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In this study, we aimed to determine the origin of the difference, in terms of anti-Müllerian hormone production, existing between the bovine and porcine ovaries. We first confirmed by quantitative real-time-Polymerase-Chain Reaction, ELISA assay and immunohistochemistry that anti-Müllerian hormone mRNA and protein production are very low in porcine ovarian growing follicles compared to bovine ones. We then have transfected porcine and bovine granulosa cells with vectors containing the luciferase gene driven by the porcine or the bovine anti-Müllerian hormone promoter. These transfection experiments showed that the porcine anti-Müllerian hormone promoter is less active and less responsive to bone morphogenetic protein stimulations than the bovine promoter in both porcine and bovine cells. Moreover, bovine but not porcine granulosa cells were responsive to bone morphogenetic protein stimulation after transfection of a plasmidic construction including a strong response element to the bone morphogenetic proteins (12 repetitions of the GCCG sequence) upstream of the luciferase reporter gene. We also showed that SMAD6, an inhibitor of the SMAD1-5-8 pathway, is strongly expressed in porcine compared to the bovine granulosa cells. Overall, these results suggest that the low expression of anti-Müllerian hormone in porcine growing follicles is due to both a lack of activity/sensitivity of the porcine anti-Müllerian hormone promoter, and to the lack of responsiveness of porcine granulosa cells to bone morphogenetic protein signaling, potentially due to an overexpression of SMAD6 compared to bovine granulosa cells. We propose that the low levels of anti-Müllerian hormone in the pig would explain the poly-ovulatory phenotype in this species.

Ankyrin proteins (ANKRD) are key mediators linking membrane and sub-membranous cytoskeletal proteins. Recent findings have highlighted a new role of ANKRD31 during spermatogenesis, elucidating its involvement in meiotic recombination and male germ cell progression. Following testicular differentiation, spermatozoa (SPZ) enter into the epididymis, where they undergo several biochemical and enzymatic changes. The epididymal epithelium is characterized by cell-to-cell junctions that are able to form the blood-epididymal barrier (BEB). This intricate epithelial structure provides the optimal microenvironment needed for epididymal sperm maturation. To date, no notions have been reported regarding a putative role of ANKRD31 in correct BEB formation. In our work, we generated an Ankrd31 knockout male mouse model (Ankrd31–/–) and characterized its reproductive phenotype. Ankrd31–/– mice were infertile and exhibited oligo-astheno-teratozoospermia (a low number of immotile SPZ with abnormal morphological features). In addition, a complete deregulation of BEB was found in Ankrd31–/–, due to cell-to-cell junction anomalies. In order to suggest that BEB deregulation may depend on Ankrd31 gene deletion, we showed the physical interaction among ANKRD31 and some epithelial junction proteins in wild-type (WT) epididymides. In conclusion, the current work shows a key role of ANKRD31 in the control of germ cell progression as well as sperm and epididymal integrity.

