Asiaticoside modulates human NK cell functional fate by mediating metabolic flexibility in the tumor microenvironment.

BACKGROUND: Transforming growth factor-beta (TGF-ß), an immunosuppressive cytokine, is often elevated in various tumors and inhibits the immune system's ability to combat tumor cells. Despite promising results from TGF-ß inhibitor therapies, their clinical efficacy remains limited. PURPOSE: This study aimed to enhance the antitumor capabilities of natural killer (NK) cells in the presence of TGF-ß by exploring the potential of asiaticoside, a natural compound with established clinical safety. STUDY DESIGN: The effects of asiaticoside on NK cells were investigated to determine its potential to counteract TGF-ß-induced immunosuppression and elucidate the underlying mechanisms. METHODS: Natural compounds were screened using a Luminex assay to identify those promoting Interferon-γ (IFN-γ) secretion from NK cells. Asiaticoside-pretreated NK cells' cytotoxicity was assessed against K562, OVCAR8, and A2780 cells using organoids from ascites-derived ovarian cancer (OC) cells. In vivo efficacy was evaluated with B16 melanoma lung metastasis and subcutaneous tumor models in C57BL/6 mice, using asiaticoside as a 50 mg/kg injection. The compound's ability to enhance NK cell-driven anti-neoplastic responses was further assessed in an OC murine model. Effects on TGF-ß/SMAD pathways and mitochondrial functions were examined through various microscopy and metabolomic techniques. The involvement of the mTOR/DRP1 axis in asiaticoside-mediated restoration of mitochondrial oxidation in NK cells after TGF-ß suppression was determined using the mTOR inhibitor rapamycin and the DRP1 inhibitor Mdivi-1. RESULTS: Asiaticoside-treated NK cells retained their ability to suppress tumor growth and metastasis despite TGF-ß presence. Asiaticoside downregulated TGF-ß receptors 1 (TGFBR1) expression, impaired the protein stability of TGFBR1 and TGF-ß receptors 2 (TGFBR2), and reduced SMAD2 phosphorylation, preventing SMAD2 translocation from the mitochondria. This preserved mitochondrial respiration and maintained NK cell antitumor activity. CONCLUSION: The study concludes that asiaticoside has significant potential as a strategy for "priming" NK cells in cellular immunotherapy. By demonstrating that asiaticoside degrades the TGF-ß receptor, leading to reduced phosphorylation of SMAD2 and preventing its mitochondrial translocation, thereby maintaining mitochondrial integrity. Meantime, asiaticoside counteracts TGF-ß-induced suppression of mitochondrial oxidative and aerobic respiration through the mTOR/DRP1 pathways. The research uncovers a previously unreported pathway for preserving mitochondrial respiration and NK cell functionality. A detailed mechanistic insight into how asiaticoside functions at the molecular level was explored. Its ability to counteract the immunosuppressive effects of TGF-ß makes it a valuable candidate for enhancing the effectiveness of immunotherapies in treating a variety of tumors with elevated TGF-ß levels.

Emerging biomolecules for practical theranostics of liver hepatocellular carcinoma.

Most cases of hepatocellular carcinoma (HCC) are able to be diagnosed through regular surveillance in an identifiable patient population with chronic hepatitis B or cirrhosis. Nevertheless, 50% of global cases might present incidentally owing to symptomatic advanced-stage HCC after worsening of liver dysfunction. A systematic search based on PUBMED was performed to identify relevant outcomes, covering newer surveillance modalities including secretory proteins, DNA methylation, miRNAs, and genome sequencing analysis which proposed molecular expression signatures as ideal tools in the early-stage HCC detection. In the face of low accuracy without harmonization on the analytical approaches and data interpretation for liquid biopsy, a more accurate incidence of HCC will be unveiled by using deep machine learning system and multiplex immunohistochemistry analysis. A combination of molecular-secretory biomarkers, high-definition imaging and bedside clinical indexes in a surveillance setting offers a comprehensive range of HCC potential indicators. In addition, the sequential use of numerous lines of systemic anti-HCC therapies will simultaneously benefit more patients in survival. This review provides an overview on the most recent developments in HCC theranostic platform.

