Vale ´rie Amarger

Dominique Gauguier

Martine Yerle

Franc ¸oise Apiou

Philippe Pinton

Fabienne Giraudeau

Sylvaine Monfouilloux

Mark Lathrop

Bernard Dutrillaux

Je ´ro ˆme Buard

Gilles Vergnaud

Analysis of Distribution in the Human, Pig, and Rat Genomes Points toward a General Subtelomeric Origin of Minisatellite Structures

Analysis of distribution in the human, pig, and rat genomes points toward a general subtelomeric origin of minisatellite structures.

INTRODUCTION

Tandem repeats are classified, according to the overall size of the repeat sequence, among satellites (largest, megabases long), minisatellites (in the kilobase size range), and microsatellites (10 to 40 nucleotides long). Satellite DNA is preferentially associated with centromeric heterochromatin, and microsatellites are distributed throughout the genome. Similarly, in the mouse [START_REF] Jeffreys | Mouse DNA 'fingerprints': Analysis of chromosome localization and germ-line stability of hypervariable loci in recombinant inbred strains[END_REF][START_REF] Julier | Minisatellite linkage maps in the mouse by cross-hybridization with human probes containing tandem repeats[END_REF][START_REF] Mariat | Genetic mapping through the use of synthetic tandem repeats in the mouse genome[END_REF], bovine [START_REF] Georges | Characterization of a set of variable number of tandem repeat markers conserved in Bovidae[END_REF], and rat genomes [START_REF] Pravenec | A genetic linkage map of the rat derived from recombinant inbred strains[END_REF] at least, minisatellites are spread over the genome. In contrast, the minisatellites characterized so far in human show a marked clustering in the terminal cytogenetic bands of each chromosome [START_REF] Royle | Clustering of hypervariable minisatellites in the proterminal regions of human autosomes[END_REF][START_REF] Wells | Simultaneous genetic mapping of multiple human minisatellite sequences using DNA fingerprinting[END_REF][START_REF] Vergnaud | The use of synthetic tandem repeats to isolate new VNTR loci: Cloning of a human hypermutable sequence[END_REF].

This apparent discrepancy may be an artifact due to the cloning procedures that have been used: clustering in human is suggested by a variety of independent approaches performed in different laboratories, whereas the work in the mouse, rat, and bovine relies in each case essentially on a single approach. Furthermore, a very limited number of the supposed minisatellites detected [START_REF] Jeffreys | Mouse DNA 'fingerprints': Analysis of chromosome localization and germ-line stability of hypervariable loci in recombinant inbred strains[END_REF][START_REF] Julier | Minisatellite linkage maps in the mouse by cross-hybridization with human probes containing tandem repeats[END_REF][START_REF] Mariat | Genetic mapping through the use of synthetic tandem repeats in the mouse genome[END_REF] or cloned [START_REF] Georges | Characterization of a set of variable number of tandem repeat markers conserved in Bovidae[END_REF] from animal genomes have been characterized at the sequence level to confirm their minisatellite nature. Alternatively, this discrepancy could reflect differences in the evolutionary forces that shape mammalian genomes.

To investigate this issue, we cloned minisatellites in human, pig, and rat using identical techniques and compared their chromosomal localization in the three genomes. Clones containing minisatellites were identified in genomic libraries using both synthetic tandem repeats as screening probes [START_REF] Vergnaud | The use of synthetic tandem repeats to isolate new VNTR loci: Cloning of a human hypermutable sequence[END_REF] and a new procedure that is based only on the structural characteristics of the minisatellites and does not require a hybridization step. The cosmids were assigned to a cytogenetic band by in situ hybridization assisted when appropriate by linkage analyses. Striking differences were observed in the distribution patterns of these sequences, with subtelomeric clustering confirmed in human, a majority of internal loci in the rat, Sequence data for this article have been deposited with the EMBL/ GenBank Data Libraries under Accession Nos. AJ001151 to AJ001156.

1 Present address: IECH, Bat 400, Institut de Ge ´ne ´tique et Microbiologie, Universite ´Paris-Sud, 91405 Orsay Cedex, France.

2 Present address: Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, UK.

3 To whom correspondence should be addressed at IECH, Bat 400, Institut de Ge ´ne ´tique et Microbiologie, Universite ´Paris-Sud, 91405 Orsay Cedex, France. Telephone: ϩ33 1 69 15 62 08. Fax: ϩ33 1 69 15 66 78. E-mail: gilles@igmors.u-psud.fr. and an intermediate situation in pig. The comparative mapping of the minisatellite-containing regions in the three genomes is consistent with the hypothesis that minisatellites have a subtelomeric origin. These results underline the interest of minisatellites for comparative genome analysis.

MATERIALS AND METHODS

Minisatellite Cloning

Cosmid clones from commercially available libraries (Clontech Refs. HL 1145y, RL1032m, and PL1008m for human, rat, and pig, respectively, on pWE15 vector) were plated at a low density and picked in 96-or 384-well microtiter plates. Replicas were made on nylon filters with a comb.

Procedure without hybridization screening. The clones were grown individually in 2-ml LB medium cultures with shaking at 37°C. After 20 h growth, 24 cultures were pooled, and the cosmid DNA was extracted by the alkaline SDS lysis method as described in [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual[END_REF]. The cosmid DNA pools were suspended in 300 l of 10 mM Tris, pH 8, 1 mM EDTA (TE).

Procedure with hybridization screening using synthetic tandem repeat probes. This procedure was described in detail in [START_REF] Vergnaud | The use of synthetic tandem repeats to isolate new VNTR loci: Cloning of a human hypermutable sequence[END_REF]. The synthetic probes used were 13C1, 14C3, 14C5, 14C21, 14C24, 14C31, 15C3, 16C2, 16C4, 16C7, 16C17, 16C20, 16C22, 16C23, 16C24, 16C26, and 16C27. DNA from positive clones was extracted by the alkaline SDS lysis method from 50 ml MLS [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual[END_REF] medium culture and suspended in 300 l of TE. In both procedures, three aliquots of 20 (for pools of clones) or 1 g (for individual clones) of cosmid DNA were digested respectively with the restriction endonucleases AluI and HaeIII, AluI and HinfI, and HinfI and HaeIII in a total volume of 100 l following the supplier's instructions (New England Biolabs or Appligene). After an overnight digestion, the samples were ethanol precipitated, suspended in 5 l of TE, and loaded for electrophoresis on a 1% horizontal agarose gel. The gel was then stained with ethidium bromide. Fragments above 1.5 kb (from pools) or above 1kb (from individual clones) were sliced out of the gel and recovered by centrifugation as described by [START_REF] Heery | A simple method for subcloning DNA fragments from gel slices[END_REF]. One to five nanograms of insert DNA was then directly [ 32 P] labeled by random priming [START_REF] Feinberg | A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity[END_REF]) ([ 32 P]dCTP from New England Nuclear; random priming kit from Boehringer) and hybridized at 65°C as described in [START_REF] Vergnaud | The use of synthetic tandem repeats to isolate new VNTR loci: Cloning of a human hypermutable sequence[END_REF] to a Southern blot carrying the DNA from two unrelated reference individuals digested by HaeIII, HinfI, and PvuII. This provided an identification pattern for each minisatellite probe, and the profile was encoded by sizing each allele in the three digests. In the case of a pool of clones, the cosmid from which a fragment originates was identified by hybridizing back to the replica of the 384well plate.

Chromosomal Mapping by Linkage Analysis

Polymorph loci were mapped by linkage analysis using the CEPH panel of human families, the PiGMaP panel of pig reference families, and a F2 (BN ϫ GK) rat population [START_REF] Gauguier | Chromosomal mapping of genetic loci associated with the GK rat[END_REF]. Genotypes were managed using GENBASE, developed by Jean-Marc Sebaoun. Linkage file output from the human or pig data was converted to CRIMAP file format using the LINK2CRI utility software written by John Attwood. CRIMAP version 2.4 was used for the analyses.

Fluorescence in Situ Hybridization

Rat chromosomes were prepared from normal rat fibroblast cultures. The probes were labeled by nick translation with digoxigenin-11-dUTP (Boehringer Mannheim) and hybridized at a final concentration varying from 2 to 10 ng/l in presence of an excess of sonicated rat DNA (between 100 and 300 ng/l) used as competitor. A standard hybridization was performed, as described in [START_REF] Apiou | Fine mapping of human HOX gene clusters[END_REF]. Immunochemical detection of hybridization was performed using mouse anti-digoxygenin antibodies and sheep FITC-conjugated anti-mouse antibodies (Boehringer Mannheim). Rat metaphases were stained with DAPI, and chromosomes were identified with computer-generated reverse DAPI banding. Metaphases were observed under a fluorescence microscope (DMRB, Leica, Germany). Images were captured using a cooled Sensys CCD camera and Quips-Smat capture software (Vysis).

Pig metaphase chromosome spreads were obtained from peripheral blood lymphocyte cultures by standard procedures. Chromosomes were G-banded before hybridization with the G-T-G technique, and pictures were taken using a video printer (Mitsubishi). They were classified according to their G-band pattern as defined by the Committee for the Standardized Karyotype of the Domestic Pig [START_REF] Gustavsson | Standard karyotype of the domestic pig. Committee for the Standardized Karyotype of the Domestic Pig[END_REF]. Whole cosmids containing minisatellite sequences were used as probes and were labeled by incorporation of biotinylated 16 -dUTP (Boehringer Mannheim) by the random priming method [START_REF] Feinberg | A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity[END_REF]. For suppression of background caused by repetitive sequences, the cosmid probe (final concentration 2 g/ml), sonicated pig genomic competitor DNA (200 g/ml), and salmon sperm DNA were ethanol precipitated together, dissolved in 25 ml of hybridization mixture, and prehybridized for 3 h at 37°C. In situ hybridization, detection of the signals, and interpretation of the results were performed as described previously [START_REF] Yerle | Localization of the pig luteinizing hormone/choriogonadotropin receptor gene (LHCGR) by nonradioactive in situ hybridization. Cytogenet[END_REF].

