Digestive Epithelium Research Articles

Differential localization of lingual antimicrobial peptide in the digestive tract mucosal epithelium of calves

Immunolocalization of lingual antimicrobial peptide (LAP), a member of the β-defensin family, in the digestive tract of calves was investigated to determine its distribution in the digestive tract of Holstein-Friesian calves. Various regions of the digestive tract were collected from slaughtered calves, and fixed in 10% formalin in PBS. Paraffin sections were stained with anti-LAP antibody, followed by visualization of immunoreactions products utilizing the avidin–biotin complex method. Expression of LAP mRNA was analyzed with reverse transcription-PCR. Immunoreactive LAP was localized in the stratum corneum of the stratified squamous epithelium of the tongue, esophagus, rumen, reticulum and omasum but not in their basal layer and lamina propria. In the gastric glands of the abomasum, only chief cells showed LAP positive reaction at the apical side of their cytoplasm. Lamina propria and Peyer's patch of the ileum had some leukocyte-like cells immunopositive for LAP. Weak immunoreaction of LAP was also detected in the mucosal epithelium of the intestinal gland of the cecum, colon and rectum. All regions of digestive tract showed LAP mRNA expression with PCR. These results indicate differential localization of LAP in the mucosal epithelium of digestive tracts in calves. The LAP expressed in stratum corneum of stratified squamous epithelium and chief cells of abomasum specifically may play role in the innate immune function in these tissues.

Micro-PIXE study of Ag in digestive glands of a nano-Ag fed arthropod (Porcellio scaber, Isopoda, Crustacea)

Micro-proton induced X-ray emission (micro-PIXE) method was applied to study the micro-localization of silver (Ag) in digestive glands of a terrestrial arthropod (Porcellio scaber) after feeding on silver nanoparticles (nano-Ag) dosed food. The aim of our work was to assess whether feeding on nano-Ag results in the assimilation of silver (Ag) in digestive gland cells. To study micro-localization and elemental distribution of Ag, the animals were fed on food dosed with nanoparticles for 14days under controlled laboratory conditions. At the end of the feeding exposure, the animals were dissected and digestive glands prepared for micro-PIXE analyses and TEM investigation. The results obtained by micro-PIXE documented high amounts of Ag inside S-cells of the digestive gland epithelium; however, TEM investigation did not show particle aggregates inside digestive gland cells. Also no adverse effect on feeding behavior was recorded what is a measure of toxic effects. We explain the presence of Ag inside the cells as a result of the assimilation of dissoluted Ag ions from ingested nano-Ag particles. Assimilation of excessive amounts of ingested metal ions in S-cells is a well known metal detoxification mechanism in isopods. We discuss the advantages of using micro-PIXE for the micro-localization of elements in biological tissue in studies of interactions between nanoparticles and biological systems.

Application of a battery of biomarkers in mussel digestive gland to assess long-term effects of the Prestige oil spill in Galicia and Bay of Biscay: Tissue-level biomarkers and histopathology

In order to assess the biological effects of the Prestige oil spill (POS), mussels, Mytilus galloprovincialis, were collected in 22 localities along the North coast of the Iberian Peninsula over 3 years (April 2003-April 2006). Different tissue-level biomarkers including cell type composition (volume density of basophilic cells, Vv(BAS)) in digestive gland epithelium, structural changes of digestive alveoli (mean luminal radius to mean epithelial thickness, MLR/MET) and histopathological alterations (prevalence and intensity) of the digestive gland were analysed. Severe alterations in the general condition of the digestive gland tissue were observed all over the study area up to 2004-2005. High Vv(BAS) values were recorded mainly in Galicia but also to a lesser extent in the Bay of Biscay in 2003-2004. Atrophy of the digestive alveoli, measured in terms of MLR/MET, was detected all along the studied area up to 2006. Inflammatory responses cannot be related to pollution due to the POS: (a) prevalence and intensity of focal hemocytic infiltration were higher in the Bay of Biscay than in Galicia but they did not show a clear temporal trend; (b) high intensities of brown cell aggregates were only sporadically recorded; and (c) granulocytomas were more frequently recorded in the Bay of Biscay than in Galicia and especially in localities (i.e. Arrigunaga) subjected to chronic pollution. Likewise, Marteilia, trematodes, intracellular ciliates, unidentified eosinophilic bodies, R/CLO and Mytilicola did not follow any recognisable pattern that could be associated to the POS. In contrast, high Nematopsis intensities recorded in several localities in 2003 might suggest some response of local interest after the POS (i.e., in combination with particular factors/conditions). More data at a regional scale are needed before histopathology may provide a reliable ecosystem health assessment but the present results suggest that the approach is worthwhile. Overall, although Vv(BAS) returned to reference values by 2004-2005, MLR/MET values indicated that the mussel health condition was affected during the whole study period up to April 2006.

