bluuebunny/crossref_metadata_2025 huggingface dataset:
Risk factors for hepatitis C virus infection among blood donors in northern Thailand
Authors: Thaikruea Lakkana , Thongsawat Satawat , Maneekarn Niwat , Netski Dale , Thomas David L. , Nelson Kenrad E.
Published: September 2004

BACKGROUND: The epidemiology, virology, and risk factors for hepatitis C virus (HCV) infection among blood donors in northern Thailand have not been extensively evaluated.

STUDY DESIGN AND METHODS: We did a prospective matched case‐control study of blood donors who tested positive for HCV and were confirmed by recombinant immunoblot assay or nucleic acid testing. Infected donors were matched with one to four HCV‐uninfected donors for sex, age ± 5 years, and donation at the same site within 15 days of the HCV‐positive donor. Married donors were invited to bring their spouse for HCV testing.

RESULTS: Among 166 matched sets, a history of intravenous drug use (IDU), reported by 58 HCV infected donors (35.5%) and 2 HCV‐negative donors, was strongly associated with HCV infection (odds ratio [OR], 107.6; 95% confidence interval, 14.8‐780.7). In multivariate analysis among donors without a history of IDU, significant risk factors included a history of a blood transfusion (OR, 28.8), immediate family with a history of hepatitis/jaundice (OR, 4.4), six or more lifetime sexual partners (OR, 2.7), and increased frequency of blood donation (OR, 0.9). Six of 45 spouses of HCV‐infected donors, and none of 44 spouses of uninfected donors, were HCV positive (p = 0.005).

CONCLUSION: Our data indicate that illicit IDU and a history of transfusion are important risk factors for HCV infection in Thailand. Also, our data suggest there may be some risk of transmission by sex or other close contact between spouses.
Daily doses of 20 mg of elemental iron compensate for iron loss in regular blood donors: a randomized, double‐blind, placebo‐controlled study
Authors: Radtke Hartmut , Tegtmeier Joanna , Röcker Lothar , Salama Abdulgabar , Kiesewetter Holger
Published: September 2004

BACKGROUND: A considerable number of regular blood donors develops an iron deficiency, and the exact amount of iron required to compensate for the iron loss from whole‐blood donation in males and females is still unknown.

STUDY DESIGN AND METHODS: A total of 526 regular blood donors (289 male and 237 female) were randomly assigned to treatment with either 40 mg, 20 mg, or 0 mg per day of elemental iron as ferrous gluconate for a period of 6 months, during which one unit of whole blood was collected on four occasions (males) or three occasions (females). Hemoglobin level, serum ferritin, and soluble transferrin receptor levels were measured before each donation.

RESULTS: Daily doses of either 40 mg or 20 mg of elemental iron adequately compensated for iron loss in males, who gave blood at 2‐month intervals, but did not result in a positive iron balance or an increase in storage iron as reflected by the logarithm of the ratio of transferrin receptor to ferritin concentration. In females, who donated at 3‐month intervals, the same daily doses not only restored the iron balance but also led to an increase in storage iron. The number of gastrointestinal side effects due to iron supplementation (12%) was only slightly higher in both iron groups than in the placebo group.

CONCLUSION: The results of this study indicate that 20 mg of elemental iron per day can adequately compensate for iron loss in males and females who donate whole blood up to four (females) or six times per year (males).
Toxicities of hemoglobin solutions: in search of in‐vitro and in‐vivo model systems
Authors: Buehler Paul W. , Alayash Abdu I.
Published: September 2004

Several hemoglobin‐based oxygen carriers (HBOCs) have been developed with a rationale focused on exploiting one or more physicochemical properties (e.g., oxygen affinity, molecular weight, viscosity, and colloid osmotic pressure) resulting from the chemical or recombinant modification of hemoglobin (Hb). Several chemically modified Hbs have reached late stages of clinical evaluation in the United States and Canada. These Hbs, in general, demonstrated mixed preclinical safety and efficacy, and reasonable safety in Phase I trials. However, as clinical development shifted into later stages, an undesirable safety and efficacy profile became clear in patient populations studied, and as a result some products were withdrawn from further clinical pursuit. Several questions still remain unanswered regarding the safety of Hb products for their proposed clinical indication(s). For example, 1) were preclinical studies predictive of clinical outcome? And, 2) were the most appropriate preclinical studies performed to predict clinical outcome?

The primary objectives of this analysis are to explore prelinical safety issues associated with HBOCs and provide an overview of the in‐vitro and in‐vivo models employed. The methods for obtaining data to serve as a basis for discussion are compiled from a literature‐based survey of safety and efficacy derived from biochemical, cellular, and whole animal assessment of HBOCs. Results from this overview of a vast body of published data may provide a means for identifying critical preclinical safety issues, which may ultimately lead to identification of potential limitations in the effective clinical use of certain HBOCs.
Patients with pertrochanteric hip fracture may benefit from preoperative intravenous iron therapy: a pilot study
Authors: Cuenca Jorge , García‐Erce José A. , Muñoz Manuel , Izuel Mónica , Martínez Angel A. , Herrera Antorio
Published: September 2004

BACKGROUND:  Patients undergoing pertrochanteric hip fracture (PHF) repair surgery often receive perioperative allogeneic blood transfusions (ABTs) to avoid the deleterious effects of anemia. Nevertheless, concerns about adverse effects of ABTs have prompted the review of transfusion practice and the search for a safer treatment of perioperative anemia.

MATERIAL AND METHODS:  The effect of preoperative 200 to 300 mg of intravenous (IV; Group 2; n = 55) iron sucrose administration on transfusion requirements and postoperative morbidity‐mortality in patients with PHF has been prospectively investigated. A previous series of 102 PHF patients served as the control group (Group 1). All patients were older than 65 years and were operated on at the third day after admission to the hospital, by the same medical team and with the same implant.

