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J C Roder

Publications and source records attributed to J C Roder.

At least 109 records · Page 6Linked to original sources
PubMed1983

Modulation of K562 cells with sodium butyrate. Association of impaired NK susceptibility with sialic acid and analysis of other parameters.

Neuraminidase treatment of parental and butyrate-induced K562 tumor cells was associated with an increase in natural killer (NK) susceptibility of these target cells. The degree of enhancement with neuraminidase was significantly greater for the NK-resistant (NRR) butyrate-differentiated K562 cells so that the relative difference between the parental NK-sensitive (NKS) K562 line and its induced NKR variants, in terms of NK sensitivity, was no longer five- or six-fold but only two-fold. The predominant reason for the altered NK susceptibilities of the target cells after neuraminidase treatment was an increase in the target-cell-binding ability of these cells as assessed by a direct conjugate-forming cell assay using Percoll-enriched NK cells and cold target competition assays. The enhancement did not appear to be due simply to an increased membrane-membrane attraction caused by a reduction of net negative cell surface charges since protamine sulphate, a positively charged molecule, had no effect on NK activity. Compared with the NKS parental K562 tumor cells, the NKR butyrate-induced cells had 3.6- to 4.0-fold higher sialo-transferase activities and were associated with significantly greater amounts of cell surface sialic acid detected both in sialyl glycoproteins (2.2- to 2.9-fold higher) and particularly within ganglioside extracts (6.2- to 13.6-fold higher). In conformity with the marked neuraminidase enhancement of NK-mediated cytolysis of the butyrate-induced targets, these NKR cells were associated with significantly enhanced levels of neuraminidase-accessible sialic acid compared to the NKS parental K562 cell line. Other parameters such as sensitivity to superoxide radicals, intrinsic superoxide dismutase levels, altered membrane repair mechanisms and transferrin competition, were not significantly different between the NKS and NKR target phenotypes. Sugar inhibition studies demonstrated an enhanced inhibition against the butyrate-induced cells with a variety of neutral sugars. The degree of inhibition with phosphorylated sugars was comparable between the parental and induced K562 tumor target cells and is consistent with our previous findings showing that these hexose phosphates may be inhibiting cytolysis at a step independent of target-cell recognition.

Butyrates↗
PubMed1983

The effect of unphosphorylated and phosphorylated sugar moieties on human and mouse natural killer cell activity: is there selective inhibition at the level of target recognition and lytic acceptor site?

A number of different sugars were investigated for their effect on human and mouse natural killer cell (NK)-mediated cytolysis. From the pool of nonphosphorylated sugars, D-mannose, N-acetyl-D-glucosamine (NAcGlc), D-glucose, and, to a lesser extent, beta-gentiobiose were found to inhibit human NK cytolysis. Mouse NK activity against YAC-1 target cells was reduced consistently in the presence of D-mannose and NAcGlc only. The sugars, NAcGlc, D-glucose, and beta-gentiobiose, were specifically inhibitory against NK-mediated cytolysis with no inhibitory effects being observed against ADCC, monocyte-mediated cytolysis, or CTL activity. Pretreatment and washing at either the target or effector cell level as well as direct target binding assays using Percoll-purified NK cells indicated that at least NAcGlc and beta-gentiobiose function at the recognition stage of NK cytolysis. D-Mannose, which was the most effective nonphosphorylated sugar inhibitor, was capable of inhibiting all cell-mediated cytotoxic mechanisms tested (NK, ADCC, monocyte, and CTL) and its action did not appear to be solely due to an impairment in the recognition event. All the phosphorylated sugars caused significant inhibition of human and mouse NK-mediated cytolysis, although repeated analyses of sugar titration curves consistently showed mannose-6-phosphate (Man-6-P) to be the most effective inhibitor. Inhibition with the phosphorylated sugars was apparent against all cytotoxic mechanisms investigated. It is possible that these sugars may function as general metabolic inhibitors or may activate a common signal which negatively regulates cell-mediated cytotoxic mechanisms. Nevertheless, the relative degree of inhibition with the majority of these sugars (particularly Man-6-P) was greater against NK and ADCC activity than against monocyte and CTL activity. Furthermore, studies with selected well-characterized human and mouse NK-resistant target cells strongly indicated that these sugars, particularly Man-6-P, compete at an acceptor site responsible for the uptake of the NK lytic factor, which is independent of the recognition structure(s).

