Arsenite and monomethylarsonous acid generate oxidative stress response in human bladder cell culture.

Arsenicals have commonly been seen to induce reactive oxygen species (ROS) which can lead to DNA damage and oxidative stress. At low levels, arsenicals still induce the formation of ROS, leading to DNA damage and protein alterations. UROtsa cells, an immortalized human urothelial cell line, were used to study the effects of arsenicals on the human bladder, a site of arsenical bioconcentration and carcinogenesis. Biotransformation of As(III) by UROtsa cells has been shown to produce methylated species, namely monomethylarsonous acid [MMA(III)], which has been shown to be 20 times more cytotoxic. Confocal fluorescence images of UROtsa cells treated with arsenicals and the ROS sensing probe, DCFDA, showed an increase of intracellular ROS within five min after 1 microM and 10 microM As(III) treatments. In contrast, 50 and 500 nM MMA(III) required pretreatment for 30 min before inducing ROS. The increase in ROS was ameliorated by preincubation with either SOD or catalase. An interesting aspect of these ROS detection studies is the noticeable difference between concentrations of As(III) and MMA(III) used, further supporting the increased cytotoxicity of MMA(III), as well as the increased amount of time required for MMA(III) to cause oxidative stress. These arsenical-induced ROS produced oxidative DNA damage as evidenced by an increase in 8-hydroxyl-2'-deoxyguanosine (8-oxo-dG) with either 50 nM or 5 microM MMA(III) exposure. These findings provide support that MMA(III) cause a genotoxic response upon generation of ROS. Both As(III) and MMA(III) were also able to induce Hsp70 and MT protein levels above control, showing that the cells recognize the ROS and respond. As(III) rapidly induces the formation of ROS, possibly through it oxidation to As(V) and further metabolism to MMA(III)/(V). These studies provide evidence for a different mechanism of MMA(III) toxicity, one that MMA(III) first interacts with cellular components before an ROS response is generated, taking longer to produce the effect, but with more substantial harm to the cell.

A factor causing stable stimulation of juvenile hormone synthesis by Diploptera punctata corpora allata in vitro.

Co-incubation of corpora allata (CA) from the cockroach, Diploptera punctata, with ovaries, fat body or muscle but not brain or testis, leads to a substantial increase in juvenile hormone synthesis. Incubation of the glands in medium pre-conditioned with ovaries also stimulates JH synthesis. The ovary was used as a convenient source of stimulatory factor for a detailed analysis of its physiological effects on the CA. The increase in JH synthesis is stable, maintained over 24h after exposure to the stimulatory factor. Stimulation is dose-dependent, and the corpora allata show an exquisite relationship between sensitivity to this factor and developmental stage. Day 0 and day 1 glands, as well as glands from post-vitellogenic females, are sensitive to stimulation, whereas glands from vitellogenic females are not sensitive. Corpora allata attached to the brain do not respond to the stimulatory factor, and denervation in vivo leads to an increase in JH synthesis by the glands and a loss in sensitivity to the factor. These data suggest that glands from pre- and post-vitellogenic females are inhibited by their nervous connection to the brain. In contrast, glands from vitellogenic females are normally responding to the endogenous stimulatory factor and are thus no longer stimulated in vitro. Co-incubation of CA with allatostatin and conditioned medium still leads to a stimulation of JH synthesis, suggesting that the restraining effect of the nervous connections to the brain is not caused by allatostatin. The CA cell number increases between emergence and day 2, then remains stable until after oviposition. The stimulatory factor accelerates the increase in cell number in young adult females. The results are interpreted as providing evidence for a constitutive change in CA activity caused by a humoral factor produced by various tissues including the ovary, and modulated by nervous connections to the brain.

An image analysis workstation designed for multiple users: application of quantitative digital imaging techniques to electron microscopy.

The purpose of the present study is to describe the setup of an image analysis workstation designed for multiple users, and to show the application of digital imaging techniques to the analysis of electron microscopic images. The image analysis system consists of a conventional light microscope mounted on a table-top, vibration-free platform, a light box for viewing negatives, two separate video cameras, a switch box, a video monitor, a digitizing tablet, a computer, and morphometric software packages. The system can quantitate the amount that each of the 256 gray levels contributes to the image, perform morphometric analysis (eg, shape and size) on individual gray level-defined subimages, and perform statistical analysis. Each operator has access to his or her own data and program setups through the use of 21.4-Mb removable Bernoulli cartridges. This setup for multiple users prevents the cluttering of the hard drive of the computer and avoids the possibility of accidentally removing the stored data of another user. The quantitative capabilities of the digital imaging system is demonstrated using an image of a normal lymphocyte and an apoptotic cell (ie, a cell which has undergone programmed cell death), both captured on the same electron microscopic negative. A comparison of the histograms of nuclear densities determined for these two cells reveals subtleties in gray level distribution not appreciated by the naked eye.

