Assessment of Lentiviral Vector Mediated CFTR Correction in Mice Using an Improved Rapid in vivo Nasal Potential Difference Measurement Protocol.

Cystic Fibrosis (CF) is caused by a defect in the CF transmembrane conductance regulator (CFTR) gene responsible for epithelial ion transport. Nasal potential difference (PD) measurement is a well established diagnostic technique for assessing the efficacy of therapies in CF patients and animal models. The aim was to establish a rapid nasal PD protocol in mice and quantify the efficacy of lentiviral (LV) vector-based CFTR gene therapy. Anaesthetised wild-type (WT) and CF mice were non-surgically intubated and nasal PD measurements were made using a range of buffer flow rates. Addition of the cAMP agonist, isoproterenol, to the buffer sequence was then examined. The optimised rapid PD technique was then used to assess CFTR function produced by second and third generation LV-CFTR vectors. V5 epitope tagged-CFTR in nasal tissue was identified by immunohistochemistry. When intubated, mice tolerated higher flow rates. Isoproterenol could discriminate between WT and CF mice. Improved chloride transport was observed for the second and third generation LV-CFTR vectors, with up to 60% correction of the cAMP-driven chloride response towards WT. V5-CFTR was located in ciliated epithelial cells. The rapid PD technique enables improved functional assessment of the bioelectrical ion transport defect for both current and potential CF therapies.

Airway gene transfer in a non-human primate: lentiviral gene expression in marmoset lungs.

Genetic therapies for cystic fibrosis (CF) must be assessed for safety and efficacy, so testing in a non-human primate (NHP) model is invaluable. In this pilot study we determined if the conducting airways of marmosets (n = 2) could be transduced using an airway pre-treatment followed by an intratracheal bolus dose of a VSV-G pseudotyped HIV-1 based lentiviral (LV) vector (LacZ reporter). LacZ gene expression (X-gal) was assessed after 7 days and found primarily in conducting airway epithelia as well as in alveolar regions. The LacZ gene was not detected in liver or spleen via qPCR. Vector p24 protein bio-distribution into blood was transient. Dosing was well tolerated. This preliminary study confirmed the transducibility of CF-relevant airway cell types. The marmoset is a promising NHP model for testing and translating genetic treatments for CF airway disease towards clinical trials.

Optimisation of a multipartite human immunodeficiency virus based vector system; control of virus infectivity and large-scale production.

BACKGROUND: We have previously described a five-plasmid HIV-1 vector system that utilises a codon-optimised gagpol gene. While this system was shown to be safer than systems using proviral type helpers, the titre of virus produced was relatively low. Therefore, a process of optimising all aspects of virus production was initiated. METHODS: A systematic approach was taken to the optimisation of virus production by transient expression using a five-plasmid packaging system. Codon-manipulation was used to reduce homology between helper and vector constructs. Ultrafiltration and ultracentrifugation were used for large-scale virus production. RESULTS: We describe codon-optimised reading frames for Tat and Rev and the optimisation of virus production. The optimisation process resulted in an increase in virus titre of 7- to 8-fold. Several other approaches to increasing viral titre described by others proved ineffective in our system after it had been optimised. In addition, we show that by varying the ratio of the GagPol helper construct to vector, the infectivity of the virus could be controlled. The use of a novel codon-optimised HIV-1 GagPol expression construct with reduced homology to vector sequences significantly reduced transfer of gagpol sequences to transduced cells. Virus could be collected in serum-free medium without a significant loss of titre, which facilitated subsequent processing. Processing using a combination of ultrafiltration and ultracentrifugation allowed efficient and rapid processing of litre volumes of virus supernatant. CONCLUSIONS: By taking a systematic approach to optimising all aspects of our five-plasmid lentiviral vector system we improved titre, safety, large-scale production, and demonstrated that infectivity could be specifically controlled.

Cefazolin does not accelerate gastric emptying in the critically ill.

