bluuebunny/crossref_metadata_2025 huggingface dataset:
What Are the Sources of Exposure to Eight Frequently Used Phthalic Acid Esters in Europeans?
Authors: Wormuth Matthias , Scheringer Martin , Vollenweider Meret , Hungerbühler Konrad
Published: June 2006

Phthalic acid esters (phthalates) are used as plasticizers in numerous consumer products, commodities, and building materials. Consequently, phthalates are found in human residential and occupational environments in high concentrations, both in air and in dust. Phthalates are also ubiquitous food and environmental contaminants. An increasing number of studies sampling human urine reveal the ubiquitous phthalate exposure of consumers in industrialized countries. At the same time, recent toxicological studies have demonstrated the potential of the most important phthalates to disturb the human hormonal system and human sexual development and reproduction. Additionally, phthalates are suspected to trigger asthma and dermal diseases in children. To find the important sources of phthalates in Europeans, a scenario‐based approach is applied here. Scenarios representing realistic exposure situations are generated to calculate the age‐specific range in daily consumer exposure to eight phthalates. The scenarios demonstrate that exposure of infant and adult consumers is caused by different sources in many cases. Infant consumers experience significantly higher daily exposure to phthalates in relation to their body weight than older consumers. The use of consumer products and different indoor sources dominate the exposure to dimethyl, diethyl, benzylbutyl, diisononyl, and diisodecyl phthalates, whereas food has a major influence on the exposure to diisobutyl, dibutyl, and di‐2‐ethylhexyl phthalates. The scenario‐based approach chosen in the present study provides a link between the knowledge on emission sources of phthalates and the concentrations of phthalate metabolites found in human urine.
ExpoFacts—An Overview of European Exposure Factors Data
Authors: Vuori V. , Zaleski R. T. , Jantunen M. J.
Published: June 2006

European exposure factor data have been collected in one centrally available, freely accessible site on the Internet: the ExpoFacts database (http://www.ktl.fi/expofacts/). The process of compiling the database required locating the exposure factor data and evaluating its general applicability and public availability. The scope of the ExpoFacts database covers 30 European countries, often each with its own approach for data generation and publication. The database includes information on food intake, time use, physiology, housing, and demographic parameters, as available. Information included in the database, as well as the challenges in collecting and compiling this information, are summarized. Data were found to be unavailable for ExpoFacts for a number of reasons: (1) data have not been collected, (2) collected data are not published, (3) the publishing format or language makes the data hard to locate and use, (4) copyright restrictions prevent presenting the data in an open access website, or (5) data exist, but are too expensive to acquire. Improving accessibility and harmonization of existing data would enhance the information base for exposure and risk assessments. In addition, the ExpoFacts project demonstrates a successful process for acquiring, storing, and sharing exposure factors data.
A Heuristic Risk Assessment Technique for Birdstrike Management at Airports
Authors: Allan John
Published: June 2006

Collisions between birds and aircraft (birdstrikes) have caused the loss of at least 88 aircraft and 243 lives in world civil aviation. Conservative estimates suggest that more routine damage and delays following birdstrikes cost the industry and its insurers US$1.2–1.5 billion per year. The majority of strikes happen close to airports and most countries have regulations that require airport managers to control the birdstrike risk on their property. Birdstrike prevention has, however, lagged behind other aspects of flight safety in the development and implementation of risk assessment protocols, possibly because of the inherent difficulty in quantifying the variability in the populations and behavior of the various bird species involved. This article presents a technique that uses both national and airport‐specific data to evaluate risk by creating a simple probability‐times–severity matrix. It uses the frequency of strikes reported for different bird species at a given airport over the preceding five years as a measure of strike probability, and the proportion of strikes with each species that result in damage to aircraft, in the national birdstrike database, as a measure of likely severity. Action thresholds for risk levels for particular bird species are then defined, above which the airport should take action to reduce the risk further. The assessment is designed for airports where the reporting and collation of birdstrike events is reasonably consistent over time and where a bird hazard management program of some sort is already in place. This risk assessment is designed to measure risk to the airport as a business rather than risk to the traveling passenger individually. It therefore takes no account of aircraft movement rate in the calculations and is aimed at minimizing the number of damaging incidents rather than concentrating on catastrophic events. Once set up at an airport, the technique is simple to implement for nonexperts, and it allows managers to focus bird control resources on the species causing the greatest risk, hence maximizing the return on investment. This protocol is now being successfully used at major airports in the United Kingdom and elsewhere in the world.
A Unified Theory and Test of Extended Immediate Deterrence
Authors: Signorino Curtis S. , Tarar Ahmer
Published: June 2006

We present a unified theory and test of extended immediate deterrence—unified in the sense that we employ our theoretical deterrence model as our statistical model in the empirical analysis. The theoretical model is a straightforward formalization of the deterrence logic in Huth (1988) and Huth and Russett (1984), coupled with private information concerning utilities. Our statistical analysis suggests that the attacker and defender's decisions are influenced by the balance of forces, nuclear weapons, defender‐protege military alliances, arms transfers, and trade, as well as the regime types of those involved. Many of these findings contradict previous research by Huth (1988) and Huth and Russett (1988). We find that many of the variables involved in the deterrence calculus are nonmonotonically linked to the probability of deterrence success or war. We illustrate the results with case studies of the Soviet‐Japanese dispute over Manchukuo (1937–1938) and the Berlin Blockade (1948).
Authoritarian Attitudes, Democracy, and Policy Preferences among Latin American Elites
Authors: Stevens Daniel , Bishin Benjamin G. , Barr Robert R.
Published: June 2006

