A Paranigral VTA Nociceptin Circuit that Constrains Motivation for Reward.

Nociceptin and its receptor are widely distributed throughout the brain in regions associated with reward behavior, yet how and when they act is unknown. Here, we dissected the role of a nociceptin peptide circuit in reward seeking. We generated a prepronociceptin (Pnoc)-Cre mouse line that revealed a unique subpopulation of paranigral ventral tegmental area (pnVTA) neurons enriched in prepronociceptin. Fiber photometry recordings during progressive ratio operant behavior revealed pnVTAPnoc neurons become most active when mice stop seeking natural rewards. Selective pnVTAPnoc neuron ablation, inhibition, and conditional VTA nociceptin receptor (NOPR) deletion increased operant responding, revealing that the pnVTAPnoc nucleus and VTA NOPR signaling are necessary for regulating reward motivation. Additionally, optogenetic and chemogenetic activation of this pnVTAPnoc nucleus caused avoidance and decreased motivation for rewards. These findings provide insight into neuromodulatory circuits that regulate motivated behaviors through identification of a previously unknown neuropeptide-containing pnVTA nucleus that limits motivation for rewards.

Investigation of heterocellular features of the mouse retinal neurovascular unit by 3D electron microscopy.

The retina has a complex structure with a diverse collection of component cells that work together to facilitate vision. The retinal capillaries supplying the nutritional requirements to the inner retina have an intricate system of neural, glial and vascular elements that interconnect to form the neurovascular unit (NVU). The retina has no autonomic nervous system and so relies on the NVU as an interdependent, physical and functional unit to alter blood flow appropriately to changes in the physiological environment. The importance of this is demonstrated by alterations in NVU function being apparent in the blinding disease diabetic retinopathy and other diseases of the retina. It is, therefore, imperative to understand the anatomy of the components of the NVU that underlie its functioning and in particular the nanoscale arrangements of its heterocellular components. However, information on this in three spatial dimensions is limited. In the present study, we utilised the technique of serial block-face scanning electron microscopy (SBF-SEM), and computational image reconstruction, to enable the first three-dimensional ultrastructural analysis of the NVU in mouse retinal capillaries. Mouse isolated retina was prepared for SBF-SEM and up to 150 serial scanning electron microscopy images (covering z-axes distances of 12-8 mm) of individual capillaries in the superficial plexus and NVU cellular components digitally aligned. Examination of the data in the x-, y- and z-planes was performed with the use of semi-automated computational image analysis tools including segmentation, 3D image reconstruction and quantitation of cell proximities. A prominent feature of the capillary arrangements in 3D was the extensive sheath-like coverage by singular pericytes. They appeared in close register to the basement membrane with which they interwove in a complex mesh-like appearance. Breaks in the basement membrane appeared to facilitate pericyte interactions with other NVU cell types. There were frequent, close (<10 nm) pericyte-endothelial interactions with direct contact points and peg-and-socket-like morphology. Macroglia typically intervened between neurons and capillary structures; however, regions were identified where neurons came into closer contact with the basement membrane. A software-generated analysis to assess the morphology of the different cellular components of the NVU, including quantifications of convexity, sphericity and cell-to-cell closeness, has enabled preliminary semi-quantitative characterisation of cell arrangements with neighbouring structures. This study presents new data on the nanoscale spatial characteristics of components of the murine retinal NVU in 3D that has implications for our understanding of structural integrity (e.g. pericyte-endothelial cell anchoring) and function (e.g. possible paracrine communication between macroglia and pericytes). It also serves as a platform to inform future studies examining changes in NVU characteristics with different biological and disease circumstances. All raw and processed image data have been deposited for public viewing.

Vitamin B6 levels do not correlate with severity of neuropathy in chronic idiopathic axonal polyneuropathy.