RESULTS:  Iron sucrose was well tolerated and reduced the transfusion rate in patients with admission hemoglobin levels of greater than 120 g per L (p < 0.05) who also received fewer units of red blood cells (p < 0.05). In addition, iron sucrose reduced postoperative infection rate (p < 0.05), but not 30‐day mortality rate or mean length of hospital stay.

CONCLUSION:  The administration of IV iron sucrose seems to reduce ABT requirements in patients with PHF and is associated to lower postoperative morbidity. The possible mechanisms involved in these effects are discussed.
The success of precaution? Managing the risk of transfusion transmission of variant Creutzfeldt‐Jakob disease
Authors: Wilson Kumanan , Ricketts Maura N.
Published: September 2004

The precautionary principle has emerged as an important new paradigm influencing decision making in the blood system. The principle has influenced decision making in several nations leading to the institution of policies to protect their blood supplies form variant Creutzfeldt‐Jakob disease (vCJD). Increasingly evidence has emerged to support the institution of these policies, which were introduced in advance of clear evidence of risk. These vCJD decisions serve as an example of the successful application of precaution and provide lesions as to how the principle should be applied in future transfusion safety decisions.
A Comparison of Arbitration Procedures for Risk‐Averse Disputants*
Authors: Armstrong Michael J.
Published: September 2004

We propose an arbitration model framework that generalizes many previous quantitative models of final offer arbitration, conventional arbitration, and some proposed alternatives to them. Our model allows the two disputants to be risk averse and assumes that the issue(s) in dispute can be summarized by a single quantifiable value. We compare the performance of the different arbitration procedures by analyzing the gap between the disputants' equilibrium offers and the width of the contract zone that these offers imply. Our results suggest that final offer arbitration should give results superior to those of conventional arbitration.
Linking Perceived Quality and Customer Satisfaction to Store Traffic and Revenue Growth*
Authors: Babakus Emin , Bienstock Carol C. , Van Scotter James R.
Published: September 2004

Effects of perceived merchandise and service quality, relative to competition, on retail store performance are investigated using store traffic and revenue growth as outcome variables. A model is proposed and tested using aggregate customer data and store performance outcomes from a group of stores owned by a national retail organization. Results suggest that both service and merchandise quality exert significant influence on store performance, measured by sales growth and customer growth, and their impact is mediated by customer satisfaction. Implications of the results and future research directions are discussed.
Is the Precautionary Principle Used to Cover Up Ignorance?
Authors: Tuomisto Jouko
Published: September 2004

Abstract: The precautionary principle is rapidly spreading in risk management in spite of vague understanding of its consequences. Some caveats are presented in this commentary, and some principles proposed for improved risk assessment/risk management procedures. Improvement is especially needed in such cases where the risks of different management options must be compared.
Unsuccessful Radioiodine Treatment of a Non‐Toxic Goiter: A Case Report
Authors: Hartoft‐Nielsen Marie‐Louise , Rasmussen Åse Krogh , Friis Esbern , Blichert‐Toft Mogens , Buhl Thora , Hesse Birger , Feldt‐Rasmussen Ulla
Published: September 2004

Abstract: A middle‐aged woman with a large right‐sided, non‐toxic goiter with low iodine uptake was admitted to the Department of Endocrinology with the purpose of volume reduction of the goiter. Thyroid pertechnetate scintigraphy showed homogenous and diffuse uptake in both lobes. Initially thyroxine treatment was given without volume‐reducing effect. Radioiodine was administered twice to deliver a total radiation dose of 70 mCi iodine (I)‐131. Subsequent pertechnetate scintigraphy showed that the normal‐sized, normally functioning left lobe had disappeared after radioiodine, whereas the enlarged right lobe appeared unchanged. During the following years the size of the right lobe increased, and compression symptoms developed. The thyroid gland finally had to be removed by surgery. A large solitary thyroid nodule was removed, but no left lobe was identified. After surgery the patient had no thyroid tissue and had to be substituted by thyroid hormones. Despite good results of iodine treatment of non‐toxic goiters, this case describes an unintended outcome leaving a patient without thyroid tissue, and a protracted course could have been avoided if the patient had undergone surgery earlier. However, this reported case should not discredit the use of radioiodine treatment of non‐toxic goiters, but focus on patients with a single large solitary adenoma in whom this treatment may be inappropriate.
Functional Analysis of the Tsh Autotransporter from an Avian Pathogenic
Escherichia coli
Strain
Authors: Kostakioti Maria , Stathopoulos Christos
Published: September 2004

The temperature-sensitive hemagglutinin (Tsh) is an autotransporter protein secreted by avian-pathogenic
Escherichia coli
strains that colonize the respiratory tract and lead to airsacculitis, pericarditis, and colisepticemia. It is synthesized as a 140-kDa precursor protein, whose processing results in a 106-kDa passenger domain (Tsh
s
) and a 33-kDa β-domain (Tsh
β
). The presence of a conserved 7-amino-acid serine protease motif within Tsh
s
classifies the protein in a subfamily of autotransporters, known as serine protease autotransporters of the
Enterobacteriaceae
. In this study, we report that purified Tsh
s
is capable of adhering to red blood cells, hemoglobin, and the extracellular matrix proteins fibronectin and collagen IV. We also demonstrate that Tsh
s
exerts proteolytic activity against casein, and we provide experimental evidence demonstrating that serine 259 is essential for the protease function. However, this residue is not required for adherence to substrates, and its replacement by an alanine does not abolish binding activity. In summary, our results demonstrate that Tsh is a bifunctional protein with both adhesive and proteolytic properties.
Polymorphism and Transcription at the
p44
-
1/p44
-
18
Genomic Locus in
Anaplasma phagocytophilum
Strains from Diverse Geographic Regions
Authors: Lin Quan , Rikihisa Yasuko , Massung Robert F. , Woldehiwet Zerai , Falco Richard C.
Published: September 2004

