Comparison of bone marrow-derived and mucosal mast cells in controlling intramacrophage Francisella tularensis replication.

Although the importance of mast cells (MCs) in response to allergens has been characterized extensively, the contribution of these cells in host defense against bacterial pathogens is not well understood. Previously, we have demonstrated that the release of interleukin-4 by bone marrow-derived MCs inhibits intramacrophage replication of Francisella tularensis live vaccine strain (LVS). Because pneumonic tularemia is one of the several manifestations of infection by Francisella, it is important to determine whether MCs present in mucosal tissues, i.

An agonist of human complement fragment C5a enhances vaccine immunity against Coccidioides infection.

Coccidioides is a fungal pathogen and causative agent of a human respiratory disease against which no clinical vaccine exists. In this study we evaluated a novel vaccine adjuvant referred to as EP67, which is a peptide agonist of the biologically active C-terminal region of human complement component C5a. The EP67 peptide was conjugated to live spores of an attenuated vaccine strain (ΔT) of Coccidioides posadasii.

Mast cell TLR2 signaling is crucial for effective killing of Francisella tularensis.

TLR signaling is critical for early host defense against pathogens, but the contributions of mast cell TLR-mediated mechanisms and subsequent effector functions during pulmonary infection are largely unknown. We have previously demonstrated that mast cells, through the production of IL-4, effectively control Francisella tularensis replication. In this study, the highly human virulent strain of F.

Investigating the function of Ddr48p in Candida albicans.

Candidiasis now represents the fourth most frequent nosocomial infection both in the United States and worldwide. Candida albicans is an increasingly common threat to human health as a consequence of AIDS, steroid therapy, organ and tissue transplantation, cancer therapy, broad-spectrum antibiotics, and other immune defects. The pathogenic potential of C.

Perforin- and granzyme-mediated cytotoxic effector functions are essential for protection against Francisella tularensis following vaccination by the defined F. tularensis subsp. novicida ΔfopC vaccine strain.

A licensed vaccine against Francisella tularensis is currently not available. Two Francisella tularensis subsp. novicida (herein referred to by its earlier name, Francisella novicida) attenuated strains, the ΔiglB and ΔfopC strains, have previously been evaluated as potential vaccine candidates against pneumonic tularemia in experimental animals.

Gene disruption in Coccidioides using hygromycin or phleomycin resistance markers.

The following transformation protocol is based on homologous recombination that occurs between a gene disruption or gene replacement construct and a target gene of Coccidioides. The DNA constructs employed contain either the gene that encodes for hygromycin B or phleomycin resistance, which are present in the pAN7.1 or pAN8.

Absence of phagocyte NADPH oxidase 2 leads to severe inflammatory response in lungs of mice infected with Coccidioides.

Production of reactive oxygen species (ROS) resulting from phagocytic NADPH oxidase (NOX2) activity has been reported to contribute to host defense against numerous microbial pathogens. In this study we explored the role of NOX2 production in experimental coccidioidomycosis, a human respiratory disease caused by a soil-borne fungal pathogen. Activated and non-activated macrophages isolated from either NOX2(-/-) knock-out or wild type (WT) mice showed comparable ROS production and killing efficiency in vitro when infected with parasitic cells of Coccidioides.

N-terminal residues of the Vibrio cholerae virulence regulatory protein ToxT involved in dimerization and modulation by fatty acids.

The regulatory protein ToxT is an AraC family protein that is responsible for activating transcription of the genes encoding cholera toxin and toxin coregulated pilus, which are required for virulence by the human pathogen Vibrio cholerae. The N terminus of ToxT contains dimerization and regulatory elements, whereas the C terminus contains the DNA binding domain. Bile and long chain fatty acids negatively regulate ToxT activity.

Validation of the tetracycline regulatable gene expression system for the study of the pathogenesis of infectious disease.

