Biomedical subjects

M Fougereau

Publications and source records attributed to M Fougereau.

At least 19 recordsLinked to original sources

PubMed2000

Responding to allegations of scientific misconduct: the procedure at the French National Medical and Health Research Institute.

Institutions in France are not yet well prepared to respond to allegations of scientific misconduct. Following a serious allegation in late 1997, INSERM, the primary organization for medical and health-related research in France, began to reflect on this subject, aided by scientists and jurists. The conclusions have resulted in establishing a procedure to be followed in cases of alleged misconduct, and also in reinforcing the application of good laboratory practices within each laboratory. Guidelines for authorship practices and scientific assessment must also be considered. Even though each institution must remain responsible for responding to allegations of scientific misconduct within its doors, INSERM would like to see national, European, and international co-ordination about the methods of such response.

Academies and Institutes↗

PubMed2000

Autosomal primary immunodeficiencies affecting human bone marrow B-cell differentiation.

Since the initial report of X-linked agammaglobulinemia by Bruton, numerous autosomal primary immune deficiencies affecting early B-cell differentiation have been described in humans. The identification of these autosomal mutations has been facilitated by phenotype comparison with knockout mice. In mice, defects in B-cell development have been observed after disruption of genes encoding transcription factors, the interleukin-7 pathways as well as structural or signaling components of the pre-B-cell receptor. In general, the phenotypes of primary immune deficiencies in humans correlate with those observed in mutant mice, validating the use of the mouse model approach. In addition, we report a follow-up analysis of an autosomal primary deficiency in a young female patient born from consanguinous parents and characterized by the absence of pre-B and B-cell compartments. The patient's gene defect was identified as a cytosine insertion at the beginning of the CH1 exon of the Ig(mu) gene, resulting in a stop codon at position 48 and the absence of Ig(mu) chain expression. The precise phenotype of this patient is compared to other autosomal primary immunodeficiencies affecting humans and mice.

Amino Acid Sequence↗

PubMed1999

The human (PsiL+mu-) proB complex: cell surface expression and biochemical structure of a putative transducing receptor.

The surrogate light chain (PsiL) associates with mu and Igalpha-Igbeta chains to form the preB-cell receptor that plays a critical role in early B-cell differentiation. Discrepancies exist in human concerning the existence of PsiL+mu- proB cells and the biochemical structure of such a proB-cell complex remains elusive. Among new antihuman VpreB monoclonal antibodies (MoAbs), 5 of the gamma kappa isotype bound to recombinant and native VpreB protein with high affinity. They recognized 4 discrete epitopes, upon which 2 were in the extra-loop fragment. Such MoAbs detected the PsiL at the cell surface of either preB or on both proB and preB cells. The previously reported SLC1/SLC2 MoAbs recognize a conformational epitope specific for the mu/PsiL association in accordance with their preB-cell reactivity. Using the proB/preB 4G7 MoAb, PsiL cell surface expression was detected on normal bone marrow, not only on CD34(-)CD19(+) preB but also on CD34(+)CD19(+) proB cells. Futhermore, this MoAb identified PsiL+mu- fresh proB leukemic cells of the TEL/AML1 type. Biochemical studies showed that, at the proB stage, the PsiL is associated noncovalently with two proteins of 105 and 130 kD. Triggering of this complex induces intracellular Ca2+ flux, suggesting that the PsiL may be involved in a new receptor at this early step of the B-cell differentiation.

Animals↗

PubMed1999

Mu-surrogate light chain physicochemical interactions of the human preB cell receptor: implications for VH repertoire selection and cell signaling at the preB cell stage.

