A role for adenosine deaminase in human monocyte maturation.

The occurrence of a deficiency of adenosine deaminase (ADA) activity in some patients with severe combined immunodeficiency suggests a possible relationship between the activity of ADA and the aberration of the immune system. To help delineate the function of ADA in the immune response we have examined its role in monocyte maturation. When incubated in vitro, peripheral blood monocytes transformed, within 3 days, to macrophagea as assessed by phase-contrast microscopy and an increase in the specific activity of the lysosomal enzyme acid phosphatase. The specific activity of ADA increased as much as ninefold, reaching a peak after the 1st day in culture, while the activities of other enzymes involved in the purine salvage pathway were not altered. Sucrose density ultracentrifugation of extracts prepared immediately after the isolation of monocytes revealed the presence of two forms of ADA with molecular weights of approximately 30,000 and 110,000. The increase in ADA specific activity during monocyte cultivation correlated with an increase in the activity of the smaller molecular species. A specific inhibitor ADA, erythro-9-(2-hydroxy-3-nonyl) adenine, prevented the increase in acid phosphatase activity, as well as the morphological changes associated with the monocyte maturation. These data suggest a role for ADA in monocyte to macrophage maturation. In view of the central role of macrophages in immune function, this observation may relate to the association of combined immunodeficiency and a deficiency of this enzyme.

Thymidine kinase isoenzymes in human malignant lymphoma.

The activities of thymidine kinase (TK) isoenzyme 1 and 2 were examined in extracts of human benign or malignant lymphoid tissue and correlated with degrees of morphological differentiation. TK2 activity occurred in peripheral blood lymphocytes of normal individuals, patients with chronic lymphocytic leukemia, or solid lymphoid tissue, exhibiting either nonneoplastic histological findings or those of diffuse well-differentiated lymphocytic lymphoma. TK1 activity occurred in solid, non-Hodgkin's lymphoma tissue, exhibiting lesser degrees of cellular differentiation, or in peripheral blood lymphocytes of patients with clinical aggressive chronic lymphocytic leukemia or lymphosarcoma leukemia. In non-Hodgkin's lymphoma tissue, the range of TK1 activities correlated broadly with the Rappaport classification, with higher values occurring in tissue exhibiting changes of diffuse poorly differentiated lymphocytic lymphoma or diffuse histiocytic lymphoma.

Kinetic mechanism and inhibition of human liver thymidine kinase.

Thymidine kinase (ATP : thymidine 5'-phosphotransferase, EC 2.7.1.21), purified to apparent homogeneity from human liver, was found to have Michaelis constants for thymidine and ATP of 5 and 90 microM, respectively. Based on studies of initial velocity and product inhibition, the enzyme kinetic mechanism is compatible with an ordered sequential reaction with thymidine binding first and thymidine monophosphate released last. The activity of various triphosphate nucleosides as phosphate donors for human liver thymidine kinase showed little specificity with ATP greater than CTP greater than UTP greater than GTP and the respective Michaelis constants ranged from 0.10 to 0.30 mM. Among various purine and pyrimidine compounds, only TTp and dCTP were effective inhibitors of the enzyme. Inhibition with TTP was competitive with respect to both thymidine and ATP with Ki values of 13.5 and 8.5 microM, respectively, while the inhibition produced by dCTP was complex. Deoxycytidine was found to be an effective nucleoside substrate for human liver thymidine kinase with a Michaelis constant of 6 microM. This finding suggests that human mitochondrial deoxycytidine and thymidine kinase activity is a single protein.

Purine and pyrimidine activities in acute and chronic lymphocytic leukaemia: relation to cellular proliferative status.

The cellular levels of the purine catabolic enzymes adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) and those for the pyrimidine activities thymidine phosphorylase and thymidine kinase isozymes have been measured concurrently in peripheral blood nucleated cells of patients with acute lymphoblastic leukaemia, chronic lymphocytic or prolymphocytic leukaemia and correlated with the spontaneous tritiated thymidine uptake of the isolated cells. Highest ADA levels occurred in T-ALL cells but considerable overlap of individual activities occurred for non-T, non-BALL, B-CLL and T-CLL cells. The levels of PNP showed no distinct discriminatory trend in cells of the lymphoid proliferative disorders examined. Thymidine phosphorylase activity was markedly reduced in T-ALL and T-CLL cells with a stepwise increase in the level of mean activities for non-T, non-B ALL, B-CLL and B-PLL cells to that of isolated normal peripheral blood lymphocytes. Spontaneous tritiated thymidine uptake of the abnormal lymphoid cells exhibited a correlation between cellular thymidine kinase isozyme 1 and elevated ADA levels. The use of ADA inhibitors together with thymidine infusion for the treatment of lymphoproliferative disorders is discussed.

