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P Sideras

Publications and source records attributed to P Sideras.

At least 37 records · Page 2Linked to original sources
PubMed1998

Independent and opposing roles for Btk and lyn in B and myeloid signaling pathways.

Transphosphorylation by Src family kinases is required for the activation of Bruton's tyrosine kinase (Btk). Differences in the phenotypes of Btk-/- and lyn-/- mice suggest that these kinases may also have independent or opposing functions. B cell development and function were examined in Btk-/-lyn-/- mice to better understand the functional interaction of Btk and Lyn in vivo. The antigen-independent phase of B lymphopoiesis was normal in Btk-/-lyn-/- mice. However, Btk-/-lyn-/- animals had a more severe immunodeficiency than Btk-/- mice. B cell numbers and response to T cell-dependent antigens were reduced. Btk and Lyn therefore play independent or partially redundant roles in the maintenance and function of peripheral B cells. Autoimmunity, hypersensitivity to B cell receptor (BCR) cross-linking, and splenomegaly caused by myeloerythroid hyperplasia were alleviated by Btk deficiency in lyn-/- mice. A transgene expressing Btk at approximately 25% of endogenous levels (Btklo) was crossed onto Btk-/- and Btk-/-lyn-/- backgrounds to demonstrate that Btk is limiting for BCR signaling in the presence but not in the absence of Lyn. These observations indicate that the net outcome of Lyn function in vivo is to inhibit Btk-dependent pathways in B and myeloid cells, and that Btklo mice are a useful sensitized system to identify regulatory components of Btk signaling pathways.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1998

Activated murine B lymphocytes and dendritic cells produce a novel CC chemokine which acts selectively on activated T cells.

Genes were isolated using the suppression subtractive hybridization method by stimulation of pro/pre B cells with anti-CD40 and interleukin (IL)-4 to mature S mu-Sepsilon-switched cells. One of the strongly upregulated genes encodes a novel murine CC chemokine we have named ABCD-1. The ABCD-1 gene has three exons separated by 1. 2- and 2.7-kb introns. It gives rise to a 2.2-kb transcript containing an open reading frame of 276 nucleotides. Two polyadenylation sites are used, giving rise to cDNAs with either 1550 or 1850 bp of 3' untranslated regions. The open reading frame encodes a 24 amino acid-long leader peptide and a 68 amino acid-long mature protein with a predicted molecular mass of 7.8 kD. ABCD-1 mRNA is found in highest quantities in activated splenic B lymphocytes and dendritic cells. Little chemokine mRNA is present in lung, in unstimulated splenic cells, in thymocytes, and in lymph node cells. No ABCD-1 mRNA is detected in bone marrow, liver, kidney, or brain, in peritoneal exudate cells as well as in the majority of all unstimulated B lineage cells tested. It is also undetectable in Concanavalin A-activated/IL-2-restimulated splenic T cells, and in bone marrow-derived IL-2-induced natural killer cells and IL-3-activated macrophages. Recombinant ABCD-1 revealed a concentration-dependent and specific migration of activated splenic T lymphoblasts in chemotaxis assays. FACS(R) analyses of migrated cells showed no preferential difference in migration of CD4(+) versus CD8(+) T cell blasts. Murine as well as human T cells responded to ABCD-1. Freshly isolated cells from bone marrow, thymus, spleen, and lymph node, IL-2-activated NK cells, and LPS-stimulated splenic cells, all did not show any chemotactic response. Thus, ABCD-1 is the first chemokine produced in large amounts by activated B cells and acting selectively on activated T lymphocytes. Therefore, ABCD-1 is expected to play an important role in the collaboration of dendritic cells and B lymphocytes with T cells in immune responses.

Amino Acid Sequence↗
PubMed1998

Absence of Bruton's tyrosine kinase (Btk) mutations in patients with acute myeloid leukaemia.

