bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of a Cytolethal Distending Toxin Gene Locus and Features of a Virulence-Associated Region inActinobacillus actinomycetemcomitans
Authors: Mayer Marcia P. A. , Bueno Lina C. , Hansen Eric J. , DiRienzo Joseph M. 
Published: December 2019

ABSTRACTA genetic locus for a cytolethal distending toxin (CDT) was identified in a polymorphic region of the chromosome ofActinobacillus actinomycetemcomitans, a predominant oral pathogen. The locus was comprised of three open reading frames (ORFs) that had significant amino acid sequence similarity and more than 90% sequence identity to thecdtABCgenes of some pathogenicEscherichia colistrains andHaemophilus ducreyi, respectively. Sonic extracts from recombinantE. coli, containing theA. actinomycetemcomitansORFs, caused the distension and killing of Chinese hamster ovary cells characteristic of a CDT. Monoclonal antibodies made reactive with the CdtA, CdtB, and CdtC proteins ofH. ducreyirecognized the corresponding gene products from the recombinant strain. CDT-like activities were no longer expressed by the recombinant strain when an ΩKan-2 interposon was inserted into thecdtAandcdtBgenes. Expression of the CDT-like activities inA. actinomycetemcomitanswas strain specific. Naturally occurring expression-negative strains had large deletions within the region of thecdtlocus. ThecdtABCgenes were flanked by an ORF (virulence plasmid protein), a partial ORF (integrase), and DNA sequences (bacteriophage integration site) characteristic of virulence-associated regions. These results provide evidence for a functional CDT in a human oral pathogen.
The Levels and Bactericidal Capacity of Antibodies Directed against the UspA1 and UspA2 Outer Membrane Proteins of
Moraxella
(
Branhamella
)
catarrhalis
in Adults and Children
Authors: Chen Dexiang , Barniak Vicki , VanDerMeid Karl R. , McMichael John C. 
Published: December 2019

ABSTRACT

The UspA1 and UspA2 proteins from
Moraxella catarrhalis
share antigenic epitopes and are promising vaccine candidates. In this study, the levels and bactericidal activities of antibodies in sera from healthy adults and children toward UspA1 and UspA2 from the O35E strain were measured. Human sera contained antibodies to both proteins, and the levels of immunoglobulin G (IgG) antibodies were age dependent. Adult sera had significantly higher titers of IgG than child sera (
P
< 0.01). The IgG3 titers to the UspA proteins were higher than the IgG1 titers in the adults’ sera, while the IgG1 titers were higher than the IgG3 titers in the children’s sera (
P
< 0.05). The IgG antibodies in the sera from 2-month-old children appeared to be maternally derived, since the mean titer was significantly higher than that in sera from 6- to 7-month-old children (
P
< 0.05). Serum IgA antibodies to both UspA1 and UspA2 were low during the first 7 months of age but thereafter gradually increased along with the IgG titers. Analysis of sera absorbed with UspA1 or UspA2 showed that the antibodies to UspA1 and UspA2 were cross-reactive with each other and associated with serum bactericidal activity. Examination of affinity-purified human antibodies confirmed that naturally acquired antibodies to UspA1 and UspA2 were bactericidal and cross-reactive. These results support using UspA1 and UspA2 in a vaccine to prevent
M. catarrhalis
infections.
Infection of Human Endothelial Cells with
Chlamydia pneumoniae
Stimulates Transendothelial Migration of Neutrophils and Monocytes
Authors: Molestina Robert E. , Miller Richard D. , Ramirez Julio A. , Summersgill James T. 
Published: December 2019

