Emma Foster

John C Mathers

Ashley Jane Adamson

E Foster J C Mathers

Packaged food intake by British children aged 0 to 16 years g per kg body weight

Keywords: Exposure assessment, Children, Food packaging, Packaging migration

The European Union (EU) approach to assessing exposure to chemical migrants from plastic Food Contact Materials (FCMs) has been to assume an intake of 1 kg of food in contact with a particular material, per 60 kg person per day, which equates to 16.7 g/kg body weight. A food packaging surface area to food mass ratio of 6dm 2 /1kg is assumed, equivalent to 0.1dm 2 /kg of body weight. Children may be at increased risk to exposure from migrants as they have higher intakes of food per kg body weight compared with adults. In addition, much of the food marketed for/to children is in small portions and therefore the FCM area to food mass ratio is relatively high. To determine if, and how, the EU model might be modified to ensure specific protection against chemical migration into food marketed for children, data on 4-day food intakes of 297 children aged 0 to 6 years were collected including information on pack size, pack type and FCM area to food mass ratio. The 297 children consumed a total of 1,646 kg of food and drink (including tap water) of which 978 kg (59%) was packaged with 67% of this packaged in plastics. Mean intakes of food packaged in plastic ranged from 27g per kg body weight (for the infants under 1 year) to 51 g per kg body weight (for the 1-4 year olds). This was higher than the 16.7 g/kg body weight derived from the EU convention. The mean area of packaging in contact with the food consumed daily per kg body weight were 0.65 dm2/kg for the infants under one, 0,81 dm2/kg for the 1-4 year olds and 0.66 dm2/kg for the 4-6 year olds. All 297 children had intakes which exceeded 0.1dm2 of packaging per kg of body weight assumption.

Introduction

Trends in food purchasing

Food purchasing patterns have changed dramatically over the past 50 years. There has been a move away from the daily purchase of fresh food, stored in boxes and baskets wrapped in paper on purchase and stored in ceramic or glass containers at home, to today's pre-prepared and/or highly packaged food provision. It is now difficult to purchase food that has not, at some time, been stored in a container such as a plastic crate or packaging made from a variety of materials such as plastic, paper and board or metal.

The consumer drive towards convenience has resulted not only in more packaged food items being sold but also reduced time allocated to food preparation and cooking [START_REF] Foster | BNF 40 th anniversary briefing paper 'Food availability and our changing diet[END_REF]. This has resulted in a demand for items which can be quickly cooked in pack such as microwave meals, part baked bread and individual portions of foods such as rice pudding [START_REF] Huxley | A Review of the UK Food Market[END_REF]].

Role of packaging

Packaging food has a number of key purposes. The packaging protects food from spoilage for example by oxidation, moisture, pests, mould and micro-organisms or contamination from foreign materials thereby prolonging the shelf life of food and protecting the health of the consumer. Packaging can aid transport and storage of foods and can also be used to inform and attract the consumer.

Migration

The transfer of constituents from the food contact material into food may occur by a process known as migration and may have an effect on the quality and safety of the food [START_REF] Barnes | Chemical Migration and Food Contact Materials[END_REF]]. The extent of migration is determined by several factors including the temperature of storage; the length of time the food is in contact with the food contact material; the type of food -aqueous, acidic, alcoholic or fatty; the nature of the food -solid, liquid or dry; whether the food is processed or cooked in the container; the type of food contact material e.g. plastic, paper or coated can; the type of possible chemical migrant including molecular weight, volatility, concentration in the material, diffusion through the food packaging material and the contact area of the food contact material:food mass ratio [Goulas et al. 1998;Anderson et al. 2003, Quinto-Fernandez et al. 2003] Examples of substances that have been found to migrate from food contact materials or articles in recent years include: semicarbazide from seals used in the 'press-on, twist off' metal lids on glass jars [START_REF] Nestmann | Safety Assessment and risk-benefit analysis of the use of azodicarbonamide in babyfood jar closure technology: Putting trace levels of semicarbazide exposure into perspective -A Review[END_REF]]; 2-isopropylthioxanthone (ITX) as a set-off from printing into infant formula and fruit juices [Rothenbacher et al. 2007]; formaldehyde from melamine ware [START_REF] Bradley | Survey of the migration of melamine and formaldehyde from melamine food contact articles available on the UK market[END_REF]; and benzophenone from printed packaging [Food Standards Agency 2006]. These are substances for which a specific safety assessment was required following notification through the EU Rapid Alert System for food and feed.

It is essential that legislation on food packaging protects the consumer from any potential harmful effects of migrants whilst ensuring that the correct use of packaging minimises any health risks due to contamination of the foodstuff.

Legislation

Within the European Union all food contact materials and articles are controlled by the Framework Regulation (EC) 1935/2004. Article 3 of this Regulation states that 'materials and articles, including active and intelligent material and articles, shall be manufactured in compliance with good manufacturing practice so that, under normal or foreseeable conditions of use, they do not transfer their constituents to food in quantities which could: a) endanger human health; or b) bring about an unacceptable change in the composition of the food; or c) bring about a deterioration in the organoleptic characteristics thereof. ' [European Regulation No. 1935/2004] replaced by the European Food Safety Authority (EFSA). To set the limit, it is assumed that, every day throughout his/her lifetime, a person of 60kg bodyweight eats 1kg of food packed in plastics containing the relevant substance at the maximum permitted quantity . This is equivalent to 16.7g/ kg body weight. A food packaging surface area to food mass ratio of 6dm 2 /1kg is assumed, equivalent to a packaging surface area of 0.1dm2/kg of body weight.

The packaged food intake of the British population is unknown. The National Diet and Nutrition Surveys provide comprehensive data on the nutritional intake of British adults and children measured by weighed food intake [Finch et al 1998, Gregory et al. 1993, Gregory and Lowe 2000, Ruston et al. 2004] but the method of collection of these data does not allow extraction of information on intakes of packaged food. No established method is available by which to measure packaged food intake. For plastics, the intake of packaged food by adults, on which the specific migration limits (SML) are based, relies on the assumptions mentioned above.

Current knowledge of packaged food intakes

In the absence of detailed data on individual intakes of packaged food, exposure estimates are often made using per capita consumption and production statistics. For example [START_REF] Dionisi | Estimates of per capita exposure to substances migrating from canned food and beverages[END_REF] used the production statistics from EU can manufacturers, together with import and export statistics, to estimate exposure to substances migrating from canned foods and beverages. This method assumes that the adjusted production is equal to consumption and gives an estimate of average individual consumption. However, without data on individual intakes it is not possible to identify and characterise high (and low) consumers which is important to ensure that consumers with high intakes are protected. Duffy et al. (2007) suggest using the upper percentiles of food intake to estimate exposure and stress the need to link the use of a particular type of food packaging to the consumption of a specific item of food.

Franz et al. discuss that statistical data on food consumption and packaging use can be used to estimate exposure more realistically [START_REF] Franz | Migration modelling from food-contact plastics into foodstuffs as a new tools for consumer exposure estimation[END_REF] a 2 dimensional probabilistic model, along with 'best estimates' of packaging materials, to provide a better estimate of the 'true' exposure of the population and to identify those at higher risk [START_REF] Holmes | Dietary exposure to chemical migrants from food contact materials: A probablistic approach[END_REF].

Why children are potentially at increased risk

An important issue in exposure assessment is the food intake of children. Children consume more food per unit of body weight than adults due to their additional energy and nutrient needs for growth. On supermarket shelves some food products are targeted specifically at/ for children. Much of this food is in small portions and thus the food packaging to food mass ratio may be higher than the conventional 6 square decimetres (dm 2 ) of packaging used for plastic materials and articles per kg of food.