Animals↗
PubMed1983

A comparative analysis of the phenotypic characteristics of available fusion partners for the construction of human hybridomas.

Of several human fusion partners available for the production of monoclonal antibodies, only SKO-007 and RPMI 8226 have phenotypic features characteristic for myeloma cells. Cells from both lines exhibited abundant rough endoplasmic reticulum (RER) with a prominent Golgi apparatus, few free ribosomes, condensed nuclear chromatin, and absence of the Epstein-Barr virus determined nuclear antigen (EBNA). However, following prolonged passage of these lines, the amount of immunoglobulin (Ig) production has declined. The other cell lines, GM 1500 6TG-A11, KR-4, B6, HS-Sultan, and Raji possessed the phenotypic characteristics of B-lymphoblastoid cell lines (LCLs) and B-lymphomas including surface Ig expression, sparse RER, free polyribosomes, a poorly developed Golgi apparatus and strong EBNA expression. Accordingly, they secreted little (nanograms) or no Ig. However, hybrids constructed with two LCLs secrete very large amounts of Ig despite their expressed morphologic similarity to the parental lines. These data indicate that morphology can still be used as an important consideration in choosing a human fusion partner but other parameters such as fusion frequency, cloning efficiencies, and growth rates may be equally important.

Antibodies, Monoclonal↗
PubMed1983

Comparison of the specific IgM and IgG antibody response in humans induced by antigen (tetanus toxoid) or a polyclonal activator (EBV) in vitro.

Culture conditions were established for the reproducible induction of specific antibody responses in vitro using peripheral blood lymphocytes from tetanus toxoid (TT) immunized donors. Maximum anti-TT antibody responses (250-350 ng/ml) were detected on day 9 of culture, with optimum quantities of antigen (0.1-1.0 micrograms/ml TT) or Epstein-Barr virus (EBV). The antigen-driven antibody response unlike the EBV-induced polyclonal response was both T-cell and monocyte-dependent. TT stimulated mainly the IgM class of anti-TT antibody whereas EBV stimulated both IgM and IgG. A combination of TT and EBV caused a partially additive IgM response but suppressed the IgG class of anti-TT antibody. Transfer of cells from 9-day EBV-stimulated cultures to fresh cultures suppressed the anti-TT antibody response, possibly due to elevated levels of OKT8+ suppressor/cytotoxic cells found in culture after EBV infection. These results provide a reliable system for studying B-cell regulation in man.

Adult↗
PubMed1983

Nerve growth factor receptors of human tumors of neural crest origin: characterization of binding site heterogeneity and alteration by theophylline.

Heterogeneous populations of saturable specific high-affinity binding sites for the biologically active subunit of the 7S nerve growth factor complex purified from mouse submaxillary gland (NGF) are detected on human tumor cells of neural crest origin. Detailed studies of the melanoma cell line MeWo demonstrate two populations of binding sites of high affinity with dissociation equilibrium constants of Kd 4 X 10(-11) and 5 X 10(-10) M, respectively. Other cell lines of neural crest origin also show high-affinity binding heterogeneity. Exposure of the cell lines to 1 mM theophylline reversibly reduces the number of available NGF binding sites without influencing the affinities of binding. On the MeWo cell line, this is not related to the stages of the cell cycle or to production and release of a NGF-like molecule by the theophylline-exposed cells. These observations complement earlier studies of theophylline-induced alterations in NK cell sensitivity on the MeWo cell line, providing further evidence for cell surface phenotypic changes induced by a compound that promotes differentiation in melanocytes and melanoma cells. Future studies of cell surface phenomena involved in theophylline-induced NGF binding site disappearance may lead to a better understanding of the NGF receptor and its disappearance from certain embryonic cells of neural crest origin during differentiation.

Animals↗
PubMed1983

The Chediak-Higashi gene in humans. III. Studies on the mechanisms of NK impairment.

