bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular Growth ofTrypanosoma cruziin Cardiac Myocytes Is Inhibited by Cytokine-Induced Nitric Oxide Release
Authors: Fichera Laura Edith , Albareda Maria Cecilia , Laucella Susana Adriana , Postan Miriam
Published: December 2003

The effect of interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) onTrypanosoma cruzimultiplication and nitric oxide (NO) production in cardiac myocytes was investigated. Cardiac myocyte cultures were obtained from neonatal Wistar rat hearts, infected withT. cruzi, and treated with IL-1β, TNF-α, IFN-γ, orN-monomethyl-l-arginine (l-NAME) for 72 h. Parasite growth was calculated from the number of infected cells in Giemsa-stained smears. Nitric oxide production was determined with the Griess reagent. Inducible nitric oxide synthase (iNOS) expression by cardiac myocytes was detected by Western blot. The results showed that the percentages of cardiac myocytes containingT. cruziamastigotes in cytokine-treated cultures were significantly lower than in nontreated cultures. The addition ofl-NAME reversed the inhibitory effect on parasite growth of IL-1β and TNF-α but not of IFN-γ. Nitrite levels released byT. cruzi-infected and noninfected cardiac myocyte cultures after 72 h of stimulation with IL-1β were significantly higher than those produced upon treatment with TNF-α, IFN-γ, or medium alone, regardless of the infection status. Nitrite levels in TNF-α-stimulated infected cultures were significantly higher than in untreated infected cultures and TNF-α-treated noninfected cultures.l-NAME inhibited IL-1β- but not TNF-α-induced NO production, indicating the presence of iNOS-dependent and iNOS-independent mechanisms for NO formation in this experimental system. iNOS expression was detected in infected and noninfected cardiac myocytes stimulated with IL-1 β and TNF-α but not with IFN-γ. These results suggest an important role for cardiac myocytes and locally secreted cytokines in the control of parasite multiplication inT. cruzi-induced myocarditis.
Plasminogen Binding by Group A Streptococcal Isolates from a Region of Hyperendemicity for Streptococcal Skin Infection and a High Incidence of Invasive Infection
Authors: McKay Fiona C. , McArthur Jason D. , Sanderson-Smith Martina L. , Gardam Sandra , Currie Bart J. , Sriprakash Kadaba S. , Fagan Peter K. , Towers Rebecca J. , Batzloff Michael R. , Chhatwal Gursharan S. , Ranson Marie , Walker Mark J.
Published: December 2003

Reports of resurgence in invasive group A streptococcal (GAS) infections come mainly from affluent populations with infrequent exposure to GAS. In the Northern Territory (NT) of Australia, high incidence of invasive GAS disease is secondary to endemic skin infection, serotype M1 clones are rare in invasive infection, the diversity and level of exposure to GAS strains are high, and no particular strains dominate. Expression of a plasminogen-binding GAS M-like protein (PAM) has been associated with skin infection in isolates elsewhere (D. Bessen, C. M. Sotir, T. M. Readdy, and S. K. Hollingshead, J. Infect. Dis.
173:
896-900, 1996), and subversion of the host plasminogen system by GAS is thought to contribute to invasion in animal models. Here, we describe the relationship between plasminogen-binding capacity of GAS isolates, PAM genotype, and invasive capacity in 29 GAS isolates belonging to 25 distinct strains from the NT. In the presence of fibrinogen and streptokinase, invasive isolates bound more plasminogen than isolates from uncomplicated infections (
P
≤ 0.004). Only PAM-positive isolates bound substantial levels of plasminogen by a fibrinogen-streptokinase-independent pathway (direct binding). Despite considerable amino acid sequence variation within the A1 repeat region of PAM where the plasminogen-binding domain maps, the critical lysine residue was conserved.
Neisseria meningitidisLipooligosaccharide Structure-Dependent Activation of the Macrophage CD14/Toll-Like Receptor 4 Pathway
Authors: Zughaier Susu M. , Tzeng Yih-Ling , Zimmer Shanta M. , Datta Anup , Carlson Russell W. , Stephens David S.
Published: December 2003

Meningococcal lipopoly(oligo)saccharide (LOS) is a major inflammatory mediator of fulminant meningococcal sepsis and meningitis. Highly purified wild-type meningococcal LOS and LOS from genetically defined mutants ofNeisseria meningitidisthat contained specific mutations in LOS biosynthesis pathways were used to confirm that meningococcal LOS activation of macrophages was CD14/Toll-like receptor 4 (TLR4)-MD-2 dependent and to elucidate the LOS structural requirement for TLR4 activation. Expression of TLR4 but not TLR2 was required, and antibodies to both TLR4 and CD14 blocked meningococcal LOS activation of macrophages. Meningococcal LOS α or β chain oligosaccharide structure did not influence CD14/TLR4-MD-2 activation. However, meningococcal lipid A, expressed by meningococci with defects in 3-deoxy-d-manno-octulosonic acid (KDO) biosynthesis or transfer, resulted in an ∼10-fold (P< 0.0001) reduction in biologic activity compared to KDO2-containing meningococcal LOS. Removal of KDO2from LOS by acid hydrolysis also dramatically attenuated cellular responses. Competitive inhibition assays showed similar binding of glycosylated and unglycosylated lipid A to CD14/TLR4-MD-2. A decrease in the number of lipid A phosphate head groups or penta-acylated meningococcal LOS modestly attenuated biologic activity. Meningococcal endotoxin is a potent agonist of the macrophage CD14/TLR4-MD-2 receptor, helping explain the fulminant presentation of meningococcal sepsis and meningitis. KDO2linked to meningococcal lipid A was structurally required for maximal activation of the human macrophage TLR4 pathway and indicates an important role for KDO-lipid A in endotoxin biologic activity.
Long-Term Effect of Heat Shock Protein 60 from
Actinobacillus actinomycetemcomitans
on Epithelial Cell Viability and Mitogen-Activated Protein Kinases
Authors: Zhang Liangxuan , Pelech Steven , Uitto Veli-Jukka
Published: December 2003

