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Purpose. The antimicrobial activity of N-chlorotaurine (NCT), an endogenous long-lived oxidant applied topically, was tested against Chlamydiae in vitro. Methodology. Elementary bodies of Chlamydia pneumoniae strain CV-6 and Chlamydia trachomatis serovars A and D were incubated in 0.01, 0.1 and 1 % (w/v) NCT solution at pH 7.1 and 37 °C. After different incubation times, aliquots were removed and grown in cell culture. The number of inclusion forming units was quantified by immunofluorescence and real-time qPCR. Results/Key findings. Chlamydia pneumoniae and Chlamydia trachomatis were inactivated by 1 and 0.1 % NCT within 1 min. Moreover, 0.025–0.1 % NCT significantly reduced the number of intracellularly growing C. pneumoniae within 30 min. Conclusions. This is the first study demonstrating the antimicrobial activity of NCT against Chlamydiae. Clinical implications of these findings have to be investigated in further trials.

Since the Peace of Westphalia, few great powers have “died”, while the “death rate” and proliferation of small states has been dramatic at times. What causes these fluctuations? In this paper, I claim that the dominant reason for the extinction, emergence and proliferation of the small state over the last three and a half centuries is to be found at the system level. Ultimately, small state survival is determined by the particular set-up of the state system. I advance this argument from the perspective of international relations theory, integrating the relevant scholarship of the English School and realism, especially structural realism. The latter’s systemic perspective provides the basis for arguing that small states are structurally irrelevant. It is this feature of the small state, its irrelevance with regard to the power-based structure of the state system, which has caused the small state to “struggle for existence” in the past, and which has allowed small states to proliferate during the bi...

The candidacy of Barack Obama created high hopes and major expectations overseas. His electoral victory in 2008 promised the return of a benevolent US. He being awarded the Nobel Peace Prize was the ultimate expression of the world’s anticipation. Such high expectations had to be disappointed, and they were. However, it is remarkable that Obama frustrated many but maintained his overall popularity worldwide until the end of his two four-year terms. The disillusionment, disappointment, and frustration over Obama’s foreign policy and global leadership remained largely sympathetic. It was, in a way, a “benevolent disappointment” which characterizes the world views of the Obama era.

In late 2008, the US chose Barack Obama as its 44th president and, as I observed then, “[t]he world approved” (Maass, The World Views of the U.S. Presidential Election: 2008. Palgrave Macmillan, 2009). This current volume is the analytical “bookend” to the previous cooperative study. Then, the task was to capture the world views on the US presidential election. The claim was that the US-American election was indeed a global election in the sense that it was watched and evaluated across the world. Its anticipated effects globally made it in fact a global event.

How did the small state fare in the 20th century? As it turns out, small states did remarkably well. Surprisingly enough, during the height of the Cold War small state proliferation doubled their total number. The 20th century’s international-political environment became highly permissive of small state survival and in fact strongly supportive of small state proliferation. As a result, the 20th century witnessed the reversal of the centuries-old downward trend in small state numbers. The early 20th century saw the introduction of collective action and security and the beginnings of global governance. After the Second World War, the system evolved further towards global governance but was challenged by the end of the Cold War and rise of global terrorism. The chapter demonstrates how these changes were critical factors in shaping a corresponding story of small state survival. The highly permissive environment of the Cold War and its era of decolonization are given particular attention.

How safe was the largely unbridled balance of power of the 17th and 18th centuries for the small state? This chapter shows that the balance of power system was in fact rather permissive and allowed small states to survive in historically large numbers. The loose and fairly unrestrained balance of power system turned out to be a surprisingly safe environment for the small state. The chapter covers the era of the classic balance of power, which is bookended by two major peace summits, the Peace of Westphalia, concluded in 1648, and the Congress of Vienna, held in 1814/15. During this era, the particular balance-of-power that defined it was also the main cause of the moderate decline in small states numbers.

In the previous chapters, it has been demonstrated how over more than 3 ½ centuries, the fate of small states has depended first of all on the states system. This chapter concludes the investigation with the key finding, that small state survival and proliferation are largely system-dependent phenomena. It is pointed out that the small state’s dependency and its structural irrelevance in a world of power are critical to properly understanding the issue of small state survival. On the one hand, small states are units that don’t matter much to the system. On the other hand, small states’ survival is to a good degree predetermined by the particular system in which they exist, but which they cannot shape.

The 4th chapter starts with the Congress of Vienna 1814/15 and moves the discussion to the eve of the First World War. At Vienna, the so-called concert system was introduced, and it structured most of 19th century international politics. But how did the small state fare in the 19th century system? During the first half of the century, small state numbers continued to erode before the all but collapsed in the later decades of the century. These historic losses of small states, it is argued, stem largely from the particular ‘oligopolistic’ features of the concert system and its key modifiers. Small state survivability decreased as great powers formed a cartel and later split into two hostile camps.

