A naturally processed mitochondrial self-peptide in complex with thymic MHC molecules functions as a selecting ligand for a viral-specific T cell receptor.

Peptide fragments of self-proteins bound to major histocompatibility complex molecules within the thymus are important for positively selecting T cell receptor (TCR)-bearing CD4(+)CD8(+) double positive (DP) thymocytes for further maturation. The relationship between naturally processed thymic self-peptides and TCR-specific cognate peptides is unknown. Here we employ HPLC purification of peptides released from H-2K(b) molecules of the C57BL/6 thymus in conjunction with mass spectrometry (MS) and functional profiling to identify a naturally processed K(b)-bound peptide positively selecting the N15 TCR specific for the vesicular stomatitis virus octapeptide (VSV8) bound to K(b). The selecting peptide was identified in 1 of 80 HPLC fractions and shown by tandem MS (MS/MS) sequencing to correspond to residues 68-75 of the MLRQ subunit of the widely expressed mitochondrial NADH ubiquinone oxidoreductase (NUbO(68-75)). Of note, the peptide differs at six of its eight residues from the cognate peptide VSV8 and functions as a weak agonist for mature CD8 single positive (SP) N15 T cells, with activity 10,000-fold less than VSV8. In N15 transgenic (tg) recombinase activating gene 2(-/)- transporter associated with antigen processing 1(-/)- fetal thymic organ culture, NUbO(68-75) induces phenotypic and functional differentiation of N15 TCR bearing CD8 SP thymocytes. Failure of NUbO(68-75) to support differentiation of a second K(b)-restricted TCR indicates that its inductive effects are not general.

Redox control of resistance to cis-diamminedichloroplatinum (II) (CDDP): protective effect of human thioredoxin against CDDP-induced cytotoxicity.

Thioredoxin is a small ubiquitous protein with multiple biological functions, including cellular defense mechanisms against oxidative stress. In the present study, we investigated the role of human thioredoxin (hTRX) in the acquisition of cellular resistance to cis-diamminedichloroplatinum (II) (CDDP). The expression and activity of hTRX in Jurkat T cells was dose-dependently enhanced by exposure to CDDP, as determined by immunoblot analysis and insulin reducing assay. Furthermore, chloramphenicol acetyltransferase analysis using the hTRX promoter-reporter gene construct revealed that treatment of Jurkat cells with CDDP caused transcriptional activation of the hTRX gene, which might be mediated through increased generation of intracellular reactive oxygen intermediates. To examine the biological significance of hTRX induction, we established hTRX-overexpressing derivatives of L929 fibrosarcoma cells by stable transfection with the hTRX cDNA. The clones, which constitutively expressed the exogenous hTRX, displayed increased resistance to CDDP-induced cytotoxicity, compared with the control clones. After exposure to CDDP, the control cells showed a significant increase in the intracellular accumulation of peroxides, whereas the hTRX-transfected cells did not. Taken together, these results suggest that overexpressed hTRX is responsible for the development of cellular resistance to CDDP, possibly by scavenging intracellular toxic oxidants generated by this anticancer agent.

Role of intracellular redox status in apoptosis induction of human T-cell leukemia virus type I-infected lymphocytes by 13-cis-retinoic acid.

We have shown that cell cycle progression of human T-cell leukemia virus type I (HTLV-I)-transformed T-cell lines was inhibited by 13-cis-retinoic acid (13cRA). In the present study, we report that 13cRA inhibited proliferation and induced cell death of peripheral blood mononuclear cells obtained from four patients with acute adult T-cell leukemia but not of mitogen- or interleukin 2-activated peripheral blood mononuclear cells from HTLV-I-negative healthy donors. Because HTLV-I-infected lymphocytes are susceptible to oxidative stress, we examined the role of the intracellular redox state in 13cRA-induced cell death using a HTLV-I-positive T-cell line, ATL2, as a model. 13cRA induced apoptosis in ATL2 cells within 48 h in a dose-dependent manner. The ability of 13cRA to induce apoptosis was more potent than that of all-trans-retinoic acid. Apoptosis induction by 13cRA was significantly enhanced by buthionine sulfoximine (BSO), which decreased the levels of intracellular reduced glutathione, although 13cRA by itself did not alter them, suggesting that intracellular reduced glutathione may modulate 13cRA-induced apoptosis. In addition, flow cytometric analysis revealed that 13cRA increased intracellular peroxides in 24 h and that the addition of BSO further enhanced them. Although N-acetylcysteine had only a marginal effect, pretreatment with catalase markedly inhibited 13cRA-induced apoptosis. These results suggest that peroxide generation, ie., oxidative stress, may play a crucial role in the induction of apoptosis by 13cRA and further demonstrate that combined treatment with 13cRA and BSO induces apoptosis of HTLV-I-positive lymphocytes even more potently.

