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Biomedical subjects

A Saxon

Publications and source records attributed to A Saxon.

At least 19 recordsLinked to original sources
PubMed1992

B cells from subjects with CVI can be driven to Ig production in response to CD40 stimulation.

The majority of patients with common variable immunodeficiency (CVI) have low to normal numbers of membrane Ig-bearing B cells; yet these cells fail to differentiate in vivo resulting in hypogammaglobulinemia. We have suggested that the differentiation failure of CVI B cells is related to a failure to respond appropriately to signals involved in terminal B cell differentiation as most CVI subjects' cells undergo activation and proliferation normally. Whether this failure relates to a direct "intrinsic" defect in the B cells or is secondary to a lack of appropriate T cell or other influences in vivo is uncertain. We have previously reported that the majority of patients with CVI have elevated circulating levels of IL-6. We now show that the IL-6 produced by these patients is functionally normal. Additionally, the display of IL-6 receptors on in vitro stimulated CVI B cells is normal. However, we found that the patients' cells do not make IgE in response to an IL-6/T-cell-dependent differentiation pathway employing exogenous interleukin-4 (IL-4). The failure to respond in the IL-6-dependent system could not be overcome by exogenous IL-6 or varying doses of IL-4. In contrast, when stimulated by CD40 plus IL-4 in a differentiation pathway that does not require IL-6, B cells from CVI patients were stimulated to produce IgE. These findings, along with our earlier data showing that 13-cis-retinoic acid can drive maturation in CVI patients, strengthen the concept that B cells in patients with CVI have the potential for terminal differentiation but do not appear to achieve this in vitro or in vivo through a polyclonal Ig differentiation pathway that employs IL-6 as one of its maturation signals.

Adult↗
PubMed1992

A novel B cell stimulation/proliferation assay using simultaneous flow cytometric detection of cell surface markers and DNA content.

Assessment of the specific B cell proliferative response by thymidine incorporation following polyclonal stimulation (e.g. with pokeweed mitogen or B cell growth factor) is problematic whenever the proportion of B cells in a particular sample is low as in blood samples. Here we describe a simple assay consisting of identifying the cultured B cells with anti-CD19/CD20 fluorescein-conjugated antibody and simultaneously using propidium iodide DNA staining to determine the percent of S/G2/M phase cells among the gated B cells. This assay can be used to determine B cell responsiveness even in individuals with very low blood B cell counts (e.g., in recipients of bone marrow transplant), does not require laborious B cell purifications and does not involve radiation hazard.

Adult↗
PubMed1992

Two unusual forms of human immunoglobulin E encoded by alternative RNA splicing of epsilon heavy chain membrane exons.

We present evidence for RNA transcripts encoding two forms of human epsilon immunoglobulin (Ig) heavy chain that differ significantly from those of other isotypes. We previously demonstrated three human epsilon mRNA species, instead of the two, corresponding to membrane and secreted proteins, seen with other heavy chain transcripts. In human genomic DNA downstream of the C epsilon gene, we identified sequences homologous to the two putative murine exons M1 (encoding a hydrophobic, presumably transmembrane region) and M2 (encoding hydrophilic residues). To determine the structures of epsilon transcripts containing these sequences, we amplified epsilon-related RNAs with the reverse transcriptase polymerase chain reaction. RNA was examined from fresh human B cells stimulated to IgE production by interleukin 4 plus anti-CD40, as well as from the human IgE-producing line AF10. Instead of the single CH4-M1-M2 splice product predicted for murine membrane IgE, we found two other RNA species. One form has the structure CH4-M1'-M2, in which M1' includes the human sequence homologous to the murine M1 as well as a unique segment of 52 codons further upstream in the genomic sequence; this RNA species apparently encodes the IgE expressed on the membrane of IgE-producing lymphocytes. The other RNA has the structure CH4-M2', in which M2' is spliced in an alternative reading frame that includes an additional 109 codons downstream of the termination codon of the CH4-M1'-M2 form. Because the CH4-M2' mRNA form does not encode a hydrophobic segment, its translated product should be secreted. A secreted epsilon protein of approximately the size predicted for this form was identified by Western blotting. This novel IgE protein could play a significant and distinctive role in allergic disorders.

