Human immunodeficiency virus and oral microbiota: mutual influence on the establishment of a viral gingival reservoir in individuals under antiretroviral therapy.

The role of the oral microbiota in the overall health and in systemic diseases has gained more importance in the recent years, mainly due to the systemic effects that are mediated by the chronic inflammation caused by oral diseases, such as periodontitis, through the microbial communities of the mouth. The chronic infection by the human immunodeficiency virus (HIV) interacts at the tissue level (e.g. gut, genital tract, brain) to create reservoirs; the modulation of the gut microbiota by HIV infection is a good example of these interactions. The purpose of the present review is to assess the state of knowledge on the oral microbiota (microbiome, mycobiome and virome) of HIV-infected patients in comparison to that of HIV-negative individuals and to discuss the reciprocal influence of HIV infection and oral microbiota in patients with periodontitis on the potential establishment of a viral gingival reservoir. The influence of different clinical and biological parameters are reviewed including age, immune and viral status, potent antiretroviral therapies, smoking, infection of the airway and viral coinfections, all factors that can modulate the oral microbiota during HIV infection. The analysis of the literature proposed in this review indicates that the comparisons of the available studies are difficult due to their great heterogeneity. However, some important findings emerge: (i) the oral microbiota is less influenced than that of the gut during HIV infection, although some recurrent changes in the microbiome are identified in many studies; (ii) severe immunosuppression is correlated with altered microbiota and potent antiretroviral therapies correct partially these modifications; (iii) periodontitis constitutes a major factor of dysbiosis, which is exacerbated in HIV-infected patients; its pathogenesis can be described as a reciprocal reinforcement of the two conditions, where the local dysbiosis present in the periodontal pocket leads to inflammation, bacterial translocation and destruction of the supporting tissues, which in turn enhances an inflammatory environment that perpetuates the periodontitis cycle. With the objective of curing viral reservoirs of HIV-infected patients in the future years, it appears important to develop further researches aimed at defining whether the inflamed gingiva can serve of viral reservoir in HIV-infected patients with periodontitis.

Detection of HPV in urine for cervical cancer screening: Feasibilty of an assay system.

The detection of urine HPV is considered as a promosing alternative to increase the screening coverage of cervical cancer. However, the validated assay of urine HPV is still scarse. We described a nouvel assay syetem for the urine-based detection of HPV in the framework of HPV screening. This sytsem consisted of Automate Nimbus extraction of DNA and Anyplex™ II HPV HR Detection PCR of HPV DNA. We validated this system by spiking HPV-infected cervical cancer cell line HeLa cells into normal urine and compared the prelimary results of cervical samples and urine samples. We found that this system could detect as few as 5 HeLa cells in normal urine model. Some discordances of HPV results between cervical samples and urine samples were observed. We concluded that this assay system could be applied for the detection of HPV in urine. A large scale study is necessary to evaluate the clinical significance of this assay system.

Urine-based detection of HPV for cervical cancer screening: Time for standardized tests.

Cervical cancer is preventable because it has an established etiology, mainly attributed to a detectable pathogen, human papillomavirus (HPV). In 2018, the world health organization issued an unprecedented call for global action to eliminate cervical cancer by 2030. The adaptation of regular screening programs is fundamental to achieve the goal of cervical cancer elimination. However, it is still difficult to achieve satisfactory coverage rates of screening in developing countries as well as in developed countries because many women are reluctant to participate in gynecologic examination. HPV detection in urine is a convenient, widely acceptable by women and relatively affordable without the necessity for clinical visits to improve the coverage rates of cervical cancer screening. Unfortunately, the clinical implementation of urine-based tests for HPV detection has been hindered by the lack of standardized tests. Further optimization of protocols and standardization of urinary HPV detection are expected to be realized. With the advantages of urine sampling to overcome cost, personal, and cultural barriers, time has come for the standardized tests to facilitate a wide clinical implementation of urinary HPV detection that will significantly contribute to the WHO's goal, that is, to eliminate the cervical cancer globally.

