ALBERT

Description: Measurements of erythropoiesis and iron balance were made in eight normal and 32 anemic subjects. The latter consisted of 12 individuals with ineffective erythropoiesis (beta-thalassemia/hemoglobin E), 13 subjects with ineffective erythropoiesis and hemolytic anemia (hemoglobin H), and seven subjects with hemolytic anemia (hereditary spherocytosis). A consistent relationship within each group existed between the degree of erythropoiesis and radioiron absorption. Although the effect of erythropoiesis on iron absorption was of similar magnitude in the two thalassemia groups, the effect in hereditary spherocytosis was much less. There was agreement between absorption and ferritin or magnetic susceptibility (SQUID) measurements of iron stores in thalassemia, but in hereditary spherocytosis a discrepancy existed between absorption and ferritin. It is concluded that, although increased erythropoiesis is associated with increased iron absorption, some additional factor associated with red cell breakdown is more directly responsible for the positive iron balance in thalassemia.

Description: Measurements of erythropoiesis and iron balance were made in eight normal and 32 anemic subjects. The latter consisted of 12 individuals with ineffective erythropoiesis (beta-thalassemia/hemoglobin E), 13 subjects with ineffective erythropoiesis and hemolytic anemia (hemoglobin H), and seven subjects with hemolytic anemia (hereditary spherocytosis). A consistent relationship within each group existed between the degree of erythropoiesis and radioiron absorption. Although the effect of erythropoiesis on iron absorption was of similar magnitude in the two thalassemia groups, the effect in hereditary spherocytosis was much less. There was agreement between absorption and ferritin or magnetic susceptibility (SQUID) measurements of iron stores in thalassemia, but in hereditary spherocytosis a discrepancy existed between absorption and ferritin. It is concluded that, although increased erythropoiesis is associated with increased iron absorption, some additional factor associated with red cell breakdown is more directly responsible for the positive iron balance in thalassemia.

Description: Two leukemia patients, refractory to chemotherapy, were treated with T101-ricin A-chain immunotoxin (T101 IT). Patient 1 (T-ALL) received a single 13.5 mg dose of T101 IT IV (12-hour infusion). Patient 2 (B-CLL) was treated with a daily 25 mg dose of T101 IT IV (two-hour infusion) over three consecutive days. Patient 2 also received 300 mg of chloroquine IM on days two and three as enhancer. In vivo binding of T101 IT was demonstrated by FACS analysis using either an antimouse Ig- FITC or anti-A-chain-FITC antibodies. Following IT therapy, the expression of T65 antigen on target cells dropped to 50% and 20% of pretreatment levels, respectively. In patient 1, circulating blast cells remained unsaturated during therapy while in patient 2, cells were fully saturated for four to six hours following each infusion. Pharmacokinetic studies showed a rapid clearance of T101 IT after IV administration. Antimouse and anti-A-chain antibodies could not be detected. There were no treatment-related adverse effects. In patient 1 a rapid but transient decrease of target cells was observed, possibly related to the administration of the antibody part of T101 IT. In contrast, patient 2 showed a 40% reduction of the lymphocyte count, which remained stable over a period of 2 weeks. Such a clinical benefit following IT therapy in patient 2 could be ascribed to the absence of circulating free antigen and the complete saturation of target cells.

Description: Recently human interleukin-3 (IL-3) produced by molecular cloning was characterized as a growth factor for basophils and eosinophils in human bone marrow cultures. Since we found a similar activity of the human factor on simian bone marrow cells, we investigated the in vivo effects of recombinant human (rh) IL-3 in healthy rhesus monkeys (n = 10). rh IL-3 was administered subcutaneously (SC) to monkeys at different doses (11, 33, and 100 micrograms/kg/d) for 14 days followed by subsequent rh GM-CSF administration (5.5 micrograms/kg/d SC) for another two weeks. During the second week of rh IL-3 administration monkeys responded with a twofold to threefold increase of WBCs caused by a dose-dependent elevation of basophils (up to 40% of WBCs) and eosinophils. rh IL-3 also induced a dose-dependent increase of histamine (up to 700-fold above normal values) in monkey blood cells. Administration of rh GM-CSF to rh IL-3 pretreated monkeys resulted in a twofold enhanced increase in WBCs (due mainly to eosinophils and neutrophils) compared with animals treated with rh GM-CSF alone. Simultaneous administration of both cytokines (100 micrograms/kg rh IL-3 + 5.5 micrograms/kg rh GM-CSF SC) to two separate monkeys for 14 days induced a WBC elevation similar to that observed in monkeys treated with rh GM-CSF alone. In conclusion, our results indicate that rh IL-3 is a differentiation factor for blood basophils and primes the hematopoietic system for subsequent rh GM-CSF actions.

