bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Analysis of Type E Botulinum Toxin-Producing
Clostridium butyricum
Strains
Authors: Wang Xingmin , Maegawa Tsuneo , Karasawa Tadahiro , Kozaki Shunji , Tsukamoto Kentaro , Gyobu Yotaku , Yamakawa Kiyotaka , Oguma Keiji , Sakaguchi Yoshihiko , Nakamura Shinichi
Published: July 2002

Type E botulinum toxin (BoNT/E)-producing
Clostridium butyricum
strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the
bont
/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the
bont
/E gene. Nucleotide sequences of the
bont
/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of the
bont
/E genes of 11
C. butyricum
isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from
C. butyricum
BL 6340 and
Clostridium botulinum
type E, respectively. The BoNT/E-producing
C. butyricum
strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with
Sma
I or
Xho
I, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for the
bont
/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the
bont
/E gene in
C. butyricum
. The present results suggest that BoNT/E-producing
C. butyricum
is clonally distributed over a vast area.
Genetic Variation among Endosymbionts of Widely Distributed Vestimentiferan Tubeworms
Authors: Di Meo Carol A. , Wilbur Ami E. , Holben William E. , Feldman Robert A. , Vrijenhoek Robert C. , Cary S. Craig
Published: July 2002

Vestimentiferan tubeworms thriving in sulfidic deep-sea hydrothermal vents and cold seeps are constrained by their nutritional reliance on chemoautotrophic endosymbionts. In a recent phylogenetic study using 16S ribosomal DNA, we found that endosymbionts from vent and seep habitats form two distinct clades with little variation within each clade. In the present study, we used two different approaches to assess the genetic variation among biogeographically distinct vestimentiferan symbionts. DNA sequences were obtained for the noncoding, internal transcribed spacer (ITS) regions of the rRNA operons of symbionts associated with six different genera of vestimentiferan tubeworms. ITS sequences from endosymbionts of host genera collected from different habitats and widely distributed vent sites were surprisingly conserved. Because the ITS region was not sufficient for distinguishing endosymbionts from different habitats or locations, we used a DNA fingerprinting technique, repetitive-extragenic-palindrome PCR (REP-PCR), to reveal differences in the distribution of repetitive sequences in the genomes of the bacterial endosymbionts. Most of the endosymbionts displayed unique REP-PCR patterns. A cladogram generated from these fingerprints reflected relationships that may be influenced by a variety of factors, including host genera, geographic location, and bottom type.
Simultaneous Detection and Differentiation of
Escherichia coli
Populations from Environmental Freshwaters by Means of Sequence Variations in a Fragment of the β-
d
-Glucuronidase Gene
Authors: Farnleitner Andreas H. , Kreuzinger Norbert , Kavka Gerhard G. , Grillenberger Sonja , Rath Johannes , Mach Robert L.
Published: July 2002

A PCR-based denaturing-gradient gel electrophoresis (DGGE) approach was applied to a partial sequence of the β-
d
-glucuronidase gene (
uidA
) for specific detection and differentiation of
Escherichia coli
populations according to their
uidA
sequence variations. Detection of sequence variations by PCR-DGGE and by PCR with direct sequencing correlated perfectly. Screening of 50
E. coli
freshwater isolates and reference strains revealed 11 sequence types, showing nine polymorphic sites and an average number of pairwise differences between alleles of the
uidA
gene fragments (screened fragment length, 126 bp) of 2.3%. Among the analyzed strains a range of dominating to more rarely and/or uniquely observed
E. coli
sequence types was revealed. PCR-DGGE applied to fecally polluted river water samples simultaneously detected
E. coli
and generated a fingerprint of the mixed populations by separating the polymorphic
uidA
amplicons. No significant differences between non-cultivation-based and cultivation-based profiles were observed, suggesting that at least some members of all occurring sequence types could be cultivated. As
E. coli
is frequently used as a fecal indicator, this work is considered an important step towards a new, practical tool for the differentiation and tracing of fecal pollution in all kinds of waters.
Use of a Single, Triplicate Arbitrarily Primed-PCR Procedure for Molecular Fingerprinting of Lactic Acid Bacteria
Authors: Cusick Sean M. , O'Sullivan Daniel J.
Published: July 2002

Arbitrarily primed (AP)-PCR can be used to generate characteristic DNA fingerprint patterns. However, small changes in reaction conditions can cause band irreproducibility. In this study, a single methodology encompassing triplicate reactions, which were intentionally exposed to three different annealing temperatures, enabled bands that were reproducibly generated to be recognized. A single triplicate AP-PCR (TAP-PCR) procedure, using an 18-mer primer, was developed and used to fingerprint representative isolates from the major genera of lactic acid bacteria and
Bifidobacterium
to the strain level.
Use of Repetitive DNA Sequences and the PCR To Differentiate
Escherichia coli
Isolates from Human and Animal Sources
Authors: Dombek Priscilla E. , Johnson LeeAnn K. , Zimmerley Sara T. , Sadowsky Michael J.
Published: July 2002

The rep-PCR DNA fingerprint technique, which uses repetitive intergenic DNA sequences, was investigated as a way to differentiate between human and animal sources of fecal pollution. BOX and REP primers were used to generate DNA fingerprints from
Escherichia coli
strains isolated from human and animal sources (geese, ducks, cows, pigs, chickens, and sheep). Our initial studies revealed that the DNA fingerprints obtained with the BOX primer were more effective for grouping
E. coli
strains than the DNA fingerprints obtained with REP primers. The BOX primer DNA fingerprints of 154
E. coli
isolates were analyzed by using the Jaccard band-matching algorithm. Jackknife analysis of the resulting similarity coefficients revealed that 100% of the chicken and cow isolates and between 78 and 90% of the human, goose, duck, pig, and sheep isolates were assigned to the correct source groups. A dendrogram constructed by using Jaccard similarity coefficients almost completely separated the human isolates from the nonhuman isolates. Multivariate analysis of variance, a form of discriminant analysis, successfully differentiated the isolates and placed them in the appropriate source groups. Taken together, our results indicate that rep-PCR performed with the BOX A1R primer may be a useful and effective tool for rapidly determining sources of fecal pollution.
Spatial Patterns in Antibiotic Resistance among Stream Bacteria: Effects of Industrial Pollution
Authors: McArthur J Vaun , Tuckfield R. Cary
Published: July 2002