Pyridoxine (vitamin B6) toxicity is known to cause a length-dependent, sensory predominant axonal polyneuropathy. There is debate regarding the threshold at which intake levels can cause neurological symptoms through pyridoxine toxicity. We asked if elevated plasma vitamin B6 levels were related to outcome measures in a well-characterized cohort of patients with chronic idiopathic axonal polyneuropathy (CIAP). We included 261 patients enrolled in the Peripheral Neuropathy Research Registry who had a complete dataset including a plasma vitamin B6 value. Patients with vitamin B6 deficiency (0-4.9 µg/L) were excluded. We performed a chi-square test for independence and analyzed the logistic relation of elevated plasma B6 level to nerve conduction studies (NCS), neurological examination findings, and patient-reported symptoms controlling for age and time elapsed since neuropathy symptom onset. Plasma B6 level was not related to neuropathy severity. There was no logistic relation of elevated plasma B6 level to NCS results, examination features including toe strength, vibration sense, and deep tendon reflexes, or patient-reported numbness or pain intensity. This study suggests that moderately elevated plasma B6 levels, even in the 100 to 200 µg/L range, are not associated with significantly worse neuropathy signs or symptoms. Although standard supplementation of B6 does not appear to have a major negative affect on CIAP, this study does not directly answer whether stopping supplementation will have a beneficial effect. Very few patients in the study had vitamin B6 levels >300 µg/L, suggesting that screening for vitamin B6 toxicity may be left to the discretion of the physician.

Diminished Utilization of in Vitro Fertilization Following Ovarian Transposition in Cervical Cancer Patients.

OBJECTIVE: To assess subsequent utilization of fertility treatment in reproductive-age women with cervical cancer (CC) who underwent ovarian transposition (OT) to preserve fertility prior to pelvic radiation. STUDY DESIGN: This is a case series of 216 CC patients seen in a comprehensive cancer center. Sixteen patients underwent OT for fertility preservation prior to pelvic radiation. Patients were assessed for utilization of fertility treatment, follicle-stimulating hormone (FSH) levels as a measure of ovarian reserve, and functional assessment of chronic illness therapy-cervix cancer (FACT-CX) to assess quality of life after OT. RESULTS: Of the patients, 94% of patients [corrected] maintained regular menstrual cycles 3 years after ovarian transposition (OT) [corrected] surgery (15/16). When measured (n = 5), serum FSH was normal at baseline and showed a transient elevation at 3 months following chemoradiation, with a return to normal levels at 6 months (means, 6.33 ± 2.94, 48.44 ± 18.63, and 12.52 ± 8.25 mIU/mL, respectively). Only 1 patient in this series attempted fertility treatment (in vitro fertilization) following OT, and she did not become pregnant. FACT-CX indicated that quality of life did not change significantly over the 6 months' duration following OT and chemoradiation therapy. CONCLUSION: OT preserves menstrual cycle regularity without negatively impacting patients' quality of life. The utility of OT as an effective fertility preservation option is hampered by the low utilization rate of in vitro fertilization and lack of ovarian reserve assessment following OT.

Salbutamol tolerance to bronchoprotection: course of onset.

BACKGROUND: Regular use of inhaled ß-agonist leads to tolerance to its bronchoprotective effect. This occurs within 12 hours with salmeterol and has been documented at 1 week for salbutamol. The course of onset after introduction of salbutamol has not been investigated. OBJECTIVE: To determine the course of onset of tolerance to the bronchoprotective effect of salbutamol against methacholine. METHODS: Thirteen individuals with mild asthma completed a randomized, double-blind, placebo-controlled, cross-over study. Each treatment period consisted of 7 twice-daily doses (2 puffs of 100 µg of salbutamol or placebo). Methacholine challenges were conducted 24 hours apart on 4 consecutive days, 10 minutes after the first, third, fifth, and seventh doses. The 2 treatment periods were separated by at least 14 days. RESULTS: Methacholine provocation concentration that caused a decrease in forced expiratory volume in 1 second of 20% (PC(20)) values during the 4 days of placebo treatment did not significantly differ (analysis of variance P = .79). A single dose of salbutamol shifted the methacholine PC(20) approximately 5-fold from a geometric mean of 2.1 mg/mL to a geometric mean of 10.7 mg/mL. Maximal bronchoprotection after the active treatment occurred on day 2 after the third dose, which was significantly higher than on day 1 after the first dose (P = .04). After the fifth dose the methacholine PC(20) was trending downward, and on day 4 the bronchoprotective effect of salbutamol had significantly decreased from its peak protection (P = .001). CONCLUSION: The detrimental effects on bronchoprotection after regular use of salbutamol manifest after 5 doses and are significantly reduced from peak protection after 7 doses. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01338311.

