Toxicological comparison of E2a-deleted and first-generation adenoviral vectors expressing alpha1-antitrypsin after systemic delivery

Second-generation adenoviral vectors, mutated in E2a, have been proposed to decrease host immune responses against transduced cells, reduce toxicity, and increase duration of expression as compared with first-generation vectors deleted only in E1. To test these hypotheses further, we have developed an E2a-deleted adenoviral vector expressing human alpha1-antitrypsin (hAAT). Toxicity of first-generation and E2a-deleted vectors, as determined by hematological indices, liver function tests, and histological analyses, was evaluated in C3H mice for 21 days after vector administration at increasing doses starting at 1 x 10(12) particles/kg. Both vectors induced dose-dependent abnormalities including transient thrombocytopenia, elevated ALT levels in serum, and increased hepatocyte proliferation followed by inflammation and then hypertrophy. Differences in the ratio of particles to plaque-forming units among vector preparations led to differences in hAAT expression at similar particle doses. There were no differences in toxicity between the two vectors when measured at matching levels of hAAT expression. However, the E2a-deleted vector was demonstrated to have slightly reduced hepatocyte toxicity at an intermediate particle dose. This suggests that hepatocyte toxicity is related primarily to viral entry and expression, rather than to the presence of noninfectious particles, and implies that vectors with complete elimination of viral gene expression, such as vectors with all viral coding sequences deleted, are likely to have substantial advantages in terms of safety and toxicity.     

An agent that increases tumor suppressor transgene product coupled with systemic transgene delivery inhibits growth of metastatic lung cancer in vivo

Low levels of gene expression following systemic delivery have impaired the effectiveness of tumor suppressor gene replacement in treating metastases. We asked whether combined treatment with 2-methoxyestradiol (2-Me), which increases levels of wild-type p53 protein in cancer cells, and the systemic administration of an adenoviral vector expressing wild-type p53 (Ad-p53) would inhibit the growth of human metastatic lung cancer cells in vivo. The simultaneous administration of p53 and 2-Me resulted in a greater than additive reduction with the lung colony count reduced to 33% of its control value. These results suggest that the synergistic effect of 2-Me and Ad-p53 in combination treatment may have application in the systemic treatment of cancer.     

Inhibition of prostaglandin E release by anti-inflammatory steroids

The aearance of tritium in plasma water was measured after i.p. injection of 2-tritio-L-alanine as an index of alanine transamination in rats. L-Cycloserine (10 mg/kg) and D-cycloserine (150 mg/kg) inhibited tritium release, whereas theophylline (100 mg/kg) stimulated tritium release.     

A study of topical and systemic prostaglandin E1 and survival of experimental skin flaps

A study has been undertaken to investigate Prostaglandin E1 administration procedure for improving flap survival. Whether the drug was administered continuously or transcutaneously using a silicone gel drug delivery system; or was topically injected into the critical zone of the flap; or was intraperitoneally administered intermittently over an hour after surgery a statistically significant improvement of flap survival occurred (P < 0.01, Student's t-test). However, no improvement of flap survival was seen when the drug was administered only once intraperitoneally immediately after flap elevation, although administered doses of the drug in those rats was equal to the doses in the rats which received intermittent administration of the drug intraperitoneally over an hour after surgery.     

Evaluation of lung function after intratracheal perfluorocarbon administration in healthy animals