Proteomic Characterization of SAS Cell-Derived Extracellular Vesicles in Relation to Both BPA and Neutron Irradiation Doses.

Boron neutron capture therapy (BNCT) is a selective radiotherapy based on nuclear reaction that occurs when 10B atoms accumulated in cancer cells are irradiated by thermal neutrons, triggering a nuclear fission response leading to cell death. Despite its growing importance in cancer treatment, molecular characterization of its effects is still lacking. In this context, proteomics investigation can be useful to study BNCT effect and identify potential biomarkers. Hence, we performed proteomic analysis with nanoLC-MS/MS (liquid chromatography coupled to tandem mass spectrometry) on extracellular vesicles (EVs) isolated from SAS cultures treated or not with 10B-boronophenylalanine (BPA) and different doses of neutron irradiation, to study the cellular response related to both boron administration and neutrons action. Despite the interference of fetal bovine serum in the medium, we were able to stratify BPA- and BPA+ conditions and to identify EVs-derived proteins characterizing pathways potentially related to a BNCT effect such as apoptosis, DNA repair and inflammatory response. In particular, KLF11, SERPINA1 and SERPINF2 were up-regulated in BPA+, while POLE and SERPINC1 were up-regulated in BPA-. These results provide the first proteomic investigation of EVs treated with BNCT in different conditions and highlight the potentiality of proteomics for improving biomarkers identification and mechanisms understanding of BNCT.

Radiosensitization to γ-Ray by Functional Inhibition of APOBEC3G.

The radiosensitization of tumor cells is one of the promising approaches for enhancing radiation damage to cancer cells and limiting radiation effects on normal tissue. In this study, we performed a comprehensive screening of radiosensitization targets in human lung cancer cell line A549 using an shRNA library and identified apolipoprotein B mRNA editing enzyme catalytic subunit 3G (APOBEC3G: A3G) as a candidate target. APOBEC3G is an innate restriction factor that inhibits HIV-1 infection as a cytidine deaminase. APOBEC3G knockdown with siRNA showed an increased radiosensitivity in several cancer cell lines, including pancreatic cancer MIAPaCa2 cells and lung cancer A549 cells. Cell cycle analysis revealed that APOBEC3G knockdown increased S-phase arrest in MIAPaCa2 and G2/M arrest in A549 cells after γ-irradiation. DNA double-strand break marker γH2AX level was increased in APOBEC3G-knocked-down MIAPaCa2 cells after γ-irradiation. Using a xenograft model of A549 in mice, enhanced radiosensitivity by a combination of X-ray irradiation and APOBEC3G knockdown was observed. These results suggest that the functional inhibition of APOBEC3G sensitizes cancer cells to radiation by attenuating the activation of the DNA repair pathway, suggesting that APOBEC3G could be useful as a target for the radiosensitization of cancer therapy.

A Predictive Model for Prognosis and Therapeutic Response in Hepatocellular Carcinoma Based on a Panel of Three MED8-Related Immunomodulators.

The current tumor-node-metastasis (TNM) system is limited in predicting the survival and guiding the treatment of hepatocellular carcinoma (HCC) patients since the TNM system only focuses on the anatomical factors, regardless of the intratumoral molecule heterogeneity. Besides, the landscape of intratumoral immune genes has emerged as a prognostic indicator. The mediator complex subunit 8 (MED8) is a major polymerase regulator and has been described as an oncogene in renal cell carcinoma, but its pathophysiological significance of HCC and its contribution to the prognosis of HCC remain unclear. Here, we aimed to discuss the expression profile and clinical correlation of MED8 in HCC and construct a predictive model based on MED8-related immunomodulators as a supplement to the TNM system. According to our analyses, MED8 was overexpressed in HCC tissues and increased expression of MED8 was an indicator of poor outcome in HCC. The knockdown of MED8 weakened the proliferation, colony forming, and migration of HepG2 and Huh7 cells. Subsequently, a predictive model was identified based on a panel of three MED8-related immunomodulators using The Cancer Genome Atlas (TCGA) database and further validated in International Cancer Genome Consortium (ICGC) database. The combination of the predictive model and the TNM system could improve the performance in predicting the survival of HCC patients. High-risk patients had poor overall survival in TCGA and ICGC databases, as well as in subgroup analysis with early clinicopathology classification. It was also found that high-risk patients had a higher probability of recurrence in TCGA cohort. Furthermore, low-risk score indicated a better response to immunotherapy and drug therapy. This predictive model can be served as a supplement to the TNM system and may have implications in prognosis stratification and therapeutic guidance for HCC.

