While stem cell cryopreservation methods have been optimized using dimethylsulfoxide (DMSO), the established techniques are not optimal when applied to unfertilized human embryonic cells. In addition, important questions remain regarding the toxicity and characteristics of DMSO for treatment of stem cells for clinical use. The objective of this study was to establish an optimal method for cryopreservation of stem cells using low concentrations (0.2 M) of trehalose, a nontoxic disaccharide of glucose, which possesses excellent protective characteristics, in place of current methods utilizing high concentrations (1-2 M) of DMSO. A human hematopoietic cell line was used in this investigation as a surrogate for human stem cells. Trehalose was loaded into cells using a genetically engineered mutant of the pore-forming protein alpha-hemolysin from Staphylococcus aureus. This method results in a nonselective pore equipped with a metal-actuated switch that is sensitive to extracellular zinc concentrations, thus permitting controlled loading of trehalose. Preliminary experiments characterized the effects of poration on TF-1 cells and established optimal conditions for trehalose loading and cell survival. TF-1 cells were frozen at 1 degrees C/min to -80 degrees C with and without intra- and extracellular trehalose. Following storage at -80 degrees C for 1 week, cells were thawed and evaluated for viability, differentiation capacity, and clonogenic activity in comparison to cells frozen with DMSO. Predictably, cells frozen without any protective agent did not survive freezing. Colony-forming units (CFU) generated from cells frozen with intra- and extracellular trehalose, however, were comparable in size, morphology, and number to those generated by cells frozen in DMSO. There was no observable alteration in phenotypic markers of differentiation in either trehalose- or DMSO-treated cells. These data demonstrate that low concentrations of trehalose can protect hematopoietic progenitors from freezing injury and support the concept that trehalose may be useful for freezing embryonic stem cells and other primitive stem cells for therapeutic and investigational use.
Biomarkers associated with asthma aetiology and exacerbation have been sought to shed light on this multifactorial disease. One candidate is the serum concentration of the Clara cell secretory protein (CC16, sometimes referred to as CC10 or uteroglobin). In this review, we examine serum CC16's relation to asthma aetiology and exacerbation. There is evidence that acute exposures to certain pulmonary irritants can cause a transient increase in serum CC16 levels, and limited evidence also suggests that a transient increase in serum CC16 levels can be caused by a localized pulmonary inflammation. Research also indicates that a transient increase in serum CC16 is not associated with measurable pulmonary damage or impairment of pulmonary function. The biological interpretation of chronic changes in serum CC16 is less clear. Changes in serum CC16 concentrations (either transient or chronic) are not specific to any one agent, disease state, or aetiology. This lack of specificity limits the use of serum CC16 as a biomarker of specific exposures. To date, many of the critical issues that must be understood before serum CC16 levels can have an application as a biomarker of effect or exposure have not been adequately addressed.
Advances in both sensitivity and specificity of analytical chemistry have made it possible to quantify substances in human biological specimens, such as blood, urine, and breast milk, in specimen volumes that are practical for collection from individuals. Research laboratories led by the Centers for Disease Control and Prevention (CDC) in its series National Report on Human Exposure to Environmental Chemicals [Centers for Disease Control and Prevention (CDC), 2005. Third National Report on Human Exposure to Environmental Chemicals. NCEH Pub. No. 05-0570.] are dedicating substantial resources to designing and conducting human biomonitoring studies and compiling biomonitoring data for the general population. However, the ability to quantitatively interpret the results of human biomonitoring in the context of a health risk assessment currently lags behind the analytical chemist's ability to make such measurements. The traditional paradigm for human health risk assessment of environmental chemicals involves comparing estimated daily doses to health-based criteria for acceptable, safe, or tolerable daily intakes (for example, reference doses [RfDs], tolerable daily intakes [TDIs], or minimal risk levels [MRLs]) to assess whether estimated doses exceed such health screening levels. However, biomonitoring efforts result in measured chemical concentrations in biological specimens (the result of absorption, distribution, metabolism and excretion of administered doses) rather than estimated intake doses. Quantitative benchmarks of acceptable or safe concentrations in biological specimens (analogous to RfDs, TDIs, or MRLs) needed to interpret these levels exist for very few chemicals of environmental interest. This paper discusses issues inherent in converting existing health screening benchmarks based on intake doses to screening levels for evaluating biomonitoring data, and presents methods and approaches that can be used to derive such screening levels (termed ''Biomonitoring Equivalents,'' or BEs) for a range of chemicals and biological media.
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