The spatial distribution of antibiotic resistance to streptomycin and kanamycin was examined in natural bacterial communities of two streams. The proportion of resistant bacteria was substantially higher (
P
< 0.05) in the midreaches of an industrially perturbed stream, but no such pattern was apparent in an undisturbed reference stream. The highest relative frequency of resistance was found at the confluence of a tributary draining a nuclear reactor and industrial complex. Antibiotic resistance increased with distance upstream from the confluence and was positively correlated (
r
2
= 0.54,
P
= 0.023) with mercury concentrations in the sediments. When the data for two years were compared, this pattern was stable for streptomycin resistance (paired
t
test,
P
< 0.05) but not for kanamycin resistance (
P
> 0.05). Our results imply that heavy metal pollution may contribute to increased antibiotic resistance through indirect selection.
Exposure of Workers to Airborne Microorganisms in Open-Air Swine Houses
Authors: Chang C. W. , Chung H. , Huang C. F. , Su H. J. J.
Published: July 2002

This study quantified the levels of airborne microorganisms in six swine farms with more than 10,000 pigs in subtropical Taiwan. We evaluated breeding, growing, and finishing stalls, which were primarily open-air buildings, as well as partially enclosed farrowing and nursery piggeries. Airborne culturable bacteria, gram-negative bacteria, and fungi were placed on appropriate media by using an all-glass impinger or single-stage Andersen microbial sampler. Results showed that mean concentrations of culturable bacteria and gram-negative bacteria were 3.3 × 10
5
and 143.7 CFU/m
3
, respectively. The concentration of airborne culturable fungi was about 10
3
CFU/m
3
, with
Cladosporium
the predominant genus. The highest airborne levels of culturable bacteria and gram-negative bacteria were identified in the finishing units. The air of the nursery stalls was the least contaminated with culturable and gram-negative bacteria. Irregular and infrequent cleaning, high pig density, no separation of wastes from pen floors, and accumulation of water as a result of the processes for cleaning and reducing pig temperature possibly compromise the benefits of the open characteristic of the finishing units with respect to airborne bacterial concentration.
Requirement of DNA Repair Mechanisms for Survival of
Burkholderia cepacia
G4 upon Degradation of Trichloroethylene
Authors: Yeager Chris M. , Bottomley Peter J. , Arp Daniel J.
Published: July 2002

A Tn
5
-based mutagenesis strategy was used to generate a collection of trichloroethylene (TCE)-sensitive (TCS) mutants in order to identify repair systems or protective mechanisms that shield
Burkholderia cepacia
G4 from the toxic effects associated with TCE oxidation. Single Tn
5
insertion sites were mapped within open reading frames putatively encoding enzymes involved in DNA repair (UvrB, RuvB, RecA, and RecG) in 7 of the 11 TCS strains obtained (4 of the TCS strains had a single Tn
5
insertion within a
uvrB
homolog). The data revealed that the
uvrB
-disrupted strains were exceptionally susceptible to killing by TCE oxidation, followed by the
recA
strain, while the
ruvB
and
recG
strains were just slightly more sensitive to TCE than the wild type. The
uvrB
and
recA
strains were also extremely sensitive to UV light and, to a lesser extent, to exposure to mitomycin C and H
2
O
2
. The data from this study establishes that there is a link between DNA repair and the ability of
B. cepacia
G4 cells to survive following TCE transformation. A possible role for nucleotide excision repair and recombination repair activities in TCE-damaged cells is discussed.
Regulation of Fumonisin B
1
Biosynthesis and Conidiation in
Fusarium verticillioides
by a Cyclin-Like (C-Type) Gene,
FCC1
Authors: Shim Won-Bo , Woloshuk Charles P.
Published: July 2002

Fumonisins are a group of mycotoxins produced in corn kernels by the plant-pathogenic fungus
Fusarium verticillioides
. A mutant of the fungus, FT536, carrying a disrupted gene named
FCC1
(for
Fusarium
cyclin C1) resulting in altered fumonisin B
1
biosynthesis was generated.
FCC1
contains an open reading frame of 1,018 bp, with one intron, and encodes a putative 319-amino-acid polypeptide. This protein is similar to UME3 (also called SRB11 or SSN8), a cyclin C of
Saccharomyces cerevisiae
, and contains three conserved motifs: a cyclin box, a PEST-rich region, and a destruction box. Also similar to the case for C-type cyclins,
FCC1
was constitutively expressed during growth. When strain FT536 was grown on corn kernels or on defined minimal medium at pH 6, conidiation was reduced and
FUM5
, the polyketide synthase gene involved in fumonisin B
1
biosynthesis, was not expressed. However, when the mutant was grown on a defined minimal medium at pH 3, conidiation was restored, and the blocks in expression of
FUM5
and fumonisin B
1
production were suppressed. Our data suggest that
FCC1
plays an important role in signal transduction regulating secondary metabolism (fumonisin biosynthesis) and fungal development (conidiation) in
F. verticillioides
.
Attached and Unattached Bacterial Communities in a 120-Meter Corehole in an Acidic, Crystalline Rock Aquifer
Authors: Lehman R. Michael , Roberto Francisco F. , Earley Drummond , Bruhn Debby F. , Brink Susan E. , O'Connell Sean P. , Delwiche Mark E. , Colwell Frederick S.
Published: July 2002