Cytomegalovirus and Inflammatory Bowel Diseases (IBD) with a Special Focus on the Link with Ulcerative Colitis (UC).

Cytomegalovirus (CMV) infects approximately 40% of adults in France and persists lifelong as a latent agent in different organs, including gut. A close relationship is observed between inflammation that favors viral expression and viral replication that exacerbates inflammation. In this context, CMV colitis may impact the prognosis of patients suffering from inflammatory bowel diseases (IBDs), and notably those with ulcerative colitis (UC). In UC, the mucosal inflammation and T helper cell (TH) 2 cytokines, together with immunomodulatory drugs used for controlling flare-ups, favor viral reactivation within the gut, which, in turn, increases mucosal inflammation, impairs corticoid and immunosuppressor efficacy (the probability of steroid resistance is multiplied by more than 20 in the case of CMV colitis), and enhances the risk for colectomy. This review emphasizes the virological tools that are recommended for exploring CMV colitis during inflammatory bowel diseases (IBD) and underlines the interest of using ganciclovir for treating flare-ups associated to CMV colitis in UC patients.

Evidence of CD40L/CD40 pathway involvement in experimental transfusion-related acute lung injury.

Platelet transfusions can cause adverse reactions in their recipients, including transfusion-related acute lung injury (TRALI). The pathophysiology of TRALI depends on a number of signaling pathways and the inflammatory role played by blood platelets remains controversial. Platelets are important in inflammation, particularly via the immunomodulator complex CD40/CD40L. We studied the specific function of the CD40/CD40L interaction in regulating an experimental TRALI Two-hit model. A mouse model of immune TRALI was triggered by injection of LPS and an anti-MHC I antibody, and the effect of injection of a neutralizing anti-CD40L antibody before induction of TRALI investigated. The characteristics of TRALI were decreased body temperature, pulmonary lesions, and immune cell infiltration into the alveolar space. Pulmonary infiltration was evaluated by blood counts of specific immune cells and their detection in lung sections. Inhibition of the CD40/CD40L immunomodulator interaction significantly reduced communication between immune and/or endothelial cells and the development of pulmonary edema. Hence, our results indicate that targeting of the CD40/CD40L interaction could be an important method to prevent TRALI. While considering that our work concerned a mouse model, we postulate that improvement of the conditions under which platelet concentrates are prepared/stored would assist in alleviating the risk of TRALI.

Inhibition of the CD40/CD40L complex protects mice against ALI-induced pancreas degradation.

BACKGROUND: Acute lung injury (ALI) is a severe complication of transfusion. In a previous study, we saw that inhibition of the CD40/CD40L complex allowed restoration of ALI lesions in an experimental mouse model. OBJECTIVES: This study focused on pancreas-associated injury development during experimental ALI pathogenesis and its limitation through CD40/CD40L complex inhibition. MATERIALS AND METHODS: An ALI mouse model was established through intraperitoneal lipopolysaccharide and intravenous anti-major histocompatibility complex class I monoclonal antibody injection. Preemption of lesions was achieved with intravenous injection of neutralizing anti-CD40L monoclonal antibody 30 minutes before the trigger, that is, anti-major histocompatibility complex class I monoclonal antibody administration. Histology and immunoassay analyses were used to evaluate pancreatic lesions. RESULTS: ALI development induced significant degradation of the lungs and pancreas and was associated with pancreatic lesions. Different scores were established showing more severe injury to the pancreas in ALI conditions; however, injury was significantly reduced through CD40/CD40L complex inhibition. CONCLUSION: This study supports the idea that several organs are exposed during ALI development, and particularly when such experimental ALI aims at mimicking transfusion-associated ALI; nevertheless, preventive treatment inhibiting CD40/CD40L (sCD40L) complex formation provides protection from lung disease as well as disease of other organs.

A serum protein factor mediates maturation and apoB-association of HCV particles in the extracellular milieu.

