Dihydrotestosterone and Leptin Regulate Gonadotropin-Releasing Hormone (GnRH) Expression and Secretion in Human GnRH-Secreting Neuroblasts

IntroductionThe reversal of hypogonadotropic hypogonadism (HH), occurring after discontinuation of testosterone therapy in adolescents with delayed puberty and in a small percentage of adults with congenital HH, suggests a role for androgens in favoring a spontaneous recovery of reproductive function.AimWe investigated the effect of androgens and leptin on gonadotropin-releasing hormone (GnRH) expression and secretion in human GnRH-secreting neuroblasts (FNC-B4).MethodsQuantitative real-time polymerase chain reaction RT-PCR for mRNA expression and radioimmunoassay for GnRH secretion were used. Immunohistochemical studies assessed GnRH protein expression. FNC-B4 migration was analyzed with multiwell Boyden chamber technique.Main Outcome MeasuresEffects of the non-aromatizable androgen dihydrotestosterone (DHT) and leptin in FNC-B4 were tested after 24 and 48 hours.ResultsExposure to increasing concentrations of DHT after 24 hours significantly stimulated GnRH mRNA in FNC-B4. This effect was still present after prolonged exposure (48 hours). Similarly, treatment with leptin significantly induced GnRH mRNA after 24 hours, but not at 48 hours. Interestingly, mRNA for leptin receptors (LEPR) was significantly reduced after 48 hours of leptin, while, at this time point, it was stimulated by DHT. Coincubation for 48 hours with leptin and DHT maintained the stimulatory effect on both GnRH and LEPR mRNA, suggesting that DHT could stabilize the leptin effect by preventing downregulation of LEPR. Similar results were obtained for GnRH protein expression analysis. Moreover, both DHT and leptin increased GnRH release into the culture medium. We also found that DHT or leptin treatment significantly increased FNC-B4 basal migration. As we previously found that GnRH stimulates FNC-B4 migration, we hypothesized that this effect could be mediated by DHT- and leptin-induced GnRH release. Accordingly, the GnRH antagonist cetrorelix inhibited DHT- and leptin-induced migration.ConclusionOur results suggest that androgens (adequate hormonal status) could have a positive effect on GnRH neuronal activity by synergizing with leptin (adequate energy status) in the regulatory mechanisms required for reproductive and sexual fitness. Morelli A, Fibbi B, Marini M, Silvestrini E, De Vita G, Chavalmane AK, Vignozzi L, Filippi S, Forti G, Vannelli GB, and Maggi M. Dihydrotestosterone and leptin regulate gonadotropin-releasing hormone (GnRH) expression and secretion in human GnRH-secreting neuroblasts. J Sex Med 2009;6:397–407.     

Mutation analysis of the RET receptor tyrosine kinase in Hirschsprung disease

Hirschsprung disease (HSCR), or congenital agangiionic megacolon,is the most common cause of congenital bowel obstruction withan incidence of 1 in 5000 live births. Recently, linkage ofan incompletely penetrant, dominant form of HSCR was reported,foilowed by identification of mutations in the RET receptortyrosine kinase. To determine the frequency of RET mutationsin HSCR and correlate genotype with phenotype, we have screenedfor mutations among 80 HSCR probands representing a wide rangeof phenotypes and family structures. Polymerase chain reaction(PCR) and single-strand conformation polymorphism (SSCP) analysisof RET's 20 exons for mutations among probands revealed eightputative mutations (10%). Sequence changes, which included missense,frameshift and complex mutations, were detected in both familialand isolated cases, among patients with both long-and short-segmentHSCR and in three kindreds with other phenotypes (maternal deafness,talipes and mairotation of the gut, respectively). Two mutations(C609Y and C620R) we identified have previously been associatedwith multiple endocrine neoplasla type 2A (MEN2A), medullarythyroid carcinoma (MTC) and, on rare occasions, HSCR. Thus,while HSCR family members may be at risk for developing neuroendocrinetumors, it follows that identical mutations in RET may be ableto participate in the pathogenesis of distinct phenotypes. Ourdata suggest that: (i) the overall frequency of RET mutationsin HSCR patients is low and therefore, other genetic and/orenvironmental determinants contribute to the majority of HSCRsusceptibility, and (ii) at present, there is no obvious relationshipbetween RET genotype and HSCR phenotype.     