A polymorphic multigene family (
p44
) of
Anaplasma phagocytophilum
encodes the immunodominant 44-kDa major outer membrane proteins. With
p44
-specific PCR and gene-specific probes,
p44
-
1
was found in all human isolates from New York State but not in isolates from Minnesota, whereas
p44
-
18
and two other
p44
species were found in isolates from both regions. We therefore sequenced the genomic locus corresponding to the
p44
-
1/p44
-
18
tandem locus of
A. phagocytophilum
HZ in 14 other geographically divergent strains from various hosts. The locus was found in all 14 strains, and
p44
-
18
was conserved among all 13 United States isolates studied. In all nine northeastern strains,
p44
-
1
was conserved. However, in three of the Minnesota strains and in one California strain,
p44
-
1
was replaced at this genomic locus by the novel gene
p44
-
61
(
p44
-
61/18
), whose hypervariable region (hv) was a chimera of
p44
-
20
hv and
p44
-
23
hv. The conserved base sequence within the hv region linked the two segments. In contrast, in the Old Sourhope strain isolated from sheep in the United Kingdom, only a single and distinct
p44
,
p44
-
OS
, was found in this locus. This suggests different rates of evolution of
p44
-
1
and
p44
-
18
at this locus and conservation of the locus within strains isolated from the same geographic region. Locus-specific reverse transcription-PCR revealed expression of
p44
-
1
by New York and
p44
-
61
by Minnesota strains at this locus. These
p44
loci provide insight into the molecular evolution and functional divergence of
p44
paralogs and may serve as markers for typing strains from different geographic regions.
Protection againstPlasmodium chabaudiMalaria Induced by Immunization with Apical Membrane Antigen 1 and Merozoite Surface Protein 1 in the Absence of Gamma Interferon or Interleukin-4
Authors: Burns James M. , Flaherty Patrick R. , Nanavati Payal , Weidanz William P.
Published: September 2004

Strategies to optimize formulations of multisubunit malaria vaccines require a basic knowledge of underlying protective immune mechanisms induced by each vaccine component. In the present study, we evaluated the contribution of antibody-mediated and cell-mediated immune mechanisms to the protection induced by immunization with two blood-stage malaria vaccine candidate antigens, apical membrane antigen 1 (AMA-1) and merozoite surface protein 1 (MSP-1). Immunologically intact or selected immunologic knockout mice were immunized with purified recombinantPlasmodium chabaudiAMA-1 (PcAMA-1) and/or the 42-kDa C-terminal processing fragment ofP. chabaudiMSP-1 (MSP-142). The efficacy of immunization in each animal model was measured as protection against blood-stageP. chabaudimalaria. Immunization of B-cell-deficient JH−/−mice indicated that PcAMA-1 vaccine-induced immunity is largely antibody dependent. In contrast, JH−/−mice immunized with PcMSP-142were partially protected againstP. chabaudimalaria, indicating a role for protective antibody-dependent and antibody-independent mechanisms of immunity. The involvement of γδ T cells in vaccine-induced PcAMA-1 and/or PcMSP-142protection was minor. Analysis of the isotypic profile of antigen-specific antibodies induced by immunization of immunologically intact mice revealed a dominant IgG1 response. However, neither interleukin-4 and the production of IgG1 antibodies nor gamma interferon and the production of IgG2a/c antibodies were essential for PcAMA-1 and/or PcMSP-142vaccine-induced protection. Therefore, for protective antibody-mediated immunity, vaccine adjuvants and delivery systems for AMA-1- and MSP-1-based vaccines can be selected for their ability to maximize responses irrespective of IgG isotype or any Th1 versus Th2 bias in the CD4+-T-cell response.
Growth, Virulence, and Immunogenicity ofListeria monocytogenes aroMutants
Authors: Stritzker Jochen , Janda Jozef , Schoen Christoph , Taupp Marcus , Pilgrim Sabine , Gentschev Ivaylo , Schreier Peter , Geginat Gernot , Goebel Werner
Published: September 2004

Mutants ofListeria monocytogeneswith deletions in genes of the common branch of the biosynthesis pathway leading to aromatic compounds were constructed as possible virulence-attenuated carrier strains for protein antigens or vaccine DNA.aroA,aroB, and in particulararoEmutants showed strongly reduced growth rates in epithelial cells and even in rich culture media. The metabolism of thearomutants under these conditions was predominantly anaerobic. Aerobic metabolism and a wild-type growth rate were, however, regained upon the addition of vitamin K2, suggesting that thearomutants are deficient in oxidative respiration due to the lack of menaquinone. Replication of thearomutants in the host cell's cytosol and cell-to-cell spread were drastically slowed down, and allaromutants showed high virulence attenuation in mice, i.e., the 50% lethal dose in BALB/c mice was increased at least 104-fold for thearoA,aroB, andaroA/Bmutants and >105-fold for thearoEmutant compared to the parent strain. Nevertheless, mice preimmunized witharomutant bacteria elicited good T-cell response and full protection against a subsequent challenge with the virulent wild-type strain. A total of 5 × 106aroA,aroB, andaroA/Bmutant bacteria were sufficient to obtain a protective T-cell response, while 5 × 108aroEoraroA/Emutants were necessary to achieve comparable numbers of antigen-specific T cells. These numbers were well tolerated without causing any signs of disease, indicating thatListeriastrains with deletions in genes of the basic branch of the aromatic amino acid pathway could be useful vaccine carriers for inducing T-cell immunity.
Serum Levels of the Proinflammatory Cytokines Interleukin-1 Beta (IL-1β), IL-6, IL-8, IL-10, Tumor Necrosis Factor Alpha, and IL-12(p70) in Malian Children with SeverePlasmodium falciparumMalaria and Matched Uncomplicated Malaria or Healthy Controls
Authors: Lyke K. E. , Burges R. , Cissoko Y. , Sangare L. , Dao M. , Diarra I. , Kone A. , Harley R. , Plowe C. V. , Doumbo O. K. , Sztein M. B.
Published: September 2004

