bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective Accumulation of Mature Asexual Stages of
Plasmodium falciparum
-Infected Erythrocytes in the Placenta
Authors: Beeson James G. , Amin Nishal , Kanjala Maxwell , Rogerson Stephen J.
Published: September 2002

A feature of malaria in pregnancy is accumulation of
P. falciparum
-infected erythrocytes (IEs) in the placenta, which is associated with adverse outcomes for mothers and infants. Infection appears to involve parasite adhesion to molecules such as chondroitin sulfate A, hyaluronic acid, and immunoglobulins. In vitro, adhesion is predominantly a property of mature asexual forms of IEs; however, adhesion of immature or ring forms has recently been reported. We have assessed the parasitemia and developmental stages of IEs in the placenta by examination of placental blood and histological sections with comparison to parasites in the peripheral blood from the same individuals. Approximately 90% of IEs in the placenta were mature forms. Compared to peripheral blood, the placental parasitemia was 10-fold higher and the density of mature IEs was over 200-fold higher. By contrast, the average peripheral and placental ring-stage parasitemias were not significantly different. In 2 of 14 cases, the density of ring forms was higher in placental than in peripheral blood. These findings demonstrate prominent selective accumulation of mature asexual-stage IEs but infrequent accumulation of ring stages in the placental blood spaces, consistent with an important role for mature-stage IE adhesion.
Identification and Characterization of
lpfABCC
′
DE
, a Fimbrial Operon of Enterohemorrhagic
Escherichia coli
O157:H7
Authors: Torres Alfredo G. , Giron Jorge A. , Perna Nicole T. , Burland Valerie , Blattner Fred R. , Avelino-Flores Fabiola , Kaper James B.
Published: September 2002

The mechanisms underlying the adherence of
Escherichia coli
O157:H7 and other enterohemorrhagic
E. coli
(EHEC) strains to intestinal epithelial cells are poorly understood. We have identified a chromosomal region (designated
lpfABCC
′
DE
) in EHEC O157:H7 containing six putative open reading frames that was found to be closely related to the long polar (LP) fimbria operon (
lpf
) of
Salmonella enterica
serovar Typhimurium, both in gene order and in conservation of the deduced amino acid sequences. We show that
lpfABCC
′
DE
is organized as an operon and that its expression is induced during the exponential growth phase. The
lpf
genes from EHEC strain EDL933 were introduced into a nonfimbriated (Fim
−
)
E. coli
K-12 strain, and the transformed strain produced fimbriae as visualized by electron microscopy and adhered to tissue culture cells. Anti-LpfA antiserum recognized a ca. 16-kDa LpfA protein when expressed under regulation of the T7 promoter system. The antiserum also cross-reacted with the LP fimbriae in immunogold electron microscopy and Western blot experiments. Isogenic
E. coli
O157:H7
lpf
mutants derived from strains 86-24 and AGT300 showed slight reductions in adherence to tissue culture cells and formed fewer microcolonies compared with their wild-type parent strains. The adherence and microcolony formation phenotypes were restored when the
lpf
operon was introduced on a plasmid. We propose that LP fimbriae participate in the interaction of
E. coli
O157:H7 with eukaryotic cells by assisting in microcolony formation.
Increased Expression of Clumping Factor and Fibronectin-Binding Proteins by
hemB
Mutants of
Staphylococcus aureus
Expressing Small Colony Variant Phenotypes
Authors: Vaudaux Pierre , Francois Patrice , Bisognano Carmelo , Kelley William L. , Lew Daniel P. , Schrenzel Jacques , Proctor Richard A. , McNamara Peter J. , Peters G. , Von Eiff Christof
Published: September 2002

Small colony variants (SCVs) of
Staphylococcus aureus
are slow-growing subpopulations that cause persistent and relapsing infections. The altered phenotype of SCV can arise from defects in menadione or hemin biosynthesis, which disrupt the electron transport chain and decrease ATP concentrations. With SCVs, virulence is altered by a decrease in exotoxin production and susceptibility to various antibiotics, allowing their intracellular survival. The expression of bacterial adhesins by SCVs is poorly documented. We tested fibrinogen- and fibronectin-mediated adhesion of a
hemB
mutant of
S. aureus
8325-4 that is defective for hemin biosynthesis and exhibits a complete SCV phenotype. In this strain, adhesion to fibrinogen and fibronectin was significantly higher than that of its isogenic, normally growing parent and correlated with the increased surface display of these adhesins as assessed by flow cytometry. Real-time quantitative reverse transcription-PCR demonstrated increased expression of
clfA
and
fnb
genes by the
hemB
mutant compared to its isogenic parent. The influence of the
hemB
mutation on altered adhesin expression was confirmed by showing complete restoration of the wild-type adhesive phenotype in the
hemB
mutant, either by complementing with intact
hemB
or by supplementing the growth medium with hemin. Increased surface display of fibrinogen and fibronectin adhesins by the
hemB
mutation occurred independently from
agr
, a major regulatory locus of virulence factors in
S. aureus
. Both
agr
-positive and
agr
-lacking
hemB
mutants were also more efficiently internalized by human embryonic kidney cells than were their isogenic controls, presumably because of increased surface display of their fibronectin adhesins.
Fimbrial
ghf
Gene Cluster of Genital Strains of
Haemophilus
spp
Authors: Bruant Guillaume , Gousset Nathalie , Quentin Roland , Rosenau Agnes
Published: September 2002