Extracellular Release of HMGB1 as an Early Potential Biomarker for the Therapeutic Response in a Xenograft Model of Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is a non-invasive therapeutic technique for treating malignant tumors, however, methods to evaluate its therapeutic efficacy and adverse reactions are lacking. High mobility group box 1 (HMGB1) is an inflammatory molecule released during cell death. Therefore, we aimed to investigate HMGB1 as a biomarker for BNCT response, by examining the early responses of tumor cells to 10B-boronophenylalanine (BPA)-based BNCT in the Kyoto University Nuclear Reactor. Extracellular HMGB1 release was significantly increased in human squamous carcinoma SAS and melanoma A375 cells 24 h after neutron irradiation but not after γ-irradiation. At 3 days post-BPA-based BNCT irradiation in a SAS xenograft mouse model, plasma HMGB1 levels were higher than those in the non-irradiation control, and HMGB1 was detected in both nuclei and cytoplasm in tumor cells. Additionally, increased plasma HMGB1 levels post-BNCT irradiation were detected even when tumors decreased in size. Collectively, these results indicate that the extracellular HMGB1 release occurs at an early stage and is persistent when tumors are reduced in size; therefore, it is a potential biomarker for evaluating the therapeutic response during BNCT.

A newly established monoclonal antibody against ERCC1 detects major isoforms of ERCC1 in gastric cancer.

Identifying patients resistant to cisplatin treatment is expected to improve cisplatin-based chemotherapy for various types of cancers. Excision repair cross-complementing group 1 (ERCC1) is involved in several repair processes of cisplatin-induced DNA crosslinks. ERCC1 overexpression is reported as a candidate prognostic factor and considered to cause cisplatin resistance in major solid cancers. However, anti-ERCC1 antibodies capable of evaluating expression levels of ERCC1 in clinical specimens were not fully optimized. A mouse monoclonal antibody against human ERCC1 was generated in this study. The developed antibody 9D11 specifically detected isoforms of 201, 202, 203 but not 204, which lacks the exon 3 coding region. To evaluate the diagnostic usefulness of this antibody, we have focused on gastric cancer because it is one of the major cancers in Japan. When ERCC1 expression was analyzed in seventeen kinds of human gastric cancer cell lines, all the cell lines were found to express either 201, 202, and/or 203 as major isoforms of ERCC1, but not 204 by Western blotting analysis. Immunohistochemical staining showed that ERCC1 protein was exclusively detected in nuclei of the cells and a moderate level of constant positivity was observed in nuclei of vascular endothelial cells. It showed a clear staining pattern in clinical specimens of gastric cancers. Antibody 9D11 may thus be useful for estimating expression levels of ERCC1 in clinical specimens.

