The influence of glutathione modulators on the course of Leishmania major infection in susceptible and resistant mice

Glutathione (GSH) has an important dual role in parasite-host relationship in Leishmania major infection. Our previous studies showed that both antioxidant systems, glutathione and trypanothione/trypanothione reductase, participate in the protection of Leishmania against the toxic effect of nitrogen-derived reactive species. On the other hand, GSH also is very important to the modulation of the effective immune response, inducting NO production and leishmanicidal activity of macrophages. In the present study, we investigated the role of host GSH during the course of L. major infection, analysing the size of footpad lesions and parasite load from mice treated with two GSH modulators, N-acethyl-l-cysteine (NAC) and buthionine sulphoximine (BSO). Resistant mice treated with BSO, which depletes GSH develop exacerbated lesions, but only harbour higher parasite load in their lesions 2 weeks post-infection. Although the NAC treatment does not affect the footpad lesions development in susceptible BALB/c mice, it significantly reduced the tissue parasitism in the lesions throughout the course of infection. Interestingly, the treatment with BSO did not change the course of L. major infection on susceptible mice when compared with nontreated mice. These results suggest that GSH is an important antioxidant modulator during anti-Leishmania immune response in vivo.     

己酮可可碱对肺结节病患者肺泡巨噬细胞释放细胞因子的作用

目的　研究己酮可可碱 (POF)对肺结节病患者肺泡巨噬细胞 (AM)产生细胞因子的作用 ,并与地塞米松 (DEX)的作用相比较。方法　收集 1 4例活动期肺结节病患者的AM ,以 1 0 %RPMI为培养液 (含有 1 0 %热灭活胎牛血清、2mmol/LL 谷氨酰胺、2 0 0kU/L青霉素及 2 0 0mg/L链霉素 ) ;或1 0 %RPMI加内毒素 (LPS ,1 0 0 μg/L) ;或分别加入浓度为 0 0 1mmol/L、0 1mmol/L和1mmol/L的POF ;或加入 0 1mmol/LDEX进行AM培养 2 4h。用酶联免疫吸附 (ELISA)法测定培养上清液中细胞因子含量。结果　POF对结节病患者AM自发释放的肿瘤坏死因子α(TNF α)有剂量依赖性抑制作用 (P <0 0 0 1 ) ,而对其他自发释放的细胞因子无影响。 0 1mmol/LDEX抑制自发释放的TNF α、可溶性肿瘤坏死因子受体 (sTNFR 2 )、白细胞介素 (IL) 1 β和IL 1 0 (P <0 0 0 1或 <0 0 5 或 <0 0 1 )。除sTNFR 1外 ,POF亦抑制这些由LPS刺激的AM释放的细胞因子 (P <0 0 5或 <0 0 0 1 )。与POF相似 ,0 1mmol/LDEX同样抑制这些LPS刺激的细胞因子释放 (P <0 0 5或 <0 0 0 1 ) ,但对sTNFR 1和IL 1 β无影响。 结论　与DEX相比 ,POF有更宽的治疗窗。用在结节病治疗上可以减少皮质激素用量或可将其替代。然而POF治疗结节病及其他肺部疾病的临床价     

Inhibition of cytokine release from alveolar macrophages in pulmonary sarcoidosis by pentoxifylline: comparison with dexamethasone

STUDY OBJECTIVES: Pentoxifylline (POF) has been shown to suppress the cytokine production from lipopolysaccharide (LPS)-stimulated monocytes/alveolar macrophages (AMs). Sarcoidosis is a granulomatous disease that is driven by the action of tumor necrosis factor (TNF)-alpha and other proinflammatory cytokines. In this study, we aimed to investigate the effects of POF on the production of TNF-alpha, interleukin (IL)-1 beta, IL-6, IL-8, IL-10, and the soluble TNF receptors (sTNFRs) 1 and 2 from AMs in sarcoidosis, and we also compared them with those of dexamethasone (DEX). METHODS: AMs from 14 patients with sarcoidosis were cultured for 24 h with RPMI medium alone or with LPS (100 ng/mL), and with POF at concentrations of 0.01, 0.1, and 1 mmol/L, or with 0.1 mmol/L DEX. Cytokines in the culture supernatants were analyzed by enzyme-linked immunosorbent assay. RESULTS: The results showed that POF induced a dose-dependent suppression of the spontaneous TNF-alpha release from AMs in sarcoidosis (p < 0.001), and that the spontaneous release of the other cytokines was unaffected by POF at all tested concentrations, but a trend for the inhibition of IL-10 production was found (p = 0.092). DEX inhibited the spontaneous release of TNF-alpha (p < 0.001), sTNFR2 (p < 0.05), IL-1 beta (p < 0.05), and IL-10 (p < 0.01). POF also suppressed the LPS-stimulated production of these cytokines except for that of sTNFR1. Similar to POF, DEX inhibited the LPS-stimulated production of these cytokines, but not that of sTNFR1 and IL-1 beta. CONCLUSIONS: Compared with DEX, POF may improve therapeutic regimens in patients with sarcoidosis either by sparing or by replacing corticosteroids. However, the precise clinical value of POF in the treatment of sarcoidosis and other lung diseases will have to be determined in further clinical trials.     

