Selection of lineage-restricted cell lines immortalized at different stages of hematopoietic differentiation from the murine cell line 32D

Erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor- (G-CSF) dependent cell lines have been derived from the murine hematopoietic cell line 32D with a selection strategy involving the culture of the cells in FBS-deprived medium supplemented only with pure recombinant Epo, GM-CSF, or G-CSF. The cells retain the diploid karyotype of the original 32D clone, do not grow in the absence of exogenous growth factor, and do not induce tumors when injected into syngeneic recipients. The morphology of the Epo-dependent cell lines (32D Epo1, -2, and -3) was heterogeneous and evolved with passage. The percent of differentiated cells also was a function of the cell line investigated. Benzidine-positive cells ranged from 1-2% (32D Epo3) to 50-60% (32D Epo1). These erythroid cells expressed carbonic anhydrase I and/or globin mRNA but not carbonic anhydrase II. The GM-CSF- and G-CSF-dependent cell lines had predominantly the morphology of undifferentiated myeloblasts or metamyelocytes, respectively. The GM-CSF-dependent cell lines were sensitive to either GM-CSF or interleukin-3 (IL-3) but did not respond to G-CSF. The G-CSF-dependent cell lines grew to a limited extent in IL-3 but did not respond to GM-CSF. These results indicate that the cell line 32D, originally described as predominantly a basophil/mast cell line, has retained the capacity to give rise to cells which proliferate and differentiate in response to Epo, GM-CSF, and/or G-CSF. These cells represent the first nontransformed cell lines which can be maintained in growth factors other than IL-3 and which differentiate in the presence of physiologic signals. As such, they may represent a model to study the molecular mechanisms underlying the process of hematopoietic differentiation, as well as sensitive targets for bioassays of specific growth factors.     

Intraventricular 6-hydroxydopamine increases thyrotropin-releasing hormone (TRH) content in regions of rat brain

Rats were given intraventricular (ivt) injections of various doses (50-400 micrograms, hydrobromide salt) of 6-hydroxydopamine (6-OHDA) and killed 1, 3 or 6 days later. Brains were removed, dissected into 11 regions, and the thyrotropin-releasing hormone (TRH) content of each region was measured by radioimmunoassay. 6-OHDA (400 micrograms) caused significant elevations in the TRH content of 6 regions: olfactory bulb, anterior cortex, brainstem, posterior cortex, hippocampus, and amygdala-piriform cortex. The magnitude of these increases ranged from 59% in olfactory bulb to 497% in hippocampus and was, in all cases, greatest at 3 days. These results suggest that the TRH content of certain brain regions may be regulated by catecholamine neurotransmitters.     

Motility and fertility of stallion spermatozoa isolated in bovine serum albumin

Semen from three mature stallions was used in an attempt to isolate a population of highly motile spermatozoa. An ejaculate of semen, collected from each stallion at 7-day intervals for 35 days, was evaluated for percentage of motile spermatozoa and rate of progressive motility (scale 1 to 4). Two milliliters of semen were layered over 6 ml of 3% bovine serum albumin (BSA) in 13 × 125 mm columns at room temperature (RMT) or in a warm water bath (WB). After a 30-minute separation period, the top semen layer and the upper and lower halves of the BSA fraction were separately withdrawn from columns and reevaluated. In both the RMT and WB separation columns, percent motile spermatozoa and progressive motility decreased in the top semen fraction as compared to initial values for these parameters. Percentage of motile spermatozoa in the lower BSA fractions increased (P<.01) to 58.7 and 65.7 following separation at RMT and in a WB, respectively. Also, there was a significant increase in rate of progressive motility rate for spermatozoa in the lower BSA fraction of both the RMT and WB treatments.In a second experiment 30 Quater Horse mares were artificially inseminated to compare fertility of spermatozoa isolated in BSA with raw semen diluted with either Tyrode's solution or BSA. The pregnancy rate for 10 mares inseminated with 100 × 10⁶ live isolated spermatozoa was not different from that of control mares inseminated with the same number of live untreated spermatozoa. Foaling rates were 70, 40 and 60% for the isolated, Tyrode and BSA treatment groups respectively.     

Coinfection of human foreskin fragments with multiple human papillomavirus types (HPV-11, -40, and -LVX82/MM7) produces regionally separate HPV infections within the same athymic mouse xenograft.

