In vivo-restricted and reversible malignancy induced by human herpesvirus-8 KSHV: a cell and animal model of virally induced Kaposi's sarcoma.

Transfection of a Kaposi's sarcoma (KS) herpesvirus (KSHV) Bacterial Artificial Chromosome (KSHVBac36) into mouse bone marrow endothelial-lineage cells generates a cell (mECK36) that forms KS-like tumors in mice. mECK36 expressed most KSHV genes and were angiogenic, but they didn't form colonies in soft agar. In nude mice, mECK36 formed KSHV-harboring vascularized spindle cell sarcomas that were LANA+/podoplanin+, overexpressed VEGF and Angiopoietin ligands and receptors, and displayed KSHV and host transcriptomes reminiscent of KS. mECK36 that lost the KSHV episome reverted to nontumorigenicity. siRNA suppression of KSHV vGPCR, an angiogenic gene upregulated in mECK36 tumors, inhibited angiogenicity and tumorigenicity. These results show that KSHV malignancy is in vivo growth restricted and reversible, defining mECK36 as a biologically sensitive animal model of KSHV-dependent KS.

Activation of the unfolded protein response by 2-deoxy-D-glucose inhibits Kaposi's sarcoma-associated herpesvirus replication and gene expression.

Lytic replication of the Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for the maintenance of both the infected state and characteristic angiogenic phenotype of Kaposi's sarcoma and thus represents a desirable therapeutic target. During the peak of herpesvirus lytic replication, viral glycoproteins are mass produced in the endoplasmic reticulum (ER). Normally, this leads to ER stress which, through an unfolded protein response (UPR), triggers phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α), resulting in inhibition of protein synthesis to maintain ER and cellular homeostasis. However, in order to replicate, herpesviruses have acquired the ability to prevent eIF2α phosphorylation. Here we show that clinically achievable nontoxic doses of the glucose analog 2-deoxy-d-glucose (2-DG) stimulate ER stress, thereby shutting down eIF2α and inhibiting KSHV and murine herpesvirus 68 replication and KSHV reactivation from latency. Viral cascade genes that are involved in reactivation, including the master transactivator (RTA) gene, glycoprotein B, K8.1, and angiogenesis-regulating genes are markedly decreased with 2-DG treatment. Overall, our data suggest that activation of UPR by 2-DG elicits an early antiviral response via eIF2α inactivation, which impairs protein synthesis required to drive viral replication and oncogenesis. Thus, induction of ER stress by 2-DG provides a new antiherpesviral strategy that may be applicable to other viruses.

Presenilin-1 detection in brain neurons and FOXp3 in peripheral blood mononuclear cells: normalizer gene selection for real time reverse transcriptase pcr using the DeltaDeltaCt method

Quantification of gene expression is important to confirm changes in levels of gene expression in disease. Prior quantification methods include standard curves, absolute quantification, and relative quantification. This paper describes an analytic method for the relative quantification of Presenilin-1 (PS-1) in neurons and Forkhead-box (FOX) p3 in PBMNCs using real-time PCR analytic techniques. A comparative Ct method (deltadelatCt) is described in which the quantity of target normalized to a normalizer gene reference is given by 2-deltadelatCt where deltadelatCt = [Ct of the gene of interest in the unknown specimen - Ct normalizer gene in the unknown specimen] - [Ct of the gene of interest in the calibrator specimen - Ct normalizer gene in the calibrator specimen]. The calibrator specimen is ideally from a non-treated control specimen and is analyzed on every assay plate with the unknown specimens of interest. The use of the deltadelatCt methodology allows for a higher throughput and a more economical approach to investigate gene expression. We applied this methodology to the quantification of PS-1 and FOXp3 genes and compare the levels of expression by normalizing to different normalizer genes using the deltadelatCt methodology. We find that use of GAPDH is the optimum normalizer gene for the genes analyzed in neurons from human brain and in PBMNCs.

HIV-associated dementia, Alzheimer's disease, multiple sclerosis, and schizophrenia: gene expression review.

RNA and protein gene expression technologies are revolutionizing our view and understanding of human diseases and enable us to analyze the concurrent expression patterns of large numbers of genes. These new technologies allow simultaneous study of thousands of genes and their changes in regulation and modulation patterns in relation to disease state, time, and tissue specificity. This review summarizes the application of this modern technology to four common neurological and psychiatric disorders: HIV-1-associated dementia, Alzheimer's disease, multiple sclerosis, and schizophrenia and is a first comparison of these diseases using this approach.