The histone deacetylase inhibitor sodium butyrate induces DNA topoisomerase II alpha expression and confers hypersensitivity to etoposide in human leukemic cell lines.

The differentiating agent and histone deacetylase inhibitor, sodium butyrate (NaB), was shown previously to cause a transient, 3-17-fold induction of human DNA topoisomerase II alpha (topo II alpha) gene promoter activity and a 2-fold increase in topo II alpha protein early in monocytic differentiation of HL-60 cells. This observation has now been extended to other short chain fatty acids and aromatic butyrate analogues, and evidence is presented that human topo II alpha promoter induction correlates closely with histone H4 acetylation status. Because increased topo II alpha expression is associated with enhanced efficacy of topo II-poisoning antitumor drugs such as etoposide, the hypothesis tested in this report was whether NaB pretreatment could sensitize HL-60 myeloid leukemia and K562 erythroleukemia cells to etoposide-triggered DNA damage and cell death. A 24-72 h NaB treatment (0.4-0.5 mM) induced topo II alpha 2-2.5-fold in both HL-60 and K562 cells and caused a dose-dependent enhancement of etoposidestimulated, protein-linked DNA complexes in both cell lines. At concentrations with minimal effects on cell cycle kinetics (0.4 mM in HL-60; 0.5 mM in K562), NaB pretreatment also modestly enhanced etoposidetriggered apoptosis in HL-60 cells, as determined morphologically after acridine orange/ethidium bromide staining, and substantially increased K562 growth inhibition and poly(ADP-ribose)polymerase cleavage after etoposide exposure. Therefore, a temporal window may exist whereby a differentiating agent may sensitize experimental leukemias to a cytotoxic antitumor agent. These results indicate that histone deacetylase inhibitors should be investigated for etoposide sensitization of other butyrate-responsive hematopoietic and nonhematopoietic tumor lines in vitro and in vivo.

Modulation of human DNA topoisomerase IIalpha function by interaction with 14-3-3epsilon.

Human DNA topoisomerase IIalpha (topo II), a ubiquitous nuclear enzyme, is essential for normal and neoplastic cellular proliferation and survival. Several common anticancer drugs exert their cytotoxic effects through interaction with topo II. In experimental systems, altered topo II expression has been associated with the appearance of drug resistance. This mechanism, however, does not adequately account for clinical cases of resistance to topo II-directed drugs. Modulation by protein-protein interactions represents one mechanism of topo II regulation that has not been extensively defined. Our laboratory has identified 14-3-3epsilon as a topo II-interacting protein. In this study, glutathione S-transferase co-precipitation, affinity column chromatography, and immunoprecipitations confirm the authenticity of these interactions. Three assays evaluate the impact of 14-3-3epsilon on distinct topo II functional properties. Using both a modified alkaline comet assay and a DNA cleavage assay, we demonstrate that 14-3-3epsilon negatively affects the ability of the chemotherapeutic, etoposide, to trap topo II in cleavable complexes with DNA, thereby preventing DNA strand breaks. By electrophoretic mobility shift assay, this appears to be due to reduced DNA binding activity. The association of topo II with 14-3-3 proteins does not extend to all 14-3-3 isoforms. No protein interaction or disruption of topo II function was observed with 14-3-3final sigma.

Galactosylation of serum IgG and autoantibodies in murine models of autoimmune haemolytic anaemia.

A number of systemic autoimmune diseases are associated with increased levels of the agalactosyl (G0) IgG isoforms that lack a terminal galactose from the CH2 domain oligosaccharide. The current aim was to determine whether the galactosylation of serum IgG is also reduced in a classic antibody-mediated, organ-specific autoimmune condition, and whether the pathogenic autoantibodies are preferentially G0. In two murine forms of autoimmune haemolytic anaemia (AIHA), sera and autoantibodies eluted from erythrocytes were obtained, and the levels of G0 measured using a lectin-binding assay. Serum IgG galactosylation was unaffected following the induction of AIHA in CBA/Igb mice by immunization with rat erythrocytes, but in all animals with the disease the IgG autoantibodies generated were more G0 than the sera. The anti-rat erythrocyte antibodies were similar to the autoantibodies in being preferentially G0, and when CBA/Igb mice were immunized with canine erythrocytes as a control foreign antigen, there was again a bias towards the production of G0 IgG antibodies. In NZB mice with chronic, spontaneous AIHA, the concentration and galactosylation of both serum IgG and autoantibodies were lower than in the induced model, and the ratio of G0 IgG in the serum and erythrocyte eluates varied markedly between different individuals. Our interpretation of these results is that changes in serum IgG or autoantibody galactosylation are not consistent in different models of AIHA, and that production of low galactosyl antibodies can be a feature of a normal immune response.

