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.     

Increased incidence of gap junctional coupling between spinal motoneurones following transient blockade of NMDA receptors in neonatal rats

Neonatal rat motoneurones are electrically coupled via gap junctions and the incidence of this coupling declines during postnatal development. The mechanisms involved in this developmental regulation of gap junctional communication are largely unknown. Here we have studied the role of NMDA receptor-mediated glutamatergic synaptic activity in the regulation of motoneurone coupling. Gap junctional coupling was demonstrated by the presence of graded, short latency depolarising potentials following ventral root stimulation, and by the transfer of the low molecular weight tracer Neurobiotin to neighbouring motoneurones. Sites of close apposition between the somata and/or dendrites of the dye-coupled motoneurones were identified as potential sites of gap junctional coupling. Early postnatal blockade of the NMDA subtype of glutamate receptors using the non-competitive antagonist dizocilpine maleate (MK801) arrested the developmental decrease in electrotonic and dye coupling during the first postnatal week. These results suggest that the postnatal increase in glutamatergic synaptic activity associated with the onset of locomotion promote the loss of gap junctional connections between developing motoneurones.     

Prediction of lactose malabsorption in referral patients

Two hundred forty-two patients referred for various gastrointestinal complaints were evaluated for clinical parameters that would predict findings of lactose malabsorption. Breath hydrogen and blood glucose lactose tests were performed after ingestion of 50 g lactose. Presenting complaints, duration of symptoms, and patient demographics such as age, sex, and ethnic heritage were not different between lactose malabsorbers and absorbers as defined by the breath hydrogen lactose test. Foodrelated symptoms in general and after specific foods such as milk, ice cream, cheese, and yogurt were also similar between groups. Prior to testing, 30% of malabsorbers (N=161) and 36% of absorbers (N=81) reported lactoserelated symptoms (P=NS). The blood glucose response to lactose was abnormal in 60% of malabsorbers and 15% of absorbers. This study confirmed our impression that it is difficult to predict lactose absorption status by clinical parameters. The majority of our lactose malabsorber patients were unaware of lactose-associated symptoms. Furthermore, symptom assessment, demographics, food history, and blood glucose testing did not predict abnormal hydrogen responses to lactose.The opinions and assertations expressed herein are those of the authors and are not to be construed as reflecting opinions of the United States Air Force or the Department of Defense.This work has been presented in part at the Annual Scientific Session of the American Gastroenterological Association, San Francisco, California, May 19, 1986, and published as an abstract (Gastroenterology 90:1562, 1986).     

Symptomatic reversible duodenal compression due to iatrogenic retroperitoneal hematoma

Selective serotonin reuptake inhibitors are frequently employed to treat depression. However, although rarely, coagulation abnormalities have been described following the use of these compounds, and these effects appear to be enhanced by simultaneous use of nonsteroidal anti-inflammatory drugs. We describe a case of reversible symptomatic duodenal compression caused by a retroperitoneal hematoma after ingestion of sertraline and nimesulide.     

The prognostic impact of fluctuating levels of C-reactive protein in Brazilian haemodialysis patients: a prospective study.

BACKGROUND: A single elevated C-reactive protein (CRP) value predicts mortality in haemodialysis (HD) patients, but the relative importance of repeated vs occasional positive systemic inflammatory response findings is not known. METHODS: To assess the influence on survival of occasional inflammation, CRP, serum albumin (S-Alb) and fibrinogen were analysed bimonthly in 180 HD patients (54% male, 49+/-14 years). Clinically significant inflammation was defined as CRP >5.1 mg/l, based on the receiver operating characteristics curve for CRP as predictor of death. Based on four consecutive measurements of CRP, patients were assigned into three groups: group 1 (n = 74; 41%), no inflammation (CRP < or = 5.1 mg/l in all measurements); group 2 (n = 65; 36%), occasional inflammation (1-3 measurements of CRP > 5.1 mg/l); and group 3 (n = 41; 23%), persistent inflammation (all measurements of CRP >5.1 mg/l). The nutritional status was evaluated by subjective global assessment (SGA) and body mass index (BMI), and the survival (21 months of follow-up) by Kaplan-Meier curve and Cox model. RESULTS: The median and range of CRP values (mg/l) for group 1, 2 and 3 were: 3.2 (3.2-5.1), 3.6 (3.2-54.9) and 13.8 (5.2-82), respectively (P<0.001), whereas the prevalence of malnutrition, assessed by SGA and BMI, did not differ significantly between the groups. The survival rate by Kaplan-Meier analysis was significantly different among the groups (chi2 = 12.34; P = 0.0004). Patients in group 3 showed the highest mortality (34%; P = 0.001), compared with group 1 (8%) and group 2 (14%; P = 0.01), respectively, whereas there was no significant difference in mortality between groups 1 and 2. Age, CRP, S-Alb level and SGA were independent predictors of mortality. CONCLUSION: The patients with a persistent elevation of CRP had a higher mortality rate than the patients with occasional CRP elevation. Thus, persistent, rather than occasional, inflammation is an important predictor of death in HD patients.     