The chapter has to major objectives: First, the chapter works towards a definition of the small states that allows for the collection of statistical data on small state survival across time. This definition is developed in a way consistent with the overall theoretical framework. The small state is defined as a formally equal but structurally irrelevant unit of the system of states. Second, the chapter presents the quantitative data put together on the basis of the definition developed previously. Surveying the data of small state survival over the course of more than 3 ½ centuries, a picture emerges of the historical rise and fall of the small state. This first cut at the picture of small state survival, together with the theoretically-derived definition of the small state provides the basis for the following investigations.

How can the wide fluctuations in the number of small states over the past 350 years be explained? Rejecting the notion that the deaths of over 150 states can be properly explained by poor statecraft, incompetent leadership or major faults in foreign policy decision making of small states, the article claims that vast numbers of state deaths and large-scale state creation are most properly explained at the system level. An argument is developed that describes small state death and proliferation as heavily impacted by the state system. By investigating the core operating principles of the international state system since 1648 in light of their impact on the survivability of small states, the argument emerges that larger rates of survival and proliferation of small states are shaped by broad systemic factors. The article contributes a new facet to the study of the small state and offers a new and general explanation of small state death, creation and proliferation. The findings also challenge International Relation Theory’s traditional focus on great powers. It is suggested that the study of the major structuring actors in international politics may be complimented fruitfully by better understanding the system’s impact on weak and small states.

During the period of Germany’s reunification in the early 1990s, disagreement between Germany and Vietnam over the return of Vietnamese individuals to Vietnam escalated into a diplomatic dispute that also spilled over into Vietnam’s negotiations with the European Union over a major eu–Vietnam treaty. In mid-1995, however, the German and Vietnamese governments finally agreed on a repatriation arrangement that allowed Germany to begin deporting about 40,000 Vietnamese who were living in Germany illegally. This article explores the episode in the wider context of diplomatic dispute resolution. While Germany was demanding full cooperation from Vietnam on the issue of returning Vietnamese nationals, the Vietnamese government initially resisted large-scale repatriation for economic and social reasons. Hanoi attempted to frame the discussion within bilateral negotiations, economic costs and human rights, whereas Bonn argued from the perspective of customary international law and applied increasingly coercive diplomacy. German authorities escalated the disagreement and made economic threats with the aim of changing Hanoi’s behaviour. In order to frame this approach analytically, this article uses a modified form of coercive diplomacy. The analysis proceeds in three stages: first, the article analyses the origins of the dispute, which had its roots in German reunification; second, it evaluates the legal arguments advanced by each side; and third, it investigates Germany’s ‘soft’ coercive diplomacy and Vietnam’s response. The article concludes with an evaluation of Germany’s approach, benchmarking 1995’s diplomatic outcome against results on the ground, namely the number of returnees to Vietnam.

Recently, President Obama stated: “Our systematic effort to dismantle terrorist organizations must continue. But this war, like all wars, must end.” I investigate America’s legal basis, its claim to self-defense in response to 9/11, and conclude that this claim is weakening rapidly due to the passage of time and military successes. Similarly, the case for drone strikes as pre-emption is fading due to the damage drone strikes have done to Al Qaeda’s capabi-lities. I conclude that overall US drone strikes follow the rules set by International Humanitarian Law (IHL). How-ever, today America’s rationale for these missions is shi-fting away from defeating Al Qaeda towards deterring its members and sympathizers. In this case, the US would most likely be in breach of IHL. I complete the analysis by claiming that the recent recognition of Obama that “this war... must end” still lacks the need for a new legal framework for America’s counterterrorism efforts.

Background Improvements in eddy current suppression are necessary to meet the demand for increasing miniaturization of inductively driven transmission systems in industrial and biomedical applications. The high magnetic permeability and the simultaneously low electrical conductivity of ferrite materials make them ideal candidates for shielding metallic surfaces. For systems like cochlear implants the transmission of data as well as energy over an inductive link is conducted within a well-defined parameter set. For these systems, the shielding can be of particular importance if the properties of the link can be preserved. Results In this work, we investigate the effect of single and double-layered substrates consisting of ferrite and/or copper on the inductance and coupling of planar spiral coils. The examined link systems represent realistic configurations for active implantable systems such as cochlear implants. Experimental measurements are complemented with analytical calculations and finite element simulations, which are in good agreement for all measured parameters. The results are then used to study the transfer efficiency of an inductive link in a series–parallel resonant topology as a function of substrate size, the number of coil turns and coil separation. Conclusions We find that ferrite sheets can be used to shield the system from unwanted metallic surfaces and to retain the inductive link parameters of the unperturbed system, particularly its transfer efficiency. The required size of the ferrite plates is comparable to the size of the coils, which makes the setup suitable for practical implementations. Since the sizes and geometries chosen for the studied inductive links are comparable to those of cochlear implants, our conclusions apply in particular to these systems.

