Protection against cell death and sustained tyrosine hydroxylase phosphorylation in hydrogen peroxide- and MPP+-treated human neuroblastoma cells with melatonin

Abstract:  Neuroprotective effects of melatonin against oxidative stress-induced neuronal cell degeneration in human SH-SY5Y neuroblastoma cells were investigated in this report. The results demonstrate that exogenous administration of H₂O₂ and 1-methyl, 4-phenyl, pyridinium ion (MPP⁺) significantly decreased cell viability in SH-SY5Y cultured cells. Desipramine, a monoamine uptake blocker was able to abolish the toxic effects of MPP⁺ but not H₂O₂ in reduction of cell viability. Conversely, melatonin reversed the toxic effects of H₂O₂ and MPP⁺ on cell viability. In addition, the reduction of phosphorylation of tyrosine hydroxylase, the rate limiting enzyme in dopamine synthesis, and phosphorylation of cyclic AMP responsive element-binding protein by H₂O₂ and MPP⁺ was also diminished by melatonin. These results demonstrate some effective roles of melatonin on neuroprotection and its action on the modulation of tyrosine hydroxylase phosphorylation.     

Adriamycin stimulates proliferation of human lymphoblastic leukaemic cells via a mechanism of hydrogen peroxide (H2O2) production

It is becoming clear that adriamycin cytotoxicity may be mediated by semiquinone-free radicals derived from the drug itself and reactive oxygen species (ROS). Recent evidence supports the concept that low concentrations of ROS are able to stimulate cell proliferation, and, based on the observation that subtoxic concentrations of adriamycin can also induce cell proliferation, we hypothesize that low concentrations of adriamycin stimulate cell proliferation by a ROS generation mechanism. We have employed spin-trapping and electron spin resonance (ESR) spectroscopy to investigate the nature of the adriamycin-generated ROS. The spin trap 3,5-dibromo-4-nitrosobenzenesulphonate (DBNBS), which is oxidized in the presence of H₂O₂ and peroxidase enzymes, was used to produce a characteristic three-line spectrum, and it was found that an identical spectrum was produced by human lymphoblastic leukaemic cells (CCRF-CEM cells) after exposure to adriamycin. We tested our hypothesis further by exposing CCRF-CEM cells to subtoxic concentrations of adriamycin (10⁻⁸, 10⁻⁹ and 10⁻¹⁰  M ) and low concentrations of H₂O₂ (10⁻⁸, 10⁻⁹ and 10⁻¹⁰  M ) and subsequently monitored cell proliferation. We found that low concentrations of both adriamycin and H₂O₂ significantly stimulate CCRF-CEM cell proliferation. We therefore conclude that subtoxic concentrations of adriamycin are likely to induce cell proliferation via an H₂O₂ mediated mechanism.     

Inflammatory markers in exhaled breath condensate from patients with asthma

Background and objective:   Evaluation of airway inflammation is important for the diagnosis and treatment of asthma. Exhaled breath condensate (EBC) is a minimally invasive method for assessing inflammation and may be useful for monitoring airway inflammation in asthma. The aims of this study were to establish an EBC collection method, to assess biomarkers reflecting asthmatic airway inflammation, and to determine the relationship of these biomarkers with asthma severity and lung function.
Methods:   Fifty-eight non-smoking healthy subjects, seven asymptomatic smokers, nine subjects with common cold and 55 asthmatics with disease severity ranging from mild intermittent to severe persistent were studied. The efficacy of a pipette method was compared with that of a commercial collecting device. pH, CRP, albumin, hydrogen peroxide (H₂O₂) and nitrite/nitrate levels were measured in EBC.
Results:   Except for the quantity of EBC collected and albumin levels, there were no differences between the commercial method and the pipette method in levels of biomarkers measured. Levels of CRP, H₂O₂ and nitrite/nitrate were significantly higher in the asthma group than that in the control group. In terms of asthma severity, pH and levels of CRP, H₂O₂ and nitrate were significantly higher in the mild persistent group than that in the other groups. In addition, H₂O₂ levels in EBC correlated significantly with the level of nitrite/nitrate. FEV₁ and PEF showed significant negative correlations with H₂O₂ and nitrite/nitrate levels.
Conclusion:   Measurement of EBC biomarkers is a non-invasive and useful way to evaluate airway inflammation in patients with asthma.     

