bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytokine Production and Monocyte HLA-DR Expression as Predictors of Outcome for Patients with Community-Acquired Severe Infections
Authors: Lekkou A. , Karakantza M. , Mouzaki A. , Kalfarentzos F. , Gogos C. A.
Published: January 2004

This study was performed to evaluate the impact of pro- and anti-inflammatory molecules and human leukocyte antigen DR (HLA-DR) expression as markers of immune status for the final outcome of septic patients. The study included 30 patients with severe sepsis due to community-acquired infections. Concentrations of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-8, IL-10, and transforming growth factor β1 (TGF-β1) in serum, as well as monocyte HLA-DR expression, were determined on admission and on days 3, 10, 13, and 17 during hospitalization. Of the 30 patients enrolled, 13 survived, while 17 died during their hospital stay. All patients had significantly lower HLA-DR expression and higher pro- and anti-inflammatory cytokine levels than healthy individuals. HLA-DR expression was significantly decreased in nonsurvivors at almost all time points. In nonsurvivors, higher levels in serum of TNF-α on days 13 and 17; IL-6 levels on day 3; and IL-10 on days 3, 10, and 13 were found. Baseline levels of TGF-β1 were significantly higher in survivors. Independent risk factors of mortality were IL-10 levels on days 3 and 10, while monocyte HLA-DR expression on admission was a good predictor for survival. Several pro- and anti-inflammatory cytokines are oversynthesized during severe infections, especially in patients with a poor outcome. Monocyte HLA-DR expression is an early and constant predictive marker for survival in severe sepsis, while serum IL-10 levels on days 3 and 10 have negative prognostic value for the final outcome.
Establishment of Adult Peripheral Blood Lymphocyte Subset Reference Range for an Asian Population by Single-Platform Flow Cytometry: Influence of Age, Sex, and Race and Comparison with Other Published Studies
Authors: Chng Wee J. , Tan Guat B. , Kuperan Ponnudurai
Published: January 2004

We established a normal reference range for peripheral blood lymphocyte subsets in a multiracial adult population by using single-platform flow cytometry. Further analysis of our cohort showed that the CD8
+
-cell counts decrease with age, there is a gender difference in NK cell percentages and counts, and there are significant differences in the CD3
+
-, CD4
+
-, and CD19
+
-cell counts between Indians and other racial groups. Overall, our results are significantly different from other published data. This difference further stresses the need for different populations to establish their own reference ranges as these may have important implications for the management of patients with human immunodeficiency virus and AIDS. The use of single-platform flow cytometry will eliminate some of the variability between different study centers, making studies more comparable. This platform should be used for future studies into the effects of age, sex, and race on lymphocyte subsets.
Mastitis Increases Mammary mRNA Abundance of β-Defensin 5, Toll-Like-Receptor 2 (TLR2), and TLR4 but Not TLR9 in Cattle
Authors: Goldammer T. , Zerbe H. , Molenaar A. , Schuberth H.-J. , Brunner R. M. , Kata S. R. , Seyfert H.-M.
Published: January 2004

Coordination of the primary defense mechanisms against pathogens relies on the appropriate expression of pathogen recognition receptors (PRRs) triggering the early release of effector molecules of the innate immune system. To analyze the impact of this system on the counteraction of infections of the mammary gland (mastitis), we characterized the bovine gene encoding the key PRR Toll-like receptor 9 (TLR9) and mapped its precise position on chromosome BTA22. The sequence information was used to establish real-time PCR quantification assays to measure the mRNA abundances of TLR9, TLR2, and TLR4 together with those of β-defensin 5 (BNBD5), an early bactericidal effector molecule of the innate system, in healthy and infected mammary glands. Mastitis strongly increased (4- to 13-fold) the mRNA abundances of all of these genes except TLR9. Slight subclinical infections already caused a substantial increase in the copy numbers, though they did so the least for TLR9. Induction was not systemic, since mRNA abundance was low in uninfected control quarters of the udder but high in the severely infected quarters of the same animal. The number of TLR2 copies correlated well with those of TLR4, indicating coordinated regulation of these two PRRs during infection of the udder. Their coordinated regulation explains our unexpected observation that pureStaphylococcus aureusinfections caused a strong increase also in TLR4 mRNA abundance. In situ hybridizations revealed that BNBD5 is expressed predominantly in the mammary epithelial cells (MEC) of the infected gland. Our data therefore suggest a significant contribution of the innate immune system to counteract mastitis and attribute a prominent effector function to the MEC.
Prevalence of Neutralizing Antibodies against Different Rotavirus Serotypes in Children with Severe Rotavirus-Induced Diarrhea and Their Mothers
Authors: Ray Pratibha G. , Kelkar Shobhana D.
Published: January 2004

Neutralizing antibody (NAb) responses to different rotavirus serotypes were compared in 64 convalescent-phase serum samples from hospitalized rotavirus-positive children less than 2 years of age and their mothers. Compared to the child patients, the mothers showed significantly higher NAb positivity to animal rotavirus serotypes G3 simian (96.88%), G6 bovine (85.94%), and G10 bovine (25.0%) and to human rotavirus serotypes G8 (79.69%) and G3 (57.81%) (
P
< 0.01 for each) but not to human serotypes G1, G2, G4, and G9 (
P
> 0.05). The overall prevalence of NAb among the child patients was low for human rotavirus serotypes G1 (20.31%) and G3 (21.8%). The comparative NAb response in individual mother-child paired serum samples was analyzed against each rotavirus serotype. A substantial number of child patients showed higher NAb titers than their mothers to serotypes G1, G2, G4, and G9, indicating that these serotypes are the major serotypes causing rotavirus diarrhea among the children of Pune, India. In these cases, the mothers were either negative or had lower titers of NAbs than their children. Correlation was observed between the infecting serotype and child patient serum that showed a homologous NAb response at a higher level than that of the mother. It appears that when the level of NAb to a particular serotype is higher among child patients than among their mothers, that serotype is the infecting serotype, and that low titers of NAb among the mothers predispose the children to infection with that serotype, if the serotype is in circulation.
Decreased Expression of the CD3ζ Chain in T Cells Infiltrating the Synovial Membrane of Patients with Osteoarthritis
Authors: Sakkas Lazaros I. , Koussidis George , Avgerinos Efthimios , Gaughan John , Platsoucas Chris D.
Published: January 2004

