European quality system for tissue banking.

AIM: The aims of this project were to analyze the factors that influence quality and safety of tissues for transplantation and to develop the method to ensure standards of quality and safety in relation to tissue banking as demanded by European Directive 2004/23/EC and its technical annexes. It is organized in 4 Working Groups, the objectives of each one being focused in a specific area. STANDARDS: The Guide of Recommendations for Tissue Banking is structured into 4 parts: (1) quality systems that apply to tissue banking and general quality system requirements, (2) regulatory framework in Europe, (3) standards available, and (4) recommendations of the fundamental quality and safety keypoints. REGISTRY: This Working Group handled design of a multinational musculoskeletal tissue registry prototype. TRAINING: This Working Group handled design and validation of a specialized training model structured into online and face-to-face courses. The model was improved with suggestions from students, and 100% certification was obtained. AUDIT: The Guide for Auditing Tissue Establishments provides guidance for auditors, a self-assessment questionnaire, and an audit report form. The effectiveness and sustainability of the outputs were assessed. Both guides are useful for experienced tissue establishments and auditors and also for professionals that are starting in the field. The registry prototype proves it is possible to exchange tissues between establishments throughout Europe. The training model has been effective in educating staff and means having professionals with excellent expertise. Member states could adapt/adopt it. The guides should be updated periodically and perhaps a European organization should take responsibility for this and even create a body of auditors.

T-cell receptors in ectothermic vertebrates.

The structure and expression of genes encoding molecules homologous to mammalian T-cell receptors (TCR) have been recently studied in ectothermic vertebrate species representative of chondrychthians, teleosts, and amphibians. The overall TCR chain structure is well conserved in phylogeny: TCR beta- and TCR alpha-like chains were detected in all the species analyzed; TCR gamma- and TCR delta-like chains were also present in a chondrychthian species. The diversity potential of the variable (V) and joining (J) segments is rather large and, as in mammals, conserved diversity (D) segments are associated to the TCR beta and TCR delta chains. An important level of junctional diversity occurred at the V-(D)-J junctions, with the potential addition of N- and P-nucleotides. Thus, the conservation of the structure and of the potential of diversity of TCR molecules have been under a permanent selective pressure during vertebrate evolution. The structure of MHC class I and class II molecules was also well conserved in jawed vertebrates. TCR and MHC molecules are strongly functionally linked and play a determinant role in the initiation and the regulation of the specific immune responses; thus, it is not surprising that their structures have been reciprocally frozen during evolution.

Genomic organization of the TcR beta-chain diversity (Dbeta) and joining (Jbeta) segments in the rainbow trout: presence of many repeated sequences.

This work describes a 5.5 kb genomic sequence of the rainbow trout T-cell receptor beta-chain locus. It includes, from 5' to 3', a Dbeta gene, 10 Jbeta genes and the 5'-end of the first Cbeta exon. The trout Dbeta-Jbeta-Cbeta locus is about the same size as the mouse, rat and human homologous loci, but it is less compact and contains 10 Jbeta segments instead of the 6-7 found in mammals. The trout Dbeta coding sequence is identical to those of the mouse, rat and human Dbeta, and the Dbeta recombination signal sequences (RSS) are also very well conserved. Each trout Jbeta segment is flanked in 5' by a 7-mer RSS, which matches with the canonical conserved 7-mer sequences of all RSS. However, 6 of the 10 Jbeta segments have no characteristic 9-mer RSS, although at least some of them are well expressed (Jbeta1 and Jbeta2). The Jbeta region of the trout TcRbeta locus contains numerous micro/minisatellite repeated DNA sequences; some of these repeats contain heptamer RSS-like sequences that could interfere with Jbeta expression. Knowledge of the germline boundaries of the trout Dbeta and Jbeta ends makes it possible to evaluate precisely the exonuclease activity and N-nucleotide addition at the Dbeta-Jbeta junctions of the rearranged TcRbeta chain genes. Many (40%) of the Dbeta-Jbeta junctions in the adult trout have no N-nucleotides, compared to 26.4% in adult mice, and 37% of the adult trout TcRbeta transcripts are out of frame. Thus, there may be major differences in the T-cell developmental kinetics and selection in fish and mammals.

Structure and diversity of the TCR alpha-chain in a teleost fish.

T cell receptor beta-chain genes are well characterized in representatives of most vertebrate phyla, from sharks to mammals, but the molecular structure of complete TCR alpha-chains has not yet been established in cold-blooded vertebrates. We used a PCR approach to isolate cDNAs encoding putative teleost fish (Oncorhynchus mykiss, rainbow trout) TCR alpha-chains. Eight V alpha segments were identified, belonging to six different families, and the best amino acid sequence identity scores for these trout V alpha were all provided by mammalian V alpha or V delta sequences. Twenty-four (60.1 %) of the 39 analyzed V alpha segments belong to the V alpha 2 family, which has limited homology with mammalian V alpha/delta sequences and with the human V pre-B sequence. A total of 32 different J alpha segments were identified from 40 J alpha regions sequenced, suggesting that a large repertoire of J alpha segments is a characteristic of most vertebrates. The structural properties of the TCR alpha-chain complementarity-determining region 3 loop are well conserved between trout and mammals, suggesting that this region has been under continuous selective pressure in jawed vertebrate evolution. The trout C alpha segment has conserved N-terminal and transmembrane domains, but the C alpha intercysteine distance contains only 40 residues, significantly smaller as compared with mammals (49-56 residues). The conserved features of teleost fish TCR beta- and alpha-chains with their mammalian equivalents suggest that TCR-alpha beta receptors were still present in the common Devonian ancestors of modern teleost fish and mammals, about 450 million years ago.

