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Biomedical subjects

F Melchers

Publications and source records attributed to F Melchers.

At least 127 records · Page 7Linked to original sources
PubMed1990

The proteins encoded by the VpreB and lambda 5 pre-B cell-specific genes can associate with each other and with mu heavy chain.

The murine pre-B cell-specific genes VpreB and lambda 5, as well as the murine gene for mu heavy chain, were introduced into Ltk- fibroblast cells which normally do not express these genes. Stable transfectants carrying these genes produced the corresponding proteins of 15.5, 21.5, and 75 kD. They secreted the three proteins as a triple complex that could be immunoprecipitated by mu heavy chain-specific antibodies, consisting of one VpreB, one lambda 5, and one mu heavy chain. The mu heavy chain and lambda 5 were disulfide-bonded with each other, while the VpreB protein was noncovalently associated. These experiments proved that the VpreB, lambda 5 and mu H chain proteins can form a heavy/light chain-like heterocomplex.

Animals↗
PubMed1989

One synchronous wave of B cell development in mouse fetal liver changes at day 16 of gestation from dependence to independence of a stromal cell environment.

Precursor cells of the B lineage can be enriched from mouse fetal liver by FACS with the aid of the pre-B cell-specific mAb G-5-2. The cells are concomitantly enriched for cells expressing the pre-B cell-specific gene lambda 5, and for cells developing to LPS-reactive mature B cells. The enriched purified precursors are not influenced by rIL-2 through -7, alone or in combination, to develop to mitogen-reactive, sIg+ cells. Marginal proliferation of the precursors is observed in response to IL-3 plus -4, and IL-6 plus -7, and this does not change in the presence of stromal cells. Development to mitogen-reactive, sIg+ cells is dependent on interactions with embryonic stromal cells from fetal liver. Two mAbs raised against the stromal cells inhibit this development. Two phases of precursor cell development can be distinguished in fetal liver. Between days 13 and 15 of gestation, it is dependent on stromal cell interactions, thereafter, from days 16 to 19, it is independent. A sudden increase in the number of mitogen-reactive, sIg+ B lineage cells occurs within 24 h between days 16 and 17. All these results indicate that B cell development occurs in one wave with synchronous steps of changes from a mitogen-insensitive, sIg-, stromal cell dependent to a mitogen-reactive, sIg+, stromal cell-independent B lineage line.

Animals↗
PubMed1989

Monoclonal antibodies reactive with the mouse interleukin 5 receptor.

The rat mAbs R52.120 and R52.625 inhibit the action of IL-5 on both IL-5-sensitive cell lines and freshly isolated splenic B lymphocytes. Neither antibody inhibits the proliferative cell responses promoted by IL-2, IL-3, or IL-4. Purified R52.120+ lymphoid spleen cells contain 15-20-fold higher numbers of B lymphocytes responding to IL-5 in the form of maturation into antibody-producing cells. By immunofluorescence staining and flow fluorocytometry, the R52.120 and R52.625 antibodies bound to all 12 IL-5-sensitive cell lines tested. Both antibodies react with 2-4% cells in the spleen, 5% lymphoid cells, and 10-15% myeloid cells in the bone marrow, and 10-14% in the peritoneum of C57BL/6, DBA/2, and BALB/c adult mice. No positive cells for either antibody were detected in the thymus and lymph nodes of these mice. Both R52.120 and R52.625 antibodies specifically inhibit the binding of radiolabeled IL-5 to its receptor. Finally, R52.120 and R52.625 antibodies precipitate from 35S-methionine-labeled IL-5-R+ cell lysates three proteins with Mr 46,000, 130,000, and 140,000. Taken together from these results, we conclude that the R52.120 and R52.625 mAbs recognize epitopes on the IL-5-R complex very close or identical to the IL-5 binding sites.

Animals↗
PubMed1989

Restriction fragment analysis of V preB and lambda 5 within the genus Mus.