The athymic mouse xenograft system was used to prepare infectious stocks of two additional anogenital tissue-targeting human papillomaviruses (HPVs) in a manner similar to that for the development of infectious stocks of HPV-11. An anal condyloma from a transplant patient was used as material for extraction of infectious virus, and human foreskin fragments were incubated with the virus suspension and transplanted subrenally into athymic mice. Partial viral sequencing indicated that two rare HPV types (HPV-40 and HPVLVX82/MM7) were concurrently present in both the patient condyloma and the foreskin xenografts, and passage of both types was achieved as a mixed infection with HPV-40 predominating. Xenografts that developed from simultaneous infection of human foreskin fragments with HPV-11, -40, and -LVX82/MM7 virions produced regionally separate areas of HPV-11 and -40 infection as determined by in situ hybridization. In addition, in situ hybridization with HPV-40 and HPVLVX82/MM7 DNA probes demonstrated that both of these HPV types were present as adjacent but separate infections within the same anal condyloma of the transplant patient. These studies indicate that multiple HPV types can simultaneously infect genital tissue and that each HPV type predominantly maintains regional separation within the same papilloma.     

Synergistic and antagonistic effects of recombinant human interleukin (IL) 3, IL-1 alpha, granulocyte and macrophage colony-stimulating factors (G-CSF and M-CSF) on the growth of GM-CSF-dependent leukemic cell lines

Three human leukemia cell lines (TALL-101, AML-193, and MV4-11) that require granulocyte/macrophage-colony stimulating factor (GM-CSF) for growth in a chemically defined medium were examined for their response to recombinant human (rh) cytokines. Either rh interleukin (IL)-3 or rhGM-CSF alone supported the long term growth of all three cell lines, and the two growth factors acted synergistically to stimulate the proliferation of the early T lymphoblastic leukemia (TALL-101) and of the monocytic leukemia (AML-193) cells. However, IL-3 antagonized the proliferation of the biphenotypic B-myelomonocytic leukemia (MV4-11) cells in the presence of GM-CSF when both factors were used at very low concentrations. The rh granulocyte (G)-CSF independently supported the long and short term growth of AML-193 and MV4-11, respectively, and synergized with GM-CSF in inducing proliferation of these cells. By contrast, G-CSF did not stimulate TALL-101 cell growth and antagonized the effect of GM-CSF such that proliferation was arrested. Although neither rh macrophage (M)-CSF nor rhIL-1 alpha independently promoted proliferation of the three leukemia cell lines, these cytokines were able to either up- or down-regulate the GM-CSF-dependent growth of these cells. Taken together, these data demonstrate that leukemic cells often require the synergistic action of several cytokines for optimal growth, whereas other combinations of factors may be growth-inhibitory. This raises the possibility that multiple hemopoietic growth factors sustain or control leukemic cell proliferation also in vivo. In addition, the observation the G-CSF, M-CSF, and IL-1 alpha can, in some cases, arrest cell proliferation without inducing differentiation suggests that the programs of proliferative arrest and differentiation in leukemic cells can be dissociated.     

Establishment and characterization of an undifferentiated human T leukemia cell line which requires granulocyte-macrophage colony stimulatory factor for growth

A human leukemia cell line (TALL-101) was established from the bone marrow of a patient with an undifferentiated acute T cell leukemia using the conditioned medium (CM) of the human T cell leukemia virus (HTLV) II-transformed human cell line J-LB1. Immunofluorescence analysis on the original leukemic cells indicated the presence of T cell markers (Leu-1, Tdt, and T11); however, the established TALL-101 cell line expressed only antigens commonly present on progenitor cells, thymocytes, and myelomonocytic cells, but not on mature T cells. A high percentage of TALL-101 cells displayed the Tac antigen which was down-regulated upon incubation in the presence of recombinant human (rH) interleukin 2 (IL 2). Interferon (IFN)-gamma induced the appearance of class II histocompatibility leukocyte antigens (HLA) and of a T cell marker (3A1), and enhanced the expression of transferrin receptors on these cells. Further evidence for a T cell lineage of the TALL-101 cell line was provided by both chromosomic and genotypic analysis showing a translocation in chromosome 14 typical of T cell leukemias, and a rearrangement of the T-beta receptor locus. The growth-promoting activity in the J-LB1-CM was identified as granulocyte-macrophage colony stimulatory factor (GM-CSF), a growth factor which stimulates proliferation of normal myelomonocytic cells and other progenitor cells, but not known to have an effect on T cells. Dose response curves of [3H]thymidine incorporation and growth indicated that TALL-101 cells were sensitive to very low concentrations of rHGM-CSF, 5 ng/ml inducing maximal proliferation in chemically defined medium. The TALL-101 cell line is strictly GM-CSF-dependent for growth: upon depletion of GM-CSF from the culture medium, the cells stop proliferating immediately and die within 1 to 2 wk. The overall data, showing that GM-CSF is able to support the growth of a highly undifferentiated T cell leukemia, strongly suggests that this factor might have similar growth promoting effects on other immature T cell leukemias, and possibly, on normal T cell progenitors.     

Laboratory production in vivo of infectious human papillomavirus type 11.

Human papillomaviruses (HPV) induce among patients natural lesions which produce small amounts of virus. Infection of human cell cultures does not lead to the multiplication of virus, which also does not replicate in experimental animals. We have developed a unique system for the laboratory production of HPV type 11 (HPV-11). Fragments of human neonatal foreskin were infected with an extract of naturally occurring human vulvar condylomata and grafted beneath the renal capsule of athymic mice. Later (3 to 5 months), condylomatous cysts developed from those grafts. Nuclei of koilocytotic cells contained large amounts of capsid antigen and intranuclear virions. The experimentally induced condylomata were homogenized, and the virions were extracted and used to infect another generation of human foreskin grafts in athymic mice. The HPV-11 DNA content and infectivity of the natural and experimental condylomata were similar. Extracts of experimental condylomata were subjected to differential ultracentrifugation and sedimentation in CsCl density gradients. A single, opalescent band was visible at a density of 1.34 g/ml. It contained HPV virions with HPV-11 DNA. This report is the first demonstration of the laboratory production of an HPV.     

Antibody-mediated neutralization in vivo of infectious papillomaviruses.

Specific antibody-mediated neutralization of infectious human papillomavirus type 11 (HPV-11) was achieved in the athymic mouse xenograft system, in which HPV-11 induced morphological transformation of human foreskin. Virus-specific neutralization was demonstrated by the ability of an HPV-11-specific polyclonal antiserum to neutralize HPV-11 infectivity and not bovine papillomavirus type 1 (BPV-1) or cottontail rabbit papillomavirus (CRPV) infectivity. In all three virus infectivity systems, neutralization was detected by the failure of the virus suspension to induce morphological transformation of the appropriate skin xenografts placed under the renal capsule of athymic mice. Rabbit polyclonal antisera were also generated against intact virions of both BPV-1 and CRPV, and neutralizing activity was tested in the xenograft system with BPV-1 and fetal bovine skin and with CRPV and rabbit ear skin. The three polyclonal antisera contained virus-specific neutralizing antibodies, demonstrating that neutralizing epitopes existed on all three papillomaviruses and that these epitopes were antigenically non-cross-reactive. The athymic mouse xenograft system was a useful model for detecting papillomavirus-specific neutralizing antibodies and offers the only opportunity for the analysis of neutralizing antibodies to human papillomaviruses.     

Induction of the granulocyte-macrophage colony-stimulating factor (CSF) receptor by granulocyte CSF increases the differentiative options of a murine hematopoietic progenitor cell.

32DC13(G) is an interleukin-3-dependent murine hematopoietic precursor cell line which differentiates into neutrophilic granulocytes upon exposure to granulocyte colony-stimulating factor (G-CSF) but ceases to proliferate and dies when exposed to granulocyte-macrophage (GM)-CSF. Surface receptors for GM-CSF are undetectable on 32DC13(G) cells but can be induced by priming the cells with G-CSF. Exposure of the G-CSF-primed cells to GM-CSF then results in the generation of monocytes as well as granulocytes. The acquired competence to respond to GM-CSF remains irreversibly encoded in the primed cells, although the GM-CSF receptor can be down regulated by interleukin-3. This phenomenon suggests a mechanism by which hematopoietic precursors may obtain additional receptors, thereby increasing their differentiative potential.     

Systemic administration of kainic acid produces elevations in TRH in rat central nervous system

Several studies have suggested that the concentration of thyrotropin releasing hormone (TRH) in the central nervous system (CNS) is influenced by the level of CNS activation. Hibernation in the ground squirrel and estivation in the lungfish result in region-specific decreases in TRH concentrations. Repeated electroconvulsive shock (ECS) and amygdaloid kindling have been shown to result in elevations of TRH in limbic brain regions. In the present study, limbic seizures induced by systemic administration of kainic acid resulted in substantial increases in the TRH content of posterior cortex and of dorsal and ventral hippocampus, and in moderate elevations in anterior cortex, amygdala/piriform cortex and corpus striatum. Maximal elevations in TRH were observed 2-4 days after kainic acid administration, and by 14 days TRH levels were similar to control values, with the exception of the dorsal hippocampus, which exhibited more prolonged elevations in TRH levels. Prior exposure to limbic seizure activity attenuated the magnitude of TRH elevation in response to a second administration of kainic acid in the posterior cortex but in no other region. These results indicate that seizure-related processes or events influence TRH systems in the CNS. Neuronal populations involved in limbic seizure induced damage may be involved in the modulation of posterior cortical TRH levels.