Paracoccidioidomicose é adquirida pela via respiratória e o óxido nitrico (NO) está envolvido na eliminação de patógenos. Nós propusemos investigar o papel do NO na doença em animais NO-/- sintase deficientes (iNOS-/-) e seu grupo WT. Na 2ª semana de infecção, a ausência de NO resultou em doença menos grave com aumento dos níveis de TNF-a acompanhado com o intenso afluxo de linfócitos T e macrófagos para os pulmões. Na 10ª semana, os animais iNOS-/- desenvolveram alta carga fúngica nos pulmões com menor afluxo de celulas T ativadas e macrófagos e presença de células T reguladoras CD4+CD25+FoxP3+ nos pulmões. Somente os animais iNOS-/- desenvolveram lesões pulmonares organizadas em granulomas, embora não foi detectado diferença na mortalidade para ambos os grupos. As diferenças morfológicas nas lesões foram abolidas pela depleção de TNF-a que induziu nos animais iNOS-/- uma mortalidade precoce e intenso afluxo inflamatório nos pulmões. Além disso, a depleção de linfócitos TCD8+ resultou em doença mais grave e menor recrutamento celular pulmonar nos animais iNOS-/-. / Paracoccidioidomycosis is acquired by the respiratory route and nitric oxide (NO) is involved in the killing of pathogens, we aimed to investigate the role of NO in the course of the disease using NO- synthase deficient (iNOS-/-) and WT mice. At week 2 postinfection, NO absence resulted in less severe infection associated with increased TNF-a levels besides a massive influx of activated T cells and macrophages to the lungs. By week 10, iNOS-/- mice developed increased fungal burdens allied with less pronounced influx of activated T cells and macrophages and increased presence of regulatory CD4+CD25+FoxP3+ T cells to the lungs. Only iNOS-/- mice developed organized pulmonary granulomas, although no differences in the mortality rates were detected. The differences in the morphology of lesions were partially abrogated by TNF-a depletion which, induced a precocious mortality of iNOS-/- and massive influx of inflammatory pulmonary cells. Indeed, the CD8+T cells depletion developed a more severe infection with less recruitment of pulmonary cells in iNOS-/- mice.

Orientador: Jose Ricardo Albergaria Barbosa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-08T02:07:16Z (GMT). No. of bitstreams: 1 Luna_AnibalHenriqueBarbosa_D.pdf: 1133521 bytes, checksum: 0b56f9a0d7a3c458d4ca3675a6467a94 (MD5) Previous issue date: 2006 / Resumo: O granuloma central de células gigantes (GCCG) é uma lesão benigna que acomete tanto a maxila como a mandíbula, representando menos de 7% de todos os tumores benignos dos maxilares. A sua etiologia é incerta, sendo implicados fatores genéticos. O GCCG pode manifestar-se como lesões de grandes dimensões com características de agressividade ¿ como crescimento rápido, reabsorções radiculares ou parestesia e tendência à recidiva, ou como lesões pequenas, uniloculares, sem aspectos de agressividade. A modalidade de tratamento mais empregada é a curetagem, associada ou não a ostectomia periférica. No entanto são relatadas outras modalidades de tratamento, como a administração de corticosteróides, calcitonina ou a-interferon. Os índices de recidiva podem ser altos, variando de 0% a 49%. A ocorrência de recidiva parece depender do comportamento clínico da lesão, da localização anatômica e da modalidade de tratamento instituída. O presente estudo retrospectivo analisou o tratamento de GCCG no período de janeiro de 1996 a julho de 2006 atendidos pela Área de Cirurgia Buco-Maxilo-Facial da FOP ¿ Unicamp, correlacionando seus aspectos clínicos. Foram analisados 14 casos (9M; 5F) com uma média de idade de 18,5 (variando de 5 ¿ 59) anos, sendo a maxila o osso mais acometido. Do total, 5 casos foram tratados cirurgicamente por meio de curetagem associada a ostectomia periférica, e 9 foram tratados clinicamente. A administração intralesional de corticosteróides foi iniciada nestes casos, sendo o tratamento com calcitonina instituído na ausência de uma resposta clínica satisfatória. O tempo médio de tratamento com corticosteróides foi de 3,84 (±3,87) meses, sendo que em dois casos foi instituída a administração de calcitonina. O tempo médio de tratamento com calcitonina foi de 18,8 (±7,94) meses, sendo que em um caso não foi observada boa evolução clínica. Nenhum caso de recidiva foi observado após um acompanhamento de 38,22 (variando de 3 ¿ 174) meses / Abstract: The central giant cell granuloma is a benign lesion of the jaws, accounting for less than 7% of all benign lesions of the jaws. Its origin is unknown, but it has been suggested that genetic factors may be implicated. The central giant cell granuloma demonstrates a variable clinical behavior, ranging from slowly growing painless swelling to rapidly expanding aggressive tumors, characterized by pain, local destruction of bone, root displacement or resorption and a significantly high recurrence rate. Surgical treatment represented by curettage with peripheral ostectomy or not is the most widely used procedure. However, other treatment options such as intralesional corticosteroids, daily calcitonin administration or a-interferon are advocated. The recurrence rate may be high (ranging form 0% to 49%), and it seems to depend on the clinical behavior, the treatment employed, and anatomic site envolved. The aim of this study was to report the results of long-term follow up of the management of central giant cell granulomas. A retrospective analysis was conducted from January 