Phase 1 dose expansion and biomarker study assessing first-in-class tumor microenvironment modulator VT1021 in patients with advanced solid tumors.

BACKGROUND: Preclinical studies have demonstrated that VT1021, a first-in-class therapeutic agent, inhibits tumor growth via stimulation of thrombospondin-1 (TSP-1) and reprograms the tumor microenvironment. We recently reported data from the dose escalation part of a phase I study of VT1021 in solid tumors. Here, we report findings from the dose expansion phase of the same study. METHODS: We analyzed the safety and tolerability, clinical response, and biomarker profile of VT1021 in the expansion portion of the phase I study (NCT03364400). Safety/tolerability is determined by adverse events related to the treatment. Clinical response is determined by RECIST v1.1 and iRECIST. Biomarkers are measured by multiplexed ion beam imaging and enzyme-linked immunoassay (ELISA). RESULTS: First, we report the safety and tolerability data as the primary outcome of this study. Adverse events (AE) suspected to be related to the study treatment (RTEAEs) are mostly grade 1-2. There are no grade 4 or 5 adverse events. VT1021 is safe and well tolerated in patients with solid tumors in this study. We report clinical responses as a secondary efficacy outcome. VT1021 demonstrates promising single-agent clinical activity in recurrent GBM (rGBM) in this study. Among 22 patients with rGBM, the overall disease control rate (DCR) is 45% (95% confidence interval, 0.24-0.67). Finally, we report the exploratory outcomes of this study. We show the clinical confirmation of TSP-1 induction and TME remodeling by VT1021. Our biomarker analysis identifies several plasmatic cytokines as potential biomarkers for future clinical studies. CONCLUSIONS: VT1021 is safe and well-tolerated in patients with solid tumors in a phase I expansion study. VT1021 has advanced to a phase II/III clinical study in glioblastoma (NCT03970447).

The network of cells that surround a tumor, the tumor microenvironment, can help cancers to grow. Therapies targeting the tumor microenvironment may help to stop tumor growth. One such therapy is VT1021. In animal models, VT1021 treatment stops tumor cells from growing by changing the tumor microenvironment. Here, we have tested VT1021 in a clinical trial and found that VT1021 treatment is safe and well tolerated in patients with cancer. We also see signs of efficacy in some patients and observe evidence that VT1021 modifies the tumor microenvironment, which may help to block tumor growth. Finally, we identified several markers from the blood that may help to predict which patients will best benefit from VT1021 treatment. With further testing in clinical trials, VT1021 may be a useful therapy for patients with cancer.

First-in-human phase I dose escalation trial of the first-in-class tumor microenvironment modulator VT1021 in advanced solid tumors.

BACKGROUND: VT1021 is a cyclic peptide that induces the expression of thrombospondin-1 (TSP-1) in myeloid-derived suppressor cells (MDSCs) recruited to the tumor microenvironment (TME). TSP-1 reprograms the TME via binding to CD36 and CD47 to induce tumor and endothelial cell apoptosis as well as immune modulation in the TME. METHODS: Study VT1021-01 (ClinicalTrials.gov ID NCT03364400) used a modified 3 + 3 design. The primary objective was to determine the recommended Phase 2 dose (RP2D) in patients with advanced solid tumors. Safety, tolerability, and pharmacokinetics (PK) were assessed. Patients were dosed twice weekly intravenously in 9 cohorts (0.5-15.6 mg/kg). Safety was evaluated using CTCAE version 5.0 and the anti-tumor activity was evaluated by RECIST version 1.1. RESULTS: The RP2D of VT1021 is established at 11.8 mg/kg. VT1021 is well tolerated with no dose-limiting toxicities reported (0/38). The most frequent drug-related adverse events are fatigue (15.8%), nausea (10.5%), and infusion-related reactions (10.5%). Exposure increases proportionally from 0.5 to 8.8 mg/kg. The disease control rate (DCR) is 42.9% with 12 of 28 patients deriving clinical benefit including a partial response (PR) in one thymoma patient (504 days). CONCLUSIONS: VT1021 is safe and well-tolerated across all doses tested. RP2D has been selected for future clinical studies. PR and SD with tumor shrinkage are observed in multiple patients underscoring the single-agent potential of VT1021. Expansion studies in GBM, pancreatic cancer and other solid tumors at the RP2D have been completed and results will be communicated in a separate report.

