Exploration of immune competencies of viral-specific CD8+T cells in MS patients using tetramers and functional CTL assays

Introduction: Epstein-Barr Virus(EBV) has been repeatedly associatedwith multiple sclerosis (MS). Wehave previously shown that there is ahigh peripheral as well as intrathecalactivation of EBV-, but not cytomegalovirus(CMV)-specific CD8+ Tcells, early in the course of MS,strengthening the link between EBVand MS. However, the trigger of thisincreased EBV-specific CD8+ T cellresponse remains obscure. It could resultfrom a higher EBV viral load. Alternatively,it could be due to an intrinsicallydeficient EBV-specificCTL response, cytotoxic granulesmediated.Thus, we performed anin-depth study of the phenotype of exvivo EBV- and CMV-specific CD8+T cells in MS patients and control patients,assessing their cytotoxic activity.Methods:We analyzed the profileof cytotoxic granules in EBV- andCMV-specific CD8+ T cells in a cohortof 13 early MS patients, 20 lateMS, 30 other neurological diseases(OND) patients and 7 healthy controlsubjects. Ex vivo analysis of EBV- orCMV-specific CD8+ T cells was performedusing HLA class I/tetramercomplexes coupled to CCR7 andCD57 markers in conjunction withperforin, granzymes A, BandKstaining.In a sub-cohort of MS patientsand controls, cytotoxic activity ofEBV- and CMV-specific CD8+ Tcells was investigated using a functionalCFSE CTL assay. Results: UsingHLA Class I tetramers for EBVand CMV, we found that the frequencyof EBV- or CMV-specificCD8+ T cells were similar in all studysubjects. Most of EBV- and CMVspecificCD8+Tcells were highly differentiated(CCR7-) and a variousproportion expressed the exhaustionmarker CD57. MS and OND patientshad increased perforin expression inEBV-specific CD8+ T cells. Most importantly,we found that MS patientswith longer disease duration tended tohave lower CTL cytotoxicity as comparedto earlyMSpatients or controls.Conclusions: Effector EBV-specificCD8+ T cells are increased in earlyMS, however their cytotoxic granuleprofile does not seem to be fully alteredand the cytotoxic activity ofthese cells is normal. However, thecytotoxic activity of CTL decreasedin late MS patients suggesting an exhaustionof EBV-specific CD8+ Tcells possibly due to EBV reactivation.This work was supported by theSwiss National Foundation PP00B3-124893, the Swiss Society for MS,and the Biaggi Foundation to RADP.

Inter-specific and developmental differences on the array of antennal chemoreceptors in four species of triatominae (Hemiptera: Reduviidae)

The aim of the work was to investigate the pattern of chemoreceptor sensilla in adults and fifth stage nymphs of Rhodnius prolixus, R. neglectus, Triatoma infestans and T. sordida in order to study differences and similarities between genera and species. Three types of sensilla were analyzed by light microscopy: thin-walled trichoidea, thick-walled trichoidea and basiconica. The number of sensilla of each three types were counted. The length of the antennal segments were also used as a variable for the analysis. The statistical analysis showed that the number of these antennal chemoreceptors had significant differences between species and between adults and nymphs of each species. Discriminant analysis separates incompletely the fifth stage nymphs of the four species and showed similarity between them. Discriminant analysis performed with 12 variables of the antennae, allowed a complete separation of the adults of the four species.

Patterns of sex work contact among men in the general population of Switzerland, 1987-2000

OBJECTIVES: To analyse the prevalence of lifetime recourse to prostitution (LRP) among men in the general population of Switzerland from a trend and cohort perspective. METHODS: Using nine repeated representative cross-sectional surveys from 1987 to 2000, age-specific estimates of LRP were computed. Trends and period effect were analysed as the evolution of cross-sectional population estimates within age groups and overall. Cohort analysis relied on cohorts constructed from the 1989 survey and followed in subsequent waves. Age and cohort effects were modelled using logistic regression and non-parametric monotone regression. RESULTS: Whereas prevalence for the younger groups was found to be logically lower, there was no consistent increasing or decreasing trend over the years; there was no significant period effect. For the 17-30 year age group, the mean estimate over 1987-2000 was 11.5% (range 8.3 to 12.7%); for the 31-45 year group, the mean was 21.5% (range over 1989-2000 20.3 to 23.0%). Regarding cohort analysis, the prevalence of LRP was found to increase steeply in the youngest ages before reaching a plateau near the age of 40 years. At the age of 43 years, the prevalence was estimated to be 22.6% (95% CI 21.1% to 24.1%). CONCLUSIONS: The steep increase in the cohort-wise prevalence of LRP in younger ages calls for a concentration of prevention activities in young people. If the plateauing at approximately 40 years of age is not followed by a further increase later in life, which is not known, then consumers of paid sex would be repeat buyers only, a fact that should be taken into account by prevention.

