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Biomedical subjects

L K Ashman

Publications and source records attributed to L K Ashman.

At least 91 records · Page 5Linked to original sources
PubMed1987

The expression of mature myeloid cell differentiation markers in acute leukemia.

Cells from 82 patients with leukemia in acute phase (40 ANLL, 1 AUL, 36 ALL, 5 CGL in blast crisis) were studied for the expression of mature cell markers of the major nonlymphocytic cell lineages (monocytes, granulocytes, erythrocytes and platelets) using monoclonal antibodies. In addition, cells were examined for the presence of HLA-A, B, C antigens, Ia antigens and common ALL antigen, as well as Fc receptors capable of binding murine immunoglobulins. Approximately one-third of ANLL specimens lacked any of the mature-cell differentiation markers studied. These were always in the relatively undifferentiated morphological subgroups (M1 and M2). Some of the specimens in these groups also expressed little or no HLA-A, B, C and/or Ia antigen. Of the lineage-specific MAb, FMC32 and FMC34, which bind to monocytes, and monocytes plus granulocytes respectively, gave the most interesting results. Together with the anti-CALLA antibody J5, they contributed to the differential diagnosis of ANLL and ALL. In addition they detected phenotypic heterogeneity within the FAB types of ANLL, particularly the M1 and M2 groups. Binding of murine IgG2a and IgG3 antibodies, apparently via Fc receptors, was commonly observed with ANLL cells. This is a potentially serious source of "false positives" in studies using murine MAb with human leukemic cells.

Antibodies, Monoclonal↗
PubMed1986

Autologous responses to human leukaemic cells in mixed leucocyte culture.

Cryopreserved leukaemic blasts and remission non-T cells from 22 patients with acute leukaemia (15 lymphocytic, 7 non-lymphocytic) were tested as stimulators of autologous remission T cells and normal allogeneic T cells in primary and secondary MLC. In most cases the autologous response elicited by leukaemic cells was less than or equal to that elicited by remission non-T cells. However, T cells from 2 patients in long-standing first remission from ANLL displayed greater proliferation in response to leukaemic blasts than to remission non-T cells in both primary and secondary MLC. The results are suggestive of sensitization of these 2 patients to leukaemia-specific antigens, but other possible explanations are discussed.

Acute Disease↗
PubMed1985

Variations in antigen expression during the cell cycle of a human B-lymphoblastoid cell line.

Expression of six surface markers during the cell cycle of a human B-lymphoblastoid cell line has been examined. Cultures were synchronised by a double thymidine block, then monitored through a complete cell cycle following release. Surface marker expression was studied by manual immunofluorescence and flow cytometry. Surface topography of the cells during the cycle was examined by scanning electron microscopy. Some variation in the expression of all markers was observed during the cell cycle. Four antigens (surface membrane immunoglobulin, common ALL antigen, an immature cell marker identified by monoclonal antibody 11D1, and an ubiquitous leucocyte antigen identified by monoclonal antibody 6B1) showed maximal expression in S and/or G2 phase and minimal expression during mitosis. These results could be due to changes in cell size. However, the individual patterns of expression of these four antigens during the phases of the cell cycle implied that changes in antigen density also occurred, at least in some cases. In contrast, expression of HLA-A, B, C and Ia antigens did not parallel cell size and was lowest during the S and G2 phases, increasing to a peak in early G1 phase.

Antigens, Neoplasm↗
PubMed1985

Surface antigen expression by a human B-lymphoblastoid cell line treated with 'differentiation' inducers, dimethysulfoxide and tetradecanoylphorbol acetate.

Changes in expression of surface antigens and morphology induced in a human B lymphoblastoid cell line by 'differentiation' inducers DMSO and TPA have been examined. Both of these agents were shown to cause arrest in the G1 phase of the cell cycle, and morphological changes including a decrease in the nuclear to cytoplasmic ratio. In addition, TPA caused a marked increase in membrane area with extensive ruffling. Treatment of cells with 1.25% DMSO for 6 d brought about decreases in the expression of Ia antigens, CALLA, and an immature cell antigen defined by the monoclonal antibody 11D1, and an increase in the expression of surface membrane immunoglobulin. This pattern is consistent with the induction of differentiation towards a more mature B cell. In contrast, treatment of cells with 5 X 10(-8)M TPA for 2 d resulted in increased expression of both Ia and HLA-A, B,C antigens, decreased expression of surface membrane immunoglobulin, and little or no change in the other markers. These changes do not indicate a maturation process and can be explained in part by accumulation of cells in the G1 phase of the cell cycle.