Effect of variable transmission rate on the dynamics of HIV in sub-Saharan Africa.

BACKGROUND: The cause of the high HIV prevalence in sub-Saharan Africa is incompletely understood, with heterosexual penile-vaginal transmission proposed as the main mechanism. Heterosexual HIV transmission has been estimated to have a very low probability; but effects of cofactors that vary in space and time may substantially alter this pattern. METHODS: To test the effect of individual variation in the HIV infectiousness generated by co-infection, we developed and analyzed a mathematical sexual network model that simulates the behavioral components of a population from Malawi, as well as the dynamics of HIV and the co-infection effect caused by other infectious diseases, including herpes simplex virus type-2, gonorrhea, syphilis and malaria. RESULTS: The analysis shows that without the amplification effect caused by co-infection, no epidemic is generated, and HIV prevalence decreases to extinction. But the model indicates that an epidemic can be generated by the amplification effect on HIV transmission caused by co-infection. CONCLUSION: The simulated sexual network demonstrated that a single value for HIV infectivity fails to describe the dynamics of the epidemic. Regardless of the low probability of heterosexual transmission per sexual contact, the inclusion of individual variation generated by transient but repeated increases in HIV viral load associated with co-infections may provide a biological basis for the accelerated spread of HIV in sub-Saharan Africa. Moreover, our work raises the possibility that the natural history of HIV in sub-Saharan Africa cannot be fully understood if individual variation in infectiousness is neglected.

Enacting Entangled Practice: Interagency Collaboration in Domestic and Family Violence Work.

Interagency collaboration in domestic and family violence (DFV) work is generally assumed to be good practice. This article questions this assumption, suggesting caution in adopting an uncritical pro-collaboration stance, arguing the need to trace the effects of working together on victims/survivors. Employing an innovative sociomaterial approach, this ethnographic study of interagency practice unravels its complexity, showing that not all ways of working together serve the interests of victims/survivors equally. Conceptualizing interagency DFV work as two distinctive, yet entangled, modes of collaboration, the findings have important implications for interagency DFV practice and policy.

Use of a bupivacaine continuous wound infusion system in gynecologic oncology: a randomized trial.

OBJECTIVE: The aim of the current study was to evaluate the safety and efficacy of a widely available bupivacaine continuous wound infusion system in gynecologic oncology patients undergoing laparotomy. METHODS: A prospective, randomized, double-blind, placebo-controlled trial was performed. After closure of the fascia, flexible soaker catheters were placed in the deep subcutaneous space. The infusion pump was filled with 290 mL of either 0.5% bupivacaine or normal saline, to infuse over 72 hours. Daily assessments of pain scores utilized the Wisconsin Brief Pain Inventory. All patients received intravenous narcotics via patient-controlled devices. RESULTS: Eighty surgeries were evaluated in a total of 79 women (40 per arm). Mean age was 56 years, with 79% having invasive gynecologic pathology. The two groups were not significantly different in terms of type of surgery, length of incision, estimated blood loss, operative time, or medical history. Postoperative outcomes, including wound toxicity, time to flatus, and hospital stay, did not differ. Study patients averaged 75 mg intravenously and 107 mg total narcotic use (morphine equivalent), whereas controls averaged 60 mg intravenously and 86 mg total (P = .40 intravenously; P = .25 total). Acetaminophen and intravenous ketorolac consumption were equal between groups. The Brief Pain Inventory score for "current pain" was 2.84 for bupivacaine patients and 3.14 for controls (P = .46; least = 0, most = 10). There was no individual postoperative day when "current pain" BPI scores differed. "Worst pain" and "least pain" Brief Pain Inventory scores showed similar results. CONCLUSION: The results suggest that although the continuous infusion system seems safe, it is not efficacious in this patient population.

