Reliability and validity of a histologic score as a marker for skin cancer chemoprevention studies

OBJECTIVE: To develop a reliable and valid scoring system for grading skin biopsies from actinic keratosis (AK) and sun-damaged skin for use in evaluating the efficacy of skin cancer chemopreventive agents. STUDY DESIGN: A panel of dermatopathologists developed histologic criteria and diagnostic definitions for the progression of lesions from early AK to AK. The criteria were then applied to a sample of 335 histologic slides from an ongoing chemoprevention study. A 10% sample of 35 slides was reread in order to assess intrarater reliability. RESULTS: Six of the 7 criteria demonstrated high reliability (> 85%). The total histologic score, calculated using the 6 criteria, was found to significantly differentiate between (blinded) biopsy location (normal, pre-AK, AK and adjacent to squamous cell carcinoma) and histologic diagnosis (normal, pre- or early AK, AK and squamous cell carcinoma). CONCLUSION: The total histologic score, having demonstrated reliability on repeated readings and validity in its association with biopsy location and histologic score, is a reliable and valid end point for judging the efficacy of agents in skin cancer chemoprevention studies. Additional interrater reliability tests utilizing larger test sets and a rigorous statistical design should be undertaken to establish its portability.     

Rates of protein synthesis in explanted embryonic chick lens epithelia: differential stimulation of delta-crystallin synthesis.

Cells in the central region of 6-day-old embryonic chick lens epithelia display morphological and biochemical changes, when cultured in medium supplemented with fetal calf serum, comparable to those of lens fiber cells differentiating in vivo. In the present study the rates of synthesis of total protein and of δ-crystallin were quantitated during the first day of culture by measuring (1) ³H-valine incorporation into bulk proteins and into δ-crystallin (isolated by quantitative immunoprecipitation), (2) the specific radioactivity of picomolar amounts of intracellular valine (determined by analysis of the ¹⁴C-dansyl-derivative of ³H-valine), (3) the amount of protein degradation occurring during the labeling period (estimated by “pulse-chase” experiments with cycloheximide), and (4) the number of cells in the explants (counted following dispersal with trypsin-EDTA). The results showed that total protein synthesis increased 1.7-fold per cell during the first 24 hrs in vitro. In contrast, δ-crystallin synthesis increased 2.8-fold per cell during this time. These experiments establish that δ-crystallin synthesis is differentially stimulated in epithelia cultured in serum-supplemented medium, and provide the basis for quantitative analysis of the mechanism controlling differential protein synthesis during lens fiber differentiation in vitro.     

Delta-crystallin mRNA in chick lens cells: mRNA accumulates during differential stimulation of delta-crystallin synthesis in cultured cells.

Increasing specialization for δ-crystallin synthesis is a prominent feature of the differentiation of chick lens epithelial cells into lens fiber cells and can be studied in cultured embryonic lens epithelia. Quantitation of δ-crystallin mRNA by molecular hybridizaton to a [³H]DNA complementary to δ-crystallin mRNA demonstrates that differentiation, both in ovo and in tissue culture, is associated with the accumulation of δ-crystallin mRNA. In the cultures, there is an overall stimulation of protein synthesis, including δ-crystallin mRNA during the first 5 hr in vitro. Between 5 and 24 hr in vitro there is a differential stimulation of δ-crystallin synthesis and an accumulation of δ-crystallin mRNA that can quantitatively account for this stimulation.     

Aberrations of chick eyes during normal growth and lens induction of myopia

Understanding the control of eye growth may lead to the prevention of nearsightedness (myopia). Chicks develop refractive errors in response to defocusing lenses by changing the rate of eye elongation. Changes in optical image quality and the optical signal in lens compensation are not understood. Monochromatic ocular aberrations were measured in 16 chicks that unilaterally developed myopia in response to unilateral goggles with −15D lenses and in 6 chicks developing naturally. There is no significant difference in higher-order root mean square aberrations (RMSA) between control eyes of the goggled birds and eyes of naturally developing chicks. Higher-order RMSA for a constant pupil size exponentially decreases in the chick eye with age more slowly than defocus. In the presence of a defocusing lens, the exponential decrease begins after day 2. In goggled eyes, asymmetric aberrations initially increase significantly, followed by an exponential decrease. Higher-order RMSA is significantly higher in goggled eyes than in controls. Equivalent blur, a new measure of image quality that accounts for increasing pupil size with age, exponentially decreases with age. In goggled eyes, this decrease also occurs after day 2. The fine optical structure, reflected in higher-order aberrations, may be important in understanding normal development and the development of myopia.     

Ageing in the chick lens: in vitro studies.

In principle, ageing may be due to the interaction of several factors, including the accumulation of random changes both genomic and non-genomic, secondary changes in a tissue contingent upon the changing function of other tissues, and programmed non-random changes in the tissue-specific expression of various genes. The use of a single tissue comprising one cell type only, in which the major gene products are well defined, in which there is a well attested series of developmental and age-related changes in cell properties and gene expression and which can be studied and compared in vivo and in vitro, offers advantages for investigation of these questions. The vertebrate eye lens possesses these advantages. The crystallins (proteins expressed at super-abundant levels in the lens) are well characterised. The lens epithelial cells (LEC) grow readily and can differentiate into the lens fibre cells in vitro, and, finally, such terminally differentiated cells may also be derived, by a process of transdifferentiation, from neural retina cells (NRC) in vitro. Thus the effect on ageing changes of the tissue of origin may also be studied. This article reviews our previous studies on long-term changes in growth potential, differentiation capacity and crystallin expression of chick lens cells in ageing cultures, their overall similarity to events in vivo and the effect on ageing changes of genotypes affecting the growth rate. It presents new information on these genetic aspects, and on crystallin expression in long-term ageing cultures of transdifferentiated neural retina, and compares the behaviour of ageing chick lens cells with that reported for mammals.     

