bluuebunny/crossref_metadata_2025 huggingface dataset:
Trypanosoma cruziInfection Modulates In Vivo Expression of Major Histocompatibility Complex Class II Molecules on Antigen-Presenting Cells and T-Cell Stimulatory Activity of Dendritic Cells in a Strain-Dependent Manner
Authors: Alba Soto Catalina D. , Mirkin Gerardo A. , Solana Maria E. , González Cappa Stella M.
Published: February 2003

A striking feature of Chagas' disease is the diversity of clinical presentations. Such variability may be due to the heterogeneity amongTrypanosoma cruziisolates or to the host immune response. Employing two strains which differ in their virulence, we investigated the effect of in vivo infection on professional antigen-presenting cells (APC). Acute infection with the virulent RA strain downregulated the expression of major histocompatibility complex (MHC) class II on splenic dendritic cells (DC) and inhibited its induction on peritoneal macrophages and splenic B cells. It also impaired the ability of DC to prime allogeneic T cells and to form homotypic clusters, suggesting a low maturation state of these cells. In contrast, the low-virulence K98 strain maintained the expression of MHC class II on DC or stimulated it on peritoneal macrophages and B cells and preserved DC's T-cell priming capacity and homotypic clustering. DC from RA-infected mice elicited a lower activation ofT. cruzi-specific T-cell proliferation than those from K98-infected mice. APC from RA-infected mice that reached the chronic phase of infection restored MHC class II levels to those found in K98-infected mice and upregulated costimulatory molecules expression, suggesting that the immunosuppression caused by this strain is only transient. Taken together, the results indicate that in vivo infection withT. cruzimodulates APC functionality and that this is accomplished in a strain-dependent manner.
Activation of Natural Killer T Cells by α-Galactosylceramide Impairs DNA Vaccine-Induced Protective Immunity againstTrypanosoma cruzi
Authors: Miyahira Yasushi , Katae Masaharu , Takeda Kazuyoshi , Yagita Hideo , Okumura Ko , Kobayashi Seiki , Takeuchi Tsutomu , Kamiyama Tsuneo , Fukuchi Yoshinosuke , Aoki Takashi
Published: February 2003

Innate immunity as a first defense is indispensable for host survival against infectious agents. We examined the roles of natural killer (NK) T cells in defense againstTrypanosoma cruziinfection. TheT. cruziparasitemia and survival of CD1d-deficient mice exhibited no differences compared to wild-type littermates. NK T-cell activation induced by administering α-galactosylceramide (α-GalCer) toT. cruzi-infected mice significantly changed the parasitemia only in the late phase of infection and slightly improved survival when mice were infected intraperitoneally. The combined usage of α-GalCer and benznidazole, a commercially available drug for Chagas' disease, did not enhance the therapeutic efficacy of benznidazole. These results suggest that NK T cells do not play a pivotal role in resistance toT. cruziinfection. In addition, we found that the coadministration of α-GalCer with DNA vaccine impaired the induction of epitope-specific CD8+T cells and undermined the DNA vaccine-induced protective immunity againstT. cruzi. Our results, in contrast to previous reports demonstrating the protective roles of NK T cells against other infectious agents, suggest that these cells might even exhibit adverse effects on vaccine-mediated protective immunity.
Congenital Exposure toPlasmodium falciparumAntigens: Prevalence and Antigenic Specificity of In Utero-Produced Antimalarial Immunoglobulin M Antibodies
Authors: Xi Guoling , Leke Rose G. F. , Thuita Lucy W. , Zhou Ainong , Leke Robert J. I. , Mbu Robinson , Taylor Diane Wallace
Published: February 2003

CongenitalPlasmodium falciparummalaria in newborns is uncommon in sub-Saharan Africa. A significant number of infants, however, become infected or exposed to malarial antigens either in utero or at delivery and have the potential to produce antimalarial antibodies and memory cells before their first natural infection. In Yaounde, Cameroon, parasite-specific immunoglobulin M (IgM) was detected in 14% of cord blood samples. The IgM antibodies reacted with a wide range of asexual-stage antigens, with each newborn having its own unique pattern of IgM reactivity. PCR-based detection and genotyping of cord blood parasites found that the prevalence, total number of parasite genotypes, and complexity of infection were higher in newborns who had produced antimalarial IgM than those who had not. Maternal placental malaria and anemia were associated with the production ofP. falciparum-specific IgM by the fetus. The effect of early immune priming on acquisition of immunity by infants is unknown and merits further investigation, since a significant proportion of Cameroonian newborns developed a humoral response to malaria before birth.
Macrophage Migration Inhibitory Factor Plays a Critical Role in Mediating Protection against the Helminth ParasiteTaenia crassiceps
Authors: Rodríguez-Sosa Miriam , Rosas Lucia E. , David John R. , Bojalil Rafael , Satoskar Abhay R. , Terrazas Luis I.
Published: February 2003

