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Covering up to September 2016 This review reports on natural compounds that derive from the isoxazolinone ring as well as the 3-nitropropanoic acid (3-NPA) moiety. These structural elements occur in compounds that have been identified in plants, insects, bacteria and fungi. In particular, plants belonging to the family of legumes produce such compounds. In the case of insects, isoxazolin-5-one and 3-NPA derivatives were found in leaf beetles of the subtribe Chrysomelina. A number of these natural products have been synthesized so far. In the case of the single compound 3-NPA, several synthetic strategies have been reported and some of the most efficient routes are reviewed. The toxicity of 3-NPA results from its ability to bind covalently to the catalytic center of succinate dehydrogenase causing irreversible inhibition of mitochondrial respiration. As a motif that is produced by many species of plants, leaf beetles and fungi, different detoxification mechanisms for 3-NPA have evolved in different species. These mechanisms are based on amide formation of 3-NPA with amino acids, reduction to β-alanine, ester formation or oxidation to malonic acid semialdehyde. The biosynthetic pathways of 3-NPA and isoxazolin-5-one moieties have been studied in fungi, plants and leaf beetles. In the case of fungi, 3-NPA derives from aspartate, while leaf beetles use essential amino acids such as valine as ultimate precursors. In the case of plants, it is supposed that malonate serves as a precursor of 3-NPA, as indicated by feeding of 14C-labeled precursors to Indigofera spicata. In other leguminous plants it is suggested that asparagine is incorporated into compounds that derive from isoxazolin-5-one, which was indicated by 14C-labeled compounds as well. In the case of leaf beetles it was demonstrated that detection of radioactivity after 14C-labeling from a few precursors is not sufficient to unravel biosynthetic pathways.

Purpose We aimed to report by light microscopy the normal histology of the A1 pulley, describe the histologic abnormalities of A1 pulleys in trigger digits, and look for possible correlations between these findings and the severity of the disease. Methods In a series of 104 trigger digits operated on in 80 adult patients, the A1 pulleys were removed and histologically studied. The findings were compared with 55 normal A1 pulleys obtained from fresh-frozen cadaveric specimens. Results The normal A1 pulley was composed of 3 layers: layer I, an inner, avascular, concave unicellular or bicellular gliding layer containing cartilage-like cells; layer II, a middle layer, also avascular, characterized by spindle-shaped fibroblasts; and layer III, an outer, richly vascularized layer, continuous with the membranous tendons sheath. We used a 3-grade classification, increasing in severity, to describe the histologic abnormalities observed in trigger digit A1 pulleys. Mild abnormalities (grade 1) were those with a fibrocartilaginous gliding surface almost intact. The margin between the fibrocartilaginous and membranous portions of the pulley was well delineated. In moderate abnormalities (grade 2), the avascular fibrocartilaginous gliding surface appeared fissured and thinner. The inner layer (I) was interrupted and replaced by fibrous tissue, with fissures that did not cross through the middle layer (II). A mild vascular network hyperplasia was observed in the outer layer (III), which began to invade the fibrocartilage. In severe abnormalities (grade 3), the fibrocartilaginous gliding surface was thin, discontinuous, or even completely destroyed. The vascular network hyperplasia became excessive and reached the synovial space of the flexor tendon sheath. The histologic features were correlated with the severity of the clinical symptoms (p Conclusions The histologic abnormalities observed in the A1 pulley of trigger digits are characteristic and not related to inflammation. As the trigger digit worsens, the gliding surface begins to wear and is gradually replaced by a secondary invasive hyperplasia from the outer layer. These abnormalities could be caused by a modification or an increase of the mechanical stresses along the flexor tendons.

Although the importance of selenium for bone metabolism is unknown, some clinical conditions such as Kashin-Beck osteoarthropathy have been associated with selenium deficiency. Although selenium deficiency induces growth retardation in rats, it has not been established whether this growth inhibition is associated with changes in bone metabolism. We investigated the effect of selenium deficiency on bone metabolism in growing male rats fed a selenium-deficient diet for two generations (Se-). In Se- rats, erythrocyte glutathione peroxidase activity and plasma selenium concentration were strongly reduced compared with pair-fed selenium-adequate rats (Se+). Weight and tail length were reduced by 31% and 13% in the Se- rats, respectively (p < 0.001). The Se- diet was associated with a 68% reduction of pituitary growth hormone (GH; p = 0.01) and a 50% reduction of plasma insulin-like growth factor I (IGF-I; p < 0.001). Plasma calcium was lower and urinary calcium concentration was greater in Se- rats. This group had a 2-fold increase in parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in plasma. Plasma osteocalcin and urinary deoxypyridoline were reduced by 25% and 57% in the Se- rats (p < 0.001). Selenium deficiency resulted in a 23% and 21% reduction in bone mineral density (BMD) of the femur and tibia (p < 0.001) and this effect persisted after adjustment for weight in a linear regression model. A 43% reduction in trabecular bone volume of the femoral metaphysis (p < 0.001) was found in Se- rats. This experimental study shows that growth retardation induced by selenium deficiency is associated with impaired bone metabolism and osteopenia in second-generation selenium-deficient rats.

Background: Epiluminescence microscopy (ELM) is a noninvasive clinical tool recently developed for the diagnosis of pigmented skin lesions (PSLs), with the aim of improving melanoma screening strategies. However, the complexity of the ELM grading protocol means that considerable expertise is required for differential diagnosis. In this paper we propose a computer-based tool able to screen ELM images of PSLs in order to aid clinicians in the detection of lesion patterns useful for differential diagnosis. Methods: The method proposed is based on the supervised classification of pixels of digitized ELM images, and leads to the construction of classes of pixels used for image segmentation. This process has two major phases, i.e., a learning phase, where several hundred pixels are used in order to train and validate a classification model, and an application step, which consists of a massive classification of billions of pixels (i.e., the full image) by means of the rules obtained in the first phase. Results: Our results show that the proposed method is suitable for lesion-from-background extraction, for complete image segmentation into several typical diagnostic patterns, and for artifact rejection. Hence, our prototype has the potential to assist in distinguishing lesion patterns which are associated with diagnostic information such as diffuse pigmentation, dark globules (black dots and brown globules), and the gray-blue veil. Conclusions: The system proposed in this paper can be considered as a tool to assist in PSL diagnosis. Cytometry 37:255‐266, 1999. r 1999 Wiley-Liss, Inc.

The aim of this study is to present a new classification of nasal polyps. This classification is based both on morphologic criteria relating to morphonuclear features from isolated Feulgen-stained nuclei and on glycohistochemical characteristics from histologic slides submitted to three lectins (peanut, wheat germ, and gorse seed agglutinins) and one neoglycoconjugate glycohistochemical stain. While the morphonuclear features (including 30 variables) relate essentially to chromatin pattern, the glycohistochemical stains (including 16 variables) are linked to the presence of specific carbohydrate moieties in cell membranes and cytoplasm. Forty-nine nasal polyps, including single polyps, diffuse polyposis, cystic fibrosis-related polyposis, and aspirin idiosyncracy-related polyposis associated with asthma, were thus characterized. All the variables were obtained quantitatively by means of computer-assisted microscopy. Two complementary methods of data classification were used to determine the actual diagnostic value contributed by each quantitative variable, namely, discriminant analysis, which forms part of multifactorial statistical analysis, and the decision tree technique, which is an artificial intelligence-related algorithm. The data so obtained show that our morphologic classification of nasal polyps fits in with the classification of nasal polyps defined on the basis of clinical criteria.