Minisatellite Subcloning and Sequencing

Minisatellite inserts were subcloned into PUC 18 or 19 and partially sequenced from both ends using [ 33 P]-labeled universal and reverse M13 primers and the cycle sequencing kit deltaTaq from USB/Amersham.

Identification of Conserved Regions

Restriction fragments from pig cosmids were purified from agarose gel as described before and used as probes to screen a human chromosome 1 library obtained from the Max-Planck Institute for Molecular Genetics: c112 Reference Library, ICRF [START_REF] Lehrach | Genome Analysis: Genetic and Physical Mapping[END_REF]. This library consists of two high-density gridded filters of 20,000 clones on each membrane. Each clone is named by the number of the library, c112, and a specific number (for example, c112 F0525). Hybridization on the library filters was at 60°C as described by [START_REF] Vergnaud | Polymers of random short oligonucleotides detect polymorphic loci in the human genome[END_REF]. Cross-hybridizing fragments from the human cosmids were identified by hybridization with the pig fragments on restriction digests of human cosmids.

Conserved fragments from both species were subcloned in PUC 18 or 19 and sequenced as described before. Direct (universal) and reverse M13 primers were used for sequencing of both ends, and specific primers were ordered to continue the sequence reaction. The accession numbers of the sequences generated in the course of this project are AJ001151 to AJ001156.

RESULTS

Cloning of Minisatellites

Minisatellites were isolated from human, pig, and rat cosmid libraries by two procedures. The first procedure screens cosmid libraries with synthetic tandem repeat probes [START_REF] Vergnaud | Polymers of random short oligonucleotides detect polymorphic loci in the human genome[END_REF] and the second ("large fragment procedure") does not need a hybridization step (see Materials and Methods). Briefly, hybridization-selected individual cosmids or pools of random cosmids are digested with two frequently cutting restriction enzymes, and fragments above 1 (from hybridization selected cosmids) or 1.5 kb (from pools of random cosmids) are tested by Southern blot hybridization for the presence of a minisatellite.

The number of minisatellites isolated with each method is given in Table 1. Sixty-six, 34, and 43 mini-satellites were isolated from human, pig, and rat libraries, respectively. The efficiency of the large fragment procedure varies from one species to another, with yields of 0.72% in human, 0.41% in rat, and only 0.2% in pig.

The efficiency of synthetic probes is different in the three species. Probes allowing the detection of minisatellites were 13C1, 14C21, 16C2, 16C4, 16C17, and 16C27 in human; 14C24, 15C3, 16C2, 16C7, 16C22, 16C23, 16C24, and 16C26 in pig; and 13C1, 14C3, 14C5, 14C31, 16C2, 16C4, 16C17, 16C20, 16C24, and 16C27 in rat. The most successful STR probes in the detection of minisatellites were 16C27 in human and 16C23 and 16C26 in pig, allowing the detection of 14, 6, and 6 minisatellites, respectively.

Identification of Minisatellite Structures and Sequences

The fragments detected were identified as minisatellites according to their hybridization pattern on Southern blots, i.e., a strong hybridization as expected for a tandem repeat probe. To confirm minisatellite structure, we subcloned and sequenced 20, 15, and 15 fragments from human, rat, and pig, respectively. A tandem repeat structure is observed in the three species for each fragment sequenced. In all but four cases, a repeat unit, with length ranging from 9 to 128 nucleotides, can be defined. Four fragments detecting multilocus hybridization patterns on Southern blots even under high stringency conditions are composed of successive di-, tri-, or tetranucleotide repeats interspersed with a large number of variant repeats. Consensus sequences of four minisatellites from each of the three species are presented in Table 2, together with the length of the repeat unit and internal variations among units. The majority of variants is due to transition events (represented by letters Y and R). Transversions (S, K, or W) and sometimes three possible variants at the same position (represented by letters D, H, V, and B) are also observed. The number of variant sites in repeat units differs between the minisatellites from 2 variants/51 nucleotides in human minisatellite CEB84 and 30 variants/50 nucleotides in pig minisatellite ADGE S1. A length heterogeneity of the units resulting from insertion/deletion is sometimes observed [repeat (AGGG) n in CEB1].

Chromosomal Assignment of the Minisatellite Loci

The cosmids were assigned to a cytogenetic band by a combination of fluorescence in situ hybridization (FISH) and segregation analyses. This second approach was most appropriate for human loci because a physical assignment of highly polymorphic loci can usually be deduced from segregation data of CEPH families [START_REF] Spurr | European Gene Mapping Project (EUROGEM): Genetic maps based on the CEPH reference families[END_REF] and the knowledge of the physical assignment of close-by markers [START_REF] Bray-Ward | Integration of the cytogenetic, genetic, and physical maps of the human genome by FISH mapping of CEPH YAC clones[END_REF] from the CEPH database. Thirty-nine, 33, and 34 cosmids were mapped by FISH in human, pig, and rat, respectively. The minisatellite localization is shown on Figs. 1A (hu- man loci), 1B (pig loci), and 1C (rat loci). A prominent subtelomeric clustering of the minisatellites is a common feature of human and pig genomes. In human, 59 among 66 loci isolated (90%) are in the terminal cytogenetic band of a chromosome. Of 23 terminal human bands with minisatellites, 10 are "major clusters," which we define as the presence of three or more minisatellites. These 10 terminal bands alone contain 60% of minisatellites reported here. The most striking cluster is found on chromosome 1p36.3. The lower proportion of subtelomeric minisatellites in pig (23 of 34; 66%) results from a few interstitial clustered or isolated loci (on chromosomes 5, 6, 7, 8, 10, 12, 13, and 17 ). In the rat genome, the situation is different, with a much lower rate of terminal band clustering (30%).

Comparative Localization of the Minisatellites in the Three Genomes

Human-Pig Comparison

Chromosome 6 alone accounts for 3 of the 11 interstitial sites detected in pig. Interestingly, the physical assignment of the minisatellite cluster at 6q2.1-q2.2 corresponds to the position of an ancestral chromosome fusion event with remnants of telomere arrays [START_REF] Gu | A signal of telomeric sequences on porcine chromosome 6q21-q22 detected by primed in situ labelling[END_REF]. The proximal side of the fusion event is homologous to part of human chromosome 19, and the distal side is homologous to human chromosome 1p36 -p31 as deduced from heterologous chromosome painting experiments [START_REF] Goureau | Human and porcine correspondence of chromosome segments using bidirectional chromosome painting[END_REF] and a number of gene assignments [START_REF] Yerle | The cytogenetic map of the domestic pig (Sus scrofa domestica)[END_REF]. We have identified conserved regions within cosmids containing the pig chr 6q minisatellites to characterize more precisely the homology between pig chr 6q minisatellite loci and human (Fig. 2).

The different fragments obtained after an EcoRI digestion of the four pig cosmids containing minisatellites ADGE S26, S37, S16, and S3 were used to screen a human chromosome 1 cosmid library at moderate stringency. Clones giving a hybridization signal with at least two fragments from the same pig cosmid and no signal with fragments from another cosmid were selected. No clone satisfying this criterion was obtained when fragments from the ADGE S26 cosmid were used. In the three other cases, cross-hybridizing fragments were identified and sequenced, and the human homologous cosmids were assigned to a chromosome 1 cytogenetic band by FISH.

Following is a characterization of the conserved sequences.

Pig ADGE S37-containing cosmid is homologous to human cosmid icrf-c112 F0525 located at chromosome 1p36. The conserved region was included in a 0.9-kb SmaI fragment in the pig cosmid (Accession No. AJ001154) and in a 1.6-kb PvuII fragment in the human cosmid c112 F0525 (Accession No. AJ001151). The fragments show two regions of high homology (88 -90%) separated by a short fragment of low homology (54%). No homology was found within the databases, and no splicing sites were observed. No open reading frame common to the two fragments was found.

Pig ADGE-S16 minisatellite-containing cosmid is homologous to human cosmid icrf-c112 B1018 and corresponds to the P5CDH locus. A part of the pig restriction fragment (PvuII-1.8 kb; Accession No. AJ001155) and a part of the human restriction fragment (BamHI-0.5 kb; Accession No. AJ001152) showed 88 -90 and 100% homology, respectively, each with a different contiguous segment of the human expressed sequence tag HSU24267, located on chromosome 1p36 and encoding the human ␦ 1-pyrroline-5-carboxylate dehydrogenase [START_REF] Hu | Cloning, characterization, and expression of cDNAs encoding human delta 1-pyrroline-5carboxylate dehydrogenase[END_REF]. Splice acceptor and donor site consensus sequences were found at the extremities of each exon.

Pig ADGE-S3 minisatellite-containing cosmid is homologous to human cosmid icrf-c112 D2131 located at chromosome 1p31-p32. The cross-hybridizing fragments in the pig cosmid (BamHI-1.6 kb; Accession No. AJ001156) and in the human cosmid (SmaI-0.7 kb; Accession No. AJ001153) showed two regions each of about 100 bp with 74% homology separated by about 200 bp of unrelated sequence and followed by a region of intermediate homology (66 -70%).

