bluuebunny/crossref_metadata_2025_split huggingface dataset:
Live
Lactobacillus reuteri
Is Essential for the Inhibitory Effect on Tumor Necrosis Factor Alpha-Induced Interleukin-8 Expression
Authors: Ma Donglai , Forsythe Paul , Bienenstock John 
Published: August 2004

ABSTRACT

The mechanism of the apparent anti-inflammatory action of probiotic organisms is unclear.
Lactobacillus reuteri
is effective in inhibiting colitis in interleukin-10 (IL-10)-deficient mice. Nerve growth factor (NGF), in addition to its activity on neuronal cell growth, has significant anti-inflammatory effects in several experimental systems in vitro and in vivo, including a model of colitis. Our experiments were designed to explore the mechanism of effect of
L. reuteri
in the human epithelial cell lines T84 and HT29 on cytokine and NGF synthesis and IL-8 response to tumor necrosis factor alpha (TNF-α). Epithelial cells were cultured for various times with live and killed
L. reuteri
and examined by reverse transcription-PCR for NGF, IL-10, and TNF-α-induced IL-8 expression. An enzyme-linked immunosorbent assay was used to quantitate intracellular IL-8 and secreted product. Western blotting and confocal microscopy were used to determine the effects on IκB and NF-κB, respectively. Live but not heat-killed or gamma-irradiated
L. reuteri
upregulated NGF and dose dependently inhibited constitutive synthesis by T84 and HT29 cells of IL-8 and that induced by TNF-α in terms of mRNA and intracellular and secreted protein. Similarly,
L. reuteri
inhibited IL-8 synthesis induced by
Salmonella enterica
serovar Typhimurium.
L. reuteri
required preincubation and adherence for effect, inhibited translocation of NF-κB to the nuclei of HeLa cells, and prevented degradation of IκB. Neither cellular lysates nor media supernatants had any effect on TNF-α-induced IL-8. The conclusion is that
L. reuteri
has potent direct anti-inflammatory activity on human epithelial cells, which is likely to be related to the activity of ingested probiotics.
L. reuteri
also upregulates an unusual anti-inflammatory molecule, NGF, and inhibits NF-κB translocation to the nucleus.
Identification ofMycobacterium tuberculosisCounterimmune (cim) Mutants in Immunodeficient Mice by Differential Screening
Authors: Hisert Katherine B. , Kirksey Meghan A. , Gomez James E. , Sousa Alexandra O. , Cox Jeffery S. , Jacobs William R. , Nathan Carl F. , McKinney John D. 
Published: August 2004

ABSTRACTTuberculosis (TB) is characterized by lifetime persistence ofMycobacterium tuberculosis. Despite the induction of a vigorous host immune response that curtails disease progression in the majority of cases, the organism is not eliminated. Subsequent immunosuppression can lead to reactivation after a prolonged period of clinical latency. Thus, while it is clear that protective immune mechanisms are engaged duringM. tuberculosisinfection, it also appears that the pathogen has evolved effective countermechanisms. Genetic studies with animal infection models and with patients have revealed a key role for the cytokine gamma interferon (IFN-γ) in resistance to TB. IFN-γ activates a large number of antimicrobial pathways. Three of these IFN-γ-dependent mechanisms have been implicated in defense againstM. tuberculosis: inducible nitric oxide synthase (iNOS), phagosome oxidase (phox), and the phagosome-associated GTPase LRG-47. In order to identify bacterial genes that provide protection against specific host immune pathways, we have developed the strategy of differential signature-tagged transposon mutagenesis. Using this approach we have identified threeM. tuberculosisgenes that are essential for progressiveM. tuberculosisgrowth and rapid lethality in iNOS-deficient mice but not in IFN-γ-deficient mice. We propose that these genes are involved in pathways that allowM. tuberculosisto counter IFN-γ-dependent immune mechanisms other than iNOS.
EncapsulatedStreptococcus suisInhibits Activation of Signaling Pathways Involved in Phagocytosis
Authors: Segura Mariela , Gottschalk Marcelo , Olivier Martin 
Published: August 2004