Melatonin represses oxidative stress-induced activation of the MAP kinase and mTOR signaling pathways in H4IIE hepatoma cells through inhibition of Ras

Abstract:  Reactive oxygen species (ROS) have been implicated in the pathogenesis of a variety of diseases, and antioxidant treatment is currently being investigated as a potential therapy to attenuate the detrimental effects of ROS-mediated oxidative stress. Melatonin is a potent naturally produced antioxidant, which acts through various mechanisms to ameliorate the toxic effects of ROS. However, little is known about the mechanisms of signaling pathways through which melatonin acts to reverse the effects of ROS. In the present study, the effect of melatonin treatment on the hydrogen peroxide (H₂O₂)-induced activation of the mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) signaling pathways was assessed in H4IIE hepatoma cells. It was found that melatonin strongly attenuated H₂O₂-induced activation of the ERK1/2 and p38 MAP kinases, as well as several of their downstream targets. Melatonin also attenuated the H₂O₂-induced phosphorylation of Akt and the Akt substrate mTOR, as well as a downstream target of mTOR action, 4E-BP1. Upregulation of ERK1/2, p38, and Akt signaling by H₂O₂ was accompanied by activation of Ras, an effect that was blocked by melatonin. Overall, the results suggest that melatonin acts to prevent many of the H₂O₂-induced alterations in the MAPK and mTOR signaling pathways through inhibition of Ras, at least in H4IIE hepatoma cells.     

Oxidative stress impairs oocyte quality and melatonin protects oocytes from free radical damage and improves fertilization rate

Abstract:  We investigated the relationship between oxidative stress and poor oocyte quality and whether the antioxidant melatonin improves oocyte quality. Follicular fluid was sampled at oocyte retrieval during in vitro fertilization and embryo transfer (IVF-ET). Intrafollicular concentrations of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in women with high rates of degenerate oocytes were significantly higher than those with low rates of degenerate oocytes. As there was a negative correlation between intrafollicular concentrations of 8-OHdG and melatonin, 18 patients undergoing IVF-ET were given melatonin (3 mg/day), vitamin E (600 mg/day) or both melatonin and vitamin E. Intrafollicular concentrations of 8-OHdG and hexanoyl-lysine adduct were significantly reduced by these antioxidant treatments. One hundred and fifteen patients who failed to become pregnant with a low fertilization rate (50%) in the previous IVF-ET cycle were divided into two groups during the next IVF-ET procedure; 56 patients with melatonin treatment (3 mg/day) and 59 patients without melatonin treatment. The fertilization rate was improved by melatonin treatment compared to the previous IVF-ET cycle. However, the fertilization rate was not significantly changed without melatonin treatment. Oocytes recovered from preovulatory follicles in mice were incubated with H₂O₂ for 12 hr. The percentage of mature oocytes with a first polar body was significantly reduced by addition of H₂O₂ (300  μ m ). The inhibitory effect of H₂O₂ was significantly blocked by simultaneous addition of melatonin. In conclusion, oxidative stress causes toxic effects on oocyte maturation and melatonin protects oocytes from oxidative stress. Melatonin is likely to improve oocyte quality and fertilization rates.     

Melatonin-induced estrogen receptor α-mediated calbindin-D9k expression plays a role in H2O2-mediated cell death in rat pituitary GH3 cells

Abstract:  Calbindin-D9k (CaBP-9k) is a 9-kDa polypeptide possessing two calcium-binding sites that is expressed in the mammalian intestine, uterus, and pituitary gland. The factors regulating the expression of the estrogen receptor (ER) and CaBP-9k in the pituitary gland are currently unknown. In this study, we investigated whether the ER and CaBP-9k expression are regulated by melatonin during H₂O₂-induced cell death in rat pituitary GH3 cells. Cell survival increased by approximately 27–36% in H₂O₂ plus melatonin compared to H₂O₂ alone, and CaBP-9k expression was augmented by treatment with H₂O₂ plus melatonin. These results suggest that the increase in cell survival and the melatonin-induced CaBP-9k expression may play a role in protecting cells against H₂O₂-mediated cell death. This result is also consistent with the increase in CaBP-9k expression leading to rises in p-ERK and p-Bad (S112). Over-expression of CaBP-9k caused an increase in p-ERK. ERα expression was higher in H₂O₂ plus melatonin-treated cells compared to those treated with H₂O₂ alone, while ERβ expression was not. Also, ERα in the nuclear fraction increased in the presence of melatonin and decreased in the presence of ICI 182 780 or ICI 182 780 plus melatonin. The relative binding affinity of ERα for melatonin was higher than that of ERβ, suggesting that melatonin has the potential to preferentially bind ERα. In conclusion, these results indicate that melatonin may increase CaBP-9k expression through ERα.     