A Combination of GM-CSF and Released Factors from Gamma-Irradiated Tumor Cells Enhances the Differentiation of Macrophages from Bone Marrow Cells and Their Antigen-Presenting Function and Polarization to Type 1.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes dendritic cell differentiation from precursors, and consequently, enhances the antigen presentation process and adaptive immune responses. With such functions, GM-CSF has been used as immunotherapy in combination with radiotherapy for cancer treatment to augment the survival and activity of immune cells. However, an immune-suppressive tumor microenvironment may cause anergy of T cells. It has also been reported that GM-CSF contributes to the development of myeloid-derived suppressor cells from the precursors. In this study, to analyze the combined effect of GM-CSF and released factors from cancer cells after gamma-ray irradiation on bone marrow cell differentiation and dynamics, we established an in vitro culture system using mouse bone marrow cells, GM-CSF, and conditioned medium from gamma ray irradiated mouse melanoma B16 cells at 24 Gy. We analyzed the gene expression changes of the bone marrow-derived cells on day 6. The results showed that GM-CSF dose-dependently enhanced the differentiation of macrophages from bone marrow cells, their antigen-presenting function and polarization to type I. The results implied the induced macrophages from the bone marrow may potentially contribute to tumor immune responses in a systemic manner when GM-CSF is boosted during photon-beam radiation therapy.

Transnitrosylation Mediated by the Non-canonical Catalase ROG1 Regulates Nitric Oxide Signaling in Plants.

The redox-based protein S-nitrosylation is a conserved mechanism modulating nitric oxide (NO) signaling and has been considered mainly as a non-enzymatic reaction. S-nitrosylation is regulated by the intracellular NO level that is tightly controlled by S-nitrosoglutathione reductase (GSNOR). However, the molecular mechanisms regulating S-nitrosylation selectivity remain elusive. Here, we characterize an Arabidopsis "repressor of" gsnor1 (rog1) mutation that specifically suppresses the gsnor1 mutant phenotype. ROG1, identical to the non-canonical catalase, CAT3, is a transnitrosylase that specifically modifies GSNOR1 at Cys-10. The transnitrosylase activity of ROG1 is regulated by a unique and highly conserved Cys-343 residue. A ROG1C343T mutant displays increased catalase but decreased transnitrosylase activities. Consistent with these results, the rog1 mutation compromises responses to NO under both normal and stress conditions. We propose that ROG1 functions as a transnitrosylase to regulate the NO-based redox signaling in plants.

Protein S-Nitrosylation in plants: Current progresses and challenges.

Nitric oxide (NO) is an important signaling molecule regulating diverse biological processes in all living organisms. A major physiological function of NO is executed via protein S-nitrosylation, a redox-based posttranslational modification by covalently adding a NO molecule to a reactive cysteine thiol of a target protein. S-nitrosylation is an evolutionarily conserved mechanism modulating multiple aspects of cellular signaling. During the past decade, significant progress has been made in functional characterization of S-nitrosylated proteins in plants. Emerging evidence indicates that protein S-nitrosylation is ubiquitously involved in the regulation of plant development and stress responses. Here we review current understanding on the regulatory mechanisms of protein S-nitrosylation in various biological processes in plants and highlight key challenges in this field.

The IL-15-AKT-XBP1s signaling pathway contributes to effector functions and survival in human NK cells.

Interleukin 15 (IL-15) is one of the most important cytokines that regulate the biology of natural killer (NK) cells1. Here we identified a signaling pathway-involving the serine-threonine kinase AKT and the transcription factor XBP1s, which regulates unfolded protein response genes2,3-that was activated in response to IL-15 in human NK cells. IL-15 induced the phosphorylation of AKT, which led to the deubiquitination, increased stability and nuclear accumulation of XBP1s protein. XBP1s bound to and recruited the transcription factor T-BET to the gene encoding granzyme B, leading to increased transcription. XBP1s positively regulated the cytolytic activity of NK cells against leukemia cells and was also required for IL-15-mediated NK cell survival through an anti-apoptotic mechanism. Thus, the newly identified IL-15-AKT-XBP1s signaling pathway contributes to enhanced effector functions and survival of human NK cells.

Crystal Structures and Human Leukemia Cell Apoptosis Inducible Activities of Parthenolide Analogues Isolated from Piptocoma rufescens.