We identified increased expression and redistribution of the intracellular protein 60-kDa human heat shock protein (hHSP60) (HSPD1) to the cell surface in human endothelial cells subjected to classical atherosclerosis risk factors and subsequent immunologic cross-reactivity against this highly conserved molecule, as key events occurring early in the process of atherosclerosis. The present study aimed at investigating the role of infectious pathogens as stress factors for vascular endothelial cells and, as such, contributors to early atherosclerotic lesion formation. Using primary donor-matched arterial and venous human endothelial cells, we show that infection with Chlamydia pneumoniae leads to marked upregulation and surface expression of hHSP60 and adhesion molecules. Moreover, we provide evidence for an increased susceptibility of arterial endothelial cells for redistribution of hHSP60 to the cellular membrane in response to C. pneumoniae infection as compared to autologous venous endothelial cells. We also show that oxidative stress has a central role to play in endothelial cell activation in response to chlamydial infection. These data provide evidence for a role of C. pneumoniae as a potent primary endothelial stressor for arterial endothelial cells leading to enrichment of hHSP60 on the cellular membrane and, as such, a potential initiator of atherosclerosis.

The year 1995 marked a major watershed for modern Vietnam. It completed its post-Cold War strategy of “multilateralizing” its foreign policy by joining ASEAN, normalizing relations with the US, and signing a comprehensive framework agreement with the EU. All three are recognized as major accomplishments for modern Vietnamese diplomacy. However, in the EU–Vietnam framework agreement, Hanoi made an unprecedented concession when it agreed to the human rights clause in the treaty. For the very first time, Vietnam had accepted an explicit, legally binding stipulation on human rights in a bilateral treaty. This remarkable development resulted from the confluence of three major dynamics. First, Hanoi had committed itself to establish sound relations with all major economic centers-of-gravity at the time, ASEAN, the US, and the EU. Second, the EU also was keenly interested in stronger relations with Asian countries but was flexible about prioritizing any particular bilateral relationship. Third, Brussels’ diplomats had to work off a treaty template when negotiating fundamental bilateral agreements. The standard EU framework agreement at the time included a human rights clause. In 1995, the EU was insisting that any treaty with Vietnam would have to include a clause on human rights. Initially, Hanoi rejected such a treaty provision, and the negotiations stalled. However, when Hanoi realized that Brussels felt no urgency to complete the treaty and was unable to compromise on human rights, Vietnam’s leadership reconsidered. In order to complete the strategy of “multilateralizing” its international affairs, Vietnam had to accept the human rights clause in the treaty with the EU.

Clostridium difficile, a Gram-positive, spore-forming, anaerobic bacterium, is the main causative agent of hospital-acquired diarrhoea worldwide. In addition to metronidazole and vancomycin, rifaximin, a rifamycin derivative, is a promising antibiotic for the treatment of recurring C. difficile infections (CDI). However, exposure of C. difficile to this antibiotic has led to the development of rifaximin-resistance due to point mutations in the β-subunit of the RNA polymerase (rpoB) gene. In the present study, 348 C. difficile strains with known PCR-ribotypes were investigated for respective single nucleotide polymorphisms (SNPs) within the proposed rpoB hot-spot region by using high-resolution melting (HRM) analysis. This method allows the detection of SNPs by comparing the altered melting behaviour of dsDNA with that of wild-type DNA. Discrimination between wild-type and mutant strains was enhanced by creating heteroduplexes by mixing sample DNA with wild-type DNA, leading to characteristic melting curve shapes from samples containing SNPs in the respective rpoB section. In the present study, we were able to identify 16 different rpoB sequence-types (ST) by sequencing analysis of a 325 bp fragment. The 16 PCR STs displayed a total of 24 different SNPs. Fifteen of these 24 SNPs were located within the proposed 151 bp SNP hot-spot region, resulting in 11 different HRM curve profiles (CP). Eleven SNPs (seven of which were within the proposed hot-spot region) led to amino acid substitutions associated with reduced susceptibility to rifaximin and 13 SNPs (eight of which were within the hot-spot region) were synonymous. This investigation clearly demonstrates that HRM analysis of the proposed SNP hot-spot region in the rpoB gene of C. difficile is a fast and cost-effective method for the identification of C. difficile samples with reduced susceptibility to rifaximin and even allows simultaneous SNP subtyping of the respective C. difficile isolates.