Our previous studies showed that bacterial heat shock protein 60 (hsp60) induces cultured epithelial cell proliferation within 24 h. Here we investigated the long-term effects of heat shock protein 60 isolated from
Actinobacillus actinomycetemcomitans
on skin keratinocyte (HaCaT cell line) viability and the cell signaling involved. Prolonged incubation in the presence of hsp60 increased the rate of epithelial cell death. The number of viable cells in hsp60-treated culture was 37% higher than the number in the control at 24 h but 27% lower at 144 h. A kinetics study of the effect of hsp60 on the phosphorylation of mitogen-activated protein kinases (MAPKs) involving Western blotting with phospho-specific antibodies showed that in addition to a transient early increase in p38 levels, a second peak appeared in keratinocytes 24 h after the addition of hsp60. In contrast, prolonged incubation with hsp60 caused a decrease in the level of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) compared with that in the controls, possibly as a result of protein phosphatase activity. We found that hsp60 increased the levels of several phosphatases, including MAP-2, which strongly dephosphorylates ERK1/2. Moreover, hsp60 increased the level of tumor necrosis factor alpha (TNF-α) in culture medium in a dose-dependent manner. TNF-α added to culture showed a cytotoxic effect on epithelial cells, particularly with longer incubation periods. TNF-α also induced the phosphorylation of p38. Finally, our results show that bacterial hsp60 inhibited stress-induced synthesis of cellular hsp60. Therefore, several cell behavior changes caused by long-term exposure to bacterial hsp60 may lead to impaired epithelial cell viability.
Immune Responses to Mycobacterial Antigens in the Gambian Population: Implications for Vaccines and Immunodiagnostic Test Design
Authors: Vekemans Johan , Ota Martin O. C. , Sillah Jackson , Fielding Katherine , Alderson Mark R. , Skeiky Yasir A. W. , Dalemans Wilfried , McAdam Keith P. W. J. , Lienhardt Christian , Marchant Arnaud
Published: December 2003

Recombinant immunodominant mycobacterial antigens are needed for the development of new vaccines and immunodiagnostic tools for use against tuberculosis. Ubiquitous exposure to mycobacteria in tropical countries could influence vaccine-induced immunity and the specificity of tuberculosis immunodiagnosis. For this study conducted in The Gambia, cellular immune responses to recombinant mycobacterial antigens were characterized inMycobacterium bovisBCG-vaccinated and nonvaccinated infants, adult community controls, household contacts, health care workers, and tuberculosis patients. Neonatal BCG vaccination induced gamma interferon (IFN-γ) responses to Mtb8.4, Mtb32-C, Mtb39A, Mtb9.9A, and Mtb32-N, but not CFP-10 (Mtb11) and α-crystallin (Mtb16). Exposure toMycobacterium tuberculosisin household contacts and health care workers was associated with high responses to CFP-10 and α-crystallin. Generally, low IFN-γ responses were found in tuberculosis patients. These results suggest that Mtb8.4, Mtb32-C, Mtb39A, Mtb9.9A, and Mtb32-N may be used in a subunit vaccine to boost BCG-induced immunity. While CFP-10 and α-crystallin are promising candidates for the immunodiagnosis ofM. tuberculosisinfection or for vaccine use, disease-associated immunosuppression may prevent IFN-γ immunodiagnosis of more advanced tuberculosis.
Induction of Interleukin-8 in T84 Cells byVibrio cholerae
Authors: Zhou Xin , Gao Da Q. , Michalski Jane , Benitez Jorge A. , Kaper James B.
Published: December 2003

The induction of interleukin-8 (IL-8) in vitro has been suggested to correlate with the reactogenicity ofVibrio choleraevaccine candidates.V. choleraevaccine candidate 638, a hemagglutinin protease/hap-defective strain, was recently reported to be well tolerated in human volunteers, suggesting a role for Hap in reactogenicity. We examined the role ofhapin the induction of IL-8 in intestinal epithelial T84 cells. Wild-typeV. choleraestrains 3038 and C7258 and a vaccine candidate strain, JBK70, induced levels of IL-8 similar to those of their isogenichapmutants. Supernatant containing Hap did not stimulate IL-8 production at a variety of concentrations tested, suggesting that Hap itself does not induce IL-8 production. Furthermore, supernatant from CVD115, which had deletions ofhapandrtxA(encoding repeats in toxin) and was derived from a reactogenic strain, CVD110, induced IL-8 production in T84 cells in a dose-dependent manner. The IL-8-stimulating activity of CVD115 culture supernatants was growth phase dependent and was strongest in stationary phase cultures. This IL-8 stimulator(s) was resistant to heat treatment but sensitive to proteinase. Protease activity in vitro did not correlate with the reactogenicity ofV. choleraevaccine candidates. Our data suggest that Hap is not an IL-8 inducer in T84 cells and that the IL-8 stimulator in the supernatant ofV. choleraeculture may play a role in reactogenicity.
Both Free-Living and Parasitic Nematodes Induce a Characteristic Th2 Response That Is Dependent on the Presence of Intact Glycans
Authors: Tawill Salah , Le Goff Laetitia , Ali Fahimeda , Blaxter Mark , Allen Judith E.
Published: December 2003