The various grading systems proposed for renal cell carcinomas all suffer from problems related to inter-observer variability. Some of these grading systems are based, either partially or wholly, on morphonuclear criteria, such as nuclear size and shape, anisonucleosis, and chromatin pattern. These criteria can be quantitatively (and thus objectively) evaluated by means of the computer-assisted microscopic analysis of Feulgen-stained nuclei. In the present work, 39 quantitative variables, including two morphometric, 28 chromatin pattern-related, and nine DNA ploidy level-related, were computed for 65 renal cell carcinomas. The actual diagnostic information contributed by each variable was determined by means of multifactorial statistical analysis (discriminant analysis) and two artificial intelligence-related methods of data classification (the decision tree and production rule methods). The results show that quantitative information, as provided by the computer-assisted microscopy of Feulgen-stained nuclei and analysed by means of artificial intelligence-related methods of data classification, contributes significant diagnostic information for the grading of renal cell carcinoma, thus reducing the problem of inter-observer reproducibility.

The hormone sensitivity of a tumor is traditionally based on the presence of steroid receptors. Other factors should be taken into consideration. Here, we studied the influence of 10 nM epidermal growth factor (EGF) or gastrin on the proliferation of human ex vivo tumor cultures by means of [3H]thymidine autoradiography. The immunohistochemical EGF-receptor expression was also quantified by means of computer-assisted microscopy. The results demonstrated that the proliferation of 6/11 astrocytic tumors and 3/16 meningiomas was sensitive to at least one factor tested, i.e. EGF or gastrin (P < 0.01), and 5 of these 9 'hormone-sensitive' tumors were sensitive to both factors. The immunohistochemical labeling index for the EGF receptor was higher than 80% in 15/16 meningiomas, but only in 6/11 gliomas (P < 0.01). These results suggest that EGF and gastrin are important for astrocytic tumor proliferation and significantly (P < 0.01) less important for meningiomas. Thus, astrocytic tumors may be steroid insensitive in term of cell growth, but are certainly not hormone insensitive.

The current classification of clinically nonfunctioning pituitary adenomas is based on immunocytochemical and ultrastructural studies. However, a number of cases have less distinctive features that cannot be easily conformed with the prevailing morphologic classifications. The diagnostic information contributed by the determination of the nuclear DNA content (DNA ploidy level) and quantitative chromatic pattern description as opposed to the morphofunctional diagnosis in clinically nonfunctioning adenomas was consequently investigated in a series of 71 pituitary adenomas, including 31 null-cell adenomas, 35 gonadotropin adenomas, and 5 nonfunctioning adenomas that were not examined by electron microscopy. DNA ploidy level (8 variables) and quantitative chromatin pattern description (30 variables) were carried out by means of the computer-assisted microscope analysis of 80-1600 Feulgen-stained nuclei analyzed/case. The diagnostic information contributed by the 38 quantitative variables was determined by multifactorial statistical analysis (i.e., Discriminant Analysis). This computer-assisted classification significantly differentiated nulLcell adenomas from gonadotropin adenomas (p = 0.0025). In addition, it was able to differentiate three major subtypes of nonfunctioning adenomas on the basis of their immunohistochemical profiles. These were the immunonegative adenomas, the follicle-stimulating hormone (FSH)-positive adenomas, and the a-subunit (a) and/or luteiwizing hormone (LH)-positive adenomas (p0.0001 to p0.001). We thus suggest that the cytometric image analysis of Feulgen-stained nuclei can contribute on the discrimination of subtypes of clinically nonfunctioning pituitary adenomas.

A 22-year-old patient with type I trichorinophalangeal Langer-Giedion syndrome presented a stage 2 osteonecrosis of the lunate associated with congenital shortening of the ulna and carpal tunnel syndrome. The patient was treated by shortening osteotomy of the radius and median nerve neurolysis, with an excellent result. This case provides an additional piece of evidence associating ulnar-minus variance with lunatomalacia, and another argument in favor of the theory that Kienböck's disease results from microfractures sustained by the lunate under an abnormal stress distribution situation.

✓ The authors investigated whether cytometry-related variables generated by means of computer-assisted microscopic analysis of Feulgen-stained nuclei can contribute significant information toward the characterization of low-grade astrocytic tumor aggressiveness. This investigation was conducted using the nearest-neighbor rule (a traditional classification method used in pattern recognition) to analyze a series of 250 supratentorial astrocytic tumors from adult patients. This series included 39 low-grade astrocytomas and 211 high-grade astrocytic tumors (including 47 anaplastic astrocytomas and 164 glioblastomas multiforme [GBMs]). The results show that the 3-nearest-neighbors rule enabled a subgroup of “atypical” astrocytomas to be distinguished from the “typical” tumors. The atypical astrocytoma species exhibited a DNA content (DNA ploidy level) and morphonuclear characteristics that were statistically more similar to the characteristics of GBMs than to those exhibited by the typical astrocytomas. An analysis of survival data revealed that patients with atypical astrocytomas survived for a significantly shorter period (p < 0.001) than patients with typical lesions of this kind. In fact, patients with atypical astrocytomas had a survival period similar to that of patients with anaplastic astrocytomas, whereas patients with typical astrocytomas had a survival period significantly longer (p < 0.0001) than those associated with anaplastic astrocytomas and GBMs.

The antitumoral effects of 30 drugs was monitored in vitro on three neoplastic cell lines. These 30 drugs belonged to various pharmacological classes which included alkylating agents, antimetabolites, Vinca alkaloids, topoisomerase II inhibitors, and intercalating agents. The aim of the present work is to use the same methodology to characterize the drug-induced effects at several biological levels. The drug-induced modifications were, therefore, monitored by means of the digital cell image analysis of Feulgen-stained nuclei. This methodology enabled the cell cycle kinetics to be studied. Furthermore, the numerical data quantitatively describing chromatin patterns were submitted to multivariate (principal-components) analyses, with the canonical transformation of the data. Statistical analysis shows that each pharmacological class of anticancer drugs induces specific modifications to the chromatin patterns. The present study, therefore, shows that it is possible to identify distinct classes of antineoplastic drugs on the basis of their mechanisms of action by means of the quantitative chromatin pattern description of Feulgen-stained nuclei.

BACKGROUND/AIMS: Compared with normal colonic mucosa, lectin receptor expression is increased in hyperplastic and neoplastic tissues; some lectins have been shown to influence human colonic epithelial cell proliferation. The aim was to assess further the influence of five lectins (Phaseolus vulgaris (PNA), Griffonia simplicifolia (GSA), concanavalin A (Con A), wheat germ (WGA), and peanut (PHA-L) agglutinins) on cellular growth in three human colorectal cancer cell lines (LoVo, HCT-15 and SW837). METHODS: Cells were cultured in four lectin concentrations (0.1, 1.0, 10, and 100 micrograms/ml) and growth assessed at days 2, 3, 5, and 7. The experiments were performed in media supplemented with either 1% or 10% fetal calf serum (FCS). Growth was assessed using the MTT (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric assay. RESULTS: Growth in each cell line was greatly affected by at least two of the lectins tested. There was some variation in the effect of a given lectin on different cell lines. Lectin effects showed a dose-response and the greatest effects generally resulted from the highest concentrations at the longest culture time. WGA and Con A induced large effects in all cell lines; the effects of Con A were partly blocked by the higher concentration of FCS. PNA had modest and uniform stimulatory effects overall. The effects of GSA and PHA-L varied between cell lines. CONCLUSIONS: The lectins studied all have the potential to affect colonic cancer growth in vitro. Many dietary lectins are resistant to digestion and may have important effects in vitro but the definition of their role in human colonic cancer biology must take into account the variability in lectin response.

