Functional changes in human leukemic cell line HL-60. A model for myeloid differentiation

Polar solvents induce terminal differentiation in the human promyelocytic leukemia cell line HL-60. The present studies describe the functional changes that accompany the morphologic progression from promyelocytes to bands and poly-morphonuclear leukocytes (PMN) over 9 d of culture in 1.3 percent dimethylsulfoxide (DMSO). As the HL-60 cells mature, the rate of O(2-) production increase 18-fold, with a progressive shortening of the lag time required for activation. Hexosemonophosphate shunt activity rises concomitantly. Ingestin of paraffin oil droplets opsonized with complement or Ig increases 10-fold over 9 d in DMSO. Latex ingestion per cell by each morphologic type does not change significantly, but total latex ingestion by groups of cells increases with the rise in the proportion of mature cells with greater ingestion capacities. Degranulation, as measured by release of β-glucuronidase, lysozyme, and peroxidase, reaches maximum after 3-6 d in DMSO, then declines. HL-60 cells contain no detectable lactoferrin, suggesting that their secondary granules are absent or defective. However, they kill staphylococci by day 6 in DMSO. Morphologically immature cells (days 1-3 in DMSO) are capable of O(2-) generation, hexosemonophosphate shunt activity, ingestion, degranulation, and bacterial killing. Maximal performance of each function by cells incubated in DMSO for longer periods of time is 50-100 percent that of normal PMN. DMSO- induced differentiation of HL-60 cells is a promising model for myeloid development.     

Detection of genetic markers of fecal indicator bacteria in Lake Michigan and determination of their relationship to Escherichia coli densities using standard microbiological methods

Lake Michigan surface waters impacted by fecal pollution were assessed to determine the occurrence of genetic markers for Bacteroides and Escherichia coli. Initial experiments with sewage treatment plant influent demonstrated that total Bacteroides spp. could be detected by PCR in a 25- to 125-fold-higher dilution series than E. coli and human-specific Bacteroides spp., which were both found in similar dilution ranges. The limit of detection for the human-specific genetic marker ranged from 0.2 CFU/100 ml to 82 CFU/100 ml culturable E. coli for four wastewater treatment plants in urban and rural areas. The spatial and temporal distributions of these markers were assessed following major rain events that introduced urban storm water, agricultural runoff, and sewage overflows into Lake Michigan. Bacteroides spp. were detected in all of these samples by PCR, including those with <1 CFU/100 ml E. coli. Human-specific Bacteroides spp. were detected as far as 2 km into Lake Michigan during sewage overflow events, with variable detection 1 to 9 days postoverflow, whereas the cow-specific Bacteroides spp. were detected in only highly contaminated samples near the river outflow. Lake Michigan beaches were also assessed throughout the summer season for the same markers. Bacteroides spp. were detected in all beach samples, including 28 of the 74 samples that did not exceed 235 CFU/100 ml of E. coli. Human-specific Bacteroides spp. were detected at three of the seven beaches; one of the sites demonstrating positive results was sampled during a reported sewage overflow, but E. coli levels were below 235 CFU/100 ml. This study demonstrates the usefulness of non-culture-based microbial-source tracking approaches and the prevalence of these genetic markers in the Great Lakes, including freshwater coastal beaches.     

Genetic analysis reveals demographic fragmentation of grizzly bears yielding vulnerably small populations

Ecosystem conservation requires the presence of native carnivores, yet in North America, the distributions of many larger carnivores have contracted. Large carnivores live at low densities and require large areas to thrive at the population level. Therefore, if human-dominated landscapes fragment remaining carnivore populations, small and demographically vulnerable populations may result. Grizzly bear range contraction in the conterminous USA has left four fragmented populations, three of which remain along the Canada-USA border. A tenet of grizzly bear conservation is that the viability of these populations requires demographic linkage (i.e. inter-population movement of both sexes) to Canadian bears. Using individual-based genetic analysis, our results suggest this demographic connection has been severed across their entire range in southern Canada by a highway and associated settlements, limiting female and reducing male movement. Two resulting populations are vulnerably small (< or =100 animals) and one of these is completely isolated. Our results suggest that these trans-border bear populations may be more threatened than previously thought and that conservation efforts must expand to include international connectivity management. They also demonstrate the ability of genetic analysis to detect gender-specific demographic population fragmentation in recently disturbed systems, a traditionally intractable yet increasingly important ecological measurement worldwide.     