Characterization of the antioxidant effects of melatonin and related indoleamines in vitro

Abstract: The antioxidant and possible pro-oxidant effects of melatonin and related indoleamines (tryptophan, serotonin, N-acetylserotonin, and 5-methoxytryptamine) were studied in vitro. In two model membrane systems, i.e., phospholipid liposomes and rat erythrocytes, lipid peroxidation induced by Fe²⁺ and H₂O₂, respectively, were reduced by the tested indoleamines except for tryptophan. The 5-hydroxy-indoleamines, serotonin, and N-acetylserotonin exhibited pro-oxidant actions in the bleomycin assay by reducing Fe³⁺ to Fe²⁺, which leads to DNA damage. The 5-methoxy-indoleamines, melatonin and 5-methoxytryptamine, were devoid of any pro-oxidant actions in this assay. Serotonin, but not N-acetylserotonin, scavenged the superoxide anion. None of the indoleamines tested had any reactivity towards H₂O₂. All the indoleamines, including tryptophan, were, however, shown to react with hypochlorous acid. These findings support an antiperoxidative and antioxidant action of melatonin which is devoid of pro-oxidant effect on non-lipid substrates.     

Fecal Hydrogen Sulfide Production in Ulcerative Colitis

Objective: Sulfide, a product of sulfate-reducing bacteria, has been proposed to play an etiologic role in ulcerative colitis. Ulcerative colitis feces have increased numbers and activity of sulfate-reducing bacteria, but only modestly increased sulfide. However, fecal sulfide exists largely in the volatile, highly toxic H₂S form that moves rapidly from feces to surrounding gas. Our aim was to quantify the fecal release of H₂S and other volatiles (CO₂, H₂, CH₂, methanethiol, and dimethylsulfide).
Methods: Fecal samples from 25 subjects with ulcerative colitis and 17 controls were incubated in 4-L containers, and gas release was assessed at intervals over 24 h.
Results: H₂S release by ulcerative colitis feces was elevated 3–4-fold at every measurement point compared with normal feces (   p < 0.003  at 24 h). The only other significant difference was increased CO₂ release by ulcerative colitis feces at 1 h. Supplementation of fecal homogenates with sulfur-containing substrates showed that organic compounds (mucin, cysteine, taurocholate) provided more readily utilizable substrate for H₂S production than did sulfate.
Conclusions: Increased H₂S release is a relatively localized metabolic aberration of ulcerative colitis feces. This increased H₂S may reflect abnormalities of the fecal bacteria and/or substrate availability.     

Cytoprotective effects of melatonin on C6 astroglial cells exposed to glutamate excitotoxicity and oxidative stress

Abstract:  To preserve the central nervous system (CNS) function after a traumatic injury, therapeutic agents must be administered to protect neurons as well as glial cells. Cell death in CNS injuries and diseases are attributed to many factors including glutamate toxicity and oxidative stress. We examined whether melatonin, a potent anti-oxidant and free radical scavenger, would attenuate apoptotic death of rat C6 astroglial cells under glutamate excitotoxicity and oxidative stress. Exposure of C6 cells to 500 μ m l -glutamic acid (LGA) and 100 μ m hydrogen peroxide (H₂O₂) for 24 hr caused significant increases in apoptosis. Apoptosis was evaluated by Wright staining and ApopTag assay. Melatonin receptor 1 appeared to be involved in the protection of these cells from excitotoxic and oxidative damage. Cells undergoing excitotoxic and oxidative stress for 15 min were then treated with 150 n m melatonin, which prevented Ca²⁺influx and cell death. Western blot analyses showed alterations in Bax and Bcl-2 expression resulting in increased Bax:Bcl-2 ratio during apoptosis. Western blot analyses also showed increases in calpain and caspase-3 activities, which cleaved 270 kD α-spectrin at specific sites to generate 145 kD spectrin breakdown product (SBDP) and 120 kD SBDP, respectively. However, 15-min post-treatment of C6 cells with melatonin dramatically reduced Bax:Bcl-2 ratio and proteolytic activities, decreasing LGA or H₂O₂-induced apoptosis. Our data showed that melatonin prevented proteolysis and apoptosis in C6 astroglial cells. The results suggest that melatonin may be an effective cytoprotective agent against glutamate excitotoxicity and oxidative stress in CNS injuries and diseases.     