OBJECTIVES: To investigate the effects of partial liquid ventilation (i.e., mechanical ventilation in combination with intratracheal administration of perfluorocarbon) on lung function, with particular attention to the integrity of the alveolocapillary membrane in healthy adult animals. DESIGN: Prospective, randomized, controlled study. SETTING: Laboratory at the Department of Experimental Anesthesiology, Erasmus University Rotterdam. SUBJECTS: Ten adult male New Zealand rabbits. INTERVENTIONS: Five rabbits were intratracheally treated with 12 mL/kg of perfluorocarbon while conventional mechanical ventilation (volume-controlled, tidal volume of 12 mL/kg, respiratory rate of 30 breaths/min, inspiration/expiration ratio of 1:2, positive end-expiratory pressure of 2 cm H2O, and an FIO2 of 1.0) was applied for 3 hrs. To assess the permeability of the alveolocapillary membrane, pulmonary clearance of inhaled technetium-99m-labeled diethylenetriamine pentaacetic acid (99mTc-DTPA) measurements were performed at 3 hrs and compared with data from the control group (n = 5) treated with mechanical ventilation only, using the same ventilatory parameters. MEASUREMENTS AND MAIN RESULTS: Pulmonary gas exchange and lung mechanical parameters were measured in both groups at 30-min intervals. Mean values for PaO2 in the perfluorocarbon group, although at adequate levels, were less than those values of the control group during the 3-hr study period (370 +/- 44 vs. 503 +/- 44 torr at 3 hrs [49.3 +/- 5.9 vs. 67.1 +/- 5.9 kPa]). Peak and mean airway pressures were higher in the perfluorocarbon group (ranging from 1.9 to 3.4 cm H2O and 0.7 to 1.3 cm H2O, respectively) compared with the control group, while end-inspiratory airway pressure was similar in both groups. The half-life of 99mTc-DTPA was 83.7 +/- 24.5 mins in the control group, which was significantly longer (p < .01) than in the perfluorocarbon group (49.8 +/- 6.1 mins). CONCLUSIONS: These findings suggest that partial liquid ventilation with perfluorocarbons lowers pulmonary gas exchange in healthy animals, and the increased pulmonary clearance of 99mTc-DTPA after 3 hrs of this type of ventilatory support may reflect minimal reversible changes in the lung surfactant system.     

Inhibition of purinergic transmission by prostaglandin E1 and E2 in the guinea-pig vas deferens: an electrophysiological study

1. The effects of prostaglandin E1 (PGE1) and E2 (PGE2) on postjunctional electrical activity in the guinea-pig vas deferens evoked by sympathetic nerve stimulation were investigated using both intracellular and focal extracellular recording techniques in vitro. 2. Bath application of PGE1 (1-100 nM) or PGE2 (0.1-100 nM) concentration-dependently inhibited the amplitudes of all excitatory junction potentials (e.j.ps) evoked during short trains of stimuli (10 stimuli at 1 Hz). Increasing the duration of nerve stimulation (100 stimuli at 1 Hz) did not overcome this inhibitory effect. At these concentrations PGE1 and PGE2 were without any apparent inhibitory effect on the amplitudes of spontaneous e.j.ps. 3. Local application of PGE1 (10-100 nM) or PGE2 (10-30 nM) markedly reduced the frequency of occurrence of excitatory junction currents (e.j.cs) evoked by trains of 20-100 stimuli at 1 to 4 Hz without changing the amplitudes of spontaneous e.j.cs or the configuration of the nerve terminal impulse. 4. In the presence of PGE1 or PGE2, raising the frequency of stimulation (from 1 to 4 Hz), increased the likelihood of e.j.c. occurrence. 5. The postjunctional electrical activity recorded in the guinea-pig vas deferens is believed to be due to ATP released from the sympathetic nerve endings. Thus the present study demonstrates that both PGE1 and PGE2 powerfully inhibit quantal ATP release in the guinea-pig vas deferens.     

Inhibition of Trimeresurus flavoviridis phospholipase A2 by lung surfactant protein A (SP-A)

A marked sequence homology has been noted between lung surfactant protein A (SP-A) and an inhibitor of phospholipase A2 (PLA2) isolated from the serum of Trimeresurus flavoviridis (Habu snake). This study evaluated the effect of SP-A on PLA2 activity from several sources. SP-A was isolated from bovine or rat lung surfactant by extraction with 1-butanol and octyl beta-D-glucopyranoside. The addition of SP-A produced a concentration-dependent inhibition of T. flavoviridis PLA2 that indicated non-competitive kinetics with Ki 5 micrograms/ml. Inhibition was reversed by heat inactivation, disulfide bond reduction or alkylation of SP-A, or by the presence of anti-SP-A antibody. Treatment of SP-A with endoglycosidase F or the presence of variation monosaccharides or lectins did not alter SP-A inhibition. Binding of PLA2 to SP-A was shown by ultrafiltration and was abolished by SP-A alkylation or the presence of SDS. The SP-A/PLA2 complex recovered from the ultrafilter had essentially no enzymatic activity, but activity was restored by treatment with mercaptoethanol. SP-A had no effect on activity of PLA2 from Naja naja, Crotalus atrox, or bovine pancreas. These results indicate that surfactant protein A selectively inhibits Trimeresurus phospholipase A2 activity and suggest that binding to the enzyme is the mechanism for inhibition.     

