DUTRA, Brunheld Maia. Efeito imunomodulador de CXCL10 em camundongos BALB/C infectados com cepa de Leishmania braziliensis refratária ao tratamento com antimônio. 2015. 78 f. Dissertação (Mestrado em Patologia) – Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2015. / Submitted by denise santos (denise.santos@ufc.br) on 2015-12-01T11:34:02Z No. of bitstreams: 1 2015_dis_bmdutra.pdf: 917921 bytes, checksum: 088bf681abe483ebcbf34475f6929795 (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2015-12-01T11:36:00Z (GMT) No. of bitstreams: 1 2015_dis_bmdutra.pdf: 917921 bytes, checksum: 088bf681abe483ebcbf34475f6929795 (MD5) / Made available in DSpace on 2015-12-01T11:36:00Z (GMT). No. of bitstreams: 1 2015_dis_bmdutra.pdf: 917921 bytes, checksum: 088bf681abe483ebcbf34475f6929795 (MD5) / Leishmania braziliensis from antimony-resistant patient is able to induce high levels of IL-4 and Arginase in mice, contributing to the increased virulence of the strain and the severity of the disease. CXCL10 is a chemokine that recruits and activates Th1 cells, NK, macrophages, dendritic cells and lymphocytes B. The objective of this study was to evaluate in vivo the effect of CXCL10 in the infection by strain of L. braziliensis refractory to treatment with antimony. BALB/c mice (n=64) were infected intradermally in the right ear 107 promastigotes (20 µl), and after appearance of lesions (5ª week), the animals were divided into four groups (16/group): 1. Control untreated; 2.Glucantime (100mg/kg/day, I.M.); 3.CXCL10 (100 ng/10μL, I.M.); 4.Glucantime+CXCL10 (100ng/10µL+100mg/kg/day, I.M.). The animals were treated for 7 days, followed by weekly measurements of lesions, and were euthanized in the first and fourth week post-treatment (w.p.t) for the evaluation of some parameters: parasite burden (lesions, and draining lymph node-LN), production of the cytokines IFN-γ, IL-4, IL-10 and TGF-β (LN) and histological changes (lesions). The results showed CXCL10 and Glucantime induced, early as the first s.p.t., non-ulcerated lesions that regressed significantly (0.35 ± 0.07, 0.047 ± 0.27, p <0.05) compared to controls (0.63 ± 0.12) and Glucantime (0.56 ± 0.12). This was associated with a significant decrease in parasite load observed in CXCL10 and CXCL10 + Glucantime groups, in the lymph node (1.17x103 ± 0.85x103, 2.05x103 ± 1.50x103, respectively) compared to control (3.2x104 ± 1.97x104) and Glucantime (1.18x105 ± 1.12x105). Regarding to cytokines, CXCL10 treated animals showed high levels of IFN-γ, IL-10 and TGF-β, and low IL-4 production. In contrast, was observed in the control and Glucantime groups increased production of IL-4. These data also corroborate the histopathological findings showing that animals treated with CXCL10, with or without Glucantime, showed a non-exacerbated inflammation, with absence of necrosis, lower parasitism, granulomas and cellular infiltrate with more activated macrophages, many lymphocytes and few plasma cells, compared to control and Glucantime. In conclusion, CXCL10 and the association CXCL10+Glucantime induced minor injuries, lesions non-ulcerated, with decreased earlier parasite load, leading to the resolution of the disease through an immune response with high IFN-γ and low IL-4 production, and with the control of inflammation mediated by IL-10 and TGF-β. / Leishmania braziliensis proveniente de paciente resistente ao antimônio é capaz de induzir altos níveis de IL-4 e de Arginase em camundongos, contribuindo para a maior virulência da cepa e gravidade da doença. CXCL10 é uma quimiocina que recruta e ativa células Th1, NK, macrófagos, células dendríticas e linfócitos B. O objetivo deste trabalho foi avaliar in vivo o efeito de CXCL10 na infecção por cepa de L. braziliensis refratária ao tratamento com antimônio. Camundongos BALB/c (n=64) foram infectados por via intradérmica na orelha direita, 107 promastigotas (20µL) e após o aparecimento das lesões (5a sem), os animais foram divididos em quatro grupos (16 animais/grupo): 1. Controle não tratado; 2. Glucantime (100mg/kg/dia, I.M); 3. CXCL10 (100ng/10µL, I.M.); 4. Glucantime+CXCL10 (100ng/10µL + 100mg/kg/dia, I.M.). Os animais foram tratados por 7 dias, acompanhados com medidas semanais das lesões e eutanasiados na 1ª e 3ª semanas pós-tratamento (s.p.t.) para a avaliação de alguns parâmetros: carga parasitária (lesões e linfonodo de drenagem-LN), produção das citocinas IFN-γ, IL-4, IL-10 e TGF-β (linfonodo) e alterações histológicas (lesões). Os resultados mostraram que CXCL10 e CXCL10+Glucantime induziram, já a partir da 1a s.p.t, lesões não ulceradas e que regrediram significativamente (0,35±0,07; 0,047±0,27; p<0,05), quando comparado ao Controle (0,63±0,12) e ao Glucantime (0,56±0,12). Isso foi relacionado com a importante diminuição da carga parasitária observada nos grupos CXCL10 e CXCL10+Glucantime, no LN (1,17x103 ± 0,85 x 103; 2,05x103 ± 1,50x103, respectivamente) quando comparado ao Controle (3,2x104 ± 1,97x104) e ao Glucantime (1,18x105 ± 1,12x105). Em relação às citocinas, o tratamento com CXCL10 mostrou altas concentrações de