bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid Virus Production and Removal as Measured with Fluorescently Labeled Viruses as Tracers
Authors: Noble Rachel T. , Fuhrman Jed A.
Published: July 2002

Pelagic marine viruses have been shown to cause significant mortality of heterotrophic bacteria, cyanobacteria, and phytoplankton. It was previously demonstrated, in nearshore California waters, that viruses contributed to up to 50% of bacterial mortality, comparable to protists. However, in less productive waters, rates of virus production and removal and estimates of virus-mediated bacterial mortality have been difficult to determine. We have measured rates of virus production and removal, in nearshore and offshore California waters, by using fluorescently labeled viruses (FLV) as tracers. Our approach is mathematically similar to the isotope dilution technique, employed in the past to simultaneously measure the release and uptake of ammonia and amino acids. The results indicated overall virus removal rates in the dark ranging from 1.8 to 6.2% h
−1
and production rates in the dark ranging from 1.9 to 6.1% h
−1
, corresponding to turnover times of virus populations of 1 to 2 days, even in oligotrophic offshore waters. Virus removal rates determined by the FLV tracer method were compared to rates of virus degradation, determined at the same locations by radiolabeling methods, and were similar even though the current FLV method is suitable for only dark incubations. Our results support previous findings that virus impacts on bacterial populations may be more important in some environments and less so in others. This new method can be used to determine rates of virus degradation, production, and turnover in eutrophic, mesotrophic, and oligotrophic waters and will provide important inputs for future investigations of microbial food webs.
Identification of
Bacillus thuringiensis
subsp.
kurstaki
Strain HD1-Like Bacteria from Environmental and Human Samples after Aerial Spraying of Victoria, British Columbia, Canada, with Foray 48B
Authors: Valadares de Amorim Giovana , Whittome Beatrixe , Shore Benjamin , Levin David B.
Published: July 2002

Aerial applications of Foray 48B, which contains
Bacillus thuringiensis
strain HD1, were carried out on 9 to 10 May, 19 to 21 May, and 8 to 9 June 1999 to control European gypsy moth (
Lymantria dispar
) populations in Victoria, British Columbia, Canada. A major assessment of the health impact of
B. thuringiensis
subsp.
kurstaki
was conducted by the Office of the Medical Health Officer of the Capital Health Region during this period. Environmental (air and water) and human (nasal swab) samples, collected before and after aerial applications of Foray 48B, both in the spray zone and outside of the spray zone, were analyzed for the presence of strain HD1-like bacteria. Random amplified polymorphic DNA analysis,
cry
gene-specific PCR, and dot blot DNA hybridization techniques were used to screen over 11,000 isolates of bacteria. We identified bacteria with genetic patterns consistent with those of
B. thuringiensis
subsp.
kurstaki
HD1 in 9,102 of 10,659 (85.4%) isolates obtained from the air samples, 13 of 440 (2.9%) isolates obtained from the water samples, and 131 of 171 (76.6%) isolates from the nasal swab samples. These analyses suggest that
B. thuringiensis
subsp.
kurstaki
HD1-like bacteria were present both in the environment and in the human population of Victoria prior to aerial applications of Foray 48B. The presence of
B. thuringiensis
subsp.
kurstaki
HD1-like bacteria in human nasal passages increased significantly after the application of Foray 48B, both inside and outside the spray zone.
Identification of Methyl Halide-Utilizing Genes in the Methyl Bromide-Utilizing Bacterial Strain IMB-1 Suggests a High Degree of Conservation of Methyl Halide-Specific Genes in Gram-Negative Bacteria
Authors: Woodall Claire A. , Warner Karen L. , Oremland Ronald S. , Murrell J. Colin , McDonald Ian R.
Published: July 2002

Strain IMB-1, an aerobic methylotrophic member of the alpha subgroup of the
Proteobacteria
, can grow with methyl bromide as a sole carbon and energy source. A single c
mu
gene cluster was identified in IMB-1 that contained six open reading frames:
cmuC, cmuA, orf146, paaE, hutI
, and partial
metF
. CmuA from IMB-1 has high sequence homology to the methyltransferase CmuA from
Methylobacterium chloromethanicum
and
Hyphomicrobium chloromethanicum
and contains a C-terminal corrinoid-binding motif and an N-terminal methyltransferase motif. However,
cmuB
, identified in
M. chloromethanicum
and
H. chloromethanicum,
was not detected in IMB-1.
Biogenic Magnetite Formation through Anaerobic Biooxidation of Fe(II)
Authors: Chaudhuri Swades K. , Lack Joseph G. , Coates John D.
Published: July 2002

The presence of isotopically light carbonates in association with fine-grained magnetite is considered to be primarily due to the reduction of Fe(III) by Fe(III)-reducing bacteria in the environment. Here, we report on magnetite formation by biooxidation of Fe(II) coupled to denitrification. This metabolism offers an alternative environmental source of biogenic magnetite.
Survival of
Escherichia coli
O157:H7 ATCC 43895 in a Model Apple Juice Medium with Different Concentrations of Proline and Caffeic Acid
Authors: Reinders Robert D. , Biesterveld Steef , Bijker Peter G. H.
Published: July 2002

The effects of proline and caffeic acid on the survival of Shiga toxin-producing
Escherichia coli
(STEC) O157:H7 strain ATCC 43895 in a model apple juice medium were studied. It is hypothesized that the inhibitory effect of caffeic acid may explain why almost all outbreaks of STEC O157:H7 infections linked to apple juice or cider have occurred in October or November.
Energy Yield of Respiration on Chloroaromatic Compounds in
Desulfitobacterium dehalogenans
Authors: van de Pas Bram A. , Jansen Stefan , Dijkema Cor , Schraa Gosse , de Vos Willem M. , Stams Alfons J. M.
Published: July 2002