A multidisciplinary approach to the diagnosis of cutaneous T-cell lymphomas.

The cutaneous T-cell lymphomas (CTCLs) comprise a spectrum of non-Hodgkin lymphomas with a predilection for the skin. This heterogeneous group of CTCLs include the prototypic CTCL mycosis fungoides (MF) and the recently described Ki-1+ lymphomas. MF is notoriously difficult to diagnose in its early stages. The histologic appearance of early MF is indistinguishable from that of chronic dermatitis. The limitations of light microscopy in the diagnosis of the CTCLs have led to the development of other diagnostic laboratory techniques. The best approach to the diagnosis of the CTCLs is a multidisciplinary one and should include ultrastructural morphometry, immunophenotyping, immunogenotyping, and histologic evaluation whenever possible. It is the purpose of this overview to point out the strengths and weaknesses of each of these techniques and, together with clinical input, to provide a comprehensive and rational approach to patient care.

Limitations of ZAF correction factors in the determination of calcium/phosphorus ratios: important forensic science considerations relevant to the analysis of bone fragments using scanning electron microscopy and energy-dispersive x-ray microanalysis.

A series of calcium phosphate standards having calcium/phosphorus (Ca/P) molar ratios of 0.50, 1.00, 1.50, and 1.67, respectively, was prepared for bulk specimen analysis using scanning electron microscopy (SEM) and energy-dispersive X-ray microanalysis (EDXA). The standards were mounted on carbon planchettes as either pure crystals or crystals embedded in epoxy resin. Ten different samples of each embedded and non-embedded standard were analyzed in a JEOL 100 CX electron microscope interfaced with a Kevex 8000 EDXA system using a lithium-drifted silicon detector and a multichannel analyzer. The Ca/P ratios were determined by calculating both net peak intensities without matrix corrections and atomic kappa-ratios using the MAGIC V computer program with ZAF correction factors for quantitative analysis. There was such extensive absorption of phosphorus X-rays in standards embedded in an epoxy matrix that the observed Ca/P ratios were statistically compatible with four different standards ranging in theoretical Ca/P ratios from 1.0 to 1.67. Although the non-embedded crystals showed a greater separation in the Ca/P ratios, both methods of preparation produced serious flaws in analysis. Direct application of the discovery of this caveat to the identification of suspected bone fragments for forensic science purposes is discussed.

A comparative mathematical evaluation of contour irregularity using form factor and PERBAS, a new analytical shape factor.

Contour irregularity was mathematically evaluated using two different analytical shape factors: (1) "form factor" (FF), a conventional type of shape analysis formula defined by the formula 4 pi A/P2, and (2) the "PErimeter Ratio Before and After Smoothing" (PERBAS). PERBAS values are obtained by first "smoothing" a contour that contains one or more concavities; surface projections are connected using straight lines and include only tangent lines that do not interest the nucleus, resulting in a convex wrap. Perimeters before and after smoothing were obtained using a MOP-3 computerized image analyzer. Using a series of computer-generated images, it was determined that FF was more sensitive than PERBAS to simple deformations of shape (i.e., having no contour concavities) whereas PERBAS was more specific for surface "roughness" (i.e., contour having at least one concavity). As an application to cancer diagnosis, 6 cases of Burkitt's lymphoma (BL) and 14 cases of Burkitt's-like lymphoma (BLL) were compared for nuclear contour roughness using both shape factors. The BLL group could be distinguished from the BL group on the basis of mean PERBAS values (P = .029), but not on the basis of mean FF values (P = .093). The BL group had a mean PERBAS value of 1.051 +/- 0.023 and a mean FF value of 0.790 +/- 0.068. The BLL group had a mean PERBAS value of 1.093 +/- 0.054 and a mean FF value of 0.723 +/- 0.094. Mean FF and PERBAS values, plotted in a bivariate graphic display, established morphometric shape domains. BL cases occupied a significantly smaller shape domain than did the BLL cases, indicating a greater heterogeneity among nuclear shapes in the latter group. Shape discrimination, in general, was greatly increased when both shape factors were used; this has a great potential for determining cancer diagnosis and patient prognosis.

Ultrastructural morphometric analysis of papillary neoplasms: biological and diagnostic relevance.