To determine the role of endogenous migration inhibitory factor (MIF) in regulation of immune response during murine cysticercosis caused by the helminth parasiteTaenia crassiceps, we analyzed the course ofT. crassicepsinfection in MIF−/−BALB/c mice. MIF−/−mice were highly susceptible toT. crassicepsand developed significantly higher parasite loads compared to similarly infected MIF+/+mice. Throughout the course of infection,Taenia crassicepssoluble antigen-stimulated spleen cells from both MIF+/+and MIF−/−mice produced significant and comparable levels of interleukin-4 (IL-4), but those from MIF−/−mice produced significantly more IL-13, as well as gamma interferon (IFN-γ), suggesting that the susceptibility of MIF−/−mice toT. crassicepswas not due to the lack of IFN-γ production. Interestingly, low levels of both total and specific immunoglobulin G2a were observed in MIF−/−cysticercotic mice despite the high IFN-γ levels; in addition, peritoneal macrophages obtained fromT. crassiceps-infected MIF−/−mice at different time points failed to respond efficiently to stimulation in vitro with lipopolysaccharide plus IFN-γ and produced significantly lower levels of IL-12, tumor necrosis factor alpha, and NO compared to those from MIF+/+mice. These findings demonstrate that MIF plays a critical role in mediating protection againstT. crassicepsin vivo. Moreover, these findings also suggest that impaired macrophage function rather than the lack of Th1 development may be responsible for mediating susceptibility toT. crassiceps.
Structural Characterization of the N Terminus of IpaC from
Shigella flexneri
Authors: Harrington Amanda T. , Hearn Patricia D. , Picking Wendy L. , Barker Jeffrey R. , Wessel Andrew , Picking William D.
Published: February 2003

The primary effector for
Shigella
invasion of epithelial cells is IpaC, which is secreted via a type III secretion system. We recently reported that the IpaC N terminus is required for type III secretion and possibly other functions. In this study, mutagenesis was used to identify an N-terminal secretion signal and to determine the functional importance of the rest of the IpaC N terminus. The 15 N-terminal amino acids target IpaC for secretion by
Shigella flexneri,
and placing additional amino acids at the N terminus does not interfere with IpaC secretion. Furthermore, amino acid sequences with no relationship to the native IpaC secretion signal can also direct its secretion. Deletions introduced beyond amino acid 20 have no effect on secretion and do not adversely affect IpaC function in vivo until they extend beyond residue 50, at which point invasion function is completely eliminated. Deletions introduced at amino acid 100 and extending toward the N terminus reduce IpaC's invasion function but do not eliminate it until they extend to the N-terminal side of residue 80, indicating that a region from amino acid 50 to 80 is critical for IpaC invasion function. To explore this further, the ability of an IpaC N-terminal peptide to associate in vitro with its translocon partner IpaB and its chaperone IpgC was studied. The N-terminal peptide binds tightly to IpaB, but the IpaC central hydrophobic region also appears to participate in this binding. The N-terminal peptide also associates with the chaperone IpgC and IpaB is competitive for this interaction. Based on additional biophysical data, we propose that a region between amino acids 50 and 80 is required for chaperone binding, and that the IpaB binding domain is located downstream from, and possibly overlapping, this region. From these data, we propose that the secretion signal, chaperone binding region, and IpaB binding domain are located at the IpaC N terminus and are essential for presentation of IpaC to host cells during bacterial entry; however, IpaC effector activity may be located elsewhere.
Phenotypic Switching in
Mycoplasma gallisepticum
Hemadsorption Is Governed by a High-Frequency, Reversible Point Mutation
Authors: Winner Florian , Markovà Ivana , Much Peter , Lugmair Albin , Siebert-Gulle Karin , Vogl Gunther , Rosengarten Renate , Citti Christine
Published: February 2003

Mycoplasma gallisepticum
is a flask-shaped organism that commonly induces chronic respiratory disease in chickens and infectious sinusitis in turkeys. Phenotypic switching in
M. gallisepticum
hemadsorption (HA) was found to correlate with phase variation of the GapA cytadhesin concurrently with that of the CrmA protein, which exhibits cytadhesin-related features and is encoded by a gene located downstream of the
gapA
gene as part of the same transcription unit. In clones derived from strain R
low
, detailed genetic analyses further revealed that on-off switching in GapA expression is governed by a reversible base substitution occurring at the beginning of the
gapA
structural gene. In HA
−
variants, this event generates a stop codon that results in the premature termination of GapA translation and consequently affects the expression of CrmA. Sequences flanking the mutation spot do not feature any repeated motifs that could account for error-prone mutation via DNA slippage and the exact mechanism underlying this high-frequency mutational event remains to be elucidated. An HA
−
mutant deficient in producing CrmA, mHAD3, was obtained by disrupting the
crmA
gene by using transposition mutagenesis. Despite a fully functional
gapA
gene, the amount of GapA detected in this mutant was considerably lower than in HA
+
clonal variants, suggesting that, in absence of CrmA, GapA might be subjected to a higher turnover.
A Surface Amebic Cysteine Proteinase Inactivates Interleukin-18
Authors: Que Xuchu , Kim Soo-Hyun , Sajid Mohammed , Eckmann Lars , Dinarello Charles A. , McKerrow James H. , Reed Sharon L.
Published: February 2003

Amebiasis is a major cause of morbidity and mortality worldwide. Invasion byEntamoeba histolyticatrophozoites causes secretion of proinflammatory cytokines from host epithelial cells, leading to a local acute inflammatory response, followed by lysis of colonic cells. Extracellular cysteine proteinases from amebic trophozoites are key virulence factors and have a number of important interactions with host defenses, including cleavage of immunoglobulin G (IgG), IgA, and complement components C3 and C5. Amebic lysates have also been shown to activate the precursor to interleukin 1-beta (proIL-1β), mimicking the action of caspase-1. IL-18 is also a central cytokine, which induces gamma interferon (IFN-γ) and activates macrophages, one of the main host defenses against invading trophozoites. Because proIL-18 is also activated by caspase-1, we evaluated whether amebic proteinases had a similar effect. Instead, we found that recombinant proIL-18 was cleaved into smaller fragments by the complex of surface-associated and released amebic proteinases. To evaluate the function of an individual proteinase from the complex pool, we expressed an active surface proteinase, EhCP5, which is functional only inE. histolytica.Recombinant EhCP5 expressed inPichia pastorishad kinetic properties similar to those of the native enzyme with respect to substrate specificity and sensitivity to proteinase inhibitors. In contrast to the activation of proIL-1β by amebic lysates, the purified proteinase cleaved proIL-18 and mature IL-18 to biologically inactive fragments. These studies suggest that the acute host response and amebic invasion result from a complex interplay of parasite virulence factors and host defenses.E. histolyticamay block the host inflammatory response by a novel mechanism, inactivation of IL-18.
Intrapulmonary Expression of Macrophage Inflammatory Protein 1α (CCL3) Induces Neutrophil and NK Cell Accumulation and Stimulates Innate Immunity in Murine Bacterial Pneumonia
Authors: Zeng Xianying , Moore Thomas A. , Newstead Michael W. , Hernandez-Alcoceba Ruben , Tsai Wan C. , Standiford Theodore J.
Published: February 2003

