bluuebunny/crossref_metadata_2025 huggingface dataset:
Ecological Aspects of
ntcA
Gene Expression and Its Use as an Indicator of the Nitrogen Status of Marine
Synechococcus
spp
Authors: Lindell Debbie , Post Anton F.
Published: July 2002

Nitrogen nutrition in cyanobacteria is regulated by NtcA, a transcriptional activator that is subject to negative control by ammonium. Using
Synechococcus
sp. strain WH7803 as a model organism, we show that
ntcA
expression was induced when cells were exposed to nitrogen stress but not when they were subjected to phosphorus or iron deprivation. Transcript levels accumulated in cells grown on a variety of inorganic and organic nitrogen sources, with the sole exception of ammonium.
ntcA
transcription was induced when ammonium levels dropped below 1 μM and reached maximal levels within 2 h. Furthermore, the addition of more than 1 μM ammonium led to a rapid decline in
ntcA
mRNA. The negative effect of ammonium was prevented by the addition of
l
-methionine-
d
,
l
-sulfoximine (MSX) and azaserine, inhibitors of ammonium assimilation. Thus, basal
ntcA
transcript levels are indicative of ammonium utilization. Conversely, the highest
ntcA
transcript levels were found in cells lacking a nitrogen source capable of supporting growth. Therefore, maximal
ntcA
expression would indicate nitrogen deprivation. This state of nitrogen deprivation was induced by a 1-h incubation with MSX. The rapid response of
ntcA
gene expression to the addition of ammonium and MSX was used to design a protocol for assessing relative
ntcA
transcript levels in field populations of cyanobacteria, from which their nitrogen status can be inferred.
ntcA
was basally expressed in
Synechococcus
at a nutrient-enriched site at the northern tip of the Gulf of Aqaba, Red Sea. Therefore, these cyanobacteria were not nitrogen stressed, and their nitrogen requirements were met by regenerated nitrogen in the form of ammonium.
The
P450-4
Gene of
Gibberella fujikuroi
Encodes
ent
-Kaurene Oxidase in the Gibberellin Biosynthesis Pathway
Authors: Tudzynski Bettina , Hedden Peter , Carrera Esther , Gaskin Paul
Published: July 2002

At least five genes of the gibberellin (GA) biosynthesis pathway are clustered on chromosome 4 of
Gibberella fujikuroi
; these genes encode the bifunctional
ent
-copalyl diphosphate synthase/
ent
-kaurene synthase, a GA-specific geranylgeranyl diphosphate synthase, and three cytochrome P450 monooxygenases. We now describe a fourth cytochrome P450 monooxygenase gene (
P450-4
). Gas chromatography-mass spectrometry analysis of extracts of mycelia and culture fluid of a
P450-4
knockout mutant identified
ent
-kaurene as the only intermediate of the GA pathway. Incubations with radiolabeled precursors showed that the metabolism of
ent
-kaurene,
ent
-kaurenol, and
ent
-kaurenal was blocked in the transformants, whereas
ent
-kaurenoic acid was metabolized efficiently to GA
4
. The GA-deficient mutant strain SG139, which lacks the 30-kb GA biosynthesis gene cluster, converted
ent
-kaurene to
ent
-kaurenoic acid after transformation with
P450-4
. The B1-41a mutant, described as blocked between
ent
-kaurenal and
ent
-kaurenoic acid, was fully complemented by
P450-4
. There is a single nucleotide difference between the sequence of the B1-41a and wild-type
P450-4
alleles at the 3′ consensus sequence of intron 2 in the mutant, resulting in reduced levels of active protein due to a splicing defect in the mutant. These data suggest that
P450-4
encodes a multifunctional
ent
-kaurene oxidase catalyzing all three oxidation steps between
ent
-kaurene and
ent
-kaurenoic acid.
Identification and Characterization of a Chitinase Antigen from
Pseudomonas aeruginosa
Strain 385
Authors: Thompson Suzanne E. , Smith Mark , Wilkinson Mark C. , Peek Keith
Published: July 2002

A chitinase antigen has been identified in
Pseudomonas aeruginosa
strain 385 using sera from animals immunized with a whole-cell vaccine. The majority of the activity was shown to be in the cytoplasm, with some activity in the membrane fraction. The chitinase was not secreted into the culture medium. Purification of the enzyme was achieved by exploiting its binding to crab shell chitin. The purified enzyme had a molecular mass of 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 5.2. NH
2
-terminal amino acid sequencing revealed two sequences of M(I/L)RID and (Q/M/V)AREDAAAAM that gave an exact match to sequences in a translated putative open reading frame from the
P. aeruginosa
genome. The chitinase was active against chitin azure, ethylene glycol chitin, and colloidal chitin. It did not display any lysozyme activity. Using synthetic 4-methylumbelliferyl chitin substrates, it was shown to be an endochitinase. The

K
m

and

k
cat

for 4-nitrophenyl-β-
d
-
N,N
′-diacetylchitobiose were 4.28 mM and 1.7 s
−1
respectively, and for 4-nitrophenyl-β-
d
-
N,N
′,
N
′′-triacetylchitotriose, they were 0.48 mM and 0.16 s
−1
respectively. The pH optimum was determined to be pH 6.75, and 90% activity was maintained over the pH range 6.5 to 7.1. The enzyme was stable over the pH range 5 to 10 for 3 h and to temperatures up to 50°C for 30 min. The chitinase bound strongly to chitin, chitin azure, colloidal chitin, lichenan, and cellulose but poorly to chitosan, xylan, and heparin. It is suggested that the chitinase functions primarily as a chitobiosidase, removing chitobiose from the nonreducing ends of chitin and chitin oligosaccharides.
Toxic Effects of Ag(I) and Hg(II) onCandida albicansandC. maltosa: a Flow Cytometric Evaluation
Authors: Zhang Shangtong , Crow Sidney A.
Published: July 2002

