Lanthanide-based laser-induced phosphorescence for spray diagnostics.

Laser-induced phosphorescence (LIP) is a relatively recent and versatile development for studying flow dynamics. This work investigates certain lanthanide-based molecular complexes for their use in LIP for high-speed sprays. Lanthanide complexes in solutions have been shown to possess long phosphorescence lifetimes (∼1-2 ms) and to emit light in the visible wavelength range. In particular, europium and terbium complexes are investigated using fluorescence/phosphorescence spectrometry, showing that europium-thenoyltrifluoracetone-trioctylphosphineoxide (Eu-TTA-TOPO) can be easily and efficiently excited using a standard frequency-tripled Nd:YAG laser. The emitted spectrum, with maximum intensity at a wavelength of 614 nm, is shown not to vary strongly with temperature (293-383 K). The decay constant of the phosphorescence, while independent of ambient pressure, decreases by approximately 12 µs/K between 323 and 373 K, with the base level of the decay constant dependent on the used solvent. The complex does not luminesce in the gas or solid state, meaning only the liquid phase is visualized, even in an evaporating spray. By using an internally excited spray containing the phosphorescent complex, the effect of vaporization is shown through the decrease in measured intensity over the length of the spray, together with droplet size measurements using interferometric particle imaging. This study shows that LIP, using the Eu-TTA-TOPO complex, can be used with different solvents, including diesel surrogates. Furthermore, it can be easily handled and used in sprays to investigate spray breakup and evaporation.

Differential orientation of 10T1/2 mesenchymal cells on non-uniform stretch environments.

Non-uniform stress and strain fields are prevalent in many tissues in vivo, and often exacerbated by disease or injury. These mechanical gradients potentially play a role in contributing to pathological conditions, presenting a need for experimental tools to allow investigation of cell behavior within non-uniformly stimulated environments. Herein, we employ two in vitro cell-stretching devices (one previously published; one newly presented) capable of subjecting cells to cyclic, non-uniform stretches upon the surface of either a circular elastomeric membrane or a cylindrical PDMS tube. After 24 hours of cyclic stretch, 10T1/2 cells on both devices showed marked changes in long-axis orientation, with tendencies to align parallel to the direction of minimal deformation. The degree of this response varied depending on location within the stretch gradients. These results demonstrated the feasibility of conducting cell mechanobiology investigations with the two novel devices, while also highlighting the experimental capabilities of non-uniform mechanical environments for these types of studies. Such capabilities include robust data collection for developing mechanobiological dose-response curves, signal threshold identification, and potential spatial targeting for drug delivery.

Active site inhibited factor VIIa attenuates myocardial ischemia/reperfusion injury in mice.

BACKGROUND: Inhibition of specific coagulation pathways such as the factor VIIa-tissue factor complex has been shown to attenuate ischemia/reperfusion (I/R) injury, but the cellular mechanisms have not been explored. OBJECTIVES: To determine the cellular mechanisms involved in the working mechanism of active site inhibited factor VIIa (ASIS) in the protection against myocardial I/R injury. METHODS: We investigated the effects of a specific mouse recombinant in a mouse model of myocardial I/R injury. One hour of ischemia was followed by 2, 6 or 24 h of reperfusion. Mouse ASIS or placebo was administered before and after induction of reperfusion. RESULTS: ASIS administration reduced myocardial I/R injury by more than 40% at three reperfusion times. Multiplex ligation dependent probe amplification (MLPA) analysis showed reduced mRNA expression in the ischemic myocardium of CD14, TLR-4, interleukin-1 (IL-1) receptor-associated kinase (IRAK) and IkappaBalpha upon ASIS administration, indicative of inhibition of toll-like receptor-4 (TLR-4) and subsequent nuclear factor-kappaB (NF-kappaB) mediated cell signaling. Levels of nuclear activated NF-kappaB and proteins influenced by the NF-kappaB pathway including tissue factor (TF) and IL-6 that were increased after I/R, were attenuated upon ASIS administration. After 6 and 24 h of reperfusion, neutrophil infiltration into the area of infarction was decreased upon ASIS administration. There was, however, no evidence of an effect of ASIS on apoptosis (Tunel staining and MLPA analysis). CONCLUSIONS: We conclude that the diminished amount of myocardial I/R injury after ASIS administration is primarily due to attenuated inflammation-related lethal I/R injury, probably mediated through the NF-kappaB mechanism.