The surrogate light chain (SL) composed of the A-like and VpreB polypeptides is organized as two Ig domains and an extra-loop structure. It associates to the mu-chain in preB cells. We have produced human VpreB, SL, two Fdmu (VH-CH1), and the two corresponding Fab-like (Fdmu-SL) recombinant proteins in baculovirus. The correctness of the general conformation of the proteins was assessed by epitope mapping and affinity measurements using a new batch of anti-VpreB mAbs. Plasmon resonance analysis showed that both VpreB and the entire SL associated with the Fdmu fragments, with Kd values of 3x10(-8) M for VpreB-Fdmu and of 10(-9) to 10(-10) M, depending upon the V(H), for SL-Fdmu. These results indicate that the A-like chain, in addition to be covalently bound to the Cmu1 domain, also interacts with the VH domain. Therefore, a dual role of the SL emerges: 1) interaction of the C-domain of A-like would release the mu-chain from its interaction with binding protein in the endoplasmic reticulum, and 2) interaction of a part of A-like and most of VpreB would bind to VH, ensuring a "quality control" of the native heavy chain that represents the first step of selection of the B cell repertoire. We also demonstrated that two Fab-like fragments did not interact with each other, suggesting that activation of the cell surface preB receptor does not involve aggregation neither in cis nor in trans of the Fab-like structures.

Antibodies, Monoclonal↗

PubMed1998

Tyrosine phosphorylation of the Wiskott-Aldrich syndrome protein by Lyn and Btk is regulated by CDC42.

The Wiskott-Aldrich syndrome (WAS) is a rare immunodeficiency disease affecting mainly platelets and lymphocytes. Here, we show that the WAS gene product, WASp, is tyrosine phosphorylated upon aggregation of the high affinity IgE receptor (Fc epsilonRI) at the surface of RBL-2H3 rat tumor mast cells. Lyn and the Bruton's tyrosine kinase (Btk), two protein tyrosine kinases involved in Fc epsilonRI-signaling phosphorylate WASp and interact with WASp in vivo. Interestingly, expression of a GTPase defective mutant form of CDC42, that interacts with WASp, is accompanied by a substantial increase in WASp tyrosine phosphorylation. This study suggests that activated CDC42 recruits WASp to the plasma membrane where it becomes phosphorylated by Lyn and Btk. We conclude that WASp represents a connection between protein tyrosine kinase signaling pathways and CDC42 function in cytoskeleton and cell growth regulation in hematopoietic cells.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1998

Construction of temperature and Zn-dependent human stromal cell lines that amplify hematopoietic precursors from cord blood CD34+ cells.

Forty-five human stromal cell lines were established from long-term bone marrow cultures transformed with a new vector, pNu MTSVts, which contains the Zn-inducible metallothionein promoter and the temperature-dependent SV40 T antigen from SV40 A58 mutant. Six of these cell lines were studied because of their growth capacity. All cell lines differed with respect to growth potential, expression of cell surface markers, and cytokine transcripts. Major histocompatibility complex (MHC) class I, CD29, CD49d, and CD51 were present on all stromal cell lines, MHC class II and CD34 were consistently absent, and CD11a (LFA-1), CD18 (ICAM-1R), CD54 (ICAM-1), CD58 (LFA-3) CD56 (N-CAM), CD106 (V-CAM), laminin, and collagen IV were diversely expressed. All cell lines contained interleukin (IL)-1alpha, IL-1beta, IL-2, IL-5, and macrophage colony-stimulating factor (M-CSF) transcripts, whereas granulocyte M-CSF, TNFalpha, IL-3, IL-4, and IL-7 were diversely expressed. The most characteristic feature of these cells was their varying capacity to expand cord blood CD34+ cells. One of these stromal cell lines ensured more than twofold expansion of the initial CD34+CD10-CD19- population in the first 2 weeks. Differentiation toward the B cell lineage was limited, producing only very small numbers of CD19+ cells after 6 weeks of culture.

Antigens, CD34↗

PubMed1997

A non-XLA primary deficiency causes the earliest known defect of B cell differentiation in humans: a comparison with an XLA case.

