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Technical delivery of myogenic cells through an endocardial injection catheter for myocardial cell implantation.

BACKGROUND: The next clinical frontier in the therapeutics of ischemic heart disease may involve the development and delivery of specific molecules and cells into the myocardium. The aim of the present study was to evaluate the efficiency and safety of the MyoStar injection catheter (Biosense-Webster Inc.) that has recently been developed to deliver molecules and cells to the myocardium. The 8 Fr (110 cm length) catheter comprises a navigation sensor with a 27 gauge needle at the distal tip. METHODS: Mouse myogenic cells (C2) were delivered to a tissue culture dish through different modalities: a standard laboratory pipette, a syringe needle (27 gauge) and the injection catheter. The cells were counted and monitored for growth and differentiation in the tissue culture immediately after delivery and two, three and six days later. Cells that were injected through a regular syringe needle or through the injection catheter demonstrated the same capacity to proliferate in tissue culture up to six days. RESULTS: The behavior of the cells in culture (fusion) was identical for the cells delivered to the tissue culture by a pipette or by the injection catheter. CONCLUSION: The results of the present study indicate that delivery of cells through the MyoStar injection catheter is a method with no significant loss or adverse effects to the cells along the path of the catheter. The catheter, which possesses both injection and navigation capabilities, can be used to deliver cell therapy to patients with ischemic heart disease.

Journal Article↗

DNA integrity and transgene expression after passage through the NOGA needle catheter used for therapeutic myocardial angiogenesis.

BACKGROUND: The NOGA (Biosense Webster, Markham, ON, Canada) injection catheter is an innovative navigational device that provides an ideal platform for intra-myocardial injection material. However, injection through a long (1.91 m), narrow (27G) nitinol needle could result in deterioration in the integrity and functionality of DNA. METHODS: To test this possibility, DNA in plasmid form (pcDNA3.1) containing the Lac Z transgene (250 micro l) was passed through the NOGA needle using a hand-held 1 cc syringe at a gentle hand injection pressure (43 +/- 3 PSI, 3.0 +/- 0.2 kg/cm(2)) or at maximal manual pressure (90 +/- 6 PSI, 6.3 +/- 0.4 kg/cm(2)), either once or 20 times. This DNA, compared to DNA not passed through the NOGA needle (control), was then used to transfect primary cultures of rat skin fibroblasts (FB) from Fisher 344 rats and the cells were subsequently stained for beta galactosidase (betagal). RESULTS: Transfection efficiency was significantly reduced by passing the DNA through the needle at both 43 +/- 3 PSI (78 +/- 4% of control, n = 10, P < 0.05 versus control) and 90 +/- 6 PSI (66 +/- 4 % of control, n = 10, P < 0.01 versus control, P < 0.02 versus 43 +/- 3 PSI). Passage of the DNA through the NOGA needle 20 times resulted in a transfection efficiency of only 5 +/- 1% of control (n = 20, P < 0.1 x 10(-11) versus control). Capillary Electrophoresis revealed that the reduction in transfection efficiency was due to a conformational change in the DNA from predominantly supercoiled to nicked and linearized DNA. Transfection efficiency as compared with control decreased as the concentration of the DNA solution which was passed through the needle was increased from 0.3 micro g/ micro l to 2.4 micro g/ micro l. Recovery experiments confirmed that the reduction in transfection efficiency was not due to loss of DNA by binding to the NOGA needle. CONCLUSION: These results suggest that DNA is susceptible to shear forces when injected through the NOGA needle even at nominal clinical injection pressures, suggesting that careful and controlled injections will be required to achieve optimal gene integrity and expression.

Journal Article↗

Double-stranded nucleic acids in liquid-crystalline dispersions as building blocks for cross-linked supramolecular structures.

Double-stranded DNA fixed in a cholesteric liquid-crystalline dispersion was used for generating an ordered supramolecular structure in the presence of anthracycline and copper (II) ions. The structure is stable in a water-salt solution and does not require poly(ethyleneglycol).The ordered network can be immobilized on the surface of a polymeric film, and may collapse in the presence of biologically and pharmacologically relevant compounds. Accordingly, the DNA-based liquid-crystalline network represents the basis to obtain novel highly sensitive biosensing units.

Animals↗

Efficient signaling platforms built from a small catalytic DNA and doubly labeled fluorogenic substrates.

