A new method to evaluate the thermal stability of lyophilized enzymes

Summary The thermal inactivation of lyophilized chymotrypsin was studied at controlled water activities. At 60 °C the enzyme showed good stability except at aw 0.97, whereas at 75 °C considerable inactivation occured at most water activities. Increasing the amount of buffer on the preparation decreased the stability significantly. The optimal temperature of enzymatic activity was increased 14 °C, when the water activity was decreased from 1 to 0.5.     

A new method to evaluate the residual activity in patients undergoing 131I thyroid therapy

The radioiodine administration is a standard therapeutic approach to both benign thyroid diseases, such as hyperthyroidism, and carcinomas. The high administered ¹³¹I activities are of radiation protection concern, due to relevant patient residual contamination. The aim of this work was to develop a new procedure based on external radiometric surveys and on a mathematical model in order to estimate the ¹³¹I activity in patients undergoing hyperthyroidism radioiodine therapy.In the first stage of this study, a suitable detector was chosen and its response vs. activity was characterized. The experimental verification was performed measuring the ambient dose equivalent rate from patients receiving radioiodine administration. The results confirm the reliability of the proposed method, as shown by the slight differences between the administered activities and the ones calculated from external measurements. Furthermore, the same procedure was applied to detect the percentage residual activity in patients at two preset time intervals: 4 hours and 4 days after the radioiodine administration. The obtained results clearly highlight that the method can ensure a level of reliability compatible with the radiation protection purposes.     

A new histochemical method to assess and evaluate potential bone marrow damage from therapeutic substances

Summary A new model is presented for assessing and evaluating the influence of bone-marrow-damaging substances in mice. Qualitative and quantitative results of histological, histochemical and enzyme histochemical studies facilitate the assessment of bone marrow damage in terms of extent and intensity. Bone marrow taken from the right femur of treated animals was embedded in renal tissue of controls for subsequent work-up in different techniques. From each of the experimental groups specimens from 10 animals were frozen in liquid nitrogen, specimens from another 10 animals were fixed in buffered formalin. Assessment and evaluation of changes was performed after the required histologic and histochemical staining (nucleic acid). Results were correlated with the cytology of bone marrow smears sampled from the left femur of each respective animal. Damage was visualized, in smear cytology or in histologic and histochemical preparations, and quantified by microphotometry and special staining for cytochrome oxidase activity.     

A new method for fast blood cell counting and partial differentiation by flow cytometry

A new blood counting method by flow cytometry is described which determines absolute counts and relative proportions of erythrocytes, reticulocytes, thrombocytes, lymphocytes and granulocytes from one sample of saline diluted human or animal blood. Staining time is 2 to 5 min and measuring time between 1 and 2 additional minutes. Measured simultaneously are the electrical cell volume, the green and optionally also the red fluorescence of the transmembrane potential sensitive dye 3,3-dihexyloxacarbocyanine DiOC6(3) and the RNA/DNA stain acridine orange (AO). Work is under way to fully automate staining, measurement and data evaluation. The use of stains by which blood cell counting and biochemical analysis can be combined offers new possibilities for routine blood cell counting without requirement for additional time. The potential of such stains is that pathologic cell conditions which are not, or not yet reflected in the cell count may be earlier detectable by biochemical stains.     

Novel application of a fibrin cell block method to evaluate melanocytic cell populations

Confirming melanocytic lineage and purity is important for experiments using cultured human melanocytes. The objective of this study was to develop a simple, reliable method to evaluate and archive cultured melanocytic cells. Melanocytes were isolated from adult skin biopsies or from neonatal foreskins using standard culturing methods. Fibrin cell blocks (FCBs) were prepared from cultured cells at passages two and six. Fibrin blocks were paraffin-embedded and sectioned for immunohistochemical (CD68, Melan-A, and HMB-45) and H & E staining. Flow cytometry was performed (Melan-A) at passage six. A mixing experiment with cultured melanocytes and fibroblasts was performed and cell population purity was determined by manual counts of positively staining cells in the FCBs and by flow cytometry. The FCB method of evaluating population purity was validated experimentally and by correlation with flow cytometry results. Preparation of a FCB followed by immunohistochemical staining is an easy and inexpensive way to confirm melanocytic lineage, estimate population purity, and provide a permanent archive of cultured cells.     

