Biomedical subjects

Antoine Blancher

Publications and source records attributed to Antoine Blancher.

16 recordsLinked to original sources

PubMed2006

Study of Cynomolgus monkey (Macaca fascicularis) MhcDRB (Mafa-DRB) polymorphism in two populations.

Cynomolgus monkey is one of the macaque species currently used as an animal model for experimental surgery and medicine, in particular, to experiment new drugs or therapy protocols designed for the prevention of allograft rejection. In this field, it is of utmost importance to select histoincompatible recipient-donor pairs. One way to ensure incompatibility between donor and recipient is to check their major histocompatibility complex (MHC) genotypes at the loci playing a determinant role in histocompatibility. We report in this paper on the cynomolgus monkey DRB polymorphism evidenced by sequencing of amplified exon 2 separated either by denaturing gradient gel electrophoresis (DGGE), or by cloning. By the study of 253 unrelated animals from two populations (Mauritius and The Philippines), we characterized 50 exon 2 sequences among which 28 were identical to sequences already reported in Macaca fascicularis or other macaque species (Macaca mulatta, Macaca nemestrina). By cloning and sequencing DRB cDNA, we revealed two additional DRB alleles. Out of the 20 haplotypes that we defined here, only two were found in both populations. The functional impact of DR incompatibility was studied in vitro by mixed lymphocyte culture.

Amino Acid Sequence↗

PubMed2006

Analysis of CCR5, CCR2, CX3CR1, and SDF1 polymorphisms in HIV-positive treated patients: impact on response to HAART and on peripheral T lymphocyte counts.

Although polymorphisms of chemokine genes (SDF1, stromal cell-derived factor-1 and RANTES, regulated on activation, normal T cell expressed and secreted) and chemokine-receptor genes (CCR5, CCR2, CX(3)CR1) were shown to be associated with sensitivity to HIV infection and untreated HIV disease progression, their association with the response to highly active antiretroviral therapy (HAART) remains unclear. To explore the possible influence of such polymorphisms on the evolution of AIDS in treated patients, we have studied SDF1-3'A, CCR5Delta32, CCR2-64I, CX(3)CR1-249I, and CX(3)CR1-280M polymorphisms in HIV-infected patients under HAART (n = 169). We studied the evolution of plasma virus load and peripheral T lymphocyte counts in these patients up to 3 years after the initiation of HAART. We observed that some of the genetic polymorphisms studied had an impact on the evolution of these two parameters. After 1 year of HAART, patients with a virological response (undetectable plasma HIV-1 RNA) have a higher frequency of the homozygous SDF1-3'A genotype than other patients (p = 0.005). Similarly, patients with a CD4 increase of over 200/mm(3) from baseline after 1 year of HAART display higher frequencies of homozygous SDF1-3'A (p = 0.035) and homozygous CX(3)CR1-280M genotypes (p = 0.04) than other patients. Moreover, we showed that the CX(3)CR1- 280M allele was associated with higher peripheral CD4+ T cell counts not only in HIV+ patients but also in healthy controls (p = 0.003).

Adult↗

PubMed2005

FOXP3 mRNA levels are decreased in peripheral blood CD4+ lymphocytes from HIV-positive patients.

The impact of HIV infection on regulatory CD4(+)CD25(high) (Treg) lymphocyte subpopulations was evaluated by FOXP3 quantitative reverse transcriptase polymerase chain reaction and by flow cytometry. FOXP3 mRNA was quantified in peripheral blood mononuclear cells or purified CD4(+) lymphocytes from HIV(+) lymphopenic patients. Patients were distributed among clinical stages A, B, and C and received highly active antiretroviral therapy. The frequency of CD4(+)CD25(high) lymphocytes, measured by flow cytometry, was decreased in HIV patients (n = 38) compared with the group of uninfected subjects (n = 39). FOXP3 mRNA levels were found decreased in HIV patients (n = 25) compared with controls (n = 17) when expression of CD3gamma or beta-actin but not that of TATA box binding protein 1 was used for data normalization. Our results are compatible with a decrease of the Treg lymphocytes during HIV infection. The consequences of a Treg decrease are discussed in the context of immunologic anomalies observed during HIV infection.

Adult↗

PubMed2005

Decrease of Lewis frequency in HIV-infected patients: possible competition of fucosylated antigens with HIV for binding to DC-SIGN.

