Addition Of Cyclosporine Research Articles

Involvement of the mitochondrial KATP+ channel in H2O2- or NO-induced programmed death of soybean suspension cell cultures

Soybean suspension cell cultures were treated by H2O2 or nitric oxide (NO), to assess the mechanism leading to programmed cell death (PCD). Hydrogen peroxide (5 mM) induced PCD. Cells become necrotic at 20 mM H2O2, with cells exhibiting intermediate hallmarks before that (necrapoptotic cells). The level of ATP and of glucose-6-phosphate remained constant in cells undergoing PCD, while it decreased significantly in the necrotic ones. Mitochondria, isolated from 5 mM H2O2-treated (apoptotic) cells, showed that succinate-dependent oxygen consumption was slightly uncoupled, and the electrical potential difference (delta psi) weakly decreased. The addition of KCl to the delta psi formed determined a partial dissipation, which was higher than the dissipation observed in mitochondria from control cells. The addition of cyclosporin A (CsA) to de-energized mitochondria also induced delta psi formation, due to a K+ efflux from the matrix, which was decreased in mitochondria from treated cells. The same pattern of response was also observed in mitochondria isolated from 1 mM sodium nitroprusside (NO)-treated cells, exhibiting apoptotic symptoms. In mitochondria isolated from 20 mM H2O2-treated (necrotic) cells, succinate-dependent oxygen consumption was completely uncoupled, delta psi generation significantly inhibited, and CsA-dependent delta psi formation prevented. In addition, mitochondria isolated from control cells still underwent swelling, which was partially or completely prevented in mitochondria isolated from apoptotic or necrotic cells, respectively. The moderate swelling was accompanied by a slight rupture of the outer membrane and by a release of cytochrome c. These results point to the involvement of a K(+)ATP channel during the manifestation of PCD induced by H2O2 or NO in plants.

Mitochondrial permeability transition induced by dinuclear gold(I)–carbene complexes: potential new antimitochondrial antitumour agents

Seven dinuclear gold(I) complexes of bidentate N-heterocyclic carbene ligands have been evaluated for their ability to induce mitochondrial membrane permeabilisation (MMP) in isolated rat liver mitochondria. Six of the compounds, at concentrations of 10 μM, induced Ca 2+-sensitive MMP as evidenced by mitochondrial swelling. In the absence of low concentrations of exogenous Ca 2+, the compounds were either inactive or their activity was significantly decreased. The mitochondrial swelling was completely blocked by the addition of cyclosporin A, a well established inhibitor of the mitochondrial permeability transition pore (MPT) that is believed to be responsible for MMP. The rates and levels of uptake of these compounds into mitochondria were estimated by measuring mitochondrial Au levels using inductively coupled plasma optical emission spectroscopy. Significant differences were found in the levels at which the different compounds accumulated in the mitochondria, but these differences did not correlate with the rate at which they induced mitochondrial swelling. These results suggest that the mechanism by which MMP is induced by these lipophilic cationic Au(I)–carbene complexes is not purely a function of the level of compound accumulation. Instead, a more specific mechanism, possibly involving disruption of the function of a particular enzyme, or interaction with a MPT component, appears to be more likely.

Addition of cyclosporin A to the combination of mitoxantrone and etoposide to overcome resistance to chemotherapy in refractory or relapsing acute myeloid leukaemia:: A randomised phase II trial from HOVON, the Dutch–Belgian Haemato-Oncology Working Group for adults

Cyclosporin A (CsA) inhibits the P-gp pump that can be responsible for failure of cytostatic treatment in acute myeloid leukaemia (AML). Eighty patients with relapsing/refractory AML were randomly assigned to mitoxantrone (M) and etoposide (VP) (MVP) in unmitigated antileukaemic doses with or without CsA, to investigate if toxicity was manageable and if antileukaemic therapy could be improved. CsA did not delay haematological recovery, but fewer CsA patients received post-induction therapy because of haematological and non-haematological toxicity. CR rate was 43% for MVP and 53% for CsA; DFS was 9 and 8 months, and OS 8 and 9 months, respectively. Seventeen of 38 CR patients proceeded to stem cell transplantation (SCT). After a median follow-up of 66 months, six patients were still alive. Addition of CsA did not improve treatment outcome, possibly due to inadequate post-induction therapy as a result of increased toxicity.

