RESUMO
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.
RESUMO
AIMS: Entomopathogenic Metarhizium fungi are widely recognized for their biological control potential. In Cuba, several fungus-based bio-insecticides have been developed and are produced as part of integrated pest management (IPM) programmes for economically relevant agricultural pests. Screening of fungal isolates from the INISAV strain collection was used for the development of bio-insecticides against important pest insects as, for example the sweet potato weevil, Cylas formicarius. METHODS AND RESULTS: Six fungal isolates from Cuba were microscopically, morphologically and molecular-taxonomically characterized using marker sequences ef1a, rpb1 and rpb2, and the 5TEF region of the ef1a gene. Five isolates were assigned to the species Metarhizium anisopliae sensu stricto and one isolate to Metarhizium robertsii. The pathogenic potential was evaluated against adults of C. formicarius, and growth and conidial production on different nutritional media were determined. Metarhizium anisopliae strain LBM-267 displayed pronounced virulence against the sweet potato weevil and abundant conidia production on several culture media. CONCLUSIONS: Entomopathogenic fungal isolates from Cuba were assigned to the taxonomic species M. anisopliae sensu stricto and M. robertsii. Virulence assessment with respect to C. formicarius led to the identification of two M. anisopliae isolates holding biocontrol potential. Isolate LBM-11 has previously been developed into the bio-insecticide METASAVE-11 that is widely used to control several species of plant pathogenic weevils, Lepidoptera and thrips in Cuba. Isolate LBM-267 has not been employed previously but is as virulent against C. formicarius as LBM-11; its growth and conidial production capacities on different nutritional media will likely facilitate economically feasible bio-insecticide development. SIGNIFICANCE AND IMPACT OF THE STUDY: Metarhizium anisopliae isolate LBM-267 has been selected as a promising candidate for biocontrol of the sweet potato weevil, an economically important agricultural pest in Cuba, and for further R&D activities within the framework of the Biological Control Program of Cuba.
Assuntos
Besouros , Inseticidas , Ipomoea batatas , Metarhizium , Gorgulhos , Animais , Cuba , Ipomoea batatas/microbiologia , Controle Biológico de Vetores/métodos , Esporos Fúngicos , Virulência , Gorgulhos/microbiologiaRESUMO
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 , MutaçãoAssuntos
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
PURPOSE: There is evidence showing that mesenchymal stromal cells (MSC) may constitute a potential therapeutic strategy to induce bone regeneration. In this work, we investigate the capacity of autologous bone marrow (BM) MSC loaded on collagen microspheres (CM) and included into autologous platelet-rich plasma (PRP) clots (MSC/CM/PRP) to induce bone formation in patients with nonunion lesions. METHODS: MSC were isolated from BM cells of patients with nonunion lesions. Phenotypical (marker expression) and functional studies (osteogenic differentiation) were performed. MSC were seeded on CM and included into autologous PRP clot (MSC/CM/PRP). The capacity of MSC/CM/PRP to induce bone formation was evaluated in three patients diagnosed with nonunion. RESULTS: MSC loaded on CM/PRP clots maintain their biological functions, in vitro. After three months, post-MSC transplantation, all patients showed evidence of osteogenesis at the site of nonunion. After one year, all patients showed a complete healing of the nonunion. CONCLUSIONS: Our results support the use of autologous MSC transplanted as MSC/CM/PRP for the treatment of nonunion fractures. Future studies incorporating a larger number of patients may confirm the results obtained in this work.
Assuntos
Regeneração Óssea/efeitos dos fármacos , Fraturas não Consolidadas/tratamento farmacológico , Transplante de Células-Tronco Mesenquimais/métodos , Cicatrização/efeitos dos fármacos , Adulto , Idoso de 80 Anos ou mais , Colágeno/metabolismo , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Microesferas , Osteogênese/efeitos dos fármacos , Plasma Rico em Plaquetas/efeitos dos fármacosRESUMO
Although the majority of B cells express surface CD20 in chronic lymphocytic leukaemia (B-CLL), only â¼50% of patients respond to treatment with rituximab. Decreased CD20 expression on these tumour B cells could be responsible for the lack of response observed in some patients treated with rituximab. Despite the potential critical role of CD20 in the biology of B cell malignancies, the mechanisms controlling its expression are poorly understood. At the bone marrow level, mesenchymal stromal cells (MSC) may regulate and support the survival of malignant cells, such as B-CLL cells. In this study, we investigated whether MSC may regulate the CD20 expression on B-CLL. For this purpose, B cells from CLL patients were isolated and co-cultured on MSC. B-CLL cells were collected from B-CLL/MSC co-cultures and examined for their expression of CD20. We demonstrate decreased CD20 expression in B-CLL cells after 2 weeks of co-culture with MSC, under contact and non-contact conditions, which was associated with a decreased susceptibility to rituximab. Additionally, B cells co-cultured with MSCs show an increase in CD59 expression. Our findings strongly suggest that the interaction between B-CLL cells and MSC may play a major role in the resistance to rituximab-induced apoptosis of B-CLL cells.
Assuntos
Antígenos CD20/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Células-Tronco Mesenquimais/metabolismo , Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Monoclonais Murinos/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos CD20/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Antígenos CD59/genética , Antígenos CD59/metabolismo , Comunicação Celular , Técnicas de Cocultura , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , RituximabRESUMO
The marine sponge Ectyoplasia ferox produces antipredatory and allelopathic triterpenoid glycosides as part of its chemical defense repertoire against predators, competitors, and fouling organisms. These molecules are responsible for the pharmacological potential found in the glycosides present in this species. In order to observe the glycochemical diversity present in E. ferox, a liquid chromatography coupled to a tandem mass spectrometry approach to analyse a complex polar fraction of this marine sponge was performed. This gave valuable information for about twenty-five compounds three of which have been previously reported and another three which were found to be composed of known aglycones. Furthermore, a group of four urabosides, sharing two uncommon substitutions with carboxyl groups at C-4 on the terpenoid core, were identified by a characteristic fragmentation pattern. The oxidized aglycones present in this group of saponins can promote instability, making the purification process difficult. Cytotoxicity, cell cycle modulation, a cell cloning efficiency assay, as well as its hemolytic activity were evaluated. The cytotoxic activity was about IC50 40 µg/mL on Jurkat and CHO-k1 cell lines without exhibiting hemolysis. Discussion on this bioactivity suggests the scanning of other biological models would be worthwhile.
Assuntos
Glicosídeos/química , Glicosídeos/farmacologia , Poríferos/química , Triterpenos/química , Triterpenos/farmacologia , Animais , Células CHO , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão/métodos , Cricetulus , Citotoxinas/química , Citotoxinas/farmacologia , Hemólise/efeitos dos fármacos , Humanos , Células Jurkat , Poríferos/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Three new triterpene glycosides, named ulososide F (1), urabosides A (2) and B (3), together with the previously reported ulososide A (4), were isolated from the Caribbean marine sponge Ectyoplasia ferox. Their structures were elucidated using extensive interpretation of 1D and 2D-NMR data, as well as HRESIMS. The aglycon of all compounds is a rare 30-norlonastane and the sugar residues were identified after acid hydrolysis and GC analyses. Cytotoxicities of the isolated compounds were evaluated against Jurkat and CHO cell lines by a MTT in vitro assay as well as a hemolysis assay. Unexpectedly, all these saponin derivatives showed very low activity in our bioassays.