We analyzed the LKP fimbrial gene clusters of six piliated strains of a cryptic genospecies of
Haemophilus
isolated from the genital tracts of adult patients (five strains) and from an infected neonate. In a group of 19 genital strains, LKP-like genes have been found in only these 6 strains. In addition to the
ghfA
,
ghfD
, and
ghfE
genes previously described, we characterized two genes, designated
ghfB
and
ghfC
, encoding the putative chaperone and assembly platform proteins. All six strains had a complete and unique LKP-like gene cluster consisting of the five genes
ghfA
to
ghfE
, homologous to genes
hifA
to
hifE
of
Haemophilus influenzae
. The sequences of the coding and intergenic regions of the
ghf
clusters of the six strains were remarkably homologous. Unlike
hif
clusters, which are inserted between
purE
and
pepN
, the
ghf
cluster was inserted between
purK
and
pepN
on the chromosome. Analysis of the flanking regions of the
ghf
cluster identified a large deletion, identical in the 5′ end regions of all strains, including the whole
purE
gene and much of the
purK
gene. Ultrastructural observations, an attempt at enriching LKP fimbriae, and hemagglutination experiments demonstrated that none of the strains had LKP-type fimbriae. Nevertheless, reverse transcription (RT)-PCR showed that
ghf
genes were transcribed in four of the six strains. Sequencing of the intergenic
ghfA
-
ghfB
regions, including the
ghf
gene promoters, showed that the absence of transcripts in the remaining two strains was due to a decrease in the number of TA repeats (4 or 9 repeats rather than 10) between the −10 and −35 boxes of the two overlapping and divergent promoters. The other four strains, which had
ghf
transcripts, had the optimal 10 TA repeats (one strain) or 5 repeats associated with putative alternative −35 boxes (three strains). The absence of 10 repeated palindromic sequences of 44 or 45 nucleotides upstream of
ghfB
induces an increased instability of mRNA, as quantified by real-time RT-PCR, and may explain why the LKP fimbrial gene cluster is not expressed in these strains.
Epitope Mapping of the Immunodominant Antigen TB10.4 and the Two Homologous Proteins TB10.3 and TB12.9, Which Constitute a Subfamily of the
esat-6
Gene Family
Authors: Skjøt Rikke Louise Vinther , Brock Inger , Arend Sandra M. , Munk Martin E. , Theisen Michael , Ottenhoff Tom H. M. , Andersen Peter
Published: September 2002

The human T-cell recognition of the low-molecular-mass culture filtrate antigen TB10.4 was evaluated in detail. The molecule was strongly recognized by T cells isolated from tuberculosis (TB) patients and from BCG-vaccinated donors. The epitopes on TB10.4 were mapped with overlapping peptides and found to be distributed throughout the molecule. The broadest response was found in TB patients, whereas the response in BCG-vaccinated donors was focused mainly toward a dominant epitope located in the N terminus (amino acids 1 to 18). The gene encoding TB10.4 was found to belong to a subfamily within the
esat
-
6
family that consists of the three highly homologous proteins TB10.4, TB10.3, and TB12.9 (Rv0288, Rv3019c, and Rv3017c, respectively). Southern blot analysis combined with database searches revealed that the three members of the TB10.4 family were present only in strains of the
Mycobacterium tuberculosis
complex, including BCG, and
M. kansasii
, whereas other atypical mycobacteria had either one (
M. avium
,
M. intracellulare
, and
M. marinum
) or none (
M. scrofulaceum
,
M. fortuitum
, and
M. szulgai
) of the genes. The fine specificity of the T-cell response to the three closely related
esat-6
family members was markedly different, with only a few epitopes shared between the molecules. Minimal differences in the amino acid sequence translated into large differences in recognition by T cells and secretion of gamma interferon. In general, the peptides from TB10.4 stimulated the largest responses, but epitopes unique to both TB10.3 and TB12.9 were found. The relevance of the findings for TB vaccine development and as a potential mechanism for immune evasion is discussed.
Role of RegM, a Homologue of the Catabolite Repressor Protein CcpA, in the Virulence of
Streptococcus pneumoniae
Authors: Giammarinaro Philippe , Paton James C.
Published: September 2002

As part of a study of virulence gene regulation in
Streptococcus pneumoniae
, we have identified a gene encoding a homologue of the staphylococcal catabolite control protein CcpA in the pneumococcal genome sequence. The pneumococcal protein, designated RegM, has significant similarity to members of the LacI/GalR family of bacterial regulatory proteins.
S. pneumoniae
D39 derivatives with insertion-duplication or deletion mutations in
regM
were significantly attenuated in virulence with respect to the wild-type strain. In defined media containing either sucrose or lactose as sole carbon sources, the in vitro growth rates of D39 and the
regM
mutants were essentially the same. However, in the presence of galactose the
regM
mutants grew significantly faster than the wild-type strain, whereas growth rates were significantly lower in the presence of glucose or maltose. These data are consistent with the involvement of
regM
in the catabolism of carbohydrates in
S. pneumoniae
. RegM was a repressor of both α-glucosidase and β-galactosidase activities in
S. pneumoniae
, but unlike the situation in certain other bacteria, it does not mediate the repression of these enzymes by glucose. The observed attenuation in virulence was not attributable to poorer growth of the
regM
mutants in mouse blood ex vivo, but nevertheless, the mutants were rapidly cleared from the blood of infected mice in vivo. The
regM
mutation had no apparent impact on expression of several confirmed pneumococcal virulence proteins, but studies employing a
lacZ
transcriptional fusion construct indicated that mutation of
regM
resulted in a significant reduction in transcription of the capsular polysaccharide biosynthesis locus (
cps
). Thus,
regM
is the first gene outside of the
cps
locus to be implicated in regulation of capsular gene expression.
Interplay between Protective and Inhibitory Antibodies Dictates the Outcome of Experimentally Disseminated Candidiasis in Recipients of aCandida albicansVaccine
Authors: Bromuro Carla , Torosantucci Antonella , Chiani Paola , Conti Stefania , Polonelli Luciano , Cassone Antonio
Published: September 2002

