bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytokine Kinetics and Other Host Factors in Response to Pneumococcal Pulmonary Infection in Mice
Authors: Bergeron Yves , Ouellet Nathalie , Deslauriers Anne-Marie , Simard Marie , Olivier Martin , Bergeron Michel G.
Published: December 2019

There is a need for more insight into the pathogenesis ofStreptococcus pneumoniaepneumonia, as the fatality rate associated with this disease remains high despite appropriate antibiotherapy. The host response to pneumococci was investigated after intranasal inoculation of CD1 mice with 107log-phase CFU of bacteria. We identified five major pathogenesis steps from initial infection to death. In step 1 (0 to 4 h), there was ineffective phagocytosis by alveolar macrophages, with concurrent release of tumor necrosis factor alpha (TNF), interleukin-6 (IL-6), and nitric oxide (NO) in bronchoalveolar lavage (BAL) fluid, TNF, IL-6, and interleukin-1 alpha (IL-1) in lung tissues, and IL-6 in serum, which were associated with tachypnea and hemoconcentration. In step 2 (4 to 24 h), bacterial growth in alveoli and polymorphonuclear cell recruitment from bloodstream to lung tissue (high myeloperoxidase levels) to alveoli were associated with high release of all three cytokines and leukotriene B4(LTB4) in tissue and BAL fluid, as well as transient spillover of IL-1 in serum. In step 3 (24 to 48 h), despite downregulation of TNF and IL-1 in BAL fluid and lungs, there was appearance of injury to alveolar ultrastructure, edema to interstitium, and increase in lung weight as well as regeneration of type II pneumocytes and increased secretion of surfactant; bacteria progressed from alveoli to tissue to blood, and body weight loss occurred. In step 4 (48 to 72 h), strong monocyte recruitment from blood to alveoli was associated with high NO release in tissue and BAL fluid, but there was also noticeable lymphocyte recruitment and leukopenia; bacteremia was associated with TNF and IL-6 release in blood and thrombocytopenia. In step 5 (72 to 96 h), severe airspace disorganization, lipid peroxidation (high malondialdehyde release in BAL fluid), and diffuse tissue damage coincided with high NO levels; there was further increase in lung weight and bacterial growth, loss in body weight, and high mortality rate. Delineation of the sequential steps that contribute to the pathogenesis of pneumococcal pneumonia may generate markers of evolution of disease and lead to better targeted intervention.
Different Classes of Proteoglycans Contribute to the Attachment of
Borrelia burgdorferi
to Cultured Endothelial and Brain Cells
Authors: Leong John M. , Wang Hong , Magoun Loranne , Field Jodie A. , Morrissey Pamela E. , Robbins Douglas , Tatro Jeffrey B. , Coburn Jenifer , Parveen Nikhat
Published: December 2019

The Lyme disease spirochete,
Borrelia burgdorferi
, infects multiple tissues, such as the heart, joint, skin, and nervous system and has been shown to recognize heparan sulfate and dermatan sulfate proteoglycans. In this study, we examined the contribution of different classes of proteoglycans to the attachment of the infectious
B. burgdorferi
strain N40 to several immortalized cell lines and primary cultured cells, including endothelial cells and brain cells. Bacterial attachment was inhibited by exogenous proteoglycans or by treatment of host cells with inhibitors of proteoglycan synthesis or sulfation, indicating that proteoglycans play a critical role in bacterial binding to diverse cell types. Binding to primary bovine capillary endothelial cells or a human endothelial cell line was also inhibited by digestion with heparinase or heparitinase but not with chondroitinase ABC. In contrast, binding to glial cell-enriched brain cell cultures or to a neuronal cell line was inhibited by all three lyases. Binding of strain N40 to immobilized heparin could be completely inhibited by dermatan sulfate, and conversely, binding to dermatan sulfate could be completely blocked by heparin. As measured by 50% inhibitory dose, heparin was a better inhibitor of binding than dermatan sulfate, regardless of whether the substrate was heparin or dermatan sulfate. These results are consistent with the hypotheses that the species of proteoglycans recognized by
B. burgdorferi
vary with cell type and that bacterial recognition of different proteoglycans is mediated by the same bacterial molecule(s).
Immune Responses of Specific-Pathogen-Free Mice to Chronic
Helicobacter pylori
(Strain SS1) Infection
Authors: Ferrero Richard L. , Thiberge Jean-Michel , Huerre Michel , Labigne Agnès
Published: December 2019

A model permitting the establishment of persistent
Helicobacter pylori
infection in mice was recently described. To evaluate murine immune responses to
H. pylori
infection, specific-pathogen-free Swiss mice (
n
= 50) were intragastrically inoculated with 1.2 × 10
7
CFU of a mouse-adapted
H. pylori
isolate (strain SS1). Control animals (
n
= 10) received sterile broth medium alone. Animals were sacrificed at various times, from 3 days to 16 weeks postinoculation (p.i.). Quantitative culture of gastric tissue samples from inoculated mice demonstrated bacterial loads of 4.0 × 10
4
to 8 × 10
6
CFU per g of tissue in the animals. Infected mice had
H. pylori
-specific immunoglobulin M (IgM) and IgG antibodies in serum (at day 3 p.i.) and IgG and IgA antibodies in their gastric contents (weeks 4 and 16 p.i.) and saliva (week 16 p.i.). Mucosal IgM antibodies were not detected. Histological examination of the gastric mucosae from control and infected mice revealed mild chronic gastritis, characterized by the presence of polymorphoneutrophil cell infiltrates and submucosal lymphoid aggregates, in infected animals at 16 weeks p.i. Differences in the quantities of IgG1 and IgG2a subclass antibodies detected in the sera of mouse strains (Swiss, BALB/c, and C57BL/6) infected by
H. pylori
suggested that host factors influence the immune responses induced against this bacterium in the host. In conclusion, immune responses to
H. pylori
infection in mice, like those in chronically infected humans, appear to be ineffective in resolving the infection.
Cytokine Involvement in Immunomodulatory Activity Affected byCandida albicansMannan
Authors: Wang Yanyun , Li Shaokang P. , Moser Stephen A. , Bost Kenneth L. , Domer Judith E.
Published: December 2019

