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H M Dosch

Publications and source records attributed to H M Dosch.

At least 73 records · Page 4Linked to original sources
PubMed1990

EBV utilizes a unique activation pathway for the transformation of human B cells.

EBV growth-transforms primate B lymphocytes and directly causes mono/multiclonal B cell lymphomas in vulnerable hosts. In this report we demonstrate that the degree of B cell transformability is not quantitatively determined at the level of either the saturable, transformation-prerequisite virus receptors or of the actual viral cell entry process. Instead, post-receptor binding events [Na+/H+ exchange, Ca2+ flux, tyrosine phosphorylation of two proteins (55-60/130-140 kd)] were identified as critical determinants of transformability. The presence of competent virus in transformable cells was per se insufficient for transformation: blockade of Ca2+ fluxes (or the antiport) generates virus-loaded cells that express viral genes but remain untransformed. Delayed induction by ionomycin of appropriately sized Ca2+ fluxes ([Ca2+]i greater than 180 less than 400 nM) re-starts transformation processes in EGTA-blocked, virus-loaded cells, perhaps providing a model for the study of virus re-activation. Overall, EBV induces unique cellular activation events different from non-oncogenic lymphocyte mitogens/activators, and, given the oncogenic potential of transformed cells in susceptible hosts, we hypothesize that these events describe a novel oncogenic transformation pathway.

B-Lymphocytes↗
PubMed1989

Properties and heterogeneity of human fetal pre-B cells transformed by EBV.

A series of 75 EBV-transformed pre-B and B-cell lines from fetal bone marrow at 14 to 18 wk of gestation was cloned for phenotypic, functional and molecular genetic studies. B-cell type volume regulation in response to hypotonic stress, low level CD9 (BA2), and, perhaps biased by our use of EBV, functional EBV receptors with expression of CD21 (B2) determinants characterized the most immature cells detected. These "B-progenitors" contained EBV genomes, maintained Ig H and L chain (as well as TCR) constant region genes in germ-line configuration and did not express other B, T, or myeloid lineage-associated surface markers including CD20 and MHC class II determinants. Such cells may represent B progenitor cells preceding classical pre-B-lymphocytes in pathways of B cell differentiation. Reminiscent of Abelson virus-induced transformation of immature murine B cells, EBV transformability together with the above properties may be the earliest markers of B lineage commitment in man. The expression of MHC class II Ag, CD20, C mu-H and then L chain rearrangements and expression followed in less immature pre-B lymphocytes and permitted a classification of lines into discrete subgroups of increasing maturity. The physical organization of the H chain locus in a given line was a stable characteristic. However, fetal pre-B cell lines showed considerable intraclonal heterogeneity with respect to H chain gene expression and that of differentiation markers such as CD20. Subcloning experiments indicated that this heterogeneity reflected clonally stable expression patterns distributed among subclones in an all-or-none fashion. The induction, by IL-6, of L chain expression in some but not all of these clones was linked to the presence of C mu transcription products, consistent with a possible regulatory role of mu protein in L chain rearrangement and expression. Although pre-B cell differentiation likely follows inherent programming, external signals seem able to hasten development along prescribed, hierarchical differentiation pathways.

B-Lymphocytes↗
PubMed1989

Concerted generation of Ig isotype diversity in human fetal bone marrow.

