considering the short half-life of neutrophils, it is difficult to explain how the inhibitory effect of CTX on circulating and peritoneal neutrophils persists for long periods after a single injection of this toxin. Thus, the aim of this study was to evaluate the in vitro and in vivo effect of CTX on the functionality of bone marrow neutrophils from mice and some of the molecular mechanisms involved on the inhibitory effect of CTX on these functions. For in vitro assays, neutrophils were incubated with CTX (0,08 μg/mL), for 1 or 24 hours. For in vivo assays, the animals were pretreated with a single administration of CTX (44 mg/kg), 1 day before the isolation of cells. Once obtained the neutrophils, the following functional parameters were evaluated: chemotaxis, adhesion to fibronectin, phagocytosis, reactive oxygen species production and degranulation. The results demonstrated that CTX, in vitro and in vivo, inhibited the processes of chemotaxis, adhesion to fibronection and phagocytosis of opsonized particles, however, it did not alter the reactive oxygen species production and degranulation. These results showed that CTX induces an inhibitory effect on the functionality of bone marrow neutrophils, particularly on functions that depend on actin polymerization and cytoskeleton rearrangement. Furthermore, to elucidate some possible mechanisms involved on this inhibitory effect, assays to analyze the expression of the receptor CR3, as well as, assays to analyze the total expression and activity of signaling proteins involved on actin polymerization were done. The results showed that CTX inhibited both subunits of CR3 (CD11b and CD18) and the activity of Syk, Vav1, Cdc42, Rac1 and RhoA and the expression of the subunit 1B from Arp2/3. Together, the results presented herein demonstrate that CTX inhibits the functionally of bone marrow neutrophils and that this effect is associated to the inhibition of CR3 receptor and inhibition of the activity of Syk and its downstream signaling proteins, which results in the decrease of F-actin. The results prove the hypothesis that the long-lasting inhibitory effect of CTX on the activity of circulating and peritoneal neutrophils is associated to functional modifications of bone marrow neutrophils. Besides, considering the central role of these cells on the inflammatory response, this study contributes to the better understanding of the long-lasting anti-inflammatory effect of CTX

A Leucemia Mielóide Crônica (LMC) é uma doença rara. No entanto, os avanços nas pesquisas básica e clínica nos últimos anos, colocaram a LMC em evidência sendo hoje uma neoplasia maligna potencialmente curável. O diagnóstico e tratamento desta doença são, no entanto, extremamente caros. Não havendo dados sistemáticos nem registros de incidência da LMC no Rio Grande do Sul ou no Brasil, o levantamento de dados baseado em registros dos centros de referência se justifica também para planejar ações em saúde. Entre 1996 e 2000, 276 casos foram diagnosticados. A estimativa de casos novos anuais foi de aproximadamente 0,6:100.000 habitantes, e a idade média no momento do diagnóstico foi 42 anos e 4 meses (±16 anos e 2 meses). Quanto ao tratamento e evolução destes pacientes, dos 257 avaliados, 56 (21,8%) foram submetidos ao transplante alogênico de medula óssea, com taxa de sobrevida em 5 anos de 75% e 27% para as fases crônica e acelerada/blástica, respectivamente. O tempo médio de sobrevida para os 257 pacientes foi de 47,7 meses (IC 43,3 - 52,1). Comparando ao relatado na literatura, encontramos um menor número anual de novos casos e também uma média de idade no diagnóstico mais baixa. Isto poderia ser explicado pela menor referência de idosos a serviços terciários de saúde. Para os pacientes transplantados, os resultados foram semelhantes aos relatados na literatura. / Although rare, the advances made in basic and clinical research throughout the last years have thrown a spotlight on CML. Diagnosis and treatment of CML is of high cost. Since there is no systematic data or information about the incidence of CML in Rio Grande do Sul or Brazil, the data obtained from reference centers serve to estimate the number of CML cases in our state to better plan health actions. Between 1996 and 2000, 276 cases were diagnosed. The annual estimate of new cases was approximately of 0,6:100,000 inhabitants, and the median age at diagnosis was 42 years and 4 months (±16 years and 2 months). The mean overall survival time for the 257 patients was 47,7 months (CI 43,3-52,1). That could be explained by the lack of referral for older patients. Regarding treatment and evolution, of the 257 valuable patients, 56 (21,8%) were submitted to allogeneic BMT with a five-year survival of 75% and 27% for chronic and accelerated/blastic phases, respectively. In conclusion, we found a lower estimated incidence and a lower median age at diagnosis. For the transplanted patients the results were similar to those reported in the literature.