Lymphocytes from six Chediak-Higashi (CH) patients were markedly depressed in their ability to lyse tumour cell targets in both 51Cr release and single cell cytotoxicity assays. The frequency of lymphocytes bearing the OKM1 marker and the frequency of T3+, T4+, T8+, Ia+, Mo1+, Mo2+ and B1+ cells was normal among sheep erythrocyte rosetting (E+) and non-rosetting (E-) peripheral blood leucocytes analysed by flow cytofluorography. Cells expressing the NK shared markers, OKM1, mac-1, FcR, and the characteristic large granular lymphocyte (LGL) morphology of NK cells were also present in normal numbers in the highly enriched NK fraction separated on Percoll density gradients. This fraction did not contain detectable numbers of cells expressing the Mo2 marker of human monocytes. Therefore most of the cells stained by monoclonal OKM1 and mac-1 in this fraction are likely NK cells, rather than monocytes, and we conclude that the size of the NK pool in CH patients is probably normal. The capacity of CH lymphocytes to recognize and bind to tumour cells was also normal as was the subsequent burst of oxygen intermediates produced by the NK cells in a chemiluminenscence assay. We have shown elsewhere that O2- generation is directly involved in activating subsequent steps in the NK cytolytic pathway. These results suggest that NK cells in CH patients are present in normal frequency but are blocked at some post-recognition, post-activation step in the cytolytic pathway subsequent to the burst of oxygen intermediates but preceding the lethal hit.

Adolescent↗
PubMed1982

Oxygen intermediates are triggered early in the cytolytic pathway of human NK cells.

The mechanism of tumour cell destruction by natural killer (NK) cells or other lymphocytes is not understood. NK cells appear to represent a primitive anti-tumour surveillance system more analogous to macrophages than lymphocytes. Free oxygen radicals (O-2, OH) and H2O2 are thought to be involved in cell destruction by macrophages and therefore we looked for similar cytocidal intermediates of oxygen in NK cells. These highly reactive molecular species can easily be detected in the presence of luminol by the emission of light. We show here that highly enriched human NK cells respond to NK-sensitive but not NK-insensitive tumour cells with a rapid burst of oxygen metabolites as detected both by chemiluminescence and cytochrome c reduction. Agents which can prevent chemiluminescence and cytochrome c reduction, such as superoxide dismutase (SOD), reduced NK-mediated cytolysis and agents which increased chemiluminescence, such as interferon, also increased NK-mediated cytolysis. These results suggest that the production of oxygen species may be the earliest event to occur in the NK cell following tumour cell contact, and these products are involved in NK-mediated cytolysis.

Animals↗
PubMed1982

Chemiluminescence response of human natural killer cells. I. The relationship between target cell binding, chemiluminescence, and cytolysis.

The binding of tumor cells or fetal fibroblasts to human natural killer (NK) cells led to a rapid chemiluminescence response within seconds of target-effector interaction. The degree of chemiluminescence was dependent on the concentration of NK-enriched lymphocytes or target cells, and plasma membrane vesicles from K562 also induced a chemiluminescence response. Mild glutaraldehyde treatment of effector cells abrogated their ability to generate chemiluminescence, whereas K562 target cells treated in the same way were almost fully able to induce a chemiluminescence response to NK-enriched lymphocytes. These results show a directionality of response with NK as the responders and tumor cells as the stimulators. A survey of eight different tumor cell lines and fetal fibroblast lines revealed a striking correlation (r greater than 0.93, P less than 0.001) between the ability of a given line to bind to NK-enriched lymphocytes, induce chemiluminescence, and to be lysed. Three differentiated sublines of K562 grown in butyrate and cloned induced little chemiluminescence compared with the K562 parent, and they were selectively resistant to NK-mediated binding and cytolysis. In addition, treatment of K562 cells with higher concentrations of glutaraldehyde for longer periods led to varying degrees of target antigen preservation, as measured in cold target competition assays and in conjugate formation. The degree of NK target antigen preservation correlated directly with the ability of the cells to induce chemiluminescence (r greater than 0.95). The degree of NK activation was also important because interferon-pretreated effectors generated more chemiluminescence upon stimulation with K562 or MeWo targets. Monocytes or granulocytes did not contribute to the chemiluminescence induced by NK-sensitive targets. Some NK-resistant tumor cell lines were sensitive to monocyte-mediated cytolysis and also induced chemiluminescence in monocytes but not NK cells. These results show that the target structures recognized by the NK cell may play a role in NK activation because the degree of chemiluminescence was directly proportional to the ability of a given target cell line to bind to the NK cell and to be lysed.