We explored the impact of human ABO glycosyltransferase and Lewis and secretor fucosyltransferase polymorphisms in HIV infection. We found that, compared with healthy blood donors, HIV-infected patients display a significant decrease in Le(a-b+) phenotype frequencies. We showed that HIV binding on DC-SIGN-transduced Jurkat cells was inhibited by fucosyl bovine serum albumin. Our results suggest a slight protective effect of Lewis b antigen on HIV infection, possibly by the competition of Lewis antigens with HIV for binding to DC-SIGN.

Cell Adhesion Molecules↗

PubMed2005

Immunomodulatory effect of human adipose tissue-derived adult stem cells: comparison with bone marrow mesenchymal stem cells.

Like mesenchymal stem cells from bone marrow (BM-MSCs), adipose tissue-derived adult stem cells (ADAS cells) can differentiate into several lineages and present therapeutical potential for repairing damaged tissues. The use of allogenic stem cells can enlarge their therapeutical interest, provided that the grafted cells could be tolerated. We investigate here, for the first time, the immunosuppressive properties of ADAS cells compared with the well-characterized immunosuppressive properties of BM-MSCs. ADAS cells did not provoke in vitro alloreactivity of incompatible lymphocytes and, moreover, suppressed mixed lymphocyte reaction (MLR) and lymphocyte proliferative response to mitogens. The impairment of inhibition when ADAS cells and BM-MSCs were separated from lymphocytes by a permeable membrane suggests that cell contact is required for a full inhibitory effect. Hepatocyte growth factor is secreted by both stem cells but, similar to interleukin-10 and transforming growth factor-beta (TGF-beta), the levels of which were undetectable in supernatants of MLR inhibited by ADAS cells or BM-MSCs, it did not seem implicated in the stem cell suppressive effect. These findings support that ADAS cells share immunosuppressive properties with BM-MSCs. Therefore, ADAS cell-based reconstructive therapy could employ allogenic cells and because of their immunosuppressive properties, ADAS cells could be an alternative source to BM-MSCs to treat allogenic conflicts.

Adipose Tissue↗

PubMed2004

Structural basis for the inactivity of human blood group O2 glycosyltransferase.

The human ABO(H) blood group antigens are carbohydrate structures generated by glycosyltransferase enzymes. Glycosyltransferase A (GTA) uses UDP-GalNAc as a donor to transfer a monosaccharide residue to Fuc alpha1-2Gal beta-R (H)-terminating acceptors. Similarly, glycosyltransferase B (GTB) catalyzes the transfer of a monosaccharide residue from UDP-Gal to the same acceptors. These are highly homologous enzymes differing in only four of 354 amino acids, Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268. Blood group O usually stems from the expression of truncated inactive forms of GTA or GTB. Recently, an O(2) enzyme was discovered that was a full-length form of GTA with three mutations, P74S, R176G, and G268R. We showed previously that the R176G mutation increased catalytic activity with minor effects on substrate binding. Enzyme kinetics and high resolution structural studies of mutant enzymes based on the O(2) blood group transferase reveal that whereas the P74S mutation in the stem region of the protein does not appear to play a role in enzyme inactivation, the G268R mutation completely blocks the donor GalNAc-binding site leaving the acceptor binding site unaffected.

ABO Blood-Group System↗

PubMed2004

Sequence, evolution and ligand binding properties of mammalian Duffy antigen/receptor for chemokines.

The Duffy antigen/ receptor for chemokine, DARC, acts as a widely expressed promiscuous chemokine receptor and as the erythrocyte receptor for Plasmodium vivax. To gain insight into the evolution and structure/function relations of DARC, we analyzed the binding of anti-human Fy monoclonal antibodies (mAbs) and human chemokines to red blood cells (RBCs) from 11 nonhuman primates and two nonprimate mammals, and we elucidated the structures of the DARC genes from gorilla, gibbon, baboon, marmoset, tamarin, night monkey and cattle. CXCL-8 and CCL-5 chemokine binding analysis indicated that the promiscuous binding profile characteristic of DARC is conserved across species. Among three mAbs that detected the Fy6 epitope by flow cytometric analysis of human and chimpanzee RBCs, only one reacted with night monkey and squirrel monkey. Only chimpanzee RBCs bound a significant amount of the anti-Fy3 mAb. Fy3 was also poorly detected on RBCs from gorilla, baboon and rhesus monkey, but not from new world monkeys. Alignment of DARC homologous sequences allowed us to construct a phylogenetic tree in which all branchings were in accordance with current knowledge of primate phylogeny. Although DARC was expected to be under strong internal and external selection pressure, in order to maintain chemokine binding and avoid Plasmodium vivax binding, respectively, our present study did not provide arguments in favor of a selection pressure on the extracellular domains involved in ligand specificity. The amino acid variability of DARC-like polypeptides was found to be well correlated with the hydrophilicity indexes, with the highest divergence on the amino-terminal extracellular domain. Analysis of the deduced amino acid sequences highlighted the conservation of some amino acid residues, which should prove to be critical for the structural and functional properties of DARC.