Osteoarthritis (OA) is a heterogeneous disease which rheumatologists consider to be noninflammatory. However, recent studies suggest that, at least in certain patients, OA is an inflammatory disease and that patients often exhibit inflammatory infiltrates in the synovial membranes (SMs) of macrophages and activated T cells expressing proinflammatory cytokines. We report here that the expression of CD3ζ is significantly decreased in T cells infiltrating the SMs of patients with OA. The CD3ζ chain is involved in the T-cell signal transduction cascade, which is initiated by the engagement of the T-cell antigen receptor and which culminates in T-cell activation. Double immunofluorescence of single-cell suspensions derived from the SMs from nine patients with OA revealed significantly increased proportions of CD3ε-positive (CD3ε+) cells compared with the proportions of CD3ζ-positive (CD3ζ+) T cells (means ± standard errors of the means, 80.48% ± 3.92% and 69.02% ± 6.51%, respectively;P= 0.0096), whereas there were no differences in the proportions of these cells in peripheral blood mononuclear cells (PBMCs) from healthy donors (94.73% ± 1.39% and 93.79% ± 1.08%, respectively; not significant). The CD3ζ+cell/CD3ε+cell ratio was also significantly decreased for T cells from the SMs of patients with OA compared with that for T cells from the PBMCs of healthy donors (0.84 ± 0.17 and 0.99 ± 0.01, respectively;P= 0.0302). The proportions of CD3ε+CD3ζ+cells were lower in the SMs of patients with OA than in the PBMCs of healthy donors (65.04% ± 6.7% and 90.81% ± 1.99%, respectively;P= 0.0047). Substantial proportions (about 15%) of CD3ε+CD3ζ-negative (CD3ζ−) and CD3ε-negative (CD3ε−) CD3ζ−cells were found in the SMs of patients with OA. Amplification of the CD3ζ and CD3δ transcripts from the SMs of patients with OA by reverse transcriptase PCR consistently exhibited stronger bands for CD3δ cDNA than for CD3ζ cDNA The CD3ζ/CD3δ transcript ratio in the SMs of patients with OA was significantly lower than that in PBMCs from healthy controls (P< 0.0001). These results were confirmed by competitive MIMIC PCR. Immunoreactivities for the CD3ζ protein were detected in the SMs of 10 of 19 patients with OA, and they were of various intensities, whereas SMs from all patients were CD3ε+(P= 0.0023). The decreased expression of the CD3ζ transcript and protein in T cells from the SMs of patients with OA relative to that of the CD3ε transcript is suggestive of chronic T-cell stimulation and supports the concept of T-cell involvement in OA.
Induction of Nitric Oxide Production Mediated by Tumor Necrosis Factor Alpha on Staphylococcal Enterotoxin C-Stimulated Bovine Mammary Gland Cells
Authors: Komine Ken-ichi , Kuroishi Toshinobu , Komine Yumiko , Watanabe Kouichi , Kobayashi Jin , Yamaguchi Takahiro , Kamata Shin-ichi , Kumagai Katsuo
Published: January 2004

Mammary gland (MG) secretions (MGS) derived from secretory cows infected with coagulase-negative staphylococci (CoNS) showed somatic cell counts and lactoferrin similar to levels found in the MGS of secretory cows infected with
Staphylococcus aureus
. However, nitrite and nitrate (NOx) and staphylococcal enterotoxin C (SEC) were found in MGS infected with
S. aureus
at much higher levels than in cows infected with CoNS. These results suggested that NOx could be intimately correlated with the production of SEC in secretory cows infected with
S. aureus
. Therefore, we examined the production of NOx and the expression of proinflammatory cytokines and microsomal cytochrome P450 (CYP450) after injection of SEC into the MGS of secretory cows. We were able to detect NOx and the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) on MG cells of SEC-injected MGS. It was also found that CYP450 in the MG cells from SEC-injected MGS was down-regulated by approximately one-third in comparison with the cells from phosphate-buffered saline-injected MGS. This in vitro system also showed that NOx could be induced in the culture of bovine macrophage-lined cells (FBM-17) with the supernatants of SEC-stimulated bovine peripheral blood lymphocytes (BoPBLs) but not in the culture of peripheral mononuclear cells with SEC-stimulated BoPBLs. The expression of the mRNA for both inducible nitric oxide synthase and TNF-α in FBM-17 was enhanced by culturing with the supernatant of SEC-stimulated BoPBLs, although CYP450 was down-regulated. These results indicate that the down-regulation of CYP450 was caused by the production of TNF-α in SEC-stimulating MG cells containing macrophages and via NOx production. Therefore, we suggest that NOx released from activated MG cells via the superantigenic activity of SEC caused oxidative damage to the MG in
S. aureus
-induced mastitis.
Phenotypic Drug Susceptibility Assay for Influenza Virus Neuraminidase Inhibitors
Authors: McSharry James J. , McDonough Ann C. , Olson Betty A. , Drusano George L.
Published: January 2004