Eukaryote plasma membranes protect cells from chemical attack. Xenobiotics, taken up through passive diffusion, accumulate in the membranes, where they are captured by transporters, among which P-glycoproteins (Pgps). In nematodes such as Haemonchus contortus, eggshells and cuticles provide additional protective barriers against xenobiotics. Little is known about the role of these structures in the transport of chemical molecules. Pgps, members of the ABC transporter family, are present in eggshells and cuticles. Changes in the activity of these proteins have also been correlated with alterations in lipids, such as cholesterol content, in eggshells. However, the cellular mechanisms underlying these effects remain unclear. We show here that an experimental decrease in the cholesterol content of eggshells of Haemonchus contortus, with Methyl-beta-CycloDextrin (MβCD), results in an increase in membrane fluidity, favouring Pgp activity and leading to an increase in resistance to anthelmintics. This effect is modulated by the initial degree of anthelminthic resistance of the eggs. These results suggest that eggshell fluidity plays a major role in the modulation of Pgp activity. They confirm that Pgp activity is highly influenced by the local microenvironment, in particular sterols, as observed in some vertebrate models. Thus, eggshell barriers could play an active role in the transport of xenobiotics.Effets de la teneur en cholestérol sur l’activité des glycoprotéines P et sur l’état physique de la membrane, et conséquences pour la résistance aux anthelminthiques chez le nématode Haemonchus contortus.Les membranes plasmiques des eucaryotes protègent les cellules contre les attaques chimiques. Les xénobiotiques, absorbés par diffusion passive, s’accumulent dans les membranes où ils sont capturés par des transporteurs, parmi lesquels les glycoprotéines P (Pgp). Chez les nématodes, les coques des œufs et les cuticules constituent des barrières de protection supplémentaires contre les xénobiotiques. On en sait peu sur le rôle de ces structures dans le transport des molécules chimiques. Les Pgp, membres de la famille des transporteurs ABC, sont présents dans les coques et les cuticules. Des changements dans l’activité de ces protéines ont également été mis en corrélation avec des altérations des lipides, tels que la teneur en cholestérol, des coques des œufs. Cependant, les mécanismes cellulaires sous-jacents à ces effets restent flous. Nous montrons ici que la diminution expérimentale de la teneur en cholestérol des coques des œufs d’Haemonchus contortus, avec la méthyl-beta-cyclodextrine (MβCD), entraîne une augmentation de la fluidité membranaire favorisant l’activité des Pgp et une augmentation de la résistance aux anthelminthiques. Cet effet est modulé par le degré initial de résistance aux anthelminthiques des œufs. Ces résultats suggèrent que la fluidité de la coque joue un rôle majeur dans la modulation de l’activité des Pgp. Ils confirment que l’activité des Pgp est fortement influencée par le microenvironnement local, en particulier les stérols, comme observé dans certains modèles de vertébrés. Ainsi, les barrières de coques des oeufs pourraient jouer un rôle actif dans le transport des xénobiotiques.

Toll-like receptor 4 (TLR4), which recognizes lipopolysaccharide from Gram-negative bacteria, plays a major role in resistance of mice and humans to Salmonella infection. In chickens, Salmonella may establish a carrier state whereby bacteria are able to persist in the host organism without triggering clinical signs. Based on cellular morphological parameters, we developed a method, without using antibodies, to separate three cecal cell subpopulations: lymphocytes, enterocytes, and a population encompassing multiple cell types. We analyzed the mRNA expression of TLR4, interleukin-1β (IL-1β), IL-8, IL-12, and lipopolysaccharide-induced tumor necrosis factor alpha factor (LITAF) in cecal subpopulations of chicks from inbred lines resistant or susceptible to the carrier state infected with Salmonella enterica serovar Enteritidis. The results showed that resistance to the carrier state in chicks is associated with a larger percentage of lymphocytes and with higher levels of expression of TLR4 and IL-8 at homeostasis in the three cell subpopulations, as well as with a higher level of expression of LITAF in lymphocytes during the carrier state. In contrast to the early phase of infection, the carrier state is characterized by no major cell recruitment differences between infected and noninfected animals and no significant modification in terms of TLR4, IL-1β, IL-8, IL-12, and LITAF expression in all cell subpopulations measured. However, TLR4 expression increased in the lymphocytes of chicks from the susceptible line, reaching the same level as that in infected chicks from the resistant line. These observations suggest that the carrier state is characterized by a lack of immune activation and highlight the interest of working at the level of the cell population rather than that of the organ.

International audience; We have studied the plasma membrane protein phenotype of human culture-amplified and native bone marrow mesenchymal stem cells (BM MSCs). We have found, using microarrays and flow cytometry, that cultured cells express specifically 113 transcripts and 17 proteins that were not detected in hematopoietic cells. These antigens define a lineage-homogenous cell population of mesenchymal cells, clearly distinct from the hematopoietic lineages, and distinguishable from other cultured skeletal mesenchymal cells (periosteal cells and synovial fibroblasts). Among the specific membrane proteins present on cultured MSCs, 9 allowed the isolation from BM mononuclear cells of a minute population of native MSCs. The enrichment in colony-forming units–fibroblasts was low for CD49b, CD90, and CD105, but high for CD73, CD130, CD146, CD200, and integrin alphaV/beta5. In addition, the expression of CD73, CD146, and CD200 was down-regulated in differentiated cells. The new marker CD200, because of its specificity and immunomodulatory properties, deserves further in-depth studies.

