bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms Involved in Apoptosis of Human Macrophages Induced by Lipopolysaccharide fromActinobacillus actinomycetemcomitansin the Presence of Cycloheximide
Authors: Suzuki Takao , Kobayashi Makoto , Isatsu Kyoko , Nishihara Tatsuji , Aiuchi Toshihiro , Nakaya Kazuyasu , Hasegawa Kohji
Published: March 2004

Actinobacillus actinomycetemcomitansis a major periodontopathic bacterium with multiple virulence factors, including lipopolysaccharide (LPS). Previous reports have demonstrated that LPS induced apoptosis in a murine macrophage-like cell line, J744.1, as well as in peritoneal macrophages from C3H/HeN mice in the presence of cycloheximide (CHX). However, the detailed molecular mechanisms involved in the apoptosis of macrophages induced by LPS and CHX are not well known. To clarify the possible role of LPS in the induction of macrophage apoptosis, we investigated cell death induced by LPS fromA. actinomycetemcomitansand CHX in human macrophage-like U937 cells, which were differentiated by 12-O-tetradecanoylphorbol 13-acetate (TPA), and also assessed the molecular mechanisms involved in the process. We found that TPA-differentiated U937 cells usually showed resistance to LPS-induced apoptosis. However, in the presence of CHX, LPS induced release of cytochromecwithout modifying steady-state levels of Bcl-2, Bcl-xL, Bax, and Bak. Treatment with LPS in the presence of CHX also led to activation of caspase-3 and apoptosis via, in part, the CD14/toll-like receptor 4 (TLR4). The induction of cytochromecrelease may have been due to dephosphorylation of Akt and Bad, which were cooperatively induced by CHX and LPS. However, endogenous tumor necrosis factor alpha- and Fas-induced signals, extracellular signal-regulated kinase kinase/mitogen-activated protein kinases and I-κBα/nuclear factor-κB (NF-κB) were not required for caspase-3-dependent apoptosis. These results emphasize the possible important role of the mitochondrial apoptotic pathway leading to caspase-3 activation in LPS-induced apoptosis of human macrophages in the presence of CHX.
N-Linked Glycosylation Is Required for C1 Inhibitor-Mediated Protection from Endotoxin Shock in Mice
Authors: Liu Dongxu , Gu Xiaogang , Scafidi Jennifer , Davis Alvin E.
Published: March 2004

C1 inhibitor (C1INH) prevents endotoxin shock in mice via a direct interaction with lipopolysaccharide (LPS). This interaction requires the heavily glycosylated amino-terminal domain of C1INH. C1INH in which N-linked carbohydrate was removed by usingN-glycosidase F was markedly less effective in protecting mice from LPS-induced lethal septic shock. N-deglycosylated C1INH also failed to suppress fluorescein isothiocyanate (FITC)-LPS binding to and LPS-induced tumor necrosis factor alpha mRNA expression by the murine macrophage-like cell line, RAW 264.7, and cells in human whole blood. In an enzyme linked immunosorbent assay, the N-deglycosylated C1INH bound to LPS very poorly. In addition, C1INH was shown to bind to diphosphoryl lipid A (dLPA) but only weakly to monophosphoryl lipid A (mLPA). As with intact LPS, binding of N-deglycosylated C1INH to dLPA and mLPA was diminished in comparison with the native protein. Removal of O-linked carbohydrate had no effect on any of these activities. Neither detoxified LPS, dLPA, nor mLPA had any effect on the rate or extent of C1INH complex formation with C1s or on cleavage of the reactive center loop by trypsin. These data demonstrate that N-linked glycosylation of C1INH is essential to mediate its interaction with the LPA moiety of LPS and to protect mice from endotoxin shock.
Involvement ofSalmonellaPathogenicity Island 2 in the Up-Regulation of Interleukin-10 Expression in Macrophages: Role of Protein Kinase A Signal Pathway
Authors: Uchiya Kei-ichi , Groisman Eduardo A. , Nikai Toshiaki
Published: March 2004

Salmonellae are facultative intracellular bacteria capable of surviving within macrophages.Salmonellapathogenicity island 2 (SPI-2) is required for growth within macrophages and for virulence in mice. In this study, we show the involvement of SPI-2 in a signal transduction pathway that induces cytokine expression inSalmonella-infected macrophages. High levels of interleukin-10 (IL-10) mRNA were induced in macrophages by infection with wild-type salmonellae compared to a strain carrying a mutation in thespiCgene, which is encoded within SPI-2. The two strains had the same effect on the expression of proinflammatory cytokines such as IL-1α, IL-6, and tumor necrosis factor alpha. IL-10 expression was dose dependently blocked by treatment of infected macrophages with the protein kinase A (PKA) inhibitor H-89, while IL-10 expression was increased by the PKA activator dibutyryl cyclic AMP. Cyclic AMP-dependent PKA activity was higher in macrophages infected with wild-type salmonellae compared to thespiCmutant, and Ser132phosphorylation of cyclic AMP response element-binding protein (CREB), which is an important mediator of PKA activation, correlated with the levels of PKA activity. Taken together, these results indicate that salmonellae cause an SPI-2-dependent increase in PKA activity that leads to CREB phosphorylation, resulting in up-regulation of IL-10 expression inSalmonella-infected macrophages. Suppression of IL-10 expression by an antisense oligonucleotide did not affect the growth of wild-type salmonellae within macrophages, whereas growth was dose dependently inhibited by H-89, suggesting that the PKA signaling pathway plays a significant role in intramacrophageSalmonellasurvival.
Effects of Gamma Interferon, Interleukin-10, and Transforming Growth Factor β on the Survival ofMycobacterium aviumsubsp.paratuberculosisin Monocyte-Derived Macrophages from Naturally Infected Cattle
Authors: Khalifeh M. S. , Stabel J. R.
Published: March 2004

