Taekyun Shin

Role of natural killer cells and TCRγ δ T cells in acute autoimmune encephalomyelitis

To elucidate the role of NK cells and TCRγ δ + T cells in acute experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats, the distribution, number and function of these cells were studied using several methods. Immunohistochemical and flow cytometric analysis revealed that a certain number of NK cells (17 % of the total inflammatory cells) infiltrated the central nervous system (CNS) at the peak stage of EAE and were mainly located in the perivascular region. On the other hand, virtually no TCRγ δ + T cells were found in the CNS. NK‐T (NKR‐P1+ TCRα β + ) cells were few and did not increase in number in the CNS and lymphoid organs. In the cytotoxic assay using YAC‐1 cells, effector cells isolated from the spleen of rats at the peak of EAE showed essentially the same cytotoxicity as those isolated from normal controls although the total number of NK cells decreased to one fifth of that of normal rats. Furthermore, in vivo administration of anti‐NK cell (3.2.3 and anti‐asialo GM1), but not of anti‐TCRγ δ (V65), antibodies exacerbated the clinical features of EAE and induced fatal EAE in some rats. These findings suggest that NK cells play a suppressive role in acute EAE whereas TCRγ δ + T cells are not involved in the development of or recovery from the disease.

Competitive PCR quantification of pro- and anti-inflammatory cytokine mRNA in the central nervous system during autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system that can be induced by immunization with myelin basic protein (MBP)/complete Freunds adjuvant and serves as a model for multiple sclerosis. Recent studies have suggested that cytokines play a crucial role in the clinical course of EAE. To clarify the roles of cytokines in EAE, we examined levels of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta1 (TGF-beta1) and interleukin-10 (IL-10) mRNA in isolates from infiltrating inflammatory cells in EAE lesions induced in Lewis rats. The non-radioactive and sensitive competitive PCR method was employed to quantify the relative amounts of cytokine mRNA. Levels of both IFN-gamma and TNF-alpha mRNA were increased at the early stage of EAE and rapidly decreased at the peak stage. On the other hand, TGF-beta1 mRNA was demonstrated throughout the course of EAE as well as under normal conditions and its amount paralleled the severity of EAE. IL-10 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR) under normal conditions, but was below the level of detection of competitive PCR. IL-10 mRNA expression peaked at the early stage of EAE and declined gradually thereafter. Taken together, these results suggest that IFN-gamma and TNF-alpha might play a crucial role in the development of EAE. Furthermore, it appears that the peak expression of IL-10 mRNA at the early stage and the following marked TGF-beta1 expression at the peak stage might represent an important endogenous mechanism to limit the extent of inflammation and to prevent relapse in the course of acute monophasic EAE.

Cyclophosphamide impairs hippocampus-dependent learning and memory in adult mice: Possible involvement of hippocampal neurogenesis in chemotherapy-induced memory deficits

Cyclophosphamide (CYP) is an anti-neoplastic agent as well as an immunosuppressive agent. In order to elucidate the alteration in adult hippocampal function following acute CYP treatment, hippocampus-related behavioral dysfunction and changes in adult hippocampal neurogenesis in CYP-treated (intraperitoneally, 40 mg/kg) mice (8-10-week-old ICR) were analyzed using hippocampus-dependent learning and memory tasks (passive avoidance and object recognition memory test) and immunohistochemical markers of neurogenesis (Ki-67 and doublecortin (DCX)). Compared to the vehicle-treated controls, mice trained at 12h after CYP injection showed significant memory deficits in passive avoidance and the object recognition memory test. The number of Ki-67- and DCX-positive cells began to decrease significantly at 12h post-injection, reaching the lowest level at 24h after CYP injection; however, this reverted gradually to the vehicle-treated control level between 2 and 10 days. We suggest that the administration of a chemotherapeutic agent in adult mice interrupts hippocampal functions, including learning and memory, possibly through the suppression of hippocampal neurogenesis.

