Immunological-based approaches for cancer therapy

The immunologic landscape of tumors has been continuously unveiled, providing a new look at the interactions between cancer cells and the immune system. Emerging tumor cells are constantly eliminated by the immune system, but some cells establish a long-term equilibrium phase leading to tumor immunoediting and, eventually, evasion. During this process, tumor cells tend to acquire more mutations. Bearing a high mutation burden leads to a greater number of neoantigens with the potential to initiate an immune response. Although many tumors evoke an immune response, tumor clearance by the immune system does not occur due to a suppressive tumor microenvironment. The mechanisms by which tumors achieve the ability to evade immunologic control vary. Understanding these differences is crucial for the improvement and application of new immune-based therapies. Much effort has been placed in developing in silico algorithms to predict tumor immunogenicity and to characterize the microenvironment via high-throughput sequencing and gene expression techniques. Each sequencing source, transcriptomics, and genomics yields a distinct level of data, helping to elucidate the tumor-based immune responses and guiding the fine-tuning of current and upcoming immune-based therapies. In this review, we explore some of the immunological concepts behind the new immunotherapies and the bioinformatic tools to study the immunological aspects of tumors, focusing on neoantigen determination and microenvironment deconvolution. We further discuss the immune-based therapies already in clinical use, those underway for future clinical application, the next steps in immunotherapy, and how the characterization of the tumor immune contexture can impact therapies aiming to promote or unleash immune-based tumor elimination

Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

Wiscott Aldrich Syndrome protein (WASP) deficiency results in defects in calcium ion signaling, cytoskeletal regulation, gene transcription and overall T cell activation. The activation of WASP constitutes a key pathway for actin filament nucleation. Yet, when WASP function is eliminated there is negligible effect on actin polymerization at the immunological synapse, leading to gaps in our understanding of the events connecting WASP and calcium ion signaling. Here, we identify a fraction of total synaptic F-actin selectively generated by WASP in the form of distinct F-actin ‘foci’. These foci are polymerized de novo as a result of the T cell receptor (TCR) proximal tyrosine kinase cascade, and facilitate distal signaling events including PLCγ1 activation and subsequent cytoplasmic calcium ion elevation. We conclude that WASP generates a dynamic F-actin architecture in the context of the immunological synapse, which then amplifies the downstream signals required for an optimal immune response. DOI: http://dx.doi.org/10.7554/eLife.04953.00

Immunological-based approaches for cancer therapy

The immunologic landscape of tumors has been continuously unveiled, providing a new look at the interactions between cancer cells and the immune system. Emerging tumor cells are constantly eliminated by the immune system, but some cells establish a long-term equilibrium phase leading to tumor immunoediting and, eventually, evasion. During this process, tumor cells tend to acquire more mutations. Bearing a high mutation burden leads to a greater number of neoantigens with the potential to initiate an immune response. Although many tumors evoke an immune response, tumor clearance by the immune system does not occur due to a suppressive tumor microenvironment. The mechanisms by which tumors achieve the ability to evade immunologic control vary. Understanding these differences is crucial for the improvement and application of new immune-based therapies. Much effort has been placed in developing in silico algorithms to predict tumor immunogenicity and to characterize the microenvironment via high-throughput sequencing and gene expression techniques. Each sequencing source, transcriptomics, and genomics yields a distinct level of data, helping to elucidate the tumor-based immune responses and guiding the fine-tuning of current and upcoming immune-based therapies. In this review, we explore some of the immunological concepts behind the new immunotherapies and the bioinformatic tools to study the immunological aspects of tumors, focusing on neoantigen determination and microenvironment deconvolution. We further discuss the immune-based therapies already in clinical use, those underway for future clinical application, the next steps in immunotherapy, and how the characterization of the tumor immune contexture can impact therapies aiming to promote or unleash immune-based tumor elimination

Composição centesimal e de minerais de casca e polpa de manga (Mangifera indica L.) cv. Tommy Atkins