It may be possible to treat cancers with therapies that modify the tumor microenvironment. This is the environment in the body in which tumors survive and grow and is composed of different types of cells. One such potential therapy is VT1021. Here, we conduct the first clinical trial to test this therapy in patients. We identify the optimal dose of the treatment to take into further studies, finding that VT1021 is safe and well tolerated by patients. We see some signs that the treatment is working in some patients and see evidence of modification of the tumor microenvironment. These findings help to inform further clinical trials of VT1021 to determine whether it is safe and effective in larger cohorts of patients.

EYA2 tyrosine phosphatase inhibition reduces MYC and prevents medulloblastoma progression.

BACKGROUND: Medulloblastoma is the most common pediatric brain malignancy. Patients with the Group 3 subtype of medulloblastoma (MB) often exhibit MYC amplification and/or overexpression and have the poorest prognosis. While Group 3 MB is known to be highly dependent on MYC, direct targeting of MYC remains elusive. METHODS: Patient gene expression data were used to identify highly expressed EYA2 in Group 3 MB samples, assess the correlation between EYA2 and MYC, and examine patient survival. Genetic and pharmacological studies were performed on EYA2 in Group 3 derived MB cell models to assess MYC regulation and viability in vitro and in vivo. RESULTS: EYA2 is more highly expressed in Group 3 MB than other MB subgroups and is essential for Group 3 MB growth in vitro and in vivo. EYA2 regulates MYC expression and protein stability in Group 3 MB, resulting in global alterations of MYC transcription. Inhibition of EYA2 tyrosine phosphatase activity, using a novel small molecule inhibitor (NCGC00249987, or 9987), significantly decreases Group 3 MB MYC expression in both flank and intracranial growth in vivo. Human MB RNA-seq data show that EYA2 and MYC are significantly positively correlated, high EYA2 expression is significantly associated with a MYC transcriptional signature, and patients with high EYA2 and MYC expression have worse prognoses than those that do not express both genes at high levels. CONCLUSIONS: Our data demonstrate that EYA2 is a critical regulator of MYC in Group 3 MB and suggest a novel therapeutic avenue to target this highly lethal disease.

SIX1 and EWS/FLI1 co-regulate an anti-metastatic gene network in Ewing Sarcoma.

Ewing sarcoma (ES), which is characterized by the presence of oncogenic fusion proteins such as EWS/FLI1, is an aggressive pediatric malignancy with a high rate of early dissemination and poor outcome after distant spread. Here we demonstrate that the SIX1 homeoprotein, which enhances metastasis in most tumor types, suppresses ES metastasis by co-regulating EWS/FLI1 target genes. Like EWS/FLI1, SIX1 promotes cell growth/transformation, yet dramatically inhibits migration and invasion, as well as metastasis in vivo. We show that EWS/FLI1 promotes SIX1 protein expression, and that the two proteins share genome-wide binding profiles and transcriptional regulatory targets, including many metastasis-associated genes such as integrins, which they co-regulate. We further show that SIX1 downregulation of integrins is critical to its ability to inhibit invasion, a key characteristic of metastatic cells. These data demonstrate an unexpected anti-metastatic function for SIX1, through coordinate gene regulation with the key oncoprotein in ES, EWS/FLI1.

Outpatient management of moderate cellulitis in children using high-dose oral cephalexin.

Objectives: To evaluate the effectiveness of a high-dose (HD) oral cephalexin treatment guideline for children with moderate cellulitis treated as outpatients. Methods: In this retrospective cohort study, we included children who presented to the emergency department (ED) with moderate cellulitis and treated according to the institution's HD oral cephalexin guideline over a 2-year period. All children had standardized follow-up at a medical day hospital (MDH). Treatment was considered effective in the absence of treatment failure, defined as admission, switch to IV treatment or ED visit within 2 weeks of discharge from the MDH. Safety was ascertained by recording adverse events and severe complications at follow-up. Results: A total of 123 children were treated as outlined in the guideline, including 117 treated with HD oral cephalexin. The success rate was 89.7% (105/117). Among 12 (10.3%) children who had treatment failure, 10 (8.5%) required admission, 1 (0.9%) received IV antibiotics at the MDH and 1 (0.9%) had a return visit to the ED without admission. No severe complications were reported; four abscesses required drainage and one patient had a rash. The mean number of visits per child at the MDH was 1.6 (SD 1.0). Conclusions: With a success rate of 89.7%, HD oral cephalexin seems effective and safe for the treatment of children with moderate cellulitis. Its use potentially reduces hospitalization rates for this condition and decreases the need for IV insertion.

