bluuebunny/crossref_metadata_2025 huggingface dataset:
Up-Regulation of Interleukin-8 by Novel Small Cytoplasmic Molecules of NontypeableHaemophilus influenzaevia p38 and Extracellular Signal-Regulated Kinase Pathways
Authors: Wang Beinan , Cleary P. Patrick , Xu Haidong , Li Jian-Dong
Published: September 2003

NontypeableHaemophilus influenzae(NTHI) is an important etiological agent of otitis media (OM) and of exacerbated chronic obstructive pulmonary diseases (COPD). Inflammation is a hallmark of both diseases. Interleukin-8 (IL-8), one of the important inflammatory mediators, is induced by NTHI and may play a significant role in the pathogenesis of these diseases. Our studies demonstrated that a soluble cytoplasmic fraction (SCF) from NTHI induced much greater IL-8 expression by human epithelial cells than did NTHI lipooligosaccharides and envelope proteins. The IL-8-inducing activity was associated with molecules of ≤3 kDa from SCF and was peptidase and lipase sensitive, suggesting that small lipopeptides are responsible for the strong IL-8 induction. Moreover, multiple intracellular signaling pathways were activated in response to cytoplasmic molecules. The results indicated that the p38 mitogen-activated protein kinase (MAPK) and Src-dependent Raf-1-Mek1/2-extracellular signal-regulated kinase mitogen-activated protein kinase (ERK MAPK) pathways are required for NTHI-induced IL-8 production. In contrast, the phosphatidylinositol 3-kinase (PI3K)-Akt pathway did not affect IL-8 expression, although this pathway was concomitantly activated upon exposure to NTHI SCF. The PI3K-Akt pathway was also directly activated by IL-8 and significantly inhibited by an antagonist of IL-8 receptors during NTHI stimulation. These results indicated that the PI3K-Akt pathway is activated in response to IL-8 that is induced by NTHI and may lead to other important epithelial cell responses. This work provides insight into essential molecular and cellular events that may impact on the pathogenesis of OM and COPD and identifies rational targets for anti-inflammatory intervention.
Differential Expression of Cytokine Genes and Inducible Nitric Oxide Synthase Induced by Opacity Phenotype Variants ofStreptococcus pneumoniaeduring Acute Otitis Media in the Rat
Authors: Long J. P. , Tong H. H. , Shannon P. A. , DeMaria T. F.
Published: September 2003

Phase variation in the colonial opacity phenotype ofStreptococcus pneumoniaehas been implicated as a factor in bacterial adherence, colonization, and invasion in the pathogenesis of pneumococcal otitis media (OM). The purpose of this study was to determine whetherS. pneumoniaeopacity variants influence the induction of gene expression for proinflammatory mediators in vivo using the rat model of OM. Both the opaque and transparent phenotype variants induced a significant up-regulation in gene expression for interleukin-1α (IL-1α), IL-1β, IL-6, IL-10, tumor necrosis factor alpha, and inducible nitric oxide synthase (iNOS) compared to saline sham-inoculated controls at both 4 and 24 h postinoculation (P< 0.05 in all cases). Furthermore, whereas a significant difference in gene expression was evident for only IL-6 (greater following challenge with the opaque variant) and IL-1β (greater following challenge with the transparent variant) at 4 h, by 24 h the opaque variant cohort demonstrated a significant increase in gene expression for IL-1α, IL-1β, IL-6, IL-10, and iNOS relative to animals inoculated with the transparent phenotype variant (P< 0.05 in all cases). Enzyme-linked immunosorbent assay results confirmed the gene expression data as determined by real-time PCR. Moreover, the concentrations of the opaque variant in the middle ear lavage fluid were a full log higher than those of the transparent variant. The aforementioned results indicate that the opaque phenotype variant is more efficient at survival and multiplication within the middle ear space, resulting in the accumulation of more inflammatory cells and the enhanced expression and production of inflammatory mediators. However, when the data were normalized to account for differences in middle ear bacterial titers, it became apparent that the transparent variant ofS. pneumoniaeis a more potent inducer of inflammation, triggering the accumulation of more inflammatory cells and substantially greater fold increases in the expression and production of inflammatory mediators. Data from this study indicate thatS. pneumoniaeopacity variants influence the temporal mRNA expression of inflammatory mediators within the middle ear.
Pneumococcal Lipoteichoic Acid (LTA) Is Not as Potent as Staphylococcal LTA in Stimulating Toll-Like Receptor 2
Authors: Han Seung Hyun , Kim Je Hak , Martin Michael , Michalek Suzanne M. , Nahm Moon H.
Published: September 2003

