Comparison of Immune Functional Parameters Following In Vitro Exposure to Natural and Synthetic Amphetamines

The potential of synthetic and natural amphetamines to modulate cellular immune effector and regulatory mechanisms was evaluated in an in vitro exposure system. Murine splenic lymphocytes and elicited peritoneal macrophages were cultured with 0.0001-100 μM of amphetamine sulfate, methamphetamine hydrochloride, or the (S) or (R) isomers of cathinone hydrochloride. T-lymphocyte regulatory function was assessed by quantitating the production of cytokines, and T-lymphocyte effector function was assessed by the induction of cytotoxic T-lymphocytes (CTL). B-lymphocyte function was measured by proliferation, and natural immunity was assessed by quantitating basal and IL-2 augmented natural killer (NK) cell activity. None of the compounds tested had any direct effect on cellular viability. Exposure to amphetamine resulted in a significant suppression of IL-2, but not IL-4, production by T-lymphocytes, as well as a suppression of B-lymphocyte proliferation only at the highest amphetamine concentration examined. NK cell function was slightly suppressed by amphetamine exposure, but was enhanced by methamphetamine exposure. Conversely, exposure to either (S) or (R) isomers of cathinone resulted in stimulation of IL-2 production, B-lymphocyte proliferation, and CTL induction. No significant effect of cathinone was noted on NK cell function. These data suggest that natural and synthetic amphetamines exhibit differential immunomodulatory activity following in vitro exposure.     

Carvedilol Modulates In-Vitro Granulocyte-Macrophage Colony-Stimulating Factor-Induced Interleukin-10 Production in U937 Cells and Human Monocytes

Both granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-10 (IL-10) are important mediators regulating inflammatory responses. Inflammatory processes have an important role in atherogenesis. In this paper, the effects of carvedilol on GM-CSF-induced IL-10 production were examined on human monocytic cell line, U937, and purified human monocytes. First, we showed that one-time carvedilol pretreatment at concentrations 0.3-10 μM dose-dependently inhibited GM-CSF-induced IL-10 production in U937 cells. In addition, we found carvedilol to be non-cytotoxic at concentrations equal to or less than 10 μM. However, at concentrations higher than 10 μM, carvedilol induced programmed cell death in U937 cells. The inhibition of GM-CSF-induced IL-10 production by carvedilol was also observed at the expression of mRNA. Furthermore, the inhibition of IL-10 production was demonstrated in GM-CSF-activated purified human peripheral blood monocytes. Finally, long-term carvedilol pretreatment of U937 cells up to 2 months at concentrations of 1.0 μM mildly enhanced the IL-10 production. Our observations that carvedilol modulated GM-CSF-induced IL-10 production may have some implication in understanding the broad-spectrum effects of carvedilol in regulating inflammatory reactions.     

Effect of chromium and cobalt ions on primary human lymphocytes in vitro

Cobalt-chromium (Co-Cr) alloy metal-on-metal hip resurfacing is increasingly common among younger more active patients suffering from osteoarthritis. Recent reports have increased awareness of metal ions leaching from metallic articulations; this ion exposure may have adverse effects on the immune system. As previous studies reported alterations in lymphocyte number and function in patients with Co-Cr implants, we investigated effects of clinically relevant concentrations of Cr(6+) and Co(2+) on primary human lymphocytes in vitro. Here, both resting and activated (anti-CD3 ± anti-CD28 antibodies) primary human lymphocytes were exposed to Cr(6+) or Co(2+) (0.1-100 μM). Following 24 or 48?h of exposure, cell viability, proliferation, cytokine [interferon-γ (IFNγ and interleukin-2 (IL-2)] release, and apoptosis (with and without pre-treatment of cells with a caspase-3 inhibitor) were assessed. Exposure to 10 and 100 μM Cr(6+) significantly decreased cell viability and increased apoptosis in both resting and activated lymphocytes. Cell proliferation and cytokine release were also significantly reduced in activated lymphocytes following exposure. The exposure of resting lymphocytes to 100 μM Co(2+) resulted in significant decreases in cell viability accompanied by a significant increase in apoptosis. Activated lymphocytes also showed this response after exposure to 100 μM Co(2+); in fact, activated cells were significantly more sensitive to Co(2+) toxicity. Exposure to 10 μM Co(2+) led to significant decreases in cell proliferation and cytokine release, but no significant increase in apoptosis, in activated cells. The results indicate that exposure to high concentrations of metal ions initiate apoptosis that results in decreased lymphocyte proliferation. IL-2 release is inhibited by both metal ions at concentrations that are not overtly toxic. However, metal ion concentrations not directly cytotoxic to lymphocytes may affect events at a molecular level, thereby impeding lymphocyte proliferation. Hence, this may contribute to altered immune system function in patients with Co-Cr implants.     

