bluuebunny/crossref_metadata_2025 huggingface dataset:
Skin Facilitates
Candida albicans
Mating
Authors: Lachke Salil A. , Lockhart Shawn R. , Daniels Karla J. , Soll David R.
Published: August 2003

Mating between natural
a/a
and α/α strains of
Candida albicans
requires that cells first switch from the white to opaque phase phenotype. However, because cells expressing the opaque phase phenotype are induced to switch back to the white phase phenotype at physiological temperature (37°C) and because opaque phase cells are highly efficient at colonizing skin, we tested whether skin, which is several degrees lower than physiological temperature, is conducive to mating. Using a model in which a mixture of
a/a
and α/α cells are incubated for 24 h under a cotton patch on the hairless skin of newborn mice and using scanning electron microscopy to visualize cells on skin, it was demonstrated that skin facilitates mating. In some regions of the skin, 40% of all cells had fused. All of the stages of mating observed in vitro were observed in vivo. However, some unique morphological characteristics of mating on skin were observed and are attributable to parent cell immobilization on the skin. In control experiments on glass, plastic, and silicone elastomer surfaces at 32°C, cells either failed to fuse or did so at an extremely low frequency, suggesting that unique features of the skin surface other than reduced temperature also facilitate fusion.
The Hag Protein ofMoraxella catarrhalisStrain O35E Is Associated with Adherence to Human Lung and Middle Ear Cells
Authors: Holm Melissa M. , Vanlerberg Serena L. , Sledjeski Darren D. , Lafontaine Eric R.
Published: August 2003

Previous studies have demonstrated that theMoraxella catarrhalissurface antigen UspA1 is an adhesin for Chang human conjunctival cells. The present report demonstrates that lack of UspA1 expression does not affect the adherence of strain O35E to A549 human lung cells or primary cultures of human middle ear epithelial (HMEE) cells. These results imply that another molecule mediates the adherence ofM. catarrhalisto these two cell lines. To identify this adhesin, strain O35E was mutagenized with a transposon and 1,000 mutants were screened in a microcolony formation assay using A549 cells. Nine independent isolates exhibited an 8- to 19-fold reduction in adherence and contained a transposon in the same locus. Nucleotide sequence data and PCR analysis indicated that the transposons were inserted in different locations in the gene encoding the surface protein Hag. Quantitative assays using one representative transposon mutant, O35E.TN2, showed considerably decreased binding to A549 as well as HMEE cells. However, this mutant adhered at wild-type levels to Chang conjunctival cells. These findings suggest that theM. catarrhalisHag protein is an adhesin for cell lines derived from human lung and middle ear tissues.
Multiple Elements Controlling Adherence of Enterohemorrhagic
Escherichia coli
O157:H7 to HeLa Cells
Authors: Torres Alfredo G. , Kaper James B.
Published: August 2003

Adherence of enterohemorrhagic
Escherichia coli
(EHEC) to the intestinal epithelium is essential for initiation of infection. Intimin is the only factor demonstrated to play a role in intestinal colonization by EHEC O157:H7. Other attempts to identify additional adhesion factors in vitro have been unsuccessful, suggesting that expression of these factors is under tight regulation. We sought to identify genes involved in the control of adherence of EHEC O157:H7 to cultured epithelial cells. A total of 5,000 independent transposon insertion mutants were screened for their ability to adhere to HeLa cells, and 7 mutants were isolated with a markedly enhanced adherence. The mutants adhered at levels 113 to 170% that of the wild-type strain, and analysis of the protein profiles of these mutants revealed several proteins differentially expressed under in vitro culture conditions. We determined the sequence of the differentially expressed proteins and further investigated the function of OmpA, whose expression was increased in a mutant with an insertionally inactivated
tcdA
gene. An isogenic
ompA
mutant showed reduced adherence compared to the parent strain. Disruption of the
ompA
gene in the
tdcA
mutant strain abolished the hyperadherent phenotype, and anti-OmpA serum inhibited adhesion of wild-type and
tdcA
mutant strains to HeLa cells. Enhanced adhesion mediated by OmpA was also observed with Caco-2 cells, and anti-OmpA serum blocked adherence to HeLa cells of other EHEC O157:H7 strains. Our results indicate that multiple elements control adherence and OmpA acts as an adhesin in EHEC O157:H7.
Schistosomiasis Decreases Central Nervous System Inflammation and Alters the Progression of Experimental Autoimmune Encephalomyelitis
Authors: La Flamme Anne Camille , Ruddenklau Kate , Bäckström B. Thomas
Published: August 2003

A preestablished infection with the parasitic helminth,Schistosoma mansoni, significantly reduced the incidence and delayed the onset of experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice immunized with myelin oligodendrocyte glycoprotein (MOG)35-55peptide. The altered disease progression was not solely due to the induction of a strong Th2 response, since intraperitoneal injection of schistosome eggs did not affect disease development. MOG-specific gamma interferon (IFN-γ), nitric oxide, and tumor necrosis factor alpha production by splenocytes was significantly reduced in schistosome-infected mice compared to uninfected mice. However, similar levels of interleukin-10 (IL-10) were produced in an antigen-specific manner, suggesting that the induction of antigen-specific responses was not inhibited. Analysis of in vivo cytokine production by real-time PCR indicated that IL-12p40, but not IFN-γ, transcript levels were dramatically reduced in the spinal cords of schistosome-infected, MOG-immunized mice. Furthermore, analysis of the cellular composition of the spinal cords and brains revealed that a preestablished infection withS. mansonidecreased central nervous system (CNS) inflammation, particularly of macrophages and CD4 T cells. These results suggest that schistosomiasis may negatively regulate the onset of EAE by downregulating the production of proinflammatory cytokines and altering CNS inflammation.
Actinobacillus actinomycetemcomitans
Serotype d-Specific Antigen Contains the O Antigen of Lipopolysaccharide
Authors: Lakio Laura , Paju Susanna , Alfthan Georg , Tiirola Terttu , Asikainen Sirkka , Pussinen Pirkko J.
Published: August 2003