Serial changes in the galactosylation of autoantibodies and serum IgG in autoimmune haemolytic anaemia.

A number of systemic autoimmune diseases are associated with increased levels of the agalactosyl (G0) IgG isoforms that lack a terminal galactose from the C(H)2 domain oligosaccharide. The aims were to determine whether there are also persistently high levels of G0 autoantibodies or serum IgG in autoimmune haemolytic anaemia (AIHA), and whether any changes in galactosylation over time are related to the course of disease. Autoantibodies eluted from red blood cells, and serum IgG, were obtained from a patient with chronic AIHA over a 21 month period, and the degree of galactosylation measured using a lectin-binding assay. There were wide fluctuations in the galactosylation of autoantibody and serum IgG, but these changes were unrelated to the severity of the anaemia. The galactosylation of autoantibody and serum IgG varied independently, and the autoantibodies were preferentially G0 in comparison with serum IgG in only half of the serial samples. We conclude that AIHA differs from other, systemic autoimmune conditions in that high levels of G0 autoantibodies or serum IgG are not persistent, and that changes in galactosylation do not parallel the course of disease.

Agalactosyl IgG in aggregates from the rheumatoid joint.

It has been postulated that agalactosyl immunoglobulin G (IgG) self-associates to form pathological aggregates in the rheumatoid joint. To examine this hypothesis, IgG aggregates from synovial fluid (SF) of 22 patients with RA were prepared by precipitation with polyethylene glycol (PEG) 6000. The PEG precipitates and SFs were reduced with 2-mercaptoethanol (2ME) and bound to protein G. This procedure isolated the IgG in the PEG precipitates from other contaminating glycosylated proteins. The levels of galactose and N-acetylglucosamine (GlcNAc) residues present on the reduced IgG were quantified by their ability to bind the lectins Ricinus communis (RCA)120 and Bandeiraea simplicifolia (BS) II. Proportionally less galactose (expressed as a ratio of bound RCA120 to BS II) was present on the IgG from the PEG precipitates than on the IgG in the paired SF (P = 0.001). However, in many cases more RCA120 as well as BS II bound to IgG from PEG precipitates than from the corresponding SF. It is considered that agalactosyl IgG occurs preferentially in RA SF PEG precipitates and that this IgG may also exhibit increased Fab glycosylation.

The glycosylation of red cell autoantibodies affects their functional activity in vitro.

Factors governing the functional activity of red cell autoantibodies are poorly defined. Here we report the presence of qualitative differences in the glycosylation of IgG autoantibodies which affect in vitro interactions with Fc gamma RIII. The following antibodies were affinity-purified by adsorption and elution from normal red cells: IgG eluted from the red cells of 27 haemolysing or non-haemolysing patients, anti-D in sera from 11 pregnant women, and IgG1 and IgG3 human monoclonal anti-D. Monoclonal antibodies with differing levels of agalactosyl IgG were produced by culturing cell lines at high or low cell density. The % IgG with oligosaccharides lacking terminal galactose residues (agalactosyl IgG) of antibodies was designated as low, medium or high according to their reactivity with a monoclonal antibody to terminal N-acetylglucosamine. Fc gamma RIII-mediated functional activity was assessed by measuring the K-cell-mediated lysis of red cells in eluates diluted to achieve comparable levels of red cells sensitization. All eluates containing allo-anti-D were lytic (range 74-100%). In contrast, lysis by autoantibodies varied from 0 to 100%; 11/13 eluates from red cells of haemolysing patients promoted > 5% lysis compared to 2/7 eluates from red cells of non-haemolysing patients (P < 0.02). The ability of autoantibodies to promote K-cell-mediated red cell lysis correlated inversely with their level of agalactosyl IgG (r = -0.56, P < 0.01, n = 23). Further, monoclonal anti-D antibodies with very low levels of agalactosyl IgG were comparatively more lytic than the same antibodies containing more agalactosyl IgG. Analysis of the ratio of kappa:lambda light chains suggested that autoantibodies from 6/19 patients were monoclonal or oligoclonal in nature. The data indicate that IgG red cell autoantibodies from different patients are functionally heterogenous, and that this may be due, at least in part, to qualitative differences in the Fc region glycosylation reflected by differences in the proportion of agalactosyl IgG. This heterogeneity is consistent with the clonally-restricted nature of the autoantibodies in some patients.

