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P D Burrows

Publications and source records attributed to P D Burrows.

At least 37 records · Page 2Linked to original sources
PubMed1994

Switch recombination in normal IgA1+ B lymphocytes.

Most B lymphocytes in normal individuals express two classes of cell-surface immunoglobulins, IgM and IgD. The specificity of the two antigen receptors is identical since they are produced by transcription and differential splicing of the same variable region gene segment to the heavy-chain constant region gene segments for both mu and delta heavy chains. B lymphocytes expressing other immunoglobulin isotypes, IgG, IgA, or IgE, are rare and not well characterized. Particularly controversial is the molecular mechanism of their isotype switch. Here we use high-gradient magnetic cell sorting and fluorescence-activated cell sorting to purify surface IgA1-bearing B lymphocytes from human blood for cellular and molecular analysis. These cells express no immunoglobulin class other than IgA1 and are a relatively uniform population with regard to expression of other cell-surface molecules. They are resting cells in terms of cell cycle and activation marker analysis. The molecular basis for class switching in the IgA1+ cells is not differential transcription or splicing. Rather, switch recombination involving deletion of DNA has occurred on both immunoglobulin heavy-chain gene loci, including the allelically excluded one, and appears to have been directed to IgA1 under normal physiological conditions.

Antigens, CD↗
PubMed1993

Helper T cell subsets for immunoglobulin A responses: oral immunization with tetanus toxoid and cholera toxin as adjuvant selectively induces Th2 cells in mucosa associated tissues.

Antigen-specific B cell responses to mucosally delivered proteins are dependent upon CD4-positive T helper (Th) cells, and the frequency of Th1 and Th2 cell responses after oral immunization may determine the level and isotype of mucosal antibody responses. We have used a protein-based vaccine, tetanus toxoid (TT), together with the mucosal adjuvant cholera toxin (CT), for oral immunization of mice to study the nature of antigen-specific Th cell subsets induced in Peyer's patches (PP) of the gastrointestinal (GI) tract and in the spleen (SP) during peak antibody responses. Mice orally immunized with TT and CT responded with antigen-specific secretory immunoglobulin A (S-IgA) antibodies in the GI tract, and with both IgG and IgA antibody responses in serum. PP and SP CD4+ T cells from mice orally immunized with TT plus CT were cultured with antigen-coated latex microspheres for induction of proliferative responses and for enumeration of cytokine producing CD4+ T cells. Interestingly, both PP and SP CD4+ T cell cultures showed increased numbers of IL-4- and IL-5 (Th2-type)-producing, spot-forming cells (SFCs) after 21 d of immunization, while essentially no interferon-gamma (IFN-gamma) or IL-2 (Th1-type) SFCs were noted. Cytokine-specific Northern blots and RT-PCR also revealed that significant IL-4 and IL-5 mRNA levels, but not IFN-gamma or IL-2 mRNA, were present in CD4+ T cells isolated from antigen-stimulated cultures. However, systemic immunization with TT and CT induced antigen-specific IgG and IgM but not IgA antibodies in serum. Further, both IL-2 and IFN-gamma-producing Th1-type cells as well as IL-4- and IL-5-secreting Th2-type cells were generated in SP. Our results show that oral immunization with TT and the mucosal adjuvant CT selectively induced antigen-specific Th2-type responses which may represent the major helper cell phenotype involved in mucosal IgA responses in the GI tract.

Administration, Oral↗
PubMed1993

A mitochondrial porin cDNA predicts the existence of multiple human porins.

