A recombinant retrovirus (DHFR*-SVADA) in which human adenosine deaminase (ADA) cDNA is transcribed from an internal SV40 promoter was used to infect murine hematopoietic stem and progenitor cells. Human ADA enzyme was not expressed in infected primary murine pluripotent stem cell-derived spleen or progenitor colonies (CFU-GM, CFU-Mix, BFU-E). In contrast, human ADA enzyme activity was readily detected in progenitor colonies derived from immortalized multipotent factor-dependent cells. The level of human enzyme was near endogenous murine enzyme levels and was equivalent in undifferentiated stem cells and differentiated myeloid, erythroid, and mixed colonies. These results indicate that cellular properties other than the stage of differentiation are important in determining the expression of foreign sequences introduced by retroviruses. Cell lines that are immortalized but still capable of induced differentiation may contain factors that abrogate blocks to expression that are manifested in primary hematopoietic stem cells.

Adenosine Deaminase↗

PubMed1987

Retrovirus-mediated gene transfer of human adenosine deaminase: expression of functional enzyme in murine hematopoietic stem cells in vivo.

Simplified Moloney murine leukemia virus-based recombinant retrovirus vectors have been constructed which transduce human adenosine deaminase (ADA) cDNA. ADA transcription is under the control of the constitutive promoter for the human X chromosome phosphoglycerate kinase (pgk) gene. In these simplified vectors, dominant selectable markers are not included and selection is dependent on overproduction of functional ADA enzyme. Primary murine hematopoietic cells were infected with helper-free recombinant ADA virus generated from Psi-2 packaging cells. Protein analysis revealed that human ADA enzyme was expressed in progenitor-derived hematopoietic colonies in vitro and CFU-S-derived spleen colonies in vivo. Enzyme expression was dependent on transcription from the pgk promoter. ADA expression in primary murine hematopoietic cells directed by the internal promoter was not adversely affected by the presence of the Moloney virus long terminal repeat enhancer sequence. Use of these vectors allows systematic evaluation of the effects of specific sequences in recombinant retrovirus vectors on expression in primary murine hematopoietic cells in vivo.

Adenosine Deaminase↗

PubMed1987

Transfer and expression of human ADA in murine hematopoietic stem cells.

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Adenosine Deaminase↗

PubMed1987

The presence of clonogenic cells in high-grade malignant lymphoma: a prognostic factor.

A culture system has been developed that promotes growth of clonogenic lymphoma cells of some patients with intermediate and high-grade malignant lymphoma. The formation of colonies in bone marrow, lymph nodes, and peripheral blood samples is best supported by human plasma. Colony formation of some patients was dependent upon growth factors, which in this study were added in the form of medium conditioned by phytohemagglutinin (PHA)-stimulated leukocytes (PHA-LCM). Some gave rise to lymphoma colonies without PHA-LCM but improved their frequency with PHA-LCM; others were completely independent of PHA-LCM. Colonies grown in primary cultures were routinely recloned and propagated as Epstein-Barr virus (EBV)-negative cell lines with stable B cell phenotype. The cell lines showed the same immunoglobulin rearrangement pattern as that observed in the primary lymphoma sample. In addition, a significant clinical correlation was observed between culture data and clinical outcome. Survival of patients who formed lymphoma colonies at any time during their clinical course was significantly shorter than survival of patients who did not give rise to colonies (P = 0.0009). The same observation was made when the survival assessment was performed for the subset of patients studied at diagnosis (P = 0.0014).

Adult↗

PubMed1986

Characterization of reticulofibroblastoid colonies (CFU-RF) derived from bone marrow and long-term marrow culture monolayers.

The maintenance of hemopoietic precursors in long-term liquid bone marrow cultures (LTBMC) is associated with the presence of an adherent stromal layer composed of heterogeneous cell populations. We have used a culture assay to promote the growth of one of its cellular components and characterize its properties. Freshly obtained bone marrow cells and cells derived from the adherent layer of LTBMC were grown in methylcellulose-clotted plasma in the presence of phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM), hydrocortisone (HC), and citrated normal human plasma. Both sources contained cells (CFU-RF) that gave rise to colonies of cells with a reticulofibroblastoid appearance. In the presence of HC, most colonies contained lipid-laden cells. Colonies could be further propagated as adherent layers when transferred into liquid cultures. These cells produced laminin, fibronectin, and collagen types I, III, IV, and V. They were negative for Von Willebrand factor VIII. The ability to synthesize laminin and collagen type IV distinguished these cells from a population of previously described bone marrow fibroblasts (CFU-F). The relationship of CFU-RF to hemopoietic precursors was investigated using patients with chronic myeloid leukemia and bone marrow transplant recipients. Cells within CFU-RF-derived colonies were uniformly negative for the Philadelphia chromosome, thus making it unlikely that they belonged to the malignant hemopoietic clone. CFU-RF-derived colonies in bone marrow transplant recipients were found to be exclusively of host origin. Both observations support the view that CFU-RF is not part of the repertoire of hemopoietic stem cells.

Antigens↗

PubMed1985

Colony growth and self renewal of plasma cell precursors in multiple myeloma.

Culture conditions that support the growth of multi-and single-lineage hemopoietic colonies are also able to give rise to large myeloma colonies from bone marrow and peripheral blood samples of some patients with multiple myeloma. The culture system was used to determine the frequency of hemopoietic precursors and clonogenic myeloma progenitors in 71 patients with multiple myeloma studied in various clinical phases of the disease. The frequency of normal hemopoietic precursors in patients with benign monoclonal gammopathy and smoldering myeloma were indistinguishable from normal controls. Myeloma colonies were not observed in these subgroups. In contrast, patients with active disease showed significantly reduced hemopoietic colony formation, even before the initiation of therapy. A further reduction was demonstrated for patients with acute phase disease. A correlation between the frequency of hemopoietic colonies and the concentration of plasma cells in the plated sample was not observed. Large myeloma colonies with recloning potential were identified in cultures of specimens derived from 14 of the studied patients. These colonies were most frequently (ten cases) obtained from patients who had entered the acute phase of the disease. These patients manifested marrow failure (pancytopenia) and their marrow had a limited capacity to generate normal hemopoietic colonies. Three of the patients that formed myeloma colonies were studied in chronic phase following chemotherapy and one patient was examined at diagnosis. The myeloma colonies were composed exclusively of cells characterized by the same M protein as the patient. Some of the cells within the colonies retained their ability to self renew extensively, as demonstrated by serial recloning studies. Colonies derived from six of the patients are now propagated in semisolid and liquid medium for as many as nine to 34 generations. Patients that form myeloma colonies under these culture conditions represent a high-risk group with significantly shorter survival than patients not able to give rise to myeloma colonies. A Cox proportional hazards model was fitted to the data to determine the prognostic role of myeloma colony growth in culture after accounting for the influence of other well established risk factors, such as concentration of plasma cells and disease status. The analysis indicated that myeloma colony growth in culture serves as a strong and independent predictive indicator of poor clinical prognosis.

Actuarial Analysis↗

PubMed1985

Heterogeneity of human peripheral blood eosinophil-type colonies: evidence for a common basophil-eosinophil progenitor.

We have recently shown that a proportion of previously designated human eosinophil "(Eo)-type" colonies in methylcellulose contain basophils and histamine (Denburg et al Blood 61:775, 1983). In the present studies, individual Eo-type colonies have been analyzed by cell morphology as well as by biochemical assays for histamine, Charcot-Leyden crystal protein (CLC), and eosinophil granule major basic protein (MBP). Clonal origin of single Eo-type colonies was confirmed by G6PD isoenzyme analysis. Morphological observations of such colonies revealed the existence of two distinct colony types: (1) Eo type containing 100% basophils and (2) Eo type containing mixtures of basophils and eosinophils, including cells with mixed basophil-eosinophil granulation. Histamine was not detected in pure, mature peripheral blood eosinophils. Immunofluorescent studies demonstrated bright staining for CLC and MBP in 95% +/- 3% of cells in Eo-type colonies but only in 5% +/- 4% of cells in GM-type colonies. Radioimmunoassay for MBP was positive in 5/9 Eo-type and 0/10 neutrophil-macrophage ("GM-type") colonies, with a mean level (nanogram/colony) of 11.6 +/- 4.2 per Eo-type colony; four of the latter colonies were doubly positive for both histamine and MBP. These and previous findings point out the morphological and biochemical heterogeneity of peripheral blood Eo-type colonies and provide direct evidence for the existence of a common, circulating basophil-eosinophil progenitor.

Basophils↗

PubMed1985

Clonal origin of human basophil/mast cells from circulating multipotent hemopoietic progenitors.

The origin of the human basophil/mast cell lineage from a pluripotent hematopoietic stem cell has been surmised but never demonstrated. By examining individual hemopoietic colonies in methylcellulose under inverted microscopy and using histochemical stains in conjunction with single-colony histamine assays, we have previously identified basophil/mast cell progenitors in human peripheral blood. We now report that a large proportion of normal human peripheral blood mixed granuloerythropoietic (GEMM) colonies contain histamine, in contrast to a significantly lower frequency of histamine positivity among normal neutrophil-macrophage, eosinophil, erythroid, macrophage, or megakaryocyte colonies. Morphological observations confirmed the presence of basophil/mast cells in the majority of GEMM colonies. In our work, the clonal derivation of basophils/mast cells from circulating multipotent (CFU-GEMM) hemopoietic stem cells was formally demonstrated, using combined histamine and G6PD isoenzyme analysis of single colonies grown in methylcellulose from a normal G6PD heterozygote.

Basophils↗

PubMed1984

Flexible association of hemopoietic differentiation programs in multilineage colonies.

Pluripotent hemopoietic progenitors lose potentialities during the process of differentiation. We have examined events that lead to lineage restriction by determining the cellular composition of 785 multilineage colonies grown from peripheral blood samples of glucose-6-phosphate-dehydrogenase (G-6-PD) heterozygous volunteers. Of these colonies, 762 contained only one isoenzyme type and were considered to be of clonal origin. A considerable heterogeneity was observed. Some colonies were composed of cells belonging to two different lineages, while other colonies contained three or more different cell types. A small number of colonies consisted--in addition to myeloid cells--of T-lymphocytes. The variable association within individual colonies of members belonging to different hemopoietic lineages suggests a flexible determination and expression of differentiation programs by early progenitors.

Bone Marrow Cells↗

PubMed1984

Immunohistochemical localization of the delta antigen associated with hepatitis B virus in liver biopsy sections embedded in Araldite.

Immunohistochemical localization of the delta antigen in the nuclei of the liver tissue embedded in Araldite was successfully carried out by an indirect immunoperoxidase technique. Deplasticization, using sodium ethoxide solution, was required prior to application of immunochemical reagents. The specificity of the staining was confirmed by the abolition of positive staining after absorption of the antibody with the delta antigen.

Biopsy↗