Azithromycin and tezacaftor/ivacaftor is associated with first-degree heart block in an adult with cystic fibrosis

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene is expressed in the heart, but this is not known to cause myocardial dysfunction or abnormalities in the ECG in people with CF.CFTR modulators such as tezacaftor/ivacaftor improve lung function and overall health in people with CF. Minor adverse events have been reported in the early clinical trials, but no consistent changes in the ECGs have been reported.This case highlights an unusual side effect of first degree heart block that occurred after more than 8 months of azithromycin and tezacaftor/ivacaftor in combination. Drug withdrawal and reintroduction confirmed that neither drug alone, but only the combination, caused this change. As tezacaftor/ivacaftor is also present in elexacaftor/tezacaftor/ivacaftor, care may be needed to exclude this delayed interaction with azithromycin.     

Temporary occlusion of the great cardiac vein and coronary sinus to facilitate radiofrequency catheter ablation of the mitral isthmus

Introduction: Ablation of the mitral isthmus to achieve bidirectional conduction block is technically challenging, and incomplete block slows isthmus conduction and is often proarrhythmic. The presence of the blood pool in the coronary venous system may act as a heat-sink, thereby attenuating transmural RF lesion formation. This porcine study tested the hypothesis that elimination of this heat-sink effect by complete air occlusion of the coronary sinus (CS) would facilitate transmural endocardial ablation at the mitral isthmus.
Methods: This study was performed in nine pigs using a 30 mm-long prototype linear CS balloon catheter able to occlude and displace the blood within the CS (the balloon was inflated with ∼5 cc of air). Using a 3.5 mm irrigated catheter (35 W, 30 cc/min, 1 minute lesions), two sets of mitral isthmus ablation lines were placed per animal: one with the balloon deflated (CS open) and one inflated (CS Occluded). After ablation, gross pathological analysis of the linear lesions was performed.
Results: A total of 17 ablation lines were placed: 7 with CS Occlusion, and 10 without occlusion. Despite similar biophysical characteristics of the individual lesions, lesion transmurality was consistently noted only when using the air-filled CS balloon.
Conclusions: Temporary displacement of the venous blood pool using an air-filled CS balloon permits transmurality of mitral isthmus ablation; this may obviate the need for ablation within the CS to achieve bidirectional mitral isthmus conduction.     

Cooled needle catheter ablation creates deeper and wider lesions than irrigated tip catheter ablation

OBJECTIVES: To design and test a catheter that could create deeper ablation lesions. BACKGROUND: Endocardial radiofrequency (RF) ablation is unable to reliably create transmural ventricular lesions. We designed an intramural needle ablation catheter with an internally cooled 1.1-mm diameter straight needle that could be advanced up to 14 mm into the myocardium. The prototype catheter was compared with an irrigated tip ablation catheter. METHODS: Ablation lesions were created under general anesthesia in 14 male sheep (weight 44 +/- 7.3 kg) with fluoroscopic guidance. Each of the catheters was used to create two ablation lesions at randomly allocated positions within the left ventricle. The irrigation rate, target temperature, and maximum power were: 20 mL/min, 85 degrees C, 50 W for the intramural needle catheter and 20 mL/min, 50 degrees C, 50 W for the irrigated tip catheter, respectively. All ablations were performed for 2 minutes. After the last ablation, blue tetrazolium (12.5 mg/kg) was infused intravenously. The heart was removed via a left thoracotomy after monitoring the sheep for one hour. RESULTS: There was no evidence of cardiac tamponade in any sheep. The intramural needle catheter lesions were significantly wider (10.9 +/- 2.8 mm vs 10.1 +/- 2.4 mm, P = 0.01), deeper (9.6 +/- 2.0 mm vs 7.0 +/- 1.3 mm, P = 0.01), and more likely to be transmural (38% vs 0%, P = 0.03). CONCLUSIONS: Cooled intramural needle ablation creates lesions that are significantly deeper and wider than endocardial RF ablation using an irrigated tip catheter in sheep hearts. This technology may be useful in treating ventricular tachycardia resistant to conventional ablation techniques.     