Gamma interferon (IFN-γ) plays a significant role in the control of mycobacterial infections, includingMycobacterium aviumsubsp.paratuberculosis. However, the contribution of other immunoregulatory cytokines, such as interleukin-10 (IL-10) and transforming growth factor β (TGF-β), in Johne's disease has not been investigated as yet. In this study, we examined the effects of in vivo and in vitro infection withM. aviumsubsp.paratuberculosison the production of IFN-γ, IL-10, and TGF-β by peripheral blood mononuclear cells (PBMC). We also examined the effects of exogenous IFN-γ, IL-10, and TGF-β onM. aviumsubsp.paratuberculosissurvival in the cell cultures. PBMC obtained from naturally infected cows, regardless of their disease status, specifically upregulated IL-10 and TGF-β in culture supernatants in response to stimulation with liveM. aviumsubsp.paratuberculosis. Nonstimulated PBMC recovered from subclinically infected animals secreted the lowest levels of TGF-β, but after stimulation with liveM. aviumsubsp.paratuberculosis, TGF-β levels in the culture supernatants increased to levels similar to that produced by PBMC from healthy animals. The numbers of viableM. aviumsubsp.paratuberculosisrecovered from cultures from naturally infected animals were higher than those from healthy cows after in vitro infection withM. aviumsubsp.paratuberculosis. The addition of exogenous IL-10 and TGF-β to PBMC isolated from healthy cows inhibited the bactericidal activity of these cells as evidenced by the increased number of viableM. aviumsubsp.paratuberculosisrecovered from these cultures compared to cell cultures containing medium alone. These data suggest important immune regulatory roles for IL-10 and TGF-β during infection withM. aviumsubsp.paratuberculosisthat may be directly related to their effects on macrophage activation and killing ofM. aviumsubsp.paratuberculosis.
Dendritic Cell Populations inLeishmania major-Infected Skin and Draining Lymph Nodes
Authors: Baldwin Tracey , Henri Sandrine , Curtis Joan , O'Keeffe Meredith , Vremec David , Shortman Ken , Handman Emanuela
Published: March 2004

Using a metacyclic promastigote ear infection model of cutaneous leishmaniasis, we examined the phenotype, parasite load, and cytokine production of dendritic cells in the skin and draining lymph nodes of resistant C57BL/6J and susceptible BALB/c mice. Five dendritic cell populations were isolated from the skin and lymph nodes, and the main difference between the groups of mice was an increased number of plasmacytoid dendritic cells in the lymph nodes of the susceptible mice. Although similar cell types were present in the skin emigrants of both strains, there was a 10-fold larger number of cells in BALB/c mouse skin early in infection than in C57BL/6J mouse skin. None of the dendritic cells in the lymph nodes harbored parasites until 3 weeks after infection, with the Langerhans cells having the largest load and the plasmacytoid dendritic cells having the smallest load but the longest lasting infection. Although parasites could be detected in the lymph nodes a few hours after infection, none of the skin emigrants harbored parasites, indicating that they are not the vehicle that ferries the parasites from the skin to the lymph nodes. The presence of larger numbers of plasmacytoid cells in infected BALB/c mice, the more protracted infection of these cells, and their production of alpha interferon point to a complex and important role for the plasmacytoid cells in leishmaniasis.
Induction of Protective Cellular Immunity againstMycobacterium tuberculosisby Recombinant Attenuated Self-DestructingListeria monocytogenesStrains Harboring Eukaryotic Expression Plasmids for Antigen 85 Complex and MPB/MPT51
Authors: Miki Keita , Nagata Toshi , Tanaka Takao , Kim Yeung-Hyen , Uchijima Masato , Ohara Naoya , Nakamura Satoshi , Okada Masaji , Koide Yukio
Published: March 2004

We report here the induction of specific protective cellular immunity againstMycobacterium tuberculosisby the employment of vaccination with recombinant attenuatedListeria monocytogenesstrains. We constructed self-destructing attenuatedL. monocytogenesΔ2 strains carrying eukaryotic expression plasmids for the antigen 85 complex (Ag85A and Ag85B) and for MPB/MPT51 (mycobacterial protein secreted byM. bovisBCG/mycobacterial protein secreted byM. tuberculosis) molecules. Infection of these recombinant bacteria allowed expression of the genes in the J774A.1 murine macrophage cell line. Intraperitoneal vaccination of C57BL/6 mice with these recombinant bacteria was capable of inducing purified protein derivative-specific cellular immune responses, such as foot pad reactions, proliferative responses of splenocytes, and gamma interferon production from splenocytes, suggesting the efficacy of vaccination against mycobacterial infection by use of these recombinantL. monocytogenesstrains. Furthermore, intravenous vaccination with recombinant bacteria carrying expression plasmids for Ag85A, Ag85B, or MPB/MPT51 in BALB/c mice elicited significant protective responses, comparable to those evoked by a liveMycobacterium bovisBCG vaccine. Notably, this is the first report to show that MPB/MPT51 is a major protective antigen in addition to Ag85A and Ag85B, which have been reported to be major mycobacterial protective antigens.
The Nature of Extracellular Iron Influences Iron Acquisition byMycobacterium tuberculosisResiding within Human Macrophages
Authors: Olakanmi Oyebode , Schlesinger Larry S. , Ahmed Ambar , Britigan Bradley E.
Published: March 2004