The bacteria colonizing geologic core sections (attached) were contrasted with those found suspended in the groundwater (unattached) by examining the microbiology of 16 depth-paired core and groundwater samples using a suite of culture-independent and culture-dependent analyses. One hundred twenty-two meters was continuously cored from a buried chalcopyrite ore hosted in a biotite-quartz-monzonite porphyry at the Mineral Park Mine near Kingman, Ariz. Every fourth 1.5-m core was acquired using microbiologically defensible methods, and these core sections were aseptically processed for characterization of the attached bacteria. Groundwater samples containing unattached bacteria were collected from the uncased corehole at depth intervals corresponding to the individual cores using an inflatable straddle packer sampler. The groundwater was acidic (pH 2.8 to 5.0), with low levels of dissolved oxygen and high concentrations of sulfate and metals, including ferrous iron. Total numbers of attached cells were less than 10
5
cells g of core material
−1
while unattached cells numbered about 10
5
cells ml of groundwater
−1
. Attached and unattached acidophilic heterotrophs were observed throughout the depth profile. In contrast, acidophilic chemolithotrophs were not found attached to the rock but were commonly observed in the groundwater. Attached communities were composed of low numbers (<40 CFU g
−1
) of neutrophilic heterotrophs that exhibited a high degree of morphologic diversity, while unattached communities contained higher numbers (ca. 10
3
CFU ml
−1
) of neutrophilic heterotrophs of limited diversity. Sulfate-reducing bacteria were restricted to the deepest samples of both core and groundwater. 16S ribosomal DNA sequence analysis of attached, acidophilic isolates indicated that organisms closely related to heterotrophic, acidophilic mesophiles such as
Acidiphilium organovorum
and, surprisingly, to the moderately thermophilic
Alicyclobacillus acidocaldarius
were present. The results indicate that viable (but possibly inactive) microorganisms were present in the buried ore and that there was substantial distinction in biomass and physiological capabilities between attached and unattached populations.
Efficacy of Common Laboratory Disinfectants on the Infectivity of
Cryptosporidium parvum
Oocysts in Cell Culture
Authors: Weir Susan C. , Pokorny Nicholas J. , Carreno Ramon A. , Trevors Jack T. , Lee Hung
Published: July 2002

Nine liquid disinfectants were tested for their ability to reduce infectivity of
Cryptosporidium parvum
oocysts in cell culture. A 4-min exposure to 6% hydrogen peroxide and a 13-min exposure to ammonium hydroxide-amended windshield washer fluid reduced infectivity 1,000-fold. Other disinfectants tested (70% ethanol, 37% methanol, 6% sodium hypochlorite, 70% isopropanol, and three commercial disinfectants) did not reduce the infectivity after a 33-min exposure. The results indicate that hydrogen peroxide and windshield washer fluid or ammonium hydroxide disinfectant may be suitable laboratory disinfectants against
C. parvum
oocysts.
Impact of Irradiation and Polycyclic Aromatic Hydrocarbon Spiking on Microbial Populations in Marine Sediment for Future Aging and Biodegradability Studies
Authors: Melcher Rebecca J. , Apitz Sabine E. , Hemmingsen Barbara B.
Published: July 2002

Experiments were carried out to develop methods to generate well-characterized, polycyclic aromatic hydrocarbon (PAH)-spiked, aged but minimally altered sediments for fate, biodegradation, and bioavailability experiments. Changes in indigenous bacterial populations were monitored in mesocosms constructed of relatively clean San Diego Bay sediments, with and without exposure to gamma radiation, and then spiked with five different PAHs and hexadecane. While phenanthrene and chrysene degraders were present in the unspiked sediments and increased during handling, PAH spiking of nonirradiated sediments led to dramatic increases in their numbers. Phenotypic characterization of isolates able to grow on phenanthrene or chrysene placed them in several genera of marine bacteria:
Vibrio
,
Marinobacter
or
Cycloclasticus
,
Pseudoalteromonas
,
Marinomonas
, and
Halomonas
. This is the first time that marine PAH degraders have been identified as the latter two genera, expanding the diversity of marine bacteria with this ability. Even at the highest irradiation dose (10 megarads), heterotrophs and endospore formers reappeared within weeks. However, while bacteria from the unirradiated sediments had the capacity to both grow on and mineralize
14
C-labeled phenanthrene and chrysene, irradiation prevented the reappearance of PAH degraders for up to 4 months, allowing spikes to age onto the sediments, which can be used to model biodegradation in marine sediments.
Production of Sepiapterin in
Escherichia coli
by Coexpression of Cyanobacterial GTP Cyclohydrolase I and Human 6-Pyruvoyltetrahydropterin Synthase
Authors: Woo Hyun Joo , Kang Jee Yun , Choi Yong Kee , Park Young Shik
Published: July 2002

Synechocystis
sp. strain PCC 6803 GTP cyclohydrolase I and human 6-pyruvoyltetrahydropterin synthase were coexpressed in
Escherichia coli
. The
E. coli
transformant produced sepiapterin, which was identified by high-performance liquid chromatography and enzymatically converted to dihydrobiopterin by sepiapterin reductase. Aldose reductase, another indispensable enzyme for sepiapterin production, may be endogenous in
E. coli
.
Evaluation of TruCount Absolute-Count Tubes for Determining CD4 and CD8 Cell Numbers in Human Immunodeficiency Virus-Positive Adults
Authors: Schnizlein-Bick Carol T. , Spritzler John , Wilkening Cynthia L. , Nicholson Janet K. A. , O'Gorman Maurice R. G. ,   Site InvestigatorsThe NIAID DAIDS New Technologies Evaluation Group
Published: July 2002

