bluuebunny/crossref_metadata_2025_split huggingface dataset:
Antibodies Reactive with the N-Terminal Domain ofPlasmodium falciparumSerine Repeat Antigen Inhibit Cell Proliferation by Agglutinating Merozoites and Schizonts
Authors: Pang Xin-Li , Mitamura Toshihide , Horii Toshihiro 
Published: March 2020

ABSTRACTThe serine repeat antigen (SERA) is a vaccine candidate antigen ofPlasmodium falciparum. Immunization of mice withEscherichia coli-produced recombinant protein of the SERA N-terminal domain (SE47′) induced an antiserum that was inhibitory to parasite growth in vitro. Affinity-purified mouse antibodies specific to the recombinant protein inhibited parasite growth between the schizont and ring stages but not between the ring and schizont stages. When Percoll-purified schizonts were cultured with the affinity-purified SE47′-specific antibodies, schizonts and merozoites were agglutinated. Indirect-immunofluorescence assays with unfixed parasite cells showed that SE47′-specific immunoglobulin G (IgG) bound to SERA molecules on rupturing schizonts and merozoites but the IgG did not react with the schizont-infected erythrocytes (RBC). Furthermore, double-fluorescence staining against SE47′-specific IgG and anti-human RBC membrane IgG showed that the RBC membrane disappeared from SE47′-specific-IgG-bound schizonts after cultivation. These observations suggest that the SE47′-specific antibodies inhibit parasite growth by cross-linking SERA molecules that are associated with merozoites in rupturing schizonts with partly broken RBC and parasitophorous vacuole membranes, blocking merozoite release.
Severe Impairment in Early Host Defense againstCandida albicansin Mice Deficient in Myeloperoxidase
Authors: Aratani Yasuaki , Koyama Hideki , Nyui Sei-ichiro , Suzuki Kazuo , Kura Fumiaki , Maeda Nobuyo 
Published: March 2020

ABSTRACTMyeloperoxidase (MPO) catalyzes the reaction of hydrogen peroxide with chloride ion to produce hypochlorous acid (HOCl), which is used for microbial killing by phagocytic cells. Despite the important role of MPO in host defense, however, MPO deficiency is relatively common in humans, and most of these individuals are in good health. To define the in vivo role of MPO, we have generated by gene targeting mice having no MPO activity in their neutrophils and monocytes. The mice without MPO developed normally, were fertile, and showed normal clearance of intraperitonealStaphylococcus aureus. However, they showed increased susceptibility to pneumonia and death following intratracheal infection withCandida albicans. Furthermore, the lack of MPO significantly enhanced the dissemination of intraperitoneally injectedC. albicansinto various organs during the first 7 days. Thus, MPO is important for early host defense against fungal infection, and the inability to generate HOCl cannot be compensated for by other oxygen-dependent systems in vivo in mice. The mutant mice serve as a model for studying pulmonary and systemic candidiasis.
Characterization of a Novel Trypanosome Lytic Factor from Human Serum
Authors: Raper Jayne , Fung Ramie , Ghiso Jorge , Nussenzweig Victor , Tomlinson Stephen 
Published: March 2020

ABSTRACT

Natural resistance of humans to the cattle pathogen
Trypanosoma brucei brucei
has been attributed to the presence in human serum of nonimmune factors that lyse the parasite. Normal human serum contains two trypanosome lytic factors (TLFs). TLF1 is a 500-kDa lipoprotein, which is reported to contain apolipoprotein A-I (apoA-I), haptoglobin-related protein (Hpr), hemoglobin, paraoxonase, and apoA-II, whereas TLF2 is a larger, poorly characterized particle. We report here a new immunoaffinity-based purification procedure for TLF2 and TLF1, as well as further characterization of the components of each purified TLF. Immunoaffinity-purified TLF1 has a specific activity 10-fold higher than that of TLF1 purified by previously described methods. Moreover, we find that TLF1 is a lipoprotein particle that contains mainly apoA-I and Hpr, trace amounts of paraoxonase, apoA-II, and haptoglobin, but no detectable hemoglobin. Characterization of TLF2 reveals that it is a 1,000-kDa protein complex containing mainly immunoglobulin M, apoA-I, and Hpr but less than 1% detectable lipid.
Immune Response to
Yersinia
Outer Proteins and Other
Yersinia pestis
Antigens after Experimental Plague Infection in Mice
Authors: Benner Gretchen E. , Andrews Gerard P. , Byrne W. Russell , Strachan Susan D. , Sample Allen K. , Heath David G. , Friedlander Arthur M. 
Published: March 2020