Actinobacillus actinomycetemcomitans
is a gram-negative, facultatively anaerobic bacterium which is associated especially with aggressive forms of periodontitis. Contradictory results on the localization of the
A. actinomycetemcomitans
serotype-specific antigen have been reported. The aim of the present study was to characterize the
A. actinomycetemcomitans
serotype d-specific antigen. The antigen was isolated by affinity chromatography. The affinity column was prepared from immunoglobulin G isolated from rabbit antiserum raised against
A. actinomycetemcomitans
serotype d. The isolated antigen was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and silver staining, all of which revealed a ladder-like structure typical for the O antigen of lipopolysaccharide (LPS). In a displacement enzyme-linked immunosorbent assay (ELISA), the isolated antigen displaced in a concentration-dependent manner the binding of the polyclonal rabbit antiserum raised against
A. actinomycetemcomitans
serotype d to the competing whole-cell serotype d antigen. The isolated antigen contained LPS, and an equal concentration of LPS isolated from
A. actinomycetemcomitans
serotype d gave a similar displacement curve in the ELISA. In order to test the immunogenic properties of the isolated antigen, it was used to immunize a rabbit. The antiserum raised against the isolated antigen displayed specificity in Western blotting and ELISA similar to that of antibody raised against LPS isolated from
A. actinomycetemcomitans
serotype d. In conclusion, our results show that the
A. actinomycetemcomitans
serotype d-specific antigen contains the O-antigenic structure of LPS.
Regulation of Expression of the Paralogous Mlp Family in
Borrelia burgdorferi
Authors: Yang Xiaofeng F. , Hübner Anette , Popova Taissia G. , Hagman Kayla E. , Norgard Michael V.
Published: August 2003

The Mlp (multicopy lipoproteins) family is one of many paralogous protein families in
Borrelia burgdorferi
. To examine the extent to which the 10 members of the Mlp family in
B. burgdorferi
strain 297 might be differentially regulated, antibodies specific for each of the Mlps were developed and used to analyze the protein expression profiles of individual Mlps when
B. burgdorferi
replicated under various cultivation conditions. All of the Mlps were upregulated coordinately when
B. burgdorferi
was cultivated at either elevated temperature, reduced culture pH, or increased spirochete cell density. Inasmuch as the expression of OspC is influenced by a novel RpoN-RpoS regulatory pathway, similar induction patterns for OspC and the Mlp paralogs prompted an assessment of whether the RpoN-RpoS pathway also was involved in Mlp expression. In contrast to wild-type
B. burgdorferi
, both RpoN- and RpoS-deficient mutants were unable to upregulate OspC or the Mlp paralogs when grown at lower pH (6.8), indicating that pH-mediated regulation of OspC and Mlp paralogs is dependent on the RpoN-RpoS pathway. However, when RpoN- or RpoS-deficient mutants were shifted from 23°C to 37°C or were cultivated to higher spirochete densities, some induction of the Mlps still occurred, whereas OspC expression was abolished. The combined findings suggest that the Mlp paralogs are coordinately regulated as a family and have an expression profile similar, but not identical, to that of OspC. Although Mlp expression as a family is influenced by the RpoN-RpoS regulatory pathway, there exists at least one additional layer of gene regulation, yet to be elucidated, contributing to Mlp expression in
B. burgdorferi
.
An
Enterococcus faecium
Secreted Antigen, SagA, Exhibits Broad-Spectrum Binding to Extracellular Matrix Proteins and Appears Essential for
E. faecium
Growth
Authors: Teng Fang , Kawalec Magdalena , Weinstock George M. , Hryniewicz Waleria , Murray Barbara E.
Published: August 2003

A gene encoding a major secreted antigen, SagA, was identified in
Enterococcus faecium
by screening an
E. faecium
genomic expression library with sera from patients with
E. faecium
-associated endocarditis. Recombinant SagA protein showed broad-spectrum binding to extracellular matrix (ECM) proteins, including fibrinogen, collagen type I, collagen type IV, fibronectin, and laminin. A fibrinogen-binding protein, purified from culture supernatants of an
E. faecium
clinical isolate, was found to match the N-terminal sequence of the predicted SagA protein and to react with the anti-SagA antibody, confirming that it was the SagA protein; this protein appeared as an 80- to 90-kDa smear on a Western blot that was sensitive to proteinase K and resistant to periodate treatment and glycoprotein staining. When overexpressed in
E. faecium
and
Escherichia coli
, the native and recombinant SagA proteins formed stable oligomers, apparently via their C-terminal domains. The SagA protein is composed of three domains: (i) a putative coiled-coil N-terminal domain that shows homology to the N-terminal domain of
Streptococcus mutans
SagA protein (42% similarity), previously shown to be involved in cell wall integrity and cell shape maintenance, and to the P45 protein of
Listeria monocytogenes
(41% similarity); (ii) a central domain containing direct repeats; and (iii) a C-terminal domain that is similar to that found in various proteins, including P45 (50% similarity) and P60 (52% similarity) of
L. monocytogenes
. The P45 and P60 proteins both have cell wall hydrolase activity, and the latter has also been shown to be involved in virulence, whereas cell wall hydrolase activity was not detected for SagA protein. The
E. faecium sagA
gene, like the
S. mutans
homologue, is located in a cluster of genes encoding proteins that appear to be involved in cell wall metabolism and could not be disrupted unless it was first transcomplemented, suggesting that the
sagA
gene is essential for
E. faecium
growth and may be involved in cell wall metabolism. In conclusion, the extracelluar
E. faecium
SagA protein is apparently essential for growth, shows broad-spectrum binding to ECM proteins, forms oligomers, and is antigenic during infection.
Analysis of RogB-Controlled Virulence Mechanisms and Gene Expression in
Streptococcus agalactiae
Authors: Gutekunst Heike , Eikmanns Bernhard J. , Reinscheid Dieter J.
Published: August 2003

