bluuebunny/crossref_metadata_2025 huggingface dataset:
Improved Repetitive-Element PCR Fingerprinting of
Salmonella enterica
with the Use of Extremely Elevated Annealing Temperatures
Authors: Johnson James R. , Clabots Connie
Published: July 2002

Modified thermal cycling conditions were explored in an effort to improve the reproducibility and resolving power of repetitive-element PCR (rep-PCR) fingerprinting. Assay performance was rigorously evaluated under standard and modified cycling conditions, using as a test set 12 strains putatively representing 12 serovars of
Salmonella enterica
. For all three fingerprint types (ERIC2, BOXA1R, and composite fingerprints), the use of extremely elevated annealing temperatures plus an initial “touchdown” cycling routine yielded significant improvements in day-to-day reproducibility and discriminating power despite the somewhat sparser appearance of the fingerprints. Modified cycling conditions markedly reduced the variability of fingerprints between cyclers, allowing fingerprints from different cyclers to be analyzed together without the degradation of assay performance that occurred with between-cycler analyses under standard cycling conditions. With modified cycling, composite fingerprints exhibited the lowest reproducibility but the highest net discriminating power of the three fingerprint types. rep-PCR fingerprints led to the discovery of a serotyping error involving one of the 12 test strains. These data demonstrate that modified cycling regimens that incorporate elevated annealing temperatures (with or without an initial touchdown routine) may markedly improve the performance of rep-PCR fingerprinting as a bacterial typing tool.
Relevance of the Viral RAK Alpha Gene in Diagnosis of Malignant Versus Nonmalignant Tumors of the Ovary and Uterus
Authors: Rakowicz-Szulczynska Eva M.
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1)-like antigens RAK (named after the inventor E. M. Rakowicz) p120, p42, and p25, as well as HIV-1-like segments of cancer DNA (RAK gene alpha), have been found before in breast and prostate cancers. The present study focused on determining the value of markers RAK in the diagnosis and prognosis of gynecological cancer. Expression of RAK antigens in ovarian, uterine, cervical, and vulvar cancer, in benign tumors, in tissues adjacent to cancer, and in normal tissues was tested by Western blot hybridization of the electrophoretically separated proteins with monoclonal antibody RAK BrI. The RAK alpha gene was PCR amplified with HIV-1-derived primers SK68 and SK69. RAK antigens p120, p42, and p25 were found in 95% of ovarian, uterine, and cervical cancer cases and in 75% of vulvar cancer cases. The RAK alpha gene was expressed in 100% of cancer cases, in approximately 25% of benign ovarian tumors, and in 40% of benign tumors of the uterus. DNA sequences amplified in all cancer cases exhibited more than 90% homology to HIV-1 gp41 and were encoded for the functional peptide. DNA sequences found in benign tumors contained frameshift mutations and encoded truncated or nonfunctional peptides. Such sequences have not been amplified in normal tissues. RAK antigens and the RAK alpha gene seem to belong to a lentivirus type that is highly related to HIV-1. Beyond the diagnostic value of RAK markers, future cloning of the full viral genome would lead to a better understanding of the etiology of malignant and nonmalignant tumors of reproductive organs and to the development of novel therapeutic approaches.
Development of an Indirect Tams1 Enzyme-Linked Immunosorbent Assay for Diagnosis ofTheileria annulataInfection in Cattle
Authors: Gubbels Marc-Jan , d'Oliveira Christine , Jongejan Frans
Published: July 2002

