bluuebunny/crossref_metadata_2025 huggingface dataset:
Soluble Factors Released byToxoplasma gondii-Infected Astrocytes Down-Modulate Nitric Oxide Production by Gamma Interferon-Activated Microglia and Prevent Neuronal Degeneration
Authors: Rozenfeld Claudia , Martinez Rodrigo , Figueiredo Rodrigo T. , Bozza Marcelo T. , Lima Flávia R. S. , Pires Ana Lúcia , Silva Patrícia M. , Bonomo Adriana , Lannes-Vieira Joseli , De Souza Wanderley , Moura-Neto Vivaldo
Published: March 2003

The maintenance of a benign chronicToxoplasma gondiiinfection is mainly dependent on the persistent presence of gamma interferon (IFN-γ) in the central nervous system (CNS). However, IFN-γ-activated microglia are paradoxically involved in parasitism control and in tissue damage during a broad range of CNS pathologies. In this way, nitric oxide (NO), the main toxic metabolite produced by IFN-γ-activated microglia, may cause neuronal injury duringT. gondiiinfection. Despite the potential NO toxicity, neurodegeneration is not a common finding during chronicT. gondiiinfection. In this work, we describe a significant down-modulation of NO production by IFN-γ-activated microglia in the presence of conditioned medium ofT. gondii-infected astrocytes (CMi). The inhibition of NO production was paralleled with recovery of neurite outgrowth when neurons were cocultured with IFN-γ-activated microglia in the presence of CMi. Moreover, the modulation of NO secretion and the neuroprotective effect were shown to be dependent on prostaglandin E2(PGE2) production byT. gondii-infected astrocytes and autocrine secretion of interleukin-10 (IL-10) by microglia. These events were partially eliminated when infected astrocytes were treated with aspirin and cocultures were treated with anti-IL-10 neutralizing antibodies and RP-8-Br cyclic AMP (cAMP), a protein kinase A inhibitor. Further, the modulatory effects of CMi were mimicked by the presence of exogenous PGE2and by forskolin, an adenylate cyclase activator. Altogether, these data point to aT. gondii-triggered regulatory mechanism involving PGE2secretion by astrocytes and cAMP-dependent IL-10 secretion by microglia. This may reduce host tissue inflammation, thus avoiding neuron damage during an established Th1 protective immune response.
Recombinant Gamma Interferon Stimulates Signal Transduction and Gene Expression in Alveolar Macrophages In Vitro and in Tuberculosis Patients
Authors: Condos Rany , Raju Bindu , Canova Antony , Zhao Ben-Yang , Weiden Michael , Rom William N. , Pine Richard
Published: March 2003

Tuberculosis is the seventh leading cause of morbidity and mortality in the world, with eight million cases per year. Animal and human studies demonstrate an enrichment of CD4 cells at sites of disease, with a more favorable clinical course when there is a Th1 response with the presence of gamma interferon (IFN-γ). We previously treated patients who had multidrug-resistant tuberculosis with recombinant IFN-γ (rIFN-γ) in aerosol form and were able to convert smear-positive cases to smear negative with 12 treatments over 1 month. We hypothesized that rIFN-γ would induce signal transducer and activator of transcription (STAT) and interferon regulatory factor (IRF) binding activity in alveolar macrophages (AM). AM treated in vitro showed clear upregulation of STAT-1 and IRF-1 by rIFN-γ. STAT-1 was not activated and IRF-1 was only weakly induced after 1 day of infection byMycobacterium tuberculosisTN913. In bronchoalveolar lavage (BAL) cells obtained from 10 of 10 tuberculosis patients 10 ± 2 days post-antituberculosis treatment, there was no detectable STAT-1 or IRF-1 DNA-binding activity. After 4 weeks of treatment with rIFN-γ aerosol in addition to the antituberculosis drugs, 10 of 10 patients had increased STAT-1, IRF-1, and/or IRF-9 DNA-binding activity in BAL cells from lung segments shown radiographically to be involved and in those shown to be uninvolved. Symptoms and chest radiographs improved, and amounts of macrophage inflammatory cytokines and human immunodeficiency virus type 1 (HIV-1) viral loads (in five of five HIV-1-coinfected patients) declined in the second BAL specimens. rIFN-γaerosol induces signal transduction and gene expression in BAL cells and should be evaluated for efficacy in a randomized, controlled clinical trial.
Exposure of Immunocompetent Adult Mice to
Pneumocystis carinii
f. sp.
muris
by Cohousing: Growth of
P
.
carinii
f. sp.
muris
and Host Immune Response
Authors: An Chun Li , Gigliotti Francis , Harmsen Allen G.
Published: March 2003

There has been emerging evidence that immunocompetent hosts can harbor
Pneumocystis
in their lungs. The purpose of this study was to determine the kinetics of
Pneumocystis carinii
f. sp.
muris
infection in adult immunocompetent mice and the host immune response to the organisms. To accomplish this, we exposed adult immunocompetent mice to SCID mice infected with
P
.
carinii
f. sp.
muris
by cohousing. We found that
P
.
carinii
f. sp.
muris
was detectable in the lungs of cohoused immunocompetent mice by PCR by 3 weeks after the beginning of cohousing. At about 4 weeks of cohousing,
P
.
carinii
f. sp.
muris
was readily detectable in the lungs of mice by microscopic techniques. Also at this time,
P
.
carinii
f. sp.
muris-
specific immunoglobulin G was found in the sera of the mice, and CD62
low
CD4- and CD8-positve T cells accumulated in the lungs. Shortly after this immune response, the
P
.
carinii
f. sp.
muris
organisms were cleared from the lungs. Adult mice cohoused for only 1 week also contained
P
.
carinii
f. sp.
muris
cysts detectable by silver staining at 5 and 6 weeks after the beginning of cohousing. We also found that the
P
.
carinii
f. sp.
muris
organisms grew to greater numbers in the lungs of BALB/c mice than in those of C57BL6 mice. This indicates that immunocompetent hosts develop a mild infection with
P
.
carinii
f. sp.
muris
which resolves in 5 to 6 weeks when there is a detectable immune response to the organism. Once an acquired immune response was initiated, the
P
.
carinii
f. sp.
muris
organisms were quickly eliminated without clinical signs of disease.
Transmission ofAnaplasma phagocytophilumtoIxodes ricinusTicks from Sheep in the Acute and Post-Acute Phases of Infection
Authors: Ogden N. H. , Casey A. N. J. , Woldehiwet Z. , French N. P.
Published: March 2003

