bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Reproductive Hormones on Experimental Vaginal Candidiasis
Authors: Fidel Paul L. , Cutright Jessica , Steele Chad
Published: July 2002

Vulvovaginal candidiasis (VVC) is an opportunistic mucosal infection caused by
Candida albicans
that affects large numbers of otherwise healthy women of childbearing age. Acute episodes of VVC often occur during pregnancy and during the luteal phase of the menstrual cycle, when levels of progesterone and estrogen are elevated. Although estrogen-dependent experimental rodent models of
C. albicans
vaginal infection are used for many applications, the role of reproductive hormones and/or their limits in the acquisition of vaginal candidiasis remain unclear. This study examined the effects of estrogen and progesterone on several aspects of an experimental infection together with relative cell-mediated immune responses. Results showed that while decreasing estrogen concentrations eventually influenced infection-induced vaginal titers of
C. albicans
and rates of infection in inoculated animals, the experimental infection could not be achieved in mice treated with various concentrations of progesterone alone. Furthermore, progesterone had no effect on (i) the induction and persistence of the infection in the presence of estrogen, (ii) delayed-type hypersensitivity in primary-infected mice, or (iii) the partial protection from a secondary vaginal infection under pseudoestrus conditions. Other results with estrogen showed that a persistent infection could be established with a wide range of
C. albicans
inocula under supraphysiologic and near-physiologic (at estrus) concentrations of estrogen and that vaginal fungus titers or rates of infection were similar if pseudoestrus was initiated several days before or after inoculation. However, the pseudoestrus state had to be maintained for the infection to persist. Finally, estrogen was found to reduce the ability of vaginal epithelial cells to inhibit the growth of
C. albicans
. These results suggest that estrogen, but not progesterone, is an important factor in hormone-associated susceptibility to
C. albicans
vaginitis.
Functional Conservation of the Polysaccharide Biosynthetic Protein WbpM and Its Homologues in
Pseudomonas aeruginosa
and Other Medically Significant Bacteria
Authors: Burrows Lori L. , Urbanic Robert V. , Lam Joseph S.
Published: July 2002

WbpM is a highly conserved protein involved in synthesis of the O antigens of
Pseudomonas aeruginosa
. Homologues of this protein have been identified in a large number of bacteria, and they can be divided into two subfamilies: subfamily 1, including WbpM, contains large proteins (∼600 amino acids), while subfamily 2, typified by HP0840 (FlaA1) of
Helicobacter pylori
, contains smaller proteins (∼350 amino acids) homologous to the C termini of proteins in subfamily 1. Analysis of knockout mutants of
wbpM
in
P. aeruginosa
serotypes O3, O10, O15, and O17 showed that although all 20 serotypes of
P. aeruginosa
possess
wbpM
, it is not universally required for O-antigen biosynthesis. Homologous genes from
Bordetella pertussis
(
wlbL
),
Staphylococcus aureus
(
cap8D
), and
H. pylori
(
flaA1
) complemented a
P. aeruginosa
O5
wbpM
mutant to various degrees. These conserved proteins may represent interesting targets for the design of inhibitors of bacterial exopolysaccharide biosynthesis.
Subunit Vaccination of Mice against New World Cutaneous Leishmaniasis: Comparison of Three Proteins Expressed in Amastigotes and Six Adjuvants
Authors: Aebischer Toni , Wolfram Markus , Patzer Silke I. , Ilg Thomas , Wiese Martin , Overath Peter
Published: July 2002

A mixture of well-defined recombinant antigens together with an adjuvant that preferentially stimulates specific gamma interferon (IFN-γ)-secreting helper type 1 CD4+T cells (Th1 cells) presents a rational option for a vaccine against leishmaniasis. The potential of this approach was investigated in murine infections withLeishmania mexicana, which are characterized by the absence of a parasite-specific Th1 response and uncontrolled parasite proliferation. A mixture of three antigens (glycoprotein 63, cysteine proteinases, and a membrane-bound acid phosphatase), which are all expressed in amastigotes, the mammalian stage of the parasite, were used for the immunization of C57BL/6 mice in combination with six adjuvants (interleukin 12 [IL-12], Detox, 4′-monophosphoryl lipid A, QS-21,Mycobacterium bovisBCG, andCorynebacterium parvum). All six vaccine formulations containing the mixture of recombinant antigens were protective against challenge infections with promastigotes, the insect stage of the parasite, in that mice controlled and healed infections but developed transient and, in certain cases, accentuated disease. The most effective adjuvants were IL-12 followed by Detox. Further studies using these two adjuvants showed that a similar protective effect was observed with a mixture of the corresponding native proteins, and mice which had controlled the infection showed a preponderance of IFN-γ-secreting CD4+T cells in the lymph nodes draining the lesion. Using the recombinant proteins individually, it is shown that the relatively abundant cysteine proteinases and glycoprotein 63, but not the acid phosphatase, are able to elicit a protective response. The results are discussed in comparison to previous studies with subunit vaccines and with respect to cell biological aspects of antigen presentation inLeishmania-infected macrophages.
Linkage of Exogenous T-cell Epitopes to the 19-Kilodalton Region ofPlasmodium yoeliiMerozoite Surface Protein 1 (MSP119) Can Enhance Protective Immunity against Malaria and Modulate the Immunoglobulin Subclass Response to MSP119
Authors: Ahlborg Niklas , Ling Irene T. , Holder Anthony A. , Riley Eleanor M.
Published: July 2002

