Biomedical subjects

J Theze

Publications and source records attributed to J Theze.

At least 19 recordsLinked to original sources

PubMed2001

The p38 MAPK pathway is involved in the IL-2 induction of TNF-beta gene via the EBS element.

As an important cytokine of the immune system, interleukin-2 (IL-2) can induce the expression of various genes, one of which is the tumor necrosis factor-beta (TNF-beta). However, the induction mechanism of TNF-beta remains to be fully explored. We have previously shown JAK-STAT pathway mediates TNF-beta gene induction upon IL-2 stimulation through an upstream -200GAS element. In this study, we further demonstrated that there is another essential -130EBS element in TNF-beta gene promoter region. Using IL-2-dependent cell line BAF/BO3beta, we found that this -130EBS element can form a specific complex with nuclear protein, which contained a novel ETS transcription factor. Furthermore, using kinase inhibitors, we revealed that p38 MAP kinase is involved in the formation of -130EBS-protein complex and the subsequent transcriptional activation of TNF-beta gene in response to IL-2 stimulation. Taken together, our results suggested that the complicated IL-2 induction of TNF-beta gene expression requires not only the activation of JAK-STAT pathway on the -200GAS element, but also the cooperation of another signal pathway on the -130EBS element.

Animals↗

PubMed2001

Critical sites for the interaction between IL-2Rgamma and JAK3 and the following signaling.

JAK3 is the only known protein tyrosine kinase associating with IL-2Rgamma. This interaction is supposed to be very important to IL-2 signaling. In order to identify the critical residues for these two molecular interactions and the following signal events, various mutants of gammac and JAK3 were constructed on the basis of computer analysis. The direct interaction was determined via the yeast two-hybrid system, while the signaling was analyzed with reporter genes under the control of the c-fos, c-myc, or tnf-beta promoters, respectively. Results showed that there are two key sites on gammac involved in this interaction and the following signal transduction: the critical one is E327 via electrostatic interaction, the other is L293 via hydrophobic interaction. As to JAK3, the data indicated that Y100 is important for the interaction with gammac. These results also document that the requirement for interaction between gammac and JAK3 is different to activate different signaling pathways mediated by gammac, such as c-fos, c-myc, and JAK-STAT.

Animals↗

PubMed2000

Increased liposome-mediated gene transfer into haematopoietic cells grown in adhesion to stromal or fibroblast cell line monolayers.

We investigated transfection rates of CD34+ haematopoietic progenitor cells (HPC) or haematopoietic cell lines (TF-1, KG1a and K562) using the LacZ gene as a reporter and cationic liposomes. The transfection efficiency of CD34+ haematopoietic progenitor cells (HPC) or TF-1, KG1a and K562 grown in suspension is very low (average percentage of 0.013 for HPC and 0.03 for cell lines). Adhesion of HPC or cell lines to plates by immunological or physical methods significantly enhances transfection efficiency; however, the percentage of transfected cells still remained low. We found that adhesion of TF-1, KG1a and K562 HC to MS-5 stroma cells or NIH-3T3 fibroblast cells increased transfection efficiency. Under these conditions transfection is achieved in 11.2-25% (mean 18.30%) for the cell lines and 13.6% (range 8.2-24.2%) for CD34+ HPC. These results indicate that liposome-mediated transfection of HC is significantly increased when cells are grown in adherence to stroma or fibroblast monolayers.

3T3 Cells↗

PubMed2000

Interleukin-12 induces gene expression in interleukin-2 stimulated human T lymphocytes.

IL-12 is a critical immunoregulatory cytokine that promotes cell-mediated immune responses by inducing the differentiation of Th1 cells. To better clarify the molecular basis of IL-12 action, we compared the gene expression in human T lymphocytes activated by IL-2 and IL-12. mRNAs from T lymphocytes activated by either IL-2 alone or IL-2 plus IL-12 were transcribed into cDNAs. A differential mRNA display was conducted. As a result, differential display of five cDNA fragments was obtained. Sequence analysis suggests that they had high homology with recorded genes as found by a computer search against GenBank. Two full genes of the five fragments were cloned, which activation-induced C-type lectin and glucose transporter-like protein. Interestingly, these proteins were expressed in the T cells stimulated by IL-2 and IL-12, but not in the T cells stimulated by IL-2 alone. These results suggest that C-type lectin and glucose transporter-like protein may play an important role in the T lymphocyte activation induced by IL-12.