Understanding the pathogenesis of infectious disease requires the examination and successful integration of parameters related to both microbial virulence and host responses. As a practical and powerful method to control microbial gene expression, including in vivo, the tetracycline-regulatable system has recently gained the favor of many investigative groups. However, some immunomodulatory effects of the tetracyclines, including doxycycline, could potentially limit its use to evaluate host responses during infection.

Oligopeptide permease A5 modulates vertebrate host-specific adaptation of Borrelia burgdorferi.

Borrelia burgdorferi, the agent of Lyme disease, undergoes rapid adaptive gene expression in response to signals unique to its arthropod vector or vertebrate hosts. Among the upregulated genes under vertebrate host conditions is one of the five annotated homologs of oligopeptide permease A (OppA5, BBA34). A mutant lacking oppA5 was constructed in an lp25-deficient isolate of B.

Non-FcεR bearing mast cells secrete sufficient interleukin-4 to control Francisella tularensis replication within macrophages.

Mast cells have classically been implicated in the triggering of allergic and anaphylactic reactions. However, recent findings have elucidated the ability of these cells to selectively release a variety of cytokines leading to bacterial clearance through neutrophil and dendritic cell mobilization, and suggest an important role in innate host defenses. Our laboratory has established a primary bone marrow derived mast cell-macrophage co-culture system and found that mast cells mediated a significant inhibition of Francisella tularensis live vaccine strain (LVS) uptake and replication within macrophages through contact and the secreted product interleukin-4 (IL-4).

Tumor necrosis factor alpha production from CD8+ T cells mediates oviduct pathological sequelae following primary genital Chlamydia muridarum infection.

The immunopathogenesis of Chlamydia trachomatis-induced oviduct pathological sequelae is not well understood. Mice genetically deficient in perforin (perforin(-/-) mice) or tumor necrosis factor alpha (TNF-α) production (TNF-α(-/-) mice) displayed comparable vaginal chlamydial clearance rates but significantly reduced oviduct pathology (hydrosalpinx) compared to that of wild-type mice. Since both perforin and TNF-α are effector mechanisms of CD8(+) T cells, we evaluated the role of CD8(+) T cells during genital Chlamydia muridarum infection and oviduct sequelae.

Effects of fluconazole, amphotericin B, and caspofungin on Candida albicans biofilms under conditions of flow and on biofilm dispersion.

We have examined the effect of continuous perfusion with antifungals on Candida albicans biofilms under conditions of flow, closely mimicking physiological conditions encountered within patients. Biofilms displayed high levels of resistance to fluconazole, and this antifungal exerted minor effects on dispersion levels. Amphotericin B proved effective in reducing viability of cells within the biofilms and dispersion, but only at high concentrations.

Nitric oxide synthase activity has limited influence on the control of Coccidioides infection in mice.

The functions of inducible nitric oxide synthase (iNOS) activity in protection against microbial insults are still controversial. In this study, we explored the role of iNOS in protection against Coccidioides infection in mice. We observed that wild type (WT) and iNOS(-/-) mice showed similar percent survival and fungal burden in their lungs at days 7 and 11 after intranasal challenge with Coccidioides.

TLR4-dependent activation of inflammatory cytokine response in macrophages by Francisella elongation factor Tu.

The bacterial determinants of pulmonary Francisella induced inflammatory responses and their interaction with host components are not clearly defined. In this study, proteomic and immunoblot analyses showed presence of a cytoplasmic protein elongation factor Tu (EF-Tu) in the membrane fractions of virulent Francisella novicida, LVS and SchuS4, but not in an attenuated F. novicida mutant.

Evasion of IFN-γ signaling by Francisella novicida is dependent upon Francisella outer membrane protein C.

Background: Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of the lethal disease tularemia. An outer membrane protein (FTT0918) of F. tularensis subsp.

Increased disease severity of parasite-infected TLR2-/- mice is correlated with decreased central nervous system inflammation and reduced numbers of cells with alternatively activated macrophage phenotypes in a murine model of neurocysticercosis.