Mice immunized with heat-inactivated, whole yeast-form cells (Y cells) ofCandida albicansdeveloped intense, specific humoral and cell-mediated immune responses. However, they were modestly protected against a lethal challenge by the fungus, and their sera did not confer passive protection upon nonimmunized animals. Surprisingly, this immune serum conferred an elevated degree of passive protection to normal and SCID mice when preadsorbed on wholeC. albicanscells. After adsorption, no antibodies specific to mannoprotein (MP)-rich extracts or secretions were detected by indirect enzyme-linked immunosorbent assay and no serum reaction with the fungal cell surface was seen in immunofluorescence assays. However, this serum had totally preserved the level of other antibodies, in particular those reacting with β-1,3 and β-1,6 glucan (GG). The hypothesis that anti-GG antibodies contributed to the passive protection was suggested by the following circumstantial evidence: (i) mice immunized withC. albicanscells treated with dithiothreitol and protease (YDP cells), which exposed GG on their surfaces and generated anti-GG but not anti-MP antibodies, were substantially protected against a lethal fungus challenge; (ii) the sera, and their immunoglobulin fractions, of mice immunized with YDP cells transferred protection to nonimmune animals; and (iii) this passive protection was substantially abolished by preadsorption on GG but not on intact cells. Overall, our findings demonstrate that some anti-Candidaantibodies can block the protective potential of immune serum, a potential to which anti-GG antibodies appear to contribute. Our observations may also help explain why subjects with elevated anti-Candidaantibody titers, inclusive of anti-MP and anti-GG antibodies, remain nonetheless susceptible to invasive candidiasis.
Mycobacterium bovisBCG Vaccination Augments Interleukin-8 mRNA Expression and Protein Production in Guinea Pig Alveolar Macrophages Infected withMycobacterium tuberculosis
Authors: Lyons Mark J. , Yoshimura Teizo , McMurray David N.
Published: September 2002

Alveolar macrophages are likely the first cell type to encounterMycobacterium tuberculosisin a pulmonary infection, resulting in the production of chemokines. In order to evaluate this response, alveolar macrophages harvested from nonvaccinated andMycobacterium bovisBCG-vaccinated guinea pigs were infected in vitro with liveM. tuberculosisH37Ra or H37Rv (multiplicity of infection, 1:1) or cultured with lipopolysaccharide (10 μg/ml) for 3, 12, and 24 h. Interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) mRNA expression was determined by real-time PCR. Culture supernatants were assayed for guinea pig IL-8 protein by using a human IL-8 enzyme-linked immunosorbent assay kit. Alveolar macrophages harvested from BCG-vaccinated guinea pigs produced significantly more mRNA and protein for IL-8 than alveolar macrophages harvested from nonvaccinated guinea pigs at 12 and 24 h poststimulation or postinfection. Infection with attenuatedM. tuberculosis(H37Ra) stimulated alveolar macrophages isolated from BCG-vaccinated guinea pigs to produce significantly more IL-8 mRNA than did alveolar macrophages infected with a virulent strain (H37Rv) at 12 and 24 h postinfection. Significant MCP-1 mRNA production was also detected in stimulated or infected alveolar macrophages; however, prior vaccination did not significantly affect levels of MCP-1 mRNA. Alveolar macrophages isolated from BCG-vaccinated guinea pigs produced significantly more IL-8 mRNA and protein when stimulated for 24 h with heat-killed H37Ra, heat-killed H37Rv, and H37Rv cell wall, but not mannose-capped lipoarabinomannan (ManLAM), than did cells stimulated with media alone. These observations indicate that prior vaccination may alter very early events in theM. tuberculosis-infected lung.
Influence of Intravenous Anesthesia on Mucosal and Systemic Antibody Responses to Nasal Vaccines
Authors: Janakova Libuse , Bakke Hilde , Haugen Inger Lise , Berstad Aud K. H. , Høiby E. Arne , Aaberge Ingeborg S. , Haneberg Bjørn
Published: September 2002

Inhalation of antigens may stimulate the immune system by way of the upper as well as the lower airways. We have shown that at least 1,000 times more live pneumococci were recovered from pulmonary tissue after being presented as drops of a liquid suspension onto the nares of anesthetized mice compared to the number of bacteria recovered from animals that were not anesthetized in the course of the challenge. Mice that were similarly immunized intranasally by inhalation of three different nonreplicating particulate vaccine formulations, i.e., a meningococcal outer membrane vesicle (OMV) vaccine, a formalin-inactivated whole-virus influenza (INV) vaccine, and the INV vaccine with OMVs as a mucosal adjuvant, during general intravenous anesthesia developed concentrations of vaccine-specific serum immunoglobulin G (IgG) antibodies that were four to nine times higher than in mice that were fully awake during immunizations. The concentrations of IgA antibodies in serum were also higher in anesthetized than in nonanesthetized mice and correlated positively with the corresponding levels of serum IgG antibodies in the anesthetized but not in the nonanesthetized mice. In saliva and feces, however, the concentrations of IgA antibodies were equally high whether or not the animals were dormant during immunizations. The results indicate that intrapulmonary antigen presentation, as a part of an intranasal immunization strategy, is of importance for systemic but not for mucosal antibody responses. A major portion of IgA antibodies in serum may thus be derived from nonmucosal sites.
Purification and Characterization of a Second Immunoreactive Mannoprotein fromCryptococcus neoformansThat Stimulates T-Cell Responses
Authors: Huang Chao , Nong Shu-hua , Mansour Michael K. , Specht Charles A. , Levitz Stuart M.
Published: September 2002

