Therapeutic DNA Vaccines: The Final Step for Success

Therapeutic DNA vaccines are mostly plasmidic constructs containing a strong promoter that allows in situ transcription and translation of one or many encoded proteins/antigens to induce protective cellular and humoral immune responses against different pathogenic organisms [1–5]. Currently, at least 114 open clinical studies are recruiting patients for distinct clinical phases using a DNAvaccine approach

Use of Wild Type or Recombinant Lactic Acid Bacteria as an Alternative Treatment for Gastrointestinal Inflammatory Diseases: A Focus on Inflammatory Bowel Diseases and Mucositis

The human gastrointestinal tract (GIT) is highly colonized by bacterial communities, which live in a symbiotic relationship with the host in normal conditions. It has been shown that a dysfunctional interaction between the intestinal microbiota and the host immune system, known as dysbiosis, is a very important factor responsible for the development of different inflammatory conditions of the GIT, such as the idiopathic inflammatory bowel diseases (IBD), a complex and multifactorial disorder of the GIT. Dysbiosis has also been implicated in the pathogenesis of other GIT inflammatory diseases such as mucositis usually caused as an adverse effect of chemotherapy. As both diseases have become a great clinical problem, many research groups have been focusing on developing new strategies for the treatment of IBD and mucositis. In this review, we show that lactic acid bacteria (LAB) have been capable in preventing and treating both disorders in animal models, suggesting they may be ready for clinical trials. In addition, we present the most current studies on the use of wild type or genetically engineered LAB strains designed to express anti-inflammatory proteins as a promising strategy in the treatment of IBD and mucositis

Síndrome colestática intra-hepática medicamentosa pelo uso de metildopa em gestante hipertensa - relato de caso/ Intrahepatic cholestatic syndrome, due to the use of methyldopa in a hypertensive pregnant woman - case report

A síndrome colestática resulta do deficiente fluxo de bile do ducto canalicular para o duodeno, cursando com alterações morfológicas, fisiológicas e clínicas¹. A colestase intra-hepática é resultado de disfunção hepatocelular, lesão nos ductos biliares intra-hepáticos de pequenos e médio calibres, que pode ser causada por vírus, álcool ou drogas². É um processo bioquímico caracterizado por aumento da fração hepatobiliar da fosfatase alcalina, além da alteração de outros parâmetros laboratoriais como gamaglutamil transferase, bilirrubinas e colesterol. Manifesta-se através de um conjunto de sinais e sintomas como icterícia, prurido, xantelasmas, em decorrência do acúmulo de produtos normalmente excretados pela bile, como a bilirrubina, ácidos biliares e colesterol, no plasma. O diagnóstico etiológico requer a utilização de marcadores virais, tumorais e imunológicos.³ Esse trabalho tem como objetivo relatar o caso de uma gestante hipertensa que desenvolveu síndrome colestática intra-hepática em decorrência do uso de metildopa

Bioestimulantes aplicados em sementes e plantas de milho doces sob condições de estresse abiótico / Bioestimulants applied to seeds and sweet corn plants under abiotic stress conditions

No intuito de melhorar a qualidade de sementes de milho super doce, novas tecnologias vêm sendo incorporadas aos sistemas de produção. Dentre elas, a utilização de bioestimulantes são consideradas estratégias agronômicas promissoras para o incremento da produtividade. Objetivou-se avaliar a qualidade das sementes de milho super doce sob condições de estresse hídrico com a utilização de bioestimulantes. As sementes dos híbridos (BRS Vivi, Tropical Plus) foram submetidas ao tratamento com os produtos Booster®, Vitakelp® e Stimulate®. Após os tratamentos a qualidade fisiológica das sementes foram avaliadas pelo teste de primeira contagem de germinação, germinação final, emergência de plântulas, índice de velocidade de emergência (IVE) e teste frio. Adotou-se um delineamento inteiramente casualizado, com quatro repetições em esquema fatorial, sendo 4x4x2para germinação, sendo três bioestimulantes mais a testemunha, quatros potenciais osmóticos (0 -0,3, -0,6, -0,9 Mpa) e 2 temperaturas (25 e 30°C). Enquanto para os demais testes foi utilizado o esquema fatorial 4x4 (três bioestimulantes mais a testemunha e 4 condições de déficit hídrico). O bioestimulante Booster® é mais eficiente para o tratamento das sementes de milho super doce. Existem diferenças das respostas das cultivares em relação aos bioestimulantes. Sob condições de estresse, os bioestimulantes têm efeito positivo no estabelecimento das plântulas