Macrophage inflammatory protein 1α (MIP-1α) (CCL3) is an important mediator of leukocyte recruitment and activation in a variety of inflammatory states, including infection. A recombinant human type 5 adenovirus containing the murine MIP-1α cDNA (AdMIP-1α) was constructed to determine the effect of transient intrapulmonary expression of MIP-1α on leukocyte recruitment, activation, and bacterial clearance in a murine model ofKlebsiella pneumoniaepneumonia. The intratracheal administration of AdMIP-1α resulted in both time- and dose-dependent expression of MIP-1α mRNA and protein within the lung. Importantly, the intrapulmonary overexpression of MIP-1α resulted in a maximal 35- and 100-fold reduction in lung and blood bacterial burden, respectively, in animals cochallenged withK. pneumoniae, which was associated with a significant increase in neutrophil and activated NK cell accumulation. Furthermore, the transgenic expression of MIP-1α during bacterial pneumonia resulted in enhanced expression of gamma interferon mRNA, compared to that observed inKlebsiella-challenged animals pretreated with control vector. These findings indicate an important role for MIP-1α in the recruitment and activation of selected leukocyte populations in vivo and identify this cytokine as a potential immunoadjuvant to be employed in the setting of localized bacterial infection.
Reduced Glutathione Is Required for Pertussis Toxin Secretion by
Bordetella pertussis
Authors: Stenson Trevor H. , Patton Angela K. , Weiss Alison A.
Published: February 2003

The abilities of cysteine-containing compounds to support growth of
Bordetella pertussis
and influence pertussis toxin transcription, assembly, and secretion were examined. Cysteine is an essential amino acid for
B. pertussis
and must be present for protein synthesis and bacterial growth. However, cysteine can be metabolized to sulfate, and high concentrations of sulfate can selectively inhibit transcription of the virulence factors, including pertussis toxin, via the BvgAS two-component regulatory system in a process called modulation. In addition, pertussis toxin possesses several disulfide bonds, and the cysteine-containing compound glutathione can influence oxidation-reduction reactions and perhaps disulfide bond formation. Bacterial growth was not observed in the absence of a source of cysteine. Oxidized glutathione, as a sole source of cysteine, also did not support bacterial growth. Cysteine, cystine, and reduced glutathione did support bacterial growth, and none of these compounds caused modulation at the concentrations tested. Similar amounts of periplasmic pertussis toxin were detected regardless of the source of cysteine; however, in the absence of reduced glutathione, pertussis toxin was not efficiently secreted. Addition of the reducing agent dithiothreitol was unable to compensate for the lack of reduced glutathione and did not promote secretion of pertussis toxin. These results suggest that reduced glutathione does not affect the accumulation of assembled active pertussis toxin in the periplasm but plays a role in efficient pertussis toxin secretion by the bacterium.
Genetic Characterization and Immunogenicity of Coli Surface Antigen 4 from Enterotoxigenic
Escherichia coli
when It Is Expressed in a
Shigella
Live-Vector Strain
Authors: Altboum Zeev , Levine Myron M. , Galen James E. , Barry Eileen M.
Published: February 2003

The genes that encode the enterotoxigenic
Escherichia coli
(ETEC) CS4 fimbriae,
csaA
, -
B
, -
C
, -
E
, and -
D
′, were isolated from strain E11881A. The
csa
operon encodes a 17-kDa major fimbrial subunit (CsaB), a 40-kDa tip-associated protein (CsaE), a 27-kDa chaperone-like protein (CsaA), a 97-kDa usher-like protein (CsaC), and a deleted regulatory protein (CsaD′). The predicted amino acid sequences of the CS4 proteins are highly homologous to structural and assembly proteins of other ETEC fimbriae, including CS1 and CS2, and to CFA/I in particular. The
csaA
, -
B
, -
C
, -
E
operon was cloned on a stabilized plasmid downstream from an osomotically regulated
ompC
promoter. pGA2-CS4 directs production of CS4 fimbriae in both
E
. coli DH5α and
Shigella flexneri
2a vaccine strain CVD 1204, as detected by Western blot analysis and bacterial agglutination with anti-CS4 immune sera. Electron-microscopic examination of
Shigella
expressing CS4 confirmed the presence of fimbriae on the bacterial surface. Guinea pigs immunized with CVD 1204(pGA2-CS4) showed serum and mucosal antibody responses to both the
Shigella
vector and the ETEC fimbria CS4. Among the seven most prevalent fimbrial antigens of human ETEC, CS4 is the last to be cloned and sequenced. These findings pave the way for CS4 to be included in multivalent ETEC vaccines, including an attenuated
Shigella
live-vector-based ETEC vaccine.
Isolation of Acid-Inducible Genes ofMycobacterium tuberculosiswith the Use of Recombinase-Based In Vivo Expression Technology
Authors: Saviola Beatrice , Woolwine Samuel C. , Bishai William R.
Published: February 2003