An enzyme-linked immunosorbent assay (ELISA) was developed based on a recombinant majorTheileria annulatamerozoite surface antigen, Tams1. Four different recombinant proteins derived from two different Tams1 alleles, both in two different truncated forms, were tested for their performance in the ELISA. Furthermore, antigen concentration, various buffers, washing protocol, and the choice of anti-total-immunoglobulin G (IgG), anti-IgG1, or anti-IgG2 as second antibody were evaluated. The performance of the resulting ELISA was analyzed by measuring the coefficient of variation (CV). A total of 22 sera were analyzed over the measurement range, resulting in a CV of ca. 10%, whereas 30% variation is the maximum acceptable. The cutoff value was determined by the two-graph receiver operating characteristic (TG-ROC), using the indirect fluorescent antibody test (IFAT) as a reference. It was shown that up to 3 months postinfection (p.i.) IFAT is more sensitive and specific, whereas beyond 3 months p.i. ELISA performed as well as IFAT. The cutoff was determined at maximal sensitivity, based on the TG-ROC after 3 months p.i. Nine calves experimentally infected with four differentT. annulatastocks remained positive in the ELISA for at least 1 year p.i. Finally, limited cross-reaction was found only withT. parvaantisera, but not with any otherTheileriaorBabesiaspecies. Since theT. parvaendemic area hardly overlaps withT. annulata, the Tams1 ELISA has the potential to become a useful tool in the epidemiology of tropical theileriosis.
Secretory Leukocyte Protease Inhibitor Inhibits Infection of Monocytes and Lymphocytes with Human Immunodeficiency Virus Type 1 but Does Not Interfere with Transcytosis of Cell-Associated Virus across Tight Epithelial Barriers
Authors: Hocini Hakim , Becquart Pierre , Bouhlal Hicham , Adle-Biassette Homa , Kazatchkine Michel D. , Bélec Laurent
Published: July 2002

In the present study, we demonstrate that recombinant human secretory leukocyte protease inhibitor (rhSLPI) inhibits infection of lymphocyte- and monocyte-derived tumor cell lines and peripheral blood lymphocytes with laboratory-adapted isolates and with the primary isolate, NDK, of free human immunodeficiency virus type 1 (HIV-1). In contrast, rhSLPI did not exhibit inhibitory activity toward transcytosis of cell-associated HIV-1 through a tight monolayer of endometrial epithelial cells. These observations indicate that the inhibitory effect of SLPI is restricted to free HIV-1 in corporal fluids.
Reactivity to p52 and CM2 Recombinant Proteins in Primary Human Cytomegalovirus Infection with a Microparticle Agglutination Assay
Authors: Nulens Eric , Bodéus Monique , Bonelli Fabrizio , Soleti Antonio , Goubau Patrick
Published: July 2002

We evaluated the reactivities of sera against p52 and CM2 recombinant antigens of human cytomegalovirus (HCMV), coated on microparticles, for the differentiation of primary HCMV infection from an established infection. Two different test formats of the CMV Multiplex Copalis assay were evaluated. The 214 serum samples tested were immunoglobulin M (IgM) positive or equivocal by our reference assay. Reactivities against p52 and CM2 antigens were tested for sera from 37 patients with a well-documented seroconversion within the preceding 3 months (119 serum specimens), 31 patients known to have had a seroconversion at least 8 months earlier (31 serum specimens), and 57 patients without a documented seroconversion (64 serum specimens). The assay had a sensitivity for the detection of a primary infection of 70 or 86% by the first test format and a sensitivity of 88 or 94% by the second test format, according to the criteria used to indicate a primary infection by this test. A good correlation of the results of the assay with our in-house avidity index was found. The specificity of the assay warrants further evaluation. With IgM-positive sera, the assay was not sufficiently specific to make a distinction between a primary infection and an established infection.
Need for an External Proficiency Testing Program for Cytokines, Chemokines, and Plasma Markers of Immune Activation
Authors: Fahey John L. , Aziz Najib , Spritzler John , Plaeger Susan , Nishanian Parunag , Lathey Janet L. , Seigel Joan , Landay Alan L. , Kilarui Rakhi , Schmitz John L. , White Carmen , Wara Diane W. , Akridge Robert , Cutili Joie , Douglas Steven D. , Reuben James , Shearer William T. , Nokta Mustafa , Polland Richard , Schooley Robert , Asthana Deshratn , Mizrachi Yaffa , Waxdal Myron
Published: July 2002

