Orientador: Márcio Zaffalon Casati / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-27T08:08:59Z (GMT). No. of bitstreams: 1 Monteiro_MabelledeFreitas_M.pdf: 1431989 bytes, checksum: 55bf42377e6ffeb3626cb5a8d6551c1c (MD5) Previous issue date: 2015 / Resumo: O Resumo poderá ser visualizado no texto completo da tese digital / Abstract: The Abstract is available with the full electronic digital document. / Mestrado / Periodontia / Mestra em Clínica Odontológica

Orientadores: Leonilda Maria Barbosa dos Santos, Alessandro dos Santos Farias / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T11:48:04Z (GMT). No. of bitstreams: 1 Santos_MarianaPeresAlmeida_M.pdf: 3287854 bytes, checksum: 80bab808b4f3e83a5512a78140733089 (MD5) Previous issue date: 2014 / Resumo: O Resumo poderá ser visualizado no texto completo da tese digital / Abstract: The Abstract is available with the full electronic digital document / Mestrado / Imunologia / Mestra em Genética e Biologia Molecular

O melanoma maligno é uma forma de câncer com alto índice de morte devido, em parte, à falta de tratamentos eficazes e à sua tendência de formar metástases. Nosso grupo tem desenvolvido vetores virais para a transferência gênica de fatores anti-tumorais e, inicialmente, foi construído um vetor adenoviral, AdPG, no qual a expressão do transgene é controlada por p53, um supressor de tumor e fator de transcrição. Sendo que aproximadamente 90% dos casos de melanoma retêm p53 selvagem, foi proposto que isto pudesse ser utilizado como uma ferramenta para dirigir a expressão do transgene codificado pelo vetor AdPG, um mecanismo apoiado por resultados anteriores de nosso grupo. Por exemplo, a transdução de células B16 (melanoma de camundongo, p53-selvagem, deleção de p19Arf) com vetores AdPG portadores de p19Arf ou interferon-<font face=\"Symbol\">b (IFN<font face=\"Symbol\">b) resultou em morte celular maciça enquanto a transferência de apenas um destes fatores isolados não causou o mesmo efeito. O trabalho descrito aqui inclui dois avanços tecnológicos críticos em comparação com trabalhos anteriores do grupo. Primeiramente, os transgenes de interesse (eGFP, p19Arf e IFN<font face=\"Symbol\">b) foram inseridos num vetor adenoviral que apresenta o tripetídeo RGD na sua proteína fibra. Essa modificação no vetor permite a eficiente transdução de um amplo espectro de células alvos sem a dependência do receptor viral do adenovírus selvagem, CAR. Além disso, foi construído um vetor bicistrônico, que contém a combinação de ambos os genes terapêuticos, como forma de garantir a transferência dos dois fatores ao mesmo tempo para as células-alvo. A inclusão de p19Arf, um supressor de tumor e inibidor de MDM2, como um gene terapêutico deve complementar as atividades do p53 celular endógeno e, como consequência, atuar na promoção da expressão a partir do vetor e também na inibição da proliferação das células tumorais. Porém, a transferência de p19Arf sozinho acarretaria efeito somente nas células que foram transduzidas e, então, seu efeito seria limitado. Por este motivo, descreve-se, além do p19Arf, a utilização de IFN<font face=\"Symbol\">b, uma proteína secretada com funções anti-tumorais, incluindo inibição de angiogênese, indução de apoptose e ativação da resposta imunológica. A estratégia do projeto contemplou vários níveis relacionados ao mecanismo do processo de transferência, incluindo a eficiência da transdução, o mecanismo de controle da expressão dos transgenes e as atividades dos transgenes. Assim, foi proposto que a combinação de p19Arf e IFN<font face=\"Symbol\">b pudesse ser uma estratégia interessante para induzir morte no tumor primário e uma resposta imunológica contra as células metastáticas. Com este projeto, foi iniciada a construção destes novos vetores aprimorados para transferência gênica nas células de melanoma. / Malignant melanoma is a type of cancer with high death rates, in part, because of a lack of efficient treatments and its tendency to generate metastases. Our group has developed viral vectors for the gene transfer of anticancer factors and, initially, we