Comparison of the direct effects of human adipose- and bone-marrow-derived stem cells on postischemic cardiomyoblasts in an in vitro simulated ischemia-reperfusion model.

Regenerative therapies hold a promising and exciting future for the cure of yet untreatable diseases, and mesenchymal stem cells are in the forefront of this approach. However, the relative efficacy and the mechanism of action of different types of mesenchymal stem cells are still incompletely understood. We aimed to evaluate the effects of human adipose- (hASC) and bone-marrow-derived stem cells (hBMSCs) and adipose-derived stem cell conditioned media (ACM) on the viability of cardiomyoblasts in an in vitro ischemia-reperfusion (I-R) model. Flow cytometric viability analysis revealed that both cell treatments led to similarly increased percentages of living cells, while treatment with ACM did not (I-R model: 12.13 +/- 0.75%; hASC: 24.66 +/- 2.49%; hBMSC: 25.41 +/- 1.99%; ACM: 13.94 +/- 1.44%). Metabolic activity measurement (I-R model: 0.065 +/- 0.033; hASC: 0.652 +/- 0.089; hBMSC: 0.607 +/- 0.059; ACM: 0.225 +/- 0.013; arbitrary units) and lactate dehydrogenase assay (I-R model: 0.225 +/- 0.006; hASC: 0.148 +/- 0.005; hBMSC: 0.146 +/- 0.004; ACM: 0.208 +/- 0.009; arbitrary units) confirmed the flow cytometric results while also indicated a slight beneficial effect of ACM. Our results highlight that mesenchymal stem cells have the same efficacy when used directly on postischemic cells, and differences found between them in preclinical and clinical investigations are rather related to other possible causes such as their immunomodulatory or angiogenic properties

Pretreatment of therapeutic cells with poly(ADP-ribose) polymerase inhibitor enhances their efficacy in an in vitro model of cell-based therapy in myocardial infarct.

The potential of cell-based therapies in diseases involving ischemia-reperfusion is greatly hampered by the excessive loss of administered cells in the harsh and oxidative environment where these cells are supposed to act. Therefore, we investigated if inhibition of poly(ADP-ribose) polymerase (PARP) in the therapeutically added cells would lead to their increased viability and, subsequently, to an enhanced effect in an in vitro simulated ischemia-reperfusion (I-R) setting. Ischemic conditions were simulated by oxygen and glucose deprivation for 160 min using H9c2 rat cardiomyoblast cells. After 30 min of reperfusion, these cells received 4 types of treatments: no added cells (I-R model), fluorescently labeled (Vybrant DiD) therapeutic H9c2 cells with vehicle (H9c2) or PARP inhibitor (10 microM or 100 microM PJ34) pretreatment. We assessed viability (live, apoptotic and necrotic) of both 'postischemic' and therapeutic cells with flow cytometric analysis using calcein-AM/ethidium homodimer-2 fluorescent staining after 24 h of co-culture. Further measurements on necrosis and metabolic activity were performed using lactate dehydrogenase (LDH) release and resazurin based assays. The percentage of surviving therapeutic cells increased significantly with PARP inhibition (untreated, 52.02+/-5.01%; 10 microM PJ34, 63.38+/-4.50%; 100 microM PJ34, 64.99+/-3.47%). The percentage of necrotic cells decreased in a similar manner (untreated, 37.23+/-4.40%; 10 microM PJ34, 26.83+/-3.49%; 100 microM PJ34, 24.96+/-2.43%). Notably, the survival of the cells that suffered I-R injury was also significantly higher when treated with PARP-inhibited therapeutic cells (I-R model, 36.44+/-5.05%; H9c2, 42.81+/-5.11%; 10 microM PJ34, 52.07+/-5.80%; 100 microM PJ34, 54.95+/-5.55%), while necrosis was inhibited (I-R model, 43.64+/-4.00%; H9c2, 37.29+/-4.55%; 10 microM PJ34, 30.18+/-4.60%; 100 microM PJ34, 25.52+/-3.47%). In subsequent experiments, PARP inhibition decreased LDH-release of the observed combined cell population and enhanced the metabolic activity. Thus, our results suggest that pretreating the therapeutically added cells with a PARP inhibitor could be beneficial in the setting of cell-based therapies

Mesenchymal stem cells rescue cardiomyoblasts from cell death in an in vitro ischemia model via direct cell-to-cell connections

<p>Abstract</p> <p>Background</p> <p>Bone marrow derived mesenchymal stem cells (MSCs) are promising candidates for cell based therapies in myocardial infarction. However, the exact underlying cellular mechanisms are still not fully understood. Our aim was to explore the possible role of direct cell-to-cell interaction between ischemic H9c2 cardiomyoblasts and normal MSCs. Using an in vitro ischemia model of 150 minutes of oxygen glucose deprivation we investigated cell viability and cell interactions with confocal microscopy and flow cytometry.</p> <p>Results</p> <p>Our model revealed that adding normal MSCs to the ischemic cell population significantly decreased the ratio of dead H9c2 cells (H9c2 only: 0.85 ± 0.086 vs. H9c2+MSCs: 0.16 ± 0.035). This effect was dependent on direct cell-to-cell contact since co-cultivation with MSCs cultured in cell inserts did not exert the same beneficial effect (ratio of dead H9c2 cells: 0.90 ± 0.055). Confocal microscopy revealed that cardiomyoblasts and MSCs frequently formed 200-500 nm wide intercellular connections and cell fusion rarely occurred between these cells.</p> <p>Conclusion</p> <p>Based on these results we hypothesize that mesenchymal stem cells may reduce the number of dead cardiomyoblasts after ischemic damage via direct cell-to-cell interactions and intercellular tubular connections may play an important role in these processes.</p

