R.A. Good

TRANSPLANTATION FOR ACUTE LEUKAEMIA WITH HLA-A AND B NONIDENTICAL PARENTAL MARROW CELLS FRACTIONATED WITH SOYBEAN AGGLUTININ AND SHEEP RED BLOOD CELLS

A new procedure for enrichment of marrow precursors and removal of T lymphocytes from large volumes of human bone marrow, involving initial differential agglutination of T lymphocytes and mature marrow elements with soybean agglutinin, followed by rosetting with sheep red blood cells, was used to fractionate marrow cells from an HLA-A, B, DR non-identical, MLC non-reactive, paternal donor for transplantation into an infant with acute leukaemia. This transplant became completely engrafted and resulted in full recovery of normal, donor-derived haematopoietic function without graft-versus-host disease, sustained for 11 weeks after transplantation, at which time the patients leukaemia recurred. Subsequently, the patient received chemotherapy and achieved a remission with regeneration of normal marrow cells of donor origin. The patients course demonstrated the potential of lectin-separated marrow grafts to restore durable haematopoiesis, without graft versus host disease, in a lethally irradiated allogeneic host.

Circulating thymic hormone levels in zinc deficiency

Abstract The effect of zinc deficiency (Zn − ) on the circulating thymic hormone (FTS) levels in A/J mice was studied. After 3 weeks of feeding the mice a Zn − diet, FTS levels were markedly reduced and after 17 weeks, FTS was undetectable. By contrast, the zinc-supplemented (Zn + ) group seemed to maintain FTS levels better than the normal diet group with aging. On the other hand, spleen spontaneous rosette-forming cells (sRFC) were studied for their azathioprine (AZ) sensitivity in A/J mice on different diets. The Zn − mice had fewer sRFC than did the normally fed or Zn + mice. The role of zinc in controlling levels of FTS and thus thymic function is discussed.

ALLOGENEIC BONE MARROW TRANSPLANTATION USING STEM CELLS FRACTIONATED BY LECTINS: VI, IN VITRO ANALYSIS OF HUMAN AND MONKEY BONE MARROW CELLS FRACTIONATED BY SHEEP RED BLOOD CELLS AND SOYBEAN AGGLUTININ

A procedure was developed for the isolation from human bone marrow of a cell fraction enriched for haematopoietic precursors and depleted of T lymphocytes. T cells are eliminated from bone marrow by rosetting with sheep red blood cells, followed by differential agglutination of residual T lymphocytes in the non-rosetting population by the lectin, soybean agglutinin. The fraction unagglutinated by the lectin contains a high proportion of colony-forming cells and non detectable T cell alloreactivity in vitro. Similar results were obtained with monkey bone-marrow cells, suggesting that monkeys can be used for evaluation of this fractionation technique for bone-marrow transplantation across histocompatibility barriers.

Defective cellular immune responsein vitro in common variable immunodeficiency

Mononuclear cells from 39 patients with hypogammaglobulinemia of the common variable type were analyzed forin vitro proliferative responses to a panel of cell activators in order to examine the lymphocyte response to mitogens and to study the capacity to generate an immunologically specific secondary response. Patient lymphocyte response to phytohemagglutinin and concanavalin A was found to be significantly lower than that of controls studied in parallel (P<0.01), and low response did not correlate with T-lymphocyte number. Response to pokeweed mitogen was significantly lower than that of controls (P<0.01), but response to zinc, tested in a few patients, was normal. Strong depressions of patient lymphocyte proliferative responses toCandida albicans, Escherichia coli, andStaphylococcus aureus were observed (P<0.01); all of these microbial activators require intact B-cell function for maximum response. Repeated testing of individual patients indicated that poor lymphocyte response could be consistently observed. Examination of changes inin vitro lymphocyte response during clinical course and disease management showed that a consistent pattern of intrinsic lymphocyte functional deficiency could be demonstrated.

CIRCULATING THYMIC-HORMONE ACTIVITY IN CONGENITAL IMMUNODEFICIENCY

Circulating thymic-hormone activity was assayed by measuring Thy 1-2 antigen induction on null lymphocytes from athymic mice incubated with human plasma or serum. Plasma from 19 normal children aged under 10 had inductive activity equivalent to 10-6-16-2 ng thymopoitin/ml. Plasma from 15 infants were severe combined immuno-deficiency, 2 of whom had appreciable immunoglobulin synthesis, and from 2 infants with DiGeorge syndrome had little or no inductive activity. Successful reconstitution with thymus or bone-marrow grafts and with red-cell infusions (if adenosine-deaminase deficiency is present) was followed by a rise in circulating thymic-hormone activity.

Defective expression of T cell-associated glycoprotein in severe combined immunodeficiency.

