Development of Schistosoma mansoni worms in mice analyzed by bright field and confocal microscopy

The blood flukes of mammals (Digenea: Schistosomatidae) are among trematodes unique whose adult worms have separeted sexes which are dissimilar in appearance. The developmental features, growth and organogenesis of Schistosoma mansoni were studied in Swiss Webster mice by a digital system for image analysis and confocal microscopy. Data so far obtained showed two phases with significative morphological changes at 3-4 weeks post-infection, and a gradual similar development onwards in the reproductive system and tegument. Our male-dependent phase demonstrated that mating occurs before sexual maturing. At week three, the majority of male worms (59%) had formed the gynaecophoric canal although testicular lobes and tegumental tubercles were absent. By this time, 33% females had an incipient ovary (without cellular differentiation). At week four, 77.2% males presented testicular lobes with few germinative cells while 26% had developing tegumental tubercles. The immature ovary was observed in 69% females. Suckers followed different pattern of growth between male and females. The size of oral and ventral suckers from six-week-old male worms grew abruptly (3.0 fold) more than that of three-week-old. In female worms, maximum growth was attained at week four, reducing in size thereafter. From sixth week onwards, all specimens showed the fully developed reproductive system. Probably, these features are morphological traits which schistosome has experienced from hermaphrodite to dioecy.

Host cell adhesion to Schistosoma mansoni larvae in the peritoneal cavity of naive mice: histological and scanning electron microscopic studies

Cercariae of Schistosoma mansoni inoculated into the peritoneal cavity of naive mice induced host cell adhesion to their surface, but after 90 minutes the number of adherent cells sharply decreased. The cell detachment is progressive and simultaneous to the cercaria-schistosomule transformation. The histological study showed mainly neutrophils in close contact with the larvae. Mononuclear cells and some eosinophils were occasionally seen surrounding the adherent neutrophils. The scanning electron microscopy showed cells displaying twisted microvilli and several microplicae contacting or spreading over the larval surface, and larvae completely surrounded by clusters of cells. These results suggest that the neutrophils recognize molecules on the cercarial surface which induce their spreading

Evaluation of follicular lymphoid depletion in the Bursa of Fabricius: an alternative methodology using digital image analysis and artificial neural networks

Fifty Bursa of Fabricius (BF) were examined by conventional optical microscopy and digital images were acquired and processed using Matlab® 6.5 software. The Artificial Neuronal Network (ANN) was generated using Neuroshell® Classifier software and the optical and digital data were compared. The ANN was able to make a comparable classification of digital and optical scores. The use of ANN was able to classify correctly the majority of the follicles, reaching sensibility and specificity of 89% and 96%, respectively. When the follicles were scored and grouped in a binary fashion the sensibility increased to 90% and obtained the maximum value for the specificity of 92%. These results demonstrate that the use of digital image analysis and ANN is a useful tool for the pathological classification of the BF lymphoid depletion. In addition it provides objective results that allow measuring the dimension of the error in the diagnosis and classification therefore making comparison between databases feasible.

Image analysis and definition of nuclear phenotypes

Some basic topics concerned with the extraction of textural and geometric information from cell nucleus images as well as description and characterization of chromatin supraorganization and consequent classification of nuclear phenotypes are presented.

Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

This paper reviews the use of confocal microscopy as it pertains to the identification of G-protein coupled receptors and the study of their dynamic properties in cell cultures and in mammalian brain following their tagging with specific fluorescent ligands. Principles that should guide the choice of suitable ligands and fluorophores are discussed. Examples are provided from the work carried out in the authors' laboratory using custom synthetized fluoresceinylated or BODIPY-tagged bioactive peptides. The results show that confocal microscopic detection of specifically bound fluorescent ligands permits high resolution appraisal of neuropeptide receptor distribution both in cell culture and in brain sections. Within the framework of time course experiments, it also allows for a dynamic assessment of the internalization and subsequent intracellular trafficking of bound fluorescent molecules. Thus, it was found that neurotensin, somatostatin and mu- and delta-selective opioid peptides are internalized in a receptor-dependent fashion and according to receptor-specific patterns into their target cells. In the case of neurotensin, this internalization process was found to be clathrin-mediated, to proceed through classical endosomal pathways and, in neurons, to result in a mobilization of newly formed endosomes from neural processes to nerve cell bodies and from the periphery of cell bodies towards the perinuclear zone. These mechanisms are likely to play an important role for ligand inactivation, receptor regulation and perhaps also transmembrane signaling.

