ALBERT

Description: The effect of recombinant human granulocyte-macrophage colony- stimulating factor (rhGM-CSF) was evaluated in 37 patients with marrow graft failure after allogeneic (n = 15), autologous (n = 21), or syngeneic (n = 1) bone marrow transplantation. rhGM-CSF was administered by 2-hour infusion at doses between 60 and 1,000 micrograms/m2/d for 14 or 21 days. At doses of less than 500 micrograms/m2, rhGM-CSF was well-tolerated and did not exacerbate graft- versus-host disease in allogeneic transplant recipients. No patient with myelogenous leukemia relapsed while receiving rhGM-CSF. Twenty-one patients reached an absolute neutrophil count (ANC) greater than or equal to 0.5 x 10(9)/L within 2 weeks of starting therapy while 16 did not. None of seven patients who received chemically purged autologous marrow grafts responded to rhGM-CSF. The survival rates of GM-CSF- treated patients were significantly better than those of a historical control group.

Description: Frommeyer, Epstein and Taylor: Refractoriness in hemophilia to coagulation-promoting agents: Whole blood and plasma derivatives. Blood 5: 401-420 (May), 1950. The authors wish to note that the following changes should have appeared in the article: Page 402, first line: "Lawrence and Johnson, 1941,8 reported" (instead of "Lawrence and Johnston, in 1946,8 reported"). Page 402, second sentence of same paragraph: "In this instance the patient had received whole blood and plasma in therapeutic management prior to the appearance of resistance to therapy." (instead of ". . . had received chemically prepared plasma fractions in the form of Fraction I of Cohn in addition to whole blood and plasma in therapeutic management prior to . . . "). Page 420, reference 8. To this reference should be added: "(Presented before the American Clinical and Climatological Society, October 1941.)".

Description: Twenty-two patients with classic hemophilia were studied from the point of view of refractoriness to the usual coagulation-promoting effect of whole blood, and of chemically prepared plasma fractions. Of these, 5 patients showed clinical and laboratory evidence of refractoriness to therapy. All had previously received by intravenous injection for therapeutic purposes during bleeding episodes large amounts of plasma fractions in addition to whole blood and plasma. Subsequently, each refractory patient exhibited a poor clinical response to therapy during hemorrhagic episodes. Thus, despite the administration of whole blood, plasma, or plasma derivatives in amounts which were usually effective, bleeding continued unabated and the blood coagulation time was not significantly reduced. An anticoagulant distinct from antithrombin, antithromboplastin, antiprothrombin and heparin was demonstrated in the plasma of each patient. Each had adequate amounts of fibrinogen and prothrombin in the circulating plasma. However, antibodies were demonstrated in the plasma of each patient by means of precipitin reactions with antigens possessing antihemophilic activity, including various plasma derivatives and Fraction I of Cohn. No such antibodies were demonstrable in a normal individual, in 2 anemic patients who had received many transfusions of whole blood, or in a patient with afibrinogenemia who had received large amounts of antihemophilic globulin because of its fibrinogen content. Prompt reduction in plasma anticoagulant activity, in precipitin titer, and coagulation time were effected only by the administration of large quantities of fresh whole blood. A secondary rise of antibodies always followed such a procedure within several days. When therapy was withheld, the anticoagulant effect and precipitin titer decreased in one patient over a period of six months and in another, had essentially disappeared by the end of a year. From these studies the following conclusions may be drawn: 1. A refractory state in hemophilia may develop following the therapeutic use of whole blood, plasma or chemically prepared plasma fractions. 2. This state is characterized by defective clinical and laboratory responses to therapy with whole blood, plasma or plasma fractions. An anticoagulant is demonstrable in the plasma as well as precipitins to various plasma derivatives possessing antihemophilic activity. 3. The formation of antibodies is apparently an immunologic response to a substance closely associated with antihemophilic activity in normal blood and in plasma derivatives previously employed in therapy. 4. The refractory state can be promptly and temporarily abolished and good clinical and laboratory responses can be effected by transfusions of very large amounts of fresh whole blood. 5. Because of their demonstrated ability to act as antigens in certain hemophilic patients, whole blood, plasma and chemically prepared plasma fractions should be withheld unless their employment as an emergency therapeutic measure is necessary.

