Yasushi Takahata

Epstein-Barr Virus (EBV) Load and Cytokine Gene Expression in Activated T Cells of Chronic Active EBV Infection

To identify the role of T cells in chronic active Epstein-Barr virus (EBV) infection, EBV and cytokine gene expression was quantified by use of real-time polymerase chain reaction (PCR) among 6 patients who fulfilled the diagnostic criteria for chronic active EBV infection. Four of these patients showed clonal expansion of EBV-infected T cells. Quantitative PCR for EBV DNA in peripheral blood of patients with symptomatic chronic active EBV infection showed higher copy numbers of virus (mean, 1.45 x 10(5) copies/mL) than were seen in blood from patients with infectious mononucleosis (3.08 x 10(3) copies/mL) or with EBV-associated hemophagocytosis (2.95 x 10(4) copies/mL). Fractionated CD3(+) HLA-DR(+) cells from patients with chronic active EBV infection contained higher copy numbers than did CD3(+) HLA-DR(-) cells. Quantitative PCR for cytokines revealed that interferon-gamma, interleukin (IL)-2, IL-10, and transforming growth factor-beta genes were expressed at higher levels in HLA-DR(+) than in HLA-DR(-) T cells. These results suggest that activated T cells in chronic active EBV infection expressed high levels of EBV DNA and both Th1 and Th2 cytokines. EBV-infected T cells may contribute to the unbalanced cytokine profiles of chronic mononucleosis.

Interleukin-18 in Human Milk

We analyzed IL-18 levels of human milk. Colostrum contained significantly higher levels of IL-18 compared with early milk and mature milk. By stepwise multiple linear regression analysis, preterm delivery and pregnancy complications of mothers significantly correlated with high levels of IL-18 in human milk (p = 0.0007 and 0.0018, respectively). There was a significant correlation between the levels of IL-18 and soluble Fas ligand in colostrum (p = 0.0003). IL-18 was detected in actively secreting epithelial cells in lactating mammary gland by immunohistochemical staining. These results suggest that IL-18 in colostrum plays an important role in host defense of high-risk neonates.

Physical Growth and Retinopathy in Preterm Infants: Involvement of IGF-I and GH

GH and IGF-I are important for physical growth. We measured serum levels of these factors in preterm infants. The study population (n = 81) was divided into three groups according to the gestational age. We evaluated differences in serum GH and IGF-I levels among groups with regard to physical growth and development of retinopathy of prematurity. Serum GH levels in extremely preterm infants born at <28 wk of gestational age were significantly higher than levels in those born between 28 and 34 wk at 1 and 2 mo of age. In contrast, serum IGF-I levels in extremely preterm infants remained low, whereas those in the other two groups gradually increased. Evaluation of the effects of GH and IGF-I on physical growth in very low birth weight infants (<1500 g) showed that IGF-I concentrations were positively related to physical growth for several months after birth, whereas no relationship was observed between GH and physical growth. Multivariate analysis demonstrated that high GH concentration at 1 mo of age was significantly associated with development of severe retinopathy of prematurity. In conclusion, persistent low serum IGF-I levels may explain the slow physical growth during neonatal life, and exposure of high GH may cause, at least in part, severe retinopathy of prematurity in preterm infants.

Increased serum levels of interferon-γ-inducible protein 10 and monokine induced by gamma interferon in patients with haemophagocytic lymphohistiocytosis

We measured serum interferon‐gamma‐inducible protein 10 (IP‐10) and monokine induced by gamma interferon (MIG) levels to investigate the role of these molecules in the pathophysiology of haemophagocytic lymphohistiocytosis (HLH). Serum IP‐10 and MIG levels were significantly increased in patients with active HLH compared with those of healthy controls. Serum MIG levels decreased gradually during the course of disease in a patient who recovered without therapy. On the other hand, rapid reduction of MIG and IP‐10 levels was observed after chemotherapy in a patient with severe HLH. IP‐10 and MIG mRNA expression was enhanced in liver and spleen, and IP‐10 mRNA expression was enhanced in bone marrow in the patients, suggesting activated macrophages that infiltrated in these organs as one of the main producers of these cytokines. Serum IP‐10 and MIG levels showed a significant correlation with serum IFN‐γ levels. In addition, these chemokines had a significant correlation with fever and serum LDH levels, which are clinical indicators of disease activity of HLH. These results suggest that IP‐10 and MIG which are produced by activated macrophages by the stimulation of IFN‐γ, play an important role in the pathophysiology of HLH, by recruitment of activated Th1 cells into the tissues or organs.