Base Sequence↗

PubMed1998

Jak-STAT pathway is involved in the induction of TNF-beta gene during stimulation by IL-2.

IL-2 is the major regulatory cytokine of the immune system. It plays a key role in T cell survival, growth and activation. IL-2 may induce the expression of multiple genes including some cytokine genes. The induction of these genes is triggered by different signal pathways, one of them being the Jak-STAT signal pathway. The genes regulated by this pathway remain to be determined. By studying IL-2-inducible genes, we have confirmed that the TNF-beta gene is one of the immediate early genes activated by IL-2. By analysis of the DNA sequences around 180-300 bases upstream of the transcription initiation point of the mouse TNF-beta gene, we demonstrate that there is a STAT5 binding site which is essential to the inducibility of the TNF-beta gene. Furthermore, in BA/F3 cells co-transfected with the STAT5A gene and IL-2R beta gene, the activation of the TNF-beta gene promoter by IL-2 was greatly promoted, whereas the TNF-beta gene promoter became IL-2-non-inducible if the STAT5A gene was substituted with a dominant negative STAT5A, i.e. a C-terminally truncated mutant. Taken together, our results show that the Jak-STAT signal pathway is involved in induction of the TNF-beta gene in cells stimulated by IL-2.

Animals↗

PubMed1998

Further characterization of the binding of human recombinant interleukin 2 to heparin and identification of putative binding sites.

We have previously provided compelling evidence that human recombinant interleukin 2 (IL-2) binds to the sulfated polysaccharides heparin, highly sulfated heparan sulfate and fucoidan. Here we show that IL-2 binding is dependent on heparin chain length, but with fragments as small as 15-mers retaining binding activity. The addition of exogenous heparin has no effect on the in vitro biological activity of IL-2. In addition soluble IL-2 receptor alpha and beta polypeptides do not compete with heparin for the binding of IL-2. IL-2 bound by heparin is still recognized by two IL-2 specific monoclonal antibodies, 3H9 and H2-8, whose epitopes lie in the amino terminal region. Murine IL-2 unlike its human counterpart fails to bind to heparin. Human IL-2 analogs with single amino acid substitutions at positions Lys43, Thr51, and Gln126 analogs no longer bind to heparin. By contrast the Arg38Ala analog retains heparin full heparin binding activity. These experimental findings together with molecular modeling studies suggest two putative heparin binding sites on human IL-2, one involving four basic residues, Lys48, Lys49, Lys54, and His55, and the other being a discontinuous site comprising Lys43, Lys64, Arg81, and Arg83. Neither of these two clusters is completely conserved in murine IL-2. Overall our data suggest that the binding of human IL-2 to heparin and heparan sulfate does not interfere with IL-2/IL-2 receptor interactions. Therefore, binding to glycosaminoglycan may be a mechanism for retaining the cytokine in an active form close to its site of secretion in the tissue, thus favoring a paracrine role for IL-2.

Animals↗

PubMed1997

Analysis of human IL-2/IL-2 receptor beta chain interactions: monoclonal antibody H2-8 and new IL-2 mutants define the critical role of alpha helix-A of IL-2.

Interleukin 2 (IL-2) interacts with a receptor (IL-2R) composed of three subunits (IL-2R alpha, IL-2R beta and IL-2R gamma). IL-2R beta plays a critical role in signal transduction. An anti-human IL-2 mAb (H2-8) produced after immunization with peptide 1-30 of IL-2 was found to recognize the region occupied by Asp20, at the exposed interface between alpha-helices A and C. Muteins at position 17 and 20 are not recognized by mAb H2-8. mAb H2-8 specifically inhibits the IL-2 proliferation of TS1beta cells which are dependent on the expression of human IL-2R beta chain for IL-2 proliferation. Substitution at internal position Leu17 demonstrates that this position is essential for IL-2 binding and IL-2 bioactivity. New IL-2 mutants at position Asp20 have been analysed. Substitutions Asp --> Asn, Asp --> Lys, Asp --> Leu, show a correlation between diminished affinity for IL-2 receptor and reduced bioactivity measured on TS1beta cells. Mutein Asp Arg lose affinity for IL-2R and bioactivity simultaneously. Furthermore, during the course of the study we have found that mutein Asp20 --> Leu is an IL-2 antagonist. The biological effects of mAb H2-8 and the properties of new mutants at positions 17 and 20 demonstrate that this region of alpha helix-A is involved in IL-2-IL-2R beta interactions.

