bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of p38 Mitogen-Activated Protein Kinase Augments Lipopolysaccharide-Induced Cell Proliferation in CD14-Expressing Chinese Hamster Ovary Cells
Authors: Chakravortty Dipshikha , Kato Yutaka , Sugiyama Tsuyoshi , Koide Naoki , Mu Mya Mya , Yoshida Tomoaki , Yokochi Takashi 
Published: July 2002

ABSTRACTCD14-expressing Chinese hamster ovary (CD14-CHO) cells, established by transfection of human CD14 DNA, acquired high responsiveness to lipopolysaccharide (LPS) through membrane-bound CD14 expression. LPS induced DNA synthesis and activated a series of mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase 1/2 (Erk1/2), p38, and c-Jun N-terminal kinase/stress-activated protein kinase, in CD14-CHO cells but not in mock-transfected CHO cells. Anti-CD14 antibody completely abrogated both LPS-induced DNA synthesis and LPS-induced phosphorylation of those MAP kinases, suggesting a critical role of membrane-bound CD14 in LPS signaling. A p38 MAP kinase inhibitor, SB203580, markedly augmented LPS-induced DNA synthesis in CD14-CHO cells, whereas an Erk1/2 inhibitor, PD98059, had no affect. On the other hand, SB203580 exhibited no effect on epidermal growth factor-induced DNA synthesis in CD14-CHO cells, although PD98059 inhibited it significantly. The activation and inactivation of p38 MAP kinase with dominant negative and dominant positive mutants also suggested the participation of p38 MAP kinase in LPS-induced DNA synthesis. It was therefore suggested that the activation of p38 MAP kinase can negatively regulate LPS-induced cell proliferation in CD14-CHO cells.
Human Neutrophils and Their Products Induce Shiga Toxin Production by Enterohemorrhagic
Escherichia coli
Authors: Wagner Patrick L. , Acheson David W. K. , Waldor Matthew K. 
Published: July 2002

ABSTRACT

The Shiga toxins (Stx) are critical virulence factors for
Escherichia coli
O157:H7 and other serotypes of enterohemorrhagic
E. coli
(EHEC). These potent toxins are encoded in the genomes of temperate lambdoid bacteriophages. We recently demonstrated that induction of the resident Stx2-encoding prophage in an O157:H7 clinical isolate is required for toxin production by this strain. Since several factors produced by human cells, including hydrogen peroxide (H
2
O
2
), are capable of inducing lambdoid prophages, we hypothesized that such molecules might also induce toxin production by EHEC. Here, we studied whether H
2
O
2
and also human neutrophils, an important endogenous source of H
2
O
2
, induced Stx2 expression by an EHEC clinical isolate. Both H
2
O
2
and neutrophils were found to augment Stx2 production, raising the possibility that these agents may lead to prophage induction in vivo and thereby contribute to EHEC pathogenesis.
Flow Cytometric Determination of Panton-Valentine Leucocidin S Component Binding
Authors: Gauduchon Valérie , Werner Sandra , Prévost Gilles , Monteil Henri , Colin Didier A. 
Published: July 2002

ABSTRACT

The binding of the S component (LukS-PV) from the bicomponent staphylococcal Panton-Valentine leucocidin to human polymorphonuclear neutrophils (PMNs) and monocytes was determined using flow cytometry and a single-cysteine substitution mutant of LukS-PV. The mutant was engineered by replacing a glycine at position 10 with a cysteine and was labeled with a fluorescein moiety. The biological activity of the mutant was identical to that of the native protein. It has been shown that LukS-PV has a high affinity for PMNs (
K

d

= 0.07 ± 0.02 nM,
n
= 5) and monocytes (

K
d

= 0.020 ± 0.003 nM,
n
= 3) with maximal binding capacities of 197,000 and 80,000 LukS-PV molecules per cell, respectively. The nonspecifically bound molecules of LukS-PV do not form pores in the presence of the F component (LukF-PV) of leucocidin. LukS-PV and HlgC share the same receptor on PMNs, but the S components of other staphylococcal leukotoxins, HlgA, LukE, and LukM, do not compete with LukS-PV for its receptor. Extracellular Ca
2+
at physiological concentrations (1 to 2 nM) has only a slight influence on the LukS-PV binding, in contrast to its complete inhibition by Zn
2+
. The down-regulation by phorbol 12-myristate 13-acetate (PMA) of the binding of LukS-PV was blocked by staurosporine, suggesting that the regulatory effect of PMA depends on protein kinase C activation. The labeled mutant form of LukS-PV has proved very useful for detailed binding studies of circulating white cells by flow cytometry. LukS-PV possesses a high specific affinity for a unique receptor on PMNs and monocytes.
Knockout of the Rodent Malaria Parasite Chitinase PbCHT1 Reduces Infectivity to Mosquitoes
Authors: Dessens Johannes T. , Mendoza Jacqui , Claudianos Charles , Vinetz Joseph M. , Khater Emad , Hassard Stuart , Ranawaka Gaya R. , Sinden Robert E. 
Published: July 2002

