bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and Distribution of Leptospiral Outer Membrane Components during Renal Infection of Hamsters
Authors: Barnett Jeanne K. , Barnett Dean , Bolin Carole A. , Summers Theresa A. , Wagar Elizabeth A. , Cheville Norman F. , Hartskeerl Rudy A. , Haake David A.
Published: December 2019

The outer membrane of pathogenicLeptospiraspecies grown in culture media contains lipopolysaccharide (LPS), a porin (OmpL1), and several lipoproteins, including LipL36 and LipL41. The purpose of this study was to characterize the expression and distribution of these outer membrane antigens during renal infection. Hamsters were challenged with host-derivedLeptospira kirschnerito generate sera which contained antibodies to antigens expressed in vivo. Immunoblotting performed with sera from animals challenged with these host-derived organisms demonstrated reactivity with OmpL1, LipL41, and several other proteins but not with LipL36. Although LipL36 is a prominent outer membrane antigen of cultivatedL. kirschneri, its expression also could not be detected in infected hamster kidney tissue by immunohistochemistry, indicating that expression of this protein is down-regulated in vivo. In contrast, LPS, OmpL1, and LipL41 were demonstrated on organisms colonizing the lumen of proximal convoluted renal tubules at both 10 and 28 days postinfection. Tubular epithelial cells around the luminal colonies had fine granular cytoplasmic LPS. When the cellular inflammatory response was present in the renal interstitium at 28 days postinfection, LPS and OmpL1 were also detectable within interstitial phagocytes. These data establish that outer membrane components expressed during infection have roles in the induction and persistence of leptospiral interstitial nephritis.
The Cell Wall and Membrane ofCryptococcus neoformansPossess a Mitogen for Human T Lymphocytes
Authors: Mody Christopher H. , Wood Cynthia J. , Syme Rachel M. , Spurrell Jason C. L.
Published: December 2019

The mechanism of human T-lymphocyte activation by the pathogenic yeastCryptococcus neoformanshas not been established. Previous investigations have suggested thatC. neoformanscontains a mitogen for T lymphocytes, while other investigators have attributed lymphocyte proliferation in vitro to a recall antigen. Because of the potential importance of the mechanism of T-cell activation for our understanding of the immune response toC. neoformans, the present studies were performed to determine whetherC. neoformanscontains a mitogen for T lymphocytes.C. neoformansstimulates fetal blood lymphocytes to proliferate and stimulates proliferation of CD45RA+cells from adults, indicating that it stimulates naive T cells. The T-cell response toC. neoformanswas dependent upon the presence of accessory cells. However, allogeneic cells were sufficient for accessory cell function, indicating that the response was not major histocompatibility complex restricted. The percentage of T cells in the cell cycle was higher than that with the recall antigen tetanus toxoid but lower than that with the mitogenic lectin phytohemagglutinin A or the superantigenStaphylococcusenterotoxin B. Precursor frequency analysis established that 1 in 7,750 ± 2,270 T cells proliferated in response to the cryptococcal cell wall and membrane. Compared to the case for most mitogens or superantigens, the proliferative response is late and the number of T cells that enter the cell cycle and the precursor frequency are low, indicating that the mitogenic effect is modest. However, the mitogenic effect ofC. neoformansshould be considered when interpreting the immune response toC. neoformans, since even weak mitogens can have profound effects on host defense.
Role of Antibodies against
Bordetella pertussis
Virulence Factors in Adherence of
Bordetella pertussis
and
Bordetella parapertussis
to Human Bronchial Epithelial cells
Authors: van den Berg Bernard M. , Beekhuizen Henry , Mooi Frits R. , van Furth Ralph
Published: December 2019