In a study of 594 Irish children Duffy et al. (2007Duffy et al. ( , 2006aDuffy et al. ( , 2006b) created the first database linking food consumption data (collected using a 7-day weighed food diary) with information on food packaging. This study included chemical characterisation of food contact materials but did not measure the surface area of the packaging in contact with the food. Their study demonstrated that intakes in this population were in excess of the assumed 16.7g/kg body weight.

Aim

The current European approach for the authorisation and control of substances used in food contact materials and articles is intended to be cautious in relation to the estimation of potential exposure of the consumer to these substances with respect to an average adult. The purpose of this study was to quantify the intake of packaged food and thereby the use of associated food contact materials by British children aged 1 to 6 years. The aim was to determine if, and how, the current EU model of 1 kg of packaged food per 60 kg person per day might need to be modified to ensure specific protection against chemical migration into food marketed for children

Materials and Methods

Recruitment

The aim was to recruit a sample of children which were representative of the UK population as a whole in terms of socio-economic status, urban-rural mix and ethnicity. Participants were selected from the volunteers based on these criteria. Socio-economic status was assessed using Index of Multiple Deprivation (IMD). The All recruitment took place in Newcastle and the North Tyneside area. Newcastle has been reported to be a representative city for the UK [START_REF] Charles | Core Cities: Key centres for regeneration synthesis report[END_REF]], the population spans the spectrum of socio-economic status and ethnicity can be represented by the inclusion of schools city wide (ranging from 0-98% ethnicity). The National Diet and Nutrition Survey of young people aged 4 to 16 years found no significant differences between the North and other regions except for higher than average consumption of carbonated drinks in the Northern region [Gregory and Lowe, 2000].

Five nurseries, nine primary schools and one family centre were recruited to take part in the study. Parental consent was sought for participation in the study. Recruitment of preschool children, cared for at home, took place through posters in libraries, leisure centres, supermarkets and local shops. The aim was to complete the study with 100 children in each of three age groups, under 1 year (infants), 1-4 years (preschool) and 4 to 6 years (primary). The distinction between 4 year olds in the middle and upper age groups was based on their attendance at primary school.

Dietary data collection

Food diary

Packaged food intake of all study participants was measured for 4 consecutive days (including both weekend days) using a specially developed food diary. Information was collected on the date and time food or drink was consumed, the brand, pack size and a description of the food. A prospectively recorded food diary with estimated weights was chosen as the method of data collection due to the extensive nature of the information required on food packaging. This required prospective recording in which the subject's carer was required to record pack size and product details at the time of preparation/ consumption of the food.

Estimation of the weight of food consumed using food photographs was selected to minimise the burden on the subject. There is extensive evidence that requiring subjects to weigh and record all food and drink consumed results in changes to habitual diet [START_REF] Livingstone | Validation of estimates of energy intake by weighed dietary record and diet history in children and adolescents[END_REF]. It was concluded that, given the additional information required on packaging, asking subjects to complete a weighed food diary would over burden them and so be likely to result in reduced response rate and in dietary change over the recording period. To quantify food intake, a combination of food photographs developed specifically for use with children [START_REF] Foster | The development and evaluation of three portion size assessment tools for use with children[END_REF]] and the adult food atlas [START_REF] Nelson | A photographic atlas of food portion sizes[END_REF]] was used to estimate the amount of food served and amount of food leftover.

Collection of packaging

In addition to the diary, subject's carers were provided with a sealable storage container and asked to keep the packaging of all foods and beverages the child consumed during the recording period. In addition, where samples of food packaging for foods consumed were not collected by the participants, these foods were purchased so that the packaging information could be obtained.

In school/ nursery observations

Children attending nursery or school on the recording days were observed by the research team at every occasion during the day when food was provided. Where possible the food was weighed as it was served to the child and any food leftover was weighed to determine actual consumption. In the school dining hall weighing the food as it was served to the child was not possible due to the number of children being served in a short space of time. In this situation school meals were purchased from the school and an example of the meal selected by each child was used to obtain a weight for each food served. The children's trays were collected at the end of the meal and any food leftover was weighed to determine consumption.

Interview

An interview was conducted with each subject's parent or guardian in their home to obtain estimates of the amount of food consumed, whilst the child was in their care, using food photographs. The interviewer reviewed all entries with the parent or guardian and collected details on the type of packaging for each food, pack size, brand and whether the item was cooked in its pack. The children's height was measured to the nearest cm using a stadiometer and weight was measured to the nearest 100g using Tanita digital scales, by trained staff following the protocol used in the National Diet and Nutrition Survey [Gregory and Lowe, 2000]. For babies, length was measured using a standard tape measure with the baby in the supine position.

Food packaging database

The type of packaging was identified where possible by visual examination including assistance from industry experts where necessary. Where possible all components of any multilayer materials were identified. A photographic record was taken of each item of food packaging collected. Surface area and food contact material to food mass ratios were calculated for all complete packaging which had been collected. The surface area calculated was the total internal surface area and included any part of the packaging which could be in contact with the food. For example for a yogurt pot the underside of the lid was included in the surface area calculation as this may come into contact with the food depending on the method of storage and transit. Details from the packaging were entered onto an ACCESS database. This included information on the brand, food name, pack size, pack type, fat content per 100g, food contact material to food mass ratio and whether the food was acidic, alcoholic, aqueous, fatty or dry. Information on packaging type was recorded on three levels:

Main category e.g. glass, plastic, metal, paper/board Sub category e.g. Polystyrenes, paper, board Product e.g. HIPS, LDPE, HDPE, PET, recycled board/paper, metal lids with or without gaskets.

These categories were developed in collaboration with industry experts. If more than one material was used in the packaging of a single item, the above information was recorded for each material.

Each unique item consumed was given a unique package food code within the database. An item was considered to be unique if it was a different food type, a different brand, a different pack type or a different pack size. For example Asda mayonnaise was given a different code from Asda tomato ketchup, and tomato ketchup from Asda was given a different code from that for tomato ketchup from Codes were established for non-packaged items including tap water and loose fruit and vegetables and deli-counter items where the food was loose at the point of purchase and transferred into a paper or plastic bag to hand to the consumer. In addition an 'unknown' category was created and used for items consumed outside the home such as at restaurants, fast food outlets and at friends' or relatives' homes where sufficient information on the packaging type and pack size could not be collected.

This code was also used for foods which had been decanted into storage jars and the original packaging discarded before the study began, and where the respondent could not recall the details required.

Coding of individual food diaries

The food diaries were coded using unique product codes in an ACCESS database so that an individual's data on the type and amount of food consumed, along with age, gender, socio-economic status (SES) and anthropometry could be linked with the product information including packaging type, FCM area:foodmass and fat content.

Data analysis

Mean daily intakes of total, packaged and non packaged foods were calculated in g/kg body weight. Mean daily intake of packaged foods was also calculated by pack type in g/kg body weight. Mean intakes were calculated first on an individual basis and then as mean values for each age group.

In order to calculate the total area of packaging to which a child was exposed per day per kg body weight the following equation was used:

AB/CD

Where A is the mass of a particular food consumed by a child during the survey, B is the FCM for that food expressed in area of contact per mass of food, C is the mass of the child and D is the duration of the survey.

Then a single numerical indicator of possible contamination from the particular food would be: This was used to give the total area of packaging to which the child was exposed per day, per kg body weight. This numerical indicator increases the greater the amount eaten, the smaller the child and the greater the food contact to food mass ratio.

