The B cell antigen CD75 is a cell surface sialytransferase.

In this work we have isolated a cDNA clone encoding the B cell antigen CD75. The amino acid sequence of CD75 is shown to be identical to that of human alpha 2,6 sialyltransferase, believed to be primarily associated with the Golgi complex. This is the first demonstration of cell surface expression of sialytransferase which, in B cells, may play an important role in intercellular adhesion and antigen presentation events.

Interleukin 4 induces selective production of interleukin 6 from normal human B lymphocytes.

In this paper we have shown that extensively purified human B lymphocytes respond to IL-4 treatment with a marked production of IL-6. Addition of anti-mu potentiated the effect of IL-4 on IL-6 production. Other cytokines tested like TNF-alpha and-beta, IFN-gamma, IL-1, IL-2, and IL-5 did not induce IL-6 secretion when given to resting B cells. Although B cells generally also produced TNF-alpha and TNF-beta upon stimulation, IL-4 did not induce TNF secretion and seemingly had a specific effect on IL-6 production.

DNA replication in Physarum polycephalum: electron microscopic analysis of patterns of DNA replication in the presence of cycloheximide.

DNA from synchronously replicating nuclei of Physarum polycephalum was studied electron microscopically after 15, 30, 60, and 90 or 120 min of replication in the presence or absence of the protein synthesis inhibitor cycloheximide. The replication-loop size-distribution showed that replication fork progression is severely retarded in the presence of cycloheximide. Analysis of replication-loop frequency showed a similar pattern in control and cyclo-heximide-treated samples, with an increase from 15 to 30 and 60 min. This suggests, surprisingly, that initiations of new replicons either may not be inhibited by cycloheximide or, alternatively, that all initiations have already taken place at the very start of S-phase. The latter conclusion is favored in the light of previous results in our laboratory, discussed here.

Cell cycle-specific expression and nuclear binding of DNA polymerase alpha.

The expression and distribution of DNA polymerase alpha was measured by cytometry and confocal laser scanning microscopy. Expression was proportional to DNA content in proliferating cells, while only S-phase cells retained DNA polymerase alpha after detergent extraction. Nuclear DNA polymerase alpha binding may be one of the key events of S-phase entry.

New indirect approach to the therapeutic use of immunotoxins.

To improve the applicability of immunotoxins (ITs), we have developed a new two-step indirect procedure. The target cells to be killed are first incubated with cell-specific mouse monoclonal antibodies (MAbs). After removal of excess unbound antibody, the cells are incubated in the presence of lactose with an indirect IT, a conjugate of whole abrin and sheep anti-mouse immunoglobulin (SAM) that binds only to cells having primary mouse MAbs on their surfaces. The SAM-abrin IT is affinity purified before use to remove molecules with exposed B-chain-binding sites; it was nontoxic in the absence of the specific mouse MAbs, demonstrating the specificity of the two-step method. We compared the indirect approach, using four different primary MAbs, with the conventional method, in which abrin is coupled directly to the mouse MAbs. In three human cell lines--the melanoma line FEMX, the Burkitt cell line Rael, and the leukemia cell line KM3--the cell kill, measured by a clonogenic assay, was consistently greater with the indirect than with the direct method. In the melanoma and Rael cells, the indirect method gave a higher cell kill than even native abrin. With a mixture of two different antibodies an additive effect was observed with the indirect but not with the direct method. The new approach greatly simplifies the therapeutic application in vitro of ITs, because it permits the use of different primary antibodies, singly or in mixtures, in conjunction with only one or a few general indirect ITs. In efforts to further improve the usefulness of the indirect method, other indirect ITs containing different toxin moieties are being examined. The possibility of employing the indirect principle in vivo is being explored.

Successful clinical use of an anti-HLA-DR monoclonal antibody for autologous bone marrow transplantation.