ABSTRACT

During mosquito transmission, malaria ookinetes must cross a chitin-containing structure known as the peritrophic matrix (PM), which surrounds the infected blood meal in the mosquito midgut. In turn, ookinetes produce multiple chitinase activities presumably aimed at disrupting this physical barrier to allow ookinete invasion of the midgut epithelium.
Plasmodium
chitinase activities are demonstrated targets for human and avian malaria transmission blockade with the chitinase inhibitor allosamidin. Here, we identify and characterize the first chitinase gene of a rodent malaria parasite,
Plasmodium berghei
. We show that the gene, named
PbCHT1
, is a structural ortholog of
PgCHT1
of the avian malaria parasite
Plasmodium gallinaceum
and a paralog of
PfCHT1
of the human malaria parasite
Plasmodium falciparum
. Targeted disruption of
PbCHT1
reduced parasite infectivity in
Anopheles stephensi
mosquitoes by up to 90%. Reductions in infectivity were also observed in ookinete feeds—an artificial situation where midgut invasion occurs before PM formation—suggesting that PbCHT1 plays a role other than PM disruption. PbCHT1 null mutants had no residual ookinete-derived chitinase activity in vitro, suggesting that
P. berghei
ookinetes express only one chitinase gene. Moreover, PbCHT1 activity appeared insensitive to allosamidin inhibition, an observation that raises questions about the use of allosamidin and components like it as potential malaria transmission-blocking drugs. Taken together, these findings suggest a fundamental divergence among rodent, avian, and human malaria parasite chitinases, with implications for the evolution of
Plasmodium
-mosquito interactions.
Coiled-Coil Domain of Enteropathogenic
Escherichia coli
Type III Secreted Protein EspD Is Involved in EspA Filament-Mediated Cell Attachment and Hemolysis
Authors: Daniell Sarah J. , Delahay Robin M. , Shaw Robert K. , Hartland Elizabeth L. , Pallen Mark J. , Booy Frank , Ebel Frank , Knutton Stuart , Frankel Gad 
Published: July 2002

ABSTRACT

Many animal and plant pathogens use type III secretion systems to secrete key virulence factors, some directly into the host cell cytosol. However, the basis for such protein translocation has yet to be fully elucidated for any type III secretion system. We have previously shown that in enteropathogenic and enterohemorrhagic
Escherichia coli
the type III secreted protein EspA is assembled into a filamentous organelle that attaches the bacterium to the plasma membrane of the host cell. Formation of EspA filaments is dependent on expression of another type III secreted protein, EspD. The carboxy terminus of EspD, a protein involved in formation of the translocation pore in the host cell membrane, is predicted to adopt a coiled-coil conformation with 99% probability. Here, we demonstrate EspD-EspD protein interaction using the yeast two-hybrid system and column overlays. Nonconservative triple amino acid substitutions of specific EspD carboxy-terminal residues generated an enteropathogenic
E. coli
mutant that was attenuated in its ability to induce attaching and effacing lesions on HEp-2 cells. Although the mutation had no effect on EspA filament biosynthesis, it also resulted in reduced binding to and reduced hemolysis of red blood cells. These results segregate, for the first time, functional domains of EspD that control EspA filament length from EspD-mediated cell attachment and pore formation.
Nitric Oxide Is Protective in Listeric Meningoencephalitis of Rats
Authors: Remer K. A. , Jungi T. W. , Fatzer R. , Täuber M. G. , Leib S. L. 
Published: July 2002

