A functional network of highly pure enteric neurons in a dish

The enteric nervous system (ENS) is the intrinsic nervous system that innervates the entire digestive tract and regulates major digestive functions. Recent evidence has shown that functions of the ENS critically rely on enteric neuronal connectivity; however, experimental models to decipher the underlying mechanisms are limited. Compared to the central nervous system, for which pure neuronal cultures have been developed for decades and are recognized as a reference in the field of neuroscience, an equivalent model for enteric neurons is lacking. In this study, we developed a novel model of highly pure rat embryonic enteric neurons with dense and functional synaptic networks. The methodology is simple and relatively fast. We characterized enteric neurons using immunohistochemical, morphological, and electrophysiological approaches. In particular, we demonstrated the applicability of this culture model to multi-electrode array technology as a new approach for monitoring enteric neuronal network activity. This in vitro model of highly pure enteric neurons represents a valuable new tool for better understanding the mechanisms involved in the establishment and maintenance of enteric neuron synaptic connectivity and functional networks

Heterogeneity of biologic responses of melanoma-specific CTL

International audienceTo better understand how Ag density influences the various biologic responses of CTL, we analyzed lysis and, at the single cell level, cytokine production by a panel of melanoma-specific CTL clones. Titration experiments done with peptide-pulsed TAP-deficient T2 cells indicated that: 1) Ag density affects both the fraction of responding cells and the amount of cytokine secreted by each cell. 2) Different responses have a relative Ag requirement that may vary between clones. Lysis and secretion of IFN-gamma, and for most clones' secretion of TNF-alpha, required lower Ag densities, by one or two logs, than IL-2 and granulocyte-macrophage CSF secretion. 3) In a significant fraction of IFN-gamma-secreting cells, IL-2 production is not induced. 4) A large fraction of cloned cells is refractory to lymphokine gene activation for about 2 wk after previous stimulation. Together these data indicate that CTL biologic responses are controlled by variable Ag thresholds and by additional parameters affecting activation requirements of each cell. A similar heterogeneity of cytokine responses was observed to Ag endogenously presented by melanoma cells. As a consequence, most melanoma lines, including those with the highest Ag expression, could trigger only low CTL fractions to secrete IL-2 and, also for most clones, granulocyte-macrophage CSF. This may be an important component of the inefficiency of specific CTL in cancer patients

Role of Heat Shock Factor 2 in proteasome subunits genes expression

Poster présenté par Sylvain LecomteHeat Shock Factors (HSF) form the main family of transcription factors involved in response to proteotoxic stress. Five HSF are currently described in vertebrate, but only HSF1 and HSF2 are for the moment, well documented. HSF1 is considered as the bona-fide stress-induced transcription factor, whereas HSF2 is more described for its implication in gametogenesis and embryonic development. However, recent studies have shown that HSF2 can have additional roles in response to protein denaturation1,2 and in bookmarking of several stress-related genes 3,4. We have studied the specific role of HSF1 and HSF2 in the cellular response to proteasome inhibition. Using immortalized Mouse Embryonic Fibroblast (iMEF) derived from knock-out Hsf1 and/or Hsf2 mice, we found that cells deleted for either HSF1 or HSF2 are more sensitive to proteasome inhibition than wild type cells. This result suggests that both HSFs are important for cellular response to proteasome inhibitor treatment. To establish the relationship between the proteasome and these transcription factors, several tests were performed using knock-out cells. First, we found a significant decrease of the chymotrypsin-like activity of the proteasome in Hsf2-/- and in Hsf1-/- & Hsf2-/- iMEF. Secondly, the basal expression of ten proteasome subunits was measured by real time PCR. In agreement with our measure of proteasome activity, we found that two catalytic subunits, PSMB2 and PSMB5 which support trypsin-like and chymotrypsin-like activity respectively, were present at lower level in HSF2 deleted iMEF. Moreover, PSMA1, PSMC4 and PSMD10 also display a lower expression in absence of HSF2, whereas the other proteasome subunits tested are not affected. In conclusion, our data showed that HSF2 is involved in some proteasome subunits basal expression. In HSF2 deficient iMEF, decrease expression of these subunits have a direct influence in cell physiology with a decrease of catalytic activity of the proteasome. These results could open a new field in cancer therapy: development of HSF2 inhibitors could improve the action and reduce chemoresistance to proteasome inhibitors such as bortezomib

Implication of HSF1 and HSF2 in cellular response to proteasome inhibition.