Antigens, Neoplasm↗
PubMed1985

A murine monoclonal antibody specific for a cell-surface antigen expressed by a subgroup of human myeloid leukaemias.

Murine monoclonal antibody YB5.B8 was raised against leukaemic blasts from a patient with M1-type acute non-lymphocytic leukaemia (ANLL). The antibody, which is of IgG1 class, bound to the majority of leukaemic blasts in the immunizing population, but not to cells of an autologous EBV-transformed B cell line. The antigen was not detected on normal blood or bone marrow cells, or on any of the eleven haemopoietic cell lines tested. It was present on some cells in peripheral blood specimens from 7/37 patients with ANLL and 1/5 patients with chronic myelomonocytic leukaemia and one patient with myelofibrosis with blastic change. In contrast, the antigen was not detected on cells in any of the 32 lymphoid leukaemic specimens tested, or on cells from four patients with chronic granulocytic leukaemia in accelerated chronic phase. In the ANLL group, expression of the antigen usually occurred on cells from types M1 or M2 according to the F.A.B. classification, and appeared to be associated with an unfavourable response to chemotherapy. The antigen was removed from the cell surface by digestion with pronase, and was re-expressed after 24 h in culture. Re-expression was prevented by the protein synthesis inhibitor, cycloheximide, but not by tunicamycin which inhibits glycosylation. Therefore, it seems likely that YB5.B8 binds to a peptide antigenic determinant.

Animals↗
PubMed1985

The Rose Bengal assay for monoclonal antibodies to cell surface antigens: comparisons with common hybridoma screening methods.

An automated colorimetric procedure for detection of antibodies specific for cell surface antigens (1) has been compared for specificity and sensitivity to other methods of hybridoma supernatant screening. The Rose Bengal colorimetric assay (RBA) compared favourably in these respects with whole cell radioimmunoassay and indirect immunofluorescence with manual or flow cytometric analysis (FACS). A major advantage of the method is that it allows a large number of samples to be screened in a comparatively short time. Unlike other semi-automated colorimetric assays, such as ELISA, the procedure does not require cell fixation, which can destroy some antigenic determinants. The original assay of O'Neill and Parish (1) has been modified to give increased sensitivity and also to enable the detection of erythrocyte specific antibodies by elimination of the dye staining step and direct measurement of haemoglobin by spectrophotometry. The RBA allows detection of monoclonal antibodies (MoAb's) binding to only a proportion of the cells in a sample, which is an important feature when hybridoma supernatants are screened for reactivity to a minor cell population, for example against leukaemic cell samples with low percentages of blast cells.

Antibodies, Monoclonal↗
PubMed1985

Specificity of the passive antibody-induced suppression of the humoral immune response of mice to surface antigens on human cells.

The effect of passively administered antibody on the humoral immune response of BALB/c mice to antigenic determinants on human cells has been examined. Antiserum raised by immunizing mice with the human leukaemic cell line K562, which lacks HLA-A,B,C antigens, was administered to mice, together with the HLA-A,B,C-positive cell line, BALM-1. The antibody response to the unique antigen was assessed by measuring the ability of the resultant antiserum to inhibit the binding to BALM-1 cells of a labelled monoclonal antibody, 7B6, which is specific for a monomorphic HLA-A,B,C determinant. As an indication of the immune response to antigens common to K562 and BALM-1, the ability of the same antiserum to inhibit the binding of monoclonal antibody 6B1, which detects an epitope common to both cell lines, was measured. Passive antibody to K562 blocked the immune response of mice to the common antigen on BALM-1 cells. However, the response to the antigen not recognized by the passive antibody was unaffected, even though the two antigens were present on the same immunizing cell. Thus, the effect of passive antibody was 'determinant specific'. Similar results were obtained, irrespective of whether the i.v. or i.p. route of immunization was used, and whether the passive antibody was adsorbed onto the immunizing cells prior to injection, or administered separately. The blocking of the immune response did not depend on simple masking of the antigenic determinants by the passive antibody, since non-saturating amounts of antibody were effective. In addition, blocking activity was dependent on antibody class and on an intact Fc region. The latter considerations also imply that the outcome of passive antibody administration in this system was determined by factors other than the ability of the antigen-antibody complexes to interact directly with B cells, and indicate the importance of antigen processing and/or a mechanism such as antigen-reactive cell opsonization.