Introduction: The Ineffectiveness of the North Korean Sanctions RegimeThe fact that nuclear weapons capability can quickly and radically alter the strategic balance is well known.3 This quality of nuclear weapons has made their acquisition particularly valuable to North Korea.4 And for the very same reason, the international community has tried to prevent the Democratic People's Republic of Korea (DPRK) from acquiring nuclear weapons and missile-based delivery systems. Since at least 1994, concerted efforts have been made to halt Pyongyang's nuclear programs and state the international community's opposition.5The major state players engaged in these international efforts are the bordering states, grouped loosely in the so-called Six Party Talks (DPRK and South Korea, China, the United States, Russia, and Japan). The denial strategy employed by these actors has been a combination of diplomacy, negotiations, and coercion in the form of sanctions.6 By 2006/2009, however, it had become clear that these attempts had failed in their objective.7 In light of this and the worsening security situation on the Korean Peninsula today, the need for effective sanctions has arguably never been greater.Below, the argument is made that the instrument of sanctions imposed on North Korea has been reduced largely to economic sanctions, although statecraft and in particular international public law conceptualizes it more broadly as "economic and other sanctions."8 With sanctions understood too narrowly as essentially economic instruments of diplomacy, too much faith has been put in the assumption that "economic costs will translate into political effects"9 of the targeted state. With respect to North Korea, such an approach has remained ineffective.10 Therefore, the focus should be shifted to other, especially diplomatic sanctions in order to increase the effect of the international sanctions regime on the DPRK.Diplomacy, Coercive Diplomacy, and SanctionsSanctions fall under the rubric of coercive diplomacy, which itself is a subspecies of general diplomacy. Diplomacy is meant to manage, limit, or prevent international problems from deteriorating into crises and open conflict.11 The choice between diplomacy and the application of physical violence is, however, often more a dilemma than a choice. Crisis diplomacy without the threat of military force may be perceived as impotent; at the same time, the actual use of physical violence may be considered politically "out of bounds" and normatively unacceptable. Sanctions promise a middle ground between diplomatic negotiations and direct violence, making them an immensely relevant tool of statecraft in general, and "probably the most common foreign policy tool democracies use to bring about policy or institutional changes in authoritarian regimes."12Combining Thomas Schelling's and Alexander George's work on the subject, compulsion is conceptualized here as using the threat of war to alter a situation proactively, coercion as applying the threat of force to reverse or stall a development, and deterrence as the threat of major military force if clearly communicated interests are violated.13 A sanctions regime, such as the one in place for North Korea, is coercion as part of coercive diplomacy. Coercive diplomacy employs the threat of force to provide the coercing state with leverage,14 but remains distinct from open, violent interstate conflict.15The common objective of any form of coercive diplomacy is to achieve a change in behavior by the targeted state. With respect to North Korea, the sanctions regime has been structured with the objective of altering North Korean policy towards its missile programs and nuclear armament. More specifically, the idea has been to use economic sanctions to create an unsustainable economic situation for North Korea, thus coercing it to adjust its military-political strategy.SanctionsAll too often sanctions are conceptualized narrowly as economic sanctions and an effort at "international economic coercion. …

Aim: Behcet’s disease (BD) is an autoimmune disorder associated with HLA-B51 positivity. Serologic/genomic findings have suggested microbes as possible causative agents and the geographical distribution suggests environmental influences. Methods: We performed comparative analyses of 40 patients with BD or related symptoms not fulfilling BD criteria. Patients originating from different regions of Iran were tested by molecular/serological methods for human herpes viruses and parvovirus B19, two Chlamydiae species, as well as Coxiella, Listeria, Yersinia, Leptospira and Mycobacterium paratuberculosis. Human leukocyte antigen-typing was performed: testing of cytokine profiles and immune mediators representative for the cellular immune system, including neopterin/kynurenine production. Results: No apparent differences in interleukin (IL)-4, 6, 8 and 10 were observed, whereas production of soluble IL-2-receptor and tumor necrosis factor (TNF)-alpha were more pronounced in the BD group. Neopterin/kynurenine production was comparable, although both groups showed twice the levels of healthy people. No significant differences of herpes simplex virus (HSV) antibody titres were observed but higher titres against Chlamydophila pneumoniae were found in the controls. In 20 BD patients and controls neither parvovirus B19 DNA was detected nor bacterial DNA. Viral DNA of Epstein–Barr virus (EBV), cytomegalovirus (CMV) and human herpes virus (HHV)8 was detected more frequently in the BD group, whereas HSV DNA was only found in the controls, indicating that stomatitis might be caused by HSV. Conclusion: Although no significant association of BD was detected with a single pathogen, our findings suggest that detection of HSV DNA or Chlamydiae would rather argue against classic BD. Whether there is a discriminative potential of the tested immune mediators/receptors has to be elucidated in further studies.