A total of 60 sheep were exposed toAnaplasma phagocytophiluminfection on an enclosed area ofIxodes ricinus-infested pasture in North Wales, United Kingdom, and rapidly acquired acuteA. phagocytophiluminfections detectable by PCR and blood smear examination. Of the ticks that had engorged in the previous instar on infected sheep, 52% of adult ticks and 28% of nymphs were PCR positive; a significant, 10-fold increase in prevalence compared to that of ticks that engorged on sheep preinfection was observed (P= 0.015). The likelihood that ticks were PCR positive, after feeding on the sheep and molting to the next instar, increased marginally with increasing numbers of infected neutrophils per milliliter of blood of their sheep host (P= 0.068) and increased significantly when they were collected from sheep carrying higher numbers of adult female ticks (P= 0.017), but increasing numbers of feeding nymphs had a significant negative effect on transmission (P= 0.049). The numbers of circulating neutrophils and of infected neutrophils also varied significantly with the numbers of ticks feeding on the sheep when the blood was collected. Our study suggests that ruminants are efficient reservoirs ofA. phagocytophilumduring the acute and post-acute phases of infection. The risk of ruminant-derived infections may, however, be strongly affected by variations in tick densities, which may influence transmission from acutely infected animals via effects on the numbers of infected cells in the blood and possibly by within-skin modulation of infection.
Major Histocompatibility Complex Heterozygote Superiority during Coinfection
Authors: McClelland Erin E. , Penn Dustin J. , Potts Wayne K.
Published: March 2003

Genes of the major histocompatibility complex (MHC) play a critical role in immune recognition, and many alleles confer susceptibility to infectious and autoimmune diseases. How these deleterious alleles persist in populations is controversial. One hypothesis postulates that MHC heterozygote superiority emerges over multiple infections because MHC-mediated resistance is generally dominant and many allele-specific susceptibilities to pathogens will be masked by the resistant allele in heterozygotes. We tested this hypothesis by using experimental coinfections withSalmonella enterica(serovar Typhimurium C5TS) and Theiler's murine encephalomyelitis virus (TMEV) in MHC-congenic mouse strains where one haplotype was resistant toSalmonellaand the other was resistant to TMEV. MHC heterozygotes were superior to both homozygotes in 7 out of 8 comparisons (P= 0.0024), and the mean standardized pathogen load of heterozygotes was reduced by 41% over that of homozygotes (P= 0.01). In contrast, no heterozygote superiority was observed when the MHC haplotype combinations had similar susceptibility profiles to the two pathogens. This is the first experimental evidence for MHC heterozygote superiority against multiple pathogens, a mechanism that would contribute to the evolution of MHC diversity and explain the persistence of alleles conferring susceptibility to disease.
Pneumolysin-Dependent and -Independent Gene Expression Identified by cDNA Microarray Analysis of THP-1 Human Mononuclear Cells Stimulated byStreptococcus pneumoniae
Authors: Rogers P. David , Thornton Justin , Barker Katherine S. , McDaniel D. Olga , Sacks Gordon S. , Swiatlo Edwin , McDaniel Larry S.
Published: March 2003

Pneumolysin is an important virulence factor ofStreptococcus pneumoniae, interacting with the membranes of host cells to elicit a multitude of inflammatory responses. We used cDNA microarrays to identify genes which are responsive toS.pneumoniaein a pneumolysin-dependent and -independent fashion. The THP-1 human monocytic cell line was coincubated for 3 h with medium alone, with the virulent type 2S.pneumoniaestrain D39, or with the isogenic strain PLN, which does not express pneumolysin. RNA was isolated from the monocytes and hybridized on cDNA microarrays. Of 4,133 genes evaluated, 142 were found to be responsive in a pneumolysin-dependent fashion, whereas 40 were found to be responsive independent of pneumolysin. Genes that were up-regulated in cells exposed to D39 relative to those exposed to PLN included genes encoding proteins such as mannose binding lectin 1, lysozyme, α-1 catenin, cadherin 17, caspases 4 and 6, macrophage inflammatory protein 1β (MIP-1β), interleukin 8 (IL-8), monocyte chemotactic protein 3 (MCP-3), IL-2 receptor β (IL-2Rβ), IL-15 receptor α (IL-15Rα), interferon receptor 2, and prostaglandin E synthase. Down-regulated genes included those encoding complement component receptor 2/CD21, platelet-activating factor acetylhydrolase, and oxidized low-density lipoprotein receptor 1 (OLR1). Pneumolysin-independent responses included down-regulation of the genes encoding CD68, CD53, CD24, transforming growth factor β2, and signal transducers and activators of transcription 1. These results demonstrate the striking effects of pneumolysin on the host cell upon exposure toS.pneumoniae.
Leishmania donovani-Induced Expression of Suppressor of Cytokine Signaling 3 in Human Macrophages: a Novel Mechanism for Intracellular Parasite Suppression of Activation
Authors: Bertholet Sylvie , Dickensheets Harold L. , Sheikh Faruk , Gam Albert A. , Donnelly Raymond P. , Kenney Richard T.
Published: March 2003