Melatonin reduces H2O2-induced lipid peroxidation in homogenates of different rat brain regions

Abstract: The ability of melatonin to modify H₂O₂-induced lipid peroxidation in brain homogenates was determined. The concentrations of brain malonaldehyde (MDA) and 4-hydroxyalkenals (4-HDA) were assayed as an index of induced membrane oxidative damage. Homogenates from five different regions of the brain (cerebral cortex, cerebellum, hippocampus, hypothalamus, and corpus striatum) derived from two different strains of rats, Sprague-Dawley and Wistar, were incubated with either H₂O₂ (5 mM) alone or H₂O₂ together with melatonin at increasing concentrations ranging from 0.1 to 4 mM. The basal level of lipid peroxidation was strain-dependent and about 100% higher in homogenates from the brain of Wistar rats than those measured in Sprague-Dawley rats. MDA + 4-HDA levels increased after H₂O₂ treatment in homogenates obtained from each region of the brain in both rat strains but the sensitivity of the homogenates from Sprague-Dawley rats was greater than that for the homogenates from Wistar rats (increases after H₂O₂from 45 to 165% compared 20 to 40% for Sprague-Dawley and Wistar rats, respectively). Melatonin co-treatment reduced H₂0₂-induced lipid peroxidation in brain homogenates in a concentration-dependent manner; the degree of protection against lipid peroxidation was similar in all brain regions.     

H2O2 Release from Filtration Leukapheresis-Procured Leukocytes

Abstract. Filtration leukapheresis-procured leukocytes (FL-leukocytes), which were collected by the elution of filtration columns with vigorous tapping, released a certain amount of H₂O₂, even in the absence of any phagocytic stimuli. Furthermore, FL-leukocytes, eluted with either gentle or vigorous tapping, exhibited a marked release of H₂O₂ during phagocytosis. The myeloperoxidase (MPO) activity of FL-leukocytes was lower than that of leukocytes collected by the dextran sedimentation method (DS-leukocytes). The data suggest that the release of both H₂O₂ and MPO from FL-leukocytes may be related to adverse transfusion reactions and abnormal post-transfusion kinetics of FL-leukocytes due to their toxic effects on living cells.     

Red Cell Membrane Response to Hydrogen Peroxide-Sensitivity in Hereditary Xerocytosis and in Other Abnormal Red Cells

S ummary. Osmotically resistant red cells associated with some haemolytic anaemias, including hereditary xerocytosis, sickle-cell disease and β thalassaemia minor, are more sensitive than normal red cells to exogenous in vitro hydrogen peroxide (H₂O₂). This sensitivity is manifested by a rapid loss of intracellular potassium, shape change, protein aggregation, and methaemoglobin formation at lower concentrations of H₂O₂ (225 μ m .) than are required to induce similar effects in normal red cells (450 μ m ). Malonyldialdehyde (MDA) formation occurs later than the other effects and can be inhibited by the antioxidant, butylated hydroxytoluene (BHT), without affecting protein aggregation or potassium leak. Incubation of normal red cells directly with MDA induces protein aggregation, but only after 1 h of incubation. Although nystatin-sucrose treated normal cells which are dehydrated with altered cation content, and therefore osmotically resistant, do not display abnormal H₂O₂ hypersentitivity as manifested by excessive potassium permeability, they do show an increase in methaemoglobin formation and protein aggregation similar to xerocytes. These data indicate that membrane protein cross-linking occurring immediately following H₂O₂ exposure seems independent of either the sulfhydryl or MDA mechanisms, and that the membrane permeability of the abnormal red cells predisposes them to oxidative damage.     