IFN-γ, IL-10 e TGF-β, e baixa produção de IL-4. Em contrapartida, nos grupos Controle e Glucantime, foi observada maior produção de IL-4. Esses dados corroboram também com os achados histopatológicos que mostraram que os animais tratados com CXCL10, associado ou não ao Glucantime, apresentaram uma inflamação não exacerbada, com ausência de necrose, menor parasitismo, presença de granulomas e infiltrado celular com mais macrófagos ativados, muitos linfócitos e poucos plasmócitos, em relação ao Controle e ao Glucantime. Em suma, CXCL10 e a associação CXCL10+Glucantime induziram lesões menores, não ulceradas, com diminuição da carga parasitária mais precocemente, conduzindo à resolução da doença através de uma resposta imunológica com alta produção de IFN-γ e baixa produção de IL-4, e controle da inflamação modulado por IL-10 e TGF-

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Leishmania braziliensis à o agente etiolÃgico mais comum da leishmaniose cutÃnea no Brasil. Camundongos BALB/c sÃo os mais susceptÃveis à infecÃÃo por L. braziliensis, embora desenvolvam lesÃes pequenas e nÃo ulceradas. As lesÃes em BALB/c infectados com L. braziliensis sÃo acompanhadas pela expressÃo de um amplo espectro de quimiocinas, entre elas CXCL10, que à capaz de atrair a ativar cÃlulas NK e Th1 com produÃÃo de IFN-&#947;. O objetivo deste trabalho foi avaliar se a quimiocina CXCL10, administrada nos estÃgios iniciais da infecÃÃo, poderia alterar o curso da doenÃa, dimunindo o tempo de cura. Para isso, camundongos BALB/c (n=24) foram infectados com L. braziliensis e divididos em dois grupos: um grupo (n=12) recebeu CXCL10 (100 ng/5ÂL) e o outro (n=12) PBS (5ÂL), nos dias 1, 3, 5 e 7 dias de infecÃÃo. Alguns parÃmetros foram avaliados: a espessura das lesÃes a cada trÃs dias; a carga parasitÃria na pata e no linfonodo de drenagem, a concentraÃÃo de NO, IFN-&#947;, IL-12p40, TNF-&#945;, IL-4, IL-10 e TGF-&#946;, 15 e 30 dias apÃs a infecÃÃo; e anÃlise histopatolÃgica das lesÃes. Os animais que receberam CXCL10 apresentaram uma reduÃÃo no tempo de duraÃÃo da doenÃa, uma vez que as lesÃes comeÃaram a regredir mais precocemente (12 dias), enquanto que nos animais controle (PBS) a doenÃa evoluiu por mais tempo (15 dias). CXCL10 induziu diferenÃas estatisticamente significativas na carga parasitÃria do linfonodo de drenagem nos dois pontos avaliados, quando comparado com o controle (15 dias: p=0,0353; 30 dias: p=0,0292). Nos animais que receberam CXCL10, nÃo foi observada mudanÃa no nÃmero de parasitos no sÃtio de inoculaÃÃo, mesmo com reduÃÃo da espessura da lesÃo, mostrando que nÃo houve correlaÃÃo entre o tamanho da lesÃo e a carga parasitÃria. NÃo houve diferenÃa na concentraÃÃo de NO entre os grupos, no 15 dia de infecÃÃo, e esta concentraÃÃo manteve-se igual no grupo que recebeu CXCL10 no 30 dia, embora tenha sido menor do que a observada no grupo controle. CXCL10 tambÃm induziu concentraÃÃes mais elevadas de IFN-&#947; e IL-12 e mais reduzidas de IL-4, IL-10, TGF-&#946; 30 dias pÃs-infecÃÃo, quando comparados aos animais controle. A anÃlise histopatolÃgica das patas mostrou uma inflamaÃÃo muito leve em ambos os grupos, com diferenÃa apenas quanto à ausÃncia de neutrÃfilos nas lesÃes dos animais que receberam CXCL10. Em conjunto, os dados mostraram que CXCL10 apresentou um efeito protetor na infecÃÃo por L. braziliensis em camundongos BALB/c, com reduÃÃo no tempo de evoluÃÃo da doenÃa, associada a maior concentraÃÃo de IFN-&#947; e menor de IL-4 e IL-10. / Leishmania braziliensis is the more common etiologic agent of the cutaneous leishmaniasis in Brazil. BALB/c mice are more susceptible to L. braziliensis infection, although develop small and not ulcerated lesions. The lesions in BALB/c mice infected by L. braziliensis are accompanied by expression of a broad chemokines spectrum, including CXCL10, which is able to attract and activate NK and Th1 cells with IFN-&#947; production. The aim of this study was to evaluate whether the chemokine CXCL10, administered in early stages of infection could influence the course of disease, leading to a faster and more effective time of healing. Groups of BALB/c mice (n=24) were infected by L. braziliensis and divided into two groups: one group (n=12) received CXCL10 (100 ng/5&#956;L) and the other (n=12) received PBS (5&#956;L), at 1, 3, 5 and 7 days postinfection. Some parameters were evaluated: thickness of the lesions every three days; parasite burden in both the footpad and the draining lymph node, NO concentration, cytokines production (IFN-&#947;, IL-12p40, TNF-&#945;, IL-4, IL-10 and TGF-&#946;), at 15 and 30 days after infection; and histopathological analysis of lesions. Animals receiving CXCL10 showed reduced disease duration, since the lesions started to regress earlier (12 days), whereas the control animals developed the disease for longer (15 days). CXCL10 induced statistically significant differences in parasite load in the draining lymph nodes compared with control in both times evaluated (15 days: p=0.0353; 30 days: p=0.0292). In the animals receiving CXCL10 was not observed