Dynamics of antigenic membrane sites relating to cell aggregation in Dictyostelium discoideum.

Membrane interaction in aggregating cells of Dictyostelium discoideum can be blocked by univalent antibodies directed against specific membrane sites. Using a quantitative technique for measuring cell association, two classes of target sites for blocking antibodies were distinguished and their developmental dynamics studied. One class of these sites is specific for aggregation-competent cells, their quantity rising from virtually 0-level during growth, with a steep increase shortly before cell aggregation. The serological activity of these structures is species specific; they are not detectable in a nonaggregating mutant, but present in a revertant undergoing normal morphogenesis. Patterns of cell assembly in the presence of antibodies show that selective blockage of these membrane sites abolishes the preference for end-to-end association which is typical for aggregating cells. A second class of target sites is present in comparable quantities in particle fractions from both growth-phase and aggregation-competent cells. Blockage of these sites leads to aggregation patterns in which the side-by-side contacts of aggregating cells are abolished. The target sites of aggregation-inhibiting antibodies are suggested to be identical or associated with the molecular units of the cell membrane that mediate cell-to-cell contacts during aggregation. The results indicate that in one cell, two independent classes of contact sites can be simultaneously active.

Expression of the X-linked agammaglobulinemia gene, btk in B-cell acute lymphoblastic leukemia.

The gene which causes X-linked agammaglobulinemia, btk, has recently been identified as a cytoplasmic tyrosine kinase expressed almost exclusively in B cells, and at all stages of B-cell differentiation. To assess the possibility of involvement of this gene in childhood B-cell malignancies, cells from 23 pediatric patients with B-cell acute lymphoblastic leukemia were examined for expression and alteration of the Btk protein and also for mutations in the btk gene. Btk proteins, similar in both molecular weight and quantity to those seen in unaffected individuals, were detected in whole cell lysates from the blasts of 12/12 patients indicating that no abnormal protein was present. cDNAs from the leukemic blasts of all 23 patients were screened with specific primers covering the coding region of the btk cDNA for mutations using single strand conformation polymorphism (SSCP) analysis. No mutations were found but a nucleotide polymorphism was identified in 4/23 patients at the 3' end of btk. Although the sample size in this study was relatively small, these data suggest that btk does not appear to play a critical role in childhood B-cell leukemias.

Near haploid acute lymphoblastic leukemia: seven new cases and a review of the literature.

Seven new cases are described of near haploid acute lymphoblastic leukemia (ALL) and the findings reviewed together with updated complete remission duration and survival data for the 21 cases already published. The patients were four males and three females, with an age range 2-19 years; all had an immunophenotype consistent with common ALL. The poor prognostic outlook for patients with near haploid ALL is confirmed by the median remission duration of 14 months for these patients, which is comparable to that for the previously published cases. The pattern of chromosome loss was marked particularly by the presence of two copies of chromosomes 10, 14, 18, 21 and both sex chromosomes. Populations of hyperdiploid cells with double the near haploid number were observed in six of the patients, one of whom demonstrated further clonal evolution, and it is proposed that some cases classified as hyperdiploid ALL with greater than 50 chromosomes may also have arisen from a near haploid stem line.

Distribution and epitope analysis of the cell membrane glycoprotein (HPCA-1) associated with human hemopoietic progenitor cells.

Monoclonal antibodies My10, BI.3C5, 12.8, and ICH3 identify a monomeric cell surface glycoprotein (HPCA-1) of 100-120 kD, which is selectively expressed on human hemopoietic progenitor cells. Other tissues are nonreactive with the exception of capillary endothelia and basement membrane in some sites. In addition, the antigen can be detected on cell lines that exhibit characteristics associated with early T cell precursors. HPCA-1 is therefore associated with myeloid, B, and T lineage precursors. Sequential immunoprecipitation and Western blotting studies demonstrate that BI.3C5, ICH3, My10, and an antibody directed against endothelial cells, 188.27, all react with the same glycoprotein species, although the epitopes involved may be distinct. The epitope recognized by BI.3C5 is sialic acid dependent, whereas that recognized by ICH3 is not. The My10 epitope has partial sensitivity to neuraminidase. Competitive/additive binding experiments suggest that these epitopes, although probably distinct, may be closely associated.

Selective expression of class-II MHC antigens during hemopoietic differentiation.