Candida albicansmannoprotein (MAN) administered intravenously to mice stimulates the production of splenic CD8+effector cells which downregulate delayed hypersensitivity (DH) in immunized mice. Cytokine involvement in the induction and/or elicitation of downregulation was studied by (i) examining murine splenocytes qualitatively for mRNA for interleukin-2 (IL-2), IL-4, IL-10, IL-12p40, and gamma interferon (IFN-γ), (ii) quantitating splenocyte mRNA for IL-12p40 by quantitative-competitive reverse transcriptase-mediated PCR, and (iii) measuring serum levels of IL-12p40 and IL-12p70 by capture enzyme-linked immunosorbent assay, each performed at selected intervals over 96 h after giving MAN. Further, the effect of in vivo administration of anti-IL-4 on the induction and elicitation of MAN-specific DH in MAN-treated mice was measured. Expression of IL-12p40 mRNA in the spleen was reduced to near 0 during the first 24 h but rebounded thereafter. Transcripts for IL-10 were present throughout the 96-h period, whereas those for IL-4 and IFN-γ were either weak or undetectable prior to 24 to 48 h. In vivo administration of anti-IL-4 partially abrogated the downregulatory effect of MAN only when given at the time of MAN administration. Serum levels of IL-12p40, but not IL-12p70, were increased by 24 h and maximal at 48 h. The antagonistic effect of IL-12p40 could contribute to the mechanism(s) for downregulation of DH. Moreover, IL-10, IL-4, and/or IFN-γ, interacting with MAN-activated cells in the absence of biologically active IL-12, may induce the production of CD8+downregulatory effector cells. Partial abrogation of downregulatory activity in animals treated with anti-IL-4 at the time of induction of such activity lends support to this hypothesis.
Mutation of
invH
, but Not
stn
, Reduces
Salmonella
-Induced Enteritis in Cattle
Authors: Watson Patricia R. , Galyov Edouard E. , Paulin Sue M. , Jones Philip W. , Wallis Tim S.
Published: December 2019

The induction of secretory and inflammatory responses in calves by
Salmonella typhimurium
and
Salmonella dublin
strains was compared, and the effects of mutations in the
invH
and
stn
genes were assessed.
S. typhimurium
induced greater secretory and inflammatory responses than
S. dublin
in bovine ileal loops, despite the fact that these serotypes were recovered from bovine ileal mucosa in comparable numbers (P. R. Watson, S. M. Paulin, A. P. Bland, P. W. Jones, and T. S. Wallis, Infect. Immun. 63:2743–2754, 1995). These results implicate serotype-specific factors other than, or in addition to, intestinal invasion in the induction of enteritis. The secretory and inflammatory responses induced by
S. typhimurium
and
S. dublin
in bovine ligated ileal loops were not significantly altered by mutation of
stn
, which suggests that
stn
does not have a major role in
Salmonella
-induced enteritis. The
invH
mutation significantly reduced the secretory and inflammatory responses induced in bovine ileal loops, and this correlated with a reduction in the severity of enteritis following oral inoculation of calves. The attenuation associated with the
invH
mutation did not appear to be due to an increased susceptibility to the innate host defense mechanisms, because the resistance of
S. typhimurium
to the bactericidal action of either bovine polymorphonuclear leukocytes or bovine serum was not significantly altered. However, lysis of macrophages following infection with
S. typhimurium
was significantly reduced by the
invH
mutation. The
invH
mutation prevented the normal secretion of several proteins, including SipC, by
S. typhimurium
, indicating that the function of the
inv-spa
-encoded type III protein secretion system was disrupted. Taken together, these observations implicate
inv-spa
-dependent effectors in mediation of
Salmonella
-induced enteritis in cattle. Clearly, however, other undefined serotype-specific virulence factors are also involved in
Salmonella
-induced enteritis.
Pseudomonas aeruginosa
Invasion and Cytotoxicity Are Independent Events, Both of Which Involve Protein Tyrosine Kinase Activity
Authors: Evans David J. , Frank Dara W. , Finck-Barbançon Viviane , Wu Christine , Fleiszig Suzanne M. J.
Published: December 2019

Pseudomonas aeruginosa
clinical isolates exhibit invasive or cytotoxic phenotypes. Cytotoxic strains acquire some of the characteristics of invasive strains when a regulatory gene,
exsA
, that controls the expression of several extracellular proteins, is inactivated.
exsA
mutants are not cytotoxic and can be detected within epithelial cells by gentamicin survival assays. The purpose of this study was to determine whether epithelial cell invasion precedes and/or is essential for cytotoxicity. This was tested by measuring invasion (gentamicin survival) and cytotoxicity (trypan blue staining) of PA103 mutants deficient in specific
exsA
-regulated proteins and by testing the effect of drugs that inhibit invasion for their effect on cytotoxicity. A transposon mutant in the
exsA
-regulated extracellular factor
exoU
was neither cytotoxic nor invasive. Furthermore, several of the drugs that inhibited invasion did not prevent cytotoxicity. These results show that invasion and cytotoxicity are mutually exclusive events, inversely regulated by an
exsA
-encoded invasion inhibitor(s). Both involve host cell protein tyrosine kinase (PTK) activity, but they differ in that invasion requires Src family tyrosine kinases and calcium-calmodulin activity. PTK inhibitor drugs such as genistein may have therapeutic potential through their ability to block both invasive and cytotoxicity pathways via an action on the host cell.
Role of Intimin and Bundle-Forming Pili in Enteropathogenic
Escherichia coli
Adhesion to Pediatric Intestinal Tissue In Vitro
Authors: Hicks Susan , Frankel Gad , Kaper James B. , Dougan Gordon , Phillips Alan D.
Published: December 2019

