Effect of Lansoprazole on Human Leukocyte Function

Recent findings on the capacity of omeprazole to mfluence various leukocyte functions, in vitro, raises the question on the potential use of protonic pump inhibitors, commonly used in the treatment of acid-secretion- related disorders, as immunomodulators. The aim of this study was to evaluate the in vitro effect of lansoprazole on human natural killer (NK) cell cytotoxix activity, chemotaxis and superoxide anion (02*^-) generation exerted by polymorphonucleated cells (PMNs). NK cytotoxicity activity was assessed by a ⁵¹Cr release assay, PMN chemotaxis was determined by an under agarose method and 02*^- generation was analyzed on the basis of reduced cytochrome C. Incubation times with lansoprazole was 30 min for PMNs and 1-4.5 hours for NK cells, respectively. Lansoprazole induced significant dose dependent bitiion of NK cell activity and PMN functions at concentrations ranging fiom 100 to 1,000μM.

Ths study demonstrate that lansoprazole, like omeprazole, inhibits several leukocyte functions, in vitro, then suggesting that protonic pump inhibitors are able to provoke these effects, at least at certain doses.     

PSK and OK-432-induced immunomodulation of inducible nitric oxide (NO) synthase gene expression in mouse peritoneal polymorphonuclear leukocytes and NO-mediated cytotoxicity

We investigated whether PSK (a polysaccharide from the mycelia of Coriolus versicolor) or OK-432 (a streptococcal preparation) can up-regulate inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production in mouse peritoneal polymorphonuclear leukocytes (PMNs). Six hrs after intraperitoneal injection of mice with PSK (2500 μg/mouse) or OK-432 (100 μg/mouse), mouse peritoneal PMNs were restimulated with PSK (500 μg/ml) or OK-432 (10 μg/ml) plus 100 U/ml of mouse interferon-gamma (IFN-γ) in vitro. Northern blot analysis showed strong synergism between both PSK and OK-432 and IFN-γ for the induction of iNOS gene expression. NO production by PMNs was increased up to 20 μM (2 μM/10⁶ PMNs/24 hrs) as measured by the Griess reagent method when PMNs were restimulated with PSK or OK-432 plus IFN-γ for 24 hrs, although tumor cell killing was not detected. NO concentrations of more than 80 μM were required for P815 tumor cell killing. These results suggest that PMNs produce NO after stimulation with PSK or OK-432 in combination with IFN-γ and may regulate the immune system in vivo, although the NO production induced by these agents is insufficient for tumor cell killing in vitro.     

Commonly used tropical medicinal plants exhibt distinct in vitro antioxidant activities against hepatotoxins in rat liver

Lipid peroxidation in biological systems has been considered as one of the major mechanisms of cell injury in aerobic organisms subjected to oxidation stress. Plants, among other functions, are considered to act as free radical scavengers and as antioxidants. Iron II (Fe²⁺), sodium nitroprusside (SNP) and nitropropionic acid stimulate the production of free radicals and lipid peroxidation. In this study, four commonly used tropical medicinal plants (Kigelia africana, Calotropis procera, Hibiscus sabdariffa and Alchornea cordifolia) were studied (in vitro) for their effects on the formation of thiobarbituric acid reactive substances (TBARS) induced by different pro-oxidants (10 μM FeSO₄, 5 μM -sodium SNP and 2 mM 3-nitropropionic acid) in rat liver homogenate. All the pro-oxidants significantly increased (P<0.05) the formation of TBARS, which indicates increased lipid peroxidation in the rat liver (in vitro). However, all the plant extracts statistically (P<0.05) reduced the production of TBARS in a concentration-dependent manner in all the tested pro-oxidant-induced oxidative stresses. Alchornea cordifolia appeared to offer the highest protection. The results of the present study suggest that the use of these plants in the treatment of various diseases, especially liver disease, is probably due to their ability to act as antioxidants.     