Glial-specific cAMP response of the glial fibrillary acidic protein gene cell lines.

Expression of the rat glial fibrillary acidic protein (GFAP) gene is responsive to the intracellular level of cAMP. We have examined the sequence 5'-upstream of the transcription start site of the rat GFAP-encoding gene to determine the elements responsible for regulating the cAMP response. The RT4 cell lines consist of a neural stem-cell type RT4-AC and its three derivative cell types, one glial-cell type, RT4-D, and two neuronal-cell types, RT4-B and RT4-E. GFAP is expressed in the stem-cell type and the glial-cell type but is not expressed in the neuronal-cell types. Luciferase expression vectors containing various areas of the 10.8-kb region upstream of the transcription start site of the GFAP gene were transiently transfected into these RT4 cells. The effect of cAMP was examined by quantitating the transient expression of luciferase. We found that (i) the 5'-upstream region alone (up to 10.8 kb) allows expression of the GFAP gene in the stem-cell type, the glial-cell type, and a neuronal-cell type; (ii) there are negative and positive cAMP-responsive elements that are juxtaposed within the region between -240 bp and -110 bp upstream and are functional in the stem-cell and glial-cell types but are not functional in the neuronal-cell type RT4-E; (iii) there may be elements that respond to dibutyryl-cAMP in all three RT4 cell types within the region from 2 kb to 10.8 kb upstream of the transcription start site; and (iv) a regulatory luciferase plasmid pRLgfap-1, containing both the upstream and downstream regulatory regions of the GFAP gene, not only expresses luciferase but also responds to forskolin in the stem-cell type and the glial-cell type. This regulatory plasmid, however, does not express in the neuronal-cell type with or without the forskolin treatment.

The functional activity of Fc gamma RII and Fc gamma RIII on subsets of human lymphocytes.

Subsets of human lymphocytes were isolated from peripheral blood using magnetic beads coated with anti-CD4, -CD8, -CD19 or -CD56 antibodies to yield T4, T8, B and natural killer (NK) cell suspensions with greater than 95% purity. The functional activity of Fc gamma receptor II (Fc gamma RII) and Fc gamma receptor III (Fc gamma RIII) on these subsets was assessed by measuring rosette formation with red cells sensitized with known levels of either rabbit IgG or human (monoclonal or polyclonal) IgG1 anti-D, IgG3 anti-D or IgG3 anti-c (E-IgG). Lysis of red cells by K cells (mediated by Fc gamma RIII) in antibody-dependent cell-mediated cytotoxicity (ADCC) assays was promoted by polyclonal and some monoclonal antibodies. Using these 'ADCC+' antibodies, minimum red cell sensitization levels required to promote rosette formation with NK cells were 2000 IgG1 or IgG3 molecules/red cell compared to 15,000 IgG1 or 4000 IgG3 molecules/red cell with 'ADCC-' monoclonal antibodies. The greater efficiency of ADCC+ antibodies is consistent with their previously reported ability to bind Fc gamma RIII via CH2 and CH3 domains whereas ADCC- antibodies bind only via CH3 domains. B cells formed rosettes only at high levels of sensitization: approximately 60,000 IgG1 or 20,000 IgG3 anti-D molecules/cell. These data reflect the low affinity of Fc gamma RII for monomeric human IgG. Although over 90% of NK cells bound anti-CD16, and 70% formed rosettes with red cells sensitized with rabbit IgG (30,000 molecules/cell), only 25% of NK cells formed rosettes with E-IgG3 at 100,000 IgG molecules/cell. Approximately 35% of B cells, 10% of T8 cells but no T4 cells formed rosettes with E-IgG (100,000 IgG3 molecules/cell). With T8, B and NK cells, IgG3 anti-D promoted greater rosette formation than IgG1 anti-D at comparable levels of sensitization. Presumably the longer hinge region of IgG3 enabled it to bridge the gap between negatively charged lymphocytes and red cells more efficiently than IgG1.