Animals↗

PubMed1996

Fine analysis of immunoreactivity of V3 peptides: antibodies specific for V3 domain of laboratory HIV type 1 strains recognize multiple V3 sequences synthesized from field HIV type 1 isolates.

Production of cross-reactive antibodies recognizing the V3 loop--that is, the principal neutralizing determinant (PND)--of various HIV-1 isolates is an important challenge in the development of passive immunotherapy or vaccinations against AIDS. We have produced two types of antibodies to the V3 domain of HIV-1: (1) antibodies against the HIV-1 MN laboratory strain generated in rabbits and (2) antibodies targeted to the HIV-1 LAI laboratory strain induced in chimpanzees. These antibodies were shown to be specific for HIV-1 subtype B. The cross-reactivity of these antibodies has been evaluated against a large panel of peptides representing different parts of the V3 loop. Seventy-five peptides, referred to as clinical peptides, were synthesized according to HIV-1 sequences recovered from PMBCs of 27 patients followed in three Parisian hospitals. Thirteen V3 peptides derived from 4 HIV-1 laboratory strains (MN, LAI, SF2, and RF) were also included in the study. The results show that both the amino-terminal and central parts of the V3 loop are immunogenic. The rabbit antibodies against the amino-terminal end of the PND proved to be highly cross-reactive against the clinical peptides. The anti-gp160 antibodies induced in one chimpanzee recognized a significant proportion of the panel of V3 clinical sequences. These antibodies cross-reacted mainly with the apex of the V3 loop. These data give some additional indications on the immunogenicity of the V3 loop and further demonstrate that extensive cross-reactivity of anti-V3 antibodies can be obtained on field HIV-1 isolates despite the high variability of the V3 loop amino acid sequence.

Animals↗

PubMed1996

V(H) gene-family representation in peripheral activated B cells from systemic lupus erythematosus (SLE) patients.

A semiquantitative polymerase chain reaction (PCR) assay described in this study has been used to analyse the V(H)1, V(H)3 and V(H)4 repertoire expressed by total IgM+ and IgG+ B cells from normal individuals and lupus patients. This approach consists of a combination of B cell selection, utilization of the anchored PCR technique to avoid technical bias in the amplification of different V(H) gene family cDNA templates, and screening of the amplified IgM or IgG cDNA rearrangements by family-specific oligonucleotide probes. In four lupus patients, V(H) family representation in IgM+ and IgG+ in vivo activated B cells, selected by anti-CD71 antibody, and in total CD19+ B cells were compared. In all patients, V(H)4 gene family segments were preferentially under-represented in IgM+ activated B cells. In IgG+ B cells the results suggest that V(H)4 expression is variable, depending on the phase of the disease. Polyclonal B cell activation, which is usually considered as being the first event in autoantibody production in SLE, cannot explain our results. The data evoke the possible involvement of a V(H)4-specific B cell superantigen in the onset or development of SLE. This hypothesis is also supported by the sequence conservation of the fourth beta loop a putative superantigen binding site of functional V(H)4 gene segments which are preferentially used by anti-dsDNA lupus antibodies of established clones and hybridomas.

Adult↗

PubMed1995

Two new human monoclonal antibodies against HIV type 1 glycoprotein 120: characterization and neutralizing activities against HIV type 1 strains.