Immunization with whole-cell pertussis vaccines (WCV) containing heat-killed
Bordetella pertussis
cells and with acellular vaccines containing genetically or chemically detoxified pertussis toxin (PT) in combination with filamentous hemagglutinin (FHA), pertactin (Prn), or fimbriae confers protection in humans and animals against
B. pertussis
infection. In an earlier study we demonstrated that FHA is involved in the adherence of these bacteria to human bronchial epithelial cells. In the present study we investigated whether mouse antibodies directed against
B. pertussis
FHA, PTg, Prn, and fimbriae, or against two other surface molecules, lipopolysaccharide (LPS) and the 40-kDa outer membrane porin protein (OMP), that are not involved in bacterial adherence, were able to block adherence of
B. pertussis
and
B. parapertussis
to human bronchial epithelial cells. All antibodies studied inhibited the adherence of
B. pertussis
to these epithelial cells and were equally effective in this respect. Only antibodies against LPS and 40-kDa OMP affected the adherence of
B. parapertussis
to epithelial cells. We conclude that antibodies which recognize surface structures on
B. pertussis
or on
B. parapertussis
can inhibit adherence of the bacteria to bronchial epithelial cells, irrespective whether these structures play a role in adherence of the bacteria to these cells.
Role ofListeria monocytogenesExotoxins Listeriolysin and Phosphatidylinositol-Specific Phospholipase C in Activation of Human Neutrophils
Authors: Sibelius Ulf , Schulz Eva-Cathrin , Rose Frank , Hattar Katja , Jacobs Thomas , Weiss Siegfried , Chakraborty Trinad , Seeger Werner , Grimminger Friedrich
Published: December 2019

Polymorphonuclear leukocytes (PMN) are essential for resolution of infections withListeria monocytogenes. The present study investigated the role of the listerial exotoxins listeriolysin (LLO) and phosphatidylinositol-specific phospholipase C (PlcA) in human neutrophil activation. DifferentListeriastrains, mutated in individual virulence genes, as well as purified LLO were used. Coincubation of human neutrophils with wild-typeL. monocytogenesprovoked PMN activation, occurring independently of phagocytosis events, with concomitant elastase secretion, leukotriene generation, platelet-activating factor (PAF) synthesis, respiratory burst, and enhanced phosphoinositide hydrolysis. Degranulation and leukotriene formation were noted to be solely dependent on LLO expression, as these features were absent when the LLO-defective mutant EGD− and the avirulent strainL. innocuawere used. These effects were fully reproduced by a recombinantL. innocuastrain expressing LLO (INN+) and by the purified LLO molecule. LLO secretion was also required for PAF synthesis. However, wild-typeL. monocytogeneswas more potent in eliciting PAF formation than mutants expressing LLO, suggesting the involvement of additional virulence factors. This was even more obvious for phosphoinositide hydrolysis and respiratory burst: these events were provoked not only by INN+ but also by the LLO-defective mutant EGD− and by a recombinantL. innocuastrain producing listerial PlcA. We conclude that human neutrophils react to extracellularly provided listerial exotoxins by rapid cell activation. Listeriolysin is centrally involved in triggering degranulation and lipid mediator generation, and further virulence factors such as PlcA apparently contribute to trigger neutrophil phosphoinositide hydrolysis and respiratory burst. In this way, listerial exotoxins may influence the host defense against infections withL. monocytogenes.
Invasion of Human Mucosal Epithelial Cells byNeisseria gonorrhoeaeUpregulates Expression of Intercellular Adhesion Molecule 1 (ICAM-1)
Authors: Jarvis Gary A. , Li Jing , V. Swanson Karen
Published: December 2019