Evaluation of strontium as a trace-element marker for dispersal studies on Heliothis armigera

A method of preparing moth specimens for strontium (Sr) analysis by atomic absorption spectrophotometry is described. A screenhouse experiment demonstrated that foliar sprays of SrCl₂ at 5 kg/ha successfully marked late instar Heliothis armigera larvae feeding on chickpea. Laboratory experiments, in which larvae were reared from the 4th instar on artificial diet containing, concentrations of SrCl₂ up to 5000 ppm,showed no adverse effects on development and even at 50 ppm moths were unequivocally marked above the highest recorded background level. Sr level did not decline appreciably after the second day of adult life, unlike rubidium which declined at a faster rate. Female moths had consistently higher Sr levels than males. Following a single field application of SrCl₂ at 5 kg/ha on pigeonpea heavily infested with H. armigera larvae, 61.8% of emergent moths were marked.

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Le strontium comme marqueur pour l'étude de la dispersion d' Heliothis armigera

Résumé La méthode de préparation des papillons pour l'analyse du strontium (Sr) en spectrophotométrie par absorption atomique est décrite. Une expérience en serre a montré que la pulvérisation des feuilles avec SrCl₂ à raison de 5 kg/ha a permis de marquer le dernier stade des chenilles de H. armigera sur Cicer arietinum. En laboratoire, des chenilles ont été élevées à partir du quatrième stade sur un régime artificiel avec des concentrations de SrCl₂ allant jusqu'à 5000 ppm: aucun effet sur la biologie n'a été observe. A 50 ppm le marquage des pappilons exclut déjà tout risque de confusion avec le bruit du fond. Le taux de Sr ne diminue pas considérablement apres le second jour de vie imaginale, contrairement au rubidium qui diminue à une plus grande vitesse. La teneur en Sr des femelles est significativement plus élevée que celle des mâles. Après un traitement unique de SrCl₂ dans la nature, à raison de 5 kg/ha, sur des C. arietinum trés attaqués par des chenilles de H. armigera, 61.8% des papillons obtenus étaient marqués.

     

Cytochemical reaction for cationic proteins as a marker of primary granules during development in chick heterophilis.

The ammoniacal silver reaction (ASR) for cationic proteins was used as a cytochemical marker for the primary or A granules in the cytoplasm of developing heterophils of chick bone marrow. The presence of the electron-dense particulate reaction product of silver, which is localized in the fully formed rod-shaped A granules, provides a marker by which the A granules could be distinguished from the B granules of similar size and by which the formation and maturation of both granule types could be followed through the developmental stages. Progressive developmental stages were ascertained on the basis of decreasing cell size, increasing condensation and margination of the chromatin, and the number and morphology of the granules; the stages were divided into promyelocyte, myelocyte, metamyelocyte and heterophil. During the promyelocyte stage, the first appearance of the electron-dense, membrane-bound, spherical granules (0.3--1.0 micrometer in diameter) is observed in the vicinity of an extensive Golgi complex. They occur in a cytoplasm containing rough-surfaced endoplasmic reticulum, ribosomal clusters, centrioles, mitochondria, microtubules, as well as the membranes, saccules, vesicles and vacuoles of the Golgi complex. These granules are considered as primary but their presence as the only granule type appears very brief. The ASR reaction product is first detected on the surface of these primary granules in late promyelocytes or myelocytes. The secondary or B granule, devoid of reaction for cationic protein at all stages, appears as a condensing vacuole in promyelocytes, but after some A granules are already present. The vacuole contents condense to form the B granules which are 0.1--0.6 micrometer in diameter, often oval-shaped, and contain a loose filamentous material surrounded by a membrane. Tertiary C granules or lysosomes appear during the myelocyte stage as dense core vesicles (0.1--0.2 micrometer in diameter) negative for cationic protein.     

Biochemical and immunological studies of lentoid formation in cultures of embryonic chick neural retina and day-old chick lens epithelium

During long-term cell culture of 8-day embryonic chick neural retina, lentoid bodies containing lens crystallins are developed. Although very low levels of crystallin can be detected in the embryonic neural retina, gross synthesis of each major crystallin class (α, anodal β, cathodal β, and δ) begins only after 12–16 days in culture. This occurs at least 10 days before lentoid bodies can be distinguished by eye. The concentration of each crystallin class was determined during lentoid development in cultures of both neural retina and lens epithelium. The proportions of crystallins in lentoid-containing cultures do not resemble those of embryonic lens fibres. Comparisons between two chick strains (N and Hy-1) differing in their growth rates revealed several differences in the crystallin compositions of lentoid bodies. These differences imply independent quantitative regulation for most or all of the crystallins.