Early Post-transplant Lymphoproliferative Disease in the Donor Ureter Without Systemic Involvement: A Case Report.

BACKGROUND: Post-transplant lymphoproliferative disease is a serious complication of renal transplantation. Major risk factors include Epstein-Barr virus (EBV) seronegativity and induction immunosuppression with lymphocyte-depleting agents. RESULTS: We present a case of a 50-year year-old woman with very early onset PTLD confined to the donor ureter. Phenotypic studies on the tumor material reveal that the lymphoma was most likely of donor origin. A complete staging workup including the kidney allograft was negative for any other sites of involvement. CONCLUSIONS: This case, which had a fatal outcome, emphasizes the risk of renal transplantation in BV-negative individuals when given induction with lymphocyte-depleting drugs.

Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells.

The bcr-abl oncogene, present in 95% of patients with chronic myelogenous leukemia (CML), has been implicated as the cause of this disease. A compound, designed to inhibit the Abl protein tyrosine kinase, was evaluated for its effects on cells containing the Bcr-Abl fusion protein. Cellular proliferation and tumor formation by Bcr-Abl-expressing cells were specifically inhibited by this compound. In colony-forming assays of peripheral blood or bone marrow from patients with CML, there was a 92-98% decrease in the number of bcr-abl colonies formed but no inhibition of normal colony formation. This compound may be useful in the treatment of bcr-abl-positive leukemias.

Repression of Fanconi anemia gene (FACC) expression inhibits growth of hematopoietic progenitor cells.

Bone marrow failure is a consistent feature of Fanconi anemia (FA) but it is not known whether the bone marrow failure is a direct and specific result of the inherited mutation or a consequence of accumulated stem cell losses resulting from nonspecific DNA damage. We tested the hypothesis that the protein encoded by the FA group C complementing gene (FACC) plays a regulatory role in hematopoiesis. We exposed normal human lymphocytes, bone marrow cells, endothelial cells, and fibroblasts to an antisense oligodeoxynucleotide (ODN) complementary to bases -4 to +14 of FACC mRNA. The mitomycin C assay demonstrated that the antisense ODN, but not missense or sense ODNs, repressed FACC gene expression in lymphocytes. Treatment with the antisense ODN substantially reduced, in a sequence-specific fashion, cytoplasmic levels of FACC mRNA in bone marrow cells and lymphocytes. Escalating doses of antisense ODN increasingly inhibited clonal growth of erythroid and granulocyte-macrophage progenitor cells but did not inhibit growth of fibroblasts or endothelial cells. The antisense ODN did not inhibit growth factor gene expression by low density bone marrow cells or marrow-derived fibroblasts. We conclude that, while the FACC gene product plays a role in defining cellular tolerance to cross-linking agents, it also functions to regulate growth, differentiation, and/or survival of normal hematopoietic progenitor cells.

Constitutive and induced expression of hematopoietic growth factor genes by fibroblasts from children with Fanconi anemia.

The pathophysiological abnormalities leading to marrow failure and leukemogenesis in children with Fanconi anemia (FA) are not understood. We tested the hypothesis that the Fanconi anemia mutation results in insufficient production of hematopoietic growth factors by stromal cells by quantifying constitutive and induced production of interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and steel factor (SF) by untransformed fibroblasts from eight patients with FA from five different families. While no abnormalities were noted in SF or M-CSF production, we noted substantial variability in IL-6, GM-CSF, and G-CSF responses of cells obtained from different FA patients. Responses ranged from blunting to augmentation when compared to normal controls. Because there was variation between fibroblast strains from affected members of two multiplex sibships, however, it is clear that neither augmentation nor blunting is a direct effect of the FA mutations. In addition, because there was discordance between the G-CSF responses and the GM-CSF and IL-6 responses, the abnormalities noted in IL-1 responsiveness must lie distal to IL-1 receptor function and to stimulus-response coupling pathways shared between the three cytokines.

Specific repression of granulocyte-macrophage and granulocyte colony-stimulating factor gene expression in interleukin-1-stimulated endothelial cells with antisense oligodeoxynucleotides.