Bruton's tyrosine kinase (Btk) is a non-receptor protein tyrosine kinase (PTK) that is expressed in all haemopoietic lineages except mature T cells and plasma cells. Despite the broad range of expression. mutations that inactivate this molecule affect primarily the development of the B-cell lineage. As a PTK, Btk could potentially be involved directly or indirectly in the processes that relate to the malignant transformation of all the cell lineages where this molecule is expressed. Previous studies have failed to demonstrate mutations in patients with B-cell origin acute lymphoblastic leukaemia (ALL). We have utilized a recently developed method that enables the rapid and convenient detection of mutations at the cDNA level, namely, the non-isotopic RNase cleavage assay (NIRCA) to analyse Btk sequences from 27 patients with different types of acute myeloid leukaemia (AML). The only alteration that we observed was a polymorphism at position 2031. This polymorphism has already been seen in previous studies. Furthermore, using the same methodology, we identified the Btk mutations in six XLA (X-linked agammaglobulinaemia) patients. Our results, although they do not exclude the involvement of Btk mutations in the development or progression of some type of AML, nevertheless suggest that such mutations do not constitute a major co-factor in the development of myeloid malignancies.

Acute Disease↗
PubMed1997

Btk dosage determines sensitivity to B cell antigen receptor cross-linking.

Mutations in Btk result in the B cell immunodeficiencies X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. Btk is a critical component of signaling pathways regulating B cell development and function. We used a genetic approach to determine whether Btk is also limiting for these processes. One allele of a murine Btk transgene expressed a dosage of Btk (25% of endogenous levels in splenic B cells) sufficient to restore normal numbers of phenotypically mature conventional B cells in xid mice. 2,4,6-trinitrophenyl-Ficoll response, anti-IgM-induced proliferation, B1 cell development, and serum IgM and IgG3 levels remained significantly impaired in these animals. B cells from Btk -/- transgenic mice also responded poorly to anti-IgM, indicating that the xid mutation does not create a dominant negative form of Btk. Response to 2,4,6-trinitrophenyl-Ficoll and B cell receptor cross-linking were increased 3- to 4-fold in xid mice homozygous for the transgene. These results demonstrate that Btk is a limiting component of B cell antigen receptor signaling pathways and suggest that B cell development and response to antigen may require different levels of Btk activity.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1997

Structural analysis of human gamma 3 intervening regions and switch regions: implication for the low frequency of switching in IgG3-deficient patients.

High and low serum concentrations of IgG3 are associated with the human G3m(b) and G3m(g) allotypes, respectively. In the present study, we analyzed the structure of the S gamma 3 and I gamma 3, the switch frequency, switch breakpoints and the levels and initiation sites of I gamma 3 transcripts both in normal blood donors expressing (b) or (g) allotypes as well as IgG3-deficient (D) patients. A low switch frequency to gamma 3 was found in the (g) allotype IgG3D patients which may be caused in part by the allotype-associated mutations in the S gamma 3 region and in part by additional individual mutations observed in the A (SNAP) and B (SNIP/ NF-kappa B) sites in the S gamma 3 repeat region. A higher I gamma 3 germ-line (GL) transcriptional rate was seen in cells from the IgG3D patient, suggesting that low levels of GL I gamma 3 transcripts are not a major contributing factor to the defect. However, individual mutations in the I gamma 3 region and differential splicing of GL I gamma 3 transcripts were found which may affect the switching process.

Base Sequence↗
PubMed1997

Impaired B cell maturation in mice lacking Bruton's tyrosine kinase (Btk) and CD40.

Mutations in Bruton's tyrosine kinase (Btk) gene, in mice, result in reduced numbers and responses of peripheral B cells. Surface Ig-mediated signaling is defective in Btk mutant B cells as they do not proliferate upon slg cross-linking and lack thymus-independent (TI) type II responses. Signals through sIg and CD40 play a critical role in B cell maturation. To investigate the consequences of the lack of both Btk and CD40 on B cell development and function, mice were generated that were homozygous for targeted mutations in the Btk and the CD40 genes (BtkMCD40M). The CD40 mutation (CD40M) had a synergistic effect on the BtkM defects. In BtkMCD40M mice the number of B cells was reduced 3- to 4-fold compared to BtkM mice and mature B cells (IgMlow/IgDhigh) were virtually absent; serum levels of all Ig isotypes were diminished; and antibody responses to TI-I TI-II and thymus-dependent antigens were impaired. Furthermore, although wild-type BtkM and CD40M mice produced germinal centers in response to TI-I antigen, the BtkMCD40M mice did not. Maturational and functional B cell defects in BtkMCD40M mice may result from a combination of intrinsic B cell defects, lack of CD40L-dependent T cell help and microenvironmental defects. These data suggest that signals through Btk and CD40 are necessary for the production and maintenance of the mature B cell.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1996

