Nasal administration of a non-viable Lactobacillus casei to infant mice modulates lung damage induced by Poly I:C and hyperreactivity in airways.

Viral respiratory infections caused by RNA viruses are one of the most important diseases around the world. The aim of this work was to study whether the nasal administration of non-viable Lactobacillus casei (LcM) was able to enhance respiratory antiviral defenses in young mice challenged with Poly I:C. Three-week-old BALB/c mice were nasally challenged with Poly I:C, used to mimic the pro-inflammatory state of lung infections caused by RNA viruses. LcM was nasally administered 2 days before Poly I:C challenge. Lactate dehydrogenase (LDH) activity, albumin concentration in broncho-alveolar lavages (BAL), wet-to-dry lung weight ratio, and total and differential leukocytes counts in blood were evaluated. Also, α, λ, γ interferons, IL-10, TNF-α, IL-4 in BAL and nasal lavages and total IgE in BAL and serum, were evaluated by ELISA. Poly I:C induced pulmonary injuries while alteration of bronchoalveolar-capillary barrier was reduced by nasal administration of LcM. Moreover, alterations in leukocyte counts induced by Poly I:C were regulated. LcM favorably modulated the cytokines responses triggered by Poly I:C challenge in nasal and lung mucosal compartments. Also, LcM decreased IgE levels in BAL and plasma compared with the Poly I:C group. LcM nasally administered reduced the lung damage induced by Poly I:C and prevented airway hyperreactivity.

Non-Viable Lactobacillus Casei Beneficially Modulates Poly I:C Immune Response in Co-Cultures of Human Cells.

BACKGROUND: Polyinosinic:polycytidylic acid (Poly-IC) has been used as a viral stimulus to mimic in vivo and in vitro infection induced by some viruses. OBJECTIVE: To determine whether non-viable Lactobacillus casei CRL431 (LcM) can modulate the immune response induced by Poly I:C in co-culture models of peripheral blood mononuclear cells (PBMC) and A549 cells. METHODS: T and NK cell activation was evaluated by flow cytometry and levels of TNF-α, IFN-γ, IL-10, IL-29, and IL-17 by ELISA. Cells in direct contact with A549 (PBMC-A549) and cells with no contact with it (PBMC//A549) were used for this purpose. PBMCs alone and both co-culture systems were stimulated for 24 h with the following stimuli: LPS (10 µg/ml), LcM (106 UFC/ml), Poly I:C (2 µg/ml), Poly I:C+LcM, and LcM (3 h)+Poly I:C. Moreover, unstimulated cells were used as a control. RESULTS: Poly I:C and LcM (3 h)+Poly I:C in PBMC-A549 showed a significant increase in the percentage of CD8+ expression (p<0.05). All stimuli induced significant activation from T CD4+, CD8+ cells compared with unstimulated PBMCs in both co-culture cells system. However, activation percentages were higher in direct co-culture. Poly I:C induced a higher level of pro-inflammatory TNF-α and IFN-γ cytokines as well as IL-17 and IL-29 with lower IL-10 levels in both co-culture systems while LcM induced a beneficial pattern of cytokines that would regulate Poly I:C effect. CONCLUSION: This in vitro model allowed us to highlight the potential of LcM as a modulator of anti-viral immune response and suggest its potential use in formulations against RNA respiratory viruses.

Decrease in lactobacilli in the intestinal microbiota of celiac children with a gluten-free diet, and selection of potentially probiotic strains.

The intestinal microbiota would be implicated in pathology associated with celiac disease caused by an abnormal immune system reaction against gluten present in cereal grains. The objectives of this work were to detect through basic methods the changes in the composition of the most common genera of bacteria from the intestinal microbiota of symptom-free celiac disease children with a gluten-free diet compared with healthy children from Tucumán and to select lactobacilli (Lb) strains with probiotic potential from the feces of healthy children. Results demonstrated that the feces of celiac children with a gluten-free diet showed significantly lower counts of Lb (P < 0.05) compared with healthy children, while enterobacteria tended to increase in celiac children. On the basis of these results, isolation of some Lb from the feces of healthy children was carried out. Thus, 5 Lb strains were selected because of their high resistance percentages to gastrointestinal tract conditions. In addition, their autoaggregation and hydrophobicity properties were evaluated: Lactobacillus rhamnosus (LC4) showed the highest percentage of autoaggregation while Lactobacillus paracasei (LC9) showed high hydrophobicity. Based on these results, LC4 and LC9 were selected, and their use as potential probiotic strains to improve signs and symptoms associated with celiac disease is discussed. This is the first study performed in Argentina concerning the relationship between intestinal microbiota and celiac disease in celiac children with a gluten-free diet. In addition, the development of a probiotic food addressed towards celiac patients and designed with Lb isolated from the feces of healthy children from our province represents a promising alternative to improve the quality of life of celiac patients.

