bluuebunny/crossref_metadata_2025_split huggingface dataset:
Optimal Tumor Necrosis Factor Induction byPlasmodium falciparumRequires the Highly Localized Release of Parasite Products
Authors: O'Dea Kieran P. , Pasvol Geoffrey 
Published: May 2003

ABSTRACTOverproduction of tumor necrosis factor (TNF) has been linked with the pathogenesis ofPlasmodium falciparummalaria. Here, we examined why the high levels of TNF-inducing activity associated withP. falciparum-parasitized erythrocytes (PE) appear to be lost after cell lysis. Static coculture of PE and peripheral blood mononuclear cells (PBMC), with or without separation by porous membranes, demonstrated that rupture of live PE in the presence of responder cells was required for optimal TNF induction. Although the insoluble fraction of lysed PE was found to partially inhibit TNF responses, supernatants prepared from large numbers of lysed PE still contained only low levels of TNF-inducing activity, which showed no evidence of instability. A dramatic reduction in TNF levels resulted when noncytoadherent PE lines were maintained under low-cell-proximity conditions by suspension coculture. This reduction was much less marked with PE capable of adhering to PBMC, despite the fact that cytoadherent and noncytoadherent parasite lines induced comparable levels of TNF in high-cell-proximity, static coculture. These results suggest that rupture of PE in a highly localized setting, facilitated by either static coculture or the more biologically relevant phenomenon of cytoadherence to PBMC, can result in considerable enhancement of theP. falciparum-induced TNF response.
Humoral and Cellular Immune Responses toTrypanosoma cruzi-Derived Paraflagellar Rod Proteins in Patients with Chagas' Disease
Authors: Michailowsky Vladimir , Luhrs Keith , Rocha Manoel Otávio C. , Fouts David , Gazzinelli Ricardo T. , Manning Jerry E. 
Published: May 2003

ABSTRACTSera and peripheral blood mononuclear cells (PBMC) from patients displaying different clinical symptoms as well as from normal uninfected individuals (NI) were used to evaluate the humoral and cellular responses of Chagas' disease patients toTrypanosoma cruzi-derived paraflagellar rod proteins (PFR). Our results show that sera from both asymptomatic Chagas' disease patients (ACP) and cardiac Chagas' disease patients (CCP) have higher levels of antibodies to PFR than sera from NI. Immunoglobulin G1 (IgG1) and IgG3 were the main Ig isotypes that recognized PFR. We also tested three recombinant forms of PFR, named rPAR-1, rPAR-2, and rPAR-3, by Western blot analysis. Sera from seven out of eight patients with Chagas' disease recognized one of the three rPAR forms. Sera from 75, 50, and 37.5% of Chagas' disease patients tested recognized rPAR-3, rPAR-2, and rPAR-1, respectively. PFR induced proliferation of 100 and 70% of PBMC from ACP and CCP, respectively. Further, stimulation of cells from Chagas' disease patients with PFR enhanced the frequencies of both small and large CD4+CD25+and CD4+CD69+lymphocytes, as well as that of small CD8+CD25+lymphocytes. Finally, we evaluated the ability of PFR to elicit the production of gamma interferon (IFN-γ) by PBMC from patients with Chagas' disease. Fifty percent of the PBMC from ACP as well as CCP produced IFN-γ upon stimulation with PFR. PFR enhanced the percentages of IFN-γ-producing cells in both CD3+and CD3−populations. Within the T-cell population, large CD4+T lymphocytes were the main source of IFN-γ.
Perforin and Gamma Interferon Are Critical CD8+T-Cell-Mediated Responses in Vaccine-Induced Immunity againstLeishmania amazonensisInfection
Authors: Colmenares María , Kima Peter E. , Samoff Erika , Soong Lynn , McMahon-Pratt Diane 
Published: May 2003