ABSTRACTThe bacteriumListeria monocytogenescauses meningoencephalitis in humans. In rodents, listeriosis is associated with granulomatous lesions in the liver and the spleen, but not with meningoencephalitis. Here, infant rats were infected intracisternally to generate experimental listeric meningoencephalitis. Dose-dependent effects of intracisternal inoculation withL. monocytogeneson survival and activity were noted; 104L. monocytogenesorganisms induced a self-limiting brain infection. Bacteria invaded the basal meninges, chorioid plexus and ependyme, spread to subependymal tissue and hippocampus, and disappeared by day 7. This was paralleled by recruitment and subsequent disappearance of macrophages expressing inducible nitric oxide synthase (iNOS) and nitrotyrosine accumulation, an indication of nitric oxide (NO⋅) production. Treatment with the spin-trapping agent α-phenyl-tert-butyl nitrone (PBN) dramatically increased mortality and led to bacterial numbers in the brain 2 orders of magnitude higher than in control animals. Treatment with the selective iNOS inhibitorl-N6-(1-iminoethyl)-lysine (L-NIL) increased mortality to a similar extent and led to 1 order of magnitude higher bacterial counts in the brain, compared with controls. The numbers of bacteria that spread to the spleen and liver did not significantly differ among L-NIL-treated, PBN-treated, and control animals. Thus, the infant rat brain is able to mobilize powerful antilisterial mechanisms, and both reactive oxygen and NO⋅contribute toListeriagrowth control.
Down-Regulated Lymphoproliferation Coincides with Parasite Maturation and with the Collapse of Both Gamma Interferon and Interleukin-4 Responses in a Bovine Model of Onchocerciasis
Authors: Graham Simon P. , Trees Alexander J. , Collins Robert A. , Moore Davina M. , Guy Francis M. , Taylor Mark J. , Bianco Albert E. 
Published: July 2002

ABSTRACTOnchocerciasis is a debilitating parasitic infection caused by the filarial nematodeOnchocerca volvulus. Infections are chronic, and persistence of the parasites for several years argues for highly adapted mechanisms of immune evasion. Due to the restricted host repertoire ofO. volvulus, we have used the cattle parasiteOnchocerca ochengito investigate the nature of immunomodulation underpinning these long-term infections. Cattle were infected with a single inoculation of 350 infective-stage larvae under laboratory conditions (n= 6). Intradermal nodules containing immature adult worms were detected from 110 days postinfection, and microfilariae in skin were detected from day 280 postinfection. Parasite-specific responses during early infection were nonpolarized with respect to the major Th cytokines (interleukin-4 [IL-4], IL-2, and gamma interferon [IFN-γ]) produced by antigen-stimulated peripheral blood mononuclear cells (PBMC) or serum antibody isotypes. Antigen-induced proliferation of PBMC peaked shortly after exposure and remained high during the prepatent infection. As the parasites matured and animals developed patent infections, there was a profound down-regulation of lymphoproliferation, accompanied by sharp falls in the expression of both IL-4 and IFN-γ and a gradual decline in IL-2. Levels of immunoglobulin G2 (IgG2) fell, while those of IgG1 remained high. We conclude that neither a classical Th2 response nor a simple Th1-to-Th2 switch is sufficient to explain the immunomodulation associated with patentOnchocercainfections. Instead, there is an initial Th0 response, which matures into a response with some, but not all of the features of a Th2 response. The natural host-parasite relationship ofO. ochengiin cattle may be useful as both a descriptive and predictive tool to test more refined models of immunomodulation in onchocerciasis.
Haemophilus ducreyi
Inhibits Phagocytosis by U-937 Cells, a Human Macrophage-Like Cell Line
Authors: Wood Gwendolyn E. , Dutro Susan M. , Totten Patricia A. 
Published: July 2002

ABSTRACT

Haemophilus ducreyi
is a gram-negative obligate human pathogen that causes the genital ulcer disease chancroid. Chancroid lesions are deep necrotic ulcers with an immune cell infiltrate that includes macrophages. Despite the presence of these phagocytic cells, chancroid ulcers can persist for months and live
H. ducreyi
can be isolated from these lesions. To analyze the interaction of
H. ducreyi
with macrophages, we investigated the ability of
H. ducreyi
strain 35000 to adhere to, invade, and survive within U-937 cells, a human macrophage-like cell line. We found that although
H. ducreyi
strain 35000 adhered efficiently to U-937 cells, few bacteria were internalized, suggesting that
H. ducreyi
avoids phagocytosis by human macrophages. The few bacteria that were phagocytosed in these experiments were rapidly killed. We also found that
H. ducreyi
inhibits the phagocytosis of a secondary target (opsonized sheep red blood cells). Antiphagocytic activity was found in logarithmic, stationary-phase, and plate-grown cultures and was associated with whole, live bacteria but not with heat-killed cultures, sonicates, or culture supernatants. Phagocytosis was significantly inhibited after a 15-min exposure to
H. ducreyi
, and a multiplicity of infection of approximately 1 CFU per macrophage was sufficient to cause a significant reduction in phagocytosis by U-937 cells. Finally, all of nine
H. ducreyi
strains tested were antiphagocytic, suggesting that this is a common virulence mechanism for this organism. This finding suggests a mechanism by which
H. ducreyi
avoids killing and clearance by macrophages in chancroid lesions and inguinal lymph nodes.
Chlamydia pneumoniae
Expresses Genes Required for DNA Replication but Not Cytokinesis during Persistent Infection of HEp-2 Cells
Authors: Byrne Gerald I. , Ouellette Scot P. , Wang Zhao , Rao J. P. , Lu Lin , Beatty Wandy L. , Hudson Alan P. 
Published: July 2002