Human-Rat Comparison

We focused our analysis on rat chromosome 1 from which six minisatellite-containing cosmids were isolated and which benefits from relatively intensive gene mapping efforts. Minisatellites R12 and R21 were localized by FISH in the centromeric region of chromosome 1 and R19 in the telomeric region, whereas R10, R15, and R30 are interstitial. The minisatellites' positions with respect to genes and other markers (Fig. 3) have been determined by typing a rat F2 cross with microsatellites isolated from the cosmids (F. Giraudeau, unpublished). Minisatellites R10, R12, R15, R19, and R30 are closely linked with genes Drd4, Prkcg, Igf2, Cyp2c12, and Pygm, respectively (Fig. 3).

DISCUSSION

Minisatellite Cloning

We present here a differential organization of minisatellite loci from the human, pig, and rat genomes. The combination of two independent procedures was highly efficient for the cloning of these loci from genomic cosmid libraries. The method involving synthetic tandem-repeat hybridization probes has already proved its efficiency in detecting polymorphic loci in complex genomes (Mariat and Vergnaud, 1992) and was initially tested on a smaller scale for the cloning of such loci in human [START_REF] Vergnaud | The use of synthetic tandem repeats to isolate new VNTR loci: Cloning of a human hypermutable sequence[END_REF]. The species-specific efficiency of a given synthetic probe in minisatellite cloning in the human, rat, and pig genomes parallels our earlier observations on Southern blots of genomic DNA in a larger range of species (Mariat et al., 1992) and might indicate the existence of species-specific minisatellite families. The second method uses a repeat structure criterion and avoids the selection bias introduced by cross-hybridization. This method showed different isolation efficiencies for human, rat, and pig (0.7, 0.4, and 0.2%, respectively, of the cosmid clones analyzed; Table 1), suggesting that the frequency of minisatellites is highest in human or, alternatively, that on average human minisatellite alleles are longer.

Chromosomal Localization

Results reported here are in agreement with earlier ones conducted in human and suggesting minisatellite clustering in subtelomeric regions of human chromosomes [START_REF] Royle | Clustering of hypervariable minisatellites in the proterminal regions of human autosomes[END_REF][START_REF] Vergnaud | The use of synthetic tandem repeats to isolate new VNTR loci: Cloning of a human hypermutable sequence[END_REF][START_REF] Vergnaud | Detection, cloning, and distribution of minisatellites in some mammalian genomes[END_REF]. Because the same methodology was here applied in a single laboratory to the three genomes, it is clear that the distribution of minisatellite localizations reported now is not an artifact introduced by cloning bias. Three different situations are observed, with 90% of subtelo-FIG. 2. Synteny relationships between pig chromosome 6 and human chromosomes 1 and 19. Large black circles represent minisatellites as localized by FISH. Small circles represent the same minisatellites (names given) as localized by linkage analysis among several genes (for reasons of convenience, only one (CEB108) representative minisatellite of the human chromosome 1p terminal band cluster is shown). Black lines along the chromosomes represent homologous segments between human and pig chromosomes as suggested by heterologous painting experiments [START_REF] Rettenberger | Visualization of the conservation of synteny between humans and pigs by heterologous chromosomal painting[END_REF][START_REF] Goureau | Human and porcine correspondence of chromosome segments using bidirectional chromosome painting[END_REF]. In addition to homologous segments between HSA 1 and 19 and SSC 6, those from HSA 18 and SSC 4, contributing to SSC 6 and HSA 1, respectively, are shown. meric clustering for human minisatellite loci, 66% for pig loci, and only 30% for rat loci. In addition, we demonstrate that minisatellites are not found equally at all chromosome ends. In this respect, human 1pter is most remarkable compared to 1qter. It may be relevant to note that 1p36 is a very strong T band [START_REF] Dutrillaux | Nouveau syste `me de marquage chromosomique: Les bandes T[END_REF] characterized by a high concentration of H3 isochores, CpG islands, and genes [START_REF] Saccone | Identification of the gene-richest bands in human chromosomes[END_REF][START_REF] Zoubak | The gene distribution of the human genome[END_REF]. The much more homogeneous distribution of minisatellites observed in rat would then also be correlated with GC level, which is distributed more homogeneously in rodent chromosomes [START_REF] Sabeur | The isochore patterns of mammalian genomes and their phylogenetic implications[END_REF]. However, a number of discrepancies exist between the location of minisatellite clusters (for instance human 6qter is a minisatellite cluster; Fig. 1) and the location of T bands (6qter is not a T band; [START_REF] Dutrillaux | Nouveau syste `me de marquage chromosomique: Les bandes T[END_REF]. More generally, what is the reason for the relative paucity of minisatellites at some chromosome ends: does it reflect a more recent telomeric location or rather a specific behavior of some subtelomeric domains promoting the creation of minisatellites (either at a higher density or at a comparable density in a larger domain). Further studies and especially sequencing of a number of subtelomeric regions may help address these issues.

Comparative Analysis of Minisatellite-Containing Regions

Comparative gene mapping data available show a correspondence between pig interstitial minisatellitecontaining regions and human chromosome extremi- ties. The genes RNR2 and VWF (Von Willebrand Factor) localized respectively on pig chr 10p12-p13 and chr 5q21 [START_REF] Miyake | Regional localization of rDNA gene on pig chromosome 10 by in situ hybridization[END_REF][START_REF] Sjoberg | FISH mapping of the porcine vWF gene to chromosome 5q21 extends synteny homology with human chromosome 12[END_REF] in the vicinity of interstitial pig minisatellites (Fig. 1) map on human terminal bands on chromosome 14p12 and chromosome 12p13.3, respectively (the VWF gene is also subtelomeric in cattle (chr 5q35), sheep (chr 3q35), and goat (chr 5q35); [START_REF] Janel | The localization of the von Willebrand factor gene on cattle, sheep and goat chromosomes illustrates karyotype evolution in mammals[END_REF].

Pig chromosome 6 results from a telomeric fusion between two chromosomes [START_REF] Gu | A signal of telomeric sequences on porcine chromosome 6q21-q22 detected by primed in situ labelling[END_REF]. Heterologous painting experiments [START_REF] Rettenberger | Visualization of the conservation of synteny between humans and pigs by heterologous chromosomal painting[END_REF][START_REF] Goureau | Human and porcine correspondence of chromosome segments using bidirectional chromosome painting[END_REF] and comparative gene mapping reveal that the two extremities involved are homologous with human chromosome 1p and chromosome 19q (Fig. 2). Pig minisatellites ADGE S26 and ADGE S37 are localized in the band containing the telomere fusion between the two synteny groups, and the human homologue of the ADGE S37 locus is indeed located in 1p36, showing that the pig interstitial chromosome 6 minisatellite cluster is conserved syntenically with the human chromosome 1pter minisatellite cluster. Strikingly, the human homologue of the 6qter pig cosmid containing minisatellite ADGE S3 is located at 1p31-p32, from which we independently characterized a minisatellite locus (Fig. 1).

Although the situation in rat is expectedly much more complex, results from the localization of rat minisatellites provide further support for the hypothesis of a subtelomeric origin of minisatellite loci. As a general rule, comparative analysis between human and rat genomes is hampered by the paucity of mapped genes in rat [START_REF] Bihoreau | A linkage map of the rat genome derived from three F2 crosses[END_REF] and by the large number of chromosomal rearrangements between rodent and human genomes. The number of conserved segments between human and mouse is estimated to be at least 180 [START_REF] Debry | Human/mouse homology relationships[END_REF], whereas it is estimated at less than 50 between human and pig [START_REF] Rettenberger | Visualization of the conservation of synteny between humans and pigs by heterologous chromosomal painting[END_REF][START_REF] Goureau | Human and porcine correspondence of chromosome segments using bidirectional chromosome painting[END_REF]. Comparative mapping of genes in human, mouse, and rat chromosomes X showed the extent of complex rearrangements within a single chromosome [START_REF] Millwood | A gene-based genetic linkage and comparative map of the rat X chromosome[END_REF]. The rat chromosome 1 provides a good model for comparative mapping and chromosomal evolution because it is the longest rat chromosome (about 10% of the genome), and the presence of susceptibility loci for multifactorial diseases [START_REF] Gauguier | Chromosomal mapping of genetic loci associated with the GK rat[END_REF] has led to a dramatic improvement in the density of the linkage map [START_REF] Bihoreau | A linkage map of the rat genome derived from three F2 crosses[END_REF].

There is evidence of conservation between rat chromosome 1 and mouse chromosome 7 with an inversion of a segment at the extremity of mouse chromosome 7 (genes Drd4 and Igf2). Starting from the extremity of the long arm, rat chr 1 is homologous to segments from human chromosomes 10q24 -q25, 11q12-q13, 11p15.5, 10q25q26, 16p13.1-p11, 11p15, and 19q13. R30 is located between PygM and Cyp2c12, which are on two different chromosomes in human. Moreover, minisatellite loci R10 and R15 are located immediately proxi-mal and immediately distal to the two genes Drd4 and Igf2, which are in the terminal 11p15.5 human band.

