Defined Sensing Mechanisms and Signaling Pathways Contribute to the Global Inflammatory Gene Expression Output Elicited by Ionizing Radiation.

Environmental insults are often detected by multiple sensors that activate diverse signaling pathways and transcriptional regulators, leading to a tailored transcriptional output. To understand how a tailored response is coordinated, we examined the inflammatory response elicited in mouse macrophages by ionizing radiation (IR). RNA-sequencing studies revealed that most radiation-induced genes were strongly dependent on only one of a small number of sensors and signaling pathways, notably the DNA damage-induced kinase ATM, which regulated many IR-response genes, including interferon response genes, via an atypical IRF1-dependent, STING-independent mechanism. Moreover, small, defined sets of genes activated by p53 and NRF2 accounted for the selective response to radiation in comparison to a microbial inducer of inflammation. Our findings reveal that genes comprising an environmental response are activated by defined sensing mechanisms with a high degree of selectivity, and they identify distinct components of the radiation response that might be susceptible to therapeutic perturbation.

Adaptation of the Tumor Antigen Presentation Machinery to Ionizing Radiation.

Ionizing radiation (IR) can reprogram proteasome structure and function in cells and tissues. In this article, we show that IR can promote immunoproteasome synthesis with important implications for Ag processing and presentation and tumor immunity. Irradiation of a murine fibrosarcoma (FSA) induced dose-dependent de novo biosynthesis of the immunoproteasome subunits LMP7, LMP2, and Mecl-1, in concert with other changes in the Ag-presentation machinery (APM) essential for CD8+ T cell-mediated immunity, including enhanced expression of MHC class I (MHC-I), ß2-microglobulin, transporters associated with Ag processing molecules, and their key transcriptional activator NOD-like receptor family CARD domain containing 5. In contrast, in another less immunogenic, murine fibrosarcoma (NFSA), LMP7 transcripts and expression of components of the immunoproteasome and the APM were muted after IR, which affected MHC-I expression and CD8+ T lymphocyte infiltration into NFSA tumors in vivo. Introduction of LMP7 into NFSA largely corrected these deficiencies, enhancing MHC-I expression and in vivo tumor immunogenicity. The immune adaptation in response to IR mirrored many aspects of the response to IFN-γ in coordinating the transcriptional MHC-I program, albeit with notable differences. Further investigations showed divergent upstream pathways in that, unlike IFN-γ, IR failed to activate STAT-1 in either FSA or NFSA cells while heavily relying on NF-κB activation. The IR-induced shift toward immunoproteasome production within a tumor indicates that proteasomal reprogramming is part of an integrated and dynamic tumor-host response that is specific to the stressor and the tumor and therefore is of clinical relevance for radiation oncology.

Radiation-induced tissue damage and response.

Normal tissue responses to ionizing radiation have been a major subject for study since the discovery of X-rays at the end of the 19th century. Shortly thereafter, time-dose relationships were established for some normal tissue endpoints that led to investigations into how the size of dose per fraction and the quality of radiation affected outcome. The assessment of the radiosensitivity of bone marrow stem cells using colony-forming assays by Till and McCulloch prompted the establishment of in situ clonogenic assays for other tissues that added to the radiobiology toolbox. These clonogenic and functional endpoints enabled mathematical modeling to be performed that elucidated how tissue structure, and in particular turnover time, impacted clinically relevant fractionated radiation schedules. More recently, lineage tracing technology, advanced imaging and single cell sequencing have shed further light on the behavior of cells within stem, and other, cellular compartments, both in homeostasis and after radiation damage. The discovery of heterogeneity within the stem cell compartment and plasticity in response to injury have added new dimensions to the consideration of radiation-induced tissue damage. Clinically, radiobiology of the 20th century garnered wisdom relevant to photon treatments delivered to a fairly wide field at around 2 Gy per fraction, 5 days per week, for 5-7 weeks. Recently, the scope of radiobiology has been extended by advances in technology, imaging and computing, as well as by the use of charged particles. These allow radiation to be delivered more precisely to tumors while minimizing the amount of normal tissue receiving high doses. One result has been an increase in the use of schedules with higher doses per fraction given in a shorter time frame (hypofractionation). We are unable to cover these new technologies in detail in this review, just as we must omit low-dose stochastic effects, and many aspects of dose, dose rate and radiation quality. We argue that structural diversity and plasticity within tissue compartments provides a general context for discussion of most radiation responses, while acknowledging many omissions. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Blood and urinary cytokine balance and renal outcomes at cardiac surgery.

