Titanium Ions Promote Exogenous Calcium-Dependent Calcium Influx in Activated Jurkat T Cells: A Possible Mechanism to Explain Its Immunostimulatory Properties.

Titanium and its alloys have been widely used in dental and orthopedic implants. Owing to the biotribocorrosion behavior of implants in simulated oral environment, Ti(IV) ions could be released into surrounding tissues. Current studies have found that Ti(IV) ions could affect the biological activities of immune cells in adjacent tissues and subsequently jeopardize the long-term performance of implant prostheses. However, the potential mechanism underlying its immunomodulatory properties remains unclear. Calcium signaling has been confirmed to be involved in regulation of lymphocyte immune function. Therefore, we hypothesize that Ti(IV) ions modulated T cell function through the change of intracellular calcium concentrations. This study is aimed at exploring the role of intracellular calcium responses in the modulatory effect of Ti(IV) ions on unactivated and phytohemagglutinin-activated Jurkat T cells. Here, we confirmed that Ti(IV) ions within a certain concentration range induced CD69 expression on both unactivated and activated T cells in our study. Additionally, the combined stimulation with Ti(IV) ions and PHA increased expression of IL-1ß, TNF-α, and RANKL. Furthermore, we found that treatment with Ti(IV) induced a transitory increase in the levels of [Ca2+]i in activated Jurkat cells, dependent on the presence of exogenous calcium. Treatment with different doses of Ti(IV) for 24 h significantly increased the levels of [Ca2+]i in the activated Jurkat cells in a dose-dependent manner, but had little effect in the unactivated cells. Treatment with Ti(IV) did not significantly affect the PLCγ1 activation and inositol-1,4,5-trisphosphate (IP3) secretion in Jurkat cells. Taken together, these data indicated that Ti(IV) enhanced calcium influx during the T cell activation, independent of IP3-mediated intracellular calcium release. Our work provides insights into the mechanism involved in the regulation of lymphocyte behaviors under the effect of Ti(IV) ions, which may help to develop therapeutic strategies for dental implant failures.

Mitogenic activity of Artocarpus lingnanensis lectin and its apoptosis induction in Jurkat T cells.

Lectins are a class of carbohydrate-binding proteins or glycoproteins and used in the purification and characterization of glycoproteins according to their specificity to carbohydrates. In the present study, the mitogenic activity of Artocarpus lingnanensis lectin (ALL) and its apoptosis induction in Jurkat T cells were explored. MTT assay revealed strong mitogenic potential of ALL. Meanwhile, the anti-cancer activity of ALL was also explored using the human leukemic Jurkat T cell line. ALL exhibited strong binding affinity (97%) to the cell membrane, which could be effectively inhibited by N-acetyl-D-galactosaminide (NAD). ALL induced time- and dose-dependent growth inhibition in Jurkat T cells. ALL could induce morphologic change and increase the hypodiploid cell population with the decreased population of S and G2/M phases. The induction of phosphatidylserine externalization and PARP cleavage further confirmed its apoptosis-inducing activity due to the activation of caspase-8 and -9. The inhibition of caspase-9 but not caspase-8 could rescue ALL-induced apoptotic cells. Further studies showed that ALL enhanced the cleavage of Bid, the release of cytochrome C, the depolarization of mitochondria and the activation of caspase-3. ALL downregulated the expression of Bcl-xl and Bcl-2 without impact on Bax and Bad. In addition, the activation of p38/JNK MAPK signaling pathways was observed to be a requisite for ALL apoptotic activity. In contrast, ALL could not induce apoptosis of normal T cells. These findings present the differential effect of ALL on Jurkat and normal T lymphocytes, suggesting its therapeutic value in leukemia.

Vitamin E synthetic derivate-TPGS-selectively induces apoptosis in jurkat t cells via oxidative stress signaling pathways: implications for acute lymphoblastic leukemia.