BACKGROUND & AIMS: In the sera of infected patients, hepatitis C virus (HCV) particles display heterogeneous forms with low-buoyant densities (<1.08), underscoring their lipidation via association with apoB-containing lipoproteins, which was proposed to occur during assembly or secretion from infected hepatocytes. However, the mechanisms inducing this association remain poorly-defined and most cell culture grown HCV (HCVcc) particles exhibit higher density (>1.08) and poor/no association with apoB. We aimed to elucidate the mechanisms of lipidation and to produce HCVcc particles resembling those in infected sera. METHODS: We produced HCVcc particles of Jc1 or H77 strains from Huh-7.5 hepatoma cells cultured in standard conditions (10%-fetal calf serum) vs. in serum-free or human serum conditions before comparing their density profiles to patient-derived virus. We also characterized wild-type and Jc1/H77 hypervariable region 1 (HVR1)-swapped mutant HCVcc particles produced in serum-free media and incubated with different serum types or with purified lipoproteins. RESULTS: Compared to serum-free or fetal calf serum conditions, production with human serum redistributed most HCVcc infectious particles to low density (<1.08) or very-low density (<1.04) ranges. In addition, short-time incubation with human serum was sufficient to shift HCVcc physical particles to low-density fractions, in time- and dose-dependent manners, which increased their specific infectivity, promoted apoB-association and induced neutralization-resistance. Moreover, compared to Jc1, we detected higher levels of H77 HCVcc infectious particles in very-low-density fractions, which could unambiguously be attributed to strain-specific features of the HVR1 sequence. Finally, all 3 lipoprotein classes, i.e., very-low-density, low-density and high-density lipoproteins, could synergistically induce low-density shift of HCV particles; yet, this required additional non-lipid serum factor(s) that include albumin. CONCLUSIONS: The association of HCV particles with lipids may occur in the extracellular milieu. The lipidation level depends on serum composition as well as on HVR1-specific properties. These simple culture conditions allow production of infectious HCV particles resembling those of chronically-infected patients. LAY SUMMARY: Hepatitis C virus (HCV) particles may associate with apoB and acquire neutral lipids after exiting cells, giving them low-buoyant density. The hypervariable region 1 (HVR1) is a majorviral determinant of E2 that controls this process. Besides lipoproteins, specific serum factors including albumin promote extracellular maturation of HCV virions. HCV particle production in vitro, with media of defined serum conditions, enables production of infectious particles resembling those of chronically infected patients.

The amino-terminus of the hepatitis C virus (HCV) p7 viroporin and its cleavage from glycoprotein E2-p7 precursor determine specific infectivity and secretion levels of HCV particle types.

Viroporins are small transmembrane proteins with ion channel activities modulating properties of intracellular membranes that have diverse proviral functions. Hepatitis C virus (HCV) encodes a viroporin, p7, acting during assembly, envelopment and secretion of viral particles (VP). HCV p7 is released from the viral polyprotein through cleavage at E2-p7 and p7-NS2 junctions by signal peptidase, but also exists as an E2p7 precursor, of poorly defined properties. Here, we found that ectopic p7 expression in HCVcc-infected cells reduced secretion of particle-associated E2 glycoproteins. Using biochemical assays, we show that p7 dose-dependently slows down the ER-to-Golgi traffic, leading to intracellular retention of E2, which suggested that timely E2p7 cleavage and p7 liberation are critical events to control E2 levels. By studying HCV mutants with accelerated E2p7 processing, we demonstrate that E2p7 cleavage controls E2 intracellular expression and secretion levels of nucleocapsid-free subviral particles and infectious virions. In addition, our imaging data reveal that, following p7 liberation, the amino-terminus of p7 is exposed towards the cytosol and coordinates the encounter between NS5A and NS2-based assembly sites loaded with E1E2 glycoproteins, which subsequently leads to nucleocapsid envelopment. We identify punctual mutants at p7 membrane interface that, by abrogating NS2/NS5A interaction, are defective for transmission of infectivity owing to decreased secretion of core and RNA and to increased secretion of non/partially-enveloped particles. Altogether, our results indicate that the retarded E2p7 precursor cleavage is essential to regulate the intracellular and secreted levels of E2 through p7-mediated modulation of the cell secretory pathway and to unmask critical novel assembly functions located at p7 amino-terminus.

