Matthieu Delannoy

Guido Rychen

Agnès Fournier

Catherine Jondreville

Cyril Feidt

Effects of condensed organic matter on PCBs bioavailability in juvenile swine, an animal model for young children

Keywords: Soil, Bioavailability, NDL-PCBs, Children, Piglets, Organic matter

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction ________________________________________________________________

The exposure assessment to pollutants from contaminated soils is a critical issue in terms of safety management. Little reliable information is available on the pollutant transfer to target population through involuntary soil ingestion. Children may exhibit hand-to-mouth activities and according to their development they are particularly susceptible to hazardous chemicals. Indeed, the infant has an immature blood-brain barrier [START_REF] Grandjean | Developmental neurotoxicity of industrialchemicals[END_REF] and at various developmental stages, his metabolism pathways may differ from those of adults [START_REF] Vieira | Developmental expression of CYP2E1 in the human liver[END_REF], NRC, 2003). Concerning PCBs, cohorts studies (on 7 months of age, and at 4 years of age) reported a possible memory deficit following exposure [START_REF] Jacobson | Effects of prenatal PCB exposure on cognitive processing efficiency and sustained attention[END_REF][START_REF] Jacobson | Intellectual impairment in children exposed to polychlorinated biphenyls in utero[END_REF][START_REF] Faroon | Effects of polychlorinated biphenyls on the nervous system[END_REF][START_REF] Grandjean | Developmental neurotoxicity of industrialchemicals[END_REF]. Similar results were found following a perinatal exposure in rats [START_REF] Lilienthal | Sensitive periods for behavioral toxicity of polychlorinated biphenyls: determination by cross-fostering in rats[END_REF][START_REF] Corey | Effects of exposure to polychlorinated biphenyl (PCB) from conception on growth, and development of endocrine, neurochemical, and cognitive measures in 60 days old rats[END_REF], ATSDR, 2000), even at environmentally low doses [START_REF] Elnar | Neurodevelopmental and behavioral toxicity via lactational exposure to the sum of six indicator non-dioxin-like-polychlorinated biphenyls (Σ6 NDLPCBs) in mice[END_REF] and in monkeys [START_REF] Levin | Delayed spatial alternation deficits resulting from perinatal PCB exposure in monkeys[END_REF][START_REF] Schantz | Effects of perinatal PCB exposure on discrimination-reversal learning in monkeys[END_REF]. These neurodevelopmental and behavioral impacts were linked to non-dioxin like congeners [START_REF] Boix | Differential long-term effects of developmental exposure to polychlorinated biphenyls 52, 138 or 180 on motor activity and neurotransmission. Gender dependence and mechanisms involved[END_REF][START_REF] Boix | Developmental exposure to polychlorinated biphenyls 52, 138 or 180 affects differentially learning or motor coordination in adult rats. Mechanisms involved[END_REF]. Six of these non-dioxin like PCBs (NDL-PCBs) constitute the most abundant part of PCBs found in environmental matrices like soils, sediments or foodstuff [START_REF] Meijer | Global distribution and budget of PCBs and HCB in background surface soils: implications for sources and environmental processes[END_REF][START_REF] Anses | Avis de l'Agence française de sécurité sanitaire des aliments relatif à l[END_REF][START_REF] Efsa | Results of the monitoring of nondioxin-like PCBs in food and feed[END_REF] and are laid down in European regulation No 1259/2011 (European Commission, 2011). In this context and in order to perform a quantitative assessment of health risks, it appears of real importance to focus on exposure to NDL-PCBs via involuntary soil ingestion. The relative bioavailability (RBA) approach which relies on the ratio of the bioavailability of a compound in one exposure context to that in another exposure context appears to be a valuable way to perform this exposure assessment (NRC, 2003, USEPA, 2007a, USEPA, 2007b).

Chemosphere (104), Pages 105-112 10.1016/j.chemosphere.2015.06.098 2 CC BY-NC-ND 4.0 However, when considering the RBA of lipophilic organic soil bound compounds, highly variable results were obtained between mammals highlighting the major importance of the animal model [START_REF] Fries | Influence of soil properties on the uptake of hexachlorobiphenyls by rats[END_REF][START_REF] Saghir | Effect of organic carbon content, clay type, and aging on the oral bioavailability of hexachlorobenzene in rats[END_REF][START_REF] Ounnas | Relative bioavailability of soil-bound polycyclic aromatic hydrocarbons in goats[END_REF][START_REF] Fournier | Relative bioavailability to laying hens of indicator polychlorobiphenyls present in soil[END_REF], Jondreville et al., 2013, Feidt et al., in press). Considering the assessment of children's exposure after ingestion it appears that three animals are particularly used and pertinent to model children: rats, monkeys and swine. Concerning juvenile swine and rats contaminated soil uptakes have been also previously reported to be different [START_REF] Budinsky | A pilot study of oral bioavailability of dioxins and furans from contaminated soils: impact of differential hepatic enzyme activity and species differences[END_REF]. Indeed, gastrointestinal absorption differs between rats and swine regarding specific mechanisms driving absorption, as noticed for metallic ions [START_REF] Oomen | Effect of bile type on the bioaccessibility of soil contaminants in an in vitro digestion model[END_REF][START_REF] Casteel | Estimation of relative bioavailability of lead in soil and soil-like materials using young swine[END_REF], USEPA, 2007a[START_REF] Denys | In vivo validation of the unified barge method to assess the bioaccessibility of arsenic, antimony, cadmium, and lead in soils[END_REF]. The juvenile swine appears to be a better model than rodent one. Indeed, the relative similarity of physiology, growth and mechanisms of absorption between young humans and swine led to an increasing use of this animal as a model of oral bioavailability and its application in risk assessment studies [START_REF] Casteel | Estimation of relative bioavailability of lead in soil and soil-like materials using young swine[END_REF], USEPA, 2007a[START_REF] Budinsky | A pilot study of oral bioavailability of dioxins and furans from contaminated soils: impact of differential hepatic enzyme activity and species differences[END_REF]. Such an approach consisting a combination of both RBA methodology and swine model has already been performed for trace metals [START_REF] Denys | In vivo validation of the unified barge method to assess the bioaccessibility of arsenic, antimony, cadmium, and lead in soils[END_REF].

Overall assessment of the risk linked to contaminated sites is mostly carried out in response to toxicity to humans and in estimating this, the oral bioavailability of the contaminants is a key parameter. Soil particles that are ingested by mammals are exposed to gastro-intestinal fluids, enzymes and salts [START_REF] Oomen | Effect of bile type on the bioaccessibility of soil contaminants in an in vitro digestion model[END_REF]. Bile acids, pancreatic enzymes, the dietary environment may play major roles in the extraction of organic compounds from soil in the gastro-intestinal lumen [START_REF] Oomen | Effect of bile type on the bioaccessibility of soil contaminants in an in vitro digestion model[END_REF]. It is also known that physical/chemical properties of the soil, like porosity, pH, clay, and organic matter (OM) content, along with aging, may impact both sequestration and/or release of contaminants [START_REF] Saghir | Effect of organic carbon content, clay type, and aging on the oral bioavailability of hexachlorobenzene in rats[END_REF]. The characteristics of the soil OM is thought to be one of the most important factors that determines the strength of interactions between the soil fractions and the organic lipophilic pollutants [START_REF] Pignatello | Mechanisms of slow sorption of organic chemicals to natural particles[END_REF]. Thus, the sorption-desorption process would be driven by OM characteristics like condensation [START_REF] Pignatello | Soil organic matter as a nanoporous sorbent of organic pollutants[END_REF]. Furthermore, ample evidence has been gathered in vitro that adsorption of lipophilic chemicals to soil particles is positively correlated to OM content [START_REF] Ping | Phenanthrene adsorption by soils treated with humic substances under different pH and temperature conditions[END_REF].

The present study was conducted to assess the effects of different OM characteristics on the oral RBA of NDL-PCBs sequestrated in two artificial soils amended with 1% of organic carbon in the juvenile swine. Assessment of the relevance of the target tissues and the range of linearity of the response to oral exposure were tested first.

Material and Methods ________________________________________________________

Experimental design

In order to assess the impact of soil OM condensation on RBA of NDL-PCBs the following study was designed (Fig. 1). The first step of the study consisted in assessing the linearity of the NDL-PCBs dose-response pattern. (Fig. 1). The following step was aimed at evaluating the impact of OM condensation on the concentration of NDL-PCBs in tissues. Two well-characterized sources of OM were selected to illustrate opposite degrees of condensation: fulvic acid and activated carbon (Fig. 1). RBA was estimated by comparing the levels obtained in target tissues (liver, adipose tissue, muscle) of spiked soilfeed or corn oil feed [START_REF] Wittsiepe | Bioavailability of PCDD/F from contaminated soil in young Goettingen minipigs[END_REF].

Dosing material

Spiked oil

Spiked corn oil containing graded levels of Aroclor 1254 (Sigma-Aldrich, Supelco, Saint-Louis, USA) were used in the first step of the study. Four doses of Aroclor 1254 were prepared in order to achieve these four exposure doses: 3000 ng Aroclor 1254 per g of spiked oil (1X), 9000 ng g -1 (3×), 15 000 ng g -1 (5×), 21 000 ng g -1 (7×) (Fig. 1). These exposure doses corresponded respectively to 950 ng of NDL-PCBs per g of spiked oil (1×), 2800 ng g -1 (3×), 4700 ng g -1 (5×) and 6550 ng g -1 (7×). The reference spiked oil was contaminated with 19 200 ng Aroclor 1254 per g of spiked oil corn corresponding to 6000 ng of NDL-PCBs per g of spiked oil. Corn oil (Sigma Aldrich) was spiked with an adequate volume of a hexane solution of Aroclor 1254 (Sigma Aldrich, Supelco). Secondly, hexane was rotary-evaporated over 2 h and let evaporating under an extractor hood overnight. The difference of weigh of spiked corn oil before and after spiking and evaporation was assessed to determine persistent hexane was less than 1:10 000.