THE SUBARACHNOID SPACE AS A SITE FOR PRECURSOR T CELL PROLIFERATION AND EFFECTOR T CELL SELECTION IN EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS

To characterize the phenotype of inflammatory cells in the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE), Lewis rats were immunized with guinea pig myelin basic protein and frozen sections of the spinal cord with EAE were examined immunohistochemically using a panel of monoclonal antibodies against T cells and adhesion molecules. In addition, double immunostaining was performed with glial and T cells markers to examine the interaction between infiltrating T cells and reactive brain cells during the course of EAE. In the early stage of EAE, inflammatory cells first appeared in the subarachnoid space (SAS) and infiltrated the subpial region. The majority of inflammatory cells in SAS expressed TCR alpha beta and either CD4 or CD8 molecules. However, only CD4+ T cells infiltrated the parenchyma while the majority of CD8+ cells remained in SAS. A similar differential localization of T cells was observed with regard to CD45RC molecules. Inflammatory cells in SAS consisted of both CD45RC+ and CD45RC- population, while those in the parenchyma were largely CD45RC-. With regard to adhesion molecules, the leptomeninges constitutively expressed fibronectin (FN) and intercellular adhesion molecule 1 (ICAM-1). Most SAS inflammatory cells expressed very late activation antigen 4 (VLA-4) and, to lesser extent, lymphocyte function-associated antigen 1 (LFA-1) in the early stage of EAE. On the other hand, parenchymal infiltrating cells expressed LFA-1 more strongly in the peak stage. Double staining for V beta 8.2 TCR and microglia demonstrated an increase in the number of microglia together with morphological changes into rod-shape cells in the vicinity of infiltrating T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and immunohistochemical localization of galectin-3 in various mouse tissues

The expression and immunohistochemical localization of galectin‐3, a β‐galactoside‐binding protein, was studied in several mouse tissues. Galectin‐3 expression was low in the cerebrum, heart, and pancreas, and moderate in the liver, ileum, kidney, and adrenal gland. High expression of galectin‐3 was found in the lung, spleen, stomach, colon, uterus, and ovary. The results of Western blot analysis largely matched the immunohistochemical findings for galectin‐3. These findings suggest that galectin‐3 is differentially expressed in a variety of organs in the mouse. This study provides valuable information for research on galectin‐3.

Expression of caveolin-1, -2, and -3 in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis

The expression of caveolin-1, -2, and -3 in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis (EAE) was analyzed. Western blot analysis showed that three isotypes of caveolins including caveolin-1, -2 and -3 increased significantly in the spinal cords of rats during the early stage of EAE, as compared with the levels in control animals (p<0.05); the elevated level of each caveolin persisted during the peak and recovery stage of EAE. Immunohistochemistry demonstrated that caveolin-1 and -2 were expressed constitutively in the vascular endothelial cells and ependymal cells of the normal rat spinal cord, whereas caveolin-3 was almost exclusively localized in astrocytes. In EAE lesions, the immunoreactivity of caveolin-1 was increased in the ependymal cells, some astrocytes, and some inflammatory cells of the spinal cord, while that of caveolin-2 showed an intense immunoreactivity. Caveolin-3 was expressed constitutively in some astrocytes, but not in endothelial cells; its immunoreactivity was increased in reactive astrocytes in EAE lesions. The results of the Western blot analysis largely confirmed the observations obtained with immunohistochemistry. Taking all the findings into consideration, we postulate that the expression levels of each caveolin begin to increase when EAE is initiated, possibly contributing to the modulation of signal transduction pathways in the affected cells.

Immunohistochemical study of arginase-1 in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis

Arginase-1, a marker for M2 phenotype alternatively activated macrophages, inhibits inflammation and is associated with phagocytosis of cell debris and apoptotic cells. We analyzed the expression of arginase-1, a competitive enzyme of inducible nitric oxide synthase (iNOS), in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis (EAE). Western blot analysis showed that both arginase-1 and iNOS significantly increased in the spinal cords of rats at the peak stage of EAE compared with the expression level in control animals (p<0.05) and declined thereafter. Immunofluorescent staining demonstrated that increased expression of arginase-1 in EAE spinal cords was confirmed in macrophages as well as in some neurons and astrocytes that were constitutively positive for arginase-1 in normal spinal cords. A semiquantitative analysis by immunofluorescence showed that in EAE lesions, an increased level of arginase-1 immunoreactivity was matched with ED1-positive macrophages, which were also positive for activin A, a marker for the M2 phenotype. Taking all of these findings into consideration, we postulate that the increased level of arginase-1, which is partly from M2 macrophages, contributes to the modulation of neuroinflammation in EAE lesions, possibly through the reduction of nitric oxide in the lesion via competition with iNOS for the use of L-arginine.