Streptococcus agalactiae
is the leading cause of bacterial sepsis and meningitis in neonates and also the causative agent of different serious infections in immunocompromised adults. The wide range of diseases that are caused by
S. agalactiae
suggests regulatory mechanisms that control the formation of specific virulence factors in these bacteria. The present study describes a gene from
S. agalactiae
, designated
rogB
, encoding a protein with significant similarity to members of the RofA-like protein (RALP) family of transcriptional regulators. Disruption of the
rogB
gene in the genome of
S. agalactiae
resulted in mutant strain RGB1, which was impaired in its ability to bind to fibrinogen and fibronectin. Mutant RGB1 also exhibited a reduced adherence to human epithelial cells but did not show an altered invasion of eukaryotic cells. By real-time PCR analysis, mutant RGB1 revealed an increased expression of the
cpsA
gene, encoding a regulator of capsule gene expression. However, strain RGB1 exhibited a reduced expression of the
rogB
gene and of two adjacent genes, encoding putative virulence factors in
S. agalactiae
. Furthermore, mutant RGB1 was impaired in the expression of the
fbsA
gene, coding for a fibrinogen receptor from
S. agalactiae
. The altered gene expression in mutant RGB1 could be restored by plasmid-mediated expression of
rogB
, confirming a RogB deficiency as the cause for the observed changes in virulence gene expression in
S. agalactiae
. Reporter gene studies with a promotorless luciferase gene fused to
fbsA
allowed a growth-dependent analysis of
fbsA
expression in
S. agalactiae
. These reporter gene studies also suggest that RogB exerts a positive effect on
fbsA
expression in
S. agalactiae
.
Cloning and Characterization of a Gene Encoding an Immunoglobulin-Binding Receptor on the Cell Surface of Some Members of the FamilyTrypanosomatidae
Authors: Campos-Neto Antonio , Suffia Isabelle , Cavassani Karen A. , Jen Shyian , Greeson Kay , Ovendale Pamela , Silva João S. , Reed Steven G. , Skeiky Yasir A. W.
Published: August 2003

Several members of theTrypanosomatidaefamily, when freshly isolated from their mammalian hosts, have immunoglobulins adsorbed to their cell surfaces. However, a significant portion of these antibody molecules is not parasite specific, i.e., the immunoglobulins are bound to the parasite's cell surface molecules via noncognitive interactions. It has been proposed that this noncognitive adsorption of immunoglobulins to the parasite is mediated by an Fc-like receptor present in several members of theTrypanosomatidaefamily. However, the molecular identification of this receptor has never been defined. Here, we describe the cloning of a gene encoding a protein that might represent this molecule. The gene, namedLmsp1, was cloned by screening aLeishmania majorcDNA expression library using a rabbit antiserum.Lmsp1is present in bothLeishmaniaandTrypanosomaand is expressed in all developmental stages of these parasites. The predicted protein has a molecular mass of 16.6 kDa and contains an RGD sequence starting at residue 104 and three cysteine residues at positions 55, 74, and 116. The purified recombinant protein strongly binds to normal immunoglobulins of various animal species (humans, rabbits, sheep, goats, guinea pigs, donkeys, rats, and mice) and the binding to human immunoglobulins appears to be immunoglobulin G (IgG) and IgM isotype specific. Moreover,Lmsp1binds to both purified Fc and Fab fragments of IgG from both humans and rabbits. The mapping of theLmsp1epitopes that bind human IgG revealed that different sequences of the molecule bind to Fc or Fab. In addition, fluorescence-activated cell sorter analyses with a specific rabbit anti-Lmsp1antiserum showed thatLmsp1is associated with the parasite's cell surface. Finally, inhibition experiments point to an active role of this molecule in the immunoglobulin-mediated attachment and penetration ofTrypanosoma cruziin its macrophage host cells, thus suggesting thatLmsp1is a putativeTrypanosomatidaeimmunoglobulin receptor.
Central Role for B Lymphocytes and CD4
+
T Cells in Immunity to Infection by the Attaching and Effacing Pathogen
Citrobacter rodentium
Authors: Simmons Cameron P. , Clare Simon , Ghaem-Maghami Marjan , Uren Tania K. , Rankin Joanna , Huett Allan , Goldin Rob , Lewis David J. , MacDonald Thomas T. , Strugnell Richard A. , Frankel Gad , Dougan Gordon
Published: August 2003