Measurement of epidermal thickness in a patient with psoriasis by computer-supported image analysis

The aim of the present study was to measure full epidermal thickness, stratum corneum thickness, rete length, dermal papilla widening and suprapapillary epidermal thickness in psoriasis patients using a light microscope and computer-supported image analysis. The data obtained were analyzed in terms of patient age, type of psoriasis, total body surface area involvement, scalp and nail involvement, duration of psoriasis, and family history of the disease. The study was conducted on 64 patients and 57 controls whose skin biopsies were examined by light microscopy. The acquired microscopic images were transferred to a computer and measurements were made using image analysis. The skin biopsies, taken from different body areas, were examined for different parameters such as epidermal, corneal and suprapapillary epidermal thickness. The most prominent increase in thickness was detected in the palmar region. Corneal thickness was more pronounced in patients with scalp involvement than in patients without scalp involvement (t = -2.651, P = 0.008). The most prominent increase in rete length was observed in the knees (median: 491 µm, t = 10.117, P = 0.000). The difference in rete length between patients with a positive and a negative family history was significant (t = -3.334, P = 0.03), being 27% greater in psoriasis patients without a family history. The differences in dermal papilla distances among patients were very small. We conclude that microscope-supported thickness measurements provide objective results.

Alternagin-C, a disintegrin-like protein from the venom of Bothrops alternatus, modulates alpha2ß1 integrin-mediated cell adhesion, migration and proliferation

The alpha2ß1 integrin is a major collagen receptor that plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Alternagin-C (ALT-C), a disintegrin-like protein purified from the venom of the Brazilian snake Bothrops alternatus, competitively interacts with the alpha2ß1 integrin, thereby inhibiting collagen binding. When immobilized in plate wells, ALT-C supports the adhesion of fibroblasts as well as of human vein endothelial cells (HUVEC) and does not detach cells previously bound to collagen I. ALT-C is a strong inducer of HUVEC proliferation in vitro. Gene expression analysis was done using an Affimetrix HU-95A probe array with probe sets of ~10,000 human genes. In human fibroblasts growing on collagen-coated plates, ALT-C up-regulates the expression of several growth factors including vascular endothelial growth factor, as well as some cell cycle control genes. Up-regulation of the vascular endothelial growth factor gene and other growth factors could explain the positive effect on HUVEC proliferation. ALT-C also strongly activates protein kinase B phosphorylation, a signaling event involved in endothelial cell survival and angiogenesis. In human neutrophils, ALT-C has a potent chemotactic effect modulated by the intracellular signaling cascade characteristic of integrin-activated pathways. Thus, ALT-C acts as a survival factor, promoting adhesion, migration and endothelial cell proliferation after binding to alpha2ß1 integrin on the cell surface. The biological activities of ALT-C may be helpful as a therapeutic strategy in tissue regeneration as well as in the design of new therapeutic agents targeting alpha2ß1 integrin.

Optimization of image analysis techniques for quality assessment of whole-wheat breads made with fat replacer

The cellular structure of healthy food products, with added dietary fiber and low in calories, is an important factor that contributes to the assessment of quality, which can be quantified by image analysis of visual texture. This study seeks to compare image analysis techniques (binarization using Otsu’s method and the default ImageJ algorithm, a variation of the iterative intermeans method) for quantification of differences in the crumb structure of breads made with different percentages of whole-wheat flour and fat replacer, and discuss the behavior of the parameters number of cells, mean cell area, cell density, and circularity using response surface methodology. Comparative analysis of the results achieved with the Otsu and default ImageJ algorithms showed a significant difference between the studied parameters. The Otsu method demonstrated the crumb structure of the analyzed breads more reliably than the default ImageJ algorithm, and is thus the most suitable in terms of structural representation of the crumb texture.