constructed an adenoviral vector, AdPG, in which transgene expression is controlled by p53, a tumor suppressor and transcription factor. As 90% of melanoma cases maintain wild-type p53, it was proposed that this could be used as a tool to drive transgene expression encoded by the AdPG vector, as evidenced by previous studies from our group. For example, transduction of B16 cells (mouse melanoma, wild-type p53, p19Arf-null) with vectors encoding p19Arf or interferon-<font face=\"Symbol\">b (IFN<font face=\"Symbol\">b) resulted in massive death cell, while transfer of just one of these factors alone did not cause the same effect. The work described here includes two critical technologic advances in comparison with our previous work. First, transgenes of interest (eGFP, p19Arf and IFN<font face=\"Symbol\">b) were inserted into an adenoviral vector which presents the RGD tripeptide in its fiber. This vector modification allows efficient transduction in a wide range of target cells without dependence on the wild type adenovirus receptor, CAR. In addition, a bicistronic vector was constructed which contains the combination of both therapeutic genes, ensuring the transfer of both factors to the target cells at the same time. Use of p19Arf, a tumor suppressor and MDM2 inhibitor, as a therapeutic gene should complement endogenous p53 activities and, as a consequence, promote expression from the AdPG vector and inhibit tumor cell proliferation. However, p19Arf gene transfer alone should have an effect only in transduced cells and, therefore, its effect would be limited. For this reason, we describe, in addition to p19Arf, the application IFN<font face=\"Symbol\">b, a secreted protein with antitumor functions, including inhibition of angiogenesis, induction of apoptosis and activation of immunologic response. This strategy involves several mechanistic levels related with the gene transfer process, including transduction efficiency, control over transgene expression and transgene activity. Therefore, it was proposed that the combination of p19Arf and IFN<font face=\"Symbol\">b could be an interesting strategy to induce primary tumor death and an immunologic response against metastatic cells. In this project, the construction of new vectors optimized for gene transfer in melanoma cells was initiated.

INTRODUÇÃO: A urticária crônica é caracterizada pelo aparecimento de placas eritêmato-edematosas, pruriginosas que perduram por mais de seis semanas. A investigação da imunidade inata é de grande importância para a compreensão da imunopatologia de doenças dermatológicas crônicas como a Urticária Crônica Idiopática (UCI). Entre os componentes da resposta inata, a avaliação das células dendríticas plasmocitóides (pDCs) e sua capacidade de ativação por agonistas de Toll Like receptor 9 (TLR9) pode contribuir para avaliar o desequilíbrio imunológico encontrado na UCI. OBJETIVOS: Investigar a imunidade inata em pacientes com UCI e os mecanismos envolvidos na modulação da produção de IFN- por pDCs induzida pela ativação do TLR9. MÉTODOS: A secreção de IFN- e IL-10 por leucócitos de pacientes UCI (n=26) e indivíduos normais (Co, n=50) induzida pelo agonista de TLR9, CpGA (1.0 e 2.0M), ou IL12p40 induzida pelo ODN inibitório (ODN S) foi realizado por ensaio imunoenzimático. Quantificação de pDCs em sangue periférico, expressão de moléculas coestimulatórias, expressão intracelular de IFN- por pDCs e fosforilação de STAT (1 e 4) foram analisados por citometria de fluxo. Expressão do mRNA de TLR9 e fator regulador de interferon-7 foi realizado por real time PCR. A presença da pDC (CD123+) em lesões de pele de pacientes UCI foi avaliada por imunoistoquímica. RESULTADOS: Os resultados mostraram uma diminuição na produção de IFN- e IL-10 por leucócitos de pacientes UCI após estímulo com CpGA. O estímulo com ODN S induziu a produção de IL12p40 e suprimiu ainda mais a produção de IL-10 e IFN- induzida pelo CpGA, até 98% de inibição. O número de pDCs no sangue periférico e a capacidade de ativação da pDC nos pacientes, avaliada pela expressão de moléculas co-estimuladoras foi