Streptococcus pneumoniaeis a leading cause of gram-positive sepsis, and lipoteichoic acid (LTA) may be important in causing gram-positive bacterial septic shock. Even though pneumococcal LTA is structurally distinct from the LTA of other gram-positive bacteria, the immunological properties of pneumococcal LTA have not been well characterized. We have investigated the ability of LTAs to stimulate human monocytes by using highly pure and structurally intact preparations of pneumococcal LTA and its two structural variants. The variants were pneumococcal LTA with only one acyl chain (LTA-1) and completely deacylated LTA (LTA-0). The target cells used in the study were peripheral blood mononuclear cells (PBMCs) and two model cell lines (CHO/CD14/TLR2 and CHO/CD14/TLR4) that express human CD25 protein in response to Toll-like receptor 2 (TLR2) and TLR4 stimulation, respectively. Intact pneumococcal LTA and LTA-1 stimulated PBMC and CHO/CD14/TLR2 cells in a dose-dependent manner but did not stimulate CHO/CD14/TLR4 cells. Pneumococcal LTA was about 100-fold less potent thanStaphylococcus aureusLTA in stimulating the CHO/CD14/TLR2 cells and PBMCs. LTA-0 (or pneumococcal teichoic acid) stimulated neither CHO/CD14/TLR2 nor CHO/CD14/TLR4 cells even at high concentrations. Excess teichoic acid, LTA-0, antibodies to phosphocholine, or antibodies to TLR4 did not inhibit the LTA-induced TLR2 stimulation. However, antibodies to CD14, TLR1, or TLR2 suppressed tumor necrosis factor alpha (TNF-α) production by PBMCs in response to LTA or LTA-1. These results suggest that pneumococcal LTA with one or both acyl chains stimulates PBMCs primarily via TLR2 with the help of CD14 and TLR1.
Immunogenicity of, and Immunologic Memory to, a Reduced Primary Schedule of Meningococcal C-Tetanus Toxoid Conjugate Vaccine in Infants in the United Kingdom
Authors: Borrow Ray , Goldblatt David , Finn Adam , Southern Joanna , Ashton Lindsey , Andrews Nick , Lal Gouri , Riley Christine , Rahim Rukhsana , Cartwright Keith , Allan Geraldine , Miller Elizabeth
Published: September 2003

It has been previously shown that one of the three meningococcal C conjugate (MCC) vaccines introduced in the United Kingdom proved highly immunogenic after the first dose of a three-dose schedule, with evidence of immune memory after dose 3. Thus, in infants a one- or two-dose schedule of this MCC vaccine, conjugated to tetanus toxoid (TT), may suffice. Healthy infants (n= 586) were randomized to receive either one (group 1), two (group 2), or three (group 3) doses of MCC-TT vaccine with a 10-μg polysaccharide booster given at 13 to 14 months of age. Serum bactericidal antibody (SBA) levels were measured by utilizing rabbit complement (rSBA), meningococcal C-specific immunoglobulin G (IgG), and avidity indices (AIs). For groups 1, 2, and 3, the percentages of infants with an rSBA level of ≥8 against strain C11 were 98.4, 100, and 99.4%, respectively. Infants in group 1 with prevaccination rSBA titers of ≥8 had post-primary MCC rSBA geometric mean titers (GMTs) significantly lower than those infants with prevaccination rSBA titers of <8. One dose of MCC-TT vaccine given to infants at 2 months of age yielded significantly lower SBA GMTs and geometric mean AIs (GMAIs) than two or three doses but elicited a significantly greater response after boosting, as reflected by rSBA levels and GMAI. This study provides the first evidence that the number of doses of MCC-TT used in infant immunization schedules could be decreased.
The Urokinase Receptor Can Be Induced byBorrelia burgdorferithrough Receptors of the Innate Immune System
Authors: Coleman James L. , Benach Jorge L.
Published: September 2003

Monocytic cells exposed toBorrelia burgdorferi, through unknown receptors, overexpress the urokinase receptor (uPAR), a key mediator of the plasminogen activation system. We show that combined blockade of CD14 and TLR2 causes a significant inhibition ofB. burgdorferi-induced uPAR in Mono Mac 6 (MM6) cells. Other pattern recognition receptors tested (CD11b/CD18, the mannose receptor, and theN-formyl-methionyl-leucyl-phenylalanine receptor) did not have demonstrated roles inB. burgdorferi-mediated uPAR induction. We dissected the result for CD14 andTLR2 by investigating the singular contributions of each. Independent functional blockade of CD14 or TLR2 failed to inhibitB. burgdorferi-mediated uPAR induction. 1,25-Dihydroxyvitamin D3differentiation of MM6 cells increased CD14 expression 12-fold but did not augmentB. burgdorferi-mediated uPAR expression. Peritoneal exudate macrophages (PEM) from CD14- or TLR2-deficient mice were not defective inB. burgdorferi-mediated synthesis of uPAR mRNA and protein. Increased uPAR mRNA or protein or both were apparent in PEM from transgenic and control mice, even at a ratio of oneBorreliaspirochete per cell. We conclude that signaling for the uPAR response, as mediated byB. burgdorferi, proceeds with CD14 and TLR2 as partial contributors. That part under control of CD14 and TLR2 represents a new link between the host plasminogen activation and innate immunity systems.
Mucin Degradation Mechanisms by DistinctPseudomonas aeruginosaIsolates In Vitro
Authors: Aristoteli Lina Panayiota , Willcox Mark D. P.
Published: September 2003

Pseudomonas aeruginosahas emerged as an important causative agent of bacterial keratitis, a rapidly progressive ocular condition that may result in blindness. Secretory mucin forms the main constituent of the precorneal tear film, a three-layer film on the ocular surface protecting the underlying corneal epithelium from potential pathogens. The purpose of the present study was to compare mucin degradation mechanisms between ocularP. aeruginosastrains. Mucin degradation was assessed by agarose electrophoresis, lectin blotting, and size exclusion chromatography. The results indicate that certainP. aeruginosastrains (Paer12, ATCC 15442, 6294, and Paer25) had depleted mucin from the culture supernatant and that this was contingent on the inherent ability of these isolates to produce proteases. Non-protease-producing strains (Paer1 and Paer3) did not appreciably degrade mucin. Further, galactosidase,N-acetylglucosaminidase, andN-acetylgalactosaminidase activities were detected in some strains, suggesting the operation of further mechanisms of mucin degradation byP. aeruginosa. Mucin degradation byP. aeruginosaalso seemed to be for the acquisition of nutrients, as a growth advantage was observed in mucin-depleting strains over nondepleting strains in the long term. It is postulated that the degradation of mucin serves to collapse the mucin barrier and its associated network containing antibacterial tear components and to provide energy for sustained bacterial growth.
A Gonococcal Efflux Pump System Enhances Bacterial Survival in a Female Mouse Model of Genital Tract Infection
Authors: Jerse Ann E. , Sharma Nirmala D. , Simms Amy N. , Crow Emily T. , Snyder Lori A. , Shafer William M.
Published: September 2003