Siu, Yeung Tung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 65-75). / Abstracts in English and Chinese. / Title Page --- p.i / Abstract in English --- p.ii / Abstract in Chinese --- p.iii / Acknowledgement --- p.iv / Table of Contents --- p.v-viii / List of Figures --- p.ix / List of Tables --- p.x / Abbreviations --- p.xi-xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Growth factors involved in hepatocytes proliferation --- p.1-6 / Chapter 1.1.1 --- Hepatocyte growth factor (HGF) --- p.1 / Chapter 1.1.2 --- Tumor necrosis factor-a (TNF-α) and interleukin-6 (IL-6) --- p.2 / Chapter 1.1.3 --- Epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) --- p.3 / Chapter 1.1.4 --- Other comitogens --- p.4 / Chapter 1.1.5 --- Transforming growth factor-β (TGF-β) --- p.5 / Chapter 1.2 --- Bone marrow stromal cells and hepatocytes proliferation --- p.7-12 / Chapter 1.2.1 --- Role of bone marrow stromal cells in bone marrow --- p.7 / Chapter 1.2.2 --- Bone marrow as a source of hepatic oval cells --- p.8 / Chapter 1.2.3 --- Growth factors secreted by bone marrow stromal cells involved in hepatocytes proliferation --- p.9 / Chapter 1.2.4 --- Endocrine in hepatocytes proliferation --- p.12 / Chapter 1.3 --- Objective of this study --- p.13-15 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Cell cultures --- p.16 / Chapter 2.2 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.17-18 / Chapter 2.2.1 --- "Enrichment of human hepatic cell lines, Hep 3B, Hep G2, C3A, SK-Hep-1 and Chang cells at G0-G1 phases by serum deprivation" --- p.17 / Chapter 2.2.2 --- "Incubation of serum deprived Hep 3B, Hep G2, C3A, SK- Hep-1 and Chang cells with mitogenic stimuli" --- p.17 / Chapter 2.2.3 --- Cell cycle analysis by flow cytometry using propidium iodide staining --- p.17 / Chapter 2.3 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.18-20 / Chapter 2.3.1 --- Partially growth arrested human SK-Hep-1 cells --- p.18 / Chapter 2.3.2 --- Human bone marrow stromal cells --- p.19 / Chapter 2.3.2.1 --- Bone marrow stromal cellular extract --- p.19 / Chapter 2.3.2.2 --- Total protein assay --- p.19 / Chapter 2.3.3 --- Incubation of SK-Hep-1 cells with bone marrow stromal cellular extracts --- p.20 / Chapter 2.4 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.21-22 / Chapter 2.4.1 --- Dialysis --- p.21 / Chapter 2.4.2 --- Temperature treatment --- p.21 / Chapter 2.4.3 --- Proteolysis --- p.22 / Chapter 2.5 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.22-26 / Chapter 2.5.1 --- Incubation of SK-Hep-1 cells with bone marrow stromal cellular extract or other growth factors --- p.22 / Chapter 2.5.2 --- Metabolic labeling of SK-Hep-1 cells with [32P]orthophosphate --- p.23 / Chapter 2.5.3 --- Incubation of labeled SK-Hep-1 cells with bone marrow stromal cellular extract or other growth factors --- p.23 / Chapter 2.5.4 --- SK-Hep-1 cells lysate extraction --- p.23 / Chapter 2.5.5 --- Two-dimensional electrophoresis --- p.24 / Chapter 2.5.5.1 --- First dimension isoelectric focusing --- p.24 / Chapter 2.5.5.2 --- Second dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis --- p.25 / Chapter 2.5.6 --- Amplification of radiolabeled signal by EN3HANCE --- p.25 / Chapter 2.5.7 --- Visualization of autoradiography --- p.26 / Chapter 2.5.8 --- Visualization by silver staining --- p.26 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.27-30 / Chapter 3.1.1 --- "Enrichment of human hepatic cell lines, Hep 3B, Hep G2, C3A, SK-Hep-1 and Chang cells at G0-G1 phases by serum deprivation" --- p.27 / Chapter 3.1.2 --- DNA synthesis of hepatic cell lines in response to 10 % FBS after serum deprivation --- p.29 / Chapter 3.2 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.31-39 / Chapter 3.2.1 --- Cell cycle distribution of partially growth arrested SK-Hep-1 cells in response to mitogens --- p.31 / Chapter 3.2.2 --- Time course on DNA synthesis of partially growth arrested SK-Hep-1 cells in response to FBS and bone marrow stromal cellular extract --- p.36 / Chapter 3.2.3 --- Dose response on DNA synthesis of partially growth arrested SK-Hep-1 cells in response to bone marrow stromal cellular extracts --- p.38 / Chapter 3.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.40-44 / Chapter 3.