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A fast, reliable, and inexpensive method was developed for titration of dengue viruses in microtiter plates with an indirect fluorescent-antibody technique. No significant differences were found in median infectious dose endpoints of samples titrated in microtiter plates as compared with titrations in multichambered slides.
Plaque assays for titrating dengue virus (DENV) are time-consuming and not suitable for strains that do not plaque. Fluorescence-activated cell sorting (FACS) has been used to detect DENV-infected cells. Here we describe a FACS-based assay for titrating DENV. We determined that at 24 h postinfection, the number of infected cells detected by FACS represented the first round of infection and therefore could be used as a readout of the number of infectious particles in the inoculum. When the titers of different laboratory and clinical strains of DENV were compared using FACS, plaque, and endpoint dilution assays, for most strains the FACS titers were comparable to titers obtained by plaque or endpoint dilution assays. The FACS assay is an improvement over the plaque assay because the infection period is reduced from 5 to 7 days to 24 h and the assay can be used to titrate clinical isolates that frequently do not form clear plaques on cell monolayers. The novel FACS-based methods described here will facilitate laboratory studies of dengue.
Recombinant adeno-associated virus (rAAV) vectors have many advantages for gene therapeutic applications compared with other vector systems. Several methods that use plasmids or helper viruses have been reported for the generation of rAAV vectors. Unfortunately, the preparation of large-scale rAAV stocks is labor-intensive. Moreover, the biological titration of rAAV is still difficult, which may limit its preclinical and clinical applications. For this study, we developed a novel strategy to generate and biologically titrate rAAV vectors. A recombinant pseudorabies virus (PrV) with defects in its gD, gE, and thymidine kinase genes was engineered to express the AAV rep and cap genes, yielding PS virus, which served as a packaging and helper virus for the generation of rAAV vectors. PS virus was useful not only for generating high-titer rAAV vectors by cotransfection with an rAAV vector plasmid, but also for amplifying rAAV stocks. Notably, the biological titration of rAAV vectors was also feasible when cells were coinfected with rAAV and PS virus. Based on this strategy, we produced an rAAV that expresses prothymosin alpha (ProT). Expression of the ProT protein in vitro and in vivo mediated by rAAV/ProT gene transfer was detected by immunohistochemistry and a bioassay. Taken together, our results demonstrate that the PrV vector-based system is useful for generating rAAV vectors carrying various transgenes.
Elective cardioversion with titrated energy was carried out under diazepam anaesthesia (38 procedures) in 26 patients on digitalis who presented with supraventricular or ventricular arrhythmias. Low energy shocks were given to establish the absence of latent digitalis intoxication, not unmasked by previous carotid sinus massage, and to restore sinus rhythm with the lowest possible energy discharge. In the absence of digitalis overdosage, titrated energy cardioversion restored sinus rhythm, at least temporarily, without dangerous ventricular arrhythmias. In one patient this precaution was neglected: a shock of 200 Ws provoked ventricular tachycardia. We present experimental and clinical evidence for the safety and effectiveness of titrated energy cardioversion in patients on digitalis.