RNA-cleaving deoxyribozyme 8-17 has been increasingly used in nanotechnology and biosensing applications. Conventional methods to equip 8-17 with fluorescent signaling property usually involve covalent attachment of two dyes at nucleotide positions that are far away from the catalytic core, such that the bulky dye structures would not affect the deoxyribozyme activity. However, the maximum fluorescent enhancement associated with these 8-17 constructs is typically < or =10-fold, due to a high fluorescent background. To find an optimal balance between signal enhancement and signaling speed, we have conducted a comprehensive study on the effects of the nature of dyes (Alexa Fluor 488, 546 and 647; QSY 9 and 21) as well as their attaching positions along the substrate strand on the catalytic and signaling performance of 8-17. Our results have indicated that 8-17 is able to cleave almost every modified substrate, including those that have chromophores only 1 nt away from the cleavage site. Most importantly, almost all of these substrates are able to generate 15- to 85-fold signal enhancement within 10 min. We have also provided guidelines for selecting substrates that could offer the best signal enhancement, the fastest signaling speed, or the best balance between signal enhancement and signaling speed.

Catalysis↗

A set of multicolored Photinus pyralis luciferase mutants for in vivo bioluminescence applications.

Error-prone PCR was used to isolate Photinus pyralis luciferase mutants producing bright light in the red-orange region of the spectrum. All mutations were clustered in the beta5-alpha10-beta6 region of N-terminal subdomain B and appear to affect bioluminescence color by modulating the position of the Ser314-Leu319 mobile loop with respect to the putative active site. Two red variants (Q283R and S284G) and one orange mutant (S293P) contained a single substitution. Although the remaining orange variant contained two mutations, L287I mainly contributed to the color change. Emission spectra collected on whole cells at pH 7.0 revealed that while a single peak of lambdamax approximately 605 nm accounts for red light production by the Q283R and S284G variants, orange light results from the contribution of two peaks of lambdamax approximately 560 and 600 nm. All spectra underwent a red-shift when cells were assayed under acidic conditions, whereas a blue-shift was observed at pH 8.0, indicating that the internal pH of Escherichia coli is close to the external pH shortly after imposition of acid or alkaline stress. In addition, changes in assay pH led to bimodal emission spectra, lending support to the idea that bioluminescence color is determined by the relative contribution of yellow-green and red-orange peaks. The set of multicolored luciferase mutants described here may prove useful for a variety of applications including biosensing, pH monitoring, and tissue and animal imaging.

Animals↗

Potato Black Scurf and Stem Canker: Pathogen Biology, Global Distribution, and Traditional and Modern Diagnostics.

Rhizoctonia solani is a soil- and seed-borne fungal pathogen of potatoes. It is a persistent threat to potato production worldwide. The symptoms appear as black scurf on tubers and stem canker, causing severe yield and quality losses of potatoes. The pathogen reproduces asexually via hyphae and sclerotia. Its genetic diversity is organized into anastomosis groups (AGs), with AG3-PT being the predominant group on potato. The global trade of seed potatoes is very important for agricultural development; however, it has facilitated the dissemination of the pathogen across regions. Moreover, disease development is affected by environmental and agronomic factors, causing variable symptom severity and differential economic impacts. Given the pathogen's genetic complexity, accurate diagnosis is very important, necessitating a transition from traditional culture-based and biochemical methods toward molecular, genomic, and emerging digital technologies. Methods such as PCR, isothermal amplification, sequencing, sensor-based biosensing, and artificial intelligence-driven imaging have improved the detection, quantification, and noninvasive monitoring of the pathogen. Combining these diagnostic methods into a tiered framework will be helpful for precision disease surveillance, informed disease management decision-making, and the development of sustainable potato production systems.

black scurf↗

Novel magnetic technology for intraoperative intracranial frameless navigation: in vivo and in vitro results.

OBJECTIVE: To characterize the accuracy of the Magellan electromagnetic navigation system (Biosense Webster, Tirat HaCarmel, Israel) and to demonstrate the feasibility of its use in image-guided neurosurgical applications. DESCRIPTION OF INSTRUMENTATION: The Magellan system was developed to provide real-time tracking of the distal tips of flexible catheters, steerable endoscopes, and other surgical instruments, using ultra-low electromagnetic fields and a novel miniature position sensor for image-correlated intraoperative navigation and mapping applications. METHODS: An image registration procedure was performed, and static and qualitative accuracies were assessed in a series of phantom, animal, and human neurosurgical studies. EXPERIENCE AND RESULTS: During the human study phase, an accuracy error of up to 5 mm was deemed acceptable. Results demonstrated that this degree of accuracy was maintained throughout all procedures. All anatomic landmarks were reached with precision and were accurately viewed on the display screen. Navigation that relied on the system was also successful. No interference with operating room equipment was noted. The accuracy of the system was maintained during regular surgical procedures, using standard surgical tools. CONCLUSION: The system provides precise lesion localization without limiting the line of vision, the mobility of the surgeon, or the flexibility of instruments. Electromagnetic navigation promises new advances in neuronavigation and frameless stereotactic surgery.