Infection of the mucosa byNeisseria gonorrhoeaeinvolves adherence to and invasion of epithelial cells. Little is known, however, about the expression by mucosal epithelial cells of molecules that mediate cellular interactions between epithelial cells and neutrophils at the site of gonococcal infection. The aim of this study was to determine the expression of intercellular adhesion molecule 1 (ICAM-1) by epithelial cells during the process of gonococcal invasion. The highly invasive strain FA1090 and the poorly invasive strain MS11 were incubated with human endometrial adenocarcinoma (HEC-1-B) or human cervical carcinoma (ME-180) epithelial cells, after which ICAM-1 expression was measured by flow cytometry. After 15 h of infection with FA1090, expression of ICAM-1 increased 4.7- and 2.1-fold for HEC-1-B and ME-180 cells, respectively, whereas 15 h of infection of HEC-1-B cells with MS11 increased ICAM-1 expression only 1.6-fold. ICAM-1 expression was restricted to the cell surface, since no soluble ICAM-1 was detected. The distribution of staining was heterogeneous and mimicked that seen after treatment of HEC-1-B cells with the ICAM-1 agonist tumor necrosis factor alpha (TNF-α) in the absence of bacteria. PCR and dot blot analyses of ICAM-1 mRNA showed no change in levels over time in response to infection. Although TNF-α was produced by HEC-1-B cells after infection, the extent of ICAM-1 upregulation was not affected by neutralizing anti-TNF-α antiserum. Dual-fluorescence flow cytometry showed that the cells with the highest levels of ICAM-1 expression were cells with associated gonococci. We conclude that epithelial cells upregulate the expression of ICAM-1 in response to infection with invasive gonococci. On the mucosa, upregulation of ICAM-1 by infected epithelial cells may function to maintain neutrophils at the site of infection, thereby reducing further invasion of the mucosa by gonococci.
A 55-Kilodalton Immunodominant Antigen of
Porphyromonas gingivalis
W50 Has Arisen via Horizontal Gene Transfer
Authors: Hanley Shirley A. , Aduse-Opoku Joseph , Curtis Michael A.
Published: December 2019

A 55-kDa outer membrane protein of
Porphyromonas gingivalis
W50 is a significant target of the serum immunoglobulin G antibody response of periodontal disease patients and hence may play an important role in host-bacterium interactions in periodontal disease. The gene encoding the 55-kDa antigen (
ragB
, for receptor antigen B) was isolated on a 9.5-kb partial
Sau
3AI fragment of
P. gingivalis
W50 chromosomal DNA in pUC18 by immunoscreening with a monoclonal antibody to this antigen. The 1.6-kb open reading frame (ORF) encoding RagB was located via subcloning and nested-deletion analysis. Sequence analysis demonstrated the presence of an upstream 3.1-kb ORF (
ragA
) which is cotranscribed with
ragB
. A number of genetic characteristics suggest that the
ragAB
locus was acquired by a horizontal gene transfer event. These include a significantly reduced G+C content relative to that of the
P. gingivalis
chromosome (42 versus 48%) and the presence of mobility elements flanking this locus in
P. gingivalis
W50. Furthermore, Southern blotting and PCR analyses showed a restricted distribution of this locus in laboratory and clinical isolates of this bacterium. The association of

ragAB
+
P. gingivalis

with clinical status was examined by PCR analysis of subgingival samples.
ragAB
+
was not detected in
P. gingivalis
-positive shallow pockets from periodontal disease patients but was present in 36% of the
P. gingivalis
-positive samples from deep pockets. These data suggest that the
ragAB
locus was acquired by certain
P. gingivalis
strains via horizontal gene transfer and that the acquisition of this locus may facilitate the survival of these strains at sites of periodontal destruction.
Repertoire of Human Antibodies against the Polysaccharide Capsule ofStreptococcus pneumoniaeSerotype 6B
Authors: Sun Yan , Park Moon K. , Kim Jihye , Diamond Betty , Solomon Alan , Nahm Moon H.
Published: December 2019