The amount of energy that can be conserved via halorespiration by
Desulfitobacterium dehalogenans
JW/IU-DC1 was determined by comparison of the growth yields of cells grown with 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) and different electron donors. Cultures that were grown with lactate, pyruvate, formate, or hydrogen as an electron donor and Cl-OHPA as an electron acceptor yielded 3.1, 6.6, 1.6, and 1.6 g (dry weight) per mol of reduction equivalents, respectively. Fermentative growth on pyruvate yielded 14 g (dry weight) per mol of pyruvate oxidized. Pyruvate was not fermented stoichiometrically to acetate and lactate, but an excess of acetate was produced. Experiments with
13
C-labeled bicarbonate showed that during pyruvate fermentation, approximately 9% of the acetate was formed from the reduction of CO
2
. Comparison of the growth yields suggests that 1 mol of ATP is produced per mol of acetate produced by substrate-level phosphorylation and that there is no contribution of electron transport phosphorylation when
D. dehalogenans
grows on lactate plus Cl-OHPA or pyruvate plus Cl-OHPA. Furthermore, the growth yields indicate that approximately 1/3 mol of ATP is conserved per mol of Cl-OHPA reduced in cultures grown in formate plus Cl-OHPA and hydrogen plus Cl-OHPA. Because neither formate nor hydrogen nor Cl-OHPA supports substrate-level phosphorylation, energy must be conserved through the establishment of a proton motive force. Pyruvate ferredoxin oxidoreductase, lactate dehydrogenase, formate dehydrogenase, and hydrogenase were localized by in vitro assays with membrane-impermeable electron acceptors and donors. The orientation of chlorophenol-reductive dehalogenase in the cytoplasmic membrane, however, could not be determined. A model is proposed, which may explain the topology analyses as well as the results obtained in the yield study.
Heteroduplex Panel Analysis, a Novel Method for Genetic Identification of
Aspergillus
Section
Flavi
Strains
Authors: Kumeda Yuko , Asao Tsutomu
Published: July 2002

For genetic identification of
Aspergillus
Section
Flavi
isolates and detection of intraspecific variation, we developed a novel method for heteroduplex panel analysis (HPA) utilizing fragments of the internal transcribed spacer (ITS) regions (ITS1-5.8S-ITS2) of the rRNA gene that was PCR amplified with universal primers. The method involves formation of heteroduplexes with a set of reference fragments amplified from
Aspergillus flavus, A. parasiticus, A. tamarii
, and
A. nomius
and subsequent minislab vinyl polymer gel electrophoresis. The test panel is compared with species-specific standard panels (F-1, P-1, T-1, and N-1) generated by pairwise reannealing among four reference fragments. Of 90 test panels, 89 succeeded in identifying the species and 74 were identical to one of the four standard panels. Of the 16 new panels, 11
A. flavus/A. oryzae
panels were identical and typed as F-2 and 4 of 5
A. nomius
panels were typed as N-2 or N-3. The other strain,
A. nomius
IMI 358749, was unable to identify the species because no single bands were formed with any of the four reference strains. DNA sequencing revealed that our HPA method has the highest sensitivity available and is able to detect as little as one nucleotide of diversity within the species. When
Penicillium
or non-Section
Flavi Aspergillus
was subjected to HPA, the resulting bands of heteroduplexes showed apparently lower mobility and poor heteroduplex formation. This indicates that HPA is a useful identification method without morphological observation and is suitable for rapid and inexpensive screening of large numbers of isolates. The HPA typing coincided with the taxonomy of Section
Flavi
and is therefore applicable as an alternative to the conventional methods (Samson, R. A., E. S. Hoekstra, J. C. Frisvad, and O. Filtenborg, p. 64–97,
in Introduction to Food- and Airborne Fungi
, 6th ed., 2000).
Nucleotide Sequence and Genetic Structure of a Novel Carbaryl Hydrolase Gene (
cehA
) from
Rhizobium
sp. Strain AC100
Authors: Hashimoto Masayuki , Fukui Mitsuru , Hayano Kouichi , Hayatsu Masahito
Published: July 2002

Rhizobium
sp. strain AC100, which is capable of degrading carbaryl (1-naphthyl-
N
-methylcarbamate), was isolated from soil treated with carbaryl. This bacterium hydrolyzed carbaryl to 1-naphthol and methylamine. Carbaryl hydrolase from the strain was purified to homogeneity, and its N-terminal sequence, molecular mass (82 kDa), and enzymatic properties were determined. The purified enzyme hydrolyzed 1-naphthyl acetate and 4-nitrophenyl acetate indicating that the enzyme is an esterase. We then cloned the carbaryl hydrolase gene (
cehA
) from the plasmid DNA of the strain and determined the nucleotide sequence of the 10-kb region containing
cehA
. No homologous sequences were found by a database homology search using the nucleotide and deduced amino acid sequences of the
cehA
gene. Six open reading frames including the
cehA
gene were found in the 10-kb region, and sequencing analysis shows that the
cehA
gene is flanked by two copies of insertion sequence-like sequence, suggesting that it makes part of a composite transposon.
Influence of Medium Buffering Capacity on Inhibition ofSaccharomyces cerevisiaeGrowth by Acetic and Lactic Acids
Authors: Thomas K. C. , Hynes S. H. , Ingledew W. M.
Published: July 2002

