Fabiola Mancini

Chlamydophila pneumoniae phospholipase D (CpPLD) drives Th17 inflammation in human atherosclerosis.

Phospholipases are produced from bacterial pathogens causing very different diseases. One of the most intriguing aspects of phospholipases is their potential to interfere with cellular signaling cascades and to modulate the host–immune response. Here, we investigated the role of the innate and acquired immune responses elicited by Chlamydophila pneumoniae phospholipase D (CpPLD) in the pathogenesis of atherosclerosis. We evaluated the cytokine and chemokine production induced by CpPLD in healthy donors’ monocytes and in vivo activated T cells specific for CpPLD that infiltrate atherosclerotic lesions of patients with C. pneumoniae antibodies. We also examined the helper function of CpPLD-specific T cells for monocyte matrix metalloproteinase (MMP)-9 and tissue factor (TF) production as well as the CpPLD-induced chemokine expression by human venular endothelial cells (HUVECs). We report here that CpPLD is a TLR4 agonist able to induce the expression of IL-23, IL-6, IL-1β, TGF-β, and CCL-20 in monocytes, as well as CXCL-9, CCL-20, CCL-4, CCL-2, ICAM-1, and VCAM-1 in HUVECs. Plaque-derived T cells produce IL-17 in response to CpPLD. Moreover, CpPLD-specific CD4+ T lymphocytes display helper function for monocyte MMP-9 and TF production. CpPLD promotes Th17 cell migration through the induction of chemokine secretion and adhesion molecule expression on endothelial cells. These findings indicate that CpPLD is able to drive the expression of IL-23, IL-6, IL-1β, TGF-β, and CCL-20 by monocytes and to elicit a Th17 immune response that plays a key role in the genesis of atherosclerosis.

Human immunodeficiency virus integrase inhibitors efficiently suppress feline immunodeficiency virus replication in vitro and provide a rationale to redesign antiretroviral treatment for feline AIDS.

BackgroundTreatment of feline immunodeficiency virus (FIV) infection has been hampered by the absence of a specific combination antiretroviral treatment (ART). Integrase strand transfer inhibitors (INSTIs) are emerging as a promising new drug class for HIV-1 treatment, and we evaluated the possibility of inhibiting FIV replication using INSTIs.MethodsPhylogenetic analysis of lentiviral integrase (IN) sequences was carried out using the PAUP* software. A theoretical three-dimensional structure of the FIV IN catalytic core domain (CCD) was obtained by homology modeling based on a crystal structure of HIV-1 IN CCD. The interaction of the transferred strand of viral DNA with the catalytic cavity of FIV IN was deduced from a crystal structure of a structurally similar transposase complexed with transposable DNA. Molecular docking simulations were conducted using a genetic algorithm (GOLD). Antiviral activity was tested in feline lymphoblastoid MBM cells acutely infected with the FIV Petaluma strain. Circular and total proviral DNA was quantified by real-time PCR.ResultsThe calculated INSTI-binding sites were found to be nearly identical in FIV and HIV-1 IN CCDs. The close similarity of primate and feline lentivirus IN CCDs was also supported by phylogenetic analysis. In line with these bioinformatic analyses, FIV replication was efficiently inhibited in acutely infected cell cultures by three investigational INSTIs, designed for HIV-1 and belonging to different classes. Of note, the naphthyridine carboxamide INSTI, L-870,810 displayed an EC50 in the low nanomolar range. Inhibition of FIV integration in situ was shown by real-time PCR experiments that revealed accumulation of circular forms of FIV DNA within cells treated with L-870,810.ConclusionWe report a drug class (other than nucleosidic reverse transcriptase inhibitors) that is capable of inhibiting FIV replication in vitro. The present study helped establish L-870,810, a compound successfully tested in human clinical trials, as one of the most potent anti-FIV agents ever tested in vitro. This finding may provide new avenues for treating FIV infection and contribute to the development of a small animal model mimicking the effects of ART in humans.

Response of Feline Immunodeficiency Virus (FIV) to Tipranavir May Provide New Clues for Development of Broad-Based Inhibitors of Retroviral Proteases Acting on Drug-Resistant HIV-1

The feline AIDS model for HIV-1 treatment failed in the 1990s, due to structural features resembling protease inhibitor (PI) resistant HIV-1 variants. Widespread drug-resistance to PIs now invokes the possibility of rescuing feline immunodeficiency virus (FIV) as a model for PI treatment. We here analyzed susceptibility of FIV to second generation PIs, lopinavir, atazanavir, and the structurally unrelated non-peptidic PI tipranavir. We found that FIV protease resembles HIV-1 protease drug resistance mutations limiting binding of lopinavir and atazanavir but not tipranavir. All three PIs were found to inhibit FIV replication in a concentration-dependent manner, but only tipranavir inhibited FIV similarly to HIV-1. This drug inhibited FIV synergistically with ritonavir. Inhibition of protease activity was confirmed by Western blot analysis. In molecular docking simulations, tipranavir displayed energetically favorable interactions with the catalytic cavity of the mature dimeric FIV protease. The calculated hydrogen bond network was similar to that found in HIV-1 protease/tipranavir complexes and involved atoms in the protein backbone. We also modeled the interaction of tipranavir with an immature protease monomer, suggesting that inhibition of protease dimerization may be a secondary modality for FIV inhibition by tipranavir. In conclusion, tipranavir is the first FDA-approved non-reverse transcriptase inhibitor of HIV-1 to show anti-FIV properties. The tipranavir response by FIV may 1) support the idea of using FIV as a small animal model for PI-resistant HIV-1, thus expanding access to animal AIDS models; and 2) pave the way for development of novel broad-based inhibitors for treatment of drug resistant HIV-1.