Addition of cyclosporin A to the combination of mitoxantrone and etoposide to overcome resistance to chemotherapy in refractory or relapsing acute myeloid leukaemia:A randomised phase II trial from HOVON, the Dutch?Belgian Haemato-Oncology Working Group for adults

Cyclosporin A (CsA) inhibits the P-gp pump that can be responsible for failure of cytostatic treatment in acute myeloid leukaemia (AML). Eighty patients with relapsing/refractory AML were randomly assigned to mitoxantrone (M) and etoposide (VP) (MVP) in unmitigated antileukaemic doses with or without CsA, to investigate if toxicity was manageable and if antileukaemic therapy could be improved. CsA did not delay haematological recovery, but fewer CsA patients received post-induction therapy because of haematological and non-haematological toxicity. CR rate was 43% for MVP and 53% for CsA; DFS was 9 and 8 months, and OS 8 and 9 months, respectively. Seventeen of 38 CR patients proceeded to stem cell transplantation (SCT). After a median follow-up of 66 months, six patients were still alive. Addition of CsA did not improve treatment outcome, possibly due to inadequate post-induction therapy as a result of increased toxicity.

Gliotoxin induces Mg 2+ efflux from intact brain mitochondria

Gliotoxin (GT) is a hydrophobic fungal metabolite of the epipolythiodioxopiperazine group which reacts with membrane thiols. When added to a suspension of energized brain mitochondria, it induces matrix swelling of low amplitude, collapse of membrane potential (Δ Ψ), and efflux of endogenous cations such as Ca 2+ and Mg 2+, typical events of mitochondrial permeability transition (MPT) induction. These effects are due to opening of the membrane transition pore. The addition of cyclosporin A (CsA) or ADP slightly reduces membrane potential collapse, matrix swelling and Ca 2+ efflux; Mg 2+ efflux is not affected at all. The presence of exogenous Mg 2+ or spermine completely preserve mitochondria against Δ Ψ collapse, matrix swelling and Ca 2+ release. Instead, Mg 2+ efflux is only slightly affected by spermine. Our results demonstrate that, besides inducing MPT, gliotoxin activates a specific Mg 2+ efflux system from brain mitochondria.

Mitochondrial dysfunction and apoptosis in myopathic mice with collagen VI deficiency.

Collagen VI is an extracellular matrix protein that forms a microfilamentous network in skeletal muscles and other organs. Inherited mutations in genes encoding collagen VI in humans cause two muscle diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy. We previously generated collagen VI-deficient (Col6a1-/-) mice and showed that they have a muscle phenotype that strongly resembles Bethlem myopathy. The pathophysiological defects and mechanisms leading to the myopathic disorder were not known. Here we show that Col6a1-/- muscles have a loss of contractile strength associated with ultrastructural alterations of sarcoplasmic reticulum (SR) and mitochondria and spontaneous apoptosis. We found a latent mitochondrial dysfunction in myofibers of Col6a1-/- mice on incubation with the selective F1F(O)-ATPase inhibitor oligomycin, which caused mitochondrial depolarization, Ca2+ deregulation and increased apoptosis. These defects were reversible, as they could be normalized by plating Col6a1-/- myofibers on collagen VI or by addition of cyclosporin A (CsA), the inhibitor of mitochondrial permeability transition pore (PTP). Treatment of Col6a1-/- mice with CsA rescued the muscle ultrastructural defects and markedly decreased the number of apoptotic nuclei in vivo. These findings indicate that collagen VI myopathies have an unexpected mitochondrial pathogenesis that could be exploited for therapeutic intervention.