Active efflux of antimicrobial substances is likely to be an important bacterial defense against inhibitory host factors inherent to different body sites. Two well-characterized multidrug resistance efflux systems (MtrCDE and FarAB-MtrE) exist in
Neisseria gonorrhoeae
, a bacterial pathogen of the human genital mucosae. In vitro studies suggest that the MtrCDE and FarAB-MtrE efflux systems protect the gonococcus from hydrophobic antimicrobial substances that are likely to be present on mucosal surfaces. Here we report that a functional MtrCDE efflux system, but not a functional FarAB-MtrE system, enhances experimental gonococcal genital tract infection in female mice. Specifically, the recovery of
mtrD
and
mtrE
mutants, but not a
farB
mutant, from mice inoculated with mutant or wild-type gonococci was reduced compared with that of the wild-type strain. Competitive-infection experiments confirmed the survival disadvantage of MtrCDE-deficient gonococci. This report is the first direct evidence that a multidrug resistance efflux system enhances survival of a bacterial pathogen in the genital tract. Additionally, experiments using ovariectomized mice showed that MtrCDE-deficient gonococci were more rapidly cleared from mice that were capable of secreting gonadal hormones. MtrCDE-deficient gonococci were more sensitive to nonphysiological concentrations of progesterone in vitro than were wild-type or FarAB-MtrE-deficient gonococci. These results suggest that progesterone may play an inhibitory role in vivo. However, hormonally regulated factors rather than progesterone itself may be responsible for the more rapid clearance of
mtr
-deficient gonococci from intact mice.
The Global Regulator ArcA Modulates Expression of Virulence Factors in
Vibrio cholerae
Authors: Sengupta Nilanjan , Paul Kalidas , Chowdhury Rukhsana
Published: September 2003

A
Vibrio cholerae arcA
mutant was constructed and used to examine the role of the global anaerobiosis response regulator ArcA in the expression of virulence factors in this important human pathogen. In
V. cholerae
, expression of the major virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) is regulated by the transcriptional activator ToxT.
toxT
expression, in turn, is controlled by the transmembrane DNA binding proteins ToxR and TcpP. In the
V. cholerae arcA
mutant, although ToxR and TcpP were unaffected, Northern blot and reverse transcription-PCR analyses indicated that the expression of
toxT
was significantly decreased with concomitant reduction in the expression of CT and TCP. CT and TCP expression was completely restored in the
V. cholerae arcA
mutant strain by expressing a cloned
toxT
gene in the mutant. These results suggest that ArcA functions as a positive regulator of
toxT
expression under both aerobic and anaerobic conditions, although as expected, the effect was more pronounced during anaerobic growth. This was reflected in a reduction of virulence of the
V. cholerae arcA
mutant strain in the infant mouse cholera model.
Activation of Human Dendritic Cells Is Modulated by Components of the Outer Membranes ofNeisseria meningitidis
Authors: Al-Bader Tamara , Christodoulides Myron , Heckels John E. , Holloway Judith , Semper Amanda E. , Friedmann Peter S.
Published: September 2003

Neisseria meningitidisserogroup B is a major cause of life-threatening meningitis and septicemia worldwide, and no effective vaccine is available. Initiation of innate and acquired immune responses toN. meningitidisis likely to be dependent on cellular responses of dendritic cells (DC) to antigens present in the outer membrane (OM) of the meningococcus. In this study, the responses of human monocyte-derived DC (mo-DC) to OM isolated from parent (lipopolysaccharide [LPS]-replete) meningococci and from a mutant deficient in LPS were investigated. Parent OM selectively up-regulated Toll-like receptor 4 (TLR4) mRNA expression and induced mo-DC maturation, as reflected by increased production of chemokines, proinflammatory cytokines, and CD83, CD80, CD86, CD40, and major histocompatibility complex (MHC) class II molecules. In contrast, LPS-deficient OM selectively up-regulated TLR2 mRNA expression and induced moderate increases in both cytokine production and expression of CD86 and MHC class II molecules. Preexposure to OM, with or without LPS, augmented the allostimulatory properties of mo-DC, which induced proliferation of naive CD4+CD45RA+T cells. In addition, LPS-replete OM induced a greater gamma interferon/interleukin-13 ratio in naive T cells, whereas LPS-deficient OM induced the reverse profile. These data demonstrate that components of the OM, other than LPS, are also likely to be involved in determining the levels of DC activation and the nature of the T-helper immune response.
Augmentation ofActinobacillus actinomycetemcomitansInvasion of Human Oral Epithelial Cells and Up-Regulation of Interleukin-8 Production by Saliva CD14
Authors: Takayama Atsuko , Satoh Aya , Ngai Tomoko , Nishimura Takashi , Ikawa Keiji , Matsuyama Takami , Shimauchi Hidetoshi , Takada Haruhiko , Sugawara Shunji
Published: September 2003

