Mary Sue Coleman

Overproduction of adenine deoxynucleosides and deoxynucletides in adenosine deaminase deficiency with severe combined immunodeficiency disease.

The deoxynucleotide, dATP, is elevated 50- to 1,000-fold above normal in erythrocytes, lymphocytes, and bone marrow from a child with adenosine deaminase deficiency and severe combined immunodeficiency disease. The child, when 17 mo of age, was also excreting approximately 30 mg of deoxyadenosine per day in urine (normal is less than 0.1 mg/day). Urinary excretion of uric acid was decreased. Elevated dATP levels in lymphocytes and bone marrow, and increased urinary excretion of deoxyadenosine, persisted despite hypertransfusion of the child with irradiated erythrocytes from a donor with normal adenosine deaminase. Overproduction of deoxynucleotides by increased salvage of adenosine appears to be the primary metabolic abnormality in patients with adenosine de aminase deficiency.

Terminal deoxynucleotidyltransferase distribution in neoplastic and hematopoietic cells.

In the present study, terminal deoxynucleotidyltransferase was examined in the peripheral blood and (or) bone marrow of 115 children with a variety of neoplastic, hematologic, and other unrelated disorders. Terminal deoxynucleotidyltransferase activity was present at 4.08+/-0.74 U/108 cells in 23 morphologicall normal bone marrow samples from childhood controls. Terminal transferase was present at greater than 23 U/108 nucleated cells and at greater than31 U/108 blasts in the bone marrow of all children with acute lymphoblastic leukemia studied at initial diagnosis and at disease relapse. Terminal deoxynucleotidyltransferase was detectable at low levels, less than 7.5 U/108 cells, in all remission marrow smaples. Bone marrow terminal transferase activity was markedly elevated in all untreated acute lymphoblastic leukemia patients, whereas low levels which were difficult to interpret were present in the peripheral blood samples of two patients at diagnosis and six patients at relapse who had low absolute lymphoblast counts. Because of greater variation in the lymphoblast content of peripheral blood, bone marrow assays are more reliable in detecting disease activity. Marrow terminal deoxynucleotidyltransferase values obtained during the active phase of acute lymphoblastic leukemia were significantly greater than those found in other types of leukemia, bone marrow malignancies, and hematologic disorders. Terminal transferase determinations in blast cells of two patients with leukemic conversion of non-Hodgkins lymphoma and in tumor cells from one patient with Burkitts lymphoma were within the control range. These dat further define the usefulness of terminal deoxynucleotidyltrnasferase assay in the differentiation and classication of hematologic malignancies.

Terminal Deoxynucleotidyl Transferase Measurements in the Differential Diagnosis of Adult Leukaemias

Summary. Terminal deoxynucleotidyl transferase (TDT) is an unusual DNA polymerase that does not use template information to synthesize new strands of DNA. It is normally found in high concentration in thymus (50 u/108 cells) and in low concentration in bone marrow (< 5 u/108). We report TDT measurements in the marrow and/or peripheral blood of 51 adult patients, 28 of whom had leukaemia. TDT is present in very high levels (> 50 u/108 cells) in leukaemic lymphoblasts and in low levels in leukaemic myeloblasts (< 9 u/108 cells). Of two patients who developed lymphosarcoma‐cell leukaemia following treatment of poorly differentiated lymphocytic lymphoma, one had high and one low levels of TDT in the leukaemic blast cells. Leukaemic cells from three of seven patients with chronic myeloid leukaemia in blast crisis had TDT levels within the range expected of acute lymphoblastic rather than acute myeloid leukaemia. High TDT in leukaemic cells probably marks them as derivatives of lymphoid progenitor, thymic or pluripotential stem cells. Quantitative assay of TDT may provide information useful in classifying haematological neoplasms.

