Feasibility of the transseptal approach for fast and unstable left ventricular tachycardia mapping and ablation with a non-contact mapping system

Background Radiofrequency ablation of fast and unstable left ventricular tachycardia (VT) usually requires non-contact mapping. The procedure is usually performed by a retrograde-transaortic route, requiring a double femoral artery puncture, for the 9F multielectrode catheter and the 7F ablation catheter which are advanced through the aorta and aortic valve into the left ventricle (LV). Reported limitations of the procedure are due to the stiffness of the balloon catheter, particularly in patients with tortuous peripheral arteries, atherosclerotic aorta, or with aortic stenosis. The aim of our study was to test the feasibility and assess the safety of a transseptal approach for left VT non-contact mapping and ablation.Materials and methods Ten patients with multiple cardiac defibrillator shocks because of fast and unstable VT were selected for non-contact mapping and ablation. After a double transseptal puncture the multielectrode catheter (Ensite Array™, St. Jude Medical) was advanced through a standard 10F introducer to a stable position in the LV apex over a 260 cm length 0.035 J-tip guidewire. The ablation catheter (Celsius™ Thermo-cool, Biosense Webster) was then inserted through the second 8F introducer. Twenty-five monomorphic sustained ventricular tachycardia were induced and ablated at the level of the diastolic pathway or exit point revealed by unipolar isopotential mapping. The total procedural and fluoroscopy times were 209 ± 32 min and 28.5 ± 9.27 min, respectively, which were comparable to those described with the traditional retrograde-transaortic approach. No major complication related with the transseptal approach were reported.Conclusion A transseptal approach can be a feasible and effective alternative approach for mapping and ablation of fast and unstable left VT with a non-contact mapping system.     

Intrasphincteric botulinum toxin injection in the treatment of chagasic achalasia.

According to the WHO, 16-18 million people in Central and South America are infected by Trypanosoma cruzi. Chagasic achalasia affects between 7.1% and 10.6% of the population. The aim of this study was to evaluate the effects of Botox injections in the clinical response and esophageal function of patients with dysphagia due to chagasic achalasia. In total, 24 symptomatic patients with chagasic achalasia were randomly chosen to receive Botulinum Toxin (BT) or saline injected by endoscopy in the lower esophageal sphincter (LES). Patients were monitored with a clinical score of dysphagia and an objective assessment (esophagograms, scintillography, manometry, and nutritional assessment) for a period of 6 months. Clinical improvement of dysphagia was statistically significant (P < 0.001) in patients receiving BT when compared with the placebo. There was no significant difference in the placebo group regarding clinical score, LES basal pressure and esophageal emptying time. Esophageal emptying time in the toxin group was significantly lower than in the placebo (P=0.04) after 90 days. There were non-significant increases in esophageal emptying of 25.36% and 17.39%, respectively, at 90 and 180 days, in the BT group (P=0.266). Gender, age, and baseline LES pressure did not influence the response to BT. Our data strongly suggests that intrasphincteric injection of BT in LES is clinically effective in the treatment of chagasic achalasia.     

Rare recombination events generate sequence diversity among balancer chromosomes in Drosophila melanogaster