A flow cytometric (fluorescence-activated cell sorter [FACS]) assay was developed for analysis of the drug susceptibilities of wild-type and drug-resistant influenza A and B virus laboratory strains and clinical isolates for the neuraminidase (NA) inhibitors oseltamivir carboxylate, zanamivir, and peramivir. The drug susceptibilities of wild-type influenza viruses and those with mutations in the hemagglutinin (HA) and/or NA genes rendering them resistant to one or more of the NA inhibitors were easily determined with the FACS assay. The drug concentrations that reduced the number of virus-infected cells or the number of PFU by 50% as determined by the FACS assay were similar to those obtained with the more time-consuming and labor-intensive virus yield reduction assay. The NA inhibition (NAI) assay confirmed the resistance patterns demonstrated by the FACS and virus yield assays for drug-resistant influenza viruses with mutations in the NA gene. However, only the FACS and virus yield assays detected NA inhibitor-resistant influenza viruses with mutations in the HA gene but not in the NA gene. The FACS assay is more rapid and less labor-intensive than the virus yield assay and just as quantitative. The FACS assay determines the drug susceptibilities of influenza viruses with mutations in either the HA or NA genes, making the assay more broadly useful than the NAI assay for measuring the in vitro susceptibilities of influenza viruses for NA inhibitors. However, since only viruses with mutations in the NA gene that lead to resistance to the NA inhibitors correlate with clinical resistance, this in vitro assay should not be used in the clinical setting to determine resistance to NA inhibitors. The assay may be useful for determining the in vivo susceptibilities of other compounds effective against influenza A and B viruses.
Molecular Cloning of a
Babesia caballi
Gene Encoding the 134-Kilodalton Protein and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay
Authors: Tamaki Yoh , Hirata Haruyuki , Takabatake Noriyuki , Bork Sabine , Yokoyama Naoaki , Xuan Xuenan , Fujisaki Kozo , Igarashi Ikuo
Published: January 2004

A
Babesia caballi
gene encoding the 134-kDa (BC134) protein was immunoscreened with
B. caballi
-infected horse serum. An enzyme-linked immunosorbent assay (ELISA) using recombinant BC134 protein could effectively differentiate
B. caballi
-infected horse sera from
Babesia equi
-infected or noninfected control horse sera. These results suggest that the recombinant BC134 protein is a potential diagnostic antigen in the detection of
B. caballi
infection.
Bacterial Expression of a Human Monoclonal Antibody-Alkaline Phosphatase Conjugate Specific for
Entamoeba histolytica
Authors: Tachibana Hiroshi , Takekoshi Masataka , Cheng Xun-Jia , Nakata Yuta , Takeuchi Tsutomu , Ihara Seiji
Published: January 2004

We previously produced human monoclonal antibody Fab fragments specific to
Entamoeba histolytica
in
Escherichia coli
. In order to use these Fab fragments for diagnostic purposes, an expression vector to produce a fusion protein of Fab and alkaline phosphatase (PhoA) in
E. coli
was designed and constructed. The
E. coli
PhoA gene was fused to the 3′ terminus of the gene encoding the heavy-chain Fd region. The kappa and Fd genes from a previously prepared antibody clone, CP33, which is specific for the 260-kDa lectin of
E. histolytica
, were used as human antibody genes. When the fusion protein of CP33 and PhoA was incubated with paraformaldehyde-fixed trophozoites of
E. histolytica
and developed with a substrate, the trophozoites appeared to be stained. These results demonstrate the feasibility of bacterial expression of a human monoclonal antibody-PhoA conjugate specific for
E. histolytica
and that the antibody can be used to detect
E. histolytica
antigen without the use of chemically conjugated secondary antibodies.
Suitability of Frozen Serum Stored in Gel Separator Primary Sampling Tubes for Serological Testing
Authors: Rosa-Fraile Manuel , Sampedro Antonio , Rodríguez-Granger Javier , Camacho Enrique , Manrique Ester
Published: January 2004

The suitability of frozen serum after storage in primary sampling tubes with a gel separator for serological enzyme-linked immunosorbent assay testing (hepatitis B virus surface antigen [HBs Ag], anti-HBs Ag, anti-
Toxoplasma gondii
immunoglobulin G [IgG], anti-rubella virus IgG, anti-cytomegalovirus IgM, and anti-Epstein-Barr virus IgM) was evaluated for 375 samples. No difference was found among test results using fresh or stored frozen serum
Recognition of ESAT-6 Sequences by Antibodies in Sera of Tuberculous Nonhuman Primates
Authors: Kanaujia G. V. , Motzel S. , Garcia M. A. , Andersen P. , Gennaro M. L.
Published: January 2004

Previous work in our laboratory showed that the ESAT-6 protein of
Mycobacterium tuberculosis
and
Mycobacterium bovis
induces strong antibody responses in a large proportion (∼90%) of experimentally or naturally infected nonhuman primates. Here, the antibody response to ESAT-6 in tuberculous monkeys was characterized at the epitope level by measuring antibodies to overlapping, synthetic peptides spanning the ESAT-6 sequence. The antibody response against the COOH-terminal portion of the protein was the strongest in both experimentally and naturally infected animals. Moreover, these antibodies became detectable the earliest during experimental infection, suggesting an ordered expansion of ESAT-6-specific B-cell clones in the course of infection. The data support use of synthetic peptides in lieu of the full-length ESAT-6 protein in diagnostic antibody detection assays.
Profile of Antibodies to the Nucleocapsid Protein of the Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus in Probable SARS Patients
Authors: Liu Xuan , Shi Yulin , Li Ping , Li Linhai , Yi Yanping , Ma Qingjun , Cao Cheng
Published: January 2004

