Cord blood transplantation (CBT) frequently can be complicated by cytomegalovirus (CMV) infection, which is required for administration of antiviral chemotherapy. Although the naïve feature of cord blood T-cells were suggested to increase the incidence of CMV infection, the underlying mechanism is not fully elucidated. CMVresponsive T-cells detected by intracellular interferon-gamma (IFN-γ) staining and CMV-specific CD8⁺ T- cells detected by MHC class I tetramers were analyzed in healthy adult blood and recipient blood post-CBT. CMV antigenemia was accompanied in thirty-two of thirty-nine recipients (82%) post-CBT. CMV-responsive CD4⁺ T-cells were detected in all recipients with CMV antigenemia within 120 days post CBT [first date of CMV- responsive CD4+ T-cell detection, median 41 days, (32-90 days); peak frequencies 18±21 cells/μl, n=22]. In contrast, CMV-responsive CD8⁺ T-cells were detected in only 38% of patients with CMV antigenemia with similar frequencies (peak frequencies 18±24 cells/μl, n=8). CMV-responsive CD4⁺ T-cells but not CMV responsive CD8⁺ T-cells were significantly induced in all recipients with CMV antigenemia. Rapid reconstitution of CMV-responsive CD4⁺ T-cells might suppress the progression of CMV disease from CMV antigenemia.

Aim. We are in charge of the central diagnosis and cell preservation as a part of childhood acute lymphoblastic leukemia treatment study in Tokyo Children’s Cancer Study Group. It is necessary to diagnose with a minimal quantity of specimen, to preserve leukemic cells effectively as possible. Therefore a diagnosis of childhood acute lymphoblastic leukemia by four-color analysis with digital flow cytometer has been examined.

Methods. We examined cell markers of childhood acute lymphoblastic leukemia cells by four-color analysis using digital flow cytometers. We selected the monoclonal antibodies for the diagnosis based on the recommendation of Japan Pediatric Lymphoma Study Group and made out a panel of antibodies which enable us to confirm aberrant antigen-expressions on the leukemic cells.

Results. Four colors that we used in this study were fluorescein isothyocyanate, phycoerythrin, phycoerythrin-cyanin 5.1, and phycoerythrin-cyanin-7. The most of childhood acute lymphoblastic leukemia cases could be diagnosed without CD45-gating. List mode compensation was useful to re-investigate specimens which was difficult to re-examine, because there were very few.

Discussion. Four-color analysis using digital flow cytometer is useful to save precious specimen of childhood acute lymphoblastic leukemia. We are intending to perform five-color analysis with CD45-gating as a next step.

Relative DNA content of tumor cells determined by flowcytometric analysis (DNA ploidy) has been reported as a prognostic factor of solid cancer. However, it is still unknown correlation between morphologic characteristics and DNA ploidy. We have evaluated the cytopathologic findings including area of nucleus, area of nucleolus, number of nucleolus, nuclear/ cytoplasm ratio, and DNA ploidy in 76 patients with uterine endometrial carcinomas. DNA aneuploid was detected in 18 patients (24%), and DNA aneuploid cases exhibited significantly larger nuclear area and area of nucleolus than that of the DNA diploid cases. However, no correlation was seen in nuclear/cytoplasm ratio, and number of nucleolus. Our findings suggest that relative DNA content would be influenced to area of the nucleus and area of the nucleolus.

Molecular targeting of hematopoietic malignancies has not been successfully developed because of difficulties in introducing genes of interest into these neoplastic cells. Recently, peptide delivery systems using protein transduction domains (PTD) have attracted attention for possibly overcoming such obstacles in a therapeutic approach. Here, we show the development of a peptide transporter system of maximum utility that can suppress growth of biologically aggressive or intractable leukemia/lymphoma cells. The transporter (Wr- T) has an enlarged hydrophobic pocket consisting of triple tryptophan-rich domains fused with nine D-enantiomer poly-arginines (D-Arg) via proline spacers, which enhance the efficiency of peptide-introduction over previous mechanisms by more than 20 times. Delivering small amounts of p16 (INK4a) functional peptide by Wr-T enables dramatic inhibition of growth of up to 80% in highly aggressive leukemia/lymphomas lacking p16 expression by recovery of their p16 function. Thus, the WR-T system exhibits high-performance cargopeptide delivery and is supposed to be useful for treating hematopoietic malignancies in combination of the p16 peptide. This system may also help treat various tumors by functional peptide selection.

The cell fractions of G₁, S and G₂ + M phases are determined from the DNA histograms. The relationship between fraction and duration of G1, S and G₂ + M phases was derived using an equation, n (t, τ) = αN₀exp (α t) exp (α τ), where n (t, τ) is cell density of phase τ at time t, N₀ is the initial cell number at time t = 0, T_c is cell-cycle time and α = ln2 / T_c.

The BrdU continuous labeling method was presented for a method that can determine the cell cycle time. In this method, the relationships are derived using an equation that n( t,τ) =αN₀exp(αt) exp (ατ), where n (t,τ) is cell density of a phase τ at time t, N₀ is the initial cell number at time t = 0, Tc is cell-cycle time and α = ln2 / Tc. BrdU is incorporated into DNA strand instead of TdR. Anti BrdU monoclonal antibody is used to distinguish labeled and unlabeled cells in a population and to determine the fraction. The plotting of labeled or unlabeled cell fraction against time reveals the cell cycle time.

The stathomokineses method was presented as the cell cycle analysis to determine cell cycle parameters. In this method, all of the relationships are derived from an equation that n(t, τ) =αN₀exp(αt)exp(ατ), where n(t, τ) is cell density of a phase τ at time t, N₀ is the initial cell number at time t = 0, T_c is cell-cycle time and α = ln2 / T_c. The stathomokinetic agents, colchicines or demecolcine (Colcemid), are added in a cell population to block cell cycle progression at the M phase. The plotting of G₁ or G₂/M cell fractions against time reveals the cell cycle parameters. A computer program of cell cycle analysis of unconstrained-type is presented to help the stathomokinetic analysis.