Perturbation in the ability of bone marrow stroma from patients with acute myeloid leukemia but not chronic myeloid leukemia to support normal early hematopoietic progenitor cells.

The ability of bone marrow (BM) stroma derived from patients with acute myeloid leukemia (AML) or chronic myeloid leukemia (CML) to support normal hematopoiesis was investigated using a two-stage long-term bone marrow culture (LTBMC) procedure. Of particular interest was whether leukemia-derived stroma were capable of supporting the very immature, uncommitted hematopoietic progenitor cells (HPC) which are considered to have a higher dependence and association with the BM stroma than the more mature committed HPC. Confluent stromal layers were recharged with selected populations of normal HPC enriched for the CD34+CD38- cells (immature, uncommitted HPC) or the CD34+CD38+ cells (mature, committed HPC). The weekly output of clonable granulocyte-macrophage progenitor cells (CFU-GM) was used as an indicator of the hematopoietic-supporting ability of the cultures. Stromal layers derived from 5/7 patients newly diagnosed with AML, showed significantly depressed ability to support the CD34+CD38- cells compared to stroma derived from normal donors. The supporting function of the AML-derived stroma for the more mature CD34+CD38+ cells was similar to that of the normal stroma (3/3 cases). Stromal layers derived from patients with chronic-phase CML showed normal or enhanced supporting function for the CD34+CD38- cells (5/6 cases), and likewise for the CD34+CD38+ cells (3/3 cases). This study revealed a selective defect in the ability of BM stroma from patients with AML to support the maturation of normal early uncommitted HPC, represented by the CD34+CD38- cells, whilst the ability to support the committed CD34+CD38+ cells was not affected. This suggests that the BM microenvironment may be implicated in the disease mechanism of AML. It does not, however, appear to be as clearly implicated in chronic-phase CML.

Common expression of the multidrug resistance marker P-glycoprotein in B-cell chronic lymphocytic leukaemia and correlation with in vitro drug resistance.

The expression of P-glycoprotein (Pgp), which is associated with multidrug resistance (MDR), was investigated in 20 B-cell chronic lymphocytic leukaemia (B-CLL) patients by flow cytometry using two Pgp-specific monoclonal antibodies (mAb), MRK-16 which recognizes an extracellular epitope, and JSB-1 which recognizes an intracellular epitope. Sixteen (80%) patients were positive with MRK-16 whereas all patients were positive with JSB-1. The proportion of Pgp-positive lymphocytes from each patient sample varied from 2-94% for MRK-16 and 20-93% for JSB-1. There was no correlation between the level of positivity and disease stage or treatment history. In vitro drug resistance to vincristine (VCR) and doxorubicin (DOX) was determined by the colorimetric MTT assay. All patients were resistant to one or both drugs being consistent with the expression of Pgp. There was no correlation between the level of resistance and disease stage or drug treatment. We investigated the expression of Pgp in the normal counterpart of the B-CLL cells, CD5+CD19+ B-lymphocytes. A minor subpopulation (3%) of CD5+CD19+ lymphocytes isolated from normal controls expressed Pgp suggesting that these cells may be the potential precursors to the B-CLL cell. We conclude that Pgp expression and drug resistance are inherent characteristics of the B-CLL lymphocyte.

Expression of CD45 isoforms in chronic B-cell leukaemias.

CD45 isoform expression was studied in 204 cases of B-cell lymphoproliferative disease, including 162 chronic lymphocytic leukaemia (CLL). In almost half the samples tested, CD45R0 was co-expressed with CD45RA, unlike normal B-cells, which express only CD45RA, except at terminal stages of differentiation. In a small number of cases CD45R0 was the dominant isoform expressed. No correlation could be discerned either with Binet or RAI staging in CLL or with disease type CLL, prolymphocytic leukaemia (PLL) or hairy cell leukaemia (HCL). Comparison of CD45 isoform expression with other markers showed no correlation with apparent maturational status of the cells involved.

Vitamin E and dapsone-induced hemolysis.