Structural and Functional Analyses of an Allosteric EYA2 Phosphatase Inhibitor That Has On-Target Effects in Human Lung Cancer Cells.

EYA proteins (EYA1-4) are critical developmental transcriptional cofactors that contain an EYA domain (ED) harboring Tyr phosphatase activity. EYA proteins are largely downregulated after embryogenesis but are reexpressed in cancers, and their Tyr phosphatase activity plays an important role in the DNA damage response and tumor progression. We previously identified a class of small-molecule allosteric inhibitors that specifically inhibit the Tyr phosphatase activity of EYA2. Herein, we determined the crystal structure of the EYA2 ED in complex with NCGC00249987 (a representative compound in this class), revealing that it binds to an induced pocket distant from the active site. NCGC00249987 binding leads to a conformational change of the active site that is unfavorable for Mg2+ binding, thereby inhibiting EYA2's Tyr phosphatase activity. We demonstrate, using genetic mutations, that migration, invadopodia formation, and invasion of lung adenocarcinoma cells are dependent on EYA2 Tyr phosphatase activity, whereas growth and survival are not. Further, we demonstrate that NCGC00249987 specifically targets migration, invadopodia formation, and invasion of lung cancer cells, but that it does not inhibit cell growth or survival. The compound has no effect on lung cancer cells carrying an EYA2 F290Y mutant that abolishes compound binding, indicating that NCGC00249987 is on target in lung cancer cells. These data suggest that the NCGC00249987 allosteric inhibitor can be used as a chemical probe to study the function of the EYA2 Tyr phosphatase activity in cells and may have the potential to be developed into an antimetastatic agent for cancers reliant on EYA2's Tyr phosphatase activity.

SIX2 Mediates Late-Stage Metastasis via Direct Regulation of SOX2 and Induction of a Cancer Stem Cell Program.

The capacity for tumor cells to metastasize efficiently is directly linked to their ability to colonize secondary sites. Here we identify Six2, a developmental transcription factor, as a critical regulator of a breast cancer stem cell program that enables metastatic colonization. In several triple-negative breast cancer (TNBC) models, Six2 enhanced the expression of genes associated with embryonic stem cell programs. Six2 directly bound the Sox2 Srr2 enhancer, promoting Sox2 expression and downstream expression of Nanog, which are both key pluripotency factors. Regulation of Sox2 by Six2 enhanced cancer stem cell properties and increased metastatic colonization. Six2 and Sox2 expression correlated highly in breast cancers including TNBC, where a Six2 expression signature was predictive of metastatic burden and poor clinical outcome. Our findings demonstrate that a SIX2/SOX2 axis is required for efficient metastatic colonization, underscoring a key role for stemness factors in outgrowth at secondary sites. SIGNIFICANCE: These findings provide novel mechanistic insight into stemness and the metastatic outgrowth of triple-negative breast cancer cells.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/4/720/F1.large.jpg.

[Nurse and dietitian: effective collaboration in rehabilitation]. / Infirmière et diététicienne : une collaboration efficace en service de rééducation.

In functional rehabilitation service, the dietician, as educator, contributes to preventing recidivism and return to independence. A hospital team demonstrates the benefits of collaboration with dietitians in coordinated care for patients with various pathologies causing functional impairment.

Eya3 promotes breast tumor-associated immune suppression via threonine phosphatase-mediated PD-L1 upregulation.

Eya proteins are critical developmental regulators that are highly expressed in embryogenesis but downregulated after development. Amplification and/or re-expression of Eyas occurs in many tumor types. In breast cancer, Eyas regulate tumor progression by acting as transcriptional cofactors and tyrosine phosphatases. Intriguingly, Eyas harbor a separate threonine (Thr) phosphatase activity, which was previously implicated in innate immunity. Here we describe what we believe to be a novel role for Eya3 in mediating triple-negative breast cancer-associated immune suppression. Eya3 loss decreases tumor growth in immune-competent mice and is associated with increased numbers of infiltrated CD8+ T cells, which, when depleted, reverse the effects of Eya3 knockdown. Mechanistically, Eya3 utilizes its Thr phosphatase activity to dephosphorylate Myc at pT58, resulting in a stabilized form. We show that Myc is required for Eya3-mediated increases in PD-L1, and that rescue of PD-L1 in Eya3-knockdown cells restores tumor progression. Finally, we demonstrate that Eya3 significantly correlates with PD-L1 in human breast tumors, and that tumors expressing high levels of Eya3 have a decreased CD8+ T cell signature. Our data uncover a role for Eya3 in mediating tumor-associated immune suppression, and suggest that its inhibition may enhance checkpoint therapies.