We have reported thatMycobacterium tuberculosisresiding within the phagosomes of human monocyte-derived macrophages (MDM) can acquire Fe from extracellular transferrin (TF) and sources within the MDM. In the lung, Fe is also bound to lactoferrin (LF) and low-molecular-weight chelates. We therefore investigated the ability of intraphagosomalM. tuberculosisto acquire Fe from these sources.M. tuberculosisacquired 30-fold and 3-fold more Fe from LF and citrate, respectively, compared to TF, in spite of similar MDM-associated Fe.M. tuberculosisinfection decreased MDM-associated Fe relative to uninfected MDM as follows: TF (38.7%), citrate (21.1%), and LF (15.3%).M. tuberculosisFe acquisition from extracellular chelates (exogenous source) and from endogenous MDM Fe initially acquired from the three chelates (endogenous source) was compared.M. tuberculosisFe acquisition was similar from exogenous and endogenous sources supplied as Fe-TF. In contrast, there was much greater intracellularM. tuberculosisFe uptake from LF and citrate from the exogenous than endogenous source. Gamma interferon (IFN-γ) reduced MDM Fe uptake from each chelate by ∼50% and augmented theM. tuberculosis-induced decrease in MDM Fe uptake from exogenous TF, but not from LF or citrate. IFN-γ minimally decreased intracellularM. tuberculosisFe acquisition from exogenous Fe-TF but significantly increased Fe uptake from LF and citrate. IntraphagosomalM. tuberculosisFe acquisition from both exogenous and endogenous MDM sources, and the effect of IFN-γ on this process, is influenced by the nature of the extracellular Fe chelate.M. tuberculosishas developed efficient mechanisms of acquiring Fe from a variety of Fe chelates that it likely encounters within the human lung.
Degenerate Peptide Recognition by
Candida albicans
Adhesins Als5p and Als1p
Authors: Klotz Stephen A. , Gaur Nand K. , Lake Douglas F. , Chan Vincent , Rauceo Jason , Lipke Peter N.
Published: March 2004

Candida albicans
and
Saccharomyces cerevisiae
expressing the adhesins Als5p or Als1p adhere to immobilized peptides and proteins that possess appropriate sequences of amino acids in addition to a sterically accessible peptide backbone. In an attempt to further define the nature of these targets, we surveyed the ability of yeast cells to adhere to 90-μm-diameter polyethylene glycol beads coated with a 7-mer peptide from a library of 19
7
unique peptide-beads.
C. albicans
bound to ca. 10% of beads from the library, whereas
S. cerevisiae
expressing Als5p or Als1p bound to ca. 0.1 to 1% of randomly selected peptide-beads.
S. cerevisiae
expressing Als1p had a distinctly different adherence phenotype than did cells expressing Als5p. The former adhered in groups or clumps of cells, whereas the latter adhered initially as single cells, an event which was followed by the build up of cell-cell aggregates. Beads with adherent cells were removed, and the peptide attached to the bead was determined by amino acid sequencing. All adhesive beads carried a three-amino-acid sequence motif (τφ+) that possessed a vast combinatorial potential. Adherence was sequence specific and was inhibited when soluble peptide identical to the immobilized peptide was added. The Als5p adhesin recognized some peptides that went unrecognized by Als1p. The sequence motif of adhesive peptides identified by this method is common in proteins and offers so many possible sequence combinations that target recognition by the Als proteins is clearly degenerate. A degenerate recognition system provides the fungi with the potential of adhering to a multitude of proteins and peptides, an advantage for any microorganism attempting to establish a commensal or pathogenic relationship with a host.
Whole-Genome DNA Array Analysis of the Response of
Borrelia burgdorferi
to a Bactericidal Monoclonal Antibody
Authors: Anderton Julie M. , Tokarz Rafal , Thill Charles D. , Kuhlow Christopher J. , Brooks Chad S. , Akins Darrin R. , Katona Laura I. , Benach Jorge L.
Published: March 2004

Identification and characterization of genes that contribute to infection with
Borrelia burgdorferi
and, of those, genes that are targets of host responses is important for understanding the pathogenesis of Lyme disease. The complement-independent bactericidal monoclonal antibody (MAb) CB2 recognizes a carboxy-terminal, hydrophilic epitope of the outer surface protein B (OspB). CB2 kills
B. burgdorferi
by an unknown bactericidal mechanism. Upon binding of CB2 to OspB, differentially expressed gene products may be responsible for, or associated with, the death of the organism. A time course of the response of
B. burgdorferi
to CB2 was completed to analyze the differential gene expression in the bacteria over a period of visual morphological changes. Bacteria were treated with a sublethal concentration in which spirochetes were visibly distressed by the antibody but not lysed. Preliminary whole-genome DNA arrays at various time points within 1 h of incubation of
B. burgdorferi
with the antibody showed that most significant changes occurred at 25 min. Circular plasmid 32 (cp32)-encoded genes were active in this period of time, including the
blyA
homologs, phage holin system genes. DNA array data show that three
blyA
homologs were upregulated significantly, ≥2 standard deviations from the mean of the log ratios, and a
P
value of ≤0.01. Quantitative real-time PCR analysis verified
blyA
and
blyB
upregulation over an 18- to 35-min time course. The hypothesis to test is whether the killing mechanism of CB2 is through uncontrolled expression of the
blyA
and
blyB
phage holin system.
Intraspleen Delivery of a DNA Vaccine Coding for Superoxide Dismutase (SOD) ofBrucella abortusInduces SOD-Specific CD4+and CD8+T Cells
Authors: Muñoz-Montesino Carola , Andrews Edilia , Rivers Rodolfo , González-Smith Andrés , Moraga-Cid Gustavo , Folch Hugo , Céspedes Sandra , Oñate Angel A.
Published: March 2004

