Linking innate and adaptive immunity to streptococcus pneumoniae

Streptococcus pneumoniae (the pneumococcus) most commonly colonizes the human nasopharyngeal mucosa without causing any symptoms. However, this organism has the potential to spread to normally sterile sites and cause pneumonia, meningitis or sepsis; diseases which are characterized by excessive inflammation. Despite the large burden of pneumococcal disease, relatively little is known about the mechanisms behind development of natural immunity to the pneumococcus. The purpose of this thesis was to study the role of human dendritic cells (DCs) in linking innate and adaptive immune responses to pneumococci. The immunological events in which DC-mediated T helper (Th) cell responses are generated were also investigated, as well as possible ways to modulate these responses. As a first part of this work, a novel role of the pneumococcal toxin pneumolysin in the evasion of DC-mediated immunosurveillance was described. Pneumolysin inhibited DC maturation, production of inflammatory cytokines and inflammasome activation, and induced caspase-dependent apoptosis of infected cells. Interestingly, murine DCs differed in their response to pneumolysin, emphasizing the need to study human responses to this human-specific pathogen In the second part of this work, we demonstrated that pneumococcus-infected monocytes and DCs efficiently promote the production of inflammatory Th1 and Th17 cytokines from autologous co-cultured memory cells. Live pneumococci and pneumococcal peptidoglycan triggered activation of DCs, which in turn induced the generation of Th cytokines via cell-to-cell contact and soluble components. Our work further revealed that the inflammatory response could be modulated with exogenous substances, such as recombinant cytokines, and cytokine- and receptor-blocking antibodies. Moreover, exposure of DCs to vitamin D skewed the response from an inflammatory Th1/Th17 phenotype towards a regulatory T cell phenotype. In the last part of this work, we focused on patients with primary immunodeficiencies (PIDs), suffering from frequent respiratory tract infections. The mechanisms behind the infectious susceptibility among these patients remain elusive and we hypothesized that it may be due to defects in the production of antimicrobial peptides (AMPs) in the nasal mucosa. We found that two patient groups, namely common variable immunodeficiency (CVID) and Hyper-IgE syndrome (HIES), had a dysregulated AMP response to bacteria in the upper respiratory tract. In addition, cells from these patients exhibited an impaired Th17 cytokine response. In order to improve management of patients with pneumococcal infections there is a need to elucidate the role of DC-mediated cytokine responses in the delicate balance between protective immunity and immunopathology. An increased understanding of these processes is also essential for the development of pneumococcal vaccines, designed to elicit cell-mediated immunity. The work presented in this thesis contributes to our understanding of the dynamic interplay between pneumococci and host cells, and provides the opportunity to explore the potential role of vitamin D in limiting the inflammatory response in pneumococcal disease

The permanency and effect of four organic sythetic insecticides on selected wool fabrics

Call number: LD2668 .T4 1948 O6Master of Scienc

Impaired Release of Antimicrobial Peptides into Nasal Fluid of Hyper-IgE and CVID Patients

Patients with primary immunodeficiency (PID) often suffer from frequent respiratory tract infections. Despite standard treatment with IgG-substitution and antibiotics many patients do not improve significantly. Therefore, we hypothesized that additional immune deficits may be present among these patients.To investigate if PID patients exhibit impaired production of antimicrobial peptides (AMPs) in nasal fluid and a possible link between AMP-expression and Th17-cells.Nasal fluid, nasopharyngeal swabs and peripheral blood mononuclear cells (PBMCs) were collected from patients and healthy controls. AMP levels were measured in nasal fluid by Western blotting. Nasal swabs were cultured for bacteria. PBMCs were stimulated with antigen and the supernatants were assessed for IL-17A release by ELISA.In healthy controls and most patients, AMP levels in nasal fluid were increased in response to pathogenic bacteria. However, this increase was absent in patients with common variable immunodeficiency (CVID) and Hyper-IgE syndrome (HIES), despite the presence of pathogenic bacteria. Furthermore, stimulation of PBMCs revealed that both HIES and CVID patients exhibited an impaired production of IL-17A.CVID and HIES patients appear to have a dysregulated AMP response to pathogenic bacteria in the upper respiratory tract, which could be linked to an aberrant Th17 cell response