Infection with parasitic nematodes is characterized by the induction of a profound type 2 immune response. We have studied the role of glycans in the induction of the skewed type 2 response by antigens of the parasitic nematodeBrugia malayias well as the free-living nematodeCaenorhabditis elegans. Lymph node cells from BALB/c mice immunized with soluble extracts of the two nematodes showed distinct antigen-specific proliferation and cytokine production; however, both nematodes induced antigen-specific interleukin 4 (IL-4) production, demonstrating that the induction of a biased type 2 response is not unique to parasitic nematodes. Sodium periodate-treated soluble extracts of both nematodes consistently induced significantly less IL-4 production than the respective mock-treated extracts, indicating that glycans play a critical role in the induction of the Th2 immune response by these nematodes. The glycan-dependent induction of the Th2-potentiating cytokine IL-4 occurs by 72 h postinoculation. Our data suggest that glycan determinants common to nematodes act as ligands, displaying distinct molecular patterns that trigger the immune system to launch a biased Th2 immune response upon exposure to these organisms or their products. Further, the similarity of our findings to those forSchistosoma mansoniegg antigen is striking considering the enormous phylogenetic distance between nematodes and trematodes. These data thus have important implications for how the mammalian host responds to widely divergent metazoan invaders and suggest that the powerfulC. elegansmodel system can be used to address these questions.
Contribution of Glutathione Peroxidase to the Virulence of
Streptococcus pyogenes
Authors: Brenot Audrey , King Katherine Y. , Janowiak Blythe , Griffith Owen , Caparon Michael G.
Published: December 2003

Glutathione peroxidases are widespread among eukaryotic organisms and function as a major defense against hydrogen peroxide and organic peroxides. However, glutathione peroxidases are not well studied among prokaryotic organisms and have not previously been shown to promote bacterial virulence. Recently, a gene with homology to glutathione peroxidase was shown to contribute to the antioxidant defenses of
Streptococcus pyogenes
(group A streptococcus). Since this bacterium causes numerous suppurative diseases that require it to thrive in highly inflamed tissue, it was of interest to determine if glutathione peroxidase is important for virulence. In this study, we report that GpoA glutathione peroxidase is the major glutathione peroxidase in
S. pyogenes
and is essential for
S. pyogenes
pathogenesis in several murine models that mimic different aspects of streptococcal suppurative disease. In contrast, glutathione peroxidase is not essential for virulence in a zebrafish model of streptococcal myositis, a disease characterized by the absence of an inflammatory cell infiltrate. Taken together, these data suggest that
S. pyogenes
requires glutathione peroxidase to adapt to oxidative stress that accompanies an inflammatory response, and the data provide the first demonstration of a role for glutathione peroxidase in bacterial virulence. The fact that genes encoding putative glutathione peroxidases are found in the genomes of many pathogenic bacterial species suggests that glutathione peroxidase may have a general role in bacterial pathogenesis.
GeneExpression in HL60 Granulocytoids and Human PolymorphonuclearLeukocytes Exposed toCandidaalbicans†
Authors: Mullick Alaka , Elias Miria , Harakidas Penelope , Marcil Anne , Whiteway Malcolm , Ge Bing , Hudson Thomas J. , Caron Antoine W. , Bourget Lucie , Picard Serge , Jovcevski Orce , Massie Bernard , Thomas David Y.
Published: December 2003

Candida albicansis an opportunistic human pathogen causing both superficial and disseminated diseases. It is a dimorphic fungus, switching between yeast and hyphal forms, depending on cues from its microenvironment. Hyphae play an important role in the pathogenesis of candidiasis. The host's response toCandidainfection is multifaceted and includes the participation of granulocytes as key effector cells. The aim of this investigation was to study host gene expression during granulocyte-Candidainteraction. Effector cells were generated by the granulocytic differentiation of HL60 cells. The resulting cell population was shown to be morphologically and functionally equivalent to granulocytes and is therefore referred to as HL60 granulocytoids for the purposes of this study. Gene expression profiles were determined 1 h after hosts were infected withC. albicans.ThreeCandida-granulocytoid ratios were chosen to reflect different degrees of HL60 granulocytoid inhibition ofC. albicans. The data demonstrate that at the high pathogen-host ratio,C. albicansmodulated the HL60 granulocytoid's response by downregulating the expression of known antimicrobial genes. In addition, looking at the expression of a large number of genes, not all of which have necessarily been implicated in candidastatic or candidacidal mechanisms, it has been possible to describe the physiological response of the HL60 granulocytoid to an infectious challenge withC. albicans. Finally, some of the observed changes in HL60 granulocytoid gene expression were investigated in freshly isolated human polymorphonuclear leukocytes infected withC. albicans. Similar changes were seen in these primary human cells, lending support to the validity of this model.
Anthrax Lethal Toxin Induces Human Endothelial Cell Apoptosis
Authors: Kirby James E.
Published: December 2003

