A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfillment of the requirements for the Degree of Master of Dentistry In the branch of Oral Pathology November 2012 / The giant cell lesions of the jaws are characterised histologically by scattered multinucleated giant cells (MNGCs) within a connective tissue stroma containing round and spindled mononuclear cells. Additional features include haemorrhage and haemosiderin deposition. Central giant cell granuloma (CGCG), peripheral giant cell granuloma (PGCG), cherubism and aneurysmal bone cyst (ABC) are thus encompassed by this term. The osteoclastic nature of the MNGCs within these lesions is well established. Microphthalmia-associated transcription factor (Mitf) is essential in the terminal differentiation of osteoclasts, the abnormal expression of which, results in dysfunctional osteoclast activity. Transcription factor E3 (Tfe3) belongs to the same transcription factor subfamily and is capable of forming co-immunoprecipitates with Mitf to function in a synergistic manner. It is abundantly expressed in physiological osteoclasts. Both factors are crucial for gene regulation in osteoclastic bone resorption. This study aimed to assess the expression of Mitf and Tfe3 within the stromal and MNGCs of the aforementioned giant cell lesions in order to enhance our understanding of the biological nature of these cells. The results showed positive nuclear staining within both the stromal and MNGCs in all four lesions with preferential expression noted in the MNGCs. This finding supports the concept of precursor stromal cell fusion. In addition, Mitf was consistently expressed at higher levels than Tfe3, in keeping with its reported principal role in the terminal differentiation process. The only exception to this was observed in ABC where Mitf and Tfe3 expression levels proved to be similar. It is thus apparent that the co-expression of Mitf and Tfe3 serves to confirm the osteoclast-like phenotype of the MNGCs within the giant cell lesions of the jaws. The degree of expression does not, however, correlate with the clinical behaviour of these cells, an observation substantiated by the minimal osteolytic potential of PGCG.

Thesis (M.S.)--University of Michigan, 1962.

Thesis (M.S.)--University of Michigan, 1962.

No description available.

A lesão central de células gigantes (LCCG) é uma afecção benigna dos maxilares, de comportamento biológico incerto, variando de discreta tumefação assintomática e de crescimento lento à uma forma agressiva, associada a dor, reabsorção radicular e óssea, com destruição cortical. Sua etiologia permanece desconhecida, havendo controvérsias entre processo reacional, neoplásico ou genético. Mutações no gene SH3BP2 foram identificadas em pacientes com querubismo, condição que compartilha várias características clínicas, radiográficas e histopatológicas com a LCCG. Para testar a hipótese de que tais mutações seriam responsáveis por, ou estariam associadas a LCCG e na tentativa de melhor entender a diferenciação microscópica/morfométrica das lesões agressivas e não agressivas, vinte e cinco pacientes portadores de LCCG foram selecionados para o estudo. O DNA foi obtido através do sangue e de espécimes em blocos de parafina, oriundos de biópsias e tratamento cirúrgico. Um estudo microscópico morfométrico foi paralelamente realizado, para avaliar o número de células gigantes e densidade de volume das mesmas nas lesões agressivas e não agressivas. O sequenciamento genético dos treze exons do gene SH3BP2 nos vinte e cinco pacientes estudados evidenciou uma alteração no códon do exon 4 em 10 pacientes. A densidade de volume de células gigantes foi maior nas lesões agressivas quando comparadas às não agressivas (p=0,013). Não houve diferença significante quanto ao número de células gigantes/mm2 em lesões agressivas e não agressivas (p =0,245). / Central giant cell lesion (CGCL) is a benign disease of the jaws, with uncertain behavior, ranging from mild asymptomatic slow-growing swelling to an aggressive form, with pain, radicular and bone resorption and cortical destruction. Its aetiology is still unknown and there is discussion whether it is a reactive, neoplastic or genetic disease. Mutations on gene SH3BP3 were identified in patients with cherubism, which shares several clinical, radiographic and histopathological features with CGCL. In order to test the hypothesis that such mutations would be responsible for or would be related to CGCL and also in order to better understand microscopic morphometric differentiation of the aggressive and non-aggressive lesions, 25 patients with CGCL were selected to this study. DNA was extracted from blood samples and from tissue samples, obtained by biopsy or surgical treatment. Microscopic morphometric assessment was also performed, in order to evaluate the number and the volume density of the giant cells in aggressive and in non-aggressive lesions. Gene sequencing of all 13 exons in gene SH3BP3, performed on each of the 25 patients, showed an alteration in one codon from exon 4, in ten patients. Volume density of giant cells was greater in aggressive lesions than in non-aggressive ones (p=0,013). There was no significant difference on the number of giant cells per mm2 when comparing aggressive and non-aggressive lesions.

