Gergely Rácz

MicroRNA expression profiling in benign (sporadic and hereditary) and recurring adrenal pheochromocytomas

MicroRNAs are involved in the pathogenesis of several tumors, however, there have been no data on microRNA expression in pheochromocytomas to date. The objective of our study was to perform microRNA expression profiling in sporadic and hereditary benign, and recurring adrenomedullary tumors. Furthermore, the applicability of formalin-fixed paraffin-embedded tissue samples for the analysis of microRNA expression in pheochromocytomas was examined. MicroRNA expression data of three matched frozen and formalin-fixed paraffin-embedded samples were correlated. A total of 21 formalin-fixed paraffin-embedded samples (sporadic benign, multiple endocrine neoplasia 2, von Hippel-Lindau disease, sporadic recurring) were subjected to microRNA expression profiling using microarrays. MicroRNAs with significant differences in expression were validated and sample sizes were extended including tumors from neurofibromatosis type 1 patients by real-time quantitative reverse-transcription PCR (n=33). MicroRNA target prediction was carried out by TargetScan and MicroCosm Targets. Pathway analysis of targets was performed by Ingenuity Pathway Analysis and DIANA mirPath. Furthermore, microRNA expression profiles of a malignant pheochromocytoma and a pair of primary and recurrent tumors were studied by TaqMan Human MicroRNA Cards. MicroRNA expression correlated well between frozen and formalin-fixed paraffin-embedded samples (70–92%). Microarray analysis revealed 16 significantly differentially expressed microRNAs. Five of these were validated by real-time RT-PCR. miR-139-3p, miR-541 and miR-765 were significantly differentially expressed between sporadic benign and von Hippel-Lindau-related pheochromocytomas. Significantly higher expression of miR-885-5p and miR-1225-3p was found in multiple endocrine neoplasia type 2 and sporadic recurring pheochromocytomas, respectively. Pathway analysis revealed the possible involvement of Notch- and G-protein-coupled receptor signaling in tumor recurrence. MicroRNA expression profiles in the primary recurrent and recurring malignant comparisons have been similar. In conclusion, we have proved that formalin-fixed paraffin-embedded samples can be used for the analysis of microRNA expression in pheochromocytomas. MicroRNA expression patterns differ between various sporadic, hereditary and recurring tumors and miR-1225-3p may be useful for identifying recurring pheochromocytomas.

Protein kinase Cγ antibodies and paraneoplastic cerebellar degeneration

Onconeural antibodies are diagnostic markers for paraneoplastic neurological syndromes and indicate the underlying tumor type. Recently, a new antibody against protein-kinase Cγ (PKCγ) was detected in a patient with paraneoplastic cerebellar degeneration (PCD). We report here a second patient. A 70-year-old woman presented with cerebellar ataxia, dysdiadochokinesia, and dysarthria. Her serum showed immunoreactivity against the cytoplasm, dendrites and axons of Purkinje cells in tissue-based screening, later confirmed by immunoblot and phage plaques as anti-PKCγ. Tumor search revealed an adenocarcinoma of hepatobiliary origin. Detection of PKCγ antibodies should be considered in PCD and adenocarcinoma without typical onconeural antibodies.

RAPID COMMUNICATION: WATER DISINFECTION BY-PRODUCTS ENHANCED APOPTOTIC ACTIVITY IN HUMAN LYMPHOCYTES

The apoptosis-inducing activity of concentrated sediments of disinfected and non-disinfected water samples from the waterworks in Budapest, Hungary, was investigated using cultures of human peripherial blood lymphocytes. Chlorine-treated water and untreated (raw) water sediments were concentrated with the use of Amberlite XAD-2 resin columns. The concentrates were dissolved in dimethyl sulfoxide and added to cultures of human peripherial blood lymphocytes. Apoptotic index was determined in lymphocytes after treatment with the raw or disinfected concentrates by flow cytomerty. Disinfected water concentrates of 100μl/ml increased the apoptotic ratio of lymphocyte culture. The same amount of raw water concentrate also enhanced apoptosis. Both raw water and disinfected water contain substances that induced a significant rate of apoptosis in lymphocyte cultures.

