Intestinal brush border assembly driven by protocadherin-based intermicrovillar adhesion.

Transporting epithelial cells build apical microvilli to increase membrane surface area and enhance absorptive capacity. The intestinal brush border provides an elaborate example with tightly packed microvilli that function in nutrient absorption and host defense. Although the brush border is essential for physiological homeostasis, its assembly is poorly understood. We found that brush border assembly is driven by the formation of Ca(2+)-dependent adhesion links between adjacent microvilli. Intermicrovillar links are composed of protocadherin-24 and mucin-like protocadherin, which target to microvillar tips and interact to form a trans-heterophilic complex. The cytoplasmic domains of microvillar protocadherins interact with the scaffolding protein, harmonin, and myosin-7b, which promote localization to microvillar tips. Finally, a mouse model of Usher syndrome lacking harmonin exhibits microvillar protocadherin mislocalization and severe defects in brush border morphology. These data reveal an adhesion-based mechanism for brush border assembly and illuminate the basis of intestinal pathology in patients with Usher syndrome. PAPERFLICK:

Gonococcal invasion into epithelial cells depends on both cell polarity and ezrin.

Neisseria gonorrhoeae (GC) establishes infection in women from the cervix, lined with heterogeneous epithelial cells from non-polarized stratified at the ectocervix to polarized columnar at the endocervix. We have previously shown that GC differentially colonize and transmigrate across the ecto and endocervical epithelia. However, whether and how GC invade into heterogeneous cervical epithelial cells is unknown. This study examined GC entry of epithelial cells with various properties, using human cervical tissue explant and non-polarized/polarized epithelial cell line models. While adhering to non-polarized and polarized epithelial cells at similar levels, GC invaded into non-polarized more efficiently than polarized epithelial cells. The enhanced GC invasion in non-polarized epithelial cells was associated with increased ezrin phosphorylation, F-actin and ezrin recruitment to GC adherent sites, and the elongation of GC-associated microvilli. Inhibition of ezrin phosphorylation inhibited F-actin and ezrin recruitment and microvilli elongation, leading to a reduction in GC invasion. The reduced GC invasion in polarized epithelial cells was associated with non-muscle myosin II-mediated F-actin disassembly and microvilli denudation at GC adherence sites. Surprisingly, intraepithelial GC were only detected inside epithelial cells shedding from the cervix by immunofluorescence microscopy, but not significantly in the ectocervical and the endocervical regions. We observed similar ezrin and F-actin recruitment in exfoliated cervical epithelial cells but not in those that remained in the ectocervical epithelium, as the luminal layer of ectocervical epithelial cells expressed ten-fold lower levels of ezrin than those beneath. However, GC inoculation induced F-actin reduction and myosin recruitment in the endocervix, similar to what was seen in polarized epithelial cells. Collectively, our results suggest that while GC invade non-polarized epithelial cells through ezrin-driven microvilli elongation, the apical polarization of ezrin and F-actin inhibits GC entry into polarized epithelial cells.

Intestinal differentiation involves cleavage of histone H3 N-terminal tails by multiple proteases.

The proteolytic cleavage of histone tails, also termed histone clipping, has been described as a mechanism for permanent removal of post-translational modifications (PTMs) from histone proteins. Such activity has been ascribed to ensure regulatory function in key cellular processes such as differentiation, senescence and transcriptional control, for which different histone-specific proteases have been described. However, all these studies were exclusively performed using cell lines cultured in vitro and no clear evidence that histone clipping is regulated in vivo has been reported. Here we show that histone H3 N-terminal tails undergo extensive cleavage in the differentiated cells of the villi in mouse intestinal epithelium. Combining biochemical methods, 3D organoid cultures and in vivo approaches, we demonstrate that intestinal H3 clipping is the result of multiple proteolytic activities. We identified Trypsins and Cathepsin L as specific H3 tail proteases active in small intestinal differentiated cells and showed that their proteolytic activity is differentially affected by the PTM pattern of histone H3 tails. Together, our findings provide in vivo evidence of H3 tail proteolysis in mammalian tissues, directly linking H3 clipping to cell differentiation.

Syndapin constricts microvillar necks to form a united rhabdomere in Drosophila photoreceptors.