This article examines the prevalence and consequences of authoritarian attitudes among elites in Argentina, Brazil, Chile, Colombia, Mexico, and Venezuela. We focus on the connection between antidemocratic elite attitudes and support for democracy; the causes and effects of authoritarian attitudes among elites and their implications for authoritarianism; and the impact of authoritarian attitudes beyond social policy preferences to other policy areas that have indirect implications for order. Contrary to some of the literature, we find that antidemocratic attitudes affect elites' support for democracy. Our analysis also speaks to the debate on the origins of authoritarianism. Much of the evidence supports Altemeyer's notion that perceived threat raises levels of authoritarianism, rather than Feldman's contention that threat strengthens the influence of authoritarian attitudes. Finally, we demonstrate that there is a broader influence of authoritarian attitudes on economic policy preferences, but only where those policies appear to have implications for social order.
The Conscientious Sovereign: Public and Private Rule in Thomas Hobbes's Early Discourses
Authors: Abosch Yishaiya
Published: June 2006

In midcareer political treatises, Hobbes trades humanism for scientific method and minimizes distinctions between conquest and mutual covenant as grounds of sovereignty. From this, many conclude that arbitrary government is the goal, or the likeliest outcome of Hobbes's science of justice. Three early discourses, however, suggest that the perils of transition from the private rule of a conqueror to the public rule of a law‐bound sovereign are of primary concern. Whether the change is taken as proof of the independent activity of natural law, or as solely attributable to the skills of exemplary sovereigns, it destabilizes any law‐bound ruler who fails to reorient the passions motivating subjects to obey. Lasting stability and comfort can be achieved only when public rule is redefined as the preservation of a cognitive balance, setting fear of the destructive effects of unregulated self‐assertion against hope of the possibility of continuous peace through obedience to law.
Tocqueville, Jansenism, and the Psychology of Freedom
Authors: McLendon Michael Locke
Published: June 2006

In American political discourse, freedom is often spoken of in terms of its inherent rationality or divine origins and is conceptualized as nothing more than a set of concrete institutions coupled with individual rights. By way of

Tocqueville's
Democracy in America,

I will attempt to broaden our political vocabulary by constructing a psychology of freedom. According to Tocqueville, the American consciousness is largely a product of two conflicting tendencies: Cartesian rationality and Pascalian existential angst. Out of the tensions created by the interplay of these two elements Tocqueville demonstrates that the motivations to sustain freedom, as well as the institutions and practices crucial for the maintenance of it, result from a complex psychological mixture of self‐interest, vanity, and a desire for solitude.
Locke on the Moral Basis of International Relations
Authors: Ward Lee
Published: June 2006

This article aims to focus analysis of Locke's theory of international relations away from the familiar discourse of sovereignty and natural law and toward a different discourse involving self‐government and international society. It argues that Locke's conception of international society balanced interrelated, overlapping, and even competing claims about sovereignty and natural law in a normative framework in which the right of self‐government replaced the principle of sovereignty as the moral basis of international relations. Thus, for Locke the norms deduced from the law of nature govern the international state of nature even as independent societies remain the primary executors of the law of nature in international society. The article concludes by considering how Locke's reflections on international relations may contribute to our understanding of contemporary debates about sovereignty, the use of force, and the ethics of intervention.
Value Choices and American Public Opinion
Authors: Jacoby William G.
Published: June 2006

Individual preferences among core values are widely believed to be an important determinant of political attitudes. However, several theoretical perspectives suggest that people experience difficulties making choices among values. This article uses data from the 1994 Multi‐Investigator Study to test for hierarchical structure in citizens' value preferences. The empirical results show that most people make transitive choices among values and that their value preferences have an impact on subsequent issue attitudes. To the extent that citizens exhibit intransitive value choices and/or apparent difficulties in the “translation process” from value preferences to issue attitudes, it is due more to low levels of political sophistication than to the existence of value conflict.
Motivated Skepticism in the Evaluation of Political Beliefs
Authors: Taber Charles S. , Lodge Milton
Published: June 2006

We propose a model of motivated skepticism that helps explain when and why citizens are biased‐information processors. Two experimental studies explore how citizens evaluate arguments about affirmative action and gun control, finding strong evidence of a prior attitude effect such that attitudinally congruent arguments are evaluated as stronger than attitudinally incongruent arguments. When reading pro and con arguments, participants (Ps) counterargue the contrary arguments and uncritically accept supporting arguments, evidence of a disconfirmation bias. We also find a confirmation bias—the seeking out of confirmatory evidence—when Ps are free to self‐select the source of the arguments they read. Both the confirmation and disconfirmation biases lead to attitude polarization—the strengthening of t2 over t1 attitudes—especially among those with the strongest priors and highest levels of political sophistication. We conclude with a discussion of the normative implications of these findings for rational behavior in a democracy.
Abundance‐Based Similarity Indices and Their Estimation When There Are Unseen Species in Samples
Authors: Chao Anne , Chazdon Robin L. , Colwell Robert K. , Shen Tsung‐Jen
Published: June 2006

