The Impact Pathway Approach (IPA) is an innovative methodology to establish links between emissions, related impacts and monetary estimates. Only few attempts have so far been presented regarding emissions of metals; in this study the external costs of airborne lead (Pb) emissions are assessed using the IPA. Exposure to Pb is known to provoke impacts especially on children's cognition. As cognitive abilities  are known to have implications for lifetime income, a pathway can be established leading from figures for Pb emissions to the implied loss in earnings, and on this basis damage costs per unit of Pb emission can be assessed.Different types of models are here linked. It is relatively straightforward to establish the relationship between Pb emissions and consequent increase in air-Pb concentration, by means of a Gaussian plume dispersion model (OML). The exposed population can then be modelled by linking the OML-output to population data nested in geo-referenced grid cells. Less straightforward is to establish the relationship between exposure to air-Pb concentrations and the resulting blood-Pb concentration. Here an Age-Dependent Biokinetic Model (ADBM) for Pb is applied. On basis of previous research which established links between increases in blood-Pb concentrations during childhood and resulting IQ-loss we arrive at our results.External costs of Pb airborne emissions, even at low doses, in our site are in the range of 41-83 €/kg emitted Pb, depending on the considered meteorological year. This estimate applies only to the initial effects of air-Pb, as our study does not address the effects due to the Pb environmental-accumulation and to the subsequent Pb re-exposure. These are likely to be between one and two orders of magnitude higher.Biokinetic modelling is a novel tool not previously included when applying the IPA to explore impacts of Pb emissions and related external costs; it allows for more fine-tuned, age-dependent figures for the external costs from low-dose exposure. Valuation of additional health effects and impacts e.g. due to exposure via ingestion appear to be feasible when extending the insights from the present pilot study. While environmental scientists tend to use physical units in order to measure and describe risks and impacts, the policy-making process is faced with trade-offs, that are often economic in nature. Attempts to express the environmental impacts of emissions in monetary terms have been presented in recent years -3. Measux and SO2, e.g. from energy sources such as power plants. More recently modern sources of emissions such as WtE plants, which emit certain persistent micro-pollutants , have been explored, but authoritative figures for the external costs are still lacking. The micro-pollutants represent a matter of concern due to their high toxicity and persistence in environmental media. WtE plants provide energy and heat from the combustion of municipal waste: they are often located in urban areas and the concentration of micro pollutants in their stack emissions depends on the municipal waste composition, which may be varying, and is therefore sometimes unpredictable ['Monetization' of the impacts of emissions is not always feasible, and is associated with a great deal of uncertainty. The Impact Pathway Approach (IPA) for assessment of the 'external costs' of air emissions, developed under the ExternE project series, has however gained recognition for reducing the biases associated with more pure, preference based willingness-to-pay estimates . 'Externdictable . For sucThis study explores the possible external costs associated with atmospheric emissions of lead (Pb) from a WtE plant located in a suburb of Copenhagen, Denmark. Pb is a substance for which the negative impacts on human health have been established in numerous studies reported in the scientific literature -9, and fWe take our point of departure in a site-specific study of Pb-emissions from a large WtE plant, but we argue that the findings have general validity beyond the specific site, provided that the specific circumstances of population density and the relative monetary values in Denmark are adjusted to other sites.The IPA structure consists of four consecutive steps : 1) Emisair. The emission of Pb from the plant raises the existing level of Pbair. The concentration contribution from the source to the background level is defined as a 'delta concentration': Δ-Pbair. It is described as a "marginal" contribution, because the source is small compared to the sum of all the other existing sources contributing both to Pbair and to Pb exposure. The Danish Gaussian plume dispersion model (OML) [air values . The plant considered in this study emitted 969 kg Pb in year 2000 in stack gases . The Pb el (OML) ,15 has b32 coordinates. A calculated Δ-Pbair value is assigned by the model to each grid cell in the model domain. Mean values of Δ-Pbair are 1 × 10-5 μg/m3 and the range comprises values of between 1 × 10-4 and 1 × 10-6 μg/m3. Values are calculated for three different years: 2000, 2001 and 2002 based on the same emissions but with different meteorological data for the three years. The concentration contribution from the point source (Δ-Pbair) is therefore, on average, two orders of magnitude lower than the background concentration of Pbair, as reported in Denmark's Air Quality Monitoring Programme [10 (atmospheric particles with aerodynamic diameter less than 10 μm) was measured for traffic and urban background conditions. Values of Pbair = 0.0079 (μg/m3) for traffic and Pbair = 0.0039 μg/m3 for urban background conditions respectively have been measured in the city of Copenhagen [The domain of the model covers 50 × 50 km around the point source giving a total number of 1 × 1 km grid cells of approx. 2,500, all geo-referenced in UTMrogramme  where thpenhagen .air data. The population data for the age distribution of the inhabitants in each grid cell have been retrieved from the Danish CPR-registry . The population under exposure can be derived by crossing the two distributions . In this way, the grid cells where no additional Pbair occurs or no population is reported will not contribute to the calculation of external costs.Population data for Denmark have been distributed on the same grid cells as applied in the calculation of the Δ-PbThe damage window for the impact of Pb explored in this study is cognitive impairment in children resulting from Pb exposure. Neurodevelopment is critical during childhood, especially the first 2-3 years of life ,16,17. Fblood). Pb is known to provoke cognitive impairment in children; such impact is measured in terms of Intelligence Quotient (IQ) loss in children and related to increases in the Pbblood [air and the corresponding increase in Pb impacts.The Pb concentrated in the air is inhaled and then absorbed in the body, and increases the blood Pb concentration  can then be calculated as the product of two functions (1):The CRF linking Pbblood - Pbair (μg/dl Pbblood/μg/m3 Pbair), the relationship between an increase in Pbair and the corresponding increase in Pbblood.1) Pbloss - Pbblood (IQ lost point/μg/dl Pbblood), the relationship between an increase in Pbblood, and a loss of IQ points.2) IQValues and references for the two functions 1) and 2) are presented in detail in the following sections (cf. sect. 2.2.2. and 2.2.3.).blood - Pbair function by means of statistical techniques assessing the relationship between measured media concentration and bio-monitoring values [blood after defined periods of exposure and for different age categories or physiological characteristics. In this specific case-study we only include the contribution from inhalation to the resulting concentration in blood. The amount of Pb that is daily absorbed in the body (Pbabsorbed), expressed in μg/day, is the input to the ADBM model and has been estimated according to equation (2):Previous studies determined the Pbg values ,21. In tg values  for Pb fg values -24. The inhalation is the breathing rate in m3/day, and AFPb is the Absorption Factor for Pb via inhalation expressed as percent of Pb intake.Where IR3) inhaled daily by children and adults have been adopted as reported by US EPA [air (cfr. sect.2.1.). The relation between Pbair and Pb intake is then, for the range of concentrations here considered, assumed to be linear for marginal increases in Pbair.The breathing rates and Pb absorption rates are the key parameters determining the actual Pb absorption needed as input for the ADBM. Reference values for age-dependent breathing rates, representing the amount of air  of exposure, and the Pbair. The model is nonlinear for constant values of Pbair . When age/time exposure conditions are instead fixed, Pbblood is a linear function of Pbair.Pbair - Pbblood varies greatly, for children, according to the age of the subject. It is then necessary to use different functions according both to the age of the children population under exposure and to the duration of the exposure.The ADBM shows that the Pbair, is the result of an annual Pb emission and because our interest is in the annualized external costs. Three separate age classes have been considered: children aged between 0 and 1 year, between 1 and 2 years, and between 2 and 3 years respectively.Three different scenarios for exposure have then been explored with reference to children. Duration of exposure is set to one year in all the scenarios, because the increment of Pb in the air, Δ-Pbblood - Pbair functions determining the level of Pbblood in children exposed for one year to a constant value of Pbair have been calculated with the ADBM as 1.97 μg/dl/μg/m3 for children aged between 0 and 1 year, 2.85 μg/dl/μg/m3 for children aged between 1 and 2 years, and 3.27 μg/dl/μg/m3 for children aged between 2 and 3 years.The age-dependent PbDue to different indications in the literature ,16,17, tblood in children, even at very low Pbblood levels (Pbblood < 10 μg/dl) [blood and is assumed to be linear without threshold [blood are estimated to be between 2-4 μg/dl in Europe [loss - Pbblood function for marginal increases of Pbblood are then assumed. Grosse [loss - Pbblood function. The author also notices how the function shows a greater slope for lower values of Pbblood in children (Pbblood < 10 μg/dl) than for higher values (10 μg/dl <Pbblood < 20 μg/dl). Following the approach already proposed by Grosse [loss - Pbblood parameter: lower bound, upper bound and base case (Table loss- Pbblood slope proposed by Schwartz [Pb has a negative impact on children's neurodevelopment. Numerous studies conducted since the late 1980s to date have shown how cognitive impairment is inversely associated with increases in Pb0 μg/dl) -19,28-30hreshold ,31. Actun Europe . In this. Grosse  reports y Grosse , three lSchwartz  is derivA single pollutant can provoke multiple impacts both with regard to the environment and humans, and not all of them can be expressed in monetary terms. In the specific case of Pb, relevant impacts on human health are those related to anaemia in adults and loss of IQ in children . Monetarair value for each selected grid cell i  is multiplied by the CRF function that relates Pbblood to Pbair in children aged between 0 and 1 year, in order to obtain the marginal increase Δ-Pbblood (3).The procedure behind IPA can be described with a number of equations. First, the Δ-Pbblood is then multiplied by the value of the IQloss - Pbblood slope and the marginal loss Δ-IQloss is obtained (4).