ABSTRACT

We have previously shown that different isolates of
Chlamydia pneumoniae
display heterogeneity in the in vitro stimulation of chemokines and adhesion molecules from infected human endothelial cells. In the present study, we examined the ability of different isolates of
C. pneumoniae
to promote transendothelial migration of neutrophils and monocytes. Human umbilical vein endothelial cells (HUVEC) were infected with low (<15)-passage
C. pneumoniae
isolates A-03, PS-32, and BR-393 and high (>40)-passage isolates BAL-16, TW-183, and T-2634, and levels of neutrophil and monocyte transendothelial migration were determined following 24 h of infection. Compared to mock-infected controls, significant increases in neutrophil migration were observed in response to most
C. pneumoniae
isolates examined (
P
< 0.001). Levels of monocyte migration were significantly increased in response to TW-183 and T-2634 (
P
< 0.001). Serial passage (>40 times) of the three low-passage isolates in HEp-2 cell cultures prior to infection of HUVEC generally resulted in the promotion of higher levels of neutrophil and monocyte transendothelial migration. These findings were compatible with differences observed in the extent of interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) stimulation between low- and high-passage A-03, PS-32, and BR-393. As opposed to
C. pneumoniae
, infection with
C. trachomatis
L2 caused only a slight increase in neutrophil transendothelial migration, which correlated with the lack of measurable IL-8 levels by this species. However, significant levels of monocyte migration were induced in response to
C. trachomatis
L2 despite a lack of measurable MCP-1 stimulation.
C. trachomatis
serovars A and E also failed to induce IL-8 and MCP-1 production in HUVEC. Results from this study indicate that the passage history of
C. pneumoniae
may play a role in the divergence of stimulatory activities observed among isolates in human endothelial cells. In addition, the differences observed between this organism and
C. trachomatis
suggest that the upregulation of IL-8 and MCP-1 in endothelial cells may be unique to
C. pneumoniae
.
Involvement of Tumor Necrosis Factor Alpha and Interleukin-1β in Enhancement of Pentylenetetrazole-Induced Seizures Caused by
Shigella dysenteriae
Authors: Yuhas Yael , Shulman Lester , Weizman Abraham , Kaminsky Elizabeth , Vanichkin Alexey , Ashkenazi Shai 
Published: December 2019

ABSTRACT

Neurologic manifestations, mainly convulsions, are the most frequent extraintestinal complications of shigellosis. We used an animal model to study the roles of tumor necrosis factor alpha (TNF-α) and interleukin-1 β (IL-1β) in
Shigella
-related seizures. Administration of
Shigella dysenteriae
60R sonicate enhanced the sensitivity of mice to the proconvulsant pentylenetetrazole (PTZ) within 7 h. This was indicated by a significantly higher mean convulsion score and an increased number of mice responding with clonic-tonic seizures in the
Shigella
-pretreated group. Preinjection of mice with anti-murine TNF-α (anti-mTNF-α) or anti-murine IL-1β (anti-mIL-1β) 30 min prior to administration of
Shigella
sonicate abolished their enhanced response to PTZ at 7 h. Mean convulsion scores were reduced by anti-mTNF-α from 1.2 to 0.8 (
P
= 0.017) and by anti-mIL-1β from 1.3 to 0.7 (
P
= 0.008). Preinjection of anti-mTNF-α also reduced the percentage of mice responding with clonic-tonic seizures, from 48 to 29% (
P
= 0.002), and preinjection of anti-mIL-1β reduced it from 53 to 21% (
P
= 0.012). Neutralization of TNF-α or IL-1β did not protect the mice from death due to
S. dysenteriae
60R. These findings indicate that TNF-α and IL-1β play a role in the very early sensitization of the central nervous system to convulsive activity after
S. dysenteriae
administration. Similar mechanisms may trigger neurologic disturbances in other infectious diseases.
Identification and Characterization of an
Escherichia coli
Invasion Gene Locus,
ibeB
, Required for Penetration of Brain Microvascular Endothelial Cells
Authors: Huang Sheng-He , Chen Yu-Hua , Fu Qi , Stins Monique , Wang Ying , Wass Carol , Kim Kwang Sik 
Published: December 2019

