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M Introna

Publications and source records attributed to M Introna.

At least 37 records · Page 2Linked to original sources
PubMed2000

Genetic modification of human T cells with CD20: a strategy to purify and lyse transduced cells with anti-CD20 antibodies.

A retroviral vector has been constructed that contains the human CD20 cDNA under the control of the Moloney murine leukemia virus (Mo-MuLV) LTR. Freshly isolated mononuclear cells are infected for three consecutive days in the presence of PHA and hrlL-2, and a mean 15.9% of the cells (range, 6.5 to 31.7%) acquire a CD3+CD20+ phenotype. Transduced T lymphocytes grow and expand in vitro for up to 3 weeks like mock-infected cells and, as observed for the T lymphoblastoid CEM cell line, CD20 expression is maintained for several months with no change in the growth curve of the cells. CD20-expressing CEM and fresh T lymphocytes can be positively immunoselected on columns using different anti-CD20 antibodies. Exposure to monoclonal chimeric anti-CD20 IgG1(kappa) Rituximab antibody (Roche), in the presence of complement, results in effective and rapid killing of the transduced CD3+CD20+ human T cells in vitro. This approach represents a new and alternative method to gene manipulation with "suicide" genes for the production of drug-responsive T cell populations, a crucial step for the future management of graft-versus-host disease in bone marrow transplant patients.

Antibodies, Monoclonal↗
PubMed2000

Nucleolin, a novel partner for the Myb transcription factor family that regulates their activity.

To unravel the mechanisms of action of transcriptional regulation by the Myb family of transcription factors, we have set out to isolate their protein partners. We identify nucleolin as one of the nuclear polypeptides that interact specifically with the A-Myb and c-Myb, but not B-Myb DNA-binding domains. We show unambiguously that this interaction is direct and takes place in vivo, as demonstrated by co-immunoprecipitation of the endogenously and exogenously expressed proteins. The minimal DNA-binding domain containing only the R2R3 c-Myb repeats is sufficient for nucleolin binding. Computer analysis of the R2R3 three-dimensional structure, as well as extensive mutational analysis within this region, reveals that the Arg(161) residue, present in c-Myb and A-Myb, but not B-Myb, is crucial for this interaction. We show that the interaction of nucleolin with Myb is functional because co-transfection of nucleolin down-regulates Myb transcriptional activity. Nucleolin is a multifunctional phosphoprotein present in both nucleoplasm and more abundantly in the nucleolus and shows helicase and chromatin decondensing activities. This is the first demonstration of nucleolin binding to a transcription factor.

Amino Acid Sequence↗
PubMed2000

Expression and production of the long pentraxin PTX3 in rheumatoid arthritis (RA).

PTX3 is a secreted molecule which consists of a C-terminal domain similar to classical pentraxins (e.g. C-reactive protein (CRP)) and of an unrelated N-terminal domain. Unlike the classical pentraxins, the long pentraxin PTX3 is expressed in response to IL-1beta and tumour necrosis factor-alpha (TNF-alpha), but not to IL-6, in various cell types. The present study was designed to investigate the expression of PTX3 in RA. Dissociated RA and osteoarthritis (OA) type B synoviocytes were cultured in the presence and in the absence of inflammatory cytokines. PTX3 mRNA expression in synoviocytes was evaluated by Northern analysis. PTX3 protein levels in synovial cell cultures and synovial fluid were estimated by ELISA, and PTX3 distribution in synovial tissues by immunohistochemical techniques. OA synoviocytes were induced to express high levels of PTX3 mRNA by TNF-alpha, but not by other cytokines including IL-1beta and IL-6. RA synoviocytes, unlike OA synoviocytes, constitutively expressed high levels of PTX3 in the absence of deliberate stimulation. The constitutive expression of PTX3 in RA synoviocytes was not modified by anti-TNF-alpha antibodies, IL-1 receptor antagonist or a combination of the two agents. In contrast, interferon-gamma and transforming growth factor-beta inhibited PTX3 constitutive expression in RA synoviocytes. The joint fluid from RA patients contained higher levels of immunoreactive PTX3 than controls and the synovial tissue contained endothelial cells and synoviocytes positive for PTX3 by immunohistochemistry. In conclusion, PTX3 may play a role in inflammatory circuits of RA, and its relevance as a marker of disease activity deserves further study.