Summary A wide variety of similarity indices for comparing two assemblages based on species incidence (i.e., presence/absence) data have been proposed in the literature. These indices are generally based on three simple incidence counts: the number of species shared by two assemblages and the number of species unique to each of them. We provide a new probabilistic derivation for any incidence‐based index that is symmetric (i.e., the index is not affected by the identity ordering of the two assemblages) and homogeneous (i.e., the index is unchanged if all counts are multiplied by a constant). The probabilistic approach is further extended to formulate abundance‐based indices. Thus any symmetric and homogeneous incidence index can be easily modified to an abundance‐type version. Applying the Laplace approximation formulas, we propose estimators that adjust for the effect of unseen shared species on our abundance‐based indices. Simulation results show that the adjusted estimators significantly reduce the biases of the corresponding unadjusted ones when a substantial fraction of species is missing from samples. Data on successional vegetation in six tropical forests are used for illustration. Advantages and disadvantages of some commonly applied indices are briefly discussed.
Apologies and Settlement Levers
Authors: Robbennolt Jennifer K.
Published: June 2006

This study uses experimental methods to explore the role of apologies in legal settlement negotiation. Specifically, the study examines the influences of apologies on disputants' perceptions, and the effects of apologies on a number of judgments that influence negotiation outcomes—settlement levers such as reservation, aspirations, and judgments of fair settlement amounts. Five‐hundred‐fifty‐six participants were asked to take the role of potential plaintiffs, to provide their reactions to an experimental scenario, and to indicate the values they would set for each settlement lever. The nature of the communication with the offender and the description of the evidentiary rule governing the admissibility of the offender's statement were manipulated. The data suggest that apologies can promote settlement by altering the injured parties' perceptions of the situation and the offender so as to make them more amenable to settlement discussions and by altering the values of the injured parties' settlement levers in ways that are likely to increase the chances of settlement. The results suggest further, however, that the nature of the apology itself, as well as the factual circumstances surrounding the incident, may play important roles in how apologies are understood.
Whale Science—and How (Not) to Use it
Authors: Hammond Philip
Published: June 2006

Few creatures carry more emotion on their broad backs than whales; and few issues arouse as much passion as whaling. Each year around this time the International Whaling Commission comes under pressure to allow the resumption of commercial catching and killing of whales and the Save the Whale lobbyists protest. But how many whales are there? Can the scientists and statisticians tell us—and how much influence do they wield in the real world of whale-politik? Philip Hammond, a former Chairman of the Scientific Committee of the IWC, explains.
Statistics and the Medicine of the Future
Authors: Harbron Chris
Published: June 2006

New drugs that are effective and safe for all become harder and harder to find. Individuals react differently to different medications. Chris Harbron points to a future where drugs will be better targeted. With the help of statisticians we will each have our own designer drugs, no longer off-the-shelf but tailored exactly to suit our own individual genome.
Protecting the Leaders—Syndromic Surveillance for the G8 Summit in Scotland
Authors: Robertson Chris
Published: June 2006

The G8 summit was held at the Gleneagles Hotel, Scotland, from July 6th to 8th, 2005. Leaders of the eight largest economies, and their entourages, assembled to discuss economic and political issues and the state of the world, including terrorism. Previous G8 summits had sparked off waves of protests and violence. Consequently, a huge security exercise took place in central Scotland for the 2005 summit. With Bush, Putin, Blair and a clutch of other premiers on hand, their safety had to be protected—as did their health, and that of those around them. Chris Robertson was charged with that task.
Driving Out Failure
Authors: Davis Tim , Champkin Julian
Published: June 2006

Product recall notices in shops, newspapers and elsewhere inform us now and then of children's toys that might be dangerous or electrical goods that could catch fire. You or I might describe that as faulty design. Tim Davis calls it an “escaped failure mode”—and he is against it. His world is not toys or electrical goods, but vehicles, where failure modes and recalls are more expensive and usually more serious. As an example: in 2001 the failure of certain Firestone tyres on Ford SUVs led to rollover accidents in which close to 300 people died. Nearly 20 million tyres needed to be recalled. Industry estimates put the cost at around †3 billion. It was Tim Davis who was called in to find out what had gone wrong. Julian Champkin interviewed him.
Real Data, Real Learning and the London Olympics
Authors: Davies Neville
Published: June 2006

The Olympic and Paralympic Games are coming to London in 2012, and there will be huge interest, especially among the young. Could the Games be used to involve students of all ages in a large scale project that involves and interests them to break down their fear of statistics and to motivate learning? Neville Davies of the Royal Statistical Society Centre for Statistical Education appeals for help in a major teaching and learning initiative based on the London Olympics. Significance is proud to announce the launch of a scheme that could bring thousands of students, nationally and internationally, to appreciate and value the usefulness of statistics.
Scale-Free Intermittent Flow in Crystal Plasticity
Authors: Dimiduk Dennis M. , Woodward Chris , LeSar Richard , Uchic Michael D.
Published: June 2006

Under stress, crystals irreversibly deform through complex dislocation processes that intermittently change the microscopic material shape through isolated slip events. These underlying processes can be revealed in the statistics of the discrete changes. Through ultraprecise nanoscale measurements on nickel microcrystals, we directly determined the size of discrete slip events. The sizes ranged over nearly three orders of magnitude and exhibited a shock-and-aftershock, earthquake-like behavior over time. Analysis of the events reveals power-law scaling between the number of events and their magnitude, or scale-free flow. We show that dislocated crystals are a model system for studying scale-free behavior as observed in many macroscopic systems. In analogy to plate tectonics, smooth macroscopic-scale crystalline glide arises from the spatial and time averages of disruptive earthquake-like events at the nanometer scale.
Carbon Nanotubes as High-Pressure Cylinders and Nanoextruders
Authors: Sun L. , Banhart F. , Krasheninnikov A. V. , Rodríguez-Manzo J. A. , Terrones M. , Ajayan P. M.
Published: June 2006

