Biomedical subjects

S Darche

Publications and source records attributed to S Darche.

15 recordsLinked to original sources

PubMed2001

Contribution of double-negative thymic precursors to CD8alpha alpha (+) intraepithelial lymphocytes of the gut in mice bearing TCR transgenes.

Using male and female RAG(-/-) mutant mice expressing TCR transgenes specific for MHC class I- or II-presented HY peptides, we performed quantitative and phenotypic comparisons between the TCR(+) lymphocytes present in the lymphoid organs and the gut mucosa in euthymic versus athymic (nude) animals. These comparisons suggest that only a minority of the TCR(+) CD8alpha alpha (+) intraepithelial lymphocytes (IEL) of the transgenic euthymic mice originate from hematopoietic precursors acquiring a TCR in the gut wall, while a majority of these CD8alpha alpha(+) IEL appear to be of thymic origin (as were all TCR(+) CD8alpha beta (+) or CD4(+) in any location); these last cells are released from the thymus as double-negative thymocytes, which are at a more immature stage (CD44(+)CD25(+)) in female mice than in males (CD44(-)). In view of previous observations that in non-transgenic athymic mice the CD8alpha alpha (+) TCR(+) IEL populations are also markedly reduced quantitatively, the possibility of a thymic contribution to these ontogenically peculiar populations may also exist in normal mice. At which stage of differentiation such precursors might leave the thymus of normal adult mice remains to be explored.

Animals↗

PubMed1999

Modulation of T-cell functions in KIR2DL3 (CD158b) transgenic mice.

In humans, a minor subset of T cells express killer cell Ig-like receptors (KIRs) at their surface. In vitro data obtained with KIR(+) alphabeta and gammadelta T-cell clones showed that engagement of KIR molecules can extinguish T-cell activation signals induced via the CD3/T-cell receptor (TCR) complex. We analyzed the T-cell compartment in mice transgenic for KIR2DL3 (Tg-KIR2DL3), an inhibitory receptor for HLA-Cw3. As expected, mixed lymphocyte reaction and anti-CD3 monoclonal antibody (MoAb)-redirected cytotoxicity exerted by freshly isolated splenocytes can be inhibited by engagement of transgenic KIR2DL3 molecules. In contrast, antigen and anti-CD3 MoAb-induced cytotoxicity exerted by alloreactive cytotoxic T lymphocytes cannot be inhibited by KIR2DL3 engagement. In double transgenic mice, Tg-KIR2DL3 x Tg-HLA-Cw3, no alteration of thymic differentiation could be documented. Immunization of double transgenic mice with Hen egg white lysozime (HEL) or Pigeon Cytochrome-C (PCC) was indistinguishable from immunization of control mice, as judged by recall antigen-induced in vitro proliferation and TCR repertoire analysis. These results indicate that KIR effect on T cells varies upon cell activation stage and show unexpected complexity in the biological function of KIRs in vivo.

Animals↗

PubMed1998

Ontogeny of the heavy chain immunoglobulin repertoire in fetal liver and bone marrow.

We studied the kinetics of maturation of B cell progenitors in the mouse embryo, from day 15 of development to birth, both in liver and bone marrow. The analysis of Ig heavy chain rearrangements at different time points of late fetal development shows that oligoclonal patterns of V(H)-D-J(H) rearrangements are detected by day 15 in fetal liver. The pattern is polyclonal and diverse by day 17; however, 80% of the rearrangements are nonproductive. In bone marrow, the pattern of rearrangements is less diverse at birth, although the percentage of nonproductive rearrangements approaches adult bone marrow levels (35-40%). After day 17 in fetal liver, there is a sudden reversal in the percentage of nonproductive rearrangements that reaches 33% at day 19 (birth). Maturation of B cells, as measured by the fraction of surface Ig+ in total B220+ cells and the presence of N sequence additions in V(H)-D-J(H) joints, occurs in the marrow before fetal liver. These results demonstrate that the lymphopoietic environment in fetal liver and bone marrow of animals at the same stage of development is functionally distinct.

Animals↗

PubMed1998

CD8 expression allows T cell signaling by monomeric peptide-MHC complexes.

Physiologically, TCR signaling is unlikely to result from the cross-linking of TCR-CD3 complexes, given the low density of specific peptide-MHC complexes on antigen-presenting cells. We therefore have tested directly an alternative model for antigen recognition. We show that monomers of soluble peptide-MHC trigger Ca2+ responses in CD8alphabeta+ T cells. This response is not observed in CD8- T cells and when either the CD8:MHC or CD8:Lck interactions are prevented. This demonstrates that an intact CD8 coreceptor is necessary for effective TCR signaling in response to monomeric peptide-MHC molecules. We propose that this heterodimerization of TCR and CD8 by peptide-MHC corresponds to the physiological event normally involved during antigen-specific signal transduction.