Multiply inverted balancer chromosomes that suppress exchange with their homologs are an essential part of the Drosophila melanogaster genetic toolkit. Despite their widespread use, the organization of balancer chromosomes has not been characterized at the molecular level, and the degree of sequence variation among copies of balancer chromosomes is unknown. To map inversion breakpoints and study potential diversity in descendants of a structurally identical balancer chromosome, we sequenced a panel of laboratory stocks containing the most widely used X chromosome balancer, First Multiple 7 (FM7). We mapped the locations of FM7 breakpoints to precise euchromatic coordinates and identified the flanking sequence of breakpoints in heterochromatic regions. Analysis of SNP variation revealed megabase-scale blocks of sequence divergence among currently used FM7 stocks. We present evidence that this divergence arose through rare double-crossover events that replaced a female-sterile allele of the singed gene (sn^X2) on FM7c with a sequence from balanced chromosomes. We propose that although double-crossover events are rare in individual crosses, many FM7c chromosomes in the Bloomington Drosophila Stock Center have lost sn^X2 by this mechanism on a historical timescale. Finally, we characterize the original allele of the Bar gene (B¹) that is carried on FM7, and validate the hypothesis that the origin and subsequent reversion of the B¹ duplication are mediated by unequal exchange. Our results reject a simple nonrecombining, clonal mode for the laboratory evolution of balancer chromosomes and have implications for how balancer chromosomes should be used in the design and interpretation of genetic experiments in Drosophila.Balancer chromosomes are genetically engineered chromosomes that suppress crossing over with their homologs and are used for many purposes in genetics, including construction of complex genotypes, maintenance of stocks, and estimation of mutation rates. Balancers typically carry multiple inversions that suppress genetic exchange or result in the formation of abnormal meiotic products if crossing over does occur (Fig. 1A); for example, single crossovers inside the inverted segment create acentric or dicentric chromosomes that will fail to segregate properly during meiosis or large deletions or duplications that will likely result in inviable gametes (1, 2). Balancers also often carry recessive lethal or sterile mutations to prevent their propagation as homozygotes as well as dominant markers for easy identification. First developed for use in Drosophila melanogaster, balancer chromosomes remain some of the most powerful tools for genetic analysis in this species (3).Open in a separate windowFig. 1.Consequences of a single or double crossover between a WT X chromosome and an X chromosome carrying a single inversion, In(1)dl-49. Euchromatin is shown in blue, heterochromatin is shown in gray, and centromeres are depicted as circles. Thin white lines mark locations of inversion breakpoints, and yellow crosses/thin lines mark locations of crossover events. (A) A single crossover event within the inverted segment results in the formation of chromosomes with deletions and zero (acentric) centromeres or duplications and two (dicentric) centromeres, neither of which will segregate properly during meiosis. (B) A double crossover within an inverted segment results in intact chromosomes with one centromere that will segregate properly during meiosis.Despite their widespread use, very little is known about the organization of Drosophila balancer chromosomes at the molecular level. Since their original syntheses decades ago, balancers have undergone many manipulations, including the addition or removal of genetic markers. Moreover, rare recombination events can cause spontaneous loss of deleterious alleles on chromosomes kept over balancers in stock, as well as loss of marker alleles on balancer chromosomes themselves (3). Likewise, recent evidence has shown that sequence variants can be exchanged between balancer chromosomes and their wild type (WT) homologs via gene conversion during stock construction or maintenance (4, 5). Thus, substantial variation may exist among structurally identical balancer chromosomes owing to various types of sequence exchange.To gain insight into the structure and evolution of balancer chromosomes, we have undertaken a genomic analysis of the most commonly used X chromosome balancer in D. melanogaster, First Multiple 7 (FM7). We have focused on FM7 because this X chromosome balancer series lacks lethal mutations and thus can be easily sequenced in a hemizygous or homozygous state. In addition, the FM7 chromosome has been shown to pair normally along most of its axis with a standard X chromosome, providing a structural basis for possible exchange events (6). Moreover, although details of how early balancers in D. melanogaster were created are not fully recorded, the synthesis and cytology of the FM7 series is reasonably well documented (3).The earliest chromosome in the FM7 series, FM7a, was constructed using two progenitor X chromosome balancers, FM1 and FM6, to create a chromosome carrying three inversions—In(1)sc⁸, In(1)dl-49, and In(1)FM6—relative to the WT configuration (7, 8) (Fig. 2A). Subsequently, a female-sterile allele of singed (sn^X2) was introduced onto FM7a to create FM7c, which prevents the loss of balanced chromosomes carrying recessive lethal or female-sterile mutations (9). More recently, versions of FM7a and FM7c have been generated that carry transgene insertions that allow the determination of balancer genotypes in embryonic or pupal stages (10–14).Open in a separate windowFig. 2.Structure of the FM7 balancer chromosome. Euchromatin is shown in blue, and heterochromatin is shown in gray. (A) Schematic view of the organization of WT and FM7 X chromosomes. FM7 contains three inversions—In(1)sc⁸, In(1)dl-49, and In(1)FM6—relative to WT. The six breakpoint junctions for the three inversions are numbered 1–6 and are shown in detail in B. (B) Location and organization of inversion breakpoints in FM7. Each inversion has two breakpoints that can be represented as A/B and C/D in the standard WT arrangement and as A/C and B/D in the inverted FM7 arrangement, where A, B, C, and D represent the sequences on either side of the breakpoints. Locations of euchromatic breakpoints are on Release 5 genome coordinates, and the identity of the best BLAST match in FlyBase is shown for heterochromatic sequences. Primers used for PCR amplification are shown above each breakpoint; details are provided in Methods and Datasets S2 and S3. Forward and reverse primers are named with respect to the orientation of the assembled breakpoint contigs, not the orientation of the WT or FM7 X chromosome.To identify the inversion breakpoints in FM7 balancers and to study patterns of sequence variation that may have arisen since the origin of the FM7 series, we sequenced genomes of eight D. melanogaster stocks carrying the FM7 chromosome (four FM7a and four FM7c). We discovered several megabase-scale regions in which FM7c chromosomes differ from one another, which presumably arose via double-crossover (DCO) events from balanced chromosomes (Fig. 1B). These DCOs eliminate the female-sterile sn^X2 allele in the centrally located In(1)dl-49 inversion and are expected to confer a fitness advantage to sn⁺ chromosomes, either by allowing propagation of sn⁺ FM7 as homozygotes in females or by sn⁺ FM7 males outcompeting sn^X2 FM7 males in culture. We found that loss of the sn^X2 allele is common in FM7c chromosomes by screening other FM7c-carrying stocks at the Bloomington Drosophila Stock Center. We also identified the breakpoints of the B¹ duplication carried on FM7, and found direct molecular evidence for the role of unequal exchange in the origin and reversion of the B¹ allele (15–19). Our results provide clear evidence that the common assumption that balancers are fully nonrecombining chromosomes is incorrect on a historical timescale, and that substantial sequence variation exists among balancer chromosomes in circulation today.     

