Spontaneous cytotoxicity mediated by invertebrate mononuclear cells toward normal and malignant vertebrate targets: Inhibition by defined mono- and disaccharides

Spontaneous non-antigen-dependent cytotoxicity is displayed in vitro by mononuclear cells from molluscs, annelids, and echinoderms. The cytotoxic potential of these cells appears to be independent of prior antigenic exposure, is easily demonstrated in vitro, and is temperature dependent. The specificity of these cells may be directed at cell-surface glycoproteins on the target cell surface since a variety of defined mono- and disaccharides can block killing. The ability of sugars to block is target cell and effector cell specific. This finding is exactly analogous to our previous finding that human spontaneous monocyte-mediated cytotoxicity is blocked in a target-specific fashion by different mono- and disaccharides. These data suggest that invertebrate as well as vertebrate mononuclear cells may “recognize” targets through a series of sugar-specific “lectin-like” molecules present on the effector cell surface.     

The role of CD18 in phorbol ester-induced human monocyte-mediated cytotoxicity

Monocyte cell surface molecules play an important role in the regulation of monocyte function. To investigate the molecular basis of monocyte-mediated cytotoxicity, we tested the ability of a variety of mediators to stimulate human monocyte-mediated cytotoxicity. Phorbol myristic acetate (PMA) stimulated significant monocyte-mediated killing of tumor cells in an 18-hr indium-111 release assay. Five other cytoactive substances did not induce monocyte-mediated cytotoxicity. The acquisition of monocyte cytotoxicity was associated with nearly a twofold increase in surface expression of three CD18-bearing cell surface molecules (CD11a, CD11b, CD11c). The direct involvement of the CD18-bearing molecules in monocyte-mediated cytotoxicity was investigated using monoclonal antibody (MAb) inhibition. MAb recognizing the CD18 subunit significantly inhibited monocyte-mediated killing. The inhibition by anti-CD18 MAb could not be attributed to LFA-1 (CD11a) alone, suggesting that CR3 (CD11b) and p150,95 (CD11c) may also participate in monocyte-mediated cytotoxicity. In contrast, seven of eight other cell surface structures were not affected by PMA treatment, and MAb to all eight cell surface structures did not inhibit killing. These findings suggest that CD18-bearing molecules are upregulated with monocyte activation and may play a functional role in monocyte-mediated killing.     

The in vivo and in vitro substrate specificity of quinoprotein glucose dehydrogenase of Acinetobacter calcoaceticus LMD79.41

Quinoprotein glucose dehydrogenase (GDH; EC 1.1.99.17) was partially purified from cell-free extracts of Acinetobacter calcoaceticus LMD79.41. The enzyme oxidized monosaccharides (d-glucose, d-allose, 2-deoxy-d-glucose, d-galactose, d-mannose, d-xylose, d-ribose and l-arabinose) as well as disaccharides (d-lactose, d-maltose and d-cellobiose).Intact cells of A. calcoaceticus LMD79.41 also oxidized these monosaccharides, but not the disaccharides.The difference in substrate specificity can not be explained by impermeability of the outer membrane for disaccharides, since right-side-out membrane vesicles did not oxidize disaccharides either. Destruction of the cytoplasmic membrane strongly affected the catalytic properties of GDH. Not only did the affinity towards some monosaccharides change substantially, but disaccharides also became good substrates upon solubilization of the enzyme. Thus, at least in A. calcoaceticus LMD79.41, the oxidation of disaccharides by GDH can be considered as an in vitro ‘artefact’ caused by the removal of the enzyme from its natural environment.     

Chemical synthesis and immunological activities of glycolipids structurally related to lipid A

Complete chemical syntheses of a number of monosaccharides derived from 2-deoxy-2-[(3R)-3-hydroxytetradecanamido]-D-glucopyranose and structurally related to the hydrophobic moiety (lipid A) of several bacterial endotoxins are described. Selected humoral (complement activation) and cellular (mitogenicity and induction of interleukin 1 production) in vitro activities of a lipid A preparation obtained from the Bordetella pertussis endotoxin were compared with those of ten of these monosaccharides and with those of previously synthesized, analogous disaccharides. Results show that each of these in vitro activities of the lipid A preparation can be efficiently induced by at least one of the monosaccharide derivatives.     