Contextualizing the impact of work organization on employee satisfaction

The paper assesses the relationship between the use of alternative workplace practices (AWP) and job satisfaction. Using a unique employeremployee data set with rich information on both firm and employee characteristics we test whether there is a positive impact of AWPs on job satisfaction (motivation hypothesis) or it is negative (intensification hypothesis). We expand a growing empirical literature focusing on small and medium size firms from a southern European area. Our results show an overall positive effect, depending on the specific practice considered. We also obtain some sort of time-dependence with the effects turning from negative to positive once the practice has been implemented for some time. Keywords: Job satisfaction, work organization, unobserved heterogeneity.

Isotype specific immune responses in murine experimental toxocariasis

In this work, a murine experimental model of toxocariasis has been developed in BALB/c, C57BL/10 and C3H murine strains orally inoculated with 4,000 Toxocara canis embryonated eggs, in order to investigate the isotype-specific immune responses against excretory-secretory antigens from larvae. T. canis specific IgG+M, IgM, IgG, IgA, IgG1, IgG2a and IgG3 were tested by ELISA. The dynamics of the specific immunoglobulins (IgG+IgM) production showed a contrasting profile regarding the murine strain. Conversely to the results obtained with the IgM isotype, the IgG antibody class showed similar patterns to those obtained with IgG+IgM antibodies, only in the case of the BALB/c strain, being different and much higher than the obtained with IgG+IgM antibodies, when the C3H murine strain was used. The antibodies IgG+IgM tested in BALB/c and C57BL/10 were both of the IgM and IgG isotypes. Conversely, in the C3H strain only IgG specific antibody levels were detected. The IgG1 subclass responses showed a similar profile in the three murine strains studied, with high values in BALB/c, as in the case of the IgG responses.

Specific situations related to acute schistosomiasis in Pernambuco, Brazil

The present work reports on two epidemiological episodes resulting in acute schistosomiasis involving wealthy persons living in the State of Pernambuco, Brazil. The authors discuss the epidemiological, clinical and serologic characteristics of the acute infections and also the way in which the conditions for transmission occurred.

Youth Homelessness Strategy

This Strategy aims to build on this work and to ensure a more co-ordinated and planned approach to tackling youth homelessness. Particular emphasis is placed on prevention and on the importance of supporting schools, communities, the young people themselves and their families in this context. Where a young person becomes homeless the Strategy stresses the need for a prompt child focused service which will address the individual needs of the young person. Download the Report here

Gender and Health: Strategies for Implemention - Seminar Proceedings

The recently published National Women's Strategy (NWS) identified the need to incorporate a gender perspective into mainstream health policy, as well as implementing positive action measures to ensure that the health of women in this country is promoted and protected (Government of Ireland, 2007). The strategyalso refers to the importance of gender as a health determinant and the recognition by the Health Service Executive of the need to work in partnership with the Women's Health Council (WHC) to develop gender mainstreaming in the planning and delivery of its services (HSE, 2005). Download document here

Ballymena Youth Bank

This project will work with 14-25 year olds to increase their understanding of health issues relevant to their age group and increase their skills to encourage other young people to become actively involved in promoting health and raising health awareness through the YouthBank grant-making scheme. 10 sessions will be carried out.

Unintentional injuries among 16 to 20 year old students in Switzerland.

In our society, accidents constitute a major public health problem, especially among youth. The objective of this paper was to describe the incidence of nonfatal injuries that required medical care among 16 to 20 year-old in Switzerland, its distribution by type of injury and whether there were differences by gender or by academic track and whether these injuries had sequels (hospitalisation, physical and psychological sequels). Overall, 28.3% of the sample reported at least one accident needing medical care in the previous 12 months, with males having more accidents than females and apprentices more than students. By type of accident, sports were the most frequently reported, followed by traffic, leisure time and work accidents. Half of males and one-third of females reported more than one accident, and 16% and 8% of them, respectively, reported four or more. Both physical and psychological sequels were more frequent among females, while hospitalisation was more frequent among males. Accident prevalence rates remain high among adolescents. Safety counselling and environmental measures need to be implemented.