The human fetal bone marrow B cell compartment of 14- to 21-wk gestational age was examined phenotypically and with respect to Ig H chain commitment and diversity. A dramatic expansion of fetal marrow B cell pools at 16- to 18-wk gestational age characterizes a rapid and concerted chain of differentiation events. Transiently up to 1/4 of nucleated marrow cells are CD20+/CD21+ cells which begin to express surface Ig other than IgM. Limiting dilution analysis of EBV-infected marrow cells delineated a virtually exclusive commitment to IgM production until 15 wk and the absolute and relative number of these cells were small (approximately 5% of comparable adult values). In parallel to the rapid increase in total B cell pools size, cells committed and able to secrete any of the five Ig isotypes are generated by 16-wk gestational age and by 18 wk the frequencies of these cells rapidly reach levels typical for adult peripheral tissue such as blood or lymph node. Fetal L chain diversity always anticipated that observed in adult serum. In addition to rising pool sizes and diverse IgH expression, EBV transformability is a major variable during this period of B cell development with up to 2/3 of B lineage cells transformable, about half of which are pre-B cells. By 21-wk gestational age transformable pre-B cells have disappeared and (as in adult tissue) approximately 10 to 20% of CD20+ cells are transformable. The rapid, concerted expression of full H chain diversity during a narrow period in fetal development is unique to marrow and implies a lymphopoietic process in a privileged site rather than an immunologic differentiation event. During this event, the relative proportions between the different IgH classes expressed, resembled that found in adult tissue, perhaps suggesting that B cell inherent programming rather than only antigenic forces determine heavy chain choice. The staggered expression, early in postnatal life, of IgH regions 3' of the C mu locus may reflect regulatory functions rather than inherent immaturity of the B lineage.

Adult↗
PubMed1989

Rapid amplification of complementary DNA from small amounts of unfractionated RNA.

We have combined, in a rapid and straightforward manner, the synthesis and subsequent amplification of individual cDNA sequences from microgram quantities of unfractionated total RNA. Taq1 polymerase, a thermostable DNA polymerase, and Moloney murine leukemia virus (MMLV) reverse transcriptase share similar buffer conditions; these reactions can be performed sequentially, in a single tube, without the need for purification or changes of buffer after the synthesis of cDNA. In this way, nonspecific losses of material are minimized and the required number of cells is reduced. Cell numbers, particularly from human tissues, can be limiting; the requirement for only small amounts of unfractionated RNA makes possible the isolation and characterization of cDNAs from biological materials available in limited quantities. As a demonstration system, we report the rapid synthesis and amplification of cDNA sequences corresponding to the first exon of human immunoglobulin E (IgE). MMLV reverse transcriptase is used with specific (i.e., IgE) or generic (i.e., oligo-[dT(12-18)]) oligomers to prime first strand cDNA synthesis from unfractionated RNA isolated from a human myeloma line, U-266. The necessary primers, deoxynucleotides and Taq1 polymerase, required for second strand cDNA synthesis and the subsequent logarithmic amplification process, are then added to the reaction mixture. This technique provides a useful means of characterizing expressed and processed gene transcripts.

DNA↗
PubMed1989

Clonal and molecular characteristics of the human IgE-committed B cell subset.

We have followed the pathway of the IgE-committed B lymphocyte from fresh, unstimulated peripheral blood, through EBV activation, transformation, and eventual cloning. Using cell sorting in conjunction with limiting dilution culture systems, we found that: (a) cells that are selected in the cell sorter and secrete IgE in culture are sIgM+/sIgD+. They secrete all three isotypes after EBV activation and continue to do so stably in culture; (b) individual IgE+ cells in culture coproduce IgM, IgD, and IgE and cytoplasmic Ig of each isotype can be detected in single cells; (c) no rearrangement was observed of VDJ to epsilon in any of six lines tested. DNA between the rearranged VDJ-mu and -epsilon appears to be overall intact, including a region 10.5 kb upstream and 18 kb downstream of the 2-kb epsilon coding region and; (d) mRNA of mu and epsilon species is of normal and comparable size. In contrast to IgG- and IgA-producing clones, multiple isotype expression appears to be both frequent and stable in cells committed to IgE production. We propose that IgE-committed cells represent a unique B cell sublineage whose differentiation pathway may be more strictly regulated than that of other isotypes with regard to the signals required for classical, deletional switch recombination that has been observed in rare IgE-producing myeloma cell lines.

B-Lymphocytes↗
PubMed1989

Fluorescence-based viability assay for studies of reactive drug intermediates.