Δ-Pbloss is multiplied by the cost of one point of IQloss to obtain the marginal monetary loss Δ-€loss (5).Δ-IQair is now associated to each considered grid cell i. This value needs to be multiplied for the number ni of children aged between 0 and 1 years present in the cell. The total cost for each single cell is obtained. This needs to be summarized across all the considered cells. The total cost of the Pb emissions Total_ €loss is then obtained (6).A monetary value related to the increase in Pbloss by the total amount of Pb emitted, in kg (7).In order to obtain a cost per kg it is necessary to divide the Total_ €blood to Pbair in children aged between 1 and 2 years. The two values for external costs are then added in order to obtain the total external cost of the Pb emission from the source via the inhalation pathway.The external cost per kg of emitted Pb for the population aged between 0 and 1 year is thereby determined. With the same procedure, the external cost aggregated over the population aged between 1 and 2 years is calculated, using the CRF that relates Pbloss- Pbblood slope, discount rate of 3%, and damage window for Pb impacts of 3 years from birth (0<age<3). The value of 83 Euros is the cost of each extra kg of Pb emitted, when the population is exposed via the inhalation pathway.Table loss- Pbblood slope, discount rate, and Pb impacts damage window. This approach on the variability of parameters and results cannot be considered a complete sensitivity analysis, but represents an indication of the sensitivity and uncertainty involved.Values of external costs of Pb emissions, in Euro per kg Pb emitted, for exposure via inhalation and for the different meteorological conditions of the years 2000, 2001 and 2002, are reported in Table The values of 83, 59 and 41 Euro/kg Pb emitted for the meteorological years 2000, 2001, 2002 respectively  based o-) Emission and dispersion: the measurements determine only "Total Pb" in flue gases. Pb is adsorbed on PM and the dispersion model does not account for differences in the dispersion between various species of Pb.-) Exposure: while the breathing rate is independent from the Pb species, the absorption rate is based only on the bio-available fraction of Pb. Not all the inhaled Pb is assumed to be absorbed. In this way a pooled estimate of the relevant Pb species appears.-) Bio-Accumulation: the ADBM parameters have been calculated from bio-monitoring data and consider "Total Pb" in the different organs/tissues as a pooled figure for the relevant Pb species.blood function: is in the literature calculated from bio monitoring data reporting the total (pooled) Pb content in blood.-) IQ loss - Pbloss - Pbblood function, several studies [blood in children aged 6 months to 3 years). The meta-review of Schwartz [loss - Pbblood function affects the final results only to some extent; the selection of damage window contributes more strongly, evidently because definition of the number of children exposed is involved. By using the ADBM we can give a good definition of the Pbblood - Pbair function for different ages and times of chronic exposure, but then we need to select the exposed population carefully. The state of the art of current research does not give precise indications of the damage window for Pb-related neurodevelopmental impacts. The above cited studies regarding the loss of IQ related to Pb in blood have been performed measuring IQ value  at the minimum age of 6 months, and focusing mainly on the influence of covariates such as gender, sex, and level of Pbblood, without trying to identify a specific moment when impact is supposed to occur. In general, a valid assumption, considering that the process of neurodevelopment in children is higher in the first 2-3 years immediately after birth, is that the impacts of potential impairment due to Pb should be greater in these early years. The scenarios considering 0 < age <2 and 0 < age < 3 years as the damage window are included to demonstrate the relative significance of the age parameter choice. In comparison the different spatial dispersion of the contaminant, pending only on meteorology and resulting in a different amount of population under exposure, displays some significance too.The topic of IQ loss from Pb exposure in childhood remains of concern, perhaps in particular in developing countries. With regard to the IQ studies ,28,29,31 studies , with a Schwartz , howeverSchwartz . Schwartblood- Pbair function of 5 μg/dl/μg/m3 for incremental exposure to Pb in ambient air, citing a reference from a UK study. The number of children under exposure is assumed to be the 1.1% of the total population, regardless of any damage window for Pb impact. The monetization is determined using the relationship between loss in IQ and decrease in income. The values for external costs reported by Spadaro are two orders of magnitude higher than the values here reported for Denmark. They are reported at an EU level and they are not limited to inhalation but include also ingestion of Pb, because the measured Pbblood- Pbairfunction implicitly accounts for the ingestion pathway also. The difference between the inhalation and the ingestion pathway is significant. If inhalation is assumed to account only for 4% of the total intake of Pb belonging from the source, as has been suggested above, then it is reasonable to expect a value for the external costs regarding the total Pb intake  that is somewhere between one and two orders of magnitude higher than when referring to inhalation only.It is interesting to compare the results obtained in the present study with findings from earlier studies. As the main references for comparison we can take the work performed in the Espreme project  and the blood, and in particular to relate such increment to the specific considered source of emission. The ADBM has been here used in order to isolate and specify the relationship existing between Pbair and Pbblood and separate this from other intake pathways, so the model allows for a better quantification of this source and this exposure pathway contribution to the overall impact. However, the total impact is likely to be much greater. Under natural background exposure conditions the ingestion of Pb is, in terms of Pb intake (μg/day), around two orders of magnitude greater than the inhalation [soil - Pbblood by using the ADBM and applying the same approach that has been explained above for inhalation. Ideally this could allow for accounting of the incremental impact relating to the ingestion of Pb emitted from the source and consecutively deposited in the soil. The problem is that, at the moment, the uncertainties in the establishment of a multimedia link between source and impact via ingestion, considering the marginality of the source, appear to be quite high. We provide only a partial result, but the methodology based on ADBM piloted here would allow for exploring also the ingestion pathway and so arrive at more robust figures for the external costs of Pb exposure. A multimedia approach will be required as an extension of the modelling framework presented here.In our study we chose to focus on the inhalation pathway, because the link between air emission and inhalation is direct and no multimedia distribution is involved. It is also difficult to distinguish the contribution of different exposure pathways to the total Pb exposure and to the increase in Pbhalation . This doloss - Pbblood function, the damage window for Pb impacts, and the discount rate has been discussed in a critical perspective. This approach considers only the initial effects via inhalation, and consequently accounts for only a part of the total costs of Pb emissions. Even these conservative values suggest that policies to reduce Pb exposure deserve careful considerations in the policy making process. The importance of assessing the contribution of the ingestion pathway, even if under conditions of greater uncertainty, has been stressed and will be the topic of further research.External costs for marginal Pb emission originating from a waste-to-energy plant have been assessed according to the impact pathway approach and extended with a detailed atmospheric dispersion model and a biokinetic model. The assessment of exposure to Pb for the most sensitive segment of the population has been determined carefully using spatially defined data as well as reference values for Pb intake and Pb impacts. The influence of some key parameters such as the IQADBM: Age-Dependent Biokinetic Model; AF: Absorption Factor; CBA: Cost Benefit Analysis; CPR: Personal Registration Code; CRF: Concentration Response Function; ICRP: International Commission for Radiological Protection; IPA: Impact Pathway Approach; IQ: Intelligence Quotient; IR: Intake (Breathing) Rate; LCA: Life Cycle Assessment; NERI: National Environmental Research Institute; OML: Operational Meteorological air-quality Model; PM: Particulate Matter; US- EPA: United States Environmental Protection Agency; WtE: Waste-to-Energy plants.The authors declare that they have no competing interests.MP carried out the determination of population under exposure, age-dependent CRF using the ADBM model and calculation of external costs, and drafted the manuscript. MSA and MT participated in the design and coordination of the study and supervised the work, then contributed to the drafting and revising of the manuscript. LMF carried out the air dispersion modelling and provided the relative data. All authors read and approved the final manuscript.