Although T-cell responses are known to be critical for effective host defenses against the fungal pathogenCryptococcus neoformans, the antigens that stimulate protective responses are poorly characterized but are thought to be comprised, at least in part, of mannoproteins. Recently, we created a panel of murine CD4+-T-cell hybridomas that react withC. neoformansantigens. A mannoprotein antigen, MP98, that stimulated one of the hybridomas was purified, and the gene encoding MP98 was cloned. In the present study, the cryptococcal antigen, MP88, that stimulated a second T-cell hybridoma, X5A3, to secrete interleukin-2 was characterized. MP88 was purified from supernatants of glass bead-disruptedC. neoformansby anion-exchange and hydrophobic interaction chromatography. A single band with an apparent molecular mass of 88 kDa was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to partial internal amino acid sequencing. The gene encoding MP88 was cloned and sequenced. MP88 features a C-terminal serine/threonine-rich region, which presumably serves as a site for extensive O glycosylation, followed by a putative glycosylphosphatidylinositol anchor site. A search ofC. neoformansgenomic databases revealed thatMP88shares this feature with at least 11 other genes, includingMP98. The mannoprotein nature of MP88 was established based upon the capacity of (i) the mannoprotein fraction ofC. neoformanssupernatants to stimulate X5A3 and (ii) mannosylated ligands to competitively inhibit this stimulation. Thus, a second cryptococcal mannoprotein has been identified which stimulates T-cell responses and is a vaccine candidate.
Gene Expression Profiling of Peripheral Blood Mononuclear Cells from Cattle Infected withMycobacterium paratuberculosis
Authors: Coussens Paul M. , Colvin Christopher J. , Wiersma Kacie , Abouzied Amy , Sipkovsky Sue
Published: September 2002

A bovine-specific cDNA microarray system containing 721 unique leukocyte expressed sequence tags (ESTs) and amplicons representing known genes was used to compare gene expression profiles of peripheral blood mononuclear cells (PBMCs) from clinical and subclinical Johne's disease-positive Holstein cows (n= 2 per group). Stimulation of PBMCs from clinically infected cows withMycobacterium paratuberculosistended to decrease expression of 83 genes (fold change, >1.5). Of these 83 genes, 16 displayed significant down regulation across both clinical cows (P< 0.1), including genes encoding microspherule protein 1, fibroblast growth factor, and the Lyn B protein kinase. Only eight genes from PBMCs of clinically infected cows exhibited a modest up regulation following stimulation withM. paratuberculosis, including those encoding bovine CD40L, gamma interferon, interleukin-10 (IL-10), and tissue inhibitor of matrix metalloproteinases (TIMP) 4. In contrast, stimulation of PBMCs from subclinically infected cows withM. paratuberculosistended to up regulate expression of 71 genes representing 68 unique transcripts. Of these, 11 genes showed significant up regulation (fold change, >1.5;P< 0.1) across both animals, including those encoding bovine CD40L, several matrix metalloproteinases, and SPARC (secreted protein, acidic and rich in cystine). Repression of gene expression was also observed in PBMCs from the subclinical cows, with 16 genes being significantly down regulated (fold change, >1.5;P< 0.1) across both animals, including those encoding the bovine orthologs of cytochrome oxidase subunit III, IL-1 receptor type I, and fibrinogen-like 2 protein. Only one clone, representing an unknown bovine EST, was similarly down regulated in PBMCs from both the clinical and subclinical cows. Thus, the most prominent change induced by exposure of PBMCs from clinical cows toM. paratuberculosisin vitro tended to be repression of gene expression, while changes in similarly treated PBMCs from subclinical cows was balanced between gene activation and repression. Comparison of gene expression profiles between PBMCs from clinical and uninfected (control) cows stimulated with the general mitogen concanavalin A were highly similar (overallr= 0.84), suggesting thatM. paratuberculosis-induced gene repression in clinically infected cow PBMCs was not due to a general failure of the immune response in these animals.
Representational Difference Analysis between Afa/Dr Diffusely Adhering
Escherichia coli
and Nonpathogenic
E. coli
K-12
Authors: Blanc-Potard Anne-Beatrice , Tinsley Colin , Scaletsky Isabel , Le Bouguenec Chantal , Guignot Julie , Servin Alain L. , Nassif Xavier , Bernet-Camard Marie-Francoise
Published: September 2002

Diffusely adhering
Escherichia coli
strains harboring Afa/Dr adhesins (Afa/Dr DAEC) have been associated with diarrhea and urinary tract infections (UTIs). The present work is the first extensive molecular study of a Afa/Dr DAEC strain using the representational difference analysis technique. We have searched for DNA sequences present in strain C1845, recovered from a diarrheagenic child, but absent from a nonpathogenic K-12 strain. Strain C1845 harbors part of a pathogenicity island (PAI
CFT073
) and several iron transport systems found in other
E. coli
pathovars. We did not find genes encoding factors known to subvert host cell proteins, such as type III secretion system or effector proteins. Several C1845-specific sequences are homologous to putative virulence genes or show no homology with known sequences, and we have analyzed their distribution among Afa/Dr and non-Afa/Dr clinical isolates and among strains from the
E. coli
Reference Collection. Three C1845-specific sequences (MO30, S109, and S111) have a high prevalence (77 to 80%) among Afa/Dr strains and a low prevalence (12 to 23%) among non-Afa/Dr strains. In addition, our results indicate that strain IH11128, an Afa/Dr DAEC strain recovered from a patient with a UTI, is genetically closely related to strain C1845.
Identification of Two Distinct Subpopulations ofLeishmania major-Specific T Helper 2 Cells
Authors: Kropf Pascale , Herath Shanti , Tewari Rita , Syed Nelofer , Klemenz Roman , Müller Ingrid
Published: September 2002