An external evaluation program for measuring the performance of laboratories testing for cytokines and immune activation markers in biological fluids was developed. Cytokines, chemokines, soluble cytokine receptors, and other soluble markers of immune activation (CSM) were measured in plasma from a healthy human immunodeficiency virus (HIV)-seronegative reference population and from HIV-seropositive individuals as well as in supernatant fluids from in vitro-stimulated human immune cells. The 14 components measured were tumor necrosis factor (TNF) alpha, gamma interferon, interleukin-1 (IL-1), IL-2, IL-4, IL-6, IL-10, Rantes, MIP-Ia, MIP-Iβ, soluble TNF receptor II, soluble IL-2 receptor alpha, β
2
-microglobulin, and neopterin. Twelve laboratories associated with the Adult and Pediatric AIDS Clinical Trial Groups participated in the study. The performance features that were evaluated included intralaboratory variability, interlaboratory variability, comparison of reagent sources, and ability to detect CSM in the plasma of normal subjects as well as the changes occurring in disease. The principal findings were as follows: (i) on initial testing, i.e., before participating in the program, laboratories frequently differed markedly in their analytic results; (ii) the quality of testing of a CSM in individual participating laboratories could be assessed; (iii) most commercial kits allowed distinction between normal and abnormal plasma CSM levels and between supernatants of stimulated and unstimulated cells; (iv) different sources of reagents and reference standards frequently provided different absolute values; (v) inexperienced laboratories can benefit from participating in the program; (vi) laboratory performance improved during active participation in the program; and (vii) comparability between analyses conducted at different sites can be ensured by an external proficiency testing program.
Development of a Recombinant Nucleoprotein-Based Enzyme-Linked Immunosorbent Assay for Quantification of Antibodies against Porcine Reproductive and Respiratory Syndrome Virus
Authors: Witte Steven B. , Chard-Bergstrom Cindy , Loughin Thomas A. , Kapil Sanjay
Published: July 2002

A rapid, inexpensive enzyme-linked immunosorbent assay (ELISA) to quantitate antibodies to porcine respiratory and reproductive syndrome virus (PRRSV) in serum was developed using a recombinant PRRSV nucleoprotein (rN). The sensitivity (85.3%) and specificity (81.7%) of the Kansas State University ELISA were good, correlating well (82.4%) with the IDEXX HerdChek ELISA.
Combined Cell Culture Enzyme-Linked Immunosorbent Assay for Quantification of Poliovirus Neutralization- Relevant Antibodies
Authors: Wahby A. F.
Published: July 2002

A combined cell culture enzyme-linked immunosorbent assay (CCC-ELISA) was developed for measuring the neutralizing antipoliovirus antibodies in human sera. The binding of different concentrations of each of the three poliovirus types to BGM cells in the presence and absence of a constant dilution from each test and reference serum was measured in the CCC-ELISA. The titers of the viruses neutralized by each serum were measured with the titration curves and used for interpretation of neutralizing titers to the three poliovirus types. Analysis of human sera revealed that the sensitivity and specificity of the CCC-ELISA and the microneutralization assay were comparable. The CCC-ELISA is nonsubjective, rapid, and highly reproducible. Furthermore, the CCC-ELISA could potentially be used as a seroepidemiologic tool for assessment of the humoral response to the cell culture infectious viruses.
Recombinant p51 as Antigen in an Immune Complex Transfer Enzyme Immunoassay of Immunoglobulin G Antibody to Human Immunodeficiency Virus Type 1
Authors: Hashinaka Kazuya , Hashida Seiichi , Nishikata Ichiro , Adachi Akio , Oka Shinichi , Ishikawa Eiji
Published: July 2002

An ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of antibody immunoglobulin G (IgG) to human immunodeficiency virus type 1 (HIV-1) has been developed using recombinant HIV-1 reverse transcriptase (rRT) as antigen. However, some disadvantages were noted in the use of rRT as antigen: rRT was produced only with low efficiency in widely used strains of
Escherichia coli
using a rather long DNA fragment (3,012 bp) of the whole HIV-1
pol
gene, and it was impossible to produce fusion proteins of RT for simple purification, since rRT is a heterodimer of p66 and p51. In this study, recombinant HIV-1 p51 and p66 with Ser-Ser at the N termini (Ser-Ser-rp51 and Ser-Ser-rp66) were produced in
E. coli
as fusion proteins with maltose binding protein containing a factor Xa site between the two proteins and were purified after digestion with factor Xa. Ser-Ser-rp51 was produced in larger amounts and purified in higher yields with less polymerization than Ser-Ser-rp66. Polymerized Ser-Ser-rp66 tended to be precipitated on mercaptoacetylation for conjugation to β-
d
-galactosidase (used as a label) and showed higher nonspecific and lower specific signals in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 than Ser-Ser-rp51. The signals for serum samples of HIV-1-seropositive subjects by immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 using Ser-Ser-rp51 as antigen (
Y
) were well correlated to those obtained using rRT as antigen (
X
) (log
Y
= 0.99 log
X
+ 0.23;
r
= 0.99). Thus, the use of rp51 as antigen was advantageous over that of rp66 and rRT in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1.
Monoclonal Antibody Binding to a Surface-Exposed Epitope on
Cowdria ruminantium
That Is Conserved among Eight Strains
Authors: Shompole Sankale , Rurangirwa Fred R. , Wambugu Anderson , Sitienei John , Mwangi Duncan M. , Musoke Anthony J. , Mahan Suman , Wells Clive W. , McGuire Travis C.
Published: July 2002

Monoclonal antibodies (MAb) binding to
Cowdria ruminantium
elementary bodies (EB) were identified by enzyme-linked immunosorbent assay, and surface binding of one MAb (446.15) to intact EB was determined by immunofluorescence, immunogold labeling, and transmission electron microscopy. MAb 446.15 bound an antigen of approximately 43 kDa in immunoblots of eight geographically distinct strains. The MAb did not react with
Ehrlichia canis
antigens or uninfected bovine endothelial cell lysate and may be useful in diagnostic assays and vaccine development.
Correlation between Enzyme-Linked Immunosorbent Assay and Immunofluorescence Assay with Lytic Antigens for Detection of Antibodies to Human Herpesvirus 8
Authors: Topino Simone , Vincenzi Laura , Mezzaroma Ivano , Nicastri Emanuele , Andreoni Massimo , Sirianni Maria C.
Published: July 2002

The purpose of this study was to evaluate, in Kaposi's sarcoma patients, the correlation between antibody titers to the lytic antigens of human herpesvirus 8, as assessed by immunofluorescence assay, and values obtained by an enzyme immunoassay. The methods showed a stringent correlation,
r
= 0.625 (
P
< 0.001).
Monoclonal Antibodies to Conformational Epitopes of the Surface Glycoprotein of Caprine Arthritis-Encephalitis Virus: Potential Application to Competitive-Inhibition Enzyme-Linked Immunosorbent Assay for Detecting Antibodies in Goat Sera
Authors: Özyörük Fuat , Cheevers William P. , Hullinger Gordon A. , McGuire Travis C. , Hutton Melinda , Knowles Donald P.
Published: July 2002