Hydrogen peroxide release from alveolar macrophages and alveolar type II cells during adaptation to hyperoxia in vivo.

The effect of hyperoxia (1-14 days, 85% O2) on rat alveolar macrophage and alveolar type II cell oxidant and antioxidant characteristics was investigated. Unstimulated control macrophages (2 h ex vivo) released hydrogen peroxide at a rate of 3.5 +/- 1.3 nmol/min mg protein-1, which was a cyanide-sensitive process. H2O2 release from alveolar macrophages decreased slightly but not significantly after 1 day in hyperoxia and increased significantly after 3 days (180%, p less than .05) and 14 days (380%, p less than .01). When H2O2 release was expressed as nmol from total macrophages per animal, the increase after 14 days in hyperoxia was 760%. H2O2 generation by hyperoxic macrophages was cyanide resistant, indicating the involvement of active NADPH oxidase. In both control and hyperoxic macrophages H2O2 release could be significantly stimulated with phorbol myristate acetate (PMA). Comparisons of H2O2 release by freshly isolated alveolar macrophages and alveolar type II cells must be cautiously interpreted because some cell functions may change during the isolation procedure. Freshly isolated (6 h ex vivo) control alveolar type II cells were found to generate H2O2 at a rate of 0.26 +/- 0.05 nmol/min mg protein-1. In type II cells H2O2 release, calculated as nmol/mg protein, decreased during the first 7 days of hyperoxia to 10% (p less than .01) of the control value and then returned back up to the control level after 14 days. A similar decrease was observed if H2O2 release was calculated as nmol/cell number. H2O2 release from control and hyperoxic type II cells was cyanide sensitive. The decrease in H2O2 release in type II cells was associated with cell membrane injury (as assessed by electron microscopy), while biochemical markers of cellular injury (trypan blue exclusion and cellular high-energy phosphates ATP, ADP) were unchanged. The ability of type II cells to scavenge extracellular H2O2 did not change in acute hyperoxia, but it increased significantly during the second week in hyperoxia. These results indicate that macrophages but not type II cells are stimulated to produce H2O2 during prolonged exposure to hyperoxia.     

Intracellular calcium release mediated by noradrenaline and acetylcholine in mammalian pineal cells

Abstract: The effects of noradrenergic and cholinergic receptor agonists on intracellular Ca²⁺ concentration ([Ca²⁺]_i) in single dissociated rat pineal cells were investigated by microfluorimetric measurements in Fura-2 acetoxymethyl ester (Fura-2/AM) loaded cells. Noradrenaline (NA) evoked characteristic biphasic increments of intracellular Ca²⁺ consisting of one or more leading spikes followed by a plateau, resulting from the release of Ca²⁺ from intracellular stores and from the influx of Ca²⁺ from the external medium, respectively. This response was reproduced by the α₁ adrenoceptor agonist, phenylephrine (PE), in the presence of the β-adrenoceptor antagonist, propranolol, and was abolished when NA or PE was applied in conjunction with the α₁-adrenoceptor antagonist, prazosin. The curve relating the peak amplitude of the Ca²⁺ increments to different PE concentrations (0.5–10 μM) showed a half-maximum response at 0.6 μM PE, and saturation at concentrations greater than 2 μM. Acetylcholine (ACh) also elicited transient Ca²⁺ increments consisting of an abrupt rise to a maximum value which decayed exponentially to the basal Ca²⁺ level. A half-maximum response was achieved at 59 μM ACh. The muscarinic cholinergic receptor agonist, carbachol (CCh), similarly activated Ca²⁺ increments while the muscarinic antagonist, atropine, abolished them. In the absence of extracellular Ca²⁺, repetitive stimuli with either α₁-adrenergic and muscarinic agonists produced a progressive decrement in the amplitude of the Ca²⁺ signals because of the depletion of intracellular stores. However, extinction of the response to muscarinic agonists did not preclude a response to adrenergic agonists, while the contrary was not true. These results suggest that these agonists liberate Ca²⁺ from two functionally distinct, caffeine-insensitive, Ca²⁺ intracellular stores.     

