Detection of Epstein-Barr virus infection in primary junctional epithelial cell cultures.

Background: Junctional epithelium (JE) provides the front-line defense against pathogens invading periodontium. The breakdown of the JE barrier is the hallmark of periodontitis. Recent studies have implicated the Epstein-Barr virus (EBV) as one of the etiopathogenetic factors of periodontitis. EBV exhibits tropism for two target cells in vivo: B cells, where it primarily remains latent, and epithelial cells, where viral replication occurs. Objective: Our knowledge of junctional epithelial cell (JEC) infection with EBV has been limited by the difficulty of generating cell cultures and the inability to infect JECs in vitro readily. Design: To study EBV infection in JECs, we developed human JEC cultures derived from a periodontitis patient. Furthermore, we established a successful contact-free co-culture infection model between the EBV-donor B95-8 cell line and the EBV-permissive JEC culture. JECs and EBV infection of JECs were detected using immunofluorescent staining of cytokeratin 19 and EBNA1, respectively. In addition, EBV infection was confirmed by RT-qPCR for EBNA1, LMP1, and BZLF1 expression. Results and conclusions: Our results suggest that the infection of JECs with EBV can occur in an in vitro experimental model. These outcomes have the potential to enhance our understanding of EBV's involvement in periodontitis and advance periodontal research.

Epstein-Barr virus-infected plasma cells in periodontitis lesions.

Growing evidence supports that the Epstein-Barr virus (EBV) is a putative periodontal pathogen, but little is known regarding EBV behavior in periodontitis. Here, EBV infection was monitored in saliva and periodontal pocket (PP), at baseline and 3 months after periodontal non-surgical therapy (p-NST) in 20 patients diagnosed with periodontitis. After the treatment, the patients with the improved periodontal condition (good responders) showed a significant decrease in salivary EBV load. In contrast, in poor responders, EBV load was slightly increased. Moreover, after the therapy, most patients showed clear signs of EBV infection in a deep PP (≥5 mm) selected as a study site. To investigate how EBV can persist in a PP, we further investigate cellular sites of viral replication in PP. We identified large amounts of infiltrated EBV-infected cells, mostly overlapping with CD138+ plasma cells (PC). EBV-infected PCs formed high-density clusters within the infiltrate and along the periodontal epithelium which were commonly associated with CD3+ T-cells and CD20+ B-cells to evoke diffuse ectopic lymphoid-like structures. Taking together, this study provides new insights to support a model where the periodontal condition may play a major role in oral EBV shedding. Since PC harbors the late productive phases of EBV replication, the periodontal condition may favor B-cell differentiation with possible amplification of periodontal EBV infection and viral spreading. PCs have long been recognized as pathogenic markers in inflammatory lesions. Our finding sheds new light on the role of EBV infection and PC in periodontitis.

Detection of Epstein-Barr Virus in Periodontitis: A Review of Methodological Approaches.

Periodontitis, an inflammatory condition that affects the structures surrounding the tooth eventually leading to tooth loss, is one of the two biggest threats to oral health. Beyond oral health, it is associated with systemic diseases and even with cancer risk. Obviously, periodontitis represents a major global health problem with significant social and economic impact. Recently, a new paradigm was proposed in the etiopathogenesis of periodontitis involving a herpesviral-bacterial combination to promote long-term chronic inflammatory disease. Periodontitis as a risk factor for other systemic diseases can also be better explained based on viral-bacterial etiology. Significant efforts have brought numerous advances in revealing the links between periodontitis and Epstein-Barr virus (EBV), a gamma herpesvirus ubiquitous in the adult human population. The strong evidence from these studies may contribute to the advancement of periodontitis research and the ultimate control of the disease. Advancing the periodontitis research will require implementing suitable methods to establish EBV involvement in periodontitis. This review evaluates and summarizes the existing methods that allow the detection and diagnosis of EBV in periodontitis (also applicable in a more general way to other EBV-related diseases), and discusses the feasibility of the application of innovative emerging technologies.

Evaluation of tetrafunctional block copolymers as synthetic vectors for lung gene transfer.

In the present study, we evaluated, in mice, the efficacy of the tetrafunctional block copolymer 704 as a nonviral gene delivery vector to the lungs. SPECT/CT molecular imaging of gene expression, biochemical assays, and immunohistochemistry were used. Our dataset shows that the formulation 704 resulted in higher levels of reporter gene expression than the GL67A formulation currently being used in a clinical trial in cystic fibrosis patients. The inflammatory response associated with this gene transfer was lower than that induced by the GL67A formulation, and the 704 formulation was amenable to repeated administrations. The cell types transfected by the 704 formulation were type I and type II pneumocytes, and transgene expression could not be detected in macrophages. These results emphasize the relevance of the 704 formulation as a nonviral gene delivery vector for lung gene therapy. Further studies will be required to validate this vector in larger animals, in which the lungs are more similar to human lungs.

Normalisation to blood activity is required for the accurate quantification of Na/I symporter ectopic expression by SPECT/CT in individual subjects.

