Objetivo: Uma série de casos sugerindo uma possível associação de pancreatite aguda (PA) e síndrome de ativação macrofágica (SAM) em lúpus eritematoso sistêmico pediátrico (LESP) foi reportada em dez crianças no nosso serviço, no entanto, não existem dados relativos à comparação entre PA e SAM em grandes populações de LESP e LES adulto (LESA). Métodos: Este estudo incluiu 362 pacientes LESP e 1.830 pacientes LESA. SAM foi diagnosticada de acordo com os critérios diagnósticos preliminares e PA de acordo com a presença de dor abdominal e/ou vômitos associados a um aumento de enzimas pancreáticas e/ou alterações radiológicas pancreáticas nos exames de ultrassonografia e/ou tomografia abdominal. Dados demográficos, características clínicas, SLEDAI-2K, SLICC/ACR-DI e tratamento foram avaliados. Resultados: A frequência de PA foi significantemente aumentada no LESP em comparação ao LESA [12/362 (3,3%) vs. 20/1830 (1,1%), p=0,003], com similar duração da PA nos dois grupos [22 (6-60) vs. 15 (4-90), dias, p=0,534]. As frequências de SAM (85% vs 30%, p=0,003) e óbito (31% vs. 0%; p=0,017) foram significantemente elevadas em crianças com PA comparadas com adultos com PA. Na análise dos pacientes com PA e SAM em comparação com os com somente PA sem SAM demonstrou que a idade dos pacientes com PA e SAM foi significantemente menor em comparação com aqueles sem SAM [15 (8,8- 55) vs. 33,5 (10,2-45,7) anos, p=0,007]. As frequências de febre (94% vs. 37%, p=0,001), leucopenia (82% vs. 19%, p=0,0001), trombocitopenia (65% vs. 19%, p=0,013), hipertrigliceridemia (87% vs. 42%, p=0,037) e hiperferritinemia (93% vs. 37%, p=0,011) foram significantemente aumentadas nos pacientes com PA e SAM comparados aos pacientes com somente PA. A concomitância de febre e hiperferritinemia foi significantemente mais freqüente no primeiro grupo (86% vs. 12%, p=0,0015). Conclusões: Este estudo forneceu novos dados que evidenciaram que SAM ocorreu na maioria dos LESP com PA com uma maior mortalidade em comparação com LESA. Além disso, foram identificados em pacientes com PA e SAM, um conjunto de parâmetros clínicos e laboratoriais associado com as duas complicações / Objective: We previously reported a case series of acute pancreatitis (AP) and macrophage activation syndrome (MAS) in childhood (cSLE) patients, however there are no data regarding the comparison of AP and MAS in large populations of cSLE and adult SLE (aSLE). Methods: This study included 362 cSLE and 1,830 aSLE patients. MAS was diagnosed according to preliminary diagnostic guidelines and AP according to the presence of abdominal pain or vomiting associated to an increase of pancreatic enzymes and/or pancreatic radiological abnormalities. Demographic data, clinical features, SLEDAI-2K, SLICC/ACR-DI and treatment were assessed. Results: Higher and significant frequency of AP in cSLE compared to aSLE patients [12/362(3.3%) vs. 20/1830(1.1%), p=0.003], with similar AP duration [22(6- 60) vs. 15(4-90) days, p=0.534]. MAS (85% vs. 30%, p=0.003) and death by MAS complication (31% vs. 0%, p=0.017) were significantly higher in children with AP compared with aSLE with AP. Further analysis of patients with AP and MAS compared with AP without MAS demonstrated that age in MAS patients was significantly lower compared with those without this complication [15(8.8-55) vs. 33.5(10.2-45.7) years, p=0.007]. The frequencies of fever (94% vs. 37%,p=0.001), leucopenia (82% vs. 19%,p=0.0001), thrombocytopenia (65% vs. 19%,p=0.013), hypertriglyceridemia (87% vs. 42%,p=0.037) and hyperferritinemia (93% vs. 37%,p=0.011) were also more frequently observed in AP patients with MAS compared in AP patients without MAS. Fever and hyperferritinemia concomitantly were more frequent in the former group (86% vs. 12%, p=0.0015). Conclusions: This study provides novel data demonstrating that MAS occur in the majority of cSLE with AP with a higher mortality compared to aSLE. In addition, we identified in AP patients, a cluster of MAS clinical and laboratorial parameters more associated with this complication

INTRODUÇÃO: A incidência de câncer da glândula tireoide é a que mais vem crescendo nas últimas décadas. Dentro desse grupo de diferentes neoplasias, o carcinoma papilífero, um dos carcinomas bem diferenciados, representa a maioria e tem prognóstico favorável, com sobrevida acima dos 90% em 5 anos. Embora sejam utilizadas diversas classificações de risco baseadas em diferentes fatores prognósticos, ainda não se consegue predizer quais pacientes terão maior chance de recorrência, metástases linfonodais e desfecho desfavorável, que se beneficiariam de um tratamento mais agressivo. Já foi demonstrado em diversos tipos de neoplasias que diferenças no perfil do infiltrado imune tumoral têm relação com prognóstico e resposta ao tratamento. Neste estudo caracterizou-se o microambiente imune do carcinoma papilífero através de marcadores imuno-histoquímicos de células inflamatórias e relacionou-se este perfil de infiltração com fatores prognósticos clínico-patológicos e com sobrevida livre de recorrência. MÉTODOS: Foram incluídos 151 casos selecionados com base em um banco de dados que incluiu todos os pacientes submetidos a tratamento cirúrgico para câncer de tireoide no A.C.Camargo Cancer Center entre 2008 e 2010. Casos com tireoidite significante foram excluídos. Estes tumores selecionados foram então submetidos a reação imuno-histoquímica