Acetic acid (167 mM) and lactic acid (548 mM) completely inhibited growth ofSaccharomyces cerevisiaeboth in minimal medium and in media which contained supplements, such as yeast extract, corn steep powder, or a mixture of amino acids. However, the yeast grew when the pH of the medium containing acetic acid or lactic acid was adjusted to 4.5, even though the medium still contained the undissociated form of either acid at a concentration of 102 mM. The results indicated that the buffer pair formed when the pH was adjusted to 4.5 stabilized the pH of the medium by sequestering protons and by lessening the negative impact of the pH drop on yeast growth, and it also decreased the difference between the extracellular and intracellular pH values (ΔpH), the driving force for the intracellular accumulation of acid. Increasing the undissociated acetic acid concentration at pH 4.5 to 163 mM by raising the concentration of the total acid to 267 mM did not increase inhibition. It is suggested that this may be the direct result of decreased acidification of the cytosol because of the intracellular buffering by the buffer pair formed from the acid already accumulated. At a concentration of 102 mM undissociated acetic acid, the yeast grew to higher cell density at pH 3.0 than at pH 4.5, suggesting that it is the total concentration of acetic acid (104 mM at pH 3.0 and 167 mM at pH 4.5) that determines the extent of growth inhibition, not the concentration of undissociated acid alone.
Statistical Analysis of
Pseudomonas aeruginosa
Biofilm Development: Impact of Mutations in Genes Involved in Twitching Motility, Cell-to-Cell Signaling, and Stationary-Phase Sigma Factor Expression
Authors: Heydorn Arne , Ersbøll Bjarne , Kato Junichi , Hentzer Morten , Parsek Matthew R. , Tolker-Nielsen Tim , Givskov Michael , Molin Søren
Published: July 2002

Four strains of
Pseudomonas aeruginosa
(wild type, Δ
pilHIJK
mutant,
lasI
mutant, and
rpoS
mutant) were genetically tagged with the green fluorescent protein, and the development of flow chamber-grown biofilms by each of them was investigated by confocal laser scanning microscopy. The structural developments of the biofilms were quantified by the computer program COMSTAT (A. Heydorn, A. T. Nielsen, M. Hentzer, C. Sternberg, M. Givskov, B. K. Ersbøll, and S. Molin, Microbiology 146:2395-2407, 2000). Two structural key variables, average thickness and roughness, formed the basis for an analysis of variance model comprising the four
P. aeruginosa
strains, five time points (55, 98, 146, 242, and 314 h), and three independent rounds of biofilm experiments. The results showed that the wild type, the Δ
pilHIJK
mutant, and the
rpoS
mutant display conspicuously different types of temporal biofilm development, whereas the
lasI
mutant was indistinguishable from the wild type at all time points. The wild type and the
lasI
mutant formed uniform, densely packed biofilms. The
rpoS
mutant formed densely packed biofilms that were significantly thicker than those of the wild type, whereas the Δ
pilHIJK
mutant formed distinct microcolonies that were regularly spaced and almost uniform in size. The results are discussed in relation to the current model of
P. aeruginosa
biofilm development.
Ability of theListeria monocytogenesStrain Scott A To Cause Systemic Infection in Mice Infected by the Intragastric Route
Authors: Czuprynski Charles J. , Faith Nancy G. , Steinberg Howard
Published: July 2002

Listeriosis is an important food-borne disease that causes high rates of morbidity and mortality. For reasons that are not clear, most large outbreaks of human listeriosis involveListeria monocytogenesserotype 4b. Relatively little is known about the pathogenesis of listeriosis following gastrointestinal exposure to food-borne disease isolates ofL. monocytogenes. In the present study, we investigated the pathogenesis of systemic infection by the food-borne isolate Scott A in an intragastric (i.g.) mouse challenge model. We found that the severity of infection withL. monocytogenesScott A was increased in mice made neutropenic by administration of monoclonal antibody RB6-8C5. This observation was similar to a previous report on a study with the laboratory strainL. monocytogenesEGD. Prior administration of sodium bicarbonate did not enhance the virulence ofL. monocytogenesstrain Scott A for i.g. inoculated mice. Following i.g. inoculation of mice, two serotype 4b strains ofL. monocytogenes(Scott A and 101M) achieved a greater bacterial burden in the spleen and liver and elicited more severe histopathological damage to those organs than did a serotype 1/2a strain (EGD) and a serotype 1/2b stain (CM). Of the four strains tested, only strain CM exhibited poor survival in synthetic gastric fluid in vitro. The other three strains exhibited similar patterns of survival at pHs of greater than 5 and relatively rapid (<30 min) loss of viability at pHs of less than 5.0. Growth ofL. monocytogenesScott A at temperatures of 12.5 to 37°C did not affect its ability to cause systemic infection in i.g. inoculated mice. These observations suggest that the serotype 4bL. monocytogenesstrains Scott A and 101M possess one or more virulence determinants that make them better able to cause systemic infection following inoculation via the g.i. tract than do the serotype 1/2 strains EGD and CM.
Evaluation of the Modified ELISPOT Assay for Gamma Interferon Production in Cancer Patients Receiving Antitumor Vaccines
Authors: Asai Tadao , Storkus Walter J. , Whiteside Theresa L.
Published: July 2002

