Infectious burden and atherosclerosis. A clinical issue

Atherosclerotic cardiovascular diseases, chronic inflammatory diseases of multifactorial etiology, are the leading cause of death worldwide. In the last decade, more infectious agents, labeled as "infectious burden", rather than any single pathogen, have been showed to contribute to the development of atherosclerosis through different mechanisms. Some microorganisms, such as Chlamydia pneumoniae (C. pneumoniae), human cytomegalovirus, etc. may act directly on the arterial wall contributing to endothelial dysfunction, foam cell formation, smooth muscle cell proliferation, platelet aggregation as well as cytokine, reactive oxygen specie, growth factor, and cellular adhesion molecule production. Others, such as Helicobacter pylori (H. pylori), influenza virus, etc. may induce a systemic inflammation which in turn may damage the vascular wall (e.g., by cytokines and proteases). Moreover, another indirect mechanism by which some infectious agents (such as H. pylori, C. pneumoniae, periodontal pathogens, etc.) may play a role in the pathogenesis of atherosclerosis is molecular mimicry. Given the complexity of the mechanisms by which each microorganism may contribute to atherosclerosis, defining the interplay of more infectious agents is far more difficult because the pro-atherogenic effect of each pathogen might be amplified. Clearly, continued research and a greater awareness will be helpful to improve our knowledge on the complex interaction between the infectious burden and atherosclerosis

Diversity of cervical microbiota in asymptomatic chlamydia trachomatis genital infection: a pilot study

Chlamydia trachomatis genital infection continues to be an important public health problem worldwide due to its increasing incidence. C. trachomatis infection can lead to severe sequelae, such as pelvic inflammatory disease, obstructive infertility, and preterm birth. Recently, it has been suggested that the cervico-vaginal microbiota may be an important defense factor toward C. trachomatis infection as well as the development of chronic sequelae. Therefore, the investigation of microbial profiles associated to chlamydial infection is of the utmost importance. Here we present a pilot study aiming to characterize, through the metagenomic analysis of sequenced 16s rRNA gene amplicons, the cervical microbiota from reproductive age women positive to C. trachomatis infection. The main finding of our study showed a marked increase in bacterial diversity in asymptomatic C. trachomatis positive women as compared to healthy controls in terms of Shannon's diversity and Shannon's evenness (P = 0.031 and P = 0.026, respectively). More importantly, the cervical microbiota from C. trachomatis positive women and from healthy controls significantly separated into two clusters in the weighted UniFrac analysis (P = 0.0027), suggesting that differences between the two groups depended entirely on the relative abundance of bacterial taxa rather than on the types of bacterial taxa present. Furthermore, C. trachomatis positive women showed an overall decrease in Lactobacillus spp. and an increase in anaerobes. These findings are part of an ongoing larger epidemiological study that will evaluate the potential role of distinct bacterial communities of the cervical microbiota in C. trachomatis infection

New insights into Chlamydiae persistence: an energy metabolism strategy?

Chlamydiaceae is a family of obligate intracellular bacteria generally considered energy parasites. Several studies have suggested that Chlamydiae are capable of independently producing energy and, more importantly, several genes involved in the energy metabolism are up-regulated during the persistent state. Thus, it has been suggested that chlamydial persistence could be a complex and flexible metabolic strategy designed to favor a lengthy survival in the host cell by evading the immune response. In conclusion, more detailed studies on the shift in the chlamydial energy metabolism, from the active to the persistent form, may be helpful in future to determine whether chlamydial persistence observed in vitro does occur in vivo and whether chronic sequelae of chlamydial diseases may be related to the persistence

In-cell western assay as a high-throughput approach for Chlamydia trachomatis quantification and susceptibility testing to antimicrobials

Chlamydia trachomatis, the leading cause of bacterial sexually transmitted diseases in developed countries, with around 127 million new cases per year, is mainly responsible for urethritis and cervicitis in women, and urethritis and epididymitis in men. Most C. trachomatis infections remain asymptomatic (>50%) and, hence, untreated, leading to severe reproductive complications in both women and men, like infertility. Therefore, the detection of C. trachomatis as well as the antimicrobial susceptibility testing becomes a priority, and, along the years, several methods have been recommended, like cell culture and direct immunofluorescence (DFA) on cell cultures. Herein, we described the application of In-Cell Western assay (ICW) via Odyssey CLx as a fast, more accessible, and high-throughput platform for the quantification of C. trachomatis and the screening of anti-chlamydial drugs. As a first step, we set up a standard curve by infecting cell monolayers with 2-fold serial dilutions of C. trachomatis Elementary Body (EB) suspension. Then, different unknown C. trachomatis EB suspensions were quantified and the chlamydial susceptibility testing to erythromycin was performed, using the DFA as comparison. Our results showed a very high concordance between these two assays, as evidenced by the enumeration of chlamydial IFUs as well as the determination of erythromycin Minimum Inhibitory Concentration (MIC). In conclusion,the ICW assay may be a promising candidate as an accurate and accessible methodology for C. trachomatis antimicrobial susceptibility testing