Overexpression of neuronal calcium sensor 1 in cortical neurons can help restore axonal plasticity and regeneration following axonal injury in adult rats, and can also improve behavioral function. Following trauma of the adult brain or spinal cord the injured axons of central neurons fail to regenerate or if intact display only limited anatomical plasticity through sprouting. Adult cortical neurons forming the corticospinal tract (CST) normally have low levels of the neuronal calcium sensor-1 (NCS1) protein. In primary cultured adult cortical neurons, the lentivector-induced overexpression of NCS1 induces neurite sprouting associated with increased phospho-Akt levels. When the PI3K/Akt signalling pathway was pharmacologically inhibited the NCS1-induced neurite sprouting was abolished. The overexpression of NCS1 in uninjured corticospinal neurons exhibited axonal sprouting across the midline into the CST-denervated side of the spinal cord following unilateral pyramidotomy. Improved forelimb function was demonstrated behaviourally and electrophysiologically. In injured corticospinal neurons, overexpression of NCS1 induced axonal sprouting and regeneration and also neuroprotection. These findings demonstrate that increasing the levels of intracellular NCS1 in injured and uninjured central neurons enhances their intrinsic anatomical plasticity within the injured adult central nervous system. Following trauma to the central nervous system , neurons show very little capacity to re-grow their axons, which can lead to a permanent loss of function in those regions. In this study, we show that this failure of axon re-growth is associated with low intracellular levels of a small molecule called neuronal calcium sensor-1 (NCS1). We modified a non-replicating virus in two ways so as to increase the level of NCS1 in neurons while simultaneously labelling them with a green fluorescent protein, which allowed us to track neuronal growth. Using this virus to increase the level of NCS1 in a particular group of neurons that communicate between the brain and spinal cord, we showed that new axonal growth occurred in the spinal cord with or without injury to the neurons. Electrophysiological assessments demonstrated that these new processes formed functional connections in the spinal cord, and behavioural experiments revealed that this recovery also helped the animals move their limbs more effectively. Furthermore, an increase in NCS1 protected these neurons, such that fewer of them died after the injury. These findings demonstrate that increasing the intracellular levels of NCS1 in neurons can aid in the recovery from central nervous system injury, and can help improve behavioural function. Spinal cord injury is a significant clinical problem that produces life long disability, although in a minority of patients some degree of recovery can occur spontaneously without any therapeutic intervention There is a growing literature suggesting pharmacological interventions can enhance both axonal regeneration Recently, we have found that following injury, the lentiviral overexpression of retinoic acid receptor β2 (RARβ2) induces regeneration in sensory and central axons NCS1 is highly conserved across species and emerges as a key intracellular calcium signalling component in a number of regulatory pathways in neurons Here we show using lentiviral vectors that NCS1 overexpressed in primary cultured adult cortical neurons increases neurite sprouting. Following corticospinal tract (CST) denervation by unilateral pyramidotomy, axons of uninjured corticospinal neurons (CSN) overexpressing NCS1 sprout across the midline to form functional connections in the CST-denervated spinal cord. In axotomized CSN, overexpression of NCS1 induces axonal sprouting and regeneration and also neuroprotection. These studies reveal NCS1 as an important intracellular molecule for promoting anatomical plasticity following CNS injuries in the adult.To transduce adult cortical neurons with NCS1 at high efficiency and to enable visualisation with an extrinsic marker, we constructed a minimal human immunodeficiency virus (HIV) based lentiviral vector expressing NCS1 and GFP under cytomegalovirus (CMV) and spleen focus-forming virus (SFFV) promoters, respectively, termed HIV-GFP-NCS1 . A contrThe type of neurite that has undergone sprouting with NCS1 overexpression was further investigated using the specific dendritic immunomarker microtubule-associated protein 2 (MAP2). MAP2 has been previously shown strongly and weakly to immunolabel dendrites and axons, respectively These data indicate that primary cultured adult cortical neurons overexpressed with NCS1 have significantly more neurites and sprouts from dendrites and axons than the control neurons.NCS1 has been shown to induce neuronal survival via the activation of the PI3K/Akt pathway These data indicate the levels of phospho-Akt are elevated in neurons with overexpressed NCS1 and blockade of Akt production reduces neurite sprouting in these neurons.In Western blots, the levels of NCS1 were significantly higher in cortical neurons transduced with HIV-GFP-NCS1 compared with the controls both in vitro and in vivo . This inWith the demonstration that NCS1 overexpression induces neurite sprouting in primary cultured adult cortical neurons, it was next determined whether this also occurred in vivo. HIV-GFP-NCS1 or control HIV-GFP lentivector was injected into the forelimb and hindlimb regions of the left sensorimotor cortex of adult rats. High efficiency was achieved of both GFP and NCS1 expression in the CSN at 3 wk with HIV-GFP-NCS1 . SimilarAdult Wistar rats received unilateral intracortical injections of either HIV-GFP-NCS1 (NCS1-transduced) or the HIV-GFP (control) lentivector 3 wk before receiving on the contralateral side a unilateral pyramidal tract lesion which, in turn, causes CST-denervation of the contralateral side of the spinal cord . The lesDuring the 6-wk period after CST-injury, behavioural testing was used to assess sensorimotor function in control (HIV-GFP) and NCS1-transduced (HIV-GFP-NCS1) rats. The sensorimotor function was assessed using the staircase reaching apparatus to test for forelimb extension and grasping movements and the grid exploration test to test for sensory motor function of fore- and hindlimbs. The CST-denervated forelimb of the control and NCS1-transduced groups showed a significant impairment in eaten or food pellet displacement compared to the sham group 2 d post-CST-injury . HoweverIn the grid exploration test, a significant difference in limb misplacement was observed in the control group compared with the sham throughout the behavioural testing period . In contTo demonstrate further that overexpressed NCS1 had induced axon collateral sprouting from the intact CST, extending into the CST-denervated side of the spinal cord, a subsequent lesion of the intact pyramidal tract (termed second lesion) was made at medullary level . This seIt is well established in the anesthetised rats that stimulation of the forelimb area of the motor cortex results in contralateral forelimb movements To investigate whether a delayed NCS1 overexpression in CSN can also promote axon collateral sprouting to the same extent as priming the CSN for 3 wk, adult Wistar rats received unilateral intracortical injections of either HIV-GFP-NCS1 or the control HIV-GFP lentivector 2 d after receiving on the contralateral side a unilateral pyramidal tract lesion . At 4 wkThese data show that delaying the overexpression of NCS1 in CSN after injury can still induce intact CST axons to sprout into the CST-denervated side of the spinal cord.To investigate whether a delayed NCS1 overexpression of axotomized CSN can promote axonal sprouting and regeneration, adult Wistar rats received unilateral intracortical injections of either HIV-GFP-NCS1 or the control HIV-GFP lentivector 2 d after receiving on the ipsilateral side a unilateral pyramidal tract lesion at the medullary level, approximately 2 mm rostral to the decussation . At 4 wkThese data show that delaying the overexpression of NCS1 in axotomized CSN can induce axonal sprouting and axonal regeneration.It has been shown that a pyramidal tract lesion in adult hamster causes CSN to become atrophied after 2 wk post-injury These data suggest NCS1 overexpression in CSN exerts a neuroprotective effect on axotomized CSN.This present study demonstrates that the intracellular levels of NCS1 in adult cortical neurons can be significantly elevated by transduction with a lentiviral vector. In culture, neurons overexpressing NCS1 develop extensive neurite sprouting which by immunocytochemistry and Western blotting was shown to be via Akt phosphorylation. Similarly, analogous experiments conducted in vivo show that CSN overexpressing NCS1 with intact CST axons can undergo distal collateral sprouting and cross the midline into the CST-denervated side of the spinal cord. This anatomical plasticity is also functional as demonstrated by the behavioural and electrophysiological outcomes in NCS1-transduced adult rats. Furthermore, studies on the axotomized CSN show that NCS1 overexpression not only induces axonal sprouting and regeneration at the lesion site but also exerts a neuroprotective affect on injured CSN.To date, several therapies for spinal cord injury models have shown both significant axonal regeneration In addition to observing neurite sprouting in vitro, we demonstrated an NCS1 mediated axon collateral sprouting in vivo following unilateral pyramidotomy. The HIV-GFP-NCS1 lentivector injected into the cortex enabled GFP labelling of neurons overexpressing NCS1. The GFP labelling allowed visualisation and quantification of sprouting of the CST axons without the need of applying an independent tracer, and such labelling of axons and their collaterals can be detected as far distal as the lumbar region.We report for cervical region the number of GFP-positive collateral fibers from uninjured CST axons that have crossed the midline into the CST-denervated side of the spinal cord, as measured per 40 µm section, is 1–2 for the control and 4–5 for the NCS1-transduced rats. It is of interest to consider the possible total number of crossing fibers over the relevant cervical region. The length of the adult rat C5–C8 cervical cord containing the majority of forelimb motoneurons is approximately 11 mm We have previously induced axonal regeneration by lentivector overexpression of retinoic acid receptor β2 (RARβ2) The successful regenerative responses of CSN after pyramidotomy with delayed post-injury NCS1 overexpression suggests axonal sprouting and regeneration can occur without the need to prime with overexpression of NCS1. These data provide promising support for NCS1 overexpression as a possible therapeutic treatment for CNS injury in a clinical setting.This study also reveals a neuroprotective feature of NCS1 overexpression in reducing cell shrinkage due to retrograde effects of axotomy. Others have shown that neurotrophic factors can prevent atrophy or death of axotomized CSN Our study establishes that NCS1 is an important intracellular component in the regulation of axonal sprouting and regeneration, and neuroprotection in central neurons of an adult mammalian nervous system, as recently shown for the peripheral nervous system in chick embryo studies on dorsal root ganglion (DRG) neurons In summary, the limited ability of adult CST neurons to undergo functional sprouting may be due to low endogenous levels of NCS1. Thus NCS1 emerges as a potential intracellular target for therapeutic intervention following injury to the central nervous system.5′-ATGGGGAAATCCAACAGCAAG-3′; and the reverse primer, 5′-CTATACCAGCCCGTCGTAGAG-3′ then cloned into a pCR2.1-TOPO vector (Invitrogen). The ncs1 gene was inserted under the control of a CMV promoter in a human immunodeficiency virus type 1 (HIV1) vector containing a 5′ central polypurine tract (cPPT) element and a 3′ woodchuck post-transcriptional regulatory