Management of cytomegalovirus infection in solid organ transplant recipients: SET/GESITRA-SEIMC/REIPI recommendations.

Cytomegalovirus (CMV) infection remains a major complication of solid organ transplantation. Because of management of CMV is variable among transplant centers, in 2011 the Spanish Transplantation Infection Study Group (GESITRA) of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) developed consensus guidelines for the prevention and treatment of CMV infection in solid organ transplant recipients. Since then, new publications have clarified or questioned the aspects covered in the previous document. For that reason, a panel of experts revised the evidence on CMV management, including immunological monitoring, diagnostics, prevention, vaccines, indirect effects, treatment, drug resistance, immunotherapy, investigational drugs, and pediatric issues. This document summarizes the recommendations.

DMRT1 expression during gonadal differentiation and spermatogenesis in the rainbow trout, Oncorhynchus mykiss.

DMRT1 has been suggested to be the first conserved gene involved in sex differentiation found from invertebrates to human. To gain insight on its implication for fish gonadal differentiation, we cloned a DMRT1 homologue in the rainbow trout, Oncorhynchus mykiss (rtDMRT1), and showed that this gene is expressed during testicular differentiation, but not during ovarian differentiation. After 10 days of steroid treatment, expression was shown to be decreased in estrogen-treated male differentiating gonads but not to be restored in androgen-treated differentiating female gonads. This clearly reinforces the hypothesis of an important implication for DMRT1 in testicular differentiation in all vertebrates. In the adults a single 1.5 kb transcript was detected by Northern blot analysis in the testis, and its expression was found to be sustained throughout spermatogenesis and declined at the end of spermatogenesis (stage VI). Along with this expression in the testis we also detected by reverse transcriptase-polymerase chain reaction a slight expression in the ovary. We also obtained new DM-domain homologous sequences in fish, and their analysis suggest that at least four different genes bearing 'DM-domain' (DMRT genes) exist in fish just as in all vertebrate genomes.

Characterization and repeat analysis of the compact genome of the freshwater pufferfish Tetraodon nigroviridis.

Tetraodon nigroviridis is a freshwater pufferfish 20-30 million years distant from Fugu rubripes. The genome of both tetraodontiforms is compact, mostly because intergenic and intronic sequences are reduced in size compared to other vertebrate genomes. The previously uncharacterized Tetraodon genome is described here together with a detailed analysis of its repeat content and organization. We report the sequencing of 46 megabases of bacterial artificial chromosome (BAC) end sequences, which represents a random DNA sample equivalent to 13% of the genome. The sequence and location of rRNA gene clusters, centromeric and subtelocentric satellite sequences have been determined. Minisatellites and microsatellites have been cataloged and notable differences were observed in comparison with microsatellites from Fugu. The genome contains homologies to all known families of transposable elements, including Ty3-gypsy, Ty1-copia, Line retrotransposons, DNA transposons, and retroviruses, although their overall abundance is <1%. This structural analysis is an important prerequisite to sequencing the Tetraodon genome.

Estimate of human gene number provided by genome-wide analysis using Tetraodon nigroviridis DNA sequence.

The number of genes in the human genome is unknown, with estimates ranging from 50,000 to 90,000 (refs 1, 2), and to more than 140,000 according to unpublished sources. We have developed 'Exofish', a procedure based on homology searches, to identify human genes quickly and reliably. This method relies on the sequence of another vertebrate, the pufferfish Tetraodon nigroviridis, to detect conserved sequences with a very low background. Similar to Fugu rubripes, a marine pufferfish proposed by Brenner et al. as a model for genomic studies, T. nigroviridis is a more practical alternative with a genome also eight times more compact than that of human. Many comparisons have been made between F. rubripes and human DNA that demonstrate the potential of comparative genomics using the pufferfish genome. Application of Exofish to the December version of the working draft sequence of the human genome and to Unigene showed that the human genome contains 28,000-34,000 genes, and that Unigene contains less than 40% of the protein-coding fraction of the human genome.

Karyotype and chromosome location of characteristic tandem repeats in the pufferfish Tetraodon nigroviridis.

