bluuebunny/crossref_metadata_2025_split huggingface dataset:
Substrate Interactions during the Biodegradation of Benzene, Toluene, Ethylbenzene, and Xylene (BTEX) Hydrocarbons by the Fungus
Cladophialophora
sp. Strain T1
Authors: Prenafeta-Boldú F. X. , Vervoort J. , Grotenhuis J. T. C. , van Groenestijn J. W. 
Published: July 2002

ABSTRACT

The soil fungus
Cladophialophora
sp. strain T1 (= ATCC MYA-2335) was capable of growth on a model water-soluble fraction of gasoline that contained all six BTEX components (benzene, toluene, ethylbenzene, and the xylene isomers). Benzene was not metabolized, but the alkylated benzenes (toluene, ethylbenzene, and xylenes) were degraded by a combination of assimilation and cometabolism. Toluene and ethylbenzene were used as sources of carbon and energy, whereas the xylenes were cometabolized to different extents.
o
-Xylene and
m
-xylene were converted to phthalates as end metabolites;
p
-xylene was not degraded in complex BTEX mixtures but, in combination with toluene, appeared to be mineralized. The metabolic profiles and the inhibitory nature of the substrate interactions indicated that toluene, ethylbenzene, and xylene were degraded at the side chain by the same monooxygenase enzyme. Our findings suggest that soil fungi could contribute significantly to bioremediation of BTEX pollution.
Pneumococcal Type 22F Polysaccharide Absorption Improves the Specificity of a Pneumococcal-Polysaccharide Enzyme-Linked Immunosorbent Assay
Authors: Concepcion Nelydia F. , Frasch Carl E. 
Published: July 2002

ABSTRACTThe specificity of the immune response to the 23-valent pneumococcal-polysaccharide (PS) vaccine in healthy adults and to a pneumococcal conjugate vaccine in infants was examined by measuring immunoglobulin G (IgG) antibody titers by enzyme-linked immunosorbent assay (ELISA) and the opsonophagocytosis assay. ELISA measures total antipneumococcal IgG titers including the titers of functional and nonfunctional antibodies, while the opsonophagocytosis assay measures only functional-antibody titers. Twenty-four pairs of pre- and post-pneumococcal vaccination sera from adults were evaluated (ELISA) for levels of IgG antibodies against serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. Twelve of the pairs were also examined (opsonophagocytosis assay) for their functional activities. The correlation coefficients between assay results for most types ranged from 0.75 to 0.90, but the correlation coefficient was only about 0.6 for serotypes 4 and 19F. The specificities of these antibodies were further examined by the use of competitive ELISA inhibition. A number of heterologous polysaccharides (types 11A, 12F, 15B, 22F, and 33A) were used as inhibitors. Most of the sera tested showed cross-reacting antibodies, in addition to those removed by pneumococcal C PS absorption. Our data suggest the presence of a common epitope that is found on most pneumococcal PS but that is not absorbed by purified C PS. Use of a heterologous pneumococcal PS (22F) to adsorb the antibodies to the common epitope increased the correlation between the IgG ELISA results and the opsonophagocytosis assay results. The correlation coefficient improve from 0.66 to 0.92 for type 4 and from 0.63 to 0.80 for type 19F. These common-epitope antibodies were largely absent in infants at 7 months of age, suggesting the carbohydrate nature of the epitope.
Quantification of Bacterial Transcripts during Infection Using Competitive Reverse Transcription-PCR (RT-PCR) and LightCycler RT-PCR
Authors: Goerke Christiane , Bayer Manfred G. , Wolz Christiane 
Published: July 2002

ABSTRACT

Bacteria have evolved sophisticated regulatory circuits to modulate their gene expression in response to disparate environments. In order to monitor bacterial gene expression and regulation in the host, methods for direct transcript analysis from clinical specimens are needed. For most bacterial infections, amplification of the mRNAs of interest is necessary due to the low numbers of cells present and the low levels of specific transcripts. Here we compare two methods of quantitative reverse transcription-PCR (RT-PCR)—competitive RT-PCR using a one-tube system followed by standard gel analysis and the real-time detection of PCR product formation by fluorescence resonance energy transfer technology using the LightCycler unit. We isolated
Staphylococcus aureus
RNA directly from clinical specimens obtained from cystic fibrosis patients with chronic
S. aureus
lung infection and from an animal model of foreign-body infection with no further cultivation of the bacteria. Competitive RT-PCR and LightCycler RT-PCR were tested for their ability to quantify the transcription of a constitutively expressed gyrase gene (
gyr
) and a highly regulated α-toxin gene (
hla
) of
S. aureus
. Reproducible results were obtained with both methods. A sensitivity of 10
4
(
gyr
) and 10
3
(
hla
) copies, respectively, was reached, which was sufficient for the quantification of transcripts during bacterial infection. Overall, the competitive RT-PCR is a robust technique which does not need special RNA purification. On the negative side, it is labor intensive and time consuming, thus limiting the numbers of samples which can be analyzed at a given time. LightCycler RT-PCR is very susceptible to even traces of inhibitors, but it allows high-throughput processing of samples.
Functional Antibody Activity Elicited by Fractional Doses ofHaemophilus influenzaeType b Conjugate Vaccine (Polyribosylribitol Phosphate–Tetanus Toxoid Conjugate)
Authors: Romero-Steiner Sandra , Fernandez Josefina , Biltoft Christel , Wohl Melissa E. , Sanchez Jacqueline , Feris Jesus , Balter Sharon , Levine Orin S. , Carlone George M. 
Published: July 2002

ABSTRACTWe evaluated the functional activities of antibodies, serum bactericidal activity (SBA), and immunoglobulin G (IgG) antibody avidity indices, using sodium thiocyanate (NaSCN) elution, elicited after vaccination with fractional doses of theHaemophilus influenzaetype b conjugate (polyribosylribitol phosphate [PRP] conjugated to tetanus toxoid [PRP-T]) vaccine. A cohort of 600 infants from the Dominican Republic were randomized to receive one of three regimens of the PRP-T vaccine at ages 2, 4, and 6 months: full doses (10 μg of PRP antigen), one-half doses (5.0 μg), and one-third doses (3.3 μg) (J. Fernandez et al., Am. J. Trop. Med. Hyg. 62:485–490, 2000). Sixty serum samples, collected at age 7 months, with ≥2.0 μg of anti-PRP IgG per ml were randomly selected for avidity determinations. Geometric mean IgG concentrations were 13, 14, and 17 μg/ml for infants who received the full-dose (n= 19), one-half-dose (n= 19), and one-third-dose (n= 22) regimens, respectively. SBA geometric mean titers (1/dilution) were 85.0, 82.0, and 76.1 in sera from infants receiving the full-, one-half-, and one-third-dose regimens, respectively. Avidity indices (mean ± standard error weighted average of NaSCN molar concentration × serum dilution factor) were 71.9 ± 9.4, 123.6 ± 26.8, and 150.9 ± 24.9 for the full-, one-half-, and one-third-dose regimens, respectively. Upon comparison, the only significant difference (P= 0.024) found was a greater avidity index for sera from infants receiving the one-third-dose regimen than for sera from infants receiving the the full-dose regimen. We conclude that fractional doses elicit similar functional antibody activities in infants with ≥2 μg of anti-PRP IgG per ml, corresponding to 89, 90, and 97% of infants receiving three doses of either the full concentration or one-half or one-third of the labeled concentration, respectively. This approach offers an alternative strategy for the prevention ofH. influenzaetype b disease in countries with limited resources.
Improved Assessment of T-Cell Receptor (TCR) VB Repertoire in Clinical Specimens: Combination of TCR-CDR3 Spectratyping with Flow Cytometry-Based TCR VB Frequency Analysis
Authors: Pilch H. , Höhn H. , Freitag K. , Neukirch C. , Necker A. , Haddad P. , Tanner B. , Knapstein P. G. , Maeurer M. J. 
Published: July 2002

ABSTRACTAntigen-specific T-cell responses may be described by combining three categories: (i) the specificity and effector functions of a T-cell population, (ii) the quantity of T-cell responses (i.e., the number of responding T cells within the CD4/CD8 population), and (iii) the “quality” of T cells (defined by the T-cell receptor [TCR] structure). Several methods to measure T-cell responses are now available including evaluation of T-cell precursors using limiting dilution, the enzyme-linked immunospot assay, ex vivo TCR variable (v)-segment analysis determined by flow cytometry, and TCR-CDR3 length analysis (spectratyping), as well as identification of peptide-specific T cells using major histocompatibility complex (MHC) class I tetramers containing appropriate peptides. Until now, only a limited set of MHC-peptide complexes have been available as tetramer complexes. We demonstrate that CD8+or CD4+T cells in patients with cancer can be molecularly defined using a combination of spectratyping (TCR structure and “molecular composition”) plus the implementation of an antibody panel directed against 21 individual VB TCR chains (“quantity” of T-cell families). This approach is instrumental in defining and comparing the magnitudes of CD4+or CD8+T-cell responses over time in individual patients, in comparing the TCR VA and VB repertoire in different anatomic compartments, and in comparing the TCR VA-VB diversity with that in normal healthy controls. This method provides the means of objectively defining and comparing the TCR repertoire in patients undergoing vaccination protocols and underlines the necessity to calibrate the TCR-CDR3 analysis with a qualitative assessment of individual TCR VB families.
Cytotoxic Necrotizing Factor Type 1 of Uropathogenic
Escherichia coli
Kills Cultured Human Uroepithelial 5637 Cells by an Apoptotic Mechanism
Authors: Mills Melody , Meysick Karen C. , O'Brien Alison D. 
Published: July 2002

ABSTRACT

Pathogenic
Escherichia coli
associated with urinary tract infections (UTIs) in otherwise healthy individuals frequently produce cytotoxic necrotizing factor type 1 (CNF1), a member of the family of bacterial toxins that target the Rho family of small GTP-binding proteins. To gain insight into the function of CNF1 in the development of
E. coli
-mediated UTIs, we examined the effects of CNF1 intoxication on a panel of human cell lines derived from physiologically relevant sites (bladder, ureters, and kidneys). We identified one uroepithelial cell line that exhibited a distinctly different CNF1 intoxication phenotype from the prototypic one of multinucleation without cell death that is seen when HEp-2 or other epithelial cells are treated with CNF1. The 5637 bladder cell line detached from the growth surface within 72 h of CNF1 intoxication, a finding that suggested frank cytotoxicity. To determine the basis for the unexpected toxic effect of CNF1 on 5637 cells, we compared the degree of toxin binding, actin fiber formation, and Rho modification with those CNF1-induced events in HEp-2 cells. We found no apparent difference in the amount of CNF1 bound to 5637 cells and HEp-2 cells. Moreover, CNF1 modified Rho, in vivo and in vitro, in both cell types. In contrast, one of the classic responses to CNF1 in HEp-2 and other epithelial cell lines, the formation of actin stress fibers, was markedly absent in 5637 cells. Indeed, actin stress fiber induction by CNF1 did not occur in any of the other human bladder cell lines that we tested (J82, SV-HUC-1, or T24). Furthermore, the appearance of lamellipodia and filopodia in 5637 cells suggested that CNF1 activated the Cdc42 and Rac proteins. Finally, apoptosis was observed in CNF1-intoxicated 5637 cells. If our results with 5637 cells reflect the interaction of CNF1 with the transitional uroepithelium in the human bladder, then CNF1 may be involved in the exfoliative process that occurs in that organ after infection with uropathogenic
E. coli
.
Phenotypic Switching in
Candida glabrata
Involves Phase-Specific Regulation of the Metallothionein Gene
MT-II
and the Newly Discovered Hemolysin Gene
HLP
Authors: Lachke Salil A. , Srikantha Thyagarajan , Tsai Luong K. , Daniels Karla , Soll David R. 
Published: July 2002

ABSTRACT

Although
Candida glabrata
has emerged in recent years as a major fungal pathogen, there have been no reports demonstrating that it undergoes either the bud-hypha transition or high-frequency phenotypic switching, two developmental programs believed to contribute to the pathogenic success of other
Candida
species. Here it is demonstrated that
C. glabrata
undergoes reversible, high-frequency phenotypic switching between a white (Wh), light brown (LB), and dark brown (DB) colony phenotype discriminated on an indicator agar containing 1 mM CuSO
4
. Switching regulates the transcript level of the
MT-II
metallothionein gene(s) and a newly discovered gene for a hemolysin-like protein,
HLP
. The relative
MT-II
transcript levels in Wh, LB, and DB cells grown in the presence of CuSO
4
are 1:27:81, and the relative transcript levels of
HLP
are 1:20:35. The relative
MT-II
and
HLP
transcript levels in cells grown in the absence of CuSO
4
are 1:20:30 and 1:20:25, respectively. In contrast, switching has little or no effect on the transcript levels of the genes
MT-I
,
AMT-I
,
TRPI
,
HIS3
,
EPAI
, and
PDHI
. Switching of
C. glabrata
is not associated with microevolutionary changes identified by the DNA fingerprinting probe Cg6 and does not involve tandem amplification of the
MT-IIa
gene, which has been shown to occur in response to elevated levels of copper. Finally, switching between Wh, LB, and DB occurred in all four clinical isolates examined in this study. As in
Candida albicans
, switching in
C. glabrata
may provide colonizing populations with phenotypic plasticity for rapid responses to the changing physiology of the host, antibiotic treatment, and the immune response, through the differential regulation of genes involved in pathogenesis. More importantly, because
C. glabrata
is haploid, a mutational analysis of switching is now feasible.
Mechanisms of the Proinflammatory Response of Endothelial Cells toCandida albicansInfection
Authors: Orozco Alison S. , Zhou Xiang , Filler Scott G. 
Published: July 2002

ABSTRACTEndothelial cells can influence significantly the host inflammatory response against blood-borne microbial pathogens. Previously, we found that endothelial cells respond to in vitro infection withCandida albicansby secreting interleukin 8 (IL-8) and expressing E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1). We have now examined the mechanisms mediating this endothelial cell response. We determined thatC. albicansstimulated endothelial cells to synthesize tumor necrosis factor alpha (TNF-α), which in turn induced these infected cells to secrete IL-8 and express E-selectin by an autocrine mechanism. Expression of VCAM-1 was mediated not only by TNF-α but also by IL-1α and IL-1β, all of which were synthesized by endothelial cells in response toC. albicans. These three cytokines remained primarily cell associated rather than being secreted. Candidal induction of ICAM-1 expression was independent of TNF-α, IL-1α, and IL-1β. These observations demonstrate that different proinflammatory endothelial cell responses toC. albicansare induced by distinct mechanisms. A clear understanding of these mechanisms is important for therapeutically modulating the endothelial cell response toC. albicansand perhaps other opportunistic pathogens that disseminate hematogenously.
Interaction between
Burkholderia pseudomallei
and
Acanthamoeba
Species Results in Coiling Phagocytosis, Endamebic Bacterial Survival, and Escape
Authors: Inglis Timothy J. J. , Rigby Paul , Robertson Terry A. , Dutton Nichole S. , Henderson Mandy , Chang Barbara J. 
Published: July 2002

ABSTRACT

Burkholderia pseudomallei
causes melioidosis, a potentially fatal disease whose clinical outcomes include rapid-onset septicemia and relapsing and delayed-onset infections. Like other facultative intracellular bacterial pathogens,
B. pseudomallei
is capable of survival in human phagocytic cells, but unlike mycobacteria,
Listeria monocytogenes
, and
Salmonella
serovar Typhimurium, the species has not been reported to survive as an endosymbiont in free-living amebae. We investigated the consequences of exposing
Acanthamoeba astronyxis
,
A. castellani
, and
A. polyphaga
to
B. pseudomallei
NCTC 10276 in a series of coculture experiments. Bacterial endocytosis was observed in all three
Acanthamoeba
species. A more extensive range of cellular interactions including bacterial adhesion, incorporation into amebic vacuoles, and separation was observed with
A. astronyxis
in timed coculture experiments. Amebic trophozoites containing motile intravacuolar bacilli were found throughout 72 h of coculture. Confocal microscopy was used to confirm the intracellular location of endamebic
B. pseudomallei
cells. Transmission electron microscopy of coculture preparations revealed clusters of intact bacilli in membrane-lined vesicles inside the trophozoite cytoplasm; 5 × 10
2
CFU of bacteria per ml were recovered from lysed amebic trophozoites after 60 min of coculture. Demonstration of an interaction between
B. pseudomallei
and free-living acanthamebae in vitro raises the possibility that a similar interaction in vivo might affect environmental survival of
B. pseudomallei
and subsequent human exposure. Endamebic passage of
B. pseudomallei
warrants further investigation as a potential in vitro model of intracellular
B. pseudomallei
infection.
Intestinal Epithelial Cell Apoptosis followingCryptosporidium parvumInfection
Authors: McCole Declan F. , Eckmann Lars , Laurent Fabrice , Kagnoff Martin F. 
Published: July 2002

ABSTRACTCryptosporidium parvuminduces moderate levels of apoptosis of cultured human intestinal epithelial cells, which are maximal at 24 h after infection. Apoptosis is further increased inC. parvum-infected cells by inhibition of NF-κB.C. parvuminfection also attenuates epithelial apoptosis induced by strongly proapoptotic agents. The data suggestC. parvumhas developed strategies to limit apoptosis in order to facilitate its growth and maturation in the early period after epithelial cell infection.
T-Cell Epitopes in Variable Segments ofChlamydia trachomatisMajor Outer Membrane Protein Elicit Serovar-Specific Immune Responses in Infected Humans
Authors: Ortiz Linette , Angevine Mark , Kim Seon-Kyeong , Watkins David , DeMars Robert 
Published: July 2002

ABSTRACTWe previously identified 18 stimulatoryChlamydia trachomatismajor outer membrane protein (MOMP) peptides containing at least 23 epitopes presented with various HLA class II allotypes. Only one peptide contained an epitope localized in a variable segment (VS2). Continued studies reported here identified a total of five VS peptides containing T-cell epitopes that are distributed among MOMPs VS1, VS2, and VS4. Only MOMP-primed T-cell cultures from subjects infected with serovar E responded to the serovar E VS peptides, while the response of such cultures to constant-segment peptides was independent of the infecting serovar. Furthermore, MOMP-primed T cells proliferated in response only to the VS peptides encoded in serovar E but not to the corresponding peptides derived from serovar F, I, or J, confirming that these responses were serovar specific.
Identification of a New Repetitive Element in
Staphylococcus aureus
Authors: Cramton Sarah E. , Schnell Norbert F. , Götz Friedrich , Brückner Reinhold 
Published: July 2002

ABSTRACT

The
Staphylococcus aureus
repeat (STAR) element is a sequence identified in two intergenic regions in
S. aureus
. The element is found in 13 to 21 copies in individual
S. aureus
strains, and elements in the homologous intergenic location are variable in length. The element sequence consists of several small and unusually GC-rich direct repeats with recurring intervening sequences. In addition, STAR-like elements may be present in related staphylococcal species.
Attenuation of and Protection Induced by a Leucine Auxotroph ofMycobacterium tuberculosis
Authors: Hondalus Mary K. , Bardarov Stoyan , Russell Robert , Chan John , Jacobs William R. , Bloom Barry R. 
Published: July 2002

ABSTRACTAttenuated mutants ofMycobacterium tuberculosisrepresent potential vaccine candidates for the prevention of tuberculosis. It is known that auxotrophs of a variety of bacteria are attenuated in vivo and yet provide protection against challenge with wild-type organisms. A leucine auxotroph ofM. tuberculosiswas created by allelic exchange, replacing wild-typeleuD(Rv2987c), encoding isopropyl malate isomerase, with a mutant copy of the gene in which 359 bp had been deleted, creating a strain requiring exogenous leucine supplementation for growth in vitro. The frequency of reversion to prototrophy was <10−11. In contrast to wild-typeM. tuberculosis, the ΔleuDmutant was unable to replicate in macrophages in vitro. Its attenuation in vivo and safety as a vaccine were established by the fact that it caused no deaths in immunodeficient SCID mice. Complementation of the mutant with wild-typeleuDabolished the requirement for leucine supplementation and restored the ability of the strain to grow both in macrophages and in SCID mice, thus confirming that the attenuated phenotype was due to the ΔleuDmutation. As a test of the vaccine potential of the leucine auxotroph, immunocompetent BALB/c mice, susceptible to fatal infection with wild-typeM. tuberculosis, were immunized with the ΔleuDmutant and subsequently challenged with virulentM. tuberculosisby both the intravenous and aerosol routes. A comparison group of mice was immunized with conventionalMycobacterium bovisBCG vaccine. Whereas all unvaccinated mice succumbed to intravenous infection within 15 weeks, mice immunized with either BCG or the ΔleuDmutant ofM. tuberculosisexhibited enhanced and statistically equivalent survival curves. However, theleuDauxotroph was less effective than live BCG in reducing organ burdens and tissue pathology of mice challenged by either route. We conclude that attenuation and protection againstM. tuberculosischallenge can be achieved with a leucine auxotroph and suggest that to induce optimal protection, attenuated strains ofM. tuberculosisshould persist long enough and be sufficiently metabolically active to synthesize relevant antigens for an extended period of time.
Interleukin-6 Induces Early Gamma Interferon Production in the Infected Lung but Is Not Required for Generation of Specific Immunity toMycobacterium tuberculosisInfection
Authors: Saunders Bernadette M. , Frank Anthony A. , Orme Ian M. , Cooper Andrea M. 
Published: July 2002

ABSTRACTImmunity toMycobacterium tuberculosisis dependent upon the generation of a protective gamma interferon (IFN-γ)-producing T-cell response. Recent studies have suggested that interleukin-6 (IL-6) is required for the induction of a protective T-cell response and that IL-4 may suppress the induction of IFN-γ. To evaluate the role of the cytokines IL-6 and IL-4 in the generation of pulmonary immunity toM. tuberculosis, IL-6 and IL-4 knockout mice were infected withM. tuberculosisvia aerosol. The absence of IL-6 led to an early increase in bacterial load with a concurrent delay in the induction of IFN-γ. However, mice were able to contain and control bacterial growth and developed a protective memory response to secondary infection. This demonstrates that while IL-6 is involved in stimulating early IFN-γ production, it is not essential for the development of protective immunity againstM. tuberculosis. In contrast, while the absence of IL-4 resulted in increased IFN-γ production, this had no significant effect upon bacterial growth.