International audience; Eosinophils are one of the major mammalian effector cells encountered by helminths during infection. In the present study, we investigated the effects of eosinophil granule exposure on the sheep parasitic nematode Haemonchus contortus as a model. H. contortus eggs exposed to eosinophil granule products showed increased rhodamine 123 efflux and this effect was not due to loss of egg integrity. Rh123 is known to be a specific P-glycoprotein (Pgp) substrate and led to the hypothesis that in addition to their critical role in xenobiotic resistance, helminth ABC transporters such as Pgp may also be involved in the detoxification of host cytotoxic products. We showed by quantitative RT-PCR that, among nine different H. contortus Pgp genes, Hco-pgp-3, Hco-pgp-9.2, Hco-pgp-11 and, Hco-pgp-16 were specifically up-regulated in parasitic life stages suggesting a potential involvement of these Pgps in the detoxification of eosinophil granule products. Using exsheathed L3 larvae that mimic the first life stage in contact with the host, we demonstrated that eosinophil granules induced a dose dependent overexpression of Hco-pgp-3 and the closely related Hco-pgp-16. Taken together, our results provide the first evidence that a subset of helminth Pgps interact with, and could be involved in the detoxification of, host products. This opens the way for further studies aiming to explore the role of helminth Pgps in the host-parasite interaction, including evasion of the host immune response.

Marek’s disease virus (MDV) is an alpha-herpesvirus causing Marek’s disease in chickens, mostly associated with T-cell lymphoma. VP22 is a tegument protein abundantly expressed in cells during the lytic cycle, which is essential for MDV spread in culture. Our aim was to generate a pathogenic MDV expressing a green fluorescent protein (EGFP) fused to the N-terminus of VP22 to better decipher the role of VP22 in vivo and monitor MDV morphogenesis in tumors cells. In culture, rRB-1B EGFP22 led to 1.6-fold smaller plaques than the parental virus. In chickens, the rRB-1B EGFP22 virus was impaired in its ability to induce lymphoma and to spread in contact birds. The MDV genome copy number in blood and feathers during the time course of infection indicated that rRB-1B EGFP22 reached its two major target cells, but had a growth defect in these two tissues. Therefore, the integrity of VP22 is critical for an efficient replication in vivo, for tumor formation and horizontal transmission. An examination of EGFP fluorescence in rRB-1B EGFP22-induced tumors showed that about 0.1% of the cells were in lytic phase. EGFP-positive tumor cells were selected by cytometry and analyzed for MDV morphogenesis by transmission electron microscopy. Only few particles were present per cell, and all types of virions (except mature enveloped virions) were detected unequivocally inside tumor lymphoid cells. These results indicate that MDV morphogenesis in tumor cells is more similar to the morphorgenesis in fibroblastic cells in culture, albeit poorly efficient, than in feather follicle epithelial cells.

International audience; Cryptosporidium parvum is a zoonotic protozoan parasite found worldwide, that develops only in the gastrointestinal epithelium and causes profuse diarrhea. Using a mouse model of C. parvum infection, we demonstrated by conditional depletion of CD11c+ cells that these cells are essential for the control of the infection both in neonates and adults. Neonates are highly susceptible to C. parvum but the infection is self-limited, whereas adults are resistant unless immunocompromised. We investigated the contribution of DC to the age-dependent susceptibility to infection. We found that neonates presented a marked deficit in intestinal CD103+ DC during the first weeks of life, before weaning, due to weak production of chemokines by neonatal intestinal epithelial cells (IEC). Increasing the number of intestinal CD103+ DC in neonates by administering FLT3-L significantly reduced susceptibility to the infection. During infections in neonates, the clearance of the parasite was preceded by a rapid recruitment of CD103+ DC mediated by CXCR3-binding chemokines produced by IEC in response to IFN gamma. In addition to this key role in CD103+ DC recruitment, IFN gamma is known to inhibit intracellular parasite development. We demonstrated that during neonatal infection CD103+ DC produce IL-12 and IFN gamma in the lamina propria and the draining lymph nodes. Thus, CD103+ DC are key players in the innate immune control of C. parvum infection in the intestinal epithelium. The relative paucity of CD103+ DC in the neonatal intestine contributes to the high susceptibility to intestinal infection.