Mutation of the surface layer protein SlpB has pleiotropic effects in the probiotic propionibacterium freudenreichii CIRM-BIA 129

Propionibacterium freudenreichii is a beneficial Gram-positive bacterium, traditionally used as a cheese-ripening starter, and currently considered as an emerging probiotic. As an example, the P. freudenreichii CIRM-BIA 129 strain recently revealed promising immunomodulatory properties. Its consumption accordingly exerts healing effects in different animal models of colitis, suggesting a potent role in the context of inflammatory bowel diseases. This anti-inflammatory effect depends on surface layer proteins (SLPs). SLPs may be involved in key functions in probiotics, such as persistence within the gut, adhesion to host cells and mucus, or immunomodulation. Several SLPs coexist in P. freudenreichii CIRM-BIA 129 and mediate immunomodulation and adhesion. A mutant P. freudenreichii CIRM-BIA 129ΔslpB (CB129ΔslpB) strain was shown to exhibit decreased adhesion to intestinal epithelial cells. In the present study, we thoroughly analyzed the impact of this mutation on cellular properties. Firstly, we investigated alterations of surface properties in CB129ΔslpB. Surface extractable proteins, surface charges (ζ-potential) and surface hydrophobicity were affected by the mutation. Whole-cell proteomics, using high definition mass spectrometry, identified 1,288 quantifiable proteins in the wild-type strain, i.e., 53% of the theoretical proteome predicted according to P. freudenreichii CIRM-BIA 129 genome sequence. In the mutant strain, we detected 1,252 proteins, including 1,227 proteins in common with the wild-type strain. Comparative quantitative analysis revealed 97 proteins with significant differences between wild-type and mutant strains. These proteins are involved in various cellular process like signaling, metabolism, and DNA repair and replication. Finally, in silico analysis predicted that slpB gene is not part of an operon, thus not affecting the downstream genes after gene knockout. This study, in accordance with the various roles attributed in the literature to SLPs, revealed a pleiotropic effect of a single slpB mutation, in the probiotic P. freudenreichii. This suggests that SlpB may be at a central node of cellular processes and confirms that both nature and amount of SLPs, which are highly variable within the P. freudenreichii species, determine the probiotic abilities of strains.Fil: do Carmo, Fillipe L. R.. Institut National de la Recherche Agronomique; Francia. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Marques Da Silva, Wanderson. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Tavares, Guilherme C.. Universidade Federal de Minas Gerais; BrasilFil: Ibraim, Izabela C.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Cordeiro, Barbara F.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Oliveira, Emiliano R.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Rabah, Houem. Institut National de la Recherche Agronomique; FranciaFil: Cauty, Chantal. Institut National de la Recherche Agronomique; FranciaFil: da Silva, Sara H.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Canário Viana, Marcus V.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Caetano, Ana C. B.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: dos Santos, Roselane G.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: de Oliveira Carvalho, Rodrigo D.. Instituto de Ciencias Da Saúde; BrasilFil: Jardin, Julien. Institut National de la Recherche Agronomique; FranciaFil: Pereira, Felipe L.. Universidade Federal de Minas Gerais; BrasilFil: Folador, Edson L.. Universidade Estadual da Paraiba; BrasilFil: Le Loir, Yves. Institut National de la Recherche Agronomique; FranciaFil: Figueiredo, Henrique C. P.. Universidade Federal de Minas Gerais; BrasilFil: Jan, Gwénaël. Institut National de la Recherche Agronomique; FranciaFil: Azevedo, Vasco. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; Brasi

Cloning, expression and characterization of the colonystimulating factor recombinant human granulocyte (rhG-CSF) in Escherichia coli