Animals↗

Catheter-based plasmid-mediated transfer of genes into ischemic myocardium using the pCOR plasmid.

BACKGROUND: Direct transfer of genes holds promise for the sustained delivery of therapeutic proteins to treat cardiovascular diseases. This can be accomplished by several approaches, including use of adenoviral vectors and naked plasmid DNA vectors. We previously demonstrated achieval of effective delivery of genes into the myocardium with a left ventricular-guided catheter-based approach using an adenoviral vector. OBJECTIVE: To evaluate the levels and duration of expression of genes induced after injection of a specific plasmid vector, using the same delivery platform as that in our previous work. METHODS: The pCOR plasmids are narrow-host-range plasmid vectors designed for nonviral gene therapy. We tested the ability of the pCOR plasmid vector to express its transgene after injection into the myocardium of pigs with chronic experimental ischemia using a catheter-based transendocardial delivery system. Four animals were subjected to transendocardial injections of the luciferase reporter pCOR gene into ischemic and nonischemic zones using the Biosense intramyocardial injection catheter. Injections (1 mg per animal, 50 micrograms per injection site) were performed at 20 sites in ischemic and nonischemic zones. Measurements of luciferase activity were performed 3 and 7 days thereafter. RESULTS: We observed high levels of expression of luciferase gene in ischemic and nonischemic regions (on days 3 and 7, respectively, in ischemic zone 58,237 and 33,709 pg; in nonischemic zone 39,928 and 46,036 pg). Control noninjected samples from the left and right ventricles contained no detectable luciferase activity. CONCLUSIONS: With a catheter-based approach, the pCOR plasmid was successfully used to deliver genes into designated myocardial regions, and provides sustained expression of protein for at least 7 days, of roughly similar magnitudes in ischemic and nonischemic myocardium.

Animals↗

Correlation between endocardial voltage mapping and myocardial perfusion: implications for the assessment of myocardial ischemia.

BACKGROUND: Recent clinical studies using a non-fluoroscopic three-dimensional (3D) left ventricular (LV) mapping system suggested reduced endocardial voltage amplitudes measured in zones sustaining myocardial ischemia or infarction. However, the direct relationship between myocardial perfusion and endomyocardial voltage amplitudes has not been fully elucidated. METHODS: In a pig model of chronic myocardial ischemia (n = 20), LV endocardial unipolar voltage (UpV) mapping was performed using the Biosense 3D navigation system (Johnson and Johnson, Warren, New Jersey, USA) 4 weeks after ameroid constrictor placement around the left circumflex coronary artery. Echocardiography was used to assess regional changes in myocardial wall thickening (MT) and fluorescent microspheres (4 x 10/injection) were used to quantify rest regional myocardial blood flow (MBF) in ischemic (left circumflex) and remote non-ischemic (left anterior descending) regions. RESULTS: UpV measurements were reduced in ischemic compared to non-ischemic zones (9.9+/-3.3 compared with 13.3+/-3.3 mV, P = 0.03). This corresponded to changes in endocardial MBF and MT, which were both noted to be significantly reduced in the ischemic compared to the non-ischemic area (MBF, 0.50+/-0.16 compared with 0.74+/-0.15 ml/g per min, P = 0.001; MT, 26.1+/-12.0 compared with 37.4+/-10.1%, P=0.003). A positive linear correlation was found between UpV at rest and endomyocardial (but not epicardial) perfusion: UpV (mV) = 7.8+5.9xMBFendocardial (r = 0.32, P = 0.05). CONCLUSIONS: Chronic myocardial ischemia, resulting in reduced perfusion and function at rest (that is, hibernating myocardium), is characterized by a significant reduction ( approximately 25%) in endocardial UpV potentials, which correlates with reduced endomyocardial blood flow and tissue perfusion at rest.

Animals↗

Microfabricated electrophoresis systems for DNA sequencing and genotyping applications: current technology and future directions.