Antibody responses to the blood group antigen D in SCID mice reconstituted with human blood mononuclear cells.

Mice with severe combined immunodeficiency (SCID) were reconstituted with peripheral blood mononuclear cells (PBMC) obtained from D-negative individuals who had been sensitized to D-positive erythrocytes. Anti-D was spontaneously secreted in mice reconstituted with PBMC obtained from donors within 14 days of re-immunization with D-positive erythrocytes, but was not detected in murine plasma when mice were reconstituted with PBMC obtained from the same donors many years after sensitization, even though anti-D was still present in the serum of these donors. In the murine plasma the anti-D titres were not related to the total human immunoglobulin concentrations. SCID mice reconstituted with PBMC from some donors immune to D-positive erythrocytes (but not immunized within 34 days of donating the sample) made a recall response to the D antigen, which in some cases was maintained for at least 84 days. Depletion of adherent cells from the reconstituting PBMC reduced the total concentration of human IgG obtained. These results show that SCID mice reconstituted with human PBMC (Hu PBMC-SCID) can make a recall response to the D antigen which cannot be attributed to non-specific polyclonal B-lymphocyte activation, and that efficient antigen processing and presentation of the integral erythrocyte membrane D polypeptide occurs in the Hu PBMC-SCID model.

Failure of blood mononuclear cells from human donors with autoimmune haemolytic anaemia to reconstitute severe combined immunodeficient mice.

The use of severe combined immunodeficient (SCID) mice to study humoral responses by peripheral blood mononuclear cells (PBMC) from patients with autoimmune haemolytic anaemia (AIHA) was assessed. Upon transfer to SCID mice, PBMC from normal donors and patients with autoimmune thyroid disease (AITD) produced substantial levels of immunoglobulin (Ig), detectable in the plasma of recipient SCID mice. In contrast, the majority of PBMC from AIHA donors did not produce Ig in recipient mice. The capacity of PBMC to reconstitute SCID mice was not related to the donor's age. In one case, remission of AIHA allowed the donor's PBMC to successfully reconstitute SCID mice, despite the fact that the donor had developed immune thrombocytopenic purpura (ITP). AIHA PBMC were viable by dye exclusion and contained cells in various states of activation, as judged by their IgG secretion profiles when cultured in vitro. The proportions of leukocytes in AIHA PBMC (T to B cell ratios, CD4+ to CD8+ cell ratios and monocytes) were highly variable compared to non-AIHA PBMC. To determine the effect of abnormal lymphocyte proportions on SCID reconstitution, depletion experiments were carried out on normal and AITD PBMC. This work demonstrated a requirement for high T cell numbers, especially CD4+ cells, and minimal B cell numbers for successful reconstitution. CD8+ depletion of PBMC led to increased levels of Ig production in some instances. It is considered that PBMC from AIHA patients have a defect different from that of other autoimmune disorders, which renders them incapable of reconstituting SCID mice.

Severe combined immunodeficient (SCID) mice: a model for investigating human thyroid autoantibody synthesis.

We have studied the ability of lymphocytes from the blood, thyroid and lymph nodes of patients with autoimmune thyroid disease (AITD) to produce autoantibodies to thyroglobulin (Tg) and/or thyroid peroxidase (TPO) in SCID mice. Human IgG class Tg and/or TPO antibodies were detectable in plasma from SCID mice 7 days after transfer of 15-25 x 10(6) cells/mouse and the highest levels were recorded 2-3 weeks later. In contrast, Tg and/or TPO antibodies were undetectable in recipients of lymphocytes from thyroid antibody negative controls. AITD thyroid lymphocytes produced the most antibody in recipient mice and lower levels were observed in recipients of AITD blood and lymph node lymphocytes. The amounts of Tg and/or TPO antibody detected were in accordance with the ability of thyroid and lymph node lymphocytes to secrete these autoantibodies spontaneously in culture (indicating the presence of cells activated in the patient) and with the capacity of blood lymphocytes (probably B memory cells) to secrete Tg and/or TPO antibodies in culture in response to pokeweed mitogen. Tg antibodies in plasma from SCID recipients of thyroid lymphocytes were of subclasses IgG1, IgG2 and IgG4 and the proportions closely resembled those of the donor's serum Tg antibodies. Blood lymphocytes transferred to SCID recipients were also able to produce Tg antibodies of subclasses 1, 2 and 4 but the subclass distribution varied between mice and the reason for this is not clear at present. Since SCID mice provide an environment in which B lymphocytes from patients with AITD can be activated without mitogen to secrete thyroid antibodies, this model will provide a powerful system for elucidating the mechanisms regulating the secretion of human antibodies to Tg and TPO.