Inflammatory cytokines play an important role in human immune responses to malarial disease. However, the role of these mediators in disease pathogenesis, and the relationship between host protection and injury remains unclear. A total of 248 cases of severePlasmodium falciparummalaria among children aged 3 months to 14 years residing in Bandiagara, Mali, were matched to cases of uncomplicated malaria and healthy controls. Using modified World Health Organization criteria for defining severe malaria, we identified 100 cases of cerebral malaria (coma, seizure, and obtundation), 17 cases of severe anemia (hemoglobin, <5 g/dl), 18 cases combined cerebral malaria with severe anemia, and 92 cases with hyperparasitemia (asexual trophozoites, >500,000/mm3). Significantly elevated levels (given as geometric mean concentrations in picograms/milliliter) of interleukin-6 (IL-6; 485.2 versus 54.1;P= <0.001), IL-10 (1,099.3 versus 14.1;P= <0.001), tumor necrosis factor alpha (10.1 versus 7.7;P= <0.001), and IL-12(p70) (48.9 versus 31.3;P= 0.004) in serum were found in severe cases versus healthy controls. Significantly elevated levels of IL-6 (485.2 versus 141.0;P= <0.001) and IL-10 (1,099.3 versus 133.9;P= <0.001) were seen in severe malaria cases versus uncomplicated malaria controls. Cerebral malaria was associated with significantly elevated levels of IL-6 (754.5 versus 311.4;P= <0.001) and IL-10 (1,405.6 versus 868.6;P= 0.006) compared to severe malaria cases without cerebral manifestations. Conversely, lower levels of IL-6 (199.2 versus 487.6;P= 0.03) and IL-10 (391.1 versus 1,160.9;P= 0.002) were noted in children with severe anemia compared to severe malaria cases with hemoglobin at >5 g/dl. Hyperparasitemia was associated with significantly lower levels of IL-6 (336.6 versus 602.1;P= 0.002). These results illustrate the complex relationships between inflammatory cytokines and disease inP. falciparummalaria.
Immunization withLeishmania majorExogenous Antigens Protects Susceptible BALB/c Mice against Challenge Infection withL. major
Authors: Tonui Willy K. , Mejia J. Santiago , Hochberg Lisa , Mbow M. Lamine , Ryan Jeffrey R. , Chan Adeline S. T. , Martin Samuel K. , Titus Richard G.
Published: September 2004

The potential ofLeishmania majorculture-derived soluble exogenous antigens (SEAgs) to induce a protective response in susceptible BALB/c mice challenged withL. majorpromastigotes was investigated. Groups of BALB/c mice were immunized withL. majorSEAgs alone,L. majorSEAgs coadministered with either alum (aluminum hydroxide gel) or recombinant murine interleukin-12 (rmIL-12),L. majorSEAgs coadministered with both alum and rmIL-12, andL. majorSEAgs coadministered with Montanide ISA 720. Importantly and surprisingly, the greatest and most consistent protection against challenge withL. majorwas seen in mice immunized withL. majorSEAgs alone, in the absence of any adjuvant. Mice immunized withL. majorSEAgs had significantly smaller lesions that at times contained more than 100-fold fewer parasites. When lymphoid cells fromL. majorSEAg-immunized mice were stimulated with leishmanial antigen in vitro, they proliferated and secreted a mixed profile of type 1 and type 2 cytokines. Finally, analyses with Western blot analyses and antibodies against three surface-expressed and secreted molecules ofL. major(lipophosphoglycan, gp46/M2/PSA-2, and gp63) revealed that two of these molecules are present inL. majorSEAgs, lipophosphoglycan and the molecules that associate with it and gp46/M2/PSA-2.
TRAF6-Dependent Mitogen-Activated Protein Kinase Activation Differentially Regulates the Production of Interleukin-12 by Macrophages in Response toToxoplasma gondii
Authors: Mason Nicola J. , Fiore Jim , Kobayashi Takashi , Masek Katherine S. , Choi Yongwon , Hunter Christopher A.
Published: September 2004

The production of interleukin-12 (IL-12) is critical to the development of innate and adaptive immune responses required for the control of intracellular pathogens. Many microbial products signal through Toll-like receptors (TLR) and activate NF-κB family members that are required for the production of IL-12. Recent studies suggest that components of the TLR pathway are required for the production of IL-12 in response to the parasiteToxoplasma gondii; however, the production of IL-12 in response to this parasite is independent of NF-κB activation. The adaptor molecule TRAF6 is involved in TLR signaling pathways and associates with serine/threonine kinases involved in the activation of both NF-κB and mitogen-activated protein kinase (MAPK). To elucidate the intracellular signaling pathways involved in the production of IL-12 in response to soluble toxoplasma antigen (STAg), wild-type and TRAF6−/−mice were inoculated with STAg, and the production of IL-12(p40) was determined. TRAF6−/−mice failed to produce IL-12(p40) in response to STAg, and TRAF6−/−macrophages stimulated with STAg also failed to produce IL-12(p40). Studies using Western blot analysis of wild-type and TRAF6−/−macrophages revealed that stimulation with STAg resulted in the rapid TRAF6-dependent phosphorylation of p38 and extracellular signal-related kinase, which differentially regulated the production of IL-12(p40). The studies presented here demonstrate for the first time that the production of IL-12(p40) in response to toxoplasma is dependent upon TRAF6 and p38 MAPK.
Wolbachia-Induced Neutrophil Activation in a Mouse Model of Ocular Onchocerciasis (River Blindness)
Authors: Gillette-Ferguson Illona , Hise Amy G. , McGarry Helen F. , Turner Joseph , Esposito Andrew , Sun Yan , Diaconu Eugenia , Taylor Mark J. , Pearlman Eric
Published: September 2004

