bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and Immunogenicity of Group AStreptococcusCulture Supernatant Proteins
Authors: Lei Benfang , Mackie Stacy , Lukomski Slawomir , Musser James M.
Published: July 2002

Extracellular proteins made by group AStreptococcus(GAS) play critical roles in the pathogenesis of human infections caused by this bacterium. Although many extracellular GAS proteins have been identified and characterized, there has been no systematic analysis of culture supernatant proteins. Proteins present in the culture supernatant of strains of serotype M1 (MGAS 5005) and M3 (MGAS 315) mutants lacking production of the major extracellular cysteine protease were separated by two-dimensional gel electrophoresis and identified by amino-terminal amino acid sequencing and interrogation of available databases, including a serotype M1 genome sequence. In the aggregate, amino-terminal amino acid sequence data for 66 protein spots were generated, 53 unique sequences were obtained, and 44 distinct proteins were identified. Sixteen of the 44 proteins had apparent secretion signal sequences and 27 proteins did not. Eight of the 16 proteins with apparent secretion signal sequences have not been previously described for GAS. Antibodies against most of the apparently secreted proteins were present in sera from mice infected subcutaneously with MGAS 5005 or MGAS 315. Humans with documented GAS infections (pharyngitis, acute rheumatic fever, and severe invasive disease) also had serum antibodies reacting with many of the apparently secreted proteins, indicating that they were synthesized in the course of GAS-human interaction. The genes encoding four of the eight previously undescribed and apparently secreted culture supernatant proteins were cloned, and the proteins were overexpressed inEscherichia coli. Western blot analysis with these recombinant proteins and sera from GAS-infected mice and humans confirmed the immunogenicity of these proteins. Taken together, the data provide new information about the molecular aspects of GAS-host interactions.
Urease as a Virulence Factor in Experimental Cryptococcosis
Authors: Cox Gary M. , Mukherjee Jean , Cole Garry T. , Casadevall Arturo , Perfect John R.
Published: July 2002

Urease catalyzes the hydrolysis of urea to ammonia and carbamate and has been found to be an important pathogenic factor for certain bacteria.
Cryptococcus neoformans
is a significant human pathogenic fungus that produces large amounts of urease; thus we wanted to investigate the importance of urease in the pathogenesis of cryptococcosis. We cloned and sequenced the genomic locus containing the single-copy
C. neoformans
urease gene (
URE1
) and used this to disrupt the native
URE1
in the serotype A strain H99. The
ure1
mutant strains were found to have in vitro growth characteristics, phenoloxidase activity, and capsule size similar to those of the wild type. Comparison of a
ure1
mutant with H99 after intracisternal inoculation into corticosteroid-treated rabbits revealed no significant differences in colony counts recovered from the cerebrospinal fluid. However, when these two strains were compared in both the murine intravenous and inhalational infection models, there were significant differences in survival. Mice infected with a
ure1
strain lived longer than mice infected with H99 in both models. The
ure1
strain was restored to urease positivity by complementation with
URE1
, and two resulting transformants were significantly more pathogenic than the
ure1
strain. Our results suggest that urease activity is involved in the pathogenesis of cryptococcosis but that the importance may be species and/or infection site specific.
Interleukin-4 Receptor Alpha-Deficient BALB/c Mice Show an Unimpaired T Helper 2 Polarization in Response toLeishmania majorInfection
Authors: Mohrs Markus , Holscher Christoph , Brombacher Frank
Published: July 2002

We recently generated interleukin-4 (IL-4) receptor alpha-deficient (IL-4Rα−/−) BALB/c mice and showed evidence for a protective role of IL-13-mediated functions in leishmaniasis. In this study, we investigated the IL-4 expression and T helper 2 (Th2) development inLeishmania major-infected IL-4Rα−/−mice. Here we show that the early burst of IL-4 expression observed inL. major-infected BALB/c mice is independent of IL-4Rα-mediated functions. Subsequently, we confirmed an impaired Th2 development in vitro. Unexpectedly, duringL. majorinfection, isolated CD4+IL-4Rα−/−T cells expressed high IL-4- but low gamma interferon (IFN-γ)-specific mRNA, comparable to Th2-polarized BALB/c CD4+cells and in contrast to Th1-polarized C57BL/6 CD4+cells. Since antigen-specific restimulated popliteal lymph node cells (PLN) of IL-4Rα−/−mice also responded with high IL-4 but low IFN-γ production, comparable to Th2-polarized cells from wild-type BALB/c mice and in contrast to Th1-polarized C57BL/6 cells, these results suggested an unimpaired Th2 polarization during an established infection withL. major. To further define the observed IL-4 receptor-independent Th2 cell phenotype, we determined an independent Th2 marker, the IL-12 receptor beta-2 (IL-12Rβ2)-specific transcript levels of CD4+T cells. Confirming Th2 polarization inL. major-infected IL-4Rα−/−mice, comparable IL-12Rβ2 message levels between CD4+T cells from infected IL-4Rα−/−mice and Th2 cells from BALB/c mice were found, whereas Th1-polarized C57BL/6 cells showed strikingly increased IL-12Rβ2 expression levels. These results indicate that signals mediated by the IL-4Rα are not necessary to induce and sustain an efficient IL-4 expression and Th2 polarization inL. major-infected BALB/c mice and suggest that IL-4Rα-independent mechanisms underlie the default Th2 development inL. major-infected BALB/c mice.
Macrophage Migration Inhibitory Factor Release by Macrophages after Ingestion ofPlasmodium chabaudi-Infected Erythrocytes: Possible Role in the Pathogenesis of Malarial Anemia
Authors: Martiney James A. , Sherry Barbara , Metz Christine N. , Espinoza Marisol , Ferrer Angel S. , Calandra Thierry , Broxmeyer Hal E. , Bucala Richard
Published: July 2002