semelhante aos indivíduos normais. Além disto, foram detectadas raras pDCs nas lesões de pele dos pacientes. Um aumento constitutivo da fosforilação de STAT1 foi detectado em linfócitos não estimulados de pacientes UCI associado a uma diminuição da expressão de mRNA de TLR9 após estímulo com CpGA e alterada expressão de mRNA de IRF-7, que podem contribuir para a baixa produção de IFN-. CONCLUSÃO: Os achados mostram uma inibição na produção de IFN- via TLR9, tendo como alvo a pDC e sua participação na resposta inata na Urticária Crônica / INTRODUCTION: Chronic urticaria is a skin disorder characterized by recurrent and transitory itchy weals occurring regularly more than 6 weeks. The investigation of the innate immunity is an important parameter to understand the immunopathology of chronic dermatological diseases, such as Chronic Idiopathic Urticaria (CIU). Among the innate immune components, such as plasmacytoid dendritic cells (pDC) and their functional study activation thorough TLR-9 ligand could contribute to evaluate the immunologic disequilibrium founded in the CIU. OBJECTIVES: We decided to investigate innate immunity in CIU and the mechanisms implicated in the modulation of IFN- production by pDCs upon TLR9 activation. METHODS: The secretion of IFN- and IL-10 secretion by leucocytes of patients with CIU (n=26) and healthy control (HC, n=50) induced by TLR9 ligand, CpG type A (1.0 and 2.0M), or IL12p40 secretion induced by inhibitory ODN (ODN S) was assessed by enzyme-linked immunosorbent assay. Enumeration of pDC, coestimulatory molecules expression, intracellular IFN- expression, and STAT (1 and 4) phosphorylation were assessed by flow cytometry. TLR9 and interferon regulatory factor-7 mRNA transcripts were evaluated by real-time PCR. The presence of pDCs (CD123+) in the skin lesion of patients with CIU were assessed by means of immunohistochemistry staining. RESULTS: The findings showed a decreased IFN- and IL-10 secretion by leucocytes of CIU patients induced by CpGA. Inhibitory ODN was able to induce IL12p40 and further inhibited the IFN- secretion in both HC and patients achieving up 98% inhibition. A normal pDC percentage and degree of activation by costimulatory molecules expression was observed in CIU in patients was similar to control group. Moreover, rare presence of pDCs was detected in the skin lesion of patients. An increased constitutive STAT1 phosphorylation on non-stimulated lymphocytes and CpGA activation induced a down-regulation of TLR9 mRNA transcripts associated with altered IRF-7 along the time of CpG activation which may contribute to the decreased IFN- secretion. CONCLUSIONS: Altogether, the findings showed an impaired IFN- secretion via TLR-9, implicating innate immunity through pDCs as target in Chronic Urticaria

Soňa Fraňková: Role of host-dependent factors in prediction of antiviral treatment response in chronic hepatitis C Abstract Hepatitis C virus infection represents a leading cause of liver disease in western countries. The primary goal of HCV therapy is elimination of the virus, i.e. sustained virological response (SVR) achievement. Genetic factors have long been suspected of playing a crucial role in determining response to IFN-α-based therapies, but pretreatment predictors of response were only poorly defined and did not allow personalization of therapy. The aim of the thesis is to describe the role of host-dependent factors in prediction of antiviral treatment response in chronic hepatitis C in specific groups of patients. First, we focused on the role of the IFNG -764G/C promoter variant in SVR achievement. We did not prove that this variant predicted SVR in Czech HCV-infected individuals. Next, we focused on the role of IL28B and IFNL4 in HCV-infected patients: we confirmed that the IL28B rs12979860 CC genotype slows down the progression of liver fibrosis in chronic HCV infection and that IFNL4 ss469415590 TT|ΔG genotyping does not bring a better prediction of treatment success than IL28B rs12979860 in the Czech population. Third, we assessed prediction of treatment response in HCV positive liver...