Chlamydia pneumoniae clinical isolate from gingival crevicular fluid: a potential atherogenic strain

Chlamydia pneumoniae has been associated to atherosclerotic cardiovascular diseases. The aim of our study was to characterize, for the first time, a C. pneumoniae strain isolated from the gingival crevicular fluid of a patient with chronic periodontitis, described as a risk factor for cardiovascular diseases. C. pneumoniae isolate was characterized and compared to the respiratory AR-39 strain by VD4-ompA genotyping and by investigating the intracellular growth in epithelial and macrophage cell lines and its ability to induce macrophage-derived foam cells. Inflammatory cytokine levels were determined in the gingival crevicular fluid sample. C. pneumoniae isolate showed a 99% similarity with the AR-39 strain in the VD4-ompA gene sequence and shared a comparable growth kinetic in epithelial cells and macrophages, as evidenced by the infectious progeny and by the number of chlamydial genomic copies. C. pneumoniae isolate significantly increased the number of foam cells as compared to uninfected and LDL-treated macrophages (45 vs. 6%, P = 0.0065) and to the AR-39 strain (45 vs. 30%, P = 0.0065). Significantly increased levels of interleukin 1-β (2.1 ± 0.3 pg/μL) and interleukin 6 (0.6 ± 0.08 pg/μL) were found. Our results suggest that C. pneumoniae may harbor inside oral cavity and potentially be atherogenic, even though further studies will be needed to clarify the involvement of C. pneumoniae in chronic periodontitis as a risk factor for cardiovascular diseases

Interferon-ε as potential inhibitor of Chlamydia trachomatis infection

Chlamydia trachomatis, the main cause of bacterial sexually transmitted diseases, is responsible for severe reproductive sequelae. Amongst all the cytokines involved in host immunity towards this pathogen, IFN-ε has recently acquired importance for its potential contribution to the female reproductive tract innate defenses. Herein, our study aimed to explore, for the first time, the activity of IFN-ε toward C. trachomatis in an in vitro infection model, by testing its effects on the different phases of chlamydial developmental cycle, as well as on the ultrastructural characteristics of chlamydial inclusions, via transmission electron microscopy. Main result is the capability of IFN-ε to alter C. trachomatis growth, as suggested by reduced infectious progenies, as well as a patchy distribution of bacteria and altered morphology of reticulate bodies within inclusions. In conclusion, our results suggest that IFN-ε could play a role in the innate and adaptive immune defenses against C. trachomatis; in the future, it will be needed to investigate its activity on an infection model more closely resembling the physiological environment of the female genital tract

Effect of bovine lactoferrin on chlamydia trachomatis infection and inflammation.

Chlamydia trachomatis is an obligate, intracellular pathogen responsible for the most common sexually transmitted bacterial disease worldwide, causing acute and chronic infections. The acute infection is susceptible to antibiotics, whereas the chronic one needs prolonged therapies, thus increasing the risk of developing antibiotic resistance. Novel alternative therapies are needed. The intracellular development of C. trachomatis requires essential nutrients, including iron. Iron-chelating drugs inhibit C. trachomatis developmental cycle. Lactoferrin (Lf), a pleiotropic iron binding glycoprotein, could be a promising candidate against C. trachomatis infection. Similarly to the efficacy against other intracellular pathogens, bovine Lf (bLf) could both interfere with C. trachomatis entry into epithelial cells and exert an anti-inflammatory activity. In vitro and in vivo effects of bLf against C. trachomatis infectious and inflammatory process has been investigated. BLf inhibits C. trachomatis entry into host cells when incubated with cell monolayers before or at the moment of the infection and down-regulates IL-6/IL-8 synthesized by infected cells. Six out of 7 pregnant women asymptomatically infected by C. trachomatis, after 30 days of bLf intravaginal administration, were negative for C. trachomatis and showed a decrease of cervical IL-6 levels. This is the first time that the bLf protective effect against C. trachomatis infection has been demonstrated

The Chlamydia muridarum plasmid revisited : new insights into growth kinetics.