DNA from a panel of inbred strains of mice and colony bred mice, isolated from different geographical locations, was hybridized to mouse V preB and lambda 5 probes under stringent conditions, indicating sequence similarities greater than 80%. The probe for lambda 5 detects one gene and the probe for V preB detects two genes (V preB1 and V preB2) in the inbred strains of mice examined under the stringency used. No restriction endonuclease fragment length polymorphisms (RFLP) were detected with the V preB and lambda 5 DNA probes among the inbred strains of mice using Bam HI and Hind III. Very few RFLP were detected among Mus musculus subspecies, and the intensity of the hybridization did not differ significantly with either DNA probe. The number of RFLP increased slightly when different species and subgenera were examined, and the intensity of the hybridization signal began to decrease in samples from the different subgenera, suggesting a slight decrease in sequence similarity for both V preB genes with increased time of divergence. Fewer RFLP were detected with the lambda 5 DNA probe. DNA from 11 different Mus species representing 4 subgenera, genetically isolated from laboratory mice for approximately 1-12 million years, continued to hybridize under high stringency conditions using both DNA probes. A comigrating lambda 5 and V preB restriction endonuclease fragment was detected in most of the samples examined, suggesting the close physical linkage of V preB1 and lambda 5 is maintained within the genus Mus. These results suggest that V preB1, V preB2 and lambda 5 have been present for over 12 million years.

Animals↗
PubMed1989

Cellular stages and molecular steps of murine B-cell development.

Development of B cells in fetal liver occurs in one synchronous wave and involves probably no more than four critical divisions. This leads us to suggest that the main pool of proliferating progenitors that replenish the peripheral B-cell pool are progenitors before Ig gene rearrangement, that the four Ig gene rearrangements (DH to JH, VH to DHJH, VK to JK, and V lambda to J lambda) might occur in four critical divisions, and that a stromal-cell-dependent phase of pre-B development in which all rearrangements are made is succeeded by a stromal-cell-independent phase of sIG+ pre-B-cell maturation to mature mitogen-reactive B cells. We speculate on the molecular nature of the tightly controlled steps of Ig rearrangements during pre-B-cell development that might involve the pre-B-cell specific genes Vpre-B and lambda 5 and the B-lineage-specific gene mb-1 in interactions with stromal cells.

Animals↗
PubMed1988

Recombinant interleukin 2 or 5, but not 3 or 4, induces maturation of resting mouse B lymphocytes and propagates proliferation of activated B cell blasts.

Plasmacytoma transformants of the X63-Ag8-653 cell line carrying an expression vector with either IL-2, -3, -4, or -5 cDNA were established that secrete the corresponding ILs at high rates. The four mouse ILs (mILs) were then tested as single ILs and in combinations for their effects on the maturation of resting and proliferation of activated normal mouse splenic B cells. mIL-3 and mIL-4 were inactive in all assays. mIL-2, as well as mIL-5, synergized with Ig-specific antibodies and B cell growth factor alpha (BCGF-alpha) to stimulate successive rounds of B cell division with LPS-activated B cells. This activity as BCGF-beta was effective at concentrations similar to those at which mIL-2 induced proliferation of the CTL-L T cell line, indicating a high-affinity interaction of both mIL-2 and mIL-5 with their corresponding receptors on activated B cells. mIL-5 and maybe IL-2 also induced maturation of resting B cells to Ig-secreting cells without proliferation. This B cell maturation factor (BMF) activity of mIL-5 was as effective as its BCGF-beta activity, while the BMF activity of mIL-2 was at least 10(2)-fold less effective. BMF activity of mIL-2, but not mIL-5, was blocked by anti-Il-2-R antibodies, indicating that mIL-2 and mIL-5 use separate receptors for B cell signaling. mIL-2, as well as mIL-5, furthermore, acted as filler activities when proliferation in the presence of Ig-specific antibodies and BCGF-alpha was measured with as little as 500 B cells. In the case of mIL-5, this was also true for maturation of that few cells. Limiting dilution analyses showed that approximately 1-2% of the resting B cells matured without division, while 30-100-fold fewer cells (0.03-0.06%) proliferated and matured in response to IL-5. A single IL, therefore, is capable of inducing maturation and of stimulating mitotic cell cycle progression of normal B cells.

Animals↗
PubMed1988

Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors.

