Dodecafluoropentane Emulsion Extends Window for tPA Therapy in a Rabbit Stroke Model.

Dodecafluoropentane emulsion (DDFPe) nanodroplets are exceptional oxygen transporters and can protect ischemic brain in stroke models 24 h without reperfusion. Current stroke therapy usually fails to reach patients because of delays following stroke onset. We tested using DDFPe to extend the time window for tissue plasminogen activator (tPA). Longer treatment windows will allow more patients more complete stroke recovery. We test DDFPe to safely extend the time window for tPA thrombolysis to 9 h after stroke. With IACUC approval, randomized New Zealand white rabbits (3.4-4.7 kg, n = 30) received angiography and 4-mm blood clot in the internal carotid artery for flow-directed middle cerebral artery occlusion. Seven failed and were discarded. Groups were IV tPA (n = 11), DDFPe + tPA (n = 7), and no therapy controls (n = 5). DDFPe (0.3 ml/kg, 2 % emulsion) IV dosing began at 1 h and continued at 90 min intervals for 6 doses in one test group; the other received saline injections. Both got standard IV tPA (0.9 mg/kg) therapy starting 9 h post stroke. At 24 h, neurological assessment scores (NAS, 0-18) were determined. Following brain removal percent stroke volume (%SV) was measured. Outcomes were compared with Kruskal-Wallis analysis. For NAS, DDFPe + tPA was improved overall, p = 0.0015, and vs. tPA alone, p = 0.0052. For %SV, DDFPe + tPA was improved overall, p = 0.0003 and vs. tPA alone, p = 0.0018. NAS controls and tPA alone were not different but %SV was, p = 0.0078. With delayed reperfusion, DDFPe + tPA was more effective than tPA alone in preserving functioning brain after stroke. DDFPe significantly extends the time window for tPA therapy.

Progress in dodecafluoropentane emulsion as a neuroprotective agent in a rabbit stroke model.

Dodecafluoropentane emulsion (DDFPe) in 250 nm nanodroplets seems to swell modestly to accept and carry large amounts of oxygen in the body at >29 °C. Small particle size allows oxygen delivery even into hypoxic tissue unreachable by erythrocytes. Using permanent cerebral embolic occlusion in rabbits, we assessed DDFPe dose response as a neuroprotectant at 7 and 24 h post-embolization without lysis of arterial obstructions and investigated blood pharmacokinetics. New Zealand White rabbits (N = 56) received cerebral angiography and embolic spheres (diameter = 700-900 µm) occluded middle and/or anterior cerebral arteries. Intravenous DDFPe dosing (2 % w/v emulsion) began at 60 min and repeated every 90 min until sacrifice at 7 or 24 h post-embolization. Seven-hour groups: (1) control (embolized without treatment, N = 6), and DDFPe treatment: (2) 0.1 ml/kg (N = 7), (3) 0.3 ml/kg (N = 9), (4) 0.6 ml/kg (N = 8). Twenty-four-hour groups: (5) control (N = 16), and DDFPe treatment: (6) 0.1 ml/kg (N = 10). Infarcts as percent of total brain volume were determined using vital stains on brain sections. Other alert normal rabbits (N = 8) received IV doses followed by rapid arterial blood sampling and GC-MS analysis. Percent infarct volume means significantly decreased for all DDFPe-treated groups compared with controls, p = <0.004 to <0.03. Blood DDFP (gas) half-life was 1.45 ± 0.17 min with R = 0.958. Mean blood clearance was 78.5 ± 24.9 ml/min/kg (mean ± SE). Intravenous DDFPe decreases ischemic stroke infarct volumes. Blood half-life values are very short. The much longer therapeutic effect, >90 min, suggests multiple compartments. Lowest effective dose and maximum effective therapy duration are not yet defined. Rapid development is warranted.

Linked linear amplification: a new method for the amplification of DNA.

BACKGROUND: Linked Linear Amplification (LLA) is a new nucleic acid amplification method that uses multiple cycles of primer extension reactions. The presence of nonreplicable elements in LLA primers renders primer extension products unusable as templates for further amplification, leading to linear accumulation of products. Through the use of nested primers, linear reactions can be "linked", providing total amplification yields comparable to those obtained by PCR. METHODS: The LLA model predicts (a) that amplification yield will approach that of PCR as the number of primers increases and (b) that the unique composition of LLA products will give lower carryover amplification efficiency compared with PCR. To test these hypotheses, the human ss-globin gene was amplified by 10-, 14-, or 18-primer LLA and the yield was compared with PCR. Carryover contamination was simulated by reamplifying a dilution series of LLA or PCR products. To demonstrate the clinical utility of the method, LLA coupled with allele-specific oligonucleotide (ASO) capture was used to detect the factor V Leiden mutation in a panel of 111 DNA samples. RESULTS: Fourteen- and 18-primer LLA gave amplification yields comparable to PCR. However, LLA carryover amplification efficiency was four orders of magnitude lower than that of PCR. The LLA-ASO assay detected the correct factor V Leiden genotype in all 111 samples. CONCLUSIONS: LLA is a robust target amplification method that is comparable to PCR in yield. However, LLA is more resistant to false results caused by carryover amplicon contamination.