Results and Discussion

Recruitment

Data on the 4-day food intakes of 297 children, approximately 100 from each age group, were collected (Table 1). The distribution of IMDs in the sample population was very similar to the national sample for age group 1. For age groups 2 and 3 we slightly over-recruited subjects from households with a high IMD indicating that our study population for these age groups has a slightly higher level of deprivation compared with the national average. However, 5.7% of the sample could not be categorised by IMD as they lived in newly built owner occupied houses -the IMD for these houses is likely to be low, that is not deprived. Data were collected on whether subjects lived in an urban or rural setting. Across all age groups 11% of our sample lived in a rural location; nationally 11.2% of people live in a rural location (Table 2) [Office for National Statistics, 2001].

We also collected data on whether our subjects were from an ethnic minority background. Across all age groups, 7.7% of our sample was from ethnic minority origins compared with 7.9 % at the national level (Table 2) [Office for National Statistics, 2001]. The information collected in terms of socio-economic status and the proportion of children from ethnic minority backgrounds clearly supports the conclusion that the sample is unbiased and that Newcastle is a typical UK city. The observed intake of packaged food can therefore be taken to represent packaged food intake in UK children.

Database of packaged foods

During the course of the study, data were collected on 6615 unique food items and entered into the database. Of the 6615 items, 6159 (93%) were packaged products with the remainder including non-packaged items such as loose fruit and vegetables, tap water, meat bought loose from butchers and deli counter items.

Children's intakes of packaged foods

The mean total food intake in kg per day for the 1 to 4 year old age group were found to be 1.4kg which is very close to the mean intakes of 1.5kg food per child per day for 1½ to 4½ year olds in the National Diet and Nutrition Survey [Gregory et al. 2000].

Table 3 shows the mean daily intakes per kg body weight for each age group of packaged foods (and drinks), total food, non-packaged items and items where packaging information was not known. As age increased, food intake/kg body weight fell. Infants aged 0-1 years consumed 136.9g/kg body weight, children of 1-4 years consumed 99.9g/kg body weight and children aged 4-6 years consumed 70.4g/kg body weight. Children aged 1-4 years had the highest intake of packaged food/kg body weight at 67.6g/kg, with infants (0-1 years) and children aged 4-6 years having very similar intakes (49.5g/kg and 48.0g/kg respectively).

For the three age groups combined, 54% of the total weight of food consumed was derived from packaged items. Non-packaged items included tap water and fruit and vegetables purchased loose. The infants (under one year) had a much lower proportion of their intake from packaged foods mostly due to their consumption of breast milk or formula reconstituted with tap water but also because a number of infants were fed puréed fruits and vegetables which had been purchased loose. The children aged 1-4 years and 4-6 years both derived 68% of their intake from packaged foods. There was a large variation in individual intakes ranging from 2.5g per kg body weight, for an infant fed formula milk and puréed fruit and vegetables purchased loose, to 182.5 g per kg body weight for a 2 year old child fed predominantly preprepared packaged foods. Overall only 3% of intake/kg body weight was of unknown packaging ranging from 1% for infants (under one year) to 4% for children aged 1-4 years.

Table 4 shows the mean daily intake of packaged food by age group and by main packaging category (e.g. plastic, glass, metal and alloys). For all age groups, foods packaged in the main category 'plastic' represented the greatest proportion of packaged foods consumed (67%). Infants (under one year) had the lowest proportion of intake from foods packaged in plastic, due mainly to high consumption of commercial baby foods which are generally packaged in glass jars, small food cans and/or multilayer packets. Children in age group 1-4 yrs had the highest intake of foods packaged in plastic/kg body weight (mean = 50.5g/kg).

Figure 1 shows that the mean intakes of foods packaged in plastic per kg body weight for children of all ages were greater than the 16.7g per kg body weight assumed in the EU model. Some individual children's intakes greatly exceeded this level and for the 1-4 year old children no child had an intake of foods packaged in plastic per kg body weight below 16.7g per kg body weight, 63% of the infants (under one year) and 97% of the 4 to 6 year olds had intakes in excess of this level.

Multilayer packaging was the second most commonly used main category of packaging for food consumed with an overall average of 12% of the packaged foods consumed/kg body weight packed in multilayer packaging. Examples of this type of packaging included fresh fruit juice cartons, packets of dry mixes for sauces and pasta meals and multilayer cylinders for snacks. Intake of foods packaged in multilayer items varied across the age groups being highest for children aged 4-6years (15% of all packaged items/kg body weight).

Metal and alloys and glass were the other main categories of packaging making a significant contribution to the children's intakes especially for the infants (under one year) where metal and alloys were the second most important main category of packaging of food consumed (17% of all packaged items/kg body weight). Across all age groups metal and alloys accounted for 9% and glass 8% of all packaged foods consumed .

Foods packaged in paper and board, paraffin and microcrystalline waxes, textiles and wood made a minimal contribution to the children's diets. Examples of items packaged in these pack types include raisins (paper and board), babybel cheese (paraffin and microcrystalline waxes), rice (textiles) and balsamic vinegar (cork stopper). Food contact material area:food mass ratio was calculated for 92.6% of the weight of food consumed by the children. For a further 3.6% FCM area:food mass ratios were imputed from mean values for that food and packaging type and pack size. This could not be done for 3.8% of the weight of food consumed and these foods were excluded from the analysis of FCM:food mass ratio presented in Table 5 which refers to 96.2% of foods consumed. Table 5 shows the average surface area of packaging in contact with the food consumed daily for each age group. For all age groups combined, an average 10.7dm 2 of packaging was in contact with the food consumed per day. This equated to 0.7dm 2 of packaging per kg body weight and 7.7dm 2 of packaging per kg of food consumed. Again, there were large variations in individual intakes with mean daily packaging in contact with the food consumed ranging from 0.8dm 2 to 37.9dm 2 .

The surface area of packaging in contact with daily food consumed, expressed per kg body weight, ranged from 0.1dm 2 to 2.5dm 2 and the area of packaging per kg food consumed ranged from 0.8dm 2 to 20.8 dm 2 . The 4-6 year old age group had the highest mean area of packaging in contact with the food consumed (14 dm 2 ) and the greatest mean surface area of packaging per kg of food consumed (9.7dm 2 ). The infants (under one year) had the lowest mean surface area of packaging in contact with the food consumed due to the large amount of non-packaged items consumed by this group. The surface area of the packaging in contact with the food exceeded 6 dm 2 per kg of food for both the 1-4 year old and the 4-6 year old age groups. All children taking part in the study had intakes which exceeded 0.1dm2 of packaging per kg of body weight assumption.

The 297 children consumed a total of 978kg of packaged food and drink between them over the 4 days during which they recorded their dietary intake. Of these foods, 38% were classified as aqueous, 27% as dry, 23% as acidic, 12% as fatty and 0.01% as alcoholic (from wine used in cooking). As fatty foods have a higher migration potential this means that 88% of the food and drink consumed had a lower migration potential.

This study has shown that, probably because of their higher intakes of food per kg body weight, children consume higher intakes of packaged foods per kg body weight than assumed adult intakes. Here we report data on intake of food per kg body weight for specific age groups and the proportion of that food which was packaged for Statistics, 2001]. Mean intakes of food packaged in plastic ranged from 27g per kg body weight (for the infants under 1 year) to 51 g per kg body weight (for the 1-4 year olds). This was significantly higher than the 16.7 g/kg body weight derived from the convention that a person of 60 kg could consume daily up to 1 kg of foodstuffs in contact with a particular plastic.