The effects of Ag(I) and Hg(II) on membrane potential and integrity of cells ofCandida albicansandC. maltosawere determined with a flow cytometric procedure that employed an anionic membrane potential-sensitive dye, bis-(1,3-dibutylbarbituric acid) trimethine oxonol, and a membrane integrity indicator, propidium iodide. The membrane potentials of cells of both species were reduced rapidly within 15 min of exposure to Ag(I). No threshold dose for Hg(II) existed, and cells of both species lost membrane potential gradually in Hg(II) solutions. Cells of both species lost membrane integrity more rapidly in Ag(I) solutions than in Hg(II) solutions. In Ag(I) solutions, the decrease in the numbers of cells recoverable in culture occurred at a rate similar to the rate of cell depolarization and membrane permeabilization. In Hg(II) solutions, loss of cell recoverability preceded the loss of membrane potential and membrane integrity. C. albicans, in contrast toC. maltosa, showed no loss of membrane integrity after exposure to Hg(II) solutions for 1 h. Different rates of binding of Ag(I) and Hg(II) between the two species suggest that the two ions target different primary sites.
Incidence of Virulence Factors and Antibiotic Resistance among Enterococci Isolated from Food
Authors: Franz Charles M. A. P. , Muscholl-Silberhorn Albrecht B. , Yousif Nuha M. K. , Vancanneyt Marc , Swings Jean , Holzapfel Wilhelm H.
Published: July 2002

The incidence of virulence factors among 48
Enterococcus faecium
and 47
Enterococcus faecalis
strains from foods and their antibiotic susceptibility were investigated. No strain was resistant to all antibiotics, and for some strains, multiple resistances were observed. Of
E. faecium
strains, 10.4% were positive for one or more virulence determinants, compared to 78.7% of
E. faecalis
strains. Strains exhibiting virulence traits were not necessarily positive for all traits; thus, the incidence of virulence factors may be considered to be strain specific.
Molecular Monitoring of Succession of Bacterial Communities in Human Neonates
Authors: Favier Christine F. , Vaughan Elaine E. , De Vos Willem M. , Akkermans Antoon D. L.
Published: July 2002

The establishment of bacterial communities in two healthy babies was examined for more than the first 10 months of life by monitoring 16S ribosomal DNA (rDNA) diversity in fecal samples by PCR and denaturing gradient gel electrophoresis (DGGE) and by analyzing the sequences of the major ribotypes. DGGE profiles of the dominant populations in the intestines of the infants were obtained by analyzing daily or weekly fecal samples. After delivery, the germfree infant gastrointestinal tracts were rapidly colonized, and the succession of bacteria in each ecosystem was monitored. During the first few days of life the profiles were simple, but they became more complex as the bacterial diversity increased with time in both babies. Clone libraries of amplified 16S rDNA fragments from baby feces were constructed, and these libraries allowed identification of the bacterial types by comparative DNA sequence analysis; the bacteria identified included members of the generaBifidobacterium,Ruminococcus,Enterococcus,Clostridium, andEnterobacter. Species most closely related to the generaBifidobacteriumandRuminococcusin particular dominated the intestinal microbiota based on the stability over time and the numbers, as estimated by the intensities of the bands. However, 19 of the 34 cloned rDNA sequences exhibited less than 97% identity with sequences of known bacteria or cloned sequences in databases. This study showed that using PCR-DGGE and 16S rDNA sequence analysis together resulted in a dynamic description of bacterial colonization in the infant intestinal ecosystem and allowed visualization of bacteria that are difficult to cultivate or to detect by other methods.
Functional Genomics Approach to Identifying Genes Required for Biofilm Development by
Streptococcus mutans
Authors: Wen Zezhang T. , Burne Robert A.
Published: July 2002

Streptococcus mutans
, the primary etiological agent of human dental caries, is an obligate biofilm-forming bacterium. The goals of this study were to identify the gene(s) required for biofilm formation by this organism and to elucidate the role(s) that some of the known global regulators of gene expression play in controlling biofilm formation. In
S. mutans
UA159, the
brpA
gene (for biofilm regulatory protein) was found to encode a novel protein of 406 amino acid residues. A strain carrying an insertionally inactivated copy of
brpA
formed longer chains than did the parental strain, aggregated in liquid culture, and was unable to form biofilms as shown by an in vitro biofilm assay. A putative homologue of the enzyme responsible for synthesis of autoinducer II (AI-2) of the bacterial quorum-sensing system was also identified in
S. mutans
UA159, but insertional inactivation of the gene (
luxS
Sm
) did not alter colony or cell morphology or diminish the capacity of
S. mutans
to form biofilms. We also examined the role of the homologue of the
Bacillus subtilis
catabolite control protein CcpA in
S. mutans
in biofilm formation, and the results showed that loss of CcpA resulted in about a 60% decrease in the ability to form biofilms on an abiotic surface. From these data, we conclude that CcpA and BrpA may regulate genes that are required for stable biofilm formation by
S. mutans
.
Reversal of Flagellar Rotation Is Important in Initial Attachment of
Escherichia coli
to Glass in a Dynamic System with High- and Low-Ionic-Strength Buffers
Authors: McClaine Jennifer W. , Ford Roseanne M.
Published: July 2002