Because of its ease of dispersal and high lethality,
Bacillus anthracis
is one of the most feared biowarfare agents. A better understanding of anthrax pathogenesis is urgently needed to develop new therapies for systemic disease that is relatively unresponsive to antibiotics. Although experimental evidence has implicated a role for macrophages in anthrax pathogenesis, clinical and pathological observations suggest that a direct insult to the host vasculature may also be important. Two bacterial toxins, lethal toxin and edema toxin, are believed to mediate the clinical sequelae of anthrax. Here, I examined whether these toxins are directly toxic to endothelial cells, the cell type that lines the interior of blood vessels. I show for the first time that lethal toxin but not edema toxin reduces the viability of cultured human endothelial cells and induces caspase-dependent endothelial apoptosis. In addition, this toxicity affects both microvascular and large vessel endothelial cells as well as endothelial cells that have differentiated into tubules within a type I collagen extracellular matrix. Finally, lethal toxin induces cleavage of mitogen-activated protein kinase kinases in endothelial cells and inhibits phosphorylation of ERK, p38, and JNK p46. Based on the contributions of these pathways to endothelial survival, I propose that lethal toxin-mediated cytotoxicity/apoptosis results primarily through inhibition of the ERK pathway. I also hypothesize that the observed endothelial toxicity contributes to vascular pathology and hemorrhage during systemic anthrax.
Brucella abortusRough Mutants Are Cytopathic for Macrophages in Culture
Authors: Pei Jianwu , Ficht Thomas A.
Published: December 2003

Rough mutants ofBrucellaspp. are attenuated for survival in animal models. However, conflicting in vitro evidence has been obtained concerning the intracellular survival of rough mutants. Transposon-derived rough mutants isolated in our laboratory were previously shown to exhibit small but significant reductions in intracellular survival in a 12-h in vitro assay. Several recent publications report that rough mutants exhibited increased macrophage uptake relative to their smooth parental strains, and a reduction in numbers at the end of the assay has been interpreted as intracellular killing. In an effort to explore the role of O antigen in the interaction betweenBrucella abortusand macrophages, we have monitored the uptake of rough mutants and survival in vitro by using the murine macrophage cell line J774.A1. The results confirm a 10- to 20-fold-increased uptake of rough mutants over that of smooth organisms under standard conditions. Recovery of the rough mutants persisted up to 8 h postinfection, but at the point when intracellular replication of the smooth organisms was observed, the number of rough organisms recovered declined. Fluorescence microscopy revealed the intracellular multiplication of both smooth and rough organisms, and assays performed in the absence of antibiotic confirmed the replication of the rough organisms. Examination by phase-contrast microscopy revealed the lytic death of macrophages infected with the rough mutants, which was confirmed by the release of lactate dehydrogenase (LDH) from the cell cytoplasm. Thus, the decline in the number of rough organisms was the result of the lysis of macrophages and not from intracellular killing. The cytopathic effect is characterized as necrotic rather than apoptotic cell death based on early LDH release, annexin V and propidium iodide staining, morphological changes of infected cells and nuclei, and glycine protection. The cytopathic effect was observed with macrophages at multiplicities of infection (MOIs) of as low as 20 and was not observed with epithelial cells at MOIs of as high as 2000. These findings suggest a role for O antigen during the early stages of host-agent interaction that is essential in establishing an intracellular niche that maintains and supports persistent intracellular infection resulting in disease.
Chlamydia-Infected Cells Continue To Undergo Mitosis and Resist Induction of Apoptosis
Authors: Greene Whitney , Xiao Yangming , Huang Yanqing , McClarty Grant , Zhong Guangming
Published: December 2003

Both anti- and proapoptotic activities have been reported to occur during chlamydial infection. To reconcile the apparent controversy, we compared host cell apoptotic responses to infection with 17 different chlamydial serovars and strains. None of the serovars caused any biologically significant apoptosis in the infected host cells. Host cells in chlamydia-infected cultures can continue to undergo DNA synthesis and mitosis. Chlamydia-infected cells are resistant to apoptosis induction, although the extent of the antiapoptotic ability varied between serovars. These observations have demonstrated that an anti- but not proapoptotic activity is the prevailing event in chlamydia-infected cultures.
Immunotherapy ofTrypanosoma cruziInfection with DNA Vaccines in Mice
Authors: Dumonteil Eric , Escobedo-Ortegon Javier , Reyes-Rodriguez Norma , Arjona-Torres Arletty , Ramirez-Sierra Maria Jesus
Published: December 2003

The mechanisms involved in the pathology of chronic chagasic cardiomyopathy are still debated, and the controversy has interfered with the development of new treatments and vaccines. Because of the potential of DNA vaccines for immunotherapy of chronic and infectious diseases, we tested if DNA vaccines could control an ongoingTrypanosoma cruziinfection. BALB/c mice were infected with a lethal dose (5 × 104parasites) as a model of acute infection, and then they were treated with two injections of 100 μg of plasmid DNA 1 week apart, beginning on day 5 postinfection. Control mice had high levels of parasitemia and mortality and severe cardiac inflammation, while mice treated with plasmid DNA encoding trypomastigote surface antigen 1 or Tc24 had reduced parasitemia and mild cardiac inflammation and >70% survived the infection. The efficacy of the immunotherapy also was significant when it was delayed until days 10 and 15 after infection. Parasitological analysis of cardiac tissue of surviving mice indicated that most mice still contained detectable parasite kinetoplast DNA but fewer mice contained live parasites, suggesting that there was efficient but not complete parasite elimination. DNA vaccine immunotherapy was also evaluated in CD1 mice infected with a low dose (5 × 102parasites) as a model of chronic infection. Immunotherapy was initiated on day 70 postinfection and resulted in improved survival and reduced cardiac tissue inflammation. These results suggest that DNA vaccines have strong potential for the immunotherapy ofT. cruziinfection and may provide new alternatives for the control of Chagas' disease.
Urokinase-Deficient Mice Fail To Generate a Type 2 Immune Response following Schistosomal Antigen Challenge
Authors: Gyetko Margaret R. , Sud Sudha , Chensue Stephen W.
Published: December 2003