Artificial soils and spiking

Three artificial soils were prepared. They were based on a standard soil (SS) which was prepared according to Organization for Economic Cooperation and Development (OECD) guideline 207 (OECD, 1984). This soil fulfilled the composition and pH of an OECD artificial soil but the sphagnum peat was not introduced to avoid sorption competition between the different OM phases. One of the studied soils was SS. To identify the incidence of OM on NDL-PCBs bioavailability, two well-characterized OMs were used. fulvic acid was purchased from the International Humic Substances Society (Suwannee River II, IHSS, St. Paul) and introduced in a part of SS to form a fulvic acid amended artificial soil (FA). Secondly activated carbon (Activated carbon, Sigma) was introduced in a part of SS to form an activated carbon amended artificial soil (AC). Fulvic acid and activated carbon were introduced in FA and AC soils respectively in order to achieve a mass concentration Chemosphere (104), Pages 105-112 10.1016/j.chemosphere.2015.06.098 3 CC BY-NC-ND 4.0 of 1% of the organic carbon regarding the whole dry matter (DM) of each soil. The soils were mixed manually thoroughly for 5 min until a homogeneous color soil was reached.

An adequate amount of water and PCBs were introduced at the same time in the three artificial soils which were stored in amber glass container. The spiking solution was prepared using milliQ water and the adequate volume of Aroclor 1254 dissolved in methanol to achieve 19 200 ng g -1 of DM and a moisture content of 17.5%. Expected exposure concentrations in NDL-PCBs are: 7.3 ng kg BW -1 (PCB 28), 206 ng kg BW -1 (PCB 52), 308 ng kg BW -1 (PCB 101), 223 ng kg BW -1 (PCB 138), 145 ng kg BW -1 (PCB 153) and 26 ng kg BW -1 (PCB180). The same amount of spiking solution was introduced in 85 g of DM of each artificial soil. The soils were mixed manually thoroughly for 10 min until a homogeneous color was reached and methanol was evaporated under an extractor hood overnight. Then, additional water was added to achieve 17.5% of moisture and soils were thoroughly mixed during 10 additional minutes. The containers were mixed on a rolling mill for 24 h and then placed in a 10 °C room in static position during 4 weeks to allow correct impregnation aging of NDL-PCBs in soil matrices.

Dough ball manufacturing

Animals were exposed to Aroclor 1254 for 10 d. The daily doses and ration were based on the individual body weight (BW). The oral delivery doses were achieved by transferring a calculated weight of contaminated soilmatrices (200 mg of DM per kg of BW ± 5%) in the depression of one dough ball. Dough ball were manufactured by squeezing repeatedly 8 g of commercial pellets (Porcelet Super VE 25 kg, Lorial, Laxou, France) moistened with an adequate amount of milliQ water to obtain dough-like consistency. For the dough ball containing spiked oil, 4 g of commercial pellets were pulverized using Analytical mill A 11 (IKA®, Staufen, Germany), mixed with spiked oil and then placed in the depression of 4 g of commercial pellets moistened by distilled water.

Animals

Each step of the experiment was performed with a distinct group of juvenile swine. Crossbred (Pietrain × Youna®) castrated male swine, weaned at 28 d were kept in individual stainless-steel metabolism cages with wire floors and nipple waterers which delivered fresh tap water ad libitum. The animals were fed an age-appropriate commercial pelleted swine diet once daily at a level of 5% of body weight (Porcelet Super VE 25 kg). All animals were acclimated to the facility during 5 d. One dough ball containing the appropriate dose for acclimation or exposure was hand fed 3 h prior to ration distribution. An ambient temperature of 26-27 °C and a nycthemeral cycle (10 h light) were maintained in the animal room. Human care of swine and administration of spiked artificial soils and oils were carried out in compliance with the guidelines of the French Ministry of Agriculture for Animal Research..

Assessment of the doseresponse linearity

Eight swine were acclimated to the facilities and adapted to progressive oil supplementation in hand fed dough balls during 5 d (BW 8.0 ± 0.5 kg). Two swine (n = 2) were assigned to each treatment groups 1×, 3×, 5× and 7×. The exposure period of 10 d consisted of a daily administration of one moistened dough ball containing the appropriate quantity of one of the four spiked oils to achieve the expected exposure doses.

Assessment of the oral bioavailability

Fourteen swine were acclimated to the facilities and adapted progressively to oil supplementation and geophagia during 5 d (body weight 8 ± 0.5 kg). Three swine (n = 3) were assigned to each treatment group exposed to one of the four exposure matrices: SS, AC, FA or spiked oil (SO). Two control swine (C) were exposed to a clean standard soil (n = 2) through clean dough balls. The exposure period consisted in a daily administration of one moistened dough ball containing the appropriate quantity of one of the four matrices in order to achieve the daily exposure dose (1200 ng of NDL-PCBs per kg BW).

Sampling and analysis of biological samples

After exposure period piglets were slaughtered by electronarcosis immediately followed by exsanguination.

After laparotomy, the whole liver was collected after exeresis of the gallbladder, as well as pericaudal adipose tissue and rib-eye muscle (Longissimus dorsi). All samples were kept in individual boxes and chopped. Then, samples were freezed-dried and pulverized thoroughly using Analytical mill (A 11, IKA®, Staufen, Germany). NDL-PCBs were analyzed by gas chromatography coupled with a mass spectrometer (GC-MS). These analyzes involved three successive steps: extraction, purification and quantification. Chemicals used in this study were pesticide residue analytical grade.

Extraction

Before extraction, 6 13 C-labeled NDL-PCBs from LGC Standard (Molsheim, France) were added to the various samples studied. After spiking, the solid samples were transferred into accelerated solvent extraction (ASE) cells. Pressure and temperature were set at 100 bar and 120 °C respectively. The extraction solvent was a mixture composed of toluene (Sigma-Aldrich, Fluka) and acetone (Sigma-Aldrich, Fluka) at 70:30 (v/v), and three successive extraction cycles (5 min each) were performed by ASE device (ASE 350, Dionex, Sunnyvale, USA). The extracts were redissolved in cyclohexane (Sigma-Aldrich, Fluka) for sample clean up after rotary evaporation.

Purification

The purification process included a glass column (length 50 cm; ID 2.5 cm) which was composed of successive layers including 6.0 g (±0.10 g) of Florisil (Promochem, Fluka) added with 20 mL of cyclohexane, 5.0 g (±0.10 g) of anhydrous sodium sulfate (Sigma-Aldrich, Wesel, Germany), 4 g (±0.1 g) of pure silica gel (Sigma-Aldrich, Fluka) added with 20 mL of cyclohexane (Sigma-Aldrich, Fluka), 8.0 g (±0.20 g) of silica gel acidified with sulfuric acid (VWR International, Analar, Poole, England) (22%) added with 20 mL of cyclohexane, 10.0 g (±0.25 g) of silica gel acidified with sulfuric acid (44%) added with 20 mL of cyclohexane and 5.0 g (±0.10 g) of anhydrous sodium sulfate.

Quantification

CC BY-NC-ND 4.0

PCBs analyzes were performed by GC-MS (7890A, Agilent Technologies, Santa Clara, USA). The mass spectrometer was set at a resolution of 10 000, in electron ionization mode (70 eV electron energy) (5975C, Agilent Technologies, Santa Clara, USA). Single Ion Monitoring (SIM) was used to record the two most abundant signals. A DB5MS (30 m × 0.25 mm × 0.251 m) capillary column from Agilent J&W (Agilent Technologies) was used in splitless mode. The GC temperature program for PCBs analysis was the following: 110 °C (1 min), 20 °C min -1 to 200 °C (0 min) and 2 °C min -1 to 230 °C and 5 °C min -1 to 270 °C and 10 °C min -1 to 300 °C (5 min). Signals were integrated using Chemstation (chemstation E.02.00, Agilent Technologies, Santa Clara, USA). To avoid any over estimation the control samples were injected at the beginning of the experiment.

Calculations and statistical analyses

Assessment of linearity

Linearity of the dose response of NDL-PCBs concentration to ingested NDL-PCBs was assessed by regression analysis performed by means of the REG procedure of SAS (9.1, SAS Institute, Cary, NC, USA). The linear and the quadratic effects of PCBs ingested (ng g -1 BW d -1 ) were tested. Where not significant (P > 0.10) the quadratic term was removed from the model and the response was declared linear.

Incidence of condensation of OM on bioavailability

In order to evaluate the incidence of contaminated matrices on bioavailability a variance analysis were performed. For each congener, values below the limit of quantification (LOQ) were replaced by LOQ/2 to avoid missing data. Statistical analyzes were carried out using SAS (9.1, SAS Institute). Each piglet was considered as an experimental unit. Concentrations of each NDL-PCB congener in liver, muscle, and adipose tissue were analyzed using the GLM procedure. The model included the exposure media (AC, FA, SS, SO, n = 3 and C, n = 2). A Tukey-Kramer test was used for comparison of means. Differences were considered significant for P < 0.05.

Relative bioavailability factors (RBA) calculation

In order to compare the bioavailability from the two exposure media (soil and oil), the relative bioavailability in a selected tissue was calculated as the ratio of the bioavailability in soil and the bioavailability in oil, adapted from a method preliminary described [START_REF] Wittsiepe | Bioavailability of PCDD/F from contaminated soil in young Goettingen minipigs[END_REF]. Estimation of bioavailability in one matrix of exposure m in a selected tissue t was calculated as the ratio of the mass of a PCB-NDL-congener p in the tissue to the administered mass of the same congener from soil or oil multiplied by 100%. Because no significant differences were found considering ratio of masses of sampled tissues on BW each treatment group, it was assumed that masses of each tissue were similar. Calculation of RBA was performed using the ratio of the adjusted means of concentrations of NDL-PCBs congener in tissues after a soil and an oil exposure after subtracting concentration founded in controls.