ABSTRACTPrevious studies have demonstrated that protection against New World leishmaniasis caused byLeishmania amazonensiscan be elicited by immunization with the developmentally regulatedLeishmaniaamastigote antigen, P-8. In this study, several independent experimental approaches were employed to investigate the protective immunological mechanisms involved. T-cell subset depletion experiments clearly indicate that elicitation of CD8+(as well as CD4+) effector responses is required for protection. Further, mice lacking β2-microglobulin (and hence deficient in major histocompatibility complex class I antigen presentation) were not able to control a challenge infection after vaccination, indicating an essential protective role for CD8+T effector responses. Analysis of the events ongoing at the cutaneous site of infection indicated a changing cellular dynamic involved in protection. Early postinfection in protectively vaccinated mice, a predominance of CD8+T cells, secreting gamma interferon (IFN-γ) and expressing perforin, was observed at the site of infection; subsequently, activated CD4+T cells producing IFN-γ were primarily found. As protection correlated with the ratio of total IFN-γ-producing cells (CD4+and CD8+T cells) to macrophages found at the site of infection, a role for IFN-γ was evident; in addition, vaccination of IFN-γ-deficient mice failed to provide protection. To further assess the effector mechanisms that mediate protection, mice deficient in perforin synthesis were examined. Perforin-deficient mice vaccinated with the P-8 antigen were unable to control infection. Thus, the elicitation of CD8+T cell effector mechanisms (perforin, IFN-γ) are clearly required in the protective immune response againstL. amazonensisinfection in vaccinated mice.
Characterization of Anthrolysin O, theBacillus anthracisCholesterol-Dependent Cytolysin
Authors: Shannon Jeffrey G. , Ross Cana L. , Koehler Theresa M. , Rest Richard F. 
Published: May 2003

ABSTRACTWe characterized the expression of a putative toxin ofBacillus anthracis, a member of the cholesterol-dependent cytolysin (CDC) family, which includes listeriolysin O, perfringolysin O, and streptolysin O. We named this cytotoxin anthrolysin O (ALO). AlthoughB. anthracisexpresses minimal hemolytic activity in clinical settings, we show that Sterne strain 7702 expresses hemolytic activity when grown in brain heart infusion broth or in other rich bacteriologic media, but it secretes barely detectable amounts of hemolysin when grown in Luria-Bertani (LB) broth. Glucose supplementation of LB broth increases the amount of secreted hemolytic activity. Expression of hemolytic activity is maximal during mid- to late-log phase and decreases in the stationary phase. These observations are supported, in part, by semiquantitative reverse transcriptase PCR ofalomRNA. Hemolytic activity in growth supernatants was increased in the presence of reducing agent and almost totally inhibited in a dose-dependent manner by cholesterol; both of these activities are characteristic of a CDC toxin. A mutant of Sterne strain 7702, strain UT231, in which thealogene was deleted and replaced by a kanamycin cassette, secreted barely detectable hemolytic activity into the growth medium. When strain UT231 was complemented intranswith nativealoon a low-copy-number plasmid [strain UT231(pUTE554)], it regained the ability to secrete hemolytic activity, indicating that ALO is the major hemolysin secreted by this strain ofB. anthracisin rich media in vitro. To further support thealogene product being a hemolysin, recombinantB. anthracisALO (rALO) purified fromEscherichia coliwas extremely active against washed human erythrocytes, with complete hemolysis detected at ∼30 molecules of rALO per erythrocyte. Considering the virulence roles of CDCs for other gram-positive bacteria, we speculate that ALO may have a role in anthrax virulence.
Expression of MultipleCPBGenes Encoding Cysteine Proteases Is Required forLeishmania mexicanaVirulence In Vivo
Authors: Denise Hubert , McNeil Kathryn , Brooks Darren R. , Alexander James , Coombs Graham H. , Mottram Jeremy C. 
Published: May 2003

ABSTRACTLeishmania mexicanamutants deficient in the multicopyCPBgene array have reduced virulence, demonstrated by poor lesion growth in BALB/c mice and induction of a protective Th1 response. Reinsertion of the amastigote-specificCPB2.8or metacyclic stage-specificCPB2gene into aCPB-deficient mutantL. mexicanafailed to restore either a Th2 response or sustained virulence. However, reexpression of multipleCPBgenes from a cosmid significantly restored virulence. This was characterized by increased lesion and parasite growth and the acquisition of a Th2 response, as determined by measuring interleukin-4 production and immunoglobulin G1 (IgG1) and IgE levels. These studies confirm thatL. mexicanacysteine proteases are important virulence factors and provide an explanation for the presence inL. mexicanaof a multicopy tandem array ofCPBgenes.
Modulation of Virulence by Two Acidified Nitrite-Responsive Loci ofSalmonella entericaSerovar Typhimurium
Authors: Kim Charles C. , Monack Denise , Falkow Stanley 
Published: May 2003