Attaching and effacing (A/E) lesion formation is central to enteropathogenic
Escherichia coli
(EPEC) pathogenesis. In vitro experiments with human epithelial cell lines have implicated virulence plasmid-encoded bundle-forming pili (BFP) in initial binding and intimin in intimate attachment and A/E lesion formation. This study investigated the role of BFP and intimin in EPEC interactions with pediatric small intestinal biopsy tissue in in vitro organ culture. Organ culture infections (2 to 8 h) were performed with E2348/69 (a wild-type EPEC O127:H6 clinical isolate) and E2348/69 derivatives including CVD206 (
eae
deficient), CVD206(pCVD438) (
eae
-complemented CVD206), CVD206(pCVD438/01) (expressing intimin, which is nonfunctional due to a single amino acid substitution), JPN15 (spontaneous EPEC adherence factor virulence plasmid-cured E2348/69), and 31-6-1(1) (E2348/69 with a Tn
phoA
insertion inactivation mutation in the virulence plasmid-encoded
bfpA
gene). Scanning and transmission electron microscopy revealed that after 8 h E2348/69 and CVD206(pCVD438) (both Int
+
BFP
+
) adhered to all specimens, causing A/E lesions with surrounding microvillous elongation. JPN15 and 31-6-1(1) (both Int
+
BFP
−
) adhered and caused A/E lesions although bacteria adhered in “flat,” two-dimensional groups. CVD206 and CVD206(pCVD438/01) (both Int
−
BFP
+
) did not adhere to any sample, and no pathological tissue changes were seen. Thus, in human intestinal organ culture, BFP do not appear to be involved in the initial stages of EPEC nonintimate adhesion but are implicated in the formation of complex, three-dimensional colonies via bacterium-bacterium interactions. Intimin appears to play an essential role in establishing colonization of EPEC on pediatric small intestinal tissue.
Phagocytosis of the Malarial Pigment, Hemozoin, Impairs Expression of Major Histocompatibility Complex Class II Antigen, CD54, and CD11c in Human Monocytes
Authors: Schwarzer Evelin , Alessio Massimo , Ulliers Daniela , Arese Paolo
Published: December 2019

InPlasmodium falciparummalaria, large proportions of resident macrophages and circulating monocytes and leukocytes contain massive amounts of the malarial pigment, hemozoin. Previous studies have shown that important functions (e.g., the generation of the oxidative burst, the ability to repeat phagocytosis, and protein kinase C activity) were severely impaired in hemozoin-loaded monocytes. Expression of membrane antigens directly involved in the immune response and in the phagocytic process, and/or under protein kinase C control, in hemozoin-loaded human monocytes was studied. Expression of major histocompatibility complex (MHC) class II after gamma interferon stimulation was blocked in hemozoin-loaded monocytes at the protein expression and gene transcription levels but was preserved in control monocytes loaded with opsonized latex beads or anti-D(Rho)-immunoglobulin G (IgG)-opsonized human erythrocytes. Expression of CD54 (intracellular adhesion molecule 1) and CD11c (p150,95 integrin) was also decreased in hemozoin-loaded monocytes. Expression of MHC class I, CD16 (low-affinity Fc receptor for aggregated IgG), CD32 (low-affinity Fc receptor for aggregated IgG), CD64 (high-affinity receptor for IgG), CD11b (receptor for complement component iC3b [CR3]), CD35 (receptor for complement components C3b and C4b [CR1]), and CD36 (non-class-A scavenger receptor) was not specifically affected by hemozoin loading. These results suggest that hemozoin loading may contribute to the impairment of the immune response and the derangement of antigen presentation reported in previous studies ofP. falciparummalaria.
Divergent Signal Transduction Responses to Infection with Attaching and Effacing
Escherichia coli
Authors: Ismaili Arif , McWhirter Elaine , Handelsman Michelle Y. C. , Brunton James L. , Sherman Philip M.
Published: December 2019

Shiga toxin-producing
Escherichia coli
(STEC) O157:H7 is an attaching and effacing pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome. Although this organism causes adhesion pedestals, the cellular signals responsible for the formation of these lesions have not been clearly defined. We have shown previously that STEC O157:H7 does not induce detectable tyrosine phosphorylation of host cell proteins upon binding to eukaryotic cells and is not internalized into nonphagocytic epithelial cells. In the present study, tyrosine-phosphorylated proteins were detected under adherent STEC O157:H7 when coincubated with the non-intimately adhering, intimin-deficient, enteropathogenic
E. coli
(EPEC) strain CVD206. The ability to be internalized into epithelial cells was also conferred on STEC O157:H7 when coincubated with CVD206 ([158 ± 21] % of control). Neither the ability to rearrange phosphotyrosine proteins nor that to be internalized into epithelial cells was evident following coincubation with another STEC O157:H7 strain or with the nonsignaling
espB
mutant of EPEC.
E. coli
JM101(pMH34/pSSS1C), which overproduces surface-localized O157 intimin, also rearranged tyrosine-phosphorylated and cytoskeletal proteins when coincubated with CVD206. In contrast, JM101(pMH34/pSSS1C) demonstrated rearrangement of cytoskeletal proteins, but not tyrosine-phosphorylated proteins, when coincubated with intimin-deficient STEC (strains CL8KO1 and CL15). These findings indicate that STEC O157:H7 forms adhesion pedestals by mechanisms that are distinct from those in attaching and effacing EPEC. Taken together, these findings point to diverging signal transduction responses to infection with attaching and effacing bacterial enteropathogens.
Enhancement of Lipopolysaccharide-Induced Neutrophil Oxygen Radical Production by Tumor Necrosis Factor Alpha
Authors: Jersmann Hubertus P. A. , Rathjen Deborah A. , Ferrante Antonio
Published: December 2019

