bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of Monocytes and Bacteria in
Staphylococcus epidermidis
Endocarditis
Authors: Bancsi Maurice J. L. M. F. , Veltrop Marcel H. A. M. , Bertina Rogier M. , Thompson Jan 
Published: December 2019

ABSTRACT

The endocardial vegetation which is formed in the course of bacterial endocarditis (BE) contains tissue factor (TF)-dependent procoagulant activity. Earlier studies showed that monocytes are the main source of TF in the vegetations. The TF activity (TFA) of vegetations isolated from
Streptococcus sanguis
-infected rabbits depended on the numbers of bacteria as well as monocytes in the vegetation. In this study, we investigated whether for
Staphylococcus epidermidis
, a frequent pathogen in BE, an effect similar to that found for
S. sanguis
could be shown. In vitro,
S. epidermidis
was found to stimulate TFA of fibrin adherent monocytes significantly. This stimulation was maximal at a bacterium-to-monocyte ratio of 7. In vivo, TFA was found to be significantly higher in
S. epidermidis
-infected than in sterile catheter-induced vegetations. Reduction of vegetational bacterial numbers by teicoplanin treatment lead to a small but significant decrease of TFA. Reduction of monocyte numbers by etoposide did not affect vegetational TFA. Comparison of data for
S. epidermidis
and
S. sanguis
revealed that at equivalent bacterial numbers, vegetational TFAs were approximately the same for both microorganisms. Combining the results of the present study with those of a previous study using
S. sanguis
, we conclude that the main factor determining monocyte-dependent vegetational TFA is the number of vegetation-associated bacteria. The lower TFA found for
S. epidermidis
-infected than for
S. sanguis
-infected vegetations can be explained by the significantly lower bacterial numbers in the infected vegetations and consequently a lower stimulation of vegetation-associated monocytes.
Vaccine Potential of Attenuated Mutants ofCorynebacterium pseudotuberculosisin Sheep
Authors: Simmons Cameron P. , Dunstan Sarah J. , Tachedjian Mary , Krywult Jolanta , Hodgson Adrian L. M. , Strugnell Richard A. 
Published: December 2019

ABSTRACTCorynebacterium pseudotuberculosis, a gram-positive facultative intracellular bacterial pathogen, is the etiological agent of the economically important disease caseous lymphadenitis (CLA) in both sheep and goats. Attenuated mutants ofC. pseudotuberculosishave the potential to act as novel vaccines against CLA and as veterinary vaccine vectors. In this report, we have assessed the virulence of botharoQandpldmutants ofC. pseudotuberculosisin sheep and concurrently their capacity to act as vaccines against homologous challenge. The results suggest thataroQmutants ofC. pseudotuberculosisare attenuated with regard to both lymph node persistence and vaccination site reactogenicity. Immunologically,aroQmutants failed to elicit detectable specific gamma interferon (IFN-γ)-secreting lymphocytes and induced low levels of antibodies toC. pseudotuberculosisculture supernatant antigens. Following subcutaneous vaccination, the immune responses induced byaroQmutants did not protect sheep from infection with the wild-type strain but did appear to reduce the clinical severity of disease resulting from challenge. Conversely, an attenuatedC. pseudotuberculosisstrain expressing an enzymatically inactive phospholipase D exotoxin, when used as a vaccine, elicited a protective immune response. Protection appeared to correlate with in vivo persistence of the vaccine strain, the induction of IFN-γ-secreting lymphocytes, and relatively high levels of antibodies to culture supernatant antigens. The results suggest thataroQmutants ofC. pseudotuberculosismay be overly attenuated for use as a CLA vaccines or as vaccine vectors.
Lactoferrin Inhibits the Endotoxin Interaction with CD14 by Competition with the Lipopolysaccharide-Binding Protein
Authors: Elass-Rochard Elisabeth , Legrand Dominique , Salmon Valerie , Roseanu Anca , Trif Mihaela , Tobias Peter S. , Mazurier Joel , Spik Genevieve 
Published: December 2019