Trichothecene Mycotoxins Depress the Mononuclear-Phagocytic System of Young Turkeys

Macrophage cells isolated from the abdominal cavity of 21-day-old turkeys after a single injection of Sephadex suspension were used to quantitate the effects of direct in vitro exposure to deoxynivalenol (DON), 3-acetyldeoxynivalenol (3ac-DON), scirpentriol (STO), or 15-acetylscirpenol (15-MAS). Macrophage monolayers were established on glass surfaces and cells were exposed to graded levels of individual mycotoxins for 1 hour: DON, 20 -640 μ9/μ1 of culture; 3ac-DON, STO, 15-MAS, 20 -1280 μg/μ1 of culture. All four mycotoxins caused dose-related effects. A concentration of 50 μg/ml DON caused a significant decrease in macrophage adherence, phagocytosis of opsonized SRBC, and number of opsonized SRBC per macrophage; at 200 μg/ml, phagocytosis of unopsonized SRBC was decreased. There were also increasing percentages of damaged macrophages with increasing DON doses as indicated by morphological alterations. Linear decreases in macrophage viability on exposure to 3-acDON and STO were observed. Moreover, STO and 15-MAS decreased macrophage adherence to glass and 3-acDON, STO, and 15-MAS induced macrophage morphological alterations. This study suggests that trichothecene mycotoxins may be immunosuppressive by affecting viability, adherence and phagocytic potential of mononuclear phagocytic cells of young turkeys.     

Inhibitory effect of mast cell-mediated immediate-type allergic reactions by sulfapyridine

We examined the effect of sulfapyridine on mast cell-mediated immediate-type allergic reactions. Sulfapyridine (1 and 10 μg/kg) significantly inhibited systemic allergic reaction induced by compound 48/80 in rats. Sulfapyridine (1 and 10 μg/kg) also inhibited significantly local mast cell-mediated immediate-type allergic reactions activated by anti-dinitrophenyl (DNP) IgE. Moreover, sulfapyridine inhibited histamine release dose-dependently in the rat peritoneal mast cells (RPMC) activated by compound 48/80 or anti-DNP IgE. When sulfapyridine was added, the level of cAMP in RPMC, transiently and significantly increased about 4-fold compared with that of basal cells. These results indicate that sulfapyridine inhibits mast cell-mediated immediate-type allergic reactions in vivo and in vitro.     

Soluble HLA class i antigens in pediatric patients with renal transplants from related living donors without acute rejection and treated with triple therapy

All HLA class I Ag—expressing cells may be the source of serum Ag sHLA I. T and B lymphocytes secrete considerable amounts of Ag sHLA I in a variety of in vitro and in vivo activation systems. The purpose of this study was to evaluate the level of Ag sHLA I in serum of children with kidney transplants from related living donors without acute rejection and with triple therapy. We studied 25 patients (2–21 years) with first kidney transplant, 19 individuals (10–20 years) undergoing hemodialysis without transplant, and 25 normal children (4–21 years). The levels of Ag sHLA in transplant patients was 0.2–3.2 μg/ml ( ). The hemodialyzed patients was 0.48-4.5 μg/ml ( ), and the normal control was 0.30-4.38 μg/ml ( ). A statistically significant reduction was observed in transplant patients compared to normal control and hemodialyzed patients (p < 0.05 in both cases), whereas between normal and hemodialyzed patients no significant difference was seen ( ). The reduced levels of Ag sHLA I in blood could be an expression of adequate immunosuppressive treatment. Human Immunology 50, 135–139 (1996)     

Chitosan-mediated stimulation of macrophage function

According to the modern definition of biocompatibility, a biocompatible material need not be inert but be bioactive. A benign reactivity implies that the reactivity has to be appropriate for the intended use. Chitosan, a non-acetylated or partially deacetylated chitin (a linear homopolymer of β(1–4)-linked N-acetylglucosamine) has been proposed as a biomaterial because of its apparent satisfactory biocompatibility. The present investigation demonstrates that chitosan has an in vitro stimulatory effect on both macrophage nitric oxide (NO) production and chemotaxis. The macrophage NO secretion is attributed to the N-acetylglucosamine unit of the chitosan molecule rather than to the glucosamine residue (28 and 15μM NO respectively). Moreover, the immune stimulatory effect of chitosan was very specific since other glycosaminoglycans, such as and , had no effects on NO production (5 and 8 respectively). In vivo experiments strengthen this hypothesis. Transmission electron microscopy analysis identifies the presence of many leucocytes in the specimens after 14d post-implantation, showing poor healing processes (i.e. fibroblast proliferation and collagen deposition) that characterize the tissue repair at this time in our animal model. Biomaterials (1994) 15, 1215–1220     

