Portal Of Entry For Pathogens Research Articles

Traffic of poly(lactic acid) nanoparticulate vaccine vehicle from intestinal mucus to sub-epithelial immune competent cells

Mucosal immunization is designed to induce strong immune responses at portal of pathogen entry. Unfortunately, mechanisms underlying the fate of the vaccine vector co-administered with antigens are still partially uncovered and limit further development of mucosal vaccines. Hence, poly(lactic acid) (PLA) nanoparticles being a versatile vaccine vehicle, we have analyzed the fate of these PLA nanoparticles during their uptake at intestinal mucosal sites, both in vivo and ex vivo, to decipher the mechanisms involved during this process. We first designed specific fluorescent PLA nanoparticles exhibiting strong colloidal stability after encapsulation of either 6-coumarin or CellTrace BODIPY ® before monitoring their transport through mucosa in the mouse ligated ileal loop model. The journey of the particles appears to follow a three-step process. Most particles are first entrapped in the mucus. Then, crossing of the epithelial barrier takes place exclusively through M-cells, leading to an accumulation in Peyer’s patches (PP). Lastly, we noticed specific interaction of these PLA nanoparticles with underlying B cells and dendritic cells (DCs) of PP. Furthermore, we could document that DCs engulfing some nanoparticles could exhibit a TLR8+ specific expression. Specific targeting of these two cell types strongly supports the use of PLA nanoparticles as a vaccine delivery system for oral use. Indeed, following oral gavage of mice with PLA nanoparticles, we were able to observe the same biodistribution patterns, indicating that these nanoparticles specifically reach immune target required for oral immunization.

Differential roles of migratory and resident DCs in T cell priming after mucosal or skin HSV-1 infection

Although mucosal surfaces represent the main portal of entry for pathogens, the mechanism of antigen presentation by dendritic cells (DCs) that patrol various mucosal tissues remains unclear. Instead, much effort has focused on the understanding of initiation of immune responses generated against antigens delivered by injection. We examined the contributions of migratory versus lymph node–resident DC populations in antigen presentation to CD4 and CD8 T cells after needle injection, epicutaneous infection, or vaginal mucosal herpes simplex virus (HSV) 1 infection. We show that upon needle injection, HSV-1 became lymph-borne and was rapidly presented by lymph node–resident DCs to CD4 and CD8 T cells. In contrast, after vaginal HSV-1 infection, antigens were largely presented by tissue-derived migrant DCs with delayed kinetics. In addition, migrant DCs made more frequent contact with HSV-specific T cells after vaginal infection compared with epicutaneous infection. Thus, both migrant and resident DCs play an important role in priming CD8 and CD4 T cell responses, and their relative importance depends on the mode of infection in vivo.

Anti-inflammatory abilities of Imupret®: Inhibition of IL-8 and human β-defensin 2 induced by LPS and IL-1β in lung epithelial A549 cells