D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) is a water-soluble derivative of natural vitamin E commonly used as a drug delivery agent. Recently, TPGS alone has been reported to induce cell death in lung, breast and prostate cancer. However, the effect of TPGS on cancer cell viability remains unclear. Thus, this study was aimed to evaluate the cytotoxic effect of TPGS on human periphral blood lymphocytes (PBL) and on T cell acute lymphocytic leukemia (ALL) Jurkat clone E6-1 cells and its possible mechanism of action. PBL and Jurkat cells were treated with TPGS (10, 20, 40, 60, and 80 µM), and morphological changes in the cell nucleus, mitochondrial membrane potential (ΔΨm), and intracellular reactive oxygen species levels were determined by immune-fluorescence microscopy and flow cytometry. Cellular apoptosis markers were also evaluated by immunocytochemistry. In this study, TPGS induced apoptotic cell death in Jurkat cells, but not in PBL, in a dose-response manner with increasing nuclear DNA fragmentation, increasing cell cycle arrest, and decreasing ΔΨm. Additionally, TPGS increased dichlorofluorescein fluorescence intensity, indicative of H2O2 production, in a dose-independent fashion. TPGS increased DJ-1 Cys(106)-sulfonate, as a marker of intracellular stress and induced the activation of NF-κB, p53 and c-Jun transcription factors. Additionally, it increased the expression of apoptotic markers Bcl-2 related pro-apoptotic proteins Bax and PUMAand activated caspase-3. The antioxidant N-acetyl-L-cysteine and known pharmacological inhibitors protected the cells from the TPGS induced effects. In conclusion, TPGS selectively induces apoptosis in Jurkat cells through two independent but complementary H2O2-mediated signaling pathways. Our findings support the use of TPGS as a potential treatment for ALL.

Clausmarin A, Potential Immunosuppressant Revealed by Yeast-Based Assay and Interleukin-2 Production Assay in Jurkat T Cells.

Small-molecule inhibitors of Ca2+-signaling pathways are of medicinal importance, as exemplified by the immunosuppressants FK506 and cyclosporin A. Using a yeast-based assay devised for the specific detection of Ca2+-signaling inhibitors, clausmarin A, a previously reported terpenoid coumarin, was identified as an active substance. Here, we investigated the likely mechanism of clausmarin A action in yeast and Jurkat T-cells. In the presence of 100 mM CaCl2 in the growth medium of Ca2+-sensitive Δzds1 strain yeast, clausmarin A exhibited a dose-dependent alleviation of various defects due to hyperactivation of Ca2+ signaling, such as growth inhibition, polarized bud growth and G2 phase cell-cycle arrest. Furthermore, clausmarin A inhibited the growth of Δmpk1 (lacking the Mpk1 MAP kinase pathway) but not Δcnb1 (lacking the calcineurin pathway) strain, suggesting that clausmarin A inhibited the calcineurin pathway as presumed from the synthetic lethality of these pathways. Furthermore, clausmarin A alleviated the serious defects of a strain expressing a constitutively active form of calcineurin. In the human Jurkat T-cell line, clausmarin A exhibited a dose-dependent inhibition of IL-2 production and IL-2 gene transcription, as well as an inhibition of NFAT dephosphorylation. The effects of clausmarin A observed in both yeast and Jurkat cells are basically similar to those of FK506. Our study revealed that clausmarin A is an inhibitor of the calcineurin pathway, and that this is probably mediated via inhibition of calcineurin phosphatase activity. As such, clausmarin A is a potential immunosuppressant.

Development of novel DIF-1 derivatives that selectively suppress innate immune responses.

The multiple pharmacological activities of differentiation-inducing factor-1 (DIF-1) of the cellular slime mold Dictyostelium discoideum led us to examine the use of DIF-1 as a 'drug template' to develop promising seed compounds for drug discovery. DIF-1 and its derivatives were synthesized and evaluated for their regulatory activities in innate immune responses. We found two new derivatives (4d and 5e) with highly selective inhibitory activities against production of the antimicrobial peptide attacin in Drosophila S2 cells and against production of interleukin-2 in Jurkat cells.

Induction of microtubule-damage, mitotic arrest, Bcl-2 phosphorylation, Bak activation, and mitochondria-dependent caspase cascade is involved in human Jurkat T-cell apoptosis by aruncin B from Aruncus dioicus var. kamtschaticus.

Exposure of human Jurkat T cells to aruncin B, purified from Aruncus dioicus, caused apoptosis along with microtubule damage, G(2)/M-arrest, Bcl-2 phosphorylation, Bak activation, mitochondrial membrane potential (Δψm) loss, cytochrome c release, activation of multiple caspases, and PARP degradation. Analyses by employing Bcl-2 overexpression and selective caspase inhibitors revealed that G(2)/M-arrest and Bcl-2 phosphorylation occurred prior to mitochondria-dependent activation of caspase-9, -3, and -8. The IC(50) values for human resting T cells, activated T cells, and Jurkat T cells were >60µg/ml, 49µg/ml, and 22µg/ml, respectively. These results demonstrate the apoptogenic activity of a novel microtubule-damaging agent aruncin B.