BACKGROUND: Increased perioperative pro-inflammatory biomarkers, renal hypoperfusion and ischemia reperfusion injury (IRI) heighten cardiac surgery acute kidney injury (CS-AKI) risk. Increased urinary anti-inflammatory cytokines attenuate risk. We evaluated whether blood and urinary anti-inflammatory biomarkers, when expressed as ratios with biomarkers of inflammation, hypoperfusion and IRI are increased in CS-AKI patients. METHODS: Preoperative and 24-h postoperative blood and urinary pro-inflammatory and anti-inflammatory cytokines, blood VEGF and H-FABP (hypoperfusion biomarkers), and MK, a biomarker for IRI, were measured in 401 cardiac surgery patients. Pre- and postoperative concentrations of biomarkers and selected ratios thereof, were compared between non-CS-AKI and CS-AKI patients. RESULTS: Compared with non-CS-AKI, blood pro-inflammatory (pre- and post-op TNFα, IP-10, IL-12p40, MIP-1α, NGAL; pre-op IL-6; post-op IL-8, MK) and anti-inflammatory (pre- and post-op sTNFsr1, sTNFsr2, IL-1RA) biomarkers together with urinary pro-inflammatory (pre- and post-op uIL-12p40; post-op uIP-10, uNGAL) and anti-inflammatory (pre- and post-op usTNFsr1, usTNFsr2, uIL-1RA) biomarkers, were significantly higher in CS-AKI patients. Urinary anti-inflammatory biomarkers, when expressed as ratios with biomarkers of inflammation (blood and urine), hypoperfusion (blood H-FABP and VEGF) and IRI (blood MK) were decreased in CS-AKI. In contrast, blood anti-inflammatory biomarkers expressed as similar ratios with blood biomarkers were increased in CS-AKI. CONCLUSIONS: The urinary anti-inflammatory response may protect against the injurious effects of perioperative inflammation, hypoperfusion and IRI. These finding may have clinical utility in bioprediction and earlier diagnosis of CS-AKI and informing future therapeutic strategies for CS-AKI patients.

The Rewarding and Anxiolytic Properties of Ethanol within the Central Nucleus of the Amygdala: Mediated by Genetic Background and Nociceptin.

In humans, alcohol is consumed for its rewarding and anxiolytic effects. The central nucleus of the amygdala (CeA) is considered a neuronal nexus that regulates fear, anxiety, and drug self-administration. Manipulations of the CeA alter ethanol (EtOH) consumption under numerous EtOH self-administration models. The experiments determined whether EtOH is reinforcing/anxiolytic within the CeA, whether selective breeding for high alcohol consumption alters the rewarding properties of EtOH in the CeA, and whether the reinforcing/anxiolytic effects of EtOH in the CeA are mediated by the neuropeptides corticotropin-releasing factor (CRF) and nociceptin. The reinforcing properties of EtOH were determined by having male Wistar and Taconic alcohol-preferring (tP) rats self-administer EtOH directly into the CeA. The expression of anxiety-like behaviors was assessed through multiple behavioral models (social interaction, acoustic startle, and open field). Coadministration of EtOH and a CRF1 antagonist (NBI35965) or nociceptin on self-administration into the CeA and anxiety-like behaviors was determined. EtOH was self-administered directly into the lateral CeA, and tP rats self-administered a lower concentration of EtOH than Wistar rats. EtOH microinjected into the lateral CeA reduced the expression of anxiety-like behaviors, indicating an anxiolytic effect. Coadministration of NBI35965 failed to alter the rewarding/anxiolytic properties of EtOH in the CeA. In contrast, coadministration of the nociceptin enhanced both EtOH reward and anxiolysis in the CeA. Overall, the data indicate that the lateral CeA is a key anatomic location that mediates the rewarding and anxiolytic effects of EtOH, and local nociceptin receptors, but not local CRF1 receptors, are involved in these behaviors. SIGNIFICANCE STATEMENT: Alcohol is consumed for the stimulatory, rewarding, and anxiolytic properties of the drug of abuse. The current data are the first to establish that alcohol is reinforcing and anxiolytic within the lateral central nucleus of the amygdala (CeA) and that the nociceptin system regulates these effects of alcohol within the CeA.

