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THE properties of the fungus metabolite, cyclosporin A, have suggested its potential as a clinically valuable immunosup-pressive agent(1,2). Experiments in animals have demonstrated that its suppressive action against cell-mediated and humoral immunity is not accompanied by any appreciable myelotoxicity(3,4). In this respect, cyclosporin A contrasts with other immunosuppressants in current use, such as antilymphocyte globulin and azathioprine. There are, however, few data to substantiate a selective toxicity of cyclosporin A against human lymphocytes. Here, we have compared its effects against human lymphoid and myeloid cells, using colony formation by the different cell types as the end point. We found that the compound exhibited far greater toxicity towards a sub-population of T cells than towards either B lymphocytes or haematopoietic precursor cells.

Repeated doses of the non-specific immunostimulant BCG were injected intraperitoneally, followed by serial measurement of serum colony stimulating factor (CSF), and CFU-C number and percent synthesizing DNA. Following primary challenge with BCG, CSF remained at low levels until day 7, rose to a peak by day 10, and remained elevated through 14 days. Secondary challenge resulted in a bi-phasic CSF response with a small peak on day 1 and a larger one by day 7. Following secondary challenge, the percent of CFU-C in 'S' phase doubled in 24 hours; CFU-C significantly increased in number by 48 hours. The studies suggest that the marrow toxicity of cycle active cytotoxic drugs might be altered by non-specific immunostimulants such as BCG.

The origin and nature of cells forming the in vitro microenvironment in long-term cultures of human marrow were studied in five patients with clonal myeloproliferative disorders who were heterozygous for glucose-6-phosphatase dehydrogenase (G6PD). The results showed that cells in the adherent stromal layer forming the in vitro microenvironment were derived from the same clonal progenitors involved by the neoplasm in the four patients whose diseases originated in multipotent stem cells. In contrast, stromal cells were derived from normal progenitors in a patient with acute non-lymphocytic leukemia whose clone showed differentiative expression confined to cells in the granulocytic lineage. Mixing experiments demonstrated that the G6PD type displayed by the adherent marrow stromal cells was not obscured by contaminating non-adherent hematopoietic cells or marrow fibroblasts. The data suggest the existence of a pluripotent cell in normal hematopoiesis that gives rise to hematopoietic cells and to their micro-environment.

Further in vitro studies of hematopoietic regulation were carried out in two patients with polycythemia vera who were also heterozygotes (Gd(B)/Gd(A)) for glucose-6-phosphate-dehydrogenase (G-6-PD). While only G-6-PD type A was detectable in circulating erythrocytes, granulocytes and platelets, cultures of peripheral blood and marrow from one patient revealed a substantial number of G-6-PD type B erythroid burst-forming units (BFU-E) and granulocyte/macrophage colony-forming units. Detailed analysis demonstrated: (a) where detectable, normal BFU-E and granulocyte/macrophage colony-forming units were found with similar frequencies; (b) the same frequencies for normal progenitors characterized cultures of peripheral blood and marrow; (c) inhibition of normal erythroid differentiation between BFU-E and the more mature erythroid colony-forming unit; (d) a decline in the prevalence of normal colony-forming units with time, suggesting that disease progression is associated with further suppression of normal hematopoiesis by products of the abnormal clone.

The response of marrow stromal cells transformed with wild-type simian virus 40 to recombinant growth factors was examined. When transformed stromal cells were plated in semisolid medium without the addition of growth factors, only 0.4% of cells formed colonies while with the addition of recombinant factors such as interleukin 1 (IL-1) or tumor necrosis factor (TNF), up to 10% of the cells formed colonies. Colonies were individually plucked and cell lines were developed that could be analyzed for expression of growth factors. The data show that unstimulated marrow stromal cells lines produced no detectable colony-stimulating activity. However, cell lines derived from "autonomously growing colonies" and from colonies grown with T cell-conditioned medium, with IL-1 alpha or beta, or with TNF alpha produced colony-stimulating activity and transcripts for granulocyte/macrophage-colony-stimulating factor (CSF), granulocyte-CSF, and IL-1 beta. A novel feature of the cell lines derived from colonies was that the production of growth factors was constitutive and persisted in excess of 4 m.