It has been widely assumed that anti-HLA-DR antibodies react with pluripotent stem cells and cannot be used in bone marrow purging. We report a case of non-Hodgkin's lymphoma in which an anti-HLA-DR antibody (AB4) was used for immunomagnetic purging and the subsequent autologous bone marrow transplantation resulted in rapid marrow engraftment with no serious complications. The results indicate that the AB4 antibody, which binds to an antigen encoded by the B3 gene of the DR region, can be safely used in the clinic in the purging of bone marrow from patients with AB4-positive tumors (non-T-cell acute lymphocytic leukemia, non-Hodgkin's lymphoma, and some cases of acute myelogenous leukemia.

Elimination of B-lymphoma cells from human bone marrow: model experiments using monodisperse magnetic particles coated with primary monoclonal antibodies.

Conditions for removing B-lymphoma cells from human bone marrow using "immunobeads" (IBs) were investigated. The IBs were prepared by coupling monoclonal antibodies directly to a new type of monodisperse magnetic polymer particles (M 450). Two monoclonal immunoglobulin M antibodies, AB-1 (CD 19), a B-cell-specific antibody, and AB-4, an HLA-DR-specific antibody, were used. The IBs were incubated with Rael Burkitt lymphoma cells admixed to fresh, mononuclear human bone marrow cells. After incubation for 30 min at 4 degrees C, the IBs were removed using cobalt samarium magnets. The number of remaining clonogenic tumor cells was assayed by the Courtenay and Mills soft agar procedure, and the clonogenic capacity of the bone marrow progenitor cells was measured by granulocyte-monocyte and granulocyte-erythroid-monocyte-megakaryocyte assays. With a ratio of tumor cells to normal bone marrow cells of 0.1 or 0.01 and a ratio of immunobeads to tumor cells in excess of 75, a tumor cell depletion of more than 3 logs was achieved with the AB-4 IBs and slightly less with the AB-1 beads. After two consecutive cycles of purification with the AB-4 beads, no colonies were found, corresponding to more than 6 logs of purification. In the case of the AB-1 beads, 4 to 5 logs of purification were achieved. The concomitant reduction in clonogenic bone marrow progenitor cells was only 30 to 40%. Flow cytometric studies showed that the tumor cell population contained appreciable proportions of cells binding only small amounts of the antibodies used. The results indicate that the IB procedure is highly efficient and capable of removing tumor cells expressing low levels of antigen. Compared to other purging methods in use the procedure described seems to offer several advantages with respect to efficacy, speed, and simplicity. By the use of a panel of suitable antibodies the new immunobead procedure may be potentially useful in autologous bone marrow transplantation of B-lymphomas and non-T-leukemias with poor prognosis.

Distinct effect of forskolin and interferon-gamma on cell proliferation and regulation of histocompatibility antigen expression in hematopoietic cells.

The adenylate cyclase activator, forskolin, was found to induce expression of class I and class II major histocompatibility complex antigens in a B precursor cell line, Reh, as well as in a B lymphoid cell line, Raji. No such effect was, however, observed when the promyelocytic cells line HL-60 was treated with either forskolin or the cAMP analogue 8-bromoadenosine cyclic monophosphate. As expected, all three cell lines showed reduced proliferation upon forskolin treatment. Forskolin induced expression of class I and class II major histocompatibility complex antigens in cell lines not affected by interferon-gamma and vice versa, indicating that cAMP is not involved in the regulation of histocompatibility antigens by interferon-gamma. We also compared the effect of interferon-gamma and 12-O-tetradecanoylphorbol 13-acetate on major histocompatibility complex class I and class II expression, and despite differences in the response on the tested cell lines, we can not at this point exclude the possibility that protein kinase C is involved in the action of interferon-gamma.

Characterization of a promoter region supporting transcription of a novel human beta-galactoside alpha-2,6-sialyltransferase transcript in HepG2 cells.

In humans, two transcripts encoding beta-galactoside alpha-2,6-sialytransferase (EC 2.4.99.1.) have previously been described. One of the transcripts is widely expressed, whereas the other is restricted to mature B-cells. In this study we demonstrate the existence of a third transcript in the hepatoma cell-line HepG2. The expression of this transcript is controlled by a promoter region which efficiently supports transcription in HepG2 cells, and which harbours putative binding sites for liver-enriched and acute phase inducible transcription factors.

