Human parvovirus B19 (B19) was discovered in 1974 and is the only member of the family Parvoviridae that is known to be pathogenic for humans.1) Nearly 50% of all people are positive for B19 antibody 2) and this virus is ubiquitous, but its manifestations vary with the immunologic and hematologic status of the host. In healthy immunocompetent children, B19 generally causes erythema infectiosum, an innocuous rash. Particularly in adults, B19 infection is occasionally associated with chronic symmetric polyarthropathy that may mimic rheumatoid arthritis, as well as with anemia and fetal hydrops, [3][4][5] which are caused by reactivation of persistent B19 infection.5) The B19 virion has a simple structure composed of only two proteins (VP1 and VP2) and a linear, single-stranded DNA molecule.6) The nonenveloped viral particles are about 22 to 24 nm in diameter and show icosahedral symmetry. After infection, two non-structural proteins (NS1 and NS2) are expressed by host cells. The effects of NS1 protein are well characterized, with the target cells of B19 being erythroid progenitor cells, liver cells, and monocyte/ macrophages, 7,8) and NS1 being expressed by these cells. The B19 capsid is composed of two capsomer proteins (VP1 and VP2), which are encoded by overlapping reading frames.9,10) VP2 is the major structural protein, accounting for 96% of all capsid protein.11) This protein is encoded by the sequence from nt 3125 to 4786 and has a molecular mass of 58 kDa.11,12) The minor capsid protein, VP1, is encoded by the sequence from nt 2444 to 4786 and is identical to VP2 apart from the addition of 227 amino acids (termed the VP1-unique region) at its amino terminus.11,12) VP1 protein has a molecular mass of 84 kDa and makes up the remaining 4% of the capsid protein. 11)The monocyte is one of the important immunocompetent cells involved in fighting viral infection, which causes the differentiation of monocytes into macrophages. Macrophages have phagocytic activity and express important cytokines such as IL-1, IL-12, TNF-a and IFN-g after viral infection. Generally, unenveloped viruses are believed to insert pores into the plasma membrane or disrupt vesicle membranes within the endocytic pathway, but the mechanisms involved in the entry of naked viruses into monocytes are not fully understood.B19 is difficult to culture in the laboratory. Bansal et al., empty capsids consisting of the two B19 structural proteins (VP1 and VP2) have already been used to mimic B19 infection in a murine model. 13)In the present study, we investigated the effect of B19 capsids on a human monocyte/macrophage cell line, including production of inflammatory cytokines and maturation. MATERIALS AND METHODS Cell CultureThe human monocyte cell line THP-1 was maintained at 37°C under an atmosphere containing 5% CO 2 in RPMI-1640 medium supplemented with 1.5 g/l sodium bicarbonate, 1 mM sodium pyruvate, 10 mM HEPES, 2.5 g/l Dglucose, 0.05 mM 2-mercaptoethanol, and 10% fetal bovine serum. THP-1 cells were kindly donated by the Cell Resource Center...
Poly(ADP-ribose) (PAR) formation is catalyzed by poly(ADP-ribose) polymerase (PARP) family proteins in nuclei as well as in cytosols. The anti-PAR antibodies that specifically detect PAR are useful for the quantitative measurement of PAR in cells, in tissue, and in the body. In clinical trials of PARP inhibitors, a pharmacodynamic (PD) assay for the measurement of PARP activity inhibition in peripheral blood mononuclear cells (PBMCs) with dot-blot assay or an ELISA assay using anti-PAR antibodies have been used. In these assays, ex vivo PARP activity and its inhibition assay have been used. For a PD assay to assess the efficacy of the treatment, the measurement of PARP activity inhibition in tumor tissues/cells has been recommended. A dot or slot blot assay may also be suitable for the measurement of such crude tissue samples. Here, we investigate the optimum conditions for a dot/slot blot assay of an ex vivo PARP activity assay by utilizing physical and chemical crosslinking methods. Using 10H monoclonal antibody to PAR, we show that use of a nylon membrane and UV crosslink at 254 nm can stably enhance the detection level of PAR. However, the limitation of this assay is that the size of PAR detectable using the 10H antibody must be around 20 ADP-ribose residues, since the antibody cannot bind PAR of lower size.
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