bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of Monoclonal Antibody in Diagnosis of Candidiasis Caused by
Candida albicans
: Detection of Circulating Aspartyl Proteinase Antigen
Authors: Na Byoung-Kuk , Song Chul-Yong 
Published: December 2019

ABSTRACT

To develop a serological diagnosis of invasive candidiasis based on detection of circulating secreted aspartyl proteinase (SAP) antigen of
Candida albicans
, three different enzyme-linked immunosorbent assays (ELISAs) were compared. The first was a standard ELISA to detect anti-SAP antibodies, and the others were an antigen capture ELISA and an inhibition ELISA to detect circulating SAP antigen with monoclonal antibody (MAb) CAP1, which is highly specific for SAP. These tests were applied to 33 serum samples retrospectively selected from 33 patients with mycologically and/or serologically proven invasive candidiasis caused by
C. albicans
. Serum samples from 12 patients with aspergillosis and serum samples from 13 healthy individuals were also included. The sensitivities and specificities were 69.7 and 76.0% for the standard ELISA and 93.9 and 92.0% for the antigen capture ELISA, respectively. However, these values reached 93.9 and 96.0%, respectively, for the inhibition ELISA. Serum samples from 31 of 33 patients had detectable SAP antigen, with concentrations ranging from 6.3 to 19.0 ng/ml. These results indicate that the inhibition ELISA with MAb CAP1 is effective in detection of circulating SAP antigen and that this assay may be useful for diagnosis and treatment monitoring of invasive candidiasis.
Increased Levels of ɛ and γ Isoforms of 14-3-3 Proteins in Cerebrospinal Fluid in Patients with Creutzfeldt-Jakob Disease
Authors: Takahashi Hidehiro , Iwata Toshinari , Kitagawa Yoshinori , Takahashi Reisuke H. , Sato Yuko , Wakabayashi Hideki , Takashima Miwa , Kido Hiroshi , Nagashima Kazuo , Kenney Kimbra , Gibbs Clarence J. , Kurata Takeshi 
Published: December 2019

ABSTRACT
We established four hybridoma cell lines producing monoclonal antibodies (MAbs) against 14-3-3 proteins. Immunoblot analysis revealed that ɛ and γ isoforms were specifically increased in premortem cerebrospinal fluid samples from patients with sporadic Creutzfeldt-Jakob disease. Furthermore, dot immunoblot analysis showed that MAbs were more specific for native antigen than polyclonal antibodies were.
Temporal Variation of the Merozoite Surface Protein-2 Gene ofPlasmodium falciparum
Authors: Eisen Damon , Billman-Jacobe Helen , Marshall Vikki F. , Fryauff Dave , Coppel Ross L. 
Published: December 2019

ABSTRACTExtensive polymorphism of key parasite antigens is likely to hamper the effectiveness of subunit vaccines againstPlasmodium falciparuminfection. However, little is known about the extent of the antigenic repertoire of naturally circulating strains in different areas where malaria is endemic. To address this question, we conducted a study in which blood samples were collected from parasitemic individuals living within a small hamlet in Western Irian Jaya and subjected to PCR amplification using primers that would allow amplification of the gene encoding merozoite surface protein-2 (MSP2). We determined the nucleotide sequence of the amplified product and compared the deduced amino acid sequences to sequences obtained from samples collected in the same hamlet 29 months previously.MSP2genes belonging to both major allelic families were observed at both time points. In the case of the FC27MSP2family, we observed that the majority of individuals were infected by parasites expressing the same form of MSP2. Infections with parasites expressing 3D7MSP2family alleles were more heterogeneous. NoMSP2alleles observed at the earlier time point were detectable at the later time point, either for the population as a whole or for individuals who were assayed at both time points. We examined a subset of the infected patients by using blood samples taken between the two major surveys. In no patients could we detect reinfection by a parasite expressing a previously encountered form of MSP2. Our results are consistent with the possibility that infection induces a form of strain-specific immune response against the MSP2 antigen that biases against reinfection by parasites bearing identical forms of MSP2.
Differential Responses of Human Mononuclear Phagocytes to Mycobacterial Lipoarabinomannans: Role of CD14 and the Mannose Receptor
Authors: Bernardo John , Billingslea Andrea M. , Blumenthal Robin L. , Seetoo Kurt F. , Simons Elizabeth R. , Fenton Matthew J. 
Published: December 2019

