Rapid and reliable quantification of minimal residual disease in acute lymphoblastic leukemia using rearranged immunoglobulin and T-cell receptor loci by LightCycler technology.
(65/1961)
The detection of minimal residual disease (MRD) using immunoglobulin and T-cell receptor (TCR) rearrangements as PCR targets provides important prognostic information on the in vivo effectiveness of treatment in acute lymphoblastic leukemia (ALL). Here we report on the real-time quantification of MRD in 25 ALL patients using LightCycler technology. We designed and adapted allele-specific oligonucleotide (ASO)-PCR protocols that enabled the detection of >90% of the IGH, IGK, TCRD, and TCRG rearrangements observed in ALL patients. In all patients, at least two suitable markers could be identified (average, 3.4 markers/patient). We applied ASO-PCR with 35 immunoglobulin and TCR rearrangements (11 IGH, 6 IGK, 12 TCRG, and 6 TCRD) and compared the sensitivity and practicability of the LightCycler strategy with conventional ASO-PCR on a block thermocycler followed by quantification with gel electrophoresis. The LightCycler measured leukemia-specific PCR products at each cycle (real-time) by staining the PCR product with the DNA-binding dye SYBR Green I. LightCycler technology showed a higher sensitivity than the conventional method in eight cases, whereas the sensitivity of the other markers matched exactly. The detection level varied between 10(-4) and 10(-6) leukemic cells. Furthermore, we determined the MRD status of 27 bone marrow follow-up samples from 15 ALL patients by both methods and revealed comparable results. However, the LightCycler also allowed accurate quantification in samples containing relatively high levels (>10(-3)) of residual leukemia cells. The conventional ASO-PCR technique comprises various laborious and time-consuming PCR experiments and post-PCR steps to determine the number of cycles with the optimal linearity and sensitivity of the PCR. Real-time quantification through LightCycler technology obviates these post-PCR steps, provides the highest sensitivity via software analysis, and therefore represents a rapid, reliable, sensitive, and cost-effective technique for the routine monitoring of MRD in ALL patients. (+info)
Quantitation of supercoiled circular content in plasmid DNA solutions using a fluorescence-based method.
(66/1961)
A method for quantifying the proportion of supercoiled circular (SC) forms in DNA solutions is described. The method (SCFluo) takes advantage of the reversible denaturation property of SC forms and the high specificity of the PicoGreen fluorochrome for double-stranded (ds)DNA. Fluorescence values of forms capable of reversible denaturation after a 5 min heating, 2 min cooling step are normalised to fluorescence values of total dsDNA present in the preparation. For samples with a SC content >20-30%, good regression fits were obtained when values derived from densitometric scanning of an agarose gel and those derived from the SCFluo method were compared. The method represents an attractive alternative to currently established methods because it is simple, rapid and quantitative. During large-scale processing and long-term storage, enzymatic, chemical and shear degradation may substantially decrease the SC content of plasmid DNA preparations. Regulations for pharmaceutical grade products for use in gene therapy and DNA vaccination may require >90% of the plasmid to be in the SC form. In the present study the SC content of 6.9, 13 and 20 kb plasmid preparations that had been subjected to chemical and shear degradation was successfully quantified using the new method. (+info)
Comparison of Cryptosporidium parvum viability and infectivity assays following ozone treatment of oocysts.