Animals↗
PubMed1982

The biology of the human natural killer cell.

Natural killer (NK) cells in the human are a population of large granular lymphocytes (LGL) with at least one unique surface antigen not expressed on cells of other lineages. NK-target-cell interaction appears to involve carbohydrate recognition and, following binding, the NK cells are induced to generate O2-, transmethylate membrane phospholipids, and activate phospholipase A2. Some or all of these activities trigger a cascade of events which ultimately leads to the secretion of a substance toxic to the target cell. A variety of genes controls various steps in this cytolytic pathway. There is a good deal of evidence in the mouse, and some in the human, that NK cells play a role in host surveillance against tumor development, resistance to viral infections, and, possibly, hematopoietic regulation.

Animals↗
PubMed1982

Human hybridomas constructed with antigen-specific Epstein-Barr virus-transformed cell lines.

A 6-thioguanine-resistant, human lymphoblastoid B-cell line (GM1500 6TG A-11; IgG secreting) was mutagen-treated with low-level gamma-irradiation and selected for ouabain resistance. One line showing 10,000-fold higher drug resistance, designated KR-4, was fused with an Epstein-Barr virus-transformed, cloned, B-lymphocyte cell line (B6) producing antitetanus toxoid (TT) antibody (IgM), and the hybrids were selected in hypoxanthine/aminopterin/thymidine medium containing 10 microM ouabain. Surviving cells, which arose at an optimal frequency of 10(-5), were subcloned by limiting dilution and screened for anti-TT production. Out of 395 final subclones, 372 were found positive for anti-TT, and seven that were selected for further study secreted specific antibody (IgM, kappa chain) at a maximum concentration of 3-6 micrograms/ml. The differential rate of anti-TT production during the logarithmic phase of cell growth was 15-fold higher in the hybridomas than in the original B6 line. The hybrid nature of the clones was confirmed by karyotype analysis, histocompatibility antigen typing, and expression of secreted and membrane-bound Ig classes. Biosynthetic labeling of the cells revealed that all hybrids secreted both IgM and IgG but that only the IgM class had specificity for TT. Because Epstein-Barr virus is a polyclonal B-lymphocyte activator, the technique we applied here may be useful for increasing the recovery of rare antigen-specific B cells in the peripheral blood and for improving the frequency and stability of hybridomas secreting a given antibody.

Antibody Formation↗
PubMed1982

Human anti-tetanus toxoid monoclonal antibody secreted by EBV-transformed human B cells fused with murine myeloma.

An Epstein-Barr virus (EBV)-transformed human B cell line (B6) producing anti-tetanus toxoid (TT) antibody was fused with a nonimmunoglobulin (Ig)-producing murine myeloma and selected in hypoxanthine-aminopterin-thymidine (HAT) medium containing 10(-5) M ouabain. Surviving cells were cloned by limiting dilution and confirmed as hybrids by karyotype analysis and G-11 staining. Hybridomas were stable and secreted 10-fold more anti-TT antibody (IgM kappa) than the human parental cell line. In addition, the hybridomas exhibited a markedly reduced growth requirement for serum (1% fetal calf serum). Since EBV can be used to expand rare antigen-specific B cells in the human, the technique described here may be at present the method of choice for producing human monoclonal antibodies.

Animals↗
PubMed1982

Comparative analysis of cell surface markers on murine NK cells and CTL target-effector conjugates.

