Simplified method to generate large quantities of dendritic cells suitable for clinical applications.

The present study describes the optimization of an in vitro culture method for generating large amounts of dendritic cells (DC) in serum-free conditions from leukapheresis containing a mixed population of peripheral blood mononuclear cells (PBMC) which are cultured in the presence of GM-CSF and IL-13. Initial comparisons between the generation of DC from bulk and monocyte-enriched leukapheresis products showed that the presence of lymphocytes during the culture favors the differentiation of monocytes into DC. DC yields obtained from mixed mononuclear cell cultures were between 38 and 54% higher than yields obtained from monocyte-enriched cultures. Both types of cultures resulted in the generation of DC with an immature phenotype (CD83- and high phagocytic activity), which have been previously shown to be good stimulators for T cell responses. DC yields of bulk cultures in serum-free conditions were significantly higher than those obtained in the presence of 2% human serum. The cytokines of the supernatants of serum-free cultures comprised a significant content of pro-inflammatory cytokines such as IL-1, IL-12 and TNF-alpha. Maturation of DC generated by this method can be induced by treatment with double-stranded RNA, LPS or TNF-alpha, resulting in enhanced surface expression of CD80, CD86, CD40, CD83 and MHC molecules on the DC. The methodology described here offers the possibility for generating large amounts of clinical grade DC from bulk leukapheresis products, thus avoiding DC precursor purification steps, and thereby minimizing the risks of contamination. This culture process may be applied to cell-based therapeutic approaches for the treatment of cancer or chronic viral infections.

Monocyte-derived dendritic cells: development of a cellular processor for clinical applications.

Since dendritic cells (DCs) are the most professional antigen-presenting cells, (Schuler et al., 1997), increasing interest in their use in clinical approaches has been observed. (Nestle et al., 1998; Murphy G. et al., 1996). We have developed an ex vivo standardized process for the generation of dendritic-like cells (MAC-DCs) from human blood circulating monocytes. Human monocytes can differentiate into very different functional cells according to the conditions of culture, media and cytokines used. In the present study, we demonstrate that both pure monocytes and mononuclear cells differentiate into DCs when they are grown in defined medium AIM-V in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) plus IL13 and in approved biocompatible non-adherent bags. Quality and functional controls of the immature DCs obtained rely on bacterial sterility, viability, morphology and recovery. The MAC-DCs also present an immature DC phenotype with a low expression of CD14 and CD64, and high expression of MHC-I, MHC-II and CD40. They also express B7 costimulatory molecules (CD80, CD86), CD83, and CD1a molecules. They induce strong allogenic T-cell proliferation (mixed lymphocyte reaction as well as proliferation of autologous memory T lymphocytes when incubated in the presence of recall antigens (tuberculosis, Candida albicans, and tetanus toxoid). They also show an increase in phagocytic uptake of yeast, tumour cells and debris. The global closed system which, under reproducible good medical practice (GMP) conditions, enables the production of dendritic cells of clinical quality, has been optimized ("Vac Cell Processor"). It contains all bags, connections, media, reagents, washing solutions, control antibodies, standard operating procedures, data management, traceability and help in the form of dedicated software.

Development and evolutionary aspects of thymic T cell education to neuroendocrine self.

Thymic epithelial cells, including nurse cells (TECs/TNCs), from various species synthesize neuroendocrine-related precursors belonging to neurohypophysial, tachykinin and insulin hormone families. The thymic repertoire of neuroendocrine-related polypeptides illustrates at the molecular level the paradoxical role of the thymus in both T cell positive and negative selection. On the one hand, these precursors are a source of signals which interact with neuroendocrine-type receptors expressed by target pre-T cells according to the cryptocrine type of cell-to-cell signaling. On the other hand, the same precursors constitute a source of self-antigens which are presented to pre-T cells by the thymic major histocompatibility complex system. Basically, the model of thymic T cell education to neuroendocrine self was established by the identification in TECs/TNCs of immunoreactive (ir) oxytocin as the self-antigen of the neurohypophysial family. Nevertheless, through the expression in TECs/TNCs of ir-neurokinin A and ir-insulin-like growth factor-II, the model also applies to the tachykinin and insulin hormone families.

Cryptocrine signaling in the thymus network and T cell education to neuroendocrine self-antigens.