Isolation and characterization of human hematopoietic progenitor cells: an effective method for positive selection of CD34+ cells.

Immunomagnetic beads are well suited for positive selection of CD34+ cells. However, both unspecific binding of beads to cells as well as the effectiveness of detachment of beads from cells may represent significant problems. We used an anti-Fab antiserum (DETACHaBEAD, Dynal) for rapid and effective detachment of immunomagnetic beads from the positively selected cells. By this detachment technique, the cells remained phenotypically unaltered. To reduce unspecific binding, we have coated various anti-CD34 monoclonal antibodies directly to paramagnetic beads M450 (Dynal). Use of beads coated with BI-3C5 was found to be optimal with regard to yield and purity of the isolated cells. The yield was on average 1.5% (range 0.5-2.5%) of bone marrow mononuclear cells and the purity was usually greater than 95% CD34+ cells of the isolated cells. Subpopulations of the cells expressed myeloid markers (CD13, CD33, and to a lesser extent CD15 and CD14) or early B-lineage markers (CD19 and CD10). Most of the cells expressed CD38, and a majority of the cells also expressed CD41. In general, most of the CD34+ cells with low forward scatter expressed B-lineage markers, as was also the case for the few contaminating CD34- cells which were found to be predominantly CD37+ mature B cells. Reactivity with antibodies against T-lineage markers (CD2, CD3, CD4, CD7, and CD8) was generally detected only on 1-2% of the cells or less. Isolated cells responded to interleukin 3, granulocyte-macrophage colony-stimulating factor, mast cell growth factor, and/or granulocyte colony-stimulating factor alone or in combinations in short-term liquid cultures. The cells were also markedly enriched for granulocyte-macrophage colony-forming units as well as for early progenitor cells capable of forming blast colonies on preformed stromal feeder layers. Moreover, the CD34- population was depleted of 70-80% of CFU-GM and cells capable of blast colony formation. Thus, we conclude that the isolated cells are phenotypically unaltered after isolation, and show a normal response in various in vitro assays.

Immunomagnetic isolation of NK and LAK cells.

The present study describes the immunomagnetic isolation of human natural killer (NK) and lymphokine activated killer (LAK) cells. Antibodies against CD56 and sheep anti-mouse IgG-coated magnetic monodisperse particles (Dynabeads M-450) were used for the positive isolation of CD56+ cells from unstimulated mononuclear cells (PBMC). A highly enriched population of CD56+ cells (less than or equal to 3% contaminating cells) was obtained with this method. The cellular yield of CD56+ cells was high (5.3% of the unseparated PBMC). The CD56+ cells remained unactivated after separation and preserved their functional characteristics, as measured by cytotoxic activity against the NK sensitive K562 cells. Incubating the CD56+ cells with IL-2 resulted in high LAK activity, as measured by cytotoxic activity against Daudi cells. Large numbers of functionally active CD56+ cells were obtained from IL-2 stimulated lymphocytes using anti-CD56 coated Dynabeads 450. A further enrichment of effector cells with LAK activity was accomplished by depleting the CD56+ cells for T-cells by anti-CD3 coated Dynabeads M450. The immunomagnetic isolation technique described was easy to perform, did not require expensive equipment and yielded NK and LAK cells of satisfactory purity.

A new method for detachment of Dynabeads from positively selected B lymphocytes.

This paper describes a method for the detachment of immunomagnetic beads from positively selected human B lymphocytes. After rosetting of B cells using anti-CD19 coated magnetic beads (Dynabeads M-450 Pan B, Dynal), the Dynabeads were rapidly detached (efficiency 80%) from the cells using goat anti-mouse-Fab antiserum (DETACHaBEAD, Dynal) at ambient temperature. Isolated B cells did not show significant differences in the expression of a number of B cell antigens when compared to B cells stained in fresh whole blood. In contrast, positively selected B cells that had detached from the beads following overnight incubation, demonstrated a significantly reduced expression of certain of the antigens examined (CD19, CD20 and CD23). It was further demonstrated that neither anti-CD19 nor anti-Fab resided on the surface of the cells after detachment. The cells were still in G0 phase (greater than 90%) at the end of the isolation procedure. Moreover, anti-IgM antibodies stimulated the vast majority of the cells to leave the G0 phase, and to progress through S phase in the presence of growth factors. The cells could also be stimulated to differentiate, further confirming the normal functional capacity of the isolated cells. The method described in this paper can also be used for the detachment of other positively selected cells, such as CD4+ T cells, CD8+ T cells and CD34+ stem cells.