Antisense oligodeoxynucleotides (ODNs) have been used to effect the specific inhibition of cellular gene expression. We have evaluated the application of this approach to the inhibition of interleukin-1 (IL-1)-induced granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) expression in cultured human umbilical vein endothelial cells. Antisense ODNs or control ODNs (sense ODNs or missense ODNs containing random base substitutions) were added to cultures of endothelial cells, the cells were induced with IL-1 alpha, and the conditioned media were assayed for GM-CSF and G-CSF by quantitative bioassays and for immunoreactive GM-CSF by enzyme immunoassay. Antisense ODNs complementary to the first 15 or 18 bases of the translation start sites of GM-CSF or G-CSF mRNAs inhibited, in a concentration-dependent fashion, the IL-1-stimulated expression of the corresponding factor, but did not affect expression of the other factor. Control ODNs did not affect GM-CSF or G-CSF expression. Exposure to a GM-CSF antisense ODN, but not a control ODN, substantially reduced cytoplasmic GM-CSF mRNA levels in IL-1-stimulated endothelial cells. Neither ODN affected levels of endothelial leukocyte adhesion molecule (ELAM)1 or glyceraldehyde-3-phosphate dehydrogenase mRNAs. We conclude that antisense ODNs complementary to the translation start sites of GM-CSF or G-CSF mRNAs inhibit expression of the corresponding factor in a sequence-specific fashion and this effect is mediated, at least in part, by reduction in the cytoplasmic level of the targeted mRNA. Moreover, IL-1-induced GM-CSF or G-CSF expression does not depend on expression of the other factor.

Comparison of granulocyte-macrophage colony stimulating factor and interleukin-1 production from human peripheral blood mononuclear cells as measured by specific radioimmunoassays.

Attention has focused on cytokine networks in which gene and protein expression of some cytokines is under the influence of other cytokines. In the present studies, we addressed the relationship between the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-1 (IL-1) in human peripheral blood mononuclear cells (PBMC) stimulated with mitogens. Since bioassays for cytokines are sensitive to more than one of these factors, it was necessary to measure the amounts of IL-1 and GM-CSF independent of bioassays. A specific and sensitive (40 pg/ml) radioimmunoassay (RIA) was developed for human GM-CSF. The sensitivity of the RIA was greater when lysine residues were iodinated with Bolton-Hunter reagent than tyrosine residues using chloramine T. After stimulating PBMC with concanavalin A (Con A), the biological activity of GM-CSF was determined in bone marrow cultures and compared to immunoreactive GM-CSF; GM-CSF levels detected by bioassays and RIAs were highly correlated in two separate sets of experiments (r2 = 0.95 and 0.43). Incubation with Con A for 48 h induced more GM-CSF than stimulation by phytohemagglutinin (PHA) despite the fact that PHA stimulates large amounts of IL-1 alpha; indomethacin had no effect on Con A stimulated synthesis of GM-CSF or IL-1 alpha. In two separate studies, PBMC from 14 donors and a second group of 12 donors were incubated with Con A for 48 h and the total amount of immunoreactive IL-1 alpha and GM-CSF was determined in the same cell cultures. There was no correlation of the amount of either cytokines in these cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Transient myeloproliferative disorder of the Down type in the normal newborn.

Two infants with congenital nonlymphoblastic leukemia were discovered to have mosaicism for trisomy 21. Both infants achieved durable spontaneous remissions. Trisomy was apparently restricted to the leukemic clone and could be detected in neither phytohemagglutinin-stimulated peripheral blood cells or bone marrow in either patient nor in myeloid progenitor cells from the second patient after resolution of the transient myeloproliferative disorder. We conclude that spontaneous remission of congenital leukemia is not confined to infants with partial or complete systemic trisomy 21 but can occur in genetically normal newborns whose leukemic cells contain a third chromosome 21.

Interleukin-1 stimulation stabilizes GM-CSF mRNA in human vascular endothelial cells: preliminary studies on the role of the 3' AU rich motif.

To date, the notion that the AU rich motif in the 3' UT of GM-CSF mRNA can function as an IL-1 response element has not been adequately examined. We suspect that it must play some role because virtually all IL-1 inducible gene have AU rich 3' untranslated regions. Thus, we will continue to address these technical problems so that the hypothesis can be more clearly tested. Nonetheless, we can state with reasonable certainty that: 1) IL-1 induces expression of GM-CSF in EC by inducing accumulation of mRNA, 2) the GM-CSF gene is constitutively transcribed but the half life of the mRNA is short, 3) IL-1 induced mRNA accumulation results from stabilization of the transcript, 4) although most IL-1 responsive genes have AU-rich domains in their 3' untranslated regions, it is not yet clear that these AU-rich regions are sufficient to function as an IL-1 response element, and 5) murine L cells cannot be used for studies on the molecular biology of cytokine induction by IL-1.

The hematopoietic microenvironment in the elderly: defects in IL-1-induced CSF expression in vitro.

The expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) genes by stromal cells of the hematopoietic microenvironment is regulated, in vitro, by interleukin 1 alpha (IL-1 alpha) and IL-1 beta. We reasoned that malfunction of this inductive mechanism in vivo might contribute to the intolerance of the aged to myelosuppressive therapy. We found that bone marrow fibroblasts from healthy elderly (ages 65-75 years) volunteers were less sensitive to the inductive effects of recombinant IL-1 beta than were marrow fibroblasts from younger volunteers, and we proposed that an age-related decline in sensitivity of marrow stroma to IL-1 may impair the ability of the elderly to increase production of hematopoietic growth factors under conditions of physiologic stress.

Human vascular endothelial cells, granulopoiesis, and the inflammatory response.

We have carried out a series of in vitro studies designed to characterize the role of mononuclear phagocytes as regulators of hematopoiesis. The results of these studies have demonstrated that mononuclear phagocytes produce factors, including interleukin-1 (IL-1), that induce the expression of multilineage hematopoietic growth factors by human vascular endothelial cells. In more recent studies we and others have identified these induced factors as G-CSF, GM-CSF, IL-6, and IL-1. Interleukin 1 stimulates expression of these genes by inducing the accumulation of gene transcripts. Moreover, transcript accumulation, at least with GM-CSF, results from prolongation of mRNA half-life. Based on preliminary studies in a cell-free system, we propose that the inductive capacity of IL-1 results from its activation of ribonuclease inhibitors in the cytoplasm of IL-1-induced cells and hypothesize that this may be a general mechanism by which IL-1 induces gene expression.

Analysis of murine megakaryocyte colony size and ploidy: effects of interleukin-3.

Megakaryocytes develop from diploid precursor cells that, after variable numbers of mitoses, cease cell division and then undergo synchronous nuclear endoreduplication (endomitosis). Megakaryocyte colony formation represents an in-vitro model of these processes in which the number and ploidy of colony cells reflect the activity of the mitotic and endomitotic pathways, respectively. We have analyzed the size and ploidization of murine megakaryocyte colonies grown in agar and examined the influence of interleukin-3 (IL-3) on these parameters. Colonies were identified in situ by staining for acetylcholinesterase and the ploidy of colony cells was determined by fluorescence cytophotometry. More than 98% of the megakaryocytes that developed in culture could be analyzed. In cultures of unfractionated marrow cells stimulated by either pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM, a crude source of megakaryocyte colony-stimulating activity) or IL-3, the modal ploidy of day-5 colony megakaryocytes was 16N (range 2N-128N). The distribution of colony size was described by an inverse exponential relationship. Colony size and geometric mean ploidy were inversely correlated under conditions of maximal stimulation with PWM-SCM and at all concentrations of IL-3 tested. Increasing concentrations of IL-3 in cultures of either unfractionated marrow cells or nonadherent T-lymphocyte-depleted (NATD) marrow cells stimulated similar dose-dependent increases in the mean size and numbers of megakaryocyte colonies but did not significantly alter their ploidy distribution. However, the mean ploidy of colony megakaryocytes in IL-3-stimulated cultures of NATD marrow cells was significantly less (P less than 0.001) than the mean ploidy of megakaryocytes in IL-3-stimulated cultures of unfractionated marrow cells. The mean ploidy of megakaryocytes, which developed in PWM-SCM-stimulated cultures, was not affected by initial accessory cell depletion. We conclude that the size and ploidy characteristics of day-5 murine megakaryocyte colonies are structured as continua and that IL-3 stimulates an increase in mean colony size and numbers without affecting ploidization. T-lymphocytes and adherent cells elaborate an activity which promotes endomitosis in vitro; factors in PWM-SCM can substitute for this activity.

Erythroid burst-promoting activity produced by interleukin-1-stimulated endothelial cells is granulocyte-macrophage colony-stimulating factor.