Two human IgGk monoclonal antibodies (HuMAbs), termed 48-16 and 50-61A, were derived by Epstein-Barr virus transformation of B cells from two HIV-1-infected donors. These HuMAbs recognized discrete, nonoverlapping, and conformational or discontinuous epitopes on the gp120 envelope protein of HIV-1. The binding affinities of 48-16 and 50-61A for recombinant gp120 from HIV-1LAI strain, reflected by their dissociation constants, were estimated to be 2-5 x 10(-9) and 2.4 x 10(-10) M, respectively. 48-16 was shown to react with a conserved determinant present on a variety of divergent laboratory isolates, residing outside the CD4-binding site and the V3 region, which remains to be determined. 48-16 did not display, however, any detectable functional activity. 50-61A exhibited a more restricted recognition pattern, but was able to completely inhibit the 2 HIV-1 laboratory strains LAI and SF2 in a concentration range of 0.5-10 micrograms/ml, as measured by an antigen capture assay. The ability of 50-61A to block the interaction between recombinant gp120LAI and recombinant as well as cellular CD4 indicated that 50-61A epitope was localized near or within the CD4-binding side. We also demonstrated that 50-61A- and 48-16-defined epitopes (or closely related epitopes) were immunogenic in infected humans, since serum samples from 45 seropositive subjects were able to inhibit both gp120LAI-HuMAb recognitions. However, the presence of "50-61A-like" antibodies in these sera could not be associated with their neutralizing activities of HIV-1LAI infection. Interest in producing such antibodies for characterization of the human B cell repertoire to HIV-1 and their potential use in passive immunotherapy or vaccination strategy against AIDS are discussed.

Antibodies, Monoclonal↗

PubMed1994

Anti-idiotypic antibodies to the third variable domain of gp120 induce an anti-HIV-1 antibody response in mice.

We tested the potential of anti-idiotypic antibodies to function as molecular mimics of a neutralizing epitope of the human immunodeficiency virus (HIV). Three monoclonal antibodies to the third variable domain (V3) of gp120 from the HIV-1LAI isolate were raised in a BALB/c mouse. They bound gp120LAI with high affinity (K congruent to 10(-11) M), immunoprecipitated viral gp120LAI, recognized HIVLAI-infected cells by immunofluorescence and neutralized HIVLAI infection in vitro. Mice and rabbits were immunized against each one of these antibodies, and one monoclonal and two polyclonal anti-idiotypic reagents were selected for further study. The three anti-idiotypes recognized binding site-related idiotopes that were not present on other V3-specific mouse sera or monoclonal antibodies. Groups of mice and rabbits were immunized against these anti-idiotypes presented in a homopolymerized form, conjugated to a protein carrier, or in an uncoupled form. In the absence of antigen, mice that received polyclonal anti-idiotypes mounted an antibody response to gp120LAI, but not to V3-deleted gp120LAI recombinant proteins. Unlike the three nominal mAbs, the induced antibodies also reacted with gp120 from the divergent HIV strain SF2. The isotypic distribution of the anti-idiotype-induced antibodies and their temporal evolution resemble the restricted pattern and the rapid rise of the antibody response obtained after administration of recombinant gp120LAI to mice. These data thus present evidence that anti-idiotypes raised against HIV-specific antibodies may substitute viral antigens for induction of a humoral immune response to HIV. They also suggest that the HIV epitope variability may be, in part, overcome with the use of anti-idiotypic reagents.

Animals↗

PubMed1992

Hyper-IgM immunodeficiency syndrome: influence of lymphokines on in vitro maturation of peripheral B cells.

Peripheral B cells from six patients affected with the hyper-IgM immunodeficiency syndrome, characterized by an absence of IgG and IgA in serum with a concomitant elevated level of IgM, were analyzed for phenotypic and functional characteristics. We report that although the membrane antigenic pattern expression was characteristic of mature B cells, B cells from most patients exhibited an impairment in their in vitro response to several lymphokines, such as recombinant interleukin 2 (rIL-2) and low molecular weight B-cell growth factor (BCGF), that induce proliferation of anti-mu-activated B cells. This impairment was also found in response to a lymphokine mixture from a CD2-activated T-cell clone. The decrease in lymphokine-induced B-cell proliferation was accompanied by a low B-cell differentiation, whether patients' B cells were stimulated by the T-cell clone supernatant or rIL-2 and rIL6, lymphokines able to support differentiation of Staphylococcus aureus Cowan I (SAC)-activated B cells. In addition, none of the lymphokines tested were able to induce patients' B cells to switch from IgM-secreting cells to IgG- and IgA-secreting cells. We conclude that this syndrome is associated with a defect in lymphokine-dependent maturation of B lymphocytes, although the T- or the B-cell origin of the defect still cannot be determined.