Arthritis, Rheumatoid↗
PubMed1999

Bacterial lipopolysaccharide causes rapid shedding, followed by inhibition of mRNA expression, of the IL-1 type II receptor, with concomitant up-regulation of the type I receptor and induction of incompletely spliced transcripts.

The IL-1 type I receptor (IL-1RI) is part of a signaling complex together with the IL-1R accessory protein, whereas available information is consistent with a "decoy" model of function for the IL-1 type II receptor (IL-1RII). The present study was designed to investigate the effect of bacterial LPS on IL-1R in human monocytes. LPS causes rapid release of the IL-1RII, an effect blocked by a metalloprotease inhibitor. Subsequently, LPS-treated monocytes showed a drastic reduction of IL-1RII mRNA. In contrast, LPS induced IL-1RI and, to a lesser extent, IL-1AcP expression. LPS-induced augmented expression of the canonical 5-kb IL-1RI mRNA was accompanied by the appearance of 2.4-kb IL-1RI transcripts. The use of probes representative of different regions of the IL-1RI mRNA, as well as cDNA cloning, revealed that the 2.4-kb inducible band includes incompletely spliced, polyadenylated transcripts potentially encoding truncated versions of the receptor. The observation that the prototypic proinflammatory molecule LPS has divergent effects on IL-1Rs, with inhibition of IL-1RII and stimulation of IL-1RI and IL-1R accessory protein, is consistent with the view that these molecules subserve opposite functions in the pathophysiology of the IL-1 system. The rapid shedding of IL-1RII by monocytes early in recruitment may serve to buffer the systemic action of IL-1 leaking from sites of inflammation. This early event, followed by prolonged inhibition of IL-1RII expression and up-regulation of IL-1RI, may render monocytes more responsive to IL-1 at sites of inflammation.

Base Sequence↗
PubMed1999

Characterization of type II intracellular IL-1 receptor antagonist (IL-1ra3): a depot IL-1ra.

Three molecular forms of the IL-1 receptor antagonist (IL-1ra) have been identified and cloned. Secreted IL-1ra (sIL-1ra or IL-1ra1) contains a classical leader peptide giving a released mature protein. Two intracellular isoforms, icIL-1ra type I (IL-1ra2) and icIL-1ra type II (IL-1ra3), have no leader sequence, thus predicting that these proteins remain intracellular. In an effort to define its biological role, we structurally and functionally characterized IL-1ra3. Endogenous immunoreactive IL-1ra3 was detected in a variety of inflammatory cells and tissues. We used a gene transfer strategy to explore the possible intracellular functions of IL-1ra3 (and IL-1ra2) and the cell-associated agonist IL-1alpha. The intracellular IL-1ra3 isoform, as well as IL-1ra2, does not block the action of exogenous and endogenous IL-1 under these conditions. Intact IL-1ra3 was released from the cells killed by NK effectors. The intracellular isoforms may represent a reservoir of IL-1ra, released upon cell death, whose function is to limit the pro-inflammatory action of cell debris.

Animals↗
PubMed1999

How can oncogenic transcription factors cause cancer: a critical review of the myb story.

Many transcription factors have been found to be oncogenic either when functionally altered through fusion with other proteins or through deregulated expression. However, still little is understood about the mechanism by which these proteins can transform cells of different origin. The Myb transcription factor is emblematic in this respect. Several forms of Myb can transform hematopoietic cells of different lineages in the chicken and Myb has been shown to be required for murine fetal hematopoiesis and to regulate the growth and differentiation of hematopoietic cells of several animal species. The role of the transcriptional activity of Myb and its possible gene targets in transformation are critically discussed.

