• IMMUNE NETWORK
• /
• v.4 no.2
• /
• pp.81-87
• /
• 2004

Background: In the thymus, developing thymocytes continually interact with thymic epithelial cell components. Self MHC restriction of mature T cells are imposed in the thymus through interaction of immature double positive thymocytes and thymic cortical epithelial cells. The site of negative selection, however, is a matter of debate. Through systemic injection of anti-TCR antibody or antigenic peptides, investigators suggested that most of the negative selection occurs in the thymic cortex. But the requirements for negative selection, i.e cellular counterparts and costimulatory molecules are more available in the medulla or cortico-medullary junction rather than in the thymic cortex. Methods: The direct and indirect pathways of thymocyte death after systemic anti-TCR antibody injection were separated through several experimental systems. B6 mice were either adrenalectomized or sham-adrenalectomized to evaluate the role of endogenous glucocorticoids from adrenal gland. Role of TNF were evaluated through using TNF receptor double knockout mice. Results: We found that without indirectly acting mediators such as $TNF-\alpha$ or corticosteroid, double positive thymocyte death were minimal by systemic injection of anti-TCR antibody in TNF receptor double knockout neonatal mice. Also by analyzing neonatal wild-type mice with adoptively transferred mature T cells, only peripheral activation of mature T cells could induce extensive double positive thymocyte death. Conclusion: Thus, systemically injected anti-TCR antibody mediated thymocyte death are mostly induced through indirect pathway.

• Journal of Ginseng Research
• /
• v.21 no.3
• /
• pp.160-168
• /
• 1997

The present study was carried out to investigate the effects of Panax ginseng saponin extracts on the dexamethasone-induced apoptosis of mouse thymus in vivo and mouse thymocytes in vitro. The saponin fractions of red ginseng (R-SAP) and white ginseng (Wl-SAP) were provided by the Korea Ginseng & Tobacco Research Institute, and the other saponin fraction of white ginseng (W2-SAP) was extracted in our laboratory. 1. The male ICR mice (3~4 wk old; weighing 15$\pm$2 g) were given by each saponin fraction of 5 mg/kg/ day for 4 days, and at one hour after the last treatment, they were injected by deuamethasone (5 mg/kg : DX). The mouse thymus was extracted at 6 hours after DX injection, and they were stained with hematoxylin-eosin reagents and an Apop-Tag kit, respectively, and the thymocytes prepared from it were labelled with anti-mouse FITC-anti-CD4 and anti-mouse PE-anti-CD8 and then analyzed by fluorescence activated cell sorter (FACS). DX-induced reduction of thymus weight was significantly attenuated by W2- SAP but was not affected by other saponin fractions. And DX-induced apoptotic death of thymocytes, appeared in the histologic findings of the thymus, was inhibited by the saponin fractions and the order of these inhibitory potencies was R-SAP》W2-SAP>Wl-SAP. However, in respect of T cell receptors, the differentiation of thymocytes seems not to be changed by treatments with DX or/and the saponin fractions. 2. In the primary thymocyte culture, the DX-induced reduction of thymocyte MTT values was rather greater in RPMI 1640 medium of IWc fetal bovine serum (FBS) or horse serum (HS). In addition, the DX-Induced MTT reduction was significantly inhibited by R-SAP or W2-SAP, in the culture using that medium of 5% FBS or HS. But these saponin fraction did not effected the DX-induced reduction of thymocyte MTT value in primary culture of 10% FBS or 10% HS. These results suggest that R-SAP and some W-SAP fractions may protect thymocyte from stress or glucocorticoisteroid-induced death of them.

• YAKHAK HOEJI
• /
• v.46 no.1
• /
• pp.69-74
• /
• 2002

The effects of genistein on murine thymocytes for inducing apoptotic cell death and phagocytic activity of peritoneal macrophage were studied in vitro. Addition of genistein (10 and 50$\mu$M) to cultured thymocytes from BALB/c mice definitely promoted DNA fragmentation. Also, cytofluorometric analysis of these cells demonstrated a reduction in mitochondrial transmembrane potential ($\Delta$Ψm). But, repeated administration of genistein (1 mg/mouse/day) to mice for 7 days did not cause any detectable DNA fragmentation. Genistein decreased lucigenin chemiluminescence and engulfment of fluorescein-conjugated E. coli particles in peritoneal macrophage. These results suggest that genistein induce an apoptosis of thymocyte via reduction in $\Delta$Ψm and decrease phagocytic activity of peritoneal macrophage in vitro.