A cross-sectional study of postmenopausal women found an association between osteoporosis and past gastric surgery or oral corticosteroids.

OBJECTIVE: To determine the relationship between osteoporosis and the presence of specific and nonspecific medical conditions in postmenopausal women. To what extent is this relationship useful in detecting osteoporosis in daily general practice. STUDY DESIGN AND SETTING: Subjects were 1,684 postmenopausal women registered with 23 general practitioners. Multivariate logistic regression analysis was done with 52 disease variables and 24 biometrical and lifestyle variables, using BMD as the dependent variable. Bivariate analysis was performed to calculate their contribution to the risk of having osteoporosis. RESULTS: Having more than one disease was associated with a lower prevalence of osteoporosis. A positive association with the presence of osteoporosis was only found for the use of corticosteroids, gastric surgery, and cervical complaints. The risk for osteoporosis in the high risk category increased from 39 to 71% in women using oral corticosteroids, from 39 to 56% in women with a history of gastric surgery, and from 39 to 63% in women with cervical complaints. CONCLUSION: The clinical relevance of medical conditions for detecting osteoporosis is limited. However, all patients using oral corticosteroids and patients with a history of gastric surgery should be checked for the presence of osteoporosis. Cervical compaints in the high risk category was associated with osteoporosis.

A continuous displacement immunoassay for human heart-type fatty acid-binding protein in plasma.

Human heart-type fatty acid-binding protein (FABP) is suggested as an early plasma marker of acute myocardial infarction (AMI), and several studies have proved that, for early diagnosis of AMI, FABP performs better than myoglobin, which is a more often used early marker protein. Because serial measurement of biochemical markers in plasma is now universally accepted as an important determinant in AMI diagnosis, a rapid and continuous measuring method for FABP would be desirable. The aim of the present study was to develop an immunoassay based on the principle of displacement and using a column for rapid and continuous measurement of FABP in plasma. Glass columns filled with Sepharose-bound FABP were loaded with a horseradish peroxidase (HRP)-labeled antibody (Ab) and equilibrated with human plasma. After reaching a stable baseline, human plasma spiked with FABP or plasma from AMI patients was added. The Ab-HRP complex dissociated due to the presence of FABP in the plasma and was subsequently quantified. For plasma from AMI patients (n=5), the Ab-HRP level thus measured correlated with the corresponding plasma FABP concentration (R=0.96). The results of this study show the feasibility of a sensor for continuous monitoring of FABP in plasma.

Development of a displacement immunoassay for human heart-type fatty acid-binding protein in plasma: the basic conditions.

To risk-stratify patients with chest pain who are admitted to emergency rooms and for whom initial evaluation is not conclusive, the use of cardiac markers has become a standard procedure. A recently introduced early plasma marker for acute myocardial infarction (AMI) is the 14.5-kDa cytoplasmic heart-type fatty acid-binding protein (FABP). To fully exploit its early release from injured myocardium, a rapid method for repeated measurements or continuous monitoring of FABP in plasma is desirable. Such an on-line method could be an immunosensor based on displacement. The aim of the present study was to further investigate the principles underlying the displacement assay of FABP, both in buffer and in plasma. Batches of sepharose-bound FABP were loaded with an antibody-horseradish peroxidase (HRP) conjugate (anti-FABP). Continuous measurement of FABP was mimicked by repeated addition of FABP containing solutions followed by several washing steps. In the presence of free FABP the antibody-HRP complex dissociated and was subsequently quantified. Significant displacement in the presence of free FABP was observed in both buffer and human plasma. Anti-FABP could be intermittently displaced in the same batch, for at least 9 h, and the displacement was concentration-dependent. These results show the feasibility of a sensor based on the displacement principle to be used for the diagnosis of AMI in emergency medicine.

