Development of a marrow transplant regimen for acute leukemia using targeted hematopoietic irradiation delivered by 131I-labeled anti-CD45 antibody, combined with cyclophosphamide and total body irradiation

In an attempt to decrease the relapse rate after bone marrow transplantation (BMT) for advanced acute leukemia, we initiated studies using 131I-labeled anti-CD45 antibody (BC8) to deliver radiation specifically to hematopoietic tissues, followed by a standard transplant preparative regimen. Biodistribution studies were performed in 23 patients using 0.5 mg/kg trace 131I-labeled BC8 antibody. The BC8 antibody was cleared rapidly from plasma with an initial disappearance half-time of 1.5 +/- 0.2 hours, presumably reflecting rapid antigen- specific binding. The mean radiation absorbed doses (cGy/mCi131I administered) were as follows: marrow, 7.1 +/- 0.8; spleen, 10.8 +/- 1.4; liver, 2.7 +/- 0.2; lungs, 2.1 +/- 0.1; kidneys, 0.7 +/- 0.1; and total body, 0.4 +/- 0.03. Patients with acute myelogenous leukemia (AML) in relapse had a higher marrow dose (11.4 cGy/mCi) than those in remission (5.2 cGy/mCi; P = .001) because of higher uptake and longer retention of radionuclide in marrow. Twenty patients were treated with a dose of 131I estimated to deliver 3.5 Gy (level 1) to 7 Gy (level 3) to liver, with marrow doses of 4 to 30 Gy and spleen doses of 7 to 60 Gy, followed by 120 mg/kg cyclophosphamide (CY) and 12 Gy total body irradiation (TBI). Nine of 13 patients with AML or refractory anemia with excess blasts (RAEB) and two of seven with acute lymphocytic leukemia (ALL) are alive disease-free at 8 to 41 months (median, 17 months) after BMT. Toxicity has not been measurably greater than that of CY/TBI alone, and the maximum tolerated dose has not been reached. This study demonstrates that with the use of 131I-BC8 substantially greater doses of radiation can be delivered to hematopoietic tissues as compared with liver, lung, or kidney, which may improve the efficacy of marrow transplantation.     

Interleukin-10 is a proliferation factor but not a differentiation factor for human myeloma cells

Because interleukin-10 (IL-10) is a potent differentiation factor of human B cells into mature plasma cells, we investigated its effect on human malignant plasma cells. IL-10 did not induce any differentiation and increase in Ig synthesis in four human IL-6-dependent malignant plasma cell lines. However, it stimulated the proliferation of two of four cytokine-dependent cell lines in the absence of IL-6 and IL-10- dependent myeloma cell lines have been obtained. The myeloma cell growth activity of IL-10 was unaffected by anti-IL-6 and anti-IL-6R antibodies. Similarly, IL-10 stimulated (P = .001) the proliferation of freshly-explanted myeloma cells in IL-6-deprived cultures of tumor samples from patients with active multiple myeloma (MM) and produced twice as many myeloma cells in these cultures. Again, this cytokine was unable to induce further differentiation (assessed by rate of Ig production) of fresh myeloma cells. A very sensitive enzyme-linked immunosorbent assay (ELISA; 1 pg/mL) only rarely detected IL-10 in the sera of MM patients (3 of 89). On the contrary, serum IL-10 was detected in 60% of patients with plasma cell leukemia (12 of 20). These data show that IL-10 is an IL-6-unrelated growth factor for malignant plasmablastic cells. This cytokine could be involved in the late phase of MM in vivo.     

Aggregation of mammalian cells expressing the platelet glycoprotein (GP) Ib-IX complex and the requirement for tyrosine sulfation of GP Ib alpha

The glycoprotein (GP) Ib-IX complex mediates platelet aggregation in response to high shear forces by binding von Willebrand factor (vWF) in the plasma. We investigated the possibility that the complex could mediate a similar phenomenon if expressed in nonhematopoietic cells. When agitated on a tabletop shaker, CHO and L cells expressing the full complex formed large aggregates in the presence of vWF and the modulator ristocetin. When the rate of agitation was increased, aggregation occurred without added ristocetin and appeared to require only the application of a physical force. The aggregation was homophilic and temperature-dependent and required a functional ligand- binding subunit of the GP Ib-IX complex, GP Ib alpha. Posttranslational tyrosine sulfation of GP Ib alpha was required for aggregate formation and stability. Thus, aggregation of cells expressing the GP Ib-IX complex is a unique example of a ligand-receptor interaction induced by mechanical forces and demonstrates an important biological role for sulfation of tyrosine residues.     