Cell specific expression of human Bruton's agammaglobulinemia tyrosine kinase gene (Btk) is regulated by Sp1- and Spi-1/PU.1-family members.

Bruton's agammaglobulinemia tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase involved in the human disease X-linked agammaglobulinemia (XLA). The gene is expressed in all hematopoietic cells with the exception of T-cells and plasma cells. For this expression pattern the first 280 bp upstream of the major transcriptional start site seems to be sufficient. In vitro footprinting analysis within this part of the promoter revealed two Sp1 binding sites as well as a PU-box. The transcription factor Spi-1/PU.1 as well as the closely related factor Spi-B bound to the PU-box in B-cells. In the erythroleukemia cell line K562, due to the absence of Spi-B, only PU.1 bound to the Btk promoter. Mutation of either site reduced the expression in transient transfection experiments. However, mutation of the PU box had no effect in the T-cell line Jurkat, where none of the Spi-1 family members is expressed. In addition Spi-B as well as PU.1 were able to transactivate Btk expression. In fetal liver of PU.1-/- mice, which lack lymphoid and myeloid cells, expression of Btk was reduced two- to threefold but not abolished. Collectively this study shows that expression of the Btk gene is regulated by the combined action of Sp1- and PU.1-family members.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1995

A Drosophila hsp70 gene contains long, antiparallel, coupled open reading frames (LAC ORFs) conserved in homologous loci.

A clone isolated from a Drosophila auraria heat-shock cDNA library presents two long, antiparallel, coupled (LAC) open reading frames (ORFs). One strand ORF is 1,929 nucleotides long and exhibits great identity (87.5% at the nucleotide level and 94% at the amino acid level) with the hsp70 gene copies of D. melanogaster, while the second strand ORF, in antiparallel in-frame register arrangement, is 1,839 nucleotides long and exhibits 32% identity with a putative, recently identified, NAD(+)-dependent glutamate dehydrogenase (NAD(+)-GDH). The overlap of the two ORFs is 1,824 nucleotides long. Computational analysis shows that this LAC ORF arrangement is conserved in other hsp70 loci in a wide range of organisms, raising questions about possible evolutionary benefits of such a peculiar genomic organization.

Amino Acid Sequence↗
PubMed1995

Identification of Btk mutations in 20 unrelated patients with X-linked agammaglobulinaemia (XLA).

X-linked agammaglobulinaemia (XLA) is an inherited immunodeficiency resulting from mutations in the gene for a cytoplasmic protein tyrosine kinase (Btk). We have utilised reverse-transcription-based PCR in combination with the chemical cleavage and mismatch technique (CCM) to screen for Btk mutations in 42 unrelated patients having classical XLA or 'leaky' XLA-like phenotypes. A variety of mutations, including point mutations, large deletions and splicing defects were detected using this strategy. In total, 20 mutations were found in these patients. All the mutations were different with the exception of three unrelated patients who all showed the same Arg-->His amino acid substitution (R641H) at a highly-conserved residue in the kinase domain. We have also used structural modelling of the Btk kinase domain to predict how two different amino acid substitution mutations at highly-conserved residues are likely to affect the Btk kinase activity.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1995

The human I alpha 1 region contains a TGF-beta 1 responsive enhancer and a putative recombination hotspot.