Carbon dioxide laser salvage surgery after radiotherapy failure in T1 and T2 glottic carcinoma.

OBJECTIVES: Recurrent glottic carcinoma after radiotherapy (RT) may be managed by open neck or endoscopic surgery. The impact of endoscopic treatment with CO(2) laser for recurrent glottic carcinoma after RT is reported. METHODS: We present the oncologic and vocal outcomes of a retrospective study based on a series of 16 patients with rT1 and rT2 glottic carcinoma who were endoscopically managed between February 1995 and December 1999 after RT failure. All patients were males with a mean age of 68.7 years (range, 50 to 87 years). Before RT, the lesions had been staged as T1 N0 in 11 patients and T2 N0 in 5, and after RT as rT1 N0 in 12 and rT2 N0 in 4. According to the European Laryngological Society classification, a total of 9 transmuscular, 3 total, and 4 extended cordectomies were performed. Mean follow-up was 45 months (range, 9 to 79 months). RESULTS: Endoscopic salvage surgery was successful in 14 patients. One of them developed a second recurrence and was definitively cured with an additional endoscopic procedure. Two of the 16 patients had recurrent disease after salvage laser surgery and died due to progression of disease. Ultimate local control with laser alone at 3 years was 87.1%, according to the Kaplan-Meier method. Laryngeal preservation was obtained in all survivors after endoscopic rescue surgery. Voice analysis showed a clear correlation between the amount of vocal cord tissue resected and decrease of the vocal outcome. CONCLUSIONS: The present series indicates that selected recurrences after primary RT for T1 and T2 glottic carcinoma are eligible for endoscopic salvage surgery with oncologic results comparable to those with open neck procedures but with a lower complication rate and a favorable functional outcome.     

Sleep complaints and their relation with drug treatment in patients suffering from Parkinson's disease.

The aim of this research was to quantify sleep problems in patients suffering from Parkinson's disease by means of the new Parkinson's Disease Sleep Scale (PDSS) and to correlate such problems with the possible influence of current drug treatment. A total of 70 patients (36 men and 34 women) with a diagnosis of Parkinson's disease were enrolled. Their mean age was 69.7 +/- 8.2 years, and duration of disease was 7.4 +/- 4.8 years. All patients completed the PDSS and the Unified Parkinson's Disease Rating Scale (UPDRS Parts I-IV). Drug consumption and doses were registered. The mean score on the PDSS scale was 109.23 +/- 19.75 and on the UPDRS III scale was 25.24 +/- 11.35. The lowest scores were obtained in Item 3 (sleep fragmentation): 5.53 (2.46); and in Item 8 (nocturia): 5.75 (2.91). There was a weak correlation between the PDSS and UPDRS III (cc = -0.355, P = 0.003), PDSS and UPDRS I (cc = -0.272, P = 0.02), and PDSS and UPDRS IV (cc = -0.416, P < 0.001). Motor conditions, mental state, and drug complications influence sleep quality. Although this effect was significant, it was not of a great magnitude. Dopaminergic drugs did not increase daytime sleepiness. As a whole, sleep quality in patients who took dopaminergic agonists did not differ from that of patients who took levodopa in monotherapy.     