Human falciparum malaria, caused byPlasmodium falciparuminfection, results in 1 to 2 million deaths per year, mostly children under the age of 5 years. The two main causes of death are severe anemia and cerebral malaria. Malarial anemia is characterized by parasite red blood cell (RBC) destruction and suppression of erythropoiesis (the mechanism of which is unknown) in the presence of a robust host erythropoietin response. The production of a host-derived erythropoiesis inhibitor in response to parasite products has been implicated in the pathogenesis of malarial anemia. The identity of this putative host factor is unknown, but antibody neutralization studies have ruled out interleukin-1β, tumor necrosis factor alpha, and gamma interferon while injection of interleukin-12 protects susceptible mice against lethalP. chabaudiinfection. In this study, we report that ingestion ofP. chabaudi-infected erythrocytes or malarial pigment (hemozoin) induces the release of macrophage migration inhibitory factor (MIF) from macrophages. MIF, a proinflammatory mediator and counter-regulator of glucocorticoid action, inhibits erythroid (BFU-E), multipotential (CFU-GEMM), and granulocyte-macrophage (CFU-GM) progenitor-derived colony formation. MIF was detected in the sera ofP. chabaudi-infected BALB/c mice, and circulating levels correlated with disease severity. Liver MIF immunoreactivity increased concomitant with extensive pigment and parasitized RBC deposition. Finally, MIF was elevated three- to fourfold in the spleen and bone marrow ofP. chabaudi-infected mice with active disease, as compared to early disease, or of uninfected controls. In summary, the present results suggest that MIF may be a host-derived factor involved in the pathophysiology of malaria anemia.
Invasion of the Brain and Chronic Central Nervous System Infection after SystemicMycobacterium aviumComplex Infection in Mice
Authors: Wu Hsiu-Shan , Kolonoski Peter , Chang Yung Yee , Bermudez Luiz E.
Published: July 2002

Central nervous system (CNS) infections caused by nontuberculous mycobacteria have been described previously, especially in patients with AIDS. To investigate specific aspects of the pathogenesis of this entity, C57BLbg+/bg−mice were infected intravenously withMycobacterium avium, and cultures of blood and brain as well as histopathology examination of brain tissue were carried out at several time points up to 6 months after infection. Low-grade inflammatory changes with small aggregates of lymphocytes and macrophages as well as perivascular cuffing were seen early in the infection. A small number of bacteria could be observed in the parenchyma of the choroid plexus. Six months after infection, numerous bacteria were present within the foamy macrophage of the granulomatous lesions along the ventricle and meninges. None of the mice developed clinical signs of meningitis or encephalitis or even died spontaneously during the period of observation. Use of CD18−/−knockout mice indicated that transport of the bacterium within neutrophils or monocytes into the brain is unlikely. Mild chronic CNS infection developed in the mice during sustained systemicM. aviuminfection, similar to what has been reported in most human cases.
Inhibition of Adhesion ofEscherichia coliK88ac Fimbria to Its Receptor, Intestinal Mucin-Type Glycoproteins, by a Monoclonal Antibody Directed against a Variable Domain of the Fimbria
Authors: Sun Ronggai , Anderson Timothy J. , Erickson Alan K. , Nelson Eric A. , Francis David H.
Published: July 2002

Strains of enterotoxigenicEscherichia colithat express K88 fimbriae are among the most common causes of diarrhea in young pigs. Adhesion of bacteria to receptors on intestinal epithelial cells, mediated by K88 fimbriae, is the initial step in the establishment of infection. Three antigenic variants of K88 fimbriae exist in nature: K88ab, K88ac, and K88ad. K88ac is the most prevalent and may be the only variant of significance in swine disease. Each K88 fimbrial variant is composed of multiple antigenic determinants. Some of these determinants are shared among the three variants and may be referred to as conserved epitopes, whereas others are unique to a specific variant and may be referred to as variable epitopes. In this study, monoclonal antibodies (MAbs) specific to either variable or conserved epitopes of K88ac fimbriae were produced. The specificity of each MAb was tested by enzyme-linked immunosorbent and immunoblot assays. Fab fragments were prepared from these MAbs and were tested for their ability to block the binding of K88-positive bacteria and purified fimbriae to porcine enterocyte brush border vesicles and purified K88 receptors, respectively. The purified receptors were intestinal mucin-type sialoglycoproteins (IMTGP) isolated from porcine enterocytes (A. K. Erickson, D. R. Baker, B. T. Bosworth, T. A. Casey, D. A. Benfield, and D. H. Francis, Infect. Immun. 62:5404–5410, 1994). Fab fragments prepared from MAbs specific for variable epitopes blocked the binding of bacteria to brush borders and of fimbriae to IMTGP. However, those from MAbs specific for a conserved epitope did not. These observations indicate that the receptor-binding domain of a K88ac fimbria is contained, at least in part, within the antigenically variable epitopes of that fimbria. Epitope mapping for one of the MAbs, which recognizes a linear epitope on K88ac fimbriae, indicated that this MAb binds to the region from amino acid no. 64 to no. 107 on the major subunit of K88ac fimbriae.
Monitoring BioluminescentStaphylococcus aureusInfections in Living Mice Using a NovelluxABCDEConstruct
Authors: Francis Kevin P. , Joh Danny , Bellinger-Kawahara Carolyn , Hawkinson Matthew J. , Purchio Tony F. , Contag Pamela R.
Published: July 2002

