Search PubMed⌕ Search

Biomedical subjects

J Schwaber

Publications and source records attributed to J Schwaber.

At least 19 recordsLinked to original sources
PubMed2000

Fas-mediated apoptosis eliminates B cells that acquire self-reactivity during the germinal center response to NP.

C57Bl/6 mice with the lpr mutation of Fas (CD95) were tested for deviation from the genetically restricted antibody response to the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). lambda1+ germinal centers (GC) with the canonical v186.2 V(H) gene element develop in lpr/lpr mice with the same time course as in wild-type (+/+) mice. In contrast to +/+ mice, however, lambda1+ GC persist in the spleens of lpr/lpr mice 25 days after immunization. Virtually all of the lambda1+ GC are reactive with NP 10 days after immunization. Sixteen days after immunization, however, many of the lambda1+ GC are not reactive with NP, and few of the lambda1+ GC are reactive with NP 25 days after immunization. The V(H) gene elements of three lambda1+NP- GC 25 days after immunization are derived by somatic mutation of v186.2, but have lost reactivity with NP. The mutated VDJs from these GC react with cells in spleen sections from +/+ and lpr/lpr mice, indicating that they represented secondary antibody responses induced by self antigens that are available as presented antigen. These data indicate that Fas-mediated apoptosis serves to eliminate a (limited) population of B cells that acquire reactivity to "self antigens" by somatic mutation of VDJs in the GC.

Animals↗
PubMed2000

BTK mutations in patients with X-linked agammaglobulinemia: lack of correlation between presence of peripheral B lymphocytes and specific mutations.

X-linked agammaglobulinemia (XLA) is a human antibody deficiency that results from mutation of the tyrosine kinase btk. We tested the hypothesis that XLA patients who varied from the classic phenotype of XLA by presence of normal or near normal number of peripheral B lymphocytes would have a set of mutations of BTK that is different from the mutations found in patients without peripheral B lymphocytes. The mutations of BTK we found in two patients with normal numbers of peripheral B lymphocytes have been previously identified in patients without peripheral B lymphocytes. A third patient, without peripheral B cells, was found to express normal levels of wild type btk. Exmination of the mutations of the BTK gene in patients in the BTKbase who were identified as having peripheral B lymphocytes found that these same mutations, or mutations of the same protein domains, were also present in patients identified as lacking peripheral B lymphocytes. Analysis of mutations in BTK has previously led to the conclusion that severity of disease in XLA cannot be predicted from the specific mutation of BTK. The results of this study suggest that whether an XLA patient will develop peripheral B lymphocytes cannot be predicted from the specific mutation of BTK.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2000

Engineering a living cell to desired metabolite concentrations and fluxes: pathways with multifunctional enzymes.

With molecular genetics enabling modulation of the concentrations of cellular enzymes, metabolic engineering becomes limited by the question of which modulations of the enzyme concentrations are required to bring about a desired pattern of cellular metabolism. In an earlier paper (Kholodenko et al. (1998). Biotechnol. Bioeng. 59, 239-247) we derived a method to determine the required modulations. This method, however, cannot be immediately applied to cellular pathways with enzymes catalyzing more than one step in metabolism (multifunctional enzymes). In the present paper we show to which extent the presence of multifunctional enzymes limits biotechological ambitions, which one might otherwise pursue in vain. In particular, it is impossible to change the concentration of a single intermediate and leave the rest of metabolism unperturbed if that intermediate interacts directly with a multifunctional enzyme. The analytical machinery of Metabolic Control Analysis is used to relate the desired and ensuing changes in the metabolic pattern. An explicit solution to this problem of engineering metabolism is then given in the form of a single matrix equation.

Animals↗
PubMed1998

Metabolic design: how to engineer a living cell to desired metabolite concentrations and fluxes.