Background The respiratory epithelium is a major portal of entry for pathogens and applies various defense mechanisms. Recruitment of neutrophils in airway inflammation may account for the generation of interleukin 8 (IL-8), which is also generated by tissue cells after stimulation by endotoxin (LPS) or proinflammatory cytokines. This study was designed to evaluate the effectiveness of the commercial herbal medicinal product (Imupret®, Bionorica AG) in the modulation of airway inflammation. For the production of IL-8 and human β-defensin 2 (hBD-2), LPS and interleukin-1β (IL-1β) activated A549 bronchial epithelial cells were analyzed. Materials and methods A549 cells, which express toll-like receptor 4 (TLR4), were used. The cytotoxicity and antiproliferative effect of Imupret® (50–2000 μg/ml) were investigated (propidium iodide uptake, WST-1 assay). LPS from Pseudomonas aeruginosa (100 μg/ml) or IL-1β (50 ng/ml) were used as stimulating agents. Dexamethasone (10−7 M) served as positive anti-inflammatory control. IL-8 and hBD-2 production were detected in the supernatants of A549 cells after 18 h with commercially available ELISA test kits (Bender MedSystems, CA; Phoenix Pharmaceuticals, CA). Imupret® dissolved in single solvents (bidest H2O, cell culture medium, 70% (v/v) ethanol and DMSO) was investigated at nontoxic concentrations in the range between 0.01 and 100 μg/ml. Results Imupret® (50–2000 μg/ml) in different solvents showed dose-dependent growth inhibitory effect on A549 cells. Comparative studies indicate quantitative differences concerning 50% growth inhibitory (GI50) concentrations ranging between 122–823 μg/ml. Viability of cells was not affected. The growth inhibitory effect of water and DMSO extracts was significantly diminished in LPS-primed cells at concentrations above 100 μg/ml in contrast to medium and ethanol preparations. Production of IL-8 after stimulation by LPS or IL-1β in A549 cells was significantly inhibited by pre-treatment with Imupret®. IL-8 level of LPS-stimulated cells was decreased about 20–40% by Imupret® (1–100 μg/ml ) treatment; however in Il-1β-primed cells 30% (100 μg/ml) and 20% (10 μg/ml) decreases were detected. IL-1β up-regulated level of hBD-2 was inhibited by Imupret® at concentrations between 0.1 and 100 μg/ml. Conclusion Imupret® may help to suppress airway inflammation by inhibiting IL-8 production and down-regulation of hBD-2 (increased level in chronic inflammatory diseases) in epithelial cells.

Comparative study of the effect of a herbal preparation (Imupret®) dissolved in different solvents on LPS-primed type II-like human lung epithelial A549 cells

Recently, there has been extensive effort to employ scientific methods to prove the efficacy of herbal extracts. The respiratory epithelium is a major portal of entry for pathogens and employes defense mechanisms to prevent colonization and infection. This study, therefore, was designed to evaluate the effect of a commercial herbal medical product (Imupret®, Bionorica AG) on the growth of A549 cells alone and in combination with non-toxic dose of LPS. For the stimulation of A549 cells LPS from Pseudomonas aeruginosa, an important respiratory pathogen, at concentration of 100µg/ml was used. Imupret® a herbal combination preparation from (Radix Althaeae, Flores Chamomillae, Herba Equiseti, Folia Juglandis, Herba Millefolii, Cortex Quercus and Herba Taraxaci) was dissolved in single solvents (bidest H2O, cell culture medium, 70% (v/v) ethanol and DMSO). The cytotoxicity and antiproliferative effect of Imupret® was investigated with propidium iodide uptake and with WST-1 assay after 24h incubation. Solutions of Imupret® in different solvents showed dose-dependent growth inhibitory effect on A549 cells in the concentrations between 50–2000µg/ml. Comparative studies indicate quantitative differences with GI50 values of 122–823µg/ml. The effect of water and DMSO extracts were about 6- and 4-fold more potent (p<0.05) than of medium and ethanol preparations (both exerted comparable effects). Viability of cells was not affected at any concentration. The growth inhibitory effect of water and DMSO extracts was significantly diminished in LPS-primed cells at concentrations above 100µg/ml, however in medium and ethanol preparations this effect was marginal. Results of this study showed differences between the herbal medical product dissolved in single solvents. These results suggested differences in the composition and/or concentration of compounds in single preparations and supported further pharmacological (chemical, analytical) analyses.