It has recently been shown that human salivary glands constitutively express CD14, an important molecule in innate immunity, and that a soluble form of CD14 is secreted in saliva. The concentration of CD14 in parotid (a serous gland) saliva was comparable to that in normal serum and 10-fold the amount in whole saliva, although the physiological function of saliva CD14 remained unclear.Actinobacillus actinomycetemcomitansis a periodontopathic bacterium and is able to invade oral epithelial cells. The present study showed that upon exposure to liveA. actinomycetemcomitansY4 for 2 h, human oral epithelial HSC-2 cells produced interleukin-8 (IL-8) for a further 24 h and whole saliva augmented the production induced byA. actinomycetemcomitansY4. Parotid saliva showed a more pronounced effect on the production of IL-8 than whole saliva. Neither saliva preparation itself had IL-8-inducing activity. Parotid saliva exhibited antibacterial activity against a low concentration ofA. actinomycetemcomitansY4, but recombinant CD14 did not show the activity. The internalization ofA. actinomycetemcomitansY4 into HSC-2 cells was inhibited by cytochalasin B, indicating that the process was actin dependent, and depletion of CD14 from parotid saliva inhibited the invasion and, as a consequence, inhibited production of IL-8. Furthermore, human recombinant CD14 augmented invasion and IL-8 production. These results suggest that saliva CD14 promoted the invasion of oral epithelial cells byA. actinomycetemcomitansand consequently augmented the production of IL-8, playing an important role in innate immunity in the oral cavity.
Protection against Syphilis Correlates with Specificity of Antibodies to the Variable Regions ofTreponema pallidumRepeat Protein K
Authors: Morgan Cecilia A. , Lukehart Sheila A. , Van Voorhis Wesley C.
Published: September 2003

Syphilis has been recognized as a disease since the late 1400s, yet there is no practical vaccine available. One impediment to the development of a vaccine is the lack of understanding of multiple reinfections in humans despite the development of robust immune responses during the first episode. It has been shown that theTreponema pallidumrepeat protein K (TprK) differs in seven discrete variable (V) regions in isolates and that the antibody response during infection is directed to these V regions. Immunization with TprK confers significant protection against infection with the homologous strain. We hypothesize that the antigenic diversity of TprK is involved in immune evasion, which contributes to the lack of heterologous protection. Here, using the rabbit model, we show a correlation between limited heterologous protection andtprKdiversity in the challenge inoculum. We demonstrate that antibody responses to the V regions of one TprK molecule show limited cross-reactivity with heterologous TprK V regions.
Presence of Active Aliphatic Amidases in
Helicobacter
Species Able To Colonize the Stomach
Authors: Bury-Moné Stéphanie , Skouloubris Stéphane , Dauga Catherine , Thiberge Jean-Michel , Dailidiene Daiva , Berg Douglas E. , Labigne Agnès , De Reuse Hilde
Published: September 2003

Ammonia production is of great importance for the gastric pathogen
Helicobacter pylori
as a nitrogen source, as a compound protecting against gastric acidity, and as a cytotoxic molecule. In addition to urease,
H. pylori
possesses two aliphatic amidases responsible for ammonia production: AmiE, a classical amidase, and AmiF, a new type of formamidase. Both enzymes are part of a regulatory network consisting of nitrogen metabolism enzymes, including urease and arginase. We examined the role of the
H. pylori
amidases in vivo by testing the gastric colonization of mice with
H. pylori
SS1 strains carrying mutations in
amiE
and/or
amiF
and in coinfection experiments with wild-type and double mutant strains. A new cassette conferring resistance to gentamicin was used in addition to the kanamycin cassette to construct the double mutation in strain SS1. Our data indicate that the amidases are not essential for colonization of mice. The search for
amiE
and
amiF
genes in 53
H. pylori
strains from different geographic origins indicated the presence of both genes in all these genomes. We tested for the presence of the
amiE
and
amiF
genes and for amidase and formamidase activities in eleven
Helicobacter
species. Among the gastric species,
H. acinonychis
possessed both
amiE
and
amiF
,
H. felis
carried only
amiF
, and
H. mustelae
was devoid of amidases.
H. muridarum
, which can colonize both mouse intestine and stomach, was the only enterohepatic species to contain
amiE
. Phylogenetic trees based upon the sequences of
H. pylori amiE
and
amiF
genes and their respective homologs from other organisms as well as the amidase gene distribution among
Helicobacter
species are strongly suggestive of amidase acquisition by horizontal gene transfer. Since amidases are found only in
Helicobacter
species able to colonize the stomach, their acquisition might be related to selective pressure in this particular gastric environment.
Enhanced CXC Chemokine Responses of Human Colonic Epithelial Cells to Locus of Enterocyte Effacement-Negative Shiga-Toxigenic
Escherichia coli
Authors: Rogers Trisha J. , Paton Adrienne W. , McColl Shaun R. , Paton James C.
Published: September 2003

There is increasing evidence that by facilitating translocation of Shiga toxin (Stx) across the intestinal epithelium and by transporting bound toxin to remote sites such as the renal endothelium, polymorphonuclear leukocytes (PMNs) play a key role in the pathogenesis of Shiga-toxigenic
Escherichia coli
(STEC) disease. Plasma levels of PMN-attracting CXC chemokines such as interleukin-8 (IL-8) also appear to correlate in humans with the severity of disease. Thus, the capacity of STEC strains to elicit CXC chemokine responses in intestinal epithelial cells may be a crucial step in pathogenesis. Accordingly, we attempted to determine which STEC factors are responsible for CXC chemokine induction in human colonic epithelial cells. Infection of Hct-8 cells with locus for enterocyte effacement (LEE)-negative STEC strains isolated from patients with severe STEC disease resulted in up-regulation of IL-8, macrophage inflammatory protein 2α (MIP-2α), MIP-2β, and ENA-78 mRNA significantly higher and earlier than that elicited by several LEE-positive STEC strains, including the O157:H7 strain EDL933. Similarly, levels of IL-8 protein in LEE-negative STEC-infected Hct-8 culture supernatants were significantly higher than in LEE-positive STEC-infected culture supernatants. The difference in responses could not be attributed to the expression or nonexpression of LEE genes, the presence or absence of an STEC megaplasmid, or differences in O serogroups or in the type or amount of Stx produced. Interestingly, however, several of the LEE-negative STEC strains eliciting the strongest chemokine responses belonged to flagellar serotype H21. Incubation of Hct-8 cells with isolated H21 flagellin elicited IL-8 and MIP-2α responses similar to those seen in the presence of the most potent LEE-negative STEC strains. Deletion of the
fliC
gene, but not the
stx
2
gene, largely abolished the capacity of O113:H21 LEE-negative STEC strain 98NK2 to elicit IL-8 and MIP-2α responses in Hct-8 cells. Taken together, these data suggest that although Stx is capable of inducing CXC chemokine responses, the elevated responses seen in cells infected with certain STEC strains are largely attributable to the production of flagellin.
TheluxSGene ofStreptococcus pyogenesRegulates Expression of Genes That Affect Internalization by Epithelial Cells
Authors: Marouni Mehran J. , Sela Shlomo
Published: September 2003

