Gregg M. Ridder

Age, sunlight, and facial skin: A histologic and quantitative study

Quantitative methods were developed to assess the interrelation between age and sunlight on the facial skin of healthy women living in the same sunny area. The women were grouped into the following categories: young versus old and low versus high solar exposure. The features evaluated were perceived age, amount of facial wrinkling, skin color, and skin elasticity. A punch biopsy specimen of cheek skin was obtained and prepared histologically for evaluation of solar elastosis. The histologic examination was complemented by quantification of collagen and elastin by computer-assessed image analysis. Perceived age was estimated by untrained women viewing high quality photographs. As expected, those with greater sun exposure looked older and had more wrinkles, more severe elastosis, increased elastin, and decreased collagen.

Characterization of p53 in Chinese hamster cell lines CHO-K1, CHO-WBL, and CHL: implications for genotoxicity testing.

Since the p53 gene function is critical to how a cell responds to DNA damage, we investigated the p53 status in Chinese hamster cell lines commonly used in genotoxicity tests for cytogenetic damage around the world. These included: Chinese hamster ovary K1 (CHO-K1), Chinese hamster ovary WBL (CHO-WBL), and Chinese hamster lung (CHL) cells. The results of DNA sequencing, protein analysis, and cell cycle analysis demonstrate that the CHO-K1 and CHO-WBL cell lines have mutant p53 sequence [a mutation in codon 211 in exon 6 resulting in a change from Thr (ACA) to Lys (AAA)], mutant protein (high spontaneous levels that are non-inducible after X-irradiation), and mutant function (lack of G1 checkpoint). Interestingly, the CHL cell line has a completely wild-type p53 DNA sequence. However, the CHL cells have an abnormally high spontaneous level of wild-type p53 protein expression that is not inducible after X-irradiation, yet there is some evidence of G1 delay after irradiation. The protein data suggests that p53 in CHL cells is not being regulated normally, and thus is probably not functioning normally. The mechanism leading to this abnormal regulation of p53 in CHL cells clearly does not involve mutation in the p53 gene. Overall, the CHL cell line may be similar to the CHO cell lines, in that they all appear to have abnormal p53 function. Further work is needed to determine whether the presence of spontaneously high levels of wild-type p53 in CHL cells results in a difference in response to DNA damage (quantitatively or qualitatively) compared to the p53 mutant CHO cell lines.

Advances in automated 3-D image analysis of cell populations imaged by confocal microscopy

Automated three-dimensional (3-D) image analysis methods are presented for rapid and effective analysis of populations of fluorescently labeled cells or nuclei in thick tissue sections that have been imaged three dimensionally using a confocal microscope. The methods presented here greatly improve upon our earlier work (Roysam et al.:J Microsc 173: 115-126, 1994). The principal advances reported are: algorithms for efficient data pre-processing and adaptive segmentation, effective handling of image anisotrophy, and fast 3-D morphological algorithms for separating overlapping or connected clusters utilizing image gradient information whenever available. A particular feature of this method is its ability to separate densely packed and connected clusters of cell nuclei. Some of the challenges overcome in this work include the efficient and effective handling of imaging noise, anisotrophy, and large variations in image parameters such as intensity, object size, and shape. The method is able to handle significant inter-cell, intra-cell, inter-image, and intra-image variations. Studies indicate that this method is rapid, robust, and adaptable. Examples were presented to illustrate the applicability of this approach to analyzing images of nuclei from densely packed regions in thick sections of rat liver, and brain that were labeled with a fluorescent Schiff reagent.

Cytokine induction of a human acute myelogenous leukemia cell line (KG-1) to a CD1a+ dendritic cell phenotype.

Abstract Dendritic cells (DC) are highly specialized antigen-presenting cells located in many nonlymphoid tissues, and Langerhans cells (LC), a specialized form of DC, are found in the skin. LC as antigen-presenting cells play a critical role in the induction of allergic contact dermatitis. LC research is difficult because few LCs can be isolated from human skin, so efforts have focused on obtaining DCs from alternative sources. Mononuclear cells from peripheral blood and CD34 + stem cells from human cord blood and marrow can be induced to form phenotypic and functional DCs, but experiments of this type are expensive and the DC yield is low. We report here the induction of the myeloid leukemia cell line (KG-1) to a DC morphology and phenotype by culturing the cells in a defined cytokine cocktail. Morphologically, the KG-1-derived DCs are large irregularly shaped cells with prominent dendritic processes and hair-like cytoplasmic projections. Phenotypically, the KG-1-derived DCs lack lineage-specific markers, and express MHC class II, costimulatory molecules CD80 and CD86, and CD83. Functionally, KG-1-derived DCs are capable of phagocytosing latex microspheres and are able to induce a potent allogeneic T-cell response. Within the KG-1-derived DCs, a subpopulation maintains the DC phenotype and morphology described above but further develops CD1a + marker expression similar to that of resident skin-derived LCs. These findings illustrate that phenotypic, morphologic and functional DCs can be derived from the KG-1 cell line.