As the linear model was the most efficient to fit experimental data for the range of doses used in each group, dose-responses were proven linear. Accordingly to this linearity 1/concentrations in adipose tissue of PCB 101, PCB 138, PCB 153 and PCB 180 2/and exposure doses were normalised by dividing them by the maximum daily dose given for one treatment (soil or oil). After this process, slope of the linear regression linking normalised concentrations in adipose tissue and normalised exposure dose was calculated for each treatment group. Relative bioavailability estimates were provided using the following equation:

𝑅𝐵𝐴 𝑚,𝑡,𝑝 = 𝐶 𝑚,𝑡,𝑝 𝑠𝑜𝑖𝑙-𝐶 𝑐𝑜𝑛𝑡𝑟𝑜𝑙𝑠,𝑡,𝑝 𝐶 𝑚,𝑡,𝑝 𝑜𝑖𝑙-𝐶 𝑐𝑜𝑛𝑡𝑟𝑜𝑙𝑠,𝑡,𝑝 .

Results ____________________________________________________________________

The animals remained healthy throughout the experiment.

During the exposure period, daily ingestion and growth performance were unaffected by treatments. Thus the average daily gain was 0.24 ± 0.03 kg of BW d -1 (mean ± SD, P > 0.10) in the first step and 0.27 ± 0.04 kg of BW d -1 for the second one (mean ± SD, P > 0.10). At the end of first experiment piglets weighed 9.04 ± 1.22 kg of BW (mean ± SD, P > 0.10), and after the follow up one 9.98 ± 0.72 kg of BW (mean ± SD, P > 0.10).

Assessment of the dose-response linearity

Limits of quantification are presented in Table 1. Since levels of PCB 28 were found to be mainly below LOQ, this congener was not further considered. Data concerning one animal (dose 5×) was not considered in the statistical analysis because of abnormally high concentrations. CC BY-NC-ND 4.0 whereas the quadratic terms were not significant (P > 0.10, Table 1). No conclusions could be reached considering PCB 180 results because of integration issues in some chromatograms. Considering the coefficient of determination (R 2 ) equal or over than 0.77 for subcutaneous fat, 0.77 for hepatic tissue and 0.71 for muscular one, linear equations provided reliable predictions of dose-response relationships in these tissues in the tested range of dose (3000-21 000 ng of Aroclor 1254 per kg of BW) (Table 1, Fig. 2). The intercepts did not differ from zero (P > 0.10) except for 3 NDL-PCBs in adipose tissue. Higher slopes of dose-responses were found in hepatic tissues when compared to muscles or adipose tissue (Table 1, Fig. 2) 1 (respective curves are fitted by full black lines, grey and white ones).

Incidence of condensed OM on RBA

Regardless of the contamination media, PCB 28 levels were below LOQs and were not included in the data set. NDL-PCBs concentrations in all analyzed tissues (hepatic, muscular and subcutaneous fat) were above LOQs in C for all other congeners. The highest levels of NDL-PCBs were recovered in tissue samples from piglets contaminated by SO, SS and FA spiked with the same amount of Aroclor 1254 (6000 ng g -1 of soil DM corresponding to a similar exposure dose of 1200 ng kg -1 of BW). Thus, the lowest levels of NDL-PCBs were observed in the AC and in the C groups.

The ratio of means by treatment indicated that NDL-PCBs levels in tissues after AC treatment were higher than after C exposure (1.3-5.2 times higher) and much lower than the SO reference tissues (5.2-9.8 times lower). Two distinct groups of treatments composed by "SO, SS, FA" on the one hand and "AC, CS" on the other one (P < 0.05) could be differentiated (Table 2). The exposure media effect was significant (P < 0.02) except in liver for PCB 52. Higher levels were found in liver than in muscles (1.4-1.7 times higher considering ratios between sum of 5 NDL-PCBs) and in adipose tissue (1.2-1.4 times) (Table 2). Calculated RBA factors were estimated for the NDL-PCBs sum (5 NDL-PCBs) (Table 2).

Discussion ________________________________________________________________

Although the historic of contamination of the experimental animals were known, most control tissues presented quantifiable levels of NDL-PCBs (above LOQs, Table 1). [START_REF] Meijer | Global distribution and budget of PCBs and HCB in background surface soils: implications for sources and environmental processes[END_REF]. Thus, noticeable levels of PCBs were found in several environmental matrices (soil and sediments) without a known history of contamination [START_REF] Meijer | Global distribution and budget of PCBs and HCB in background surface soils: implications for sources and environmental processes[END_REF].

The experimental design of this RBA study required a validation step. Indeed, the need to determine the doseresponse pattern in order to evaluate the representativeness of final concentrations of NDL-PCBs in tissues related to an ingested dose of contaminants appeared as a major point. In fact, it is known that ample variability exists between individuals among applied doses (USEPA, 2007a). This variability tends to increase as the dose increases, resulting to a rise of variance. Among factors that could lead to the increase of the variance, metabolism induction which is depending on individual susceptibility and the applied dose, is known to be one of the most important [START_REF] Devito | Comparative ability of various PCBs, PCDFs, and TCDD to induce cytochrome P450 1A1 and 1A2 activity following 4 weeks of treatment[END_REF][START_REF] Tang | Assessment of the bioaccessibility of polycyclic aromatic hydrocarbons in soils from Beijing using an in vitro test[END_REF]. The linearity of the dose-response in the tested range of doses validates the correspondence between the ingested doses and the final concentrations of NDL-PCBs in the studied organs.

No significant different behaviors could be noticed between PCBs from SS, FA, SO whereas far lower concentrations were found after AC treatment. Since OC content were equal for FA and AC treatments, this study reveals that the quality of the OM pilots the retention of PCBs during the digestive process in piglets. It demonstrates therefore that carbon organic content should be analyzed in regards with characteristics of this OM to determine potentialities of bioavailability reduction of this media. This study also highlights that poorly condensed OM, like fulvic acids, does not significantly reduce bioavailability compared to the reference matrix, SO. Some hindsight concerning bioavailability of pharmaceutical apolar compounds like furosemide tends to show that this sort of OM should even increase bioavailability of such compounds [START_REF] Agarwal | Complexation of furosemide with fulvic acid extracted from shilajit: a novel approach[END_REF][START_REF] Agarwal | Enhancement of the dissolution and diuretic effect of furosemide through a novel complexation with humic acid extracted from shilajit[END_REF]. However no statistical evidence was achieved to confirm or infirm this hypothesis comparing SS and FA treatments. Activated carbon is a manufactured and clean type of black carbon and is often used to model this carbonaceaous, ubiquitous and highly condensed organic material [START_REF] Zimmerman | Addition of carbon sorbents to reduce PCB and PAH bioavailability in marine sediments: physicochemical tests[END_REF], Cornelissen et al., 2006[START_REF] Saghir | Effect of organic carbon content, clay type, and aging on the oral bioavailability of hexachlorobenzene in rats[END_REF]. Black carbon is already known to provide strong binding with apolar contaminants like PAHs resulting in a physical trapping and reducing desorption processes and bioavailability to limbic and mammals [START_REF] Pikaar | Sorption of organic compounds to activated carbons. Evaluation of isotherm models[END_REF]. Activated carbon is also suggested as an efficient way to remediate contaminated areas by geosorbing apolar compounds [START_REF] Zimmerman | Addition of carbon sorbents to reduce PCB and PAH bioavailability in marine sediments: physicochemical tests[END_REF], Cornelissen et al., 2006[START_REF] Jakob | PAH-sequestration capacity of granular and powder activated carbon amendments in soil, and their effects on earthworms and plants[END_REF]. Nanoporosity of black carbon has been previously demonstrated as responsible for sorption of pollutants [START_REF] Pignatello | Soil organic matter as a nanoporous sorbent of organic pollutants[END_REF]). An industrial and highly nanoporous activated carbon introduced in a simple artificial soil, was employed during the current experiment to evaluate potentialities of reduction offered by this kind of media during the digestive process. It appeared that activated carbon dramatically reduced RBA of NDL-PCBs resulting to less considerably exposition to these pollutants through direct ingestion of contaminated soil. However this However this apparently heavy sequestration correspond to an activated carbon particularly well-known for its notable surface specific area, far higher than commonly observed in black carbon [START_REF] Koelmans | Black carbon: the reverse of its dark side[END_REF] and simple artificial soils. The total organic carbon chosen (1%) ranges in the limit low concentrations found in soils whatever land use [START_REF] References _________________________________________________________________ Agarwal | Is black carbon a better predictor of polycyclic aromatic hydrocarbon distribution in soils than total organic carbon?[END_REF]. Median black carbon as a fraction of total organic carbon content (BC/TOC) has been previously reported to be 4% in soils [START_REF] Cornelissen | Extensive sorption of organic compounds to balck carbon, coal, and Kerogen in sediments and soils: mechanisms and consequences for distribution, bioaccumulation, and biodegradation[END_REF]. However much higher levels were found in fire-impacted soils where black carbon reached 30-45% of total organic carbon [START_REF] Cornelissen | Extensive sorption of organic compounds to balck carbon, coal, and Kerogen in sediments and soils: mechanisms and consequences for distribution, bioaccumulation, and biodegradation[END_REF]. These considerations let us considerate as possible such levels of organic carbon and black carbon in natural soils. PCBs concentrations employed correspond to 1000 folds medium known-levels concentrations found in soils without historic of contamination (19 500 pg g-1) [START_REF] Meijer | Global distribution and budget of PCBs and HCB in background surface soils: implications for sources and environmental processes[END_REF]. These concentrations correspond also to those found in some industrial areas [START_REF] Schuhmacher | Levels of PCDD/Fs, PCBs, and PCNs in soils and vegetation in an area with chemical and petrochemical industries[END_REF]. Therefore this huge reduction of bioavailability may be overestimated comparing to what may occur in naturally contaminated soils.