element (WPRE) enhancer. To allow for coexpression of NCS1 and the enhanced green fluorescent protein (GFP), the eGFP gene was inserted into the Cla1 site under the control of a SFFV promoter. Viral vector stocks, pseudotyped with the VSV-G envelope glycoprotein, were prepared by triple plasmid transient transfection of HEK293T cells as previously described 8 TU ml−1 and pRRL-CMV-eGFP (for simplicity termed HIV-GFP) was 4.7–4.8×108 TU ml−1 determined by transient transfection of the HEK 293T cell line and analysed by flow cytometry.The complete cDNA sequence of the rat NCS1 was generated by PCR from adult rat cortex using the following forward primer; Adult cortical neurons were cultured as previously described For phospho-Akt studies, the reversible PI3K/Akt inhibitor LY294002  or DMSO alone at a final concentration of 0.01% was added to the cultures at 1 DIV and 2 DIV. The neurons were harvested at 3 DIV.After 3 d in culture, neurons were fixed with 4% paraformaldehyde for 20 min and then permeablized with cold methanol for 3 min. The neurons were washed three times for 5 min with 0.01 M phosphate-buffered saline (PBS) before 2 h incubation with chicken anti-GFP , rabbit anti-NCS1 , rabbit anti-phospho-Akt , or rabbit anti-MAP2 . The cover slips were washed 3×5 min with PBS and then incubated with donkey anti-rabbit Alexa Fluor 546 and goat anti-chicken Alexa Fluor 488 secondary antibodies  for 45 min. After 3×5 min PBS washes, they were mounted with FluorSave reagent containing 0.5 µl DAPI (10 µg/ml) to visualise cell nuclei. The staining with phalloidin-TRITC  was carried out 1 h before immunostaining with other antibodies was carried out.Image analysis and quantification was made with the observer blinded to the group assignment as previously described 3VO4, 5 mM EDTA, 1% Nonidet P-40 and 1× protease inhibitor cocktail; Roche). To obtain sufficient protein, the same 250 µl lysis buffer was used in three cultured wells and the lysates rotated for 2 h at 4°C. After centrifugation at 13,500 g for 15 min at 4°C, the supernatant was collected and total protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce).Western blots were carried out as previously described n = 4–5 per group) were carried out as described below. To determine the role of Akt activation, the PI3K/Akt inhibitor LY294002 (100 mM) or vehicle (DMSO) was injected intracerebroventricularly via an externalised catheter on every other day of the third post-injection week. At the end of the third week, rats were sacrificed and the injected region of the cortex was freshly and quickly removed and stored at −80°C until further processed. The protein obtained for Western blotting was extracted as described above.Intracortical injections of either HIV-GFP-NCS1 or HIV-GFP lentivector in adult male Wistar rats (β-mercaptoethanol) before re-blotting with rabbit anti-Akt . Western blotting was carried out with 3–5 independent samples.Fifteen micrograms of total protein were electrophoresed on 12% acrylamide gel before transfer onto Hybond P membranes (Amersham) and incubated overnight at 4°C with rabbit anti-phospho-Akt , rabbit anti-NCS1 , or mouse anti-β III tubulin . Visualisation was performed using secondary antibodies, donkey anti-rabbit IRDye-800CW, and goat anti-mouse IRdye-680CW (LI-COR Biosciences). Fluorescent blots were imaged on the Odyssey Infrared Imaging System (LI-COR Biosciences). To allow for visualisation of the total Akt on the same blot as phospho-Akt (both antibodies were raised in the same species), the blot was first stripped with buffer  were anaesthetized using a combination of ketamine and medetomidine, then fixed in a stereotaxic frame. The skull was exposed and injections were made at a depth of 2 mm dorsoventrally into the sensorimotor cortex region using the injection coordinates as determined from a microstimulation mapping study The surgery was performed aseptically in accordance with UK Home Office regulations as previously described n = 5–6 per group) received intracortical lentiviral injections 2 d after a unilateral pyramidal tract lesion as described above. To study the sprouting effect of delayed lentivector transduction on uninjured and injured CST axons, the lentiviral injections were administered into the sensorimotor cortex corresponding to the unlesioned and lesioned pyramidal tract, respectively. After 4 wk post-surgery, the rats were perfused transcardially with 4% paraformaldehyde and tissue collected for histology.In the delayed lentivector transduction studies, adult Wistar rats (n = 4–5 per group) received intracortical lentiviral injections 1 wk before receiving on the ipsilateral side a unilateral pyramidal tract lesion as described above. Using a microinfusion pump, Fast Blue tracer  was administered at a rate of 0.2 µl/min into the lesion site via a finely pulled glass micropipette and left in situ for a further 1 min. Unlesioned rats without intracortical lentiviral injection had Fast Blue tracer  injected into the pyramidal tract at the medullary region. Care was taken to minimise axonal damage by the injection process. After 2 wk post-injection of tracer, the rats were perfused transcardially with 4% paraformaldehyde and tissue collected for histology.In the neuroprotection study, adult Wistar rats . The next day, the sections were washed in 3×5 min PBS-T before incubation with goat anti-rabbit biotin  for 1 h 30 min. After 3×5 min PBS-T washes, sections were incubated in avidin-biotin-peroxidase complex  for 30 min at room temperature. Following 3×5 min washes in PBS-T, sections were incubated in tyramide  for 10 min. After a further 3×5 min wash in PBS-T, the sections were incubated with extra-avidin FITC  for 2 h before the sections were washed, mounted, and cover slipped. For quantitative analysis, five to six sections per animal (experimenter was blind to group assignment) stained for GFP were selected and captured as a complete spinal cord image at 10× magnification with five z-axis planes  using a Zeiss Axioplan 2 microscope and Axiovision V4.6 program. The number of GFP positive fibers in the CST was taken as the number of stained axons. The number of CST axon collaterals in the gray matter of the CST-innervated and -denervated side was assessed by counting the number of fibers crossing a virtual line at each 100 µm interval lateral from the midline.Axon collateral sprouting of the intact CST across the midline into the CST-denervated side of the cord was assessed by staining for the GFP immunopositive fibers. Forty µm transverse sections of the cervical (C5–C8 level) and lumbar (L3–L6 level) spinal cord were cut using a vibratome. Sections were collected in cold 0.01 M PBS with 0.1% sodium azide with the format of one section in each of 32 wells and this was repeated so each well had at least six sections containing the corresponding spinal cord levels. Sections chosen from randomly selected wells were rinsed in 3×5 min PBS-T (0.01 M PBS with 0.3% Triton X-100), then incubated with 0.3% HThe immunostaining for PKCγ in the spinal cord involved washing the sections in 3×5 min PBS-T before overnight incubation at room temperature with rabbit anti-PKCγ . The next day, sections were washed with 3×5 min PBS-T, then incubated with goat anti-rabbit Alexa Fluor 488  for 3 h. Following a 3×5 min PBS-T wash, sections were mounted and cover slipped.Axon collateral sprouting of the injured CST across the lesion site at the caudal medullary level was assessed by staining for the GFP positive fibers. Forty µm horizontal sections of the caudal medulla oblongata including the lesion site were cut using a vibratome. The immunostaining for GFP positive fibers in the brainstem was carried out with the tyramide signal amplification procedure as mentioned above. After the sections were immunostained for GFP, they were then incubated overnight with rabbit anti-GFAP antibody  to further highlight the lesion site with immunostained reactive astrocytes. The next day, sections were washed 3×5 min in PBS and then incubated in donkey anti-rabbit Alexa Fluor 568  for 3 h at room temperature. Following a further 3×5 min wash in PBS, sections were mounted and cover slipped.z-axis planes  using a Zeiss Axioplan 2 microscope and Axiovision V4.6 program. The number of CST axon collaterals was assessed by counting the number of fibers crossing a virtual line at each 200 µm interval rostral and caudal to the lesion site and within the ipsilateral side demarcated by the reactive astrocytes and midline, respectively.For quantitative analysis, five to six horizontal sections per animal at the level of the pyramidal tract lesion stained for GFP and GFAP were selected (experimenter was blind to group assignment) and captured as a complete horizontal section of the medulla oblongata at 10× magnification with four n = 4–7 per group).The expression of NCS1 and GFP in the cortex of whole rats was assessed by immunohistochemistry. Forty µm coronal sections of the cortex between bregma −2.5 mm and +3.0 mm were cut using a vibratome. Sections were collected and processed as described above. To visualise NCS1 using rabbit anti-NCS1  in histological sections, an antigen retrieval step was required using the antigen unmasking solution  according to manufacturer's instructions. Chicken anti-GFP  was used to detect GFP positive neurons. Standard immunohistochemical procedures were followed with donkey anti-rabbit Alexa Fluor 568 and goat anti-chicken Alexa Fluor 488 secondary antibodies. Dual or triple colour images of the layer V cortical region were captured at 20× magnification and analysed using the AxioVision V4.6 program. At least six transduced sections were analysed and quantified per animal (n = 4–5 per group). A total of over 2,400 neurons were analyzed.The analysis of atrophy in CSN was carried out as previously described Following unilateral pyramidotomy, functional recovery was assessed behaviourally using the staircase reaching and grid exploration tests at 2 d post-lesion and then weekly for 6 wk as described previously n = 4) received intracortical injections of HIV-GFP while NCS1-transduced rats (n = 4) received HIV-GFP-NCS1 followed by a unilateral pyramidotomy on the right side 3 wk later as described above. All electrophysiological measurements were performed at least 6 wk post-injury under urethane  anesthesia. Following tracheotomy, the rat was fixed into a frame by ear bars and spinal clamps such that the forelimbs were fully pendent. A pair of hooked stainless steel wires insulated to the tip was inserted into the tricep brachii of both forelimbs approximately 6 mm apart for EMG recording. The area of the sensorimotor cortex where lentiviral vectors were injected was exposed by craniotomy, covered by mineral oil, and stimulated through a flat ended silver wire electrode (0.5 mm diameter) ensheathed with plastic to its tip to minimize surface spread of the stimulating current. A 2 mm diameter anode was placed on the skull periosteum rostral to the stimulating electrode. The stimuli repeated at 1 Hz consisted of 1 to 4 pulses, 3 ms apart, and 0.1 ms duration from an isolated stimulator (Digitimer DS2A). The final stimulation site was selected after systematic mapping with varying stimulus parameters until a discrete contralateral (left) forelimb movement was observed with a clear EMG response and a threshold below 25 V for the least number of effective pulses. The EMG was amplified  and digitized using a CED 1401 interface with a sampling rate of 10 kHz. The area of the EMG response (Vs) was measured from 20 averaged responses using Spike 2 V5.0 software.Control rats  Primary cultured adult cortical neurons transduced with control HIV-GFP lentivector showing low levels of NCS1 immunostaining and limited neurite sprouts after 3 d in vitro. (G–L) Neurons transduced with HIV-GFP-NCS1 have higher levels of NCS1 immunostaining and a greater number of neurite sprouts . (M–N) Quantification data show NCS1-transduced neurons (red bar) have a larger number of neurites from cell body and sprouts from longest neurites compared to control HIV-GFP-transduced neurons (black bar). Data are expressed as mean ± SEM from 3 independent experiments. Scale bars:  100 µm;  20 µm.