Alternative splicing and nonsense-mediated decay regulate telomerase reverse transcriptase (TERT) expression during virus-induced lymphomagenesis in vivo

<p>Abstract</p> <p>Background</p> <p>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 <it>in vivo</it>, from the start point to tumor establishment.</p> <p>Results</p> <p>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 <it>in vitro </it>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.</p> <p>Conclusions</p> <p>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.</p

Hypoosmotic swelling test: a new approach by multi-hypotonic steps.

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Evaluation of the hepatic NK cell response during the early phase of Fasciola hepatica infection in rats

The in situ distribution of NK cells in rat liver during the first 28 days of an experimental infection with F. hepatica was investigated. NK cells were distributed homogeneously throughout the hepatic parenchyma in uninfected animals. The total number of hepatic mononuclear cells increased significantly following infection, but the proportion of NK cells did not change. After infection, these cells were found around the portal space, around the centrolobular vein, in the periportal fibrosis and in the band of collagen. However, no NK cells could be detected in or around the granuloma during infection. The frequency of both IL-2- and IFN$\gamma$-producing NK cells was higher on day 7 postinfection (pi) but only the percentage of IFN$\gamma$ $^+$CD161$^+$ subsets remained elevated thereafter, whereas the percentage of both IL-2$^+$CD161$^+$ and IL-4$^+$CD161$^+$ subsets returned to the baseline. The number of CD161$^+$IL10$^+$ cells did not change significantly. These results suggest that NK cells could be another source for the early production of IFN$\gamma$ but provide no evidence that these cells are involved in early events associated with granuloma formation.Évaluation de la réponse des cellules NK hépatiques pendant la phase précoce d'infestation par Fasciola hepatica chez le rat. Nous avons suivi la localisation et la distribution hépatique des cellules NK durant les 28 premiers jours d'une fasciolose expérimentale chez le rat. Chez les animaux non infestés, les cellules NK sont distribuées de façon homogène dans tout le foie. Chez les animaux infestés, ces cellules sont localisées préférentiellement au niveau des espaces portes, autour des veines centrolobulaires, dans les bandes de collagène et dans la zone de fibrose périportale. En revanche, aucune de ces cellules n'a été détectée ni autour ni au sein du granulome. Le pourcentage des cellules NK n'a pas changé durant l'infestation, en dépit de leur augmentation significative en nombre absolu. Tandis que la fréquence des cellules NK productrices d'IL-2 et d'IFN-$\gamma$ a augmenté au 7$^{\rm e}$ jour après infestation, seule l'augmentation de celles qui produisent de l'IFN-$\gamma$ a persisté, la fréquence des cellules produisant l'IL-2 diminuant par la suite. Les cellules NK productrices d'IL-4 présentaient la même cinétique que l'IL-2, alors que celles qui produisent de l'IL-10 n'ont montré aucun changement. L'ensemble de ces résultats montre que les cellules NK constituent une autre source précoce d'IFN-$\gamma$, mais que leur participation à la formation précoce du granulome apparaît peu probable

Early hepatic immune response in rats infected with Fasciola hepatica

We investigated the phenotype of the T cells (CD4$^+$ and CD8$^+$) that produced Th1 (IFN-$\gamma$) 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 animals 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-$\gamma$ on 7 days postinfection (pi) was similar to that in control animals. However, the frequency of CP IFN-$\gamma$ 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 pronounced on day 14. In contrast, IFN-$\gamma$ 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-$\gamma$ 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 response and the induction of a Th2 cytokine profile.Réponse immunitaire hépatique précoce chez le rat infesté par Fasciola hepatica. Nous avons caractérisé le phénotype des sous-populations lymphocytaires T (CD4$^+$, CD8$^+$) qui produisent des cytokines de type Th1 (IFN-$\gamma$) et Th2 (IL-4, IL-10) au cours de l'infestation expérimentale par F. hepatica chez le rat (phase précoce). Nous avons aussi suivi la production des cytokines et la réponse proliférative des cellules mononucléaires hépatiques (CMNH) après une stimulation in vitro par des mitogènes (phorbol myristate acetate) et par des antigènes spécifiques (les produits d'excrétion-sécrétion de F. hepatica). Le pourcentage des cellules CD4$^+$ augmente après infestation tandis que celui des cellules CD8$^+$ demeure inchangé. Après infestation, la fréquence des cellules productrices (CP) de cytokine change. Tandis qu'au 7$^{\rm e}$ jour après infestation (JAI) la fréquence des CP d'IFN-$\gamma$ reste identique à celles des animaux témoins, elle est nettement plus basse au 14$^{\rm e}$ JAI ; la fréquence des CP d'IL-4 et des CP d'IL-10 augmente (surtout les CD4$^+$ IL-10$^+$, notamment au 14$^{\rm e}$ JAI). En outre, dans les surnageants de culture des CMNH, la production de cytokines de type Th2 est accrue au 7$^{\rm e}$ JAI et plus importante encore au 14$^{\rm e}$ JAI. Le niveau de production d'IFN-$\gamma$ augmente au 7$^{\rm e}$ JAI mais décroît jusqu'à devenir inférieure à celle du témoin au 14$^{\rm e}$ JAI. De plus, la réponse proliférative est plus importante au 7$^{\rm e}$ JAI mais est réduite au 14$^{\rm e}$ JAI. L'ensemble de ces résultats montre que durant la période précoce de l'infestation, le parasite induit une réponse transitoire de type Th0 (au 7$^{\rm e}$ JAI), suivie d'une réduction de la réponse cellulaire et d'une induction d'une réponse de type Th2 (au 14$^{\rm e}$ JAI)

Morphogenesis of a Highly Replicative EGFPVP22 Recombinant Marek's Disease Virus in Cell Culture▿

Marek's disease virus (MDV) is an alphaherpesvirus for which infection is strictly cell associated in permissive cell culture systems. In contrast to most other alphaherpesviruses, no comprehensive ultrastructural study has been published to date describing the different stages of MDV morphogenesis. To circumvent problems linked to nonsynchronized infection and low infectivity titers, we generated a recombinant MDV expressing an enhanced green fluorescent protein fused to VP22, a major tegument protein that is not implicated in virion morphogenesis. Growth of this recombinant virus in cell culture was decreased threefold compared to that of the parental Bac20 virus, but this mutant was still highly replicative. The recombinant virus allowed us to select infected cells by cell-sorting cytometry at late stages of infection for subsequent transmission electron microscopy analysis. Under these conditions, all of the stages of assembly and virion morphogenesis could be observed except extracellular enveloped virions, even at the cell surface. We observed 10-fold fewer naked cytoplasmic capsids than nuclear capsids, and intracellular enveloped virions were very rare. The partial envelopment of capsids in the cytoplasm supports the hypothesis of the acquisition of the final envelope in this cellular compartment. We demonstrate for the first time that, compared to other alphaherpesviruses, MDV seems deficient in three crucial steps of viral morphogenesis, i.e., release from the nucleus, secondary envelopment, and the exocytosis process. The discrepancy between the efficiency with which this MDV mutant spreads in cell culture and the relatively inefficient process of its envelopment and virion release raises the question of the MDV cell-to-cell spreading mechanism

Effect of storage and temperature on in vitro stallion sperm parameters and fertility rate

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