Biomedical subjects
Publications and source records attributed to B E Roberts.
The results of a case-control study of chronic myeloid leukaemia in adults are reported. 122 cases and 241 controls were interviewed. Excess risks associates with heart disease and related drugs were revealed. In addition a weak association with occupational and/or hobby exposure to irradiation emerged.
Using the combined techniques of immunomagnetic depletion and multiple colour flow cytometry, the expression of HLA-ABC (W6/32) by normal T-cell subpopulations, defined by 2H4 (CD45RA) expression, was examined. It is thought that a CD45RA+CD45RO- phenotype defines the 'virgin' T-cell fraction, whereas a CD45RA-CD45RO+ phenotype defines the 'primed' or memory T-cell population. In addition, an intermediate phenotype (CD45RA+CD45RO+) appears to correspond to a transitional stage of development. In this study, these three phenotypic stages were represented by distinct levels of 2H4 staining defined as 2H4+, 2H4int and 2H4-, respectively. The results of this current investigation are of importance in two main areas. Firstly, when compared to the 2H4+ component, the HLA-ABC expression of 2H4- cells was significantly higher. This was true for both CD4+CD8- and CD4-CD8+ lymphocytes, but was not the case for CD4-CD8dim+, CD3+CD4-CD8- and CD3-CD4-CD8- fractions. Additionally, when HLA-ABC expression was examined as a function of 2H4 staining intensity, it was found that, for the CD4+ fraction, the greatest increase in HLA-ABC expression occurred between the 2H4int and 2H4- stages. In contrast, the increase in HLA-ABC expression by CD8+ lymphocytes was associated with transition from 2H4+ to 2H4int status, which suggests that increased HLA-ABC expression occurs at an earlier stage in the acquisition of CD45RO in CD8+ cells than for CD4+ cells. Secondly, for each individual blood examined, a close and highly significant correlation (P = 0.002) for membrane HLA-ABC expression was found between (i) CD4+2H4+ and CD8+2H4+ and (ii) CD4+2H4- and CD8+2H4- subpopulations. This suggests that modulation of HLA-ABC expression in CD4+ and CD8+ cells is subject to common control mechanisms and remains proportionate for these lymphocyte fractions in any given individual.
Enriched CD4+CD8-/CD4-CD8-, CD4-CD8+/CD4-CD8- and CD4-CD8- cell suspensions were prepared from normal peripheral blood by selective immunomagnetic depletion of monoclonal antibody-defined lymphocyte populations. Subsequent examination of these modified cell fractions by two-colour flow cytometry provided a means of determining the expression of membrane T-cell receptor (TcR)alpha beta and TcR gamma delta chains by both major (CD4+ and CD8+) and minor (CD3+CD4-CD8dim+ and CD3+CD4-CD8-) lymphocyte subpopulations. Normal CD4+CD8- lymphocytes were almost invariably (greater than 99%) TcR alpha beta+, whereas lymphocytes expressing membrane CD8, which could be further subdivided according to differences in fluorescent staining intensity into CD3+CD4-CD8+, CD3+CD4-CD8dim+ and CD3-CD4-CD8dim+ components, were characterized by distinct differences in patterns of TcR chain expression. In contrast to CD3+CD4-CD8+ cells, which were predominantly (99%) TcR alpha beta+, CD3+CD4-CD8dim+ lymphocytes showed a significant proportion (33%) of TcR gamma delta+ cells (natural killer-associated CD3-CD4-CD8dim+ cells were uniformly TcR-). The highest proportion (62%) of TcR gamma delta+ cells was associated with the CD3+CD4-CD8- fraction, but these studies also revealed that a significant minority of this population was TcR alpha beta+. Despite some evidence for normal inter-individual variation, further analysis of membrane CD8 fluorescent intensities confirmed clear differential relationships for TcR alpha beta and TcR gamma delta chain expression.
Control of oral anticoagulant therapy in outpatients is often unsatisfactory. The contribution of poor compliance with prescribed warfarin to unstable anticoagulant control was investigated prospectively using low-dose phenobarbitone as an indicator of compliance in 30 out-patients, 15 with stable and 15 with unstable control. Following entry to the study, there was no significant change in anticoagulation (p = 0.36) in the group with stable control. In the group who previously had unstable control, there was a significant change in INR (p = 0.0045) and anticoagulant control greatly improved. It appears that the considerable fluctuation in INR seen in many of the latter patients before the study was due to poor compliance and that entering them into the study modified their behavior. Two patients in this group who continued to have unstable anticoagulant control were shown to be poorly compliant using the phenobarbitone indicator. The results suggest that, in outpatients, poor compliance is the major cause of unstable anticoagulation with warfarin.