Resting T cells negatively regulate osteoclast generation from peripheral blood monocytes

There is accumulating evidence that T cells may be involved in osteoclastogenesis in a variety of murine systems. However, the precise role of human T cells in the regulation of osteoclast generation is still unclear. To address this issue, we investigated the effect of resting peripheral T cells on receptor activator of NF-κB ligand (RANKL)-induced osteoclast generation from human peripheral monocytes. Although osteoclasts were not generated in the culture of human peripheral blood mononuclear cells (PBMC) in the presence of RANKL and macrophage colony-stimulating factor (M-CSF), the addition of cyclosporine A (CsA), a potent inhibitor of T-cell function, resulted in the formation of an increasing number of lacunae resorption on dentine, suggesting T cells may inhibit osteoclast formation. In a coculture of T cells and monocytes, which were isolated from PBMC, T cells inhibited the osteoclast generation from monocytes, as determined by tartrate-resistant acid phosphatase (TRAP) staining and a pit assay using dentine. This inhibition of osteoclast generation by T cells was also observed in a culture of the parathyroid hormone-stimulated SaOS4/3 osteoblast cell line and monocytes. The culture in Transwell plates revealed that the cell-to-cell interaction was not required for the inhibition, suggesting that T-cell cytokines may be responsible for the inhibition. Among inhibitory T-cell cytokines on osteoclastogenesis, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-γ (IFN-γ) were actively produced by CD4 T cells but not CD8 T cells in the coculture of T cells with monocytes, and the neutralizing antibodies to these cytokines partially rescued the T-cell-induced inhibition of osteoclast formation. Although CsA did not affect RANKL-induced osteoclast generation in the culture of monocytes alone, it completely rescued the T-cell-induced inhibition of osteoclast formation and strongly inhibited the production of GM-CSF and IFN-γ. Thus, we demonstrate that resting T cells negatively regulate the osteoclast generation via production of GM-CSF and IFN-γ by CD4 T cells and that CsA stimulates the osteoclast generation through the inhibition of the production of these cytokines. These findings provide new insight into therapeutic strategies for immunosuppression-induced bone loss in transplant and other diseases.

Zinc causes loss of membrane potential and alters production of reactive oxygen species in isolated brain mitochondria

Excessive accumulation of intracellular free zinc is lethal to neurons. The precise mechanism by which zinc kills neurons remains unclear, however, a number of reports suggest that zinc may impair neuronal metabolism. The present study examined how zinc affects the activity of isolated rat brain mitochondria. Using spectrofluorometry and polarography, we determined the effects of zinc on mitochondrial membrane potential (Δψ), reactive oxygen species (ROS) generation, and respiration. In mitochondria respiring on either glutamate + malate (G + M), succinate, or glycerol 3‐phosphate (G3P), addition of submicromolar levels of zinc caused dissipation of delta psi. Zinc‐induced depolarization was rapidly reversed by the addition of the membrane‐impermeant metal chelator EGTA. In all three substrate conditions, ruthenium red (200 nm) partially protected mitochondria from zinc‐induced depolarization, suggesting that the calcium uniporter may mediate mitochondrial zinc uptake. Conversely, addition of cyclosporin A (1 µM) afforded no protection, arguing against zinc‐induced permeability transition. ROS production was enhanced by zinc in mitochondria fueled by either G + M or G3P, but zinc inhibited succinate‐driven ROS production. Zinc inhibited respiration in phosphorylating mitochondria regardless of substrate conditions, and zinc also inhibited respiration in uncoupled mitochondria using G + M. Together, these results suggest that zinc has several deleterious effects on mitochondria that could contribute to neuronal injury.Acknowledgements: Supported by NS34138 and the American Heart Association.