Spontaneous Early Withdrawal Behaviors after Chronic 24-hour Free-Choice Access to Ethanol.

AIMS: Abstinence after chronic alcohol consumption leads to withdrawal symptoms, which are exacerbated after repeated cycles of relapse. This study examined withdrawal-like behaviors after chronic ethanol drinking, with or without repeated cycles of deprivation. METHODS: Male alcohol-preferring (P) rats had access to continuous ethanol (CE), chronic ethanol with repeated deprivation (RD), or remained ethanol naïve (EN). The RD group experienced seven cycles of 2 weeks of deprivation and 2 weeks of re-exposure to ethanol after an initial 6 weeks of ethanol access. Withdrawal was measured after an initial 24 h of ethanol re-exposure in the RD group, which coincided with the same day of ethanol access in the CE group. Withdrawal-like behavior was measured by (a) ethanol intake during the initial 24 h of re-exposure, (b) locomotor activity (LMA) in a novel field 9-13 h after removal of ethanol at the beginning of the fifth re-exposure cycle and (c) acoustic startle responding (ASR) 8-15 h after removal of ethanol at the beginning of the sixth re-exposure cycle. RESULTS: The RD rats displayed a 1-h alcohol deprivation effect (ADE) (temporary ethanol increase), relative to CE rats, during the first to fourth and seventh re-exposure cycles. RD and CE rats displayed significant increases in LMA than EN rats. Regarding ASR, RD rats displayed significantly greater ASR relative to EN rats. CONCLUSION: This study confirms that P rats meet the animal model criterion for ethanol-associated dependence, without a reliance on either behavioral (limited fluid access) or pharmacological (seizure threshold manipulation) challenges.

Interleukin 32 expression in human melanoma.

BACKGROUND: Various proinflammatory cytokines can be detected within the melanoma tumor microenvironment. Interleukin 32 (IL32) is produced by T cells, NK cells and monocytes/macrophages, but also by a subset of melanoma cells. We sought to better understand the biology of IL32 in human melanoma. METHODS: We analyzed RNA sequencing data from 53 in-house established human melanoma cell lines and 479 melanoma tumors from The Cancer Genome Atlas dataset. We evaluated global gene expression patterns associated with IL32 expression. We also evaluated the impact of proinflammatory molecules TNFα and IFNγ on IL32 expression and dedifferentiation in melanoma cell lines in vitro. In order to study the transcriptional regulation of IL32 in these cell lines, we cloned up to 10.5 kb of the 5' upstream region of the human IL32 gene into a luciferase reporter vector. RESULTS: A significant proportion of established human melanoma cell lines express IL32, with its expression being highly correlated with a dedifferentiation genetic signature (high AXL/low MITF). Non IL32-expressing differentiated melanoma cell lines exposed to TNFα or IFNγ can be induced to express the three predominant isoforms (α, ß, γ) of IL32. Cis-acting elements within this 5' upstream region of the human IL32 gene appear to govern both induced and constitutive gene expression. In the tumor microenvironment, IL32 expression is highly correlated with genes related to T cell infiltration, and also positively correlates with high AXL/low MITF dedifferentiated gene signature. CONCLUSIONS: Expression of IL32 in human melanoma can be induced by TNFα or IFNγ and correlates with a treatment-resistant dedifferentiated genetic signature. Constitutive and induced expression are regulated, in part, by cis-acting sequences within the 5' upstream region.

Position of lipidation influences anticancer activity of Smac analogs.

A small group of lipid-conjugated Smac mimetics was synthesized to probe the influence of the position of lipidation on overall anti-cancer activity. Specifically, new compounds were modified with lipid(s) in position 3 and C-terminus. Previously described position 2 lipidated analog M11 was also synthesized. The resulting mini library of Smacs lipidated in positions 2, 3 and C-terminus was screened extensively in vitro against a total number of 50 diverse cancer cell lines revealing that both the position of lipidation as well as the type of lipid, influence their anti-cancer activity and cancer type specificity. Moreover, when used in combination therapy with inhibitor of menin-MLL1 protein interactions, position 2 modified analog SM2 showed strong synergistic anti-cancer properties. The most promising lipid-conjugated analogs SM2 and SM6, showed favorable pharmacokinetics and in vivo activity while administered subcutaneously in the preclinical mouse model. Collectively, our findings suggest that lipid modification of Smacs may be a viable approach in the development of anti-cancer therapeutic leads.