Karyotype analysis of Tetraodon nigroviridis, a pufferfish of the family Tetraodontidae with a small compact genome (385 Mb) which is currently being investigated in our laboratory, indicates that this species has 2n = 42 chromosomes. The small chromosome size (the largest pair measuring less than 3 microm) has complicated accurate chromosome pairing based on morphology alone. DAPI staining, however, provides a banding-like pattern. Because of quantitative variations of some heterochromatin classes, the chromosome formula can not be established precisely, but is estimated to include approximately 20 meta- or submetacentric chromosomes and 22 subtelocentric chromosomes. A centromeric satellite, telomeric repeats, and the major and minor rRNA clusters have been localized unequivocally by FISH. As a result, the 28S and 5S rDNA sequences can be used as chromosome-specific probes.

Concerted evolution of two Mhc class II B loci in pheasants and domestic chickens.

The major histocompatibility complex (Mhc) of the ring-necked pheasant contains two polymorphic Mhc class II B genes. We show here, by screening of a cDNA library and RT-PCR from RNA, that both of these loci, Phco-DAB1 and Phco-DAB2, normally are transcribed in the spleen. They differ mainly in the 3' untranslated (UT) region, with the transcript lengths, not including the poly(A) tails, being 1,100 nt for DAB1 and 955 nt for DAB2. These two loci are orthologous to the B-LBI and B-LBII loci of the domestic chicken, respectively. DAB1 and DAB2 therefore seem to have evolved from a duplication before the split of the evolutionary lineages leading to the pheasant and the domestic chicken ca. 20 MYA. This is the first report of an orthologous relationship between avian Mhc genes. Yet, the third exons of DAB1 and DAB2 were identical in all available sequences and differed at 10 positions from the exon 3 sequences of B-LBI/B-LBII. The species-specific exon 3 suggests that DAB1 and DAB2 are subject to concerted evolution, i.e., interlocus genetic exchange. The exon 2 sequences show characteristic polymorphism, with hypervariable segments occurring in different combinations in different alleles. Given the divergence in the 3'UT region, the finding of the same exon 2 sequence at both the DAB1 and the DAB2 loci in one of the pheasant haplotypes also suggests that interlocus genetic exchange does occur. Accordingly, the exon 2 sequences tended to cluster irrespective of locus in the phylogenetic analyses. Genetic exchange simultaneously involving both exon 2 and exon 3 may be facilitated by the short length of the intervening intron (< 100 bp) in pheasants and domestic chickens compared with, e.g., humans (about 3 kb).

Analysis of the spermine synthase gene region in Fugu rubripes, Tetraodon fluviatilis, and Danio rerio.

A prerequisite to understanding the evolution of the human X chromosome is the analysis of synteny of X-linked genes in different species. We have focused on the spermine synthase gene in human Xp22. 1. We show that whereas the human gene spans a genomic region of 54 kb, the Fugu rubripes gene is encompassed in a 4.7-kb region. However, we could not find conserved synteny between this region of human Xp22 and the equivalent F. rubripes region. A cosmid clone containing the F. rubripes gene does not contain other X-linked genes. Instead we identified homologs of human genes that are autosomally localized: the ryanodine receptor type I (RYRI), which is implicated in malignant hyperthermia and central core disease, and the HE6 gene. Comparison of the F. rubripes, Tetraodon fluviatilis, mouse, human, and Danio rerio 5'UTRs of spermine synthase highlights conserved sequences potentially involved in regulation. Interestingly, pseudogenes of this gene that are present in the human and mouse genomes seem to be absent in the compact F. rubripes genome. Analysis of a D. rerio PAC clone containing spermine synthase shows an intermediate genomic size in this fish. Sequence analysis of this PAC clone did not reveal other known genes: neither the RYRI gene, nor the HE6 gene, nor other human Xp22 genes were identified.

Phenotype-genotype correlation in Jewish patients suffering from familial Mediterranean fever (FMF).

Familial Mediterranean Fever is one of the most frequent recessive disease in non-Ashkenazi Jews. The gene responsible for the disease (MEFV) has very recently been identified. The M694V ('MED') mutation was found in about 80% of the FMF Jewish (Iraqi and North African) chromosomes. To see if the presence of this mutation could be correlated with particular traits of the disease, we examined a number of clinical features in a panel of 109 Jewish FMF patients with 0, 1 or 2 MED mutations. We showed that homozygosity for this mutation was significantly associated with a more severe form of the disease. In homozygous patients, the disease started earlier (mean age 6.4 +/- 5 vs 13.6 +/- 8.9) and both arthritis and pleuritis were twice as frequent as in patients with one or no M694V mutation. Moreover, 3/3 patients with amyloidosis displayed two MED mutations. No association was found with fever, peritonitis, response to colchicine and erysipeloid eruption. The present result strongly suggests the potential prognostic value of the presence of this mutation.