ABSTRACT

Escherichia coli
K1 is the most common gram-negative organism causing neonatal meningitis, but the mechanism by which
E. coli
K1 crosses the blood-brain barrier is incompletely understood. We have previously described the cloning and molecular characterization of a determinant,
ibeA
(also called
ibe10
), from the chromosome of an invasive cerebrospinal fluid isolate of
E. coli
K1 strain RS218 (O18:K1:H7). Here we report the identification of another chromosomal locus,
ibeB
, which allows RS218 to invade brain microvascular endothelial cells (BMEC). The noninvasive Tn
phoA
mutant 7A-33 exhibited <1% the invasive ability of the parent strain in vitro in BMEC and was significantly less invasive in the central nervous system in the newborn rat model of hematogenous
E. coli
meningitis than the parent strain. The Tn
phoA
insert with flanking sequences was cloned and sequenced. A 1,383-nucleotide open reading frame (ORF) encoding a 50-kDa protein was identified and termed
ibeB
. This ORF was found to be 97% identical to a gene encoding a 50-kDa hypothetical protein (p77211) and located in the 13-min region of the
E. coli
K-12 genome. However, no homology was observed between
ibeB
and other known invasion genes when DNA and protein databases in GenBank were searched. Like the Tn
phoA
insertion mutant 7A-33, an isogenic
ibeB
deletion mutant (IB7D5) was unable to invade BMEC. A 7.0-kb locus containing
ibeB
was isolated from a LambdaGEM-12 genomic library of
E. coli
RS218 and subcloned into a pBluescript KS vector (pKS7-7B). pKS7-7B was capable of completely restoring the BMEC invasion of the noninvasive Tn
phoA
mutant 7A-33 and the
ibeB
deletion mutant IB7D5 to the level of the parent strain. More importantly, the
ibeB
deletion mutant IB7D5 was fully complemented by pFN476 carrying the
ibeB
ORF (pFN7C), indicating that
ibeB
is required for
E. coli
K1 invasion of BMEC. Taken together, these findings indicate that several
E. coli
determinants, including
ibeA
and
ibeB
, contribute to crossing of the blood-brain barrier.
Autoregulatory Effect of Interleukin-10 on Proinflammatory Cytokine Production byPorphyromonas gingivalisLipopolysaccharide-Tolerant Human Monocytes
Authors: Shimauchi Hidetoshi , Ogawa Tomohiko , Okuda Kozo , Kusumoto Yutaka , Okada Hirohi 
Published: December 2019

ABSTRACTPretreatment of human peripheral blood monocytes with a very low concentration (0.1 ng/ml) ofPorphyromonas gingivalislipopolysaccharides (LPS) resulted in a significant decrease of interleukin-6 (IL-6) production, but not IL-8 production, by restimulation of a high concentration (1 μg/ml) of the same LPS. In contrast, the same pretreatment withEscherichia coliLPS resulted in the enhanced production of both IL-6 and IL-8 after restimulation. The selective induction byP. gingivalisLPS tolerance of IL-6 production developed in a time-dependent manner during the primary culture.P. gingivalisLPS-pretreated cells were also refractory to a high-doseE. coliLPS restimulation in terms of IL-6 production. The expression of IL-6 mRNA decreased 10 h after restimulation ofP. gingivalisLPS-pretreated monocytes. Furthermore, an up-regulation of anti-inflammatory cytokine IL-10 upon a second high-dose LPS rechallenge occurred at the same time point in the pretreated cells. We studied the role of IL-10 in the process of IL-6 down-regulation. Neutralization by an anti-IL-10 polyclonal antibody prevented IL-6 down-regulation inP. gingivalisLPS-pretreated monocytes, whereas IL-8 production was not affected. Addition of exogenous IL-10 during the high-dose LPS stimulation of untreated cells substituted for the LPS pretreatment and resulted in the inhibition of IL-6 production in a dose-dependent manner. A higher dose of IL-10 was required to suppress IL-8 synthesis from monocytes. Our data suggest that IL-10 mediates IL-6 down-regulation inP. gingivalisLPS-tolerant monocytes in an autocrine manner.
Cytoskeletal Effects Induced by Pet, the Serine Protease Enterotoxin of Enteroaggregative
Escherichia coli
Authors: Navarro-García Fernando , Sears Cynthia , Eslava Carlos , Cravioto Alejandro , Nataro James P. 
Published: December 2019