The tubercular badger and the uncertain curve:- The need for a multiple stressor approach in environmental radiation protection.

This article presents the results of a workshop held in Stirling, Scotland in June 2018, called to examine critically the effects of low-dose ionising radiation on the ecosphere. The meeting brought together participants from the fields of low- and high-dose radiobiology and those working in radioecology to discuss the effects that low doses of radiation have on non-human biota. In particular, the shape of the low-dose response relationship and the extent to which the effects of low-dose and chronic exposure may be predicted from high dose rate exposures were discussed. It was concluded that high dose effects were not predictive of low dose effects. It followed that the tools presently available were deemed insufficient to reliably predict risk of low dose exposures in ecosystems. The workshop participants agreed on three major recommendations for a path forward. First, as treating radiation as a single or unique stressor was considered insufficient, the development of a multidisciplinary approach is suggested to address key concerns about multiple stressors in the ecosphere. Second, agreed definitions are needed to deal with the multiplicity of factors determining outcome to low dose exposures as a term can have different meanings in different disciplines. Third, appropriate tools need to be developed to deal with the different time, space and organisation level scales. These recommendations permit a more accurate picture of prospective risks.

Plasticity of Myeloid Cells during Oral Barrier Wound Healing and the Development of Bisphosphonate-related Osteonecrosis of the Jaw.

Injury to the barrier tissue initiates a rapid distribution of myeloid immune cells from bone marrow, which guide sound wound healing. Bisphosphonates, a widely used anti-bone resorptive drug with minimal systemic side effects, have been linked to an abnormal wound healing in the oral barrier tissue leading to, in some cases, osteonecrosis of the jaw (ONJ). Here we report that the development of ONJ may involve abnormal phenotypic plasticity of Ly6G+/Gr1+ myeloid cells in the oral barrier tissue undergoing tooth extraction wound healing. A bolus intravenous zoledronate (ZOL) injection to female C57Bl/6 mice followed by maxillary first molar extraction resulted in the development of ONJ-like lesion during the second week of wound healing. The multiplex assay of dissociated oral barrier cells exhibited the secretion of cytokines and chemokines, which was significantly modulated in ZOL mice. Tooth extraction-induced distribution of Ly6G+/Gr1+ cells in the oral barrier tissue increased in ZOL mice at week 2. ONJ-like lesion in ZOL mice contained Ly6G+/Gr1+ cells with abnormal size and morphology as well as different flow cytometric staining intensity. When anti-Ly6G (Gr1) antibody was intraperitoneally injected for 5 days during the second week of tooth extraction, CD11b+GR1(hi) cells in bone marrow and Ly6G+ cells in the oral barrier tissue were depleted, and the development of ONJ-like lesion was significantly attenuated. This study suggests that local modulation of myeloid cell plasticity in the oral barrier tissue may provide the basis for pathogenesis and thus therapeutic as well as preventive strategy of ONJ.

Reduced Levels of mGlu2 Receptors within the Prelimbic Cortex Are Not Associated with Elevated Glutamate Transmission or High Alcohol Drinking.

BACKGROUND: A Grm2 cys407* stop codon mutation, which results in a loss of the metabotropic glutamate 2 (mGlu2) receptor protein, was identified as being associated with high alcohol drinking by alcohol-preferring (P) rats. The objectives of the current study were to characterize the effects of reduced levels of mGlu2 receptors on glutamate transmission and alcohol drinking. METHODS: Quantitative no-net-flux microdialysis was used to test the hypothesis that basal extracellular glutamate levels in the prelimbic (PL) cortex and nucleus accumbens shell (NACsh) will be higher in P than Wistar rats. A lentiviral-delivered short-hairpin RNA (shRNA)-mediated knockdown was used to test the hypothesis that reduced levels of mGlu2 receptors within the PL cortex will increase voluntary alcohol drinking by Wistar rats. A linear regression analysis was used to test the hypothesis that there will be a significant correlation between the Grm2 cys407* mutation and level of alcohol intake. RESULTS: Extracellular glutamate concentrations within the PL cortex (3.6 ± 0.6 vs. 6.4 ± 0.6 µM) and NACsh (3.2 ± 0.4 vs. 6.6 ± 0.6 µM) were significantly lower in female P than female Wistar rats. Western blot detected the presence of mGlu2 receptors in these regions of female Wistar rats, but not female P rats. Micro-infusion of shRNAs into the PL cortex significantly reduced local mGlu2 receptor levels (by 40%), but did not alter voluntary alcohol drinking in male Wistar rats. In addition, there was no significant correlation between the Grm2 mutation and alcohol intake in 36 rodent lines (r = 0.29, p > 0.05). CONCLUSIONS: Collectively, these results suggest a lack of association between the loss of mGlu2 receptors and glutamate transmission in the NACsh and PL cortex of female P rats, and between the level of mGlu2 receptors in the PL cortex and alcohol drinking of male Wistar rats.