Four immunoglobulin G1 monoclonal antibodies (MAbs) to the gp135 surface envelope glycoprotein (SU) of the 79–63 isolate of caprine arthritis-encephalitis virus (CAEV), referred to as CAEV-63, were characterized and evaluated for their ability to compete with antibody from CAEV-infected goats. Three murine MAbs (MAbs GPB16A, 29A, and 74A) and one caprine MAb (MAb F7-299) were examined. All MAbs reacted in nitrocellulose dot blots with native CAEV-63 SU purified by MAb F7-299 affinity chromatography, whereas none reacted with denatured and reduced SU. All MAbs reacted in Western blots with purified CAEV-63 SU or the SU component of whole-virus lysate following denaturation in the absence of reducing agent, indicating that intramolecular disulfide bonding was essential for epitope integrity. Peptide-
N
-glycosidase F digestion of SU abolished the reactivities of MAbs 74A and F7-299, whereas treatment of SU with
N
-acetylneuraminate glycohydrolase (sialidase A) under nonreducing conditions enhanced the reactivities of all MAbs as well as polyclonal goat sera. MAbs 29A and F7-299 were cross-reactive with the SU of an independent strain of CAEV (CAEV-Co). By enzyme-linked immunosorbent assay (ELISA), the reactivities of horseradish peroxidase (HRP)-conjugated MAbs 16A and 29A with homologous CAEV-63 SU were <10% of that of HRP-conjugated MAb 74A. The reactivity of HRP-conjugated MAb 74A was blocked by sera from goats immunized with CAEV-63 SU or infected with CAEV-63. The reactivity of MAb 74A was also blocked by sera from goats infected with a CAEV-Co molecular clone, although MAb 74A did not react with CAEV-Co SU in Western blots. Thus, goats infected with either CAEV-63 or CAEV-Co make antibodies that inhibit binding of MAb 74A to CAEV-63 SU. A competitive-inhibition ELISA based on displacement of MAb 74A reactivity has potential applicability for the serologic diagnosis of CAEV infection.
Development of an Immunoglobulin M (IgM) Capture Enzyme-Linked Immunosorbent Assay for Detection of Equine and Swine IgM Antibodies to Vesicular Stomatitis Virus
Authors: Zhou En-min , Riva Jose , Clavijo Alfonso
Published: July 2002

An immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (MC-ELISA) was developed for the detection of primary infection of vesicular stomatitis virus (VSV) in equine and swine sera. The test was based on the use of biotinylated sheep antibodies against equine or swine IgM molecules bound to a streptavidin-coated ELISA plate. The captured IgM antibodies were detected by application of antigens prepared from the New Jersey and the Indiana VSV serotypes (VSV-NJ and VSV-IN, respectively) and mouse polyclonal antibodies against VSV-NJ and VSV-IN. The MC-ELISA was compared to a competitive ELISA (C-ELISA) and the standard microtiter serum neutralization (MTSN) assay by testing serum samples from horses and pigs experimentally infected with VSV-NJ or VSV-IN. The MC-ELISA detected specific homologous IgM antibodies from equine and swine sera as early as 5 and 4 days postinfection (DPI), respectively, and as late as 35 DPI. The MTSN test also detected antibodies as early as 5 DPI and as late as 160 DPI. In a similar fashion, the C-ELISA detected antibodies from 6 to 7 DPI and as late as 160 DPI. These results demonstrated that the MC-ELISA is a useful test for serodiagnosis of primary VSV infection in horses and pigs.
Chromogenic Assay Measuring Opsonophagocytic Killing Capacities of Antipneumococcal Antisera
Authors: Lin J. S. , Park M. K. , Nahm M. H.
Published: July 2002

Assays measuring opsonophagocytic killing capacity of immune sera are good surrogate assays for assessing pneumococcal vaccine responses, but they are tedious to perform primarily because the enumeration of surviving bacteria requires the counting of individual bacterial colonies. To overcome this limitation, we have developed a simple and rapid chromogenic assay for estimating the number of surviving bacteria. In this method, the conventional opsonophagocytic killing assays were performed in microtiter wells with differentiated HL-60 cells as phagocytes. At the end of the assay the reaction mixture was cultured for an additional 4.5 h to increase the number of bacteria. After the short culture, XTT (3,3′-[1{(phenylamino)carbonyl}-3,4-tetrazolium]-bis[4-methoxy-6-nitro] benzene sulfonic acid hydrate) and coenzyme Q were added to the wells and the optical density at 450 nm was measured. Our study shows that changes in the optical density were proportional to the number of CFU of live bacteria in the wells. Also, the number of bacteria at the end of the 4.5-h culture was found to be proportional to the original number of bacteria in the wells. When the performance of the chromogenic assay was evaluated by measuring the opsonizing titers of
Streptococcus pneumoniae
serotypes 6B and 19F, the sensitivity and precision of the new method were similar to those of the conventional opsonization assay employing the colony counting method. Furthermore, the results of this chromogenic assay obtained with 33 human sera correlate well with those obtained with the conventional colony counting method (
R
> 0.90) for the two serotypes (6B and 19F). Thus, this simple chromogenic assay would be useful in rapidly measuring the capacities of antisera to opsonize pneumococci.
Antenatal Screening for Hepatitis B and Antibodies to
Toxoplasma gondii
and Rubella Virus: Evaluation of Two Commercial Immunoassay Systems
Authors: Diepersloot Robert J. A. , Dunnewold-Hoekstra Hiske , Kruit-den Hollander Jorien , Vlaspolder Fer
Published: July 2002

