Prolonged activity of the transposase helper may raise safety concerns during DNA transposon-based gene therapy.

DNA transposon-based gene delivery vectors represent a promising new branch of randomly integrating vector development for gene therapy. For the side-by-side evaluation of the piggyBac and Sleeping Beauty systems-the only DNA transposons currently employed in clinical trials-during therapeutic intervention, we treated the mouse model of tyrosinemia type I with liver-targeted gene delivery using both transposon vectors. For genome-wide mapping of transposon insertion sites we developed a new next-generation sequencing procedure called streptavidin-based enrichment sequencing, which allowed us to identify approximately one million integration sites for both systems. We revealed that a high proportion of piggyBac integrations are clustered in hot regions and found that they are frequently recurring at the same genomic positions among treated animals, indicating that the genome-wide distribution of Sleeping Beauty-generated integrations is closer to random. We also revealed that the piggyBac transposase protein exhibits prolonged activity, which predicts the risk of oncogenesis by generating chromosomal double-strand breaks. Safety concerns associated with prolonged transpositional activity draw attention to the importance of squeezing the active state of the transposase enzymes into a narrower time window.

A versatile transposon-based technology to generate loss- and gain-of-function phenotypes in the mouse liver.

BACKGROUND: Understanding the contribution of gene function in distinct organ systems to the pathogenesis of human diseases in biomedical research requires modifying gene expression through the generation of gain- and loss-of-function phenotypes in model organisms, for instance, the mouse. However, methods to modify both germline and somatic genomes have important limitations that prevent easy, strong, and stable expression of transgenes. For instance, while the liver is remarkably easy to target, nucleic acids introduced to modify the genome of hepatocytes are rapidly lost, or the transgene expression they mediate becomes inhibited due to the action of effector pathways for the elimination of exogenous DNA. Novel methods are required to overcome these challenges, and here we develop a somatic gene delivery technology enabling long-lasting high-level transgene expression in the entire hepatocyte population of mice. RESULTS: We exploit the fumarylacetoacetate hydrolase (Fah) gene correction-induced regeneration in Fah-deficient livers, to demonstrate that such approach stabilizes luciferase expression more than 5000-fold above the level detected in WT animals, following plasmid DNA introduction complemented by transposon-mediated chromosomal gene transfer. Building on this advancement, we created a versatile technology platform for performing gene function analysis in vivo in the mouse liver. Our technology allows the tag-free expression of proteins of interest and silencing of any arbitrary gene in the mouse genome. This was achieved by applying the HADHA/B endogenous bidirectional promoter capable of driving well-balanced bidirectional expression and by optimizing in vivo intronic artificial microRNA-based gene silencing. We demonstrated the particular usefulness of the technology in cancer research by creating a p53-silenced and hRas G12V-overexpressing tumor model. CONCLUSIONS: We developed a versatile technology platform for in vivo somatic genome editing in the mouse liver, which meets multiple requirements for long-lasting high-level transgene expression. We believe that this technology will contribute to the development of a more accurate new generation of tools for gene function analysis in mice.

Preterm Intraventricular Hemorrhage-Induced Inflammatory Response in Human Choroid Plexus Epithelial Cells.

Following an intraventricular hemorrhage (IVH), red blood cell lysis and hemoglobin (Hb) oxidation with the release of heme can cause sterile neuroinflammation. In this study, we measured Hb derivates and cellular adhesion molecules ICAM-1 and VCAM-1 with cell-free miRNAs in cerebrospinal fluid (CSF) samples obtained from Grade-III and Grade-IV preterm IVH infants (IVH-III and IVH-IV, respectively) at multiple time points between days 0-60 after the onset of IVH. Furthermore, human choroid plexus epithelial cells (HCPEpiCs) were incubated with IVH and non-IVH CSF (10 v/v %) for 24 h in vitro to investigate the IVH-induced inflammatory response that was investigated via: (i) HMOX1, IL8, VCAM1, and ICAM1 mRNAs as well as miR-155, miR-223, and miR-181b levels by RT-qPCR; (ii) nuclear translocation of the NF-κB p65 subunit by fluorescence microscopy; and (iii) reactive oxygen species (ROS) measurement. We found a time-dependent alteration of heme, IL-8, and adhesion molecules which revealed a prolonged elevation in IVH-IV vs. IVH-III with higher miR-155 and miR-181b expression at days 41-60. Exposure of HCPEpiCs to IVH CSF samples induced HMOX1, IL8, and ICAM1 mRNA levels along with increased ROS production via the NF-κB pathway activation but without cell death, as confirmed by the cell viability assay. Additionally, the enhanced intracellular miR-155 level was accompanied by lower miR-223 and miR-181b expression in HCPEpiCs after CSF treatment. Overall, choroid plexus epithelial cells exhibit an abnormal cell phenotype after interaction with pro-inflammatory CSF of IVH origin which may contribute to the development of later clinical complications in preterm IVH.