International audience; Dendritic cells (DCs) are pivotal in the development of specific T-cell responses to control pathogens, as they govern both the initiation and the polarization of adaptive immunity. To investigate the capacities of migrating DCs to respond to pathogens, we used physiologically generated lymph DCs (L-DCs). The flexible polarization of L-DCs was analysed in response to Salmonella or helminth secretions known to induce different T cell responses. Mature conventional CD1b(+) L-DCs showed a predisposition to promote pro-inflammatory (IL-6), pro-Th1 (IL-12p40) and anti-inflammatory (IL-10) responses which were amplified by Salmonella, and limited to only IL-6 induction by helminth secretions. The other major population of L-DCs did not express the CD1b molecule and displayed phenotypic features of immaturity compared to CD1b(+) L-DCs. Salmonella infection reduced the constitutive expression of TNF-alpha and IL-4 mRNA in CD1b(-) L-DCs, whereas this expression was not affected by helminth secretions. The cytokine response of T cells promoted by L-DCs was analysed in T cell subsets after co-culture with Salmonella or helminth secretion-driven CD1b(+) or CD1b(-) L-DCs. T cells preferentially expressed the IL-17 gene, and to a lesser extent the IFN-gamma and IL-10 genes, in response to Salmonella-driven CD1b(+) L-DCs, whereas a preferential IL-10, IFN-gamma and IL-17 gene expression was observed in response to Salmonella-driven CD1b(-) L-DCs. In contrast, a predominant IL-4 and IL-13 gene expression by CD4(+) and CD8(+) T cells was observed after stimulation of CD1b(+) and CD1b(-) L-DCs with helminth secretions. These results show that mature conventional CD1b(+) L-DCs maintain a flexible capacity to respond differently to pathogens, that the predisposition of CD1b(-) L-DCs to promote a Th2 response can be oriented towards other Th responses, and finally that the modulation of migrating L-DCs responses is controlled more by the pathogen encountered than the L-DC subsets.

In nematodes as in other eukaryotes, there is increasing evidence that drug resistance depends on both changes in the drug cellular targets and in nonspecific mechanisms, involving cellular detoxification by efflux pumps. In vertebrates, P-glycoproteins (Pgp) are membrane efflux pumps responsible for the elimination of xenobiotic agents, especially drugs. We previously reported the presence of Pgp pumps in eggshells and cuticles of the nematode Haemonchus contortus. Eggshells and cuticles are different from cell membranes, in particular they include a chitin layer. Nevertheless these structures present some common biological features with cell membranes and play a role in xenobiotic transport. Pgp activity has been shown to depend on the lipid environment and, in particular, on the cholesterol content in both vertebrate and nematode models. In vertebrates, Pgp is in part located in membrane cholesterol-enriched microdomains, the rafts. We describe here, for the first time, lipid microdomains in eggshells that could correspond with raft-like structures (RLSs). Moreover, a large proportion of the Pgp was shown to colocalize with these RLSs. The functional consequences of the colocalization for xenobiotic transport and thus drug resistance in nematodes were analyzed and compared with results obtained in vertebrates. An understanding of such mechanisms is crucial in overcoming the failure of drug treatments due to the development of resistance.