Size, charge and concentration dependent uptake of iron oxide particles by non-phagocytic cells.

A promising new direction for contrast-enhanced magnetic resonance (MR) imaging involves tracking the migration and biodistribution of superparamagnetic iron oxide (SPIO)-labeled cells in vivo. Despite the large number of cell labeling studies that have been performed with SPIO particles of differing size and surface charge, it remains unclear which SPIO configuration provides optimal contrast in non-phagocytic cells. This is largely because contradictory findings have stemmed from the variability and imprecise control over surface charge, the general need and complexity of transfection and/or targeting agents, and the limited number of particle configurations examined in any given study. In the present study, we systematically evaluated the cellular uptake of SPIO in non-phagocytic T cells over a continuum of particle sizes ranging from 33nm to nearly 1.5microm, with precisely controlled surface properties, and without the need for transfection agents. SPIO labeling of T cells was analyzed by flow cytometry and contrast enhancement was determined by relaxometry. SPIO uptake was dose-dependent and exhibited sigmoidal charge dependence, which was shown to saturate at different levels of functionalization. Efficient labeling of cells was observed for particles up to 300nm, however, micron-sized particle uptake was limited. Our results show that an unconventional highly cationic particle configuration at 107nm maximized MR contrast of T cells, outperforming the widely utilized USPIO (<50nm).

New constituents from the leaves of Morinda citrifolia.

A new iridoid glycoside, citrifoside (1), and a new anthraquinone, 1,5,15-trimethylmorindol (2), together with 24 known compounds, were isolated from the leaves of Morinda citrifolia. The structures of the new compounds were elucidated by spectral data. 1,5,15-Trimethylmorindol (2) did not show significant cytotoxic activity by itself but showed cytotoxicity when combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), while citrifoside (1) did not show any activity even with TRAIL.

An aqueous stem bark extract of Mangifera indica (Vimang) inhibits T cell proliferation and TNF-induced activation of nuclear transcription factor NF-kappaB.

A commercial aqueous stem bark extract of Mangifera indica L. (Vimang) has been reported to have antiinflammatory, immunomodulatory and antioxidant activities. The molecular basis for these diverse properties is still unknown. This study shows that a stem bark extract of M. indica inhibits early and late events in T cell activation, including CD25 cell surface expression, progression to the S-phase of the cell cycle and proliferation in response to T cell receptor (TCR) stimulation. Moreover, the extract prevented TNFalpha-induced IkappaBalpha degradation and the binding of NF-kappaB to the DNA. This study may help to explain at the molecular level some of the biological activities attributed to the aqueous stem bark extract of M. indica (Vimang).

Citrulline can preserve proliferation and prevent the loss of CD3 zeta chain under conditions of low arginine.

BACKGROUND: Arginine depletion by the enzyme Arginase I, decreases expression of the TCR zeta chain preventing T-cell activation and causing T-cell dysfunction. We hypothesized that citrulline could substitute for arginine under conditions of increased arginase expression. Thus, the goal was to establish a possible mechanism of how citrulline could overcome arginine depletion caused by arginase. METHODS: Jurkat cells were cultured, with or without arginase, in media containing different amino-acid constituents: complete RPMI containing arginine (C-RPMI) (arginine), Arginine-Free-RPMI (Arg-Free RPMI) and Citrulline-containing RPMI (Cit RPMI). Incorporation of citrulline was measured via uptake of 3H-citrulline, whereas proliferation was measured via 3H-thymidine incorporation. zeta Chain was analyzed by 2-color flow cytometry. Argininosuccinate synthase (AS) and argininosuccinate lyase expression was detected using Northern blots, RT-PCR, and Western blots. RESULTS: Jurkat cells exhibited a significant decrease in proliferation and 5 chain expression when cultured in the presence of arginase or in the absence of arginine. With citrulline, zeta chain expression and proliferation were maintained in the absence of arginine or in the presence of the enzyme arginase. Jurkat cells, cultured in the absence of arginine, were associated with a 5-fold increase in citrulline uptake. The absence of arginine was also associated with increased expression of AS. CONCLUSIONS: T cells exhibit the molecular capability of increasing citrulline membrane transport and up-regulating AS expression, thus exhibiting the necessary mechanisms for converting citrulline into arginine and escaping the ill effects of arginine depletion. Therefore, citrulline has the potential to be a substitute for supplemental arginine in diseases associated with arginase-mediated T cell dysfunction.