Lung cancer is one of the main causes of death worldwide, with most patients suffering from an advanced unresectable or metastatic non-small cell lung cancer. The mortality trends are mostly related to patterns of tobacco use, specifically from cigarettes. Tobacco is the basic etiological agent found as a consequence of the inhalation of tobacco smoke. Published data show the use of interferons (IFNs) in the treatment of lung tumours due to their potential in displaying antiproliferative, anti-angiogenic, immunoregulatory, and proapoptotic effects. Type1 IFNs have been employed as treatments for many types of cancer, both for haematological cancers and solid tumours. The IFN-γ (naked) functions as an anticancer agent against various forms of cancer. Hence, this study aimed to investigate the genoprotective and genotoxic effects of IFN-γ liposome (nano) on 42 blood samples from lung cancer patients, compared to the same sample size from healthy individuals. The effectiveness of IFN- γ liposome against oxidative stress was also evaluated in this study. A concentration of 100U/ml of IFN-γ liposome was used to treat the lymphocytes in: Comet and micronucleus assays, Comet repair, Western blotting and real-time polymerase chain reaction (qPCR) were based on a preliminary test for the optimal dose. The lymphocytes from lung cancer patients presented with higher DNA damage levels than those of healthy individuals. IFN-γ liposome was not found to induce any DNA damage in the lymphocytes. Also, it caused a significant reduction in DNA damage in the lymphocytes from lung cancer patients in; Comet, Comet repair and micronucleus assays. Furthermore, the 100U/ml of IFN-γ liposome significantly reduced the oxidative stress caused by H2O2 and appeared to be effective in both groups using the Comet and micronucleus assays. Results from; Comet, Comet repair and micronucleus assays were consistent. The data obtained indicated that IFN-γ in both forms (naked INF-γ and INF-γ nano-liposome) may potentially be effective for the treatment of lung cancer and showed the ability of IFN-γ liposome to reduce the DNA damage more than the naked form. The IFN-γ in both forms has also shown anti-cancer potential in the lymphocytes from lung cancer patients by regulating the expression of p53, p21, Bcl-2 at mRNA and protein levels by up-regulating the p53 and p21 to mediate cell cycle arrest and DNA repair in lung cancer patients. The findings of this study are consistent with the view that the naked IFN-γ and liposome could have a significant role in cancer treatment, including lung cancer. / Mutah University in Jordan

Die myotone Dystrophie ist eine Multisystemerkrankung, die sich hauptsächlich durch Myotonie, Muskelschwäche, sowie einen früh beginnenden Katarakt und kardiale Erregungsleitungsstörungen auszeichnet. Im Laufe der Zeit wurden zwei Typen dieser Erkrankung definiert, welche auf unterschiedlichen genetischen Defekten basieren. Dem Typ 1 der myotonen Dystrophie (DM1) liegt eine Expansion der CTG-Wiederholungssequenz im 3‘-untranslatierten Bereich des myotonic dystrophy protein kinase (DMPK) Gens auf dem Chromosom 19q13.3 zugrunde. Der myotone Dystrophie Typ 2 (DM2) wird hingegen durch eine Expansion der CCTG-Wiederholungssequenz im Intron 1 des CCHC-type zinc finger nucleic acid binding protein (CNBP) Gens auf Chromosom 3q21 verursacht. Trotz ähnlicher klinischer Symptome und pathologischer Mechanismen konnten wichtige Unterschiede zwischen den beiden Typen aufgedeckt werden. Daher müssen beide Formen der myotonen Dystrophie als eigenständige Erkrankungen betrachtet werden. Vorarbeiten der Günther-Arbeitsgruppe sowie zwei unabhängige Studien konnten ein erhöhtes Vorkommen von Autoimmunerkrankungen bei DM2 Patienten im Vergleich zur gesunden Population und zu DM1 Patienten feststellen. In dieser Arbeit wurde zudem eine erhöhte Expression von Interferon stimulierten Genen (ISGs) in Fibroblasten von DM2 Patienten nachgewiesen. Eine chronische Interferon Produktion kann zu der Entwicklung von Autoimmunerkrankungen führen, bei denen das körpereigene Immunsystem nicht nur pathogene, sondern auch endogene Strukturen angreift. Warum Autoimmunerkrankungen bei DM2 Patienten häufiger auftreten, war bisher nicht bekannt. Daher war das Ziel dieser Arbeit den zugrundeliegenden Mechanismus für dieses Phänomen zu untersuchen. Rezeptoren des Immunsystems können modifizierte Nukleinsäuren