ABSTRACTTwo acidified nitrite-inducible genes ofSalmonella entericaserovar Typhimurium were identified with a green fluorescent protein-based promoter-trap screen. The nitrite-inducible promoters were located upstream of loci that we designatednipABandnipC, which correspond tohcp-hcr(hybrid cluster protein) ofEscherichia coliandnorAofAlcaligenes eutrophus, respectively. Maximal induction of the promoters by nitrite was dependent on pH. ThenipABpromoter was regulated by oxygen in an Fnr-dependent manner. ThenipCpromoter was also regulated by oxygen but in an Fnr-independent manner. The promoters were upregulated in activated RAW264.7 macrophage-like cells, which produce NO via the inducible nitric oxide synthase (iNOS), and the induction was inhibited by aminoguanidine, an inhibitor of iNOS. Although thenipABandnipCmutants displayed no defects under a variety of in vitro conditions or in tissue culture infections, they exhibited lower oral 50% lethal doses (LD50s) than did the wild type in C57BL/6J mouse infections. The lower LD50s reflected an unexpected increased ability of small inoculating doses of the mutant bacteria to cause lethal infection 2 to 3 weeks after challenge, compared to a similar challenge dose of wild-type bacteria. We conclude that these genes are regulated by physiological nitrogen oxides and that the absence of these bacterial genes in some way diminishes the ability of mice to clear a low dose infection.
Mutation ofluxSofStreptococcus pneumoniaeAffects Virulence in a Mouse Model
Authors: Stroeher Uwe H. , Paton Adrienne W. , Ogunniyi A. David , Paton James C. 
Published: May 2003

ABSTRACTThe LuxS protein is required for the biosynthesis of the type 2 autoinducer (AI-2), which is involved in quorum sensing in a wide range of bacterial species. We have determined the effects of a definedluxSmutation on the virulence ofStreptococcus pneumoniae. Although theluxSmutant displayed reduced virulence relative to its wild-type parent, the type 2 strain D39, it was by no means avirulent in a mouse model. After intranasal administration, theluxSmutant was able to colonize the nasopharynx of the mouse as efficiently as the wild type. However, it was less able to spread from the nasopharynx to the lungs or the blood. Intraperitoneal coadministration studies indicated that theluxSmutant was less fit and was readily outcompeted by wild-type D39. However, when administered on its own by this route, the mutant was able to proliferate and cause fatal systemic disease, albeit at a lower rate than the wild type. Western blot analysis of whole-cell lysates of the mutant and its parent did not reveal any differences in the levels of several well-characterized virulence proteins. However, analysis of Coomassie blue-stained protein profiles after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that mutation ofluxShad pleiotropic effects on protein expression in all cellular compartments. This is consistent with the product ofluxShaving a regulatory role inS.pneumoniae. This is the first report of a direct role forluxS(and by inference, AI-2) in the virulence of a gram-positive pathogen. However, the fact that mutagenesis ofluxSdoes not completely attenuateS.pneumoniaehas implications for the possible use of AI-2 antagonists for treatment of pneumococcal infections.
Purification and Characterization of Enterotoxigenic El Tor-Like Hemolysin Produced byVibrio fluvialis
Authors: Kothary Mahendra H. , Lowman Heather , McCardell Barbara A. , Tall Ben D. 
Published: May 2003

ABSTRACTThe halophilic bacteriumVibrio fluvialisis an enteric pathogen that produces an extracellular hemolysin. This hemolysin was purified to homogeneity by using sequential hydrophobic-interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. It has a molecular weight of 63,000 and an isoelectric point of 4.6, and its hemolytic activity is sensitive to heat, proteases, and preincubation with zinc ions. The hemolysin lyses erythrocytes of the eight different animal species that we tested, is cytotoxic against Chinese hamster ovary cells in tissue culture, and elicits fluid accumulation in suckling mice. Lysis of erythrocytes occurs by a temperature-dependent binding step followed by a temperature- and pH-dependent lytic step. Fourteen of the first 20 N-terminal amino acid residues (Val-Ser-Gly-Gly-Glu-Ala-Asn-Thr-Leu-Pro-His-Val-Ala-Phe-Tyr-Ile-Asn-Val-Asn-Arg) are identical to those of the El Tor hemolysin ofVibrio choleraeand the heat-labile hemolysin ofVibrio mimicus. This homology was further confirmed by PCR analysis using a 5′ primer derived from the amino-terminal sequence of the hemolysin and a 3′ primer derived from the El Tor hemolysin structural gene. The hemolysin also reacts with antibodies to the El Tor-like hemolysin of non-O1V. cholerae.
Lipopolysaccharide-Mimetic Activities of a Toll-Like Receptor 2-Stimulatory Substance(s) in Enterobacterial Lipopolysaccharide Preparations
Authors: Muroi Masashi , Ohnishi Takahiro , Azumi-Mayuzumi Satoko , Tanamoto Ken-ichi 
Published: May 2003