Pores formed in the outer membrane of mitochondria by mitochondrial porin make it permeable to water-soluble metabolites smaller than approximately 5 kDa. We have isolated a full-length cDNA encoding a human porin. This probe detects a single approximately 1.5-kilobase mRNA species on Northern blots, but multiple hybridizing bands on genomic Southern blots. The open reading frame predicts a 38.1-kDa protein with a pI of 6.59 that is homologous but not identical to a previously reported protein sequence of a 31-kDa porin isolated from human lymphocytes (porin 31HL). The most striking difference between the two porins is that the sequence predicted by the cDNA is longer than the 31HL porin by 27 amino acids at the amino terminus and 38 amino acids at the carboxyl terminus. The porin cDNA directs the in vitro translation of two protein species of approximately 32 and approximately 36 kDa, which appear to result from alternate usage of AUG initiation codons. The 32-kDa protein is the predominant species imported into both rat and yeast mitochondria in vitro. Taken together, these results suggest that multiple porin proteins can be expressed in humans. Additionally, a porin consensus protein sequence has been identified that is conserved in eukaryotic organisms as diverse as yeast and man.

Amino Acid Sequence↗
PubMed1993

B-cell development in man.

The development of B-lineage cells requires a series of complex interactions with hemopoietic stromal cell elements during the generative phase, and with antigen and T lymphocytes during the subsequent proliferative/differentiative phases in lymphoid tissues. Recent advances have been made in defining developmental changes in structure and assembly of the antigen receptors and in identifying protein kinases involved in signal transduction via these receptors. The mechanism of T-cell help has also come into much clearer focus through elucidation of the interaction between CD40 on B cells and the CD40 ligand on activated T cells. Finally, progress has been made with the recent identification of defects in a cytoplasmic protein tyrosine kinase and in the CD40 ligand as causes of two B-cell immunodeficiencies in man.

Antigens, CD↗
PubMed1993

Normal B lymphocyte differentiation.

Normal differentiation of B lineage cells has been the subject of intensive investigation over the past three decades. Current models of this process in humans are melded from the results of studies in a variety of organisms, including humans, mice and birds. Several recent developments have significantly reshaped and refined these models. The technique of homologous recombination in embryonic stem cells has allowed the production of mice with selectively disrupted genes that are important for B cell development in mice. At the same time, functional studies of human B cell differentiation, together with analysis of naturally occurring mutations that disrupt this process, have progressed rapidly. This has provided insight into the pathogenesis of lymphoproliferative and immunodeficiency diseases as well as a clearer view of normal developmental events. In this chapter we have reviewed human B cell differentiation with particular emphasis on newly emerging concepts. We also discussed CD5, a pan-T cell antigen that is expressed in low levels on a subpopulation of B cells implicated in the pathogenesis of chronic lymphocytic leukaemia (CLL). Finally, we discussed the issue of restricted variable region gene usage during B cell ontogeny and in CLL.

Animals↗
PubMed1992

Molecular cloning and expression pattern of a human gene homologous to the murine mb-1 gene.

The mouse mb-1 gene was originally identified based on its restricted expression in B lineage cells. Predicted structural homology with the gamma chain of the CD3 complex on T cells led to the suggestion that the MB-1 protein might associate with surface Ig(sIg) on B cells and be involved in signal transduction. Other studies identified at least two proteins that are noncovalently associated with sIgM, one of which has recently been shown to be the product of the mb-1 gene. To identify genes specifically expressed in normal human B cells we constructed a B minus T lymphocyte subtraction library and isolated a cDNA clone highly homologous to murine mb-1 (m-mb-1). A full-length cDNA was subsequently isolated and found to encode a membrane glycoprotein of 226 amino acids. It included a leader sequence (32 amino acids), an extracytoplasmic domain (111 amino acids) containing six potential N-glycosylation sites and three cysteine residues for potential inter- or intrachain disulfide linkages, a transmembrane domain (22 amino acids), and an intracytoplasmic domain (61 amino acids). The amino acid sequence homology between human and mouse mb-1 was especially striking (approximately 92%) in the intracytoplasmic, transmembrane, and membrane-proximal extracellular domains but was less marked (approximately 42%) in the remaining extracytoplasmic portion. Interestingly, part of the 3'-untranslated region was also highly conserved between species, suggesting an important role for this region in the regulation of mb-1 expression. The human mb-1 (h-mb-1) cDNA hybridized with a mRNA species of approximately 1.2 kb on Northern blots. Similar to m-mb-1, the h-mb-1 transcripts could be detected in pre-B cell lines and fetal bone marrow, in normal, mitogen activated- and transformed B cells but not in myeloma plasma cells. h-mb-1 was not expressed in peripheral T cells nor by cells of other hemopoietic lineages or in brain, heart, muscle, lung, and kidney. Surprisingly, however, low levels of h-mb-1 transcripts were detectable in two early T lineage cell lines and in the fetal thymus. This suggests that mb-1 may have other functions in addition to its role in signal transduction in B lineage cells.