We examined the repertoire of antibodies toStreptococcus pneumoniae6B capsular polysaccharide induced with the conventional polysaccharide vaccine in adults at the molecular level two ways. In the first, we purified from the sera of seven vaccinees antipneumococcal antibodies and determined their amino acid sequences. Their VH regions are mainly the products of VH3 family genes (candidate genes, 3-23, 3-07, 3-66, and 3-74), but the product of a VH1 family gene (candidate gene, 1-03) is occasionally used. All seven individuals have small amounts of polyclonal κ+antibodies (Vκ1 to Vκ4 families), although κ+antibodies are occasionally dominated by antibodies formed with the product of the A27 Vκ gene. In contrast, λ+anti-6B antibodies are dominated by the antibodies derived from one of 3 very similar Vλ2 family genes (candidate genes, 2c, 2e, and 2a2) and Cλ1 gene product. The Vλ2+antibodies express the 8.12 idiotype, which is expressed on anti-double-stranded-DNA antibodies. In one case, Vλ is derived from a rarely expressed Vλ gene, 10a. In the second approach, we studied a human hybridoma (Dob1) producing anti-6B antibody. Its VH region sequence is closely related to those of the 3-15 VH gene (88% nucleotide homology) and JH4 (92% homology). Its VL region is homologous to the 2a2 Vλ2 gene (91%) and Jλ1/Cλ1. Taken together, the V region of human anti-6B antibodies is commonly formed by a VH3 and a Vλ2 family gene product.
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

A 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

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

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

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

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

Pretreatment 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

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

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

Asp 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

Staphylococcal 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

Phagocytosis 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

Previous 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

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.
In Vitro Adhesion and Platelet Aggregation Properties of Bacteremia-Associated Lactobacilli
Authors: Kirjavainen Pirkka V. , Tuomola Elina M. , Crittenden Ross G. , Ouwehand Arthur C. , Harty Derek W. S. , Morris Leone F. , Rautelin Hilpi , Playne Martin J. , Donohue Diana C. , Salminen Seppo J.
Published: December 2019

Eight bacteremia-associated
Lactobacillus
strains were evaluated in vitro for the ability to adhere to human intestinal mucosa and to aggregate platelets. Adherence varied significantly among the strains, and platelet aggregation was induced by three strains. In conclusion, strong binding ability does not appear to be a prerequisite for the involvement of lactobacilli in bacteremia or to their ability to aggregate platelets.
Deficiency in Tumor Necrosis Factor Alpha Activity Does Not Impair Early Protective Th1 Responses against Blood-Stage Malaria
Authors: Sam Hakeem , Su Zhong , Stevenson Mary M.
Published: December 2019

Blood-stagePlasmodium chabaudiAS infection was controlled by 4 weeks in mice with deletion of tumor necrosis factor p55 and p75 receptors (TNFR-knockout [KO]) and control wild-type (WT) mice, although female TNFR-KO mice showed slightly but significantly higher parasitemia immediately following the peak. Serum interleukin 12 (IL-12) p70 and gamma interferon (IFN-γ) levels were similar but tumor necrosis factor alpha levels were significantly higher in TNFR-KO mice than in WT controls. Splenic IL-12 receptor β1 and β2 and IFN-γ mRNA expression, as well as spleen cell production of IFN-γ and IL-4, were comparable in both mouse types, but IL-10 production was significantly higher in cells from TNFR-KO mice than in cells from WT mice. Lipopolysaccharide-induced NO secretion by splenic macrophages in vitro was significantly reduced but systemic NO3−levels were similar in infected TNFR-KO and WT mice.
Lipopolysaccharide (LPS)-Binding Synthetic Peptides Derived from Serum Amyloid P Component Neutralize LPS
Authors: de Haas Carla J. C. , van der Zee Ruurd , Benaissa-Trouw Barry , van Kessel Kok P. M. , Verhoef Jan , van Strijp Jos A. G.
Published: December 2019

