Killer cells such as CTL, NK cells, and monocytes play important roles in immune surveillance against transformed cells and virus-infected cells. CTL and NK cells directly kill target cells via two major effector pathways, the perforin-mediated and FasL-mediated pathways. In addition to these pathways, however, some studies by using the gene-targeting mice suggested the possible existence of some other effector mechanisms for the cytotoxicity. Monocytes not only mediate inflammatory responses via production of various cytokines and chemical mediators, but also kill directly some transformed cells. However the effector mechanisms remain largely unknown. Recently, some death-inducing TNF family members, such as TNF-related apoptosis-inducing ligand (TRAIL) and TWEAK were identified and it has been reported that TRAIL- and TWEAK-dependent mechanisms constitute novel pathways of killer cell cytotoxicity. FasL and TRAIL have also been implicated in the tissue damage associated with some inflammatory diseases, such as hepatitis and rheumatoid arthritis. This review describes the expression of FasL, TRAIL, and TWEAK on killer cells and discusses the pathophysiological roles of these molecules in immune system.

View full abstract

Imbalance of Th1/Th2 responses is associated with the development of human autoimmune diseases and allergic diseases. We have studied a role of Txk, a member of Tec family non-receptor tyrosine kinase for the Th1/Th2 cell development.
We found that Txk is expressed Th1/Th0 cells with IFN-γ producing potential. Txk transfection leads to severalfold increase in IFN-γ production; neither IL-2 nor IL-4 production were affected. Thus, Txk acts as a Th1 cell specific transcription factor.
Furthermore, we found that Txk expression is enhanced by Th1 cytokines but is inhibited by Th2 cytokines. Txk expression of the peripheral blood T cells is decreased in patients with allergic diseases. Collectively, Txk expression is modulated by with Th1/Th2 cytokines and is importantly involved in immune aberration in patients with immunological disorders. These results suggest that Txk is a possible therapeutic target of the diseases with Th1/Th2 imbalance.

View full abstract

As the result of the human genome project, the entire sequence of human genome was determined. Unfortunately, it is still not a trivial task to identify genes from these genome sequences only by the computational methods. Thus, full-length cDNAs are indispensable for the identification of genes and the determination of their structures. In addition, cDNAs will be a valuable resource for the functional analysis of the gene. We have performed so called FLJ project of collecting and determining the entire sequences of putative full-length cDNA clones. In this project, we extensively used cDNA libraries made by oligo-capping method. In this review, I will describe the current status of FLJ project and high-through-put functional analysis using full-length cDNA clones obtained by FLJ project.

View full abstract

Glucosamine, an amino monosaccharide naturally occurring in the connective and cartilage tissues, contributes to maintaining the strength, flexibility and elasticity of these tissues. In recent years, glucosamine has been widely used to treat osteoarthritis in humans, because glucosamine increases proteoglycan synthesis and prevents cartilage degradation. Recently, we have revealed that glucosamine suppresses neutrophil functions, thereby possibly exhibiting anti-inflammatory actions in arthritis. Alternatively, we have found that glucosamine improves the fluidity of blood (hemorheology) analyzed by microchannel array. Therefore, in this study, we evaluated the effects of glucosamine on the functions of platelets, which play an essential role in thrombosis. Glucosamine suppressed platelet aggregation in response to ADP-stimulation, but not collagen-and thrombin-stimulation. Furthermore, glucosamine inhibited the extracellular release of granular constituents (ATP and platelet factor 4), and the production of thromboxane from ADP-stimulated platelets. In addition, glucosamine significantly inhibited the mobilization of intracellular calcium and the phosphorylation of Syk associated with platelet activation. Together these observations suggest that glucosamine suppresses platelet aggregation via the inhibition of calcium mobilization, Syk phosphorylation, granular content release and thromboxane production. Thus, glucosamine may have preventive effects on thrombosis by its suppressive actions on platelet functions.