ABSTRACT

We have previously described enteroaggregative
Escherichia coli
(EAEC) strains that induce cytotoxic effects on T84 cells, ligated rat ileal loops, and human intestine in culture. Such strains secrete a 104-kDa protein termed Pet (for plasmid-encoded toxin). We have also shown previously that the Pet toxin induces rises in short-circuit current and decreases the electrical resistance in rat jejunum mounted in an Ussing chamber. The nucleotide sequence of the
pet
gene revealed that Pet is a member of the autotransporter class of secreted proteins. Here we show that a concentrated supernatant of
E. coli
HB101 harboring the minimal
pet
clone pCEFN1 induces temperature-, time- and dose-dependent cytopathic effects on HEp-2 cells and HT29 C
1
cells in culture. The effects were characterized by release of the cellular focal contacts from the glass substratum, followed by complete rounding of the cells and detachment from the glass. Staining of the Pet-treated cells with Live/Dead viability stain revealed that >90% of rounded cells were viable. Pet-intoxicated HEp-2 and HT29 cells stained with fluorescein-labeled phalloidin revealed contraction of the cytoskeleton and loss of actin stress fibers. However, the effects of Pet were not inhibited by cytoskeleton-altering drugs, including colchicine, taxol, cytochalasin D, and phallicidin. The Pet protein induced proteolysis in zymogram gels, and preincubation with the serine protease inhibitor phenylmethylsulfonyl fluoride resulted in complete abrogation of Pet cytopathic effects. We introduced a mutation in a predicted catalytic serine residue and found that the mutant (Pet S260I) was deficient in protease activity and did not produce cytopathic effects, cytoskeletal damage, or enterotoxic effects in Ussing chambers. These data suggest that Pet is a cytoskeleton-altering toxin and that its protease activity is involved in each of the observed phenotypes.
Structural and Antigenic Properties of Merozoite Surface Protein 4 of
Plasmodium falciparum
Authors: Wang Lina , Black Casilda G. , Marshall Vikki M. , Coppel Ross L. 
Published: December 2019

ABSTRACT

Merozoite surface protein 4 (MSP4) of
Plasmodium falciparum
is a glycosylphosphatidylinositol-anchored integral membrane protein of 272 residues that possesses a single epidermal growth factor (EGF)-like domain near the carboxyl terminus. We have expressed both full-length MSP4 and a number of fragments in
Escherichia coli
and have used these recombinant proteins to raise experimental antisera. All recombinant proteins elicited specific antibodies that reacted with parasite-derived MSP4 by immunoblotting. Antibody reactivity was highly dependent on the protein conformation. For example, reduction and alkylation of MSP4 almost completely abolished the reactivity of several antibody preparations, including specificities directed to regions of the protein that do not contain cysteine residues and are far removed from the cysteine-containing EGF-like domain. This indicated the presence of conformation-dependent epitopes in MSP4 and demonstrated that proper folding of the EGF-like domain influenced the antigenicity of the entire molecule. The recombinant proteins were used to map epitopes recognized by individuals living in areas where malaria is endemic, and at least four distinct regions are naturally antigenic during infection. Binding of human antibodies to the EGF-like domain was essentially abrogated after reduction of the recombinant protein, indicating the recognition of conformational epitopes by the human immune responses. This observation led us to examine the importance of conformation dependence in responses to other integral membrane proteins of asexual stages. We analyzed the natural immune responses to a subset of these antigens and demonstrated that there is diminished reactivity to several antigens after reduction. These studies demonstrate the importance of reduction-sensitive structures in the maintenance of the antigenicity of several asexual-stage antigens and in particular the importance of the EGF-like domain in the antigenicity of MSP4.
Conformational and Linear B-Cell Epitopes of Asp f 2, a Major Allergen ofAspergillus fumigatus, Bind Differently to Immunoglobulin E Antibody in the Sera of Allergic Bronchopulmonary Aspergillosis Patients
Authors: Banerjee Banani , Greenberger Paul A. , Fink Jordan N. , Kurup Viswanath P. 
Published: December 2019