com marcadores de células inflamatórias e foram analisados por dois patologistas experientes. As características clínicas e patológicas foram avaliadas, assim como as recorrências e sobrevida, relacionando-as com as leituras de células marcadas. As análises de sobrevida global e livre de doença foram realizadas pelo método de Kaplan-Meier, com comparação de curvas de sobrevida pelo teste de Logrank. RESULTADOS: Cento e cinquenta e um pacientes foram incluídos, sendo 130 (86,1%) mulheres e 21 (13.9%) homens. Multifocalidade foi encontrada em 41 (27.2%), extensão extratireoidiana em 43 (28.5%) e metástase linfonodal em 36 (23.8%) casos. Apenas dois pacientes apresentaram metástase a distância. O tempo de seguimento médio foi 65,1 meses e observou-se nove (6%) pacientes com recorrências de neoplasia. Os pacientes com tumores com metástase linfonodal e/ou extensão extratireoidiana apresentaram maior risco de recorrência. Dos marcadores analisados, uma maior densidade de CD8 (que marca linfócitos citotóxicos) na área peritumoral esteve associada a uma tendência a melhor sobrevida livre de recorrência: 97,1% versus 87,5% (p=0,057), além de significativos menores índices de multifocalidade tumoral e de metástase linfonodal. A maior infiltração de linfócitos T CD8+ no tumor também se relacionou com menor ocorrência de metástase linfonodal nesta amostra (18,4% versus 38,1%, p=0,011). Além disso, a densidade desta marcação, tanto no interior da lesão neoplásica como em área peritumoral foi significativamente maior nos casos de carcinomas papilíferos restritos à tireoide, ou seja, sem extensão extratireoidiana e sem metástases. Os demais marcadores analisados não apresentaram relação significativa e consistente com recorrência ou outros fatores prognósticos. CONCLUSÕES: Nas neoplasias malignas de tireoide, o microambiente imune parece ter uma relação com características patológicas de agressividade. Este estudo mostrou que em carcinoma papilífero de tireoide quando não associado à tireoidite significativa, a densidade do infiltrado tumoral e peritumoral por linfócitos T CD8+ está inversamente relacionada com chance de disseminação metastática linfonodal e, provavelmente, com recidiva da doença, sendo, portanto, um marcador de melhor prognóstico. Este dado sugere que estes linfócitos exercem efeito antitumoral no carcinoma papilífero de tireoide, corroborando a importância da resposta imune na evolução desta neoplasia / INTRODUCTION: Within the last few decades thyroid cancer has the fastest rising incidence rate among all malignancies. In this group of different neoplasms, the papillary carcinoma, one of the well-differentiated carcinomas, represents the great majority and has favorable prognosis, with overall survival rates above 90% in five years. Although several risk classifications based on different prognostic features have been used, they are not accurate to predict which patients will have higher chance of recurrence, lymphatic metastasis and worse outcome, benefiting from more aggressive treatment. It has been described in several kinds of malignancies that tumor related immune infiltration has relation with prognosis and response to treatment. In this study, we characterize the immune microenvironment in papillary thyroid carcinomas, using immunohistochemical markers to inflammatory cells, and relate it with clinical and pathological prognostic features and with recurrence free survival rates. METHODS: The 151 included cases were selected from a database that included all patients who underwent surgical treatment for thyroid cancer at A.C.Camargo Cancer Center between the years 2008 and 2010. Tumor with significant thyroiditis were excluded. The selected tumors were submitted to immunohistochemical reactions with markers of inflammatory cells and analysis in complete slides by two experienced pathologists. Clinical and pathological features were evaluated, as well as recurrence and survival, relating them with the reading of marked cells. Survival analysis were made using Kaplan-Meier method, comparing survival curves with the Logrank test. RESULTS: One hundred and fifty one patients were included, of which 130 (86.1%) were females and 21 (13.9%) males. Multifocal disease was found in 41 cases (27.2%), extrathyroidal extension in 43 (28.5%) and lymph node metastasis in 36 (23.8%). Only two patients had distant metastasis. The mean follow-up time was 65.1 months and we observed nine (6%) tumor recurrences. Tumors with lymph node metastasis and/or extrathyroidal extension showed significantly higher recurrence rates. Of the analyzed markers, the cases with a higher density of CD8 (which marks cytotoxic T lymphocytes) in peritumoral areas presented a trend to better recurrence-free survival: 97.1% versus 87.5% (p=0.057), in addition to lower rates of mutifocal tumors and lymph node metastasis. A higher infiltration rate of CD8+ T lymphocytes in the tumor also correlated with less risk of lymph node metastasis in this sample (18.4% versus 38.1%, p=0.011). Besides that, the density of this marking both in the tumor and in peritumoral areas, was significantly higher in the papillary carcinomas limited to the thyroid gland (without extrathyroidal extension or metastasis). The other markers analyzed did not presented significant or consistent relation with recurrence or other prognostic factors. CONCLUSIONS: In thyroid cancer, the immune microenvironment seems to relate with pathological features of aggressiveness. This study showed that in papillary thyroid carcinomas without significant thyroiditis, the density of tumoral and peritumoral infiltration by CD8+ T lymphocytes is inversely related with lymph node metastasis rate and probably with recurrence, being therefore a marker of better prognosis. These data suggest that these lymphocytes play an anti-tumoral role in papillary thyroid carcinoma, supporting the implication of immune response in the progression of this neoplasm