Description: The syndecans comprise a family of integral membrane proteoglycans that regulate cell behaviors by binding to extracellular matrix and binding growth factors. In mouse blood cells, syndecan expression is restricted to cells of the B-cell lineage where it is expressed by pre-B cells and plasma cells, but is absent from circulating B cells. In the present study, we examined the expression, structure, and function of syndecan on human myeloma cell lines and myeloma patient bone marrow cells. On myeloma cells, syndecan is a small (modal relative molecular mass [M(r)] = 120 Kd) heparan sulfate proteoglycan localized at the cell surface. Syndecan was detected by immunodot blotting on 7 of 10 human myeloma cell lines and by reverse transcriptase polymerase chain reaction on 10 of 14 patient samples. Cell binding assays show that myeloma cells expressing syndecan bind to type I collagen via heparan sulfate chains, while those cell lines not expressing syndecan do not bind to collagen. Furthermore, the cell lines expressing syndecan were negative for CD19 and CD45 staining, indicating that syndecan expression is restricted to tumors having a well-differentiated phenotype. We conclude that syndecan acts as a matrix receptor on human myeloma cells but is not expressed by all tumors, suggesting that syndecan may participate in regulating myeloma cell adhesion to the bone marrow stromal matrix.

Description: Anti-B-blocked ricin (anti-B4-bR) combines the specificity of the anti- B4 (CD19) monoclonal antibody with the protein toxin “blocked ricin.” In blocked ricin, affinity ligands are attached to the ricin B-chain to attenuate its lectin binding capacity. In a phase I trial, Anti-B4-bR was administered by 7-day continuous infusion to 12 patients in complete remission after autologous bone marrow transplantation (ABMT) for relapsed B-cell non-Hodgkin's lymphoma (NHL). Patients were treated at 20, 40, and 50 micrograms/kg/d for 7 days. Potentially therapeutic serum levels could be sustained for 3 to 4 days. The maximum tolerated dose was 40 micrograms/kg/d for 7 days (total 280 micrograms/kg). The dose-limiting toxicities were reversible grade IV thrombocytopenia and elevation of hepatic transaminases. Mild capillary leak syndrome was manifested by hypoalbuminemia, peripheral edema (4 patients), and dyspnea (1 patient). Anti-immunotoxin antibodies developed in 7 patients. Eleven patients remain in complete remission between 13 and 26 months post-ABMT (median 17 months). These results show that Anti-B4- bR can be administered with tolerable, reversible toxicities to patients with B-cell NHL in complete remission following ABMT.

Description: Forty-seven patients with hematologic neoplasia received recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) by daily 2-hour infusion following allogeneic bone marrow transplantation from HLA-identical sibling donors in a phase I-II dose-escalation trial. Dose levels ranged from 30 to 500 micrograms/m2/d. At doses at or below 250 micrograms/m2/d, toxicity felt to be caused by rhGM-CSF was negligible. However, three of five patients treated with 500 micrograms/m2/d had unacceptable side effects caused by rhGM-CSF. Two different graft-versus-host disease (GVHD) prophylactic regimens were administered. Twenty-seven evaluable patients were administered regimens that did not contain methotrexate (MTX) (Group I) and reached an absolute neutrophil count of 1,000/microL by a median of day 14. In contrast, 18 patients who received GVHD prophylactic regimens containing MTX (Group II) reached an absolute neutrophil count of 1,000/microL on a median of day 20. Patients in Group I had fewer febrile days and, of those discharged, had shorter initial hospitalizations than patients in Group II. The overall incidence of severe acute GVHD (grade 2 or greater) in the rhGM-CSF-treated patients was 28% and was similar to that in historical “good risk” patients who did not receive rhGM-CSF. These preliminary data suggest rhGM-CSF is unlikely to exacerbate GVHD in HLA-identical sibling donor transplants and indicate the need for randomized trials of rhGM-CSF in allogeneic marrow transplant patients.

Description: IL-6-PE4E is a recombinant protein consisting of interleukin-6 (IL-6) fused to a mutant form of Pseudomonas exotoxin in which four basic amino acids are changed to glutamate (PE4E). The chimeric toxin has been previously shown to specifically kill malignant hepatic, prostatic, epidermoid, and myeloma cell lines in vitro. To explore the possible clinical utility of IL-6-PE4E, particularly as an agent for ex vivo purging of marrow for autologous bone marrow transplantation (ABMT), we tested malignant cells from patients with multiple myeloma for sensitivity to this chimeric toxin. Ficoll-purified bone marrow cells were incubated with and without IL-6-toxin for 2 to 3 days. Eight of the 15 myeloma patients had cells that were sensitive to IL-6-toxin as measured by a decrease in the level of protein synthesis. Cells from five patients were very sensitive to IL-6-PE4E, with 50% inhibition of protein synthesis (ID50) achieved at or below 6 ng/mL (7 x 10(-11) mol/L). Cells from three additional patients showed moderate sensitivity, with ID50s between 30 and 140 ng/mL. The remaining seven samples showed little or no sensitivity, with ID50s greater than or equal to 400 ng/mL. Normal bone marrow cells or normal BFU-E and CFU-GM were resistant to the IL-6-toxin even at 1,000 ng/mL. Neither IL-6, IL- 2-PE4E, nor an enzymatically deficient mutant of IL-6-PE4E was cytotoxic toward the myeloma cells, indicating that the cytotoxic effect of IL-6-PE4E required the adenosine diphosphate-ribosylation function as well as the specific ligand. Our data suggest that IL-6- toxin could be effective in ex vivo marrow purging in selected multiple myeloma patients who are candidates for ABMT, and that this toxin should also be investigated further for in vivo therapy.