OBJECTIVE: To evaluate the effect of intravenous cefazolin on gastric emptying measured by the C-13 octanoic acid breath test. DESIGN: Prospective, double-blind, cross-over, randomised, placebo-controlled trial. SETTING: Mixed multidisciplinary intensive care unit in a university hospital. PATIENTS: Fourteen critically ill, mechanically ventilated patients. INTERVENTIONS: After a 4-h fast patients received either 50 mg cefazolin or 20 ml saline over 20 min immediately prior to measurement of gastric emptying. The next day the study was repeated with the alternative therapy. MEASUREMENTS AND RESULTS: Breath samples were analysed for the concentration of (13)CO2 by mass spectrometer, and the gastric emptying coefficient (GEC) and half-emptying time (t(50)) were calculated. Results are mean (standard deviation). Data were analysed with the paired t-test (saline vs cefazolin). Two patients were excluded for technical problems. Twelve patients remained (six male/six female), aged 57 (+/-16) years, with an APACHE II score of 20 (+/-8). Both GEC and t(50) were unchanged after administration of cefazolin compared with placebo (t(50) cefazolin, 138 (+/-54) vs saline 122 (+/-46) min, P=0.32; GEC cefazolin 3.27 (+/-0.83) vs saline 3.55 (+/-0.6), P=0.24). Two patients had abnormal t(50) after saline and five after cefazolin. There was no order effect of the study day. CONCLUSION: In mechanically ventilated patients, cefazolin had no effect on gastric emptying. These data do not support the use of low-dose cefazolin as a pro-kinetic agent in critically ill patients.

Delayed gastric emptying in ventilated critically ill patients: measurement by 13 C-octanoic acid breath test.

OBJECTIVE: To measure gastric emptying in ventilated critically ill patients with a new noninvasive breath test. DESIGN: Single-center, open study. SETTING: Combined medical and surgical intensive care unit of a university hospital. SUBJECTS: Thirty unselected mechanically ventilated critically ill patients receiving gastric feeding and 22 healthy volunteers. INTERVENTIONS: None. PATIENTS: After 4 hrs without feeding, intragastric infusion of 100 mL of a liquid meal (Ensure) labeled with 100 microL 13C-octanoic acid. End-expiratory breath samples were collected into evacuated tubes from the respirator circuit every 5 mins for the first hour, then every 15 mins for 3 hrs. End-expiratory breath samples were also collected from volunteers studied supine after an overnight fast following an identical infusion via a nasogastric tube. Breath 13CO2 was measured with an isotope ratio mass spectrometer. MEASUREMENTS AND MAIN RESULTS: Performance of the breath test posed no difficulty or interference with patient care. The CO2 level was >1% in 1297/1300 breath samples, indicating satisfactory end-expiratory timing. Data are median and interquartile range. Gastric emptying was slower in patients compared with volunteers: gastric emptying coefficient 2.93 (2.17-3.39) vs. 3.58 (3.18-3.79), p <.001 and gastric half emptying time, derived from the area under the 13CO2 curve, 155 min (130-220) vs. 133 min (120-145), p <.008. Fourteen of the 30 patients had a gastric emptying coefficient <95% of all volunteers and 11 had a gastric half emptying time longer than 95% of all volunteers. The Acute Physiology and Chronic Health Evaluation (APACHE II) score (median 22, range 13-43) either at admission or on the day of the study did not correlate with gastric emptying coefficient. CONCLUSION: Gastric emptying of a calorie-dense liquid meal is slow in 40% to 45% of unselected mechanically ventilated patients in a combined medical and surgical intensive care unit. The 13C-octanoic acid breath test is a novel and useful bedside technique to measure gastric emptying in these patients.

A technique for obtaining repeated liver biopsies from rats.

Many morphological, pharmacological and toxicological studies of hepatotoxicity require frequent sampling of liver over time. In the past this has been achieved by including large numbers of animals in the study and killing subgroups at different times. In this paper we describe a technique for repeated liver biopsy that procures sufficient liver tissue for histopathological assessment and for additional studies, for example measurement of hepatic iron concentration or vitamin A measurement. The advantages and disadvantages of this technique are discussed.

Use of artificial sweeteners to promote alcohol consumption by rats.

