Expression of cytokines, chemokines receptors in peripheral blood mononuclear cells from patients with coronary arthery disease

Orientador: Maria Heloisa Souza Lima BlottaDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias MedicasResumo: Pesquisas recentes têm demonstrado que a aterosclerose é uma doença inflamatória crônica. Diversos componentes que participam da resposta imunológica inata e adaptativa foram descritos nas lesões e estão relacionados com o início e progressão das mesmas. Monócitos/macrófagos, linfócitos e células endoteliais possuem papel central na patogênese da aterosclerose por meio da produção de citocinas inflamatórias e anti-inflamatórias, como a IL-1ß, IL-6, TNF-a, IFN-?, IL-10, TGF-ß e quimiocinas. Quimiocinas são citocinas de baixo peso molecular (8-10 kDa) que promovem quimiotaxia e ativação celular no local da lesão. A resposta ao estímulo promovido pelas quimiocinas é resultado de sua ligação a receptores que sinalizam via proteína G trimérica. Este trabalho teve como objetivo analisar a resposta imunológica sistêmica de pacientes com doença arterial coronariana (DAC) comparada a controles saudáveis. Para tal, avaliamos a expressão do RNA mensageiro (RNAm) e a produção in vitro das quimiocinas CCL2 (MCP-1), CXCL8 (IL-8), CXCL9 (Mig), CXCL10 (IP-10), dos receptores CCR2 e CXCR3 e das citocinas IFN-?, IL-10 e TGF-ß em células mononucleares do sangue periférico de pacientes com angina estável (AE), instável (AI) e controles saudáveis estimuladas ou não com LDL oxidada (LDLox) ou lipopolissacarídeo (LPS). Os resultados mostraram que pacientes com DAC apresentam maior expressão constitutiva do RNAm para CCL2, CXCL8, CXCL9, CXCL10, IFN-? e, após estimulação com LDLox ou LPS, de CCL2 e CXCL8, em comparação ao grupo controle. A análise da expressão protéica (ou de proteínas) revelou maiores concentrações de CCL2 e CXCL8 nos pacientes, em relação aos controles. A expressão dos receptores mostrou que pacientes com DAC apresentam maior porcentagem de células CCR2+ de forma constitutiva e de células CXCR3+ após estimulação com LDLox ou LPS. As diferenças mais marcantes entre pacientes com AI e AE foram a maior produção da proteína CXCL8 e maior expressão de RNAm para CXCL9 no primeiro grupo, em contrapartida a maior expressão do RNAm para IL-10 e da citocina TGF-ß no grupo com AE. Em conjunto, estes dados sugerem que os pacientes com DAC dispõem de mecanismos que possibilitam a contínua migração de células do sangue periférico para o local da placa. Após a migração e ativação, estas células estariam aptas a responder rapidamente ao antígeno (LDLox) produzindo grandes quantidades de mediadores imunológicos, criando um ambiente inflamatório propenso ao desenvolvimento da lesão, com possível influência na ruptura e conseqüente surgimento de eventos isquêmicosAbstract: Atherosclerosis is now recognized to involve chronic inflammatory and immune response. Several components of innate and adaptive immune responses were described in the lesions and are involved in the initiation and progression of the atheroma. Monocytes/macrophages and lymphocytes have a key role in the pathogenesis of atherosclerosis through the production of inflammatory and antiinflammatory cytokines such as IL-1ß, IL-6, TNF-a, IFN-?, IL-10, TGF-ß and chemokines. Chemokines are low molecular mass cytokines (8 ¿ 10 kDa), which promote chemotaxis and cellular activation in the local of the lesion. The response to the stimulus promoted by the chemokines results from its link to receptors that signals through the G protein. This study aimed to analyze the systemic immune response of patients with coronary artery disease (CAD) by comparing them to healthy controls. To this purpose, we evaluated mRNA expression and the in vitro production CCL2 (MCP-1), CXCL8 (IL-8), CXCL9 (Mig), CXCL10 (IP-10), IFN-?, IL-10 and TGF-ß, in addition to the expression CCR2 and the CXCR3 in peripheral blood mononuclear cells (PBMC) of patients with stable (SA), unstable angina (UA) and healthy controls stimulated or not with oxidized LDL (LDLox) or with LPS. Our results showed that patients with coronary arterial disease (CAD) present higher constitutive expression of mRNA for CCL2, CXCL8, CXCL9, CXCL10, IFN-?? and, after stimulation with oxidized LDL or LPS, of CCL2 and CXCL8, as compared to the control group. We also detected higher ammounts of CCL2 and CXCL8 in the patients, when compared to the controls. The chemokine receptors expression showed that patients with CAD present higher percentage of cells CCR2+ in a constitutive manner and CXCR3+ cells after stimulation with LDLox or LPS. The main differences between SA and UA were higher expression of CXCL8 (protein) and CXCL9 mRNA in UA patients and higher IL-10 mRNA and TGF-ß (protein) in SA. All together our data suggest that PBMC of CAD patients are able to produce chemokines and cytokines involved in the regulation of monocytes and lymphocyte migration and retention into atherosclerotic lesions. After migration and activation, these cells would be ready to quickly respond to the antigen (LDLox) producing high amounts of inflammatory mediators, which may contribute to plaque instability, and the development of acute coronary syndromesMestradoCiencias BiomedicasDoutor em Ciências Médica

