Early biochemical events in lymphocyte activation. I. Investigations on the nature and significance of early calcium fluxes observed in mitogen-induced T and B lymphocytes.
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Amino Acids↗
Biomedical subjects
M H Freedman
Publications and source records attributed to M H Freedman.
A rapid technique for measuring calcium uptake in mitogen-induced T and B lymphocytes.
The procedures for lymphocyte activation and for removing the cells from the radioactive loading solution in incubation medium were modified to routinely obtain significant and reproducible 45Ca2+ uptakes in mitogen-induced mouse T and B lymphocytes. Factors such as mouse strain, lymphocyte origin, and media pH were not critical to the 45Ca2+ uptake measurements. In contrast, factors such as lymphocyte cell concentration during mitogenic activation, filtering the 45Ca2+:3H2O mixtures, and the nature and purity of the B-cell mitogens were critical for obtaining maximal and reproducible 45Ca2+ uptakes. Centrifugation through silicone oil into sucrose was an efficient and rapid procedure for separating the cells from the radioactive loading solution in the incubation medium. Using optimal conditions, an approximate twofold increase in 45Ca2+ uptake (representing an influx of approximately 97 amol per lymphocyte and an increase in average cellular Ca2+ of approximately 0.72 mM) was routinely obtained with purified mouse lymphocytes activated with a variety of T- and B-cell mitogens (using concentrations resulting in maximal [3H]thymidine incorporation). A larger 45Ca2+ uptake was routinely obtained with mitogenic concentrations of A23187, a divalent cation ionophore stimulating T cells. Experiments employing [14C]sucrose and [14C]inulin with control and mitogen-induced lymphocytes showed that the trapped extracellular fluid measurements in the cell pellets should be used to correct the magnitude of the 45Ca2+ uptake measurements.
Animals↗
Granulopoiesis in Shwachman's syndrome (pancreatic insufficiency and bone marrow dysfunction).
Granulopoiesis was studied in 10 children with Shwachman's syndrome (chronic neutropenia and exocrine pancreatic insufficiency). Marrow proliferative activity assessed by determination of mitotic indices and tritiated thymidine uptake into granulocytic cells was normal. Assay of bone marrow granulocyte colony-forming cells (CFU-C) in a methylcellulose tissue culture system demonstrated normal CFU-C numbers in four patients and reduced numbers in five. The granulocyte colonies formed were indistinguishable from normal colonies morphologically. Production of colony-stimulating activity (CSA) from patients' peripheral blood leukocytes appeared normal when tested on control marrow. No serum inhibitors against CFU-C or CSA could be demonstrated using both control and autologous marrow, and co-culture of patients' peripheral blood lymphocytes with control marrow did not inhibit CFU-C growth. We conclude that in Shwachman's syndrome committed granulocytic stem cells are present, and the numbers detected in vitro vary widely as does the clinical neutropenia. The proliferative activity of recognizable granulocytic cells is normal and neither a deficiency of humoral stimulators nor the presence of serum or cellular inhibitors of granulopoiesis can be demonstrated.
Adolescent↗
Nocturnal intragastric infusion of glucose in management of defective gluconeogenesis with hypoglycemia.
Three children with defective gluconeogenesis and hypoglycemia were treated with frequent daytime feeding and continuous intragastric infusion of glucose at night. By this technique, the blood glucose level was maintained at or slightly above the physiological range. Secondary lacticacidemia, hyperlipidemia, hyperuricacidemia, and coagulation defects all improved. Weight and height velocity increased dramatically. Strength and sense of well-being improved. Nocturnal intragastric infusion of glucose is now the management of choice for children with defective gluconeogenesis and hypoglycemia.
Body Weight↗
Constitutional aplastic anaemia: defective haematopoietic stem cell growth in vitro.
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Anemia, Aplastic↗
Diamond-Blackfan syndrome: evidence against cell-mediated erythropoietic suppression.
The profound anemia of Diamond-Blackfan syndrome (DBS) is due to marrow red cell failure, but the pathogenesis is not understood. Studies by others indicated cell-mediated erythropoietic suppression in this condition. To explore this mechanism further, Ficoll-Hypaque--separated peripheral blood lymphocytes (PBL) from four anemic untreated patients with DBS, or from normals were cocultured with control marrow in vitro and the growth of erythropoietin-responsive stem cell colonies (CFU-E) was dermined. CFU-E numbers obtained from cultures with added normal PBL were not significantly different from the number without PBL. Similarly, CFU-E from cultures with added DBS PBL were not significantly different from the number without PBL (215 versus 220, 229 versus 220 and 84 versus 60, 74 versus 94/10(5) cells, respectively). Mixing marrows from a control and one DBS patient in ratios of 2:1, 1:1, or 1:2 prior to culture failed to disclose a decrease of colony growth. We could not show cellular inhibition of erythropoiesis in these patients with DBS. The mechanism of anemia in this disorder remains an open question.