ABSTRACT

There is limited information concerning the nature and extent of the immune response to the virulence determinants of
Yersinia pestis
during the course of plague infection. In this study, we evaluated the humoral immune response of mice that survived lethal
Y. pestis
aerosol challenge after antibiotic treatment. Such a model may replicate the clinical situation in humans and indicate which virulence determinants are expressed in vivo. Immunoglobulin G enzyme-linked immunosorbent assay and immunoblotting were performed by using purified, recombinant antigens including F1, V antigen, YpkA, YopH, YopM, YopB, YopD, YopN, YopE, YopK, plasminogen activator protease (Pla), and pH 6 antigen as well as purified lipopolysaccharide. The major antigens recognized by murine convalescent sera were F1, V antigen, YopH, YopM, YopD, and Pla. Early treatment with antibiotics tended to reduce the immune response and differences between antibiotic treatment regimens were noted. These results may indicate that only some virulence factors are expressed and/or immunogenic during infection. This information may prove useful for selecting potential vaccine candidates and for developing improved serologic diagnostic assays.
Middle Ear Fluid Cytokine and Inflammatory Cell Kinetics in the Chinchilla Otitis Media Model
Authors: Sato Katsuro , Liebeler Carol L. , Quartey Moses K. , Le Chap T. , Giebink G. Scott 
Published: March 2020

ABSTRACT

Streptococcus pneumoniae
is the most frequent microbe causing middle ear infection. The pathophysiology of pneumococcal otitis media has been characterized by measurement of local inflammatory mediators such as inflammatory cells, lysozyme, oxidative metabolic products, and inflammatory cytokines. The role of cytokines in bacterial infection has been elucidated with animal models, and interleukin (IL)-1β, IL-6, and IL-8 and tumor necrosis factor alpha (TNF-α) are recognized as being important local mediators in acute inflammation. We characterized middle ear inflammatory responses in the chinchilla otitis media model after injecting a very small number of viable pneumococci into the middle ear, similar to the natural course of infection. Middle ear fluid (MEF) concentrations of IL-1β, IL-6, IL-8, and TNF-α were measured by using anti-human cytokine enzyme-linked immunosorbent assay reagents. IL-1β showed the earliest peak, at 6 h after inoculation, whereas IL-6, IL-8, and TNF-α concentrations were increasing 72 h after pneumococcal inoculation. IL-6, IL-8, and TNF-α but not IL-1β concentrations correlated significantly with total inflammatory cell numbers in MEF, and all four cytokines correlated significantly with MEF neutrophil concentration. Several intercytokine correlations were significant. Cytokines, therefore, participate in the early middle ear inflammatory response to
S. pneumoniae.
Integration Host Factor and Cyclic AMP Receptor Protein Are Required for TyrR-Mediated Activation of
tpl
in
Citrobacter freundii
Authors: Bai Qing , Somerville Ronald L. 
Published: March 2020

ABSTRACT

The
tpl
gene of
Citrobacter freundii
encodes an enzyme that catalyzes the conversion of
l
-tyrosine to phenol, pyruvate, and ammonia. This gene is known to be positively regulated by TyrR. The amplitude of regulation attributable to this transcription factor is at least 20-fold. Three TyrR binding sites, designated boxes A, B, and C, centered at coordinates −272.5, −158.5, and −49.5, respectively, were identified in the upstream region of the
tpl
promoter. The results of mutational experiments suggest that TyrR binds in cooperative fashion to these sites. The nonavailability of any TyrR site impairs transcription. Full TyrR-mediated activation of
tpl
required integration host factor (IHF) and the cAMP receptor protein (CRP). By DNase I footprinting, it was shown that the IHF binding site is centered at coordinate −85 and that there are CRP binding sites centered at coordinates −220 and −250. Mutational alteration of the IHF binding site reduced the efficiency of the
tpl
promoter by at least eightfold. The proposed roles of CRP and IHF are to introduce bends into
tpl
promoter DNA between boxes A and B or B and C. Multimeric TyrR dimers were demonstrated by a chemical cross-linking method. The formation of hexameric TyrR increased when
tpl
DNA was present. The participation of both IHF and CRP in the activation of the
tpl
promoter suggests that molecular mechanisms quite different from those that affect other TyrR-activated promoters apply to this system. A model wherein TyrR, IHF, and CRP collaborate to regulate the expression of the
tpl
promoter is presented.
Role of SarA in Virulence Determinant Production and Environmental Signal Transduction in
Staphylococcus aureus
Authors: Chan Pan F. , Foster Simon J. 
Published: March 2020