Implications of male circumcision for women in Papua New Guinea: a transformational grounded theory study.

BACKGROUND: Male circumcision reduces the risk of female-to-male transmission of human immunodeficiency virus (HIV) and is being explored for HIV prevention in Papua New Guinea (PNG). PNG has a concentrated HIV epidemic which is largely heterosexually transmitted. There are a diverse range of male circumcision and penile modification practices across PNG. Exploring the implications of male circumcision for women in PNG is important to inform evidence-based health policy that will result in positive, intended consequences. METHODS: The transformational grounded theory study incorporated participatory action research and decolonizing methodologies. In Phase One, an existing data set from a male circumcision study of 861 male and 519 female participants was theoretically sampled and analyzed for women's understanding and experience of male circumcision. In Phase Two of the study, primary data were co-generated with 64 women in seven interpretive focus group discussions and 11 semi-structured interviews to develop a theoretical model of the processes used by women to manage the outcomes of male circumcision. In Phase Three participants assisted to refine the developing transformational grounded theory and identify actions required to improve health. RESULTS: Many women know a lot about male circumcision and penile modification and the consequences for themselves, their families and communities. Their ability to act on this knowledge is determined by numerous social, cultural and economic factors. A transformational grounded theory was developed with connecting categories of: Women Know a Lot, Increasing Knowledge; Increasing Options; and Acting on Choices. Properties and dimensions of each category are represented in the model, along with the intervening condition of Safety. The condition of Safety contextualises the overarching lived realty for women in PNG, enables the inclusion of men in the transformational grounded theory model, and helps to explain relationships between men and women. The theory presents the core category as Power of Choice. CONCLUSIONS: This transformational grounded theory provides a means to explore how women experience male circumcision and penile modification in PNG, including for HIV prevention. Women who have had opportunities for education have a greater range of choices and an increased opportunity to act upon these choices. However, women can only exercise their power of choice in the context of safety. The concept of Peace drawn from the Social Determinants of Health is applied in order to extend the explanatory power of the transformational grounded theory. This study shows that women's ambivalence about male circumcision is often related to lack of safety, a consequence of gender inequality in PNG.

Oral Conditioned Cues Can Enhance or Inhibit Ethanol (EtOH)-Seeking and EtOH-Relapse Drinking by Alcohol-Preferring (P) Rats.

BACKGROUND: Conditioned cues can elicit drug-seeking in both humans and rodents. The majority of preclinical research has employed excitatory conditioned cues (stimuli present throughout the availability of a reinforcer), but oral consumption of alcohol is similar to a conditional stimuli (presence of stimuli is paired with the delivery of the reinforcer) approach. The current experiments attempted to determine the effects of conditional stimuli (both excitatory and inhibitory) on the expression of context-induced ethanol (EtOH)-seeking. METHODS: Alcohol-preferring (P) rats self-administered EtOH and water in standard 2-lever operant chambers. A flavor was added to the EtOH solution (CS+) during the EtOH self-administration sessions. After 10 weeks, rats underwent extinction training (7 sessions), followed by a 2-week home cage period. Another flavor was present during extinction (CS-). Rats were exposed to a third flavor in a non-drug-paired environment (CS(0)). EtOH-seeking was assessed in the presence of no cue, CS+, CS-, or CS(0) in the dipper previously associated with EtOH self-administration (no EtOH available). Rats were maintained a week in their home cage before being returned to the operant chambers with access to EtOH (flavored with no cue, CS+, CS-, or CS(0)). RESULTS: The results indicated that the presence of the CS+ enhanced EtOH-seeking, while the presence of the CS- suppressed EtOH-seeking. Similarly, adding the CS- flavor to 15% EtOH reduced responding for EtOH while the CS+ enhanced responding for EtOH during relapse testing. CONCLUSIONS: Overall, the data indicate that conditional stimuli are effective at altering both EtOH-seeking behavior and EtOH-relapse drinking.