Sixteen patients, each receiving 100 mg of dapsone per day, were studied for evidence of hemolysis. Vitamin E (dl-alpha tocopherol acetate), 800 mg/day, was then administered for up to three months, and dapsone therapy was continued at the same dose. Hemolysis factors were reexamined immediately prior to cessation of vitamin E therapy. No substantial change was demonstrable for levels of hemoglobin, reticulocyte count, and haptoglobin at the end of vitamin E therapy, despite a significant rise in serum vitamin E levels. Erythrocyte survival measured in four patients before and at the end of vitamin E therapy also showed no substantial change. Erythrocyte Heinz body count, however, fell in nine of 15 patients studied, and none showed an increase in this measurement while receiving vitamin E. We conclude that in patients receiving dapsone at 100 mg/day, vitamin E therapy at 800 mg/day does not substantially ameliorate the hemolytic effect of this drug.

A micromethod for determining adenosine deaminase and purine nucleoside phosphorylase activity in cells from human peripheral blood.

A radiochromatographic method is described for measuring adenosine deaminase and purine nucleoside phosphorylase activity in cells from human peripheral blood. The respective substrates, [8-14C]adenosine or [8-14C]inosine, are converted either to inosine and hypoxanthine or hypoxanthine, respectively. A single simple and rapid chromatographic procedure is used to isolate the products of both reactions. The mean normal activity (nmol h-1mg-1) of ADA for erythrocytes is 63 +/- 24 (+/- 1 S.D.) for leukocytes, 750 +/- 280 and for lymphocytes, 2105 +/- 1170. Corresponding activities for purine nucleoside phosphorylase are 1850 +/- 490, 3665 +/- 1170 and 5890 +/- 2030. With the described methods a further patient with severe combined immuno-deficiency and adenosine deaminase deficiency has been identified.

An improved method for the determination of human erythrocyte pyrimidine 5'-nucleotidase activity.

A radiometric method is described for measuring pyrimidine 5'-nucleotidase activity in human peripheral blood haemolysate. The substrate [14C]uridine monophosphate is converted to [14C]uridine. Two simple and rapid methods to isolate the product of the reaction employing either descending chromatography or elution from DEAE cellulose paper have been developed. With the described methods the specific activity of pyrimidine 5'-nucleotidase of human erythrocytes is 6.7 +/- 2.7 (+/- 1 S.D.) nmol/h/mg protein and this activity appears to be widely distributed in human tissue.

A rapid and simple radiometric assay for thymidine phosphorylase of human peripheral blood cells.

A radiometric method is described for measuring thymidine phosphorylase activity in human peripheral blood cells. The substrates [14C]thymidine or [14C]thymine are converted to the base or deoxynucleoside, respectively, and two alternative chromatographic methods to isolate the products of the reaction have been employed. With the described methods the specific activity for thymidine phosphorylase in human lymphocytes is 0.21 +/- 0.08; monocytes 0.21 +/- 0.16 and granulocytes 0.17 +/- 0.02 mu mol.h-1.mg-1 protein. For human T- or null lymphoblasts, thymidine phosphorylase activity was found to be approximately 10% of that of B lymphoblasts.

Hematological, biochemical and bone scan findings in patients with marrow carcinoma.

The peripheral blood hematological and biochemical parameters in 60 patients with marrow involvement with carcinoma are described. In all patients the hematologic findings were of anemia (46%), thrombocytopenia (36%), leukocytosis (28%) and leukoerythroblastosis (22%). Elevated serum lactate dehydrogenase (sLDH) and alkaline phosphatase (sALP) occurred in 78% of patients and hypercalcemia in 28% and these abnormal biochemical parameters occurred more frequently with marrow fibrosis. In patients with small cell carcinoma of lung (SCCL) the sLDH was normal in 24% and sALP in 32% and abnormal hematological findings or a raised sLDH occurred more frequently with liver involvement. Bone scan was positive in 49% of patients and the hematological and biochemical parameters in these patients were similar to those of patients with a normal scan. Ten percent of patients with SCCL and marrow involvement had both a normal sLDH and bone scan.

Clinching the diagnosis: macrocytic anemia.

Macrocytosis is a common laboratory finding. Whether this change requires further attention is dictated by clinical circumstances and concomitant cytopenias or aberrant erythrocyte and leukocyte morphology. The utility of these changes for dictating further investigation and the appropriate "modus operandi" in diagnostic strategies for the adult and the younger patient are outlined.

Heparin induced thrombocytopenia. Variable platelet-rich plasma reactivity to heparin dependent platelet aggregating factor.

