Reversal of CPT-11 resistance of lung cancer cells by adenovirus-mediated gene transfer of the human carboxylesterase cDNA

To evaluate the concept that transfer of the human carboxylesterase (CE) gene will overcome the drug resistance of a solid tumor to CPT-11 (irinotecan), we used an adenovirus vector (AdCMV.CE) carrying human CE cDNA to infect CPT-11-resistant A549 human adenocarcinoma cells (A549/CPT) in vitro and in vivo and evaluated cell growth over time. The A549/CPT cells, selected by stepwise and continuous exposure of parental A549 cells to CPT-11 over 10 months, had a 6-fold resistance to CPT-11 and 42% CE activity in comparison with parental A549 cells. AdCMV.CE infection resulted in an increase in functional CE protein in resistant cells in vitro that was sufficient to convert CPT-11 to its active metabolite, SN-38, and effectively suppressed resistant cell growth in vitro in the presence of CPT-11. When AdCMV.CE was directly injected into established s.c. resistant A549-based tumors in nude mice receiving CPT-11, there was a 1.8-fold reduction in tumor size at day 20 compared to that of controls (P < 0.05). These observations suggest that adenovirus-mediated gene transfer of the human CE gene and concomitant administration of CPT-11 may have potential as a strategy for local control of acquired CPT-11 resistance of solid tumors.     

Imbalance of morphologically addressed telophases reflects interphase DNA aneuploidy in tumorigenesis

Chromosome division figures (CDFs) are quantitatively different from normal mitoses and represent a novel cytogenetic phenomenon. This investigation was focused on morphologically addressed bipolar telophases in histologically defined human biopsies and in the tumour breast cell-line MDA231. Single cell nuclei were recorded by image microphotometry on inflamed and premalignant lesions of skin (49 cases), oral mucosa (43) and colon mucosa (46). DNA content and replication status were analysed in interphase nuclei as well as in mitoses and in CDFs. In contrast to inflamed lesions, premalignancies were characterised by pronounced endoreplication, when the rate exceeding 5 c was > or = 10% in interphase nuclei. CDFs from the corresponding lesions showed an aberrant DNA content beyond 5 c even more frequently. DNA profiles of metaphases and telophases resembled those of prophases. Therefore, the DNA content of corresponding telophase hemispheres was measured. Severe differences averaged 0.3 c in MDA231 and up to 0.5 c in premalignant lesions. The mean difference between two corresponding hemispheres was 0.39 +/- 0.09 c in Bowenoid keratosis (n = 31), 0.40 +/- 0.08 c in high-grade dysplasia of oral mucosa (n = 16) and 0.21 +/- 0.03 c in high-grade dysplasia of colon adenoma (n = 65 telophases). As a control, the telophase difference was only 0.07 +/- 0.02 c (n = 23) in foetal liver and 0.06 +/- 0.01 c in 24 amnion cells. Thus, genomic instability and, in consequence, genomic imbalance can best be quantified from the DNA profiles of telophase CDFs and from the various DNA amounts in their hemispheres. A strong selection against telophases was observed in neoplasias developing DNA aneuploidy. Those aberrant telophases which escape selection are thought to enhance tumour progression.     

Determination of extent of formaldehyde-induced crosslinking in hard gelatin capsules by near-infrared spectrophotometry

PURPOSE: To predict the degree of crosslinking from formaldehyde-stressed hard gelatin capsules (HGCs) using near-infrared spectrophotometry (NIR). METHODS: HGCs were exposed to a 150 ppb atmosphere of formaldehyde for 2.25, 4.60, 9.42, 16.0 and 24.0 hours. The capsules were filled with fresh amoxicillin, placed in a 90 degrees conical reflector cone, and scanned in a NIR spectrophotometer. Principal component regression (PCR) was employed to analyze the spectra of the intact capsules. Dissolution profiles were then obtained for each experimental group. RESULTS: The dissolution of amoxicillin from the capsules at pH 1.2 was found to decrease with increasing time of exposure to the formaldehyde atmosphere. A set of principal components (PCs) was formed by a linear combination of the absorbance values at each wavelength scanned. A good correlation was established (r2 = 0.963) when PC values from the NIR spectra of the HGCs were regressed against percentage of amoxicillin dissolved at 45 minutes, at pH 1.2. Water content of the capsules was found to be the largest determinant in the variation between HGC spectra at each exposure time. CONCLUSIONS: NIR spectrophotometry, combined with PCR, was successful at not only predicting dissolution of HGCs exposed to formaldehyde, but also at determining which wavelengths contributed most to spectral variation of these stressed HGCs.     