Animals↗
PubMed1984

Proliferative response of normal and activated human lymphocytes to tetradecanoyl phorbol acetate.

The mitogenicity of 12-O-tetradecanoyl phorbol-13-acetate (TPA) for normal human peripheral blood mononuclear cells was investigated. TPA was a weak mitogen giving simulation indices in the range 2.5 to 10.5 at the optimum concentration (10 ng/ml) compared with 39 to 95 for phytohemagglutinin (PHA) at its optimum concentration (1 microgram/ml). No absolute requirement for a comitogen could be demonstrated, however TPA and PHA were synergistic in their action at low concentrations, and additive at optimum concentrations. Cell fractionation by rosetting with sheep erythrocytes showed that most of the proliferative response to TPA occurred in the T-cell fraction, however some proliferation of non-T cells was also observed. Surface marker studies showed that this could not have been due to residual T cells in the non-T fraction. A small number of monocytes was required for optimal proliferation of T cells in response to TPA. After a 3-day incubation with mitogen, the responding cell populations were tested for binding of a range of antibodies specific for T-cell (OKT3, OKT4, OKT8, and OKT11), "natural killer" (NK) cell (anti-Leu-7), monocyte (FMC17), and B-cell (anti-human immunoglobulin) surface markers. These experiments indicated that the responding cell types were T cells and B cells, but not NK cells or monocytes. Marked modulation of the antigen detected by OKT4, and to a lesser extent that detected by OKT3, in the presence of TPA precluded determination of which subpopulations of T cells proliferated in response to TPA. TPA was also tested for its ability to "maintain" activated T-cell blasts in a standard assay for interleukin 2 (IL-2). Mitogen-activated T cells were strongly responsive to TPA in this assay, but progressively lost responsiveness when maintained in crude IL-2 for about 2 weeks. Thus TPA does not have "maintenance" (i.e., IL-2-like) activity. However, small amounts of TPA acted synergistically with PHA in maintaining blast populations which were not responsive to TPA alone. This illustrates the importance of using long term IL-2-dependent cell lines for quantitation of IL-2 in supernatants prepared by stimulating T cells with these agents.

Cell Division↗
PubMed1983

Stimulatory ability of human leukaemic cells in the allogeneic mixed leucocyte reaction.

Cryopreserved mononuclear cells from 34 patients with a high proportion of blasts in the peripheral blood were tested for their ability to stimulate normal allogeneic lymphocytes in the one-way mixed leucocyte reaction (MLR). Of these, more than half failed to stimulate allogeneic responders, despite the fact that many of them carried Ia-like antigens. In general, acute myeloid leukaemia cells were non-stimulatory whereas acute myelomonocytic leukaemia cells were effective stimulators. Thus, MLR stimulatory ability appears to be a differentiation marker in acute non-lymphocytic leukaemias. Acute lymphocytic leukaemia cells were divided between the two categories, although this division did not parallel conventional classification. As reported by other workers, chronic lymphocytic leukaemia cells were non-stimulatory. The implications of these results for host-leukaemia relationships are discussed.

Histocompatibility Antigens Class II↗
PubMed1983

Binding of mouse monoclonal antibodies to human leukaemic cells via the Fc receptor: a possible source of 'false positive' reactions in specificity screening.