Animals↗

PubMed2004

A prevalent POLG CAG microsatellite length allele in humans and African great apes.

The human nuclear gene for the catalytic subunit of mitochondrial DNA polymerase gamma ( POLG) contains within its coding region a CAG microsatellite encoding a polyglutamine repeat. Previous studies demonstrated an association between length variation at this repeat and male infertility, suggesting a mechanism whereby the prevalent (CAG)(10) allele, which occurs at a frequency of >80% in different populations, could be maintained by selection. Sequence analysis of the POLG CAG microsatellite region of more than 1000 human chromosomes reveals that virtually all allelic variation at the locus is accounted for by length variation of the CAG repeat. Analysis of POLG from African great apes shows that a prevalent length allele is present in each species, although its exact length is species-specific. In common chimpanzee ( Pan troglodytes) a number of different sequence variants contribute to the prevalent length allele, strongly supporting the idea that the length of the POLG microsatellite region, rather than its exact nucleotide or amino acid sequence, is what is maintained. Analysis of POLG in other primates indicates that the repeat has expanded from a shorter, glutamine-rich sequence, present in the common ancestor of Old and New World monkeys.

Amino Acid Sequence↗

PubMed2004

Evolution of the O alleles of the human ABO blood group gene.

BACKGROUND: To date, at least 40 different alleles O have been characterized on the basis of exon 6 and exon 7 sequences but not always for intron 6. STUDY DESIGN AND METHODS: Among 415 individuals, from four continents (Africa, Europe, South America, and Asia), studied for exon 6 and exon 7 sequences, we selected 46 individuals (of respective phenotypes O [39], AB [3], B [3], or A [1]) for sequencing 1800-bp amplicons spanning exon 6, intron 6, and exon 7. The amplicons were characterized either by direct sequencing or after cloning when required. RESULTS: We defined 14 new intron 6 O allele sequences, including four recombinant alleles. Based on sequence comparison, a phylogenetic network was constructed. It confirmed recombinant allele origins and that most O alleles are derived by point mutations from the two worldwide distributed alleles O01 and O02. CONCLUSION: Allele O phylogenetic analysis suggests that the most frequent silencing mutation (deletion of a G in exon 6) appeared once in human evolution in the ancient O02 allele lineage and that allele O01 resulted from an interallele exchange between O02 and A101. Assuming constancy of evolutionary rate, diversification of the representative alleles of the three human ABO lineages (A101, B101, and O02) was estimated at 4.5 to 6 million years ago.

ABO Blood-Group System↗

PubMed2003

Polymorphism of human and primate RANTES, CX3CR1, CCR2 and CXCR4 genes with regard to HIV/SIV infection.

Among genes that influence human susceptibility to HIV (human immunodeficiency virus) infection or AIDS (acquired immunodeficiency syndrome) progression, chemokine-receptor and chemokine genes were extensively studied because of their role as HIV co-receptors or co-receptor competitors, respectively. We have studied in non-human primates (chimpanzee, gorilla, gibbon, orang-utan, crab-eating and rhesus macaque, baboon and marmoset) the RANTES, CCR2 and CX3CR1 gene sequences in regions surrounding human mutations that were associated with susceptibility to HIV or AIDS progression: RANTES G-403A and C-28G, CCR2 V64I, CX3CR1 V249I and CX3CR1 T280M. Among these five dimorphisms, only RANTES G-403A is observed in one of the eight primate species studied here (gibbon). This suggests that these mutations appeared recently in humans and probably do not account for variable HIV/SIV disease progression in primates. It is noteworthy that chimpanzees, which are naturally resistant to HIV-1- and HIV-2-induced AIDS, do not have the human mutations associated with delayed disease progression. Inter-species and intra-species polymorphic positions are observed in primates and we discuss the potential impact of these mutations on HIV/SIV disease progression. Particularly, we identified polymorphisms in old-world monkey (OWM) genes, and it could be of great importance to analyse the possible association between these polymorphisms and disease progression in OWM species that are currently used in research for HIV vaccine and therapy.