The attachment rates of wild-type, smooth-swimming, tumbly, and paralyzed
Escherichia coli
to glass was measured at fluid velocities of 0.0044 and 0.044 cms
−1
(corresponding to shear rates of 0.34 and 3.4 s
−1
, respectively), in 0.02 and 0.2 M buffer solutions. At the highest ionic strength, we did not observe a significant difference in the attachment rate of wild-type and paralyzed cells at either fluid velocity. However, when the ionic strength was reduced, paralyzed bacteria attached at rates 4 and 10 times lower than that of the wild type under fluid velocities of 0.0044 and 0.044 cms
−1
, respectively. This suggested that the rotation of the flagella assisted in attachment. We then compared the attachment rates of smooth-swimming (counterclockwise rotation only) and tumbly (clockwise rotation only) cells to the wild type to determine whether the direction of rotation was important to cell attachment. At 0.0044 cms
−1
, the smooth-swimming cells attached at rates similar to that of the wild type in both buffer solutions but significantly less at the higher fluid velocity. Tumbly cells attached at much lower rates under all conditions. Thus, the combination of clockwise and counterclockwise flagellar rotation and their coupling appeared to be important in cell attachment. We considered a number of hypotheses to interpret these observations, including a residence time analysis and a comparison of traditional Derjaguin-Landau-Verwey-Overbeek (DLVO) theory to soft-particle theory.
Response of Endophytic Bacterial Communities in Potato Plants to Infection with
Erwinia carotovora
subsp.
atroseptica
Authors: Reiter Birgit , Pfeifer Ulrike , Schwab Helmut , Sessitsch Angela
Published: July 2002

The term endophyte refers to interior colonization of plants by microorganisms that do not have pathogenic effects on their hosts, and various endophytes have been found to play important roles in plant vitality. In this study, cultivation-independent terminal restriction fragment length polymorphism analysis of 16S ribosomal DNA directly amplified from plant tissue DNA was used in combination with molecular characterization of isolates to examine the influence of plant stress, achieved by infection with the blackleg pathogen
Erwinia carotovora
subsp.
atroseptica
, on the endophytic population in two different potato varieties. Community analysis clearly demonstrated increased bacterial diversity in infected plants compared to that in control plants. The results also indicated that the pathogen stress had a greater impact on the bacteria population than the plant genotype had. Partial sequencing of the 16S rRNA genes of isolated endophytes revealed a broad phylogenetic spectrum of bacteria, including members of the α, β, and γ subgroups of the
Proteobacteria
, high- and low-G+C-content gram-positive organisms, and microbes belonging to the
Flexibacter-Cytophaga-Bacteroides
group. Screening of the isolates for antagonistic activity against
E. carotovora
subsp.
atroseptica
revealed that 38% of the endophytes protected tissue culture plants from blackleg disease.
Extensive Profiling of a Complex Microbial Community by High-Throughput Sequencing
Authors: Hill Janet E. , Seipp Robyn P. , Betts Martin , Hawkins Lindsay , Van Kessel Andrew G. , Crosby William L. , Hemmingsen Sean M.
Published: July 2002

Complex microbial communities remain poorly characterized despite their ubiquity and importance to human and animal health, agriculture, and industry. Attempts to describe microbial communities by either traditional microbiological methods or molecular methods have been limited in both scale and precision. The availability of genomics technologies offers an unprecedented opportunity to conduct more comprehensive characterizations of microbial communities. Here we describe the application of an established molecular diagnostic method based on the chaperonin-60 sequence, in combination with high-throughput sequencing, to the profiling of a microbial community: the pig intestinal microbial community. Four libraries of cloned
cpn60
sequences were generated by two genomic DNA extraction procedures in combination with two PCR protocols. A total of 1,125 cloned
cpn60
sequences from the four libraries were sequenced. Among the 1,125 cloned
cpn60
sequences, we identified 398 different nucleotide sequences encoding 280 unique peptide sequences. Pairwise comparisons of the 398 unique nucleotide sequences revealed a high degree of sequence diversity within the library. Identification of the likely taxonomic origins of cloned sequences ranged from imprecise, with clones assigned to a taxonomic subclass, to precise, for cloned sequences with 100% DNA sequence identity with a species in our reference database. The compositions of the four libraries were compared and differences related to library construction parameters were observed. Our results indicate that this method is an alternative to 16S rRNA sequence-based studies which can be scaled up for the purpose of performing a potentially comprehensive assessment of a given microbial community or for comparative studies.
Preservation of Lymphocyte Immunophenotype and Proliferative Responses in Cryopreserved Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus Type 1-Infected Donors: Implications for Multicenter Clinical Trials
Authors: Reimann Keith A. , Chernoff Miriam , Wilkening Cynthia L. , Nickerson Christine E. , Landay Alan L. ,   The ACTG Immunology Advanced Technology Laboratories
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) infection results in impaired immune function that can be measured by changes in immunophenotypically defined lymphocyte subsets and other in vitro functional assays. These in vitro assays may also serve as early indicators of efficacy when new therapeutic strategies for HIV-1 infection are being evaluated. However, the use of in vitro assays of immune function in multicenter clinical trials has been hindered by their need to be performed on fresh specimens. We assessed the feasibility of using cryopreserved peripheral blood mononuclear cells (PBMC) for lymphocyte immunophenotyping and for lymphocyte proliferation at nine laboratories. In HIV-1-infected patients with moderate CD4
+
lymphocyte loss, the procedures of density gradient isolation, cryopreservation, and thawing of PBMC resulted in significant loss of CD19
+
B cells but no measurable loss of total T cells or CD4
+
or CD8
+
T cells. No significant changes were seen in CD28
−
CD95
+
lymphocytes after cell isolation and cryopreservation. However, small decreases in HLA-DR
+
CD38
+
lymphocytes and of CD45RA
+
CD62L
+
were observed within both the CD4
+
and CD8
+
subsets. Fewer than 10% of those specimens that showed positive PBMC proliferative responses to mitogens or microbial antigens lost their responsiveness after cryopreservation. These results support the feasibility of cryopreserving PBMC for immunophenotyping and functional testing in multicenter AIDS clinical trials. However, small changes in selected lymphocyte subsets that may occur after PBMC isolation and cryopreservation will need to be assessed and considered in the design of each clinical trial.
Development of the Antinuclear and Anticytoplasmic Antibody Consensus Panel by the Association of Medical Laboratory Immunologists
Authors: James Karen , Carpenter A. Betts , Cook Linda , Marchand Richard , Nakamura Robert M.
Published: July 2002