Analysis of the inhibition of mammalian thioredoxin, thioredoxin reductase, and glutaredoxin by cis-diamminedichloroplatinum (II) and its major metabolite, the glutathione-platinum complex.

Several studies have demonstrated a correlation between cellular toxicity of cis-diamminedichloroplatinum (II) (cisplatin, CDDP) and inhibited intracellular activity of the thioredoxin system, i.e., thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH. Conversely, increased cellular activity of the Trx system confers resistance to CDDP. In this study, we have analyzed the interaction of CDDP with Trx and TrxR in order to clarify the mechanism. The inhibition with time-dependent kinetics by CDDP of NADPH-reduced (but not oxidized) TrxR was irreversible, strongly suggesting covalent modification of the reduced selenocysteine-containing active site. Assuming second order kinetics, the rate constant of TrxR inhibition by CDDP was 21 +/- 3 M(-1) x s(-1). Transplatin was found to be an even more efficient inhibitor, with a second order rate constant of 84 +/- 22 M(-1) x s(-1), whereas carboplatin (up to 1 mM) gave no inhibition of the enzyme under the same conditions. Escherichia coli Trx or human or bacterial glutaredoxin (Grx) activities were in comparison only slightly or not at all inhibited by either CDDP, transplatin, or carboplatin. However, glutaredoxins were found to be inhibited by the purified glutathione adduct of cisplatin, bis-(glutathionato)platinum(II) (GS-Platinum complex, GS-Pt), with an IC50 = 350 microM in the standard beta-hydroxyethyl disulfide-coupled assay for human Grx. Also the mammalian Trx system was inhibited by GS-Pt with similar efficiency (IC(50) = 325 microM), whereas neither the E. coli Trx system nor glutathione reductase were inhibited. Formation of GS-Pt is a major route for cellular elimination of CDDP. The fact that GS-Pt inhibits the mammalian Trx as well as Grx systems shows that CDDP may exert effects at several stages of its metabolism, including after conjugation with GSH, which are intimately linked with the cellular disulfide/dithiol redox regulatory systems.

Possible involvement of thioredoxin reductase as well as thioredoxin in cellular sensitivity to cis-diamminedichloroplatinum (II).

The thioredoxin (TRX) system, composed of nicotinamide adenine dinucleotide phosphate (reduced form), TRX, and TRX reductase (TRXR), has multiple biologic functions via thiol-mediated redox control. In this study, we investigated the relationship between intracellular TRXR levels and cellular sensitivity to cis-diamminedichloroplatinum (II) (CDDP). HeLa, a human cervical carcinoma cell line, cultured with CDDP showed a time- and dose-dependent reduction of intracellular TRXR activity, which was well correlated with the decrease in cell viability after exposure to CDDP. In a cell-free system, CDDP was found to directly inactivate the reduced form of purified human TRXR. The CDDP-resistant variants of HeLa cells, established by continuous exposure to CDDP, exhibited an increased expression and activity of TRXR as well as TRX compared with the parental cells. In addition, sodium selenate, an inhibitor of TRXR, was found to increase the susceptibility to CDDP in the CDDP-resistant cells. Moreover, the HeLa cells transfected with an antisense TRXR RNA expression vector to reduce the intracellular enzyme activity displayed an enhanced sensitivity to CDDP. Taken together with previous reports on TRX, these results indicate the possible involvement of TRXR as well as TRX in the cellular sensitivity and resistance to CDDP.

Secretion of thioredoxin enhances cellular resistance to cis-diamminedichloroplatinum (II).

Thioredoxin (TRX) is a redox-active protein induced by a variety of stress and secreted from cells. Collecting evidence revealed that extracellular TRX shows cytokine- and chemokine-like activities. In the present study, we studied the role of secreted TRX on cellular resistance to cis-diamminedichloroplatinum (II) (CDDP). The CDDP-resistant variants of HeLa cells not only have enhanced expression of intracellular TRX, but also show increased secretion of TRX into the culture medium, compared to the parental cells. The CDDP-resistant cells also exhibit an enhanced L-cystine uptake capability, which results in a significant increase in the intracellular sulfhydryl content, including glutathione (GSH). Exogenous administration of recombinant TRX (rTRX) increases cellular resistance to CDDP and augments the L-cystine uptake in the parental HeLa cells. Moreover, depletion of L-cystine from the culture medium or combined treatment with L-cystine uptake inhibitors increases cellular sensitivity to CDDP in the CDDP-resistant cells. These findings suggest that secreted TRX may play an important role in the acquisition of cellular CDDP resistance through enhancement of the L-cystine uptake activity, and that the L-cystine transport system, as well as the TRX system, may be a novel therapeutic target in CDDP-resistant cancer cells.