A common polymorphism of the human cardiac sodium channel alpha subunit (SCN5A) gene is associated with sudden cardiac death in chronic ischemic heart disease

Cardiac death remains one of the leading causes of mortality worldwide. Recent research has shed light on pathophysiological mechanisms underlying cardiac death, and several genetic variants in novel candidate genes have been identified as risk factors. However, the vast majority of studies performed so far investigated genetic associations with specific forms of cardiac death only (sudden, arrhythmogenic, ischemic etc.). The aim of the present investigation was to find a genetic marker that can be used as a general, powerful predictor of cardiac death risk. To this end, a case-control association study was performed on a heterogeneous cohort of cardiac death victims (n=360) and age-matched controls (n=300). Five single nucleotide polymorphisms (SNPs) from five candidate genes (beta2 adrenergic receptor, nitric oxide synthase 1 adaptor protein, ryanodine receptor 2, sodium channel type V alpha subunit and transforming growth factor-beta receptor 2) that had previously been shown to associate with certain forms of cardiac death were genotyped using sequence-specific real-time PCR probes. Logistic regression analysis revealed that the CC genotype of the rs11720524 polymorphism in the SCN5A gene encoding a subunit of the cardiac voltage-gated sodium channel occurred more frequently in the highly heterogeneous cardiac death cohort compared to the control population (p=0.019, odds ratio: 1.351). A detailed subgroup analysis uncovered that this effect was due to an association of this variant with cardiac death in chronic ischemic heart disease (p=0.012, odds ratio = 1.455). None of the other investigated polymorphisms showed association with cardiac death in this context. In conclusion, our results shed light on the role of this non-coding polymorphism in cardiac death in ischemic cardiomyopathy. Functional studies are needed to explore the pathophysiological background of this association.

Presence of cardiomyocytes exhibiting Purkinje-type morphology and prominent connexin45 immunoreactivity in the myocardial sleeves of cardiac veins

BACKGROUND Pulmonary vein (PV) myocardium is a known source of atrial fibrillation. A debated question is whether myocardial extensions into caval veins and coronary sinus (CS) have similar properties. No studies have documented specific pacemaker and/or conducting properties of human extracardiac myocardium. OBJECTIVE The purpose of this study was to characterize the histology and immunohistochemical features of myocardial sleeves in the wall of cardiac veins. METHODS Sections of 32 human hearts were investigated. Specimens of PVs, superior caval vein (SVC), CS, sinoatrial and atrioventricular nodes, and left ventricle were stained with Bests Carmine for selective staining of intracellular glycogen. Anti-connexin45 (Cx45)- and Cx43-specific antibodies were used to determine the conduction properties of extracardiac myocardium. RESULTS Myocardial sleeve was found in the wall of PVs of 15 of 16 hearts, 21 of 22 SVCs, and 8 of 8 CSs. Bundles of glycogen-positive cardiomyocytes exhibiting pale cytoplasm and peripheral myofibrils were observed in the venous sleeves. Strong Cx45 and weak Cx43 labeling was detected in the extracardiac myocardium. Similar staining pattern was observed for the pacemaker and conduction system, whereas ventricular myocardium exhibited prominent Cx43 and no Cx45 immunoreactivity. CONCLUSION Myocardial fibers of PVs, SVC, and CS exhibit morphology similar to that of Purkinje fibers and are enriched in glycogen. We provide data for the first time on prominent positive staining for Cx45 in the extracardiac myocardium, indicating its potential pacemaker and/or conducting nature.

Primary Obstruction of the Foramen of Luschka: Anatomy, Histology, and Clinical Significance

BACKGROUND The foramen of Luschka is a natural aperture between the fourth ventricle and the subarachnoid space at the cerebellopontine angle. Membranous closure of this foramen is referred to as primary obstruction. Available information about this variant and its role in the development of the cysts of the posterior fossa is contradictory. METHODS The macroscopic and histologic features of the obstructed foramina were examined in 61 formalin-fixed human brains (122 foramina). Three rhomboid lips of various sizes with lateral recess were used for comparison. Five postoperative cases of diverticulum of the foramen of Luschka were included in this study, with 1 case presented in detail to illustrate anatomic and histologic findings. RESULTS Primary obstruction was present in 11 of 122 cases. In 1 case, an enlarged rigid pouch with a thick wall was found. The wall of the membrane in primary obstruction and the rhomboid lip were composed of an inner ependymal, a middle glial, and an outer leptomeningeal layer. CONCLUSIONS The rhomboid lip is a remnant of the roof of the fourth ventricle. Imperforation of the foramen of Luschka results in a pouch in the cerebellopontine angle that contains choroid plexus (Bochdaleks flower basket) and communicates with the fourth ventricle. This pouch has the potential to grow to a diverticulum and cause clinical symptoms. Based on our clinical observations, detailed radiologic and surgical-anatomic criteria were proposed to support the differential diagnosis of a diverticulum of the foramen of Luschka. Treatment strategies were also suggested.