Natural killer (NK) cells have a key role in the innate immune response against pathogens because of their cytotoxic properties and production of interferon-gamma (IFN-gamma). Some insight into ruminant NK cell biology has been gained through the characterization of bovine NK cells as NKp46(+)/CD3(-) cells. However, ovine NK cells have been little studied because of the lack of specific antibodies. Most NK cells in humans and cattle express CD16. We found that an antibody against human CD16 that cross-reacts with bovine NK cells also recognizes cell populations in ovine peripheral blood mononuclear cells. Using double labelling with CD14 revealed the same profile as described in other species, and we identified a putative NK cell population. We therefore sorted this ovine CD16(+)/CD14(-) cell population and tested it for NK cell characteristics. More than 80% of sorted CD16(+)/CD14(-) cells expressed perforin. After a week of culture in the presence of IL-2 and IL-15, ovine CD16(+)/CD14(-) cells had become large cells with intra-cytoplasmic granules containing perforin, and the vast majority displayed an activated CD2(-/low)/CD25(+)/CD8(+) phenotype, as observed for bovine NKp46(+)/CD3(-) cells. Moreover, these cells expressed transcripts for the NKp46 receptor, and were cytotoxic in a CD16-mediated redirected lysis assay against a murine cell line, P815, and in a direct lysis assay against the ovine cell line, IDO5. Finally, ovine CD16(+)/CD14(-) cells having expanded for 7 days in culture secreted IFN-gamma in response to IL-12 in a dose-dependent manner. Taken together, these findings led us to conclude that the ovine CD16(+)/CD14(-) lymphocyte sub-population displays the phenotype and functional characteristics of NK cells.

International audience; Orientation of the immune response toward Th1, Th2, Th17 or Treg plays all important role in self-tolerance and defence against pathogens and tumors. However, this orientation has not been fully characterised in the pig and little is known about the influence of maturation stimulus Oil the capacity of dendritic cells selectively to direct different types of Th cell responses. Dendritic cell (DC) Maturation Call be induced by different agents such as inflammatory cytokines, TLR ligands and CD40L. However, the role of the latter in the maturation of pig DC has never been reported. In this study we analysed how different maturation agents influence the capacity of DC to skew the immune response. Monocyte-derived porcine DCs were matured with human CD40L-transfected L-cells, Lipopolysaccharide (LIPS) alone or LIPS in combination with Tumor necrosis factor-alpha (TNF alpha) and interferon-alpha (IFN alpha). We found that human CD40L induced DC maturation characterised by increased expression of co-stimulatory CD80/86 molecules, high production of IL-12p40 in DC and induction of IFN-gamma and t-bet mRNA in T cells, suggesting a Th I orientation. Moreover we report for the first time the appearance of CD25 after activation of porcine DC. Futhermore, DC activated with TNF + LPS + IFN showed the highest allo-stimulatory capacity of allogeneic lymphocytes and induced IL-17 mRNA in T lymphocytes, Suggesting a Th17 orientation that has never been previously reported in the pig. We also showed that immature DCs did not produce any IL-10 or IL-12 and induced both GATA-3 and IL-13 transcription in allogeneic MLR Suggesting a Th2 orientation. This study therefore underlines that the nature of the stimulus Strongly influences the capacity of DC to steer the immune response in the pig. (C) 2008 Elsevier Ltd. All rights reserved.

Brucella,the causative agent of brucellosis in animals and humans, can survive and proliferate within macrophages. Macrophages mediate mouse resistance to various pathogens through the expression of theNramp1gene. The role of this gene in the control ofBrucellainfection was investigated. When BALB/c mice (Nramp1s) and C.CB congenic mice (Nramp1r) were infected withBrucella melitensis,the number ofBrucellaorganisms per spleen was significantly larger in the C.CB mice than in the BALB/c mice during the first week postinfection (p.i.). ThisNramp1-linked susceptibility toBrucellawas temporary, since similar numbers ofBrucellawere recovered from the two strains of mice 2 weeks p.i. The effect ofNramp1expression occurred within splenocytes intracellularly infected byBrucella.However, there was no difference between in vitro replication rates ofBrucellain macrophages isolated from the two strains of mice infected in vivo or inNramp1RAW264 transfectants. In mice, infection withBrucellainduced an inflammatory response, resulting in splenomegaly and recruitment of phagocytes in the spleen, which was amplified in C.CB mice. Reverse transcription-PCR (RT-PCR), performed 5 days p.i., showed that inducible nitric oxide synthase, tumor necrosis factor alpha (TNF-α), interleukin-12 p40 (IL-12p40), gamma interferon (IFN-γ), and IL-10 mRNAs were similarly induced in spleens of the two strains. In contrast, the mRNA of KC, a C-X-C chemokine, was induced only in infected C.CB mice at this time. This pattern of mRNA expression was maintained at 14 days p.i., with IFN-γ and IL-12p40 mRNAs being more intensively induced in the infected C.CB mice, but TNF-α mRNA was no longer induced. The higher recruitment of neutrophils observed in the spleens of infected C.CB mice could explain the temporary susceptibility of C.CB mice toB. melitensisinfection. In contrast to infections withSalmonella, Leishmania,andMycobacterium,the expression of theNramp1gene appears to be of limited importance for the natural resistance of mice toBrucella.