Suppression of cell-mediated immune reactions by monosaccharides.

Various monosaccharides have been shown capable of inhibiting lymphokine activity in in vitro assay systems. In this study, we demonstrate that L-fucose can inhibit the ability of lymphokine-containing supernatants to induce skin reactions or cause reductions in the macrophage content of peritoneal exudates. Moreover, L-fucose can inhibit the cutaneous delayed hyoersensitivity reaction and the peritoneal macrophage disappearance reaction (MDR) induced by antigen in actively immunized guinea pigs. The effect of L-rhamnose, another sugar with in vitro inhibitory activity, was investigated in the MDR, and was also shown to be inhibitory. L-arabinose, which has no in vitro effects on lymphokines, had no suppressive effect on any of the in vivo systems studied. No monosaccharide inhibition of Arthus reactions or nonspecific inflammation could be found in these studies. The results demonstrate that monosaccharides capable of inhibiting lymphokine activity in vitro are effective in suppressing in vivo manifestations of cellular immunity.     

A manual method for reducing sugar determinations with 2,2′-bicinchoninate reagent

A method was developed to analyze reducing sugars manually by use of a 2,2′-bicinchoninate reagent. Potassium phosphate was used to buffer the reagent. Ethylene glycol increased the sensitivity of the assay for several sugars. Sugar determinations can be performed in the presence of borate ion. In comparative assays of a number of monosaccharides and disaccharides, the bicinchoninate reagent was about as sensitive as the Nelson reagent but more convenient to use. The effects of ethylene glycol concentration, reagent pH, heating time, and borate ion on color development were evaluated.     

Lipopolysaccharide-induced immunomodulation of the generation of cell-mediated cytotoxicity. I. Suppression of the development of cytotoxic lymphocytes

Bacterial lipopolysaccharide from a variety of Gram-negative organisms suppresses the development of cytotoxic killer cells in the murine MLC. Cytotoxic T lymphocytes were generated in vitro by incubating BALB/c responder spleen cells with irradiated C57BL/6 stimulator cells for 5 days in mixed lymphocyte culture (MLC). The addition of LPS at the initiation of MLC suppressed killing of 51Cr-labeled target cells in a dose-dependent manner. LPS was active only during the afferent phase of CMC, since it did not interfere with the efferent phase of the assay. Furthermore, timed addition and timed removal studies suggested that the presence of LPS during the first 48 hr of MLC was critical for maximal suppression of CMC. Lipid A extracted from LPS, which had been shown to be highly suppressive when added to the sensitization phase of the CMC assay, was also inhibitory. Moreover, when LPS was added to MLC in the presence of tritiated thymidine, the proliferative activity of the responder cells increased markedly after 72 hr of culture. These data suggest that LPS, a known B cell mitogen, can modulate the complex sequence of cellular interactions that leads to the generation of cell-mediated cytotoxicity.     

Dendrons with spermine surface groups as potential building blocks for nonviral vectors in gene therapy

This paper investigates a series of dendrons based on the Newkome dendritic scaffold that displays a naturally occurring polyamine (spermine) on their surface. These dendrons have previously been shown to interact with DNA in a generation dependent manner with the more highly branched dendrons exhibiting a strong multivalency effect for the spermine surface groups. In this paper, we investigate the ability of these dendrons to transfect DNA into cells (human breast carcinoma cells, MDA-MB-231, and murine myoblast cells, C2C12) as determined by the luciferase assay. Although the dendrons are unable to transfect DNA in their own right, they are capable of delivering DNA in vitro when administered with chloroquine, which assists with escape from endocytic vesicles. The cytotoxicity of the dendrons was determined using the XTT assay, and it was shown that the dendrons were nontoxic either alone or in the presence of DNA. However, when administered with DNA and chloroquine, the most highly branched dendron did exhibit some cytotoxicity. This paper elucidates the relationship between in vitro transfection efficiency and toxicity. While transfection efficiencies are modest, the low toxicity of the dendrons, both in their own right, and in the presence of DNA, provides encouragement that this type of building block, which has a relatively high affinity for DNA, will provide a useful starting point for the further synthetic development of more effective gene transfection agents.     