OBJECTIVES: To investigate the efficacy of transdermal glyceryl trinitrate given continuously and with a nocturnal nitrate free period. DESIGN: Double blind placebo controlled study with two parallel limbs. SETTING: Multicentre trial. PATIENTS: 52 patients randomised to receive either continuous treatment (23 patients) or intermittent treatment with an individually titrated dose (29 patients) for 14 days: both treatments were compared with placebo in a cross-over fashion. INTERVENTION: Continuous treatment with 10 mg per 24 hours of transdermal glyceryl trinitrate or intermittent transdermal glyceryl trinitrate titrated to give an arbitrary 10 mm Hg drop in systolic blood pressure (mean dose 18.2 mg) given over approximately 16 hours. MAIN OUTCOME MEASURE: Treadmill exercise stress testing and ambulatory monitoring of the ST segment after 14 days' treatment. RESULTS: After 14 days' intermittent treatment resting supine and standing systolic blood pressure fell by 7.5 mm Hg (95% confidence interval 2.7 to 12.2) and 9.0 mm Hg (95% CI 3.4 to 14.5) respectively (p less than 0.01); resting heart rate was unchanged. Mean heart rate at 1 mm ST segment depression rose by 11.9 beats/min (CI 1.1 to 23.7) (p less than 0.05), mean time to onset of angina increased by 59 seconds (CI 10.8 to 108) (p less than 0.05), and total exercise duration increased by 40 seconds (p less than 0.05). These changes were not seen after continuous treatment. The frequency of ischaemic episodes was not reduced with either regimen nor was the circadian distribution of these episodes altered, in particular nocturnal episodes did not increase during intermittent treatment. CONCLUSION: Tolerance to glyceryl trinitrate was avoided by the use of individually titrated doses administered with a nocturnal nitrate free period. There was no evidence of "rebound" on ambulatory monitoring during this treatment.
We have used two-dimensional 1H-15N heteronuclear single quantum correlation spectroscopy to measure the pH dependence of backbone amide group chemical shifts in the actin binding protein hisactophilin over the pH range 5.7-11.1. Most of the resonances can be analyzed using a simple equation involving a single apparent ionization constant, pK(app). The majority of resonances in the protein titrate with pK(app) values of 5.6-7.4. The results can be rationalized in terms of titration of many histidine residues in hisactophilin. The titration data provide direct experimental support for the proposed models of the atomic basis of actin and membrane binding by hisactophilin.
Acute in vivo CO2 titration curves were performed on rats anaesthetized with pentobarbitone. The slope of the in vivo CO2 titration curve in the rat was found to be similar to that previously reported in the dog and in man. Removal of approximately 30% of the haemoglobin of the body did not influence significantly the slope of the in vivo CO2 titration curve in the rat.
A microelectrometric titration method is described to measure picomole amounts of Cl- present in solutions at micromolar concentrations. This method was used to measure total intracellular chloride concentration ([Cl-]i) in leukocytes. Through the use of submicroliter samples, [Cl-] can be measured in the range of 5-500 microM. There is no measurable interference from other ions normally present in the cell and no intracellular ion is falsely measured as Cl-. [Cl-]i determined by the conventional coulometric titration and the new microelectrometric titration method was the same. Among the commonly used substituting anions, thiocyanate was the only one falsely measured as Cl- and should be avoided in experiments using this method. Because only picomole amounts of Cl- are required, measurements of intracellular pH and total concentrations of other intracellular ions can be done in the same lysate of as few as 5 X 10(5) cells. This feature should make it possible to study the time course of changes in [Cl-]i together with measurements of other intracellular ions following various physiological or experimental maneuvers.