It is widely accepted that a strong Th2 response is responsible for nonhealingLeishmania majorinfections in BALB/c mice. This Th2 response has been thoroughly documented by measuring the levels of Th2 cytokines produced by CD4+T cells present in the lymphoid organs by enzyme-linked immunosorbent assay and PCR. However, the cytokine profile ofL. major-specific Th2 cells has never been determined. In this study, we used the recently described Th2 marker T1/ST2 to characterize Th2 cells during the course of nonhealingL. majorinfection. We analyzed the intracellular cytokine profile of CD4+T1/ST2+T cells and showed that they clearly displayed a Th2 phenotype, as they expressed interleukin 4 (IL-4), IL-10, and IL-5. In addition, we detected another population of Th2 cells among the CD4+T1/ST2−T cells that expressed IL-4 and IL-10 but excluded IL-5. In summary, we show here that two type 2 subpopulations are present in the lymphoid organs ofL. major-infected BALB/c mice; Th2 cells from both subsets expressed IL-4 and IL-10, but they could be distinguished by their expression of IL-5 and T1/ST2.
Major Histocompatibility Complex Class II DR-Restricted Memory CD4+T Lymphocytes Recognize Conserved Immunodominant Epitopes ofAnaplasma marginaleMajor Surface Protein 1a
Authors: Brown Wendy C. , McGuire Travis C. , Mwangi Waithaka , Kegerreis Kimberly A. , Macmillan Henriette , Lewin Harris A. , Palmer Guy H.
Published: September 2002

Native major surface protein 1 (MSP1) ofAnaplasma marginale, composed of covalently associated MSP1a and MSP1b proteins, stimulates protective immunity in cattle against homologous and heterologous strain challenge. Protective immunity against pathogens in the familyAnaplasmataceaeinvolves both CD4+T cells and neutralizing immunoglobulin G. Thus, an effective vaccine should contain both CD4+T- and B-lymphocyte epitopes that will elicit strong memory responses upon infection with homologous and heterologous strains. Previous studies demonstrated that the predominant CD4+T-cell response in MSP1 vaccinates is directed against the MSP1a subunit. The present study was designed to identify conserved CD4+T-cell epitopes in MSP1a presented by a broadly represented subset of major histocompatibility complex (MHC) class II molecules that would be suitable for inclusion in a recombinant vaccine. Transmembrane protein prediction analysis of MSP1a from the Virginia strain revealed a large hydrophilic domain (HD), extending from amino acids (aa) 1 to 366, and a hydrophobic region extending from aa 367 to 593. The N terminus (aa 1 to 67) includes one 28-aa form A repeat and one 29-aa form B repeat, which each contain an antibody neutralization-sensitive epitope [Q(E)ASTSS]. In MSP1 vaccinates, recombinant MSP1a HD (aa 1 to 366) stimulated recall proliferative responses that were comparable to those against whole MSP1a excluding the repeat region (aa 68 to 593). Peptide mapping determined a minimum of five conserved epitopes in aa 151 to 359 that stimulated CD4+T cells from cattle expressingDR-DQhaplotypes common in Holstein-Friesian breeds. Peptides representing three epitopes (aa 231 to 266, aa 270 to 279, and aa 290 to 319) were stimulatory for CD4+T-cell clones and restricted by DR. A DQ-restricted CD4+T-cell epitope, present in the N-terminal form B repeat (VSSQSDQASTSSQLG), was also mapped using T-cell clones from one vaccinate. Although form B repeat-specific T cells did not recognize the form A repeat peptide (VSSQS_EASTSSQLG), induction of T-cell anergy by this peptide was ruled out. The presence of multiple CD4+T-cell epitopes in the MSP1a HD, in addition to the neutralization-sensitive epitope, supports the testing of this immunogen for induction of protective immunity againstA. marginalechallenge.
Cholera Toxin and Heat-Labile Enterotoxin Activate Human Monocyte-Derived Dendritic Cells and Dominantly Inhibit Cytokine Production through a Cyclic AMP-Dependent Pathway
Authors: Bagley Kenneth C. , Abdelwahab Sayed F. , Tuskan Robert G. , Fouts Timothy R. , Lewis George K.
Published: September 2002

Cholera toxin (CT) and heat-labile enterotoxin (LT) are powerful mucosal adjuvants whose cellular targets and mechanism of action are unknown. There is emerging evidence that dendritic cells (DC) are one of the principal cell types that mediate the adjuvant effects of these toxins in vivo. Here we investigate the effects of CT and LT on the maturation of human monocyte-derived DC (MDDC) in vitro. We found that an enzymatically active A domain is necessary for both CT and LT to induce the maturation of MDDC and that this activation is strictly cyclic AMP (cAMP) dependent. ADP-ribosylation-defective derivatives of these toxins failed to induce maturation of MDDC, whereas dibutyryl-cyclic-3′,5′-AMP and Forskolin mimic the maturation of MDDC induced by CT and LT. In addition, an inhibitor of cAMP-dependent kinases, Rp-8-Br-cAMPs, blocked the ability of CT, LT, and Forskolin to activate MDDC. CT, LT, dibutyryl-cyclic-3′,5′-AMP, and Forskolin also dominantly inhibit interleukin 12 and tumor necrosis factor alpha production by MDDC in the presence of saturating concentrations of lipopolysaccharide. Taken together, these results show that the effects of CT and LT on MDDC are mediated by cAMP.
Effect of
omp10
or
omp19
Deletion on
Brucella abortus
Outer Membrane Properties and Virulence in Mice
Authors: Tibor Anne , Wansard Valérie , Bielartz Valery , Delrue Rose-May , Danese Isabelle , Michel Patrick , Walravens Karl , Godfroid Jacques , Letesson Jean-Jacques
Published: September 2002