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.45-49 / Chapter 3.4.1 --- Mitogenic response of SK-Hep-1 cells in response to bone marrow stromal cellular extract and other growth factors --- p.45 / Chapter 3.4.2 --- Early intracellular signaling of SK-Hep-1 cells in response to bone marrow stromal cellular extract and other growth factors --- p.47 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.50 / Chapter 4.2 --- "Mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.51 / Chapter 4.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.52 / Chapter 4.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.53 / Chapter 4.5 --- Possible directions for future investigation --- p.55 / Chapter 4.6 --- Conclusions --- p.56 / Chapter Chapter 5 --- Appendices / Chapter 5.1 --- Reagents and solutiuons --- p.57-64 / Chapter 5.1.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.57 / Chapter 5.1.2 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.59 / Chapter 5.1.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.60 / Chapter 5.1.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.61 / Chapter Chapter 6 --- References --- p.65-75

A síndrome mielodisplásica primária (SMD) compreende um grupo de doenças hematopoéticas clonal de célula tronco pluripotente cacacterizada por vários graus de pancitopenia e alterações morfológica das células hematopoeticas e risco aumentado de transformação para leucemia mielóide aguda. A citogenética e a morfologia da medula óssea desempenham um papel fundamental para o diagnóstico e o prognóstico desses pacientes. Alterações cromossômicas são encontradas em aproximadamente 30-50% dos casos. Devido à importância da análise desses fatores para escolha terapêutica, torna-se necessário definir as alterações morfológicas e citogenéticas que possam contribuir para o prognóstico. Esse trabalho visa correlacionar as características morfológicas e citogenéticas da medula óssea em pacientes com SMD primária com as classificações OMS e FAB e com o IPSS. Foram estudados 32 pacientes com SMD primária diagnosticados entre 2000 e 2009 no HUPE-UERJ. As características clínicas foram analisadas através do levantamento de prontuários. A análise citogenética foi feita pela técnica de bandeamento GTG em células da medula ossea. A análise morfológica da biópsia de medula óssea e do mielograma foram realizadas através da revisão de lâminas. Vinte e três pacientes foram classificados em estágios iniciais da doença (22 AR, 1 ARSA) e 9 em estágio avançado AREB de acordo com a FAB. Alterações cromossômicas foram detectadas em 16 pacientes (50%). As mais frequentes foram: del(11)(q23) e del(17p). Dos pacientes com doença avançada, seis (66%) apresentaram aumento significativo da relação M:E (p=0,003) e sete (77%) possuíam alterações arquiteturais acentuadas (p<0,001) em comparação ao grupo de doença inicial. Pacientes classificados como intermediário 2 e alto risco pelo IPSS tiveram importante perda arquitetural (p<0,001), número significativamente maior de micromegacariócitos (p=0,017) e seis (85%) sofreram transformação leucêmica (p=0,006). ALIP foi significantemente aumentada nos pacientes de pior prognóstico (p=0,0 1) e naqueles com doença avançada (p=0,001). Nossos resultados apresentaram implicações potenciais para o diagnóstico e o prognóstico da SMD primária. As alterações morfológicas foram associadas com as classificações FAB, OMS e com os grupos de risco segundo o IPSS. / The primary myelodysplastic syndrome (MDS) comprises a heterogeneous group of clonal bone marrow disorders characterized by varying degrees of pancytopenia and morphological abnormalities of hematopoietic cells, and an increased risk of transformation into acute myeloid leukemia. The morphology and the cytogenetic of bone marrow play a key role in the diagnosis and the prognosis. Chromosomal abnormalities are found in 30-50% of cases. Due to the importance of analyzing these factors for therapeutic choice, it becomes necessary to define the morphological and the cytogenetic changes which could contribute to the prognosis.The aim of this study was to analyze the morphological and cytogenetic features of bone marrow and their correlations with FAB and WHO clasification and IPSS. We studied 32 patients with primary MDS between 2000 - 2009. The clinical data was