Applications of Fecal Microbiota Transplantation: Emphasis on Clostridioides difficile Infections

Objective: This study aimed to perform a comprehensive review of clinical trials using fecal microbiota transplantation in cases of Clostridioides difficile infection. Methods: This manuscript reviews clinical studies published from 2003 to 2020 at the Scientific Electronic Library Online (SciELO Brazil), Latin American and Caribbean Health Sciences Literature (LILACS) and US National Library of Medicine (MedLine/PubMed) databases using the descriptors antibiotic/antimicrobial, Clostridium difficile/Clostridioides difficile, intestinal microbiota/intestinal microbiome and fecal transplantation. Results: Interventions on microbiota include the use of probiotics, prebiotics, and fecal microbiota transplantation as therapeutic methods. Results show that fecal microbiota transplantation is an excellent alternative for the treatment of recurrent C. difficile infections

Identification of TH17 related cytokines in human atherosclerotic plaques

Orientadores: Maria Heloisa Souza Lima Blotta, Fábio Hüssemann MenezesTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: A presença de células T ativadas em lesões ateroscleróticas confirma a sua participação na progressão da doença. Recentemente, um novo subtipo de linfócitos TCD4+ produtores de IL-17 (TH17) foi descrito e a sua associação a doenças de caráter inflamatório, bem como a sua capacidade de induzir a produção de mediadores frequentemente encontrados em lesões ateroscleróticas (IL-1?? TNF-? ??TGF-? ??CCL2 e MMP) sugerem que possam ter uma participação na aterosclerose. O objetivo do presente estudo foi investigar componentes da resposta TH17 em lesões ateroscleróticas humanas e relacioná-los a outros integrantes da resposta imunológica. Como controle foram utilizados fragmentos da artéria aorta de 3 doadores de órgãos. Para tal, avaliamos fragmentos de placas ateroscleróticas obtidas de 41 pacientes com estenose crítica de carótida, submetidos à endarterectomia. A expressão gênica e protéica de diversos mediadores foi avaliada pelo método de RT-PCR em tempo real e imunoistoquímica, respectivamente. Em alguns experimentos fragmentos de placa aterosclerótica foram submetidos à estimulação in vitro com IL-23, IL-2, LPS, PHA ou ?CD3+?CD28, para posterior análise da expressão gênica (RT-PCR) e protéica (ELISA e citometria de fluxo) de mediadores relacionados à resposta TH17. A detecção do RNAm de IL-17 se restringiu a um pequeno número de amostras e em baixa concentração, enquanto a marcação imunoistoquímica foi negativa em todas as placas analisadas. Entretanto, observamos maior expressão relativa do RNAm de mediadores relacionados à resposta TH17 (IL-23, RorC, CCR4, IL-1?, TNF-? MMP-9), à resposta TH1 e TH2 (IFN-? e GATA-3), à resposta anti-inflamatória (FoxP3, IL-10, EBI3 e IL-27R) e a mediadores com potencial citotóxico (granzima A, perforina e granulisina) em placas de pacientes do que em amostras controle. Células positivas para IL-23, IL-1?, IL-18, ICAM-1, IL-10 e TGF- ? foram encontradas com maior frequência em placas classificadas como instáveis, ricas em macrófagos, células espumosas, cristais de colesterol e focos de necrose. Finalmente, a análise da suspensão celular obtida de fragmento de lesão revelou a presença de 1% de células IL-17+, enquanto a estimulação in vitro resultou em pequena produção de IL-17 apenas após estímulo com anti?CD3+ anti-CD28. Em conjunto, os resultados mostraram a expressão de componentes da resposta TH17, em associação com mediadores da resposta TH1, TH2 e Treg em lesões ateroscleróticas humanasAbstract: The detection of activated T cells in human atherosclerotic plaques confirms their participation in the disease progression. In this scenario, IFN-? -producing T cells (TH1) are well recognized as having a pro-atherogenic role. Recently, a new subtype of IL-17-producing T helper (TH17) lineage of