A better understanding of mycobacterial gene regulation under certain stress conditions (e.g., low pH) may provide insight into mechanisms of adaptation during infection. To identify mycobacterial promoters induced at low pH, we adapted the recombinase-based in vivo expression technology (RIVET) promoter trap system for use with mycobacteria. Our results show that the TnpR recombinase of transposon γδ is active inMycobacterium smegmatisandMycobacterium tuberculosis. We developed a method to perform sequential double selection with mycobacteria by using RIVET, with a kanamycin preselection and a sucrose postselection. A library ofM. tuberculosisDNA inserted upstream oftnpRwas created, and using the double selection, we identified two promoters which are upregulated at low pH. The promoter regions drive the expression of a gene encoding a putative lipase,lipF(Rv3487c), as well as a PE-PGRS gene,Rv0834c, in a pH-dependent manner in bothM. smegmatisandM. tuberculosis. The acid inducibility oflipFandRv0834cwas independent of the stress response sigma factor, SigF, as acid induction of the two genes in anM. tuberculosis sigFmutant strain was similar to that in the wild-type strain. No induction oflipForRv0834cwas observed during infection of J774 murine macrophages, an observation which is in agreement with previous reports on the failure of phagosomes containingM. tuberculosisto acidify.
CD40-CD40 Ligand Costimulation Is Not Required for Initiation and Maintenance of a Th1-Type Response toLeishmania majorInfection
Authors: Padigel Udaikumar M. , Farrell Jay P.
Published: February 2003

Although previous studies demonstrated a requirement for CD40-CD40 ligand (CD40L) interaction in the development of resistance toLeishmaniainfection, we recently showed that mice lacking the gene for CD40L (CD40L−/−mice) can controlLeishmania majorinfection when they are infected with reduced numbers of parasites. In this study, we examine the cytokine pattern in healing versus nonhealing CD40L−/−mice and investigated whether CD40 activation is required for resistance to reinfection. We observed that CD4+cells in healed CD40L−/−mice produce high levels of gamma interferon compared to cells from nonhealing, high-dose-inoculated mice. In addition, we observed a higher frequency of interleukin-12 (IL-12)- producing cells and a reduced number of IL-4-producing cells in mice infected with reduced numbers of parasites. Importantly, we found that healed CD40L−/−mice are highly resistant to reinfection with a large parasite inoculum. In addition, by comparing the cytokine patterns at an early and late stage of infection in nonhealing CD40L−/−mice, we demonstrated that nonhealing CD40L−/−mice produce a weak Th1-type response during the early stage of infection, but this response wanes as a Th2-type response emerges during late stages of infection. Anti-IL-4 antibody treatment, starting either at the beginning of infection or at week 4 postinfection enabled CD40L−/−mice to control a high-dose infection. Together, these results show that CD40-CD40L interaction, although important for IL-12 production in high-dose infections, is not required for either the development or maintenance of resistance in mice infected with reduced numbers of parasites.
Geographical Structure of Diversity and Differences between Symptomatic and Asymptomatic Infections for
Plasmodium falciparum
Vaccine Candidate AMA1
Authors: Cortés Alfred , Mellombo Mata , Mueller Ivo , Benet Ariadna , Reeder John C. , Anders Robin F.
Published: February 2003

Plasmodium falciparum
apical membrane antigen 1 (AMA1) is a prime malaria vaccine candidate. Antigenic diversity within parasite populations is one of the main factors potentially limiting the efficacy of any asexual-stage vaccine, including one based on AMA1. The DNA coding for the most variable region of this antigen, domain I, was sequenced in 168 samples from the Wosera region of Papua New Guinea, including samples from symptomatic and asymptomatic infections. Neutrality tests applied to these sequences provided strong evidence of selective pressure operating on the sequence of
ama1
domain I, consistent with AMA1 being a target of protective immunity. Similarly, a peculiar pattern of geographical diversity and the particular substitutions found were suggestive of strong constraints acting on the evolution of AMA1 at the population level, probably as a result of immune pressure. In addition, a strong imbalance between symptomatic and asymptomatic infections was detected in the frequency of particular residues at certain polymorphic positions, pointing to AMA1 as being one of the determinants of the morbidity associated with a particular strain. The information yielded by this study has implications for the design and assessment of AMA1-based vaccines and provides additional data supporting the importance of AMA1 as a malaria vaccine candidate.
CD14- and Toll-Like Receptor-Dependent Activation of Bladder Epithelial Cells by Lipopolysaccharide and Type 1 PiliatedEscherichia coli
Authors: Schilling Joel D. , Martin Steven M. , Hunstad David A. , Patel Kunal P. , Mulvey Matthew A. , Justice Sheryl S. , Lorenz Robin G. , Hultgren Scott J.
Published: February 2003

The gram-negative bacteriumEscherichia coliis the leading cause of urinary tract infection. The interaction between type 1 piliatedE. coliand bladder epithelial cells leads to the rapid production of inflammatory mediators, such as interleukin-6 (IL-6) and IL-8. Conflicting reports have been published in the literature regarding the mechanism by which uroepithelial cells are activated by type 1 piliatedE. coli. In particular, the role of lipopolysaccharide (LPS) in these responses has been an area of significant debate. Much of the data arguing against LPS-mediated activation of bladder epithelial cells have come from studies using a renal epithelial cell line as an in vitro model of the urinary epithelium. In this report, we analyzed three bladder epithelial cell lines and demonstrated that they all respond to LPS. Furthermore, the LPS responsivity of the cell lines directly correlated with their ability to generate IL-6 afterE. colistimulation. The LPS receptor complex utilized by the bladder epithelial cell lines included CD14 and Toll-like receptors, and signaling involved the activation of NF-κB and p38 mitogen-activated protein kinase. Also, reverse transcription-PCR analysis demonstrated that bladder epithelial cells express CD14 mRNA. Thus, the molecular machinery utilized by bladder epithelial cells for the recognition ofE. coliis very similar to that described for traditional innate immune cells, such as macrophages. In contrast, the A498 renal epithelial cell line did not express CD14, was hyporesponsive to LPS stimulation, and demonstrated poor IL-6 responses toE. coli.
Role of the
Brucella suis
Lipopolysaccharide O Antigen in Phagosomal Genesis and in Inhibition of Phagosome-Lysosome Fusion in Murine Macrophages
Authors: Porte Françoise , Naroeni Aroem , Ouahrani-Bettache Safia , Liautard Jean-Pierre
Published: February 2003

