bluuebunny/crossref_metadata_2025 huggingface dataset:
Combined Use of 16S Ribosomal DNA and 16S rRNA To Study the Bacterial Community of Polychlorinated Biphenyl-Polluted Soil
Authors: Nogales Balbina , Moore Edward R. B. , Llobet-Brossa Enrique , Rossello-Mora Ramon , Amann Rudolf , Timmis Kenneth N.
Published: July 2002

The bacterial diversity assessed from clone libraries prepared from rRNA (two libraries) and ribosomal DNA (rDNA) (one library) from polychlorinated biphenyl (PCB)-polluted soil has been analyzed. A good correspondence of the community composition found in the two types of library was observed. Nearly 29% of the cloned sequences in the rDNA library were identical to sequences in the rRNA libraries. More than 60% of the total cloned sequence types analyzed were grouped in phylogenetic groups (a clone group with sequence similarity higher than 97% [98% for
Burkholderia
and
Pseudomonas
-type clones]) represented in both types of libraries. Some of those phylogenetic groups, mostly represented by a single (or pair) of cloned sequence type(s), were observed in only one of the types of library. An important difference between the libraries was the lack of clones representative of the
Actinobacteria
in the rDNA library. The PCB-polluted soil exhibited a high bacterial diversity which included representatives of two novel lineages. The apparent abundance of bacteria affiliated to the beta-subclass of the
Proteobacteria
, and to the genus
Burkholderia
in particular, was confirmed by fluorescence in situ hybridization analysis. The possible influence on apparent diversity of low template concentrations was assessed by dilution of the RNA template prior to amplification by reverse transcription-PCR. Although differences in the composition of the two rRNA libraries obtained from high and low RNA concentrations were observed, the main components of the bacterial community were represented in both libraries, and therefore their detection was not compromised by the lower concentrations of template used in this study.
Enhanced Detection of Surface-Associated Bacteria in Indoor Environments by Quantitative PCR
Authors: Buttner Mark P. , Cruz-Perez Patricia , Stetzenbach Linda D.
Published: July 2002

Methods for detecting microorganisms on surfaces are needed to locate biocontamination sources and to relate surface and airborne concentrations. Research was conducted in an experimental room to evaluate surface sampling methods and quantitative PCR (QPCR) for enhanced detection of a target biocontaminant present on flooring materials. QPCR and culture analyses were used to quantitate
Bacillus subtilis
(
Bacillus globigii
) endospores on vinyl tile, commercial carpet, and new and soiled residential carpet with samples obtained by four surface sampling methods: a swab kit, a sponge swipe, a cotton swab, and a bulk method. The initial data showed that greater overall sensitivity was obtained with the QPCR than with culture analysis; however, the QPCR results for bulk samples from residential carpet were negative. The swab kit and the sponge swipe methods were then tested with two levels of background biological contamination consisting of
Penicillium chrysogenum
spores. The
B. subtilis
values obtained by the QPCR method were greater than those obtained by culture analysis. The differences between the QPCR and culture data were significant for the samples obtained with the swab kit for all flooring materials except soiled residential carpet and with the sponge swipe for commercial carpet. The QPCR data showed that there were no significant differences between the swab kit and sponge swipe sampling methods for any of the flooring materials. Inhibition of QPCR due solely to biological contamination of flooring materials was not evident. However, some degree of inhibition was observed with the soiled residential carpet, which may have been caused by the presence of abiotic contaminants, alone or in combination with biological contaminants. The results of this research demonstrate the ability of QPCR to enhance detection and enumeration of biocontaminants on surface materials and provide information concerning the comparability of currently available surface sampling methods.
Reductive Precipitation of Gold by Dissimilatory Fe(III)-Reducing
Bacteria
and
Archaea
Authors: Kashefi Kazem , Tor Jason M. , Nevin Kelly P. , Lovley Derek R.
Published: July 2002

Studies with a diversity of hyperthermophilic and mesophilic dissimilatory Fe(III)-reducing
Bacteria
and
Archaea
demonstrated that some of these organisms are capable of precipitating gold by reducing Au(III) to Au(0) with hydrogen as the electron donor. These studies suggest that models for the formation of gold deposits in both hydrothermal and cooler environments should consider the possibility that dissimilatory metal-reducing microorganisms can reductively precipitate gold from solution.
Sequence Analysis and Molecular Characterization of the
Lactococcus lactis
Temperate Bacteriophage BK5-T
Authors: Mahanivong Chitladda , Boyce John D. , Davidson Barrie E. , Hillier Alan J.
Published: July 2002

The
Lactococcus lactis
temperate bacteriophage BK5-T is one of twelve type phages that define
L. lactis
phage species. This paper describes the nucleotide sequence and analysis of a 21-kbp region of the BK5-T genome and completes the nucleotide sequence of the genome of this phage. The 40,003-nucleotide linear genome encodes 63 open reading frames. Sequence runoff experiments showed that the cohesive ends of the BK5-T genome contained a 12-bp 3′ single-stranded overhang with the sequence 5′-CACACACATAGG-3′. Two major BK5-T structural proteins, of approximately 30 and 20 kDa, were identified, and N-terminal sequence analysis determined that they were encoded by
orf7
and
orf12
, respectively. A 169-bp fragment containing a 37-bp direct repeat and several smaller repeat sequences conferred resistance to BK5-T infection when introduced in
trans
to the host cell and is likely a part of the BK5-T origin of replication (
ori
).
Elucidation of the Flavonoid Catabolism Pathway in
Pseudomonas putida
PML2 by Comparative Metabolic Profiling
Authors: Pillai Bhinu V. S. , Swarup Sanjay
Published: July 2002