Strains ofStaphylococcus aureuswere transformed with plasmid DNA containing aPhotorhabdus luminescens luxoperon (luxABCDE) that was genetically modified to be functional in both gram-positive and gram-negative bacteria.S. aureuscells containing this novelluxconstruct, downstream of an appropriate promoter sequence, are highly bioluminescent, allowing the detection of fewer than 100 CFU in vitro (direct detection of exponentially dividing cells in liquid culture). Furthermore, these bacteria produce light stably at 37°C and do not require exogenous aldehyde substrate, thus allowingS. aureusinfections in living animals to be monitored by bioluminescence. Two strains ofS. aureus8325-4 that produce high levels of constitutive bioluminescence were injected into the thigh muscles of mice, and the animals were then either treated with the antibiotic amoxicillin or left untreated. Bioluminescence from bacteria present in the thighs of the mice was monitored in vivo over a period of 24 h. The effectiveness of the antibiotic in the treated animals could be measured by a decrease in the light signal. At 8 h, the infection in both groups of treated animals had begun to clear, as judged by a decrease in bioluminescence, and by 24 h no light signal could be detected. In contrast, both groups of untreated mice had strong bioluminescent signals at 24 h. Quantification of CFU from bacteria extracted from the thigh muscles of the mice correlated well with the bioluminescence data. This paper shows for the first time that bioluminescence offers a method for monitoringS. aureusinfections in vivo that is sensitive and noninvasive and requires fewer animals than conventional methodologies.
Adjuvant Activity of a Nontoxic Mutant ofEscherichia coliHeat-Labile Enterotoxin on Systemic and Mucosal Immune Responses Elicited against a Heterologous Antigen Carried by a LiveSalmonella entericaSerovar Typhimurium Vaccine Strain
Authors: Guillobel Heloisa C. R. , Carinhanha Joana I. , Cárdenas Lucia , Clements John D. , de Almeida Darcy F. , Ferreira Luís C. S.
Published: July 2002

Systemic and mucosal antibody responses against both the major subunit of colonization factor antigen I (CFA/I) of enterotoxigenicEscherichia coli(ETEC) and the somatic lipopolysaccharide expressed by recombinant bivalentSalmonellavaccine strains were significantly enhanced by coadministration of a detoxified derivative with preserved adjuvant effects of the ETEC heat-labile toxin, LT(R192G). The results further support the adjuvant effects of LT(R192G)and represent a simple alternative to improve responses against passenger antigens expressed by orally deliveredSalmonellavaccine strains.
Cloning of Genes of Nontypeable
Haemophilus influenzae
Involved in Penetration between Human Lung Epithelial Cells
Authors: van Schilfgaarde Muriel , van Ulsen Peter , van der Steeg Wim , Winter Victor , Eijk Paul , Everts Vincent , Dankert Jacob , van Alphen Loek
Published: July 2002

Haemophilus influenzae
penetrates between epithelial cells via an unknown mechanism. A chromosomal library of nonencapsulated
H. influenzae
strain A960053 DNA was constructed in
Escherichia coli
DH5α to identify bacterial genes contributing to this paracytosis. Two
E. coli
clones that contained open reading frames (ORFs) homologous to HI0636 to HI0641 of
H. influenzae
strain Rd and that showed an increased penetration in epithelial cell layers of the human bronchial epithelial cell line NCI-H292 were identified. ORFs HI0636 and HI0638, encoding two small proteins of unknown functions, were further investigated. The clone containing ORFs HI0636 and HI0637 as well as the clone containing ORF HI0638 showed a significant increase in penetration. Disruption of HI0638 by kanamycin box insertion in
H. influenzae
strain A960053 resulted in loss of penetration into the epithelial cell layers. Disruption of HI0636 had no effect on penetration in this model system. Since a role for HI0637 in the paracytosis of
H. influenzae
is very unlikely because it encodes TrpS, we conclude that the protein encoded by ORF HI0638 may function as a paracytin, while that encoded by HI0636 may have an auxiliary function.
Regulation of Human β-Defensins by Gastric Epithelial Cells in Response to Infection withHelicobacter pylorior Stimulation with Interleukin-1
Authors: O'Neil Deborah A. , Cole Sheri P. , Martin-Porter Edith , Housley Michael P. , Liu Lide , Ganz Tomas , Kagnoff Martin F.
Published: July 2002

Gastric epithelial cells in vitro and in vivo are shown to constitutively express the peptide antibiotic human β-defensin type 1 (hBD-1). In contrast, hBD-2 expression is regulated in gastric epithelial cells and increases in response to infection withHelicobacter pylorior stimulation with the proinflammatory cytokine interleukin-1. These data suggest that hBD-2 is a component of the regulated host gastric epithelial cell response toH. pyloriinfection and proinflammatory mediators.
Cyclophosphamide Decreases Nitrotyrosine Formation and Inhibits Nitric Oxide Production by Alveolar Macrophages in Mycoplasmosis
Authors: Hickman-Davis Judy M. , Lindsey J. Russell , Matalon Sadis
Published: July 2002