L-arginine uptake and metabolism by lung macrophages and neutrophils following intratracheal instillation of silica in vivo

Cold air inhalation and exercise-induced bronchoconstriction (EIB) have both been used as measures of bronchial responsiveness. Both stimuli are often combined in the Nordic climate. The main objective of the present study was to investigate the climatic influence of cold temperatures upon exercise-induced asthma. The secondary aims were: (a) to assess metacholine bronchial hyper-responsiveness and EIB in children with bronchial asthma (n = 32; mean age 10.8 years) compared to children with other chronic lung diseases (CLD) (n = 26, mean age 10.1 years); and (b) to assess the influence of cold air inhalation upon EIB in the two groups of children. Methods used were: (a) the metacholine concentration causing a reduction in FEV1 of 20% (PC20-M), (b) maximum FEV1 fall (delta FEV1) after submaximal treadmill run (EIB test); and (c) delta FEV1 after submaximal treadmill run while inhaling cold (-20 degrees C) dry air (CA-EIB test). Geometric mean PC20-M did not differ significantly between the asthma children (1.28 mg ml-1) and the CLD children (2.90 mg ml-1). In the asthma children, mean delta FEV1 after EIB test was 12.8% vs 21.8% after adding cold air (P < 0.0001), compared to 5.2 and 7.4%, respectively (P = 0.03), in the CLD group. Maximum sensitivity and specificity for the EIB test were 69.8% at a fall in FEV1 of 6.8%; for the CA-EIB test, 72% at a fall in FEV1 of 10.2%; and for metacholine provocation, 56% at a PC20-M of 1.5 mg ml-1. In conclusion, children with bronchial asthma are substantially more sensitive to cold air than children with CLD, and EIB is markedly increased by cold air inhalation in asthmatic children, maintaining the specificity of the EIB test and increasing the sensitivity. The low sensitivity of the EIB test is probably influenced by the use of inhaled steroids. Metacholine inhalation test has less specificity and sensitivity in discriminating asthma from other chronic lung diseases.     

p53 mutation spectrum in relation to GSTM1, CYP1A1 and CYP2E1 in surgically treated patients with non-small cell lung cancer

OBJECTIVE: The purpose of this study was to assess the feasibility of using a prototype split-specimen design to assess integrity of a portion of the total testing process in medical clinics and laboratories. DESIGN: Two or three tubes of venous blood were collected from 177 patients for analysis of one of three analytes (serum potassium, serum total cholesterol, and whole-blood hemoglobin). Patients were seen at one of the nine clinics participating in this study. In all cases, one tube of blood from each patient was sent to a commercial referral laboratory, and the other tube(s) forwarded to the laboratory that routinely tested specimens for the clinic (participating laboratory) for analysis. Each participating laboratory removed a preanalysis and sometimes a post-analysis aliquot from each specimen and forwarded these to the referral laboratory for analysis. SETTING: The study was conducted in six physician office laboratories (three serving 1 to 4 [mean, 2.7] internists and three serving 3 to 24 [mean, 12] family physicians) and three hospital laboratories (serving hospitals with 100 to more than 700 beds). PATIENTS: Study patients were voluntary participants and provided informed consent. Patient age ranged from 18 to 80 years, and for all the laboratory test was specifically ordered for clinical reasons. Patients who were unable or unwilling to provide informed consent, those for whom testing would require that they provide more than 100 mL of blood, those whose blood was being collected by fingerstick, and those with results that were part of a laboratory test profile were excluded. MAIN OUTCOME MEASURES: Two main outcome measures were assessed: (1) percent differences between split-specimen results exceeding the maximum allowable imprecision level, which was based on published biological variation data (defined as one-half of the intraindividual percent coefficient of variation), for each analyte (result discrepancies); and (2) all "problems" (defined as departures from standard operating procedures) that could be documented by retrospective review of all relevant medical and laboratory records. RESULTS: The rate of result discrepancies was 1 in 20 (5%) for patients in whom hemoglobin was analyzed, 12 in 57 (21%) for patients in whom potassium was analyzed, and 1 in 60 (2%) for patients in whom total cholesterol was analyzed. Results of samples obtained during the aliquoting and storage phases of the total testing process were subject to study-induced problems and were generally not useful in tracing problems to specific stages of the testing process. A total of 28 problems (involving 26 patients) were documented, but only 6 problems were due to routine testing processes. CONCLUSIONS: The feasibility and limitations of a split-specimen design to detect result discrepancies were demonstrated. Most documented problems (22 of 28, or 79%) were study induced. To assess integrity of the total testing process, such problems need to be avoided.     