Description: The effect of recombinant human (rh) cytokines, interleukin-1 alpha (IL- 1 alpha), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL- 4), granulocyte/macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), monocyte/macrophage colony stimulating factor (M-CSF), interferon-alpha (IF-alpha), interferon-gamma (IF-gamma), and the tumor necrosis factor-alpha (TNF- alpha) on differentiation and function of metachromatic cells (MCS) was studied. Among all cytokines tested, rh interleukin-3 (rhIL-3) selectively induced a significant formation of MCS (IL-3: 1.1 +/- 0.6 x 10(5) v control: 0.02 +/- 0.15 x 10(5) MCS/mL suspension) and dose dependent increase in formation of intracellular histamine (IL-3, 100 U/mL: 95 +/- 23 ng/mL v control: 1.8 +/- 0.8 ng/mL) in a bone marrow suspension culture system (analyzed on day 14 of culture). Besides MCS, formation of eosinophils was observed in this culture system in the continuous presence of rhIL-3, whereas IL-3 pulse-stimulation for three hours and subsequent exposure to control medium induced growth of MCS but not of eosinophils. By combined immunofluorescence/toluidine blue staining, MCS were found to express a cell surface marker profile that corresponds to the immunological phenotype of peripheral blood basophils (MY-7(CD13)+, VIM12(CD11b)+, VIM2+, MAX1-, MAX24- and YB5B8- ). Furthermore, cultured MCS expressed surface membrane receptors for IgE and could be triggered for nontoxic histamine release by a monoclonal anti-IgE antibody. To evaluate a possible influence of IL-3 on basophil function, studies were extended to freshly obtained blood basophils (healthy volunteers, n = 3). However, like all other cytokines tested, rhIL-3 failed to induce basophil histamine release. Taken together, our studies demonstrate that IL-3 is a differentiation factor for human basophils.

Description: Two leukemia patients, refractory to chemotherapy, were treated with T101-ricin A-chain immunotoxin (T101 IT). Patient 1 (T-ALL) received a single 13.5 mg dose of T101 IT IV (12-hour infusion). Patient 2 (B-CLL) was treated with a daily 25 mg dose of T101 IT IV (two-hour infusion) over three consecutive days. Patient 2 also received 300 mg of chloroquine IM on days two and three as enhancer. In vivo binding of T101 IT was demonstrated by FACS analysis using either an antimouse Ig- FITC or anti-A-chain-FITC antibodies. Following IT therapy, the expression of T65 antigen on target cells dropped to 50% and 20% of pretreatment levels, respectively. In patient 1, circulating blast cells remained unsaturated during therapy while in patient 2, cells were fully saturated for four to six hours following each infusion. Pharmacokinetic studies showed a rapid clearance of T101 IT after IV administration. Antimouse and anti-A-chain antibodies could not be detected. There were no treatment-related adverse effects. In patient 1 a rapid but transient decrease of target cells was observed, possibly related to the administration of the antibody part of T101 IT. In contrast, patient 2 showed a 40% reduction of the lymphocyte count, which remained stable over a period of 2 weeks. Such a clinical benefit following IT therapy in patient 2 could be ascribed to the absence of circulating free antigen and the complete saturation of target cells.

Description: Recently human interleukin-3 (IL-3) produced by molecular cloning was characterized as a growth factor for basophils and eosinophils in human bone marrow cultures. Since we found a similar activity of the human factor on simian bone marrow cells, we investigated the in vivo effects of recombinant human (rh) IL-3 in healthy rhesus monkeys (n = 10). rh IL-3 was administered subcutaneously (SC) to monkeys at different doses (11, 33, and 100 micrograms/kg/d) for 14 days followed by subsequent rh GM-CSF administration (5.5 micrograms/kg/d SC) for another two weeks. During the second week of rh IL-3 administration monkeys responded with a twofold to threefold increase of WBCs caused by a dose-dependent elevation of basophils (up to 40% of WBCs) and eosinophils. rh IL-3 also induced a dose-dependent increase of histamine (up to 700-fold above normal values) in monkey blood cells. Administration of rh GM-CSF to rh IL-3 pretreated monkeys resulted in a twofold enhanced increase in WBCs (due mainly to eosinophils and neutrophils) compared with animals treated with rh GM-CSF alone. Simultaneous administration of both cytokines (100 micrograms/kg rh IL-3 + 5.5 micrograms/kg rh GM-CSF SC) to two separate monkeys for 14 days induced a WBC elevation similar to that observed in monkeys treated with rh GM-CSF alone. In conclusion, our results indicate that rh IL-3 is a differentiation factor for blood basophils and primes the hematopoietic system for subsequent rh GM-CSF actions.