Both during phylogeny and ontogeny the thymus appears as a nodal point between the two major systems of cell-to-cell signaling, the neuroendocrine and immune systems. This review presents the experimental observations which support a dual role in T cell selection played by the thymic repertoire of neuroendocrine polypeptide precursors. Through the mode of cryptocrine intercellular signaling thymic neuroendocrine-related precursors synthesized in thymic epithelial cells have been shown to influence the early steps in T cell differentiation. In addition, thymic neuroendocrine-related polypeptides are a source of self-antigens which are presented by the major histocompatibility system of the thymic epithelium. Preliminary data also suggest that the intrathymic T cell education to neuroendocrine self-antigens is not strictly superimposible to the antigen presentation by dedicated presenting cells. Insulin-like growth factor-II (IGF-II) was identified as one dominant member of the insulin family expressed by thymic epithelial and nurse cells. The intrathymic presentation of IGF-II or IGF-II derived self-antigens is under current investigation. If further confirmed, the central tolerogenic properties of IGF-II could be considered in the elaboration of a strategy for an efficient and safe prevention of insulin-dependent diabetes.

RGD-mediated adhesion of porcine granulosa cells modulates their differentiation response to FSH in vitro.

Porcine granulosa cells cultured in serum-free medium undergo metabolic and morphologic changes after follicle stimulating hormone (FSH) stimulation. Under these conditions, granulosa cells differentiate and tend to round up and their links with the plastic support are reduced. Coating of culture substratum with PepTite-2000, an integrin-binding synthetic peptide containing RGD (Arg-Gly-Asp) sequences enhanced the plating of granulosa cells. Whether the peptide be present or not, cells cultivated in basal synthetic medium (without FSH) were flattened and attached to the substratum by stress fibers at focal contacts where integrin beta 1, extracellular fibronectin, and urokinase plasminogen activator colocalized. After FSH stimulation, part of the cells rounded up and F-actin took a more uniform, cortical localization. Correlatively, extracellular fibronectin aggregated in a clump, while integrin beta 1 and urokinase plasminogen activator spread over rounded cells. These morphological changes elicited by FSH were little affected by the presence of PepTite-2000, yet a larger number of cells remained flattened. However, concerning steroidogenesis, increasing concentrations of peptide seemed to favor progesterone rather than estrogen production, and to restrain luteinizing hormone (LH) receptor expression, suggesting a premature commitment of cells towards luteinization rather than completion of follicular preovulatory differentiation.

Suppression of fertility in male mice by immunization against LH receptor.

We have investigated the potential contraceptive effects of immunization against the luteinizing hormone (LH) receptor in male mice at the prepubertal stage. Two N-terminal fragments of the porcine LH receptor encoding amino acids 1-297 and 1-370 were produced in large quantities through the Baculovirus insect cell system. We have immunized three-week-old mice from two Balb/c stocks of differing fecundity with Sf9 insect cells producing the short (1-297) or long (1-370) recombinant LH receptor. A booster injection was performed at six weeks using purified antigens. Ten days later, the immunized male mice were mated over a period of two weeks with adult untreated females. After weaning of the first litters, the same partners were mated once again under the same conditions. There was no decrease in the antiserum titers against the antigens over a two-month period. The circulating testosterone decreased as the anti-LH receptor antibodies increased. The fertility of the treated male mice was reduced up to 75%, depending on the mouse stock, the antigen used and the time separating immunization and mating. The impaired fertility was mostly due to male sterilization (up to 60% of sterile mates). The delay between mating and birth was enhanced by the treatment, reflecting delayed fertility and/or delayed male behaviour acquisition.

Ontogeny of gonadal luteinizing hormone and follicle-stimulating hormone receptors in the fetal pig and related changes in gonadotropin and testosterone secretion.

Fetuses of Large White and Meishan sows were collected at 28, 35, 49, 56, 75, 90, 103, and 113 days of gestation. LH, FSH, and testosterone concentrations were measured either in amniotic fluid (Days 28-56) or in arterial umbilical blood (Days 75-113). Gonads were analyzed for their content of LH and FSH receptors and RNA transcripts. Most of these parameters were similar in the two breeds, except that the mean testosterone concentration was higher (p < 0.05) in the plasma of 75-113-day Meishan fetuses (309 and 136 pg/ml in males and females, respectively) than in Large White fetuses (152 and 109 pg/ml). Higher testosterone concentrations were detected in males than in females, either in amniotic fluid (114 vs. 81 pg/ml, p < 0.05) or in plasma (230 vs. 122 pg/ml, p < 0.01). In contrast, higher gonadotropin concentrations were found in the plasma of females than in that of males (2 vs. 1.6 ng/ml LH and 4.2 vs. 1.4 ng/ml FSH, p < 0.05). In the testis, 2-3 pmol/g gonad of LH and FSH receptors were detected as early as Day 28. This value increased to 10-13 pmol/g between 35 and 56 days before declining to 3-5 pmol/g after Day 90. In ovaries, LH receptors were detected in the earlier period (Days 28-56) at 1-3 pmol/g before diminishing to 0.1-0.4 pmol/g. FSH receptors were higher at Day 28 (1 pmol/g) than at any subsequent stage (0.1-0.4 pmol/g). The gonadal content of RNA transcripts was significantly higher in testes than in ovaries.(ABSTRACT TRUNCATED AT 250 WORDS)

Reconstitution of a high-affinity functional lutropin receptor by coexpression of its extracellular and membrane domains.