Decreasing levels of tumour necrosis factor alpha and interleukin 6 during lowering of body mass index with orlistat or placebo in obese subjects with cardiovascular risk factors

Aim:   Obesity is associated with increased levels of inflammatory mediators. The objective of this study was to evaluate changes in the leucocyte derived inflammatory mediators tumour necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and the isoprostane 8-epi-prostaglandin (PG) F_2α during BMI lowering with orlistat (Xenical^®, Roche) or placebo.
Methods:   TNF-α, IL-6, and 8-epi PGF_2α evaluated in 376 subjects aged 18–75 years with BMI 28–38 kg/m² before and after 1 year of double-blind, randomized treatment with orlistat 120 mg or placebo three times daily.
Results:   Weight reduction was associated with decreasing (p < 0.001) levels of TNF-α and IL-6 in both orlistat and placebo groups. After 12 months, TNF-α was lower (p < 0.05) in the orlistat compared with the placebo group. In the orlistat group, the change in TNF-α correlated with change in s-glucose (r = 0.22; p = 0.01), and the change in 8-epi-PGF_2α correlated with changes in s-cholesterol (r = 0.27; p < 0.001) and s-LDL-cholesterol (r = 0.28; p < 0.001).
Conclusion:   Weight reduction was associated with decreasing levels of both TNF-α and IL-6. After 12 months of treatment, TNF-α levels were lower in orlistat than in placebo-treated subjects. Whether these results translate into reduced incidence of cardiovascular disease remains to be elucidated.     

HBcAg-pulsed dendritic cell vaccine induces Th1 polarization and production of hepatitis B virus-specific cytotoxic T lymphocytes

Aim:  Dendritic cells (DCs) pulsed with HBsAg efficiently reverse the immune tolerance to hepatitis B virus (HBV) and induce HBV-specific cytotoxic T lymphocyte (CTL) responses in transgenic mice and healthy volunteers. However, it is not clear whether HBV core antigen (HBcAg)-pulsed DCs can effectively induce CD4⁺ helper T cells polarization into Th1, which contribute to the induction and maintenance of HBV-specific CD8⁺ T cells in chronic hepatitis B (CHB) patients. To address this issue, we conducted this study and investigated whether HBcAg-pulsed DCs could polarize Th1 cells and induce an HBcAg-specific CTL response.
Methods:  HBcAg-pulsed DCs were generated from 21 CHB patients. The capacity of the HBcAg-pulsed DC vaccine to stimulate CD4⁺ and CD8⁺ T cells to produce IFN-γ and IL-4 was estimated by intercellular cytokine staining, and the HBcAg-pulsed DCs derived from 10 humam leucocyte antigen (HLA)-A2⁺ CHB patients were tested for the induction of HBV-specific CTLs from autologous T cells by pentamer staining. The cytotoxicity of these CTLs was evaluated in vitro by flow cytometry.
Results:  The HBcAg-pulsed DCs derived from CHB patients exhibited a stronger capacity to stimulate autologous CD4⁺ and CD8⁺ T cells to release IFN-γ rather than IL-4, which could induce HBV core 18-27 specific CTLs, suggesting a specific cytotoxicity against T2 cells that had been loaded with the HBV core 18-27 peptide in vitro .
Conclusion:  HBcAg-pulsed DC vaccine derived from CHB patients efficiently induced autologous T cell polarization to Th1 and generation of HBV core 18-27 specific CTLs.     

Effects of all-trans-retinoic acid on superoxide generation in intact neutrophils and a cell-free system

To determine the pathophysiology of the retinoic acid syndrome which occurs during all- trans -retinoic acid (ATRA) treatment of acute promyelocytic leukaemia patients, we investigated the direct effects of ATRA on the function of human neutrophils. We found that ATRA (10–200 μ M ) dose-dependently stimulated superoxide (O⁻₂) generation in intact neutrophils. The maximal activity of ATRA-stimulated O⁻₂ generation was 3.0 nmol/min/10⁶ cells. Adding EGTA to the assay mixture did not affect the activity nor was the intracellular free calcium concentration changed upon stimulation. The treatment of neutrophils with 0.1 μ M staurosporine, an antagonist of protein kinase C, for 10 min, enhanced the activity of ATRA-stimulated O⁻₂ generation up to 186% of that for control samples. Wortmannin (1 μ M ), an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), reduced this stimulatory activity by 67%. These results suggest that ATRA activates the signalling pathway related to PI 3-kinase rather than that utilizing calcium and protein kinase C. ATRA enhanced the O⁻₂ generated in a sodium-dodecyl-sulphate (SDS) cell-free system, resulting in rates up to 288% higher than that seen with SDS alone. This enhancement was not affected by pretreatment with staurosporine or wortmannin. ATRA may thus directly activate and/or enhance the function of neutrophils.     