ABSTRACT

Chlamydia pneumoniae
causes community-acquired pneumonia and is associated with several chronic diseases, including asthma and atherosclerosis. The intracellular growth rate of
C. pneumoniae
slows dramatically during chronic infection, and such persistence leads to attenuated production of new elementary bodies, appearance of morphologically aberrant reticulate bodies, and altered expression of several chlamydial genes. We used an in vitro system to further characterize persistent
C. pneumoniae
infection, employing both ultrastructural and transcriptional activity measurements. HEp-2 cells were infected with
C. pneumoniae
(TW-183) at a multiplicity of infection of 3:1, and at 2 h postinfection gamma interferon (IFN-γ) was added to the medium at 0.15 or 0.50 ng/ml. Treated and untreated cultures were harvested at several times postinfection. RNA was isolated and reverse transcribed, and reverse transcription (RT)-PCR analyses targeting primary transcripts from chlamydial rRNA operons as well as
dnaA
,
polA
,
mutS
,
minD
,
ftsK
, and
ftsW
mRNA were done. Some cultures were fixed and stained for electron microscopic analysis, and a real-time PCR assay was used to assess relative chlamydial chromosome accumulation under each culture condition. The latter assays showed that bacterial chromosome copies accumulated severalfold during IFN-γ treatment of infected HEp-2 cells, although less accumulation was observed in cells treated with the higher dose. Electron microscopy demonstrated that high-dose IFN-γ treatment elicited aberrant forms of the bacterium. RT-PCR showed that chlamydial primary rRNA transcripts were present in all IFN-γ-treated and untreated cell cultures, indicating bacterial metabolic activity. Transcripts from
dnaA
,
polA
,
mutS
, and
minD
, all of which encode products for bacterial chromosome replication and partition, were expressed in IFN-γ-treated and untreated cells. In contrast,
ftsK
and
ftsW
, encoding products for bacterial cell division, were expressed in untreated cells, but expression was attenuated in cells treated with low-dose IFN-γ and absent in cells given the high dose of cytokine. Thus, the development of persistence included production of transcripts for DNA replication-related, but not cell division-related, genes. These results provide new insight regarding molecular activities that accompany persistence of
C. pneumoniae
, as well as suggesting requirements for reactivation from persistent to productive growth.
Strain-Dependent Differences in the Regulatory Roles of
sarA
and
agr
in
Staphylococcus aureus
Authors: Blevins Jon S. , Beenken Karen E. , Elasri Mohamed O. , Hurlburt Barry K. , Smeltzer Mark S. 
Published: July 2002

ABSTRACT

The accessory gene regulator (
agr
) and the staphylococcal accessory regulator (
sar
) are central regulatory elements that control the production of
Staphylococcus aureus
virulence factors. To date, the functions of these loci have been defined almost exclusively using RN6390, which is representative of the laboratory strain 8325-4. However, RN6390 was recently shown to have a mutation in
rsbU
that results in a phenotype resembling that of a
sigB
mutant (I. Kullik et al., J. Bacteriol. 180:4814–4820, 1998). For that reason, it remains unclear whether the regulatory events defined in RN6390 are representative of the events that take place in clinical isolates of
S. aureus
. To address this issue, we generated mutations in the
sarA
and
agr
loci of three laboratory strains (RN6390, Newman, and S6C) and four clinical isolates (UAMS-1, UAMS-601, DB, and SC-1). Mutation of
sarA
in the
cna
-positive strains UAMS-1 and UAMS-601 resulted in an increased capacity to bind collagen, while mutation of
agr
had little impact. Northern blot analysis confirmed that the increase in collagen binding was due to increased
cna
transcription. Without exception, mutation of
sarA
resulted in increased production of proteases and a decreased capacity to bind fibronectin. Mutation of
agr
had the opposite effect. Although mutation of
sarA
resulted in a slight reduction in
fnbA
transcription, changes in the ability to bind fibronectin appeared to be more directly correlated with changes in protease activity. Lipase production was reduced in both
sarA
and
agr
mutants. While mutation of
sarA
in RN6390 resulted in reduced hemolytic activity, it had the opposite effect in all other strains. There appeared to be reduced levels of the
sarC
transcript in RN6390, but there was no difference in the overall pattern of
sar
transcription or the production of SarA. Although mutation of
sarA
resulted in decreased RNAIII transcription, this effect was not evident under all growth conditions. Taken together, these results suggest that studies defining the regulatory roles of
sarA
and
agr
by using RN6390 are not always representative of the events that occur in clinical isolates of
S. aureus
.
Increased Susceptibility of C1q-Deficient Mice toSalmonella entericaSerovar Typhimurium Infection
Authors: Warren Joanna , Mastroeni Pietro , Dougan Gordon , Noursadeghi Mahdad , Cohen Jonathan , Walport Mark J. , Botto Marina 
Published: July 2002

