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Biomedical subjects

P Chartrand

Publications and source records attributed to P Chartrand.

At least 37 records · Page 2Linked to original sources
PubMed1996

Presence in peripheral blood of healthy individuals of autoreactive T cells to a membrane antigen present on bone marrow-derived cells.

Intrathymic clonal deletion is thought to be the major mechanism responsible for tolerance to nonsequestered antigens such as the ones expressed by bone marrow-derived cells. In the case of sequestered antigens that potentially do not come in contact with T cells in the thymus, it is thought that autoreactive T cells are present in periphery but are tightly regulated to prevent autoimmune disease. Indeed, autoreactive T cells to sequestered antigens can be isolated in healthy individuals. However, the presence of autoreactive T cells to nonsequestered circulating antigens had not been observed. In this report, we present evidence for the presence, in the periphery of all healthy individuals tested (n = 25), of autoreactive T cells to GpIIb-IIIa, a membrane antigen present on bone marrow-derived cells that is expressed on circulating platelets and on the cell surface of the epithelial cells of the thymic stroma early in intrauterine life. Using an in vitro T-cell proliferation assay, we have demonstrated that activation of these specific GpIIb-IIIa autoreactive alpha beta TCR+ CD4+ CD8- T cells requires internalization and processing of the GpIIb-IIIa by antigen-presenting cells and its presentation by HLA-DR class II molecules in the presence of exogenous interleukin 2 (IL-2). This indicates that some autoreactive T cells directed against membrane antigens present on bone marrow-derived cells and also expressed in the thymus are not necessarily eliminated by intrathymic deletion.

Antigen-Presenting Cells↗
PubMed1996

pGATA: a positive selection vector based on the toxicity of the transcription factor GATA-1 to bacteria.

The transcription factor GATA-1 is a zinc finger DNA-binding protein essential for the development of red blood cells. When we expressed different regions of the zinc finger domain in bacteria using an isopropyl-beta-D-thiogalactoside (IPTG) inducible system, growth of bacteria harboring the active DNA-binding domain of GATA-1 was rapidly inhibited upon IPTG induction. The growth inhibition pattern suggested it may be occurring at the level of the initiation of replication, and GATA-1 was found to bind to three of the four DNA A protein-binding sites in the origin of replication. This toxicity was used to develop a positive selection vector system in which cloned DNA fragments interfered with the production of the GATA-1 DNA-binding domain. Thus, vector molecules containing the insert of interest are selected for when bacteria are grown in the presence of IPTG. With this system, the vector does not need to be dephosphorylated, purified or completely digested with a restriction enzyme for the efficient cloning of DNA fragments even when the vector-to-insert DNA molar ratio in ligation reactions is 10 to 1. Moreover, no special strain of Escherichia coli is required, and the selection might also be applicable to other species of bacteria if the toxicity of GATA-1 relates to inhibition of the DNA A protein.

Bacterial Proteins↗
PubMed1995

An oligodeoxyribonucleotide that supports catalytic activity in the hammerhead ribozyme domain.

A study of the activity of deoxyribonucleotide-substituted analogs of the hammerhead domain of RNA catalysis has led to the design of a 14mer oligomer composed entirely of deoxyribonucleotides that promotes the cleavage of an RNA substrate. Characterization of this reaction with sequence variants and mixed DNA/RNA oligomers shows that, although the all-deoxyribonucleotide oligomer is less efficient in catalysis, the DNA/substrate complex shares many of the properties of the all-RNA hammerhead domain such as multiple turnover kinetics and dependence on Mg2+ concentration. On the other hand, the values of kinetic parameters distinguish the DNA oligomer from the all-RNA oligomer. In addition, an analog of the oligomer having a single ribonucleotide in a strongly conserved position of the hammerhead domain is associated with more efficient catalysis than the all-RNA oligomer.

Base Sequence↗
PubMed1995

Autoreactive T cells in healthy individuals show tolerance in vitro with characteristics similar to but distinct from clonal anergy.

Peripheral tolerance to self antigens has been suspected to play an important role in the regulation of the immune response in humans since autoreactive T cells can be isolated from the peripheral blood of healthy individuals. The mechanism of this tolerance is not known, but a number of groups have shown that autoreactive T cells can be induced to proliferate in vitro by the addition of their specific antigen and exogenous interleukin (IL)-2. In this report, we present the analysis of autoreactive T cells, isolated from healthy individuals, to the autoantigen GpIIb-IIIa present on circulating bone-marrow-derived cells and on thymic epithelial cells. We found that the response of GpIIb-IIIa autoreactive T cells in vitro, when stimulated with GpIIb-IIIa, shares characteristics with the response found for anergic T cells. In response to GpIIb-IIIa, the GpIIb-IIIa-autoreactive T cells are neither able to proliferate nor produce IL-2 on their own, but do express IL-2 receptors alpha on their cell surface and produce IFN-gamma. This state of unresponsiveness can be broken by the addition of exogenous IL-2 and IL-7, as in the case of anergic T cells. However, GpIIb-IIIa-autoreactive T cells differ from anergic T cells in their capacity to be stimulated by IL-12 and by their production of IL-2 mRNA. Interestingly, once the unresponsive state to GpIIb-IIIa has been broken by the addition of IL-2, GpIIb-IIIa autoreactive T cells can produce IL-2 and proliferate when restimulated by GpIIb-IIIa alone. Altogether, these results suggest that the tolerance of GpIIb-IIIa autoreactive T cells from healthy individuals could involve post-transcriptional regulation of IL-2 expression.