Ten papillary adenocarcinomas of thyroid origin (P-Thy), ten papillary adenocarcinomas of ovarian origin (P-Ov), and eight papillary neoplasms of non-thyroid/non-ovarian origin (P-Other) were morphometrically compared using 19 distinct quantitative nuclear and nucleolar parameters as a database for diagnosis. The selected cases consisted of 16 primary and 12 metastatic neoplasms. It was determined that the P-Thy group had a significantly smaller nucleolar area (NuA) and nucleolar perimeter (NuP), and smaller SDs of nuclear area (NA), NuA, and NuP compared with the P-Ov and P-Other groups (P less than .05). The P-Ov group had a significantly smaller SD of NA compared with the P-Other group (P less than .05). The P-Ov group exhibited the greatest variability among the papillary neoplasms. Linear regression analysis indicated that in the P-Thy group alone there was a significant correlation between mean nuclear form factor (4 pi A/P2) and mean NuA (r = -.82; P less than .01), and mean NP and mean NuA (r = +.77; P less than .01). Linear regression analysis also indicated that in the P-Ov group alone, there was a significant correlation between mean NA and mean NuA (r = +.75; P less than .02). Morphometric domains were established using statistically significant sets of variables that distinguished between the groups. The application of three-dimensional computerized cluster analysis techniques indicated that the P-Thy group consistently had the smallest morphometric domains. It was concluded that ultrastructural morphometric analysis of papillary neoplasms has diagnostic potential and reveals interesting biological relationships among distinct nuclear features in the different groups of neoplasms.

Nuclear contour irregularity correlates with Leu-9-, Leu-8- cells in benign lymphoid infiltrates of skin. An ultrastructural morphometric and quantitative immunophenotypic analysis suggesting the normal T-cell counterpart to the malignant mycosis fungoides/Sézary cell.

The purpose of the present study was to determine if a nonmalignant skin-associated T-cell might exhibit nuclear irregularity and represent the normal counterpart to the malignant Sézary T-cell found in mycosis fungoides (MF) and the Sézary syndrome (SS). Punch skin biopsies from ten benign lymphoid infiltrates of skin were subjected to ultrastructural morphometric analysis and quantitative immunohistochemistry (on serial frozen sections) using a battery of monoclonal antibodies. Form factor (FF) (4 pi A/p2) was used to assess nuclear contour irregularity. The lymphoid infiltrates were morphometrically heterogeneous (range of FF values: 0.482-0.704). Immunophenotypically, there was marked variation in host response to the variety of antigens presented. Linear regression analysis was used to determine if nuclear contour irregularity showed any statistical relationship to immunophenotypically defined lymphocyte subpopulations. It was determined that nuclear contour irregularity (mean FF values) did not correlate with the presence of any surface antigen tested. This included the antigens Leu-2a, Leu-3a + b, Leu-4, Leu-6, Leu-7, Leu-8, Leu-9, Leu-14, and Ia. There was, however, significant correlation of increased nuclear contour irregularity with the presence of Leu-9- and Leu-8- cells (r = 0.7 and 0.6, respectively), lymphocyte subsets reportedly deficient in MF and the SS. This finding leads to the speculation that the Leu-9-, Leu-8- reactive T-cell with an irregular nucleus might represent the normal counterpart to the malignant clonally expanded T-cell found in MF and the SS. It was also determined that helper to suppressor ratios varied 68-fold from 0.2 to 13.5 among these ten benign lymphoid infiltrates of skin. This finding underscores the futility of using helper to suppressor ratios as a diagnostic tool in defining T-cell malignancies.

An ultrastructural morphometric and immunophenotypic evaluation of Burkitt's and Burkitt's-like lymphomas.

Six cases of Burkitt's lymphoma (BL) and 12 cases of Burkitt's-like lymphoma (BLL) were classified by using strict histologic and cytologic criteria. These cases were processed for electron microscopy and analyzed by using computerized image analysis techniques. Form factor (4 pi A/P2) was used to measure nuclear contour irregularity. The mean of the standard deviation (SD) of nuclear area and form factor was used to assess pleomorphism. Overall, there were 8 similarities and 10 statistically significant dissimilarities out of 18 parameters analyzed. The similarities (p greater than 0.05) between the BL and BLL groups included the means of form factor, nuclear area/cytoplasmic area, SD of nuclear area/cytoplasmic area, number of nuclear pockets/100 nuclei, percentage of cells with nuclear pockets, number of lipid droplets/micron 2 of cytoplasm, percentage of cells with lipid droplets and number of mitochondria/micron 2 of cytoplasm. The dissimilarities (p less than 0.05) included the means of nuclear perimeter, SD of nuclear perimeter, nuclear area, SD of nuclear area, cellular area, SD of cellular area, cytoplasmic area, SD of cytoplasmic area, SD of form factor, and nucleolar frequency. Multiparameter analysis clearly separated these 18 patients into two distinct groups and confirms that the subtleties used in the histologic classification of these lymphoma subtypes are meaningful. Sixteen cases of BL and BLL were snap-frozen in isopentane and analyzed by using 16 lymphoid surface markers. All of the immunoglobulin-positive BL were of the mu isotype, whereas the BLL cases were divided between mu (6 cases) and gamma expression (4 cases). All 4 of the BL evaluated manifested CALLA expression, whereas 3 of the 11 BLL evaluated coexpressed CALLA. One BL case was of a pre-pre-B phenotype and one BLL case was of pre-B phenotype. The BL and BLL were compared to 49 cases of SIg (+) large cell lymphomas. The high incidence of coexpression of lambda, CALLA, and Ki-67 in BL and BLL separates these lymphomas, as a group, from the large cell lymphomas. We have also determined from this study that the separation of patients into distinct BL and BLL subtypes is clinically relevant. The BL group were all children (median of 6.5 years) compared with the BLL group who were all adults (median of 63 years). The complete remission rate was higher in the BL (67%) than in the BLL group (25%).4off