Amino Acid Sequence↗
PubMed1992

Human mature T cells that are anergic in vivo prevail in SCID mice reconstituted with human peripheral blood.

In these studies we have characterized the human cells that repopulate severe combined immunodeficient (SCID) mice after injection of adult peripheral blood or cord blood (hu-PBL-SCID mice). In all organs of the chimeras, and at any time point tested, single-positive (CD4+ or CD8+) T cells that expressed the alpha/beta T cell receptor (TCR) prevailed. All T cells were CD45RO+ and the majority were also HLA-DR+. Thus, the human T cells in the chimeras exhibited the phenotype of mature, memory cells that showed signs of recent activation. Cell cycle studies revealed a mitotically active human T cell population in the murine host. However, when freshly isolated from the chimeras, the human T cells were refractory to stimulation by anti-CD3 antibody but proliferated in response to exogenous interleukin 2. Chimera-derived human T cell lines retained this state of unresponsiveness to TCR-triggered proliferation for 4-6 wk in vitro. Subsequently, the T cell lines developed responses to anti-CD3 stimulation and 9 of 11 of the lines also proliferated in response to splenic stimulator cells of SCID mice. These data demonstrate that the human T cells are in a state of reversible anergy in the murine host and that xenoreactivity might play a critical role in hu-PBL-SCID mice. Mechanisms that may determine repopulation of SCID mice with human peripheral blood mononuclear cells are discussed.

Animals↗
PubMed1992

Recovery of mononuclear cell subsets after bone marrow transplantation: overabundance of CD4+CD8+ dual-positive T cells reminiscent of ontogeny.

Patients after bone marrow transplantation are immunodeficient for months to years. To understand better the pathogenesis of this immunodeficiency, we studied quantitative reconstitution of blood monocytes, natural killer (NK) cells, T cells, and B cells at 2-22 months post-transplant. The results indicate monocyte and NK counts generally recover within 2 months, followed by CD4-CD8+ T cell, B cell, and finally (after > 1 year) CD4+CD8- T cell numbers. Dual-positive CD4+CD8+ T cells (which were barely detectable in normal adults), CD4-CD8+ T cells and B cells transiently reached supranormal levels during recovery. Both CD4+CD8- and CD4-CD8+ T cells were larger than controls throughout the 2-year follow up. Comparison with neonatal and infant mononuclear cell subsets suggested the reconstitution of CD4+CD8+ T cells and B cells is similar to ontogeny. In contrast, the reconstitution of CD4+CD8- T cells did not resemble ontogeny.

B-Lymphocytes↗
PubMed1992

The second century of the antibody. Molecular perspectives in regulation, pathophysiology, and therapeutic applications.

The modern age of immunology began in 1890 with the discovery of antibodies as a major component of protective immunity. The 2nd century of the antibody begins with a focus on the molecular physiology and pathophysiology of immunoglobulin production. Numerous human variable-region antibody genes have been identified through advances in molecular cloning and anti-variable-region monoclonal antibodies. Some of these variable-region genes are now known to be involved in specific stages of B-lymphocyte differentiation and immune development. This connection has yielded new insights into the pathogenesis of immune dyscrasias and lymphoid neoplasia; common variable immunodeficiency and cryoglobulinemia are highlighted here. The molecular regulation of immunoglobulin expression suggests new targets for pathogenesis and clinical intervention. Finally, genetically engineered antibodies offer novel opportunities for diagnostic and therapeutic applications.

Agammaglobulinemia↗
PubMed1992

Results of the National Institute of Allergy and Infectious Diseases Collaborative Clinical Trial to test the predictive value of skin testing with major and minor penicillin derivatives in hospitalized adults.