Mouse cell lines of different lineages have been established which constitutively secrete large quantities of recombinant mouse interleukins (mIL2, mIL3, mIL4 or mIL5). An existing bovine papilloma virus-based expression vector, pBV-1MTHA, was modified to allow transformed X63Ag8-653 myeloma cells, NIH 3T3 fibroblasts and C127 mammary tumor cells to stably carry multiple copies of the vector, to express the inserted cDNA encoding a single interleukin constitutively, and to secrete the interleukin in high quantities. Cell lines transformed with mIL2 cDNA stably carried 30-100 copies of the plasmid per cell and constitutively secreted biologically active mIL2 in quantities similar to those produced by murine EL4 thymoma cells or rat spleen cells stimulated with mitogens. Deletion of the 3' untranslated region containing AT-rich sequences from the mIL2 cDNA resulted in a 100-fold increase in the constitutive production and secretion of mIL2 by the transformants. Addition of a heavy metal further increased the production 2 to 6-fold. Cells transformed with 3'-deleted mIL3 cDNA constitutively secreted 300-1000 times higher activities of mIL3 than the myelomonocytic leukemia line WEHI3. mIL4 produced by the similar transformants induced [3H]thymidine uptake of a T cell line, a mast cell line and B leukemia cells, and enhanced the production of IgG1 by B cells. IL4 titers were 150 times higher than those produced by the concanavalin A-stimulated T cell line 2.19. mIL5 was secreted by similar transformants at 10-fold higher titers than those produced by concanavalin A-stimulated 2.19 T cells, as judged by the proliferation and maturation of B cell leukemia BCL1. The expression vectors should be useful in establishing eukaryotic cell lines producing proteins from full length cDNA clones at higher rates. The established cell lines secreting IL2, 3, 4 or 5 at high rate should be useful sources for these interleukins in the investigation of their function in the immune system.

Animals↗
PubMed1988

Structure and pre-B lymphocyte restricted expression of the VpreB in humans and conservation of its structure in other mammalian species.

DNA from several mammals, including humans, was found to contain one or more restriction enzyme digested DNA fragments which hybridized to the mouse VpreB gene under stringencies demonstrating at least 70% nucleotide sequence homologies, indicating that the VpreB locus may be widespread and highly conserved among mammals. A human VpreB genomic clone was isolated and sequenced. Two exons and the intervening intron are spaced almost identically as in the mouse VpreB1 gene, and show 76% sequence homology to the mouse gene. As in the mouse VpreB1 gene, the 5' end of the human VpreB gene contains characteristic features of Ig domains, while the 3' end is Ig non-related. This 3' Ig non-related structure of the VpreB gene(s) may, therefore, have existed before the speciation of humans and mice over 65 million years ago. Sequences encoding the entire putative second framework region and a stretch in the third framework region are identical in human and mouse VpreB. the human VpreB gene appears to be selectively expressed in human pre-B cell lines as an 0.85 kb poly(A)+ RNA. Its expression promises to be a useful marker for the detection of normal and malignant human pre-B lymphocytes.

Amino Acid Sequence↗
PubMed1988

B lymphocyte lineage-restricted expression of mb-1, a gene with CD3-like structural properties.

A gene, called m-mb-1, was isolated from a murine pre-B-minus T lymphocyte subtracted library. It was found expressed as mRNA at low to medium abundance in early progenitors of the B lineage, in pre-B and mature B lineage cell lines, in normal resting B lymphocytes and in polyclonally activated B cell blasts. The gene was not expressed in plasmacytomas, in cell lines of the monocyte/macrophage, the T lymphocyte or the fibroblast lineages, nor in thymus, liver, heart, kidney, lung or brain. The nucleotide sequence of the m-mb-1 gene encodes a putative membrane glycoprotein with 220 amino acids, which includes a leader sequence, a putative extracellular domain with two potential N-glycosylation sites, a transmembrane portion and a putative intracellular domain. The partial sequence of a human homologue, h-mb-1, shows nearly 90% homology in nucleotide as well as amino acid sequences to the murine form of a stretch of the putative intracytoplasmic domain. Antibodies raised against a fusion protein of m-mb-1 with protein A, affinity purified for their m-mb-1 specificity, stained pre-B and mature B cell lines on their surface, but did not stain T cell lines and fibroblasts. Antibodies raised against a stretch of 20 amino acids of the putative intracellular domain with 90% homology between the mouse and human protein did not stain the surface of any cell lines tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

The human Vpre B gene is located on chromosome 22 near a cluster of V lambda gene segments.

The chromosomal location of the human VpreB gene was determined by Southern blotting analysis of restriction enzyme-digested DNAs from a panel of 17 mouse-human somatic cell hybrids. The pattern of hybridization of a Vpre B-specific probe in conjunction with earlier analysis of several marker genes allowed the following conclusions: 1) Vpre B is on human chromosome 22 within band 22q11.2 distal to the bcr-like gene, bcr-2 and proximal to the bcr-like gene, bcr-4. 2) Vpre B has been localized relative to several constitutional and tumor-specific breakpoints within 22q11.2, segregates in hybrids retaining 22q- chromosomes with some but not with all members of the V lambda 1 subgroup of the V lambda genes, and is amplified with these genes in K562 cells. 3) The order of the loci on chromosome 22 is centromere----bcr-2, Vpre B, V lambda 1----bcr-4----C lambda----bcr-1----bcr-3----sis.

