227 research outputs found

    Regulação da ativação epigenética do gene MMP2 diante da sinalização da fibronectina em linhagens tumorais de mama

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    Orientadora : Profª Drª Giseli KlassenCo-orientadora : Profª Drª Edneia A. S. R. CavalieriDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Microbiologia, Parasitologia e Patologia. Defesa: Curitiba, 02/12/2014Inclui referênciasÁrea de concentraçãoResumo: O câncer de mama é o tipo de câncer mais frequente entre as mulheres em todo o mundo. A enzima MMP-2 é uma metaloprotease capaz de degradar o colágeno tipo IV, um dos principais constituintes da matriz extracelular (MEC). Sua função é amplamente descrita por atuar no mecanismo de metástases em diversos tipos de câncer. Estudos recentes demonstraram que a linhagem tumoral de mama MCF7 possui o gene MMP2 regulado por metilação do DNA. Além disso, alguns estudos têm demonstrado que a fibronectina, uma importante proteína constituinte da MEC, é capaz de promover a expressão de MMP-2 em linhagens tumorais de mama. Resultados recentes do nosso grupo mostraram que a linhagem tumoral MCF7 quando tratada com fibronectina por 5 horas sofre 30% de desmetilação da região promotora do gene MMP2. Entretanto, observou-se também que tal processo foi transitório, porque houve a remetilação parcial do promotor quando o estímulo foi retirado. Ainda, a fibronectina induziu o aumento de uma marca de histona no promotor de MMP2 que sinaliza para a ativação da transcrição gênica. Diante disso, o objetivo deste trabalho foi dar continuidade a esse estudo, avaliando a regulação epigenética do gene MMP2 em linhagens tumorais de mama após o cultivo com fibronectina em diferentes tempos. Para isso, células da linhagem MCF7 submetidas ao tratamento com fibronectina por 8, 12 e 24 horas, e da linhagem MDA-MB-436 por 24 horas, tiveram seus níveis de expressão de MMP2, metilação do DNA e modificações de histonas do promotor do gene avaliados. Na linhagem MCF7, o tratamento por 8, 12 e 24 horas foi capaz de promover o aumento da expressão de MMP2 em 7, 25 e 9 vezes, respectivamente, comparado com o controle, assim como a redução da metilação do promotor de 90% (controle) para 70%, 40% e 52%, respectivamente. O tratamento por 24h também promoveu o aumento da marca de ativação gênica H3K4me3. Ainda, após 12h de cultivo com fibronectina, a linhagem MCF7 aumentou sua capacidade migratória. A fim de verificar se o efeito observado não era exclusivo da linhagem MCF7, foi incluída no estudo a linhagem MDA-MB-436 que foi submetida ao tratamento por 24h, e apresentou aumento na expressão gênica de MMP2 (4 vezes) e redução da metilação do promotor (de 90% para 22%). Por fim, as células tratadas foram mantidas em cultivo sem a fibronectina para avaliar a estabilidade das modificações. Essas células, chamadas de recultivo, apresentaram uma redução na expressão gênica e aumento na metilação do promotor quando comparadas com as células logo após os tratamentos, confirmando o efeito transitório da fibronectina nos eventos epigenéticos, que já tinha sido observado após 5 horas de tratamento. Esses dados corroboram e complementam os mecanismos observados pelo nosso grupo de pesquisa e contribuem para a compreensão dos mecanismos epigenéticos que regulam a expressão de um importante gene associado às metástases tumorais. Palavras-chave: Câncer de mama, MMP-2, epigenética, metilação do DNA.Abstract: Breast cancer is the most common cancer worldwide among women. The MMP- 2 enzyme is a metalloprotease capable of degrading type IV collagen, a major constituent of the extracellular matrix (ECM). Its function is widely described by acting in the mechanism of metastasis in various cancers. Recent data has shown that the MCF7 breast tumor cell line has the MMP2 gene regulated by DNA methylation. In addition, some studies have shown that fibronectin, an important constituent of the extracellular matrix, is capable of promoting MMP-2 expression in breast tumor cell lines. Recent results from our group showed that fibronectin was able to induce MMP2 expression by a 30% decrease in its promoter methylation in MCF7 tumor cell line. However, it was also noted that this process was transient, because a partial promoter remethylation was observed when the stimulus was removed. Moreover, a histone marker for an open chromatin conformation was significantly increased. Therefore, the aim of this work was to continue evaluating the epigenetic regulation of the MMP2 gene after cultivation of breast tumor cell lines with fibronectin at different times. To this reason, MCF7 cells subjected to treatment with fibronectin for 8, 12 and 24 hours and MDA-MB-436 cells subjected to treatment for 24 hours, were evaluated by the levels of MMP2 expression, gene promoter DNA methylation and histone modifications. In the MCF7 cell line, treatments for 8, 12 and 24 hours were able to promote 7, 9 and 25-fold increase in MMP2 expression, respectively, compared with the mock. In addition, promoter methylation was reduced from 90% (control) to 70%, 40% and 52%, respectively. Treatment for 24h also promoted an increase of the histone open chromatin conformation marker H3K4me3. Moreover, the migratory capacity of MCF7 cells treated for 12h was increased. In order to confirm the fibronectin effect, the MDA-MB-436 cell line was subjected to 24h fibronectin treatment, which showed an increase in MMP2 gene expression (4-fold) and a reduction of promoter methylation (from 90% to 22%). Finally, the treated cells were kept in culture without fibronectin to evaluate the modifications stability. These cells (recultivation) showed a gene expression reduction and promoter methylation increased when compared to the cells after treatments, confirming the fibronectin transient effect observed after the 5h treatment. These data support and complement the mechanisms observed by our research group and contribute to understand the epigenetic mechanisms that regulate the expression of an important gene associated with tumor metastasis. Keywords: Breast cancer, MMP-2, epigenetics, DNA methylation

