Development of a human cell line by selection and drug-metabolizing gene transfection with increased capacity to activate promutagens

We have isolated a human lymphoblastoid cell line with higher levels of native cytochrome P450IA1 activity and by DNA transfection introduced human cDNAs for a putative cytochrome P450IIA2 and epoxide hydrolase (E.C. 3.3.2.3). The resultant cell line, designated MCL-1, was substantially more sensitive to the mutagenicity of dimethylnitrosamine and benzo[a]pyrene than the AHH-1 cell line and was found to have increased metabolism of benzo[a]pyrene to dihydrodiols. The increase in native cytochrome P450IA1 activity was achieved by mutation and selection based on resistance to the phototoxicity of benzo[ghi]perylene. One resistant clone, designated L3, was used for subsequent studies. Two complete cDNAs, one encoding a putative cytochrome P450IIA2 and the other a microsomal epoxide hydrolase, were isolated from a human liver cDNA library. After introduction of the cDNAs into an expression vector and transfection into AHH-1 cells, gene expression was detected at the level of enzyme activity (epoxide hydrolase) or by increased sensitivity to dimethylnitrosamine cytotoxicity/mutagenicity (putative P450IIA2). A vector containing both cDNAs was then constructed and transfected into L3 cells to produce MCL-1 cells. The potential usefulness of drug-metabolizing gene transfection and of the MCL-1 cell line, in particular, for genetic toxicity testing is discussed.     

A critical period for functional vestibular development in zebrafish.

We have determined a critical period for vestibular development in zebrafish by using a bioreactor designed by NASA to simulate microgravity for cells in culture. A critical period is defined as the briefest period of time during development when stimulus deprivation results in long lasting or permanent sensory deficits. Zebrafish eggs were collected within 3 hours of being laid and fertilized. In experiment 1, eggs were placed in the bioreactor at 3, 24, 30, 36, 48, or 72 hours postfertilization (hPF) and maintained in the bioreactor until 96 hPF. In experiment 2, eggs were placed in the bioreactor immediately after they were collected and maintained in the bioreactor until 24, 36, 48, 60, 66, 72, or 96 hPF. Beginning at 96 hPF, all larvae had their vestibulo-ocular reflexes (VOR) evaluated once each day for 5 days. Only larvae that hatched from eggs that were placed in the bioreactor before 30 hPF in experiment 1 or removed from the bioreactor later than 66 hPF in experiment 2 had VOR deficits that persisted for at least 5 days. These data suggest a critical period for vestibular development in the zebrafish that begins before 30 hPF and ends after 66 hPF. To confirm this, zebrafish eggs were placed in the bioreactor at 24 hPF and removed at 72 hPF. VORs were evaluated in these larvae once each day for 5 days beginning at 96 hPF. These larvae had VOR deficits that persisted for at least 5 days. In addition, larvae that had been maintained in the bioreactor from 24 to 66 hPF or from 30 to 72 hPF, had only temporary VOR deficits. In a final experiment, zebrafish eggs were placed in the bioreactor at 3 hPF and removed at 96 hPF but the bioreactor was turned off from 24 hPF to 72 hPF. These larvae had normal VORs when they were removed from the bioreactor at 96 hPF. Taken as a whole, these data support the idea that there is a critical period for functional maturation of the zebrafish vestibular system. The developmental period identified includes the timeframe during which the vestibular primary afferent neurons are born, innervate their central and peripheral targets, and remodel their central projections.     

Does supervised exercise offer adjuvant benefit over exercise advice alone for the treatment of intermittent claudication? A randomised trial.

OBJECTIVE: Exercise advice is the main treatment for symptom relief in the UK for patients with mild to moderate Intermittent Claudication (IC). Would a weekly exercise and motivation class for 6 months offer adjuvant benefit over written and verbal exercise advice alone? PATIENTS AND METHODS: Fifty-nine patients attending a regional vascular centre for whom IC was the main factor affecting mobility were randomised to either exercise advice alone (n=30) or exercise advice with a once a week 45 min supervised exercise/motivation class (n=29). The mean age was 68 years. Baseline and 6-month assessment included a Quality of Life Questionnaire--the Short-Form-36, the Charing Cross Symptom Specific Claudication Questionnaire (CCCQ) and treadmill walking distance (3.5 km/h 12%). RESULTS: At 6-month follow-up the supervised exercise group had improved their treadmill walking by 129% compared to 69% in the advice alone group (p=0.001). This significant improvement was maintained at the subsequent 9 and 12-month follow-up assessments. By the 9-month stage the advice only group CCCQ score had improved 16% from baseline, while the supervised exercise group had a significantly better 43% improvement in base line score (p<0.05). Self reported frequency of walks was higher in the exercise class group being significant for improvement in CCCQ score. CONCLUSION: A weekly, supervised exercise and motivation class for a 6-month period provides a significant improvement in patients' symptoms, quality of life, and distance walked compared with advice alone and this improvement continues after attendance at class has ceased.     