SDS-PAGE analysis of the protein layers adsorbing in vivo and in vitro to bone substituting materials

The composition of the protein layer adsorbed to the bone substituting materials, hydroxyapatite, β-whitlockite, titanium and aluminium, in vivo (intramuscularly in guinea pig) and in vitro, was investigated using SOS-gel electrophoresis (SDS-PAGE). After in vivo implantation for 1 d mainly proteins with molecular weights between 10 000 and 20 000 were adsorbed. After 3 months the biolayer of the implanted biomaterials also contained proteins with molecular weights 35 000, 45 000, 60 000 and 200 000. No large qualitative differences in protein composition of the biolayers on the various implanted materials were found.

In vitro incubation with human serum resulted in binding of proteins with estimated molecular weights of 30 000, 60 000 (albumin), 200 000 and > 200 000. It is suggested that the differences between in vivo and in vitro protein adsorption are due to proteolysis occurring in vivo in the vicinity of the implanted material.     

Bombycis corpus Extract (BCE) Protects Hippocampal Neurons Against Excitatory Amino Acid-Induced Neurotoxicity

Bombycis corpus (BC) or Bombyx Batryticatus, a batryticated silkworm and white-stiff silkworm, is a drug consisting of the dried larva of silkworm, Mobyz mori L., dead and stiffened due to the infection of Beauveria (Bals.) Vuill. In a previous paper (Kim et al., Pharmacol. Res., 43, 12-16, 2001), BC was shown to protect amyloid-β-induced cytotoxicity. In the present study, we have found that BCE can prevent or reduce the neurotoxic actions in the hippocampus of the glutamate agonists N-methyl-D-aspartic acid (NMDA) in vitro or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainic acid in vitro. Pre-treatment with BCE (0, 1, 2, 5, and 10 μg/ml for 6-8 h) protected primary hippocampal cultures from embryonic day 18 (E18) embryos against NMDA-induced toxicity (0.1, 1, 10, and 50 mM/ml). BCE added either with NMDA (1 mM) or 1 h later had lesser, but still significant, protective actions. BCE also reduced NMDA-induced toxicity (1 mM). BCE (10 μg/ml) protected cultured neurons against the neurotoxic actions of either AMPA (25 μM) or kainic acid (1 mM) as well. Because the release of glutamate has been implicated in the neural damage after cerebral ischemia and other neural insults, these results suggest that BCE may contribute significantly to protect human brain to such damage.     

Corynebacterium parvum can Reverse the Depression of Macrophage Hydrogen Peroxide Production Caused by Erythrocyte Phagocytosis

Our previous studies have shown that the phagocytosis of IgG-coated erythrocytes (EIgG) in vivo increases the mortality rate with bacterial infection, and EIgG phagocytosis in vitro depresses phorbol myristate acetate (PMA)-triggered H₂O₂ production. The present study was undertaken to determine if the depression of H₂O₂ production caused by EIgG phagocytosis could be reversed by exposing macrophages to priming agents. Macrophages exposed to 100 μg/ml of C. parvum, it's pyridine-soluble extract (PE), or the pyridine extract residue (PER) for 1 hr showed an enhanced production of H₂O₂ in response to PMA triggering. The priming effect of C. parvum, PE, and PER lasted for 3-6 hr. 18 hr after exposure to C parvum or PER, PMA-triggered H₂O₂ production was depressed, however PE did not have this effect. The priming effect of C parvum was not prevented by cycloheximide. EIgG phagocytosis caused a dose dependent depression of PMA-triggered H₂O₂ production. When macrophages were exposed to C. parvum, PE, or PER following EIgG phagocytosis, the priming of PMA-triggered H₂O₂ production was reduced but H₂O₂ production was maintained at levels equal to or greater than that of control macrophages. These results show that phagocytosis did not prevent the action of priming agents on macrophage respiratory burst capacity, and suggests that such agents may preserve macrophage bactericidal function following phagocytosis.     