BACKGROUND: A history (or lack thereof) of penicillin allergy is known to be unreliable in predicting reactions on subsequent administration of the drug. This study tests the usefulness of four penicillin allergen skin tests in the prediction of IgE-mediated reactions subsequent to administration of penicillin. METHODS: Eight centers cooperated in the National Institute of Allergy and Infectious Diseases trial of the predictive value of skin testing with major and minor penicillin derivatives. Hospitalized adults were tested with a major determinant (octa-benzylpenicilloyl-ocytalysine) and a minor determinant mixture and its components (potassium benzylpenicillin, benzylpenicilloate, and benzylpenicilloyl-N-propylamine). Patients then received a therapeutic course of penicillin and were observed, for 48 hours, for adverse reactions compatible with an IgE-mediated immediate or accelerated allergy. RESULTS: Among 726 history-positive patients, 566 with negative skin tests received penicillin and only seven (1.2%) had possibly IgE-mediated reactions. Among 600 history-negative patients, 568 with negative skin tests received penicillin and none had a reaction. Only nine of the 167 positive skin test reactors received a penicillin agent and then usually by cautious incremental dosing. Two (22%) of these nine patients had reactions compatible with IgE-mediated immediate or accelerated penicillin allergy; both were positive to the two determinants. CONCLUSIONS: These data corroborate previous data about the negative predictive value of negative skin tests to these materials. The reaction rate in skin test-positive patients was significantly higher than in those with negative skin tests, demonstrating the positive predictive value of positive tests to both major and minor determinants. The number of patients positive only to the major determinant or only to the minor determinant mix was too small to draw conclusions about the positive predictive value of either reagent alone.

Adult↗
PubMed1992

Reconstitution of B cell immunity following bone marrow transplantation.

Bone marrow transplant recipients are known to be immunodeficient in cellular and humoral immune responses for months to years. Mechanisms underlying the post-transplant immunodeficiency are mostly unknown and therefore it has been difficult to design therapeutic strategies to address this problem. Here, we review studies of post-transplant B cell (humoral) immunity including B cell blood counts, phenotype, function and origin, and the morphology of lymphoid organs. We postulate that the aetiology of post-transplant humoral immunodeficiency is multifactorial, involving: B cell immaturity due to recapitulation of their ontogenesis, and, in some patients, insufficient T cell help and/or exaggerated CD8+ T cell/NK cell suppression. Implications for clinical practice are outlined, e.g. hypersensitivity reactions and autoimmune diseases as part of the differential diagnosis of post-transplant disorders, questionable diagnostic benefit of serologic studies, and usefulness of prophylactic intravenous immunoglobulin.

Antibody Formation↗
PubMed1991

Inhibition of human IgE production via Fc epsilon R-II stimulation results from a decrease in the mRNA for secreted but not membrane epsilon H chains.

We have previously shown, using IgE-anti-IgE immune complexes or mAb directed against CD23, that ongoing production of secreted IgE from the human plasma cell line AF-10 can be inhibited via the low affinity FcR for IgE (CD23). Changes occurring in the various forms of epsilon messenger RNA (epsilon mRNA) during this suppression were investigated. mRNA levels of lambda L chain were also assessed as well as beta-actin controls. Changes in membrane IgE and secreted IgE and lambda protein were simultaneously measured. Using a genomic probe corresponding to the human epsilon C region domains, three sets of epsilon-mRNA bands were identified (2.1, 3.0, and 3.8 kb). Only the largest of these (3.8 kb) contained the full epsilon membrane sequence and coded for true membrane epsilon protein. The 2.1-kb species of epsilon-mRNA contained no membrane sequence and coded for classical secreted epsilon protein. The intermediate epsilon-mRNA species (3.0 kb) was shown to contain membrane sequence but did not contain the full epsilon membrane sequence. The product of this mRNA would, in fact, function as a secreted protein. When IgE production by AF-10 cells was suppressed via Fc epsilon R-II, there was a 50% fall in the steady state levels of both forms of mRNA (2.1 and 3.0 kb) that code for secreted epsilon protein. Similarly, there was a fall in lambda-mRNA corresponding to the observed decrease in free lambda secretion. In marked contrast, levels of both membrane IgE protein and mRNA coding for membrane epsilon were unaltered on the suppressed AF-10 cells. These data suggest that inhibition of IgE production via Fc epsilon R-II is related to a fall in mRNA for secreted proteins (epsilon and lambda) and probably reflects a post-transcriptional mechanism effecting mRNA for secreted vs membrane protein mRNA.

Antigen-Antibody Complex↗
PubMed1991

Retinoic acid-induced growth inhibition of a human myeloma cell line via down-regulation of IL-6 receptors.