Citrobacter rodentium
, an attaching-effacing bacterial pathogen, establishes an acute infection of the murine colonic epithelium and induces a mild colitis in immunocompetent mice. This study describes the role of T-cell subsets and B lymphocytes in immunity to
C. rodentium
. C57Bl/6 mice orally infected with
C. rodentium
resolved infection within 3 to 4 weeks. Conversely, systemic and colonic tissues of RAG1
−/−
mice orally infected with
C. rodentium
contained high and sustained pathogen loads, and in the colon this resulted in a severe colitis. C57Bl/6 mice depleted of CD4
+
T cells, but not CD8
+
T cells, were highly susceptible to infection and also developed severe colitis. Mice depleted of CD4
+
T cells also had diminished immunoglobulin G (IgG) and IgA antibody responses to two
C. rodentium
virulence-associated determinants, i.e., EspA and intimin, despite having a massively increased pathogen burden. Mice with an intact T-cell compartment, but lacking B cells (μMT mice), were highly susceptible to
C. rodentium
infection. Systemic immunity, but not mucosal immunity, could be restored by adoptive transfer of convalescent immune sera to infected μMT mice. Adoptive transfer of immune B cells, but not naïve B cells, provided highly variable immunity to recipient μMT mice. The results suggest that B-cell-mediated immune responses are central to resolution of a
C. rodentium
infection but that the mechanism through which this occurs requires further investigation. These data are relevant to understanding immunity to enteric attaching and effacing bacterial pathogens of humans.
Serum Opacity Factor (SOF) ofStreptococcus pyogenesEvokes Antibodies That Opsonize Homologous and Heterologous SOF-Positive Serotypes of Group A Streptococci
Authors: Courtney Harry S. , Hasty David L. , Dale James B.
Published: August 2003

Serum opacity factor (SOF) is a protein expressed byStreptococcus pyogenesthat opacifies mammalian serum. SOF is also a virulence factor ofS. pyogenes, but it has not been previously shown to elicit a protective immune response. Herein, we report that SOF evokes bactericidal antibodies againstS. pyogenesin humans, rabbits, and mice. Rabbit antiserum against purified recombinant SOF2 opsonized SOF-positive M type 2, 4, and 28S. pyogenesin human blood but had no effect on SOF-negative M type 5S. pyogenes. Furthermore, affinity-purified human antibodies against SOF2 also opsonized SOF-positive streptococci. A combination of antisera against M2 and SOF2 proteins was dramatically more effective in killing streptococci than either antiserum alone, indicating that antibodies against SOF2 enhance the opsonic efficiency of M protein antibodies. Mice tolerated an intravenous injection of 100 μg of SOF without overt signs of toxicity, and immunization with SOF protected mice against challenge infections with M type 2S. pyogenes. These data indicate that SOF evokes opsonic antibodies that may protect against infections by SOF-positive serotypes of group A streptococci and suggest that different serotypes of SOF have common epitopes that may be useful vaccine candidates to protect against group A streptococcal infections.
Characterization of Antibody Responses to
Wolbachia
Surface Protein in Humans with Lymphatic Filariasis
Authors: Punkosdy George A. , Addiss David G. , Lammie Patrick J.
Published: August 2003

Symbiotic
Wolbachia
organisms of filarial nematodes have received much attention as possible chemotherapy targets and disease-causing organisms. In order to further investigate the association between anti-
Wolbachia
immune responses and chronic filarial disease in humans, antibody responses to
Wolbachia
surface protein (WSP) were assayed in serum samples collected from 232 individuals living in Leogane, Haiti, an area where
Wuchereria bancrofti
infection is endemic, and from 67 North Americans with no history of lymphatic filariasis. As opposed to antifilarial antibody responses, which were largely influenced by the patient's infection status, the prevalence and levels of anti-WSP immunoglobulin G (IgG) antibodies among individuals with lymphedema or hydrocele were significantly greater than those in gender- and infection-matched individuals without disease. In at least one case, the anti-WSP IgG response was coincident with the onset of lymphedema development, and among anti-WSP-positive women with lymphedema, anti-WSP IgG levels were negatively correlated with the duration of lymphedema. The presence of anti-WSP IgG was also associated with the severity of inguinal adenopathy among men with hydrocele. In addition to the presence of anti-WSP antibodies among Haitians, 15 of 67 (22%) serum samples collected from individuals from North America, where filariasis is not endemic, were also positive for anti-WSP antibodies. In comparison to those from Haitians, anti-WSP antibodies from North Americans primarily recognized a distinct region of WSP located within the highly conserved second transmembrane domain. The results of this study demonstrate that anti-WSP antibody responses are associated with the presence of chronic filarial morbidity and not filarial infection status in humans and suggest that WSP should be further studied as a potential trigger for the development of filarial disease.
Coinjection with CpG-Containing Immunostimulatory Oligodeoxynucleotides Reduces the Pathogenicity of a Live Vaccine against Cutaneous Leishmaniasis but Maintains Its Potency and Durability
Authors: Mendez Susana , Tabbara Khaled , Belkaid Yasmine , Bertholet Sylvie , Verthelyi Daniela , Klinman Dennis , Seder Robert A. , Sacks David L.
Published: August 2003

The inoculation of live, nonattenuatedLeishmania majorto produce a lesion in a selected site that heals, referred to as leishmanization, is to date the only vaccine against leishmaniasis that has proven to be effective in humans. Its use has been restricted or abandoned entirely, however, due to safety concerns. In an attempt to develop a leishmanization protocol that minimizes pathology while maintaining long-term protection, live parasites were coinjected with CpG-containing immunostimulatory oligodeoxynucleotides (CpG ODNs) alone or in combination with whole-cell lysates of heat-killedL. majorpromastigotes bound to alum (ALM). C57BL/6 mice infected intradermally by usingL. majorplus CpG ODN with or without ALM developed few or no dermal lesions and showed an early containment of parasite growth, while mice infected withL. majorwith or without ALM developed sizable dermal lesions that required up to 10 weeks to heal. The CpG ODNs provoked a transient inflammation that included an early recruitment and accumulation of gamma interferon-producing CD4+lymphocytes in the site. Attenuation of the live vaccine did not compromise its ability to confer long-term immunity, as mice receivingL. majorand CpG ODN plus ALM were totally protected against reinfection withL. majorfor up to 6 months. By comparison, the immunity elicited by two efficient nonlive vaccines began to wane by 6 months. Our results suggest that immune modulation using CpG ODNs might be a practical approach to improving the safety of a highly effective live vaccine that has already been widely applied.
Early Induction of Humoral and Cellular Immune Responses during ExperimentalMycobacterium aviumsubsp.paratuberculosisInfection of Calves
Authors: Waters W. R. , Miller J. M. , Palmer M. V. , Stabel J. R. , Jones D. E. , Koistinen K. A. , Steadham E. M. , Hamilton M. J. , Davis W. C. , Bannantine J. P.
Published: August 2003