Redox control of Epstein-Barr virus replication by human thioredoxin/ATL-derived factor: differential regulation of lytic and latent infection.

Human thioredoxin (hTRX)/adult T-cell leukemia (ATL)-derived factor (ADF) was originally reported as an interleukin-2 (IL-2) receptor-alpha-inducing factor produced by human T-cell lymphotropic virus-1-positive (HTLV-1+) cell lines. Growing evidence indicates that hTRX/ADF plays important roles in cellular responses against oxidative stress and is involved in a variety of cellular functions. A high level of hTRX/ADF expression is also observed in other human virus-infected cell lines including those of Epstein-Barr virus (EBV) and human papillomavirus. In this report, we analyzed the effect of hTRX/ADF on lytic amplification and persistent replication of EBV as a model for lytic versus latent phase of viral replication in host cells. Addition of hTRX/ADF clearly suppressed lytic replication of EBV in Raji cells and B95-8 cells induced to the lytic phase of 12-O-tetradecanoylphorbol-13-acetate (TPA), and it prevented the death of these cells evoked by the lytic induction. In contrast, hTRX/ADF did not have any effect on persistent replication in the latent phase. These data indicated that hTRX/ADF prevents EBV-transformed cells from proceeding into the lytic phase and regulates cohabitation of EBV and its host cells.

Transactivation of an inducible anti-oxidative stress protein, human thioredoxin by HTLV-I Tax.

Adult T-cell leukemia derived factor (ADF)/human thioredoxin (TRX), which has thiol reducing and radical scavenging activities, plays an essential role on cellular protection against oxidative stress and cell death. TRX itself is induced by various oxidative stress as well as the Human T-cell lymphotropic virus type I (HTLV-I) Tax protein. To investigate the mechanism of this induction, the promoter region of the TRX gene was analyzed. Chloramphenicol acetyltransferase (CAT) reporter constructs containing the TRX promoter sequences responded to the overexpression of the Tax protein, whereas various oxidative agents activated the TRX promoter through a newly identified oxidative responsive element. Moreover, TRX was translocated from the cytoplasm into the nucleus by ultraviolet irradiation, suggesting its possible role on sensing and transducing oxidative signals.

Expression of thioredoxin in bleomycin-injured airway epithelium: possible role of protection against bleomycin induced epithelial injury.

Bleomycin (BLM) is an anticancer drug, administration of which leads to severe lung injury, in which the generation of intracellular reactive oxygen species (ROS) is thought to participate in that. Thioredoxin (TRX) has been found to function as a powerful antioxidant by reducing ROS, and thus protecting against ROS-mediated cytotoxicity. However, a protective role of TRX in BLM-induced lung injury has not been determined. In the present study, we therefore attempted to clarify this issue. Human TRX-transfected L929 murine fibrosarcoma cells were more resistant to BLM-induced cytotoxicity than the parental and the control transfected cells, indicating that TRX plays the protective role in BLM-induced cytotoxicity. Next, we examined TRX expression in the lung of in vivo model of BLM-induced lung injury and BLM-stimulated bronchial epithelial cells in vitro to clarify the role of TRX in BLM-induced lung injury. In the lungs of BLM-treated mice, the expression of TRX was strongly induced in bronchial epithelial cells. TRX expression was also up-regulated at both the mRNA and protein levels in cultured BEC with the treatment with BLM. However, the expression of other major antioxidants, such as Cu/Zn-SOD, Mn-SOD, catalase and glutathione peroxidase, was not affected by BLM. These observations suggest that the cellular reduction and oxidation (redox) state modified by TRX is involved in the BLM resistancy and the induction of TRX in bronchial epithelial cells might play a protective role in BLM-induced lung injury.

[Accuracy of the measurements of left ventricular volume and ejection fraction determined by multigated blood pool tomography].