change in the number of parasites in the site of inoculation, even with reduced thickness of the lesion, showing no correlation between lesion size and parasite load. There was no difference in NO concentration between groups with 15 days of infection, and this concentration remained the same in the group receiving CXCL10 at day 30, although it was lower than that observed in the control group. CXCL10 also induced higher levels of IFN-&#947; and IL-12, and reduced IL-4, IL-10, and TGF-&#946; at 30 days postinfection, when compared to control animals. Histopathology analyses of lesions after 15 days postinfection showed a very mild inflammation in both groups, differing only for the presence of neutrophils, which were practically not observed in lesions of animals receiving CXCL10. Taken together, the data from this study showed CXCL10 chemokine presented a protective effect on L. braziliensis infection in BALB/c mice, with a reduction in the progression of the disease, associated with a higher concentration of IFN-&#947; and lesser of IL-4 and IL-10.

In der vorliegenden Arbeit wurde die Rolle der Transkriptionsfaktoren NFATc1/αA bzw. NFATc1/ßC und NFATc2 in NK-Zellen und die Rolle der Faktoren NFATc1-4 und NFAT5 in Keratinozyten analysiert. Die Familie der Nuclear Factor of Activated T-cell (NFAT) Transkriptionsfaktoren besteht aus fünf Mitgliedern, welche entscheidend die Gentranskription bei Immunantworten beeinflussen. Nach Antigenaktivierung wird in Lymphozyten die Expression des Nfatc1-Gens stark induziert. Die Akkumulation der dabei gebildeten kurzen Isoform NFATc1/αA ist für die Zytokinproduktion als Effektorfunktion sowie für die Proliferation und das Überleben aktivierter Zellen verantwortlich. Das kurze NFATc1-Protein unterscheidet sich nicht nur strukturell, sondern auch funktionell von fast allen anderen NFAT-Proteinen. Um neue Erkenntnisse über die Funktion der Isoform NFATc1/αA in Lymphozyten zu gewinnen, wurden KT12 NK-Zellen mit NFATc1/αA bzw. NFATc1/ßC und NFATc2- exprimierenden Vektoren transfiziert, geklont, stimuliert und anschließend auf ihre jeweilige Apoptoserate und die Zytokinsynthese bzw. -expression hin untersucht. Die gewählten KT12-Hybridoma-Zellen erwiesen sich allerdings in ihrer intendierten Funktion als Testzellen für die Über-Expression von NFATc-Proteinen in NK-Zellen als ungeeignet. Fehlregulationen von NFAT-Signalwegen werden mit einer fehlerhaften Entwicklung des Immunsystems, mit der Entstehung von Autoimmunerkrankungen und mit Krebs in Verbindung gebracht. Um die Rolle von NFAT-Faktoren in Keratinozyten besser zu verstehen, wurden HaCaT-Zellen und primäre humane Keratinozyten mit Differenzierungssignalen bzw. UVB-Licht stimuliert. Änderungen der Transkription von NFAT-Faktoren, Keratinozyten-spezifischen Proteinen und Chemokinen wurden mittels qRT-PCR-Assays detektiert und analysiert. Insgesamt konnte die Beteiligung von NFAT-Faktoren am Differenzierungsprozess und an der UV-Antwort von Keratinozyten gezeigt werden. Es zeigte sich tendenziell eine stärkere Induktion der kurzen NFATc1-Isoform im Vergleich zu langen NFATc1-Isoformen, was die Frage nach einer besonderen Funktion der kurzen NFATc1-Isoform in Keratinozyten aufwirft. Generell ließen sich besonders hohe Expressionslevel des Transkriptionsfaktors NFAT5 - verglichen mit anderen NFAT-Faktoren - messen. Für die Entwicklung von Therapien, welche die Ursachen und Folgen einer dysregulierten Hautbarrierenbildung behandeln, könnten sich weitere Studien zu einzelnen NFAT-Faktoren bzw. NFATc1-Isoformen als zielführend erweisen. / In the present work the role of the transcription factors NFATc1/αA or NFATc1/ßC and NFATc2 in NK cells and the role of the factors NFATc1-4 and NFAT5 in keratinocytes were investigated. The nuclear factor of activated T-cell (NFAT) transcription factor family consists of five members that critically influence gene transcription in immune responses. Following antigen activation, expression of the Nfatc1 gene is strongly induced in lymphocytes. Accumulation of the short NFATc1/αA isoform - formed in this process - is responsible for cytokine production as an effector function and for proliferation along with the survival of activated cells. The short NFATc1 protein differs not only structurally but also functionally from almost all other NFAT proteins. To gain new insights into the function of the NFATc1/αA isoform in lymphocytes, KT12 NK cells were transfected with NFATc1/αA, NFATc1/ßC or NFATc2- expressing vectors. The KT12 cells were cloned, stimulated and subsequently analyzed for their respective apoptosis rates together with cytokine synthesis or expression. However, the selected KT12 hybridoma cells proved to be unsuitable in their function as test cells for the overexpression of NFATc proteins in NK cells. Misregulation of NFAT signaling pathways has been associated with defective development of the immune system and autoimmune diseases and cancer. To better understand the role of NFAT factors in keratinocytes, HaCaT cells and primary human keratinocytes were stimulated with