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Light density human bone marrow cells were cryopreserved, then thawed and tested for their ability to form myeloid (CFUGM), erythroid (BFUE) and mixed myeloid-erythroid colonies in vitro. Proliferation of mixed hematopoietic colonies was supported by conditioned media from a human T-lymphocyte cell line and erythropoietin. All mixed colonies contained neutrophil and erythroid precursors and approximately 25% had mononuclear phagocytes. Following cryopreservation the bone marrow retained its capacity to form mixed colonies without apparent loss. The recovery of CFUGM and BFUE was 76% and 72% respectively.

We have examined procedures for maintaining human bone marrow in a viable state for a period of 1 to 3 days. Various fractionation methods and environmental conditions were compared, and these results indicate that unfractionated, washed bone marrow cells stored at 25 C in a CO2-charged incubator gave the highest total recovery of granulopoietic stem cells capable of colony formation in vitro. After 24- and 48-hr incubation, a mean of 93 and 89% were preserved. Room temperature storage of bone marrow may have clinical applicability in autologous marrow infusion after high-dose chemoradiotherapy.

Hemopoietic cells from human fetal livers of 9-21 weeks gestation were cultured in semisolid media in vitro and in diffusion chambers implanted in mice. Myeloid colony formation in agar (CFU-C) was detected only in the presence of colony-stimulating factor. Formation of erythroid bursts in vitro was enhanced by addition of erythropoietin or erythroid-potentiating activity derived from a human T cell line (Mo). Fetal liver cells implanted in diffusion chambers gave rise to myeloid (CFU-DG) and occasionally to erythroid colonies. Administration of colony-stimulating factor derived from the T cell line to the host mice did not effect CFU-DG numbers. In contrast, a partially purified preparation containing erythroid-potentiating activity, caused a significant increase in the formation of CFU-DG. Similar stimulation was achieved by pretreatment of the diffusion chamber hosts with cyclophosphamide. None of these manipulations effected erythroid colony formation in diffusion chambers in mice.

Human multipotential hematopoietic progenitor cells can be assayed by their ability to form colonies of mixed cell lineages in vitro. These cells display la-like antigens and HLA-A,B,C antigens as evidenced by inhibition of colony formation by specific monoclonal antibodies and complement.

A liquid bone marrow culture technique was used to study erythropoiesis in polycythaemia vera. Three patients with the diagnosis of polycythaemia vera treated only by phlebotomy were compared to three healthy volunteers. Erythropoiesis in vitro was assessed by counts of nucleated red cells and 3H-thymidine incorporation into erythroid precursors. Human urinary erythropoietin was found to stimulate erythropoiesis in cultures from the polycythaemia patients as well as normals. The erythropoietin effect was blocked by a rabbit anti-erythropoietin antibody. These results suggest that some erythroid precursor cells maintain an intact erythropoietin response mechanism in polycythaemia vera. They also support clinical studies of this disorder indicating that some regulation by erythropoietin may occur in vivo.

Proto-oncogenes are phylogenetically ancient genes which are involved in the normal controls of cell proliferation. Under some circumstances, these normal genes may be altered and generate qualitatively or quantitatively abnormal gene products. Under these circumstances the proto-oncogenes may be involved in the pathogenesis of malignant diseases.

Mice treated with high-dose busulfan develop a 'latent' form of bone marrow failure characterized by near-normal peripheral blood counts and marrow cellularity, but marked reductions in marrow pluripotent stem cells (CFUs) and myeloid progenitor cells (CFUc). Spleen cell suspensions from control and 'latent' mice were placed in liquid culture in the presence of colony-stimulating activity. Cells were harvested at intervals up to 14 days and sub-cultured in agar to assay for CFUc. Baseline splenic CFUc did not differ significantly between control and 'latent' mice. Splenic CFUc from control mice increased 50-fold and reached a peak at day 10 in liquid culture. In contrast, splenic CFUc from 'latent' mice increased only 7-fold and reached a peak at day 3. Our results indicate that although splenic CFUc are present in normal numbers in 'latent' mice, their proliferative capacity is markedly reduced, either as the result of defective CFUc self-renewal or defective feed-in from CFUs or both.