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A Echinodorus macrophyllus (Alismataceae), conhecida como chapéu de couro no Brasil, é usada popularmente para tratar doenças reumáticas e inflamatórias. Neste trabalho, foram avaliados os efeitos antiinflamatórios do extrato aquoso de E. macrophyllus (EAEm) e suas frações etanólicas no modelo murino de air pouch. Para a obtenção das frações, 7 g do EAEm foram aplicadas em uma coluna cromatográfica aberta de sílica gel eluída com diferentes concentrações de etanol. Os cromatogramas do EAEm/frações foram obtidos usando um sistema de HPLC. Foram obtidas quatro frações, duas delas com maior rendimento. Resumidamente, a bolha de ar foi induzida pela injeção de 5 mL de ar estéril (s.c) no dorso de camundongos SW machos (25-35 g). Após 3 dias, mas 3 mL de ar estéril foram injetados para manter a bolha. No sexto dia, cada grupo (n = 4) foi tratado intraperitoneal (ip) ou oralmente (v.o) com EAEm (25 ou 250 mg/kg), Fr20 ou Fr40 (2,5, 25, 50 ou 100 mg/kg) e os controles com indometacina (10 mg/kg, v.o.) ou veículo (salina). Uma hora depois, 1 mL de salina ou de carragenina 1% estéril foi injetada dentro da bolha. Após 4 h, a cavidade foi lavada com NaCl 0,9%, EDTA 2 mM (1 mL), para a determinação do número de leucócitos, volume do exsudato e concentração de proteínas. Células do exsudato foram preparadas em citocentrífuga e coradas pelo método do Panótico para a contagem diferencial dos leucócitos. Cortes histológicos coletados dos diferentes grupos foram fixados com formol tamponado 10% (pH 7,4) por 7 dias, corados com HE e analisados em MO. A análise da expressão da iNOS e da COX-2 foi realizada em células do exsudato por RT-PCR. O acúmulo de nitrito (NO2−) no sobrenadante do cultivo de células RAW 264.7 foi determinado usando um ensaio colorimétrico baseado na reação de Griess. Os resultados foram expressos como média EP e comparados usando ANOVA seguido de teste de Dunnet. Os experimentos foram realizados em triplicata. No modelo air pouch, a injeção de carragenina 1% aumentou tanto a migração celular quanto a concentração de proteína no exsudato. Contudo, enquanto o pré-tratamento com a Fr40 aumentou a resposta inflamatória, o pré-tratamento com o EAEm e a Fr20, sobretudo por via i.p., inibiu esta resposta quando comparado ao grupo controle tratado apenas com o veículo. Assim, foram observadas as seguintes razões de inibição da migração de células: EAEm, i.p. a 25 mg/kg (66,44%) e a 250 mg/kg (87,27%) e Fr20 a 2,5 mg/kg (26,89%), 25 mg/kg (60,06%), 50 mg/kg (63,13%) e a 100 mg/kg (77,47%). Em relação à contagem diferencial, o EAEm e a Fr20 afetaram principalmente o número de neutrófilos, inibindo sua migração no exsudato. O EAEm e a Fr20 também reduziram a concentração total de proteínas no exsudato principalmente no tratamento i.p.; EAEm a 25 e 250 mg/kg mostrou 3,33 0,55 e 2,05 0,51 mg/mL, respectivamente, quando comparado aos grupos controles (Indometacina 2.88 0.64 mg/mL; Veículo 5.48 0.88 mg/mL). A Fr20 a 2,5, 25, 50 e 100 mg/kg mostrou 4,788 0,444, 1,417 0,519, 2,474 0,529 e 2,215 0, 361 mg/mL. A análise histológica mostrou infiltrado celular, principalmente composto de leucócitos polimorfonucleares ao longo da derme inflamada de animais tratados com veículo. O tratamento com o EAEm ou Fr20 reduziu a infiltração de leucócitos no tecido inflamado. Além disso, o tratamento com o EAEm e a Fr20 mostrou atividade supressora sobre a expressão de iNOS e COX-2, e mostrou efeitos inibitórios na produção de NO induzida por LPS. Concluindo, todos estes resultados confirmam o potencial antiinflamatório sugerido para esta planta e fornecem uma base para a compreensão de seus mecanismos moleculares de ação. Contudo, outros estudos devem ser realizados para melhor elucidar as vias pelas quais o EAEm e a Fr20 exercem seus efeitos antiinflamatórios. Além disso, estudos fitoquímicos devem ser realizados para identificar os compostos ativos no EAEm e na Fr20. / Echinodorus macrophyllus (Alismataceae), known as "chapéu de couro" in Brazil, is used popularly to treat rheumatic and inflammatory diseases. In this work we have evaluated the anti-inflammatory effects of the aqueous extract of E. macrophyllus (AEEm) and of its ethanolic fractions in mice air pouch model. Fractions were obtained by applying 7 g AEEm on a silica gel chromatography open column eluted with different ethanol concentrations. The fractions so obtained were evaporated under vacuum and lyophilized. Representatives chromatograms of EAEm/fractions were obtained using a HPLC system. We obtained four fractions, two with higher-yielding. Briefly, the air pouch was induced by 5 mL of sterile air injection (s.c.) on the back of male SW mice (25-35 g). After 3 days, 3 mL of sterile air has been injected again to keep it. After six days each group (n = 4) received