differentiation signals and UVB light, respectively. Changes in transcription of NFAT factors, keratinocyte-specific proteins and chemokines were detected and analyzed by qRT-PCR assays. Overall, the involvement of NFAT factors in the differentiation process and UV response of keratinocytes was demonstrated. The short NFATc1 isoform tended to be more strongly induced compared with long NFATc1 isoforms, raising the question of a special function of the short NFATc1 isoform in keratinocytes. Particularly high expression levels of the transcription factor NFAT5 - compared to other NFAT factors - could be measured. For the development of therapies that treat the causes and consequences of dysregulated skin barrier formation further studies on individual NFAT factors or NFATc1 isoforms will prove useful.

Viral infection stimulates the immune response to produce many cytokines and chemokines, the proteins imperative to fight a brewing infection. This response begins through recognition of pathogen-associated molecular patterns (PAMPs) from the virus, or from other signatures characteristic of tissue damage, damage-associated molecular patterns (DAMPs), by pattern recognition receptors (PRRs) that in turn stimulate pro-inflammatory signaling cascades. The results of these signaling pathways include the release of cytokines and chemokines that work to further upregulate immune responses and attract immune cells to the site of infection, respectively. In the case of SARS-CoV-2 infection, these responses can become problematic if they go unmitigated or unresolved, resulting in the severe COVID-19 manifestation of the 'cytokine storm,' or multisystem inflammatory syndrome in children (MIS-C). One classically increased protein in cytokine storm of COVID-19 patients is C-X-C motif chemokine 10 (CXCL10), which has been explored as a prognostic marker as it is shown to be predictive of disease outcome in hospitalized patients. To prevent severe outcomes like cytokine storm, a delicate balance must be struck, to ensure that this inflammation does not result in high levels of diffuse tissue damage. To achieve this, anti-inflammatory pathways exist within the immune system and help dampen the signals being induced. One such unique anti-inflammatory protein is a pattern recognition receptor known as NLRX1 (Nucleotide binding oligomerization domain, leucine rich repeat containing X1), that can interact with two main pathways involved with anti-viral immunity, the NFB and interferon pathways, downregulating them to keep off-target tissue damage at bay. NLRX1 is also involved in several other cellular processes, including modulating cell death processes and cellular metabolism which can also impact viral replication and clearance indirectly. In this work, we investigated both the pro- and anti-inflammatory arms of the anti-SARS-CoV-2 response focusing on two key proteins – pro-inflammatory chemokine CXCL10 and immunoregulatory PRR NLRX1. The roles of these two proteins were explored utilizing transcriptomic analysis of both human and mouse RNA samples, immortalized cell culture work, humanized mouse models of SARS-CoV-2 infection, and mouse-adapted virus models to be able to utilize deficient mouse models. In this work we better characterize the immune response to SARS-CoV-2 and its related immune-driven pathobiology of disease. The data presented in this work continues to elucidate CXCL10's role as an important driver of viral clearance of SARS-CoV-2, translating data from human patient nasal swabs to the animal model of disease, exploring differential inflammation and immune responses in the absence of CXCL10. Additionally, the work shown here provides further understanding of NLRX1 and its role in antiviral immunity with the context of SARS-CoV-2 infection. The interactions between this protein and the virus remains to be fully characterized, however, it appears they have some degree of mutual inhibition as determined by animal and cell culture models. The culmination of work here emphasizes the importance for both the pro- and anti-inflammatory responses in SARS-CoV-2 infection and offers insight into two possible related targets for future drug development. / Doctor of Philosophy / When a virus invades the body, the immune system kicks off many signaling cascades to keep the virus from replicating, clear virus already established in cells, and clean up the tissues surrounding the infected area of the remnants of cells that already succumbed to the virus. While this immune response is important to fight off the virus that has made its way into the body, overactive immune responses can result in hospital stays requiring supportive care to aid recovery from possible off-target tissue damage. One such case of this happening is when SARS-CoV-2 induces such a strong response, the immune system becomes overzealous and results in overproduction of pro-inflammatory cytokines and chemokines, signaling proteins in the immune system, which can lead to the characteristic 'cytokine storm' of severe COVID-19 disease. One of the proteins most