ABSTRACTLipopolysaccharide (LPS) preparations are known to often contain substances which activate cells through Toll-like receptor 2 (TLR2), and it is suspected that bacterial lipoproteins are responsible for this activation. We compared the mode of action of the TLR2-stimulatory substances with that of a synthetic bacterial lipopeptide (tripalmitoyl-Cys-Ser-Ser-Asn-Ala [Pam3CSSNA]), as well as with that of peptidoglycan. Six out of eight LPS preparations tested induced NF-κB-dependent reporter activity in 293 cells expressing CD14 and TLR2. Phenol extract (PEX) prepared fromEscherichia coliLPS by modified phenol extraction induced reporter activity in 293 cells expressing TLR2, and this activity was enhanced by coexpression of CD14, whereas the activity of Pam3CSSNA was not dependent on CD14. The activity of PEX, but not that of Pam3CSSNA or peptidoglycan, was also enhanced by LPS binding protein or serum and blocked by polymyxin B. In addition, the activity of PEX was inhibited by a lipid A precursor (compound 406) in 293 cells expressing CD14 and TLR2. These results indicate thatE. coliLPS preparations contain LPS-mimetic TLR2-stimulatory substances which differ from bacterial lipopeptides or peptidoglycan.
The Secreted Aspartyl Proteinases Sap1 and Sap2 Cause Tissue Damage in an In Vitro Model of Vaginal Candidiasis Based on Reconstituted Human Vaginal Epithelium
Authors: Schaller Martin , Bein Matthias , Korting Hans C. , Baur Stefan , Hamm Gerald , Monod Michel , Beinhauer Sabine , Hube Bernhard 
Published: May 2003

ABSTRACT

Secreted aspartyl proteinases (Saps) contribute to the ability of
Candida albicans
to cause mucosal and disseminated infections. A model of vaginal candidiasis based on reconstituted human vaginal epithelium (RHVE) was used to study the expression and role of these
C. albicans
proteinases during infection and tissue damage of vaginal epithelium. Colonization of the RHVE by
C. albicans
SC5314 did not cause any visible epithelial damage 6 h after inoculation, although expression of
SAP2
,
SAP9
, and
SAP10
was detected by reverse transcriptase PCR. However, significant epithelial damage was observed after 12 h, concomitant with the additional expression of
SAP1
,
SAP4
, and
SAP5
. Additional transcripts of
SAP6
and
SAP7
were detected at a later stage of the artificial infection (24 h). Similar
SAP
expression profiles were observed in three samples isolated from human patients with vaginal candidiasis. In experimental infection, secretion of antigens Sap1 to Sap6 by
C. albicans
was confirmed at the ultrastructural level by using polyclonal antisera raised against Sap1 to Sap6. Addition of the aspartyl proteinase inhibitors pepstatin A and the human immunodeficiency virus proteinase inhibitors ritonavir and amprenavir strongly reduced the tissue damage of the vaginal epithelia by
C. albicans
cells. Furthermore,
SAP
null mutants lacking either
SAP1
or
SAP2
had a drastically reduced potential to cause tissue damage even though
SAP3
,
SAP4
, and
SAP7
were up-regulated in these mutants. In contrast the vaginopathic potential of mutants lacking
SAP3
or
SAP4
to
SAP6
was not reduced compared to wild-type cells. These data provide further evidence for a crucial role of Sap1 and Sap2 in
C. albicans
vaginal infections.
Protection against Shiga Toxin 1 Challenge by Immunization of Mice with Purified Mutant Shiga Toxin 1
Authors: Ishikawa Satoshi , Kawahara Kazuyoshi , Kagami Yutaka , Isshiki Yasunori , Kaneko Aki , Matsui Hidenori , Okada Nobuhiko , Danbara Hirofumi 
Published: May 2003

ABSTRACT

Shiga toxin 1 (Stx1) of enterohemorrhagic
Escherichia coli
O157:H7 was cloned, and four mutant Stx1s were constructed by site-directed mutagenesis with PCR. The wild-type and mutant Stx1s with amino acid replacements at positions 167 and 170 of the A subunit were purified by one-step affinity chromatography with commercially available Globotriose Fractogel, and the mutant Stxs were used for the immunization of mice. The mutant toxins were nontoxic to Vero cells in vitro and to mice in vivo and induced the immunoglobulin G antibody against the wild-type Stx1, which neutralized the cytotoxicity of Stx1. The induced antibody titers depended on the mutation at position 170 of the A subunit. The mice immunized with the mutant Stx1s were protected against a challenge of approximately 100 times the 50% lethal dose of the wild-type Stx1, suggesting that the mutant toxins are good candidates for toxoid vaccines for infection by Stx1-producing
E. coli.
Antigenic Organization of the N-Terminal Part of the Polymorphic Outer Membrane Proteins 90, 91A, and 91B ofChlamydophilaabortus
Authors: Vretou Evangelia , Giannikopoulou Panagiota , Longbottom David , Psarrou Evgenia 
Published: May 2003