intraperitoneal (i.p.) or oral (p.o.) treatment with AEEm (25 or 250 mg/kg), Fr20 or Fr40 (2.5, 25, 50 or 100 mg/kg) or controls indomethacin (10 mg/kg, p.o.) and vehicle (saline). One hour later 1 mL saline or carrageenan 1% sterile was injected into the pouch. After 4 h, the cavity was washed with NaCl 0.9%, EDTA 2 mM (1 mL), for determination of leukocyte numbers, final exudate volume and protein concentration. Cytospin preparations of exudates were stained with Panotic method for differential leukocyte count. Histological sections of tissue collected from different groups were fixed with 10% buffered formalin (pH 7.4) for 7 days and stained with HE and analyzed by MO. The iNOS and COX-2 expression analyses were performed on the exudate cells by RT-PCR. Accumulated nitrite (NO2−) in the media obtained from the RAW 264.6 cell cultures was determined using a colorimetric assay based on the Griess reaction. Results were expressed as mean SEM and compared using ANOVA and Dunnet‟s test. Experiments were performed in triplicate. In air pouch model, carrageenan 1% increased both the cell migration and the exudate protein level. However, while pretreatment with Fr40 increased inflammatory response, the pretreatment with AEEm and Fr20, mainly i.p. inhibited its when compared to the control group treated only the vehicle. So, the following rates of inhibition of cell migration were observed: AEEm, i.p. at 25 mg/kg (66.44%) and at 250 mg/kg (87.27%) and Fr20 at 2.5 mg/kg (26.89%), at 25 mg/kg (60.06%), at 50 mg/kg (63.13%) and at 100 mg/kg (77.47%). Regarding the differential count, the EAEm and Fr20 affected mainly the content of neutrophils, inhibiting the neutrophils migration in exudate. AEEm and Fr20 also reduced the total protein level in exudates mainly in the i.p. treatment. AEEm at 25 and 250 mg/kg showed 3.33 0.55 and 2.05 0.51 mg/mL, respectively, when compared to controls groups (Indomethacin 2.88 0.64 mg/mL; vehicle 5.48 0.88 mg/mL). Fr20 at 2.5, 25, 50 and 100 mg/kg showed 4.788 0.444, 1.417 0.519, 2.474 0.529 and 2.215 0.361 mg/mL. The histological analysis showed cellular infiltrate, mainly composed by polymorphonuclear leukocytes throughout the inflamed dermis of animals treated with vehicle. Treatment with AEEm or Fr20 reduced the leukocyte infiltrate on inflamed tissue. In addition, treatment with AEEm and Fr20 showed suppressive activity on iNOS and COX-2 expression, and showed inhibitory effects on LPS-induced nitric oxide production. In conclusion, all these findings support an anti-inflammatory potential suggested for this plant and provides a basis for understanding their action molecular mechanism. However, further studies should be undertaken to better elucidate the pathways by which AEEm and Fr20 exert their anti-inflammatory effects. In addition, phytochemical studies must be underway to identify active compounds in AEEm and Fr20.

Die Leber nimmt als Hauptzielorgan des metastasierenden Kolorektalkarzinoms (CRC) eine exponierte Rolle ein; bei mehr als 50 % der betroffenen Patienten werden im Laufe ihrer Tumorerkrankung Lebermetastasen diagnostiziert, die trotz multimodaler Therapiekonzepte immer noch den fatalen Kranksheitsverlauf bestimmen. Zwecks Entwicklung neuer Therapiestrategien ist ein fundiertes Verständnis der Entstehungsmechanismen und des Genexpressionsprofils der Metastasen erforderlich. Makrophagen wird in diesem Kontext ein großer Einfluss auf die Tumorentstehung und -progression zugeschrieben, weshalb von einer klinischen Relevanz der Makrophagenpolarisation auszugehen ist. Im Rahmen der vorliegenden Arbeit wurde mit der CRC Zelllinie CMT-93 ein syngenes, orthotopes Lebermetastasenmodell in C57BL/6N Mäusen etabliert. Basierend auf RNA Sequenzierungsdaten und bioinformatischen Analysemethoden wurde eine Entwicklung und fein abgestimmte Adaptation der CMT-93 Zellen im Zuge ihrer Propagation im hepatischen Milieu nachgewiesen. Diese resultierte in divergierenden Genexpressionsprofilen zwischen Zelllinie und Metastasen. Von insgesamt 3329 differentiell exprimierten Genen wurden mittels einer Selektionsliste 32 signifikant exprimierte Gene identifiziert. Insbesondere Matrix-Metalloproteasen (MMP-2, -7, -9), Chemokinrezeptoren (CXCR2, CXCR4), Zelladhäsionsgene (ITGA6, ITGB3) sowie Wif1 als Feinregulator des kanonischen Wnt Signalweges nehmen eine bedeutende Rolle ein und tragen zum Invasions- und Metastasierungsprofil von CMT-93 Zellen unter dem Einfluss des Lebermilieus bei. Invasive und aggressive Eigenschaften der CMT-93 induzierten Lebermetastasen wurden insbesondere im frühen und späten Metastasierungsstadium nachgewiesen. Hier wurde u.a. die Expression der EMT Marker Vimentin, MMP-7 und CD44, Ki-67, NFkb1 und Stat3 untersucht. Eine Gene Ontology Analyse und RNA Sequenzierungsdaten verschiedener Leberareale zeigten, dass die vielschichtige Interaktion zwischen CMT-93 Zellen und der hepatischen Mikroumgebung u.a. durch immunregulatorische Prozesse gesteuert wird, die in veränderten Genexpressionsprofilen zwischen Zelllinie, Metastase und tumorumgebendem Gewebe resultiert. In Lebermetastasen konnte eine Mischpopulation aus M1 und M2 Makrophagen nachgewiesen werden, die einen tendenziell M2 geprägten Charakter aufweisen. Dieser ist höchstwahrscheinlich an einer Stimulation der invasiven Eigenschaften der Tumorzellen beteiligt. Durch qRT-PCR und immunhistochemische Analysen konnten