Safety of a nasal vaccine against Streptococcus pneumoniae using heat-killed Lactobacillus casei as adjuvant.

Streptococcus pneumoniae is a highly important respiratory pathogen that causes infections in children, elderly people and immunocompromised people around the world. Pneumococcal vaccines licensed did not reach to eradicate the pneumococcal infection. In a previous study was demonstrated the effectiveness of a nasal experimental vaccine that consisted in a pneumococcal protective protein A (PppA) co-administrated with heat-killed-Lactobacillus casei (LcM), in mice model of respiratory pneumococcal challenge. In the present work the safety of the experimental vaccine LcM+PppA and its components were evaluated through hematological, biochemical and immune parameters in a model infection with S. pneumoniae. Thus, alanine transaminase activity, creatinine levels, lactate dehydrogenase activity, C reactive protein levels, corticosterone levels in serum, total and differential leukocyte counts in blood and bronchoalveolar lavages (BAL) and IgE in BAL, were evaluated. Experimental vaccine: LcM+PppA nasally administered does not induce harmful effects in our vaccination-infection model. Studied parameters showed LcM+PppA's safety in liver, kidney, pulmonary and systemic levels. Although studies in experimental animals do not guarantee security for the application of the vaccine on humans, they are important evidence for the planning and subsequent clinical trials in humans.

Immune response in nasopharynx, lung, and blood elicited by experimental nasal pneumococcal vaccines containing live or heat-killed lactobacilli as mucosal adjuvants.

This work analyzes the humoral and cellular immune responses induced by live (LcV) and heat-killed (LcM) Lactobacillus casei associated with the pneumococcal antigen (P-Ag) at the nasopharynx level, considering nasal-associated lymphoid tissue (NALT) as the primary inductive site of the mucosal immune system, and lung and blood as effector sites. Levels of P-Ag IgA and IgG antibodies, main types of B and T cells, and cytokines in mucosal and systemic compartments were evaluated. The results showed that both LcM+P-Ag and LcV+P-Ag vaccines effectively induced IgA and IgG anti-P-Ag Abs in the upper and lower respiratory tract and plasma. These results correlated with increased IgA+ cells in NALT and lung that was induced by the experimental vaccines. Moreover, numbers of IgG+ cells increased in the blood. Profiles of inflammatory and regulatory cytokines were evaluated and their possible implications for the defense against pneumococci was assessed. Considering the overall results, the potential mechanisms of immune stimulation induced by LcM and LcV used as adjuvants are discussed. LcV and LcM showed similar effects on the immune system. Strain viability is not crucial for the stimulation of the antigen-specific immune response, and LcM is a convenient and effective mucosal adjuvant.

Host immunity in the protective response to nasal immunization with a pneumococcal antigen associated to live and heat-killed Lactobacillus casei.

BACKGROUND: At present, available pneumococcal vaccines have failed to eradicate infections caused by S. pneumoniae. Search for effective vaccine continues and some serotype independent pneumococcal proteins are considered as candidates for the design of new vaccines, especially a mucosal vaccine, since pneumococci enter the body through mucosal surfaces. Selection of the appropriate adjuvant is important for mucosal vaccines, and lactic acid bacteria (LAB) with immunostimulant properties are promissory candidates. In this work, we assessed the adjuvant effect of a probiotic strain, Lactobacillus casei (L. casei), when nasally administered with a pneumococcal antigen (pneumococcal protective protein A: PppA) for the prevention of pneumococcal infection. Adjuvanticity of both live (LcV) and heat-killed (LcM) was evaluated and humoral and cellular antigen-specific immune response was assessed in mucosal and systemic compartments. The potential mechanisms induced by nasal immunization were discussed. RESULTS: Nasal immunization of young mice with PppA+LcV and PppA+LcM induced anti-PppA IgA and IgG antibodies in mucosal and systemic compartments and levels of these specific antibodies remained high even at day 45 after the 3rd Immunization (3rd I). These results were correlated with IL-4 induction by the mixture of antigen plus LcV and LcM. Also, PppA+Lc (V and M) induced stimulation of Th1 and Th17 cells involved in the defence against pneumococci. The protection against pneumococcal respiratory challenge at day 30 after the 3rd I showed that PppA+LcV and PppA+LcM immunizations significantly reduced pathogen counts in nasal lavages while prventing their passage into lung and blood. Survival of mice immunized with the co-application of PppA plus LcV and LcM was significantly higher than in mice immunized with PppA alone and control mice when intraperitoneal challenge was performed. No significant differences between the treatments involving LcV and LcM were found. CONCLUSIONS: Live and heat-killed L. casei enhanced the antigen-specific immune response when administered nasally with a pneumococcal antigen. Considering the potential risk associated with live bacteria, the design of a nasal vaccine based on pneumococcal antigens and heat-killed L. casei emerges as a safe and effective strategy for the prevention of pneumococcal infections and opens new possibilities of application of dead LAB as adjuvants in vaccine formulations against other pathogens.

