Recombinant Probiotics and Microbiota Modulation as a Good Therapy for Diseases Related to the GIT

Many diseases that affect the gastrointestinal tract (GIT) have great influence on the quality of life of the majority of patients. Many probiotic strains are being highly studied as a promising candidate due to their beneficial effect reported in the GIT. With the purpose of increasing the beneficial characteristics of some probiotics strains and, consequently, to improve further the reported results, many probiotic strains expressing or encoding different proteins, with anti-inflammatory activities, have been developed. These recombinant strains have been reported as good candidates for the treatment of different pathological conditions, especially colitis and mucositis disease since they have been shown to have positive results and good perspectives for GIT inflammation. Thus, this chapter will first address the aspects of the gastrointestinal tract in humans as well as its microbiota. In a second moment, it will discuss about chronic diseases, mainly the intestinal ones. Finally, it will discuss about probiotics, especially concerning on lactic acid bacteria (LAB), and its action in the prevention and treatment of these diseases. At the final part, we will point out aspects on the development of recombinant strains and the results found in the literature on disease models

Relação da infecção por clostridium difficile e doenças inflamatórias intestinais associada a fatores de risco e transplante de microbiota fecal: revisão sistemática / Relation between the Clostridium difficile infection and inflammatory bowel diseases associated with risk factors and fetal microbiote transplantation: systematic review

Portadores de Doença Inflamatória Intestinal (DII) são mais propensos a infecções por Clostridium difficile e suas complicações. Dessa forma, a busca por novas formas terapêuticas torna-se imprescindível, como, por exemplo, o Transplante de Microbiota Fecal (TMF). Revisão de literatura na base de dados PUBMED, e foram escolhidos alguns critérios de inclusão para esses artigos. Foram encontrados 723 artigos sobre a temática. Dentre esses artigos, foram selecionados 28 para a extração de dados e leitura prévia. E por fim, entre esses, 10 foram escolhidos para a revisão sistemática. É evidente a relação entre o aumento da incidência em infecção por Clostridium difficile em pacientes portadores de DII, e suas repercussões negativas. Além disso, é necessário analisar as opções de tratamento, e como alternativa a elas, a utilização do Transplante de Microbiota Fecal (TMF), a fim de minimizar essas ações negativas da bactéria. Conclusão: É evidente que o Transplante de Microbiota Fecal é uma alternativa eficaz aos portadores de DII infectados por Clostridium difficile, mas sua utilização, indicação e viabilidade devem ser mais discutidas

New Approaches for the design of DNA vaccine vectors delivered by lactic acid bacteria