Brucella
species are gram-negative, facultative intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment inside professional and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both cell types. However, the molecular mechanisms and the microbial factors involved are poorly understood. Smooth lipopolysaccharide (LPS) of
Brucella
has been reported to be an important virulence factor, although its precise role in pathogenesis is not yet clear. In this study, we show that the LPS O side chain is involved in inhibition of the early fusion between
Brucella suis
-containing phagosomes and lysosomes in murine macrophages. In contrast, the phagosomes containing rough mutants, which fail to express the O antigen, rapidly fuse with lysosomes. In addition, we show that rough mutants do not enter host cells by using lipid rafts, contrary to smooth strains. Thus, we propose that the LPS O chain might be a major factor that governs the early behavior of bacteria inside macrophages.
Lymphoid Follicle-Dense Mucosa at the Terminal Rectum Is the Principal Site of Colonization of Enterohemorrhagic
Escherichia coli
O157:H7 in the Bovine Host
Authors: Naylor Stuart W. , Low J. Christopher , Besser Thomas E. , Mahajan Arvind , Gunn George J. , Pearce Michael C. , McKendrick Iain J. , Smith David G. E. , Gally David L.
Published: February 2003

Escherichia coli
O157:H7 causes bloody diarrhea and potentially fatal systemic sequelae in humans. Cattle are most frequently identified as the primary source of infection, and
E. coli
O157:H7 generally colonizes the gastrointestinal tracts of cattle without causing disease. In this study, persistence and tropism were assessed for four different
E. coli
O157:H7 strains. Experimentally infected calves shed the organism for at least 14 days prior to necropsy. For the majority of these animals, as well as for a naturally colonized animal obtained from a commercial beef farm, the highest numbers of
E. coli
O157:H7 were found in the feces, with negative or significantly lower levels detected in lumen contents taken from the gastrointestinal tract. Detailed examination demonstrated that in these individuals the majority of tissue-associated bacteria were adherent to mucosal epithelium within a defined region extending up to 5 cm proximally from the recto-anal junction. The tissue targeted by
E. coli
O157:H7 was characterized by a high density of lymphoid follicles. Microcolonies of the bacterium were readily detected on the epithelium of this region by immunofluorescence microscopy. As a consequence of this specific distribution,
E. coli
O157:H7 was present predominately on the surface of the fecal stool. In contrast, other
E. coli
serotypes were present at consistent levels throughout the large intestine and were equally distributed in the stool. This is a novel tropism that may enhance dissemination both between animals and from animals to humans. The accessibility of this site may facilitate simple intervention strategies.
Effects of Respiratory
Mycoplasma pneumoniae
Infection on Allergen-Induced Bronchial Hyperresponsiveness and Lung Inflammation in Mice
Authors: Chu Hong Wei , Honour Joyce M. , Rawlinson Catherine A. , Harbeck Ronald J. , Martin Richard J.
Published: February 2003

Airway mycoplasma infection may be associated with asthma pathophysiology. However, the direct effects of mycoplasma infection on asthma remain unknown. Using a murine allergic-asthma model, we evaluated the effects of different timing of airway
Mycoplasma pneumoniae
infection on bronchial hyperresponsiveness (BHR), lung inflammation, and the protein levels of Th1 (gamma interferon [IFN-γ]) and Th2 (interleukin 4 [IL-4]) cytokines in bronchoalveolar lavage fluid. When mycoplasma infection occurred 3 days before allergen (ovalbumin) sensitization and challenge, the infection reduced the BHR and inflammatory-cell influx into the lung. This was accompanied by a significant induction of Th1 responses (increased IFN-γ and decreased IL-4 production). Conversely, when mycoplasma infection occurred 2 days after allergen sensitization and challenge, the infection initially caused a temporary reduction of BHR and then increased BHR, lung inflammation, and IL-4 levels. Our data suggest that mycoplasma infection could modulate both physiological and immunological responses in the murine asthma model. Our animal models may also provide a new means to understand the role of infection in asthma pathogenesis and give evidence for the asthma hygiene hypothesis.
Role ofPLB1in Pulmonary Inflammation and Cryptococcal Eicosanoid Production
Authors: Noverr Mairi C. , Cox Gary M. , Perfect John R. , Huffnagle Gary B.
Published: February 2003