Although tissues become exposed to both exogenous and endogenous cell-activating mediators during infection, there is little appreciation of the effects of subjecting cells to multiple mediators. We examined the hypothesis that the response of neutrophils to bacterial lipopolysaccharide (LPS) is significantly altered in the presence of the endogenous mediator tumor necrosis factor alpha (TNF). The data showed that human neutrophils pretreated with TNF for 10 to 30 min, displayed significantly enhanced superoxide production in response to LPS (from eitherEscherichia coliK-235 orE. coli0127:B8), measured as lucigenin-dependent chemiluminescence (CL), seen as an increase in the initial peak rate as well as the total CL accumulated over the incubation period. TNF amplified the response to LPS at 1 to 100 U of TNF/106neutrophils and was able to enhance the response to a wide range of concentrations of LPS (0.01 to 1,000 ng/ml). The TNF-induced increase in the LPS response was paralleled by an increase in LPS binding to the neutrophils, which could be abrogated by an anti-CD14 monoclonal antibody. The results demonstrate that TNF significantly increases the LPS-induced release of oxygen radicals in neutrophils through the upregulation of cell surface CD14.
Adherence to and Penetration of Human Intestinal Caco-2 Epithelial Cell Monolayers by
Pseudomonas aeruginosa
Authors: Hirakata Yoichi , Izumikawa Kohichi , Yamaguchi Toshiyuki , Igimi Shizunobu , Furuya Nobuhiko , Maesaki Shigefumi , Tomono Kazunori , Yamada Yasuaki , Kohno Shigeru , Yamaguchi Keizo , Kamihira Shimeru
Published: December 2019

Clinical isolates of
Pseudomonas aeruginosa
from blood adhered to and penetrated intestinal Caco-2 cell monolayers to a greater degree than did isolates from sputum, with a concomitant drastic decrease in transepithelial electrical resistance. PAO-PR1, an avirulent exotoxin A mutant of PAO1, did not cause a decrease in the resistance. The Caco-2 monolayer system may be useful for the evaluation of certain
P. aeruginosa
virulence factor activities.
Expression of the
Candida albicans
Gene
ALS1
in
Saccharomyces cerevisiae
Induces Adherence to Endothelial and Epithelial Cells
Authors: Fu Yue , Rieg Günter , Fonzi William A. , Belanger Paul H. , Edwards John E. , Filler Scott G.
Published: December 2019

To identify genes encoding adhesins that mediate the binding of
Candida albicans
to endothelial cells, a genomic library from this organism was constructed and used to transform
Saccharomyces cerevisiae
. These transformed organisms were screened for adherence to endothelial cells, and a highly adherent clone was identified. The adherence of this clone to endothelial cells was over 100-fold greater than that of control
S. cerevisiae
transformed with the empty plasmid. This clone also exhibited enhanced adherence to epithelial cells. The
C. albicans
gene contained within this clone was found to be
ALS1
. These results indicate that
ALS1
may encode a candidal adhesin.
Hemoglobin-Induced Binding of
Candida albicans
to the Cell-Binding Domain of Fibronectin Is Independent of the Arg-Gly-Asp Sequence
Authors: Yan Sizhuang , Rodrigues Rui G. , Roberts David D.
Published: December 2019

Hemoglobin specifically induces fibronectin (FN) binding to the pathogenic yeast
Candida albicans
. When grown in the complex medium Sabouraud broth,
C. albicans
expresses receptors that bind to several domains of FN. Growth in defined medium supplemented with 0.1% hemoglobin, however, enhanced the binding of FN to a single class of receptors, with a

K
d

= 4.6 × 10
−8
M. Competitive binding assays using recombinant and proteolytic fragments of FN revealed that the cell-binding domain mediated this interaction. A recombinant 40-kDa fragment of FN consisting of type III repeats 9 to 13 had an inhibitory activity similar to that of the entire 120-kDa cell-binding domain, indicating that the C-terminal portion of the cell-binding domain contains the binding site. A recombinant 33-kDa fragment of the cell-binding domain and a 33-kDa fragment with the RGD sequence deleted had the same inhibitory activities, demonstrating that the RGD sequence recognized by some mammalian integrins is not required. The addition of hemoglobin to the culture medium also enhanced
Candida
cell adhesion to immobilized FN and to 120- and 40-kDa fragments of FN but not to the collagen-binding or fibrin I domains. Using ligand protection, we identified a surface protein from
C. albicans
with an apparent molecular mass of 55 kDa that was protected by both FN and the 40-kDa fragment derived from the cell-binding domain. Therefore, hemoglobin both induces FN binding and changes the relative affinities of
C. albicans
for the cell- and collagen-binding domains of FN.
Comparisons between Colony Phase Variation of
Neisseria gonorrhoeae
FA1090 and Pilus, Pilin, and S-Pilin Expression
Authors: Long Cynthia D. , Madraswala Rashida N. , Seifert H. Steven
Published: December 2019