Prevalence of tick-borne pathogens in an urban park in Rome, Italy

INTRODUCTION Limited information is available about the presence of tick-borne pathogens in urban parks in Italy. To fill this gap, ticks were collected in a public park in Rome over a 1-year period and screened by molecular methods for tick-borne pathogens. RESULTS AND CONCLUSION The most abundant tick species were Rhipicephalus turanicus and Ixodes ricinus. The predominant pathogens detected were Borrelia. burgdorferi sensu lato (36%), Rickettsia spp. (36%), and Coxiella burnetii (22%). Among less frequently detected pathogens, Babesia microti was detected for the first time in Italy, with a prevalence of 4%. Neither Bartonella spp. nor Francisella tularensis were detected. With regard to co-infections, the most frequent double and triple infections involved Rickettsia spp., B. burgdorferi sl., and C. burnetii.. A positive correlation was detected between pathogens and I. ricinus. Further studies are needed in order to assess risk associated with tick-borne pathogens in urban areas.

Identification and molecular discrimination of toxigenic and nontoxigenic diphtheria Corynebacterium strains by combined real-time polymerase chain reaction assays☆

With the recognition of several diphtheria outbreaks and the emergence of nontoxigenic corynebacteria strains, there has been renewed interest in the development of laboratory diagnostic methods. Previously reported polymerase chain reaction (PCR) assays can have low diagnostic sensitivity or give species misidentifications among clinical isolates. The aim of the present study was the development of combined real-time PCR assays, based on the tox and rpoB genes, for the detection and differentiation of toxigenic and nontoxigenic corynebacteria. By the PCR tox assay, it was possible to perform the direct identification of DT tox gene of Corynebacterium diphtheriae and Corynebacterium ulcerans, while the PCR rpoB assay differentiated C. diphtheriae from C. ulcerans, irrespective of their toxigenic status. In addition, we detected the DT toxin of Corynebacterium pseudotuberculosis for the first time. These assays revealed high sensitivity, specificity, and reproducibility, and the availability of plasmid controls will facilitate further research into the diagnostics of diphtheria corynebacteria.

Real-time polymerase chain reaction and laser capture microdissection: an efficient combination tool for Chlamydophila pneumoniae DNA quantification and localization of infection in atherosclerotic lesions.

Chlamydophila pneumoniae has been implicated in atherosclerosis, but the role of this obligate intracellular pathogen in the development of the above pathology is still unclear. In particular, its presence and quantitative distribution within lesional areas has not yet been defined. We studied 18 carotid biopsies obtained from patients undergoing endoartherectomy. By laser microdissection (LCM), two different sites (intra-plaque and plaque-adjacent areas) were taken from each lesion, and the presence and quantity of the pathogen DNA were determined by real-time polymerase chain reaction (Real-time PCR). A total of 8 plaques, exclusively, from patients with unstable angina, were positive in real-time PCR. The bacterial DNA was detected in both lesional areas of 3 plaques which contained the highest number of DNA copies (1,900 to 2,200 copy numbers), while C. pneumoniae DNA was detected only in the intra-plaque area of the other 5 positive (500 to 1,600 copy numbers). No C. pneumoniae DNA was found in the other 10 plaques of which 6 were from patients with unstable angina and 4 from stable angina patients. No DNA from Helicobacter pylori or Cytomegalovirus was found in any plaque. This is the first report where both the target lesion and an adjacent reference site were evaluated for the presence of C. pneumoniae DNA by the combination of LCM and Real-time PCR assays. The integration of these two methodologies offer an excellent tool for in situ studies and may help to elucidate the putative role of C. pneumoniae in atherosclerosis.

Chlamydophila pneumoniae, Mycoplasma pneumoniae, and influenza in children with respiratory infections in Alexandria, Egypt.