Interleukin-1 (IL-1) induces cultured human umbilical vein endothelial cells to elaborate heterogeneous hematopoietic growth factors, including granulocyte-macrophage and granulocyte colony-stimulating factors (GM-CSF and G-CSF, respectively). Because erythroid burst-promoting activity (BPA) is also elaborated by endothelial cells exposed to IL-1, we sought to determine whether the BPA released by IL-1-induced endothelial cells simply reflects the known erythropoietic activity of GM-CSF or whether other uncharacterized factors might be involved. Media conditioned by multiply passaged endothelial cells cultured for three days with recombinant IL-1 alpha (ECMIL-1) stimulated erythroid burst and GM colony formation in cultures of human nonadherent T-lymphocyte-depleted marrow mononuclear cells. Pretreatment with an anti-GM-CSF antiserum neutralized all the BPA and 56% of the GM colony-stimulating activity (GM-CSA) in ECMIL-1. The antiserum used in these studies did not inhibit IL-3 or G-CSF activity and did not inhibit ECMIL-1-induced murine GM colony growth (a measure of human G-CSF). To examine whether GM-CSF induces BPA release by accessory cells, media conditioned by marrow cells cultured for three days with GM-CSF were tested in the colony growth assays. Pretreatment with anti-GM-CSF antiserum completely neutralized the BPA and GM-CSA of the marrow cell-conditioned medium. We conclude that GM-CSF is the BPA elaborated by IL-1-induced endothelial cells. The in vitro erythropoietic activity of GM-CSF is not dependent on induced BPA release by accessory cells and therefore likely results from a direct effect of GM-CSF on progenitor cells.

Vascular endothelial cells and hematopoietic regulation.

Vascular endothelial cells elaborate growth factors which support the proliferation and differentiation of hematopoietic progenitor cells. Both the regulation and potential biological significance of growth factor production by endothelial cells are reviewed.

The anemia of end-stage renal disease: hematopoietic progenitor cell response.

Patients with the anemia of end-stage renal disease (ESRD) fail to display an appropriate compensatory increase in red cell production. In order to investigate the extent to which the impaired erythropoietic response is determined at the progenitor cell level, we determined the frequencies of marrow colony-forming cells in 11 anemic and 3 non-anemic, dialysis-dependent ESRD patients and 10 healthy individuals. In addition, we measured serum levels of erythropoietin (Epo) by radioimmunoassay. There were no significant differences (P greater than 0.1) between normal and ESRD groups in the frequencies of primitive or late erythroid (BFU-E and CFU-E, respectively), granulocyte-macrophage, and megakaryocyte progenitors, CFU-E/BFU-E ratios, or serum Epo levels. In contrast, 5 non-uremic patients with chronic anemia comparable in severity to the anemic ESRD patients had serum Epo levels and CFU-E/BFU-E ratios that were significantly increased (P less than 0.05 and P less than 0.001, respectively) in comparison to the normal controls and ESRD patients. Pre-dialysis serum and plasma from both ESRD groups were as supportive of autologous erythroid and non-erythroid colony growth in vitro as normal serum and plasma; inhibition was not observed. We conclude that the relative numbers of erythroid and non-erythroid progenitors and the majority of serum Epo levels are unchanged from normal in patients with the anemia of ESRD. However, their normal CFU-E/BFU-E ratio reflects an inadequate compensatory erythropoietic response due to their inability to appropriately increase Epo production in response to anemia. Inhibitors of autologous erythroid colony formation were not detected in ESRD serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 induces human bone marrow-derived fibroblasts to produce multilineage hematopoietic growth factors.

Interleukin-1 (IL-1) has been shown to induce stromal cells, including endothelial cells and fibroblasts, to produce multilineage hematopoietic growth factors. Although both of these cell types are well-described elements of the hematopoietic microenvironment, previous studies of IL-1-inducible colony-stimulating factor responses have utilized fibroblasts and endothelial cells from nonhematopoietic sites. Since we hypothesize that this intercellular growth network is active in the hematopoietic microenvironment, we sought to determine the responsiveness of bone marrow fibroblasts to IL-1. We demonstrate here that recombinant human IL-1 alpha and beta stimulate the dose-dependent release of granulocyte-macrophage colony-stimulating activity (GM-CSA) and burst-promoting activity (BPA) by cultured human bone marrow fibroblasts. We conclude that bone marrow fibroblasts produce hematopoietic growth factors in response to interleukin-1, and that this may be a mechanism by which stromal cells regulate cellular growth and differentiation within the hematopoietic microenvironment.

Mechanisms of thrombocytopenia in chronic autoimmune thrombocytopenic purpura. Evidence of both impaired platelet production and increased platelet clearance.

Mechanisms of thrombocytopenia were studied in 38 patients with mild to moderately severe chronic autoimmune thrombocytopenia (AITP). 51Cr and 111In-labeled autologous platelet turnover studies and in vitro analysis of committed megakaryocyte progenitors (CFU-Meg) were used as independent measures of platelet production. Autologous 111In-labeled platelet localization studies were performed to assess platelet clearance. Although there was no increase in the frequency of marrow CFU-Meg, a specific increase in the CFU-Meg [3H]TdR suicide rate was seen which was inversely correlated with the platelet count (P less than 0.001). Platelet turnover studies showed significant numbers of patients had inappropriate thrombopoietic responses to their reduced platelet counts. Platelet-associated antibody levels correlated inversely with platelet turnover suggesting that antiplatelet antibody impairs platelet production. The circulating platelet count was best predicted by an index relating platelet production (i.e., turnover) to the spleen-liver platelet clearance that correlated directly with platelet survival (P less than 0.001). In summary, both depressed platelet production and increased platelet clearance by the liver and spleen contribute to the thrombocytopenia of AITP.