Lactic Acid Bacteria (LAB) have been bioengineered for mucosal delivery of prophylactic, pro-inflammatory and useful molecules that include bioactive peptides, cytokines, enzymes, allergens and DNA. One of the many benefits that have been attributed to these has been the modulation of immune responses. Regarding LAB as a means of delivery, it is important to highlight that this bacteria have the ability to resist gastric and bile juices allowing their survival and transit through the gastrointestinal tract [1, 2] and conferring them the capability to delivery molecules directly at the intestinal mucosal surface. The Food and Drug Administration (FDA) of the U.S.A. consider Lactobacilli and Lactococcus lactis safe or "GRAS" (Generally Recognized As Safe), and according to with European Food Safety Authority (EFSA), these species have fulfilled the criteria of the competent Qualified Presumption of Safety (QPS) status. Increase knowledge in genetic engineering technologies have made it possible to incorporating new genes, as well as modifying the metabolic functions of these important microorganisms which has lead to improvements in health and food technology [3, 4].The oral administration of live recombinant microorganisms such as LAB can be considered as bio-drugs or bio-pharmaceuticals. This strategy can be used not only in the prevention or in the treatment of different diseases, but also in the development of innovative drugs and vaccines [5, 6]. DNA vaccines are constructions based on plasmids that combine sequences that allows its replication in different bacterial species (including E. coli and LAB) with others that are necessary to express the transgene of interest in vertebrate cells after delivery (Williams, 2013). These kind of vaccines using LAB as delivery vehicles are extremely safe and because they cannot revert to a disease causing form (as occurs with viral vector) since they only encode and express the target antigens and not virulence factors (Premenko-Lanier et al, 2004).Only a handful of groups are performing DNA vaccine design vectors that can be delivered by LAB. One is being led by Dr. Desai; who recently published the construction of the pPERDBY plasmid, in which the reporter gene is in the backbone of the plasmid allowing the cloning of the gene of interest in frame with a reporter gene [11]. Our group, led by Dr. Azevedo developed in 2009, the pValac vector, the first vector designed for DNA vaccines to be delivered by LAB [12] Recently we have published a now DNA vaccine vector called pExu [13]. The pExu vector has some attractive characteristics, such as containing the theta origin replication which offers a higher structural and segregational stability, and has showed exceptional results in in vivo test [13], and can also stably maintain large heterologous DNA inserts [14, 15]. The most common promoter and polyA signals used in DNA vaccine vectors are the constitutive human Cytomegalovirus (CMV) promoter and the bovine growth hormone gene because these increase transcription of mRNA that improves transgene expression (Williams, 2013).All of these plasmids are only being used in ?proof- of- concept? studies since these vectors having antibiotic resistance markers that for legal and ethical reasons are not acceptable which might compromise their applications in health treatments. For genetically modified LAB to be used, the development of food-grade cloning systems and biological containment systems are part of the solution for these to be approved for use in human trials.Johansen (1999) defined food-grade recombinant microorganism as those that contain DNA from the same genus or from other GRAS microorganisms. Some food-grade vectors have been constructed for LAB, but only for the production of heterologous proteins (Henrich et al., 2002, Leenhouts et al 1998; Simoes-Barbosa et al, 2004), not to deliver foreign DNA. The design of these vectors is based on the replacement of antibiotics resistance selection markers; however, other properties need to be considered in order to construct effective food-grade vectors to be used as DNA vaccines. The size, the number of copies of the plasmid, the ability to correctly translate the protein of interest in host cell, as well as their low capacity of recombination with host?s DNA are some characteristics that need to be considered. The use DNA delivery vectors by LAB does not require complex technologies and can even replace the industrial production of certain proteinsthat require expensive purification protocols since the host cells would produce the protein of interest for themselves.Fil: Mancha Agresti, Pamela. Universidade Federal de Minas Gerais; BrasilFil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Azevedo, Vasco. Universidade Federal de Minas Gerais; BrasilFil: Martins Drumond, Mariana. Universidade Federal de Minas Gerais; Brasil. Centro Federal de Educação Tecnológica de Minas Gerais; Brasi

DETERMINAÇÃO DA FUNÇÃO DE CONDUTIVIDADE HIDRÁULICA DE UM LATOSSOLO DO CERRADO

O estudo dos solos não saturados tem aplicação direta no uso racional da água, sendo essenciais em projetos e manejo da irrigação. Objetivou-se neste trabalho, determinar a função de condutividade hidráulica dos solos (FCH) pelo método indireto ajustada pelo modelo de Van Genuchten-Mualem para um latossolo da região do Alto Paranaíba. A FCH foi obtida por meio da curva de retenção de água nos solos (CRA) pela técnica do papel filtro e a condutividade hidráulica saturada do solo (Ksat) através do permeâmetro de Guelph, em 5 pontos e duas profundidades (0,2-0,4; 0,4-0,6 m). As CRA’s possuem comportamento bimodal devido a macro e microporosidade deste solo. Os valores de Ksat foram condizentes aos encontrados na literatura. As CRA’s e Ksat não apresentaram variabilidade entre os pontos e profundidades, mostrando a boa homogeneidade física deste latossolo ao longo do perfil do solo. A FCH ajustou-se bem ao modelo, sendo o primeiro trecho da CRA (0-8000 kPa) suficiente em projetos de irrigaçã

