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The term histiocyte refers to cells of either the macrophage or Langerhans cell lineages. The histiocytic disorders are characterized by the proliferation of cells of these lineages. With recent advances in knowledge of the developmental biology of histiocytic cells, it is now possible to formulate a reasonable catalogue of histiocytic diseases based on ultra-structural and phenotypic markers of cellular origins and molecular or chromosomal markers of malignancy. The catalogue includes the following groups of diseases. Nonmalignant reactive macrophage disorders include (1) macrophage storage diseases, (2) several benign proliferative macrophage disorders that predominantly involve skin and bone, and (3) several hemophagocytic syndromes that vary from indolent and benign to fulminant and fatal. In some of the latter disorders, viruses have been identified as the inciting stimulus. The malignant macrophage disorders include (1) acute monocytic leukemia and (2) chronic myelomonocytic leukemia. A rare disorder that gave rise to a permanent cell line with an anomaly of chromosomal segment 5q35 may also be an example of a histiocytic malignancy. The existence of a separate category of true histiocytic lymphoma of macrophage type is uncertain. Reactive Langerhans cell disorders include (1) congenital self-healing histiocytosis, (2) the many variants of eosinophilic granuloma, and (3) a related disorder designated as relapsing Langerhans cell histiocytosis that is characterized by a relapsing course and infiltration of bone and soft tissues by Langerhans cells. Presumptively neoplastic diseases of Langerhans and dendritic cells include (1) progressive Langerhans cell histiocytosis, a disease with prominent involvement of blood and BM as well as skin and viscera; (2) Langerhans cell lymphoma, and (3) dendritic cell lymphoma. However, clonality as a marker of malignancy has not been proven in these disorders.

Summary A patient with typical Ph1-positive CML was studied during sequential phases: (1) initial chronic phase, (2) first myeloid blast crisis, (3) second chronic phase, and (4) accelerated disease leading to a second blast crisis. A point mutation in codon 239 of the p53 gene and a novel chromosome 17 alteration appeared concomitantly with the first blast crisis and then disappeared with re-establishment of a second chronic phase. They did not reappear with the second acute phase, indicating that the clone responsible for the original blast crisis had been suppressed and supplanted by another clone of malignant cells. This observation suggests that in at least some CML patients drug therapy can suppress or eliminate an aggressive malignant cell clone, but that the underlying molecular defect in haemopoietic cells (in this case the c-ABL translocation) persists and other aggressive clones with different molecular lesions eventually arise. Our observations and inferences are consistent with the hypothesis advanced by Fialkow et al (1991) to explain clonal remissions in acute non-lymphocytic leukaemia.

A patient with typical Philadelphia chromosome (Ph1)-positive chronic myelocytic leukemia (CML) was studied during sequential phases of disease: (1) initial chronic phase; (2) myeloid blast crisis; (3) second chronic phase; and (4) accelerated disease. A point mutation in the coding sequence of the p53 gene first appeared concomitantly with the blast crisis and then disappeared with the re-establishment of a second chronic phase. The chromosomal concomitant of the molecular alteration was a deletion of 17p. These observations suggest that abnormalities of the p53 anti-oncogene are temporally related to the clinical progression of some cases of CML and are probably responsible for the development of blast crisis in these cases.

(1991). Oncogenes and Anti-oncogenes in the Evolution of Human Leukemia/Lymphoma. Leukemia & Lymphoma: Vol. 4, No. 3, pp. 153-158.

DNA from 161 patients with various forms of hematologic malignancies were investigated for mutations in exons 1 and 2 of the N-RAS, K-RAS and Ha-RAS gene by direct sequencing of DNA amplified in vitro by the polymerase chain reaction. Mutations involving either codons 11, 12, or 13 of the N-RAS gene were identified in 18 of the 161 patients. The relative frequencies of N-RAS gene mutations in these hematologic disorders was as follows: acute myelogenous leukemia (AML), 15%; acute lymphoblastic leukemia (ALL), 14%; myelodysplastic syndromes, 24%; and myeloid and lymphoid blast crisis of chronic myelogenous leukemia (CML), 3%. No correlation was observed between the presence of mutations and cytologic features or immunophenotype of these malignancies. Mutations involving codons 12 or 13 were equally prevalent, with a glycine to aspartic acid substitution being the most frequently encountered change. A single T-ALL case had a codon 11 mutation resulting in substitution of alanine with threonine. We failed to find mutations in exons 1 and 2 of the K-RAS or Ha-RAS genes in any case except a single AML with a mutation in codon 61 of the K-RAS gene. Also, no mutations were identified in chronic phase of CML, chronic lymphocytic leukemia. Ph1 positive ALL, non-Hodgkin's lymphoma, Hodgkin's disease, or multiple myeloma. These results indicate that RAS mutations, especially those involving exon 1 of the N-RAS gene, are frequent only in a subset of hematologic malignancies.