Antibody-Producing Cells↗

PubMed1992

Molecular area involved in the in-vitro dimerization of the murine p55 IL-2 receptor.

In order to study structure-function relationships of the M(r) 55,000 subunit of the murine IL-2R (p55 IL-2R) by epitope mapping, we have expressed the p55 IL-2R molecule in a cell-free translation system. Under these in vitro conditions, we detected the expected p55 IL-2R polypeptide initiated at Met 1 and, in addition, two internally initiated molecules at Met 64 and Met 105. In this report we describe that from such a mixture, containing three molecular species of p55 IL-2R, mAb 135D5 immunoprecipitated the polypeptide initiated at Met 105 although this N-terminally truncated form of p55 IL-2R does not contain its epitope located between amino acids 72-88. This observation can be taken as a further evidence for the formation of p55 IL-2R dimers. Finally, we identified the region implicated in the formation of p55 IL-2R dimers close to the transmembrane region of the molecule.

Animals↗

PubMed1992

Antibodies of HIV-1 positive subjects and experimentally immunized primates and rodents bind to sequence divergent regions of the third variable domain (V3) of gp120.

Several motifs have been found to be the target of the neutralizing antibody response to HIV, the human immunodeficiency virus. One of the well characterized motifs maps to a loop within the third hypervariable region (V3) of the exterior envelope glycoprotein gp120 at amino acid positions 308-331 and is referred to as the principal neutralizing determinant (PND). The sequence of this V3 loop raises the question of the immunogenicity and the degree of diversity of the antibody response to the PND. We show here that this neutralization-related motif is highly immunogenic in HIV-positive subjects and in experimentally immunized primates and rodents submitted to various anti-HIV immunization regimens. In probing the diversity of the antibody response to PNDs corresponding to 11 HIV sequence-divergent isolates in serum samples of 101 HIV-positive individuals we found that human antibodies exhibit binding affinity to up to nine PND synthetic peptides. This antibody binding was in all cases tested inhibitable by the homologous PND synthetic peptide. We additionally demonstrate that this antibody cross-reactivity towards sequence-divergent PNDs is detectable in the sera of mice and chimpanzees experimentally immunized against a single HIV-1 isolate. Finally, we noticed that there is a hierarchy of reactivity among the various PNDs wherein the synthetic peptide corresponding to the MN isolate was generally the most prominently recognized by antibodies of human, non-human primate, and rodent origins. Based on these findings and on features of the sequences analyzed we suggest that, despite its overall sequence variability, the PND encompasses conserved amino acid positions or epitopes that are the targets of antibodies recognizing sequence-divergent isolates. We also propose that the high positive charge density of the most frequently recognized PNDs and the high antigenicity value of some of their residues are critical to the broad immunoreactivity of this neutralization-related motif.

Acquired Immunodeficiency Syndrome↗

PubMed1992

Monomorphic organization of human diversity (DH) heavy chain variable genes.

While recent evidence indicates that human immunoglobulin heavy chain variable (VH) genes exhibit a high degree of heterogeneity, little is known concerning the polymorphism of the diversity (DH) gene complex. This locus comprises two clusters, major and minor, which are physically linked with the VH locus. In assessing the variability of the human major and minor DH clusters, we found no evidence for a substantial restriction site polymorphism. We also noted that, in contrast to what was found in the Japanese population, the DH1 gene is not deleted in an appreciable proportion of the European population. We propose that, because DH genes impart the critical functions associated with the third complementarity-determining region, the genomic organization of the human DH locus has been evolutionarily conserved through selection pressure mechanisms.

Antibody Diversity↗

PubMed1991

Structure-function study of the p55 subunit of murine IL-2 receptor by epitope mapping.