We report a detailed comparison of B cell defects in two patients, one XLA and one non-XLA. Both had severe agammaglobulinemia with a total absence of CD19+ cells in the periphery. In the non-XLA case, CD19 expression was also highly impaired in the bone marrow, resulting in the absence of both B and preB compartments. Early proB cells were present since CD34+CD10+ and some CD19+CD10+ mostly CD34+ were identified, although diminished. By contrast, in the XLA patient the CD34+CD19+ proB cells were increased whereas the CD34-CD19+ preB cell population was low. Semi-quantitative RT-PCR analysis performed on mononuclear bone marrow cells from the non-XLA patient indicated that lambda-like, VpreB, Rag-1, Rag-2 and TdT transcripts expressed during proB cell stages were found at normal levels whereas E2A, CD10, Syk, Pax-5, CD19, Ig alpha, Ig beta, VH-C mu and V kappa-C kappa transcripts characteristic of later stages were severely depressed. By contrast in the XLA patient most of these transcripts were observed in normal amounts. The phenotype of the non-XLA patient resembles that of Pax-5 or Ig beta knock-out mice, but since the coding sequence of both cDNAs were shown to be normal, the blockage might rather result from an altered regulation of one of these genes or from defect of other genes. All these data indicate that the non-XLA patient suffers from a new genetic defect that results in an arrest of differentiation within the proB cell compartment, before the onset of Ig gene rearrangements. From all agammaglobulinemias reported so far, including XLA cases and those resulting from C mu gene defects, the non-XLA patient exhibits the earliest blockage in the B cell differentiation pathway.

Agammaglobulinemia↗

PubMed1997

The SH3 domain of Bruton's tyrosine kinase interacts with Vav, Sam68 and EWS.

Bruton tyrosine kinase (BTK) is a cytoplasmic protein tyrosine kinase which controls crucial steps of differentiation of B lymphocytes. Mutations affecting either the PH, SH3, SH2 or kinase domain of BTK all give rise to X linked agammaglobulinaemia (XLA) in humans. In this study, the authors report that the BTK-SH3 domain binds to a set of proteins expressed in pro-B, pre-B and B cell lines. Three of them were characterized as Vav, Sam68 and EWS. The authors show that a Pro-->Leu substitution in a region of the SH3 domain, which is deleted in an XLA patient, is sufficient to abolish BTK-SH3 binding potential. The authors also report that several of the BTK-SH3 binding proteins, including Sam68, EWS and Vav, are tyrosine phosphorylated in conditions that also promote BTK kinase activity. For EWS and Sam68 this tyrosine phosphorylation was cell cycle dependent.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Human thymic B cells largely overexpress the VH4 Ig gene family. A possible role in the control of tolerance in situ?

Human thymus contains a small population of B cells, a fraction of which expresses CD2 and/or CD5, as revealed by FACS analysis. Human thymic B cells enriched on CD19 panning revealed a constant co-expression of CD19 and Ig light chains. In the absence of detectable B precursors, these cells had reached at least the stage of IgM+ immature B lymphocytes. Analysis of Ig transcripts by PCR amplification first revealed a strong bias in favor of the VH4 and sequencing of 45 VH4-D-J cDNA clones isolated from two thymuses indicated that this thymic repertoire significantly differed from that of peripheral blood lymphocytes. Most genes of the VH4 family were used with various D-J combinations and a relatively high frequency of somatic mutations. This repertoire thus appears clearly selected in the thymus. This is of particular interest since the VH4 gene family is frequently encountered in autoimmunity a situation in which the number of thymic B cells is largely increased. They could play a major role in the control of tolerance in situ.

Antigens, CD19↗

PubMed1996

A human non-XLA immunodeficiency disease characterized by blockage of B cell development at an early proB cell stage.