A rapid and quantitative safranin‐based fluorescent microscopy method to evaluate cell wall lignification

One of the main characteristics of plant cells is the presence of the cell wall located outside the plasma membrane. In particular cells, this wall can be reinforced by lignin, a polyphenolic polymer that plays a central role for vascular plants, conferring hydrophobicity to conducting tissues and mechanical support for upright growth. Lignin has been studied extensively by a range of different techniques, including anatomical and morphological analyses using dyes to characterize the polymer localization in situ. With the constant improvement of imaging techniques, it is now possible to revisit old qualitative techniques and adapt them to obtain efficient, highly resolutive, quantitative, fast and safe methodologies. In this study, we revisit and exploit the potential of fluorescent microscopy coupled to safranin‐O staining to develop a quantitative approach for lignin content determination. The developed approach is based on ratiometric emission measurements and the development of an imagej macro. To demonstrate the potential of our methodology compared with other commonly used lignin reagents, we demonstrated the use of safranin‐O staining to evaluate and compare lignin contents in previously characterized Arabidopsis thaliana lignin biosynthesis mutants. In addition, the analysis of lignin content and spatial distribution in the Arabidopsis laccase mutant also provided new biological insights into the effects of laccase gene downregulation in different cell types. Our safranin‐O‐based methodology, also validated for Linum usitatissimum (flax), Zea mays (maize) and Populus tremula x alba (poplar), significantly improves and speeds up anatomical and developmental investigations of lignin, which we hope will contribute to new discoveries in many areas of cell wall plant research.     

Regional extravascular and interstitial lung water in normal dogs

We measured the regional distribution of pulmonary extravascular and interstitial water to examine the possibility that regional differences in microvascular pressure or tissue stress may cause regional differences in lung water. We placed chloralose-anesthetized dogs in an upright (n = 6) or supine (n = 7) position for 180 min. We injected 51Cr-labeled EDTA to equilibrate to the extracellular space and 125I-labeled albumin to equilibrate with plasma. At the end of the experiment, the lungs were removed, passively drained of blood, and inflated before rapid freezing. Lungs were divided into horizontal slices, and extravascular, interstitial, and plasma water, red cell volume, and dry lung weight were determined for each slice. We found that regional extravascular and interstitial water were constant throughout the lungs in both groups and that there were no significant differences between upright and supine dogs. There were no significant differences in hematocrit between slices. We conclude that gravity and body position have no measurable effect on either the total size of the extravascular and interstitial compartments or their regional distribution.     

A method to evaluate dynamics and periodicity of hormone secretion

Spontaneous hormone secretory dynamics include tonic and pulsatile components and a number of periodic processes. Circadian variations are usually found for melatonin, TSH and GH, with peak secretions at night, and in cortisol secretion, which peaks in the morning. Free thyroxine (FT4) and insulin-like growth factor (IGF)1 levels do not always change with circadian rhythmicity or show only minor fluctuations. Fractional variations explore the dynamics of secretion related to time intervals, and the rate of change in serum levels represents a signal for the receptorial system and the target organ. We evaluated time-related variations and change dynamics for melatonin, cortisol, TSH, FT4, GH and IGF1 levels in blood samples obtained every 4 h for 24 h from eleven healthy males, ages 35-53 years (mean ? SE 43.6 ± 1.7). Nyctohemeral (i.e., day-night) patterns of hormone secretion levels and the fractional rate of variation between consecutive 4-hourly time-qualified hormone serum levels (calculated as percent change from time 1 to time 2) were evaluated for circadian periodicity using a 24 and 12-h cosine model. A circadian rhythm was validated for serum level changes in cortisol with peaks of the 24-h cosine model at 07:48 h, and melatonin, TSH and GH, with phases at 01:35 h, 23:32 h, and 00:00 h, respectively. A weak, but significant, 12-h periodicity was found for FT4 serum levels, with minor peaks in the morning (10:00 h) and evening (22:00 h), and for IGF1, with minor peaks in the morning (07:40 h) and evening (19:40 h). Circadian rhythmicity was found in the 4-hourly fractional variations with phases of increase or surge at 02:00 h for cortisol, 22:29 h for melatonin, 05:14 h for FT4, and 21:19 h for GH. A significant 12-h periodicity was found for the 4-hourly fractional variations of TSH with two peaks in the morning (decrease or drop at 04:42 h) and afternoon (surge at 16:28 h), whereas IGF1 fractional variation changes did not show a significant rhythmic pattern. In conclusion, the calculation of the time-qualified fractional rate of variation allows evaluation of the dynamics of secretion and the specification of the timepoint(s) of maximal change of secretion, not only for hormones whose secretion is characterized by a circadian pattern of variation, but also for hormones that show no circadian or only weak ultradian (12 h) variations (i.e., FT4).     