For Irish children, Duffy et al reported similar intakes of total food and drink (overall mean, 1.5kg for 5-12 year old children) compared with those (overall mean, 1.4kg) in this sample of slightly younger UK children [Duffy et al. 2006a]. The fall in intakes of food packaged in plastics (g/kg body weight) with increasing age from 68g/kg body weight for the 1-4 year olds to 48g/kg body weight for the 4-6 year olds in the current study is in line with the mean intakes of 39g/kg body weight reported for the 5-12 year old Irish children [Duffy et al. 2006a]. However, the highest consumers had intakes very much greater than these values -up to169g/kg body weight for the 4-6 year old age group.

The area of food packaging in contact with the foods consumed was measured and the ratio of food contact material area:food mass was calculated. For infants (less than one year old), the mean area of packaging per 1 kg food was less than 6 dm 2 compared with 8.3 dm 2 and 9.7 dm 2 for children aged 1-4 and 4-6 years respectively.. For all age groups together, the mean surface area of packaging associated with 1 kg of food was 7.7 dm 2 . There was large variation in the surface area of packaging associated with 1 kg of food ranging from 0.8 dm 2 to 20.8 dm 2 .

The results of this study together with those of Duffy et al.(2006a) suggest that the assumptions of the EU legislation in terms of intakes of packaged food per kg body weight and mean surface area of food contact material to food mass ratio may not be as conservative for young children.

This study focused on packaged food as sold to the customer and did not take account of any additional potential exposure due to the transfer of food into food contact materials such as Tupperware, cling film or foil in the home. The database derived from this study will be used as a source of intake information in the European Commission FP7& FACET project which is looking at consumer exposure to food additives, flavourings and substances migrating from food contact materials. *This is the average surface area of packaging (dm 2 ) in contact with the food consumed daily + This is the average surface area of packaging (dm 2 ) in contact with the food consumed daily expressed per kg body weight † This is the area (dm 2 ) of packaging per kg of food consumed

F o r P e e r R e v i e w O n l y

  . To ensure the protection of the health of the consumer and avoid adulteration of the foodstuff two types of migration limits have been established in the area of plastic materials [European Directive 2002/72/EC]: an Overall Migration Limit (OML) of 60mg (of substances)/kg (of foodstuff or food simulants) that applies to all substances that can migrate from the food contact material to the foodstuff and a Specific Migration Limit (SML) which applies to individual authorised substances and is fixed on the basis of the toxicological evaluation of the substance. The SML is generally established according to Acceptable Daily Intake (ADI) or the Tolerable Daily Intake (TDI) set by the Scientific Committee on Food (SCF) now

  . Holmes et al. used dietary records collected during the National Diet and Nutrition Survey to provide data on actual body weight and variation in food consumption amongst individuals for use in

  , barriers to entering society, employment and the local environment[Communities and Local Government, 2007].

  plastic bottle of tomato ketchup from Tesco was given a different code from a glass bottle and a 500g bottle was given a different code from an 800g bottle.

  ...+AkBk)/C}/D.

  children who were representative of the wider UK population in terms of ethnic origin, socioeconomic status and rural:urban mix [Office for National

  Baumann M and Fügel D. (2007) 1-isopropylthioxanthone (2-ITX) in food and food packaging materials on the German market, Food Additives and Contaminants, 24 (4), 438-444 Ruston D, Hoare J, Henderson L, Gregory J, Bates CJ, Prentice A, Birch M, Swan G, and Farron M (2004) The National Diet and Nutrition Survey: Adults Aged 19 to 64 Years. The Stationery Office, London. Smithers G; Finch S, Doyle W; Lowe C; Bates C.J; Prentice A and Clarke P.C. (1998) National diet and Nutrition Survey: people aged 65 years and older. Volume 1: Report of the diet and nutrition survey. Stationery Office: London.

  Figure 1 -Mean and range of intakes in g per kg body weight of foods packaged in plastic

Table 1 -Recruitment and Completion

 1

	Page 21 of 27			Food Additives and Contaminants
	Age group	Target	No. consenting	No. included	Completers (%)
	(years)
	<1	100	98	98	96	(98%)
	1-4	100	131	103	99	(96%)
	4-6	100	139	113	102	(90%)
	Total	300	368	314	297	(95%)

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Table 2 -Characteristics of study sample

 2 IMD is Index of Multiple Deprivation. The IMD is based on lower level super output areas and incorporates aspects such as health, crime, barriers to entering society, employment and the local environment[Communities and Local Government, 2007].

	Age group (years) <1 1-4 4-6 All children National levels* † -Food Additives and Contaminants Male:Female Urban:Rural %Rural Ethnicity Majority:Minority 53:43 89:7 7.3 92:4 39:60 81:18 18.0 93:6 52:50 94:8 7.8 89:13 144:153 264:33 11.0 275:23 11.2	% minority ethnic group 4.2 6.1 12.7 7.7 7.9	IMD † (National Mn =21.8) 21.9 29.5 23.9 25.1 21.8

* -[Office for National Statistics, 2001]. Page 22 of 27 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table 3 -Food intake (g per kg body weight) of packaged and non-packaged foods Food intake (g per kg body weight) Age group

 3

	Page 23 of 27			Food Additives and Contaminants
	(years)		Total packaged	Non-Packaged	Unknown	Deli counter items Total food	% packaged food
			food	food	packaging
	<1	Mean	49.5	85.0	1.6	0.8	136.9	36%
		(SD)	(24.6)	(41.5)	(3.6)	(2.4)	(35.7)
		Range	2.5-142.9	11.1-200.8	0.0-21.1	0.0-18.3	64.9-238.2
	1-4	Mean	67.6	26.9	4.2	1.1	99.9	68%
		(SD)	(23.4)	(18.6)	(5.0)	(2.2)	(31.5)
		Range	24.0-182.5	0.0-90.1	0.0-28.3	0.0-14.1	46.0-200.9
	4-6	Mean	48.0	17.4	3.6	1.5	70.4	68%
		(SD)	(16.3)	(10.4)	(4.9)	(2.5)	(21.4)
		Range	22.9-110.2	1.1-57.9	0.0-35.8	0.0-16.0	36.8-143.5
	Total	Mean	55.0	42.4	3.2	1.1	101.7	54%
		(SD)	(23.4)	(39.8)	(4.7)	(2.4)	(40.4)
		Range	2.5-182.5	0.0-200.8	0.0-35.8	0.0-18.3	36.8-238.2

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Table 4 -Intake (g per kg body weight of foods) from foods packaged in different packaging types Mean intake (g) per kg body weight of packaged foods Age group

 4

	(years)		Glass	Metal and	Paper and	Plastic	Waxes	Textiles	Wood	Multilayer
				alloys	board
	<1	Mean (%)	6.7 (14%) 8.4 (17%) 0.7 (1.4%) 26.7 (54%)	0 (0%)	0 (0%)	0 (0%)	6.9 (14%)
		(SD)	(8.9)	(8.9)	(1.3)	(19.5)			(0.005)	(13.0)
		Range	0.0-47.8	0.0-43.8	0.0-7.1	0.0-87.6	N/A	N/A	0.0-0.1	0.0-97.9
	1-4	Mean (%)	4.0 (6%)	4.5 (7%)	2.2 (3%)	50.5 (75%)	0.1 (0%)	0 (0%)	0.01 (0%)	6.3 (9%)
		(SD)	(6.9)	(3.6)	(2.0)	(23.6)	(0.2)		(0.04)	(6.2)
		Range	0.0-35.4	0.0-15.7	0.0-9.7	14.7-169.8	0-1.3	N/A	0.0-0.2	0.0-25.3
	4-6	Mean (%)	2.3 (5%)	2.6 (5%)	1.9 (4%)	33.8 (70%) 0.02 (0%) 0.2 (0%) 0.02 (0%)	7.1 (15%)
		(SD)	(4.4)	2.3	2.0	12.4	(0.07)	(1.6)	(0.04)	(8.4)
		Range	0.0-22.2	0.0-8.9	0.0-12.5	9.1-78.0	0.0-0.4	0.0-15.8	0.0-0.2	0.0-46.7
	Total	Mean (%)	4.3 (8%)	5.1 (9%)	1.6 (3%)	37.1 (67%) 0.03 (0%) 0.1 (0%) 0.01 (0%)	6.8 (12%)
		(SD)	(7.2)	(6.1)	(1.9)	(21.