The gonococcal pilus is a primary virulence factor, providing the initial attachment of the bacterial cell to human mucosal tissues. Pilin, the major subunit of the pilus, can carry a wide spectrum of primary amino acid sequences which are generated by the action of a complex antigenic variation system. Changes in the pilin amino acid sequence can produce different pilus-dependent colony morphotypes, which have been previously shown to reflect phase variation of pili on the bacterial cell surface. In this study, we further examined the relationships between changes in pilus-dependent colony morphology, pilin sequence, pilus expression, and pilus function in
Neisseria gonorrhoeae
FA1090. A group of FA1090 colony variants expressed different pilin sequences and demonstrated different levels of pilin, S-pilin, and pilus expression. The analysis of these colony variants shows that they do not represent two distinct phases of pilus expression, but that changes in pilin protein sequence produce a spectrum of S-pilin production, pilus expression, and pilus aggregation levels. These different levels of pilus expression and aggregation influence not only colony morphology but also DNA transformation efficiency and epithelial cell adherence.
Participation of Parasite Surface Glycoproteins in Antibody-Mediated Protection of Epithelial Cells against
Trichinella spiralis
Authors: McVay Catherine S. , Tsung Allan , Appleton Judith
Published: December 2019

The L1 stage of the parasitic nematode
Trichinella spiralis
displays on its surface glycoproteins that are immunologically cross-reactive with several larval excretory-secretory (ES) products. The basis for the cross-reactivity is tyvelose, the terminal residue on the complex glycans shared by these surface and ES glycoproteins. In neonatal rats, tyvelose-specific monoclonal antibodies mediate the expulsion of larvae from the intestine. The aim of the studies described in this report was to determine how antibody binding to larval surfaces contributes to expulsion. In these experiments, which involve an in vitro assay of epithelial cell invasion, surface proteins on living larvae were biotinylated to distinguish them from ES products. Biotinylated and nonbiotinylated larvae were cocultured with avidin, biotin-specific antibodies, or anti-tyvelose monoclonal antibodies. Biotinylated larvae cultured with avidin or biotin-specific antibodies invaded Madin-Darby canine kidney (MDCK) cells equally as well as biotinylated larvae cultured with medium alone. Anti-tyvelose monoclonal antibodies were highly protective in this assay; however, biotinylation of larval surfaces hindered the ability of anti-tyvelose monoclonal antibodies to prevent larval invasion of epithelial cells. This correlated with a reduction in the binding of anti-tyvelose antibody to biotinylated larval surfaces. Our results indicate that antibody binding to surface glycoproteins contributes to protection against
T. spiralis
invasion but that surface binding alone is not sufficient for protection. Our findings support the notion that protection is effected by cross-linking of ES products to surface antigens.
The Cell Wall-Associated Glyceraldehyde-3-Phosphate Dehydrogenase of
Candida albicans
Is Also a Fibronectin and Laminin Binding Protein
Authors: Gozalbo Daniel , Gil-Navarro Inés , Azorín Inmaculada , Renau-Piqueras Jaime , Martínez José P. , Gil M. Luisa
Published: December 2019

By immunoelectron microscopy with a polyclonal antibody against the cytosolic glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from
Candida albicans
(anti-GAPDH PAb), the protein was clearly detected at the outer surface of the cell wall, particularly on blastoconidia, as well as in the cytoplasm. Intact blastoconidia were able to adhere to fibronectin and laminin immobilized on microtiter plates, and this adhesion was markedly reduced by both the anti-GAPDH PAb and soluble GAPDH from
Saccharomyces cerevisiae
. In addition, semiquantitative flow cytometry analysis with the anti-GAPDH PAb showed a decrease in antibody binding to cells in the presence of soluble fibronectin and laminin. Purified cytosolic
C. albicans
GAPDH was found to bind to fibronectin and laminin in a ligand Western blot assay. These observations suggest that the cell wall-associated form of the GAPDH in
C. albicans
could be involved in mediating adhesion of fungal cells to fibronectin and laminin, thus contributing to the attachment of the microorganism to host tissues and to the dissemination of
Candida
infection.
Nitric Oxide is Produced byCowdria ruminantium-Infected Bovine Pulmonary Endothelial Cells In Vitro and Is Stimulated by Gamma Interferon
Authors: Mutunga Mbithe , Preston Patricia M. , Sumption Keith J.
Published: December 2019

Nitric oxide (NO) is a labile inorganic free radical produced by NO synthase from the substratel-arginine in various cells and tissues including endothelial cells. A substantial elevation of nitrite levels indicative of NO production occurred in cultures ofCowdria ruminantium-infected bovine pulmonary endothelial cells (BPEC) incubated in medium alone. Exposure of the infected cultures to recombinant bovine gamma interferon (BorIFN-γ) resulted in more rapid production of NO, reduced viability ofC. ruminantium, and induction of endothelial cell death. Significant inhibition of NO production was noted after addition of the NO synthase inhibitorN-monomethyl-l-arginine (l-NMMA), indicating that the increase in production occurred via the inducible NO synthase pathway. Reduction in the infectivity ofC. ruminantiumelementary bodies (EBs) occurred in a dose-dependent manner after incubation with the NO donor moleculeS-nitroso-N-acetyl-dl-penicillamine (SNAP) prior to infection of endothelial cells. The level of infection in cultures maintained in SNAP was reduced in a dose-dependent manner with significant negative correlation between the final level of infection on day 7 and the level of SNAP (r= −0.96). It was established that pretreatment and cultivation ofC. ruminantiumEBs with the NO donor molecule SNAP reduced infectivity to cultures and viability of EBs with the implication that release of NO in vivo following infection of endothelial cells may have an effect upon the multiplication of the agent in the host animal and may be involved in the pathogenesis of heartwater through the effect of this molecule upon circulation.
Active and Passive Immunity againstBorrelia burgdorferiDecorin Binding Protein A (DbpA) Protects against Infection
Authors: Hanson Mark S. , Cassatt David R. , Guo Betty P. , Patel Nita K. , McCarthy Michael P. , Dorward David W. , Höök Magnus
Published: December 2019