Identification and immune regulation of 25-hydroxyvitamin D-1-α-hydroxylase in murine macrophages

Receptors for 1,25(OH)2vitaminD3 are found in most immune cells and important immunological effects have been described in vitro, reflected by its capacity to prevent autoimmunity and to prolong graft survival. The aim of this study was to examine the presence and nature of the enzyme responsible for final activation of the molecule, 1-alpha-hydroxylase, in murine macrophages and to analyse its regulation and possible role in the immune system. Peritoneal macrophages from C57Bl/6 mice were incubated with lipopolysaccharide (LPS; 100 microg/ml), interferon-gamma (IFN-gamma; 500 U/ml) or a combination of both. By quantitative reverse transcriptase-polymerase chain reaction, using primers based on the murine renal cDNA sequence, low levels of 1-alpha-hydroxylase mRNA were detected in freshly isolated cells (18 +/- 7 x 10-6 copies/beta-actin copies). Analysis of the cDNA sequence of the gene revealed identical coding sequences for the macrophage and renal enzymes. mRNA levels rose three-fold with LPS (NS), but a six-fold increase was seen after IFN-gamma stimulation (P < 0.05). Combining LPS and IFN-gamma did not result in a major additional increase, but addition of cyclosporin A further increased levels 2.5-fold both in IFN-gamma- and combination-stimulated cells (P < 0.05). Time course analysis revealed that up-regulation of 1-alpha-hydroxylase was a late phenomenon, preceded by the up-regulation of activating macrophage products such as IL-1 and tumour necrosis factor-alpha. Finally, a defect in 1-alpha-hydroxylase up-regulation by immune stimuli was found in autoimmune non-obese diabetic mice. In conclusion, we propose that the up-regulation of 1-alpha-hydroxylase in activated macrophages, resulting in the synthesis of 1,25(OH)2D3, might be a negative feedback loop in inflammation. A defect in this system might be an additional element in tipping the balance towards autoimmunity.

HLA-identical renal transplant recipients: immunosuppression, long-term complications, and survival

HLA-IDENTICAL living donor renal transplant recipients have superior long-term graft and patient survival. They require less immunosuppression than other living-donor and cadaver-donor recipients. However, early acute rejection episodes occurring in up to 55% of recipients have been reported with azathioprine (AZA) and prednisone immunosuppression. Although acute rejection (AR) rates are significantly lowered by the addition of cyclosporine (CyA) to the immunosuppression regimen, concerns arise regarding the safety of long-term CyA in this group of patients with low immunologic risk and excellent long-term results. The aim of this study is to analyze the AR rate, immunosuppression requirements, posttransplant complications, long-term allograft function, and graft and patient survival rates in HLA-identical living-donor renal transplant recipients. The effects of two immunosuppressive regimens (AZA and prednisone or AZA, CyA, and prednisone) are compared, and the outcome of CyA and/or prednisone withdrawal is also analyzed.

Etoposide induction of tumor immunity in Lewis lung cancer.