ABSTRACTStreptococcus suiscapsular type 2 is an important zoonotic agent of meningitis. Previous studies reported that, in contrast to nonencapsulated mutants, encapsulatedS. suisis able to resist phagocytosis. However, the mechanisms by whichS. suisavoids phagocytosis are unknown. To elucidate the signaling pathway(s) involved inS. suisantiphagocytosis, we compared the ability of an encapsulated strain and its nonencapsulated mutant to induce the activation of Akt and protein kinase C (PKC), which are downstream kinases of the phosphatidylinositol 3-kinase (PI-3K) pathway, known to be involved in the phagocytosis processes. The results demonstrated high levels of Akt and PKCα phosphorylation after infection of J774 macrophages with the nonencapsulated mutant, whereas the encapsulated strain showed reduced activation of PI-3K/Akt/PKCα signaling pathway, as well as several protein tyrosine events. These results correlated with the number of intracellular bacteria. Macrophages pretreated with specific PI-3K or PKC inhibitors showed reduced levels of Akt and PKCα phosphorylation, resulting in 50% reduction of phagocytosis. The role of phosphatases in the antiphagocytic mechanisms was evaluated by using phosphatase inhibitors, as well as SHP-1-deficient macrophages. Only in the absence of SHP-1 did the phagocytosis of encapsulatedS. suissignificantly increase, leading to Akt phosphorylation levels similar to those observed with the nonencapsulated strain, indicating activation of this important SH2 domain-containing tyrosine phosphatase by encapsulatedS. suis. Finally, when purifiedS. suiscapsular polysaccharide (CPS) was added to macrophages, no phosphorylation events were observed. In addition, CPS and encapsulatedS. suiswere able to inhibit the uptake of the nonencapsulated mutant. These results suggest the importance of CPS in the mechanisms, wherebyS. suisdownmodulates phagocytosis.
Dendritic Cells Induce Immunity and Long-Lasting Protection against Blood-Stage Malaria despite an In Vitro Parasite-Induced Maturation Defect
Authors: Pouniotis Dodie S. , Proudfoot Owen , Bogdanoska Violeta , Apostolopoulos Vasso , Fifis Theodora , Plebanski Magdalena 
Published: August 2004

ABSTRACTDendritic cells (DC) suffer a maturation defect following interaction with erythrocytes infected with malaria parasites and become unable to induce protective malaria liver-stage immunity. Here we show that, by contrast, maturation-arrested DC in vitro are capable of the successful induction of antigen-specific gamma interferon (IFN-γ) and interleukin 4 (IL-4) T-cell responses, antibody responses, and potent protection against lethal blood-stage malaria challenge in vivo. Similar results were found with DC pulsed with intact parasitizedPlasmodium yoeliiorPlasmodium chabaudierythrocytes. Cross-strain protection was also induced. High levels of protection (80 to 100%) against lethal challenge were evident from 10 days after a single immunization and maintained up to 120 days. Interestingly, correlation studies versus blood-stage protection at different time points suggest that the immune effector mechanisms associated with protection could change over time. Antibody-independent, T-cell- and IL-12-associated protection was observed early after immunization, followed by antibody and IL-4-associated, IFN-γ-independent protection in long-term studies. These results indicate that DC, even when clearly susceptible to parasite-induced maturation defect effects in vitro, can be central to the induction of protection against blood-stage malaria in vivo.
Novel Modification of Lipid A of
Francisella tularensis
Authors: Phillips Nancy J. , Schilling Birgit , McLendon Molly K. , Apicella Michael A. , Gibson Bradford W. 
Published: August 2004

ABSTRACT

We have investigated the lipid A of
Francisella tularensis
subsp.
holarctica
strain 1547-57, a type B strain, by using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, nanoelectrospray quadrupole ion-trap mass spectrometry, and chemical methods. In accordance with the previously published structures of the lipid A from
F. tularensis
live vaccine strain (LVS) (ATCC 29684) (E. Vinogradov et al., Eur. J. Biochem.
269:
6112-6118, 2002), all of the major lipid A forms from strain 1547-57 were tetraacylated. As in the LVS strain, the major fatty acids detected in the
F. tularensis
1547-57 lipid A sample included 3-hydroxyoctadecanoic acid, 3-hydroxyhexadecanoic acid, hexadecanoic acid, and tetradecanoic acid. However, several of the lipid A components present in strain 1547-57 were of higher molecular weight than the previously published structures. A major component with an
M
r
of 1,666 was found to contain three C
18:0
(3-OH) fatty acids, one C
16:0
fatty acid, one phosphate group, and one 161-Da moiety. This 161-Da moiety could be removed from the lipid A by treatment with aqueous hydrofluoric acid and was identified as galactosamine following peracetylation and analysis by gas chromatography-mass spectrometry. Detailed investigations of the
M
r
-1,666 species by ion-trap mass spectrometry with multiple stages of fragmentation suggested that the galactosamine-1-phosphate was linked to the reducing terminus of the lipid A. Similar to the modification of lipid A with arabinosamine, lipopolysaccharide species from
F. tularensis
containing a phosphate-linked galactosamine could potentially influence its intracellular survival by conferring resistance to antimicrobial peptides.
Involvement of Raft-Like Plasma Membrane Domains of
Entamoeba histolytica
in Pinocytosis and Adhesion
Authors: Laughlin Richard C. , McGugan Glen C. , Powell Rhonda R. , Welter Brenda H. , Temesvari Lesly A. 
Published: August 2004