Needle in the heart: a rare case of cardiac tamponade caused by a migrated foreign body and mimicking ST segment elevation myocardial infarction.

BACKGROUND: Pericardial tamponade is a serious condition which may eventually lead to severe haemodynamic disturbances and cardiac arrest. It is most often caused by the accumulation of fluid inside the pericardium, as a result of different aetiological factors such as pericarditis, neoplastic diseases, lymphatic dysfunctions, or idiopathic pericardial disease. Pericardial tamponade can develop after cardiac surgical procedures or as a complication of myocardial infarction. Collection of blood inside the pericardial sack can be the result of pericardial or cardiac trauma. It is exceedingly rare for the injury to be caused by a migrating foreign body. Although a typical picture of pericardial tamponade has been previously described, the disorder may clinically resemble an acute myocardial infarction. CASE PRESENTATION: We report the case of a 58-year-old female patient complaining of new onset thoracic pain and shortness of breath. Electrocardiographic examination results were suggestive of an acute inferior myocardial infarction. However, echocardiography revealed significant pericardial tamponade. The cause was found to be a needle which remained inside the pelvis following a previous cesarean delivery, which the patient had undergone 18 years prior. In emergency setting, the needle was removed and the pericardial tamponade was resolved. Due to the prompt and efficient management, the patient had an uneventful postoperative recovery and presented no recurrence at the follow-up examinations. CONCLUSIONS: The migration of foreign bodies through tissues is exceedingly rare. If present, it may cause life-threatening complications. Since the aetiology of pericardial tamponade is vast, a thorough assessment is highly important. Therefore, echocardiography is the imaging modality of choice. We wish to highlight the possibility of migrating foreign bodies as probable cause for pericardial tamponade, as well as the importance of echocardiographic methods in the fast-track evaluation of such critical conditions.

Clinical and genetic characteristics of craniosynostosis in Hungary.

Craniosynostosis, the premature closure of cranial sutures, is a common craniofacial disorder with heterogeneous etiology and appearance. The purpose of this study was to investigate the clinical and molecular characteristics of craniosynostoses in Hungary, including the classification of patients and the genetic analysis of the syndromic forms. Between 2006 and 2012, 200 patients with craniosynostosis were studied. Classification was based on the suture(s) involved and the associated clinical features. In syndromic cases, genetic analyses, including mutational screening of the hotspot regions of the FGFR1, FGFR2, FGFR3, and TWIST1 genes, karyotyping and FISH study of TWIST1, were performed. The majority (88%) of all patients with craniosynostosis were nonsyndromic. The sagittal suture was most commonly involved, followed by the coronal, metopic, and lambdoid sutures. Male, twin gestation, and very low birth weight were risk factors for craniosynostosis. Syndromic craniosynostosis was detected in 24 patients. In 17 of these patients, Apert, Crouzon, Pfeiffer, Muenke, or Saethre-Chotzen syndromes were identified. In one patient, multiple-suture craniosynostosis was associated with achondroplasia. Clinical signs were not typical for any particular syndrome in six patients. Genetic abnormalities were detected in 18 syndromic patients and in 8 relatives. In addition to 10 different, known mutations in FGFR1,FGFR2 or FGFR3, one novel missense mutation, c.528C>G(p.Ser176Arg), was detected in the TWIST1 gene of a patient with Saethre-Chotzen syndrome. Our results indicate that detailed clinical assessment is of paramount importance in the classification of patients and allows indication of targeted molecular testing with the highest possible diagnostic yield.

Characterization of pericardial and plasma ghrelin levels in patients with ischemic and non-ischemic heart disease.