Lactococcus lactis as an adjuvant and delivery vehicle of antigens against pneumococcal respiratory infections.

Most studies of Lactococcus lactis as delivery vehicles of pneumococcal antigens are focused on the effectiveness of mucosal recombinant vaccines against Streptococcus pneumoniae in animal models. At present, there are three types of pneumococcal vaccines: capsular polysaccharide pneumococcal vaccines (PPV), protein-polysaccharide conjugate pneumococcal vaccines (PCV) and protein-based pneumococcal vaccines (PBPV). Only PPV and PCV have been licensed. These vaccines, however, do not represent a definitive solution. Novel, safe and inexpensive vaccines are necessary, especially in developing countries. Probiotic microorganisms such as lactic acid bacteria (LAB) are an interesting alternative for their use as vehicles in pneumococcal vaccines due to their GRAS (Generally Recognized As Safe) status. Thus, the adjuvanticity of Lactococcus lactis by itself represents added value over the use of other bacteria, a question dealt with in this review. In addition, the expression of different pneumococcal antigens as well as the use of oral and nasal mucosal routes of administration of lactococcal vaccines is considered. The advantages of nasal live vaccines are evident; nonetheless, oral vaccines can be a good alternative when the adequate dose is used. Another point addressed here is the use of live versus inactivated vaccines. In this sense, few researchers have focused on inactivated strains to be used as vaccines against pneumoccoccus. The immunogenicity of live vaccines is better than the one afforded by inactivated ones; however, the probiotic-inactivated vaccine combination has improved this matter considerably. The progress made so far in the protective immune response induced by recombinant vaccines, the successful trials in animal models and the safety considerations of their application in humans suggest that the use of recombinant vaccines represents a good short-term option in the control of pneumococcal diseases.

Stimulation of respiratory immunity by oral administration of Lactococcus lactis.

This work demonstrates that non-recombinant Lactococcus lactis NZ, administered by the oral route at the proper dose, is able to improve resistance against pneumococcal infection. Lactococcus lactis NZ oral administration was able to improve pathogen lung clearance, increased survival of infected mice, and reduced lung injuries. This effect was related to an upregulation of the respiratory innate and specific immune responses. Administration of L. lactis NZ improved production of bronchoalveolar lavage (BAL) fluid TNF-alpha, enhanced recruitment of neutrophils into the alveolar spaces, and induced a higher activation of BAL phagocytes compared with the control group. Lactococcus lactis NZ administered orally stimulated the IgA cycle, increased IgA+ cells in intestine and bronchus, and improved production of BAL IL-4 and IL-10 during infection. Moreover, mice treated with L. lactis NZ showed higher levels of BAL anti-pneumococcal IgA and IgG. Taking into consideration that orally administered L. lactis NZ stimulates both the innate and the specific immune responses in the respiratory tract and that bacterial and viral antigens have been efficiently produced in this strain, L. lactis NZ is an excellent candidate for the development of an effective pneumococcal oral vaccine.

Nasal administration of Lactococcus lactis improves local and systemic immune responses against Streptococcus pneumoniae.