The degree of protection againstPlasmodium yoeliiasexual blood stages induced by immunization of mice with the 19-kDa region of merozoite surface protein 1 (MSP119) is H-2 dependent. As a strategy to improve the protection, mouse strains with disparate H-2 haplotypes were immunized with glutathioneS-transferase (GST)–MSP119proteins including either a universal T-cell epitope from tetanus toxin (P2) or an I-Ak-restricted T-cell epitope (P8) fromPlasmodium falciparumPf332. In H-2kmice which are poorly protected following immunization with GST-MSP119, GST-P2-MSP119significantly improved the protection. In mice partially (H-2k/b) or well protected by GST-MSP119(H-2dand H-2b), P2 did not further increase the protection. However, the protection of H-2k/bmice and to some extent H-2kmice was improved by immunization with GST-P8-MSP119. The magnitudes of immunoglobulin G1 (IgG1) and IgG2a responses in mice immunized with the GST-MSP119variants correlated with low peak parasitemia, indicating a protective capacity of these IgG subclasses. In H-2kmice immunized with GST-P2-MSP119, both IgG1 and IgG2a responses were significantly enhanced. The epitope P2 appeared to have a general ability to modulate the IgG subclass response since all four mouse strains displayed elevated IgG2a and/or IgG2b levels after immunization with GST-P2-MSP119. In contrast, GST-P8-MSP119induced a slight enhancement of IgG responses in H-2k/band H-2kmice without any major shift in IgG subclass patterns. The ability to improve the protective immunity elicited byP. yoeliiMSP119may have implications for improvement of human vaccines based onP. falciparumMSP119.
Detection of
Chlamydia pneumoniae
DNA and Antigen in the Circulating Mononuclear Cell Fractions of Humans and Koalas
Authors: Bodetti Tracey J. , Timms Peter
Published: July 2002

Chlamydia pneumoniae
is a common respiratory pathogen of humans which, in addition to causing disease at the respiratory site, has recently been linked to disease at other body sites. If
C. pneumoniae
does contribute to disease at nonrespiratory sites, then it must have a mechanism by which it reaches these sites. We analyzed the peripheral blood mononuclear cell (PBMC) fractions from 60 healthy human blood donors for the presence of
C. pneumoniae
DNA (by
ompA
PCR) and chlamydial antigens (by genus- and species-specific monoclonal antibody staining). Ten of the sixty (16.7%) blood donors were
C. pneumoniae
positive by PCR, and all 10 of these PCR-positive individuals' samples demonstrated specific staining with anti-
C. pneumoniae
monoclonal antibodies. The only other host naturally infected with
C. pneumoniae
is the koala, in which the bacterium also causes respiratory infections. We demonstrated the presence of
C. pneumoniae
DNA and antigens in the PBMC fractions of 30% of 20 koalas tested. Our finding of
C. pneumoniae
-infected PBMCs in koalas as well as humans suggests that the ability to infect PBMCs and to disseminate from the respiratory site is not restricted to the human biovar of
C. pneumoniae
but is a general characteristic of this chlamydial species.
Role of Eosinophils in the Pathogenesis of
Mycobacterium bovis
BCG Infection in Gamma Interferon Receptor-Deficient Mice
Authors: Kirman Joanna , Zakaria Zamsyari , McCoy Kathy , Delahunt Brett , Le Gros Graham
Published: July 2002

A profound eosinophil infiltration of granulomas is observed in the lungs of
Mycobacterium bovis
bacillus Calmette Guérin-infected gamma interferon receptor-deficient mice. Blockade of eosinophil proliferation and recruitment into the lung by treatment with anti-interleukin-5 monoclonal antibody marginally reduced mycobacterial growth within the lung but did not affect dissemination of the infection to other tissues.
Salmonella enterica
Serovar Typhi Uses Type IVB Pili To Enter Human Intestinal Epithelial Cells
Authors: Zhang Xiao-Lian , Tsui Inez S. M. , Yip Cecilia M. C. , Fung Ada W. Y. , Wong Danny K.-H. , Dai Xiaoyun , Yang Yanhua , Hackett Jim , Morris Christina
Published: July 2002

DNA sequencing upstream of the
Salmonella enterica
serovar Typhi
pilV
and
rci
genes previously identified in the ca. 118-kb major pathogenicity island (X.-L. Zhang, C. Morris, and J. Hackett, Gene 202:139–146, 1997) identified a further 10
pil
genes apparently forming a
pil
operon. The product of the
pilS
gene, prePilS protein (a putative type IVB structural prepilin) was purified, and an anti-prePilS antiserum was raised in mice. Mutants of serovar Typhi either lacking the whole
pil
operon or with an insertion mutation in the
pilS
gene were constructed, as was a strain in which the
pilN
to
pilV
genes were driven by the
tac
promoter. The
pil
+
strains synthesized type IVB pili, as judged by (i) visualization in the electron microscope of thin pili in culture supernatants of one such strain and (ii) the presence of PilS protein (smaller than the prePilS protein by removal of the leader peptide) on immunoblotting of material pelleted by high-speed centrifugation of either the culture supernatant or sonicates of
pil
+
strains. Control
pil
mutants did not express the PilS protein. A
pilS
mutant of serovar Typhi entered human intestinal INT407 cells in culture to levels only 5 to 25% of those of the wild-type strain, and serovar Typhi entry was strongly inhibited by soluble prePilS protein (50% inhibition of entry at 1.4 μM prePilS).
Innate Immune Responses in Children and Adults with Shigellosis
Authors: Raqib Rubhana , Mia S. M. Shahjahan , Qadri Firdausi , Alam Tanfis I. , Alam Nur H. , Chowdhury Ashish K. , Mathan Minnie M. , Andersson Jan
Published: July 2002