A biotechnological aim of genetic engineering is to increase the intracellular concentration or secretion of valuable compounds, while making the other concentrations and fluxes optimal for viability and productivity. Efforts to accomplish this based on over-expression of the enzyme, catalyzing the so-called "rate-limiting step," have not been successful. Here we develop a method to determine the enzyme concentrations that are required to achieve such an aim. This method is called Metabolic Design Analysis and is based on the perturbation method and the modular ("top-down") approach-formalisms that were first developed for the analysis of biochemical regulation such as, Metabolic Control Analysis. Contrary to earlier methods, the desired alterations of cellular metabolism need not be small or confined to a single metabolite or flux. The limits to the alterations of fluxes and metabolite concentrations are identified. To employ Metabolic Design Analysis, only limited kinetic information concerning the pathway enzymes is needed.

Biotechnology↗
PubMed1997

Transcription of germline VH gene elements by normal human fetal liver.

Transcription of the gene elements that form the variable region of immunoglobulin heavy chains has been proposed to represent the process that controls access for the recombination enzymes in their sequential steps of catalysis. Evidence for germline transcription of VH gene elements, as part of VH to DJH recombination, has been limited to transcripts of only a few gene elements. We have examined normal fetal liver mRNA by Northern blotting and present evidence for germline transcripts from six human VH gene families. The candidate VH4 transcripts have been confirmed as germline transcripts by hybridization with 3' flanking sequences that would have been removed by recombination from mature VHDJH genes. The candidate transcripts for VH1, VH3, VH4 and VH6 have been confirmed by polymerase chain reaction amplification with primers from the 3' flanking sequences of these gene families and determination of the sequence of these products. Determination of sequence from two clones of VH1, VH3 and VH4 indicates that more than one gene from each of these families is transcribed. PCR amplification of VH4 and VH6 with primers specific for the leader sequence (exon 1) and 3' flanking sequence indicate that these transcripts are spliced, representing RNA processing. Germline transcripts from these families are also present in normal human bone marrow. These results indicate that transcriptional activation of germline VH gene elements is a general phenomenon in tissues undergoing V to DJ recombination.

Adult↗
PubMed1996

VH and VL gene elements that encode human antibodies to DNA.

We have determined the cDNA sequence of variable regions of heavy and light chains of three antibodies with low affinity to DNA. The variable heavy chains were found to result from utilization of VH gene elements that have been identified previously in other low-affinity anti-DNAs. These VH gene elements, VH26 and VH1.9III, are expressed in association with different D gene-encoded CDR3s than in the other antibodies. The variable light chains were found to be encoded by VL gene elements that have not previously been identified in anti-DNAs. The recurrent identification of only 10 germ line VH genes in 22 low-affinity anti-DNAs indicates that there is a high probability that all of the VH gene elements that confer reactivity with DNA have been identified. Previous studies have suggested that high-affinity antibodies to DNA result from an antigen-driven process of affinity maturation. However, only 6 of 13 high-affinity antibodies to DNA are derived from this set of low-affinity VHs, indicating that DNA is unlikely to be the driving antigen.

Antibodies, Antinuclear↗
PubMed1992

Germ line transcription of the immunoglobulin heavy chain locus directs production of mu chain without VDJ.

Immunoglobulin VDJ recombination is associated with transcriptional activation of the Ig variable region elements. We have previously described a novel Ig mu chain protein and mRNA produced by pre-B cell hybrids from normal and X-linked agammaglobulinemic bone marrow. We have now characterized the mRNA encoding this protein and find that it is composed of a 5' leader sequence spliced to C mu (LS-C mu), lacking the variable (V), diversity (D), and joining (J) gene sequences. The leader sequence is encoded by a novel exon 16 kb upstream of the JH locus. Transcription of the germ line heavy chain locus from this LS exon results in transcriptional activation of the JH locus, apparently the initial step in commitment to B lymphoid development. Polymerase chain reaction amplification of normal bone marrow shows that these germ line LS-C mu transcripts are a product of bone marrow pre-B cells. Production of LS-C mu commences a sequential process of transcriptional activation, with concordant translation of Ig rearrangement intermediates, in the process of creating a productive VDJ rearrangement.

