Recombinant human interleukin-1 receptor antagonist in the treatment of steroid-resistant graft-versus-host disease

Acute graft-versus-host disease (GVHD) that is resistant to therapy is a highly lethal complication of marrow transplantation. Inflammatory cytokines such as interleukin-1 (IL-1) may be critical mediators of this process. If so, specific inhibition of IL-1 activity with recombinant human IL-1 receptor antagonist (IL-1Ra), a naturally occurring competitive inhibitor of IL-1, may ameliorate acute GVHD. We performed an open-label, phase I/II trial to evaluate the safety and efficacy of IL-1Ra in 17 patients with steroid-resistant GVHD. The IL- 1Ra was administered as a 24-hour continuous infusion over 7 days. The dose was escalated in cohorts of patients from 400 to 3,200 mg/d. Acute GVHD was evaluated in each affected organ and as an overall grade. Stage-specific improvement of acute GVHD occurred in the skin (8 of 14, 57%), gut (9 of 11, 82%), and liver (2 of 11, 18%). Overall, acute GVHD improved by at least one grade in 10 of 16 (63%) patients. Response to therapy was associated with a reduction of tumor necrosis factor-alpha (TNF-alpha) mRNA levels in blood mononuclear cells (P = .001). The only toxicity attributable to IL-1Ra was reversible transaminase elevation in two patients. Inhibition of IL-1 activity with IL-1Ra is safe and has demonstrable efficacy in acute GVHD that failed to respond to conventional treatment. These data provide further evidence that IL-1 is a mediator of GVHD.     

Interleukin-3 expression by activated T cells involves an inducible, T- cell-specific factor and an octamer binding protein

Interleukin-3 (IL-3) is exclusively expressed by activated T and natural killer cells, a function that is tightly controlled both in a lineage-specific and in a stimulation-dependent manner. We have investigated the protein binding characteristics and functional importance of the ACT-1-activating region of the IL-3 promoter. This region binds an inducible, T-cell-specific factor over its 5' end, a site that is necessary for the expression of IL-3 in the absence of other upstream elements. Over its 3' end, it binds a factor that is ubiquitously and constitutively expressed. This factor is Oct-1 or an immunologically related octamer-binding protein, and it plays a role in coordinating the activity of several regulatory elements. These characteristics make the ACT-1 site analogous to the activating ARRE-1 site in the IL-2 promoter. Furthermore, and despite a lack of sequence homology, the promoters of IL-3 and IL-2 share an organizational pattern of regulatory elements that is likely to be important for the T- cell-specific expression of these genes.     

Lymphoma regression induced by monoclonal anti-idiotypic antibodies correlates with their ability to induce Ig signal transduction and is not prevented by tumor expression of high levels of bcl-2 protein

Custom-made monoclonal anti-idiotype antibodies (anti-Id MoAbs) have been tested as a treatment modality in 34 non-Hodgkin's lymphoma (NHL) patients. Partial or complete tumor remissions have been induced with this treatment in 68% of these patients. One mechanism by which anti- idiotype antibodies may have induced these tumor responses is via a direct antiproliferative effect on the tumor cells, resulting in apoptosis. Primary NHL cells do not proliferate well enough in vitro to test this hypothesis directly. Therefore, we studied the effect of anti- idiotype antibodies on signal transduction through the surface Ig receptor as measured by the induction of cellular protein tyrosine phosphorylation. To assess whether bcl-2 protein could protect lymphoma cells from death induced by anti-Id MoAb, we also measured the level of bcl-2 protein in the same tumor cells. We found a strong correlation between the ability of an anti-Id MoAb to induce an increase in tyrosine phosphorylation in vitro and its ability to induce a tumor regression in the patient. By contrast, the level of bcl-2 expressed by the tumor cells was not correlated with clinical response to anti-Id MoAb treatment.     