recovered from medical records. Morphological characteristics were analyzed through a review of bone marrow biopsies and mielogram slides. Cytogenetic analysis was performed by GTG banding from bone marrow cells. The patients were classified according to FAB in initial stages: RA (22 patients) and RARS (1 patient) and nine in advanced stages - RAEB. Chromosomal abnormalities were detected in 16 patients (50%). The most frequent abnormalities were: del(11)(q23) and del(17p). Patients with advanced disease, six (66%) had significant increased M:E ratio (p=0.003) and seven patients (77%) had increased architectural changes (p<0.001). Patients classified as intermediary 2 and high risk according IPSS had important architectural loss (p<0.001), presence of micromegakaryocyte was considered significant higher (p=0.017) and six patients (85%) underwent leukemia transformation (p=0.006). ALIP was significant higher in the patients with a worse prognosis (p=0.021) and advanced stage of disease (p=0.001). Our results showed potential implications for diagnosis and prognosis of MDS. The morphological findings in this study were associated with FAB and WHO groups and prognostic risk (IPSS).

Les propriétés microvasculaires de la moëlle osseuse (MO) sont mal connues chez l’être humain. L’IRM de perfusion en permet une évaluation quantitative non invasive. La hanche a été choisie, car elle est la cible de pathologies fréquentes et handicapantes, qu’il est nécessaire de diagnostiquer plus précocement telle la coxarthrose. Nous avons d’abord implémenté une séquence IRM volumique à voxels isotropiques, avec une couverture large et une résolution spatiale élevée. Celle-ci a ensuite permis l’étude d’une série de 60 patients âgés de 18 à 60 ans, sans antécédent de pathologie osseuse et présentant une MO d’aspect normal en IRM. Les paramètres de perfusion semi-quantitatifs et pharmacocinétiques ont été mesurés dans 15 régions d’intérêt chez chaque patient. Tous les paramètres de perfusion diffèrent entre les zones de MO rouge et de MO jaune. La perfusion est différente entre les MO acétabulaire (squelette axial) et fémorale intertrochantérienne (squelette appendiculaire). Plusieurs paramètres sont corrélés de manière négative à l’âge. Plusieurs paramètres sont différents entre les hommes et les femmes. La perfusion de la tête fémorale est hétérogène, probablement en raison de l’exposition aux contraintes mécaniques. Les paramètres Ktrans, Kep et TTP sont corrélés à l’indice de masse corporelle, ce qui suggère que l’obésité influence le métabolisme de la MO. Enfin, le tabagisme et l’hypercholestérolémie ont une incidence sur ces mêmes paramètres dans certaines zones. Ils pourraient donc être le reflet d’altérations de la microvascularisation osseuse. Ces travaux ouvrent de nouvelles perspectives de recherche sur la physiologie et la pathologie de la MO. / Bone Marrow (BM) microvascular properties are insufficently known in humans. Dynamic-Contrast Enhanced (DCE) MRI allows its non-invasive quantitative assessment. We concentrated on the hip because this joint is frequently affected by debilitating pathologies such as osteoarthritis. Their early diagnosis is a current medical challenge. We implemented a 3D DCE-MRI sequence with isotropic voxels, high spatial resolution and a large coverage. It was used in a study of 60 patients aged 18 to 60, with no previous history of bone disease and with normal-appearing BM on MR images. Semi-quantitative and pharmacokinetic parameters were measured in 15 regions of interest in each patient. All the parameters were different between red and yellow BM. Perfusion was different between acetabular (axial skeleton) and femoral intertrochanteric (appendicular skeleton) BMs. Several parameters were negatively correlated with age. Perfusion was different in men and women. The femoral head perfusion was heterogeneous, likely because of mechanical load exposure. Ktrans, Kep and TTP were correlated with body mass index. This suggests that obesity influences BM metabolism. Smoking and hypercholesterolemia influenced these same parameters in several zones. We hypothesized that these parameters might reflect BM microvascular aletrations. Our results open new research perspectives both in the physiology and pathology of BM.