Flavonoids are 15-carbon plant secondary metabolites exuded in the rhizosphere that hosts several flavonoid-degrading bacteria. We studied flavonoid catabolism in a plant growth-promoting rhizobacterial strain of
Pseudomonas
by using a combination of biochemical and genetic approaches. Transposants carrying mini-Tn
5gfp
insertions were screened for flavonoid auxotrophy, and these mutant strains were found to be unable to grow in the flavonols naringenin and quercetin, while their growth in glycerol was comparable to that of the parental strain. In order to understand flavonoid catabolism, culture supernatants, whole-cell fractions, cell lysate, and cell debris of the wild-type and mutant strains were analyzed. Intermediates that accumulated intracellularly and those secreted in the medium were identified by a combination of reversed-phase high-pressure liquid chromatography and electrospray ionization-mass spectrometry. Structures of four key intermediates were confirmed by one-dimensional nuclear magnetic resonance spectroscopy. Comparative metabolic profiling of the compounds in the wild-type and mutant strains allowed us to understand the degradation events and to identify six metabolic intermediates. The first step in the pathway involves 3,3′-didehydroxylation, followed by hydrolysis and cleavage of the C-ring, leading via subsequent oxidations to the formation of protocatechuate. This is the first report on quercetin dehydroxylation in aerobic conditions leading to naringenin accumulation.
Flux through Citrate Synthase Limits the Growth of EthanologenicEscherichia coliKO11 during Xylose Fermentation
Authors: Underwood S. A. , Buszko M. L. , Shanmugam K. T. , Ingram L. O.
Published: July 2002

Previous studies have shown that high levels of complex nutrients (Luria broth or 5% corn steep liquor) were necessary for rapid ethanol production by the ethanologenic strainEscherichia coliKO11. Although this strain is prototrophic, cell density and ethanol production remained low in mineral salts media (10% xylose) unless complex nutrients were added. The basis for this nutrient requirement was identified as a regulatory problem created by metabolic engineering of an ethanol pathway. Cells must partition pyruvate between competing needs for biosynthesis and regeneration of NAD+. Expression of low-KmZymomonas mobilis pdc(pyruvate decarboxylase) in KO11 reduced the flow of pyruvate carbon into native fermentation pathways as desired, but it also restricted the flow of carbon skeletons into the 2-ketoglutarate arm of the tricarboxylic acid pathway (biosynthesis). In mineral salts medium containing 1% corn steep liquor and 10% xylose, the detrimental effect of metabolic engineering was substantially reduced by addition of pyruvate. A similar benefit was also observed when acetaldehyde, 2-ketoglutarate, or glutamate was added. InE. coli, citrate synthase links the cellular abundance of NADH to the supply of 2-ketoglutarate for glutamate biosynthesis. This enzyme is allosterically regulated and inhibited by high NADH concentrations. In addition, citrate synthase catalyzes the first committed step in 2-ketoglutarate synthesis. Oxidation of NADH by added acetaldehyde (or pyruvate) would be expected to increase the activity ofE. colicitrate synthase and direct more carbon into 2-ketoglutarate, and this may explain the stimulation of growth. This hypothesis was tested, in part, by cloning theBacillus subtilis citZgene encoding an NADH-insensitive citrate synthase. Expression of recombinantcitZin KO11 was accompanied by increases in cell growth and ethanol production, which substantially reduced the need for complex nutrients.
Cloning of
Escherichia coli lacZ
and
lacY
Genes and Their Expression in
Gluconobacter oxydans
and
Acetobacter liquefaciens
Authors: Mostafa Hesham E. , Heller Knut J. , Geis Arnold
Published: July 2002

An efficient transformation protocol for
Gluconobacter oxydans
and
Acetobacter liquefaciens
strains was developed by preparation of electrocompetent cells grown on yeast extract-ethanol medium. Plasmid pBBR122 was used as broad-host-range vector to clone the
Escherichia coli lacZY
genes in
G. oxydans
and
A. liquefaciens.
Although both
lac
genes were functionally expressed in both acetic acid bacteria
,
only a few transformants were able to grow on lactose. However, this ability strictly depended on the presence of a plasmid expressing both
lac
genes. Mutations in the plasmids and/or in the chromosome were excluded as the cause of growth ability on lactose.
Identification of OpuC as a Chill-Activated and Osmotically Activated Carnitine Transporter in
Listeria monocytogenes
Authors: Angelidis Apostolos S. , Smith Linda Tombras , Hoffman Les M. , Smith Gary M.
Published: July 2002

The food-borne pathogen
Listeria monocytogenes
is notable for its ability to grow under osmotic stress and at low temperatures. It is known to accumulate the compatible solutes glycine betaine and carnitine from the medium in response to osmotic or chill stress, and this accumulation confers tolerance to these stresses. Two permeases that transport glycine betaine have been identified, both of which are activated by hyperosmotic stress and one of which is activated by low temperature. An osmotically activated transporter for carnitine, OpuC, has also been identified. We have isolated a Tn
917
-LTV3 insertional mutant that could not be rescued from hyperosmotic stress by exogenous carnitine. The mutant, LTS4a, grew indistinguishably from a control strain (DP-L1044) in the absence of stress or in the absence of carnitine, but DP-L1044 grew substantially faster under osmotic or chill stress in the presence of carnitine. LTS4a was found to be strongly impaired in KCl-activated as well as chill-activated carnitine transport.
13
C nuclear magnetic resonance spectroscopy of perchloric acid extracts showed that accumulation of carnitine by LTS4a was negligible under all conditions tested. Direct sequencing of LTS4a genomic DNA with a primer based on Tn
917
-LTV3 yielded a 487-bp sequence, which allowed us to determine that the
opuC
operon had been interrupted by the transposon. It can be concluded that
opuC
encodes a carnitine transporter that can be activated by either hyperosmotic stress or chill and that the transport system plays a significant role in the tolerance of
L. monocytogenes
to both forms of environmental stress.
CD4+Lymphocyte-Mediated Suppression of Cytomegalovirus Expression in Human Astrocytes
Authors: Cheeran Maxim C.-J. , Gekker Genya , Hu Shuxian , Yager Stephanie L. , Peterson Phillip K. , Lokensgard James R.
Published: July 2002