Autoimmunity↗
PubMed1994

Interference of DNA sequence divergence with precise recombinational DNA repair in mammalian cells.

Studies done in prokaryotes and eukaryotes have indicated that DNA sequence divergence decreases the frequency of homologous recombination. To determine which step(s) of homologous recombination is sensitive to DNA sequence divergence in mammalian cells we have used an assay that does not rely on the recovery of functional products. The assay is based on the acquisition by homologous recombination of endogenous LINE-1 sequences by exogenous LINE-1 sequences. In parallel experiments, we introduced into mouse cells two gapped exogenous LINE-1 sequences, one from the mouse, L1Md-A2, and the other from the rat, L1Rn-3. Although L1Rn-3 is on average less than 85% homologous to the LINE-1 elements of the mouse, the frequency of homologous recombination with endogenous LINE-1 elements obtained with L1Rn-3 was the same as the one obtained with L1Md-A2 which is on average 95% homologous to the LINE-1 elements of the mouse. The endogenous LINE-1 sequences rescued by L1Rn-3 were 8-18% divergent from L1Rn-3 sequences, whereas those rescued by L1Md-A2 were 2-5% divergent from L1Md-A2 sequences. The gap which had been introduced into the exogenous LINE-1 sequences had been precisely repaired in 50% of the recombinants obtained with L1Md-A2. None of the L1Rn-3 recombinants showed precise gap repair.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

KIN17, a mouse nuclear protein, binds to bent DNA fragments that are found at illegitimate recombination junctions in mammalian cells.

Illegitimate recombination is the dominant mechanism of recombination in mammalian somatic cells. It is responsible for most genome rearrangements such as translocations, deletions and integrations. Little is known as yet about the mechanism of illegitimate recombination and the enzymes involved. Recently, it has been shown that intrinsically bent DNA, also known as curved DNA, is present at chromosomal sites of illegitimate recombination events. Here we report that KIN17, a new mouse nuclear protein, binds to the curved DNA fragments found at illegitimate recombination sites.

Animals↗
PubMed1994

Association of a host DNA structure with retroviral integration sites in chromosomal DNA.

Integration of retroviral genomes is a site-specific process with respect to the virus but not the host genome. Numerous chromosomal sites and various sequences can be used as targets. Nevertheless, preferential regions and integration patterns have been observed. Using a functional assay, we investigated if host structural DNA elements could be associated with retroviral integration sites. The results were that 9 of 10 distinct retroviral integration events occurred in close proximity of structural elements behaving like intrinsically bent DNA.

Animals↗
PubMed1994

One-sided invasion events in homologous recombination at double-strand breaks.

Two classes of homologous recombination mechanism for repair of double-strand breaks (DSBs) have been described in eukaryotes so far. One is conservative and has been explained by the double-strand break repair model (Szostak et al., 1983), whereas the other one is non-conservative and has been explained by the single-strand annealing model (Lin et al., 1984). Here, we will review data supporting the existence of another homologous recombination mechanism for double-strand break repair. We will present the one-sided invasion model that we have proposed to explain this mechanism and discuss its potential implication in various homologous recombination events.

Animals↗
PubMed1994

Analysis of immunoglobulin class, IgG subclass and titre of HPA-1a antibodies in alloimmunized mothers giving birth to babies with or without neonatal alloimmune thrombocytopenia.

We analysed the titre and isotype composition of antibodies produced by mothers giving birth to babies with or without neonatal alloimmune thrombocytopenic purpura (NAITP) and patients with post-transfusion purpura (PTP). All these individuals produced an antibody specific for the HPA-1a allotype present on the platelet glycoprotein IIb-IIIa (GPIIb-IIIa). Sera from mothers who gave birth to thrombocytopenic babies (group 1, n = 36), non-thrombocytopenic babies (group 2, n = 4) or from PTP patients (group 3, n = 3) were tested by an indirect-ELISA. Results indicated no evident differences in the isotype composition or titre of the antibodies from the three groups of sera. The antibody titre ranged from 1:120 to 1:3500. Antibodies with the IgG1 subclass were present in all sera. Most sera contained IgG1 alone (24/43 sera tested) or in combination with IgG3 (10/43). IgG2 was never present and only three sera showed intermediate reactivity with anti-IgG4 MAb. Few sera (nine sera from groups 1 and 2) were weakly positive when tested with the anti-IgM antibodies. These results suggest that neither the titre nor the isotype composition can be used to predict the severity or the occurrence of thrombocytopenia in newborns.

