Mesenchymal stromal cells: a promising cell source for the treatment of inflammatory cardiomyopathy.

Inflammatory cardiomyopathy is associated with a diffuse inflammation in the heart accompanied with cardiac fibrosis, cardiomyocyte apoptosis, and reduced capillary density. On the other hand, mesenchymal stromal cells (MSCs) have immunomodulatory, anti-fibrotic, anti-apoptotic, and pro-angiogenic features, making them attractive candidates for the treatment of inflammatory cardiomyopathy. The potential of MSC application for the treatment of myocarditis is supported by their beneficial effects in experimental models of acute and chronic inflammatory cardiomopathy. This review summarizes the cardioprotective features of MSCs and describes how MSCs are primed by the inflammatory environment to exert their protective effects. In view of clinical translation, searching for the optimal source of MSC and delivery route, allowing efficient and non-invasive cell application, the differences between MSCs of distinct origin and between diverse routes of application are outlined.

Mesenchymal stem cells improve murine acute coxsackievirus B3-induced myocarditis.

AIMS: Coxsackievirus B3 (CVB3)-induced myocarditis, initially considered a sole immune-mediated disease, also results from a direct CVB3-mediated injury of the cardiomyocytes. Mesenchymal stem cells (MSCs) have, besides immunomodulatory, also anti-apoptotic features. In view of clinical translation, we first analysed whether MSCs can be infected by CVB3. Next, we explored whether and how MSCs could reduce the direct CVB3-mediated cardiomyocyte injury and viral progeny release, in vitro, in the absence of immune cells. Finally, we investigated whether MSC application could improve murine acute CVB3-induced myocarditis. METHODS AND RESULTS: Phase contrast pictures and MTS viability assay demonstrated that MSCs did not suffer from CVB3 infection 4-12-24-48 h after CVB3 infection. Coxsackievirus B3 RNA copy number decreased in this time frame, suggesting that no CVB3 replication took place. Co-culture of MSCs with CVB3-infected HL-1 cardiomyocytes resulted in a reduction of CVB3-induced HL-1 apoptosis, oxidative stress, intracellular viral particle production, and viral progeny release in a nitric oxide (NO)-dependent manner. Moreover, MSCs required priming via interferon-γ (IFN-γ) to exert their protective effects. In vivo, MSC application improved the contractility and relaxation parameters in CVB3-induced myocarditis, which was paralleled with a reduction in cardiac apoptosis, cardiomyocyte damage, left ventricular tumour necrosis factor-α mRNA expression, and cardiac mononuclear cell activation. Mesenchymal stem cells reduced the CVB3-induced CD4- and CD8- T cell activation in an NO-dependent way and required IFN-γ priming. CONCLUSION: We conclude that MSCs improve murine acute CVB3-induced myocarditis via their anti-apoptotic and immunomodulatory properties, which occur in an NO-dependent manner and require priming via IFN-γ.

Influence of dopamine energic pharmacology drugs on secretion of the adrenocorticotropic hormone from the hypophysis.

To elucidate the role of dopamine as a neuromediator in the adrenocorticotropic hormone (ACTH) secretion, investigations were carried out with dopaminergic pharmacology drugs on male white Wistar rats. In the first series of experiments, the effects of 200 mg/kg body wt L-DOPA, of the combination of 200 mg/kg L-DOPA and 50 mg/kg body wt carbidopa, and of 2.5 mg/kg body wt bromocriptine, after a single intraperitoneal injection of ACTH in the serum of rats after 30, 90 and 120 min, following the injection, were studied. In the second series of experiments, the effect of 200 mg/kg body wt L-DOPA, of the combination of 200 mg/kg body wt L-DOPA and 50 mg/kg body wt carbidopa, of 1 mg/kg body wt bromocriptine, after intraperitoneal injection, on the concentration of ACTH in the serum within 7 days, were assessed. The inhibition of agonists of dopamine after ACTH secretion with repeated application has been shown. Using a radioimmunology assay with test kits, the amount of ACTH in the serum was determined.

Effect of cryopreservation on lipids and some physiological features of spermatozoa from rams pastured in highlands and in valleys.