Early menopause: increased fracture risk at older age.

The aim of this study was primarily to determine the relationship between early menopause and the presence of fractures later in life, and secondly, to check for the significance of confounding factors (such as smoking habits, body mass index (BMI), weight and use of hormones). In this cross-sectional population based study, the subjects were 4725 postmenopausal women, 50-80 years of age, registered with 23 general practitioners (GPs). For the purpose of the present study, the total population was analyzed as well as the subgroup of 2757 women (the study population) with a natural menopause. Medical history questionnaire, weight, height and bone mineral density measurements were taken. Bivariate and multivariate analyses were carried out with documented fractures in three categories: during lifetime; after menopause and after age 50 years) as dependent variable and age, BMI, bone mineral density, weight, smoking habits, use of hormones and early menopause as independent variables. The total study population as well as the subgroups "early" and "normal menopause", stratified in three 10-year and in six 5-year categories, were analyzed. Results are expressed as odds ratio and 95% confidence intervals (CI). Multivariate logistic regression analysis revealed that over 70 years of age, BMD< or =0.800 ( t-score<2.5) and early menopause were the only systemic independent predictors of all three fracture categories. Comparing the subgroups normal menopause and early menopause, the early menopause group showed a statistically significant higher overall fracture rate (OR=1.5; CI 1.2-1.8). Over age 70, the difference in the prevalence of fractures reached statistical significance in each age category (OR: 1.8 and 2.1, respectively). Smoking was found to be associated with early menopause (OR=1.5; CI 1.2-1.8) but not with the presence of fractures. Height above 165 cm was found to be associated with a higher prevalence of fractures during lifetime. The present study shows that early menopause is statistically significant associated with the presence of fractures during lifetime, after age 50 years and after menopause. Especially at older age, early menopause is an important predictor of fractures.

Risk factors for osteoporosis related to their outcome: fractures.

The aim of the study was to determine to what extent easy obtainable bone mineral density (BMD)-related risk factors are associated with the occurrence of fractures and to what extent changes in these determinants during a patient's lifetime are relevant. A cross-sectional population-based study was carried out on 4725 postmenopausal women, 50-80 years of age, registered with 23 general practitioners (GPs). The women were questioned and examined. BMD of the lumbar spine was measured using dual-energy X-ray absorptiometry (QDR-1000, Hologic). We analyzed the total population as well as a random sample of 1155 women for whom additional data were collected on recalled weight at age 20-30 years and on self-reported height. Body mass index (BMI) was estimated in two ways: (2) objective BMI [= measured weight/(measured height)2]; (2) recalled BMI [= recalled body weight at age 20-30/(self-reported height)2]. Fractures (dependent variable) were categorized as: (1) fractures sustained during the patient's lifetime; (2) fractures after the age of 50 years; (3) fractures that had occurred during the 5 years before BMD measurement took place. Multivariate stepwise backward and forward logistic regression analyses, using fractures as the dependent variable, were performed with all discrete and non-discrete variables (divided into quartiles). The relationship between the presence of osteoporosis and the presence of fractures was related to the changes in BMI (recalled BMI versus objective BMI). More advanced age, positive family history of fractures and BMD had a positive association with the presence of fractures. Low recalled BMI was a statistically significant predictor of 'fractures during the patient's lifetime' and of 'fractures after the age of 50'. Hysterectomy was associated with a higher prevalence of 'fractures during the patient's lifetime'. Perimenopausal complaints in the history seemed to be associated with a lower prevalence of 'fractures after the age of 50'. Moderate (and heavy) occupational exercise in the past were associated with the presence of fractures 'after the age of 50' and 'fractures during the past 5 years'. Sporting activities in the past showed a slightly positive relationship with the presence of 'fractures during the patient's lifetime' and 'fractures after the age of 50'. Bivariate analysis revealed that current smokers had not sustained significantly more fractures than current nonsmokers, but within the subgroup of current smokers, the prevalence of fractures was significantly higher among those women who had smoked for more than 35 years. Smoking was statistically significantly associated with early menopause. Early menopause was not statistically significantly related to the presence of osteoporosis but appeared to be statistically significantly associated with the prevalence of fractures in the age categories over 65 years. The absolute risks of sustaining one or more fractures ranged from 3% to 44%. Women in the lowest quartile of recalled and objective BMI were often osteoporotic (40%). In this category, women with normal BMD had a statistically significant lower fracture risk than osteoporotic women. Women with a possibly decreased BMI were most often osteoporotic and had sustained more 'fractures during the past 5 years' than expected. Women who had (probably) always been obese were less often osteoporotic and had a much lower fracture risk. It is concluded that decreased BMI is associated with a higher risk of developing fractures at an older age. Prevention of fractures should include fall prevention. In addition, in lean women treatment of low BMD is important.

