Comparison of Insulin Sensitivity of Horses Adapted to Different Exercise Intensities

AbstractDiets high in concentrates and soluble carbohydrates are associated with reduced insulin sensitivity in horses. Exercise training could protect against diet-induced insulin resistance. The objective of this study was to determine the intensity of exercise training required to affect insulin sensitivity in stabled horses fed a diet high in concentrates but moderate in soluble carbohydrates. In all, 31 stabled horses underwent three different exercise regimens: turnout, light exercise, and moderate exercise, while being fed a diet containing 60% concentrate. Blood was sampled monthly and analyzed for insulin. Insulin sensitivity was assessed using basal insulin concentrations and calculated insulin sensitivity (reciprocal of the square root of insulin) and compared across months by analysis of variance with repeated measures. Insulin sensitivity (reciprocal of the square root of insulin) was higher during periods of moderate and light physical activity as compared with turnout. These results indicate that turnout alone may not be adequate to improve insulin sensitivity in horses fed high amounts of concentrate

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Decidualization and syndecan-1 knock down sensitize endometrial stromal cells to apoptosis induced by embryonic stimuli.

Human embryo invasion and implantation into the inner wall of the maternal uterus, the endometrium, is the pivotal process for a successful pregnancy. Whereas disruption of the endometrial epithelial layer was already correlated with the programmed cell death, the role of apoptosis of the subjacent endometrial stromal cells during implantation is indistinct. The aim was to clarify whether apoptosis plays a role in the stromal invasion and to characterize if the apoptotic susceptibility of endometrial stromal cells to embryonic stimuli is influenced by decidualization and Syndecan-1. Therefore, the immortalized human endometrial stromal cell line St-T1 was used to first generate a new cell line with a stable Syndecan-1 knock down (KdS1), and second to further decidualize the cells with progesterone. As a replacement for the ethically inapplicable embryo all cells were treated with the embryonic factors and secretion products interleukin-1β, interferon-γ, tumor necrosis factor-α, transforming growth factor-β1 and anti-Fas antibody to mimic the embryo contact. Detection of apoptosis was verified via Caspase ELISAs, PARP cleavage and Annexin V staining. Apoptosis-related proteins were investigated via antibody arrays and underlying signaling pathways were analyzed by Western blot. Non-decidualized endometrial stromal cells showed a resistance towards apoptosis which was rescinded by decidualization and Syndecan-1 knock down independent of decidualization. This was correlated with an altered expression of several pro- and anti-apoptotic proteins and connected to a higher activation of pro-survival Akt in non-differentiated St-T1 as an upstream mediator of apoptotis-related proteins. This study provides insight into the largely elusive process of implantation, proposing an important role for stromal cell apoptosis to successfully establish a pregnancy. The impact of Syndecan-1 in attenuating the apoptotic signal is particularly interesting in the light of an already described influence on pregnancy disorders and therefore might provide a useful clinical tool in the future to prevent pregnancy complications provoked by inadequate implantation

Expression of apoptosis-related proteins before and after IITT+F treatment.

<p>Antibody array analysis of apoptosis-related proteins in ESCs of (A) untreated, non-differentiated (St-T1, red bar; KdS1, blue bar) and decidualized (dSt-T1, bright red bar; dKdS1, bright blue bar) ESCs, n = 4 ± SEM, *p<0.05 Sdc-1 wildtype vs. Sdc-1 kd cells, #p<0.05 non-differentiated vs. decidualized cells. (B) Antibody Array with protein from IITT+F treated, non-differentiated (St-T1, red bar; KdS1, blue bar) and decidualized (dSt-T1, bright red bar; dKdS1, bright blue bar) ESCs. Pixel density is given as mean±SEM of n = 4 independent experiments, *p<0.05 Sdc-1 wildtype vs. Sdc-1 kd cells, #p<0.05 non-differentiated vs. decidualized cells, ✝p<0.05 untreated vs. IITT+F treated.</p

Quantification of active Caspase-3 in ESCs treated with embryonic stimuli.

<p>Non-differentiated (St-T1, red bar; KdS1, blue bar) and decidualized (dSt-T1, bright red bar; dKdS1, bright blue bar) ESCs were treated with IITT and F for 24h individually or in combination as indicated and the amount of active Caspase-3 was analyzed in ng/ml and displayed as mean±SEM of n = 3 independent experiments; *p<0.05 Sdc-1 wildtype vs. Sdc-1 kd cells, #p<0.05 non-differentiated vs. decidualized cells, ✝p<0.05 untreated controls vs. treated cells.</p

Induction of FasR expression after IITT treatment.

<p>Fold change of FasR mRNA in non-differentiated (St-T1, red bar; KdS1, blue bar) and decidualized (dSt-T1, bright red bar; dKdS1, bright blue bar) ESCs after treatment with IITT 24h. 2^-ΔΔCt is are given as mean±SEM of n = 5 independent experiments, ✝p<0.05 untreated vs. IITT treated.</p

Activation of the pro-survival protein Akt after IITT and F treatment.

<p>Western blot analysis of pAkt and Akt in non-differentiated (St-T1, first line; KdS1, second line) and decidualized (dSt-T1, third line; dKdS1, fourth line) ESCs after treatments with F 15min, IITT 15min and IITT 24h + F 15min vs. untreated controls. (A) Representative blot of pAkt (60kDa), Akt (60kDa) and β-Actin (42kDa) as loading control. (B) Pixel densitiy evaluation of pAkt normalized to Akt is given as mean±SEM of n = 6 independent experiments, *p<0.05 wildtype vs. Sdc-1 kd cells, #p<0.05 undifferentiated vs. decidualized cells, ✝p<0.05 untreated control vs. treated.</p

Investigation of the extrinsic and intrinsic apoptosis pathway after IITT+F treatment.

<p>Analysis of Caspase-8 and -9 activation of treated cells vs. untreated controls in non-differentiated (St-T1, red bar; KdS1, blue bar) and decidualized dSt-T1, bright red bar; dKdS1, bright blue bar) ESCs after treatment with IITT+F. Untreated controls were assigned being 1 and enzymatic activity of caspases after treatment was determined as fold induction vs. controls and given as mean±SEM of n = 3 independent experiments, *p<0.05 Sdc-1 wildtype vs. Sdc-1 kd cells, ✝p<0.05 untreated controls vs. treated.</p

Activation of the pro-apoptotic JNK pathway after IITT and F treatment.

<p>Western blot analysis of pJNK and JNK in non-differentiated (St-T1, first line; KdS1, second line) and decidualized (dSt-T1, third line; dKdS1, fourth line) ESCs after treatments with F 15min, IITT 15min and IITT 24h + F 15min vs. untreated controls. (A) Representative blot of pJNK (46/54kDa), JNK (46/54kDa) and β-Actin (42kDa) as loading control. (b) Pixel densitiy evaluation of pJNK normalized to JNK is given as mean±SEM of n = 6 independent experiments, *p<0.05 wildtype vs. Sdc-1 kd cells, ✝p<0.05 untreated control vs. treated.</p