The Association of Medical Laboratory Immunologists (AMLI) have developed a panel of antinuclear and anticytoplasmic antibody consensus sera that can be useful for enzyme immunoassay (EIA), Ouchterlony, and immunofluorescence assay methods. It was developed to assist in the evaluation of newly available EIA methods for the detection of autoantibodies. The panel of sera was evaluated in several clinical laboratories and a large number of laboratories owned by manufacturers of clinical autoantibody testing kits. The majority of sera performed well for the EIAs in both the clinical laboratories and the manufacturers' laboratories, but some samples had discrepant results. A major source of discrepancy is the current inability of the EIA results to be directly compared in a quantitative way as no standardization exists. The evaluation demonstrated lower sensitivity of detection by the Ouchterlony method. The limited evaluation of the sera with immunoblotting and Western blotting did not show good agreement with other methods. Further work must be done to standardize blotting methods prior to their use in routine clinical testing. The sera are now available to vendors and clinical laboratories for use in the detection of SS-A, SS-B, Sm, U1-RNP, Scl-70, Jo-1, double-stranded DNA, and centromere antibodies. The availability of the consensus sera will help evaluate and improve the EIA methods currently being used.
Comparison of Commercial Latex Agglutination and Sandwich Enzyme Immunoassays with a Competitive Binding Inhibition Enzyme Immunoassay for Detection of Antigenemia and Antigenuria in a Rabbit Model of Invasive Aspergillosis
Authors: Hurst Steven F. , Reyes Guadalupe H. , McLaughlin David W. , Reiss Errol , Morrison Christine J.
Published: July 2002

A commercial latex agglutination assay (LA) and a sandwich enzyme immunoassay (SEIA) (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) were compared with a competitive binding inhibition assay (enzyme immunoassay [EIA]) to determine the potential uses and limitations of these antigen detection tests for the sensitive, specific, and rapid diagnosis of invasive aspergillosis (IA). Toward this end, well-characterized serum and urine specimens were obtained by using a rabbit model of IA. Serially collected serum or urine specimens were obtained daily from control rabbits or from rabbits immunosuppressed and infected systemically with
Aspergillus fumigatus
. By 4 days after infection, EIA, LA, and SEIA detected antigen in the sera of 93, 93, and 100% of
A. fumigatus
-infected rabbits, respectively, whereas antigen was detected in the urine of 93, 100, and 100% of the rabbits, respectively. False-positive results for non-
A. fumigatus
-infected rabbits for EIA, LA, and SEIA were as follows: for serum, 14, 11, and 23%, respectively; for urine, 14, 84, and 90%, respectively. Therefore, although the sensitivities of all three tests were similar, the specificity was generally greater for EIA than for LA or SEIA. Infection was also detected earlier by EIA, by which the serum of 53% of
A. fumigatus
-infected rabbits was positive as early as 1 day after infection, whereas the serum of only 27% of the rabbits tested by LA was positive. Although the serum of 92% of
A. fumigatus
-infected rabbits was positive by SEIA as early as 1 day after infection, the serum of a high percentage (50%) was false positive before infection. The urine of 21% of
A. fumigatus
-infected rabbits was positive by EIA as early as 1 day after infection, and the urine of none of the rabbits was false positive before infection. When EIA results for urine specimens were combined with those for serum, sensitivity was improved (i.e., 67% of rabbits were positive by 1 day after infection and only one rabbit gave a false-positive result). A total of 93% of
A. fumigatus
-infected rabbits were positive for antigen in urine as early as 1 day after infection and the urine of 100% of the rabbits was positive by SEIA. However, before infection, 79% of
A. fumigatus
-infected rabbits were false positive for antigen in urine by LA and 90% were false positive for antigen in urine by SEIA. These data indicate that the EIA has the potential to be used to diagnose IA with both serum and urine specimens and to detect a greater number of infections earlier with greater specificity than the specificities achieved with the commercial tests.
Differentiation of
Bartonella
Species by a Microimmunofluorescence Assay, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, and Western Immunoblotting
Authors: Liang Zhongxing , Raoult Didier
Published: July 2002