Profiles of antibodies to the nucleocapsid protein of the severe acute respiratory syndrome (SARS)-associated coronavirus in 445 probable SARS patients and 3,749 healthy people or non-SARS patients were analyzed by antigen-capturing enzyme-linked immunosorbent assay. Antinucleocapsid antibodies were elucidated in 17.5% of the probable SARS patients 1 to 7 days after the onset of symptoms and in 80% of the patients 8 to 14 days after the onset. About 90% of the probable SARS patients were positive 15 or more days after illness. Antibody titers increased up to 70 days, and high antibody titers were maintained at least for another 3 months. Of the healthy people and non-SARS patients, only seven (0.187%) were weakly positive.
Age-Related Changes in Expression of CXCR4 and CCR5 on Peripheral Blood Leukocytes from Uninfected Infants Born to Human Immunodeficiency Virus Type 1-Infected Mothers
Authors: Shalekoff Sharon , Gray Glenda E. , Tiemessen Caroline T.
Published: January 2004

Cross-sectional analysis of human immunodeficiency virus-exposed, uninfected infants revealed high proportions of CXCR4-expressing cells in their cord blood, which declined at 4.5 months and increased between 9 and 15 months to levels approaching those of uninfected adults. Proportions of CCR5-expressing cells, however, were very low in cord blood and subsequently increased with age.
Population-Based Hematologic and Immunologic Reference Values for a Healthy Ugandan Population
Authors: Lugada Eric S. , Mermin Jonathan , Kaharuza Frank , Ulvestad Elling , Were Willy , Langeland Nina , Asjo Birgitta , Malamba Sam , Downing Robert
Published: January 2004

To assess the validity of the reference values for hematologic and immunologic indices currently used in Africa, we evaluated blood samples from 3,311 human immunodeficiency virus (HIV)-negative Ugandans aged 1 week to 92 years. Erythrocyte, hemoglobin, and hematocrit levels and mean corpuscular volume all significantly increased with age (P< 0.001) and were independent of gender until the age of 13 years, after which the levels were higher in males than in females (P< 0.001). White blood cell, neutrophil, lymphocyte, basophil, and monocyte counts significantly declined with age until the age of 13 years (P< 0.001), with no differences by gender, while platelet counts declined with age (P< 0.001) and showed differences by gender only among adults older than age 24 years. CD4+- and CD8+-cell counts declined with age until the age of 18 years; thereafter, females had higher counts than males. The absolute values for many of these parameters differed from those reported for populations outside Africa, suggesting that it may be necessary to develop tables of reference values for hematologic and immunologic indices specific for the African population. This may be particularly important with regard to CD4+-cell counts among children because significant differences in absolute and percent CD4+-cell counts exist between the values for Western populations and the values for the population evaluated in our study. These differences could influence the decision to initiate antiretroviral therapy among children infected with HIV.
Neutralization of Gamma Interferon Augments Borreliacidal Antibody Production and Severe Destructive Lyme Arthritis in C3H/HeJ Mice
Authors: Munson Erik L. , DeCoster David J. , Nardelli Dean T. , England Douglas M. , Callister Steven M. , Schell Ronald F.
Published: January 2004

Development of a high level of sustained borreliacidal antibody is paramount for maintaining protection against infection withBorrelia burgdorferi. We show that production of borreliacidal antibody can be enhanced by preventing the effects of gamma interferon (IFN-γ). When lymph node cells capable of producing borreliacidal antibody were cultured with anti-murine IFN-γ, an eightfold increase in borreliacidal antibody production was obtained. However, anti-IFN-γ treatment of these cells also enhanced their ability to adaptively induce arthritis. When anti-IFN-γ-treated lymph node cells producing borreliacidal antibody were infused into C3H/HeJ mice and the mice were then challenged withB. burgdorferi, the mice developed severe destructive Lyme arthritis. Additional studies are needed to delineate the immune response responsible for the induction of arthritis and production of borreliacidal antibody. These studies are needed to ensure an effective and safe vaccine against infection withB. burgdorferi.
Performance and Use of a Ribonucleotide Reductase Herpes Simplex Virus Type-Specific Serological Assay
Authors: Wales S. Q. , Smith C. C. , Wachsman M. , Calton G. , Aurelian L.
Published: January 2004

In response to the increasingly evident need for herpes simplex virus (HSV) serotype-specific serologic assays that rely on proteins other than glycoprotein-G (gG), we developed a rapid serologic assay that is based on type-specific epitopes within the large subunit of HSV ribonucleotide reductase (R1). The assay (Au-2 enzyme-linked immunosorbent assay [ELISA]) uses an HSV type 2 (HSV-2) R1 peptide antigen. It provides a reliable method for detecting serotype-specific antibody to a protein other than gG-2. The Au-2 ELISA has high sensitivity and specificity as determined by direct comparison to Western blotting, a widely accepted “gold standard,” and to ELISA with an HSV-1 R1 peptide (Au-1). The use of the Au-2 ELISA in conjunction with the gG-2-based assays will improve the sensitivity and specificity of serologic diagnosis and patient management.
Comparison of a Multiplexed Fluorescent Covalent Microsphere Immunoassay and an Enzyme-Linked Immunosorbent Assay for Measurement of Human Immunoglobulin G Antibodies to Anthrax Toxins
Authors: Biagini Raymond E. , Sammons Deborah L. , Smith Jerome P. , MacKenzie Barbara A. , Striley Cynthia A. F. , Semenova Vera , Steward-Clark Evelen , Stamey Karen , Freeman Alison E. , Quinn Conrad P. , Snawder John E.
Published: January 2004