A number of techniques are available for insertion of new genetic information into mammalian cells. some of these have been used successfully for genetic modification of germ line cells and somatic cells of living animals. Some of these techniques may be applicable to treatment of some of the genetic diseases of man, once problems related to the control of expression of introduced genes are solved.

Thyroid hormones have important effects on erythropoiesis in man and animals. We performed in vitro culture studies with murine and human bone marrow in order to define the interaction of these hormones with erythroid and granulocyte-monocyte progenitor cells. The methylcellulose clonogenic assay was used with the appropriate addition of erythropoietin or colony-stimulating activity. L-thyroxine, D-thyroxine and L-triiodothyronine potentiated erythropoientin-stimulated erythroid colony formation in concentrations of 50-100 ng/ml. These hormones had no effect on granulocyte-monocyte colony formation at concentrations up to 500 ng/ml. Testing of various thyroid analogues showed no clear correlation between potentiation of erythropoiesis and known calorigenic potency. Reverse triiodothyronine also had potentiating activity in this system. The active thyroid hormones stimulated erythroid colony formation at several concentrations of erythropoietin but could not substitute for erythropoietin. These data suggest that thyroid hormones have a direct effect on erythroid precursor proliferative capacity, a finding which may have relevance to the mechanism of erythropoietic dysfunction in human thyroid disease.

Murine int-2 is one of the genetic loci implicated in the induction of mouse breast cancers by murine mammary tumor virus. An homologous gene has recently been identified in the human genome (Casey et al., 1986). The human int-2 locus was found to be amplified 7- to 25-fold in 4 of 46 infiltrating ductal breast cancers and 30- to 60-fold in 2 of 8 squamous carcinomas of the head and neck region, but not in other cancers. All of the involved tumors had metastasized to regional lymphatics at the time of analysis and five exhibited clinically aggressive behavior.

We derived a permanent human T lymphocyte cell line that elaborates a potent colony-stimulating activity (CSA). The line was established with spleen cells from a patient with a T lymphocyte variant of hairy-cell leukemia. These cells form rosettes with sheep erythrocytes, show a proliferative response to phytohemagglutinin, and are lysed by antithymocyte globulin. They do not synthesize immunoglobulin, nor do they contain Epstein-Barr virus. CSA is regularly detected in the supernatant medium after 3 days culture. In the presence of PHA there is augmented elaboration of CSA; maximal activity is reached by 2 days and is 20% greater than that produced by a feeder layer of 1 X 10(6) peripheral blood leukocytes. One microliter of the supernatant material stimulated colony formation from the light-density nonadherent fraction of human bone marrow; there was maximal activity between 10 and 50 microliter/ml. Conditioned medium from these cells has little effect in stimulating CFU-C from murine bone marrow. The availability of a human T lymphocyte line producing CSA will provide a source for large quantities of the lymphocyte-derived hormone and permit a definition of factors modulating the interaction of T lymphocytes with granulocyte and monocyte stem cells.

Human myeloid colony formation in diffusion chambers in mice (CFU-DG) was enhanced following administration of a human T-cell-line-derived conditioned medium (Mo). The Mo cell line also elaborates activities stimulating human myeloid colony formation in vitro in agar (CSF) and potentiating erythroid colony formation in vitro in methylcellulose (EPA). Depletion of CSF from Mo conditioned medium by heat inactivation or gel exclusion chromatography did not affect CFU-DG formation. EPA and CFU-DG stimulating activities are heat stable and have approximately the same molecular weight. Culture of human bone marrow cells in diffusion chambers in mice for 4 days under the influence of Mo conditioned medium resulted in significant increment of BFU-E and CFU-DG as judged by subculture of diffusion chamber contents. No effect on CFU-C could be detected.