Table 5 Mean surface area of packaging in contact with the food consumed daily.

 5

	Mean daily

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Acknowledgements. This work was funded by the Food Standards Agency (A03051).

Thanks to the Newcastle schools, teachers, parents and children who participated in this study. Special thanks to Laurence Castle (CSL, York), John Fairweather (Amcor Flexibles), Richard Whitaker (Crown Packaging), Julian Stocker (H.J.Heinz) and Peter Oldring (Valspar) for their invaluable assistance in identification of packaging type. A full report is available on the Food Standards Agency's Open Access Repository at: www.foodbase.org,uk


Aniruddha ; Choudhury

Katja Derkow

Amir Hossein Daneshmanesh

Eva Mikaelsson

Shahryar Kiaii

Parviz Kokhaei

Anders Österborg

Professor Håkan Mellstedt
email: hakan.mellstedt@karolinska.se

Silencing of Ror-1 and Fibromodulin with siRNA results in apoptosis of CLL cells

Keywords: APOPTOSIS, CHRONIC LYMPHOCYTIC LEUKAEMIA, TUMOUR ANTIGENS Ror1, fibromodulin, siRNA, apoptosis, CLL, silencing

We have previously demonstrated that Ror1 and fibromodulin (FMOD) are two genes upregulated in CLL cells compared to normal blood B cells. In this study, we have used siRNAs to specifically silence Ror1 and fibromodulin gene expression in CLL cells, healthy B cells and human fibroblast cell lines. siRNA treatment induced a specific reduction (75-95%) in fibromodulin and Ror1 mRNA. Western blot analysis with specific antibodies for fibromodulin and Ror1 demonstrated that the proteins were significantly downregulated 48 hours after siRNA treatment. Silencing of fibromodulin and Ror1 resulted in statistically significant (p ≤ 0.05-0.001) apoptosis of CLL cells but not of B cells from normal donors. Human fibroblast cell lines treated with fibromodulin and Ror1 siRNA did not undergo apoptosis. This is the first report demonstrating that Ror1 and fibromodulin may be involved in the survival of CLL cells.

Ror-1 in particular is further explored as potential target for therapy in CLL.

Introduction

Targeted anticancer therapies depend on the discovery of molecules that are uniquely expressed or overexpressed by malignant cells and are critical for the growth and survival of the cancerous cells. Gene array analyses have previously demonstrated that the genes Ror1 and fibromodulin (FMOD) are upregulated in chronic lymphocytic leukemia (CLL) cells [START_REF] Jelinek | Identification of a global gene expression signature of B-chronic lymphocytic leukemia[END_REF][START_REF] Klein | Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells[END_REF][START_REF] Vallat | The resistance of B-CLL cells to DNA damage-induced apoptosis defined by DNA microarrays[END_REF]. Several studies, including our own have subsequently confirmed that Ror1 and fibromodulin are expressed by CLL cells but not by normal nonmalignant B cells [START_REF] Baskar | Unique cell surface expression of receptor tyrosine kinase ROR1 in human B-cell chronic lymphocytic leukemia[END_REF][START_REF] Daneshmanesh | Ror1, a cell surface receptor tyrosine kinase is expressed in chronic lymphocytic leukemia and may serve as a putative target for therapy[END_REF][START_REF] Fukuda | Antisera induced by infusions of autologous Ad-CD154-leukemia B cells identify ROR1 as an oncofetal antigen and receptor for Wnt5a[END_REF][START_REF] Mayr | Fibromodulin as a novel tumor-associated antigen (TAA) in chronic lymphocytic leukemia (CLL), which allows expansion of specific CD8+ autologous T lymphocytes[END_REF][START_REF] Mikaelsson | Fibromodulin, an extracellular matrix protein: characterization of its unique gene and protein expression in Bcell chronic lymphocytic leukemia and mantle cell lymphoma[END_REF].

Fibromodulin is a 59 kDa collagen-binding protein of the leucine-rich repeat family of proteins located on chromosome 1q32.1. It is found in many types of connective tissues such as cartilage, sclera, tendon, skin and cornea [START_REF] Oldberg | A collagen-binding 59-kd protein (fibromodulin) is structurally related to the small interstitial proteoglycans PG-S1 and PG-S2 (decorin)[END_REF]. This protein is thought to be involved with extracellular matrix organization by means of its ability to interact with collagen [START_REF] Hedbom | Interaction of a 59-kDa connective tissue matrix protein with collagen I and collagen II[END_REF]. fibromodulin is also known to bind TGF-β and modulate its biological activity [START_REF] Hildebrand | Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor beta[END_REF][START_REF] Soo | Differential expression of fibromodulin, a transforming growth factor-beta modulator, in fetal skin development and scarless repair[END_REF].

Ror1 is located on the chromosomal region 1p31.3 (http://www.ensembl.org) and is a 105 kDa protein with potential tyrosine kinase function [START_REF] Yoda | Expression and function of the Ror-family receptor tyrosine kinases during development: lessons from genetic analyses of nematodes, mice, and humans[END_REF]. Ror1 mRNA is normally expressed in heart, lung and kidneys in adult humans and to a lesser extent in the placenta, pancreas and skeletal muscles and a truncated Ror1 is known to be expressed in human leukemia and lymphoma cell lines [START_REF] Reddy | Human neural tissues express a truncated Ror1 receptor tyrosine kinase, lacking both extracellular and transmembrane domains[END_REF]. Recent studies have demonstrated that Ror1 can bind Wnt5a in CLL cells [START_REF] Fukuda | Antisera induced by infusions of autologous Ad-CD154-leukemia B cells identify ROR1 as an oncofetal antigen and receptor for Wnt5a[END_REF] and is currently being actively explored in vitro by several research groups as a potential target for therapy of CLL.

Using a large cohort of patients we have previously demonstrated that Ror-1 and fibromodulin are overexpressed at the protein level in virtually all patients with CLL tested and is not detected in circulating B cells and other peripheral blood cells in normal donors [START_REF] Daneshmanesh | Ror1, a cell surface receptor tyrosine kinase is expressed in chronic lymphocytic leukemia and may serve as a putative target for therapy[END_REF][START_REF] Mikaelsson | Fibromodulin, an extracellular matrix protein: characterization of its unique gene and protein expression in Bcell chronic lymphocytic leukemia and mantle cell lymphoma[END_REF]. In the present study we have used siRNAs to specifically silence these two genes in CLL cells and examine their role in survival of CLL cells.

Materials and Methods

Blood samples: Blood was obtained from totally thirteen CLL patients and two healthy donors with informed consent. Peripheral blood was drawn in heparinized tubes according to protocols approved by the regional ethics committee and in accordance with the Declaration of Helsinki.

None of the patients were receiving anti-leukemic therapy when their blood was collected.