Accuracy of the measurements of LV volumes and ejection fraction determined from multigated blood pool tomography (MGBPT) was tested in 23 patients with various heart diseases who underwent cineventriculography (CV). Preliminary phantom studies showed that a 42% threshold value was found to provide the best relationship between measured and actual volumes (r = 0.99, standard error of the estimate (SEE) = 4.7 ml). The patients studies were performed at 16 frames/cardiac cycle at 18 angles over a 180 degrees rotation for one minute per angle. Long-axis horizontal views were reconstructed. The numbers of LV voxel with counts above the threshold value were summed and multiplied by the known volume of a voxel. Measurements of LV volume (r = 0.82, SEE = 29 ml), end-systolic LV volume (r = 0.83, SEE = 25 ml), end-diastolic LV volume (r = 0.68, SEE = 33 ml), and ejection fraction (r = 0.73, SEE = 10%) determined from MGBPT correlated well with those determined by CV. However, the ratios (Y) of the difference of the MGBPT-volume from the CV-volume to the CV-volume were decreased progressively and negatively as the CV-volumes (X) were smaller (Y = 38-3,996/X). Thus, we conclude that the determinations of volume by MGBPT would lead to more errors with the smaller volumes.

Effect of shear rate on clot growth at foreign surfaces.

The hydrodynamic effect on clot growth at foreign surfaces was investigated quantitatively in vitro. Shear rates from 2 to 1,000 s-1 were applied to a blood sample contained in a cone and plate viscometer. Four different artificial materials were used for cone and plate combination, namely, stainless steel, polytetrafluoroethylene, polycarbonate, and polymethylmethacrylate. Evaluation of clot growth was derived from the clotting ratio (the volumetric fraction of clot in the whole blood), which was experimentally determined from the rate of increase of frictional torque between the rotating cone and the stationary plate. The results show that the clotting ratio decreases markedly as the shear rate increases to 400 s-1, regardless of material used. This study demonstrates that at a shear rate of greater than 400 s-1, clot growth at foreign surfaces is considerably inhibited.

Recurrent splenic artery aneurysms developing after aneurysmectomy without splenectomy: report of a case.

We report herein the case of a 47-year-old man who developed a huge splenic artery aneurysm (SAA) with splenomegaly, for which ligation of the splenic artery and partial aneurysmectomy was performed. A celiac arteriogram taken 2 months postoperatively revealed that two small aneurysms had developed in the collateral vessels, indicating that increased blood flow through the collateral circulation could be responsible for the formation of secondary aneurysms. This postoperative change suggests that the etiology is related to the SAA and thus, the possibility that aneurysms may develop in the collateral vessels following spleen-preserving procedures for SAA must be borne in mind and careful follow-up performed at regular intervals.

A critical role for CD2 in both thymic selection events and mature T cell function.

To examine the function of CD2 in vivo, N15 TCR transgenic (tg) RAG-2(-/-) H-2(b) mice bearing a single TCR specific for the vesicular stomatitis virus octapeptide bound to the H-2K(b) molecule were compared on a wild-type or CD2(-/-) background. In N15tg RAG-2(-/-) CD2(-/-) mice, thymic dysfunction is evident by 6 wk with a pre-TCR block in the CD4(-)CD8(-) double-negative thymocytes at the CD25(+)CD44(-) stage. Moreover, mature N15tg RAG-2(-/-) CD2(-/-) T cells are approximately 100-fold less responsive to vesicular stomatitis virus octapeptide and unresponsive to weak peptide agonists, as judged by IFN-gamma production. Repertoire analysis shows substantial differences in Valpha usage between non-tg C57BL/6 (B6) and B6 CD2(-/-) mice. Collectively, these findings show that CD2 plays a role in pre-TCR function in double-negative thymocytes, TCR selection events during thymocyte development, and TCR-stimulated cytokine production in mature T cells.

Relationship between the conformity and the lubricating ability of synovial joints.

OBJECTIVE: To relate conformity of sliding surfaces with the lubricating ability of synovial joints. DESIGN: Measurement of start-up friction in the stifles of various animals. Assessment of conformity by Hertzian contact area. BACKGROUND: Past studies showed that the start-up friction in synovial joints sharply increased with the loading duration. The reasons why the friction increased and why the increasing rate is different in different joints were, however, not found. METHODS: Nine stifle joints from various animals were used. A robotic arm was used to give the compressive force and the sliding motion to the joint. Start-up friction was measured by a universal force sensor. The principal curvatures of the sliding surfaces were directly measured by a radius-gauge. Hertzian contact area was calculated from the principal curvatures of the sliding surfaces. RESULTS: The duration until the frictional coefficient reached 0.1 was related to the Hertzian contact area. CONCLUSION: The conformity of sliding surfaces is related to the lubrication ability in synovial joints. The squeeze-film mechanism plays an important role in joint lubrication.