(67/1961)
Several in vitro surrogates have been developed as convenient, user-friendly alternatives to mouse infectivity assays for determining the viability of Cryptosporidium parvum oocysts. Such viability assays have been used increasingly to determine oocyst inactivation following treatment with chemical, physical, or environmental stresses. Defining the relationship between in vitro viability assays and oocyst infectivity in susceptible hosts is critical for determining the significance of existing oocyst inactivation data for these in vitro assays and their suitability in future studies. In this study, four viability assays were compared with mouse infectivity assays, using neonatal CD-1 mice. Studies were conducted in the United States and United Kingdom using fresh (<1 month) or environmentally aged (3 months at 4 degrees C) oocysts, which were partially inactivated by ozonation before viability and/or infectivity analyses. High levels of variability were noted within and between the viability and infectivity assays in the U.S. and United Kingdom studies despite rigorous control over oocyst conditions and disinfection experiments. Based on the viability analysis of oocyst subsamples from each ozonation experiment, SYTO-59 assays demonstrated minimal change in oocyst viability, whereas 4',6'-diamidino-2-phenylindole-propidium iodide assays, in vitro excystation, and SYTO-9 assays showed a marginal reduction in oocyst viability. In contrast, the neonatal mouse infectivity assay demonstrated significantly higher levels of oocyst inactivation in the U.S. and United Kingdom experiments. These comparisons illustrate that four in vitro viability assays cannot be used to reliably predict oocyst inactivation following treatment with low levels of ozone. Neonatal mouse infectivity assays should continue to be regarded as a "gold standard" until suitable alternative viability surrogates are identified for disinfection studies. (+info)
Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission.
(68/1961)
The diffraction barrier responsible for a finite focal spot size and limited resolution in far-field fluorescence microscopy has been fundamentally broken. This is accomplished by quenching excited organic molecules at the rim of the focal spot through stimulated emission. Along the optic axis, the spot size was reduced by up to 6 times beyond the diffraction barrier. The simultaneous 2-fold improvement in the radial direction rendered a nearly spherical fluorescence spot with a diameter of 90-110 nm. The spot volume of down to 0.67 attoliters is 18 times smaller than that of confocal microscopy, thus making our results also relevant to three-dimensional photochemistry and single molecule spectroscopy. Images of live cells reveal greater details. (+info)
Toxic evaluation of organic extracts from airborne particulate matter in Puerto Rico.
(69/1961)
In recent years, several hypotheses have emerged to explain the toxicologic activity of particulate matter. Organic compounds, ultrafine particles, biologic components, and transition metals are some of the constituents that reportedly exert some type of adverse effect on human health. A considerable fraction of the urban particulate matter consists of carbon compounds, which originate mostly from anthropogenic sources. The toxicity of organic fractions from particulate matter have been mainly evaluated by considering their mutagenic activity. This research expands on the toxicologic profile of organic compounds adsorbed to particulate matter, specifically in Puerto Rico, by using the cytotoxic neutral red bioassay (NRB). The NRB uses normal human epidermal keratinocytes or other types of cells to measure the effect on cell viability when exposed to organic compounds associated to the particles in the air. We validated the NRB for particulate matter by using a standard reference material (SRM 1649). We used the NRB to determine toxicologic differences of extracts between an urban industrialized site with anthropogenic activity versus a coastal region with less human activity. The cytotoxicity associated with organic compounds in particulate matter collected at the urban industrialized site was detected in both the particulate matter (3/4) 10 microm in aerodynamic diameter (PM(10)) and particulate matter (3/4) 100 microm in aerodynamic diameter (PM(100)). Greater toxic effects were observed in PM(10) extracts than in PM(100) extracts, but PM(10) toxic effects were not significantly different from those in PM(100). The extracts from the industrialized site were more cytotoxic than the extracts from coastal reference site, although in the summer, extracts from both sites were significantly cytotoxic to normal human epidermal keratinocytes. In addition, the nonpolar extracts of both PM(10) and PM(100) exerted the greatest cytotoxicity, followed by the polar, and, finally, the moderately polar extract. This study demonstrates that extracts from the Guaynabo industrialized site were more toxic than similar extracts obtained from a reference coastal site in Fajardo, Puerto Rico. (+info)
The effect of a T cell-specific NF-kappa B inhibitor on in vitro cytokine production and collagen-induced arthritis.