ABSTRACTAsp f 2 is a majorAspergillus fumigatusallergen involved in allergic bronchopulmonary aspergillosis. Knowledge of the B-cell epitopes may contribute to the understanding of immunoregulation and immunodiagnosis. To elucidate the immunoglobulin E (IgE) binding epitopes in the linear sequence of Asp f 2, we synthesized decamer peptides spanning the whole molecule of Asp f 2 on derivatized cellulose membranes and evaluated IgE binding in ABPA patient and control sera. Peptides three to five amino acids long were synthesized based on amino acid sequences within the IgE binding regions and evaluated for the specificity of epitope antibody interactions. Nine IgE binding regions were recognized in this protein of 268 amino acid residues. Of the nine epitopes, seven (ATQRRQI, RKYFG, HWR, YTTRR, DHFAD, ALEAYA, and THEGGQ) are present in the hydrophilic regions of Asp f 2. Immunologic evaluation of the three recombinant fragments, Asp f 2A encompassing the N-terminal epitope region, Asp f 2B without N- and C-terminal regions of the protein, and Asp f 2C representing C-terminal epitopes, revealed that either the N- or C-terminal region of the protein is essential for the correct folding and conformation for IgE antibody binding.
Effects of Staphylococcal Enterotoxins on Human Neutrophil Functions and Apoptosis
Authors: Moulding Dale A. , Walter Catherine , Hart C. Anthony , Edwards Steven W. 
Published: December 2019

ABSTRACTStaphylococcal enterotoxins have marked effects on the properties of T cells and monocytes and have recently been reported to affect neutrophil function. In this study, we investigated the abilities of staphylococcal enterotoxins A and B and toxic shock syndrome toxin 1 to affect respiratory burst activity and to delay apoptosis in human neutrophils. When cultures containing approximately 97% neutrophils were tested, the toxins all delayed neutrophil apoptosis in a dose-dependent manner and induced the expression of FcγRI on the neutrophil cell surface. These effects on apoptosis and expression of FcγRI were largely abrogated by the addition of a neutralizing anti-gamma interferon antibody. Similarly, the effects of these toxins on phorbol ester-induced chemiluminescence were decreased after neutralization of gamma interferon. These effects on neutrophil function were mimicked by the addition of conditioned medium from peripheral blood mononuclear cells incubated with the toxins, and again, neutralizing anti-gamma interferon antibodies largely negated the effects. However, when highly purified neutrophils prepared by immunodepletion of T cells and major histocompatibility complex class II-expressing cells were analyzed, the toxins were without effect on apoptosis and FcγRI expression, but granulocyte-macrophage colony-stimulating factor and gamma interferon could still delay apoptosis. These data indicate that these toxins have no direct effect on neutrophil apoptosis but can act indirectly via the production of T-cell-derived and monocyte-derived cytokines. It is noteworthy that such effects are detected in neutrophil suspensions containing only 3% contamination with T cells and other mononuclear cells.
Phagocytosis Does Not Play a Major Role in Naturally Acquired Transmission-Blocking Immunity toPlasmodium falciparumMalaria
Authors: Healer J. , Graszynski A. , Riley E. 
Published: December 2019