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.     

Cytokine-stimulated chemotaxis of human neutrophils in a 3-D conjoined fibrin gel assay

The ability of neutrophils to migrate through three-dimensional (3-D) tissues in response to chemical stimuli is critical to their host defense function. However, studies characterizing stimulated migration in vitro have been largely limited to two-dimensional (2-D) surfaces. In this study, we have employed direct observation methods to quantify human neutrophil migration in 3-D fibrin gel using time-lapse video microscopy and automated cell tracking methods. A novel 3-D conjoined gel assay was developed to establish experimentally quantifiable and theoretically predictable diffusion gradients of chemotactic factors. This assay was used to measure objective migration parameters, namely the random motility and chemotaxis coefficients, in response to the cytokine, interleukin-8 (IL-8). The random motility coefficient, μ, showed a biphasic dependence on IL-8 concentration with a maximum of 1.1 × 10⁻⁸ cm²/s at 5 × 10⁻⁸ M IL-8; no significant motility was observed in the absence of IL-8. We further established the dependence of cell orientation bias, φ, on the concentration and gradient steepness (i.e., specific gradient, SG) of IL-8. Results indicate that φ increases with increasing SG, provided the concentration is maintained sufficiently low, which we conjecture to result from minimizing IL-8 receptor down-regulation. The chemotaxis coefficient, χ, was maximum at an intermediate SG for both IL-8 concentrations studied. We also examined the applicability of this assay to estimate μ and χ from indirect measurements of chemotaxis, namely the simpler measurement of cell redistribution after a prescribed incubation time, as opposed to direct cell tracking measurements. By virtue of measuring χ, this is the first quantitatively objective study of mammalian cell chemotaxis in a physiologically relevant 3-D gel and, in particular, of neutrophil chemotaxis on any substratum in response to the physiologically relevant chemotactic factor, IL-8.     

Polyethylene particles of a ‘critical size’ are necessary for the induction of cytokines by macrophages in vitro

Particulate wear debris from total hip prosthetic components can stimulate macrophages to produce mediators of osteolysis which may cause aseptic implant loosening. This study evaluated the in vitro response of murine peritoneal macrophages to polyethylene particles of difinitive size distributions at varying volume doses. Ceridust 3615 polyethylene particles with a mean size of 0.21, 0.49, 4.3 and 7.2 μm and GUR1120 polyethylene resin with a mean size of 88 μm were co-cultured with C3H murine peritoneal macrophages at volume (μm)³ to cell number ratios of 100 : 1, 10 : 1, 1 : 1 and 0.1 : 1. The secretion of IL-6, IL-1β and TNF- was determined by ELISA. Significantly elevated levels of TNF- and IL-1β were determined at 100 : 1 ratios when the macrophages were challenged with particles with a mean size of 0.49, 4.3 and 7.2 μm, and at 10 : 1 ratios for particles with a mean size of 0.49 and 4.3 μm. IL-6 production was significantly elevated at 100 : 1 ratios for mean particle sizes of 0.49 and 4.3 μm. Particles outside this range produced considerably less cytokine suggesting that both the size and volume (or number) of polyethylene particles are critical factors in macrophage activation. Therefore particles in the phagocytosable size range of 0.3–10 μm appear to be the most biologically active.     