Dorsal raphé nucleus glucocorticoid receptors inhibit tph2 gene expression in male C57BL/6J mice.

The serotonergic dorsal raphé nucleus (DRN) expresses glucocorticoid receptors (GR), and systemic glucocorticoids have been shown to regulate expression and activity of tryptophan hydroxylase isoform 2, the rate-limiting enzyme for serotonin synthesis in brain. We have used intra-DRN injection of pseudotyped adeno-associated virus AAV2/9 transducing either green fluorescent protein (GFP control) or Cre recombinase (DRN GR deletion) in floxed GR mice to determine if DRN GR directly regulate DRN mRNA levels of tryptophan hydroxylase 2 (tph2). In a separate set of similarly-treated floxed GR mice, we also measured limbic forebrain region concentrations of serotonin (5-hydroxytryptamine; 5-HT) and its major metabolite, 5-hydroxyindoleacetic acid (5-HIAA). DRN GR deletion increased tph2 mRNA levels in the dorsal, lateral wing, and caudal parts of the DRN without altering tissue concentrations of 5-HT, 5-HIAA, or the 5-HIAA/5-HT ratio in limbic forebrain regions. We conclude that DRN GR inhibit DRN tph2 gene expression in mice without marked effects on serotonin metabolism, at least under basal conditions at the circadian nadir. These data provide the first evidence of localized control of DRN tph2 mRNA expression by DRN GR in mice.

Variability of Care in Infants with Severe Bronchiolitis: Less-Invasive Respiratory Management Leads to Similar Outcomes.

OBJECTIVE: To compare the management of children with severe bronchiolitis requiring intensive care (based on duration of ventilatory support and duration of pediatric intensive care unit [PICU] stay) in 2 countries with differing pediatric transport and PICU organizations. STUDY DESIGN: This was a prospective observational care study in 2 PICUs of tertiary care university hospitals, 1 in France and 1 in Canada. All children with bronchiolitis who required admission to the PICU between November 1, 2013, and March 31, 2014, were included. RESULTS: A total of 194 children were included. Baseline characteristics and illness severity were similar at the 2 sites. There was a significant difference between centers in the use of invasive ventilation (3% in France vs 26% in Canada; P < .0001). The number of investigations performed from admission to emergency department presentation and during the PICU stay was significantly higher in Canada for both chest radiographs and blood tests (P < .001). The use of antibiotics was significantly higher in Canada both before (60% vs 28%; P < .001) and during (72% vs 33%; P < .0001) the PICU stay. The duration of ventilatory support, median length of stay, and rate of PICU readmission were similar in the 2 centers. CONCLUSION: Important differences in the management of children with severe bronchiolitis were observed during both prehospital transport and PICU treatment. Less invasive management resulted in similar outcomes with in fewer complications.

Federal and State Responses to Domestic Minor Sex Trafficking: The Evolution of Policy.

Domestic minor sex trafficking (DMST) is a rapidly growing problem in the United States, yet legislative efforts to address victim needs have begun only recently. DMST is an issue that spans several areas of social work practice, as emerging research shows that most children and youths exploited in commercial sex have typically experienced prior abuse, neglect, or other forms of trauma. Many have been involved with the child welfare and juvenile justice systems and are often lured by promises of love, security, protection, and belonging. Policy development to address DMST is still relatively new and evolving at both federal and state levels, but the general trend is to recognize such minors as victims rather than perpetrators of sex crimes. In this article the authors trace the development of legislation addressing DMST at the federal and state levels, with a particular focus on states' "safe harbor laws" that provide limited or total criminal immunity and a varying range of services to victims. Although space limitation precludes a detailed discussion of specific state laws, comparative analysis of representative provisions are discussed, highlighting social work application and further policy and research implications.

Comparison of the efficacy of five adeno-associated virus vectors for transducing dorsal raphé nucleus cells in the mouse.