Visualization of X4- and R5-Tropic HIV-1 Viruses Expressing Fluorescent Proteins in Human Endometrial Cells: Application to Tropism Study.

Worldwide most HIV infections occur through heterosexual transmission, involving complex interactions of cell-free and cell-associated particles with cells of the female genital tract mucosa. The ability of HIV-1 to "infect" epithelial cells remains poorly understood. To address this question, replicative-competent chimeric constructs expressing fluorescent proteins and harboring the envelope of X4- or R5-tropic HIV-1 strains were used to "infect" endometrial HEC1-A cells. The virus-cell interactions were visualized using confocal microscopy (CM) at various times post infection. Combined with quantification of viral RNA and total HIV DNA in infected cells, the CM pictures suggest that epithelial cells do not support a complete viral replication cycle: X4-tropic viruses are imported into the nucleus in a non-productive way, whereas R5-tropic viruses transit through the cytoplasm without replication and are preferentially transmitted to susceptible activated peripheral blood mononuclear cells. Within the limit of experiments conducted in vitro on a continued cell line, these results indicate that the epithelial mucosa may participate to the selection of HIV-1 strains at the mucosal level.

Brief Report: A High Rate of ß7+ Gut-Homing Lymphocytes in HIV-Infected Immunological Nonresponders is Associated With Poor CD4 T-Cell Recovery During Suppressive HAART.

OBJECTIVE: Correlation between GALT homing markers on lymphocytes and the low blood CD4 T-cell reconstitution in immunological nonresponders (INRs) has been studied. DESIGN: Thirty-one INRs, 19 immunological responders (IRs), and 12 noninfected controls were enrolled in this study. INRs were defined by an undetectable plasma viral load RNA less than 40 copies per milliliter and CD4 T-cell count <500 cells per cubic milliliter in at least 3 years. METHODS: A complete peripheral and mucosal lymphocyte immunophenotyping was performed on these patients with a focus on the CCR9, CCR6, and α4ß7 gut-homing markers. RESULTS: A highly significant upregulation of α4ß7 on INRs peripheral lymphocytes compared with that of IRs has been observed. This upregulation impacts different lymphocyte subsets namely CD4, CD8, and B lymphocytes. The frequency of ß7 Th17 and Treg cells are increased compared with IRs and healthy controls. The frequency of ß7 CD8 T cells in the blood is negatively correlated with integrated proviral DNA in rectal lymphoid cells in contrast to ß7 CD4 T cells associated with HIV integration. CONCLUSIONS: Alteration of lymphocyte homing abilities would have deleterious effects on GALT reconstitution and could participate to HIV reservoir constitution. These results emphasize the great interest to consider α4ß7-targeted therapy in INR patients to block homing of lymphocytes and/or to directly impair gp120-α4ß7 interactions.

Pivotal Role of the Genital Epithelial Cells in HIV-1 Transmission.

AIDS remains one of the world's most serious health challenges, with 35 million people living with HIV worldwide at the end of 2013. HIV sexual transmission accounts for the overwhelming majority of people newly infected, making genital and rectal mucosal tissues the major sites of infection. This review focuses on the role of the female genital epithelial cells in the establishment of HIV infection.

Transforming growth factor beta 1 up-regulates CD169 (sialoadhesin) expression on monocyte-derived dendritic cells: role in HIV sexual transmission.