Exposure to the electrofusion process can increase the immunogenicity of human cells

The cellular products obtained following electrofusion (EF) of dendritic cells (DC) and tumour cells have shown promise as cancer vaccines. The immunogenicity of these preparations has been attributed to the presence of small numbers of DC-tumour hybrids and the contribution of the non-hybrid tumour cells present has received little attention. In this report, we investigated the effect of the EF process on the immunogenicity of allogeneic human cells, in particular the colorectal cell line, SW620. EF conditions were optimised to yield the maximum number of DC-SW620 hybrids co-expressing tumour associated antigen (TAA) and DC associated antigens. Exposure of SW620 to EF induced significant increases (P<0.05) in apoptosis and necrosis. Pre-exposure of SW620 to the EF buffer alone [0.3 M glucose, 0.1 mM Ca(CH₃COO)₂ and 0.5 mM Mg(CH₃COO)₂] resulted in significant increases in TAA uptake by DC during co-culture (P<0.05). DC phenotype was, however, not altered by exposure to EF treated tumour cells. In co-cultures of PBMC responders with SW620, the levels of IFN release and cytotoxic activity were significantly increased (P<0.05) by pre-exposure of the SW620 to EF. Pre-exposure of allogeneic non-T cells, the colorectal cell line Lovo and a breast cancer cell line (MCF7) to EF also significantly (P<0.05) increased the levels of IFN release by responding PBMC. These results demonstrate that the EF process itself can increase the immunogenicity of at least some human cell types independently of hybrid formation. These findings suggest that EF protocols should be evaluated with regard to the possibility that DC-tumour hybrids may not contribute all, or even most, of the immunostimulatory capacity present in preparations of EF treated cells.BH and GR contributed equally as senior authors     

Attention and the detectability of weak taste stimuli

Subjects detected weak solutions of sucrose or citric acid under conditions in which attention was directed toward one of the tastants or the other. Detection thresholds were measured using an adaptive, forced-choice procedure, with a three-down one-up rule, which computer simulations suggest should be more reliable than the popular two-down one-up rule. The thresholds were modestly but systematically lower for attended tastants than for unattended ones. Similar results have been reported in other sense modalities, including vision (greater sensitivity to stimuli presented to attended versus unattended spatial locations) and hearing (greater sensitivity to stimuli presented at attended versus unattended sound frequencies). Taken together, the findings are consistent with a general hypothesis regarding attention in sensory systems: gains or losses in detectability occur when a central attentional mechanism (or, conceivably, a preattentive mechanism) selectively and preferentially monitors signals arising from particular subsets of peripheral neural inputs.      

CONFIDENCE ELLIPSES APPLIED TO THE COMPARISON OF SENSORY PROFILES

Our experiment involved seven panels and six chocolates – five dark chocolates and one milk chocolate. The aim of the study was to compare the sensory profiles of the chocolates. A natural question to ask is “Did the panelists detect any differences among the five dark chocolates or did they systematically contrast them with the milk chocolate?” The scatter plot of the chocolates obtained by principal component analysis was useless to answer that question, because of the proximity of the points. To overcome that, we used confidence ellipses calculated using bootstrap. The originality of the study lies in the fact that we applied those ellipses to hierarchical multiple factor analysis (HMFA): among the seven panels, six were composed of trained professionals and the last one was composed of untrained students, and through that method, we managed to compare the two types of panels and balance the role of each trained panel. HMFA provides in a single scatter plot a representation of the six chocolates for each panel, the trained panels and all the panels. Confidence ellipses around each chocolate show that the combined panels – the six trained panels and also the untrained panel – differentiate the five dark chocolates. They also show how much larger the untrained panel's variability is than that of the trained panels, and how comparable are the trained panels' variability to each other.     

Microbial Communities and Fecal Indicator Bacteria Associated with Cladophora Mats on Beach Sites along Lake Michigan Shores

A high biomasses of Cladophora, a filamentous green alga, is found mainly during the summer along the shores of Lake Michigan. In this study, the abundance and persistence of the fecal indicator bacterium Escherichia coli and sulfate-reducing bacteria (SRB) on Cladophora mats collected at Lake Michigan beaches were evaluated using both culture-based and molecular analyses. Additionally, 16S rRNA gene cloning and sequencing were used to examine the bacterial community composition. Overall, E. coli was detected in all 63 samples obtained from 11 sites, and the average levels at most beaches ranged from 2,700 CFU/100 g (wet weight) of Cladophora to 7,500 CFU/100 g of Cladophora. However, three beaches were found to have site average E. coli densities of 12,800, 21,130, and 27,950 CFU/100 g of Cladophora. The E. coli levels in the lake water collected at the same time from these three sites were less than the recommended U.S. Environmental Protection Agency limit, 235 CFU/100 ml. E. coli also persisted on Cladophora mats in microcosms at room temperature for more than 7 days, and in some experiments it persisted for as long as 28 days. The SRB densities on Cladophora mats were relatively high, ranging from 4.4 × 10⁶ cells/g (6.64 log CFU/g) to 5.73 × 10⁶ cells/g (6.76 log CFU/g) and accounting for between 20% and 27% of the total bacterial counts. Partial sequences of the 16S rRNA gene clones revealed a phylogenetically diverse community, in which the Cytophaga-Flavobacterium-Bacteroides cluster and the low-G+C-content gram-positive bacteria were the dominant organisms, accounting for 40% and 12.8%, respectively, of the total clone library. These results further reveal the potential public health and ecological significance of Cladophora mats that are commonly found along the shoreline of Lake Michigan, especially with regard to the potential to harbor microorganisms associated with fecal pollution and odor-causing bacteria.