Cellular oncogenes have been implicated in the induction of malignant transformation in some model systems in vitro and may be related to malignancies in vivo in some vertebrate species. This article describes a study of the expression of 15 cellular oncogenes in fresh human tumors from 54 patients, representing 20 different tumor types. More than one cellular oncogene was transcriptionally active in all of the tumors examined. In 14 patients it was possible to study normal and malignant tissue from the same organ. In many of these patients, the transcriptional activity of certain oncogenes was greater in the malignant than the normal tissue. The cellular fes (feline sarcoma) oncogene, not previously known to be transcribed in mammalian tissue, was found to be active in lung and hematopoietic malignancies.

Haemopoietic cells with pluripotent capabilities, were grown in soft-gel cultures from the peripheral blood of a glucose-6-phosphate dehydrogenase (G6PD) electrophoretic A,B heterozygous female with chronic myelocytic and granulocytic progeny. Only type B isoenzyme of G6PD was found in mixed colonies, in mature neutrophils, erythrocytes, and committed granulocyte and isoenzymes of G6PD. This study provides direct evidence that a pluripotent haemopoietic stem cell is involved in the malignant clone in CML.

The human T-cell leukemia viruses HTLV-I and HTLV-II are unique among the transforming retroviruses of vertebrates in their ability to transform human T cells in vitro and in their close association with human malignancies (T-cell lymphomas and leukemia). Their genomes are relatively simple, containing the genes gag, pol, env, and a 3' region termed "X." This 3' region may be responsible for the transforming potential of the viruses. The existence of proteins encoded by the 3' region has been postulated on the basis of multiple open reading frames. In the present study this region is shown to contain a gene encoding a protein of 40 kilodaltons in HTLV-I and 37 kilodaltons in HTLV-II. It is proposed that these proteins be called, respectively, p40xI and p37xII.

We have used cryopreserved autologous hematopoietic cells to repopulate bone marrow of patients receiving intensive chemotherapy and radiation. We employed the technique of density step centrifugation to concentrate stem cells from whole bone marrow. The concentrate of cells was frozen in vials using dimethyl sulphoxide (DMSO) as a cryo-protective agent and was stored in the vapor phase of liquid nitrogen. Test vials were thawed and assayed for myeloid stem cells (CFU-C) to determine the potential of the larger marrow aliquot. These test values were compared to the recovery of cells and CFU-C when the marrow was used for autotransplantation. The return of hematopoietic function after autotransplantation was evaluated and correlated with the dosage of cells and CFU-C infused.

In order to investigate the effect of human growth hormone on T lymphocytes, we utilized a clonogenic assay for mitogen-responsive human peripheral blood T lymphocytes. Lymphocytes were purified by density gradient centrifugation and incubated in the presence of phytohemagglutinin using a two-layer agar technique for CFU- T-lymphocyte culture. Nanogram concentrations of human growth hormone, ovine prolactin, human chorionic somatomammotropin, or growth hormone fragment were added to cell cultures. Growth hormone potentiated normal T-cell colony formation in a species-specific manner. Cells from a homogeneous T-lymphoblast line derived from a patient with a T-cell variant of hairy cell leukemia also showed augmentation of colony growth in the presence of human growth hormone. These studies provide evidence for a direct effect of growth hormone on normal and some neoplastic human T cells.

A continuously growing human myeloid leukemia cell line (K562) produced a potent high-molecular-weight inhibitor of hematopoietic cell proliferation. It was most active against myeloid stem cells (CFU-C) and proliferating T lymphocytes; it was less active against erythroid precursors (CFU-E) and did not inhibit fibroblasts or established lines of epithelioid cells or B lymphocytes. Inhibition of CFU-C was by direct interaction rather than by modulation of production of colony-stimulating activity and probably occurred at restricted points in the cell cycle. Inhibition could, within limits, be reversed by washing the target cells. Production of inhibitors of hematopoiesis is not a general property of established cell lines, and only two have thus far been identified in screening of 30 such lines.