Effect of chromium and cobalt ions on primary human lymphocytes in vitro

Cobalt-chromium (Co-Cr) alloy metal-on-metal hip resurfacing is increasingly common among younger more active patients suffering from osteoarthritis. Recent reports have increased awareness of metal ions leaching from metallic articulations; this ion exposure may have adverse effects on the immune system. As previous studies reported alterations in lymphocyte number and function in patients with Co-Cr implants, we investigated effects of clinically relevant concentrations of Cr⁶⁺ and Co²⁺ on primary human lymphocytes in vitro. Here, both resting and activated (anti-CD3 ± anti-CD28 antibodies) primary human lymphocytes were exposed to Cr⁶⁺ or Co²⁺ (0.1–100 µM). Following 24 or 48?h of exposure, cell viability, proliferation, cytokine [interferon-γ (IFNγ and interleukin-2 (IL-2)] release, and apoptosis (with and without pre-treatment of cells with a caspase-3 inhibitor) were assessed. Exposure to 10 and 100 µM Cr⁶⁺ significantly decreased cell viability and increased apoptosis in both resting and activated lymphocytes. Cell proliferation and cytokine release were also significantly reduced in activated lymphocytes following exposure. The exposure of resting lymphocytes to 100 µM Co²⁺ resulted in significant decreases in cell viability accompanied by a significant increase in apoptosis. Activated lymphocytes also showed this response after exposure to 100 µM Co²⁺; in fact, activated cells were significantly more sensitive to Co²⁺ toxicity. Exposure to 10 µM Co²⁺ led to significant decreases in cell proliferation and cytokine release, but no significant increase in apoptosis, in activated cells. The results indicate that exposure to high concentrations of metal ions initiate apoptosis that results in decreased lymphocyte proliferation. IL-2 release is inhibited by both metal ions at concentrations that are not overtly toxic. However, metal ion concentrations not directly cytotoxic to lymphocytes may affect events at a molecular level, thereby impeding lymphocyte proliferation. Hence, this may contribute to altered immune system function in patients with Co-Cr implants.     

Immune Function in Mice Exposed to the Adenosine Deaminase Inhibitor 2'-Deoxycoformycin During Immune System Development

Pregnant mice were administered 2'-deoxycoformycin (2dCF), a potent inhibitor of adenosine deaminase activity, by intraperitoneal injection on day 7 or 15 of gestation or from day 8-12 or 14-18 of gestation. A total dose of 0.5 or 2.0 μg 2dCF/g of maternal body weight was given to the dams. In a seperate study, pups born to nontreated dams were given 5 intraperitoneal injections totaling 0.5, 2.0 or 4.0 μg 2dCF/g beginning at 4 weeks of age. Administered doses of 2dCF were at levels known to profoundly suppress adenosine deaminase levels in adult mice. Pups born to dams injected with 2dCF from day 14-18 all died within 48 h of birth whereas other injection schedules had no effect on birth rate or survival of pups. In utero 2dCF exposure had little effect on immune function in offspring. On the other hand, body, spleen and thymus weight, and splenic cellularity were decreased in weanling mice 24 h after the last injection of 4 μg/g 2dCF. Proliferative responses of splenocytes to T cell mitogens and alloantigens were likewise suppressed at both 2.0 and 4.0 μg/g 2dCF. Suppression of proliferative responses in treated weanling mice were no longer apparent at 7 weeks of age although splenic cellularity and weight remained lower than control values. These results are similar to those we have reported for 8 week old mice given similar doses of 2dCF, with the exception of elevated levels of NK cell activity in older 2dCF-treated mice and suggest that there may be age-related differences in the sensitivity of certain cell populations to the effects of 2dCF.     

The Effects of Methimazole on Haematopoiesis in Mice

The effects of anti-thyroid drug, methimazole (MMI), on haematopoiesis in inbred C57BL/6 mice were studied. The in vitro proliferative response of bone marrow (BM) cells to interleukin-3 (IL-3) was significantly increased when mice were provided with 0.1% MMI in water (w/v) ad librium for 4 to 6 weeks. Using soft agar agar colony assay, the numbers of myeloid cell colonies were also significantly increased in mice treated with MMI. However, the proliferative response of BM cells to IL-3 was found to be greatly reduced 10 weeks after MMI treatment. In vitro studies showed that MMI alone at the concentrations of 500μM or above inhibited both the growth of normal BM cells in liquid culture and the formation of macrophage (M) - / granulocyte (G) - colonies in soft agar culture in a dose dependent manner. Direct cytotoxic effect of MMI (0 - 1250 μM) to normal BM cells was not observed. Results from this study suggested that MMI can modulate the development of myeloid haematopoietic cells.     