(3.10 MB TIF)Click here for additional data file.Figure S2Further examples of NCS1 overexpression promoting neurite sprouting via Akt activation in vitro. (A–D) Primary cultured adult cortical neurons transduced with HIV-GFP-NCS1 have high phospho-Akt levels and more extensive neurite sprouting when treated with vehicle (0.01% DMSO). (E–H) Control GFP-transduced neurons have low phospho-Akt levels and few neurite sprouts when treated with vehicle. Phospho-Akt, red; GFP, green; DAPI, blue. Inserts are higher magnification of corresponding panels. Scale bars: 50 µm.(1.90 MB TIF)Click here for additional data file.Figure S3Further examples of NCS1-induced neurite sprouting reduced by inhibiting Akt activation in vitro. (A–D) Primary cultured adult cortical neurons transduced with HIV-GFP-NCS1 have reduced phospho-Akt levels and fewer neurite sprouts in the presence of LY249002. (E–H) Control HIV-GFP-transduced neurons continue to express low phospho-Akt levels and fewer neurite sprouts in the presence of LY249002. Phospho-Akt, red; GFP, green; DAPI, blue. Inserts are higher magnification of corresponding panels. Scale bars: 50 µm.(1.63 MB TIF)Click here for additional data file.Figure S4Schematic diagrams illustrating the design and sequences of the in vivo experimental studies. The green circle and line indicate the lentiviral vector transduced neurons and GFP positive labelled axons and collaterals, respectively. The white circle and line indicate untransduced neurons and axons. The black circle and line indicate the CST, which when transected rostral to the pyramidal decussation leads to degeneration of the main dorsal CST on the opposite side and a corresponding CST-denervation of the spinal cord (black dashed line). (A) A 3 wk primed NCS1 overexpression in the transduced non-axotomized corticospinal neurons (CSN) is predicted to promote axon collateral sprouting on the same side of the intact CST and into the CST-denervated side of the spinal cord, but not in the control group (thin green lines). To establish if the functional recovery is dependent on collateral sprouting from the intact CST into the CST-denervated side, a second lesion of the intact pyramidal tract then causes CST-denervation of the postulated source of sprouting resulting in loss of the previously achieved recovery in behaviour and EMG activity (dashed green lines). (B) A 2 d delayed NCS1 overexpression in the transduced non-axotomized CSN after a pyramidotomy is predicted to promote axon collateral sprouting on the same side of the intact CST and into the CST-denervated side of the spinal cord, but not in the control group (thin green lines). (C) A 2 d delayed NCS1 overexpression in the transduced axotomized CSN after a pyramidotomy is predicted to promote axonal sprouting and regeneration into and around the lesion site at the medullary level, but not in the control group (thin green lines). (D) A 1 wk primed NCS1 overexpression in the transduced axotomized CSN after a pyramidotomy is predicted to prevent cell atrophy, but not in the control group.(2.00 MB TIF)Click here for additional data file.Figure S5Assessment of the pyramidotomy lesion. (A–B) Macroscopic images of a unilateral left pyramidal tract lesion rostral to the decussation in the lower medulla. The lesion (arrow) generates contralaterally a CST-denervated side of the spinal cord. (C–D) Photomicrographs of reactive astrocytes (GFAP immunopositive) demarcating the lesion site and the absence of PKCγ immunostaining caudal to the lesion (arrow). Arrowhead and arrow indicate the intact and lesioned pyramidal tract, respectively. (E–F) Macroscopic images from a rat containing a previous unilateral pyramidotomy (arrow) with a subsequent lesion of the intact pyramid (arrowhead) resulting in complete CST-denervation of both sides of the spinal cord. (G–H) Macroscopic images of a sham operated rat that received a unilateral pyramidal tract lesion in a subsequent operation (arrowhead). Higher magnification in panels B, F, and H are indicated by dashed boxes in panels A, E, and G, respectively. Scale bars:  5 mm,  1 mm.(8.28 MB TIF)Click here for additional data file.Figure S6Complete transverse section of cervical spinal cord from a pyramidotomized control GFP-transduced rat. (A–C) Rat transduced with the control HIV-GFP lentivector has GFP positive fibers present in the CST-innervated side of the spinal cord (CST (A), dorsal horn (B), ventral horn (C)). (D–F) In contrast, few GFP positive fibers were detected in the CST-denervated side of the spinal cord , intermediate laminae (E), ventral horn (F)). Scale bars: (A–F) 200 µm.(5.70 MB TIF)Click here for additional data file.Figure S7Complete transverse section of cervical spinal cord from a pyramidotomized NCS1-transduced rat. (A–C) Animal transduced with the HIV-GFP-NCS1 lentivector has GFP positive fibers present in the CST-innervated side of the spinal cord (CST (A), dorsal horn (B), and ventral horn (C)). (D–F) On the CST-denervated side of the spinal cord, extensive amounts of GFP positive fibers have crossed the midline with axon collaterals detected in the dorsal horn (D), intermediate laminae (E), and ventral horn (F). Scale bars: (A–F) 200 m.(5.13 MB TIF)Click here for additional data file.Figure S8Confirmation of pyramidotomy lesion using PKCγ immunostaining. (A–D) Photomicrographs show the presence of bilateral PKCγ immunostaining in the intact dorsal CST in sham rats (arrows). (E–L) Following a unilateral pyramidotomy, PKCγ immunostaining is only present in the intact dorsal CST (arrow) and absent in the lesioned dorsal CST (arrowhead) in rats injected with either the control (GFP) or NCS1 overexpressing lentivector. The numbers at bottom left indicate the rat identity from which the tissues were taken. Scale bars: 200 µm.(3.32 MB TIF)Click here for additional data file.Figure S9Unilateral pyramidotomy does not affect the CST-innervated limbs in behavioural tasks. (A–B) In the staircase reaching test, the CST-innervated forelimb for both transduced groups showed no significant reduction in numbers of food pellets displaced or eaten compared to sham group following unilateral pyramidotomy. (C–D) In the grid exploration test, the CST-innervated fore- and hindlimbs showed no significant difference in the number of misplacements compared to the sham operated group following unilateral pyramidotomy. (A–D) One week after a subsequent operation to lesion the intact pyramidal tract, the previously CST-innervated forelimb showed a reduced capability to displace or to eat food pellets in the staircase reaching test, and the number of misplacements in the grid exploration test was increased. Black line, CST-innervated limb of GFP rats; red line, CST-innervated limb of NCS1 rats; blue line, limb of sham rats.(0.37 MB TIF)Click here for additional data file.Figure S10The effects of increasing the number of stimulating pulses to the NCS1-transduced sensorimotor cortex on the EMG activities of the forelimbs. (A–B) With 1 pulse, neither movement nor little or no EMG activity was observed in either forelimbs (ripple is attributed to low threshold neck muscle activity). (C–D) With 2 pulses, forelimb movements occurred and EMG responses were observed in both forelimbs, with delayed latency on the CST-denervated side. (E–H) With 3 or 4 pulses, the EMG responses further increased in size in both forelimbs. (I–J) After transection of the intact dorsal CST at C4 spinal level, the EMG response from the forelimb on the now acutely CST-denervated side was all but abolished and it was lost altogether from the chronically CST-denervated forelimb.(0.59 MB TIF)Click here for additional data file.


In March 2006, Australia introduced graphic pictorial warnings on cigarette packets. For the first time, packs include the Quitline number.To measure the combined effect of graphic cigarette pack warnings and printing the Quitline number on packs on calls to the Australian Quitline service.Calls to the Australian Quitline were monitored over 4 years, 2 years before and after the new packets were introduced.There were twice as many calls to the Quitline in 2006 (the year of introduction), as there were in each of the preceding 2 years. The observed increase in calls exceeds that explained by the accompanying television advertising alone. While call volume tapered back in 2007, it remained at a level higher than before the introduction of new packets. No change was observed in the proportion of first time callers.Introducing graphic cigarette packet warnings and the Quitline number on cigarette packets boosts demand for Quitline services, with likely flow on effects to cessation. In March 2006, graphic health warnings were included on cigarette and other tobacco packs in Australia. In addition, and for the first time, the Australian Quitline number was printed on packets. Prior to 2006, Australia had text-based warnings. There was an infoline number printed in small text on the side of the pack. This number diverted to the Quitline.www.quitnow.info.au” is also included on the back of all packs. The Quitline number is also “stamped” on top of the graphic image on the backs of packs. Regulations prescribe the details of the size of the elements.Like the text-based warnings that preceded them, the graphic health warnings are mandated under Australia’s Trade Practices Act,A series of mass media campaign activities accompanied the introduction of the new cigarette packet warnings. The Australian Government screened an awareness raising campaign in February 2006.Australia is not the first country to introduce a Quitline or smokers’ helpline number on cigarette packets. In 2002, a smoking cessation message and quit line number were included on Dutch cigarette packets, along with prominent text warnings. This led to a 3.5-fold increase in calls to the Dutch Quitline.–It is well established that television advertising to promote quitting can increase calls to Quitlines8The Australian Quitline can be accessed from anywhere in Australia by dialling 131848 or 13 QUIT (137848) for the price of a local call. The Telstra Analyser , software of the telecommunications provider, provides data on volume of calls, call source (broken down by state and region), time and duration of calls.Individual states and territories have their own databases of caller details. These data were examined in one jurisdiction , where callers who spoke to a counsellor  were asked routinely whether they had called the Quitline before.Television anti-smoking advertising is quantified using target audience rating points (TARPs), provided by a media agency ACNielsen . TARPs are a standard measure of television advertising weight. TARPs are used to indicate the number of people within a certain demographic group that were exposed to an advertisement within a given period of time. For example, 100 TARPs for 1 week is equal to an average of 1 exposure per person in the target population within that week of the campaign. In the present study, the TARPs relate to the target audience of Australians aged ⩾18 years.Data analyses were conducted with SPSS V. 15 . Linear regression analyses were used to estimate the effect on calls to the Quitline of television advertising and the introduction of graphic pack warnings using data from January 2004 to December 2007 inclusive. In regression modelling, calls to the Quitline were the dependent variable, TARPs were a continuous independent variable and separate dummy variables were created for 2006 and 2007. Although data were not distributed normally, data were not transformed as this did nothing to strengthen the resulting model.Calls increased markedly when new cigarette packet warnings were first introduced. Call volume levelled off in the weeks following the initial launch