The glycoprotein hormone receptors differ from other G protein-coupled receptors by their large extracellular domain which mediates ligand binding. Cooperation between the G-protein coupled membrane domain, the extracellular domain and the hormone in establishing high-affinity binding and efficient transduction is likely to exist. Expression plasmids encoding the full-length porcine LH-hCG receptor (1-696), its extracellular (1-297) and membrane domain (298-696), as well as the alpha and beta subunits of hCG were constructed. We report that coexpression in COS cells of the two LH-hCG receptor domains restores cell surface high-affinity hormone binding and hormone dependent adenylyl cyclase activation, suggesting sufficient interactions between the two receptor domains to reconstitute a complete functional molecule. Moreover, the two hormone subunits and the two receptor domains are able to associate within coexpressing COS cells into an active complex.

LH receptor RNA and protein levels after hormonal treatment of porcine granulosa cells in primary culture.

Granulosa cells were prepared from small follicles (less than 3 mm) from the ovaries of 5-month-old gilts. They were cultured in plastic dishes coated with a synthetic adhesion peptide in a chemically defined medium supplemented with 2% serum substitute. After 3 days of culture, the cells reached confluence and expression of the LH receptor could be stimulated in a hormonally defined medium. LH receptor RNAs were estimated by autoradiography using Northern blots and dot blots of total cell RNA. LH receptor RNAs were hybridized with a homologous 32P-labelled random-primed DNA probe. The LH receptor was measured using 125I-labelled human chorionic gonadotrophin (hCG) as tracer. Northern blots of LH receptor RNAs revealed a predominant signal of 4.4 kb and two less-intense hybridization bands of 7.5 and 1.9 kb. The 4.4 kb band was used for quantification of LH receptor RNAs because it was the most intense and may be attributed to the full-length messenger RNA. In these conditions, after 72 h stimulation, FSH (0.6 nM), insulin (5 micrograms/ml), oestradiol (30 nM) and deoxycorticosterone (0.3 nM) yielded high LH receptor RNA levels (eight times unstimulated cell level), while dibutyryl cyclic AMP (1 mM), cortisol (5.4 nM), thyroxine (100 nM) and epidermal growth factor (16 pM) gave low LH receptor RNA levels (one to five times). However, the respective amounts of the receptor RNA did not give yield to the same proportion of LH receptor for every factor, indicating some post-transcriptional regulations. The kinetic study of the production of the LH receptor obtained in a defined medium supplemented with FSH, oestradiol and insulin showed that the receptor appeared after 48 h of stimulation and reached a maximum of about 7000 receptors per cell at 72 h. The three hybridization bands on Northern blots evolved in parallel and appeared as early as 24 h. They were at maximal level from 24 to 48 h of stimulation. When the granulosa cells were pulse-treated for 2 h with cycloheximide (10 micrograms/ml), they exhibited a transient rise in LH receptor RNA content which was followed by a delayed receptor increase especially at 72 h of stimulation. Taken together, these results indicate that the LH receptor in primary culture of granulosa cells seems to be regulated by different physiological factors both at the transcriptional and the translational levels.

High titers of ecdysteroids are associated with the secretory process of embryonic envelopes in the european lobster.

The newly laid egg of the lobster Homarus gammarus is surrounded by a vitelline coat. Just after fertilization, a new subjacent envelope (2), originating from the cortical reaction, is deposited beneath the vitelline coat. In the course of embryonic development, five new coatings (envelopes 3 to 7) are secreted successively from the ectodermal embryonic cells. These will remain until hatching, freeing the mysis larva in concentric order without exuviation. The concentration of both the two major ecdysteroids (ponasterone A and 20-hydroxyecdysone) and their respective precursors (25-deoxyecdysone and ecdysone) were determined as a function of the secretory phase for three embryonic envelopes (2, 3 and 6). We determined that the secretory processes proceed in the presence of high titers of 20-hydroxyecdysone during the onset of envelope secretion and of ponasterone A in the last phase of secretion.