Parameters of Context-Induced Ethanol (EtOH)-Seeking in Alcohol-Preferring (P) Rats: Temporal Analysis, Effects of Repeated Deprivation, and EtOH Priming Injections.

BACKGROUND: Drug-paired environments can act as stimuli that elicit drug craving. In humans, drug craving is influenced by the amount of time abstinent, number of past periods of abstinence, and inadvertent exposure to the previously abused drug. The current experiments were designed to determine the effects of (i) the duration of abstinence on expression of ethanol (EtOH)-seeking; (ii) EtOH priming following a short and long abstinence period; and (iii) repeated deprivation cycles on relapse drinking and EtOH-seeking. METHODS: Rats were allowed to self-administer 15% EtOH, processed through extinction training, maintained in a home cage for a designated EtOH-free period, and then reintroduced to the operant context in the absence of EtOH. The experiments examined the effects of: (i) various home-cage duration periods (1 to 8 weeks), (ii) priming injections of EtOH in the Pavlovian spontaneous recovery (PSR; 14 days after extinction) and reinstatement of responding (RoR; 1 day after extinction) models, and (iii) exposure to repeated cycles of EtOH access-deprivation on relapse drinking and EtOH-seeking behavior. RESULTS: Highest expression of EtOH-seeking was observed following 6 weeks of home-cage maintenance. Priming injections of EtOH were more efficacious at stimulating/enhancing EtOH-seeking in the PSR than RoR model. Exposure to repeated cycles of EtOH deprivation and access enhanced and prolonged relapse drinking and the expression of EtOH-seeking (318 ± 22 responses), which was not observed in rats given equivalent consistent exposure to EtOH (66 ± 11 responses). CONCLUSIONS: Overall, the data indicated that the PSR model has ecological validity; factors that enhance EtOH craving in humans enhance the expression of EtOH-seeking in the PSR test. The data also detail factors that need to be examined to determine the biological basis of EtOH-seeking (e.g., neuroadaptations that occur during the incubation period and following repeated cycles of EtOH drinking and abstinence).

Gene Expression Changes in Glutamate and GABA-A Receptors, Neuropeptides, Ion Channels, and Cholesterol Synthesis in the Periaqueductal Gray Following Binge-Like Alcohol Drinking by Adolescent Alcohol-Preferring (P) Rats.

BACKGROUND: Binge drinking of alcohol during adolescence is a serious public health concern with long-term consequences, including increased pain, fear, and anxiety. The periaqueductal gray (PAG) is involved in processing pain, fear, and anxiety. The effects of adolescent binge drinking on gene expression in this region have yet to be studied. METHODS: Male adolescent alcohol-preferring (P) rats were exposed to repeated binge drinking (three 1-hour sessions/d during the dark/cycle, 5 days/wk for 3 weeks starting at 28 days of age; ethanol intakes of 2.5 to 3 g/kg/session). We used RNA sequencing to assess the effects of ethanol intake on gene expression. RESULTS: Ethanol significantly altered the expression of 1,670 of the 12,123 detected genes: 877 (53%) decreased. In the glutamate system, 23 genes were found to be altered, including reduction in 7 of 10 genes for metabotropic and NMDA receptors. Subunit changes in the NMDA receptor may make it less sensitive to ethanol. Changes in GABAA genes would most likely increase the ability of the PAG to produce tonic inhibition. Five serotonin receptor genes, 6 acetylcholine receptor genes, and 4 glycine receptor genes showed decreased expression in the alcohol-drinking rats. Opioid genes (e.g., Oprk1, Oprm1) and genes for neuropeptides linked to anxiety and panic behaviors (e.g., Npy1r) had mostly decreased expression. Genes for 27 potassium, 10 sodium, and 5 calcium ion channels were found to be differentially expressed. Nine genes in the cholesterol synthesis pathway had decreased expression, including Hmgcr, encoding the rate-limiting enzyme. Genes involved in the production of myelin also had decreased expression. CONCLUSIONS: The results demonstrate that binge alcohol drinking during adolescence produces developmental changes in the expression of key genes within the PAG; many of these changes point to increased susceptibility to pain, fear, and anxiety, which could contribute to excessive drinking to relieve these negative effects.