Analysis of protooncogene RNA expression in marrow stromal cells from long-term marrow culture demonstrated high levels of c-abl 5-, 6-, and 7-kilobase (kb) RNA transcripts. In experiments on three independently derived simian virus 40-transformed marrow stromal cell lines, the expression of these c-abl transcripts was further increased in response to recombinant tumor necrosis factor alpha (1000 units/ml) and interleukin 1 alpha (10 units/ml). Although lymphocyte-conditioned medium predominantly up-regulated the 5-kb transcript, interleukin 1 alpha primarily affected the 6-kb transcript. The up-regulation of the 5-kb c-abl message correlated with up-regulation of the granulocyte/macrophage colony-stimulating factor transcript and down-regulation of procollagen I transcripts in transformed cells. These data suggest that c-abl plays roles in the regulation of extracellular matrix expression and in the regulation of hematopoietic growth factors by stromal cells.

Recent investigations have shown a therapeutic and cytogenetic response in chronic myelogenous leukemia (CML) patients treated with recombinant alpha 2-interferon (IFN alpha 2). Philadelphia chromosome-positive (and many Ph1-negative) chronic myelogenous leukemia cells transcribe a novel bcr-abl fusion mRNA which may confer a growth advantage upon these cells. We investigated the effect of IFN alpha 2 on the levels of bcr-abl transcript expression in three Ph1-positive CML cell lines, EM2, KCL22, and K562. Although IFN alpha 2 inhibited cell proliferation in all three CML cell lines, IFN alpha 2 had no effect on the level of bcr-abl mRNA expression in any of the CML cell lines. In contrast, IFN alpha 2 increased the expression of class I HLA gene products. We conclude that while the bcr-abl fusion gene and its transcript undoubtedly play key roles in the pathogenesis of CML, the antiproliferative effect of IFN alpha 2 in CML cell lines relies upon genetic mechanisms other than modulation of bcr-abl expression.

Two patients with acute nonlymphocytic leukemia who were heterozygous for the X-chromosome-linked enzyme glucose-6-phosphate dehydrogenase were studied to determine the number and type of cells in which the disease arises. Both type A and B isoenzymes were found in normal tissues, but the myeloblasts showed only one enzyme type, indicating that at the time of study, the disease had a clonal origin. The observation in one patient that erythroid cells did not arise from this clone contrasts with conclusions reached in patients previously studied with chromosomal markers. The results suggest that in this patient, the leukemic clone suppressed expression of normal granulopoiesis but did not inhibit erythroid differentiation from normal progenitors. They suggest also that the disease is heterogeneous. In some patients, the disease is expressed in cells with differentiation restricted to the granulocyte-macrophage pathway; in others, it involves stem cells that also differentiate into erythrocytes. This heterogeneity may reflect differences in causation and could have prognostic importance.

To determine whether acute nonlymphocytic leukemia develops clonally, to study the pattern of differentiation of the involved stem cells, and to determine whether clinical remissions are true remissions, we studied 27 patients with acute nonlymphocytic leukemia who were heterozygous for the X-chromosome-linked glucose-6-phosphate dehydrogenase. In each case, leukemic blast cells manifested only one type of glucose-6-phosphate dehydrogenase, indicating that the malignant process had developed from a single cell. In six elderly patients, circulating erythrocytes, platelets, or both expressed only the glucose-6-phosphate dehydrogenase found in blast cells, indicating that these leukemias had arisen from stem cells with multipotent differentiative expression. In 16 younger adults and children, erythroid cells and platelets were predominantly derived from normal stem cells. In three other cases, the stem cell that gave rise to leukemic blasts apparently also gave rise to erythroid progenitors but not to mature erythrocytes. Heterogeneity was also found during remissions. In 8 of 13 patients, restoration of nonclonal hemopoiesis and repopulation of the marrow by normal stem cells was observed during remission. In the other five patients, marrow stem cells remained partially or completely clonal, even during remission. These data indicate that acute nonlymphocytic leukemia is a heterogeneous disease with respect to differentiation of the stem cells involved by leukemia and the nature of remissions.