In the development of vaccines capable of providing immunity against brucellosis, Cu-Zn superoxide dismutase (SOD) has been demonstrated to be one of the protective immunogens ofBrucella abortus. In an earlier study, we provided strong evidence that intramuscular injection with a plasmid DNA carrying the SOD gene (pcDNA-SOD) was able to induce a protective immune response. The present study was designed to characterize T-cell immune responses after an intraspleen (i.s.) vaccination of BALB/c mice with pcDNA-SOD. Animals vaccinated with pcDNA-SOD did not develop SOD-specific antibodies, at least until week 4 after immunization (the end of the experiment), and in vitro stimulation of their splenocytes with either recombinant Cu-Zn SOD or crudeBrucellaprotein induced the secretion of gamma interferon (IFN-γ), but not interleukin-4, and elicited the induction of cytotoxic-T-lymphocyte activity. Upon analyzing the SOD-specific T-cell responses, the pcDNA-SOD vaccination was found to be stimulating both CD4+- and CD8+-T-cell populations. However, only the CD4+population was able to produce IFN-γ and only the CD8+population was able to induce cytotoxic activity. Nevertheless, although i.s. route vaccination induces a significant level of protection in BALB/c mice against challenge with the virulentB. abortusstrain 2308, vaccination by the intramuscular route with a similar amount of plasmid DNA does not protect. Based on these results, we conclude that i.s. immunization with pcDNA-SOD vaccine efficiently induced a Th1 type of immune response and a protective response that could be related to IFN-γ production and cytotoxic activity against infected cells by SOD-specific CD4+and CD8+T cells, respectively.
Inhibition of Major Histocompatibility Complex II Expression and Antigen Processing in Murine Alveolar Macrophages byMycobacterium bovisBCG and the 19-Kilodalton Mycobacterial Lipoprotein
Authors: Fulton Scott A. , Reba Scott M. , Pai Rish K. , Pennini Meghan , Torres Martha , Harding Clifford V. , Boom W. Henry
Published: March 2004

Alveolar macrophages constitute a primary defense againstMycobacterium tuberculosis, but they are unable to controlM. tuberculosiswithout acquired T-cell immunity. This study determined the antigen-presenting cell function of murine alveolar macrophages and the ability of the model mycobacterium,Mycobacterium bovisBCG, to modulate it. The majority (80 to 85%) of alveolar macrophages expressed both CD80 (B7.1) and CD11c, and 20 to 30% coexpressed major histocompatibility complex II (MHC-II). Gamma interferon (IFN-γ) enhanced MHC-II but not B7.1 expression. Naive or IFN-γ-treated alveolar macrophages did not express CD86 (B7.2), CD11b, Mac-3, CD40, or F4/80.M. bovisBCG and the 19-kDa mycobacterial lipoprotein inhibited IFN-γ-regulated MHC-II expression on alveolar macrophages, and inhibition was dependent on Toll-like receptor 2. The inhibition of MHC-II expression by the 19-kDa lipoprotein was associated with decreased presentation of soluble antigen to T cells. Thus, susceptibility to tuberculosis may result from the ability of mycobacteria to interfere with MHC-II expression and antigen presentation by alveolar macrophages.
Novel Program of Macrophage Gene Expression Induced by Phagocytosis ofLeishmania chagasi
Authors: Rodriguez Nilda E. , Chang Haeok K. , Wilson Mary E.
Published: March 2004

Leishmaniaspp. are protozoans that survive and replicate intracellularly in mammalian macrophages. Antileishmanial immunity requires gamma interferon (IFN-γ)-mediated macrophage activation and generation of microbicidal effector molecules. The presence of intracellularLeishmaniasp. impairs macrophage responses to IFN-γ, which has led to the description of macrophages as deactivated. It has recently become apparent that in addition to classical activation, macrophages can be activated by distinct triggers to express noninflammatory or anti-inflammatory genes. These nonclassical activation programs have been called alternative or type II pathways. We hypothesized that during initial contact with a phagocyte, leishmaniae activate one of these nonclassical pathways, resulting in expression of genes whose products suppress microbicidal responses. Using DNA microarrays, we studied gene expression in RNAs from BALB/c bone marrow macrophages with and withoutLeishmania chagasiinfection. Some changes were verified by an RNase protection assay, reverse transcription-PCR, immunoblotting, or a bioassay. The pattern of genes activated by leishmania phagocytosis differed from the pattern of genes activated by bacteria or lipopolysaccharide and IFN-γ. Genes encoding some proinflammatory cytokines, receptors, and Th1-type immune response genes were down-modulated, and some genes associated with anti-inflammatory or Th2-like immune responses were up-regulated. Nonetheless, some markers of alternative (arginase) or type II activation (interleukin-10, tumor necrosis factor alpha) were unchanged. These data suggest that macrophages infected withL. chagasiexhibit a hybrid activation profile that is more characteristic of alternative or type II activation than of classical activation but does not strictly fall into either of these categories. We speculate that the pattern of genes upregulated by leishmania phagocytosis optimizes the chance of parasite survival in this hostile environment.
Cell Surface-Associated Elongation Factor Tu Mediates the Attachment of
Lactobacillus johnsonii
NCC533 (La1) to Human Intestinal Cells and Mucins
Authors: Granato Dominique , Bergonzelli Gabriela E. , Pridmore Raymond David , Marvin Laure , Rouvet Martine , Corthésy-Theulaz Irène E.
Published: March 2004