An array of pro- and anti-inflammatory mediators of the innate immune system was analyzed in stool, urine, and rectal mucosa samples from adults and children with shigellosis to better understand their role in recovery from and in the immunopathogenesis of the disease. Increased concentrations of lactoferrin (Lf), myeloperoxidase (MPO), prostaglandin E2, and leukotriene B4(LTB4) in stool during acute shigellosis in both children and adults indicated that activated cells of the innate defense system at the mucosal site were secreting the mediators. Increased concentration of MPO and 8-iso-prostaglandin F2αand lower levels of superoxide dismutase (SOD) activity in stool during acuteShigellainfection suggested increased formation of reactive oxygen species, free radical-catalyzed peroxidation of membrane lipids, and decreased scavenging of the reactive oxygen radicals. In children, lower expression of SOD in tissue with severe inflammation and lower levels of SOD activity in stool for longer periods compared to adults may further worsen the tissue damage and predispose the children to a lowered defense. Both adult and pediatric patients had significantly higher expression of inducible nitric oxide synthase (iNOS) in the rectum with severe inflammation, compared to that seen with mild inflammation, accompanied by persistently up-regulated iNOS mRNA, reflecting increased production of nitric oxide at the local site. However, in contrast to adults, reduced urinary nitrate levels in pediatric patients during acute shigellosis suggested lower production of nitric oxide in the renal compartment. Persistent production of Lf in pediatric patients may contribute to chronic inflammation in the rectum. In addition, increased production of proinflammatory mediators in the rectum of patients with severe histology suggested contribution of these molecules to the immunopathogenesis of severe colitis caused by shigellae.
The CXC Chemokines Gamma Interferon (IFN-γ)-Inducible Protein 10 and Monokine Induced by IFN-γ Are Released during Severe Melioidosis
Authors: Lauw Fanny N. , Simpson Andrew J. H. , Prins Jan M. , van Deventer Sander J. H. , Chaowagul Wipada , White Nicholas J. , van der Poll Tom
Published: July 2002

Gamma interferon (IFN-γ)-inducible protein 10 (IP-10) and monokine induced by IFN-γ (Mig) are related CXC chemokines which bind to the CXCR3 receptor and specifically target activated T lymphocytes and natural killer (NK) cells. The production of IP-10 and Mig by various cell types in vitro is strongly dependent on IFN-γ. To determine whether IP-10 and Mig are released during bacterial infection in humans, we measured plasma levels of IP-10 and Mig in patients with melioidosis, a severe gram-negative infection caused byBurkholderia pseudomallei. IP-10 and Mig were markedly elevated in patients with melioidosis on admission, particularly in blood culture-positive patients, and remained elevated during the 72-h study period. Levels of IP-10 and Mig showed a positive correlation with IFN-γ concentrations and also correlated with clinical outcome. In whole blood stimulated with heat-killedB. pseudomallei, neutralization of IFN-γ and tumor necrosis factor alpha (TNF-α) partly attenuated IP-10 and Mig release, while anti-interleukin-12 (IL-12) and anti-IL-18 had a synergistic effect. Stimulation with other bacteria or endotoxin also induced strong secretion of IP-10 and Mig. These data suggest that IP-10 and Mig are part of the innate immune response to bacterial infection. IP-10 and Mig may contribute to host defense in Th1-mediated host defense during infections by attracting CXCR3+Th1 cells to the site of inflammation.
Identification of Glycosaminoglycan Binding Domains in
Plasmodium falciparum
Erythrocyte Membrane Protein 1 of a Chondroitin Sulfate A-Adherent Parasite
Authors: Reeder John C. , Hodder Anthony N. , Beeson James G. , Brown Graham V.
Published: July 2002

Accumulation of
Plasmodium falciparum
-infected erythrocytes in the placenta is a key feature of maternal malaria. This process is mediated in part by the parasite ligand
P. falciparum
erythrocyte membrane protein 1 (PfEMP1) at the surface of the infected erythrocyte interacting with the host receptor chondroitin sulfate A (CSA) on the placental lining. We have localized CSA binding activity to two adjacent domains in PfEMP1 of an adherent parasite line and shown the presence of at least three active glycosaminoglycan binding sites. A putative CSA binding sequence was identified in one domain, but nonlinear binding motifs are also likely to be present, since binding activity in the region was shown to be dependent on conformation. Characterization of this binding region provides an opportunity to investigate further its potential as a target for antiadhesion therapy.
The C Terminus of Component C2II of
Clostridium botulinum
C2 Toxin Is Essential for Receptor Binding
Authors: Blöcker Dagmar , Barth Holger , Maier Elke , Benz Roland , Barbieri Joseph T. , Aktories Klaus
Published: July 2002

The binary
Clostridium botulinum
C2 toxin consists of two separate proteins, the binding component C2II (80.5 kDa) and the actin-ADP-ribosylating enzyme component C2I (49.4 kDa). For its cytotoxic action, C2II binds to a cell membrane receptor and induces cell entry of C2I via receptor-mediated endocytosis. Here we studied the structure-function relationship of C2II by constructing truncated C2II proteins and producing polyclonal antisera against selective regions of C2II. An antibody raised against the C terminus (amino acids 592 to 721) of C2II inhibited binding of C2II to cells. The antibody prevented pore formation by C2II oligomers in artificial membranes but did not influence the properties of existing channels. To further define the region responsible for receptor binding, we constructed proteins with deletions in C2II; specifically, they lacked amino acid residues 592 to 721 and the 7 C-terminal amino acid residues. The truncated proteins still formed sodium dodecyl sulfate-stable oligomers but were unable to bind to cells. Our data indicate that the C terminus of C2II mediates binding of the protein to cells and that the 7 C-terminal amino acids are structurally important for receptor binding.
Switching of Flagellar Motility in
Helicobacter pylori
by Reversible Length Variation of a Short Homopolymeric Sequence Repeat in
fliP
, a Gene Encoding a Basal Body Protein
Authors: Josenhans Christine , Eaton Kathryn A. , Thevenot Tracy , Suerbaum Sebastian
Published: July 2002