ABSTRACTHuman lactoferrin (hLf), a glycoprotein released from neutrophil granules during inflammation, and the lipopolysaccharide (LPS)-binding protein (LBP), an acute-phase serum protein, are known to bind to the lipid A of LPS. The LPS-binding sites are located in the N-terminal regions of both proteins, at amino acid residues 28 to 34 of hLf and 91 to 108 of LBP. Both of these proteins modulate endotoxin activities, but they possess biologically antagonistic properties. In this study, we have investigated the competition between hLf and recombinant human LBP (rhLBP) for the binding ofEscherichia coli055:B5 LPS to the differentiated monocytic THP-1 cell line. Our studies revealed that hLf prevented the rhLBP-mediated binding of LPS to the CD14 receptor on cells. Maximal inhibition of LPS-cell interactions by hLf was raised when both hLf and rhLBP were simultaneously added to LPS or when hLf and LPS were mixed with cells 30 min prior to the incubation with rhLBP. However, when hLf was added 30 min after the interaction of rhLBP with LPS, the binding of the rhLPS-LBP complex to CD14 could not be reversed. These observations indicate that hLf competes with rhLBP for the LPS binding and therefore interferes with the interaction of LPS with CD14. Furthermore, experiments involving competitive binding of the rhLBP-LPS complex to cells with two recombinant mutated hLfs show that in addition to residues 28 to 34, another basic cluster which contains residues 1 to 5 of hLf competes for the binding to LPS. Basic sequences homologous to residues 28 to 34 of hLf were evidenced on LPS-binding proteins such as LBP, bactericidal/permeability-increasing protein, andLimulusanti-LPS factor.
Dynamics of the Murine Humoral Immune Response toNeisseria meningitidisGroup B Capsular Polysaccharide
Authors: Colino Jesús , Outschoorn Ingrid 
Published: December 2019

ABSTRACTImmunization withNeisseria meningitidisgroup B capsular polysaccharide (CpsB) elicited responses in adult mice that showed the typical dynamic characteristics of the response to a thymus-independent antigen, in contrast to the thymus-dependent behavior of antibody responses to CpsC. The former had a short latent period and showed a rapid increase in serum antibodies that peaked at day 5, and immunoglobulin M (IgM) was the major isotype even though IgG (mainly IgG2a and IgG2b) was also detectable. This response was of short duration, and the specific antibodies were rapidly cleared from the circulation. The secondary responses were similar in magnitude, kinetics, IgM predominance, and IgG distribution. Nevertheless, a threefold IgG increase, a correlation between IgM and IgG levels, and dose-dependent secondary responses were observed. Hyperimmunization considerably reinforced these responses: 10-fold for IgM and 300-fold for IgG. This favored isotype switch was accompanied by a progressive change in the subclass distribution to IgG3 (62%) and IgG1 (28%), along with the possible generation of B-cell memory. The results indicate that CpsB is being strictly thymus independent and suggest that unresponsiveness to purified CpsB is due to tolerance.
Analysis of theYersinia pestisV Protein for the Presence of Linear Antibody Epitopes
Authors: Pullen Jeffrey K. , Anderson George W. , Welkos Susan L. , Friedlander Arthur M. 
Published: December 2019

ABSTRACTThe V protein expressed by pathogenicYersinia pestisis an important virulence factor and protective immunogen. The presence of linear B-cell epitopes in the V protein was investigated by using a series of 17 overlapping linear peptides. Groups of 10 mice were immunized intraperitoneally with 30 μg of each peptide on days 0, 30, and 60. Although the V protein-specific antibody response to the peptides varied, most of the peptides elicited high antibody titers. The immunized mice were challenged subcutaneously with 60 50% lethal doses (LD50) (1 LD50= 1.9 CFU) of a virulentY. pestisstrain, CO92. None of the peptide-immunized mice survived challenge. The animals immunized with the V protein were completely protected against challenge. The immunogenicity of some of the V peptides was increased by conjugating them to keyhole limpet hemocyanin. Only one peptide (encompassing amino acids 1 to 30) conjugate demonstrated some protection; the others were not protective. In additional experiments, V peptides that reacted well with sera from mice survivingY. pestisinfection were combined and used to immunize mice. Although the combined peptides appeared to be very immunogenic, they were not protective. Therefore, the protective B-lymphocyte epitope(s) in the V protein is most likely to be conformational.
Identification and Characterization of the PutP Proline Permease That Contributes to In Vivo Survival ofStaphylococcus aureusin Animal Models
Authors: Schwan William R. , Coulter Silvija N. , Ng Eva Y. W. , Langhorne Michael H. , Ritchie Heather D. , Brody Linnea L. , Westbrock-Wadman Shannon , Bayer Arnold S. , Folger Kim R. , Stover C. Kendall 
Published: December 2019