Cytomegalovirus-stimulated CD4+lymphocytes from seropositive but not seronegative donors suppressed viral gene expression in primary human astrocytes. This suppressive activity was mediated through soluble factors. These findings suggest that CD4+lymphocytes play a role in defense of the brain against cytomegalovirus.
Immunization of BALB/c Mice with KilledNeospora caninumTachyzoite Antigen Induces a Type 2 Immune Response and Exacerbates Encephalitis and Neurological Disease
Authors: Baszler Timothy V. , McElwain Terry F. , Mathison Bruce A.
Published: July 2002

BALB/c mice were immunized subcutaneously with solubleNeospora caninumtachyzoite antigen (NSO) entrapped in nonionic surfactant vesicles (NISVs) or administered with Freund's complete adjuvant (FCA). Following virulent parasite challenge, groups of mice immunized with NSO and either NISVs or FCA had clinical neurological disease and increased numbers of brain lesions compared to groups of mice inoculated with FCA, NISVs, or phosphate-buffered saline (PBS) alone. Increased numbers of brain lesions were statistically significant only between mice immunized with NISV-NSO and NISV- or PBS-treated mice. Following parasite challenge, brain inflammatory infiltrates in all experimental and control groups of mice were relatively similar and consisted of compact infiltrates of macrophages admixed with various numbers of lymphoid cells. Increased brain lesions in NSO-immunized mice were associated with increased antigen-specific interleukin 4 (IL-4) secretion and increased IL-4:gamma interferon secretion ratios from splenocytes in vitro and increased antigen-specific immunoglobulin G1 (IgG1):IgG2a ratios in vivo. Thus, immunization with whole killedN. caninumantigen and either liposoidal or Freund's adjuvant induced a type 2 immune response that was associated with worsened disease. The present studies emphasize the need to identify specificN. caninumantigens or other delivery systems that will elicit protective immune responses to neosporosis.
In Vitro Hydroxyurea Decreases Th1 Cell-Mediated Immunity
Authors: Weinberg Adriana
Published: July 2002

Hydroxyurea (HU) is used in the treatment of hematologic disorders and is sometimes added to antiretroviral combination therapy to potentiate human immunodeficiency virus (HIV) suppression. However, HU has toxic effects on rapidly dividing cells, including the effectors of the immune response. To determine whether HU affects specific T-cell responses, we measured lymphocyte proliferation and cytokine production in response to microbial antigen and mitogen stimulation in the presence of added HU (10 to 1,000 μM). HU treatment of peripheral blood mononuclear cells obtained from HIV-infected patients and uninfected controls decreased lymphocyte proliferation and gamma interferon production compared with untreated cells. Interleukin-2 (IL-2) and IL-10 production was not affected by HU. The HU-mediated decrease of lymphocyte proliferation was similar in peripheral blood mononuclear cells from HIV-infected patients and from uninfected controls. The inhibitory effect of HU required continuous exposure to the drug and could be reverted by washing the drug out of the culture environment. These findings suggest that HU-containing therapeutic regimens might decrease Th1-cell-mediated immune responses in vivo.
Activation of the Alternative Complement Pathway by Fungal Melanins
Authors: Rosas Á. L. , MacGill R. S. , Nosanchuk J. D. , Kozel T. R. , Casadevall A.
Published: July 2002

Melanins are complex biological pigments formed by the oxidative polymerization of phenolic and/or indolic compounds. These pigments have been implicated in the pathogenesis of some microbial infections, malignancies, degenerative disorders, and autoimmune diseases. Recent studies have demonstrated that melanins have antigenic and anti-inflammatory properties. These findings led us to further explore the interaction of melanins with the immune system. Melanin particles (“ghosts”) were isolated from in vitro-melanizedCryptococcus neoformanscells andAspergillus nigerconidia and then incubated in normal human serum containing125I-labeled complement C3. The results demonstrated deposition of C3 fragments onto the melanin ghosts as early as 1 min after incubation, with maximum deposition occurring after 12 min forC. neoformans-derived melanin ghosts and after 25 min forA. niger-derived melanin ghosts. The blocking of classical pathway activation did not affect the kinetics or total deposition of C3 onto the melanin ghosts, indicating that melanins activate complement through the alternative pathway. Immunofluorescence analysis of lungs from BALB/c mice injected intratracheally withC. neoformans-derived melanin ghosts demonstrated deposition of C3 fragments onto the ghosts. Small granulomas were also observed surrounding the ghosts. However, melanization of theC. neoformanscell wall did not alter the kinetics or total deposition of C3 fragments onto the fungal cells. The finding that melanin surfaces can activate the complement system suggests a potential mechanism for the pathogenesis of some degenerative and/or autoimmune processes that involve melanized cells as well as another potential role for melanin in the virulence of melanin-producing microorganisms.
Sodium Bicarbonate Enhances the Severity of Infection in Neutropenic Mice Orally Inoculated withListeria monocytogenesEGD
Authors: Czuprynski C. J. , Faith N. G.
Published: July 2002

Epidemiological studies have suggested an association between antacid therapy and development of listeriosis in humans. In this study we used a neutropenic mouse model to demonstrate that oral administration of sodium bicarbonate shortly before intragastric (i.g.) inoculation withListeria monocytogenesEGD (serotype 1/2a) significantly increased the severity of the resulting systemic infection. An explanation for this observation is provided by evidence thatL. monocytogenesEGD is rapidly inactivated in synthetic gastric fluid at pH below 5. A second strain ofL. monocytogenes(CM [serotype 1/2b]) exhibited little ability to cause systemic infection following i.g. inoculation and was not significantly enhanced by administration of sodium bicarbonate. Strain CM was readily inactivated in synthetic gastric fluid even at pH 7. These data suggest that gastric acidity and enzymes provide some innate defense against gastrointestinal listeriosis in neutropenic mice.
Human C-Reactive Protein Is Protective against FatalSalmonella entericaSerovar Typhimurium Infection in Transgenic Mice
Authors: Szalai Alexander J. , VanCott J. L. , McGhee Jerry R. , Volanakis John E. , Benjamin William H.
Published: July 2002

C-reactive protein (CRP) is an acute-phase protein with a well-known association with infection and other inflammatory conditions. We have shown that expression of human CRP by CRP transgenic (CRPtg) mice is protective against lethal infection byStreptococcus pneumoniae, an effect likely mediated by CRP's ability to bind to this gram-positive pathogen. In the present study we tested whether CRPtg mice are resistant to infection withSalmonella entericaserovar Typhimurium, a gram-negative pathogen that causes the murine equivalent of typhoid fever. CRPtg mice experimentally infected with a virulent Typhimurium strain lived longer and had significantly lower mortality than their non-tg littermates. The greater resistance of CRPtg mice could be attributed to significantly increased early (0 to 4 h) blood clearance of salmonellae and significantly decreased numbers of bacteria in the liver and spleen on day 7 postinfection. In addition, 14 days after infection with an avirulentSalmonellastrain, the serum titer of anti-Salmonellaimmunoglobulin G antibodies was higher in CRPtg than non-tg mice. This study provides unequivocal evidence that CRP plays an important role in vivo in host defense against salmonellae during the early stages of infection. In addition, as the beneficial effect of CRP includes enhancement of the host's humoral immune response, CRP may also contribute indirectly to host defense during later stages of infection.
Pet Toxin from Enteroaggregative
Escherichia coli
Produces Cellular Damage Associated with Fodrin Disruption
Authors: Villaseca Jorge M. , Navarro-García Fernando , Mendoza-Hernández Guillermo , Nataro James P. , Cravioto Alejandro , Eslava Carlos
Published: July 2002

Pet toxin is a serine protease from enteroaggregative
Escherichia coli
which has been described as causing enterotoxic and cytotoxic effects. In this paper we show that Pet produces spectrin and fodrin (nonerythroid spectrin) disruption. Using purified erythrocyte membranes treated with Pet toxin, we observed degradation of α- and β-spectrin chains; this effect was dose and time dependent, and a 120-kDa protein fraction was observed as a breakdown product. Spectrin degradation and production of the 120-kDa subproduct were confirmed using specific antibodies against the α- and β-spectrin chains. The same degradation effect was observed in α-fodrin from epithelial HEp-2 cells, both in purified cell membranes and in cultured cells which had been held in suspension for 36 h; these effects were confirmed using antifodrin rabbit antibodies. The spectrin and fodrin degradation caused by Pet is related to the Pet serine protease motif. Fluorescence and light microscopy of HEp-2 Pet-treated cells showed morphological alterations, which were associated with irregular distribution of fodrin in situ. Spectrin and fodrin degradation by Pet toxin were inhibited by anti-Pet antibodies and by phenylmethylsulfonyl fluoride. A site-directed Pet mutant, which had been shown to abolish the enterotoxic and cytotoxic effects of Pet, was unable to degrade spectrin in erythrocyte membranes or purified spectrin or fodrin in epithelial cell assays. This is a new system of cellular damage identified in bacterial toxins which includes the internalization of the protease, induction of some unknown intermediate signaling steps, and finally the fodrin degradation to destroy the cell.
Hemin-Binding Surface Protein from
Bartonella quintana
Authors: Carroll James A. , Coleman Sherry A. , Smitherman Laura S. , Minnick Michael F.
Published: July 2002

Bartonella quintana
, the agent of trench fever and a cause of endocarditis and bacillary angiomatosis in humans, has the highest reported in vitro hemin requirement for any bacterium. We determined that eight membrane-associated proteins from
B. quintana
bind hemin and that a ∼25-kDa protein (HbpA) was the dominant hemin-binding protein. Like many outer membrane proteins, HbpA partitions to the detergent phase of a Triton X-114 extract of the cell and is heat modifiable, displaying an apparent molecular mass shift from approximately 25 to 30 kDa when solubilized at 100°C. Immunoblots of purified outer and inner membranes and immunoelectron microscopy with whole cells show that HbpA is strictly located in the outer membrane and surface exposed, respectively. The N-terminal sequence of mature HbpA was determined and used to clone the HbpA-encoding gene (
hbpA
) from a lambda genomic library. The
hbpA
gene is 816 bp in length, encoding a predicted immature protein of approximately 29.3 kDa and a mature protein of 27.1 kDa. A Fur box homolog with 53% identity to the
Escherichia coli
Fur consensus is located upstream of
hbpA
and may be involved in regulating expression. BLAST searches indicate that the closest homologs to HbpA include the
Bartonella henselae
phage-associated membrane protein, Pap31 (58.4% identity), and the OMP31 porin from
Brucella melitensis
(31.7% identity). High-stringency Southern blots indicate that all five pathogenic
Bartonella
spp. possess
hbpA
homologs. Recombinant HbpA can bind hemin in vitro; however, it does not confer a hemin-binding phenotype upon
E. coli
. Intact
B. quintana
treated with purified anti-HbpA Fab fragments show a significant (
P
< 0.004) dose-dependent decrease in hemin binding relative to controls, suggesting that HbpA plays an active role in hemin acquisition and therefore pathogenesis. HbpA is the first potential virulence determinant characterized from
B. quintana
.
Emerging Family of Proline-Specific Peptidases of
Porphyromonas gingivalis
: Purification and Characterization of Serine Dipeptidyl Peptidase, a Structural and Functional Homologue of Mammalian Prolyl Dipeptidyl Peptidase IV
Authors: Banbula Agnieszka , Bugno Marcin , Goldstein Jason , Yen Jane , Nelson Daniel , Travis James , Potempa Jan
Published: July 2002

Porphyromonas gingivalis
is an asaccharolytic and anaerobic bacterium that possesses a complex proteolytic system which is essential for its growth and evasion of host defense mechanisms. In this report, we show the purification and characterization of prolyl dipeptidyl peptidase IV (DPPIV) produced by this organism. The enzyme was purified to homogeneity, and its enzymatic activity and biochemical properties were investigated.
P. gingivalis
DPPIV, like its human counterpart, is able to cleave the N terminus of synthetic oligopeptides with sequences analogous to those of interleukins 1β and 2. Additionally, this protease hydrolyzes biologically active peptides including substance P, fibrin inhibitory peptide, and β-casomorphin. Southern blot analysis of genomic DNA isolated from several
P. gingivalis
strains reveal that a single copy of the DPPIV gene was present in all strains tested.
Comparable Endotoxic Properties of Lipopolysaccharides Are Manifest in Diverse Clinical Isolates of Gram-Negative Bacteria
Authors: Luchi Michael , Morrison David C.
Published: July 2002

In general there is a poor correlation between serum lipopolysaccharide (LPS; the biologically active constituent of endotoxin) levels and mortality in septic patients. The objective of this study was to determine if chemical, structural, or biological differences among LPS from different clinical isolates of gram-negative bacteria might explain this discrepancy. LPS preparations were made using the hot phenol-water extraction method from eight clinical isolates of gram-negative bacteria. As a percentage of the total weight of the LPS, the phosphate content ranged from 3.0 to 13.8% (average, 6.7 ± 3.6%), and the 2-keto-3-deoxyoctonate content ranged from 1.9 to 27.4% (average, 8.9 ± 8.5%). These values were not dissimilar to those obtained for a reference endotoxin. In a standard measure of LPS activity, theLimulusamoebocyte lysate assay, there was approximately a twofold difference between the least and most active preparations. The two preparations with the greatest difference in their ability to elicit the secretion of tumor necrosis factor alpha from a mouse peritoneal macrophage cell line were similar in lethality when administered to mice sensitized to the effects of LPS byd(+)-galactosamine. These relatively minor differences in LPS activity seem unlikely to explain the generally observed discrepancy between serum endotoxin levels and mortality in patients with gram-negative sepsis.
Survival of Group BStreptococcusType III in Mononuclear Phagocytes: Differential Regulation of Bacterial Killing in Cord Macrophages by Human Recombinant Gamma Interferon and Granulocyte-Macrophage Colony-Stimulating Factor
Authors: Maródi László , Káposzta Rita , Nemes Éva
Published: July 2002

Phagocytic and killing capacities of resident and cytokine-activated human macrophages against group BStreptococcus(GBS) type III were studied. Evidence is presented that monocyte-derived macrophages from cord and adults ingest serum-opsonized GBS but that killing of bacteria was negligible in resident cells. Treatment of adult macrophages with recombinant human gamma interferon (rhIFN-γ; 100 U/ml) or recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; 200 U/ml) resulted in significant increases of killing of GBS (P< 0.01 for each). The killing capacity of cord macrophages treated with rhGM-CSF was also enhanced compared to that of untreated cells (P< 0.01). However, treatment with rhIFN-γ resulted in only a moderate increase in the capacity of cord macrophages to kill GBS (P> 0.1). These results mirrored the effect of rhIFN-γ on candidacidal capacities of cord and adult macrophages, reported earlier from our laboratory. These data indicate differential modulation of neonatal macrophages by rhGM-CSF and rhIFN-γ. We suggest that administration of rhGM-CSF to neonates with invasive GBS disease may enhance host resistance to these bacteria.
Mucosal Immune Responses to Meningococcal Group C Conjugate and Group A and C Polysaccharide Vaccines in Adolescents
Authors: Zhang Q. , Choo S. , Everard J. , Jennings R. , Finn A.
Published: July 2002

Previous studies in children have shown thatHaemophilus influenzaetype b (Hib) polysaccharide conjugate vaccines can reduce nasopharyngeal carriage ofH. influenzaeand provide herd immunity and suggest that this effect is mediated through mucosal antibodies. As this phenomenon may operate in other invasive bacterial infections which are propagated by nasopharyngeal carriage, mucosal antibody responses to meningococcal C conjugate and A/C polysaccharide vaccines were investigated. A total of 106 school children aged 11 to 17 years were randomized to receive a single dose of either conjugate or polysaccharide vaccine in an observer-blind study. Before and at 1, 6, and 12 months after immunization, samples of unstimulated saliva were collected and assayed by enzyme-linked immunosorbent assay for group C polysaccharide-specific immunoglobulin A (IgA), IgA1, IgA2 and secretory component, IgG antibodies, and total IgG and IgA. A subset of serum samples were also assayed for specific IgA and IgG antibodies. The concentrations of specific IgA and IgG in saliva were expressed both as nanograms per milliliter and as nanograms per microgram of total IgA or IgG. One month after immunization, significant increases in antibody titers (both IgA and IgG) were observed in saliva in both groups. There were significant subsequent falls in antibody titers by 6 months. Anti-meningococcal C-specific secretory component and IgA antibody titers were closely correlated (r= 0.85,P< 0.001), but there was no significant correlation between salivary and serum IgA titers, suggesting that IgA antibodies are locally produced. Significant correlation was found between salivary and serum IgG titers (r= 0.52,P< 0.01), suggesting that salivary IgG may be serum derived. Compared with polysaccharide vaccine, the conjugate vaccine induced significantly higher salivary IgG responses (P< 0.05), although there were no significant differences between salivary IgA responses to the two vaccines. The conjugate vaccine induced greater salivary IgG responses than a polysaccharide vaccine. Both vaccines induced significant salivary IgA antibodies. Further studies are needed to establish the functional significance of these mucosal responses.
Mucosal and Systemic Immune Responses to Chimeric Fimbriae Expressed bySalmonella entericaSerovar Typhimurium Vaccine Strains
Authors: Chen Huaiqing , Schifferli Dieter M.
Published: July 2002

Recombinant live oral vaccines expressing pathogen-derived antigens offer a unique set of attractive properties. Among these are the simplicity of administration, the capacity to induce mucosal and systemic immunity, and the advantage of permitting genetic manipulation for optimal antigen presentation. In this study, the benefit of having a heterologous antigen expressed on the surface of a live vector rather than intracellularly was evaluated. Accordingly, the immune response of mice immunized with aSalmonella entericaserovar Typhimurium vaccine strain expressing theEscherichia coli987P fimbrial antigen on its surface (Fas+) was compared with the expression in the periplasmic compartment (Fas−). Orally immunized BALB/c mice showed that 987P fimbriatedSalmonellaserovar Typhimurium CS3263 (aroA asd) with pCS151 (fas+asd+) elicited a significantly higher level of 987P-specific systemic immunoglobulin G (IgG) and mucosal IgA than serovar Typhimurium CS3263 with pCS152 (fasDmutant,asd+) expressing 987P periplasmic antigen. Further studies were aimed at determining whether the 987P fimbriae expressed by serovar Typhimurium χ4550 (cya crp asd) could be used as carriers of foreign epitopes. For this, the vaccine strain was genetically engineered to express chimeric fimbriae carrying the transmissible gastroenteritis virus (TGEV) C (379-388) and A (521-531) epitopes of the spike protein inserted into the 987P major fimbrial subunit FasA. BALB/c mice administered orally serovar Typhimurium χ4550 expressing the chimeric fimbriae from thetetpromoter in pCS154 (fas+asd+) produced systemic antibodies against both fimbria and the TGEV C epitope but not against the TGEV A epitope. To improve the immunogenicity of the chimeric fimbriae, the in vivo induciblenirBpromoter was inserted into pCS154, upstream of thefasgenes, to create pCS155. In comparison with the previously used vaccine, BALB/c mice immunized orally with serovar Typhimurium χ4550/pCS155 demonstrated significantly higher levels of serum IgG and mucosal IgA against 987P fimbria. Moreover, mucosal IgA against the TGEV C epitope was only detected with serovar Typhimurium χ4550/pCS155. The induced antibodies also recognized the epitopes in the context of the full-length TGEV spike protein. Hence, immune responses to heterologous chimeric fimbriae onSalmonellavaccine vectors can be optimized by using promoters known to be activated in vivo.
Cytopathogenic Effect of
Trichomonas vaginalis
on Human Vaginal Epithelial Cells Cultured In Vitro
Authors: Gilbert R. O. , Elia G. , Beach D. H. , Klaessig Suzanne , Singh B. N.
Published: July 2002

In this study we established human vaginal epithelial cells (hVECs) in culture and evaluated their interaction with
Trichomonas vaginalis
parasites to complement previous studies using other cell types. Primary cultures of hVECs were established. Contaminating fibroblasts were separated from epithelial cells by differential trypsinization. Specific antibody staining revealed that over 92% of cells in hVEC monolayers were epithelial cells.
T. vaginalis
adhered to hVECs and produced severe cytotoxic effects resulting in obliteration of the monolayer within 24 h. Adherence and cytotoxicity were not observed when
T. vaginalis
was exposed to human vaginal fibroblasts or bovine vaginal epithelial cells. Likewise, the bovine parasite
Tritrichomonas foetus
had no cytotoxic effects on hVECs. We concluded that the interaction between
T. vaginalis
and hVECs is both cell specific (limited to epithelial cells and not vaginal fibroblasts) and species specific (limited to human vaginal cells and not bovine cells). Pretreatment of
T. vaginalis
with metronidazole or periodate abolished the adhesion of parasites to cell monolayers and the cytotoxic effect, suggesting involvement of carbohydrate-containing molecules in these processes. Different clinical isolates of
T. vaginalis
caused damage to cultured cells at different rates. Parasites separated from the vaginal cell monolayer by a permeable membrane did not produce a cytopathic effect, suggesting contact-dependent cytotoxicity.
The
Entamoeba histolytica
Mitochondrion-Derived Organelle (Crypton) Contains Double-Stranded DNA and Appears To Be Bound by a Double Membrane
Authors: Ghosh Sudip , Field Jessica , Rogers Rick , Hickman Mark , Samuelson John
Published: July 2002

Amebae have an Hsp60-associated, mitochondrion-derived organelle (crypton). In this study, the crypton was stained with multiple DNA-binding fluorochromes and a monoclonal anti-double-stranded DNA antibody. Transmission microscopy of partially purified cryptons revealed organelles bound by a double membrane.
Macrophage Damage byLeishmania amazonensisCytolysin: Evidence of Pore Formation on Cell Membrane
Authors: Noronha Fátima S. M. , Cruz Jader S. , Beirão Paulo S. L. , Horta M. Fátima
Published: July 2002

We have previously shown that both promastigotes and amastigotes ofLeishmania amazonensiscontain a lytic protein that damages erythrocytes and nucleated cells, including macrophages (F. S. M. Noronha, F. J. Ramalho-Pinto, and M. F. Horta, Infect. Immun. 64:3975–3982, 1996). Using the patch-clamp technique, we show here that cell damage by parasite extracts is mediated by the formation of nonselective pores on the target membrane. This demonstrates thatL. amazonensiscytolysin is a pore-forming protein (PFP), here named leishporin. We show that the diameters of the pores formed by parasite extracts are heterogeneous, varying from ∼1.6 to >6.1 nm according to cytolysin concentration or time. We also show that pore formation involves the binding of the PFP to the target cell membrane, a temperature-independent event that is necessary but not sufficient to lyse cells. This is followed by a temperature-dependent step that triggers lysis, probably the insertion and the polymerization of protein subunits in the lipid bilayer. We provide evidence that suggests that polymerization of single subunits must occur for pore formation. We show, in addition, thatL. amazonensisexpresses molecules antigenically homologous to other PFPs.
CTLA-4 Down-Regulates the Protective Anticryptococcal Cell-Mediated Immune Response
Authors: McGaha Tracy , Murphy Juneann W.
Published: July 2002

Cell-mediated immune (CMI) responses defined by delayed-type hypersensitivity (DTH) reactivity to cryptococcal culture filtrate antigen (CneF) can be either protective or nonprotective against an infection withCryptococcus neoformans. The protective and nonprotective anticryptococcal DTH responses are induced by different immunogens and have differing activated-T-cell profiles. This study examined the effects of blockade of the interaction between cytotoxic T lymphocyte antigen 4 (CTLA-4) and its ligands B7-1 (CD80) and B7-2 (CD86) on the anticryptococcal DTH responses and protection. We found that CTLA-4 blockade at the time of immunization with the immunogen that induces the protective response, CneF, in complete Freund's adjuvant (CFA) or the immunogen that induces the nonprotective response, heat-killed cryptococcal cells (HKC), enhanced anticryptococcal DTH reactivity. In contrast, blocking CTLA-4 after the immune response was induced failed to enhance responses. Blockade of CTLA-4 in an infection model resulted in earlier development of the anticryptococcal CMI response than in control mice. Concomitant with increases in DTH reactivity in mice treated with anti-CTLA-4 Fab fragments at the time of immunization, there were decreases in cryptococcal CFU in lungs, spleens, and brains compared to controls. Blockade of CTLA-4 resulted in long-term protection, as measured by significantly increased survival times, only in mice given the protective immunogen, CneF-CFA. Anti-CTLA-4 treatment did not shift the response induced by the nonprotective immunogen, HKC, to a long-term protective one. Our data indicate that blockade of CTLA-4 interactions with its ligands may be useful in enhancing host defenses againstC. neoformans.
Constitutive Expression of the Vi Polysaccharide Capsular Antigen in AttenuatedSalmonella entericaSerovar Typhi Oral Vaccine Strain CVD 909
Authors: Wang Jin Yuan , Noriega Fernando R. , Galen James E. , Barry Eileen , Levine Myron M.
Published: July 2002

Live oral Ty21a and parenteral Vi polysaccharide vaccines provide significant protection against typhoid fever, albeit by distinct immune mechanisms. Vi stimulates serum immunoglobulin G Vi antibodies, whereas Ty21a, which does not express Vi, elicits humoral and cell-mediated immune responses other than Vi antibodies. Protection may be enhanced if serum Vi antibody as well as cell-mediated and humoral responses can be stimulated. Disappointingly, several new attenuatedSalmonella entericaserovar Typhi oral vaccines (e.g., CVD 908-htrAand Ty800) that elicit serum O and H antibody and cell-mediated responses following a single dose do not stimulate serum Vi antibody. Vi expression is regulated in response to environmental signals such as osmolarity by controlling the transcription oftviAin theviaBlocus. To investigate if Vi antibodies can be stimulated if Vi expression is rendered constitutive, we replaced PtviAin serovar Typhi vaccine CVD 908-htrAwith the constitutive promoter Ptac, resulting in CVD 909. CVD 909 expresses Vi even under high-osmolarity conditions and is less invasive for Henle 407 cells. In mice immunized with a single intranasal dose, CVD 909 was more immunogenic than CVD 908-htrAin eliciting serum Vi antibodies (geometric mean titer of 160 versus 49,P= 0.0007), whereas O antibody responses were virtually identical (geometric mean titer of 87 versus 80). In mice challenged intraperitoneally with wild-type serovar Typhi 4 weeks after a single intranasal immunization, the mortality of those immunized with CVD 909 (3 of 8) was significantly lower than that of control mice (10 of 10,P= 0.043) or mice given CVD 908-htrA(9 of 10,P= 0.0065).
Characterization of Adherence of Nontypeable
Haemophilus influenzae
to Human Epithelial Cells
Authors: van Schilfgaarde Muriel , van Ulsen Peter , Eijk Paul , Brand Michiel , Stam Martin , Kouame Jamal , van Alphen Loek , Dankert Jacob
Published: July 2002

The adherence of 58 nontypeable
Haemophilus influenzae
isolates obtained from patients with otitis media or chronic obstructive pulmonary disease (COPD) and obtained from the throats of healthy individuals to Chang and NCI-H292 epithelial cells was compared. Otitis media isolates, but not COPD isolates, adhered significantly more to both cell lines than did throat isolates. Since high-molecular-weight (HMW) proteins are major adhesins of nontypeable
H. influenzae
, the isolates were screened for HMW protein expression by Western blotting with two polyclonal sera and PCR with
hmw
-specific primers. Twenty-three of the 32 adhering isolates (72%) and only 1 of the 26 nonadherent strains were HMW protein or
hmw
gene positive. Among the 32 isolates adhering to either cell line, 5 different adherence patterns were distinguished based on the inhibiting effect of dextran sulfate. Using
H. influenzae
strain 12 expressing two well-defined HMW proteins (HMW1 and HMW2) and its isogenic mutants as a reference, we observed HMW1-like adherence to both cell lines for 16 of the 32 adherent isolates. Four others showed HMW2-like adherence to NCI-H292. Of the three other patterns of adherence, one probably also involved HMW protein. Screening of the isolates with six HMW-specific monoclonal antibodies in a whole-cell enzyme-linked immunosorbent assay showed that the HMW proteins of COPD isolates and carrier isolates were more distinct from the HMW proteins from
H. influenzae
strain 12 than those from otitis media isolates. Characterization of the HMW protein of a COPD isolate by adherence and DNA sequence analysis showed that despite large sequence diversity in the
hmwA
gene, probably resulting in the antigenic differences, the HMW protein mediated the HMW2-like adherence of this strain.
Down-Regulation of GATA-2 Transcription during
Pneumocystis carinii
Infection
Authors: Tang Xing , Lasbury Mark E. , Davidson Darrell D. , Bartlett Marilyn S. , Smith James W. , Lee Chao-Hung
Published: July 2002

Differences in gene expression between
Pneumocystis carinii
-infected and noninfected rats were examined. Total RNA was isolated from homogenized rat lungs and then subjected to differential display with combinations of oligo(dT) and various arbitrary PCR primers. Approximately 50 differentially expressed bands were observed. Several of these DNA bands were isolated, reamplified, and cloned. The cloned DNA fragments were used as probes to perform Northern hybridization on RNA from
P. carinii
-infected and noninfected rat lungs. One clone was found to react with a 3-kb mRNA from noninfected but not from
P. carinii
-infected rat lung, suggesting that the gene represented by this clone was down-regulated during
P. carinii
infection. The nucleotide sequence of this clone was determined and found to be 97% homologous to the mouse GATA-2 transcription factor. In situ hybridization using RNA probes derived from this clone revealed that alveolar macrophages, resident lung monocytes, and bronchial epithelial cells express the GATA-2 gene in the lung.
Antigenic and Sequence Diversity in Gonococcal Transferrin-Binding Protein A
Authors: Cornelissen Cynthia Nau , Anderson James E. , Boulton Ian C. , Sparling P. Frederick
Published: July 2002

Neisseria gonorrhoeaeis a gram-negative pathogen that is capable of satisfying its iron requirement with human iron-binding proteins such as transferrin and lactoferrin. Transferrin-iron utilization involves specific binding of human transferrin at the cell surface to what is believed to be a complex of two iron-regulated, transferrin-binding proteins, TbpA and TbpB. The genes encoding these proteins have been cloned and sequenced from a number of pathogenic, gram-negative bacteria. In the current study, we sequenced four additionaltbpAgenes from otherN. gonorrhoeaestrains to begin to assess the sequence diversity among gonococci. We compared these sequences to those from other pathogenic bacteria to identify conserved regions that might be important for the structure and function of these receptors. We generated polyclonal mouse sera against synthetic peptides deduced from the TbpA sequence from gonococcal strain FA19. Most of these synthetic peptides were predicted to correspond to surface-exposed regions of TbpA. We found that, while most reacted with denatured TbpA in Western blots, only one antipeptide serum reacted with native TbpA in the context of intact gonococci, consistent with surface exposure of the peptide to which this serum was raised. In addition, we evaluated a panel of gonococcal strains for antigenic diversity using these antipeptide sera.
Streptococcus pneumoniaeInduces Secretion of Vascular Endothelial Growth Factor by Human Neutrophils
Authors: van der Flier Michiel , Coenjaerts Frank , Kimpen Jan L. L. , Hoepelman Andy M. , Geelen Sibyl P. M.
Published: July 2002

Infection by pneumococci causes an acute inflammatory response associated with neutrophil influx, increased vascular permeability, and edema. Vascular endothelial growth factor (VEGF) is one of the most potent regulators of endothelial permeability. In vitro stimulation of neutrophils showed that pneumococci and purified pneumococcal cell wall induce VEGF secretion, independent of the presence of pneumolysin or polysaccharide capsule. The results of this study indicate VEGF is secreted in pneumococcal disease, suggesting a role as a mediator of increased vascular permeability.
Invasion of Human Vascular Endothelial Cells by
Actinobacillus actinomycetemcomitans
via the Receptor for Platelet-Activating Factor
Authors: Schenkein Harvey A. , Barbour Suzanne E. , Berry C. R. , Kipps Barbara , Tew John G.
Published: July 2002

Strains of the periodontal pathogen
Actinobacillus actinomycetemcomitans
are variable with respect to display of phosphorylcholine (PC)-bearing antigens. We have examined strains of
A. actinomycetemcomitans
with and without PC to assess their ability to invade endothelial cells via the receptor for platelet-activating factor (PAF). Results of antibiotic protection assays indicate that PC-bearing
A. actinomycetemcomitans
invade human vascular endothelial cells by a mechanism inhibitable by CV3988, a PAF receptor antagonist, and by PAF itself. The invasive phenotype was verified by transmission electron microscopy. A PC-deficient strain of this organism was not invasive. This property, in addition to the established ability of
A. actinomycetemcomitans
to invade epithelial cells, may provide this organism with access to the systemic circulation. The ability of PC-bearing oral bacteria to access the circulation may also explain the elevated levels of anti-PC antibody in serum found in patients with periodontitis.