Animals↗
PubMed1999

C-myb, but not B-myb, upregulates type I collagen gene expression in human fibroblasts.

C-myb and B-myb belong to the myb family of transcription factors. We have shown previously that c-myb is deregulated in fibroblasts from systemic sclerosis (scleroderma) patients relative to normal fibroblasts. Scleroderma fibroblasts are known to express elevated levels of collagen genes and transforming growth factor beta is known to be a pro-fibrotic cytokine and to induce transcription of type I collagen genes. We have therefore investigated the role of c-myb and B-myb in the regulation of type I collagen genes in response to transforming growth factor beta in normal human fibroblasts. We show that, in these cells, transforming growth factor beta treatment induces c-myb as well as collagen alpha1(I) and alpha2(I) gene expression, but not B-myb. Furthermore we demonstrate by cotransfection assays that c-myb can upregulate alpha1(I) and alpha2(I) collagen promoters by 6-10-fold whereas B-myb is inactive. The activity of c-myb on both type I collagen promoters requires a functional c-myb DNA binding domain suggesting a direct interaction between c-myb and these promoters. Indeed c-myb is active also on a 500 bp fragment of the alpha2(I) collagen promoter and can bind to this fragment in electrophoretic mobility shift assays. Finally, we show that anti-c-myb anti-sense treatment reduces alpha1(I) and to a lesser extent alpha2(I) collagen gene expression. These data strongly suggest that c-myb, but not B-myb, plays a direct role in the upregulation of type I collagen gene expression in response to transforming growth factor beta.

Cell Cycle Proteins↗
PubMed1998

Regulation of inhibitory pathways of the interleukin-1 system.

The IL-1 system includes two agonists, converting enzymes, antagonists, and two receptors (R). New elements and functions in the system will be discussed, including (a) cloning of a new isoform of the receptor antagonist; (b) further analysis of the type II IL-1-binding molecule as a decoy R. The modulation of IL-1R by chemotactic signals was recently investigated. It was found that chemoattractants cause rapid release of the type II decoy R from myelomonocytic cells with a t1/2 of 30 sec. Induction of decoy R release represents an early event in the multistep process of recruitment. It may serve to block the systemic action of IL-1 leaking from sites of inflammation, while preserving responsiveness in situ. We recently cloned the first long pentraxin, PTX3 (human and mouse, cDNA and genomic) as an IL-1-inducible gene. The structural and functional features of this molecule as well as initial evidence of involvement in human pathology will be discussed.

Animals↗
PubMed1998

The A-Myb transcription factor is a marker of centroblasts in vivo.

The A-Myb transcription factor is structurally related to the c-myb proto-oncogene and is involved in the control of proliferation and/or differentiation of mature B lymphocytes. We have shown previously by PCR analysis that A-myb is preferentially expressed in CD38+ CD39- sIgM- mature B cells. We demonstrate here, using in situ hybridization, that A-myb expression is restricted to the dark zone of human tonsils and lymph nodes. Furthermore, we show that A-Myb expression is cell cycle regulated both in tonsillar B cells and in Burkitt's lymphoma cell lines, being detectable only in the S and G2/M phases of the cell cycle and not in G0/G1 phase. Strong proliferation of resting human B cells induced in vitro by a variety of physiologic signals, including anti-mu, CD40 ligand, IL-2, IL-4, IL-6, IL-13, IFN-gamma, TNF-alpha, anti-CD19, and anti-CD20, failed to induce A-myb expression, suggesting that proliferation alone is not sufficient for A-myb expression in the absence of induction of a true centroblast phenotype. Finally, we show that differentiation of germinal center B cells in vitro toward either memory or plasma cells is accompanied by rapid down-regulation of A-myb expression. We conclude that A-myb is a marker of centroblasts generated in vivo.

B-Lymphocytes↗
PubMed1998

Interferon-gamma inhibits expression of the long pentraxin PTX3 in human monocytes.

