Ionophore-mediated uptake of ciprofloxacin and vincristine into large unilamellar vesicles exhibiting transmembrane ion gradients

A new method, based on the ion-translocating properties of the ionophores nigericin and A23187, is described for loading large unilamellar vesicles (LUVs) with the drugs vincristine and ciprofloxacin. LUVs composed of distearoylphosphatidylcholine/cholesterol (DSPC/Chol) (55:45 mol/mol) or sphingomyelin (SPM)/Chol (55:45 mol/mol) exhibiting a transmembrane salt gradient (for example, internal solution 300 mM MnSO4 or K2SO4; external solution 300 mM sucrose) are incubated in the presence of drug and, for experiments involving divalent cations, the chelator EDTA. The addition of ionophore couples the outward movement of the entrapped cation to the inward movement of protons, thus acidifying the vesicle interior. External drugs that are weak bases can be taken up in response to this induced transmembrane pH gradient. It is shown that both nigericin and A23187 facilitate the rapid uptake of vincristine and ciprofloxacin, with entrapment levels approaching 100% and excellent retention in vitro. Following drug loading, the ionophores can be removed by gel exclusion chromatography, dialysis, or treatment with biobeads. In vitro leakage assays (addition of 50% mouse serum) and in vivo pharmacokinetic studies (in mice) reveal that the A23187/Mn2+ system exhibits superior drug retention over the nigericin/K+ system, and compares favorably with vesicles loaded by the standard DeltapH or amine methods. The unique features of this methodology and possible benefits are discussed.     

Comparison of PF4/heparin ELISA assay with the 14C-serotonin release assay in the diagnosis of heparin-induced thrombocytopenia

The diagnosis of heparin-induced thrombocytopenia (HIT) may be affirmed by demonstrating heparin-dependent anti-platelet antibodies using the 14C-serotonin release assay (SRA). In this study, results of the SRA was compared with the recently described platelet factor 4 (PF4)/heparin enzyme-linked immunosorbent assay (ELISA). Compared with the SRA, the sensitivity and specificity of a PF4/heparin ELISA was 87% and 92%, respectively, using an assay developed in our laboratory; and 90% and 98%, respectively, using a commercially developed kit (Diagnostica Stago, Asnieres, France). However, antibodies to PF4/heparin were also detected in up to 8% of patients whose plasma was negative by SRA, and 23% of patients receiving heparin who were not thrombocytopenic. These data indicate that results obtained with the PF4/heparin ELISA and the SRA are generally in accord in patients with a clinical diagnosis of HIT. However, discrepant results occur in approximately 20% of cases because of the greater sensitivity of ELISA and the possible involvement of other heparin-binding proteins. The fact that each assay contributes independent information in some cases must be considered in the sequence of test performance and in providing consultation to the practicing hematologist.     

The kappa opioid receptor expressed on the mouse R1.1 thymoma cell line down-regulates without desensitizing during chronic opioid exposure

The R1.1 mouse thymoma cell line expresses a single class of kappa opioid receptors that is negatively coupled to adenylyl cyclase through a Bordetella pertussis toxin-sensitive inhibitory guanine nucleotide-binding protein. The aim of the present study was to determine whether chronic opioid treatment of R1.1 cells altered either the binding properties or the functional response associated with the kappa opioid receptor. Culturing of R1.1 cells with the kappa-selective agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methane-sulfonate hydrate (U50,488) for 3 hr and longer, followed by extensive washing of R1.1 cell membranes, produced a concentration- and time-dependent reduction in the binding of the kappa-selective ligand (5 alpha,7 alpha,8 beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1- oxaspiro(4,5)dec-8-yl) benzeneacetamide ([3H]U69,593). Culturing of R1.1 cells with 100 nM U50,488 for 24 hr produced approximately a 50% reduction in the Bmax value for [3H]U69,593 and [3H]naloxone binding. In contrast to the reduction in binding, there was no change in the inhibition of adenylyl cyclase activity by (-)-U50,488. To determine whether kappa opioid receptor function was maintained by spare receptors after agonist-induced down-regulation, membranes from untreated R1.1 cells were incubated with 400 nM of the irreversible opioid antagonist beta-chlornaltrexamine (beta-CNA) followed by extensive washing. beta-CNA produced a 50% reduction in the [3H]U69,593 binding and a 6-fold increase in the IC50 value for (-)-U50,488 inhibition of adenylyl cyclase activity, with no change in the maximal inhibition of cyclic AMP levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