View full abstract

Today nonviral gene transfer vectors attract more attention as a therapeutic strategy for human diseases, because viral vectors have problems, especially in immunogenicity and cytotoxicity. However, main limitation of nonviral vectors has been low efficiency of gene expression. To overcome this defect, we have developed a new class of gene transfer system, HVJ-cationic liposomes. The use of cationic lipid, DC-cholesterol, facilitates efficient entrapment of negatively charged macromolecules and efficient interaction with negatively charged plasma membrane. Moreover, the fusogenic envelope proteins of HVJ enhance delivery of genes and oligonucleotides. We have succeeded in obtaining 100-800 times higher gene expression than the conventional HVJ-liposome method. On the other hand, although several gene transfer methods have been utilized in clinical gene therapy trials, there is no almighty method. Thinking of problems in all methods including nonviral methods, the one thing that is common in all methods is we cannot target the specific organ or area. To improve the specificity of gene transfer we have succeeded in developing an immunoliposome (HVJ-liposome + Anti Thy 1 antibody). With this newly developed method, we performed specific gene transfer into the kidney especially to glomeruli. Taken together we have developed a novel nonviral vector system based on HVJ-liposome method.

View full abstract

Previous studies have shown that apoptosis of neutrophils represents a physiologic clearance mechanism in the circulation and in the tissue to create and maintain homeostasis of neutrophil numbers. It is well known that spontaneous and Fas-mediated apoptosis of neutrophils can be regulated by proinflammatory mediators. The effects of a selective cyclooxygenase-2 (COX-2) inhibitor, NS-398, on the Fas-mediated apoptosis in inflammatory stimuli-activated neutrophils were examined. Tumor necrosis factor-α(TNF-α) and granulocyte-macrophage stimulating factor (GM-CSF) enhanced PGE₂ release through induction of COX-2, but not interleukin-1β(IL-1β) and IL-8. TNF-α- and GM-CSF-induced PGE₂ release was abolished by the addition of NS-398 (1 μM), nevertheless NS-398 did not change the TNF-α- and GM-CSF-induced expression of COX-2. Although not only GM-CSF but also IL-1β and IL-8 delayed Fas-mediated apoptosis in neutrophils, this effect was suppressed by the addition of NS-398 (100 μM). On the contrary, TNF-α-treated neutrophils did not change the susceptibility to Fas-mediated apoptosis. These results suggest that selective COX-2 inhibitor not only suppresses PGE₂ release, but also enhances Fas-mediated apoptosis of cytokine activated-neutrophils. Recent studies have provided evidence that COX-2 inhibitor acts through a COX-2-independednt mode of various cell functions. Therefore, the proapoptotic activity of selective COX-2 inhibitor may be independent of COX-2 activity. Considering these studies, selective COX-2 inhibitor may contribute to come to an end of acute inflammation via enhanced apoptosis of neutrophils.

View full abstract

Human hematopoietic stem cells (HSC) in cord blood (CB) are increasingly being used as an alternative to those in adult bone marrow (BM) for treating patients with various hematological disorders. However, the difference in the hematopoietic activity of HSC between CB and adult BM is still unclear. We then compared CD34⁺ cells, a hematopoietic cell population, in CB with those in adult BM, using their phenotypic subpopulations, colony-forming activity and long-term repopulating ability. Although the proportion of CD34⁺ cells was higher in adult BM than CB mononuclear cells in flow cytometry, more immature subpopulations, CD34⁺CD33^- and CD34⁺CD38^- cells, were present in CB CD34⁺ cells. Clonal culture assay showed that more multipotential progenitors were present in CB CD34⁺ cells. When transplanted into immunodeficient NOD/SCID mice, adult BM CD34⁺ cells could not reconstitute human hematopoiesis in recipient BM, but CB CD34⁺ cells achieved a high level of engraftment, indicating that CB CD34⁺ cells possess greater repopulating ability. Thus, CB CD34⁺ cells contain more primitive hematopoietic cells including HSC, suggesting the usefulness of CB CD34⁺ cells not only as a graft for therapeutic HSC transplantation, but as a target cell population of ex vivo expansion of transplantable HSC.

View full abstract