A patient with episodic panleukopenia involving neutrophilic granulocytes, monocytes and lymphocytes had recurrent bacterial infections and a circulating cytotoxin that injured primitive myeloid cells as well as differentiated granulocytes and mononuclear cells. The cytotoxin was an immunoglobulin with activity against mouse, as well as human, myeloid stem cells. Clinical remission during cyclophosphamide therapy was associated with disappearance of the circulating leukocytotoxin. Serum from a period of active disease was cytotoxic for the patient's own lymphocytes obtained during clinical remission. Absorption of cytotoxic serum with granulocytes removed activity against both granulocytes and lymphocytes. These studies suggest an autoimmune disease in man characterized by episodic granulocytopenia, monocytopenia and lymphocytopenia due to humoral antibody against non-HLA antigens shared by several hematopoietic cell lines.

Mutations in codons 12 or 13 of the first exon of the N-RAS gene have been reported in myelodysplastic syndromes (MDS) in frequencies that vary between 9% and 40% depending on the techniques used in analysis. Gene amplification and direct sequencing provides the only unambiguous method of detecting those mutations that induce amino acid alterations. Using this technique, we analyzed 21 MDS patients for mutations in exon-1 of N-RAS. Codon 12 mutations substituting aspartic acid (GAT) for glycine (GGT) were found in four cases, and a codon 13 mutation substituting alanine (GCT) for glycine (GGT) was detected in one patient. We conclude that N-RAS exon-1 mutations in one patient. We conclude that N-RAS exon-1 mutations producing amino acid changes occur in about 20% to 25% of MDS cases.

The incidence of c-ERBB-2 amplification in breast cancers and its usefulness as a predictor of tumor recurrence after treatment have been subjects of controversy (Ali et al., 1988, Slamon et al., 1987). We re-examined this subject by analysing 157 primary and 14 metastatic breast cancers with c-ERBB-2 and 18 other molecular probes as controls. Five proto-oncogenes were found to be occasionally amplified in primary breast cancers: c-ERBB-2 (11%), c-MYB (3%), c-RAS-Ki (3%), INT-2 (4%) and c-MYC (6%). No statistically significant correlation between amplification of c-ERBB-2 and recurrence of tumors was observed. The only significant correlation observed was between early recurrence of advanced (stage III) tumors and amplification of one or another of the above five proto-oncogenes. We conclude that breast cancer utilize multiple genetic mechanisms in their progression and metastasis, and that analysis of c-ERBB-2 alone is not a useful guide.

Mononuclear phagocytes are a widely distributed system of cells involved in host defense functions, immunologic reactions, disposal of unwanted organic and inorganic materials, and synthesis of biologically active compounds such as complement components and prostaglandins. The system includes early precursor cells in the bone marrow, blood monocytes, alveolar and peritoneal macrophages, sinusoidal lining cells in the spleen, and Kupffer cells in the liver. We have recently come to understand the biology of these interesting cells and the diseases they produce.

Murine spleen cells were cultured in vitro to study the induction of committed granulopoietic stem cell (CFU-C) proliferation and maturation. Marbrook-type diffusion cultures were established with and without the addition of colony-stimulating activity (CSA) and harvested at intervals up to 14 days for viable and differential cell counts, [3H]TdR autoradiography, and quantitation of CFU-C by the agar plate method. Without CSA there was poor cell viability and little proliferative capacity. In CSA-stimulated cultures there was a prominent rise in viable cell counts and [3H]TdR labeling indices rose from a mean of 2% at 0 time to 47% after 5 days in vitro. CFU-C increased by 70-fold in these cultures. Peak numbers of CFU-C, immature cells, and [3H]TdR-labeled cells occurred at about 7 days. Thereafter, mature granulocytes and macrophages predominated in culture. Because the liquid spleen cell culture system begins in a resting state and undergoes a wave of proliferative activity in response to CSA, it can provide a useful model system for studying phenomena associated with stem cell activation and differentiation in vitro.