Bone Marrow Cells↗
The morbidity and mortality of splenectomy in childhood.
One hundred and eighty-two patients undergoing splenectomy in infancy and childhood were followed for periods of 2 to 15 years. Serious infections occurred in 11 patients (6%) with death in 6 (3.3%). In 10 patients the infection was sepsis, and in all but one patient the infection occurred within 2 years of splenectomy. Among children over 2 years of age the risk of infection was still appreciable except when the spleen was removed incidentally or for traumatic rupture. Splenectomy for thalassemia and portal hypertension resulted in an increased risk of serious infections when compared with removal of the spleen for hereditary spherocytosis, idiopathic thrombocytopenic purpura, trauma, or for technical reasons in the course of another operation. Post-splenectomy infections tended to follow a characteristic pattern. The infecting organism was predominantly pneumococcus, the course was fulminating and the mortality high.
Adolescent↗
Factors affecting erythroid colony growth (CFU-E) from human marrow.
Human marrow grown in a plasma clot gives rise to discrete colonies of nucleated red cells in response to erythropoietin (EPO). In 10 studies of human marrow with normal erythropoiesis, human EPO in doses up to 1 unit/ml produced a significant increase in colonies when compared to sheep EPO. To achieve 50% of maximum colony growth, only 0.1 units/ml of human EPO was needed in contrast to 0.5 units/ml of sheep EPO. In seven other experiments, nandrolone decanoate, 10(-7) to 10(-9) M, yielded a mean increase in colonies of 38% when compared to cultures without added androgen. This effect was entirely dependent on the presence of EPO. We conclude that the method is useful for the invitro study of factors affecting early events in erythropoiesis.
Animals↗
A complement independent erythropoietic inhibitor acting on the progenitor cell in refractory anemia.
An erythropoietic inhibitor was detected in the serum of a patient with refractory anemia. Using an in vitro heme synthesis method, the patient's serum produced tenfold inhibition of erythropoietin-stimulated radioactive iron (Fe59) incorporation into heme of normal human marrow at 72 hours, as compared with AB serum. In a separate experiment the patient's serum produced threefold inhibition, whereas immunoglobulin G (IgG) prepared from the same serum sample produced 12-fold inhibition. To identify the site of action of the inhibitor, serum was tested in a cell culture system whereby human marrow cells, grown in a plasma clot, respond to exogenous erythropoietin with the appearance of nucleated erythroid colonies. Each colony arises from a committed erythroid progenitor. The patient's serum produced a two- or tenfold reduction in the number of colonies from normal human marrow. The effect was also demonstrated on autologous marrow obtained when the patient was in "partial clinical remission". Serum samples obtained at various times during the course of the patient's illness all demonstrated a suppressive effect on colony growth. All serums were heat-inactivated, and total hemolytic complement could not be detected in either culture system. It is concluded that the anemia is due to an inhibitor, probably of IgG class, that acts on the erythroid progenitor cell. The absence of heat-labile complement components in the culture systems suggests that the mechanism is not due to immune cytolysis.
ABO Blood-Group System↗
Hemoglobin-oxygen affinity in hypophosphatemic rickets.
In 14 patients with simple X-linked hypophosphatemic rickets, 5 were below the third percentile in height and 9 were between the third and twenty-fifth percentile. Although the mean serum inorganic phosphorus level was only 2.01 +/- 0.65 (normal range for all age groups is 3.8 to 6.0 mg/100 ml), both the mean values for red cell 2,3-diphosphoglycerate (2,3-DPG) and adenosine triphosphate (ATP) were normal at 4.78 +/- 1.23 and 1.02 +/- 0.17 mumol/ml of red blood cells respectively. Moreover, the mean P50 value was normal at 26.4 +/- 0.9 mmHg. These normal oxygen transport data make unlikely any proposal that short stature seen in these patients is secondary to chronic tissue hypoxia. They also indicate that the intra-erythrocytic organic phosphate levels are maintained at normal levels despite profound chronic hypophosphatemia.
Adenosine Triphosphate↗
Erythroid colony growth in congenital hypoplastic anemia.