often overproduced is the chemokine CXCL10, and this protein has been used as a biomarker in clinical practice to successfully predict severe disease outcomes in COVID-19 patients. To help combat severe disease outcomes and high levels of tissue damage, the immune system has inborn checks and balances to ensure that proteins like CXCL10 do not reach the level of overproduction as in the cytokine storm of COVID-19. One of these natural checkpoints is a protein called NLRX1, which interacts with two of the main pathways that can lead to the overproduction of cytokines seen overproduced in the case of cytokine storm. NLRX1 also has other roles in other interesting facets important for viral infections, including the metabolism of the cell and cellular death processes. The culmination of these roles could offer up NLRX1 as a possible target for treatments in the future. The work put together here explores both sides of the immune response, turning it 'on' with pro-inflammatory signaling, and turning it 'off' with anti-inflammatory signaling, trying to find just the right amount of inflammation to clear a viral invader while also impeding off target and diffuse tissue damage as the body fights the virus. This work focuses on two key proteins, CXCL10 to represent pro-inflammatory responses, and NLRX1 to represent the anti-inflammatory signaling. Understanding both arms of the immune response to SARS-CoV-2 infection is crucial to being able to identify potential targets for future treatments to help combat severe outcomes of SARS-CoV-2 infection. Using multiple levels across the translational spectrum, including cell culture, animal models, and human patient RNA from COVID test swabs, we explore both facets of SARS-CoV-2 immunity, focusing on these two proteins. Utilizing mouse models bearing deletions of the genes required to make these proteins and a mouse-adapted strain of SARS-CoV-2, this work characterizes how important these individual proteins are in the immune response to SARS-CoV-2, and work as proxies to understand the broader impacts of either the positive or negative regulation of immune signaling. Because of the work culminated here, these two tangentially related proteins are also offered up as possible future drug targets for the development of treatments in severe COVID-19 disease with cytokine storm presentation.

O líquen plano oral (LPO) é uma doença inflamatória crônica que afeta em torno de 1 a 2% da população mundial adulta, entre 30 e 60 anos, principalmente mulheres. As lesões podem estar presentes em diversos sítios, porém tem predileção pela mucosa jugal, gengiva e bordas laterais da língua, todas bilateralmente. As manifestações de LPO são frequentes, e podem se caracterizar clinicamente em seis tipos: reticular, em placas, papular, atrófico, erosivo-ulcerativo e bolhoso, porém o mais comum é o reticular. O presente estudo teve como objetivo avaliar a expressão de genes relacionados à resposta inflamatória e autoimunidade no líquen plano oral (LPO) em pacientes com a variante exclusivamente reticular comparado a controle saudável. Foram incluídos consecutivamente 10 pacientes com LPO que preencheram os critérios de inclusão adotados, sendo 9 mulheres e um homem, e 6 indivíduos controle, sendo 4 mulheres e 2 homens pareados por sexo, idade e uso de medicações sistêmicas. Amostras de mucosa bucal foram coletadas por meio de biópsia incisional em ambos os grupos de pacientes e submetidas à extração de RNA para posterior análise da expressão gênica por PCR-array selecionada para este estudo. Os resultados obtidos foram o aumento da expressão de 8 genes, CXCL 9 e CXCL 10 vão de acordo com a literatura, corroborando com nosso estudo e de certa forma reafirmar mais necessidade de estudos bem desenhados . / The oral lichen planus (OLP) is a chronic inflammatory disease that affects around 1-2% of the adult population between 30 and 60 years, mainly women. The lesions may be present in several sites, but has a predilection for the buccal mucosa, gums and lateral borders of the tongue, all bilaterally. The manifestations of OLP are frequent, and may be characterized clinically in six types: reticular, plaque-like, papular, atrophic, erosive-ulcerative and bullous, but the most common is the reticular . The present study aimed to evaluate the expression of genes related to inflammatory response and autoimmunity in oral lichen planus (OLP) in patients with exclusively reticular variant compared to healthy control. 10 consecutive patients with OLP who met the inclusion criteria, 9 women and one man, and six control subjects were included were 4 women and 2 being proportionally matched by sex, age and use of systemic medications men. Samples of oral mucosa were collected by incisional biopsy in both groups of patients and subjected to RNA extraction for analysis of gene expression by selected for this study-PCR array. The results were the overexpression of genes 8, where only two of them go according to the literature, corroborating our study and somehow reaffirm need for more well-designed studies.