Amino Acid Sequence↗
PubMed1992

Evidence for failure of V(D)J recombination in bone marrow pre-B cells from X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) results from a failure of B lymphoid development. We have previously examined pre-B cell hybrids from three patients with XLA and found them to be limited to production of a novel germ line transcript of the Ig H chain locus composed of a leader sequence (LS) spliced to the constant region of mu chain (C mu) as mRNA and polypeptide. These transcripts result from transcriptional activation of the germ line heavy chain locus from an LS exon upstream of the embryonic JH locus. Germ line LS-C mu transcripts are produced by pre-B cells from normal bone marrow and fetal liver, indicating that they are products of normal pre-B cell development, as part of the process of transcriptional activation to provide access for the recombinase. Bone marrow from three patients with XLA has been examined directly by polymerase chain reaction amplification to determine whether the exclusive production of LS-C mu by XLA pre-B cell hybrids is representative of XLA pre-B cells. I report that LS-C mu is the predominant Ig molecule produced by XLA pre-B cells, with limited production of the D mu product of DJH intermediate stage of V(D)J recombination. Mature VHDJH recombinations were not detected with a variety of primers that amplify VH sequences. I conclude that XLA is associated with a limitation in V(D)J recombination that may cause the failure of pre-B cell development.

Agammaglobulinemia↗
PubMed1990

X chromosome linked immunodeficiency.

Six human immunodeficiency diseases have been associated with the X chromosome by family studies. Genetic mapping with restriction fragment length polymorphisms (RFLPs) has permitted assignment of these diseases to specific loci on the X chromosome. Each of the disease entities maps to a single locus, confirming that the diagnostic criteria describe single diseases. X-linked chronic granulomatous disease and Wiskott-Aldrich syndrome map to loci on the short arm of the X chromosome; X-linked severe combined immunodeficiency, X-linked agammaglobulinemia, X-linked immunodeficiency with hyper-IgM, and X-linked lymphoproliferative syndrome map to loci on the long arm. Lyon's hypothesis predicts that these X-linked immunodeficiencies may be detectable in carriers of the diseases as a result of X chromosome inactivation of the normal disease gene. Four of the immunodeficiency diseases, X-linked agammaglobulinemia, X-linked severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, and X-linked chronic granulomatous disease, affect cellular development so that carriers have a monomorphic population of immunocytes. The specific immunocyte development affected in carriers varies according to the disease. Genetic mapping of the diseases, with a collection of informative RFLPs, provides a tool that permits probability-based prenatal diagnosis. Carrier detection complements the RFLP-based genetic mapping, serving to confirm X-linkage in carriers.

Chromosome Mapping↗
PubMed1988

B lymphocytes from X-linked agammaglobulinemia. Delayed expression of light chain and demonstration of Lyonization in carriers.

We report an unusual phenotype of B cells in a patient with X-linked agammaglobulinemia (XLA), and cellular evidence for Lyonization of B cells from his mother and sister. The patient has a failure of B cell maturation at the stage of early B lymphocytes, associated with production of D(mu delta) H chain. The phenotype of his B cells includes: (a) limitation to expression of the mu and delta H chain isotypes, (b) production of mu and delta H chains of reduced size and (c) delayed expression of L chain. Peripheral blood and B cell lines from the patient's mother and sister include 50% cells that express H chain without L chain. B cell lines from the mother and sister produce full-length mu and gamma H chains and truncated mu and delta chains corresponding to the H chains produced by the patient's B cells. Clones with normal and XLA phenotype have been isolated from B cell lines derived from the patient's mother. We conclude that the dimorphism of mother's and sister's B cells results from Lyonization, implying that the gene defect in XLA is intrinsic to B lymphocytes.