The rosetting of sheep erythrocytes (SRBC) coated with non-haemagglutinating monoclonal antibodies rather than conventional haemagglutinating antisera revealed readily detectable FcR on most splenic natural killer (NK) cells since 76% of splenic lymphocytes forming conjugates with YAC also rosetted with SRBC coated with high concentrations of monoclonal anti-SRBC antibody of the IgG2b subclass and since Ficoll depletion or enrichment of splenic lymphocytes rosetting with IgG2b-coated SRBC resulted in a corresponding 4-fold decrease or increase in conjugate-forming cells and a 10-fold decrease or increase in NK cytolytic activity. NK cells bound much less readily to monoclonal IgG2a and not at all to monoclonal IgG1 or IgM, but the degree of binding was directly proportional to the amount of antibody on the erythrocytes and was not isotope-restricted. In addition, immunofluorescent studies revealed that YAC-1-conjugated lymphocytes were Lyt-1-, Lyt-2-, partially Thy-1+ (60%), asialo(GM1+ (80%). Qa-4+ (77%), Qa-5+ (79%), and Ly-5+ (94%). In comparison, a proportion (39%) of alloimmune peritoneal exudate cells which conjugated with P815-2 also stained by immunofluorescence with anti-asialo GM1 antisera. Most (greater than 90%) P815-conjugated cells were Thy-1+, Lyt-2%, and a subpopulation of Lyt-1+2+ conjugates was observed (25%). Qa-5 and Ly-5 were also expressed on most (two-thirds) cytolytic T lymphocytes (CTL) conjugates, whereas Qa-4 and FcR for IgG2b were not detected. The best phenotype distinctions between NK cells and CTL were therefore based on the presence or absence of Lyt-2, Qa-4, and FcR for IgG2b on most effector cells. Anti-asialo-GM1 or monoclonal anti-Qa-4 and complement treatment greatly diminished both the frequency of NK conjugates and the percentage of conjugates with detectable IgG2b FcR or asialo-GM1. These results confirm that NK cells co-express asialo-GM1 and Fc receptors, at the single-cell level, and provide a simple method for greatly enriching NK populations at least 10-fold.

Animals↗
PubMed1982

Natural killer (HNK-1+) cells in Chediak-Higashi patients are present in normal numbers but are abnormal in function and morphology.

Children with the Chediak-Higashi (CH) syndrome are known to have abnormalities of natural killer (NK) cell function. We used the HNK-1 monoclonal antibody that reacts specifically with human NK and K cells to distinguish whether this abnormality was due either to a numerical deficiency of NK cells or a defect in their ability to function. In eight CH patients, a significant proportion of their blood mononuclear cells (10--19%) expressed the HNK-1 differentiation antigen. The level of NK cells in the five children with CH syndrome was higher than for age-matched normal controls (15.8% vs. 5.8%, P less than 0.001). When HNK-1+ cells were isolated with a fluorescence-activated cell sorter, the NK cells from CH patients were a homogeneous population of lymphocytes with a single large granule rather than the multiple small granules seen in Nk cells from normal individuals. The purified HNK-1+ cells from the CH patients had minimal NK or K cell function. The CH syndrome thus includes a functionally defective population of NK cells that retain the capability of expressing the HNK-1 differentiation antigen.

Adolescent↗
PubMed1982

Further studies of natural killer cell function in Chediak-Higashi patients.

Spontaneous natural killer (NK) activity and antibody-dependent cellular cytotoxicity (ADCC) of blood lymphocytes against five human tumour cell lines (K562, Molt-4, HL-60, Chang, Daudi) and three mouse tumour lines (YAC, P815, RBL-5) were ten- to 100-fold lower than normal in six patients with Chediak-Higashi (CH) disease. NK and ADCC were defective at 4 hr, and less so at 18 hr. The NK activity in normals and CH patients was mediated in part by FcR+, E- effector cells. ADCC against human erythrocytes was normal in CH patients, as were lectin-dependent cytolysis and mixed lymphocyte proliferative responses. Phagocytosis of antibody-coated ox erythrocytes was normal in CH patients as well. These observations confirm that the CH syndrome is associated with a profound and selective defect in NK and ADCC activity against tumour cells, whereas other mononuclear cell-mediated functions are normal.

Adult↗