OBJECTIVE: Recent data describe CD169 (also called sialoadhesin or Siglec-1) as the main HIV-1 receptor expressed by mucosal dendritic cells involved in the capture of the virus and its transmission to target cells. In this study, we investigated the effect of transforming growth factor beta 1 (TGF-ß1), a cytokine found in abundance in semen, on the expression of CD169 on dendritic cells in order to characterize its potential role in the capture of HIV-1 particles by these antigen-presenting cells. METHODS: Monocyte-derived dendritic cells (MDDCs) were cultured in the presence of lipopolysaccharide, pro-inflammatory cytokines [interleukin (IL)-1ß and tumor necrosis factor alpha (TNF-α)] or different concentrations of TGF-ß1, and analyzed for maturation marker and Siglec expression. The ability of MDDCs to capture HIV particles following the different treatments was also analyzed. RESULTS: TGF-ß1 treatment promotes a significant increase of CD169 expression on MDDCs. This effect was specific since neither DC-SIGN nor other Siglec expressions were changed. The CD169 increase was due to a de-novo synthesis as evidenced by Western blot experiment. This up-regulation was well correlated to the concentration of TGF-ß1 and associated with an increase of the MDDC ability to bind HIV particles. Interestingly, this phenomenon was independent of the maturation status of MDDCs. CONCLUSION: This study demonstrates that the most abundant cytokine present in semen (TGF-ß1) is able to enhance specifically the expression of an important molecule (CD169) involved in the capture and transmission of HIV-1 particles from the mucosal lumen to the submucosal compartment. Our results suggest that this mechanism may play a relevant role in sexual HIV transmission.

Potential contribution of saliva to the sexual transmission of HIV through the secretion of CCL20 by genital epithelial cells.

Saliva can be considered as an important actor during sexual intercourse. However, there is no data concerning its influence on HIV sexual transmission. The aim of this study was to evaluate the role of whole saliva on the in vitro secretion of CCL20 by monolayered HEC-1A endocervical epithelium cells. HEC-1A cells were cultivated in 96-well microplates and incubated with specimens of whole saliva collected from 57 subjects tested seropositive (n = 34) or seronegative (n = 23) for HIV and presenting different oral conditions (healthy periodontally, n = 22, and gingivitis/periodontitis, n = 35). The production of CCL20 in the supernatants of HEC-1A cells after overnight incubation at 37°C was quantified using ELISA. The salivary concentration of lactoferrin (Lf) and IL-1ß was tested by ELISA. Saliva samples were found able to stimulate dramatically the production of CCL20 by epithelial cells, increasing this synthesis by a mean factor of 38.1 with reference to untreated cells. This stimulation was equivalent to that observed with IL-1ß used as positive control. Although no difference was observed according to oral condition, HIV status or salivary concentration of Lf and IL-1ß, the high salivary concentration of the latter protein could acknowledge in large part for the overproduction of CCL20 by HEC-1A cells when stimulated by saliva. Saliva was shown to significantly increase CCL20 secretion and may be responsible for an enhanced recruitment of dendritic/Langerhans cells at the genital level. These results suggest that saliva could facilitate HIV entry and possibly other pathogens through the genital mucosa during heterosexual intercourse.

Anti-Trypanosoma cruzi cross-reactive antibodies detected at high rate in non-exposed individuals living in non-endemic regions: seroprevalence and association to other viral serologies.