The molecular structures of three parthenolide analogues, (-)-goyazensolide (1), (-)-15-deoxygoyazensolide (2), and (-)-ereglomerulide (3), isolated from the leaves of Piptocoma rufescens in a previous study were determined by X-ray analysis, and the absolute configuration of (-)-goyazensolide (1) was confirmed crystallographically using Cu Kα radiation at low temperature. Compounds 1-3, (+)-rufesolide A (4), and commercial parthenolide were found to be growth inhibitory toward MOLM-13 and EOL-1 human acute myeloid leukemia cells using PKC412 (midostaurin) as the positive control, with 1-3 being more active than parthenolide. Also, compounds 1-4 exhibited synergistic effects when tested with PKC412, but parthenolide did not show this type of activity. At a concentration lower than 2.0 µM, both 1 and 2 induced approximately 50% of the cells to become apoptotic at a late stage of the cell cycle, but no similar apoptotic effects were observed for 3, 4, or parthenolide. Leukemia cell apoptosis was induced by these compounds through the activation of caspase-3 and the inhibition of NF-κB, as indicated by immunoblotting analysis, and compounds 1 and 2 seem to be promising leads for development as potential antileukemic agents.

TGFß Treatment Enhances Glioblastoma Virotherapy by Inhibiting the Innate Immune Response.

Oncolytic viruses, including oncolytic herpes simplex virus (oHSV), have produced provocative therapeutic responses in patients with glioblastoma, the most aggressive brain tumor. Paradoxically, innate immune responses mediated by natural killer (NK) cells and macrophages/microglia appear to limit oHSV efficacy. Therefore, we investigated whether pretreatment with an immunosuppressive cytokine, TGFß, might reverse these effects and thereby potentiate oHSV efficacy. TGFß treatment of NK cells rendered them less cytolytic against oHSV-infected glioblastoma cells and stem-like cells in vitro. Furthermore, TGFß treatment of NK cells, macrophages, or microglia increased viral titers of oHSV in cocultures with glioblastoma cells. In a syngeneic mouse model of glioblastoma, administering TGFß prior to oHSV injection inhibited intracranial infiltration and activation of NK cells and macrophages. Notably, a single administration of TGFß prior to oHSV therapy was sufficient to phenocopy NK-cell depletion and suppress tumor growth and prolong survival in both xenograft and syngeneic models of glioblastoma. Collectively, our findings show how administering a single dose of TGFß prior to oncolytic virus treatment of glioblastoma can transiently inhibit innate immune cells that limit efficacy, thereby improving therapeutic responses and survival outcomes.

Synergistic antidepressant-like effect of ferulic acid in combination with piperine: involvement of monoaminergic system.

The lifetime prevalence rate for major depressive disorder (MDD) is approximately 17 % for most developed countries around the world. Dietary polyphenols are currently used as an adjuvant therapy to accelerate the therapeutic efficacy on depression. Ferulic acid (FA) or 4-hydroxy-3-methoxy-cinnamic acid (Fig. 1a) is a main polyphenolic component of Chinese herb Radix Angelicae Sinensis, which is found to have antidepressant-like effects through regulating serotonergic and noradrenergic function. The present study examined the synergistic effect of low doses of FA combined with subthreshold dose of piperine, a bioavailability enhancer, on depression-like behaviors in mice, and investigated the possible mechanism. The administration of FA, even in the highest dose tested, reduced immobility time by 60 % in the tail suspension and forced swimming tests (TST and FST) in mice when compared to control. The maximal antidepressant-like effect of FA was obtained with 200 mg/kg. In addition, piperine only produced a weak antidepressant-like effect in the TST and FST. However, the evidence from the interaction analysis suggested a synergistic effect when low doses of FA were combined with a subthreshold dose of piperine. Further neurochemical evidence such as monoamine levels in the frontal cortex, hippocampus, and hypothalamus and measurements of monoamine oxidase activity also supported a synergistic effect of FA and piperine in the enhancement of monoaminergic function. This finding supports the concept that the combination strategy might be an alternative therapy in the treatment of psychiatric disorders with high efficacy and low side effects.

The effect of trans-resveratrol on post-stroke depression via regulation of hypothalamus-pituitary-adrenal axis.