Agammaglobulinemia↗
PubMed1988

Premature termination of variable gene rearrangement in B lymphocytes from X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) results from failure of B lymphocyte development. Immature B cells from a patient with XLA were found to produce truncated mu and delta immunoglobulin H chains encoded by D-JH-C (mu delta). The 5' terminal sequence of cDNA encoding the H chains is composed of D-JH with the characteristic GGTTTGAAG/CACTGTG consensus sequence utilized for VH gene rearrangement upstream, and a leader sequence that serves for translation of this intermediate stage of rearrangement. Failure of variable region gene rearrangement may underlie the failure of B lymphoid development in XLA.

Agammaglobulinemia↗
PubMed1988

Correction of the molecular defect in B lymphocytes from X-linked agammaglobulinemia by cell fusion.

The X chromosome-linked antibody deficiency disease, X-linked agammaglobulinemia (XLA), results from failure of B lymphoid development. In the minor form of XLA, B lymphoid development terminates at the stage of immature B lymphocytes that produce truncated Ig heavy (H) chains composed of D-J-C(mu/delta), resulting from failure of VH gene rearrangement. Fusion of B cells from a patient with the minor form of XLA with mouse myeloma results in complementation of this defect; hybrid cells produce full-length H chains composed of VH-D-JH-C. The VH gene is of human origin. Complementation occurs independent of retention or loss of the human X (XLA) chromosome in the hybrid cells. These results indicate that the D-JH-C structure of the XLA B cells is fully functional for the subsequent rearrangement of a VH gene element, and that failure of immunoglobulin expression is susceptible to correction.

Agammaglobulinemia↗
PubMed1987

Identification and sequence of the VH gene elements encoding a human anti-DNA antibody.

Antibodies to DNA similar to those found in patients with systemic lupus erythematosus (SLE) and autoimmune mice can be derived from the lymphocytes of normal individuals. It is not known whether these normal derived anti-DNA antibodies are made from the same VH gene elements as the anti-DNA antibodies made by SLE patients. To begin to answer this question, we examined mu chain cDNA clones from human hybrid clone C6B2 producing anti-DNA antibodies. The sequence of the 500 base pair restriction fragment containing the variable region (5' terminus) was determined and was sequenced. This antibody uses a VHII heavy chain subgroup gene, a J3 joining segment, a hitherto unknown D segment, and a previously reported leader sequence. Significant homology was found to a mouse anti-DNA antibody sequence in the use of VH subgroup in J3, and in the hypervariable regions with a shared Ser-Tyr construction in CDR1 and an identical five amino acid residue stretch in CDR2. Comparison with the limited sequence data of published SLE monoclonal anti-DNA antibodies, both human and mouse, suggests that this shared Ser-Tyr may be important in some but not all antibodies to DNA. Comparison of C6B2 antibody is made with other known antibody sequences with identification of those residues likely to be part of the antigen binding site.

Amino Acid Sequence↗
PubMed1986

Specificity analysis of human anti-DNA antibodies.

Human hybrids producing anti-DNA antibodies were generated by the fusion of pokeweed mitogen-stimulated splenic lymphocytes from a child with sickle cell anemia to GM4672. Of 19 hybrids, three (15%) produced anti-DNA antibody as detected by an enzyme linked immunosorbent assay. One subclone from each of these three hybrids was then characterized. All produced IgM antibody in large amounts ranging from 22 to 266 micrograms/ml per million cells per 24 hr. All three antibodies bound both double- and single-stranded DNA. Competitive inhibition assays revealed the greatest inhibition of DNA binding with the ribohomopolymers polyinosinic and polyguanylic acid. A complex pattern of cross-reactivity with various other polynucleotides and with some phospholipids was observed. Subtle differences were found among the three antibodies in light chain class and some of the binding specificities. By using a modified Farr assay, all three monoclonals were found to be of low to intermediate affinity. These results confirm that anti-DNA antibodies apparently equivalent to those seen in patients with SLE can be derived from "normal" nonautoimmune individuals.