Monocyte- and natural killer cell-mediated spontaneous cytotoxicity against human noncultured solid tumor cells

Unstimulated human peripheral blood mononuclear cells from healthy donors exhibited spontaneous cytotoxicity against noncultured solid tumor targets in a 12- to 24-hr 51Cr release or 111In release assay. Both purified monocytes (greater than 99% monocytes) and natural killer (NK)-enriched lymphocytes exhibited comparable levels of spontaneous cytotoxicity against fresh melanoma tumor targets. This cytotoxicity was observed under endotoxin-free conditions. NK-depleted lymphocytes did not lyse the melanoma targets. Culture supernatants of monocytes incubated with the melanoma tumor cells did not exhibit cytotoxic activity against these targets. Purified monocytes lacked NK activity against the K562 targets in a 4-hr 51Cr release assay. Treatment of the monocytes with anti-Leu 1 1b and anti-Leu7 monoclonal antibodies plus complement did not reduce monocyte-mediated lysis of the melanoma targets, demonstrating that contaminating NK cells, if any, were not responsible for the lysis of noncultured melanoma targets by monocytes. In contrast, Leu 1 1b+ NK cells were responsible for the lysis of the melanoma targets by NK-enriched lymphocytes. The addition of recombinant interferon-gamma (rIFN-gamma), but not lipopolysaccharide, into the 51Cr release assay or pretreatment of monocytes with rIFN-gamma significantly increased their cytotoxicity against noncultured solid tumor cells. Monocytes cultured for 3 days with medium alone lost their cytotoxic activity. The addition of rIFN-gamma from the beginning of these cultures prevented the loss of the cytotoxic activity of monocytes. In summary, both unstimulated monocytes and NK-enriched lymphocytes exhibit comparable levels of spontaneous cytotoxicity against fresh solid tumor targets.     

The Design and Characterization of Oligospecific Antibodies for Simultaneous Targeting of Multiple Disease Mediators

Monoclonal antibodies are traditionally used to block the function of a specific target in a given disease. However, some diseases are the consequence of multiple components or pathways and not the result of a single mediator; thus, blocking at a single point may not optimally control disease. Antibodies that simultaneously block the functions of two or more disease-associated targets are now being developed. Herein, we describe the design, expression, and characterization of several oligospecific antibody formats that are capable of binding simultaneously to two or three different antigens. These constructs were generated by genetically linking single-chain Fv fragments to the N-terminus of the antibody heavy and light chains and to the C-terminus of the antibody C_H3 domain. The oligospecific antibodies were expressed in mammalian cells, purified to homogeneity, and characterized for binding to antigens, Fcγ receptors, FcRn, and C1q. In addition, the oligospecific antibodies were assayed for effector function, protease susceptibility, thermal stability, and size distribution. We demonstrate that these oligospecific antibody formats maintain high expression level, thermostability, and protease resistance. The in vivo half-life, antibody-dependent cellular cytotoxicity function, and binding ability to Fcγ receptors and C1q of the test oligospecific antibodies remain similar to the corresponding properties of their parental IgG antibodies. The excellent expression, biophysical stability, and potential manufacturing feasibility of these multispecific antibody formats suggest that they will provide a scaffold template for the construction of similar molecules to target multiple antigens in complex diseases.     

Role of interferon γ and tumour necrosis factor α in monocyte-mediated cytostasis and cytotoxicity against a human histiocytic lymphoma cell line

Summary In view of cellular adoptive immunotherapy we have studied monocyte-mediated cytostasis and cytotoxicity against U 937 cells, a human histiocytic lymphoma cell line. Highly purified human monocytes and monocytederived macrophages were activated with interferon (IFN) or tumour necrosis factor (TNF) to antileukemic immune effector cells. Antileukemic activity of human monocytes was dependent on monocyte differentiation into macrophages and on a dose- and time-dependent activation with IFN or TNF. Maximum cytostasis of 97.0±0.7% (mean ± SEM) (conventional [³H]dT uptake assay) and 81.9±5.3% cytotoxicity (modified MTT assay) of U 937 cells was obtained by monocytes activated with 100 U/ml IFN for at least 24 h at an effector-to-target-cell ratio of 10. U 937 cells premodified with IFN showed an increase in susceptibility to monocyte-mediated cytotoxicity. U 937 cells premodified with TNF were almost resistant to monocyte-mediated cytotoxicity while activated monocytes maintained their cytotoxic potential. These data show that IFN and TNF are potent activators of monocyte-mediated cytotoxicity. Furthermore, IFN and TNF might be involved in the regulation of the susceptibility of leukemic cells to lysis by interactions with monocytes or macrophages.     