Age-related changes in the turnover rates of D1-dopamine receptors in the retina and in distinct areas of the rat brain

The steady-state density and the turnover rates of D₁-dopamine receptors were investigated in the striatum, nucleus accumbens, substantia nigra, and retina of adult (3-month-old) and aged (23-month-old) rats. The turnover rates were measured by monitoring the repopulation kinetics of D₁-dopamine receptors labeled with [³H]-SCH 23390 after the irreversible inactivation induced by a single dose of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 10 mg/kg, s.c.). In all the neural tissues examined, the repopulation of D₁ dopamine receptors could be adequately described by a theoretical model that assumes a constant rate of receptor production (i.e. zero order) and a rate of degradation that is dependent on the receptor density at any time (i.e. first order). The results obtained indicate that the reduction in the density of D₁-dopamine receptors in the striatum, nucleus accumbens and substantia nigra of aged rats is the result of a larger decrease in the receptor production rate (−44 to −60%) than in the receptor degradation rate (−21 to −46%). By contrast, the production rate of D₁-dopamine receptors in the retina of aged rats remains unchanged, whilst the degradation rate is reduced by 25%. This results in an age-related increase in the density of D₁-dopamine receptors in the rat retina.     

Chronic peritoneal dialysis in paediatrics: Experience of a national registry

The results of the first 3 year' collaboration of the Italian Registry of Paediatric Chronic Peritoneal Dialysis (CPD) (1986–1988) are presented. This Registry acquired data on the majority of the paediatric patients treated with CPD in Italy, thus providing a national picture in a field where few nationwide surveys are available. Patients of less than 15 years of age at the start of dialysis were enrolled and clinical data collected until the age of 19 years. The number of nephrological paediatric centres participating in the Registry increased from 7 in 1986 to 11 in 1988. The total number of patients on CPD was 70 and the percentage of dialysed children treated with CPD ranged from 40.2% to 43.6%. Data on 89 peritoneal catheters were collected: during 1417 dialysis-months 70 catheter-related complications were observed (1:20.8 dialysis-months); actuarial catheter survival was 92.7% at 6 months, 84.8% at 1 year and 68.8% at 2 years. The incidence of peritonitis changed from 1 episode every 10.9 patient-months in 1986 to 1 every 19.8 in 1988. Abdominal hernias were the other main clinical complication observed. The survival of patients was 92.5% at 3 years, while the technique survival at the same time was 84%.     

In vivo selective monitoring of basal levels of cerebral dopamine using voltammetry with Nafion modified (NA-CRO) carbon fibre micro-electrodes.

The electrochemical technique of differential pulse voltammetry (DPV) with micro-biosensors has been used for a number of years to monitor in vivo and in situ changes in the extracellular concentration of cerebral ascorbic acid, as well as that of the metabolites of dopamine (DA) and serotonin (5-HT). We have recently prepared a carbon fibre micro-electrode (mCFE) which specifically pretreated and coated with Nafion (a negatively charged polymer which repels acids such as 3,4-dihydroxyphenylacetic acid (DOPAC)) allows the direct selective detection of the oxidation of DA and 5-HT in nanomolar concentration in vitro and that of extracellular basal levels of cerebral 5-HT in vivo (peak B at +240 mV). We describe here a modified version of this micro-biosensor now called NA-CRO mCFE as its active tip (30 microns in diameter) is coated with a 50/50 (v:v) mixture of Nafion and dibenzo-18-crown-6 (Aldrich). In vitro this newly reported electrode shows insensitivity to acids (e.g., DOPAC) up to 100 microns and sensitivity to 0.5-1 nM DA. In vivo, in the striatum of anaesthetised rats, a basal oxidation peak at +80 mV (peak A, on average 0.6 nA in height), which corresponds to the oxidation potential of DA in vitro, is consistently detectable with the NA-CRO mCFE (corresponding to an estimated concentration of 1.5 nM). Experiments performed in vivo in anaesthetised rats implanted in the striatum with uncoated (normal) mCFE to measure extracellular DOPAC or with NA-CRO mCFE have been performed in order to analyse the chemical nature of peak A in vivo. It is concluded that the addition of the crown-ether compound to the Nafion coat improves the sensitivity of the micro-biosensor for DA in vitro and allows the detection of its basal extracellular levels in vivo.