Three patients had progressive CLL and had previously received fludarabine-based chemotherapy (9-26 mo before sampling). Their leukemic cell count ranged from 90-236 x 10 9 /L. Ten patients had indolent CLL, (Rai stage 0-1) with leukemic cells between 17 and 53 x 10 9 /L. Mononuclear cells were separated by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) densitygradient centrifugation. B cells from the blood of healthy donors were further enriched by filtration through nylon wool. [START_REF] Rezvany | Autologous T lymphocytes may specifically recognize leukaemic B cells in patients with chronic lymphocytic leukaemia[END_REF] Cell lines: The adherent human fibroblast cell lines HFSF-PI3, HSF-PI19, HFFF PI6, and HSF-PI18 were gifts from the National Cell Bank of Iran. These, as well as NIH-3T3 fibroblasts stably transfected to express membrane-bound human CD40L (Microbiology and Tumor Biology Centrum, Karolinska Institute, Sweden) was cultured in RPMI 1640 (HFFF PI6, and HSF-PI18, 3T3) or in a 1:1 mixture of DMEM and IMDM (Gibco, Paisley, Scotland). Media were supplemented with 10 or 20% FBS (Gibco) 1% L-Glutamine (2mM) and 1% Pen-Strep (Gibco).

Adherent cells were removed with trypsin-EDTA (Gibco), washed and resuspended in fresh media prior to use in experiments. Viability of cells was determined by quantifying the number of cells in a Buerker Chamber (hemocytometer), mixed in a dilution of 1:10 with 0.4% trypan blue (Gibco) Oligonucleotides and primers siRNA against fibromodulin and Ror1 were designed using a commercially available algorithm (www.dharmacon.com). Three separate siRNA were designed against distinct regions of each gene. Control non-silencing siRNA were also purchased from Dharmacon Inc. (Lafayette, CO, USA). All siRNA were supplied as dried pellet in presence of buffer by the vendor. The siRNA 1A-B. CLL cells were transfected with three different siRNAs each against fibromodulin or Ror1.

Additionally, the cells were also transfected with a pool of the fibromodulin or Ror1 siRNA containing the three at equal concentrations. Two controls were included in every experiment; untransfected cells and cells transfected with a pool of 4 different non-silencing control siRNA.

Transfection of cells with siRNA

The day before transfection, CD40L + -3T3 fibroblasts were added to 24 well plates at a concentration of 250,000 cells/well and incubated at 37 o C in 5% CO 2 and 98% humidity. For every experiment PBMC from normal donors or CLL patients were freshly ficolled, counted and suspended in Optimem® (Invitrogen, Carlsbad, CA) at a concentration of 10x10 6 viable cells/ml. 2 x 10 6 cells in 200 μls of Optimem® were added to each well of 48-well plates for siRNA transfection.

Cells were transfected using the cationic, nonliposomal transfection reagent TransIT-TKO ® (Mirus Bio LLC, Madison, WI). Details of the quantities of reagents utilized are provided in table 2 and are adapted from the published protocol provided by the manufacturer of the transfection reagents. In brief, the diluted transfection reagent was mixed with an appropriate amount of siRNA, incubated for 10 minutes at RT. The siRNA/transfection reagent complex was then added dropwise to the cells in the well of the 48-well plate. The plate was incubated at 37 o C in 5% CO 2 and 98% humidity for 6 hours with gentle rocking every hour. At the end of 6 hours the cells were transferred to the washed CD40L + -3T3 fibroblast monolayers and further incubated with an additional 1 ml of AIM V® till the termination of the experiment.

The human fibroblast cell lines were transfected with siRNA using a procedure that is essentially similar to the one described above with minor modifications. The fibroblast cell lines were added at the concentration of 50,000 cells/well in a 24-well plate and 240,000 cells/ well in a 6well plate on the day prior to transfection with siRNA. Transfection was performed as described above and the cells were subsequently cultured by themselves rather than with CD40L + -3T3 fibroblast.

Apoptosis quantification by flow cytometry

Apoptosis in cells was measured by flow cytometry following staining with Annexin-V and propidium iodide. Cells were stained with anti-CD3 antibody conjugated with APC (BD Biosciences, San Jose, CA) prior to staining with Annexin-V and propidium iodide. A commercially available kit was used for staining cells (BD). Cells were gently detached from the CD40L + -3T3 fibroblast layer, collected in a tube, washed with PBS and 100μls of the cell suspension was stained with 5μls of Annexin V-FITC and 5μls of propidium iodide. Cells were examined using a FACScalibur (BD) flow cytometer and analyzed using the CellQuest™ software.

Polymerase chain reaction (PCR)

RT-PCR and quantitative RT-PCR was performed as described previously [START_REF] Daneshmanesh | Ror1, a cell surface receptor tyrosine kinase is expressed in chronic lymphocytic leukemia and may serve as a putative target for therapy[END_REF][START_REF] Mikaelsson | Fibromodulin, an extracellular matrix protein: characterization of its unique gene and protein expression in Bcell chronic lymphocytic leukemia and mantle cell lymphoma[END_REF] with minor modifications. RNA was extracted using a commercial kit (RNeasy, Qiagen Inc., Valencia, CA, USA). Cells were homogenized by repeated passage through a 22G needle. DNA contamination was minimized by DNase1 treatment (Qiagen). RNA samples were stored at -80 o C. Following reverse transcription as described previously [START_REF] Mikaelsson | Fibromodulin, an extracellular matrix protein: characterization of its unique gene and protein expression in Bcell chronic lymphocytic leukemia and mantle cell lymphoma[END_REF], the cDNA was amplified for 30 cycles for the housekeeping genes βactin and RPLP0 and 35 cycles for fibromodulin and Ror1.

Cell lysis and Western Blotting

Cells were lysed directly in the 24-well plate adding 60 µL of lysis buffer containing 0.2% triton-X, 130 mM HEPES, 4 mM MgCl2, 10 mM EGTA with 2% proteinase inhibitor cocktail (Sigma, St Louis, MO, USA)fibromodulin to each well. After 1 h incubation on ice, lysates were harvested and centrifuged at 2500 rpm for 5 min and the soluble fraction was collected. 30 µL of each sample was loaded onto a 10% NuPAGE Bis-Tris gel (Invitrogen, Carlsbad, CA, USA) and run at 120 V for 3 h under reducing conditions. Resolved proteins were transferred onto Immobilon-P PVDF membrane (Millipore Corporation, Bedford, MA, USA) in a Mini-Transblot Cell (Invitrogen). The membranes were blocked at room temperature for one hour with 5% non-fat (Semper, Stockholm, Sweden) in PBS plus 0.05% Tween 20 (PBS-T). Filters were incubated with 2 µg/ml of anti-FMOD mouse monoclonal antibody (R&D, Minneapolis, MN, USA) over night at +4 o C. Following extensive washings in PBS-T, filters were incubated with a secondary horseradish peroxidise (HRP)-conjugated rabbit anti-mouse antibody (DakoCytomation, Glostrup, Denmark) for 1.5 h at room temperature. Filters were developed using Amersham Enhanced Chemiluminescence ECLTM system (GE Healthcare, Bio-sciences AB, Buckinghamshire, UK). To verify equal loading of samples, filters were stripped in a buffer containing 62.5 mM Tris-HCL, 2% SDS, 100 mM Mercaptoethanol (Sigma) at 50 o C for 30 min.

Following 3x15 min washing in PBS-T, the membranes were re-probed with 2.5 µg/ml of a mouse anti-β-actin monoclonal antibody (Sigma).

Western blot for Ror1 was performed essentially as described above with minor modifications [START_REF] Daneshmanesh | Ror1, a cell surface receptor tyrosine kinase is expressed in chronic lymphocytic leukemia and may serve as a putative target for therapy[END_REF]. Ror1 affinity purified antibody (R&D Systems) was used at a concentration of 1µg/ml as the primary antibody.