The heparin dependent platelet aggregating factor in the serum from an individual with heparin induced thrombocytopenia exhibited reactivity with only 50% of an extensive panel of normal platelet rich plasmas, and this variability was independent of the platelet rich plasma platelet Factor 4 content. Individual sera of 3 other patients with heparin induced thrombocytopenia showed considerable variation in the lag phase and rate of platelet aggregation when tested with different normal platelet rich plasma preparations. These findings suggest that in the appropriate clinical setting the documentation of heparin dependent platelet aggregating factor should employ a panel of platelet rich plasmas if the initial screen is negative.

Exaggerated reaction to insect bites in patients with chronic lymphocytic leukemia. Clinical and histological findings.

We report three patients with chronic lymphocytic leukemia with unusual vesiculo-bullous skin eruptions on exposed areas which were more commonly noted in spring and summer. The lesions appeared to be due to insect bites and could be differentiated from other causes on the basis of histological, immunohistological and clinical features. There was no correlation between the occurrence of the skin eruptions, disease status and treatment modalities. Exaggerated cutaneous reaction to insect bites should be considered as a cause of vesiculo-bullous lesions in patients with chronic lymphocytic leukemia.

Basic ferritin content of red cells of patients with anemia and polycythemia vera.

Basic ferritin content of red cells has been evaluated with a simplified assay in subjects with various erythroid disorders. In 39 patients with iron deficiency anemia, red cell ferritin was significantly reduced compared with that of normal individuals. Thirty percent of these patients had low normal red cell ferritin content and the MCV for this group was significantly higher than that of patients with reduced red cell ferritin. The mean red cell ferritin of 30 subjects with the anemia of chronic disease was significantly reduced and patients in this group with normal red cell ferritin had higher plasma ferritin levels. In 14 patients with polycythemia vera, the mean red cell ferritin was significantly reduced and showed a positive correlation with the hemoglobin level and percent transferrin saturation. The red cell ferritin content of 9 individuals with acquired immune hemolytic anemia and 10 with acquired sideroblastic anemia was significantly elevated and, in subjects with immune hemolysis, showed a positive correlation with the reticulocyte count. These findings suggest a lack of discriminatory function for red cell ferritin in iron deficiency anemia and anemia of chronic disease. Evaluation of this parameter in the individual patient should take into account the presence of reticulocytosis.

Diagnostic information derived from immunotyping 1000 patients with leukemia and lymphoma.

Immunotyping analysis has been performed on cells from 1000 patients with leukemia or lymphoma using 14 markers of cell lineage or differentiation stage over a 4 yr period. Results showed considerable heterogeneity of cell type among these groups of malignant diseases not readily apparent by morphology and histochemistry. Immunotyping contributed additional diagnostic information in 30% of patients and should be a routine procedure in 8 disease categories. These are: acute leukemia, cell type not determined; acute lymphoblastic leukemia; lymphocytosis of undetermined origin; chronic myeloid leukemia-terminal blast crisis; chronic lymphocytic leukemia; malignant lymphoma-leukemic phase; Sézary syndrome, mycosis fungoides and chronic T cell leukemia; malignant lymphoma and lymphadenopathy- ? lymphoma. Immunotyping provided information on cell lineage and differentiation stage of major leukemic cell populations. Abnormal monoclonal proliferations of B lymphocytes and the presence of primitive cells amongst normally mature tissue cells were identified. Disturbances in normal lymphoid and monocytic cell populations in blood, marrow or tissues could also be demonstrated. Many of the reagents used in this period are now replaced by monoclonal antibody reagents to human lineage and differentiation antigens. These are expected to increase diagnostic usefulness of these techniques.

[Registration of clinical trials: a statement from the International Committee of Medical Journal Editors]. / Registratie van klinische trials: een verklaring van het International Committee of Medical Journal Editors.

Altruistic motives and trust are central to scientific investigations involving people. These prompt volunteers to participate in clinical trials. However, publication bias and other causes of the failure to report trial results may lead to an overly positive view of medical interventions in the published evidence available. Registration of randomised controlled trials right from the start is therefore warranted. The International Committee of Medical Journal Editors has issued a statement to the effect that the 11 journals represented in the Committee will not consider publication of the results of trials that have not been registered in a publicly accessible register such as www.clinicaltrials.gov. Patients who voluntarily participate in clinical trials need to know that their contribution to better human healthcare is available for decision making in clinical practice.