ABSTRACT

Lipid rafts are highly ordered, cholesterol-rich, and detergent-resistant microdomains found in the plasma membrane of many eukaryotic cells. These domains play important roles in endocytosis, secretion, and adhesion in a variety of cell types. The parasitic protozoan
Entamoeba histolytica
, the causative agent of amoebic dysentery, was determined to have raft-like plasma membrane domains by use of fluorescent lipid analogs that specifically partition into raft and nonraft regions of the membrane. Disruption of raft-like membrane domains in
Entamoeba
with the cholesterol-binding agents filipin and methyl-β-cyclodextrin resulted in the inhibition of several important virulence functions, fluid-phase pinocytosis, and adhesion to host cell monolayers. However, disruption of raft-like domains did not inhibit constitutive secretion of cysteine proteases, another important virulence function of
Entamoeba
. Flotation of the cold Triton X-100-insoluble portion of membranes on sucrose gradients revealed that the heavy, intermediate, and light subunits of the galactose-
N
-acetylgalactosamine-inhibitible lectin, an important cell surface adhesion molecule of
Entamoeba
, were enriched in cholesterol-rich (raft-like) fractions, whereas EhCP5, another cell surface molecule, was not enriched in these fractions. The subunits of the lectin were also observed in high-density, actin-rich fractions of the sucrose gradient. Together, these data suggest that pinocytosis and adhesion are raft-dependent functions in this pathogen. This is the first report describing the existence and physiological relevance of raft-like membrane domains in
E. histolytica
.
Development of a Noninvasive Method for Detecting and Monitoring the Time Course of
Helicobacter pylori
Infection
Authors: Nyan Dougbeh C. , Welch Anthony R. , Dubois Andre , Coleman William G. 
Published: August 2004

ABSTRACT

Helicobacter pylori
infection status following experimental inoculation of mice presently requires euthanasia. The purpose of this study was to develop a method for following the time course of
H. pylori
infection in live experimental animals. Twenty-six C57BL/6,
Helicobacter
-free female mice were inoculated with
H. pylori
Sydney strain 1, and 16 mice were sham inoculated. The mice were repeatedly tested during a period of about 1 year with an
H. pylori
species-specific primer-based PCR analysis of DNA extracted from fecal pellets of mice. The mice were euthanized at 6 months (
n
= 15) and 10 months (
n
= 15) to determine their infection status by histology, culture, and PCR of gastric specimens.
H. pylori
-inoculated mice were tested via the PCR method at 6 and 10 months prior to necropsy. Nine of 13 (69%) and 10 of 13 (77%) mice tested at 6 and 10 months, respectively, were positive. All sham-inoculated mice were negative. These two PCR results suggested a specificity of 100% with a sensitivity range between 69 and 77%. In contrast, sensitivity and specificity rose to 90 and 100% if groups of mice were tested once daily for 4 days. Seventy-seven to 85% of the experimental mice were also positive for
H. pylori
by culture. The histopathology demonstrated mild to severe gastritis. These findings demonstrate that the persistence or transience of
H. pylori
infection in live mice can be repeatedly evaluated over time. This method could allow the determination of the time course of infection and the efficacy of medications and/or vaccine without necropsy.
Analysis of Subassemblies of Pertussis Toxin Subunits In Vivo and Their Interaction with the Ptl Transport Apparatus
Authors: Burns Drusilla L. , Fiddner Stefanie , Cheung Anissa M. , Verma Anita 
Published: August 2004