Long-term marrow cultures (LTMCs) provide a selective growth advantage for cytogenetically normal cells in patients with acute and chronic myeloid leukemias. In the present study, LTMCs were established from two patients with newly diagnosed acute myeloid leukemia (AML) who were heterozygous for the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD). Initially only leukemic clusters grew from cells plated in semisolid medium, but after 1 or more weeks in LTMC, morphologically normal granulocyte-macrophage colonies were detected. Nonetheless, in one of the patients, more than 80% of these colonies expressed the G6PD type observed in the leukemic blast cells, indicating a probable neoplastic derivation for many of them. In the second patient, colonies cultured during the first 3 weeks of the LTMC were predominantly derived from clonal progenitors, whereas after week 4 the colonies were derived from normal stem cells. Colonies derived from clonal or normal stem cells were not morphologically distinguishable. These data support the conclusion that LTMC has a selective anti-leukemic effect on marrow cells from some patients. However, normalization of colony growth is by itself not a sufficient criterion for determination of whether committed progenitor cells from patients with AML are derived from normal or leukemic stem cells.

Cardiac toxicity due to anthracycline therapy is dose related, and congestive failure is a major limiting factor to therapy. Radionuclide cardiac evaluation provides a sensitive noninvasive method for detecting changes in cardiac function. Fifteen patients receiving either doxorubicin or daunomycin were evaluated with radionuclide ejection fractions (EFrn). The data indicate that the EFrn can detect an acute depressant cardiac action of these drugs as early as 24 hr after drug administration. In addition, a cumulative or chronic cardiac depression was noted; cumulative dosage of doxorubicin or daunomycin correlated with a reduced EFrn (p less than 0.001). We conclude that (1) the EFrn can noninvasively detect significant changes in cardiac function at low cumulative doses of doxorubicin or daunomycin and (2) the EFrn may be useful in evaluating cardiac function in patients during doxorubicin or daunomycin therapy.

Few established cell lines derived from patients with chronic myelogenous leukemia have been reported. Cytogenetic examinations of two independently derived Philadelphia chromosome (Ph)-positive cell lines from a patient with chronic myelogenous leukemia were performed serially from their initiation in 1982 to the present. Subcultures of each of these lines maintained separately in two laboratories for over 2 years were compared for degree of divergence. The modal chromosome number declined substantially within the first few months and slowly thereafter. Despite hyperploidy, these lines have remained remarkably stable cytogenetically. For each line, the modal chromosome number is hypotetraploid, multiple copies of Ph are present and no normal chromosome #9 remains. Only a few marker chromosomes have arisen. Despite the multiple copies of Ph, a single bcr restriction pattern was seen, suggesting duplication of a single Ph, rather than independent translocation events. These lines should be very useful for in vitro studies of chronic myelogenous leukemia.

When substituted steroids of several classes are added to cultures of rat bone marrow cells in the presence of erythropoietin a consistent enhancement of the number of colonies of hemoglobin synthesizing cells is obtained. Maximum steroid effectiveness was found to be between 10(-6) and 10(-7) M. Representative compounds of several classes of steroids were examined for their ability to enhance colony growth, including delta 4-estrenes, delta 4-androstenes, 5alpha-H androstanes and estranes, 5beta-H estranes, pregnanes and androstanes. While testosterone and its 5alpha-H derivatives had little or no activity, many synthetic derivatives of testosterone were highly active in increasing erythroid colony growth. All 5beta-H androstanes, estranes, and all but one 5beta-H pregnane were active. Cortisol consistently inhibited colony growth and estradiol and progesterone had no significant effect.

The Philadelphia chromosome (Ph1), observed in greater than 90% of chronic myelogenous leukemia (CML) patients, results from a specific chromosomal translocation involving the c-abl gene. The translocation breakpoint occurs near c-abl and correlates with the production of an altered c-abl mRNA. In the CML-derived cell line K562, Ph1 is accompanied by a structurally altered c-abl protein (P210c-abl) with in vitro tyrosine kinase activity not detected with the normal c-abl protein (P145c-abl). We have examined c-abl proteins in other Ph1-positive CML cell lines and found that they all express P210c-abl. P210c-abl was also detected in bone marrow cells from CML patients with Ph1 in the accelerated and blast crisis phases of the disease. Comparison of the [35S]methionine-labeled tryptic peptides generated from the normal P145c-abl and P210c-abl showed that they have closely related structures, but additional polypeptide sequences are present in P210c-abl. Based on these results we propose that translocation of c-abl in Ph1-positive CML results in the creation of a chimeric gene leading to the production of a structurally altered c-abl protein with activated tyrosine kinase activity. The altered P210 c-abl protein is strongly implicated in the pathogenesis of CML.