O líquen plano oral (LPO) é uma doença inflamatória crônica que afeta em torno de 1 a 2% da população mundial adulta, entre 30 e 60 anos, principalmente mulheres. As lesões podem estar presentes em diversos sítios, porém tem predileção pela mucosa jugal, gengiva e bordas laterais da língua, todas bilateralmente. As manifestações de LPO são frequentes, e podem se caracterizar clinicamente em seis tipos: reticular, em placas, papular, atrófico, erosivo-ulcerativo e bolhoso, porém o mais comum é o reticular. O presente estudo teve como objetivo avaliar a expressão de genes relacionados à resposta inflamatória e autoimunidade no líquen plano oral (LPO) em pacientes com a variante exclusivamente reticular comparado a controle saudável. Foram incluídos consecutivamente 10 pacientes com LPO que preencheram os critérios de inclusão adotados, sendo 9 mulheres e um homem, e 6 indivíduos controle, sendo 4 mulheres e 2 homens pareados por sexo, idade e uso de medicações sistêmicas. Amostras de mucosa bucal foram coletadas por meio de biópsia incisional em ambos os grupos de pacientes e submetidas à extração de RNA para posterior análise da expressão gênica por PCR-array selecionada para este estudo. Os resultados obtidos foram o aumento da expressão de 8 genes, CXCL 9 e CXCL 10 vão de acordo com a literatura, corroborando com nosso estudo e de certa forma reafirmar mais necessidade de estudos bem desenhados . / The oral lichen planus (OLP) is a chronic inflammatory disease that affects around 1-2% of the adult population between 30 and 60 years, mainly women. The lesions may be present in several sites, but has a predilection for the buccal mucosa, gums and lateral borders of the tongue, all bilaterally. The manifestations of OLP are frequent, and may be characterized clinically in six types: reticular, plaque-like, papular, atrophic, erosive-ulcerative and bullous, but the most common is the reticular . The present study aimed to evaluate the expression of genes related to inflammatory response and autoimmunity in oral lichen planus (OLP) in patients with exclusively reticular variant compared to healthy control. 10 consecutive patients with OLP who met the inclusion criteria, 9 women and one man, and six control subjects were included were 4 women and 2 being proportionally matched by sex, age and use of systemic medications men. Samples of oral mucosa were collected by incisional biopsy in both groups of patients and subjected to RNA extraction for analysis of gene expression by selected for this study-PCR array. The results were the overexpression of genes 8, where only two of them go according to the literature, corroborating our study and somehow reaffirm need for more well-designed studies.

Die Diagnostik eines Glioblastoms (GBM) stützt sich z.Z. neben klinischer Symptomatik auf bildgebende Diagnostik sowie die histologische Untersuchung. In letzter Zeit werden zuneh-mend Serumproteine beschrieben und untersucht, die mit einer GBM-Erkrankung assoziiert sind. Elstner, Stockhammer und Kollegen haben 2011 ein Serumproteinprofil identifiziert, das aus CXCL10/IP-10, BMP-2 und HSP70 besteht und in einem Kollektiv von 23 GBM-Erkrankten und 9 Gesunden eine Sensitivität von 89% und eine Spezifität von 96% besaß. Dieses Profil wurde nun in einer unizentrischen klinischen Studie an 35 GBM-Erkrankten und 37 Patienten mit differenzialdiagnostisch relevanten Erkrankungen (v. a. Hirnmetastasen und primären ZNS-Lymphomen) überprüft. Dabei wurden die präoperativ abgenommenen Blut-proben mittels des ELISA-Nachweisverfahrens untersucht und die jeweiligen Konzentratio-nen in die von Elstner et al. (2011) entwickelte Regel eingesetzt. In diesem Kollektiv konnte das Profil nicht zwischen einem GBM und seinen Differenzialdiagnosen unterscheiden (Sensitivität 31%, Spezifität 54%). Es ist nicht als Hilfsmittel zur Diagnostik von Glioblastomen geeignet.