Post-stroke depression (PSD) occurs about 40% among all stroke survivors, but the effective pharmacotherapy is inadequately understood. The present study investigated the effects of a natural polyphenol trans-resveratrol (RES) on behavioral changes after middle cerebral artery occlusion (MCAO) and examined what its molecular targets may be. RES was shown to decrease the infarct size and neurological scores after MCAO, suggesting the amelioration of brain damage and motor activity. RES also reversed the depressive-like behaviors 13 days after MCAO, both in the forced swimming and sucrose consumption tests. Moreover, MCAO-induced series abnormalities related to depressive-like behaviors, such as an abnormal adrenal gland weight to body weight ratio, an increased expression of the corticotropin-releasing factor (CRF) in the frontal cortex, hippocampus and hypothalamus, the differential expression of glucocorticoid receptor (GR) in these three brain regions, and a decreased brain-derived neurotrophic factor (BDNF) level, were ameliorated after treatment with increasing doses of RES at 10, 20 and 40 mg/kg via gavage. These findings provide compelling evidence that RES protects the brain against focal cerebral ischemia-induced injury, but most of all is its antidepressant-like effect on PSD, which might at least in part be mediated by regulation of hypothalamus-pituitary-adrenal axis function.

Site-specific nitrosoproteomic identification of endogenously S-nitrosylated proteins in Arabidopsis.

Nitric oxide (NO) regulates multiple developmental events and stress responses in plants. A major biologically active species of NO is S-nitrosoglutathione (GSNO), which is irreversibly degraded by GSNO reductase (GSNOR). The major physiological effect of NO is protein S-nitrosylation, a redox-based posttranslational modification mechanism by covalently linking an NO molecule to a cysteine thiol. However, little is known about the mechanisms of S-nitrosylation-regulated signaling, partly due to limited S-nitrosylated proteins being identified. In this study, we identified 1,195 endogenously S-nitrosylated peptides in 926 proteins from the Arabidopsis (Arabidopsis thaliana) by a site-specific nitrosoproteomic approach, which, to date, is the largest data set of S-nitrosylated proteins among all organisms. Consensus sequence analysis of these peptides identified several motifs that contain acidic, but not basic, amino acid residues flanking the S-nitrosylated cysteine residues. These S-nitrosylated proteins are involved in a wide range of biological processes and are significantly enriched in chlorophyll metabolism, photosynthesis, carbohydrate metabolism, and stress responses. Consistently, the gsnor1-3 mutant shows the decreased chlorophyll content and altered photosynthetic properties, suggesting that S-nitrosylation is an important regulatory mechanism in these processes. These results have provided valuable resources and new clues to the studies on S-nitrosylation-regulated signaling in plants.

S-nitrosylation positively regulates ascorbate peroxidase activity during plant stress responses.

Nitric oxide (NO) and reactive oxygen species (ROS) are two classes of key signaling molecules involved in various developmental processes and stress responses in plants. The burst of NO and ROS triggered by various stimuli activates downstream signaling pathways to cope with abiotic and biotic stresses. Emerging evidence suggests that the interplay of NO and ROS plays a critical role in regulating stress responses. However, the underpinning molecular mechanism remains poorly understood. Here, we show that NO positively regulates the activity of the Arabidopsis (Arabidopsis thaliana) cytosolic ascorbate peroxidase1 (APX1). We found that S-nitrosylation of APX1 at cysteine (Cys)-32 enhances its enzymatic activity of scavenging hydrogen peroxide, leading to the increased resistance to oxidative stress, whereas a substitution mutation at Cys-32 causes the reduction of ascorbate peroxidase activity and abolishes its responsiveness to the NO-enhanced enzymatic activity. Moreover, S-nitrosylation of APX1 at Cys-32 also plays an important role in regulating immune responses. These findings illustrate a unique mechanism by which NO regulates hydrogen peroxide homeostasis in plants, thereby establishing a molecular link between NO and ROS signaling pathways.

Decoupling of sleepiness from sleep time and intensity during chronic sleep restriction: evidence for a role of the adenosine system.