We previously reported that congenic C57BL/6 inducible nitric oxide synthase
−/−
(iNOS
−/−
) mice infected with
Mycoplasma pulmonis
developed higher bacterial numbers and lung lesion scores than C57BL/6 iNOS
+/+
controls but had similar lung nitrotyrosine levels. The present studies investigated the role of inflammatory cells in nitrotyrosine formation during mycoplasmal infection. iNOS
+/+
and iNOS
−/−
mice were injected with cyclophosphamide (CYP) and inoculated with 10
7
CFU of
M. pulmonis
. CYP pretreatment of
M. pulmonis
-infected iNOS
+/+
and iNOS
−/−
mice reduced polymorphonuclear cells (PMNs) within bronchoalveolar lavages (BALs) by 88 and 72%, respectively, and whole-lung myeloperoxidase levels by 80 and 78%, respectively, at 72 h postinfection but did not alter the number of alveolar macrophages (AMs) in BALs. CYP treatment also significantly decreased nitrate and nitrite (NOx) levels in BALs and plasma of infected iNOS
+/+
mice, whereas neither CYP nor mycoplasmal infection altered NOx in iNOS
−/−
mice. CYP reduced lung nitrotyrosine levels in both iNOS
+/+
and iNOS
−/−
mice to uninfected-control levels as shown by immunohistochemical staining and enzyme-linked immunosorbent assay and inhibited mycoplasmal killing by iNOS
+/+
mice in vivo. CYP inhibited the production of gamma interferon-inducible NOx by iNOS
+/+
AMs in vitro but did not alter the number of iNOS-positive AMs, as detected by immunocytochemistry. In addition, AMs from CYP-treated iNOS
+/+
mice had significantly decreased ability to kill mycoplasmas in vitro. These results demonstrate that reactive species generated by inflammatory cells as well as PMN myeloperoxidase are important contributors to nitrotyrosine formation during mycoplasmal infection and that treatment with CYP decreases NO
⋅
production by AMs and inhibits mycoplasmal killing.
Characterization of FasG Segments Required for 987P Fimbria-Mediated Binding to Piglet Glycoprotein Receptors
Authors: Choi Byung-Kwon , Schifferli Dieter M.
Published: July 2002

The 987P fimbriae of enterotoxigenic strains of
Escherichia coli
bind to both glycoprotein and glycolipid receptors on the brush borders of piglet enterocytes. A mutation in lysine residue 117 of the adhesive subunit FasG [
fasG
(K117A)] previously shown to abrogate 987P binding to the lipid receptor sulfatide did not affect the interaction with the glycoprotein receptors. Both the fimbriae and the FasG subunits of the wild type and the
fasG
(K117A) mutant bound to the glycoprotein receptors, confirming that lysine 117 was not required for binding to the glycoprotein receptors. Truncated FasG molecules were used to identify domains required for glycoprotein receptor recognition. At least two segments which did not include lysine117, namely, residues 211 (glutamine) to 220 (serine) and 20 (aspartic acid) to 41 (serine), were shown to be involved in the FasG-glycoprotein receptor interactions by ligand-blotting assays. Changing isoleucine 217 or leucine 215 of FasG to alanine abolished the property of a truncated FasG fusion protein to inhibit 987P recognition of its glycoprotein receptors. Thus, the K117 residue of FasG is required only for binding to the glycolipid receptor, whereas the newly identified hydrophobic residues of the FasG subunit are required specifically for the recognition of the glycoprotein receptor. Taken together, our data indicate that different residues of the FasG adhesin are important in 987P fimbrial binding to sulfatide and glycoprotein receptors, suggesting different mechanisms of interaction.
Dual-Function Vaccine for
Pseudomonas aeruginosa
: Characterization of Chimeric Exotoxin A-Pilin Protein
Authors: Hertle Ralf , Mrsny Randall , Fitzgerald David J.
Published: July 2002

Pseudomonas aeruginosa
is the major infectious agent of concern for cystic fibrosis patients. Strategies to prevent colonization by this bacterium and/or neutralize its virulence factors are clearly needed. Here we characterize a dual-function vaccine designed to generate antibodies to reduce bacterial adherence and to neutralize the cytotoxic activity of exotoxin A. To construct the vaccine, key sequences from type IV pilin were inserted into a vector encoding a nontoxic (active-site deletion) version of exotoxin A. The chimeric protein, termed PE64Δ553pil, was expressed in
Escherichia coli
, refolded to a near-native conformation, and then characterized by various biochemical and immunological assays. PE64Δ553pil bound specifically to asialo-GM1, and, when injected into rabbits, produced antibodies that reduced bacterial adherence and neutralized the cell-killing activity of exotoxin A. Results support further evaluation of this chimeric protein as a vaccine to prevent
Pseudomonas
colonization in susceptible individuals.
How Giardia Swim and Divide
Authors: Ghosh Sudip , Frisardi Marta , Rogers Rick , Samuelson John
Published: July 2002

To determine how binuclear giardia swim, we used video microscopy to observe trophozoites of
Giardia intestinalis
, which were labeled with an amino-specific Alexa Fluor dye that highlighted the flagella and adherence disc. Giardia swam forward by means of the synchronous beating of anterior, posterolateral, and ventral flagella in the plane of the ventral disc, while caudal flagella swam in a plane perpendicular to the disc. Giardia turned in the plane of the disc by means of a rudder-like motion of its tail, which was constant rather than beating. To determine how giardia divide, we used three-dimensional confocal microscopy, the same surface label, nuclear stains, and antitubulin antibodies. Giardia divided with mirror-image symmetry in the plane of the adherence disc, so that the right nucleus of the mother became the left nucleus of the daughter. Pairs of nuclei were tethered together by microtubules which surrounded nuclei and prevented mother or daughter giardia from receiving two copies of the same nucleus. New adherence discs formed upon a spiral backbone of microtubules, which had a clockwise rotation when viewed from the ventral surface. These dynamic observations of the parasite begin to reveal how giardia swim and divide.
Infection of Synovial Fibroblasts in Culture by
Yersinia enterocolitica
and
Salmonella enterica
Serovar Enteritidis: Ultrastructural Investigation with Respect to the Pathogenesis of Reactive Arthritis
Authors: Meyer-Bahlburg Almut , Brinkhoff Jörg , Krenn Veit , Trebesius Karlheinz , Heesemann Jürgen , Huppertz Hans-Iko
Published: July 2002