1996 to July 2006, analyzing all cases of the Oral and Maxillofacial Area, Piracicaba Dental School. The sample was represented by 14 patients (9 M; 5 F) with a mean age of 18.5 (ranging from 5 ¿ 59) years, and the maxilla was involved in most of the cases. Regarding the treatment modality, 5 cases were treated by curettage with peripheral ostectomy, and a medical treatment was instituted in the others. In these cases, intralesional injections with corticosteroids were initiated, and the treatment with calcitonin was employed only if proper resolution was not achieved. The mean time of treatment with corticosteroids was 3.84 (±3.87) months, but in two cases calcitonin daily administration was initiated. The mean time of treatment with calcitonin was 18.8 (±7.94) months, but in one case calcitonin did not seem to be effective. No case of recurrence was observed after a mean follow-up of 38.22 (ranging from 3 ¿ 174) months / Doutorado / Cirurgia e Traumatologia Buco-Maxilo-Faciais / Doutor em Clínica Odontológica

Orientador: Regina Helena Garcia Martins / Coorientador: Daniele Cristina Cataneo / Resumo: Introdução: os granulomas laríngeos são lesões benignas, não neoplásicas, uni ou bilaterais, de etiologia variável, que ocorrem no terço posterior das pregas vocais ou na região aritenoídea. Os sintomas são diversos, sendo o mais comum a rouquidão. Os granulomas decorrentes de intubação são altamente recidivantes e não existe consenso quanto ao melhor tratamento. Objetivo: comparar a efetividade dos tratamentos dos granulomas laríngeos decorrentes de intubação endotraqueal. Métodos: foram realizadas revisão sistemática e metanálise proporcional de estudos sobre o tratamento de granulomas laríngeos decorrentes de intubação endotraqueal, seja ele primário ou recidivante. Os critérios de elegibilidade foram: ensaios clínicos randomizados e estudos prospectivos controlados, e na ausência destes, aceitos também estudos retrospectivos e prospectivos não controlados com no mínimo cinco participantes. Os desfechos estudados foram resolução, recidiva e tempo para resolução do granuloma. Os estudos foram identificados na base de dados Pubmed, Embase, Lilacs e Cochrane. Para a análise dos dados e metanálise, utilizou-se o programa StatsDirect 3.0.121. Resultados: dentre os 578 artigos encontrados, 61 foram lidos na íntegra e seis selecionados para a revisão, totalizando 85 pacientes, com idade variando de 21 a 86 anos. Os tratamentos encontrados foram: antirrefluxo, fonoterapia, anti-inflamatórios, corticoterapia, antibioticoterapia, sulfato de zinco e cirurgia. Para o tratamento primár... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Laryngeal granulomas are benign, non-neoplastic lesions that can occur unilaterally or bilaterally for various causes. They are usually located in the posterior third of the vocal folds or in the arytenoid region. Patients may present a number of symptoms, the main one being hoarseness. Post-intubation granulomas are highly recurrent and there is no consensus on the best treatment. Objective: to compare the effectiveness of treatments of laryngeal granulomas secondary to endotracheal intubation. Methods: systematic review and proportion meta-analysis of studies that address the treatment of laryngeal granulomas caused by endotracheal intubation. The eligibility criteria were: randomized controlled trials and controlled prospective studies, and in the absence of these, retrospective and prospective uncontrolled studies were also accepted, with at least five participants. The outcomes that were measured were resolution, recurrence and time to resolve the granuloma. Databases searched were Pubmed, Embase, Lilacs and Cochrane. Statistical analysis was performed with the StatsDirect version 3.0.121 software. Results: among the 578 articles found, 61 were eligible for full reading and 11 articles were included, involving 85 patients, with ages varying from 21 to 86 years). The treatments were: anti-reflux, speech therapy, anti-inflammatory drugs, corticosteroids, antibiotics, zinc sulfate and surgery. For the primary treatment, 85 patients were investigated in six studies, divided into two groups: surgical ± associations (41 patients), with chance of resolution of 75% (95% CI: 0,3% to 100%, I2= 90%), and absolute risk of recurrence of 25% (95% CI: 0,2% to 71%) and clinical (44 patients), with chance of resolution of 86% (95% CI: 67% to 97%), and absolute risk of recurrence of 14% (95% CI: 3 to 33%). In the interpretation of the meta-analysis, there was no statistical significance... (Complete abstract click electronic access below) / Mestre

Thesis (PhD (Pathology. Anatgomical Pathology))--University of Stellenbosch, 2005. / The formation of granulomas at the site of antigen presentation in both tuberculosis and sarcoidosis is an essential component of host immunity for controlling inflammation. Granuloma formation is a complex process that also requires recruitment and activation of lymphocytes and macrophages to the site of infection and arrangement into a granuloma. It is dependant on the activation of especially IFNγ secreting CD4+ T cells, resulting in a Th1 profile. However, it is suggested that a persistently high IFNγ is responsible for the damage caused by granulomatous disease and that moderating cytokines, resulting in a Th0 profile, are necessary to down-regulate the IFNγ response to more appropriate levels later in the disease process, after the antigen