Ghrelin is an endocrine regulatory peptide with multiple functions including cardioprotective effects. It is produced in various tissues among others in the myocardium. Pericardial fluid has been proven to be a biologically active compartment of the heart that communicates with the myocardial interstitium. Thus, pericardial level of certain agents may reflect their concentration in the myocardium well. In our study we measured acylated (active) and total (acylated and non-acylated) pericardial and plasma ghrelin levels of patients with ischemic and non-ischemic heart disease. Pericardial fluid and plasma samples were obtained from patients with coronary artery disease (ISCH, n=54) or valvular heart disease (VHD, n=41) undergoing cardiac surgery. Acylated pericardial ghrelin concentrations were found to be significantly higher in patients with ischemic heart disease (ISCH vs. VHD, 32±3 vs. 16±2pg/ml, p<0.01), whereas plasma levels of the peptide showed no difference between patient groups. Pericardial-to-plasma ratio, an index abolishing systemic effects on local ghrelin level was also significantly higher in ISCH group for both acylated and total ghrelin. Plasma total ghrelin showed negative correlation to BMI, plasma insulin and insulin resistance index HOMA-A. Pericardial acylated and total ghrelin concentrations were negatively correlated with posterior wall thickness (R=-0.31, p<0.05 and R=-0.35, p<0.01, respectively). Plasma insulin concentration and HOMA-A showed significant negative correlation with pericardial ghrelin levels. In conclusion, increased pericardial active ghrelin content and higher pericardial-to-plasma ghrelin ratio were found in ischemic heart disease as compared to non-ischemic patients suggesting an increased ghrelin production of the chronically ischemic myocardium. According to our results, pericardial ghrelin content is negatively influenced by left ventricular hypertrophy and insulin resistance.

Achondroplasia with multiple-suture craniosynostosis: a report of a new case of this rare association.

We report on a female patient with an exceedingly rare combination of achondroplasia and multiple-suture craniosynostosis. Besides the specific features of achondroplasia, synostosis of the metopic, coronal, lambdoid, and squamosal sutures was found. Series of neurosurgical interventions were carried out, principally for acrocephaly and posterior plagiocephaly. The most common achondroplasia mutation, a p.Gly380Arg in the fibroblast growth factor receptor 3 (FGFR3) gene, was detected. Cytogenetic and array CGH analyses, as well as molecular genetic testing of FGFR1, 2, 3 and TWIST1 genes failed to identify any additional genetic alteration. It is suggested that this unusual phenotype is a result of variable expressivity of the common achondroplasia mutation.

Shunt insufficiency due to knot formation in the peritoneal catheter.

The authors report a rare case of the peripheral obstruction of a ventriculoperitoneal shunt. Premature baby was operated on hydrocephalus due to germinal matrix bleeding. After two months of implantation of venticuloperitoneal shunt peripheral insufficiency of the system was emerged. During the shunt revision extensive knot formation became visible. We simply cut the catheter above the knot and the working shunt was replaced into the abdominal cavity. The postoperative course was uneventful and the baby was free of complaints for more than one year. The pathomechanism of knot formation is not clear thus the discovery of the problem during the operation is an unexpected event. In our opinion tight knot cannot be spontaneously formed intraabdominally. Loose knots can be developed and can reduce the capacity of liquor flow. We think that the knot tightens during pulling out. Longer peritoneal catheters can precipitate multiple looping and/or axial torquations and increase the peripheral resistance of the shunt. In such cases when the pulling out is challenged conversion to laparotomy is suggested.

Hmgb1 can facilitate activation of the matrilin-1 gene promoter by Sox9 and L-Sox5/Sox6 in early steps of chondrogenesis.