Effect of Metals on Interleukin-6 (IL-6) Mitogenic Stimulation of Murine Hybridoma Cells

The effect of several divalent metal cations (Co, Cu, Zn, Hg and Pb) on the proliferative response of B9 hybridoma cells to IL-6 has been investigated. At concentrations which are not cytotoxic all the metals inhibited proliferation. The inhibition by Cd and Pb was dependent on both time of addition of the metal and IL-6 concentration. Cd (10μM) and Pb (50μM) added at the same time as IL-6 were inhibitory but added 24h later had no effect. Increasing the concentration of IL-6 overcame the inhibitory effect of Cd (10μM) and Pb (50μM). Inhibition caused by the other metals was independent of either time of addition or IL-6 concentration. IL-6 did not stimulate an increased intracellular concentration of metallothionein suggesting that the protective effect of IL-6 is not mediated via induction of metallothionein. The results suggest that there are at least two distinct metal sensitive events In B9 proliferation, i) IL-6 reversible inhibition by Cd and Pb ii) IL-6 Independent inhibition by Co, Cu and Hg.     

Selective Inhibitory Effects of Stress Hormones on Natural Killer (NK) Cell Activity of Lymphocytes from AIDS Patients

To examine the potential role of stress hormones in the progression of HIV infections, we developed an in vitro model system that investigates the effects of cortisol, adreno-corticotropin-releasing hormone (ACTH) and β-endorphin on the natural killer cell activity of lymphocytes from normal subjects and AIDS patients. The system employs a 4 hr⁵¹Cr release assay and K562 target cells. Direct addition of cortisol (0.05, 0.1, and 0.2 μg/ml) or ACTH (10^-6 to 10^-8 M) to the mixture of effector and prelabeled target cells did not produce any significant immunoregulatory effects on the NK cell activity of normal lymphocytes. Direct addition of β-endorphin (10^-13 to 10^-17 M) to the mixture of effector and prelabeled target cells did not produce any significant immunoregulatory effects on the NK cell activity of lymphocytes from normal or AIDS subjects. However, cortisol and ACTH significantly inhibited the NK activity of lymphocytes from AIDS patients. The selective inhibitory effects of cortisol and ACTH in patients with HIV infections are consistent with a model which proposes that stress related neurohormones and/or neuropeptides may be involved in the progression of HIV infections.     

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.     

Vanadium pentoxide increased PTEN and decreased SHP1 expression in NK-92MI cells,affecting PI3K-AKT-mTOR and Ras-MAPK pathways

Vanadium is an air pollutant that imparts immunosuppressive effects on NK cell immune responses, in part, by dysregulating interleukin (IL)-2/IL-2R-mediated JAK signaling pathways and inducing apoptosis. The aim of the present study was to evaluate effects of vanadium pentoxide (V₂O₅) on other IL-2 receptor-mediated signaling pathways, i.e. PI3K-AKT-mTOR and Ras-MAPK. Here, IL-2-independent NK-92MI cells were exposed to different V₂O₅ doses for 24?h periods. Expression of PI3K, Akt, mTOR, ERK1/2, MEK1, PTEN, SHP1, BAD and phosphorylated forms, as well as caspases-3, -8, -9, BAX and BAK in/on the cells were then determined by flow cytometry. The results show that V₂O₅ was cytotoxic to NK cells in a dose-related manner. Exposure increased BAD and pBAD expression and decreased that of BAK and BAX, but cell death was not related to caspase activation. At 400?µM V₂O₅, expression of PI3K-p85 regulatory subunit increased 20% and pPI3K 50%, while that of the non-pPI3K 110α catalytic subunit decreased by 20%. At 200?μM, V₂O₅ showed significant decrease in non-pAkt expression (p?2O₅. No differences were found with non-phosphorylated ERK-1/2. PTEN expression increased significantly at 100?μM V₂O₅ exposure whereas pPTEN decreased by 18% at 25?μM V₂O₅ concentrations, but remained unchanged thereafter. Lastly, V₂O₅ at all doses decreased SHP1 expression and increased expression of its phosphorylated form. These results indicated a toxic effect of V₂O₅ on NK cells that was due in part to dysregulation of signaling pathways mediated by IL-2 via increased PTEN and decreased SHP1 expression. These results can help to explain some of the known deleterious effects of this particular form of vanadium on innate immune responses_.     