The effect of low temperature preservation on the motility and morphology of acrosomes, acrosomal proteolytic activity, phospholipid and fatty acid composition of phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE), and the cholesterol/phospholipid molar ratio in sperm from rams housed in the highlands or in the valleys, were studied. The indices of motility and morphological integrity of sperm from highland rams were much greater compared with those of valley rams. Phosphatidyl choline (PC) of the highland rams was more unsaturated, while PE was more saturated compared with those of valley rams. Cryopreservation of the sperm from highland rams significantly increased the content of choline plasmalogen, accompanied by a slight rise in the levels of lysophosphatidyl choline (LPC) and phosphatidyl inositol (PI) in their sperm. The fatty acid composition altered following cryopreservation. These variations were mainly due to a decrease in the amount of docosahexaenic acid and an increase in the amounts of linoleic and palmitic fatty acids. The results may be indicative of the fact that the alterations in the sperm of the valley rams were more pronounced and they may be attributed to the structural features of the sperm, as well as a reduced concentration of oxygen in the organs and tissues of the highland rams.

The effect of prostaglandin E1 on in vitro transcription of sperm chromatin, isolated from patients with azoospermia, teratospermia and chronic prostatitis.

We have investigated the influence of Prostaglandin E1 on the in vitro transcription of chromatin, isolated from spermatozoa of patients suffering from different pathologies, leading to infertility, namely, azoospermia, teratospermia and chronic prostatitis. Our studies indicate that prostaglandin E1 has a stimulatory effect both on in vitro transcription, on the number of RNA polymerase molecules and the polyribonucleotide elongation rates as compared to sperm chromatin from healthy patients. The results on the incorporation of alpha-32P-ATP in to RNA in the presence and absence of Prostaglandin E1 correlate well with the data on the number of actively transcribing RNA polymerase molecules and the rate of RNA elongation, which might be due to low levels of prostaglandin E1 in human semen.

The influence of prostaglandin E1 on in vitro transcription of sperm chromatin from healthy males.

Run-on transcription experiments with sonicated chromatin from healthy individuals in the presence and absence of prostaglandin E1 showed different levels of transcription. Thus the level of incorporation of 14C-UTP into pre-mRNA proved to be about twice as high in the presence of postaglandin E1 as in the controls. The kinetic data of the in vitro transcription indicated that there were two peaks of incorporation of 14C-UTP into RNA both in the controls and in prostaglandin E1 treated probes but at different time intervals. The results show that prostaglandin E1 has a stimulatory effect on in vitro transcription of chromatin, isolated from normal patients.

Quantification of actively transcribing RNA polymerase B molecules in human sperm chromatin and the effect of prostaglandin E1.

Run-on transcription experiments with sonicated chromatin from spermatozoa of healthy men revealed the presence of in vitro transcription. The number of actively transcribing RNA polymerase B molecules in sperm chromatin in vitro, isolated from healthy individuals, and the effect of prostaglandin El, were investigated. The results indicate that the number of RNA polymerase B molecules actively engaged in transcription increased one and a half times when 5 units of prostaglandin E1 was added to the reaction mixtures. These results correlate well with the authors' previous findings that the incorporation of labelled uridine triphosphate into pre-mRNA was higher when prostaglandin E1 was added to the reaction mixtures in run-on transcription experiments, and when sperm chromatin from healthy individuals was used as a template.

Design and synthesis of highly selective in vitro and in vivo uterine receptor antagonists of oxytocin: comparisons with Atosiban.

