Optimising adjacent membrane segmentation and parameterisation in multicellular aggregates by piecewise active contours.

In fluorescence microscopy imaging, the segmentation of adjacent cell membranes within cell aggregates, multicellular samples, tissue, organs, or whole organisms remains a challenging task. The lipid bilayer is a very thin membrane when compared to the wavelength of photons in the visual spectra. Fluorescent molecules or proteins used for labelling membranes provide a limited signal intensity, and light scattering in combination with sample dynamics during in vivo imaging lead to poor or ambivalent signal patterns that hinder precise localisation of the membrane sheets. In the proximity of cells, membranes approach and distance each other. Here, the presence of membrane protrusions such as blebs; filopodia and lamellipodia; microvilli; or membrane vesicle trafficking, lead to a plurality of signal patterns, and the accurate localisation of two adjacent membranes becomes difficult. Several computational methods for membrane segmentation have been introduced. However, few of them specifically consider the accurate detection of adjacent membranes. In this article we present ALPACA (ALgorithm for Piecewise Adjacent Contour Adjustment), a novel method based on 2D piecewise parametric active contours that allows: (i) a definition of proximity for adjacent contours, (ii) a precise detection of adjacent, nonadjacent, and overlapping contour sections, (iii) the definition of a polyline for an optimised shared contour within adjacent sections and (iv) a solution for connecting adjacent and nonadjacent sections under the constraint of preserving the inherent cell morphology. We show that ALPACA leads to a precise quantification of adjacent and nonadjacent membrane zones in regular hexagons and live image sequences of cells of the parapineal organ during zebrafish embryo development. The algorithm detects and corrects adjacent, nonadjacent, and overlapping contour sections within a selected adjacency distance d, calculates shared contour sections for neighbouring cells with minimum alterations of the contour characteristics, and presents piecewise active contour solutions, preserving the contour shape and the overall cell morphology. ALPACA quantifies adjacent contours and can improve the meshing of 3D surfaces, the determination of forces, or tracking of contours in combination with previously published algorithms. We discuss pitfalls, strengths, and limits of our approach, and present a guideline to take the best decision for varying experimental conditions for in vivo microscopy.

Prion Function and Pathophysiology in Non-Mammalian Models.

More than thirty years have passed since the discovery of the prion protein (PrP) and its causative role in transmissible spongiform encephalopathy. Since a combination of both gain- and loss-of-function mechanisms may underlay prion pathogenesis, understanding the physiological role of PrP may give important clues about disease mechanisms. Historically, the primary strategy for prion research has involved the use of human tissue, cell cultures and mammalian animal models. Nevertheless, experimental difficulties of in vivo studies and controversial observations obtained in these systems have stimulated the search for alternative animal models. PrPC is highly conserved in mammals, and PrPC-related orthologs are expressed in zebrafish, a vertebrate model organism suitable to study the mechanisms associated with human diseases. Invertebrate models, as they do not express PrPC have served to investigate the neurotoxic mechanisms of mammalian PrP. Here we overview most recent advances in the study of PrP function in normal and pathogenic conditions based on non-mammalian studies, highlighting the contribution of zebrafish, fly and worms to our current understanding of PrP biology.

Computational methods for analysis of dynamic events in cell migration.

Cell migration is a complex biological process that involves changes in shape and organization at the sub-cellular, cellular, and supra-cellular levels. Individual and collective cell migration can be assessed in vitro and in vivo starting from the flagellar driven movement of single sperm cells or bacteria, bacterial gliding and swarming, and amoeboid movement to the orchestrated movement of collective cell migration. One key technology to access migration phenomena is the combination of optical microscopy with image processing algorithms. This approach resolves simple motion estimation (e.g. preferred direction of migrating cells or path characteristics), but can also reveal more complex descriptors (e.g. protrusions or cellular deformations). In order to ensure an accurate quantification, the phenomena under study, their complexity, and the required level of description need to be addressed by an adequate experimental setup and processing pipeline. Here, we review typical workflows for processing starting with image acquisition, restoration (noise and artifact removal, signal enhancement), registration, analysis (object detection, segmentation and characterization) and interpretation (high level understanding). Image processing approaches for quantitative description of cell migration in 2- and 3-dimensional image series, including registration, segmentation, shape and topology description, tracking and motion fields are presented. We discuss advantages, limitations and suitability for different approaches and levels of description.