but built up again in subsequent months when the accompanying quit campaign TVCs were launched.2 = 0.657). The model predicted a base number of calls ; a significant linear relationship between every 100 TARPs and calls to the Quitline ; and separate independent increases in calls were observed for years 2006 ; and 2007 , above what was explained by TARPs alone. Call volume was still elevated in 2007, compared to 2004 and 2005, although there was erosion in call volume from 2006.The linear regression model showed significant relationships between the independent variables and the dependent variable overall and had good overall explanatory value , the year in which new pack warnings including the Quitline number were introduced, compared to 2005 (78%).Australia is a “mature” tobacco control market where most forms of tobacco promotion are banned, increasing the significance of the packet as a medium for marketing.Many countries are moving to introduce graphic cigarette packet warnings; some with a Quitline or helpline number.However, the impact on calls to the Quitline of graphic (in contrast to text-only) warnings with accompanying Quitline number has not previously been quantified.This study shows that even in a “mature” tobacco control environment such as Australia, such an intervention has considerable positive impact on demand for a Quitline, with positive implications for quitting.Since the 1980s, most Australian state and territories have established strong anti-tobacco (quit) mass media campaigns, supported by the Australian Quitline. Because of the clear relationships between high-quality mass media campaigns, calls to the Quitline and quitting behaviour,However, the rise in calls to the Australian Quitline service observed in this study was substantial and sustained. The size and timing of the rise in calls, compared to the previous 2 years, indicates that this is highly likely to be due to the introduction of the new graphic cigarette packet warnings that included the Quitline number. The regression analysis also demonstrates that it is very unlikely that mass media alone explained the observed increase in calls because the introduction of the warnings had an independent effect. Further evidence that mass media quit campaigns were not the primary cause of increased calls is the fact that some of the increase in calls was observed prior to the launch of the quit campaigns. The Quitline number is a prominent but integrated component of the new-style warnings on Australian cigarette packets. There was no prominent display of the Quitline number on Australian cigarette packets prior to this, only the low-profile infoline number. Therefore it is not possible to separate the contributions of the components of the new warnings: namely the visual image, the large warning text, the detailed warning on the back of the packet or the Quitline number. Their impact has been measured as a whole.There was no change in the proportion of first time callers, compared to the previous year, indicating that the intervention had a positive effect upon new quitters and repeat callers.The observed increase in call volume did persist in the year following the introduction of the warnings (2007). Although there are 14 different warnings, with a scheduled rotation mechanism, it is likely that the reduction in call volume was due to a degree of “wear out”. This provides another example of a health promotion intervention having a positive effect more akin to a spring than a screw.In conclusion, the Australian Quitline experienced a doubling of calls upon introduction of graphic cigarette packet warnings that included a prominent Quitline number. Other countries with mature tobacco markets could expect a similar impact upon introduction of graphic warnings, especially if accompanied by reinforcing mass media activities. The flow-on effects in terms of quitting are likely to be substantial. Previous research has demonstrated that at 12 months, around 30% of callers to the Australian Quitline have succeeded in quitting smoking,


Alzheimer's disease (AD) is characterized by the deposition of beta-amyloid (Aβ) peptides in the brain extracellular matrix, resulting in pathological changes and neurobehavioral deficits. Previous work from this laboratory demonstrated that the choroid plexus (CP) possesses the capacity to remove Aβ from the cerebrospinal fluid (CSF), and exposure to lead (Pb) compromises this function. Since metalloendopeptidase insulin-degrading enzyme (IDE), has been implicated in the metabolism of Aβ, we sought to investigate whether accumulation of Aβ following Pb exposure was due to the effect of Pb on IDE.in vitro studies, choroidal epithelial Z310 cells were treated with Pb for 24 h in the presence or absence of a known IDE inhibitor, N-ethylmaleimide (NEM) to assess IDE enzymatic activity and subsequent metabolic clearance of Aβ. Additionally, the expression of IDE mRNA and protein were determined using real time PCR and western blots respectively.Rats were injected with a single dose of Pb acetate or an equivalent concentration of Na-acetate; CP tissues were processed to detect the location of IDE by immunohistochemistry. For Immunohistochemistry and confocal imaging revealed the presence of IDE towards the apical surface of the CP tissue with no visible alteration in either its intensity or location following Pb exposure. There was no significant difference in the expressions of either IDE mRNA or protein following Pb exposure compared to controls either in CP tissues or in Z310 cells. However, our findings revealed a significant decrease in the IDE activity following Pb exposure; this inhibition was similar to that seen in the cells treated with NEM alone. Interestingly, treatment with Pb or NEM alone significantly increased the levels of intracellular Aβ, and a greater accumulation of Aβ was seen when the cells were exposed to a combination of both.These data suggest that Pb exposure inhibits IDE activity but does not affect its expression in the CP. This, in turn, leads to a disrupted metabolism of Aβ resulting in its accumulation at the blood-CSF barrier. Studies in literature have shown that more than 90% of Alzheimer's disease (AD) cases are sporadic in nature , suggestOne of the target regions in the brain where Pb has shown to accumulate substantially is the choroid plexus (CP), a tissue lining the brain ventricles and separating the blood from the CSF. This finding is supported by evidence from an autopsy study which showed an age-related accumulation of Pb in the CP of the brains of 51 human subjects who had lived in New York City . This obRecent studies from this laboratory have demonstrated that Pb accumulation in the CP results in a marked increase of Aβ within the tissue . Since tIDE, a 110-KD zinc metallopeptidase, is known to participate extensively in the clearance of insulin, glucagon and Aβ -21. The in vivo affected the subcellular distribution of IDE, (3) investigate whether Pb exposure altered the IDE activity and/or the expression of mRNA or protein levels in the CP, and (4) determine whether altered IDE function led to the abnormal accumulation of Aβ in the BCB. The outcomes of this study will establish the role of IDE in the metabolic clearance of Aβ at the BCB and illustrate a mechanism by which Pb alters Aβ homeostasis in the CSF, potentially contributing to the etiology of AD.Considering the substantial accumulation of Pb and the abundant presence of IDE in the CP, there was sound reason to hypothesize that accumulation of Pb may alter the enzymatic activity or expression levels of IDE in the CP, leading to alterations in Aβ metabolism at the blood-CSF barrier (BCB). Thus, the purpose of this study was to (1) identify the subcellular location of IDE in the CP, (2) determine whether Pb exposure Chemicals and assay kits were purchased as follows: ELISA kit and ultra purified Aβ (1-40) ; mouse anti -IDE antibody ; IDE activity kit ; Alexa-labeled secondary anti- mouse antibody ; enhanced chemiluminescene reagent (ECL) and ECL films ; Dulbecco's modified essential medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and gentamycin ; PCR buffer, dNTP, Oligo dT and MuLV reverse transcriptase ; the ABsolute QPCR SYBR green Mix kit ; the primers for real time RT-PCR ; β-actin, 2-mercaptoethanol, phenylmethylsulfonyl fluoride (PMSF), polyacrylamide, tetramethyl-ethylenediamine (TEMED), n-ethylmaeimide (NEM) and all other chemicals . All reagents were of analytical grade, HPLC grade or the best available pharmaceutical grade.Male Sprague-Dawley rats were purchased from Harlan Laboratories  and were 8-9 weeks old (250-300 g) at the time they were used. The animals were housed in a temperature-controlled, 12 h:12 h light/dark room, and were allowed free access to tap water and food. Rats received an i.p. injection of 50 mg/kg Pb acetate  or an equivalent molar concentration of Na-acetate  as control. Twenty-four h post injection, the rats were anesthetized with an injection of ketamine/xylazine  and euthanized by exsanguination to remove excess blood. The lateral ventricle CP was then isolated for further experimentation. Animal protocols pertinent to this study were approved by the Purdue University Animal Care and Use Committee.2 at 37°C and were passaged twice a week. The cells were seeded at a density of 1 × 106 in a 100-mm culture dish. Twenty-four h after initial seeding, the cells were treated with 0 or 10 μM Pb for 24 or 48 h, and the following studies were performed.The methods and characteristics of immortalized rat choroidal epithelial Z310 cells have been described previously . Brieflyin vitro studies, Z310 cells were seeded on a 35-mm dish, and treated with 10 μM Pb for 24 or 48 h. At the end of Pb treatment, the culture medium was removed and replaced with 0.5-1 mL PBS. The tissues or cells were washed with PBS three times, fixed with 3% paraformaldehyde/0.25% glutaraldehyde in PBS for 10 min, and permeabilized with 0.5% Triton X-100 for 30 min at room temperature, followed by 3-5 washes of PBS. After blocking with 1% bovine serum albumin (BSA) in PBS for 30 min at room temperature, the tissues or cells were double-immunostained with mouse anti-IDE (1:500) in 1% BSA overnight at 4°C, washed the next day with PBS in 1% BSA, and then incubated with goat anti- mouse Alexa-488 conjugated secondary antibody (1:7500) in 1% BSA at 37°C for 60 min. After further washing in PBS with 1% BSA, the tissues or cells were observed using an inverted confocal fluorescent microscope. Negative controls were treated similarly except that they were not exposed to any of the primary antibodies.Immunohistochemistry was performed on both rat CP tissues and the Z310 cells following Pb exposure. For fresh tissue studies, the CPs were isolated from the rat following the exposure condition described above and transferred to a 35-mm glass bottomed dish containing PBS. For To acquire images, the dish containing the CP specimen was mounted on the stage of an Olympus, FV1000 inverted confocal laser-scanning microscope and viewed through a 40× water-immersion objective (numeric aperture = 1.2), with a 488-nm laser line for excitation (Ar-ion laser). Low laser intensity was used to avoid photo bleaching. The CP was examined under a reduced transmitted-light illumination. An area containing undamaged epithelium with underlying vasculature was selected. For each image acquired , four areas of the specimen were selected for image collection. Data reported, unless otherwise stated, are representative of 3-4 replicate experiments.6 in 100-mm plates for 24 h; they were then incubated with 0, 5, 10, 25 or 50 μM of NEM overnight for 16 h and the cell viability was determined using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Based on our results and previously published data [To screen for a suitable NEM concentration at which cell death was minimal, Z310 cells were seeded at a density of 1 × 10hed data , a conce6and treated with Pb for 24 or 48 h in the presence or absence of NEM. The four treatment conditions were as follows: Group 1 served as an untreated control (Pb-/NEM-), group 2 was treated with 10 μM Pb for 24 h in the absence of NEM (Pb+/NEM-), group 3 was treated with only 10 μM NEM for 16 h in the absence of Pb (Pb-/NEM+), and group 4 with 10 μM NEM for 16 h followed by 10 μM Pb for 24 h (Pb+/NEM+).To assess the IDE activity, the cells were seeded at a density of 1 × 10The principle of the assay is based on measuring the IDE activity using a fluorescence resonance energy transfer (FRET) substrate which is cleaved by IDE to release the fluorophore from the quenching molecule, resulting in an increase in fluorescence. The activity was determined as per manufacturer's protocol . Briefly, Z310 cells were lysed with RIPA buffer and centrifuged at 10,000 rpm for 10 min and the supernatant was used for analysis. Total protein (80 μg/well) was loaded into in a 96-well plate, which contained an affinity-purified polyclonal antibody that recognizes human, mouse, and rat insulysin; the antibody was immobilized on the plate in order to capture the IDE enzyme. Following 1 h incubation, the plate was washed with sample buffer, and an aliquot (100 μL) of substrate was added and incubated for 2-4 h at 37°C in the dark. The fluorescence was measured using an excitation wavelength of 320 nm and an emission wavelength of 405 nm. The results are reported as IDE fluorescence intensity in arbitrary units (a.u.)