Animals↗

PubMed2003

Cloning of a rat gene encoding the histo-blood group B enzyme: rats have more than one Abo gene.

A genomic DNA fragment corresponding to exon 7 of the human ABO gene was amplified from rats of several inbred and outbred strains. Five different sequences were obtained, four of them corresponding to A-type sequences and one to a B-type sequence based on the amino acids equivalent to residues at positions 266 and 268 of the human enzymes. In rats from inbred strains, a single A-type sequence and the unique B-type sequence were found, whereas some animals of outbred strains presented two or three A-type sequences along with the B-type sequence. The complete coding sequence of the B-type gene was obtained; identification of the exon-intron boundaries, determined by comparison with rat genomic sequences from data banks, revealed that the rat B-type gene structure is identical with that of the mouse Abo gene. Compared with the human ABO gene and the rat A gene, it lacks exon 4. Like the rat A gene (symbol: Abo), the rat B gene (symbol: Abo2) is located on chromosome 3q11-q12. It could be shown by transfection experiments that the B-type cDNA encodes an active B transferase. A transcript of the B gene was found ubiquitously, whereas the B antigen was only detected in a restricted set of tissues. These data indicate that rats have at least two distinct Abo genes, one monomorphic gene encoding a B-specific enzyme and one or more genes in some cases encoding an A-specific enzyme.

ABO Blood-Group System↗

PubMed2003

A single point mutation reverses the donor specificity of human blood group B-synthesizing galactosyltransferase.

Blood group A and B antigens are carbohydrate structures that are synthesized by glycosyltransferase enzymes. The final step in B antigen synthesis is carried out by an alpha1-3 galactosyltransferase (GTB) that transfers galactose from UDP-Gal to type 1 or type 2, alphaFuc1-->2betaGal-R (H)-terminating acceptors. Similarly the A antigen is produced by an alpha1-3 N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine from UDP-GalNAc to H-acceptors. Human alpha1-3 N-acetylgalactosaminyltransferase and GTB are highly homologous enzymes differing in only four of 354 amino acids (R176G, G235S, L266M, and G268A). Single crystal x-ray diffraction studies have shown that the latter two of these amino acids are responsible for the difference in donor specificity, while the other residues have roles in acceptor binding and turnover. Recently a novel cis-AB allele was discovered that produced A and B cell surface structures. It had codons corresponding to GTB with a single point mutation that replaced the conserved amino acid proline 234 with serine. Active enzyme expressed from a synthetic gene corresponding to GTB with a P234S mutation shows a dramatic and complete reversal of donor specificity. Although this enzyme contains all four "critical" amino acids associated with the production of blood group B antigen, it preferentially utilizes the blood group A donor UDP-GalNAc and shows only marginal transfer of UDP-Gal. The crystal structure of the mutant reveals the basis for the shift in donor specificity.

ABO Blood-Group System↗

PubMed2002

Virological and immunological effects of salvage therapy following treatment interruption and a shift in HIV-1 resistance genotype.

The circulating human immunodeficiency virus type 1 (HIV-1) population of patients in whom many prior therapy regimens have failed often undergo a shift from a drug-resistant virus to a wild-type virus following interruption of treatment. This study analyses the virological and immunological effects of salvage therapy following treatment interruption and a shift from a drug-resistance genotype. Twenty-one HIV-1 infected patients who had genotype reversion by population-based sequencing after 3 months of treatment interruption were given a new salvage regimen consisting of 3-5 drugs selected according to their treatment history. Seven (33%) of 21 patients who had fewer than 200 HIV-1 RNA copies/ml until month 12 were defined as virological responders. Four patients were transient responders and 10 were nonresponders. The virological responders were more frequently CDC group A and had higher CD4 + T lymphocyte counts at the time of treatment resumption. The peripheral blood T CD4 + and T CD8 + lymphocyte populations of the patients declined significantly during treatment interruption. Only virological responders showed significant increases in their CD4 + T lymphocyte count 12 months after treatment resumption and these counts rapidly returned to pre-interruption baseline values in most of these patients. Treatment interruption could be useful for optimising salvage therapy for patients previously given many failing regimens. However, controlled trials are needed to assess the clinical benefit of this strategy.

