Adjuvant properties and colonization potential of adhering and non-adhering Lactobacillus spp following oral administration to mice

This study aimed to determine whether adhesive strains of Lactobacillus possessed an increased ability to colonize the gastrointestinal tract and to examine the adjuvant capacities of these strains for the 50000 molecular-mass fragment C of tetanus toxin (TTFC) following oral administration. The three strains used in this study showed different patterns of adhesion to tissue from all regions of the gastrointestinal tract, with two strains adhering in high numbers and one strain showing negligible association with all tissue types. The colonization patterns in the gastrointestinal tract of C57BL/6 mice following oro-gastric dosing was also monitored, and it was found that adhesive Lactobacillus strains could be detected for at least 24 h, in association with either fecal material and/or with gastrointestinal tissue or contents. In addition, mice were given an oro-gastric dose of the lactobacilli (5 x 10(8) colony forming units) with TTFC (10 and 50 micro g), and the serum-specific IgM and IgG antibody responses monitored in serum. The adhesive strains, which persisted within the gastrointestinal tract for at least 24 h, showed enhanced antigen-specific serum IgG and IgM antibody responses in comparison to a non-adhesive strain that failed to be detected in the gastrointestinal tract. Adhesion to the gastrointestinal tract is a factor affecting the capacity of lactobacilli to persist within the gastrointestinal tract and to act as an adjuvant for orally administered antigen.     

Expression and delivery of tetanus toxin fragment C fused to the N-terminal domain of SipB enhances specific immune responses in mice

Live attenuated bacteria can be used as a carrier for the delivery of foreign antigens to a host's immune system. The N-terminal domain of SipB, a translocon protein of the type III secretion system of Salmonella enterica serovar Typhimurium, is required for secretion and outer membrane localization. In the present study, vaccine plasmids for antigen delivery in which the non-toxic tetanus toxin fragment C (TTFC), which contains a T cell epitope, is fused to the N-terminal 160 amino acids of SipB were developed. It was found that the recombinant proteins are secreted into the culture media and localized to the bacterial surface. TTFC-specific antibody responses are significantly increased in mice orally immunized with attenuated S. Typhimurium BRD509 strains carrying TTFC delivery plasmids. When the TTFC delivery cassettes were introduced into a low copy vector, the plasmid was stably maintained in the BRD509 strain and induced an immune response to the TTFC antigen in mice. These results suggest that expression and delivery of heterologous antigens fused to the N-terminus of SipB enhance the induction of antigen-specific immune responses, and that the N-terminal domain of SipB can be used as a versatile delivery system for foreign antigens.     

Adaptation of the nisin-controlled expression system in Lactobacillus plantarum: a tool to study in vivo biological effects

The potential of lactic acid bacteria as live vehicles for the production and delivery of therapeutic molecules is being actively investigated today. For future applications it is essential to be able to establish dose-response curves for the targeted biological effect and thus to control the production of a heterologous biopeptide by a live lactobacillus. We therefore implemented in Lactobacillus plantarum NCIMB8826 the powerful nisin-controlled expression (NICE) system based on the autoregulatory properties of the bacteriocin nisin, which is produced by Lactococcus lactis. The original two-plasmid NICE system turned out to be poorly suited to L. plantarum. In order to obtain a stable and reproducible nisin dose-dependent synthesis of a reporter protein (beta-glucuronidase) or a model antigen (the C subunit of the tetanus toxin, TTFC), the lactococcal nisRK regulatory genes were integrated into the chromosome of L. plantarum NCIMB8826. Moreover, recombinant L. plantarum producing increasing amounts of TTFC was used to establish a dose-response curve after subcutaneous administration to mice. The induced serum immunoglobulin G response was correlated with the dose of antigen delivered by the live lactobacilli.     