Activated lymphocytes express urokinase-type plasminogen activator (uPA). Previous work suggests that uPA modulates T-lymphocyte responses. Mice deficient in uPA (uPA−/−) fail to generate type 1 (T1) immune responses during infection withCryptococcus neoformans. Failure to generate either a T1 or a T2 immune response is not predictive of defects in the alternative response. Conversely, down-regulation of one type of immune response may result in inappropriate overactivation of the other. It is not known whether the immune defect in uPA−/−mice affects only T1 responses or whether T2 responses are also impaired. Impairment of both T1 and T2 responses would suggest a global T-cell defect in the absence of uPA. To determine the role of uPA in T2 immune responses, wild-type (WT) and uPA−/−mice were primed and challenged with schistosomal egg antigen (SEA). This elicits strong polarization to T2 immune responses in immunocompetent mice. The challenged WT mice developed delayed-type hypersensitivity (DTH) to SEA; high levels of serum immunoglobulin E (IgE); a strong T2 cytokine phenotype with markedly elevated levels of interleukin-4 (IL-4), IL-5, and IL-13; and eosinophil-rich pulmonary granulomas. uPA−/−mice failed to develop DTH to SEA; did not polarize Ig production to IgE; did not produce high levels of IL-4, IL-5, or IL-13; and had markedly reduced numbers of granuloma-associated eosinophils. uPA−/−mice fail to generate polarized T2 immune responses to a T2-inducing pathogen. These findings, in conjunction with our previous work, demonstrate that mice deficient in uPA have profoundly impaired immunity involving both T1 and T2 polarization and are largely immunologically unresponsive.
Role of Neutrophils in Murine Salmonellosis
Authors: Cheminay Cédric , Chakravortty Dipshikha , Hensel Michael
Published: December 2003

Gastrointestinal infections withSalmonella entericaserovars have different clinical outcomes that range from localized inflammation to a life-threatening systemic disease in the case of typhoid fever. Using a mouse model of systemic salmonellosis, we investigated the contribution of neutrophils to the innate immune defense againstSalmonellaafter oral infection. Neutrophil infiltration was dependent on the bacterial burden in various infected organs (Peyer's patches, mesenteric lymph nodes, spleen, and liver). However, the massive infiltration of neutrophils did not allow clearance of an infection with wild-typeSalmonella, presumably due to protection of intracellularSalmonellaagainst neutrophil activities. ASalmonellamutant strain deficient inSalmonellapathogenicity island 2 (SPI2) was able to infect systemic sites, but its replication was highly restricted and it did not cause detectable attraction of neutrophils. Neutrophil depletion by antibody treatment of mice did not restore the virulence of SPI2 or auxotrophic mutant strains, supporting the hypothesis that attenuation of the strains is not due to greater susceptibility to neutrophil killing. Our observations reveal that neutrophils have completely different roles during systemic salmonellosis and localized gastrointestinal infections. In the latter conditions, rapid neutrophil attraction efficiently prevents the spread of the pathogen, whereas the neutrophil influx is delayed during systemic infections and cannot protect against lethal bacteremia.
TraJ-DependentEscherichia coliK1 Interactions with Professional Phagocytes Are Important for Early Systemic Dissemination of Infection in the Neonatal Rat
Authors: Hill Val T. , Townsend Stacy M. , Arias Robyn S. , Jenabi Jasmine M. , Gomez-Gonzalez Ignacio , Shimada Hiroyuki , Badger Julie L.
Published: December 2003

Escherichia coliis a major cause of neonatal bacterial sepsis and meningitis. We recently identified a gene,traJ, which contributes to the ability ofE. coliK1 to penetrate the blood-brain barrier in the neonatal rat. Because very little is known regarding the most critical step in disease progression, translocation to the gut and dissemination to the lymphoid tissues after a natural route of infection, we assessed the ability of atraJmutant to cause systemic disease in the neonatal rat. Our studies determined that thetraJmutant is significantly less virulent than the wild type in the neonatal rat due to a decreased ability to disseminate from the mesenteric lymph nodes to the deeper tissues of the liver and spleen and to the blood during the early stages of systemic disease. Histopathologic studies determined that although significantly less or no mutant bacteria were recovered from the spleen and livers of infected neonatal rats, the inflammatory response was considerably greater than that in wild-type-colonized tissues. In vitro studies revealed that macrophages internalize thetraJmutant less frequently than they do the wild type and by a morphologically distinct process. Furthermore, we determined that tissue macrophages and dendritic cells within the liver and spleen are the major cellular targets ofE. coliK1 and that TraJ significantly contributes to the predominantly intracellular nature ofE. coliK1 within these professional phagocytes exclusively during the early stages of systemic disease. These data indicate that, contrary to earlier indications,E. coliK1 resides within professional phagocytes, and this is essential for the efficient progression of systemic disease.
Listeriosis in the Pregnant Guinea Pig: a Model of Vertical Transmission
Authors: Bakardjiev Anna I. , Stacy Brian A. , Fisher Susan J. , Portnoy Daniel A.
Published: December 2003

Feto-placental infections represent a major cause of pregnancy complications, and yet the underlying molecular and cellular mechanisms of vertical transmission are poorly understood.Listeria monocytogenes, a facultative intracellular pathogen, is one of a group of pathogens that are known to cause feto-placental infections in humans and other mammals. The purpose of this study was to evaluate possible mechanisms of vertical transmission ofL. monocytogenes. Humans and guinea pigs have a hemochorial placenta, where a single layer of fetally derived trophoblasts separates maternal from fetal circulation. We characterizedL. monocytogenesinfection of the feto-placental unit in a pregnant guinea pig model and in primary human trophoblasts and trophoblast-derived cell lines. The clinical manifestations of listeriosis in the pregnant guinea pigs and the tropism ofL. monocytogenesto the guinea pig placenta resembled those in humans. Trophoblast cell culture systems were permissive for listerial growth and cell-to-cell spread and revealed thatL. monocytogenesdeficient in internalin A, a virulence factor that mediates invasion of nonphagocytic cells, was 100-fold defective in invasion. However, crossing of the feto-placental barrier in the guinea pig model was independent of internalin A, suggesting a negligible role for internalin-mediated direct invasion of trophoblasts in vivo. Further understanding of vertical transmission ofL. monocytogeneswill help in designing more effective means of treatment and disease prevention.
Survival Strategy of Obligately IntracellularEhrlichia chaffeensis: Novel Modulation of Immune Response andHost CellCycles
Authors: Zhang Jian-zhi , Sinha Mala , Luxon Bruce A. , Yu Xue-jie
Published: December 2003