    Trajectory of Spike-Specific B Cells Elicited by Two Doses of BNT162b2 mRNA Vaccine

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    : The mRNA vaccines for SARS-CoV-2 have demonstrated efficacy and immunogenicity in the real-world setting. However, most of the research on vaccine immunogenicity has been centered on characterizing the antibody response, with limited exploration into the persistence of spike-specific memory B cells. Here we monitored the durability of the memory B cell response up to 9 months post-vaccination, and characterized the trajectory of spike-specific B cell phenotypes in healthy individuals who received two doses of the BNT162b2 vaccine. To profile the spike-specific B cell response, we applied the tSNE and Cytotree automated approaches. Spike-specific IgA+ and IgG+ plasmablasts and IgA+ activated cells were observed 7 days after the second dose and disappeared 3 months later, while subsets of spike-specific IgG+ resting memory B cells became predominant 9 months after vaccination, and they were capable of differentiating into spike-specific IgG secreting cells when restimulated in vitro. Other subsets of spike-specific B cells, such as IgM+ or unswitched IgM+IgD+ or IgG+ double negative/atypical cells, were also elicited by the BNT162b2 vaccine and persisted up to month 9. The analysis of circulating spike-specific IgG, IgA, and IgM was in line with the plasmablasts observed. The longitudinal analysis of the antigen-specific B cell response elicited by mRNA-based vaccines provides valuable insights into our understanding of the immunogenicity of this novel vaccine platform destined for future widespread use, and it can help in guiding future decisions and vaccination schedules

    B cell response after SARS-CoV-2 mRNA vaccination in people living with HIV

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    Background: Limited longitudinal data are available on immune response to mRNA SARS-CoV-2 vaccination in people living with HIV (PLWHIV); therefore, new evidence on induction and persistence of spike-specific antibodies and B cells is needed. Methods: In this pilot study we investigated the spike-specific humoral and B cell responses up to six months after vaccination with two doses of mRNA vaccines in 84 PLWHIV under antiretroviral therapy compared to 79 healthy controls (HCs). Results: Spike-specific IgG persisted six months in PLWHIV with no significant differences compared to HCs, even though a significantly lower IgG response was observed in patients with CD4+ T cells < 350/mmc. The frequency of subjects with antibodies capable of inhibiting ACE2/RBD binding was comparable between PLWHIV and HCs a month after the second vaccine dose, then a higher drop was observed in PLWHIV. A comparable percentage of spike-specific memory B cells was observed at month six in PLWHIV and HCs. However, PLWHIV showed a higher frequency of spike-specific IgD- CD27- double-negative memory B cells and a significantly lower rate of IgD- CD27+ Ig-switched memory B cells compared to HCs, suggesting a reduced functionality of the antigen-specific memory B population. Conclusions: The mRNA vaccination against SARS-CoV-2 elicits humoral and B cell responses quantitatively similar between PLWHIV and HCs, but there are important differences in terms of antibody functionality and phenotypes of memory B cells, reinforcing the notion that tailored vaccination policies should be considered for these patients