Human keratin 8 variants predispose to acetaminophen toxicity

Background and Aims: Keratins 8 and 18 (K8/K18) are the intermediate filaments of digestive epithelia and display an important cytoprotective function. Accordingly, K8/K18 variants predispose humans to chronic liver disease development/progression and to acute acetaminophen toxicity. Given that K8 G62C and K8 R341H are the most common K8 variants in Caucasians, we studied their hitherto unknown molecular effects in the newly generated transgenic K8 R341H overexpressors as well as in the previously described K8 G62C mouse. Methods: Alterations in gene expression profile were quantified by microarray analysis. Analyses in disease-relevant mouse models (acetaminophen, concavacain A and Fas induced liver injury) were complemented by experiments in primary hepatocyte cultures. Immunostaining, histological staining, immunoblotting, real time RT-PCR as well as serological analysis were used to evaluate the results of the experiments. Results: Under basal conditions, K8 G62C/K8 R341H mice did not show any obvious liver phenotype or altered keratin distribution pattern. Unlike in K18 R90C mice, isolated hepatocytes did not display any mechanical fragility. The presence of K8 G62C/R341H variants resulted in profoundly different gene expression profile alterations, while both variants predisposed to acetaminophen liver toxicity. On the other hand, the extent of concavacain A-induced liver injury was comparable to non-transgenic littermates and K8 G62C, but not K8 R341H predisposed to Fas-induced liver injury. Conclusion: Transgenic mice overexpressing K8 G62C/R341H represent a great tool to validate the findings of human association studies as well as to evaluate the underlying pathomechanisms. Our results suggest that K8 G62C/R341H variants exhibit some common properties, but also result in distinct liver phenotypes.

Integrated biomarker assessment of the effects exerted by treated produced water from an onshore natural gas processing plant in the North Sea on the mussel Mytilus edulis

The biological impact of a treated produced water (PW) was investigated under controlled laboratory conditions in the blue mussel, Mytilus edulis. Mussel health status was assessed using an integrated biomarker approach in combination with chemical analysis of both water (with SPMDs), and mussel tissues. Acyl-CoA oxidase activity, neutral lipid accumulation, catalase activity, micronuclei formation, lysosomal membrane stability in digestive cells and haemocytes, cell-type composition in digestive gland epithelium, and the integrity of the digestive gland tissue were measured after 5week exposure to 0%, 0.01%, 0.1%, 0.5% and 1% PW. The suite of biomarkers employed were sensitive to treated PW exposure with significant sublethal responses found at 0.01–0.5% PW, even though individual chemical compounds of PW were at extremely low concentrations in both water and mussel tissues. The study highlights the benefits of an integrated biomarker approach for determining the potential effects of exposure to complex mixtures at low concentrations. Biomarkers were integrated in the Integrative Biological Response (IBR/n) index.

Stem Cells of Adult Organisms in Biology and Medicine

SummaryThe life span of adult cells is restricted, becoming senescent and eventually undergoing apoptosis. Cells are replaced by new ones which originate from a population of stem cells. An example is provided by the hemopoietic system in the bone marrow. In an adult body, stem cells may be identified by markers present in both embryonic and tissue-specific stem cells. Oct3/4, CXCR4, Nanog, CD133 and CD34 are examples of such markers. The stem cells share several properties, such as they may: 1) replicate and give rise to new stem cells, 2) differentiate into heterogenous tissue and organ specific cells and, finally, 3) stem cells are self-protected against various toxic agents and radiation. In adult tissues, stem cells reside in specialized sites (niches), for example in the bone marrow, skin, digestive tract epithelium, and skeletal muscles. Stem cells residing in these stem cell niches are anchored there by chemokines and adhesion molecules. Stroma-Derived Factor-1 (SDF-1), α-chemokine and stem-cell expressed receptor CXCR4 play an important role in the retention of stem cells in such niches. Several stress factors may attenuate the SDF-1-CXCR4 axis in the bone marrow, which leads to the release of stem cells from their niches into circulation. Another important stem cell niche-anchoring mechanism is the interaction of Wnt (a ligand) with Frizzled LRP (receptor). The recently accumulated evidence suggests that malignancy arises from an arrest in maturation and mutation of stem cells. Cancer stem cells are responsible for tumor growth, its relapse and metastasis.The first direct evidence for the existence of cancer stem cells came from observations of acute myeloblastic leukemia. Currently, cell populations enriched for cancer stem cells have been isolated from several tumors. The cancer stem cells represent less than 1% of tumor cells in the mouse models.