ABSTRACTA series of overlapping recombinant antigens, 61 to 74 residues in length, representing polymorphic outer membrane protein 90 (POMP90) ofChlamydophilaabortusand two recombinant peptides spanning gene fragmentp91Bf99of POMP91B were assessed by immunoblotting to determine the antigen-binding sites of 20 monoclonal antibodies to POMP90, -91A, and -91B. The epitopes were further restricted by scanning 52 overlapping synthetic 12-mer peptides representing the N-terminal part of POMP90, and the 12-mer epitopes were then analyzed by using hexapeptides to the resolution of a single amino acid. Ten epitopes were defined: 1, TSEEFQVKETSSGT; 2, SGAIYTCEGNVCISYAGKDSPL; 3, SLVFHKNCSTAE; 4, AIYADKLTIVSGGPTLFS; 5, SPKGGAISIKDS; 6, ITFDGNKIIKTS; 7, LRAKDGFGIFFY; 7a, DGFGIF; 7b, GIFFYD; 8, IFFYDPITGGGS; 8a, FFYDPIT; 9, GKIVFSGE; and 10, DLGTTL. The 20-mer peptide LRAKDGFGIFFYDPITGGGS was a major epitope that was recognized by seven antibodies. Epitopes 7 to 10 were conserved in reference strains of the former speciesC. psittaci, whereas the strong antigenic peptides FYDPIT and IVFSGE were conserved among members of the genusChlamydophila. Epitopes 3 to 8 were located within the best-scoring beta-helical wrap (residues 148 to 293) predicted for POMP91B by the program BETAWRAP. Other studies have suggested an association of the POMPs with type V secretory autotransporter proteins. The results presented in this study provide some evidence for a passenger domain that is folded as a beta-helix pyramid with compact antigenic organization.
Killing of
Candida albicans
by Human Salivary Histatin 5 Is Modulated, but Not Determined, by the Potassium Channel TOK1
Authors: Baev Didi , Rivetta Alberto , Li Xuewei S. , Vylkova Slavena , Bashi Esther , Slayman Clifford L. , Edgerton Mira 
Published: May 2003

ABSTRACT

Salivary histatin 5 (Hst 5), a potent toxin for the human fungal pathogen
Candida albicans
, induces noncytolytic efflux of cellular ATP, potassium, and magnesium in the absence of cytolysis, implicating these ion movements in the toxin's fungicidal activity. Hst 5 action on
Candida
resembles, in many respects, the action of the K1 killer toxin on
Saccharomyces cerevisiae
, and in that system the yeast plasma membrane potassium channel, Tok1p, has recently been reported to be a primary target of toxin action. The question of whether the
Candida
homologue of
Saccharomyces
Tok1p might be a primary target of Hst 5 action has now been investigated by disruption of the
C. albicans TOK1
gene. The resultant strains (
TOK1/tok1
) and (
tok1/tok1
) were compared with wild-type
Candida
(
TOK1/TOK1
) for relative ATP leakage and killing in response to Hst 5. Patch-clamp measurements on
Candida
protoplasts were used to verify the functional deletion of Tok1p and to provide its first description in
Candida
. Tok1p is an outwardly rectifying, noisily gated, 40-pS channel, very similar to that described in
Saccharomyces
. Knockout of
CaTOK1
(
tok1/tok1
) completely abolishes the currents and gating events characteristic of Tok1p. Also, knockout (
tok1/tok1
) increases residual viability of
Candida
after Hst 5 treatment to 27%, from 7% in the wild type, while the single allele deletion (
TOK1/tok1
) increases viability to 18%. Comparable results were obtained for Hst-induced ATP efflux, but quantitative features of ATP loss suggest that wild-type
TOK1
genes function cooperatively. Overall, very substantial killing and ATP efflux are produced by Hst 5 treatment after complete knockout of wild-type
TOK1
, making clear that Tok1p channels are not the primary site of Hst 5 action, even though they do play a modulating role.