Thus, the main interest of this study was to simulate extreme conditions with a same amount of organic carbon (OC) either in the form of AC or in the form of FA. No other OM was used during processing of the artificial soils in order to ensure no competition effect between different phases of OM [START_REF] Burgess | Effects of different forms of organic carbon on the partitioning and bioavailability of nonylphenol[END_REF]. Indeed, sorption of activated carbon in environmental matrices could be weaker than those reported using activated carbon alone. Blockage of sorption of lipophilic apolar contaminants sites in complex matrices by several compounds like natural organic matter [START_REF] Pignatello | Effect of natural organic substances on the surface and adsorptive properties of environmental black carbon (char): attenuation of surface activity by humic and fulvic acids[END_REF] or lipids components [START_REF] Kwon | Effect of natural organic substances on the surface and adsorptive properties of environmental black carbon (char): pseudo pore blockage by model lipid components and its implications for N2-probed surface properties of natural sorbents[END_REF] are known to decrease sorption in environmental matrices.

Conclusion ________________________________________________________________

Characteristic of the OM pilots the retention of PCBs during the digestive process in piglets. Indeed, it appears that condensed OM (AC) strongly reduces bioavailability whereas the less condensed one (FA) does not seem to have a significant effect. These elements are of great interest for further investigations in terms of risk assessment in contaminated areas

Figure 1 .

 1 Figure 1. Animal management and experimental design of the study

Figure 1 .

 1 Figure 1. Dose-response relationships of NDL-PCBs in liver, muscle and adipose tissue (values are presented respectively by full triangles ▴, grey squares ■, empty diamonds ♢; n = 2) to the amount ingested originating from spiked oil. Parameters of the linear models are presented inTable 1 (respective curves are fitted by full black lines, grey and white ones).

Chemosphere

  (104), Pages 105-112 10.1016/j.chemosphere.2015.06.098 9 CC BY-NC-ND 4.0

Table 1 . Parameters of the linear dose-response relationships between NDL-PCBs doses (ng kg BW -1 d -1 ) and retrieved concentration in adipose, muscular and hepatic tissues (ng g -1 of fat).

 1

	Chemosphere (104), Pages 105-112
	10.1016/j.chemosphere.2015.06.098

Regression analysis for PCBs 52, 101, 138 and 153 demonstrated significant linear relationships (P < 0.01) between the administrated doses and the levels of NDL-PCBs analyzed in adipose, hepatic and muscular tissues CC BY-NC-ND 4.0 A sequential fitting was performed in order to suppress insignificant parameters. P-value and resulting model originated of parameters values are presented. RMSE: root means square error. P-value of parameters are presented in front of each value: NS > 0.10.

Table 2 . Assessment of dose-response relationship, linear parameters and relative bioavailability (RBA) factors.

 2 Values are adjusted means (n = 3 for AC, FA, SS and SO groups; n = 2 for C).Letters (a, b, c) indicates values that do not statistically differ from other values within column presenting a common letter (P < 0.05). Numbers into brackets indicate values below LOQ replaced by LOQ/2. RMSE: root means square error.

			C	AC	FA	SS	SO	Exposure media	RMSE
		Adipose tissue	0.934 a	1.37 a	8.70 b	11.4 bc	15.0 c	0.001	2.1
	PCB 52	Muscle	0.583 a(2)	1.83 a	6.21 ab	7.88 b	9.11 b	0.003	2.0
		Liver	2.29	3.10 A	12.9	12.1 A	20.4	0.351	10.3
		Adipose tissue	0.505 a	1.83 a	13.8 b	13.6 b	16.8 b	<0.001	1.7
	PCB 101	Muscle	0.407 a(2)	2.24 a	9.73 b	13.2 b	12.2 b	<0.001	1.5
		Liver	1.27 a	2.37 a	10.1 ab	17.4 bc	23.5 c	<0.001	4.1
		Adipose tissue	0.467 a(2)	1.75 a	14.6 b	11.8 b	14.4 b	0.003	3.6
	PCB 138	Muscle	1.76 ab(2)	1.76 ab(3)	11.7 bc	15.5 c	14.8 c	0.001	3.3
		Liver	1.56 ab	2.57 a	24.9 abc	27.9 bc	34.9 c	0.005	8.9
		Adipose tissue	1.19 a	1.97 a	12.5 b	10.8 b	15.0 b	<0.001	2.3
	PCB 153	Muscle	0.626 a(1)	2.59 a	9.06 ab	13.4 b	12.9 b	0.004	3.3
		Liver	4.35 a	3.19 a	23.3 b	23.3 b	27.0 b	0.002	5.7
		Adipose tissue	0.154 a(2)	0.493 a	2.37 b	2.45 b	2.45 b	<0.001	0.5
	PCB 180	Muscle	0.821 ab(2)	0.821 a(3)	1.78 b	2.59 c	2.45 bc	0.010	0.6
		Liver	0.530 abA	0.618 a	4.31 b	4.21 b	4.56 b	0.010	1.2
		Adipose tissue	3.38 a	8.96 a	72.6 b	68.3 b	89.9 b	<0.001	12.5
	∑PCB	Muscle	4.54 a	10.6 a	51.5 b	66.7 b	65.6 b	0.001	10.7
		Liver	11.1 aA	14.5 a	87.6 b	95.5 b	124 b	<0.001	20.6
	RBA factors	Adipose tissue		6.4% B	80.0% C	75.0% C
	∑PCB	Muscle Liver		9.9% B 3.0% B	76.9% C 67.8% C	101.8% C 74.8% C

Acknowledgements ___________________________________________________________

The authors are grateful to the French Ministry for Agriculture and Agence de l'Environnement et de la Maîtrise de l'Energie for their financial support. They thank S. Denys (ANSES) for scientific comments, T. Duval (URAFPA) and C. Grandclaudon (URAFPA) for NDL-PCBs analyzes and P. Hartmeyer (URAFPA) and H. Toussaint (URAFPA) for providing care to piglets.


Vanessa Brochard

Béhazine Combadière

Annick Prigent

Yasmina Laouar

Aline Perrin

Virginie Beray-Berthat

Olivia Bonduelle

Daniel Alvarez-Fischer

Jacques Callebert

Jean-Marie Launay

Charles Duyckaerts

Richard A Flavell

Etienne C Hirsch
email: etienne.hirsch@upmc.fr

Stéphane Hunot
email: stephane.hunot@upmc.fr

Infiltration of CD4+ lymphocytes into the

Keywords: α-Syn: α-synuclein BBB: blood brain barrier DN: dopamine-containing neurons GFAP: glial fibrillary acidic protein Gld: Fas ligand-mutated generalized lymphoproliferative MPTP: 1-methyl-4-phenyl-1, 2, 3, 6

INTRODUCTION

Parkinson's disease is a neurodegenerative disorder characterized by the loss of dopamine-containing neurons (DNs) in the substantia nigra pars compacta (SNpc). Except for rare genetic forms, PD is a sporadic condition of unknown cause. Yet, several scenarios regarding the mechanisms by which DNs degenerate have been suggested including mitochondrial dysfunction, oxidative stress and the impairment of protein degradation machinery [START_REF] Dauer | Parkinson's disease: mechanisms and models[END_REF]. In addition to these well established pathomechanisms, there is mounting evidence from epidemiological, postmortem and animal studies to suggest that innate neuroinflammatory processes associated with glial cell activation participate in the progression of DN cell death [START_REF] Chen | Nonsteroidal antiinflammatory drug use and the risk for Parkinson's disease[END_REF][START_REF] Wahner | Nonsteroidal antiinflammatory drugs may protect against Parkinson disease[END_REF][START_REF] Nagatsu | Changes in cytokines and neurotrophins in Parkinson's disease[END_REF][START_REF] Czlonkowska | Immune processes in the pathogenesis of Parkinson's disease -a potential role for microglia and nitric oxide[END_REF][START_REF] Whitton | Inflammation as a causative factor in the aetiology of Parkinson's disease[END_REF].

Far more enigmatic is the putative role of the adaptive immune system in PD pathogenesis.

While several changes in cellular and humoral immune responses have been described in the peripheral immune system of PD patients, it remains unclear whether these alterations are primarily involved in the etiogenesis of PD or simply reflect secondary consequences of nigrostriatal pathway injury [START_REF] Czlonkowska | Immune processes in the pathogenesis of Parkinson's disease -a potential role for microglia and nitric oxide[END_REF]. Up to now, the hypothesis that the cellular arm of the adaptive immune system plays a role in neurodegeneration has been hampered by the fact that no clear demonstration of a prominent involvement of leukocytes at the site of neuronal injury has been provided in PD. Yet, the reports many years ago that 1) cytotoxic T-cells (T C ) can be massively found in PD SN (7) and 2) the density of IFN-γ-positive cells is markedly increased in PD brains [START_REF] Hunot | FcεRII/CD23 is expressed in Parkinson's disease and induces, in vitro, production of nitric oxide and tumor necrosis factor-α in glial cells[END_REF] suggest that T-cell brain recruitment may be associated with nigrostriatal pathway injury in PD. In support of this view is the demonstration that the accumulation of T-cells in the brain can be stimulated in mouse by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced DN injury [START_REF] Kurkowska-Jastrzebska | The inflammatory reaction following 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine intoxication in mouse[END_REF]. Yet, whether this immune response truly contributes to neurodegeneration, and, if so, by what mechanisms, remains to be established.