Células de Langerhans CD1a+ en la epidermis peritumoral del carcinoma basocelular / CD1a+ Langerhans Cells in the Peritumoral Epidermis of Basal Cell Carcinoma

Introducción. El carcinoma basocelular (CBC) es un tumor maligno frecuente y su incidencia ha aumentado en las últimas décadas. Investigaciones han demostrado la relación entre radiación ultravioleta (RUV), sistema inmune cutáneo y CBC. La función de las células de Langerhans (CL) en la respuesta inmune antitumoral ha motivado la investigación de su densidad y morfología frente a la RUV y al CBC. Sin embargo, los resultados publicados presentan discordancias debido a diferencias en la metodología científica. Objetivo. Estudiar la densidad y morfología de las CL en la epidermis peritumoral comparada con la epidermis supratumoral mediante inmunohistoquímica y un programa computacional de imágenes digitales. Material y métodos. Doce casos de CBC fueron teñidos con CD1a. Se utilizó microscopia óptica y el programa computacional Image J para calcular las áreas epidérmicas sobre el tumor y adyacente a él. Se contabilizaron el número de CL de cada área y se determinaron y compararon las densidades celulares. Finalmente, se analizó la morfología de las CL en ambas áreas epidérmicas. Resultados. Los resultados mostraron una menor densidad celular en la epidermis supratumoral respecto a la peritumoral (p < 0,05). Las CL entre ambas áreas presentaron diferencias en el tamaño, forma celular y patrón dendrítico. Discusión. La menor densidad y alteraciones estructurales de las CL supratumorales podrían dar lugar a variaciones de la respuesta inmune en el CBC. El uso de tecnología de análisis de imágenes digitales sería un método fiable en el estudio morfométrico de las CL (AU)

Background. Basal cell carcinoma (BCC) is a common malignant tumor and its incidence has risen in recent decades. Research has shown the relationship between ultraviolet (UV) radiation, the skin immune system, and BCC. The role of Langerhans cells (LC) in the immune response to tumors has prompted research into LC density and morphology in response to UV radiation and BCC. However, the data are inconsistent due to differences in research methodology. Objective. To study the density and morphology of LCs in the peritumoral epidermis of BCC using immunohistochemistry and image processing software and compare the results with those from the epidermis overlying the tumor. Material and methods. Twelve samples from patients with BCC were prepared with a CD1a stain. Areas of epidermis overlying and adjacent to the tumor were defined using light microscopy and the Image J image processing software. The LCs in each area were counted and the cell densities were calculated and compared. Morphological features of LCs were also evaluated in each epidermal areas. Results. The results showed a lower density of LCs in the epidermis overlying the tumor than in the peritumoral epidermis (p < 0.05). There were also differences in the size, shape, and dendritic pattern of the LCs between the epidermal areas. Conclusions. The lower density and fewer morphological changes of LCs in the epidermis overlying BCC may give rise to alterations in the immune response to BCC. Digital image analysis is a reliable method for the morphometric evaluation of LCs (AU)

[CD1a+ Langerhans cells in the peritumoral epidermis of basal cell carcinoma]. / Células de Langerhans CD1a+ en la epidermis peritumoral del carcinoma basocelular.

BACKGROUND: Basal cell carcinoma (BCC) is a common malignant tumor and its incidence has risen in recent decades. Research has shown the relationship between ultraviolet (UV) radiation, the skin immune system, and BCC. The role of Langerhans cells (LC) in the immune response to tumors has prompted research into LC density and morphology in response to UV radiation and BCC. However, the data are inconsistent due to differences in research methodology. OBJECTIVE. To study the density and morphology of LCs in the peritumoral epidermis of BCC using immunohistochemistry and image processing software and compare the results with those from the epidermis overlying the tumor. MATERIAL AND METHODS: Twelve samples from patients with BCC were prepared with a CD1a stain. Areas of epidermis overlying and adjacent to the tumor were defined using light microscopy and the Image J image processing software. The LCs in each area were counted and the cell densities were calculated and compared. Morphological features of LCs were also evaluated in each epidermal areas. RESULTS: The results showed a lower density of LCs in the epidermis overlying the tumor than in the peritumoral epidermis (p < 0.05). There were also differences in the size, shape, and dendritic pattern of the LCs between the epidermal areas. CONCLUSIONS: The lower density and fewer morphological changes of LCs in the epidermis overlying BCC may give rise to alterations in the immune response to BCC. Digital image analysis is a reliable method for the morphometric evaluation of LCs.

Inoculation of Sarcocystis neurona merozoites into the central nervous system of horses.

Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome in horses from the Americas and is usually caused by infection with the apicomplexan parasite, Sarcocystis neurona. A horse model of EPM is needed to test the efficacy of chemotherapeutic agents and potential vaccines. Five horses that were negative for antibodies to S. neurona in their serum and cerebrospinal fluid (CSF) were injected in the subarachnoid space with living merozoites of the SN2 isolate of S. neurona. None of the horses developed clinical disease or died over a 132-day observation period. All five horses developed antibodies to S. neurona in their CSF and serum 3-4 weeks after injection. Two of the horses were examined at necropsy and no parasite induced lesions were observed in their tissues and no parasites were recovered from portions of their spinal cords inoculated on to cell cultures. Results of this study demonstrate that merozoites of the SN2 isolate of S. neurona will induce seroconversion but not clinical disease when inoculated directly into the CSF of nonimmune horses.

Chorion formation and ultrastructure of the egg of the cat flea (Siphonaptera: Pulicidae).

Oocyte development in adult female cat fleas, Ctenocephalides felis (Bouché), was studied by light and electron microscopy to determine the formation and ultrastructural morphology of the eggshell. As oocytes develop, somatic follicle cells from the lining of the ovariole migrate around the oocytes. The follicle cells produce electron-dense granules that form the vitelline membrane around the developing oocyte. Subsequently, electron-lucent granules containing an electron-dense core (precursors of the chorion) are produced from the rough endoplasmic reticulum that appear as dilated and clear linear clefts in the cytoplasm of the follicle cells. Exocytosis and coalescence of the granules around the oocyte as the follicle cells disintegrate give rise to the chorion. The chorion was found to consist of 4 distinct layers. The external surface of the egg shell consists of a particulate layer approximately 760 nm thick, composed of an electron-lucent layer of widely dispersed granules. Embedded in this layer are electron-dense spheres that project above the surface of this granular layer. Beneath this outermost layer is a band of electron-dense material, consisting of densely packed granules and is half as thick as the outer particulate layer. The 3rd layer consists of relatively thick, weakly laminated chorion, with a felt-like appearance due to a meshwork of microfibrils. Projections of this network of microfibrils form pillars that attach this layer to a thin relatively compact 4th or basal layer. The pillars and the air-filled cavities lying between the 3rd and 4th chorionic layers constitute the chorionic meshwork known as the palisades or trabecular layer that form the major respiratory organ of the eggshell. The trabecular layer is connected to the external environment by means of the lateral and anterior aeroplyes. The vitelline membrane lies between the chorion and oocyte and is a granular, uniform, moderately electron-dense layer measuring approximately 260 nm thick. The micropyle at the posterior of the flea egg consists of a rosette of 50-80 apertures and possesses an internal electron-dense plug between the chorion and the vitelline membrane. An aeropyle at the anterior end of the egg consists of a rosette of 40-50 apertures. An inconspicuous aeropyle appears as a cluster of hexagonal or polygonal-shaped plaques on the lateral surface of the chorion. Each plaque contains 3-8 pores.

Mode of action of pyriproxyfen and methoprene on eggs of Ctenocephalides felis (Siphonaptera: Pulicidae).

Adult cat fleas were exposed to residues of pyriproxyfen and methoprene in glass vials, then fed on a cat 24 h later to investigate the mode of action of juvenoid growth regulators on embryonic development in flea eggs. Eggs laid by pyriproxyfen-treated fleas within 70 h after exposure to this juvenoid were often devoid of yolk and frequently collapsed after oviposition. Minimal amounts of yolk were deposited in eggs laid after 70 h, and no blastoderm was formed. These results are significant because both modes of action were different than those observed earlier by investigators studying ovicidal effects in adult insects treated with juvenile hormone. In contrast to the pyriproxyfen results, eggs laid by methoprene-treated fleas showed no gross morphological effects, and these eggs remained turgid during embryogenesis. However, the eggs either did not hatch or the larvae died within hours after hatching. Histological examination of the eggs revealed that most of the eggs contained segmented embryos which had apparently died during blastokinesis. Although eggs of some insects exposed to juvenile hormone during oogenesis fail to undergo germ band formation, there was no evidence of this effect in methoprene-treated cat fleas.

Field evaluation of Nylar for control of cat fleas (Siphonaptera: Pulicidae) in home yards.

Procedures were developed to evaluate juvenoid insect growth regulators in home yards as part of an ongoing program to identify photostable insecticides for control of the cat flea, Ctenocephalides felis (Bouché). Nylar, an emulsifiable concentrate formulation of pyriproxyfen, was selected as the experimental insect growth regulator. Efficacy studies, yard survey, and pretreatment sampling techniques are described. Soil samples collected from the yards at intervals after treatment were taken to the laboratory, infested with flea larvae, and tested for juvenoid activity. Results indicated that Nylar applied at a concentration of 32 mg/m2 in 0.82 liter of water/m2 (20 gal/1,000 ft2) prevented development of approximately 80% of the fleas for a period of 3 wk.