Ex vivo cytotoxic drug evaluation by DiSC assay to expedite identification of clinical targets: results with 8-chloro-cAMP

There is a pressing need to reduce the time and cost of developing new cytotoxic agents and to accurately identify clinically active agents at an early stage. In this study, the differential staining cytotoxicity (DiSC) assay was used to assess the efficacy of the novel antitumour cAMP analogue, 8-chloro-cAMP (8-Cl-cAMP) (and its metabolite 8-Cl-adenosine) against 107 fresh specimens of human neoplastic and normal cells. Diagnoses included chronic and acute leukaemias, myeloma, non-Hodgkin's lymphoma (NHL) and miscellaneous solid tumours. The aim was to identify targets for subsequent phase I, II and III trials. 8-Cl-cAMP was tested at 4-985 microM, along with standard chemotherapeutic drugs. 8-Cl-cAMP and its metabolite caused no morphologically observable cell differentiation but induced dose-dependent cytotoxicity. Compared with untreated patients, previously treated chronic lymphocytic leukaemia (CLL) patients showed no increase in ex vivo resistance to 8-Cl-cAMP (P = 0.878); minimal cross-resistance with other cytotoxic drugs was detected. Compared with normal cells (mean LC90 = 1803 microM), 8-Cl-cAMP showed significant ex vivo activity against CLL (117.0 microM; P < 0.0001) and NHL (140.0 microM; P < 0.0001), of which eight were mantle cell NHL (84.7 microM), and greatest activity against cells from patients with acute myeloid leukaemia (AML; mean LC90 = 24.3 microM; in vitro therapeutic index 74-fold, P < 0.0001). Solid tumour specimens were comparatively resistant to 8-Cl-cAMP. The results highlight the clinical potential of 8-Cl-cAMP, point to several new phase I, II and III trial possibilities and provide a rationale for the inclusion of ex vivo cytotoxic drug evaluation in the drug development process.     

Carbamazepine and its metabolites in human perfused placenta and in maternal and cord blood

Placental transfer and metabolism of carbamazepine (CBZ) was studied in a dual recirculating placental cotyledon perfusion system and was also evaluated in 16 pairs of maternal venous and cord blood samples. Among the parameters studied as possible indicators of a successful perfusion, volume changes in perfusate divided the perfusions into two groups, whereas no significant differences between perfusions were noted in blood gas analysis or in antipyrine transfer. CBZ added into the maternal circulation crosses the placenta in the beginning quicker than antipyrine which is in agreement with the different lipid solubilities of these compounds. Because the transfer rates of antipyrine and CBZ were about the same, the mechanism of transfer of CBZ is probably similar to that of antipyrine (passive diffusion). No metabolites of CBZ could be detected in the perfusate by high-performance liquid chromatography (HPLC) or gas chromatography/mass spectrometry. With the improved HPLC methodology for CBZ metabolites, six metabolites were detected in clinical samples, including 10-hydroxy-10,11-dihydro-CBZ (10-OH-CBZ), which has been described earlier in only 1 uremic patient. Relative levels of metabolites showed significant individual differences. CBZ crosses perfused placenta rapidly, but this does not contribute to CBZ metabolites detected in maternal and fetal circulation.     

Evaluation of an educational program for asthma control in adults

We have developed a 6-month educational plan associated with outpatient follow-up and special clinical care for asthmatic patients in a deprived population, with serious socioeconomic problems and a very low level of education. The objective was to determine the effects of the program on clinical asthma outcomes, lung function, and quality of life. Forty patients were enrolled in the program with a regular schedule of outpatient visits, and 31 finished the 6-month intervention, which included information about asthma, instruction in appropriate use of medication, training in metered-dose inhaler technique, how to identify and control asthma triggers, how to use symptom diary cards, and how to recognize early signs of deterioration. Patients included 8 males and 23 females, 47.8 +/- 16.5 years old, with 77.4% elementary school education and 22.6% illiterate, and an average monthly income of around $450. After the 6-month program there was a significant change in asthma control with a reduction in asthma emergency visits and hospitalization, reduction of score symptoms, and improvement in quality of life. Based on the results, educational programs are recommended and should be adapted to the socioeconomic and cultural characteristics of the target population.     