Androgenic steroids and their non-androgenic 5beta-H metabolites enhance the number of colonies of hemoglobin synthesizing cells grown from rat bone marrow in response to a standard (0.25 unit/ml) concentration of erythropoietin. The target cells for two steroids were found to be different. Cells influenced by the androgen, fluoxymesterone (fluoxy), resembled cells responding to erythropoietin in their cycle characteristics, as measured by tritiated thymidine suicide, and in their physical characteristics, as determined by velocity sedimentation gradient separation. Cells responding to etiocholanolone (etio) had a much lower tritiated thymidine suicide rate and different sedimentation velocities. Preincubation of marrow cells with etio for two hours was sufficient to enhance erythroid colony growth by 84%, whereas a similar incubation with fluoxy produced no increment. These studies demonstrate that different classes of steroids may influence in vitro erythropoiesis by acting on distinct populations of marrow cells. Fluoxymesterone appears to act through cells already committed to respond to erythropoietin, while etiocholanolone appears to act on a separate, perhaps more primitive population of marrow cells.

A 23-year-old man developed Ph1-positive chronic myelogenous leukemia in 1966. After two short courses of busulfan not associated with severe myelosuppression, the frequency of Ph1-positive metaphases in his bone marrow was 40%. Over the next eight years, he remained hematologically stable without further therapy. During that time there was a progressive decline in the frequency of Ph1-positive metaphases in his bone marrow to 3% by 1976. His disease subsequently transformed to an acute lymphoblastic leukemia. Cytogenetic studies of the marrow at the onset of blast crisis and of cultured marrow blast cells suggested that the blasts were a mixture of Ph1-positive and Ph1-negative cells. This patient was thus unique in that he demonstrated spontaneous decline in the frequency of Ph1-positive cells in his bone marrow and he developed blast crisis with an apparent mixture of Ph1-positive and Ph1-negative blasts. These findings demonstrate that a Ph1-positive cell clone may lose its proliferative advantage over Ph1-negative cells and raise the possibility that Ph1-negative cells persisting in patients with Ph1-positive chronic myelogenous leukemia may be abnormal and, like the Ph1-positive cells, may be susceptible to acute leukemic transformation.

Eight recipients of marrow transplants with cytomegalovirus (CMV) pneumonia diagnosed by open lung biopsy were treated with doses of human leukocyte interferon of 2 X 10(4)--6.4 X 10(5) units/kg per day to evaluate its toxicity after marrow transplant and its effectiveness against CMV infection. All eight patients died from pneumonia, and virus was still present in lung tissue from seven patients cultured after death. Interferon doses of less than or equal to 1.6 X 10(5) units/kg per day did not affect the circulating granulocyte count. Patients treated with higher doses had a decrease in circulating granulocyte count, but study of granulocyte-macrophage colony-forming cells in culture showed no evidence of toxicity to granulocyte progenitor cells. The effect on in vitro lymphocyte function was variable. Antibody production was not impaired. Interferon was not effective against established CMV infection. However, there was less hematologic toxicity than was anticipated, and the prophylactic use of interferon after marrow transplant is feasible.

A 34-year-old man with diffuse eosinophilic fasciitis and a hypocellular myelodysplastic syndrome underwent marrow transplantation from an HLA-identical brother. Prompt hematopoietic reconstitution was observed, strongly suggesting that the marrow hypocellularity was caused by neither a serum inhibitory factor nor a microenvironmental disorder. The patient died of disseminated cytomegalovirus infection too early to evaluate the impact of hematopoietic reconstitution on the eosinophilic fasciitis. Nevertheless, marrow transplantation may offer a therapeutic option for those patients with this disorder who develop severe hematopoietic dysfunction and who have a suitable marrow donor.