Production of granulocyte-macrophage colony-stimulating factor by primary cultures of unstimulated rat microvascular endothelial cells

Small vessel (microvascular) endothelial cells are in close contact with hematopoietic progenitor cells in the bone marrow and therefore may have an important role in hematopoiesis. Although other studies have shown that endothelial cells produce various colony-stimulating factors (CSFs), these studies examined large vessel endothelial cells, which are different in many respects from microvascular endothelial cells and which do not contact cells in the bone marrow. We show in this study that primary cultures of unstimulated rat fat capillary endothelial cells grown in serum-free medium produce a substantial amount of granulocyte-macrophage CSF (GM-CSF). The medium conditioned by these cells stimulated proliferation of two different lines of GM- CSF-responsive cells--PT-18 mast cells and FDC-P1 cells--and supported the growth of cells of the granulocyte and macrophage lines in cultures of rat bone marrow cells. The factor responsible for this activity had physical properties consistent with those of GM-CSF, namely, a similar apparent mol wt by gel filtration, resistance to repeated freeze-thaws, resistance to boiling for ten minutes but not for 30 minutes, and resistance to heating to 56 degrees C for one hour. The factor causing target cell stimulation was not B cell-stimulating factor-1 (BSF-1, or IL 4), since it failed to stimulate a BSF-1-responsive cell line HT2- JH, and target cells (PT-18) did not respond appreciably to recombinant BSF-1. Northern blot analysis of mRNA from rat fat capillary endothelial cells showed high levels of expression of GM-CSF, confirming that this factor is produced by microvascular endothelial cells. This is the first report of CSF production by unstimulated microvascular endothelial cells, demonstrating that these ubiquitous cells are capable of producing sizable amounts of at least one growth factor for hematopoietic progenitor cells.     

High fetal hemoglobin production in sickle cell anemia in the eastern province of Saudi Arabia is genetically determined

Homozygous sickle cell disease in the eastern province of Saudi Arabia is clinically mild. Circulating fetal hemoglobin levels of 16.0 +/- 7.4% were found in these anemic patients, but only 1.09 +/- 0.97% in their sickle trait parents. To determine whether these sickle cell anemia patients inherit an increased capacity to synthesize fetal hemoglobin, a radioimmunoassay of fetal and adult hemoglobin was performed on erythroid progenitor (BFU-E)-derived erythroblasts from Saudi Arabian sickle cell patients and their parents. Mean fetal hemoglobin content per BFU-E-derived erythroblast from Saudi Arabian sickle cell patients was 6.2 +/- 2.4 pg/cell or 30.4 +/- 8.6% fetal hemoglobin (normal 1.1 +/- 0.7 pg/cell and 5.1 +/- 1.8%). Linear regression analysis of % HbF in peripheral blood versus % HbF per BFU-E- derived cell showed a positive correlation with an r of 0.65. The variance of the intrinsic capacity to produce HbF may account for almost 40% (r2) of the variance of circulating fetal hemoglobin but other factors, particularly selective survival of F cells, must also contribute significantly. Despite virtually normal HbF levels in sickle trait parents of these Saudi patients, mean fetal hemoglobin production per BFU-E-derived erythroblast in these individuals was elevated to 3.42 +/- 1.79 pg/cell or 16.1 +/- 6.4% fetal hemoglobin, and the magnitude of fetal hemoglobin production found in parents correlated with that of the patients. These data indicate that the high fetal hemoglobin in Saudi sickle cell disease is genetically determined but expressed only during accelerated erythropoiesis. Further evidence of such genetic determination was provided by analysis of DNA polymorphisms within the beta-globin gene cluster on chromosome 11. This revealed a distinctive 5' globin haplotype (+ + - + +) on at least one chromosome 11 in all high F SS and AS tested. The precise relationship of this haplotype to HbF production in this population remains to be defined.     

Busulfan and total body irradiation as antihematopoietic stem cell agents in the preparation of patients with congenital bone marrow disorders for allogenic bone marrow transplantation

The capacity of busulfan and total body irradiation to ablate hematopoietic stem cells as preparation for the allogeneic bone marrow transplantation of patients with congenital bone marrow disorders was studied. Fourteen patients received 18 transplants; busulfan was used in the preparatory regimen of eight transplants and total body irradiation in the regimens of six transplants. Sustained hematopoietic ablation was achieved in six of eight patients prepared with busulfan and in all six patients prepared with total body irradiation. Three patients prepared with total body irradiation died with idiopathic interstitial pneumonitis, whereas no patients receiving busulfan developed interstitial pneumonitis. The optimal antihematopoietic stem cell agent to be used for the preparation of patients with congenital bone marrow disorder for bone marrow transplantation is not certain.     

