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Severe COVID-19 is associated with hyperinflammation and weak T cell responses against SARS-CoV-2. However, the links between those processes remain partially characterized. Moreover, whether and how therapeutically manipulating T cells may benefit patients are unknown. Our genetic and pharmacological evidence demonstrates that the ion channel TMEM176B inhibited inflammasome activation triggered by SARS-CoV-2 and SARS-CoV-2-related murine ß-coronavirus. Tmem176b-/- mice infected with murine ß-coronavirus developed inflammasome-dependent T cell dysfunction and critical disease, which was controlled by modulating dysfunctional T cells with PD-1 blockers. In critical COVID-19, inflammasome activation correlated with dysfunctional T cells and low monocytic TMEM176B expression, whereas PD-L1 blockade rescued T cell functionality. Here, we mechanistically link T cell dysfunction and inflammation, supporting a cancer immunotherapy to reinforce T cell immunity in critical ß-coronavirus disease.
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Clinical and molecular heterogeneity are hallmarks of chronic lymphocytic leukemia (CLL), a neoplasm characterized by accumulation of mature and clonal long-lived CD5 + B-lymphocytes. Mutational status of the IgHV gene of leukemic clones is a powerful prognostic tool in CLL, and it is well established that unmutated CLLs (U-CLLs) have worse evolution than mutated cases. Nevertheless, progression and treatment requirement of patients can evolve independently from the mutational status. Microenvironment signaling or epigenetic changes partially explain this different behavior. Thus, we think that detailed characterization of the miRNAs landscape from patients with different clinical evolution could facilitate the understanding of this heterogeneity. Since miRNAs are key players in leukemia pathogenesis and evolution, we aim to better characterize different CLL behaviors by comparing the miRNome of clinically progressive U-CLLs vs. stable U-CLLs. Our data show up-regulation of miR-26b-5p, miR-106b-5p, and miR-142-5p in progressive cases and indicate a key role for miR-26b-5p during CLL progression. Specifically, up-regulation of miR-26b-5p in CLL cells blocks TGF-ß/SMAD pathway by down-modulation of SMAD-4, resulting in lower expression of p21-Cip1 kinase inhibitor and higher expression of c-Myc oncogene. This work describes a new molecular mechanism linking CLL progression with TGF-ß modulation and proposes an alternative strategy to explore in CLL therapy.
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Despite the importance of recombinant protein production in the academy and industrial fields, many issues concerning the expression of soluble and homogeneous products are still unsolved. Several strategies were developed to overcome these obstacles; however, at present, there is no magic bullet that can be applied for all cases. Indeed, several key expression parameters need to be evaluated for each protein. Among the different hosts for protein expression, Escherichia coli is by far the most widely used. In this chapter, we review many of the different tools employed to circumvent protein insolubility problems.
Assuntos
Escherichia coli , Proteômica , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
Chronic lymphocytic leukemia (CLL) is the most common mature B-cell neoplasm in the West. IGHV4-34 is one of the most frequently used genes in CLL patients, which usually display an indolent outcome. In this study, we explored the mutational profile of CLL patients expressing IGHV4-34 within different stereotypes and their association with prognostic factors and clinical outcome. A multi-institutional cohort of unselected 1444 CLL patients was analyzed by RT-PCR and bidirectional sequencing. Cytogenetics and molecular cytogenetics analyses were also performed. We identified 144 (10%) IGHV4-34 expressing cases, 119 mutated (M), 44 of them with stereotyped B-cell receptors. Subset #4 was the most frequent (56.8% of cases) followed by subsets #16 (13.6%), #29 (6.8%), and #201 (2.3%), with different distribution among countries. Analysis of somatic hypermutation profile showed significant differences among stereotyped subsets for G28>D/E, P45>S, E55>Q, and S64>I changes (p < 0.01) and high frequency of disruption of the glycosylation motif in the VH CDR2 region. All stereotyped IGHV4-34 cases showed normal karyotypes. Deletion 13q14 as a sole alteration was present in 42.8% of stereotyped cases with a different distribution among subsets. A shorter time to first treatment was found in non-stereotyped vs. stereotyped M-IGHV4-34 patients (p = 0.034). Our results add new information supporting the importance of recurrent amino acid changes at particular positions, contributing to refine the molecular characterization of South American CLL patients.
Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Leucemia Linfocítica Crônica de Células B/genética , Idoso , Estudos de Coortes , Feminino , Rearranjo Gênico , Humanos , Leucemia Linfocítica Crônica de Células B/epidemiologia , Masculino , Pessoa de Meia-Idade , Mutação , Receptores de Antígenos de Linfócitos B/genética , Hipermutação Somática de Imunoglobulina , América do Sul/epidemiologiaRESUMO
Most cancers become more dangerous by the outgrowth of malignant subclones with additional DNA mutations that favor proliferation or survival. Using chronic lymphocytic leukemia (CLL), a disease that exemplifies this process and is a model for neoplasms in general, we created transgenic mice overexpressing the enzyme activation-induced deaminase (AID), which has a normal function of inducing DNA mutations in B lymphocytes. AID not only allows normal B lymphocytes to develop more effective immunoglobulin-mediated immunity, but is also able to mutate nonimmunoglobulin genes, predisposing to cancer. In CLL, AID expression correlates with poor prognosis, suggesting a role for this enzyme in disease progression. Nevertheless, direct experimental evidence identifying the specific genes that are mutated by AID and indicating that those genes are associated with disease progression is not available. To address this point, we overexpressed Aicda in a murine model of CLL (Eµ-TCL1). Analyses of TCL1/AID mice demonstrate a role for AID in disease kinetics, CLL cell proliferation, and the development of cancer-related target mutations with canonical AID signatures in nonimmunoglobulin genes. Notably, our mouse models can accumulate mutations in the same genes that are mutated in human cancers. Moreover, some of these mutations occur at homologous positions, leading to identical or chemically similar amino acid substitutions as in human CLL and lymphoma. Together, these findings support a direct link between aberrant AID activity and CLL driver mutations that are then selected for their oncogenic effects, whereby AID promotes aggressiveness in CLL and other B-cell neoplasms.
Assuntos
Citidina Desaminase/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Regulação para Cima , Animais , Modelos Animais de Doenças , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MutaçãoRESUMO
The enzyme activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes, critical actions for an effective adaptive immune response. However, in addition to the benefits generated by its physiological roles, AID is an etiological factor for the development of human and murine leukemias and lymphomas. This review highlights the pathological role of AID and the consequences of its actions on the development, progression, and therapeutic refractoriness of chronic lymphocytic leukemia (CLL) as a model disease for mature lymphoid malignancies. First, we summarize pertinent aspects of the expression and function of AID in normal B lymphocytes. Then, we assess putative causes for AID expression in leukemic cells emphasizing the role of an activated microenvironment. Thirdly, we discuss the role of AID in lymphomagenesis, in light of recent data obtained by NGS analyses on the genomic landscape of leukemia and lymphomas, concentrating on the frequency of AID signatures in these cancers and correlating previously described tumor-gene drivers with the presence of AID off-target mutations. Finally, we discuss how these changes could affect tumor suppressor and proto-oncogene targets and how they could be associated with disease progression. Collectively, we hope that these sections will help to better understand the complex paradox between the physiological role of AID in adaptive immunity and its potential causative activity in B-cell malignancies.
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The aim of this work was to evaluate the physicochemical and biological properties of docetaxel (DCX) loaded chitosan nanocapsules (CS Nc) functionalized with the monoclonal antibody Chi-Tn (CS-PEG-ChiTn mAb Nc) as a potential improvement treatment for cancer therapy. The Tn antigen is highly specific for carcinomas, and this is the first time that such structure is targeted for drug delivery. The nanocapsules (Ncs), formed as a polymeric shell around an oily core, allowed a 99.9% encapsulation efficiency of DCX with a monodispersity particle size in the range of 200 nm and a high positive surface charge that provide substantial stability to the nanosystems. Release profile of DCX from Ncs showed a sustained and pH dependent behavior with a faster release at acidic pH, which could be favorable in the intracellular drug delivery. We have designed PEGylated CS Nc modified with a monoclonal antibody which recognize Tn antigen, one of the most specific tumor associated antigen. A biotin-avidin approach achieved the successful attachment of the antibody to the nanocapsules. Uptake studies and viability assay conducted in A549 human lung cancer cell line in vitro demonstrate that ChiTn mAb enhance nanoparticles internalization and cell viability reduction. Consequently, these ChiTn functionalized nanocapsules are promising carriers for the active targeting of DCX to Tn expressing carcinomas.
Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Antineoplásicos/administração & dosagem , Quitosana/análogos & derivados , Docetaxel/administração & dosagem , Imunoconjugados/administração & dosagem , Nanocápsulas/química , Células A549 , Animais , Anticorpos Monoclonais/imunologia , Antineoplásicos/química , Células Cultivadas , Docetaxel/química , Proteína Duplacortina , Liberação Controlada de Fármacos , Humanos , Imunoconjugados/química , CamundongosRESUMO
Despite significant therapeutic improvements chronic lymphocytic leukemia (CLL) remains an incurable disease and there is a persistent pursuit of new treatment alternatives. Lurbinectedin, a selective inhibitor of active transcription of protein-coding genes, is currently in phase II/III clinical trials for solid tumors such as small-cell lung cancer (SCLC). In this study, we aimed to evaluate the activity of Lurbinectedin on circulating mononuclear cells from CLL patients and to determine whether Lurbinectedin could affect the cross-talk between B-CLL cells and the tumor microenvironment. We found that Lurbinectedin induced a dose- and time-dependent death in all cell types evaluated, with B cells, monocytes and monocytic myeloid derived suppressor cells (Mo-MDSC) being the most susceptible populations. At sub-apoptotic doses, Lurbinectedin decreased the expression of CCR7 in B-CLL cells and impaired their migration towards CCL19 and CCL21. Furthermore, low concentrations of Lurbinectedin stimulated the synthesis of pro-IL1ß in monocytes and nurse-like cells, without inducing the inflammasome activation. Altogether, these results indicate that Lurbinectedin might have antitumor activity in CLL due to its direct action on leukemic cells in combination with its effects on the tumor microenvironment. Our findings encourage further investigation of Lurbinectedin as a potential therapy for CLL.
Assuntos
Carbolinas/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Microambiente Tumoral/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Quimiocina CCL19/imunologia , Quimiocina CCL19/metabolismo , Quimiocina CCL21/imunologia , Quimiocina CCL21/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Células Supressoras Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Cultura Primária de Células , Receptores CCR7/imunologia , Receptores CCR7/metabolismo , Células Tumorais Cultivadas , Microambiente Tumoral/imunologiaAssuntos
Etnicidade/genética , Região Variável de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/genética , Receptores de Antígenos de Linfócitos B/genética , Biomarcadores Tumorais , Humanos , Leucemia Linfocítica Crônica de Células B/epidemiologia , Leucemia Linfocítica Crônica de Células B/etnologia , Mutação , América do Sul/epidemiologiaRESUMO
The advent of new DNA sequencing technologies leads to a dramatic increase in the number of available genome sequences and therefore of target genes with potential for functional analysis. The insertion of these sequences into proper expression vectors requires a simple an efficient cloning method. In addition, when expressing a target protein, quite often it is necessary to evaluate different DNA constructs to achieve a soluble and homogeneous expression of the target with satisfactory yields. The development of new molecular methods made possible the cloning of a huge number of DNA sequences in a high-throughput manner, necessary for meeting the increasing demands for soluble protein expression and characterization. In this chapter several molecular methods suitable for high-throughput cloning are reviewed.
Assuntos
Reação em Cadeia da Polimerase/métodos , Clonagem Molecular , Enzimas de Restrição do DNA/genéticaRESUMO
Activation-induced cytidine deaminase (AID) initiates somatic hypermutation and class switch recombination of the immunoglobulin genes. As a trade-off for its physiological function, AID also contributes to tumor development through its mutagenic activity. In chronic lymphocytic leukemia (CLL), AID is overexpressed in the proliferative fractions (PFs) of the malignant B lymphocytes, and its anomalous expression has been associated with a clinical poor outcome. Recent preclinical data suggested that ibrutinib and idelalisib, 2 clinically approved kinase inhibitors, increase AID expression and genomic instability in normal and neoplastic B cells. These results raise concerns about a potential mutagenic risk in patients receiving long-term therapy. To corroborate these findings in the clinical setting, we analyzed AID expression and PFs in a CLL cohort before and during ibrutinib treatment. We found that ibrutinib decreases the CLL PFs and, interestingly, also reduces AID expression, which correlates with dampened AKT and Janus Kinase 1 signaling. Moreover, although ibrutinib increases AID expression in a CLL cell line, it is unable to do so in primary CLL samples. Our results uncover a differential response to ibrutinib between cell lines and the CLL clone and imply that ibrutinib could differ from idelalisib in their potential to induce AID in treated patients. Possible reasons for the discrepancy between preclinical and clinical findings, and their effect on treatment safety, are discussed.