A clinical syndrome of severe cardiomyopathy often accompanies administration of high doses of anthracycline agents. We studied 36 patients serially with radionuclide angiography. At three weeks following drug administration, 8 of 36 patients showed depression of ejection fraction (EF). All had received at least 280 mg/m2 of the drug and 7 had received more than 380 mg/M2. Definite clinical syndromes of congestive cardiomyopathy developed only in patients showing EF depression and in some patients, EF depression developed without signs of congestive heart failure. Ejection fraction studies at 5 minutes, 1 hour, 4 hours, 24 hours, 72 hours, and one week following drug administration showed no changes when compared to immediate preg-drug EF. Seven patients who died during the study underwent histologic examination. Only the single patient with a depressed EF showed histologic evidence of athracycline cardiotoxicity, although all but 1 of these patients had received at least 400 mg/M2. We conclude that serial radionuclide EF just prior to anthracycline administration is a potentially useful predictor of cardiac toxicity, and that EF depression and/or preservation of a normal EF should be weighed in the decision for administering a drug of this type at high dosage levels.

In previous studies of two patients with polycythemia vera (PV) and heterozygous at the X-linked locus for glucose-6-phosphate dehydrogenase (G-6-PD), only type A isoenzyme was found in non-lymphoid hematopoietic cells. However, some granulocytic and erythrocytic colonies grown in vitro had type B G-6-PD and therefore arose from presumably normal progenitors. In this study we exposed marrow cells from these same two patients to high-specific activity tritiated thymidine (3HTdR) before culture to kill cells actively synthesizing DNA. Individual granulocytic colonies were plucked and tested for G-6-PD after 14 d of culture. The frequency of type B colonies rose after exposure to 3HTdR from 8/101 to 11/36 in patient 1 and from 0/32 to 6/31 in patient 2 (P less than 0.003). No increase in the frequency of normal erythroid bursts after 3HTdR exposure was seen, implying that in PV, early granulopoiesis, and erythropoiesis are regulated differently. The results demonstrated that only type A granulocytic colonies, arising from the abnormal clone, were removed by the 3HTdR. In addition, for patient 2, statistical analysis indicated there was an absolute increase in normal granulocytic colonies detected in culture. Thus, PV clonal colony-forming units in culture (CFU-C) cycle more rapidly than do normal CFU-C and may suppress proliferation of normal CFU-C in vitro.

We evaluated the karyotypes of marrow-derived stromal cell lines established by simian virus-40 (SV-40) transformation of long-term cultures from four men who received marrow transplants from women donors. In all cases a normal female karyotype was found in marrow cells. In contrast, a Y chromosome was identified in metaphase cells from the stromal lines, suggesting that following successful marrow transplantation, cells in the marrow microenvironment susceptible to SV-40 transformation remain host in origin.

The method for long-term culture of marrow cells in vitro as described by Dexter has recently been successfully applied to human marrow and is dependent on the development of an adherent stromal cell layer consisting of cells described as "endothelial-like cells, fat cells, and macrophages". The present study was designed to determine the origin and composition of the stromal cells forming the in vitro 'microenvironment' and maintaining haematopoiesis in long-term cultures grown from marrows of 14 patients who received marrow transplants from HLA identical siblings of the opposite sex. The presence of a Y chromosome was used as a marker to establish the donor or recipient origin of the cells. We found that the stromal cells became progressively donor in origin with time after transplantation and some reacted with antibody directed against factor VIII-associated antigen. In addition, donor-derived in vitro stromal cells synthesized both interstitial and basal lamina collagen types, indicating that the in vitro microenvironment is transplantable and composed in part of endothelial-like cells.

A patient with angioimmunoblastic lymphadenopathy with dysproteinemia was followed over a three year period from diagnosis to death. He presented with arthralgias, uveitis and respiratory insufficiency and developed hyperuricemic renal failure upon institution of treatment. Aggressive combination chemotherapy was required to reverse progressive thrombocytopenia and pulmonary involvement. A complete remission was achieved twice. There was a striking temporal relationship between the administration of antibiotics or allopurinol and exacerbations of the disease. Hypocomplementemia and transient evidence of vasculitis suggested the presence of immunecomplexes. Serial lymph node biopsies showed the progression of this disorder from a pleomorphic immunoblastic proliferation to a lymphocyte-depleted, fibrotic process, in parallel with a decline from hyper- to hypogammaglobulinemia. This case illustrates the broad clinical spectrum of angioimmunoblastic lymphadenopathy with dysproteinemia and suggests that aggressive treatment is necessary in selected patients.