Chlamydia pneumoniae is an intracellular bacterium that responds poorly to antibiotic treatment. Insufficient antibiotic usage leads to chronic infection, which is linked to disease processes of asthma, atherosclerosis, and Alzheimer's disease. The Chlamydia research lacks genetic tools exploited by other antimicrobial research, and thus other approaches to drug discovery must be applied. A set of 2-arylbenzimidazoles was designed based on our earlier findings, and 33 derivatives were synthesized. Derivatives were assayed against C. pneumoniae strain CWL-029 in an acute infection model using TR-FIA method at a concentration of 10 μM, and the effects of the derivatives on the host cell viability were evaluated at the same concentration. Fourteen compounds showed at least 80% inhibition, with only minor changes in host cell viability. Nine most potential compounds were evaluated using immunofluorescence microscopy on two different strains of C. pneumoniae CWL-029 and CV-6. The N-[3-(1H-benzimidazol-2-yl)phenyl]-3-methylbenzamide (42) had minimal inhibitory concentration (MIC) of 10 μM against CWL-029 and 6.3 μM against the clinical strain CV-6. This study shows the high antichlamydial potential of 2-arylbenzimidazoles, which also seem to have good characteristics for lead compounds.

The “Roosevelt Corollary to the Monroe Doctrine of 1904/05” constitutes a landmark in United States foreign policy. However, the 1902/03 Venezuela Crisis—in particular, the arbitration process between the South American country and Germany, Great Britain, and Italy that settled the crisis—led to President Theodore Roosevelt's decision to amend the Monroe Doctrine. The arbitrational award was an important impetus for the corollary because its decision appeared to encourage future European interventions in the western hemisphere. The Roosevelt Corollary was needed to prevent a situation similar to the 1902/03 Venezuela Crisis from occurring again. Strategically speaking, Roosevelt felt the corollary was necessary to uphold the Monroe Doctrine under new circumstances.

Since 1648, the number of small states has varied significantly. There have been more than one "rise and fall" in the number of small states. This study begins a broader analysis into causality by investigating this phenomenon. By setting out the changes in the composition of the international states system since its inception in its modem form with the Peace of Westphalia, the study intends to discuss the proliferation of small states over time as a significant phenomenon in the history of international relations. The study then continues by linking the changes in the number of small states to major systemic changes, arguing that different types of states system correspond to different levels of "systemic resilience" of small states.

The small state has so far escaped a consensus definition because ‘the’ small state has in fact been conceived of very differently. Different studies of ‘the’ small state have characterized it quite distinctly. In fact, there is substantial disagreement even over what type of criteria, quantifiable or qualitative, are most appropriate to characterize the small state. However, I argue that such fundamental disagreement over what makes a state small has actually benefited the area of small states studies by providing it with conceptual flexibility to match different research designs as well as the quite substantial variations among actual small states in the world. In short, in the discipline of international relations as well as in reality, more than one definition of the small state does and should exist.

The controversial discussion about the role of Chlamydia pneumoniae in atherosclerosis cannot be solved without a reliable diagnosis that allows discrimination between past and persistent infections. Using a proteomic approach and immunoblotting with human sera, we identified 31 major C. pneumoniae Ags originating from 27 different C. pneumoniae proteins. More than half of the proteins represent Chlamydia Ags not described previously. Using a comparative analysis of spot reactivity Pmp6, OMP2, GroEL, DnaK, RpoA, EF-Tu, as well as CpB0704 and CpB0837, were found to be immunodominant. The comparison of Ab-response patterns of sera from subjects with and without evidence for persisting C. pneumoniae, determined by multiple PCR analysis of PBMC and vasculatory samples, resulted in differential reactivity for 12 proteins, which is not reflected by reactivity of the sera in the microimmunofluorescence test, the current gold standard for serodiagnosis. Although reactivity of sera from PCR-positive donors was increased toward RpoA, MOMP, YscC, Pmp10, PorB, Pmp21, GroEL, and Cpaf, the reactivity toward YscL, Rho, LCrE, and CpB0837 was decreased, reflecting the altered protein expression of persisting C. pneumoniae in vitro. Our data provide the first evidence of a unique Ab-response pattern associated with persistent C. pneumoniae infections, which is a prerequisite for the serological determination of persistently infected patients.