Daily administration of the GIP-R antagonist (Pro3)GIP in streptozotocin-induced diabetes suggests that insulin-dependent mechanisms are critical to anti-obesity-diabetes actions of (Pro3)GIP

Aim:  Glucose-dependent insulinotropic polypeptide-receptor (GIP-R) antagonism using (Pro³)GIP improves glucose tolerance and ameliorates insulin resistance and abnormalities of islet structure and function in a commonly used model of obesity-diabetes, namely ob/ob mice. The effect of GIP-R antagonism in a streptozotocin (STZ)-induced model of insulin deficiency has not been evaluated. The present study has investigated the effects of daily administration of (Pro³)GIP to STZ-treated mice.
Methods:  Swiss TO mice received once-daily injection of (Pro³)GIP (25 nmol/kg body weight) or saline 4 days prior to and 16 days after injection of STZ, and effects on metabolic parameters and islet architecture were assessed.
Results:  (Pro³)GIP treatment had no significant effect on hyperphagia or body weight loss. However, hyperglycaemia and glycated haemoglobin were worsened, glucose tolerance further decreased and insulin sensitivity was impaired by (Pro³)GIP. These effects were observed on an STZ-induced background characterized by severe reductions of circulating insulin, beta-cell mass and pancreatic insulin stores.
Conclusions:  These data indicate that the beneficial actions of the GIP-R antagonist, (Pro³)GIP, in obesity-diabetes appear to be largely mediated through insulin-dependent mechanisms that merit further investigation.     

Ventricular arrhythmias following exposure of failing hearts to oxidative stress in vitro

Introduction: There is experimental evidence that heart failure (HF) is an oxidative stress and that HF myocytes may be damaged by oxygen-derived free radicals. However, the arrhythmogenicity of these radicals has not been studied in HF.
Methods and Results: Isolated perfused hearts were obtained from sham-operated (SHAM, n = 6), and fast pacing (250 ms, 2 weeks)-induced heart failure porcines (HF, n = 8). Epicardial conduction was mapped in the longitudinal and transverse directions and ventricular arrhythmias were closely monitored after perfusion of 100, 300, and 1000 μmol/L H ₂ O ₂. Left ventricular epicardium was sampled for action potentials recordings in the same conditions. Myocardial levels of thiobarbituric acid reactive substances and antioxidant enzymatic capacity were also assessed. Epicardial conduction velocities were unaffected by H ₂ O ₂ in both groups. Isolated ventricular premature beats and runs of slow ventricular rhythm with H ₂ O ₂ more frequently occurred in HF compared to SHAM despite an increased antioxidant capacity including Cu/Zn and Mn superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase. Sustained arrhythmias were not observed. Higher thiobarbituric acid reactive substances levels were found in HF confirming endogenous oxidative stress. Action potential duration at plateau level was increased following H ₂ O ₂ in SHAM but not in HF epicardial fibers where a toxic effect developed at 1000 μmol/L.
Conclusion: Oxidative stress with concomitant increase in antioxidant capacity develops in this HF model. There is a greater proclivity to oxidative stress-mediated arrhythmias in HF. These arrhythmias are mainly extrasystoles or slow ventricular rhythms and not dependent on abnormal myocardial conduction.     