Ehrlichia chaffeensisis an obligatory intracellular bacterium which resides in an early endosome in monocytes.E. chaffeensisinfection in a human monocyte cell line (THP1) significantly altered the transcriptional levels of 4.5% of host genes, including those coding for apoptosis inhibitors, proteins regulating cell differentiation, signal transduction, proinflammatory cytokines, biosynthetic and metabolic proteins, and membrane trafficking proteins. The transcriptional profile of the host cell revealed key themes in the pathogenesis ofEhrlichia. First,E. chaffeensisavoided stimulation of or repressed the transcription of cytokines involved in the early innate immune response and cell-mediated immune response to intracellular microbes, such as the interleukin-12 (IL-12), IL-15, and IL-18 genes, which might makeEhrlichiaa stealth organism for the macrophage. Second,E. chaffeensisup-regulated NF-κB and apoptosis inhibitors and differentially regulated cell cyclins and CDK expression, which may enhance host cell survival. Third,E. chaffeensisalso inhibited the gene transcription of RAB5A, SNAP23, and STX16, which are involved in membrane trafficking. By comparing the transcriptional response of macrophages infected with other bacteria and that of macrophages infected withE. chaffeensis, we have identified few genes that are commonly induced and no commonly repressed genes. These results illustrate the stereotyped macrophage response to other pathogens, in contrast with the novel host response to obligate intracellularEhrlichia, whose survival depends entirely on a long evolutionary process of outmaneuvering macrophages.
MulticlonalLeishmania braziliensisPopulation Structure and Its Clinical Implication in a Region of Endemicity for American Tegumentary Leishmaniasis
Authors: Schriefer A. , Schriefer A. L. F. , Góes-Neto A. , Guimarães L. H. , Carvalho L. P. , Almeida R. P. , Machado P. R. , Lessa H. A. , de Jesus A. Ribeiro , Riley L. W. , Carvalho E. M.
Published: December 2003

In Corte de Pedra (CP), northeastern Brazil,Leishmania braziliensiscauses three distinct forms of American tegumentary leishmaniasis (ATL). To test the hypothesis that strain polymorphism may be involved in this disease spectrum and accurately characterize the parasite population structure in CP, we compared oneL. major, two non-CPL. braziliensis, one CPL. amazonensis, and 45 CPL. braziliensisisolates, obtained over a 10-year period from localized cutaneous, mucosal, and disseminated leishmaniasis patients, with randomly amplified polymorphic DNA (RAPD). Electrophoretic profiles were mostly unique across species. All typing protocols revealed polymorphism among the 45 CPL. braziliensisisolates, which displayed eight different RAPD patterns and greater than 80% overall fingerprint identity, attesting to the adequacy of the tools to assess strain variability in CP's geographically limited population of parasites. The dendrogram based on the sum of RAPD profiles of each isolate unveiled nine discrete typing units clustered into five clades. Global positioning showed extensive overlap of these clades in CP, precluding geographic sequestration as the mechanism of the observed structuralization. Finally, all forms of ATL presented a statistically significant difference in their frequencies among the clades, suggesting thatL. braziliensisgenotypes may be accompanied by specific disease manifestation after infection.
IndividualMycobacterium tuberculosisResuscitation-Promoting Factor Homologues Are Dispensable for Growth In Vitro and In Vivo
Authors: Tufariello JoAnn M. , Jacobs, William R. , Chan John
Published: December 2003

Mycobacterium tuberculosispossesses five genes with significant homology to the resuscitation-promoting factor (Rpf) ofMicrococcus luteus. TheM. luteusRpf is a secreted ∼16-kDa protein which restores active growth to cultures ofM. luteusrendered dormant by prolonged incubation in stationary phase. More recently, the Rpf-like proteins ofM. tuberculosishave been shown to stimulate the growth of extended-stationary-phase cultures ofMycobacterium bovisBCG. These data suggest that the Rpf proteins can influence the growth of mycobacteria; however, the studies do not demonstrate specific functions for the various members of this protein family, nor do they assess the function ofM. tuberculosisRpf homologues in vivo. To address these questions, we have disrupted each of the fiverpf-like genes inM. tuberculosisErdman, and analyzed the mutants for their growth in vitro and in vivo. In contrast toM. luteus, for whichrpfis an essential gene, we find that all of theM. tuberculosis rpfdeletion mutant strains are viable; in addition, all show growth kinetics similar to Erdman wild type both in vitro and in mouse organs following aerosol infection. Analysis ofrpfexpression inM. tuberculosiscultures from early log phase through late stationary phase indicates that expression of therpf-like genes is growth phase-dependent, and that the expression patterns of the fiveM. tuberculosis rpfgenes, while overlapping to various degrees, are not uniform. We also provide evidence that mycobacterialrpfgenes are expressed in vivo in the lungs of mice acutely infected with virulentM. tuberculosis.
Isolation and Characterization of a Secretory Component ofEchinococcus multilocularisMetacestodes Potentially Involved in Modulating the Host-Parasite Interface
Authors: Walker Mirjam , Baz Adriana , Dematteis Sylvia , Stettler Marianne , Gottstein Bruno , Schaller Johann , Hemphill Andrew
Published: December 2003