We have recently shown that background presence of chronic metabolic acid-base disorder markedly alters in vivo acute CO2 titration curve. These studies were carried out to assess the influence of chronic respiratory acid-base disorders on response to acute hypercapnia and to explore whether the chronic level of plasma pH is the factor responsible for alterations in the CO2 titration curve. We compared whole-body responses to acute hypercapnia of dogs with preexisting chronic respiratory alkalosis (n = 8) with that of normal animals (n = 4) and animals with chronic respiratory acidosis (n = 13). Chronic respiratory alkalosis and acidosis, as well as the acute CO2 titrations, were produced in unanesthetized dogs within a large environmental chamber. For comparison with our data on chronic metabolic acidosis and alkalosis, plasma bicarbonate levels, which are secondarily altered in chronic respiratory acid-base disorders, were used as an index of chronic acid-base status of the animals. Results indicate that, as with chronic metabolic acid-base disorders, a larger increment in plasma bicarbonate occurs during acute hypercapnia when steady-state plasma bicarbonate is low (respiratory alkalosis) than when it is high (respiratory acidosis). Yet, in further analogy with the metabolic studies, plasma hydrogen ion concentration is better defended at higher plasma bicarbonate levels in accordance with mathematical relationships defined by the Henderson-Hasselbalch equation. Combined results demonstrate that the influence of chronic acid-base status on whole-body response to acute hypercapnia is independent of initial plasma pH.(ABSTRACT TRUNCATED AT 250 WORDS)
PURPOSE: BMS-247550 is a semisynthetic derivative of epothilone B with mechanism of action analogous to paclitaxel. It has shown impressive antitumor activity in preclinical studies including in taxane-resistant models. We conducted a phase I trial, based on accelerated titration "2B" design, of BMS-247550 given as a 1-hour infusion every 3 weeks. EXPERIMENTAL DESIGN: Seventeen patients (M:F, 10:7; median age, 54 years; performance status, 0-2) were treated on the trial. Forty-five cycles (1-9 cycles) of BMS-247550 were given at dosages ranging from 7.4 to 56 mg/m2. All patients received prophylaxis for hypersensitivity reactions, related to Cremophor-EL, with steroids and histamine antagonists. RESULTS: First-course dose-limiting toxicity (DLT) was observed in two of three patients at 56 mg/m2 (neutropenic sepsis, prolonged grade 4 neutropenia) and in one of six patients at 40 mg/m2. Nonhematologic grade 3 to 4 toxicities observed were emesis and fatigue and they occurred only at 56 mg/m2. Grade 1 to 2 peripheral neuropathy was also observed. Other grade 1 to 2 toxicities were myalgias, arthralgias, rash, hand/foot syndrome, and mucositis. AUC and C(max) seemed proportional to the dose and the DLT. Development of neutropenia with BMS-247550 is related to the duration of drug exposure above a threshold. CONCLUSIONS: The maximum tolerated dose (MTD) of BMS-247550 is 40 mg/m2 given every 3 weeks. Neutropenia is the DLT. The accelerated titration "2B" design may help in determining MTD with fewer patients enrolled and more being treated closer to the MTD. However, the accelerated titration design did not seem to shorten the study duration.
Esophageal pressure amplitude (DeltaPes), inspiratory pulmonary resistance (RLI) and inspiratory flow limitation score (FS) are used as indices of upper airway obstruction for the titration of nasal continuous positive airway pressure (nCPAP) in patients with obstructive sleep apnea syndrome (OSAS). This study was designed to determine whether oscillatory respiratory resistive impedance at 16 Hz (RFO) might be proposed as an alternative index. Eleven OSAS patients were studied during a night of polysomnography-controlled nCPAP titration. Nasal flow (V) and airway opening and esophageal pressures (Pao and Pes, respectively) were continuously measured during nasal breathing, and forced-flow oscillations (FO) were applied for 5 min at each nCPAP level. RLI was calculated by linear regression analysis of resistive pressure versus V over inspiration. R FO was obtained by linear regression analysis of respiratory resistive impedance versus frequency. Application of FO affected neither sleep nor pulmonary mechanics. RFO correlated with RLI in all patients. RFO did not correlate with DeltaPes in two patients, and was not significantly related to FS in five patients. This study demonstrates the applicability of the FO technique in sleeping patients receiving nCPAP, and the reliability of RFO for assessing pulmonary resistance. RFO might therefore be proposed as a quantitative index of airway obstruction for nCPAP titration.