Cryptococcal phospholipase (PLB1) is a secreted enzyme with lysophospholipase hydrolase and lysophospholipase transacylase activities. To investigate the role ofPLB1in the evasion of host immune responses, we characterized pulmonary immune responses to the parental (H99), theplb1mutant, and theplb1recreconstituted mutant strains ofCryptococcus neoformansin mice.PLB1was required for virulence during infection acquired via the respiratory tract. Mice infected with either H99 or theplb1recstrain generated a nonprotective inflammatory response with subsequent eosinophilia, while mice infected with theplb1mutant generated a protective immune response that controlled the infection. BecausePLB1is believed to facilitate virulence through host cell lysis, we examined the interaction of these strains with macrophages. Theplb1recmutant exhibited decreased survival during coculture with macrophages. One factor which may be involved in the survival of yeast in the presence of macrophages is fungal eicosanoid production. Host eicosanoids have been shown to down-modulate macrophage functions.plb1exhibited a defect in eicosanoid production derived from exogenous arachidonoyl-phosphatidylcholine, suggesting thatPLB1is required for the release of arachidonic acid from phospholipids. These data suggest that cryptococcalPLB1may act as a virulence factor by enhancing the ability to survive macrophage antifungal defenses, possibly by facilitating fungal eicosanoid production.
The Zinc-Dependent Major Histocompatibility Complex Class II Binding Site of Streptococcal Pyrogenic Exotoxin C Is Critical for Maximal Superantigen Function and Toxic Activity
Authors: Tripp Timothy J. , McCormick John K. , Webb Jennifer M. , Schlievert Patrick M.
Published: February 2003

The cocrystal structure of streptococcal pyrogenic exotoxin C (SPE C) with HLA-DR2a (DRA*0101,DRB5*0101) revealed a zinc-dependent interaction site through residues 167, 201, and 203 on SPE C and residue 81 on the β-chain of HLA-DR2a (DRA*0101,DRB5*0101). Mutation of these SPE C residues resulted in dramatically reduced biological activities. Thus, the zinc-dependent major histocompatibility complex II binding site is critical for maximal biological function of SPE C.
Role of Interleukin-6 in the Control of Acute and Chronic
Giardia lamblia
Infections in Mice
Authors: Zhou Ping , Li Erqiu , Zhu Nannan , Robertson Jason , Nash Theodore , Singer Steven M.
Published: February 2003

In this study, we investigated the role of interleukin-6 (IL-6) in
Giardia lamblia
infections in mice. Elevated IL-6 expression was found in wild-type mice 15 days postinfection. Furthermore, IL-6-deficient mice controlled infections only slowly although normal immunoglobulin A production was observed. Thus, IL-6 is necessary for early control of acute
G. lamblia
infections.
N-Terminal E-Cadherin Peptides Act as Decoy Receptors for
Listeria monocytogenes
Authors: da Silva Tatley Fernanda , Aldwell Frank E. , Dunbier Anita K. , Guilford Parry J.
Published: February 2003

The observation that E-cadherin is the principal epithelial receptor for the bacterial pathogen
Listeria monocytogenes
led us to investigate whether N-terminal fragments of E-cadherin containing the
L. monocytogenes
binding domain could inhibit entry of the bacteria into cultured epithelial cells. Here we demonstrate that a conditioned medium from a gastric cancer cell line (Kato III) that carries a truncating
CDH-1
mutation 3′ of the
L. monocytogenes
binding domain can inhibit the uptake of the bacteria into Caco-2 cells. The inhibitory activity of the Kato III conditioned medium could be mimicked by incubation of the bacteria with a recombinant 26-kDa N-terminal E-cadherin peptide prior to infection. Furthermore, these data suggest that cleavage of the 80-kDa extracellular domain of E-cadherin from the cell surface may provide an innate form of pathogen defense by acting as a decoy receptor for
L. monocytogenes
.
Purification of Fully ActivatedClostridium botulinumSerotype B Toxin for Treatment of Patients with Dystonia
Authors: Arimitsu Hideyuki , Inoue Kaoru , Sakaguchi Yoshihiko , Lee Jaechul , Fujinaga Yukako , Watanabe Toshihiro , Ohyama Tohru , Hirst Robert , Oguma Keiji
Published: February 2003

Clostridium botulinumserotype B toxins 12S and 16S were separated by using a beta-lactose gel column at pH 6.0; toxin 12S passed through the column, whereas toxin 16S bound to the column and eluted with lactose. The fully activated neurotoxin was obtained by applying the trypsin-treated 16S toxin on the same column at pH 8.0; the neurotoxin passed through the column, whereas remaining nontoxic components bound to the column. The toxicity of this purified fully activated neurotoxin was retained for a long period by addition of albumin in the preparation.
Clostridium difficile
Vaccine and Serum Immunoglobulin G Antibody Response to Toxin A
Authors: Aboudola Samer , Kotloff Karen L. , Kyne Lorraine , Warny Michel , Kelly Eoin C. , Sougioultzis Stavros , Giannasca Paul J. , Monath Thomas P. , Kelly Ciarán P.
Published: February 2003

There is a strong association between serum antibody responses to toxin A and protection against
Clostridium difficile
diarrhea. A parenteral
C. difficile
toxoid vaccine induced very-high-level responses to anti-toxin A immunoglobulin G (IgG) in the sera of healthy volunteers. After vaccination, the concentrations of anti-toxin A IgG in the sera of all 30 recipients exceeded the concentrations that were associated with protection in previous clinical studies. Furthermore, the median concentration of serum anti-toxin A IgG in the test group was 50-fold higher than the previous threshold. These findings support the feasibility of using a vaccine to protect high-risk individuals against
C. difficile
-associated diarrhea and colitis.
A NONLINEAR RICHARDSON ALGORITHM FOR THE SOLUTION OF ELLIPTIC PDE'S
Authors: BERTOLUZZA S. , MAZET S. , VERANI M.
Published: February 2003

We prove convergence of a computable adaptive wavelet algorithm for the solution of elliptic PDE's, which combines Richardson type iterations with nonlinear projection steps.
ANALYSIS AND COMPARISON OF TWO APPROXIMATION SCHEMES FOR A RADIATIVE TRANSFER SYSTEM
Authors: FALCONE MAURIZIO , LÓPEZ-POUSO ÓSCAR
Published: February 2003