Mitochondrial glutathione replacement restores surfactant synthesis and secretion in alveolar epithelial cells of ethanol-fed rats

BACKGROUND: Chronic alcohol abuse increases the incidence and severity of acute lung injury in critically ill patients. Previously we determined that ethanol ingestion in rats dramatically decreased alveolar epithelial cellular levels of glutathione and surfactant synthesis and secretion in vitro. Previous studies in alcoholic liver disease suggest that mitochondrial glutathione levels, and not cellular levels per se, are involved in the pathogenesis of ethanol-mediated hepatotoxicity. Therefore, we hypothesized that alveolar epithelial mitochondrial glutathione depletion mediates the observed defects in surfactant synthesis and secretion in ethanol-fed rats. METHODS: Male Sprague-Dawley rats were fed the Lieber-DeCarli liquid diet with or without ethanol (36% of total calories) for 6 weeks. In some experiments, ethanol-fed rats were then switched to the control diet for 1 week, with or without glutathione supplementation with either N-acetylcysteine (NAC) or procysteine (PRO). Alveolar epithelial type II cells were then isolated and glutathione levels (cytosolic and mitochondrial) and surfactant production (synthesis and secretion) were determined. RESULTS: Ethanol ingestion decreased (p < 0.05) mitochondrial and cytosolic levels of glutathione, and surfactant synthesis and secretion in isolated type II cells when compared to cells from control-fed rats. NAC treatment restored (p < 0.05) cytosolic but not mitochondrial glutathione levels (p > 0.05), and had no effect (p > 0.05) on surfactant synthesis and secretion in type II cells isolated from ethanol-fed rats. In contrast, PRO treatment restored (p < 0.05) cytosolic and mitochondrial glutathione levels, and normalized (p < 0.05) surfactant synthesis and secretion in type II cells isolated from ethanol-fed rats. CONCLUSIONS: These results suggest that mitochondrial, and not simply cytosolic, replacement of glutathione is necessary to improve surfactant function in critically ill patients with a history of alcohol abuse.     

巨噬细胞刺激蛋白对烟熏大鼠肺泡巨噬细胞氧化应激和细胞因子产生的影响

目的 探讨巨噬细胞刺激蛋白(MSP)对烟熏大鼠肺泡巨噬细胞氧化应激和细胞因子产生的影响.方法 培养正常和烟熏不同时间(1个月、2个月、3个月)的大鼠肺泡巨噬细胞,给予不同浓度MSP处理24 h,采用酶联免疫法检测细胞上清液中细胞因子肿瘤坏死因子α(TNF-α)、白介索8(IL-8)和IL-1β的浓度,比色法检测细胞上清液中丙二醛(MDA)和超氧化物歧化酶(SOD)的水平.结果 ①MSP呈浓度依赖性促进正常组和各烟熏组大鼠肺泡巨噬细胞分泌TNF-α、IL-8和IL-1β;经MSP处理后,各烟熏组大鼠肺泡巨噬细胞上清液中TNF-α、IL-8和n-1β浓度均高于正常组(P＜0.05);大鼠肺泡巨噬细胞上清液中TNF-α、IL-8和IL-1β浓度随烟熏时间延长呈时间依赖性增加.②MSP呈浓度依赖性促进正常组和各烟熏组大鼠肺泡巨噬细胞分泌MDA,抑制其产生SOD;烟熏2个月组和烟熏3个月组大鼠肺泡巨噬细胞上清液中MDA水平均高于正常组(P＜0.05),SOD水平均低于正常组(P＜0.05);随着烟熏时间延长,大鼠肺泡巨噬细胞上清液中MDA水平呈时间依赖性增加,SOD水平呈时间依赖性降低.结论 MSP呈浓度依赖性促进正常和烟熏大鼠肺泡巨噬细胞分泌TNF-α、IL-8、IL-1β和MDA,抑制其产生SOD.MSP促烟熏大鼠肺泡巨噬细胞分泌TNF-α、IL-8、IL-1β、MDA及抑制其产生SOD的作用较正常大鼠更显著,且烟熏时间越长此作用越明显.     