In this study we sought to determine whether PD is associated with leukocyte brain infiltration within the affected SN region and, if so, whether this process contributes to DN degeneration. We found that both CD8

RESULTS

Lymphocyte brain infiltration in PD patients

To test the possibility that leukocytes infiltrate the brain parenchyma of PD patients, we performed immunohistochemical staining for various leukocyte-specific markers [START_REF] Janeway | Immunobiology: the immune system in health and disease[END_REF] on postmortem human brain (for antibody description, see Supplemental Table 1). Positive staining was observed for CD8 and CD4 but not for B-cell and natural killer cell markers.

Within the SNpc, CD8 + and CD4 + T-cells were found either in close contact with blood vessels or in the vicinity of melanized DNs (Figure 1, A andB). These cells displayed a typical lymphocyte morphology that was further confirmed by electron microscopy analysis.

At the ultrastructural level, parenchymal CD8+ T-cells displayed small cytoplasmic volume, membrane-type CD8 staining and, in some cases, uropod-like cytoplasmic extension usually involved in T-cell motility (Figure 1C). Importantly, quantitative analysis revealed a significant increase (10 fold in average) in the density of CD8 + and CD4 + T-cells in the SNpc, but not in the non-lesioned red nucleus, from PD patients compared to age-matched control subjects (Figure 1D, and Supplemental Table 2).

MPTP-induced DN lesion stimulates extravasation of lymphocytes into the brain

The above data suggest that migration of peripheral lymphocytes within the central nervous system is associated with nigrostrital pathway injury in PD. Yet, although postmortem studies in humans are essential to establish that immune cell infiltration is characteristic of the disease, they cannot provide information about the dynamic and functional relevance of such mechanisms. To address this issue, we conducted in vivo experiments using the wellcharacterized MPTP-intoxicated mouse model of PD [START_REF] Dauer | Parkinson's disease: mechanisms and models[END_REF]. Although shortcoming, this non-invasive model provides a unique mean to study non-cell autonomous pathomechanisms as it recapitulates many of the hallmarks of PD including DN loss, attenuation of striatal dopamine and glial cell-associated inflammatory processes. To easily track the migration of peripheral leukocyte independently of their phenotypic traits, we first used a passive transfer strategy which consisted in transferring GFP-tagged T-or B-cells into recombinase activating gene 1 (Rag1)-deficient mice which lack mature lymphocytes. Recipient animals with reconstituted lymphoid organs were then treated with MPTP or saline solution (Figure 2A, and Supplemental Figure 2). Unlike GFP 2D). Furthermore, infiltrated Tcells had clearly transmigrated through the brain parenchyma since most of them were found to be excluded from the lumen of blood vessels identified by Glut-1 immunostaining (Figure 2D). Finally, both CD8 + and CD4 + T-cell subtypes were found to be recruited (Figure 2D).

To ensure that the brain infiltration of T-cells observed in our graft-based experimental model was not merely an artefact related to the methodology, we looked at leukocyte transmigration in MPTP-intoxicated C57Bl/6J inbred mice using immunohistochemistry for the T-cell specific marker CD3. In these conditions, a similar T-cell infiltration process was observed (Figure 2E). When compared to the time course of glial cell activation, CD3 + Tcell brain infiltration was found to arise after the increase of CD11b + microglial cells (i.e. 12 hours post-MPTP intoxication) but concomitantly with astrogliosis (Supplemental Figure 4). Consistent with our postmortem findings, nigral CD8+ T-cells were more abundant than CD4 + T-cells and transmigration occurred at day 2 post-treatment, increased continuously for up to 7 days and had totally ceased at 21 days (Figure 2E). Such dynamics are therefore compatible with a possible role of activated microglial cells in the brain regionspecific recruitment of T cells.

The continuous nigral accumulation of T-cells indicates that lymphocytes could clonally

expand into the brain parenchyma. To test such a possibility, we surveyed the expression of proliferating cell nuclear antigen (PCNA), taken as a marker of dividing cells, at 0.5, 1, 2, 4 and 7 days after the last MPTP injection. As shown in Figure 2F, while we observed numerous PCNA-positive cells in the SNpc after MPTP intoxication, none of them were found to co-localize with either GFP + T-cells or glial fibrillary acidic protein (GFAP)positive astrocytes at all the time points analyzed. Instead, most of these cells also expressed the myeloid marker CD11b, suggesting that infiltrated macrophages and/or resident microglial cells multiply at the site of injury. Thus, accumulation of T-cells may indicate a continuous extravasation process at least up to 7 days after neurotoxin exposure. Yet, the number of CD3+ T cells within the SNpc was found to dramatically decrease shortly after this accumulation i.e. at day 9 (Supplemental Figure 5A) raising the possibility that they could be rapidly eliminated by apoptotic cell death due to regulatory mechanisms aimed at terminating the immune response. To test this, we looked for morphological criteria as well as molecular changes indicative of apoptosis in the entire SNpc and VTA of animals sacrificed 7 and 9 days after systemic MPTP administration.

Immunohistochemistry for CD3 coupled with thionin counterstaining revealed the presence of apoptotic cells characterized by shrinkage of cellular body, chromatin condensation, and presence of distinct, round, well-defined chromatin clumps (Supplemental Figure 5B). Whereas apoptotic cells were found in all MPTP-treated mice analyzed at day 7 and 9 (n=10 and 5, respectively), they were not observed in saline-injected controls. In few instances, these cells have retained some CD3 staining attesting their T cell origin (Supplemental Figure 5C). Additional evidences for T cell apoptosis were obtained from double immunofluorescent staining for CD3 and activated caspase-3. Thus, some CD3+ T cells were found to be positive for the p17 fragment of activated caspase-3 (Supplemental Figure 5D).

Since previous report indicated that the blood-brain barrier (BBB) may be dysfunctional in PD patients [START_REF] Kortekaas | Blood-brain barrier dysfunction in parkinsonian midbrain in vivo[END_REF], we examined whether MPTP-associated BBB disruption could account for a passive entry of lymphocytes into the brain. A time course of BBB leakage from 6 hours to 7 days after MPTP administration was determined by assessing serum albumin and immunoglobulin extravasation. Our data reveal that MPTP induces a widespread and transitory serum protein leakage (detectable at 6 hours but absent from 12 hours after MPTP) contrasting therefore with the cell type-and region-specific leukocyte extravasation process (Figure 3A). To further explore the mechanism of T-cell extravasation we surveyed the expression of intercellular adhesion molecule-1 (ICAM-1/CD54), an immunoglobulinlike cell adhesion molecule involved in leukocyte adherence and transendothelial migration at sites of inflammation [START_REF] Engelhardt | Molecular mechanisms involved in T cell migration across the blood-brain barrier[END_REF]. Whereas CD54 was barely detectable in controls (data not shown), we observed a marked increase in CD54 immunostaining within the SNpc at the site of T-cell infiltration following MPTP intoxication (Figure 3B). Blood vessels, glial cells and T-cells were all intensely positive, indicating possible cross-interactions between these cellular actors.

Mice deficient in cell-mediated immunity are more resistant to MPTP Investigations dedicated to unravel the role of infiltrated T-cells in various models of neuronal injury have led to opposite conclusions [START_REF] Serpe | Exacerbation of facial motoneuron loss after facial nerve transection in severe combined immunodeficient (scid) mice[END_REF][START_REF] Chen | Reduced susceptibility to kainic acid-induced excitotoxicity in T-cell deficient CD4/CD8 (-/-) and middle-aged C57BL/6 mice[END_REF]. To determine whether T-cell accumulation in the SNpc following MPTP intoxication is beneficial or harmful to DNs, we compared the effects of the toxin in T-cell antigen receptor beta gene mutant mice lacking mature T lymphocytes (Tcrb -/-) [START_REF] Mombaerts | Mutations in T-cell antigen receptor genes alpha and beta block thymocyte development at different stages[END_REF] and in their wild-type (WT) littermates. We found that whereas only 72% of the nigral TH+ DNs survived MPTP injection in WT mice, 91% of these neurons survived in Tcrb -/- mice treated with a similar MPTP regimen (Figure 4, A andB). Importantly, the decrease in MPTP-induced DN loss in Tcrb -/- mice correlated with a reduction in the number of infiltrated T-cells to a level similar to that of saline-injected animals (Figure 4C). Residual T-cell infiltration in Tcrb -/- mice may come from the small number (about 8% of the WT level) of cells remaining in the thymus of these mutants [START_REF] Mombaerts | Mutations in T-cell antigen receptor genes alpha and beta block thymocyte development at different stages[END_REF].

Given the apparent harmful behavior of infiltrated T-cells, we next investigated whether CD8 + and/or CD4 + T-cells could mediate such a detrimental function. To that end, we intoxicated mice homozygous for either a Cd8a or a Cd4 targeted mutation characterized by a deficiency in functional cytotoxic and helper T-cells (T H ), respectively (16, 17).

Whereas MPTP induced a comparable level of DN cell death in Cd8a -/- mice as in WT littermates, we observed a marked reduction in TH + cell loss in Cd4 -/- mice (Figure 4, A andB). Interestingly, the rate of DN survival in Cd4 -/- mice was comparable to that of Tcrb -/- animals, suggesting that most of the deleterious outcome associated with infiltrated T-cells is mediated by the CD4 + T H cell subset. In support of this idea, susceptible WT and Cd8a -/- mice exhibited a similar number of infiltrated CD4 + T-cells following MPTP exposure (Figure 4C). However, there were no significant differences in the extent of loss in striatal levels of dopamine, DOPAC (3-4-dihydroxyphenylacetic acid) and HVA (homovanillic acid)

between mutant mice and their wild-type littermates after the administration of MPTP implying that striatal T-cell infiltration is not a major component of dopaminergic fibber injury (Table 1). Importantly, reduced MPTP metabolism in mutant mice is not likely to account for the observed neuroprotection as striatal MPP+ content after a single systemic MPTP injection did not differ between genotypes (Table 2). Moreover, consistent with the partial resistance of Tcrb -/- and Cd4 -/- mice to MPTP, mid-term (2-7 days) microglial cell activation was almost completely abolished in mutant mice (Figure 4B).