Functional interactions of aglycosylated monoclonal anti-D with Fc gamma RI+ and Fc gamma RIII+ cells.

Four human monoclonal antibodies (mAb) to the Rh antigen D were produced in aglycosylated forms by culture of B-cell lines in medium containing tunicamycin (Tm-mAb). Erythrocytes sensitized with these or control mAb were compared in U937 rosette and monocyte chemiluminescence assays to determine Fc gamma receptor I (Fc gamma RI)-mediated functional activity, and in lymphocyte rosette and lymphocyte antibody-dependent cell-mediated cytotoxicity (ADCC) assays to study Fc gamma RIII-mediated binding and lysis. Fc gamma RI-mediated interactions with Tm-mAb were greatly reduced compared with control mAb. All Tm-mAb failed to promote ADCC, although lymphocyte rosette formation was unaltered. The anti-D titre of Tm-mAb and their interaction with mAb JL512 (recognizing an epitope in the CH2 domain) were unchanged. These data suggest that glycosylation of IgG is required for CH2 domain interactions with both Fc gamma RI and Fc gamma RIII, but not for CH3 domain interactions with Fc gamma RIII.

A rapid solid-phase rosette-inhibition assay for the detection of Fc receptor (FcRII)-blocking alloantibodies [corrected].

A procedure for the detection of FcR-blocking alloantibodies is described which uses human B lymphocytes immobilised on plastic by poly-L-lysine. Antibodies which inhibited rosette formation between B lymphocytes or Daudi cells and ox erythrocytes coated with rabbit antibodies (EA) were detected in 10 out of 10 sera containing anti-HLA A2 antibodies and 3 out of 3 sera containing anti-HLA class II antibodies. Inhibition of rosette formation (EAI activity) was mediated by protein G-separated IgG. Analysis of rosette formation using these 13 sera and lymphocytes from 39 donors revealed that the degree of inhibition was bimodal; most sera were either clearly inhibitory or non-inhibitory in the assay. However, there was no correlation between inhibition of rosette formation (EAI activity) and lymphocytotoxicity. Four pairs of sera showed similar patterns of reactivity (r greater than 0.6, p less than 0.01; 2 x 2 chi-square test), and cells from 2 donors showed antithetical reactions with 12 of 13 sera (r = -0.86, p less than 0.001). These data suggest that the solid-phase rosette inhibition assay is a rapid and reproducible means of detecting antibodies reactive with non-HLA class I or class II antigens on human B cells.

Correlation of serological, quantitative and cell-mediated functional assays of maternal alloantibodies with the severity of haemolytic disease of the newborn.

Serum samples containing IgG red blood cell (RBC) antibodies were collected without reference to clinical information from 131 pregnant alloimmunized women. Anti-D and anti-K were present in sera from 75 and 20 patients respectively. Antibody titres were determined by indirect antiglobulin test (IAGT), anti-D levels were measured by AutoAnalyzer, RBC-binding IgG was quantified using an enzyme-linked immunosorbent assay (SOL-ELISA), and functional activities were measured using the monocyte chemiluminescence (CL) test, antibody-dependent monocyte-mediated and K cell-mediated cytotoxicity (ADCC) assays, and rosette formation with U937 cells. Details of clinical outcomes were obtained retrospectively from 104 pregnancies. Forty-one babies were 'antigen-negative', and of the remainder, four required top-up transfusions, 12 required exchange transfusions, three received intrauterine transfusions, and two died in utero. A comparison of test results with severity of haemolytic disease of the newborn (HDN) showed that, provided sera tested were collected within 8 weeks of the expected delivery date, the CL test and the monocyte-mediated ADCC assay differentiated those D-positive babies which required exchange transfusions from those unaffected or only mildly affected. The usefulness of results from the AutoAnalyzer and IAGT in predicting disease severity was compromised by the wide range of results from mothers of unaffected babies. This variability was less apparent in the SOL-ELISA which predicted severe HDN with greater precision. Results from the U937 rosette assay and the K cell-mediated ADCC assay failed to correlate with disease severity.