Suppression by Candida albicans beta-glucan of cytokine release from activated human monocytes and from T cells in the presence of monocytes

The effect of a soluble beta-glucan from Candida albicans (CSBG) on cytokine production by cultured human peripheral blood mononuclear cells (PBMC) was assessed. CSBG induced a slight increase in the spontaneous release of proinflammatory cytokines, such as interleukin (IL)-6, but significantly suppressed endotoxin-induced IL-6 production in cultures of PBMC and monocytes isolated from PBMC. CSBG also suppressed the release of type 1 cytokines, IL-2, and interferon-gamma. These findings suggest that CSBG suppresses monocyte functions directly and thus suppresses T cell function indirectly. CSBG may play a role in the development of candidiasis.     

Intracellular mechanisms involved in the stimulation of growth hormone release from rat anterior pituitary cells by Russell's viper venom.

We have previously shown that a heat-stable component of Russell's viper venom (RVV) releases GH in a dose-dependent manner from cultured rat anterior pituitary cells. We have now investigated the intracellular mechanisms involved in RVV-stimulated GH release by concomitant administration of RVV with known intracellular mediators in rat pituitary cells. 3-Isobutyl-1-methylxanthine (IBMX; 0.5 mmol/l), added to cultured rat anterior pituitary cells simultaneously with RVV, at concentrations up to a maximally effective dose of 10 micrograms/ml, increased GH release (3.7-fold, 4.0-fold and 2.0-fold; P less than 0.001) compared with the effect of venom alone. These effects were additive, indicating that RVV and IBMX stimulate through different intracellular messengers. RVV failed to increase the formation of basal or IBMX-stimulated intracellular cyclic AMP (cAMP), confirming that RVV affects GH release through a cAMP-independent pathway. 12-0-Tetradecanoylphorbol-13-acetate (TPA; 0.1 mumol/l), added simultaneously with various doses of RVV (0.1-10 micrograms/ml), did not increase GH release beyond the maximal effect of RVV. This result indicates that RVV might be stimulating GH release through a similar mechanism to that of TPA (by activating protein kinase C). When pituitary cells were perifused with Ca(2+)-free medium or verapamil (50 mumol/l), RVV-stimulated GH release was inhibited by 65 and 42% respectively. This reflects the recognized requirement of Ca2+ for secretory processes. However, RVV (10 micrograms/ml) had no significant effect on intracellular free Ca2+ concentrations as measured using the fluorescent Ca2+ probe quin-2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-related defects in TLR2 signaling diminish the cytokine response by alveolar macrophages during murine pneumococcal pneumonia

Alveolar macrophages (AMs) are the first immune cells to respond to an invading pathogen and coordinate the inflammatory response within the lungs. Studies suggest that macrophages exhibit age-related deficiencies in Toll-like receptor (TLR) function; however, the impact of this dysfunction during pneumonia, the leading cause of infectious death in the elderly, and the underlying mechanisms responsible remain unclear. We examined disease severity in young, mature, and aged BALB/cBy mice following intratracheal infection with the Gram-positive bacteria Streptococcus pneumoniae (Spn). Both mature and aged mice failed to clear bacteria and as a result had increased mortality, tissue damage and vascular leakage. Early production of TNFα, IL-1β, and IL-6 during pneumonia declined with age and was associated with an inability of isolated AMs to respond to pneumococcal cell wall (CW) and ethanol-killed Spn ex vivo. Total levels of TLR1 were unaffected by age and TLR2 surface expression was slightly yet significantly increased on aged AMs suggesting that intracellular TLR signaling defects were responsible for the age-related decline in cytokine responsiveness. Following infection of isolated AMs with live Spn, a significant age-related decline in TLR2-induced phosphorylation of p65 NFκB, JNK and p38 MAPK, and an increase in ERK phosphorylation was observed by immunoblotting. These data are the first to demonstrate that TLR2-dependent recognition of Spn by aged AMs is impaired and is associated with a delayed pro-inflammatory cytokine response in vivo along with enhanced susceptibility to pneumococcal pneumonia.     