Increased hyaline droplet formation in male rats exposed to decalin is dependent on the presence of α2u-globulin

A peculiar decalin-induced male rat nephropathy associated with the altered renal handling of filtered protein appears limited to the accumulation of the protein, alpha 2u-globulin. Several strains of male rats that produce alpha 2u-globulin (Fischer-344, Sprague-Dawley, Buffalo, and Norway Brown) demonstrate spontaneous renal cortical hyaline droplets which are exacerbated after exposure to decalin. In all cases, a close correlation exists between hyaline droplet formation observed histologically and alpha 2u-globulin accumulation measured biochemically. In stark contrast, the NCI-Black-Reiter strain, which does not produce measurable quantities of alpha 2u-globulin, neither forms hyaline droplets nor accumulates any filtered protein in its kidney cortex either spontaneously or after exposure to decalin. Also, female rats injected ip with male rat alpha 2u-globulin exhibit increased hyaline droplet formation and alpha 2u-globulin accumulation when treated with decalin. These data provide evidence that the presence of alpha 2u-globulin is key in understanding why this nephropathy appears unique to the male rat.

Evaluation in rats of the dose-response relationship among colonic mucosal growth, colonic fermentation, and dietary fiber

The dose-response relationship among dietary fiber, colonic fermentation, fecal weight, and mucosal growth were evaluated in this study. The morphometric parameter of total mucosal volume was used to assess diet-induced differences in colonic mucosal growth. Dietary fibers with a wide range of fermentability and that have previously been shown to inhibit the development of colonic neoplasia in rats were used. Sprague-Dawley rats were fed Purina Rodent Chow, AIN-76a fiber-free diet, or an AIN-76a diet supplemented with three different dietary fibers, (cellulose, guar gum, or wheat bran) at 2, 5, 10, or 15% of the diet. Diets were fed for 28 days. Total colonic mucosal volume was determined using sterologic principles and computerized image analysis; 48-hr fecal weight was measured; and the concentration of short-chain fatty acids (SCFA) in colonic contents was determined at study termination. Each type of fiber induced a dose-dependent increase in total mucosal volume of the colon and fecal weight. Mucosal volume and fecal weight were closely correlated (R2>0.95). Total mucosal volume was not correlated with the concentration of total SCFA or butyrate in the colon. These results indicate that diet-induced change in colonic mucosal growth, as measured by total mucosal volume, is positively correlated with fecal weight and not related to alterations in colonic fermentation. Enhanced colonic mucosal growth occurs in rats fed dietary fibers that have previously been shown to inhibit the development of genotoxin-induced colonic neoplasia in rats.

Automated quartz injector/trap for fused-silica capillary columns

Abstract An automated quartz injector/trap was developed for a Perkin-Elmer 3920 gas chromatograph. The injector/trap serves to concentrate gaseous or liquid samples without introducing organic contaminants. The concentrated sample is volatilized and transferred to a second trap by cooling a small section of a fused-silica capillary column. Reconcentration in the capillary column preserves band-shape and provides a system which delivers quantitative results on a variety of samples. Liquid samples show relatives standard deviations of 1% and 2%, respectively, for isothermal and temperaure-programmed analyses. A detection limit of less than 1 ppb is expected, with a flame ionization detector, for samples of n-octane in air.

An improved watershed algorithm for counting objects in noisy, anisotropic 3-D biological images

Effective 3-D image processing algorithms are presented for automatic counting and analysis of cells in anisotropic 3-D biological images that are collected by laser-scanning confocal microscopes. In these instruments, the x-y resolution is much better than the resolution along the z axis, hence the voxels (pixels in 3-D) are anisotropic. In this work, the images are pre-processed by a 3-D extension of an anisotropic diffusion algorithm, and the resulting images are binarized by a clustering based segmentation algorithm. As a result of binary segmentation, some regions consist of individual objects while others are multi-object clusters. An extension of Vincent and Soilles watershed algorithm (1991) to anisotropic 3D spaces is used to separate such cell clusters. The watershed algorithm is applied on marker functions that are generated using a combination of 3-D morphological inverse distance functions and 3-D image gradients. Cell measurements, such as volume, average intensity and locations, are calculated on the result of watershed segmentation. This algorithm has been successfully applied to the automated analysis of cell populations from a variety of biological studies involving large numbers of tissue samples.