Frequencies of vaccine-responsive T-lymphocyte precursors in peripheral blood mononuclear cells (PBMC) prior to and after administration of peptide-based vaccines in patients with cancer can be measured by limiting-dilution assays (LDA) or by ELISPOT assays. We have used a modified version of the ELISPOT assay to monitor changes in the frequency of gamma interferon (IFN-γ)-producing T cells in a population of lymphocytes responding to a relevant peptide or a nonspecific stimulator, such as phorbol myristate acetate-ionomycin. Prior to its use for monitoring of patient samples, the assay was validated and found to be comparable to the LDA performed in parallel, using tumor-reactive cytolytic T-lymphocyte (CTL) lines. The sensitivity of the ELISPOT assay was found to be 1/100,000 cells, with an interassay coefficient of variation of 15%, indicating that it could be reliably used for monitoring of changes in the frequency of IFN-γ-secreting responder cells in noncultured or cultured lymphocyte populations. To establish that the assay is able to detect the T-cell precursor cells responsive to the vaccine, we used CD8+T-cell populations positively selected from PBMC of HLA-A2+patients with metastatic melanoma, who were treated with dendritic cell-based vaccines containing gp100, MELAN-A/MART-1, tyrosinase, and influenza virus matrix peptides. The frequency of peptide-specific responder T cells ranged from 0 to 1/2,600 before vaccination and increased by at least 1 log unit after vaccination in two patients, one of whom had a clinical response to the vaccine. However, no increases in the frequency of peptide-responsive T cells were observed in noncultured PBMC or PBMC cultured in the presence of the relevant peptides after the melanoma patients enrolled in another trial were treated with the intramuscular peptide vaccine plus MF59 adjuvant. Thus, while the ELISPOT assay was found to be readily applicable to assessments of frequencies of CTL precursors of established CTL lines and ex vivo-amplified PBMC, its usefulness for monitoring of fresh PBMC in patients with cancer was limited. In many of these patients antitumor effector T cells are present at frequencies of lower than 1/100,000 in the peripheral circulation. Serial monitoring of such patients may require prior ex vivo amplification of specific precursor cells.
CD8+T-Cell Gamma Interferon Production Specific for Human Immunodeficiency Virus Type 1 (HIV-1) in HIV-1-Infected Subjects
Authors: Huang Xiao-Li , Fan Zheng , Kalinyak Christine , Mellors John W. , Rinaldo Charles R.
Published: July 2002

The CD8+-T-cell response to human immunodeficiency virus type 1 (HIV-1) is considered to be important in host control of infection and prevention of AIDS. We have developed a single-cell enzyme immunoassay (enzyme-linked immunospot assay) specific for gamma interferon (IFN-γ) production stimulated by either autologous B-lymphoblastoid cell lines (B-LCL) infected with vaccinia virus vectors expressing HIV-1 proteins or synthetic peptides representing known HIV-1 CD8+cytotoxic T-lymphocyte (CTL) epitopes. Single-cell IFN-γ production stimulated by HIV-1 Gag-, Pol-, and Env-expressing B-LCL was a reliable measure of HIV-1-specific T-cell immunity in peripheral blood CD8+T cells from HIV-1 infected individuals. This method was more sensitive than stimulation of IFN-γ by direct infection of the cultures with HIV-1–vaccinia virus vectors. Comparable results were found for IFN-γ production in CD8+T cells from HIV-1-negative, cytomegalovirus (CMV)-seropositive, healthy donors stimulated with B-LCL expressing the CMV pp65 lower matrix protein. HIV-1 peptides were immunodominant for both CD8+single-cell IFN-γ production and CTL precursor frequencies. The number of cells producing IFN-γ decreased in individuals with late-stage HIV-1 infection and was temporally enhanced during combination antiretroviral therapy with two reverse transcriptase nucleoside inhibitors and a protease inhibitor.
Diagnosis of
Enterocytozoon bieneusi
by PCR in Stool Samples Eluted from Filter Paper Disks
Authors: Carnevale Silvana , Velásquez Jorge N. , Labbé Jorge H. , Chertcoff Agustín , Cabrera Marta G. , Rodríguez Mónica I.
Published: July 2002

We report a PCR-based assay for the detection of
Enterocytozoon bieneusi
. We extracted DNA from feces which had been applied to filter paper disks and evaluated four preserving solutions. Infected specimens were identified by electrophoresis of amplicons from concentrated formalin-fixed samples and unconcentrated fresh feces. Our findings demonstrate that this methodology is effective for sample collection, mailing, and diagnosis of this pathogen.
Relationship between Serum Bactericidal Activity and Serogroup-Specific Immunoglobulin G Concentration for Adults, Toddlers, and Infants Immunized withNeisseria meningitidisSerogroup C Vaccines
Authors: Sikkema Daniel J. , Friedman Keith E. , Corsaro Bartholomew , Kimura Alan , Hildreth Stephen W. , Madore Dace V. , Quataert Sally A.
Published: July 2002

A new meningococcal group C-CRM197conjugate vaccine (MnCC; Meningitec) has been evaluated in multiple clinical trials in the United States and most recently has been approved for routine administration in the United Kingdom. Meningococcal serogroup C (MnC)-specific immunoglobulin G (IgG) antibodies in pre- and postimmunization sera obtained from healthy U.S. adults, toddlers, and infants were quantitated by enzyme-linked immunosorbent assay (ELISA) and by an antibody-dependent, complement-mediated serum bactericidal assay (SBA). Serogroup-specific IgG antibody (micrograms per milliliter) in adults immunized either with the quadrivalent polysaccharide (A, C, Y, and W-135) vaccine or with MnCC showed a strong correlation (r= 0.848 and 0.934, respectively) by linear regression analysis with SBA. Sera from infants immunized with the MnCC (n= 30) and an age-matched unimmunized control group (n= 15) were also analyzed. Linear regression analysis of serum bactericidal and IgG ELISA data from sera obtained at 2 months of age (preimmunization) showed no correlation; however, a high degree of correlation was observed at time points after two (r= 0.877) and three (r= 0.951) immunizations, where significant rises in anti-MnC polysaccharide antibodies occurred relative to the age-matched control group. Infants previously primed with 3 doses of MnCC were given a booster dose of conjugate vaccine at 12 to 15 months of age. The correlation coefficient of ELISA to SBA for combined pre- and postbooster data wasr= 0.836 (n= 48 pairs). In conclusion, increases in serum bactericidal activity in immunized adult, toddler, and infant populations were found to correlate very well with increases in serogroup-specific IgG concentrations, whereas the correlation between these two assays in nonimmunized 2-month-old infants was poor. Characterizing the relationship between these methods is important for understanding the significance of antigen-specific antibody concentrations relative to vaccine performance and protection from disease.
Serodiagnosis of Recently Acquired
Toxoplasma gondii
Infection Using an Enzyme-Linked Immunosorbent Assay with a Combination of Recombinant Antigens
Authors: Li Shuli , Galvan Gina , Araujo Fausto G. , Suzuki Yasuhiro , Remington Jack S. , Parmley Stephen
Published: July 2002