Background: Research in chlamydial genetics is challenging because of its obligate intracellular developmental cycle. In vivo systems exist that allow studies of different aspects of basic biology of chlamydiae, the murine Chlamydia muridarum model is one of great importance and thus an essential research tool. C. muridarum carries a plasmid that has a role in virulence.  Our aim was to compare and contrast the C. muridarum plasmid-free phenotype with that of a chromosomally isogenic plasmid-bearing strain, through the inclusion phase of the developmental cycle. Methods: We measured infectivity for plasmid bearing and plasmid-cured C. muridarum by inclusion forming assays in McCoy cells and in parallel bacterial chromosome replication by quantitative PCR, throughout the developmental cycle. In addition to these studies, we have carefully monitored chlamydial inclusion formation by confocal microscopy and transmission electron microscopy. A new E.coli/chlamydial shuttle vector (pNigg::GFP) was constructed using standard cloning technology and used to transform C. muridarum for further phenotypic studies. Results: We have advanced the definition of the chlamydial phenotype away from the simple static observation of mature inclusions and redefined the C. muridarum plasmid-based phenotype on growth profile and inclusion morphology. Our observations on the growth properties of plasmid-cured C. muridarum challenge the established interpretations, especially with regard to inclusion growth kinetics. Introduction of the shuttle plasmid pNigg::GFP into plasmid-cured C. muridarum restored the wild-type plasmid-bearing phenotype and confirmed that loss of the plasmid was the sole cause for the changes in growth and chromosomal replication. Conclusions: Accurate growth curves and sampling at multiple time points throughout the developmental cycle is necessary to define plasmid phenotypes.  There are subtle but important (previously unnoticed) differences in the overall growth profile of plasmid-bearing and plasmid-free C. muridarum.  We have proven that the differences described are solely due to the plasmid pNigg

Prospective double-blind randomised controlled trial protocol comparing bone marrow aspirate concentrate intra-articular injection combined with subchondral injection versus intra-articular injection alone for the treatment of symptomatic knee osteoarthritis

Introduction: Subchondral and intra-articular injections of bone marrow aspirate concentrate (BMAC) showed promising results for knee osteoarthritis (OA) patients. To date, there is no evidence to demonstrate whether the combination of these treatments provides higher benefits than the intra-articular injection alone. Methods and analysis: Eighty-six patients with symptomatic knee OA (aged between 40 and 70 years) are randomised to BMAC intra-articular injection combined with subchondral BMAC injection or BMAC intra-articular injection alone in a ratio of 1:1. The primary outcome is the total Western Ontario and McMaster Universities Osteoarthritis Index, the secondary outcomes are the International Knee Documentation Committee Subjective and Objective Knee Evaluation Form, the Tegner activity scale, the EuroQol-Visual Analogue Scale, and the health questionnaire European Quality of Life Five Dimension score. Additional CT and MRI evaluations are performed at the baseline assessment and at the final 12-month follow-up. The hypothesis is that the combined injections provide higher knee pain and function improvement compared with BMAC intra-articular injection alone. The primary analysis follows an intention to treat principle. Ethics and dissemination: The study protocol has been approved by the Emilia Wide Area Ethical Committee of the Emilia-Romagna Region (CE-AVEC), Bologna, Italy. Written informed consent is obtained from all the participants. Findings of this study will be disseminated through peer-reviewed publications and conference presentations. Protocol version: Version 1 (14 May 2018). Trial registration number: NCT03876795

Chlamydia pneumoniae and oxidative stress in cardiovascular disease. State of the art and prevention strategies.

Chlamydia pneumoniae, a pathogenic bacteria responsible for respiratory tract infections, is known as the most implicated infectious agent in atherosclerotic cardiovascular diseases (CVDs). Accumulating evidence suggests that C. pneumoniae-induced oxidative stress may play a critical role in the pathogenesis of CVDs. Indeed, the overproduction of reactive oxygen species (ROS) within macrophages, endothelial cells, platelets and vascular smooth muscle cells (VSMCs) after C. pneumoniae exposure, has been shown to cause low density lipoprotein oxidation, foam cell formation, endothelial dysfunction, platelet adhesion and aggregation, and VSMC proliferation and migration, all responsible for the typical pathological changes of atherosclerotic plaque. The aim of this review is to improve our insight into C. pneumoniae-induced oxidative stress in order to suggest potential strategies for CVD prevention. Several antioxidants, acting on multi-enzymatic targets related to ROS production induced by C. pneumoniae, have been discussed. A future strategy for the prevention of C. pneumoniae-associated CVDs will be to target chlamydial HSP60, involved in oxidative stress