This collection of observations, together with the striking distribution of minisatellites in human, supports the hypothesis of a subtelomeric formation of minisatellites followed by internalization through chromosomal rearrangements. This hypothesis predicts that interstitial minisatellites are markers of ancestrally telomeric regions of the genome. Not exclusively, one could postulate movements of minisatellites within the genome by a number of mechanisms, as reported, for instance, in [START_REF] Wong | A novel human DNA polymorphism resulting from transfer of DNA from chromosome 6 to chromosome 16[END_REF], [START_REF] Eichler | Duplication of a gene-rich cluster between 16p11.1 and Xq28: A novel pericentromeric-directed mechanism for paralogous genome evolution[END_REF][START_REF] Monfouilloux | Recent human-specific spreading of a subtelomeric domain[END_REF]. However, the events described in these reports produce duplications, triplications, etc., of relatively small domains (less than a few hundred kilobases in [START_REF] Monfouilloux | Recent human-specific spreading of a subtelomeric domain[END_REF] and 26 kb in [START_REF] Eichler | Duplication of a gene-rich cluster between 16p11.1 and Xq28: A novel pericentromeric-directed mechanism for paralogous genome evolution[END_REF]), whereas the interstitial pig minisatellite cluster on chromosome 6 described here is part of a very large conserved segment between human and pig and has not been duplicated. Interestingly, in the two independent examples reported in [START_REF] Wong | A novel human DNA polymorphism resulting from transfer of DNA from chromosome 6 to chromosome 16[END_REF] and [START_REF] Eichler | Duplication of a gene-rich cluster between 16p11.1 and Xq28: A novel pericentromeric-directed mechanism for paralogous genome evolution[END_REF], one of which contains a minisatellite, the ancestral site of the transposition event is telomeric (at respectively 6pter and Xq28) and both target sites are within band 16p11.

No general function has been attributed to minisatellites, but some of them do have a biological role. They can be coding sequences [START_REF] Boguski | Evolution of the apolipoproteins[END_REF][START_REF] Paulsson | Balbiani ring 1 gene in Chironomus tentans. Sequence organisation and dynamics of a coding minisatellite[END_REF] or regulatory elements [START_REF] Krontiris | An association between the risk of cancer and mutations in the HRAS1 minisatellite locus[END_REF][START_REF] Bennett | Susceptibility to human type 1 diabetes at IDDM2 is determined by tandem repeat variation at the insulin gene minisatellite locus[END_REF]. The abnormal expansion of a minisatellite can create a fragile site [START_REF] Virtaneva | Unstable minisatellite expansion causing recessively inherited myoclonus epilepsy, EPM1[END_REF][START_REF] Yu | Human chromosomal fragile site FRA16B is an amplified AT-rich minisatellite repeat[END_REF]. The analysis of neomutated alleles at other, nonpathological hypermutable minisatellites has shown that they evolve predominantly by gene-conversion-like mechanisms, presumably initiated by double-strand breaks [START_REF] Buard | Complex recombination events at the hypermutable minisatellite CEB1 (D2S90)[END_REF][START_REF] Jeffreys | Complex gene conversion events in germline mutation at human minisatellites[END_REF], whereas microsatellites most likely mutate by replication slippage mechanisms [START_REF] Heale | The stabilization of repetitive tracts of DNA by variant repeats requires a functional mismatch repair system[END_REF]. Therefore, a good knowledge of domains in which minisatellites are likely to be found within the human genome may accelerate the identification of genetic disorders associated with this type of tandem repeat element.

a

  FIG. 1. Chromosomal localization of minisatellites loci on human (A), pig (B), and rat (C) chromosomes (Giemsa banding). Minisatellites localized by both FISH and linkage analyses are represented by black circles. Minisatellites localized only by linkage analysis to physically assigned polymorphic markers are represented by open circles. The two loci represented at the tip of the short arm of the X chromosome are

FIG. 3 .

 3 FIG. 3. Comparative mapping between rat chromosome 1 and human and mouse chromosomes. (a) Localization of rat minisatellites (represented by black circles) by FISH. (b) Localization of rat minisatellites (small black circle and name R-) among genes as determined by linkage analysis. Genetic distances in cM between minisatellites and closest genes are indicated. (c) Localization of the homologous genes in human (physical localization). (d) Localization of the homologous genes in mouse (genetic localization: on the right of the black line are gene names and chromosome numbers and on the left the position of the gene in centimorgans on the genetic map). The relative positions of genes in human and mouse were obtained from NCBI (http://www.ncbi.nlm.nih.gov/SCIENCE 96/) for human and from The Jackson Laboratory (http://www.jax.org/) for mouse.

TABLE 1 Number of Minisatellite-Containing Cosmids Isolated with Each of the Two Methods

 1 Method 2: the large fragment procedure with no hybridization screening of clones (indicating total number of cosmid clones analyzed in pools).

		Number of minisatellite-
		containing cosmids isolated
				Number of
			Method 2 b	cosmids localized
	Species	Method 1 a	(clones analyzed)	by FISH c
	Human	30	36 (5000)	39
	Pig	27	7 (3500)	33
	Rat	15	28 (7400)	34

a Method 1: screening with synthetic tandem repeat probes. b c Number of clones localized by FISH.

TABLE 2 Nucleotide Sequence of a Set of Minisatellites from Human, Pig, and Rat Genomes

 2

	Name a	Unit sequence b	Unit length c
	CEB 1	GRGGGAGGGARGG(AGGG) n TGG(C)CTGCRGAGGTCCCYGGGYTGA	37-43 bp
	CEB 8	GGGGRAGBDT	10 bp
	CEB 62	AYCTACCCCRGGS(C)AGCAGGSAGGAGGCWGC	30-31 bp
	CEB 84	GGTCACATGTGGGGGGGCTGGRGTCAGASAGACCAAGGTTGGAACCCGGCA	51 bp
	ADGE S1	AYGWGDKYTYRARYCHCARTMTRKHTTYWKRYTGKGWTYRAGTYCYRRS(Y)	49-50 bp
	ADGE S2	GGATGRGGWCRGGGACR(GGG(R)CA)	17-23 bp
	ADGE S12	HAG(G)TTCAGGTGATGGRVG	19-20 bp
	ADGE S40	Y(A)GARR(R)CAGGVCWGTGTYTCYCYTCAGCCYGGGG(Y)TC(YC)	35-40 bp
	CEB R10	CCC(AAT)AT	5-8 bp
	CEB R15	AYBRKGBH(C)T(T)CC	11-13 bp
	CEB R21	AGRYRCCASRGYYRRR	16 bp
	CEB R31	DGCYCATCAGTGGGAGYGTCC(G)AKTGAGA	28-29 bp

ACKNOWLEDGMENTS

The authors thank Professor Howard Cann for helpful comments on the manuscript. This work was supported by the PiGMaP project (EC Contract BIO2-CT94-3044), by the EUROGEM project (EC Contract GENE-CT93-0101), and by a rat genome project (EC Contract BI04-CT96-0372). Research at Oxford was supported by the Wellcome Trust. M.L. holds a Wellcome Trust Principal Fellowship. More detailed information on individual markers can be accessed at our Web site (http://www.igmors.u-psud.fr/iech).


Michel Arock

Alexandra Le Nours

Odile Malbec

MD Marc Daëron
email: daeron@pasteur.fr

Michel Arock #

Alexandra Le Nours #

Ex vivo and in vitro Primary Mast Cells

Keywords: Mouse, rat, human, mast cells, purification, culture

Ex vivo and in vitro primary mast cells.

INTRODUCTION

Like many other cells of the myeloid lineage, mast cells stand at the interface between innate and adaptive immunity. They contribute to anti-infectious defense when pathogens interact directly with specific receptors [1] or indirectly through the binding of bacterial products to Tolllike receptors (TLRs) [2,3]. They initiate the allergic reaction when their high-affinity IgE receptors (FcεRI) [4], which are constitutively occupied by IgE antibodies, are aggregated by a multivalent allergen [5]. They also initiate IgG-dependent inflammatory responses when immune complexes aggregate their low-affinity IgG receptors (FcγRIIIA in mice) in murine models of autoimmune diseases [6]. Mast cell biology has therefore been actively investigated, in mice, rats and humans.

Mast cells are widely distributed among tissues, in virtually all organs [7]. In all cases, however, mast cells represent a minor population. Sources of homogenous mast cells have therefore been searched for and used for experimental studies. Methods based on differential sedimentation in high-density medium have proved useful to obtain reasonably pure ex vivo mature tissue mast cells [START_REF] Salari | Novel technique for isolation of human lung mast cells[END_REF][START_REF] Hachisuka | Purification of rat cutaneous mast cells with Percoll density centrifugation[END_REF][START_REF] He | Mast-cell heterogeneity: functional comparison of purified mouse cutaneous and peritoneal mast cells[END_REF]. One such method is described here. The major limitation of these techniques is their poor yield, making biochemical analyses difficult. For this reason, transformed mast cell lines of various origins have been extensively used. The most popular has been the Rat Basophilic Leukemia (RBL) cell line, which was understood to be a mucosal-type immature rat mast cell line [11,[START_REF] Seldin | Homology of the rat basophilic leukemia cell and the rat mucosal mast cell[END_REF]. RBL cells have been invaluable for the biochemical characterization and the cloning of FcεRI subunits [START_REF] Blank | Complete structure and expression in transfected cells of high affinity IgE receptor[END_REF], as well as the dissection of proximal signaling following FcεRI aggregation [START_REF] Turner | Signalling through the high-affinity IgE receptor Fc epsilonRI[END_REF]. RBL has also been widely used for transfection of cDNAs and for the mutagenesis analysis of the functional properties of a variety of membrane receptors. A few mouse and human tumor mast cell lines have been described, often lacking FcεRI [START_REF] Dunn | A transplantable mast-cell neoplasm in the mouse[END_REF][START_REF] Barsumian | Establishment of four mouse mastocytoma cell lines[END_REF][START_REF] Butterfield | Establishment of an immature mast cell line from a patient with mast cell leukemia[END_REF][START_REF] Kirshenbaum | Characterization of novel stem cell factor responsive human mast cell lines LAD 1 and 2 established from a patient with mast cell sarcoma/leukemia; activation following aggregation of FcepsilonRI or FcgammaRI[END_REF][START_REF] Ko | Differential regulation of CC chemokine receptors by 9-cis retinoic acid in the human mast cell line[END_REF][START_REF] Demehri | Establishment of a murine model of aggressive systemic mastocytosis/mast cell leukemia[END_REF]. In any case, tumor mast cells are not suitable for addressing issues such as differentiation and proliferation. Cultured mast cells, derived from various sources of precursor cells permitted such studies. Hematopoietic stem cell-derived Mast Cells from mice (mBMMC) [21], rats (rBMMC) [22] or from humans (HMC) [START_REF] Saito | Selective growth of human mast cells induced by Steel factor, IL-6, and prostaglandin E2 from cord blood mononuclear cells[END_REF] have been instrumental for analyzing growth factor requirements and intracellular signaling leading to mast cell proliferation and activation. Considerable numbers of pure primary mouse mast cells can indeed be generated and kept in culture for several months, enabling in vitro biochemical analyses and in vivo studies, especially by reconstituting mast cell-deficient mice with cultured mast cells derived from genetically modified mice [START_REF] Abe | Reconstitution of mucosal mast cells in W/WV mice by adoptive transfer of bone marrow-derived cultured mast cells and its effects on the protective capacity to Strongyloides ratti-infection[END_REF].