Objectives: The efficiency of the anthelmintics used to treat small domestic ruminants infected with nema- todes is compromised by the emergence of resistant parasites. Both specific and non-specific mechanisms of resistance exist. The non-specific mechanisms involve multiple resistance phenomena and are depend- ent on the multidrug resistance (MDR) system, which is also responsible for the development of chemo- therapy-resistant tumour cells. We showed previously that the system also exists in nematodes. Membrane 'pumps', known as P-glycoproteins (Pgp), are activated in the MDR system. The nature of the membrane, in particular the lipids, appears to condition the activity of the pumps. Thus, we studied the effects of choles- terol on drug transport activity in the nematode Haemonchus contortus. Materials and methods: We used methyl-β-cyclodextrin to carry out cholesterol depletion and cholesterol loading experiments. The resulting changes in resistance were estimated by measuring changes in drug transport (a) by means of in vitro egg hatch assays in the presence of a benzimidazole anthelmintic, thia- bendazole and (b) by measuring the transport of rhodamine 123 (R123), a specific substrate of Pgp. We used biochemical assays to estimate the cholesterol concentration in the parasites. Results: Changes in the cholesterol content induced changes in anthelmintic resistance; cholesterol depletion gave increased resistance and cholesterol loading gave decreased resistance. These changes also altered the transport of R123. Conclusion: Cholesterol depletion or cholesterol loading allow modulation of xenobiotic resistance in nematode eggs as they do in tumour cells. The effect appears to be correlated with changes in the function of membrane P-glycoproteins. The lipid environment thus influences the nematode Pgp activity.

We investigated the phenotype of the T cells (CD4 + and CD8 + ) that produced Th1 (IFN-γ ) and Th2 cytokines (IL-4 and IL-10) during the first two weeks of experimental fasciolosis in rats. We also followed the kinetics of the cytokine and proliferative responses of hepatic mononuclear cells (HMNC) over the same period. We found that HMNC were more numerous in the infected ani- mals than in the controls. The percentage of CD4 + cells increased significantly after infection, whereas the percentage of CD8 + cells did not change. Moreover, the frequency of the cells producing (CP) cytokine changed after infection. The frequency of CP IFN-γ on 7 days postinfection (pi) was similar to that in control animals. However, the frequency of CP IFN-γ was clearly lower on day 14 pi, whereas the frequency of CP IL-4 and CP IL-10 had increased. The CP IL-10-were mostly CD4 + . Mitogenic stimulation (phorbol myristate acetate/ionomycin) of HMNC led to an increase in the amounts of the Th2 cytokines in the supernatant on days 7 and 14 pi, with the increase more pro- nounced on day 14. In contrast, IFN-γ levels also increased by day 7 pi but then decreased to below control levels by day 14. In addition, HMNC proliferation in response to mitogen followed a similar pattern to IFN-γ production. These findings suggested that, during the first 2 weeks of infection, F. hepatica induced a transient Th0 cytokine profile followed by downregulation of the cellular re- sponse and the induction of a Th2 cytokine profile. Fasciola hepatica / rat / cytokine / liver / lymphocytes