Bartonella
species can be differentiated by microimmunofluorescence assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting with murine polyclonal antisera to
Bartonella henselae
,
B. quintana
,
B. elizabethae
, and
B. bacilliformis
. A pairwise comparison on the basis of SDS-PAGE protein profiles demonstrated similarity values for proteins of different
Bartonella
species ranging from 28.6 to 86.4%. Antigenic relationships revealed by immunoblotting with murine antisera were equivalent to those of proteins observed by SDS-PAGE. A dendrogram obtained on the basis of protein bands of SDS-polyacrylamide gels showed that
Bartonella
species could be divided into three groups.
B. bacilliformis
was distinct from all other
Bartonella
species;
B. grahamii
,
B. taylorii
,
B. doshiae
, and
B. vinsonii
formed a cluster, as did
B. henselae
,
B. quintana
,
B. elizabethae
, and
B. clarridgeiae
. These relationships were consistent with those revealed by parsimony trees derived from 16S rRNA and
gltA
gene sequencing. SDS-PAGE analysis showed that 120-, 104-, 85-, 71-, 54-, 47-, 40-, 33-, 30-, and 19-kDa proteins were present in all species, with the 54-kDa protein being the most dominant. Proteins with a molecular mass of less than 54 kDa allow the differentiation of species and are a possible target for future species-specific antibodies and antigens.
Identifying Diagnostic Peptides for Lyme Disease through Epitope Discovery
Authors: Kouzmitcheva Galina A. , Petrenko Valery A. , Smith George P.
Published: July 2002

Serum antibodies from patients with Lyme disease (LD) were used to affinity select peptide epitopes from 12 large random peptide libraries in phage display format. The selected peptides were surveyed for reactivity with a panel of positive sera (from LD patients) and negative sera (from subjects without LD), thus identifying 17 peptides with a diagnostically useful binding pattern: reactivity with at least three positive sera and no reactivity with any of the negative sera. The peptides define eight sequence motifs, none of which can be matched convincingly with segments of proteins fromBorrelia burgdorferi, the LD pathogen; evidently, then, they are “mimotopes,” mimicking natural pathogen epitopes without matching contiguous amino acids of pathogen proteins. Peptides like these could be the basis of a new diagnostic enzyme-linked immunosorbent assay for LD, with sufficient specificity and sensitivity to replace expensive immunoblotting tests that are currently required for definitive serological diagnosis. Moreover, the method used to discover these peptides did not require any knowledge of the pathogen and involved generic procedures that are applicable to almost any infectious disease, including emerging diseases for which no pathogen has yet been identified.
Lateral-Flow Assay for Rapid Serodiagnosis of Human Leptospirosis
Authors: Smits Henk L. , Eapen C. K. , Sugathan Sheela , Kuriakose Mariamma , Gasem M. Hussein , Yersin Claude , Sasaki David , Pujianto Bambang , Vestering Marc , Abdoel Theresia H. , Gussenhoven George C.
Published: July 2002

An assay device for the rapid detection of
Leptospira
-specific immunoglobulin M (IgM) antibodies in human sera is presented. The sensitivity (85.8%) and specificity (93.6%) of the assay compared well (91.9% agreement) with those of an IgM enzyme-linked immunosorbent assay routinely used in the serodiagnosis of leptospirosis. The sensitivity of the assay varied with the stage of the disease. The assay uses stabilized components and is simply performed by the addition of serum and sample fluid to the sample well of the assay device. The assay is read after 10 min, and a positive result is obtained when staining of the test line is observed.
Early Diagnosis of Scrub Typhus with a Rapid Flow Assay Using Recombinant Major Outer Membrane Protein Antigen (r56) of
Orientia tsutsugamushi
Authors: Ching W.-M. , Rowland D. , Zhang Z. , Bourgeois A. L. , Kelly D. , Dasch G. A. , Devine P. L.
Published: July 2002

The variable 56-kDa major outer membrane protein of
Orientia tsutsugamushi
is the immunodominant antigen in human scrub typhus infections. We developed a rapid immunochromatographic flow assay (RFA) for the detection of immunoglobulin M (IgM) and IgG antibodies to
O. tsutsugamushi
. The RFA employs a truncated recombinant 56-kDa protein from the Karp strain as the antigen. The performance of the RFA was evaluated with a panel of 321 sera (serial bleedings of 85 individuals suspected of scrub typhus) which were collected in the Pescadore Islands, Taiwan, from 1976 to 1977. Among these 85 individuals, IgM tests were negative for 7 cases by both RFA and indirect fluorescence assay (IFA) using Karp whole-cell antigen. In 29 cases specific responses were detected by the RFA earlier than by IFA, 44 cases had the same detection time, and 5 cases were detected earlier by IFA than by RFA. For IgG responses, 4 individuals were negative with both methods, 37 cases exhibited earlier detection by RFA than IFA, 42 cases were detected at the same time, and 2 cases were detected earlier by IFA than by RFA. The sensitivities of RFA detection of antibody in sera from confirmed cases were 74 and 86% for IgM and IgG, respectively. When IgM and IgG results were combined, the sensitivity was 89%. A panel of 78 individual sera collected from patients with no evidence of scrub typhus was used to evaluate the specificity of the RFA. The specificities of the RFA were 99% for IgM and 97% for IgG. The sensitivities of IFA were 53 and 73% for IgM and IgG, respectively, and were 78% when the results of IgM and IgG were combined. The RFA test was significantly better than the IFA test for the early detection of antibody to scrub typhus in primary infections, while both tests were equally sensitive with reinfected individuals.
Posttreatment Follow-Up of
Helicobacter pylori
Infection Using a Stool Antigen Immunoassay
Authors: Roth Daniel E. , Taylor David N. , Gilman Robert H. , Meza Rina , Katz Uriel , Bautista Christian , Cabrera Lilia , Velapatiño Billie , Lebron Carlos , Razúri Manuel , Watanabe J. , Monath T. ,   The Gastrointestinal Physiology Working Group
Published: July 2002