The aim of this work was to identify
Lactobacillus johnsonii
NCC533 (La1) surface molecules mediating attachment to intestinal epithelial cells and mucins. Incubation of Caco-2 intestinal epithelial cells with an
L. johnsonii
La1 cell wall extract led to the recognition of elongation factor Tu (EF-Tu) as a novel La1 adhesin-like factor. The presence of EF-Tu at the surface of La1 was confirmed by analysis of purified outer surface protein extract by immunoblotting experiments, by electron microscopy, and by enzyme-linked immunosorbent assays of live bacteria. Furthermore, tandem mass spectrometry analysis proved that EF-TU was expressed at the La1 surface as an intact molecule. Using recombinant La1 EF-Tu protein, we were able to determine that its binding to intestinal cells and to mucins is pH dependent. Competition experiments suggested that EF-Tu has an important role in La1 mucin binding capacity. In addition, immunomodulation studies performed on HT29 cells showed that EF-Tu recombinant protein can induce a proinflammatory response in the presence of soluble CD14. Our in vitro results indicate that EF-Tu, through its binding to the intestinal mucosa, might participate in gut homeostasis.
Purification and Molecular Cloning of and Immunization with
Ancylostoma ceylanicum
Excretory-Secretory Protein 2, an Immunoreactive Protein Produced by Adult Hookworms
Authors: Bungiro Richard D. , Solis Carolina V. , Harrison Lisa M. , Cappello Michael
Published: March 2004

Hookworms remain major agents of global morbidity, and vaccination against these bloodfeeding parasites may be an attractive complement to conventional control methods. Here we describe the cloning of
Ancylostoma ceylanicum
excretory-secretory protein 2 (AceES-2), a novel immunoreactive protein produced by adult worms. Native AceES-2 was purified from excretory-secretory (ES) products by reverse-phase high-pressure liquid chromatography, subjected to amino-terminal sequencing, and cloned from adult worm RNA by using reverse transcription-PCR. The translated AceES-2 cDNA predicts that the mature protein consists of 102 amino acids and has a molecular mass of 11.66 kDa. Western immunoblot and enzyme-linked immunosorbent assay analyses demonstrated that recombinant AceES-2 (rAceES-2) reacted strongly with antibodies from
A. ceylanicum
-infected hamsters. Immunization of hamsters with native ES products adsorbed to alum induced antibodies that recognized rAceES-2, while rAceES-2-alum vaccination resulted in antibodies that reacted with a single protein band in ES products that closely approximated the size predicted for the native molecule. Infected hamsters that were passively immunized with hyperimmune rabbit anti-rAceES-2 serum exhibited more rapid and complete recovery from anemia than controls that received normal serum. Oral immunization with rAceES-2 was associated with significantly reduced anemia upon challenge, an outcome similar to the outcome observed in hamsters that were orally vaccinated with soluble hookworm extract (the latter animals were also resistant to weight loss). These data suggest that AceES-2 plays an important role in the host-parasite interaction and that vaccination against this protein may represent a useful strategy for controlling hookworm anemia.
Molecular Characterization ofAncylostoma ceylanicumKunitz-Type Serine Protease Inhibitor: Evidence for a Role in Hookworm-Associated Growth Delay
Authors: Chu Daniel , Bungiro Richard D. , Ibanez Maureen , Harrison Lisa M. , Campodonico Eva , Jones Brian F. , Mieszczanek Juliusz , Kuzmic Petr , Cappello Michael
Published: March 2004

Hookworm infection is a major cause of iron deficiency anemia and malnutrition in developing countries. TheAncylostoma ceylanicumKunitz-type inhibitor (AceKI) is a 7.9-kDa broad-spectrum inhibitor of trypsin, chymotrypsin, and pancreatic elastase that has previously been isolated from adult hookworms. Site-directed mutagenesis of the predicted P1 inhibitory reactive site amino acid confirmed the role of Met26in mediating inhibition of the three target serine proteases. By using reverse transcription-PCR, it was demonstrated that the level of AceKI gene expression increased following activation of third-stage larvae with serum and that the highest level of expression was reached in the adult stage of the parasite. Immunohistochemistry studies performed with polyclonal immunoglobulin G raised against recombinant AceKI showed that the inhibitor localized to the subcuticle of the adult hookworm, suggesting that it has a potential in vivo role in neutralizing intestinal proteases at the surface of the parasite. Immunization with recombinant AceKI was shown to confer partial protection against hookworm-associated growth delay without a measurable effect on anemia. Taken together, the data suggest that AceKI plays a role in the pathogenesis of hookworm-associated malnutrition and growth delay, perhaps through inhibition of nutrient absorption in infected hosts.
Role of Extracellular Phospholipases and Mononuclear Phagocytes in Dissemination of Cryptococcosis in a Murine Model
Authors: Santangelo Rosemary , Zoellner Hans , Sorrell Tania , Wilson Christabel , Donald Christine , Djordjevic Julianne , Shounan Yi , Wright Lesley
Published: March 2004

Secreted phospholipase B (PLB) activity promotes the survival and replication of
Cryptococcus neoformans
in macrophages in vitro. We therefore investigated the role of mononuclear phagocytes and cryptococcal PLB in the dissemination of infection in a mouse model, using
C. neoformans
var.
grubii
wild-type strain H99, a
PLB1
deletion mutant (Δ
plb1
), and a reconstituted strain (Δ

plb1
rec

). PLB facilitated the entry of endotracheally administered cryptococci into lung IM. PLB was also required for lymphatic spread from the lung to regional lymph nodes and for entry into the blood. Langhans-type giant cells containing budding cryptococci were seen free in the lymphatic sinuses of hilar nodes of H99- and Δ

plb1
rec

-infected mice, suggesting that they may have a role in the dissemination of cryptococcal infection. The transfer of infected lung macrophages to recipient mice by tail vein injections demonstrated that these cells can facilitate hematogenous dissemination of cryptococci to the brain, independent of cryptococcal PLB secretion. PLB activities of cryptococci isolated from lung macrophages or infected brains were not persistently increased. We conclude that mononuclear phagocytes are a vehicle for cryptococcal dissemination and that PLB activity is necessary for the initiation of interstitial pulmonary infections and for dissemination from the lung via the lymphatics and blood. PLB is not, however, essential for the establishment of neurological infections when cryptococci are presented within, or after passage through, mononuclear phagocytes.
Probiotic Bifidobacteria Protect Mice from Lethal Infection with Shiga Toxin-Producing
Escherichia coli
O157:H7
Authors: Asahara Takashi , Shimizu Kensuke , Nomoto Koji , Hamabata Takashi , Ozawa Ayako , Takeda Yoshifumi
Published: March 2004