Hinweise gesammelt werden, dass nicht explizit die Quantität oder spezifische Lokalisation von Makrophagenphänotypen, sondern mutmaßlich eher das Zytokinprofil des Tumors und der Mikroumgebung und damit einhergehend der Polarisationsstatus der Makrophagen zum Metastasierungserfolg beiträgt. Eine Inkubation von CMT-93 Zellen mit konditionierten Medien der verschiedenen Makrophagenphänotypen bestätigte die Rolle sezernierter Faktoren. Eine indirekte Interaktion zwischen Tumorzelle und M2 Makrophagen reicht offensichtlich aus, um tumorstimulierende Eigenschaften, Aggressivität und Proliferationsfähigkeit der Zelllinie zu beeinflussen. Somit wurden Gene und Signalwege identifiziert, die relevant sind für eine erfolgreiche Induktion und Progression von Lebermetastasen infolge der Implantation von CRC-Zellen. Eine Adaptation des Genexpressionsprofils der CMT-93 Zelllinie im Zuge der Leberkolonisation konnte nachgewiesen werden. Das molekulare Profil bzw. die Gensignatur der Metastasen korrelierte dabei in hohem Maße mit der Migrations- und Invasionsfähigkeit der Tumorzellen. Insbesondere die Anwendung bioinformatischer Methoden erwies sich dabei als nützliches Werkzeug zur Analyse von großen Datensätzen, die mittels RNA Sequenzierung generiert wurden. Diese werden nun zur Formulierung prädiktiver Modelle der Metastasierungsvorgänge genutzt, um deregulierte Gene und damit einhergehend die Aggressivität von Tumorzellen gezielt identifizieren und konsekutiv beeinflussen zu können. Eine Analyse der Makrophagenphänotypen und ihrer Interaktion mit Tumorzellen verdeutlichte den durchaus tumorstimulierend geprägten Charakter der CMT-93 induzierten Lebermetastasen und den Einfluss des Zytokinprofils auf die Tumorprogression. Eine weiterführende Untersuchung der Makrophagenpolarisation bedarf jedoch eine rigorose Charakterisierung der Phänotypen anhand weiterer spezifischer Marker. Die subtile Analyse wird Rückschlüsse der organspezifischen Einflüsse des Zytokinprofils auf die kolorektale Karzinogenese erlauben.

Le chondrosarcome (CHS) est caractérisé par une grande chimio et radiorésistance ; il y a un besoin urgent de nouvelles stratégies thérapeutiques pour cette tumeur. Parmi celles-ci, certaines approches d'immunothérapie pourraient être d'un grand intérêt. Nous étudions actuellement l'implication du système immunitaire dans la progression du CHS et la réponse thérapeutique à la fois sur des échantillons humains et dans le modèle de chondrosarcome de rat (SRC).Dans le CHS humain et de rat, des infiltrats immunitaires composés de lymphocytes et macrophages ont été identifiés dans la zone péritumorale. L’infiltration immunitaire est en corrélation avec l’évolution de la tumeur (grade, envahissement et taille). L'expression de PD1 et PDL1 ont été détectée dans les infiltrats immunitaires et cellules tumorales du CHS chez l’homme et le rat. Le niveau d'expression PD-L1 en corrélation avec la survie des patients et le taux de rechute. Dans le model SRC, la déplétion sélective de lymphocytes T a entrainé une accélération de la progression tumorale, tandis que la déplétion de macrophages l’a ralenti. Les splénocytes isolés de rats porteurs de CHS ont montré une cytotoxicité spécifique dirigée contre les cellules de chondrosarcome (27%), qui a diminué de manière significative avec des rats appauvrie en CD3 (11%). La voie de signalisation PI3K/mTOR ne peut pas être associée à une immunothérapie car elle induit une action immunosuppressive in vivo.L'environnement immunitaire contribue à la progression du CHS à la fois chez l’homme et chez le rat, ce qui suggère que une approche immunomodulatrice avec des anticorps bloquant PDL1 pourrait être testée pour le CHS / Chondrosarcoma is highly resistant to chemotherapy and radiation and there is an urgent need in developing new therapeutic strategies for this malignancy; among these, some immunotherapy approaches could be of great interest. We are currently investigating the immune system implication in chondrosarcoma progression and therapeutic response both on human samples and in rat chondrosarcoma model (SRC). In human and rat chondrosarcoma, immune infiltrates composed of lymphocytes and macrophages were identified in the peritumoral area. Immune infiltrates composition was found correlated with tumors characteristics and evolution (grade, invasiveness and size). Expression of PD-1 and PD-L1 was detected in CHS immune infiltrates, both in human and rat (and on tumor cells). PD-L1 expression level correlated with patients survival and relapse rate. In SRC, T lymphocytes depletion resulted in an accelerated tumor progression, while CD163+ macrophages depletion slowed down tumor progression. Splenocytes isolated from CHS bearing SRC showed a specific cytotoxicity directed against chondrosarcoma cells (27%), which significantly decreased in CD3 depleted SRC (11%). The immune environment contributes to CHS progression in both human and animal models, this associated with expression of immune checkpoint PD1/PDL1 suggest that immunomodulatory approaches with PD-L1 blocking antibody could be applied in CHS; this approach is currently being tested in SRC