Studies of drug toxicity, toxicologic structure-function relationships, screening of idiosyncratic drug reactions, and a variety of cytotoxic events and cellular functions in immunology and cell biology require the sensitive and rapid processing of often large numbers of cell samples. This report describes the development of a high-sensitivity, high-throughput viability assay based on (a) the carboxyfluorescein derivative 2'-7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) as a vital dye, (b) instrumentation capable of processing multiple small (less than 100 cells) samples, and (c) a 96-well unidirectional vacuum filtration plate. Double staining of cultured peripheral blood mononuclear cells with BCECF and propidium iodide (PI) showed no overlap between PI+ (nonviable) and BCECF+ (viable) cells by flow cytometric analysis. Optimal conditions were developed for dye loading and minimizing physical cell damage and fluorescence quench during the assay procedure. The ratio of BCECF fluorescence to internal standard fluorescent particles was linear from 40 to greater than 20,000 cells with a signal:noise ratio of approximately 3 at 40 cells/well. Sulfamethoxazole hydroxylamine (SMX-HA) was used as a model toxic drug metabolite to explore the validity of the BCECF procedure. SMX-HA, but not its parent compound sulfamethoxazole, resulted in a dose dependent loss of cellular fluorescence and the parallel accumulation of PI+ nonviable cells. When compared to the currently used tetrazolium dye reduction viability assay, the BCECF method was 3-fold more sensitive, greater than 10-fold faster, and required 1/10-1/100 the cell numbers.

Cell Survival↗
PubMed1989

Human IgE response: virus-activated IgE secretors are interleukin-2-dependent cells.

The activation of human IgE-secreting B lymphocytes was difficult until the use of low cell density limiting dilution cultures, which are more efficient and eliminate regulatory growth restraints, became possible. The transformation of IgE-producing cells by Epstein-Barr virus unexpectedly required the presence of small supplements of mature T lymphocytes early in culture. Interleukin 2 (IL2, 10 U/ml) was able to completely replace T-cell supplements when added during a brief (1-2 cell cycles) but critical period, 5-8 days after infection with the virus. The observation that IgE-committed B cells are principally able to express receptors for and respond to IL2 is of experimental utility and implies the possibility that this T-cell product could be part of the complex T-cell controls which govern IgE-committed B cells.

Adult↗
PubMed1988

Extracellular nucleotide catabolism in human B and T lymphocytes. The source of adenosine production.

Extracellular nucleotide degradation was studied in intact human B and T lymphocyte subpopulations and in lymphoblastoid cell lines. Cells of B lymphocyte lineage showed high nucleotide degrading activity, whereas T lymphocytes were unable to degrade extracellular nucleotides. The external surface of B cells contained active sites of ecto-triphosphonucleotidase (ecto-ATPase), ecto-diphosphonucleotidase (ecto-ADPase), and ecto-monophosphonucleotidase (ecto-AMPase). The expression of all three ectoenzyme activities seemed closely associated with B cell development. ATPase and ADPase activities increase continuously during B cell maturation, ecto-AMPase activity, on the other hand, reaches maximal activity in late pre-B cells. These results combined with our previous studies of intracellular ATP catabolism (Barankiewicz, J., and Cohen, A. (1984) J. Biol. Chem. 259, 15178-15181) provide evidence that extracellular ATP catabolism may represent exclusive source for adenosine in lymphocytes. It is suggested that adenosine may serve as a means of communication between B and T cells in lymphoid organs, B lymphocytes being the sole producers of adenosine and T lymphocytes being the recipients of this signal.

Adenosine↗
PubMed1988

Activation of human B lymphocytes by 8' substituted guanosine derivatives.

The 8-substituted guanosine derivatives (8-sGs), mercaptoguanosine and bromoguanosine, are shown to induce immunoglobulin production and proliferation in a subset of normal human B lymphocytes. Limiting dilution analysis indicated that the 8-sG-responsive B cell pool is approximately 10 times larger than that activated by pokeweed mitogen (PWM), 10 times smaller than that activated by Epstein-Barr virus (EBV), and contains approximately equal proportions of cells committed to the expression of IgG, IgA or IgM isotypes. Although some B cells seem able to respond to 8-sGs in the absence of T helper cells, maximal immunoglobulin production is only observed in the presence of T cells. The 8-sG response pattern of human B lymphocytes appeared similar but not identical to that reported for rodent cells. The mitogenic 8-sGs are unique activators as they bypass surface membrane interactions obligatory for other agents including anti-IgM, Staphylococcus aureus particles, PWM, and EBV.