A expressão da conexina 32 e 43 nas células gigantes trofoblásticas foi analisada em condições de cultura celular com e sem influência de hormônios sexuais. Placentônios bovinos foram coletados de vacas prenhes em abatedouro nos diferentes períodos gestacionais e divididos em três grupos, primeiro terço (I), segundo terço (II), terceiro terço (III) e transportados ao laboratório em condições assépticas à temperatura de 4ºC em solução de PBS com antibiótico. No laboratório as células foram isoladas e cultivadas em meio D-MEM com 10% SFB por cinco dias. As detecções das conexinas 32 e 43 foram realizadas através de munofluorescencia, pelo método de amplificação da tiramida-fluoresceina, utilizando anticorpo primário policlonal a imunoglobulina de coelho, anti-conexina 32 e 43 de camundongo. Os resultados mostraram que as células gigantes trofoblásticas expressam conexinas 32 e 43 nos três períodos gestacionais com exceção da Cx32 no primeiro terço gestacional sem adição de hormônios, a qual passou a expressar fluorescência após a adição de hormônios. A distribuição da Cx43 evoluiu com a progressão da gestação, permanecendo limitada ao interior das células gigantes trofoblásticas, sem formar junções comunicantes. O terceiro terço gestacional mostra a Cx43 na CGTT localizada no interior do núcleo. A adição de hormônios ao meio de cultura vem confirmar que a progesterona e o estrógeno podem ter um papel no controle da Cx32. Para Cx43 onde se adicionou progesterona os níveis de expressão foram baixos no início aumentando no decorrer da gestação, o mesmo foi encontrado para o conjugado de progesterona/ estrógeno. Para o estrógeno no grupo I os níveis de expressão foram menores se comparados aos três grupos diminuindo no grupo II e voltando a aumentar no grupo III. / The trophoblast cells expression of connexins 32 and 43 was studied in cell culture conditions with or without influence of sexual hormones. Bovine placentomes were collected from pregnant cows in slaughterhouses in different gestational periods and so divided into three groups, first term (I), second term (II), and third term (III), and transported to the laboratory in aseptic conditions at a temperature of 40C in PBS solution with antibiotics. At the laboratory the cells were isolated and cultivated in D-MEM environment with 10% SFB for five days. The detections of connexins 32 and 43 were achieved through immunofluorescence, by the tyramide-fluorescein amplification method, using a rabbit polyclonal primary antibody anti-connexin 32 and 43 of mice. The results showed that the trophoblast cells expressed connexins 32 and 43 in the three gestational periods with an exception of the Cx32 at the first gestational period. However, after the addition of sexual hormones, they began to express the connexins in the cytoplasm in the first stage. The distribution of the Cx43 evolved with the progression of the gestation, remaining limited to the trophoblast`s cells interior, without formation of gap junctions. The third gestational term shows the Cx43 located inside the nuclei of the CGTT. Addition of hormones in the culture environment confirmed that estrogen and progesterone may have an important action controlling Cx32. For Cx43, the expression was low after prosterone was added to the culture medium, but increased as the gestation evolved, the same was found for a combined compost of estrogen\\ progesterone. For estrogen, in group I the expression levels were inferior when compared to the three groups decreasing in group II and increasing again in group III. We may conclude that the sexual hormes, specially estrogen, affect the expression of connexins in trophoblastic cells.

O presente trabalho estudou a expressão da conexina 43 nas células trofoblásticas gigantes bovinas, em especial nas células trofoblásticas gigantes binucleadas. Foram utilizadas 12 amostras de placenta bovina, divididas em três grupos segundo a fase gestacional, primeiro terço, segundo terço e terceiro terço da gestação. O tecido placentário foi fixado em solução metacarn e, posteriormente, submetido ao método tradicional de inclusão em parafina. A detecção da conexina 43 foi realizada através de imuno-histoquímica, pelo método de amplificação da tyramida, utilizando como anticorpo primário policlonal a imunoglobulina de coelho anti-conexina 43 de camundongo. Os resultados deste trabalho indicaram que as células trofoblásticas gigantes bovinas são capazes de expressar a conexina 43 nas três fases da gestação. A distribuição da conexina 43 evoluiu com a progressão da gestação, mas ficou limitada ao interior das células trofoblásticas gigantes bovinas, sem formar marcações pontuais na membrana citoplasmática que pudessem indicar a formação de junções comunicantes. A avaliação de diferentes protocolos histológicos e imuno-histoquímicos permitiu estabelecer a melhor metodologia para a preservação do arranjo tecidual da placenta bovina e a identificação da conexina 43 nas células trofoblásticas gigantes bovinas / The present study has researched the expression of Connexin-43 in bovine trophoblast giant cells, especially in binucleate trophoblast giant cells. Twelve samples of bovine placenta were used, divided into three groups according to the gestational phase, first part, second part and third part of gestation. The placental tissue was fixed in Methacarn solution and later submitted to the traditional method, that is, embedded in paraffin. The detection of Connexin-43 was done through immunohistochemistry by the tyramide amplification method, using as polyclonal primary antibody, rabbit immunoglobuline anti-mouse Connexin-43. The results of this study have revealed that the bovine trophoblast giant cells are capable of the expression of Connexin-43 in the three phases of gestation. The distribution of Connexin-43 evolved as gestation progressed, but was limited to the interior of the bovine trophoblast giant cells without punctate markings in the cytoplasmic membrane which could indicate the formation of communicating junctions. The assessment of different histological and immunohistochemical protocols allowed us to establish the best methodology for the preservation of the tissue arrangement of bovine placenta and the identification of Connexin-43 in the bovine trophoblast giant cells