MEMS-Based Dual-Axes Confocal Microendoscopy

We demonstrate a miniature, near-infrared microscope (λ = 785 nm) that uses a novel dual axes confocal architecture. Scalability is achieved with post-objective scanning, and a MEMS mirror provides real time (>4 Hz) in vivo imaging. This instrument can achieve sub-cellular resolution with deep tissue penetration and large field of view. An endoscope-compatible version can image digestive tract epithelium to guide tissue biopsy and monitor therapy.

Seasonality in cell and tissue-level biomarkers in Mytilus galloprovincialis: relevance for long-term pollution monitoring

Mussels Mytilus galloprovincialis were collected monthly in 2 localities in the Northern Iberian Peninsula, Oia (Galicia) and Mundaka (Basque Coast), from April 2005 to May 2006, to inves- tigate whether seasonal variability in biomarkers follows the same pattern at both geographical areas. The following battery of biomarkers was analysed: lysosomal membrane stability and struc- tural changes, intracellular neutral lipid accumulation, cell type composition in the digestive gland epithelium, structural changes of digestive alveoli and digestive gland histopathology. Additionally, gonad index was measured as a supporting parameter. Overall, results demonstrate that most stud- ied biomarkers showed similar seasonal variability pattern in both localities. Lysosomal membrane stability (labilisation period (LP)) and most histopathological conditions showed similar seasonal vari- ability patterns in both localities, although values of LP and prevalences of some parasites were higher in Mundaka. Data provide baseline values to be used as reference values for the biomonitor- ing programmes carried out in coastal areas from Galicia to the Basque Coast. It is concluded that for monitoring programmes based on biomarkers, samples must be taken at least twice per year (spring and autumn) to avoid the effect on biomarkers of the late stages of each reproductive cycle.

Microscopic Anatomy of the Digestive System in Normal and Regenerating Specimens of the Brittlestar Amphipholis kochii

The morphology and regeneration of the digestive system of the ophiuroid Amphipholis kochii were investigated. The epithelia of the esophagus and stomach of A. kochii were composed of typical enterocytes and mucous cells. The digestive epithelium of the stomach contained two types of granular secretory cells. After autotomy of the disk, the animals retained the esophagus and a small part of the stomach. The dedifferentiation of enterocytes and mucous cells began on the first day after autotomy. On day 3 the cells formed an anlage of stomach around the mouth opening. Later, the stomach anlage grew as a result of cell proliferation. The opening on the aboral side of the body was closed by day 7. By this time differentiating cells were already observed in the stomach lining. The stomach mesothelium was formed by peritoneocytes and myoepithelial cells, which migrated from other coelomic epithelia of the body. Our study showed that the formation of the digestive system in A. kochii during regeneration depended on cells from the esophagus and the stomach remnant. Both enterocytes and mucous cells were able to dedifferentiate, migrate, and proliferate to give rise to the luminal epithelium. The basic mechanism of stomach formation was epithelial morphogenesis.