An enzyme-linked immunosorbent assay (ELISA) using four recombinant antigens of
Toxoplasma gondii
(rP22, rP25, rP29, and rP35) was used in an attempt to differentiate pregnant women with toxoplasma serologic profiles (TSPs) indicative of recently acquired infections (acute profile) from those with TSPs indicative of infections acquired in the distant past (chronic profile). In general, immunoglobulin G antibodies in sera from women with the acute profile reacted more strongly with the recombinant antigens than did those in sera from women with the chronic profile. However, reactivities differed significantly between antigens that reacted with a single serum and between sera that reacted with a single antigen. Because of these variations, we employed a combination of the four antigens in an ELISA (Comb-ELISA) and evaluated its ability to distinguish pregnant women with the acute profile from those with the chronic profile. Eighteen of 20 (90%) sera from acute-profile women were positive in the Comb-ELISA, whereas 69 of 70 (98.6%) sera from the chronic-profile women were negative. Thus, the Comb-ELISA may be useful for diagnosis of toxoplasmosis in pregnant women and for differentiation between recently acquired infections and infections acquired in the more distant past.
Detection and Quantification of Antibodies to Newcastle Disease Virus in Ostrich and Rhea Sera Using a Liquid Phase Blocking Enzyme-Linked Immunosorbent Assay
Authors: de Sousa Ricardo Luiz Moro , Montassier Helio José , Pinto Aramis Augusto
Published: July 2002

A liquid phase blocking ELISA (LPB-ELISA) was adapted for the detection and quantification of antibodies to Newcastle disease virus. Sera from vaccinated and unvaccinated commercial flocks of ostriches (
Struthio camelus
) and rheas (
Rhea americana
) were tested. The purified and nonpurified virus used as the antigen and the capture and detector antibodies were prepared and standardized for this purpose. The hemagglutination-inhibition (HI) test was regarded as the reference method. The cutoff point for the LPB-ELISA was determined by a two-graph receiver operating characteristic analysis. The LPB-ELISA titers regressed significantly (
P
< 0.0001) on the HI titers with a high correlation coefficient (
r
= 0.875). The two tests showed good agreement (κ = 0.82;
P
< 0.0001), relative sensitivity (90.91%) and specificity (91.18%), and accuracy (91.02%), suggesting that they are interchangeable.
Cross-Reactivity of Epstein-Barr Virus-Specific Immunoglobulin M Antibodies with Cytomegalovirus Antigens Containing Glycine Homopolymers
Authors: Lang Dieter , Vornhagen Rolf , Rothe Markus , Hinderer Walter , Sonneborn Hans-H. , Plachter Bodo
Published: July 2002

Timely and reliable detection of acute primary human cytomegalovirus (HCMV) infection is important in prenatal screening programs and for differential diagnosis of infectious mononucleosis-like disease. Enzyme-linked immunosorbent assays (ELISAs) based on HCMV proteins enable the sensitive detection of immunoglobulin M (IgM) antibodies during primary infection. However, concerns have been raised about possible cross-reactivities of the HCMV antigens used for the design of such ELISAs with IgM antibodies induced by Epstein-Barr Virus (EBV). In this study we investigated whether IgM antibodies generated during acute EBV infection reacted with recombinant HCMV antigens. Serum samples from patients with primary EBV infection frequently scored positive when tested in different HCMV IgM ELISAs, irrespective of whether conventional or recombinant antigens were used for the design of the HCMV IgM assays. Such cross-reactive IgM antibodies were found to be directed against short glycine-rich motifs contained within the nonstructural HCMV proteins pUL44 and pUL57. Further analyses revealed that these glycine-rich motifs were major antigenic domains for IgM antibodies induced during HCMV infection. Their deletion from recombinant proteins abrogated reactivity with IgM synthesized during HCMV infection. EBV-induced IgM antibodies that reacted with HCMV antigens showed similar kinetics of reactivity in HCMV- or EBV-specific assays in the course of primary EBV infection, indicating that the two populations of antibodies were highly overlapping. The results demonstrate that primary EBV infection leads to the induction of IgM antibodies that specifically bind to widely used diagnostic antigens of HCMV. This has to be considered in the interpretation of HCMV-specific IgM assays.
Enzyme-Linked Immunosorbent Assay for Recombinant K39 Antigen in Diagnosis and Prognosis of Indian Visceral Leishmaniasis
Authors: Kumar R. , Pai K. , Pathak K. , Sundar S.
Published: July 2002

The recombinant product (rK39) of the 39-amino-acid repeats encoded by a kinesin-like protein-encoding gene ofLeishmania chagasiwas evaluated by enzyme-linked immunosorbent assay (ELISA) for diagnostic potential and the ability to predict the response to therapy in Indian kala-azar or visceral leishmaniasis (VL); we also compared its performance with that of crude soluble antigen (CSA). At the diagnosis of VL, the anti-rK39 antibody titer was 59-fold higher than the anti-CSA antibody titer. With successful therapy, antibody titers declined steeply at the end of treatment and during follow-up. In contrast, patients who relapsed showed increased titers of antibodies to rK39. The extremely high levels of anti-rK39 antibodies in VL cases suggest the application of rK39 for sensitive and specific serodiagnosis, and rK39 ELISA is also valuable in monitoring drug therapy and detecting relapse of the disease.
Cervical Cat Scratch Disease Lymphadenitis in a Patient with Immunoglobulin M Antibodies to
Toxoplasma gondii
Authors: Arvand Mardjan , Kazak Ilkay , Jovanovic Sergije , Foss Hans-Dieter , Liesenfeld Oliver
Published: July 2002