Synergistic interaction of the histone deacetylase inhibitor SAHA with the proteasome inhibitor bortezomib in mantle cell lymphoma

Objectives:  Mantle cell lymphoma (MCL) is an incurable B cell lymphoma, and novel treatment strategies are urgently needed. We evaluated the effects of combined treatment with the proteasome inhibitor bortezomib and the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) on MCL. Bortezomib acts by targeting the proteasome, and – among other mechanisms – results in a reduced nuclear factor-kappa B (NF-κB) activity. HDACi promote histone acetylation, and also interfere with NF-κB signaling.
Methods:  Human MCL cell lines (JeKo-1, Granta-519 and Hbl-2) were exposed to bortezomib and/or SAHA. Cell viability and apoptosis were quantified by the MTT and annexin-V assay, respectively. Reactive oxygen species (ROS) were analyzed using the fluorophore H₂DCFDA. In addition, activated caspases, proteasome- and NF-κB activity were quantified.
Results:  Combined incubation with bortezomib and SAHA resulted in synergistic cytotoxic effects, as indicated by combination index values <1 using the median effect method of Chou and Talalay. The combination of both inhibitors led to a strong increase in apoptosis as compared to single agents and was accompanied by enhanced ROS generation, while each agent alone only modestly induced ROS. The free radical scavenger N -acetyl- l -cysteine blocked the ROS generation and reduced the apoptosis significantly. In addition, coexposure of bortezomib and SAHA led to increased caspase-3, -8 and -9 activity, marked reduction of proteasome activity and decrease of NF-κB activity.
Conclusions:  This is the first report giving evidence that SAHA and bortezomib synergistically induce apoptosis in MCL cells. These data build the framework for clinical trials using combined proteasome and histone deacetylase inhibition in the treatment of MCL.     

Monitoring of CD95 and CD38 expression in peripheral blood T lymphocytes during active human cytomegalovirus infection after orthotopic liver transplantation

Aim:  The aim of the present study was to quantitatively monitor the response of CD95 molecules expressed on CD3⁺ T cells (CD95⁺CD3⁺ cells) and CD38 molecules expressed on CD8⁺ T cells (CD38⁺CD8⁺ cells) to ganciclovir treatment after orthotopic liver transplant (OLT) in recipients with active human cytomegalovirus (HCMV) infection.
Methods:  Blood samples were collected from 20 liver transplanted recipients with active HCMV infection and 24 recipients without HCMV infection. CD95⁺CD3⁺ cells and CD38⁺CD8⁺ cells were quantitatively detected with QuantiBRITE bead methods by dual-color flow cytometry analysis during the post-transplantation period.
Results:  CD95⁺CD3⁺ cells and CD38⁺CD8⁺ cells were not significantly different among different ages of healthy adults ( P  > 0.05). CD95⁺CD3⁺ cells and CD38⁺CD8⁺ cells were drastically increased in the active HCMV infection group compared with that in the stable group or in the healthy group ( P  < 0.001), and then they were gradually decreased within the next several weeks after ganciclovir treatment when compared with active HCMV infection recipients ( P  < 0.001).
Conclusions:  The present study showed that CD38⁺CD8⁺ T cells can be an appropriate immunological marker for early detection and antiviral therapeutic monitoring of HCMV infection. The evaluation of CD95 molecule levels may be used routinely in clinical practice to assess the level of immunosuppression.     

Antimony in plasma and skin of patients with cutaneous leishmaniasis – relationship with side effects after treatment with meglumine antimoniate

Objective  To evaluate the levels of antimony in plasma and skin of patients being treated with pentavalent antimonials (Glucantime^®) and their relationship with side effects.
Methods  We evaluated 19 patients treated endovenously at the conventional dose (20 mg Sb^v/kg/day), two at a smaller dose (5 mg Sb^v/kg/day) and three treated intralesionally (up to 4.0 ml/week). During treatment, patients underwent periodic blood exams and were interviewed weekly about the incidence of adverse symptoms. The levels of antimony in plasma and skin samples were determined by Inductively Coupled Plasma with Mass Spectrometry (ICP-MS).
Results  The patients under conventional treatment presented a mean initial antimony plasma concentration of 3.39 μg/l; at the end of treatment, these levels were 0.21 before Glucantime^® application and 125.8 mg after Glucantime^® application. The mean antimony level in their skin at the end of the treatment was 9.24 μg/g. The main adverse symptoms were arthralgia and myalgia; laboratory results showed mainly lymphocytosis and eosinophilia.
Conclusions  We found some significant correlations between antimony concentrations, adverse symptoms and laboratory alterations, strengthening the hypothesis of a dose–dependent relationship between antimony concentration in plasma and skin and side effects.