ABSTRACTPhagocytosis ofPlasmodium falciparumsexual stages in vitro and within the mosquito midgut was assayed in order to assess its role in transmission-blocking immunity to malaria. Both monocytes/macrophages (MM) and polymorphonuclear neutrophils (PMN) phagocytosed malarial gametes in vitro, but levels of phagocytosis were low. Intraerythrocytic gametocytes were not susceptible to phagocytosis. In vitro phagocytosis was positively correlated with levels of antibodies against the gamete surface proteins Pfs230 and Pfs48/45. Immunoglobulin G (IgG) subclass analysis revealed that phagocytosis was correlated with levels of antigamete IgG1. In vivo membrane-feeding experiments were performed in the presence of both pooled and individual malaria immune sera. The phagocytic process proceeded less efficiently in vivo than in vitro, which may be related to the lower ambient temperature (26°C, compared with 37°C). Finally, although we found a correlation between the ability of a serum to promote phagocytosis in vitro and the presence of antibodies against transmission-blocking target antigens, we were unable to demonstrate a role for MM- or PMN-mediated phagocytosis in reduction of infectivity of the malarial parasite to mosquitoes.
Attachment Ligands of ViableToxoplasma gondiiInduce Soluble Immunosuppressive Factors in Human Monocytes
Authors: Channon Jacqueline Y. , Suh Edward I. , Seguin Rosanne M. , Kasper Lloyd H. 
Published: December 2019

ABSTRACTPrevious studies have demonstrated that surface antigen proteins, in particular SAG-1, ofToxoplasma gondiiare important to this parasite as attachment ligands for the host cell. An in vitro assay was developed to test whether these ligands and other secretory proteins are involved in the immune response of human cells to toxoplasma. Human monocytes were infected with tachyzoites in the presence of antiparasite antibodies, and their effect on mitogen-induced lymphoproliferation was examined. The presence of antibody to either parasite-excreted proteins (MIC-1 and MIC-2) or surface proteins (SAG-1 and SAG-2) during infection neutralized the marked decrease seen in mitogen-induced lymphoproliferation in the presence of infected monocytes. Conversely, antibodies to other secreted proteins (ROP-1) and cytoplasmic molecules had no effect on parasite-induced, monocyte-mediated downregulation. Fluorescence microscope analysis detected microneme and surface antigen proteins on the monocyte cell surface during infection. These results suggest that microneme and surface antigen proteins trigger monocytes to downregulate mitogen-induced lymphoproliferation.
Host Cell Death due to Enteropathogenic
Escherichia coli
Has Features of Apoptosis
Authors: Crane John K. , Majumdar Swastika , Pickhardt Donald F. 
Published: December 2019

ABSTRACT

Enteropathogenic
Escherichia coli
(EPEC) is a cause of prolonged watery diarrhea in children in developing countries. The ability of EPEC to kill host cells was investigated in vitro in assays using two human cultured cell lines, HeLa (cervical) and T84 (colonic). EPEC killed epithelial cells as assessed by permeability to the vital dyes trypan blue and propidium iodide. In addition, EPEC triggered changes in the host cell, suggesting apoptosis as the mode of death; such changes included early expression of phosphatidylserine on the host cell surface and internucleosomal cleavage of host cell DNA. Genistein, an inhibitor of tyrosine kinases, and wortmannin, an inhibitor of host phosphatidylinositol 3-kinase, markedly increased EPEC-induced cell death and enhanced the features of apoptosis. EPEC-induced cell death was contact dependent and required adherence of live bacteria to the host cell. A quantitative assay for EPEC-induced cell death was developed by using the propidium iodide uptake method adapted to a fluorescence plate reader. With EPEC, the rate and extent of host cell death were less that what has been reported for
Salmonella
,
Shigella
, and
Yersinia
, three other genera of enteric bacteria known to cause apoptosis. However, rapid apoptosis of the host cell may not favor the pathogenic strategy of EPEC, a mucosa-adhering, noninvasive pathogen.