We report on a young patient with chronic cervical lymphadenopathy and serological and histological evidence for infection with
Bartonella henselae
and
Toxoplasma gondii
. Serological follow-up studies, including testing for avidity of
Toxoplasma
-specific immunoglobulin G antibodies, assisted in the determination of the cause of the acute lymphadenitis. Our results suggest that the clinical symptoms were most likely due to cat scratch disease rather than to acute toxoplasmosis.
Experimental Oral Candidiasis in Animal Models
Authors: Samaranayake Yuthika H. , Samaranayake Lakshman P.
Published: July 2002

Oral candidiasis is as much the final outcome of the vulnerability of the host as of the virulence of the invading organism. We review here the extensive literature on animal experiments mainly appertaining to the host predisposing factors that initiate and perpetuate these infections. The monkey, rat, and mouse are the choice models for investigating oral candidiasis, but comparisons between the same or different models appear difficult, because of variables such as the study design, the number of animals used, their diet, the differences in Candida strains, and the duration of the studies. These variables notwithstanding, the following could be concluded. (i) The primate model is ideal for investigating Candida-associated denture stomatitis since both erythematous and pseudomembranous lesions have been produced in monkeys with prosthetic plates; they are, however, expensive and difficult to obtain and maintain. (ii) The rat model (both Sprague-Dawley and Wistar) is well proven for observing chronic oral candidal colonization and infection, due to the ease of breeding and handling and their ready availability. (iii) Mice are similar, but in addition there are well characterized variants simulating immunologic and genetic abnormalities (e.g., athymic, euthymic, murine-acquired immune deficiency syndrome, and severe combined immunodeficient models) and hence are used for short-term studies relating the host immune response and oral candidiasis. Nonetheless, an ideal, relatively inexpensive model representative of the human oral environment in ecological and microbiological terms is yet to be described. Until such a model is developed, researchers should pay attention to standardization of the experimental protocols described here to obtain broadly comparable and meaningful data.
Sequence Analysis of Tn
phoA
Insertion Sites in
Vibrio cholerae
Mutants Defective in Rugose Polysaccharide Production
Authors: Ali Afsar , Hayat Mahmud Zahid , Morris J. Glenn , Sozhamannan Shanmuga , Johnson Judith A.
Published: July 2002

Vibrio cholerae
can switch from a smooth to a wrinkled or rugose colony phenotype characterized by the secretion of a polysaccharide that enables the bacteria to survive harsh environmental conditions. In order to understand the genetic basis of rugosity, we isolated Tn
phoA
-induced stable, smooth mutants of two O1 El Tor rugose strains and mapped the insertion sites in several of the mutants using a modified Y-adapter PCR technique. One of the Tn
phoA
insertions was mapped to the first gene of the
vps
region that was previously shown to encode the rugose polysaccharide biosynthesis cluster. Three insertions were mapped to a previously unknown
hlyA
-like gene, also in the
vps
region. Five other insertions were found in loci unlinked to the
vps
region: (i) in the
epsD
gene (encodes the “secretin” of the extracellular protein secretion apparatus), (ii) in a
hydG
-like gene (encodes a ς
54
-dependent transcriptional activator similar to HydG involved in labile hydrogenase production in
Escherichia coli
, (iii) in a gene encoding malic acid transport protein upstream of a gene similar to
yeiE
of
E. coli
(encodes a protein with similarities to LysR-type transcriptional activators), (iv) in
dxr
(encodes 1-deoxy-
d
-xylulose 5-phosphate reductoisomerase), and (v) in the intergenic region of
lpd
and
odp
(encode enzymes involved in the pyruvate dehydrogenase complex formation). These data suggest the involvement of a complex regulatory network in rugose polysaccharide production and highlight the general utility of the Y-adapter PCR technique described here for rapid mapping of transposon insertion sites.
Inhibition of T-cell Response byEscherichia coliHeat-Labile Enterotoxin-Treated Epithelial Cells
Authors: Lopes Luciene M. , Maroof Asher , Dougan Gordon , Chain Benjamin M.
Published: July 2002

Escherichia coliheat-labile enterotoxin (LT) is an extensively studied adjuvant of mucosal responses. Nevertheless, its mode of action as an adjuvant remains incompletely understood. In this study, we describe a simplified in vitro model with which to look at some aspects of immunoregulation by LT. The interaction of LT with the apical surface of a monolayer of CaCo-2 epithelial cells induces the release of a soluble factor which inhibits the antigen-induced release of interleukin-2 by T cells cultured at the basolateral side of the cells. The release of this factor requires the ADP-ribosylating activity of LT since the isolated B subunit, as well as an enzymatically silent LT mutant, loses biological activity in this model. The inhibitory activity is likely to be due to prostaglandin release, since it is blocked by indomethacin. The contribution of LT-induced prostaglandin release to the complex immunoregulatory activity of LT is discussed.
Occurrence of Severe Destructive Lyme Arthritis in Hamsters Vaccinated with Outer Surface Protein A and Challenged withBorrelia burgdorferi
Authors: Croke Cindy L. , Munson Erik L. , Lovrich Steven D. , Christopherson John A. , Remington Monica C. , England Douglas M. , Callister Steven M. , Schell Ronald F.
Published: July 2002

Arthritis is a frequent and major complication of infection withBorrelia burgdorferisensu stricto. The antigens responsible for the induction of arthritis are unknown. Here we provide direct evidence that a major surface protein, outer surface protein A (OspA), can induce arthritis. Hamsters were vaccinated with 30, 60, or 120 μg of recombinant OspA (rOspA) in aluminum hydroxide and challenged withB. burgdorferisensu stricto isolate 297 or C-1-11. Swelling of the hind paws was detected in 100, 100, and 50% of hamsters vaccinated with 30, 60, or 120 μg of rOspA, respectively. In addition, arthritis developed in 57% of hamsters vaccinated with a canine rOspA vaccine after infection withB. burgdorferisensu stricto. When the canine rOspA vaccine was combined with aluminum hydroxide, all vaccinated hamsters developed arthritis after challenge withB. burgdorferisensu stricto. Histopathologic examination confirmed the development of severe destructive arthritis in rOspA-vaccinated hamsters challenged withB. burgdorferisensu stricto. These findings suggest that rOspA vaccines should be modified to eliminate epitopes of OspA responsible for the induction of arthritis. Our results are important because an rOspA vaccine in aluminum hydroxide was approved by the Food and Drug Administration for use in humans.
Bacterial Phosphorylcholine Decreases Susceptibility to the Antimicrobial Peptide LL-37/hCAP18 Expressed in the Upper Respiratory Tract
Authors: Lysenko Elena S. , Gould Jane , Bals Robert , Wilson James M. , Weiser Jeffrey N.
Published: July 2002