ABSTRACTStaphylococcus aureusis an important pathogen of humans and other animals, causing bacteremia, abscesses, endocarditis, and other infectious syndromes. A signature-tagged mutagenesis (STM) system was adapted for use in studying the genes required for in vivo survival ofS. aureus. An STM library was ultimately created inS. aureusRN6390, with Tn917being used to create the transposon mutations. Pools ofS. aureusRN6390 mutants were screened in mouse abscess, bacteremia, and wound infection models for growth attenuation after in vivo passage. One of the mutants that was identified displayed marked attenuation following large-pool screening in all three animal models, which was confirmed in bacteremia and endocarditis models of infection with a smaller pool of mutants. Sequence analysis of the entire open reading frame showed a 99% identity to the high-affinity proline permease (putP) gene characterized in another strain ofS. aureus. In wound and murine abscess infection models, theputPmutant was approximately 10-fold more attenuated than was wild-type strain RN6390. AnotherS. aureusstrain transduced with theputPmutation also displayed an attenuated phenotype after passage in the wound model. A [3H]proline uptake assay showed that less proline was specifically transported into theputPmutant than into strain RN6390. The reduced viability of the bacteria possessing the mutation in theS. aureushigh-affinity proline permease suggests that proline scavenging by the bacteria is important for in vivo growth and proliferation and that analogs of proline may serve as potential antistaphylococcal therapeutic agents.
Activation of Bovine Lymphocyte Subpopulations by Staphylococcal Enterotoxin C
Authors: Ferens Witold A. , Davis William C. , Hamilton Mary Jo , Park Yong H. , Deobald Claudia F. , Fox Lawrence , Bohach Gregory 
Published: December 2019

ABSTRACTStaphylococcus aureusis a major mastitis-causing pathogen in cattle. The chronic nature of bovine staphylococcal mastitis suggests that some products or components ofS. aureusmay interfere with the development of protective immunity. One class of molecules that could be involved are superantigens (SAgs). Although a significant number of mastitis isolates produce SAgs, the effect of these molecules on the bovine immune system is unresolved. To determine if immunosuppression caused by SAgs could play a role in pathogenesis, we monitored bovine lymphocytes exposed to staphylococcal enterotoxin C1 (SEC1). Activation of bovine lymphocytes by either SEC1 or concanavalin A (ConA) was influenced by the γδ/αβ T-cell ratio in the culture. Compared to ConA-induced stimulation, cultures stimulated with SEC1 generated small numbers of CD4+αβ T cells expressing high levels of interleukin-2 receptor α chain (IL-2Rα) and major histocompatibility complex class II (MHCII), suggesting that SAg exposure does not lead to full activation of these cells. This state of partial activation was most pronounced in cultures with a high γδ/αβ ratio. In contrast, significant numbers of CD8+αβ T cells expressed high levels of IL-2Rα and MHCII, regardless of the γδ/αβ ratio and the stimulant used. CD8+blasts in cultures stimulated with SEC1 also expressed another activation marker, ACT3, previously detected predominantly on thymocytes and CD4+T cells. Although γδ CD2−and CD2+T cells expressed MHCII and IL-2Rα following stimulation with SEC1, only a few cells increased to blast size, suggesting that they were only partially activated. The results suggest ways in which SAgs might facilitate immunosuppression that promotes the persistence of bacteria in cattle and contributes to chronic intramammary infection.
Coiling Phagocytosis Discriminates between Different Spirochetes and Is Enhanced by Phorbol Myristate Acetate and Granulocyte- Macrophage Colony-Stimulating Factor
Authors: Rittig M. G. , Jagoda J. C. , Wilske B. , Murgia R. , Cinco M. , Repp R. , Burmester G. R. , Krause A. 
Published: December 2019