/mg of total proteins in the cells.4 cells/well and treated as the four groups described above. The cells were washed with PBS and incubated with 200 μL of 2 μM ultrapure Aβ1-40 solution in serum free medium for 1 h. The medium was then removed, cells washed 3 times with PBS, and fresh serum-free medium added for an additional hour to allow for metabolic clearance of Aβ by IDE. The cells were then washed with PBS, scraped and lysed using 60 μL of RIPA buffer. The intracellular Aβ was determined using a colorimetric ELISA kit (Invitrogen KHB3481). All data reported from the ELISA were normalized to the total amount of protein in the cells as determined by the Bradford assay.Cells were grown on 24 well plates at a density of ~4 × 10NM_013159) and rat glyceraldehydes-3-phosphate dehydrogenase (GAPDH) used as an internal control, had a forward primer 5'-CCT GGA GAA ACC TGC CAA GTA T-3' and a reverse primer 5'-AGC CCA GGA TGC CCT TTA GT-3' (Genbank Accession No. NM_017008).The transcription of the gene encoding IDE was quantified using RT-PCR as described in . BrieflyThe plexus tissues or Z310 cells were homogenized  on ice in a buffer containing 20 mM Tris (pH 7.5), 5 mM EGTA, 1% TritonX-100, 0.1% SDS, 50 μM phenylmethylsulphonylfluoride (PMSF), 15 mM 2-mercaptoethanol and a protease inhibitor cocktail containing 500 μM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), 150 nM aprotinin, 1 μM E-64, 0.5 mM EDTA, 1 μM leupeptin . Samples were sonicated using a Model 500 Sonic Dismembrator (Fisher Scientific) at duty cycle 20% and output 4-6 for 30 pulses. Following centrifugation at 10,000 g at 4°C for 10 min, aliquots of supernatants were assayed for protein concentrations by the Bradford method. A volume of protein extract (40 μg of protein) was mixed with an equal volume of 2× sample buffer , loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and then transferred to a PVDF membrane. The membrane was blocked with 5% dry milk in TBST  at room temperature for 1 h and immunoblotted with an antibody directly against IDE (1:250). The membrane was stained with a horse-radish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (1:5000) at room temperature for 1 h and developed using ECL reagent. The exposure time varied from 30 sec to several min depending on the signal strength. β-actin (42 kD) (1:2000) was used as a loading control; the corresponding secondary antibody (1:2000) for β-actin was HRP-conjugated goat anti-mouse IgG. All band intensities were quantified using Scion Image software .To determine the Pb concentration at which it altered Aβ metabolism in the CP in the absence of nonspecific cytotoxicity, three general cytotoxicity assays were conducted including the MTT cell viability assay, cell membrane permeability assessment (lactate dehydrogenase or LDH assay), and cellular oxidative stress estimation (superoxide dismutase or SOD assay). The MTT assay was performed by culturing Z310 cells at a density of 15,000 cells/well in a 96 well plate for 2-3 days until they reached 70% confluence. The medium was then replaced with fresh medium containing different concentrations of Pb as Pb-acetate . The cells were incubated for an additional 24 h, followed by adding an aliquot of MTT stock solution (2 mg/mL in PBS) to each well. The absorbance of the converted dye was measured at a wavelength of 570 nm. To determine the LDH activity, Z310 cells were treated as described above and LDH released into the culture medium was determined using the assay kit according to manufacturer's protocol. Finally, to determine oxidative stress, the cells were exposed to Pb (10 μM) for 24 h and SOD activity was determined according to the instructions in the assay kit.post hoc comparisons using the Dunnett's test or paired t-tests . All data are expressed as mean ± SD. Differences between two means were considered significant when the p value was equal or less than 0.05.Statistical analyses were carried out by a one-way ANOVA with in vivo exposure to Pb did not alter the subcellular localization of IDE, nor did it seem to affect the intensity of IDE staining  compared to untreated controls   and was comparable to the group treated with Pb in the absence of the inhibitor . When the cells were exposed to a combination of Pb and NEM , the IDE activity was reduced by a significant 29.3% (p < 0.001) relative to untreated controls , by 11.7% (p < 0.05) relative to the cells exposed to Pb alone , and by 11.8% (p < 0.05) relative to the NEM alone group . NEM did not cause any significant cell death at the concentrations used, as determined by the MTT assay. These results suggested that exposure to Pb (10 μM) results in a decrease in IDE activity; the effect of Pb on the enzymatic activity of IDE was exacerbated in the presence of IDE inhibitor, NEM.For the IDE activity study, NEM, an effective IDE inhibitor, was used as a positive control in the presence or absence of Pb exposure. Cells exposed to Pb for 24 h in the absence of NEM  showed a significant decrease of 17.6% in their IDE activity (M-) Fig. . Treatmep < 0.01, Fig. p < 0.05). Treatment with NEM alone  resulted in a 7 fold increase in intracellular Aβ concentrations compared to untreated controls (p < 0.01).To determine if the inhibition of IDE activity led to cellular accumulation of Aβ, Z310 cells were incubated with Aβ (2 μM) following treatment with Pb and/or NEM. Analysis using a one way ANOVA revealed an overall significant difference between the groups (p < 0.01). This finding supports a synergistic effect between Pb and NEM; both may possibly alter the IDE activity and subsequently decrease Aβ metabolism in the CP.When both Pb and NEM  were added to the culture system, the intracellular Aβ levels were increased by nearly 12 fold  IDE is located towards the apical membrane in CP tissues and the distribution does not appear to be altered by Pb exposure; (ii) Pb exposure results in a significant decrease in IDE activity; (iii) the decrease in IDE activity by Pb may lead to an accumulation of Aβ in the CP possibly due to a reduced metabolic clearance at the BCB; and (iv) Pb exposure does not alter the mRNA or protein expression of IDE.The observation that the subcellular location of IDE was concentrated towards the apical membrane has significant implications. Earlier studies in our laboratory have demonstrated that the direction of Aβ transport by the CP is predominantly from the CSF to the blood . The preRecent studies in our laboratory have shown that exposure to Pb results in the accumulation of intracellular Aβ ; the stuin vivo Pb exposure. As a future direction, it would be of interest to know how our in vitro studies correlate with in vivo IDE activity in the CP.Results from this study indicated that while Pb exposure caused a 1.5 fold increase in Aβ, the group treated with NEM alone caused a 7 fold increase in Aβ. This may be, in part, due to a non-IDE-related effect of NEM, since NEM action involves a general covalent binding to cysteine residues. One strategy for future studies would be to knock-down IDE using short interference RNA and to more specifically determine the contribution of IDE. Another approach would be to up-regulate IDE in these cells and analyze Aβ accumulation in the presence or absence of Pb exposure. We would also like to point out that due to a small tissue mass, we were unable to assess the IDE activity in the CP tissue following The current study raises several interesting questions. By what potential mechanism does Pb exert its effects on IDE? Extensive literature suggests that Pb is capable of binding to Zn finger proteins and subsequently altering their function, specifically at the sp1 binding domain -40. IDE Upstream of binding to the Zn finger, Pb might also influence the protein kinase C (PKC) pathway. Studies in literature suggest that IDE activity is decreased by an energy-dependent ATP mechanism . PKC is Another question is how much of a role does IDE play in Pb-mediated accumulation of Aα? We should point out that this study does not imply that IDE is the single most important factor in Aβ regulation at the BCB. Other mechanisms, particularly receptor-mediated Aβ uptake and clearance in the CP including the involvement of Aα transporters such as low density lipoprotein receptor protein 1 (LRP1), receptor for advanced glycosylation end products (RAGE) and p-glycoprotein must also be considered.Finally, what is the consequence of Aβ accumulation in the CP following Pb exposure? A build up of Aβ in the CP could either be a result of overproduction of Aβ in the CNS under the interference of Pb, or maybe due to a compromised function of the CP in clearing Aβ from the CSF. Data from the current study indicate that Pb may alter the Aβ breakdown mechanism at a critical site in the brain that is often overlooked. A disruption in Aβ clearance, in turn, might lead to elevated Aβ accumulation in the brain's internal milieu. Although this study provides some evidence for an IDE-mediated metabolic clearance, further studies are warranted to explore this mechanism in-depth.In summary, our results provide evidence that Pb exposure increases Aβ accumulation at the BCB at least in part, by altering the activity of IDE and thereby potentially decreasing the metabolic clearance of Aβ. In addition, we provide evidence that IDE in the CP is localized towards the apical membrane, which is consistent with the large capacity of the CP to take up Aβ from the CSF. Finally, our study suggests that Pb is a potential environmental trigger in the dysregulation of Aβ homeostasis, and may subsequently contribute to the etiology of AD.Aβ: beta-amyloid; AD: Alzheimer's disease; BCB: blood-CSF barrier; CP: choroid plexus; CSF: cerebrospinal fluid; ELISA: enzyme-linked immunosorbent assay; IDE: insulin degrading enzyme; LDH: lactate dehydrogenase; MRI: magnetic resonance imaging; MTT: methylthiazolyldiphenyl-tetrazolium bromide; NEM: n-ethylmaleimide; Pb: lead; PKC: protein kinase C; RT-PCR: reverse transcription polymerase chain reaction; SOD: superoxide dismutase.The authors declare that they have no competing interests.MB participated in designing the study, performing the experiments, analyzing and interpreting the data and drafting the manuscript. YZ participated in designing the study, assisted in carrying out the experiments and analyzing the data. WZ (senior author) participated in designing the study, interpreting the data and revising the manuscript. All authors have read and approved the final manuscript.


Malaria remains a major public health problem in Sub Saharan Africa, where widespread poverty also contribute to the burden of the disease. This study was designed to investigate the relationship between the prevalence of childhood fever and socioeconomic factors including poverty in Nigeria, and to examine these effects at the regional levels.Determinants of fever in the last two weeks among children under five years were examined from the 25004 children records extracted from the Nigeria Demographic and Health Survey 2008 data set. A two-level random effects logistic model was fitted. About 16% of children reported having fever in the two weeks preceding the survey. The prevalence of fever was highest among children from the poorest households (17%), compared to 15.8% among the middle households and lowest among the wealthiest (13%) (p<0.0001). Of the 3,110 respondents who had bed nets in their households, 506(16.3%) children had fever, while 2,604(83.7%) did not. (p=0.082). In a multilevel model adjusting for demographic variables, fever was associated with rural place of residence , sex of child: female  and all age categories (>6months), whereas the effect of wealth no longer reached statistical significance.While, overall bednet possession was low, less fever was reported in households that possessed bednets. Malaria control strategies and interventions should be designed that will target the poor and make an impact on poverty. The mechanism through which wealth may affect malaria occurrence needs further investigation.  Malaria is one of the most important challenges to public health with about 300 to 500 million cases reported annually. More than 1 million people die from the disease, most of them children under age 5 years. Over 90.0% of the cases and 75% of the deaths occur in sub-Saharan Africa (SSA). These childhood deaths, resulting mainly from cerebral malaria and anaemia, constitute somewhere between 20% and 25% of child mortality in Africa [African countries south of the Sahara bear the heaviest burden of malaria. These countries are among the poorest in the world and widespread poverty on the continent continues to play a role in the burden of the disease. Malaria cases and deaths have risen steadily in sub-Saharan Africa since the late 1970s, especially in Nigeria. The emergence of resistance to insecticides and chloroquine, the cheap anti-malarial treatment widely used for clinical management of uncomplicated malaria, has been held as a major factor in this trend, aided by a general weakening of health systems. This effect was exacerbated by economic stagnation and decline, which has implications for growth and welfare. For instance, malaria is responsible for about a 1.3 per cent reduction in the average annual rate of economic growth for those countries with the highest burden. In Nigeria, malaria is the major cause of morbidity and mortality, especially among children below age five .Given the above scenarios, the drive of the government to achieve the targets of the National Economic Empowerment and Development Strategy (NEEDS), Seven-Point Agenda and the MDGs is currently threatened by the seemingly intractable trend of the ancient scourge which has compounded national and household poverty through intensive loss of productive time to attack and death in extreme cases. This is compounded by the development of resistance to the traditional first-line drug (chloroquine), while the newly introduced therapy (ACT) would be largely beyond the reach of many due to poverty, if it was not heavily sponsored by international organizations. Given that malaria is endemic throughout Nigeria, and that over 50.0% of the country's population are living below poverty line, malaria incidence may increase significantly in Nigeria because many may not be able to afford the newly introduced drugs due to poverty.There are quite a good number of attempts to analyse the economic effects of malaria in the literature ,3,5-7. MThere are many pathways through which the relationship between malaria and poverty operates. At the household level, poor housing can expose people to contact with infective mosquitoes. Simple preventive measures such as insecticide-treated bed nets are unaffordable to the poorest if they must pay for them. Lack of resources prevents people from seeking timely health care. Often, peak malaria transmission coincides with harvesting time, which is a factor that affects risks at the village level. A few days of work lost to illness can mean food insecurity for the entire family.A meaningful relationship between malaria and poverty should consider the effect of both individual and cluster where individual belongs. This approach requires a multilevel analysis incorporating variables at different levels of aggregation. In this report, the relationship between poverty and malaria in Nigeria was analyzed using a multi-level logistic model. The independent variables considered were bed net ownership, type of place of residence, sex and age of child, and literacy level of mother.This report utilizes secondary data from the Nigerian Demographic and Health Survey . DemograThe study population in this analysis was the 25,004 under five children extracted from the records of 104,808 respondents who responded to the household questionnaire. The outcome measure in this analysis was occurrence of fever in children less than five years of age in the last two weeks preceding the survey. The explanatory variables were sex of child, age of child, whether or not household possess a bed net, whether child slept under bed net night before survey, type of bed net (treated or untreated), type of place of residence  and wealth index; a measure of poverty. This index serves as a proxy for measuring the long-term standard of living. It was based on data from the household's ownership of consumer goods; dwelling characteristics; type of drinking water source; toilet facilities and other characteristics that are related to a household socio-economic status such as car, radio, television, gas cooker, iron, boat/ship, telephone, source of water, electricity supplies, and type of wall, floor and roof, etc. To construct the index, each of these assets was assigned a weight (factor score) generated through principal component analysis, and the resulting asset scores were standardized in relation to a standard normal distribution with a mean of zero and standard deviation of one . Each hoFrequency tables were generated for relevant variables. Descriptive statistics such as means, medians and standard deviations were used to summarize continuous variables. Relationships between the outcome measure (occurrence of fever) and each explanatory variable was first explored using the chi squared test in bivariable analysis before a multilevel logistic regression was carried out. Explanatory variables found to be associated with the outcome were included in the logistic model based on the magnitude of the chi squared values. Odd ratios and 95% CIs were computed. A random effect logistic model was fitted that included fixed effects and group-level intercepts as random effects . This alMulti-level models allow the additional information provided by knowing which cluster a child comes from to be taken into account in modeling the relationship between independent and dependent variables. This model was run using the GLLAMM routine in Stata. In using this approach, the correlation between children from the same cluster or region arises from their sharing specific but unobserved properties of the respective regions. A random effect logistic regression model for the data is given by1, b2, b3 and b4 represents fixed effects and Ui represents a random effect. Ui is a random variable with a mean of zero and constant variance. Ui, is the estimation of the variance across all of the regions involved in the study. If the variance is large then the outcome of interest is dependent on the region, if the variance is small then the variations in outcome of interest may be explained by the measured characteristics alone.Where bi, the responses from the same regions are mutually independent that is the correlation between children from the same regions is completely explained by them having been observed in the same regions. The variance τ2 measures the degree of heterogeneity in the probability of experiencing fever that cannot be explained by the classification into the 2 categories (fever: yes Vs no)Given UAn important measure that describes these dependences in the data is called the intra-class correlation coefficient (ICC); this statistic measures the extent to which individuals within the same group are more similar to each other than they are to individuals in different groups. The intraclass correlation (ICC), ρ was calculated using:τ = estimated variance and π = 3.142Where This is a stata procedure for fitting generalized linear models, a class of regression models for univariate responses with density from an exponential family . The logxi: gllamm fever i.place of residence i.sex of child i.childsage i.wealthnw, i(region ) family link(logit) adapt nip(5), eformThe study sample consisted of 25,004 children. In this sample, 3,965(15.9%) children had fever in the two weeks prior to the survey. The mean age of the children was 27 months (SD = 17.2 months).Table A total of 2,068(16.3%) male children had fever compared to 1,897(15.4%) females. (p = 0.034). More children had fever in the rural areas compared with the urban areas (16.8% and 13.3% respectively) (p < 0.0001). Children aged 6-11 months and 12-23 months reported more fevers (19.9% and 21% respectively) than other children (p < 0.0001). In addition, fever prevalence decreased significantly as wealth quintile increased. A total of 1,106(17.04%) children from the poorest  household had fever, 978(16.6%) from poorer households  had fever, while 460(13.1%) from the richest households had fever (p < 0.0001).After controlling for all the predictors simultaneously in a multi-level model, fewer predictors remained significant Table . Only agThe overall prevalence of fever found in this survey is quite high when compared with the prevalence of fever in other SSA countries. Studies have shown that the prevalence of fever is less than 4.2% in the developed countries . The regThe analyses also showed that fever is higher in rural than urban areas. This may be associated with the high level of poverty in the rural areas. Poor people live in dwellings prone to mosquito proliferation. It has been shown that the characteristics of wall construction are associated with malaria prevalence . In anotThe results of this analysis suggest that the possession of bed nets is not associated with fever. In addition, no association exists between "whether child slept under bed net and type of bed net child slept under". However, households that had bed nets reported less fever than those who did not though the difference did not achieve significance. This result is in line with other findings in other parts of Africa on the use of insecticide treated bed nets, and their effectiveness when studied on a population basis. A study conducted by Lengeler and Snow  showed tIn the bivariate analysis, fever was found to be highest among the poor, followed by middle group, followed by the wealthiest group. On the basis of country-level data it was estimated that 57.9% of all deaths from malaria in the world in 1990 occurred among the poorest 20% of the world's population . In TanzWhile, overall bed net possession was low, less fever was reported in households that possessed bed nets. Malaria control strategies and interventions should be designed that will target the poor and make an impact on poverty. The results suggest that regional factors in Nigeria may be more influential in the risk of developing childhood fever than risks related to household wealth status. The mechanism through which wealth may affect malaria occurrence needs further investigation.The authors declare that they have no competing interests.All the authors conceived the study. OO was the principal investigator for Nigeria. DP and DB were collaborators from John Hopkins School of Public Health and contributed to the revising of the manuscript. OY conducted the analysis, BA and OO, contributed to data analysis and interpretation. OY and BA prepared the initial draft. All authors contributed to the draft. All authors read and approved the final manuscript.