The distribution of a novel difucoganglioside (6B ganglioside, NeuAc alpha 2----3Gal beta 1----4[Fuc alpha 1----3]GlcNAc beta 1----3Gal beta 1----4[Fuc alpha 1----3]GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1Cer) in various normal adult and fetal tissues, as well as in cancer tissues, has been studied by immunoperoxidase staining with a specific monoclonal antibody, FH6, directed to this antigen. A large variety of embryonic and fetal tissues (stomach, colon, small intestine, pancreas, esophagus, lung, and heart) showed a diffuse, weakly positive staining, particularly in the epithelial layer, up to the 70th to 80th day of gestation. However, no staining was observed in various normal adult tissues, including gastrointestinal and glandular epithelial tissues which were stained positively by antibody N-19-9 (directed to sialyl-Lea) or CSLEXI (directed to sialyl-Lex). FH6-positive loci were limited to the proximal convoluted tubuli in kidney and granulocytes. In contrast, 44 of 76 cases of cancer tissue tested, including gastric, colonic, lung, breast, and renal cancers, showed clearly positive staining. The intensity of staining in gastric and colonic cancer tissues by FH6 antibody was weaker and less frequent, although the incidence of positive staining for lung (50%) and breast cancer (86%) was significantly higher than that of the antigen stained by monoclonal antibody FH4 (Y. Fukushi, S. Hakomori, and T. Shepard, J. Exp. Med., 159: 506-520, 1984), which is directed to the asialo core of the FH6 antigen. The antigen levels in the serum of patients with various cancers, inflammatory diseases, and normal subjects were determined by radioimmunoassay. The antigen level was found to be significantly higher in the serum of some patients with cancer, particularly lung, liver, and pancreatic cancers, as compared with the serum levels in other types of cancer, noncancerous diseases, and normal subjects.

In order to study the pattern of B cell involvement in acute nonlymphocytic leukemia (ANLL), multiple B lymphoid cell lines were established by Epstein-Barr virus transformation of peripheral blood mononuclear cells from two patients with the disease who were heterozygous for the X chromosome-linked glucose-6-phosphate dehydrogenase (G6PD). In one patient, the progenitor cells involved by the leukemia exhibited multipotent differentiative expression, whereas in the other patient the cells showed differentiative expression restricted to the granulocytic pathway. In the patient whose abnormal clone showed multipotent expression, the ratio of B-A G6PD in B lymphoid cell lines was skewed in the direction of type B (the enzyme characteristic of the leukemia clone) and significantly different from the 1:1 ratio expected. It is, therefore, likely that the neoplastic event occurred in a stem cell common to the lymphoid series as well as to the myeloid series. In contrast, evidence for B cell involvement was not detected in the patient whose ANLL progenitor cells exhibited restricted differentiative expression. These findings underscore the heterogeneity of ANLL. Clinically and morphologically similar malignancies in these two patients originated in progenitors with different patterns of stem cell differentiative expression. This difference may reflect differences in cause and pathogenesis.

The adherent cell layer in the human long-term marrow culture system provides an in vitro model for the hematopoietic microenvironment. We have devised a two-stage long-term marrow culture system to examine the effect of adherent cell layers from normal and neoplastic cultures on early hematopoietic progenitors. We found that irradiated layers from normal long-term marrow cultures supported granulopoiesis in normal allogeneic marrow cells. This effect, manifested by increased CFU-C production, was amplified when the irradiated layers were overlaid with normal allogeneic marrow initially depleted of these stem cells with cytotoxic anti-Ia (HLA-DR) antibodies. In contrast, there was no regeneration of CFU-C from normal marrow depleted of these progenitors after incubation with irradiated layers from chronic myelogenous leukemia cultures or with passaged marrow fibroblasts. Our data suggest that a functional abnormality may exist in the in vitro microenvironment in chronic myelogenous leukemia and that the two-stage long-term marrow culture system may provide a means of assessing the capacity of adherent cells to support hematopoiesis and an indirect assay for stem cells not measurable with semisolid clonal methods.

When exposed to 5-azacytidine, marrow stromal cells from active long-term marrow cultures and cell lines derived from simian virus 40-transformed stromal cells rapidly upregulated c-abl and interleukin-6 transcripts while downregulating the expression of collagen I, a major matrix protein. Similar effects occurred with interleukin-1 alpha and tumor necrosis factor alpha, although the time course was considerably prolonged.