The
Helicobacter pylori
stool antigen enzyme immunoassay (HpSA) was evaluated during posttreatment follow-up of patients in a country with a very high prevalence of
H. pylori
infection. From among 273 dyspeptic individuals (18 to 55 years) initially recruited from a shantytown in Lima, Peru, 238 participants who met the inclusion criteria and were suspected to be
H. pylori
positive based on
14
C urea breath test (UBT) results underwent endoscopy. Participants with endoscopy-proven infections received standard eradication therapy and were monitored by UBT and HpSA at 1 month following treatment and at 3-month intervals for 9 months posttreatment. A second endoscopy was performed if UBT results showed evidence of treatment failure or
H. pylori
recurrence. Biopsy results were considered the “gold standard” in all analyses. Among patients who underwent endoscopy, HpSA had a pretreatment sensitivity of 93%. Two-hundred thirty patients completed the treatment regimen, of whom 201 (93%) were considered to have had successful treatment outcomes based on a negative follow-up UBT. Thirty-two patients with UBT-defined treatment failures or
H. pylori
recurrences at any point during the 9-month follow-up underwent a second endoscopy. In the posttreatment setting, HpSA had an overall sensitivity of 73% and a specificity of 67%. Agreement between UBT and HpSA diminished throughout the follow-up. Among 14 participants in whom HpSA remained positive at 1 month following treatment despite UBT evidence of treatment success, 12 (86%) became HpSA negative within 3 months posttreatment. Although this study confirmed the validity of the HpSA in the initial assessment of dyspeptic patients, the test demonstrated a reduced overall accuracy in the detection of treatment failures and
H. pylori
recurrences during 9 months of posttreatment follow-up. Furthermore, in some patients it may take up to 3 months after successful eradication for antigen shedding to diminish to levels within the negative HpSA range.
Expression of Natural Peptide Antibiotics in Human Articular Cartilage and Synovial Membrane
Authors: Paulsen Friedrich , Pufe Thomas , Petersen Wolf , Tillmann Bernhard
Published: July 2002

In view of frequent present use of invasive procedures on limb joints, it is astonishing that articular joint inflammation is a rare event. We questioned whether antimicrobial peptides play a role in protecting human articular cartilage and synovial membrane against inflammatory agents. Our results implicate defensins in the protection of human articular joints against pathogens.
Quantification of Substance P mRNA in Human Immune Cells by Real-Time Reverse Transcriptase PCR Assay
Authors: Lai Jian-Ping , Douglas Steven D. , Shaheen Farida , Pleasure David E. , Ho Wen-Zhe
Published: July 2002

We have applied a newly developed real-time reverse transcriptase (RT) PCR (RT-PCR) assay for quantification of substance P (SP) mRNA expression (the SP real-time RT-PCR assay) in human blood monocyte-derived macrophages, peripheral blood lymphocytes, and microglia isolated from fetal brain. The SP real-time RT-PCR assay had a sensitivity of 60 mRNA copies, with a dynamic range of detection between 60 and 600,000 copies of the SP gene transcript per reaction mixture. The coefficient of variation of the threshold cycle number between the SP real-time RT-PCR assays was less than 1.16%. This assay with an SP-specific primer pair efficiently recognizes all four isoforms of preprotachykinin A (the SP precursor) gene transcripts. In order to use this assay to measure the levels of SP mRNA in the human immune cells quantitatively, we designed a specific probe (molecular beacon) derived from exon 3 of the SP gene. We demonstrated that the real-time RT-PCR quantitatively detected SP mRNA in the human immune cells, among which the microglia isolated from fetal brain had the highest levels of SP mRNA. The SP real-time PCR assay yielded reproducible data, as the intra-assay variation was less than 1%. Thus, it is feasible to apply the real-time RT-PCR assay for quantification of SP mRNA levels in human immune cells, as well as in other nonneuronal cells. Since SP is a major modulator of neuroimmunoregulation, this assay has the potential for widespread application for basic and clinical investigations.
Influence of Specimen Age and Use of Different Negative Controls in Determination of Intracytoplasmic Levels of Cytokines after Whole-Blood Culture Assay
Authors: Schultz Christian , Rott Christina , Temming Petra , von Puttkammer Julia , Bucsky Peter
Published: July 2002

Intracytoplasmic detection of cytokines by flow cytometry has become a powerful tool in the characterization of cytokine-producing cells. However, it is not known to what extent specimen age and the use of various negative controls may influence the amount of cytokine-positive cells. We therefore compared different times of storage and the use of several negative controls in the determination of intracytoplasmic levels of cytokines. There was a substantial decline of interleukin-2- and gamma interferon-positive lymphocytes after 20 h and especially after 48 h of storage. The precision of intracytoplasmic interleukin-6 determination decreases after long-term storage compared to 2 h of storage, whereas the amount of interleukin-8-positive monocytes remained rather stable. Therefore, we recommend performing the analysis as fast as possible after the blood sample is drawn. Under consideration of isotype-matched antibodies and nonstimulated cells as negative controls instead of the purified antibody-blocking control, strikingly higher amounts of interleukin-2-, gamma interferon, interleukin-6-, and interleukin-8-positive cells were found. For a meaningful interpretation of data these differences have to be kept in mind. Further studies should evaluate the exact specificity of these controls.
Predicting Kala-Azar Disease Manifestations in Asymptomatic Patients with LatentLeishmania donovaniInfection by Detection of Antibody against Recombinant K39 Antigen
Authors: Singh Sarman , Kumari Veena , Singh Niti
Published: July 2002