A leishmaniose é um termo global para doenças causadas por parasitos do gênero Leishmania, sendo a Leishmaniose Visceral (LV) a forma mais grave da doença. No Brasil é causada pelo parasita Leishmania (Leishmania) infantum chagasi. Para garantir a sua sobrevivência, alguns parasitas são capazes de manipular respostas de defesa das células do sistema imune. Recentes estudos demonstraram a participação da proteína imunomodulatória CD200 durante o processo de infecção de L. (L.) amazonenses. O presente estudo teve como objetivo investigar se os parasitos L. (L.) infantum chagasi são capazes de induzir a expressão da proteína CD200 durante o processo infeccioso. Em ensaios de infecção ex vivo, não foi observado proliferação de parasitas intracelulares. Apesar disso, L. (L.) infantum chagasi foi capaz de induzir a expressão do gene CD200. De maneira interessante, diferente de infecções por L. (L.) amazonenses, a indução de CD200 nessas células foi observada em tempos mais tardios de infecção. Ensaios de imunoprecipitação e Western blot indicaram a síntese da proteína, que atingiu os seus maiores níveis a 120 horas pós-infecção. A presença de CD200 sugere o envolvimento dessa molécula em tempos mais tardios de infecção por L. (L.) infantum chagasi. / Leishmaniasis is a global term for diseases caused by parasites of the genus Leishmania, and Visceral Leishmaniasis (VL) are the most severe form of the disease. In Brazil is caused by the parasite Leishmania (Leishmania) infantum chagasi. To ensure their survival, some parasites can handle defensive responses of the cells of the immune system. Recent studies have demonstrated the participation of immunomodulatory protein CD200 during the infection process of L. (L.) amazonenses. This study aimed to investigate whether the parasites L. (L.) infantum chagasi are capable of inducing the expression of CD200 protein during the infectious process. In trials of ex vivo infection, there was no proliferation of intracellular parasites. Nevertheless, L. (L.) infantum chagasi was able to induce the expression of CD200 gene. Interestingly, unlike infection by L. (L.) amazonenses, CD200 induction of these cells was observed at later times in infection. Immunoprecipitation assays and Western blot indicated protein synthesis, which reached their highest levels at 120 hours post-infection. The presence of CD200 suggests the involvement of this molecule at later times of infection with L. (L.) infantum chagasi.

Introdução Os fumantes inalam grandes quantidades de partículas de carbono, o que pode contribuir para efeitos adversos pulmonares e sistêmicos. É sabido que os macrófagos alveolares (MA) desempenham um papel extremamente importante no reconhecimento e processamento de qualquer material estranho inalado e são as células predominantes que processam e removem partículas inaladas. Existe também a deposição superficial a longo prazo do carbono observado nos pulmões de fumantes em autópsias. Atualmente, a distribuição e retenção de partículas de fumo derivadas de cigarros quando a pessoa também está exposta a níveis elevados de poluição do ar ainda não é clara. Portanto, procurou-se avaliar a carga de carbono nos MA e a deposição de superfície pulmonar em uma população exposta a alta poluição atmosférica (São Paulo), tanto em fumantes como não-fumantes. Métodos Uma coorte de 72 sujeitos post mortem foi obtida do Serviço de Verificação de Óbitos da Capital da Universidade de São Paulo (SVOC). As imagens das superfícies pulmonares foram obtidas sob condições padrão e pequenos fragmentos de tecido pulmonar foram coletados para análise de macrófagos usando a técnica de esfregaço. A superfície total de negro de carbono foi analisada utilizando o programa Imagem J (National Institute of Health, MD, EUA), teste cego ao fumo. A absorção interna de carbono nos MA foi medida utilizando o programa Image Pro Plus (The Proven Solution, Media Cybernetics Inc., EUA). A aprovação ética foi obtida. A média de negro de carbono de macrófagos tanto em fumantes como em não-fumantes foi analisada utilizando teste de Mann Whitney e expressa como intervalo interquartil (IQR). Resultados Os fumantes têm um nível significativamente mais elevado de negro de carbono nos macrófagos alveolares (103.4 (IQR 29.44 to 226.3) vs. 9.27 (IQR 3.1 to 85.13) um2, P < 0.001)103.4um2. Não houve diferença significativa entre a área média de deposição superficial de carbono nos pulmões de fumantes e não fumantes de 6, 74 cm2 (IQR 3, 47 a 10, 02) versus 5, 20 cm2 (IQR 2, 29 a 7, 54) P=NS. Conclusão O teor de carbono nos MA é claramente muito maior nos fumantes do que os nãofumantes. No entanto, a análise da superfície pulmonar não mostrou diferença significativa. Isso pode indicar que, em uma área de alta poluição do ar, o principal fator que contribui para a deposição de carbono no pulmão a longo prazo é a exposição à poluição com efeitos limitados da exposição à fumaça de cigarro. O preto de carbono nos MA ainda aparece significativamente influenciado pela exposição à fumaça de cigarro / Rationale Smokers inhale large amounts of carbonaceous particulate matter, which may contribute to pulmonary and systemic adverse effects. It is clear that alveolar macrophages (AM) play a critically important role in the recognition and processing of any inhaled foreign material and are the predominant cells that process and remove inhaled particulate matter from the lung. There is also long-term surface deposition of carbon seen in the lungs of smokers at post-mortem. At present the distribution and retention of cigarette smoke-derived particulate matter when the person is also exposed to high