Conférence invitée présentée par Yves Le DréanInternational audienceInhibition of proteasome induces the intracellular accumulation of misfolded proteins that would otherwise be degraded. Cells adapt to proteasome inhibitor treatment by overexpressing chaperone proteins and proteasome subunits. Using clusterin, a secreted chaperone protein, as a model; we found that its overexpression involves interplay between HSF1 and HSF2. Proteasome inhibition activated HSF1 and HSF2 by different mechanisms involving phosphorylation and stabilization, respectively, then these two transcription factors interact and form heterotrimer on the heat shock element within the clusterin gene promoter. We have further studied the role of HSF1 and HSF2 interplay in the cellular response to proteasome inhibition, by using immortalized Mouse Embryonic Fibroblast (iMEF) derived from knock-out Hsf1 and/or Hsf2 mice. We found that cells deleted for either HSF1 or HSF2 are more sensitive to proteasome inhibition than wild type cells. Moreover, using real-time reverse transcription-PCR analyses, we assessed the level of expression of HSFs target genes, and found that cells deleted in HSF1 and/or HSF2 present defect in transcriptional response to proteasome inhibitor. All together, these results confirm that both HSFs are important for cellular response to proteasome inhibitor treatment. Furthermore, to establish the relationship between proteasome and HSFs, the basal expression of ten proteasome subunits was measured in KO iMEF. We found that two catalytic subunits, beta 2 and beta 5, which support trypsin-like and chymotrypsin-like activity respectively, were present at lower level in Hsf2-/- and Hsf1-/- & Hsf2-/- iMEF. Moreover, the subunits alpha 1, PSMC4 and PSMD10 also display a lower expression in absence of HSF2, whereas the other proteasome subunits tested are not affected. In agreement with this observation, we found a significant decrease of the proteasome activity in HSF2 deleted cells. In conclusion, our data show that HSF1 and HSF2 form a functional complex in response to proteasome inhibition. Moreover, we found that HSF2 is more directly involved in some proteasome subunits basal expression. In HSF2 deficient iMEF, decrease expression of these subunits have a direct influence in cell physiology, with a decrease of catalytic activity of the proteasome

IgG1 variations in the colostrum of Holstein dairy cows

International audienceHigh-immune quality colostrum (IgG1 concentration ⩾50 g/l) is crucial for the health and development of the young calf. Studies oncolostrum quality tend to focus on external factors such as breed, parity or dry period length, but few have focused on within-cowvariations. Here we ran experiments to gain a deeper insight into within-cow variation in IgG1 concentrations in dairy cow colostrum.Trials were performed in an experimental farm, located in the Western part of France. Colostrum from each quarter and a compositesample (mix of four quarters) were concomitantly collected on 77 Holstein dairy cows just after calving to assess the influence of sampletype on IgG1 concentrations. Variation in IgG1 concentrations during the first milking was studied on samples from nine cows collectedevery minute from the start of milking. Repeatability of colostral IgG1 concentration was estimated from 2009 and 2010 data on16 healthy cows. IgG1 concentrations were tested using a radial immunodiffusion method. Sensitivity and specificity were similarregardless of sample type tested (individual quarter or composite milk). Mean average IgG1 concentration was 54.1 g/l in compositecolostrum, and was significantly higher in hind quarter teats (56.2 g/l) than front quarter teats (53.1 g/l). Average IgG1 concentration didnot change significantly during colostrum milking, and the variations observed (15% or less) were likely due to the laboratory method(CV 15%). IgG1 concentrations in dam colostrum increased slightly from 2009 to 2010 due to BW and parity effects. In 56% of cases,colostrum quality could have been assessed on either individual or composite colostrum samples collected at any time during the firstmilking without affecting the reliability of the measurement. However, in other cases, differences were significant enough to mean thatestimates of average IgG1 concentration in colostrum from any one quarter would not be reliable. It is concluded that colostrum quality,from an IgG1 concentration point of view, could be assessed with a composite sample taken at any time during the first milking

Comparison of three culture media for the establishment of melanoma cell lines

Melanoma cell lines are useful tools for the analysis of tumor-specific lymphocytes which are injected to patients treated by adoptive immunotherapy. So they have been established previously (with an efficacy of 47%) in Roswell Park Memorial Institute (RPMI) medium enriched with fetal calf serum (FCS). In order to improve the probability of establishing melanoma cell lines, we compared two FCS-free media with the original FCS medium. Ten melanoma-invaded lymph nodes were tested for their ability to grow in three different culture media: RPMI with FCS; RPMI with human serum (HS); serum-free X-vivo 15 (X15). For each medium, we compared the following criteria: percentage of lines obtained; period of establishment; cell morphology; expression of melanoma-associated antigens and surface molecules. More cell lines were obtained with HS and X15 media compared to FCS medium (7/10, 5/10 and 4/10, respectively). The time period to establish a stable line was similar for the three media. No morphological differences were observed in cells derived from the same tumor sample in the different media. With the X15 medium, cells generally expressed lower levels of melanocytic differentiation antigens and surface molecules. The growth of melanoma cell lines in FCS-free culture media appears possible and advantageous, with an increased probability of obtaining autologous tumor cell lines. Furthermore the cells obtained could be used as multiple antigenic sources in active or adoptive immunotherapy protocols