Low levels of fractional exhaled nitric oxide and deep inhalation bronchoprotection are associated with mannitol non-responsiveness in asthma.

BACKGROUND: Airway hyperresponsiveness (AHR) to indirect agents like mannitol is thought to be dependent on concurrent airway inflammation as these stimuli exert their effects via the release of bronchoconstricting mediators from inflammatory cells. Airway inflammation correlates negatively with deep inhalation bronchoprotection against direct stimuli like methacholine. We hypothesised that deep inhalation bronchoprotection to methacholine would be absent and airway inflammation would be present in individuals with AHR to inhaled mannitol. METHODS: Twenty asthmatic, otherwise healthy individuals, either gender, aged 18-65 years, with a Visit 1 (screening) methacholine two-minute tidal breathing PC20 of 16 mg/mL or less completed the study. Visits 2 and 3 consisted of either mannitol or deep inhalation methacholine challenge in random order, at least 24 h apart. All visits were completed within a period of two weeks. RESULTS: Eleven of the twenty participants had AHR to mannitol (PD15 ≤ 635 mg, the "responders") and nine did not (the "non-responders"). Responders did not bronchoprotect to methacholine via deep inhalation (doubling dose shift = 0.7; p = 0.13) and had high levels of exhaled nitric oxide (geometric mean 49 ppb; range 16-109 ppb). Conversely, significant deep inhalation bronchoprotection to methacholine occurred in the non-responder group (doubling dose shift = 1.6; p = 0.013). This group also had significantly lower levels of exhaled nitric oxide (geometric mean 23 ppb (range 16-45 ppb; p = 0.015). CONCLUSIONS: Deep inhalation bronchoprotection to methacholine and low levels of exhaled nitric oxide coincide with mannitol non-responsiveness in an asthmatic population. Clinical Trials Registration #NCT01642745 (clinicaltrials.gov).

Regulation of pituitary tumor transforming gene (PTTG) expression and phosphorylation in thyroid cells.

Human pituitary tumor transforming gene (hPTTG) is a multifunctional proto-oncogene implicated in the initiation and progression of several tumors. Phosphorylation of hPTTG is mediated by cyclin-dependent kinase 2 (CDC2), whereas cellular expression is regulated by specificity protein 1 (SP1). The mechanisms underlying hPTTG propagation of aberrant thyroid cell growth have not been fully defined. We set out to investigate the interplay between hPTTG and growth factors, as well as the effects of phosphorylation and SP1 regulation on hPTTG expression and function. In our study, epidermal growth factor (EGF), TGFα, and IGF-1 induced hPTTG expression and phosphorylation in thyroid cells, which was associated with activation of MAPK and phosphoinositide 3-kinase. Growth factors induced hPTTG independently of CDC2 and SP1 in thyroid carcinoma cells. Strikingly, CDC2 depletion in TPC-1 cells resulted in enhanced expression and phosphorylation of hPTTG and reduced cellular proliferation. In reciprocal experiments, hPTTG overexpression induced EGF, IGF-1, and TGFα mRNAs in primary human thyrocytes. Treatment of primary human thyrocytes with conditioned media derived from hPTTG-transfected cells resulted in autocrine upregulation of hPTTG protein, which was ameliorated by growth factor depletion or growth factor receptor tyrosine kinase inhibitors. A transgenic murine model of thyroid targeted hPTTG overexpression (hPTTG-Tg) (FVB/N strain, both sexes) demonstrated smaller thyroids with reduced cellular proliferation and enhanced secretion of Egf. In contrast, Pttg(-/-) knockout mice (c57BL6 strain, both sexes) showed reduced thyroidal Egf mRNA expression. These results define hPTTG as having a central role in thyroid autocrine signaling mechanisms via growth factors, with profound implications for promotion of transformed cell growth.