Echinococcus multilocularismetacestodes are fluid-filled, vesicle-like organisms, which are characterized by continuous asexual proliferation via external budding of daughter vesicles, predominantly in the livers of infected individuals. Tumor-like growth eventually leads to the disease alveolar echinococcosis (AE). We employed the monoclonal antibody (MAb) E492/G1, previously shown to be directed against a carbohydrate-rich, immunomodulatory fraction ofEchinococcus granulosus, to characterize potentially related components inE. multilocularis. Immunofluorescence studies demonstrated that MAb E492/G1-reactive epitopes were found predominantly on the laminated layer and in the periphery of developing brood capsules. The respective molecules were continuously released into the exterior medium and were also found in the parasite vesicle fluid. The MAb E492/G1-reactive fraction inE. multilocularis, named Em492 antigen, was isolated by immunoaffinity chromatography. Em492 antigen had a protein/carbohydrate ratio of 0.25, reacted with a series of lectins, and is related to the laminated layer-associated Em2(G11) antigen. The epitope recognized by MAb E492/G1 was sensitive to sodium periodate but was not affected by protease treatment. Anti-Em492 immunoglobulin G1 (IgG1) and IgG2 and, at lower levels, IgG3 were found in sera of mice suffering from experimentally induced secondary, but not primary, AE. However, with regard to cellular immunity, a suppressive effect on concanavalin A- or crude parasite extract-induced splenocyte proliferation in these mice was observed upon addition of Em492 antigen, but trypan blue exclusion tests and transmission electron microscopy failed to reveal any cytotoxic effect in Em492 antigen-treated spleen cells. This indicated that Em492 antigen could be modulating the periparasitic cellular environment duringE. multilocularisinfection through as yet unidentified mechanisms and could be one of the factors contributing to immunosuppressive events that occur at the host-parasite interface.
Helicobacter pyloriDisrupts STAT1-Mediated Gamma Interferon-Induced Signal Transduction in Epithelial Cells
Authors: Mitchell David J. , Huynh Hien Q. , Ceponis Peter J. M. , Jones Nicola L. , Sherman Philip M.
Published: December 2003

Infection withHelicobacter pyloriis chronic despite a vigorous mucosal immune response characterized by gastric T-helper type 1 cell expansion and gamma interferon (IFN-γ) production. IFN-γ signals by activation and nuclear translocation of signal transducer and activator of transcription 1 (STAT1); however, the effect ofH. pyloriinfection on IFN-γ-STAT1 signaling is unknown. We infected human gastric (MKN45 and AGS) and laryngeal (HEp-2) epithelial cell lines with type 1 and type 2H. pyloristrains and then stimulated them with IFN-γ. Western blotting of whole-cell protein extracts revealed that infection with live, but not heat-killed,H. pyloritime-dependently decreased IFN-γ-induced STAT1 tyrosine phosphorylation. Electrophoretic mobility shift assay of nuclear protein extracts demonstrated thatH. pyloriinfection reduced IFN-γ-induced STAT1 DNA binding. STAT1 was unable to translocate from the cytoplasm to the nucleus inH. pylori-infected HEp-2 cells examined by immunofluorescence, and reverse transcription-PCR showed that IFN-γ-induced interferon regulatory factor 1 expression was inhibited. These effects were independent of thecagA,cagE, and VacA status of the infectingH. pyloristrain. Furthermore, neitherH. pyloriculture supernatants nor conditioned medium fromH. pylori-infected MKN45 cells inhibited IFN-γ-induced STAT1 tyrosine phosphorylation, suggesting that inhibition is independent of a soluble epithelial or bacterial factor but is dependent on bacterial contact with epithelial cells.H. pyloridisruption of IFN-γ-STAT1 signaling in epithelial cells may represent a mechanism by which the bacterium modifies mucosal immune responses to promote its survival in the human host.
A Gene,
uge
, Is Essential for
Klebsiella pneumoniae
Virulence
Authors: Regué Miguel , Hita Beatriz , Piqué Nuria , Izquierdo Luis , Merino Susana , Fresno Sandra , Benedí Vicente Javier , Tomás Juan M.
Published: December 2003

Klebsiella pneumoniae
strains typically express both smooth lipopolysaccharide (LPS) with O antigen molecules and capsule polysaccharide (K antigen) on the surface. A single mutation in a gene that codes for a UDP galacturonate 4-epimerase (
uge
) renders a strain with the O
−
:K
−
phenotype (lack of capsule and LPS without O antigen molecules and outer core oligosaccharide). The
uge
gene was present in all the
K. pneumoniae
strains tested. The
K. pneumoniae uge
mutants were unable to produce experimental urinary tract infections in rats and were completely avirulent in two different animal models (septicemia and pneumonia). Reintroduction of the single
uge
wild-type gene in the corresponding mutants completely restored the wild-type phenotype (presence of capsule and smooth LPS) independently of the O or K serotype of the wild type. Furthermore, complemented
uge
mutants recovered the ability to produce experimental urinary tract infections in rats and virulence in the septicemia and pneumonia animal models.
The ADP Ribosyltransferase Domain of
Pseudomonas aeruginosa
ExoT Contributes to Its Biological Activities
Authors: Garrity-Ryan L. , Shafikhani S. , Balachandran P. , Nguyen L. , Oza J. , Jakobsen T. , Sargent J. , Fang X. , Cordwell S. , Matthay M. A. , Engel J. N.
Published: December 2003