EndosymbioticWolbachiabacteria are abundant in the filarial nematodes that cause onchocerciasis (river blindness), including the larvae (microfilariae) that migrate into the cornea. Using a mouse model of ocular onchocerciasis, we recently demonstrated that it is these endosymbiotic bacteria rather than the nematodes per se that induce neutrophil infiltration to the corneal stroma and loss of corneal clarity (Saint Andre et al., Science295:1892-1895, 2002). To better understand the role ofWolbachiaorganisms in the pathogenesis of this disease, we examined the fate of these bacteria in the cornea by immunoelectron microscopy. Microfilariae harboringWolbachiaorganisms were injected into mouse corneas, and bacteria were detected with antibody toWolbachiasurface protein. Within 18 h of injection, neutrophils completely surrounded the nematodes and were in close proximity toWolbachiaorganisms.Wolbachiasurface protein labeling was also prominent in neutrophil phagosomes, indicating neutrophil ingestion ofWolbachiaorganisms. Furthermore, the presence of numerous electron-dense granules around the phagosomes indicated that neutrophils were activated. To determine ifWolbachiaorganisms directly activate neutrophils, peritoneal neutrophils were incubated with either parasite extracts containingWolbachiaorganisms, parasite extracts depleted ofWolbachiaorganisms (by antibiotic treatment of worms), orWolbachiaorganisms isolated from filarial nematodes. After 18 h of incubation, we found that isolatedWolbachiaorganisms stimulated production of tumor necrosis factor alpha and CXC chemokines macrophage inflammatory protein 2 and KC by neutrophils in a dose-dependent manner. Similarly, these cytokines were induced by filarial extracts containingWolbachiaorganisms but not byWolbachia-depleted extracts. Taken together, these findings indicate that neutrophil activation is an important mechanism by whichWolbachiaorganisms contribute to the pathogenesis of ocular onchocerciasis.
Release of Periplasmic Proteins of
Brucella suis
upon Acidic Shock Involves the Outer Membrane Protein Omp25
Authors: Boigegrain Rose-Anne , Salhi Imed , Alvarez-Martinez Maria-Teresa , Machold Jan , Fedon Yann , Arpagaus Martine , Weise Christoph , Rittig Michael , Rouot Bruno
Published: September 2004

The survival and replication of
Brucella
in macrophages is initially triggered by a low intraphagosomal pH. In order to identify proteins released by
Brucella
during this early acidification step, we analyzed
Brucella suis
conditioned medium at various pH levels. No significant proteins were released at pH 4.0 in minimal medium or citrate buffer, whereas in acetate buffer,
B. suis
released a substantial amount of soluble proteins. Comparison of 13 N-terminal amino acid sequences determined by Edman degradation with their corresponding genomic sequences revealed that all of these proteins possessed a signal peptide indicative of their periplasmic location. Ten proteins are putative substrate binding proteins, including a homologue of the nopaline binding protein of
Agrobacterium tumefaciens
. The absence of this homologue in
Brucella melitensis
was due to the deletion of a 7.7-kb DNA fragment in its genome. We also characterized for the first time a hypothetical 9.8-kDa basic protein composed of five amino acid repeats. In
B. suis
, this protein contained 9 repeats, while 12 were present in the
B. melitensis
orthologue.
B. suis
in acetate buffer depended on neither the
virB
type IV secretory system nor the
omp31
gene product. However, the integrity of the
omp25
gene was required for release at acidic pH, while the absence of
omp25b
or
omp25c
displayed smaller effects. Together, these results suggest that Omp25 is involved in the membrane permeability of
Brucella
in acidic medium.
The Phosphatidylinositol 3-Kinase/Protein Kinase B Signaling Pathway Is Activated by Lipoteichoic Acid and Plays a Role in Kupffer Cell Production of Interleukin-6 (IL-6) and IL-10
Authors: Dahle Maria K. , Øverland Gunhild , Myhre Anders E. , Stuestøl Jon Fredrik , Hartung Thomas , Krohn Claus Danckert , Mathiesen Øystein , Wang Jacob E. , Aasen Ansgar O.
Published: September 2004

Sepsis caused by gram-positive bacteria lacking lipopolysaccharide (LPS) has become a major and increasing cause of mortality in intensive-care units. We have recently demonstrated that the gram-positive-specific bacterial cell wall component lipoteichoic acid (LTA) stimulates the release of the proinflammatory cytokines in Kupffer cells in culture. In the present study, we have started to assess the signal transduction events by which LTA induces the production of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and the anti-inflammatory cytokine IL-10 in rat Kupffer cells. LTA was found to trigger phosphorylation of mitogen-activated protein kinases (MAPK) (p38 MAPK and ERK 1/2) and protein kinase B (PKB). Compared to LPS, LTA was more potent in inducing PKB phosphorylation after 40 min, although we found that the cytokine responses were similar. For both bacterial molecules, blocking phosphatidylinositol 3-kinase (PI3-K; Ly294002) or Janus kinase 2 (JAK-2; AG490) particularly affected the induction of IL-6 and IL-10 release, whereas TNF-α levels were strongly reduced by inhibition of Src family tyrosine kinases (PP2). All three cytokines were reduced by inhibition of p38 MAPK (SB202190) or the broad-range tyrosine kinase inhibitor genistein, whereas IL-6 release was particularly blocked by inhibition of ERK 1/2 (PD98059). Divergences in the regulatory pathways controlling TNF-α, IL-10, and IL-6 production in Kupffer cells following LPS or LTA stimulation may create a basis for understanding how the balance between pro- and anti-inflammatory cytokines is regulated in the liver following infections by gram-positive or gram-negative bacteria.
Escherichia coli
Cytotoxic Necrotizing Factor 1 Inhibits Intestinal Epithelial Wound Healing In Vitro after Mechanical Injury
Authors: Brest Patrick , Turchi Laurent , Le'Negrate Gaëlle , Berto Frédérick , Moreilhon Chimène , Mari Bernard , Ponzio Gilles , Hofman Paul
Published: September 2004