Recently, the Centers for Disease Control and Prevention reported an accurate, sensitive, specific, reproducible, and quantitative enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to
Bacillus anthracis
protective antigen (PA) in human serum (C. P. Quinn, V. A. Semenova, C. M. Elie et al., Emerg. Infect. Dis. 8:1103-1110, 2002). The ELISA had a minimum detectable concentration (MDC) of 0.06 μg/ml, which, when dilution adjusted, yielded a whole-serum MDC of 3.0 μg of anti-PA IgG per ml. The reliable detection limit (RDL) was 0.09 μg/ml, while the dynamic range was 0.06 to 1.7 μg/ml. The diagnostic sensitivity of the assay was 97.6% and the diagnostic specificity was 94.2% for clinically verified cases of anthrax. A competitive inhibition anti-PA IgG ELISA was also developed to enhance the diagnostic specificity to 100%. We report a newly developed fluorescence covalent microbead immunosorbent assay (FCMIA) for
B. anthracis
PA which was Luminex xMap technology. The FCMIA MDC was 0.006 μg of anti-PA IgG per ml, the RDL was 0.016 μg/ml, and the whole-serum equivalent MDC was 1.5 μg/ml. The dynamic range was 0.006 to 6.8 μg/ml. Using this system, we analyzed 20 serum samples for anti-PA IgG and compared our results to those measured by ELISA in a double-masked analysis. The two methods had a high positive correlation (
r
2
= 0.852;
P
< 0.001). The FCMIA appears to have benefits over the ELISA for the measurement of anti-PA IgG, including greater sensitivity and speed, enhanced dynamic range and reagent stability, the use of smaller sample volumes, and the ability to be multiplexed (measurement of more than one analyte simultaneously), as evidenced by the multiplexed measurement in the present report of anti-PA and anti-lethal factor IgG in serum from a confirmed clinical anthrax infection.
Important Role of P-Selectin for Leukocyte Recruitment, Hepatocellular Injury, and Apoptosis in Endotoxemic Mice
Authors: Klintman Daniel , Li Xiang , Thorlacius Henrik
Published: January 2004

Leukocyte recruitment in the liver includes a two-step procedure in which selectin-dependent leukocyte rolling is a prerequisite for subsequent CD18-dependent leukocyte firm adhesion in postsinusoidal venules. However, the roles of the individual selectins in leukocyte rolling and adhesion, hepatocellular injury, and apoptosis remain elusive. Therefore, we examined the pathophysiological role of P-, E-, and L-selectin in male C57BL/6 mice challenged with lipopolysaccharide (LPS) andd-galactosamine (Gal) by use of intravital microscopy of the liver microcirculation. In control animals, administration of LPS-Gal provoked reproducible hepatic damage, including marked increases of leukocyte recruitment, liver enzymes, and hepatocyte apoptosis and reduced sinusoidal perfusion. Interestingly, pretreatment with an anti-P-selectin antibody (RB40.34) markedly reduced leukocyte rolling and firm adhesion by 65 and 71%, respectively. Moreover, interference with P-selectin function significantly improved sinusoidal perfusion and reduced the increase in liver enzymes by 49 to 84% in endotoxemic mice. Moreover, the activity of caspase-3 and the number of apoptotic hepatocytes were significantly reduced by 55 and 54%, respectively, in RB40.34-treated animals. In contrast, administration of an anti-E-selectin antibody (10E9.6) and an anti-L-selectin antibody (Mel-14) did not protect against endotoxin-induced leukocyte responses or hepatic injury. In conclusion, our novel findings document a principal role of P-selectin in mediating leukocyte rolling, a precondition to the subsequent firm adhesion of leukocytes in liver injury. Furthermore, our novel data demonstrate that inhibition of P-selectin function reduces hepatocellular injury and apoptosis, suggesting a causal relationship between leukocyte recruitment on one hand and hepatocellular injury and apoptosis on the other hand. Based on these findings, it is suggested that P-selectin may be an important therapeutic target in endotoxin-induced liver injury.
Relationship of Binding of Immunoglobulin G to
Plasmodium falciparum
-Infected Erythrocytes with Parasite Endemicity and Antibody Responses to Conserved Antigen in Immune Individuals
Authors: Diatta Antoine-Marie , Marrama Laurence , Tall Adama , Trape Jean-François , Dieye Alioune , Garraud Olivier , Mercereau-Puijalon Odile , Perraut Ronald
Published: January 2004

To investigate the potential for use of a well-established strain of
Plasmodium falciparum
as a reference strain for infected red blood cell (IRBC) surface reactivity, we monitored the binding of specific immunoglobulin G (IgG) from immune individuals to the reference Knob-positive FCR3 strain by flow cytometry. To permit interassay comparison for 162 plasma samples drawn after the rainy season, a labeling index (LI) was defined as the percentage of labeled parasites multiplied by the mean peak intensity. An LI ratio (LIR) was then calculated as the LI of the sample divided by the LI of the control. LIRs were calculated for individuals living in Dielmo and Ndiop, two Senegalese villages where
P. falciparum
is transmitted holoendemically and mesoendemically, respectively. The incidence (persons with an LIR of >3) observed in Dielmo was lower than that observed in Ndiop. Significantly higher LIRs were observed (i) for samples from Ndiop than for samples from Dielmo (
P
< 0.01) and (ii) in Ndiop, in subjects with hemoglobin AS (HbAS) than in those with hemoglobin AA (
P
= 0.03). No correlation with the cumulative age-associated immune status of the villagers was evidenced, contrary to antibody (Ab) responses against conserved IRBC-associated antigen (Ag) measured by enzyme-linked immunosorbent assay. These results are consistent with the notions that protection in HbAS individuals may relate to an increased IgG response to IRBC membrane Ags and that cell surface reactivity parallels IgG responses even though it is in itself a distinct indicator of the anti-
P. falciparum
Ab response. Measures of IgG binding to live IRBC are thus relevant for the functional screening of conserved IRBC-associated Ags that contribute to parasite destruction in vivo, as these Ags might be included in a multitarget vaccine.
Inhibition of Prostate Cancer Cell Colony Formation by the Flavonoid Quercetin Correlates with Modulation of Specific Regulatory Genes
Authors: Nair Hari Krishnan , Rao Kesava V. K. , Aalinkeel Ravikumar , Mahajan Supriya , Chawda Ram , Schwartz Stanley A.
Published: January 2004