Detection of interferon-γ-inducible chemokines in human milk

Aim: To assess the immunological role of human milk by analysing the concentrations of interferon‐γ‐inducible protein of 10kda (IP‐10) and monokine induced by interferon‐γ (MIG) in human milk from mothers of preterm and term infants. Methods: IP‐10 and MIG levels of colostrum, early milk, mature milk and sera were measured by enzyme‐linked immunosorbent assay (ELISA). IP‐10 and MIG mRNA expression levels in cellular components of human milk were determined by RT‐PCR. IP‐10 and MIG protein expression in mammary gland tissues was analysed by immunohistochemistry. Results: Significant amounts of IP‐10 and MIG were detected in human milk. The concentrations of IP‐10 and MIG in colostrum and early milk were significantly higher than those of sera from healthy controls or lactating mothers. These chemokine concentrations in colostrum and early milk were significantly higher than those of mature milk. Premature delivery or pregnancy complications of mothers had no significant correlation with these chemokine concentrations in breast milk. There were significant correlations between MIG and interferon‐γ (IFN‐γ) or IP‐10 levels (p < 0.001) in human milk. Expression of IP‐10 and MIG genes and proteins in the milk cells as well as in mammary gland epithelial tissues was detected by RT‐PCR and immunohistochemistry.

Dominant expression of interleukin 10 but not interferon γ in CD4–CD8–αβT cells of autoimmune lymphoproliferative syndrome

Summary. Cytokine expression in CD4–CD8– double‐negative (DN) T cells of autoimmune lymphoproliferative syndrome (ALPS) was analysed. Two patients with DN αβT‐cell expansion showed higher serum interleukin 10 (IL‐10) levels than one patient without it. Intracellular flow‐cytometric analysis indicated the IL‐10‐expressing CD3+CD4–CD8– cells in patients with lymphoproliferation. Quantitative real‐time polymerase chain reaction revealed ∼100 times higher IL‐10, but not interferon‐γ or transforming growth factor‐β in DN than in single‐positive T cells. IL‐10 was exclusively expressed in DN αβ but not γδΤ cells. Circulating DN αβT cells may constitutively express IL‐10 and contribute to the ALPS phenotype.

Cytokine imbalance in hyper-IgE syndrome: reduced expression of transforming growth factor β and interferon γ genes in circulating activated T cells

Summary. Hyper‐IgE syndrome (HIES) is a primary immunodeficiency disease characterized by recurrent infections and marked immunoglobulin (Ig)E elevation. To assess the proper T‐cell defects of HIES, the cytokine profile of naturally activated T cells was compared between HIES, atopic dermatitis and chronic granulomatous disease (CGD). Intracellular flow cytometric analysis after in vitro stimulation showed no difference in the proportion of interferon (IFN)γ‐ or interleukin 4 (IL‐4)‐producing T cells among these diseases. Quantitative polymerase chain reaction (PCR) for the cytokine genes was performed using circulating highly fractionated HLA‐DR+ and HLA‐DR– T cells. The IFNγ/IL‐4 or IFNγ/IL‐10 ratios were lower in HLA‐DR+ T cells of HIES than in CGD (P = 0·0106, 0·0445), but did not differ between HIES and atopy. The transforming growth factor‐β (TGFβ)/IL‐4 ratio in HLA‐DR+ T cells of HIES was lower than that of atopy (0·0106) or CGD (0·0062). The TGFβ/IL‐4 ratio in HLA‐DR– T cells of HIES was also lower than that of atopy (0·0285). Stepwise logistic regression analysis identified TGFβ/IL‐4 ratios in HLA‐DR+ (0·0001) or HLA‐DR– (0·0086) T cells as the most powerful parameters to distinguish HIES from atopy and/or CGD. Serum IgE levels negatively correlated with IFNγ/IL‐4 (0·0108), IFNγ/IL‐10 (0·0254), or TGFβ/IL‐4 (0·0163) ratios in HLA‐DR+, but not HLA‐DR–, T cells. These results suggested that the in vivo activated T cells of HIES did not sufficiently express the IFNγ and TGFβ genes, which could affect IL‐4‐dependent IgE production. The reduced TGFβ expression may involve the indigenous T‐cell defects of HIES.