Peripheral Administration of Ethanol Results in a Correlated Increase in Dopamine and Serotonin Within the Posterior Ventral Tegmental Area.

AIMS: Two critical neurotransmitter systems regulating ethanol (EtOH) reward are serotonin (5-HT) and dopamine (DA). Within the posterior ventral tegmental area (pVTA), 5-HT receptors have been shown to regulate DA neuronal activity. Increased pVTA neuronal activity has been linked to drug reinforcement. The current experiment sought to determine the effect of EtOH on 5-HT and DA levels within the pVTA. METHODS: Wistar rats were implanted with cannula aimed at the pVTA. Neurochemical levels were determined using standard microdialysis procedures with concentric probes. Rats were randomly assigned to one of the five groups (n = 41; 7-9 per group) that were treated with 0-3.0 g/kg EtOH (intraperitoneally). RESULTS: Ethanol produced increased extracellular DA levels in the pVTA that resembled an inverted U-shape dose-response curve with peak levels (~200% of baseline) at the 2.25 g/kg dose. The increase in DA levels was observed for an extended period of time (~100 minutes). The effects of EtOH on extracellular 5-HT levels in the pVTA also resembled an inverted U-shape dose-response curve. However, increased 5-HT levels were only observed during the initial post-injection sample. The increases in extracellular DA and 5-HT levels were significantly correlated. CONCLUSION: The data indicate intraperitoneal EtOH administration stimulated the release of both 5-HT and DA within the pVTA, the levels of which were significantly correlated. Overall, the current findings suggest that the ability of EtOH to stimulate DA activity within the mesolimbic system may be modulated by increases in 5-HT release within the pVTA. SHORT SUMMARY: Two critical neurotransmitter systems regulating ethanol reward are serotonin and dopamine. The current experiment determined that intraperitoneal ethanol administration increased serotonin and dopamine levels within the pVTA (levels were significantly correlated). The current findings suggest the ability of EtOH to stimulate serotonin and dopamine activity within the mesolimbic system.

Bone morphogenetic protein 7 sensitizes O6-methylguanine methyltransferase expressing-glioblastoma stem cells to clinically relevant dose of temozolomide.

BACKGROUND: Temozolomide (TMZ) is an oral DNA-alkylating agent used for treating patients with glioblastoma. However, therapeutic benefits of TMZ can be compromised by the expression of O6-methylguanine methyltransferase (MGMT) in tumor tissue. Here we used MGMT-expressing glioblastoma stem cells (GSC) lines as a model for investigating the molecular mechanism underlying TMZ resistance, while aiming to explore a new treatment strategy designed to possibly overcome resistance to the clinically relevant dose of TMZ (35 µM). METHODS: MGMT-expressing GSC cultures are resistant to TMZ, and IC50 (half maximal inhibitory concentration) is estimated at around 500 µM. Clonogenic GSC surviving 500 µM TMZ (GSC-500 µM TMZ), were isolated. Molecular signatures were identified via comparative analysis of expression microarray against parental GSC (GSC-parental). The recombinant protein of top downregulated signature was used as a single agent or in combination with TMZ, for evaluating therapeutic effects of treatment of GSC. RESULTS: The molecular signatures characterized an activation of protective stress responses in GSC-500 µM TMZ, mainly including biotransformation/detoxification of xenobiotics, blocked endoplasmic reticulum stress-mediated apoptosis, epithelial-to-mesenchymal transition (EMT), and inhibited growth/differentiation. Bone morphogenetic protein 7 (BMP7) was identified as the top down-regulated gene in GSC-500 µM TMZ. Although augmenting BMP7 signaling in GSC by exogenous BMP7 treatment did not effectively stop GSC growth, it markedly sensitized both GSC-500 µM TMZ and GSC-parental to 35 µM TMZ treatment, leading to loss of self-renewal and migration capacity. BMP7 treatment induced senescence of GSC cultures and suppressed mRNA expression of CD133, MGMT, and ATP-binding cassette drug efflux transporters (ABCB1, ABCG2), as well as reconfigured transcriptional profiles in GSC by downregulating genes associated with EMT/migration/invasion, stemness, inflammation/immune response, and cell proliferation/tumorigenesis. BMP7 treatment significantly prolonged survival time of animals intracranially inoculated with GSC when compared to those untreated or treated with TMZ alone (p = 0.0017), whereas combination of two agents further extended animal survival compared to BMP7 alone (p = 0.0489). CONCLUSIONS: These data support the view that reduced endogenous BMP7 expression/signaling in GSC may contribute to maintained stemness, EMT, and chemoresistant phenotype, suggesting that BMP7 treatment may provide a novel strategy in combination with TMZ for an effective treatment of glioblastoma exhibiting unmethylated MGMT.