Schistosoma mansoni: host cell adhesion to the different stages of the parasite, in vivo

The peritoneal cavity of laboratory mice was used to study the phenomenon of host cell adhesion to different evolutive stages of the Schistosoma mansoni (cercaria, adult worm, developing and mature eggs, miracidium, young and mature daughter sporocysts). Material recovered from the peritoneal cavity 30 and 180 min after the inoculation of each evolutive form was examined with the help of a stereomicroscope. The free swimming larvae (cercaria and miracidium), and the evolutive forms producing such larvae (mature egg and mature daughter sporocyst) elicited the host cell adhesion phenomenon. In all forms but cercariae the adherent cells remained as so till 180 minutes after inoculation

Effect of oxamniquine on cell adhesion to Schistosoma mansoni larvae in the peritoneal cavity of naive mice

The treatment of naive mice with high closes of oxamniquine, 1 hour before the intraperitoneal inoculation of Schistosoma mansoni cercariae, induces a delay in the transformation process resulting in a longer host cell adhesion.

Activated peripheral lymphocytes with increased expression of cell adhesion molecules and cytotoxic markers are associated with dengue fever disease

The immune mechanisms involved in dengue fever and dengue hemorrhagic/dengue shock syndrome are not well understood. The ex vivo activation status of immune cells during the dengue disease in patients was examined. CD4and CD8 T cells were reduced during the acute phase. Interestingly, CD8 T cells co-expressing activation marker HLA-DR, Q, P, and cytolytic granule protein-Tia-1 were significantly higher in dengue patients than in controls. Detection of adhesion molecules indicated that in dengue patients the majority of T cells (CD4 and CD8) express the activation/memory phenotype, characterized as CD44HIGH and lack the expression of the naïve cell marker, CD62L LOW. Also, the levels of T cells co-expressing ICAM-1 (CD54), VLA-4, and LFA-1 (CD11a) were significantly increased. CD8 T lymphocytes expressed predominantly low levels of anti-apoptotic molecule Bcl-2 in the acute phase, possibly leading to the exhibition of a phenotype of activated/effector cells. Circulating levels of IL-18, TGF-b1 and sICAM-1 were significantly elevated in dengue patients. Early activation events occur during acute dengue infection which might contribute to viral clearance. Differences in expression of adhesion molecules among CD4 and CD8 T cells might underlie the selective extravasation of these subsets from blood circulation into lymphoid organs and/or tissues. In addition, activated CD8 T cells would be more susceptible to apoptosis as shown by the alteration in Bcl-2 expression. Cytokines such as IL-18, TGF-b1, and sICAM-1 may be contributing by either stimulating or suppressing the adaptative immune response, during dengue infection, thereby perhaps establishing a relationship with disease severity.

Chronic Trypanosoma cruzi-elicited cardiomyopathy: from the discovery to the proposal of rational therapeutic interventions targeting cell adhesion molecules and chemokine receptors - how to make a dream come true

One hundred years ago, Carlos Chagas discovered a new disease, the American trypanosomiasis. Chagas and co-workers later characterised the disease's common manifestation, chronic cardiomyopathy, and suggested that parasitic persistence coupled with inflammation was the key underlying pathogenic mechanism. Better comprehension of the molecular mechanisms leading to clinical heart afflictions is a prerequisite to developing new therapies that ameliorate inflammation and improve heart function without hampering parasite control. Here, we review recent data showing that distinct cell adhesion molecules, chemokines and chemokine receptors participate in anti-parasite immunity and/or detrimental leukocyte trafficking to the heart. Moreover, we offer evidence that CC-chemokine receptors may be attractive therapeutic targets aiming to regain homeostatic balance in parasite/host interaction thereby improving prognosis, supporting that it is becoming a non-phantasious proposal.