Selected androgenic and nonandrogenic steroids enhance in vitro granulocytic and erythroid colony formation by mouse marrow cells, but do so by influencing either different target cells or cells in different states of cell cycle. Etiocholanolone, a naturally occurring nonandrogenic testosterone metabolite, permits cells not in active cycle to respond to colony-stimulating factor or erythropoietin. Fluoxymesterone, a synthetic androgen, appears to enhance colony growth by increasing the responsiveness of target cells to tropic stimuli. The majority of cells responding to this androgen are in active DNA synthesis. Direct comparison, however, of etiocholanolone-dependent erythroid or granulocytic colony-forming cells demonstrates nonidentity of the target cells. Thus colony-forming units responding to different classes of steroids are in different states of cell cycle and are physically separable. The enhancement of the in vitro response of colony-forming cells to regulating hormones by steroids such as etiocholanolane suggests a mechanism by which such agents may be therapeutically effective in certain cases of marrow failure in man.

In a preliminary manner, the data presented here characterize some features of MSC and their progenitors. The progenitors, at least in chronic myelogenous leukemia, are derived from the neoplastic pluripotent stem cell that also differentiates along lymphoid and myeloid pathways. In addition, we have demonstrated that the precursor for MSC is lacking both the Ia and CALLA determinants. Several antigenic and functional characteristics of the mature stromal cell population have also been identified. Stromal cells express CALLA, synthesize types I, III, and IV collagen, and may express factor VIII associated antigen. It is of interest that fibroblasts do not express factor VIII associated antigen, do not synthesize type IV collagen in measurable quantities, but do express CALLA [9]. Endothelial cells express factor VIII associated antigen, synthesize type IV collagen, but are not CALLA positive. Thus, MSC have some features in common with fibroblasts and others with endothelial cells. The unique characteristics of MSC are that they are transplantable and are derived from a common progenitor with other hematopoietic cells. These features clearly distinguish this cell population from fibroblasts, which are neither transplantable nor derived from the neoplastic clone in CML.

Four patients with acute nonlymphocytic leukemia who were heterozygous for the X-chromosome-linked enzyme glucose-6-phosphate dehydrogenase (G6PD) were studied to determine the numbers and types of progenitor cells in which the disease arose. Both forms of enzyme were found in normal tissues, but the malignant blast cells showed only one G6PD, indicating that the disease was clonal at the time of testing. The observations that normal erythroid cells were present in two young patients at diagnosis and relapse indicate that the clone suppressed expression of normal granulopoiesis but did not prevent normal erythroid differentiation. In contrast to this situation, in two elderly patients, the disease involved stem cells multipotent for granulocytes, red cells, and platelets. These results indicate that acute nonlymphocytic leukemia is heterogeneous. In some patients, the disease is expressed in cells with differentiation restricted to the granulocyte-monocyte pathway; in others, it involves stem cells capable of differentiating to granulocytes-monocytes, platelets, and erythrocytes. This heterogeneity may reflect differences in causation and could have prognostic and therapeutic importance.

Production of interleukin-6 (IL-6) by marrow stromal cells from human long-term marrow cultures and from stromal cells transformed with simian virus 40 was examined. As with other cultured mesenchymal cells, unstimulated stromal cells produced undetectable amounts of IL-6 mRNA when assayed by Northern blots. However, within 30 minutes after exposure of transformed marrow stromal cells to the inflammatory mediators, recombinant human interleukin-1 alpha (IL-1 alpha) or recombinant human tumor necrosis factor alpha (TNF alpha), significant increases in IL-6 expression were observed. The time course of IL-6 mRNA upregulation in transformed marrow stromal cells with IL-1 alpha and TNF alpha differed: The maximal response to TNF alpha was observed at 30 minutes whereas that to IL-1 alpha occurred at 8 hours. Although IL-6 at a concentration of 500 U/mL was inhibitory to adherent transformed marrow stromal cell proliferation, a concentration-dependent stimulation of anchorage-independent colony growth was observed when the cells were plated in semisolid medium with IL-6. The stromal cell colony-stimulating effect of IL-6 was abrogated by a neutralizing antibody to IL-6. Moreover, the heteroserum with anti-IL-6 activity and two anti-IL-6 monoclonal antibodies partially blocked autonomous and IL-1 alpha-induced colony formation, suggesting that colony formation by transformed marrow stromal cells may require IL-6. Clonal-transformed stromal cell lines were derived from the anchorage-independent stromal cell colonies. Both IL-6 mRNA and protein were constitutively produced at high levels. The addition of IL-6 to either long-term marrow culture adherent cells or transformed marrow stromal cells downregulated the expression of collagen I, a major stromal cell matrix protein. Thus, IL-6 affects proliferation of stromal cells and influences their production of extracellular matrix, suggesting that IL-6 may have indirect as well as direct influences on hematopoietic cell proliferation.