Leishmania donovaniprotozoan parasites, the causative agent of visceral leishmaniasis, establish an infection partly by interfering with cytokine signaling in the host macrophages. Therefore, we investigated the expression of the suppressor of cytokine signaling (SOCS) genes in human macrophages infected withL.donovani. The expression of SOCS3 mRNA was induced transiently after exposure to live or heat-killed parasites, but not purified lipophosphoglycan, while that of other SOCS genes remained unchanged. SOCS3 gene expression was not dependent on phagocytosis or on cytokines released byL.donovani-infected macrophages, such as interleukin-1β or tumor necrosis factor alpha. In addition,Leishmaniaused a different signaling pathway(s) than bacterial lipopolysaccharide to induce SOCS3 mRNA, as indicated by the kinetics of induction and sensitivity to polymyxin B inhibition. Finally, phosphorylation of the STAT1 transcription factor was significantly reduced inL.donovani-infected macrophages and required de novo transcription. The induction of SOCS3 provides a potent inhibitory mechanism by which intracellular microorganisms may suppress macrophage activation and interfere with the host immune response.
Salmonella
Infection Induces a Hypersecretory Phenotype in Human Intestinal Xenografts by Inducing Cyclooxygenase 2
Authors: Bertelsen Lone S. , Paesold Guenther , Eckmann Lars , Barrett Kim E.
Published: March 2003

Enteric
Salmonella
infection is accompanied by inflammation and diarrhea, and yet little is known about its effects on intestinal epithelial physiology. Since species differences limit the utility of animal tissues and cell lines lack relevant cell-cell interactions, we have used a human model of fetal intestine grown as xenografts in SCID mice. We investigated here the effects of
Salmonella
enterica
serovar Typhimurium SL1344 on xenograft ion transport. Harvested xenografts were stripped of seromuscular layers by blunt dissection, infected with
Salmonella
, and mounted in Ussing chambers.
Salmonella
infection for 1 h increased baseline ion transport without altering tissue conductance or morphology. The increased transport was blocked by the cyclooxygenase inhibitor, indomethacin, or the specific Cox-2 inhibitor, NS-398. Further, xenografts infected for 2 h showed increased secretory responses to the calcium-dependent agonist, carbachol, and the cyclic AMP-dependent agonists prostaglandin E
2
(PGE
2
) and forskolin, which were blocked by indomethacin. Western blot experiments revealed that infection was accompanied by increased cyclooxygenase 2 (Cox-2) expression, with no change in Cox-1 levels. Immunoassay demonstrated basolateral PGE
2
release, which was inhibited by indomethacin. Histological examination of infected xenografts illustrated that upregulated Cox-2 expression was restricted to the epithelium and that little or no invasion of the tissue by
Salmonella
occurred for up to 2 h. In summary,
Salmonella
infection rapidly increases Cox-2 expression in human intestinal tissue, accounting for increased epithelial ion transport characteristic of infectious diarrhea.
Interactions betweenBrucella melitensisand Human Phagocytes: Bacterial Surface O-Polysaccharide Inhibits Phagocytosis, Bacterial Killing, and Subsequent Host Cell Apoptosis
Authors: Fernandez-Prada Carmen M. , Zelazowska Elzbieta B. , Nikolich Mikeljon , Hadfield Ted L. , Roop R. Martin , Robertson Gregory L. , Hoover David L.
Published: March 2003

Brucellae are gram-negative intracellular pathogens that survive and multiply within host phagocytic cells. Smooth organisms present O-polysaccharides (OPS) on their surface. ThewboAgene, which codes for the enzyme glycosyl transferase, is essential for the assembly of O-chain inBrucella. Deletion ofwboAin smooth, virulentB. melitensis16M results in a rough mutant designated WRR51. UnlikeB. abortus, both smooth and rough strains ofB. melitensisare resistant to complement-mediated killing. To determine the role of surface OPS in the interactions ofB. melitensiswith monocytes/macrophages (M/M), 16M and WRR51 were transformed with the plasmid pBBR1MCS-6y encoding green fluorescent protein, and the transformants were used to infect human mononuclear phagocytes with and without fresh human serum as a source of complement. Human monocytes were cultured in the presence of macrophage colony-stimulating factor to allow their differentiation into macrophages during the course of infection. Intracellular bacteria were easily visualized using fluorescence microscopy. Infection in M/M, identified by surface staining and fate of infected phagocytes, was quantitated by flow cytometry. Rough bacteria were internalized, with no requirement for opsonization by serum, at a higher rate than smooth organisms. SmoothB. melitensissurvived and multiplied for at least 6 days inside M/M, but rough organisms were eliminated by death of the infected cells. In human monocytes cultured for 1 day without serum in order to trigger the apoptotic pathway, infection by rough brucellae accelerated phagocyte death; smooth brucellae inhibited apoptosis. This study suggests that the presence of surface OPS on liveB. melitensisbenefits the bacterium by preventing the death of macrophages,Brucella's preferred target for intracellular replication.
Flagellin of EnteropathogenicEscherichia coliStimulates Interleukin-8 Production in T84 Cells
Authors: Zhou Xin , Girón Jorge A. , Torres Alfredo G. , Crawford J. Adam , Negrete Erasmo , Vogel Stefanie N. , Kaper James B.
Published: March 2003