Lipopolysaccharide (LPS) is the major mediator of gram-negative septic shock. Molecules that bind LPS and neutralize its toxic effects could have important clinical applications. We showed that serum amyloid P component (SAP) neutralizes LPS. A SAP-derived peptide, consisting of amino acids 27 to 39, inhibited LPS-mediated effects in the presence of human blood. In this study, we used a pepscan of overlapping 15-mer peptides and distinguished two additional LPS-binding regions within the SAP molecule, identified in the regions spanning amino acids 61 to 75 and 186 to 200. The corresponding SAP-derived peptides, pep61-75 and pep186-200, inhibited the binding of fluorescein isothiocyanate-labeled LPS to monocytes as efficiently as a bactericidal/permeability-increasing protein (BPI)-derived 15-mer peptide comprising amino acids 85 to 99. The same SAP-derived peptides very potently inhibited LPS-induced priming of phagocytes in human blood. Also, SAP-derived pep186-200 caused a prolonged survival of actinomycin D-sensitized mice treated with LPS to induce septic shock, indicating a potential use of this peptide in the defense against serious gram-negative sepsis in humans.
Evaluation ofSalmonella typhimuriumMutants in a Model of Experimental Gastroenteritis
Authors: Everest Paul , Ketley Julian , Hardy Simon , Douce Gill , Khan Shahid , Shea Jacqui , Holden David , Maskell Duncan , Dougan Gordon
Published: December 2019

Salmonella typhimuriumstrains harboring independent, defined mutations inaroA,invA,ssrA, ormsbBwere assessed for their ability to induce fluid accumulation, tissue damage, and local inflammation in rabbit ileal loops. Three wild-type strains ofS. typhimurium, TML, HWSH, and SL1344, and two mutant strains,S. typhimuriumSL1344ssrAandS. typhimuriumSL1344msbB, consistently induced fluid accumulation in the lumen of loops and inflammation of loop-associated tissues. In contrast, three differentS. typhimurium aroAstrains and aninvAmutant of SL1344 did not induce significant fluid accumulation in the rabbit ileal loops. However, theS. typhimurium aroAstrains did induce an inflammatory infiltrate and some local villus-associated damage, but theinvAmutant did not. Histologically, wild-typeS. typhimurium,S. typhimuriumSL1344ssrA, andS. typhimuriumSL1344msbBdemonstrated more severe effects on villus architecture thanS. typhimurium aroAstrains, whereasS. typhimurium invA-infected loops showed no detectable damage. This suggests that villus damage most likely contributes to fluid accumulation within the loop.
Effects of Cytokines and Endotoxin on the Intracellular Growth of Bacteria
Authors: Kanangat Siva , Meduri G. Umberto , Tolley Elizabeth A. , Patterson David R. , Meduri Christopher U. , Pak Chol , Griffin John P. , Bronze Michael S. , Schaberg Dennis R.
Published: December 2019

Patients with unresolving acute respiratory distress syndrome (ARDS) have persistently elevated levels of proinflammatory cytokines in the lungs and circulation and increased rates of bacterial infections. Phagocytic cells hyperactivated with lipopolysaccharide (LPS), which induces high levels of proinflammatory cytokines in monocytic cells, are inefficient in killing ingested bacteria despite having intact phagocytic activity. On the other hand, phagocytic cells that are activated with an analogue of LPS that does not induce the expression of proinflammatory cytokines effectively ingest and kill bacteria. We hypothesized that in the presence of high concentrations of proinflammatory cytokines, bacteria may adapt and utilize cytokines to their growth advantage. To test our hypothesis, we primed a human monocytic cell line (U937) with escalating concentrations of the proinflammatory cytokines tumor necrosis factor alpha, interleukin-1β (IL-1β), and IL-6 and with LPS. These cells were then exposed to fresh isolates of three common nosocomial pathogens:Staphylococcus aureus,Pseudomonas aeruginosa, and anAcinetobactersp. In human monocytes primed with lower concentrations of proinflammatory cytokines (10 to 250 pg) or LPS (1 and 10 ng), intracellular bacterial growth decreased. However, when human monocytes were primed with higher concentrations of proinflammatory cytokines (1 to 10 ng) or LPS (1 to 10 μg), intracellular growth of the tested bacteria increased significantly (P<0.0001). These results were reproduced with peripheral blood monocytes obtained from normal healthy volunteers. The specificity of the cytokine activity was demonstrated by neutralizing the cytokines with specific antibodies. Our findings provide a possible mechanism to explain the frequent development of bacterial infections in patients with an intense and protracted inflammatory response.
Role of Pneumolysin’s Complement-Activating Activity during Pneumococcal Bacteremia in Cirrhotic Rats
Authors: Alcantara Rosemarie B. , Preheim Laurel C. , Gentry Martha J.
Published: December 2019