Drosophila photoreceptors develop from polarized epithelial cells that have apical and basolateral membranes. During morphogenesis, the apical membranes subdivide into a united bundle of photosensory microvilli (rhabdomeres) and a surrounding supporting membrane (stalk). By EMS-induced mutagenesis screening, we found that the F-Bin/Amphiphysin/Rvs (F-BAR) protein syndapin is essential for apical membrane segregation. The analysis of the super-resolution microscopy, STORM and the electron microscopy suggest that syndapin localizes to the neck of the microvilli at the base of the rhabdomere. Syndapin and moesin are required to constrict the neck of the microvilli to organize the membrane architecture at the base of the rhabdomere, to exclude the stalk membrane. Simultaneous loss of syndapin along with the microvilli adhesion molecule chaoptin significantly enhanced the disruption of stalk-rhabdomere segregation. However, loss of the factors involving endocytosis do not interfere. These results indicated syndapin is most likely functioning through its membrane curvature properties, and not through endocytic processes for stalk-rhabdomere segregation. Elucidation of the mechanism of this unconventional domain formation will provide novel insights into the field of cell biology.

Prominin-1 (CD133) modulates the architecture and dynamics of microvilli.

Prominin-1 is a cell surface biomarker that allows the identification of stem and cancer stem cells from different organs. It is also expressed in several differentiated epithelial and non-epithelial cells. Irrespective of the cell type, prominin-1 is associated with plasma membrane protrusions. Here, we investigate its impact on the architecture of membrane protrusions using microvilli of Madin-Darby canine kidney cells as the main model. Our high-resolution analysis revealed that upon the overexpression of prominin-1 the number of microvilli and clusters of them increased. Microvilli with branched and/or knob-like morphologies were observed and stimulated by mutations in the ganglioside-binding site of prominin-1. The altered phenotypes were caused by the interaction of prominin-1 with phosphoinositide 3-kinase and Arp2/3 complex. Mutation of tyrosine 828 of prominin-1 impaired its phosphorylation and thereby inhibited the aforementioned interactions abolishing altered microvilli. This suggests that the interplay of prominin-1-ganglioside membrane complexes, phosphoinositide 3-kinase and cytoskeleton components regulates microvillar architecture. Lastly, the expression of prominin-1 and its mutants modified the structure of filopodia emerging from fibroblast-like cells and silencing human prominin-1 in primary hematopoietic stem cells resulted in the loss of uropod-associated microvilli. Altogether, these findings strengthen the role of prominin-1 as an organizer of cellular protrusions.

Cytoskeleton induced the changes of microvilli and mechanical properties in living cells by atomic force microscopy.

The cytoskeleton acts as a scaffold for membrane protrusion, such as microvilli. However, the relationship between the characteristics of microvilli and cytoskeleton remains poorly understood under the physiological state. To investigate the role of the cytoskeleton in regulating microvilli and cellular mechanical properties, atomic force microscopy (AFM) was used to detect the dynamic characteristics of microvillus morphology and elastic modulus of living HeLa cells. First, HeLa and MCF-7 cell lines were stained with Fluor-488-phalloidin and microtubules antibody. Then, the microvilli morphology was analyzed by high-resolution images of AFM in situ. Furthermore, changes in elastic modulus were investigated by the force curve of AFM. Fluorescence microscopy and AFM results revealed that destroyed microfilaments led to a smaller microvilli size, whereas the increase in the aggregation and number of microfilaments led to a larger microvilli size. The destruction and aggregation of microfilaments remarkably affected the mechanical properties of HeLa cells. Microtubule-related drugs induced the change of microtubule, but we failed to note significant differences in microvilli morphology and mechanical properties of cells. In summary, our results unraveled the relationship between microfilaments and the structure of microvilli and Young's modulus in living HeLa cells, which would contribute to the further understanding of the physiological function of the cytoskeleton in vivo.

SARS-CoV-2: preliminary study of infected human nasopharyngeal tissue by high resolution microscopy.

BACKGROUND: The novel coronavirus SARS-CoV-2 is the etiological agent of COVID-19. This virus has become one of the most dangerous in recent times with a very high rate of transmission. At present, several publications show the typical crown-shape of the novel coronavirus grown in cell cultures. However, an integral ultramicroscopy study done directly from clinical specimens has not been published. METHODS: Nasopharyngeal swabs were collected from 12 Cuban individuals, six asymptomatic and RT-PCR negative (negative control) and six others from a COVID-19 symptomatic and RT-PCR positive for SARS CoV-2. Samples were treated with an aldehyde solution and processed by scanning electron microscopy (SEM), confocal microscopy (CM) and, atomic force microscopy. Improvement and segmentation of coronavirus images were performed by a novel mathematical image enhancement algorithm. RESULTS: The images of the negative control sample showed the characteristic healthy microvilli morphology at the apical region of the nasal epithelial cells. As expected, they do not display virus-like structures. The images of the positive sample showed characteristic coronavirus-like particles and evident destruction of microvilli. In some regions, virions budding through the cell membrane were observed. Microvilli destruction could explain the anosmia reported by some patients. Virus-particles emerging from the cell-surface with a variable size ranging from 80 to 400 nm were observed by SEM. Viral antigen was identified in the apical cells zone by CM. CONCLUSIONS: The integral microscopy study showed that SARS-CoV-2 has a similar image to SARS-CoV. The application of several high-resolution microscopy techniques to nasopharyngeal samples awaits future use.