Synovial fibroblasts were infected with
Yersinia enterocolitica
or
Salmonella enterica
serovar Enteritidis and analyzed by electron microscopy and fluorescence in situ hybridization. Intracellular bacterial replication was followed by degradation leading to “ghosts” possessing lipopolysaccharides but not DNA. However, single bacteria survived for more than 2 weeks. Therefore, transient intra-articular infection might be the missing link between initial intestinal infection and late synovial inflammation in the pathogenesis of reactive arthritis.
Human Infection withAscaris lumbricoidesIs Associated with Suppression of the Interleukin-2 Response to Recombinant Cholera Toxin B Subunit following Vaccination with the Live Oral Cholera Vaccine CVD 103-HgR
Authors: Cooper Philip J. , Chico Martha , Sandoval Carlos , Espinel Ivan , Guevara Angel , Levine Myron M. , Griffin George E. , Nutman Thomas B.
Published: July 2002

To investigate the potential immunomodulatory effects of concurrent ascariasis on the cytokine response to a live oral vaccine, we measured cytokine responses to cholera toxin B subunit (CT-B) following vaccination with the live oral cholera vaccine CVD 103-HgR inAscaris lumbricoides-infected subjects randomized in a double-blind study to receive two doses of either albendazole or placebo prior to vaccination and in a group of healthy U.S. controls. Postvaccination cytokine responses to CT-B were characterized by transient increases in the production of interleukin-2 (IL-2;P= 0.02) and gamma interferon (IFN-γ;P= 0.001) in the three study groups combined; however, postvaccination increases in IFN-γ were significant only in the albendazole-treatedA. lumbricoidesinfection group (P= 0.008). Postvaccination levels of IL-2 were significantly greater in the albendazole-treated group compared with the placebo group (P= 0.03). No changes in levels of Th1 and Th2 cytokines in response to control ascaris antigens were observed over the same period. These findings indicate that vaccination with CVD 103-HgR is associated with a Th1 cytokine response (IL-2 and IFN-γ) to CT-B, that infection withA. lumbricoidesdiminishes the magnitude of this response, and that albendazole treatment prior to vaccination was able to partially reverse the deficit in IL-2. The potential modulation of the immune response to oral vaccines by geohelminth parasites has important implications for the design of vaccination campaigns in geohelminth-endemic areas.
Rat Mannose-Binding Protein A Binds CD14
Authors: Chiba Hirofumi , Sano Hitomi , Iwaki Daisuke , Murakami Seiji , Mitsuzawa Hiroaki , Takahashi Toru , Konishi Masanori , Takahashi Hiroki , Kuroki Yoshio
Published: July 2002

Lipopolysaccharide (LPS) has been known to induce inflammation by interacting with CD14, which serves as a receptor for LPS. Mannose-binding protein (MBP) belongs to the collectin subgroup of the C-type lectin superfamily, along with surfactant proteins SP-A and SP-D. We have recently demonstrated that SP-A modulates LPS-induced cellular responses by interaction with CD14 (H. Sano, H. Sohma, T. Muta, S. Nomura, D. R. Voelker, and Y. Kuroki, J. Immunol. 163:387–395, 2000) and that SP-D also interacts with CD14 (H. Sano, H. Chiba, D. Iwaki, H. Sohma, D. R. Voelker, and Y. Kuroki, J. Biol. Chem. 275:22442–22451, 2000). In this study, we examined whether MBP, a collectin highly homologous to SP-A and SP-D, could bind CD14. Recombinant rat MBP-A bound recombinant human soluble CD14 in a concentration-dependent manner. Its binding was not inhibited in the presence of excess mannose or EDTA. MBP-A bound deglycosylated CD14 treated withN-glycosidase F, neuraminidase, andO-glycosidase, indicating that MBP-A interacts with the peptide portion of CD14. Since LPS was also a ligand for the collectins, we compared the characteristics of binding of MBP-A to LPS with those of binding to CD14. MBP-A bound to lipid A fromSalmonella entericaserovar Minnesota and rough LPS (S. entericaserovar Minnesota Re595 andEscherichia coliJ5, Rc), but not to smooth LPS (E. coliO26:B6 and O111:B4). Unlike CD14 binding, EDTA and excess mannose attenuated the binding of MBP-A to rough LPS. From these results, we conclude that CD14 is a novel ligand for MBP-A and that MBP-A utilizes a different mechanism for CD14 recognition from that for LPS.
Acid-Induced Gene Expression in
Helicobacter pylori
: Study in Genomic Scale by Microarray
Authors: Ang Sunny , Lee Cha-Ze , Peck Konan , Sindici Michelle , Matrubutham Udayakumar , Gleeson Martin A. , Wang Jin-Town
Published: July 2002