has been effectively contained. I propose that: “Cytokine profiles determine clinical and histopathological phenotypes of disease. This thesis tests the hypothesis that it will be reflected by cytokine expression profiles in granulomas in different forms of tuberculosis and in sarcoidosis.” To examine this, biopsy tissue was obtained from patients with pulmonary cavitary tuberculosis, pleural tuberculosis in HIV sero-negative and sero-positive patients, and sarcoidosis. The diagnosis of tuberculosis or sarcoidosis was confirmed, granulomas were characterised as necrotic or non-necrotic, sarcoidosis cases were graded histologically and in situ hybridisation was performed for IL-12-, IFNγ-, TNFα- and IL-4-mRNA. In all patients with pleural tuberculosis, a Th0 profile was noted, while necrotic granulomas were more evident in HIV positive than HIV negative patients. There was a clear association between TNFα and necrosis in tuberculous granulomas that may be ascribed to the increased apoptotic activity of TNFα. An increase in IFNγ correlated with an increase in necrosis, supporting the theory that high IFNγ levels later in disease is detrimental. This effect may be enhanced by a strong presence of TNFα positive cells. An increase in both Th1 and Th2 cytokine mRNA in HIV positive patients supports the theory that an overproduction of cytokines may be a mechanism to compensate for the failure of another immune effector mechanism. Findings in pulmonary tuberculosis were similar to those in pleural tuberculosis. In all sarcoidosis cases the presence of a very strong Th1 and TNFα, but no Th0 response was confirmed. None of the differences in either the histological grading, or the clinical outcome of patients were reflected in the cytokine profile. It is possible that this profile does not reflect the histological grade of disease or that it may reflect various stages of disease. These findings support the theory that a strong Th1 presence later in disease, in conjunction with TNFα may induce fibrosis, as most of these cases showed signs of at least focal fibrosis. Numerous aspects, including a T helper response are involved in granulomatous inflammation. The earlier dogma of good, beneficial (Th1) versus evil, detrimental (Th2), is an oversimplification of a very complex process. It is clear that the effect of a cytokine depends at least partially on the stage of disease. The balance between the various cytokines, and the levels of these cytokines contribute to their role in resolution or disease progression. An early, pure Th1 response may be beneficial if effectively clearing the granuloma-inducing antigen. At this stage, a Th2 presence will be harmful as clearing of the antigen will not be as effective. In chronic disease where failure to remove the antigen results in progression of granulomas with subsequent necrosis and/or fibrosis, a proinflammatory Th1 response may be detrimental and minimising of this effect is needed. An overly strong presence of the various cytokines may also be detrimental, while lower levels will be beneficial.

Orientador: Flávio Ruas de Moraes / Coorientador: Maria Isabel Quiroga Berdeal / Banca: Eduardo Juan Gimeno / Banca: Rogério Salvador / Banca: Laura Satiko Okada Nakaghi / Banca: Marco Antonio de Andrade Belo / Resumo: A piscicultura está caracterizada pela alta densidade de estocagem, transporte e altos níveis de arraçoamento, alterando a qualidade de água e a homeostase dos peixes. Uma das principais características frente a estas alterações é a resposta geral de adaptação, aumentando a susceptibilidade dos peixes às doenças infecciosas e parasitárias ao mesmo tempo em que a má qualidade da água particularmente quando rica em matéria orgânica, favorece a proliferação de agentes com potencial. Um dos processos fisiopatológicos de grande importância para manter a saúde do hospedeiro é a resposta inflamatória aguda que se cronifica com a persistência do agente. O implante de lamínulas de vidro no tecido subcutâneo de peixes induz a resposta inflamatória crônica, apresentando acúmulo de macrófagos e formação de células gigantes multinucleadas. O inóculo do BCG induz intenso infiltrado mononuclear com desorganização do tecido causado por edema com dissociação das fibras musculares e necrose. O surgimento de células gigantes ocorre de forma progressiva e com o tempo estas aumentam de tamanho e número de núcleos formando células gigantes tipo corpo estranho. Com o avanço do processo a disposição dos macrófagos ao redor do inóculo é maior e surgem fibroblastos, linfócitos e na maioria dos casos presença de melanomacrófagos formando o granuloma. Neste trabalho objetivou-se caracterizar morfologicamente o processo inflamatório crônico por corpo estranho induzido pelo implante de lamínulas de vidro no tecido subcutâneo e o epitelióide induzido pelo inóculo de BCG em Piaractus mesopotamicus e Oreochromis niloticus. 