erkennen, wenn diese in hoher Konzentration in der Umgebung des Rezeptors vorliegen. Daher wurde zunächst die Lokalisation der in DM2 Patienten Fibroblasten zahlreich vorkommenden CCTG Wiederholungssequenzen untersucht. Dabei zeigte sich, dass die DM2 spezifischen RNA Wiederholungssequenzen nicht nur im Nukleus, sondern auch im Zytoplasma auftraten. Eine direkte Erkennung dieser zytosolischen RNA Wiederholungssequenzen durch die im Zytoplasma lokalisierten RNA Rezeptoren konnte jedoch nicht nachgewiesen werden. Allerdings konnte die Translation dieser RNA Wiederholungssequenzen durch den Prozess der Repeat assoziierten non-ATG (RAN) Translation mittels Nachweis der RAN Proteine LPAC und QAGR festgestellt werden. Diese RAN Proteine, sowie die Akkumulation der RNA Wiederholungssequenzen können einen zellulären Stress in den DM2 Patienten Fibroblasten auslösen, der sich in einem chronischen endoplasmatischen Retikulum (ER) Stress manifestierte. Der chronische ER Stress in den Fibroblasten von DM2 Patienten zeichnet sich vor allem durch eine Aktivierung des ATF6 Signalweges aus, um die Anpassung der Zellen an langanhaltenden Stress zu unterstützen. Interessanterweise zeigte sich eine Verbindung zwischen der erhöhten ISG Expression und der Aktivierung des ATF6 Signalweges, da eine Herunterregulierung von ATF6 in DM2 Patienten Fibroblasten zu einer Verringerung der erhöhten ISG Expression führte. Der chronische Stress innerhalb der Patienten Fibroblasten erfasste auch die Mitochondrien, was durch eine erhöhte Menge von mitochondrialen reaktiven Sauerstoffspezies (ROS), sowie eine Herunterregulierung von wichtigen mitochondrialen Genen gezeigt wurde. Bemerkenswerterweise war ein erhöhtes Vorkommen mitochondrialer DNA (mtDNA) im Zytoplasma der DM2 Patienten detektierbar, wobei eine erhöhte Apoptose Rate als Auslöser für die mtDNA Freilassung ausgeschlossen werden konnte. Durch eine Depletierung der mtDNA wurde ebenfalls eine Verringerung der ISG Expression in DM2 Patienten Fibroblasten erreicht. Dies wies nicht nur daraufhin, dass die mtDNA in die erhöhte Interferon Produktion in den Patientenzellen beteiligt ist, sondern auch auf eine Verbindung zwischen dem ER und den Mitochondrien. Um zu verstehen, wie mtDNA zu einer erhöhten Interferon Produktion führen kann, wurde der zytosolische DNA Rezeptor cGAS sowie das nachfolgende Adapterprotein STING herunterreguliert. Erstaunlicherweise führten diese Herunterregulierungen zu einer Verringerung der ISG Expression in Fibroblasten von DM2 Patienten. Dies deutet daraufhin, dass die erhöhte Produktion von Interferon in den Zellen der DM2 Patienten durch die Aktivierung des cGAS-STING Signalweges ausgelöst wird. In dieser Arbeit konnte eine erhöhte Stressantwort in Fibroblasten von DM2 Patienten festgestellt werden, die möglicherweise durch die gemeinsame Akkumulierung von RNA Wiederholungssequenzen und RAN Proteinen im Zytoplasma ausgelöst wird. Dieser Stress äußert sich in chronischem ER- und mitochondrialem Stress und führt zu einer Permeabilisierung der mitochondrialen Membran einzelner Mitochondrien. Dadurch können geringe Mengen mtDNA in das Zytoplasma freigesetzt werden, ohne den Prozess der Apoptose auszulösen. Die zytosolische mtDNA aktiviert den cGAS-STING Signalweg und führt zu einer erhöhten Produktion von Interferon, welche die Patienten für die Entwicklung von Autoimmunerkrankung prädisponiert. Durch den in dieser Arbeit aufgedeckten Mechanismus eröffnen sich potentielle therapeutische Ansatzpunkte für die bisher nicht behandelbare Erkrankung. Eine Hemmung des cGAS-STING Signalweges könnte die Entwicklung von Autoimmunerkrankungen bei den DM2 Patienten reduzieren. Dies ließe sich durch die Einbeziehung von DM2 Patienten in klinische Studien mit cGAS oder STING Inhibitoren prüfen. / Myotonic dystrophy, a multi-systemic disorder, is primarily characterised by myotonia, muscle weakness, early-onset cataracts, and cardiac conduction defects. Over time, two distinct types of this disease have been defined, each with unique genetic defects. Myotonic dystrophy type 1 (DM1) is caused by a CTG repeat expansion in the 3’ untranslated region of the myotonic dystrophy protein kinase (DMPK) gene on chromosome 19q13.3. In contrast, myotonic dystrophy type 2 (DM2) is caused by a CCTG repeat expansion in intron 1 of the CCHC-type zinc finger nucleic acid binding protein (CNBP, former known as ZNF9) gene on chromosome 