PTX3 is a prototypic long pentraxin expressed by various cell types, most prominently monocytes and endothelial cells, in response to interleukin-1 (IL-1), tumor necrosis factor (TNF) and bacterial products. In the present report, we show that interferon-gamma (IFN-gamma) inhibits the expression of the PTX3 gene induced by exposure to IL-1, TNF or lipopolysaccharide in human monocytes. This effect is dose dependent and observable when IFN-gamma is added from 24 h before up to 3 h after the addition of IL-1. While the time course of the IL-1-induced PTX3 mRNA expression is not affected, IFN-gamma reduces the stability of the PTX3 mRNA as well as its transcription. The inhibition of PTX3 expression is restricted to monocytes in that no inhibition occurs in cytokine-stimulated fibroblasts and endothelial cells. Under the same conditions, as expected, IFN-gamma augmented monocyte chemotactic protein-1 expression in the same cell preparations. PTX3 protein secretion by activated monocytes is also suppressed by exposure to IFN-gamma. Altogether, these data identify a negative pathway of regulation mediated by IFN-gamma, which may occur under inflammatory conditions.

C-Reactive Protein↗
PubMed1998

Rapid retroviral infection of human haemopoietic cells of different lineages: efficient transfer in fresh T cells.

In order to develop a clinically feasible gene marking approach, we have used the recently described PINCO retroviral expression system, composed of the enhanced green fluorescence protein (EGFP) cDNA driven by Moloney MLV LTR and packaged in the Phoenix amphotropic cell line. Two T, five B, one erythromyeloid and three myeloid cell lines were successfully infected with % GFP+ cells ranging from 4% to 79%, showing a lineage-dependent difference in infection susceptibility, with the myeloid cells being the least efficiently infected. We also infected normal mononuclear peripheral cells cultured in PHA and rhIL-2 for 2 d, and obtained an average of 30% GFP+ cells, all present within the CD3+ population, with CD4+ and CD8+ cells being equally infected. Finally, the tonsillar purified B population showed lower levels of infectivity (6%) whereas high susceptibility was shown by normal human umbilical vein endothelial cells (57%). Highly purified CD34+ cells were also susceptible, varying from 6% to 10% GFP+ cells. Immature myeloid/erythroid progenitors have been infected which stably expressed the GFP protein during further differentiation in culture. The GFP+ T cells were FACS-sorted rapidly upon infection, subsequently cultured and the fluorescence intensity monitored. In all cases the difference in percentage of GFP+ cells did not correlate with the percentage of S/G2/M cycling cells as determined at the moment of infection or with the expression levels of Ram-1 amphotrophic receptor. The improved safety of this retroviral system, the rapidity of the technique, the high efficiency of infection with respect to normal T lymphocytes (in this last case higher than previously reported) and the lack of need for in vitro selection make this system favourable for clinical development.

Antigens, CD34↗
PubMed1997

Multimer formation and ligand recognition by the long pentraxin PTX3. Similarities and differences with the short pentraxins C-reactive protein and serum amyloid P component.