Production of lactose-free galacto-oligosaccharide mixtures: comparison of two cellobiose dehydrogenases for the selective oxidation of lactose to lactobionic acid

Galacto-oligosaccharides, complex mixtures of various sugars, are produced by transgalactosylation from lactose using beta-galactosidase and are of great interest for food and feed applications because of their prebiotic properties. Most galacto-oligosaccharide preparations currently available in the market contain a significant amount of monosaccharides and lactose. The mixture of galacto-oligosaccharides (GalOS) in this study produced from lactose using recombinant beta-galactosidase from Lactobacillus reuteri contains 48% monosaccharides, 26.5% lactose and 25.5% GalOS. To remove efficiently both monosaccharides and lactose from this GalOS mixture containing significant amounts of prebiotic non-lactose disaccharides, a biocatalytic approach coupled with subsequent chromatographic steps was used. Lactose was first oxidised to lactobionic acid using fungal cellobiose dehydrogenases, and then lactobionic acid and monosaccharides were removed by ion-exchange and size-exclusion chromatography. Two different cellobiose dehydrogenases (CDH), originating from Sclerotium rolfsii and Myriococcum thermophilum, were compared with respect to their applicability for this process. CDH from S. rolfsii showed higher specificity for the substrate lactose, and only few other components of the GalOS mixture were oxidised during prolonged incubation. Since these sugars were only converted once lactose oxidation was almost complete, careful control of the CDH-catalysed reaction will significantly reduce the undesired oxidation, and hence subsequent removal, of any GalOS components. Removal of ions and monosaccharides by the chromatographic steps gave an essentially pure GalOS product, containing less than 0.3% lactose and monosaccharides, in a yield of 60.3%.     

Spontaneous cytotoxicity of human peripheral blood mononuclear cells toward red blood cell targets. II. Time-dependent loss of suppressor cell activity.

In a previous paper we demonstrated that human peripheral blood mononuclear cells become strikingly cytotoxic toward a wide variety of red blood cell targets after 7 days of in vitro culture. The cell responsible for cytotoxicity does not rosette with SRBC and demonstrates both surface adherence and phagocytic properties. In this paper we wish to show that development of spontaneous cytotoxicity is due to a time-dependent loss of suppressor cell function. Fresh autologous lymphocytes, when added to cultured cells, abrogate the subsequent expression of spontaneous cytotoxicity toward RBC targets. The suppressor cell is radioresistant; requires 24 hr to suppress optimally; is inactivated by heating at 56 degrees C for 15 min, and is enriched in the non-T interface after SRBC rosette depletion over a discontinuous Ficoll-Hypaque gradient. Furthermore, the addition of a cell-free sonicate of fresh lymphocytes is capable of inhibiting spontaneous cytotoxicity toward RBC targets. However, if mononuclear cells are allowed to incubate in tissue culture medium for 7 days they are no longer suppressive after sonication. These data suggest that fresh mononuclear cells exert a potent negative regulatory influence on monocyte killing. Our culture conditions by removing this negative influence have produced a new model of spontaneous nonspecific killing by monocytes.     

Modulation of a human immunosuppressive lymphokine by monosaccharides

Soluble suppressor factor (SSF) is a recently purified human lymphokine produced by peripheral blood lymphocytes (PBL) in serum-free medium as a likely consequence of an autologous mixed lymphocyte reaction. Immunoregulatory actions of SSF include suppression of: polyclonal B cell activation, proliferative responses of normal PBL, and natural killer (NK) and antibody-dependent cellular cytotoxicity. We examined the ability of the monosaccharides fucose (Fuc), galactose (Gal), glucose (Glc), and mannose (Man) to reverse SSF-mediated suppression of NK activity. Fuc and Gal can partially or completely reverse SSF-mediated suppression at four effector:target cell ratios. Man and Glc were unable to significantly reverse SSF-mediated suppression. Fuc or Gal was added to PBL at various times after addition of SSF. SSF-mediated suppression of NK cytotoxicity becomes irreversible with respect to these monosaccharides during the first 24 hr of PBL exposure to SSF. To explore the mechanism behind this block of SSF-mediated suppression. Fuc or Gal (50 mM) was cultured with PBL for 24 hr before addition of SSF, or with SSF for 24 hr before addition to PBL. Our experiments indicate that SSF is directly interacting with these monosaccharides, and may function by recognizing specific sugar moieties on the surface of effector cells.     