The anti-infectious activity of probiotic Bifidobacteria against Shiga toxin-producing
Escherichia coli
(STEC) O157:H7 was examined in a fatal mouse STEC infection model. Stable colonization of the murine intestines was achieved by the oral administration of
Bifidobacterium breve
strain Yakult (naturally resistant to streptomycin sulfate) as long as the mice were treated with streptomycin in their drinking water (5 mg/ml). The pathogenicity of STEC infection, characterized by marked body weight loss and subsequent death, observed in the infected controls was dramatically inhibited in the
B. breve
-colonized group. Moreover, Stx production by STEC cells in the intestine was almost completely inhibited in the
B. breve
-colonized group. A comparison of anti-STEC activity among several
Bifidobacterium
strains with natural resistance to streptomycin revealed that strains such as
Bifidobacterium bifidum
ATCC 15696 and
Bifidobacterium catenulatum
ATCC 27539
T
did not confer an anti-infectious activity, despite achieving high population levels similar to those of effective strains, such as
B. breve
strain Yakult and
Bifidobacterium pseudocatenulatum
DSM 20439. The effective strains produced a high concentration of acetic acid (56 mM) and lowered the pH of the intestine (to pH 6.75) compared to the infected control group (acetic acid concentration, 28 mM; pH, 7.15); these effects were thought to be related to the anti-infectious activity of these strains because the combination of a high concentration of acetic acid and a low pH was found to inhibit Stx production during STEC growth in vitro.
Impairment of IntramacrophagicBrucella suisMultiplication by Human Natural Killer Cells through a Contact-Dependent Mechanism
Authors: Dornand Jacques , Lafont Virginie , Oliaro Jane , Terraza Annie , Castaneda-Roldan Elsa , Liautard Jean-Pierre
Published: March 2004

Brucellaspp. are facultative intracellular bacteria that can establish themselves and cause chronic disease in humans and animals. NK cells play a key role in host defense. They are implicated in an early immune response to a variety of pathogens. However, it was shown that they do not controlBrucellainfection in mice. On the other hand, NK cell activity is impaired in patients with acute brucellosis, and recently it was demonstrated that human NK cells mediate the killing of intramacrophagicMycobacterium tuberculosisin in vitro infection. Therefore, we have analyzed the behavior ofBrucella suisinfecting isolated human macrophages in the presence of syngeneic NK cells. We show that (i) NK cells impair the intramacrophagic development ofB. suis, a phenomenon enhanced by NK cell activators, such as interleukin-2; (ii) NK cells cultured in the presence of infected macrophages are highly activated and secrete gamma interferon and tumor necrosis factor alpha; (iii) impairment of bacterial multiplication inside infected cells is marginally associated with the cytokines produced during the early phase of macrophage-NK cell cocultures; (iv) direct cell-to-cell contact is required for NK cells to mediate the inhibition ofB. suisdevelopment; and (v) inhibition ofB. suisdevelopment results from an induction of NK cell cytotoxicity against infected macrophages. Altogether, these findings show that NK cells could participate early in controlling the intramacrophagic development ofB. suisin humans. It seems thus reasonable to hypothesize a role for NK cells in the control of human brucellosis. However, by impairing the activity of these cells in the acute phase of the illness, the pathogen should avoid this control.
Role for Moesin in Lipopolysaccharide-Stimulated Signal Transduction
Authors: Iontcheva Iveta , Amar Salomon , Zawawi Khalid H. , Kantarci Alpdogan , Van Dyke Thomas E.
Published: March 2004

Moesin is a 78-kDa protein with diverse functions in linking the cytoskeleton to the membrane while controlling cell shape, adhesion, locomotion, and signaling. The aim of this study was to characterize the expression and localization of moesin in mononuclear phagocytes by using confocal microscopy, flow cytometry, immunoprecipitation, and Western blotting and to analyze the function of moesin as a lipopolysaccharide receptor, utilizing an antisense oligonucleotide approach to knock down the moesin gene. Results revealed that moesin is expressed on the surface of monocytes/macrophages and surface expression is increased after lipopolysaccharide stimulation. The total protein mass of moesin is increased in monocytes after lipopolysaccharide stimulation. Immunoprecipitation showed that moesin coprecipitates with TLR4, a well-known lipopolysaccharide receptor, suggesting an early role of moesin in the formation of the initiation complex for lipopolysaccharide signaling. Two antisense and two control sense oligonucleotides were synthesized and introduced every 4 h for 48 h in adherent macrophage-like cells. Cells were then stimulated with lipopolysaccharide for 4 h, and the supernatants were assayed for tumor necrosis factor alpha (TNF-α) production. Cell lysates were assayed for moesin expression by Western blotting immediately after the 48-h treatment period and also after 116 h of recovery to assess the return of moesin expression and function. Moesin gene expression was completely suppressed after 48 h of incubation with antisense oligonucleotides. The antisense elimination of moesin gene expression led to a significant reduction of lipopolysaccharide-induced TNF-α secretion. Restoration of moesin gene expression led to restoration of TNF-α production. These data suggest an important role for moesin in lipopolysaccharide-induced TNF-α production, highlighting its importance in lipopolysaccharide-mediated signal transduction.
Plasmodium falciparum
Merozoite Surface Protein 6 Is a Dimorphic Antigen
Authors: Pearce J. Andrew , Triglia Tony , Hodder Anthony N. , Jackson David C. , Cowman Alan F. , Anders Robin F.
Published: March 2004