This paper analyzes two algorithms based on the S2 approximation for the evolutive, gray, 1D radiative transfer equations in the slab with isotropic scattering. It is known that simple fixed point iteration does not work when the medium is optically thick, and that this difficulty can be overcome by using a Newton method. In this paper we quantify the first observation, giving a criterium to decide a priori whether fixed point converges or not. We also observe that the Newton method can be implemented faster than the fixed point iteration, but this implementation strongly relies on the particular discretization schemes used here. Existence theorems for the discrete nonlinear problem are also proved.
QUALITATIVE ANALYSIS OF A MEAN FIELD MODEL OF TUMOR-IMMUNE SYSTEM COMPETITION
Authors: DE ANGELIS ELENA , JABIN PIERRE-EMMANUEL
Published: February 2003

This paper deals with the qualitative analysis of a model related to the immune response to the evolution of the progression of endothelial cells which have lost their differentiation and start their evolution toward methastatic states. We prove the existence of solutions to the Cauchy problem related to the model. The asymptotic behavior in time of our solutions is also investigated.
UNCONDITIONAL NONLINEAR STABILITY FOR TEMPERATURE-DEPENDENT DENSITY FLOW IN A POROUS MEDIUM
Authors: CARR MAGDA
Published: February 2003

A nonlinear stability analysis of thermal convection in a saturated porous medium is presented. Density is assumed to have a cubic temperature dependence and the equations of flow in the porous medium are described via Darcy's law with a Forchheimer drag term. Unconditional stability is established using L3 and L4 norms and it is shown that L2 theory is insufficient to obtain similar results. Previous authors have established conditional nonlinear stability but we believe this is the first analysis that addresses the important problem of unconditional stability for the system in hand.
ANALYSIS AND APPROXIMATION OF A SCALAR CONSERVATION LAW WITH A FLUX FUNCTION WITH DISCONTINUOUS COEFFICIENTS
Authors: SEGUIN NICOLAS , VOVELLE JULIEN
Published: February 2003

We study here a model of conservative nonlinear conservation law with a flux function with discontinuous coefficients, namely the equation ut + (k(x)u(1 - u))x = 0. It is a particular entropy condition on the line of discontinuity of the coefficient k which ensures the uniqueness of the entropy solution. This condition is discussed and justified. On the other hand, we perform a numerical analysis of the problem. Two finite volume schemes, the Godunov scheme and the VFRoe-ncv scheme, are proposed to simulate the conservation law. They are compared with two finite volume methods classically used in an industrial context. Several tests confirm the good behavior of both new schemes, especially through the discontinuity of permeability k (whereas a loss of accuracy may be detected when industrial methods are performed). Moreover, a modified MUSCL method which accounts for stationary states is introduced.
Oxidative Stress–Induced Iron Signaling Is Responsible for Peroxide-Dependent Oxidation of Dichlorodihydrofluorescein in Endothelial Cells
Authors: Tampo Yoshiko , Kotamraju Srigiridhar , Chitambar Christopher R. , Kalivendi Shasi V. , Keszler Agnes , Joseph Joy , Kalyanaraman B.
Published: February 2003

Dichlorodihydrofluorescein (DCFH) is one of the most frequently used probes for detecting intracellular oxidative stress. In this study, we report that H
2
O
2
-dependent intracellular oxidation of DCFH to a green fluorescent product, 2′,7′-dichlorofluorescein (DCF), required the uptake of extracellular iron transported through a transferrin receptor (TfR) in endothelial cells. H
2
O
2
-induced DCF fluorescence was inhibited by the monoclonal IgA–class anti-TfR antibody (42/6) that blocked TfR endocytosis and the iron uptake. H
2
O
2
-mediated inactivation of cytosolic aconitase was responsible for activation of iron regulatory protein-1 and increased expression of TfR, resulting in an increased iron uptake into endothelial cells. H
2
O
2
-mediated caspase-3 proteolytic activation was inhibited by anti-TfR antibody. Similar results were obtained in the presence of a lipid hydroperoxide. We conclude that hydroperoxide-induced DCFH oxidation and endothelial cell apoptosis required the uptake of extracellular iron by the TfR-dependent iron transport mechanism and that the peroxide-induced iron signaling, in general, has broader implications in oxidative vascular biology.
α-Tocopherol Induces Expression of Connective Tissue Growth Factor and Antagonizes Tumor Necrosis Factor-α–Mediated Downregulation in Human Smooth Muscle Cells
Authors: Villacorta Luis , Graça-Souza Aurélio V. , Ricciarelli Roberta , Zingg Jean-Marc , Azzi Angelo
Published: February 2003

The effect of α-tocopherol treatment on gene expression in human aortic vascular smooth muscle cells was analyzed by gene expression arrays. The expression of the connective tissue growth factor (CTGF) gene was induced by α-tocopherol 1.8-fold in gene array experiments, and similar results were also obtained by RT-PCR (1.7-fold) and at the protein level (1.4-fold). The antioxidants β-tocopherol and
N
-acetylcysteine did not induce CTGF gene expression, suggesting a nonantioxidant mechanism for α-tocopherol action. Protein kinase C (PKC) inhibition by α-tocopherol has been previously described. However, PKC downregulation did not prevent CTGF induction by α-tocopherol, and inhibition of PKC activity with several inhibitors did not increase its expression, suggesting an alternative pathway for the α-tocopherol effect. On the other hand, tumor necrosis factor-α reduced the expression of CTGF, an effect that was reversed by antioxidants. The data suggest that tumor necrosis factor-α inhibition of CTGF gene expression is prevented in an antioxidant-sensitive process and that α-tocopherol increases CTGF expression by a PKC-independent, nonantioxidant mechanism. Because CTGF stimulates the synthesis of extracellular matrix, the normalization of CTGF gene expression by α-tocopherol may accelerate wound repair and tissue regeneration during atherosclerosis.
Critical Role of Cyclin D1 Nuclear Import in Cardiomyocyte Proliferation
Authors: Tamamori-Adachi Mimi , Ito Hiroshi , Sumrejkanchanakij Piyamas , Adachi Susumu , Hiroe Michiaki , Shimizu Masato , Kawauchi Junya , Sunamori Makoto , Marumo Fumiaki , Kitajima Shigetaka , Ikeda Masa-Aki
Published: February 2003