ABSTRACTThe mechanisms involved in coiling phagocytosis are not yet known, and it is not even clear whether this phenomenon is either an incidental event or a specific response. Therefore, the phagocytic uptake ofBorrelia burgdorferiand other spirochetes by human monocytes in vitro was used to investigate the involvement of both sides—microbes and phagocytes—in coiling phagocytosis. As seen with electron microscopy, morphologically similarBorrelia,LeptospiraandTreponemastrains induced markedly different frequencies of coiling phagocytosis. The monocytes used coiling phagocytosis for both live (motile) and killed (nonmotile)B. burgdorferi, but pseudopod coils were observed neither with fragmentedB. burgdorferinor with cell-free supernatant fromB. burgdorfericultures. Investigation of the relationship of coiling phagocytosis with other pseudopod-based cellular mechanisms revealed that the use of bioreagents that inhibit conventional phagocytosis also inhibited coiling phagocytis but did not affect membrane ruffling. Bioreagents that increase membrane ruffling did not affect phagocytosis ofB. burgdorferi, except for granulocyte-macrophage colony-stimulating factor and phorbol myristate acetate, which increased coiling phagocytosis selectively. These results demonstrate that coiling phagocytosis is not induced by microbial motility, viability, or a certain morphology and that it is not a random event. Rather, it is a selective uptake mechanism actively driven by the phagocytes. However, whether coiling phagocytosis represents an independent alternative to conventional phagocytosis or, alternatively, a fault in conventional phagocytosis remains to be determined.
Apoptosis of Renal Cortical Cells in the Hemolytic-Uremic Syndrome: In Vivo and In Vitro Studies
Authors: Karpman Diana , Håkansson Anders , Perez Maria-Thereza R. , Isaksson Christina , Carlemalm Eric , Caprioli Alfredo , Svanborg Catharina 
Published: December 2019

ABSTRACT

This study examined apoptotic cell death associated with Shiga-like toxin (Stx)-producing
Escherichia coli
. Renal cortices from three children with postenteropathic hemolytic-uremic syndrome (HUS) and from mice infected with
E. coli
O157:H7 and pediatric renal tubular epithelial cells stimulated with Stx and
E. coli
O157:H7 extracts were examined for apoptotic changes. Apoptotic cells were detected by terminal dUTP nick end labeling of tubuli and glomeruli from HUS patients and from mice inoculated with Stx-2-positive and Stx-negative strains. Apoptosis was more extensive and severe ultramorphological nuclear and cytoplasmic changes were seen in the Stx-2-positive group. Stx caused DNA fragmentation and ultramorphological changes indicating apoptosis in cultured pediatric tubular cells. DNA fragmentation increased when cells were prestimulated with tumor necrosis factor alpha. Polymyxin extracts from Stx-2-positive and Stx-negative strains induced DNA fragmentation, but only extracts from Stx-2-positive strains caused ultramorphological changes and extensive DNA fragmentation. The results indicate that HUS is accompanied by increased apoptosis of kidney cells and that bacterial factors, possibly together with host cytokines in vivo, may activate apoptotic tissue injury.
Filamentous Actin Disruption and Diminished Inositol Phosphate Response in Gingival Fibroblasts Caused by
Treponema denticola
Authors: Yang Po Fong , Song Meja , Grove David A. , Ellen Richard P. 
Published: December 2019