180 peixes de cada gênero foram utilizados no estudo, divididos em quatro grupos de 45 cada. Os tratamentos foram: Inoculados, Implantados, Inoculado e Implantados e grupo controle. Nos grupos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Fish farming is characterized by high stocking density, transport and high levels of feeding, changing water quality and fish homeostasis. A major characteristic of these changes is opposite the overall response adjustment, increasing the susceptibility of fish to infectious and parasitic diseases at the same time that poor water quality particularly when rich in organic matter, favors the proliferation of agents with potential. One of the pathophysiological processes of major importance for maintaining the health of the host is the acute inflammatory response that chronic the persistence of the agent. The implantation of glass coverslips in the subcutaneous tissue of fish induces chronic inflammatory response, with accumulation of macrophages and multinucleated giant cell formation. The inoculum of BCG induces intense mononuclear infiltrate with disruption caused by tissue dissociation with edema and necrosis of muscle fibers. The emergence of giant cells occurs gradually and over time they increase in size and number of cores forming giant cells of foreign body type. With the progress of the process layout of macrophages around the inoculum is larger and appear fibroblasts, lymphocytes and in most cases the presence of melanomacrophages forming the granuloma. This study aimed to characterize morphologically the chronic inflammatory process induced by foreign body implant glass slides in the subcutaneous tissue and epithelioid induced by inoculation of BCG in Piaractus mesopotamicus and Oreochromis niloticus. 180 fish in each sex were used in the study were divided into four groups of 45 each. The treatments were: inoculated, deployed, deployed and Inoculated and control group). In inoculated groups were given 20 μl of BCG vaccine (40 mg/mL) (n° bacilli than 2.0 x 106 CFU / mg of BCG strain Mureau Rio de Janeiro) in skeletal striated... (Complete abstract click electronic access below) / Doutor

Mycobacterium tuberculosis (Mtb) remains a pathogen of significant importance with respect to global health. Although approximately one third of the world is infected with TB, only 5-10% develop clinical manifestations of active TB within 2 years post exposure. Infection with Mtb can cause active tuberculosis (ATB), inactive latent infection (LTBI) and be reactivated. The immune response is contained within the formation of a collection of immune cells, a granuloma, in the infected individual’s lungs, which is seen in all disease states. The granuloma functions both as an immune response to contain the bacterium from surrounding lung parenchyma as well as a site for the bacterium to remain in the individual; therefore, providing an environment in which the bacterium maintains the ability to reactivate. We currently lack a complete understanding of the physiology and the metabolic state of Mtb in this granulomatous environment during different states of infection. Leveraging a novel technique known as mesodisection, we microdissect TB granulomas from various infective stages as well as from different sections of the granuloma from non-human primate (NHP) derived formalin fixed paraffin embedded (FFPE) lung tissue. From these extracted tissue sections, RNA is extracted, amplified and subsequent microarray and nCounter analysis is performed; consequently, allowing us to uncover the Mtb specific transcriptomic profiles. First, our findings reveal statistically significant Mtb genes induced in ATB and LTBI in various granuloma types. Of the genes induced, many belong to the following categories: PE/ PPE, sigma factors, toxin-antitoxin complexes and the DosR regulon. In addition, our findings reveal a core group of genes commonly identified in both ATB and LTBI induced in ATB and LTBI in various granuloma types. Of the genes induced, many belong to the following categories: PE/ PPE, sigma factors, toxin-antitoxin complexes and the DosR regulon. In addition, our findings reveal a core group of genes commonly identified in both ATB and LTBI in all lesion types. Further, we propose that these findings improve our understanding of the physiology of the pathogen as well as its virulence which in turn can be used for the development of improved therapeutics, diagnostics and vaccines. / acase@tulane.edu

Die Wegener'sche Granulomatose (WG) ist eine Autoimmunerkrankung, die sich typischerweise als chronische Entzündung des oberen Respirationstraktes, Vaskulitis und Glomerulonephritis manifestiert. WG gehört zur Gruppe der sog. “pauci-immunen” Vaskulitiden, die mit Anti-Neutrophilen-Antikörpern (ANCA, anti neutrophil cytoplasmic antibody) assoziiert sind. Mit Hilfe der indirekten Immunfluoreszenz lassen sich ANCA im Serum der meisten WG-Patienten nachweisen. Die mit WG assoziierten sog. “klassischen” ANCA (c-ANCA) erkennen spezifisch Konformationsepitope der Serinprotease Proteinase 3 (PR3), die in den azurophilen Granula neutrophiler Granulozyten gespeichert wird. Die enge Korrelation PR3-spezifischer Antikörperspiegel mit dem Krankheitsverlauf läßt vermuten, daß sie bei der Pathogenese eine zentrale Rolle spielen könnten. Diese Hypothese wird von Daten aus in vitro Experimenten gestützt: werden Zytokin-stimulierte neutrophile Granulozyten mit Patientenserum oder isolierten c-ANCA inkubiert, erfolgt eine Aktivierung der Neutrophilen, die sich durch Degranulation und Freisetzung von Sauerstoffradikalen äußert. c-ANCA können so indirekt - aber vermutlich auch direkt - zur Endothelschädigung beitragen. Jedoch konnte bisher kein direkter Beweis des pathogenen Potentials von c-ANCA am Tiermodell erbracht werden. Um die Wirkung von c-ANCA an einem Mausmodell zu testen, war zunächst ein murines Maus-PR3- (mPR3) spezifisches Antiserum notwendig. Da humane c-ANCA nicht mit mPR3 kreuzreagieren, wurde für die Immunisierung von