The intranasal adjuvant Endocine((TM)) enhances both systemic and mucosal immune responses in aged mice immunized with influenza antigen

Despite availability of annual influenza vaccines, influenza causes significant morbidity and mortality in the elderly. This is at least in part a result of immunosenescence; the age-dependent decrease in immunological competence that results in greater susceptibility to infections and reduced responses to vaccination. To improve protective immune responses in this age group, new vaccines strategies, such as the use of adjuvants, are needed. Here, we evaluated the mucosal vaccine adjuvant Endocine(TM), formulated with split influenza antigen and administered intranasally in aged (20-month old) mice. Humoral immune responses were assessed and compared to unadjuvanted intranasal and subcutaneous vaccines. We show that formulation with Endocine(TM) significantly enhances hemagglutination inhibition (HI) titers, as well as serum IgG and mucosal IgA antibody titers, compared to both types of unadjuvanted vaccines. Thus, our results indicate that intranasal vaccination with Endocine(TM) is a possible approach for the development of mucosal influenza vaccines for the elderly

Petrobacter succinatimandens gen. nov., sp. nov., a moderately thermophilic, nitrate-reducing bacterium isolated from Australian oil well

A novel Gram-negative, aerobic and moderately thermophilic bacterium, strain 4BONT, was isolated from a non-water-flooded Australian terrestrial oil reservoir. Cells were non-spore-forming straight rods, which were motile by means of a polar flagellum. The optimum growth conditions were 55 °C, pH 6·9 and 0·5 % NaCl. Strain 4BONT was oxidase- and catalase-positive; it grew on fumarate, pyruvate, succinate, formate, ethanol and yeast extract in the presence of oxygen or nitrate as terminal electron acceptor. Nitrate was reduced to nitrous oxide. The DNA G+C content of the strain was 58·6 mol%. The closest phylogenetic relative of strain 4BONT was Hydrogenophilus thermoluteolus (similarity of 91·8 %), of the β-Proteobacteria. As strain 4BONT is physiologically and phylogenetically different from H. thermoluteolus, it is proposed that it be assigned to a novel species of a novel genus, Petrobacter succinatimandens gen. nov., sp. nov. The type strain is 4BONT (=DSM 15512T=CIP 107790T)

Cinobufagin Modulates Human Innate Immune Responses and Triggers Antibacterial Activity

<div><p>The traditional Chinese medicine Chan-Su is widely used for treatment of cancer and cardiovascular diseases, but also as a remedy for infections such as furunculosis, tonsillitis and acute pharyngitis. The clinical use of Chan-Su suggests that it has anti-infective effects, however, the mechanism of action is incompletely understood. In particular, the effect on the human immune system is poorly defined. Here, we describe previously unrecognized immunomodulatory activities of cinobufagin (CBG), a major bioactive component of Chan-Su. Using human monocyte-derived dendritic cells (DCs), we show that LPS-induced maturation and production of a number of cytokines was potently inhibited by CBG, which also had a pro-apoptotic effect, associated with activation of caspase-3. Interestingly, CBG triggered caspase-1 activation and significantly enhanced IL-1β production in LPS-stimulated cells. Finally, we demonstrate that CBG upregulates gene expression of the antimicrobial peptides (AMPs) hBD-2 and hBD-3 in DCs, and induces secretion of HNP1-3 and hCAP-18/LL-37 from neutrophils, potentiating neutrophil antibacterial activity. Taken together, our data indicate that CBG modulates the inflammatory phenotype of DCs in response to LPS, and triggers an antibacterial innate immune response, thus proposing possible mechanisms for the clinical effects of Chan-Su in anti-infective therapy.</p></div

Induction of AMPs in DCs and neutrophils.