Factors associated with schistosomiasis mansoni in a population from the municipality of Jaboticatubas, State of Minas Gerais, Brazil

Jaboticatubas is a municipality in the metropolitan region of Belo Horizonte which has been a target of a wide media release as "the capital of schistosomiasis" since the 1960's. In order to give support to a work based on an integrated control, we sought to identify the disease determinants at the site. A transversal study was carried out aimed at identifying prevalence rates of the disease and factors associated with the infection in the district of São José de Almeida, and two close localities, Cipó Velho and São José da Serra, all of them located in the municipality of Jaboticatubas. A parasitological survey was performed, applying the Kato-Katz method with two slides per sample in 1186 schoolchildren which represents 77% of all registered pupils in four public schools in 2001. Among these schoolchildren a number of 101 (8.6%) prooved positive for Schistosoma mansoni eggs in their stool samples. A total of 64 families, whose schoolchildren had shown to be positive for schistosomiasis, also undertook examinations. As negative control, a random sample was collected from the 206 families, whose children had proven negative for schistosomiasis. The prevalence among 270 families (1304 people) was 12%. To assess those who continued to have contact with possibly contaminated water, 1061 (81.4%) people of the 270 families were interviewed. A multivariate analysis identified the following factors associated with the infection: time of residence in the area (short period), garbage disposal (use of deserted areas), gender (male), age (from 10 to 29 years), and water contact (daily and weekly). Further analysis of these factors revealed a close correlation between water contact and the disease, with a positive significant frequency concerning almost all those items. Depending on gender and age significant variations of water contact patterns associated with leisure and professional activities were found. A malacological survey on water collections in the area identified snails of the species Biomphalaria straminea and B. glabrata. The latter showed 17 (0.6%) specimens positive for S. mansoni. Qualitative studies have complemented such evidences, which allowed us to design a reference picture and specific indicators of the disease for the local population. Those data provided the essential information to continue the development of an already ongoing educative process, as well as projects on environmental improvements.

Assessing attachment cognitions and their associations with depression in youth with eating or drug misuse disorders.

The study investigates associations between attachment cognitions and depression symptoms in 71 15-25-year-olds, 26 of whom have eating disorders, and 20 of whom are drug misusers. Attachment cognitions were measured with the CaMir Q-sort, which provides indexes for secure, avoidant, and preoccupied attachment, as well as scores on 13 dimensions. The BDI-13 was used to measure depressive symptomatology. Consistent with the literature, BDI scores were associated with cognitions of preoccupied attachment. They were also related to cognitions of avoidant attachment, confirming Bowlby's theory on defensive exclusion. For participants with eating disorders, depressive symptomatology was related to preoccupation and parental interference, whereas for drug misusers, it was negatively related to security, preoccupation, parental support, and lack of parental concern. These findings help understand how attachment cognitions may participate in depressive symptomatology, namely in youth whose behavior problems may be associated with specific attachment experiences.