The possible influence of the menstrual cycle on the adherence of Candida albicans to human buccal epithelial cells in vitro

Although the influence of the menstrual cycle on both vaginal candidosis and Candida albicans adherence to vaginal epithelial cells in vitro has been shown to be significant, similar studies have not been made on oral candidosis and adherence to buccal epithelial cells. The aim of this study was therefore to use an in vitro adherence assay to investigate the possible influence of the menstrual cycle on the adherence of C. albicans to buccal epithelial cells. Epithelial cells were collected from a single, healthy, female volunteer on days 5, 15, 22 and 28 of six menstrual cycles. Adherence of C. albicans was significantly higher to buccal epithelial cells collected on day 5 of the menstrual cycle when compared with days 15, 22 and 28, both in terms of the percentage of buccal epithelial cells with adherent C. albicans and the number of C. albicans adhering per 200 buccal epithelial cells in four out of six menstrual cycles (p < 0.001). This result indicates that hormonal influences should be considered when buccal epithelial cells are used in vitro to assess candidal adherence and may implicate hormonal factors in the aetiology of oral candidosis.     

Randomized trial of breast self-examination in Shanghai: methodology and preliminary results

BACKGROUND: The efficacy of breast self-examination in helping to reduce mortality from breast cancer has not been rigorously demonstrated. PURPOSE: To assess efficacy, a large, randomized trial was initiated in Shanghai, China. METHODS: From October 1989 to October 1991, 267040 current and retired female employees associated with 520 factories in the Shanghai Textile Industry Bureau were randomly assigned on the basis of factory to either a self-examination instruction group (133375 women) or a control group (133665 women). The women were born within the period from 1925 through 1958. Women in the instruction group were given intensive training in breast self-examination, including the use of silicone breast models and personalized instruction, plus two subsequent reinforcement sessions and multiple reminders to practice the technique. Women in the control group were asked to attend training sessions on the prevention of low back pain. All women have been followed for the development of breast diseases and for death from breast cancer. RESULTS: A high level of participation during the first 4-5 years of the trial was documented among women in the instruction group. Randomly sampled women in this group demonstrated greater proficiency in detecting lumps in breast models than did randomly sampled women in the control group. Approximately equal numbers of breast cancers were detected in the two groups (331 in the instruction group and 322 in the control group) through 1994, which is the last year for which case-finding efforts have been completed. The breast cancers detected in the instruction group were not diagnosed at an appreciably earlier stage or smaller size than those in the control group. More benign breast lesions were detected in the instruction group than in the control group (1457 versus 623, respectively), suggesting a higher index of suspicion for women who received training. Cumulative breast cancer mortality rates through 5 years from entry into the study were nearly equivalent for the two groups. CONCLUSIONS: Breast self-examination has not led to a reduction in mortality from breast cancer in this study cohort in the first several years since the trial began. A shift toward the diagnosis of disease at a less advanced stage in women given instruction has also not been demonstrated. Longer follow-up of participants in this trial is required before final assessment can be made of the efficacy of breast self-examination. IMPLICATIONS: At this time, there is insufficient evidence to recommend for or against the teaching of breast self-examination.     