The role of lectins as biosignalling molecules or as markers of human astrocytic tumors remains relatively unexplored. The aim of the present work is to investigate (1) whether or not human astrocytic tumors express specific glycans, evidenced experimentally by means of lectin histochemistry, and (2) whether, in turn, these lectins can significantly modulate astrocytic tumor cell proliferation. Using a cell image processor, we therefore began by quantitatively measuring the histochemical binding pattern of 5 lectins (WGA, PNA, PHA-L, GSA-IA4 and Con A) in 5 astrocytomas, 5 anaplastic astrocytomas and 5 glioblastomas. Secondly, we measured the influence of these 5 lectins on the proliferation of 3 astrocytic tumor cell lines (SW1088, U373 and U87) growing in vitro as monolayers. Cell proliferation was assessed by means of the colorimetric MTT assay. The histochemical lectin staining markedly varied intra- and inter-group. However, some constant results were obtained. Indeed, the staining increased markedly from GSA-IA4 and PHA-L through WGA and PNA to ConA in the three histopathological groups. The assessment of cell proliferation demonstrated that WGA, Con A and PHA-L very significantly decreased proliferation in the 3 astrocytic cell lines in a dose-dependent manner. Astrocytic tumor cells in the confluent growth phase were less sensitive to the WGA, Con A and PHA-L lectin-induced effects than cells in the log growth phase. The GSA-IA4 and PNA lectins had globally very weak effects on the proliferation of the astrocytic tumor cell lines. Increasing the fetal calf serum from 1% to 10% in the culture media significantly antagonized the WGA-, Con A- and PHA-L-induced cell proliferation decrease in the 3 astrocytic cell lines. In conclusion, the present data strongly suggest that some lectins (including WGA, Con A and PHA-L) significantly influence the proliferation of astrocytic tumor cells.

BACKGROUNDA variety of hormones have demonstrated effects on prostatic tissue growth dynamics. Our goal was to define the effect of dihydrotestosterone (DHT), epidermal growth factor (EGF), and prolactin (PRL) on prostate cellular proliferation.METHODSThirty benign human prostatic hyperplasias (BPH) were maintained 48 hr as in vitro cultures. Culture media were supplemented with EGF, DHT, and PRL alone and in combinations. Proliferation was assessed by labeling with tritiated thymidine.RESULTSThe proliferative response of individual BPH cultures was heterogeneous. DHT and EGF tended to have a greater proliferative effect than PRL, both in terms of the percent cultures responding and the magnitude of the response. PRL antagonized EGF-induced proliferative effects. EGF- and PRL-mediated effects correlated with each other, while DHT-mediated effects did not correlate with either those of PRL or EGF.CONCLUSIONSThe proliferative response of individual BPH to DHT, EGF, and PRL, alone or in combination, is too variable to define a predictable response to their influence. Our methodology represents a technique with the capacity to define therapeutic potential for individual cases. Prostate 30:47–52, 1997 © 1997 Wiley-Liss, Inc.

While lectins are known to influence the cell growth of several types of normal and neoplastic tissues, their roles in the case of prostatic cancer cells remain relatively unexplored. In the present work, we report thein vitro influence of five lectins, namely peanut (PNA), wheat germ (WGA), Concanavalin A (Con A),Griffonia simplicifolia (GSA-IA4), andPhaseolus vulgaris (PHA-L) agglutinins, on the cell proliferation of one androgen-sensitive (LNCaP) and two androgen-insensitive (PC-3 and DU 145) human prostatic cancer cell lines cultured in either 10% or 1% fetal bovine serum (FBS)-supplemented media. The cell proliferation was assessed by means of the colorimetric 3-(4,5-dimethythiazol-2-yle)2,5-diphenyltetrazolium bromide. (MTT) assay. Four lectin concentrations were tested (i.e., 0.1, 1, 10, and 100 μg/ml) at five experimental states (i.e., 2, 3, 5, 7, and 9 d following the addition of each lectin to the culture media). Our results demonstrated that the five lectins under study had a globally significant dose-dependent toxic effect on prostatic cancer cell proliferation. Nevertheless, low doses of GSA-IA4 and PHA-L significantly (P

Well-differentiated lipomatous tumors constitute a histopathologic category whose nomenclature has been controversial, particularly with respect to the distinction between atypical lipomas of the extremities and well-differentiated liposarcomas of the retroperitoneum. To determine whether there were differences in image analytic parameters between these neoplasms, 72 lesions including 21 typical lipomas, 7 atypical lipomas, 16 retroperitoneal and 5 nonretroperitoneal well-differentiated, 9 dedifferentiated, and 14 pleomorphic liposarcomas were submitted to the computer-assisted microscopic analysis of Feulgen-stained nuclei. This methodology enabled four groups of variables to be calculated. These included: (1) quantitative chromatin pattern description (14 variables); (2) the measurement of proliferative activity (1 variable); (3) nuclear DNA content (DNA ploidy level, 5 variables); and (4) the measurement of cell density and topographical cell nuclei organization (2 variables). The results strongly suggest that atypical lipomas, whether superficial or deep, and well-differentiated liposarcomas, whether retroperitoneal or not, belong to the same category in terms of the variables analyzed.

A computer-assisted microscope analysis of Feulgen-stained nuclei was carried out on a series of 50 nasal polyps in order to try to identify specific biological subgroups. The present series of 50 nasal polyps includes single polyps both associated (n=9) and unassociated (n=9) with allergy and diffuse polyposis both associated (n=7) and unassociated (n=9) with allergy, cystic fibrosis (n=9) and ASA (aspirin-sinusitis-asthma) related polyposis (n=7). The computer-assisted microscope analysis provides 36 quantitative variables which include 1 variable describing proliferative activity, 9 describing the nuclear desoxyribonucleic acid distribution (DNA ploidy level) and 26 describing nucleus morphology, i.e. its size and chromatin pattern. The results show that the methodology proposed here enabled four major groups of nasal polyps to be identified, i.e. diffuse polyposis associated with allergy, cystic fibrosis-related polyposis, single polyps both associated and unassociated either with allergy and a fourth group including diffuse polyposis not associated with allergy and ASA-related polyposis. These four groups of nasal polyps differed markedly in their morphonuclear characteristics, but not in the proliferative activity- and DNA ploidy- related variables.

