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Summary. The perforin gene was analysed in 15 Japanese patients with primary haemophagocytic lymphohistiocytosis (HLH). Perforin gene defects were found in two out of eight patients with familial HLH (FHL), and one out of seven without affected siblings. Four novel mutations were identi®ed. Compound heterozygous mutations (one FHL and one sporadic HLH) and only one allele mutation (one FHL) were de®ned. Flow cytometry revealed no perforin expression in CD8 + or CD56 + cells from a surviving patient with a mutation. The frequency of mutation was at least 20% of FHL in Japan. Flow cytometry for intracellular perforin may be useful for the screening of FHL2.

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.

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SUMMARYWe 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-g 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 IFNg , play an important role in the pathophysiology of HLH, by recruitment of activated Th1 cells into the tissues or organs.

SUMMARYTo continue the search for immunological roles of breast milk, cDNA microarray analysis on cytokines and growth factors was performed for human milk cells. Among the 240 cytokine-related genes, osteopontin (OPN) gene ranked top of the expression. Real-time PCR revealed that the OPN mRNA levels in colostrum cells were approximately 100 times higher than those in PHA-stimulated peripheral blood mononuclear cells (PBMNCs), and 10 000 times higher than those in PB CD14+ cells. The median levels of OPN mRNA in early milk or mature milk cells were more than three times higher than those in colostrum cells. Western blot analysis of human milk showed appreciable expression of full-length and short form proteins of OPN. The concentrations of full-length OPN in early milk or mature milk whey continued to be higher than those in colostrum whey and plasma as assessed by ELISA. The early milk (3-7 days postpartum) contained the highest concentrations of OPN protein, while the late mature milk cells (1 years postpartum) had the highest expression of OPN mRNA of all the lactating periods. The results of immunohistochemical and immunocytochemical staining indicated that OPN-producing epithelial cells and macrophages are found in actively lactating mammary glands. These results suggest that the persistently and extraordinarily high expression of OPN in human milk cells plays a potential role in the immunological development of breast-fed infants.

SUMMARYThl and Thl-inducing cytokines and T cell responses were investigated in human salmonellosis. Serum IFN-g , IL-12 and IL-18 levels were increased significantly in patients with salmonellosis. The increase in serum IL-15 and IL-18 levels was more significant and prolonged in patients with the systemic form of salmonellosis than in those with the gastroenteric form. The serum IFN-g level was correlated significantly with IL-12 and IL18 levels, and the IL-15 level was correlated significantly with IL-18. Upon stimulation with Salmonella in vitro , mononuclear cells from salmonellosis patients produced significantly higher amounts of IFN-g and IL-12 compared with those from healthy controls. Anti-IL-12 moAb or anti-IL18 MoAb significantly inhibited Salmonella -induced IFN-g production in vitro . gd T cells expressed significantly higher levels of IFN-g mRNA in salmonellosis patients than in healthy controls. The results suggest that Th1-inducing cytokines appear to be involved in the in vivo response against Salmonella infection, promoting IFN-g production by ab and gd T cells which plays a protective role against Salmonella .

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