Borrelia burgdorferi, the spirochete that causes Lyme disease, binds decorin, a collagen-associated extracellular matrix proteoglycan found in the skin (the site of entry for the spirochete) and in many other tissues. Two borrelial adhesins that recognize this proteoglycan, decorin binding proteins A and B (DbpA and DbpB, respectively), have recently been identified. Infection of mice by low-doseB. burgdorferichallenge elicited antibodies against DbpA and DbpB that were sustained at high levels, suggesting that these antigens are expressed in vivo. Scanning immunoelectron microscopy showed that DbpA was surface accessible on intact borreliae. Passive administration of DbpA antiserum protected mice from infection following challenge with heterologousB. burgdorferisensu stricto isolates, even when serum administration was delayed for up to 4 days after challenge. DbpA is the first antigen target identified that is capable of mediating immune resolution of early, localizedB. burgdorferiinfections. DbpA immunization also protected mice fromB. burgdorferichallenge; DbpB immunization was much less effective. DbpA antiserum inhibited in vitro growth of manyB. burgdorferisensu lato isolates of diverse geographic, phylogenetic, and clinical origins. In combination, these findings support a role for DbpA in the immunoprophylaxis of Lyme disease and suggest that DbpA vaccines have the potential to eliminate early-stageB. burgdorferiinfections.
Production of Proinflammatory Cytokines and Inflammatory Mediators in Human Intestinal Epithelial Cells after Invasion by
Trichinella spiralis
Authors: Li Chris K. F. , Seth Rashmi , Gray Trevor , Bayston Roger , Mahida Yashwant R. , Wakelin Derek
Published: December 2019

Epithelial cells are the first point of host contact for invasive intestinal pathogens and may initiate mucosal inflammatory responses via production of proinflammatory cytokines and mediators. The aim of the present study was to investigate in vitro the initial invasion of a parasitic nematode (
Trichinella spiralis
), to measure the early production of specific epithelial cytokines and inflammatory mediators after invasion, and to compare these responses with those to invasive bacteria. Monolayers of human colonic epithelial cell lines (HT29, T84, and Caco-2) were infected by
T. spiralis
or
Listeria monocytogenes
. Bile-activated infective larvae of
T. spiralis
invaded and migrated into the epithelial cell monolayers, leaving trails of dead cells. Transmission electron microscopy studies of damaged cells along the trail showed a progressive increase in size, disruption of cell membranes, loss or dilution of cytoplasmic proteins, and swelling of mitochondria and nuclei. However, no nuclear fragmentation was observed. With reverse transcription-PCR and an enzyme-linked oligonucleotide chemiluminescent assay, mRNA transcripts of interleukin-1β (IL-1β), IL-8, and epithelial neutrophil-activating peptide 78 were shown to increase in epithelial cells invaded by
T. spiralis
or
L. monocytogenes
, but only
L. monocytogenes
elicited increased inducible nitric oxide synthase (iNOS) mRNA. No increase in tumor necrosis factor alpha or transforming growth factor β mRNA was seen after
T. spiralis
invasion. Increased levels of IL-8 were also released from the basolateral surfaces of infected monolayers as detected by sandwich enzyme-linked immunosorbent assay. Induction and secretion of proinflammatory cytokines in epithelial cells after nematode or bacterial invasion may initiate the acute inflammatory response of the small intestine. The upregulation of iNOS in bacterial infections may contribute to mucosal defense and may also be associated with subsequent cell death, whereas different mechanisms appear to operate after nematode invasion.
TheLegionella pneumophila icmGCDJBFGenes Are Required for Killing of Human Macrophages
Authors: Purcell Mary , Shuman Howard A.
Published: December 2019

Previously, a collection of mutants ofLegionella pneumophilathat had lost the ability to multiply within and kill human macrophages was generated by Tn903dIIlacZ transposon mutagenesis and classified into DNA hybridization groups. A subset of these mutants was complemented by a plasmid, pMW100, containing a 13.5-kb genomic DNA insert. This plasmid restored the ability to multiply within and produce cytopathic effects on human macrophages to members of DNA hybridization groups II, IV, VI, and XVII. A region of the genomic insert of pMW100 was sequenced, and eight potential genes were identified and namedicmE,icmG,icmC,icmD,icmJ,icmB,icmF, andtphA. None of the genes encode potential protein products with significant homology to previously characterized proteins, except fortphA, whose product has significant homology to a family of metabolite/H+symport proteins from gram-negative bacteria. The positions of the Tn903dIIlacZ insertions within the genes were determined by nucleotide sequencing. No Tn903dIIlacZ insertions mapped toicmG,icmJ, ortphA; therefore, these loci were mutated to test whether they were required for macrophage killing. Complementation analysis was used to evaluate the roles of the potential gene products and provide information on the organization of transcriptional units within the region. The results indicate that all identified open reading frames excepttphAare required for killing of human macrophages.
Identification and Characterization of Heparin Binding Regions of the Fim2 Subunit ofBordetella pertussis
Authors: Geuijen Cecile A. W. , Willems Rob J. L. , Hoogerhout Peter , Puijk Wouter C. , Meloen Rob H. , Mooi Frits R.
Published: December 2019