The expression of membrane CD11c by leukaemic blast cells was examined (indirect immunorosetting) in 75 cases of acute leukaemia (myeloid, n = 60; lymphoid, n = 15) and evaluated as a potential marker for the diagnostic discrimination between monocytic (AMML-M4 and AMoL-M5) and non-monocytic (M1, M2 and M3) AML subtypes. Preliminary studies of normal bone marrow cells indicated that CD11c expression was not restricted to cells of monocytic lineage but was also present, with apparent lower density, on significant proportions of mature and immature granulocytes. Examination of acute myeloid leukaemia (AML) subtypes revealed that the non-monocytic leukaemias (n = 33) were CD11c-, defined as less than 30% positive cells, whereas all but one of the AMML-M4 (n = 13) and AMoL-M5 (n = 14) cases were CD11c+. All 15 cases of lymphoblastic leukaemia (ALL) showed less than 5% CD11c+ blasts. Membrane CD11c expression was also compared to the more widely used markers of monocytic differentiation; cytoplasmic alpha-naphthyl acetate esterase (ANAE) and membrane CD14 expression. This analysis showed that all 13 AMML-M4 leukaemias studied, including seven cases that were CD14- and eight that were ANAE-, were CD11c+. In addition, the AMoL-M5 cases (all of which were ANAE+) could be phenotypically subdivided into CD11c+ CD14+ (n = 9), CD11c+ CD14- (n = 4) and CD11c- CD14- (n = 1) subgroups. The study also confirmed that the discriminitive ability and sensitivity of the immunorosetting procedure for the detection of membrane CD11c compared favourably to immunofluorescent staining intensities as measured by flow cytometry.
The expression of membrane CD1c, as defined by monoclonal antibody L161, was examined on malignant lymphoid cells from 191 cases of chronic lymphoproliferative disease and on eight 'normal' enriched tonsil B-cell extracts. Of 79 cases of chronic lymphocytic leukaemia (CLL) studied, 77 showed low (less than 20% positive cells) CD1c expression whereas 63/71 (89%) cases of B-PLL, HCL and B-NHL showed increased CD1c+ (but not CD1a or CD1b) components. In contrast, malignancies corresponding to terminal stages of B-cell differentiation (immunocytoma and myeloma) generally showed low CD1c expression as did lymphoid cells from 10 cases of post-thymic malignancy. Although there was some correlation between the expression of membrane CD1c and immunoglobulin (SIg) light chain densities (P less than 0.001), it is relevant in diagnostic terms that seven cases of B-NHL with low SIg staining intensities more typically associated with CLL were CD1c+. CD1c expression was not, however, correlated with the presence of CD23 or FMC7 determinants but did show a similar pattern of expression to that previously reported for beta-2 microglobulin. Determination of cellular CD1c by APAAP immunocytochemistry confirmed the presence of higher antigen densities in malignant B-cells at intermediate/late stages of differentiation and this interpretation was further supported by the finding that the majority of phenotypically mature tonsil B-cells were also CD1c+. The determination of CD1c expression by malignant B-cells may therefore be of particular value in the diagnostic differentiation of chronic lymphoproliferative disorders.
Leukaemic promyelocytes from 30 cases of hypergranular and 14 cases of hypogranular acute promyelocytic leukaemia (M3) were analysed for the presence of monocyte-associated characteristics to determine whether there was any evidence of mixed (hybrid) granulocytic-monocytic differentiation. Cytochemically, a high proportion of hypergranular cases showed significant alpha-naphthyl acetate esterase (ANAE) staining and simultaneous chloroacetate esterase, and ANAE expression by single cells was commonly seen. These atypical staining patterns were, however, not a feature of hypogranular cases. Immunophenotypic studies revealed that most hypergranular M3 cases were HLA-DR- and that monocyte-associated membrane CD14 expression was low in all cases tested. In addition, serum lysozyme concentrations (20 cases) were generally within the normal range and thus inconsistent with monocytic involvement in the leukaemic process. The significance of atypical ANAE staining of leukaemic promyelocytes was further examined by analysing ANAE isoenzyme components (defined by isoelectric focusing) in 11 cases. The patterns obtained (G1 and G2) were identical to those found in normal granulocytes and did not show any evidence of monocyte-associated esterase isoenzyme expression. On the basis of these findings, it is considered that the differentiation process in acute promyelocytic leukaemia is relatively well conserved and that the atypical esterase cytochemistry of hypergranular promyelocytes does not reflect their mixed lineage nature but is simply a consequence of increased granulation.