Closed-shell carbon nanostructures, such as carbon onions, have been shown to act as self-contracting high-pressure cells under electron irradiation. We report that controlled irradiation of multiwalled carbon nanotubes can cause large pressure buildup within the nanotube cores that can plastically deform, extrude, and break solid materials that are encapsulated inside the core. We further showed by atomistic simulations that the internal pressure inside nanotubes can reach values higher than 40 gigapascals. Nanotubes can thus be used as robust nanoscale jigs for extruding and deforming hard nanomaterials and for modifying their properties, as well as templates for the study of individual nanometer-sized crystals under high pressure.
Response to Comment on "Heterogeneous Hadean Hafnium: Evidence of Continental Crust at 4.4 to 4.5 Ga"
Authors: Harrison T. M. , Blichert-Toft J. , Müller W. , Albarede F. , Holden P. , Mojzsis S. J.
Published: June 2006

Valley
et al
. review the lines of evidence on which we drew to conclude that continental crust formed much earlier than previously thought. Their comment contains some misrepresentations that we correct, but new information they provide appears to bolster our hypothesis. Nothing in their comment refutes the presence of continental crust or plate boundary processes prior to 4 billion years ago.
Earthquake Rupture Stalled by a Subducting Fracture Zone
Authors: Robinson D. P. , Das S. , Watts A. B.
Published: June 2006

We showed that the rupture produced by the great Peru earthquake (moment magnitude 8.4) on 23 June 2001 propagated for ∼70 kilometers before encountering a 6000-square-kilometer area of fault that acted as a barrier. The rupture continued around this barrier, which remained unbroken for ∼30 seconds and then began to break when the main rupture front was ∼200 kilometers from the epicenter. The barrier had relatively low rupture speed, slip, and aftershock density as compared to its surroundings, and the time of the main energy release in the earthquake coincided with the barrier's rupture. We associate this barrier with a fracture zone feature on the subducting oceanic plate.
Regulation of Adult Bone Mass by the Zinc Finger Adapter Protein Schnurri-3
Authors: Jones Dallas C. , Wein Marc N. , Oukka Mohamed , Hofstaetter Jochen G. , Glimcher Melvin J. , Glimcher Laurie H.
Published: June 2006

Genetic mutations that disrupt osteoblast function can result in skeletal dysmorphogenesis or, more rarely, in increased postnatal bone formation. Here we show that Schnurri-3 (Shn3), a mammalian homolog of the
Drosophila
zinc finger adapter protein Shn, is an essential regulator of adult bone formation. Mice lacking Shn3 display adult-onset osteosclerosis with increased bone mass due to augmented osteoblast activity. Shn3 was found to control protein levels of Runx2, the principal transcriptional regulator of osteoblast differentiation, by promoting its degradation through recruitment of the E3 ubiquitin ligase WWP1 to Runx2. By this means, Runx2-mediated extracellular matrix mineralization was antagonized, revealing an essential role for Shn3 as a central regulator of postnatal bone mass.
The New Era in Cancer Research
Authors: Varmus Harold
Published: June 2006

For many years, discoveries about the genetic determinants of cancer appeared to be having only minor effects on efforts to control the disease in the clinic. Following advances made over the past decade, however, a description of cancer in molecular terms seems increasingly likely to improve the ways in which human cancers are detected, classified, monitored, and (especially) treated. Achieving the medical promise of this new era in cancer research will require a deeper understanding of the biology of cancer and imaginative application of new knowledge in the clinic, as well as political, social, and cultural changes.
Yersinia
YopJ Acetylates and Inhibits Kinase Activation by Blocking Phosphorylation
Authors: Mukherjee Sohini , Keitany Gladys , Li Yan , Wang Yong , Ball Haydn L. , Goldsmith Elizabeth J. , Orth Kim
Published: June 2006

Yersinia
species use a variety of type III effector proteins to target eukaryotic signaling systems. The effector YopJ inhibits mitogen-activated protein kinase (MAPK) and the nuclear factor κB (NFκB) signaling pathways used in innate immune response by preventing activation of the family of MAPK kinases (MAPKK). We show that YopJ acted as an acetyltransferase, using acetyl–coenzyme A (CoA) to modify the critical serine and threonine residues in the activation loop of MAPKK6 and thereby blocking phosphorylation. The acetylation on MAPKK6 directly competed with phosphorylation, preventing activation of the modified protein. This covalent modification may be used as a general regulatory mechanism in biological signaling.
Molecular Cloning of a
Trypanosoma cruzi
Cell Surface Casein Kinase II Substrate, Tc-1, Involved in Cellular Infection
Authors: Augustine Swinburne A. J. , Kleshchenko Yuliya Y. , Nde Pius N. , Pratap Siddharth , Ager Edward A. , Burns James M. , Lima Maria F. , Villalta Fernando
Published: June 2006