Four children with congenital hypoplastic anemia (Diamond-Blackfan syndrome) and 30 control children with normal erythropoiesis were studied by a cell culture method in which human marrow, grown in a plasma clot, responds to added erythropoietin (EPO) with the appearance of discrete colonies of nucleated erythroid cells. The colonies arise from EPO-responsive stem cells and are not related to the number of morphologically identifiable marrow erythroids plated. Results of studies on control marrow indicated that without EPO there was little or no colony formation. Increasing EPO doses or nucleated marrow cells per culture resulted in a linear increase in colony numbers. The optimal EPO concentration of 2.5 U/ml yielded a mean of 158 +/- 79 colonies/1 x 10(5) nucleated cells on day 7 of incubation. Even in the absence of recognizable erythroids, marrows of all four patients with anemia grew erythroid colonies. Two patients on no therapy had decreased colony numbers. The other two, on prednisone, had normal numbers. Sera from patients did not inhibit colony formation from either autologous or control marrow. In contrast, serum from an adult with acquired pure red cell aplasia produced striking inhibition of colony growth. It appears that the red cell failure in this disorder is not due to an absence of erythroid stem cells, and a serum inhibitor to erythropoiesis as seen in the acquired disease is unlikely.
Anemia, Aplastic↗
Post recognition ion dependent events in mitogen induced lymphocyte proliferation and in cytotoxic effector cell responses.
This review discusses the increased permeability and enhanced uptake transport of monovalent and divalent cations following mitogen-induced lymphocyte proliferation. The observed Ca2+ uptake is discussed in terms of gated Ca2+ channels. The importance of divalent cations, particularly Ca2+, is discussed in terms of triggering of cytotoxic effector cell responses for three model systems (antibody dependent cytotoxicity, mitogen-induced cytotoxicity and cell mediated cytotoxicity); (85).
Antibodies↗
Management of intracerebral hemorrhage in idiopathic thrombocytopenic purpura. Report of four cases.
There has been little comment on the specific management of intracerebral bleeding occurring in patients suffering idiopathic thrombocytopenic purpura. The authors present the cases of four children with intracerebral hemorrhage due to this coagulation disturbance. A plan of management is described based on this experience; it includes immediate control of cerebral edema, emergency splenectomy, supportive care with platelet transfusions and corticosteroids, cerebral angiogtaphy, and a definitive neurosurgical procedure. If necessary, the radiological investigation and surgical therapy can be performed with a single general anesthetic. Three of the patients have survived without major neurological sequelae.
Adolescent↗
Granulopoiesis in severe congenital neutropenia.
The pathogenesis of the granulopoietic failure in three children with severe congenital neutropenia was studied. Mature neutrophils were absent from both peripheral blood and bone marrow. Assay of bone marrow granulocyte colony-forming cells (CFU-C) in a methylcellulose tissue culture system using colony-stimulating activity (CSA) from peripheral blood leukocytes demonstrated normal or increased concentrations of CFU-C compared to those from marrows of 60 age-matched controls. Colonies were of normal size and by light microscopy appeared to contain granulocytes in all stages of maturation including the mature polymorphonuclear neutrophil. CFU-C from peripheral blood of two patients were normal. Production and activity of CSA from the patients' peripheral blood leukocytes and urinary CSA excretion were normal. No serum inhibitors against CFU-C or CSA could be demonstrated using both control and autologous marrow. The defect did not appear to be due to a lack of granulocytic stem cells, a reduction of humoral stimulators of granulopoiesis, nor the presence of an inhibitor as measured by these techniques.
Agranulocytosis↗
Induction of increased calcium uptake in mouse T lymphocytes by concanavalin A and its modulation by cyclic nucleotides.
The binding of concanavqlin A to T but not B mouse spleen lymphocytes increases Ca-2+ uptake in these cells which is measurable by 45 s and complete by 1 min. Dibutyrl cyclic AMP, but not sodium azide inhibits induced Ca-2+ uptake, wheras dibutyryl cyclic GMP enhances it. B cell mitogens do not cause a similar Ca-2+ uptake in mouse B lymphocytes. The induction of increased Ca-2+ uptake by T cells is discussed in terms of gated membrane channels for Ca-2+.
Animals↗
Effect of altitude exposure on platelets.
Since decompression from depth is known to produce a fall in platelet count, the effect of altitude decompression and high-altitude exposure on platelets was investigated. Sixteen subjects decompressed without hypoxia to 20,000 ft simulated altitude for two hours showed a significant (P less than 0.01) drop in circulating platelet count of approximately 10% for three days following decompression. Four of five subjects similarly exposed had a shortened autologous platelet survival compared to that prior to exposure. Subjects exposed to 9,800 ft and then 17,600 ft in a mountain environment showed a significant mean decrease in platelet count on day 2 of 7% and 25% respectively, which had returned to control by day 5. Nonhypoxic and hypoxic decompressed rabbits which received homologous chromium-51-labeled platelets had an increase in lung radioactivity compared with sea-level controls. It is postulated that altitude decompression produces platelet reductions similar to these seen after decompression from depth, and that platelets sequester in the pulmonary vascular bed.
Altitude↗
Residual abnormalities in acquired aplastic anemia of childhood.
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Adolescent↗
Carbon-13 nuclear magnetic resonance titration shifts in amino acids.
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Amino Acids↗