The aim of the present work is to present the potential uses of a classification technique labeled the "decision tree" for tumor characterisation when faced with a large number of features. The decision tree technique enables multifeature logical classification rules to be produced by determining discriminatory values for each feature selected. In this report, we propose a methodology that used decision trees to compare and evaluate the information contributed by different types of features for tumor characterisation. This methodology is able to produce a set of hypotheses related to a diagnosis and or prognosis problem. For example, hypotheses can be producted (on the basis of a set of descriptive features) to explain why tumor cases belong to a given histopathological group. To illustrate our purpose, this methodology was applied to the difficult problem of leiomyomatous tumour diagnosis. The aim was to illustrate what kind of diagnostic information can be extracted from a sample data set including 23 smooth muscle tumors (14 benign leiomyomas and 9 malignant leiomyosarcomas) described by a large set of computer-assisted, microscope-generated features. Three groups of features were used relating to: (1) ploidy level determination (10 features), (2) quantitative chromatin pattern description (15 features), and (3) immunohistochemically related antigen specificities (6 features). All these features were quantified by digital cell image analysis. The results suggest that an objective distinction between leiomyomas and leiomyosarcomas can be established by means of simple logical rules depending on only a few features among which the immunohistochemically revealed antigen expression of desmin plays a preponderant part. One of the combinations of features proposed by the methodology is interesting for pathologists, because it includes two features describing the appearance of a nucleus in terms of chromatin distribution homogeneity and density, two features widely used by pathologists in tumor-grading systems.

The aim of our study is to characterize the disturbance induced by repeated BCNU treatments in 12 human brain tumor cell lines in terms of their collective behavior. This collective behavior was characterized by means of the Delaunay triangulation and Voronoi mathematical paving techniques combined with the computer-assisted microscope analysis of Feulgen-stained nuclei. This methodology enabled growth to be characterized in terms of cell colony size and density. In addition to this colony pattern characterization, the DNA ploidy level was assessed by means of DNA histogram typing. The cell proliferation level was also determined. Ten astrocytic and two medulloblastoma cell lines treated weekly with BCNU were analyzed. Study of the cell colony architecture and cell proliferation revealed specific BCNU-induced modifications in connection with the origins of the cell lines, i.e. astrocytoma (AST), glioblastoma (GBM), or medulloblastoma (MED). The BCNU-induced effect on GBM (the more malignant of the cell lines) was very different in that proliferation was weakened, but the cell colony density increased after a latency phase. The decrease in cell colony density and cell proliferation of MED seems to indicate that they are more sensitive to BCNU than GBM, but relatively tolerant of this type of chemotherapy in comparison with AST.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) test was employed as a means of studying the cross resistance and collateral sensitivities of two bladder cell lines able to grow in the presence of several antineoplastic drugs and/or despite the effects of radiotherapy. This cross resistance and collateral sensitivities were investigated in the context of two antineoplastic drugs (i.e. doxorubicin (an anthracycline) and vinorelbine (a Vinca alkaloid) and radiotherapy. The results show that cell lines able to grow in the presence of anticancer drugs develop a significant degree of resistance to antineoplastic compounds and may also develop resistance to radiotherapy. On the other hand, most cell lines treated first with radiotherapy develop a significant degree of resistance towards ionizing radiation and may also display increased sensitivity towards anticancer drugs. If these results obtained in vitro are clinically relevant, they may have important applications in the treatment of patients. Nevertheless, it will be necessary to validate the results on extensive series of chemo- and/or radioresistant cell lines exhibiting different mechanisms of resistance.

Neurotensin (NT) and neurotensin receptors (NTRs) are widely found in the brain, NT may be considered as a mitogen factor in some tissues. However, no NT-mediated effects on glioma cell proliferation have been reported so far. In our present study we investigated the influence of NT on the proliferation of astrocytic tumor cell lines. To this end we used a synthetic NT agonist (JMV-449), a protease inhibitor which blocks the natural degradation of NT (JMV-531), and NT. The in vitro biological models used in the present study included the low grade SW1088, and the high grade U87, U373 and A172 astrocytic tumor cell lines. The peptide-induced influence on astrocytic tumor cell proliferation was investigated by means of the colorimetric MTT assay. Our results show that the NT and the NT agonist significantly stimulated the proliferation in 2/4 and 3/4 of the astrocytic cell lines respectively. Similarly, compound JMV-531 also induced an increase in the proliferation of 2/4 of the astrocytic cell lines. This marked influence of the NT and NT agonists, or the enzyme-endogenous prevention of its degradation on the regulation of astrocytic tumor growth therefore suggests that NT antagonists might be used to treat certain patients with high grade astrocytic tumors that do not respond to chemotherapy and/or radiotherapy.

The nuclear DNA content (DNA ploidy) level was determined in a series of 83 human brain metastases, for which 35 complete clinical follow-ups were available. The DNA ploidy level determination was carried out by means of DNA histogram types. The results show that certain brain metastases were diploid, while others exhibited aneuploidy levels ranging from low to very high. The present study also shows that a significant proportion, i.e. 18%, of the 83 brain metastases, exhibited very high levels of aneuploidy, i.e. hypertetraploidy, hyperpentaploidy and octoploidy. We have previously observed that this feature appeared only rarely, i.e. in less than 2% of primary nervous tumours. Furthermore, the present study shows that DNA ploidy level in brain metastases is related significantly (P0.001) to patient survival. Indeed, while 9/13 (69%) patients with diploid brain metastases survived longer than 9 months, none (0%) of the 22 patients with aneuploid brain metastases survived longer than the 9 months following the diagnosis of their brain metastases.

Cell death is an essential event in the functioning of multicellular organisms. It plays a role opposite to that of mitosis in the regulation of cell populations. In the present work, we describe an original methodology which permits the easy detection and count of apoptotic cells in a given tissue. This methodology is based on the digital cell image analysis of Feulgen-stained nuclei, which also permits the calculation of the proliferation index, i.e. the percentage of cells in the S phase of the cell cycle. This percentage of cells in the S phase is strongly related to the mitotic index. Our methodology, which involves the multivariate analysis of 14 morphonuclear parameters computed by means of the digitized cell image analysis of Feulgen-stained nuclei, was applied here to a well-known biological apoptosis model, namely glucocorticoid-treated rat thymocytes. The parameters that permitted the detection of apoptotic cells were the integrated optical density, a parameter that describes the nuclear DNA content, and the run length percentage and long run length parameters which are related to the pattern of chromatin condensation. This determination can be carried out on a relatively small number of cells.

The cell kinetics (percentage of cells in the S+G2 phases of the cell cycle) and the DNA ploidy levels (nuclear DNA content) were determined in 108 samples each of the PC3, DU145, and LNCaP prostate cancer models. This was carried out by means of the digital cell image analysis of Feulgen-stained nuclei. Two to three hundred cell nuclei were analyzed for each of the 324 samples under study. The three cell lines were submitted to experimental conditions including the addition of dihydrotestosterone (DHT), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF), either alone or in combination, to the culture media. The results show that under the present culture conditions, the PC3 cell line was DHT-, EGF- and bFGF-insensitive. In contrast to what is generally reported in the literature, the DU145 cell line was DHT- and EGF-sensitive under the present culture conditions, but bFGF-insensitive. The LNCaP cell line was DHT-sensitive, but EGF- and bFGF-insensitive. While mainly tetraploid, the three cell lines nevertheless exhibited a significant level of heterogeneity in their nuclear DNA content distributions. Indeed, the proportions of non-tetraploid (diploid, hyperdiploid, triploid, hypertriploid, hypertetraploid, polymorphic) DNA histograms were 14% in the PC3, 16% in the DU145, and 29% in the LNCaP cell lines. These results suggest that the DNA ploidy level would not influence the hormone sensitivity level in the cell lines since they had significantly distinct hormone sensitivity profiles while remaining mainly tetraploid.