Animals↗
PubMed1985

The human T cell antigen Leu-2 (T8) is encoded on chromosome 2.

The locus encoding the human T lymphocyte cell surface antigen Leu-2 has been assigned to chromosome 2 with a DNA mapping panel derived from somatic cell hybrids. The two genomic components identified by a cDNA clone for Leu-2 segregated with human chromosome 2 in all 24 independent hybrid clones examined. The cosegregation of the Leu-2 and immunoglobulin kappa (IgK) loci in hybrids with spontaneous rearrangements of chromosome 2 is consistent with the possibility that the Leu-2 locus is on proximal human 2p near IgK. In the mouse, a locus for a T lymphocyte cell surface antigen with properties similar to Leu-2 is closely linked to the IgK locus on mouse chromosome 6. Hence the syntenic relationship of a gene implicated in T cell killing with the immunoglobulin kappa locus would then be conserved in the mouse and human genomes.

Animals↗
PubMed1984

Lymphoid cell lines from patients with "non-secretory" agammaglobulinemia produce glycosylated heavy chains which are reduced in molecular weight.

B lymphocytes from patients with "non-secretory" agammaglobulinemia synthesize but do not secrete Ig. A previous study attributed this secretion failure to a failure of the cells to glycosylate Ig. We examined four B cell lines from three patients with "non-secretory" agammaglobulinemia as a model of this disease. All four cell lines synthesized IgG or IgM in quantities comparable to that produced by normal cell lines, but failed to secrete Ig of either isotype. Molecular weight determination in SDS-polyacrylamide slab gels showed that the heavy chains produced by these cell lines were reduced in size compared to normal: gamma of 49,000 daltons and mu of 59,000 daltons (compared to 55,000 and 68,000 daltons for normal, respectively). Radioactive precursors of the Ig carbohydrate moiety were specifically incorporated into these Ig molecules, suggesting that the reduction in size was not due to failure to glycosylate the Ig. Tunicamycin treatment of the B cell lines resulted in an apparent reduction in size of these already small heavy chains, confirming that the observed reduction in size was not due to the absence of carbohydrate from the Ig molecules. Electrophoresis of cellular IgG and IgM under nonreducing conditions indicated that the molecules were incompletely assembled, lacking disulfide bridging between H (both gamma and mu) chains and L chains. The discrepancy in incorporation of carbohydrate between our studies with "non-secretory" B cell lines and a previous study of "non-secretory" lymphocytes in short term culture led us to reexamine Ig glycosylation in short term cultures. We found that mitogenically stimulated lymphocytes from three patients incorporated radioactive carbohydrate precursors into Ig. These results indicate that the failure of "non-secretory" B cells to secrete Ig is not secondary to a failure of glycosylation, either in short term cultures of B lymphocytes or in B cell lines. Rather, our studies of "non-secretory" B cell lines suggest that there is a polypeptide deletion of the gamma and mu heavy chains, indicated by reduced molecular weight, incomplete assembly of H and L chains, with consequent failure to be secreted, resulting in "non-secretory" agammaglobulinemia.

Agammaglobulinemia↗
PubMed1983

Frequency of anti-DNA antibody producing cells from normals and patients with systemic lupus erythematosus.

The frequency of anti-DNA antibody producing cells from normals and patients with systemic lupus erythematosus (SLE) was determined. Peripheral blood lymphocytes (PBL) from normals and patients with SLE were cultured for 8 and 15 days with and without transformation by Epstein-Barr virus (EBV). Culture supernatants were examined for the presence of anti-DNA antibody using an enzyme-linked immunosorbent assay. We found that PBL from patients with SLE spontaneously produce anti-DNA antibodies whereas PBL from normals do not. After EBV transformation, anti-DNA antibody producing cells were detected in both cultures from patients with SLE as well as from normals. These data suggest that the high levels of anti-DNA antibody observed in patients with SLE represent activation of B cells committed to anti-DNA antibody production and that such cells are present but are not activated in normal individuals.

Adult↗