A number of pathogens of the upper respiratory tract express an unusual prokaryotic structure, phosphorylcholine (ChoP), on their cell surface. We tested the hypothesis that ChoP, also found on host membrane lipids in the form of phosphatidylcholine, acts so as to decrease killing by antimicrobial peptides that target differences between bacterial and host membranes. In
Haemophilus influenzae
, ChoP is a phase-variable structure on the oligosaccharide portion of the lipopolysaccharide (LPS). There was a bactericidal effect of the peptide LL-37/hCAP18 on a nontypeable
H. influenzae
strain, with an increasing selection for the ChoP
+
phase as the concentration of the peptide was raised from 0 to 10 μg/ml. Moreover, constitutive ChoP-expressing mutants of unrelated strains showed up to 1,000-fold-greater survival compared to mutants without ChoP. The effect of ChoP on resistance to killing by LL-37/hCAP18 was dependent on the salt concentration and was observed only when bacteria were grown in the presence of environmental choline, a requirement for the expression of ChoP on the LPS. Further studies established that there is transcription of the LL-37/hCAP18 gene on the epithelial surface of the human nasopharynx in situ and inducible transcription in epithelial cells derived from the upper airway. The presence of highly variable amounts of LL-37/hCAP18 in normal nasal secretions (<1.2 to >80 μg/ml) was demonstrated with an antibody against this peptide. It was concluded that ChoP alters the bacterial cell surface so as mimic host membrane lipids and decrease killing by LL-37/hCAP18, an antimicrobial peptide that may be expressed on the mucosal surface of the nasopharynx in bactericidal concentrations.
Dual Role for Transforming Growth Factor β-Dependent Signaling in
Trypanosoma cruzi
Infection of Mammalian Cells
Authors: Hall Belinda S. , Pereira Miercio A.
Published: July 2002

Expression of functional transforming growth factor β (TGF-β) receptors (TβR) is required for the invasion of mammalian cells by the protozoan parasite
Trypanosoma cruzi
. However, the precise role of this host cell signaling complex in
T. cruzi
infection is unknown. To investigate the role of the TGF-β signaling pathway, infection levels were studied in the mink lung epithelial cell lines JD1, JM2, and JM3. These cells express inducible mutant TβR1 proteins that cannot induce growth arrest in response to TGF-β but still transmit the signal for TGF-β-dependent gene expression. In the absence of mutant receptor expression, trypomastigotes invaded the cells at a low level. Induction of the mutant receptors caused an increase in infection in all three cell lines, showing that the requirement for TGF-β signaling at invasion can be divorced from TGF-β-induced growth arrest. TGF-β pretreatment of mink lung cells expressing wild-type TβR1 caused a marked enhancement of infection, but no enhancement was seen in JD1, JM2, and JM3 cells, showing that the ability of TGF-β to stimulate infection is associated with growth arrest. Likewise, expression of SMAD7 or SMAD2SA, inhibitors of TGF-β signaling, did not block infection by
T. cruzi
but did block the enhancement of infection by TGF-β. Taken together, these results show that there is a dual role for TGF-β signaling in
T. cruzi
infection. The initial invasion of the host cell is independent of both TGF-β-dependent gene expression and growth arrest, but TGF-β stimulation of infection requires a fully functional TGF-β signaling pathway.
Upregulation of Monocyte Urokinase Plasminogen Activator Receptor during Human Endotoxemia
Authors: Dekkers Pascale E. P. , ten Hove Tessa , te Velde Anje A. , van Deventer Sander J. H. , van der Poll Tom
Published: July 2002

The receptor for urokinase-type plasminogen activator (uPAR) (CD87) plays an important role in leukocyte adhesion and migration. To assess the effect of endotoxin on cellular uPAR, uPAR expression was determined on leukocytes by fluorescence-activated cell sorter analysis in seven healthy subjects following intravenous injection of endotoxin (lot G; 4 ng/kg). Endotoxin induced a transient increase in uPAR expression on monocytes, reaching a 92% ± 46% increase over baseline expression after 6 h (P< 0.05). Endotoxin did not influence uPAR expression on granulocytes, while uPAR remained undetectable on lymphocytes. Endotoxin also increased soluble uPAR levels in plasma (P< 0.05). Stimulation of human whole blood with endotoxin or gram-positive stimuli in vitro also resulted in an upregulation of monocyte uPAR expression. Although tumor necrosis factor alpha (TNF) upregulated monocyte uPAR expression, anti-TNF did not influence the endotoxin-induced increase in monocyte uPAR expression. These data suggest that infectious stimuli may influence monocyte function in vivo by enhancing the expression of uPAR.
Motility Is Required To Initiate Host Cell Invasion by
Yersinia enterocolitica
Authors: Young Glenn M. , Badger Julie L. , Miller Virginia L.
Published: July 2002