Differences in muscle endurance and recovery between fallers and nonfallers, and between young and older women

BACKGROUND: Significant morbidity and mortality are associated with falls in older adults. We tested the hypothesis that older women with a history of falls demonstrate decreased muscle endurance and longer recovery times following fatiguing exercise. METHODS: We evaluated dynamic endurance and recoverability of the quadriceps femoris of 29 young women (YW) (M age = 21.7), 26 older women with a history of falls (FA) (M age = 73.3), and 27 older women with no history of falls (NF) (M age = 71.2) using an isokinetic dynamometer. Subjects performed repeated maximal concentric knee extensions until the force output of two consecutive repetitions fell below 50% of their maximal voluntary contraction (MVC). Recovery was defined as the time required for the return of force output > or = 80% MVC for 2 consecutive repetitions, within a set consisting of 3 maximal contractions. One minute rest was allowed between sets. We collected electromyographic (EMG) data from the quadriceps during all testing to evaluate spectral shifts. RESULTS: ANOVA with a post-hoc Bonferroni-Dunn test revealed time to fatigue was significantly faster in FA than YW (p < .02) and in FA than NF (p < .05), but not different between YW and NF. Time to recovery was significantly slower in FA than YW (p = .01), but not different between YW and NF, or between FA and NF, EMG median frequency power shift (from the beginning to the end of the test) was significantly less in FA (p < .001) than either YW (p < .002) or NF (p < .05). CONCLUSIONS: Older women with a history of falls demonstrate decreased muscular endurance compared to YW and NF, and increased time to recover from fatiguing exercise when compared to young women.     

Treatment with alendronate prevents fractures in women at highest risk: results from the Fracture Intervention Trial

BACKGROUND: The efficacy of antiresorptive therapy in preventing fractures in women at highest fracture risk, such as very elderly women or those with severe osteoporosis, is uncertain. PARTICIPANTS AND METHODS: Using data from a double-blind, randomized, placebo-controlled clinical trial that enrolled 2027 postmenopausal women aged 55 to 81 years with low femoral neck bone mineral density (BMD) and existing vertebral fractures, we examined the consistency of the effect of treatment with alendronate sodium in preventing fractures within a priori-specified risk subgroups defined at baseline by age, bone density, number of preexisting vertebral fractures, and history of postmenopausal fracture. The women were randomized to oral administration of alendronate or placebo and followed up for an average of 2.9 years. The initial dose of alendronate sodium was 5 mg/d; the dosage was increased from 5 to 10 mg/d at 24 months. New vertebral fractures, the primary end point of this arm of the trial, were defined by morphometry as a decrease of 20% and at least 4 mm in any vertebral height between baseline and a follow-up radiograph at 36 months. Incident clinical fractures, the secondary end point, included nonspine and clinical (symptomatic) vertebral fractures. All clinical fractures were confirmed with x-ray film reports or, in the case of clinical vertebral fractures, x-ray films. RESULTS: Overall, there was a 47% significant reduction in risk of new vertebral fractures in the alendronate group compared with the placebo group. The reduction in risk of new vertebral fracture was consistent across fracture risk categories including age (relative risk [RR], 0.49 in women < 75 years compared with 0.62 in those > or = 75 years), BMD (RR, 0.54 in women with a femoral neck BMD < 0.59 g/cm2 [median] compared with 0.53 in those with a BMD > or = 0.59 g/cm2), and number of preexisting vertebral fractures (RR, 0.58 in women with 1 vertebral fracture compared with 0.52 in those with > or = 2). The overall significant 28% reduction in risk of incident clinical fractures in the alendronate group compared with the placebo group was also observed within these subgroups. Compared with the number of lower-risk women, a similar or smaller number of high-risk women needed to be treated to prevent 1 fracture. For example, 8 women aged 75 years or older compared with 9 women younger than 75 years, or 4 women with 2 or more existing vertebral fractures compared with 16 women with 1 existing vertebral fracture, needed to be treated with alendronate for 5 years to prevent 1 new vertebral fracture. CONCLUSIONS: Alendronate effectively reduces fracture risk in postmenopausal women with vertebral fractures and low BMD, including those women at highest risk because of advanced age or severe osteoporosis. Since the risk reductions observed with alendronate treatment were consistent within fracture risk categories, more fractures were prevented by treating women at highest risk.