Immunomodulatory mechanisms of lactobacilli

Over the past decade it has become clear that lactobacilli and other probiotic and commensal organisms can interact with mucosal immune cells or epithelial cells lining the mucosa to modulate specific functions of the mucosal immune system. The most well understood signalling mechanisms involve the innate pattern recognition receptors such as Toll-like receptors, nucleotide oligomerization domain-like receptors and C-type lectin receptors. Binding of microbe-associated molecular patterns with these receptors can activate antigen presenting cells and modulate their function through the expression of surface receptors, secreted cytokines and chemokines. In vitro the cytokine response of human peripheral blood mononuclear cells and dendritic cells to lactobacilli can be strikingly different depending on both the bacterial species and the strain. Several factors have been identified in lactobacilli that influence the immune response in vitro and in vivo including cell surface carbohydrates, enzymes modifying the structure of lipoteichoic acids and metabolites. In mice mechanistic studies point to a role for the homeostatic control of inducible T regulatory cells in the mucosal tissues as one possible immunomodulatory mechanism. Increasing evidence also suggests that induction of epithelial signalling by intestinal lactobacilli can modulate barrier functions, defensin production and regulate inflammatory signalling. Other probiotic mechanisms include modulation of the T cell effector subsets, enhancement of humoral immunity and interactions with the epithelial-associated dendritic cells and macrophages. A major challenge for the future will be to gain more knowledge about the interactions occurring between lactobacilli and the host in vivo and to understand the molecular basis of innate signalling in response to whole bacteria which trigger multiple signalling pathways.     

Cross‐talk between probiotic lactobacilli and host immune system

The mechanism by which probiotic lactobacilli affect the immune system is strain specific. As the immune system is a multicompartmental system, each strain has its way to interact with it and induce a visible and quantifiable effect. This review summarizes the interplay existing between the host immune system and probiotic lactobacilli, that is, with emphasis on lactobacilli as a prototype probiotic genus. Several aspects including the bacterial‐host cross‐talk with the mucosal and systemic immune system are presented, as well as short sections on the competing effect towards pathogenic bacteria and their uses as delivery vehicle for antigens.     

Adaptation of the Nisin-Controlled Expression System in Lactobacillus plantarum: a Tool To Study In Vivo Biological Effects

The potential of lactic acid bacteria as live vehicles for the production and delivery of therapeutic molecules is being actively investigated today. For future applications it is essential to be able to establish dose-response curves for the targeted biological effect and thus to control the production of a heterologous biopeptide by a live lactobacillus. We therefore implemented in Lactobacillus plantarum NCIMB8826 the powerful nisin-controlled expression (NICE) system based on the autoregulatory properties of the bacteriocin nisin, which is produced by Lactococcus lactis. The original two-plasmid NICE system turned out to be poorly suited to L. plantarum. In order to obtain a stable and reproducible nisin dose-dependent synthesis of a reporter protein (β-glucuronidase) or a model antigen (the C subunit of the tetanus toxin, TTFC), the lactococcal nisRK regulatory genes were integrated into the chromosome of L. plantarum NCIMB8826. Moreover, recombinant L. plantarum producing increasing amounts of TTFC was used to establish a dose-response curve after subcutaneous administration to mice. The induced serum immunoglobulin G response was correlated with the dose of antigen delivered by the live lactobacilli.     

Influence of bacteria of Lactobaccilus genera on cytotoxic activity of splenocytes in experimental animals

Influence of lactobacilli on cytotoxic activity of splenocytes from mice of CBA line was studied in vivo and in vitro (after cultivation of splenocytes with chloroform-killed bacteria, homologous lysates obtained by ultrasound, and with native filtrates). Data on increase of splenocytes cytotoxic functions induced by lactobacilli has been obtained. Mechanisms of lactobacilli-mediated activation of cellular factors of antitumor immunity (cytotoxic lymphocytes and natural killers) were discussed. Information on the ability of cell wall components, cytoplasmic fractions, and substances secreted by lactobacilli to stimulate the immune response of a microorganism is presented.     

Identification and Characterization of Bacterial Vaginosis-Associated Pathogens Using a Comprehensive Cervical-Vaginal Epithelial Coculture Assay

Bacterial vaginosis (BV) is the most commonly treated female reproductive tract affliction, characterized by the displacement of healthy lactobacilli by an overgrowth of pathogenic bacteria. BV can contribute to pathogenic inflammation, preterm birth, and susceptibility to sexually transmitted infections. As the bacteria responsible for BV pathogenicity and their interactions with host immunity are not understood, we sought to evaluate the effects of BV-associated bacteria on reproductive epithelia. Here we have characterized the interaction between BV-associated bacteria and the female reproductive tract by measuring cytokine and defensin induction in three types of FRT epithelial cells following bacterial inoculation. Four BV-associated bacteria were evaluated alongside six lactobacilli for a comparative assessment. While responses differed between epithelial cell types, our model showed good agreement with clinical BV trends. We observed a distinct cytokine and human β-defensin 2 response to BV-associated bacteria, especially Atopobium vaginae, compared to most lactobacilli. One lactobacillus species, Lactobacillus vaginalis, induced an immune response similar to that elicited by BV-associated bacteria, stimulating significantly higher levels of cytokines and human β-defensin 2 than other lactobacilli. These data provide an important prioritization of BV-associated bacteria and support further characterization of reproductive bacteria and their interactions with host epithelia. Additionally, they demonstrate the distinct immune response potentials of epithelial cells from different locations along the female reproductive tract.     