To investigate the possibility that the recently recognised syndrome, leukaemia of large granular lymphocytes, could be associated with human T-cell leukaemia/lymphoma virus type I (HTLV-I), sera from 12 patients with this type of leukaemia were tested by the use of western-blot techniques for IgG antibodies to proteins related to human T-cell leukaemia/lymphoma virus type I (HTLV-I). Sera from 6 patients, including 2 patients with rheumatoid arthritis, reacted with p19 or p24 retroviral proteins or both. In contrast, no sample from 32 patients with uncomplicated rheumatoid arthritis, 27 with Felty's syndrome, 11 with other connective tissue disorders, or 21 normal individuals reacted with HTLV-I. The results suggest that leukaemia of large granular lymphocytes may be associated with a retrovirus related to HTLV-I.

Co-culture studies have two potential values: (1) In untransfused patients with AA they may be able to detect the small population of patients who have immunologically mediated disease and who therefore may respond to immunosuppressive therapy; (2) In transfused AA patients under consideration for allogenic marrow transplantation, inhibition in the coculture assay may indicate sensitization to minor histocompatibility antigens. Preliminary data indicates that this test may be more sensitive than the chromium release assay and should be added to a battery of prospective in vitro tests designed to detect those patients at risk for marrow graft rejection.

Thirteen recipients of bone marrow transplants were given high-dose acyclovir and alpha-interferon (Cantell interferon) for the treatment of biopsy-proven cytomegaloviral pneumonia. Three patients survived. Doses of acyclovir between 500 and 1,000 mg/m2 of body surface area (peak plasma levels, 7-86 micrograms/ml) and doses of interferon between 2 X 10(4) and 40 X 10(4) units/kg per day (peak serum levels, 5-608 units/ml) were given. No consistent antiviral effect was seen despite the large doses employed. Possible marrow toxicity associated with this regimen occurred in five patients, neurologic symptoms in two, and nephrotoxicity in one. Thus, treatment with high-dose acyclovir plus alpha-interferon was moderately toxic but ineffective against cytomegaloviral pneumonia after bone marrow transplantation.

This report describes an IgG1 mouse monoclonal antibody derived after immunization of mice with washed stromal cells from human, long-term bone marrow cultures. The antigen recognized by the antibody (BMS-1) is a carbohydrate-containing prosthetic group that is common to and specific for multiple horse serum proteins. These proteins are avidly ingested by stromal cells and concentrated in endocytic vesicles. Cultured smooth muscle cells took up the horse proteins in a similar manner to marrow stromal cells while cultured marrow fibroblasts, endothelial cells, and hepatoma cells did not. These data indicate that marrow stromal cells specifically accumulate horse serum proteins which might partially explain the horse serum requirement for long-term marrow culture maintenance. The data also suggest further similarities between marrow stromal and smooth muscle cells and additional differences between marrow fibroblasts and marrow stromal cells.

Twenty patients receiving anthracycline chemotherapy were studied by the technique of radionuclide ejection fraction (EFrn). This technique is capable of detecting noninvasively small changes in left ventricular function. Two patterns of anthracycline toxicity emerged: (a) an acute toxicity observable between 24 and 72 hours after administration of greater than 60 mg/m2 of anthracycline with some recovery noted by 72-96 hours (no changes were observed within the first 4 hours after administration of the drug), and (b) a chronic dose-dependent decrease in left ventricular function when studies were performed 3 weeks after the last dose of anthracycline. After anthracyclines were stopped, four of four patients showed significant recovery in left ventricular function. We concluded that the radionuclide EFrn is a sensitive noninvasive index of left ventricular function which can be used to serially study patients receiving anthracycline therapy and can potentially be used to evaluate pharmacologic means for preventing anthracycline cardiotoxicity.