Cross-reactive antibodies are characterized by their recognition of antigens that are different from the trigger immunogen. This happens when the similarity between two different antigenic determinants becomes adequate enough to enable a specific binding with such cross-reactive antibodies. In the present manuscript, we report the presence, at an "abnormal" high frequency, of antibodies in blood samples from French human subjects cross-reacting with a synthetic-peptide antigen derived from a Trypanosoma cruzi (T. cruzi) protein sequence. As the vector of T. cruzi is virtually confined to South America, the parasite is unlikely to be the trigger immunogen of the cross-reactive antibodies detected in France. At present, the cross-reactive antibodies are measured by using an in-house ELISA method that employs the T. cruzi -peptide antigen. However, to underline their cross-reactive characteristics, we called these antibodies "Trypanosoma cruzi Cross Reactive Antibodies" or TcCRA. To validate their cross-reactive nature, these antibodies were affinity-purified from plasma of healthy blood donor and were then shown to specifically react with the T. cruzi parasite by immunofluorescence. Seroprevalence of TcCRA was estimated at 45% in serum samples of French blood donors while the same peptide-antigen reacts with about 96% of T. cruzi -infected Brazilian individuals. In addition, we compared the serology of TcCRA to other serologies such as HSV 1/2, EBV, HHV-6, CMV, VZV, adenovirus, parvovirus B19, mumps virus, rubella virus, respiratory syncytial virus, measles and enterovirus. No association was identified to any of the tested viruses. Furthermore, we tested sera from different age groups for TcCRA and found a progressive acquisition starting from early childhood. Our findings show a large seroprevalence of cross-reactive antibodies to a well-defined T. cruzi antigen and suggest they are induced by a widely spread immunogen, acquired from childhood. The etiology of TcCRA and their clinical relevance still need to be investigated.

Molecular HIV screening.

Nuclear acid testing is more and more used for the diagnosis of infectious diseases. This paper focuses on the use of molecular tools for HIV screening. The term 'screening' will be used under the meaning of first-line HIV molecular techniques performed on a routine basis, which excludes HIV molecular tests designed to confirm or infirm a newly discovered HIV-seropositive patient or other molecular tests performed for the follow-up of HIV-infected patients. The following items are developed successively: i) presentation of the variety of molecular tools used for molecular HIV screening, ii) use of HIV molecular tools for the screening of blood products, iii) use of HIV molecular tools for the screening of organs and tissue from human origin, iv) use of HIV molecular tools in medically assisted procreation and v) use of HIV molecular tools in neonates from HIV-infected mothers.

Selective transmigration of monocyte-associated HIV-1 across a human cervical monolayer and its modulation by seminal plasma.

OBJECTIVE: To analyse the transmigration of immune cells infected by HIV-1 across the epithelial monolayer using the endometrial human endometrial carcinoma (HEC)-1A cell line and to study the influence of seminal plasma in this process. DESIGN: After sexual intercourse involving a male partner infected by HIV-1, a selection process has been shown to lead to a predominant transmission of the R5 phenotype despite the presence of X4 and R5 strains in semen. Transmigration of HIV-infected monocytes present in semen may represent a pertinent mechanism that could explain this tropism selection. METHODS: Epithelial monolayer crossing was studied by using HEC-1A epithelial cells cultured on permeable support and monocyte-enriched or lymphocyte-enriched populations of cells infected or not by HIV R5 or X4 strains. Transmigrating cells were quantified and analysed for their ability to transmit HIV infection to immune target cells. The effect of HIV-negative seminal plasma on cell transmigration was analysed. RESULTS: A preferential passage of the R5 strain associated with monocyte-enriched populations was observed together with the ability of this strain to transmit infection. Seminal plasma was found able to decrease the epithelial crossing of immune cells by enhancing transepithelial resistance and by increasing the adherence of immune cells to the monolayer. CONCLUSION: The preferential transmigration of HIV R5 strains associated with monocytes across the endocervical monolayer may explain the predominant transmission of the R5 strains after sexual intercourse. By its capacity to modulate the tightness of the epithelial structure, seminal plasma reinforces this selection process.

Current and future microbicide approaches aimed at preventing HIV infection in women.

Women from developing countries, in which the prevalence of HIV infection is very high, are at risk of becoming infected without having the possibility of personally controlling this risk. Therefore, there is an urgent need to develop anti-HIV vaginal microbicide strategies. This review considers the modes of entry of HIV through the mucosa of the female genital tract, the different classes of vaginal microbicide compounds, the mode of delivery of these drugs, the aims and methods of in vitro and animal experiments at the preclinical stage, the results of the Phase III trials conducted in different countries, including the ongoing assays, and the future orientations for the next 5 years with a discussion relative to antiviral resistance, combination strategies and development of new-generation compounds.