BACKGROUND: Delivery of genes to various brain regions can be accomplished using serotype 2 of the adeno-associated virus (AAV). Pseudotype AAV2 vectors, composed of the AAV2 genome packaged in the capsid of an alternative serotype, have increased efficiency of viral transduction. Transduction of pseudotype AAV2 vectors depends on cell type, brain region and stage of development. The dorsal raphé nucleus (DRN) and median raphé provides the majority of serotonin to forebrain regions and are implicated in the pathology and treatment of depression and anxiety. Viral vector technology in combination with stereotaxic surgery in mice provides a means to differentiate gene function in the DRN compared to the median raphé nucleus. NEW METHOD: Since AAV transduction efficiency has not yet been characterized for the DRN, we tested if AAV2 pseudotypes are more efficient than a standard serotype (AAV2/2) in transducing DRN cells in adult male mice on a C57BL/6J background. RESULTS: Although transduction did not differ significantly among vectors by 15 days post-injection, pseudotype AAV2/9 and AAV2/rh.10 vectors achieved significantly greater transduction of the DRN than did AAV2/2 and AAV2/1 vectors by 30 days post-injection. Pseudotypes AAV2/1 and AAV2/5 tended, although not significantly, to transduce DRN cells more efficiently than did AAV2/2. COMPARISON WITH EXISTING METHODS: At the same titer, all pseudotype AAV tested tended to transduce the DRN more efficiently than standard AAV2/2 serotype at 30 days post-injection. CONCLUSIONS: Our results support the use of pseudotype AAV2/9 and AAV2/rh.10 for studying gene deletion or overexpression in the DRN.

Glucocorticoid receptor deletion from the dorsal raphé nucleus of mice reduces dysphoria-like behavior and impairs hypothalamic-pituitary-adrenocortical axis feedback inhibition.

Glucocorticoids can cause depression and anxiety. Mechanisms for glucocorticoid effects on mood are largely undefined. The dorsal raphé nucleus (DRN) produces the majority of serotonin in the brain, and expresses glucocorticoid receptors (GR). Because we previously showed that antidepressants used to treat depression and anxiety decrease DRN GR expression, we hypothesized that deleting DRN GR would have anxiolytic- and antidepressant-like effects. We also hypothesized that DRN GR deletion would disinhibit activity of the hypothalamic-pituitary-adrenal (HPA) axis. Adeno-associated virus pseudotype AAV2/9 expressing either Cre recombinase (DRNGRKO mice) or GFP (DRN-GFP mice) was injected into the DRN of floxed GR mice to test these hypotheses. Three weeks after injection, mice underwent 21 days of social defeat or control handling and were tested for anxiety-like behavior (open-field test, elevated-plus maze), depression-like behavior [sucrose preference, forced-swim test (FST), tail-suspension test (TST)], social interaction, and circadian and stress-induced HPA activity. DRN GR deletion decreased anxiety-like behavior in control but not in defeated mice. DRN GR deletion decreased FST and tended to decrease TST despair-like behavior in both control and defeated mice, but did not affect sucrose preference. Exploration of social (a novel mouse) as well as neutral (an empty box) targets was increased in DRNGRKO mice, suggesting that DRN GR deletion also promotes active coping. DRN GR deletion increased stress-induced HPA activity without strongly altering circadian HPA activity. We have shown a novel role for DRN GR to mediate anxiety- and despair-like behavior and to regulate HPA negative feedback during acute stress.

Sensitivity of depression-like behavior to glucocorticoids and antidepressants is independent of forebrain glucocorticoid receptors.

The location of glucocorticoid receptors (GR) implicated in depression symptoms and antidepressant action remains unclear. Forebrain glucocorticoid receptor deletion on a C57B/6×129×CBA background (FBGRKO-T50) reportedly produces increased depression-like behavior and elevated glucocorticoids. We further hypothesized that forebrain GR deletion would reduce behavioral sensitivity to glucocorticoids and to antidepressants. We have tested this hypothesis in mice with calcium calmodulin kinase IIα-Cre-mediated forebrain GR deletion derived from a new founder on a pure C57BL/6 background (FBGRKO-T29-1). We measured immobility in forced swim or tail suspension tests after manipulating glucocorticoids or after dose response experiments with tricyclic or monoamine oxidase inhibitor antidepressants. Despite forebrain GR deletion that was at least as rapid and more extensive than reported in the mixed-strain FBGRKO-T50 mice (Boyle et al. 2005), and possibly because of their different founder, our FBGRKO-T29-1 mice did not exhibit increases in depression-like behavior or adrenocortical axis hormones. Nevertheless, FBGRKO-T29-1 mice were at least as sensitive as floxed GR controls to the depressive effects of glucocorticoids and the effects of two different classes of antidepressants. FBGRKO-T29-1 mice also unexpectedly exhibited increased mineralocorticoid receptor (MR) gene expression. Our results reinforce prior evidence that antidepressant action does not require forebrain GR, and suggest a correlation between the absence of depression-like phenotype and combined MR up-regulation and central amygdala GR deficiency. Our findings demonstrate that GR outside the areas targeted in FBGRKO-T29-1 mice are involved in the depressive effects of glucocorticoids, and leave open the possibility that these GR populations also contribute to antidepressant action.