ABSTRACT

Pertussis toxin (PT) has an AB
5
structure that is typical of many bacterial protein toxins; however, this toxin is more complex than many toxins since it is composed of five different subunit types, subunits S1 to S5. Little is known about how PT assembles in vivo and how and when it interacts with its secretion apparatus, known as the Ptl transporter. In order to better understand these events, we expressed subsets of the genes encoding the S1, S2, and/or S4 subunits of PT in strains of
Bordetella pertussis
that either did or did not produce the Ptl proteins. We found evidence to suggest that certain subassemblies of the toxin, including subassemblies consisting of the S1 subunit and incomplete forms of the B oligomer, can form in vivo, at least transiently. These results suggest that the B oligomer of the toxin does not need to completely form before interactions between the S1 subunit and B-oligomer subunits can occur in vivo. All subassemblies localized primarily to the membrane fraction of the cell. Moreover, we found that Ptl-mediated secretion occurs in a strain that produces S1 and an incomplete complement of B-oligomer subunits. These results indicate that subassemblies of the toxin consisting of the S1 subunit and a partial B oligomer can interact with the Ptl system.
Involvement of CD14, Toll-Like Receptors 2 and 4, and MyD88 in the Host Response to the Fungal PathogenCryptococcus neoformansIn Vivo
Authors: Yauch Lauren E. , Mansour Michael K. , Shoham Shmuel , Rottman James B. , Levitz Stuart M. 
Published: August 2004

ABSTRACTThe major capsular polysaccharide ofCryptococcus neoformans, glucuronoxylomannan (GXM), is recognized by Toll-like receptor 2 (TLR2), TLR4, and CD14. In these studies, mice deficient in CD14, TLR2, TLR4, and the TLR-associated adaptor protein, MyD88, were utilized to investigate the contribution of TLRs and CD14 to in vivo host defenses againstC. neoformans. MyD88−/−mice had significantly reduced survival compared with wild-type C57BL/6 mice after intranasal (i.n.) and intravenous (i.v.) infection with liveC. neoformans. CD14−/−mice had reduced survival when infected i.v., while TLR2−/−mice died significantly earlier after i.n. infection. Mortality was similar comparing TLR4 mutant C3H/HeJ mice and control C3H/HeOuJ mice following i.v. or i.n. challenge withC. neoformans. The course of pulmonary cryptococcosis was studied in more detail in the CD14−/−, TLR2−/−, and MyD88−/−mice. MyD88−/−mice infected i.n. had higher numbers of CFU in the lungs as well as higher GXM levels in the sera and lungs 7 days after infection than wild-type mice did. Surprisingly, there were no major differences in the levels of tumor necrosis factor alpha, interleukin-4 (IL-4), IL-10, IL-12p70, or gamma interferon in the lungs ofC. neoformans-infected knockout mice compared with wild-type mice. Histopathologic analysis of the lungs on day 7 postinfection revealed minimal inflammation in all mouse groups. These studies demonstrate a major role for MyD88 and relatively minor roles for CD14 and TLR2 in the response to cryptococcal infection, with the decreased survival of MyD88−/−mice correlating with increased numbers of lung CFU and serum and lung GXM levels.
Reduction of Antibody Response to an 11-Valent Pneumococcal Vaccine Coadministered with a Vaccine Containing Acellular Pertussis Components
Authors: Dagan Ron , Goldblatt David , Maleckar James R. , Yaïch Mansour , Eskola Juhani 
Published: August 2004

ABSTRACTIn pneumococcal conjugate vaccines (PCVs), polysaccharide antigens are often conjugated to protein carriers related to other common vaccines. It is therefore important to test PCV interaction with other pediatric vaccines when administered simultaneously. We assessed the immune response to an 11-valent PCV conjugated to diphtheria and tetanus carriers (PncD/T11), administered concomitantly, but in separate sites, with a combined vaccine containing epitopes related antigenically to the carriers: polyribosylribitol phosphate-tetanus tox oid (PRP-T), diphtheria toxoid (DT), and tetanus toxoid (TT). In addition, these combinations contained inactivated poliovirus vaccine (IPV) and either whole-cell pertussis (wP) or acellular pertussis (aP) components. After coadministration of PncD/T11 with the combined vaccine containing wP (DTwP/IPV/PRP-T), the responses to all polysaccharides in the PncD/T11 were satisfactory. In contrast, when coadministered with an aP-containing combination (DTaP/IPV/PRP-T), the response to all seven pneumococcal conjugates to TT was significantly reduced after primary and booster immunization. The pneumococcal conjugates to DT were not significantly reduced after the primary series, but were somewhat reduced after booster. It is likely that some suppression of the tetanus-mediated response occurred even when the PncD/T11 was coadministered with wP, but this suppression was masked by the adjuvant effect of wP. By replacing wP with aP, this adjuvant effect was removed, unmasking the suppression of the tetanus-mediated response. With the increasing use of multiple aP-containing vaccines in infancy, novel approaches to adjuvants and carrier protein technology are likely to be required.
Neutrophil NADPH Oxidase Is Reduced at theAnaplasma phagocytophilumPhagosome
Authors: IJdo Jacob W. , Mueller Angel C. 
Published: August 2004