The innate immune response in calves to Boophilus microplus tick transmitted Babesia bovis involves type-1 cytokine induction and NK-like cells in the spleen

The innate immune response to Babesia bovis infection in cattle is age-related, spleen-dependent and, in stabilate inoculated calves, has type-1 characteristics, including the early induction of IL-12 and IFN-gamma. In this study with three calves, parameters of innate immunity were followed for 2 weeks after tick transmission of B. bovis. Each calf survived the acute disease episode without drug intervention, and responded with increased levels of plasma interferon-gamma and type-1 cytokine expression, monocyte/macrophage activation, and CD8+ cellular proliferation in the spleen. The proliferating CD8+ population consisted primarily of NK-like cells, and the expansion occurred in parallel with an increase in IL-15 mRNA expression in the spleen.     

Primary cilia and the reciprocal activation of AKT and SMAD2/3 regulate stretch-induced autophagy in trabecular meshwork cells

Activation of autophagy is one of the responses elicited by high intraocular pressure (IOP) and mechanical stretch in trabecular meshwork (TM) cells. However, the mechanosensor and the molecular mechanisms by which autophagy is induced by mechanical stretch in these or other cell types is largely unknown. Here, we have investigated the mechanosensor and downstream signaling pathway that regulate cyclic mechanical stretch (CMS)-induced autophagy in TM cells. We report that primary cilia act as a mechanosensor for CMS-induced autophagy and identified a cross-regulatory talk between AKT1 and noncanonical SMAD2/3 signaling as critical components of primary cilia-mediated activation of autophagy by mechanical stretch. Furthermore, we demonstrated the physiological significance of our findings in ex vivo perfused eyes. Removal of primary cilia disrupted the homeostatic IOP compensatory response and prevented the increase in LC3-II protein levels in response to elevated pressure challenge, strongly supporting a role of primary cilia-mediated autophagy in regulating IOP homeostasis.