Paeoniflorin induces apoptosis of lymphocytes through a redox-linked mechanism.

Paeoniflorin (PF), isolated from paeony root, has been used as a herbal medicine for more than 1,200 years in China, Korea, and Japan for its anti-allergic, anti-inflammatory, and immunoregulatory effects. In this study, we found that PF induces apoptosis in both murine T-lineage cells and human T-cell leukemia Jurkat cells. This apoptosis was mediated through the reduction of mitochondrial membrane potential, activation of caspase, and fragmentation of DNA. Interestingly, PF induced generation of reactive oxygen species (ROS) and a reducing agent, dithiothreitol (DTT), and a ROS scavenger, N-acetyl cysteine (NAC), successfully attenuated the PF-induced apoptosis. Additionally, PF induced the phosphorylation of three mitogen-activated protein (MAP) family kinases, extracellular signal-regulated kinase, c-Jun amino-terminal kinase (JNK), and p38 MAP kinase. Curcumin, an anti-oxidant and JNK inhibitor, inhibited PF-induced apoptosis, suggesting the possible involvement of curcumin-sensitive JNK or other redox-sensitive elements in PF-induced apoptosis. These results partially explain the action mechanism of PF-containing paeony root as a herbal medicine.

Identification of genes differentially expressed in T cells following stimulation with the chemokines CXCL12 and CXCL10.

BACKGROUND: Chemokines are involved in many biological activities ranging from leukocyte differentiation to neuronal morphogenesis. Despite numerous reports describing chemokine function, little is known about the molecular changes induced by cytokines. METHODS: We have isolated and identified by differential display analysis 182 differentially expressed cDNAs from CXCR3-transfected Jurkat T cells following treatment with CXCL12 or CXCL10. These chemokine-modulated genes were further verified using quantitative RT-PCR and Western blot analysis. RESULTS: One hundred and forty-six of the cDNAs were successfully cloned, sequenced, and identified by BLAST. Following removal of redundant and non-informative clones, seventeen mRNAs were found to be differentially expressed post treatment with either chemokine ligand with several representing known genes with established functions. Twenty-one genes were upregulated in these transfected Jurkat cells following both CXCL12 and CXCL10, four genes displayed a discordant response and seven genes were downregulated upon treatment with either chemokine. Identified genes include geminin (GEM), thioredoxin (TXN), DEAD/H box polypeptide 1 (DDX1), growth hormone inducible transmembrane protein (GHITM), and transcription elongation regulator 1 (TCERG1). Subsequent analysis of several of these genes using semi-quantitative PCR and western blot analysis confirmed their differential expression post ligand treatment. CONCLUSIONS: Together, these results provide insight into chemokine-induced gene activation and identify potentially novel functions for known genes in chemokine biology.

[Establishment of high-throughput drug screening cell models based on JAK-STAT signal pathway].

AIM: To discover new drugs which may be applied to diseases of the immune system, hemogenesis system diseases and tumors, several high-throughput drug screening cell models based on JAK-STAT signal pathway have been established. METHODS: Four repeats of STAT DNA binding conserved sequences were synthesized, subcloned into pGL-Luc reporter vector and stably transfected into cell lines in vitro. Cell clones with high copy numbers of STAT binding sites and reporter genes were chosen as high-throughput drug screening cell models. The cell models were tested with known anti-allergic drugs and anti-tumor drugs by determining luciferase activity. The reaction was performed in 96 well micro-plates with a final volume of 50 microL. RESULTS: The cell models by performing rapid fluorescence assay were shown to be highly sensitive and stable after testing with cytokine and drugs. The modification of the expression plasmid simplified this method and made it more practical. It also provided good linear correlation, wide range of assay, highly sensitive and good reproducibility. CONCLUSION: The method can be performed by high-throughput drug screening for effective extraction of Chinese traditional herbs.

Domain structure of bi-functional selenoprotein P.

