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Beneficial effects of Lactiplantibacillus plantarum strains have been widely reported. Knowing that the effects of probiotic bacteria are strain-dependent, this study aimed to characterize the probiotic properties and investigate the gastrointestinal protective effects of nine novel L. plantarum strains isolated from Bahia, Brazil. The probiotic functionality was first evaluated in vitro by characterizing bile salt and acidic tolerance, antibacterial activity, and adhesion to Caco-2 cells. Antibiotic resistance profile, mucin degradation, and hemolytic activity assays were also performed to evaluate safety features. In vivo analyses were conducted to investigate the anti-inflammatory effects of the strains on a mouse model of 5-Fluorouracil-induced mucositis. Our results suggest that the used L. plantarum strains have good tolerance to bile salts and low pH and can inhibit commonly gastrointestinal pathogens. Lp2 and Lpl1 strains also exhibited high adhesion rates to Caco-2 cells (13.64 and 9.05%, respectively). Phenotypical resistance to aminoglycosides, vancomycin, and tetracycline was observed for most strains. No strain showed hemolytic or mucolytic activity. Seven strains had a protective effect against histopathological and inflammatory damage induced by 5-FU. Gene expression analysis of inflammatory markers showed that five strains upregulated interleukin 10 (Il10), while four downregulated both interleukin 6 (Il6) and interleukin 1b (Il1b). Additionally, all strains reduced eosinophilic and neutrophilic infiltration; however, they could not prevent weight loss or reduced liquid/ food intake. Altogether, our study suggests these Brazilian L. plantarum strains present good probiotic characteristics and safety levels for future applications and can be therapeutically adjuvant alternatives to prevent/treat intestinal mucositis.

Several studies have shown the probiotic potential of Lactiplantibacillus plantarum strains. However, the efficacy of this strain in treating intestinal mucositis is little explored. Here, we investigated the gastrointestinal protective effects of nine novel L. plantarum strains isolated from Bahia, Brazil. The probiotic functionality was first evaluated, in vitro, by characterization of bile salt and low pH tolerance, antibacterial activity, and adhesion to Caco-2 cells. Antibiotic resistance profile, mucin degradation, and haemolytic activity assays were also carried out to evaluate safety features. In vivo analyses were conducted to investigate the anti-inflammatory effects of the strains using a mouse model of 5-FU-induced mucositis. Our results suggest that the used L. plantarum strains have good tolerance to bile salts and low pH and are able to inhibit commonly gastrointestinal pathogens. Additionally, Lp11 and Lpl2 strains exhibit high adhesion rates to Caco-2 cells (9.05 and 13.64% respectively) and no strain show haemolytic or mucolytic activity. Phenotypical resistance to aminoglycosides, vancomycin, and tetracycline is observed for most strains. Seven strains can protect against histopathological damage in a mouse model of mucositis. Gene expression analysis of immune response markers shows that five strains upregulate Il10, while four downregulate both Il6 and Il1b to inhibit mucositis. Additionally, all strains were found to reduce eosinophilic and neutrophilic infiltration; however, they are not able to prevent weight loss or reduced liquid/ food intake. Taken together, our study suggests these Brazilian L. plantarum strains are safe for human use and are potential alternatives to treat intestinal mucositis.

Lactobacillus delbrueckii subsp. lactis CIDCA is a new potential probiotic strain whose molecular basis attributed to the host’s benefit has been reported. This study investigated the safety aspects of Lactobacillus delbrueckii subsp. lactis CIDCA 133 based on whole-genome sequence and phenotypic analysis to avoid future questions about the harmful effects of this strain consumption. Genomic analysis showed that L. delbrueckii subsp. lactis CIDCA 133 harbors virulence, harmful metabolites, and antimicrobial resistance-associated genes. However, none of these genetic elements is flanked or located within prophage regions and plasmid sequence. At a phenotypic level, it was observed L. delbrueckii subsp. lactis CIDCA 133 antimicrobial resistance to aminoglycosides streptomycin and gentamicin antibiotics, but no hemolytic and mucin degradation activity was exhibited by strain. Furthermore, no adverse effects were observed regarding mice clinical and histopathological analysis after the strain consumption (5 × 107 CFU/mL). Overall, these findings reveal the safety of Lactobacillus delbrueckii subsp. lactis CIDCA 133 for consumption and future probiotic applications.