The architectural high mobility group box 1 (Hmgb1) protein acts as both a nuclear and an extracellular regulator of various biological processes, including skeletogenesis. Here we report its contribution to the evolutionarily conserved, distinctive regulation of the matrilin-1 gene (Matn1) expression in amniotes. We previously demonstrated that uniquely assembled proximal promoter elements restrict Matn1 expression to specific growth plate cartilage zones by allowing varying doses of L-Sox5/Sox6 and Nfi proteins to fine-tune their Sox9-mediated transactivation. Here, we dissected the regulatory mechanisms underlying the activity of a conserved distal promoter element 1. We show that this element carries three Sox-binding sites, works as an enhancer in vivo, and allows promoter activation by the Sox5/6/9 chondrogenic trio. In early steps of chondrogenesis, declining Hmgb1 expression overlaps with the onset of Sox9 expression. Unlike repression in late steps, Hmgb1 overexpression in early chondrogenesis increases Matn1 promoter activation by the Sox trio, and forced Hmgb1 expression in COS-7 cells facilitates induction of Matn1 expression by the Sox trio. The conserved Matn1 control elements bind Hmgb1 and SOX9 with opposite efficiency in vitro. They show higher HMGB1 than SOX trio occupancy in established chondrogenic cell lines, and HMGB1 silencing greatly increases MATN1 and COL2A1 expression. Together, these data thus suggest a model whereby Hmgb1 helps recruit the Sox trio to the Matn1 promoter and thereby facilitates activation of the gene in early chondrogenesis. We anticipate that Hmgb1 may similarly affect transcription of other cartilage-specific genes.

Occurrence and distribution of polycyclic aromatic hydrocarbons in surface water of the Raba River, Hungary.

The objective of this study was to investigate the concentration level and distribution of polycyclic aromatic hydrocarbons (PAHs) in surface water of the Raba River; the largest Danube tributary in Hungary. A total of 54 water samples were collected in the period of 2008-2011 and analysed for PAHs by gas chromatography-mass spectrometry (GC/MS) method. The total PAH concentrations (sum of the concentrations of 17 individual PAH compounds) ranged from 41 to 437 ng/L with the mean value of 111 ±  69.4 ng/L. Phenanthrene and naphthalene were the dominant species in the surface water. Using TEF approaches on the mean concentration PAH data, benzo[a]pyrene and dibenz[ah]anthracene contributed the highest carcinogenic exposure equivalent. A selected number of concentration ratios of specific PAH compounds were calculated to evaluate the possible sources of PAH contamination. The ratios reflected a pattern of pyrogenic input as a major source of PAHs. The comparison of the total PAH concentrations observed for Raba River with other surface waters of the world confirmed that the Raba River could not be regarded as a contaminated river according to the levels of PAHs.

Recycling of bowel content: the importance of the right timing.

INTRODUCTION: Extracorporeal stool transport (recycling of chyme discharged from the proximal stoma end to the distal end of a high jejunostomy or ileostomy) is thought to be beneficial in preventing malabsoprtion, sodium loss, cholestasis and atrophy of the distal intestine until restoration of the intestinal continuity becomes possible. However little is known about its adverse effects. Our aim was to investigate the microbiological safety of recycling. MATERIAL AND METHOD: Native samples were taken from the proximal stoma in 5 premature neonates who underwent an ileostomy or a jejunostomy due to necrotising enterocolitis, for qualitative culture. The first sample was drawn immediately after the change of the stoma bag, further samples were sent from the stoma bag at 30, 60, 90, 120, 150, and 180min later. The samples were inoculated by calibrated (10 µl) loops onto blood agar (5% sheep blood), eosin-methylene blue agar and anaerobic blood agar, respectively (Oxoid). The aerobic plates were incubated for 18-20 h at 5% CO2, whereas the anaerobic plates were incubated for 24-48 h in an anaerobic chamber (Concept 400). The bacterial strains were identified to species level by specific biochemical reactions, RapID-ANA II system (Oxoid) and ID32E, Rapid ID 32 Strep ATB automatic system cards (bioMérieux). RESULTS: The number of colony forming unit (CFU) of Gram-negative bacteria (mainly E. coli) exponentially increased after 30 min and reached 10(5)/ml after 120 min. Gram-positive strains (primarily E. faecalis) were detected after 60 min and CFU increased to 10(5)/ml after 120 min. The number of anaerobic (principally Bacteroides fragilis) CFU started to increase after 120 min. In two cases coagulase negative Staphylococcus strains were isolated the earliest in the chyme. The average of total CFU approached 10(5)/ml after 90 min and exceeded 10(5)/ml after 120 min. CONCLUSION: The chyme in the stoma bag is colonized by commensal facultative pathogenic enteral/colonic as well as skin flora species after 120 min. Recycling of stoma bag content may be dangerous after 90 min.

Tumor cell expression of heat shock protein (HSP) 72 is influenced by HSP72 [HSPA1B A(1267)G] polymorphism and predicts survival in small Cell lung cancer (SCLC) patients.