We investigated the role of pneumolysin’s complement-activating activity during
Streptococcus pneumoniae
bacteremia in a hypocomplementemic, cirrhotic host. Isogenic mutant pneumococcal strains, in which pneumolysin was expressed from a plasmid, were used. These strains included H+C+, expressing wild-type pneumolysin with both cytolytic and complement-activating activity; PLY−, carrying the plasmid without the pneumolysin gene; and, H+C−, expressing pneumolysin with cytolytic activity only. In control rats, intravenous infection with 2.0 × 10
7
CFU of H+C+ per ml of blood resulted in a decrease in bacteremia of 3.5 log units by 18 h postinfection and 55% mortality. By contrast, cirrhotic rats infected similarly with the H+C+ strain demonstrated a 0.2-log-unit increase in bacteremia by 18 h postinfection and 100% mortality. Both control and cirrhotic rats cleared the PLY− strain more effectively from their bloodstreams by 18 h postinfection (6.2 and 5.6 log unit decreases, respectively). Infection with the PLY− strain also resulted in low mortality (0 and 14%, respectively) for control and cirrhotic rats. When infected with the H+C− strain (without complement-activating activity), both groups cleared the organism from their bloodstreams nearly as well as they did the PLY− strain. Furthermore, the mortality rate for control and cirrhotic rats was identical after infection with the H+C− strain. These studies suggest that pneumolysin production contributes to decreased pneumococcal clearance from the bloodstream and higher mortality in both control and cirrhotic rats. However, pneumolysin’s complement-activating activity may uniquely enhance pneumococcal virulence in the hypocomplementemic, cirrhotic host.
Antibody Responses in the Lower Respiratory Tract and Male Urogenital Tract in Humans after Nasal and Oral Vaccination with Cholera Toxin B Subunit
Authors: Rudin Anna , Riise Gerdt C. , Holmgren Jan
Published: December 2019

Nasal vaccine delivery is superior to oral delivery in inducing specific immunoglobulin A (IgA) and IgG antibody responses in the upper respiratory tract. Although an antibody response in the nasal passages is important in protecting against primary colonization with lung pathogens, antibodies in the lungs are usually required as well. We immunized 15 male volunteers twice nasally or orally with cholera toxin B subunit (CTB) and determined the specific antibody levels in serum, bronchoalveolar lavage (BAL) fluid, and urine before and 2 weeks after immunization. Nasal immunization induced fivefold increases in the levels of specific IgA antibodies in BAL fluid of most volunteers, whereas there were no significant specific IgA responses after oral immunization. The specific IgG antibody level increased eightfold in BAL fluid in the nasally vaccinated subjects, and the major part of IgG had most probably been transferred from serum. Since the specific IgG response in serum was lower in the individuals vaccinated orally, the IgG response in BAL fluid in this group was also lower and not significant. In conclusion, nasal immunization is also preferable to the oral route when vaccinating against lower respiratory tract infections, and a systemic immune response is considerably more important in the lower than in the upper respiratory tract. Moreover, both nasal and oral immunizations were able to stimulate 6- to 10-fold specific IgA and IgG responses in urine in about half of the individuals, which indicates that distant mucosal vaccination might be used to prevent adhesion of pathogens to the urogenital tract.