It appears that the switch recombination machinery of a B lymphocyte targets preferentially unrearranged heavy chain genes that have been rendered transcriptionally active. Transcriptional activation of the 'germline' human C alpha 1 and C alpha 2 genes is triggered by TGF-beta 1 and is controlled by proximal positive and distal negative regulatory elements residing upstream of the alpha 1 and alpha 2 switch regions respectively. In this report we characterize the positive proximal regulatory elements and analyse their interaction with DNA binding proteins. Our data demonstrate that a 100 bp fragment that contains a cAMP responsive element (CRE)/activating transcription factor (ATF) motif, a putative Ets binding site and an element that is created by two previously described neighbouring direct repeats (DRE), can increase the basal level of transcription and confer TGF-beta 1 inducibility to a heterologous promoter in an orientation- and position-independent manner. Ubiquitously expressed DNA binding proteins interact specifically with the CRE/ATF, the Ets site and the DRE element. Additionally, nuclear proteins interact with sequences which are located downstream of this enhancer are not essential for transcription in the transient expression assays utilized; however, they contain motifs that have been previously implicated in regulating DNA recombination events. These motifs include a Chi motif and a Chi-like element previously found in the recombination hotspot region of the Bcl-2 proto-oncogene and close to chromosomal breakpoints in T-ALL lines. Our findings raise the possibility that the intervening region associated regulatory elements in addition to regulating the transcriptional activation of the Ig heavy chain genes could also facilitate the physical interaction of transcription and recombination controlling molecular mechanisms.

Base Sequence↗
PubMed1994

Genomic organization of mouse and human Bruton's agammaglobulinemia tyrosine kinase (Btk) loci.

Btk is a cytoplasmic protein tyrosine kinase (PTK) that has been directly implicated in the pathogenesis of X-linked agammaglobulinaemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. We have isolated phage and cosmid clones that allowed us to deduce the genomic structure of mouse and human Btk loci. The mouse and human genes are contained within genomic regions that span approximately 43.5 kb and 37.5 kb, respectively. Both loci contain 18 coding exons ranging between 55 and 560 bp in size with introns ranging in size from 164 bp to approximately 9 kb. The 5'-untranslated regions are encoded by single exons located approximately 9 kb upstream of the first coding exon. Exon 18 encodes for the last 23 carboxyl-terminal amino acids and the entire 3'-untranslated region. The location of intron/exon boundaries in the catalytic domains of the mouse and human Btk loci differs from that found in other described sub-families of intracellular PTKs, namely that of Src, Fes/Fer, Csk, and Abl/Arg. This observation is consistent with the classification of Btk together with the recently characterized kinases, Tec and Itk, into a separate sub-family of cytoplasmic PTKs. Putative transcription initiation sites in the mouse and human Btk loci have been determined by using the rapid amplification of cDNA ends assay. Similar to many other PTK specific genes, the putative Btk promoters lack obvious TATAA and CAAAT motifs. Putative initiator elements and potential binding sites for Ets (PEA-3), zeste, and PuF transcription factors are located within the 300 bp which are located upstream of the major transcription start site in both species. These sequences can mediate promoter activity when placed upstream of a promotorless chloramphenicol acetyl transferase reporter gene in an orientation-dependent manner. The present analysis will significantly facilitate the mutational analyses of patients with XLA and the further characterization of the function and regulation of the Btk molecule.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1994

Molecular analysis of IgA deficiency. Evidence for impaired switching to IgA.

The most common form of primary immunodeficiency is IgA deficiency (IgAD). However, the molecular basis of this disease remains elusive. Therefore, to address this issue we made a systematic analysis of the molecular events leading to IgA production. B lymphocytes that produce IgA have undergone somatic rearrangement that joins the switch (S) mu to S alpha region with deletion of the intervening sequences. Examination of the resulting S mu/S alpha junctions in unstimulated PBMC from IgAD patients by nested primer PCR revealed a significant decrease in the number of the S mu/S alpha fragments. To obtain the antisense primers to generate the S mu/S alpha fragments, we sequenced the human S alpha 1 and the downstream region extending to the C alpha 1 locus. Similar to previously reported switch sequences, we also found the S alpha 1 to be predominantly composed of pentameric repeats GAGCT and GGGCT. The decrease in the number of S mu/S alpha fragments is consistent with a profound decrease in the C alpha membrane mRNA expression in unstimulated PBMC, as well as in the C alpha mRNA levels and IgA production in PWM-stimulated PBMC. Sequence analysis of the switch junctions from IgA-producing cell lines, control donors, and an IgAD patient showed direct joining in 8 of 9 cases examined. TGF-beta 1, previously shown to be the switch factor for human and mouse IgA, was also examined. No difference in the TGF-beta 1 mRNA levels in unstimulated PBMC between the control subjects and the IgAD patients were detected. Our findings indicate that the failure to switch to IgA-producing B lymphocytes, or an impaired survival of such cells, may be an important molecular mechanism in IgAD.