Interleukin 1 stimulates endothelial cells to release multilineage human colony-stimulating activity.

Cultured human umbilical vein endothelial cells, when exposed to soluble products of peripheral blood monocytes, elaborate granulocyte-macrophage colony-stimulating activity (GM-CSA) and erythroid burst-promoting activity (BPA). We have performed studies to determine if the monokine IL 1 can stimulate endothelial cells to release hematopoietic growth factors and whether such factors can also support human megakaryocyte (Meg) and mixed-cell colony growth. Various concentrations of recombinant human IL 1 beta (rIL 1) and media conditioned by monocytes (MCM), endothelial cells (ECM), and endothelial cells cultured for 3 days in 50% MCM (ECMM) or rIL 1 (ECMrIL 1) were added to marrow mononuclear cells cultured in methylcellulose. ECMM and ECMrIL 1 stimulated, in a dose-dependent fashion, the growth of Meg, mixed-cell, and GM colonies and erythroid bursts. In contrast, ECM, MCM, and rIL 1 displayed little or no activity in the colony-forming assays. Preincubation with specific antisera to native human IL 1 or rIL 1 reduced by 75 to 100% the activity of MCM in stimulating endothelial cell release of BPA, GM-CSA, Meg-CSA, and mixed-cell CSA. Meg-CSA, although readily detectable at ECMM and ECMrIL 1 concentrations in culture of 1 to 5%, was partially masked by lineage-specific inhibitors of Meg colony growth. When ECMM was analyzed by gel filtration chromatography, the megakaryocytopoietic inhibitory activity eluted in the high Mr fractions (greater than 75 kD). Meg-CSA co-eluted with GM-CSA and BPA in a single peak of 30 kD. We conclude that endothelial cells, in response to IL 1, produce one or more growth factors that probably act on multiple classes of progenitor cells.

Spermine and spermidine are non-specific inhibitors of in vitro hematopoiesis.

The polyamine spermine has been reported to be the inhibitor of in vitro erythropoiesis present in uremic serum. We have employed a panel of hematopoietic colony-forming assays to evaluate the specificity of the inhibitory activity. Spermine and its precursor spermidine when added to culture inhibited mouse and human erythroid (CFU-E and BFU-E), granulocyte-macrophage, and megakaryocyte colony growth in a non-specific, dose-dependent fashion. Erythroid and non-erythroid colony growth were equally sensitive to spermine- and spermidine-induced inhibition. Increasing concentrations in culture of erythropoietin and mitogen-stimulated leukocyte-conditioned medium, a source of colony-stimulating activity, failed to overcome the in vitro inhibition. Although anemia is characteristic of chronic renal failure (CRF), leukopenia and thrombocytopenia are not. Therefore, we conclude that the non-specific inhibitory activity of spermine and spermidine, as defined by in vitro colony assays, is either of no pathophysiologic significance in the anemia of CRF, or else there are unrecognized repair mechanisms in vivo which maintain granulopoiesis and thrombopoiesis at normal levels.

The L4F3 antigen is expressed by unipotent and multipotent colony-forming cells but not by their precursors.

Antibody L4F3 is a murine monoclonal antibody that recognizes an antigen expressed on in vitro colony-forming cells, including virtually all CFU-GM, CFU-Meg, BFU-E, and CFU-Mix. In the present study we examined whether cells that do not express the L4F3 antigen include precursors of hematopoietic colony-forming cells. Colony-forming cells were depleted from marrow by treatment with L4F3 and complement. The remaining cells generated CFU-GM, BFU-E, and CFU-Mix when cultured in the presence of irradiated adherent cell layers from long-term marrow cultures. Marrow cells not expressing the L4F3 antigen, which were separated by cell-sorting techniques, were depleted of colony-forming cells but nevertheless generated CFU-GM when cultured over irradiated adherent cell layers. These data suggest that there are marrow precursors that do not express the L4F3 antigen and that give rise to colony-forming cells of multiple types. Negative selection techniques should allow the enrichment of these precursors of colony-forming cells, thereby enabling direct studies of these immature stem cells.