"Cytoplasmic" expression of nuclear antigens.
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Antigens, Nuclear↗
Biomedical subjects
E Coustan-Smith
Publications and source records attributed to E Coustan-Smith.
Double and triple staining methods for studying the proliferative activity of human B and T lymphoid cells.
New methods for double and triple colour labelling using monoclonal antibodies to the proliferation-associated markers 5-bromo-2'-deoxyuridine (BrdU) and Ki67 are described. In order to make incorporated BrdU accessible to most anti-BrdU antibodies, mild denaturation of the DNA is needed, and this is usually obtained by exposing the cells to acid or base. This procedure destroys most cellular antigens, including nuclear TdT and Ki67. In this study we show that fixation in cold methanol, instead of 70% ethanol, for 30 min followed by immersion in 7 X 10(-3) N NaOH for 10-15 s allows BrdU staining with the simultaneous detection of nuclear, cytoplasmic and membrane antigens as well as preservation of morphological detail. This method is optimal for detection of nuclear Ki67 and TdT. These reagents together with antibodies to membrane antigens can be included in triple colour labelling using second layers conjugated to FITC, TRITC and colloidal gold. With these methods it is now possible to characterize the phenotype of dividing cell populations such as precursors in central lymphoid tissues and germinal centre blasts in peripheral lymphoid organs.
Antibodies, Monoclonal↗
Independent clonal origin of coexisting chronic lymphocytic leukaemia and multiple myeloma.
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Aged↗
Differential expression of MHC class II antigens in chronic B-cell disorders.
Cells from the peripheral blood of 22 patients with chronic B-cell disorders were examined for the expression of surface MHC class II antigens. We made use of well-characterized monoclonal antibodies (McAbs) specific for HLA-DP, -DQ and -DR molecules (B7/21, Tü22, RFDR1 and RFDR2) and of another McAb, RFD1, associated with the class II system. By using indirect immunofluorescence and flow cytometry we found both non-coordination and heterogeneity in expression of MHC class II antigens but generally with a hierarchy of positivity: DR greater than DQ greater than DP. This suggests a sequence of gradual acquisition of HLA-D antigens and indicates distinct differences in maturation arrest of the individual cases. However, after treatment with phorbol ester TPA and calcium ionophore A23187, all cases expressed the previously absent molecules indicating that the structural genes for these products remained intact. TPA and A23187 increased both the number of positive cells in most cases and the fluorescence staining intensities of all class II markers including RFD1. Thus, leukaemic cells may express different combinations of class II antigens reflecting: (i) a predetermined order of gradual acquisition of class II molecules; (ii) differences in the stages of maturation arrest; and (iii) in the cases of disordered expression a desynchronized regulation of these markers.
Adult↗
Autologous bone marrow transplantation in acute lymphoblastic leukemia--preclinical immunologic studies.
Immunologic aspects of autologous bone marrow transplantation (ABMT), immunodiagnosis, patient monitoring, and the purging of bone marrow have been studied in individual patients. It was demonstrated that the most sensitive method for detecting lymphoid cells which show the phenotypes of ALLs of B or T lineage was double immunofluorescence staining for nuclear terminal transferase (TdT) and B or T lineage antigens. With the help of these sensitive tests in the presence of rabbit complement (C'), MAbs CD10 (RFAL3 of IgM class), CD19 (SB4 of IgM class), and their cocktail were capable of eliminating greater than 3 log blast cells of B lineage ALL in 84%, 75.5%, and 90% of cases, respectively. The same reagents lysed 26.8%, 0%, and 45% of blasts in the presence of human C'. CD7 (RFT2, IgG2) eliminated greater than 3 log T-ALL blast cells in 73% of cases. The proliferative fractions of leukemic blasts were also TdT+ and sensitive to lysis with MAb and C'. On the basis of these observations MABs were selected for purging in 36 patients undergoing ABMT in first remission (10 patients considered to be at a high risk of relapse), second and third remissions (23 and 2 patients), and without entering into remission (1 patient). The efficacy of eliminating the MAb-reactive cells from the bone marrow inoculum was also documented in five patients. By the use of sensitive immunologic assay (TdT/cytoplasmic CD3 double staining) in patients with T-ALL, no residual leukemia (less than 10(-4] could be detected at the time of transplantation. Following an observation period of 5-34 months, 24 of the 36 patients are alive and well with no procedure-related mortality.
Antibodies, Monoclonal↗
Analysis of signal transduction in B chronic lymphocytic leukemia cells.
We report here experiments on the analysis of cellular signal transduction in a series of patients with chronic B cell disorders (B cell chronic lymphocytic leukemia [B-CLL] and prolymphocytic leukemia). We compared the response of the leukemic cells with primary external signals (interleukin 2 [IL-2] or B cell differentiation factors [BCDF or IL-6]) with their response to secondary inducers (the phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA] or the calcium ionophore A23187) that circumvent the first part of the signal transduction pathway by directly activating the key enzyme protein kinase C. One BCDF was synthesized by mitogen-activated peripheral blood B lymphocytes; a second BCDF was constitutively produced by the human bladder carcinoma cell line T24. Changes in morphology, Tac (IL-2 receptor) expression, RNA synthesis measured by 3H-uridine uptake, and immunoglobulin production tested by enzyme-linked immunosorbent assay were used as parameters of successful signal transduction. TPA alone and TPA plus A23187 (synergistically) effectively initiated differentiation in all the leukemia cases. Neither IL-2 nor BCDF (singly or in combinations) caused equivalent responses. On the other hand, IL-2 and BCDF produced a substantial differentiation effect on normal B lymphocytes. Our data suggest that (a) B-CLL cells are able to respond to direct stimulation of the second messenger pathway (through protein kinase C) but not to the physiological stimuli IL-2 or BCDF; (b) the defect in signal transduction appears to be located upstream of protein kinase C (a possible candidate is a G protein); (c) malignant B cells may spontaneously or after treatment with inducers express the IL-2 receptor (Tac antigen) in the absence of a functional differentiating response to IL-2; and (d) signs of proliferation/differentiation in B-CLL samples after incubation with IL-2 or BCDF might be due to contamination of the cell populations with residual normal B cells.