Introdução: A diarréia por Clostridium difficile é uma das principais causas de diarréia infecciosa associada à assistência à saúde. Objetivo: Descrever os casos e a incidência de diarréia por C. difficile em pacientes hematológicos e TCTH internados na enfermaria da Hematologia e do Transplante de Medula óssea do Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP). Métodos: Foi utilizada uma ficha para coleta de dados de todos os pacientes na unidade de hematologia e TCTH do HC-FMUSP que tinham resultado positivo para toxina A/B do C. difficile durante o período de janeiro 2007 a junho de 2011 e criado um banco de dados no programa Epiinfo. Os desfechos avaliados foram forma grave de doença e óbito até 14 dias da diarréia. Qui-quadrado para tendência foi usado para avaliar a incidência de diarréia por C. difficile durante o período do estudo. Resultados: Um total de 69 episódios foi identificado em 64 pacientes em 439 pacientes suspeitos. Na distribuição temporal o qui-quadrado para tendência evidenciou que o número de casos suspeitos de diarréia por C. difficile permaneceu estável (p=0,418), em contraste com o aumento do número de casos confirmados (p=0,0006). A incidência de diarréia por C. difficile por 1.000 pacientes variou de 0,65 a 5,45 durante o período do estudo. O qui-quadrado para tendência evidenciou aumento do número de casos confirmados de C. difficile nas unidades de TMO (p=0,00004) e Hematologia (p=0,006). Já, a incidência de diarréia por C. difficile por 1.000 neutropênicos dia variou de 0,78 a 5,45 durante o período do estudo. A maioria 68% dos pacientes era do gênero masculino, a média de idade foi 38 anos. A doença de base mais comum foi Leucemia mielóide aguda (44%), destes 46% estavam neutopênicos, 45% TCTH alogênicos, 88% dos pacientes usaram antibióticos prévios. A maioria dos pacientes 88% recebeu tratamento com metronidazol via oral, 37% tiveram resposta parcial nos primeiros sete dias de tratamento, 57% terminaram o tratamento e três pacientes tiveram recidiva, 24% evoluíram para óbito. A análise bivariada dos fatores de risco associados com a forma grave de diarréia por C. difficile, identificou o tipo de transplante alogênico como o único fator de risco significantivo, já a análise multivariada não identificou nenhum fator de risco independente. A análise bivariada de fatores de risco associados com óbito até 14 dias do diagnóstico de diarréia por C. difficile, identificou o tipo de transplante alogênico, forma grave de doença, internação em UTI e uso de linezolida como fatores de risco para óbito e Linfoma como protetor. Os fatores de risco independentes associados com óbito foram forma grave de doença, internação em UTi e uso de linezolida. Conclusão: A incidência de infecção por C. difficile durante o período do estudo aumentou, entretanto, a manifestação da forma grave da doença se manteve estável. Os fatores de risco independentes associados com óbito foram forma grave de doença, internação em UTI e uso de linezolida / Introduction: Diarrhea caused by Clostridium difficile is one of the main causes of infectious diarrhea associated with healthcare. Objective: To describe cases and the incidence of diarrhea by C.difficile in hematological and HSCT inpatients of the infirmary of Hematology and Bone Marrow Transplant of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP). Methods: A form was used to collect data from all patients in the hematology and HSCT unit of the HCFMUSP who showed positive results for toxin A/B of the C. difficile during the period from January 2007 to June 2011, and to create a databank in the Epiinfo program. The outcomes evaluated were the severe form of the disease and death within 14 days of the [onset of] diarrhea. The Chi-squared test for tendency was used to evaluate the incidence of diarrhea by C. difficile during the study period. Results: A total of 69 episodes were identified in 64 patients in 439 suspect patients. In the temporal distribution, the chi-squared test for tendency evidenced that the number of cases of suspected diarrhea due to C. difficile remained stable (p=0.418), in contrast with the increase in number of confirmed cases (p=0.0006). The incidence of diarrhea by C. difficile