Identification of cDNA clones encoding HMG 2, a major protein of the mexican axolotl hydrocortisone-sensitive thymocytes.

We have identified and analyzed cDNA clones encoding a major 26 kDa protein of the HMG1-2 family which is abundant in the cytoplasm and nucleus of axolotl hydrocortisone-sensitive thymocytes. The axolotl HMG2 protein is very similar to proteins belonging to the HMG1-2 family, from teleost fish to mammals. All the molecular features of the HMG1-2 proteins are conserved, including the high proportion of basic and aromatic residues, and the characteristic acidic C-terminus tail. The 3'-untranslated region of the HMG2 axolotl cDNA is also similar to the avian and mammalian HMG2 3'-UT sequences, suggesting that some selective events have acted at the DNA level to conserve this region, which could be important in the differential expression of the HMG1 and HMG2 genes. The axolotl HMG2 protein contains the two well conserved HMG boxes which are thought to be the DNA-binding domains of the molecule. Axolotl thymocytes and spleen cells contain almost identical amounts of HMG2 mRNAs but HMG2 polypeptide is undetectable in spleen cells using anti-26 kDa antibodies. The reason for the accumulation of HMG1-2 molecules in vertebrate hydrocortisone-sensitive thymocytes is discussed, as well as their possible role in apoptosis.

Structure and diversity of the T cell antigen receptor beta-chain in a teleost fish.

Cell-mediated immunity (e.g., allograft rejection) is found in all vertebrates, and these reactions are known to depend on thymus-derived cells in amphibian, avian, and mammalian species. The participation of peripheral T cell-like lymphocytes subpopulations to fish immunity is now well documented, but the developmental origin, migration, and peripheral tissue distribution of these cells remain practically unknown. This is mainly due to the difficulty of efficiently thymectomizing fish at an early stage of development and to the lack of Ab strictly specific for thymocytes and T cell surface Ag. One strategy for analyzing T cell biology in fish would be to characterize the genes encoding polypeptides homologous to the TCR molecules. This report describes cDNA clones from the rainbow trout (Oncorhynchus mykiss) that have sequences very similar to amphibian, avian, and mammalian TCR beta-chains. Three complete trout V beta segments belonging to different families were analyzed; one of them had limited amino acid sequence similarity to the human V beta 20 family. The 10 trout beta-chain-joining segments all retain the invariant mammalian J beta residues, and comparison of 66 V beta-J beta junctions led to the identification of a D beta-like sequence (GGACAGGG) that is shorter than but very similar to the chicken D beta and mammalian D beta 1 sequences. There is considerable diversity at the V beta-D beta and D beta-J beta junctions, suggesting the presence of N-nucleotides. The trout C beta extracellular domain is shorter than mammalian C beta, and the hinge region has no cysteine residue. The transmembrane C beta domain contains a lysine residue that in mammals is thought to be involved in charged interactions with members of the CD3 complex.

Evolution of specific antigen recognition: size reduction and restricted length distribution of the CDRH3 regions in the rainbow trout.

The immunoglobulin heavy chain repertoire in fish was investigated by cloning a total of 88 rearranged VDJ junctions from the head kidney B cell mRNA of a salmonid, the rainbow trout (Oncorhynchus mykiss). Trout DH segments are short and cannot be classified into independent DH families. Several of the ten identified putative DH segments had stretches of nucleotide sequence identity with mouse (DQ52, DFL 16.2 and Dsp 2.1), human (DM1) and chicken (DH4) DH. There was a clear preference for one or two of the three putative DH reading frames and a stop codon is often present in the less used reading frame. Four of the six JH segments are preferentially used, and analysis of the VH-DH and DH-JH junctions suggest the presence of N-nucleotides. The absolute size and size heterogeneity of the rainbow trout CDRH3 are smaller than those of the Xenopus, mouse and human CDRH3. About 75% of the 84 in-frame trout CDRH3 have 8, 9 or 10 residues and none of them have more than 11 residues. This homogeneization of the CDRH3 loop size may partly explain the restricted antibody diversity in lower vertebrates.

[Characterization of cDNA of T-cell receptor beta chain in rainbow trout]. / Caractérisation d'ADNc de la chaîne beta du récepteur des lymphocytes T chez la truite arc-en-ciel.

Using a two-step PCR strategy, we have cloned several cDNA segments encoding the T-cell receptor beta chain in a Teleost fish, the rainbow trout (Oncorhynchus mykiss). The nine clones analyzed encode identical N-terminal-truncated V beta regions which present limited sequence similarities with several mammalian TcR V beta chains, from residue Tyr-35 to residue Ser-95. These V beta regions are followed by V beta-D beta-J beta-like regions which are different in all the sequenced clones, and by identical C beta regions. The trout C beta domain (156 amino acids) is most related to the chicken and to amphibian (axolotl) C beta domains but no cysteine residue appears in the hinge region. Like in other vertebrate C beta s, the TM region carries a positively charged lysine residue (Lys-271). The intracytoplasmic domain is virtually absent. The possibility to analyze the structure, expression and diversity of a T-cell receptor chain in a Teleost fish model will be important for our future understanding of the evolution of specific immune recognition in vertebrates.