B-Lymphocytes↗
PubMed1988

Autoreactive B-cell repertoire in mice with chronic graft versus host disease.

A quantitative analysis of the frequencies of autoantibody producing B-cells has been undertaken by producing and analyzing random hybridoma collections generated in fusions with activated B-cells. Activated B-cells were derived from mice injected with LPS and SRBC and normal mice. They were compared to those derived from mice undergoing chronic GVHD. The frequencies of successful fusion events correlate well with the number of activated B-cells used in the fusions, so that it is reasonable to conclude that the hybridoma collections reflect the activated B-cell repertoires in the different animals. The frequencies of hybridomas producing autoantibodies as well as their specificities for self-antigens, were not significantly different between the different collections of hybridomas. Moreover, no difference in VH gene family expression was found in the different collections of autoantibody producing hybridomas. So, the activated autoreactive B-cell repertoires in GVHF1 mice and in normal mice is similar. In contrast to the normal activated autoreactive B-cell repertoires, which make predominantly IgM antibodies, the GVH-activated autoreactive B-cells make predominantly antibodies of the IgG class. Therefore, we conclude that T-cell mediated graft versus host activation does not generally lead to selective expansion of autoreactive B-cells, but appears to play a crucial role in the switch from IgM to IgG production.

Animals↗
PubMed1988

Monoclonal autoantibodies specific for kidney proximal tubular brush border from mice with experimentally induced chronic graft-versus-host disease.

Nine hybridomas producing monoclonal autoantibodies specific for kidney proximal tubular brush border were found in 600 hybridomas derived from (C57BL/6J X DBA/2)F1 mice injected with DBA/2 T cells. None of the 1100 hybridomas derived from nonautoimmune (C57BL/6J X DBA/2)F1 mice produced antibodies with a similar specificity. Four of these nine monoclonal antibodies were characterized further. They did not bind to cryosections of liver, lung, stomach, or intestine. Three bound to kidney proximal tubular brush border of mouse, cattle, sheep, pigs, rabbits, rats, and humans, whereas the fourth was specific only for murine brush border. All four precipitated from mouse kidney microvilli, a protein with an apparent molecular weight of 160,000 under reducing as well as nonreducing conditions. Removal of asparagine-linked carbohydrate with EndoF reduced the molecular weight of the 160,000 protein by about 20,000. One of the three multi-species-specific antibodies bound to pig kidney aminopeptidase, a glycosylated enzyme located on the microvilli of kidney proximal tubular brush border. Three antibodies have a heavy-chain variable region encoded by VH genes of the J558 family, whereas the heavy-chain variable region of the fourth is encoded by a VH gene of the 7183 family. Attempts to passively transfer immune complex glomerulonephritis to normal mice by injection of the purified monoclonal antibodies or by growth of the corresponding hybridoma cells in mice have so far been unsuccessful. However, antibodies recognizing the 160,000 molecule are present in the serum of mice with chronic graft-versus-host disease and can be eluted from kidneys with immune complex glomerulonephritis.

Aminopeptidases↗
PubMed1988

The role of C3 and its fragments in the control of S phase entry of activated mouse B lymphocytes via the complement receptor type 2.

Complement component C3 and receptors for its activated fragments have been recognized as regulators of humoral immune responses. Cross-linked C3b and C3d will substitute for alpha B cell growth factors (alpha BCGF) produced by macrophages by stimulating activated murine B lymphocytes to enter S phase. In contrast, soluble C3d and synthetic oligopeptides--containing the amino acid sequence of the binding site of C3d to its receptor, the complement receptor type 2--inhibit the entry into S phase of alpha BCGF-stimulated B cells. There is evidence supporting the hypothesis that a portion of the C3 gene at the 3' end, encoding parts of the alpha chain, encodes an alternate C3 product with alpha BCGF activity.

Amino Acid Sequence↗
PubMed1987

The autoantigen-binding B cell repertoires of normal and of chronically graft-versus-host-diseased mice.