Description: The response of megakaryocytes and platelets to the administration of recombinant human interleukin-6 (IL-6) was investigated in normal and sublethally irradiated dogs. IL-6 was administered for 2 weeks at doses of 10 to 160 micrograms/kg/d to normal animals to assess dose-response and toxicity. Subsequently, 40, 80, or 160 micrograms/kg/d for 2 weeks was administered to animals treated with 200 cG total body irradiation. Analysis of normal dogs showed a significant increment in the platelet count detectable approximately 11 days after initiation of IL-6 at all administered doses. Large platelets greater than 6.3 microns in diameter were observed 1 day after beginning IL-6, progressively increasing to as many as 19.1% of the total circulating platelets by day 10. The ploidy distribution of the marrow megakaryocytes did not differ from the normal at doses of less than or equal to 80 micrograms/kg/d, but at 160 micrograms/kg/d, a shift toward higher ploidy cells was noted. No change in total white count was noted; however, a decrease in hematocrit was seen at all doses. In the irradiated animals, the platelet count recovered earlier in the IL-6- treated dogs than in the controls, but no consistent change in the ploidy distribution was observed irrespective of dose. Large platelets were also noted in the treated animals, comprising up to 6.9% of the total platelet count. Fibrinogen levels were elevated to greater than 4 times normal. A significant decrease in hematocrit was seen in all animals, while no consistent change was noted in the white count. Elevations in serum cholesterol, triglycerides, and alkaline phosphatase, together with a decline in serum albumin were observed in all the treated animals (both normal and irradiated), but clinical symptoms were observed only in the dogs receiving greater than or equal to 80 micrograms/kg/d. The data show that IL-6 alone is capable of enhancing platelet recovery in dogs with bone marrow suppression.

Description: The syndecans comprise a family of integral membrane proteoglycans that regulate cell behaviors by binding to extracellular matrix and binding growth factors. In mouse blood cells, syndecan expression is restricted to cells of the B-cell lineage where it is expressed by pre-B cells and plasma cells, but is absent from circulating B cells. In the present study, we examined the expression, structure, and function of syndecan on human myeloma cell lines and myeloma patient bone marrow cells. On myeloma cells, syndecan is a small (modal relative molecular mass [M(r)] = 120 Kd) heparan sulfate proteoglycan localized at the cell surface. Syndecan was detected by immunodot blotting on 7 of 10 human myeloma cell lines and by reverse transcriptase polymerase chain reaction on 10 of 14 patient samples. Cell binding assays show that myeloma cells expressing syndecan bind to type I collagen via heparan sulfate chains, while those cell lines not expressing syndecan do not bind to collagen. Furthermore, the cell lines expressing syndecan were negative for CD19 and CD45 staining, indicating that syndecan expression is restricted to tumors having a well-differentiated phenotype. We conclude that syndecan acts as a matrix receptor on human myeloma cells but is not expressed by all tumors, suggesting that syndecan may participate in regulating myeloma cell adhesion to the bone marrow stromal matrix.

Description: The genetic defect in the p67phox-deficient form of chronic granulomatous disease (CGD) follows an autosomal recessive pattern of inheritance. When genomic DNA from normal individuals is digested with HindIII and probed with p67phox cDNA an allelic restriction fragment length polymorphism (RFLP) of 4.0 kb or 2.3 kb is detected. We cloned and characterized the p67phox gene using the cDNA and sequenced the exon/intron boundaries, mapping 16 exons on the 40-kb gene. The polymorphic region was then sequenced to identify the inheritance pattern of amniocentesis-derived fetal cells by genomic amplification. The proband, a 9-year-old female patient with p67phox-deficient CGD, and her phenotypically normal mother are homozygous for the RFLP marker, whereas the father and two brothers are heterozygous. The fetus was shown to be heterozygous as well, showing it had inherited at least one normal p67phox gene from the father and that it was predicted to have a normal phenotype. Cord blood samples at birth showed normal oxidative function. Amplification allows rapid detection of the inheritance pattern for fetal diagnosis in informative families. We report the genomic structure of p67phox and an amplification-based method for detection of the marker on chromosome 1q25, used here for prenatal diagnosis of CGD.