INTRODUCTION Acute respiratory infections (ARI) are the leading cause of pediatric morbidity and mortality worldwide. Information about etiological agents of ARI in developing countries is still limited. METHODOLOGY Throat swabs collected from children hospitalized with ARI between December 2009 and May 2010 were investigated for Chlamydophila pneumoniae, Mycoplasma pneumoniae, and influenza viruses by molecular analyses. RESULTS This study conducted in Alexandria, Egypt, was designed to determine the prevalence of several microorganisms in 156 children hospitalized with ARI. Overall, samples from 76 individuals (49%) were found to be positive for at least one pathogen, and 10 of them were positive for two agents. C. pneumoniae was the most commonly detected agent, followed by M. pneumonia and H1N1 pandemic influenza virus. Positivity for C. pneumoniae was associated with colder months and mild disease of the upper respiratory tract such as laryngitis. CONCLUSIONS Further studies are needed to identify other possible agents of ARI (e.g., RSV, adenoviruses, other bacterial infections) in this population and to better understand the causal role of atypical bacteria detected in respiratory samples.

Chlamydia pneumoniae modulates human monocyte-derived dendritic cells functions driving the induction of a Type 1/Type 17 inflammatory response.

Chlamydia pneumoniae is a respiratory pathogen involved in the onset of chronic inflammatory pathologies. Dendritic cells (DC), are major players in spreading of C. pneumoniae from the lungs, a crucial step leading to disseminated infections. Less is known concerning modulation of DC functions consequent to encounter with the bacterium. In order to address this aspect, human monocyte-derived (MD)DC were infected with C. pneumoniae. After internalization bacterial counts increased in MDDC, as well as the expression of CPn1046, a gene involved in bacterial metabolism, with a peak 48 h after the infection. Infected MDDC switched to the mature stage, produced IL-12p70, IL-1β, IL-6, and IL-10, and drove a mixed Type 1/Type 17 polarization. Intracellular pathways triggered by C. pneumoniae involved Toll-like receptor (TLR) 2. Indeed, TLR2 was activated by C. pneumoniae in transfected HEK 293 cells, and C. pneumoniae-mediated phosphorylation of ERK1/2 was inhibited by an anti-TLR2 antibody in MDDC. When an ERK1/2 inhibitor was used, IL-12p70 and IL-10 release by MDDC was reduced and T cell polarization shifted towards a Type 2 profile. Overall, our findings unveiled the role played by TLR2 and ERK1/2 induced by C. pneumoniae to affect DC functions in a way that contributes to a Type 1/Type 17 pro-inflammatory response.

Characterization of spotted fever group Rickettsiae in ticks from a city park of Rome, Italy

BACKGROUND Ticks are vectors and important reservoirs for microbial agents that cause disease in humans and animals. Among these pathogens, the members of Rickettsia species play an important role in public health. AIM AND METHODS One hundred twenty-nine ticks belonging to four tick species (Ixodes ricinus, Rhipicephalus turanicus, Dermacentor marginatus, and Haemaphysalis punctata) were collected at different sites of the Insugherata Natural Reserve, localized in the urban area of Rome, Italy. Questing ticks were tested by PCR for Rickettsia spp., amplifying partial gene of ompA. RESULTS Forty-six ticks were found to be infected with Rickettsia species. Five SFG rickettsiae were identified: three human pathogens Rickettsia conorii, Rickettsia massiliae and Rickettsia aeschlimannii, and two putative new strains Rickettsia sp. strain RM1 and Rickettsia sp. strain RM2. The phylogenetic analysis of partial gene sequences of ompA, gltA, and 17-kd antigen showed that they clustered with several rickettsiae with unidentified pathogenicity. However, Rickettsia sp. strain RM1 and Rickettsia sp. strain RM2 clustered in a statistically supported clade with R. massiliae, and R. monacensis, respectively. CONCLUSION Our findings suggest that Rickettsia species other than R. conorii are implicated in human disease in Italy.

Seasonal dynamics of tick species in an urban park of Rome

Regular collections were obtained in the Natural Reserve of the Insugherata of Rome during 2011 in order to obtain the tick species composition and the respective seasonal dynamics of the area. A total of 325 ticks was collected in selected sites by means of drag sampling. Among the identified species, Rhipicephalus turanicus was the most abundant (72.3%), followed by Ixodes ricinus (19.7%), Dermacentor marginatus (6.5%), Haemaphysalis punctata (1.2%), and Rhipicephalus bursa (0.3%). R. turanicus occurred mainly in pastures, showing a mono-modal seasonal activity pattern from spring to early summer. Questing I. ricinus were prevalent in woodland from October to May, and the seasonal trend of specimens showed a weak peak in winter. Although adult D. marginatus exhibited seasonal dynamics similar to I. ricinus, with an activity period from October to April, this species occurred in a different environment (pasture) and with considerably lower densities. Haemaphysalis punctata and R. bursa were rare, with an apparent autumn and autumn-winter seasonal activity, respectively. While the species diversity recorded appears as an unequivocal consequence of the natural state of the park, the remarkable R. turanicus density could be a direct effect of the recent introduction of wild boar, as carriers, from the close Veio Park. The presence of the species, a proven vector of various diseases in humans and domestic animals, is discussed in the light of the possible risk of tick-bite exposure of park workers and visitors.