    TRAIL receptors are expressed in both malignant and stromal cells in pancreatic ductal adenocarcinoma

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    : This study assesses the expression of all TNF-related apoptosis-inducing ligand (TRAIL) receptors in pancreatic ductal adenocarcinoma (PDAC) tumor tissue. We aimed to include TRAIL receptor expression as an inclusion parameter in a future clinical study using a TRAIL-based therapy approach for PDAC patients. Considering the emerging influence of PDAC desmoplastic stroma on the efficacy of anti-PDAC therapies, this analysis was extended to tumor stromal cells. Additionally, we performed PDAC stroma characterization. Our retrospective cohort study (N=50) included patients with histologically confirmed PDAC who underwent surgery. The expression of TRAIL receptors (DR4, DR5, DcR1, DcR2, and OPG) in tumor and stromal cells was evaluated by immunohistochemistry (IHC). The amount of tumor stroma was assessed by anti-vimentin IHC and Mallory's trichrome staining. The prognostic impact was determined by the univariate Cox proportional hazards regression model. An extensive expression of functional receptors DR4 and DR5 and a variable expression of decoy receptors were detected in PDAC tumor and stromal cells. Functional receptors were detected also in metastatic tumor and stromal cells. A poor prognosis was associated with low or absent expression of decoy receptors in tumor cells of primary PDAC. After assessing that almost 80% of tumor mass was composed of stroma, we correlated a cellular-dense stroma in primary PDAC with reduced relapse-free survival. We demonstrated that TRAIL functional receptors are widely expressed in PDAC, representing a promising target for TRAIL-based therapies. Further, we demonstrated that a low expression of DcR1 and the absence of OPG in tumor cells, as well as a cellular-dense tumor stroma, could negatively impact the prognosis of PDAC patients

    Results from a European multi-cohort study

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    Publisher Copyright: © 2021 The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.Background: INSTIs have become a pillar of first-line ART. Real-world data are needed to assess their effectiveness in routine care. Objectives: We analysed ART-naive patients who started INSTI-based regimens in 2012-19 whose data were collected by INTEGRATE, a European collaborative study including seven national cohorts. Methods: Kaplan-Meier analyses assessed time to virological failure (VF), defined as one viral load (VL) ≥1000 copies/mL, two consecutive VLs ≥50 copies/mL, or one VL ≥50 copies/mL followed by treatment change after ≥24 weeks of follow-up, and time to INSTIs discontinuation (INSTI-DC) for any reason. Factors associated with VF and INSTI-DC were explored by logistic regression analysis. Results: Of 2976 regimens started, 1901 (63.9%) contained dolutegravir, 631 (21.2%) elvitegravir and 444 (14.9%) raltegravir. The 1 year estimated probabilities of VF and INSTI-DC were 5.6% (95% CI 4.5-6.7) and 16.2% (95% CI 14.9-17.6), respectively, and were higher for raltegravir versus both elvitegravir and dolutegravir. A baseline VL ≥100 000 copies/mL [adjusted HR (aHR) 2.17, 95% CI 1.55-3.04, P 3 drugs versus 3 drugs (aHR 2.73, 95% CI 1.55-4.79, P < 0.001) and starting ART following availability of dolutegravir (aHR 0.64, 95% CI 0.48-0.83, P = 0.001). Major INSTI mutations indicative of transmitted drug resistance occurred in 2/1114 (0.2%) individuals. Conclusions: This large multi-cohort study indicates high effectiveness of elvitegravir- or dolutegravir-based first-line ART in routine practice across Europe.publishersversionpublishe

    Inhibition of ERK1/2 signaling prevents bone marrow fibrosis by reducing osteopontin plasma levels in a myelofibrosis mouse model

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    Clonal myeloproliferation and development of bone marrow (BM) fibrosis are the major pathogenetic events in myelofibrosis (MF). The identification of novel antifibrotic strategies is of utmost importance since the effectiveness of current therapies in reverting BM fibrosis is debated. We previously demonstrated that osteopontin (OPN) has a profibrotic role in MF by promoting mesenchymal stromal cells proliferation and collagen production. Moreover, increased plasma OPN correlated with higher BM fibrosis grade and inferior overall survival in MF patients. To understand whether OPN is a druggable target in MF, we assessed putative inhibitors of OPN expression in vitro and identified ERK1/2 as a major regulator of OPN production. Increased OPN plasma levels were associated with BM fibrosis development in the Romiplostim-induced MF mouse model. Moreover, ERK1/2 inhibition led to a remarkable reduction of OPN production and BM fibrosis in Romiplostim-treated mice. Strikingly, the antifibrotic effect of ERK1/2 inhibition can be mainly ascribed to the reduced OPN production since it could be recapitulated through the administration of anti-OPN neutralizing antibody. Our results demonstrate that OPN is a novel druggable target in MF and pave the way to antifibrotic therapies based on the inhibition of ERK1/2-driven OPN production or the neutralization of OPN activity