In a murine model for neurocysticercosis (NCC), intracranial inoculation of the helminth parasite Mesocestoides corti induces multiple Toll-like receptors (TLRs), among which TLR2 is upregulated first and to a relatively high extent. Here, we report that TLR2(-/-) mice displayed significantly increased susceptibility to parasite infection accompanied by increased numbers of parasites in the brain parenchyma compared to infection in wild-type (WT) mice. This coincided with an increased display of microglial nodule formations and greater neuropathology than in the WT.

Tryptophan prototrophy contributes to Francisella tularensis evasion of gamma interferon-mediated host defense.

Francisella tularensis is able to survive and replicate within host macrophages, a trait that is associated with the high virulence of this bacterium. The trpAB genes encode the enzymes required for the final two steps in tryptophan biosynthesis, with TrpB being responsible for the conversion of indole to tryptophan. Consistent with this function, an F.

Features of sepsis caused by pulmonary infection with Francisella tularensis Type A strain.

The virulence mechanisms of Francisella tularensis, the causative agent of severe pneumonia in humans and a CDC category A bioterrorism agent, are not fully defined. As sepsis is the leading cause of mortality associated with respiratory infections, we determined whether, in the absence of any known bacterial toxins, a deregulated host response resulting in sepsis syndrome is associated with lethality of respiratory infection with the virulent human Type A strain SchuS4 of F. tularensis.

Candida albicans adhesin Als3p is dispensable for virulence in the mouse model of disseminated candidiasis.

The presence of specific proteins, including Ece1p, Hwp1p and Als3p, distinguishes the Candida albicans hyphal cell wall from that of yeast-form cells. These proteins are thought to be important for the ability of C. albicans cells to adhere to living and non-living surfaces and for the cell-to-cell adhesion necessary for biofilm formation, and also to be pivotal in mediating C.

The complexity of ToxT-dependent transcription in Vibrio cholerae.

Vibrio cholerae is the causative agent of the disease cholera, characterized by profuse watery diarrhoea. Two of the main virulence factors associated with the disease are cholera toxin (CT) and toxin-coregulated pilus (TCP). Expression of CT and TCP is regulated via a complex cascade of factors that respond to environmental signals, but ultimately ToxT is the direct transcriptional activator of the genes encoding CT and TCP.

Mannose-containing oligosaccharides of non-specific human secretory immunoglobulin A mediate inhibition of Vibrio cholerae biofilm formation.

The role of antigen-specific secretory IgA (SIgA) has been studied extensively, whereas there is a limited body of evidence regarding the contribution of non-specific SIgA to innate immune defenses against invading pathogens. In this study, we evaluated the effects of non-specific SIgA against infection with Vibrio cholerae O139 strain MO10 and biofilm formation. Seven day old infant mice deficient in IgA (IgA(-/-) mice) displayed significantly greater intestinal MO10 burden at 24 hr post-challenge when compared to IgA(+/+) pups.

Isolation of brain and spinal cord mononuclear cells using percoll gradients.

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue.

Vaccination with the defined chlamydial secreted protein CPAF induces robust protection against female infertility following repeated genital chlamydial challenge.

We previously have shown the efficacy of recombinant (r) chlamydial protease-like activity factor (CPAF) vaccination against hydrosalpinx development following primary genital chlamydial challenge. In this study, we evaluated further the protection induced by rCPAF vaccination against infertility. Following primary challenge, fertility levels were not significantly different between the mock- and CPAF-vaccinated and Chlamydia alone challenged mice.

Alpha-1 antitrypsin is markedly decreased following pulmonary F. tularensis challenge.

Neutrophils form the first line of defense during infection and are indispensable in this function. The neutrophil elastase is a key effector molecule of the innate immune system with potent antimicrobial activity against Gram-negative bacteria, spirochaetes, and fungi. However, the release of neutrophil elastase during bacterial infection must be checked otherwise its release in the extracellular milieu will result in damage to surrounding tissues.