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A Radbruch

Publications and source records attributed to A Radbruch.

15 recordsLinked to original sources
PubMed1992

The murine IgG1/IgE class switch program.

Immunoglobulin class switching is controlled by cytokines. Thus, interleukin-4 (IL-4) directs class switching to both IgG1 and IgE. Consistent with this are the results reported here on restriction endonuclease analysis of active and inactive alleles of the IgH locus in IgE-producing cells. In cells that were stimulated in vitro by lipopolysaccharide and IL-4 the silent alleles preferentially switched to gamma 1, whereas in cells that were stimulated by antigen in vivo both active and inactive alleles switched to epsilon. Thirty percent of the recombined switch regions (S mu/S epsilon) contain S gamma 1 sequences, which we interpret as footprints of a previous switch to gamma 1. Since this percentage is a minimum estimate, between 30% and 100% of switching to epsilon must occur sequentially via gamma 1.

Animals↗
PubMed1992

Demethylation of the constant region genes of immunoglobulins reflects the differentiation state of the B cell.

Previous results showed a developmentally regulated, strong linkage between demethylation and transcriptional activity for the light chain kappa locus in the mouse (Kelley et al., Molec. cell. Biol. 8, 930-937, 1988). These results indicate the existence of a stage of development of the B cell in which permanent expression (which may be enhancer independent) of a gene is associated with its demethylation. According to this result, demethylation could mirror terminal differentiation of a cell. We tested this hypothesis by analyzing the methylation status of immunoglobulin (Ig) genes in normal B cells before and after their activation with lipopolysaccharide (LPS) to induce IgM secretion and an immunoglobulin class switch. This pattern of methylation has been compared with that of Ig genes in nonlymphoid tissues and in transformed cell lines. In general, transformed cells are terminally differentiated cells. Our results show, that in normal splenic B cells only regions proximal to the heavy chain enhancer are demethylated. The coding regions of the c mu, c delta and the c gamma 1 genes remain methylated regardless of transcription. Demethylation of the coding regions is only detectable in transformed cell lines. Hence demethylation of immunoglobulin genes may reflect a stage of terminal differentiation in which the transcription pattern of the cell is fixed. Methylation of the genes before terminal differentiation may be necessary to allow controlled expression of genes on the transcriptional level, such as by splicing and differential termination.

Animals↗
PubMed1992

DNA binding sites 5' of the IgG1 switch region comprising IL4 inducibility and B cell specificity.

Immunoglobulin class switch recombination is directed to the same switch region on both chromosomes of a B cell by an as yet unknown mechanism. The cytokine interleukin 4 (IL4) targets recombination in activated B lymphocytes to the gamma 1 switch region (s gamma 1). Here we report two DNA-binding-proteins which bind to a sequence 5' of s gamma 1. One protein is B cell specific, while binding of the other one is induced by IL4. These two proteins bind to a region 700 bp upstream of the putative promoter region of the gamma 1 germline transcripts and may be involved in the process of recombination and/or transcription.

Animals↗
PubMed1992

Distinct antigen presenting cell-derived signals induce TH cell proliferation and expression of effector cytokines.

In addition to the stimulus delivered by the specific interaction of the T cell receptor (TCR) and the antigen - MHC class II complex, activation of resting helper T lymphocytes (TH) requires several poorly defined accessory cell-derived co-stimulatory signals. Here we provide evidence that proliferation and expression of effector cytokine genes by TH cells are induced by distinct co-stimulatory signals. Normal murine splenic TH cells were activated by Staphylococcus aureus enterotoxin B (SEB) superantigen and various antigen presenting cells (APCs) to proliferate, differentiate into TH cells blasts, and secrete cytokines. Blast transformation and proliferation of TH cells is achieved with macrophages and other splenic APCs, like B cells. Expression of the cytokines interferon gamma (IFN gamma), IL-5, and IL-2 by TH cells, however, is to various degrees dependent on the presentation of SEB by macrophages. The requirement for macrophages is particularly striking for the expression of IFN gamma. Thus macrophages provide distinct co-stimulatory signals for cytokine secretion and proliferation. The results suggest that B cells induce clonal expansion of TH cells whereas macrophages additionally promote terminal differentiation of activated TH cells into TH effector cells.

Animals↗
PubMed1991

DNA methylation profiles in the human genes for tumor necrosis factors alpha and beta in subpopulations of leukocytes and in leukemias.