Immunosuppressive Factors in Mouse Amniotic Fluid and Neonate Serum

Alpha-fetoprotein (AFP) isolated from neonatal mouse serum (NNS) and mouse amniotic fluid (MAF) was suppressive in both the mixed lymphocyte reaction (MLR) and the lipopolysaccharide-induced (LPS) mitogenesis. On the other hand, AFP-depleted NNS and MAF showed a stronger immunosuppressive effect compared to the oriqinal fluids at the same total protein concentration. These results suggest that proteins other than AFP may also be involved in the net suppressive effect of NNS and MAF in both the T-cell-dependent and T-cell-independent in vitro immune responses.

Various AFP preparations resulted in either 1) suppression at concentrations of 5-100 μq: 2) suppression only at high concentrations (100-200 μg); and 3) nonsuppression even at 100-200 μg levels. MLR enhancement was noted with some preparations and was dose dependent; at higher concentrations some enhancing preparations would suppress. Mitogenicity, when present, may obscure suppression. Whether enhancement is a property of a particular species of AFP or a contaminant is unknown. However, the latter seems a distinct possibility since mitogenic substances are shed from columns and some apparently homogeneous AFP preparations are not mitoqenic. AFP from NNS was somewhat more suppressive than AFP from MAF and showed a faster electrophoretic mobility at pH 9.5. These findings suggest that certain subfractions of AFP may be more suppressive than others

The net effect of NNS or MAF is dependent on the dose, the test system, the supplemental serum employed, the relative abundance of different AFP variants in the preparation and the presence of a non-AFP suppressive factor(s).     

Effect of Macrophage Colony-Stimulating Factor (M-CSF) on Mouse Immune Responses in Vivo

We examined the effects of recombinant human M-CSF (rhM-CSF) on mouse macrophages and immune responses in vivo. Intraperitoneal administration of rhM-CSF (20-500 μg/ml) increased Mac-l⁺ cell numbers in the peritoneal cavity. The tumoricidal activities of the macrophages from vehicle-administered (V-MΦ) and from rhM-CSF-administered (M-MΦ) mice were the same as those observed in vitro. However, when activated by lipopolysaccharide (LPS), the tumoricidal activity of M-MΦ was stronger than that of V-MΦ. Intravenous administration of rhM-CSF (500 μ g/gk) increased the number of spleen cells. Flow cytometric analysis showed that administration of rhM-CSF increased Mac-1⁺, B220+ and NK 1.1⁺ cell counts in the spleen. However, CD4⁺ and CD8⁺ cell numbers did not change. Concomitant increases were observed in levels of IL-4 and IL-10 in mouse serum following rhM-CSF administration, but no significant changes were observed in the serum level of IFN-γ.

In experiments involving mouse immune responses, the administration of rhM-CSF reduced the contact sensitivity (CS) reaction against picryl chloride (PC) and augmented IgE production in response to 2,4-dinitrophenyl (DNP), but did not affect the production of either IgM or IgG 1.

These results suggest that administration of rhM-CSF not only activates murine macrophages, but modulates antigen-specific immune responses in vivo.     

Protein A Potentiates Lymphokine-Activated Killer Cell Induction in Normal and Melanoma Patient Lymphocytes

The effects of purified protein A from Staphylococcus aureus Cowan I strain on induction of lymphokine (IL-2) activated killer (LAK) activity were studied in normal as well as melanoma patient's lymphocyte. The coculture of peripheral blood mononuclear cells (PBMC) with various doses of protein A (0.001, 0.01 and 0.1 μmlg/ml) and IL-2 (100 U/ml) for 4 days produced synergistic effect on the LAK cells mediated cytotoxicity. The potentiation of cytotoxicity and lytic ability of LAK cells against NK sensitive (K-562) and NK-resistant (M1 4) tumor cells were observed. Further there was potentiation of DNA synthesis in PBMC after 4 days culture. Similar results were found when PBMC from melanoma patients were cultured with PA and IL-2. The potentiation of LAK cell induction associated with its cytotoxic and lytic potential by low doses of IL-2/PA regiment may be helpful in the development of LAK immunotherapy of the cancer patients.     