Johne's disease (paratuberculosis) of cattle is widespread and causes significant economic losses for producers due to decreased production and poor health of affected animals. The chronic nature of the disease and the lack of a reproducible model of infection hinder research efforts. In the present study, instillation ofMycobacterium aviumsubsp.paratuberculosisinto the tonsillar crypts of neonatal calves resulted in peripheral colonization as detected by antemortem culture of feces and postmortem (320 days postchallenge) culture of intestinal tissues. Antigen-specific blastogenic, gamma interferon (IFN-γ), and nitric oxide responses by blood mononuclear cells from infected calves exceeded prechallenge responses beginning 194 days postchallenge. Upon in vitro stimulation with paratuberculosis antigens, CD4+cells from infected calves proliferated, produced IFN-γ, and increased expression of CD26 and CD45RO (indicative of an activated memory phenotype). Utilizing a lipoarabinomannan-based enzyme-linked immunosorbent assay, specific serum immunoglobulin was detected as early as 134 days postchallenge and generally increased after this time point. Two antigens of ∼50 and ∼60 kDa were particularly immunodominant early in infection, as shown by immunoblot with serum collected within 2 weeks postchallenge. Findings indicate that the intratonsillar inoculation route will prove useful as an experimental model for paratuberculosis infection. Additionally, this study confirms that mycobacteria-specific antibody is detectable early in the course of experimental Johne's disease, even preceding the development of specific cell-mediated responses.
Lactoferrin Impairs Type III Secretory System Function in EnteropathogenicEscherichia coli
Authors: Ochoa Theresa J. , Noguera-Obenza Marita , Ebel Frank , Guzman Carlos A. , Gomez Henry F. , Cleary Thomas G.
Published: August 2003

EnteropathogenicEscherichia coli(EPEC) is an important cause of infant diarrhea in developing countries. EPEC uses a type III secretory system to deliver effector proteins into the host cell. These proteins cause the characteristic attaching and effacing lesion on enterocytes. Lactoferrin, a glycoprotein present in human milk, inhibits EPEC adherence to mammalian cells. To determine the effect of lactoferrin on the initial host cell attachment step that is mediated by the type III secretory system, we focused on EPEC-induced actin polymerization in HEp2 cells, on the hemolytic activity, and on measurement ofE. colisecreted proteins A, B, and D (EspABD). Lactoferrin blocked EPEC-mediated actin polymerization in HEp2 cells and blocked EPEC-induced hemolysis. The mechanism of this inhibition was lactoferrin-mediated degradation of secreted proteins necessary for bacterial contact and pore formation, particularly EspB. The proteolytic effect of lactoferrin was prevented by serine protease inhibitors. This disruption of the type III secretory system implies that lactoferrin could provide broad cross protection against the enteropathogens that share this mechanism.
Porphyromonas gingivalisInfection during Pregnancy Increases Maternal Tumor Necrosis Factor Alpha, Suppresses Maternal Interleukin-10, and Enhances Fetal Growth Restriction and Resorption in Mice
Authors: Lin Dongming , Smith Mary Alice , Champagne Catherine , Elter John , Beck James , Offenbacher Steven
Published: August 2003

Epidemiological studies have shown a potential association between maternal periodontitis and pregnancy complications. We used a pregnant murine model to study the effect of infection with the periodontal pathogenPorphyromonas gingivalison pregnancy outcomes. Female BALB/c mice were inoculated with heat-killedP. gingivalis(109CFU) in a subcutaneous chamber and mated 2 weeks later. At gestation day (GD) 7.5, mice were challenged with liveP. gingivalis(107CFU) (n= 20) or broth (control;n= 8) and sacrificed at GD 16.5. Fetal growth restriction (FGR, <0.46 g) was defined as fetuses with weights 2 standard deviations (SD) smaller than controls (0.56 ± 0.05 g [mean ± SD]). Among the 20 challenged mice, 8 had both normal-weight (0.51 ± 0.11 g) and FGR (0.34 ± 0.1 g) fetuses within the same litter. All other challenged dams had normal-weight fetuses (0.57 ± 0.04 g). Maternal liver, uterus, and spleen samples were examined forP. gingivalisDNA using a PCR technique. Of the eight challenged mice with FGR fetuses, three had PCR signals forP. gingivalisin liver and uterus, but not in the spleen. Liver, uterus, and spleen were negative forP. gingivalisDNA among all other challenged and control mice. In serum of dams with FGR fetuses, tumor necrosis factor alpha levels were elevated significantly, while interluekin-10 levels were significantly reduced compared to levels in dams with normal fetuses.P. gingivalis-specific serum immunoglobulin G levels were significantly elevated in dams with FGR fetuses compared to dams without any FGR fetuses. These data demonstrate thatP. gingivalis-induced murine FGR is associated with systemic dissemination of the organism and activated maternal immune and inflammatory responses.
Insertional Inactivation ofpacandrmlBGenes Reduces the Release of Tumor Necrosis Factor Alpha, Interleukin-6, and Interleukin-8 Induced byStreptococcus mutansin Monocytic, Dental Pulp, and Periodontal Ligament Cells
Authors: Engels-Deutsch Marc , Pini Annelise , Yamashita Yoshihisa , Shibata Yukie , Haikel Youssef , Schöller-Guinard Marie , Klein Jean-Paul
Published: August 2003