A comparative evaluation of the Abbott AxSYM and DPC Immulite random-access analyzers was performed using 497 prospectively collected serum samples. These samples were sent to the laboratory for routine antenatal screening for hepatitis B surface antigen and immunoglobulin G (IgG) and IgM antibodies to
Toxoplasma gondii
and rubella virus. The overall agreement between the two assay systems ranged from 97.4 to 100%. After discrepancy analysis, the outcome in terms of sensitivity and specificity varied from 98.2 to 100% for all but one of the assays tested. The AxSYM rubella virus IgG assay tended to report protective or indeterminate antibody levels in 1% of the samples. This shortcoming might be overcome by raising the cutoff of the microparticle enzyme immunoassay system.
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

The 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

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

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

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

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

The 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

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

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

Infection 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

The 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

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

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

Pulmonary 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

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.
Cellular Internalization of Cytolethal Distending Toxin fromHaemophilus ducreyi
Authors: Cortes-Bratti Ximena , Chaves-Olarte Esteban , Lagergård Teresa , Thelestam Monica
Published: July 2002

The chancroid bacteriumHaemophilus ducreyiproduces a toxin (HdCDT) which is a member of the recently discovered family of cytolethal distending toxins (CDTs). These protein toxins prevent the cyclin-dependent kinase cdc2 from being activated, thus blocking the transition of cells from the G2phase into mitosis, with the consequent arrest of intoxicated cells in G2. It is not known whether these toxins act by signaling from the cell surface or intracellularly only. Here we report that HdCDT has to undergo at least internalization before being able to act. Cellular intoxication was inhibited (i) by removal of clathrin coats via K+depletion, (ii) by treatment with drugs that inhibit receptor clustering into coated pits, and (iii) in cells genetically manipulated to fail in clathrin-dependent endocytosis. Intoxication was also completely inhibited in cells treated with bafilomycin A1 or nocodazole and in cells incubated at 18°C, i.e., under conditions known to block the fusion of early endosomes with downstream compartments. Moreover, disruption of the Golgi complex by treatment with brefeldin A or ilimaquinone blocked intoxication. In conclusion, our data indicate that HdCDT enters cells via clathrin-coated pits and has to be transported via the Golgi complex in order to intoxicate cells. This is the first member of the family of CDTs for which cellular internalization and some details of the pathway have been demonstrated.
Early Induction of Interleukin-12 by Human Monocytes Exposed toCryptococcus neoformansMannoproteins
Authors: Pitzurra Lucia , Cherniak Robert , Giammarioli Monica , Perito Stefano , Bistoni Francesco , Vecchiarelli Anna
Published: July 2002

Interleukin-12 (IL-12) production by human monocytes stimulated with mannoproteins (MPs) ofCryptococcus neoformanswas investigated. The results reported show that secreted or cell-associated MPs induce an early and significant production of IL-12. MPs show different capabilities to quantitatively affect IL-12 production; MP2, an 8.2-kDa MP purified from the culture supernatant ofC. neoformans, appears to be the most potent stimulator. Cytochalasin B inhibits both internalization and IL-12 induction by MP. In addition, a drastic reduction of IL-12 was observed when monocytes were cultured in the absence of normal human serum or treated with soluble mannan. Early production of IL-12 promotes early secretion of gamma interferon by T cells but does not influence the magnitude of the MP-induced lymphoproliferative response. Overall our results identify cryptococcal antigens responsible for rapid and potent induction of IL-12 in monocytes. MPs appear to regulate IL-12 secretion by internalization via the endocytic pathway and by interaction with monocyte receptors or serum factors.