Serial electron microscopic reconstruction of the drosophila larval eye: Photoreceptors with a rudimentary rhabdomere of microvillar-like processes.

Photoreceptor cells (PRCs) across the animal kingdom are characterized by a stacking of apical membranes to accommodate the high abundance of photopigment. In arthropods and many other invertebrate phyla PRC membrane stacks adopt the shape of densely packed microvilli that form a structure called rhabdomere. PRCs and surrounding accessory cells, including pigment cells and lens-forming cells, are grouped in stereotyped units, the ommatidia. In larvae of holometabolan insects, eyes (called stemmata) are reduced in terms of number and composition of ommatidia. The stemma of Drosophila (Bolwig organ) is reduced to a bilateral cluster of subepidermal PRCs, lacking all other cell types. In the present paper we have analyzed the development and fine structure of the Drosophila larval PRCs. Shortly after their appearance in the embryonic head ectoderm, PRC precursors delaminate and lose expression of apical markers of epithelial cells, including Crumbs and several centrosome-associated proteins. In the early first instar larva, PRCs show an expanded, irregularly shaped apical surface that is folded into multiple horizontal microvillar-like processes (MLPs). Apical PRC membranes and MLPs are covered with a layer of extracellular matrix. MLPs are predominantly aligned along an axis that extends ventro-anteriorly to dorso-posteriorly, but vary in length, diameter, and spacing. Individual MLPs present a "beaded" shape, with thick segments (0.2-0.3 µm diameter) alternating with thin segments (>0.1 µm). We show that loss of the glycoprotein Chaoptin, which is absolutely essential for rhabdomere formation in the adult PRCs, does not lead to severe abnormalities in larval PRCs.

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.

Modes of carbon dioxide delivery during laparoscopy generate distinct differences in peritoneal damage and hypoxia in a porcine model.

BACKGROUND: Insufflation with CO2 can employ continuous flow, recirculated gas and/or additional warming and humidification. The ability to compare these modes of delivery depends upon the assays employed and opportunities to minimize subject variation. The use of pigs to train colorectal surgeons provided an opportunity to compare three modes of CO2 delivery under controlled circumstances. METHODS: Sixteen pigs were subjected to rectal resection, insufflated with dry-cold CO2 (DC-CO2) (n = 5), recirculated CO2 by an AirSeal device (n = 5) and humidification and warming (HW-CO2) by a HumiGard device (n = 6). Peritoneal biopsies were harvested from the same region of the peritoneum for fixation for immunohistochemistry for hypoxia-inducible factor 1 alpha (HIF-1α) and scanning electron microscopy (SEM) to evaluate hypoxia induction or tissue/cellular damage, respectively. RESULTS: DC-CO2 insufflation by both modes leads to significant damage to mesothelial cells as measured by cellular bulging and retraction as well as microvillus shortening compared with HW-CO2 at 1 to 1.5 h. DC-CO2 also leads to a rapid and significant induction of HIF-1α compared with HW-CO2. CONCLUSIONS: DC-CO2 insufflation induces substantive cellular damage and hypoxia responses within the first hour of application. The use of HW-CO2 insufflation ameliorates these processes for the first one to one and half hours in a large mammal used to replicate surgery in humans.

Acting target of toosendanin locates in the midgut epithelium cells of Mythimna separate Walker larvae (lepidoptera: Noctuidae).