Cell population sociology was studied in two medulloblastomas and 10 astrocytic human tumor cell lines by means of the characterization of the structure of neoplastic cell colonies growing on histological slides. This was carried out via digital cell image analysis of Feulgen-stained nuclei, to which the Delaunay triangulation and Voronoi paving mathematical techniques were applied. Such assessments were compared to the DNA polidy level (assessed by means of DNA histogram typing). The results show that the cell colony architecture characteristics differed markedly according to whether the cell lines were euploid (diploid or tetraploid) or aneuploid (hyperdiploid, triploid, hypertriploid, or polymorphic). In fact, the cell colonies from the euploid cell nuclei populations were larger and more dense than those from the aneuploid ones. Furthermore, for an identical period of culture, the cell lines from high-grade malignant astrocytic tumors (glioblastomas) exhibited cell colonies that were larger and more dense than those in cell lines from low-grade astrocytic tumors (astrocytomas). In each of these two groups, the diploid cell nuclei populations exhibited cell colonies larger and more dense than the nondiploid colonies. The present methodology is now being applied in vivo to histological sections of surgically removed human brain tumors in order to distinguish between high-risk clinical subgroups and medium-risk subgroups in clearly circumscribed histopathological groups.

Intermediate and high grade subtypes of non-Hodgkin's large cell (LCL) and immunoblastic lymphomas exhibit considerable variability, and histologic morphology alone may not adequately characterize those features important for prognosis. The relationship between nuclear morphology and survival was assessed in a series of 50 cases of large cell lymphomas in which ploidy, proliferation, and nuclear area (NA) were measured. Ploidy was calculated by both DNA index (DI) and DNA histogram type (DHT). Proliferation was calculated from the proportion of S phase (SPF) cells present in the DHT. These four parameters were measured using image cytometry of Feulgen-stained nuclei from fine-needle aspirations. To characterize the relationship with survival, these parameters were associated with the clinical follow-up of the patients. The results show that of the 50 LCL cases, only 5 were clearly aneuploid, whereas the remaining 45 were either diploid (29 cases), tetraploid/hypotetraploid (13 cases), or weakly aneuploid (hyperdiploid, 3 cases). Of the 34 patients who died from their disease, both smaller NA and DI correlated with longer survival in an equivalent fashion; neither conferred greater sensitivity when combined with the other. The SPF did not correlate with survival. In LCL, aneuploidy seems to be a relatively uncommon event, but when present ploidy measurement appears useful to define prognosis.

Numerous data from published reports prove that the proliferation of gastrointestinal tumour cell lines are under the control of many hormones or growth factors, or both. Most of these publications report the influence on a very small number of cell lines of one or two such factors only. This work deals with the in vitro characterisation of the influence of the anti-gastrin, the anti-epidermal growth factor (EGF), the anti-oestradiol (E2), and the anti-luteinising hormone releasing hormone (LHRH) antibodies on the proliferation of a large series of gastrointestinal cell lines. Cell proliferation was assessed by means of the colorimetric MTT assay on a series of 27 gastrointestinal cell lines obtained from the American Type Culture Collection (ATCC). Of the 27 cell lines, the anti-gastrin, the anti-EGF, the anti-E2, and the anti-LHRH neutralising antibodies considerably influenced the proliferation of 13, 25, 12, and 16. No gastrointestinal cell line was unresponsive to the four antibodies simultaneously. The anti-gastrin and anti-EGF antibody induced effects on the 27 gastrointestinal cell line proliferation were significantly correlated, as was also the case for the anti-E2 and anti-LHRH antibody induced effects. Of the anti-gastrin, the anti-EGF, the anti-E2, and the anti-LHRH antibodies, it was the anti-EGF one that had the greatest influence, both quantitatively and qualitatively, on gastrointestinal cell proliferation. The correlation of the effects of definite anti-hormone antibodies is suggestive of a common mechanism of action for the corresponding hormones and casts some doubt on the efficiency of anti-hormone monotherapy.

Background. Identifying a marker enabling prediction of recurrence in the group of superficial transitional cell carcinomas (sTCCs) of the bladder remains an important challenge today. This report quantitatively describes chromatin patterns with respect to such sTCC recurrence. Materials and Methods. Twenty-nine patients with sTCCs who did not exhibit tumor recurrence within a minimum of 24 months were compared with 21 patients with sTCCs who exhibited tumor recurrence two or three times in a 24-month period, for a total of 74 sTCCs. Quantitative chromatin pattern description was performed by the digital cell image analyses of Feulgen-stained nuclei. Six morphonuclear parameters were thus described and subsequently used to determine a score, allowing biological behavior of sTCCs to be described, i.e., recurrence versus non-recurrence in one calculation step. DNA ploidy level was also determined in each sTCC by assessing its DNA histogram type. Results. Of 32 patients with Grade 1 pathologically classified pTa/pT1 tumors, DNA ploidy level determination permitted correct prediction of tumor nonrecurrence or recurrence of 13 (41%), whereas determination of the score values enabled prediction of nonrecurrence or recurrence of 25 (78%). Combining DNA ploidy level data and the score values enabled recurrence or nonrecurrence to be predicted for 29/32 of the patients (91%). Conclusions. The quantitative description of chromatin patterns by digital cell image analysis of Feulgenstained nuclei can provide helpful information, in addition to DNA ploidy level determination, in predicting tumor recurrence of low grade superficial transitional cell carcinomas of the bladder. Cancer 1995;75:560-8.

Purpose. The influence of three alkylating and three intercalating anticancer drugs on cell survival, cell cycle kinetics and chromatin patterns was monitored in vitro on three neoplastic cell lines.

Image cytometry was carried out on 281 superficial (Ta and T1) and 33 invasive (T2 to T4) bladder cancers. The parameters used to characterize these bladder tumors were: (1) histopathological grading, (2) clinical staging, (3) tumor size, (4) deoxyribonucleic acid (DNA) index (DI), (5) DNA histogram type (DHT), (6) percentage of euploid (diploid plus tetraploid) cells, (7) percentage of polyploid cells (> 5C DNA content), (8) proliferative activity (S phase fraction value), and (9) nuclear area (NA). The proliferative activity of the tumors was not related to either histopathological grade or to clinical stage, but it was related to the DHT parameter, which made it possible to identify diploid, hyperdiploid, triploid, hypertriploid, tetraploid, and polymorphic tumors. The hypertriploid tumors exhibited a significantly lower proliferative activity than the nonhypertriploid ones. Although both the DI and the NA values correlated significantly with histopathological grading, only the NA values correlated significantly with clinical staging. We further observed that some grade III bladder tumors were definitely diploid, whereas some grade I tumors were highly aneuploid. We thus hypothesize that the ploidy level of a given tumor reflects its age directly and its aggressiveness only very indirectly. In our opinion aneuploidy is only an indirect marker of aggressiveness because it reflects the fact that a malignant tumor is old, ie, has been present in a patient over a long period of time and has had ample time to express its malignancy at the clinical level. A significant relationship was accordingly obtained between tumor size and ploidy level with the highest proportion of aneuploid tumors and the highest percentage of polyploid cell nuclei being observed among the largest bladder tumors.