ABSTRACTCD14 is a signaling receptor for both gram-negative bacterial lipopolysaccharide (LPS) and mycobacterial lipoarabinomannan (LAM) that lacks terminal mannosyl units (AraLAM). In contrast, terminally mannosylated LAM (ManLAM) binds the macrophage mannose receptor (MMRc), although the ability of the MMRc to serve as a signaling receptor has not been previously reported. We compared the abilities of AraLAM and ManLAM to induce distinct responses in two monocytic cell populations, freshly isolated human peripheral blood monocytes (PBM) and monocyte-derived macrophages (MDM). The responses examined were chemotaxis and transient changes in free cytosolic calcium ([Ca2+]in). We found that AraLAM but not ManLAM was chemotactic for both PBM and MDM. Migration of these cells in vitro to AraLAM was specifically blocked by an anti-CD14 monoclonal antibody, suggesting that CD14 mediates the chemotactic response to AraLAM. Subsequently, we found that AraLAM induced a transient rise in [Ca2+]inlevels within a subpopulation of PBM but not MDM. This response was blocked by anti-CD14 antibodies. In contrast, ManLAM induced a transient rise in [Ca2+]inlevels within a subpopulation of MDM but not PBM. This response was blocked by either anti-CD14 or anti-MMRc antibodies. These data suggest that the MMRc can serve as a signaling receptor and that coligation of both CD14 and the MMRc is required to elicit a specific response. Thus, one response to LAM (chemotaxis) can be elicited solely by engaging CD14, whereas a different response (changes in [Ca2+]inlevels) depends on both the differentiation state of the cells and concomitant engagement of CD14 and the MMRc.
Role of Flagella in Pathogenesis ofPseudomonas aeruginosaPulmonary Infection
Authors: Feldman Matthew , Bryan Ruth , Rajan Sujatha , Scheffler Lee , Brunnert Steven , Tang Hope , Prince Alice 
Published: December 2019

ABSTRACTPseudomonas aeruginosastrains are opportunistic pathogens associated with infections in immunocompromised hosts and patients with cystic fibrosis. Like many other mucosal pathogens,P. aeruginosacells express flagella which provide motility and chemotaxis toward preferred substrates but also provide a ligand for clearance by phagocytic cells. We tested the role of flagella in the initial stages of respiratory tract infection by comparing the virulence offliCmutants in a neonatal mouse model of pneumonia. In the absence offliC, there was no mortality, compared with 30% mortality attributed to the parental strain PAK or 15% mortality associated with infection due to apilAmutant PAK/NP (P< 0.0001). ThefliCmutants caused pneumonia in only 25% of the mice inoculated, regardless of whether there was expression of the pilus, whereas the parental strain was associated with an 80% rate of pneumonia. Histopathological studies demonstrated that thefliCmutants caused very focal inflammation and that the organisms did not spread through the lungs as seen in infection due to either PAK or PAK/NP. Purified flagellin elicited an intense inflammatory response in the mouse lung.125I-labeled flagellin bound to the glycolipids GM1 and GD1aand to asialoGM1 in an in vitro binding assay. However, flagellin-mediated binding to epithelial gangliosides was a relatively unusual event, as quantified by binding assays of wild-type orfliCmutant organisms to CHO Lec-2 cells with membrane-incorporated GM1. Fla+organisms but notfliCmutants were efficiently taken up by murine macrophages.P. aeruginosaflagella are important in the establishment of respiratory tract infection and may act as a tether in initial interactions with epithelial membranes. This function is offset by the contribution of flagella to host clearance mechanisms facilitating phagocytic clearance and the role of flagellar genes in mucin binding and clearance.
Phage Antibodies Obtained by Competitive Selection on Complement-ResistantMoraxella(Branhamella)catarrhalisRecognize the High-Molecular-Weight Outer Membrane Protein
Authors: Boel Edwin , Bootsma Hester , de Kruif John , Jansze Margriet , Klingman Karin L. , van Dijk Hans , Logtenberg Ton 
Published: December 2019