MATERIALS

Mouse mast cells

Ex vivo mouse mast cells

1. 6-9 week-old mice 2. Metrizamide 3. Tris-A-EDTA buffer containing 0.025 M Tris-HCl, 0.12 M NaCl, 0.005 M KCl, 0.01 M EDTA and 0.5 mg/mL human serum albumin 4. siliconized 10 mL glass tubes 5. Toluidine blue solution containing 0.5% toluidine blue, 30% ethanol pH 1

Cultured mouse mast cells

Mouse Bone Marrow-derived Mast Cells (mBMMC)

1. 6-9 week-old mice 2. RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 IU/mL) and streptomycin (l00 µg/mL) (complete RPMI) 3. Recombinant mouse IL-3 1

Wright-Giemsa stain

Hal-Pasteur author manuscript pasteur-00269312, version 1 5. Mota's fixative prepared as follows: add 4 g of lead acetate (basic) to 50 mL of distilled deionized water; stir at slow speed and add 2-4 mL of glacial acetic acid to dissolve the lead acetate and make the solution clear; add 50 mL of absolute ethanol; keep tightly closed and store at room temperature; prepare fresh every 1-2 month.

Rat mast cells

Ex vivo rat mast cells

1. 10-15 week-old rats 2. Metrizamide 3. Tris-A-EDTA buffer containing 0.025 M Tris-HCl, 0.12 M NaCl, 0.005 M KCl, 0.01 M EDTA and 0.5 mg/mL human serum albumin 4. siliconized 10 mL glass tubes 5. Toluidine blue solution containing 0.5% toluidine blue, 30% ethanol pH 1

Cultured rat mast cells

1. 10-15 week-old rats 2. Iscove's modified dulbecco's medium supplemented with 5 x 10 -5 M 2-Mercaptoethanol, penicillin (100 IU/mL), streptomycin (l00 µg/mL), 20% heat-inactivated fetal calf serum serum and 2-10 ng/mL of recombinant rat IL-3 (rRIL-3) 2 (complete IMDM) 3. Hayem's solution

Human mast cells

Ex vivo human mast cells

METHODS

Mouse mast cells

Ex vivo mouse mast cells

Mouse mast cells can be purified from cells recovered by peritoneal washing, among which they represent 3-4% of total cells. 1. Lethally anesthetized 6-9 week-old mice are injected intraperitoneally with 2 mL Tris-A-EDTA buffer. Following careful massage of the abdomen, the abdominal cavity is opened and the fluid is recovered with a Pasteur pipette.

2. Cells are washed once and resuspended at 1 x 10 7 /mL in the same buffer at room temperature.

3. Aliquots of 2 mL peritoneal cell suspension are gently layered over 1.5 mL of 22.5% metrizamide dissolved in Tris-A-EDTA buffer, in siliconized tubes, and centrifuged at 270 g for 10 min at room temperature. Most cells sediment at the interface above metrizamide, while mast cells sediment at the bottom of the tube.

4. The upper layer, the interface (containing cells) and most of the metrizamide solution are VERY carefully discarded, leaving 50-100 µL above the small mast cell pellet 4 . Pelleted cells are gently resuspended and transferred into new tubes. 5. Under these conditions, 1 x 10 5 mast cells (>95% pure) can be recovered per mouse.

Cultured mouse mast cells

Hal-Pasteur author manuscript pasteur-00269312, version 1

Mouse Bone Marrow-derived Mast Cells (mBMMC)

Bone marrow (BM) cells are harvested from the femurs of 6-9 week-old mice.

1. After lethal anesthesia of the animals, femurs are carefully dissected to eliminate any muscle fragment.

2. One extremity is cut with sterile scissors, and the needle of a 10-mL syringe, previously filled with sterile RPMI is inserted in the medullar canal. Bone marrow cells are flushed out and collected in a sterile 15-mL tube.

3. After sedimentation of aggregates, cells in suspension are collected in another 15-mL tube, centrifuged, and the equivalent of cells from two femurs are resuspended in 60 mL complete OptiMEM supplemented with 1ng/ml recombinant murine IL-3.

4. The cell suspension is transferred into culture flasks and placed at 37°C in a humidified C0 2 incubator.

5. Every 3-4 days (i.e. twice a week), non-adherent cells are centrifuged, resuspended at 3x10 5 viable nucleated cells/mL in fresh IL-3-containing culture medium, and placed in new culture flasks.

6. After 3 weeks, cultures consist of virtually pure (>90%) FcεRI + , Kit + mast cells as judged by staining and immunofluorescence.

7. mBMMC thus obtained keep growing in IL-3 containing medium for at least 2-3 months 5,6 .

Identifying and phenotyping mouse mast cells

Wright-Giemsa staining

1. Cells are resuspended at 2x10 5 cells/mL in RPMI.

2. One hundred µL suspension are placed in cytospin chambers, mounted onto clean slides.

Slides are spun at 14 g for 5 min.

3. They are air-dried and stained with Wright-Giemsa before they are mounted in 1-2 drops Permount under a cover slip.

Toluidine Blue staining

1. Cytospin slides prepared as above are fixed by adding several drops of Mota's fixative to cover the cells for 10 min. NB: Mota's evaporates quickly, so additional drops must be added once or twice to prevent crystal formation.

2. Fixative is removed by slowly running water down, not directly on cells, and slides are dried.

3. Cells are stained for 20 min with 2-3 drops of acidic toluidine blue.

4. Stain is removed by running water down the slide.

5. Slides are air dried and mounted in 1-2 drops Permount under a cover slip 6 . 2. They are washed twice in PBS and incubated for 30 min at 0°C in 50µL complete RPMI containing an appropriate dilution of F(ab') 2 fragments of FITC-labeled rat anti-mouse immunoglobulin (for mouse IgE) or FITC-labeled mouse anti-rat immunoglobulin (for ACK2). Negative controls are incubated with FITC-labeled secondary F(ab') 2 only.

Analysis of the Expression of FcεRI and Kit by Immunofluorescence

3. Cells are washed once and resuspended in 500 µL PBS. 4. Fluorescence is analysed by flow cytometry.

Rat mast cells

Hal-Pasteur author manuscript pasteur-00269312, version 1

Ex vivo rat mast cells

The same procedure as that described for mouse mast cells can be applied to rat peritoneal cells.

Due to the body weight of rats, mast cells can be obtained in relatively high numbers 4 .

Cultured rat mast cells

1. Bone marrow cells are obtained from outbred male Wistar rats, 10-15 week-old (weight 225-250 g) with the same method as for mouse mast bone marrow cells except for the following points.

2. Complete IMDM is used as culture medium; 3. Mononuclear cell counts are performed by dilution of the cell suspension (1:10) in Hayem's solution and numeration in a hemocytometer; cell suspension is therefore adjusted to 2.5 x 10 5 viable nucleated BM cells/mL in complete IMDM. 4. A final concentration of 2.5 x 10 5 viable nucleated BM cells/mL is suspended in complete Iscove medium in 175 cm 2 flasks that are kept at 37°C in a humidified incubator with 5% CO 2 in air.

5. These cultures are refed and restimulated with fresh complete medium containing recombinant rat IL-3 every 4-6 days.

6. Percentage of mast cells is determined by differential counts (400 cells) on Wright-Giemsa and toluidine blue stained cytocentrifuge preparations.

7. Rat BMMC proliferate rapidly initially, representing 85% of cells after approximately 15 days. After this, the percentage of BMMC increases slowly to almost 100% before cell viability declines around the fifth week 7 8 .

Human mast cells

Ex vivo human mast cells

Mast cells with different phenotypes can be purified from various human tissues, including lung, intestine, uterus, skin or foreskin. The general protocol comprises a first step of enzymatic treatment of the desired tissue, in order to obtain a monocellular suspension. This enzymatic treatment is followed by a first step of purification by centrifugation over Percoll gradient and mast cells are further by separation from contaminating cells using anti c-kit immunomagnetic beads 9 .

Tissue processing

Human mast cells are isolated under sterile conditions by a four-step enzymatic tissue dispersion method.

1. Macroscopically normal human tissue is obtained from surgical specimens.

2. The tissue is placed in TE buffer at 4°C immediately after resection until dissection is started.

3. If present (human intestine) the mucosa is separated mechanically from the submucosa/muscular layers. Always for intestine specimen, mucus is removed by incubation with acetylcysteine at 1 mg/mL, and epithelial cells are detached with 5 mM EDTA. 4. Whatever the nature of the specimen used, the tissue is enzymatically digested by a fourstep incubation (each for 30 min) with four enzymes (3 mg/mL pronase corresponding to 21 U/mL, 0.75 mg/mL chymopapain corresponding to 0.39 U/mL, 1.5 mg/mL collagenaseD corresponding to 0.405 U/mL, and 0.15 mg/mL elastase corresponding to 15.75 U/mL).