Mouse monoclonal antibodies (MoAbs) of different classes and subclasses directed against antigens not expected to be present on human cells have been screened by indirect immunofluorescence using flow cytometry for binding to human non-lymphocytic leukaemic cells and normal peripheral blood leucocytes. Antibodies of the IgG2a and IgG3 subclass, but not of the IgG1, IgG2b or IgM class bound to the blast cell and monocyte populations in a peripheral blood mononuclear cell preparation from a patient with acute myelomonocytic leukaemia. F(ab')2 fragments of an anti-salmonella antibody of IgG2a subclass failed to bind, indicating that the results were not due to cross-reactivity with antigens of the cell membrane, thus implicating the Fc region in binding. Furthermore, binding was partly blocked by inclusion of 10% heat-inactivated normal rabbit serum in the assays. IgG2a bound to varying degrees to the leukaemic cell populations in seven of the nine non-lymphocytic leukaemic specimens tested, but no binding to normal peripheral blood mononuclear cells or granulocytes was detected. The results emphasize the importance of including appropriate controls when screening MoAbs for binding to various types of human cells.

Acute Disease↗
PubMed1982

Application of the nitrocellulose transfer technique and alkaline phosphatase conjugated anti-immunoglobulin for determination of the specificity of monoclonal antibodies to protein mixtures.

The target antigens of a series of monoclonal antibodies raised against a complex mixture of proteins have been identified. The protein mixture was fractionated by polyacrylamide gel electrophoresis in SDS, and the fractionated proteins electrophoretically transferred to nitrocellulose sheets. Protein bands reacting with antibody were identified by incubating the nitrocellulose strips with the 'hybridoma' culture supernatants, followed by detection of bound antibody using alkaline phosphatase conjugated to rabbit anti-mouse F(ab')2 antibody, and histochemical substrates. With this technique, the antigens reacting with 4 of a series of 5 monoclonal antibodies have been identified.

Alkaline Phosphatase↗
PubMed1982

Two-stage skin carcinogenesis in sensitive and resistant mouse strains.

Strains of laboratory mice vary markedly in their susceptibility to two-stage skin carcinogenesis using 7,12-dimethylbenz[a]anthracene (DMBA) as the initiating carcinogen and croton oil as promoter. This study has been undertaken in order to clarify the basis of the strain differences. LACA mice were used as the susceptible strain and BALB/c mice as the resistant strain. DMBA was a more effective complete carcinogen in LACA mice than in BALB/c mice. However, dose-response studies with respect to DMBA in the two strains in two-stage carcinogenesis suggested that metabolic activation of DMBA to the active carcinogen was not limiting in the resistant strain. The observed strain differences in response to DMBA in one and two stage carcinogenesis may reflect the ability of DMBA to also act as a promoter in the two strains. The possibility that the strains vary in their ability to repair damaged DNA has, however, not been eliminated. Unlike polycyclic aromatic hydrocarbon carcinogens such as DMBA, phorbol ester promoters (the active components in croton oil) do not appear to require metabolic activation. However, they are degraded and inactivated by epidermal and other cells. Experiments in which the dose and frequency of application of croton oil were increased, and the unrelated promoter anthralin was substituted for croton oil, failed to produce any evidence that differences in promoter degradation contributed to the differences in susceptibility between strains.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1980

Factors affecting the ability of various strains of Enterobacteriaceae to induce tumour resistance in mice.

Primary infection of mice with Salmonella enteritidis 11RX (11RX) confers resistance to challenge with 10(4) LD50 doses of Ehrlich Ascites tumour (EAT). A lipopolysaccharide-free protein extract of 11RX is capable of eliciting delayed type hypersensitivity (DTH) reactions in these mice and of "recalling" tumour resistance in long-term 11RX immunised mice, which no longer exhibit any resistance to tumour challenge. In the present study, we have examined the ability of five other strains of Enterobacteriaceae to induce similar effects. Primary i.p. injection of S. chester, S. luton or S. typhimurium G30 into mice resulted in persisting infections and the induction of peritoneal exudate cells (PEC) which were tumouricidal in vitro. DTH reactions could also be elicited in these animals with protein extracts of the homologous or the 11RX strain of salmonella. S. friedenan and E. coli K12, which did not persist in mice, did not elicit tumouricidal PEC and did not sensitize mice for DTH reactions. However, protein extracts from all the five strains could elicit tumouricidal PEC and DTH reactions in long-term 11RX-immunised mice (but not in normal mice). The results imply that a wide range of Enterobacteriaceae may possess antigen(s) which can be involved in tumour resistance, provided that these antigen(s) are presented in such a way that a cellular immune response develops.