The utilisation of the Na/I symporter (NIS) and associated radiotracers as a reporter system for imaging gene expression is now reaching the clinical setting in cancer gene therapy applications. However, a formal assessment of the methodology in terms of normalisation of the data still remains to be performed, particularly in the context of the assessment of activities in individual subjects in longitudinal studies. In this context, we administered to mice a recombinant, replication-incompetent adenovirus encoding rat NIS, or a human colorectal carcinoma cell line (HT29) encoding mouse NIS. We used (99m)Tc pertechnetate as a radiotracer for SPECT/CT imaging to determine the pattern of ectopic NIS expression in longitudinal kinetic studies. Some animals of the cohort were culled and NIS expression was measured by quantitative RT-PCR and immunohistochemistry. The radioactive content of some liver biopsies was also measured ex vivo. Our results show that in longitudinal studies involving datasets taken from individual mice, the presentation of non-normalised data (activity expressed as %ID/g or %ID/cc) leads to 'noisy', and sometimes incoherent, results. This variability is due to the fact that the blood pertechnetate concentration can vary up to three-fold from day to day. Normalisation of these data with blood activities corrects for these inconsistencies. We advocate that, blood pertechnetate activity should be determined and used to normalise the activity measured in the organ/region of interest that expresses NIS ectopically. Considering that NIS imaging has already reached the clinical setting in the context of cancer gene therapy, this normalisation may be essential in order to obtain accurate and predictive information in future longitudinal clinical studies in biotherapy.

Immunoanalysis indicates that the sodium iodide symporter is not overexpressed in intracellular compartments in thyroid and breast cancers.

OBJECTIVE: The active transport of iodide into thyroid cells is mediated by the Na(+)/I(-) symporter (NIS) located in the basolateral membrane. Strong intracellular staining with anti-NIS antibodies has been reported in thyroid and breast cancers. Our initial objective was to screen tumour samples for intracellular NIS staining and then to study the mechanisms underlying the altered subcellular localization of the transporters. METHODS: Immunostaining using three different anti-NIS antibodies was performed on paraffin-embedded tissue sections from 93 thyroid or breast cancers. Western blot experiments were carried out to determine the amount of NIS protein in 20 samples. RESULTS: Using three different anti-NIS antibodies, we observed intracellular staining in a majority of thyroid tumour samples. Control immunohistochemistry and western blot experiments indicated that this intracellular staining was due to non-specific binding of the antibodies. In breast tumours, very weak intracellular staining was observed in some samples. Western blot experiments suggest that this labelling is also non-specific. CONCLUSIONS: Our results strongly indicate that the NIS protein level is low in thyroid and breast cancers and that the intracellular staining obtained with anti-NIS antibodies corresponds to a non-specific signal. Accordingly, to increase the efficiency of radiotherapy for thyroid cancers and to enable the use of radioiodine in the diagnosis and therapy of breast tumours, improving NIS targeting to the plasma membrane will not be sufficient. Instead, increasing the expression level of NIS should remain the major goal of this field.

Comparison of expressed human and mouse sodium/iodide symporters reveals differences in transport properties and subcellular localization.

The active transport of iodide from the bloodstream into thyroid follicular cells is mediated by the Na+/I- symporter (NIS). We studied mouse NIS (mNIS) and found that it catalyzes iodide transport into transfected cells more efficiently than human NIS (hNIS). To further characterize this difference, we compared (125)I uptake in the transiently transfected human embryonic kidney (HEK) 293 cells. We found that the V(max) for mNIS was four times higher than that for hNIS, and that the iodide transport constant (K(m)) was 2.5-fold lower for hNIS than mNIS. We also performed immunocytolocalization studies and observed that the subcellular distribution of the two orthologs differed. While the mouse protein was predominantly found at the plasma membrane, its human ortholog was intracellular in approximately 40% of the expressing cells. Using cell surface protein-labeling assays, we found that the plasma membrane localization frequency of the mouse protein was only 2.5-fold higher than that of the human protein, and therefore cannot alone account for the difference in the obtained V(max) values. We reasoned that the observed difference could also be caused by a higher turnover number for iodide transport in the mouse protein. We then expressed and analyzed chimeric proteins. The data obtained with these constructs suggest that the iodide recognition site could be located in the region extending from the N-terminus to transmembrane domain 8, and that the region between transmembrane domain 5 and the C-terminus could play a role in the subcellular localization of the protein.

Prefrontal infusion of PD098059 immediately after fear extinction training blocks extinction-associated prefrontal synaptic plasticity and decreases prefrontal ERK2 phosphorylation.

A previous study has demonstrated that disruption of fear extinction-induced long-term potentiation (LTP) in the medial prefrontal cortex (mPFC) is associated with the return of fear responding. Given that immediate posttraining infusion of PD098059, an inhibitor of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) cascade, into the mPFC also promotes recovery of fear, we investigated whether impairment of mPFC ERK/MAPK cascade also interferes with development of extinction-related LTP in the mPFC in rats. In Experiment 1, extinction training consisting of repetitive presentations of a tone previously associated with eyelid-shock application induced LTP-like changes at hippocampal inputs to the mPFC that were evident for approximately 2 h following fear extinction. Infusion of PD098059 into the mPFC immediately after extinction training abolished training-related prefrontal LTP and impaired retention of extinction memory tested on the following day. In Experiment 2, immunoblotting assays revealed that posttraining infusion of PD098059 into the mPFC produced a significant reduction of mPFC ERK2. These data, along with previous findings, suggest that low levels of ERK2 phosphorylation in the mPFC may interfere with mechanisms of retention of extinction training. The involvement of mPFC LTP in fear extinction is discussed.