cells has been described and their association with inflammatory diseases, as well as their capacity to induce mediators frequently found in atherosclerotic lesions (IL-1? ? TNF-? ,?TGF-?,??CCL2 e MMP) suggest that they may have a role in atherosclerosis. The aim of this study was to investigate whether components of the immune response mediated by TH17 cells are present in human atherosclerotic lesions and their association with other immune mediators. For this purpose, 41 patients with critical stenosis of carotid submitted to endarterectomy were evaluated. Aortic fragments from organ donors were used as control. Carotid specimens were analyzed for mRNA and protein expression by real time RT-PCR and immunohistochemistry, respectively. In some experiments fragments of atherosclerotic plaques were incubated in vitro with IL-23, IL-2, LPS, PHA or ?CD3+?CD28 to further evaluate mRNA and protein expression. A low number of samples showed a small expression of IL-17 mRNA, while no staining for IL-17 was detected in any of the analyzed plaques. Up-regulation of IL-17 related genes (IL-23, RorC, CCR4, CCR4, IL-1?, TNF-?, MMP-9), as well as TH1 e TH2 (IFN-? e GATA-3), anti-inflammatory mediators (FoxP3, IL-10, EBI1 e IL-27R) and cytotoxic molecules (granzyme A, perforin e granulysin) mRNA expression were detected in patients plaques compared to control arteries. In addition, unstable plaques showed a high number of IL-23, IL-1?, IL-18, ICAM-1, IL-10 e TGF- ? ??granzyme B and perforin positive cells. Finally, the analysis of plaque derived cell suspensions revealed only 1% of IL-17+ cells, while in vitro stimulation resulted in IL-17 production only in the presence of anti-CD3+anti-CD28. Altogether our results showed components of TH17 response in association with mediators of TH1, TH2 and Treg response in human atherosclerotic lesions. Further studies are ongoing to elucidate the interaction, balance and collaborative potential among these effectors lineages in atherosclerosis controlDoutoradoCiencias BiomedicasDoutor em Ciências Médica

Involvement of Regulatory T Cells in the Immunosuppression Characteristic of Patients with Paracoccidioidomycosis▿

Patients with paracoccidioidomycosis (PCM) exhibit a suppression of the cellular immune response characterized by negative delayed-type hypersensitivity (DTH) to Paracoccidioides brasiliensis antigens, the apoptosis of lymphocytes, and high levels of expression of cytotoxic-T-lymphocyte-associated antigen 4 (CTLA-4), interleukin-10 (IL-10), and transforming growth factor β (TGF-β). The aim of this study was to investigate whether and how regulatory T cells (Treg cells) are involved in this immunosuppression by analyzing the number, phenotype, and activity of these cells in patients with active disease (AD group) and patients who had received treatment (TD group). Our results showed that the AD patients had more Treg cells than the TD patients or controls (C group) and also had elevated levels of expression of regulatory markers (glucocorticoid-induced tumor necrosis factor [TNF] receptor-related protein [GITR], CTLA-4, CD95L, LAP-1, and CD38). An analysis of regulatory activity showed that Treg cells from the AD group had greater activity than did cells from the other groups and that cell-cell contact is mandatory for this activity in the C group but was only partially involved in the regulatory activity of cells from AD patients. The addition of anti-IL-10 and anti-TGF-β neutralizing antibodies to the cultures showed that the production of cytokines may be another mechanism used by Treg cells. In conclusion, the elevated numbers of these cells with an increased regulatory phenotype and strong suppressive activity suggest a potential role for them in the immunosuppression characteristic of paracoccidioidomycosis. In addition, our results indicate that while Treg cells act by cell-cell contact, cytokine production also plays an important role