Animals↗
PubMed1980

Involvement of donor lymphoreticular cells in the rejection of B16 melanoma by allogeneic mice.

B16 melanoma maintained by in vivo passage in syngeneic (C57B1/6) mice was rejected when transplanted into H-2 incompatible BALB/c mice. However, after passage in tissue culture the tumour grew in BALB/c mice and, in the case of intraperitoneal (i.p.) inoculation, was fatal. Administration of lymphoreticular cells (resident peritoneal cells, PC) bearing C57B1/6 alloantigens at the same time as the cultured tumour prevented or greatly diminished tumour growth in BALB/c mice. This occurred when the PC were given either at the same site as the tumour or at a remote site, implying that tumour rejection involved specific sensitization of the BALB/c mice to C57B1/6 alloantigens presented on the PC. Conversely, administration of PC from a strain (C3H) with a H-2 haplotype unrelated to either the tumour or the allogeneic recipient mice diminished tumour growth when given at the same site, but had no effect when given at a remote site. This result is consistent with the involvement in sensitization of a soluble factor produced by or in response to the allogeneic PC, which is not antigen-specific, and which operates over a short range. These results indicate that, as for mouse thyroid allograft rejection (Lafferty and Woodnough, 1977), rejection of a B16 melanoma allograft was dependent on the presence of donor stimulator cells of lymphoreticular origin. The implications for the generation of immunity in syngeneic and autochthonous tumour systems are discussed.

Animals↗
PubMed1980

A comparison of three blood tests for cancer.

Plasma carcinoembryonic antigen concentration, serum phosphohexose isomerase activity and the erythrocyte sedimentation rate were determined in patients with cancer, patients being treated for various benign conditions and healthy young persons. This was to make an intercomparison between the ability of these assays to distinguish patients who have cancer from those who do not and from normal persons. The upper boundaries of the assays were defined by the one-sided, distribution-free, upper tolerance limits of the reference populations. Only by means of the assay for carcinoembryonic antigen was it possible to distinguish patients who had cancer from those who did not. Twenty-two per cent of patients with cancer could be identified by this assay with a false-positive error rate of less than 5 per cent.

Adult↗
PubMed1979

Inhibition of the growth of Lewis lung carcinoma in syngeneic mice by Salmonella antigens.

Administration of a protein antigen preparation from Salmonella enteritidis 11RX at the site of challenge with Lewis lung carcinoma inhibited tumour development in mice previously immunised with live bacteria, but not control mice. The kinetics of tumour growth indicated that the inhibition of tumour development was due to a reduction of the initial tumour inoculum rather than the development of specific anti-tumour immunity.

Animals↗
PubMed1979

Protective effect of oral Salmonella enteritidis 11RX infection against colon tumor induction by 1,2-dimethylhydrazine in mice.

Infection of mice with Salmonella enteritidis 11RX has been shown previously to cause nonspecific immune stimulation and, consequently, resistance to subsequent challenge with a variety of transplantable tumors. The present study has examined the effect of infection with this organism in a chemical carcinogenesis system. Colonic tumors were induced in LACA and BALB/c x C57BL/6JF1 mice by weekly s.c. injection of 1,2-dimethylhydrazine (15 mg/kg) for 28 weeks. Infection of mice p.o. with live S. enteritidis 11RX at 8-week intervals during 1,2-dimethylhydrazine administration protected both strains against colon tumorigenesis. Significantly fewer infected than control BALB/c x C57BL/6JF1 mice had colonic tumors at or before termination of the experiment (34 or 40 weeks) (p less than 0.001 in all cases). Comparable results were obtained with both male and female mice. The difference in tumor incidence between control and infected LACA mice was not statistically significant, however; the number and size of the lesions was greater in control mice (p less than 0.02). Although it has not been proven that the protective effect is mediated by the immune system, the results are consistent with the operation of a macrophage-mediated surveillance system. It is suggested that enteric infections should be considered as a possible contributing factor in the epidemiology of human colonic cancer.

Adenocarcinoma↗