ABSTRACTWe used competitive panning to select a panel of 10 different human antibodies from a large semisynthetic phage display library that distinguish between serum complement-resistant and complement-sensitive strains of the gram-negative diplococcusMoraxella(Branhamella)catarrhalis. Western blotting analyses and inhibition enzyme-linked immunosorbent assays showed that all phage antibodies were directed against the same or closely spaced epitopes on the target protein, which is the high-molecular-weight outer membrane protein (HMW-OMP) ofM. catarrhalis. HMW-OMP was found in multiple isolates of complement-resistant but not complement-sensitiveM. catarrhalisstrains. Nucleotide sequence analysis demonstrated that the immunoglobulin heavy- and light-chain variable-region genes encoding the 10 phage antibodies were remarkably similar, with a strong preference for basic amino acid residues in the heavy-chain CDR3 regions. This is the first report showing that competitive panning is a successful procedure to obtain phage antibodies against differentially expressed structures on phenotypically dissimilar strains of prokaryotic cells.
Processing ofMycobacterium tuberculosisBacilli by Human Monocytes for CD4+αβ and γδ T Cells: Role of Particulate Antigen
Authors: Balaji Kithiganahalli N. , Boom W. Henry 
Published: December 2019

ABSTRACTMycobacterium tuberculosisreadily activates both CD4+and Vδ2+γδ T cells. Despite similarity in function, these T-cell subsets differ in the antigens they recognize and the manners in which these antigens are presented byM. tuberculosis-infected monocytes. We investigated mechanisms of antigen processing ofM. tuberculosisantigens to human CD4 and γδ T cells by monocytes. Initial uptake ofM. tuberculosisbacilli and subsequent processing were required for efficient presentation not only to CD4 T cells but also to Vδ2+γδ T cells. For γδ T cells, recognition ofM. tuberculosis-infected monocytes was dependent on Vδ2+T-cell-receptor expression. Recognition ofM. tuberculosisantigens by CD4+T cells was restricted by the class II major histocompatibility complex molecule HLA-DR. Processing ofM. tuberculosisbacilli for Vδ2+γδ T cells was inhibitable by Brefeldin A, whereas processing of soluble mycobacterial antigens for γδ T cells was not sensitive to Brefeldin A. Processing ofM. tuberculosisbacilli for CD4+T cells was unaffected by Brefeldin A. Lysosomotropic agents such as chloroquine and ammonium chloride did not affect the processing ofM. tuberculosisbacilli for CD4+and γδ T cells. In contrast, both inhibitors blocked processing of soluble mycobacterial antigens for CD4+T cells. Chloroquine and ammonium chloride insensitivity of processing ofM. tuberculosisbacilli was not dependent on the viability of the bacteria, since processing of both formaldehyde-fixed dead bacteria and mycobacterial antigens covalently coupled to latex beads was chloroquine insensitive. Thus, the manner in which mycobacterial antigens were taken up by monocytes (particulate versus soluble) influenced the antigen processing pathway for CD4+and γδ T cells.
Filament Tip-Associated Antigens Involved in Adherence to and Invasion of Murine Pulmonary Epithelial Cells In Vivo and HeLa Cells In Vitro by
Nocardia asteroides
Authors: Beaman Blaine L. , Beaman LoVelle 
Published: December 2019