The distinctive properties of
Brucella
outer membrane have been considered to be critical for
Brucella
sp. virulence. Among the outer membrane molecules possibly related to these properties, Omp10 and Omp19 are immunoreactive outer membrane lipoproteins. Moreover, these proteins of
Brucella
could constitute a new family of outer membrane proteins specifically encountered in the family
Rhizobiaceae
. We evaluated the impact of
omp10
or
omp19
deletion on
Brucella abortus
outer membrane properties and virulence in mice. The
omp10
mutant was dramatically attenuated for survival in mice and was defective for growth in minimal medium but was not impaired in intracellular growth in vitro, nor does it display clear modification of the outer membrane properties. Significantly fewer brucellae were recovered from the spleens of mice infected with the
omp19
mutant than from those of mice infected with the parent strain at 4 and 8 weeks postinfection. The
omp19
mutant exhibited an increase in sensitivity to the polycation polymyxin B and to sodium deoxycholate. These results indicate that inactivation of the
omp19
gene alters the outer membrane properties of
B. abortus
.
Genetic Immunization Elicits Antigen-Specific Protective Immune Responses and Decreases Disease Severity inTrypanosoma cruziInfection
Authors: Garg Nisha , Tarleton Rick L.
Published: September 2002

Immunity toTrypanosoma cruzirequires elicitation of humoral and cell-mediated immune responses to extracellular trypomastigotes and intracellular amastigotes. In this study, the effectiveness of theT. cruzitrans-sialidase family (ts) genesASP-1,ASP-2, andTSA-1as genetic vaccines was assessed. Immunization of mice with plasmids encodingASP-1,ASP-2, orTSA-1elicited poor antigen-specific cytotoxic-T-lymphocyte (CTL) activity andT. cruzi-specific antibody responses. Codelivery of interleukin-12 and granulocyte-macrophage colony-stimulating factor plasmids with antigen-encoding plasmids resulted in a substantial increase in CTL activity and antibody production and in increased resistance toT. cruziinfection. In pooled results from two to four experiments, 30 to 60% of mice immunized with antigen-encoding plasmids and 60 to 80% of mice immunized with antigen-encoding plasmids plus cytokine adjuvants survived a lethal challenge withT. cruzi. In comparison, 90% of control mice injected with empty plasmid DNA died during the acute phase of infection. However, the pool of three ts genes provided no greater protection than the most effective single gene (ASP-2) either with or without coadministration of cytokine plasmids. Importantly, the extent of tissue parasitism, inflammation, and associated tissue damage in skeletal muscles during the chronic phase ofT. cruziinfection in mice immunized with antigen-encoding plasmids plus cytokine adjuvants was remarkably reduced compared to mice immunized with only cytokine adjuvants or empty plasmid DNA. These results identify new vaccine candidates and establish some of the methodologies that might be needed to develop effective vaccine-mediated control ofT. cruziinfection. In addition, this work provides the first evidence that prophylactic genetic immunization can prevent the development of Chagas’ disease.
Differential Responses of Bovine Macrophages toMycobacterium aviumsubsp.paratuberculosisandMycobacterium aviumsubsp.avium
Authors: Weiss Douglas J. , Evanson Oral A. , Moritz Andreas , Deng Ming Qi , Abrahamsen Mitchell S.
Published: September 2002

Mycobacterium aviumsubsp.paratuberculosisandMycobacterium aviumsubsp.aviumare antigenically and genetically similar organisms; however, they differ in their virulence for cattle.M. aviumsubsp.paratuberculosiscauses a chronic intestinal infection leading to a chronic wasting disease termed paratuberculosis or Johne's disease, whereasM. aviumsubsp.aviumcauses only a transient infection. We compared the response of bovine monocyte-derived macrophages to ingestion ofM. aviumsubsp.paratuberculosisandM. aviumsubsp.aviumorganisms by determining organism survival, superoxide and nitric oxide production, and expression of the cytokines tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), interleukin-8 (IL-8), IL-10, IL-12, and granulocyte-monocyte colony-stimulating factor (GM-CSF). UnlikeM. aviumsubsp.paratuberculosis, macrophages were able to kill approximately half of theM. aviumsubsp.aviumorganisms after 96 h of incubation. This difference in killing efficiency was not related to differences in nitric oxide or superoxide production. Compared to macrophages activated with IFN-γ and lipopolysaccharide, macrophages incubated withM. aviumsubsp.paratuberculosisshowed greater expression of IL-10 and GM-CSF (all time points) and IL-8 (72 h) and less expression of IL-12 (72 h), IFN-γ (6 h), and TNF-α (6 h). When cytokine expression by macrophages incubated withM. aviumsubsp.paratuberculosiswas compared to those of macrophages incubated withM. aviumsubsp.avium,M. aviumsubsp.paratuberculosis-infected cells showed greater expression of IL-10 (6 and 24 h) and less expression of TNF-α (6 h). Therefore, the combination of inherent resistance to intracellular degradation and suppression of macrophage activation through oversecretion of IL-10 may contribute to the virulence ofM. aviumsubsp.paratuberculosisin cattle.
Cytokine Profiles of Patients Infected withMycobacterium ulceransand Unaffected Household Contacts
Authors: Gooding Travis M. , Johnson Paul D. R. , Smith May , Kemp Andrew S. , Robins-Browne Roy M.
Published: September 2002

Mycobacterium ulcerans, the cause of Buruli ulcer, is an environmental mycobacterium with a distinct geographic distribution. The reasons why only some individuals who are exposed toM. ulceransdevelop ulcers are not known but are likely to reflect individual differences in the immune response to infections with this bacterium. In this study, we investigated cytokine profiles of peripheral blood mononuclear cells (PBMC) from 23 Buruli ulcer patients and 25 household contacts in a region of Australia where Buruli ulcer is endemic. The results showed that following stimulation withM. ulceransorMycobacterium bovisBCG, PBMC from Buruli ulcer patients mounted a Th2-type response, which was manifested by the production of mRNA for interleukin 4 (IL-4), IL-5, IL-6, and IL-10, whereas unaffected contacts responded mainly with the Th1 cytokines gamma interferon (IFN-γ) and IL-12. For example, mRNA for IL-4 was detected in 18 of 23 patients but in only 3 of 25 control subjects (P< 0.0001). By contrast, PBMC from 21 of 25 unaffected individuals produced IFN-γ compared with 3 of 23 patients (P< 0.0001). IFN-γ release following stimulation with mycobacteria was markedly reduced in affected subjects. Frequencies of antibodies toM. ulceransin serum samples from affected and unaffected subjects were similar, indicating that many of the control subjects had been exposed to this bacterium. Together, these findings suggest that a Th1-type immune response toM. ulceransmay prevent the development of Buruli ulcer in people exposed toM. ulcerans, but a Th-2 response does not.
Bacterial Artificial Chromosome-Based Comparative Genomic Analysis Identifies
Mycobacterium microti
as a Natural ESAT-6 Deletion Mutant
Authors: Brodin Priscille , Eiglmeier Karin , Marmiesse Magali , Billault Alain , Garnier Thierry , Niemann Stefan , Cole Stewart T. , Brosch Roland
Published: September 2002

Mycobacterium microti
is a member of the
Mycobacterium tuberculosis
complex that causes tuberculosis in voles. Most strains of
M. microti
are harmless for humans, and some have been successfully used as live tuberculosis vaccines. In an attempt to identify putative virulence factors of the tubercle bacilli, genes that are absent from the avirulent
M. microti
but present in human pathogen
M. tuberculosis
or
Mycobacterium bovis
were searched for. A minimal set of 50 bacterial artificial chromosome (BAC) clones that covers almost all of the genome of
M. microti
OV254 was constructed, and individual BACs were compared to the corresponding BACs from
M. bovis
AF2122/97 and
M. tuberculosis
H37Rv. Comparison of pulsed-field gel-separated DNA digests of BAC clones led to the identification of 10 regions of difference (RD) between
M. microti
OV254 and
M. tuberculosis
. A 14-kb chromosomal region (RD1
mic
) that partly overlaps the RD1 deletion in the BCG vaccine strain was missing from the genomes of all nine tested
M. microti
strains. This region covers 13 genes, Rv3864 to Rv3876, in
M. tuberculosis
, including those encoding the potent ESAT-6 and CFP-10 antigens. In contrast, RD5
mic
, a region that contains three phospholipase C genes (
plcA
to -
C
), was missing from only the vole isolates and was present in
M. microti
strains isolated from humans. Apart from RD1
mic
and RD5
mic
other
M. microti
-specific deleted regions have been identified (MiD1 to MiD3). Deletion of MiD1 has removed parts of the direct repeat region in
M. microti
and thus contributes to the characteristic spoligotype of
M. microti
strains.
Tissue Selectivity of Interferon-Stimulated Gene Expression in Mice Infected with Dam+versus Dam−Salmonella entericaSerovar Typhimurium Strains
Authors: Shtrichman Ronit , Heithoff Douglas M. , Mahan Michael J. , Samuel Charles E.
Published: September 2002

The host interferon (IFN) system plays an important role in protection against microbial infections.Salmonella entericaserovar Typhimurium is highly virulent in the mouse model, whereas mutants that lack DNA adenine methylase (Dam−) are highly attenuated and elicit fully protective immune responses against murine typhoid fever. We examined the expression of IFN-responsive genes in several mouse tissues following infection with Dam+or Dam−Salmonella.Infection of mice with Dam+Salmonellaresulted in the induction of host genes known to be indicators of IFN bioactivity and regulated by either IFN-α/β (Mx1) or IFN-γ (class II transactivator protein [CIITA] and inducible nitric oxide synthase [iNOS]) or by both IFN-α/β and IFN-γ (RNA-specific adenosine deaminase [ADAR1] and RNA-dependent protein kinase [PKR]) in a tissue-specific manner compared to uninfected animals. Since the Mx1 promoter is IFN-α/β specific and theMx1gene is not inducible directly by IFN-γ, these data suggest a role of IFN-α/β in the host response toSalmonellainfection. Mice infected with Dam−Salmonellashowed reduced expression of the same set of IFN-stimulated genes (ISGs) as that observed after infection with wild-typeSalmonella.The reduced capacity to induce ISGs persisted in Dam−-vaccinated mice after challenge with the virulent (Dam+) strain. Finally, although no Dam−organisms were recovered from the liver or spleen after oral infection of mice, ADAR, PKR, Mx, and CIITA expression levels were elevated in these tissues relative to those in uninfected mice, suggestive of the distant action of a signaling molecule(s) in the activation of ISG expression.
Enhanced Immunogenicity of Pneumococcal Surface Adhesin A by Genetic Fusion to Cytokines and Evaluation of Protective Immunity in Mice
Authors: Gor Dennis O. , Ding Xuedong , Li Qing , Schreiber John R. , Dubinsky Michael , Greenspan Neil S.
Published: September 2002