Light and electron microscopic studies on turbot Psetta maxima infected with Enteromyxum scophthalmi: histopathology of turbot enteromyxosis

In the last decade, a new parasite that causes severe losses has been detected in farmed turbot Psetta maxima (L.), in north-western Spain. The parasite was classified as a myxosporean and named Enteromyxum scophthalmi. The aim of this study was to characterize the main histological changes that occur in E. scophthalmi-infected turbot. The parasite provoked catarrhal enteritis, and the intensity of the lesions was correlated with the progression of the infection and with the development of the parasite. Infected fish were classified into 3 groups, according to the lesional degree they showed (slight, moderate and severe infections). In fish with slight infections, early parasitic stages were observed populating the epithelial lining of the digestive tract, without eliciting an evident host response. As the disease progressed, catarrhal enteritis was observed, the digestive epithelium showed a typical scalloped shape and the number of both goblet and rodlet cells was increased. Fish with severe infections suffered desquamation of the epithelium, with the subsequent release of parasitic forms to the lumen. The dislodged enterocytes underwent anoikis, a mode of apoptosis triggered by the loss of anchorage, which might facilitate spreading of the parasite. Lymphohaematopoietic depletion was also observed, mainly in head kidney and spleen, which could contribute to the high virulence of this parasite.

Health Status of Olympia Oysters (Ostrea lurida) in British Columbia, Canada

ABSTRACT The Olympia oyster (Ostrea lurida) is the only oyster species native to British Columbia, Canada. Despite management regulations designed to protect this species, populations continue to decline in many locations. In an effort to determine whether parasites or disease are contributing to the population decline, oysters were collected from 5 locations and examined for the presence of pathogens and disease using histology and polymerase chain reaction (PCR) assays. Five parasite/symbiotic organisms were detected by histology, including Rickettsia-like prokaryotes, Mytilicola sp., Rhynchodida-like ciliates, encapsulated copepods, and an unknown protist. Six pathologies of unknown etiology were also detected, including hemocytic neoplasia, basophilic inclusions in the digestive gland epithelium, metaplasia of digestive gland tubules, diapedesis, and diffuse and focal hemocyte infiltration. Despite the variety of histological findings, most were detected at low prevalence and intensity, and are not be...

Human oral keratinocyte E-cadherin degradation byCandida albicansandCandida glabrata

E-cadherin (E-Cad) is a 120-kDa adhesive protein found in adherens junctions of the digestive tract epithelium. We tested the ability of two Candida strains to degrade human E-Cad in the Candida virulence factor perspective. We set out to study oral mucosal E-Cad degradation by clinical and reference strains of Candida albicans and Candida glabrata. We also included hyphal and secreted aspartic proteinase (Sap) mutants of C. albicans to test the effect of yeast/hyphal transition on the ability to degrade E-Cad. The tests were performed at pH 4 and pH 6 to determine the effect of local tissue acidity on the activation of Saps. The C. albicans strains used were: CCUG 32723; clinical strain SC5314 which is known to be strongly invasive; hyphal mutants of SC5314: HLC52 (efg1/efg1), HLC54 (cph1/cph1 efg1/efg1) and JKC19 (cph1/cph1); clinical strain B1134; Sap 1-3 and Sap 4-6 mutants of SC5314. The C. glabrata strains used were ATCC 90030, and the clinical strains 5WT and G212. The sonicated yeast cells of C. albicans JKC19 and SC5314, both in hyphal form, degraded E-Cad at pH 4. The 10x concentrated growth media of the strains HLC-52, HLC-54, 32723 and B1134; all in yeast form, caused degradation at pH 4, HLC-52 and HLC-54 also at pH 6. The C. glabrata strains did not degrade E-Cad. pH is a strain dependent triggering factor in activating yeast or hyphal form related Candida Saps in degrading epithelial cell associated E-Cads.

Surface characteristics of isopod digestive gland epithelium studied by SEM

The structure of the digestive gland epithelium of a terrestrial isopod Porcellio scaber has been investigated by conventional scanning electron microscopy (SEM), focused ion beam-scanning electron microscopy (FIB/SEM), and light microscopy in order to provide evidence on morphology of the gland epithelial surface in animals from a stock culture. We investigated the shape of cells, extrusion of lipid droplets, shape and distribution of microvilli, and the presence of bacteria on the cell surface. A total of 22 animals were investigated and we found some variability in the appearance of the gland epithelial surface. Seventeen of the animals had dome-shaped digestive gland "normal" epithelial cells, which were densely and homogeneously covered by microvilli and varying proportions of which extruded lipid droplets. On the surface of microvilli we routinely observed sparsely distributed bacteria of different shapes. Five of the 22 animals had "abnormal" epithelial cells with a significantly altered shape. In three of these animals, the cells were much smaller, partly or completely flat or sometimes pyramid-like. A thick layer of bacteria was detected on the microvillous border, and in places, the shape and size of microvilli were altered. In two animals, hypertrophic cells containing large vacuoles were observed indicating a characteristic intracellular infection. The potential of SEM in morphological investigations of epithelial surfaces is discussed.