The Fas/FasL pathway is required for CD4 T cell-dependent DN toxicity

The finding that CD4-deficient mice are partially protected against MPTP-induced injury suggests that T H cell-mediated deleterious mechanisms are engaged and instrumental in DN demise. Cytokines, including IFN-γ, and membrane bound ligands, such as FasL, represent two major classes of molecules mediating effector functions of CD4 T H cells (e.g. macrophage activation). Interestingly, it has been shown that IFN-γ was a contributing factor in the death of DNs by regulating microglial activity [START_REF] Mount | Involvement of interferon-γ in microglial-mediated loss of dopaminergic neurons[END_REF]. In fact, we found that IFN-γ expression was up-regulated in the ventral midbrain 2 days after MPTP exposure, which coincided with the first wave of CD4 + T-cell brain infiltration (Supplemental Figure 6A). To analyze the putative deleterious role of lymphocyte-derived IFN-γ, we passively transferred total splenocytes isolated from either WT or IFN-γ-deficient mice into recipient Rag1 -/- animals. Consistent with the deleterious role of T-cells as previously evidenced in Tcrb -/- mice, we noticed that MPTP-induced injury in immunodeficient Rag1 -/- mice was significantly less severe than in WT littermates (Figure 5, A and B). Importantly, this partial resistance was completely reversed when spleen cells from either WT or Ifn-γ -/- mice were passively transferred prior to MPTP intoxication (Figure 5A). Thus, lymphocyte-derived INF-γ is not likely to be required for T cell-mediated DN cell death. In support to this assertion, Ifn-γ -/- mice and their WT littermates were found to be equally sensitive to MPTPinduced DA cell death using identical intoxication paradigm (Supplemental Figure 6B).

Therefore, we next considered the involvement of a Fas-based mechanism. To test this, we took advantage of the Fas ligand-mutated generalized lymphoproliferative (gld) mice that bear a point mutation in the extracellular domain of Fas ligand (FasL/CD95L) resulting in a dramatic decrease of affinity to its receptor Fas [START_REF] Takahashi | Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand[END_REF]. Thus, we compared the MPTP susceptibility of Rag1 -/- recipients that received spleen cells from either gld or C57Bl/6J donors (designated gld rec. Rag1 -/- and WT rec. Rag1 -/- , respectively). In WT rec. Rag1 -/- mice, MPTP caused as much DN loss as in WT animals (Figure 5, A, B andC). In sharp contrast, both non-reconstituted Rag1 -/- and gld rec. Rag1 -/- mice exposed to a similar dose of MPTP exhibited a significantly smaller reduction in the number of nigral DNs (Figure 5, B and C). The protection gained in gld rec. Rag1 -/- mice was consistently within a similar range to that observed in Cd4 -/- animals (Figure 5B and Figure 4A). Furthermore, this protective effect was not likely due to altered extravasation potency of gld-derived splenocytes as similar number of brain infiltrated CD4+ T cells were observed between MPTP-treated WT rec. Rag1

-/-

DISCUSSION

Mounting evidence support the notion that innate immunity significantly contributes to dopaminergic neurodegeneration in PD [START_REF] Dauer | Parkinson's disease: mechanisms and models[END_REF]. By contrast, although altered cellular and humoral functions have been reported in the peripheral immune system of PD patients, the role of adaptive immunity in the pathogenesis of this disorder has remained much more elusive [START_REF] Czlonkowska | Immune processes in the pathogenesis of Parkinson's disease -a potential role for microglia and nitric oxide[END_REF][START_REF] Hunot | Neuroinflammatory processes in Parkinson's disease[END_REF]. Among these peripheral immune changes, the significant increased ratio of CD8 + T C to CD4 + T H cells and IFN-γ-producing to IL-4-producing T cells suggests the existence of a disease-associated shift to a T C 1-and T H 1-type immune response which may reflect and/or contribute to the harmful brain inflammatory reaction [START_REF] Baba | Alterations of T-lymphocyte populations in Parkinson disease[END_REF]. Nonetheless, a role for the cellular arm of the adaptive immune system in neurodegeneration is curbed by the fact that no clear demonstration of a prominent involvement of leukocytes at the site of neuronal injury has been provided in PD. In this study, we present evidence that peripheral T-cells migrate to and accumulate in the brain parenchyma during parkinsonism.

Importantly, we have shown that CD4 + , but not CD8 + T-cells, are deleterious to DNs, which suggests that the adaptive immune system may contribute to disease progression in PD.

It is now well established that the CNS is not only continuously monitored by T-cells [START_REF] Cose | Evidence that a significant number of naive T cells enter non-lymphoid organs as part of a normal migratory pathway[END_REF] but also massively invaded by peripheral leukocytes in neuropathological circumstances Consistent with a previous report [START_REF] Kurkowska-Jastrzebska | The inflammatory reaction following 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine intoxication in mouse[END_REF], we found that experimental lesion of the nigrostriatal pathway in MPTP-intoxicated mice results in a similar brain accumulation of T-cells. Yet, brain accumulation of T-cells following MPTP intoxication was not restricted to the SNpc.

Indeed, other catecholaminergic areas known to be moderately injured following MPTPtreatment [START_REF] German | The neurotoxin MPTP causes degeneration of specific nucleus A8, A9 and A10 dopaminergic neurons in the mouse[END_REF] were also invaded albeit to a lower extend. This extravasation of lymphocyte is not likely to be a generalized non-specific leukocyte response: 1) other leukocyte subsets including B-cells and NK cells were not detected in the lesioned SNpc, 2) an increased occurrence of T-cells was not observed in non-lesioned brain areas suggesting that this process does not simply reflect an overall enhancement of lymphocyte patrolling.

Altogether, these findings indicate that, as with the glial cell reaction previously described, T-cell brain infiltration in PD most certainly represents a secondary and highly regulated pathogenic event associated with neuronal cell death. Such a cellular and site-specific brain immune response is not likely to be caused by a massive disruption of the BBB, for which little evidence exists in PD [START_REF] Farkas | Pathological features of cerebral cortical capillaries are double in Alzheimer's disease and Parkinson's disease[END_REF][START_REF] Haussermann | Integrity of the bloodcerebrospinal fluid barrier in early Parkinson's disease[END_REF] and which remains controversial in MPTPintoxicated mice [START_REF] Adams | Effects of MPTP on the cerebrovasculature[END_REF][START_REF] Zhao | TNF-α knockout and minocycline treatment attenuates blood-brain barrier leakage in MPTP-treated mice[END_REF]. We showed a widespread BBB leakage that occurred shortly (6 hours) after neurotoxin exposure and was rapidly resolved (by 12 hours after MPTP), well before lymphocytic invasion. Taken together, these findings argue that lymphocyte brain infiltration is not a passive phenomenon. Instead, molecular and cellular changes associated with neuronal injury are likely to regulate this site-specific brain recruitment of Tcells. Possible mechanisms may involve early microglial cell activation and innate neuroinflammatory processes which could modify the local microenvironment. In line with this, we found an up-regulated expression of the ICAM-1 adhesion molecule on both capillaries and glial cells, which may participate in the attachment of leukocytes to the vascular endothelium and their diapedesis [START_REF] Springer | Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm[END_REF]. Interestingly, a similar ICAM-1 overexpression was recently described in both PD SN and MPTP-intoxicated monkeys [START_REF] Miklossy | Role of ICAM-1 in persisting inflammation in Parkinson disease and MPTP monkeys[END_REF].

Although the level of T cell brain accumulation was found to be higher in disease model than in PD patients, one has to consider that the human disease progresses over several decades whereas it only last one to two weeks in mouse model. Moreover, investigations performed on human postmortem tissue most often address late pathomechanisms and can not therefore predict the intensity of the lymphocyte infiltration process which might be greater during earlier stages of the disease. Given that incidental Lewy body disease is considered by some authors to be a presymptomatic form of PD [START_REF] Jenner | Understanding cell death in Parkinson's disease[END_REF], it might be valuable to explore such lymphocytic invasion in those patients. Finally, it is worth noting that the distribution of T cell accumulation within the SNpc from PD patients is much more heterogeneous than that in MPTP mice. Indeed, in few instances, as shown in Figure 1 18). Yet, using two different experimental approaches, i.e. total and cell-specific IFN-γ deletion, we were unable to show a major role of this pro-inflammatory cytokine in DA cell demise. While the reason for such discrepancy is still not clear, a lack of IFN-γ expression in our disease model could not be incriminated. Indeed, in agreement with a previous report [START_REF] Ciesielska | Dynamics of expression of the mRNA for cytokines and inducible nitric synthase in a murine model of the Parkinson's disease[END_REF], we were able to detect a rise in IFN-γ following MPTP-induced nigrostriatal pathway injury. Whether different treatment protocols lead to distinct involvement of IFN-γ in disease models will need further clarifications.