FATOR DE CONDIÇÃO RELACIONADO A ASPECTOS REPRODUTIVOS DA PIAPARA (Leporinus obtusidens) (Characiformes: Anostomidae) COLETADAS A JUSANTE DA USINA HIDRELÉTRICA DO FUNIL, MINAS GERAIS, BRASIL

This study aimed to describe the seasonal variation of condition factor (K) correlated with gonadossomatic (GSI) and hepatossomatic index (HSI) of Leporinus obtusidens (Valenciennes, 1837), collected downstream of the Hidroelectric Power Station of Funil, state of Minas Gerais, Brazil, and to evaluate if these indices can indicate the reproductive period of this species. The animals were captured between September/2006 and August/2007 and grouped seasonally. The length and the weight of 110 individuals were measured to calculate K, the weight of the liver was determined to calculate HSI and the weight of the gonad to calculate GSI. The K of females and malesgrouped and of males alone were significantly lower (p<0.05) in the summer. As for the females alone, no significant variation of the K was observed throughout the year. The values of HSI were significantly greater during the spring and summer for both sexes, whereas the GSI of males and females was superior (p<0.05) in the summer. In the correlation tests, K was not related with the GSI andHSI factors; however, strong correlation between GSI and HSI was found. K is not an index of the reproductive period of Leporinus obtusidens (Valenciennes, 1837), whereas GSI and HIS can be used for indicating reproduction

Metabolic and histologic responses of pacu (Piaractus mesopotamicus) fed diets supplemented with increasing concentrations of ractopamine

ABSTRACT An experiment was conducted during 60 days with forty pacu males fed diets supplemented with increasing concentrations of ractopamine (0.00, 11.25, 22.50, 33.75, and 45.00 mg kg−1). Eight fish were evaluated for each experimental diet. Performance and survival rate of the fish were measured. At the end of the experiment, blood was collected to determine the levels of cortisol, triacylglycerol, and protein. Moreover, the liver was collected to determine the activities of glucose-6-phosphate dehydrogenase and malic enzymes. The fillets were collected to determine chemical composition, and histologic cuts were analyzed to verify muscle growth and deposition of adipose tissue between muscle fibers. Increasing concentrations of dietary ractopamine did not change feed intake, fillet yield, fillet content of protein and ash, and frequency of relative distribution of muscle fibers. By increasing the dietary ractopamine concentration, the serum cortisol level was elevated. Ractopamine supplementation (45.00 mg kg−1) increased serum levels of triacylglycerol and protein and reduced the activity of hepatic lipogenic enzymes and the survival rate of the fish, probably in response to the high concentration of circulating cortisol. In addition, the higher level of ractopamine supplementation evaluated in this research impaired the weight gain and feed conversion. However, 11.25 mg kg−1 ractopamine reduced the ether extract level determined in the fillet and the fat deposition between muscle fibers, improving the nutritional quality of meat

A New Broad Range Plasmid for DNA Delivery in Eukaryotic Cells Using Lactic Acid Bacteria: In Vitro and In Vivo Assays

Lactococcus lactis is well documented as a promising candidate for development of novel oral live vaccines. It has been broadly engineered for heterologous expression, as well as for plasmid expression vector delivery, directly inside eukaryotic cells, for DNA vaccine, or as therapeutic vehicle. This work describes the characteristics of a new plasmid, pExu (extra chromosomal unit), for DNA delivery using L. lactis and evaluates its functionality both by in vitro and in vivo assays. This plasmid exhibits the following features: (1) a theta origin of replication and (2) an expression cassette containing a multiple cloning site and a eukaryotic promoter, the cytomegalovirus (pCMV). The functionality of pExu:egfp was evaluated by fluorescence microscopy. The L. lactis MG1363 (pExu:egfp) strains were administered by gavage to Balb/C mice and the eGFP expression was monitored by fluorescence microscopy. The pExu vector has demonstrated an excellent stability either in L. lactis or in Escherichia coli. The eGFP expression at different times in in vitro assay showed that 15.8% of CHO cells were able to express the protein after transfection. The enterocytes of mice showed the expression of eGFP protein. Thus, L. lactis carrying the pExu is a good candidate to deliver genes into eukaryotic cells