We report a detailed analysis of a B cell defect affecting a patient girl born from first cousin parents, characterized by a severe non-X-linked agammaglobulinemia with a total absence of CD19- cells in the periphery. In the bone marrow, CD19 expression was also highly impaired, resulting in the absence of both B and preB compartments. By contrast, CD34+CD10+, CD34psiL+, and some CD19+CD10+ mostly CD34+ early proB cells were present, although diminished. Semiquantitative RT-PCR analysis performed on mononuclear bone marrow cells indicated that lambda-like, VpreB, Rag-1, Rag-2, and TdT transcripts expressed during proB cell stages were found at normal levels whereas E2A, CD10, Syk, Pax-5, CD19, Igalpha, Igbeta, VH-Cmu, and Vkappa-Ckappa transcripts characteristic of later stages were severely depressed. This phenotype resembles that of Pax-5 knock-out mice, but since the coding sequence of the patient Pax-5 cDNA was shown to be normal, the defect might rather result from an altered regulation of this gene. All these data indicate that the patient suffers from a new genetic defect that results in an arrest of differentiation within the proB cell compartment, i.e., earlier than X-linked agammaglobulinemia, before the onset of Ig gene rearrangements.

Agammaglobulinemia↗

PubMed1996

The VDJ repertoire expressed in human preB cells reflects the selection of bona fide heavy chains.

In early steps of B cell differentiation, mu chains are transiently expressed in association with a surrogate light chain (psi L) composed of the lambda-like and VpreB monomorphic polypeptides, thus forming a putative preB receptor. Using a monoclonal anti-VpreB antibody, preB cells were isolated from two adult human bone marrow samples and their VDJ repertoire analyzed at the transcription level. All VH families were identified and further analysis focused on VH3 sequence analysis of 37 distinct VDJ cDNA clones. The VH3 genes expressed in the two bone marrow samples were also encountered in fetal liver and adult peripheral blood lymphocytes with a roughly similar contribution of 3.30, 3.23, 3.9 and 3.53. The characteristic features of the preB repertoire as compared to the activation B repertoire include the quasi absence of somatic mutations, limited N diversity and a shorter third complementarity-determining region (CDR3). It also significantly differs from the fetal repertoire, which makes higher usage of DQ52 and has CDR3 of even shorter lengths. The almost constant presence of glycine residues in the CDR3 and predominance of JH4 with a low level of DQ52 DH usage, suggest that preB cell clones are submitted to an initial selective pressure which should be antigen independent. The bona fide heavy chains would be merely selected for their ability to interact with the surrogate light chains, thus shaping the repertoire that will be co-expressed with immunoglobulin light chains in IgM molecules.

Adult↗

PubMed1996

Cell surface expression of surrogate light chain (psi L) in the absence of mu on human pro-B cell lines and normal pro-B cells.

Surrogate light chains (psi L) encoded by lambda-like (lambda 5) and VpreB genes play a critical role in controlling the early steps of B cell differentiation. We prepared new anti-VpreB monoclonal antibodies (mAb) (3C7/6F6) which preferentially recognize the VpreB epitope at the cell surface of human cell lines that do not express the mu chain. These mAb provide the first characterization of human pro-B cell lines expressing surface psi L. We demonstrate that surface psi L expression is considerably enhanced upon interleukin-7 stimulation and that the psi L complex is formed independently of the Ig alpha/Ig beta heterodimer. Finally, using these antibodies, we confirm the existence of a normal pro-B cell population in human adult bone marrow. These cells are CD34+ CD38+ psi L+, do or do not express CD19, CD10, or both epitopes, and may represent the earliest cell population committed to B cell differentiation.

Adult↗

PubMed1996

Composite exon structure of an unusual Ig lambda-like gene located at human 22q11 position.

The surrogate light chain, composed of the VpreB and the lambda-like proteins, plays a critical role in controlling the early stages of B lymphocyte development. The lambda-like locus, located on the q11. 2-q11.3 region of human Chromosome (Chr) 22, contains three genes (14.1 Flambda-1, and 16.1) among which only the 14.1 is functional. This gene contains three exons, whereas the others lack exon 1. We have isolated in fetal liver a transcript of the Flambda-1 gene that contains the exon 3 sequence and a long non-Ig related sequence upstream. We show that this sequence resulted from the splicing of three new exons located telomeric to the Flambda-1 gene, highly homologous to beta-glucuronidase exon 11 (Chr 7), to the ABR exon 8 (Chr 17), and to an Expressed Sequence Tag (EST), respectively. We also show that this chimeric transcript is expressed in cells or tissues from various origins. This composite gene structure appears to be a new example of human genome flexibility, which can be explained by mechanisms such as exon shuffling and which results in the emergence of new transcription units inserted in regions involved in translocations.

Animals↗

PubMed1995

Fetal versus adult PreB or B cells: the human VH repertoire.

At the preB stage, when only the IGH locus has rearranged, mu chains become expressed in association with the psi L chains, lambda-like and VpreB, thus forming the preB receptor. By the use of a monoclonal anti VpreB antibody, preB cells were isolated from two adult bone marrow samples, and the VH repertoire was analyzed and compared to fetal, XLA (X-linked agammaglobulinemia), and adult B repertoires. Most VH genes identified were also expressed in fetal liver, XLA bone marrow, and adult PBLs, with similar predominant usage of certain germline genes. Multiple D/D fusions, limited N diversity, and preferential use of JH4 with a low level of DQ52 usage were also identified. Few mutations could be observed, not specifically localized in CDR regions, that could be interpreted as not positively selected. Conversely, a shorter length of CDR3 appeared to be the hallmark of the preB step. Thus, the association of psi L chains with mu does not bring about a bias in the VH gene usage, but a first selection on the CDR3 region could be the result of recognition by given autoantigens or ligands different for preB cells and B cells.

Adult↗

PubMed1995

Internalization of B cell and pre-B cell receptors is regulated by tyrosine kinase and phosphatase activities.

Prior to the expression of the B cell antigen receptor, the mu heavy chain associates with two non-polymorphic polypeptides, lambda like and VpreB, which form a pseudo-light chain complex in pre-B cells and pre-B cell lines. Surface expression of the so-called pre-B cell receptor (pre-BCR) occurs only in the presence of Ig alpha and Ig beta, known to be involved both in B cell antigen receptor (BCR) signaling and trafficking. Although the pre-BCR organization is consistent with an efficient transport to the cell surface, most of the newly synthesized receptor remains within the cells, and so far, no data are available concerning the rate of exit from the endoplasmic reticulum. Using the human pre-B cell line Nalm-6, we found that only a small fraction (2%) of newly synthesized pre-BCR is transported to the cell surface within 4-6 h after synthesis, where it is constitutively re-internalized. Membrane Ig-heavy chain cross-linking induced internalization of surface pre-BCR within a few minutes, and the mechanisms underlying endocytosis were analyzed by immunofluorescence and confocal microscopy. Preincubation of the cells with either genistein or orthovanadate, which inhibit, respectively, tyrosine kinases and tyrosine phosphatases, blocked pre-BCR internalization in a dose-dependent manner, indicating that both activities are required for endocytosis. BCR internalization was also inhibited in a reversible manner by the drugs. In contrast, neither drug affected the size of the steady-state pool of internalized transferrin receptors. Thus, our data show that tyrosine phosphorylation and dephosphorylation are both required for cross-linking-induced pre-BCR and BCR internalization.

B-Lymphocytes↗

PubMed1994

Activation of src family kinases in human pre-B cells by IL-7.