Time to First Morning Cigarette and Risk of Chronic Obstructive Pulmonary Disease: Smokers in the PLCO Cancer Screening Trial

BackgroundTime to first cigarette (TTFC) after waking is an indicator of nicotine dependence. The association between TTFC and chronic obstructive pulmonary disease (COPD), the third leading cause of death in the United States, has not yet been reported.MethodsWe investigated the cross-sectional association between TTFC and prevalent COPD among 6,108 current smokers in the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial. COPD was defined as a self-reported diagnosis of emphysema, chronic bronchitis, or both. Current smokers in PLCO reported TTFC, the amount of time they typically waited before smoking their first cigarette of the day after waking, in four categories: ≤5, 6-30, 31-60, or >60 minutes. We used logistic regression models to investigate the association between TTFC and prevalent COPD with adjustments for age, gender, race, education, and smoking (cigarettes/day, years smoked during lifetime, pack-years, age at smoking initiation), and prior lung cancer diagnosis.ResultsCOPD was reported by 19% of these 6,108 smokers. Individuals with the shortest TTFC had the greatest risk of COPD; compared to those with the longest TTFC (>60 minutes) the adjusted odds ratios (OR) and 95% confidence intervals (CI) for COPD were 1.48 (95% CI, 1.15-1.91), 1.64 (95% CI, 1.29-2.08), 2.18 (95% CI, 1.65-2.87) for those with TTFC 31-60 minutes, 6-30 minutes, and ≤5 minutes, respectively (P-trend <0.0001). The association between TTFC and emphysema was similar to that for bronchitis, albeit the ORs were slightly stronger for chronic bronchitis; comparing TTFC ≤5 minutes to >60 minutes, the adjusted OR (95% CI) was 2.29 (1.69-3.12) for emphysema and 2.99 (1.95-4.59) for chronic bronchitis.ConclusionsCurrent smokers with shorter TTFC have increased risk of COPD compared to those with longer TTFC, even after comprehensive adjustment for established smoking covariates. Future epidemiologic studies, including prospective designs, should incorporate TTFC to better assess disease risk and evaluate the potential utility of TTFC as a COPD screening tool for smokers in the clinical setting.     

A model system for the investigation of heterologous protein secretion pathways in Lactococcus lactis.

The capacity of recombinant strains of Lactococcus lactis to secrete a heterologous protein was investigated by constructing two expression-secretion vectors (pLET2 and pLET3) for use with a lactococcal gene expression system driven by the highly active T7 RNA polymerase. The vectors incorporated different lactococcal secretion leaders and translation initiation sequences. When tetanus toxin fragment C (TTFC) was used as a test protein, the quantities of TTFC produced by the pLET2-TTFC strain exceeded the rate of secretion of TTFC into the growth medium. However, nearly all of the soluble TTFC associated with the cell (3.4%) was translocated through the cell membrane. The pLET3-TTFC strain did not accumulate TTFC intracellularly and exhibited growth characteristics and viability identical to the growth characteristics and viability of the control strain. This strain secreted approximately 2.9 mg of TTFC per liter into the growth medium after 6 h of growth under test tube conditions. Our results indicate that L. lactis is capable of secreting substantial amounts of heterologous protein and also confirm the findings of other workers that the cell wall may serve as a functional barrier to the diffusion of some secreted proteins into the growth medium.     