ExoT is a type III secreted effector protein found in almost all strains of
Pseudomonas aeruginosa
and is required for full virulence in an animal model of acute pneumonia. It is comprised of an N-terminal domain with GTPase activating protein (GAP) activity towards Rho family GTPases and a C-terminal ADP ribosyltransferase (ADPRT) domain with minimal activity towards a synthetic substrate in vitro. Consistent with its activity as a Rho family GTPase, ExoT has been shown to inhibit
P. aeruginosa
internalization into epithelial cells and macrophages, disrupt the actin cytoskeleton through a Rho-dependent pathway, and inhibit wound repair in a scrape model of injured epithelium. We have previously shown that mutation of the invariant arginine of the GAP domain to lysine (R149K) results in complete loss of GAP activity in vitro but only partially inhibits ExoT anti-internalization and cell rounding activity. We have constructed in-frame deletions and point mutations within the ADPRT domain in order to test whether this domain might account for the residual activity observed in ExoT GAP mutants. Deletion of a majority of the ADPRT domain (residues 234 to 438) or point mutations of the ADPRT catalytic site (residues 383 to 385) led to distinct changes in host cell morphology and substantially reduced the ability of ExoT to inhibit in vitro epithelial wound healing over a 24-h period. In contrast, only subtle effects on the efficiency of ExoT-induced bacterial internalization were observed in the ADPRT mutant forms. Expression of each domain individually in
Saccharomyces cerevisiae
was toxic, whereas expression of each of the catalytically inactive mutant domains was not. Collectively, these data demonstrate that the ADPRT domain of ExoT is active in vivo and contributes to the pathogenesis of
P. aeruginosa
infections.
Development, Characterization, and Functional Activity of a Panel of Specific Monoclonal Antibodies to Inner Core Lipopolysaccharide Epitopes inNeisseria meningitidis
Authors: Gidney Margaret Anne J. , Plested Joyce S. , Lacelle Suzanne , Coull Philip A. , Wright J. Claire , Makepeace Katherine , Brisson Jean-Robert , Cox Andrew D. , Moxon E. Richard , Richards James C.
Published: December 2003

A panel of six murine monoclonal antibodies (MAbs) recognizing inner core lipopolysaccharide (LPS) epitopes ofNeisseria meningitidiswas prepared and characterized in order to determine the diversity of inner core LPS glycoforms among disease and carrier isolates. Two of these MAbs, L2-16 (immunoglobulin G2b [IgG2b]) and LPT3-1 (IgG2a), together with a third, previously described MAb, L3B5 (IgG3), showed reactivity, either individually or in combination, with all except 3 of 143 disease and carriage isolates (125 of 126 strains from blood, cerebrospinal fluid, or skin biopsy samples and 15 of 17 from nasopharyngeal cultures). MAbs L3B5, L2-16, and LPT3-1 were further characterized in an indirect immunofluorescence assay. All three MAbs bound to the bacterial cell surface, findings that correlated strongly with whole-cell enzyme-linked immunosorbent assay and immunodot blots. However, in contrast to our findings with L3B5, cell surface binding of L2-16 or LPT 3-1 did not correlate with functional activity as determined by bactericidal or infant rat passive protection assays against wild-typeN. meningitidisstrains. These findings are provocative with respect to the requirements for protective activity of antibodies and the development of inner core LPS vaccines against invasive meningococcal disease.
Stable T-Cell Population Expressing an Effector Cell Surface Phenotype in the Lungs of Mice Chronically Infected with
Mycobacterium tuberculosis
Authors: Junqueira-Kipnis Ana Paula , Turner Joanne , Gonzalez-Juarrero Mercedes , Turner Oliver C. , Orme Ian M.
Published: December 2003

Analysis of T-cell subsets accumulating in the lungs of C57BL/6 mice chronically infected with
Mycobacterium tuberculosis
revealed that both CD4 and CD8 T-cell populations expressed a cell surface phenotype consistent with that of effector T cells and that a significant proportion of these cells were in the process of secreting gamma interferon.
In Vitro Induction of
Neospora caninum
Bradyzoites in Vero Cells Reveals Differential Antigen Expression, Localization, and Host-Cell Recognition of Tachyzoites and Bradyzoites
Authors: Vonlaufen Nathalie , Guetg Nicole , Naguleswaran Arunasalam , Müller Norbert , Björkman Camilla , Schares Gereon , von Blumroeder Daniela , Ellis John , Hemphill Andrew
Published: December 2003

We report on an optimized method for the in vitro culture of tissue cyst-forming
Neospora caninum
bradyzoites in Vero cells and the separation of viable parasites from host cells. Treatment of tachyzoite-infected Vero cell cultures with 17 μM sodium nitroprusside for 8 days severely scaled down parasite proliferation, led to reduced expression of tachyzoite surface antigens, and induced the expression of the bradyzoite marker NcBAG1 and the cyst wall antigen recognized by the monoclonal antibody MAbCC2. Transmission electron microscopy demonstrated that intracellular parasites were located within parasitophorous vacuoles that were surrounded by a cyst wall-like structure, and the dense granule antigens NcGRA1, NcGRA2, and NcGRA7 were incorporated into the cyst wall. Adhesion-invasion assays employing purified tachyzoites and bradyzoites showed that tachyzoites adhered to, and invaded, Vero cells with higher efficiency than bradyzoites. However, removal of terminal sialic acid residues from either the host cell or the parasite surface increased the invasion of Vero cells by bradyzoites, but not tachyzoites.