Positional identification of hypertension susceptibility genes on chromosome 2

Chromosome 2 has been consistently identified as a genomic region with genetic linkage evidence suggesting that one or more loci contributes to blood pressure and hypertension status. As with all complex disease traits, following-up linkage evidence to identify the underlying susceptibility gene(s) is an arduous yet biologically and clinically important task. Using combined positional candidate gene methods, the Family Blood Pressure Program (FBPP) has concentrated efforts in narrowing a large region of chromosome 2, demonstrating evidence for linkage in several populations, and identifying underlying candidate hypertension susceptibility gene(s). Initial informatics efforts identified the boundaries of the region and the known genes within it. A total of 82 polymorphic sites in 8 genes were genotyped in a large hypothesis-generating sample consisting of 1640 African Americans, 1339 whites, and 1616 Mexican Americans. After resampling-based false discovery adjustment, SLC4A5, a sodium bicarbonate transporter, was identified as a primary candidate gene for hypertension. Polymorphisms in SLC4A5 were subsequently genotyped and analyzed for validation in two other subcomponents of the FBPP, each contributing African Americans (N=461; N=778) and whites (N=550; N=967). Again, single nucleotide polymorphisms within this gene were significantly associated with blood pressure levels and hypertension status. Although not identifying a single causal gene variant that is significantly associated with blood pressure levels and hypertension status across all samples, the results further implicate SLC4A5 as a candidate hypertension susceptibility gene. Moreover, the present study validates previous evidence for one or more genes on chromosome 2 that influence hypertension-related phenotypes in the population-at-large.     

Direct N-heterocyclization of hydrazines to access styrylated pyrazoles: synthesis of 1,3,5-trisubstituted pyrazoles and dihydropyrazoles

A microwave-assisted method has been developed for the synthesis of tri-substituted pyrazoles via direct N-heterocyclization of hydrazines with metal-acetylacetonate and -dibenzylideneacetonate without using any base or additives. Most importantly, the synthesis of 1-aryl-5-phenyl-3-styryl-1H-pyrazoles was achieved in a single step using hydrochloride salt of various phenylhydrazines and this is the first report for direct construction of these molecules. The reaction medium and microwave conditions play a critical role for their selective product formation during the reaction. The present reaction explored the usage of metal-diketonic complexes as reaction substrates providing acetylacetone and dibenzylideneacetone moieties to directly participate in cyclization with hydrazines to form the corresponding pyrazoles in excellent yields. The present protocol introduces the important N-heterocyclic moieties in the final structures, giving the reaction great applications from a medicinal chemistry perspective, particularly in the late stage modification strategies in drug discovery.

The synthesis of tri-substituted pyrazoles was achieved via direct N-heterocyclization of hydrazines with metal-acetylacetonate and -dibenzylideneacetonate without using any base or additives under microwave irradiation in a single step.     

Fusion imaging: a bipartite approach

Image fusion is the process of registering and combining multiple images from single or multiple imaging modalities to improve the imaging quality and applicability. It reduces randomness and redundancy to increase the diagnostic value of images for better assessment of medical problems. Fusion imaging was designed to overcome the disadvantages of morphological and/or functional imaging, and attempts to provide inputs that improve treatment planning, resulting in better prognostication. This review attempts to summarize the techniques and their applications in head and neck imaging.     

Full-color direct visualization of the atrial septum to guide transseptal puncture

Introduction: Transseptal puncture is required for many interventional procedures but has a serious complication rate of ～1%—primarily related to misidentification of the fossa ovalis resulting in inadvertent puncture of other cardiac structures. We investigated the utility of a full color visualization catheter to correctly position and guide transseptal puncture of the fossa ovalis. Methods and Results: Transseptal puncture and left atrial cannulation were performed after visualization of the atrial septum and fossa ovalis with the visualization catheter (IRIS, Voyage Medical Inc.) on six swine. For each animal, the transseptal puncture was performed twice and the catheter was examined for clot after each puncture. The 12 transseptal punctures required 6.8 ± 3.6 minutes procedural time and 300 ± 94 mL of fluid administered per procedure (i.e., two punctures). IRIS visualization of the atrial septum correlated well with postmortem examination of the atrial septum. In the three animals in which a patent foramen ovale was present (as confirmed by pathological examination), it was also correctly identified by in vivo visualization using the IRIS catheter. Conclusion: The IRIS catheter allows direct in vivo visualization of the interatrial septum to guide transseptal puncture of previous punctures.