The gram-positive pathogenStreptococcus pyogeneswas recently reported to possess a homologue of theluxSgene that is responsible for the production of autoinducer 2, which participates in quorum sensing of both gram-positive and gram-negative bacteria. To test the effect of LuxS on streptococcal internalization, a LuxS mutant was constructed in strain SP268, an invasive M3 serotype. Functional analysis of the mutant revealed that it was internalized by HEp-2 cells with higher efficiency than the wild type (wt). Several genes, includinghasA(hyaluronic acid synthesis),speB(streptococcal pyrogenic exotoxin B), andcsrR(capsule synthesis regulator), a part of a two-component regulatory system, are known to affect the internalization of strain SP268 (J. Jadoun, O. Eyal, and S. Sela, Infect. Immun.70:462-469, 2002). Therefore, the expression of these genes in the mutant and in the wt was examined. LuxS mutation significantly reduced the mRNA level ofspeBand increased the mRNA level ofemm3. No substantial effect was observed on transcription ofhasAandcsrR. Yet less hyaluronic acid capsule was expressed in the mutant. Further analysis revealed thatluxSis under the regulation of the two-component global regulator CsrR. Our results indicate that LuxS activity in strain SP268 plays an important role in the expression of virulence factors associated with epithelial cell internalization.
Positive and Negativecis-Acting Regulatory Sequences Control Expression of Leukotoxin inActinobacillus actinomycetemcomitans652
Authors: Mitchell Christine , Gao Ling , Demuth Donald R.
Published: September 2003

Integration of IS1301into an AT-rich inverted repeat located upstream of theltxoperon was previously shown to confer a hyperleukotoxic phenotype inActinobacillus actinomycetemcomitansIS1 (T. He, T. Nishihara, D. R. Demuth, and I. Ishikawa, J. Periodontol.70:1261-1268, 1999), but the mechanism leading to increased leukotoxin production was not determined. We show that an IS1ltxpromoter::lacZreporter construct expresses 12-fold higher levels of β-galactosidase activity than a reporter containing theltxpromoter fromA. actinomycetemcomitans652, suggesting that IS1301increases transcription of theltxoperon. Examination of the IS1301sequence identified a potential outwardly directed promoter. However, site-specific mutagenesis of the −35 element of the putative promoter had no effect on the transcriptional activity of the IS1 reporter construct. Furthermore, reverse transcriptase PCR and real-time PCR experiments did not detect a transcript that was initiated within IS1301. These results suggest that increased expression of leukotoxin in strain IS1 does not arise from an outwardly directed IS1301promoter. To determine how IS1301alters transcriptional regulation of theltxoperon,cis-acting sequences that regulate leukotoxin expression were identified. The AT-rich sequence that resides downstream from the site of IS1301insertion was shown to function as a positivecis-acting regulator of leukotoxin expression. This sequence resembles an UP element in its location, AT-rich content, and activity and is homologous to the consensus UP element sequence. In addition, a negativecis-acting sequence was identified upstream from the site of IS1301insertion, and deletion of this region increased promoter activity by fourfold. Mobility shift experiments showed that this region bound to a protein(s) in extracts fromA. actinomycetemcomitans652. The specific sequences required for this interaction were localized to a 26-nucleotide segment of theltxpromoter that resides 17 bp upstream from the site of IS1301insertion. Together, these results suggest that positive and negativecis-acting sequences regulate leukotoxin expression and that IS1301may increase transcription of theltxoperon inA. actinomycetemcomitansIS1 by displacing a negativecis-acting regulator approximately 900 bp upstream from the basal elements of theltxpromoter.
Mechanisms of Variable
p44
Expression by
Anaplasma phagocytophilum
Authors: Lin Quan , Rikihisa Yasuko , Ohashi Norio , Zhi Ning
Published: September 2003

The human intragranulocytic bacterium
Anaplasma phagocytophilum
promotes variation of P44s, which are surface-exposed proteins encoded by a
p44
multigene family. In the present study, the specific
p44
gene expression loci in four strains of
A. phagocytophilum
were identified and it was determined that each consisted of four tandem genes,
tr1
,
omp-1X
,
omp-1N
, and
p44
. A putative σ
70
-type promoter was found upstream of
tr1
. The
p44
genes include a central hypervariable region flanked by conserved regions. The hypervariable region sequence in the
p44
expression locus was duplicated and, regardless of the expression status, conserved at another locus in both low- and high-passage cell cultures of strain NY-37. No significant differences in the hypervariable region were found when we compared
p44
sequences, at the level of cDNA, within the expression locus and within other loci in the genomes of strains NY-37 and HZ. Similarly, in cDNA isolated from patients and from assorted cultures of strains NY-31, NY-36, and NY-37, hypervariable regions of 450 deduced amino acid sequences of various
p44
s within each strain were found to be identical, as were those of
p44
sequences in the genome of strain HZ. These data suggest that variations in
p44
sequences at the level of the
p44
expression locus occur through unidirectional conversion of the entire (nonsegmental)
p44
hypervariable region including flanking regions with a corresponding sequence copied from one of the conserved donor
p44
genomic loci. The data suggest that the P44 antigenic repertoire within the hypervariable region is restricted.
Oral Immunization of BALB/c Mice with
Giardia duodenalis
Recombinant Cyst Wall Protein Inhibits Shedding of Cysts
Authors: Larocque R. , Nakagaki K. , Lee P. , Abdul-Wahid A. , Faubert G. M.
Published: September 2003