During the first digestion step, the tissue (mucosa, in case of intestinal specimen) is chopped finely with scissors. For the third and fourth digestion steps, the incubation buffer is supplemented with DNase at 15 µg/mL corresponding to 15 U/mL. 5. The cells freed after the last two digestion steps are separated from tissue fragments by filtration through a polyamide Nybolt filter (pore size, 300 µm), washed, pooled, and counted after staining with Turk's solution.

The viability of cells is measured by dye exclusion using trypan blue staining

Hal-Pasteur author manuscript pasteur-00269312, version 1

Enrichment of mast cells

Cells are fractionated on discontinuous Percoll gradients and further enriched in mast cells by separation from contaminating cells using immunomagnetic beads.

1. Five mL of cell suspension resuspended in HA buffer are layered carefully over 20 mL of Percoll solution (density = 1.037 g/mL) in a 50 mL polypropylene tube.

2. After centrifugation (500 X g, 20°C, 15 min), the cell sediment is harvested, washed twice with HA buffer and counted.

3. For immunomagnetic separation, cells are resuspended in 1 mL of HA buffer containing 1 mg/mL albumin and 5 µg/mL mAbYB5.B8 (directed against human c-kit) and incubated for 30 min at 4°C with gentle rolling.

4. The cells are then washed in HA buffer and resuspended in 500 µL of HA buffer containing 1 mg/mL albumin. 5. Finally, the cell suspension is incubated with a goat-anti mouse IgG Ab coupled to paramagnetic beads for 30 min at 4°C. During incubation the tubes are gently rolled.

6. After washing in HA buffer, mast cells are enriched by magnetic separation of the cells using a MACS C1 column placed in magnetic field. 7. After separation, cells are counted, and washed in culture medium without antibiotics.

Cultured human mast cells derived from hematopoietic progenitors

Pure populations of human mast cells (HMC) are obtained by long-term culture of human normal hematopoietic progenitors (CD34+ cells) in the continuous presence of Stem Cell Factor (SCF).

Sources of these CD34+ cells are mainly bone marrow, cord blood or peripheral blood. Usually, mononuclear cells from bone marrow and cord blood contain 0.5-1% of CD34+ cells, whereas peripheral blood contains less than 0.1% of CD34+ cells. Hematopoietic progenitor cells can be rapidly and efficiently enriched to a purity of about 85-98 % using positive selection of magnetically labeled CD34+ cells. Mononuclear cells from peripheral blood (PBMC), cord blood, or bone marrow are obtained by density gradient centrifugation over Ficoll Paque®. For immunomagnetic separation, CD34+ hematopoietic progenitor cells are magnetically labeled using MACS CD34 MicroBeads. The magnetically labeled cells are enriched on positive selection columns in the magnetic field 10 . total cells to the cell suspension to inhibit unspecific or Fc-receptor mediated binding of CD34 MicroBeads to non-target cells. Label cells by adding 100 µl CD34 MicroBeads per 10 8 total cells, mix well and incubate for 30 min at 6°-12°C. Wash cells carefully and resuspend in appropriate amount of buffer (MS+/RS+ column: 500-1000 µl; LS+/VS+ column: 1-10 mL, max. 2x10 8 cells per mL). Proceed to magnetic separation. 9 Mononuclear Cells: Choose a column type (MS+/RS+ or LS+/VS+) according to the number of total unseparated cells and place it (with column adapter) in the magnetic field of the MACS separator. Fill and rinse with buffer (MS+/RS+: 500 µl; LS+/VS+: 3 mL). Pass mononuclear cells through 30 µm nylon mesh or Pre-Separation Filter to remove clumps. Wet filter with buffer before use. Apply cells to the column, allow cells to pass through the column and wash with buffer (MS+/RS+: 3x500 µl; LS+/VS+: 3x3 mL). Remove column from separator, place column on a suitable tube and pipette buffer on top of column (MS+/RS+: 1 mL; LS+/VS+: 5 mL). Firmly flush out retained cells with pressure using the plunger supplied with the column. Repeat magnetic separation step: apply the eluted cells to a new prefilled positive selection column (for <10 7 CD34+ cells: MS+/RS+; for <10 8 CD34+ cells: LS+/VS+), wash, and elute retained cells in buffer (MS+/RS+: 500 µl; LS+/VS+: 2.5 mL).

Purification of CD34 positive progenitors from human cord blood, bone marrow or peripheral blood cells

Magnetic Separation of <2x10

5. Magnetic Separation of 2x10 9 -2x10 10 Mononuclear Cells: Assemble XS+ column and place it in the column holder of the SuperMACS using XS+ column adapter. Turn 3-way-stopcock to position "fill". Fill the column from the bottom with buffer from the syringe until the buffer reaches the syringe cylinder. Turn the 3-way-stopcock to position "run" and rinse column by filling from the top with buffer. Allow buffer to run into the column. Then, add more buffer. Rinse with 50 mL of buffer. Close 3-way-stopcock; leave the syringe attached during separation, except when refilling with buffer. Move column in the magnetic field of the SuperMACS by turning the handle. Pass cells through 30 µm nylon mesh or filter to remove cell clumps. Apply cells into the syringe cylinder that is set up on the XS+ column and turn 3-way-stopcock to position "run". Allow the cells to pass through the column.

Remove flow resistor and wash with 4x30 mL buffer. Close 3-way-stopcock and remove column out of the magnetic field of the SuperMACS by turning the XS+ adapter handle backward. Detach syringe from the 3-way-stopcock, fill with buffer and attach to port A of the XS+ column. Elute retained cells with 20 mL buffer using the syringe. Repeat magnetic separation step: apply the eluted cells to a new prefilled XS+ column or VS+ column, wash, and elute retained cells in buffer.

6. Evaluation of Hematopoietic Progenitor Cell Purity: The purity of the isolated hematopoietic progenitor cells can be evaluated by flow cytometry or fluorescence microscopy. Fluorescent staining of CD34+ cells can be accomplished by direct immunofluorescent staining using an antibody recognizing an epitope different from that recognized by the CD34 monoclonal antibody QBEND/10 (e.g. CD34-PE). For optimal discrimination of CD34+ cells from other leukocytes, counterstain cells with an antibody against CD45 (e.g. CD45-FITC). CD34+ cells express CD45 at a lower level as compared to lymphocytes. Use the antibodies in appropriate concentrations recommended by the manufacturers. Typically, staining for 5 min at 6°-12°C should be sufficient. After fluorescence staining, cells should be washed and resuspended in buffer.

Culture conditions

Hal-Pasteur author manuscript pasteur-00269312, version 1 1. CD34 positive cells are cultured at 37°C in a humidified atmosphere containing 5% CO2 at a starting density of 1 x 10 5 cells/mL in SFCM.

2. CD34 + cells may initially proliferate 100 times or more the starting number of cells if rhSCF is combined with rhIL-3 and rhIL-6. Luxurious growth is observed during the first 2-3 wk, although debris will begin to accumulate because of non-mast cell lineage apoptosis and necrosis.

3. Adherent macrophages also may start increasing in number by 2 wk. Check cultures weekly and separate nonadherent mast cell-committed progenitors from adherent cells and debris.

Gently pipette and remove nonadherent cells to a new flask or centrifuge nonadherent cells and supernatant at 150g for 5 min, resuspend cells in complete media with growth factors every 5 to 7 days.

The number and percentage of mast cells (MC) in the cultures are assessed weekly using

Wright-Giemsa and acidic toluidine blue (see mouse mast cell culture section) staining of cytocentrifuged samples. Usually, 10 6 CD34+ cells at 8-10 wk may give rise to 5-15 x10 7 HHPMC with less than 5% contamination with other cell types.

5. Immunostaining for tryptase can be also used to further identify mast cells in the cultures.

Briefly, cytocentrifuged preparations are fixed in cold acetone for 30 min, air-dried, then stored at -20°C. Fixed samples are then treated with mouse IgG1 anti-tryptase monoclonal antibodies, and finally submitted to a Fast-Red immunostaining.

6. After >10 weeks in culture, more than 95 % of the cells are identified as mast cells by their morphological features, the presence of metachromatic granules and their positivity for tryptase. These cells can then used for subsequent experiments as pure normal HMC 11 12 .

7.

Since CD34 positive-derived mast cells usually do not express spontaneously a significant amount of FcεRI at their membrane, they can be incubated for 5 additional days with 2-10 µg/mL of human myeloma IgE and 20 ng/mL of recombinant human IL-4 (rhIL-4) in order to enhance the expression of FcεRI on their membrane. MUST be discarded. This is why siliconized tubes are used. Carefully wiping tube walls before collecting mast cell pellets may also improve purity.

5. Bone marrow cells can be kept frozen in liquid nitrogen before they are cultured. When frozen, mBMMC quickly die upon thawing. 7. Rat bone marrow cells can be kept frozen in liquid nitrogen before they are cultured, but it is impossible to keep rBMMC frozen. 8. Mast cell numbers are calculated exactly as described above for mBMMC cultures. 9. Isolated human mast cells cannot be frozen using classical techniques 10. Once purified, CD34+ cells can be kept frozen in liquid nitrogen before they are cultured, whereas it is impossible to keep mature HMC frozen.

11. Isolated human MC can be maintained in culture for up to 3 months in the continuous presence of recombinant human SCF. MC purity increases during culture and reached nearly 100%. During the first week of culture, MC numbers decrease, but after that time they start to proliferate. Cultured MC do not change their histamine content, phenotype or morphology. They are even more responsive towards IgE-dependent stimulation, which causes the release of high amounts of histamine, leukotrienes and cytokines such as TNFalpha and IL-5.