ABSTRACT

Previous reports have shown that
Treponema denticola
causes rearrangement of filamentous actin (F-actin) in human gingival fibroblasts (HGF). The purpose of this investigation was to determine the effect of
T. denticola
on the generation of inositol phosphates (IPs) in relation to a time course for F-actin disruption in HGF. Cultured HGF were exposed to washed cells of
T. denticola
ATCC 35405 for 140 min. Changes in the fluorescence intensity of rhodamine-phalloidin-labeled F-actin in serial optical sections of single HGF were quantified by confocal microscopy image analysis. The percentage of cells with stress fiber disruption was also determined by fluorescence microscopy. Challenge with
T. denticola
caused a significant reduction in F-actin within the first hour, especially at the expense of F-actin in the ventral third of the cells, and a significant increase in the percentage of HGF with altered stress fiber patterns. Significant concentration-dependent disruption of stress fibers was also caused by HGF exposure to a Triton X-100 extract of
T. denticola
outer membrane (OM). IPs were measured by a radiotracer assay based on the incorporation of
myo
-[
3
H]inositol into IPs in HGF incubated with LiCl to inhibit endogenous phosphatases. HGF challenge with several strains of
T. denticola
and the OM extract of
T. denticola
ATCC 35405 resulted in a diminished accumulation of radiolabeled IPs relative to both 15 and 1% fetal bovine serum, which served as strongly positive and background control agonists, respectively. The significantly diminished IP response to
T. denticola
ATCC 35405 occurred within 60 min, concomitant with significant reduction of total F-actin and disruption of stress fibers. Pretreatment with the proteinase inhibitor phenylmethylsulfonyl fluoride, which had previously been found to block
T. denticola
’s degradation of endogenous fibronectin and detachment of HGF from the extracellular matrix, had little effect on F-actin stress fiber disruption and the IP response. Therefore, in addition to its major surface chymotrypsin-like properties,
T. denticola
expresses cytopathogenic activities that diminish the generation of IPs during the time course associated with significant cytoskeletal disruption in fibroblasts.
Treponema denticola
Outer Membrane Inhibits Calcium Flux in Gingival Fibroblasts
Authors: Ko Kevin S.-C. , Glogauer Michael , McCulloch Christopher A. G. , Ellen Richard P. 
Published: December 2019

ABSTRACT

Treponema denticola
is a cultivable oral spirochete which perturbs the cytoskeleton in cultured cells of oral origin, but intracellular signalling pathways by which it affects actin assembly are largely unknown. As the outer membrane (OM) of
Treponema denticola
disrupts actin-dependent processes that normally require precise control of intracellular calcium, we studied the effects of an OM extract on internal calcium release, ligand-gated and calcium release-activated calcium channels, and related mechanosensitive cation fluxes in human gingival fibroblasts (HGF). Single-cell ratio fluorimetry demonstrated that in resting cells loaded with Fura-2, baseline intracellular Ca
2+
concentration ([Ca
2+
]
i
) was not affected by treatment with OM extract, but normal spontaneous [Ca
2+
]
i
oscillations were dramatically increased in frequency for 20 to 30 min followed by complete blockade. OM extract inhibited ATP-induced and thapsigargin-induced release of calcium from intracellular stores by 40 and 30%, respectively. Addition of Ca
2+
to the extracellular pool following depletion of intracellular Ca
2+
by thapsigargin and extracellular Ca
2+
by EGTA yielded 59% less replenishment of [Ca
2+
]
i
in OM extract-treated than in control HGF. In cells loaded with collagen-coated ferric oxide beads to stimulate integrin-dependent calcium release, baseline [Ca
2+
]
i
was nearly doubled but was not significantly different in control and OM extract-treated cells. Magnetically generated tensile forces on the beads induced >300% increases of [Ca
2+
]
i
above baseline. Cells preincubated with OM extract exhibited dose-dependent and time-dependent reductions in stretch-induced [Ca
2+
]
i
transients, which were due to neither loss of beads from the cells nor cell death. The
T. denticola
OM inhibitory activity was eliminated by heating the OM extract to 60°C and by boiling but not by phenylmethylsulfonyl fluoride treatment. Thus nonlipopolysaccharide, nonchymotrypsin, heat-sensitive protein(s) in
T. denticola
OM can evidently inhibit both release of calcium from internal stores and uptake of calcium through the plasma membrane, possibly by interference with calcium release-activated channels.
B-Cell Epitopes and Quantification of the ESAT-6 Protein ofMycobacterium tuberculosis
Authors: Harboe Morten , Malin Adam S. , Dockrell Hazel S. , Gotten Wiker Harald , Ulvund Gunni , Holm Arne , Jørgensen Mikala Clok , Andersen Peter 
Published: December 2019

ABSTRACTESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection withMycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 inM. tuberculosisantigen preparations, showing no reactivity withM. bovisBCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing theesat-6gene; this expression could not be identified by standard immunoblotting.