O Brasil produz cerca de 140 milhões de toneladas de alimento por ano; entretanto, a fome ainda é um dos maiores problemas enfrentados por grande parte da sua população. O aproveitamento integral de alimentos é uma alternativa para suprir as necessidades nutricionais e contribuir para reduzir o lixo orgânico. Nesse sentido, cascas, talos, sementes e outras partes, tradicionalmente não utilizadas como alimentos, podem ser incorporadas na dieta alimentar. Estudos revelam que cascas de muitas hortifrutícolas possuem mais nutrientes que determinadas polpas, a parte tradicionalmente consumida. No entanto, o conhecimento sobre a concentração de metais pesados nessas frações também é importante, uma vez que podem provocar intoxicações alimentares, se consumidos, mesmo em concentrações reduzidas. O Brasil é o sétimo produtor mundial de manga e dentre as cultivares de importância comercial, a cv. Tommy Atkins é a mais plantada e exportada pelo Brasil. A pesquisa teve como objetivo determinar a composição centesimal e o perfil de macro e microminerais, inclusive, os elementos-traço, da casca e da polpa de manga cv. Tommy Atkins. A maior fração da composição centesimal da casca estudada foi a umidade, seguida dos carboidratos totais, com destaque para o teor de fibra alimentar total (FAT), que representou cerca 11% dessa quantidade. Já os teores de proteína e de cinzas apresentaram cerca de 2,5%, enquanto a fração lipídica foi inferior aos demais componentes. O perfil de minerais da casca revelou que as concentrações desses elementos foram superiores aos encontrados na polpa, exceto para zinco e ferro. As concentrações de metais pesados presentes na casca, em função das concentrações menores que 0,1 mg/100g, possibilitam a sua utilização na alimentação humana. A composição centesimal e perfil mineral da casca da manga demonstraram a sua importância nutricional e a possibilidade da utilização dessa parte, até então considerada não comestível na dieta brasileira, visando a contribuir para a melhora do estado nutricional da população e a reduzir os resíduos do processo industrial

Composição centesimal e de minerais de casca e polpa de manga (Mangifera indica L.) cv. Tommy Atkins Mango rind and pulp (Mangifera indica L.) cv. Tommy Atkins centesimal composition and minerals contents

O Brasil produz cerca de 140 milhões de toneladas de alimento por ano; entretanto, a fome ainda é um dos maiores problemas enfrentados por grande parte da sua população. O aproveitamento integral de alimentos é uma alternativa para suprir as necessidades nutricionais e contribuir para reduzir o lixo orgânico. Nesse sentido, cascas, talos, sementes e outras partes, tradicionalmente não utilizadas como alimentos, podem ser incorporadas na dieta alimentar. Estudos revelam que cascas de muitas hortifrutícolas possuem mais nutrientes que determinadas polpas, a parte tradicionalmente consumida. No entanto, o conhecimento sobre a concentração de metais pesados nessas frações também é importante, uma vez que podem provocar intoxicações alimentares, se consumidos, mesmo em concentrações reduzidas. O Brasil é o sétimo produtor mundial de manga e dentre as cultivares de importância comercial, a cv. Tommy Atkins é a mais plantada e exportada pelo Brasil. A pesquisa teve como objetivo determinar a composição centesimal e o perfil de macro e microminerais, inclusive, os elementos-traço, da casca e da polpa de manga cv. Tommy Atkins. A maior fração da composição centesimal da casca estudada foi a umidade, seguida dos carboidratos totais, com destaque para o teor de fibra alimentar total (FAT), que representou cerca 11% dessa quantidade. Já os teores de proteína e de cinzas apresentaram cerca de 2,5%, enquanto a fração lipídica foi inferior aos demais componentes. O perfil de minerais da casca revelou que as concentrações desses elementos foram superiores aos encontrados na polpa, exceto para zinco e ferro. As concentrações de metais pesados presentes na casca, em função das concentrações menores que 0,1 mg/100g, possibilitam a sua utilização na alimentação humana. A composição centesimal e perfil mineral da casca da manga demonstraram a sua importância nutricional e a possibilidade da utilização dessa parte, até então considerada não comestível na dieta brasileira, visando a contribuir para a melhora do estado nutricional da população e a reduzir os resíduos do processo industrial.<br>Brazil produces about 140 million tons of food per year, however, the hunger is one of the biggest problems faced for great part of its population. The integral food exploitation is an alternative to supply the nutrition necessities of this population pursuing and to contribute to reduce the organic waste. In this direction, rind, stems, seeds and other parts traditionally not used as foods can be incorporated in the alimentary diet. Studies have verified that rind of many fruits and vegetable possess more nutrient than the pulp, the available part. However, the knowledge of the heavy metal concentration in these fractions also becomes important, as these can provoke alimentary poisonings, even when are consumed in low concentrations. Brazil is the seventh greater world-wide mango producer and the main planted and exported variety is Tommy Atkins. The objective of research was to determine the percent composition and the mineral profile of the macro and micro minerals, including heavy metals of the rind of Tommy Atkins mango. The biggest fraction of the percent composition of the rind was the moisture, followed of the total carbohydrates, with a proeminent level of total dietary fiber (TDF) that represented around 11% of this amount. The ashes and protein levels presented about 2,5%, while the lipid fraction was inferior in relation to other component. The mineral profile of the rind showed that the concentrations of these elements superior to the found in pulp, except for zinc and iron. The low heavy metal concentrations found in the rind make possible its use in the human dietary. The percent composition and mineral profile of the rind had showed its nutritional importance and the possibility of the use of this parts, not edible, in the brazilian diet could contribute for improvement of the nutrition situation of the population and to reduce the residues of the industry processing