Cytotoxic necrotizing factor type 1 (CNF1) from
Escherichia coli
activates the small GTP-binding proteins of the Rho family (Rho, Rac, and Cdc42) by catalyzing their deamidation at a specific glutamine residue. Since RhoA, Rac, and Cdc42 play a pivotal role in cell migration during the early phase of wound repair, we investigated whether CNF1 was able to interfere with wound healing in intestinal epithelial monolayers (T84 cells). After mechanical injury, we found that CNF1 blocks epithelial wound repair within 48 h. This effect was characterized by cell elongation and filopodium formation on the leading edge, in association with permanent phosphorylation of the focal adhesion kinase (FAK) via Rho activation. Moreover, inhibition of Rho kinase with Y-27632 decreased CNF1-mediated permanent FAK phosphorylation, leading to complete restitution of wound repair within 24 h. In addition, we found that CNF1 induced upregulation of mitogen-activated protein kinases (MAPK) activation. Moreover, activation of Rac and MAPK by CNF1 increased matrix metalloproteinase 9 expression in wounded T84 monolayers. Taken together, these results provide evidence that CNF1 strongly impairs intestinal epithelial wound healing.
Increased Rate of Apoptosis and Diminished Phagocytic Ability of Human Neutrophils Infected with Afa/Dr Diffusely AdheringEscherichia coliStrains
Authors: Brest Patrick , Bétis Frédéric , Çuburu Nicolas , Selva Eric , Herrant Magali , Servin Alain , Auberger Patrick , Hofman Paul
Published: September 2004

The proinflammatory effect of Afa/Dr diffusely adheringEscherichia coli(Afa/Dr DAEC) strains have been recently demonstrated in vitro by showing that polymorphonuclear leukocyte (PMN) transepithelial migration is induced after bacterial colonization of apical intestinal monolayers. The effect of Afa/Dr DAEC-PMN interaction on PMN behavior has been not investigated. Because of the putative virulence mechanism of PMN apoptosis during infectious diseases and taking into account the high level of expression of the decay-accelerating factor (DAF, or CD55), the receptor of Afa/Dr DAEC on PMNs, we sought to determine whether infection of PMNs by Afa/Dr DAEC strains could promote cell apoptosis. We looked at the behavior of PMNs incubated with Afa/Dr DAEC strains once they had transmigrated across polarized monolayers of intestinal (T84) cells. Infection of PMNs by Afa/Dr DAEC strains induced PMN apoptosis characterized by morphological nuclear changes, DNA fragmentation, caspase activation, and a high level of annexin V expression. However, transmigrated and nontransmigrated PMNs incubated with Afa/Dr DAEC strains showed similar elevated global caspase activities. PMN apoptosis depended on their agglutination, induced by Afa/Dr DAEC, and was still observed after preincubation of PMNs with anti-CD55 and/or anti-CD66 antibodies. Low levels of phagocytosis of Afa/Dr DAEC strains were observed both in nontransmigrated and in transmigrated PMNs compared to that observed with the controlE. coliDH5α strain. Taken together, these data strongly suggest that interaction of Afa/Dr DAEC with PMNs may increase the bacterial virulence both by inducing PMN apoptosis through an agglutination process and by diminishing their phagocytic capacity.
Borrelia burgdorferiChanges Its Surface Antigenic Expression in Response to Host Immune Responses
Authors: Liang Fang Ting , Yan Jun , Mbow M. Lamine , Sviat Steven L. , Gilmore Robert D. , Mamula Mark , Fikrig Erol
Published: September 2004

The Lyme disease spirochete,Borrelia burgdorferi, causes persistent mammalian infection despite the development of vigorous immune responses against the pathogen. To examine spirochetal phenotypes that dominate in the hostile immune environment, the mRNA transcripts of four prototypic surface lipoproteins, decorin-binding protein A (DbpA), outer surface protein C (OspC), BBF01, and VlsE, were analyzed by quantitative reverse transcription-PCR under various immune conditions. We demonstrate thatB. burgdorferichanges its surface antigenic expression in response to immune attack.dbpAexpression was unchanged while the spirochetes decreasedospCexpression by 446 times and increased BBF01 andvlsEexpression up to 20 and 32 times, respectively, under the influence of immune pressure generated in immunocompetent mice during infection. This change in antigenic expression could be induced by passively immunizing infected severe combined immunodeficiency mice with specificBorreliaantisera or OspC antibody and appears to allowB. burgdorferito resist immune attack.
Comparison of Immunogenicities of RecombinantPlasmodium vivaxMerozoite Surface Protein 1 19- and 42-Kilodalton Fragments Expressed inEscherichia coli
Authors: Sachdeva Suraksha , Ahmad Gul , Malhotra Pawan , Mukherjee Paushali , Chauhan V. S.
Published: September 2004