3q21. Despite the similarity in clinical presentation and pathological mechanisms, crucial differences between the two types have been uncovered, necessitating their consideration as distinct disease entities. Preliminary investigations by the Günther group, along with two independent studies have revealed a higher prevalence of autoimmune disorders in DM2 patients compared to the healthy population and DM1 patients. Furthermore, the current study has demonstrated an upregulation of interferon stimulated genes (ISGs) in fibroblasts derived from DM2 patients. Chronic interferon production can lead to the development of autoimmune disorders, causing the body´s immune system to mistakenly attack not only pathogens but also endogenous structures. Such misdirected immune responses can result in severe symptoms, significantly compromising the patients’ quality of life. The underlying mechanism for the increased incidence of autoimmune disorders in DM2 patients remains elusive, and this study aimed to unravel the mechanism responsible for this phenomenon. Innate receptors of the immune system can recognise modified nucleic acids when present in high concentrations in the receptor’s environment. Therefore, the localisation of the abundant CCTG repeats in DM2 patient fibroblast was investigated. Accumulation of these repeats was observed not only in the nucleus but also in the cytoplasm. However, there was no evidence for direct recognition of these cytosolic RNA repeats by cytoplasmic RNA receptors. Nevertheless, the translation of these RNA repeats through the process of repeat-associated non-ATG (RAN) translation was confirmed by the detection of the RAN proteins LPAC and QAGR. These RAN proteins and the accumulation of RNA repeats may contribute to cellular stress response, manifesting as chronic endoplasmic reticulum (ER) stress in fibroblasts derived from DM2 patients. Chronic ER stress in DM2 patient fibroblasts is characterised by the activation of the ATF6 signalling pathway, which is thought to support the adaptation of cells to prolonged stress. Interestingly, a connection between the increased ISG expression levels and ATF6 pathway activation was established, as a reduction of ATF6 in DM2 patient fibroblasts led to a reduction of ISG levels. The chronic stress within the patient fibroblasts also extended to the mitochondria. Evidence of mitochondrial stress was found in the form of increased mitochondrial reactive oxygen species (ROS) and downregulation of essential mitochondrial genes. Notably, an increased presence of mitochondrial DNA (mtDNA) in the cytoplasm of DM2 patient fibroblasts was detected, although its release due to a high apoptosis rate was ruled out. Remarkably, depletion of this mtDNA also resulted in a reduction of ISG expression levels in DM2 patient fibroblasts, indicating the involvement of mtDNA in the increased interferon production in these patients and a connection between the ER and mitochondria. To elucidate how mtDNA can lead to increased interferon production, knockdowns of the cytoplasmic DNA sensing receptor cGAS and the downstream adaptor protein STING were performed. Surprisingly, this genetic manipulation led to a reduction of ISG expression levels in DM2 patient fibroblasts, suggesting that the increased interferon production in DM2 patients is triggered by the activation of the cGAS-STING signalling pathway. This study unveils a heightened stress response in fibroblast derived from DM2 patients. This elevated stress is likely triggered by the combined effect of the RNA repeat accumulation and the presence of RAN proteins in the cytoplasm, manifesting as chronic ER and mitochondrial stress. This persistent stress may lead to the selective permeabilization of mitochondrial membranes, allowing the release of mtDNA into the cytoplasm without inducing apoptosis. Remarkably, this cytoplasmic mtDNA activates the cGAS-STING signalling pathway, resulting in increased interferon production. This cascade of events predisposes DM2 patients to developing autoimmune disorders. The mechanism revealed in this study opens up a new perspective on potential therapies for DM2 patients, as effective treatments have been lacking thus far. The involvement of the cGAS-STING pathway provides the opportunity to explore the use of cGAS or STING inhibitors, which are currently in clinical trials, as a therapeutic approach for DM2 patients.