In this work, we report the cloning and characterization of the first cell surface casein kinase II (CKII) substrate (Tc-1) of
Trypanosoma cruzi
, the causative agent of Chagas' disease. Analysis of the gene sequence revealed a 1,653-bp open reading frame coding for 550 amino acid residues. Northern blot analysis showed a 4.5-kb transcript that is expressed in invasive trypomastigotes but not in noninvasive epimastigote forms of
T. cruzi
. Southern blot analysis indicates that Tc-1 is a single-copy gene. At the amino acid level, Tc-1 displayed 95% and 99% identity to two hypothetical proteins recently reported by the
T. cruzi
genome project. Analysis of the translated amino acid sequence indicates that the Tc-1 gene has a putative transmembrane domain with multiple cytoplasmic and extracellular CKII phosphosites. Exogenous human CKII was able to phosphorylate serine residues on both recombinant Tc-1 and Tc-1 of intact trypomastigotes. This phosphorylation was inhibited by the CKII inhibitors heparin and 4,5,6,7,-tetrabromo-2-azabenzimidazole. Immunoblots of solubilized trypomastigotes, epimastigotes, and amastigotes probed with anti-recombinant Tc-1 immunoglobulin G revealed a 62-kDa protein that is expressed only in infective trypomastigotes. Immunoprecipitation of labeled surface proteins of trypomastigotes indicated that the 62-kDa protein is a surface protein, and we found that the protein is uniformly distributed on the surface of trypomastigotes by direct immunofluorescence. Antibodies to Tc-1 effectively blocked trypomastigote invasion of host cells and consequently reduced parasite load. Preincubation of either trypomastigotes or myoblasts with CKII inhibitors blocked
T. cruzi
infection. Thus, for the first time, we describe a cell surface CKII substrate of a protozoan parasite that is phosphorylated by human CKII and that is involved in cellular infection.
Genetically Determined Susceptibility to Tuberculosis in Mice Causally Involves Accelerated and Enhanced Recruitment of Granulocytes
Authors: Keller Christine , Hoffmann Reinhard , Lang Roland , Brandau Sven , Hermann Corinna , Ehlers Stefan
Published: June 2006

Classical twin studies and recent linkage analyses of African populations have revealed a potential involvement of host genetic factors in susceptibility or resistance toMycobacterium tuberculosisinfection. In order to identify the candidate genes involved and test their causal implication, we capitalized on the mouse model of tuberculosis, since inbred mouse strains also differ substantially in their susceptibility to infection. Two susceptible and two resistant mouse strains were aerogenically infected with 1,000 CFU ofM. tuberculosis, and the regulation of gene expression was examined by Affymetrix GeneChip U74A array with total lung RNA 2 and 4 weeks postinfection. Four weeks after infection, 96 genes, many of which are involved in inflammatory cell recruitment and activation, were regulated in common. One hundred seven genes were differentially regulated in susceptible mouse strains, whereas 43 genes were differentially expressed only in resistant mice. Data mining revealed a bias towards the expression of genes involved in granulocyte pathophysiology in susceptible mice, such as an upregulation of those for the neutrophil chemoattractant LIX (CXCL5), interleukin 17 receptor, phosphoinositide kinase 3 delta, or gamma interferon-inducible protein 10. FollowingM. tuberculosischallenge in both airways or peritoneum, granulocytes were recruited significantly faster and at higher numbers in susceptible than in resistant mice. When granulocytes were efficiently depleted by either of two regimens at the onset of infection, only susceptible mice survived aerosol challenge withM. tuberculosissignificantly longer than control mice. We conclude that initially enhanced recruitment of granulocytes contributes to susceptibility to tuberculosis.
Early Events in
Mycobacterium tuberculosis
Infection in Cynomolgus Macaques
Authors: Lin Philana Ling , Pawar Santosh , Myers Amy , Pegu Amarenda , Fuhrman Carl , Reinhart Todd A. , Capuano Saverio V. , Klein Edwin , Flynn JoAnne L.
Published: June 2006

Little is known regarding the early events of infection of humans with
Mycobacterium tuberculosis
. The cynomolgus macaque is a useful model of tuberculosis, with strong similarities to human tuberculosis. In this study, eight cynomolgus macaques were infected bronchoscopically with low-dose
M. tuberculosis
; clinical, immunologic, microbiologic, and pathologic events were assessed 3 to 6 weeks postinfection. Gross pathological abnormalities were observed as early as 3 weeks, including Ghon complex formation by 5 weeks postinfection. Caseous granulomas were observed in the lung as early as 4 weeks postinfection. Only caseous granulomas were observed in the lungs at these early time points, reflecting a rigorous initial response. T-cell activation (CD29 and CD69) and chemokine receptor (CXCR3 and CCR5) expression appeared localized to different anatomic sites. Activation markers were increased on cells from airways and only at modest levels on cells in peripheral blood. The priming of mycobacterium-specific T cells, characterized by the production of gamma interferon occurred slowly, with responses seen only after 4 weeks of infection. These responses were observed from T lymphocytes in blood, airways, and hilar lymph node, with responses predominantly localized to the site of infection. From these studies, we conclude that immune responses to
M. tuberculosis
are relatively slow in the local and peripheral compartments and that necrosis occurs surprisingly quickly during granuloma formation.
Cloning, Characterization, and Serodiagnostic Evaluation ofLeishmania infantumTandem Repeat Proteins
Authors: Goto Yasuyuki , Coler Rhea N. , Guderian Jeffrey , Mohamath Raodoh , Reed Steven G.
Published: June 2006