Public Policy and Ageing in Northern Ireland: Identifying Levers for Change

Public Policy and Ageing in Northern Ireland: Identifying Levers for Change Judith Cross, Policy Officer with the Centre for Ageing Research Development in Ireland (CARDI)��������Introduction Identifying a broad range of key public policy initiatives as they relate to age can facilitate discussion and create new knowledge within and across government to maximise the opportunities afforded by an ageing population. This article looks at how examining the current public policy frameworks in Northern Ireland can present opportunities for those working in this field for the benefit of older people. Good policy formulation needs to be evidence-based, flexible, innovative and look beyond institutional boundaries. Bringing together architects and occupational therapists, for example, has the potential to create better and more effective ways relevant to health, housing, social services and government departments. Traditional assumptions of social policy towards older people have tended to be medically focused with an emphasis on care and dependency. This in turn has consequences for the design and delivery of services for older people. It is important that these assumptions are challenged as changes in thinking and attitudes can lead to a redefinition of ageing, resulting in policies and practices that benefit older people now and in the future. Older people, their voices and experiences, need to be central to these developments. The Centre for Ageing Research and Development in Ireland The Centre for Ageing Research and Development in Ireland (CARDI) (1) is a not for profit organisation developed by leaders from the ageing field across Ireland (North and South) including age sector focused researchers and academics, statutory and voluntary, and is co-chaired by Professor Robert Stout and Professor Davis Coakley. CARDI has been established to provide a mechanism for greater collaboration among age researchers, for wider dissemination of ageing research information and to advance a research agenda relevant to the needs of older people in Ireland, North and South. Operating at a strategic level and in an advisory capacity, CARDI�۪s work focuses on promoting research co-operation across sectors and disciplines and concentrates on influencing the strategic direction of research into older people and ageing in Ireland. It has been strategically positioned around the following four areas: Identifying and establishing ageing research priorities relevant to policy and practice in Ireland, North and South;Promoting greater collaboration and co-operation on ageing research in order to build an ageing research community in Ireland, North and South;Stimulating research in priority areas that can inform policy and practice relating to ageing and older people in Ireland, North and South;Communicating strategic research issues on ageing to raise the profile of ageing research in Ireland, North and South, and its role in informing policy and practice. Context of Ageing in Ireland Ireland �۪s population is ageing. One million people aged 60 and over now live on the island of Ireland. By 2031, it is expected that Northern Ireland�۪s percentage of older people will increase to 28% and the Republic of Ireland�۪s to 23%. The largest increase will be in the older old; the number aged 80+ is expected to triple by the same date. However while life expectancy has increased, it is not clear that life without disability and ill health has increased to the same extent. A growing number of older people may face the combined effects of a decline in physical and mental function, isolation and poverty. Policymakers, service providers and older people alike recognise the need to create a high quality of life for our ageing population. This challenge can be meet by addressing the problems relating to healthy ageing, reducing inequalities in later life and creating services that are shaped by, and appropriate for, older people. Devolution and Structures of Government in Northern Ireland The Agreement (2) reached in the Multi-Party Negotiations in Belfast 1998 established the Northern Ireland Assembly which has full legislative authority for all transferred matters. The majority of social and economic public policy such as; agriculture, arts, education, health, environment and planning is determined by the Northern Ireland Assembly at Stormont. There are 11 Government Departments covering the main areas of responsibility with 108 elected Members of the Legislative Assembly (MLA�۪s). The powers of the Northern Ireland Assembly do not cover ��� reserved�۪ matters or ��� excepted�۪ matters . These are the responsibility of Westminster and include issues such as, tax, social security, policing, justice, defence, immigration and foreign affairs. Northern Ireland has 18 elected Members of Parliament (MP�۪s) to the House of Commons. Public Policy Context in Northern Ireland The economic, social and political consequence of an ageing population is a challenge for policy makers across government. Considering the complex and diverse causal factors that contribute to ageing in Northern Ireland, there are a number of areas of government policy at regional, national and international levels that are likely to impact in this area. International The Madrid International Plan of Action on Ageing (3) and the Research Agenda on Ageing for the 21st Century (4) provide important mechanisms for furthering research into ageing. The United Kingdom has signed up to these. The Madrid International Plan of Action on Ageing commits member states to a systematic review of the Plan of Action through Regional Implementation Strategies. The United Kingdom�۪s Regional Implementation Strategy covers Northern Ireland. National At National level, pension and social security are high on the agenda. The Pensions Act (5) became law in 2007 and links pensions increases with earnings as opposed to prices from 2012. Additional credits for people raising children and caring for older people to boost their pensions were introduced. Some protections are included for those who lost occupational pensions as a result of underfunded schemes being wound up before April 2005. In relation to State Pensions and benefits, this Act will bring changes to state pensions in future. The Act now places the Pension Credit element which is up-rated in line with or above earnings, on a permanent, statutory footing. Regional At regional level there are a number of age related public policy initiatives that have the potential to impact positively on the lives of older people in Northern Ireland. Some are specific to ageing such as the Ageing in an Inclusive Society (6) and others by their nature are cross-cutting such as Lifetime Opportunities: Governments Anti-Poverty Strategy for Northern Ireland (7). The main public policy framework in Northern Ireland is the Programme for Government: Building a Better Future, 2008-2011(PfG) (8) . The PfG, is the overarching high level policy framework for Northern Ireland and provides useful principles for ageing research and public policy in Northern Ireland. The PfG vision is to build a peaceful, fair and prosperous society in Northern