ABSTRACTThe intracellular organismAnaplasma phagocytophilumcauses human granulocytic ehrlichiosis and specifically infects and multiplies in neutrophilic granulocytes. Previous reports have suggested that, for its survival, this bacterium suppresses the neutrophil respiratory burst. To investigate the mechanism of survival, we first assessed the kinetics ofA. phagocytophilumentry into neutrophils by using double-labeling confocal microscopy. At 30, 60, 120, and 240 min of incubation, 25, 50, 55, and 70% of neutrophils contained bacteria, respectively. The neutrophil respiratory burst in the presence ofA. phagocytophilumwas assessed by a kinetic cytochromecassay and by measurement of oxygen consumption. Neutrophils in the presence ofA. phagocytophilumdid not produce a significant respiratory burst, butA. phagocytophilumdid not inhibit the neutrophil respiratory burst when phorbol myristate acetate was added. Immunoelectron microscopy of neutrophils infected withA. phagocytophilumorEscherichia colirevealed that NADPH oxidase subunits gp91phoxand p22phoxwere significantly reduced at theA. phagocytophilumphagosome after 1 and 4 h of incubation. In neutrophils incubated simultaneously withA. phagocytophilumandE. colifor 30, 60, and 90 min, gp91phoxwas present on 20, 14, and 10% of theA. phagocytophilumphagosomes, whereas p22phoxwas present in 11, 5, and 4% of the phagosomes, respectively. Similarly, onE. coliphagosomes, gp91phoxwas present in 62, 64, and 65%, whereas p22phoxwas detected in 54, 48, and 48%. We conclude thatA. phagocytophilumdoes not suppress a global respiratory burst and that, under identical conditions in the same cells,A. phagocytophilum, but notE. coli, significantly reduces gp91phoxand p22phoxfrom its phagosome membrane.
Mutation of
toxB
and a Truncated Version of the
efa-1
Gene in
Escherichia coli
O157:H7 Influences the Expression and Secretion of Locus of Enterocyte Effacement-Encoded Proteins but not Intestinal Colonization in Calves or Sheep
Authors: Stevens Mark P. , Roe Andrew J. , Vlisidou Isabella , van Diemen Pauline M. , La Ragione Roberto M. , Best Angus , Woodward Martin J. , Gally David L. , Wallis Timothy S. 
Published: August 2004

ABSTRACT

Enterohemorrhagic
Escherichia coli
(EHEC) strains comprise a broad group of bacteria, some of which cause attaching and effacing (AE) lesions and enteritis in humans and animals. Non-O157:H7 EHEC strains contain the gene
efa-1
(referred to in previous publications as
efa1
), which influences adherence to cultured epithelial cells. An almost identical gene in enteropathogenic
E. coli
(
lifA
) mediates the inhibition of lymphocyte proliferation and proinflammatory cytokine synthesis. We have shown previously that significantly lower numbers of EHEC O5 and O111
efa-1
mutants are shed in feces following experimental infection in calves and that these mutants exhibit reduced adherence to intestinal epithelia compared with isogenic wild-type strains.
E. coli
O157:H7 strains lack
efa-1
but encode a homolog on the pO157 plasmid (
toxB
/
l7095
) and contain a truncated version of the
efa-1
gene (
efa-1
′/
z4332
in O island 122 of the EDL933 chromosome). Here we report that
E. coli
O157:H7
toxB
and
efa-1
′ single and double mutants exhibit reduced adherence to cultured epithelial cells and show reduced expression and secretion of proteins encoded by the locus of enterocyte effacement (LEE), which plays a key role in the host-cell interactions of EHEC. The activity of LEE1, LEE4, and LEE5 promoters was not significantly altered in
E. coli
O157:H7 strains harboring
toxB
or
efa-1
′ mutations, indicating that the effect on the expression of LEE-encoded secreted proteins occurs at a posttranscriptional level. Despite affecting type III secretion, mutation of
toxB
and
efa-1
′ did not significantly affect the course of fecal shedding of
E. coli
O157:H7 following experimental inoculation of 10- to 14-day-old calves or 6-week-old sheep. Mutation of
tir
caused a significant reduction in fecal shedding of
E. coli
O157:H7 in calves, indicating that the formation of AE lesions is important for colonization of the bovine intestine.