Human monoclonal anti-D with reactivity against category DVI cells used in blood grouping and determination of the incidence of the category DVI phenotype in the DU population.

B-lymphoblastoid cell lines transformed by Epstein-Barr virus were produced from cells obtained from a hyperimmunised donor with serum anti-D activity against category DVI red cells and enriched for this activity by rosetting with category DVI red cells. Three clones produced IgG1 anti-D and had stable cell growth and continuous secretion of antibody in prolonged culture. The monoclonal antibodies reacted with category DVI red cells, when assessed manually and in an automated blood grouping system, and are useful blood grouping reagents for the detection of the category DVI phenotype. Using a radiometric technique, the number of antibody molecules bound to category DVI red cells from 5 individuals was estimated to range from 2,800 to 11,200 per cell. Five percent of blood donors classed as Du in the south western region were found to have the category DVI phenotype.

Rosette formation between immobilised human lymphocytes and erythrocytes sensitised with monoclonal anti-D.

A rapid, reproducible and sensitive assay was developed to investigate the ability of human lymphocytes to form rosettes with erythrocytes sensitised with human monoclonal anti-D. Erythrocytes sensitised with a known number of anti-D molecules per cell were incubated with lymphocytes immobilised on plastic by poly(L-lysine), the resulting rosettes fixed, unbound erythrocytes removed by washing and the cell preparation stained. IgG1 and IgG3 anti-D-coated erythrocytes gave similar rosette formation at sensitisation levels in the range of 5000-15,000 molecules per cell, although at lower sensitisation levels IgG3 gave greater rosette formation than IgG1. A minimum of 500 IgG3 and 1000 IgG1 anti-D molecules per erythrocyte were required for rosetting.

Heterogeneity in the ability of IgG1 monoclonal anti-D to promote lymphocyte-mediated red cell lysis.

Thirty-four IgG anti-D human monoclonal antibodies (mAb) derived from 18 donor were assessed for their ability to mediate lysis of D+ red cells by lymphocytes in antibody-dependent cell-mediated cytotoxicity assays. Cell-bound antibody was quantified and the mAb were compared at similar levels of sensitization. The majority (23/31) of IgG1 and all (3/3) IgG3 mAb were ineffective; two donors produced both lytic and non-lytic anti-D mAb. Greater sensitivity was achieved using fluid-phase antibody (as culture supernatants) in the assay than was obtained with pre-sensitized red cells. Minimum levels of 2000 anti-D molecules per cell were required for lysis using pre-sensitized cells. Partial D red cells (DIVa, DVa and DVI) were lysed by three mAb that were lytic with normal D+ cells. There was no relationship between lytic ability and Gm allotype or D epitope specificity of the antibodies. Four mAb to other blood group specificities were tested: two (anti-E and anti-G) were lytic and two (anti-c and anti-Kell) were not lytic. Possible reasons for the heterogeneity of the lytic activity by the mAb are discussed.

An in-vitro assessment of the functional activity of monoclonal anti-D.

The response of human monocytes to red cells sensitized with known levels of monoclonal antibody to the Rh antigen D (anti-D) was compared with that of polyclonal anti-D. Monocyte response was determined by measuring red cell adherence, erythrophagocytosis, monocyte-mediated red cell lysis and luminol-dependent chemiluminescence. By all criteria, monoclonal and polyclonal antibodies showed comparable activity, with IgG3 antibodies promoting a greater monocyte-red cell interaction than IgG1 antibodies. It is suggested that monoclonal anti-D may be effective in the prophylaxis of haemolytic disease of the newborn, providing such material is clinically acceptable.