Streptococcus mutanspossesses different cell wall molecules, such as protein of the I/II family, the serotype f polysaccharide rhamnose glucose polymer (RGP), and lipoteichoic acid (LTA), which act as adhesins and modulins, allowingS. mutansto colonize teeth and cause dental caries and pulpitis. We tested several isogenic mutants ofS. mutansdefective in protein I/II and/or RGP, as well as purified modulins such as protein I/II, RGP, and LTA, for their binding and activation abilities on monocytic, dental pulp (DP), and periodontal ligament (PDL) cells. Our results demonstrate that both protein I/II and RGP play important roles in streptococcal adherence to human monocytic and fibroblastic cells, whereas LTA is only a minor adhesin. In the activation process, the cytokine response elicited is polarized toward a Th1 response which seems principally due to protein I/II and RGP. Even if protein I/II seems to be more efficient in its purified form in triggering cells to release interleukin-8 (IL-8), RGP is the most efficient cytokine-stimulating component in intact bacteria, while LTA plays only a minor role. In cell activation, we showed, by using either cytochalasin D or coated ligands, that internalization of eitherS. mutans,S. mutansisogenic mutants, or purified ligands is not necessary to trigger cells to release IL-8. We also showed that, besides the implication of monocytes in pulpal inflammation, fibroblast-like cells such as DP and PDL cells are also actively implicated in local inflammation and in the generation of a Th1 response after stimulation withS. mutanscells or antigens.
Binding of Shiga Toxin 2e to Porcine Erythrocytes In Vivo and In Vitro
Authors: Matise Ilze , Cornick Nancy A. , Samuel James E. , Moon Harley W.
Published: August 2003

Shiga toxin 2e (Stx2e), produced by host-adapted Shiga toxin-producing
Escherichia coli
(STEC) strains, causes edema disease in weaned pigs. Edema disease is manifested as vascular necrosis, edema, neurologic signs, and death. In this study we sought to determine the correlation between the presence of Stx2e in the blood of STEC-inoculated pigs and the disease outcome. Eleven of 15 (73%) pigs with clinical and 5 of 35 (14%) pigs with subclinical edema disease had detectable levels of Stx2e in the red-blood-cell (RBC) fraction of their blood but not in serum or plasma. The presence of Stx2e in the RBC fraction was strongly associated with the development of clinical disease (relative risk, 5.8;
P
< 0.0001). Subclinical pigs with Stx2e in their blood developed more-extensive vascular lesions than pigs without detectable Stx2e in their blood (average proportions of necrotic arterioles, 63 and 27.5%, respectively;
P
= 0.001). Variations in RBC-bound Stx2e levels could in part reflect variations in the binding capacity of RBCs. As an initial step toward addressing this possibility, assays were conducted to determine if pigs vary in the Stx2e binding capacity of their RBCs. An in vitro study of noninoculated pigs demonstrated two phenotypes based on the capacity of the RBCs to bind Stx2e. While RBCs from most of the pigs consistently bound high levels of Stx2e (high-binding phenotype), consistently low Stx2e binding was detected in RBCs from a few pigs (low-binding phenotype). The low- and high-binding phenotypes of individual pigs remained consistent throughout repeated samplings over 2 months.
Liposomal Meningococcal B Vaccination: Role of Dendritic Cell Targeting in the Development of a Protective Immune Response
Authors: Arigita Carmen , Bevaart Lisette , Everse Linda A. , Koning Gerben A. , Hennink Wim E. , Crommelin Daan J. A. , van de Winkel Jan G. J. , van Vugt Martine J. , Kersten Gideon F. A. , Jiskoot Wim
Published: August 2003

The effect of targeting strategies for improving the interaction of liposomal PorA with dendritic cells (DC) on the immunogenicity of PorA was investigated. PorA, a major antigen ofNeisseria meningitidis, was purified and reconstituted in different types of (targeted) liposomes, i.e., by using mannose or phosphatidylserine as targeting moieties, or with positively charged liposomes. We studied the efficiency of liposome uptake and its effect on the maturation of and interleukin 12 (IL-12) production by murine DC. Moreover, mice were immunized subcutaneously to study the localization and immunogenicity of PorA liposomes. Uptake of liposomes by DC was significantly increased for targeted liposomes and resulted in the maturation of DC, but to various degrees. Maturation markers (i.e., CD80, CD86, major histocompatibility complex class II, and CD40) showed enhanced expression on DC incubated with targeted PorA liposomes relative to those incubated with nontargeted PorA liposomes. Moreover, only the uptake of targeted PorA liposomes induced production of IL-12 by DC, with levels similar to those produced by lipopolysaccharide (LPS)-pulsed DC. Mannose-targeted PorA liposomes administered subcutaneously had an increased localization in draining lymph nodes compared to nontargeted PorA liposomes. Liposomes in draining lymph nodes interacted preferentially with antigen-presenting cells, an effect that was enhanced with targeted PorA liposomes. Immunization studies showed an improvement of the bactericidal antibody response (i.e., increased number of responders) generated by targeted PorA liposomes compared to that generated by nontargeted ones or LPS-containing outer membrane vesicles. In conclusion, the use of targeted PorA liposomes results in an improved uptake by and activation of DC and an increased localization in draining lymph nodes. These effects correlate with an enhanced immune response toward the vaccine.
Unusual Interaction of a Lipopolysaccharide Isolated fromBurkholderia cepaciawith Polymyxin B
Authors: Shimomura Hirofumi , Matsuura Motohiro , Saito Shinji , Hirai Yoshikazu , Isshiki Yasunori , Kawahara Kazuyoshi
Published: August 2003