Ireland, with respect for the rule of law. A number of Public Service Agreements (PSA) aligned to the PfG confirm key actions that will be taken to support the priorities that the Government aim to achieve over the next three years. For example objective 2 of PSA 7: Making Peoples�۪ Lives Better: Drive a programme across Government to reduce poverty and address inequality and disadvantage, refers to taking forward strategic action to promote social inclusion for older people; and to deliver a strong independent voice for older people. The Office of the First Minister and deputy First Minister (OFMDFM) have recently appointed an Interim Older People�۪s Advocate, Dame Joan Harbison to provide a focus for older peoples issues across Government. Ageing in an Inclusive Society is the cross-departmental strategy for older people in Northern Ireland and was launched in March 2005. It sets out the approach to be taken across Government to promote and support the inclusion of older people. The vision coupled with six strategic objectives form the basis of the action plans accompanying the strategy. The vision is: ���To ensure that age related policies and practices create an enabling environment, which offers everyone the opportunity to make informed choices so that they may pursue healthy, active and positive ageing.�۝ (Ageing in an Inclusive Society, Office of the First Minister and Deputy First Minister, 2005) Action planning and maintaining momentum across government in relation to this strategy has proved to be slower than anticipated. It is proposed to refresh this Strategy in line with Opportunity Age ��� meeting the challenges of ageing in the 21st Century (9). There are a number of policy levers elsewhere which can also be used to promote the positive aspects of an ageing society. The Investing for Health (10) and A Healthier Future:A 20 Year Vision for Health and Well-being in Northern Ireland (11), seek to ensure that the overall vision for health and wellbeing is achievable and provides a useful framework for ageing policy and research in the health area. These health initiatives have the potential to positively impact on the quality of life of older people and provide a useful framework for improving current policy and practice. In addition to public policy initiatives, the anti-discrimination frameworks in terms of employment in Northern Ireland cover age as well as a range of other grounds. Goods facilitates and services are currently excluded from the Employment Equality (age) Regulations (NI) 2006 (12). Supplementing the anti-discrimination measures, Section 75 of the Northern Ireland Act 1998 (13), unique to Northern Ireland, places a statutory obligation on public authorities in fulfilling their functions to promote equality of opportunity across nine grounds, one of which is age(14). This positive duty has the potential to make a real difference to the lives of older people in Northern Ireland. Those affected by policy decisions must be consulted and their interests taken into account. This provides an opportunity for older people and their representatives to participate in public policy-making, right from the start of the process. Policy and Research Interface ���Ageing research is vital as decisions in relation to policy and practice and resource allocation will be made on the best available information�۝. (CARDI�۪s Strategic Plan 2008-2011) As outlined earlier, CARDI has been established to bridge the gap to ensure that research reaches those involved in making policy decisions. CARDI is stimulating the ageing research agenda in Ireland through a specific research fund that has a policy and practice focus. My work is presently focusing on helping to build a greater awareness of the key policy levers and providing opportunities for those within research and policy to develop closer links. The development of this shared understanding by establishing these links between researchers and policy makers is seen as the best predictor for research utilization. It is important to acknowledge and recognise that researchers and policy makers operate in different institutional, political and cultural contexts. Research however needs to ���resonate�۪ with the contextual factors in which policy makers operate. Conclusions Those working within the public policy field recognise all too often that the development of government policies and initiatives in respect of age does not guarantee that they will result in changes in actual provision of services, despite Government recommendations and commitments. The identification of public policy initiatives as they relate to age has the potential to highlight persistent and entrenched difficulties that social policy has previously failed to address. Furthermore, the identification of these difficulties can maximise the opportunities for progressing these across government. A focus on developing effective and meaningful targets to ensure measurable outcomes in public policy for older people can assist in this. Access to sound, credible and up-to-date evidence will be vital in this respect. As well as a commitment to working across departmental boundaries to effect change. Further details: If you would like to discuss this paper or for further information about CARDI please contact: Judith Cross, Policy Officer, Centre for Ageing Research and Development in Ireland CARDI). t: +44 (0) 28 9069 0066; m: +353 (0) 867 904 171; e: judith@cardi.ie ; or visit our website at: www.cardi.ie References 1) Centre for Ageing Research and Development in Ireland (2008) Strategic Plan 2008-2011. Belfast. CARDI 2) The Agreement: Agreement Reached in the Multi-Party Negotiations. Belfast 1998 3) Madrid International Plan of Action on Ageing. http://www.un.org/ageing/ 4) UN Programme on Ageing (2007) Research Agenda on Ageing for the 21st Century: 2007 Update. New York. New York. UN Programme on Ageing and the International Association of Gerontology and Geriatrics. 5) The Pensions Act 2007 Chapter 22 6) Office of the First Minister and deputy First Minister (2005). Ageing in an Inclusive Society. Belfast. OFMDFM Central Anti-Poverty Unit. 7) Office of the First Minister and deputy First Minister (2005). Lifetime Opportunities: Government�۪s Anti-Poverty and Social Inclusion Strategy for Northern Ireland. Belfast. OFMDFM Central Anti-Poverty Unit. 8) Northern Ireland Executive (2008) Building a Better Future: Programme for Government 2008-2011. Belfast. OFMDFM Economic Policy Unit. 9) Department for Work and Pensions, (2005) Opportunity Age: Meeting the Challenges of Ageing in the 21 st Century. London. DWP. 10) Department of Health, Social Services and Public Safety (DHSS&PS) (2002) Investing for Health. Belfast. DHSS&PS. 11) Department of Health, Social Services and Public Safety (DHSS&PS) (2005) A Healthier Future:A 20 Year Vision for Health and Well-being in Northern Ireland Belfast. DHSS&PS. �� 12) The Employment Equality (Age) Regulations (Northern Ireland) 2006 SR2006 No.261 13) The Northern Ireland Act 1998, Part VII, S75 14) The nine grounds covered under S75 of the Northern Ireland Act are: gender, religion, race, sexual orientation, those with dependents, disability, political opinion, marital status and age.