The process of encystation is a key step in the
Giardia duodenalis
life cycle that allows this intestinal protozoan to survive between hosts during person-to-person, animal-to-person, waterborne, or food-borne transmission. The release of cysts from infected persons and animals is the main contributing factor to contamination of the environment. Genes coding for cyst wall proteins (CWPs), which could be used for developing a transmission-blocking vaccine, have been cloned. Since the immunogenicity of recombinant
Giardia
CWP is unknown, we have investigated the immunogenicity of recombinant CWP2 (rCWP2) and its efficacy in interfering with the phenomenon of encystation taking place in the small bowels of BALB/c mice vaccinated with the recombinant protein. Here we report that the immunization of BALB/c mice with rCWP2 stimulated the immune system in a manner comparable to that for a live infection with
Giardia muris
cysts. Fecal and serum anti-rCWP2 immunoglobulin A (IgA) antibodies were detected in the immunized mice. In addition, anti-rCWP2 IgG1 and IgG2a antibodies were detected in the serum. mRNAs coding for Th1 and Th2 types of cytokines were detected in spleen and Peyer's patch cells from immunized mice. When the vaccinated mice were challenged with live cysts, the animals shed fewer cysts. We conclude that rCWP2 is a possible candidate antigen for the development of a transmission-blocking vaccine.
Helicobacter pyloriOuter Membrane Vesicles Modulate Proliferation and Interleukin-8 Production by Gastric Epithelial Cells
Authors: Ismail Salim , Hampton Mark B. , Keenan Jacqueline I.
Published: September 2003

Helicobacter pyloriinfection, which is always associated with gastritis, can progress to ulceration or malignancy. The diversity in clinical outcomes is partly attributed to the expression of virulence factors and adhesins byH. pylori. However,H. pylorimay not have to adhere to the epithelium to cause gastritis. We hypothesize that outer membrane vesicles (OMV), which are constantly shed from the surface ofH. pylori, play a role as independent activators of host cell responses. In this study, we found that low doses of OMV fromcagPAI+toxigenic andcagPAI−nontoxigenic strains increased proliferation of AGS gastric epithelial cells. At higher doses, we detected growth arrest, increased toxicity, and interleukin-8 (IL-8) production. The only strain differences detected were vacuolation with the toxigenic strain and higher levels of IL-8 production with OMV from thecagPAI−nontoxigenic strain. In summary, we suggest that constitutively shed OMV play a role in promoting the low-grade gastritis associated withH. pyloriinfection.
Specific Antibody Responses to Three Schistosome-Related Carbohydrate Structures in Recently Exposed Immigrants and Established Residents in an Area ofSchistosoma mansoniEndemicity
Authors: Naus Cynthia W. A. , van Remoortere Alexandra , Ouma John H. , Kimani Gachuhi , Dunne David W. , Kamerling Johannis P. , Deelder André M. , Hokke Cornelis H.
Published: September 2003

By the use of surface plasmon resonance spectroscopy, immunoglobulin G (IgG) subclass and IgM antibodies against three schistosome-derived carbohydrate structures, FLDN (Fucα1-3GalNAcβ1-4GlcNAcβ1-3Galα1), LDN-DF [GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAcβ1], and LDNF [GalNAcβ1-4(Fucα1-3)GlcNAcβ1-3Galα1], were measured in 184 previously unexposed Kenyan immigrants who moved into the Masongaleni area, whereSchistosoma mansoniis endemic. They were sampled within their first year of exposure and again 2 years later. A cohort selected out of the original residents of the area, who had been exposed for many years, served as controls. Associations with responses toS. mansoniworm, egg (SEA), and cercarial (CERC) antigens were examined. In addition, we measured responses to keyhole limpet hemocyanin, a glycoprotein which carries glycan epitopes that are also expressed by schistosomes. Specific IgG1 responses were most pronounced against FLDN and LDN-DF and strongly associated with those previously measured to SEA and CERC. Similarly to previously published age profiles of IgG1 and IgG2 responses to SEA, levels of IgG1 against LDN-DF decreased with age. In contrast, specific IgM responses against the three schistosome-derived carbohydrate structures were most marked against LDNF. Our results indicate that, of the three glycan structures tested, the acute response against schistosome glycoconjugate antigens in young children is mainly directed against the LDN-DF epitope. The response to LDN-DF in older individuals and the responses to the two other epitopes were similar in the two cohorts, suggesting that these antigens are recognized in the early stages of infection and that the immune response persists. The biological significance of these observations needs further elucidation.
The Aggregation Domain of Aggregation Substance, Not the RGD Motifs, Is Critical for Efficient Internalization by HT-29 Enterocytes
Authors: Waters Christopher M. , Wells Carol L. , Dunny Gary M.
Published: September 2003