IL-7 was identified originally as a specific pre-B cell growth factor. We have investigated its signal transduction mechanism by using the human pre-B cell line Nalm-6, and have found that it stimulates tyrosine phosphorylation of various proteins: pp27, pp43, pp54, pp64, pp78, pp90, pp105, and pp120. Antiphosphotyrosine immunoprecipitates from IL-7-stimulated Nalm-6 showed two major proteins of M(r) = 60,000 and 55,000, capable of autophosphorylation. Autophosphorylation was maximal 10 min after the cells were challenged with the cytokine. Antiphosphotyrosine immunoprecipitates from IL-7-stimulated cells also increased tyrosine phosphorylation of the exogenously added substrate histone H2B. Furthermore, by using a polyclonal anti-IL-7 receptor (IL-7R) Ab in Western blotting analysis, we observed that antiphosphotyrosine immunoprecipitates were associated with the IL-7R in a transient manner. These data indicate that the IL-7R associates with tyrosine-phosphorylated proteins as its amino acid sequence is devoid of a putative site of tyrosine phosphorylation. These results were confirmed as several 32P-labeled proteins were visualized after immunoprecipitation by using anti-IL-7R Ab. Anti-IL-7R immunoprecipitates from IL-7-stimulated cells revealed a unique band of M(r) = 60,000 associated with the receptor able to autophosphorylate in the presence of ATP and Mn2+. Hence, we identified p59fyn and p53/56lyn to be stimulated by IL-7. In contrast to p53/56lyn, p59fyn was found to be associated constitutively with the cloned IL-7R. These data emphasize the role of the src family in hematopoiesis.

Amino Acid Sequence↗

PubMed1994

Discrete early pro-B and pre-B stages in normal human bone marrow as defined by surface pseudo-light chain expression.

Vpre-B and lambda-like genes are selectively expressed in B cell precursors and encode polypeptide chains associated in a mu-pseudo light chain (mu-psi L) complex which is thought to regulate some early steps of B cell differentiation. We have generated anti-Vpre-B monoclonal antibodies which allowed us to identify different steps of differentiation from the pro-B to the immature B cells by following surface expression of Vpre-B, mu and light chains in normal adult human bone marrow. Already present at the surface of a small fraction of B cell progenitors (CD34+/CD19+) the Vpre-B molecule was consistently found coexpressed with CD19 and was also found with the sequentially occurring CD10, CD20, CD21, CD22 and CD5 markers. Three discrete cell types were identified: (i) a subpopulation expressing Vpre-B without mu and which represented an early stage of differentiation, (ii) a minor subpopulation co-expressing Vpre-B and mu without the conventional light chains and (iii) a major subpopulation co-expressing Vpre-B, mu and kappa or lambda chains, considered an intermediate pre-B/B stage. The presence of the psi L chain in various cell subpopulations, in possible association with discrete molecules and/or different contexts, suggests its involvement at several steps of early B cell differentiation.

Adult↗

PubMed1994

Interleukin-7 regulates c-myc expression in murine T cells and thymocytes: a role for tyrosine kinase(s) and calcium mobilization.

Interleukin-7 (IL-7) was originally identified as a pre-B cell growth factor whose proliferating activity has been extended to numerous target cells including T lymphocytes. We investigated c-myc mRNA expression, an oncogene associated with proliferation, in the murine T cell line D10 G4.1 and freshly isolated thymocytes since both target cells proliferate in response to IL-7. We find that blockade of the tyrosine kinase pathway by genistein, a potent tyrosine kinase inhibitor, inhibits both IL-7-dependent D10 G4.1 cell proliferation and c-myc mRNA expression which appears to involve de novo mRNA synthesis and to be under the control of short-lived protein repressor(s). We have also examined possible signal transduction pathways which might regulate c-myc mRNA expression in the murine T cell line. IL-7 biological activity is not affected by stimulation of the protein kinase C pathway by phorbol esters. Thus, IL-7 regulates c-myc mRNA expression in a protein kinase C-independent manner and these data are strengthened by protein kinase C depletion which does not modify IL-7 c-myc mRNA responsiveness. In contrast and independent of protein kinase C activation, intracellular calcium mobilization by means of ionomycin reduces IL-7 induction of c-myc mRNA expression and may represent a physiological mechanism whereby IL-7 bioactivity is regulated. The activity of IL-7 on c-myc mRNA expression has been extended to freshly isolated thymocytes and we find a synergistic effect of IL-7 with concanavalin A. Taken together our results illuminate the molecular mechanism of IL-7 c-myc induction in the T lineage by ascribing a role for tyrosine kinase and increase in intracellular calcium in both IL-7 induced gene induction and cell proliferation.

Animals↗