O sistema de expressão em Escherichia coli foi o primeiro a ser utilizado para produzir produtos farmacêuticos recombinantes e tem muitas vantagens quando comparado com sistemas eucarióticos, como o fácil cultivo, baixo custo e alto potencial de produção. O fator estimulador de colônias de granulócito (G-CSF) atua principalmente promovendo a maturação dos neutrófilos e estimulando sua atividade fagocítica e quimiotática, além de estar envolvido com o processo de segmentação nuclear dessas células. O fator estimulador de colônias de granulócitos humano recombinante (rhG-CSF) tem sido produzido por engenharia genética em Escherichia coli, e é usado no tratamento de diversas patologias, sobretudo em neutropenias provocadas pela quimioterapia usada no tratamento de tumores, pela radioterapia e pelo uso de drogas que suprimem a produção de células mieloides. Desse modo, o presente estudo teve como objetivo a expressão da proteína rhGCSF em bactérias Escherichia coli. A clonagem do gene rhG-CSF no vetor de expressão pET-28a(+) foi realizada nos sítios de restrição das enzimas EcoRI e XhoI, e a expressão da proteína recombinante em cepas de bactéria Escherichia coli BL21DE3 foi obtida com sucesso. A proteína rhG-CSF, fundida à cauda de seis histidinas, foi purificada com êxito e identificada pelas técnicas de Western Blotting e por espectrometria de massas. São necessários estudos para avaliar a integridade estrutural e atividade biológica da proteína produzida, que se confirmada, possibilita que esta seja produzida em escala piloto.The expression system in Escherichia coli was the first to be used to produce recombinant pharmaceuticals and has many advantages compared to eukaryotic systems, such as easy cultivation and high production potential at low costs. The granulocyte colony (G-CSF) stimulating factor acts primarily by promoting the maturation of neutrophils and stimulating their phagocytic and chemotactic activity. G-CSF is also involved with the process of neutrophils nuclear segmentation. The recombinant human granulocyte colonies stimulating factor (rhG-CSF) has been produced by genetic engineering in Escherichia coli, and it is used to treat of several conditions, especially neutropenia caused by chemotherapy used in the treatment of tumors, by radiotherapy and by the use of drugs that suppress the production of myeloid cells. The present study aimed the expression of rhG-CSF protein in Escherichia coli bacteria. The cloning of rhG-CSF gene in the expression vector pET- 28a (+) was carried out on the restriction sites of the EcoRI and XhoI enzymes. Expression of the recombinant protein in Escherichia coli BL21DE3 was successfully achieved. The rhG-CSF protein, fused with a six histidine tag, was obtained and successfully purified and identified by the Western Blotting and by mass spectrometry techniques. Studies are needed to assess the structural integrity and biological activity of the protein produced, which, if confirmed, enables the production on a pilot scale

Surface layer protein SlpB mediates immunodulation and adhesion in the probiotic Propionibacterium freudenreichii CIRM-BIA 129.