Chlamydiaceae are obligate intracellular bacteria that cause endemic trachoma, sexually transmitted diseases and respiratory infections. The course of the diseases is determined by local inflammatory immune responses and the propensity of the pathogen to replicate within infected host cells. Both features require energy which is inseparably coupled to oxygen availability in the microenvironment. Hypoxia-inducible factor-1 (HIF-1) regulates crucial genes involved in the adaptation to low oxygen concentrations, cell metabolism and the innate immune response. Here we report that Chlamydia pneumoniae directly interferes with host cell HIF-1alpha regulation in a biphasic manner. In hypoxia, C. pneumoniae infection had an additive effect on HIF-1alpha stabilization resulting in enhanced glucose uptake during the early phase of infection. During the late phase of intracellular chlamydial replication, host cell adaptation to hypoxia was actively silenced by pathogen-induced HIF-1alpha degradation. HIF-1alpha was targeted by the chlamydial protease-like activity factor, which was secreted into the cytoplasm of infected cells. Direct interference with HIF-1alpha stabilization was essential for efficient C. pneumoniae replication in hypoxia and highlights a novel strategy of adaptive pathogen-host interaction in chlamydial diseases.

Chlamydophila pneumoniae is an important respiratory pathogen. In this study we characterized C. pneumoniae strain TW183-mediated activation of human small airway epithelial cells (SAEC) and the bronchial epithelial cell line BEAS-2B and demonstrated time-dependent secretion of granulocyte macrophage colony-stimulating factor (GM-CSF) upon stimulation. TW183 activated p38 mitogen-activated protein kinase (MAPK) in epithelial cells. Kinase inhibition by SB202190 blocked Chlamydia-mediated GM-CSF release on mRNA and protein levels. In addition, the chemical inhibitor as well as dominant-negative mutants of p38 MAPK isoforms p38alpha, beta2, and gamma inhibited C. pneumoniae-related NF-kappaB activation. In contrast, blocking of MAPK ERK, c-Jun kinase/JNK, or PI-3 Kinase showed no effect on Chlamydia-related epithelial cell GM-CSF release. Ultraviolet-inactivated pathogens as compared with viable bacteria induced a smaller GM-CSF release, suggesting that viable Chlamydiae were only partly required for a full effect. Presence of an antichlamydial outer membrane protein-A (OmpA) antibody reduced and addition of recombinant heat-shock protein 60 from C. pneumoniae (cHsp60, GroEL-1)-enhanced GM-CSF release, suggesting a role of these proteins in epithelial cell activation. Our data demonstrate that C. pneumoniae triggers an early proinflammatory signaling cascade involving p38 MAPK-dependent NF-kappaB activation, resulting in subsequent GM-CSF release. C. pneumoniae-induced epithelial cytokine liberation may contribute significantly to inflammatory airway diseases like chronic obstructive pulmonary disease (COPD) or bronchial asthma.

Interleukin-6 (IL-6) is required for the clearance of bacteria in pneumococcal pneumonia. The abundance of endogenous IL-6 production on infectious stimuli is associated with genotypic differences in the −174 promoter region of IL-6 (−174 G→C), showing increased IL-6 levels in patients carrying the GG genotype. One hundred patients with culturally proven pneumococcal disease were analyzed for distribution of the G-/C-alleles in the IL-6 −174 promoter region in comparison to 50 age-matched controls. Extrapulmonary pneumococcal dissemination, including septic metastasis, endocardial and meningeal infection, was used as parameter for impaired clearance of the bacteria. No significant differences in the allele distribution were observed between patients and controls. Within the patient group, the interleukin-6 GG homozygous carriers were less likely to develop extrapulmonary pneumococcal infection (10.3% versus 30.9%; OR 0.26, 95% CI 0.07–0.94, p = 0.04). The IL-6 GG genotype, encoding for enhanced IL-6 secretion on bacterial stimuli, reduces the risk of bacterial spread to extrapulmonary sites in pneumococcal infection, possibly due to a more effective clearance of the pathogen from the blood and the respiratory tract.

Chlamydia pneumoniae uses blood monocytes (PBMC) for systemic dissemination, persists in atherosclerotic lesions, and has been implicated in the pathogenesis of atherosclerosis. During transmigration in a newly developed transendothelial migration model (TEM) C pneumoniae-infected PBMC spread their infection to endothelial cells. Transmigrated PBMC retained their infectivity and transmitted the pathogen to smooth muscle cells in the lower chamber of the TEM. Detection of chlamydial HSP60 mRNA proved pathogen viability and virulence. We conclude that PBMC can spread chlamydial infection to vascular wall cells and we suggest the TEM as a novel tool to analyze host-pathogen interactions in vascular chlamydial infections. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Summary Chlamydophila (Chlamydia) pneumoniae, a gram-negative obligate intracellular bacterium, is a widespread respiratory pathogen. Chronic C. pneumoniae infection has been suggested as a trigger/ promoter of inflammation that may result in vascular lesions. Although the genome of C. pneumoniae has been sequenced completely this information has not yet led to an understanding of the mechanisms of acute infection and target cell activation nor to the identification of potential chlamydial virulence factors. Intriguingly, current antibiotic treatment options for acute chlamy- dial infection were proven to be ineffective with respect to clinical outcome in different groups of atherosclerotic patients. The reason might be that primary infection of vascular smooth muscle cells and blood monocytes with C. pneumoniae resembles rather a persistent, antibiotic-resistant, than an active infection. In this review we will focus on the importance of putative host cell receptors for C.pneumoniae and subsequently activated signal transduction pathways.