This article presents an exceptional clinical manifestation of gout in the wrist. The patient suffered from an association of septic and urate-crystal-induced arthritis. At the time, the serum uric acid concentration was still normal. The association of septic and urate-crystal-induced arthritis has been reported in other locations. The pathophysiology of the precipitation of urate crystals in the presence of an infectious process is discussed.

The chromatin pattern in Feulgen-stained nuclei from soft tissue tumors was quantitatively described by means of computer-assisted microscope analysis. The morphonuclear parameters described densitometric, run length, and co-occurrence matrix features. The present series of cases, which relied upon archival (ie, formalin-fixed, paraffin-embedded), tissues, included 19 benign (9 lipomas and 10 leiomyomas) cases, and 49 malignant (31 primitive non-recurrent, 14 primitive locally recurrent, and 4 metastatic) cases. The 31 primitive nonrecurrent cases included 12 liposarcomas, 11 leiomyosarcomas, 4 rhabdomyosarcomas, and 4 malignant fibrohistiocytomas. The results show that the quantitative description of chromatin patterns in Feulgen-stained nuclei made it possible to distinguish between certain benign and malignant soft tissue tumors. However, this was true only when specific histopathologic groups were taken into consideration. Indeed, the lipomas were markedly different from the liposarcomas, whereas the leiomyomas closely resembled the leiomyosarcomas. The quantitative description of the chromatin patterns also made it possible to identify certain clinically aggressive soft tissue tumors. In this context, the authors observed that the chromatin pattern in the cell nuclei from the group of patients whose tumors had recurred less than 10 months after first surgery was significantly more heterogeneous and less condensed than in the cell nuclei from patients whose tumors had recurred more than 10 months after this surgery. In the same manner, the morphonuclear parameters under study made it possible to establish a more marked distinction between the primitive and recurrent soft tissue tumors that developed a metastasis between 3 and 48 months after the diagnosis, and those tumors free of metastasis until 38 months after the diagnosis. The former group exhibited cell nuclei with a chromatin pattern markedly more condensed and heterogeneous than in the case of the cell nuclei belonging to the latter group.

The chromatin patterns of Feulgen-stained nuclei in a series of six specimens of normal mucosa and 331 transitional bladder carcinomas, including 293 superficial (Ta and T1) and 38 invasive (T2-T4) cases, were quantitatively described by means of eight parameters relating to densitometric, run-length distribution, and co-occurrence matrix features. The results show that the chromatin texture of the superficial lesions was markedly different from that of the invasive tumours, which exhibited a distinctly more dense and heterogeneous chromatin pattern. The data also show that the increasing level of malignancy, as revealed by the increasing clinical stage, was accompanied by an increase in the overall chromatin condensation level. Only some areas of the nucleus actually increased in density; other pale areas appeared concomitantly with these increasingly denser chromatin areas. This chromatin density increase corresponded to a marked increase in the frequency of small dense chromatin clumps; these joined together into very large dense chromatin clumps, which were distributed more and more heterogeneously in the nucleus as the clinical stage of the tumour increased.

The concept of differentiation of prostate cancer in terms of morphonuclear characteristics and population dynamics was investigated on the PC-3 and DU145 cell lines. A software based on the concept of Voronoi paving was set up in order to characterize the structure of these cell lines growing in vitro on histological slides. The morphonuclear characteristics were assessed by means of the digital cell image analyses of Feulgen-stained nuclei. The in vitro "morphonuclear" and "pseudo-tissular" differentiations of the PC-3 and DU145 cells were described in terms of the use of various culture media, i.e., media supplemented with either 10% (F10 medium) or 1% (F1 medium) fetal calf serum and with (or without) platelet-derived growth factor and dihydrotestosterone (PA10 and PA1 media). The present data reveal that the PC-3 cell line would be more hormone-sensitive than the DU145 one. Indeed, decreasing the FCS concentration in the culture medium while adding DHT and PDGF led to marked modifications to the morphonuclear characteristics of the PC-3 cells, but not to the DU145 cells. These modifications corresponded to an increase in nuclear size occurring concomitantly with chromatin decondensation. In the same way, spectacular modifications in terms of medium-induced pseudo-tissular differentiation were observed in the PC-3 cell line, but not in the DU145 one. Such modifications corresponded to an increase in clone size related to an increase in the mean distances between neighboring cell nuclei in a given clone. Thus, according to the criteria defined in this study, the PC-3 cell line would seem to maintain a higher degree of differentiation than the DU145 cell line.

The influence of five anti-hormone and/or anti-growth factor neutralizing antibodies on the in vitro proliferation of four human astrocytic tumor cell lines (U87, U138, U373, H4) is quantitatively described by means of a new tool which makes it possible to evaluate cell growth and cell clone architecture concomitantly. This tool relies upon the combined use of the digital cell image analyses of Feulgen-stained nuclei and the Delaunay and Voronoi mathematical triangulation and paving techniques. Of the five anti-hormone and/or anti-growth factors tested here, the anti-luteinizing hormone-releasing hormone (LHRH) antibody induced the most marked perturbation in the U138 and U373 cell lines, whereas this role was played by the anti-epidermal growth factor (EGF) antibody in the U87 and H4 cell lines. The anti-gastrin (G) antibody significantly modified the growth and/or cell clone architecture of the U138, U87 and H4 cell lines, as did the anti-transforming growth factor alpha (TGFalpha) antibody. The anti-transforming growth factor beta (TGFbeta) antibody modified the growth and/or cell clone architecture of the four cell lines under study. If the five antibodies are taken into consideration, the results strongly suggest that four (the anti-G, the anti-EGF, the anti-LHRH and the anti-TGFalpha) act as inhibitory agents on some glioma cell line proliferation, while the fifth one, i.e. the anti-TGFbeta, act as a stimulator of cell proliferation, perhaps by abrogating the inhibitory effects of TGFbeta on proliferation. A comparison of cell growth data with cell clone architecture characteristics provided further evidence of some specific influence exercised by a given hormone and/or growth factor on glioma cell proliferation. Indeed, the anti-LHRH antibody caused the most pronounced perturbations in the U138 and U373 cell clone architecture; this feature was observed in the H4 cell line and, to a lesser extent in the U87 one after the anti-EGF antibody had been used.

The influence of dihydrotestosterone (DHT), the epithelial growth factor (EGF) and the basic fibroblast growth factor (bFGF) was investigated on LNCaP, DU145 and PC3 cell growth, which represents the ratio between cell gain (cell proliferation) and cell loss (cell death). In the present study, cell growth was assessed by means of the computer-assisted microscope analysis of Feulgen-stained nuclei combined with the mathematical Delaunay triangulation and Voronoi paving techniques, which enabled the cell colony patterns, i.e. their density and level of organisation, to be determined. The results from a previous study (Janssen et al, Prostate, in press) combined with those of the present one show that DHT was found to activate proliferation of the LNCaP model, as evidenced by increase in size of colonies, increase in number of cells within colonies, increase in cell colony density and, accordingly, decrease in mean segment length value (which is the distance between adjacent cell nuclei). Using the same criteria, DHT was found inhibitory on growth of DU145 cell line, and devoid of significant effect on PC3 cell line. Basic FGF was found to be a powerful stimulator of growth of PC3 cell Line and to induce a weaker stimulation of DU145 cell line. On LNCaP cell line, it increased the size of colonies without increase of the number of cells per colony. This feature can be explained by a decrease in cell colony density. With respect to the same colonies, the proliferation index (percentage of cells in the S+G2 phases of the cell cycle) was found similar to that of the controls. This suggests that the increase in the size of the colonies is due to a difference of spreading of the cells on their supports. EGF had no significant effect on LNCaP and PC3 models, and was decreasing cell density of DU145 colonies.