Mammalian cardiomyocytes irreversibly lose their capacity to proliferate soon after birth, yet the underlying mechanisms have been unclear. Cyclin D1 and its partner, cyclin-dependent kinase 4 (CDK4), are important for promoting the G1-to-S phase progression via phosphorylation of the retinoblastoma (Rb) protein. Mitogenic stimulation induces hypertrophic cell growth and upregulates expression of cyclin D1 in postmitotic cardiomyocytes. In the present study, we show that, in neonatal rat cardiomyocytes, D-type cyclins and CDK4 were predominantly cytoplasmic, whereas Rb remained in an underphosphorylated state. Ectopically expressed cyclin D1 localized in the nucleus of fetal but not neonatal cardiomyocytes. To target cyclin D1 to the nucleus efficiently, we constructed a variant of cyclin D1 (D1NLS), which directly linked to nuclear localization signals (NLSs). Coinfection of recombinant adenoviruses expressing D1NLS and CDK4 induced Rb phosphorylation and CDK2 kinase activity. Furthermore, D1NLS/CDK4 was sufficient to promote the reentry into the cell cycle, leading to cell division. The number of cardiomyocytes coinfected with these viruses increased 3-fold 5 days after infection. Finally, D1NLS/CDK4 promoted cell cycle reentry of cardiomyocytes in adult hearts injected with these viruses, evaluated by the expression of Ki-67, which is expressed in proliferating cells in all phases of the cell cycle, and BrdU incorporation. Thus, postmitotic cardiomyocytes have the potential to proliferate provided that cyclin D1/CDK4 accumulate in the nucleus, and the prevention of their nuclear import plays a critical role as a physical barrier to prevent cardiomyocyte proliferation. Our results provide new insights into the development of therapeutics strategies to induce regeneration of cardiomyocytes. The full text of this article is available at http://www.circresaha.org.
Long-Term Vitamin C Treatment Increases Vascular Tetrahydrobiopterin Levels and Nitric Oxide Synthase Activity
Authors: d’Uscio Livius V. , Milstien Sheldon , Richardson Darcy , Smith Leslie , Katusic Zvonimir S.
Published: February 2003

In cultured endothelial cells, the antioxidant,
l
-ascorbic acid (vitamin C), increases nitric oxide synthase (NOS) enzyme activity via chemical stabilization of tetrahydrobiopterin. Our objective was to determine the effect of vitamin C on NOS function and tetrahydrobiopterin metabolism in vivo. Twenty-six to twenty-eight weeks of diet supplementation with vitamin C (1%/kg chow) significantly increased circulating levels of vitamin C in wild-type (C57BL/6J) and apolipoprotein E (apoE)–deficient mice. Measurements of NOS enzymatic activity in aortas of apoE-deficient mice indicated a significant increase in total NOS activity. However, this increase was mainly due to high activity of inducible NOS, whereas eNOS activity was reduced. Significantly higher tetrahydrobiopterin levels were detected in aortas of apoE-deficient mice. Long-term treatment with vitamin C restored endothelial NOS activity in aortas of apoE-deficient mice, but did not affect activity of inducible NOS. In addition, 7,8-dihydrobiopterin levels, an oxidized form of tetrahydrobiopterin, were decreased and vascular endothelial function of aortas was significantly improved in apoE-deficient mice. Interestingly, vitamin C also increased tetrahydrobiopterin and NOS activity in aortas of C57BL/6J mice. In contrast, long-term treatment with vitamin E (2000 U/kg chow) did not affect vascular NOS activity or metabolism of tetrahydrobiopterin. In vivo, beneficial effect of vitamin C on vascular endothelial function appears to be mediated in part by protection of tetrahydrobiopterin and restoration of eNOS enzymatic activity.
The Homeobox Gene Lbx1 Specifies a Subpopulation of Cardiac Neural Crest Necessary for Normal Heart Development
Authors: Schäfer Konstanze , Neuhaus Petra , Kruse Julia , Braun Thomas
Published: February 2003

Cardiac neural crest cells are known to play multiple roles during development of the inflow and outflow tract of the heart and the aortic arch. In addition, cardiac neural crest is required for normal heart tube looping and regulation of myocardial cell proliferation, as well as differentiation and function of the myocardium. We show that the homeobox gene Lbx1 is expressed in a subpopulation of the cardiac neural crest during tubular heart formation. Inactivation of the Lbx1 gene in mice resulted in defects in heart looping, changes in gene expression pattern, and increased cell proliferation ensuing in myocardial hyperplasia. We found that the activity of the Lbx1 promoter, as indicated by a LacZ reporter gene, is upregulated in the hearts of Lbx1
+/−
:splotch
1H
/splotch
1H
and Lbx1
−/−
mice, indicating that Pax3 and Lbx1 participate in a negative regulatory feedback that might be necessary for normal differentiation and function of the myocardium during early heart development. Because migration of Lbx1-expressing neural crest cells was not altered in Lbx1
−/−
embryos, we postulate that Lbx1 gene function is critical for specification of a subpopulation of cardiac neural crest subsequent to migration.