B-Lymphocytes↗
PubMed1987

IgE-enhancing activity directly and selectively affects activated B cells: evidence for a human IgE differentiation factor.

T cells from highly atopic individuals spontaneously secrete in vitro a factor that specifically induces IgE synthesis from normal human B cells. We investigated the effects of such T cell supernatants derived from atopic individuals (TCSN-A) on functionally distinct B cell subsets to determine at what developmental stage B cells become responsive to this IgE-enhancing activity. B cells from normal and allergic donors were separated into subsets of small resting and large activated cells by density centrifugation or unit gravity sedimentation. When stimulated by TCSN-A, large activated B cells made more IgE than small resting B cells. The difference was as much as 3300% in comparing these subsets from allergic donors. Similarly, resting B cells stimulated by Staphylococcus aureus Cowan I (SAC) made 52 to 125% more IgE in response to TCSN-A than unstimulated small resting B cells. However, IgE production from large B cells, already activated in vivo, was not enhanced by the addition of SAC. Notably, the IgE level synthesized by in vivo large activated B cells from allergic persons was markedly greater than that seen with similar cells from normal donors, whereas resting B cells purified from allergic and normal donors produced comparable levels of IgE in response to TCSN-A. These results suggest that this enhancing activity functions as an IgE differentiation factor for activated B cells. This was further confirmed by the effects of TCSN-A on the IgM- and IgE-secreting EBV-transformed human B cell line K1D5. TCSN-A specifically enhanced IgE synthesis from these cells; TCSN from normal donors, IL 2, IFN-gamma, and BCGF did not. These results confirm that this activity functions as an IgE-specific differentiation factor, directly influencing activated B cells to synthesize IgE.

B-Lymphocytes↗
PubMed1986

Phenotype, frequency, and EBV responsiveness of human marrow B and pre-B cells.

We have purified subpopulations of B lineage cells from human adult (rib) bone marrow by cell sorting and panning. Limiting dilution analysis was then used for a clonal analysis of cells able to secrete IgG, IgA, or IgM spontaneously or after infection with EBV. Nonproliferating, high rate IgG or IgA producers occurred at frequencies of about one per 1000 marrow mononuclear cells. Their frequency and Ig production was unaffected by EBV, and they appeared not to express EBNA after exposure to EBV. These cells were Ia+, B1+, and over 85% expressed sIg of the IgM/D (up to 75%) and/or IgG/A isotypes (40 to 60%). B cells committed to the secretion of IgM represent 2 to 10% of marrow B lymphocytes. They were found to be Ia+/B1+/B2+/CALLA- and C3b receptor (CR3)-cells, and most (greater than 90%) required infection with EBV and proliferation to develop into IgM-producing lymphocytes. Thirty to 40% of these cells did not express Ig (H or L chain) on their surface, and therefore resembled pre-B cells at the beginning of the 4- to 5-wk culture period. Proliferating pre-B cells from adult human marrow have been described, but their conversion into IgM-producing cells has not been formally demonstrated. Although EBV induces IgM production, the expression of EBNA, and several rounds of cell division in these cells, the induction of stable (greater than 5 wk) growth transformation represents a rare event in these pre-B cells: in several thousand limiting dilution wells, not a single culture of sIg-cells showed stable growth transformation. The dichotomy between EBV-induced high-rate IgM responses and absent growth transformation discriminates activation and transformation as distinct aspects of EBV-induced B cell "responses", and suggests that cellular properties play critical roles for viral transformation. We propose a model in which cellular target genes for transforming sequences in the EBV genome are transiently expressed during B cell differentiation.

Adult↗
PubMed1986

Limiting dilution analysis of the B cell compartment in human bone marrow.