Animals↗
PubMed1993

Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes.

Mice that bear the X-linked immunodeficiency (xid) mutation have a B lymphocyte-specific defect resulting in an inability to make antibody responses to polysaccharide antigens. A backcross of 1114 progeny revealed the colocalization of xid with Bruton's agammaglobulinemia tyrosine kinase (btk) gene, which is implicated in the human immune deficiency, X-linked agammaglobulinemia. Mice that carry xid have a missense mutation that alters a highly conserved arginine near the amino-terminus of the btk protein, Btk. Because this region of Btk lies outside any obvious kinase domain, the xid mutation may define another aspect of tyrosine kinase function.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1993

The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases.

X-linked agammaglobulinaemia (XLA) is a human immunodeficiency caused by failure of pre-B cells in the bone marrow to develop into circulating mature B cells. A novel gene has been isolated which maps to the XLA locus, is expressed in B cells, and shows mutations in families with the disorder. The gene is a member of the src family of proto-oncogenes which encode protein-tyrosine kinases. This is, to our knowledge, the first evidence that mutations in a src-related gene are involved in human genetic disease.

Agammaglobulinemia↗
PubMed1993

Isolation of cDNA clones mapping around DXS178: a search for human X-linked agammaglobulinaemia gene using yeast artificial chromosomes, cosmids and direct cDNA selection.

X-linked agammaglobulinaemia (XLA) was previously mapped using genetic linkage analysis to Xq22. No recombinants have been found so far between the XLA locus and DXS178 in over 30 informative meioses. Two overlapping yeast artificial chromosomes (YACs), which cover a region of approximately 1000 kb around DXS178 and form part of a larger YAC contig, were hybridised to an ordered cosmid library constructed from a human fibroblast cell line with karyotype 49,XXXXX. Positive cosmids were gridded in high density arrays onto nylon filters and rescreened with a series of YACs, YAC end probes and additional markers from the region to assign cosmids into intervals. A 640 kb-YAC hybridising to p212 (DXS178) was used for direct selection of PCR-amplified cDNA from two cDNA libraries. To identify the cosmids containing transcribed sequences the enriched cDNA from each library was radiolabelled and hybridised back to positive clones which mapped to the region of interest. These clones were then hybridised to the subcloned enriched cDNA sublibraries. Candidate cDNA clones were isolated, grouped within the region of interest and their position was confirmed by mapping back to the cosmids.

Agammaglobulinemia↗
PubMed1993

Human immunoglobulin germline alpha(IgA) transcripts are expressed in vivo and their level correlates to class switching.

Immunoglobulin isotype switching is preceded by transcription of exons located 5' of the immunoglobulin switch regions. These exons are referred to as 'I-exons' and transcription of these regions is believed to be an essential step in switch recombination. Such mRNA species lack a variable portion and are denoted 'germline' transcripts. We have previously identified I-regions in man and we have now investigated the in vivo expression of human germline transcripts. Germline alpha transcripts (containing an I alpha exon) were expressed in various lymphoid organs, including peripheral blood mononuclear cells. Decreased levels of germline transcripts were found in immunoglobulin deficiency diseases. These findings are compatible with an in vivo role of germline transcription in isotype switching and may contribute to the understanding of mechanisms underlying deficiency diseases.

Agammaglobulinemia↗