PR3/Neutrophilen-Elastase (NE)-defizienten Mäusen ein mPR3-Zymogen rekombinant in E. coli als Einschlußkörpermaterial (IB, inclusion bodies) hergestellt. Nach Renaturierung des IB-Materials in vitro wurde mit der Dipeptidylaminopeptidase Cathepsin C das N-terminale Propeptid abgespalten. Das gewonnene Enzym besaß die für PR3 und NE spezifische katalytische Aktivität, die durch den physiologischen Inhibitor humaner PR3, a1-Antitrypsin, inhibiert werden konnte. Es ist daher anzunehmen, daß das gewonnene rekombinante Material die korrekte Konformation durch Renaturierung in vitro erhalten hatte. Um nun die pathologische Wirkung von Anti-rmPR3-Antikörpern zu testen, wurden PR3/NE-defiziente Mäuse mit dem rekombinanten Zymogen (pro-rmPR3) oder der N-terminal prozessierten Form (rmPR3) immunisiert. Die Spezifität der gewonnenen Antiseren wurde durch Festphasenimmunoassay, Western Blotting und indirekte Immunfluoreszenzfärbung überprüft. Weiterhin konnte fluoreszenzzytometrisch die Bindung von Anti-mPR3-IgG an die Plasmamembran Zytokin-stimulierter Granulozyten nachgewiesen werden. Die hergestellten Antiseren erfüllten somit die für c-ANCA-positive Seren von WG-Patienten typischen Eigenschaften hinsichtlich der Antigenspezifität. Wenn c-ANCA alleine hinreichend für die Induktion der für WG charakteristischen Symptome sind, sollte der Antiserumtransfer auf Wildtyp-Mäuse WG-ähnliche Symptome in den Rezipienten hervorrufen. Nach wiederholter intravenöser Injektion von Serum pro-rmPR3-immunisierter Mäuse ließ sich ein signifikanter Antikörperspiegel bei Verdünnungen von 1:2000 über den gesamten Behandlungszeitraum von 10 Wochen in den Rezipienten nachweisen. Der anschließende histologische Befund von Niere und Lunge ergab jedoch keine pathologischen Veränderungen. Dieses Ergebnis legt nahe, daß c-ANCA alleine keine Krankheitssymptome hervorrufen. In dem gegenwärtigen Modell für c-ANCA-vermittelte Vaskulitis entfaltet sich die pathogene Wirkung von c-ANCA vor allem dann, wenn neutrophile Granulozyten zusätzlich durch proinflammatorische Zytokine wie Tumornekrosefaktor alpha (TNF alpha) stimuliert werden. Erst die Stimulation der Granulozyten ermöglicht die Bindung von c-ANCA an die Plasmamembran und deren anschließende Aktivierung. Deshalb wurde dieses Modell, das vorwiegend aus Ergebnissen von Experimenten in vitro abgeleitet ist, auf ein lokales Entzündungsmodell der Maus übertragen: Durch wiederholte Injektion von TNF alpha in die Haut wurde eine leichte Entzündungsreaktion ausgelöst. Diese Entzündungsreaktion ließ sich schließlich durch intravenöse Verabreichung von pro-rmPR3- oder rmPR3-Antiserum verstärken. Dieser Befund ist ein wichtiger Beweis für die verbreitete Ansicht, daß c-ANCA nicht nur ein Epihänomen der WG darstellen, sondern selbst ein pathogenes Potential besitzen. Im zweiten Teil der vorliegenden Arbeit wurde die Beteiligung der beiden Serinproteasen NE und PR3 an der Entstehung inflammatorischer Prozesse untersucht. Im Hinblick auf die bei der Pathogenese der WG beteiligten Mechanismen könnten PR3 und NE eine wichtige Rolle spielen. PR3 und NE können Proteine der extrazellulären Matrix abbauen, Apoptose in Endothelzellen induzieren und sind an der Regulation entzündlicher Prozesse über verschiedene Wirkmechanismen beteiligt. Um quantitative Unterschiede bei Entzündungsreaktionen zwischen NE/PR3-defizienten Mäusen und kongenen Wildtyp-Tieren zu untersuchen, wurde als Modell einer Typ III Hypersensitivitätsreaktion eine lokale passive Arthus-Reaktion induziert. Wildtyp-Mäuse entwickelten dabei eine deutlich stärkere lokale Entzündung als NE/PR3-defiziente Mäuse. Weitere Studien sind nötig um die Frage zu klären, ob eine der beiden Serinproteasen alleine oder in Kooperation mit der anderen diesen Phänotyp hervorruft. Für einen direkten synergistischen Effekt sprechen indes die Ergebnisse eines in vitro Experiments mit pro-rmPR3 und humaner NE: Bei Inkubation von pro-rmPR3 mit hNE wurde eine Spaltung des Proenzyms beobachtet, die mit einer Verstärkung der enzymatischen Bruttoaktivität einherging. Die physiologische Relevanz dieser Beobachtung muß allerdings noch geklärt werden. Die Ergebnisse der vorliegenden Arbeit stehen im Einklang mit den neuesten Erkenntnissen über die Rolle der PR3 bei der Wegener’schen Granulomatose: PR3 dürfte sowohl aufgrund pleiotroper Effekte auf entzündliche Reaktionen als auch wegen seiner lytischen Eigenschaften zur Gewebeschädigung beitragen. Darüberhinaus konnte eine pathogene Wirkung von mPR3-spezifischen Antikörpern in der Maus nachgewiesen werden. / Wegener's granulomatosis (WG) is an autoimmune disorder typically characterized by chronic inflammmation of the upper respiratory tract, vasculitis and glomerulonephritis. WG belongs to the group of anti-neutrophil cytoplasmic autoantibody (ANCA) associated vasculitides with no or very few immune complex deposits (“pauci-immune”) in affected organs. “Classic” ANCA (c-ANCA) that produce a cytoplasmic staining pattern on neutrophils are a specific seromarker for this disease entity. They recognize conformational epitopes of proteinase 3 (PR3) from azurophil granules of neutrophil granulocytes. The correlation of PR3-specific antibody titers and disease course suggests that they are an important pathogenic factor for this autoimmune disease. The hypothesis is supported by in vitro experiments demonstrating an interaction of c-ANCA with cytokine primed neutrophils resulting in full activation manifested by degranulation