Respiratory epithelial cells require Toll-like receptor 4 for induction of Human β-defensin 2 by Lipopolysaccharide

BackgroundThe respiratory epithelium is a major portal of entry for pathogens and employs innate defense mechanisms to prevent colonization and infection. Induced expression of human β-defensin 2 (HBD2) represents a direct response by the epithelium to potential infection. Here we provide evidence for the critical role of Toll-like receptor 4 (TLR4) in lipopolysaccharide (LPS)-induced HBD2 expression by human A549 epithelial cells.MethodsUsing RTPCR, fluorescence microscopy, ELISA and luciferase reporter gene assays we quantified interleukin-8, TLR4 and HBD2 expression in unstimulated or agonist-treated A549 and/or HEK293 cells. We also assessed the effect of over expressing wild type and/or mutant TLR4, MyD88 and/or Mal transgenes on LPS-induced HBD2 expression in these cells.ResultsWe demonstrate that A549 cells express TLR4 on their surface and respond directly to Pseudomonas LPS with increased HBD2 gene and protein expression. These effects are blocked by a TLR4 neutralizing antibody or functionally inactive TLR4, MyD88 and/or Mal transgenes. We further implicate TLR4 in LPS-induced HBD2 production by demonstrating HBD2 expression in LPS non-responsive HEK293 cells transfected with a TLR4 expression plasmid.ConclusionThis data defines an additional role for TLR4 in the host defense in the lung.

A review of porcine tonsils in immunity and disease

The porcine tonsils are a group of lymphoepithelial tissues located at the common openings of the gastrointestinal and respiratory tracts. The tonsils participate in a variety of functions involving innate, cellular and humoral immunity at the local and systemic levels. Among these immunological functions is the continuous surveillance for the presence of foreign antigens at the openings of the gastrointestinal and respiratory tracts. Within the pig, the movement of lymphocytes, cytokines and chemotactic molecules from the tonsils to other lymphoid organs confers immunity to other portals of pathogen entry and facilitates an efficient and rapid systemic immune response. In spite of the immunological nature of the tonsils, some microorganisms have acquired adaptations that allow them to circumvent the tonsillar immune defenses and utilize the tonsils as a site of entry, replication and colonization. Several bacterial and viral pathogens persist asymptomatically within the tonsils, making identification of asymptomatic carrier animals difficult in disease control and/or pathogen elimination. This paper reviews the current information on the anatomy, immunology and pathobiology of porcine tonsils and discusses the tonsils as a site of pathogen entry, replication and colonization using Salmonella spp., classical swine fever virus and porcine reproductive and respiratory syndrome virus as examples.

A review of porcine tonsils in immunity and disease.

The porcine tonsils are a group of lymphoepithelial tissues located at the common openings of the gastrointestinal and respiratory tracts. The tonsils participate in a variety of functions involving innate, cellular and humoral immunity at the local and systemic levels. Among these immunological functions is the continuous surveillance for the presence of foreign antigens at the openings of the gastrointestinal and respiratory tracts. Within the pig, the movement of lymphocytes, cytokines and chemotactic molecules from the tonsils to other lymphoid organs confers immunity to other portals of pathogen entry and facilitates an efficient and rapid systemic immune response. In spite of the immunological nature of the tonsils, some microorganisms have acquired adaptations that allow them to circumvent the tonsillar immune defenses and utilize the tonsils as a site of entry, replication and colonization. Several bacterial and viral pathogens persist asymptomatically within the tonsils, making identification of asymptomatic carrier animals difficult in disease control and/or pathogen elimination. This paper reviews the current information on the anatomy, immunology and pathobiology of porcine tonsils and discusses the tonsils as a site of pathogen entry, replication and colonization using Salmonella spp., classical swine fever virus and porcine reproductive and respiratory syndrome virus as examples.

Early nutrition and the development of immune function in the neonate.