In vitro activities of candidate microbicides against cell-associated HIV.

Most research on HIV transmission and microbicides focuses on the inhibition of cell-free virus (CFV) present in genital secretions. However, an effective microbicide should also block the transmission of cell-associated virus (CAV) originating from seminal T cells and macrophages. Because inhibition of CAV remains controversial, especially for viral entry inhibitors, we developed a novel in vitro assay to evaluate the activities of different classes of candidate microbicides against cell-free HIV and HIV-infected leukocytes (i.e., resting peripheral blood mononuclear cells [PBMC], activated PBMC, and monocyte-derived macrophages). The assay is based on two CD4(+) CXCR4(+) T-cell lines (R5MaRBLE and X4MaRBLE) that both contain a firefly luciferase reporter gene but differ in the expression of the CCR5 coreceptor. Consequently, the quantification of the luciferase activities and the Gag p24 concentrations in cocultures of R5-tropic HIV-infected leukocytes with each cell line separately allowed us to discriminate between the infection of the cell lines (i.e., target cells), the ongoing infection in the HIV-infected leukocytes (i.e., effector cells), and the total infection of the coculture (i.e., effector plus target cells). All 14 antiretrovirals tested were able to block target cell infection by all three sources of CAV, although a small decrease in activity (2- to 18-fold) was observed for all entry inhibitors. On the other hand, the production of Gag p24 by the infected effector cells could be blocked only by protease inhibitors. Overall, these results show that entry and protease inhibitors are eligible drug classes for inclusion in future combination microbicides.

Enterovirus-related activation of the cardiomyocyte mitochondrial apoptotic pathway in patients with acute myocarditis.

AIMS: We examined the impact of enterovirus (EV) cardiac replication activity on the endomyocardial mitochondrial pathway in patients with acute myocarditis. METHODS AND RESULTS: Levels of apoptotic cardiomyocytes were determined by TUNEL and ligation-mediated polymerase chain reaction (PCR) assays and EV replication activity was assessed by immunostaining of EV VP1 capsid protein in ventricular myocytes of patients with acute myocarditis (n = 25), and healthy heart controls (n = 15). Ratio of cytosolic/mitochondrial cytochrome c concentrations was determined by ELISA assay, levels of active caspase-9 were determined by western blot analysis and Bax/Bcl2 mRNA ratio was assessed by real-time reverse transcription-polymerase chain reaction (RT-PCR) in the same cardiac tissues. Patients with EV-associated acute myocarditis (n = 15) exhibited a significantly higher number of apoptotic cardiomyocytes than those with non-EV-associated acute myocarditis (n = 10) and controls (n = 15) (P < 0.001). Endomyocardial ratio of cytosolic/mitochondrial cytochrome c concentrations and levels of active caspase-9 protein were significantly increased in EV than in non-EV-related myocarditis patients (P < 0.001). Moreover, Bax/Bcl2 mRNA ratio was significantly increased in EV than in non-EV-related myocarditis patients (P < 0.001). CONCLUSION: Our findings evidence an EV-related activation of the cardiomyocyte mitochondrial apoptotic pathway in patients with acute myocarditis. Moreover, our results indicate that this EV-induced pro-apoptotic mechanism could be partly related to an up-regulation of Bax expression, and suggest that inhibition of this cell death process may constitute the basis for novel therapies.

Comparative evaluation of a commercially available automated system for extraction of viral DNA from whole blood: application to monitoring of epstein-barr virus and cytomegalovirus load.

The NucliSENS easyMAG automated system was compared to the column-based Qiagen method for Epstein-Barr virus (EBV) or cytomegalovirus (CMV) DNA extraction from whole blood before viral load determination using the corresponding R-gene amplification kits. Both extraction techniques exhibited a total agreement of 81.3% for EBV and 87.2% for CMV.