Autologous bone marrow transplantation in acute myelogenous leukemia: in vitro treatment with myeloid cell-specific monoclonal antibodies

Second or third chemotherapy-induced remissions in acute myelogenous leukemia (AML) are limited by early relapse of the leukemia. We developed monoclonal antibodies (MoAbs) that are cytotoxic to myeloid leukemia cells to treat bone marrow from these patients ex vivo for autologous transplantation. In this pilot study, bone marrow was harvested from ten patients with AML in remission, treated with one or two complement-fixing MoAbs, PM-81 and AML-2-23, which react with myeloid differentiation antigens, incubated with rabbit complement, and cryopreserved. These MoAbs were chosen because they have broad reactivity with AML cells but not with pluripotent progenitor cells. At the time of transplant, 6 patients were in second complete remission, 1 each was in third complete or partial remission, and 2 were in early first relapse. The patients were treated with cyclophosphamide (60 mg/kg a day for 2 days) and total body irradiation (200 cGy twice a day for 3 days) and given infusions of MoAb-treated bone marrow. Full bone marrow reconstitution was observed in eight patients; two patients did not recover platelets. Seven of the ten patients are surviving and disease-free at 21.0, 15.0, 13.0, 10.0, 6.0, 3.0, and 2.0 months posttransplant. Treating bone marrow with MoAbs to myeloid differentiation antigens does not interfere with pluripotential stem cell engraftment. Longer follow-up and a controlled study are necessary to prove that the apparent efficacy of this therapeutic approach in some patients is attributable to MoAb-mediated killing of leukemia cells.     

A novel 37-Kd adhesive membrane protein from cloned murine bone marrow stromal cells and cloned murine hematopoietic progenitor cells

The adhesion of hematopoietic progenitor cells to bone marrow stromal cells is critical to hematopoiesis and involves multiple effector molecules. Stromal cell molecules that participate in this interaction were sought by analyzing the detergent-soluble membrane proteins of GBI/6 stromal cells that could be adsorbed by intact FDCP-1 progenitor cells. A single-chain protein from GBI/6 cells having an apparent molecular weight of 37 Kd was selectively adsorbed by FDCP-1 cells. This protein, designated p37, could be surface-radiolabeled and thus appeared to be exposed on the cell membrane. An apparently identical 37- Kd protein was expressed by three stromal cell lines, by Swiss 3T3 fibroblastic cells, and by FDCP-1 and FDCP-2 progenitor cells. p37 was selectively adsorbed from membrane lysates by a variety of murine hematopoietic cells, including erythrocytes, but not by human erythrocytes. Binding of p37 to cells was calcium-dependent, and was not affected by inhibitors of the hematopoietic homing receptor or the cell-binding or heparin-binding functions of fibronectin. It is proposed that p37 may be a novel adhesive molecule expressed on the surface of a variety of hematopoietic cells that could participate in both homotypic and heterotypic interactions of stromal and progenitor cells.     

Testing logistic regression coefficients with clustered data and few positive outcomes

Applications frequently involve logistic regression analysis with clustered data where there are few positive outcomes in some of the independent variable categories. For example, an application is given here that analyzes the association of asthma with various demographic variables and risk factors using data from the third National Health and Nutrition Examination Survey, a weighted multi stage cluster sample. Although there are 742 asthma cases in all (out of 18,395 individuals), for one of the categories of one of the independent variables there are only 25 asthma cases (out of 695 individuals). Generalized Wald and score hypothesis tests, which use appropriate cluster-level variance estimators, and a bootstrap hypothesis test have been proposed for testing logistic regression coefficients with cluster samples. When there are few positive outcomes, simulations presented in this paper show that these tests can sometimes have either inflated or very conservative levels. A simulation-based method is proposed for testing logistic regression coefficients with cluster samples when there are few positive outcomes. This testing methodology is shown to compare favorably with the generalized Wald and score tests and the bootstrap hypothesis test in terms of maintaining nominal levels. The proposed method is also useful when testing goodness-of-fit of logistic regression models using deciles-of-risk tables.     

Cell-mediated immunity in rheumatic disease

Rheumatoid arthritis, systemic lupus erythematosus, and systemic sclerosis are the three most common systemic rheumatic diseases in which disordered immune function is thought to play a pathogenetic role. Each disease has different and characteristic abnormalities of the cellular immune system. In rheumatoid arthritis the identified abnormalities of immunoregulation are largely limited to specific antigens: Epstein-Barr virus and collagen. Systemic lupus erythematosus is characterized by exuberant B-cell activity with exaggerated humoral response, a diversity of autoantibodies, non-antigen-specific loss of suppressor cell function, and general suppression of cell-mediated immunity. In systemic sclerosis systemic defects of cellular and humoral immune function are mild, but the release of lymphokines and monokines at sites of inflammatory lesions is thought to be important in the pathogenesis of the disease. Similar immune cell-connetive tissue cell interactions are probably important in the propagation of rheumatoid synovitis. Thus, despite the many shared clinical and serologic features of these diseases as well as the presence of many patients who have clinically overlapping features of more than one of these entities, the immune defects and the immunopathogenesis of these disorders appear to be distinct.     