Visceral leishmaniasis (VL) is a form of leishmaniasis, which is caused by infection with the protozoan parasiteLeishmania, and is often fatal unless it is treated. Rapid and accurate diagnosis of VL is important for effective treatment. Here we report the cloning of previously undescribed tandem repeat (TR) proteins ofLeishmania infantumand an evaluation of VL patient antibody responses to the corresponding proteins. By screening anL. infantumexpression library with sera from human VL patients or infected hamsters, we identified 43 genes encoding B-cell antigens. Surprisingly, 19 of the 43 genes (44%) were TR proteins, and that percentage was significantly higher than that for genes picked randomly from the database. We then expressed the TR regions of LinJ16.1750, LinJ22.1590, and LinJ33.2870 and the entire LinJ28.2310 protein. These recombinant proteins were all recognized by Sudanese VL patient sera in an enzyme-linked immunosorbent assay. Recombinant LinJ16.1750 (rLinJ16.1750) showed the best performance among these antigens in terms of both sensitivity and specificity. Serological evaluation revealed that 97% (34 of 35) of Sudanese VL patients had significantly elevated antibody levels to rLinJ16.1750. Furthermore, when eight of the patient sera which had low reactivities to rK39 were tested with the novel recombinant antigens, some of the sera showed stronger antibody responses to these antigens than to rK39. Our results suggest that TR regions from the novelL. infantumproteins identified in this study are immunodominant B-cell epitopes and may represent good candidates for serodiagnosis of VL.
Alanine Esters of Enterococcal Lipoteichoic Acid Play a Role in Biofilm Formation and Resistance to Antimicrobial Peptides
Authors: Fabretti Francesca , Theilacker Christian , Baldassarri Lucilla , Kaczynski Zbigniew , Kropec Andrea , Holst Otto , Huebner Johannes
Published: June 2006

Enterococcus faecalis
is among the predominant causes of nosocomial infections. Surface molecules like
d
-alanine lipoteichoic acid (LTA) perform several functions in gram-positive bacteria, such as maintenance of cationic homeostasis and modulation of autolytic activities. The aim of the present study was to evaluate the effect of
d
-alanine esters of teichoic acids on biofilm production and adhesion, autolysis, antimicrobial peptide sensitivity, and opsonic killing. A deletion mutant of the
dltA
gene was created in a clinical
E. faecalis
isolate. The absence of
d
-alanine in the LTA of the
dltA
deletion mutant was confirmed by nuclear magnetic resonance spectroscopy. The wild-type strain and the deletion mutant did not show any significant differences in growth curve, morphology, or autolysis. However, the mutant produced significantly less biofilm when grown in the presence of 1% glucose (51.1% compared to that of the wild type); adhesion to eukaryotic cells was diminished. The mutant absorbed 71.1% of the opsonic antibodies, while absorption with the wild type resulted in a 93.2% reduction in killing. Sensitivity to several cationic antimicrobial peptides (polymyxin B, colistin, and nisin) was considerably increased in the mutant strain, confirming similar results from other studies of gram-positive bacteria. Our data suggest that the absence of
d
-alanine in LTA plays a role in environmental interactions, probably by modulating the net negative charge of the bacterial cell surface, and therefore it may be involved in the pathogenesis of this organism.
Comparison of Different Live Vaccine Strategies In Vivo for Delivery of Protein Antigen or Antigen-Encoding DNA and mRNA by Virulence-AttenuatedListeria monocytogenes
Authors: Loeffler Daniela I. M. , Schoen Christoph U. , Goebel Werner , Pilgrim Sabine
Published: June 2006

Listeria monocytogenescan be used to deliver protein antigens or DNA and mRNA encoding such antigens directly into the cytosol of host cells because of its intracellular lifestyle. In this study, we compare the in vivo efficiencies of activation of antigen-specific CD8 and CD4 T cells when the antigen is secreted byL. monocytogenesor when antigen-encoding plasmid DNA or mRNA is released by self-destructing strains ofL. monocytogenes. Infection of mice with self-destructingL. monocytogenescarriers delivering mRNA that encodes a nonsecreted form of ovalbumin (OVA) resulted in a significant OVA-specific CD8 T-cell response. In contrast, infection withL. monocytogenesdelivering OVA-encoding DNA failed to generate specific T cells. Secretion of OVA by the carrier bacteria yielded the strongest immune response involving OVA-specific CD8 and CD4 T cells. In addition, we investigated the antigen delivery capacity of a self-destructing, virulence-attenuatedL. monocytogenes aroA/Bmutant. In contrast to the wild-type strain, this mutant exhibited only marginal liver toxicity when high doses (5 × 107CFU per animal administered intravenously) were used, and it was also able to deliver sufficient amounts of secreted OVA into mice. Therefore, the results presented here could lay the groundwork for a rational combination ofL. monocytogenesas an attenuated carrier for the delivery of protein and nucleic acid vaccines in novel vaccination strategies.
Use of anActinobacillus pleuropneumoniaeMultiple Mutant as a Vaccine That Allows Differentiation of Vaccinated and Infected Animals
Authors: Maas Alexander , Jacobsen Ilse D. , Meens Jochen , Gerlach Gerald-F.
Published: June 2006