The type III secretion system (TTSS) of enteropathogenicEscherichia coli(EPEC) has been associated with the ability of these bacteria to induce secretion of proinflammatory cytokines, including interleukin-8 (IL-8), in cultured epithelial cells. However, the identity of the effector molecule directly involved in this event is unknown. In this study, we determined that the native flagellar filament and its flagellin monomer are activators of IL-8 release in T84 epithelial cells. Supernatants of wild-type EPEC strain E2348/69 and its isogenic mutants deficient in TTSS (escN) and in production of intimin (eae), grown in Luria-Bertani broth, elicited similar amounts of IL-8 secretion by T84 cells. In contrast, supernatants of EPECfliCmutants and of B171, a nonflagellated EPEC strain, were defective in inducing IL-8 release, a phenotype that was largely restored by complementation of thefliCgene in the mutant lacking flagella. Purified flagella fromE. coliK-12, EPEC serotypes H6 and H34, and enterohemorrhagicE. coliserotype H7 all induced IL-8 release in T84 cells. Induction of IL-8 by purified flagella or His-tagged FliC from EPEC strain E2348/69 was dose dependent and was blocked by a polyclonal anti-H6 antibody. Finally, the mitogen-activated protein kinases (Erk1 and -2 and Jnk) were phosphorylated in flagellin-treated T84 cells, and inhibition of the p38 and Erk pathways significantly decreased the IL-8 response induced by EPEC flagellin. Our data clearly indicate that FliC of EPEC is sufficient to induce IL-8 release in T84 cells and that activation of the Erk and p38 pathways is required for IL-8 induction.
CesD2 of Enteropathogenic
Escherichia coli
Is a Second Chaperone for the Type III Secretion Translocator Protein EspD
Authors: Neves Bianca C. , Mundy Rosanna , Petrovska Liljana , Dougan Gordon , Knutton Stuart , Frankel Gad
Published: March 2003

Enteropathogenic
Escherichia coli
(EPEC) and enterohemorrhagic
E. coli
are extracellular pathogens that employ a type III secretion system to export translocator and effector proteins, proteins which facilitates colonization of the mucosal surface of the intestine via formation of attaching and effacing (A/E) lesions. The genes encoding the proteins for A/E lesion formation are located on a pathogenicity island, termed the locus of enterocyte effacement (LEE), which contains
eae
encoding intimin as well as the type III secretion system and effector genes. Many type III secreted proteins are stabilized and maintained in a secretion-competent conformation in the bacterial cytosol by specific chaperone proteins. Three type III chaperones have been described thus far within the EPEC LEE region: CesD, for the translocator proteins EspB and EspD; CesT, for the effector proteins Tir and Map; and CesF, for EspF. In this study we report the characterization of CesD2 (previously Orf27), a second LEE-encoded chaperone for EspD. We show specific CesD2-EspD protein interaction which appears to be necessary for proper EspD secretion in vitro and pathogenesis in vivo as demonstrated in the A/E-lesion-forming mouse pathogen
Citrobacter rodentium
.
AnEscherichia coliMG1655 Lipopolysaccharide Deep-Rough Core Mutant Grows and Survives in Mouse Cecal Mucus but Fails To Colonize the Mouse Large Intestine
Authors: Møller Annette K. , Leatham Mary P. , Conway Tyrrell , Nuijten Piet J. M. , de Haan Louise A. M. , Krogfelt Karen A. , Cohen Paul S.
Published: March 2003

The ability ofE. colistrains to colonize the mouse large intestine has been correlated with their ability to grow in cecal and colonic mucus. In the present study, anE. coliMG1655 strain was mutagenized with a mini-Tn5Km (kanamycin) transposon, and mutants were tested for the ability to grow on agar plates with mouse cecal mucus as the sole source of carbon and nitrogen. One mutant, designated MD42 (for mucus defective), grew poorly on cecal-mucus agar plates but grew well on Luria agar plates and on glucose minimal-agar plates. Sequencing revealed that the insertion in MD42 was in thewaaQgene, which is involved in lipopolysaccharide (LPS) core biosynthesis. Like “deep-rough”E. colimutants, MD42 was hypersensitive to sodium dodecyl sulfate (SDS), bile salts, and the hydrophobic antibiotic novobiocin. Furthermore, its LPS core oligosaccharide was truncated, like that of a deep-rough mutant. MD42 initially grew in the large intestines of streptomycin-treated mice but then failed to colonize (<102CFU per g of feces), whereas its parent colonized at levels between 107and 108CFU per g of feces. When mouse cecal mucosal sections were hybridized with anE. coli-specific rRNA probe, MD42 was observed in cecal mucus as clumps 24 h postfeeding, whereas its parent was present almost exclusively as single cells, suggesting that clumping may play a role in preventing MD42 colonization. Surprisingly, MD42 grew nearly as well as its parent during growth in undiluted, highly viscous cecal mucus isolated directly from the mouse cecum and, like its parent, survived well after reaching stationary phase, suggesting that there are no antimicrobials in mucus that prevent MD42 colonization. After mini-marinertransposon mutagenesis, an SDS-resistant suppressor mutant of MD42 was isolated. The mini-marinerinsertion was shown to be in thebipAgene, a known regulator ofE. colisurface components. When grown in Luria broth, the LPS core of the suppressor mutant remained truncated; however, the LPS core was not truncated when the suppressor mutant was grown in the presence of SDS. Moreover, when the suppressor mutant was grown in the presence of SDS and fed to mice, it colonized the mouse large intestine. Collectively, the data presented here suggest that BipA may play a role inE. coliMG1655 LPS core biosynthesis and that because MD42 forms clumps in intestinal mucus, it is unable to colonize the mouse large intestine.
Involvement of Myeloid Dendritic Cells in the Development of Gastric Secondary Lymphoid Follicles inHelicobacter pylori-Infected Neonatally Thymectomized BALB/c Mice
Authors: Nishi Toshiki , Okazaki Kazuichi , Kawasaki Kimio , Fukui Toshiro , Tamaki Hiroyuki , Matsuura Minoru , Asada Masanori , Watanabe Tomohiro , Uchida Kazushige , Watanabe Norihiko , Nakase Hiroshi , Ohana Masaya , Hiai Hiroshi , Chiba Tsutomu
Published: March 2003