We have demonstrated that lipopolysaccharide (LPS) obtained fromBurkholderia cepacia, an important opportunistic pathogen, has unique characteristics in both structure and activity. One of the structural characteristics is that theB. cepaciaLPS has 4-amino-4-deoxy-l-arabinose (Ara4N) in its inner core region. Polymyxin B (PmxB) is known to act as an LPS antagonist, but LPS with Ara4N is suggested to be PmxB resistant by decreasing the binding capability of PmxB. Interaction ofB. cepaciaLPS with PmxB was investigated and compared with that of a reference LPS ofSalmonella entericaserovar Abortusequi, referred to hereafter as the reference LPS.B. cepaciaLPS suffered no suppressive effect of PmxB in its activity to stimulate murine peritoneal macrophages for induction of tumor necrosis factor alpha (TNF-α) and IL-6 even when the activity of the reference LPS was completely suppressed. A characteristic ofB. cepaciaLPS is that it has selectively weak interleukin-1β (IL-1β)-inducing activity while activity to induce TNF-α and IL-6 has been shown to be as strong as that of the reference LPS. Remarkably, PmxB augmented the IL-1β-inducing activity ofB. cepaciaLPS to the level of that of the reference LPS and, in contrast, completely suppressed the strong activity of the reference LPS. Using PmxB-immobilized beads, the adsorbances of these LPSs to the beads were compared, and it was found thatB. cepaciaLPS bound to PmxB with a high affinity similar to that of the reference LPS. These results indicate an unusual interaction ofB. cepaciaLPS with PmxB wherebyB. cepaciaLPS not only allows the binding of PmxB with high affinity, even though it contains Ara4N, but also suffers no suppressive effect of PmxB on its activity. Moreover, a remarkable increase in its IL-1β-inducing activity was also observed.
Influence of Maternal Filariasis on Childhood Infection and Immunity toWuchereria bancroftiin Kenya
Authors: Malhotra Indu , Ouma John H. , Wamachi Alex , Kioko John , Mungai Peter , Njzovu Malik , Kazura James W. , King Christopher L.
Published: August 2003

To determine whether maternal filariasis influences the risk of infection by and immunity toWuchereria bancroftiin children, we performed a cross-sectional study in an area of Kenya where filariasis is endemic. Residents of 211 households were enrolled; 376 parents and 938 of their offspring between the ages of 2 and 17 years were examined for filarial infection status as determined by blood-borne microfilariae and filarial antigenemia. Children of infected mothers had a three- to fourfold increased risk of filarial infection, as ascertained by circulating filarial antigen, relative to children of uninfected mothers (P< 0.001). Paternal infection did not correlate with childhood infection status, indicating a specific maternal effect. Peripheral blood mononuclear cells from children of filaria-infected mothers (n= 33) had higher levels of constitutive interleukin-5 (IL-5) and IL-10, increased microfilarial antigen-specific IL-5 production, and diminished microfilarial antigen-driven lymphocyte proliferation than cells from children of uninfected mothers (n= 46;P< 0.05). In contrast, there were no differences between the two groups in adult worm antigen-driven gamma interferon, IL-2, IL-4, IL-5, and IL-10 production and lymphocyte proliferation. These data indicate that maternal filarial infection increases childhood susceptibility toW. bancroftiand skews filaria-specific immunity toward a Th2-type cytokine response. The results support the notion that in utero exposure to filarial antigens affects the natural history of filariasis during childhood.
Protective Mucosal Th2 Immune Response againstToxoplasma gondiiby Murine Mesenteric Lymph Node Dendritic Cells
Authors: Dimier-Poisson Isabelle , Aline Fleur , Mévélec Marie-Noëlle , Beauvillain Céline , Buzoni-Gatel Dominique , Bout Daniel
Published: August 2003

Toxoplasma gondii, an obligate intracellular parasite pathogen which initially invades the intestinal epithelium before disseminating throughout the body, may cause severe sequelae in fetuses and life-threatening neuropathy in immunocompromised patients. Immune protection is usually thought to be performed through a systemic Th1 response; considering the route of parasite entry it is important to study and characterize the local mucosal immune response toT. gondii. Despite considerable effort,Toxoplasma-targeted vaccines have proven to be elusive using conventional strategies. We report the use of mesenteric lymph node dendritic cells (MLNDCs) pulsed ex vivo withT. gondiiantigens (TAg) as a novel investigation approach to vaccination againstT. gondii-driven pathogenic processes. Using a murine model, we demonstrate in two genetically distinct mouse strains (C57BL/6 and CBA/J) that adoptively transferred TAg-pulsed MLNDCs elicit a mucosalToxoplasma-specific Th2-biased immune response in vivo and confer strong protection against infection. We also observe that MLNDCs mostly traffic to the intestine where they enhance resistance by reduction in the mortality and in the number of brain cysts. Thus, ex vivo TAg-pulsed MLNDCs represent a powerful tool for the study of protective immunity toT. gondii, delivered through its natural route of entry. These findings might impact the design of vaccine strategies against other invasive microorganisms known to be delivered through digestive tract.
A Monoclonal Antibody Directed against aCandida albicansCell Wall Mannoprotein Exerts Three Anti-C. albicansActivities
Authors: Moragues María D. , Omaetxebarria Miren J. , Elguezabal Natalia , Sevilla María J. , Conti Stefania , Polonelli Luciano , Pontón José
Published: August 2003