The trabecular meshwork (TM) is a pressure-sensitive tissue located in the anterior segment of the eye and key regulator of intraocular pressure (IOP). Malfunction of this tissue results in improper drainage of aqueous humor outflow, leading to ocular hypertension, the major risk factor for developing glaucoma (1–3). The TM consists of an irregular lattice of collagen beams lined by TM endothelial-like cells, followed by a zone of loose connective tissue containing TM cells, through which aqueous humor must pass before leaving the eye. Changes in pressure gradients and fluid flow associated with eye movement, circadian rhythm, or the ocular pulse cause small and high variations in IOP, which are translated in continuous cycles of tissue deformation and relaxation. Cells in the TM are known to be able to sense these deformations as mechanical forces and respond to them by eliciting a variety of responses, including reorganization of actin cytoskeleton, changes in gene expression, secretion of cytokines, modulation of matrix metalloproteinases, and extracellular-matrix remodeling (reviewed in ref. 4). It is believed that these mechanoresponses are critical regulators of IOP homeostasis; however, the mechanosensors and the downstream mechanotransduction signaling in TM have still not been characterized.Our laboratory has identified the activation of macroautophagy (hereafter autophagy) as one of the responses elicited in TM cells following application of static or cyclic mechanical stretch (CMS) (5–7). Activation of autophagy was also observed quickly after pressure elevation in porcine perfused eyes (5), which prompted us to propose autophagy as a crucial physiological response to adapt to mechanical forces and maintain cellular homeostasis. The exact roles of autophagy in TM cells and tissue function are still under investigation. Most recently, we have shown the CMS-induced translocation of the autophagy marker LC3 (microtubule-associated protein 1 light chain 3) to the nuclear compartment, where it associates with the nucleolus and interacts with the ribophagy receptor NUFIP1 (nuclear FMR1 interacting protein 1), suggesting a potential role of CMS-induced autophagy in surveillance of the nucleolus activity (6). Furthermore, we have also recently provided direct evidence demonstrating autophagy as a regulator of TGF-β/SMAD-induced fibrogenesis in TM cells (8).Autophagy is a fundamental process for degradation or recycling of intracellular components, which is essential to maintain cellular homeostasis. Autophagy occurs constitutively at basal levels, but it is quickly activated upon several types of stress, such as nutrient depletion, pathogen infections, drug treatment, accumulation of aggregated proteins and damaged organelles, and mechanical stress (7, 9–11). The molecular mechanisms by which cells recognize stress and regulate autophagic activity are very complex and differ based on the stimuli. A variety of components, for example, receptor tyrosine kinases, second messengers (Ca²⁺ or cAMP [cyclic adenosine monophosphate]), protein kinases, and downstream autophagy-related (ATG) complexes, participate in the regulation of autophagy (10). Among them, the best characterized is MTOR (mechanistic target of rapamycin kinase), a negative regulator of autophagy (10, 12, 13). Although MTOR acts as a core sensor in autophagic regulation, numerous studies have shown the MTOR-independent autophagy activation upon different stresses (10). Indeed, our own study and that of King et al. showed that the induction of autophagy in response to mechanical stress occurs in an MTOR-independent manner (5, 14). The mechanosensor and the downstream signaling pathway responsible for the activation of autophagy in response to stretching have still not been identified.Primary cilium (PC) is a nonmotile cell-surface projection found almost ubiquitously in vertebrate cells, which acts as a cellular antenna that senses a wide variety of signals, including chemical and mechanical stimuli (15–19). PC plays a critical role in smell, sight, and mechanosensation. PC defects are associated with a number of human diseases termed ciliopathies. The most common feature in patients affected with ciliopathies include visual dysfunction (16). In particular, Lowe syndrome patients often develop ocular hypertension and glaucoma (20). Structurally, the PC consists of a microtubule-based core structure, called axoneme, and a basal body, which is a derivative of the centriole of centrosome from which axoneme is extended and surrounded by ciliary membrane (21). The ciliary membrane is a specialized domain extension of the plasma membrane enriched on signaling receptors and channels, including hedgehog (Hh) and Ca²⁺ pathways, which enables the PC to function as a signaling hub (16, 22, 23). Cargo trafficking into and out of the cilium is mediated by a specialized form of vesicle trafficking, named intraflagellar transport (IFT), that is composed of a multiprotein complex (16, 23).Recent studies have demonstrated the reciprocal relationship between PC and autophagy. Autophagy has been shown to both positively and negatively regulate ciliogenesis. Under nutrient-rich conditions, basal autophagy inhibits cilia growth by limiting trafficking of PC components to the basal body through direct degradation of IFT20 (24). In contrast, nutrient starvation triggers the autophagic degradation of oral-facial-digital syndrome 1 and promotes cilia growth (24, 25). Conversely, functional PC are required for activation of autophagy in response to starvation and fluid flow. In both cases, autophagy was initiated by the recruitment of ATG16L to the basal body, suggesting that this event is a hallmark for PC-induced autophagy. Intriguingly, the signaling pathway mediating PC-induced autophagy activation differed based on the stimuli. While Hh/smoothened (SMO) was reported to mediate activation of autophagy in response to starvation (24), the LKB1–AMPK–MTOR signaling pathway was found to regulate PC-induced autophagy and cell volume in kidney epithelial cells under shear stress (26, 27). Whether PC are also involved in the regulation of autophagy triggered by mechanical stretching has not yet been explored.The purpose of this study is to investigate a potential role of PC in stretch-induced autophagy in TM cells. Here, we report that PC acts as a mechanosensor for CMS-induced autophagy, and we identified AKT1 and SMAD2/3 as critical components of the signal mechanotransduction.     