Perivascular Expression and Potent Vasoconstrictor Effect of Dynorphin A in Cerebral Arteries

BACKGROUND: Numerous literary data indicate that dynorphin A (DYN-A) has a significant impact on cerebral circulation, especially under pathophysiological conditions, but its potential direct influence on the tone of cerebral vessels is obscure. The aim of the present study was threefold: 1) to clarify if DYN-A is present in cerebral vessels, 2) to determine if it exerts any direct effect on cerebrovascular tone, and if so, 3) to analyze the role of κ-opiate receptors in mediating the effect. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemical analysis revealed the expression of DYN-A in perivascular nerves of rat pial arteries as well as in both rat and human intraparenchymal vessels of the cerebral cortex. In isolated rat basilar and middle cerebral arteries (BAs and MCAs) DYN-A (1-13) and DYN-A (1-17) but not DYN-A (1-8) or dynorphin B (DYN-B) induced strong vasoconstriction in micromolar concentrations. The maximal effects, compared to a reference contraction induced by 124 mM K(+), were 115±6% and 104±10% in BAs and 113±3% and 125±9% in MCAs for 10 µM of DYN-A (1-13) and DYN-A (1-17), respectively. The vasoconstrictor effects of DYN-A (1-13) could be inhibited but not abolished by both the κ-opiate receptor antagonist nor-Binaltorphimine dihydrochloride (NORBI) and blockade of G(i/o)-protein mediated signaling by pertussis toxin. Finally, des-Tyr(1) DYN-A (2-13), which reportedly fails to activate κ-opiate receptors, induced vasoconstriction of 45±11% in BAs and 50±5% in MCAs at 10 µM, which effects were resistant to NORBI. CONCLUSION/SIGNIFICANCE: DYN-A is present in rat and human cerebral perivascular nerves and induces sustained contraction of rat cerebral arteries. This vasoconstrictor effect is only partly mediated by κ-opiate receptors and heterotrimeric G(i/o)-proteins. To our knowledge our present findings are the first to indicate that DYN-A has a direct cerebral vasoconstrictor effect and that a dynorphin-induced vascular action may be, at least in part, independent of κ-opiate receptors

A brojlerfeldolgozás technológiája és állatjóléti vonatkozásai

A dolgozat a brojlerfeldolgozás technológiáját mutatja be. Kitér az egyes technológiai lépések során alkalmazandó paraméterekre, a műveletek során tapasztalható összefüggésekre egészen az élő állat kezeléstől a végtermék elkészüléséig. A dolgozat első része a vágóhídi állatjólétről szól , ami szigorú EU-s jogi szabályozás alatt áll.BSc/BAÉlelmiszermérnö

1-Metil-L-triptofán és a peroxomonoszulfát-ion közötti reakció kinetikája és mechanizmusa

Szakdolgozatomban az 1-Metil-L-triptofán aminosav és a peroxomonoszulfát-ion közötti redoxireakció kinetikáját és mechanizmusát tanulmányoztam. A 287 nm-es hullámhosszon végzett spektrális elemzésből bizonyosan állítható, hogy egynél több reakció játszódik le a rendszerben. A sztöchiometria vizsgálatok alapján azt tapasztaltam, hogy a folyamat első, és minden bizonnyal leggyorsabb lépése során a reaktánsok mol aránya 1:1, vagyis a triptofán kételektronos oxidációja történik meg. A kinetika alapján két egymást követő reakcióról van szó, amelyek közül a gyorsabb elsőrendű az 1-metil-triptofánra nézve, míg a második elsőrendű a belőle keletkező köztitermékre nézve, viszont mindkét folyamat elsőrendű az oxon koncentrációra nézve.BSc/BAKémia BSCg

Dynorphin A (DYN-A) induced cerebral vasoconstriction is partly mediated by heterotrimeric G_i/o-proteins.

<p>Effect of the inhibition of G_i/o-signaling with pertussis toxin (PTX, applied as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037798#s2" target="_blank">Methods</a>”) on the responsiveness of rat basilar (A) and middle cerebral (B) arteries to DYN-A (1–13). In control vessels the slightly weaker reactions to DYN-A (as compared to the previous figures) were probably the consequence of overnight incubation in the Krebs solution containing the vehicle of PTX. PTX inhibited the vasoconstrictor effects of DYN-A. Values are expressed as mean±SEM percentage of the reference contraction induced by 124 mmol/L K⁺ Krebs solution, n = 6–8. Asterisks indicate significant (*<i>P</i><0.05, **<i>P</i><0.01) differences between PTX-treated and vehicle-treated control vessels.</p