The natural product quercetin is a flavonoid found in many fruits and vegetables. Previous research has shown that quercetin has antitumor, anti-inflammatory, antiallergic, and antiviral activities. In the present investigation we studied the effect of quercetin on the ability of prostate cancer cell lines with various degrees of aggressive potential to form colonies in vitro. Specifically, we examined the molecular mechanisms underlying this effect, including the expression of cell cycle and tumor suppressor genes as well as oncogenes. We observed that quercetin at concentrations of 25 and 50 μM significantly inhibited the growth of the highly aggressive PC-3 prostate cancer cell line and the moderately aggressive DU-145 prostate cancer cell line, whereas it did not affect colony formation by the poorly aggressive LNCaP prostate cancer cell line or the normal fibroblast cell line BG-9. Using the gene array methodology, we found that quercetin significantly inhibited the expression of specific oncogenes and genes controlling G1, S, G2, and M phases of the cell cycle. Moreover, quercetin reciprocally up-regulated the expression of several tumor suppressor genes. In conclusion, our results demonstrate that the antitumor effects of quercetin directly correlate with the aggressive potential of prostate cancer cells and that the mechanism(s) of quercetin-mediated antitumor effects may involve up-regulation of tumor suppressor genes and reciprocal down-regulation of oncogenes and cell cycle genes. The results of these studies provide a scientific basis for the potential use of flavonoids as nutraceuticals in the chemoprevention of cancer.
Comparison of Antibody Responses to a Potential Combination of Specific Glycolipids and Proteins for Test Sensitivity Improvement in Tuberculosis Serodiagnosis
Authors: Julián Esther , Matas Lurdes , Alcaide José , Luquin Marina
Published: January 2004

The humoral response to different proteinaceous antigens of
Mycobacterium tuberculosis
is heterogeneous among patients with active disease, and this has originated in the proposal to use a combination of several specific antigens to find an efficient serodiagnostic test for tuberculosis (TB). However, to date, comparisons of antibody responses to several antigens in the same population have been carried out without consideration of antigenic cell wall glycolipids. In the present study the presence of immunoglobulin G (IgG), IgM, and IgA antibodies to
M. tuberculosis
glycolipids (sulfolipid I, diacyltrehaloses, triacyltrehaloses, and cord factor) was compared with the response to four commercially available tests based on the 38-kDa protein mixed with the 16-kDa protein or lipoarabinomannan. Fifty-two serum samples from TB patients and 83 serum samples from control individuals (48 healthy individuals and 35 non-TB pneumonia patients) were studied. Three relevant results were obtained. (i) Smear-negative TB patients presented low humoral responses, but the sera which did react principally showed IgA antibodies to some glycolipidic antigens. (ii) TB patients exhibit heterogeneous humoral responses against glycolipidic antigens. (iii) Finally, test sensitivity is improved (from 23 to 62%) when IgG and IgA antibodies are detected together in tests based on different antigens (proteins and glycolipids). We conclude that it is possible to include glycolipidic antigens in a cocktail of specific antigens from
M. tuberculosis
to develop a serodiagnostic test.
Calcium Signaling through Phospholipase C Activates Dendritic Cells To Mature and Is Necessary for the Activation and Maturation of Dendritic Cells Induced by Diverse Agonists
Authors: Bagley Kenneth C. , Abdelwahab Sayed F. , Tuskan Robert G. , Lewis George K.
Published: January 2004

Calcium is an important second messenger in the phospholipase C (PLC) signal transduction pathway. Calcium signaling is involved in many biological processes, including muscle contraction, cellular activation, and cellular proliferation. Dendritic cell (DC) maturation is induced by many different stimuli, including bacterial lipopolysaccharide (LPS), bacterial toxins, inflammatory cytokines, prostaglandins, as well as calcium mobilization. In the present study, we determined the role of the PLC signal transduction pathway in the activation and maturation of human monocyte-derived DCs (MDDCs) induced by diverse agonists. We found that signaling through PLC activates MDDCs to mature and is necessary for LPS, cholera toxin, dibutyryl-cyclic AMP, prostaglandin E2, and the calcium ionophore A23187 to induce MDDC maturation. The results of the present study along with the results of other studies indicate that multiple signaling pathways are involved in the activation of DCs and that inhibition of any of these pathways inhibits the maturation of DCs.
Immunologic Hyporesponsiveness to Serogroup C but Not Serogroup A following Repeated Meningococcal A/C Polysaccharide Vaccination in Saudi Arabia
Authors: Jokhdar Hani , Borrow Ray , Sultan Abdulrazaq , Adi Mousaed , Riley Christine , Fuller Emily , Baxter David
Published: January 2004