(70/1961)
NF-kappa B plays a key role in the production of cytokines in inflammatory diseases. The effects of a novel T cell-specific NF-kappa B inhibitor, SP100030, were evaluated in cultured Jurkat cells and in murine collagen-induced arthritis (CIA). Chemical libraries were screened for NF-kappa B-inhibitory activity. SP100030, a compound identified in this process, inhibited NF-kappa B activation in PMA/PHA-activated Jurkat cells by EMSA at a concentration of 1 microM. Jurkat cells and the monocytic cell line THP-1 were transfected with an NF-kappa B promotor/luciferase construct and activated. SP100030 inhibited luciferase production in the Jurkat cells (IC50 = 30 nM). ELISA and RT-PCR confirmed that IL-2, IL-8, and TNF-alpha production by activated Jurkat and other T cell lines were inhibited by SP100030. However, cytokine expression was not blocked by the compound in THP-1 cells, fibroblasts, endothelial cells, or epithelial cells. Subsequently, DBA/1J mice were immunized with type II collagen. Treatment with SP100030 (10 mg/kg/day i.p. beginning on day 21) significantly decreased arthritis severity from onset of clinical signs to the end of the study on day 34 (arthritis score, 5.6 +/- 1.7 for SP100030 and 9.8 +/- 1.5 for control; p < 0.001). Histologic evaluation demonstrated a trend toward improvement in SP100030-treated animals. EMSA of arthritic mouse ankles in CIA showed that synovial NF-kappa B binding was suppressed in the SP100030-treated mice. SP100030 inhibits NF-kappa B activation in T cells, resulting in reduced NF-kappa B-regulated gene expression and decreased CIA. Its selectivity for T cells could provide potent immunosuppression with less toxicity than other NF-kappa B inhibitors. (+info)
Mechanisms underlying InsP3-evoked global Ca2+ signals in mouse pancreatic acinar cells.
(71/1961)
In secretory epithelial cells, complex patterns of Ca2+ signals regulate physiological processes. How these patterns are generated is still not fully understood. In particular, the basis of global Ca2+ waves is not clear. We have studied regional differences in InsP3-evoked Ca2+ release in single mouse pancreatic acinar cells, using high-speed (approximately 90 frames s-1), high-sensitivity Ca2+ imaging combined with rapid (10 ms) spot photolysis (2 micrometer diameter) of caged InsP3. Within a single region we measured Ca2+ response latency and rate of rise to construct an InsP3 dose-response relationship. Spot InsP3 liberation in the secretory pole region consistently elicited a dose-dependent, rapid release of Ca2+. Spot InsP3 liberation in the basal pole region of approximately 50% of cells elicited a similar dose-response relationship but with a lower apparent InsP3 affinity than in the secretory pole. In the other cells, basal pole InsP3 liberation did not elicit active Ca2+ release, even at the highest stimulus intensities we employed, although these same cells did respond when the stimulus spot was moved to different regions. We conclude that in the basal pole active sites of rapid Ca2+ release have a lower functional affinity for InsP3 than those in the secretory pole and are spread out in discrete sites across the basal pole. These properties explain the propagation of Ca2+ waves across the basal pole that are only observed at higher stimulus levels. (+info)
Giant vesicles as models to study the interactions between membranes and proteins.
(72/1961)
The interaction between polypeptides and membranes is a fundamental aspect of cell biochemistry. Liposomes have been used in this context as in vitro systems to study such interactions. We present here the case of giant vesicles (GVs), which, due to their size (radius larger than 10 microns), mimic more closely the situation observed in cell membranes and furthermore permit to study protein-membrane interactions by direct optical monitoring. It is shown that GVs formed from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine by electroformation are permeable to certain low molecular weight molecules such as the nucleic acid dye YO-PRO-1 and fluorescein diphosphate whereas conventional liposomes (large or small unilamellar liposomes) are not. In addition, it is shown that non-membrane proteins, such as DNases or RNases, added to the selected GVs from the outside, are able to convert their substrate, which is strictly localized on the internal side of the membrane. This effect is only seen in GVs (also when they are removed from the original electroformation environment) and is absent in conventional liposomes. The fact that these effects are only present in GVs obtained by electroformation and not in conventional small liposomes is taken as an indication that certain physico-chemical properties of the bilayer are affected by the membrane curvature, although the mechanism underlying such differences could not be established as yet. (+info)