Merozoite surface protein 1 (MSP1) is a highly polymorphic
Plasmodium falciparum
merozoite surface protein implicated in the invasion of human erythrocytes during the asexual cycle. It forms a complex with MSP6 and MSP7 on the merozoite surface, and this complex is released from the parasite around the time of erythrocyte invasion. MSP1 and many other merozoite surface proteins contain dimorphic elements in their protein structures, and here we show that MSP6 is also dimorphic. The sequences of eight
MSP6
genes indicate that the alleles of each dimorphic form of MSP6 are highly conserved. The smaller 3D7-type MSP6 alleles are detected in parasites from all malarious regions of the world, whereas K1-type MSP6 alleles have only been detected in parasites from mainland Southeast Asia. Cleavage of MSP6, which produces the p36 fragment in 3D7-type MSP6 and associates with MSP1, also occurs in K1-type MSP6 but at a different site in the protein. Anti-3D7 MSP6 antibodies weakly inhibited erythrocyte invasion by homologous 3D7 merozoites but did not inhibit a parasite line expressing the K1-type MSP6 allele. Antibodies from hyperimmune individuals affinity purified on an MSP3 peptide cross-reacted with MSP6; therefore, MSP6 may also be a target of antibody-dependent cellular inhibition.
Modulation of Enteropathogenic
Escherichia coli
Virulence by Quorum Sensing
Authors: Sircili Marcelo P. , Walters Matthew , Trabulsi Luis R. , Sperandio Vanessa
Published: March 2004

Enteropathogenic
Escherichia coli
(EPEC) produces a lesion on epithelial cells called the attaching and effacing (AE) lesion. All genes necessary for AE are encoded within the locus of enterocyte effacement (LEE). EPEC also adheres in a characteristic pattern to epithelial cells by forming microcolonies, usually referred to as localized adherence (LA). LA is mediated by the bundle-forming pilus and flagella. The LEE genes are directly activated by the LEE-encoded regulator (Ler). Transcription of Ler is under the control of Per, integration host factor, Fis, BipA, and quorum sensing (QS), specifically through the
luxS
system. QS activates expression of the LEE genes in EPEC, with QseA activating transcription of
ler
. Here we report that transcription of the LEE genes and type III secretion are diminished in both
luxS
and
qseA
mutants. Transcription of the LEE genes is affected in both mutants mostly during the mid-exponential phase of growth. Transcription of
qseA
itself is diminished throughout growth in a
luxS
mutant and is under autorepression. Furthermore, QS activation of type III secretion is independent of
per
, given that QseA still activates type III secretion in a
per
mutant strain. Both mutants are deficient in adherence to epithelial cells and form smaller microcolonies. Several factors may contribute to this abnormal behavior: transcription of LEE genes and type III secretion are diminished, and expression of flagella and Per is altered in both mutants. These results suggest that QS is involved in modulating the regulation of the EPEC virulence genes.
The Hydrophobic Domain of the Mycobacterial Erp Protein Is Not Essential for the Virulence of
Mycobacterium tuberculosis
Authors: Kocíncová Dana , Sondén Berit , Bordat Yann , Pivert Elisabeth , de Mendonça-Lima Leila , Gicquel Brigitte , Reyrat Jean-Marc
Published: March 2004

Erp (exported repetitive protein) is a member of a mycobacterium-specific family of extracellular proteins. A hydrophobic region that is localized at the C-terminal domain and that represents a quarter of the protein is highly conserved across species. Here we show that this hydrophobic region is not essential for restoring the virulence and tissue damage of an
erp
::
aph
mutant strain of
M. tuberculosis
as assessed by bacterial counts and lung histology analysis in a mouse model of tuberculosis.
Helicobacter pylori-Mediated Gastritis Induces Local Downregulation of Secretory Leukocyte Protease Inhibitor in the Antrum
Authors: Wex Thomas , Treiber Gerhard , Nilius Manfred , Vieth Michael , Roessner Albert , Malfertheiner Peter
Published: March 2004

Helicobacter pylori-infected subjects exhibited a strong decline in antral secretory leukocyte protease inhibitor (SLPI) levels compared toH. pylori-negative subjects and subjects from whomH. pylorihad been eradicated (P= 0.002). This reduction was specific for the antrum, whereas SLPI expression in corpus and duodenum was not affected. Antral SLPI levels were inversely correlated with inflammatory scores of antrum-predominant gastritis.
SKN7
of
Candida albicans
: Mutant Construction and Phenotype Analysis
Authors: Singh Praveen , Chauhan Neeraj , Ghosh Anup , Dixon Freddie , Calderone Richard
Published: March 2004

The
SKN7
two-component response regulator gene of
Candida albicans
was deleted, and the phenotype of the mutant was established. This mutant exhibited impaired growth on Spider agar and 10% serum agar compared to wild-type and gene-reconstituted strains. The
skn7
mutant was sensitive to H
2
O
2
in vitro, but its virulence was only mildly attenuated. A comparison of the Skn7p and Ssk1p response regulators of
C. albicans
is discussed.
Genetic Control of Natural Resistance of Mouse Macrophages to
Coxiella burnetii
Infection In Vitro: Macrophages from Restrictive Strains Control Parasitophorous Vacuole Maturation
Authors: Zamboni Dario S.
Published: March 2004

Macrophages from A/J and BALB/c mice were more susceptible to
Coxiella burnetii
phase II infection than were those from C57BL/6, C57BL/10, B10.A, C3H/HePas, and Swiss mice. Resistant macrophages effectively controlled the development of large replication vacuoles (LRVs), which accounted for the restriction of bacterial multiplication within the cultures. However, compared to fibroblasts, all macrophages controlled bacterial multiplication within LRVs.
Effect of Growth State on Transcription Levels of Genes Encoding Major Secreted Antigens of
Mycobacterium tuberculosis
in the Mouse Lung
Authors: Shi Lanbo , North Robert , Gennaro Maria Laura
Published: March 2004