Characterization of CD4+ T Cell Subsets in Patients with Abdominal Aortic Aneurysms

Background. The mediators produced by CD4+ T lymphocytes are involved in the pathogenesis of aneurysmal lesions in abdominal aortic aneurysm (AAA) patients. The aim of this study was to identify and characterize the CD4+ T cell subsets involved in human AAA. Methods. The CD4+ T cell subsets in 30 human aneurysmal lesions were determined using flow cytometry (FC) and immunohistochemistry (IHC). The peripheral blood mononuclear cells (PBMCs) from patients with AAA were also analyzed by FC and compared with control subjects. Results. Human aneurysmal lesions contained IFN-γ, IL-12p35, IL-4, IL-23p19, IL-17R, and IL-22 positive cells. PBMCs from AAA patients had higher expression levels of IFN-γ, TNF-α, IL-4, and IL-22 when compared to controls. Conclusions. Our results show the presence of TH1, TH2, TH17, and TH22 subsets in aneurysmal lesions of AAA patients and suggest that these cells may be mainly activated in situ, where they can induce tissue degradation and contribute to the pathogenesis of AAA

Differential expression of cytokines, chemokines and chemokine receptors in patients with coronary artery disease

Monocytes/macrophages and lymphocytes have a key role in the pathogenesis of atherosclerosis through the production of inflammatory and anti-inflammatory cytokines. We evaluated mRNA expression and protein production of CCL2, CXCL8, CXCL9, CXCL10, IFN-γ and IL-10 in vitro as well as the expression of the CCR2 and CXCR3 receptors in peripheral blood mononuclear cells (PBMCs) of patients with coronary artery disease (CAD) and healthy controls in the presence or absence of oxidized LDL (oxLDL). Patients with CAD showed higher constitutive expression of CCL2, CXCL8, CXCL9, CXCL10 and IFN-γ mRNA and, after stimulation with oxLDL, higher expression of CCL2 and CXCL8 mRNA than the control group. We also detected higher levels of CCL2 and CXCL8 in supernatants of oxLDL-stimulated PBMCs from CAD patients than in corresponding supernatants from controls. Patients with CAD had a higher percentage of constitutive CCR2+ and CXCR3+ cells after stimulation with oxLDL. Among CAD patients, the main differences between the stable (SA) and unstable angina (UA) groups were lower IL-10 mRNA production in the latter group. Altogether, our data suggest that PBMCs from CAD patients are able to produce higher concentrations of chemokines and cytokines involved in the regulation of monocyte and lymphocyte migration and retention in atherosclerotic lesions13611726CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP04/01697-6Não te

ClCPI, a cysteine protease inhibitor purified from Cassia leiandra seeds has antifungal activity against Candida tropicalis by inducing disruption of the cell surface

Infections caused by Candida tropicalis have increased significantly worldwide in parallel with resistance to antifungal drugs. To overcome resistance novel drugs have to be discovered. The objective of this work was to purify and characterize a cysteine protease inhibitor from the seeds of the Amazon rainforest tree Cassia leiandra and test its inhibitory effect against C. tropicalis growth. The inhibitor, named ClCPI, was purified after ion exchange and affinity chromatography followed by ultrafiltration. ClCPI is composed of a single polypeptide chain and is not a glycoprotein. The molecular mass determined by SDS-PAGE in the absence or presence of β-mercaptoethanol and ESI-MS were 16.63 kDa and 18.362 kDa, respectively. ClCPI was stable in the pH range of 7.0–9.0 and thermostable up to 60 °C for 20 min. ClCPI inhibited cysteine proteases, but not trypsin, chymotrypsin neither alpha-amylase. Inhibition of papain was uncompetitive with a Ki of 4.1 × 10 −7 M and IC 50 of 8.5 × 10 −7 M. ClCPI at 2.6 × 10 −6 M reduced 50% C. tropicalis growth. ClCPI induced damages and morphological alterations in C. tropicalis cell surface, which led to death. These results suggest that ClCPI have great potential for the development of an antifungal drug against C. tropicalis. © 201