Omeprazole inhibits phagocytosis and acidification of phagolysosomes of normal human neutrophils in vitro

We postulated omeprazole inhibition of the neutrophil proton pump, impairing phagocytosis and phagolysosomal acidification. Neutrophils from healthy human beings were treated with omeprazole prodrug 0.5 mM/1 or acid activated omeprazole 0.5 mM/1 then incubated with killed Saccharomyces cerevisiae stained with bromcresol purple. Wet mounts were done at 10, 30 and 60 minutes. Percent neutrophils phagocytosing, percent yeast phagocytosed, and yeast per phagocytosing neutrophil were significantly decreased in acid activated omeprazole compared to controls and omeprazole prodrug. In contrast, percent acidification of intracellular yeast was significantly lower in both omeprazole prodrug and acid activated omeprazole compared to controls. Over time, control neutrophils showed an increase in percent yeast phagocytosed and yeast per phagocytosing neutrophil. When treated with acid activated omeprazole, the percent of neutrophils phagocytosing progressively decreased over time. We observed 1) omeprazole prodrug does not inhibit neutrophil phagocytosis but does inhibit phagolysosomal acidification, whereas 2) acid activated omeprazole inhibits both neutrophil phagocytosis and phagolysosome acidification. We conclude that omeprazole impairs these neutrophil functions in vitro.     

Differential immunotoxic effects of the environmental chemical benzo[a]pyrene in young and aged mice

Young (3–6 months), middle-aged (16–18 months) and aged (23–26 months) mice were exposed in vitro and in vivo to the immunotoxic environmental chemical benzo[a]-pyrene. The generation of antibody producing cells to the T-dependent antigens of sheep erythrocytes was observed to be suppressed in all age groups. Significantly, aged mice were shown to exhibit a greater percent suppression of antibody responses than young or middle-aged mice both in vitro and in vivo. The results presented provide the first evidence that the degree of immunological toxicity of environmental chemicals may be partially dependent upon the chronological and immunological age of the animal.     

Effect of Δ9-Tetrahydrocannabinol on in Vitro Sensitization of Mouse Splenic Lymphocytes

The effects of Δ⁹-tetrahydrocannabinol (Δ⁹-THC) on the immune response of murine cells sensitized in vitro was determined using a plaque-forming cell (PFC) assay. Splenic lymphocytes from mice injected with Δ⁹-THC showed a depressed immunologic response when compared with cells from control animals which were identically semitized in vitro with sheep erythrocytes (SRBC). The direct addition of Δ⁹-THC to the culture media altered the im-munological response as demonstrated by a reduction in the number of PFC.     

HEMOPOIESIS-STIMULATING ACTION OF ADAMANTYLAMIDE DIPEPTIDE: KINETICS OF INCREASE OF GM-CFC IN FEMUR AND CO-STIMULATING ACTIVITY OF SERUM, ROLE OF BONE MARROW STROMAL CELLS

Chief part of hemopoietic stromal cells in mediating hemopoiesis-stimulating effects of adamantylamide dipeptide (AdDP), a synthetic immunomodulatory compound, has been determined in a series of combined in vivo/in vitro studies. Indirect stimulatory effect of AdDP on proliferation of hemopoietic progenitor cells for granulocytes and macrophages (GM-CFC) was proved to be mediated by the cells of hemopoietic microenvironment growing as adherent stromal cell populations in vitro. These results supplement previously reported findings of a positive role which is played by AdDP at modulating the interplay among stimulatory cytokines and their cellular sources, and are in consent with the idea to introduce AdDP as a constituent of the hemopoiesis- and immunity-stimulating supportive medical care.     

In vivo validation of signaling pathways regulating human monocyte chemotaxis

Identification of novel signal transduction pathways regulating monocyte chemotaxis can indicate unique targets for preventive therapies for treatment of chronic inflammatory diseases. To aid in this endeavor we report conditions for optimal transfection of primary human monocytes coupled with a new model system for assessing their chemotactic activity in vivo. This method can be used as a tool to identify the relevant signal transduction pathways regulating human monocyte chemotaxis to MCP-1 in the complex in vivo environment that were previously identified to regulate chemotaxis in vitro. MCP-1-dependent chemotaxis of monocytes is studied in an adoptive transfer model where human monocytes transfected with mutant cDNAs are transferred to mice followed by initiation of peritonitis. Harvesting peritoneal cells at 24 h diminishes the contribution of immunologic responses to the cross-species transfer. Validation of relevant regulatory molecules in vivo is critical for understanding the most relevant therapeutic targets for drug development.     