Clinically visceral leishmaniasis is suspected in only a fraction of infected persons, as the majority of these may not have clinical manifestations and remain asymptomatic. There is scanty information on diagnosing latent infections and predicting disease in asymptomatic persons. We therefore carried out a study on asymptomatic contacts of patients with visceral leishmaniasis and post-kala-azar dermal leishmaniasis by using methods for detection of antibody to recombinant K39 (rK39) antigen. A total of 240 patients with leishmaniasis and 150 asymptomatic contacts were tested for anti-rK39 immunoglobulin G (IgG) and IgA antibodies. Fifty-five asymptomatic persons were found to be seropositive. These individuals were monitored every 3 months for 1 year. On follow-up, 43.9% of the asymptomatic seropositive contacts developed kala-azar within the first 3 months, and a cumulative total of 69% developed kala-azar within 1 year. The rest remained asymptomatic and self-healed the infection. The sensitivity and specificity of rK39 enzyme-linked immunosorbent assay (ELISA) and dipstick tests were 100%, while an in-house-developed latex agglutination test had 80% sensitivity. The antibody profile showed that the IgG anti-rK39 antibodies reached a titer of up to 10−6within 6 months of infection, started declining thereafter, and completely disappeared in 2 to 3 years in successfully treated cases. Significant titers of IgA antibodies were detectable a little earlier than those of IgG antibodies and were undetectable after 6 months. The study showed that mass screening of family members and contacts by using anti-rK39 ELISA could be a highly reliable tool for early diagnosis and to plan prophylactic treatment of latently infected asymptomatic carriers to eradicate kala-azar.
Leishmaniasis: Current Status of Vaccine Development
Authors: Handman Emanuela
Published: July 2002

Leishmaniae are obligatory intracellular protozoa in mononuclear phagocytes. They cause a spectrum of diseases, ranging in severity from spontaneously healing skin lesions to fatal visceral disease. Worldwide, there are 2 million new cases each year and 1/10 of the world's population is at risk of infection. To date, there are no vaccines against leishmaniasis and control measures rely on chemotherapy to alleviate disease and on vector control to reduce transmission. However, a major vaccine development program aimed initially at cutaneous leishmaniasis is under way. Studies in animal models and humans are evaluating the potential of genetically modified live attenuated vaccines, as well as a variety of recombinant antigens or the DNA encoding them. The program also focuses on new adjuvants, including cytokines, and delivery systems to target the T helper type 1 immune responses required for the elimination of this intracellular organism. The availability, in the near future, of the DNA sequences of the human and Leishmania genomes will extend the vaccine program. New vaccine candidates such as parasite virulence factors will be identified. Host susceptibility genes will be mapped to allow the vaccine to be targeted to the population most in need of protection.
Phase Variation in H Type I and Lewis a Epitopes of
Helicobacter pylori
Lipopolysaccharide
Authors: Appelmelk Ben J. , Martino M. Celeste , Veenhof Eveline , Monteiro Mario A. , Maaskant Janneke J. , Negrini Riccardo , Lindh Frank , Perry Malcolm , Del Giudice Giuseppe , Vandenbroucke-Grauls Christina M. J. E.
Published: July 2002

Helicobacter pylori
NCTC 11637 lipopolysaccharide (LPS) expresses the human blood group antigens Lewis x (Le
x
), Le
y
, and H type I. In this report, we demonstrate that the H type I epitope displays high-frequency phase variation. One variant expressed Le
x
and Le
y
and no H type I as determined by serology; this switch was reversible. Insertional mutagenesis in NCTC 11637 of JHP563 (a poly(C) tract containing an open reading frame homologous to glycosyltransferases) yielded a transformant with a serotype similar to the phase variant. Structural analysis of the NCTC 11637 LPS confirmed the loss of the H type I epitope. Sequencing of JHP563 in strains NCTC 11637, an H type I-negative variant, and an H type I-positive switchback variant showed a C14 (gene on), C13 (gene off), and C14 tract, respectively. Inactivation of strain G27, which expresses Le
x
, Le
y
, H type I, and Le
a
, yielded a transformant that expressed Le
x
and Le
y
. We conclude that JHP563 encodes a β3-galactosyltransferase involved in the biosynthesis of H type I and Le
a
and that phase variation in H type I is due to C-tract changes in this gene. A second H type I-negative variant (variant 3a) expressed Le
x
and Le
a
and had lost both H type I and Le
y
expression. Inactivation of HP093-HP094 resulted in a transformant expressing Le
x
and lacking Le
y
and H type I. Structural analysis of a mutant LPS confirmed the serological data. We conclude that the HP093-HP094 α2-fucosyltransferase (α2-FucT) gene product is involved in the biosynthesis of both Le
y
and Le
x
. Finally, we inactivated HP0379 in strain 3a. The transformant had lost both Le
x
and Le
a
expression, which demonstrates that the HP0379 gene product is both an α3- and an α4-FucT. Our data provide understanding at the molecular level of how
H. pylori
is able to diversify in the host, a requirement likely essential for successful colonization and transmission.
ClpC ATPase Is Required for Cell Adhesion and Invasion of
Listeria monocytogenes
Authors: Nair Shamila , Milohanic Eliane , Berche Patrick
Published: July 2002

We studied the role of two members of the 100-kDa heat shock protein family, the ClpC and ClpE ATPases, in cell adhesion and invasion of the intracellular pathogen
Listeria monocytogenes
. During the early phase of infection, a
clpC
mutant failed to disseminate to hepatocytes in the livers of infected mice whereas the invasive capacity of a
clpE
mutant remained unchanged. This was confirmed by a confocal microscopy study on infected cultured hepatocyte and epithelial cell lines, showing a strong reduction of cell invasion only by the
clpC
mutant. Western blot analysis with specific antisera showed that the absence of ClpC, but not that of ClpE, reduced expression of the virulence factors InlA, InlB, and ActA. ClpC-dependent modulation of these factors occurs at the transcriptional level with a reduction in the transcription of
inlA
,
inlB
, and
actA
in the
clpC
mutant, in contrast to the
clpE
mutant. This work provides the first evidence that, in addition to promoting escape from the phagosomes, ClpC is required for adhesion and invasion and modulates the expression of InlA, InlB, and ActA, further supporting the major role of the Clp chaperones in the virulence of intracellular pathogens.
Effect of
Chlamydia trachomatis
Infection on Atherosclerosis in Apolipoprotein E-Deficient Mice
Authors: Blessing Erwin , Nagano Sanae , Campbell Lee Ann , Rosenfeld Michael E. , Kuo Cho-chou
Published: July 2002

We have previously demonstrated that
Chlamydia pneumoniae
accelerates plaque formation in apolipoprotein E-deficient (ApoE
−/−
) mice following intranasal inoculations. In this study, we evaluated the effect of respiratory tract infection with
Chlamydia trachomatis
on the progression of atherosclerosis in ApoE
−/−
mice. The study showed that in contrast to infection with
Chlamydia pneumoniae
, infection of the lung and aorta with
C. trachomatis
was mild and transient and did not significantly accelerate plaque development.
Requirement for CD4+T Lymphocytes in Host Resistance againstCryptococcus neoformansin the Central Nervous System of Immunized Mice
Authors: Buchanan Kent L. , Doyle Hester A.
Published: July 2002

The importance of cell-mediated immunity (CMI) and CD4+T lymphocytes in host resistance againstCryptococcus neoformansis well documented and is exemplified by the high susceptibility to progressive infection with this pathogen of AIDS patients with reduced CD4+T-cell numbers. Although much has been learned about the role of CMI in the clearance ofC. neoformansfrom the lungs and other internal organs, less is known about the protective mechanisms in the brain, the organ most frequently involved with a fatal outcome of cryptococcosis. We hypothesized that host resistance mechanisms againstC. neoformansin the central nervous system (CNS) were similar to those outside the CNS (i.e., gamma interferon [IFN-γ], CD4+T cells, and others). To test this hypothesis, we used a murine model of cryptococcal meningitis whereby cryptococci are introduced directly into the CNS. In experiments where mice were immunized to mount an anticryptococcal CMI response, our results indicate that immunization induced protective mechanisms that could be detected in the CNS by inhibition of the growth of viable yeast cells. Flow cytometric analyses of leukocytes in brain and spinal cord homogenates revealed that T lymphocytes, macrophages, and neutrophils accumulated inC. neoformans-infected brains of immune mice. In vivo depletion of CD4+T cells, but not CD8+T cells, resulted in significantly reduced leukocyte accumulation in the brains of immune mice. Furthermore, depletion of CD4+T cells or neutralization of IFN-γ exacerbated CNS infection in immune mice, suggesting a critical role for CMI mechanisms in acquired protection in the CNS.
Febrile Core Temperature Is Essential for Optimal Host Defense in Bacterial Peritonitis
Authors: Jiang Qinqqi , Cross Alan S. , Singh Ishwar S. , Chen T. Timothy , Viscardi Rose M. , Hasday Jeffrey D.
Published: July 2002

Fever, a nonspecific acute-phase response, has been associated with improved survival and shortened disease duration in infections, but the mechanisms of these beneficial responses are poorly understood. We previously reported that increasing core temperature of bacterial endotoxin (LPS)-challenged mice to the normal febrile range modified expression of tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), and IL-6, three cytokines critical to mounting an initial defense against microbial pathogens, but survival was not improved in the warmer animals. We speculated that our inability to show a survival benefit of optimized cytokine expression in the warmer animals reflected our use of LPS, a nonreplicating agonist, rather than an infection with viable pathogens. The objective of this study was to determine if increasing murine core temperature altered cytokine expression and improved survival in an experimental bacterial peritonitis model. We showed that housing mice at 35.5°C rather than 23°C increased core temperature from 36.5 to 37.5°C to 39.2 to 39.7°C, suppressed plasma TNF-α expression for the initial 48 h, delayed gamma interferon expression, improved survival, and reduced the bacterial load in mice infected withKlebsiella pneumoniaeperitonitis. We showed that the reduced bacterial load was not caused by a direct effect on bacterial proliferation and probably reflected enhanced host defense. These data suggest that the increase in core temperature that occurs during bacterial infections is essential for optimal antimicrobial host defense.