Development of real-time NASBA assays with molecular beacon detection to quantify mRNA coding for HHV-8 lytic and latent genes

BACKGROUND: Human herpesvirus-8 (HHV-8) is linked to the pathogenesis of Kaposi's sarcoma (KS), and the HHV-8 DNA load in peripheral blood mononuclear cells (PBMC) is associated with the clinical stage of KS. To examine the expression of HHV-8 in PBMC, four HHV-8 mRNA specific NASBA assays were developed METHODS: We have developed four quantitative nucleic acid sequence-based amplification assays (NASBA-QT) specifically to detect mRNA coding for ORF 73 (latency-associated nuclear antigen, LANA), vGCR (a membrane receptor), vBcl-2 (a viral inhibitor of apoptosis) and vIL-6 (a viral growth factor). The NASBA technique amplifies nucleic acids without thermocycling and mRNA can be amplified in a dsDNA background. A molecular beacon is used during amplification to enable real-time detection of the product. The assays were tested on PBMC samples of two AIDS-KS patients from the Amsterdam Cohort. RESULTS: For all four assays, the limit of detection (LOD) of 50 molecules and the limit of quantification (LOQ) of 100 molecules were determined using in vitro transcribed RNA. The linear dynamic range was 50 to 10(7) molecules of HHV-8 mRNA. We found HHV-8 mRNA expression in 9 out of the 10 tested samples. CONCLUSION: These real-time NASBA assays with beacon detection provide tools for further study of HHV-8 expression in patient material

Platelet-Rich Plasma Promotes the Proliferation of Human Muscle Derived Progenitor Cells and Maintains Their Stemness

Human muscle-derived progenitor cells (hMDPCs) offer great promise for muscle cell-based regenerative medicine; however, prolonged ex-vivo expansion using animal sera is necessary to acquire sufficient cells for transplantation. Due to the risks associated with the use of animal sera, the development of a strategy for the ex vivo expansion of hMDPCs is required. The purpose of this study was to investigate the efficacy of using platelet-rich plasma (PRP) for the ex-vivo expansion of hMDPCs. Pre-plated MDPCs, myoendothelial cells, and pericytes are three populations of hMDPCs that we isolated by the modified pre-plate technique and Fluorescence Activated Cell Sorting (FACS), respectively. Pooled allogeneic human PRP was obtained from a local blood bank, and the effect that thrombin-activated PRP-releasate supplemented media had on the ex-vivo expansion of the hMDPCs was tested against FBS supplemented media, both in vitro and in vivo. PRP significantly enhanced short and long-term cell proliferation, with or without FBS supplementation. Antibody-neutralization of PDGF significantly blocked the mitogenic/proliferative effects that PRP had on the hMDPCs. A more stable and sustained expression of markers associated with stemness, and a decreased expression of lineage specific markers was observed in the PRP-expanded cells when compared with the FBS-expanded cells. The in vitro osteogenic, chondrogenic, and myogenic differentiation capacities of the hMDPCs were not altered when expanded in media supplemented with PRP. All populations of hMDPCs that were expanded in PRP supplemented media retained their ability to regenerate myofibers in vivo. Our data demonstrated that PRP promoted the proliferation and maintained the multi-differentiation capacities of the hMDPCs during ex-vivo expansion by maintaining the cells in an undifferentiated state. Moreover, PDGF appears to be a key contributing factor to the beneficial effect that PRP has on the proliferation of hMDPCs. © 2013 Li et al

Type-1 and type-2 CD8+ T-cell subsets isolated from chronic adult periodontitis tissue differ in surface phenotype and biological functions.

Cloning of CD8+ T cells expressing the alpha beta T-cell receptor from inflamed human gingiva revealed that at least two different subsets were found within the tissue and that these subsets were able to interact with each other. One subset produced high levels of interferon-gamma (IFN-gamma) and no interleukin-4 (IL-4) or IL-5, exhibited phytohaemagglutinin (PHA)- or anti-CD3-mediated cytolytic activity, and were CD28+. The other subset produced high levels of IL-4 in combination with IL-5, displayed no cytotoxicity and were CD28-. From the latter subset CD8+ T-cell clones were able to suppress the proliferative response of cytotoxic CD8+ T-cell clones. This suppression could be abolished by anti-IL-4 monoclonal antibodies. However, IL-4 alone was not able to induce the suppression. Our results indicate that CD8+ T cells might participate in local immune responses by the suppression of IFN-gamma-producing cells and by favouring humoral responses via the production of IL-4 and IL-5

Cloning, characterization, and antigen specificity of T-lymphocyte subsets extracted from gingival tissue of chronic adult periodontitis patients.

Chronic periodontitis is characterized by dense infiltrations of B and T lymphocytes within the gingival connective tissue. Distinct anaerobic gram-negative bacteria as well as autoimmunity to collagen have been reported to play a role in the etiology and the pathogenesis of this disease. Here we describe the cloning and characterization of CD4+ and CD8+ T lymphocytes isolated from inflamed gingival tissue obtained from four patients with chronic periodontitis. Clones were raised with phytohemagglutinin and interleukin-2 and tested for proliferation in response to whole-cell antigens of Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, human collagen type I, and two bacterial heat shock proteins. CD4+ T-cell clones reactive with collagen type I were obtained from all four patients. Eighty percent of these clones had phenotypes resembling the mouse type 2 T helper (Th) phenotype, i.e., they produced high levels of interleukin-4 and low levels of gamma interferon. No collagen-type-I-reactive CD8+ clones were obtained. Bacterial-antigen-reactive CD4+ and/or CD8+ T-cell clones were also obtained from each patient, and the majority of the clones showed a Th0-like cytokine pattern and produced equal amounts of interleukin-4 and gamma interferon. Although most clones were reactive with P. intermedia, it seems that the immune response is not strictly directed against this particular microorganism, as clones reactive with one of the other bacteria were also obtained from two patients. We propose that collagen-specific CD4+ Th2-like T cells contribute to the chronicity of periodontitis but that their modes of activation might be controlled by Th0-like T cells specific for periodontitis-associated bacteria