The genome of
Helicobacter pylori
contains numerous simple nucleotide repeats that have been proposed to have regulatory functions and to compensate for the conspicuous dearth of master regulatory pathways in this highly host-adapted bacterium.
H. pylori
strain 26695, whose genomic sequence was determined by The Institute for Genomic Research (TIGR), contains a repeat of nine cytidines in the
fliP
flagellar basal body gene that splits the open reading frame in two parts. In this work, we demonstrate that the 26695
C9
strain with a split
fliP
gene as sequenced by TIGR was nonflagellated and nonmotile. In contrast, earlier isolates of strain 26695 selected by positive motility testing as well as pig-passaged derivatives of 26695 were all flagellated and highly motile. All of these motile strains had a C
8
repeat and consequently a contiguous
fliP
reading frame. By screening approximately 50,000 colonies of 26695
C9
for motility in soft agar, a motile revertant with a C
8
repeat could be isolated, proving that the described switch is reversible. The
fliP
genes of 20 motile clinical
H. pylori
isolates from different geographic regions possessed intact
fliP
genes with repeats of eight cytidines or the sequence CCCCACCC in its place. Isogenic
fliP
mutants of a motile, C
8
repeat isolate of strain 26695 were constructed by allelic exchange mutagenesis and found to be defective in flagellum biogenesis. Mutants produced only small amounts of flagellins, while the transcription of flagellin genes appeared unchanged. These results strongly suggest a unique mechanism regulating motility in
H. pylori
which relies on slipped-strand mispairing-mediated mutagenesis of
fliP
.
Apically Exposed, Tight Junction-Associated β1-Integrins Allow Binding and YopE-Mediated Perturbation of Epithelial Barriers by Wild-Type
Yersinia
Bacteria
Authors: Tafazoli Farideh , Holmström Anna , Forsberg Åke , Magnusson Karl-Eric
Published: July 2002

Using polarized epithelial cells, primarily MDCK-1, we assessed the mode of binding and effects on epithelial cell structure and permeability of
Yersinia pseudotuberculosis yadA
-deficient mutants. Initially, all bacteria except the invasin-deficient (
inv
) mutant adhered apically to the tight junction areas. These contact points of adjacent cells displayed β1-integrins together with tight junction-associated ZO-1 and occludin proteins. Indeed, β1-integrin expression was maximal in the tight junction area and then gradually decreased along the basolateral membranes. Wild-type bacteria also opened gradually the tight junction to paracellular permeation of different-sized markers, viz., 20-, 40-, and 70-kDa dextrans and 45-kDa ovalbumin, as well as to their own translocation between adjacent cells in intimate contact with β1-integrins. The effects on the epithelial cells and their barrier properties could primarily be attributed to expression of the
Yersinia
outer membrane protein YopE, as the
yopE
mutant bound but caused no cytotoxicity. Moreover, the apical structure of filamentous actin (F-actin) was disturbed and tight junction-associated proteins (ZO-1 and occludin) were dispersed along the basolateral membranes. It is concluded that the
Yersinia
bacteria attach to β1-integrins at tight junctions. Via this localized injection of YopE, they perturb the F-actin structure and distribution of proteins forming and regulating tight junctions. Thereby they promote paracellular translocation of bacteria and soluble compounds.
Detection of Bacterial Virulence Genes by Subtractive Hybridization: Identification of Capsular Polysaccharide of
Burkholderia pseudomallei
as a Major Virulence Determinant
Authors: Reckseidler Shauna L. , DeShazer David , Sokol Pamela A. , Woods Donald E.
Published: July 2002

Burkholderia pseudomallei
, the etiologic agent of melioidosis, is responsible for a broad spectrum of illnesses in humans and animals particularly in Southeast Asia and northern Australia, where it is endemic.
Burkholderia thailandensis
is a nonpathogenic environmental organism closely related to
B. pseudomallei
. Subtractive hybridization was carried out between these two species to identify genes encoding virulence determinants in
B. pseudomallei
. Screening of the subtraction library revealed A-T-rich DNA sequences unique to
B. pseudomallei
, suggesting they may have been acquired by horizontal transfer. One of the subtraction clones, pDD1015, encoded a protein with homology to a glycosyltransferase from
Pseudomonas aeruginosa
. This gene was insertionally inactivated in wild-type
B. pseudomallei
to create SR1015. It was determined by enzyme-linked immunosorbent assay and immunoelectron microscopy that the inactivated gene was involved in the production of a major surface polysaccharide. The 50% lethal dose (LD
50
) for wild-type
B. pseudomallei
is <10 CFU; the LD
50
for SR1015 was determined to be 3.5 × 10
5
CFU, similar to that of
B. thailandensis
(6.8 × 10
5
CFU). DNA sequencing of the region flanking the glycosyltransferase gene revealed open reading frames similar to capsular polysaccharide genes in
Haemophilus influenzae
,
Escherichia coli
, and
Neisseria meningitidis
. In addition, DNA from
Burkholderia mallei
and
Burkholderia stabilis
hybridized to a glycosyltransferase fragment probe, and a capsular structure was identified on the surface of
B. stabilis
via immunoelectron microscopy. Thus, the combination of PCR-based subtractive hybridization, insertional inactivation, and animal virulence studies has facilitated the identification of an important virulence determinant in
B. pseudomallei
.
Characterization of
Vibrio cholerae
O1 El Tor
galU
and
galE
Mutants: Influence on Lipopolysaccharide Structure, Colonization, and Biofilm Formation
Authors: Nesper Jutta , Lauriano Crystal M. , Klose Karl E. , Kapfhammer Dagmar , Kraiß Anita , Reidl Joachim
Published: July 2002

