No abstract

Human herpesvirus 7 (HHV-7) is a new herpesvirus recently isolated from CD4+ T cells. I Recently, we reported that HHV-7 was a causal agent for exanthem subitum.2 In this report, we examined the clinical manifestations associated with HHV-7, especially exanthem subitum.

We investigated apoptosis induced by herpes simplex virus type 1 (HSV-1) in cord blood T lymphocytes by using agarose gel electrophoresis, DNA content analysis and the terminal deoxytransferase (TdT)-mediated dUTP nick end-labelling (TUNEL) method. DNA fragmentation and the hypodiploid fraction in the cell cycle were both increased in HSV-1-infected CD4 and CD8 lymphocytes stimulated with phytohaemagglutinin (PHA) compared to mock-infected lymphocytes. The percentage of cells in the S phase was decreased in HSV-1-infected CD4 and CD8 lymphocytes. HSV-1 antigen, glycoprotein D (gD) and regulatory protein ICP27 were detected in 8-18 % of the hypodiploid fraction of PHA-stimulated, HSV-1-infected lymphocytes. Apoptosis was induced not only in HSV-1 antigenexpressing cells but also in cells not expressing detectable viral proteins. Addition of anti-Fas antibody, anti-Fas-ligand antibody or a mixture of both had no effect on HSV-1-induced apoptosis, indicating that the Fas-Fas-ligand pathway did not contribute to HSV-1-induced apoptosis.Herpes simplex virus (HSV) causes severe infection with high mortality in human neonates (Whitley et al., 1991). The low frequency of specific T lymphocytes responding to HSV at birth is thought to be responsible for the severity of primary HSV infection in neonates (Hayward et al., 1984). Human herpesviruses, such as HSV-1 (Tropea et al., 1995), varicellazoster virus (Sadzot-Delvaux et al., 1995) and Epstein-Barr virus (EBV ; Kawanishi et al., 1993), induce apoptosis. In addition virus-induced apoptosis of lymphoid cells may be a major cause of immunosuppression (Groux et al., 1992 ;Razvi et al., 1993). The effect of HSV-1 infection on lymphocytes from neonates has not been fully investigated. In this study, we have investigated the induction of apoptosis by HSV-1 in T Author for correspondence : Masahiro Ito.Fax j81 59 231 5213. e-mail virus!clin.medic.mie-u.ac.jp lymphocytes from neonates and the relationship between apoptosis and impaired immune function.HSV-1 (KOS strain) was grown at an m.o.i. of 0n1 in Vero cells that were cultured in minimum essential medium (MEM, Gibco BRL) supplemented with 10 % foetal bovine serum (FBS, Gibco BRL) at 37 mC. After 48 h, the supernatant was collected and cell debris was removed by centrifugation. The supernatant virus stock was stored at k70 mC. The titre of HSV-1 in the virus stock was 2i10' p.f.u.\ml. Supernatant medium from uninfected Vero cells was used for mock-infected controls.Cord blood was obtained from the placental end of the cord at full-term birth, and mononuclear cells (MNC) were separated by Ficoll-Hypaque (Histopaque 1077, Sigma) gradient centrifugation. MNC at the interface were collected and washed three times with RPMI-1640 (Gibco BRL) and suspended in RPMI-1640 supplemented with 10 % FBS. CD4-and CD8-rich lymphocytes were obtained by using Dynabeads M-450 CD4 and M-450 CD8 (Dynal). These procedures resulted in yields of 95 % CD4 and CD8 cells.Lymphocytes (1i10' cells) were centrifuged at 1500 r.p.m. for 5 min. The pellete...

We investigated the immune reaction of lymphocytes in response to human herpes virus-6 (HHV-6) in normal children and adults. Cell proliferation was assayed by measurement of the incorporation of 5-bromo-2'-deoxyuridine (BrdU) by enzyme-linked immunosorbent assay (ELISA), and changes in expression of interleukin-2 receptors (IL-2Rs) (alpha-, beta3-, and gamma-chain) were assayed by flow cytometry. Incorporation of BrdU and expression of IL-2Rs (alpha-, beta-, and gamma-chain) in CD4+, CD8+, and CD45RO+ lymphocytes were increased when peripheral blood mononuclear cells (PBMC) from HHV-6 seropositive children aged 3 to 12 years and adults were cultured with HHV-6 antigen compared with control antigen. In contrast, cord blood mononuclear cells (CBMC) and PBMC from seronegative children did not show cell proliferation and changes in expression of IL-2Rs. In seropositive children less than 2 years of age, the magnitude of cell proliferation was low and IL-2Rs (alpha-, beta-, and gamma-chain) in CD8+ cells and IL-2Rs (alpha-chain) in CD45RO+ cells were increased. These data suggest that children below the age of 2 had immature lymphocytic response to HHV-6 antigen. Deletion of monocytes from PBMC and the addition of a mixture of anti-IL-2Rs (alpha-, beta-, and gamma-chain) antibodies reduced cell proliferation in response to HHV-6, suggesting the requirement of the presence of monocytes and expression of IL-2Rs.

Changes in the expression of intracellular interleukin-2 (IL-2), interleukin-4 (IL-4), interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha in newborn and adult lymphocytes induced by herpes simplex virus (HSV)-1 were examined. Cord blood mononuclear cells (CBMC) or adult peripheral blood mononuclear cells (PBMC) were infected with HSV-1 and cultured with phorbol 2-myristate 13-acetate (PMA) plus ionomycin in the presence of monensin for 4 hr. Surface antigen and intracellular cytokines were stained simultaneously and analyzed by flow cytometry. The percentage of cells that expressed IL-2, IFN-gamma, and TNF-alpha was significantly increased in HSV-1-infected CD3+, CD4+, CD8+, CD45RA+, and CD45RO+ lymphocytes compared with uninfected lymphocytes from adult PBMC. The percentage of cells that expressed IL-2 and TNF-alpha was increased significantly in HSV-1-infected CD3+, CD4+, CD8+, and CD45RA+ lymphocytes compared with uninfected lymphocytes from CBMC. IFN-gamma was under the detectable level in HSV-1-infected and uninfected lymphocytes from CBMC. Intracellular IL-4 was not detected in HSV-1 or in uninfected lymphocytes from PBMC and CBMC. These results demonstrate that HSV-1 enhances intracellular levels of IL-2, IFN-gamma, and TNF-alpha in adult lymphocytes and defective IFN-gamma production in cord blood.