Pathological changes of isolated spinal cord axons in response to mechanical stretch

White matter strips extracted from adult guinea-pig spinal cords were maintained in vitro and studied physiologically using a double sucrose gap technique and anatomically using a horseradish peroxidase assay. The amplitude of compound action potentials was monitored continuously before, during, and after elongation. Three types of conduction blocks resulting from stretch injury were identified: an immediate, spontaneously reversible component, which may result from a transient increase in membrane permeability and consequent disturbance of ionic distribution; a second component that was irreversible within 30–60 min of recording, perhaps resulting from profound axolemmal disruption; and a third component, which may be due to perturbation of the myelin sheath, that was reversible with application of 100 μM of the potassium channel blocker, 4-aminopyridine. The intensity of the conduction deficits correlated with the extent of initial stretch over a full range of severity. Stimulus–response data indicate that mechanical damage to axons in stretch was evenly distributed across the caliber spectrum. Morphological examinations revealed that a small portion of axons exhibited membrane damage at 2 min following stretch and appeared to be largely sealed at 30 min after injury. Further, in the entire length of the cord strip subjected to stretch, axons closer to the surface were found to be more likely to suffer membrane damage, which distinguished stretch injury from compression injury.

In summary, we have developed an in vitro model of axonal stretch that provides the ability to monitor changes in the properties of central myelinated axons following stretch injury in the absence of pathological variables related to vascular damage. This initial investigation found no evidence of secondary deterioration of axons in the first 30 min after stretch in vitro, although there was evidence of both transient and lasting physiological and anatomical damage to axons and their myelin sheaths.     

Antioxidant and anti-inflammatory activities of the plant Andrographis paniculata Nees

In this study, we explored the antioxidant and anti-inflammatory properties of the medicinal herb Andrographis paniculata using in vitro as well as in vivo systems. Methanolic extract of Andrographis paniculata was found to inhibit formation of oxygen derived free radicals such as superoxide (32%) hydroxyl radicals (80%) lipid peroxidation (80%) and nitric oxide (42.8%) in in vitro system. In vivo studies using BALB/c mice models also showed significant inhibition in PMA induced superoxide (32.4%) and nitric oxide (65.3%) formation. Interestingly we also found that, administration of Andrographis paniculata extract produced complete inhibition of carageenan induced inflammation compared with control models.     

Use of gene chip technology for the characterisation of the regulation of renal transport processes and of nephrotoxicity in rats

Gene expression profiling using microarrays (rat-specific array RG-U34A, Affymetrix, U.S.A.) was employed for the investigation of: (1) hormonal regulation of renal function and (2) nephrotoxicity. For this purpose about 8,800 genes were analysed in kidney and, additionally, in liver tissue.

Ad 1.) Kidney functions develop during postnatal life. Thus, in vivo transport and accumulation of p-aminohippurate (PAH) was investigated on renal cortical slices (RCS) from 10- and 55-day-old rats. The animals were treated with dexamethasone (DEXA; 60 μg/100 g b.wt./day) for 3 days, which caused a significant reduction in the accumulation of PAH in 10-day-old rats (42 ± 5% whereas it was only slightly reduced in 55-day-old rats (70 ± 8%). To further clarify the regulation of renal function by DEXA, results were compared with those obtained previously after in vitro stimulation with DEXA. RCS were incubated for 24 hours in DEXA-containing medium (10⁻⁹ M). Under these conditions DEXA significantly increased the PAH uptake capacity in RCS obtained from 10- and 55-day-old rats up to 126 and 136%, respectively. Thus a stimulation of tubular transport capacity is possible in vitro. The effect of DEXA treatment on the gene expression of the kidney (in vivo) was moderate. Focussing especially on transporters, ion channels, ATPases, glucuronyltransferases, glutathione-S-transferase and cytochrome P450, the expression of only few genes were significantly changed (3 to 50-fold up- or down-regulation). Moreover, distinct age differences were found after in vivo administration of DEXA. The investigation of in vitro effects of DEXA is currently been performed.