Impact of home oxygen therapy on hospital stay for infants with acute bronchiolitis.

UNLABELLED: Acute bronchiolitis has been associated with an increasing hospitalization rate over the past decades. The aim of this paper was to estimate the impact of home oxygen therapy (HOT) on hospital stay for infants with acute bronchiolitis. A retrospective cohort study was done including all children aged ≤ 12 months discharged from a pediatric tertiary-care center with a diagnosis of bronchiolitis, between November 2007 and March 2008. Oxygen was administered according to a standardized protocol. We assumed children with the following criteria could have been sent home with O(2), instead of being kept in hospital: age ≥ 2 months, distance between home and hospital <50 km, in-hospital observation ≥ 24 h, O(2) requirement ≤ 1.0 L/min, stable clinical condition, no enteral tube feeding, and intravenous fluids <50 mL/kg/day. Children with significant underlying disease were excluded. A total of 177 children were included. Median age was 2.0 months (range 0-11), and median length of stay was 3.0 days (range 0-18). Forty-eight percent of patients (85/177) received oxygen during their hospital stay. Criteria for discharge with HOT were met in 7.1 % of patients, a mean of 1.8 days (SD 1.8) prior to real discharge. The number of patient-days of hospitalization which would have been saved had HOT been available was 21, representing 3.0 % of total patient-days of hospitalization for bronchiolitis over the study period (21/701). CONCLUSIONS: In this study setting, few children were eligible for an early discharge with HOT. Home oxygen therapy would not significantly decrease the overall burden of hospitalization for bronchiolitis.

A simple method for measuring intracellular activities of glutamine synthetase and glutaminase in glial cells.

Here we report and validate a simple method for measuring intracellular activities of glial glutamine synthetase (GS) and glutaminase (GLNase) in intact glial cells. These enzymes are responsible for glutamate and glutamine recycling in the brain, where glutamate and glutamine transport from the blood stream is strongly limited by the blood-brain barrier. The intracellular levels of glutamate and glutamine are dependent on activities of numerous enzymatic processes, including 1) cytosolic production of glutamine from glutamate by GS, 2) production of glutamate from glutamine by GLNase that is primarily localized between mitochondrial membranes, and 3) mitochondrial conversion of glutamate to the tricarboxylic cycle intermediate α-ketoglutarate in the reactions of oxidative deamination and transamination. We measured intracellular activities of GS and GLNase by quantifying enzymatic interconversions of L-[(3)H]glutamate and L-[(3)H]glutamine in cultured rat astrocytes. The intracellular substrate and the products of enzymatic reactions were separated in one step using commercially available anion exchange columns and quantified using a scintillation counter. The involvement of GS and GLNase in the conversion of (3)H-labeled substrates was verified using irreversible pharmacological inhibitors for each of the enzymes and additionally validated by measuring intracellular amino acid levels using an HPLC. Overall, this paper describes optimized conditions and pharmacological controls for measuring GS and GLNase activities in intact glial cells.

Hypo-osmotic swelling modifies glutamate-glutamine cycle in the cerebral cortex and in astrocyte cultures.