Adult↗

PubMed2002

A novel cis AB allele derived from a B allele through a single point mutation.

BACKGROUND: The very rare cis AB phenotype, first described in 1964, corresponds to a special ABO allele encoding a glycosyltransferase that is capable of synthesizing both A and B substances. Until now, gene sequences of only two cis AB alleles were partially characterized. One involved the A1*02 allele with a single nonsynonymous substitution at codon 268, whereas the second arose from a single nonsynonymous substitution at codon 266 in exon 7 of a B1*01 allele. STUDY DESIGN AND METHODS: A cis AB phenotype was identified in a French family. The serologic characteristics of this phenotype were determined. The cis AB allele was characterized from exon 6 to exon 7 by cloning and sequencing. RESULTS: The cis AB.tlse(*)01 allele is identical to B(1*)01 except for a single point mutation at nucleotide position 700, where a T replaces a C, implying a change of amino acid 234 (the B(1*)01 proline being replaced by a serine). CONCLUSION: The cis AB.tlse(*)01 allele clearly differs from all previously reported ABO, including the two previous cis AB described.

ABO Blood-Group System↗

PubMed2002

Alpha1,4-fucosyltransferase activity: a significant function in the primate lineage has appeared twice independently.

In the animal kingdom the enzymes that catalyze the formation of alpha1,4 fucosylated-glycoconjugates are known only in apes (chimpanzee) and humans. They are encoded by FUT3 and FUT5 genes, two members of the Lewis FUT5-FUT3-FUT6 gene cluster, which had originated by duplications of an alpha3 ancestor gene. In order to explore more precisely the emergence of the alpha1,4 fucosylation, new Lewis-like fucosyltransferase genes were studied in species belonging to the three main primate groups. Two Lewis-like genes were found in brown and ruffed lemurs (prosimians) as well as in squirrel monkey (New World monkey). In the latter, one gene encodes an enzyme which transfers fucose only in alpha1,3 linkage, whereas the other is a pseudogene. Three genes homologous to chimpanzee and human Lewis genes were identified in rhesus macaque (Old World monkey), and only one encodes an alpha3/4-fucosyltransferase. The ability of new primate enzymes to transfer fucose in alpha1,3 or alpha1,3/4 linkage confirms that the amino acid R or W in the acceptor-binding motif "HH(R/W)(D/E)" is required for the type 1/type 2 acceptor specificity. Expression of rhesus macaque genes proved that fucose transfer in alpha1,4 linkage is not restricted to the hominoid family and may be extended to other Old World monkeys. Moreover, the presence of only one enzyme supporting the alpha1,4 fucosylation in rhesus macaque versus two enzymes in hominoids suggests that this function occurred twice independently during primate evolution.

Amino Acid Sequence↗

PubMedDate not supplied

A simple method to optimize peripheral blood mononuclear cell preparation from cynomolgus monkeys and improve mixed lymphocyte reactions.

INTRODUCTION: The standard Ficoll-Hypaque method used to isolate peripheral blood mononuclear cells (PBMC) gives very variable results when used with cynomolgus monkey blood. We have improved the method by using special cell processing tubes (CP-tubes), originally developed for clinical use. METHODS: Blood samples were collected from cynomolgus monkeys and processed for PBMC preparation using either the classical Ficoll-Hypaque method or CP-tubes following various centrifugation protocols. The preparations were compared according to their cellular content as well as their response in the mixed lymphocyte reaction (MLR). RESULTS: Good PBMC separation was achieved in >90% of samples by centrifugation of blood samples in CP-tubes for 40-45 min at 1650 x g and 20 degrees C. For the remaining samples, poor PBMC separation was probably due to low corpuscular hemoglobin concentrations (< 28 g/dl), but this could be rectified by using one to two additional centrifugations. The PBMC preparations thus obtained showed lower red blood cell (RBC) and polymorphonuclear (PMN) cell contamination, reacted well to mitogen and showed improved MLR stimulation indices vs. standard Ficoll-Hypaque-PBMC-derived preparations. The inhibitory effect of Cyclosporine-A (CsA) was within the low nanomolar range with both methods. DISCUSSION: These results demonstrate that the use of CP-tubes is a practical way of improving PBMC separation and MLR responses with cynomolgus monkey blood.

Animals↗