Amino Acid Sequence↗
PubMed1991

Regulation of IgM and IgD expression in human B-lineage cells.

IgD is thought to function primarily as an Ag receptor that is expressed, together with IgM, only on mature B lymphocytes. This differentiation stage-specific expression of IgD has been well characterized in mice, where delta mRNA is detected only in mature IgM/IgD B cells. Humans, in contrast to mice, have significant levels of serum IgD, suggesting that the regulation of this isotype might differ between the two species. Therefore, we examined the regulation of both IgM and IgD expression in cell lines encompassing the spectrum of human B lineage development. Surprisingly, two species of delta mRNA could be found at all differentiation stages -from mu+ pre-B cell to IgM-secreting plasmablast. These mRNA are translated to yield the membrane and secretory forms of delta. The membrane delta-chain: secretory delta-chain ratio did not necessarily reflect the membrane mu-chain:secretory mu-chain ratio in the same cell line, implying that different mechanisms are involved in the selection of membrane vs secretory mu- and delta-chains. The delta-chains synthesized in pre-B cells were degraded, but in more mature cell types IgD could be stably expressed and secreted. Exceptions to this panlineage synthesis of delta-chains were, however, observed in two of the B cell lymphomas, where delta expression was prevented by transcriptional and posttranscriptional mechanisms. The presence of delta-chain in pre-B cells and the secretion of IgD by more mature cells suggest that IgD may have immunoregulatory roles throughout B cell differentiation. These studies also indicated that the bias toward secretory mu-chain production that occurs in human IgM secreting cells results from posttranscriptional regulation. In addition, we have identified a B cell line that synthesizes both normal-sized mu-chains and those with smaller apparent m.w. translation products of truncated mu mRNA.

B-Lymphocytes↗
PubMed1991

Stage-specific transcription of germline IgH C gamma and C alpha regions during human B cell differentiation.

Previous studies have suggested that transcription of germline heavy chain constant region (CH) genes in murine B cells may determine the potential of their different CH regions to undergo isotype switch recombination. We have examined the transcriptional activity across the immunoglobulin heavy chain (IgH) locus in human B lineage cells. Transcription of germline C gamma and C alpha was observed in every surface IgM+ or surface IgM+/IgD+ B cell stage cell line and malignancy. In contrast, such transcription could not be detected in pre-B cells and only low levels of C alpha but not C gamma transcription were evident in IgM-secreting plasmablast cells. Transcriptional activity of germline IgH C epsilon was singularly absent at all stages of B cell development. Our results suggest that germline transcription of the C gamma and C alpha regions may be a constitutive feature of the human B cell differentiation program. Because this transcriptional activity is limited primarily to the B cell stage and occurs prior to the actual isotype switch, the induction of C gamma and C alpha transcription may represent preparation of the downstream IgH chromatin for potential switch recombination.

B-Lymphocytes↗
PubMed1990

Molecular cloning of the murine BP-1/6C3 antigen: a member of the zinc-dependent metallopeptidase family.