Differences in the architecture of cytoplasmic and intracytoplasmic membranes of three chemotrophically and phototrophically grown species of the Rhodospirillaceae

Chondroitin sulfate lyase (EC 4.2.2.4) was present constitutively at low levels (0.06 to 0.08 U/mg of protein) in cells of Bacteroides thetaiotaomicron which were growing on glucose or other monosaccharides. When these uninduced bacteria were incubated with chondroitin sulfate A (5 mg/ml), chondroitin sulfate lyase specific activity increased more than 10-fold within 90 min. Synthesis of ribonucleic acid and of protein was required for induction, and induction was sensitive to oxygen. The disaccharides which resulted from chondroitinase action did not act as inducers, nor did tetrasaccharides or hexasaccharides obtained by digestion of chondroitin sulfate with bovine testicular hyaluronidase. None of these substances was taken up by uninduced cells; they may not have been able to penetrate the outer membrane. The smallest oligomer capable of acting as an inducer was the outer membrane. The smallest oligomer capable of acting as an inducer was the octassacharide. Oligomers larger than the octassacharide induced chondroitin lyase activity nearly as well as intact chondroitin sulfate.     

2-Aminoanthracene, diethylstilboestrol and vinblastine tested in the in vitro mammalian cell micronucleus test at British American Tobacco UK in support of the OECD draft Test Guideline 487

Reference genotoxic compounds 2-aminoanthracene, diethylstilboestrol and vinblastine were tested in the in vitro micronucleus assay using Chinese hamster V79 derived cells in the laboratories of British American Tobacco in the UK. The work was conducted in support of the cytotoxicity measures recommended in the 2007 version of the OECD Test Guideline 487. The three compounds were positive in the assay in the presence and absence of the cytokinesis blocking agent cytochalasin B at concentrations that did not exceed the recommended cytotoxic limits determined by relative population doubling, relative increase in cell counts, relative cell counts and cytokinesis block proliferation index. Consequently, this work supports the hypothesis that relative population doubling, relative increase in cell counts and relative cell counts are appropriate measures of cytotoxicity for the non-cytokinesis blocked in vitro micronucleus assay.     

Cytokine effects and role of adhesive proteins and Fc receptors in human macrophage-mediated antibody dependent cellular cytotoxicity.

Mononuclear phagocytes participate in host immunological defense against tumors. We have investigated the role of selected recombinant cytokines on human macrophage-mediated tumor cytotoxicity in vitro utilizing a human colon cancer cell line target, SW1116, and murine monoclonal antibody 17-1A. Blood monocytes were kept in continuous culture to allow differentiation into macrophages. Maximum antibody dependent cellular cytotoxicity (ADCC) as measured in a 3H-thymidine release assay occurred after culturing the monocytes for 5-7 days. Human recombinant macrophage colony stimulating factor (CSF) (1,000 U/ml) did not increase ADCC above control levels whereas recombinant human granulocyte-macrophage colony stimulating factor, interleukin 4, and interleukin 3 were all capable of increasing ADCC. Antibodies to the CD11/CD18 integrin receptors did not significantly inhibit ADCC. When the ADCC incubation occurred in the presence of antibodies to the human Fc receptors, ADCC was inhibited significantly only by anti-FcRIII (3G8). A role for tumor necrosis factor alpha or other soluble mediators of cytotoxicity was not demonstrable in this system. These studies suggest avenues for manipulation and augmentation of macrophage-mediated antitumor ADCC.     

Protection by sugars against phase transition-induced leak in hydrated dimyristoylphosphatidylcholine liposomes

The disaccharides trehalose and sucrose have small effects on temperature and enthalpy of the pre- and main phase transition in hydrated DMPC bilayers. In contrast, these sugars cause a considerable retention of carboxyfluorescein when large unilamellar vesicles of DMPC are heated through the main transition. This effect is sugar specific, as the monosaccharides glucose and fructose are less effective and ethyleneglycol has no effect at all.