To understand the RNA expression in response to acid stress of
Helicobacter pylori
in genomic scale, a microarray membrane containing 1,534 open reading frames (ORFs) from strain 26695 was used. Total RNAs of
H. pylori
under growth conditions of pH 7.2 and 5.5 were extracted, reverse transcribed into cDNA, and labeled with biotin. Each microarray membrane was hybridized with cDNA probe from the same strain under two different pH conditions and developed by a catalyzed reporter deposition method. Gene expression of all ORFs was measured by densitometry. Among the 1,534 ORFs, 53 ORFs were highly expressed (≧30% of rRNA control in densitometry ratios). There were 445 ORFs which were stably expressed (<30% of rRNA in densitometry) under both pH conditions without significant variation. A total of 80 ORFs had significantly increased expression levels at low pH, while expressions of 4 ORFs were suppressed under acidic condition. The remaining 952 ORFs were not detectable under either pH condition. These data were highly reproducible and comparable to those obtained by the RNA slot blot method. Our results suggest that microarray can be used in monitoring prokaryotic gene expression in genomic scale.
Expression of Cytolethal Distending Toxin and Hemolysin Is Not Required for Pustule Formation by
Haemophilus ducreyi
in Human Volunteers
Authors: Young Royden S. , Fortney Kate R. , Gelfanova Valentina , Phillips Carrie L. , Katz Barry P. , Hood Antoinette F. , Latimer Jo L. , Munson Robert S. , Hansen Eric J. , Spinola Stanley M.
Published: July 2002

Haemophilus ducreyi
makes cytolethal distending toxin (CDT) and hemolysin. In a previous human challenge trial, an isogenic hemolysin-deficient mutant caused pustules with a rate similar to that of its parent. To test whether CDT was required for pustule formation, six human subjects were inoculated with a CDT mutant and parent at multiple sites. The pustule formation rates were similar at both parent and mutant sites. A CDT and hemolysin double mutant was constructed and tested in five additional subjects. The pustule formation rates were similar for the parent and double mutant. These results indicate that neither the expression of CDT, nor that of hemolysin, nor both are required for pustule formation by
H. ducreyi
in humans.
Plesiomonas shigelloides
Enters Polarized Human Intestinal Caco-2 Cells in an In Vitro Model System
Authors: Theodoropoulos Christina , Wong Toh Hee , O'Brien Mark , Stenzel Deborah
Published: July 2002

This study provides the first definitive evidence that the gram-negative bacterium
Plesiomonas shigelloides
adheres to and enters eukaryotic intestinal host cells in vitro.
P. shigelloides
is increasingly regarded as an emerging enteric pathogen and has been implicated in intestinal and extraintestinal infections in humans. However, the establishment of its true role in enteric disease has been hindered by inadequacies in experimental design, deficiencies in clinical diagnosis, and the lack of an appropriate animal model. In this investigation, an in vitro system was used to evaluate plesiomonad pathogenesis. Differentiated epithelium-derived Caco-2 cell monolayers inoculated apically with 12 isolates of
P. shigelloides
from clinical (intestinal) origins were examined at high resolution using transmission electron microscopy. Bacterial cells were observed adhering to intact microvilli and to the plasma membrane on both the apical and the basal surfaces of the monolayer. The bacteria entered the Caco-2 cells and were observed enclosed in single and multiple membrane-bound vacuoles within the host cell cytoplasm. This observation suggests that initial uptake may occur through a phagocytic-like process, as has been documented for many other enteropathogens.
P. shigelloides
also was noted free in the cytosol of Caco-2 cells, suggesting escape from cytoplasmic vacuoles. Differences in invasion phenotypes were revealed, suggesting the possibility that, like
Escherichia coli, P. shigelloides
comprises different pathogenic phenotypes.
Differential Interleukin-10 and Gamma Interferon mRNA Expression in Lungs of Cilium-Associated Respiratory Bacillus-Infected Mice
Authors: Kendall Lon V. , Riley Lela K. , Hook Reuel R. , Besch-Williford Cynthia L. , Franklin Craig L.
Published: July 2002

The cilium-associated respiratory (CAR) bacillus is a gram-negative, extracellular bacterium that causes persistent respiratory tract infections in rodents. We have previously demonstrated that BALB/c mice are more susceptible to CAR bacillus-induced disease than resistant C57BL/6 mice, with elevations in pulmonary gamma interferon (IFN-γ) and interleukin (IL)-4. IL-10 is a type 2 cytokine that can increase host susceptibility to bacterial diseases through its anti-inflammatory effects, including suppression of macrophage function. The purpose of this study was to further describe the cytokine profiles associated with histologic lesions in CAR bacillus-infected mice and to assess the effects of cytokine depletion on the pathogenesis of disease. Six-week-old female BALB/c and C57BL/6 mice and mice with targeted mutations in IFN-γ and IL-4 were inoculated intratracheally with 105CAR bacillus organisms, and samples were collected at 6 to 7 weeks postinoculation. Lung samples were collected for histopathologic examination and analysis of cytokine mRNA. IFN-γ, IL-10, and IL-4 mRNA levels in the lungs of infected mice were semiquantitatively measured using a reverse transcriptase-mediated PCR assay and compared to those in uninfected control animals of each strain. BALB/c mice infected with CAR bacillus had a median lung lesion score of 6 and IL-10 and IL-4 mRNA levels were significantly elevated. The majority of C57BL/6 mice were resistant to disease characterized by lung lesions scores of 2 or less and a dominant IFN-γ mRNA cytokine profile. A few C57BL/6 mice with lesions scores of 5 or greater had elevations in all three cytokines and were susceptible to disease. C57BL/6 IFN-γ knockout mice had increased disease with elevations in IL-10 and IL-4 mRNA, while BALB/c IL-4 knockout mice infected with CAR bacillus had a mild decrease in lesion severity and an attenuated IL-10 mRNA expression compared to wild-type BALB/c mice. These data indicate that IL-10 and IL-4 predominate in CAR bacillus-induced histologic lesions in mice, while IFN-γ may play a role in resistance to disease.
Legionella pneumophilaReplication in Macrophages Inhibited by Selective Immunomodulatory Effects on Cytokine Formation by Epigallocatechin Gallate, a Major Form of Tea Catechins
Authors: Matsunaga Kazuto , Klein Thomas W. , Friedman Herman , Yamamoto Yoshimasa
Published: July 2002