PTX3 is a prototypic long pentraxin consisting of a C-terminal 203-amino acid pentraxin-like domain coupled with an N-terminal 178-amino acid unrelated portion. The present study was designed to characterize the structure and ligand binding properties of human PTX3, in comparison with the classical pentraxins C-reactive protein and serum amyloid P component. Sequencing of Chinese hamster ovary cell-expressed PTX3 revealed that the mature secreted protein starts at residue 18 (Glu). Lectin binding and treatment with N-glycosidase F showed that PTX3 is N-glycosylated, sugars accounting for 5 kDa of the monomer mass (45 kDa). Circular dichroism analysis indicated that the protein consists predominantly of beta-sheets with a minor alpha-helical component. While in gel filtration the protein is eluted with a molecular mass of congruent with900 kDa, gel electrophoresis using nondenaturing, nonreducing conditions revealed that PTX3 forms multimers predominantly of 440 kDa apparent molecular mass, corresponding to decamers, and that disulfide bonds are required for multimer formation. The ligand binding properties of PTX3 were then examined. As predicted based on modeling, inductive coupled plasma/atomic emission spectroscopy showed that PTX3 does not have coordinated Ca2+. Unlike the classical pentraxins CRP and SAP, PTX3 did not bind phosphoethanolamine, phosphocholine, or high pyruvate agarose. PTX3 in solution, bound to immobilized C1q, but not C1s, and, reciprocally, C1q bound to immobilized PTX3. Binding of PTX3 to C1q is specific and saturable with a Kd 7.4 x 10(-8) M as determined by solid phase binding assay. The Chinese hamster ovary cell-expressed pentraxin domain bound C1q when multimerized. Thus, as predicted on the basis of computer modeling, the prototypic long pentraxin PTX3 forms multimers, which differ from those formed by classical pentraxins in terms of protomer composition and requirement for disulfide bonds, and does not recognize CRP/SAP ligands. The capacity to bind C1q, mediated by the pentraxin domain, is consistent with the view that PTX3, produced in tissues by endothelial cells or macrophages in response to interleukin-1 and tumor necrosis factor, may act as a local regulator of innate immunity.

Animals↗
PubMed1997

The DNA binding domain of the A-MYB transcription factor is responsible for its B cell-specific activity and binds to a B cell 110-kDa nuclear protein.

Expression studies as well as the use of transgenic animals have demonstrated that the A-MYB transcription factor plays central and specific role in the regulation of mature B cell proliferation and/or differentiation. Furthermore, it is highly expressed in Burkitt's lymphoma cells and may participate in the pathogenesis of this disease. We have therefore investigated the transcriptional activity of A-MYB and its regulation in several human lymphoid cell lines using co-transfection assays and show that A-MYB is transcriptionally active in all the B cell lines studied, but not in T cells. In particular the best responder cell line was the Burkitt's cell line Namalwa. The activity of A-MYB in B and not T cells was observed when either an artificial construct or the c-MYC promoter was used as a reporter. Furthermore, the functional domains responsible for DNA binding, transactivation, and negative regulation, previously characterized in a fibroblast context, were found to have similar activity in B cells. The region of A-MYB responsible for the B cell specific activity was defined to be the N-terminal 218 amino acids containing the DNA binding domain. Finally, a 110-kDa protein has been identified in the nuclei of all the B, but not T, cell lines that specifically binds to this A-MYB N-terminal domain. We hypothesize that this 110-kDa protein may be a functionally important B cell-specific co-activator of A-MYB.

B-Lymphocytes↗
PubMed1997

Regulatory domains of the A-Myb transcription factor and its interaction with the CBP/p300 adaptor molecules.

The A-Myb transcription factor belongs to the Myb family of oncoproteins and is likely to be involved in the regulation of proliferation and/or differentiation of normal B cells and Burkitt's lymphoma cells. To characterize in detail the domains of A-Myb that regulate its function, we have generated a series of deletion mutants and have investigated their trans-activation potential as well as their DNA-binding activity. Our results have allowed us to delineate the trans-activation domain as well as two separate regulatory regions. The boundaries of the trans-activation domain (amino acid residues 218-319) are centred on a sequence rich in charged amino acids (residues 259-281). A region (residues 320-482) localized immediately downstream of the trans-activation domain and containing a newly identified conserved stretch of 48 residues markedly inhibits specific DNA binding. Finally the last 110 residues of A-Myb (residues 643-752), which include a sequence conserved in all mammalian myb genes (region III), negatively regulate the maximal trans-activation potential of A-Myb. We have also investigated the functional interaction between A-Myb and the nuclear adaptor molecule CBP [cAMP response element-binding protein (CREB)-binding protein]. We demonstrate that CBP synergizes with A-Myb in a dose-dependent fashion, and that this co-operative effect can be inhibited by E1A and can also be observed with the CBP homologue p300. We show that this functional synergism requires the presence of the A-Myb charged sequence and that it involves physical interaction between A-Myb and the CREB-binding domain of CBP.