Ad 2.) The kidney is threatened by nephrotoxins because of its ability to accumulate them. We used a single administration of uranyl nitrate (UN; 0.5 mg/100 g b.wt.) as a model for chronic renal failure (CRF). Clearance experiments were performed 10 weeks after UN administration (maximal symptoms of CRF) in adult female rats. As expected, UN induced interstitial cicatrices with reduced GFR and diminished PAH transport capacity. Despite the impressive morphological and functional changes in the kidney after exposure to UN, the gene expression profiles in the kidneys were only minimally affected: we found significantly changed expression levels for only 20 genes (5 genes were up-regulated [e.g. transgelin], 15 down-regulated [among these the Na-K-Cl-symporter, insulin-like growth factor, kallikrein, and ornithine decarboxylase). The lack of agreement between gene expression data and the nephrotoxic effects of UN can probably be explained by the long time interval between dosing and the assessment of the effect. The results confirm that primary genomic responses are likely to be strongest transiently after exposure and then decrease in intensity.     

Carbenoxolone depresses spontaneous epileptiform activity in the CA1 region of rat hippocampal slices

An important contributor to the generation of epileptiform activity is the synchronization of burst firing in a group of neurons. The aim of this study was to investigate whether gap junctions are involved in this synchrony using an in vitro model of epileptiform activity. Hippocampal slices (400 μm) were prepared from female Sprague–Dawley rats (120–170 g). The perfusion of slices with a medium containing no added magnesium and 4-aminopyridine (50 μM) resulted in the generation of spontaneous bursts of population spikes of a fast frequency along with less frequent negative-going bursts. The frequency of the bursts produced was consistent over a 3 h period. Carbenoxolone (100 μM), a gap junction blocker and mineralocorticoid agonist, perfused for 75 min, reduced the frequency of both types of spontaneous burst activity. Perfusion of spironolactone (1 μM), a mineralocorticosteroid antagonist, for 15 min prior to and during carbenoxolone perfusion did not alter the ability of carbenoxolone to depress the frequency of spontaneous activity. The incubation of hippocampal slices in carbenoxolone prior to recording increased the time taken for the spontaneous activity to start on change to the zero magnesium/4-aminopyridine medium and decreased the total number of spontaneous bursts over the first 60 min period.

The ability of carbenoxolone to delay induction of epileptiform activity and reduce established epileptiform activity suggests that gap junctions contribute to the synchronization of neuronal firing in this model.     

Brain oxidation is an initial process in sleep induction

CNS activity is generally coupled to the vigilance state, being primarily active during wakefulness and primarily inactive during deep sleep. During periods of high neuronal activity, a significant volume of oxygen is used to maintain neuronal membrane potentials, which subsequently produces cytotoxic reactive oxygen species (ROS). Glutathione, a major endogenous antioxidant, is an important factor protecting against ROS-mediated neuronal degeneration. Glutathione has also been proposed to be a sleep-promoting substance, yet the relationship between sleep and cerebral oxidation remains unclear. Here we report that i.c.v. infusion of the organic peroxide t-butyl-hydroperoxide at a concentration below that triggering neurodegeneration (0.1 μmol/100 μl/10 h) promotes sleep in rats. Also, microinjection (2 nmol, 2 μl) or microdialysis (100 μM, 20 min) oft-butyl-hydroperoxide into the preoptic/anterior hypothalamus (POAH) induces the release of the sleep-inducing neuromodulators, nitric oxide and adenosine, without causing neurodegeneration. Nitric oxide and adenosine release was inhibited by co-dialysis of the N-methyl-d-aspartate receptor antagonist, d(−)-2-amino-5-phosphonopentanoic acid (D-AP5; 1 mM), suggesting that glutamate-induced neuronal excitation mediates the peroxide-induced release of nitric oxide and adenosine. Indeed, Ca²⁺ release from mitochondria and delayed-onset Ca²⁺ influx via N-methyl-d-aspartate receptors was visualized during peroxide exposure using Ca²⁺ indicator proteins (YC-2.1 and mitochondrial-targeted Pericam) expressed in organotypic cultures of the POAH. In the in vitro models, t-butyl-hydroperoxide (50 μM) causes dendritic swelling followed by the intracellular Ca²⁺ mobilization, and D-AP5 (100 μM) or glutathione (500 μM) inhibited t-butyl-hydroperoxide-induced intracellular Ca²⁺ mobilization and protected POAH neurons from oxidative stress.

These data suggest that low-level subcortical oxidation under the control of an antioxidant system may trigger sleep via the Ca²⁺-dependent release of sleep-inducing neuromodulators in the POAH, and thus we propose that a moderate increase of ROS during wakefulness in the neuronal circuits regulating sleep may be an initial trigger in sleep induction.