<p>(A) DCs were stimulated with vehicle or CBG in the absence or presence of LPS for 24 hours. Quantitative polymerase chain reaction (qPCR) for hBD-2 and hBD-3, normalized to GAPDH, was performed, and data shown represent mean gene expression (fold change compared to vehicle) + SEM for 3 donors. (B-D) Neutrophils were stimulated with vehicle or CBG in the absence or presence of LPS for 6 hours. (B) Neutrophil supernatants analyzed for release of HNP1-3 by ELISA. Cytochalasin B and fMLP (c/fMLP) treated cells served as positive control. Data shown represent means + SEM for 3 donors. *P<0.05. **P<0.01. (C) Neutrophil supernatants analyzed for release of proform hCAP-18 by semi-quantitative Western blot. Cytochalasin B/ fMLP (c/fMLP) served as positive control. Human serum containing 1 μg/ml hCAP-18 was used as a reference for relative determination of supernatant hCAP-18 levels. One representative donor out of two is shown (left) and data represent means + SD for 2 donors (right). (D) Bacterial killing was measured following incubation of neutrophils with live <i>S</i>. <i>pneumoniae</i> (strain T4R) at an MOI of 0.1 for 1 hour. Percentage live bacteria was calculated based on colony forming units (CFU) per ml obtained relative to vehicle-treated neutrophils. Data shown represent mean + SEM for 3 donors. *P<0.05.</p

CBG triggers IL-1β production and caspase-1 activation.

<p>DCs were stimulated with vehicle or CBG in the absence or presence of LPS (100 ng/ml) for 24 hours. (A) Supernatants were collected and analyzed for IL-1β production. Data shown represent means + SEM of IL-1β production for 17 donors. (B) Inflammasome activation was examined by analyzing the percentage of caspase-1 positive cells. Data shown represent means + SEM for 3 donors. (C) Whole-cell extracts were prepared, and equal amounts of lysates were analyzed by Western blot using an antibody against caspase-1. β-actin was used as a loading control. The results shown are representative of three independent experiments. (D) Cells were pretreated with the caspase-1 inhibitor Z-YVAD-FMK for 1 hour. Supernatants were collected at 24 hours and analyzed for IL-1β production. Data shown represent means + SEM of IL-1β production for 3 donors. **P<0.01. *P<0.05.</p

Inhibited cytokine release of LPS-stimulated DCs.

<p>DCs were stimulated with LPS (100 ng/ml) in the presence of vehicle or CBG for 24 hours. Supernatants were collected and analyzed for IL-6, IL-8, IL-12p40, TNF-α and IL-10. Data shown represent cytokine production for each donor and mean values for 6–8 donors. *P<0.05. **P<0.01.</p

Detection and diagnostics of tropical Meloidogyne spp. within the Euphresco project MeloTrop

Tropical root-knot nematodes (RKN), Meloidogyne spp., are considered as an emerging phytosanitary problem within Europe for the future. The Euphresco initiative entitled «Global warming and distribution of root-knot nematode species of the tropical group (MeloTrop)» was launched as a result of the potential damage these species can cause on economically important crops, especially under a climate change scenario. Six research partners joined forces in the consortium to reach the following objectives: to generate distribution maps of the tropical RKN in Slovenia, France, Portugal and Serbia; to assess the survival ability of M. incognita and M. arenaria in the continental European climate conditions; to validate biochemical and molecular methods for the diagnosis of tropical RKN, and to generate geographical maps of the possible open field spreading for each tropical RKN species occurring in Europe. Species identification within the group was based on a combination of morphological, morphometrical, biochemical and molecular methods. However, distinguishing tropical RKN species is very difficult due to inter-specific morphometrical similarity and intra-specific morphological and molecular variability. Esterase (Est, EC 3.1.1.1) and malate dehydrogenase (Mdh, EC 1.1.1.37) isozyme phenotyping as well as molecular identification approach based on multi locus sequencing of four mtDNA genes (nad2, nad5, cox2 and cox3) were validated within the project with inter-laboratory test performance studies. Additionally, several tropical RKN species were detected in partnering countries at the open field plant production. Open field RKN occurrence represents additional risk for several agricultural crops, especially due to predicted climate change effects and the fact that infestations at larger acreages are much more difficult to manage. The data of tropical RKN species occurrence will be presented and discussed