Solid-phase method for differential display of genes expressed in hematopoietic stem cells.

A solid-phase differential display method was designed to analyze differential gene expression in samples with low amounts of mRNA. The principle was based on using a biotinylated probe to capture the mRNA and priming both the first-strand synthesis and the subsequent polymerase chain reaction step. Coupling the mRNA to a solid phase during the procedure simplified the purification steps, limited sample loss and enabled rapid handling of mRNA. DNA contamination was also minimized when the mRNA was bound to a solid phase. Optimization of the differential display method was achieved by analyzing both the enzymatic conditions and the required cell amounts. The approach was used for the characterization of genes expressed in the most immature hematopoietic progenitor cells (CD34+CD38-). The majority of the differentially expressed fragments represented previously uncharacterized sequences.

Immunomagnetic removal of B-lymphoma cells using a novel mono-sized magnetizable polymer bead, M-280, in conjunction with primary IgM and IgG antibodies.

The efficacy of a novel monosized and magnetizable polymer bead, denoted M-280, in immunomagnetic removal of Rael B-lymphoma cells was compared with that of M-450 beads, previously used in bone marrow purging. The M-280 beads which are smaller (diameter 2.8 microns) and contain less iron than the M-450 beads were coated with polyclonal IgG sheep antimouse (SAM) antibody. The two types of immunobeads were equally efficacious when used together with the mouse monoclonal IgG antibodies HH1 or FN1, giving tumor cell depletions of about 3 logs in one cycle of operation. However, when used together with the primary IgM monoclonal antibodies (MoAbs) AB1 or HH2, the new immunobeads were significantly more efficacious than the M-450 immunobeads. To elucidate the underlying mechanism flow cytometric studies and measurements of the binding of the labeled primary MoAbs to the cellular antigens as well as to the immunobeads were carried out. Competition experiments showed that in the case of IgG MoAbs, the SAM beads bind predominantly to the Fc portion, whereas in the case of the IgM MoAbs, the Fab part plays a relatively greater role in the binding. The results indicate that if M-280 immunobeads are used, IgM MoAbs may profitably be included in antibody cocktails together with IgG antibodies in immunomagnetic purging of B-lymphoma cells. They suggest that in the case of cell bound MoAbs, the epitopes on IgG are more accessible to SAM beads than those of surface bound IgM molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunomagnetic removal of B-lymphoma cells from human bone marrow: a procedure for clinical use.

B-lymphoma cells were purged from human bone marrow by incubating the cell suspension with a cocktail of three different pan-B cell mouse IgG1 monoclonal antibodies, and then with immunobeads charged with sheep anti-mouse antibody, followed by magnetic separation. The primary antibodies used, HD37 (CD19), HD6 (CD22), and HH1 (CD37), bind to a very high percentage of the cells in non-Hodgkin's lymphomas of poor prognosis. The secondary antibody is directed against the Fc portion of the IgG antibodies. In model experiments Burkitt's lymphoma cells (Rael) were admixed to mononuclear bone marrow cells in the ratio 1/9. With a ratio of immunobeads/total antibody-binding B cells of 50/1 in a first treatment cycle and repeating the procedure with the same number of beads in a second cycle, a tumor cell depletion of more than 5 logs was achieved, as judged by a clonogenic assay. The concomitant reduction of CFU-GM and CFU-GEMM was about 20%. The purging procedure has been scaled up to clinical use. Equipment suitable for purging patients' marrow specimens, employing standard transfusion facilities, is described. With this equipment the efficacy of tumor cell removal was the same as in the model experiments, and the whole magnetic separation could be completed in 2 hours.