Cirrhosis may be reliably produced in rats by exposing them intermittently to low levels of carbon tetrachloride vapour while feeding alcohol in the Lieber-DeCarli liquid diet. Providing the alcohol in drinking water that has been sweetened with sucrose is a cheaper and more convenient method but it does not yield reliable results. This study aimed to determine whether alcohol in drinking water sweetened with artificial sweeteners would give adequate alcohol intake to achieve the desired hepatic effects. Rats were fed alcohol (8% v/v) in drinking water sweetened with sucrose (5% w/v) (n = 12), or with one of the artificial sweeteners aspartame (0.025%), saccharin (0.025%) or cyclamate (0.05%) (n = 8 per agent). During the alcohol treatment the animals were exposed to carbon tetrachloride vapour, 40 ppm, six hours per night for five nights per week, over a period of 14 weeks. All groups achieved good alcohol intakes of 5-6 g/kg/day. Only one rat, in the aspartame group, became cirrhotic; all the others had varying degrees of fibrosis which did not differ significantly among the treatments. Although it was not effective in reliably achieving cirrhosis, sweetening the alcohol solution with artificial sweeteners led to reasonable alcohol intakes with resultant hepatic fibrosis, and without the high carbohydrate intake which occurs when sucrose is used.

Dose-related effects of dietary iron supplementation in producing hepatic iron overload in rats.

The influence of varying the level of supplemental dietary iron on the development of hepatic iron overload was examined in rats. Two days after giving birth, Porton rats were fed a diet supplemented with 0, 0.5, 1 or 2% carbonyl iron, to institute dietary iron supplementation to the young via breast milk. After weaning, the offspring continued to receive the assigned diet until 32 weeks of age. Liver biopsies were taken from some rats at 8, 16 and 24 weeks of age and from all rats at 32 weeks of age, for assessment of iron overload. For both male and female rats, hepatic iron content was increased in a dose-related manner by feeding supplemented diet. Hepatic iron content of male rats tended to reach a plateau after 8, 16 weeks of supplementation, while that of female rats continued to rise throughout the experimental period, such that the hepatic iron content of female rats was 2.8-fold that of similarly treated males at 32 weeks of age. Iron supplementation was associated with only moderate retardation of growth. By choosing an appropriate level of iron supplementation, good (grade III-IV) hepatic iron loading can be achieved with minimal adverse effects on the animals' overall health.

Development of tolerance to antinociceptive effects of an intrathecal morphine/clonidine combination in rats.

Previous animal studies have shown the antinociceptive effects of intrathecal clonidine and intrathecal morphine to be synergistic. This study investigated the intrathecal administration of multiple doses of this drug combination to examine the rate of development of tolerance and to determine whether there was any toxic effect on the spinal cord. Rats with indwelling intrathecal catheters were given saline, morphine (2.5-7.5 micrograms), clonidine (17.5 micrograms), or clonidine (17.5 micrograms) plus morphine (1 microgram) intrathecally twice daily for 4 1/2 days (total of 9 doses). Hot plate and tail flick tests were conducted after the first, fifth and ninth doses. After the ninth dose animals were killed and their spinal cords were removed for histological examination. Tolerance developed to the antinociceptive effects of the drug combination, but at a slower rate than to morphine alone. No evidence of toxicity or injury to the spinal cord was observed other than changes which could be ascribed to the presence of the catheter.

Iron overload facilitates hepatic fibrosis in the rat alcohol/low-dose carbon tetrachloride model.