Many routine genomic-analysis assays rely on gel electrophoresis to perform size-selective fractionation of DNA fragments in the size range below 1 kb in length. Over the past decade, impressive progress has been made towards the development of microfabricated electrophoresis systems to conduct these assays in a microfluidic lab-on-a-chip format. Since these devices are inexpensive, require only nanolitre sample volumes, and do not rely on the availability of a pre-existing laboratory infrastructure, they are readily deployable in remote field locations for use in a variety of medical and biosensing applications. The design and construction of microfabricated electrophoresis devices poses a variety of challenges, including the need to achieve high-resolution separations over distances of a few centimetres or less, and the need to easily interface with additional microfluidic components to produce self-contained integrated DNA-analysis systems. In this paper, we review recent efforts to develop devices to satisfy these requirements and live up to the promise of fulfilling the growing need for inexpensive portable genomic-analysis equipment.

DNA↗

Biosensors in process control.

Improvement in bioprocess control will require development of monitors for a wide range of cell-growth and downstream-process parameters. These requirements are examined in terms of the development and application of biosensor devices and physical measurement techniques. Acoustic, dielectric and laser light scattering techniques are discussed primarily as monitors and analysers for biomass parameters. Developments with biosensor devices are discussed in terms of their application in membrane sampling-flow analysis probes and the potential of more direct biosensing principles.

Animals↗

Light-induced in situ patterning of DNA-tagged biomolecules and nanoparticles.

We present an in situ method for the selective manipulation of DNA-tagged nano-objects such as vesicles or gold colloids in aqueous solution, at neutral pH. The method makes use of the photosensitizer concept found in photodynamic therapy. Here, single-stranded DNA is immobilized onto a surface via the biotin/streptavidin linkage. If the streptavidin is fluorescently labeled, reactive species will be created during laser-induced photobleaching of the label. These reactive species can then completely or partly suppress the DNA hybridization and cause the removal of the streptavidin. The technique thereby enables a dynamic on-off control over surface density of immobilized DNA-tagged nano-objects. Furthermore, combining this in situ manipulation of DNA with prepatterning of single-stranded DNA in the micro and later in the nano range provides a means for the dynamic patterning required for applications in biosensing and nanotechnology.

Adsorption↗

Incidence, time course, and characteristics of microbubble formation during radiofrequency ablation of pulmonary veins with an 8-mm ablation catheter.

BACKGROUND: Microbubble formation during pulmonary vein (PV) radiofrequency (RF) ablation of atrial fibrillation (AF) occurs relatively frequently. Prior studies have shown that microbubble formation may be associated with an increased risk of complications. However, the incidence, time course, and temperature characteristics of microbubble formation during AF ablation with an 8-mm catheter have not been prospectively described in humans. METHODS: We studied 46 (30 men, age 56+/-10 years) patients with AF who underwent RF ablation of PVs between January 2005 and December 2005 using an 8F, 8-mm Biosensetrade mark ablation catheter (Biosense-Webster, Diamond Bar, CA, USA). All patients underwent continuous intracardiac echocardiography (ICE). Microbubble patterns were classified as either type 1 (intermittent, scattered microbubble formation) or type 2 (explosive shower of dense microbubbles). Formation of any microbubbles was detected by ICE and the time, PV location, and electrode temperature were recorded. RESULT: A total of 1,479 (32+/-13, range 12-73) RF lesions were delivered to 167 veins. Twenty (2%) lesions were classified as type 2. Since the number of lesions resulting in type 2 bubbles was very small, only type 1 lesions were included in the final analysis. Thirty-nine (85%) patients had at least one lesion associated with bubble formation during ablation (mean: 7+/-7 lesions, range 1-28 lesions). Twenty-three percent (327) of the RF lesions resulted in bubble formation. RF generator power setting during lesions resulting in bubble formation was lower than lesions which did not result in bubble formation (47.9+/-7.4 W vs 49.7+/-7.1 W, P<0.001). Logistic regression analysis revealed a significant negative correlation (P<0.001) between RF generator power settings and a positive correlation between the generator temperature settings and formation of bubbles (both P<0.02). However, the maximum temperature attained was not different between lesions resulting in bubble formation (n=327) and those which did not result in bubble formation (n=1,139). Fifty-three (16%) of the lesions associated with bubble formation occurred within 2-10 seconds after RF was begun. Bubble formation was significantly more frequent in left superior PVs compared to the other PVs (left superior PV 27.3% left inferior PV 18.6%, right superior PV 20.5%, and right inferior PV 18.8%, P=0.005, left superior PV vs other PVs, P<0.001) even after adjustment for the other factors including generator power settings and the temperature setting. CONCLUSION: Bubble formation is common during RF ablation of PV with 8-mm tip catheter and can occur as early as 2 seconds after starting RF. RF generator power is negatively correlated with bubble formation while generator temperature settings are positively correlated with formation of bubbles. Microbubble formation is also more frequent with ablation of the left superior PV probably due to better catheter contact in that area.