NO is produced by macrophages through activation of the inducible enzyme NOS and its production is triggered as an antiviral and antitumoral immune mechanism. Replication of Marek's disease herpes virus (MDV) is inhibited by NO in vitro. MDV induces T-lymphomas in the chicken and a genetic resistance to tumor development has been linked to the B21 major histocompatibility complex. During the first initial week of viral replication after inoculation of the highly virulent RB-1B MDV strain, histocompatible B21/B21 chickens developed strong iNOS expression and NO production capacity in the spleen, in parallel with strong systemic NO production in the serum. Comparable NO response was not seen with the vaccinal strain HVT. In contrast, reduction in spleen macrophage number and delay in iNOS gene expression was observed in genetically susceptible B13/B13 chickens after MDV infection, in addition to suppression of IFN-gamma-inducible NO production. However, vaccination with HVT 3 days before RB-1B inoculation restored strong iNOS gene expression in the spleen 1 week later and inducible NO production 3 weeks later. Following the pattern of iNOS gene expression, early strong expression of cytokines with powerful iNOS-inducing activity such as IFN-gamma and CC chemokines from the MIP family (MIP-1beta, K203) was observed in genetic resistance and resistance acquired after vaccination with HVT. In conclusion, resistance to MDV appeared preferentially linked in both types of resistance to the early establishment of cytokine induction characteristic of a Th1 immune response, thus favoring the development of an early and strong NO response.

Primary cultures of Sertoli cells provide an interesting model to study how signalling pathways induced by a single hormone in a single cell type evolve, depending on the developmental stage. In vivo, follicle-stimulating hormone (FSH) induces proliferation of Sertoli cells in neonate and controls the subsequent differentiation of the entire population. Molecular mechanisms underlying Sertoli cell pleiotropic responses to FSH have long been investigated. But to date, only cAMP-dependent kinase (PKA) activation has been reported to account for most FSH biological activities in male. Here, we demonstrate that FSH activates the ERK MAP kinase pathway following dual coupling of the FSH-R both to Gs and to Gi heterotrimeric proteins, in a PKA- and also Src-dependent manner. This activation is required for FSH-induced proliferation of Sertoli cells isolated 5 days after birth. Consistently, we show that the ERK-mediated FSH mitogenic effect triggers upregulation of cyclin D1. In sharp contrast, at 19 days after birth, as cells proceed through their differentiation program, the ERK pathway is dramatically inhibited by FSH treatment. Taken together, these results show that FSH can exert opposite effects on the ERK signalling cascade during the maturation process of Sertoli cells. Thus, signalling modules triggered by the FSH-R evolve dynamically throughout development of FSH natural target cells.

This study was designed to identify an extra-intestinal route of migration of Eimeria coecicola sporozoites and the types of cell harbouring the parasite during the invasion of the intestine. The presence of E. coecicola in blood, spleen and mesenteric lymph nodes of infected donor rabbits was demonstrated by immunohistology on donor organs and measurement of oocyst excretion by coccidia-free recipient rabbits injected with whole-cell suspensions prepared from donor tissues. Two types of donor lymphocyte, B (IgM+) and T (CD5+), were labelled using a two-colour immunofluorescence-labelling technique and separated with a cell-sorter (FACStar(Plus)). The presence of parasites in the sorted cells was assessed by direct examination and by using the same in vivo test after intravenous injection of IgM+ B or CD5+ T lymphocytes collected from donors at different times after inoculation. This test provided evidence that the parasites were alive and still infectious within the sorted lymphocytes. It was demonstrated that both B and T lymphocytes were infected.

The role of membrane drug-transport mechanisms in resistance to anthelmintics was examined using a flow cytometry method. This method was adapted from assays developed for the study of similar mechanisms in tumor cells. Rhodamine 123, a P-glycoprotein transport probe, associated with the reversal agent verapamil gave a significantly higher level of green fluorescence in Haemonchus contortus-resistant eggs as compared with that of susceptible eggs. In the same way, verapamil-bodipy, a new fluorescent probe for the detection of multidrug resistance in cells, showed a significantly higher degree of binding to resistant eggs. The results confirm those obtained with biological drug assays using both anthelmintics and verapamil and provide a quantitative and effective methodology for the functional study of multidrug resistance in nematodes.