In Saudi Arabia, vaccination with the meningococcal A/C polysaccharide (MACP) vaccine is advised every 3 years. A clinical outcome study was performed to test the effect of repeat vaccination with the MACP vaccine on the immune responses among Saudi nationals who live in the Makkah and Jeddah areas. Subjects (
n
= 230) aged 10 to 29 years were selected: 113 subjects with two or more prior vaccinations with the MACP vaccine, 79 subjects with one prior vaccination with the MACP vaccine, and 38 subjects naïve to vaccination with the MACP vaccine. All subjects received the MACP vaccine in 2002, and serum bactericidal antibody (SBA) titers were measured before and 1 month after vaccination with the MACP vaccine. For serogroup C, geometric mean SBA titers 1 month following vaccination with the MACP vaccine were 708.6 (95% confidence interval [CI], 217.5 to 2,308.9) for those naïve to prior vaccination with the MACP vaccine, and they were significantly higher (
P
< 0.0001) than 25.0 (95% CI, 12.4 to 50.2) for those who had received one prior vaccination with the MACP vaccine and 32.4 (95% CI, 18.7 to 56.4) for those who had received two or more doses of the MACP vaccine. For serogroup A, the geometric mean SBA titer 1 month after receipt of the MACP vaccine was 1,649.3 (95% CI, 835.2 to 3,256.9) for those naïve to prior vaccination, and the titers were lower (
P
= 0.67) than 2,185.7 (95% CI, 1,489.4 to 3,207.7) for those who had received one prior dose of the MACP vaccine and significantly lower (
P
= 0.042) than 3,540.8 (95% CI, 2,705.2 to 4,634.5) for those who had received two or more doses of the MACP vaccine. For serogroup C, the proportions of nonresponders (SBA titers, <8) were 19% for the naïve cohort, 52% for the cohort with one prior vaccination, and 49% for the cohort with two or more prior vaccinations. Following repeated doses of the MACP vaccine, hyporesponsiveness to serogroup C is evident, with high percentages of MACP vaccinees having SBA titers below the putative protective SBA titer. Serogroup A responses following vaccination with the MACP vaccine were boosted. Introduction of the serogroup C conjugate vaccine would provide long-term protection against serogroup C disease; however, quadrivalent conjugate vaccines are required to provide long-time protection against disease caused by serogroups A, W135, and Y.
Measurement of Serum Bactericidal Activity Specific forHaemophilus influenzaeType b by Using a Chromogenic and Fluorescent Metabolic Indicator
Authors: Romero-Steiner Sandra , Spear Willie , Brown Nekeidra , Holder Patricia , Hennessy Thomas , Gomez de Leon Patricia , Carlone George M.
Published: January 2004

We evaluated alamarBlue as a metabolic indicator in a standardized assay for the measurement of serum bactericidal activity (SBA) toHaemophilus influenzaetype b (Hib) using sera containing natural and vaccine-induced anticapsular (polyribosylribitol phosphate) antibodies. SBA assays with a colorimetric and a fluorometric end point in the presence of alamarBlue were developed and compared to a standard SBA assay, where colony counts are performed to determine the titer (12). A colorimetric end point required a spectrophotometer, whereas a fluorometric end point required a fluorometer. Prevaccination sera (n= 27) and postvaccination sera (n= 13) were tested by all three methodologies, and the SBA titers obtained in the presence of alamarBlue were compared to those from the standard method. Both the colorimetric and the fluorometric SBA titers were significantly correlated (r= 0.87 andr= 0.95, respectively) with those of the standard assay (≥50% killing as the SBA titer end point), and titers were not significantly different when compared to those of the standard assay (P> 0.68). However, the fluorometric end point had superior performance and ease of titer determination compared to the colorimetric end point (95 versus 87% of SBA titers were within 2 dilutions of the standard titer). Hib SBA assays with alamarBlue are reproducible, faster (same-day assay), and easier to perform than the standardized assay, which requires manual or automated colony counts. These semiautomated methodologies result in increased sample throughput and collection of data in digital formats that can be exported to data analysis programs for determination of SBA titers.
Immunoglobulin A (IgA) and IgG Immune Responses against P-90 Antigen for Diagnosis of Pulmonary Tuberculosis and Screening for
Mycobacterium tuberculosis
Infection
Authors: Conde Marcus B. , Suffys Philip , Lapa e Silva Jose Roberto , Kritski Afranio L. , Dorman Susan E.
Published: January 2004

The purpose of the present study was to evaluate the usefulness of detection of serum immunoglobulin A (IgA) and IgG antibodies directed against the mycobacterial P-90 antigen for the diagnosis of active pulmonary tuberculosis (PTB) among symptomatic individuals and for the detection of
Mycobacterium tuberculosis
infections among close contacts of PTB patients. Two commercially available enzyme immunoassay (EIA) kits (IgA EIA-TB [EIA-IgA] and IgG EIA-TB [EIA-IgG]; Kreatech Diagnostics) were evaluated in a blinded fashion by using stored serum samples from 268 individuals, including 69 patients with PTB, 41 patients with diseases other than tuberculosis (TB), 12 subjects with healed PTB, 39 close contacts of PTB patients, and 107 healthy volunteers. For the EIA-IgA, the sensitivity was 74% and the specificity was 68% when a cutoff determined by a receiver operator characteristic curve was used. For the EIA-IgG, the sensitivity was 69% and the specificity was 64%. The EIA-IgA was positive for 54% of healthy close contacts of PTB patients but only 8% of healthy controls without contact with a PTB patient or a prior personal history of TB (
P
< 0.001). The relatively low sensitivities and specificities of these serologic tests make them poor tools for the diagnosis of PTB among patients with suspected PTB. However, the relatively high prevalence of positive EIA-IgA results among healthy close contacts of PTB patients warrants further evaluation of this test with close contacts and other populations at risk for recent
M. tuberculosis
exposure and development of disease.
Evaluation of the Determine Rapid Syphilis TP Assay Using Sera
Authors: Diaz Theresa , de Gloria Bonecini Almeida Maria , Georg Ingebourg , de Carvalho Maia Suely , Valls de Souza Rogerio , Markowitz Lauri E.
Published: January 2004