A new tool is described which makes it possible to evaluate directly the influence of various growth factors on in vitro neoplastic cell growth on the one hand and to look at a concept of differentiation in terms of population dynamics, on the other. This tool relies upon the digital cell image analyses of Feulgen-stained nuclei and the mathematical method of Voronoi paving. This technique enabled us to characterize the influence on the proliferation and the differentiation of the HCT-15 and LoVo colorectal cell lines of anti-gastrin (G), anti-estradiol (E(2)), anti-epidermal growth factor (EGF), anti-luteinizing hormone-releasing hormone (LHRH), and anti-transforming growth factor alpha (TGF alpha) and beta (TGF beta) antibodies. Two variants were set up with respect to each of the two cell lines, i.e, one growing in culture medium supplemented with 5% fetal calf serum (FCS) and another supplemented with 1% FCS+10 nM G+10 nM E(2). The data show that it is possible to characterize the cell clone structure and to assess growth rate concomitantly by direct cell counts. It further appears that while the anti-hormone and/or anti-growth factor antibody-induced effects on growth were relatively similar, these effects were in sharp contrast at the level of cell clone architecture.

In this paper we study the feasibility of measuring the diverse biological parameters in stereotaxic biopsies from human brain lesions. These biological parameters are the nuclear area (NA), the proliferation index (PI) and the ploidy level, the latter of which was evaluated by means of the DNA index (DI) and histogram type (DHT). These parameters were assessed by means of the digital cell image analysis of Feulgen-stained nuclei. This analysis was performed on 124 samples from 22 computed tomography (CT)-guided stereotaxic biopsies. The data show that the methodology used here enables the above parameters to be assessed on small samples without limiting the classical anatomopathological diagnosis. The data also reveal that the DHT corresponded more accurately to the ploidy level in the sample analysed than the DI. Lastly, it appears that supratentorial astrocytic tumours of the adult, which constituted the majority of the cases analysed here, are strongly heterogeneous at a biological level.

The diagnostic value of ploidy level was investigated in a series of 707 tumors (from 707 patients) of the central and peripheral nervous system. The series contained 91 nerve sheath tumors (NSTs), 222 meningiomas (MNGs), 37 primitive neuroectodermal tumors (PNETs), 293 glial tumors of the adult (ATTs), and 64 brain metastases (MTTs). The ploidy level of each tumor was obtained by means of its DNA histogram type (DHT), which was computed (digital cell image analysis) on Feulgen-stained nuclei from archival formalin-fixed paraffin-embedded materials. The data reveal that the measurement of the ploidy level showed a diagnostic value in some of these tumor cases. Indeed, the proportion of highly aneuploid cases significantly (p0.05-0.01) decreased according to the sequence PNETs (72%)--MTTs (64%)--ATTs (40%)--NSTs (23%)--MNGs (9%). Nevertheless, this significant diagnostic value in terms of a distinction between benignity and malignancy only appears when all the tumors are taken into consideration. Indeed, at the level of one individual patient, our study shows that, like traumatic neuromas, schwannomas, or classical meningiomas, completely benign tumors can exhibit a high aneuploidy level. In contrast, some tumors from histopathological groups like brain metastases or PNETs, which are known to be clinically aggressive, could be completely diploid.

This paper describes differentiation in terms of population dynamics through the medium of Voronoi paving which enables (via digital cell image analysis) the structure of human LOVO and HCT-15 colorectal neoplastic cell colonies growing on histological slides to be characterized. Two other tests were also used, i.e., the colorimetric MTT assay that enables the cell growth level to be determined, and a test allowing the assessment of the proliferation index, i.e., the percentage of cells in the S phase of the cell cycle. The results show that these colorectal neoplastic cells exhibited a comparatively high level of organisation in terms of the topographical distribution of nuclei within the clones when the cells were cultivated in media containing even small amounts of fetal calf serum. On the other hand, certain chemically defined media completely overturned this “pseudo-tissular” architecture. Furthermore, the colorectal cells growing in media including fetal calf serum exhibited relatively large and dense clones, undergoing an increase in the density of these clones when hormones were added to the culture medium and, concomitantly, a decrease in their proliferation. In contrast, the cells growing in chemically defined media generally exhibited smaller clones whose cell proliferation was paradoxically greater than that of the cells referred to above. This seems to bring out the importance of the part played by the cell loss factor in this cell population dynamic. © 1993 Wiley-Liss, Inc.

Purpose : We describe by means of digital cell image analysis the influence of X-ray radiation on three in vitro cultured cell lines for which we set up chemosensitive and chemoresistant variants. Methods and Materials : The three cell lines correspond to the MXT mouse mammary and the T24 and J82 neoplastic human bladder cells. The digital cell image analysis was carried out by computing morphometric (nuclear size), densitometric (proportion of cells in the G2 cell cycle phase), and textural features (chromatin pattern characteristics) on Feulgen-stained nuclei. Results : The results show that such digital cell image analyses make it possible to monitor radiotherapy-induced effects on these morphonuclear characteristics accurately. X-ray radiotherapy induces a dose-dependent increase in the proportion of cells in the G2 phase of the cell cycle along with a decrease in the overall chromatin condensation level. These two concomitant phenomena lead to a marked radiotherapy-induced increase in nuclear size. We also obseved that radiotherapy-induced effects at the morphonuclear level are not only highly specific to the cell type analyzed, that is MXT mouse mammary or J82 or T24 human bladder carcinoma cells, but also to the fact that the cells are either chemosensitive or chemoresistant. Conclusion : The digital cell image analyses of Feulgen-stained nuclei is helpful in monitoring the irradiation-induced morphonuclear modifications.

We investigated the parameters that could affect the cytophotometric analysis of cell nuclei stained by the Feulgen reaction. These parameters included: the hydrolysis temperature (in the normal "room temperature" range); the composition of the Schiff's reagent; the speed of centrifugation of the cell suspensions; the mode of preservation [air-drying or ethanol-formalin-acetic acid (EFA) fixation]; the fixation time; the pronase digestion time; and the concentration of pronase used to obtain cell suspensions from archival (formalin-fixed, paraffin-embedded) materials. Relatively homogeneous material was studied: the MXT mouse mammary adenocarcinoma growing in vivo as tumors with both small and hyperchromatic cell nuclei and in vitro as monolayers with larger and less hyperchromatic cell nuclei. The results of these investigations demonstrate the necessity for the precise definition of a protocol for such procedures as sampling, fixation, and staining of cell nuclei if computerized cell image analyses are to be objective and reproducible. For present purposes this protocol differs depending on whether fresh or archival material is studied. For fresh tissue the protocol is immersion of the sample in EFA within 10 sec, fixation for 30 min, and staining by the Feulgen reaction in which hydrolysis is performed with 6 N HCl at 24 degrees C for 60 min. For archival tissue, the protocol becomes fixation in formol (or EFA), embedding, sectioning at 80 microns, digestion with 0.05% pronase for 2 hr, centrifugation at 1200 x g on glass slides, and staining by the Feulgen reaction in which hydrolysis is performed with 6 N HCl for 60 min at 24 degrees C.