Atherosclerosis is typically associated with a low-grade vascular inflammation that can be measured with the highly sensitive C-reactive protein (hs-CRP) assay. Actually, acute coronary syndromes are thought to result from plaque rupture which is induced by the inflammatory process in the atherosclerotic tissue. Therefore, it is interesting to discuss the value of follow-up measurements of hs-CRP in patients with coronary artery disease (CAD).The hs-CRP concentration of 133 patients consecutively admitted with an acute coronary syndrome (ACS) was measured on admission and after 6 months. The final assessment after 3 years was structured by questionnaire.17 cardiac events occurred within 6 months, 30 during the following 3 years. The hs-CRP levels (median +/- SEM) decreased significantly (p0.001) from baseline (3.9 +/- 1.3 mg/l) to follow-up (2.9 +/- 0.9 mg/l). Subdivision according to cardiac events during the first observation period confirmed this decrease in patients without events (baseline: 3.9 +/- 1.5 mg/l, follow-up: 2.9 +/- 0.9 mg/l; p0.001), whereas patients with events showed a persistent elevation (baseline: 3.8 +/- 0.9 mg/l, follow-up: 4.1 +/- 1.0 mg/l; p = 0.426). Patients who developed a further event during the 6-month period showed hs-CRP levels at follow-up that were60% of the initial level. Interestingly, 80% of the events within the following 3 years occurred in patients with an hs-CRP level above this discriminator. With a follow-up hs-CRP value above this discriminator the relative risk of suffering an event was 3.4 (95% confidence interval 1.27-8.9; p0.05).Patients with a non-ST elevation ACS who showed no event within 6 months are characterized by a decrease in hs-CRP levels from baseline to follow-up. Most events in the observation period of 3 years occurred in patients with follow-up hs-CRP levels60% of the initial level. Therefore, it was hypothesized that a repeated measurement of hs-CRP levels in CAD patients could help to discriminate those at high risk of further events.

Peripheral blood monocytes (PBMC) promote vascular inflammation and atherosclerosis. Chlamydia pneumoniae (Cp) infection of PBMC is found in atherosclerotic patients, appears refractory to antibiotics, and may predispose to vascular damage. In Cp-infected human PBMC we analyzed the role of cyclooxygenase-2 (Cox-2) for the proatherosclerotic key mediators prostaglandin E2 (PGE2) and interstitial collagenase (MMP-1). Cp infection resulted in rapid and sustained Cox-2 mRNA and protein stimulation depending on p38 and p44/42 MAPkinases. Subsequent upregulation of PGE synthase and MMP-1 was completely abrogated by the selective Cox-2 inhibitor NS398. Enhanced synthesis of PGE2 and MMP-1 in Cp infected PBMC is mediated through initiation of the p38 and p44/42 MAPK pathways and requires sustained Cox-2 activation. Selective Cox-2 inhibitors, currently under investigation for cardiovascular risk reduction, may represent a novel therapeutic option for patients with endovascular Cp infection as they target the actuated pathological signal transduction cascade in persistently infected PBMC.

The bacterium Chlamydia pneumoniae chronically infects atheromatous lesions and is linked to atherosclerosis by modifying inflammation, proliferation, and the lipid metabolism of blood monocytes. As continuous LDL modification in the vascular intima is crucial for atherogenesis we investigated the impact of endothelial infection on LDL oxidation. HUVEC were infected with a vascular C. pneumoniae strain. Supernatants of infected cells but not cell lysates increased lipid peroxidation products (6.44 vs 6.14 nmol/ml, p

Atherosclerosis is characterized by chronic inflammation and vascular cell proliferation. The intracellular bacterium Chlamydia pneumoniae has been implicated in atherogenesis as it has been recovered from atheromatous plaques. In vitro, C. pneumoniae infection initiates activation of the transcriptional factor NF-κB via small GTPases Rac1 and RhoA. This signaling cascade results in the synthesis of numerous proinflammatory and pro-coagulatory mediators (e.g., IL-6, IL-8, RANTES, MCP-1, PAI-1, TF) that are also characteristically produced in the plaque. In parallel, chlamydiae stimulate vascular cell proliferation via ERK 1/2 MAPkinase and transcription factor Egr-1. Thus, chlamydial infection may have a role in the inflammatory, as well as in the angiogenic, component of atherogenesis. The proinflammatory activity of chlamydial infection is reduced by statins, via suppression of Rho and Rac signalling, indicating a beneficial anti-inflammatory effect of lipid-lowering drugs in vascular infection. C. pneumoniae uses blood monocytes as transport vehicles for systemic dissemination. In monocytes the pathogen enters a non-replicative, but viable, state characterized by continuous production of mRNA transcripts. Release of proinflammatory mediators by circulating or transendothelially migrating infected monocytes might promote chronic inflammation and atherogenesis. Interestingly, persistent chlamydiae in monocytes appear to be refractory to conventional antichlamydial treatment, thus posing a challenge to the ongoing experimental treatment studies. The persistent state is likely to have a key role in the pathogenesis of chronic chlamydial infections and requires further examination on a molecular level.

Chlamydia pneumoniae, a major cause of community-acquired pneumonia, primarily infects the respiratory tract. Chronic infection of nonrespiratory sites, such as the vascular wall, the brain or blood monocytes, requires evasion from the lungs and spreading via the bloodstream. The cell types involved in dissemination are insufficiently characterised. In this study, New Zealand White rabbits were infected intratracheally with C. pneumoniae, and lung manifestation and systemic dissemination were monitored by polymerase chain reaction and immunohistochemistry. Infection of the lungs was characterised by an early phase dominated by granulocytes and a late phase dominated by alveolar macrophages (AM). Granulocytes, AM and alveolar epithelial cells acted as host cells for chlamydiae, which remained detectable for up to 8 weeks. AM transported the pathogen to the peribronchiolar lymphatic tissue, and subsequently C. pneumoniae entered the spleen and the aorta via dissemination by peripheral blood monocytes. In conclusion, Chlamydia pneumoniae-infected alveolar macrophages transmigrate through the mucosal barrier, and give the pathogen access to the lymphatic system and the systemic circulation. Infected peripheral blood monocytes are the vector system within the bloodstream and transmit the infection to the vascular wall. This is the first description of granulocytes acting as a reservoir for Chlamydia pneumoniae early in infection.

Chlamydia pneumoniae is an obligate intracellular pathogen that causes respiratory infections and has been associated with cardiovascular disease. We compared respiratory and cardiovascular isolates to find genetic differences associated with pathogenicity. A polymorphic region encoding a tyrosine/tryptophan permease was found to differ between disease isolates. Respiratory strains contained multiple copies of the tyrP gene, and vascular strains contained a single copy. Single-nucleotide polymorphism analysis revealed the duplication to be a phylogenetically old event. Gene amplification was associated with higher mRNA levels and higher uptake of the substrate tyrosine, indicating an amino-acid transport-related phenotype associated with the tyrP genotype. Vascular strains, despite their reduced ability to transport tyrosine, do not appear to have a reduced growth rate in vitro. We hypothesize that the important difference between strains of vascular and respiratory origin may lie in the increased tendency of vascular strains to elicit persistent infection that is triggered by amino-acid starvation.

The Chlamydia trachomatis inclusion is fragile, rendering it incompatible to micromanipulation. We show that the Chlamydia pneumoniae inclusion differs, being resistant to micromanipulation as shown by direct microinjection of the infected host cytosol or the inclusion itself. We have used micromanipulation to clone C. pneumoniae and to free it from mycoplasma contamination.

Balanced secretion of pro- and anti-inflammatory cytokines is essential in limiting pulmonary inflammation in respiratory infections. It was hypothesised that, in acute infection with Chlamydia pneumoniae, mononuclear cells from chronic obstructive pulmonary disease (COPD) patients lack the opportunity to compensate for the inflammatory immune response by secreting adequate amounts of anti-inflammatory cytokines. Alveolar macrophages (AMs) and peripheral blood mononuclear cells (PBMCs) from eight COPD patients and eight healthy controls were infected with C. pneumoniae in order to determine interleukin (IL)-1beta, IL-1 receptor antagonist (IL-1RA) and IL-8 expression and messenger ribonucleic acid levels. Secretion of IL-1beta was significantly enhanced in AMs (six-fold) and PBMCs (four-fold) from COPD patients after infection with C. pneumoniae. Compared to the control group, release of its anti-inflammatory counterpart IL-1RA was diminished in COPD patients, resulting in a significantly higher IL-1beta/IL-1RA ratio in C. pneumoniae-infected AMs and PBMCs from COPD patients. Mononuclear cells from chronic obstructive pulmonary disease patients have less capacity for balancing the pro-inflammatory immune response caused by Chlamydia pneumoniae infection than those from healthy controls. These findings suggest that, during acute exacerbation with intracellular pathogens, chronic obstructive pulmonary disease patients are predisposed to inflammatory changes in the lungs.