The Abbott Determine Rapid Syphilis TP assay is a treponemal test that can be used in resource-poor settings that lack laboratory facilities. However, this test has not been extensively evaluated. We measured its sensitivity and specificity by using stored serum specimens (
n
= 567) from all persons who tested
Treponema pallidum
hemagglutination assay (TPHA) positive (
n
= 250) or TPHA indeterminate (
n
= 17) in the year 2001 and the first 300 patients in 2001 who tested TPHA negative at the Evandro Chagas Research Institute in Rio de Janeiro, Brazil. This rapid assay was independently interpreted by three different observers. With TPHA results as the reference, sensitivity ranged between readers from 95.6 to 98.4% and specificity ranged from 97.3 to 95.7%. There was little interreader variability in the interpretation of results, with approximately 98% agreement for all reader combinations. Of samples from persons with human immunodeficiency virus (HIV) infection (
n
= 198), sensitivity was 96.9 to 99.2% and it was 94.4 to 96.3% among HIV-negative persons (
n
= 127). Specificity was 92.4 to 95.5% among HIV-positive persons and 97.2 to 100% among HIV-negative persons. We found this test to have high sensitivity and specificity and little interreader variability, indicating that it may be easily used in resource-poor settings without laboratory facilities. Further studies are needed using this test on whole blood and under the clinical conditions for which it is intended.
Expression and Immunogenicity of Proteins Encoded by Sequences Specific toMycobacterium aviumsubsp.paratuberculosis
Authors: Bannantine John P. , Hansen Janis K. , Paustian Michael L. , Amonsin Alongkorn , Li Ling-Ling , Stabel Judith R. , Kapur Vivek
Published: January 2004

The development of immunoassays specific for the diagnosis of Johne's disease in cattle requires antigens specific toMycobacterium aviumsubsp.paratuberculosis. However, because of genetic similarity to other mycobacteria comprising theM. aviumcomplex, no such antigens have been found. Through a comparative genomics approach, 21 potential coding sequences ofM. aviumsubsp.paratuberculosisthat are not represented in any other mycobacterial species tested (n= 9) were previously identified (J. P. Bannantine, E. Baechler, Q. Zhang, L. Li, and V. Kapur, J. Clin. Microbiol.40:1303-1310, 2002). Here we describe the cloning, heterologous expression, and antigenic analysis of theseM. aviumsubsp.paratuberculosis-specific sequences inEscherichia coli. Nucleotide sequences representing each unique predicted coding region were amplified and cloned into two differentE. coliexpression vectors encoding polyhistidine or maltose binding protein (MBP) affinity purification tags. All 21 of the MBP fusion proteins were successfully purified under denaturing conditions and were evaluated in immunoblotting studies with sera from rabbits and mice immunized withM. aviumsubsp.paratuberculosis. These studies showed that 5 of the 21 gene products are produced byM. aviumsubsp.paratuberculosisand are antigenic. Immunoblot analysis with a panel of sera from 9 healthy cattle and 10 cattle with clinical disease shows that the same fiveM. aviumsubsp.paratuberculosisproteins are also detected within the context of infection. Collectively, these studies have used a genomic approach to identify novelM. aviumsubsp.paratuberculosisantigens that are not present in any other mycobacteria. These findings may have a major impact on improved diagnostics for Johne's disease.
Direct Comparison of the BACTEC 9240 and BacT/ALERT 3D Automated Blood Culture Systems for
Candida
Growth Detection
Authors: Horvath Lynn L. , George Benjamin J. , Murray Clinton K. , Harrison Linda S. , Hospenthal Duane R.
Published: January 2004

A direct comparison of two automated blood culture systems was conducted to compare their ability to detect
Candida
growth. The systems evaluated were the BACTEC 9240 (Bactec) and BacT/ALERT 3D (BacT). The aerobic, anaerobic, and mycology media for each system were evaluated: Bactec Plus Aerobic/F, Plus Anaerobic/F, and Myco/F Lytic bottles,, respectively, and BacT FA, SN, and MB bottles, respectively. Each blood culture bottle was inoculated with fresh blood from healthy donors. Fifty isolates of
Candida
spp. were used. The six different blood culture bottles were each inoculated with 1,000 yeasts per bottle and then incubated in the corresponding automated system. The BacT detected growth of 90% (135 of 150) of
Candida
pathogens, while Bactec detected 66% (100 of 150). Growth was detected in all BacT and Bactec mycology bottles, all BacT aerobic bottles, and by terminal subculture of all bottles. Sixty-five of 300 (22%) bottles had no growth detected; 50 from the Bactec (5 aerobic and 45 anaerobic) and 15 from the BacT (all anaerobic). Terminal subculture of “negative” bottles demonstrated viable yeast growth from all 65 bottles, representing 65 false-negatives. The mean time to growth detection in the BacT system was 25.62 h while the Bactec was 27.30 h (
P
< 0.01). Both automated blood culture systems detected all episodes of simulated candidemia when specialized mycology media were used. However, when only standard aerobic and anaerobic media were used, the BacT performed better than the Bactec in overall growth detection, time to growth detection, and number of false-negatives.