The 42- and 19-kDa C-terminal fragments of merozoite surface protein 1 (MSP-142and MSP-119, respectively) are both promising blood-stage vaccine candidate antigens. At present, it is not clear which of the two antigens will be more suitable for inclusion in a cocktail malaria vaccine. In the present study, we expressed the two C-terminal fragments ofPlasmodium vivaxMSP-1 (PvMSP-1) in anEscherichia coliexpression system and purified them by using a rapid two-step protocol. Both of the products were recognized by monoclonal antibodies against PvMSP-1 as well as by immune sera from several individuals exposed toP. vivax. We analyzed and compared the immunological responses to recombinant PvMSP-119and PvMSP-142in mice by using six different adjuvant formulations. Moderate to high antibody responses were observed with both of the antigens in different adjuvant formulations. Surprisingly, alum, which is generally considered to be a poor adjuvant for recombinant malaria antigens, was found to be as good an adjuvant as Montanide ISA 720, ASO2A, and other adjuvant formulations. Most adjuvant formulations induced high levels of immunoglobulin G1 (IgG1), followed by IgG3 and IgG2. Lymphocytes from animals in the PvMSP-142- and PvMSP-119-immunized groups showed proliferative responses upon stimulation with the respective antigens, and high levels of interleukin-4 (IL-4), IL-5, and gamma interferon were detected in the culture supernatants. Immunodepletion studies with sera from mice immunized with these two antigens showed that while immunization with PvMSP-142does produce a PvMSP-119-specific response, a substantial portion is also focused on structures in PvMSP-142not represented by the epidermal growth factor-like domains of PvMSP-119. These findings may have implications for the design of MSP-1-based vaccine constructs.
The Ton System, an ABC Transporter, and a Universally Conserved GTPase Are Involved in Iron Utilization by
Brucella melitensis
16M
Authors: Danese Isabelle , Haine Valerie , Delrue Rose-May , Tibor Anne , Lestrate Pascal , Stevaux Olivier , Mertens Pascal , Paquet Jean-Yves , Godfroid Jacques , De Bolle Xavier , Letesson Jean-Jacques
Published: September 2004

Brucella
spp. are gram-negative intracellular facultative pathogens that are known to produce 2,3-dihydroxybenzoic acid (DHBA), a catechol siderophore that is essential for full virulence in the natural host. The mechanism of DHBA entry into
Brucella
and other gram-negative bacteria is poorly understood. Using mini-Tn
5Kmcat
mutagenesis, we created a transposon library of
Brucella melitensis
16M and isolated 32 mutants with a defect in iron acquisition or assimilation. Three of these transposon mutants are deficient in utilization of DHBA. Analysis of these three mutants indicated that the ExbB, DstC, and DugA proteins are required for optimal assimilation of DHBA and/or citrate. ExbB is part of the Ton complex, and DstC is a permease homologue of an iron(III) ABC transporter; in gram-negative bacteria these two complexes are involved in the uptake of iron through the outer and inner membranes, respectively. DugA is a new partner in iron utilization that exhibits homology with the bacterial conserved GTPase YchF. Based on this homology, DugA could have a putative regulatory function in iron assimilation in
Brucella
. None of the three mutants was attenuated in cellular models or in the mouse model of infection, which is consistent with the previous suggestion that DHBA utilization is not required in these models.
Limited Role of Antibody in Clearance of
Streptococcus pneumoniae
in a Murine Model of Colonization
Authors: McCool Tera L. , Weiser Jeffrey N.
Published: September 2004

Colonization is the first step in the interaction between
Streptococcus pneumoniae
and its human host. To better understand the mechanisms contributing to natural carriage, a mouse model of pneumococcal colonization was developed with a clinical isolate of
S. pneumoniae
previously characterized in experimental colonization of humans. Similar to carriage events in humans, colonization of mice was self-limited and there was no evidence of lower respiratory tract or invasive disease. Carriage induced a serum antibody response to whole pneumococci that was associated temporally with clearance of colonization in three inbred strains of mice. Individual mice, however, did not demonstrate a correlation between the density of colonization and amounts of serum or of mucosal antibodies, including antibodies of different isotypes and antigenic specificities. The role of antibody in the clearance of carriage was then examined in mice with genetic defects in humoral immunity.
xid
mice, which have deficient responses to polysaccharide antigens, cleared colonization at the same rate as the parent strain. Finally, we showed that μMT mice, which lack mature B cells and fail to produce antibody, were unaffected in the density or duration of colonization. These results demonstrate that antibody is not required for clearance of pneumococcal colonization in mice.
Role of B7 Costimulatory Molecules in Mediating Systemic and Mucosal Antibody Responses to AttenuatedSalmonella entericaSerovar Typhimurium and Its Cloned Antigen
Authors: Garcia Carlos A. , Martin Michael , Michalek Suzanne M.
Published: September 2004

The purpose of the present study was to evaluate the ability of an attenuatedSalmonella entericaserovar Typhimurium vaccine strain to up-regulate B7-1 and B7-2 on antigen-presenting cells and to examine the functional roles these costimulatory molecules play in mediating immune responses toSalmonellaand to an expressed cloned antigen, the saliva-binding region (SBR) of antigen I/II. In vitro stimulation of B cells (B220+), macrophages (CD11b+), and dendritic cells (CD11c+) withS. entericaserovar Typhimurium induced an up-regulation of B7-2 and, especially, B7-1 expression. The in vivo functional roles of B7-1, B7-2, and B7-1/2 were evaluated in BALB/c wild-type and B7-1, B7-2, and B7-1/2 knockout (KO) mice following intranasal immunization with theSalmonellaexpressing the cloned SBR. Differential requirements for B7-1 and B7-2 were observed upon primary and secondary immunizations. Compared to wild-type controls, B7-1 and B7-2 KO mice had reduced mucosal and systemic anti-Salmonellaantibody responses after a single immunization, while only B7-1 KO mice exhibited suppressed anti-Salmonellaantibody responses following the second immunization. Mucosal and systemic antibody responses to SBR were reduced following the primary immunization, whereas a compensatory role for either B7-1 or B7-2 was observed after the second immunization. B7-1/2 double KO mice failed to induce detectable levels of mucosal or systemic immunoglobulin A (IgA) or IgG antibody responses to eitherSalmonellaor SBR. These findings demonstrate that B7-1 and B7-2 can play distinct as well as redundant roles for mediating mucosal and systemic antibody responses, which are likely dependent upon the nature of the antigen.
Bacteroides fragilisEnterotoxin Induces Intestinal Epithelial Cell Secretion of Interleukin-8 through Mitogen-Activated Protein Kinases and a Tyrosine Kinase-Regulated Nuclear Factor-κB Pathway
Authors: Wu Shaoguang , Powell Jan , Mathioudakis Nes , Kane Sheryl , Fernandez Ellen , Sears Cynthia L.
Published: September 2004