Results

CLL cells can be transfected with siRNA at a high efficiency

In initial experiments we validated the efficiency of our siRNA transfection method. The siGLO® (Dharmacon) is a stable, fluorescent, non-targeting control siRNA used to establish optimum conditions for transfection for maximum siRNA uptake. CLL cells from 6 different patients were transfected with siGLo® siRNA and examined by flow cytometry at various time points after transfection for percentage of fluorescent cells. The results are provided as supplementary information. Using the transfection procedure described in the Materials and Methods section, >90% of CLL cells had initial uptake of siGLO® siRNA and the percentage of fluorescent cells diminished slowly over a period of 72 hours.

Silencing of Ror1 and fibromodulin and their effects on CLL cells CLL cells in vivo are characterized by accumulation of mature lymphocytes that are resistant to apoptosis, but they tend to rapidly undergo spontaneous apoptosis in vitro [START_REF] Collins | Spontaneous programmed death (apoptosis) of B-chronic lymphocytic leukaemia cells following their culture in vitro[END_REF].

CD40 expression has been found on 90% to 100% of CLL cells and in vitro, activation of CD40 by [START_REF] Grdisa | Influence of CD40 ligation on survival and apoptosis of B-CLL cells in vitro[END_REF][START_REF] Pedersen | Protection of CLL B cells by a follicular dendritic cell line is dependent on induction of Mcl-1[END_REF]. We transferred the CLL cells on to a monolayer of CD40L-transfected fibroblasts, 6 hours after transfection, to minimize spontaneous apoptosis that tended to obscure the effects of Ror1 and fibromodulin silencing. We used the same primers as for the human cells for RT-PCR analysis of the NIH-3T3 murine fibroblast cell lines, transfected with human CD40L. With these primers, no PCR product corresponding to Ror1 and fibromodulin was identified, ensuring that the expression of these genes was detected from the CLL cells rather than the feeder cells (data not shown). The feeder layer therefore facilitated investigating the effect of Ror1 and fibromodulin silencing without spontaneous apoptosis in vitro.

Transfection of CLL cells with siRNA against fibromodulin or Ror-1 resulted in a significant decrease in the expression of Ror1 or fibromodulin gene as measured by RT-PCR. Figure 1 demonstrates the silencing of Ror1 and fibromodulin as detected by RT-PCR. A major downregulation of Ror1 and fibromodulin mRNA but not of the housekeeping (control) gene was observed at 24 hours following siRNA transfection. The siRNA-mediated downregulation was quantified by real-time PCR. Figure 2 represents CLL cells from three patients and in each case the gene expression of untransfected and siRNA-transfected cells are compared to that of by cells transfected with the control, non-silencing siRNA, which is considered to be 100%. The real-time PCR results demonstrated that the siRNA constructs silenced Ror1 and fibromodulin gene expression by 75->95% (Figure 2). Downregulation of fibromodulin and Ror1 could also be demonstrated at the protein level by Western blotting. Figure 3a demonstrates downregulation of the fibromodulin bands in 3 CLL patients following silencing with FMOD-specific siRNA (lanes 3-5) compared to untransfected (lane 1) or control, non-silencing siRNA (lane 2) treated cells. Beta-actin levels, used as a control is below. Similar results were also noted for Ror1 following transfection of CLL cells from three patients with Ror1-specific siRNA (Figure 3b). The 105KDa band corresponding to Ror1 was detected in untransfected and control-transfected CLL cells (Lanes 1 and 2 respectively) but was absent in CLL cells treated with Ror1-specific siRNA. Levels of β-actin are The level of apoptosis induction following siRNA treatment varied between different patients as was the extent of spontaneous apoptosis but in all 7 patients level of apoptosis was markedly and significantly (p≤ 0.05-0.001; Student's paired, two-tailed t-test) higher with all 3 siRNAs than apoptosis with the control non-silencing siRNA (Figure 5). Time kinetics indicated that apoptosis was noted as early as 4 hours after transfection and was continuing at the end of 48 hours (data not shown).

To examine whether the effect of silencing Ror1 and fibromodulin was specific to CLL cells we tested the effect of siRNA transfection on PBMC of healthy donors and four human fibroblast cell lines. PBMC from two healthy donors were subjected to nylon wool filtration and both the B cell rich fraction recovered from the column and the T cell rich fraction in the effluent were transfected with siRNA. As seen in Figure 6, neither the siRNA pool against fibromodulin nor Ror1 induced apoptosis (<10%) in normal B and T cell enriched cell fractions. These results were in keeping with our earlier report that expression of fibromodulin and Ror1 is virtually absent on normal PBMC [START_REF] Daneshmanesh | Ror1, a cell surface receptor tyrosine kinase is expressed in chronic lymphocytic leukemia and may serve as a putative target for therapy[END_REF][START_REF] Mikaelsson | Fibromodulin, an extracellular matrix protein: characterization of its unique gene and protein expression in Bcell chronic lymphocytic leukemia and mantle cell lymphoma[END_REF]. Of the four FMOD+ Ror1+ human fibroblast cell lines tested only HFFF-PI 6 demonstrated induction of apoptosis following transfection with pooled siRNA against fibromodulin and Ror1 (Figure 7). Interestingly, this is the only cell line of the four tested that is of fetal origin.

Discussion

Our previous studies have demonstrated that Ror1 and fibromodulin are differentially expressed by CLL cells [START_REF] Daneshmanesh | Ror1, a cell surface receptor tyrosine kinase is expressed in chronic lymphocytic leukemia and may serve as a putative target for therapy[END_REF][START_REF] Mikaelsson | Fibromodulin, an extracellular matrix protein: characterization of its unique gene and protein expression in Bcell chronic lymphocytic leukemia and mantle cell lymphoma[END_REF]. Studies by other investigators have revealed that fibromodulin may serve as a tumor -associated antigen that is naturally processed and presented in CLL. Mayr et al demonstrated that stimulation of autologous lymphocytes with native or CD40-ligand treated CLL cells resulted in the expansion of T cells capable of recognizing antigen peptides derived from fibromodulin [START_REF] Mayr | Fibromodulin as a novel tumor-associated antigen (TAA) in chronic lymphocytic leukemia (CLL), which allows expansion of specific CD8+ autologous T lymphocytes[END_REF].

RT-PCR examination by Giannopolous et al demonstrated that fibromodulin is one of the prominent TAAs expressed by CLL cells [START_REF] Giannopoulos | Expression of RHAMM/CD168 and other tumor-associated antigens in patients with B-cell chronic lymphocytic leukemia[END_REF] while Hus et al demonstrated that vaccination with autologous dendritic cells loaded with CLL cell lysate increased the frequency of fibromodulin-reactive T cells [START_REF] Hus | Vaccination of B-CLL patients with autologous dendritic cells can change the frequency of leukemia antigen-specific CD8+ T cells as well as CD4+CD25+FoxP3+ regulatory T cells toward an antileukemia response[END_REF]. These reports indicate that fibromodulin may be relevant as a target for active immunotherapy which is further reinforced by the findings of our present study that fibromodulin appears to be important for the survival of CLL cells. However, the ubiquitous expression of fibromodulin in connective tissues necessitates caution due to the risk of autoimmune reactions. In spite of these reports however, the role of fibromodulin in the pathophysiology of CLL remains unclear. One of the putative mechanisms maybe through the sequestration of TGF-β [START_REF] Lotz | Transforming growth factor beta as endogenous growth inhibitor of chronic lymphocytic leukemia B cells[END_REF]. Our preliminary experiments with the addition of neutralizing antibody to TGF-β or fibromodulinpositive fibroblasts could not confirm this hypothesis (data not shown). Alternatively, fibromodulin may facilitate the interaction of CLL cells with cells of the marrow stroma, peripheral blood derived "nurse-like" cells and extramedullary mesenchymal cells in vivo (Burger andKipps 2002, Burger, et al 2000) that was exemplified by the interaction of the CLL cells with the CD40L-transfected 3T3 fibroblast monolayer in our experiments.