Oxidation of phenolic compounds by lactoperoxidase. Evidence for the presence of a low-potential compound II during catalytic turnover

The lactoperoxidase (LPO)-catalyzed oxidation of p-phenols by hydrogen peroxide has been studied. The behavior of the enzyme differs from that of other peroxidases in this reaction. In particular LPO shows several catalytic intermediates during the catalytic cycle because of its capability to delocalize an oxidizing equivalent on a protein amino acid residue. In the phenol oxidation the enzyme Compound I species, containing an iron-oxo and a protein radical, uses the iron-oxo group at acidic pH and the protein radical in neutral or basic medium. Kinetic and spectroscopic studies indicate that the ionization state of an amino acid residue with pKa 5.8 +/- 0.2, probably the distal histidine, controls the enzyme intermediate forms at different pH. LPO undergoes inactivation during the oxidation of phenols. The inactivation is reversible and depends on the easy formation of Compound III even at low oxidant concentration. The inactivation is due to the substrate redox potential since the best substrate is that with lowest redox potential, while the worst substrate has the highest potential. This strongly indicates that Compound II, formed during catalytic turnover, has a low redox potential, making easier its oxidation by hydrogen peroxide to Compound III. The dependence of LPO activity on the phenols redox potential suggests that the protein radical where an oxidizing equivalent can be localized is a tyrosyl residue.     

A study examining intravenous ethanol-conditioned place preference in C57BL/6J mice

The reinforcing effects of intravenous (I.V.) ethanol were examined in C57BL/6J (C57) mice with a conditioned-place-preference (CPP) paradigm. Before CPP testing, adult mice underwent jugular catheterization. On the following day, subjects were acclimated to a two-compartment CPP chamber. A 15-min nondrug pretest was conducted to determine compartment preference. For the treatment group, I.V. ethanol [30% (v/v), 3.4 microl/min, 25 min] was paired with the nonpreferred compartment, whereas I.V. saline was paired with the preferred compartment. The control group received I.V. saline in both compartments. Two conditioning sessions were conducted per day (0900 and 1500), and the order of the infusions was counterbalanced across subjects. The drug-free posttest was identical to the pretest, except that it occurred on the day after the final drug/compartment pairing. The entire procedure required 6 days. After just two pairings with ethanol, with a cumulative ethanol dose of only 0.82 g/kg/day, significant CPP was noted in the treatment group, whereas no change in compartment preference was noted for the control group. A separate group of C57 mice were trained to discriminate intraperitoneal ethanol (1.5 g/kg) from saline using a two-lever drug discrimination paradigm. After training was complete, these mice also underwent jugular catheterization. Substitution testing was conducted with I.V. ethanol [30% (v/v), 6.4 microl/min, 12 min] and saline. The results indicate that the subjective effects of ethanol did not differ according to the route of administration. Together, these experiments provide evidence that ethanol is rewarding for C57 mice, as indexed by ethanol CPP, and that the subjective effects of intravenously and intraperitoneally administered ethanol are similar.     

Bone metabolism following bladder substitution with the ileal urethral Kock reservoir

OBJECTIVES: To assess bone metabolism following bladder substitution with the ileal Kock reservoir. PATIENTS, SUBJECTS AND METHODS: The investigation comprised two separate studies, one with baseline measurements before and after surgery, and the second after surgery only, of bone mass, made using single-photon absorptiometry and dual-energy X-ray absorptiometry, biochemical variables of bone turnover, plasma analyses and measurements of renal calcium and phosphate excretion. After inclusion, both groups of patients were observed longitudinally for 2 years. The post-surgery study included 25 patients who had undergone bladder substitution (median age 67 years, range 44-75), with a median post-operative follow-up of 1.0 year (range 0.3-3.7), and 16 control subjects (either healthy or with other minor urological complaints; median age 62 years, range 34-80), and the pre-surgery study comprised seven patients who had undergone bladder substitution (median age 57 years, range 42-68). RESULTS: Total body, forearm and spinal bone mineral contents were similar in patients with an ileal bladder substitute measured 1 year after surgery and in control subjects. There were equivalent significant changes in both the patients and control subjects during the 2-year observation period, with a 2-3% decrease in total body and forearm bone mineral content. The values were similar in patients with and without a mild metabolic acidosis. Plasma calcium, phosphate, total alkaline phosphatase, intact parathyroid hormone, vitamin D and osteocalcin were normal in both patients and control subjects. Renal excretion of calcium and phosphate was also similar in patients and in control subjects. CONCLUSIONS: Ileal urethral Kock bladder substitution does not lead to accelerated bone mineral loss in elderly men, despite a mild metabolic acidosis in half of the patients.     