ABSTRACT

The staphylococcal accessory regulator (encoded by
sarA
) is an important global regulator of virulence factor biosynthesis in
Staphylococcus aureus
. To further characterize its role in virulence determinant production, an
sarA
knockout mutant was created by insertion of a kanamycin antibiotic resistance cassette into the
sarA
gene. N-terminal sequencing of exoproteins down-regulated by
sarA
identified several putative proteases, including a V8 serine protease and a novel metalloprotease, as the major extracellular proteins repressed by
sarA
. In kinetic studies, the
sarA
mutation delays the onset of α-hemolysin (encoded by
hla
) expression and reduces levels of
hla
to approximately 40% of the parent strain level. Furthermore, SarA plays a role in signal transduction in response to microaerobic growth since levels of
hla
were much lower in a microaerobic environment than after aerobic growth in the
sarA
mutant. An exoprotein exhibiting hemolysin activity on sheep blood, and up-regulated by
sarA
independently of the accessory gene regulator (encoded by
agr
), was specifically induced microaerobically. Transcriptional gene fusion and Western analysis revealed that
sarA
up-regulates both toxic shock syndrome toxin 1 gene (
tst
) expression and staphylococcal enterotoxin B production, respectively. This study demonstrates the role of
sarA
as a signal transduction regulatory component in response to aeration stimuli and suggests that
sarA
functions as a major repressor of protease activity. The possible role of proteases as regulators of virulence determinant stability is discussed.
Corepression of the P1 Addiction Operon by Phd and Doc
Authors: Magnuson Roy , Yarmolinsky Michael B. 
Published: March 2020

ABSTRACT

The P1 plasmid addiction operon encodes Doc, a toxin that kills plasmid-free segregants, and Phd, an unstable antidote that neutralizes the toxin. Additionally, these products repress transcription of the operon. The antidote binds to two adjacent sites in the promoter. Here we present evidence concerning the regulatory role of the toxin, which we studied with the aid of a mutation,
docH66Y
. The DocH66Y protein retained the regulatory properties of the wild-type protein, but not its toxicity. In vivo, DocH66Y enhanced repression by Phd but failed to affect repression in the absence of Phd, suggesting that DocH66Y contacts Phd. In vitro, a MalE-DocH66Y fusion protein was found to bind Phd. Binding of toxin to antidote may be the physical basis for the neutralization of toxin. DocH66Y failed to bind DNA in vitro yet enhanced the affinity, cooperativity, and specificity with which Phd bound the operator. Although DocH66Y enhanced the binding of Phd to two adjacent Phd-binding sites, DocH66Y had relatively little effect on the binding of Phd to a single Phd-binding site, indicating that DocH66Y mediates cooperative interactions between adjacent Phd-binding sites. Several electrophoretically distinct protein-DNA complexes were observed with different amounts of DocH66Y relative to Phd. Maximal repression and specificity of DNA binding were observed with subsaturating amounts of DocH66Y relative to Phd. Analogous antidote-toxin pairs appear to have similar autoregulatory circuits. Autoregulation, by dampening fluctuations in the levels of toxin and antidote, may prevent the inappropriate activation of the toxin.
An Analytical Model Applied to a Multicenter Pneumococcal Enzyme-Linked Immunosorbent Assay Study
Authors: Plikaytis Brian D. , Goldblatt David , Frasch Carl E. , Blondeau Christine , Bybel Michael J. , Giebink G. Scott , Jonsdottir Ingileif , Käyhty Helena , Konradsen Helle Bossen , Madore Dace V. , Nahm Moon H. , Schulman Cheryl A. , Holder Patricia F. , Lezhava Tamar , Elie Cheryl M. , Carlone George M. 
Published: March 2020