To determine if the antineoplastic effect of etoposide includes alteration in Lewis lung cancer cells which evoke an immunologic response in C57B1/6 host mice. Of C57B1/6 mice injected with 10(6) Lewis lung cancer (3LL) cells followed by treatment with a single 50 mg/kg dose of etoposide (VP-16), 60% survived over 60 days, in contrast to untreated control mice which died within 30 days. Approximately 40% of surviving mice rejected a subsequent challenge with 3LL. Their splenocytes protected naive mice injected with 3LL. To test if VP-16 treatment produced alterations in 3LL cells, which induce host immunity, leading to tumor rejection, C57B1/6 mice were injected with 3LL cells that had survived an 80-90% lethal concentration of VP-16 in vitro. These cells killed 75% of recipient mice but 60% of the surviving mice rejected challenge with 3LL. Splenocytes harvested from tumor-rejecting mice protected naive mice injected with 3LL. These results support the hypothesis that in addition to its antineoplastic cytotoxic effect, VP-16 induces changes in 3LL cells which are recognized by the host immune system resulting in immune rejection of 3LL. often immunosuppressive and therapeutic advantage is generally based on the tumor cytotoxicity of individual drugs or combinations of drugs [13]. Our earlier work showed a link between the use of cytotoxic chemotherapy with etoposide (VP-16) and the induction of an immune response against syngeneic murine leukemia in the intact host [16]. VP-16 is an immunosuppressive topoisomerase II-inhibiting drug which induces tumor cell apoptosis and is frequently used clinically to treat a variety of tumors [1, 3, 9, 10]. We have noted that the addition of cyclosporin A to VP-16 produces CD8 T lymphocyte-mediated tumor-specific immunity in mice bearing L1210 leukemia [17]. We have extended these experiments to a spontaneously arising non-carcinogen-induced neoplasm, Lewis lung cancer (3LL), and now report that surviving mice successfully treated with VP-16, in the absence of cyclosporin A, reject challenge with 3LL. In addition, results are presented to show that VP-16 modifies 3LL cells rendering them immunogenic. These findings are submitted to support the hypothesis that VP-16-induced cytotoxic changes include cellular membrane alterations in 3LL cells which are recognized by the immune system and cause rejection of this syngeneic lung tumor.

Graft survival in a rhesus renal transplant model after immunotoxin-mediated T-cell depletion is enhanced by mycophenolate and steroids.

Anti-CD3 immunotoxin (IT), a T-cell-depleting agent, prolongs survival of renal allografts in a rhesus monkey model without the need for long-term immunosuppression. In this study we sought to further prolong allograft survival by giving short-term conventional immunosuppression simultaneous with IT administration. MHC class II mismatched, juvenile rhesus monkeys were paired as donor and recipient for renal transplantation. Recipients received two to three daily doses of IT starting on the day of transplantation. Additional immunosuppression was given for no more than 60 days. Graft function was monitored by serum creatinine and renal biopsies. Flow cytometry was used to monitor T-cell recovery. Graft survival time (GST) in animals receiving IT was prolonged compared with controls with 50% of IT-treated monkeys surviving >100 days. Animals treated with IT plus mycophenolate mofetil (MMF) and steroids had significantly enhanced GST (mean GST, 305 days) compared with those treated with IT alone (mean GST, 94 days). In contrast, addition of cyclosporine or 40-O-[2-Hydroxyethyl]rapamycin did not significantly increase graft survival time. A comparison among animals from all treatment groups with short (<100 days) and long (>100 days) GST demonstrated that those with the shorter GST had a higher blood T-cell count 2 weeks after transplantation. Full recovery of CD4+ T cells required longer than 6 months. A combination with MMF and steroids given for 4 days after renal allograft transplantation significantly increases GST in IT-treated monkeys. We hypothesize that MMF and steroids suppress the initial T-cell activation mediated by IT.

Effect of cyclosporine, a P-glycoprotein inhibitor, on the pharmacokinetics of cefepime in rat blood and brain: A microdialysis study

In clinical application, cefepime and cyclosporine are regularly combined in the treatment of organ transplant patients, so the interaction of these two drugs can be hypothesized. Therefore, the pharmacokinetics of cefepime alone and in combination with cyclosporine in rat using microdialysis coupled with HPLC-UV on-line system was evaluated in the study. Cefepime at three doses (20, 50, and 100 mg/kg) showed linear kinetics. After addition of cyclosporine, the mean residence time was increased from 34.9 min to 48.6 min (p<0.05, n=6), and the area under the concentration versus time curve (AUC) increased from 4775 min μg/ml to 6960 min μg/ml (p<0.01, n=6). While in the brain, AUC increased from 64.3 min μg/ml to 110.2 min μg/ml. In summary, cyclosporine (20 mg/kg) could significantly alter the simultaneously administered cefepime (50 mg/kg) unbound drug pharmacokinetic parameters in both blood and brain.