Aggregation substance (AS), a surface protein encoded on the pheromone-inducible plasmids of
Enterococcus faecalis
, has been shown to increase adherence and internalization into a number of different cell types, presumably through integrin binding mediated by the N-terminal RGD motif of AS. Here, defined mutations constructed in Asc10, the AS encoded by the plasmid pCF10, are analyzed for their ability to promote increased internalization levels into HT-29 enterocytes. The results clearly show that the previously identified Asc10 functional domain, not the RGD motifs, is critical for Asc10-directed internalization of
E. faecalis
into HT-29 enterocytes. Also, expression of Asc10 in the nonaggregating
E. faecalis
strain INY3000 is unable to mediate HT-29 internalization. However, Asc10-expressing
E. faecalis
cells are not internalized as bacterial aggregates, suggesting bacterial aggregation is not a prerequisite for HT-29 internalization. These data show that Asc10 directs internalization of
E. faecalis
into HT-29 enterocytes through a non-RGD-dependent mechanism.
Deregulated Production of Protective Cytokines in Response toCandida albicansInfection in Patients with Chronic Mucocutaneous Candidiasis
Authors: Lilic Desa , Gravenor Ian , Robson Neil , Lammas David A. , Drysdale Pam , Calvert Jane E. , Cant Andrew J. , Abinun Mario
Published: September 2003

Patients with chronic mucocutaneous candidiasis (CMC) are selectively unable to clear the yeastCandida, which results in persistent debilitating infections affecting the skin, nails, and mucous membranes. The underlying defect is unknown. Recent animal studies highlighted the importance of type 1 cytokines in protection againstCandida, and previous work suggested that CMC patients may exhibit altered cytokine production in response toCandida.Based on these findings, in this study we investigated cytokine production in CMC patients by assessing a range of inflammatory, anti-inflammatory, type 1, and type 2 cytokines (interleukin-2 [IL-2], IL-4, IL-5, IL-6, IL-10, IL-12, gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α]) in whole-blood cultures in response to five different fractions ofCandida albicans(carbohydrate, purified mannan, and protein-rich fractions, etc.), as well as non-Candidaantigens. Our results demonstrate that cytokine production is deregulated in aCandida-specific way for some cytokines (IL-2, IL-10), is deregulated more generally for other cytokines (IL-12, IL-6, IFN-γ), and is not markedly altered for still other cytokines (TNF-α, IL-4, IL-5). The most notable finding in CMC patients was the markedly impaired production of IL-12 in parallel with dramatically increased levels of IL-6 and IL-10 that occurred selectively in response toCandida.These results suggest that patients with CMC have impaired production of type 1-inducing cytokines (possibly a macrophage or dendritic cell defect?), which could result in an inability to mount protective cell-mediated responses and a failure to clearCandida.Continued tissue damage and inflammation may trigger production of high levels of inhibitory cytokines, such as the IL-10 production seen in our study, which would further reduce production of type 1-inducing cytokines in a positive feedback loop leading to persistent infection.
Analysis of Immunological Nonresponsiveness to the 19-Kilodalton Fragment of Merozoite Surface Protein 1 ofPlasmodium yoelii: Rescue by Chemical Conjugation to Diphtheria Toxoid (DT) and Enhancement of Immunogenicity by Prior DT Vaccination
Authors: Stanisic Danielle I. , Martin Laura B. , Liu Xue Q. , Jackson David , Cooper Juan , Good Michael F.
Published: September 2003

ThePlasmodiummerozoite surface protein 1 (MSP1) is a leading vaccine candidate for protecting against the blood stage of malaria. Previous studies have shown that the 19-kDa carboxyl terminus of this protein is able to induce protective immunity in some monkey and mouse strains. We show that immunization with the recombinantPlasmodium yoelii19-kDa fragment of MSP1 (MSP119) expressed inSaccharomyces cerevisiae(yMSP119) can induce protective antibodies in several inbred mouse strains and one outbred mouse strain. However, mice expressing theH-2smajor histocompatibility complex haplotype are unable to generate yMSP119-specific antibodies. While synthetic peptides derived from MSP119are immunogenic in B10.S mice, they cannot function as helper epitopes, and immunization with yMSP119does not induce T cells that recognize the recombinant protein or synthetic peptides corresponding to its sequence. Nonresponsiveness could be overcome by using chemical linkers to conjugate yMSP119to diphtheria toxoid (DT), resulting in immunogens capable of inducing protective yMSP119-specific antibodies in both MSP119-responsive and otherwise nonresponsive mouse strains. The ability of sera from mice immunized with the conjugate to inhibit binding of a protective monoclonal antibody (MAb 302) to yMSP119correlated strongly with a delay in the prepatent period. Chemical conjugation of yMSP119to DT may be a preferred method to enhance immunogenicity, as carrier priming experiments demonstrated that an existing immune response to DT enhanced a subsequent antibody response to yMSP119after vaccination with yMSP119-DT. These results have important implications for the development of a malaria vaccine to protect a population with diverse HLAs.
Construction and Functional Activities of Chimeric Mouse-Human Immunoglobulin G and Immunoglobulin M Antibodies against theNeisseria meningitidisPorA P1.7 and P1.16 Epitopes
Authors: Michaelsen Terje E. , Ihle Øistein , Beckstrøm Karen Johanne , Herstad Tove K. , Kolberg Jan , Høiby E. Arne , Aase Audun
Published: September 2003

We studied the in vitro protective activities of human immunoglobulin G1 (IgG1), IgG3, and IgM antibodies against group B meningococci by constructing sets of chimeric mouse-human antibodies (chIgG1, chIgG3, and chIgM, respectively) with identical binding regions against the P1.7 and P1.16 epitopes on PorA. This was done by cloning the V genes of three mouse hybridoma antibodies and subsequently transfecting vectors containing the homologous heavy- and light-chain genes into NSO cells. Cell clones secreting intact human chIgG1, chIgG3, or chIgM antibodies originating from three parent mouse antibodies were isolated. The functional affinities appeared to be similar for all human isotypes and surprisingly also for the pentameric chIgM antibody. chIgG1 exhibited greater serum bactericidal activity (SBA) than chIgG3, while chIgG3 was more efficient in inducing a respiratory burst (RB) associated with opsonophagocytosis than chIgG1 was. On the other hand, chIgM exhibited SBA similar to that of chIgG1, but it exhibited much higher RB activity than chIgG3 and chIgG1 exhibited. The antibodies against the P1.16 epitope were more efficient in terms of SBA than the antibodies against the P1.7 epitope were; thus, 10- to 40-fold-lower concentrations of antibodies against P1.16 than of antibodies against P1.7 were needed to induce SBA. On the other hand, antibodies against these epitopes were equally effective in inducing RB. Our results revealed differences in the functional activities of human chIgG1, chIgG3, and chIgM antibodies against meningococci, which might influence their protective effects against meningococcal disease.
Serum Immunoglobulin G Response to Candidate Vaccine Antigens during Experimental Human Pneumococcal Colonization
Authors: McCool Tera L. , Cate Thomas R. , Tuomanen Elaine I. , Adrian Peter , Mitchell Tim J. , Weiser Jeffrey N.
Published: September 2003

The immune response to pneumococcal surface structures during colonization was examined in a model of experimental human pneumococcal carriage. Healthy uncolonized adults were given a type 23F or 6B pneumococcus, and a portion of these subjects became colonized (6 of 14 with type 23F and 6 of 8 with type 6B). Sera from colonized and uncolonized subjects were used to determine the titer of antibody specific to pneumococcal surface components under consideration in development of noncapsular polysaccharide-based vaccines. These vaccine candidates included pneumococcal surface protein A (PspA), choline binding protein A (CbpA), lipoteichoic acid, immunoglobulin A1 (IgA1) protease, pneumolysin, proteinase maturation protein A, and pneumococcal surface adhesin A. Only the two related choline binding proteins, PspA and CbpA, were immunogenic in colonized subjects as determined by a statistically significant rise in the serum IgG titer. The serum IgG response to PspA was shown previously to correlate inversely with susceptibility to carriage and was localized to a region within the N-terminal portion of PspA. This region is highly variable in amino acid sequence between pneumococcal strains. Despite the sequence diversity in the immunodominant regions of both PspA and CbpA, a significant strain-to-strain cross-reactivity in the serum IgG response following experimental human carriage was observed. These findings support the need for further investigation of the human antibody response to PspA and CbpA and the potential use of one or both of these proteins as novel vaccine antigens for the prevention of pneumococcal colonization.
The Vsa Proteins Modulate Susceptibility of
Mycoplasma pulmonis
to Complement Killing, Hemadsorption, and Adherence to Polystyrene
Authors: Simmons Warren L. , Dybvig Kevin
Published: September 2003

The variable surface antigens (Vsa) of the murine respiratory pathogen
Mycoplasma pulmonis
are associated with the virulence of the microorganism in the lung. In strain UAB CT, the antigens consist of an N-terminal region that is combined with one of seven different C-terminal variable regions comprised of tandem repeats.
M. pulmonis
producing a VsaA protein with about 40 tandem repeats (R40) does not adhere to red blood cells or polystyrene. Strains that produce VsaH contain a short C-terminal region that lacks tandem repeats and adhere to red blood cells and plastic. We isolated and analyzed
M. pulmonis
strain CT variants (CT182 and derivatives) that produced a VsaA protein with only three tandem repeats (R3). These variants adhered to plastic and red blood cells similarly to the VsaH-producing strain. When the R3-producing CT182 strain or the VsaH-producing strains were incubated with normal guinea pig serum, they were efficiently killed. Killing was abolished when the serum was heat inactivated. In contrast, the
M. pulmonis
strains that produced VsaA R40 were highly resistant to complement killing. CT182R3 variants that survived the complement killing reactions all produced the R40 form of VsaA and were resistant to complement killing. VsaA R40 is the first mycoplasmal protein shown to be associated with resistance to complement. As both VsaH and VsaA can mediate adherence to plastic, cytadherence, and susceptibility to complement, we propose that Vsa modulates these phenotypes by nonspecific interactions.
gp63 Homologues inTrypanosoma cruzi: Surface Antigens with Metalloprotease Activity and a Possible Role in Host Cell Infection
Authors: Cuevas Ileana C. , Cazzulo Juan J. , Sánchez Daniel O.
Published: September 2003

gp63 is a highly abundant glycosylphosphatidylinositol (GPI)-anchored membrane protein expressed predominantly in the promastigote but also in the amastigote stage ofLeishmaniaspecies. InLeishmaniaspp., gp63 has been implicated in a number of steps in establishment of infection. Here we demonstrate thatTrypanosoma cruzi, the etiological agent of Chagas' disease, has a family of gp63 genes composed of multiple groups. Two of these groups,Tcgp63-Iand-II, are present as high-copy-number genes. The genomic organization and mRNA expression pattern were specific for each group.Tcgp63-Iwas widely expressed, while theTcgp63-IIgroup was scarcely detected in Northern blots, even though it is well represented in theT. cruzigenome. Western blots using sera directed against a synthetic peptide indicated that theTcgp63-Igroup produced proteins of ∼78 kDa, differentially expressed during the life cycle. Immunofluorescence staining and phosphatidylinositol-specific phospholipase C digestion confirmed that Tcgp63-I group members are surface proteins bound to the membrane by a GPI anchor. We also demonstrate the presence of metalloprotease activity which is attributable, at least in part, to Tcgp63-I group. Since antibodies against Tcgp63-I partially blocked infection of Vero cells by trypomastigotes, a possible role for this group in infection is suggested.