Epigallocatechin gallate (EGCg) is a major form of tea catechin and has a variety of biological activities, including antitumor as well as antimicrobial activity against some pathogens. Although the biological activities of EGCg have been extensively studied, its immunological effects are not well known. In the present study, the ability of EGCg to modulate macrophage immune functions in an in vitroLegionella pneumophilainfection model of macrophages was examined. The study showed that EGCg inhibited the growth ofL. pneumophilain macrophages at a concentration as low as 0.5 μg/ml without any direct antibacterial effect on the organisms. The EGCg selectively upregulated the production of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α) and downregulated IL-10 production of macrophages induced byL. pneumophilainfection in a dose-dependent manner, but did not alter IL-6 production even at a high dose. The upregulation of the levels of macrophage gamma interferon (IFN-γ) mRNA by EGCg was also demonstrated. Treatment of macrophage cultures with anti-TNF-α and anti-IFN-γ monoclonal antibodies markedly abolished the anti-L. pneumophilaactivity of macrophages induced by the EGCg treatment. These results indicate that EGCg selectively alters the immune responses of macrophages toL. pneumophilaand leads to an enhanced anti-L. pneumophilaactivity of macrophages mediated by enhanced production of both TNF-α and IFN-γ. However, the enhancement of in vitro anti-L. pneumophilaactivity by EGCg may not be directly mediated by IL-10 and IL-12 production modulation. Thus, the results of this study revealed the immunomodulatory effect of EGCg on macrophages, which have a critical role in infections.
Contribution of Choline-Binding Proteins to Cell Surface Properties of
Streptococcus pneumoniae
Authors: Swiatlo Edwin , Champlin Franklin R. , Holman Steven C. , Wilson W. William , Watt James M.
Published: July 2002

Nonspecific interactions related to physicochemical properties of bacterial cell surfaces, such as hydrophobicity and electrostatic charge, are known to have important roles in bacterium-host cell encounters.
Streptococcus pneumoniae
(pneumococcus) expresses multiple, surface-exposed, choline-binding proteins (CBPs) which have been associated with adhesion and virulence. The purpose of this study was to determine the contribution of CBPs to the surface characteristics of pneumococci and, consequently, to learn how CBPs may affect nonspecific interactions with host cells. Pneumococcal strains lacking CBPs were derived by adapting bacteria to a defined medium that substituted ethanolamine for choline. Such strains do not anchor CBPs to their surface. Cell surface hydrophobicity was tested for the wild-type and adapted strains by using a biphasic hydrocarbon adherence assay, and electrostatic charge was determined by zeta potential measurement. Adherence of pneumococci to human-derived cells was assessed by fluorescence-activated cell sorter analysis. Strains lacking both capsule and CBPs were significantly more hydrophobic than nonencapsulated strains with a normal complement of CBPs. The effect of CBPs on hydrophobicity was attenuated in the presence of capsule. Removal of CBPs conferred a greater electronegative net surface charge than that which cells with CBPs possessed, regardless of the presence of capsule. Strains that lack CBPs were poorly adherent to human monocyte-like cells when compared with wild-type bacteria with a full complement of CBPs. These results suggest that CBPs contribute significantly to the hydrophobic and electrostatic surface characteristics of pneumococci and may facilitate adherence to host cells partially through nonspecific, physicochemical interactions.
Chemokine and Chemokine Receptor Dynamics during Genital Chlamydial Infection
Authors: Belay Tesfaye , Eko Francis O. , Ananaba Godwin A. , Bowers Samera , Moore Terri , Lyn Deborah , Igietseme Joseph U.
Published: July 2002

Current design strategies for vaccines against certain microbial pathogens, includingChlamydia trachomatis, require the induction and targeting of specific immune effectors to the local sites of infection known as the mucosal effector sites. Chemokines and their receptors are important mediators of leukocyte trafficking and of the controlled recruitment of specific leukocyte clonotypes during host defense against infections and during inflammation. We analyzed the dynamics of chemokine and chemokine receptor expression in genital mucosae during genital chlamydial infection in a murine model to determine how these molecular entities influence the development of immunity and the clearance of infection. A time course study revealed an increase of up to threefold in the levels of expression of RANTES, monocyte chemotactic protein 1 (MCP-1), gamma-interferon-inducible protein 10 (IP-10), macrophage inflammatory protein 1α (MIP-1α), and intercellular adhesion molecule type 1 (ICAM-1) after genital infection with theC. trachomatisagent of mouse pneumonitis. Peak levels of expression of RANTES, MCP-1, and MIP-1α occurred by day 7 after primary infection, while those of IP-10 and ICAM-1 peaked by day 21. Expression levels of these molecules decreased by day 42 after primary infection, by which time all animals had resolved the infection, suggesting an infection-driven regulation of expression. A rapid upregulation of expression of these molecules was observed after secondary infection. The presence of cells bearing the chemokine receptors CCR5 and CXCR3, known to be preferentially expressed on Th1 and dendritic cells, was also synchronous with the kinetics of immune induction in the genital tract and clearance of infection. Results demonstrated that genital chlamydial infection is associated with a significant induction of chemokines and chemokine receptors that are involved in the recruitment of Th1 cells into the site of infection. Future studies will focus on how selective modulation of chemokines and their receptors can be used to optimize long-term immunity againstChlamydia.
Retrieving Biological Activity from LukF-PV Mutants Combined with Different S Components Implies Compatibility between the Stem Domains of These Staphylococcal Bicomponent Leucotoxins
Authors: Werner S. , Colin D. A. , Coraiola M. , Menestrina G. , Monteil H. , Prévost G.
Published: July 2002