levels of background air pollution is unclear. Therefore we sought to assess both AM carbon loading and lung surface deposition in a population exposed to high background air pollution (São Paulo) in both smokers and non-smokers. Methods A cohort of 72 post-mortem subjects was obtained from São Paulo Autopsy Centre (SVOC). Images of lung surfaces were obtained under standard conditions and small fragments of lung tissue were collected for macrophage analysis using smear technique. The total surface black carbon was analysed using Image J (National Institute of Health, MD, USA), blinded to smoking status. Internal AM carbon uptake was measured using Image Pro Plus (The Proven Solution, Media Cybernetics Inc., USA). Ethical approval was obtained. Mean macrophage black carbon in both smokers and non-smokers was analysed using Mann Whitney and expressed as median (IQR). Results Smokers have a significantly higher level of mean macrophage black carbon (103.4 (IQR 29.44 to 226.3) vs. 9.27 (IQR 3.1 to 85.13) um2, P < 0.001)103.4um2. There was no significant difference between the mean area of surface deposition of carbon in the lungs of smokers and non-smokers 6.74 cm2 (IQR 3.47 to 10.02) versus 5.20cm2 (IQR 2.29 to 7.54) P=NS. Conclusion AM carbon content is clearly much higher in the smokers than the non-smokers. However the lung surface analysis showed no significant difference. This could indicate that in an area of high air pollution the main contributing factor to long term lung carbon deposition is pollution exposure with limited effects from cigarette smoke exposure. AM black carbon still appears significantly influenced by cigarette smoke exposure

Obesity-associated metabolic disorders are amongst the most prevalent causes of death worldwide. Understanding how obesity leads to the development of the Metabolic Syndrome (MetS) and cardiovascular disease (CVD) will enable the development of novel therapies that dissociate obesity from its cardiometabolic complications. Our laboratory views the functional capacity of white adipose tissue (WAT), the organ designed for safe lipid storage, as a key factor in the development of MetS and CVD. At a genetically-defined stage of the aberrant WAT expansion that occurs during obesity, adipocytes undergo a functional failure, resulting in an impaired control of serum free fatty acid (FFA) concentration. In such setting, FFAs and their metabolic derivatives accumulate in other organs, where they cause lipotoxicity, leading to the development of insulin resistance and CVD. We therefore aim to understand the pathophysiological mechanisms that induce adipocyte dysfunction. The past two decades of research have established the immune system as an important regulator of WAT function. The number of adipose tissue macrophages (ATMs), the most abundant immune cell type in WAT, increases during obesity, resulting in WAT inflammation. Multiple genetic and pharmacological intervention studies of murine models of obesity have assigned a causal link between ATM pro-inflammatory activation and WAT dysfunction. However, while the propagation of inflammation in ATMs during obesity has been extensively studied, factors triggering ATM inflammatory activation are less clear. Recently, our lab has observed lipid accumulation in the ATMs isolated from obese mice. Lipid-laden ATMs were pro-inflammatory, leading us to hypothesise that aberrant lipid build-up in macrophages triggers WAT inflammation during obesity. This thesis expands on the initial findings from our lab and describes two novel mechanisms that potentially contribute to lipid-induced inflammatory activation of ATMs. In chapter 3, the role of de novo phosphatidylcholine (PC) synthesis pathway during lipotoxicity in macrophages is addressed. The first part of the chapter demonstrates that lipotoxic environment increased de novo PC synthesis rate in bone marrow-derived macrophages (BMDMs) and ATMs, and that loss of rate-limiting enzyme in de novo PC synthesis pathway, CTP:phosphocholine cytidylyltransferase a (CCTa) diminished saturated FFA-induced inflammation in BMDMs. In the second part, I show that macrophage-specific CCTa deletion did not impact on the development of WAT inflammation or systemic insulin resistance, but had a minor benefitial effect on hepatic gene transcription during obesity. Chapter 4 develops on recent observations of interactions between sympathetic nerves and macrophages in WAT. In the first part of the chapter, I demonstrate that stimulating B2-adrenergic receptor (B2AR), the main receptor for sympathetic neurotransmitter norepinephrine in macrophages, enhanced intracellular triglyceride storage by up-regulating diacylglycerol O-acyltransferase 1 (Dgat1) gene expression in BMDMs. The second part of the chapter shows that macrophage-specific B2AR deletion did not modulate systemic glucose and lipid metabolism during obesity, but mice lacking B2ARs in macrophages demonstrated augmented hepatic glucose production on a chow diet. Furthermore, systemic B2AR blockade or macrophage-specific B2AR deletion in mice did not affect the thermogenic response to cold exposure. Chapter 5 includes the characterisation of B2AR stimulation-induced changes to the global cellular proteome of BMDMs, and a subsequent validation of the role of candidate transcription factors in regulating B2AR agonism-induced gene expression in BMDMs.