ABSTRACT

The interactions of
Nocardia asteroides
GUH-2 with pulmonary epithelial cells of C57BL/6 mice and with HeLa cells were studied. Electron microscopy demonstrated that only the tips of log-phase cells penetrated pulmonary epithelial cells following intranasal administration, and nocardiae were recovered from the brain. Coccobacillary cells neither invaded nor disseminated. Serum from immunized mice (IMS) decreased attachment to and penetration of pulmonary epithelial cell surfaces by log-phase GUH-2 and inhibited spread to the brain. IMS was adsorbed against stationary-phase cells. Western immunoblots suggested that this adsorbed IMS was reactive primarily with 43- and 62-kDa proteins. Immunofluorescence showed that adsorbed IMS preferentially labeled the tips of log-phase GUH-2 cells. Since this IMS was reactive to culture filtrate antigens, several of these proteins were cut from gels, and mice were immunized. Sera against 62-, 55-, 43-, 36-, 31-, and 25-kDa antigens were obtained. The antisera against the 43- and 36-kDa proteins labeled the filament tips of GUH-2 cells. Only the antiserum against the 43-kDa antigen increased pulmonary clearance, inhibited apical attachment to and penetration of pulmonary epithelial cells, and prevented spread to the brain. An in vitro model with HeLa cells demonstrated that the tips of log-phase cells of GUH-2 adhered to and penetrated the surface of HeLa cells. Invasion assays with amikacin treatment demonstrated that nocardiae were internalized. Adsorbed IMS blocked attachment to and invasion of these cells. These data suggested that a filament tip-associated 43-kDa protein was involved in attachment to and invasion of pulmonary epithelial cells and HeLa cells by
N. asteroides
GUH-2.
Anthrax Toxin as a Molecular Tool for Stimulation of Cytotoxic T Lymphocytes: Disulfide-Linked Epitopes, Multiple Injections, and Role of CD4+Cells
Authors: Ballard Jimmy D. , Collier R. John , Starnbach Michael N. 
Published: December 2019

ABSTRACTWe have previously demonstrated that anthrax toxin-derived proteins, protective antigen (PA) and the amino-terminal portion of lethal factor (LFn), can be used in combination to deliver heterologous molecules to the cytosol of mammalian cells. In this study we examined the ability of an LFn-peptide disulfide-linked heterodimer to prime cytotoxic T lymphocytes (CTL) in the presence of PA. A mutant of LFn that contains a carboxy-terminal reactive cysteine was generated. This form of LFn could be oxidized with a synthetic cysteine containing peptide to form a heterodimer of the protein and peptide. Mice injected with the heterodimer plus PA mounted a peptide-specific CTL response, indicating that this molecule functioned similarly to the genetically fused forms used previously. We also report the results of an analysis of two aspects of this system important for the development of experimental vaccines. First, CD4 knockout mice were unable to generate a CTL response when treated with PA plus an LFn-epitope fusion protein, suggesting that CD4+helper responses are essential for stimulating specific CTL with the PA-LFn system. Second, we now show that primary injection with this system does not generate any detectable antibody response to the vaccine components and that prior immunization has no effect on priming a CTL response to an unrelated epitope upon subsequent injection.
Characterization of Outer Membrane Protein OmpU of
Vibrio cholerae
O1
Authors: Nakasone Noboru , Iwanaga Masaaki 
Published: December 2019

ABSTRACT

The outer membrane protein OmpU of
Vibrio cholerae
O1 strain 86B3 was characterized with reference to colonization of the intestine by the organism. The purified OmpU exhibited a pI of 3.6. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it migrated to 38, 32, and 110 kDa when the sample was heated at 100°C for 2 min, 50°C for 15 min, and room temperature for 30 min, respectively. The purified OmpU was not hemagglutinative. Anti-OmpU serum did not agglutinate strain 86B3 or other
V. cholerae
organisms. OmpU adhered to the brush border of the rabbit small intestine; adhesion of the organisms to the intestine treated in advance with OmpU was not inhibited. Treating the organisms in advance with anti-OmpU Fab did not inhibit adhesion to the intestine. These results obtained in vitro suggest that OmpU is not involved in the adhesion of
V. cholerae
to the intestinal epithelium.
Structure and Specific Activity of Macrophage-Stimulating Lipopeptides fromMycoplasma hyorhinis
Authors: Mühlradt Peter F. , Kiess Michael , Meyer Holger , Süssmuth Roderich , Jung Günther 
Published: December 2019