Human selenoprotein P (SeP), a selenium-rich plasma glycoprotein, is presumed to contain ten selenocysteine residues; one of which is located at the 40th residue in the N-terminal region and the remaining nine localized in the C-terminal third part. We have shown that SeP not only catalyses the reduction of phosphatidylcholine hydroperoxide by glutathione [Saito, Hayashi, Tanaka, Watanabe, Suzuki, Saito and Takahashi (1999) J. Biol. Chem. 274, 2866-2871], but also supplies its selenium to proliferating cells [Saito and Takahashi (2002) Eur. J. Biochem. 269, 5746-5751]. Treatment of SeP with plasma kallikrein resulted in a sequential limited proteolysis (Arg-235-Gln-236 and Arg-242-Asp-243). The N-terminal (residues 1-235) and C-terminal (residues 243-361) fragments exhibited enzyme activity and selenium-supply activity respectively. These results confirm that SeP is a bi-functional protein and suggest that the first selenocysteine residue is the active site of the enzyme and the remaining nine residues function as a selenium supplier.

Black tea extract supplementation decreases oxidative damage in Jurkat T cells.

The purpose of this study was to investigate the protective effect of black tea (BT) extract against induced oxidative damage in Jurkat T-cell line. Cells supplemented with 10 or 25 mg/L BT were subjected to oxidation with ferrous ions. Malondialdehyde (MDA) production as marker of lipid peroxidation, DNA single strand breaks as marker of DNA damage, and modification of the antioxidant enzyme activity, glutathione peroxidase (GPX) were measured. Results show the efficacy of BT polyphenols to decrease DNA oxidative damage and to affect GPX activity (P<0.05), while no effect was shown on MDA production. The succeeding investigation of the activity of caffeine and epigallocatechin gallate demonstrated their antioxidant potential with respect to the cellular markers evaluated. In conclusion, this study supports the protective effect of BT against ferrous ions induced oxidative damage to DNA and the ability of BT to affect the enzyme antioxidant system of Jurkat cells.

Characterization of a real time H2O2 monitor for use in studies on H2O2 production by antibodies and cells.

It was recently shown that antibodies catalyze a reaction between water and ultraviolet light (UV) creating singlet oxygen and ultimately H2O2. Although the in vivo relevance of these antibody reactions is unclear, it is interesting that among a wide variety of non-antibody proteins tested, the T cell receptor is the only protein with similar capabilities. In clinical settings UV is believed to exert therapeutic effects by eliminating inflammatory epidermal T cells and we hypothesized that UV-triggered H2O2 production is involved in this process. To test the hypothesis we developed tools to study production of H2O2 by T cell receptors with the long-term goal of understanding, and improving, UV phototherapy. Here, we report the development of an inexpensive, real time H2O2 monitoring system having broad applicability. The detector is a Clark oxygen electrode (Pt, Ag/AgCl) modified to detect UV-driven H2O2 production. Modifications include painting the electrode black to minimize UV effects on the Ag/AgCl electrode and the use of hydrophilic, large pore Gelnots electrode membranes. Electrode current was converted to voltage and then amplified and recorded using a digital multimeter coupled to a PC. A reaction vessel with a quartz window was developed to maintain constant temperature while permitting UV irradiation of the samples. The sensitivity and specificity of the system and its use in cell-free and cell-based assays will be presented. In a cellfree system, production of H2O2 by CD3 antibodies was confirmed using our real time H2O2 monitoring method. Additionally we report the finding that splenocytes and Jurkat T cells also produce H2O2 when exposed to UV light.

Low-dose doxorubicin-induced necrosis in Jurkat cells and its acceleration and conversion to apoptosis by antioxidants.

We treated rapidly growing Jurkat cells with 40 nmol/l of doxorubicin for 72 h. After 36 h, the G2-arrested cells became larger and some of them started endoreplication. Nuclear staining with Hoechst 33342 combined with propidium iodide (PI) exclusion revealed that about 90% of the cells were necrotic at 72 h, although apoptotic cells accounted for only 8%. Incubation with 40 nmol/l of aclarubicin or cytosine beta-d-arabinofuranoside for 60 h induced necrosis both in Jurkat and ml-1 cells. Pre-necrotic Jurkat cells incubated with 40 nmol/l of doxorubicin had much higher intracellular reactive oxygen species (ROS) levels than pre-apoptotic ones. Addition of Tempol or Desferal accelerated doxorubicin-induced necrosis and partially converted it into apoptosis. Both antioxidants reduced surviving colony numbers of prenecrotic Jurkat cells. n-acetyl-l-cysteine had little effect on the apoptotic conversion but profoundly accelerated necrosis. Because an apoptosis-resistant Jurkat subclone was also refractory to doxorubicin-induced necrosis, apoptosis and necrosis might share some common pathways. Low-dose doxorubicin increased micronuclei-positive cell percentages and also suppressed high-dose doxorubicin-induced apoptosis in Jurkat and ml-1 cells. Some of the prenecrotic cells, therefore, might survive and obtain genomic instability. Antioxidants may be useful to suppress, at least to some extent, this vicious consequence.