The inducible heat shock protein (HSP)72 plays a central role in antitumor immunomodulation. HSP72 expression was assessed on tumor samples of 43 patients with advanced and metastatic small cell lung cancer (SCLC) by immunohistochemistry and HSP72 [HSPA1B A(1267)G] polymorphism was determined. HSP72 expression of SCLC cells was significantly decreased in GG as compared to cells of AA or AG genotype patients, and was associated with significantly shorter survival in GG patients as compared to carriers of the A allele. Decreased HSP72 expression of SCLC cells associated with HSP72 GG genotype is a negative prognostic factor for survival in SCLC patients.

Pyloric atresia associated with Dieulafoy lesion and gastric dysmotility in a neonate.

In this case study, we report a male infant with pyloric atresia, extreme gastric distension, and a caliber-persistent gastric artery (Dieulafoy lesion) with massive gastric bleeding. After a transverse pyloroplasty and endoscopic hemoclip application to the caliber-persistent gastric artery, very slow gastric emptying developed, which required repeated surgical interventions. Gastroduodenostomy failed to promote gastric emptying. The intraoperative and postmortem histologic examinations of the gastric wall revealed a loss of interstitial cells of Cajal, which possibly explains the extreme motility disorder.

In utero incarceration of congenital diaphragmatic hernia.

In utero diagnosis of incarcerated congenital diaphragmatic hernia has never been reported. In our case, congenital diaphragmatic hernia presented at 34 weeks of gestation with dilated bowel loops, pleural effusion, and ascites on fetal ultrasound. Preterm delivery and emergency exploration revealed a tight posterolateral diaphragmatic defect with extensive bowel infarction.

Plant peptides govern terminal differentiation of bacteria in symbiosis.

Legume plants host nitrogen-fixing endosymbiotic Rhizobium bacteria in root nodules. In Medicago truncatula, the bacteria undergo an irreversible (terminal) differentiation mediated by hitherto unidentified plant factors. We demonstrated that these factors are nodule-specific cysteine-rich (NCR) peptides that are targeted to the bacteria and enter the bacterial membrane and cytosol. Obstruction of NCR transport in the dnf1-1 signal peptidase mutant correlated with the absence of terminal bacterial differentiation. On the contrary, ectopic expression of NCRs in legumes devoid of NCRs or challenge of cultured rhizobia with peptides provoked symptoms of terminal differentiation. Because NCRs resemble antimicrobial peptides, our findings reveal a previously unknown innovation of the host plant, which adopts effectors of the innate immune system for symbiosis to manipulate the cell fate of endosymbiotic bacteria.

Significance of off-hours in centralized primary percutaneous coronary intervention network.

AIM: To analyze the efficacy of a regionally organized primary percutaneous coronary intervention (PCI) network at the Heart Center, Semmelweis University Budapest, part of the "Budapest model," and the factors that influence it. METHODS: In order to investigate the differences between regular and off-hours patient care in a 24-hour myocardial infarction primary care system, we included 1890 consecutive, unselected patients with ST-segment elevation myocardial infarction and followed them until at least one year. The follow-up was complete for all participants. RESULTS: The difference between regular hours and off-hours mortality was not significant either after 30 days (8.6% vs 8.8%, respectively) or after 1 year (15.3% vs 14.7%, respectively). The rate of patients with re-infarction, frequency of re-intervention, and major adverse cardiac events, including death, re-infarction, re-intervention, and coronary artery bypass graft surgery, were similar in both patient groups. The time delay between the onset of chest pain and arrival to the clinic was 5.9+/-5.8 hours (mean+/- standard deviation) during regular hours and 5.2+/-4.6 hours during off-hours (P=0.235). Direct transport caused significant decrease in the 30-day and 1-year mortality independent of duty time (7.2% vs 9.9%, P=0.027; 12.6% vs 16.7%, P=0.028; respectively). CONCLUSION: Centralized primary PCI network of the "Budapest model" achieved the same level of patient care during both off-hours and regular hours.

Comparison of elimination and cardiovascular effects of adenine nucleosides administered intrapericardially or intravenously in anesthetized dog.