Protective effect of Lactobacillus delbrueckii subsp. Lactis CIDCA 133 in a model of 5 Fluorouracil-Induced intestinal mucositis

Mucositis is a cytotoxic side effect caused by chemotherapy drugs, such as 5-Fluorouracil (5-FU), being a serious clinical issue. Lactobacillus spp. could be a helpful strategy to alleviate 5-FU chemotherapy-caused intestinal damage, due to their ability to contribute to intestinal homeostasis through improvement of microbiota balance and immunomodulation. In this work we evaluated the effect of Lactobacillus delbrueckii subsp. lactis CIDCA 133 fermented milk in 5-FU-induced experimental mucositis. Intestinal histology, permeability and biochemical parameters showed that animals administrated with 5-FU and treated with CIDCA 133 fermented milk presented reduced intestinal IgA secretion and lower permeability in the small bowel. We showed that this strain preserves villus/crypt ratio, reduces the loss of goblet cells and inflammatory infiltration in ileum sections of 5-FU-treated animals. In conclusion, CIDCA 133 is able to prevent the intestinal mucosa damage caused by 5-FU revealing to be a promising strategy for intestinal mucositis treatment.Fil: De Jesus, Luís Cláudio Lima. Universidade Federal de Minas Gerais; BrasilFil: Martins Drumond, Mariana. Universidade Federal de Minas Gerais; BrasilFil: de Carvalho, André. Universidade Federal de Minas Gerais; BrasilFil: Santos, Spencer S.. Universidade Federal de Minas Gerais; BrasilFil: Martins, Flaviano S.. Universidade Federal de Minas Gerais; BrasilFil: Ferreira, Ênio. Universidade Federal de Minas Gerais; BrasilFil: Salgado Fernandes, Renata. Universidade Federal de Minas Gerais; BrasilFil: Branco de Barros, André Luís. Universidade Federal de Minas Gerais; BrasilFil: do Carmo, Fillipe Luis R. Universidade Federal de Minas Gerais; BrasilFil: Perez, Pablo Fernando. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; ArgentinaFil: Carvalho Azevedo, Vasco Ariston. Universidade Federal de Minas Gerais; BrasilFil: Mancha Agresti, Pamela. Universidade Federal de Minas Gerais; Brasi

Intake of Lactobacillus delbrueckii (pExu:hsp65) Prevents the Inflammation and the Disorganization of the Intestinal Mucosa in a Mouse Model of Mucositis

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

Neuroprotective effects of p62(SQSTM1)-engineered lactic acid bacteria in Alzheimer’s disease: a pre-clinical study

Alzheimer’s disease (AD) is a progressive neurodegeneration characterized by neuron death ending in memory and cognitive decline. A major concern in AD research is the identification of new therapeutics that could prevent or delay the onset of the disorder, with current treatments being effective only in reducing symptoms. In this perspective, the use of engineered probiotics as therapeutic tools for the delivery of molecules to eukaryotic cells is finding application in several disorders. This work introduces a new strategy for AD treatment based on the use of a Lactobacillus lactis strain carrying one plasmid (pExu) that contains a eukaryotic expression cassette encoding the human p62 protein. 3xTg-AD mice orally administered with these bacteria for two months showed an increased expression of endogenous p62 in the brain, with a protein delivery mechanism involving both lymphatic vessels and neural terminations, and positive effects on the major AD hallmarks. Mice showed ameliorated memory, modulation of the ubiquitin-proteasome system and autophagy, reduced levels of amyloid peptides, and diminished neuronal oxidative and inflammatory processes. Globally, we demonstrate that these extremely safe, non-pathogenic and non-invasive bacteria used as delivery vehicles for the p62 protein represent an innovative and realistic therapeutic approach in AD