A breech delivery and induction with prostaglandin E2

Induction of labour with local application of PgE2 in breech presentation is still a much discussed problem. We aimed at studying the effect of local application of PgE2 (Prostin E2--3 mg) vaginal tablets for induction of labour in breech presentation. 15 pregnancies were studied and induced--8 for postdate, 6 for prae-eclampsia and 1 for fetal demise. Sonographic biometry and cardiotocographic examinations were conducted as well as a precise bishop score. Labour began within the interval of 3-13 hours after the application on the first tablet. The length of delivery was 4-24 hours. Effective labour was instituted in 93.33% on the parturiens, while in 1 (6.67%) there was a need for augmentation with oxytocin. There were no cases of hyperstimulation. Vaginal delivery was accomplished in 100% of the patients among which the breech was delivered with the classical manoeuvre in 10 cases and by the Bracht manoeuvre in 5 cases. Our results show, despite is small case number, that with favourable pelvic scores and absence of cephalopelvic disproportion and fetal distress PgE2 induction can be done locally even for breech presentation.     

Inhibition of NNK-induced lung tumorigenesis by modulators of NNK activation

Xenografts originated from human tumours offer the most appropriate research material for in vivo experimental research. However, primary human breast carcinomas are difficult to grow when transplanted in athymic mice: tumour take is less than 15%. Recently, we have achieved 60% tumour take by injecting tumour cell suspensions mixed with Matrigel. Human breast xenografts originated from primary breast carcinoma also frequently show the potential to metastasize spontaneously. In the present study, we generated a human breast carcinoma xenograft line (UISO-BCA-NMT-18) that shows 100% tumorigenicity and 80-100% lung metastasis when transplanted s.c. in athymic mice. We have studied in detail the characteristics of the xenograft and the patient's tumour from which the xenograft line originated. Both the xenograft and the patient's tumour showed intense staining for mutant p53 nuclear protein, and high expression of U-PA, PAI and u-PAR. In vivo growth of the xenograft is stimulated by exogenous supplementation of oestrogen. This xenograft is continuously growing in mice and has shown 80-100% metastasis for the last three successive in vivo passages. This well-characterized, oestrogen-responsive, metastatic breast carcinoma xenograft line will provide excellent research material for metastasis-related research.     

Inhibition of neutrophil elastase by the alpha1-proteinase inhibitor-immunoglobulin A complex

Effect of spaying on liver and plasma phospholipid components was studied in 4-day cyclic rats (Haffkin Institute Strain). Rats were ovariectomized during diestrous stage of estrous cycle. Ovariectomy (OVX) was shown to increase the total phospholipid content in both the liver lobes after 24 hr interval, it remained higher in spigelian lobe up to 72 hr but it was lowered in the right lobe at 48 hr interval. Concentration of sphingomyelin (SHP) in total phospholipids, as assessed by TLC, was slightly increased in both lobes after 48 hr of spaying. It declined significantly by 72 hr only in the spigelian lobe. Phosphotidylcholin, phosphatidylserine and phosphatidylethanolamine concentrations showed fluctuating pattern up to 48 hr of spaying, but most of these changes were seen to be restored to the normal level by 72 hr interval. Results indicate that the relative lack of ovarian hormones due to ovariectomy have obvious influence on hepatic lipid metabolism particularly that of phospholipid components. There appears to be an inverse relationship between hepatic lipid components and blood plasma. Spigelian lobe exhibits distinctly different responses to ovariectomy as compared to those of the right lobe. Maximal alterations are observable by 48 hr post-OVX interval.