We previously described an animal model ofHelicobacter pylori-induced follicular gastritis in neonatally thymectomized (nTx) mice. However, it is still not clear whether antigen-presenting dendritic cells (DCs) in the stomach have a role in the development of secondary follicles inH. pylori-infected nTx mice. We investigated the distribution of DC subsets using this model and examined their roles. To identify lymphoid and myeloid DCs, sections were stained with anti-CD11c (pan-DC marker) in combination with anti-CD8α (lymphoid DC marker) or anti-CD11b (myeloid DC marker) and were examined with a confocal microscope. Expression of macrophage inflammatory protein 3α (MIP-3α), which chemoattracts immature DCs, was analyzed by real-time PCR and immunohistochemistry. Follicular dendritic cells (FDCs) were stained with anti-SKY28 antibodies. In noninfected nTx mice, a few myeloid and lymphoid DCs were observed in the bottom portion of the lamina propria, whereas inH. pylori-infected nTx mice, there was an increased influx of myeloid DCs throughout the lamina propria. FDC staining was also observed in the stomachs of members of the infected group. MIP-3α gene expression was upregulated in the infected nTx group, and the immunohistochemistry analysis revealed MIP-3α-positive epithelial cells. These data suggest thatH. pyloriinfection upregulates MIP-3α gene expression in gastric epithelial cells and induces an influx of myeloid DCs in the lamina propria of the gastric mucosa in nTx mice. Myeloid DCs and FDCs might contribute to the development of gastric secondary lymphoid follicles inH. pylori-infected nTx mice.
Mapping the Binding Domain of the F18 Fimbrial Adhesin
Authors: Smeds A. , Pertovaara M. , Timonen T. , Pohjanvirta T. , Pelkonen S. , Palva A.
Published: March 2003

F18 fimbrial
Esherichia coli
strains are associated with porcine postweaning diarrhea and pig edema disease. Recently, the FedF subunit was identified as the adhesin of the F18 fimbriae. In this study, adhesion domains of FedF were further studied by constructing deletions within the
fedF
gene and expressing FedF proteins with deletions either together with the other F18 fimbrial subunits or as fusion proteins tagged with maltose binding protein. The region essential for adhesion to porcine intestinal epithelial cells was mapped between amino acid residues 60 and 109 of FedF. To map the binding domain even more closely, all eight charged amino acid residues within this region were independently replaced by alanine. Three of these single point mutants expressing F18 fimbriae exhibited significantly diminished capabilities to adhere to porcine epithelial cells in vitro. In addition, a triple point mutation and a double point mutation completely abolished receptor adhesiveness. The result further confirmed that the region between amino acid residues 60 and 109 is essential for the binding of F18 fimbriae to their receptor. In addition, the adhesion capability of the binding domain was eliminated after treatment with iodoacetamide, suggesting the formation of a disulfide bridge between Cys-63 and Cys-83, whereas Cys-111 and Cys-116 could be deleted without affecting the binding ability of FedF.
Identification and Characterization of a Conserved, Stage-Specific Gene Product of
Plasmodium falciparum
Recognized by Parasite Growth Inhibitory Antibodies
Authors: Daubenberger Claudia A. , Diaz Diana , Curcic Marija , Mueller Markus S. , Spielmann Tobias , Certa Ulrich , Lipp Joachim , Pluschke Gerd
Published: March 2003

We have identified a novel conserved protein of
Plasmodium falciparum
, designated D13, that is stage-specifically expressed in asexual blood stages of the parasite. The predicted open reading frame (ORF) D13 contains 863 amino acids with a calculated molecular mass of 99.7 kDa and displays a repeat region composed of pentapeptide motives. Northern blot analysis with lysates of synchronized blood stage parasites showed that D13 is highly expressed at the mRNA level during schizogony. The first N′-terminal 138 amino acids of D13 were expressed in
Escherichia coli
and the purified protein was used to generate anti-D13 monoclonal antibodies (MAbs). Using total lysates of blood stage parasites and Western blot analysis, these MAbs stained one single band of ∼100 kDa, corresponding to the predicted molecular mass of ORF D13. Western blot analysis demonstrated further that D13 is expressed during schizogony, declines rapidly in early ring stages and is undetectable in trophozoites. D13 protein is localized in individual merozoites in a distinct area, as demonstrated by indirect immunofluorescence analysis. After subcellular fractionation, D13 was confined to the pelleted fraction of the parasite lysate and its extraction by alkaline carbonate buffer treatment indicated that D13 is not a membrane-integral protein. Inclusion of certain anti-D13 MAbs into in vitro cultures of blood stage parasites resulted in considerable reduction in parasite growth. The N′-terminal domain encompassing 158 amino acids is 94 and 95%, respectively, identical at the amino acid level between
Plasmodium knowlesi
,
Plasmodium yoelii
, and
P. falciparum
. In summary, we describe a novel stage-specifically expressed, highly conserved gene product of
P. falciparum
that is recognized by parasite growth inhibitory antibodies.
Rapid Protection of Gnotobiotic Pigs against Experimental Salmonellosis following Induction of Polymorphonuclear Leukocytes by AvirulentSalmonella enterica
Authors: Foster N. , Lovell M. A. , Marston K. L. , Hulme S. D. , Frost A. J. , Bland P. , Barrow P. A.
Published: March 2003