Recently we described the isolation of spontaneous bacteriophage K139-resistant
Vibrio cholerae
O1 El Tor mutants. In this study, we identified phage-resistant isolates with intact O antigen but altered core oligosaccharide which were also affected in galactose catabolism; this strains have mutations in the
galU
gene. We inactivated another
gal
gene,
galE
, and the mutant was also found to be defective in the catabolism of exogenous galactose but synthesized an apparently normal lipopolysaccharide (LPS). Both
gal
mutants as well as a rough LPS (R-LPS) mutant were investigated for the ability to colonize the mouse small intestine. The
galU
and R-LPS mutants, but not the
galE
mutant, were defective in colonization, a phenotype also associated with O-antigen-negative mutants. By investigating several parameters in vitro, we could show that
galU
and R-LPS mutants were more sensitive to short-chain organic acids, cationic antimicrobial peptides, the complement system, and bile salts as well as other hydrophobic agents, indicating that their outer membrane no longer provides an effective barrier function. O-antigen-negative strains were found to be sensitive to complement and cationic peptides, but they displayed significant resistance to bile salts and short-chain organic acids. Furthermore, we found that
galU
and
galE
are essential for the formation of a biofilm in a spontaneous phage-resistant rugose variant, suggesting that the synthesis of UDP-galactose via UDP-glucose is necessary for biosynthesis of the exopolysaccharide. In addition, we provide evidence that the production of exopolysaccharide limits the access of phage K139 to its receptor, the O antigen. In conclusion, our results indicate involvement of
galU
in
V. cholerae
virulence, correlated with the observed change in LPS structure, and a role for
galU
and
galE
in environmental survival of
V. cholerae
.
Both Th1 and Th2 Cytokines Affect the Ability of Monoclonal Antibodies To Protect Mice againstCryptococcus neoformans
Authors: Beenhouwer David O. , Shapiro Scott , Feldmesser Marta , Casadevall Arturo , Scharff Matthew D.
Published: July 2002

Variable-region-identical mouse immunoglobulin G1 (IgG1), IgG2b, and IgG2a monoclonal antibodies to the capsular polysaccharide ofCryptococcus neoformansprolong the lives of mice infected with this fungus, while IgG3 is either not protective or enhances infection. CD4+T cells are required for IgG1-mediated protection, and CD8+T cells are required for IgG3-mediated enhancement. Gamma interferon is required for both effects. These findings revealed that T cells and cytokines play a role in the modulation of cryptococcal infection by antibodies and suggested that it was important to more fully define the cytokine requirements of each of the antibody isotypes. We therefore investigated the efficacy of passively administered variable-region-identical IgG1, IgG2a, IgG2b, and IgG3 monoclonal antibodies against intravenous infection withC. neoformansin mice genetically deficient in interleukin-12 (IL-12), IL-6, IL-4, or IL-10, as well as in the parental C57BL/6J strain. The relative inherent susceptibilities of these mouse strains toC. neoformanswere as follows: IL-12−/−> IL-6−/−> C57BL/6J ≈ IL-4−/−≫ IL-10−/−. This is consistent with the notion that a Th1 response is necessary for natural immunity against cryptococcal infection. However, none of the IgG isotypes prolonged survival in IL-12−/−, IL-6−/−, or IL-4−/−mice, and all isotypes significantly enhanced infection in IL-10−/−mice. These results indicate that passive antibody-mediated protection againstC. neoformansrequires both Th1- and Th2-associated cytokines and reveal the complexity of the mechanisms through which antibodies modulate infection with this organism.
Determination of the Infectious Dose of
Helicobacter pylori
during Primary and Secondary Infection in Rhesus Monkeys (
Macaca mulatta
)
Authors: Solnick Jay V. , Hansen Lori M. , Canfield Don R. , Parsonnet Julie
Published: July 2002

We sought to determine the infectious dose of
Helicobacter pylori
during primary and secondary infection in the rhesus monkey and to determine whether preinoculation acid suppression is necessary to produce colonization. Mixed inoculation with three human-derived strains showed that
H. pylori
J166 is particularly adapted to colonization of rhesus monkeys, since it outcompeted two other strains. The minimum infectious dose of
H. pylori
J166 was 10
4
bacteria in specific-pathogen (
H. pylori
)-free monkeys. Rechallenge of these monkeys after antibiotic therapy was characterized by a 10- to 100-fold decrease in bacterial load compared to primary infection, but with little change in the infectious dose. Acid suppression prior to inoculation was not necessary for colonization to occur. These results provide a basis for future animal experiments using more ecologically relevant conditions of inoculation and suggest that reduction in bacterial load rather than complete protection may be a more realistic goal for
H. pylori
vaccination.
Temporal Sequence and Kinetics of Proinflammatory and Anti-Inflammatory Cytokine Secretion Induced by Toxic Shock Syndrome Toxin 1 in Human Peripheral Blood Mononuclear Cells
Authors: Kum Winnie W. S. , Cameron Scott B. , Hung Ryan W. Y. , Kalyan Shirin , Chow Anthony W.
Published: July 2002