In this report we demonstrate that retinoic acid (RA) down-regulated the number of IL-6R on human leukocyte cell lines, including the myeloma cell line AF10, and two B cell hybridomas that correspond to cells at earlier stages of B cell development. Using AF10 cells, whose growth was determined to be mediated by the autocrine action of IL-6, we found that RA reduction of IL-6R was concentration-dependent over a range of 10(-11) to 10(-5) M and corresponded to the ability of the retinoid to inhibit cell proliferation. The down-regulation of IL-6R number by RA was accompanied by reduced IL-6R mRNA expression. RA did not affect endogeneous IL-6 synthesis or secretion from AF10 cells. However, addition of exogenous rIL-6 could overcome RA-induced growth inhibition. Menthol, a structurally unrelated compound to RA, also suppressed IL-6R expression and, correspondingly, inhibited cell growth. Taken together, our results suggest that the antiproliferative action of RA on AF10 cells is caused by reduction of IL-6R expression and subsequent inhibition of IL-6-mediated autocrine growth. These findings suggest the possibility that down-regulation of IL-6R is a means by which RA can modulate immune function.

Cell Division↗
PubMed1991

CD40 stimulation provides an IFN-gamma-independent and IL-4-dependent differentiation signal directly to human B cells for IgE production.

IgE induction from human cells has generally been considered to be T cell dependent and to require at least two signals: IL-4 stimulation and T cell/B cell interaction. In the present study we report a human system of T cell-independent IgE production from highly purified B cells. When human cells were co-stimulated with a mAb directed against CD40 (mAb G28-5), there was induction of IgE secretion from purified blood and tonsil B cells as well as unfractionated lymphocytes. Anti-CD40 alone failed to induce IgE from blood mononuclear cells or purified B cells. The effect of the combination of anti-CD40 and IL-4 on IgE production was very IgE isotype specific as IgG, IgM, and IgA were not increased. Furthermore, anti-CD40 with IL-5 or PWM did not co-stimulate IgG, IgM, or IgA and in fact strongly inhibited PWM-stimulated IgG, IgM and IgA production from blood or tonsil cells. IgE synthesis induced by anti-CD40 plus IL-4 was IFN-gamma independent as is the in vivo production of IgE in humans; the doses of IFN-gamma that profoundly suppressed IgG synthesis induced by IL-4, or IL-4 plus IL-6, had no inhibitory effect on anti-CD40-induced IgE production. Anti-CD23 and anti-IL-6 also could not block anti-CD40 plus IL-4-induced IgE production, but anti-IL-4 totally blocked their effect. IgE production via CD40 was not due to IL-5, IL-6 or nerve growth factor as none of these synergized with IL-4 to induce IgE synthesis by purified B cells. Finally, we observed that CD40 stimulation alone could enhance IgE production from in vivo-driven IgE-producing cells from patients with very high IgE levels; cells that did not increase IgE production in response to IL-4. Taken together, our data suggest that the signals delivered for IgE production by IL-4 and CD40 stimulation may mimic the pathway for IgE production seen in vivo in human allergic disease.

Antigens, CD↗
PubMed1991

13-cis retinoic acid enhances in vivo B-lymphocyte differentiation in patients with common variable immunodeficiency.

Retinoic acid (RA) has been demonstrated to drive both phenotypic and functional in vitro differentiation of B cell hybridomas from patients with common variable immunodeficiency (CVI) who manifest an "intrinsic" defect in terminal B cell differentiation (J Exp Med 1988;168: 55-71). Therefore, we conducted an open trial to determine the effects of oral 13-cis RA (0.5 mg/kg/day; 12 weeks receiving and 12 weeks without drug) on in vivo B cell differentiation in subjects with CVI. At various times before, during, and after drug administration, patients' B cells were tested for changes in cell-surface phenotype and in vitro immunoglobulin production in response to recombinant cytokines. Before 13-cis RA, all patients had decreased Leu-8 coexpression on CD20+ cells. Seven of eight subjects demonstrated "normalization" of this phenotype after 8 to 16 weeks of 13-cis RA administration. Patients whose B cells demonstrated more than normal CD20 display also had a fall toward normal in this parameter. These effects persisted for 6 to 12 weeks after drug was stopped. It appears that 13-cis RA drives B cells of patients with CVI to express a more differentiated cell-surface phenotype and may promote functional differentiation in some patients.