Novel strategies for enhancing tissue integration in cartilage repair

Introduction Articular cartilage is unable to initiate a spontaneous repair response when injured due to its avascular and aneural properties. Within adult cartilage, chondrocytes are entrapped within an extensive extracellular matrix and are unable to migrate to sights of injury to regulate tissue repair. Injury to this tissue therefore inevitably leads to degeneration of the cartilage and the development of degenerative diseases such as osteoarthritis. The surgical technique of autologous chondrocyte transplantation (ACT) was developed for the treatment of full‐thickness cartilage defects ( Brittberg et al. 1994 ). Implantation of chondrocytes into the defect site repairs the injury site with a mixture of fibrocartilaginous and hyaline‐like tissue that poorly integrates with the existing cartilage and frequently degenerates with time. In this current study, we have developed an in vitro model to investigate methods for enhancing this integration and the development of a more biomechanically stable repair tissue. Materials and methods Bovine articular cartilage explants from the metacarpalphalangeal joint were experimentally injured using a stainless steel trephine and cultured for a period of 28 days. Autologous chondrocytes in an agarose suspension were injected into the interface region at the injury site. Media was collected and analysed for proteoglycan and collagen content using the DMMB and hydroxyproline assays, respectively. Matrix metalloproteinase (MMP) expression was also analysed using zymography and an adapted collagen fibril assay. Results Morphological analyses indicate attempts at repair and integration within both control and experimental treatment groups, although the presence of autologous chondrocytes appeared to amplify this repair response. Although not statistically significant, considerable differences in proteoglycan release between injured explants and the intact control group were seen. Collagen release into the media was only seen at day 28 within experimental cultures. An up‐regulation of MMP‐2 and MMP‐9 was seen within the experimental cultures compared to the controls. Preliminary data also suggest up‐regulation of collagenases in the experimental group when compared to controls. Discussion As seen with clinical ACT treatment, the presence of autologous chondrocytes appears to enhance repair and integration attempts; however, morphologically, this repair tissue appears to be fibrocartilaginous. Further analysis will establish whether the repair tissue is true hyaline cartilage and monitor the synthesis and turnover of macromolecules within the established culture system.     

Airway surface pH in subjects with cystic fibrosis.

The cystic fibrosis (CF) transmembrane conductance regulator protein can transport bicarbonate and may therefore regulate airway surface (AS) pH. Disturbances of AS pH could contribute to the pathophysiology of CF lung disease. Five studies were carried out including the following: study 1) nasal pH measurements were made in 25 CF and 10 non-CF adults using an antimony pH probe. Mean nasal pH was significantly lower in the CF group. Nasal potential difference may have been a confounding factor; study 2) in a fresh cohort of CF and non-CF subjects, no significant difference was found between the two groups using a gold pH probe; study 3) simultaneous nasal pH measurements were made in 15 CF and 15 non-CF adults using both probes. In the CF group, there was a trend for the antimony probe to read lower than the gold probe. In the non-CF group, the antimony probe read higher. The pH difference noted in study 1 related to technical factors; study 4) the effect of acute changes in serum acid/base balance on nasal pH was assessed in five non-CF adults. Nasal pH was not altered by either acute respiratory acidosis or alkalosis; study 5) nasal and lower airway pH was measured in five CF and six non-CF children. No difference was found between the groups. There was a correlation between nasal and lower airway pH. The authors conclude that airway surface pH does not differ between cystic fibrosis and noncystic fibrosis subjects and therefore, cystic fibrosis transmembrane conductance regulator may not play a major role in airway surface pH in vivo.     

Effects of intrathecal and intraperitoneal morphine on gastrointestinal motility in the rat.

A method has been developed to compare gastrointestinal (GI) transit time after intrathecal (i.t.) drug injection in the rat. Each animal had a catheter implanted in the i.t. space. Eight rats, on three separate occasions, had either i.t. morphine 16 micrograms kg-1 (in 50 microliters) or intraperitoneal (i.p.) morphine (0.1%) 7.5 mg kg-1 or i.t. saline (50 microliters). The dose of morphine was the ED50 for analgesia by each route. After halothane and oxygen anaesthesia, 10 steel balls and 1 ml of contrast medium were placed into the stomach, the whole procedure being completed within 5 min. Radiographs were taken at 5 min, 3, 6 and 24 h, and the number of balls in the stomach, small and large intestine were counted. The inhibitory effect of i.t. or i.p. morphine on gut motility caused an equally significant delay at 6 h. In a separate series of eight rats the delay by i.t. morphine could be completely antagonized by i.p. naloxone 1 mg kg-1. Thus, i.t. morphine in an analgesic dose even though smaller than the i.p. dose has a similar inhibitory effect on GI tract motility in the rat. This method would enable comparisons on GI transit to be made between a variety of intrathecally administered drugs.