We report the solid phase synthesis and some pharmacological properties of seven position two analogues (peptides 1-7) of one of our lead oxytocin antagonists, des-9-glycinamide[1-(beta-mercapto-beta,beta-pentamethylenepropionic+ ++ acid),2-O-methyltyrosine,4-threonine]ornithinevasotocin(desGly+ ++-NH2, d(CH2)5-[Tyr(Me)2,Thr4]OVT) (A). Peptides 1-7 have the following substituents at position two (1) D-Tyr(Me); (2) L-Tyr(Et); (3) D-Tyr(Et); (4) L-Tyr; (5) D-Tyr; (6) D-Phe and (7) D-Trp. These were evaluated for agonistic and antagonistic activities in in vitro and in vivo OT assays, in vivo vasopressor (V1a-receptor) assays and in vivo antidiuretic (V2-receptor) assays. None of the seven peptides exhibits oxytocic or vasopressor agonism. Peptides 1, 2, 4, 6 and 7 are extremely weak V2 agonists (V2 activities range from 0.001 to 0.02 U/mg). Peptides 3 and 5 exhibit weak V2 antagonism (pA2 < 6.0 and < 5.5, respectively). Peptides 1-7 exhibit potent in vitro (no Mg2+) OT antagonism (anti-OT pA2 values range from 7.66 to 8.03). Peptides 1 and 4-7 exhibit potent in vivo OT antagonism. Estimated in vivo anti-OT pA2 values range from 7.06 to 7.79 (peptides 2 and 3 were not tested). With anti-V1a pA2 values of 5.17-6.25 all seven peptides exhibit reduced anti-V1a potencies relative to the parent peptide (A) (anti-V1a pA2 = 6.46). Four of these peptides (4-7) exhibit striking gains in in vitro and in vivo anti-OT/anti-V1a selectivities compared to (A) which has an in vitro selectivity of 30 and an in vivo selectivity of 18. The D-Tyr2 (5), D-Trp2 (7), D-Phe2 (6) and L-Tyr2 (4) analogues of (A) exhibit anti-OT (in vitro)/anti-V1a selectivities = 240, 390, 404 and 540, respectively. The L-Tyr2 (4), D-Trp2 (7), D-Phe2 (6) and D-Tyr2 (5) analogues exhibited anti-OT (in vivo)/anti-V1a selectivities of 72, 80, 88 and 95, respectively. Peptides 4-7 appear to be the most selective peptide OT antagonists reported to date. In this regard it may be noted that they appear to be as or more potent and much more selective than the closely related OT antagonist 1-deamino[D-Tyr(Et)2,Thr4]OVT (Atosiban) which is currently undergoing clinical trial as a potential therapeutic agent for the prevention of premature labor. Atosiban (peptide 8) was resynthesized and pharmacologically evaluated in our laboratories. Atosiban exhibits the following antagonistic potencies. Anti-OT (in vitro, no Mg2+) pA2 = 7.71; anti-OT in vivo pA2 = 7.05; anti-V1a pA2 = 6.14 and anti-V2 pA2 approximately 5.9. Its anti-OT (in vivo)/anti-V1a selectivity is 8. Some of these antagonists may be suitable candidates for evaluation as potential tocolytic agents for use in the treatment of pre-term labor. They could also serve as useful new pharmacological tools for studies on the physiological roles of oxytocin. Finally, the findings presented here provide useful clues for the design of more potent and more selective OT antagonists.

Electron microscopic demonstration of transcription of ram sperm chromatin.

Transcription of ram sperm chromatin was examined by two electron microscopic techniques, namely spreading and thin sections. Labelling by Streptavidin-gold particles permits identification of transcription complexes, that have previously incorporated biotinylated uridine triphosphate. This supports previous electron microscopic data for randomly distributed transcription complexes and the presence of polymerase II molecules, documented by means of specific antibody using immunoelectron microscopy. Labelling of transcripts with biotin permits exact visualization of the transcription process, as well as identification of regions, where transcription occurs.

Influence of freezing at different temperatures on the transcription activity of buffalo sperm chromatin.

The effect of freezing at different temperatures, -20 degrees C, -70 degrees C and -196 degrees C, on the transcription activity of buffalo sperm chromatin was investigated. The kinetic data of incorporation of 3H-UTP in pre-mRNA indicate that temperatures of -70 degrees C and -196 degrees C are the most favourable for preservation, since transcription activity gradually increases with time, reaching maximum values within 30 min. Freezing at -20 degrees C or preservation at 4 degrees C leads to a rapid decrease of transcriptional activity within 10 and 20 min, respectively. It is suggested that the optimal temperature for storage of buffalo spermatozoa is below -20 degrees C as judged by the transcription assay.

Electron microscopic data for the presence of post-meiotic gene expression in isolated ram sperm chromatin.

A whole mount electron microscopic technique facilitated a direct visualization of transcripts in ram spermatozoa in run-on experiments. The localization of transcripts in sperm chromatin by spreading enabled identification of regions where transcription complexes, presumably pre-mRNA species, were seen related to chromatin. In another series of experiments the localization of polymerase II was demonstrated using a specific antibody against the conservative tail domain of the polymerase II molecule, followed by protein A-gold visualization on spread chromatin and on thin sections from mature spermatozoa. Incorporation of bio-UTP in transcripts was visualized by streptavidin-gold on spreading and on thin sections. The data suggest that transcription occurs at the periphery of the mature spermatozoa.

The effect of three cryoprotective diluents on the in vitro transcription of ram sperm cells.

The effect of three different solvents was investigated in run-on experiments, using purified ram sperm nuclei. The best cryoprotective diluent proved to be Nagase-Niwa, which had the most positive effect on transcription. The kinetic data of incorporation of 32P-ATP in pre-mRNA indicated that there was a stepwise increase in the level of transcription during the first 20 min of incubation, being lowest in the control and highest in the presence of the diluent Nagase-Niwa.