Bicomponent leucotoxins, such as Panton-Valentine leucocidin, are composed of two classes of proteins, a class S protein such as LukS-PV, which bears the cell membrane binding function, and a class F protein such as LukF-PV, which interacts to form a bipartite hexameric pore. These leucotoxins induce cell activation, linked to a Ca
2+
influx, and pore formation as two consecutive and independently inhibitable events. Knowledge of the LukF-PV monomer structure has indicated that the stem domain is folded into three antiparallel β-strands in the water-soluble form and has to refold into a transmembrane β-hairpin during pore formation. To investigate the requirements for the cooperative assembly of the stems of the S and F components to produce biological activity, we introduced multiple deletions or single point mutations into the stem domains of LukF-PV and HlgB. While the binding of the mutated proteins was weakly dependent on these changes, Ca
2+
influx and pore formation were affected differently, confirming that they are independent events. Ca
2+
entry into human polymorphonuclear cells requires oligomerization and may follow the formation of a prepore. The activity of some of the LukF-PV mutants, carrying the shorter deletions, was actually improved. This demonstrated that a crucial event in the action of these toxins is the transition of the prefolded stem into the extended β-hairpins and that this step may be facilitated by small deletions that remove some of the interactions stabilizing the folded structure.
Intranasal Immunization with Pneumococcal Conjugate Vaccines with LT-K63, a Nontoxic Mutant of Heat-Labile Enterotoxin, as Adjuvant Rapidly Induces Protective Immunity against Lethal Pneumococcal Infections in Neonatal Mice
Authors: Jakobsen Håvard , Bjarnarson Stefania , Del Giudice Giuseppe , Moreau Monique , Siegrist Claire-Anne , Jonsdottir Ingileif
Published: July 2002

Immunization with pneumococcal polysaccharides (PPS) conjugated to tetanus toxoid (TT) (Pnc-TT) elicits protective immunity in an adult murine pneumococcal infection model. To assess immunogenicity and protective immunity in early life, neonatal (1 week old) and infant (3 weeks old) mice were immunized intranasally (i.n.) or subcutaneously (s.c.) with Pnc-TT of serotype 1 (Pnc1-TT). Anti-PPS-1 and anti-TT immunoglobulin G (IgG) and IgM antibodies were measured in serum and saliva, and vaccine-induced protection was evaluated by i.n. challenge with serotype 1 pneumococci. Pnc1-TT was immunogenic in neonatal and infant mice when administered s.c. without adjuvant: a majority of the young mice were protected from bacteremia and a reduction of pneumococcal density in the lungs was observed, although antibody responses and protective efficacy remained lower than in adults. The addition of LT-K63, a nontoxic mutant of heat-labile enterotoxin, as adjuvant significantly enhanced PPS-1-specific IgG responses and protective efficacy following either s.c. or i.n. Pnc1-TT immunization. Mucosal immunization was particularly efficient in neonates, as a single i.n. dose of Pnc1-TT and LT-K63 induced significantly higher PPS-1-specific IgG responses than s.c. immunization and was sufficient to protect neonatal mice against pneumococcal infections, whereas two s.c. doses were required to induce complete protection. In addition, i.n. immunization with Pnc1-TT and LT-K63 induced a vigorous salivary IgA response. This suggests that mucosal immunization with pneumococcal conjugate vaccines and LT-K63 may be able to circumvent some of the limitations of neonatal antibody responses, which are required for protective immunity in early life.
Mucosal or Parenteral Administration of Microsphere-Associated
Bacillus anthracis
Protective Antigen Protects against Anthrax Infection in Mice
Authors: Flick-Smith Helen C. , Eyles Jim E. , Hebdon Richard , Waters Emma L. , Beedham Richard J. , Stagg Tony J. , Miller Julie , Alpar H. Oya , Baillie Les W. J. , Williamson E. Diane
Published: July 2002

Existing licensed anthrax vaccines are administered parenterally and require multiple doses to induce protective immunity. This requires trained personnel and is not the optimum route for stimulating a mucosal immune response. Microencapsulation of vaccine antigens offers a number of advantages over traditional vaccine formulations, including stability without refrigeration and the potential for utilizing less invasive routes of administration. Recombinant protective antigen (rPA), the dominant antigen for protection against anthrax infection, was encapsulated in poly-
l
-lactide 100-kDa microspheres. Alternatively, rPA was loosely attached to the surfaces of microspheres by lyophilization. All of the microspheric formulations were administered to A/J mice with a two-dose schedule by either the intramuscular route, the intranasal route, or a combination of these two routes, and immunogenicity and protective efficacy were assessed. An intramuscular priming immunization followed by either an intramuscular or intranasal boost gave optimum anti-rPA immunoglobulin G titers. Despite differences in rPA-specific antibody titers, all immunized mice survived an injected challenge consisting of 10
3
median lethal doses of
Bacillus anthracis
STI spores. Immunization with microencapsulated and microsphere-associated formulations of rPA also protected against aerosol challenge with 30 median lethal doses of STI spores. These results show that rPA can be encapsulated and surface bound to polymeric microspheres without impairing its immunogenicity and also that mucosal or parenteral administration of microspheric formulations of rPA efficiently protects mice against both injected and aerosol challenges with
B. anthracis
spores. Microspheric formulations of rPA could represent the next generation of anthrax vaccines, which could require fewer doses because they are more potent, are less reactogenic than currently available human anthrax vaccines, and could be self-administered without injection.