Bordetella pertussisfimbriae bind to sulfated sugars such as heparin through the major subunit Fim2. The Fim2 subunit contains two regions, designated H1 and H2, which show sequence similarity with heparin binding regions of fibronectin, and the role of these regions in heparin binding was investigated with maltose binding protein (MBP)-Fim2 fusion proteins. Deletion derivatives of MBP-Fim2 showed that both regions are important for binding to heparin. The role of H2 in heparin binding was confirmed by site-directed mutagenesis in which basic amino acids were replaced by alanine. These studies revealed that Lys-186 and Lys-187 are important for heparin binding of MBP-Fim2, whereas Arg-179 is not required. Peptides derived from H1 and H2 (pepH1 and pepH2) also showed heparin binding activity. Using a series of peptides, in each of which a different basic amino acid was substituted for alanine, we demonstrated that the structural requirements for heparin binding differ significantly among pepH1 and pepH2 peptides. A Pepscan analysis of Fim2 revealed regions outside H1 and H2 which bind heparin and showed that not only basic amino acids but also tyrosines may be important for binding to sulfated sugars. A comparison of the heparin binding regions of Fim2 with homologous regions of Fim3 and FimX, two closely related but antigenically distinct fimbrial subunits, showed that basic amino acids and tyrosines are generally conserved. The major heparin binding regions identified in Fim2 are part of epitopes recognized by human antibodies, suggesting that the heparin binding regions are exposed at the fimbrial surface and are immunodominant. SinceB. pertussisfimbriae show weak serological cross-reactivity, the differences in primary structure in the heparin binding regions of Fim2, Fim3, and FimX may affect antibody binding but not heparin binding, allowing the bacteria to evade antibody-mediated immunity by switching the fimbrial gene expressed.
Soluble CD14 Activates Monocytic Cells Independently of Lipopolysaccharide
Authors: Landmann Regine , Link Susanne , Sansano Sebastiano , Rajacic Zarko , Zimmerli Werner
Published: December 2019

The glycoprotein CD14 acts as a receptor for lipopolysaccharide (LPS), either when anchored in the myeloid cell membrane (mCD14) or as a soluble molecule (sCD14) in serum. sCD14-LPS complexes activate cells devoid of mCD14. However, the role of sCD14 independent of LPS is unknown. Therefore, the effect of sCD14 on monocyte functions was investigated in the monocytic cell lines THP1 and Mono Mac 6 and in fresh human monocytes. Under serum-free conditions, endotoxin-free human recombinant sCD141–348(rsCD141–348) induced tumor necrosis factor alpha (TNF-α). The TNF-α effect was stronger in THP1 cells than in Mono Mac 6 cells or monocytes. It was dose dependent, with a maximum at 1 μg/ml, and time dependent, with a maximum after 2 h. sCD14 purified from urine had the same cytokine-activating capacity. In contrast, C-terminally truncated rsCD141–152was inactive. The rsCD14 effect was not due to LPS contamination, since it was resistant to polymyxin and lipid IVa but sensitive to heat and trypsin. The rsCD14-induced cytokine induction was blocked by preincubation of rsCD14 with a monoclonal anti-CD14 antibody that did not recognize the LPS-binding site. Release of the TNF-α disappeared upon pretreatment of rsCD14 in 50% plasma or in complete, heat-inactivated or sCD14-depleted serum. Moreover, cytokine production was no longer observed when rsCD14 was pretreated with thrombocytes. The thrombocyte effect was dose and time dependent. In conclusion, sCD14 is able to activate myeloid cells, and the effect is prevented by the presence of plasma, serum, or thrombocytes.
Effects of the Antioxidant α-Lipoic Acid on Human Umbilical Vein Endothelial Cells Infected with
Rickettsia rickettsii
Authors: Eremeeva Marina E. , Silverman David J.
Published: December 2019

Rickettsia rickettsii
infection of endothelial cells is manifested in very distinctive changes in cell morphology, consisting of extensive dilatation of the membranes of the endoplasmic reticulum and outer nuclear envelope and blebbing of the plasma membrane, as seen by transmission electron microscopy (D. J. Silverman, Infect. Immun. 44:545–553, 1984). These changes in cellular architecture are thought to be due to oxidant-mediated cell injury, since their occurrence correlates with dramatic alterations in cellular metabolism, particularly with regard to antioxidant systems. In this study, it was shown that
R. rickettsii
infection of human umbilical vein endothelial cells resulted in a significant depletion of intracellular reduced glutathione (thiol) content at 72 and 96 h and decreased glutathione peroxidase activity at 72 h postinfection. Infected cells displayed a dramatic increase in the concentration of intracellular peroxides by 72 h. Supplementation of the cell culture medium with 100, 200, or 500 μM α-lipoic acid, a metabolic antioxidant, after inoculation with
R. rickettsii
restored the intracellular levels of thiols and glutathione peroxidase and reduced the intracellular peroxide levels in infected cells. These effects were dose dependent. Treated infected monolayers maintained better viability at 96 h after inoculation with
R. rickettsii
than did untreated infected cells. Moreover, supplementation of the cell culture medium with 100 μM α-lipoic acid for 72 h after infection prevented the occurrence of morphological changes in the infected cells. The presence of 100 or 200 μM α-lipoic acid did not influence rickettsial growth in endothelial cells, nor did it affect the ability of
R. rickettsii
to form lytic plaques in Vero cells. Treatment with 500 μM α-lipoic acid decreased by 50% both the number and size of lytic plaques in Vero cells, and it also decreased the recovery of viable rickettsiae from endothelial cells. However, under all treatment conditions, a significant number of rickettsiae could be detected microscopically. Furthermore, the rickettsiae apparently retained their capacity for intracellular movement, since they possessed long polymerized actin tails after 72 and 96 h of treatment regardless of the concentration of α-lipoic acid used. Since α-lipoic acid does not seem to exhibit direct antirickettsial activity except with long-term exposure at very high concentrations, the mechanism of its protective activity for endothelial cells infected with rickettsiae may involve complex changes in cellular metabolism that only indirectly affect rickettsiae.
Protection against Lethal Murine Coccidioidomycosis by a Soluble Vaccine from Spherules
Authors: Zimmermann C. Roger , Johnson Suzanne M. , Martens Gregory W. , White Andrew G. , Zimmer Barbara L. , Pappagianis Demosthenes
Published: December 2019