5-Fluorouracil (5-FU) is an antineoplastic drug that causes, as a side effect, intestinal mucositis, acute inflammation in the small bowel. The Heat Shock Protein (Hsp) are highly expressed in inflammatory conditions, developing an important role in immune modulation. Thus, they are potential candidates for the treatment of inflammatory diseases. In the mucositis mouse model, the present study aimed to evaluate the beneficial effect of oral administration of milk fermented by Lactobacillus delbrueckii CIDCA 133 (pExu:hsp65), a recombinant strain. This approach showed increased levels of sIgA in the intestinal fluid, reducing inflammatory infiltrate and intestinal permeability. Additionally, the histological score was improved. Protection was associated with a reduction in the gene expression of pro-inflammatory cytokines such as Tnf, Il6, Il12, and Il1b, and an increase in Il10, Muc2, and claudin 1 (Cldn1) and 2 (Cldn2) gene expression in ileum tissue. These findings are corroborated with the increased number of goblet cells, the electronic microscopy images, and the reduction of intestinal permeability. The administration of milk fermented by this recombinant probiotic strain was also able to reverse the high levels of gene expression of Tlrs caused by the 5-FU. Thus, the rCIDCA 133:Hsp65 strain was revealed to be a promising preventive strategy for small bowel inflammation.

DNA vaccines have been used as a promising strategy for delivery of immunogenic and immunomodulatory molecules into the host cells. Although, there are some obstacles involving the capability of the plasmid vector to reach the cell nucleus in great number to promote the expected benefits. In order to improve the delivery and, consequently, increase the expression levels of the target proteins carried by DNA vaccines, alternative methodologies have been explored, including the use of non-pathogenic bacteria as delivery vectors to carry, deliver, and protect the DNA from degradation, enhancing plasmid expression.

DNA vaccines are the third generation vaccines based on purified plasmid preparations containing transgenes that encode antigenic/therapeutic proteins or peptides capable of triggering an immune response against a wide range of diseases. This vaccine platform presents several attributes that confer distinct advantages over other vaccine technologies in terms of safety, ease of fabrication and stability. Many aspects, such as antigen expression and especially vector design, are under study because of their great influence on immunogenicity and efficacy of DNA vaccines. In this regard, with the attempt of improving the efficiency of DNA vaccines, co-expression of stimulatory sequences and diverse vector delivery systems are being optimized. With this in mind, this review aims to giving a conceptual approach of DNA vaccines, explaining their mechanisms of action and listing the already licensed veterinary DNA vaccines presented in the market.