EnterotoxigenicBacteroides fragilis(ETBF) secretes a 20-kDa metalloprotease toxin termedB. fragilistoxin (BFT). ETBF disease in animals is associated with an acute inflammatory response in the intestinal mucosa, and lethal hemorrhagic colitis may occur in rabbits. In this study, we confirmed recent reports (J. M. Kim, Y. K. Oh, Y. J. Kim, H. B. Oh, and Y. J. Cho, Clin. Exp. Immunol. 123:421-427, 2001; L. Sanfilippo, C. K. Li, R. Seth, T. J. Balwin, M. J. Menozzi, and Y. R. Mahida, Clin. Exp. Immunol. 119:456-463, 2000) that purified BFT stimulates interleukin-8 (IL-8) secretion by human intestinal epithelial cells (HT29/C1 cells) and demonstrate that stimulation of IL-8 production is dependent on biologically active BFT and independent of serum. Induction of IL-8 mRNA expression occurs rapidly and ceases by 6 h after BFT treatment, whereas IL-8 secretion continues to increase for at least 18 h. Our data suggest that BFT-stimulated IL-8 secretion involves tyrosine kinase-dependent activation of nuclear factor-κB (NF-κB) as well as activation of the mitogen-activated protein kinases (MAPKs), p38 and extracellular signal-related kinase. Simultaneous activation of NF-κB and MAPKs appears necessary for secretion of IL-8 by HT29/C1 cells treated with BFT.
Merozoite Surface Protein 4/5 Provides Protection against Lethal Challenge with a Heterologous Malaria Parasite Strain
Authors: Goschnick M. W. , Black C. G. , Kedzierski L. , Holder A. A. , Coppel R. L.
Published: September 2004

Immunization with merozoite surface protein 4/5 (MSP4/5), the murine malaria homologue ofPlasmodium falciparumMSP4 and MSP5, has been shown to protect mice against challenge by parasites expressing the homologous form of the protein. The gene encoding MSP4/5 was sequenced from a number ofPlasmodium yoeliiisolates in order to assess the level of polymorphism in the protein. The gene was found to be highly conserved among the 13P. yoeliiisolates sequenced, even though many of the same isolates showed pronounced variability in their MSP119sequences. Nonsynonymous mutations were detected only for the isolatesPlasmodium yoelii nigeriensisN67 andPlasmodium yoelii killicki193L and 194ZZ. Immunization and challenge of BALB/c mice showed that the heterologous MSP4/5 proteins were able to confer a level of protection against lethalPlasmodium yoelii yoeliiYM challenge infection similar to that induced by immunization with the homologous MSP4/5 protein. To explore the limits of heterologous protection, mice were immunized with recombinant MSP4/5 protein fromPlasmodium bergheiANKA andPlasmodium chabaudi adamiDS and challenged withP. y. yoeliiYM. Interestingly, significant protection was afforded byP. bergheiANKA MSP4/5, which shows 81% sequence identity withP. y. yoeliiYM MSP4/5, but it was abolished upon reduction and alkylation. Significant protection was not observed for mice immunized with recombinantP. c. adamiDS MSP4/5, which shows 55.7% sequence identity withP. y. yoeliiYM MSP4/5. This study demonstrates the robustness of MSP4/5 in conferring protection against variant forms of the protein in a murine challenge system, in contrast to the situation found for other asexual-stage proteins, such as MSP119and AMA1.
Lipoprotein PsaA in Virulence ofStreptococcus pneumoniae: Surface Accessibility and Role in Protection from Superoxide
Authors: Johnston Jason W. , Myers Lisa E. , Ochs Martina M. , Benjamin William H. , Briles David E. , Hollingshead Susan K.
Published: September 2004

PsaA ofStreptococcus pneumoniae, originally believed to be an adhesin, is the lipoprotein component of an Mn2+transporter. Mutations inpsaAcause deficiencies in growth, virulence, adherence, and the oxidative stress response. Immunofluorescence microscopy shows that PsaA is hidden beneath the cell wall and the polysaccharide capsule and only exposed to antibodies upon cell wall removal. ApsaBCdeletion mutant, expressing PsaA normally, was as deficient in adherence to Detroit 562 cells as were strains lacking PsaA. Thus, PsaA does not appear to act directly as an adhesin, but rather,psaAmutations indirectly affect this process through the disruption of Mn2+transport. The deficiency in Mn2+transport also causes hypersensitivity to oxidative stress from H2O2and superoxide. In a chemically defined medium, growth of the wild-type strain was possible in the absence of Fe2+and Mn2+cations after a lag of about 15 h. Addition of Mn2+alone or together with Fe2+allowed prompt and rapid growth. In the absence of Mn2+, the addition of Fe2+alone extended the 15-h lag phase to 25 h. Thus, while Fe2+adversely affects the transition from lag phase to log phase, perhaps through increasing oxidative stress, this effect is relieved by the presence of Mn2+. A scavenger specific for superoxides but not those specific for hydroxyl radicals or H2O2was able to eliminate the inhibition of growth caused by iron supplementation in the absence of Mn2+. This implies that superoxides are a key player in oxidative stress generated in the presence of iron.