A single-platform technology that uses an internal bead standard and three-color flow cytometry to determine CD4 and CD8 absolute counts was evaluated for reproducibility and agreement. Values obtained using TruCount absolute-count tubes were compared to those obtained using a two-color predicate methodology. Sixty specimens from human immunodeficiency virus type 1-infected donors were shipped to five laboratories. Each site also analyzed replicates of 14 human immunodeficiency virus type 1-infected local specimens at 6 h and again at 24 h. The interlaboratory variability was significantly less with TruCount (median difference in percent coefficient of variation [%CV] between the two methods was −8% and −3% for CD4 and CD8, respectively) than with the predicate method. Intralaboratory variability was smaller, with a median difference in %CV of −1% for both CD4 and CD8 with 6-h samples and −2% and −3% for CD4 and CD8, respectively, with 24-h samples. Use of TruCount for shipped samples resulted in a median CD4 count change of 7 cells (50th estimated percentile) when all laboratories and CD4 strata were combined. For on-site samples, the median CD4 count change was 10 CD4 cells for 6-h samples and 2 CD4 cells for 24-h samples. Individual site biases occurred in both directions and cancelled each other when the data were combined for all laboratories. Thus, the combined data showed a smaller change in median CD4 count than what may have occurred at an individual site. In summary, the use of TruCount decreased both the inter- and intralaboratory variability in determining absolute CD4 and CD8 counts.
Cytopathic Changes in Rat Microglial Cells Induced by PathogenicAcanthamoeba culbertsoni: Morphology and Cytokine Release
Authors: Shin Ho-Joon , Cho Myung-Soo , Jung Suk-Yul , Kim Hyung-Il , Park Sun , Seo Jang-Hoon , Yoo Jung-Chil , Im Kyung-Il
Published: July 2002

To determine whether pathogenicAcanthamoeba culbertsonitrophozoites and lysate can induce cytopathic changes in primary-culture microglial cells, morphological changes were observed by transmission electron microscopy (TEM). In addition, the secretion of two kinds of cytokines, tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), from microglial cells was observed. Trophozoites of pathogenicA. culbertsonimade contact with microglial cells and produced digipodia. TEM revealed that microglial cells cocultured with amoebic trophozoites underwent a necrotic process, accompanied by lysis of the cell membrane. TEM of microglial cells cocultured with amoebic lysate showed that the membranes of the small cytoplasmic vacuoles as well as the cell membrane were lysed. The amounts of TNF-α secreted from microglial cells cocultured withA. culbertsonitrophozoites or lysate increased at 6 h of incubation. The amounts of IL-1β secreted from microglial cells cocultured withA. culbertsonitrophozoites at 6 h of incubation was similar to those secreted from the control group, but the amounts decreased during cultivation withA. culbertsonilysate. These results suggest that pathogenicA. culbertsoniinduces the cytopathic effects in primary-culture rat microglial cells, with the effects characterized by necrosis of microglial cells and changes in levels of secretion of TNF-α and IL-1β from microglial cells.
Heartwater (
Cowdria ruminantium
Infection) as a Cause of Postrestocking Mortality of Goats in Mozambique
Authors: Bekker Cornelis P. J. , Vink Daan , Lopes Pereira Carlos M. , Wapenaar Wendela , Langa Afonso , Jongejan Frans
Published: July 2002

A serological survey in Mozambique to detect antibodies to
Cowdria ruminantium
, the etiologic agent of heartwater, revealed a seroprevalence of 8.1% (
n
= 332) for goats in the northern province of Tete and of 65.6% (
n
= 326) for goats in the southern provinces. Translocation of 10 serologically negative goats from Tete to farms in the south resulted in two clinical cases of heartwater that were fatal. In addition, four goats seroconverted within the study period of 5 weeks. One goat showed no symptoms. Two goats died of other causes, whereas the remaining goat went missing after 1 week. Experimental needle infections of goats and sheep were conducted to confirm results and to isolate different strains of
C. ruminantium
. These data indicate that translocation of goats from the north to the south of Mozambique bears a high risk of
C. ruminantium
infection, which can cause fatal disease.
Modulation ofMycobacterium bovis-Specific Responses of Bovine Peripheral Blood Mononuclear Cells by 1,25-Dihydroxyvitamin D3
Authors: Waters W. R. , Nonnecke B. J. , Rahner T. E. , Palmer M. V. , Whipple D. L. , Horst R. L.
Published: July 2002

Historically, administration of vitamin D has been considered beneficial in the treatment of tuberculosis. The interaction of this vitamin {i.e., 1,25-dihdroxyvitamin D3[1,25(OH)2D3]} with the antitubercular immune response, however, is not clear. In the present study, in vitro recall responses of peripheral blood mononuclear cells (PBMC) from cattle infected withMycobacterium boviswere used to study the immune-modulatory effects of 1,25(OH)2D3onM. bovis-specific responses in vitro. Addition of 1 or 10 nM 1,25(OH)2D3inhibitedM. bovis-specific proliferative responses of PBMC fromM. bovis-infected cattle, affecting predominately the CD4+cell subset. In addition, 1,25(OH)2D3inhibitedM. bovis-specific gamma interferon (IFN-γ) production yet enhancedM. bovis-specific nitric oxide (NO) production. Lymphocyte apoptosis, measured by flow cytometry using annexin-V staining, was diminished by addition of 1,25(OH)2D3to PBMC cultures. These findings support the current hypothesis that 1,25(OH)2D3enhances mycobacterial killing by increasing NO production, a potent antimicrobial mechanism of activated macrophages, and suggest that 1,25(OH)2D3limits host damage by decreasingM. bovis-induced IFN-γ production.
Depletion of Human NK and CD8 Cells prior to In Vitro H1N1 Flu Vaccine Stimulation Increases the Number of Gamma Interferon-Secreting Cells Compared to the Initial Undepleted Population in an ELISPOT Assay
Authors: Dercamp Christophe , Sanchez Violette , Barrier Julie , Trannoy Emanuelle , Guy Bruno
Published: July 2002