The staphylococcal superantigen toxic shock syndrome toxin 1 (TSST-1) induces massive cytokine production, which is believed to be the key factor in the pathogenesis of TSS. The temporal sequence and kinetics of both proinflammatory and anti-inflammatory cytokines induced by TSST-1 in human peripheral blood mononuclear cells were investigated. A panel of loss-of-function single-amino-acid-substitution mutants of TSST-1, previously demonstrated to be defective in either major histocompatibility complex (MHC) class II binding (G31R) or T-cell receptor (TCR) interaction (H135A, S14N), was studied in parallel to further elucidate the mechanisms of cytokine secretion. Wild-type recombinant (WT r) TSST-1 induced a biphasic pattern of cytokine secretion: an early phase with rapid release of proinflammatory cytokines (especially gamma interferon, interleukin-2 [IL-2], and tumor necrosis factor alpha [TNF-α]) within 3 to 4 h poststimulation, and a later phase with more gradual production of both proinflammatory (IL-1β, IL-12, and TNF-β) and anti-inflammatory (IL-6, IL-10) cytokines within 16 to 72 h poststimulation. G31R, which is defective in MHC class II binding, induced a cytokine profile similar to that of WT rTSST-1, except that secretion of the early-phase proinflammatory cytokines was delayed and production of IL-1β and IL-12 was markedly reduced. In contrast, mutant toxins defective in TCR interaction either demonstrated complete absence of any cytokine secretion during the entire observation period (H135A) or resulted in complete abolishment of IL-2 and other early-phase proinflammatory cytokines, while secretion of IL-10 appeared unaffected (S14N). Neither WT rTSST-1 nor the mutant toxins induced IL-4 or transforming growth factor β. Our data indicate that effective TCR interaction is critical for the induction of the early-phase proinflammatory cytokine response, thus underscoring the importance of T-cell signaling in TSS.
Characterization of Murine Lung Dendritic Cells Infected withMycobacterium tuberculosis
Authors: Gonzalez-Juarrero Mercedes , Orme Ian M.
Published: July 2002

Lung dendritic cells were identified by immunohistochemistry in lung tissue sections from C57BL/6 mice. Following isolation from the lungs using CD11c magnetic beads, the flow cytometric analysis of I-Ab+and CD11c+cells indicated a mixed population of dendritic cells at different stages of maturation, with most expressing an immature phenotype. When cultured for 7 days with recombinant murine granulocyte-macrophage colony-stimulating factor, 99% of cells were CD11c+and had a morphology typical of immature dendritic cells. These cells were negative for CD34, CD14, and CD8α antigens but expressed low levels of the myeloid marker F4/80 and moderate levels of MAC3. All expressed high levels of CD11a (LFA-1), CD11b (Mac1), and CD54 antigens, with low levels of class II major histocompatibility complex. Most cells expressed CD80 but only a small percentage of cells were positive for CD40 and CD86. Both overnight and 7-day cultures of lung dendritic cells were able to phagocytoseMycobacterium tuberculosis, and this was associated with the production of interleukin-12 and stimulation of both naı̈ve and immune T cells to produce gamma interferon.
Influence of Costimulatory Molecules on Immune Response toLeishmania majorby Human Cells In Vitro
Authors: Brodskyn Claudia I. , DeKrey Gregory K. , Titus Richard G.
Published: July 2002

The importance of CD40, CD80, and CD86 costimulatory molecules in anti-Leishmaniaimmune responses has been established in murine models. A role for these costimulatory molecules in human anti-Leishmaniaimmune responses was investigated in this study. Autologous macrophages and peripheral blood leukocytes (PBL) were prepared from peripheral blood mononuclear cells ofLeishmania-naive donors and cultured with or withoutLeishmania majorin various combinations. After 7 days of culture, high levels of CD40 and CD86 were expressed on macrophages in the presence or absence ofL. major. When macrophages were cultured for an additional 7 days with PBL, expression of all three costimulatory molecules was detected. WhenL. majorwas present in these cultures, the expression of CD80, and to a lesser extent CD40, on macrophages was enhanced. Blockade of CD80, CD86, or both molecules (in the order of greatest effect) in cultures containing macrophages, PBL, andL. majorsignificantly inhibited the production of gamma interferon, interleukin-5 (IL-5), and IL-12. Blockade of CD40-CD154 interactions also significantly inhibited production of these cytokines in response toL. major. Production of IL-10 was unaltered by the blockade of these costimulatory molecules. Thus, these data suggest that CD40, CD80, and CD86 expression and regulation may significantly impact anti-Leishmaniaimmune responses in humans.
afa-8
Gene Cluster Is Carried by a Pathogenicity Island Inserted into the tRNA
Phe
of Human and Bovine Pathogenic
Escherichia coli
Isolates
Authors: Lalioui Lila , Le Bouguénec Chantal
Published: July 2002

We recently described a new afimbrial adhesin, AfaE-VIII, produced by animal strains associated with diarrhea and septicemia and by human isolates associated with extraintestinal infections. Here, we report that the
afa-8
operon, encoding AfaE-VIII adhesin, from the human blood isolate
Escherichia coli
AL862 is carried by a 61-kb genomic region with characteristics typical of a pathogenicity island (PAI), including a size larger than 10 kb, the presence of an integrase-encoding gene, the insertion into a tRNA locus (
pheR
), and the presence of a small direct repeat at each extremity. Moreover, the G+C content of the
afa-8
operon (46.4%) is lower than that of the
E. coli
K-12/MG1655 chromosome (50.8%). Within this PAI, designated PAI I
AL862
, we identified open reading frames able to code for products similar to proteins involved in sugar utilization. Four probes spanning these sequences hybridized with 74.3% of pathogenic
afa-8
-positive
E. coli
strains isolated from humans and animals, 25% of human pathogenic
afa-8
-negative
E. coli
strains, and only 8% of fecal strains (
P
= 0.05), indicating that these sequences are strongly associated with the
afa-8
operon and that this genetic association may define a PAI widely distributed among human and animal
afa-8
-positive strains. One of the distinctive features of this study is that
E. coli
AL862 also carries another
afa-8
-containing PAI (PAI II
AL862
), which appeared to be similar in size and genetic organization to PAI I
AL862
and was inserted into the
pheV
gene. We investigated the insertion sites of
afa-8
-containing PAI in human and bovine pathogenic
E. coli
strains and found that this PAI preferentially inserted into the
pheV
gene.
Listeria monocytogenes-Infected Phagocytes Can Initiate Central Nervous System Infection in Mice
Authors: Drevets Douglas A. , Jelinek Todd A. , Freitag Nancy E.
Published: July 2002