Tuberculosis, caused by Mycobacterium tuberculosis infection, results in the development of granulomas in affected tissues. These structures are formed by a myeloid cell core including multinucleated giant cells and surrounded by T lymphocytes. We studied mechanisms of survival, formation and functions of giant cells in Mycobacterium granulomas. Previously, our group showed that the cytokine IL-17A induces the fusion of dendritic cells (DC). Here, we identified molecules induced by the IL-17A genetic program in myeloid cells: BFL1 regulated DC survival, while the chemokines CCL2 and CCL20 directed clustering required for DC fusion. In situ, in human TB granulomas, we found that IL-17A was expressed by T lymphocytes while BFL1, CCL2 and CCL20 were expressed by the mono- and multi-nucleated myeloid cells. Then we characterized phenotype, immune functions and microbicidal activity of IL-17A-treated DC and their derived giant cells. They expressed a mixed DC-macrophage phenotype, retained classical DC functions, synthesized several destructive enzymes and had increased and differential microbicidal activities against Mycobacterium species. We named GMIC (giant myeloid inflammatory cells) these IL-17A-dependent giant cells, and propose that they constitute a new inflammatory myeloid effector with potent microbicidal activities. Altogether, our results show that IL-17A may participate in the maintenance of the myeloid core of human tuberculosis granuloma by promoting the formation of GMIC with potent destructive and microbicidal functions. The molecular mechanisms we have documented should help the development of new tuberculosis therapeutic and vaccination strategies.

Cholesterol crystals have been linked to activation of the NLRP3 inflammasome and the formation of foreign body giant cells (FBGCs). It has been hypothesized that FBGCs have a role in advanced atherosclerotic plaque formation. This thesis examined the feasibility of producing stable cultures of FBGCs starting with human monocytes with the goal to examine pterin production by these cells in comparison to human monocyte derived macrophages (HMDMs). The study also investigated the effect of cholesterol crystals on 7,8-dihydroneopterin (7,8-NP) production and modulation of IL-1β levels in macrophages. 7,8-Dihydroneopterin is a potent antioxidant generated by macrophages which also down regulates the expression of macrophage scavenger receptor CD36. The use of alpha-tocopherol and IL-4 as FBGC fusion mediators was explored. Using these mediators, large numbers of FBGC were successfully cultured. The rates of fusion achieved in the cultures were low, and the cells had poor adhesion, which prevented pterin measurement. FBGC, which are thought to remove crystallized cholesterol from the plaque, cleared 21% of cholesterol crystal compared to 50% cleared by HMDM cells. Due to this result, the effect of cholesterol crystals on pterin production in monocytes and macrophages was explored. Cholesterol crystals cause inflammation through the activation of the NLRP3 inflammasome, however, it was unknown whether they could modulate 7,8-NP production. Cholesterol crystals caused an intracellular dose-dependent loss of 7,8-NP to its oxidized form, neopterin, in HMDM cells. Cholesterol crystals induced intracellular synthesis of 7,8-NP in HMDMs. 7,8-NP was released into the supernatant and oxidized to neopterin in media. Monocytes treated with cholesterol crystals released up to 100 nM of neopterin and 120 nM of 7,8-NP in the media after 48 hours. The combination of IFN- and cholesterol crystals appeared to inhibit the release of 7,8-NP into the media for the first 48 hours, after this time 7,8-NP release rapidly increased. The addition of exogenous 200 μM 7,8-NP showed that in the presence of monocytes, cholesterol crystals did not cause the oxidation of 7,8-NP to neopterin, as seen in HMDMs but possibly to 7,8-dihydroxanthopterin or xanthopterin. The presence of 7,8-NP increased IL-1β expression in the presence of cholesterol crystals after 24 hours incubation. FBGCs and the removal of cholesterol crystals may be a key process in the resolution of atherosclerotic plaques. It appears that cholesterol crystals are able to modulate inflammatory processes including activation of the inflammasome and balance of 7,8-dihydroneopterin to the oxidized neopterin. The infiltrating monocytes may provide antioxidant protection against the inflammation induced by cholesterol crystals and the activity of the infammasome.

Thesis (118-130)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 118-130).