La maladie de Parkinson (PD) est considérée comme la deuxième maladie neurodégénérative la plus fréquente. L'examen post-mortem de patients parkinsoniens et des modèles physiologiques d’études de la maladie de Parkinson suggèrent la participation de la mort cellulaire programmée, l'inflammation et l'autophagie dues au stress oxydatif, à des mutations ou l’agrégation de protéines au sein des neurones DA. Les aptamères peptidiques sont de petites protéines combinatoires, consistitués d’une plateforme (dans notre cas, la thiorédoxine humaine, hTRX) et une boucle variable insérée dans le domaine actif de hTRX. Deux aptamères peptidiques ont été identifiés par la sélection fonctionnelle. L’aptamère peptide 32 (Apta-32) ,est spécifique liant deux paralogues T32 impliqués dans le processus d'endocytose. L’aptamère peptidique 34(Apta-34) lie à une cible "T34", une protéine pro-apoptotique ayant un rôle dans la voie apoptotique provenant du noyau. Le travail de cette thèse visait à étudier la fonction anti-apoptotique de nos deux aptamères peptidiques dans deux modèles d’étude de la maladie de Parkinson: un modèle cellulaire (in vitro) et un modèle transgénique D. melanogaster (in vivo). Deux toxines majeures ont été appliquées dans ce travail, 6-hydroxindopamine (6-OHDA) et le paraquat, un pesticide couramment utilisé. Nos observations montrent que la drosophile exprimant Apta-32 dans tous les neurones ont montré une meilleure résistance après 48h de traitement avec le paraquat comparé à deux autre aptamères peptidiques, Apta-34 et Apta-TRX (sans boucle de contrôle variable). Une autre étude a révélé un défaut dans la phagocytose des corps apoptotiques au cours du développement embryonnaire de la drosophile exprimant Apta-32 dans les macrophages, ce qui suggère qu’Apta-32 pourrait participer à et peut-être interférer avec le processus de l’autophygie, et que Apta-32 pourrait protéger contre l'autophagie induite par paraquat dans les neurones. / Parkinson’s disease is considered as the second most common neurodegenerative disease. Although the cause of the progressive cell loss of PD remains unclear to date, programmed cell death, inflammation and autophagy due to oxidative stress, gene mutations or protein aggregations within DA neuron have been suggested as potential causes. Peptide aptamers are small combinatorial proteins, with a variable loop inserted into a scaffold protein, human thioredoxin, hTRX. They are used to facilitate dissection of signaling networks by modulating specific protein interactions and functions. Two peptide aptamers were identified by functional selection which inhibit Bax-dependent cell death in mammalian models. One peptide aptamer (Apta-32) is binding two paralogues involved in endocytotic trafficking T32. The second peptide aptamer (Apta-34) is binding to a target "T34", a pro-apoptotic protein mediating apoptosis emanating from the nucleus. The work of my PhD thesis aimed to investigate the anti-apoptotic function of our two peptide aptamers in different PD models including cell model (in vitro), brain tissue slice and D. melanogaster (in vivo) ; in particular their impact on neuron survival after exposure to specific toxins. Two major toxins were applied in this work, 6-hydroxindopamine (6-OHDA) and Paraquat, a commonly used pesticide. Our observations indicated that Drosophila expressing Apta-32 in all neurons showed more resistance 48h after treatment with Paraquat, compared to drosophila expressing Apta-34 or TRX. Another study revealed a defect in phagocytosis of apoptotic bodies in drosophila embryo’s expressing Apta-32 in macrophage, suggesting Apta-32 could be involved in, and perhaps interfere with, the process of autophagy. This suggests that Apta-32 could protect against paraquat induced autophagy in neurons.

Considerando a importância do perfil do macrófago na infecção por Leishmania e o potencial papel de CD100 na modulação da ativação do m<font face=\"Symbol\">j, postulamos que essa molécula pode afetar a infectividade dessa célula por este parasita. Portanto, analisou-se e comparou-se a expressão de CD100 em diferentes tipos de m<font face=\"Symbol\">j (BALB/c e C57BL/6) e seus efeitos em termos de ativação e infecção com L. amazonensis. Avaliamos a expressão de CD100 e observamos que m<font face=\"Symbol\">js peritoneais de BALB/c expressam mais CD100 do que os de C57BL/6 e que esta expressão diminui ao longo da infecção. M<font face=\"Symbol\">j de BALB/c pré-tratados com sCD100 antes da infecção tem maior índice no tempo de 4h e quando em contato com o sCD100 antes, durante e após a infecção, o índice aumenta em 4 e 24h. Em m<font face=\"Symbol\">j de C57BL/6 pré-tratados com sCD100 ou no período de infecção, o índice aumenta em 4h. Quando permanecem durante todo o tempo em contato com o sCD100 o índice aumenta nos períodos de 4 e 24h. Concluímos que o CD100 pode estar relacionado com a modulação da infecção, e que a infecção afeta a expressão dessa molécula pelo m<font face=\"Symbol\">j. / Considering the importance of m<font face=\"Symbol\">j in Leishmania´s infection and the potential role of CD100 in the modulation of m<font face=\"Symbol\">j activation, we postulated that this molecule may affect m<font face=\"Symbol\">j infectivity. Therefore, we analyzed and compared the expression of CD100 on different types of m<font face=\"Symbol\">j (BALB/c and C57BL/6) and their effects in terms of m<font face=\"Symbol\">j activation and infectivity by L. amazonensis. We analyzed the expression of CD100 and the results suggest that m<font face=\"Symbol\">j from BALB/c express more CD100 than C57BL/6 and this expression decrease along the infection. M<font face=\"Symbol\">j from BALB/c pre-treated with sCD100 have augmented infection rates in 4h and in contact with the sCD100 before, during and after infection, increase rates in periods of 4 and 24h. In m<font face=\"Symbol\">j from C57BL/6 pre-treated with sCD100 or infection period, the rates increase in 4h. When macrophages remained in contact with sCD100 throughout the infection, rates increase after 4 and 24h. We can therefore conclude that CD100 may be related to modulation of Leishmania´s infection and that the infection itself affects CD100 expression in m<font face=\"Symbol\">j.