Modulation of megakaryocytopoiesis by thrombopoietin: the c-Mpl ligand

We have further characterized the biological activities, mechanism of action, and target cell populations of recombinant human and murine thrombopoietin (rhTPO and rmTPO) in in vitro human and murine model systems. Alone, hTPO or mTPO stimulated the maturation of immature murine megakaryoblasts as measured in a single cell assay. The combination of hTPO or mTPO and interleukin-6 (IL-6) resulted in a further increase in megakaryocyte differentiation in this system. Murine TPO stimulated mouse megakaryocyte progenitor development. Human megakaryocyte progenitor development was potentiated by hTPO alone and further augmented in the presence of the early-acting cytokines (IL-3) or kit ligand/stem cell factor (KL/SCF). To further define the mechanism of action of TPO, neutralization studies were performed with antisera to IL-3, granulocyte-macrophage colony-stimulating factor (GM- CSF), IL-1 beta, and IL-11. No diminution in TPO activity was observed in the presence of these antisera. Moreover, because adhesive interactions are known to modulate hematopoiesis, we studied whether hTPO might alter such interactions between human bone marrow (BM) megakaryocytes and human BM stromal fibroblasts. No changes were observed in either megakaryocyte expression of the surface molecules lymphocyte function-associated antigen-1, very late activation antigen- 4, or intercellular adhesion molecule-1 or the adhesion of megakaryocytes to stromal fibroblasts after treatment with the growth factor. Furthermore, no induction of secretion of the cytokines IL-1 alpha, IL-1 beta, GM-CSF, IL-6, granulocyte-CSF, tumor necrosis factor- alpha, transforming growth factor-beta 1, or transforming growth factor- beta 2 by primary human BM megakaryocytes was noted after treatment of the cells with hTPO. To address whether TPO affects very primitive hematopoietic progenitors, we studied the residual cells from the BMs of mice treated with high doses of 5-fluorouracil. Although no effect of mTPO alone was noted on the viability or replication of such primitive murine progenitor populations, the triple combination of IL-3 + KL/SCF + TPO stimulated growth of megakaryocyte progenitors. These results indicate that TPO is a highly lineage-specific growth factor whose primary biological effects are likely to be direct modulation of the growth and maturation of committed megakaryocyte precursors and immature megakaryoblasts.     

Effect of calcium ion concentration on the ability of fibrinogen and von Willebrand factor to support the ADP-induced aggregation of human platelets

To investigate the suggestion that von Willebrand factor (vWf) can substitute for fibrinogen in supporting ADP-induced aggregation of human platelets, we studied platelet reactions in two media: (1) a high calcium medium, Tyrode-albumin solution containing calcium ions in the physiological range of 2 mmol/L, and (2) a low calcium medium, modified Tyrode-albumin solution from which calcium salt was omitted (calcium ion concentration approximately 20 mumol/L). In the high calcium medium vWf even at concentrations up to six times as high as physiological, showed little or no potentiation of ADP-induced platelet aggregation, whereas fibrinogen strongly potentiated reversible aggregation without thromboxane formation or release of granule contents. In the low calcium medium, either vWf or fibrinogen supported biphasic aggregation in response to ADP, with thromboxane formation and release of granule contents. Aspirin and the thromboxane receptor blocker BM 13.177 inhibited these secondary responses to von Willebrand factor, indicating that they require thromboxane A2 formation and feedback amplification by thromboxane A2. A monoclonal antibody, 10E5, to the platelet glycoprotein IIb/IIIa complex inhibited both primary and secondary aggregation. Although vWf supports ADP-induced aggregation when the concentration of ionized calcium is in the micromolar range, it does not support ADP-induced aggregation in the presence of a concentration of ionized calcium in the physiological range, indicating that vWf probably cannot substitute for fibrinogen in supporting ADP- induced aggregation in vivo.     