Survival and Neurodevelopmental Outcome of Preterm Infants Born at 22-24 Weeks of Gestational Age

Background: The limits of viability in extremely premature infants are challenging for any neonatologists in developed countries. The neurological development and growth of extremely preterm infants have come to be the emerging issue following the management in the neonatal intensive care unit. Objective: To assess potential associations between changes in practice and survival/neurodevelopmental outcome, and clinical outcomes of extremely preterm infants born at the limit of viability studied in a tertiary center. Study Design: A retrospective study enrolled 51 infants who had no congenital disorders, and were born at 22-24 weeks of gestational age (GA) in 2000-2009 in our institution. Clinical variables and interventions were studied with regard to one-year survival and developmental quotient (DQ) at 3 years of age. Results: The one-year survival rate of 24 preterm infants born in 2005-2009 (79%) was higher than that of the 27 infants born in 2000-2004 (52%, p = 0.04). Infants born after 2005 underwent less tocolysis (54 vs. 94%, p < 0.01) and more frequently antenatal steroid therapy (32 vs. 6%, p = 0.01) than those born before 2004. The post-2005 survivors (n = 19) received more frequently indomethacin therapy (89 vs. 50%, p = 0.03) and early parenteral nutrition (95 vs. 36%, p < 0.01) than the pre-2004 survivors (n = 14). There were no differences in the proportion of infants who attained a DQ of >50 at 3 years of age between pre-2004 (9/13, 69%) and post-2005 groups (10/17, 59%). Multivariate analysis indicated that extremely premature birth at GA <24 weeks was the sole critical factor for a DQ of >50 in survivors. Conclusions: The perinatal care after 2005 improved the overall survival rate, but not the neurological outcome of preterm survivors at the limit of viability. Neurodevelopmental impairments were associated with extremely premature birth at GA <24 weeks.

Prenatal findings in a case of massive fetomaternal hemorrhage associated with intraplacental choriocarcinoma.

We describe biochemical assessment of maternal circulation in a case of massive fetomaternal hemorrhage at term associated with intraplacental choriocarcinoma. Markedly elevated maternal serum hCG level at 37 weeks of gestation suggested choriocarcinoma as a cause of fetomaternal hemorrhage in this case. Measurement of maternal hCG may be a useful parameter when intraplacental choriocarcinoma is in the differential diagnosis. In addition, the placenta should be examined in all cases of fetomaternal hemorrhage.

Immunophenotypic and functional characterization of CD33+CD34+ cells in human cord blood of preterm neonates

OBJECTIVE To characterize CD33(+)CD34(+) cells, a major population in human cord blood (CB) CD34(+) cells of preterm neonates. MATERIALS The proportion of CD33(+) cells was analyzed on CB CD34(+) cells from preterm and full-term neonates. CD33(+)CD34(+) cells were purified by cell sorting and analyzed on their clonogenic activity, proliferative activity in short-time liquid suspension culture, and GATA-2 mRNA expression by RT-PCR and Southern blot. RESULTS The absolute numbers and proportion of CD34(+) cells in mononuclear cells inversely correlated with gestational age. CD33 was expressed on a majority of CB CD34(+) cells of preterm neonates but on only a minor population of them in full-term neonates. In addition, CD33 was dominantly expressed on CD38(-)CD34(+) cells or CD117(low)CD34(+) cells in CB of preterm neonates. CD33(+)CD34(+) cells of preterm cord blood had high proliferative and reproducible potentials compared with CD33(-)CD34(+) cells. CD33(+)CD34(+) cells as well as CD33(-)CD34(+) cells from preterm CB highly expressed GATA-2, in contrast to those from BM. CONCLUSIONS These results suggest that CD33(+)CD34(+) cells, which are a major population in CB CD34(+) cells of preterm neonates, do not simply represent relatively mature myeloid lineage hematopoietic progenitor cells as those in adult BM CD34(+) cells, and may contain hematopoietic stem cells or primitive progenitor cells as in fetal liver.