A T cell surface membrane-associated glycoprotein, Tp40 (40,000 mol wt), also designated as CD-7, was not expressed by the T cells of a patient with severe combined immunodeficiency. In addition to this abnormality, T cell proliferative responses to mitogens were defective and the IL-2 receptor expression was deficient on the patients T lymphocytes. However, his T cells were found to provide help for the differentiation of normal B cells to Ig-secreting cells. Abundant circulating B cells were detected. These B cells proliferated normally in the presence of anti-mu antibodies and B cell growth factors, but did not differentiate into antibody-secreting cells when provided with the help of normal T cells. In addition, his activated B cells did not proliferate to IL-2 even though IL-2 receptors were expressed. A successful allogeneic histocompatible bone marrow transplantation resulting in T cell engraftment corrected both the T and B cell immunodeficiencies. These findings support the hypothesis that the Tp40 deficiency present in this patient is related to a defect of the T cell precursors, and that Tp40 plays important roles not only essential to T cell interactions but also to certain aspects of T-B cell interaction during the early lymphoid development.

Zinc deficiency, depressed thymic hormones, and T lymphocyte dysfunction in patients with hypogammaglobulinemia

Abstract Zinc deficient humans and animals have depressed thymic mass and increased susceptibility to infection. In the present studies, we investigated the relationship between cellular immunity, thymic hormones, and serum zinc levels in 19 patients with common varied immunodeficiency. Five (26%) had serum zinc levels 2 SD below normal and 11 (58%) had abnormally low lymphocyte proliferation to at least one mitogen. A significant statistical correlation between zinc levels and lymphocyte proliferation to phytohemagglutinin and concanavalin A was identified. Forty-two percent had abnormally low levels of facteur thymique serique and 74% had low levels of thymopoietin, although no statistical relationship between the levels of these hormones, zinc levels, or lymphocyte proliferation could be identified. Three patients with the most profound zinc deficiency had substantial increases in thymic hormones after zinc repletion, and two had complete resolution of intractable diarrhea. A therapeutic potential of zinc for certain patients with hypogammaglobulinemia is suggested.

The differential sensitivity of T-Cell and B-Cell mitogenesis to in vitro zinc deficiency

Abstract The relative requirement for zinc in B-cell and T-cell mitogenesis was investigated utilizing a tritiated thymidine uptake assay. When ethylene diaminetetraacetate (EDTA) was added to the culture medium at concentrations less than 0.02 m M , B-cell mitogenesis, induced by lipopolysaccharide B (LPS), was not affected. However, T-cell mitogenesis, induced by either phytohemagglutinin P (PHA) or concanavalin A (Con A), was inhibited. When fetal calf serum (FCS) dialyzed against EDTA was used in the medium, B-cell mitogenesis was again unaffected, whereas T-cell mitogenesis was reduced by nearly one-half, even in the absence of EDTA in the medium. Cell viability was practically unaffected by the dialysis procedure. However, the addition of EDTA at concentrations of 0.01 to 0.05 m M reduced the percentage of viable cells by about one-third. Cell viability was reduced to practically zero at a concentration of 0.5 m M EDTA. The inhibitory effect of EDTA on Con A-stimulated mitogenesis was completely reversed by zinc, only partially reversed by nickel, and unaffected by either calcium or magnesium. Thus it appears that B-cell mitogenesis is not affected by zinc deficiency in vitro , whereas T-cell mitogenesis is substantially inhibited, indicating the requirement of zinc for T-cell function.

Changes in macrophages and their functions with aging in c57bl/6j, akr/j and sjl/j mice.

Abstract Changes in phagocytosis, chemotaxis, and nonspecific esterase staining of peritoneal exudate mononuclear cells occurred with aging in B6, AKR/J, and SJL/J mice. Different patterns of change with aging were observed with each of these three parameters in each of the strains. In the long-lived B6 mice, a progressive increase with aging in all three parameters was seen. In AKR/J mice, progressive increase in phagocytosis and nonspecific esterase staining was observed but a decline of chemotaxis occurred with aging. In SJL/J mice, all three parameters were very low early in life and each showed a significant increment upon reaching the age when the characteristic malignant lymphoreticular disease occurs.

A micromethod for determination of terminal deoxynucleotidyl transferase (TdT) in the diagnostic evaluation of acute leukemias

SummaryA micromethod for the determination of TdT in peripheral leukocytes and bone marrow cells has been developed that allows unequivocal identification and quantitation of TdT in less than 1 x 106 leukocytes from ALL patients, i.e., in 1 ml of peripheral blood and/or 0.5 ml of bone marrow obtained during routine clinical sampling. The method involves disruption of cell pellet with high salt and detergent followed by centrifugation of extracts at 12,000 x g and partial purification on phosphocellulose matrix by a batch elution technique using a standard laboratory microcentrifuge. Using this microassay, TdT activities have been determined in 500 samples of peripheral blood and bone marrow of 240 adult patients with acute leukemias (86 ALL, 108 ANLL, 44 blastic CML, two acute leukemias following P. vera). From an analysis of our data based on TdT activity, cell surface markers and growth patterns in soft agar and observations published in the literature, it can be concluded that the frequencies of TdT + phenotypes in the various clinicalmorphological diagnostic groups are approximately 95% in ALL, 10% in ANLL, 50% in AUL, and 35% in blastic CML. Since the presence of high TdT activity is clearly associated with clinical response to specific forms of chemotherapy in blastic CML and most probably, also in ANLL, the determination of TdT should be considered in all cases of acute leukemias to objectively define prognostically important subgroups which can not be diagnosed by conventional means.