The effect of a dental appliance (DA) is usually evaluated in a single mandibular position reached after several weeks and corresponding to either improvement of symptoms or intolerance to any further advancement. The purpose of this study was to test the feasibility of one-night evaluation of the efficacy of a DA. The study population consisted of seven patients (six men) with obstructive sleep apnea syndrome (66.9 +/- 32.4 apneas and/or hypopneas per hour). Patients underwent two consecutive polysomnographies; first with a temporary DA (two arches connected by a hydraulic system) progressively adjusted during the night to correct sleep disordered breathing and second with a permanent DA (two arches connected by Herbst attachments) set to the effective degree of advancement during the titration night. All patients completed the protocol. The mean mandibular advancement reached during the titration night was 12.6 +/- 2.7 mm. Arousal was never observed during or for 60 seconds following advancement. The apnea-hypopnea index (AHI) was significantly reduced from 66.9 +/- 32.4 to 26.1 +/- 20.7 per hour during the titration night from the diagnostic night (p < 0.01). During the second night, the AHI was 19.6 +/- 20.2 per hour and was less than 20 per hour in 71.4% of patients and less than 10 per hour in 42.9% of patients. The efficacy of a DA can be evaluated during a single night of polysomnography.
Presented is a clinical study comparing the result of immunotherapy based on allergy testing with three different test modalities: skin end-point titration, prick testing, and scratch testing. Thirty patients with allergic rhinitis were randomly assigned to one of the three test groups. Treatment vials were made on test results according to the instructions for each test. For skin end-point titration, each antigen was included at a concentration based on the patient's sensitivity, found with the fivefold dilution testing procedure. For the prick test, treatment vials were based on a two-level test response scale of stronger or lesser positive results. The scratch test yields vials used in one concentration based simply on a positive vs. negative test result. Patients in all three groups were treated at weekly intervals with immunotherapy, progressing in volume until the maximum tolerated dose was achieved. Relief of symptoms was evaluated after 6 months of treatment. Nine of the 10 patients receiving skin end-point titration based immunotherapy had complete relief of symptoms, and one had partial relief. None of the patients in the prick or scratch test groups achieved relief of symptoms after 6 months of treatment. Results dramatically demonstrate the need to base allergy immunotherapy treatment on a test that quantifies the patient's level of sensitivity to each antigen.
BACKGROUND: Detailed polysomnography (PSG) and overnight continuous positive airway pressure (CPAP) titration as the basis for investigation and treatment of obstructive sleep apnoea/hypopnoea syndrome (OSAHS) incurs high costs from inpatient stays and technician attendance, even when split night studies are employed. Responding to rapidly increasing demand, from 1996 the sleep service at Glasgow Royal Infirmary adopted limited sleep studies and daycase CPAP titration as first line management. Here we describe the outcomes of this service between 1996 and 1999. METHODS: Data were collected from prospective records made at follow up by sleep technicians and by casenote review. RESULTS: Results were available for 158 subjects (97.5%) of 162 commenced on CPAP during this period. One hundred and forty-eight (91%) were diagnosed by limited sleep studies and 80 (50%) were diagnosed at home. The median follow up was 23 months (interquartile range (IQR), 13-35). Median use of CPAP in those continuing therapy was 5.3 hours/night (IQR, 3.1 to 6.5) and 77% were still using CPAP at three years. Only initial low CPAP compliance (< 2 hours/night) significantly predicted early CPAP cessation. In those still on CPAP in 1999, the mean drop in Epworth Sleepiness Scale score was 4.6 points (95% confidence intervals, 3.2 to 6.0). CONCLUSION: These data from the CPAP population at Glasgow Royal Infirmary provide information on longterm outcomes using a diagnostic protocol based on limited sleep studies and daycase CPAP titration.