ABSTRACT
Pneumococcal conjugate vaccines will eventually be licensed after favorable results from phase III efficacy trials. After licensure of a conjugate vaccine for invasive pneumococcal disease in infants, new conjugate vaccines will likely be licensed primarily on the basis of immunogenicity data rather than clinical efficacy. Analytical methods must therefore be developed, evaluated, and validated to compare immunogenicity results accurately within and between laboratories for different vaccines. At present no analytical technique is uniformly accepted and used in vaccine evaluation studies to determine the acceptable level of agreement between a laboratory result and the assigned value for a given serum sample. This multicenter study describes the magnitude of agreement among 12 laboratories quantifying an identical series of 48 pneumococcal serum specimens from 24 individuals (quality-control sera) by a consensus immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) developed for this study. After provisional or trial antibody concentrations were assigned to the quality-control serum samples for this study, four methods for comparison of a series of laboratory-determined values with the assigned concentrations were evaluated. The percent error between assigned values and laboratory-determined concentrations proved to be the most informative of the four methods. We present guidelines that a laboratory may follow to analyze a series of quality-control sera to determine if it can reproduce the assigned antibody concentrations within an acceptable level of tolerance. While this study focused on a pneumococcal IgG ELISA, the methods that we describe are easily generalizable to other immunological assays.
PCR and Restriction Endonuclease Analysis for Rapid Identification of Human Adenovirus Subgenera
Authors: Elnifro Elfath M. , Cooper Robert J. , Klapper Paul E. , Bailey Andrew S. 
Published: March 2020

ABSTRACT
Subgenus identification of adenoviruses is of clinical importance and is as informative as identification by serotype in most clinical situations. A PCR-based identification of adenovirus subgenera A, B, C, D, E, and F and sometimes serotypes is described. The PCR uses nonnested primer pair ADRJC1-ADRJC2, which targets a highly conserved region of the adenovirus hexon gene, has a sensitivity of 10 to 40 copies of adenovirus type 2 (Ad2) DNA, and generates 140-bp PCR products from adenovirus serotypes representative of all the subgroups. The PCR products of all subgroups can be differentiated on the basis of the restriction fragment patterns produced by a total of five restriction endonucleases. In addition, serotypes Ad40 and Ad41 (subgroup F) and important serotypes of subgroup D (Ad8, Ad10, Ad19, and Ad37) can easily be differentiated, but serotypes within subgroups B and C cannot. The method was assessed by blind subgenus identification of 56 miscellaneous clinical isolates of adenoviruses. The identities of these isolates at the subgenus level by the PCR correlated 91% (51 of 56) with the results of serotyping by the neutralization test, and 9% (5 of 56) of clinical isolates produced discordant results.
rpoB
Gene Analysis as a Novel Strategy for Identification of Spirochetes from the Genera
Borrelia
,
Treponema
, and
Leptospira
Authors: Renesto Patricia , Lorvellec-Guillon Katell , Drancourt Michel , Raoult Didier 
Published: March 2020

ABSTRACT

Spirochetes are emerging pathogens for which culture and identification are partly unresolved. In fact, 16S rRNA-based sequencing is by far the most widely used PCR methodology that is able to detect such uncultivable pathogens. However, this assay actually has some limitations linked to potential problems of contamination, which hampers diagnosis. To circumvent this, we have devised a simple PCR strategy involving targeting of the gene encoding the RNA polymerase beta subunit (
rpoB
), a highly conserved enzyme. The complete sequence of the
Leptospira biflexa
(serovar patoc)
rpoB
gene was determined and compared with the published sequences for
Borrelia burgdorferi
and
Treponema pallidum
. From the resulting analysis, degenerate nucleotide primers were designed and tested for their ability to amplify a portion of the
rpoB
gene from various spirochetes. Using two different pairs of these primers, we succeeded in obtaining specific
rpoB
-amplified fragments for all members of the genera
Leptospira
,
Treponema
, and
Borrelia
tested and no other bacteria. Our findings may have significant implications for the development of a new tool for the identification of spirochetes, especially if clinical samples are contaminated or when the infecting strain is uncultivable.
Performance of the COBAS AMPLICOR HCV MONITOR Test, Version 2.0, an Automated Reverse Transcription-PCR Quantitative System for Hepatitis C Virus Load Determination
Authors: Gerken G. , Rothaar T. , Rumi M. G. , Soffredini R. , Trippler M. , Blunk M. J. , Butcher A. , Soviero S. , Colucci G. 
Published: March 2020