cAMP-dependent protein kinase (PKA) and phospholipid-dependent protein kinase (PKC) play a role in nerve growth factor (NGF)-mediated differentiation. In PC12 cells, NGF causes neurite outgrowth and increases the number of voltage-gated Na+ channels. Neurite outgrowth involves in part activation of PKC. How NGF regulates Na+ channel number is unknown. Using patch-clamp techniques, we find that agents activating PKC, including phorbol esters and a ras oncogene product (p21) that induces neurites, caused little increase in channel number. In contrast, agents increasing intracellular cAMP were as effective as NGF. A specific protein inhibitor of the PKA catalytic subunit blocked increases by NGF or cAMP. Thus, NGF increases Na+ channel number in PC12 cells in part by activating PKA but apparently not PKC.

At least 13 of 34 patients with acute myeloid leukemia (AML) of varying FAB types were heterozygous for a BamHI restriction fragment length polymorphism (RFLP) of the Ha-ras gene on chromosome 11. In 4 of these 13 patients, one allele of the Ha-ras gene was deleted. Two of these cases had an informative heterozygosity for an RFLP on the long arm of chromosome 11. Analysis of these cases indicated that loss of genes from chromosome 11 was restricted to the short arm. In three cases with loss of one Ha-ras gene, the remaining gene had no mutations in critical areas of exons 1 and 2. With the exception of one AML case with amplification of MYC, no gross structural abnormalities in 12 other oncogenes were detected.

Alterations of the p53 anti-oncogene have recently been found to occur frequently in the blast crisis of chronic myelocytic leukaemia. The p53 gene may be altered by gross structural alterations or by point mutations in the coding sequence. We now report a novel mechanism of gene inactivation in a blast crisis cell line where a mutation in a splice donor site at the 5' end of the fifth intron of the gene interrupts RNA processing and gene expression.

Genes involved in the pathogenesis of cancer are thought to act by two general mechanisms. The first involves the structural alteration of a normal gene (a proto-oncogene) to generate a novel gene (an oncogene) whose protein product acts on the host cell to induce characteristics of malignancy. This protein product is usually involved in cellular proliferation, differentiation, or survival. The second mechanism involves the loss or inactivation of genes whose proteins suppress cancer. Genes of this class are known as tumor-suppressor genes or anti-oncogenes. Alterations in members of specific gene families are consistently associated with some types of leukemia. Table 1 lists . . .

Chronic myelocytic or Ph1-positive acute lymphoblastic leukemias have been analyzed for alterations in a variety of proto-oncogenes and anti-oncogenes implicated in the progression of chronic myeloid leukemia (CML) from its chronic phase to blast crisis. The most frequent genetic change found in disease evolution is an alteration of the p53 gene involving a point mutation, a rearrangement or a deletion. These gene changes are common in myeloid and undifferentiated variants of blast crisis but are usually undetectable in lymphoid leukemic transformants. Other molecular changes also occur in the clonal evolution of CML. The retinoblastoma-susceptibility (Rb) gene is an anti-oncogene. Structural abnormalities of Rb are frequent in all types of human acute leukemia, but are particularly common in Ph1-positive leukemia of lymphoid phenotype including both Ph1-positive ALL and lymphoid blast crisis of CML. Changes in Rb occur early in the transition to blast crisis with loss of Rb protein being the common factor. Mutations in the N-RAS gene also occur, but are rare in typical blast crisis. They are sometimes seen in Ph1-negative myeloid blast crisis. Since changes in the p53 gene are generally associated with progression of disease of a myeloid phenotype and changes in the Rb gene occur more often with a lymphoid phenotype, a particular molecular alteration may influence the character of disease evolution in CML.

The discovery of oncogenes introduced an era in which it is possible to identify genetic elements involved in the initiation and progression of malignant diseases (9, 10, 36, 61, 75, 126, 167). In some cases, these genetic elements can be used as molecular markers of human cancers.