Propionibacterium freudenreichii est une bactérie Gram-positive bénéfique, traditionnellement utilisée comme levain d’affinage fromager, qui bénéficie du statut GRAS (Generally Recognized As Safe). P. freudenreichii a révélé un effet immunomodulateur qui a été confirmé in vivo par la capacité à protéger des souris d’une colite aigüe induite. L’effet anti-inflammatoire est cependant hautement souche-dépendant. Il est dû, au moins en partie, à des composés de surface clés qui favorisent ces effets probiotiques. Les bactéries Gram-positives, y compris P. freudenreichii, peuvent être recouvertes d’une couche extérieure protéique, appelée « surface-layer », paracristalline, et formée par l’autoassemblage de protéines dites de S-layer (Slps). Les Slps, dans différentes bactéries, sont impliquées dans plusieurs caractéristiques probiotiques, telles que l’adhésion aux cellules de l’hôte et au mucus, la persistance dans l’intestin, ou encore l’immunomodulation. Le but de cette étude est d’étudil’immunomodulation. Le but de cette étude est d’étudier, chez une souche probiotique de P. freudenreichii, la protéine de surface qui joue le principal rôle dans les interactions probiotiques avec l’hôte. La souche P. freudenreichii CIRM-BIA 129, récemment reconnue comme immunomodulatrice prometteuse, possède plusieurs protéines de surface Slps, y compris SlpB. Dans la présente étude, l’inactivation du gène correspondant, dans la souche mutante CB129¿slpB, a provoqué une baisse drastique de l’adhésion aux cellules intestinales épithéliales HT-29, confirmant le rôle clé des Slps dans l’adhésiPropionibacterium freudenreichii is a beneficial Gram-positive bacterium, traditionally used as a cheese ripening starter, with the GRAS status (Generally Recognized As Safe). P. freudenreichii has revealed an immunomodulatory effect confirmed in vivo by the ability to protect mice from induced acute colitis. The anti-inflammatory effect is however highly strain-dependent and due, at least in part, to key surface compounds favouring probiotic effects. Gram-positive bacteria, including P. freudenreichii, can be covered with an external proteinaceous layer called a surface-layer paracrystalin layer and formed by the self-assembly of surface-layer-proteins (Slps). Slps were shown, in different bacteria, to be involved in several probiotics traits, such as adhesion to host cells and mucus, persistence within the gut, or immunomodulation. The aim of this study is to investigate, in a P. freudenreichii probiotic strain, the surface protein that plays the main role in the probioticinteraction with the host. The P. freudenreichii CIRM-BIA 129 strain recently revealed promising immunomodulatory properties and possesses several Slps, including SlpB. In the presented work, inactivation of the corresponding gene, CB129¿slpBa mutant strain, caused a drastic decrease in adhesion to intestinal epithelial HT-29 cells, further evidencing the key role of Slps in cell adhesion. we investigated immune response of HT-29 cells towards P. freudenreichii CIRM-BIA 129 and CB129¿slpB. The wild type strain mainly induced expression of the immunomodulatory IL-10 by the cells. Interestingly, t

La protéine de couche de surface SlpB assure la médiation de l’immunomodulation et de l’adhésion chez le probiotique Propionibacterium freudenreichii CIRM-BIA 129.

Propionibacterium freudenreichii is a beneficial Gram-positive bacterium, traditionally used as a cheese ripening starter, with the GRAS status (Generally Recognized As Safe). P. freudenreichii has revealed an immunomodulatory effect confirmed in vivo by the ability to protect mice from induced acute colitis. The anti-inflammatory effect is however highly strain-dependent and due, at least in part, to key surface compounds favouring probiotic effects. Gram-positive bacteria, including P. freudenreichii, can be covered with an external proteinaceous layer called a surface-layer paracrystalin layer and formed by the self-assembly of surface-layer-proteins (Slps). Slps were shown, in different bacteria, to be involved in several probiotics traits, such as adhesion to host cells and mucus, persistence within the gut, or immunomodulation. The aim of this study is to investigate, in a P. freudenreichii probiotic strain, the surface protein that plays the main role in the probioticinteraction with the host. The P. freudenreichii CIRM-BIA 129 strain recently revealed promising immunomodulatory properties and possesses several Slps, including SlpB. In the presented work, inactivation of the corresponding gene, CB129¿slpBa mutant strain, caused a drastic decrease in adhesion to intestinal epithelial HT-29 cells, further evidencing the key role of Slps in cell adhesion. we investigated immune response of HT-29 cells towards P. freudenreichii CIRM-BIA 129 and CB129¿slpB. The wild type strain mainly induced expression of the immunomodulatory IL-10 by the cells. Interestingly, thPropionibacterium freudenreichii est une bactérie Gram-positive bénéfique, traditionnellement utilisée comme levain d’affinage fromager, qui bénéficie du statut GRAS (Generally Recognized As Safe). P. freudenreichii a révélé un effet immunomodulateur qui a été confirmé in vivo par la capacité à protéger des souris d’une colite aigüe induite. L’effet anti-inflammatoire est cependant hautement souche-dépendant. Il est dû, au moins en partie, à des composés de surface clés qui favorisent ces effets probiotiques. Les bactéries Gram-positives, y compris P. freudenreichii, peuvent être recouvertes d’une couche extérieure protéique, appelée « surface-layer », paracristalline, et formée par l’autoassemblage de protéines dites de S-layer (Slps). Les Slps, dans différentes bactéries, sont impliquées dans plusieurs caractéristiques probiotiques, telles que l’adhésion aux cellules de l’hôte et au mucus, la persistance dans l’intestin, ou encore l’immunomodulation. Le but de cette étude est d’étudil’immunomodulation. Le but de cette étude est d’étudier, chez une souche probiotique de P. freudenreichii, la protéine de surface qui joue le principal rôle dans les interactions probiotiques avec l’hôte. La souche P. freudenreichii CIRM-BIA 129, récemment reconnue comme immunomodulatrice prometteuse, possède plusieurs protéines de surface Slps, y compris SlpB. Dans la présente étude, l’inactivation du gène correspondant, dans la souche mutante CB129¿slpB, a provoqué une baisse drastique de l’adhésion aux cellules intestinales épithéliales HT-29, confirmant le rôle clé des Slps dans l’adhés