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A tuberculose (TB) é uma doença infecto-contagiosa obtida a partir da inalação de aerossóis contendo seu agente etiológico, o Mycobacterium tuberculosis. A TB acomete principalmente os pulmões e é a patologia bacteriana líder em causar mortes no mundo. No Brasil, por ano, são notificados 69 mil casos de tuberculose, dos quais 4,6 mil evoluem para o óbito. Durante a infecção pelo M. tuberculosis, 90% dos indivíduos permanece na forma latente assintomática, e aproximadamente 10% evolui para doença. Este trabalho estudou parâmetros de resposta imune e inflamatória, em indivíduos de ambos os sexos, com idades de 18 a 65 anos, com diferentes graus de exposição ao M. tuberculosis (indivíduos não-expostos ao M. tuberculosis, TST < 5 mm, n= 30; indivíduos com tuberculose latente, TST &#8805; 5 mm, n=29; pacientes com tuberculose pulmonar n= 22). Nossos resultados mostraram que o TST isoladamente falhou em detectar todos os indivíduos expostos ao M. tuberculosis, e em 1/3 dos TST positivos não foi observada resposta in vitro a antígenos específicos de M. tuberculosis, avaliada com os biomarcadores IFN-&#947; e CXCL10. Houve uma alta correlação entre os biomarcadores IFN-&#947; e CXCL10 em culturas de sangue não fracionado estimuladas com antígenos específicos de M. tuberculosis. A utilização combinada destes 2 biomarcadores mostrou positividade para M. tuberculosis em 94,4% dos pacientes. Foram observadas diferenças marcantes de nível de expressão de RNA mensageiro específicos para CD64, GTPase associada a Ras, lactoferrina, PDL-1 e CXCL10, mas não para OASL em leucócitos sanguíneos, quando os pacientes com tuberculose pulmonar foram comparados com os dois outros grupos de voluntários. Da mesma forma, os níveis de expressão dos receptores CD64 e CD163 foram significativamente mais elevados em neutrófilos dos pacientes quando comparados com os grupos-controle. Tomadas em conjunto, nossas observações sugerem que o uso de mais de um biomarcador aumenta a sensibilidade e especificidade dos métodos para detecção de infecção latente por M. tuberculosis e tuberculose.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A tuberculose (TB) é uma doença infecto-contagiosa obtida a partir da inalação de aerossóis contendo seu agente etiológico, o Mycobacterium tuberculosis. A TB acomete principalmente os pulmões e é a patologia bacteriana líder em causar mortes no mundo. No Brasil, por ano, são notificados 69 mil casos de tuberculose, dos quais 4,6 mil evoluem para o óbito. Durante a infecção pelo M. tuberculosis, 90% dos indivíduos permanece na forma latente assintomática, e aproximadamente 10% evolui para doença. Este trabalho estudou parâmetros de resposta imune e inflamatória, em indivíduos de ambos os sexos, com idades de 18 a 65 anos, com diferentes graus de exposição ao M. tuberculosis (indivíduos não-expostos ao M. tuberculosis, TST < 5 mm, n= 30; indivíduos com tuberculose latente, TST &#8805; 5 mm, n=29; pacientes com tuberculose pulmonar n= 22). Nossos resultados mostraram que o TST isoladamente falhou em detectar todos os indivíduos expostos ao M. tuberculosis, e em 1/3 dos TST positivos não foi observada resposta in vitro a antígenos específicos de M. tuberculosis, avaliada com os biomarcadores IFN-&#947; e CXCL10. Houve uma alta correlação entre os biomarcadores IFN-&#947; e CXCL10 em culturas de sangue não fracionado estimuladas com antígenos específicos de M. tuberculosis. A utilização combinada destes 2 biomarcadores mostrou positividade para M. tuberculosis em 94,4% dos pacientes. Foram observadas diferenças marcantes de nível de expressão de RNA mensageiro específicos para CD64, GTPase associada a Ras, lactoferrina, PDL-1 e CXCL10, mas não para OASL em leucócitos sanguíneos, quando os pacientes com tuberculose pulmonar foram comparados com os dois outros grupos de voluntários. Da mesma forma, os níveis de expressão dos receptores CD64 e CD163 foram significativamente mais elevados em neutrófilos dos pacientes quando comparados com os grupos-controle. Tomadas em conjunto, nossas observações sugerem que o uso de mais de um biomarcador aumenta a sensibilidade e especificidade dos métodos para detecção de infecção latente por M. tuberculosis e tuberculose.