Suicide risk in relation to social class : A national register-based study of adult suicides in Korea, 1999-2001

Background: Few controlled studies have examined social class as a risk factor for suicide in Korea. Aim: The objective of the present study was to investigate the effects of social class on suicide risk in Korea. Methods: A case-control design was constructed from cause-of-death statistics for the period 1999 to 2001, in Korea, as published by the Korean National Statistical Office. The cases were defined as people aged between 20 and 64 who died by suicide, while the controls were defined as those who died of natural causes in the same age groups. Results and conclusions: The proportions and odds ratios for suicide were higher in young people than in elderly people, and higher for divorced subjects than for cohabitants. They were also higher for residents of rural areas, as opposed to residents of Seoul and other metropolitan areas, and for people in social classes III and IV, than they were for those in social class I. To control the variables that influence risk of suicide, such as age, marital status and area of residence, we used multiple logistic regression. Compared with class I, risk of suicide was higher in social classes III and IV, in both sexes. The principal conclusion of this study is that, regardless of sex, lower social class constitutes a high risk for suicide in Korea, even after controlling for variables such as age, marital status and area of residence. We conclude that a well-controlled and balanced social welfare system could reduce suicide risk, especially among people in lower social class.

Immunomodulatory Activity of Ginsan, a Polysaccharide of Panax Ginseng, on Dendritic Cells

Ginsan, a Panax ginseng polysaccharide that contains glucopyranoside and fructofuranoside, has immunomodulatory effects. Although several biologic studies of ginsan have been performed, its effects on dendritic cells (DCs), which are antigen-presenting cells of the immune system, have not been studied. We investigated the immunomodulatory effects of ginsan on DCs. Ginsan had little effect on DC viability, even when used at high concentrations. Ginsan markedly increased the levels of production by DCs of IL-12 and TNF-alpha, as measured by ELISA. To examine the maturation-inducing activity of ginsan, we measured the surface expression levels of the maturation markers MHC class II and CD86 (B7.2) on DCs. It is interesting that ginsan profoundly enhanced the expression of CD86 on DC surfaces, whereas it increased that of MHC class II only marginally. In (3)H-thymidine incorporation assays, ginsan-treated DCs stimulated significantly higher proliferation of allogeneic CD4(+) T lymphocytes than did medium-treated DCs. Taken together, our data demonstrate that ginsan stimulates DCs by inducing maturation. Because DCs are critical antigen-presenting cells in immune responses, this study provides valuable information on the activities of ginsan.

Differences in immunoreactivities of Ki-67 and doublecortin in the adult hippocampus in three strains of mice.

Neurogenesis in the adult hippocampus is differentially influenced by the genetic background. We examined the differences in Ki-67 (a proliferating cell marker) and doublecortin (DCX; an immature progenitor cell marker) immunolabelling in the dentate gyrus (DG) of the adult hippocampus in three strains of mice (ICR, C57BL/6, and BALB/c) to evaluate the effect of genetic background on adult hippocampal neurogenesis. All strains showed constitutive immunoreactivity of either Ki-67 or DCX in the DG of the adult hippocampus. C57BL/6 mice showed significantly higher levels of Ki-67-immunopositive cells in the subgranular zone (SGZ) of the DG (approximately 2.2-fold) compared to ICR and BALB/c mice. The greatest number of DCX-immunopositive cells was found in C57BL/6 (approximately 1.6-fold), which differed significantly from ICR and BALB/c mice. However, there was no significant difference in the number of Ki-67- and DCX-immunopositive cells between BALB/c and ICR mice. Genetic differences with respect to certain aspects of hippocampal neurogenesis in adult mice may influence hippocampal functions, including learning and memory.