First report of Perkinsus sp. infecting mangrove oysters Crassostrea rhizophorae from the Brazilian coast

Protozoan parasites of the genus Perkinsus are considered important pathogens responsible for mass mortalities in several mollusk species worldwide. In the present study we describe for the first time a parasite of the genus Perkinsus infecting the mangrove oyster Crassostrea rhizophorae from the Brazilian coast. Prevalence of this parasite was low in the Pacoti River estuary (Ceará, northeast Brazil) and absent in oysters from southern Brazil. Oyster gill and rectum tissues incubated in Ray's fluid thioglycollate medium (RFTM) revealed the presence of spherical hypnospores (5 to 55 microm diam.). Histological analysis showed the occurrence of typical signet-ring trophozoites and schizonts (3 to 6 microm diam.) infecting connective tissues of several organs and digestive epithelia. PCR assays specific to the genus Perkinsus, followed by cloning and sequencing of the internal transcribed spacer (ITS) region of the ribosomal ribonucleic acid (rRNA) gene complex, confirmed a close phylogenetic relationship between Brazilian Perkinsus sp. and P. beihaiensis infecting Chinese oysters.

Parasites of three commercially exploited bivalve mollusc species of the estuarine region of the Cachoeira river (Ilhéus, Bahia, Brazil)

This paper reports the parasites found in three commercially exploited bivalve molluscs ( Mytella guyanensis, Anomalocardia brasiliana and Iphigenia brasiliana) of an estuarine region of Ilhéus, south of Bahia, Brazil (14°48′23′′S; 39°02′47′′W). Samples of 20 individuals of each species were collected fortnightly from August 2005 to August 2006. A total of 1480 individuals was collected and processed by standard histologic techniques; the histologic sections were stained with Harris haematoxylin and eosin and examined with light microscope. The water temperature in the study area varied from 24 to 30.5 °C and the salinity from 0 to 23 ppt. Remarkable differences were found in the parasitic community between the three mollusc species involved in the study, which occupied different habitats in the estuarine region of the Cachoeira river. The following parasites were found: intracellular rickettsia-like colonies in digestive epithelia; intracellular gregarine Nematopsis sp. in gills, mantle, gonad, digestive gland and foot muscle; sporocysts of a Bucephalidae trematode in gonads, mantle, gills, digestive gland and foot; unidentified digenetic metacercariae in digestive gland and gonad; metacestodes of Tylocephalum sp. in connective tissue in the digestive gland and in gonad; and an unidentified metazoan in mantle and intestinal lumen. No significant temporal variation in the prevalence of any parasite was detected, which could be due to the narrow temperature range of the region and the absence of patterns of salinity and rainfall variation through the year. The infestation by sporocyst was the only pathological threat detected for the studied populations because of its potential for host castration. The low infection intensity and/or prevalence of the other parasites and the lack of obvious lesions suggest that there is no other serious pathological risk for the studied mollusc populations.

Bioavailability of heavy metals and apoptotic effects on the midgut cells of Pseudaletia unipuncta (Lepidoptera: Noctuidae) inhabiting volcanic environments

In Sao Miguel Island (Azores, Portugal) volcanic activity is manifested by the occurrence of soil degassing, hydrothermal vents and gas emissions on geothermal grounds; consequently, organisms living in such volcanic environments are chronically exposed to metals, as particles or associated with gases, being therefore potential sentinels of the effects derived from such exposure. Concentrations of seven trace elements (Ca, Cd, Cu, Mg, Mn, Pb, Rb, and Zn) were measured in soil, grass leaves (Lolium perenne), and larvae of Pseudaletia unipuncta from sites exposed and non-exposed to volcanic activity. Morphometry and semi-quantification of apoptotic nuclei (TUNEL test) of the midgut epithelial cells of P. unipuncta larvae were also analyzed. Soils and grass not exposed to volcanic activity showed higher levels for most of the analyzed elements with the exception of Rb. The higher levels of Cd, Zn and Mg in soils and grass from the site with no volcanic activity are probably related to the severe artificial fertilization in the studied pastures. Contrarily to soil and grass, larvae from the site with volcanic activity showed higher levels of Cu, Mn, Rb and Zn, suggesting that the volcanic environment (characterized by a lower pH, etc.) could contribute to an increase in the bioavailability of these elements to the larvae. Apoptosis was significantly higher in cells from digestive epithelium of larvae exposed to volcanic activity, probably as a cellular response and adaptation of these organisms when inhabiting active volcanic environments.