Influence of loading duration on the start-up friction in synovial joints: measurements using a robotic system.

OBJECTIVE: To determine how much and why static load influences friction in synovial joints. DESIGN: Start-up coefficient of friction in canine stifles was measured after different duration of static load. BACKGROUND: Previous investigators have shown that friction of cartilage on cartilage contact configurations sharply increases with stationary load duration. This phenomenon has not been confirmed in the entire synovial joint. METHODS:: A system to measure joint friction was designed using a robotic arm. Ten canine stifles from six animals were used. Start-up friction of the femoral condyle on the tibial plateau and femoral condyle on glass plate contact configurations was measured. The glass plate was chosen as a rigid surface where ploughing effect cannot occur. RESULTS: The mean value of the start-up frictional coefficient from femoral condyle on tibial plateau was 0.112 (SD 0.005) at 0 s stationary loading, and sharply increased with the stationary loading duration to 0.313 (SD 0.095) at 1800 s. Those from femoral condyles on glass plate were 0.005 (SD 0.003) at 0 s and 0.457 (SD 0.128) at 1800 s. CONCLUSIONS: Friction in synovial joints sharply increases with duration under static load. The ploughing effect on this increase is slight in friction in canine stifles. RELEVANCE: The lubrication mechanism is worth investigating to understand the pathology of joint diseases. Determining friction behaviour is necessary for the investigation of the lubrication mechanism.

Physicochemical and biomechanical examination of surfaces of retrieved polyethylene heads from total hip prostheses with rotating polyethylene head system.

In order to assess whether hydrodynamic lubrication occurs in total hip prostheses with a rotating polyethylene (PE) head system (R-THP), several physicochemical, morphological, and biomechanical tests were carried out. R-THPs have been used in more than 1000 patients since 1970, and 12 PE heads, retrieved from 10 patients after an average of 24.5 years since total hip arthroplasty (THA), were employed for the tests. The weight-bearing area of the PE surface was light yellow in color and considerably oxidized, but no wear scars were observed. In the non-weight-bearing area, in contrast, discoloration and oxidation were hard to detect. The weight-bearing surface of the PE head became smoother with time after THA, and the friction coefficient did not differ significantly from that of an unused PE head. The radial clearance between head and socket decreased at a temperature of 17 degrees C, which is equivalent to the difference between room temperature and the temperature of the human body. Scanning electron microscopy showed a fine undulating pattern, which suggested that hydrodynamic lubrication could occur in the rotating PE head system.

The corrosion fatigue properties of surgical implants in a living body.

Fatigue fracture of artificial implants in the human body, caused by the repeated application of stress, is well documented. It is known that the fatigue strength of implant materials decreases when they are exposed under in vivo corrosion conditions. There are, however, no investigations concerning the effect of body fluids on the fatigue characteristics of commonly used biomaterials. Accordingly, fatigue tests on machined stainless-steel AISI 316, and COP alloy rods have been conducted in the right lower leg of rabbit. These specimens were pierced through the hole drilled at the middle of the tibial bone. A cyclic tensile stress of frequency 5 or 10 Hz was applied to the rods. From the results, it was found that the fatigue strength at 5 x 10(6) cycles for AISI 316 under the in vivo environment was 680 MPa compared to 830 MPa in air and similarly for COP alloy, was 680 MPa in the living body compared to 800 MPa in air. These remarkable changes in fatigue strength associated with the in vivo environments are considered to be due to the corrosive action of body fluids on the biomaterials.

Progression of osteoporosis in cancellous bone depending on trabecular structure.

Progression of osteoporosis is caused by a decline in bone formation activity relative to the resorption activity. In this paper, the authors carried out a theoretical analysis of the progression of osteoporosis to estimate the osteoporotic change in the upper end of the femur. According to this analysis, the progression rate of osteoporosis in cancellous bone depends on the product of remodeling activity, Ract, and the trabecular structure parameter, Ktr. To confirm that the theoretical results were reasonably comparable to actual osteoporotic change, these two factors were measured in rabbits. From the results, it was concluded that the highest progression rate was shown in bar/bar-like trabecular structure (type 3); the next highest rate, was shown in plate/bar-like structure (type 2); and the plate/plate-like structure (type 1) was the most insensible. Furthermore, the bone volume fractions of cancellous bone were measured at the upper end of human femurs with and without osteoporosis. Then the measured value was compared with the theoretical value for each type of trabecular structure. Results showed that the decrease in bone volume fraction predicted by Eq. 7 was well in accord with the actual decrease.