The role of iron deposition in initiating hepatic fibrosis in iron overload disorders is not clearly established, and it is becoming increasingly recognized that iron may be interacting with other potential liver-damaging agents. The authors therefore examined the interplay of iron and alcohol in rats administered subtoxic doses of carbon tetrachloride (CCl4) vapor at 20 ppm in customized chambers. At birth, the offspring of seven pregnant Porton rats were divided into two groups: one group was fed a normal rat chow diet and the other a diet supplemented with 3% (w/w) carbonyl iron for 10 weeks after weaning. In this latter group, the mothers were fed an iron supplement while breastfeeding. At 10 weeks, the animals from the first group (normal chow) were divided into two groups of six animals and fed a Lieber-DeCarli liquid diet with daily exposure to CCl4 vapor: group 1, liquid diet+CCl4; group 2, liquid diet+alcohol 150 kcal/l+CCl4. The animals from the second iron-supplemented group were divided into two groups of six animals and fed a liquid diet with 3% (w/v) carbonyl iron and exposed to CCl4 vapor for 10 weeks: group 3, liquid diet+iron+CCl4; group 4, liquid diet+iron+alcohol supplement+CCl4. Two animals from each group of six had a liver biopsy at 4, 6, and 8 weeks, and all animals were killed after 10 weeks of CCl4 exposure. After the first 10-week iron loading period, the rats fed the carbonyl iron-supplemented diet had a 10-fold elevation in hepatic iron concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Behavioural effects of norpethidine, a metabolite of pethidine, in rats.

This study investigated behavioural effects of the toxic pethidine metabolite, norpethidine, in rats and its interactions with reserpine, apomorphine and physostigmine. Following intraperitoneal administration, brain concentrations of norpethidine reached a plateau after 20-40 min and remained elevated for 2 h. In the dose range 0.06-0.18 mmol/kg, norpethidine induced myoclonic jerks, a characteristic splayed posture, and episodes of exaggerated shivering. Forward locomotion, grooming, yawning and rearing were suppressed. Seizures and reverse locomotion occurred occasionally. Administration of reserpine 1 h prior to norpethidine, or of apomorphine or physostigmine 15 min after norpethidine, did not alter the norpethidine-induced behaviours; neither did norpethidine block the effects of apomorphine or physostigmine. The characteristic profile of behaviours induced by norpethidine make this toxicant readily amenable to animal studies. Our results indicate that its mechanism of action is unlikely to involve dopaminergic or cholinergic pathways.

Alcohol/"low-dose" carbon tetrachloride-induced cirrhosis in rats using different methods of alcohol feeding.

Cirrhosis may be reliably produced in rats by exposing them to low levels of carbon tetrachloride vapor while feeding alcohol in the Lieber-DeCarli liquid diet. This study aimed to determine whether alternative cheaper and more convenient ways of feeding alcohol would also allow the production of cirrhosis. Animals were fed alcohol in the Lieber-DeCarli diet, in a gel diet, or by addition of alcohol + sucrose to their drinking water, and were exposed to carbon tetrachloride vapor 6 hr/night, 5 nights/week. After 12 weeks of treatment, all animals (4 of 4) receiving alcohol in the Lieber-DeCarli diet, but only two in each of the gel and drinking water groups, were cirrhotic. The variable results with the gel diet may be due to loss of alcohol by evaporation from the gel. Alcohol intake in the group receiving alcohol in drinking water was greater than in those receiving Lieber-DeCarli diet. We suggest that the increased carbohydrate intake due to addition to sucrose to the water exerted a protective effect on the liver.

Dose-response relationships in hepatic injury produced by alcohol and carbon tetrachloride.

Dose-response relationships were examined for the production of hepatic fibrosis and cirrhosis by combined exposure of male Porton rats to alcohol and carbon tetrachloride. Alcohol was administered orally in Lieber-DeCarli liquid diet at levels of 75, 150, or 300 kcal/liter, giving mean daily intakes of 2.29, 4.61, and 8.16 g/kg/day, respectively. Carbon tetrachloride was administered by inhalation at concentrations of 10, 20, or 40 ppm, 6 hr/night, 5 nights/week. Liver biopsies were taken at intervals up to a maximum treatment period of 20 weeks. All four rats that received the high dose of both agents, and 1 of 4 that received the medium alcohol and high carbon tetrachloride treatments, were cirrhotic by 10 weeks. Two of the 4 rats that received the low alcohol and high carbon tetrachloride dose were cirrhotic at 20 weeks. Cirrhosis was not observed in rats that received the low or medium carbon tetrachloride dose, but some degree of hepatic fibrosis was observed in all treatment groups. Severity of fibrosis was significantly associated with both dose of alcohol and dose of carbon tetrachloride received. It is concluded that, in the alcohol-carbon tetrachloride rat model for chronic liver injury, both alcohol and carbon tetrachloride contribute to the response in a dose-related manner. Hepatic injury was observed even when relatively low doses of these agents are administered together.