Prolonged CD4+ cell/virus load discordance during treatment with protease inhibitor-based highly active antiretroviral therapy: immune response and viral control

Mechanisms that underly discordant CD4+ cell/virus load (VL) responses in patients who receive highly active antiretroviral therapy (HAART) were studied in 30 human immunodeficiency virus (HIV)-positive patients, in 3 groups. Discordant responders maintained CD4+ cell levels >200/mm(3) with stable or increasing trend, despite sustained VLs of 500-5000 copies/mL, for >2 years. Treatment-success patients had CD4+ cell counts >200/mm(3) with stable or increasing trend and VLs <50 copies/mL, for >2 years. Treatment-failure patients initially responded to HAART, followed by decreasing CD4+ cell counts and increasing VLs. Interferon-gamma production to gag and noncytolytic CD8+ cell suppressive activity were greater in discordant responders. Cellular activation was greatest in patients with treatment failure. All discordant responders had non-syncytium-inducing (CCR5-tropic) viruses. Viruses from discordant responders and from patients with treatment failure had extensive resistance mutations; discordant responders had significantly lower viral replication capacities. These findings suggest that discordant responses may be related to enhanced HIV-directed immune responses, diminished cellular activation, decreased viral replication capacity, and preservation of non-syncytium-inducing virus strains.     

An update on chemotherapy for osteosarcoma

Introduction: In the early seventies chemotherapy significantly improved survival in osteosarcoma. Since then minor innovations have occurred although recent years have offered insights of clinical and scientific relevance.Areas covered: This review focuses on the most recent results of phase 3 and 2 studies. Published data or presentations at International meetings are discussed. A specific section discusses recent insights from studies supporting the hypothesis of a possible personalized chemotherapy approach.Expert opinion: Osteosarcoma is a rare tumor and any effort should be made to improve the level of International collaboration. The MAP (methotrexate, doxorubicin and cisplatin) regimen has become the treatment of choice. Poor pathological response to primary chemotherapy is confirmed as a predictive factor of survival and, presently with the available drugs, it is not recommended to intensify or change post-operative treatment on the basis of pathological response to primary chemotherapy. The genomic complexity and the heterogeneity of osteosarcoma makes active and effective molecularly targeted therapies unlikely to be available in the near future. A relation between pharmacogenetic profile and chemotherapy toxicity and prognosis has been reported. The new frontier for clinical research in osteosarcoma is to optimize the use of the active drugs available by personalizing chemotherapy treatment.     

Real-time myocardial contrast echocardiography for the detection of stress-induced myocardial ischemia. Comparison with 99mTc-sestamibi single photon emission computed tomography

BACKGROUND: Real-time contrast echocardiography (MCE) is a new promising technique for assessing myocardial perfusion. The purpose of this study was to test whether realtime MCE can be used to detect functionally significant coronary artery stenosis in patients with known or suspected coronary artery disease. Myocardial contrast echocardiographic studies were compared with nearly simultaneous 99mTc-sestamibi single photon emission computed tomography (SPECT) as a clinical standard reference to evaluate regional myocardial perfusion defects. METHODS: Real-time MCE based on continuous infusion of Optison (8-10 ml/h) was performed in 66 patients during standard 99mTc-SPECT dipyridamole (0.56 mg/kg x 4 min) stress testing. Images were obtained in apical 4- and 2-chamber views, each divided into 6 segments. Tracer uptake and myocardial opacification were visually analyzed for each segment by two pairs of blinded observers and graded as normal, mildly reduced, severely reduced, or absent. In 792 myocardial segments, myocardial opacification by MCE was uninterpretable in 143 (18%) segments and tracer uptake by SPECT was not clearly defined in 92 (12%) segments. Interobserver variability for MCE was good with concordance rates of 83% (kappa=0.72) for rest- and 86% (kappa=0.76) for stress images. Overall concordance between MCE and SPECT was good (83%, kappa=0.63) at a segmental level. In the diagnosis of fixed and reversible defects, and of normal perfusion, concordance rates were 73, 65 and 83%, respectively. When analysis was performed at the regional level, we found comparable levels of concordance rates for LAD (83%, kappa=0.59), LCX (86%, kappa=0.64) and RCA (80%, kappa=0.68) perfusion territories. CONCLUSIONS: These findings suggest that realtime MCE is a clinically acceptable method to evaluate myocardial perfusion defects during dipyridamole stress testing.