Cocaine effects on immunocompetent cells: an observation of in vitro cocaine exposure.

This study investigated in vitro effects of cocaine on the function of T and B lymphocytes, natural killer cells and macrophages in a mouse model. In mature C57BL/6J mice (60-90 day-old), splenocytes were cultured with cocaine at different concentrations ranging from 4 to 64 microg/ml for 24 h. The exposure to cocaine in vitro was found to affect (1) T cell function, with reduced responses to stimulation of Con-A, PHA and Interleukin 2, and decreased production of gamma-IFN; (2) B cell function, with reduced response to LPS; (3) natural killer cell function, with attenuated killing capacity; (4) monocyte-macrophage function, with decreased ability to inhibit the growth of tumor cells. The results of the study indicated a direct cocaine effect on four major immune competent cells, and the generally suppressive effects of in vitro cocaine exposure may be related to its in vivo action on the immune system.     

Natural killer cell-mediated response to human cytomegalovirus-infected macrophages is modulated by their functional polarization

MΦ comprise a heterogeneous population of cells, which contribute to host defense and maintenance of immune homeostasis. MΦ may be infected by human cytomegalovirus (HCMV), which has evolved different strategies to subvert the immune response. In the present study, we comparatively analyzed the natural killer (NK) cell response against HCMV (TB40E)-infected proinflammatory (M1) and antinflammatory (M2) MΦ, derived from autologous monocytes, cultured in the presence of GM-CSF and M-CSF, respectively. M1 MΦ were more resistant to infection and secreted IL-6, TNF-α, IFN-α, and IL-12; by contrast, in HCMV-infected M2 MΦ, proinflammatory cytokines, IL-10, and IFN-α production were limited and IL-12 was undetectable. NK cell degranulation was triggered by interaction with HCMV-infected M1 and M2 MΦ at 48 h postinfection. The response was partially inhibited by specific anti-NKp46, anti-DNAM-1, and anti-2B4 mAb, thus supporting a dominant role of these activating receptors. By contrast, only HCMV-infected M1 MΦ efficiently promoted NK cell-mediated IFN-γ secretion, an effect partially related to IL-12 production. These observations reveal differences in the NK cell response triggered by distinct, HCMV-infected, monocyte-derived cell types, which may be relevant in the immunopathology of this viral infection.     

Photoperiodic regulation of behavioral responsiveness to proinflammatory cytokines

Symptoms of bacterial infection include decreases in body mass (cachexia), induction of depressive-like hedonic tone (anhedonia), decreases in food intake (anorexia), and increases in body temperature (fever). Recognition of bacteria by the innate immune system triggers the release of proinflammatory cytokines which induce these sickness behaviors via actions at central and peripheral targets. In Siberian hamsters, exposure to short day lengths decreases both the production of proinflammatory cytokines and the magnitude of the symptoms of infection. Short-day attenuation of sickness behaviors may arise solely from decreased production of cytokines; alternatively, substrates responsible for the generation of sickness behaviors may be less responsive to cytokines in short days. To discriminate among these hypotheses, Siberian hamsters were treated with either bacterial lipopolysaccharide (LPS; 25 μg) or recombinant mouse IL-1β (rIL-1β; 100 ng) following 11 weeks of exposure to long (15 h light/day) and short (9 h light/day) photoperiods. Replicating earlier work, the magnitude and/or duration of LPS-induced anorexia, anhedonia, cachexia, and fever were greater in long-day relative to short-day hamsters. A comparable short-day attenuation of sickness behaviors and fever was obtained in response to rIL-1β treatment, despite treatment with identical concentrations of cytokine. These data suggest that hamsters subjected to immunoenhancing short days exhibit diminished symptoms of infection not solely because infections elicit lower levels of cytokine production, but also because the substrates upon which cytokines act become relatively refractory.     

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.