Arrest of the multiplication of
Mycobacterium tuberculosis
caused by expression of adaptive immunity in mouse lung was accompanied by a 10- to 20-fold decrease in levels of mRNAs encoding the secreted Ag85 complex and 38-kDa lipoprotein.
esat-6
mRNA levels were high throughout infection. The data imply that multiplying and nonreplicating tubercle bacilli have different antigen compositions.
Different Responses of Macrophages to Smooth and RoughBrucellaspp.: Relationship to Virulence
Authors: Jiménez de Bagüés María P. , Terraza Annie , Gross Antoine , Dornand Jacques
Published: March 2004

By comparing smooth wild-typeBrucellastrains to their rough mutants, we show that the lipopolysaccharide (LPS) O side chain of pathogenicBrucellahas a dramatic impact on macrophage activation. It favors the development of virulentBrucellaby preventing the synthesis of immune mediators, important for host defense. We conclude that this O chain property is firmly linked toBrucellavirulence.
Enterococcal Aggregation Substance and Binding Substance Are Not Major Contributors to Urinary Tract Colonization by
Enterococcus faecalis
in a Mouse Model of Ascending Unobstructed Urinary Tract Infection
Authors: Johnson James R. , Clabots Connie , Hirt Helmut , Waters Christopher , Dunny Gary
Published: March 2004

Isogenic
Enterococcus faecalis
strains that differ in their expression of aggregation substance (AS) and its cognate receptor, enterococcal binding substance (EBS), were compared for urovirulence in mice. Strain OG1SSp/pCF500 (inducible AS
+
, constitutive EBS
+
) failed to outcompete isogenic derivative INY3000 (AS
−
EBS
−
) in the urine, bladders, or kidneys of mice harvested at 48 h postinoculation. Neither mouse nor human urine induced AS expression by OG1SSp/pCF500. Recombinant strain OG1SSp/pINY1801 (constitutive AS
+
, EBS
+
) exhibited plasmid segregation that was as extensive in vivo as in vitro. These data suggest that AS and EBS do not contribute to upper or lower urinary tract colonization by
E. faecalis
and that growth in urine does not induce AS expression by strains carrying plasmids in the pCF10 family.
Salmonella
Flagellin Is Not a Dominant Protective Antigen in Oral Immunization with Attenuated Live Vaccine Strains
Authors: Kodama Chie , Matsui Hidenori
Published: March 2004

We found that oral immunization with flagellum-defective mutant strains of
Salmonella enterica
serovar Typhimurium with the ClpXP-deficient background protected mice against oral challenge with the virulent strain. These data indicate that
Salmonella
flagellin is not a dominant protective antigen in oral immunization with attenuated live vaccine strains.
A longitudinal study on the ammonia threshold in junior cyclists
Authors: Yuan Y , Chan K-M
Published: March 2004

Objectives:To identify the effect of a one year non-specific training programme on the ammonia threshold of a group of junior cyclists and to correlate ammonia threshold with other common physiological variables.

Methods:The cyclists performed tests at three time points (T1, T2, T3) during the year. Follow up tests were conducted every six months after the original test. Ammonia threshold was obtained from a graded exercise with four minute steps.

Results:The relatively non-specific one year training programme was effective in inducing an increase in peak Vo2(60.6 (5.9), 65.9 (7.4), and 64.6 (6.5) ml/min/kg at T1, T2, and T3 respectively) and endurance time (18.3 (4.5), 20.1 (5.2), and 27.0 (6.1) minutes at T1, T2, and T3 respectively), but was not effective for the sprint related variables. Ammonia threshold, together with lactate threshold and ventilatory threshold, was not significantly different at the three test times. Only endurance time correlated significantly with ammonia threshold (r  =  0.915, p  =  0.001).

Conclusions:The findings suggest that a relatively non-specific one year training programme does not modify the ammonia threshold of junior cyclists. The significant correlation between ammonia threshold and endurance time further confirms that ammonia threshold is a measure of the ability to sustain exercise at submaximal intensities.
Modification of plasma glycosaminoglycans in long distance runners
Authors: Contini M , Pacini S , Ibba-Manneschi L , Boddi V , Ruggiero M , Liguri G , Gulisano M , Catini C
Published: March 2004

Background:It is well documented that exercise reduces the risk of thromboembolic disease, possibly by increasing the plasma concentration of anticoagulant-antithrombotic compounds.

Objectives:As plasma glycosaminoglycans (GAGs) play a role in the anticoagulant-antithrombotic potential of plasma, to examine the concentration and profile of these compounds in well trained, long distance runners and sedentary subjects.

Methods:Plasma GAGs were measured in 10 male, long distance runners and 10 sedentary counterparts before and after ergometric tests. GAGs were extracted, purified, and identified by electrophoretic and enzymatic methods, and measured as hexosamine.

Results:Plasma GAGs found in sedentary subjects were slow migrating heparan sulphates I and II, keratan sulphate I, and chondroitin 4–6-sulphate. Those found in trained athletes were slow migrating heparan sulphate I, chondroitin 4–6-sulphate (or keratan sulphate I), and fast migrating heparan sulphate. Total plasma concentrations of GAGs were higher in athletes than in sedentary subjects at rest. In sedentary subjects, plasma GAGs did not change after cycle ergometric exercise at 80% of their anaerobic threshold. However, the appearance of a novel band of heparan sulphate migrating faster than fast migrating heparan sulphate was observed in athletes after exercise.

Conclusions:Exercise changes the amount and profile of plasma GAGs; these changes may play a role in protecting subjects who practise aerobic sports against developing cardiovascular disease.