12. Once pure, HMC cultures stop growing at 8-10 weeks. They nevertheless keep alive without any significant changes in their phenotype for another 2-5 months provided that they are maintained in SCF-containing medium.

Hal-Pasteur author manuscript pasteur-00269312, version 1

1 .

 1 Aliquots of 5 x 10 5 cells are incubated in 50µL complete RPMI containing 10µg/mL mouse IgE or rat anti-mouse Kit mAb (ACK2) for 1 hr at 0°C in Eppendorf tubes.

3. 2 2 .

 22 .3.1.1. Sampling of Cord Blood Cells 1. Dilute anticoagulated (preservative-free heparin sodium, 1.0 mL for 50 mL of sample) cord blood 1:4 with PBS. 3.2.3.1.2. Sampling of Bone Marrow Cells 1. Collect bone marrow in 50 mL tubes containing 5 mL PBS containing 2 mM EDTA or 0.6 % ACD-A or 200 U/mL heparin and store at 4°C if the cells cannot be processed immediately. For release of the cells from aggregates, dilute in 10x excess of RPMI 1640 medium containing 0.02 % collagenase B and 100 U/mL DNAse and shake gently at room temperature for 45 min. 3. Pass cells through 30 µm nylon mesh or filter. Wet filter with buffer before use. 3.2.3.1.3. Sampling of Peripheral Blood Cells 1. Start with fresh human blood treated with an anticoagulant, e.g. heparin, citrate, ACD-A or citrate phosphate dextrose (CPD) or leukocyte-rich buffy coat not older than 8 hrs. Dilute cells with 2-4 volumes of PBS containing 2 mM EDTA or 0.6 % ACD-A. 2. Obtention of a mononuclear cell suspension: Carefully layer 35 mL of diluted cell suspension over 15 mL of Ficoll-Paque®. Centrifuge for 35 min at 400xg at 20°C in a swinging-bucket rotor (without brake). Aspirate the upper layer leaving the mononuclear cell layer undisturbed at the interphase. Carefully collect interphase cells and wash twice in PBS containing 2 mM EDTA or 0.6 % ACD-A. Centrifuge for 10 min at 300xg at 20°C. Resuspend cell pellet in a minimal volume of buffer. Mononuclear cell counts are performed by dilution of the cell suspension (1:100) in Hayem's solution and numeration in a hemocytometer. Adjust the cell suspension at 300 µl of buffer per 10 8 total cells. For less than 10 8 total cells, use 300 µl. Proceed to magnetic labeling. 3. Magnetic Labeling of CD34+ Progenitor Cells: Add 100µl FcR Blocking Reagent per 10 8

6 .

 6 Mast cell numbers are calculated by determining the percentage of acidic toluidine blue positive cells out of total Wright-Giemsa-positive cells. Toluidine blue should stain mast cells red-purple (metachromatic staining) and the background blue (orthochromatic staining). Metachromasia, tissue elements staining a different color from the dye solution, is due to the pH, dye concentration and temperature of the basic dye. Blue or violet dyes will show a red color shift, and red dyes will show a yellow color shift with metachromatic tissue elements.

Hal-Pasteur author manuscript pasteur-00269312, version 1


Fahem Kebair
email: fahem.kebair@univ-lehavre.fr

Frédéric Serin
email: frederic.serin@univ-lehavre.fr

Cyrille Bertelle
email: cyrille.bertelle@univ-lehavre.fr

Agent-Based Perception of an Environment in an Emergency Situation

Keywords: Factual agent, Multiagent system, Ontology, Semantic feature, Taxonomy

We are interested in the problem of multiagent systems development for risk detecting and emergency response in an uncertain and partially perceived environment. The evaluation of the current situation passes by three stages inside the multiagent system. In a first time, the situation is represented in a dynamic way. The second step, consists to characterise the situation and finally, it is compared with other similar known situations. In this paper, we present an information modelling of an observed environment, that we have applied on the RoboCupRescue Simulation System. Information coming from the environment are formatted according to a taxonomy and using semantic features. The latter are defined thanks to a fine ontology of the domain and are managed by factual agents that aim to represent dynamically the current situation.

Introduction

Recent catastrophic disasters have brought urgent needs for diverse technologies for disaster relief. Currently, there is an overwhelming need for better information technology to help support the efficient and the effective management of the disaster management (also known as emergency response). In particular, actors and agencies need an assistance to help them to make a decision in a fashion time and to be able to coordinate their efforts in a flexible way in order to prevent further problems or effectively manage the aftermath of a disaster. Our project is situated in this context and consists to develop a generic Decision Support System (DSS), able to detect a risk in an uncertain and partially perceived environment and to prevent its evolution. The DSS kernel is a multiagent system with three layers, where each one has a specific role. The role of the lower layer, that we call the representation layer, is to represent the environment state and its evolution over the time. The environment is perceived as a whole of entities, directly or indirectly observable and of which states change permanently. These entities are modeled according to a taxonomic organisa-tion and information that describe them are formatted according to a model of "semantic features", inspired by the memento design pattern rules [START_REF] Gamma | Design Patterns : Elements of Reusable Object Oriented Software[END_REF]. Moreover, the system apprehends these information via software agents (called factual agents) and according to an ontology of the studied domain. The collaboration of these agents and their comparisons with each other, form dynamic agents clusters. The latter are compared by past known scenarios. The final object of the study is to permit to prevent the occur of a crisis situation and to provide an emergency management planning. This modelling was elaborate starting from the game of Risk [START_REF] Person | From Three MultiAgent Systems to One Decision Support System[END_REF]] and tested on the RoboCupRescue Simulation System (RCRSS) [RoboCupRescue]. In this paper, we provide a modelling of information extracted from an observed environment in an emergency context. Inside the system, information are managed thanks to factual agents that interact by comparing each other. The modelling includes a definition of a taxonomy. The latter was applied to the RCRSS environment, for which we have defined an ontology of the domain. The structure of the paper is as follows: first we present the general architecture of the DSS and its internal kernel. Then, we define the taxonomic organisation of the perceived environment. After that, we present the RCRSS environment and the ontology of the domain. Finally, we present factual agents and some tests using graphic tools.

Decision Support System

The role of the Decision Support System is quite wide. In general, the purpose is "to improve the decision making ability of managers (and operating personnel) by allowing more or better decisions within the constraints of cognitive, time, and economic limits" [START_REF] Holspace | Decision support systems[END_REF]]. More specifically, the purposes of a DSS are:

• Supplementing the decision maker,

• Allowing better intelligence, design, or choice,

• Facilitating problem solving,

• Providing aid for non structured decisions,

• Managing knowledge.

Decision makers need quick responses to events that take place at a continually increasing rate and they should incorporate an enormous amount of knowledge such as data, choices and consequences. Also they must have fast access to consistent, high-quality knowledge to compete [Kim 2005].

In our context, the DSS is used as an emergency management system, able to assist actors in urban disasters mitigation and to prevent them about potential future critical consequences. The system includes a body of knowledge which describes some aspects of the decisionmaker's world and that comprises the ontology of the domain and past known scenarios. The kernel of the DSS is a multiagent system with three layers. Agents of each layer have their own way of behaving and communicating.

Representation layer : This layer is composed by factual agents and has as essential aim to represent dynamically and in real time the information of the current situation. Each new entering information is dealt by a factual agent that intends to reflect a partial part of an observed situation. Agents interactions and more precisely, aggressions and mutual aids reinforce some agents and weaken some other.

Characterisation layer : This layer has as aim to gather factual agents, emerged from the precedent layer, using clustering algorithms. We consider a cluster of agents, a group of which agents are close from dynamic and evolution manner point of view. The goal here, is to form dynamic structures, where each one is managed by a characterisation agent.

Prediction layer : This layer is made up of prediction agents. Each one represents an observed scenario originally from the current situation. The task of the prediction agents is to compare their scenarios by past ones to provide a closed one of which result may be a potential consequence. This mechanism is based on the case base reasoning, the latter differs from a classic one by its ability to manage a dynamic and an incremental development.

Taxonomic Organisation of the Studied Environment

Our perception of the environment focuses on two aspects: on the one hand, we observe the concrete objects of the world, the changes of their states and their interaction. On the other hand, we observe the events and the actions that may be created naturally or artificially. We have defined therefore, three categories of objects (Figure 2): Concrete object, Action object and Message object.

Concrete object : Three types of concrete objects are distinguished. The first type is the Person object, which represents an actor of the environment. It is the only object that has the ability to act and to interact and of which behaviour and state evolution are usually predictable. The second type is the Passive object. Two subcategories are identified: immobile objects as buildings and roads networks, and mobile objects like the means of transport. The observation of these objects is the simplest one, because they do not have any behaviour.

Their observation is reduced only to the description of their current state. The third type is the Mean object.

It is created at a given time and for a particular purpose. Its existence duration varies in time, according to the objective for which it is created. For example, a car is considered as a mean since it is driven by a driver, otherwise, it is considered as an immobile object.

Action object : This type is divided into activities and phenomena objects. Both are created at a given time and are limited temporally without a priory knowledge of the bounds. Phenomena are unpredictable events that start at a given time. Their observation is the most complex because of their uncertainties and their rapid evolutions. Activities are the actions sequences performed by actors. Generally, they are ordered and emitted for a particular purpose.

Message : Messages represent the interactions between persons and more precisely the information flows exchanged between the actors. The impact of a message is not easily measurable and it depends on its sender, its receiver and its performative. If the message is stored, its impact will be deferred.

