Clicky

Injection of human herpesvirus-8 in human skin engrafted on SCID mice induces Kaposi’s sarcoma-like lesions.

(B) Effects of vIL-6 on CatD protein levels were also tested by overexpression, achieved by lentiviral vector-mediated transduction of a vIL-6 expression cassette into BCBL-1 and JSC-1 cells. vIRF-1 binds to membrane lipids, including cardiolipin.To further understand how the vIRF-1 PD associates with mDRM, we tested if vIRF-1 PD binds directly to membrane lipids using the protein lipid overlay (PLO) assay, in which membrane lipid strips were probed with purified recombinant control GFP and GFP-fused vIRF-1 PD proteins (Fig. A., Russo, J. Elongation factor-1alpha is a novel substrate ofrho-associated kinase. VKORC1v2 contributes to PEL cell growth and survival.To test the potential relevance of VKORC1v2 to PEL cell proliferation and viability, shown previously to be promoted by vIL-6 from the ER compartment (5), shRNA-mediated depletion was again employed. (F) A similar experiment in HHV-8+ TIME-TRE/RTA cells, demonstrating mitochondrial localization of Bid, in addition to vIRF-1, in lytically reactivated cultures. This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Health and Welfare, Japan.

Supported by National Institutes of Health grants P30AR42689 (Pilot Project), R01DE12186, and K24CA85083, and by an Established Investigator Award from the American Heart Association. Northern analysis of total RNA isolated at 48 h posttransfection was carried out with the PAN probe (upper panel). Conceived and designed the experiments: MDS MLC EM JRF. E-mail: reitz{at}umbi.umd.edu. Tumor cell lines GR1 and GR3 but not the normal GR5 cell line tested positive for VEGF-C mRNA by RT-PCR (Fig. Some studies have used different types of humanized immunodeficient mice to establish a more physiologically relevant in vivo model of KSHV infection of human cells, but these models fail to generate KS-like tumours172, 173. To verify this hypothesis, HUVEC monolayers were infected with HHV-8 and treated with 5 μM cycloheximide or monensin immediately after the adsorption period or 8 hours after infection, and MCP-1 levels were determined in culture supernatants at 8 and 24 hours after infection by ELISA.

(B) gp130 binding and dimerization interactions by C-helix point variants. One hundred nanograms of DNA samples, HHV-8 ORF73 gene TaqMan probe (25), and Quantitect PCR mix was used. It has been suggested that HHV-8 has a tropism for pemphigus lesions. Expression of vIRF-2 and K11 ORFs in BCBL-1 cells. Specificity and interassay variability.Various HHV-8-infected and noninfected cells were subjected to real-time PCR. Membranes were probed with primary antibodies to phospho-Akt (ser473), Akt (New England Biolabs, Beverly, Mass.), β-actin (Sigma), or ORF74 (rabbit polyclonal—gift of Gary Hayward, Johns Hopkins University). In other words, the area is proportional to the quality of the performance (accuracy) of a diagnostic test; an area of 0.5 represents a worthless test, and an area of 1.0 represents a perfect test.

Although there was a slight disproportion in whether the KS was the presenting AIDS illness (7 [87.5%] of the 8 cases in the HHV-8 after HIV-1 group, compared with 23 [67.7%] of the 34 cases in the HHV-8 before HIV-1 group), this difference was not statistically significant. KS occurs in very young children in Africa, suggesting early infection with HHV-8. However, viral antigens other than LANA and vIL-6 were not detected within this period (data not shown). The fusion assay described here should allow further investigation into the functional domains of HHV-8 glycoproteins and possibly the cellular factors that are important in mediating cell fusion. In contrast, ORF26, which is characterized by relatively conserved nucleotide sequences, was an easier target for PCR than ORF K1; we were able to obtain by nested PCR the 336-bp ORF26 fragment from PBMCs from all 59 patients who were either seropositive for LANA or had previous or present histories of KS. Figure 2 Amplification and calibration curves. Red emission was observed with 540 ± 12.5 nm excitation and 590 LP nm emission filters.

In situ hybridization probes used were from Leica: EBV, κ, and λ. All HIV-positive patients were men who reported having had sex with other men at least once. Kaposi sarcoma is a vascular lesion which frequently develops in mucocutaneous sites including the ocular surface [15–17]. Despite this recent elucidation of the core transacting factors, the discovery of a lytic origin for HHV8 has not been discovered. HHV8 sequences have also been identified in primary effusion lymphomas (PEL), a subset of B-cell lymphomas largely confined to AIDS patients (7), and a rare lymphoproliferative disorder known as multicentric Castleman’s disease (50). Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV), or human herpesvirus 8, is the etiologic agent responsible for the endothelial cell neoplasm KS, the B-cell lymphoproliferative disorder primary effusion lymphoma (PEL), and some forms of multicentric Castleman’s disease (14, 17, 27, 34, 61, 76). Essentially all KS lesions are positive for HHV-8 DNA.

Here, we have used in vitro vIL-6–receptor binding assays to demonstrate direct binding of vIL-6 to both gp130 and IL-6R and vIL-6-induced gp130–IL-6R complex formation, and we have extended our functional analyses of the vIL-6 variants to identify residues important for IL-6R-independent and IL-6R-dependent signaling through native gp130 and gp130.PM5, respectively.