Using a cell-free translation system we have expressed the Mr 55,000 subunit of the murine IL-2R (p55 IL-2R), which binds IL-2 with low affinity (Kd = 10 nM). Mutants and truncated forms of p55 IL-2R have been used to map the epitopes recognized by three anti-p55 IL-2R mAb: 135D5, 7D4, and 2E4. The mAb 135D5 inhibits IL-2 binding to p55 IL-2R and recognizes an epitope located between amino acids 64 to 125. This epitope can be mimicked by a synthetic peptide corresponding to the region defined by residues 72 to 88. However, the mAb 7D4 and 2E4 do not affect the IL-2 binding to p55 IL-2R. These mAb recognize an epitope of p55 IL-2R lying between residues 125 to 212 that can be mimicked with a peptide corresponding to amino acids 188 to 208. A strong correlation emerged between the experimental results on epitope mapping and predictions of potential antigenicity of murine p55 IL-2R. In addition, we described two internal initiation sites of p55 IL-2R mRNA under the in vitro conditions used leading to the production of significant amounts of N-terminal truncated p55 IL-2R proteins.

Amino Acid Sequence↗

PubMed1991

Probing VH gene-family utilization in human peripheral B cells by in situ hybridization.

Recent studies on the genomic organization of the human Ig VH locus have revealed the presence of an important proportion of VH pseudogenes and a high degree of interspersion among VH gene-family members. The mechanisms of selection of VH genes expressed by differentiating B cells remain to be elucidated. We have made use of RNA-RNA in situ hybridization to probe the repertoire of VH gene-families assembled in peripheral B cells of normal adults. Cells were in vitro activated with mitogens and then hybridized to [35S]-labeled anti-sense RNA probes specific for C mu and C gamma genes, and for the six known human VH gene-families. We found that the numbers of cells expressing C mu and C gamma mRNA were similar to the total numbers of cells expressing members of the six VH gene-families. Therefore, the six known VH gene-families represent essentially the human VH locus. We also found that expression of VH gene-families does not closely correlate with their relative genomic complexity. This apparently biased expression may suggest that in some VH gene-families the ratio of pseudogenes/functional genes is particularly high, and/or that regulatory mechanisms play a major role in shaping the available VH gene repertoire in differentiating B cells.

Antibody Specificity↗

PubMed1991

Concomitant histamine, interleukin 4, and interleukin 6 production by hematopoietic progenitor subsets in response to interleukin 3.

Murine interleukin 3 (IL-3) induces a strong, concomitant increase in histamine, interleukin 6 (IL-6), and interleukin 4 (IL-4) synthesis by progenitor-enriched bone marrow cell populations, whereas interleukin 2 (IL-2) or interferon-gamma (IFN-gamma) are undetectable. This phenomenon is observed between 4 and 12 h after exposure to the growth factor and attains maximal cytokine and histamine levels within 24 and 48 h, respectively. None of these mediators is produced by lymphoid populations such as lymph node cells or by granulocytes. Splenocytes secrete only low histamine and IL-6 levels, in accordance with the lower incidence of progenitors in the spleen, whereas total bone marrow cells generate substantial amounts of the three mediators even before enrichment. Histamine, IL-4-, and IL-6-producing cells copurify with immature cells and cannot be separated from each other throughout the sorting procedures used herein. They are concentrated in the low-density layers (buoyant density 1.069-1.086 g/cm3) of a discontinuous Ficoll gradient (less than 4% of the total bone marrow) together with the majority of hematopoietic progenitors (marrow-repopulating ability [MRA] cells, spleen colony-forming units [CFU-S] day-8 and day-12, granulocyte-macrophage colony-forming units [CFU-GM], and mast cell precursors). Their lightscatter characteristics are those of relatively large, granular cells. They do not belong to the most primitive stem cell subset (MRA and part of CFU-S day-12), but to a population with high mitochondrial activity identified by their important rhodamine retention (colony-forming unit cells [CFU-C], blast cells). In addition, we provide evidence that histamine, IL-4, and IL-6 do not depend on each other for their respective expression. Taken together, our data are consistent with the notion that in certain conditions, immature hematopoietic cells are a potent source of histamine and cytokines.

Animals↗