In order to study the respective roles of CD4, CD8, and CD56 (NK) cells in gamma interferon (IFN-γ) production after in vitro stimulation with flu vaccine in a healthy adult human population, we depleted these cellular subtypes before stimulation with antigen (inactivated split vaccine, A/Texas H1N1, or A/Sydney H3N2). We observed that while CD4 cells were required for IFN-γ secretion in both conditions in vitro, CD56 (NK) cells and, to a lesser extent, CD8 cells had a negative effect on such synthesis upon H1N1 stimulation, as judged by an increased number of spots compared to the initial undepleted population. This regulation of IFN-γ secretion was associated with an increase in ICAM-1 expression, in particular on T and B cells. This study points out the importance of evaluating in vitro immune responses on a whole-cell population in addition to isolated subtypes if one needs to address potential cellular interactions occurring in vivo in some situations (H1N1 stimulation in the present case). Such cross-regulations occur even in vitro during the antigenic stimulation step.
Differential Modulation of Surface and Intracellular Protein Expression by T Cells after Stimulation in the Presence of Monensin or Brefeldin A
Authors: O'Neil-Andersen Nancy J. , Lawrence David A.
Published: July 2002

Intracellular cytokine staining is an increasingly popular analytical tool that can be used to define the profile of cytokines in various disease states. One important requirement for this assay is the inclusion of a protein transport inhibitor in stimulated cell cultures to trap the cytokine, thus allowing a brighter signal. Two compounds commonly used for this purpose are brefeldin A (BFA) and monensin (MN). Flow cytometry was used to assess the differential effects of BFA and MN on surface CD3, -4, -8, and -69 expression and the intracellular expression of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) following stimulation with phorbol myristate acetate and ionomycin. We found that BFA blocked the majority of CD3+cells from expressing surface CD69, but BFA did not inhibit intracellular CD69 expression. MN did not significantly inhibit surface CD69 expression. With regard to lymphocyte marker expression following activation, surface CD4 expression was significantly downregulated; however, less downregulation was observed with BFA treatment than with MN treatment. Analyzing intracellular cytokine expression, BFA trapped a greater percentage of TNF-α inside activated cells than MN. An analysis of the cytokine concentration in culture supernatants indicated that cells treated with MN released TNF-α and IFN-γ from the cells, while the BFA-treated cells released IFN-γ only. With prolonged (18-h) stimulation, the cells treated with MN were less viable than those treated with BFA. We conclude that the choice of a protein transport inhibitor is an important variable in this assay. When developing this method as a tool for clinical immunology laboratory analysis, investigators should consider the differential effects of BFA and MN on results.
Heterologous Expression, Purification, and Immunological Reactivity of a Recombinant HSP60 from
Paracoccidioides brasiliensis
Authors: Cunha Daniela A. , Zancopé-Oliveira Roseli M. , Sueli M. , Felipe S. , Salem-Izacc Silvia M. , Deepe George S. , Soares Célia M. A.
Published: July 2002

The complete coding cDNA of HSP60 from
Paracoccidioides brasiliensis
was overexpressed in an
Escherichia coli
host to produce high levels of recombinant protein. The protein was purified by affinity chromatography. A total of 169 human serum samples were tested for reactivity by Western blot analysis with the purified HSP60 recombinant protein. Immunoblots indicated that the recombinant
P. brasiliensis
HSP60 was recognized by antibodies in 72 of 75 sera from paracoccidioidomycosis patients. No cross-reactivity was detected with individual sera from patients with aspergillosis, sporotrichosis, cryptococcosis, and tuberculosis. Reactivity to HSP60 was observed in sera from 9.52% of control healthy individuals and 11.5% of patients with histoplasmosis. The high sensitivity and specificity (97.3 and 92.5%, respectively) for HSP60 suggested that the recombinant protein can be used singly or in association with other recombinant antigens to detect antibody responses in
P. brasiliensis
-infected patients.
YKL-40 Is Elevated in Cerebrospinal Fluid from Patients with Purulent Meningitis
Authors: Østergaard Christian , Johansen Julia S. , Benfield Thomas , Price Paul A. , Lundgren Jens D.
Published: July 2002

YKL-40, a member of the family 18 glycosyl hydrolases, is secreted by activated neutrophils and macrophages. It is a growth factor for connective tissue cells and a potent migration factor for endothelial cells and may function in inflammation and tissue remodeling. YKL-40 was determined in 134 cerebrospinal fluid (CSF) samples taken on admission from patients suspected of having meningitis (48 with purulent meningitis, 49 with lymphocytic meningitis, 5 with encephalitis, and 32 without evidence of meningitis). YKL-40 levels in CSF were significantly higher in patients with purulent meningitis (median, 663 μg/liter [range, 20 to 8,960]) and encephalitis (5,430 μg/liter [620 to 11,600]) than in patients with lymphocytic meningitis (137 μg/liter [41 to 1,865]) or patients without meningitis (167 μg/liter [24 to 630]) (Kruskal-Wallis and Dunn multiple comparison tests,P< 0.001). CSF YKL-40 levels were also determined for 26 patients with purulent meningitis having a repuncture, and patients who died (n= 5) had significantly higher YKL-40 levels than patients who survived (n= 21) (2,100 μg/liter [1,160 to 7,050] versus 885 μg/liter [192 to 15,400], respectively; Mann-Whitney test,P= 0.018). YKL-40 was most likely locally produced, since patients with infections of the central nervous system had CSF YKL-40 levels that were at least 10-fold higher than the corresponding levels in serum (2,033 μg/liter [470 to 11,600] versus 80 μg/liter [19 to 195]). The CSF neopterin level was the biochemical parameter in CSF and blood that correlated best with CSF YKL-40 levels, indicating that YKL-40 may be produced by activated macrophages within the central nervous system. In conclusion, high levels of YKL-40 in CSF are found in patients with purulent meningitis.
Cloning and Expression of aHelicobacter bilisImmunoreactive Protein
Authors: Feng Sunlian , Hodzic Emir , Kendall Lon V. , Smith Amy , Freet Kimberly , Barthold Stephen W.
Published: July 2002