In our previous work, we found that perfusion of the rat cerebral cortex with hypo-osmotic medium triggers massive release of the excitatory amino acid L-glutamate but decreases extracellular levels of L-glutamine (R. E. Haskew-Layton et al., PLoS ONE, 3: e3543). The release of glutamate was linked to activation of volume-regulated anion channels, whereas mechanism(s) responsible for alterations in extracellular glutamine remained unclear. When mannitol was added to the hypo-osmotic medium to reverse reductions in osmolarity, changes in microdialysate levels of glutamine were prevented, indicating an involvement of cellular swelling. As the main source of brain glutamine is astrocytic synthesis and export, we explored the impact of hypo-osmotic medium on glutamine synthesis and transport in rat primary astrocyte cultures. In astrocytes, a 40% reduction in medium osmolarity moderately stimulated the release of L-[(3) H]glutamine by ∼twofold and produced no changes in L-[(3) H]glutamine uptake. In comparison, hypo-osmotic medium stimulated the release of glutamate (traced with D-[(3) H]aspartate) by more than 20-fold. In whole-cell enzymatic assays, we discovered that hypo-osmotic medium caused a 20% inhibition of astrocytic conversion of L-[(3) H]glutamate into L-[(3) H]glutamine by glutamine synthetase. Using an HPLC assay, we further found a 35% reduction in intracellular levels of endogenous glutamine. Overall, our findings suggest that cellular swelling (i) inhibits astrocytic glutamine synthetase activity, and (ii) reduces substrate availability for this enzyme because of the activation of volume-regulated anion channels. These combined effects likely lead to reductions in astrocytic glutamine export in vivo and may partially explain occurrence of hyperexcitability and seizures in human hyponatremia.

Effects of 18-methoxycoronaridine on ghrelin-induced increases in sucrose intake and accumbal dopamine overflow in female rats.

RATIONALE: 18-methoxycoronaridine (18-MC), a selective antagonist of α3ß4 nicotinic receptors, has been previously shown, in rats, to reduce the self-administration of several drugs of abuse, reduce operant responding for sucrose, and prevent the development of sucrose-induced obesity. It has become increasingly apparent that there is a significant overlap between the systems regulating drug reward and food intake, therefore, we investigated whether 18-MC might modulate the effects of ghrelin, one of several orexigenic peptides recently implicated in both feeding and drug reward. OBJECTIVES: In female Sprague-Dawley rats, we determined whether acute 18-MC treatment would reduce both ghrelin-induced increases in sucrose intake and ghrelin-elicited increases in accumbal dopamine levels. RESULTS: Pretreatment with 18-MC (20 mg/kg, i.p.), given prior to the administration of ghrelin (1 µg, lateral ventricle), blocked ghrelin-induced increases in sucrose (5%) intake in a two-bottle open access paradigm. Using in vivo microdialysis, 18-MC (both 20 and 40 mg/kg) prevented ghrelin (2 µg, intraventral tegmental area)-induced increases in extracellular dopamine in the nucleus accumbens. 18-MC had no effect on deposition of fat or on serum levels of glucose, triglycerides, and cholesterol in ghrelin-treated rats. CONCLUSIONS: The present results suggest that one potential mechanism by which 18-MC exerts its effects on palatable food consumption is via modulation of ghrelin's effects.

Evaluation of an anti-tumor necrosis factor therapeutic in a mouse model of Niemann-Pick C liver disease.

BACKGROUND: Niemann-Pick type C (NPC) disease is a lysosomal storage disease characterized by the accumulation of cholesterol and glycosphingolipids. The majority of NPC patients die in their teen years due to progressive neurodegeneration; however, half of NPC patients also suffer from cholestasis, prolonged jaundice, and hepatosplenomegaly. We previously showed that a key mediator of NPC liver disease is tumor necrosis factor (TNF) α, which is involved in both proinflammatory and apoptotic signaling cascades. In this study, we tested the hypothesis that blocking TNF action with an anti-TNF monoclonal antibody (CNTO5048) will slow the progression of NPC liver disease. METHODOLOGY/PRINCIPAL FINDINGS: Treatment of wild-type C57BL/6 mice with NPC1-specific antisense oligonucleotides led to knockdown of NPC1 protein expression in the liver. This caused classical symptoms of NPC liver disease, including hepatic cholesterol accumulation, hepatomegaly, elevated serum liver enzymes, and lipid laden macrophage accumulation. In addition, there was a significant increase in the number of apoptotic cells and a proliferation of stellate cells. Concurrent treatment of NPC1 knockdown mice with anti-TNF had no effect on the primary lipid storage or accumulation of lipid-laden macrophages. However, anti-TNF treatment slightly blunted the increase in hepatic apoptosis and stellate cell activation that was seen with NPC1 knockdown. CONCLUSIONS/SIGNIFICANCE: Current therapeutic options for NPC disease are limited. Our results provide proof of principle that pharmacologically blocking the TNF-α inflammatory cascade can slightly reduce certain markers of NPC disease. Small molecule inhibitors of TNF that penetrate tissues and cross the blood-brain barrier may prove even more beneficial.