Intrapericardial (IP) administration of certain cardioactive agents allows investigation of local pharmacological actions on the heart and may carry potential benefit to influence myocardial function. The cardioprotective adenosine (ADO) and inosine (INO) may be the most representative candidates. Elimination and cardiovascular effects of IP and intravenously (IV) applied ADO and INO were compared on anesthetized dogs. Their pericardial and systemic concentrations were measured after consecutive administration of increasing ADO and INO doses. In the case of IP administration at the end of the incubation period, pericardial concentrations of adenine nucleosides significantly exceeded the control values. However, the IV applied ADO and INO were rapidly metabolized in the systemic plasma. As characteristic hemodynamic effects, small but sustained decrease in heart rate (IP ADO) and increase in myocardial contractility (IP INO) were observed. During IV administration, ADO and INO exerted remarkable effects on all hemodynamic variables, which then gradually disappeared in 15 minutes. In summary, the elimination of ADO and INO was significantly slower in the pericardial fluid than in the plasma. Considering the balanced cardiac actions and lack of strong systemic hemodynamic effects, IP administration of adenine nucleosides may suggest a promising approach in the local treatment of the diseased heart.

Comparison of virtual and fiberoptic bronchoscopy in the management of airway stenosis.

Noninvasive imaging methods can be valuable tools for diagnosing thoracic diseases, especially malignancies. The aim of this study was to compare the effectiveness of conventional and virtual bronchoscopy in the follow-up of patients with large airway stenosis. Twenty-three consecutive patients with stenoses of the trachea and/or the main bronchi were enrolled in this prospective observer study. The causes of stenosis included malignant or benign tumours, goiter, and postintubation stenoses. Patients were evaluated before and after treatment (which included mechanical dilation, laser photocoagulation, stent implantation, radiotherapy, chemotherapy, and surgical resection). The mean time between baseline and follow-up endoscopy was 140 days. No significant differences were observed between the estimated and measured data from bronchofibroscopy and virtual bronchoscopy. Exact measurement of stenoses was performed with virtual bronchoscopy.

[Virtual bronchoscopy for follow-up investigation of main airway stenoses]. / A virtuális bronchoszkópia alkalmazása nagylégúti szukületek követéses vizsgálatára.

The aim of the study was to compare the utility and effectivity of conventional and virtual bronchoscopy in the follow up of patients with main airway stenosis. 23 patients with trachea or main bronchi stenosis were enrolled in the study. The causes of the stenosis were malignant or benign tumor, struma and stent. The stenoses were evaluated before and after the different interventions (i.e. mechanical dilatation, laser photocoagulation, stent implantation, radiotherapy, chemotherapy or surgical resection.) The mean time between the first and second endoscopic and virtual bronchoscopic examinations was 140 days. The authors found good correlation between bronchofiberoscopy and virtual bronchoscopy findings. The data of virtual bronchoscopy were more exact. Based on this study the virtual bronchocopy can replace the traditional invasive method to follow-up patients with main airway stenosis.

Intrapericardial adenine nucleosides induce elevation of endothelin-1 concentration in the pericardial space of the dog heart.

The adenine nucleosides, adenosine (ADO) and inosine (INO), and the endothelin-1 (ET-1) play an important role in the regulation of coronary and myocardial functions. The pericardial fluid accumulates these regulatory agents and reflects well their levels in the myocardial interstitium. This offers a possibility to examine the local adenine nucleoside-endothelin interactions in heart tissue. We have previously demonstrated that increased levels of intrapericardial (i.p.) ET-1 enhanced significantly the adenine nucleoside concentrations of the pericardial fluid samples. In this study the effects of i.p.-administered ADO and INO were investigated on the pericardial concentrations of ET-1 in the in situ dog heart. The ET-1 concentrations were determined (enzymelinked immunosorbent assay) in the samples obtained from the pericardial fluid and from the arterial plasma before and after i.p. administration of increasing doses of ADO (10, 100 and 1000 muM, n = 8) and INO (1, 10 and 100 mM, n = 8). Standard hemodynamic variables were recorded continuously. We found that i.p. ADO and INO induced dose-dependent increases of pericardial ET-1 levels after a 15-minute incubation period in pericardial (ET-1max,ADO, +121 +/- 26%, P < 0.02; and ET-1max,INO, +84 +/- 27%, P < 0.05) but not in the plasma samples. The i.p. nucleosides elicited slight but characteristic alterations in the heart rate and ventricular contractility (dP/dt). The results suggest that the pericardial adenine nucleosides interact with the myocardial ET-1 and this effect may be provoked from, and can also be detected in, the pericardium.