In vitro and in vivo evaluation of the radiosensitizing effect of a selective FGFR inhibitor (JNJ-42756493) for rectal cancer.

BACKGROUND: We examined the anti-tumor effect and radiosensitizing potential of a small molecule inhibitor of fibroblast growth factor receptor (FGFR) in colorectal cancer (CRC) in vitro and in vivo. METHODS: Effects of in vitro drug treatment on cell survival, proliferation, FGFR signaling, cell cycle distribution, apoptosis and radiosensitivity were assessed using various CRC cell lines with FGFR wild type (Caco2 and HCA7) and FGFR2 amplification (HCT116, NCI-H716). In vivo tumor responses to FGFR inhibition with and without radiation therapy were evaluated by growth delay assays in two colorectal xenograft mouse models (NMRI nu/nu mice injected with NCI-H716 or CaCo2 cells). Mechanistic studies were conducted using Western blot analysis, immunohistochemistry and qPCR. RESULTS: In the tested cell lines, the FGFR inhibitor (JNJ-42756493) was effective in vitro and in vivo in CRC tumors with highest expression of FGFR2 (NCI-H716). In vitro, cell proliferation in this line was decreased, associated with increased apoptotic death and decreased cell survival. In vivo, growth of NCI-H716 tumors was delayed by 5 days by drug treatment alone, although when drug delivery was stopped the relative tumor volume increased compared to control. The FGFR inhibitor did not radiosensitize NCI-H716 tumors either in vitro or in vivo. CONCLUSIONS: Among tested CRC cell lines, the growth inhibitory activity of this FGFR inhibitor was evident in cell lines with high constitutive FGFR2 expression, suggesting that FGFR addiction may provide a window for therapeutic intervention, though caution is advised. Preclinical study with NCI-H716 and Caco2 tumor demonstrated that continued presence of drug could be essential for tumor growth control, especially in cells with aberrant FGFR expression. In the tested set-up, the inhibitor showed no radiosensitizing effect.

Improving the therapeutic index in cancer therapy by using antibody-drug conjugates designed with a moderately cytotoxic drug.

The antibody-drug conjugate (ADC), IMMU-130, of the moderately cytotoxic topoisomerase I inhibitor, SN-38, and the CEACAM5-targeted humanized antibody (mAb), labetuzumab, was evaluated in model systems of human colon carcinoma and in phase I clinical trials of heavily pretreated patients with metastatic colorectal cancer. The conjugate, designed with a near-homogeneous drug substitution of 7-8 SN-38/mAb and with a linker that released 50% of the drug in ∼20 h, showed significant antitumor effects compared to a nontargeted ADC in human tumor xenografts, which could be augmented in combination with bevacizumab. The advantage of fractionated dosing was demonstrated, with potential implications for the clinical dosing schedule. Biodistribution comparing IMMU-130 with labetuzumab showed that the conjugate cleared somewhat faster from the blood, but this did not affect tumor uptake and retention. The use of an ultrastable linker in the conjugate design abrogated antitumor effects. A tolerability study in rabbits showed a high safety margin, with no-observed-adverse-effect level (NOAEL) corresponding to a cumulative human-equivalent protein dose of 40-60 mg/kg. The preclinical findings appear to be corroborated in two phase I clinical trials, with high tolerability and evidence of antitumor activity, including objective responses. The impact of the ADC design on the utility of IMMU-130, tailored to a poorly internalizing target, is discussed.