As superfícies de mucosa são os principais sítios de interação entre o corpo e o meio externo constituindo uma das primeiras linhas de defesa do organismo, sendo consideradas um alvo atraente para a entrega de drogas biológicas. Por isso, nas últimas décadas o desenvolvimento de estratégias mais eficientes para entrega de plasmídeos vacinais e/outerapêuticos às mucosas tem recebido considerável atenção. Assim, o uso de bactérias carreadoras desses plasmídeos, pela rota oral, constitui uma estratégia promissora. Para tanto, a utilização de bactérias não patogênicas como as Bactérias Lácticas (BL), constitui uma opção segura para este propósito. Lactococcus lactis é a BL modelo e vem sendo muito utilizada para a produção e entrega de antígenos e citocinas ao nível de mucosas. Neste contexto, com o objetivo de otimizar a entrega de plasmídeos para células epiteliais humanas, foram construídas linhagens de L. lactis invasivas e também um plasmídeo replicativo em L. lactis, contendo um cassete de expressão eucariótica denominado pValac. Assim, a utilização da linhagem invasiva de L. lactis capaz de entregar o vetor pValac codificando a Interleucina 10 (IL-10) de Mus musculus, uma potente citocina antiinflamatória, poderia representar uma nova estratégia para o tratamento de doenças inflamatórias como as doenças inflamatórias intestinais (IBDs). Assim, este trabalho teve como objetivo construir o plasmídeo terapêutico pValac::IL-10 e verificar a sua funcionalidade, in vitro, assim como a clonagem deste plasmídeo em uma linhagem invasiva de L. lactis, visando o desenvolvimento de uma terapêutica alternativa e mais eficaz contra as IBDs. A sequência codificadora da IL-10 foi obtida a partir de um plasmídeo sintético contendo a ORF da IL-10 de M. musculus e clonada no vetor Zero Blunt® TOPO® e subclonada no vetor pValac. Para avaliação da funcionalidade do plasmídeo pValac::IL-10, células da linhagem CHO (Chinese Hamster Ovary) foram transfectadas com o plasmídeo construído e a expressão da IL-10 por essas células foi avaliada por ELISA, Western Blot, Microscopia Confocal e FACS. Por fim, o plasmídeo pValac::IL-10 foi transformado na linhagem invasiva de L. lactis e a mesma foi então utilizada em um ensaio piloto, in vivo,para avaliar a capacidade terapêutica e imunomoduladora em camundongos com IBD experimental. Enfim, este trabalho constitui um primeiro passo rumo ao desenvolvimento de uma nova estratégia para o controle de doenças inflamatórias além da validação da eficáciae efetividade da linhagem invasiva de L. lactis como veículo para novas terapias e vacinas de DNA. Mucosal surfaces are the principal sites of interaction between the body and the external environment, constituting one of the first lines of defense of an organism, and as such, are considered to be an attractive target for the delivery of biological drugs. In this regard, special attention has been given, in the last decades, to the development of more efficient strategies for delivery of vaccine and/or therapeutic plasmids to the mucosa. Theuse of bacteria carrying these plasmids, by oral route, constitutes a promising strategy; hereupon, the use of non pathogenic bacteria, such as Lactic Acid Bacteria (LAB), constitutes a safe choice for such purposes. Lactococcus lactis is the LAB model and has been extensively used for the production and delivery of antigens and cytokines to themucosal level. In this context, with the goal of optimizing plasmid delivery to human epithelial cells, invasive strains of L. lactis as well as a replicative plasmid in L. lactis, containing a eukaryotic expression cassette called pValac were constructed. Thus, the use of the invasiveL. lactis strain capable of delivering the pValac vector coding for Interleukin 10 (IL-10) of Mus musculus, a powerful anti-inflammatory cytokine, could represent a new strategy for the treatment of inflammatory diseases such as inflammatory bowel diseases (IBDs). So, thegoal of this project is to construct a therapeutic plasmid, pValac::IL-10, and verify its functionality in vitro, as well as its cloning into the invasive L. lactis strain, aiming at developing an alternative and more efficient therapeutic treatment against IBDs. The coding sequence of IL-10 was obtained from a synthetic plasmid containing the ORF of IL-10 fromM. musculus, which was first cloned into the Zero Blunt® TOPO® and subsequently into the pValac vector. To evaluate the functionality of the pValac::IL-10 plasmid, cells of the CHO (Chinese Hamster Ovary) cell line were transfected with the constructed plasmid and expression of IL-10 by these cells was evaluated by ELISA, Western Blot, Confocal Microscopy and FACS. The pValac::IL-10 plasmid was finally transformed into the invasive L. lactis strain and the same was then used in an in vivo pilot trial to evaluate its therapeutic and immunomodulatory capacity in mice with experimental IBD. Ultimately, this work constitutes a first step towards the development of a new strategy to control inflammatory diseases, besides validating the efficiency and effectiveness of the invasive strain of L. lactisas vehicle for new DNA therapies and vaccines.

Inflammatory bowel disease (IBD) is a term used to describe a group of intestinal disorders in which inflammation is a major feature. Although rare forms of IBD exist, these diseases normally pertain to ulcerative colitis (UC) (Head & Jurenka, 2003) and Crohn’s disease (CD) (Baumgart & Sandborn, 2007). There is evidence that these do not represent distinct conditions but rather are the same disease with shared etiological factors (Price, 1992); however, clinical manifestations (such as the exact location of the pathology or the affected individual’s immunological and constitutional endowment) are distinctive between both. Despite many years of study, the exact etiology and pathogenesis of these disorders remain unclear but great advances have been made using experimental animal models and have provided insights into the complex, multi-factorial processes and mechanisms that can result in chronic intestinal inflammation (Elson & Weaver, 2003). The aim of this chapter is to present an overview of the current expanding knowledge of the mechanisms by which lactic acid bacteria and other probiotic microorganisms participate in the prevention and treatment of IBD and how genetic engineering techniques can be used to improve their effectiveness or create novel therapeutic strains. In the following sections, the mechanisms by which these beneficial microorganisms exert their therapeutic effects, which include changes in the gut microbiota, stimulation of the host immune responses, enhancement of intestinal barrier function and reduction of the oxidative stress due to their antioxidant properties will be discussed. Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: del Carmen, Silvina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Alvarenga Lima, Fernanda. Universidade Federal de Minas Gerais; Brasil Fil: Zurita Turk, Meritxell. Universidade Federal de Minas Gerais; Brasil Fil: Miyoshi, Anderson. Universidade Federal de Minas Gerais; Brasil Fil: Azevedo, Vasco. Universidade Federal de Minas Gerais; Brasil Fil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina