bluuebunny/crossref_metadata_2025_split huggingface dataset:
Determination of Human Immunodeficiency Virus Type 1 Subtypes by a Rapid Method Useful for the Routine Diagnostic Laboratory
Authors: Kessler Harald H. , Deuretzbacher Doris , Stelzl Evelyn , Daghofer Elisabeth , Santner Brigitte I. , Marth Egon 
Published: July 2002

ABSTRACTThe existence of human immunodeficiency virus type 1 (HIV-1) subtypes has many important implications for the global evolution of HIV and for the evaluation of pathogenicity, transmissibility, and candidate HIV vaccines. The aim of this study was to establish a rapid method for determination of HIV-1 subtypes useful for a routine diagnostic laboratory and to investigate the distribution of HIV-1 subtypes in Austrian patients. Samples were tested by a subtyping method based on a 1.3-kb sequence of the polymerase gene generated by a commercially available drug resistance assay. The generated sequence was subtyped by means of an HIV sequence database. Results of 74 routine samples revealed subtype B (71.6%) as the predominant subtype, followed by subtype A (13.5%) and subtype C (6.8%). Subtypes E, F, G, and AE (CM240) were also detected. This subtyping method was found to be very easy to handle, rapid, and inexpensive and has proved suitable for high-throughput routine diagnostic laboratories. The specific polymerase gene sequence, however, must be existent.
Tuberculin Skin Testing Compared with T-Cell Responses to
Mycobacterium tuberculosis
-Specific and Nonspecific Antigens for Detection of Latent Infection in Persons with Recent Tuberculosis Contact
Authors: Arend Sandra M. , Engelhard Anrik C. F. , Groot Gertjan , de Boer Kirsten , Andersen Peter , Ottenhoff Tom H. M. , van Dissel Jaap T. 
Published: July 2002

ABSTRACT

The tuberculin skin test (TST) is used for the identification of latent tuberculosis (TB) infection (LTBI) but lacks specificity in
Mycobacterium bovis
BCG-vaccinated individuals, who constitute an increasing proportion of TB patients and their contacts from regions where TB is endemic. In previous studies, T-cell responses to ESAT-6 and CFP-10,
M. tuberculosis
-specific antigens that are absent from BCG, were sensitive and specific for detection of active TB. We studied 44 close contacts of a patient with smear-positive pulmonary TB and compared the standard screening procedure for LTBI by TST or chest radiographs with T-cell responses to
M. tuberculosis
-specific and nonspecific antigens. Peripheral blood mononuclear cells were cocultured with ESAT-6, CFP-10, TB10.4 (each as recombinant antigen and as a mixture of overlapping synthetic peptides),
M. tuberculosis
sonicate, purified protein derivative (PPD), and short-term culture filtrate, using gamma interferon production as the response measure. LTBI screening was by TST in 36 participants and by chest radiographs in 8 persons. Nineteen contacts were categorized as TST negative, 12 were categorized as TST positive, and 5 had indeterminate TST results. Recombinant antigens and peptide mixtures gave similar results. Responses to TB10.4 were neither sensitive nor specific for LTBI. T-cell responses to ESAT-6 and CFP-10 were less sensitive for detection of LTBI than those to PPD (67 versus 100%) but considerably more specific (100 versus 72%). The specificity of the TST or in vitro responses to PPD will be even less when the proportion of BCG-vaccinated persons among TB contacts evaluated for LTBI increases.
Mapping of
Escherichia coli
H27-Specific Epitope from H-Specific Polypeptides
Authors: Seah J. N. , Kwang J. 
Published: July 2002

ABSTRACT

A murine monoclonal antibody (MAb) reactive to H27 flagellin antigen was produced and characterized. Forty-nine partially purified native H-type flagellins were used to evaluate the specificity of the MAb. The
fliC
gene of H27 is 1,464 bp in length (487 amino acids [aa]; 50.88 kDa). The central variable region (CVR) of the H27 flagellin gene was defined by comparison with flagellin sequences derived from H8, H34, and H49. To study the distribution of antigenic epitopes, the CVR covering amino acid residues 70 to 457 (388 aa) was dissected into seven overlapping fragments. Fragments carrying the H-type-specific antigenic determinants were identified by H27-specific antiserum. Polyclonal antibodies raised against different H-type flagellin proteins were used to determine the cross-reactive determinants. Three fragments, spanning amino acid residues 240 to 380, which carried the potential H-specific determinants were used for MAb production. A MAb specific to H27 was produced, and the specific epitope was mapped to amino acid residues 330 to 340. In this study, we produced MAbs from predetermined H27-specific polypeptides and used whole flagellin in enzyme-linked immunosorbent assays to circumvent the interference of anti-glutathione
S
-transferase antibodies. These factors when combined could help to improve the identification of the desired MAb.
Evaluation of
Chlamydia pneumoniae
43- and 53-Kilodalton Recombinant Proteins for Serodiagnosis by Western Blot
Authors: Campbell Lee Ann , Roberts Shane , Inoue Shiuichiro , Kong Lilly , Kuo Cho-chou 
Published: July 2002

ABSTRACT

Chlamydia pneumoniae
is a common cause of respiratory infection. It has also been shown to be associated with coronary heart disease. Two proteins that have been reported to be recognized frequently during human infection are proteins having molecular masses of 43 and 53 kDa. In order to develop a useful alternative serological test to the microimmunofluorescence (micro-IF) assay, recombinant 43-kDa and 53-kDa chlamydia-specific proteins were evaluated in dot blot and/or for comparison to the standard micro-IF test. Primers for amplification were derived from genome sequence information for two
C. pneumoniae
genes (CPn0809 and CPn0980) encoding 53-kDa proteins and four
C. pneumoniae
genes (CPn0562, CPn0927, CPn0928, and Cpn0929) encoding 43-kDa proteins of unknown function, which were
Chlamydia
specific and not found in
Chlamydia trachomatis
. The 53-kDa protein product of CPn0809 or the N-terminal 18-kDa portion had better specificity than any of the 43-kDa recombinants but was much less sensitive than micro-IF. In contrast, the 53-kDa protein encoded by CPn0980 was recognized by 11 of 12 (92%) acute-phase sera, 35 of 46 (76%) chronic sera, 0 of 12 micro-IF-negative sera (
C. pneumoniae
and
C. trachomatis
negative), and 1 of 12 (8%)
C. pneumoniae
negative,
C. trachomatis
positive sera. Thus, it appears that the 53-kDa protein encoded by CPn0980 has potential use for serodiagnosis of
C. pneumoniae
infection.
Efficacy of a Less-Sensitive Enzyme Immunoassay (3A11-LS) for Early Diagnosis of Human Immunodeficiency Virus Type 1 Infection in Infants
Authors: Candal Debra , Bulterys Marc , Abrams Elaine J. , Steketee Richard W. , Parekh Bharat S. 
Published: July 2002

ABSTRACT
We evaluated a less-sensitive enzyme immunoassay (3A11-LS) for its possible use for early diagnosis of human immunodeficiency virus type 1 (HIV-1) infection in infants. The results were compared with those from the immunoglobulin G-capture enzyme immunoassay. A total of 239 sera from 77 infants were tested. All 25 sera from the 10 infants born to seronegative mothers were found to be negative by both assays. Forty-one seroreverting infants showed a complete decay of maternal antibodies by 4 months by the 3A11-LS assay. However, the assay detected HIV antibodies in only 9 (36%) of 25 sera collected from infected infants between 4 and 6 months and in 27 (63%) of 43 sera collected after 6 months of age. Further analysis with alternative cutoff values indicated that the 3A11-LS had a sensitivity of 12 to 44% and a specificity of 90 to 100% for infants between 4–6 months of age. This data suggest that a diagnosis of HIV infection in some of the infants could be made after 4 months of age by the 3A11-LS assay, although a negative 3A11-LS test result may not rule out infection and may require a further followup.
Immunoglobulin G, A, and M Responses in Serum and Circulating Immune Complexes Elicited by the 16-Kilodalton Antigen ofMycobacterium tuberculosis
Authors: Raja Alamelu , Uma Devi K. R. , Ramalingam B. , Brennan Patrick J. 
Published: July 2002

ABSTRACTThe 16-kDa cytosolic antigen ofM. tuberculosiswas purified to homogeneity by molecular sieving chromatography, and the diagnostic potential of the antigen was evaluated in various categories of patients by enzyme-linked immunosorbent assay (ELISA). The immunoglobulin G (IgG), IgA, and IgM antibody levels to 16-kDa antigen were estimated in the two polar groups, namely, smear- and culture-positive pulmonary tuberculosis (S+C+) patients and healthy subjects (HS). Sensitivities of 62, 52 and 11% with specificities of 100, 97, and 95% were obtained for the three isotypes, respectively. The total number of positives by a combination of the three isotypes was analyzed in the polar groups, and the sensitivity improved to 83% with a specificity of 93%. Even when a combination of IgG and IgA alone was considered, the sensitivity was 82% with a specificity of 97%. Polyethylene glycol precipitation of the circulating immune complex (CIC) in sera was carried out. The CIC-bound antibodies to 16-kDa antigen were assessed by ELISA in the S+C+, S−C+, and S−C−categories of patients. Measuring the IgG-IgA-IgM combination positivities of the CIC-bound antibodies gave sensitivities of 97.5, 100, and 45.3%, respectively. The specificity of the assay with these combinations was maintained at 95.4%.
Performance Characteristics of the PolyTiter Immunofluorescent Titration System for Determination of Antinuclear Antibody Endpoint Dilution
Authors: Flessland Karen A. , Landicho Helen R. , Borden Kimberlee K. , Prince Harry E. 
Published: July 2002

ABSTRACT
Conventional screening for circulating antinuclear antibodies (ANA) is generally performed by immunofluorescent (IF) microscopy with a 1:40 dilution of serum. Intensity of IF staining is then semiquantitated by using twofold serial dilutions, where the highest dilution in which staining intensity equals the endpoint control is expressed as an endpoint titer. The PolyTiter Immunofluorescent Titration system (Polymedco, Inc.) facilitates ANA-IF assay (IFA) testing by relating the intensity of IF staining to reference calibrators (defined in PolyTiter units), providing an endpoint titer directly from a 1:40 dilution. This study was conducted to assess the performance characteristics of the PolyTiter system. Two technologists each evaluated 10 replicates of three specimens and two controls on five sequential days. Endpoint dilution agreement (defined as ±2 dilutions) with the reference was 100% for all controls and for all specimens by one technologist. The second reader reported agreement of 98, 88, and 100% for the low, medium, and high specimens, respectively. Analysis of PolyTiter unit values yielded between-reader, between-run, and within-run precision coefficients of variation of less than 10%. The variance component in the lot-to-lot analysis was zero, indicating all of the variation was due to run-to-run differences. Overall endpoint dilution agreement between PolyTiter and serial dilution in the evaluation of 125 specimens at three sites was 90, 93, and 86%. Pattern identification with the PolyTiter was similar to that with serial dilution. The PolyTiter system demonstrates acceptable performance for routine ANA-IFA testing in the clinical laboratory.
Molecular Detection of Antimicrobial Resistance
Authors: Fluit Ad C. , Visser Maarten R. , Schmitz Franz-Josef 
Published: July 2002

SUMMARY
The determination of antimicrobial susceptibility of a clinical isolate, especially with increasing resistance, is often crucial for the optimal antimicrobial therapy of infected patients. Nucleic acid-based assays for the detection of resistance may offer advantages over phenotypic assays. Examples are the detection of the methicillin resistance-encoding mecA gene in staphylococci, rifampin resistance in Mycobacterium tuberculosis, and the spread of resistance determinants across the globe. However, molecular assays for the detection of resistance have a number of limitations. New resistance mechanisms may be missed, and in some cases the number of different genes makes generating an assay too costly to compete with phenotypic assays. In addition, proper quality control for molecular assays poses a problem for many laboratories, and this results in questionable results at best. The development of new molecular techniques, e.g., PCR using molecular beacons and DNA chips, expands the possibilities for monitoring resistance. Although molecular techniques for the detection of antimicrobial resistance clearly are winning a place in routine diagnostics, phenotypic assays are still the method of choice for most resistance determinations. In this review, we describe the applications of molecular techniques for the detection of antimicrobial resistance and the current state of the art.
Activation of Host Phospholipases C and D in Macrophages after Infection withListeria monocytogenes
Authors: Goldfine Howard , Wadsworth Sandra J. , Johnston Norah C. 
Published: July 2002

ABSTRACTInfection of the J774 murine macrophage-derived cell line withListeria monocytogenesresults in several elevations of intracellular calcium during the first 15 min of infection. These appear to result from the actions of secreted bacterial proteins, including phosphatidylinositol-specific phospholipase C (PI-PLC), a broad-range phospholipase C, and listeriolysin O (LLO) (S. J. Wadsworth and H. Goldfine, Infect. Immun. 67:1770–1778, 1999). We have measured hydrolysis of host PI and the activation of host polyphosphoinositide-specific PLC and host phospholipase D (PLD) during infection with wild-type and mutantL. monocytogenes. Elevated hydrolysis of host PI occurred within the first 10 min of infection and was dependent on both bacterial PI-PLC and LLO, both of which were required for the earliest elevations of intracellular calcium in the host cell. A more rapid hydrolysis of host PI was observed at 30 min after infection, at the time when wild-type bacteria have been internalized. Activation of host PLC, also occurred in the first 10 min of infection but was not dependent on the presence of bacterial PI-PLC. Similar observations were made in murine bone marrow-derived macrophages. In J774 cells, activation of host PLD was observed after 20 min of infection and was dependent on bacterial LLO. Mutants in the bacterial phospholipases produced levels of PLD activation similar to those produced by the wild type. Phorbol myristate acetate (PMA) also activated host PLD, while long-term treatment with PMA resulted in loss of the ability ofL. monocytogenesto activate host PLD, suggesting an involvement of protein kinase C (PKC) in the activation of PLD. Rottlerin, an inhibitor of PKC δ in J774 cells, also inhibited the activation of PLD, but hispidin, an inhibitor of PKC βI and βII, did not. Pretreatment of J774 cells with the PLD inhibitor, 2,3-diphosphoglycerate partially inhibited escape of the bacteria from the primary phagocytic vacuole.
Interaction ofMycobacterium aviumwith Human Monocyte-Derived Dendritic Cells
Authors: Mohagheghpour Nahid , van Vollenhoven Annika , Goodman Joseph , Bermudez Luiz E. 
Published: July 2002

ABSTRACTThe mechanism by which mycobacteria elicit class I-restricted T-cell responses remains undefined because these organisms have been shown to reside exclusively within membrane-bound vesicles in macrophages (Mφ), their primary host cells. We studied the interaction ofM. aviumwith dendritic cells (DC) because they are the most potent antigen-presenting cells and are abundant atM. aviuminfection sites. We observed that both DC and Mφ, generated from human peripheral blood monocytes by short-term culture, internalizedM. avium. The onset of programmed cell death and the percentage of apoptotic cells in infected DC and Mφ were comparable. However, following infection, DC secreted significantly larger amounts of interleukin-12, but not interleukin-1β, than infected autologous Mφ. Further analysis of infected cells showed that while phagosomes failed to acidify in bothM. avium-infected DC and Mφ, bacilli grew more slowly in DC. Electron microscopy studies revealed thatM. aviumresided within endocytic vacuoles in both cell types. The vacuolar membrane surrounding some bacilli in approximately 10% of the vacuoles in DC possessed several breaks. The importance of this finding will have to be addressed in future studies.
Disruption of the Gene Which Encodes a Serodiagnostic Antigen and Chitinase of the Human Fungal Pathogen
Coccidioides immitis
Authors: Reichard Utz , Hung Chiung-Yu , Thomas Pei W. , Cole Garry T. 
Published: July 2002

ABSTRACT

Disruption of genes in medically important fungi has proved to be a powerful tool for evaluation of putative virulence factors and identification of potential protein targets for novel antifungal drugs. Chitinase has been suggested to play a pivotal role in autolysis of the parasitic cell wall of
Coccidioides immitis
during the asexual reproductive cycle (endosporulation) of this systemic pathogen. Two chitinase genes (
CTS1
and
CTS2
) of
C. immitis
have been cloned. Preliminary evidence has suggested that expression of
CTS1
is markedly increased during endospore formation. The secreted CTS1 chitinase has also been shown to react with patient anti-
Coccidioides
complement-fixing (CF) antibody and is a valuable aid in the serodiagnosis of coccidioidomycosis. To examine the role of CTS1 in the morphogenesis of parasitic cells, the
CTS1
gene was disrupted by a single, locus-specific crossover event. This resulted in homologous integration of a pAN7.1 plasmid construct that contained a 1.1-kb fragment of the chitinase gene into the chromosomal DNA of
C. immitis
. Results of Southern hybridizations, immunoblot analyses of culture filtrates using both CTS1-specific murine antiserum and serum from a patient with confirmed coccidioidal infection, an immunodiffusion test for CF antigenicity, and substrate gel electrophoresis assays of chitinase activity confirmed that the
CTS1
gene was disrupted and nonfunctional. This is the first report of a successful targeted gene disruption in
C. immitis
. However, loss of
CTS1
function had no effect on virulence or endosporulation. Comparative assays of chitinase activity in the parental and Δ
cts1
strains suggested that the absence of a functional
CTS1
gene can be compensated for by elevated expression of the
CTS2
gene. Current investigations are focused on disruption of
CTS2
in the Δ
cts1
host to further evaluate the significance of chitinase activity in the parasitic cycle of
C. immitis
.
Transcriptional Activation of the
htrA
(High-Temperature Requirement A) Gene from
Bartonella henselae
Authors: Resto-Ruiz Sandra I. , Sweger Debra , Widen Raymond H. , Valkov Nikola , Anderson Burt E. 
Published: July 2002

ABSTRACT

Bacterial
htrA
genes are typically activated as part of the periplasmic stress response and are dependent on the extracytoplasmic sigma factor
rpoE
. A putative promoter region, P1, of the ς
E
-type heat-inducible promoters has previously been identified upstream of the
htrA
gene of
Bartonella henselae
. Further analysis of the
htrA
mRNA by primer extension demonstrated that transcription initiates from P1 and a second region downstream of P1. This second promoter region, termed P2, had no sequence identity to ς
E
-type heat-inducible promoters. Promoter regions were cloned individually and in tandem into pANT3 upstream of a promoterless version of the green fluorescent protein (GFP) gene (
gfpmut3
) and transformed into
B. henselae
by electroporation. The contiguous promoter region containing both P1 and P2 were necessary for the optimal transcriptional activation of the
htrA
gene. Promoter activity at 37°C was distinctively higher than at 27°C. However, thermal induction at 47°C did not increase expression of
gfpmut3
. Invasion of human microvascular endothelial cells (HMEC-1) by
B. henselae
resulted in the formation of well-defined vacuoles containing clusters of bacteria exhibiting marked expression of
gfpmut3
transcribed from the P1-P2 region. In addition, a moderate yet significant increase in the ratio of bacterial GFP to DNA was detected for intracellular bacteria compared to extracellular bacteria, indicating upregulation of
htrA
upon invasion of HMEC-1. The activation of specific genes in the intracellular environment may help us better understand the novel pathogenic mechanisms used by this bacterium.
Interaction of Surfactant Protein A with Lipopolysaccharide and Regulation of Inflammatory Cytokines in the THP-1 Monocytic Cell Line
Authors: Song Mingchen , Phelps David S. 
Published: July 2002

ABSTRACTPulmonary surfactant protein A (SP-A) is involved in innate immunity in the lung. In this study we investigated the interaction of SP-A with different serotypes of lipopolysaccharide (LPS) on the regulation of inflammatory cytokines in vitro. In the human monocytic cell line, THP-1, combining SP-A with lipid A or rough LPS further enhanced lipid A- or rough LPS-stimulated tumor necrosis factor alpha (TNF-α) mRNA levels, while SP-A-elicited increases in TNF-α mRNA levels were partially neutralized. In contrast, the combination of smooth LPS and SP-A resulted in additive effects on TNF-α mRNA levels. We also demonstrated that there was cross-tolerance between SP-A and LPS in THP-1 cells. Pretreatment of THP-1 cells with LPS modestly inhibited the response of these cells to subsequent challenge with SP-A, with regard to the production of TNF-α, whereas there was no or little effect on the production of interleukin-1β (IL-1β) and IL-8. Conversely, pretreatment of THP-1 cells with SP-A markedly increased the response to subsequent challenge with LPS with regard to the production of IL-1β and IL-8, although the production of TNF-α was modestly decreased. However, a synergistic stimulatory effect was observed when the two agents were added simultaneously to the cells. NF-κB formation was downregulated in SP-A- but not in LPS-induced tolerant cells. These results suggested that SP-A exhibits different interactions with distinct serotypes of LPS. In addition, SP-A is different from LPS with regard to the induction of cross-tolerance, and these actions may be mediated, at least in part, through different mechanisms.
Nucleotide Sequence Analysis of Hypervariable Junctions of
Haemophilus influenzae
Pilus Gene Clusters
Authors: Read Timothy D. , Satola Sarah W. , Farley Monica M. 
Published: July 2002

ABSTRACT

Haemophilus influenzae
pili are surface structures that promote attachment to human epithelial cells. The five genes that encode pili,
hifABCDE
, are found inserted in genomes either between
pmbA
and
hpt
(
hif-1
) or between
purE
and
pepN
(
hif-2
). We determined the sequence between the ends of the pilus clusters and bordering genes in a number of
H. influenzae
strains. The junctions of the
hif-1
cluster (limited to biogroup aegyptius isolates) are structurally simple. In contrast,
hif-2
junctions are highly diverse, complex assemblies of conserved intergenic sequences (including genes
hicA
and
hicB
) with evidence of frequent recombination. Variation at
hif-2
junctions seems to be tied to multiple copies of a 23-bp
Haemophilus
intergenic dyad sequence. The
hif-1
cluster appears to have originated in biogroup aegyptius strains from invasion of the
hpt-pmbA
region by a DNA template containing the
hif-2
genes with termini in the hairpin loop of flanking intergenic dyad sequences. The pilus gene clusters are an interesting model of a mobile “pathogenicity island” not associated with a phage, transposon, or insertion element.