Oral cyclophosphamide versus chlorambucil in the treatment of patients with membranous nephropathy and renal insufficiency

We treated patients with idiopathic membranous nephropathy (iMGN) and renal insufficiency, using: (i) (n = 15) monthly cycles of steroids (1 g methyl-prednisolone i.v. on three consecutive days, followed by oral prednisone 0.5 mg/kg/day months 1, 3 and 5) and chlorambucil (0.15 mg/kg/day months 2, 4 and 6); or (ii) (n = 17) oral cyclophosphamide (1.5-2.0 mg/kg/day for 1 year) and steroids in a comparable dose. The groups were comparable in age, renal function and levels of proteinuria. During the 6 months preceding treatment, serum creatinine levels increased from 148 +/- 50 to 219 +/- 73 mumol/l in the chlorambucil group and from 164 +/- 86 to 274 +/- 126 mumol/l in the cyclophosphamide group. Median (range) follow-ups were: chlorambucil 38 months (8-71); cyclophosphamide 26 months (5-68) (NS). Renal function improved in both groups, but the improvement was short-lived in the chlorambucil group; 12 months after starting treatment, mean serum creatinine was 6.3 mumol/l lower in the chlorambucil group and 121 mumol/l lower in the cyclophosphamide group (p < 0.01). Four chlorambucil-treated patients developed ESRD, and five needed a second course of therapy, whereas only one cyclophosphamide-treated patient developed ESRD (p < 0.05). Remissions of proteinuria occurred more frequently after cyclophosphamide treatment (15/17 vs. 5/15; p < 0.01). Side-effects necessitated interruption of treatment in six patients on cyclophosphamide and in 11 on chlorambucil (p < 0.05). In our patients, oral cyclophosphamide was better tolerated than oral chlorambucil. The suggested greater efficacy of the oral cyclophosphamide regimen needs to be ascertained by longer follow-up.      

A prophylactic and therapeutic AIDS vaccine containing as a component the innocuous Tat Toxoid

Extracellular Tat can act as a viral toxin on uninfected cells of different tissues, including the CNS and the immune system, thus in order to immunize humans against Tat we have prepared a biologically inactivated but immunogenic Tat (Tat Toxoid). Tat Toxoid is not toxic in mice even at high doses. It triggers high levels of specific Tat Abs in the mouse and rabbit. Furthermore, in humans Tat Toxoid immunization was safe and induced in seronegatives persistent high levels of Tat Abs and in immunodeficient patients a significant rise of these specific Abs. Facing acute HIV-1 infection, the presence of high level of circulating Tat Abs promoted by Tat Toxoid vaccine should prevent Tatinduced immunosuppression and allow anti-HIV-1 cellular response to develop. As a consequence, early release of β-chemokines could enhance host resistance towards HIV-1, and, in infected people, inhibit viral replication and evolution towards AIDS.     

Outcome for patients who relapse after allogeneic bone marrow transplantation for chronic myeloid leukemia. Chronic Leukemia Working Party. European Bone Marrow Transplantation Group

We studied the clinical course of 130 chronic myeloid leukemia (CML) patients (89 males and 41 females) in the European Bone Marrow Transplantation Group (EBMT) registry who received transplants before January 1, 1988 and who subsequently had evidence of recurrent leukemia. All patients had received a pretransplant conditioning regimen including total body irradiation (TBI). The first evidence of relapse was cytogenetic only in 74 (57%) patients and hematologic in 56 (43%). The overall actuarial survival from relapse was 36% at 6 years, with a significantly higher proportion of survivors among female patients (53% v 30%; P < .002). In univariate analysis, the 6-year probability of survival was 52% for patients with cytogenetic relapse and 30% for patients relapsing in chronic phase (CP), while no patient who relapsed in advanced phase (AP or BC) survived more than 3.5 years from relapse (P < .0001). The actuarial survival of patients relapsing before 6 months, between 6 and 12 months, and later than 12 months after transplant was 27%, 26%, and 45%, respectively (P < .002). Among patients with cytogenetic relapse, partial or complete disappearance of Ph-positive cells occurred in 40% of untreated patients and in 42% of those treated with interferon (IFN). However, IFN therapy significantly delayed progression toward hematologic disease. Cytogenetic responses were observed in 25% of patients who received IFN for relapse into CP, while only one minor cytogenetic response was reported in patients on conventional chemotherapy. For patients presenting with cytogenetic relapse as well as for those in hematologic relapse, IFN therapy significantly improved the 2-year probability of survival. However, long-term survival for IFN-treated patients in either group was not different from long-term survival in comparable patients not receiving IFN therapy. Twenty-nine patients of this series underwent a second bone marrow transplant (BMT) and the projected survival at 4 years after the second transplant is 28%. In multivariate Cox regression analysis, four factors remained significantly associated with survival: disease phase at relapse (P < .0001), duration of time interval from BMT to relapse (P = .0001), interferon therapy at relapse (P = .0024), and patient sex (P = .0032). This retrospective study provides evidence that some patients who relapse after BMT may benefit from treatment with IFN; a second BMT may offer the chance of cure. Data from this analysis may be useful in designing future prospective trials on posttransplant CML relapse.     

