Biomedical subjects
Publications and source records attributed to H D Flad.
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Human peripheral blood T-lymphocytes were fractionated by discontinuous density gradient centrifugation on Percoll. T-cells of low density were capable of proliferation in the presence of the mitogen phytohemagglutinin (PHA) as measured by colony formation, whereas cells of high density were not. Addition of supernatant of PHA-stimulated cultures to cells of high density resulted in proliferation which suggested that these cell fractions lacked interleukin-2 (II-2) producing T-helper cells. This finding was supported by the demonstration that T-cells of high density did not produce II-2 but could be induced to do so by adding II-1 producing monocytes. The data area explained on the basis of the present model of intercellular events leading to activation and proliferation of lymphocytes.
The effect of X-irradiation on growth of T-lymphocyte colony forming units (TL-CFU) from human peripheral blood was investigated. The results indicate that not the number of activated TL-CFU but the number of cell cycles of colony forming cells was reduced by X-irradiation. Therefore we presume that TL-CFU belong to a relatively radio-resistant cell population within the PHA-responsive lymphocytes. Kinetic studies revealed that colony growth following irradiation was delayed mainly during the phase of the first cell cycle. Preculture of the cells for 24 hours after irradiation with 1,200 R in the absence of PHA caused total inhibition of colony growth in the subsequent agar culture. In the presence of PHA no inhibition was observed. This finding appears to reflect a repair mechanism from radiation damage of lymphocytes stimulated by PHA.
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A patient with severe combined immunodeficiency received three transplants of bone marrow from the HLA-B- and -D-identical mother. The first transplantation led to a severe graft-versus-host reaction followed by immunological reconstitution. A split chimerism was found with engraftment only of the maternal lymphocytes. Five months after the transplantation an autoimmune hemolytic anemia was observed which was due to rhesus incompatibility as well as polyspecific antibodies. At the same time agranulocytosis developed and 9 mth after the first transplantation the child suffered from aplastic anemia. Two further attempts failed to engraft the maternal hematopoiesis. The child died during the treatment with cyclophosphamide as conditioning for a third transplantation.
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A patient with an inborn cellular immunodeficiency syndrome developed a graft-versus-host reaction after a transfusion with packed red cells. This diagnosis was confirmed by skin biopsy and finally proved by tissue typing.
The use of a new commerically available medium (Percoll) for fractionation of human mononuclear leucocytes is described. Cells can be fractionated on the basis of their densities with high reproducibility. The separated cells were characterized by morphological and functional criteria. Monocytes can be obtained in the low density fractions with a purity of 70%--90%. Lymphocytes were found in high density fractions with a purity up to 99%. No separation between E-rosette forming (E-RFC) and surface immunoglobulin-bearing lymphocytes was obtained. However, a reduced number of high avidity E-rosette forming lymphocytes (HAE-RFC) was found within low density lymphocytes. Best spontaneous DNA synthesis and reaction in mixed leucocyte culture (MLC) were obtained with cells isolated from the 1.066/1.068 density interface, whereas the stimulation with phytohemagglutinin (PHA) or pokeweed mitogen (PWM) had a peak response with cells from the 1.064/1.066 density interface. Colony forming myelopoietic stem cells and colony forming T-lymphocytes were detected in fractions of low density.
In the presence of serum, 2-mercaptoethanol-treated bovine serum albumin enhances T cell colony formation as does 2-mercaptoethanol. The factor only partially substitutes for FCS, but neither for the mitogen phytohemagglutinin nor for conditioned medium derived from cultures of adherent cells.
A one-stage micro-culture assay in glass capillary tubes was developed for colony growth of human T lymphocytes. Colony growth by this method did not depend on the addition of growth factors, human serum and erythrocytes. Advantages of this method are its low consumption of materials and the fact that high numbers of cultures can be prepared and evaluated within a relatively short period of time. Compared to the two-stage method, the one-stage method requires a higher number of seeded cells and a reduced agar concentration, suggesting that cell interactions are required for human T-lymphocyte colony formation.
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The mechanism of the macrophage-regulated proliferation of the murine lymphoma cell line FIO 30 has been further investigated. It appears that the macrophage is alone in its ability to support FIO 30 growth; a macrophage-like cell line, however, is unable to provide the stimulus required by the FIO 30 cells for their proliferation. Investigations into the nature of this stimulus indicate that the serum factor pro-MaSF and a macrophage cell surface component act synergistically to support the growth of the FIO 30 cell, but only when the two cell types are in close promixity. MaSF has also been further characterized and is shown to be closely associated with serum albumin.
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