3T3 Cells↗
PubMed1997

Identification of two novel isoforms of the ZNF162 gene: a growing family of signal transduction and activator of RNA proteins.

By differential screening of a cDNA library obtained from a GM-CSF-dependent human myeloid leukemia cell line (GF-D8), we identified two novel isoforms of the recently described ZNF162 gene, which is apparently linked to multiple endocrine neoplasia type 1. The shorter of these new isoforms, called B3, presents an open reading frame (ORF) of 1713 bp coding for 571 amino acids. Its nucleotide sequence is homologous to the cDNA coding for the ABCDF isoform of ZNF162, except for a 4-nucleotide insertion that results in a frame shift of the ORF starting from nucleotide 1725 of the ZNF162 sequence. As a consequence, the predicted translation product of B3 contains the consensus sequence of the A motif (G-X-X-X-X-G-K-S) of the "ATP/ GTP binding site," which is characteristic of several protein families including protein kinases. Moreover, B3 shows the use of a different stop codon and contains a different tyrosine-rich COOH terminus. The longer isoform, called B4, differs from the ABCDEF isoform of ZNF162 by the insertion, at position 2137, of 383 nucleotides leading to a different, proline-rich COOH terminus. The complex transcription pattern of the ZNF162 gene is characterized by four transcripts, of approximately 3.9, 3.7, 3.2, and 2.9 kb, in GF-D8 cells. The 3.7- and 2.9-kb transcripts are expressed in resting GF-D8 cells. Upon stimulation with GM-CSF the expression of these mRNAs is up-regulated in parallel with the induction of two additional transcripts of 3.9 and 3.2 kb. The same pattern of expression has also been observed in freshly isolated myeloid leukemia cells and normal CD34+ stem cells. In light of these data, and since GM-CSF is known to stimulate signal transduction pathways, it becomes relevant that all the different isoforms of ZNF162 contain the KH module, which is a sequence motif present in proteins playing a major role in regulating cellular RNA metabolism. A search for functional domains demonstrates that ZNF162 belongs to a new and growing family of genes dubbed STAR (signal transduction and activator of RNA) proteins that are thought to play a downstream role in cell signaling and also in RNA binding. The mammalian members include Sam68, which is a target of Src, Fyn, and Grb2, and the newly cloned mouse quaking proteins (qkI) necessary in early embryogenesis and myelination. Moreover, since ZNF162 is highly conserved from yeast to humans, it implies that this new pathway has a significant function.

Amino Acid Sequence↗
PubMed1997

The cytolytically inactive terminal complement complex activates endothelial cells to express adhesion molecules and tissue factor procoagulant activity.

The membrane attack complex of complement (C) in sublytic concentrations stimulates endothelial cells (EC) to express adhesion molecules and to release biologically active products. We have examined the ability of a cytolytically inactive form of this complex, which is incapable of inserting into the cell membrane, to upregulate the expression of adhesion molecules and of tissue factor (TF) procoagulant activity. The inactive terminal C complex (iTCC) was prepared by mixing C5b6, C7, C8, and C9 and was purified by fast protein liquid chromatography on a Superose 12 column. Binding of this complex to EC was found to be dose dependent and was inhibited by anti-C9 antibodies, as assessed both by ELISA using an mAb anti-C9 neoantigen and by measuring cell-bound 125I-labeled iTCC. Exposure of EC to iTCC resulted in a dose- and time-dependent expression of endothelial leukocyte adhesion molecule 1, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 accompanied by increased levels of the corresponding mRNA, but not in the rapid expression of P-selectin. Inactive TCC also induced increased TF activity evaluated by a chromogenic assay that measures the formation of factor Xa. These effects were inhibited by anti-C9 antibodies. The data support the conclusion that iTCC may induce proinflammatory and procoagulant activities on EC.

Cell Adhesion Molecules↗