The effect of aliphatic, naphthenic, and aromatic hydrocarbons on production of reactive oxygen species and reactive nitrogen species in rat brain synaptosome fraction: the involvement of calcium, nitric oxide synthase, mitochondria, and phospholipase A

This study investigated the effects of C7 and C9 aliphatic ( n-heptane, n-nonane), naphthenic (methylcyclohexane, 1,2,4-trimethylcyclohexane (TMCH)) and aromatic (toluene, 1,2,4-trimethylbenzene (TMB)) hydrocarbons on the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in rat brain synaptosome fraction. Methyl mercury (MeHg) was included as a positive control. Exposure of the synaptosomes to the hydrocarbons produced a concentration-dependent linear increase in the formation of the fluorescence of 2′,7′-dichlorofluorescein (DCF) as a measure of the production of ROS and RNS. Formation of RNS was demonstrated by preincubation of the synaptosome fraction with the neuronal nitric oxide synthase (nNOS) inhibitor Nω-nitro- l-arginine methyl ester (L-NAME), which reduced the MeHg and TMCH-stimulated fluorescence by 51% and 65%, respectively. The naphthenic hydrocarbon TMCH showed the strongest potential for ROS and RNS formation in rat brain synaptosomes, followed by TMB, toluene, n-nonane, n-heptane, and methylcyclohexane, respectively. TMCH was selected for mechanistic studies of the formation of ROS. Both MeHg and TMCH induced an increase in intracellular calcium concentration [Ca 2+] i as measured with Fura-2. Blockade of voltage-dependent Ca 2+ channels with lanthanum prior to stimulation with MeHg and TMCH led to a reduction in the ROS/RNS formation of 72% and 70%, respectively. Furthermore, addition of cyclosporin A (CSA), a blocker of the mitochondrial permeability transition pore (MTP), lowered both the MeHg and TMCH-elevated DCF fluorescence by 72% and 59%. Preincubation of the synaptosome fraction with the protein tyrosine kinase inhibitor genistein lowered the MeHg and TMCH-stimulated fluorescence by 85% and 91%, respectively. Addition of the extracellular signal-regulated protein kinase (MEK)-1 and -2 inhibitor U0126 reduced the fluorescence stimulated by MeHg and TMCH by 62% and 63%. Furthermore, the protein kinase C inhibitor bisindolylmaleimide reduced the fluorescence stimulated by MeHg and TMCH by 52% and 56%. The compound 1-(6-[17beta-3-methoxyestra- 1,3,5(10)-trien- 17-yl]-aminohexyl)-1 H-pyrrole-2,5-dione (U73122), which inhibits phospholipase C, was shown to decrease the ROS and RNS formation induced by MeHg and TMCH by 49% and 64%, respectively. The phospholipase A 2 (PLA 2) inhibitor 7,7-dimethyl eicosadienoic acid (DEDA) reduced fluorescence in response to MeHg and TMCH by 49% and 54%. Simultaneous addition of L-NAME, CSA, and DEDA to the synaptosome fraction totally abolished the DCF fluorescence. In conclusion, C7 and C9 aliphatic, naphthenic, and aromatic hydrocarbons stimulated formation of ROS and RNS in rat brain synaptosomes. The naphthenic hydrocarbon TMCH stimulated formation of ROS and RNS in the synaptosomes through Ca 2+-dependent activation of PLA 2 and nNOS, and through increased transition permeability of the MTP. Exposure of humans to the naphthenic hydrocarbon TMCH may stimulate formation of free radicals in the brain, which may be a key factor leading to neurotoxicity.

Calcineurin regulates slow myosin, but not fast myosin or metabolic enzymes, during fast-to-slow transformation in rabbit skeletal muscle cell culture.