    Enhanced immunological recovery with early start of antiretroviral therapy during acute or early HIV infection–results of Italian Network of ACuTe HIV InfectiON (INACTION) retrospective study

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    ABSTRACT Background: Viral load peak and immune activation occur shortly after exposure during acute or early HIV infection (AEHI). We aimed to define the benefit of early start of antiretroviral treatment (ART) during AEHI in terms of immunological recovery, virological suppression, and treatment discontinuation. Setting: Patients diagnosed with AEHI (Fiebig stages I-V) during 2008-2014 from an analysis of 20 Italian centers. Methods: This was an observational, retrospective, and multicenter study. We investigated the ef- fect of early ART (defined as initiation within 3 months from AEHI diagnosis) on time to virolog- ical suppression, optimal immunological recovery (defined as CD4 count ≥ 500/μL, CD4 ≥ 30%, and CD4/CD8 ≥ 1), and first-line ART regimen discontinuation by Cox regression analysis. Results: There were 321 patients with AEHI included in the study (82.9% in Fiebig stage III-V). At diagnosis, the median viral load was 5.67 log10 copies/mL and the median CD4 count was 456 cells/μL. Overall, 70.6% of patients started early ART (median time from HIV diagnosis to ART initiation 12 days, IQR 6-27). Higher baseline viral load and AEHI diagnosis during 2012-2014 were independently associated with early ART. HBV co-infection, baseline CD4/CD8 ≥ 1, lower baseline HIV-RNA, and AEHI diagnosis in recent years (2012-2014) were independently associ- ated with a shorter time to virological suppression. Early ART emerged as an independent predic- tor of optimal immunological recovery after adjustment for baseline CD4 (absolute and percent- age count) and CD4/CD8 ratio. The only independent predictor of first-line ART discontinuation was an initial ART regimen including &gt; 3 drugs. Conclusions: In a large cohort of well-characterized patients with AEHI, we confirmed the ben- eficial role of early ART on CD4+ T-cell recovery and on rates of CD4/CD8 ratio normalization. Moreover, we recognized baseline CD4/CD8 ratio as an independent factor influencing time to virological response in the setting of AEHI, thus giving new insights into research of immunolog- ical markers associated with virological control

    Outcomes and Predictors of Mortality in Patients With KPC-Kp Infections Treated With Meropenem Vaborbactam: An Observational Multicenter Study

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    Background Meropenem-vaborbactam is a recent and promising option for the treatment of KPC-producing Klebsiella pneumoniae (KPC-Kp) infections, including those resistant to ceftazidime-avibactam.Methods We conducted a retrospective analysis of observational data from 19 Italian hospitals on use and outcomes of patients treated with meropenem-vaborbactam for at least &gt;= 24 hours for KPC-Kp infections. Crude and propensity-weighted multiple Cox regression models were performed to ascertain risk factors independently associated with 30-day mortality.Results The cohort included 342 adults with bloodstream infections (n = 172) and nonbacteremic infections (n = 170), of which 107 were lower respiratory tract infections, 30 were complicated urinary tract infections, and 33 were infections involving other sites. Most infections (62.3%) were managed with meropenem-vaborbactam monotherapy, or in combination with at least 1 other active drug (usually fosfomycin, tigecycline, or gentamicin) (37.7%). The 30-day mortality rate was 31.6% (108/342). In multiple Cox regression model, 30-day mortality was independently associated with septic shock at infection onset, Charlson comorbidity index &gt;= 3, dialysis, concomitant COVID-19, and INCREMENT score &gt;= 8. Administration of meropenem-vaborbactam within 48 hours from infection onset was a negative predictor of mortality. All predictors, except administration of meropenem-vaborbactam within 48 hours, remained significant when the multiple Cox regression model was repeated after adjustment for the propensity score for receipt of combination therapy.Conclusions Despite the limits of a retrospective study, the data derived from this multicenter cohort provide additional evidence on the efficacy of meropenem-vaborbactam in treating severe KPC-Kp infections, even when used as monotherapy
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