Micromethod for quantitation of adenosine deaminase activity in cells from human peripheral blood

Abstract Adenosine deaminase (ADA) converts adenosine to inosine. The enzyme can be assayed simply and conveniently by utilizing [ 14 C]adenosine as substrate and separating the [ 14 C]inosine from the reaction mixture by chromatography on short strips of DE-81 paper. The method is particularly useful in assay and characterization of ADA in small numbers of cells. Lymphocytes, granulocytes, and erythrocytes were isolated from 10 ml samples of human peripheral blood, and ADA activity was measured in each cell type. Extracts of erythrocytes typically contain specific activities of ADA ranging from 1.5 to 2.7 nmoles [ 14 C]inosine produced/10 8 cells/min, while lymphocyte and granulocyte extracts contain 50 to 100 times as much ADA activity. The radiometric assay can be used to obtain quantitative information about the behavior of ADA during velocity sedimentation and acrylamide gel electrophoresis. Lymphocytes have both a low and high molecular weight species of ADA. Granulocyte and erythrocyte extracts have a single low molecular weight species. Isoelectric focusing of extracts from the three cell types reveals one major enzyme activity peak with a pI of about 5. These methods should prove useful in studies of the association of quantitative and qualitative variation in ADA activity with human immunodeficiency diseases.

Terminal deoxynucleotidyl transferase and DNA polymerase in classes of cells from rat thymus.

Summary Cells from rat thymus have been fractionated on albumin density gradients. The activities of terminal deoxynucleotidyl transferase, DNA dependent DNA polymerase, thymidine kinase, and the rate of incorporation of thymidine into DNA vary among the classes of cells. Terminal transferase activity present in thymus is quantitatively recovered from the gradient and is highest in fractions rich in small lymphoid cells or thymocytes.

Biochemical and Functional Abnormalities in Lymphocytes from an Adenosine Deaminase-deficient Patient during Enzyme Replacement Therapy

Biochemical and immunological properties of lymphocytes were measured repetitively over a period of 40 mo during enzyme replacement by transfusion in a child with adenosine deaminase (ADA) deficiency and severe combined immunodeficiency disease. Catalytically defective ADA protein is present in the childs cells. ADA activity in his lymphocytes is 7 nmol/min per 10(8) cells with 51 ng of ADA protein/10(8) cells by radioimmunoassay. ADA activities in normal cord and adult lymphocytes average 193 and 92 nmol/min per 10(8) cells, respectively, with 429 and 223 ng of ADA protein/10(8) cells. Deoxy(d)ATP accumulates in the patients erythrocytes and lymphocytes. Transfusion of irradiated packed erythrocytes partially corrects the metabolic defects. Frank metabolic relapse occurs if transfusions are discontinued for several months. The amounts of dATP in erythrocytes and lymphocytes averaged 13 and 2 times normal, respectively, during periods when transfusions were administered every 2-4 wk. Deoxyguanosine triphosphate and deoxycytidine triphosphate in lymphocytes were normal on 11 occasions, but deoxyribosylthymine triphosphate was ninefold increased. On 11 occasions dATP was measured in lymphocytes and erythrocytes isolated simultaneously. There was a positive, but statistically insignificant, correlation between amounts of dATP in the two types of cells (r = 0.25,P > 0.1). The absolute peripheral lymphocyte count was correlated with the activity of ADA in circulating erythrocytes and with the response of lymphocytes to phytohemagglutinin (r = 0.64, P < 0.01; r = 0.49, P < 0.05). Response of lymphocytes to stimulation by phytohemagglutinin in vitro and absolute peripheral lymphocyte counts were not significantly correlated with levels of dATP in the erythrocyte or lymphocyte during periods of intensive therapy. Although there was objective improvement during enzyme replacement, the child remained immunodeficient and biochemically abnormal.

The relationship of hair zinc concentrations to height, weight, age, and sex in the normal population.

Hair samples from forty-nine normal individuals (both children and adults) were assessed for concentrations of zinc. Pearson correlation coefficients were computed between zinc values and the variables: height, weight, and age (see Table 3). In children (under 240 months), all these relationships were linear, positive, and statistically significant. A linear regression equation using all these variables was found to account for 47.7% of the variance in hair zinc concentrations. In adults (over 240 months), the correlation coefficients between hair zinc and height, weight, and age were not found to be significant, with the exception of the negative correlation between hair zinc and weight (r = -0.464; P less than 0.047). Nevertheless, a multivariate linear regression equation accounted for about 24.6% of the variability of hair zinc values. In both children and adults, tests for sex differences in means and standard deviations using both raw score and residual values failed to reveal any significant differences. Similarly, no significant sex differences were observed between corresponding correlation coefficients. Results indicate that future studies utilizing hair must systematically or mathematically control for individual variation in zinc concentrations due to differences in age, weight, and height.