A major internal initiation site for the in vitro translation of the adenovirus DNA polymerase

An open reading frame which encodes at least 90% of the adenovirus type 2 DNA polymerase gene was cloned behind the SP6 promoter and transcribed in vitro using the SP6 RNA polymerase. The resultant RNA was translated in a rabbit reticulocyte cell free system. In addition to the translation of a 120-kDa protein corresponding to the size of the complete open reading frame, the synthesis of a 62-kDa polypeptide was demonstrated. Data is presented to show that the synthesis of the 62-kDa polypeptide resulted from internal initiation of translation in frame in the middle of the message at the 11th or 12th AUG. Capping of the mRNA resulted in an increase in synthesis of the 120-kDa protein and a concordant decrease of the internally initiated polypeptide. We propose that there may be competition between the binding of the translational preinitiation complex at or near the 5' end of the mRNA and at the internal initiation site. Because of inhibition of synthesis of the 120-kDa but not the 62-kDa polypeptide by hybrid arrested translation using DNA complementary to approximately one third of the 5' Ad Pol mRNA sequences, scanning of the ribosome from the 5' end of the mRNA to the internal initiation site seemed unlikely. The sequence proximal to the 12th AUG is ACCCACCCCAUG which is similar to a noncontinuous sequence 5'AUCCACC(X)nAUG complementary to the 3' end of the 18 S rRNA. This sequence is a favored ribosome binding site based on the observation that it is the most commonly observed one at or near the 5' end of 162 mRNA's analyzed (D. R. Sargan, S. P. Gregory, and P. H. W. Butterworth, 1982, FEBS Lett. 147, 133-136).     

Marrow transplantation for chronic myeloid leukemia: a randomized study comparing cyclophosphamide and total body irradiation with busulfan and cyclophosphamide

A prospective randomized study was conducted comparing two conditioning regimens for the treatment of patients with chronic myeloid leukemia in chronic phase by marrow transplantation from HLA identical siblings. Sixty-nine patients received 60 mg/kg of cyclophosphamide on each of 2 successive days followed by 6 fractions of total body irradiation each of 2.0 Gy (CY-TBI), and 73 patients received 16 mg/kg of busulfan delivered over 4 days followed by 60 mg/kg CY on each of 2 successive days (BU-CY). There was no significant difference between the CY-TBI and the BU-CY groups in the 3-year probabilities of survival (0.80 for both), relapse (0.13 for both), or event-free survival (CY-TBI, 0.68; BU-CY, 0.71) or in speed of engraftment or incidence of venocclusive disease of the liver. The 4-year probabilities of survival and event- free survival for patients transplanted within 1 year of diagnosis were 0.86 and 0.72, respectively, for each group. Significantly more patients in the CY-TBI group experienced major creatinine elevations. There was significantly more acute graft-versus-host disease in the CY- TBI group. Fever days, positive blood cultures, hospitalizations, and inpatient hospital days were significantly more common in the CY-TBI group than in the BU-CY group. In conclusion, the BU-CY regimen was better tolerated than, and associated with survival and relapse probabilities that compare favorably with, the CY-TBI regimen.     

Fanconi's anemia treated by allogeneic marrow transplantation

Eight patients with Fanconi's anemia were given cyclophosphamide alone (seven patients) or combined with procarbazine and antithymocyte globulin (one patient) followed by marrow grafts from HLA-identical siblings. All patients had engraftment. Seven developed acute and three chronic graft-versus-host disease (GVHD). Three patients died with GVHD and infectious complications (days 19, 56, and 82) and one with an intracerebral hemorrhage (day 540). Four patients are surviving 647- 3435 days after grafting, two are well, and two have chronic GVHD that is improving. These results show that Fanconi's anemia can be treated successfully by allogeneic marrow transplantation.     

Effect of hepatic dysfunction on oral cyclosporin pharmacokinetics in marrow transplant patients

The effect of hepatic dysfunction, defined as abnormal serum bilirubin level, on oral cyclosporin (CSP) pharmacokinetics was examined in 28 marrow transplant patients who received CSP for prophylaxis of graft-v- host disease. Serum CSP concentrations were measured by radioimmunoassay. Forty-one concentration-time courses were studied, divided among patients with no (less than 1.2 mg/dL), mild (1.2 to 2.0 mg/dL), and moderate (2.0 to 5.0 mg/dL) hepatic dysfunction. CSP elimination, as determined by elimination rate constant and clearance, was delayed in patients with moderate hepatic dysfunction compared to those with no hepatic dysfunction (P less than .05). The volume of distribution, lag time for absorption, maximum serum concentration, and time at which the maximum concentration was achieved was not affected by hepatic function. These data indicate that patients with moderate hepatic dysfunction have delayed CSP or CSP metabolite elimination and may be at higher risk for developing CSP-related toxicity.     

Sephadex-gel filtration (SGF) in infant and adult Gunn rats

SGF was compared in infant and adult homozygous Gunn rats. Without any drug application, the test was negative in the adult animals, whereas a remarkable percentage of positive SGF was obtained in 5-7-day-old rats, especially in those undernourished and/or intensely icteric. Unexpectedly, in infant rats the rate of positive SGF after sulfadimethoxine injection was lower than in the untreated control group. Moreover, in 9-10-day-old animals who had positive SGF before the injection, the test became negative 15-20 min after sulfadimethoxine application in vivo. No convincing explanation could be given, but drug interference with the binding capacities of the Sephadex column could be excluded by appropriate in vitro tests.