Evaluation of the BeTha gene 1 kit for the qualitative detection of the eight most common Mediterranean beta-thalassemia mutations.

We describe the evaluation of the Bio-Rad BeTha Gene 1 kit (Bio-Rad Laboratories, Hercules, CA), a DNA-probe assay designed for the qualitative determination of the eight most common Mediterranean beta-thalassemia mutations. The kit utilizes the principle of allele-specific oligonucleotide (ASO) hybridization. Following sample preparation and in vitro DNA amplification by the polymerase chain reaction (PCR), an allele-specific detection of the amplified products by a nonradioactive enzymatic assay is performed. Genomic DNA is prepared from an individual's whole blood with a DNA purification matrix. In a second step, the beta-globin gene is amplified in a multiplex PCR reaction containing four 5' biotinylated oligonucleotide primers. In a final step, an aliquot of the PCR reaction is first chemically denatured and then captured in two eight-well strips of a 96-well enzyme-linked immunosorbent assay (ELISA) plate by hybridization to an immobilized ASO probe. Each DNA sequence at each of the eight mutation sites is represented by one normal and one mutant ASO. During this capture/hybridization step, which is performed at 37 degrees C, only perfectly matched PCR products will be captured by an ASO. Subsequently, the allele-specific captured biotin-labeled PCR products are detected by a colorimetric enzymatic reaction. The system permits the detection of 16 beta-thalassemia alleles using a high-throughput format that can be automated easily. A clinical feasibility study was performed to evaluate the functionality (method comparison study, assay validity using samples previously collected and stored at various temperatures for different periods of time, interference on kit performance, and assay validity for prenatal diagnosis) and the usability (ease of use, sample throughput) of the kit. The analysis of 110 samples previously studied with reference methods showed 100% clinical sensitivity and specificity. We demonstrate here that the procedure not only increases the throughput of beta-thalassemia allele genotyping but also provides an accurate, rapid, reliable, and nonisotopic diagnostic tool.

Ligase chain reaction assay for human mutations: the Sickle Cell by LCR assay.

We can detect the beta-globin gene sickle cell mutation by using an assay based on the ligase chain reaction. The simultaneous amplification of the human growth hormone gene in the same reaction serves as a control for the amount of template DNA or amplification efficiency. Ligation products, which are biotinylated at one end and tagged with an arbitrary "tail" sequence at the other, are captured by hybridization to "tail"-complementary oligonucleotides immobilized on polystyrene microwells. The captured ligation products are detected colorimetrically by use of streptavidin-alkaline phosphatase conjugate. In a study of 24 subjects, the assay unequivocally discriminated among normal, carrier, and sickle cell genotypes.

Application of capillary electrophoresis to the measurement of oligonucleotide concentration and purity over a wide dynamic range.

Synthetic oligonucleotides rarely contain 100% of the full-length sequence due, in part, to the failure sequences produced during synthesis. In this paper, a method is described for the determination of both the concentration and the purity of oligonucleotides, utilizing capillary electrophoresis with a deoxyribo-nucleoside triphosphate as an internal standard. This method is advantageous for several reasons: (a) the wide dynamic range allows for the analysis of samples without the need for dilutions; (b) a small sample size is used for analysis; (c) capillary electrophoresis is automatable which allows for high throughput; and (d) all of the samples are analyzed at the same run temperature which aids in reproducibility and consistency between runs performed at different times.

Indirect laryngoscopic approach for injection of botulinum toxin in spasmodic dysphonia.

Spasmodic dysphonia is a focal dystonia that causes a loss of the fine control of intrinsic laryngeal muscles and produces a strained staccato voice. Temporary relief from symptoms has been reported in patients treated with botulinum toxin percutaneously injected into the thyroarytenoid muscle. A newly developed method of treatment differs from reported methods by increasing the accuracy of botulinum toxin placement, reducing soft tissue trauma, and applying basic scientific information about the functional histology of intrinsic laryngeal musculature. Sixteen patients with primarily adductor spasmodic dysphonia were treated. Initial assessment included laryngeal examination by indirect laryngoscopy, videoendoscopy, and stroboscopy, neurology examination (including laryngeal EMG), and vocal function studies with acoustic analysis and aerodynamic studies. A device originally designed for collagen injection allowed the precise microdelivery of toxin to the thyroarytenoid muscle. Indirect laryngoscopy was used to direct the needle, in an attempt to cover a broad area of motor end plates. The minimally effective dose was titrated for each patient, to avoid paralysis and preserve laryngeal function. All patients showed improved voices after treatment. There were no major complications. The basic technique can be performed in the otolaryngologist's office and does not require electromyography equipment or expertise.