乳酸乳球菌作为黏膜免疫活载体疫苗传递抗原的研究进展

乳酸菌是人和动物肠道内的常见细菌,被公认为安全级(generally recognized as safe,GRAS)微生物。近年来,对于乳酸菌作为宿主菌表达外源蛋白或抗原的研究取得了一定进展。乳酸乳球菌(Lactococcus lactis)是乳酸菌的代表菌种,以其生长迅速、易于操作等优点成为表达外源抗原,作为黏膜免疫活载体疫苗的理想菌株。随着对乳酸乳球菌基因工程的研究逐渐深入,已发展了一系列组成型和诱导型乳酸乳球菌表达系统以及蛋白定位系统。破伤风毒素片段C、布氏杆菌L7/L12蛋白等多种病原微生物抗原已成功在乳酸乳球菌中表达,并已证明部分重组乳酸乳球菌作为黏膜免疫疫苗可以同时刺激局部黏膜免疫应答和系统免疫应答。目前,如何使活载体乳酸乳球菌以最佳方式向黏膜免疫系统提呈抗原继而诱导有效免疫反应是该领域的研究热点,也是巨大挑战。实现外源抗原在乳酸乳球菌中的准确定位及与细胞因子的共表达是未来研究的重要方向之一。乳酸乳球菌作为黏膜免疫活载体疫苗传递外源抗原具有广阔的应用前景。     

Chronic HIV Infection Enhances the Responsiveness of Antigen Presenting Cells to Commensal Lactobacillus

Chronic immune activation despite long-term therapy poses an obstacle to immune recovery in HIV infection. The role of antigen presenting cells (APCs) in chronic immune activation during HIV infection remains to be fully determined. APCs, the frontline of immune defense against pathogens, are capable of distinguishing between pathogens and non-pathogenic, commensal bacteria. We hypothesized that HIV infection induces dysfunction in APC immune recognition and response to some commensal bacteria and that this may promote chronic immune activation. Therefore we examined APC inflammatory cytokine responses to commensal lactobacilli. We found that APCs from HIV-infected patients produced an enhanced inflammatory response to Lactobacillus plantarum WCFS1 as compared to APCs from healthy, HIV-negative controls. Increased APC expression of TLR2 and CD36, signaling through p38-MAPK, and decreased expression of MAP kinase phosphatase-1 (MKP-1) in HIV infection was associated with this heightened immune response. Our findings suggest that chronic HIV infection enhances the responsiveness of APCs to commensal lactobacilli, a mechanism that may partly contribute to chronic immune activation.     

Progress in the development ofLactococcus lactis as a recombinant mucosal vaccine delivery system

The non-pathogenic, non-colonising Gram-positive organismLactobacillus lactis is beeing developed as an antigen delivery system for mucosal vaccination. A high level expression system has been developed which allows loading of the bacterium with high levels of a heterologous antigen (TTFC) prior to inoculaton. Mucosal inoculaton of one such recombinant strain results in a protective serum antibody response and production of TTFC-specific IgA at mucosal sites.     

Lactobacilli-Host mutualism: &quot;learning on the fly&quot;

Metazoans establish with microorganisms complex interactions for their mutual benefits. Drosophila, which has already proven useful host model to study several aspects of innate immunity and host-bacteria pathogenic associations has become a powerful model to dissect the mechanisms behind mutualistic host-microbe interactions. Drosophila microbiota is composed of simple and aerotolerant bacterial communities mostly composed of Lactobacillaceae and Acetobactereaceae. Drosophila mono- or poly-associated with lactobacilli strains constitutes a powerful model to dissect the complex interplay between lactobacilli and host biologic traits. Thanks to the genetic tractability of both Drosophila and lactobacilli this association model offers a great opportunity to reveal the underlying molecular mechanisms. Here, we review our current knowledge about how the Drosophila model is helping our understanding of how lactobacilli shapes host biology.     

Intestinal microflora and homeostasis of the mucosal immune response: implications for probiotic bacteria?

The intestinal microflora can be considered a postnatally acquired organ that is composed of a large diversity of bacteria that perform important functions for the host and can be modulated by environmental factors, such as nutrition. Specific components of the intestinal microflora, including lactobacilli and bifidobacteria, have been associated with beneficial effects on the host, such as promotion of gut maturation and integrity, antagonisms against pathogens and immune modulation. Beyond this, the microflora seems to play a significant role in the maintenance of intestinal immune homeostasis and prevention of inflammation. The contribution of the intestinal epithelial cell in the first line of defense against pathogenic bacteria and microbial antigens has been recognized. However, the interactions of intestinal epithelial cells with indigenous bacteria are less well understood. This review will summarize the increasing scientific attention to mechanisms of the innate immune response of the host towards different components of the microflora, and suggest a potential role for selected probiotic bacteria in the regulation of intestinal inflammation.     