The genomic sequencing technique has been applied to assess the state of methylation in the DNA from human leukocyte subpopulations from healthy individuals and in the DNA from several individuals with myeloid or lymphatic leukemias or non-Hodgkin lymphomas. Leukocyte populations were purified by the high-gradient magnetic cell sorting technique. In the human tumor necrosis factor alpha (TNF-alpha) gene segment between nucleotides 300 and 1150, the specific methylation profile in the DNA from human granulocytes and monocytes is maintained in three cases of myeloid leukemia. In one such case, all 5-methyl-2'-deoxycytidine residues have been replaced by cytidine. In a chronic lymphatic T-cell leukemia, all 5-methyl-2'-deoxycytidine residues have been substituted by cytidine. In normal B lymphocytes, in two cases of chronic lymphatic B-cell leukemias and two cases of non-Hodgkin lymphomas, all 5'-CG-3' sequences in this gene segment are devoid of methylation. In the TNF-beta gene, DNA methylation is decreased in several examples of acute or chronic myeloid leukemias in comparison to normal human granulocytes or monocytes, whose DNA is almost completely methylated between nucleotides 700 and 900. In human T and B lymphocytes, the main producers of TNF-beta, in three instances of chronic lymphatic leukemias and two cases of non-Hodgkin lymphomas, all 5'-CG-3' sequences are unmethylated in this region. The DNA from the human HeLa cell line is highly methylated at all 5'-CG-3' sequences in the TNF-alpha and -beta genes. The TNF-alpha gene is transcribed in the cells of one case of acute myeloid leukemia in which the analyzed region of the TNF-alpha gene is completely unmethylated. The TNF-beta gene is not transcribed in any of the malignant cells tested.

Adult↗
PubMed1991

Isotype commitment of human B cells that are transformed by Epstein-Barr virus.

Epstein-Barr virus (EBV) can transform a subpopulation of preactivated B cells thus promoting their growth and differentiation into plasma cells. In EBV-transformed clones of IgM-producing cells, the heavy chain constant region (CH) genes on the productive allele are fixed in germ-line configuration, whereas in isotype-switched clones the CH genes proximal to the expressed CH gene are deleted. In order to define more precisely the EBV-susceptible B cells, we sorted subpopulations of B cells on the basis of their cell surface Ig (sIg) isotypes, infected them with EBV, and determined which isotypes they could produce following transformation. Most precursors of IgM-producing plasma cells expressed both IgM and IgD on their surface, while a minority expressed IgM alone. Some B cell precursors of IgG- and IgA-producing cells also expressed sIgM, but surprisingly none expressed IgD. Those precursors of IgG and IgA producers, which bore sIgM, expressed it in relatively low levels, whereas B cells expressing high levels of sIgM were incapable of generating IgG and IgA producers. All of the precursors of IgG and IgA plasma cells expressed these isotypes on their cell surface. Interestingly, precursor B cells capable of producing the IgG3 and IgA2 subclasses could be respectively enriched on the basis of the presence or absence of cell sIgM. These results demonstrate the isotype precommitment of EBV-transformable B cells. They further suggest that residual IgM is transiently expressed on the surface of the IgG- and IgA-committed B cell precursors, whereas sIgD expression is extinguished earlier in the process of isotype switching via CH gene deletion.

B-Lymphocyte Subsets↗
PubMed1990

High gradient magnetic cell separation with MACS.

A flexible, fast and simple magnetic cell sorting system for separation of large numbers of cells according to specific cell surface markers was developed and tested. Cells stained sequentially with biotinylated antibodies, fluorochrome-conjugated avidin, and superparamagnetic biotinylated-microparticles (about 100 nm diameter) are separated on high gradient magnetic (HGM) columns. Unlabelled cells pass through the column, while labelled cells are retained. The retained cells can be easily eluted. More than 10(9) cells can be processed in about 15 min. Enrichment rates of more than 100-fold and depletion rates of several 1,000-fold can be achieved. The simultaneous tagging of cells with fluorochromes and very small, invisible magnetic beads makes this system an ideal complement to flow cytometry. Light scatter and fluorescent parameters of the cells are not changed by the bound particles. Magnetically separated cells can be analysed by fluorescence microscopy or flow cytometry or sorted by fluorescence-activated cell sorting without further treatment. Magnetic tagging and separation does not affect cell viability and proliferation.

Animals↗
PubMed1990

Protective methylation of immunoglobulin and T cell receptor (TcR) gene loci prior to induction of class switch and TcR recombination.