The BP-1/6C3 antigen is a phosphorylated cell surface glycoprotein that can be identified by monoclonal antibodies on mouse pre-B cells, immature B cells, and certain stromal cell lines from bone marrow. Expression of this antigen is increased in stromal-dependent pre-B cell lines and retrovirally transformed pre-B cells. Expression of the BP-1/6C3 antigen thus correlates with proliferation and transformation of immature B-lineage cells. In this study, we report the isolation and characterization of cDNAs encoding the BP-1/6C3 antigen. Northern blot analysis revealed a major 4.1-kilobase mRNA in all BP-1/6C3+ mouse pre-B lines and in a human pre-B cell line. BP-1/6C3 mRNA was either absent or truncated in BP-1/6C3- cell lines. The cDNA sequence predicts a type II integral membrane protein of 945 amino acids with an intracytoplasmic amino terminus of only 17 amino acids and a typical zinc-binding motif in its extracellular domain. BP-1/6C3 has significant homology to aminopeptidase N and is the second member of the zinc-dependent metallopeptidase gene family to be found on the surface of early B-lineage cells.

Amino Acid Sequence↗
PubMed1990

Bone marrow stromal cells and interleukin-7 induce coordinate expression of the BP-1/6C3 antigen and pre-B cell growth.

The BP-1/6C3 molecule expressed by early B lineage cells and some stromal cells is a type II integral membrane glycoprotein that belongs to the zinc-dependent family of metallopeptidases. In order to explore the potential role of this cell surface molecule in precursor B cell proliferation, we established a stromal cell line (BHM) from long-term bone marrow cultures of the Whitlock - Witte type and developed a rapid bioassay for the detection of responsive target cells. When non-adherent bone marrow cells were cultured with BHM stroma, we observed an up-regulation of BP-1 expression by the B cell precursors that coincided with the induction of proliferation. The precursor B-cell targets included bone marrow cells that lacked detectable BP-1/6C3, B220, and Ia antigens. They could be identified in fetal liver and in the bone marrow, but not the thymus, spleen, or lymph nodes, of normal BALB/c mice, and were also present in the bone marrow of mice with the nu/nu, CBA/N, and SCID defects. Because the inductive effects on precursor B cells could be reproduced with BHM supernatant, soluble growth factors were evaluated in the assay. Interleukin 7 (IL-7) appeared to be unique in its ability to induce BP-1/6C3 expression and concomitant cell growth. Northern blot analysis revealed that the BHM stromal cells, which themselves express the BP-1 antigen, constitutively expressed high levels of variable length transcripts for IL-7. The data suggest that the IL-7 product of stromal cells selectively induces BP-1/6C3 expression on B cell precursors and may implicate this cell surface glycoprotein in the control of IL-7 induced proliferation of early B lineage cells.

Animals↗
PubMed1990

Regulated expression of cell surface antigens during B cell development.

The progression of lymphocytes along the B cell developmental pathway is marked by the regulated appearance and disappearance of a variety of cell surface markers including immunoglobulin. In this review, several of these antigens are discussed in the context of their possible functions during normal B cell differentiation.

Animals↗
PubMed1989

Light-chain gene expression before heavy-chain gene rearrangement in pre-B cells transformed by Epstein-Barr virus.

Epstein-Barr virus transformation of B-cell-depleted bone marrow cells from human fetuses allowed us to identify novel cell types characterized by the expression of immunoglobulin kappa or lambda light chain without heavy chains. Four kappa-only clones with normal karyotype were obtained and examined for their immunoglobulin gene configurations and expression. All four clones had kappa-chain gene rearrangements at either one or both alleles, but the heavy-chain gene loci in these clones either were in germ-line context or had undergone only D-JH rearrangements (D and JH represent diversity and joining gene segments). All clones contained kappa mRNA of normal size at levels consistent with the protein level, except for one clone that no longer produced kappa protein. No mu mRNA or immunoglobulin heavy-chain molecules were detected in any of the kappa+ clones. The results suggest that the mu heavy-chain protein is not an obligatory prerequisite for light-chain gene rearrangements.

Antibodies, Monoclonal↗
PubMed1989

Mu heavy chains can associate with a pseudo-light chain complex (psi L) in human pre-B cell lines.