Non-founder mutations in the MEFV gene establish this gene as the cause of familial Mediterranean fever (FMF).

Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by recurring attacks of fever and serositis. It affects primarily North African Jews, Armenians, Turks and Arabs, in which a founder effect has been demonstrated. The marenostrin-pyrin-encoding gene has been proposed as a candidate gene for the disease ( MEFV ), on the basis of the identification of putative mutations clustered in exon 10 (M680V, M694I, M694V and V726A), each segregating with one ancestral haplotype. In a search for additional MEFV mutations in 120 apparently non-founder FMF chromosomes, we observed eight novel mutations in exon 2 (E148Q, E167D and T267I), exon 5 (F479L) and exon 10 (I692del K695R, A744S and R761H). Except for E148Q and K695R, all mutations were found in a single chromosome. Mutation E148Q was found in all ethnic groups studied and in association with a novel ancestral haplotype in non-Ashkenazi Jews (S2). Altogether, these new findings definitively establish the marenostrin/pyrin-encoding gene as the MEFV locus.

A transcriptional Map of the FMF region.

Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by attacks of fever and serositis, which affects primarily non-Ashkenazi Jews, Armenians, Turks, and Arabs. We present here a transcriptional map covering the FMF locus that we constructed in the course of the positional cloning of the gene responsible for this disease. This map was established from a contig constructed with YAC, BAC, and cosmid clones and covers about 500 kb of 16p13.3. It contains nine transcriptional units corresponding to known genes or to genes belonging to known gene families, 23 gene fragments characterized by partial sequences, and an endogenous retrovirus sequence. It thus considerably increases the number of genes in this interval and improves our knowledge concerning some of the genes or gene families present in this region. Data accumulated in this region were also used in a comparative study of different methods of exon detection.

Characterization of avian natural killer cells and their intracellular CD3 protein complex.

Natural killer (NK) cell activity appears to be conserved throughout vertebrate development but NK cells have only been well characterized in mammals. Candidate NK cells have been identified in the chicken as cytoplasmic CD3+ and surface T cell receptor (TCR)/CD3- (TCRO) lymphocytes that often express CD8. The fact that the TCRO cells are abundant in the embryonic spleen before T cells enter this organ allowed us to cultivate the embryonic TCRO cells using growth factors derived from activated adult lymphocytes. These TCRO cells were cytotoxic for an NK target cell line. They expressed cell surface CD8, a putative interleukin-2 receptor, CD45 and a receptor for IgG, but did not express CD4, major histocompatibility complex class II or immunoglobulin. Biochemical analysis of the cytoplasmic CD3 antigen revealed two of the three CD3 gamma, delta and epsilon homologues, and RNA transcripts for the third. The CD3 monoclonal antibody also precipitated a 32-kDa dimer that may represent a heterodimer of different CD3 constituents. TCR alpha and beta gene transcripts were not detected in the TCRO cells. These results indicate that the avian TCRO cell is the mammalian NK cell homologue. The shared evolutionary features of T cells and NK cells in birds and mammals support the idea that they derive from a common progenitor.

Two Mhc class I and two Mhc class II genes map to the chicken Rfp-Y system outside the B complex.

Gene sequences highly similar to major histocompatibility complex (Mhc) class I and class II genes were recently recognized as mapping to a site in the genome of the chicken separate from the Mhc class I, class II, and B-G genes of the major histocompatibility (B) complex. The present study was undertaken to see whether this complex of Mhc-like genes designated as restriction fragment pattern Y (Rfp-Y) might reside in one of three clusters of cosmid clones contained within the molecular map of chicken Mhc genes, since only two of the three clusters can be assigned to the B system. To determine whether the third cluster (cluster II/IV) might contain Rfp-Y, a subclone (18.1) from within cluster II/IV near a polymorphic lectin gene was used to analyze the DNA of families in which Rfp-Y haplotypes are known to be segregating. The restriction fragment polymorphisms revealed by the 18.1 probe were found to segregate in parallel with the restriction fragment polymorphisms defining the Rfp-Y haplotypes, thus establishing the location of Rfp-Y within cosmid cluster II/IV. Two of six Mhc class I genes and two of five Mhc class II genes map to cosmid cluster II/IV, so a substantial fraction of chicken Mhc genes, including at least one that may be expressed, are located in a chromosomal region separate from the B system. In further linkage analyses, Rfp-Y was found to assort independently from more than 400 markers in the present linkage map of the chicken genome.