Methylation of the S gamma 1 switch region and C gamma 1 constant region gene from the immunoglobulin heavy chain locus and of the J beta 2 and C beta regions from the T cell receptor beta chain (TcR beta) locus is compared here in murine germ-line cells, nonlymphoid cells and lymphocytes. In germ-line cells and in lymphocytes prior to recombination all four regions show strong methylation, i.e. most Msp I sites are methylated. After activation of lymphocytes, demethylation is observed for those regions which are activated for recombination, at specific sites 5' of S gamma 1 in B cells activated with bacterial lipopolysaccharide and interleukin 4, and for J beta 2 in thymocytes. In nonlymphoid cells, where these regions cannot be used for recombination, considerable demethylation is observed for all four regions analyzed as compared to lymphocytes. The result implies an important role for methylation of recombinatorial regions. Methylation may be involved in protecting them from uninduced recombination, thus allowing regulated expression of distinct genes in lymphocyte ontogeny.

Animals↗
PubMed1990

Molecular components of the B cell antigen receptor complex of class IgD differ partly from those of IgM.

Two classes of immunoglobulin, IgM and IgD, are present as antigen receptors on the surface of mature B lymphocytes. We show here that IgD molecules are noncovalently associated in the B cell membrane with a heterodimer consisting of two proteins of 35 kd (IgD-alpha) and 39 kd (Ig-beta), respectively. The two novel proteins are not found in the IgD-expressing myeloma J558L delta m, which fails to bring IgD antigen receptor onto the cell surface. In a surface IgD positive variant line of this myeloma, however, membrane-bound IgD molecules are associated with the heterodimer, suggesting that the formation of an antigen receptor complex is required for surface IgD expression. We further demonstrate that the IgD-associated heterodimer differs partly from that of the IgM antigen receptor and that its binding to the heavy chain only requires the presence of the last constant domain and the transmembrane part of the delta m chain.

Animals↗
PubMed1989

CD20 positive human B lymphocytes separated with the magnetic cell sorter (MACS) can be induced to proliferation and antibody secretion in vitro.

A method is described for the efficient purification of human B lymphocytes from peripheral blood by magnetic separation. Biotinylated, superparamagnetic particles were coupled to target cells by fluorescein isothiocyanate conjugated avidin and biotinylated monoclonal antibodies directed against cell surface antigens. This combination permitted flow cytometric control of the magnetic separation. Ficoll-Paque-separated peripheral blood mononuclear cells were first eliminated from monocytes by leucine-methyl ester treatment. B cells were enriched to 97% after magnetic depletion of CD3-positive T cells and magnetic enrichment of CD20-positive B cells. The separated B cells could be induced to proliferation and antibody production by various in vitro stimuli.

Antibody-Producing Cells↗
PubMed1979

A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines.

We have isolated a subclone of the mouse myeloma cell line P3-X63-Ag8 that does not express immunoglobulin heavy or light chains. This clone X63-Ag8.653 can be used for efficient fusion with antibody-forming cells to obtain hybrid cell lines producing pure monoclonal antibodies. Screening of hybrid cell lines for specificity and immunoglobulin classes was done with a modified enzyme-linked immunosorbent assay.

Animals↗
PubMed1978

Isolation of myeloma variants with predefined variant surface immunoglobulin by cell sorting.

We describe a procedure for the isolation of somatic cell variants in which gene products are expressed on the cell surface that are not expressed in the wild type. Cloned cells of the myeloma line MPC 11, which expresses an IgG2b protein, were incubated with an antiserum specific for IgGI and IgG2a. Cells reacting with this antiserum were stained with a fluorescent anti-antiserum and enriched in three cycles of sorting in the fluorescence-activated cell sorter and subsequent growth in vitro. From the enriched population two variants were isolated by cloning in soft agar. One of them expressed a variant immunoglobulin that types serologically as an IgG2a but whose variable portion was idiotypically related to that of the MPC 11 wild-type protein.

B-Lymphocytes↗
PubMed1977

Robertsonian translocations in Mus musculus from Sicily.

The karyotypes of 6 mice from different places in Sicily have been determined. 3 of them had abnormal chromosome numbers of 2n = 26, 2n = 27 and 2n = 29, caused by Robersonian translocations of one acrocentric chromosome to another resulting in metacentric chromosomes. The newly described metacentric chromosomes are Rb(4.3)1Sic, Rb(15.2)2SIC, Rb(12.6)3SIC, Rb(13.5)4Sic, Rb(14.10)5Sic, Rb(17.8)6Sic and Rb(16.9)7Sic.

Animals↗