研究背景及實驗目的: 非酒精性脂肪性肝炎(NASH)使得肥胖和2 型糖尿病變得複雜，肝臟炎症的持續產生是其主要的發病機理。CXCL10 是一種促進炎症的細胞因數，其在肥胖和2 型糖尿病中的表達顯著升高。CXCL10 以及其受體CXCR3 是否在NASH 的發生發展中起作用尚不清楚。在本研究中，我們探索了CXCL10 以及其受體CXCR3 在脂肪性肝炎中的功能， 並評估了CXCL10 在NASH 中的臨床價值。 / 實驗方法：CXCL10 基因敲除鼠，CXCR3 敲除鼠以及野生型C57BL/6 小鼠給予蛋氨酸膽鹼缺乏食(MCD)4 周或者8 周。CXCL10 的信號通路以及下游靶點通過細胞因數分析，cDNA array, 蛋白DNA 結合實驗，自噬溶酶體系統分析進行檢測。為了闡明CXCL10 抑制對NASH 的預防治療作用，我們給MCD 餵養的小鼠注射抗CXCL10 抗體。用不同濃度的CXCL10 抗體以及CXCR3 抑制劑NIBR2130 幹預MCD 培養的肝細胞株AML-12。臨床研究中，我們收集了147個非酒精性脂肪肝患者以及73 個健康對照的血清，用酶聯免疫吸附試驗檢測血清中CXCL10 的水準。 / 結果：野生型小鼠給予MCD 餵養後，CXCL10 以及CXCR3 的表達升高，並出現脂肪性肝炎的表現。然而，MCD 飼養的CXCL10 以及CXCR3 基因敲除鼠中，脂肪性肝炎明顯減輕。CXCL10 通過促炎細胞因數的產生以及NK-κB 信號通路促進MCD 飼養的小鼠NASH 的發生。CXCL10 通過促進脂質合成的基因SREBP-1c, ChREBP 和 SCD-1 引起脂肪變性，並通過CYP2E1 以及 C/EBPβ 的上調引起氧化應激。值得注意的是，自噬的損傷在CXCL10 以及CXCR3 導致的脂肪性肝炎的進展中起重要作用。 MCD 飼養的野生型小鼠中p62 以及LC3-II 表達明顯高於CXCL10 以及CXCR3 基因敲除鼠。通過抗CXCL10 抗體中和CXCL10 可以減輕MCD 食引起的小鼠脂肪性肝炎以及MCD 培養液引起的AML-12 細胞損傷。高選擇性的CXCR3 抑制劑NIBR2130 也可以抑制MCD 引起的肝細胞損傷。我們進一步研究了CXCL10 的臨床應用價值，發現NASH 患者血清以及肝臟中CXCL10 的水準明顯升高。更重要的是，血液中CXCL10 的水準與肝小葉炎症程度有關，是NASH 的獨立危險因素。 / 結論：我們的研究首次發現CXCL10 以及其受體CXCR3 通過促進炎症，脂質聚集，氧化應激以及自噬缺乏在NASH 的發病中起重要作用。抑制CXCL10 或者CXCR3 為NASH 患者的治療提供了新的方法。CXCL10 可作為NASH 患者非侵入性診斷的標誌物。 / Background and aims: Non-alcoholic steatoheaptitis (NASH) complicates obesity and type 2 diabetes, while recruitment and perpetuation of liver inflammation is central to its pathogenesis. Expression of C-X-C motif chemokine 10 (CXCL10), a proinflammatory cytokine, correlates positively with obesity and type 2 diabetes. Whether CXCL10 and its receptor CXCR3 play a role in NASH is unknown. In this study, we investigated the functional significance of CXCL10 and its receptor CXCR3 in steatoheaptitis. Moreover, the clinical impact of CXCL10 in NASH was examined. / Methods: Gene-deleted CXCL10 (CXCL10-/-), CXCL10 receptor CXCR3 (CXCR3-/-) and C57BL/6 wildtype (WT) mice were fed methionine and choline-deficient (MCD) diet for 4 or 8 weeks. Cytokine profiling assay, cDNA array, protein-DNA binding activity assay and autophagosome-lysosome system analysis of CXCL10 signaling and downstream targets were performed. In other experiments, we injected neutralizing anti-CXCL10 monoclonal antibodies (mAb) into MCD diet-fed WT mice, while AML-12 cells were cultured in MCD medium in the presence of anti-CXCL10 mAb or CXCR3 inhibitor (NIBR2130) for 24 hours. Human serum was obtained from 147 patients with biopsy-proven non-alcoholic fatty liver disease and 73 controls. Circulating CXCL10 levels were determined by enzyme-linked immunosorbent assay. / Results: MCD-fed WT mice developed steatohepatitis with higher hepatic CXCL10 and CXCR3 expression. CXCL10-/- and CXCR3-/- mice were refractory to MCDinduced steatohepatitis. In WT mice with steatohepatitis, but not in CXCL10-/- mice, CXCL10 was associated with the induction of pro-inflammatory chemokines and cytokines, as well as activation of nuclear factor-κB (NF-κB) signaling. CXCL10 expression was linked to steatosis through lipogenic factors, including liver X receptors and its downstream targets (SREBP-1c, ChREBP and SCD-1), and also to oxidative stress (up-regulation of CYP2E1 and C/EBPβ). In particular, autophagy deficiency was involved in CXCL10- and CXCR3-induced steatohepatitis as indicated by p62 and LC3-I/II protein accumulation in MCD-fed WT mice than in CXCL10-/- and CXCR3-/- mice. Moreover, the impaired autophagic function was related to the reduction of lysosomal function in CXCL10- or CXCR3-induced NASH. Blockade of CXCL10 by anti-CXCL10 mAb protected against MCD-induced steatohepatitis in vivo and against MCD-mediated injury to AML-12 cells in vitro. The highly selective CXCR3 antagonist NIBR2130 also inhibited MCD-induced injury in AML-12 hepatocytes. We further investigated the clinical impact of CXCL10 and found circulating and hepatic CXCL10 levels were significantly higher in human NASH. Importantly, circulating CXCL10 level was correlated with the degree of lobular inflammation and was an independent risk factor for NASH patients. / Conclusions: We demonstrate for the first time that CXCL10 and its receptor CXCR3 plays a pivotal role in the pathogenesis of NASH by promoting inflammation, fatty acid accumulation, oxidative stress and autophagy deficiency. Blockade of CXCL10 or CXCR3 is a potential novel approach for NASH intervention. CXCL10 is a noninvasive biomarker for NASH patients. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Xiang. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 145-167). / Abstracts also in Chinese.