A role for oxidative stress in apoptosis: oxidation and externalization of phosphatidylserine is required for macrophage clearance of cells undergoing Fas-mediated apoptosis.

Exposure of phosphatidylserine (PS) on the surface of apoptotic cells has been suggested to serve as an important recognition signal for macrophages. In this work we show that triggering of the death receptor Fas on Jurkat cells results in the generation of reactive oxygen species with oxidation and externalization of PS but not of the other major aminophospholipid, phosphatidylethanolamine. These cells were readily ingested by several classes of macrophages, whereas Raji cells, which are defective for Fas-induced PS exposure, remained unengulfed. However, when Raji cells were incubated with the thiol-reactive agent N-ethylmaleimide to induce PS exposure in the absence of other features of apoptosis, these cells were also engulfed by macrophages. Phagocytosis of Fas-triggered Jurkat cells was inhibited by superoxide dismutase and catalase, which prevent oxidation of PS while allowing PS to remain externalized on these cells. Moreover, liposomes containing oxidized PS (PS-OX) were more potent inhibitors of phagocytosis than those containing its nonoxidized counterpart. Finally, enrichment of the plasma membrane of Jurkat or Raji cells, or myeloid leukemic HL-60 cells, with exogenous PS resulted in phagocytic cell clearance, and this process was further enhanced when PS was substituted for by PS-OX. Taken together, our data suggest that the presence of PS-OX in conjunction with nonoxidized PS on the cell surface is an important signal for macrophage clearance of apoptotic cells.

Ajoene, an experimental anti-leukemic drug: mechanism of cell death.

The organosulfur compound ajoene, a constitutent of garlic, has been shown to induce apoptosis in a leukemic cell line as well as in blood cells of a leukemic patient. The mechanisms of action of ajoene, however, are unknown. The present study aims to characterize the molecular events leading to ajoene-triggered apoptosis. We show here that ajoene (20 microM) leads to a time-dependent activation of caspase-3-like activity as well as to the proteolytic processing of procaspase-3 and -8. Activation of caspases was necessary for ajoene-induced apoptosis since the broad-range caspase inhibitor zVAD-fmk completely abrogated ajoene-mediated DNA fragmentation. Although the initiator caspase-8 was activated, the CD95 death receptor was not involved in death signaling since the HL-60 clone used was shown to express a functionally inactive CD95 receptor. Furthermore, ajoene induced the release of cytochrome c, which was not inhibited by zVAD-fmk indicating that cytochrome c release precedes caspase activation. Ajoene also led to a dissipation of the mitochondrial transmembrane potential. Overexpression of Bcl-x(L) clearly diminished ajoene-induced caspase activation as well as apoptosis. These results indicate that apoptosis in leukemia cells triggered by ajoene is based on the activation of a mitochondria-dependent caspase cascade which includes also the activation of the initiator caspase-8.

Delayed neurodegeneration in neonatal rat thalamus after hypoxia-ischemia is apoptosis.

Brain injury in newborns can cause deficits in motor and sensory function. In most models of neonatal brain injury, thalamic damage often occurs. Using the Rice-Vannucci model of neonatal hypoxic-ischemic brain injury, we have shown that neuronal degeneration in somatosensory thalamus is delayed in onset ( approximately 24 hr) compared with cortical and striatal injury and exhibits prominent structural features of apoptosis. In the present study, we examined whether cell death in the thalamus has molecular features of apoptosis. Fas death receptor protein expression increased rapidly after neonatal hypoxia-ischemia, in concert with cleavage of procaspase 8 to its active form. Concurrently, the levels of Bax in mitochondrial-enriched cell fractions increase, and cytochrome c accumulates in the soluble fraction. Mitochondria accumulate in a perinuclear distribution by 6 hr after hypoxia-ischemia. Cytochrome oxidase subunit 1 protein levels also increase at 6 hr after hypoxia-ischemia. Increased levels of Fas death receptor, Bax, and cytochrome c, activation of caspase 8, and abnormalities in mitochondria in the thalamus significantly precede the activation of caspase 3 and the appearance of neuronal apoptosis at 24 hr. We conclude that the delayed neurodegeneration in neonatal rat ventral basal thalamus after hypoxic-ischemic injury is apoptosis mediated by death receptor activation.