Human multipotential or "mixed" bone marrow colony-forming cells and lineage-restricted colony-forming progenitors have been analyzed by cell sorting (FACS) using a series of monoclonal antibodies. The latter all react with monomorphic class-II MHC glycoprotein determinants but differ in their reactivity or cross-reactivity with products of different loci, i.e., SB (DP), DC (DQ), DR. The results confirm that very few progenitor cells detectably express DC. Anti-DR monoclonals varied in their reactivity with progenitor cells. The majority of progenitors in all lineage categories express determinants that are shared or cross-reactive between DR and SB while fewer progenitors can be shown to bind antibodies specific for DR or SB.

Identification of a membrane glycoprotein associated with haemopoietic progenitor cells.

A monoclonal antibody (3C5) is described which selectively binds to progenitor cell populations in human bone marrow and foetal liver. Mature, lymphoid (T,B) colony forming cells do not express the antigen. The antibody identifies a cell surface glycoprotein of mol. wt approximately 100,000 which might have an important regulatory role in early haemopoietic differentiation.

Recombinant interleukin 2 and anti-Tac influence the growth of enriched multipotent hemopoietic progenitors: proposed hypotheses for different responses in early and late progenitors.

Experiments were designed to evaluate the effect of recombinant IL-2 on growth of hemopoietic precursors from different sources (normal cord blood and bone marrow, and PB from CGL patients). For this purpose, combined cell sorting techniques and multipotent colony forming cell assays were used. A monoclonal antibody BI-3C5, which recognizes an antigen present on early lympho-myeloid cells as well as on all colony forming cells (CFU-GEMM assay), was used to enrich the studied populations. Double colour immunofluorescence techniques were performed to analyse the expression of Tac antigen on early progenitors. The results showed that rIL-2 had a stimulatory effect on growth of enriched progenitors from the three sources and surprisingly that addition of anti-Tac did not abolish this effect. On the contrary, anti-Tac enhanced even more growth of these sorted BI-3C5 precursors, suggesting a ligand action of the antibody. More interestingly, a low percentage of cord cells (1 in 1000) expressed both BI-3C5 and Tac antigens. The vast majority of cells did not concomitantly express both markers. The double labelled cells had a lymphoid-like morphology, high nucleus/cytoplasmic ratio and 2-3 nucleoli. The results will be discussed focusing on early and late "stem" cell growth.

Effect of platelet-derived growth factor on enriched populations of haemopoietic progenitors from patients with chronic myeloid leukaemia.

The effect of pure platelet-derived growth factor and fresh serum on the in-vitro growth of purified haemopoietic progenitors from the peripheral blood of 12 patients with CML was studied. Purified haemopoietic progenitors were prepared using Percoll separation followed by cell sorting with the monoclonal antibody BI.3C5. Both pure PDGF at a concentration of 20 ng/ml and fresh serum significantly increased the numbers of BFU-E (p less than 0.01) and CFU-GEMM (p less than 0.014), but not the CFU-GM. That the PDGF effect was not mediated to any significant extent via prostaglandins, was shown by the lack of inhibitory effect of indomethacin on the growth of purified progenitor cells in the presence of fresh serum. Increased amounts of pure PDGF were required to give maximal stimulation of purified CML peripheral blood progenitors compared to normal bone marrow progenitors. These results show that CML progenitors are capable of responding to PDGF. Whether the quantitative difference in response is due to a reduced proportion of mesenchymal cells in CML peripheral blood compared to normal marrow, or whether CML progenitors are most likely already stimulated by autocrime PDGF or other growth factors remains to be elucidated.

Co-ordinate expression of BI.3C5 and HLA-DR antigens on haemopoietic progenitors from chronic myeloid leukaemia.

Haemopoietic cells isolated from the peripheral blood of patients with chronic myeloid leukaemia (CML), have been extensively purified and enriched using either Percoll density gradients or Percoll density gradients combined with elutriation. The quantitative expression of the BI.3C5 associated antigen and the co-expression of BI.3C5 and HLA-DR antigens on these two populations has been studied using either single or simultaneous two colour FACS sorting, following by in-vitro culture for single and multilineage haemopoietic progenitors thus obtained. The data show that the CFU-GEMM are always found in the most strongly BI.3C5 positive fraction, irrespective of the separation procedure and that the bulk of the CFU-GEMM co-express BI.3C5 and HLA-DR. The cell types initiating these CFU-GEMM are morphologically immature blasts. The more mature cells of the myelomonocytic and erythroid lineages forming single lineage colony types show variable BI.3C5 expression, although most are HLA-DR positive. Such enriched populations of malignant progenitors could provide a useful source of material to study both gene expression and the molecular mechanisms underlying malignant transformation.

JM, a Thy-ALL cell line produces both inhibitory and stimulatory lymphokines for bone marrow progenitor cells.