Adult↗
PubMed1994

Integration of a vector containing a repetitive LINE-1 element in the human genome.

Mammalian cells contain numerous nonallelic repeated sequences, such as multicopy genes, gene families, and repeated elements. One common feature of nonallelic repeated sequences is that they are homeologous (not perfectly identical). Our laboratory has been studying recombination between homeologous sequences by using LINE-1 (L1) elements as substrates. We showed previously that an exogenous L1 element could readily acquire endogenous L1 sequences by nonreciprocal homologous recombination. In the study presented here, we have investigated the propensity of exogenous L1 elements to be involved in a reciprocal process, namely, crossing-overs. This would result in the integration of the exogenous L1 element into an endogenous L1 element. Of over 400 distinct integration events analyzed, only 2% involved homologous recombination between exogenous and endogenous L1 elements. These homologous recombination events were imprecise, with the integrated vector being flanked by one homologous and one illegitimate junction. This type of structure is not consistent with classical crossing-overs that would result in two homologous junctions but rather is consistent with one-sided homologous recombination followed by illegitimate integration. Contrary to what has been found for reciprocal homologous integration, the degree of homology between the exogenous and endogenous L1 elements did not seem to play an important role in the choice of recombination partners. These results suggest that although exogenous and endogenous L1 elements are capable of homologous recombination, this seldom leads to crossing-overs. This observation could have implications for the stability of mammalian genomes.

Cell Line↗
PubMed1992

Minimum ribonucleotide requirement for catalysis by the RNA hammerhead domain.

Several mixed DNA/RNA and 2'-O-methylribonucleotide/RNA analogues derived from the "hammerhead" domain of RNA catalysis have been prepared to study the minimum ribonucleotide requirement for catalytic activity. Oligodeoxyribonucleotides containing from seven to as few as four ribonucleotides are active in cleaving a substrate RNA. Predominantly deoxyribonucleotide-containing analogues have kcat values 20-300 and kcat/KM values approximately 100-2000 times lower than those of all-RNA ribozyme. In the case of predominantly 2'-O-methyl analogues, at least five ribonucleotides are needed to assure catalytic activity. In addition, both predominantly deoxyribonucleotide and 2'-O-methyl oligomers are at least 3 orders of magnitude more stable than an all-RNA ribozyme in incubations with RNase A and a yeast extract. These results suggest that the ribophosphate backbone is not a strict requirement for ribozyme-type catalysis. The identification of the four required ribonucleotides in the hammerhead catalytic domain provides valuable information for the rational design of chemical species having ribonuclease activities.

Base Sequence↗
PubMed1992

Chromosomal illegitimate recombination in mammalian cells is associated with intrinsically bent DNA elements.

Illegitimate recombination is the most frequent mechanism for chromosomal rearrangements in mammalian cells, yet little is known about this process. Most of the studies to date have looked at the sequences present at illegitimate junctions. These revealed the presence of recurrent DNA motifs, none of which was consistently found. We have undertaken to determine if intrinsic DNA structures such as bent DNA elements could be a major determinant in chromosomal illegitimate recombination. Using a two dimensional electrophoretic assay we found that eight out of eight junctions, resulting from various types of chromosomal rearrangements, had migration behaviour characteristic of DNA containing intrinsically bent DNA elements. In all cases, these occurred within one kilobase of the junctions, and in most cases could be found in both participating DNA segments. We also found that these bent DNA elements were present before the recombination event. When we analysed the frequency of intrinsically bent DNA elements in random chromosomal fragments, we found it to be about one per 11 kilobases. Thus these results suggest that bent DNA is associated with chromosomal illegitimate recombination.

Animals↗
PubMed1992

Alloimmunization to platelet antigen HPA-1a (PIA1) is strongly associated with both HLA-DRB3*0101 and HLA-DQB1*0201.

Antibodies to the platelet HPA-1a antigen can elicit in the newborn a condition known as neonatal alloimmune thrombocytopenic purpura (NAITP). Previous studies based on RFLP analysis showed that 100% of HPA-1a-negative women who produced anti-HPA-1a antibodies (responders) were HLA-DRw52a (DRB3*0101). However, this specificity could also be found in some HPA-1a-negative women not producing anti-HPA-1a antibodies (nonresponders). We have analyzed in detail by PCR-SSOP the HLA-DR, -DQ, and -DP loci of 36 responders and 10 nonresponders. We found that while the allele DRB3*0101 was present in the vast majority of responders (91%), there were exceptions. Furthermore, the DQB1*0201 allele was found to be present in almost all responders (94%), but again was also found in nonresponders. The risk of alloimmunization to HPA-1a in an HPA-1b homozygous mother significantly increases with the presence of either allele, the odds ratio being 39.7 for DQB1*0201 and 24.9 for DRB3*0101. Sequencing of exon 2 of these two alleles from responders indicated no sequence difference when compared with the consensus sequences. This indicates that they do not represent variants when compared with the same alleles found in some nonresponders.

Alleles↗