Linkage of a new member of the lectin supergene family to chicken Mhc genes.

With the use of tissue-specific cDNA probes, several genes, which do not correspond to the class I (B-F), class II (B-L), or class IV (B-G) genes, were detected within the cosmid clusters containing the chicken major histocompatibility genes. We isolated cDNA clones with a probe corresponding to one of them, the 17.5 gene, located between two class I genes. The 17.5.3 cDNA, isolated from a chicken spleen cDNA library, encodes a 257-residue-long protein. This sequence shows significant similarity with several members of the C-type animal lectin superfamily and is probably a type II transmembrane protein. Analysis of several cDNA clones, together with Southern blot experiments, strongly suggest that this gene belongs to a multigene family, with at least some of its members being polymorphic. Several arguments lend support to the possibility that, together with the linked Mhc genes, the 17.5 gene is part of the recently described Rfp-Y system.

Chicken major histocompatibility complex class II B genes: analysis of interallelic and interlocus sequence variance.

Five different chicken B-LB genes were cloned and sequenced. The comparison of these sequences shows that they can be classified as members of two different families, the B-LBII family (containing the B-LBI and B-LBII genes) and the B-LBIII family (containing the B-LBIII, B-LBIV, and B-LBV genes). The extent of polymorphism within each of these families was assessed by in vitro amplification of DNA fragments encompassing exon 2 in several haplotypes. The nucleotide sequences were determined, and pairwise relationships were evaluated. In the course of this work, a sixth gene termed B-LBVI was identified, defining a third family (B-LBVI family). Polymorphism of the B-LBIII or B-LBVI families is far less extensive than that of the B-LBII family. In this latter, the distribution of conserved and polymorphic residues is similar to what has been described in mammals. These families seem to have been generated by gene duplication events giving rise to several isotypes, as observed in mammals. However, phylogenetic analyses indicate that these families are not homologous to their mammalian counterparts. Evaluation of the level of transcription of these different genes showed that genes from the B-LBII family are predominantly transcribed over those of the other families.

Primary structure and ontogeny of an avian CD3 transcript.

The structure of a chicken CD3 chain has been determined by isolating a cDNA clone (T11.15) that encodes a 175-amino-acid-long protein, including the NH2-terminal signal peptide. In Northern blot experiments, the earliest expression of the T11.15 transcript was detected in the thymus at embryonic day 10 (i.e., 1 day after cytoplasmic expression of a CD3 epitope recognized by a specific monoclonal antibody [CT3; Chen, C.L.H., Ager, L.L., Gartland, G.L. & Cooper, M.D. (1986) J. Exp. Med. 164, 375-380], but 2 days before the appearance of clonotypic components of the T-cell antigen receptor). Sequence similarity of this chicken protein sequence compared with that of the known mammalian CD3 gamma and delta polypeptides was 36-39% and 39-40%, respectively. Amino acid sequence alignments between avian and mammalian CD3 revealed maximum conservation in the transmembrane and cytoplasmic domains as well as in the regions flanking the cysteine residues in the extracellular domain, underlining their functional importance. The difficulty of unambiguously assigning this chain to a single mammalian CD3 subunit on the basis of sequence comparison raises the possibility that this polypeptide represents a derivative from an ancestral form of the gamma and delta chains. It is thus possible that a single chain may play the role of both CD3 gamma and delta subunits in the chicken CD3 complex or, alternatively, that gene duplications occurred independently in the avian and mammalian lineages.

Evolutionary relationship between human and mouse immunoglobulin kappa light chain variable region genes.

Similar to the Igh-V multigene family, the human or mouse Igk-V repertoire is a distorted continuum of homologous genes that may be grouped into families displaying greater than 80% nucleic acid sequence similarity among their members. Systematic interspecies sequence comparisons reveal that most human Igk-V gene families exhibit clear homology to mouse Igk-V families (sequence similarity generally greater than 74%). A hypothetical phylogenetic tree of Igk-V genes predicts that a minimum of seven Igk-V genes/families predate mammalian radiation. In two cases, several interrelated mouse Igk-V families exhibit phylogenetic equidistance with just one human Igk-V family, implying a more pronounced divergence for the elevated number of Igk-V gene families in the mouse. Mouse-human Igk-V comparisons, moreover, illustrate how expansion, contraction, and perhaps deletion of Igk-V gene families shape the Igk-V repertoire during mammalian evolution.