Construction of an algorithm for quick detection of patients with low bone mineral density and its applicability in daily general practice.

OBJECTIVE: To construct a quick algorithm to detect patients with low bone mineral density (BMD) and osteoporosis and determine its applicability in daily general practice. DESIGN: Cross-sectional study in all 9107 postmenopausal women, aged 50-80, registered at 12 general practice centers. SUBJECTS AND MEASUREMENTS: All healthy women (5303) and 25% of the remaining group (943/3804) were invited to participate. Of 6246 invited women, 4725 (76%) participated. The women were questioned (state of health, medical history, family history, and food questionnaire) and examined [weight, height, body mass index (BMI), and BMD of the lumbar spine]. STATISTICS: Multivariable, stepwise backward and forward logistic regression analyses were performed, with BMD of the lumbar spine (L2-L4, cut-off points at 0.800 g/cm(2) for osteoporosis and 0.970 g/cm(2) for low BMD) as the dependent variable. An algorithm was constructed with those variables that correlated statistically significantly and clinically relevant with the presence of both osteoporosis and low BMD. RESULTS: The prevalence of osteoporosis was 23%, that of low BMD was 65%. Only three variables (age, BMI, and fractures) were statistically significant and clinically relevant correlated with the presence of both osteoporosis and low BMD. Age (OR 2.70 for osteoporosis and OR 1.77 for low BMD) and fractures during the past five years (OR 3.60 for osteoporosis and OR 2.85 for low BMD) were found to be the key predictors. From the algorithm the absolute risks varied from 9% to 51% for osteoporosis and from 48% to 84% for low BMD. The corresponding relative risks varied from 1.0 to 5.7 and from 1.0 to 1.8. CONCLUSIONS: Using an algorithm with age, BMI, and fracture history subgroups at high risk could be identified. However, in whatever combination, many women with osteoporosis could not be identified. Despite the differences in methods, we found predictors for osteoporosis which were comparable with the results of other cross-sectional studies, meaning that the first selection of patients at high risk for low BMD can be done adequately by both specialists and general practitioners.

Increased post-thaw viability and phase I and II biotransformation activity in cryopreserved rat liver slices after improvement of a fast-freezing method.

An existing cryopreservation method for liver slices applies 12% dimethylsulfoxide and rapid freezing. We found that cells in rat liver slices cryopreserved in this manner deteriorated rapidly upon culturing. To improve this cryopreservation method, we varied the dimethylsulfoxide concentration (0, 12, 18, and 30%), the cryopreservation medium (Williams medium E, fetal calf serum, and University of Wisconsin medium), slice thickness, and the storage period at 4 degrees C during slice preparation before cryopreservation. After thawing, slices were cultured for 4 h at 37 degrees C before their viability was evaluated by their potassium content and the number of intact cells determined histomorphologically. The biotransformation capacity of liver slices cryopreserved by the improved method was assessed by testosterone oxidation, hydroxycoumarin sulfation, and glucuronidation. Best results were obtained with 18% dimethylsulfoxide in Williams medium E: the potassium content of cryopreserved slices was higher than 65%, and the number of intact cells was higher than 60% of that in fresh slices; with 12% dimethylsulfoxide, potassium content was less than 40%, and the number of intact cells was less than 30%. Results did not differ between the three cryopreservation media. Viability of thin slices (8-10 cell layers) was better maintained than that of thicker slices (>14 cell layers). Storage at 4 degrees C of slices before cryopreservation decreased viability after cryopreservation. Both oxidative and conjugation activities were better than 60% of fresh values. Although results varied, slices cryopreserved with this improved method and cultured for 4 h retained viability between 50 and 80%, and biotransformation activity between 60 and 90% of fresh slices.

Screening for osteoporosis using easily obtainable biometrical data: diagnostic accuracy of measured, self-reported and recalled BMI, and related costs of bone mineral density measurements.

The aims of the present study were: to determine the diagnostic accuracy of objectively measured, self-reported and recalled body mass index (BMI) for osteoporosis and osteopenia; to determine the diagnostic costs, in terms of bone mineral density (BMD) measurements, per osteoporotic or osteopenic patient detected, using different BMI tests; and to determine the extent to which the results can be used within the framework of the current screening program for breast cancer in The Netherlands. Within the framework of a cross-sectional study on the prevalence of osteoporosis in the south of The Netherlands, 1155 postmenopausal women aged 50-80 years were asked for their present height and their weight at age 20-30 years. Subsequently their actual weight, height and BMD of the lumbar spine (DXA) were measured. The BMD cutoff was 0.800 g/cm2 for osteoporosis and 0.970 g/cm2 for low BMD (osteoporosis + osteopenia). After receiver operating characteristic analysis, age was cut off at 60 years and BMI at 27 kg/m2. Diagnostic accuracies of objectively measured, self-reported and recalled BMI were evaluated using predictive values (PV) and odds ratios. The resulting 'true positive' and 'false positive' rates were used to calculate diagnostic costs (i.e., DXA) for each osteoporotic patient or low-BMD patient detected. The prevalence of osteoporosis in the study population was 25%, that of low BMD 65%. Only the age-BMI tests 'age > or = 60, BMI < or = 27' showed PVs for osteoporosis (31-41%) and for low BMD (71-81%) that were higher than the prior probabilities for these conditions. Related odds ratios were 2.14-3.18 (osteoporosis) and 1.87-3.04 (low BMD). The objective BMI test detected 50% of the osteoporotic patients. Using the self-reported BMI test and the recalled BMI test, detection rates increased to 55% and 69%, respectively. Concomitant costs per osteoporotic patient detected rose by 24%. Detection of patients with a low BMD increased from 38% for objective BMI and 42% for self-reported BMI to 60% for recalled BMI. Related costs increased by 11%. If all women over 50 years of age (irrespective of their BMI) were to be referred for BMD measurement, costs per osteoporotic patient or low-BMD patient detected would be 304 and 116 Euros, respectively. Only in women over 60 years does a BMI below 27 kg/m2 provide a better prediction of the presence of osteoporosis or low BMD than could be expected solely on the basis of the relevant prevalences in postmenopausal women aged 50-80 years. If the use of BMI for the detection of osteoporotic or low-BMD patients is still considered, measuring weight and just asking for a person's height will do. Although age and BMI are the strongest risk factors for osteoporosis, they are of less significance when used for screening the population for osteoporosis. More research is needed before age and BMI can be included in any screening program. As regards practical considerations alone, measurements of BMD could be implemented within the screening program for breast cancer.