and respiratory burst. It has been shown that c-ANCA can also directly induce the loss of endothelial barrier functions. However, direct evidence for the pathogenic potential of c-ANCA in vivo has not been published yet. The central aim of this study is to clarify the role of c-ANCA in WG. Since antibodies directed against human PR3 do not crossreact with the murine homolog, mPR3-specific antibodies were necessary to provide direct proof for c-ANCA mediated tisssue damage in a murine disease model. Hence, sufficient amounts of recombinant murine PR3 (mPR3) were necessary to generate mPR3 specific murine antibodies. Several attempts have been made to produce recombinant PR3 in prokaryotic and eukaryotic host systems. But conformational epitopes recognized by c-ANCA are not well preserved on recombinant PR3 derived from E. coli, P. pastoris, or baculovirus-infected insect cells described in the literature so far. While recombinant human PR3 expressed in eukaryotes is well recognized by c-ANCA, the yield of both active human and murine PR3 generated in eukaryotic expression systems is very low. To obtain sufficient amounts of correctly folded recombinant murine proteinase 3 (rmPR3) for multiple immunizations of PR3/NE-deficient mice, rmPR3 was produced as inclusion body material in E. coli as a catalytically inactive precursor molecule. After in vitro refolding the N-terminal propeptide was removed by limited proteolysis with the dipeptidylaminopeptidase cathepsin C yielding catalytically active enzyme. Due to its catalytic activity that could be inhibited by the physiologic inhibitor of human PR3, α1-antitrypsin, but not by secretory leukocyte protease inhibitor (SLPI), the recombinant material was assumed to harbour the correct conformation after refolding. To test, if anti-rmPR3 antibodies were sufficient to induce symptoms characteristic for WG, anti-PR3 antiserum was generated by immunization of PR3/neutrophil elastase (NE)-deficient mice with recombinant, refolded mPR3 or its zymogen. Specificity of the obtained sera was confirmed by ELISA, Western Blotting and indirect immunofluorescence. Moreover, antisera bound to the membranes of primed murine neutrophils as determined by flow cytometry. The generated antisera thus fulfilled the criteria defining c-ANCA positive sera of WG patients with respect to antigen specificity. If anti-PR3 antibodies are sufficient to induce symptoms characteristic for WG, transfer of antiserum to wildtype mice should induce vasculitis and/or glomerulonephritis. By repetitive intravenous injection of antiserum from immunized mice a persisting antibody titer was generated in naïve wild type mice over a treatment period of 10 weeks. Lung and kidney of these mice were analyzed histologically but neither granuloma or vasculitis were found in the lungs nor glomerulonephritis or vasculitis was observed in the kidneys. This result suggests that c-ANCA alone are not sufficient to induce WG symptoms in the mouse. Our initial observations were not surprising since the current hypothetical concept of c-ANCA-induced vasculitis implies that a primary inflammatory stimulus provided by cytokines like TNF alpha is required for the target antigen to be expressed on the plasma membrane of neutrophils. Exposure of the target antigen enables the binding of c-ANCA and subsequently triggers neutrophil activation. Consequently, this model, that was primarily deduced from in vitro experiments, was adapted to a model of local inflammation in the mouse: A mild inflammation was induced by repetitive local injection of TNF alpha into the skin. This inflammatory reaction increased significantly in the presence of rmPR3-antibodies. Our experimental data thus confirm the current concept that ascribes a pathogenic potential to c-ANCA. A second aspect adressed in this work is the contribution of the two neutrophil serine proteases NE and PR3 to the generation of inflammatory processes. With respect to the mechanisms involved in the pathogenesis of WG, NE and PR3 may be of particular importance due to their ability to degrade extracellular matrix proteins, induction of apoptosis in endothelial cells, and the regulation of inflammatory processes by a variety of mechanisms. A local reverse passive Arthus reaction (RPA) was chosen as a model of a type III hypersensitivity reaction to reveal quantitative differences of inflammation in PR3/NE-deficient mice and congenic wild type mice. Wild type mice reacted significantly stronger than PR3/NE-deficient mice as determined by examination of local edema and hemorrhage intensity. It remains to be determined if the observed phenotype in vivo reveals a concerted effect of both serine proteases or if deficiency of one of the proteases alone accounts already for this phenotype. Experimental data presented in this work are consistent with the hypothesis that PR3 and NE may directly interact: When recombinant mPR3 was incubated with hNE, a cleavage of rmPR3 was observed that is apparently associated with changes in enzymatic activity. The physiologic relevance of this finding has to be defined in further studies. In summary, this work adds to the current understanding of the role of PR3 in WG: PR3 is not only the relevant autoantigen whose interaction with c-ANCA contributes to the fatal outcome of the disease but also contributes together with neutrophil elastase to tissue damage by its lytic activity and pleiotropic effects on inflammatory reactions.