Cutaneous myiasis. Immunohistologic and ultrastructural morphometric features of a human botfly lesion.

This study documents the immunohistologic and ultrastructural morphometric features of a case of cutaneous myiasis due to Dermatobia hominis. The mixed host inflammatory response surrounding the larvae included lymphoblasts, eosinophils, activated fibroblasts, mature histiocytes, mast cells, plasma cells, and Langerhans cells, indicating a complex interactive immunologic response to the botfly parasite. Immunotyping revealed that the dominant dermal cells were activated (Ia+) T-helper cells. Although the strikingly elevated T-helper/T-suppressor cell ratio might suggest a monoclonal infiltrate, a mixture of Leu 8+ and Leu 8- T-cells indicates both immunoregulatory subsets of T-helper cells as found in a reactive process. Furthermore, our ultrastructural histogram found the lymphoid nuclear shapes were oval with few nuclear invaginations as found in inflammatory infiltrates. As indicated by electron microscopy, there were abundant activated fibroblasts elaborating collagen which may relate to larval containment.

Ultrastructural similarities between Chlorohydra viridissima and human neurosecretory granules: a cytochemical study using the uranaffin reaction.

The uranaffin reaction is an ultrastructural cytochemical procedure which, in humans, stains the cores of true neurosecretory granules of all morphological types, and will not stain the granules of exocrine-type glands or lysosomes found in a variety of cell types. Positively-charged uranyl ions appear to react with concentrated polyphosphate groups and stain the nucleotides known to be present in the neurosecretory granules of higher organisms. In Chlorohydra viridissima, two morphological types of neurosecretory granules were identified within the epidermis and gastrodermis by routine electron microscopy; one granule type was small and displayed a central dense core with a surrounding clear halo, whereas, the other was larger and displayed an eccentric core with a loosely-associated granule membrane. Both types stained with the uranaffin reaction, but the characteristic granules of the exocrine-type digestive glandular cells and the mucous cells of the gastrodermis were uniformly uranaffin-negative. In the gastrodermis, a third granule type was identified which showed a larger uranaffin-positive core. The discovery of uranaffin-positive neurosecretory granules in Hydra suggests that formation of nucleotide-neurohormone complexes as a means for packaging and storage of neurohormones within the granule matrix, may have existed in primitive nervous systems.

Pathogenesis of diabetic neuropathy.

Samples of lumbosacral trunk, posterior tibial nerve, and sural nerve obtained at autopsy from diabetic and nondiabetic patients without mononeuropathy multiplex were evaluated using 1-mu-thick epoxy sections and teased nerve fiber preparations. Focal fascicular lesions characterized by reduced density of myelinated axons within fascicles were found predominantly in the specimens from diabetics, mainly in the posterior tibial nerve and lumbosacral trunk. In severe examples, the perineurium and even surrounding epineurium were damaged, stamping the lesions as ischemic. In addition, identical lesions were found in biopsies of nerves of nondiabetics with vasculitis. Density of myelinated fibers at the three sites demonstrated a proximal-distal graded loss that was significantly greater in the diabetic samples. The loss from the lumbosacral trunk to the posterior tibial nerve was correlated with the density of focal lesions in the lumbosacral trunk in the diabetic (p = 0.025), indicating that distal fiber loss was partly due to the focal lesions. Teased nerve fiber abnormalities were common only in sural nerves of diabetics, suggesting that they are secondary. We conclude that beyond the possible metabolic abnormalities involved in the genesis of diabetic polyneuropathy, focal fascicular lesions, likely due to diabetic microangiopathy, are also important in the development of diabetic neuropathy.