Normal and malignant T cells as well as T-cell hybridomas have frequently been reported to produce factors which stimulate the growth of committed hemopoietic progenitors. One previous report described a lymphokine produced by a T-cell clone which inhibited hemopoietic progenitor cell proliferation. We now describe the simultaneous production of two activities by a Thy-ALL cell line (JM), a sub-line of Jurkat. Two sets of culture conditions were used: the Fauser & Messner and Iscove's assays. We have been able to separate both inhibitory and stimulatory factors for the growth of multipotent and committed bone marrow progenitors (CFU-GEMM, BFU-E, CFU-E and CFU-GM). The stimulatory factor has an apparent mol. wt of less than 30,000 and the inhibitor an apparent mol. wt of 65-80,000. The growth promoting activity for BFU-E and CFU-GEMM could replace that of phytohemagglutinin stimulated leucocyte conditioned medium (PHA-LCM). We do not know if the production of both activities is due to the malignant phenotype or if there is a normal counterpart to JM that could produce both inhibitory and stimulatory factors.

Effects of cocaine prior to and during bupropion maintenance in cocaine-abusing volunteers.

The effects of cocaine were examined prior to and during bupropion maintenance in nonopioid-dependent cocaine abusers. Prior to bupropion maintenance, subjects underwent an experimental session during which repeated cocaine doses (0, 50, 100 mg/70 kg) were administered intranasally. Then subjects were maintained on bupropion (150 and 300 mg per day) and underwent experimental sessions as before. Cocaine, regardless of bupropion, produced dose-related increases in several stimulant-like self-reports, performance and cardiovascular measures. Bupropion decreased POMS ratings of friendliness and vigor, regardless of cocaine dose. Bupropion enhanced and attenuated cocaine-induced increases in ratings on the LSD and BG subscales of the ARCI, respectively. These results suggest that bupropion does not alter the acute subjective or cardiovascular effects of cocaine in a robust manner.

Studies on the genetic control of antibody affinity: the independent control of antibody levels and affinity in Biozzi mice.

The levels (Abt) and relative affinity (KR) of antibody produced to protein antigens injected in saline have been measured in the 22nd generation of the Biozzi high (Ab/H) and low (Ab/L) antibody-producing mice. No significant differences in affinity were observed between the two lines of mice (p 0.10) but the levels of antibody (Abt) differed significantly (p 0.0025) when immunized with antigen in saline; however, both Ab/H and Ab/L mice were able to mount a high affinity response to protein antigens injected in Freund's complete adjuvant. These results substantiate earlier observations that in mice, antibody affinity (KR) and antibody level (Abt) are under independent genetic control.

The genetic control of antibody affinity in mice.

Random-bred TO mice have been selectively bred into two lines on the basis of the relative affinity (KR) of antibody produced to protein antigens, one line producing high and the other low KR antibody. After four generations of selective breeding the difference in KR between the two lines was highly significant (P less than 0-001). The selection on the basis of KR did not result in a corresponding selection for antibody levels (Abt), which were not significantly different in the two lines. These results indicate that antibody affinity is a genetically controlled parameter of the immune response. Furthermore, this control appears to be expressed by a mechanism which is independent of the amount of antibody produced.

The human leucocyte-common antigen: differential expression of framework and restricted antigenic determinants on early haemopoietic progenitors.

Monoclonal antibodies have been raised against human LC determinants. One, F10.89.4, recognizes a 'framework' epitope on all LC molecules; the other, F8.11.13, recognizes a 'restricted' epitope present on only a subset of these molecules which are found mainly on B and a subpopulation of T cells. A previous study of leukaemias showed that some early lymphoid and myeloid leukaemic cells totally lack LC (35% of ALLs and AMLs are F10.89.4-, F8.11.13-). In contrast, a proportion of myeloid leukaemias carried both 'framework' and 'restricted' epitopes (30% AMLs and AMMLs are F10.89.4+, F8.11.13+). To determine whether comparable heterogeneity exists in normal bone marrow we have analysed LC expression during haemopoiesis, using FACS separated populations and in vitro progenitor assays. Our data show that the great majority of haemopoietic progenitors express the LC 'framework' epitope. These can be separated by size into myeloid (large) and lymphoid (small) progenitor populations. However, very few myeloid progenitors (11% CFU-GM, 6% CFU-GEMM) express the additional 'restricted' LC F8.11.13 epitope. Most F8.11.13+ progenitors are CFU-lymphoid; these generate both T and B lymphocytes, but show a preference for the B lineage. Thus there is some molecular heterogeneity of LC during normal haemopoiesis, but this is far less extensive than that found in leukaemias.

Azathioprine-induced lymphocytic neoplasms of NZB mice lack ecotropic murine leukaemia virus.