Specificity of detection of Chlamydia pneumoniae in cardiovascular atheroma: evaluation of the innocent bystander hypothesis

Chlamydia pneumoniae has been detected in atherosclerotic plaque, raising the question of whether this detection is specific to atheromatous tissue. To evaluate this question, we tested cardiovascular and non-cardiovascular tissue samples from 38 autopsy cases by polymerase chain reaction and immunocytochemistry. We also tested 33 granuloma biopsy specimens, as the organism has been detected in macrophages. C. pneumoniae was detected in coronary artery tissue from 13 (34%), lung from 5 (13%), liver from 4 (10%), and spleen from 2 (5%) of the 38 autopsy cases (P < 0.05 for comparison of proportion of positive coronary arteries with that of each of the other types of tissue). Of the 21 cases with at least one positive tissue sample, 11 had only a positive cardiovascular tissue (coronary artery, venous bypass graft, or myocardium), 7 had both cardiovascular and non-cardiovascular positive tissues, and 3 had only a non-cardiovascular positive tissue. C. pneumoniae was thus detected relatively infrequently in non-cardiovascular tissues, and its detection in these tissues was usually in association with its detection in cardiovascular tissue from the same patient. The organism was also infrequently detected in granulomatous tissue (3/33 specimens). These findings demonstrate that C. pneumoniae is more frequently found in atherosclerotic than normal tissue and support the hypothesis that C. pneumoniae has a role in the pathogenesis of atherosclerosis.     

Vagal regulation during bottle feeding in low-birthweight neonates: support for the gustatory-vagal hypothesis

BACKGROUND AND PURPOSE: Linear accelerators equipped with multileaf collimators (MLCs) are becoming more common and are widely available from most commercial manufacturers. There is a need to ensure they retain their commissioning specification using a preventative maintenance and quality control (QC) programme. This paper considers the design criteria of the Philips MLC which are important to the production of a comprehensive quality control programme. MATERIALS AND METHODS: The specific QC problems related to MLCs are identified as the positional accuracy of the leaves and their relationship to the back-up collimators, leakage considerations, the relationship of X-ray to light field and the influence of gravity on the positioning and leakage characteristics of the leaves. These problems are considered in relation to the general design considerations of the MLC, and methods of performing routine quality control checks are discussed. RESULTS AND CONCLUSIONS: The introduction of MLCs into clinical use results in new QC procedures being developed but it can be concluded that for the Philips MLC only an extra 30 min of QC time is needed per month and that its use has added little to the general down-time of this department.     

Differences in the UV-crosslinking patterns of the poliovirus 5''-untranslated region with cell proteins from poliovirus-susceptible and -resistant tissues

Specific polyclonal antibody was raised against D-aspartate (D-Asp) which had been conjugated to glutaraldehyde and was purified by affinity chromatography. Immunohistochemical staining of rat pineal gland with the antibody demonstrated the presence of D-Asp in the cytoplasm of pinealocytes, the predominant cell type in this gland. D-Asp immunoreactivity was more evident in the distal region than in the proximal region of the gland. Pinealocytes in the distal region are presumably involved in the synthesis and secretion of the pineal hormone, melatonin, and the results of staining may indicate some yet unknown role of D-Asp in the regulation of melatonin secretion.