Atrial Fibrillation↗

Use of multisite electroanatomic mapping to facilitate ablation of intra-atrial reentry following the Mustard procedure.

Ablation of intra-atrial reentrant tachycardia following Mustard or Senning procedures has low success rates. The Biosense Carto system was used to map intra-atrial reentry in a 22-year-old woman who had undergone a Mustard procedure. A line of block was created connecting a Mustard baffle suture line to the tricuspid valve annulus, which terminated the arrhythmia and prevented its reinitiation. Multisite electroanatomic mapping was invaluable in defining atrial anatomy and the intra-atrial reentrant pathway, and in creating a contiguous line of block. This mapping may improve ablation success rates in patients following the Mustard or Senning repair.

Adult↗

An innovative application of anatomic electromagnetic voltage mapping in a patient with Ebstein's anomaly undergoing permanent pacemaker implantation.

A novel application of the Biosense CARTO System anatomic electromagnetic voltage mapping is presented, utilized as a guide for permanent pacemaker placement. The technique is illustrated in the successful implantation of an atrial lead in a patient with Ebstein's anomaly characterized by a severely dilated right atrium and extremely low-amplitude voltage signals, requiring a DDD pacemaker. Electromagnetic voltage mapping can be used in selected patients with structural heart disease to determine the optimal site for permanent pacemaker lead placement.

Adult↗

Ablation of atrial fibrillation in humans using a balloon-based ablation system: identification of the site of phrenic nerve damage using pacing maneuvers and CARTO.

Injury of the phrenic nerve during pulmonary vein isolation for the treatment of atrial fibrillation is a well-recognized complication, especially when performing ostial ablations or using balloon-based technologies. This report describes the exact anatomical location of phrenic nerve injury during an attempt of right superior pulmonary vein isolation using a balloon that delivered high intensity focused ultrasound. Electroanatomical three-dimensional CARTO (Biosense Webster, Diamond Bar, CA, USA) mapping of the superior caval vein, the right and left atrium, as well as the right superior pulmonary vein was performed in conjunction with meticulous phrenic nerve pacing maneuvers before and after ablation and showed that the nerve was damaged at the level of the antero-inferior ostium of the right superior pulmonary vein. Diaphragmatic denervation occurred despite using an oversized balloon fluoroscopically placed at the atrial side of the pulmonary vein ostium.

Atrial Fibrillation↗

A chamber to permit invasive manipulation of adherent cells in laminar flow with minimal disturbance of the flow field.

An obstacle to real-time in vitro measurements of endothelial cell responses to hemodynamic forces is the inaccessibility of the cells to instruments of measurement and manipulation. We have designed a parallel plate laminar flow chamber that permits access to adherent cells during exposure to flow. The "minimally invasive flow device" (MIF device) has longitudinal slits (1 mm wide) cut in the top plate of the chamber to allow insertion of a recording, measurement, or stimulating instrument (e.g., micropipette) into the flow field. Surface tension forces at the slit openings are sufficient to counteract the hydrostatic pressure generated in the chamber and thus prevent overflow. The invasive probe is brought near to the cell surface, makes direct contact with the cell membrane, or enters the cell. The slits provide access to a large number (and choice) of cells. The MIF device can maintain physiological levels of shear stress (<1-15 dyn/cm2) without overflow in the absence and presence of fine instruments such as micropipettes used in electrophysiology, membrane aspiration, and microinjection. Microbead trajectory profiles demonstrated negligible deviations in laminar flow near the surface of target cells in the presence of microscale instruments. Patch-clamp electrophysiological recordings of flow-induced changes in membrane potential were demonstrated. The MIF device offers numerous possibilities to investigate real-time endothelial responses to well-defined flow conditions in vitro including electrophysiology, cell surface mechanical probing, local controlled chemical release, biosensing, microinjection, and amperometric techniques.

Animals↗

The propagation of Lamb waves in a plate bordered with layers of a liquid.

The influence of liquid layers on the propagation of Lamb waves in a plate of finite thickness is studied theoretically. The dispersion equations of Lamb waves in a plate bordered with layers of liquids are derived. Numerical solutions of the equations show that the phase velocity of Lamb waves changes with the thickness of the liquid layers. For the lowest antisymmetrical mode of very thin plates, the numerical results calculated from the dispersion equations are compared with those derived from the bending wave acoustic impedance approach. The limitation of the latter is discussed. Applications of Lamb waves pertinent to biosensing are also presented.

Acoustics↗