The uptake of hypericin from the foliage of Hypericum perforatum by four species of adult chrysomelid beetles, Chrysolina brunsvicensis, C. geminata, C. hyperici and C. varians, was investigated under laboratory conditions. In no species were detectable levels of hypericin passed from the larval to the pupal or adult stages. The total amount of hypericin found in the adult beetles was positively related to the rate of ingestion of foliage. When feeding activity lessened or ceased, body levels of hypericin declined. In adults of C. brunsvicensis, C. hyperici and C. varians maximal body levels of hypericin occurred after 1–10 days of feeding. In C. brunsvicensis a maximal average level of c. 0.96 μg/beetle was obtained. After 20 days body levels in C. brunsvicensis declined to nearly zero. Adults of C. brunsvicensis contained more hypericin than the other species of beetles, but in no case was enough hypericin sequestered to match the original values (c. 13.3 μg/beetle) published by Rees (1969). Our results caste doubt on the role of hypericin as a sequestered defence against predators. Analysis of body distribution of hypericin indicated the majority was in the gut lumen (80%). An input-output analysis of hypericin ingested by adults of C. brunsvicensis showed that the greatest majority of hypericin was eliminated in the faeces (up to 85%). Between 15% and 35% of the ingested hypericin was unaccounted for. This loss is the result of a post-defecational degradation of hypericin (perhaps bacterial).

A 15-year-old patient with severe bone disease (with bilateral fractures of hips and shoulders) due to primary hyperoxaluria type 1 (PH1) was treated with combined liver-kidney transplantation after a 4-year hemodialysis period. Normalization of excessive oxalate synthesis brought in by the liver graft combined with the slow excretion of skeletal oxalate stores by the renal graft led to progressive improvement of clinical, radiological, and histological evidence of oxalate osteopathy. This allowed bilateral hip replacement 3 years after transplantation, which led to complete physical rehabilitation of the crippled patient. Combined liver-kidney transplantation constitutes the treatment of choice for end-stage renal failure due to PH1, even in the face of severe oxalate bone disease.

We report the morphonuclear characteristics of normal (13 cases), benign (ie, chronic) pancreatitis (six cases), and neoplastic (ie, ductal) adenocarcinoma (22 cases) tissues of the pancreas. This description is based on computerized cell image analysis, which permits the determination of parameters related to the morphometric (nuclear area), densitometric (nuclear DNA content), and chromatin texture features of Feulgen-stained nuclei from paraffin-embedded archival material. We observed that nuclear area discriminates between normal and benign (ie, chronic pancreatitis) as opposed to neoplastic cell nuclei. Morphonuclear parameters describing chromatin pattern characteristics made it possible to discriminate between grade I pancreatic carcinoma and normal and benign cell nuclei on the one hand, and grades I and III carcinoma on the other hand. The nuclear DNA content increased in a continuous manner from normal and benign through low-grade to high-grade neoplastic tissues of the pancreas. Combining the morphometric, densitometric, and textural parameters into one equation, we were able to calculate a score (ie, the malignancy level index) that showed a close relationship to conventional histopathologic grading. Thus, the computer-aided diagnosis of cytologic specimens from pancreatic lesions offers information of the same significance as that obtained by conventional histopathologic grading.

BACKGROUND: The authors studied the benefit of performing histopathologic grading and DNA ploidy characterization with respect to patient survival in a series of 206 astrocytomas (AST) for which they obtained 134 complete clinical follow-ups. METHODS: The material analyzed came from archival material, i.e., formalin-fixed paraffin-embedded tissues. DNA ploidy was assessed by means of a cell image processor computing the integrated optical density (IOD) on Feulgen-stained nuclei. RESULTS: Results showed that histopathologic diagnosis in three grades, i.e., AST, anaplastic astrocytoma (ANA), and glioblastoma multiforme (GBM), had a significant prognostic value. Patients with AST showed a mean survival time (between histopathologic diagnosis and death) of more than 36 +/- 6 months (AST versus ANA or GBM) (P less than 0.001). Patients with ANA and GBM showed a mean survival time of 15 +/- 2 and 10 +/- 1 months, respectively, (ANA versus GBM) (P less than 0.05). Patient age strongly correlated with survival. Patients younger than 40 years of age had a mean survival time of 20 +/- 4 months. Patients between 41 and 60 years of age had a mean survival time of 12 +/- 2 months, and patients older than 60 years of age had a mean survival time of 11 +/- 1 months. CONCLUSIONS: Considering DNA ploidy characterization, the authors noticed that aneuploid ANA (DNA index [DI] more than 1.30) were associated with a significantly higher mean patient survival time compared with that associated with euploid ANA. In contrast, the authors did not find this in either of the groups with AST and GBM. Recognizing six DNA histogram types (diploid, triploid, tetraploid, hyperdiploid, hypertriploid, and polymorphic), the authors observed that hypertriploid tumors were associated with greater patient survival compared with what happened in the cases of the five other DNA histogram types. This was true with respect to the three AST histopathologic types. Thus, DNA ploidy determination seemed helpful in characterizing aggressiveness in adult AST.

We set up in vitro several human colorectal neoplastic cell lines that we labelled "hormone-sensitive" (HS) in comparison to the original cell lines which appeared to be rather "hormone-insensitive" (HI). We used LoVo and HCT-15 human colorectal neoplastic cell lines and studied the influence of 17 beta-oestradiol (E2), gastrin and two gonadotropin-releasing hormone (GnRH) analogues, HRF and buserelin, on the proliferation of the HS and HI variants of the LoVo and HCT-15 cell lines. Cell proliferation was evaluated by a colorimetric assay, the MTT test. Our results show that E2, gastrin, HRF and buserelin did not induce a significant stimulatory influence on the HI variants of the LoVo and HCT-15 cells, i.e. the cells that were cultured in a hormone-free 10% FCS-supplemented medium. In sharp contrast, the colorectal cells cultured for 30 passages in an E2 and/or gastrin + 1% FCS-supplemented medium showed a marked tropic response to E2, gastrin, HRF and buserelin. However, the HS variants of the HCT-15 cells appeared less sensitive to the two GnRH analogues than did the HS variants of the LoVo cells.

Sixty-three sections of Feulgen-stained thyroid cell nuclei from paraffin-embedded material, including five multinodular goiters, 10 adenomas, 36 papillary carcinomas, seven follicular carcinomas, and five medullary carcinomas were analyzed by means of the SAMBA 200 (TITN, Grenoble, France) cell image processor. This was done in order to obtain nuclear characteristics of papillary versus follicular carcinomas. The nuclear features were assessed by morphometric, densitometric, and textural parameters. Our preliminary results indicate that the cell nuclei from typical histopathologic specimens of follicular thyroid cancers belong to a larger thyroid cell nuclei population corresponding to the histopathologic family of papillary thyroid cancers. This follicular neoplastic cell nuclei population appears to be quite distinct from the typical medullary neoplastic cell nuclei population which also belongs to the papillary neoplastic cell nuclei population. It appears that there is a specific papillary cell nuclei subpopulation containing typical hypochromatic cell nuclei. We also observed a dramatic increase in nuclear size and hyperchromatism between normal (multinodular goiters) and neoplastic (carcinomas) thyroid tissues, with the benign tissues (adenomas) showing intermediate nuclear characteristics.