Vaccination againstActinobacillus pleuropneumoniaeis hampered by the lack of vaccines inducing reliable cross-serotype protection. In contrast, pigs surviving natural infection are at least partially protected from clinical symptoms upon reinfection with any serotype. Thus, we set out to construct an attenuatedA. pleuropneumoniaelive vaccine allowing the differentiation of vaccinated from infected animals (the DIVA concept) by successively deleting virulence-associated genes. Based on anA. pleuropneumoniaeserotype 2 prototype live negative marker vaccine (W. Tonpitak, N. Baltes, I. Hennig-Pauka, and G.-F. Gerlach, Infect. Immun. 70:7120-7125, 2002), genes encoding three enzymes involved in anaerobic respiration and the ferric uptake regulator Fur were deleted, resulting in a highly attenuated sixfold mutant; this mutant was still able to colonize the lower respiratory tract and induced a detectable immune response. Upon a single aerosol application, this mutant provided significant protection from clinical symptoms upon heterologous infection with an antigenically distinctA. pleuropneumoniaeserotype 9 challenge strain and allowed the serological discrimination between infected and vaccinated groups.
Serologic Proteome Analysis ofBorrelia burgdorferiMembrane-Associated Proteins
Authors: Nowalk Andrew J. , Gilmore Robert D. , Carroll James A.
Published: June 2006

Lyme disease, a global health concern, is caused by infection withBorrelia burgdorferi,B. afzelii, orB. garinii. The spirochete responsible for the disease in the United States isB. burgdorferiand is spread by the bite of an infectedIxodestick. We utilized multiple two-dimensional gel techniques combined with proteomics to reveal the full humoral immune response of mice and Lyme patients to membrane-associated proteins isolated fromBorrelia burgdorferi. Our studies indicated that a subset of immunogenic membrane-associated proteins (some new and some previously identified) was recognized by mice experimentally infected withBorrelia burgdorferieither by low-dose needle inoculation or by tick infestation. Moreover, the majority of these immunogenic membrane-associated proteins were recognized by sera from patients diagnosed with early-disseminated Lyme disease. These included RevA, ErpA, ErpP, DbpA, BmpA, FtsZ, ErpB, LA7, OppA I, OppA II, OppA IV, FlhF, BBA64, BBA66, and BB0323. Some immunogens (i.e., BBI36/38) were more reactive with sera from mice than Lyme patients, while additional membrane proteins (i.e., FlaB, P66, LA7, and Hsp90) were recognized more strongly with sera from patients diagnosed with early-localized, early-disseminated, or late (chronic)-stage Lyme disease. We were able to examine the humoral response in Lyme patients in a temporal fashion and to identify the majority of immunoreactive proteins as the disease progresses from early to late stages. This serologic proteome analysis enabled the identification of novel membrane-associated proteins that may serve as new diagnostic markers and, more importantly, as second-generation vaccine candidates for protection against Lyme disease.
Low-Phosphate-Dependent Invasion Resembles a General Way for
Neisseria gonorrhoeae
To Enter Host Cells
Authors: Kühlewein Christiane , Rechner Cindy , Meyer Thomas F. , Rudel Thomas
Published: June 2006

Obligate human-pathogenic
Neisseria gonorrhoeae
expresses numerous variant surface proteins mediating adherence to and invasion of target cells. The invariant major outer membrane porin PorB of serotype A (P.IA) gonococci triggers invasion into Chang cells only if the medium is devoid of phosphate. Since gonococci expressing PorB
IA
are frequently isolated from patients with severe disseminating infections, the interaction initiated by the porin may be of major relevance for the development of this serious disease. Here, we investigated the low-phosphate-dependent invasion and compared it to the well-known pathways of entry initiated by Opa proteins. P.IA-triggered invasion requires clathrin-coated pit formation and the action of actin and Rho GTPases. However, in contrast to Opa-initiated invasion via heparan sulfate proteoglycans, microtubules, acidic sphingomyelinase, phosphatidylinositol 3-kinase, and myosin light chain kinase are not involved in this entry pathway. Nor are Src kinases required, as they are in invasion, e.g., via the CEACAM3 receptor. Invasion by PorB
IA
occurs in a wide spectrum of cell types, such as primary human epithelial and endothelial cells and in cancer cells of human and animal origin. Low-phosphate-dependent invasion is thus a pathway of gonococcal entry distinct from Opa-mediated invasion.
Hybridoma Passage In Vitro May Result in Reduced Ability of Antimannan Antibody To Protect against Disseminated Candidiasis
Authors: Xin Hong , Cutler Jim E.
Published: June 2006

We previously reported the enhanced resistance of monoclonal antibodies B6.1 (an immunoglobulin M [IgM]) and C3.1 (an IgG3) against experimental candidiasis. Both MAbs recognize the same fungal epitope. We have since found that a highly passaged B6.1 hybridoma (hp-B6.1) resulted in antibody that has little protective potential. The potential clinical applicability of the antibody and our interest in understanding antibody protection against candidiasis led us to investigate an explanation for this phenomenon. Antibody genetic structure of hp-B6.1, the original hybridoma clone (ori-B6.1) stored frozen since 1995, a subclone of hp-B6.1 that produces protective antibody, the IgG3-producing hybridoma, and a nonprotective IgG1-producing hybridoma were compared. Variable region gene sequences of heavy (VH) and light chains showed genetic instability of VHchains with only the hp-B6.1; the VHsequences from ori-B6.1 and the subclone were, however, identical. Activation-induced cytidine deaminase levels were greatest in the B6.1 hybridomas, which may explain the instability. The constant region CH3 domain remained unchanged, implying normalN-glycation and complement-fixing potential, and antibody binding affinities appeared unchanged. Complement fixation assays surprisingly showed that ori-B6.1 antibody fixes C3 more rapidly than does hp-B6.1 antibody. The VHregion primary structure may affect complement activation, which could explain our result. Indeed, antibody from the hp-B6.1 subclone fixed complement like antibody from ori-B6.1. These results show that the greatest protection occurs when antimannan antibodies possess the dual abilities of recognizing the appropriate carbohydrate epitope and rapidly fixing complement; loss of the latter property results in the loss of protective potential by the antibody.
TheYersinia enterocoliticaInvasin Protein Promotes Major Histocompatibility Complex Class I- and Class II-Restricted T-Cell Responses
Authors: Bühler O. T. , Wiedig C. A. , Schmid Y. , Grassl G. A. , Bohn E. , Autenrieth I. B.
Published: June 2006