The formaldehyde-killed, whole-spherule vaccine, which is protective against lethal challenge of laboratory animals withCoccidioides immitis, was fractionated. It yielded a soluble, multicomponent, subcellular fraction termed the 27K vaccine. This vaccine, when it was accompanied by adjuvant, protected mice against lethal intranasal and intravenous challenge withC. immitis.
Chlamydia trachomatisInfection of Human Mesothelial Cells Alters Proinflammatory, Procoagulant, and Fibrinolytic Responses
Authors: van Westreenen Mireille , Pronk Apollo , Diepersloot Rob J. A. , de Groot Philip G. , Leguit Piet
Published: December 2019

In this study we demonstrate the capability ofChlamydia trachomatisto infect cultured human mesothelial cell (MC) monolayers and to induce the production of the proinflammatory cytokines interleukin 1β (IL-1β) and IL-8. Seventy-two hours after initial infection, both the procoagulant activity of MC and the activity of the fibrinolytic inhibitor (plasminogen activator inhibitor 1) in the supernatants were enhanced. These findings support the hypothesis that provoked proinflammatory responses contribute to the development of complications after chlamydial infection.
Clot Formation by Group A Streptococci
Authors: Donabedian Haig , Boyle Michael D. P.
Published: December 2019

Group A streptococci of several different M serotypes can cause human plasma to clot in nutrient-poor media. Addition of glucose to the medium prevents clot formation. Once formed, clots are stable for several days and can be lysed on addition of exogenous streptokinase or urokinase. Clot lysis can also be achieved by addition of glucose to a clot containing wild-type group A streptococci but not clots containing an isogenic mutant in which the
ska
gene was inactivated.
Lipoprotein(a) Inhibits Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Production by Human Mononuclear Cells
Authors: Netea Mihai G. , de Bont Natasja , Demacker Pierre N. M. , Kullberg Bart Jan , Jacobs Liesbeth E. H. , Verver-Jansen Trees J. G. , Stalenhoef Anton F. H. , Van der Meer Jos W. M.
Published: December 2019

Lipoproteins can bind lipopolysaccharide (LPS) and decrease LPS-stimulated cytokine production. Lipoprotein(a) [Lp(a)] was as potent as low-density lipoproteins (LDL) in inhibiting LPS-stimulated tumor necrosis factor synthesis by human mononuclear cells. The kinetics of LPS inhibition by Lp(a) was similar to that of LDL. This suggests that circulating Lp(a) may be an important factor determining the amplitude of the response to LPS in humans.
Roles for Tumor Necrosis Factor and Gamma Interferon in Resistance to Enteric Listeriosis
Authors: Beretich Guy R. , Carter Philip B. , Havell Edward A.
Published: December 2019

Listeria monocytogenesnormally infects the host by translocating from the intestinal lumen. Experiments were carried out to determine if, when, and where tumor necrosis factor (TNF) and gamma interferon (IFN-γ) function in antibacterial resistance during enteric listeriosis. Groups of normal mice and severe combined immunodeficient (SCID) mice were injected with neutralizing monoclonal antibodies (MAb) specific for each cytokine and then inoculated intragastrically withL. monocytogenes. The course of infection was monitored by enumerating listeriae in gut-associated lymphoid tissues, livers, and spleens. By the third day of infection, bacterial numbers in infected tissues and organs were greatly exacerbated in all mice treated with anti-TNF MAb, whereas bacterial numbers in the organs of mice treated with anti-IFN-γ MAb did not differ from those present in the respective organs of control mice. However, by the fifth day of infection, bacterial numbers in the organs of anti-IFN-γ MAb-treated normal mice and SCID mice were much greater than in the corresponding organs of control mice. Experiments withListeria-immune mice revealed that TNF and IFN-γ are involved in the expression of anti-Listeriamemory immunity; however, it was also found that the anti-IFN-γ MAb was relatively ineffective in inhibiting the expression of anti-Listeriaimmunity, whereas a polyclonal anti-IFN-γ was quite effective.
Mapping and Identification of the Major Cell Wall-Associated Components of
Mycobacterium leprae
Authors: M. Marques Maria Angela , Chitale Sadhana , Brennan Patrick J. , Pessolani Maria Cristina V.
Published: December 2019

Mycobacterium leprae
, an obligate intracellular pathogen, can be derived only from host tissue and thus affords the opportunity to study in vivo-expressed products responsible for the particular pathogenesis of leprosy. Despite considerable progress in the characterization of the proteins and secondary gene products of
M. leprae
, there is little information on the nature of the proteins associated with the cell envelope.
M. leprae
has been fractionated into its major subcellular components, cell wall, cytoplasmic membrane, and soluble cytosol. A number of biochemical markers, including diaminopimelic acid content, monosaccharide composition, mycolic acid, and glycolipid distribution, were applied to their characterization, and two-dimensional gel electrophoresis was used to map the component proteins. A total of 391 major proteins spots were resolved, and 8 proteins were identified based on their reactivity to a panel of monoclonal antibodies and/or relative pI size. Microsequencing of six protein spots present in the cell wall fraction allowed identification of new proteins, including the protein elongation factor EF-Tu and a homolog for the
Mycobacterium tuberculosis
MtrA response regulator. These results, together with previous studies, contribute to the progressive knowledge of the composition of the in vivo-expressed proteins of
M. leprae.