Dairy Propionibacteria: Versatile Probiotics

Dairy propionibacteria are used as cheese ripening starters, as biopreservative and as beneficial additives, in the food industry. The main species, Propionibacterium freudenreichii, is known as GRAS (Generally Recognized As Safe, USA, FDA). In addition to another dairy species, Propionibacterium acidipropionici, they are included in QPS (Qualified Presumption of Safety) list. Additional to their well-known technological application, dairy propionibacteria increasingly attract attention for their promising probiotic properties. The purpose of this review is to summarize the probiotic characteristics of dairy propionibacteria reported by the updated literature. Indeed, they meet the selection criteria for probiotic bacteria, such as the ability to endure digestive stressing conditions and to adhere to intestinal epithelial cells. This is a prerequisite to bacterial persistence within the gut. The reported beneficial effects are ranked according to property’s type: microbiota modulation, immunomodulation, and cancer modulation. The proposed molecular mechanisms are discussed. Dairy propionibacteria are described as producers of nutraceuticals and beneficial metabolites that are responsible for their versatile probiotic attributes include short chain fatty acids (SCFAs), conjugated fatty acids, surface proteins, and 1,4-dihydroxy-2-naphtoic acid (DHNA). These metabolites possess beneficial properties and their production depends on the strain and on the growth medium. The choice of the fermented food matrix may thus determine the probiotic properties of the ingested product. This review approaches dairy propionibacteria, with an interest in both technological abilities and probiotic attributes

Cheese matrix effect on proteolysis of P. freudenreichii immunomodulatory proteins

Propionibacterium freudenreichii is an emergent probiotic, presenting several beneficial effects including anti-inflammatory properties, mediated by surface layer proteins (SLPs) belonged to the S-layer lattice, in particularly the protein SlpB. We hypothesize cheese matrix may be the best delivery vehicle for P. freudenreichii’s anti-inflammatory potential, by enhancing propionibacteria survival to digestives stresses, and by allowing undamaged SLPs to reach the digestive tract. Firstly, we compared the immunomodulatory effects of P. freudenreichii and intact SLPs, to SLPs digested by trypsin, i.e SLPs-peptides on HT29-cells. In contrast to P. freudenreichii and SLPs, SLPs peptides don’t reduce pro-inflammatory cytokines expression during cells co-stimulation with lipopolysaccharide. This result confirmed the importance to protect SLPs from proteolysis to permit them to trigger gut immune system. Secondly, we evaluated propionibacteria resistance and slpB proteolysis during in vitro static digestion, in different delivery vehicles increasingly concentrated in dairy proteins: milk ultrafiltrate, milk and cheese. The experiment clearly showed the protective effect of the Slp proteins by the cheese matrix and better bacterial viability. Then, we carried out in vitro a dynamic digestion using Didgi® system. We selected two matrices only: the milk ultrafiltrate and the cheese. We decided to apply fixed digestion parameters for both matrices: the digestive parameters of cheese. The results show a protective effect of the cheese matrix on the viability of the propionic bacteria and on SLPs against digestive proteolysis. Taken together, those results show that cheese is an adequate delivery vehicle for P. freudenreichii immunomodulatory proteins