Quantitative Changes in Digestive Gland Cells and Oocytes of Helix aspersa, as Biomarkers of Copper Oxychloride Exposure Under Field Conditions

This study investigated the effects of accumulated copper, on digestive epithelium height and percentage area, and on oocyte numbers of the snail Helix aspersa, in a vineyard where copper oxychloride is sprayed. The ultimate aim was to determine the usefulness of these cellular responses as biomarkers. Results showed that snails collected 2 months after fungicide application, had a significantly lower mean digestive epithelium height and percentage area, as well as significantly fewer oocytes per 1 mm(2) of ovotestis, compared to snails collected only 1 week after fungicide application and those from a control vineyard. It was concluded that these cellular responses are clear, measurable responses to copper oxychloride exposure and copper accumulation. However, they do not provide an early warning of copper exposure, which impacts on their usefulness as biomarkers.

Real-Time Intraductal Confocal Microscopy During ERCP: Feasibility and Technical Considerations

Background: Despite advancements in ERCP technology (digital fluoroscopy, choledochoscopy, intraductal ultrasound, and tissue acquisition devices), accurate diagnosis of lesions remains a fundamental problem. Confocal laser microscopy has been reported to be a useful tool for in vivo imaging of cellular structures and can be used during endoscopy using a miniprobe (Cellvizio, Mauna Kea Technologies, Paris, France) passed through the working channel of an endoscope. This system enables in vivo histopathologic imaging of the digestive epithelium.

The effect of starvation on the ultrastructure of the digestive cells of Dolops ranarum (Stuhlmann, 1891) (Crustacea: Branchiura)

Transmission electron microscopy was conducted on the digestive epithelium of the crustacean ectoparasite Dolops ranarum to elucidate its ultrastructure for the first time, both in a nourished and starved condition. Specimens were collected from the Limpopo Drainage System in South Africa, and the specimens were killed and dissected in Todd's fixative. The anterior midgut is composed mostly of absorptive cells or R cells, while the diverticula are composed of R cells and of F cells, which are moderately abundant in rough endoplasmic reticulum. They are probably responsible for producing digestive enzymes. The posterior midgut is composed of papilliform B cells with large apically located vesicles and R′ cells devoid of cell inclusions. Under starvation, specimens survive for a maximum of 12 days; R cells show the most conspicuous changes in ultrastructural characteristics. It is concluded that D. ranarum has adapted to short-term survival only without a host.

Comparison of different preparation methods of biological samples for FIB milling and SEM investigation

When a new approach in microscopy is introduced, broad interest is attracted only when the sample preparation procedure is elaborated and the results compared with the outcome of the existing methods. In the work presented here we tested different preparation procedures for focused ion beam (FIB) milling and scanning electron microscopy (SEM) of biological samples. The digestive gland epithelium of a terrestrial crustacean was prepared in a parallel for FIB/SEM and transmission electron microscope (TEM). All samples were aldehyde-fixed but followed by different further preparation steps. The results demonstrate that the FIB/SEM samples prepared for conventional scanning electron microscopy (dried) is suited for characterization of those intracellular morphological features, which have membranous/lamellar appearance and structures with composition of different density as the rest of the cell. The FIB/SEM of dried samples did not allow unambiguous recognition of cellular organelles. However, cellular organelles can be recognized by FIB/SEM when samples are embedded in plastic as for TEM and imaged by backscattered electrons. The best results in terms of topographical contrast on FIB milled dried samples were obtained when samples were aldehyde-fixed and conductively stained with the OTOTO method (osmium tetroxide/thiocarbohydrazide/osmium tetroxide/thiocarbohydrazide/osmium tetroxide). In the work presented here we provide evidence that FIB/SEM enables both, detailed recognition of cell ultrastructure, when samples are plastic embedded as for TEM or investigation of sample surface morphology and subcellular composition, when samples are dried as for conventional SEM.