Assuntos
Antineoplásicos Imunológicos/efeitos adversos , Citidina Desaminase/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Pirazóis/efeitos adversos , Pirimidinas/efeitos adversos , Adenina/análogos & derivados , Idoso , Proliferação de Células/efeitos dos fármacos , Citidina Desaminase/biossíntese , Regulação para Baixo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , PiperidinasRESUMO
Recombinant protein expression has become an invaluable tool in basic and applied research. The accumulated knowledge in this field allowed the expression of thousands of protein targets in a soluble, pure, and homogeneous state, essential for biochemical and structural analyses. A lot of progress has been achieved in the last decades, where challenging proteins were expressed in a soluble manner after evaluating different parameters such as host, strain, and fusion partner or promoter strength, among others. In this regard, we have previously developed a vector suite that allows the evaluation of different promoters and solubility enhancer-proteins, through an easy and efficient cloning strategy. Nonetheless, the proper expression of many targets remains elusive, requiring, for example, the addition of complex post-translation modifications and/or passage through specialized compartments. In order to overcome the limitations found when working with a single subcellular localization and a single host type, we herein expanded our previously developed vector suite to include the evaluation of recombinant protein expression in different cell compartments and cell hosts. In addition, these vectors also allow the assessment of alternative purification strategies for the improvement of target protein yields.
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Lipoprotein lipase (LPL) mRNA expression in chronic lymphocytic leukaemia (CLL) is associated with an unmutated immunoglobulin profile and poor clinical outcome. We evaluated the subcellular localization of LPL protein in CLL cells that did or did not express LPL mRNA. Our results show that LPL protein is differently located in CLL cells depending on whether it is incorporated from the extracellular medium in mutated CLL or generated de novo by leukaemic cells of unmutated patients. The specific quantification of endogenous LPL protein correlates with mRNA expression levels and mutational IGHV status, suggesting LPL protein as a possible reliable prognostic marker in CLL.
Assuntos
Biomarcadores Tumorais/biossíntese , Regulação Enzimológica da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/enzimologia , Lipase Lipoproteica/biossíntese , Proteínas de Neoplasias/biossíntese , Idoso , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/biossíntese , RNA Neoplásico/biossínteseRESUMO
Lipoprotein lipase (LPL) is a central enzyme in lipid metabolism. Due to its catalytic activity, LPL is involved in metabolic pathways exploited by various solid and hematologic malignancies to provide an extra energy source to the tumor cell. We and others described a link between the expression of LPL in the tumor cell and a poor clinical outcome of patients suffering Chronic Lymphocytic Leukemia (CLL). This leukemia is characterized by a slow accumulation of mainly quiescent clonal CD5 positive B cells that infiltrates secondary lymphoid organs, bone marrow and peripheral blood. Despite LPL being found to be a reliable molecular marker for CLL prognosis, its functional role and the molecular mechanisms regulating its expression are still matter of debate. Herein we address some of these questions reviewing the current state of the art of LPL research in CLL and providing some insights into where currently unexplored questions may lead to.