Laparoscopic sentinel lymph node mapping for cervix cancer--a detailed evaluation and time analysis.

OBJECTIVE: To provide data from a US center on laparoscopic (LSC) approach to sentinel lymph node (SLN) detection in cervix cancer with detailed time analysis. METHODS: This prospective trial enrolled patients with stage IA2-IIA cervix cancer undergoing primary radical surgery. Tc-99 radiocolloid was injected the morning of surgery, followed by hybrid SPECT/CT lymphoscintigraphy. Blue dye injection occurred just prior to incision. After bilateral LSC SLN detection, all patients received complete LSC pelvic lymphadenectomy. Institutional SLN protocol was followed for frozen section, hematoxylin and eosin, and cytokeratin staining. RESULTS: Between December 2003 and February 2006, 20 enrolled patients received 9 LSC-assisted radical vaginal hysterectomies, 7 radical abdominal hysterectomies, 2 LSC-assisted radical vaginal trachelectomies, and 2 LSC lymphadenectomies alone (secondary to positive lymph nodes). Mean tumor size was 2.5 cm. Nineteen percent of the 64 SLNs were found in unusual sites, including common iliac (11%), presacral (5%) and para-aortic (3%). The negative predictive value was 100%. The combined technique detected SLNs bilaterally in all patients. If blue dye alone was used, this rate would have dropped to 67.5% and was negatively correlated with elapsed surgical time (-0.7; p=0.002). The ability to visualize blue SLNs remained steady for 30 min and was completely gone by 50 min. CONCLUSIONS: Laparoscopic SLN mapping can be newly introduced into gynecologic oncology centers with high detection rates and negative predictive values. The visualization of blue dye in SLNs is transient, and this negative time correlation may explain the previously reported inferior detection rates with this technique. CLINICAL TRIAL REGISTRATION.: ClinicalTrials.gov, http://www.clinicaltrials.gov, NCT 00205010.

Weekly docetaxel and carboplatin for recurrent ovarian and peritoneal cancer: a phase II trial.

OBJECTIVE: Results of the ICON4/AGO-OVAR-2.2 trial suggest that a platinum/taxane combination provides a survival benefit in relapsed, platinum-sensitive ovarian cancer compared to platinum alone. The optimal specific combination has yet to be determined. The current study evaluates weekly docetaxel and carboplatin in this setting. METHODS: Using a prospective phase II design, patients received weekly docetaxel (35 mg/m2) and carboplatin (AUC=2) administered days 1, 8, and 15 of a 28-day cycle. Initial treatment with a platinum-based regimen was required, with a treatment-free interval of at least 3 months. Patients could have received one prior regimen for recurrence. Biologically evaluable disease (CA-125) could be followed only if measurable disease was not present. Quality of life analysis utilized the FACT-O and FACT/GOG-Ntx scales. RESULTS: Thirty-six patients enrolled in the trial over 29 months. The majority had ovarian cancer (89%) and stage III/IV (97%) disease, with a median initial disease-free interval of 12 months. Most subjects were treated for first recurrence (81%) and had measurable disease (58%). The overall response rate was 67% (PR=52%, CR=15%), with 22% stable disease. Grade 3/4 neutropenia was common (48%) while serious anemia and thrombocytopenia were not. Neuropathy was generally mild and manageable. Carboplatin hypersensitivity led to 11 subjects coming off trial (31%). Diphenhydramine premedication produced a nonsignificant decrease in reaction rate. There was no detectable difference in quality of life due to therapy. CONCLUSION: The weekly regimen of carboplatin and docetaxel has a good response rate with an acceptable toxicity profile.