ABSTRACTMycoplasmas are potent macrophage stimulators. We describe the isolation of macrophage-stimulatory lipopeptidesS-[2,3-bisacyl(C16:0/C18:0)oxypropyl]cysteinyl-GQTDNNSSQSQQPGSGTTNT andS-[2,3-bisacyl(C16:0/C18:0)oxypropyl]cysteinyl-GQTN derived from theMycoplasma hyorhinisvariable lipoproteins VlpA and VlpC, respectively. These lipopeptides were characterized by amino acid sequence and composition analysis and by mass spectrometry. The lipopeptidesS-[2,3-bis(palmitoyloxy)propyl]cysteinyl-GQTNT andS-[2,3-bis(palmitoyloxy)propyl]cysteinyl-SKKKK and the N-palmitoylated derivative of the latter were synthesized, and their macrophage-stimulatory activities were compared in a nitric oxide release assay with peritoneal macrophages from C3H/HeJ mice. The lipopeptides with the free amino terminus showed half-maximal activity at 3 pM regardless of their amino acid sequence; i.e., they were as active as the previously isolatedM. fermentans-derived lipopeptide MALP-2. The macrophage-stimulatory activity of the additionally N-palmitoylated lipopeptide or of the murein lipoprotein fromEscherichia coli, however, was lower by orders of magnitude. It is concluded that the lack of N-acyl groups in mycoplasmal lipoproteins explains their exceptionally high in vitro macrophage-stimulatory capacity. Certain features that lipopolysaccharide endotoxin and mycoplasmal lipopeptides have in common are discussed. Lipoproteins and lipopeptides are likely to be the main causative agents of inflammatory reactions to mycoplasmas. This may be relevant in the context of mycoplasmas as arthritogenic pathogens and their association with AIDS.
Purification, Potency, and Efficacy of the Botulinum Neurotoxin Type A Binding Domain fromPichia pastorisas a Recombinant Vaccine Candidate
Authors: Byrne Michael P. , Smith Theresa J. , Montgomery Vicki A. , Smith Leonard A. 
Published: December 2019

ABSTRACTRecombinant botulinum neurotoxin serotype A binding domain [BoNT/A(Hc)], expressed inPichia pastoris, was developed as a vaccine candidate for preventing botulinum neurotoxin type A (BoNT/A) intoxication. After fermentation and cell disruption, BoNT/A(Hc) was purified by using a three-step chromatographic process consisting of expanded-bed chromatography, Mono S cation-exchange chromatography, and hydrophobic interaction chromatography. Two pools of immunogenic product were separated on the Mono S column and processed individually. Both products were more than 95% pure and indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and enzyme-linked immunosorbent assay (ELISA). Each protein was assayed for potency in mice at immunogen doses ranging from 2.4 ng to 10 μg, followed by challenge with 1,000 mouse intraperitoneal 50% lethal doses (i.p. LD50) of BoNT/A. The calculated 50% effective dose for both peaks was approximately 0.1 μg/mouse. Peak 1 was evaluated further in a mouse efficacy assay. Mice were injected either once, twice, or three times at five different doses and subsequently challenged with 100,000 mouse i.p. LD50of BoNT/A. In general, multiple injections protected better than one, with complete or nearly complete protection realized at doses of ≥0.5 μg/mouse. Serum neutralization and ELISA titers were also determined. Tellingly, 82 of 83 mice with antibody titers of ≥1,600, as measured by ELISA, survived, but only 6 of 42 mice with titers of ≤100 survived. This work shows that the purified BoNT/A(Hc) produced was a highly effective immunogen, able to protect against a high challenge dose of neurotoxin.
Characterization of Group B Streptococcal Invasion of Human Chorion and Amnion Epithelial Cells In Vitro
Authors: Winram Scott B. , Jonas Mechthild , Chi Emil , Rubens Craig E. 
Published: December 2019