A quantitative analysis of the frequencies of autoantibody-producing B cells in GVHD and in normal mice has been undertaken by generating collections of hybridomas of activated B cells. These hybridomas secreted sufficient quantities of Ig to allow binding analyses on a panel of autoantigens. B cells have been activated in a variety of ways. In vivo they were activated by injection of alloreactive T cells of one parent, leading to GVHD by a foreign antigen, sheep erythrocytes, in a secondary response, or by the polyclonal activator LPS. B cells from an experimentally unstimulated animal were used for an analysis of the normal background. In vitro B cells were activated by alloreactive T cells or by LPS. The frequencies of hybridomas and, therefore, of activated B cells producing autoantibodies to DNA or to kidney were not significantly different in mice activated by a graft-vs.-host T cell response as compared with B cell populations activated by any of the other procedures. They were found to compose 7.1-17.1% of the total repertoire of activated B cells. Moreover, the frequencies of autoantibody-producing activated B cells does not change with time after induction of the graft-vs.-host reaction. The pattern and frequencies of autoantigen-binding specificities to cytoskeleton, smooth muscle, nuclei, mitochondria, and DNA were not found to be different in any of the groups of hybridomas. The single notable exception, found in GVHD mice, were hybridomas producing autoantibodies to kidney proximal tubular brush border. These results allow the conclusion that autoantigen-binding B cells exist in an activated state in GVHD mice, as well as in mice activated by a foreign antigen or by a polyclonal activator, in B cell populations activated in vitro either by alloreactive T cells or by a polyclonal activator, and even in the background of experimentally unstimulated animals. T cell-mediated graft-vs.-host activation, in large part, does not lead to a selective expansion of autoantigen-binding B cells. The main difference between the graft-vs.-host-activated B cell repertoire and all others is that approximately 90% of teh autoantibodies were of the IgG class, whereas al autoantibodies found in the other groups were IgM.

Animals↗
PubMed1987

A second gene, VpreB in the lambda 5 locus of the mouse, which appears to be selectively expressed in pre-B lymphocytes.

The murine gene lambda 5 is selectively expressed in pre-B lymphocytes. Of the three exons encoding lambda 5, exons II and III show strong homologies to immunoglobulin lambda light (L) chain gene segments, i.e. to J lambda intron and exon, and C lambda exon sequences respectively. We have now found, 4.6 kb upstream of lambda 5, another gene composed of two exons which is selectively expressed in pre-B cell lines as a 0.85 kb mRNA potentially coding for a protein of 142 amino acids including a 19 amino acid-long signal peptide. The 5' sequences of this gene show homologies to sequences encoding the variable regions of kappa and lambda L chains and of heavy (H) chains. The deduced amino acid sequence contains the consensus cysteine residues as well as other consensus amino acids at positions which characterize immunoglobulin (Ig) domains. We call the second gene VpreB. The 3' end of VpreB encoding the 26 carboxyl terminal amino acids shows no homology to any known nucleotide sequence. The putative protein encoded by VpreB is a potential candidate for association with the putative protein encoded by lambda 5, and thereby a candidate for association with H chains in pre-B cells. Southern blot analysis of DNA from liver (germ line) and 70Z/3 pre-B cell lines reveals two genes which hybridize to the VpreB gene. We call VpreB1 the gene which is found 5' of lambda 5. The other gene, called VpreB2, which has not yet been located within the genome, shows 97% nucleotide sequence homology to VpreB1 in an area of 1 kb which covers the coding region of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Organization of the murine Ig-related lambda 5 gene transcribed selectively in pre-B lymphocytes.

lambda 5 is an immunoglobulin lambda light chain-related gene which is selectively transcribed in murine pre-B lymphocytes to yield a 1.2 kb poly(A)+ mRNA. Comparison of the nucleotide sequence of a 1 kb cDNA clone with the sequence of a genomic clone isolated from 70Z/3 murine pre-B lymphoma cells shows lambda 5 is composed of three exons spanning a 3.75 kb DNA segment. Conserved splice signal sequences at all exon/intron boundaries and the presence of a long open reading frame indicate that a functional mRNA molecule can be made. Exon I contains a cap-site and a potential ATG start codon as well as sequences encoding a signal peptide. This gene could encode a lambda 5 protein of 209 amino acids which has, however, not yet been identified. The 3' portion of exon II and all of exon III shows strong sequence homologies to J lambda L and C lambda L exons. Homology to the lambda L chain genes is lost in the 5' portion of exon II and throughout exon I. In exon I short homologies to leader sequences and to VH framework 1 sequences are seen.

Amino Acid Sequence↗