The addition of cyclosporin A (500 ng ml(-1)) - an inhibitor of the Ca2+-calmodulin-regulated serine/threonine phosphatase calcineurin - to primary cultures of rabbit skeletal muscle cells had no influence on the expression of fast myosin heavy chain (MHC) isoforms MHCIIa and MHCIId at the level of protein and mRNA, but reduced the expression of slow MHCI mRNA. In addition, no influence of cyclosporin A on the expression of citrate synthase (CS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was found. The level of enzyme activity of CS was also not affected. When the Ca2+ ionophore A23187 (4 x 10(-7) M) was added to the medium, a partial fast-to-slow transformation occurred. The level of MHCI mRNA increased, and the level of MHCIId mRNA decreased. Cotreatment with cyclosporin A was able to prevent the upregulation of MHCI at the level of mRNA as well as protein, but did not reverse the decrease in MHCIId expression. The expression of MHCIIa was also not influenced by cyclosporin A. Cyclosporin A was not able to prevent the upregulation of CS mRNA under Ca2+ ionophore treatment and failed to reduce the increased enzyme activity of CS. The expression of GAPDH mRNA was reduced under Ca2+ ionophore treatment and was not altered under cotreatment with cyclosporin A. When the myotubes in the primary muscle culture were electrostimulated at 1 Hz for 15 min periods followed by pauses of 30 min, a partial fast-to-slow transformation was induced. Again, cotreatment with cyclosporin A prevented the upregulation of MHCI at the level of mRNA and protein without affecting MHCIId expression. The nuclear translocation of the calcineurin-regulated transcription factor nuclear factor of activated thymocytes (NFATc1) during treatment with Ca2+ ionophore, and the prevention of the translocation in the presence of cyclosporin A, were demonstrated immunocytochemically in the myotubes of the primary culture. The effects of cyclosporin A demonstrate the involvement of calcineurin-dependent signalling pathways in controlling the expression of MHCI, but not of MHCIIa, MHCIId, CS and GAPDH, during Ca2+ ionophore- and electrostimulation-induced fast-to-slow transformations. The data indicate a differential regulation of MHCI, of MHCII and of metabolism. Calcineurin alone is not sufficient to mediate the complete transformation.

Transcriptional activation of the interleukin-2 promoter by hepatitis C virus core protein.

Most patients infected with hepatitis C virus (HCV) become chronic carriers. Viruses that efficiently establish persistent infections must have effective ways of evading host defenses. In the case of HCV, little is known about how chronic infections are established or maintained. Besides hepatocytes, several reports suggest that HCV can infect T and B lymphocytes. Since T cells are essential for viral clearance, direct or indirect effects of HCV on T-cell function could influence the outcome of infection. Given that T-cell growth and differentiation require the cytokine interleukin 2 (IL-2), we asked whether HCV might modulate synthesis of IL-2. Portions of the HCV polyprotein were expressed in Jurkat cells under a variety of conditions. We found that the highly conserved HCV core protein, in combination with other stimuli, was able to dramatically activate transcription from the IL-2 promoter. The carboxy-terminal hydrophobic portion of the core protein was required for this activity. Activation was dependent on nuclear factor of activated T cells (NFAT), occurred in cells deficient in the tyrosine kinase p56(lck), and could be blocked by addition of cyclosporin A and by depletion of calcium. These results suggest that the HCV core protein can activate transcription of the IL-2 promoter through the NFAT pathway. This novel activity may have consequences for T-cell development and establishment of persistent infections.

Assembly of heterodimeric luciferase after de novo synthesis of subunits in rabbit reticulocyte lysate involves hsc70 and hsp40 at a post-translational stage.