STUDY OBJECTIVE: Sleep responses to chronic sleep restriction (CSR) might be very different from those observed after short-term total sleep deprivation. For example, after sleep restriction continues for several consecutive days, animals no longer express compensatory increases in daily sleep time and sleep intensity. However, it is unknown if these allostatic, or adaptive, sleep responses to CSR are paralleled by behavioral and neurochemical measures of sleepiness. DESIGN: This study was designed to investigate CSR-induced changes in (1) sleep time and intensity as a measure of electrophysiological sleepiness, (2) sleep latency as a measure of behavioral sleepiness, and (3) brain adenosine A1 (A1R) and A2a receptor (A2aR) mRNA levels as a putative neurochemical correlate of sleepiness. SUBJECTS: Male Sprague-Dawley rats INTERVENTIONS: A 5-day sleep restriction (SR) protocol consisting of 18-h sleep deprivation and 6-h sleep opportunity each day. MEASUREMENT AND RESULTS: Unlike the first SR day, rats did not sleep longer or deeper on days 2 through 5, even though they exhibited significant elevations of behavioral sleepiness throughout all 5 SR days. For all SR days and recovery day 1, A1R mRNA in the basal forebrain was maintained at elevated levels, whereas A2aR mRNA in the frontal cortex was maintained at reduced levels. CONCLUSION: CSR LEADS TO A DECOUPLING OF SLEEPINESS FROM SLEEP TIME AND SLEEP INTENSITY, SUGGESTING THAT THERE ARE AT LEAST TWO DIFFERENT SLEEP REGULATORY SYSTEMS: one mediating sleepiness (homeostatic) and the other mediating sleep time/intensity (allostatic). The time course of changes observed in adenosine receptor mRNA levels suggests that the basal forebrain and cortical adenosine system might mediate sleepiness rather than sleep time or intensity.

Brainstem prolactin mRNA is enhanced in mice with suppressed neuronal nitric oxide synthase activity.

Prolactin (PRL) and vasoactive intestinal polypeptide (VIP) mRNA levels were elevated in the brainstem of neuronal nitric oxide synthase (nNOS) gene knockout (KO) mice compared to the levels in nNOS control mice. In addition, PRL mRNA levels increased in the hypothalamus and the brainstem of nNOS control mice after administration of 7-nitro-indazole (7-NI), a relatively selective nNOS inhibitor. The results suggest that NO inhibits PRL. No differences in the genes measured were observed in inducible NOS KO mice.

Influenza virus-induced sleep responses in mice with targeted disruptions in neuronal or inducible nitric oxide synthases.

Influenza viral infection induces increases in non-rapid eye movement sleep and decreases in rapid eye movement sleep in normal mice. An array of cytokines is produced during the infection, and some of them, such as IL-1beta and TNF-alpha, are well-defined somnogenic substances. It is suggested that nitric oxide (NO) may mediate the sleep-promoting effects of these cytokines. In this study, we use mice with targeted disruptions of either the neuronal NO synthase (nNOS) or the inducible NO synthase (iNOS) gene, commonly referred to as nNOS or iNOS knockouts (KOs), to investigate sleep changes after influenza viral challenge. We report that the magnitude of viral-induced non-rapid eye movement sleep responses in both nNOS KOs and iNOS KOs was less than that of their respective controls. In addition, the duration of rapid eye movement sleep in nNOS KO mice did not decrease compared with baseline values. All strains of mice had similar viral titers and cytokine gene expression profiles in the lungs. Virus was not isolated from the brains of any strain. However, gene expression in the brain stem differed between nNOS KOs and their controls: mRNA for the interferon-induced gene 2',5'-oligoadenylate synthase 1a was elevated in nNOS KOs relative to their controls at 15 h, and IL-1beta mRNA was elevated in nNOS KOs relative to their controls at 48 h. Our results suggest that NO synthesized by both nNOS and iNOS plays a role in virus-induced sleep changes and that nNOS may modulate cytokine expression in the brain.