Assuntos
Linfócitos B/imunologia , Biomarcadores Tumorais/genética , Antígenos CD5/genética , Leucemia Linfocítica Crônica de Células B/diagnóstico , Lipase Lipoproteica/genética , Linfócitos B/patologia , Biomarcadores Tumorais/imunologia , Medula Óssea/imunologia , Medula Óssea/patologia , Antígenos CD5/imunologia , Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/mortalidade , Lipase Lipoproteica/imunologia , Prognóstico , Análise de SobrevidaRESUMO
Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by accumulation of clonal B lymphocytes, resulting from a complex balance between cell proliferation and apoptotic death. Continuous crosstalk between cancer cells and local/distant host environment is required for effective tumor growth. Among the main actors of this dynamic interplay between tumoral cells and their microenvironment are the nano-sized vesicles called exosomes. Emerging evidence indicates that secretion, composition, and functional capacity of exosomes are altered as tumors progress to an aggressive phenotype. In CLL, no data exist exploring the specific changes in the proteomic profile of plasma-derived exosomes from patients during disease evolution. We hereby report for the first time different proteomic profiles of plasma exosomes, both between indolent and progressive CLLs as well as within the individual patients at the onset of disease and during its progression. Next, we focus on the changes of the exosome protein cargoes, which are found exclusively in patients with progressive CLL after disease progression. The alterations in the proteomic cargoes underline different networks specific for leukemia progression related to inflammation, oxidative stress, and NF-κB and phosphatidylinositol 3-kinase/AKT pathway activation. Finally, our results suggest a preponderant role for the protein S100-A9 as an activator of the NFκB pathway during CLL progression and suggest that the leukemic clone can generate an autoactivation loop through S100-A9 expression, NF-κB activation, and exosome secretion. Collectively, our data propose a new pathway for NF-κB activation in CLL and highlight the importance of exosomes as extracellular mediators promoting tumor progression in CLL.
Assuntos
Calgranulina B/imunologia , Exossomos/patologia , Leucemia Linfocítica Crônica de Células B/patologia , NF-kappa B/imunologia , Basigina/análise , Basigina/imunologia , Calgranulina B/análise , Progressão da Doença , Exossomos/imunologia , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/imunologia , NF-kappa B/análise , Proteoma/análise , Proteoma/imunologiaAssuntos
Leucemia Linfocítica Crônica de Células B/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Biomarcadores , Proliferação de Células/genética , Sobrevivência Celular/genética , Progressão da Doença , Feminino , Humanos , Imunofenotipagem , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transdução de SinaisRESUMO
Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonal B cells arrested in G0/G1 stages that coexist with proliferative B cells. We identified one of these proliferative subsets in the peripheral blood from patients with unmutated disease (UM). Aiming to characterize the molecular mechanism underlying this proliferative behavior, we performed gene expression analysis of the mRNA and microRNAs in this leukemic subpopulation and compared results with those for the quiescent counterpart. Our results suggest that proliferation of this subset mainly depends on microRNA-22 overexpression, which induces phosphatase and tensin homolog (PTEN) down-regulation and phosphoinositide 3-kinase (PI3K)/AKT pathway activation. These results underline the role of the PI3K/AKT pathway at the origin of this proliferative pool in patients with UM CLL and provide additional rationale for the use of PI3K inhibitors.
Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Mutação , Transdução de Sinais , Microambiente Tumoral/genética , Antígenos CD40/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Análise por Conglomerados , Feminino , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose/genética , Masculino , MicroRNAs/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Survivina , TranscriptomaRESUMO
Despite the importance of recombinant protein production in academy and industrial fields, many issues concerning the expression of soluble and homogeneous product are still unsolved. Although several strategies were developed to overcome these obstacles, at present there is no magic bullet that can be applied for all cases. Indeed, several key expression parameters need to be evaluated for each protein. Among the different hosts for protein expression, Escherichia coli is by far the most widely used. In this chapter, we review many of the different tools employed to circumvent protein insolubility problems.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Animais , Humanos , SolubilidadeRESUMO
Chronic lymphocytic leukemia (CLL) is the main cause of autoimmune hemolytic anemia (AHA). However, the cellular basis underlying this strong association remains unclear. We previously demonstrated that leukemic B cells from patients with CLL recognize the erythrocyte protein Band 3, a prevalent autoantigen in AHA. Here we show that the major binding site of Band 3 on leukemic cells is an extrinsic protein identified as high-mobility group nucleosome binding protein 2 (HMGN2), a nucleosome-interacting factor which has not been previously reported at the cell surface. T lymphocytes do not express HMGN2 or bind Band 3. Removal of HMGN2 from the cell membrane abrogated the capacity of Band 3-pulsed CLL cells to induce CD4 + T cell proliferation. We conclude that surface HMGN2 in leukemic B cells is involved in Band 3 binding, uptake and presentation to CD4 + T lymphocytes, and as such may favor the initiation of AHA secondary to CLL.