The pathology of liver injury induced by the chronic administration of alcohol and 'low-dose' carbon tetrachloride in Porton rats.

We have previously established a model for micronodular cirrhosis by feeding Wistar rats alcohol, in the Lieber-DeCarli liquid diet, and exposing them to 'low-dose' carbon tetrachloride (CCl4) vapour for 10 weeks. This study reports the spectrum of liver pathology seen in male Porton rats exposed to 'low-dose' CCl4 vapour 5 nights/week, 6 h/night while being fed alcohol (300 kcal/L) in the Lieber-DeCarli diet. Micronodular cirrhosis developed in all animals after 5-7 weeks of treatment. The simultaneous administration of silymarin, a putative hepatoprotective agent, in the liquid diet, did not alleviate or prevent the chronic liver injury. The histopathological features of the liver injury are described, with particular emphasis on the presence of small epithelial cells ('progenitor or stem cell'), which appear to be playing a role in liver regeneration.

Assessment of antinociceptive drug effects in the presence of impaired motor performance.

The hot-plate (HP) and tail-flick (TF) tests are widely used to assess analgesic activity of drugs. These tests do not directly measure the intensity of the noxious stimulus perceived by the animal, but only the animal's response to it, and so may be affected by non-analgesic drugs. Sedatives and muscle relaxants, for example, may impair the ability to respond and hence be wrongly considered to have analgesic activity. We examined response of rats in the HP (55 degrees C, cutoff time 25 sec) and TF (cutoff time 5 sec) tests following administration of pentobarbitone, diazepam or pancuronium. These drugs all impaired motor performance as assessed by reduction in mean rotarod performance times to 6-32% of predrug values. However, HP and TF latencies were not appreciably prolonged. We also found that pancuronium did not alter effects of morphine on HP or TF latencies, despite reduction in rotarod performance to 38% of predrug values. Our results support the validity of HP and TF tests as analgesic assays even in the presence of substantial impairment of motor performance.

Leakage of fluid administered epidurally to rats into subcutaneous tissue.

Epidural catheters were implanted in rats under halothane/nitrous oxide anaesthesia. Contrast medium (Iopamidol) was injected via the catheter under fluoroscopic control 24-48 h after implantation. In 15 of 20 rats contrast could be seen leaking out of the epidural space, usually after only 25 microliters was administered. Leakage was associated with diminished antinociceptive response to morphine administered via the catheter. Both leakage and decreased response to morphine could be largely prevented by applying a drop of Supa-Glue over the site of entry of the catheter to the epidural space at the time of catheter implantation. Investigators using epidurally cannulated rats should document that leakage does not occur or discard results from rats showing evidence of leakage.

Effects of treatment with phenobarbitone or isoniazid on hepatotoxicity due to prolonged subanaesthetic halothane inhalation.

Rats were exposed to halothane vapour, 50 p.p.m., or air for a period of four weeks. Within each exposure group, some animals drank plain water, some received water plus phenobarbitone, while some received water plus isoniazid. Halothane exposure resulted in increased serum bromide concentrations and liver injury evidenced by increased serum alanine aminotransferase activity, focal hepatocellular necrosis and fatty change. Administration of isoniazid reduced halothane metabolism by 33% as assessed by serum bromide concentrations, and completely blocked the injurious effects of halothane on the liver, suggesting that halothane metabolism plays a role in halothane hepatotoxicity under these conditions. Administration of phenobarbitone partially prevented the increase in serum alanine aminotransferase activity and hepatocellular necrosis due to halothane. In contrast to isoniazid, phenobarbitone led to a slight increase in halothane metabolism. However, phenobarbitone also caused an increase in liver size, such that the amount of halothane metabolised per gram of liver was reduced by phenobarbitone treatment. These results suggest that metabolism of halothane is an important factor in liver injury due to prolonged, subanaesthetic halothane exposure.