Amino Acid Sequence↗

PubMed1997

Maternal immunoglobulins have no effect on the rate of maturation of the B cell compartment of the offspring.

To elucidate a general role of maternal immunoglobulins (Ig) on the kinetics of B cell development of the offspring, we studied non-genetic influences of maternal Ig on the developing immune system of B cell-competent mice. These animals were the offsprings of either B cell-deprived microMT or of normal C57BL/6 females. In these mice, we have compared the kinetics of Ig production, the numbers of B cell progenitors, the expression of surface markers specific of the B lineage and the progression of Ig variable gene expression. We show that the absence of maternal Ig has no detectable effect on the kinetics of IgM and IgG production by the offspring's immune system. The number of B cell precursors, the kinetics of generation of B cells and their pattern of surface markers expression is identical in both types of mice. The acquisition of diversity in the B cell repertoire and the changes in the ratios of variable gene family expression are also indistinguishable. We conclude that maternally derived Ig has no influence on the rate of development and maturation of the B cell compartment of the offspring.

Animals↗

PubMed1995

PCR-based analysis of the murine immunoglobulin heavy-chain repertoire.

The immunoglobulin heavy-chain repertoire has been mainly analysed by studying the proportion of genes belonging to each of the 14 described families, in terms of the expressed immunoglobulin molecules. Although the proportion of each variable gene family is kept stable throughout adult life and in different mice of the same strain, little information is available on the clonal representation in the repertoire of activated B cells. We describe here a new method that permits an approach to this question by separating the products of a polymerase chain reaction covering the VH-D-JH junction of the immunoglobulin heavy chain gene in a sequencing gel, thereby allowing discrimination of different rearrangements (according to their length) using a given JH and one of the member of a given VH family. Using this method, we show that it is possible to obtain a precise overview of the repertoire of activated B cells, at the mRNA level, as well as the potential repertoire, from a study of the DNA. We also show that this approach permits the detection of an induced immune B cell response and studies of emerging dominant specific B cell clones.

Animals↗

PubMed1994

Differential cytokine expression in maternal blood and placenta during murine gestation.

To evaluate the influence of gestation on peripheral blood T cells, we measured the mRNA production of several cytokines (IL-2, IL-4, IL-10, IFN-gamma) and the p55 subunit of IL-2R at different time points in the blood of pregnant mice and in the placenta. This was made possible by the use of a new PCR technique that is precise and quantitative. Our results show that pregnancy induces profound changes in the expression of these genes in peripheral blood cells. During the first week of gestation, there is an increase in the levels of all the cytokines, followed by a state of immunodepression characterized by levels of cytokines below normal (nonpregnant mice). In the placenta low levels of IL-2 and IL-10 are detected. IFN-gamma mRNA production is higher than the blood IFN-gamma mRNA in the last week of pregnancy. However, the main difference is found for IL-4 mRNA expression where the placenta levels are 5- to 10-fold higher than the blood mRNA expression. We discuss these results in the context of the placenta as a privileged immune site, where IL-4, being the main cytokine, may play a major regulatory role.

Animals↗

PubMed1993

The sizes of the CDR3 hypervariable regions of the murine T-cell receptor beta chains vary as a function of the recombined germ-line segments.

A method using PCR amplification and primer extension with fluorescent oligonucleotides was developed to analyze T-cell repertoires. The sizes of the hypervariable CDR3-like regions of the murine T-cell antigen receptor beta chains were measured for all possible V beta-J beta combinations. This analysis shows that beta chains are distributed into at least 2000 groups, a value that provides a lower limit to their complexity. The CDR3 sizes appear to be dependent on the J beta and especially the V beta segment used and correlates with amino acid sequence motifs in the corresponding CDR1 region. This feature of T-cell receptors is discussed.

Amino Acid Sequence↗

PubMed1993

Quantitative titration of nucleic acids by enzymatic amplification reactions run to saturation.

In vitro enzymatic amplification of nucleic acids by PCR or other techniques is a very sensitive method to detect rare DNA segments. We present here a protocol that allows the rapid, sensitive and precise quantification of DNA molecules using PCR amplification run to saturation. The DNA (or cDNA) to be assayed is co-amplified with known amounts of an internal standard DNA. We show that the latter must be almost identical to the assayed DNA, otherwise quantification at the plateau is unreliable. The read-out of the amplification involves one or two additional oligonucleotides. Using fluorescent oligonucleotides as primers in run-off reactions together with an automated DNA sequencer, we could measure the level of expression of several genes, like the murine MHC class I H-2Kd or a specific T cell receptor beta chain transcript in the course of an immunization. mRNA levels were normalized by measuring in a similar manner the number of transcripts encoding the housekeeping gene HPRT. Finally, our procedure might allow the rapid analysis of a large number of samples at the same time, as illustrated by the simultaneous analysis of the mRNAs encoding the CD4 and CD8 murine T cell markers.