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Diagnostic features (cytochemistry, immunophenotyping and serum biochemistry) were examined in 51 cases of acute monocytic leukaemia (AMoL). Peroxidase, Sudan black B and alpha naphthyl acetate esterase (ANAE) cytochemical reactions were unrelated to morphological (FAB groups M5a and M5b) or immunological subtype. ANAE cytochemistry, however, indicated that AMoL cases could be subdivided into those with typical (M-type) reactions and those with insignificant staining or monocytic ANAE isoenzymes (defined by IEF). All cases were phenotypically CD13/CD33 positive and, with one exception, had greater than 30% HLA-DR positive cells. Membrane CD14 expression was insignificant or variable in 33% of M5a cases in contrast to 23/24 M5b cases which showed high proportions of CD14-staining cells with at least two monoclonal antibodies. Serum lysozyme, LDH and beta-2 microglobulin (beta 2m) were increased in 88%, 68% and 81% of cases respectively but, with the exception of statistically higher lysozyme levels in CD14+ cases, were unrelated to the morphological, cytochemical or immunological diagnostic subgroups. Clinical and diagnostic features were also examined as possible prognostic indicators. The morphological, cytochemical and immunological subgroups of AMoL were not found to be of prognostic relevance but age (P = 0.004), renal failure (P = 0.005) and serum beta 2m levels (P = 0.002) were related to patient survival. Moreover, renal failure and serum beta 2m remained significant (P = 0.012 respectively) when age was taken into account and were shown to be independent prognostic variables.
Plasmids containing the vaccinia virus thymidine kinase gene, its flanking DNA sequences, and the Escherichia coli beta-galactosidase gene were used in conjunction with a thymidine kinase-deficient virus to examine the viral products of recombination. Progeny derived from single-crossover events could be distinguished from those generated by gene conversion or double-crossover events when the beta-galactosidase gene was separated from the thymidine kinase gene by the flanking sequences. Using methotrexate to select for recombinant virus and a chromogenic indicator to detect beta-galactosidase, the generation of viral recombinants was measured over a 48-h period. Recombinant progeny were first observed at 12 h and increased to a maximum of 2.5% at 48 h. Single-crossover products, as determined by beta-galactosidase expression, reached a maximum of 57% of the recombinant population at 24 h and thereafter declined. DNA hybridization analysis was used to examine genomic structures of the progeny of the initial viral plaques, plaques purified three times, and those subject to a 10(4)-fold amplification. These analyses confirmed that single-crossover events within either the 5'- or 3'-homologous flanking sequences generated unstable recombinant structures. These structures were shown to contain a single copy of the intact thymidine kinase gene within the corresponding copy of the duplicated thymidine kinase flanking sequences, separated by the beta-galactosidase gene and plasmid DNA. Significantly, these duplicated structures could undergo further recombination to produce repeats of either the intact or the deleted thymidine kinase sequences. These intermediate structures ultimately degenerated to produce either the parental thymidine kinase-deleted or the wild-type genome. The wild-type genome was also shown to be generated directly by gene conversion or double-crossover events.
Adenovirus E1A gene products are capable of modulating the expression of a variety of integrated genes. To study the mechanisms by which this regulation occurs, recombinant retroviruses have been utilized to establish cell lines containing an integrated copy of either the adenovirus E2 or E3 promoter adjacent to the bacterial guanine phosphoribosyl transferase (GPT) gene. These cell lines have been characterized with respect to both basal and E1A-induced levels of GPT gene expression. Cell lines with low levels of GPT gene expression showed increased expression in the presence of E1A, whereas cell lines with high basal levels of GPT gene expression had decreased GPT RNA levels in the presence of E1A. Further characterization of these cell lines revealed E1A modulation of the accumulation of RNA initiating at a retrovirus promoter adjacent to the E2 or E3 promoter. The use of the GPT gene as a marker of E2 or E3 promoter activity has allowed the isolation of cell lines which have spontaneously increased their levels of GPT RNA. A preliminary characterization of four of these cell lines has indicated that GPT gene expression is increased as a result of cis activation of the E2 promoter.