HIV-1 testing: product development strategies.

Strategies for the development of diagnostic products for acquired immune deficiency syndrome (AIDS) are inextricably linked to the status of our knowledge of the human immunodeficiency virus (HIV) and the events associated with the pathogenesis of AIDS. This review traces product development strategies from 1984 to the present day. Product development activities in the HIV-1 antibody screening test market were a response to the need to remove contaminated units from the blood supply. With the successes in screening blood and blood products, there has been a shift towards product development for personal health care and applicant suitability. Identification of markers for disease progression and the need to monitor therapeutic efficacy is now leading to tests for patient disease staging, monitoring and prognosis.     

Mutations and regulatory anomalies effecting tumor cell immune functions

The immune system is capable of interacting with tumor cells in such a way as to lead to tumor cell death, and this knowledge has inspired therapies to manipulate patient immune systems to eradicate cancer. However, tumor cells are able to mitigate the antitumor immune response, a fact that has rarely been addressed in the design of immunotherapies. There are many different tumor cell immune functions that play a role in mitigating the antitumor immune response. In some cases, these functions appear to be intimately associated with the tumor cell abnormalities that lead to loss of growth control, such as the cases where classical tumor suppressor proteins regulate tumor cell immune function genes. In other cases, tumor cell mutations appear to affect only the antitumor response, such as tumor cell mutations that eliminate MHC class I expression. Here I review the bases for tumor cell immune functions, noting in particular where tumor cell mutations, the gold standard for identifying a tumor-specific function, are known to be responsible for the tumor cell immune function. This review also discusses other known regulatory anomalies, in the absence of a known mutation, that are apparently important for tumor development and that regulate tumor cell immune functions. Surprisingly, in many cases where the tumor cell immune function is well understood in terms of its effect on the antitumor immune response, the tumor abnormality underlying the tumor cell immune function is completely uncharacterized.     

Lymphatic vascular morphogenesis in development, physiology, and disease

The lymphatic vasculature constitutes a highly specialized part of the vascular system that is essential for the maintenance of interstitial fluid balance, uptake of dietary fat, and immune response. Recently, there has been an increased awareness of the importance of lymphatic vessels in many common pathological conditions, such as tumor cell dissemination and chronic inflammation. Studies of embryonic development and genetically engineered animal models coupled with the discovery of mutations underlying human lymphedema syndromes have contributed to our understanding of mechanisms regulating normal and pathological lymphatic morphogenesis. It is now crucial to use this knowledge for the development of novel therapies for human diseases.     

Modulation of immune system cells by lactobacilli

The influence of lactobacilli on the proliferative potential of immune system cells after the intragastral administration of viable microbial cells and their native filtrates to mice CBA is evaluated. The data have been obtained on the modulating influence of lactobacilli on the formation of T- and B-cell immune response--their role in maintaining homeostatsis and specific features of cell mediated immune reactions after the intragastral administration of virulent Shigella dysenteriae for modeling experimental infection in CBA mice. The mechanisms of the immunomodulating action of lactobacilli on local and systemic reactions of the host as well as realization of the protective properties of lactobacilli against the causative agents of acute enteric infections are discussed.     

Exploiting natural immunity to helminth parasites for the development of veterinary vaccines

The development of subunit vaccines against most parasitic helminth infections will require a better understanding of the different components of a natural rejection process including (1) recognition of parasite antigens; (2) induction of protective immune response phenotypes; and (3) activation of appropriate immune effector mechanisms. While novel technologies have allowed significant progress to be made in the identification of candidate vaccine antigens, the large scale production of these antigens and their presentation to the host with appropriate adjuvant systems remains a major problem in vaccine research. Identification of the molecular interactions involved in the innate immune response to helminth infections and the application of new genomic and proteomic technologies are likely to lead to major advances in these research fields. Gastrointestinal nematode parasites and liver fluke are the most important helminth parasites of production animals. In recent years, a lot of new knowledge has been gathered on the immunobiology of the host-parasite interactions in these two infection systems, which has allowed new vaccination strategies to be considered. Functional genomic technologies such as gene expression analysis by microarrays, promise to further advance our understanding of the molecular pathways leading to protection against parasite infections. This will not only have implications for vaccine research, but also provide novel targets for drug development and genetic selection.