Listeria monocytogenes-infected phagocytes are present in the bloodstream of experimentally infected mice, but whether they play a role in central nervous system (CNS) invasion is unclear. To test whether bacteria within infected leukocytes could establish CNS infection, experimentally infected mice were treated with gentamicin delivered by surgically implanted osmotic pumps. Bacterial inhibitory titers in serum and plasma ranged from 1:16 to 1:256, and essentially all viable bacteria in the bloodstream of treated mice were leukocyte associated. Nevertheless, CNS infection developed in gentamicin-treated animals infected intraperitoneally or by gastric lavage, suggesting that intracellular bacteria could be responsible for neuroinvasion. This was supported by data showing that 43.5% of bacteria found with blood leukocytes were intracellular and some colocalized with F-actin, indicating productive intracellular parasitism. Experiments using anL. monocytogenesstrain containing a chromosomalactA-gfpuv-plcBtranscriptional fusion showed that blood leukocytes were associated with intracellular and extracellularly bound green fluorescent protein-expressing (GFP+) bacteria. Treatment with gentamicin decreased the numbers of extracellularly bound GFP+bacteria significantly but did not affect the numbers of intracellular GFP+bacteria, suggesting that the latter were the result of intercellular spread of GFP+bacteria to leukocytes. These data demonstrate that infected leukocytes and the intracellularL. monocytogenesharbored within them play key roles in neuroinvasion. Moreover, they suggest that phagocytes recruited to infected organs such as the liver or spleen are themselves parasitized by intercellular spread ofL. monocytogenesand then reenter the bloodstream and contribute to the systemic dissemination of bacteria.
Salivary Histatin 5 Is an Inhibitor of Both Host and Bacterial Enzymes Implicated in Periodontal Disease
Authors: Gusman Heloisa , Travis James , Helmerhorst Eva J. , Potempa Jan , Troxler Robert F. , Oppenheim Frank G.
Published: July 2002

One of the salient features of periodontitis and gingivitis is the increase in the levels of bacterial and host-derived proteolytic enzymes in oral inflammatory exudates. This study evaluated the potential of histatin 5, a 24-residue histidine-rich salivary antimicrobial protein, to inhibit these enzymes. Using biotinylated gelatin as a substrate, histatin 5 was found to inhibit the activity of the host matrix metalloproteinases MMP-2 and MMP-9 with 50% inhibitory concentrations (IC
50
s) of 0.57 and 0.25 μM, respectively. To localize the domain responsible for this inhibition, three peptides containing different regions of histatin 5 were synthesized and tested as inhibitors of MMP-9. Peptides comprising residues 1 to 14 and residues 4 to 15 of histatin 5 showed much lower inhibitory activities (IC
50
, 21.4 and 20.5 μM, respectively), while a peptide comprising residues 9 to 22 showed identical activity to histatin 5 against MMP-9. These results point to a functional domain localized in the C-terminal part of histatin 5. To evaluate the effect of histatin 5 on bacterial proteases, a detailed characterization of histatin 5 inhibition of gingipains from
Porphyromonas gingivalis
was carried out using purified Arg- and Lys-specific enzymes. Kinetic analysis of the inhibition of the Arg-gingipain revealed that histatin 5 is a competitive inhibitor, affecting only the

K
m

with a

K
i

of 15 μM. In contrast, inhibition of Lys-gingipain affected both the

K
m

and
V
max
, suggesting that both competitive and noncompetitive competitive processes underlie this inhibition. The inhibitory activity of histatin 5 against host and bacterial proteases at physiological concentrations points to a new potential biological function of histatin in the oral cavity.
Immunomodulatory Role of Interleukin-10 in Visceral Leishmaniasis: Defective Activation of Protein Kinase C-Mediated Signal Transduction Events
Authors: Bhattacharyya Sandip , Ghosh Sanjukta , Jhonson Patti L. , Bhattacharya Syamal K. , Majumdar Subrata
Published: July 2002

Leishmania donovani, an intracellular protozoan parasite, challenges host defense mechanisms by impairing the signal transduction of macrophages. In this study we investigated whether interleukin-10 (IL-10)-mediated alteration of signaling events in a murine model of visceral leishmaniasis is associated with macrophage deactivation. Primary in vitro cultures of macrophages infected with leishmanial parasites markedly elevated the endogenous release of IL-10. Treatment with eitherL. donovanior recombinant IL-10 (rIL-10) inhibited both the activity and expression of the Ca2+-dependent protein kinase C (PKC) isoform. However, preincubation with neutralizing anti-IL-10 monoclonal antibody (MAb) restored the PKC activity in the parasitized macrophage. Furthermore, we observed that coincubation of macrophages with rIL-10 andL. donovaniincreased the intracellular parasite burden, which was abrogated by anti-IL-10 MAb. Consistent with these observations, generation of superoxide (O2−) and nitric oxide and the release of murine tumor necrosis factor-α were attenuated in response toL. donovanior rIL-10 treatment. On the other hand, preincubation of the infected macrophages with neutralizing anti-IL-10 MAb significantly blocked the inhibition of nitric oxide and murine tumor necrosis factor-α release by the infected macrophages. These findings imply that infection withL. donovaniinduces endogenous secretion of murine IL-10, which in turn facilitates the intracellular survival of the protozoan and orchestrates several immunomodulatory roles via selective impairment of PKC-mediated signal transduction.
Epitope Mapping oftrans-Sialidase fromTrypanosoma cruziReveals the Presence of Several Cross-Reactive Determinants
Authors: Pitcovsky Tamara A. , Mucci Juan , Alvarez Paula , Leguizamón M. Susana , Burrone Oscar , Alzari Pedro M. , Campetella Oscar
Published: July 2002