Antibodies, Neoplasm↗
Variable Philadelphia breakpoints and potential lineage restriction of bcr rearrangement in acute lymphoblastic leukemia.
Philadelphia (Ph1) chromosome breakpoints in acute lymphoblastic leukemia (ALL) are of two kinds: those within the breakpoint cluster region (bcr+), as in chronic myeloid leukemia (CML), and those outside it (bcr-). These encode different c-abl messenger RNAs (mRNAs), p210 and p190, respectively. It has been suggested that one class of Ph+ ALL (bcr+) may be a variant of CML arising in a multipotent stem cell, the other (bcr-) de novo ALL initiated in a lymphoid-committed progenitor. Thirty-two cases of ALL (12 Ph1+, ten chromosomally normal, and ten non-mitotic cases) were investigated for bcr involvement. Breakpoints were found within five Ph1+ and in one normal case. There was no difference in clinical features, common ALL antigen (CALLA) positivity, cytogenetics, or response to treatment between the 6 bcr+ and 7 Ph1+ bcr- patients. Myeloid antigen expression was found in 2 bcr+ cases. Bcr rearrangement appeared to be restricted to the lymphoblastic component of marrow or blood in at least four bcr+ cases. In one case, separated myeloid and lymphoid cell fractions were both bcr+. Potential heterogeneity of the Ph1+ target cell, as seen in this study, may be more important in determining disease outcome than the precise location of the Ph breakpoint.
Genes, Immunoglobulin↗
In vitro induction of myeloid surface markers in a rare case of acute leukemia.
The phenotypic marker profile of a case of acute leukemia is described; its immunophenotype is unique in that the blast cells were initially negative for a wide panel of monoclonal antibodies (McAbs) to surface and intracytoplasmic antigens and for the nuclear enzyme terminal deoxynucleotidyl transferase. Morphological and cytochemical examination suggested an acute myeloid leukemia (AML), but the cells were unreactive with anti-myeloid McAbs. Treatment with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) induced the cells to differentiate morphologically to macrophage-like cells and led to the expression of surface antigens that could be detected by antimyeloid McAbs. It is not clear whether these cells represent a rare subclass of leukemic cells that are void of any characteristic surface markers but have the potential to differentiate along the myeloid axis, or whether the antigens were masked by an unknown process.
Acute Disease↗
Synergistic action of calcium ionophore A23187 and phorbol ester TPA on B-chronic lymphocytic leukemia cells.
The peripheral blood mononuclear cells from 16 patients with B-chronic lymphocytic leukemia (B-CLL, n = 13), B-prolymphocytic leukemia (B-PLL, n = 2) or hairy cell leukemia (n = 1) were incubated in the presence of the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) and the calcium ionophore A23187. A synergy between these inducers was found with respect to morphological changes and B cell proliferation and differentiation. A23187 used alone did not activate the cells. B-CLL cells treated with the double stimulus acquired a plasmacytoid morphology, showed significantly higher incorporation of 3H-thymidine and 3H-uridine, and produced significantly higher amounts of monoclonal immunoglobulin compared with the same cells exposed to either of the inducers alone. These results indicate that phorbol ester and calcium ionophore act synergistically on B-CLL cells to induce proliferation and differentiation. B-PLL cells responded more vigorously to the signals provided by TPA and A23187. Previous studies showed that TPA and A23187 can mimic the two physiological second messengers diacylglycerol and inositol trisphosphate in the transduction of signals leading to cell activation, proliferation, and differentiation in normal B cells. The present findings suggest that the capacity of B-CLL and B-PLL cells to differentiate in response to signals of the second messenger pathway is intact.
Antibody Formation↗
Rearrangement of immunoglobulin and T cell antigen receptor genes in acute myeloid leukemia with lymphoid-associated markers.
Ten cases of adult acute myeloid leukemia (AML) displaying lymphoid-associated markers CD7 and/or terminal deoxynucleotidyl transferase (TdT) have been investigated for rearrangement of immunoglobulin and T cell antigen receptor beta and gamma genes. Two of six TdT+ cases had clonally rearranged Ig genes, whereas six of eight CD7+ AMLs, including three that were TdT+, had a germ line configuration of both immunoglobulin and T cell receptor beta and gamma genes. A single case of CD7+ TdT- AML had clonal rearrangement of all three genes. These results indicate that expression of TdT and/or CD7 is not accompanied by gene rearrangement in most cases of adult AML. A minority of cases, displaying lymphoid-associated phenotypic markers and accompanying gene rearrangement, may represent a distinct subgroup of AML that arises from a rare, primitive stem cell, possessing extensive multilineage potential.
Adult↗