Human granulosa cells play an important role in the follicle, providing the oocyte with nutrients and growth factors to ensure successful ovulation. Normal granulosa cell functioning is thus crucial to human fertility. By studying their transcriptome, the mechanisms underpinning follicle development and infertility will be better understood. In this study, granulosa cells were retrieved at the time of oocyte removal for in vitro fertilization (IVF). Cells were purified through a combination of a Percoll gradient to remove red blood cells and positive selection of granulosa cell aggregations. On average, contamination by white blood cells was 2% as measured by FACS analysis using specific white blood cell markers. Histological and electron microscopy of granulosa cell aggregations did not detect evidence of resident ovarian white blood cells. This technique provided a good source of pure, healthy granulosa cells for RNA extraction and subsequent gene expression studies. The construction of a human granulosa cell SAGE library derived 1689 SAGEtags and 1289 discrete mRNA transcripts. SAGEtags for a number of well recognized granulosa cell genes (FSH receptor, follistatin, connexin 43) were found in addition to hormone receptors, multiple kinases, structural genes, apoptosis related genes and secreted proteins. A variety of other SAGE libraries were downloaded from SAGEmap (two ovary epithelium, ovary, white blood cell, brain, cerebellum, heart, liver, lung, kidney, pancreas and universal human reference) and compared to the human granulosa cell library. This was based on two measures: a gene specificity score (GSS) and a tissue specificity score (TSS). Three SAGEtags were identified with high levels of expression in granulosa cells, but no or low expression in the other libraries. These were retinol binding protein 1 (RBP 1), scavenger receptor class B member 1 (SCARB 1) and hydroxysteroid (11-beta) dehydrogenase 1 (11-β-HSD). Library comparisons were validated by real time RT-PCR. The TSS score revealed granulosa cells were most similar to the universal reference library and least similar to liver. Granulosa cell samples were collected from woman undergoing IVF for a range of infertility disorders. These included polycystic ovary syndrome (PCOS), tubal disease, endometriosis and idiopathic (unknown). Gene expression was compared between these groups using real time RT-PCR. Candidate genes included RBP 1, 11-β-HSD, SCARB 1, FSH receptor, follistatin, decidual protein induced by progesterone, and progesterone membrane receptor component 1 and 2. Granulosa cell gene expression was significantly different (p<0.05) from human white blood cells. No differences were found in gene expression levels between the infertility disorders. Analysis of each patient, however, revealed individuals with marked over-expression of selected genes. The technique of Generation of Longer cDNA fragments for Gene Identification (GLGI) was used to investigate seven SAGEtags that did match known genes or ESTs. Although no novel genes were characterized, a further 14 granulosa cell transcripts were identified by this technique. This thesis used an integrated approach to the study of human granulosa cell gene expression. This has involved development of a purification method, the use of a high-throughput technique (SAGE), bioinformatics tools to identify candidates genes, real time RT-PCR to investigate gene expression of particular genes in infertility disorders and finally a technique with the potential to characterize unknown SAGEtags (GLGI). This systematic approach has advanced the understanding of gene expression in human granulosa cells and identified avenues for future research into folliculogenesis and human infertility.

La dermatoscopia es una técnica diagnóstica y no invasiva que permite observar estructuras de la epidermis y de la dermis que son invisibles a simple vista. La dermatoscopia ha demostrado incrementar la precisión diagnóstica del melanoma. En la práctica dermatológica, existen numerosos tumores no melanocíticos que deben ser tenidos en cuenta en el diagnóstico diferencial del melanoma y que deben ser caracterizados dermatoscópicamente. En la presente tesis, se describen los hallazgos dermatoscópicos de las siguientes lesiones no melanocíticas: queratosis liquenoides, dermatofibromas, angioqueratomas y granulomas piogénicos. El primer trabajo es el estudio publicado donde se han evaluado, dermatoscópicamente, un mayor número de casos de queratosis seborreicas en regresión a queratosis liquenoides. En dicho trabajo, se ha demostrado que la dermatoscopia es una técnica que permite aumentar la precisión diagnóstica de estas lesiones (100% con el uso de la técnica y sólo el 25%, sin ella) y que el patrón granular grueso azul-grisáceo o marrón-grisáceo es muy característico de este tipo de lesiones. En el segundo trabajo se ha estudiado la evolución de la transformación de las queratosis seborreicas en queratosis liquenoides, lo que ha permitido comprobar la hipótesis de que las queratosis liquenoides son un fenómeno regresivo que aparece en algunas lesiones intra-epidérmicas. Otra conclusión que se obtiene del estudio es que la dermatoscopia es una técnica que, no sólo puede mejorar nuestra precisión diagnóstica, sino que también puede ayudarnos a estudiar la evolución de lesiones dinámicas, como las queratosis liquenoides, y