Animals↗

PubMed1992

Molecular detection and in vivo analysis of the specific T cell response to a protein antigen.

We have analyzed in detail the repertoire of transcripts encoding the V beta chains of the T cell receptor and investigated the T cell response of B10.A mice to pigeon cytochrome c. We were thus able to follow the specific T cell response in vivo after immunization with this protein antigen. The response is first detectable in the draining lymph nodes, then in the spleen and in the blood. It is qualitatively similar in individual animals. It is dominated by a major category of specific T cells harboring a V beta 3-J beta 1.2 rearrangement, and a limited and well-defined set of nucleotide sequences, previously found in several specific T cell hybridomas and clones. This predominance is observed from the onset of the immune response strongly suggesting the notion that there is no variation and, therefore, no maturation of the T cell response in the course of immunization.

Animals↗

PubMed1992

[Quantitative determination method of nucleic acids by enzymatic amplification (PCR method) to saturation].

We describe here conditions under which the enzymatic amplification of DNA using the polymerase chain reaction (PCR) is quantitative, even when the amplification reaction is run to saturation. DNA in the sample to analyze is co-amplified with known quantities of an internal standard, namely a DNA molecule whose sequence or length differs from that of the sample DNA by only a few base pairs. The two amplification products are detected as run-off products elongated from one or several additional labelled, primers. The ratio between the two signals provides a precise estimate of the amount of specific DNA in the sample to analyze.

Base Sequence↗

PubMed1987

Intramolecular recombination between partially homologous sequences in Escherichia coli and Xenopus laevis oocytes.

We describe a system to analyze the individual contribution of a single physical DNA end on intramolecular recombination between partially homologous sequences. We took advantage of this partial sequence divergence to measure the distance separating the DNA end from the final recombination event. We show that a single physical DNA end stimulates recombination when located in a region of homology. Recombination frequency decreases gradually with the distance from the DNA end. A recombinational hot spot is found at the end of the region of homology. A large insertion of unrelated DNA interferes asymmetrically with this process, suggesting that a recombinogenic signal propagates along the region of homology.

Animals↗

PubMed1985

Expression of class I genes in the major histocompatibility complex: identification of eight distinct mRNAs in DBA/2 mouse liver.

The mouse H-2 multigene family includes the genes coding for the major transplantation antigens and for genes located in the Qa-TIa region. We have studied a collection of class I cDNA clones made from liver mRNA of DBA/2 mice (H-2d haplotype) and found that at least six distinct class I genes are transcribed, including three genes of the Qa-TIa region. Two of these six genes each yield two distinct mRNAs, resulting from alternate splicing. Altogether, liver cells may express at least eight distinct class I polypeptides, of which three might be secreted, while one may be a new presumptive nonpolymorphic surface antigen.

Amino Acid Sequence↗

PubMedDate not supplied

A monoclonal antibody recognizes a subset of the H-2Dd mouse major class I antigens.

Binding studies and competition experiments have shown that a monoclonal antibody (mAb) named 28-8-6 recognizes only 5 to 10% of the cell surface Dd molecules. The molecules detected by 28-8-6 mAb appear to be genuine H-2Dd antigens on the basis of their MW and isolectric points. In addition, the detectability of the subset of cell surface Dd molecules by 28-8-6 does not depend on their degree of glycosylation nor on the presence of mouse beta-2-microglobulin. Several interpretations are discussed. mAb 28-8-6 might detect a particular conformation or a particular chemical derivatization of otherwise normal H-2Dd molecules. Also, because the epitope recognized by 28-8-6 lies close to the peptide binding site, it is possible that mAb 28-8-6 recognizes a subset of Dd molecules bearing a certain category of self peptides.

Animals↗

PubMedDate not supplied

Quantitative PCR with colorimetric detection.

We describe a quantitative polymerase chain reaction (PCR) technique with a colorimetric read-out, which enables quantitation of various sequences on a single microplate within one day. Using the quantitation of human IL10 and beta-actin transcripts, we show that this technique provides better reproducibility, as results are derived from a series of PCRs made with various amounts of standard. This also enables the control of the consistency of the data and elimination of some artifactual results.

Actins↗