An Efficient Low Cost Method for Gene Transfer to T Lymphocytes

<div><p></p><p>Gene transfer to T lymphocytes has historically relied on retro and lentivirus, but recently transposon-based gene transfer is rising as a simpler and straight forward approach to achieve stable transgene expression. Transfer of expression cassettes to T lymphocytes remains challenging, being based mainly on commercial kits.</p> <p>Aims</p><p>We herein report a convenient and affordable method based on <i>in house</i> made buffers, generic cuvettes and utilization of the widely available Lonza nucleofector II device to promote efficient gene transfer to T lymphocytes.</p> <p>Results</p><p>This approach renders high transgene expression levels in primary human T lymphocytes (mean 45%, 41–59%), the hard to transfect murine T cells (mean 38%, 36–42% for C57/BL6 strain) and human Jurkat T cell line. Cell viability levels after electroporation allowed further manipulations such as <i>in vitro</i> expansion and Chimeric Antigen Receptor (CAR) mediated gain of function for target cell lysis.</p> <p>Conclusions</p><p>We describe here an efficient general protocol for electroporation based modification of T lymphocytes. By opening access to this protocol, we expect that efficient gene transfer to T lymphocytes, for transient or stable expression, may be achieved by an increased number of laboratories at lower and affordable costs.</p> </div

Electroporation of the Jurkat T cell line.

<p>(<b>A</b>) Jurkat cells were electroporated with pT2-GFP in the presence of each of the 7 different buffers. Viability and GFP expression were evaluated by flow cytometry after 24 h. Cell viability is expressed as % of the control mock electroporated condition (100%). Electroporation scores were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060298#s2" target="_blank">materials and methods</a>. Statistical analysis was performed using One Way ANOVA and Tukey post test (* = P<0.05). (<b>B</b>) Jurkat cells were electroporated with buffer 3P. Cell viability and GFP expression were observed until day 20. Values in this figure are the average of two separate experiments in triplicate and are expressed as mean±SEM. Data were analyzed by unpaired Student t test; p<0.05 (*);p<0.01 (**).</p

Electroporation of resting vs activated mouse primary T lymphocytes.

<p>Total lymphocytes from lymph nodes of C57BL/6 mice were isolated and either directly electroporated or activated for 24 h with anti-CD3/anti-CD28 and then electroporated. Buffer 2S and 4 µg of pT2-GFP plasmid were used. Cell viability and GFP expression were analyzed after 24 h by flow cytometry. Cell viability is normalized to control (not electroporated) cells. Data are representative of two experiments in triplicate and expressed as mean±SEM. Data were analyzed by unpaired Student t test; p<0.0001 (**) or p = 0.002 (*).</p