Removal of glutathione produces apoptosis and necrosis in HepG2 cells overexpressing CYP2E1

BACKGROUND: Previous studies have shown that addition of ethanol, iron, or arachidonic acid to HepG2 cells expressing CYP2E1 produced a loss in cell viability and caused apoptosis. These effects were enhanced when cellular reduced glutathione (GSH) levels were lowered by treatment with buthionine sulfoximine (BSO). Overexpression of CYP2E1 in HepG2 cells could produce toxicity even in the absence of added toxin after BSO treatment. Studies were carried out to characterize this CYP2E1-and BSO-dependent toxicity. METHODS: HepG2 cells expressing CYP2E1 were treated with BSO for 1 to 4 days, and various parameters associated with apoptosis and cell viability were assayed. RESULTS: Treatment of cells expressing CYP2E1 (E47 cells) with BSO resulted in apoptosis as well as necrosis. The apoptosis and necrosis were independent of each other. No toxicity was found with control HepG2 cells or HepG2 cells expressing CYP3A4 instead of CYP2E1 under these conditions. The antioxidant trolox partially prevented the apoptosis and necrosis, whereas diallylsulfide, a CYP2E1 inhibitor, was fully protective. The activity of caspase 3, but not caspases 1, 8, or 9, was increased in the BSO-treated E47 cells, and an inhibitor of caspase 3 prevented apoptosis. Damage to mitochondria appears to play a role in the CYP2E1- and BSO-dependent toxicity, because mitochondrial membrane potential was decreased and cyclosporin A, an inhibitor of the mitochondrial membrane permeability transition, prevented the apoptosis and the necrosis. The fall in membrane potential was prevented by trolox and diallylsulfide, suggesting damage to the mitochondria by CYP2E1-derived reactive oxygen species. CONCLUSIONS: These results indicate the critical role of GSH in protecting against CYP2E1-mediated oxidative stress and that mitochondria may be a target for CYP2E1-derived reactive oxygen species, and suggest that interactions between CYP2E1, mitochondria, and altered GSH homeostasis may play a role in alcohol-induced liver injury.     

Reactive oxygen species–independent activation of the IL-1β inflammasome in cells from patients with chronic granulomatous disease

Humans with chronic granulomatous diseases (CGDs) due to mutations in p47-phox have defective NADPH activity and thus cannot generate NADPH-dependent reactive oxygen species (ROS). The role of ROS in inflammation is controversial; some in vitro studies suggest that ROS are crucial for secretion of IL-1β via inflammasome activation, whereas mice defective for ROS and patients with CGD have a proinflammatory phenotype. In this study, we evaluated activation of the IL-1β inflammasome in cells from CGD patients. In contrast to previous studies using the small molecule diphenylene iodonium (DPI) as a ROS inhibitor, we found no decrease in either caspase-1 activation or secretion of IL-1β and IL-18 in primary CGD monocytes. Moreover, activation of CGD monocytes by uric acid crystals induced a 4-fold higher level of IL-1β secretion compared with that seen in monocytes from unaffected subjects, and this increase was not due to increased synthesis of the IL-1β precursor. In addition, Western blot analysis of CGD cells revealed that caspase-1 activation was not decreased, but rather was increased compared with control cells. Examination of the effects exerted by the inhibition of ROS activity by DPI revealed that the decrease in IL-1β secretion by DPI was actually due to inhibition of IL-1β gene expression. Thus, inconsistent with the proinflammatory role of ROS, the present findings support the concept that ROS likely dampen inflammasome activation. The absence of ROS in CGD monocytes may explain the presence of an inflammatory phenotype characterized by granulomas and inflammatory bowel disease occurring in CGD patients.     

Melatonin stimulates inositol-1,4,5-trisphosphate and Ca2+ release from INS1 insulinoma cells

The effects of melatonin in mammalian cells are exerted via specific receptors or are related to its free radical scavenging activity. It has previously been reported that melatonin inhibits insulin secretion in the pancreatic islets of the rat and in rat insulinoma INS1 cells via Gi-protein-coupled MT1 receptors and the cyclic adenosine 3',5'-monophosphate pathway. However, the inositol-1,4,5-trisphosphate (IP3) pathway is involved in the insulin secretory response as well, and the melatonin signal may play a part in its regulation. This paper addresses the involvement of the second messengers IP3 and intracellular Ca2+ ([Ca2+]i) in the signalling cascade of melatonin in the rat insulinoma INS1 cell, a model for the pancreatic beta-cell. For this purpose melatonin at concentrations ranging from 1 to 100 nmol/L, carbachol and the nonselective melatonin receptor antagonist luzindole were used to stimulate INS1 cell batches, followed by an IP3-mass assay and Ca2+ imaging. Molecular biological studies relating to the mRNA of IP3 receptor (IP3R) subtypes and their relative abundance in INS1 cells showed expression of IP3R-1, IP3R-2 and IP3R-3 mRNA. In conclusion, we found that in rat insulinoma INS1 cells there is a dose-dependent stimulation of IP3 release by melatonin, which is accompanied by a likewise transient increase in [Ca2+]i concentrations. The melatonin effect observed mimics carbachol action. It can be abolished by 30 micromol/L luzindole and is sustained in Ca2+-free medium, suggesting a mechanism that includes the depletion of Ca2+ from intracellular stores.     