There is controversy about the effect of replacement GH on insulin action in adult hypopituitary patients. GH replacement calculated from weight leads to unacceptable side effects in some patients. Recent studies suggest it should be individually titrated in adults using serum IGF-I levels. We have assessed the effect of titrated GH replacement on peripheral and hepatic insulin action in 13 adult-onset hypopituitary patients (8 males and 5 females; ages 47 +/- 10 yr, mean duration of hypopituitarism 6 yr) with confirmed GH deficiency (GHD; maximum GH <5 mU/liter during insulin induced hypoglycemia), ACTH deficiency, and normal glucose tolerance. All patients were on stable hydrocortisone replacement (15 mg with breakfast, 5 mg with evening meal) for at least 2 months before the trial. Insulin action was assessed by the euglycemic hyperinsulinemic glucose clamp technique (1 mU/kg x min) before and after 6 months of GH therapy. GH was started at 0.8 IU sc daily and titrated monthly until the serum IGF-I increased to within 1-2 SD of the mean of normal age-matched controls. Body mass index did not change significantly during the 6 months of GH therapy. Fasting plasma glucose and HbA1c increased significantly after 6 months (5.2 +/- 0.0 vs. 5.5 +/- 0.0 mmol/liter, P < 0.0001, and 4.5 +/- 0.1 vs. 4.7 +/- 0.1%, P < 0.0005, respectively). There was no increase in fasting serum insulin (51.6 +/- 10.2 vs. 60.0 +/- 10.2 pmol/liter, P = 0.12). Exogenous glucose infusion rates required to maintain euglycemia were similar after GH (23.0 +/- 0.4 vs. 21.1 +/- 0.3 micromol/kg x min, P = 0.6). Endogenous glucose production in the fasting state was also unchanged following GH (11.8 +/- 0.7 vs.12.3 +/- 0.9 micromol/kg x min, P = 0.5) and suppressed to a similar extent following insulin (4.4 +/- 0.8 vs. 5.5 +/- 0.8 micromol/kg x min, P = 0.3). In summary, GH therapy for 6 months, with serum IGF-I maintained in the upper physiological range, increased fasting plasma glucose and HbA1c. There was no effect on peripheral or hepatic insulin sensitivity. Patients receiving GH therapy require long-term monitoring of glucose tolerance.
H+ titrations were conducted on the separated haemoglobin components of eel Anguilla anguilla in both the oxygenated and deoxygenated states. In anodic haemoglobin, the addition of GTP, and to a lesser extent C1-, increased the magnitude of the Haldane effect and shifted its maximum value into the in vivo pH range. Of the 22 histidine residues in the anodic component, only approximately seven were titratable, presumably the beta-chain residues at positions 41, 97, 109 and 146 (helical positions C7, FG4, G11 and HC3, respectively). In cathodic haemoglobin, a small negative Haldane effect was observed at pH values between 6.8 and 8.5 which disappeared in the presence of GTP (molar ratio 3:1 GTP:haemoglobin tetramer). GTP had virtually no effect on the buffer value at fixed oxygenation status, and the lowest buffer value was observed at in vivo pH values. No titratable histidine residues were observed in the cathodic component, indicating that all 14 histidines in this component are buried. We conclude that the anodic component, which constitutes two-thirds of the haemoglobin in the eel, plays the predominant role in CO2 transport and pH homeostasis in vivo.
A simple and rapid indirect potentiometric titration of sulphamethoxazole in the presence of trimethoprim contained in co-trimazole tablets is described. The method is based on the formation of a complex of sulphamethoxazole with a known excess of silver ions and the titration of unreacted silver ion potentiometrically using an inexpensive lab-made copper based mercury film electrode (CBMFE). The titration conditions have been optimized for the determination of 1.0-10.0 mg of sulphamethoxazole in pure and dosage forms. The precision and accuracy of the method have been assessed by the application of lack of fit test and other statistical methods. Overall mean recovery and relative standard deviations obtained were 99.88% and 1.32% (n=7) respectively. No interference was caused by other excipients present in pharmaceutical dosage forms. The application of this method for sulphamethoxazole assay in the presence of trimethoprim in tablets was validated by the comparison of results obtained by the proposed method with that of the British Pharmacopoeia (BP) method using F- and t-statistical tests of significance.
A highly accurate nephelometric titration method was developed for the quantitative analysis of phenytoin sodium injection and tablets. The titration operating conditions and the validation of the method were studied. Five batches of phenytoin sodium injection and tablets were determined by the proposed method and the control experiment methods, respectively. The results of the titration are comparable to those of control experiments. The proposed method is accurate and reproducible, which is considered suitable for the quantitative analysis of a large number of samples.