Beta-thalassemia due to two novel nucleotide substitutions in consensus acceptor splice sequences of the beta-globin gene

We have identified two novel RNA-splicing mutations affecting a critical nucleotide (nt) in the acceptor consensus sequences at both the IVS-1/exon 2 and IVS-2/exon 3 junctions of the human beta-globin gene. Both mutations are single nt substitutions, T to G and C to A, at position -3 adjacent to the invariant AG dinucleotide. For the IVS- 2/exon 3 mutation abnormal splicing into the cryptic splice site at IVS- 2 nt 579 is documented. Identification of these two mutations provides further support for the importance of the location of specific nucleotides within the consensus sequences in splice site selection and RNA processing.     

Alteration in bone marrow adherent layer growth factor expression: a novel mechanism of chronic myelogenous leukemia progression

Philadelphia chromosome1 positive (Ph1) chronic myelogenous leukemia (CML) is characterized by metamorphosis of the chronic phase to blastic crisis. However, cellular events associated with this transition are poorly understood. To examine the possible participation of hematopoietic growth factors in this process, we studied growth factor expression in adherent layers of bone marrows derived from CML Ph1 patients in various stages of the disease. Interleukin-1 beta (IL-1 beta) and IL-6 mRNA were expressed in five of six patients, and granulocyte-macrophage colony-stimulating factor (GM-CSF) in one of six patients with myeloid/undifferentiated blast crisis. In addition, leukemia inhibitory factor (LIF) expression was increased in four of six patients with myeloid/undifferentiated blast crisis phase of the disease. IL-1 beta was also detected in bone marrow adherent layer conditioned medium from two of these patients. These results were in sharp contrast to the lack of detectable levels of uninduced IL-1 beta, IL-6, and GM-CSF mRNA, in samples derived from 4 patients in lymphoid blastic crisis, 3 in accelerated, and 11 in chronic phases of the disease, or from normal controls. The possibility of a paracrine loop formation, whereby the adherent layers representing the bone marrow stroma are induced to express hematopoietic growth factors, was supported by our finding IL-1 beta mRNA expression in the leukemic blast cells in three of four studied patients in blast crisis and IL-1 beta protein production in seven of eight patients studied. Finally, coculturing CML blast crisis cells onto pre-established adherent layers induced the expression of both IL-1 beta and IL-6 genes. From this preliminary study, it appears that abnormal expression of growth factors is a common event with CML Ph1 progression. We hypothesize that IL-1 beta generated by the transformed malignant clone stimulates the marrow stroma to produce various growth factors, and that this process may play a role in disease progression.     

Measurement of ploidy distribution in megakaryocyte colonies obtained from culture: with studies of the effects of thrombocytopenia

Microdensitometric measurement of the DNA content of individual megakaryocytes was performed using megakaryocyte colonies obtained following culture, in soft agar, of hematopoietic cells from C57BL/6J mice. Two types of colonies were detected. After 7 days of culture, the big cell type contained 16 /+- 2.3 acetylcholinesterase (AChE) positive cells/colony, with a mean ploidy level of 16.8 /+- 0.8/cell and the ploidy distribution characteristic of recognizable megakaryocytes in bone marrow. The heterogeneous type contained 44 /+- 9.6 cells/colony (some of which were AChE negative), with a mean ploidy level of 6.8 /+- 0.7/cell. The ploidy distribution of heterogeneous colonies differed markedly from big cell colonies, with preponderance of 2N and 4N cells. Colony-forming cells, obtained 4-5 days after induction of acute thrombocytopenia, gave big cell colonies with a marked increase in DNA content. Mean ploidy level increased to 21.5 /%- 1.8/cell; the frequency of 32N cells increased from 17% to 30% and 64N cells from 0% to 6%. This is the pattern of change observed in bone marrow, in vivo, 24 to 48 hr after induction of acute thrombocytopenia. The number of cells/colony did not increase. In contrast, acute thrombocytopenia did not alter the ploidy of heterogeneous colonies. The different responses to the stimulus of acute thrombocytopenia suggest that there are at least two types of Meg-CFC. The delayed appearance of altered Meg-CFC that produced big cell colonies indicates that the pool of stem cells, from which committed megakaryocyte precursors are derived, may respond indirectly to the stimulus of platelet depletion.