Biochemical and Immunological Properties of Human Terminal Deoxynucleotidyl Transferase Purified from Blasts of Acute Lymphoblastic and Chronic Myelogenous Leukemia

Terminal deoxynucleotidyl transferase was purified to homogeneity from the blasts of eight patients with leukemia and compared with purified transferase from normal human and calf thymus. In two cases phenylmethanesulfonylfluoride was added during purification to reduce proteolysis. Comparative kinetic analyses of the purified enzymes indicated no differences in catalytic properties. There was substantial variation in the molecular structure of terminal transferase on denaturing polyacrylamide gels: (a) a protein that migrated as a single polypeptide with M(r) = 62,000 was isolated from two patients with acute lymphoblastic leukemia and from MOLT-4 cells; (b) a protein that migrated as a single polypeptide with M(r) = 42,500 was isolated from two patients with acute lymphoblastic leukemia; (c) a protein that migrated as a single polypeptide with M(r) = 42,500 was isolated from two patients with chronic myelogenous leukemia in blast crisis; (d) a protein that migrated as two non-identical subunits of M(r) = 27,000 and 10,000, respectively, was isolated from two additional patients with chronic myelogenous leukemia in blast crisis. The subunit structure of d is characteristic of the homogeneous enzymes purified from human and calf thymus. Neutralizing and precipitating antibodies to terminal transferase from human lymphoblasts and calf thymus have been produced in rabbits and goats. Antisera directed against either human or calf antigens neutralize enzymatic activity and precipitate all forms of human terminal transferase. The multiple human forms give reactions of antigenic identity by immunodiffusion, but differ antigenically from the calf enzyme. The multiple forms of terminal transferase could represent physiological processing, artifactual degradation, or isozymes coded by several genes.

Terminal deoxynucleotidyl transferase: characterization of extraction and assay conditions from human and calf tissue.

Abstract Methods of extraction and assay of terminal deoxynucleotidyl transferase (TdT) from human lymphoblasts and calf thymus were compared. A high salt concentration was mandatory for complete enzyme extraction, while dialysis of the crude extract resulted in a major loss of enzyme activity. In addition, TdT was partially purified from lymphoblasts of patients with acute lymphoblastic leukemia. The Km for the monomer, deoxy-guanosine 5′-triphosphate (dGTP), is high (~0.1 m m ) in the presence of either Mg2+ or Mn2+, whereas the Km for the initiator, poly(deoxyadenylic acid [poly(d(pA) 50 )] , with an average chain length of 50 residues, is 2.5 μm in the presence of Mg2+ and 0.3 μm in the presence of Mn2+. The maximum velocity is higher for the calf thymus TdT in the presence of Mg2+ than in Mn2+. Human TdT catalyzes the polymerization of dGTP at a higher rate in the presence of Mn2+ than with Mg2+. These data illustrate that partially purified human TdT differs in catalytic properties from the purified calf thymus enzyme. Therefore, optimal conditions for assay of TdT in extracts from calf and human tissues differ.

Immunological and biochemical profiles in response to transfusion therapy in an adenosine deaminase-deficient patient with severe combined immunodeficiency disease.

Abstract A child with severe combined immunodeficiency (SCID) and adenosine deaminase (ADA) deficiency has been treated with irradiated frozen red blood cells and irradiated cryoprecipitate-removed fresh frozen plasma. This therapy has been beneficial clinically and the patient has been free of infection for 24 months. Partial immune reconstitution has occurred. The cellular immune response has varied greatly between markedly decreased and near normal function. In spite of a progressive increase of immunoglobulins to normal levels, specific antibody production has been very poor and in vitro immune functions have been variable with low or transiently normal responses. Activities of ADA in red cells, lymphocytes, and granulocytes were low at the time of diagnosis and have remained low in lymphocytes and granulocytes despite transfusion of red cells with normal levels of ADA.