ABSTRACT

Group B streptococci (GBS) have been cultured from the chorioamnionic membrane of pregnant women, usually in association with chorioamnionitis and premature labor (K. A. Boggess, D. H. Watts, S. L. Hillier, M. A. Krohn, T. J. Benedetti, and D. A. Eschenbach, Obstet. Gynecol.
87:
779–784, 1996). Colonization and infection of placental membranes can be a prelude to neonatal GBS infections even in the presence of intact membranes (R. L. Naeye and E. C. Peters, Pediatrics
61:
171–177, 1978), suggesting that GBS cause chorioamnionitis or establish amniotic fluid infections by partial or complete penetration of the placental membranes. We have isolated and grown cultures of primary chorion and amnion cells from human cesarean-section placentas. This has provided a biologically relevant model for investigating GBS adherence to and invasion of the two epithelial barriers of the placental membrane. GBS adhered to chorion cell monolayers to a high degree. Pretreatment of GBS with trypsin reduced adherence up to 10-fold, which suggested that the bacterial ligand(s) was a protein. GBS invaded chorion cells at a high rate in vitro, and invasion was dependent on cellular actin polymerization. GBS could be seen within intracellular vacuoles of chorion cells by transmission electron microscopy. We also demonstrated that GBS were capable of transcytosing through intact chorion cell monolayers without disruption of intracellular junctions. GBS also adhered to amnion cells; in contrast, however, these bacteria failed to invade amnion cells under a variety of assay conditions. GBS interactions with the chorion epithelial cell layer shown here correlate well with epidemiological and pathological studies of GBS chorioamnionitis. Our data also suggest that the amnion cell layer may provide an effective barrier against infection of the amniotic fluid.
Biological and Biochemical Characteristics of Cytoadhesion of
Plasmodium falciparum
-Infected Erythrocytes to Chondroitin-4-Sulfate
Authors: Pouvelle Bruno , Fusaï Thierry , Lépolard Catherine , Gysin Jürg 
Published: December 2019

ABSTRACT

The cytoadhesion of
Plasmodium falciparum
laboratory strains and clones to
Saimiri
brain microvascular endothelial cells (SBEC 17), with chondroitin-4-sulfate (CSA) as the only adhesion receptor, was tested. Only one strain had significant cytoadhesion. However, CSA-specific infected erythrocytes (IRBCs) were detected in all strains after selection of a CSA-specific subpopulation by culturing the few adherent IRBCs. This demonstrates the lack of sensitivity of cytoadhesion microassays for detecting small quantities of CSA-specific IRBCs in cultures or field isolates. Cytoadhesion to CSA is maximal at 24 h of the cycle and decreases with the onset of schizogony, reaching a minimum just before reinvasion. This fluctuation must be taken into account in comparisons of the cytoadhesion of different strains or isolates. The minimum size of CSA for active inhibition was 4 kDa, and a mass of 9 kDa was required for inhibition similar to that obtained with the 50-kDa CSA. In contrast to cytoadhesion to CSA, which is pH independent or maximal at physiological pH (depending on the target endothelial cells), adhesion to CD36 and intercellular adhesion molecule 1 was pH dependent, requiring acidic conditions to be maximal in all cases. Cytoadhesion to CSA may trigger the occlusion of microvessels and cause the acidosis necessary for the other receptors to be fully efficient. If this key role in the mechanisms of sequestration were to be confirmed in vivo, prevalence studies of the CSA cytoadhesion phenotype would have to be reevaluated, because simple cytoadhesion assays do not detect CSA-specific parasites present in very low numbers, and these parasites might then be undetected in the peripheral blood but present in organs in which sequestration occurs, such as the placenta (M. Fried and P. E. Duffy, Science 272:1502–1504, 1996).