Spatial discrimination against background with different optical systems for collection of fluorescence in laser-excited atomic fluorescence spectrometry with a graphite tube electrothermal atomizer

A single 90 degrees off-axis ellipsoidal mirror fragment was used in a dispersive detection system for electrothermal atomization laser-excited atomic fluorescence spectrometry. The performance of the new optical arrangement was compared with those of optical arrangements that employed a plane mirror in combination with biconvex or plano-convex lenses. All the optical arrangements collected fluorescence in a scheme called front surface illustration. BEAM-4, an optical ray tracing program, was used for calculations of spatial ray distributions and optical collection efficiency for the various optical configurations. Experimentally, the best collection efficiency was obtained by use of the ellipsoidal mirror, in qualitative agreement with simulations done by use of the BEAM-4 software. The best detection limit for cobalt with the new optical arrangement was 20 fg, which was a factor of 5 better than that obtained with conventional optical arrangements with otherwise the same instrumentation. The signal-to-background ratio and the fluorescence collection efficiency were also studied as a function of position of the optical components for the various optical arrangements. For both cobalt and phosphorus, the signal-to-background ratio with the new optical arrangement remained stable within 10-20% during +/- 8 mm shifts in the position of the detection system from the focal plane of the optics. Overall, the new optical arrangement offered high collection efficiency, excellent sensitivity, and facile optical alignment due to efficient spatial separation between the fluorescence signal and the background radiation. The advantages of the new optical arrangement were particularly important during measurements in the presence of high levels of blackbody radiation.     

Detection of clonal immunoglobulin gene rearrangements in the PBPC harvests of patients with acute lymphoblastic leukaemia

We have used a fluorescently based PCR technique to detect rearrangements in the immunoglobulin heavy chain (IgH) gene in the presentation BM of five patients with adult ALL and have looked for similar rearrangements in their PBPC. Using this approach we have been able to demonstrate clonal rearrangements in the PBPC of two of five patients. Remission BM samples taken 6-12 weeks prior to leucapheresis failed to show a clonal rearrangement in either patient. The significance of these results is discussed.     

How sleep and mental disorders are related to complaints of daytime sleepiness

BACKGROUND: Daytime sleepiness is widespread and has negative impacts on the public sector. OBJECTIVE: To ascertain the incidence and prevalence of daytime sleepiness and associated risk factors in the general population. METHOD: In 1994, a representative sample of the non-institutionalized British population aged 15 years or older was interviewed via telephone using an expert computer-assisted program designed to facilitate surveys of this type (Sleep-Eval, M. M. Ohayon, Montreal, Quebec). Subjects were classified into 3 groups based on the severity of their daytime sleepiness. We completed 4972 interviews (acceptance rate, 79.6%). RESULTS: Severe daytime sleepiness was reported in 5.5% (95% confidence interval, 4.9%-6.1%) of the sample, and moderate daytime sleepiness in another 15.2% (95% confidence interval, 14.2%-16.2%). Associated factors with severe daytime sleepiness included female sex, middle age, napping, insomnia symptoms, high daily caffeine consumption, breathing pauses or leg pain in sleep, depressive disorder (based on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, criteria), falling asleep while reading or watching television, and motor vehicle crashes or accidents involving use of machinery. Moderate daytime sleepiness was associated with female sex, napping, insomnia symptoms, arthritis or heart disease, and gross motor movements during sleep. CONCLUSIONS: It is likely that daytime sleepiness deleteriously affects work activities, social and/or marital life, and exhibits a negative socioeconomic impact. In addition, the risk of a motor vehicle crash appears to be higher in this specific population: twice as many subjects operating a motor vehicle or using machine tools reported having a crash or accident, respectively, in the previous year in the groups with severe daytime sleepiness or moderate daytime sleepiness than did the general population with no daytime sleepiness. The high prevalence rates of daytime sleepiness and multiplicity of related factors mandate further scrutiny by public health officials.