Invasin-mediated invasion of host cells by the pathogen
Yersinia enterocolitica
was shown to be affected by flagellar-dependent motility. Motility appears to be required to ensure the bacterium migrates to and contacts the host cell. Nonmotile strains of
Y. enterocolitica
were less invasive than motile strains, but the reduction in invasion could be overcome by artificially bringing the bacteria into host cell contact by centrifugation. Mutations in known regulatory genes of the flagellar regulon,
flhDC
and
fliA
, resulted in less
inv
expression but did not have a significant effect on invasin levels. However, invasin levels were reduced for strains that harbored
flhDC
on a multicopy plasmid, apparently as a result of increased proteolysis of invasin.
Protection of Gerbils from Amebic Liver Abscess by Vaccination with a 25-mer Peptide Derived from the Cysteine-Rich Region ofEntamoeba histolyticaGalactose-Specific Adherence Lectin
Authors: Lotter Hannelore , Khajawa Fareed , Stanley Samuel L. , Tannich Egbert
Published: July 2002

The protozoan parasiteEntamoeba histolyticacauses extensive morbidity and mortality through intestinal infection and amebic liver abscess. Here we show that immunization of gerbils with a single keyhole limpet hemocyanin-coupled 25-mer peptide derived from the 170-kDa subunit of theE. histolyticagalactose-binding adhesin is sufficient to confer substantial protection against experimentally induced amebic liver abscesses. Vaccination provided total protection in 5 of 15 immunized gerbils, and abscesses were significantly smaller (P< 0.01) in the remaining vaccinated animals. The degree of protection correlated with the titer of antibodies to the peptide, and results of passive transfer experiments performed with SCID mice were consistent with a role for antibodies in protection. In addition, parenteral or oral vaccination of gerbils with 13-amino-acid subfragments of the peptide N-terminally fused to the B subunit of cholera toxin also significantly inhibited liver abscess formation (P< 0.05). These data indicate that small peptides derived from the galactose-binding adhesin administered by the parenteral or oral route can provide protection against amebic liver abscess and should be considered as components of a subunit vaccine against invasive amoebiasis.
Induction of Proinflammatory Cytokines from Human Respiratory Epithelial Cells after Stimulation by NontypeableHaemophilus influenzae
Authors: Clemans Daniel L. , Bauer Richard J. , Hanson Julie A. , Hobbs Monte V. , St. Geme Joseph W. , Marrs Carl F. , Gilsdorf Janet R.
Published: July 2002

NontypeableHaemophilus influenzae(NTHi) causes repeated respiratory infections in patients with chronic lung diseases. These infections are characterized by a brisk inflammatory response which results in the accumulation of polymorphonucleated cells in the lungs and is dependent on the expression and secretion of proinflammatory cytokines. We hypothesize that multiple NTHi molecules, including lipooligosaccharide (LOS), mediate cellular interactions with respiratory epithelial cells, leading to the production of proinflammatory cytokines. To address this hypothesis, we exposed 9HTEo− human tracheal epithelial cells to NTHi and compared the resulting profiles of cytokine gene expression and secretion using multiprobe RNase protection assays and enzyme-linked immunosorbent assays (ELISA), respectively. Dose-response experiments demonstrated a maximum stimulation of most cytokines tested, using a ratio of 100 NTHi bacterial cells to 1 9HTEo− tracheal epithelial cell. Compared with purified LOS, NTHi bacterial cells stimulated 3.6- and 4.5-fold increases in epithelial cell expression of interleukin-8 (IL-8) and IL-6 genes, respectively. Similar results were seen with epithelial cell macrophage chemotactic protein 1, IL-1α, IL-1β, and tumor necrosis factor alpha expression. Polymyxin B completely inhibited LOS stimulation but only partially reduced NTHi whole cell stimulation. Taken together, these results suggest that multiple bacterial molecules including LOS contribute to the NTHi stimulation of respiratory epithelial cell cytokine production. Moreover, no correlation was seen between NTHi adherence to epithelial cells mediated by hemagglutinating pili, Hia, HMW1, HMW2, and Hap and epithelial cytokine secretion. These data suggest that bacterial molecules beyond previously described NTHi cell surface adhesins and LOS play a role in the induction of proinflammatory cytokines from respiratory epithelial cells.
Antibody and Cytokine Responses to the Cilium-Associated Respiratory Bacillus in BALB/c and C57BL/6 Mice
Authors: Kendall Lon V. , Riley Lela K. , Hook Reuel R. , Besch-Williford Cynthia L. , Franklin Craig L.
Published: July 2002

The cilium-associated respiratory (CAR) bacillus is a gram-negative, gliding bacterium that causes persistent respiratory tract infections in rodents despite histologic and serologic evidence of a marked immune response. To assess humoral immunity and cytokine responses in CAR bacillus disease, 6-week-old female BALB/c and C57BL/6 mice were inoculated intratracheally with 105CAR bacillus organisms. CAR bacillus-specific serum immunoglobulins (immunoglobulin M [IgM], IgG1, IgG2a, IgG2b, IgG3, and IgA) and local pulmonary cytokines (tumor necrosis factor alpha [TNF-α], gamma interferon [IFN-γ], and interleukin-4 [IL-4]) were evaluated by enzyme-linked immunosorbent assay every 7 days for 49 days. BALB/c mice developed CAR bacillus-induced lesions early in the course of disease that became more severe with time. Correlating with increasing disease severity, BALB/c mice had elevations in all antibody isotypes tested, and elevations in pulmonary TNF-α, IFN-γ, and IL-4. C57BL/6 mice developed mild lesions with mild increases in serum IgM, IgG1, IgG2b, and IgG3 levels and minimally detectable IgG2a and IgA. Cytokine perturbations were not detected in C57BL/6 mice. The persistence of infection in BALB/c mice with vigorous serum antibody responses and increased IFN-γ and IL-4 responses suggests that humoral immunity and T-cell responses are ineffective at preventing CAR bacillus disease. Furthermore, the lackluster antibody responses and undetectable cytokine responses in C57BL/6 mice suggest that humoral immunity and T-cell responses are not critical in resistance to CAR bacillus-induced disease.