ABSTRACT

A clinical evaluation of an automated quantitative PCR assay, the COBAS AMPLICOR HCV MONITOR test, version 2.0 (v2.0), was carried out to assess the performance of this test in comparison with that of the previous, manual version, the AMPLICOR HCV MONITOR test, and with that of nested PCR. Serial dilutions of serum samples infected with genotype 1b, 2a, or 3, as well as synthetic RNA transcripts and serum samples derived from 87 patients with chronic hepatitis C and infected with genotype 1a, 1b, 2a, 2b, 3a, 3b, 4, or 5, were analyzed to determine the ability of the system to efficiently quantify various hepatitis C virus (HCV) genotypes. These experiments showed that the COBAS AMPLICOR HCV MONITOR test, v2.0, has mean intra-assay, interassay, and interoperator coefficients of variation that range from 22 to 34.5% and a 3-logarithm dynamic range, which spans from 10
3
to 10
6
copies/ml. Compared to the previous, manual version of the test, the COBAS AMPLICOR HCV MONITOR test, v2.0, showed an improved efficacy for all genotypes, especially genotypes 2, 3, and 4, whose estimated concentrations were on average 1 logarithm higher. When used to monitor patients under treatment, however, both versions showed the same patterns of viremia, indicating that the COBAS AMPLICOR HCV MONITOR test, v2.0, and the AMPLICOR HCV MONITOR test were equally effective at detecting relative viremia changes in serial samples. As expected, the automated test was less sensitive than nested PCR; among specimens from a cohort of patients treated with interferon, nested PCR identified three more viremic specimens, which probably contained very low concentrations of HCV RNA.
Role of
vacA
and
cagA
in
Helicobacter pylori
Inhibition of Mucin Synthesis in Gastric Mucous Cells
Authors: Beil Winfried , Enss Marie Luise , Müller Simone , Obst Barbara , Sewing Karl-Friedrich , Wagner Siegfried 
Published: March 2020

ABSTRACT

The aim of this study was to investigate the effect of
Helicobacter pylori
on the function of gastric mucous cells.
H. pylori
(10
4
to 10
7
CFU/well) was incubated with the mucin-producing gastric cell line HM02 for 12 and 24 h. Mucin synthesis and secretion were determined by the incorporation of
d
-
N
-[
acetyl
-
14
C]glucosamine into intracellular and released high-molecular-weight glycoproteins.
cagA
-positive, cytotoxin-producing and non-cytotoxin-producing
H. pylori
strains impaired the incorporation of
d
-
N
-[
acetyl
-
14
C]glucosamine into intracellular glycoproteins. Significant inhibition of mucin synthesis was noted after 12 and 24 h of cocultivation with a bacterial load of ≥10
5
bacteria (bacterium/cell ratio = 0.25). The
cagA
-positive, cytotoxin-producing strains (HP64, HP57, and HP87) caused significantly stronger inhibition of intracellular mucin synthesis than the
cagA
-positive, non-cytotoxin-producing strains (HP05, HP83, and HP84). The
cagA
-negative, non-cytotoxin-producing strains (HP01, HP04, and HP85) did not affect intracellular mucin synthesis. The results indicate that
H. pylori
directly impairs mucin synthesis in gastric mucous cells and that cytotoxic
cagA
-positive strains cause more profound inhibition of mucin synthesis. We suggest that the increased inhibitory effect of
cagA
-positive, cytotoxin-producing strains on mucin synthesis can be considered one possible factor responsible for the increased risk of developing peptic ulceration with these
H. pylori
strains.
Evaluation of Immunoglobulin M (IgM) and IgG Enzyme Immunoassays in Serologic Diagnosis of West Nile Virus Infection
Authors: Tardei G. , Ruta S. , Chitu V. , Rossi C. , Tsai T. F. , Cernescu C. 
Published: March 2020

ABSTRACT
A unique urban encephalitis epidemic in Romania signaled the emergence of neurological infection due to West Nile (WN) virus as a novel public health threat in Eastern Europe and provided an opportunity to evaluate patterns of immunoglobulin G (IgG) and IgM reactivity in IgM capture and IgG enzyme-linked immunosorbent assays (ELISAs). WN virus infection was diagnosed serologically in 236 of 290 patients from whom acute serum or cerebrospinal fluid (CSF) samples were available. In 37% of serum samples and in 25% of CSF samples collected in the first week of illness, anti-WN virus IgM antibody was detected in the absence of virus-specific IgG. The switch to an IgG antibody response occurred after 4 to 5 days of illness and earlier in CSF than in serum. A specific humoral immune response was detected in the CSF before the serum in some patients for whom paired CSF and serum samples from the same day were available. IgM antibody in convalescent serum samples persisted beyond 2 months after the onset of illness in more than 50% of patients. ELISA optical density values and antibody concentrations were well correlated for both IgM and IgG immunoassays. Anti-WN virus IgM antibody in acute-phase samples did not cross-react significantly with flaviviruses in other antigenic groups.