Complement-mediated permeabilization of platelets by monoclonal antibodies to CD9: inhibition by leupeptin, and effects on the GP Ib-actin-binding protein system.

Two monoclonal antibodies to CD9 of the IgM and IgG2a categories (FN 52 and FN 99), reproducibly induced platelet alterations in platelet-rich plasma by activation of the complement system with membrane incorporation of the pore-forming C5b-9 complex. The permeabilization could be monitored by measurements of extracellular ATP and observed as a shape change followed by an increase in light transmission in the aggregometer, and was associated with formation of tiny platelet aggregates. This could be accomplished by only minor lysis observed as extracellular lactate dehydrogenase (LDH). When leupeptin was added prior to, or immediately after the antibody, a total inhibition of the platelet alterations could be obtained. When added soon after the shape change, leupeptin had little effect on the liberation of ATP. However, whereas the ability of the platelets to become agglutinated by ristocetin was lost during the complement-mediated platelet alterations, addition of leupeptin immediately after the shape change, prevented this loss. The lost ability of the permeabilized platelets to undergo ristocetin-induced agglutination is not ascribed to degradation of GP Ib as this was relatively little affected in these studies as compared to the actin-binding protein (ABP) which was profoundly degraded. This protein represents a link between GP Ib and the submembraneous cytoskeleton, and the inhibition of its degradation by leupeptin, was clearly demonstrated. Experiments with digitonin-induced permeabilization showed that leupeptin did not inhibit permeabilization as such, but it did prevent the loss of ristocetin-induced agglutination even with this inducer.

CDw75 antigen expression in breast lesions.

Alterations on the cell surface of the oligosaccharide portion of glycoproteins and glycolipids are thought to play a role in tumorigenesis. Sialyltransferase catalyzes the incorporation of sialic acid to the carbohydrate group of glycoconjugates. Sialyltransferase has been found elevated in different tumour tissues and in the serum of cancer patients. In the present study we have examined the expression of the beta-galactosyl alpha 2,6-sialyltransferase requiring epitope CDw75, with the monoclonal antibody HH2. 142 breast lesions were included. 21% of the carcinomas in situ and 35% of the invasive carcinomas showed a diffuse cytoplasmic staining. Seven cases of invasive carcinomas also showed a distinct membrane immunoreactivity. We found no correlation between reactivity for CDw75 in malignant lesions and their metastatic potential. Only five out of 11 primary tumours with metastases expressed CDw75 in the primary tumour. In the benign lesions, there was a positive reaction in proliferating lesions, e.g. intraductal papillomas (2 out of 3 cases) and in epithelial proliferations in fibrocystic disease (10 out of 14 cases). None of the four fibroadenomas and phyllodes tumours and only one out of 22 cases of normal breast tissues showed immunoreactivity for HH2. In the malignant lesions, CDw75 was more frequently expressed in the carcinomas of high malignancy grade. The high frequency of immunoreactivity among the benign breast lesions can be indicative of activation of the epithelial cells.

DNA replication in Physarum polycephalum: characterization of DNA replication products made in vivo in the presence of cycloheximide in strains sensitive and resistant to cycloheximide.

Synchronous plasmodia of cycloheximide-sensitive and cycloheximide-resistant strains of Physarum polycephalum were labelled with 3[H]-deoxyadenosine in pulse and pulse-chase experiments in presence and absence of cycloheximide. The replication products were studied with alkaline sucrose gradient sedimentation analysis. We show that the action of cycloheximide on DNA replication in Physarum is mediated through the ribosome, since the ribosomally located resistance also makes the plasmodial DNA replication refractile to the action of cycloheximide. Cycloheximide caused inhibition of three stages in DNA replication in the wild type: first, the formation of primary replication units ("Okazaki" size fragments), secondly, the ligation of primary units into secondary ("Replicon" size) units and thirdly, the ligation of secondary units into mature DNA.