Public-domain software for root image analysis

In the search for high efficiency in root studies, computational systems have been developed to analyze digital images. ImageJ and Safira are public-domain systems that may be used for image analysis of washed roots. However, differences in root properties measured using ImageJ and Safira are supposed. This study compared values of root length and surface area obtained with public-domain systems with values obtained by a reference method. Root samples were collected in a banana plantation in an area of a shallower Typic Carbonatic Haplic Cambisol (CXk), and an area of a deeper Typic Haplic Ta Eutrophic Cambisol (CXve), at six depths in five replications. Root images were digitized and the systems ImageJ and Safira used to determine root length and surface area. The line-intersect method modified by Tennant was used as reference; values of root length and surface area measured with the different systems were analyzed by Pearson's correlation coefficient and compared by the confidence interval and t-test. Both systems ImageJ and Safira had positive correlation coefficients with the reference method for root length and surface area data in CXk and CXve. The correlation coefficient ranged from 0.54 to 0.80, with lowest value observed for ImageJ in the measurement of surface area of roots sampled in CXve. The IC (95 %) revealed that root length measurements with Safira did not differ from that with the reference method in CXk (-77.3 to 244.0 mm). Regarding surface area measurements, Safira did not differ from the reference method for samples collected in CXk (-530.6 to 565.8 mm²) as well as in CXve (-4231 to 612.1 mm²). However, measurements with ImageJ were different from those obtained by the reference method, underestimating length and surface area in samples collected in CXk and CXve. Both ImageJ and Safira allow an identification of increases or decreases in root length and surface area. However, Safira results for root length and surface area are closer to the results obtained with the reference method.

Sample size for measurement of root traits on common bean by image analysis

Evaluation of root traits may be facilitated if they are assessed on samples of the root system. The objective of this work was to determine the sample size of the root system in order to estimate root traits of common bean (Phaseolus vulgaris L.) cultivars by digital image analysis. One plant was grown per pot and harvested at pod setting, with 64 and 16 pots corresponding to two and four cultivars in the first and second experiments, respectively. Root samples were scanned up to the completeness of the root system and the root area and length were estimated. Scanning a root sample demanded 21 minutes, and scanning the entire root system demanded 4 hours and 53 minutes. In the first experiment, root area and length estimated with two samples showed, respectively, a correlation of 0.977 and 0.860, with these traits measured in the entire root. In the second experiment, the correlation was 0.889 and 0.915. The increase in the correlation with more than two samples was negligible. The two samples corresponded to 13.4% and 16.9% of total root mass (excluding taproot and nodules) in the first and second experiments. Taproot stands for a high proportion of root mass and must be deducted on root trait estimations. Samples with nearly 15% of total root mass produce reliable root trait estimates.

Image analysis for assessing broiler breeder behavior response to thermal environment

The research proposes a methodology for assessing broiler breeder response to changes in rearing thermal environment. The continuous video recording of a flock analyzed may offer compelling evidences of thermal comfort, as well as other indications of welfare. An algorithm for classifying specific broiler breeder behavior was developed. Videos were recorded over three boxes where 30 breeders were reared. The boxes were mounted inside an environmental chamber were ambient temperature varied from cold to hot. Digital images were processed based on the number of pixels, according to their light intensity variation and binary contrast allowing a sequence of behaviors related to welfare. The system used the default of x, y coordinates, where x represents the horizontal distance from the top left of the work area to the point P, and y is the vertical distance. The video images were observed, and a grid was developed for identifying the area the birds stayed and the time they spent at that place. The sequence was analyzed frame by frame confronting the data with specific adopted thermal neutral rearing standards. The grid mask overlapped the real bird image. The resulting image allows the visualization of clusters, as birds in flock behave in certain patterns. An algorithm indicating the breeder response to thermal environment was developed.