Lactococcus lactis NZ9000 is a non-pathogenic non-invasive bacterium extensively used for the delivery of antigens and cytokines at the mucosal level. However, there are no reports concerning the per se immunomodulatory capacity of this strain. The aim of the present study was to investigate the intrinsic immunostimulating properties of the nasal administration of L. lactis NZ9000 in a pneumococcal infection model. Mice were preventively treated with L. lactis (2, 5 or 7 days with 10(8) cells/day per mouse) and then challenged with Streptococcus pneumoniae. The local and the systemic immune responses were evaluated. Our results showed that nasal administration of L. lactis for 5 days (LLN5d) increased the clearance rate of S. pneumoniae from lung and prevented the dissemination of pneumococci into blood. This effect coincided with an upregulation of the innate and specific immune responses in both local and systemic compartments. LLN5d increased phagocyte activation in lung, blood and bone marrow, determined by NBT and peroxidase tests. Anti-pneumococcal immunoglobulin (Ig)A in bronchoalveolar lavages (BAL) and IgG in BAL and serum were increased in the LLN5d group. Lung tissue injury was reduced by LLN5d treatment as revealed by histopathological examination and albumin concentration and lactate dehydrogenase activity in BAL. The adjuvant effect of L. lactis in our infection model would be an important advantage for its use as a delivery vehicle of pneumococcal proteins and nasal immunization with recombinant L. lactis emerges as an effective route of vaccination for both systemic and mucosal immunity against pneumococcal infection.

Nasal immunization with Lactococcus lactis expressing the pneumococcal protective protein A induces protective immunity in mice.

Nisin-controlled gene expression was used to develop a recombinant strain of Lactococcus lactis that is able to express the pneumococcal protective protein A (PppA) on its surface. Immunodetection assays confirmed that after the induction with nisin, the PppA antigen was predictably and efficiently displayed on the cell surface of the recombinant strain, which was termed L. lactis PppA. The production of mucosal and systemically specific antibodies in adult and young mice was evaluated after mice were nasally immunized with L. lactis PppA. Immunoglobulin M (IgM), IgG, and IgA anti-PppA antibodies were detected in the serum and bronchoalveolar lavage fluid of adult and young mice, which showed that PppA expressed in L. lactis was able to induce a strong mucosal and systemic immune response. Challenge survival experiments demonstrated that immunization with L. lactis PppA was able to increase resistance to systemic and respiratory infection with different pneumococcal serotypes, and passive immunization assays of naïve young mice demonstrated a direct correlation between anti-PppA antibodies and protection. The results presented in this study demonstrate three major characteristics of the effectiveness of nasal immunization with PppA expressed as a protein anchored to the cell wall of L. lactis: it elicited cross-protective immunity against different pneumococcal serotypes, it afforded protection against both systemic and respiratory challenges, and it induced protective immunity in mice of different ages.

Effect of lactobacilli administration in the vaginal tract of mice: evaluation of side effects and local immune response by local administration of selected strains.

Lactobacilli are the predominant microorganisms in the vaginal tract of human and some homeothermic animals. They can maintain the ecological equilibrium of the tract by protecting against pathogenic microorganisms. In the last few years, there has been an increased tendency to use probiotic microorganisms to restore the ecological equilibrium and to protect against infections. This principle has been widely applied to the gastrointestinal tract. More recently, some other studies have reported the application of probiotics in different tracts, for example, the urogenital or respiratory tract. One of the objectives of our group is to design probiotic products for the urogenital tract. With this purpose, lactobacilli were isolated from the human vagina, and later some of them were selected for their probiotic characteristics (production of antagonistic substances or adhesion capability). The application of probiotic products in the vaginal tract has been approached empirically; some pharmaceuticals containing these microorganisms are available in the United States or Europe or are protected under the patent process or intellectual property rights. There are not enough studies in humans or animals to determine whether their administration can produce some type of collateral or adverse effect. Using Balb/c mice as the experimental model, the object of the present work was to study (1) whether intravaginal administration of human lactobacilli can produce colonization of the tract; (2) whether such administration produces some type of adverse or collateral effect; and (3) whether probiotics are able to stimulate the local immune system. Keeping in mind that hormones can affect the colonization or persistence ability of microorganisms, and with the purpose of having all animals at the same point in the sexual cycle, animals were cycled with estradiol 48 h before inoculation with lactobacilli. They were then inoculated im with hormones 48 h before beginning microorganism inoculations. Later they were intravaginally inoculated with the appropriate dose of each Lactobacillus strains. The animals were sacrificed on different days after inoculation to perform the following studies: 1. Microbiological assays: To determine the number of lactobacilli in the tract (in vaginal washes or in organ homogenates), by plating the samples in selective media containing antibiotic (to differentiate the resident flora from those administered experimentally).