The present review will concentrate on the development of the gut-associated lymphoid tissue and the role of early nutrition in promoting immune function. The intestine is the largest immune organ in the body, and as such is the location for the majority of lymphocytes and other immune effector cells. The intestine is exposed to vast quantities of dietary and microbial antigens, and is the most common portal of entry for pathogens, some of which are potentially lethal. The development of normal immune function of the intestine is therefore vital for survival, and is dependent on appropriate antigen exposure and processing, and also an intact intestinal barrier. In early life innate mechanisms of defence are probably more important than active or adaptive mechanisms in responding to an infectious challenge, since the healthy neonate is immunologically naïve (has not seen antigen) and has not acquired immunological memory. During this period maternal colostrum and milk can significantly augment resistance to enteric infections. The mechanisms of enhancing disease resistance are thought to be passive, involving a direct supply of anti-microbial factors, and active, by promoting the development of specific immune function. A tolerance response to dietary and non-invasive antigens is generally induced in the gut. However, it must also be able to mount an adequate immune response to ensure clearance of foreign antigens. It is now recognized that regulation of tolerance and active immune responses is critical to health, and failure to regulate these responses can lead to recurrent infections, inflammatory diseases and allergies. The education of the immune system in early life is thought to be critical in minimizing the occurrence of these immune-based disorders. During this phase of development maternal milk provides signals to the immune system that generate appropriate response and memory. One factor that has been proposed to contribute to the increase in the incidence of immune-based disorders, e.g. atopic diseases in Western countries, is thought to be the increased prevalence of formula-feeding.

The Effect of Application of Aquaphor on Skin Condition, Fluid Requirements, and Bacterial Colonization in Very Low Birth Weight Infants

To determine the effects of repeated application of an occlusive ointment on the skin of very low birth weight infants. Nineteen neonates of 26 to 30 weeks gestational age were randomly assigned to receive topical Aquaphor ointment twice daily for 2 weeks or to receive standard skin care. Skin quality, fluid requirements, and skin bacterial colonization counts were assessed. Infants treated with Aquaphor had significantly improved skin condition scores versus controls (p = 0.002). Aquaphor improved skin scores over time (p = 0.012) in treated infants, whereas skin scores of untreated infants worsened before eventually healing. There were no significant differences in total fluid requirements, urine output, serum sodium concentrations, skin bacterial counts, fungal counts, or colonization patterns between treated and control infants in either gestational age cohort. Aquaphor ointment, used during the first two postnatal weeks, improved skin condition in infants of 26 to 30 weeks' gestation without changing skin bacterial flora. We speculate that improved skin condition may limit transepidermal water loss and decrease portals of entry for pathogens, thereby potentially decreasing fluid and electrolyte imbalances and sepsis in very low birth weight infants.

Cell adhesion: more than just glue (review).

The ability of cells to interact with each other and their surroundings in a co-ordinated manner depends on multiple adhesive interactions between neighbouring cells and their extracellular environment. These adhesive interactions are mediated by a family of cell surface proteins, termed cell adhesion molecules. Fortunately these adhesion molecules fall into distinct families with adhesive interactions varying in strength from strong binding involved in the maintenance of tissue architecture to more transient, less avid, dynamic interactions observed in leukocyte biology. Adhesion molecules are extremely versatile cell surface receptors which not only stick cells together but provide biochemical and physical signals that regulate a range of diverse functions, such as cell proliferation, gene expression, differentiation, apoptosis and migration. In addition, like many other cell surface molecules, they have been usurped as portals of entry for pathogens, including prions. How the mechanical and chemical messages generated from adhesion molecules are integrated with other signalling pathways (such as receptor tyrosine kinases and phosphatases) and the role that aberrant cell adhesion plays in developmental defects and disease pathology are currently very active areas of research. This review focuses on the biochemical features that define whether a cell surface molecule can act as an adhesion molecule, and discusses five specific examples of how cell adhesion molecules function as more than just 'sticky' receptors. The discussion is confined to the signalling events mediated by members of the integrin, cadherin and immunoglobulin gene superfamilies. It is suggested that, by controlling the membrane organization of signalling receptors, by imposing spatial organization, and by regulating the local concentration of cytosolic adapter proteins, intercellular and cell-matrix adhesion is more than just glue holding cells together. Rather dynamic 'conversations' and the formation of multi-protein complexes between adhesion molecules, growth factor receptors and matrix macromolecules can now provide a molecular explanation for the long-observed but poorly understood requirement for a number of seemingly distinct cell surface molecules to be engaged for efficient cell function to occur.