<p>In sera remitted for anti-nuclear antibody (ANA) analysis, the supplement of a sensitive anti-SSA/Ro ELISA to the conventional ANA screening by immunofluorescence (IF) revealed that one fourth of the individuals with IF-ANA negative, but SSA/Ro ELISA positive sera, had systemic lupus erythematosus (SLE) or cutaneous LE. Consequently, adding a sensitive anti-SSA/Ro ELISA to the ANA screening is valuable for the serological detection of ANA negative SLE/LE patients.</p><p>SLE patients often have measurable interferon-alpha (IFN-α) levels in serum, and IFN-α treatment of patients with non-autoimmune diseases can induce SLE. Thus, the type I IFN system seems to be important in SLE and was therefore investigated. Initially, a decreased IFN-α producing capacity, due to a 70-fold reduction in the number of circulating natural IFN-α producing cells (NIPC), was noted in peripheral blood mononuclear cells (PBMC) from SLE patients. SLE-sera contained an endogenous IFN-α inducing factor (SLE-IIF), consisting of IgG and DNA in the form of small immune complexes (300-1000 kD). The SLE-IIF selectively activated NIPC and was more common in sera from patients with active disease compared to individuals with inactive disease. IFN-α producing cells could be detected by immunohistochemistry in both lesional and unaffected skin from SLE patients, and IFN-α gene transcription could be verified by in situ hybridisation in some of the skin biopsies. A reduced number of NIPC, detected by expression of the blood dendritic cell antigen (BDCA)-2, was noted among SLE-PBMC. The IFN-α production triggered by SLE-IIF in SLE-PBMC was inhibited by monoclonal antibodies (mAbs) to BDCA-2 and markedly decreased by anti-BDCA-4 mAbs. </p><p>The observations in the present thesis may explain the ongoing IFN-α production in SLE patients, indicate an important role for the activated type I IFN system in the pathogenesis, and suggest that direct targeting of SLE-NIPC may constitute a new therapeutic principle in SLE.</p>

In sera remitted for anti-nuclear antibody (ANA) analysis, the supplement of a sensitive anti-SSA/Ro ELISA to the conventional ANA screening by immunofluorescence (IF) revealed that one fourth of the individuals with IF-ANA negative, but SSA/Ro ELISA positive sera, had systemic lupus erythematosus (SLE) or cutaneous LE. Consequently, adding a sensitive anti-SSA/Ro ELISA to the ANA screening is valuable for the serological detection of ANA negative SLE/LE patients. SLE patients often have measurable interferon-alpha (IFN-α) levels in serum, and IFN-α treatment of patients with non-autoimmune diseases can induce SLE. Thus, the type I IFN system seems to be important in SLE and was therefore investigated. Initially, a decreased IFN-α producing capacity, due to a 70-fold reduction in the number of circulating natural IFN-α producing cells (NIPC), was noted in peripheral blood mononuclear cells (PBMC) from SLE patients. SLE-sera contained an endogenous IFN-α inducing factor (SLE-IIF), consisting of IgG and DNA in the form of small immune complexes (300-1000 kD). The SLE-IIF selectively activated NIPC and was more common in sera from patients with active disease compared to individuals with inactive disease. IFN-α producing cells could be detected by immunohistochemistry in both lesional and unaffected skin from SLE patients, and IFN-α gene transcription could be verified by in situ hybridisation in some of the skin biopsies. A reduced number of NIPC, detected by expression of the blood dendritic cell antigen (BDCA)-2, was noted among SLE-PBMC. The IFN-α production triggered by SLE-IIF in SLE-PBMC was inhibited by monoclonal antibodies (mAbs) to BDCA-2 and markedly decreased by anti-BDCA-4 mAbs. The observations in the present thesis may explain the ongoing IFN-α production in SLE patients, indicate an important role for the activated type I IFN system in the pathogenesis, and suggest that direct targeting of SLE-NIPC may constitute a new therapeutic principle in SLE.