The observation of an object in the environment may concern a persistent, temporary or punctual state. A persistent state can become invalid following a rupture. For example, a building with ten floors is a persistent state as long as is not destroyed. A blocked road is also a persistent state until it will be unblocked. However, a fire is a temporary state, because it is foreseeable that it will cease, fault of combustible. Finally, a punctual state is immediate and instantaneous, like sending a message.

RoboCupRescue Simulation Project

RoboCupRescue (RCR) is an annual international competition within the framework of the RoboCup [RoboCup]. This project intends to promote research and development in the disaster rescue domain by creating a standard simulator and forum for researchers and participators. RoboCupRescue project intends to simulate a urban disaster caused by an earthquake. The simulation disaster integrates various of disasters. These includes, fire, housing and building damages, disruption of roads, electricity, water supply, gas, and other infrastructures, movements of refuges, status of victims, hospital operations, etc. RCRSS is composed by several distributed modules: a kernel, a geographic information system, simulators (fire, traffic and collapse simulators), a viewer and an RCR agents module. We are interested in our work in the RCR agents module. The work consists in designing rescue teams that have as mission to save civilians and mitigate disaster consequences. The final goal is to set up a strategy planning that permits teams coordination.

The picture Figure 3 shows the hierarchy classes of the RCR disaster space. Each object in the world has properties such as its position, its shape ans its state. We distinguish two main objects categories: moving objects and motionless objects. First ones represent actors of the disaster world and they are modelled by Person object in our taxonomy. The second category consists of both buildings and networks roads and they are modelled by Passive object in the taxonomy.

Seven RCR agents types exist in the RCR simulation world: three platoon agents which are fire brigade, police force and ambulance team, three center agents which are fire station, police office and ambulance center and civilian agents. We will not develop the behaviours of the latter, because they are simulated independently with the other simulators. Each RCR agent has a partial knowledge of the whole environment state. This knowledge is updated thanks to two capacities: visual and auditory capacities. These capacities permit agents to receive information send by the kernel of the simulator each cycle (one second in the simulation which represents one minute in the reality). Agent centers represent in reality persons inside, so they can only see their surrounding area of the world and exchange messages with other agents. The role of these centers is to coordinate the communication between the three agents types. Platoon agents have more capacities, they can act by performing seven different actions: rescue, load and unload actions for ambulance team agents, extinguish for fire brigade agents, clear for police force agents and move for all agents.

Ontology of the Domain

The definition of the ontology of the domain is the result of the taxonomy application on the RCR simulation environment. The determination of the concepts is based on the object modelling of the RCR environment and respects the taxonomic organisation. The next picture (Figure 4) shows the ontology that we have implemented using protégé [Protégé].

The abstract class Object is situated on the top level of Finally, Message class is a concrete class and has two additional attributes: receiver and sender. In RoboCupRescue, a message content has the following format: "action name object name". For example, "clear road#ID", "extinguish building#ID", or "rescue civilian#ID", etc. The localisation attribute means therefore more precisely, the localisation of the target object in the message content as the road#ID, building#ID and civilian#ID in these examples.

Semantic Features

Information coming from the environment are written in the form of semantic features. The latter will be managed thereafter by factual agents in the representation layer. The idea to use a semantic feature is inspired from the memento design pattern and consists to store information, that describe the internal state of an observed object originally from the taxonomy. The structure of a semantic feature is generic and composed by a key and a set of couples <qualifier,value>. The key is defined from the taxonomy and the qualifiers are defined from the ontology.

In RoboCupRescue, information are sent by RCR agents each cycle and may be visual or auditive information.

The system treats these data in order to extract the important ones. For example, an RCR agent who sends an information describing an intact building will not be taken into account. However, an information about a burning building is interesting, the system interprets it and creates thereafter new semantic features, related to objects defined by the taxonomy. As example, a Building#14 has a property "fieryness = 25", this means that a fire has just started in this building. The system creates therefore, a semantic feature: (Phenomenon#14, type, fire, intensity, starting, localisation, 20|25, time, 7). This semantic feature is related to a phenomenon object, that means a fire is located in 20|25 coordinates at the seventh cycle of the simulation. In the case of an auditive information, the system creates semantic features according to messages contents. For example, an RCR agent sends a message "clear road#15". From this message, a semantic feature (Phenomenon#22, type, blockade, intensity, unknown, localisation, 30|40, time, 11) is created. This semantic feature is related to a blockade phenomenon, a priory we do not know the intensity of the blockade, but we can determine the coordinates of the blocked road from the world map, using its identifier (15). Thus, by treating the messages and the visual information sent by RCR agents, the system gathers the partial knowledges of these agents to build a global knowledge that can provide a clearer idea about the situation.

Semantic features are related with each other, that means they have a semantic dependencies. We defined therefore proximity measures in order to compare between them. The proximity value is comprised between [-1,1]. Two semantic features are opposite in their subjects if the proximity measure is negative, they are closed if it is positive and independent if it equals zero. More the proximity is near to 1 (-1), more the two semantic features are closed (opposite). We distinguish three types of proximities: a semantic proximity which is determined thanks to the on-tology, a spatial and a time proximities that are related to specific scales. As example, a break and a block are closed semantically, because if a building is broken, the nearest road will be certainly blocked. Moreover, to give more precision to this confrontation, we compare the localisations and the times of observation of the two events. If they are distant, we consider the two events are independent, and inversely.

5 Factual Agents of the Representation Layer

Structure and Role

The representation layer is composed by factual agents.

Each agent aims to represent a partial part of the observed situation, thanks to the semantic feature that it carries. Factual agent is a reactive and proactive agent [Wooldridge, 2002]. Its reactivity is ensured by a generic internal behavioural automaton of Augmented Transition Network (ATN) type [Woods 1970]. This automaton is composed by four states [Cardon 2004]: initialisation, deliberation, decision and action. ATN Transitions are stamped by a set of conditions and a sequence of actions. Conditions represent thresholds, defined according to three internal indicators of the agent, which are: Pseu-doPosition (PP), PseudoSpeed (PS) and PseudoAcceleration (PA). The agent has two other indicators: a satisfaction indicator and a constancy indicator, which represent respectively the satisfaction degree of the agent about its progression and its stability in its ATN. The definition of these indicators allow the factual agent to progress in its ATN, this characteristic ensures the proactivity of the agent, of which purpose is to achieve the most important state, that is the action state. In addition, the factual agent is a social agent. It interacts with the other agents in order to form a coalition with other ones, this permits it to acquire more force and power. The agent can also be attacked by other ones, with which it is opposite semantically. The list of opposites agents and closes agents is stored in an acquaintances network, which is constructed and updated dynamically.

Tests and Graphic Tools

We started to make tests on a part of the ontology. We localised our tests especially on the detection of the different events signalled by the RCR agents and the actions that they perform. We have designed some graphic tools in order to follow and study the evolution of the factual agents.

The graphic tool is composed by a grid that shows in real time points flow representing factual agents. Agents are projected on three axis: PP, PS and PA. Factual agents progress extremely quickly, so it is too hard to follow their evolution. We have created therefore, an interactive interface (agent interface). This interface has two essential functionalities. The first one permits to select a given factual agent and to show all its information: its semantic feature, its current state and its current indicators values. The second one permits to freeze all the factual agents at a given time and to reanimate them thereafter. This allows us to obtain an instantaneous view of all the agents during their evolution and to study consequently, information about any agent.

Picture Figure 6 shows an instantaneous image of the current situation of the RCRSS disaster space in the eighth cycle of the simulation. Information shown in the table, in the right, are related to the blue building, that is burning. A new factual agent, carrying the semantic feature (Phenomenon#67068017, type, fire, intensity, starting, localisation 22989100|3755100, time, 8), is created and updated according to information sent by the fire brigade agent, situated just near to the building. This factual agent is represented by the green ellipse in the grid and has as coordinates (PP=207,PS=3,PA=1). In the agent interface, we can see all information about this agent, notably, its indicators and its state which is the decision state. We note, that all indicators are strictly positive and the agent is in advanced state in its ATN. This means the agent has acquired importance and the event that it represents is more and more significant. This evolution is the result of information sent by the fire brigade agent and the interaction of the factual agent with other factual agents. The latter carry other related information, that can be messages announcing the fire, or actions performed to extinguish it.

Conclusion

This paper has presented an information modelling of a perceived environment in an emergency context. This modelling is used to represent the evolution of the current situation thanks to factual agents. Our final goal is to build a generic mutliagent system that intends to detect a risk an to deal with it. Information entering to the system are structured in the form of semantic features. The latter are defined thanks to a taxonomic organisation and to an ontology related to the domain. We choose the RCRSS as application to test this modelling.

We have implemented therefore, the ontology of the studied domain and started the representation of the RCRSS disaster space state, using a part of the ontology. This test allowed us to study the behaviour of factual agents and especially ATN thresholds and proximity measures that are very dependant on the application and that require more control of the environment in order to validate them. Our future work consists in finishing both, the implementation of the ontology and the representation layer that are still under realisation. We have the intention thereafter, to connect this layer with the characterisation layer in order to test some observed scenarios of which definition constitutes also a future subject of study.

Figure 1 :

 1 Figure 1: Kernel structure

Figure 2 :

 2 Figure 2: Taxonomy of the observed environment

Figure 3 :

 3 Figure 3: Class hierarchy of the RCR objects in the disaster space

Figure 4 :

 4 Figure 4: Ontology of the RoboCupRescue environment

Figure 5 :

 5 Figure 5: Internal structure of a factual agent

Figure 6 :

 6 Figure 6: View of the RCR disaster space

Figure 7 :

 7 Figure 7: Graphic tools to visualise factual agents