Immunization of mice with pneumococcal surface adhesin A (PsaA) emulsified in complete Freund's adjuvant (CFA) provides protection against systemic infection withStreptococcus pneumoniae. Because the use of CFA is not acceptable in humans, we sought to develop alternative means of enhancing the immunogenicity of protein antigens of potential use in pneumococcal vaccines. We designed a series of genetic constructs in which coding sequences for PsaA were linked to sequences encoding either murine interleukin-2 (mIL-2), mIL-4, or two copies of an immunostimulatory nonapeptide derived from mIL-1β. The PsaA-cytokine constructs were cloned and expressed inEscherichia coli. Mice immunized twice with PsaA-IL-2, or PsaA-IL-4 responded with PsaA-specific antibody production comparable in magnitude to that of mice primed with PsaA in CFA and boosted with PsaA in incomplete Freund's adjuvant (PsaA-Adj). Antibodies elicited by PsaA-Adj were predominantly of the immunoglobulin G1 (IgG1) subclass, while PsaA-IL-2 and PsaA-IL-4 elicited substantial amounts of IgG2a in addition to IgG1. Mice immunized with PsaA-Adj or PsaA-IL-4 were partially protected against intraperitoneal challenge with virulentS. pneumoniae(30% overall survival beyond 15 days postchallenge). Mice immunized with PsaA and no adjuvant or PsaA-IL-2 exhibited 0 or 5% survival rates, respectively, following challenge. In contrast, mice immunized twice with capsular polysaccharide were 100% protected. The modest levels of protection seen in mice immunized with PsaA and its more immunogenic derivatives may be explained in part by the relative inaccessibility of antibody to PsaA on the surface of encapsulatedS. pneumoniae.
Beneficial or Deleterious Effects of a Preexisting Hypersensitivity to Bacterial Components on the Course and Outcome of Infection
Authors: Gumenscheimer Marina , Mitov Ivan , Galanos Chris , Freudenberg Marina A.
Published: September 2002

Priming with heat-killedPropionibacterium acnesenhances the sensitivity of mice to lipopolysaccharide (LPS) and other biologically active bacterial components. We show thatP. acnespriming has protective and deleterious effects on a subsequent serovar Typhimurium infection. It may result in a complete protection or prolonged survival, or it may accelerate mortality of the infected mice, depending on the number of serovar Typhimurium bacteria administered and on the degree of LPS hypersensitivity at the time of infection. Both effects ofP. acnes-induced hypersensitivity are mediated by gamma interferon (IFN-γ) and are based on a differential activation of the innate immune mechanisms which recognize and react against the LPS present in infecting bacteria. InP. acnes-primed mice null for LPS-binding protein (LBP−/−mice), the impaired LPS recognition, due to the absence of LBP, resulted in a higher resistance to serovar Typhimurium infection. A similarP. acnespriming of mice had a protective, but no deleterious effect on a subsequentL. monocytogenesinfection. This effect was IFN-γ dependent but independent of LBP.
The Human Complement Regulator Factor H Binds Pneumococcal Surface Protein PspC via Short Consensus Repeats 13 to 15
Authors: Duthy Thomas G. , Ormsby Rebecca J. , Giannakis Eleni , Ogunniyi A. David , Stroeher Uwe H. , Paton James C. , Gordon David L.
Published: September 2002

The innate ability ofStreptococcus pneumoniaeto resist complement activation and complement-mediated phagocytosis may be a direct consequence of the ability of the bacteria to bind components of the complement regulatory system. One such component, factor H (fH), is a crucial fluid-phase negative regulator of the alternative pathway of complement and is utilized by a number of pathogenic organisms to resist complement attack. The pneumococcal surface protein C (PspC [also known as CbpA] and SpsA) has been shown to bind fH, although the exact binding site within one or more of the 20 short consensus repeats (SCRs) of the molecule is not known. The purpose of the current study was to map specific SCRs on fH responsible for this binding. Initial experiments utilizing type 2 pneumococcal strain D39 and its isogenic PspC-negative derivative (D39/pspCmutant) showed that fH binding was PspC dependent. A purified recombinant protein derivative of PspC that lacked the proline-rich region (PspCΔPro) had a reduced binding efficiency for fH, thereby directly showing the importance of this region for the fH interaction. We have specifically shown by inhibition experiments that SCRs responsible for heparin and C3b binding of fH are not involved in binding PspC and the interaction between fH and PspC is largely hydrophobic, since no inhibition was observed in the presence of high concentrations of NaCl. Construction of SCR proteins encompassing the whole fH molecule showed that SCRs 8 to 15 (SCR 8-15) mediated binding to PspC. Further localization experiments revealed that SCR 13 and SCR 15 were required for full binding, although partial binding was retained when either SCR was removed.
Characterization of P40, a Cytadhesin of
Mycoplasma agalactiae
Authors: Fleury Bénédicte , Bergonier Dominique , Berthelot Xavier , Peterhans Ernst , Frey Joachim , Vilei Edy M.
Published: September 2002

An immunodominant protein, P40, of
Mycoplasma agalactiae
was analyzed genetically and functionally. The gene encoding P40 was cloned from type strain PG2, sequenced, submitted to point mutagenesis in order to convert mycoplasma-specific TGA
Trp
codon to the universal TGG
Trp
codon, and subsequently expressed in
Escherichia coli
. Nucleotide sequence-derived amino acid sequence comparisons revealed a similarity of P40 to the adhesin P50 of
Mycoplasma hominis
and to protein P89 of
Spiroplasma citri
, which is expected to be involved in adhesion. The amino acid sequence of P40 revealed a recognition site for a signal peptidase and strong antigenic and hydrophilic motifs in the C-terminal domain. Triton X-114 phase partitioning confirmed that P40 is a membrane protein. Fab fragments of antibodies directed against recombinant purified P40 significantly inhibited adherence of
M. agalactiae
strains PG2 to lamb joint synovial cells LSM 192. Sera taken sequentially from sheep infected with PG2 revealed that P40 induced a strong and persistent immune response that gave strong signals on immunoblots containing recombinant P40 even 3 months after infection. The gene encoding P40 was present in a single copy in all of the 26 field strains of
M. agalactiae
analyzed and was not detected in closely related mycoplasma species. P40 was expressed as a protein with an apparent molecular mass of 37 kDa on sodium dodecyl sulfate-acrylamide gels by all
M. agalactiae
strains except for serotype C strains, which showed nonsense mutations in their
p40
genes.