The requirement of FasL in CD4 + T cell-mediated cytotoxicity is consistent with the finding that Fas-deficient mice display attenuated MPTP-induced DN loss [START_REF] Hayley | Regulation of dopaminergic loss by Fas in a 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease[END_REF] although another study has come to a different conclusion [START_REF] Landau | Defective Fas expression exacerbates neurotoxicity in a model of Parkinson's disease[END_REF]. Whereas, in the context of antigen presentation, the Fas/FasL pathway has been implicated in the deletion of activated macrophages thereby contributing to the resolution of inflammation [START_REF] Ashany | Th1 CD4+ lymphocytes delete activated macrophages through the Fas/APO-1 antigen pathway[END_REF], recent evidence suggests that this pathway may instead induce proinflammatory cytokine responses in tissue macrophages [START_REF] Park | Fas (CD95) induces proinflammatory cytokine responses by human monocytes and monocyte-derived macrophages[END_REF]. CD4

+ T H FasL-mediated activation of microglial cells could therefore participate in the inflammatory reaction and DN degeneration. T-cell-derived FasL may also mediate inflammatory responses in astrocytes which are known to be particularly resistant to Fas-mediated cell death and express proinflammatory cytokines and chemokines upon Fas ligation [START_REF] Choi | Fas ligand/fas system in the brain : regulator of immune and apoptosis responses[END_REF]. In line with this, Fas expression has been shown to be upregulated on these glial cells both in PD patients and in the MPTP model [START_REF] Hayley | Regulation of dopaminergic loss by Fas in a 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease[END_REF][START_REF] Ferrer | Fas and Fas-L expression in Huntington's disease and Parkinson's disease[END_REF].

Alternatively, infiltrated CD4 +

T-cells may also induce DN cell demise through cell-cell contact. Such a mechanism has previously been evidenced in vitro, where antigen primed CD4 + or CD8 + T-cells induced neuronal death independently of antigen presentation and involving cell surface expression of FasL by activated T-cells [START_REF] Giuliani | Vulnerability of human neurons to T cell-mediated cytotoxicity[END_REF]. Further investigations will be required to determine whether or not CD4 + T cell-mediated cytotoxicity relies strictly on MHC-dependent antigen presentation.

In our disease model, the finding that harmful lymphocyte response is CD4 T celldependent but IFN-γ-independent raises important but still unresolved concern about the phenotypic characteristics of these CD4 T cells. Although these observations would not favor the contribution of a T H 1-type response, one can not exclude the possibility that CD4+ T H 1 cells may mediate cytotoxicity independently of IFN-γ as previously reported (40, 41). In particular, the role of other proinflammatory T H cytokines such as TNF-α in T cell- mediated DN cell death should also be considered as a potential deleterious mechanism even though the role of this factor is still debated [START_REF] Rousselet | Role of TNF-alpha receptors in mice intoxicated with the parkinsonian toxin MPTP[END_REF][START_REF] Ferger | Genetic ablation of tumor necrosis factor-alpha (TNF-alpha) and pharmacological inhibition of TNFsynthesis attenuates MPTP toxicity in mouse striatum[END_REF][START_REF] Sriram | Minocycline attenuates microglial activation but fails to mitigate striatal dopaminergic neurotoxicity: role of tumor necrosis factor-alpha[END_REF]. Finally, another tantalizing possibility could be the involvement of an alternative inflammatory CD4 T cell population namely the recently described T H 17 cells. A growing body of evidence indicates that T H 17 cells play a critical function not only in protection against microbial challenges but also in many organ-specific autoimmune diseases including multiple sclerosis and its disease model experimental autoimmune encephalomyelitis [START_REF] Zhu | CD4 T cells: fates, functions, and faults[END_REF]. In line with this, it has recently been shown that T H 17 cells have the potential of killing neurons probably through the granzyme B cytolytic enzyme system [START_REF] Kebir | Human Th17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation[END_REF]. Whether such CD4 T-cell subset is implicated in parkinsonism as well merits further considerations.

It is worth noting that the CD4 + T cell response to neurodegenerative processes can differently affect disease outcome. Indeed, while in the MPTP mouse model of PD CD4+ T cells are likely harmful to DNs (present data and reference 32), it has recently been documented that this T cell population can provide supportive neuroprotection in animal model of inherited amyotrophic lateral sclerosis (ALS) by stimulating the trophic properties of glia [START_REF] Beers | CD4+ T cells support glial neuroprotection, slow disease progression, and modify glial morphology in an animal model of inherited ALS[END_REF]. Interestingly, such pathological improvement can be recapitulated by delivering activated regulatory or effector T cells to ALS mice but not by Copolimer-1 immunization indicating that antigen-driving T H 2/T H 3 responses are impaired in this animal model (48). In summary, we have shown that peripheral T-cells infiltrate the brain parenchyma at the site of neuronal injury both in PD patients and in experimental parkinsonism. We have demonstrated that this cell-mediated immune response contributes to DN cell degeneration through a CD4 +

T cell-dependent Fas/FasL cytotoxic pathway. Our results together with recent investigations [START_REF] Benner | Nitrated α-synuclein immunity accelerates degeneration of nigral dopaminergic neurons[END_REF] provide further rationales for targeting the adaptive arm of the immune system in PD. Therapeutic strategies may involve vaccination for antigens that promote cell-mediated anti-inflammatory responses [START_REF] Benner | Therapeutic immunization protects dopaminergic neurons in a mouse model of Parkinson's disease[END_REF][START_REF] Reynolds | Neuroprotective activities of CD4+CD25+ regulatory T cells in an animal model of Parkinson's disease[END_REF] as well as blocking the migration of immune cells across the BBB [START_REF] Luster | Immune cell migration in inflammation: present and future therapeutic targets[END_REF]. Besides providing potential neuroprotective benefits for remaining DNs, these immunomodulatory strategies may also be of interest for cell transplantation therapies as long-term survival of grafted DNs, which eventually undergo PD-related pathological changes over time, could be jeopardize by such an immune-related hostile microenvironment [START_REF] Braak | Assessing fetal nerve cell grafts in Parkinson's disease[END_REF].

METHODS

Human samples. transcardially perfused first with 50 ml of heparin solution (5U/ml) then with 100 ml of 4% paraformaldehyde. Brain and spleen free-floating sections were prepared as described elsewhere [START_REF] Hunot | JNK-mediated induction of cyclooxygenase 2 is required for neurodegeneration in a mouse model of Parkinson's disease[END_REF]. For HPLC analysis of MPP + levels, brains were rapidly removed from the skull and striata dissected on humidified filters at 4°C. Tissues were then immersed in appropriate buffer.

Passive transfers. Single-cell suspensions were prepared from spleen and/or lymph nodes isolated from either Tie2-Cre Immunohistochemistry. Human sections were first deparaffinized in 2 changes of xylene for 5 min each. They were hydrated in two changes of 100%, 95% then 75% ethanol and rinsed in distilled water. Antigen demasking was performed at 90-95°C for 30 min in citrate buffer pH=6 (for CD8, CD20, CD79a and CD57 staining) or EDTA buffer pH=8 for CD4 staining. Sections were then allowed to cool down for 20 min before being rinsed in 0.25 M Tris buffer.

Human sections were pretreated with 0.01% H 2 O 2 /20% methanol, followed by 0.5% Electron microscopy. Ultrastructural analysis of T-cells in the SNpc was performed as previously described with minor modifications [START_REF] Hunot | Nuclear translocation of NF-kappaB is increased in dopaminergic neurons of patients with Parkinson disease[END_REF]. In brief, small blocks of mesencephalon containing the SNpc were fixed in a mixture of 4% paraformaldehyde and 2.5% glutaraldehyde. Sections 50 µm thick were cut and labeled for CD8 as described above.

Following identification of CD8 + cells, small areas of sections were then excised and postfixed in 1% osmium tetroxide for 30 min, rinsed in distilled water, dehydrated in a graded series of ethanol solutions, and embedded in Epon. Ultrathin (70 nm) sections were cut, counterstained with conventional techniques and analysed with a JEOL 1200EX II electron microscope at 80 kV.

Measurement of striatal dopamine. Striatal levels of dopamine and its metabolites (DOPAC, HVA) were determined by high-performance liquid chromatography (HPLC) as previously reported [START_REF] Rousselet | Role of TNF-alpha receptors in mice intoxicated with the parkinsonian toxin MPTP[END_REF].

Measurement of striatal MPP + . Mice were killed 90 min after one i.p. injection of 30 mg/kg MPTP-HCl, and their striata were recovered and processed for HPLC using UV detection (295-nm wavelength) to measure MPP + , as described elsewhere [START_REF] Liberatore | Inducible nitric oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of Parkinson disease[END_REF].

Antibody array. C57BL/6J mice (n = 5 per group) were intoxicated with MPTP and sacrificed from 12 hours to 7 days after the last injection. between multiple data sets were analyzed using one-or two-way ANOVA with time, treatment or genotype as the independent factors. When ANOVA showed significant differences, pair-wise comparisons between means were tested by Tukey post hoc analysis. When data were not normally distributed, ANOVA on ranks was used (Kruskal-Wallis test followed by pairwise comparison using Dunn's method). In all analyses, the null hypothesis was rejected at the 0.05 level (SigmaStat Statistical Software, Jendel, St.

Raphael, CA). Striatal MPP+ levels in wild-type, Tcrb -/-, Cd8a -/-and Cd4 -/-mice at 90 min after a single MPTP injection do not differ (P = 0.87; Kruskal-Wallis test) between groups. Data represent means ± SEM for five mice per group.

Supplemental Table 1 Antibodies used for the detection of leukocyte subsets in the human brain

Antigen

  significantly invade the SN in postmortem PD specimens and in MPTP-intoxicated mice. We also show that removal of CD4 + but not of CD8 + T-cells in mouse greatly reduced MPTP-induced nigrostriatal DN cell death. Finally, we further demonstrate that the deleterious activity of infiltrated CD4 + T-cells involved the Fas/FasL pathway but not IFN-γ production.