per 1,000 patients varied from 0.65 to 5.45 during the study period. The chi-squared test for tendency demonstrated an increase in number of cases confirmed of C. difficile in the BMT TMO (p=0.00004) and Hematology (p=0.006) units. On the other hand, the incidence of diarrhea by C. difficile per 1,000 neutropenic individualsday varied from 0.78 to 5.45 during the study period. Most of the 68% of patients were of the male gender; the average age was 38 years. The most common underlying disease was acute myeloid leukemia (44%); of these, 46% were neutropenic, 45% HSCT allogeneic, and 88% of the patients had previously used antibiotics. Most of the patients (88%) received treatment with oral metronidazole, 37% had a response in the first seven days of treatment, 57% finished the treatment, three patients experienced relapse, and 24% died. The bivariate analysis of the risk factors associated with the severe form of diarrhea by C. difficile, identified the type of allogeneic transplant as the only significant risk factor, whereas the multivariate analysis did not identify any independent risk factor. Bivariate analysis of risk factors associated with death within 14 days of the diagnosis of diarrhea due to C. difficile, identified the type of allogeneic transplant, severe form of the disease, stay in the ICU, and use of linezolide as risk factors for death, and Lymphoma as a protector. Independent risk factors associated with death were the severe form of the disease, stay in the ICU, and use of linezolide. Conclusion: The incidence of infection by C. difficile during the study period increased; nevertheless, the manifestation of the severe form of the disease remained stable. The independent risk factors associated with death were the severe form of the disease, stay in the ICU, and use of linezolide

Nesta pesquisa foi avaliada a incidência e a severidade da mucosite oral em pacientes que foram submetidos à adequação bucal previamente ao Transplante de Medula Óssea. Foi realizado exame clínico e radiográfico dos pacientes portadores de Leucemia Mielóide Crônica que estavam se preparando para o transplante. Após a avaliação inicial dos pacientes foram realizados os tratamentos odontológicos necessários para a remoção de quaisquer focos de infecção bucal. Todos os pacientes receberam o mesmo tipo de condicionamento quimioterápico e de profilaxia para Doença do Enxerto-Contra-Hospedeiro. Todos os pacientes receberam adequação bucal e foram avaliados durante o período de internação hospitalar para o transplante pelo mesmo Cirurgião Dentista. Os resultados mostraram que a mucosite grau 0 foi encontrada em 5 pacientes (14,29%), grau 1 em 8 pacientes (22,85%), grau 2 em 17 pacientes (48,57%), grau 3 em 3 pacientes (8,57%) e grau 4 em 2 pacientes (5,71%). Estes dados sugerem que a adequação bucal realizada previamente ao Transplante de Medula Óssea reduziu a severidade da mucosite oral, salientando a importância da presença do Cirurgião Dentista na equipe multidisciplinar em um serviço de Transplante de Medula Óssea. / The aim of this research was to evaluate the incidence and the severity of the oral mucositis in patients submitted to dental treatment before the Bone Marrow Transplantation. Clinical and radiographical examination was performed in patients with Chronic Myeloid Leukemia who would be submitted to transplantation. After patient’s initial evaluation, dental treatment was performed in order to remove any site of oral infection. All patients received the same chemotherapy conditioning regimen and the Graft-versus-host-disease prophylaxis. All patients received oral care during the hospital intern period and were evaluated by the same dentist. The results showed that 5 patients (14,29%) presented level 0 of oral mucositis, 8 patients (22,85%) presented level 1 of oral mucositis were, 17 patients (48,57%) presented level 3 of oral mucositis and 2 patients (5,71%) presented level 4 of oral mucositis. These data suggest that oral care and dental treatment previous to the Bone Marrow Transplantation reduced the severity of oral mucositis and emphasizes the importance of the Dentist as a member of a multidisciplinary team in a Bone Marrow Transplantation unit.