Oral inoculation of 5-day-old gnotobiotic pigs withSalmonella entericaserovar Typhimurium strain F98 resulted in severe enteritis and invasive disease. Preinoculation 24 h earlier with an avirulent mutant ofSalmonella entericaserovar Infantis (1326/28) completely prevented disease for up to 14 days (when the experiment was terminated).S. entericaserovar Infantis colonized the alimentary tract well, with high bacterial counts in the intestinal lumen but with almost no invasion into the tissues. Unprotected pigs had highS. entericaserovar Typhimurium counts in the intestines, blood, and major nonintestinal organs. Recovery of this strain from the blood and major organs inS. entericaserovar Infantis-protected pigs was substantially reduced despite the fact that intestinal counts were also very high. Protection against disease thus did not involve a colonization exclusion phenomenon. Significant (P< 0.05) infiltration of monocytes/macrophages was observed in the submucosal regions of the intestines of bothS. entericaserovar Infantis-protectedS. entericaserovar Typhimurium-challenged pigs and unprotectedS. entericaserovar Typhimurium-challenged pigs. However, only polymorphonuclear neutrophils (PMNs) were observed throughout the villus, where significant (P< 0.05) numbers infiltrated the lamina propria and the subnuclear and supranuclear regions of the epithelia, indicating that PMN induction and positioning followingS. entericaserovar Infantis inoculation was consistent with rapid protection against the challenge strain. Similarly, in vitro experiments using a human fetal intestinal epithelial cell line (INT 407) demonstrated that, although significantly (P< 0.05) fewerS. entericaserovar Infantis thanS. entericaserovar Typhimurium organisms invaded the monolayers,S. entericaserovar Infantis induced an NF-κB response and significantly (P< 0.05) raised interleukin 8 levels and transmigration of porcine PMN. The results of this study suggest that attenuatedSalmonellastrains can protect the immature intestine against clinical salmonellosis by PMN induction. They also demonstrate that PMN induction is not necessarily associated with clinical symptoms and/or intestinal pathology.
Pulmonary Necrosis Resulting from DNA Vaccination against Tuberculosis
Authors: Taylor Jennifer L. , Turner Oliver C. , Basaraba Randall J. , Belisle John T. , Huygen Kris , Orme Ian M.
Published: March 2003

The use of DNA constructs encoding mycobacterial proteins is a promising new approach to vaccination against tuberculosis. A DNA vaccine encoding the hsp60 molecule ofMycobacterium lepraehas previously been shown to protect against intravenous infection of mice withMycobacterium tuberculosisin both the prophylactic and immunotherapeutic modes. It is shown here, however, that this vaccine was not effective in a more realistic aerosol infection model or in a model of latent tuberculosis in the lungs. Moreover, when given in an immunotherapeutic model the immunized mice developed classical Koch reactions characterized by multifocal discrete regions of cellular necrosis throughout the lung granulomas. Similar and equally severe reactions were seen in mice given a vaccine with DNA coding for the Ag85 antigen ofM. tuberculosis. This previously unanticipated safety problem indicates that DNA vaccines should be used with caution in individuals who may have already been exposed to tuberculosis.
Group AStreptococcusGene Expression in Humans and Cynomolgus Macaques with Acute Pharyngitis
Authors: Virtaneva Kimmo , Graham Morag R. , Porcella Stephen F. , Hoe Nancy P. , Su Hua , Graviss Edward A. , Gardner Tracie J. , Allison James E. , Lemon William J. , Bailey John R. , Parnell Michael J. , Musser James M.
Published: March 2003

The molecular mechanisms used by group AStreptococcus(GAS) to survive on the host mucosal surface and cause acute pharyngitis are poorly understood. To provide new information about GAS host-pathogen interactions, we used real-time reverse transcription-PCR (RT-PCR) to analyze transcripts of 17 GAS genes in throat swab specimens taken from 18 pediatric patients with pharyngitis. The expression of known and putative virulence genes and regulatory genes (including genes in seven two-component regulatory systems) was studied. Several known and previously uncharacterized GAS virulence gene regulators were highly expressed compared to the constitutively expressed control geneproS. To examine in vivo gene transcription in a controlled setting, three cynomolgus macaques were infected with strain MGAS5005, an organism that is genetically representative of most serotype M1 strains recovered from pharyngitis and invasive disease episodes in North America and Western Europe. These three animals developed clinical signs and symptoms of GAS pharyngitis and seroconverted to several GAS extracellular proteins. Real-time RT-PCR analysis of throat swab material collected at intervals throughout a 12-day infection protocol indicated that expression profiles of a subset of GAS genes accurately reflected the profiles observed in the human pediatric patients. The results of our study demonstrate that analysis of in vivo GAS gene expression is feasible in throat swab specimens obtained from infected human and nonhuman primates. In addition, we conclude that the cynomolgus macaque is a useful nonhuman primate model for the study of molecular events contributing to acute pharyngitis caused by GAS.
Caenorhabditis elegansas a Model Host forStaphylococcus aureusPathogenesis
Authors: Sifri Costi D. , Begun Jakob , Ausubel Frederick M. , Calderwood Stephen B.
Published: March 2003

Staphylococcus aureus, an important pathogen of humans and other warm-blooded animals, is also capable of killing the nematodeCaenorhabditis elegans. Here, we show thatC. elegansorganisms that are fedS. aureusdie over the course of several days in a process that is correlated with the accumulation of bacteria within the nematode digestive tract. SeveralS. aureusvirulence determinants known or speculated to be important in mammalian pathogenesis, including the quorum-sensing global virulence regulatory systemagrand the global virulence regulatorsarA, the alternative sigma factor σB, alpha-hemolysin, and V8 serine protease, are required for full pathogenicity in nematodes. In addition, several definedC. elegansmutants were examined for susceptibility toS. aureusinfection. Enhanced susceptibility toS. aureuskilling was observed with loss-of-function mutations in theC. elegansgenesesp-2/sek-1andesp-8/nsy-1, which encode components of a conserved p38 MAP kinase signaling pathway involved in nematode defense against multiple pathogens. These results suggest that key aspects ofS. aureuspathogenesis have been conserved, irrespective of the host, and that specificC. eleganshost factors can alter susceptibility to this gram-positive human pathogen.
Cloning and Characterization of anEhrlichia canisGene Encoding a Protein Localized to the Morula Membrane
Authors: Teng Ching-Hao , Palaniappan Raghavan U. M. , Chang Yung-Fu
Published: March 2003

A gene encoding a 23.5-kDa ehrlichial morula membrane protein designated MmpA was cloned by screening anEhrlichia canisexpression library with convalescent dog sera, which resulted in three positive clones. Sequence analysis of the insert DNAs from all three clones indicated an open reading frame with a size of 666 bp that encodes MmpA. The structural analysis of MmpA indicated that it is a transmembrane protein with extreme hydrophobicity. Southern blot analysis of theHindIII-digested chromosomal DNA demonstrated the presence of a single copy of themmpAgene inE. canisandEhrlichia chaffeensisbut not in the human granulocytic ehrlichiosis agent. ThemmpAgene was amplified, cloned, and expressed as a fusion protein. Polyclonal antibodies to the recombinant protein (rMmpA) were raised in rabbits. Western blot analysis ofE. canisandE. chaffeensislysates with the anti-rMmpA serum resulted in the presence of an MmpA band only inE. canis, not inE. chaffeenesis. Sera from dogs which were either naturally or experimentally infected withE. canisrecognized the recombinant protein. Double immunofluorescence confocal microscopy studies demonstrated that MmpA was localized mainly on the morula membrane ofE. canis. Since the morula membrane is the interface between the ehrlichial growing environment and the host cytoplasm, MmpA may play a role in bacterium-host cell interactions.
Passive Immunity in
Helicobacter
-Challenged Neonatal Mice Conferred by Immunized Dams Lasts until Weaning
Authors: Corthésy-Theulaz Irène , Corthésy Blaise , Bachmann Daniel , Velin Dominique , Kraehenbuhl Jean-Pierre
Published: March 2003

The objective of this study was to examine the effect of breast-feeding by immunized dams on
Helicobacter
colonization in newborns. Urease-based immunization regimens failed to protect nursing pups against
H. felis
, whereas
H. felis
lysate-cholera toxin resulted in protection. This observation correlated with a high recognition of cell surface-expressed bacterial antigens by milk antibodies. Protection lasted until weaning, indicating that infection is maintained at undetectable levels by passive immunity but then resumes when breast-feeding stops.
Protein Binding between PcrG-PcrV and PcrH-PopB/PopD Encoded by thepcrGVH-popBDOperon of thePseudomonas aeruginosaType III Secretion System
Authors: Allmond Leonard R. , Karaca Timur J. , Nguyen Vinh N. , Nguyen Thong , Wiener-Kronish Jeanine P. , Sawa Teiji
Published: March 2003

Of the proteins encoded by thepcrGVH-popBDoperon of thePseudomonas aeruginosatype III secretion system, PcrG bound to PcrV and PcrH bound to PopB/PopD. In addition,YersiniaLcrG bound to PcrV, andYersiniaLcrH bound to PopD. The results imply a highly functional conservation of type III secretion betweenP.aeruginosaandYersiniaspecies.
Synergistic Protection of Mice against Plague with Monoclonal Antibodies Specific for the F1 and V Antigens of
Yersinia pestis
Authors: Hill Jim , Copse Catherine , Leary Sophie , Stagg Anthony J. , Williamson E. Diane , Titball Richard W.
Published: March 2003

Monoclonal antibodies specific for
Yersinia pestis
V antigen and F1 antigen, administered singly or in combination, protected mice in models of bubonic and pneumonic plague. Antibodies showed synergy when administered prophylactically and as a therapy 48 h postinfection. Monoclonal antibodies therefore have potential as a treatment for plague.
Enhanced Murine Antigen-Specific Gamma Interferon and Immunoglobulin G2a Responses by Using Mycobacterial ESAT-6 Sequences in DNA Vaccines
Authors: Minion F. Chris , Menon Sreekumar A. , Mahairas Gregory G. , Wannemuehler M. J.
Published: March 2003

TheMycobacterium tuberculosisprotein ESAT-6 has unusual immune stimulating activities, has been implicated in the recall of long-lived immunity, and induces protection against tuberculosis in mice. For many diseases caused by bacterial or viral pathogens, a strong cell-mediated immune (i.e., type 1) response is often required for recovery or protection. Therefore, it is important to design immunization regimens that induce agent-specific type 1 immunity. We have shown in previous studies that ESAT-6 could enhance antigen-specific type 1 immune responses in BALB/c mice against a second antigen when presented as a purified fusion protein. It was also of interest to determine if ESAT-6 could enhance the type 1 response against a second antigen beyond that afforded by DNA vaccination through CpG motifs. This was tested by using gene fusions of ESAT-6 and theMycoplasma hyopneumoniaesurface antigen P71. Modified P71 gene sequences were cloned with or without ESAT-6 sequences into a DNA vaccine vector and were used to immunize mice. Splenic lymphocytes from vaccinated mice were tested for gamma interferon (IFN-γ) and interleukin-10 (IL-10) secretion. Serum antibodies were examined for P71 antigen-specific isotype responses. When stimulated in vitro with purified P71 antigen, splenocytes from the ESAT-6:P71 vaccinates secreted higher levels of IFN-γ and lower levels of IL-10 compared to those of vaccinates receiving the P71 construct alone. Furthermore, the immunoglobulin G2a serum antibody levels were significantly higher in the ESAT-6:P71 vaccinates compared to those of the vaccinates receiving P71 alone. In conclusion, ESAT-6 was shown to enhance antigen-specific type 1 immune responses in BALB/c mice when used in DNA vaccines.
Th2 Responses Predominate during the Early Phases of Infection in Patients with Localized Cutaneous Leishmaniasis and Precede the Development of Th1 Responses
Authors: Bourreau Eliane , Gardon Jacques , Pradinaud Roger , Pascalis Hervé , Prévot-Linguet Ghislaine , Kariminia Amina , Pascal Launois
Published: March 2003

Intralesional Th2 responses preceded the development of Th1 responses in localized cutaneous leishmaniasis due to
Leishmania guyanensis
. Although the number of parasites increased in Th2 lesions, no correlation was found between the levels of cytokine expression and the number of parasites. In contrast, the decreased number of parasites in Th1 lesions is negatively correlated to gamma interferon expression.
Molecular Characterization of the Prototrophic
Salmonella
Mutants Defective for Intraepithelial Replication
Authors: Suvarnapunya Akamol E. , Zurawski Daniel V. , Guy Rebecca L. , Stein Murry A.
Published: March 2003

Three Mu
d
J prototrophs demonstrated that intracellular replication is a
Salmonella
virulence trait (K. Y. Leung and B. B. Finlay, Proc. Natl. Acad. Sci. USA,
88:
11470-11474, 1991).
mutS
and
mutH
are disrupted in mutants 3-11 and 12-23, and
ssaQ
is disrupted in mutant 17-21. Further analysis revealed that loss of
Salmonella
pathogenicity island 2 function underlies the intracellular replication defect of 3-11 and 17-21.
Neisseria meningitidis
RTX Proteins Are Not Required for Virulence in Infant Rats
Authors: Forman Stanislav , Linhartova Irena , Osicka Radim , Nassif Xavier , Sebo Peter , Pelicic Vladimir
Published: March 2003

RTX cytotoxins play an important role in virulence of numerous gram-negative pathogens. Unexpectedly, however, we show here that the RTX proteins of
Neisseria meningitidis
are dispensable for virulence in the infant rat model of infection.
Vector Priming Reduces the Immunogenicity ofSalmonella-Based Vaccines inNramp1+/+Mice
Authors: Vindurampulle Christofer J. , Attridge Stephen R.
Published: March 2003

The present studies inNramp1−/−BALB/c andNramp1+/+CBA mice question the significance of this genotype as a determinant of the level of gut colonization following oral administration of naturally attenuated or highly virulentSalmonellastrains. In line with previous results in BALB/c hosts, vector priming of CBA mice withSalmonella entericaserovar Stanley was found to significantly compromise the immunogenicity of a recombinant construct expressing a foreign pilus protein.
Polymorphisms within EspA Filaments of Enteropathogenic and Enterohemorrhagic
Escherichia coli
Authors: Neves Bianca C. , Shaw Robert K. , Frankel Gad , Knutton Stuart
Published: March 2003

Enteropathogenic
Escherichia coli
(EPEC) and enterohemorrhagic
E
.
coli
(EHEC) possess a filamentous type III secretion system (TTSS) employed to deliver effector proteins into host cells. EspA is a type III secreted protein which forms the filamentous extension to the TTSS and which interacts with host cells during early stages of attaching and effacing (A/E) lesion formation. By immunofluorescence, a polyclonal antibody previously raised to EspA from EPEC strain E2348/69 (O127:H6) stained ∼12-nm-diameter EspA filaments produced by this strain but did not stain similar filaments produced by EHEC serotype O157:H7. Similarly, an antibody that we subsequently raised to EHEC strain 85-170 (O157:H7) EspA stained ∼12-nm-diameter EspA filaments produced by strain 85-170 but did not stain E2348/69 EspA filaments. Given such heterogeneity between EPEC and EHEC EspA filaments, we examined polymorphisms of functional EspA filaments among different EPEC and EHEC serotypes. With use of the EPEC EspA antiserum, EspA filaments were observed only with EPEC serotypes O127:H6 and O55:H6, serotypes which encode an identical EspA protein. When stained with the EHEC EspA antiserum, EspA filaments were detected only on EHEC strains belonging to serotype O157:H7; the EHEC antiserum did, however, stain EspA filaments produced by the closely related EPEC serotype O55:H7 but not filaments of any other EPEC serotype tested. Such polymorphisms among functional EspA filaments of EPEC and EHEC would be expected to have important implications for the development of broad-range EspA-based vaccines.
Expression of
Helicobacter pylori
Vacuolating Toxin in
Escherichia coli
Authors: McClain Mark S. , Cover Timothy L.
Published: March 2003

VacA is a secreted toxin that plays a role in
Helicobacter pylori
colonization of the stomach and that contributes to the pathogenesis of peptic ulcer disease. Studies of VacA structure and function have been hindered by the lack of an efficient system for expression and genetic manipulation of this toxin. In this study, we developed methodology for expression of a functionally active VacA toxin in
Escherichia coli
. We then used a high-throughput screen to analyze a library of mutant toxins with pentapeptide insertions and identified six mutants that lacked the capacity to induce vacuolation of HeLa cells. The capacity to analyze VacA in this heterologous-expression system should greatly facilitate efforts to elucidate the structure and function of this toxin.
Mucosal Immunization with a Genetically Engineered Pertussis Toxin S1 Fragment-Cholera Toxin Subunit B Chimeric Protein
Authors: Lee Song F. , Halperin Scott A. , Salloum Danny F. , MacMillan Ann , Morris Annette
Published: March 2003

A chimeric protein consisting of a divalent pertussis toxin (PT) S1 fragment linked to the cholera toxin (Ctx) A2B fragment was constructed. The chimera induced a mucosal immunoglobulin A (IgA) and a serum IgG immune response to PT and CtxB in BALB/c mice following intranasal immunization. The immune sera neutralized PT in vitro. In the mouse model ofBordetella pertussisrespiratory infection, the chimera-immunized animals showed a significant reduction in bacterial lung counts (P= 0.01) from that of the sham control group. Thus, a divalent S1 fragment CtxA2B chimera is an immunogenic antigen and can elicit a protective immunity.
Interleukin-10 Negatively Regulates Local Cytokine and Chemokine Production but Does Not Influence Antibacterial Host Defense during Murine Pneumococcal Meningitis
Authors: Zwijnenburg Petra J. G. , van der Poll Tom , Florquin Sandrine , Roord John J. , van Furth A. Marceline
Published: March 2003

To determine the role of endogenous interleukin-10 (IL-10) in local host defense during pneumococcal meningitis, the inflammatory responses of IL-10-gene-deficient and wild-type mice after the induction of meningitis were compared. The absence of IL-10 was associated with higher cytokine and chemokine concentrations and a more pronounced infiltrate, but antibacterial defense or survival was not influenced.