+B

  -cells (data not shown), we found GFP + Tcells in the SNpc and striatum after MPTP treatment (Figure 2, B and C). Most, if not all, other brain structures but DN-containing regions were devoid of T-cell infiltrate, indicating the region specific extravasation of these immune cells (Supplemental Figure 3). Brain sections immunostained for tyrosine hydroxylase (TH) show GFP + T-cells within the SNpc clustered in close proximity to TH-expressing DNs (Figure

( 23 )

 23 . In agreement with the seminal but rather preliminary observation by McGeer and colleagues showing a substantial presence of CD8 + T-cells in the brain from one PD case (7), we now provide strong quantitative evidence from a large number of individuals that both accumulate in the SNpc of PD patients.

  , infiltrating T cells in PD brains were found to be clustered around the few remaining pigmented DA neurons whereas they were virtually absent in advanced depigmented areas. Such distribution suggests that the number of T cell reaching the target neurons is conceivably substantial and strengthens their possible involvement in DA cell demise. A major contribution of our study is the demonstration that brain infiltration of T-cells actively participates in DN degeneration. A decreased vulnerability of DNs to MPTP toxicity associated with the removal of mature T lymphocytes was achieved by two different genetic immune defects (Rag1 -/and Tcrb -/- ), thus supporting a role for the cellular arm of the adaptive immune system in experimental parkinsonism. Our results are in agreement with a recent study showing a similar neuropathological improvement in MPTP-intoxicated severe combined immunodeficient (SCID) mice (32). Notably, our data further reveal that different T-cell subsets do not contribute equally to DN cell death. Indeed, while the predominance of infiltrated CD8 + over CD4 + T-cells was consistently observed both in PD patients and MPTP-intoxicated mice, removal of the CD8 + T-cell subset in Cd8a -/- mice did not mitigate MPTP injury. In sharp contrast, the T H cell deficiency achieved in Cd4 -/- mice conferred as much neuroprotection as in Rag1 -/- and Tcrb -/- animals, suggesting that CD4 + T-cells mediate most if not all the deleterious activity associated with the adaptive immune response. Since infiltrated T-cells highly express ICAM-1 as well as Lymphocyte function-associated antigen-1 (LFA-1) and CD44 (9, 30), we believe that most of them could be recruited from an activated/memory population. A mechanism of peripheral leukocyte activation secondary to MPTP intoxication has recently been proposed (32). In this model, nitrotyrosine modification within α-synuclein (α-Syn) promotes an antigen-specific T-cell response in draining lymph nodes. After migrating into the injured brain areas, these antigen-specific T-cells could undergo further activation and promote microglial cellin this deleterious immune response was not addressed by the authors, their prediction analysis of α-Syn-specific T-cell epitopes suggested a significant potential for respond to nitrated epitopes of α-Syn. These data, taken in conjunction with our present findings, support a model in which MPTP-induced brain antigen modification most likely generates a secondary and harmful T H cell response contributing to DN degeneration. Looking at the cytotoxic mechanism mediated by the CD4 + T-cell response following nigrostriatal pathway injury, we found that T-cell expression of FasL but not IFN-γ was required. It has been shown that IFN-γ is critical in microglial-mediated loss of DNs in MPTP-intoxicated mice (

  Thus, different neurodegenerative contexts could result in distinct T cell responses which in turn may positively or negatively influence disease progression. Undoubtedly, role of the adaptive immune system in various neurodegenerative conditions.

  Ifn-γ -/- or C57BL/6J mice. GFP + T-and B-cells were purified with a cell magnetic separator (MACS, Miltenyi Biotech, CA) according to the manufacturer's procedure using anti-TCRβ (H57-597), anti-CD3ε (145-2C11), anti-CD4 (H-129.[START_REF] Takahashi | Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand[END_REF]), anti-CD8α (53-6.7) and anti-B220 conjugated PE antibodies (Pharmingen) and anti-PE-conjugated MicroBeads. Cells labeled with MicroBeads were retained on the MACS Column while unlabeled cells passed through. The column was removed from the seperator and the retained cells were eluted as the enriched, positively selected cell fraction. The enrichment of cells was confirmed by flow cytometry analysis using a FACS-Calibur (e.g. more than 95% for T-cells, Supplemental Figure2A). Recipient Rag1 -/- mice received a single i.v. injection of either 10 either gld, Ifn-γ -/- or WT) in 0.2 ml phosphate buffered saline solution. Reconstitutions of GFP-expressing T and B lymphocytes in Rag1 -/- recipient mice were checked by fluorescence-activated cell-sorter (FACS) analysis of blood samples collected from reconstituted Rag1 -/- recipient mice prior to MPTP treatment or from lymph nodes after their sacrifice (Supplemental Figure2C). Three to four weeks after transfer, mice were intoxicated with MPTP or received an equal volume of saline solution.

Figure 1

 1 Figure 1 Lymphocyte infiltration in PD brain. (A) CD8 and (B) CD4 immunostaining with

Figure 2 MPTP

 2 Figure 2 MPTP-induced nigrostriatal pathway injury stimulates T-cell brain infiltration in

Figure 3

 3 Figure 3 Mechanism of lymphocyte entry into the brain. (A) Immunofluorescent staining for

  Student's t test) or the mean interval from death to freezing of tissue (mean ± s.e.m.: PD, 40.55 ± 3.80; controls, 28.10 ± 7.08 hours; P = 0.11, Student's t test). Due to limited accessibility to tissue sections, different numbers of patient were analysed between leukocyte markers. Tissue was fixed in formaldehyde and then embedded in paraffin. Tg) in which the GFP reporter gene is expressed in early hematopoietic cell precursors (53). Animal handling was carried out according to ethical regulations and guidelines contained in the Guide for the Care and Use of Laboratory Animals (National Research Council 1996) and the European Communities Council Directive 86/609/EEC. Treatment and tissue preparation. Groups of mice received four i.p. injections of MPTP-HCl (20 mg/kg, except where otherwise stated) at 2 h intervals and were sacrificed from 6 hours to 21 days after the last injection. Control mice received saline solution only. For

			The study was performed on autopsy brainstem tissue from 8 control
	subjects	and	14	parkinsonian	patients,	well	characterized	clinically	and
	neuropathologically. PD patients and control subjects did not differ significantly in terms of
	their mean age at death (mean ± s.e.m.: PD, 77.85 ± 2.11; controls, 76.62 ± 3.72 years: P
	= 0.76, Animals. Ten-to twelve-week-old male C57BL/6J mice, weighing 25-30 g, (CERJ, Le-Genest-St-Isle, France) were used. The following strains were obtained from Jackson Laboratories (Bar Harbor, ME): B6.129S6-Cd4 tm1Knw /J, B6.129S2-Cd8a tm1Mak /J, B6.129P2-Tcrb tm1Mom /J, B6.129S7-Rag1 tm1Mom /J, B6.129S7-Ifng tm1Ts /J, B6Smn.C3-Faslgld/J and corresponding parental wild-type inbred C57BL/6J. Mice were kept in a temperature-controlled room (23° ± 1°C) under a 12-h light/dark cycle and had ad libitum access to food and water. All animals were further genotyped after their sacrifice according to Jackson Laboratories protocols. GFP + cells were isolated from double Tie2-Cre + /ZEG + transgenic mice (designated GFP-immunohistochemistry, mice were euthanized with 100 mg/kg pentobarbital, and

Paraffin-embedded tissues were cut in serial 8-µm-thick slices on a microtome, and sections were recovered on SuperFrost Plus slides (Kindler O GmbH, Freiburg, Germany), before incubation at 56°C for 3 days. Human tonsil slices from Dako (Trappes, France) were used as tissue control for lymphoid marker immunohistochemistry.

Table 1

 1 Striatal monoamine levels (pM/mg tissue) at 7 days after the last MPTP injection do not differ (P > 0.05; Kruskal-Wallis test) between groups. Data represent means ± SEM for the indicated number of mice.

					Dopamine	DOPAC	HVA
	Saline
	Wild-type (n=5)	82.3 ± 7.5	15.4 ± 1.9	10.1 ± 1.0
	Tcrb -/-	(n=3)	85.4 ± 12.7	15.8 ± 3.0	9.5 ± 1.6
	Cd8a -/-	(n=3)	84.6 ± 4.1	16.0 ± 1.0	9.9 ± 0.9
	Cd4 -/-	(n=3)	65.0 ± 8.9	15.7 ± 2.3	8.7 ± 1.4
	MPTP
	Wild-type (n=5)	1.7 ± 0.3	1.8 ± 0.2	3.2 ± 0.4
	Tcrb -/-	(n=4)	2.8 ± 0.4	2.5 ± 0.3	3.7 ± 0.5
	Cd8a -/-	(n=4)	4.9 ± 1.6	2.2 ± 0.3	3.2 ± 0.1
	Cd4 -/-	(n=4)	3.5 ± 0.5	3.0 ± 0.5	4.5 ± 0.6
	Striatal dopamine, DOPAC and HVA levels in wild-type, Tcrb -/-, Cd8a -/-

and Cd4 -/- mice

Table 2

 2

	Striatal MPP +	levels (ng/mg tissue)
	Wild-type	7.09 ± 2.95
	Tcrb -/-	6.00 ± 2.55
	Cd8a -/-	7.89 ± 2.17
	Cd4 -/-	5.29 ± 2.16

ACKNOWLEDGMENTS

We thank Drs. C. Combadière and C. Lobsiger for helpful discussions, Dr. V. Sazdovitch for assistance in collecting postmortem material and Dr. T. Welte for providing Tie2-Cre + /ZEG + double transgenic mice. This work was supported by grants from The Michael J. Fox Foundation (SH and ECH), Fondation pour la Recherche sur le Cerveau (SH), Fondation France Parkinson (VB) and the German Academic Exchange Service (DA-F).

RAF is an investigator at the Howard Hughes Medical Institute. ECH and SH are investigators at the Centre National de la Recherche Scientifique (CNRS).