Beyond sepsis pathophysiology with cytokines: what is their value as biomarkers for disease severity?

Sepsis is a major challenge in medicine. It is a common and frequently fatal infectious condition. The incidence continues to increase, with unacceptably high mortality rates, despite the use of specific antibiotics, aggressive operative intervention, nutritional support, and anti-inflammatory therapies. Typically, septic patients exhibit a high degree of heterogeneity due to variables such as age, weight, gender, the presence of secondary disease, the state of the immune system, and the severity of the infection. We are at urgent need for biomarkers and reliable measurements that can be applied to risk stratification of septic patients and that would easily identify those patients at the highest risk of a poor outcome. Such markers would be of fundamental importance to decision making for early intervention therapy or for the design of septic clinical trials. In the present work, we will review current biomarkers for sepsis severity and especially the use of cytokines as biomarkers with important pathophysiological role.

Sex, drugs and chronic illness: health behaviours among chronically ill youth.

BACKGROUND: A growing body of literature indicates that adolescents with chronic conditions are as likely, or more likely, to take risky behaviours than their healthy peers. The objective of this research was to assess whether adolescents with chronic illness in Catalonia differ from their healthy peers in risk-taking behaviour. METHODS: Data were drawn from the Catalonia Adolescent Health database, a survey including a random school-based sample of 6952 young people, aged 14-19 years. The index group (IG) included 665 adolescents (450 females) reporting several chronic conditions. The comparison group (CG) comprised 6287 healthy adolescents (3306 females). Personal, family and school-related variables were analysed to ensure comparability between groups. Sexual behaviour, drug use (tobacco, alcohol, cannabis, cocaine and synthetic drugs) and perception of drug use among peers and in school were compared. Analysis was carried out separately by gender. chi-square, Fisher's and Student's tests were used to compare categorical and continuous variables. RESULTS: The prevalence of chronic conditions was 9.6%, with females showing a higher prevalence than males. The IG showed similar or higher rates of sexual intercourse and risky sexual behaviour. For most studied drugs, IG males reported slightly lower rates of use than CG males, while IG females showed higher rates for every drug studied. No differences were found in the perceptions of drug use among peers or in their school. CONCLUSIONS: Similar to previous research, chronically ill adolescents in our sample are as likely, or more likely, to take risky behaviours than their healthy counterparts and should receive the same anticipatory guidance.