Description: The effect of recombinant human (rh) cytokines, interleukin-1 alpha (IL- 1 alpha), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL- 4), granulocyte/macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), monocyte/macrophage colony stimulating factor (M-CSF), interferon-alpha (IF-alpha), interferon-gamma (IF-gamma), and the tumor necrosis factor-alpha (TNF- alpha) on differentiation and function of metachromatic cells (MCS) was studied. Among all cytokines tested, rh interleukin-3 (rhIL-3) selectively induced a significant formation of MCS (IL-3: 1.1 +/- 0.6 x 10(5) v control: 0.02 +/- 0.15 x 10(5) MCS/mL suspension) and dose dependent increase in formation of intracellular histamine (IL-3, 100 U/mL: 95 +/- 23 ng/mL v control: 1.8 +/- 0.8 ng/mL) in a bone marrow suspension culture system (analyzed on day 14 of culture). Besides MCS, formation of eosinophils was observed in this culture system in the continuous presence of rhIL-3, whereas IL-3 pulse-stimulation for three hours and subsequent exposure to control medium induced growth of MCS but not of eosinophils. By combined immunofluorescence/toluidine blue staining, MCS were found to express a cell surface marker profile that corresponds to the immunological phenotype of peripheral blood basophils (MY-7(CD13)+, VIM12(CD11b)+, VIM2+, MAX1-, MAX24- and YB5B8- ). Furthermore, cultured MCS expressed surface membrane receptors for IgE and could be triggered for nontoxic histamine release by a monoclonal anti-IgE antibody. To evaluate a possible influence of IL-3 on basophil function, studies were extended to freshly obtained blood basophils (healthy volunteers, n = 3). However, like all other cytokines tested, rhIL-3 failed to induce basophil histamine release. Taken together, our studies demonstrate that IL-3 is a differentiation factor for human basophils.

Description: A human monoclonal anti-Rh(D) antibody produced by an Epstein-Barr virus (EBV)-transformed B-cell line (IgG1(lambda), clone H2D5D2) has been purified on protein A-Sepharose column and used for binding studies and immune precipitation of the blood group rhesus (Rh) antigens. Scatchard plot analyses show that the 125I-labeled antibody (iodo-gen procedure), binds to 1.09 X 10(5), 0.43 X 10(5), and 0.32 X 10(5) antigen sites on each D--/D--, R2R2 and R1R1 RBC, respectively, with an association constant of approximately 0.6 X 10(8) mol/L-1. Immune precipitation studies indicate also that the Rh(D) antigen of the Rh(D)-positive RBCs is carried by a 29 kd polypeptide as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE). No material could be precipitated from Rh(D)-negative or Rhnull RBCs. These results indicate that the monoclonal and the polyclonal human anti-Rh(D) behave similarly. A sample (Blo., presumed genotype R2r or R0R2) showing an increased number of antigen sites (0.76 X 10(5)/cell) and a high binding constant (5.7 X 10(8) mol/L-1) was used, as well as D--/D-- RBCs, for further purification of the 29-kd component. Extraction by Triton X-100 (0.1% to 5%) of the immune complexes formed between the membrane-bound Rh(D) antigens and the monoclonal antibody as well as a direct quantitative estimate of the 29- kd component, suggest that the Rh(D) polypeptide is loosely bound to the skeleton, since less than or equal to 80% can be solubilized from the membrane. In similar conditions, glycophorin A showed a slight association with the Triton-insoluble residue, whereas glycophorin B was easily and completely extracted. In contrast, both the minor RBC sialoglycoproteins, glycophorin C and glycoprotein gamma, remained predominantly bound to the membrane skeleton. The purified Rh(D) polypeptide obtained from Blo. and D--/D-- RBCs by immunoprecipitation and preparative gel electrophoresis was homogenous as judged by SDS- PAGE. Amino acid composition indicated that the Rh(D) protein contained sulfhydryl groups which are essential for biological activity.

Description: A human monoclonal anti-Kell (K1) antibody secreted by an Epstein-Barr virus (EBV)-transformed B-cell line was used for binding studies and immunopurification of the K1 blood group antigen. The 125I-labeled antibody bound to 4 to 5 x 10(3) and 2.5 to 3 x 10(3) antigenic sites on K1K1 and K1K2 erythrocytes, respectively, with an affinity constant of 5 x 10(8) mol/L-1. Immunoprecipitation analysis showed that the K1 antigen is carried by a 93 Kd glycoprotein containing several cysteine residues, and approximately six N-glycosidically linked sugar chains but no detectable O-linked sugar. A minor labeled component of 32 Kd was also immunoprecipitated from K1K1 RBCs but the 93- and 32-Kd components were absent from K2K2 and Kell null erythrocytes. Under nonreducing conditions, three bands were detected at 200 (weak), 120, and 93 Kd. We suggest that the 120-Kd component represents a heterodimer of the 93- and 32-Kd proteins covalently linked by disulfide bridge(s). The 93-Kd glycoprotein is a transmembrane component which interacts with the membrane skeleton but is distinct from band 3 as shown by one-dimensional peptide mapping. The site density of K1 antigen blood group on Gerbich-negative RBCs (Ge:-2,-3) was threefold lower than on K1K1 erythrocytes, but the qualitative properties of the 93-Kd component were not modified.