A simple technique to evaluate model sensitivity in the continual reassessment method

The continual reassessment method (CRM) is a sequential design used in phase I cancer trials to determine the maximal dose with acceptable toxicity. It has been established that the CRM is consistent under model misspecification but not generally. When the method does not converge to the target percentile, some dose-response models will be more sensitive than others in terms of how close the converged recommendation is to the target. In this article, we interpret the main condition under which the CRM is consistent and apply it to evaluate the sensitivity of the model used with the CRM. The technique presented is found to be a useful supplement to simulation when planning a phase I trial.     

A method to evaluate the antioxidant system for radicals in erythrocyte membranes

The erythrocyte defense system against cellular oxidants is complex and efficient. Free radicals generated in cell membranes, however, are relatively sequestered from the cell's antioxidant mechanisms. When an oxidant challenge exceeds the capacity of the erythrocyte's antioxidant system, membrane damage may occur, causing red cell destruction and hemolytic anemia. In this study, we present a method for monitoring radical reduction in erythrocyte membranes, using fatty acid spin labels with nitroxide radicals on the hydrocarbon chains. About 50 microL of packed (about 5-6 x 10(8)), carbon monoxide (CO)-gassed red blood cells are used. The electron paramagnetic resonance signals of the 5-doxylstearic acid spin labels in the intact cells are obtained as a function of time, at 37 degrees C over a period of 2 h. The pseudo first-order rate constant for reduction of the spin label in normal adult intact cells under our experimental conditions is 4.3 +/- 1.8 x 10(-3)/min. The reproducibility and variability of the measurements are discussed. Since the measurements we describe reflect the extent of radical reductions occurring in cell membranes, we suggest that this method can be used to measure the ability to defend oxidants in membranes of erythrocytes with defective antioxidant systems. This method is particularly useful for measuring the modification of the antioxidant system toward radicals in membranes by drugs, chemicals, or environmental toxins.     

A quantitative method to evaluate mesenchymal stem cell lipofection using real‐time PCR

Genetic modification of human mesenchymal stem cells (MSC) is a powerful tool to improve the therapeutic utility of these cells and to increase the knowledge on their regulation mechanisms. In this context, strong efforts have been made recently to develop efficient nonviral gene delivery systems. Although several studies addressed this question most of them use the end product of a reporter gene instead of the DNA uptake quantification to test the transfection efficiency. In this study, we established a method based on quantitative real‐time PCR (RT‐PCR) to determine the intracellular plasmid DNA copy number in human MSC after lipofection. The procedure requires neither specific cell lysis nor DNA purification. The influence of cell number on the RT‐PCR sensitivity was evaluated. The method showed good reproducibility, high sensitivity, and a wide linear range of 75–2.5 × 10⁶ plasmid DNA copies per cell. RT‐PCR results were then compared with the percentage of transfected cells assessed by flow cytometry analysis, which showed that flow cytometry‐based results are not always proportional to plasmid cellular uptake determined by RT‐PCR. This work contributed for the establishment of a rapid quantitative assay to determine intracellular plasmid DNA in stem cells, which will be extremely beneficial for the optimization of gene delivery strategies. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A synergistic effect of albumin and fibrinogen on immunoglobulin-induced red blood cell aggregation

Therapeutic administration of immunoglobulins (Ig) has the potential to precipitate thrombotic events. This phenomenon may be explained by red blood cell (RBC) aggregation, which can be potentiated by Ig. The contribution of plasma albumin and fibrinogen to Ig-induced RBC aggregation is unclear. We examined RBC aggregation in three settings: 1) patients receiving therapeutic infusions of Ig; 2) patients receiving plasma supplemented in vitro with Ig; and 3) patients receiving RBC suspensions in standard buffer with varying concentrations of albumin, Ig, and fibrinogen. Ig infusion augmented aggregation of RBCs from patients with normal or high plasma levels of albumin but decreased aggregation in those with lower plasma albumin concentrations. In vitro, RBC aggregation was significantly increased only when all three components, fibrinogen, albumin, and Ig, were present at or above normal concentrations in the suspension but was unaffected when any one of the components was absent from the suspension. Our results suggest a three-way interaction among fibrinogen, Ig, and albumin that synergistically induces RBC aggregation in plasma. Understanding these interactions may help predict clinically important phenomena related to RBC aggregation, such as thrombotic complications of Ig infusion.