Like fibromodulin, little is presently known about the role of Ror1 in the pathophysiology of CLL.

However, a recent study by Fukuda et al [START_REF] Fukuda | Antisera induced by infusions of autologous Ad-CD154-leukemia B cells identify ROR1 as an oncofetal antigen and receptor for Wnt5a[END_REF] provides evidence that Ror1 may serve as a receptor for Wnt5a signaling in CLL cells. In this report, the authors demonstrated that the extracellular domain of Ror1 could bind Wnt5a and coexpression of Ror1 and Wnt5a in HEK cells lead to the activation of NF-κB. Additionally the viability of CLL cells cocultured with Wnt5a-expressing CHO cells was significantly greater than those cultured with CHO cells alone. This survival advantage was neutralized by the addition of antisera against Ror1. In yet another study, high-throughput RNAi screening was utilized to identify kinases and phosphatases in the human genome that are essential to the survival of cancer cells [START_REF] Mackeigan | Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance[END_REF]. In the screen, 650 known and putative kinases were targeted by RNAi in HeLa cervival carcinoma cells.

Of the 73 genes that were overall identified as survival kinases, Ror1 was one of four most potent survival kinases. Greater than three-fold increase in apoptosis was noted in HeLa cells, 72 hours after silencing of Ror1. These results taken cumulatively with those of the present study suggest that Ror1 may be an important survival kinase and targeting Ror1 using small molecules, therapeutic monoclonal antibodies or active immunotherapy approaches may be of utility for the treatment of CLL.

  aliquoted and cryopreserved at -20 o C according to manufacturer's instructions. Primers for polymerase chain reaction (PCR) were obtained from CyberGene AB, Novum Research Park, Sweden. The sequence of siRNA and PCR primers are provided in Table

  natural ligand, CD40L, or by activating monoclonal antibodies, has been shown to support the survival and expansion of B-CLL cells

  Figure5).

Figure legends Supplemental Figure 1 :

 1 Figure legends

Figure 1 :

 1 Figure 1: Downregulation of fibromodulin and Ror1 mRNA as examined by RT-PCR: CLL cells were transfected with siRNA against fibromodulin (FMOD) or Ror1 as described in Materials and Methods. Six hours later they were cultured on a monolayer of CD40L-transfected murine NIH 3T3 fibroblasts. After 24 hours, the CLL cells were collected and examined for expression of target mRNA by RT-PCR. Results of one representative experiment are shown. RPLP0 is a housekeeping gene that has been used as a control.

Figure 2 :

 2 Figure 2: Relative quantitation of fibromodulin and Ror1 silencing by real-time PCR: CLL cells from 3 patients (black, grey and white bar, respectively) were transfected with siRNA and cultured as described in Figure 1. Downregulation of fibromodulin (FMOD) (A) and Ror1 (B) mRNA was examined by real-time PCR. The samples were subject to 35 cycles for fibromodulin and Ror1 and 30 cycles for the housekeeping keeping gene (β-actin). The expression of fibromodulin and Ror1 genes were normalized against the housekeeping gene as an internal standard. Gene expression by CLL cells transfected with control, non-silencing siRNA was regarded as 100% and gene expression of all other cells were expressed relative to it. Results of one representative experiment in each patient are depicted. The star denotes a sample in which fibromodulin mRNA could not be detected even after 40 cycles.

Figure 3

 3 Figure3CLL cells treated with siRNA specific for fibromodulin (3A) or Ror1 (3B) show down regulation of the specific protein: CLL cells from three patients were transfected with FMODspecific (3A) or Ror1-specific (3B) siRNA. 48 hours later, the cells were lysed and examined by Western blotting as described in Materials and Methods. In each case, lane 1 and lane 2 represent untransfected and control siRNA-transfected cells while lanes 3-5 represent CLL cells

Figure 4 :

 4 Figure 4: Changes in morphology of CLL cells following silencing of fibromodulin and Ror1: CLL cells were transfected with pools of three siRNAs against fibromodulin (FMOD) and Ror1 respectively. Six hours after transfection, the cells were transferred to monolayers of CD40Ltransfected NIH 3T3 fibroblasts. Cells were visualized by light microscopy (Carl Zeiss Axiovert, Germany) after 24 hours. Results of a representative experiment are shown. A-untransfected; B-control siRNA-transfected; C-Cells transfected with pooled FMOD-specific siRNA; D-Cells transfected with pooled Ror1-specific siRNA.

Figure 5 :

 5 Figure 5: Silencing of fibromodulin and Ror1 genes results in apoptosis of CLL cells: CLL cells were transfected with fibromodulin (A) and Ror1 (B) siRNA and examined for apoptosis by flow cytometry. Results with 6 patients are presented. Percentage of apoptotic cells (Annexin-V or Annexin-V/PI dual positive) is depicted on the Y-axis. The individual shaded bars each represent one patient's cells. The difference between each siRNA and untransfected cells or control siRNA was statistically significant (p≤ 0.05-0.001; Student's paired, two-tailed t-test).

Figure 6 :

 6 Figure 6: Viability of peripheral blood cells from normal donors is not affected by siRNA against fibromodulin and Ror1: Peripheral blood mononuclear cells of normal donors were separated into a B cell-enriched and T cell-enriched fraction by nylon wool filtration. Cells were transfected with pools of three control, non-silencing siRNAs or pools of three siRNAs against fibromodulin (FMOD) and Ror1 respectively. Cells were labelled with anti-CD3 antibody to confirm enrichment into CD3+ and CD3-fractions, respectively prior staining with Annexin-V and propidium iodide. Results with 2 normal donors are presented. Percentage of apoptotic cells (Annexin-V or Annexin-V/PI dual positive) is depicted on the Y-axis.

Figure 7 :Figure 1 :Figure 2 :Figure 3 Figure 3 Figure 4 :Figure 5 :Figure 6 :Figure 7 :

 712334567 Figure 7: Viability of human fibroblast cell lines after transfection with siRNA against fibromodulin and Ror1: Human fibroblast cells were transfected with pools of three control, non-silencing siRNAs or pools of three siRNAs against fibromodulin (FMOD) and Ror1 respectively. Cells were stained with Annexin-V and propidium iodide and analyzed by flow

Table 2 :

 2 Volumes and concentrations of reagents used for siRNA transfection

	Culture Dish	6-well plate	24-well plate	48-well plate
	Complete growth	800 μls	250 μls	200 μls
	medium
	Opti-MEM®	64 μls	32 μls	36 μls
	TransIT-TKO®	18 μls	9 μls	7 μls
	2 μM stock siRNA	18 μls	9 μls	7 μls

British Journal of Haematology

Acknowledgements

This work was supported by the EUCAAD 200755 funded by the European Community's Seventh Framework Programme, The Swedish Cancer Society, CLL Global Research Foundation, Cancer Society of Stockholm, Cancer and Allergy Foundation and the King Gustaf V Jubilee Fund.

author for peer review, but marked to be sent in Off-Line.

Conflict of interest

The authors declare that there is no conflict of interest.

Tables Table 1A: