The interaction of the Chrna5 D398N variant with developmental nicotine exposure.

A single nucleotide polymorphism (SNP) in CHRNA5 (rs16969968, change from an aspartic acid [D] to asparagine [N] at position 398 of the human α5 nicotinic acetylcholine receptor subunit) has been associated with increased risk for nicotine dependence. Consequently, carriers of the risk variant may be at elevated risk for in utero nicotine exposure. To assess whether this gene-environment interaction might impact nicotine intake in developmental nicotine-exposed offspring, we utilized a mouse expressing this human SNP. D and N dams drank nicotine (100 µg/mL) in 0.2% saccharin water or 0.2% saccharin water alone (vehicle) as their sole source of fluid from 30 days prior to breeding until weaning of offspring. The nicotine (D Nic, N Nic) or vehicle (D Veh, N Veh) exposed offspring underwent a 2-bottle choice test between postnatal ages of 30 to 46 days. N Nic offspring consumed the most nicotine at the highest concentration (400 µg/mL) compared with all other groups. In contrast, D Nic offspring drank the least amount of nicotine at all concentrations tested. Nicotine-stimulated dopamine (DA) release measured from striatal synaptosomes was increased in D Nic offspring, while decreased in N Nic offspring relative to their genotype-matched controls. These data suggest that the α5 variant influences the effect of developmental nicotine exposure on nicotine intake of exposed offspring. This gene-environment interaction on striatal DA release may provide motivation for increased nicotine seeking in N Nic offspring and reduced consumption in D Nic offspring.

Distinct In Vitro Myelopoiesis is Dependent on the Self-Renewal of Hematopoietic Progenitors.

Spleen and bone marrow (BM) have been shown to contain the progenitors of a novel dendritic-like antigen-presenting cell type (L-DC). These progenitors are also maintained in both long-term spleen cultures and co-cultures of spleen or BM over the stromal cell line STX3. We examined mouse foetal liver (FL), rich in hematopoietic stem/progenitor cells (HSC/HPC) after embryonic day (E) 12.5, for the presence of L-DC progenitors by testing their capacity to colonize STX3 and produce L-DC. E14.5 FL from wild-type C57BL/6J mice was found to colonize STX3 and produce L-DC for 28 days. By contrast, E14.5 FL from Ikaros Plastic mice gave only short-term production of low numbers of L-DC between 7 and 14 days of co-culture. The transient and weak production of L-DC by FL from Plastic E14.5 mice maps to the loss of self-renewal capacity amongst HSC. L-DC progenitors are, therefore, closely aligned with a subset of self-renewing HSC/HPC in FL.

Dendritic cells as immune regulators: the mouse model.

Dendritic cells (DC) are central to the immune system because of their role in antigen presentation leading to either tolerance or immunity among cells of the adaptive immune response. It is becoming increasingly evident that DC show extensive plasticity in terms of their origin and function, giving rise to a number of subsets represented differentially in all lymphoid organs. This article considers the tolerogenic capacity of murine DC and draws a distinction between DC that induce tolerance in the immature state and immunity in an inflammatory context, and those that act as regulatory cells inducing immunosuppression in the presence of inflammation.

The 5-HT(1A) receptor active compounds (R)-8-OH-DPAT and (S)-UH-301 modulate auditory evoked EEG responses in rats.

Schizophrenics commonly demonstrate abnormalities in central filtering capability following repetitive sensory stimuli. Such sensory inhibition deficits can be mirrored in rodents following administration of psycho-stimulatory drugs. In the present study, male Sprague-Dawley rats were implanted with brain surface electrodes to record auditory evoked EEG potentials in a paired-stimulus paradigm, using 87 dB clicks delivered 0.5 s apart. Amphetamine (1.83 mg/kg, i.p.) produced the expected loss of sensory inhibition, as defined by an increase in the ratio between test (T) and conditioning (C) amplitudes at N40, a mid-latency peak of the evoked potentials. Also, the 5-HT(1A) agonist (R)-8-OH-DPAT caused a significant increase in the TC ratio at the highest dose studied (0.5 mg/kg s.c.), while the 5-HT(1A) antagonist (S)-UH-301 did not significantly affect the TC ratio at any dose studied (0.1-5 mg/kg s.c.). When administered with amphetamine, a lower dose of 8-OH-DPAT (0.1 mg/kg) and the highest dose of UH-301 tested (5 mg/kg, s.c.) were able to reverse the amphetamine-induced increase in TC ratio. The findings suggest that 5-HT(1A) signaling is involved in sensory inhibition and support the evaluation of 5-HT(1A) receptor active compounds in conditions with central filtering deficits, such as schizophrenia.

Development of dendritic cells from GM-CSF-/- mice in vitro : GM-CSF enhances production and survival of cells.

The production of dendritic cells (DC) from haemopoietic progenitors maintained in long term stroma-dependent cultures (LTC) of spleen or bone marrow (BM) occurs independently of added granulocyte/macrophage colony stimulating factor (GM-CSF). The possibility that cultures depend on endogenous GM-CSF produced in low levels was tested by attempting to generate LTC from spleen and BM of GM-CSF-/- mice. Multiple cultures from GM-CSF-/- and wild type mice were established and compared for cell production. GM-CSF-/- LTC developed more slowly, but by 16 weeks produced cells resembling DC in numbers comparable to wild type cultures. LTC maintained distinct populations of small and large cells, the latter resembling DC. Cells collected from GM-CSF-/- LTC were capable antigen presenting cells (APC) for T cell stimulation and morphologically resembled DC. Large cells expressed the CD11b, CD11c, CD86, 33D1 and Dec-205 markers of DC. Addition of GM-CSF to GM-CSF-/- LTC increased the proportion of large, mature DC present in culture. Stromal cells from GM-CSF-/- LTC could support the differentiation of DC from early progenitors maintained in LTC without addition of GM-CSF. However, GM-CSF is not a critical factor in the in vitro generation of DC from progenitors. It can, however, substitute for stromal cells in increasing the survival of mature DC.

Improved FACS analysis confirms generation of immature dendritic cells in long-term stromal-dependent spleen cultures.

Cells produced in spleen stroma-dependent long-term cultures (LTC) have now been clearly defined as dendritic cells (DC). Characterization of cells by antibody staining and FACS analysis has only been possible using a procedure to quench the high autofluorescence of DC produced in LTC (LTC-DC). The population of large cells produced by the established LTC-X1 culture are homogeneously positive for a number of cell-surface markers expressed by DC. These include CD11c, CD11b, Dec-205, Fc receptor and CD86. They also express markers detectable with the F4/80 and 33D1 antibodies. Cells produced in LTC do not uniformly express the MHC II marker, consistent with an immature DC phenotype. Most cells are weakly positive for MHC II with a small subset of highly positive cells. The quenching method involves staining cells with crystal violet dye, which is taken up within the cell. The importance of optimizimg fluorescent antibody staining assays for delineating DC subsets is indicated and the LTC system is established as a valuable and continuous source of DC precursors.

Review: the application of dendritic cell-derived exosomes in tumour immunotherapy.

Cancer arises from the aberrant proliferation of a single transformed cell. This population acquires the ability to metastasis. An effective way to remove cancer cells from the body is to activate tumour-specific cytotoxic T cells (CTL). Activation of naive T cells depends on the unique antigen presenting capacity of DC. Activated tumour antigen-specific CTL can destroy cancer cells without harm to normal tissue. Their ability to stimulate antigen specific T cell responses makes DC attractive candidates to potentiate anti-tumour immunity. Several studies have demonstrated the efficacy of DC based anti-tumour immunotherapy and the goal now is to optimise immune responses induced by DC, so that effective strategies in treating cancer may be realised. One way to do this is to identify DC characteristics which make them more effective in T cell stimulation. Another is to use exosomes, the antigen presenting vesicles secreted by DC, in order to induce potent anti-tumour immune responses. The non-cellular nature of exosomes offers several advantages for use in tumour immunotherapy.

Identification of progenitor cells in long-term spleen stromal cultures that produce immature dendritic cells.

Dendritic cells (DC) are produced continuously by a unique, long-term culture (LTC) system in which hemopoiesis is supported by a splenic stromal cell layer in the absence of added growth factors. Flow cytometric analysis reveals the production of two distinct cell subsets. The more predominant large-cell subset resembles highly endocytic DC that are large, granular, and possess membrane extensions. They also express high levels of the DC markers CD11c, CD11b, DEC-205, and CD80 on their cell surface. They do not resemble mature DC because they express low levels of MHC type II and CD86 molecules, as well as c-kit and Fc receptor (FcR). These are known characteristics of immature DC. Small cells are smaller and less granular than large cells, with negative to low expression of CD11c, DEC-205, and CD86. A majority of small cells express varying levels of CD11b and CD80. Subpopulations of small cells express low levels of c-kit, FcR, and MHC type II, and only a 20% subpopulation is weakly endocytic. Upon transfer to an irradiated stromal layer, cells within the small subset proliferate and differentiate to resemble the large cells in size, complexity, membrane extensions, and CD11c and CD86 expression. The two cell subsets produced in LTC are developmentally linked, with the heterogeneous small-cell subset containing progenitors of the larger homogeneous, immature DC subset. LTC represent a valuable model system for studying DC development from hemopoietic progenitors.

Proliferation of dendritic cell progenitors in long term culture is not dependent on granulocyte macrophage-colony stimulating factor.

UNLABELLED: A unique long term culture (LTC) system has been developed which supports the production of dendritic cells (DC). Cell production is dependent on a stromal cell layer derived from murine spleen. This LTC system produces a high turnover of non-adherent cells that express DC morphology, cell-surface markers, and antigen-presenting capacity. OBJECTIVE: The long term production of these cells suggests that the LTC system supports hemopoiesis. It was of interest to examine the number and nature of hemopoietic progenitors present in LTC. MATERIALS AND METHODS: A combination of approaches, including FACS analysis, spleen colony-forming unit assays, and in vitro colony assays were undertaken. RESULTS: Pluripotent haemopoietic stem cells are not detectable among the non-adherent cell population produced in LTC. Instead, LTC support a replicating c-kit+ progenitor population, which generates only dendritic-like colonies in in vitro colony assays. In addition, this population does not respond to combinations of growth factors thought to stimulate DC proliferation, including granulocyte macrophage-colony stimulating factor (GM-CSF) and Flt3L. Production of DC occurs only in the presence of LTC-derived culture supernatant or a confluent stromal cell layer. CONCLUSIONS: These results suggest that LTC contain a dendritic progenitor that is dependent upon the stromal cell network for proliferation and differentiation. The development of only DC within LTC allows easy collection of cells for experimentation. This, in combination with the fact that DC development occurs in the absence of exogenous growth factors, makes the LTC system a practical model for the study of DC function and development.

Long-term stroma-dependent cultures are a consistent source of immunostimulatory dendritic cells.

A long-term culture (LTC) system has been established that supports the continuous production of dendritic cells (DC) from haemopoietic cells present in the culture. The production of cells depends on the presence of an intact stromal cell layer containing a mixture of fibroblasts and endothelial cells. Cells are shed from foci of dividing cells in contact with the stromal cell matrix. They resemble DC in terms of morphology and cell surface marker expression. The LTC can be derived from different lymphoid tissues, but most success has been achieved with murine spleen. Different LTC vary in capacity to produce immunostimulatory DC. Some LTC produce DC that are very effective APC and can stimulate both mixed lymphocyte and antigen-specific T cell responses. The DC produced in others are weak APC. Different LTC appear to produce DC reflecting different stages of maturation or development, reflected by different phenotypic and functional characteristics. The production of cells within LTC occurs independently of added cytokines and is dependent on maintenance of the stromal cell layer and the presence of a subset of smaller progenitor cells. Long-term cultures remain a valuable source of cells for study of DC development and function.

Immunotherapeutic potential of dendritic cells generated in long-term stroma-dependent cultures.

Long term cultures (LTC) producing dendritic cells (DC) have been established from spleen. A well developed stromal cell layer supported production of DC in numbers suitable for experimentation. Cells had obvious membrane pseudopodia and could be collected from culture every 2-3 days. Large cells produced in LTC stained with fluorescently labelled monoclonal antibodies specific for DC such as 33D1, and M1/70 which is specific for DC and myeloid cells. These staining patterns confirmed the presence of DC within the LTC population. LTC-DC were tested and shown capable of migration in vivo in B10.A(2R) mice following footpad inoculation. Most cells entered the spleen and a small number entered popliteal lymph node. LTC-DC have migratory capacity comparable with control spleen lymphocytes. LTC-DC were tested for capacity to induce an anti-tumour immune response after exposing cells to tumour cell membranes. LTC-DC pulsed with BL/VL3 tumour antigens were able to induce a BL/VL3-specific primary cytotoxic T lymphocyte (CTL) response detectable in popliteal lymph nodes and spleen of C57BL/6J mice within 6 days of priming. BL/VL3 tumour cells grew in sublethally irradiated C57BL/6J mice giving 100% mortality. Adoptive transfer of spleen cells from mice given BL/VL3 antigen-pulsed LTC-DC, two weeks previously, significantly slowed the growth of BL/VL3 tumour cells in mice. DC produced in LTC can function as antigen presenting cells (APC) when adoptively transferred into animals. Their capacity to migrate effectively, to induce a CTL response and to reduce tumour load suggests that DC grown using this in vitro system may have valuable clinical potential in humans.

Hemopoiesis in long-term stroma-dependent cultures from lymphoid tissue: production of cells with myeloid/dendritic characteristics.

A long-term stroma-dependent culture system (LTC) has been developed which continuously produces hemopoietic cells providing an in vitro system for the study of cell differentiation. These nonadherent cell populations contain a large subpopulation of dendritic cells (DC). LTC producing DC were easily generated from spleen, but could also be established from bone marrow (BM) and lymph node with less success. It was difficult to establish DC-producing LTC from thymus. The properties of splenic and thymic stroma have been compared. Spleen stroma developed more complicated networks of fibroblasts, endothelial cells, macrophages, and DC. Thymic stromal monolayers were dominated by epithelial cells and fibroblasts, with a lower proportion of macrophages and endothelial cells. They had a relatively sparse structure of cell networks compared with spleen stroma. Cells with dendritiform morphology first appeared in cultures by 2-3 wk. The majority of cells produced were large cells which expressed DC-specific cell surface markers, major histocompatibility complex (MHC) Class II molecules, and the CD80/CD86(B7) costimulator. A high proportion of cells also expressed myeloid cell markers. No T or B lymphoid cells or granulocytes were present in the cultures. LTC continued to produce nonadherent cells resembling myeloid/DC for long periods, even after passage of stromal cells and stem cells at about 3-4 mo. after culture establishment. The LTC system offers potential to study the in vitro differentiation of myeloid/DC.

The role of dendritic cells in T cell activation.

Dendritic cells (DC) are distinguishable from other antigen-presenting cells by their potent antigen-presenting capacity. They are not only efficient at presenting peptide antigen but can also process and present soluble protein antigen sto antigen-specific T cells and cloned T cell lines. They are very strong stimulators of both allogeneic and syngeneic mixed lymphocyte reactions and have a unique capacity to stimulate naive T cells. The potent functional capacity of DC is related to a high-level expression of major histocompatibility complex class I/II molecules and constitutive expression of costimulatory molecules, such as CD80/CD86, as well as heat stable antigen, CD40 and the leucocyte function antigen (LFA) family of adhesion molecules. Recent studies have shown that DC are also involved in regulation of the immune response via induction of both central and peripheral tolerance.

Long-term stromal cultures produce dendritic-like cells.

A stromal cell-dependent long-term culture (LTC) system has been developed from spleen which continuously generates non-adherent cells with dendritic cell (DC) characteristics. Bioassays using factor-dependent cell lines have revealed that both spleen and thymic stromal cultures secrete interleukin (IL)-3 and IL-6-like growth factors. Conditioned medium from LTC also contains factors which appear to be unrelated to IL-3 and induces growth of stromal cells from bone marrow. Non-adherent cells generated in LTC were not T or B lymphoid cells or granulocytes, nor were mast cells detectable. Morphological and electron microscopic examination has also excluded the presence of macrophages (Mo). Cells with DC morphology have been detected by both light and electron microscopy. The majority of cells in the non-adherent population were found to have multiple membrane pseudopodia, with a small acentric nucleus. These appear to be the precursors of DC. They expressed cell surface markers detectable with DC-specific antibodies and antibody specific for major histocompatibility complex Class II molecules. A proportion of cells also expressed myeloid markers, but since this expression was not supported by histochemical staining for myeloperoxidase or non-specific esterase, it was concluded that the cells produced are not typical of the myeloid lineage. Cells generated in LTC were shown to be potent stimulators for allogeneic and syngeneic MLR and for antigen-specific T-cell proliferation.

Specific transcription of the unrearranged TCR V beta 8.2 gene in lymphoid tissues occurs independently of V(D)J rearrangement.

A truncated T cell receptor (TCR) V beta 8.2 polypeptide expressed on the surface of a precursor lymphoid cell line and on a subset of mesenteric lymph node cells has previously been shown to be encoded by transcripts from unrearranged V beta 8 genes. Germline V beta 8 transcription has now been demonstrated in multiple lymphoid and non-lymphoid tissues in mice of varying ages and in cultured cell lines by reverse transcription-polymerase chain reaction (RT-PCR). Significant levels of V beta 8 germline transcription were found in thymus, spleen, liver and bone marrow and in all lymphoid cell lines studied. Germline V beta 8 transcription in the liver dropped as mice aged, and increased in the bone marrow. Germline V beta 8 transcription was also detectable in thymus, spleen, liver and bone marrow of RAG-1-/- mice. This indicated that it is not dependent upon the presence of mature lymphoid cells, nor necessarily related to V(D)J rearrangement events. Semi-quantitative polymerase chain reaction (PCR) and hybridization with oligonucleotides specific for V beta 8.1, 8.2 and 8.3 showed that the V beta 8.2 gene produced at least 90% of all the germline V beta 8 transcripts in all of the tissues examined. The significance of these results in lymphoid cell development and for models of the regulation of V(D)J rearrangement are discussed.

Association of repeat sequences with integrated retroviruses in a murine leukaemia cell line.

An analysis was made of the retroviral integration sites for retroviruses in a murine lymphoid precursor cell line, C1-V13D, derived following in vitro infection with RadLV, an ecotropic murine retrovirus. A genomic library was constructed and lambda clones were selected for their capacity to hybridize with the specific RadLV gp70 ecotropic env probe. Analysis of these clones by a combination of approaches, including subcloning, partial restriction mapping and sequencing, has confirmed the existence of multiple recombinant and defective viruses in C1-V13D. To check for the presence of coding sequences in flanking genomic DNA, 32P-labelled cDNA from C1-V13D was used to probe HindIII- and Psti-digested virus-positive lambda clones by Southern analysis. Regions hybridizing specifically with 32P-labelled C1-V13D cDNA were subcloned and analysed. A notable feature of these cDNA+ regions was the frequent presence of B1, B2 and simple repeats. These repeat elements were found to be present in high frequency in the genomic regions flanking the proviruses, in numbers higher than expected for the genome as a whole. All full-length viruses isolated appeared to represent integration events into regions rich in repeat elements. Some B1 and B2 repeats have been shown to code for functional proteins and to play regulatory roles. Viral integration in the vicinity of these genetic elements could contribute to oncogenesis if the integration event were to disrupt normal gene function.

Expression of a TCR V beta 8.2 polypeptide from the unrearranged gene in a murine lymphoid precursor cell line.

The TCR in a mature T cell is a multimeric complex of TCR alpha and beta chains, and CD3 subunits. Functional TCR alpha and beta chains are encoded by genes that result from developmentally controlled somatic rearrangement events. By FACS analysis, we have detected a TCR V beta 8 protein on the surface of an immature lymphoid cell line, C1-V13D, that has all of its TCR genes in germline (unrearranged) configuration. RNA blot analysis detected a 1.4 kb polydenylated V beta 8 RNA in C1-V13D cells, but no expression of C beta was detected. Rapid amplification of 3' cDNA ends was used to clone an RNA that was initiated from the leader exon of the V beta 5.1 gene and spliced to the V exon of the V beta 8.2 gene. The putative sequence of the mature 10.8 kDa protein was entirely encoded by the V beta 8.2 exon. RT-PCR analysis confirmed that 97% of the V beta 8 RNA detected in C1-V13D cells was encoded by the V beta 8.2 gene, and only 3% by V beta 8.1 and V beta 8.3 genes. Furthermore, most of the V beta 8.2 RNA was spliced to the leader exon of the V beta 5.1 gene and not to the leader exon of the V beta 8.2 gene. The implications of preferential transcription from particular germline TCR genes for repertoire diversity and possible functions for proteins translated from germline TCR V beta genes are discussed.

Leukaemogenic progression: the importance of differentiation and associated genetic events.

A feature common to many blood cell cancers is the uncoupling of normal proliferative and differentiative events, both of which are intimately linked in the cell's developmental programme. In some cancers, further differentiative events have been associated with oncogenic progression and, in other cancers, terminal differentiation of cells has been shown to result in reversal of malignancy and death of the cancer cell. Clearly the development of cancer is not the result of a single oncogenic event, but rather a myriad of events which appear to proceed in concert in a step-wise fashion and which are likely to be influenced by the cellular environment. Here we review some of the major genetic changes which occur in leukaemogenesis and discuss the possible role of differentiative events in the development of leukaemia.

Induction of leukemia in mice using a radiation leukemia virus-induced cell line: a model system for studying oncogenic progression.

Leukemogenesis induced by slowly transforming retroviruses is a multistep process which is difficult to dissect because of its long latency and the problem of distinguishing oncogenic from differentiative events. A method for leukemia induction in mice has been developed using a cell line isolated following in vitro infection with the slowly transforming murine radiation leukemia virus (RadLV). The CI-V13D cell line represents a lymphoid precursor cell type at an early stage in cell transformation and can develop subcutaneous tumors in irradiated syngeneic hosts but not in allogeneic mice even after sublethal irradiation. Selective growth in allogeneic (CBA/H) mouse thymus has been demonstrated, but this requires preirradiation of the recipient. Upon reisolation from CBA/H thymus, C1-V13D progeny clones displayed increased tumorigenic potential in comparison to the 'parental' CI-V13D cell line. Tumorigenicity was shown to increase with serial passage through thymus and electron micrographs of clones also revealed increased production of C-type retroviruses. This new model for oncogenic progression should be more amenable to analysis of early genetic changes occurring during replication of leukemia in the thymus.

Germline transcription and expression of Tcrb-V8 genes in peripheral mouse lymphoid tissues.

Germline transcription of the Tcrb-V8.2 gene has been recently shown to occur in a lymphoid precursor cell line and to result in weak expression of an aberrant T-cell receptor (Tcr) beta chain on the cell surface. An investigation into the expression of a similar Tcr structure in normal lymphoid sites has involved antibody staining and sorting to identify a minor subset of Tcr-Vbeta+ Cbeta- cells in mesenteric lymph node and thymus. These cells have been analyzed by reverse transcriptase-polymerase chain reaction for the presence of transcripts encoded by bV8 genes in rearranged vs germline configuration in subsets of cells sorted on the basis of Tcrb-V8 and Tcrb-C gene expression. Germline transcripts were found in the Tcr-Vbeta- Cbeta- subset of cells in bone marrow, thymus, and mesenteric lymph node. They were also found to be present in the Tcr-Vbeta8(+) Cbeta+ subset of cells in thymus and mesenteric lymph node. Cells in the Tcr-Vbeta8(+) Cbeta- subset of mesenteric lymph node contained germline but no rearranged bV8 transcripts. The same thymus subset expressed high levels of both rearranged and germline bV8 transcripts. The presence of germline bV8 transcripts in Tcr-alphabeta- cells in bone marrow and mesenteric lymph node suggests that germline Tcrb-V8 gene transcription is unrelated to the differentiation of mature T cells. The possible function and significance of the expression of a truncated Tcr beta chain is discussed.