Increase in survival time of liver transplants by protease inhibitors and a calcium channel blocker, nisoldipine

Kupffer cells are activated by calcium and release a variety of toxic mediators, including proteases. The purpose of these studies, therefore, was to determine if protease inhibitors and a calcium channel blocker could increase survival time in the rat model of orthotopic liver transplantation. Survival for 30 days was greater than 90% in this model when livers were stored for 1 hr in Ringer's solution (survival conditions)--however, grafts stored for 4 hr in Euro-Collins solution or 8 hr in University of Wisconsin (UW) solution survived postoperatively only 1.2 and 0.7 days, respectively (nonsurvival conditions). When livers were stored for 4 hr in Euro-Collins containing a cocktail of protease inhibitors (leupeptin, pepstatin A, phenylmethylsulfonyl fluoride, 20 ng/ml each; diisopropyl fluorophosphate, 100 microM) and subsequently transplanted, however, survival time was increased significantly to 11.5 days. Inclusion of a calcium channel blocker, nisoldipine (1.4 microM), in the protease inhibitor cocktail increased survival time to 23 days. Actually, nisoldipine alone increased survival time to 25 days. Nisoldipine alone also increased survival time in livers stored for 8 or 16 hr in UW solution to between 15 and 20 days. Serum transaminase levels reached peak values greater than 2400 U/L one day postoperatively in the nonsurvival groups, and liver injury assessed histologically was apparent. Under these conditions, pulmonary infiltration of inflammatory cells was observed in about 60% of the lungs examined and was associated with massive bleeding. Inclusion of the protease cocktail, nisoldipine, or both in the storage solutions decreased maximal SGOT levels and injury to both liver and lung significantly by about 50% postoperatively. Nisoldipine also decreased phagocytosis of carbon particles by the perfused liver 2- to 3-fold following storage under nonsurvival conditions (half-maximal effect = 0.3-0.4 microM nisoldipine). Moreover, nisoldipine improved hepatic microcirculation. It accelerated blood flow into the liver, as indexed by hemoglobin reflectance from the liver surface. These data support the hypothesis that Kupffer cells are activated early in the sequence of events that causes graft failure leading to endothelial cell-mediated alterations in the microcirculation. This work demonstrates clearly that dihydropyridine-type calcium channel blockers such as nisoldipine may be clinically useful in storage solutions for liver prior to transplantation.     

Thrombocytopoietic properties of oncostatin M

Oncostatin M (OM) is a 28-kD glycoprotein that exhibits a panoply of biologic effects. Based on histologic observations of increased splenic megakaryocytes in nude mice implanted with an OM-secreting cell line, the thrombocytopoietic properties of OM in mice were investigated in culture and in vivo. Alone, OM did not induce megakaryocytic colony formation, but in combination with murine interleukin-3 (IL-3), OM markedly enhanced colony formation. The effects of OM on colony formation were similar to those of IL-6. OM alone augmented acetylcholinesterase in short-term marrow cultures. In normal mice, the administration of OM augmented platelet counts without increasing other circulating blood cell counts. The increment in counts exceeded that observed with IL-6. The kinetics of the OM response suggested that maximal increases in platelets occurred 3 days after the cessation of OM administration, irrespective of the duration of administration. In irradiated mice, OM administration accelerated platelet recovery and prevented the decrease in red blood cells observed in irradiated control animals. The data show that OM behaves as a megakaryocytic maturation factor in vitro and augments platelet production in vivo. Based on these animal data, OM may have potential clinical utility as a thrombocytopoietic agent.