Radiolabeled or biologically functional DNA molecules were introduced into cells by electroporation in a variety of forms: double stranded circles, linearized double stranded fragments and single stranded circular molecules. Molecules rapidly entered cells after exposure to a high field-strength electric pulse and then redistributed between the cytoplasm and the nucleus. Maximal intranuclear levels approximated 10(4) molecules per cell. Introduced DNA persisted in a biologically active form with a half-life of 15-24 h. There was no evidence for biologically significant alteration of two double stranded gene sequences. Single stranded DNA molecules also retained biological activity.

The monocyte-macrophage cell line is an important member of the host defense system. This report describes a series of assays that can be applied routinely in the evaluation of human monocyte function and gives information as to the activity of normal monocytes in these systems. Tests were chosen to assess various aspects of monocyte function that give some insight into the host defense status and the degree of “activation” of the monocyte. The assays outlined in this report are relatively simple to perform and include measurements of human monocyte chemotaxis, phagocytosis, fungal and bacterial killing, adhesion, and spreading.

Twenty-seven primary non-small cell (NSC) lung cancers were analyzed for alterations of protooncogenes by DNA hybridization techniques. Abnormalities were found in 56% of tumors including ten of 16 adenocarcinomas, three of nine squamous cell cancers and two of two larger cell cancers. Five protooncogenes were found to be commonly altered in tumors at frequencies between 12% and 60%. These were c-myc, c-myb, c-ras-Ha, c-erbB-1 and c-erb-B-2. Alterations in c-erbB-1 and c-erbB-2 correlated with histologic type of tumor and were more common in advanced cancers. Allelic deletions of c-ras-Ha or c-myb were frequently observed in primary tumors which recurred or progressed after surgery (five of six). Analysis of protooncogenes may provide insights into the pathogenesis of lung cancer and may aid in predicting clinical behavior.

There is considerable evidence supporting a role for colony-stimulating factor (CSF) as a humoral regulator of leukopoiesis. Data on CSF levels in the serum and urine of patients with leukemia and on the in vitro responsiveness of leukemic cells to CSF have suggested a basis for considering leukemia as a primary disorder of leukopoietic regulation. We examined the question of leukemic cell production of CSF. Conditioned medium from cultured leukemic cells was tested for colony-stimulating activity against normal human bone marrow using a two-layer agar colony assay technique. The cells from patients with acute myelogenous leukemia and a patient with chronic myelogenous leukemia in blast crisis did not elaborate CSF nor did acute lymphocytic leukemia cells. CSF production was documented with cells obtained from patients with chronic myelogenous leukemia in the chronic phase and two patients with acute myelomonocytic leukemia. In acute leukemia the cellular production of CSF correlated closely with morphologic and functional maturation along the monocyte-macrophage line. Evidence was obtained that the adherent cells within the leukemic population were primarily responsible for CSF production. We interpret these data to indicate that neoplastic hematopoietic cells may produce CSF in relation to their capacity for mononuclear leukocyte differentiation.

A system for insertion of new genetic information into mouse hematopoietic cells is described. Two selectable genes were examined: herpesvirus thymidine kinase and a mutant mouse dihydrofolate reductase. The DHFR system appears to be superior in terms of the frequency and stability of gene insertion and expression in hematopoietic tissues. About 70% of mice had indirect (karyotypic) evidence of gene insertion; of these, about 60% (three of five) had stable expression of the inserted mutant DHFR. In contrast, only 13% of mice demonstrated stable karyotypic transformation by HSVtk, and of those with stable transformation five of seven showed persistent viral gene sequences in hematopoietic tissues.

SummarySix healthy adult volunteers were injected intravenously with Pseudomonas endotoxin (Piromen) to determine effects on serum colony-stimulating activity (CSA). Serum was assayed with a double-layer agar cloning technique using normal human bone marrow as the target cell preparation. A marked increase in serum CSA was noted after endotoxin administration lasting up to 180 min. The colonies formed in response to postendo-toxin serum were smaller than those seen with leukocyte feeder layers but were otherwise morphologically normal. These studies suggest that granulopoietic humoral factors may be modulated in man by endotoxin administration.The authors thank Lisken Byers, Judy Price and Ann Sullivan for excellent technical assistance.

Normal human bone marrow contains cells capable of forming myeloid colonies (CFU-DG) in fibrin clots in diffusion chambers placed in the peritoneal cavity of cyclophosphamide-treated mice. Evidence has accumulated indicating that these colony-forming cells represent an earlier stem cell than the granulocyte-monocyte precursor cell (CFU-C) assayed in soft agar. We provide data showing that these stem cells express the “Ia-like” or DR antigen. In the presence of rabbit Ia antiserum at a titer of 1:300, all CFU-DG were inhibited. Cytotoxicity was complement-dependent. Data are also presented that suggest that the megakaryocyte stem cell also expresses the Ia antigen.

Several malignant hematopoietic cells from mice, previously shown to require sulfhydryl compounds and serum for proliferation in vitro, are shown to grow well in medium lacking both of these components if methylthio compounds are provided at appropriate concentrations. The methylthio groups can be provided as the mixed disulfides of methyl mercaptan and various thiols. The optimal concentration of the various disulfides is between 1×10−5 and 1×10−4 M. The cells can be maintained indefinitely in serum-free medium containing cysteine-methyl disulfide and one of several purified proteins. The evidence is consistent with the theory that these cells require an exogenous source of alkylthio groups for division, that serum is a source of these groups, and that sulfhydryl compounds act as carriers to transport the groups into the cells. A new diffusion analysis is described for quantitative measurement of disulfides of volatile mercaptans.

Human peripheral blood contains two types of stem cells that differentiate along the granulocytic pathway. They are separable by their ability to form colonies in agar in vitro (CFU-C) and in plasma clots in diffusion chambers in vivo (CFU-DG). Kinetic studies suggest that CFU-DG represents an intermediate between the still hypothetical human pluripotent stem cell and CFU-C.

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.

THE surface antigenic structure of hematopoietic cells changes during the course of differentiation from proliferating precursors to nondividing, functionally mature cells. In a given cell lineage, some antigens are expressed and others are lost during the maturation process. Knowledge of the antigenic structure of hematopoietic progenitor cells and their mature progeny can help us to understand the process of cell differentiation and the function of specific surface antigens. This knowledge can aid in the identification of cells and cell lineages, in the classification of hematologic neoplasia, and potentially in the diagnosis and treatment of hematologic diseases. We shall summarize current . . .

The development since 1966 of a technology for growing stem cells in vitro has provided new insights into the controls of blood cell production. Hematopoietic hormones have been purified and important cellular interactions in hematopoiesis have been defined.

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.

Acute lymphoblastic leukemia (ALL) is a heterogeneous disease as defined by clinical characteristics and immunologic techniques. The standard cell surface markers are sheep erythrocyte receptors for T lymphocytes and surface membrane immunoglobulin for B lymphocytes. Utilizing these markers, three subtypes of ALL designated T-ALL, B-ALL and non-B, non-T or null ALL have been defined. We have studied 70 patients with ALL utilizing these standard cell surface markers. In addition, we have further subclassified these patients by testing each cell for an ALL-associated antigen, the la-like antigen, and thymocyte antigen(s) all defined by well-characterized antisera. We can define 12 subgroups of ALL by their surface antigenic characteristics. These subgroups may have relevance to the clinical expression of disease and may define identifiable stages of normal lymphocyte development.

Various investigators have proposed the existence of a “chalone” or product of mature granulocytes which inhibits the replication of granulocytic progenitors in a tissue-specific, species-nonspecific regulatory system. We tested this system utilizing purified populations of human leukocytes and a variety of proliferating target cell populations. Granulocyte-conditioned medium potently inhibited 3Hthymidine incorporation in normal mouse bone marrow, in mouse erythroid precursors from spleens of phenylhydrazinetreated animals, and in human leukemic myeloblasts. This effect occurred without demonstrable cytotoxicity. Human marrow was only slightly inhibited and mitogen-stimulated proliferating lymphocytes were unaffected. Granulocyte-conditioned medium was also inhibitory in colony-forming assays for erythroid precursors but not for granulocyte precursors. It also reduced the mitotic index of proliferating erythroid progenitors in mouse spleen. Inhibitory activity was separable into at least two components on the basis of stability during heating and ultrafiltration, and in susceptibility to digestion by proteolytic enzymes. On the basis of these observations, we concluded that human granulocytes contain potent inhibitors of growth and 3H-thymidine uptake of some proliferating hematopoietic cells, but these are not true granulocyte “chalones.” I N 1960, Bullough and Lawrence provided experimental evidence for the control of epidermal cell mitosis by products of mature cells in culture in vitro.’ They evolved the concept of a tissue-specific product of differentiated cells that served as a mitotic inhibitor of early cells of the same line. They termed the inhibitor a “chalone” (from the Greek, “to loosen” or “make slack”). Since then, a number of experimenters have suggested the existence of chalones specific for various tissues.2 More recently, various investigators have emphasized theoretical reasons why granulopoiesis should be under negative feedback control and have adduced evidence for the existence of granulocyte chalones.38 However, the diversity of model systems, methodologic limitations, and frequently inadequate experimental design have made interpretations difficult. RytOmaa and Kiviniemi,7’8 Paukovitz,5’6 and Bateman3 have presented evidence suggesting that rat and calf serum solutions “conditioned” by granulocyte-rich cell suspensions and spleen homogenates inhibit DNA synthesis in developing myeloid precursors in short-term cultures of rat bone marrow cells. This “chalone” material has been partially purified by gel filtration, ultrafiltration, and electrophoresis.57 It is reported to be a polypeptide of a molecular weight of approximately 4000-5000 daltons, which is unstable at 4#{176}C,8 with

The putative transforming protein of the type I human T-cell leukemia virus (HTLV-1) is a 40-kilodalton protein encoded by the X region and is termed p40XI. On the basis of both subcellular fractionation techniques and immunocytochemical analysis, it is now shown that p40XI is a nuclear protein with a relatively short half-life (120 minutes). It is synthesized de novo in considerable quantities in a human T-cell line infected with and transformed by the virus in vitro, and it is not packaged in detectable amounts in the extracellular virus.

Previous studies using the in vitro diffusion chamber (Marbrook) have shown that bone marrow grown in this system will undergo limited stem cell replication and differentiation to mature granulocytes and mononuclear phagocytes. A series of studies with modified culture systems was initiated to improve cell production and committed stem cell (CFU-C) proliferation in vitro. Introduction of a continuous-flow system and a migration technique providing means of egress for mature neutrophils resulted in substantially improved performance. CFU-C were found to be capable of migration through a 3-mu pore membrane. These studies indicated that membrane surface area, culture medium circulation, and mature cell egress were among the conditions that could be optimized for maximum hematopoietic cell proliferation in suspension culture. The present observations also suggested that large- scale in vitro growth of mammalian bone marrow may be feasible.

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.

Fourteen patients with resistant cancers received high-dose chemotherapy and total body irradiation followed by rescue with autologous cryopreserved bone marrow cells. In seven patients, disease has remained in remission for periods up to two years. These data indicate the effectiveness of high-dose combined-modality therapy and bone marrow autotransplantation in patients with resistant cancer. The high incidence of non-marrow toxicity may be reduced by the use of this modality before patients have received extensive therapy.

Bone marrow from hematologically normal parents of congenitally neutropenic children showed defective myeloid differentiation in vitro. In one family only the mother's marrow differentiated abnormally; in the second family both parents demonstrated abnormal myeloid differentiation. In vitro culture of bone marrow may be useful in establishing the mode of inheritance of some forms of familial neutropenia.

Bone marrow transplantation from an HLA-identical sibling is increasingly used in the treatment of severe aplastic anemia. One major problem with this approach is graft rejection that occurs in 25%-60% of patients conditioned for transplantation with cyclophosphamide. At most transplant centers it has been difficult to accurately identify patients at high risk for graft rejection. We studied a conditioning regimen of cyclophosphamide (200 mg per kg) and low-dose total body irradiation (3 Gy; equivalent to 300 rad) in 23 consecutive unselected patients with aplastic anemia followed for a minimum of 6 mo. There was only one episode of graft rejection. Graft-versus-host disease and interstitial pneumonitis were not increased by the more intensive conditioning regimen. Actuarial survival was 61% at 1 yr and 49% at 2.5 yr. Cyclophosphamide and low-dose total body irradiation is an effective conditioning regimen in patients with aplastic anemia. It may be particularly useful when accurate predictive tests of graft rejection are not available as is the case in most transplant centers.

Three patients with chronic myelocytic leukemia in different phases of the natural history of the disease were studied. Their bone marrow cells were cultured under conditions favoring macrophage proliferation, and parallel cytogenetic and cytochemical studies were performed. All cell metaphases examined contained the Ph1 chromosome at a time when more than 80% of these metaphases were in identifiable macrophages. We conclude that the mononuclear phagocyte cell line contains the abnormal chromosome in Ph1-positive chronic myelocytic leukemia.

Alveolar and peritoneal macrophages differ in their energy metabolism. Alveolar macrophages are mainly aerobic whereas peritoneal macrophages are mainly anaerobic in their energy generation. We investigated the question of whether these differences in metabolism are preprogrammed in subsets of macrophage precursors in the bone marrow, or develop in proliferating cells as a consequence of exposure to different tissue environments. The progeny of single mouse macrophage progenitor cells were grown in vitro for 4 days; the resultant colonies were divided into two roughly equal populations, which were cultured in either a high or low oxygen environment corresponding to that of the alveoli or tissues. Following 4 days incubation at 5% or 20% O2, the activities of the two glycolytic enzymes lactate dehydrogenase (LDH) and pyruvate kinase (PK) were two- to threefold higher in the half of the colonies grown in the low O2 environment, whereas the activity of the oxidative phosphorylative enzyme glutamate dehydrogenase (GDH) was two- to threefold higher in the half colony grown in the aerobic environment. Re-exposure of the cells from the low O2 environment to high O2 conditions for an additional 4 days caused a rise in the GDH activity and a decrease in the LDH and PK. The recovery of the GDH activity after the re-exposure was time dependent. Our results support the theory that macrophages arising from a single progenitor cell can develop different metabolic features depending on the O2 environment in which they mature. A single precursor cell can give rise to mature cells with metabolic characteristic of either alveolar or tissue macrophages.

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.

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.

Summary. A novel mutation of the N-RAS gene of T-ALL blast cells was detected by a direct sequencing of in vitro amplified exon-1 of the N-RAS gene. Threonine (ACA) was substituted for alanine (GCA) at codon 11. This mutation would have been overlooked by conventional probe hybridization techniques. A search for other mutations in N-RAS exon-1 in T-ALL revealed a codon 13 mutation substituting aspartic acid (GAT) for glycine (GGT) in one of 18 patients. No mutations at codon 12 were detected.

An unusual case of a patient diagnosed as having the histiocytic proliferative disorder characterized by the lesion eosinophilic granuloma (histiocytosis X) is presented. Fifteen-year follow-up revealed a progression from a solitary eosinophilic granuloma of the bone to multifocal involvement of many body structures including the vulva, vagina, and cervix. This progression occurred after nine asymptomatic years and was temporally related to the patient's first pregnancy. After successful treatment and remission, rapid appearance and growth of observable genital lesions were noted during a second pregnancy. Pregnancy termination in the first trimester was advised due to signs of dissemination and local symptoms.

Twelve cases of ovarian adenocarcinoma were studied for alterations in proto-oncogenes, and a unique pattern of altered ras proto-oncogenes was observed. Amplification of ras-Ki was found in three of seven ovarian tumors and amplification of ras-Ha in one of 12. In contrast, ras-Ha amplification was not found in any of the 334 other tumors and ras-Ki amplification was only seen in breast cancer at a frequency of 3%. Other proto-oncogenes altered in ovarian adenocarcinomas included c-myc and c-erbb-2. Proto-oncogene abnormalities were more frequent in aggressive tumors of high histologic grade.

Growth of human bone marrow in liquid suspension cultures has been used to study normal hematopoietic cell differentiation and abnormalities in blood diseases. A variety of cytochemical stains were applied to human marrow cells cultured in vitro for up to 14 days. AS-D- CHLOROACETATE ESTERASE AND ALPHA-NAPHYHYL BUTYrate esterase were most useful in distinguishing different cell lines in culture. Peroxidase activity disappeared with mononuclear phagocyte morphogenesis and diminished with culture in intermediate and mature granulocytes. Acid phosphatase activity and methyl greed pyronin staining intensity increased with macrophage maturation.

To examine the efficiency of generating protein-reactive antipeptide antibodies, 35 peptides encoded by retroviral or cellular oncogenes were used to immunize rabbits. Thirty-two peptides elicited antipeptide antibodies, of which 56% reacted with their respective oncoproteins. The length of the immunizing peptide was an important factor in generating antibodies reactive with native protein. Similar peptides differing in a single or a few amino acids could elicite antisera of markedly different reactivities.

Bone marrow cells from seven patients with preleukemia and two with erythroleukemia were cultured in vitro to characterize erythropoiesis and erythropoietin responsiveness in these disorders. The methylcellulose plate technique was used to clone erythroid progenitors, and the results were compared to data obtained from studies on five healthy volunteers. Erythroid colonies formed in cultures from all patients and controls, and a clear erythropoietin dose-response effect was observable in each case. The erythroid cloning efficiency of bone marrow cells from the preleukemia patients varied considerably and in some cases was greater than normal. The erythropoietin dose-response relationships were linear on a log-log plot over the erythropoietin concentrations tested (0.05-2.0 U/ml), and linear regression equations generated for each patient and patient group had excellent fits. The mean slopes of the regression lines for preleukemia, polycythemia vera, and normal were not statistically different, suggesting that erythroid precursors in these disorders have similar erythropoietin responsiveness in vitro. Increased cloning was observed with the addition of dexamethasone (10-9 M) to cultures, indicating that glucocorticoid sensitivity was retained by erythroid colony-forming cells in these disorders. These studies show that red cell precursors in preleukemia and erythroleukemia are capable of colony formation in vitro and show erythropoietin responsiveness. The findings also suggest that the marrow pool of erythroid colony-forming cells may be expanded in some cases of preleukemia and that erythroid colony formation in vitro is not correlated with effective erythropoiesis in vivo.

Peritoneal macrophages elicited in C3H/HJ mice by the i.p. injection of Corynebacterium parvum were cytotoxic to allogeneic virus-transformed fibroblasts in vitro. Cytotoxicity was demonstrated in a morphologic (plaque) assay, and quantitated by measuring macrophage-mediated inhibition of incorporation of 3H-thymidine by the target cells. The cytotoxic effect was well established by 6 hr of macrophage-fibroblast interaction, and was retained in cultures from which the supernatant was removed before the addition of 3H-thymidine. Cytotoxic activity of macrophages diminished rapidly after 22 hr of cultivation in vitro. Maximal cytotoxic effect could be prolonged by addition of C. parvum, 50 µg/ml, to macrophage monolayers preincubated in vitro for 22 hr. It could neither be retained nor regenerated when C. parvum was added to monolayers greater than 22-hr old. C. parvum-activated macrophages, grown under anaerobic conditions for 8 hr, retained the ability to phagocytize heat-killed Candida albicans and to exclude trypan blue dye. There was a small but significant reduction in the ability of macrophages to inhibit 3H-thymidine incorporation by target fibroblasts under anaerobic conditions. The cytotoxic effect of activated macrophages in air was not altered by the presence of catalase and was enhanced by enzymatically active superoxide dismutase. We conclude that the processes involved in macrophage-mediated cytotoxicity against allogeneic fibroblasts in this system are largely independent of oxygen.

A 36 year old black woman with serologic evidence of systemic lupus erythematosus had aplastic anemia that failed to respond to corticosteroid or androgen therapy. Bone marrow culture studies were performed to determine if the marrow aplasia was immunologically mediated. A potent serum inhibitor of in vitro myeloid and erythroid colony formation was demonstrated. Incubation of normal bone marrow cells with the patient's serum and a source of complement caused a 95 per cent reduction in myeloid colony formation. Under these conditions, bone marrow from 35 normal volunteer subjects formed a mean of 5 ± 1 colonies per 2 × 10 5 marrow cells plated (control = 95 ± 8 colonies). The addition of the patient's serum plus complement to normal marrow cultured in methylcellulose also caused a 98 per cent reduction in erythroid colony numbers. Assay of serum immunoglobulin fractions obtained by gel filtration demonstrated that the inhibitory activity was contained in the immunoglobulin G (IgG) fraction. In association with a series of plasma exchanges, the titer of inhibitor fell, and marrow cellularity and peripheral blood counts returned to normal. Additional studies indicated that the inhibitor of allogeneic marrow was not directed against HLA or "B-cell" antigens. Incubation of the patient's recovery marrow with stored preplasmapheresis serum and complement did not produce a reduction in myeloid colony formation. It seems most likely that the inhibitor represents alloantibody directed against heretofore unrecognized antigens widely distributed on normal human hematopoietic progenitor cells.

Data presently available suggest that polycythemia vera is an acquired clonal disorder of the pluripotent stem cell resulting in expansion of committed stem cell pools, most prominently in the erythroid line. In vitro and in vivo studies suggest that erythropoiesis in polycythemia vera is at least partially modulated by erythropoietin.