Mononuclear cells (MNC) obtained from adult rib specimen were investigated for their capacity to produce immunoglobulin (Ig) in vitro. Using limiting dilution analysis 3 populations of B lineage cells could be distinguished. The first produces Ig in culture without any intentional activation. These cells are strictly nonproliferating and sustain extraordinary secretory rates of 5 X 10(7)-10 X 10(7) molecules IgG, IgA or IgM/cell/h for at least 2 weeks. They occur at frequencies of 1 X 10(-4)-10 X 10(-4) (marrow MNC) or represent 1-5% of marrow B cells. Following exposure to infectious Epstein-Barr virus (EBV), these cells do not (a) proliferate, (b) express EBV-determined nuclear antigens (EBNA), (c) enhance their secretory rates or (d) show secretory activity for prolonged time. These cells are therefore EBV resistant. If these cells produced their antibody at similar rates in vivo, then their frequencies would suggest that they could provide up to 2/3 of daily synthetic rates for IgG and IgA and 20-30% of the daily IgM production. The second population of marrow B cells is exclusively committed to IgM production. Following exposure to EBV these cells proliferate, express EBNA and begin to secrete IgM. The third population represents 90% of marrow B cells. In our hands, these cells are unable to produce Ig in vitro.

Antibody-Producing Cells↗
PubMed1986

Epstein-Barr virus-induced IgE production in limiting dilution cultures of normal human B cells.

The induction of in vitro IgE production in human B cells from normal, nonatopic donors has been difficult and somewhat controversial. We report that IgE production is consistently observed in limiting dilution cultures of in vitro Epstein-Barr virus (EBV)-infected normal human B lymphocytes. The frequency of IgE-committed, EBV-responsive cells ranged from 1/810 to 1/10 000 B lymphocytes, and it was similar in peripheral (blood, tonsil) and central (bone marrow) tissue sites. Poisson distribution analysis of these limiting dilution cultures suggested that IgE-committed B cells comprise 0.1-1% of all EBV-responsive B lymphocytes.

B-Lymphocytes↗
PubMed1986

Lymphocyte function in human bone marrow. III. Isotype commitment, metabolic and secretory characteristics of immunoglobulin producing cells.

We have examined the functional and metabolic properties of immunoglobulin (Ig)-secreting cells in adult (rib) bone marrow, the tissue which provides the major proportion of serum Igs. In the absence of polyclonal activators, high rate Ig production (1-2 micrograms/day/10(6) marrow mononuclear cells) was sustained from the beginning of culture throughout 2 weeks and then declined. Ten percent of the Ig secreted was of the IgM isotype and IgG/A made up the remainder at equal proportions. Infection of marrow cells with Epstein-Barr virus (EBV) induced the production of large amounts of IgM, but virtually all IgG/A-committed cells were refractory to stimulation with EBV. Both EBV-induced and the "spontaneous" Ig production was inhibited by cycloheximide, but only EBV-induced IgM production was blocked by hydroxyurea and gamma-irradiation. The polyclonal activators PHA and PWM induce suppressor-T-cell activity in marrow cultures. This suppressor function involves nonproliferating cells which acquire suppressive activity 3-4 days after mitogenic activation. Prednisolone and cyclosporine A modulate Ig production in cultures of peripheral lymphocytes but had no effect on Ig secretion in marrow cell cultures. This observation was reminiscent of the absent or at best marginal short-term effects on in vivo serum Ig levels which is typical for these drugs. Our observations suggest that the marrow Ig-producing B-lymphoid cell compartment shows major differences to other tissue sites with respect to properties of the Ig-secreting cells the immunoregulatory activities able to control their function, and the response of these cells to clinically important drugs.

Adult↗
PubMed1986

Volume regulation of natural killer cells under hypotonic stress: comparison with T and B cell subpopulations.

In response to hypotonic stress, human T and B cells vary in the ability to adjust their volume. Whereas T cells exhibit a regulatory volume decrease (RVD) under these conditions, B cells do not. We studied the ability of peripheral blood-derived natural killer (NK) cells to regulate their volume in this way. Percoll density gradient purified NK subpopulation showed variable RVD which suggested the presence of mixtures of regulatory and non-regulatory cells within these subgroups. Further purification by flow cytometry into Leu 7+ and Leu 11+ cells showed that these NK cells displayed RVD similar to thymocytes but distinct from B cells and more mature T cells. These data support the hypothesis that NK cells may be derived from T cell precursors which, upon differentiation to NK cells, retain the RVD characteristic of pre-T cells. This finding may also be useful in further characterizing neoplastic clones which display NK-like activity but phenotypic heterogeneity.

B-Lymphocytes↗