In an effort to identify immunoreactiveHelicobacter bilisantigens with potential for serodiagnosis, sera from mice experimentally infected withH. biliswere used to screen anH. bilisgenomic DNA expression library. Among 17 immunoreactive clones, several contained sequences that encoded a predicted 167-kDa protein (P167). Five overlapping P167 peptides (P167A to P167E) of approximately 40 kDa each were generated and tested. Immune sera reacted with fragments P167C and P167D at dilutions of 1:1,600 and 1:6,400, respectively, and reacted with anH. bilismembrane extract at a dilution of 1:800 in an enzyme-linked immunosorbent assay. Sera from mice experimentally infected withH. hepaticusdid not react with P167C and P167D. Sera from mice naturally infected withH. bilisbut not sera from mice naturally infected withH. hepaticusreacted with P167C and P167D. Hyperimmune sera against P167C peptide reacted with recombinant P167C and with a 120-kDa band inH. bilislysates but did not react with a protein of the same size on immunoblots prepared fromH. hepaticus,H. muridarum, or unrelatedBorrelia burgdorferiandCampylobacter jejuniwhole-cell lysates. Nevertheless, the P167A, P167B, P167C, and P167D primers, but not the P167E primers, amplified DNA fromH. hepaticus, and all five primer sets amplified DNA fromH. muridarum. These results suggest that P167 is an immunodominant,H. bilis-specific antigen that may have potential for use in serodiagnosis.
Angiogenic Effects of Extracellular Human Immunodeficiency Virus Type 1 Tat Protein and Its Role in the Pathogenesis of AIDS-Associated Kaposi's Sarcoma
Authors: Barillari Giovanni , Ensoli Barbara
Published: July 2002

The Tat protein of human immunodeficiency virus (HIV) type 1 is a transactivator of viral gene expression that is required for virus replication and spread. Moreover, Tat is released by acutely HIV-infected cells via a leaderless secretory pathway and in a biologically active form that exerts effects on both HIV-infected and uninfected cells from different organs and systems. This review focuses on the activities of extracellular Tat protein on endothelial cells, on angiogenesis, and on the pathogenesis of AIDS-associated angioproliferative diseases such as Kaposi's sarcoma. In particular, we discuss results from different groups indicating that Tat mimics the proangiogenic activities of extracellular matrix molecules and that it enhances the effects of angiogenic factors.
Characterization of the Cell Adhesion Site ofTrypanosoma cruziMetacyclic Stage Surface Glycoprotein gp82
Authors: Manque Patricio M. , Eichinger Daniel , Juliano Maria A. , Juliano Luiz , Araya Jorge E. , Yoshida Nobuko
Published: July 2002

The surface glycoprotein gp82, expressed in the insect-stage metacyclic trypomastigotes ofTrypanosoma cruzi, has been implicated in mammalian cell invasion. Here we have characterized the cell adhesion site of gp82 by using recombinant proteins and synthetic peptides based on gp82. The recombinant protein Del-4/8, lacking 65 amino acids of gp82 central domain (at positions 257 to 321), was virtually devoid of cell-binding activity and lacked the ability to inhibit parasite invasion, in contrast to J18, the construct containing the full-length gp82 sequence (amino acids 1 to 516). Constructs with shorter deletions, i.e., Del-4 (deleted from 257 to 271) and Del-8 (deleted from 293 to 321), bound to target cells to a significantly lesser degree than did J18. The sites deleted in recombinant proteins Del-4 and Del-8 contained acidic amino acids critical for cell adhesion. Thus, the cell-binding capacity of protein Del-E/D, lacking the glutamic acid (259/260) and aspartic acid (303/304) pairs, was negligible, as was its capacity to inhibit parasite internalization. Of a set of synthetic peptides spanning the gp82 central domain, a 22-mer hybrid peptide, p4/8, formed by two noncontiguous sequences (at positions 257 to 273 and 302 to 306) and containing the four acidic residues, competed with the binding of J18 protein to target cells and significantly inhibited (∼60%) the penetration of parasites. This peptide, generated by the juxtaposition of sequences that are separated by a hydrophobic stretch in the linear molecule, appears to be mimicking a conformation-dependent cell-binding site of gp82. Experiments of antibody competition with a set of 20-mer overlapping peptides mapped the epitope for 3F6, a monoclonal antibody directed to gp82 that inhibits parasite invasion, to the sequence represented by peptide p3 (244 to 263), which has a partial overlap with the cell adhesion site.
Production of Basic Fibroblast Growth Factor and Interleukin 6 by Human Smooth Muscle Cells following Infection with
Chlamydia pneumoniae
Authors: Rödel Jürgen , Woytas Marcus , Groh Annemarie , Schmidt Karl-Hermann , Hartmann Matthias , Lehmann Marc , Straube Eberhard
Published: July 2002