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-11-21T16:17:01Z No. of bitstreams: 1 carla-franca-wolanski-de-almeida.pdf: 1099876 bytes, checksum: e454fb4f1e8e4f2a5e837e602abcf47e (MD5) / Made available in DSpace on 2012-11-21T16:17:01Z (GMT). No. of bitstreams: 1 carla-franca-wolanski-de-almeida.pdf: 1099876 bytes, checksum: e454fb4f1e8e4f2a5e837e602abcf47e (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / Controle em processo é parte das ações para garantia da qualidade dos produtos a fim de que eles atendam aos requisitos mínimos estabelecidos para seu uso. Os resultados obtidos nos ensaios orientam na tomada de decisões, bem como em ajustes ao processo de produção. Muitas vezes tais resultados são condições imperativas para as etapas subsequentes. O monitoramento do processo produtivo se dá a partir do estabelecimento dos pontos críticos de controle, que precisam ser verificados a cada lote, pois interferem diretamente na qualidade e rendimentos da produção. Portanto, a confiabilidade e agilidade deresposta são fundamentais para correta tomada de decisão e/ou ajuste. O objetivo deste trabalho é propor um modelo de laboratório de controle em processo nos aspectos gerenciais e principalmente nos aspectos técnicos aplicados ao processo de produção do biofármaco Interferon alfa 2b humano recombinante. Este produto, oferecido no portfólio de Bio-Manguinhos encontra-se em processode transferência de tecnologia com o Instituto cubano CIGB. Com base nos dados da literatura foram estabelecidas as principais etapas do processo produtivo a para expressão de proteínas heterólogas, em sistema recombinante utilizando-se a bactéria Escherichia coli. Os pontos críticos de controle foram estabelecidos utilizando-se a metodologia de análise de riscos HACCP (Hazard Analisys Critical Control Point). Para as análises de controle foram propostas metodologias analíticas que proporcionem a consistência de resultados e que possam garantir a qualidade final do processo de manufatura do produto. O modelo proposto também enfatiza a conduta de ações rotineiras de forma que qualquer desvio possa ser imediatamente identificado e suas causas apuradas além de executar as análises com segurança. Os critérios gerenciais e técnicos abordados foram avaliados criticamente, utilizando-se os procedimentos do sistema de garantia da qualidade vigentes no Instituto, bem como na estrutura do laboratório de controle em processodestinado à vacina contra Hib, que foi adotado como premissa básica, fruto da transferência de tecnologia bem sucedida em Bio-Manguinhos. A proposta elaborada poderá ser utilizada em outros laboratórios destinados ao controle em processo de novos produtos em Bio-Manguinhos e enfatiza os critérios técnicos como condicionais para confiabilidade de medição. / In-process control is a part of the actions to guarantee the quality for final products in order to attend the minimum requirements established for their using. The results obtained from analytical assays guide on decision-making as well as production process adjustments. Often, these results are conditioned to the follow the subsequent stages. The process monitoring occurs from the establishment of critical control points to verify in each batch because theydeterminate product quality and process yield. Therefore, the reliability and speedof response are critical for correct decision-making and adjustment. The objective of this work is to propose a model in process control laboratory in management aspects and especially the technical aspects applied to the production of human recombinant interferon alpha 2b biopharmaceutical, object of this study. Bio-Manguinhos supplies this product in its portfolio, which production technology is being transferred from the CIGB, a Cuban Institution. Based on literature data the main steps were established for a production process of heterologous proteins expressed using a recombinant system with the Escherichia colibacteria. The critical control points were established using the risk assessment methodology HACCP, Hazard analysis Critical Control, HACCP. Analytical methodologies were proposed to improve results consistency and assure the final quality of production process. The proposed model emphasizes the routine actions, so that, any deviation could be immediately identified, their causes verified and activities performed reliably. The management and technical criteria described have been critically evaluated using the Bio-Manguinhos institute quality assurance procedures as well as compared to the structure of the in-process control laboratory for Hib vaccine. This laboratory, adopted as basic premise, resulted from a successful technology transfer in Bio-Manguinhos. The proposal drawn up can be used in other laboratories for in- process control in new products in Bio-Manguinhos and emphasizes the technical criteria for conditional measurement reliability.