The expression of membrane transferrin receptors (TfRs), as defined by monoclonal antibody OKT9, and the nuclear proliferation-associated antigen Ki-67 were examined in 159 cases of hematological malignancy. Of the "chronic" B and T cell leukemias studied (n = 85), 61% showed less than 5% OKT9-positive cells and only 7% of cases were TfR+ (defined as greater than 20% positive cells). For comparison, the acute leukemias (n = 62) showed higher (p less than 0.001) TfR expression with 39% TfR+ cases, although there was considerable variation within diagnostic subgroups. Nuclear Ki-67 expression was generally insignificant (less than 1%) in chronic leukemias (78 of 88 cases), although two of eight B cell-type prolymphocytic leukemia and four of four cases of plasma cell leukemia showed greater than 10% Ki-67+ components. In contrast, 47% (31 of 66) acute leukemias had greater than 10% Ki-67+ cells, although there appeared to be no relationship between Ki-67 expression and leukemic type. Combined assessments of TfR and Ki-67 expression revealed a Ki-67- TfR- phenotype in 82% of chronic leukemias, compared with 28% of acute type, and a Ki-67+ TfR+ pattern was found in 27% of acute proliferations but not in any case of chronic leukemia. The determination of membrane TfR expression appears to have little value in the diagnostic differentiation of leukemias, whereas Ki-67 is considered to be a useful supplementary investigation in defining high grade tumors, in the recognition of prognostically poor cases of otherwise well defined low grade malignancy, and of potential value in resolving discrepancies between morphological and immunophenotypic features in leukemias of "intermediate" type.
Any extensive analysis of Hodgkin (H) and Reed-Sternberg (RS) cells is limited by the scarcity of available material and by the common contamination with "by-stander" cells. The establishment of Hodgkin's disease-derived cell lines provides the opportunity to undertake studies in which large numbers of cells are required, as these cell lines are by definition monoclonal populations with unlimited cell growth. In this study, we analyzed the enzyme profiles of eight Hodgkin's disease cell lines (Co, Ho, Fox, HDLM-2, KM-H2, L428, L540, and L591) whereby cellular alpha-naphthyl acetate esterases, acid phosphatases, and dipeptidylpeptidase IV were examined quantitatively or qualitatively by IEF or chromatographic techniques. The results indicate that all of the H-RS cell lines examined had enzymatic features typical for lymphoid cells and, in particular, a monocyte/histiocyte origin of the cell lines could be excluded. Extrapolation of the available immunological, molecular biological, and enzymological evidence gained in vitro on cultured representatives of H-RS cells suggests a lymphoid origin for in vivo H-RS cells.
Of 417 cases of chronic lymphocytic leukaemia (CLL) examined morphologically, 346 (83%) showed low (less than 10%) numbers of cells with prolymphocytoid (PLC) morphology. Immunological studies in 267 of these cases, with particular reference to membrane surface immunoglobulin (SIg) densities and FMC7 expression, revealed that of 223 cases with insignificant prolymphocytoid components, 185 were phenotypically consistent with typical CLL whilst the other 38 cases showed significant (greater than 20% positive cells) FMC7 expression and/or increased SIg density. In contrast, 40 of the 44 cases with greater than 10% PLC showed atypical immunophenotypic features even though the expression of individual membrane components associated with B-cell differentiation appeared to be unrelated. MRBC receptor expression showed little correlation with the degree of prolymphocytoid change although all TU1- cases showed greater than 10% PLC. The results suggest that abnormal phenotypic features, similar to those observed in "prolymphocytoid" transformation, may be found in cases of CLL in the absence of morphological change.
Leukaemic myeloid and lymphoid cell N-acetyl beta-D glucosaminidase (hexosaminidase) enzymes were fractionated by Fast Protein Liquid Chromatography (FPLC) using high-resolution ion exchange (Mono-S and Mono-Q), gel filtration (Superose-6) and chromatofocusing (Mono-P) columns. Although only one molecular weight species was detected in haemopoietic cells, with an apparent mass of 129 kD, "isoenzyme" variants defined by differences in molecular charge were considerably more diverse than previously thought. Separation of the major Hex forms (A and B) by chromatofocusing indicated that intermediate (I) components were present in most acute leukaemias irrespective of lineage supporting the concept that the I form is a non-specific marker of haemopoietic immaturity. Substrate and inhibitor studies further revealed that leukaemic cell hexosaminidases hydrolyse galactopyranosides at significantly lower rates than glucopyranosides and that the hydrolysis of N-acetyl beta-D glucosamine is inhibited by both glucosamine and galactosamine products.
Ninety-seven cases of chronic myelomonocytic leukaemia (CMML) were examined retrospectively for survival and possible prognostic factors including age, total white cell count, peripheral blood and bone marrow monocyte counts, % double esterase (DE) positive cells in bone marrow and serum lysozyme. Age, absolute monocyte counts and serum lysozyme proved to be significant independent prognostic indicators but Cox model analyses showed serum lysozyme to be the most important factor whether taken as a continuous or discrete (two groups) variable. Twelve cases of second malignancy were found, including 2 cases of multiple myeloma, but this was not significantly greater than expected when compared with an age and sex matched group.
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