Heterodimeric luciferase from Vibrio harveyi had been established as a unique model enzyme for direct measurements of the effects of molecular chaperones and folding catalysts on protein folding and subunit assembly after de novo synthesis of subunits in rabbit reticulocyte lysate. It was observed that luciferase assembly can be separated in time from synthesis of the two subunits and that under these post-translational conditions assembly was inhibited by either ATP depletion or inhibition of peptidylprolyl cis/trans isomerases, that is, by addition of cyclosporin A or FK506. Furthermore, it was observed that the inhibitory effect of FK506 on luciferase assembly can be suppressed by addition of purified cyclophilin, thereby providing the first direct evidence for the involvement of peptidylprolyl cis/trans isomerases in protein biogenesis in the eukaryotic cytosol. Here the ATP requirement in luciferase assembly has been characterized. Depletion of either Hsp90 or CCT from reticulocyte lysate did not interfere with luciferase assembly. However, addition of purified Hsc70 stimulated luciferase assembly. While addition of purified Hsp40 did not have any effect on luciferase assembly, the stimulatory effect of Hsc70 was further increased by Hsp40. Thus, after synthesis of the two subunits in reticulocyte lysate assembly of heterodimeric luciferase involves Hsc70 and its co-chaperone Hsp40. Therefore, Hsc70 aids protein biogenesis in the eukaryotic cytosol not only at the levels of nascent polypeptide chains and precursor proteins that have to be kept competent for transport into cell organelles, but also at the level of subunits that have to be kept competent for assembly.

SOCS-1 can suppress CD3ζ- and Syk-mediated NF-AT activation in a non-lymphoid cell line

To elucidate T cell antigen receptor (TCR) signaling leading to activation nuclear factor of activated T cells (NF-AT), we reconstituted TCR signaling to activate NF-AT in a non-lymphoid cell line, 293T. We demonstrated that co-expression of CD8/ζ and Syk were necessary for NF-AT activation in 293T. This NF-AT response was completely inhibited by the addition of cyclosporin A or FK506, but markedly enhanced by the additional expression of Tec protein tyrosine kinase. We also show that the cytokine signaling suppressor, suppressor of cytokine signaling 1, potently inhibited this response by interacting with Syk and immunoreceptor tyrosine-based activation motifs in CD8/ζ. These results imply that this novel system may provide a useful tool to delineate or identify the regulatory molecules for CD3ζ/Syk-mediated NF-AT activation.

Immunosuppressive treatment of aplastic anemia with antithymocyte globulin and cyclosporine

Immunosuppression is the treatment modality for the majority of patients with aplastic anemia, most of whom are not candidates for allogeneic stem-cell transplantation. Antithymocyte globulin (ATG) or antilymphocyte globulin (ALG) have proven to be essential components of all regimens. Initial response rates can be improved by the addition of cyclosporine A (CsA), and this combination has become the standard of care for appropriate patients. Several new approaches to immunosuppression are being studied, including the optimal timing of administration of these drugs, the use of novel immunosuppressive agents, and the addition of early- and late-acting hematopoietic growth factors.

IGF-1 induces skeletal myocyte hypertrophy through calcineurin in association with GATA-2 and NF-ATc1.

Localized synthesis of insulin-like growth factors (IGFs) has been broadly implicated in skeletal muscle growth, hypertrophy and regeneration. Virally delivered IGF-1 genes induce local skeletal muscle hypertrophy and attenuate age-related skeletal muscle atrophy, restoring and improving muscle mass and strength in mice. Here we show that the molecular pathways underlying the hypertrophic action of IGF-1 in skeletal muscle are similar to those responsible for cardiac hypertrophy. Transfected IGF-1 gene expression in postmitotic skeletal myocytes activates calcineurin-mediated calcium signalling by inducing calcineurin transcripts and nuclear localization of calcineurin protein. Expression of activated calcineurin mimics the effects of IGF-1, whereas expression of a dominant-negative calcineurin mutant or addition of cyclosporin, a calcineurin inhibitor, represses myocyte differentiation and hypertrophy. Either IGF-1 or activated calcineurin induces expression of the transcription factor GATA-2, which accumulates in a subset of myocyte nuclei, where it associates with calcineurin and a specific dephosphorylated isoform of the transcription factor NF-ATc1. Thus, IGF-1 induces calcineurin-mediated signalling and activation of GATA-2, a marker of skeletal muscle hypertrophy, which cooperates with selected NF-ATc isoforms to activate gene expression programs.