Antibodies are believed to play a role in the protection againstCandida albicansinfections by a number of mechanisms, including the inhibition of adhesion or germ tube formation, opsonization, neutralization of virulence-related enzymes, and direct candidacidal activity. Although some of these biological activities have been demonstrated individually in monoclonal antibodies (MAbs), it is not clear if all these anti-C. albicansactivities can be displayed by a single antibody. In this report, we characterized a monoclonal antibody raised against the main target of salivary secretory immunoglobulin A in the cell wall ofC. albicans, which exerts three anti-C. albicansactivities: (i) inhibition of adherence to HEp-2 cells, (ii) inhibition of germination, and (iii) direct candidacidal activity. MAb C7 reacted with a proteinic epitope from a mannoprotein with a molecular mass of >200 kDa predominantly expressed on theC. albicansgerm tube cell wall surface as well as with a number of antigens fromCandida lusitaniae,Cryptococcus neoformans,Aspergillus fumigatus, andScedosporium prolificans.MAb C7 caused a 31.1% inhibition in the adhesion ofC. albicansto HEp-2 monolayers and a 55.3% inhibition in the adhesion ofC. albicansto buccal epithelial cells, produced a 38.5% decrease in the filamentation ofC. albicans, and exhibited a potent fungicidal effect againstC. albicans,C. lusitaniae,Cryptococcus neoformans,A. fumigatus, andS. prolificans, showing reductions in fungal growth ranging from 34.2 to 88.7%. The fungicidal activity showed by MAb C7 seems to be related to that reported by antibodies mimicking the activity of a killer toxin produced by the yeastPichia anomala, since one of these MAbs also reacted with theC. albicansmannoprotein with a molecular mass of >200 kDa. Results presented in this study support the concept of a family of microbicidal antibodies that could be useful in the treatment of a wide range of microbial infections when used alone or in combination with current antimicrobial agents.
Comparative Analysis of Plant and Animal Models for Characterization of
Burkholderia cepacia
Virulence
Authors: Bernier Steve P. , Silo-Suh Laura , Woods Donald E. , Ohman Dennis E. , Sokol Pamela A.
Published: August 2003

A simple alfalfa model was developed as an alternative infection model for virulence studies of the
Burkholderia cepacia
complex. Symptoms of disease were observed in wounded alfalfa seedlings within 7 days following inoculation of 10
1
to 10
5
CFU of most strains of the
B. cepacia
complex. Strains from seven genomovars of the
B. cepacia
complex were tested for virulence in the alfalfa model, and the degree of virulence was generally similar in strains belonging to the same genomovar. Strains of
Burkholderia multivorans
and some strains of
Burkholderia stabilis
did not cause symptoms of disease in alfalfa seedlings. Representative strains were also tested for virulence using the rat agar bead model. Most of the strains tested were able to establish chronic lung infections;
B. stabilis
strains were the exception. Most of the strains that were virulent in the alfalfa infection model were also virulent in the lung infection model. The
B. cepacia
genomovar III mutants K56
pvdA
::tp and K56-H15 were significantly less virulent in the alfalfa infection model than their parent strain. Therefore, this alfalfa infection model may be a useful tool for assessing virulence of strains of the
B. cepacia
complex and identifying new virulence-associated genes.
Mice Intranasally Immunized with a Recombinant 16-Kilodalton Antigen from RoundwormAscarisParasites Are Protected against Larval Migration ofAscaris suum
Authors: Tsuji Naotoshi , Suzuki Kayo , Kasuga-Aoki Harue , Isobe Takashi , Arakawa Takeshi , Matsumoto Yasunobu
Published: August 2003

Protective immunity to the pig roundworm,Ascaris suum, has been demonstrated by immunization of pigs with antigens derived from the parasite's larval stages. We identified a protective antigen commonly expressed in the human and pigAscarisinfections as a 16-kDa protein (As16), which has no similarity at the amino acid level to mammalian proteins but has some similarity to those of the filarial parasites andCaenorhabditis elegansgene product. Localization analysis revealed that the native As16 was highly expressed in the adult worm intestine, hypodermis, and cuticles. In addition, As16 was detected in the parasite excretory and secretory products. Mice intranasally vaccinated withEscherichia coli-expressed recombinant As16 (rAs16), coupled with cholera toxin B subunit, generated a significant increase in the level of rAs16-specific immunoglobulin G (IgG) and IgE in serum. Mucosal IgA levels were also increased. The recombinant protein evoked a mixed (both Th1 and Th2) type of immune response characterized by elevated levels of gamma interferon and interleukin-10 in the culture supernatants of activated spleen cells. An increased level of IgG1 and IgG2a in serum was also observed. The vaccinated mice showed a reduction by 58% in the recovery of challenged larvae compared to a nonvaccinated control. These results suggest the possibility of developing a mucosal vaccine for human and pig ascariasis.
Tumor Necrosis Factor Alpha Is a Key Mediator of Gut Inflammation Seen in Amebic Colitis in Human Intestine in the SCID Mouse-Human Intestinal Xenograft Model of Disease
Authors: Zhang Zhi , Mahajan Sajan , Zhang Xiaochung , Stanley Samuel L.
Published: August 2003

We usedEntamoeba histolyticainfection in human intestinal xenografts to study the roles interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-α) in the pathogenesis of amebic colitis. We found that blockade of TNF-α reduced inflammation and intestinal damage in amebic infection, while inhibition of IL-1 reduced cytokine production but had less marked effects on inflammation and disease.
Nicotinamide Ribosyl Uptake Mutants in
Haemophilus influenzae
Authors: Herbert Mark , Sauer Elizabeta , Smethurst Graeme , Kraiβ Anita , Hilpert Anna-Karina , Reidl Joachim
Published: August 2003

The gene for the nicotinamide riboside (NR) transporter (
pnuC
) was identified in
Haemophilus influenzae
. A
pnuC
mutant had only residual NR uptake and could survive in vitro with high concentrations of NR, but could not survive in vivo. PnuC may represent a target for the development of inhibitors for preventing
H. influenzae
disease.