• The Korean Journal of Pharmacology
• /
• v.32 no.1
• /
• pp.113-123
• /
• 1996

It has been well known that polyamines ensure the stability of chromatin structure and the fidelity of DNA transcription. This study was carried out to evaluate the effect of polyamines on the apoptosis of mouse thymocytes induced by dexamethasone and polyamine synthesis inhibitors. 1) In the histological death findings of thymocytes double-stained with acridine orange and ethidium bromide, the apoptotic and the necrotic fractions (AF; NF) in the control group were $9.4{\pm}4.2%$ and $4.5{\pm}5.3%$, respectively. Dexamethasone $(3\;{\times}\;10^{-8}\;M:\;DX)$ in creased AF upto $52.0{\pm}8.1%$ and did not change NF, but A23187 $(5\;{\times}\;10^{-7}\;M:\;A2)$ increased AF and NF upto $45.0{\pm}8.9%$ and $20.5{\pm}10.6%$, respectively. 2) The thymocyte viability was significantly reduced by DX, DHEA $(1\;{\times}\;10^{-4}\;M)$, A2, DFMO $(1\;{\times}\;10^{-4}\;M)$, and $MGBG\;(1\;{\times}\;10^{-4}\;M)$, respectively. It was, however, little affected by $aminoguanidine\;(1\;{\times}\;10^{-4}\;M:\;AG)$, $putrescine\;(1\;{\times}\;10^{-5}\;M:\;PT)$, $spermidine\;(1\;{\times}\;10^{-5}\;M:\;SD)$, and $spermine\;(1\;{\times}\;10^{-5}\;M:\;SM)$. 3) The genomic DNA of mouse thymocyte was markedly fragmented by DX and A2, respectively, and to a lesser extent, by DHEA, but was little affected by MGBG, DFMO, AG, and each of polyamines. 4) The DX induced reduction of thymocyte viability was moderately attenuated by DHEA, but little affected by DFMO, MGBC, and AG. However, SM significantly attenuated the viability reduction induced by A2 as well as DX. 5) The thymocyte viability reduction by MGBG and DFMO was significantly attenuated by only SM among three polyamines applied in this study. 6) The thymocyte viability redution by combined treatments of DX with DFMO and MGBG, respectively, was significantly attenuated by SM, and moderately by PT. But the viability reduction by combined treatment of DX with AG or DHEA was not affected by polyamines. These results suggest that polyamines, particularly spermine, might play the inhibitory role in thymocyte apoptosis and the inhibitory effect can be ascribed in part to the increase of polyamine uptake by thymocytes pretreated with DFMO and MGBG.

• Toxicological Research
• /
• v.13 no.1_2
• /
• pp.39-48
• /
• 1997

Cyclophosphamide(25, 50 or 100 mg/kg), orally administered to male Sprague-Dawley rats, caused a time- and dose-dependent thymic atrophy. In the light microscopic examination of the atrophic thymus, thymocytes with condensed or fragmented nucleus were multifocally observed in the cortical region, started to increase 8 hr after CPA treatment and reached to the maximal level at 16 hr, although such cells were not seen after 48 hr when the severe depletion of thymocytes were marked. In agarose gel electrophoresis to analyze the DNA changes, DNA extracted from atrophic thymus showed a oligonucleosomal laddering at the corresponding time to morphological changes. In an additional supportive experiment, thymocytes showing morphological changes, nuclear condensation or apoptotic body, exhibited a positive reaction to immunoperoxidase staining using in situ apoptosis detection kit. Separately, agarose gel electrophoresis of DNA from bone .marrow cells was performed to investigate the involvement of bone marrow cells in the process of thymocyte apoptosis. Although DNA laddering was slightly increased 2 and 4 hr after treatment, no clear correlation was inferred. Taken togather, it is concluded that thymocytes showing morphological changes in thymic atrophy induced by cyclophosphamide administration represent an apoptosis having biochemical nature of programmed cell death.

• Animal cells and systems
• /
• v.5 no.4
• /
• pp.267-274
• /
• 2001

Calpains are a family of cysteine proteases existing primarily in two forms designated by the $Ca^{2+}$ concentration needed for activation in vitro, $\mu$-calpain (calpain-I) and m-calpain (calpain-II). The physiologica1 roles of calpains remain unclear. Many groups have proposed a role for calpains In apoptosis, but their patterns of activation are not well characterized. Calpains have been implicated in neutrophil apoptosis, glucocorticoid-induced thymocyte apoptosis, as well as many other apoptotic pathways. Calpain activation in apoptosis is usually linked upstream or downstream to caspase activation, or in a parallel pathway alongside caspase activation. Calpains have been suggested to be involved in DNA fragmentation (via endonuclease activation), but also as effector proteases that cleave cellular proteins involved in DNA repair, membrane associated proteins and other homeostatic regulatory proteins. Recently, our laboratory demonstrated $\mu$-calpain activation in NAD(P)H: quinone oxidoreducatse 1 (NQO1)-expressing cells after exposure to $\beta$-lapachone, a novel quinone and potential chemo- and radio-therapeutic agent. Increased cytosolic $Ca^{2+}$ in NQO1-expressing cells after $\beta$-lapachone exposures were shown to lead to $\mu$-calpain activation. In turn, $\mu$-calpain activation was important for substrate proteolysis and DNA fragmentation associated with apoptosis. Upon activation, $\mu$-calpain translocated to the nucleus where it could proteolytically cleave PARP and p53. We provided evidence that $\beta$-lapachone-induced, $\mu$-calpain stimulated, apoptosis did not involve any of the known caspases; known apoptotic caspases were not activated after $\beta$-lapachone treatment of NQO1-expressing cells, nor did caspase inhibitors have any effect on $\beta$-1apachone-induced cell death. Elucidation of processes by which $\beta$-1apachone-stimulated $\mu$-calpain activation and calpains ability to activate endonucleases and induce apoptosis independent of caspase activity will be needed to further develop/modulate $\beta$-lapachone for treatment of human cancers that over-express NQO1.