In pre-B cells, the earliest identifiable stage of B cell differentiation, there is an asynchrony of immunoglobulin chain expression in that mu heavy chains are synthesized in the absence of light chain synthesis. These mu chains largely remain intracellular and are degraded. Here we demonstrate that a fraction of mu chains in human pre-B cell lines can reach the surface in association with three pre-B-specific proteins with relative molecular masses of 22, 18, and 16 kd, which we term collectively the pseudo-light chain complex, psi L. This association generates a multimeric complex, mu 2-psi L. Two of the psi L proteins (22 and 16 kd) are lambda-immunoreactive and form disulfide bonds with mu chains, suggesting that they are closely related to conventional lambda light chains. The 18 kd psi L species is a non-covalently-associated member of the complex. The expression of mu-psi L complexes on the surface of pre-B cells could have a functional role in the control of pre-B growth and differentiation by the hematopoietic microenvironment.

B-Lymphocytes↗
PubMed1988

Lineage and stage specificity of isotype switching in humans.

The lineage and stage specificity of human isotype switch recombination was investigated by examining the IgH gene configuration in 61 hemopoietic malignancies representing different stages of B and T cell development. An unexpectedly high frequency (20%) of IgM-producing B cell leukemias and lymphomas had undergone CH gene rearrangements and deletions consistent with attempted switch recombination. These CH gene alterations were found on productive, non-productive, and 14q+ chromosomes. These data support the concept of a non-specific (common) switch recombinase activity that is often ineffective. No evidence of such switch recombination was found in 25 mu- or mu+ pre-B cell leukemias with the single exception of a mu- pre-B leukemia in which subsets of the cells were producing gamma- or alpha-H chains. The switch recombinase activity gamma- or alpha-H chains. The switch recombinase activity may be restricted to the B cell lineage, inasmuch as CH gene deletions were not observed in T lineage malignancies.

Cell Transformation, Neoplastic↗
PubMed1988

Precursor B cells transformed by Epstein-Barr virus undergo sterile plasma-cell differentiation: J-chain expression without immunoglobulin.

Human bone marrow cells were depleted of B lymphocytes to enrich for precursor B cells that could be transformed with Epstein-Barr virus. Transformed immunoglobulin-negative precursors either maintained their immunoglobulin genes in the germ-line configuration or had undergone DJ or abortive VDJ rearrangements (V, D, and J represent variable, diversity, and joining gene segments). All cell lines and their derivative clones, even those with no detectable immunoglobulin gene rearrangements, generated subpopulations of cells that produced high levels of joining (J) chain when analyzed by immunoprecipitation after biosynthetic labeling and by blot hybridization of cytoplasmic RNA. Morphologic and immunofluorescence analyses revealed that J-chain production was confined to clonal progeny that had exited the cell cycle to undergo plasma-cell differentiation. Analysis of cell surface antigens revealed expression of several B-cell maturational markers, including complement receptor type 2 (CR2) and plasma cell antigen 1 (PCA-1). Epstein-Barr virus can thus transform B-cell progenitors, allowing them to proliferate and undergo terminal B-cell differentiation coupled with J-chain expression. These events appear to occur independently of the immunoglobulin gene status of the transformed cells.

Agammaglobulinemia↗
PubMed1988

Bone marrow origin of a B-cell lymphoma.

To search for precursors of the neoplastic B cells in a patient with a nodular lymphoma, we produced a monoclonal antibody to a variable region idiotope on the lymphoma IgM heavy chain. Clonal ancestors of the lymphoma cells were identified by this marker among bone marrow pre-B cells (5% to 26%). A second antiidiotype (anti-Id) antibody specific for the complete lymphoma IgM kappa recognized 10% of B cells in bone marrow and blood and greater than 95% of B cells in lymphomatous lymph nodes, including one obtained after tumor conversion to a diffuse large cell lymphoma. Immunoglobulin gene analysis surprisingly revealed expansion of multiple clones of early B lineage cells in bone marrow, including members of the neoplastic clone. The data suggest that this lymphoma arose through a progression of transformational events beginning in bone marrow: first, creation of an oligoclonal pre-neoplastic pool of pre-B cells, subsequent conversion of a single subclone into low grade neoplastic B cells that homed to the lymph node follicles, and later progression to a more invasive form of the B-cell lymphoma.

Adult↗