ABSTRACTThe role of the complement system in host defense againstSalmonellainfection is poorly defined. Bacterial cell wall O-antigen polysaccharide can activate the alternative pathway in vitro. No studies, however, have elucidated the role of the classical pathway in immunity toSalmonellaspp. in vivo. C1q-deficient mice (C1qa−/−) on a 129/Sv genetic background and strain-matched controls were infected intraperitoneally and intravenously withSalmonella entericaserovar Typhimurium and monitored over a 14-day period. After inoculation by either route, theC1qa−/−mice were found to be significantly more susceptible toSalmonellainfection. Hepatic and splenic bacterial counts, performed at various time points, showed increased numbers of colonies in complement-deficient mice compared to controls. Analysis of blood clearance showed no difference between the two experimental groups during the first 15 min. However, after 20 min and until 6 h postinfection, numbers of circulating bacteria were significantly higher in complement-deficient mice. In vitro experiments using either resident or thioglycolate-elicited peritoneal macrophages showed a significant increase in the number of bacteria inside C1q-deficient macrophages compared to controls irrespective of the serum used for opsonizing the bacteria. These findings could not be explained either by an increased bacterial uptake, analyzed in vitro and in vivo using green fluorescent protein-tagged salmonellae, or by a defect in the respiratory burst or in NO production. The data presented here suggest the possibility of novel pathways by which C1q may modulate the pathogenesis of infectious diseases caused by intracellular pathogens.
Investigation of Role of Nitric Oxide in Protection fromBordetella pertussisRespiratory Challenge
Authors: Canthaboo C. , Xing D. , Wei X. Q. , Corbel M. J. 
Published: July 2002

ABSTRACTThe mechanism whereby whole-cell pertussis vaccines (WCV) confer protection againstBordetella pertussisis still not fully understood. We have previously reported that macrophage activation produced by vaccination with WCV is associated with induction of NO synthesis by macrophages in response to in vitro stimulation withB. pertussisantigens. To determine whether NO production is an effector of protection or simply a marker of activation, the susceptibility of inducible nitric oxide synthase (type II, iNOS) knockout mice to infection withB. pertussiswas examined. We showed that iNOS knockout mice were more susceptible toB. pertussisrespiratory challenge than wild-type mice. iNOS-deficient mice also developed a less effective protective response than wild-type mice after the same immunization with WCV. This suggests that NO plays an important role in effecting protection againstB. pertussischallenge.
Onchocerca volvulusGlycolytic Enzyme Fructose-1,6-Bisphosphate Aldolase as a Target for a Protective Immune Response in Humans
Authors: McCarthy James S. , Wieseman Moira , Tropea Joe , Kaslow David , Abraham David , Lustigman Sara , Tuan Rocky , Guderian Ronald H. , Nutman Thomas B. 
Published: July 2002

ABSTRACTTo identify potential vaccine candidates for the prevention of infection with the filarial nematodeOnchocerca volvulus, we screened anO. volvulusL3 stage cDNA library with sera from putatively immune (PI) subjects, and a prominent immunogenic clone of 1,184 nucleotides was identified. It contained an open reading frame of 363 amino acids encoding the glycolytic enzyme fructose 1,6 bisphosphate aldolase (Ov-fba-1). Immunolocalization experiments demonstrated that the protein was most abundantly expressed in metabolically active tissues, including body wall muscle and the reproductive tract of adult female worms. Immunoelectron microscopy of L3 demonstrated binding in the region where the cuticle separates during molting, in the channels connecting the esophagus to the cuticle, and in the basal lamina surrounding the esophagus and the body cavity. Among subjects from areas where this organism is endemic specific humoral and cellular immune responses to recombinant protein were observed in both PI and infected subjects, whereas responses were not observed among subjects who had not been exposed toO. volvulus. Despite the absence of differential responsiveness in parasite-exposed human populations, when the recombinant was tested for protective efficacy in a mouse chamber model, a reduction in survival of larvae by ca. 50% was seen. This observation provides support for the further study of this parasite enzyme as a vaccine candidate in larger animal models.