The protease hypodermin A (HA) is produced by the parasitic warble-fly larva and is implicated in the modulation of the bovine immune system. This study examines the effect of this enzyme on the cell surface markers of bovine lymphocytes. HA interfered with the binding of all anti-lymphocyte receptor antibodies tested. Anti-BoCD2 and CD5 staining was completely abolished. But the mean fluorescence intensity (MFI) only was diminished for antibodies against BoCD4, CD8 and CD18. On the contrary, the MFI for anti-MHC Cl I molecules staining was increased. This effect of HA began as early as one h, and was reversed by removal of HA. Heating or PMSF treatment, which both inhibit protease activity, abolished the action of HA on the surface antigens. The HA concentrations (100 micrograms/ml) needed to alter antibody binding were similar to those that inhibited phytohaemagglutinin (PHA)-induced proliferation. These results show that enzymatic activity of HA on lymphocyte surface markers may be implicated in the inhibition of lymphocyte proliferation.

International audience; Dendritic cells (DCs) are well known as professional antigen-presenting cells (APC) able to initiate specific T-cell responses to pathogens in lymph nodes (LN) draining the site of infection. However, the respective contribution of migratory and LNresident DCs in this process remains unclear. As DC subsets represent important targets for vaccination strategies, more precise knowledge of DC subsets able to present vaccine antigens to T cells efficiently is required. To investigate the capacities of DCs migrating in the lymph (L-DCs) to initiate a specific T-cell response, we used physiologically generated DCs collected from a pseudoafferent lymphatic cannulation model in sheep. The CD1b+ L-DCs were assessed for presenting antigens from the vaccine attenuated strain of Salmonella enterica serovar Abortusovis. CD1b+ L-DCs were able to phagocytose, process and to present efficiently Salmonella antigens to effector/memory T cells in vitro. They were shown to be efficient APC for the priming of allogeneic naive T cells associated with inducing both IFN-c and IL-4 responses. They were also efficient in presenting Salmonella antigens to autologous naive T cells associated with inducing both IFN-c and IL-10 responses. The capacities of L-DCs to process and present Salmonella antigens to T cells were investigated in vivo after conjunctival inoculation of Salmonella. The CD1b+ L-DCs collected after inoculation were able to induce the proliferative response of CD4+ T cells suggesting the in vivo capture of Salmonella antigens by the CD1b+ L-DCs, and their potential to present them directly to CD4+ T cells. In this study, CD1b+ L-DCs present potential characteristics of APC to initiate by themselves T cell priming in the LN. They could be used as target cells for driving immune activation in vaccinal strategies.

Background Telomerase activation, a critical step in cell immortalization and oncogenesis, is partly regulated by alternative splicing. In this study, we aimed to use the Marek's disease virus (MDV) T-cell lymphoma model to evaluate TERT regulation by splicing during lymphomagenesis in vivo, from the start point to tumor establishment. Results We first screened cDNA libraries from the chicken MDV lymphoma-derived MSB-1 T- cell line, which we compared with B (DT40) and hepatocyte (LMH) cell lines. The chTERT splicing pattern was cell line-specific, despite similar high levels of telomerase activity. We identified 27 alternative transcripts of chicken TERT (chTERT). Five were in-frame alternative transcripts without in vitro telomerase activity in the presence of viral or chicken telomerase RNA (vTR or chTR), unlike the full-length transcript. Nineteen of the 22 transcripts with a premature termination codon (PTC) harbored a PTC more than 50 nucleotides upstream from the 3' splice junction, and were therefore predicted targets for nonsense-mediated decay (NMD). The major PTC-containing alternatively spliced form identified in MSB1 (ie10) was targeted to the NMD pathway, as demonstrated by UPF1 silencing. We then studied three splicing events separately, and the balance between in-frame alternative splice variants (d5f and d10f) plus the NMD target i10ec and constitutively spliced chTERT transcripts during lymphomagenesis induced by MDV indicated that basal telomerase activity in normal T cells was associated with a high proportion of in-frame non functional isoforms and a low proportion of constitutively spliced chTERT. Telomerase upregulation depended on an increase in active constitutively spliced chTERT levels and coincided with a switch in alternative splicing from an in-frame variant to NMD-targeted variants. Conclusions TERT regulation by splicing plays a key role in telomerase upregulation during lymphomagenesis, through the sophisticated control of constitutive and alternative splicing. Using the MDV T-cell lymphoma model, we identified a chTERT splice variant as a new NMD target.