A black man with a prolonged partial thromboplastin time has a severe deficiency of plasma thromboplastin antecedent (PTA) (factor XI) measured both in clotting assays and immunoassays, suggesting a diagnosis of homozygous PTA deficiency. His offspring seemed to be heterozygous carriers of PTA deficiency. Additionally, the proband and two of his children had decreased Hageman factor (factor XII) levels consistent with those of heterozygous carriers of Hageman trait. To our knowledge, this is the first case known of PTA deficiency in a black person. Its pattern of inheritance was independent of that of factor VII deficiency.

The mononuclear phagocytic system is a continuum of cells beginning with the bone marrow monoblast and promonocyte, through the monocyte to the larger tissue macrophages and multinucleate giant cells. This system of cells is widely distributed throughout the body in the blood and bone marrow; the pleural, peritoneal, and alveolar spaces; the lymph nodes, spleen, liver, and other parenchymal organs. The activity and composition of the cell varies with the level of maturation, changes in cellular environment, and with various cellular activities. The monocyte-macrophage group of cells plays an active role in defense reactions against certain microorganisms, and in the removal of dying cells and cell debris. They are an integral part of both the inductive phase of the immune response, and of cell-mediated immune reactions. In addition, they probably play a role in the defence against spontaneously arising tumours, in the control of granulopoiesis, and possibly in erythropoiesis.

Amplification of c-erbB-2 was at least three times more frequent in breast cancer than in most other types of carcinoma, and was not found in sarcomas or hematological malignancies. Amplification of c-erbB-2 was found in 15 of 86 primary breast cancers and in 3 of 12 secondary breast cancers. Amplification was more common in breast tumors of advanced stage, and in tumors which had metastasized to regional lymph nodes sites. Gene amplification was observed in 21% (4 of 19) of primary tumors which recurred within 3 years of mastectomy and in 6% (2 of 32) of nonrecurrent tumors.

Pseudomonas endotoxin was administered to three normal subjects in order to assess possible effects on stem cell circulation. Increased numbers of granulopoietic stem cells (CFU-C) transiently appeared in the peripheral blood, reaching a maximum after the time of maximal leukopenia. There was a mean 4-fold increase in CFU-C per ml of blood following endotoxin. A high proportion of these CFU-C did not require an added source of colony-stimulating activity for growth in agar.

28 adult patients with acute myeloid leukaemia (A.M.L.) received T.A.D., a high-dose sequential chemotherapeutic remission-induction regimen consisting of 7-day courses of cytosine arabinoside, 6-thioguanine, and daunorubicin. Overall response-rate was 82%. 22 patients (79%) achieved complete remission, and 1 had a partial remission. Median remission duration was 280 days and median survival 375 days. 10 patients remain in remission. These induction results are superior to those reported in most studies and indicate that disease remission can be achieved in a high proportion of patients with A.M.L. treated with an intensive multi-agent chemotherapeutic regimen, provided support facilities are adequate.

Expression of proteins encoded by the ras proto-oncogenes was examined in extracts from normal rat organs using anti-ras p21 antibodies generated against synthetic peptides. The highest level of ras p21 was found in brain (cerebrum) and was predominantly of c-Ha-ras origin. Levels of brain ras p21 did not vary from the newborn period of 3 months of age. Moderate levels of ras p21s were detected in lung, spleen and thymus. In contrast to the p21 in brain, these levels varied with the age of the rats and were encoded by other members of ras proto-oncogene family (Ki-ras or N-ras). This organ specific expression of different ras genes might be related to developmental control of gene expressions.

Proto-oncogenes are normal genes which affect normal cell growth and proliferation, but which have the potential to contribute to cancer development if their expression is altered. A variety of events may activate proto-oncogenes and convert them from benign genes to cancer genes. These events include mutations of nucleic acid sequences, chromosomal rearrangements, and amplification of the number of genes. The observations that alterations in specific gene sequences frequently accompany neoplasia have provided the first insights into the pathogenesis of human cancer at the molecular level. These insights offer new opportunities for developing cancer diagnostics and therapeutics.

Recent studies using cytotoxic and cell separation techniques have identified differentiation-related antigens on human hematopoietic cells. These results, combined with bone marrow transplantation studies, have yielded a picture of antigenic modulation from the pluripotent hematopoietic stem cell through the differentiated end cells.

A modification of the microcytotoxicity assay of Terasaki & McClelland was used to screen for serum inhibitors of myeloid progenitor cells (CFU-C). Sera from 104 patients with neutropenia or bone marrow failure and from 104 controls, including 30 normal subjects and 74 patients with various disorders or multiple transfusions, were studied. Inhibitors of CFU-C were found in 19 of the 104 patients with neutropenia or marrow failure: three with acquited neutropenia and 16 with aplastic anaemia. In 17 of the 19 patients the inhibitor was either complement dependent or chavincingly associated with clinical remission of disease in one patient. In another patient, the inhibitor was an IgG antibody directed against non-HL1 antigens present on CFU-C of all 50 normal target marrows tested; however, it was not clearly autoreactive. Inhibitors were also found in one of nine patients with lupus erythematosus, one with breast carcinoma, one patient with renal transplant rejection, and six of 46 multiply transfused patients without marrow failure. Our findings indicate that most serum inhibitors are associated with multiple transfusion: convincing evidence of pathogenetically important autoreactive antibody was present in only one of 19 patients with inhibitors.

Hematopoietic cell differentiation is influenced by organ-dependent microenvironmental factors as well as humoral regulators. A technique is described for examining certain aspects of the hemopoietic inductive microenvironment in vitro. Suspension and agar cultures of mouse bone marrow were used to study the effects of organ stromal factors on cellular proliferation and differentiation. Bone, spleen, and thymus fragments from irradiated mice were placed in direct contact with or separated by a Nuclepore membrane from syngeneic marrow cells growing in suspension cultures. Normal adult mouse bone and spleen influenced granulocytic differentiation as well as cell proliferation. In this system, bone marrow and organ fragments from W/Wv and SlSld mice behaved like those of their non-anemic littermates. The most prominent difference between W/Wv and Sl/Sla mice and their normal counterparts was observed in the inductionof CFU-C from splenic precursors un-er the influence of CSA. In both types of anemic mice, in vitro generation of CFU-C from spleen was abnormal in young animals but was corrected by four months of age.

A patient with poorly differentiated monocytic malignancy had extensive lytic lesions of bone and hypercalcemia. The patient's bone margins showed little evidence of osteoclast activity. Serum parathormone concentrations were slightly increased relative to the degree of hypercalcemia, consistent with impaired renal function. Serum concentrations of the relevant prostaglandins were normal. Supernatant medium from cultured tumor cells caused prominent bone resorption in vitro. The studies on this patient provide evidence that malignant cells of the monocyte-macrophage line are capable of direct bone resorption.

Pretreatment of mice with glucan, a potent macrophage activator, resulted in enhanced myeloid cluster and colony formation by bone marrow cells in diffusion chambers implanted into the peritoneal cavity. Simultaneously, erythroid colony formation was also augmented. In some experiments the plasma clots formed inside the chambers were dissolved, and the number of hematopoietic cells was determined. An increased yield of early proliferative cells, granulocytes, and macrophages was found in glucan-treated hosts. Concomitantly, higher leukocyte counts were noted in the peripheral blood of treated animals. These results suggest that glucan has a strong stimulatory effect on hematopoiesis. This stimulation is probably mediated by humoral factors of host animal origin rather than by direct interaction with proliferating hematopoietic precursors enclosed within the chambers.

Discrete aggregates of hematopoietic cells were observed in the bone marrows of patients with aplastic anemia or acute leukemia 14 days after marrow transplantation. The great majority of such colonies were of a single cell type, and less than 3% contained two or more cell types. Erythroid, myeloid, and undifferentiated hematopoietic colonies were approximately equal in frequency.