The role of chemically induced glutathione and glutathione S-transferase in protecting against 4-hydroxy-2-nonenal-mediated cytotoxicity in vascular smooth muscle cells

4-Hydroxy-2-nonenal (HNE) has been suggested to contribute to the pathogenesis of atherosclerosis. One of the major metabolic transformation pathways of HNE involves conjugation with glutathione (GSH) catalyzed by GSH S-transferase (GST). In this study, we have characterized the induction of GSH and GST by 3H-1,2-dithiole-3-thione (D3T) and the protective effects of the D3T-elevated cellular defenses on HNE-mediated toxicity in rat aortic smooth muscle A10 cells. Incubation of A10 cells with D3T resulted in a marked concentration-dependent induction of both GSH and GST. The induction of cellular GST by D3T also exhibited a time-dependent response. Pretreatment of A10 cells with D3T led to a dramatic decrease of HNE-induced cytotoxicity, as assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay and scanning electron microscopy. Incubation of A10 cells with HNE for 0.5 h and 1 h resulted in a significant depletion of cellular GSH, which preceded the decrease of cell viability. To further demonstrate the involvement of GSH and GST in protecting against HNE-induced cytotoxicity, buthionine sulfoximine (BSO) and sulfasalazine were used to inhibit cellular GSH biosynthesis and GST activity, respectively. Either depletion of GSH by BSO or inhibition of GST by sulfasalazine caused great potentiation of HNE-mediated cytotoxicity. Moreover, cotreatment of A10 cells with BSO was found to completely block the D3T-mediated GSH induction and to largely reverse the cytoprotective effects of D3T on HNE-induced toxicity. Taken together, this study demonstrates that D3T can induce both GSH and GST in aortic smooth muscle cells, and that the D3T-augmented cellular defenses afford a marked protection against HNE-induced vascular cell injury.     

Altered pattern of cytokine production by peripheral blood CD2+ cells from B chronic lymphocytic leukemia patients

To determine if activation-induced cytokine production is altered in CD2+ lymphocytes from B-CLL patients, cytokine levels were determined by ELISA in supernatants of PHA-stimulated cultures of CD2+ cells from 33 B-CLL patients and 22 healthy controls. The production of Interferon γ (IFN-γ) and Tumor Necrosis Factor (TNF-α) by mitogen-activated CD2+ lymphocytes from B-CLL patients was higher than that found in healthy controls, while no differences were found in TNF-β production. IFN-γ and TNF-α levels determined at 72 h in PHA-stimulated CD2+ cell cultures from B-CLL patients statistically correlated with the percentages of CD3+CD45RO+ and CD3−CD56+ lymphocytes, respectively. Although there were differences in the production kinetics of interleukins (ILs) 2 and 4 between B-CLL patients and the healthy controls, no differences were found at the time when the levels of both interleukins peak. The production of both IFN-γ and IL-4 by PHA-stimulated CD2+ lymphocytes from non-smouldering B-CLL patients was significantly higher than that from smouldering B-CLL patients while no significant differences were found in the production of IL-2, TNF-α, and TNF-β between the two B-CLL patient groups. These data suggest that functional alterations in the production of cytokines by CD2+ cells from B-CLL patients could help to explain the expansion of leukemic cells in B-CLL patients. Am. J. Hematol. 57:93–100, 1998. © 1998 Wiley-Liss, Inc.     

Sarilumab (IL-6R antagonist) in critically ill patients with cytokine release syndrome by SARS-CoV2

Blocking IL-6 pathways with sarilumab, a fully human anti–IL-6R antagonist may potentially curb the inflammatory storm of SARS-CoV2. In the present emergency scenario, we used “off-label” sarilumab in 5 elderly patients in life-threatening condition not candidates to further active measures. We suggest that sarilumab can modulate severe COVID-19-associated Cytokine Release Syndrome.