Yersinia enterocoliticainvasin (Inv) protein confers internalization into and expression of proinflammatory cytokines by host cells. Both events require binding of Inv to β1 integrins, which initiates signaling cascades including activation of focal adhesion complexes, Rac1, mitogen-activated protein kinase, and NF-κB. Here we tested whether Inv might be suitable as a delivery molecule and adjuvant if used as a component of a vaccine. For this purpose, hybrid proteins composed of Inv and ovalbumin (OVA) were prepared, applied as a coating to microparticles, and used for vaccination. Fusion of OVA to Inv did not significantly disturb the ability of Inv to promote host cell binding, internalization, and interleukin-8 (IL-8) secretion when applied as a coating to microparticles. The microparticles were used for vaccination of mice adoptively transferred with OVA-specific T cells from OT-1 or DO11.10 mice. Administration of OVA-Inv-coated microparticles induced OVA-specific T-cell responses. OVA-specific CD4 T cells produced both gamma interferon (IFN-γ) and IL-4 as determined by enzyme-linked immunosorbent assay. Likewise, pronounced OVA-specific CD8 T-cell responses associated with IFN-γ production were observed. Together, these results suggest that Inv might be an attractive tool in vaccination as it confers both host cell uptake and adjuvant activity by engagement of β1 integrins of host cells, which leads to CD4 as well as CD8 T-cell responses.
Responses toLeishmania donovaniin Mice Deficient in Interleukin-12 (IL-12), IL-12/IL-23, or IL-18
Authors: Murray Henry W. , Tsai Christine W. , Liu Jianguo , Ma Xiaojing
Published: June 2006

Interleukin-12 (IL-12) orchestrates acquired resistance in intracellularLeishmania donovaniinfection in the liver, inducing gamma interferon and, in turn, macrophage activation and parasite killing. Nevertheless, testing in IL-18−/−mice compared to wild-type mice and in IL-12p40−/−compared to IL-12p35−/−mice also suggested both early-acting (IL-18) and late-acting (IL-23) antileishmanial effects independent of IL-12.
Stabilization of a Plasmid Coding for a Heterologous Antigen in
Salmonella enterica
Serotype Typhi Vaccine Strain CVD908-
htrA
by Using Site-Specific Recombination
Authors: Stephens Jonathan C. , Darsley Michael J. , Turner Arthur K.
Published: June 2006

A gene cassette incorporating the
crs-rsd
site-specific recombination system from the
Salmonella enterica
subsp.
enterica
serovar Dublin virulence plasmid improved the inheritance in
S. enterica
serotype Typhi strain CVD908-
htrA
of a multicopy plasmid expression vector. Use of this recombination cassette may improve expression of heterologous antigens from multicopy plasmid expression vectors in attenuated bacterial vaccine strains.
Proinflammatory and Immunomodulatory Cytokine mRNA Time Course Profiles in Hamsters Infected with a Virulent Variant of
Leptospirainterrogans
Authors: Vernel-Pauillac Frédérique , Merien Fabrice
Published: June 2006

In order to quantify in vivo the mRNAs of cytokines which play important
roles in leptospirosis, we have developed quantitative real-time PCR
assays for interleukin-2 (IL-2), IL-4, IL-10, IL-12p40, tumor necrosis
factor alpha (TNF-α), gamma interferon (IFN-γ),
transforming growth factor β, and two housekeeping genes
(encoding β-actin and hypoxanthine phosphoribosyltransferase).
We used a lethal hamster model reflecting severe leptospirosis in
humans. The LightCycler system was used to quantify the gene expression
levels with the SYBR green I detection format using external standard
curves for each target. We compared the expression levels of cytokine
mRNA in the peripheral blood mononuclear cells of both control
(uninfected) hamsters and
Leptospira interrogans
-inoculated
hamsters from 1 to 24 h and then 1 to 4 days postinfection.
In this kinetic study, there was pronounced expression of Th1 cytokine
mRNA (TNF-α, IFN-γ, and IL-12), with transcripts being
detected as early as 1 h postinfection. Expression of
anti-inflammatory cytokines, such as IL-4 and IL-10, was prominent in
delayed samples from 1 to 4 days postinfection in response to infection
with
Leptospira interrogans
. Our data are the first to
establish that pathogenic leptospires can stimulate in vivo the
production of type 1 cytokines involved in cellular immunity by using
this informative animal model. Measuring and assessing cytokine
profiles may provide a useful method for accurate study of the
mechanisms of anti-
Leptospira
immunity, indications of
prognosis factors, and prospective evaluation of leptospirosis vaccine
efficacy in
humans.