conocer mejor su etiopatogenia. El tercer trabajo es el estudio publicado en el que se ha evaluado dermatoscópicamente un mayor número de dermatofibromas (412). De entre los hallazgos más relevantes, destacan la presencia de estructuras vasculares en el 49.5% de los dermatofibromas, hallazgo no descrito anteriormente; la comprobación de que el patrón formado por un retículo pigmentado discreto en periferia y un parche blanco central es el más frecuente en los dermatofibromas, aunque con un porcentaje más discreto (35%); la existencia de 10 patrones nuevos asociados a los dermatofibromas, de entre los que destaca el patrón atípico, indistinguible del melanoma, en el 6% de los dermatofibromas. El cuarto trabajo es el estudio dermatoscópico publicado sobre angioqueratomas solitarios con un mayor número de casos recogidos (32). El estudio estadístico de las estructuras dermatoscópicas reveló que la estructura “lagunas oscuras” era la más característica del angioqueratoma solitario, con una sensibilidad del 93.8%, una especificidad del 99.1%, un valor predictivo positivo de 93.8% y un valor predictivo negativo del 99.1%. No se encontraron lagunas oscuras en ningún melanoma ni en ningún carcinoma basocelular. El patrón que demostró ser más característico fue el patrón 1, aunque estadísticamente no fue superior a la estructura aislada “lagunas oscuras”. La dermatoscopia ha demostrado ser especialmente útil en el diagnóstico del angioqueratoma solitario, un tumor que crea importantes dificultades diagnósticas en la clínica. Finalmente, el quinto trabajo es el estudio dermatoscópico publicado sobre granulomas piogénicos con un mayor número de casos recogidos (122). El estudio estadístico de las estructuras dermatoscópicas reveló que ninguna es 100% específica del granuloma piogénico; sin embargo, las estructuras “collarete blanquecino” y “líneas blancas en rail” demostraron una alta especificidad (91% y 81%, respectivamente). Existen 3 patrones que no se han hallado en los melanomas amelanóticos del estudio y que representan el 52% de los patrones de presentación de los granulomas piogénicos. Sin embargo, los autores del estudio consideramos que la biopsia o la exéresis de todos los granulomas piogénicos debe ser recomendada; puesto que, aunque los patrones descritos no se hayan encontrado en los melanomas del estudio, las estructuras que lo forman, sí; pudiéndose dar el caso de su coexistencia en un melanoma. / DESCRIPTION AND EVALUATION OF THE DERMOSCOPIC STRUCTURES AND PATTERNS OF NONMELANOCYTIC CUTANEOUS LESIONS AND THE DIFFERENTIAL DIAGNOSIS WITH MELANOMA Dermoscopy is an in vivo, non-invasive technique that has revealed a new dimension of clinical morphology in pigmented and non-pigmented skin lesions. Previous studies have demonstrated that the use of dermoscopy improves the clinical accuracy in diagnosing melanoma and other skin lesions and may be used to clarify patterns in order to differentiate benign from malignant lesions. This work includes five studies that tries to describe dermoscopically 4 non-melanocytic skin tumours (lichenoid keratoses, dermatofibromas, solitary angiokeratomas and pyogenic granulomas). In the first study, we evaluated dermoscopically 24 cases of seborrheic keratoses regressing to lichenoid keratoses that illustrate the intermediate stage of this phenomenon. The two most common dermoscopic patterns associated with lichenoid keratoses were the localized and diffused granular patterns that are characterized by the presence of brownish grey, reddish brown, bluish grey or whitish grey coarse granules. In the second work, we studied the natural evolution of 22 other cases of intermediate stage of the regression of seborrheic keratoses in lichenoid keratoses using sequential dermoscopy imaging in time and we observed the complete or partial disappearing of the seborrehic keratosis part in all lesions. This finding support the proposal that lichenoid keratosis represents a regressive response to a pre-existent epidermal lesion. In the third study, we evaluated dermoscopically 412 dermatofibromas. Ten dermoscopic patterns were observed. The most common pattern seen in our series (34.7% of cases) was central white patch and peripheral pigment network, but in 65.3% of the cases, dermatofibromas presented different patterns including simulators of melanoma. Moreover, we observed vascular structures in 49.5% of dermatofibromas. In the fourth work, we evaluated dermoscopically 32 solitary angiokeratomas and 224 other tumours. The dermoscopic structure known as “dark lacunas” and the pattern consisting of “dark lacunas plus whitish veil” demonstrated a high sensitivity, specificity, positive predictive value, negative predictive value and reproducibility of solitary angiokeratomas. Finally, in the fifth study, we evaluated dermoscopically 122 pyogenic granulomas and 140 other reddish or pinkish tumours. Even though some dermoscopic patterns were useful in the recognition of pyogenic granulomas, dermoscopy does not substitute histology, mostly when vessels are present, since melanoma should not be ruled out. In conclusion, the present work confirms that dermoscopy is a helpful tool which may increase the clinician´s diagnostic accuracy of some non-melanocytic skin tumours and allows the observer to differentiate it from other cutaneous tumors, including malignant melanoma.

Würzburg, Univ., Diss., 2002.