NZB mice injected intramuscularly throughout a 6-month period with the immunosuppressant azathioprine (Imuran) developed lymphocytic lymphomas 6--7 months after treatment was initiated. These malignancies were quite distinct from the reticulum-cell neoplasia which occurs spontaneously in the strain, and were readily transplantable to NZB or histocompatible BALB/c recipients. Xenotropic, but not ecotropic murine leukaemia virus (MuLV) was detected in leukaemic tissues of some donor and recipient NZBs when tested in vitro by co-cultivation with permissive cell lines, genome rescue, XC and viral polymerase assays. Virus filtrates prepared from donor leukaemic tissues were non-pathogenic when injected into newborn C3H mice. These results are evidence against a mandatory ecotropic MuLV genome in lymphocytic neoplasia.

The role of low affinity antibody in immune complex disease. The quantity of anti-DNA antibodies in NZB/W F1 hybrid mice.

The level and avidity of anti-DNA antibody in the serum of New Zealand Black/White (NZB/W F1) hybrid mice has been determined. The results show that there is an age and sex-related variation in the avidity of this antibody. In mice of both sexes, the avidity of circulating anti-DNA antibody increases up to 5 months of age; thereafter the avidity falls with increasing age. These variations are more marked in males, but female mice consistently have lower avidity anti-DNA antibody than males. Thus the time of onset, time course and severity of the murine lupus syndrome in NZB/W F1 mice are associated with the presence of increasing levels of low avidity anti-DNA antibody in the serum. These results are discussed in the context of the possible role of low avidity antibody in immune complex disease.

Quantitation of membrane sites in aggregating Dictyostelium cells by use of tritiated univalent antibody.

Cell-to-cell adhesion during aggregation of Dictyostelium discoideum cells is completely blocked by univalent antibody (Fab) directed against two classes of target sites: surface structures characteristic for aggregation-competent cells ("contact sites A") and others present also on growth-phase cells ("contact sites B"). 3 x 10(5) Fab molecules bound per cell are sufficient to block contact sites A completely, although the Fab fragments cover not more than 2% of the total cell surface. Up to 8-fold this value can be bound per cell when Fab fragments of another specificity are used, without affecting activity of contact sites A. Blockage of cell-to-cell adhesion therefore depends on the binding of Fab fragments to specific target sites, rather than on the total number of Fab molecules bound per cell. This conclusion is also valid for cell adhesion attributed to contact sites B. Contact sites therefore represent a special class of cell-surface sites which, in cell homogenates as well as in vivo, can be traced by Fab, and which are not identical with the bulk of cell-surface antigens present on aggregating cells.

A cell line secreting stimulating factors for CFU-GEMM culture.

The multipotent hemopoietic stem cell has fastidious growth requirements in vitro. Traditionally, phytohemagglutinin-stimulated leukocyte conditioned medium has been used to supply the undefined growth factors required for culture of the human multipotent hemopoietic progenitor. We describe the use of medium conditioned by the bladder carcinoma cell line, 5637, to replace PHA-LCM in CFU-GEMM cultures and show that the properties of this conditioned medium closely mimic those of PHA-LCM in two separate CFU-GEMM culture systems.

MHC class I and class I-like gene product expression by malignant T cells: relationships between CD1a, HLA-ABC and beta 2-microglobulin.

Beta 2-microglobulin (beta 2m) forms the invariant light chain of the MHC-encoded HLA-ABC and the non-MHC-encoded CD1 molecules. While HLA-ABC (MHC Class I) molecules are virtually ubiquitous in tissue distribution, CD1 determinants by contrast are more restricted. We have assessed, by indirect immunoenzymeassay, the relative membrane densities of these molecules on malignant thymic and post-thymic T cells. It was found that the T cells of mature post-thymic proliferations expressed significantly more beta 2m-associated protein, predominantly HLA-ABC in nature, than thymic-ALL blasts. This parallels the situation found in normal peripheral T cells and thymocytes. In contrast to post-thymic T cells, thymic-ALL blasts showed considerable case to case variation with respect to non-HLA-associated beta 2m and, of particular interest, not all of this excess beta 2m could be accounted for by CD1a. We therefore conclude that other beta 2m-containing molecules may be expressed on thymic-ALL blasts and possibly also on post-thymic leukaemic T cells. In addition, it was found that T cells from CD4+ cases of post-thymic proliferations expressed more beta 2m-associated determinants than other T cells, whether of either normal or malignant origin, and that certain post-thymic malignancies express significantly increased levels of beta 2m-associated protein relative to normal peripheral T-cells. This is in direct contrast to the situation seen in many solid malignancies.