Trypanosoma cruzi, the agent of Chagas' disease, expressestrans-sialidase, a unique enzyme activity that enables the parasite to invade host cells by transferring sialyl residues from host glyconjugates to the parasite's surface acceptor molecules. The enzyme is also shed into the surrounding environment, causing apoptosis in cells from the immune system. During infections, an antibody response against the catalytic region of thetrans-sialidase that is coincident with the control of the parasitemia and survival of the host is observed. This low-titer humoral response is characterized by its persistence for many years in benznidazole-treated patients. Here we analyzed the antigenic structure of the molecule by phage-displayed peptide combinatorial libraries and SPOT synthesis. Several epitopes were defined and located on the three-dimensional model of the enzyme. Unexpectedly, cross-reaction was found among several epitopes distributed in different locations displaying nonconsensus sequences. This finding was confirmed by the reactivity of three monoclonal antibodies able to recognize non-sequence-related peptides that together constitute the surface surrounding the catalytic site of the enzyme. The presence of cross-reacting epitopes within a single molecule suggests a mechanism developed to avoid a strong humoral response by displaying an undefined target to the immune system.
Membrane-Associated Proteins of a Lipopolysaccharide-Deficient Mutant ofNeisseria meningitidisActivate the Inflammatory Response through Toll-Like Receptor 2
Authors: Ingalls Robin R. , Lien Egil , Golenbock Douglas T.
Published: July 2002

The recent isolation of a lipopolysaccharide (LPS)-deficient mutant ofNeisseria meningitidishas allowed us to explore the roles of other gram-negative cell wall components in the host response to infection. The experiments in this study were designed to examine the ability of this mutant strain to activate cells. Although it was clearly less potent than the parental strain, we found the LPS-deficient mutant to be a capable inducer of the inflammatory response in monocytic cells, inducing a response similar to that seen withStaphylococcus aureus. Cellular activation by the LPS mutant was related to expression of CD14, a high-affinity receptor for LPS and other microbial products, as well as Toll-like receptor 2, a member of the Toll family of receptors recently implicated in host responses to gram-positive bacteria. In contrast to the parental strain, the synthetic LPS antagonist E5564 did not inhibit the LPS-deficient mutant. We conclude that even in the absence of LPS, the gram-negative cell wall remains a potent inflammatory stimulant, utilizing signaling pathways independent of those involved in LPS signaling.
Gamma Interferon-Producing CD4+T Lymphocytes in the Lung Correlate with Resistance to Infection withMycobacterium tuberculosis
Authors: Chackerian Alissa A. , Perera Thushara V. , Behar Samuel M.
Published: July 2002

The human immune system efficiently limits the replication ofMycobacterium tuberculosisin most infected individuals. Only 5 to 10% of infected people develop clinical tuberculosis, a sign of the inability of the immune system to control the infection. We have studied the C3H/HeJ (C3H) and C57BL/6 (B6) inbred mouse strains, which differ in their susceptibility to tuberculosis, in order to ascertain the immunological determinants of a successful immune response againstM. tuberculosisand to establish a system to identify genes that influence susceptibility to tuberculosis. We found that the resistant B6 mice were able to control infection in both the lung and spleen, while susceptible C3H mice were incapable of limiting bacteria growth, especially in the lung, and succumbed to infection within 4 weeks. We determined that the susceptibility of C3H mice was independent of the Toll-like receptor 4 (tlr4) genetic locus and allelic major histocompatibility complex differences. Although the splenic immune responses were similar in the two mouse strains, the local immune responses in the lungs of the infected mice differed greatly. The pulmonary immune response in resistant B6 mice was characterized by an early influx of both CD4+and CD8+lymphocytes that produced gamma interferon (IFN-γ). In contrast, the immune response of C3H mice in the lung was characterized by a delayed and decreased influx of lymphocytes, which produced little IFN-γ. These results suggest an important role for the early appearance of IFN-γ-producing lymphocytes in the lung in resistance to infection withM. tuberculosis.
Intracellular
Actinobacillus actinomycetemcomitans
and
Porphyromonas gingivalis
in Buccal Epithelial Cells Collected from Human Subjects
Authors: Rudney Joel D. , Chen Ruoqiong , Sedgewick Gerald J.
Published: July 2002

The mouth may provide an accessible model for studying bacterial interactions with human cells in vivo. Using fluorescent in situ hybridization and laser scanning confocal microscopy, we found that human buccal epithelial cells from 23 of 24 subjects were infected with intracellular bacteria, including the periodontal pathogens
Actinobacillus actinomycetemcomitans
and
Porphyromonas gingivalis
, as well as other species which have yet to be identified. Buccal cell invasion may allow fastidious anaerobes to establish themselves in aerobic sites that otherwise present an unfavorable environment. Exfoliated buccal epithelial cells might provide a protected route for bacterial transmission between different oral sites within and between hosts.