Chlamydia pneumoniae
infection has been associated with asthma and atherosclerosis. Smooth muscle cells represent host cells for chlamydiae during chronic infection. In this study we demonstrated that
C. pneumoniae
infection of human smooth muscle cells in vitro increased production of interleukin 6 (IL-6) and basic fibroblast growth factor (bFGF) as shown by reverse transcription-PCR, immunoblotting, and enzyme-linked immunosorbent assay. In contrast, levels of platelet-derived growth factor A-chain mRNA were not affected after infection. The stimulation of bFGF and IL-6 production was most effective when viable chlamydiae were used as inoculum. Furthermore, inhibition of bacterial protein synthesis with chloramphenicol prevented up-regulation of IL-6 and bFGF in infected cells. Addition of IL-6 antibody to infected cultures diminished bFGF expression, indicating involvement of produced IL-6. These findings suggest that chlamydial infection of smooth muscle cells elicits a cytokine response that may contribute to structural remodeling of the airway wall in chronic asthma and to fibrous plaque formation in atherosclerosis.
Delayed Mortality and Attenuated Thrombocytopenia Associated with Severe Malaria in Urokinase- and Urokinase Receptor-Deficient Mice
Authors: Piguet Pierre Francois , Da Laperrousaz Chen , Vesin Christian , Tacchini-Cottier Fabienne , Senaldi Giorgio , Grau Georges Emile
Published: July 2002

We explored the role of urokinase and tissue-type plasminogen activators (uPA and tPA), as well as the uPA receptor (uPAR; CD87) in mouse severe malaria (SM), using genetically deficient (−/−) mice. The mortality resulting fromPlasmodium bergheiANKA infection was delayed in uPA−/−and uPAR−/−mice but was similar to that of the wild type (+/+) in tPA−/−mice. Parasitemia levels were similar in uPA−/−, uPAR−/−, and +/+ mice. Production of tumor necrosis factor, as judged from the plasma level and the mRNA levels in brain and lung, was markedly increased by infection in both +/+ and uPAR−/−mice. Breakdown of the blood-brain barrier, as evidenced by the leakage of Evans Blue, was similar in +/+ and uPAR−/−mice. SM was associated with a profound thrombocytopenia, which was attenuated in uPA−/−and uPAR−/−mice. Administration of aprotinin, a plasmin antagonist, also delayed mortality and attenuated thrombocytopenia. Platelet trapping in cerebral venules or alveolar capillaries was evident in +/+ mice but absent in uPAR−/−mice. In contrast, macrophage sequestration in cerebral venules or alveolar capillaries was evident in both +/+ and uPAR−/−mice. Polymorphonuclear leukocyte sequestration in alveolar capillaries was similar in +/+ and uPAR−/−mice. These results demonstrate that the uPAR deficiency attenuates the severity of SM, probably by its important role in platelet kinetics and trapping. These results therefore suggest that platelet sequestration contributes to the pathogenesis of SM.
Anti-HSP70 Autoantibody Formation by B-1 Cells inToxoplasma gondii-Infected Mice
Authors: Chen Mei , Aosai Fumie , Mun Hye-Seong , Norose Kazumi , Hata Hidekazu , Yano Akihiko
Published: July 2002

Formation of anti-Toxoplasma gondiiHSP70 (TgHSP70) antibody cross-reactive to mouse HSP70 (mHSP70) was observed in the sera of BALB/c (a resistant strain) and C57BL/6 (B6; a susceptible strain) mice after peroral infection withT. gondiicysts of the Fukaya strain. The levels of anti-mHSP70 immunoglobulin G (IgG) autoantibody production in B6 mice were higher than those in BALB/c mice. The isotype and subclass of IgG of anti-TgHSP70 monoclonal antibodies cross-reactive to mHSP70 were μ and γ3. Anti-mHSP70 autoantibody inT. gondii-infected BALB/c and B6 mice was shown to be produced by the CD5+subset of B cells (B-1a cells) but not by conventional B cells (B-2 cells). The epitopes recognized by anti-mHSP70 autoantibody were located primarily in the C-terminal fragment of mHSP70.
Helicobacter hepaticusDoes Not Induce or Potentiate Colitis in Interleukin-10-Deficient Mice
Authors: Dieleman Levinus A. , Arends Annemarie , Tonkonogy Susan L. , Goerres Marije S. , Craft David W. , Grenther Wetonia , Sellon Rance K. , Balish Ed , Sartor R. Balfour
Published: July 2002

Helicobacter hepaticushas been reported to induce colitis, hepatitis, and hepatocellular carcinoma in several different murine models. The aim of this study was to determine ifH. hepaticuswill cause colitis in monoassociated mice lacking the interleukin-10 gene (IL-10−/−mice) and potentiate colitis in specific-pathogen-free (SPF) IL-10−/−mice. Germfree IL-10−/−mice on either a mixed (C57BL/6 × 129/Ola) or inbred (129/SvEv) genetic background were monoassociated withH. hepaticusATCC 51448 by oral feeding and rectal enemas. In a second experiment, germfree IL-10−/−mice were colonized with stool from SPF mice that harbored or did not harbor endogenousH. hepaticus. After 7 to 9 weeks of colonization, weight loss and mortality were assessed, the colon was isolated for histology and IL-12 secretion, and mesenteric lymph node cells were assessed for T-cell activation markers. It was found that IL-10−/−mice monoassociated withH. hepaticusfor up to 16 weeks showed almost no histologic colitis or increased IL-12 production. SPF IL-10-knockout mice had no significant difference in weight loss, mortality rate, histologic scores, colonic IL-12 secretion, or T-cell activation with or withoutH. hepaticus. We conclude thatH. hepaticusdoes not induce or potentiate disease in our IL-10−/−mice and therefore is not required to induce colitis in genetically susceptible hosts.
Mycobacterium tuberculosisandLegionella pneumophilaPhagosomes Exhibit Arrested Maturation despite Acquisition of Rab7
Authors: Clemens Daniel L. , Lee Bai-Yu , Horwitz Marcus A.
Published: July 2002

Rab7 is a small GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway. Phagosomes containing inert particles have also been shown to transiently acquire Rab7 as they mature. Disruption in the pathway prior to the acquisition of Rab7 has been suggested as playing a role in the altered maturation ofMycobacterium bovisBCG phagosomes. As a first step to determine whether disruption in the delivery or function of Rab7 could play a role in the altered maturation ofLegionella pneumophilaandM. tuberculosisphagosomes, we have examined the distribution of wild-type Rab7 and the GTPase-deficient, constitutively active mutant form of Rab7 in HeLa cells infected withL. pneumophilaorM. tuberculosis. We have found that the majority ofL. pneumophilaandM. tuberculosisphagosomes acquire relatively abundant staining for Rab7 and for the constitutively active mutant Rab7 in HeLa cells that overexpress these proteins. Nevertheless, despite acquisition of wild-type or constitutively active Rab7, both theL. pneumophilaand theM. tuberculosisphagosomes continue to exhibit altered maturation as manifested by a failure to acquire lysosome-associated membrane glycoprotein 1. These results demonstrate thatL. pneumophilaandM. tuberculosisphagosomes have receptors for Rab7 and that the altered maturation of these phagosomes is not due to a failure to acquire Rab7.
Immunopathology of Cerebral Malaria: Morphological Evidence of Parasite Sequestration in Murine Brain Microvasculature
Authors: Hearn Jocelyn , Rayment Neil , Landon David N. , Katz David R. , de Souza J. Brian
Published: July 2002

A murine model that closely resembles human cerebral malaria is presented, in which characteristic features of parasite sequestration and inflammation in the brain are clearly demonstrable. “Young” (BALB/c × C57BL/6)F1mice infected withPlasmodium berghei(ANKA) developed typical neurological symptoms 7 to 8 days later and then died, although their parasitemias were below 20%. Older animals were less susceptible. Immunohistopathology and ultrastructure demonstrated that neurological symptoms were associated with sequestration of both parasitized erythrocytes and leukocytes and with clogging and rupture of vessels in both cerebral and cerebellar regions. Increases in tumor necrosis factor alpha and CD54 expression were also present. Similar phenomena were absent or substantially reduced in older infected but asymptomatic animals. These findings suggest that this murine model is suitable both for determining precise pathogenetic features of the cerebral form of the disease and for evaluating circumventive interventions.
Serotype AD Strains of
Cryptococcus neoformans
Are Diploid or Aneuploid and Are Heterozygous at the Mating-Type Locus
Authors: Lengeler Klaus B. , Cox Gary M. , Heitman Joseph
Published: July 2002

Cryptococcus neoformans
is a pathogenic basidiomycete with a defined sexual cycle involving mating between haploid yeast cells with a transient diploid state.
C. neoformans
occurs in four predominant serotypes (A, B, C, and D), which represent different varieties or species. Rare clinical and environmental isolates with an unusual AD serotype have been reported and suggested to be diploid. We found by fluorescence-activated cell sorter analysis that serotype AD strains are aneuploid or diploid. PCR analysis with primers specific for serotype A or D alleles of the
CNA1
,
CLA4
, and
GPA1
genes revealed that both alleles are often present in serotype AD strains. PCR analysis with primers specific for genes in the
MAT
a or
MAT
α mating-type loci revealed that serotype AD strains are heterozygous for the mating-type locus. Interestingly, in several serotype AD strains, the
MAT
α locus was derived from the serotype D parent and the
MAT
a locus was inherited from a serotype A parent that has been thought to be extinct. Basidiospores from a self-fertile serotype AD strain bearing the putative serotype A
MAT
a locus showed a very low viability (∼5%), and no fertile serotype A
MAT
a strain could be recovered. Serotype AD strains were virulent in a murine model. Hybrid AD strains could readily be isolated following a laboratory cross between a serotype A strain and a serotype D strain. In summary, serotype AD strains of
C. neoformans
are unusual aneuploid or diploid strains that result from matings between serotype A and D strains. Self-fertile isolates fail to undergo normal meiosis because of genetic divergence. Our findings further suggest that serotype A
MAT
a strains may exist in nature.
Natural Antibody Affects Survival of the SpirocheteBorrelia burgdorferiwithin Feeding Ticks
Authors: Belperron Alexia A. , Bockenstedt Linda K.
Published: July 2002

Natural antibodies are those immunoglobulin molecules found in mammalian serum that arise in the absence of exposure to environmental pathogens and may comprise an early host defense against invading pathogens. The spirocheteBorrelia burgdorferifirst encounters natural antibodies when its arthropod vector,Ixodes scapularis, begins feeding on a mammalian host. Natural antibodies may therefore have an impact on pathogens within blood-sucking vectors, prior to pathogen transmission to the mammal. In this study, we investigated whether natural antibodies influenced the number and/or phenotype ofB. burgdorferiorganisms within feedingI. scapularisnymphs. Using a competitive PCR, we found that ticks ingesting a blood meal from B-cell-deficient mice, which lack all immunoglobulins, contained fivefold more spirochete DNA than ticks feeding on control mice. Spirochete DNA levels could be reduced to that of controls with passive transfer of normal mouse serum or polyclonal immunoglobulin M (IgM), but not IgG, into B-cell-deficient mice prior to placement of infected ticks. At 48 h of tick feeding, 90% of spirochetes within salivary glands of ticks removed from B-cell-deficient mice were found by confocal immunofluorescence microscopy to express outer surface protein A (OspA), compared to only 5% of salivary gland spirochetes from ticks detached from control mice. Taken together, these results show that ingestion of natural antibodies limits the spirochete burden within feeding ticks. Because OspA is normally downregulated when spirochetes moved from the tick midgut to the salivary gland, our findings suggest that OspA-expressing midgut spirochetes may be particularly susceptible to the borrelicidal effects of these molecules.