Administration, Oral↗
PubMed1991

Human immunodeficiency resulting from a maturational arrest of germinal center B cells.

Common variable immunodeficiency (CVI) is an acquired human disorder involving a striking and heterogeneous maturational defect of B lymphocytes. In this study, we used a recently developed VH gene utilization assay to analyze the abundance of developmentally restricted and unrestricted V genes in blood B cells from nine CVI patients. Unrestricted clones (bearing rearranged VH5, VH4, or VH6 genes) were present in normal abundance in this group of CVI patients. However, clones bearing VH3L, a subgroup of the VH3 family normally abundant in blood B cells but absent in B cells at the germinal center stage, were deficient in seven of nine CVI patients. Based on these findings and a reconsideration of previously reported B cell features in CVI, we propose that the disorder represents in most cases a maturational arrest of B cells at the germinal center stage.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1991

Limited B cell repertoire in severe combined immunodeficient mice engrafted with peripheral blood mononuclear cells derived from immunodeficient or normal humans.

The ability to engraft human PBMC or fetal tissue immune cells in the severe combined immunodeficient (SCID) mouse has created a need for characterization of these systems and their application to disease models. We demonstrate that SCID mice reconstituted with PBMC support the growth and differentiation of a restricted set of B cells. Human IgG levels of 1-2 mg/ml (10-20% of normal human serum levels) were routinely achieved in spite of a serum half life of only 12 d. Ig levels peaked around 50 d and Ig production was maintained for greater than 100 d. The Ig was greater than 85% IgG though some IgM, IgA, IgD, and even IgE could be detected. However, the human IgG produced in hu-PBL-SCID mice was pauci-clonal when analyzed by isoelectric focusing and by kappa/lambda light chain usage. Using a new polymerase chain reaction based analysis capable of monitoring individual VH family utilization, we found that the engrafted B cells showed skewed and restricted human VH subfamily utilization. These parameters were markedly variable among hu-PBL-SCID mice reconstituted from the same donor cell population at both early (21-50 d) and late stages (greater than 100 d). Hu-PBL/CVI-SCID mice constructed with cells from patients with common variable immunodeficiency with an in vitro block in terminal B cell differentiation produced human Ig responses that were quantitatively the same as those produced by hu-PBL-SCID mice from normal donors. The hu-PBL-SCID system using PBMC appears to lead to growth and Ig production by a small number of B cells and results in a restricted B cell repertoire.

Animals↗
PubMed1990

Interleukin 6 is essential for antibody secretion by human in vivo antigen-induced lymphoblastoid B cells.

In vivo immunization of normal subjects with a variety of antigens generates circulating lymphoblastoid (LB) B cells, which in vitro spontaneously secrete significant levels of specific antibody. Since activation and initial differentiation of these cells occurs in vivo, they provide a useful model for the study of the later stages of B cell maturation. In the present study, we investigated the requirement of interleukin 6 (IL-6) for the "spontaneous" in vitro production of IgG-Tet by LB B cells. Addition of IL-6 to cultures of LB B cells in medium supplemented with 10% fetal calf serum failed to increase the levels of IgG-Tet produced in vitro. However, addition of anti-IL-6 antibodies decreased IgG-Tet production as much as 70%, and this inhibition could be reversed by the addition of IL-6. LB B cells cultured in serum-free medium in order to restrict endogenous IL-6 production secreted only low levels of antibody, unless exogenous IL-6 was added. Addition of 2.5 units/ml of IL-6 to serum-free cultures induced an increase in IgG-Tet secretion nearly comparable to that seen in cultures supplied with serum. The magnitude of the increase in IgG-Tet secretion in response to exogenous IL-6 was inversely related to the number of cells in culture, which was due in part to increased endogenous IL-6 production in cultures with higher cell concentrations. Experiments including hydroxyurea in serum-free cultures indicated that IL-6-dependent enhancement of LB B cells' IgG-Tet secretion was not primarily mediated by cell growth. These observations suggest that in vivo generated LB B cells are not totally committed to antibody secretion, and that IL-6 is essential for in vivo antigen-induced LB B cells to reach the antibody-secreting stage.

Antibodies↗