Toosendanin (TSN), which is extracted from the root bark of Melia toosendan Siebold and Zuccarini, has multiple modes of action against insects. Especially, this compound has a potent stomach poisoning activity against several lepidoptera pests. In this paper, the signs of toxicity, digestive enzymes activity, the histopathological changes and immuno-electron microscopic localization of TSN in the midgut epithelium of Mythimna separate Walker larvae were investigated for better understanding its action mechanism against insects. The bioassay results indicated that TSN has strong stomach poisoning against the fifth-instar larvae of M. separata (LC50 = 252.23 µg/mL). The typical poisoned symptom were regurgitation and paralysis. Activities of digestive enzymes had no obvious changes after treatment with LC80 dose of TSN. The midgut epithelial cells of insect were damaged by TSN, showing the degeneration of microvilli, hyperplasia of smooth endoplasmic reticulum and condensation of chromatin. Immunohistochemical analysis revealed that the gold particles existed on the microvilli of columnar cells and goblet cells, and gradually accumulated with the exacerbation of poisoning symptoms, showing that TSN targets on the microvilli of the midgutcells. Therefore, TSN acts on digestive system and locates in the microvilli of midgutcells of M. separata.

Dietary supplementation with Lactobacillus acidophilus enhanced the growth, gut morphometry, antioxidant capacity, and the immune response in juveniles of the common carp, Cyprinus carpio.

This study investigated the effect of Lactobacillus acidophilus-fortified diets on growth performance, antioxidant profiles, and immunity-related gene expressions of common carp, Cyprinus carpio juveniles. Four isonitrogenous diets containing 0, 102, 104, or 106 cfu/mL/kg L. acidophilus were fed to 240 Cyprinus carpio juveniles (mean = 21.34 ± 1.85 g), allotted to 12 rectangular tanks in a completely randomized designed at 3% body weight for 56 days. Growth performance and nutrient utilization were evaluated using standard procedures. Intestinal villi were measured, antioxidant profiles were evaluated from blood sera, and immunity-related gene expressions were evaluated. The results revealed that fish fed dietary 106 had significantly higher weight gain, SGR, feed intake, and lower FCR. Also, villi length, width, and areas of absorption were significantly improved in a dose-dependent manner. Furthermore, innate immune profiles, superoxide dismutase, catalase, respiratory bust activity as well as transforming growth factor beta (TGFß), interleukin 8 (IL-8), and tumor necrosis factor alpha (TNF-alpha) were significantly stimulated. This study evoked that Lactobacillus acidophilus supplementation enhanced better growth performance, improved antioxidant profiles, and modulated expression of immune-related genes of common carp, Cyprinus carpio, than the fish fed control diet. Results show that fish fed fortified diets had better growth performance, improved antioxidant profiles, and modulated expression of immune-related genes.

The scaffolding protein ZO-1 coordinates actomyosin and epithelial apical specializations in vitro and in vivo.

Polarized epithelia assemble into sheets that compartmentalize organs and generate tissue barriers by integrating apical surfaces into a single, unified structure. This tissue organization is shared across organs, species, and developmental stages. The processes that regulate development and maintenance of apical epithelial surfaces are, however, undefined. Here, using an intestinal epithelial-specific knockout (KO) mouse and cultured epithelial cells, we show that the tight junction scaffolding protein zonula occludens-1 (ZO-1) is essential for development of unified apical surfaces in vivo and in vitro We found that U5 and GuK domains of ZO-1 are necessary for proper apical surface assembly, including organization of microvilli and cortical F-actin; however, direct interactions with F-actin through the ZO-1 actin-binding region (ABR) are not required. ZO-1 lacking the PDZ1 domain, which binds claudins, rescued apical structure in ZO-1-deficient epithelia, but not in cells lacking both ZO-1 and ZO-2, suggesting that heterodimerization with ZO-2 restores PDZ1-dependent ZO-1 interactions that are vital to apical surface organization. Pharmacologic F-actin disruption, myosin II motor inhibition, or dynamin inactivation restored apical epithelial structure in vitro and in vivo, indicating that ZO-1 directs epithelial organization by regulating actomyosin contraction and membrane traffic. We conclude that multiple ZO-1-mediated interactions contribute to coordination of epithelial actomyosin function and genesis of unified apical surfaces.

Inhibition of Sodium-Hydrogen Antiport by Antibodies to NHA1 in Brush Border Membrane Vesicles from Whole Aedes aegypti Larvae.

The present research report describes Na+/H+ antiport by brush border membrane vesicles isolated from whole larvae of Aedes aegypti (AeBBMVw). Our hypothesis is that acid quenching of acridine orange by AeBBMVw is predominantly mediated by Na+/H+ antiport via the NHA1 component of the AeBBMVw in the absence of amino acids and ATP. AeNHA1 is a Na+/H+ antiporter that has been postulated to exchange Na+ and H+ across the apical plasma membrane in posterior midgut of A. aegypti larvae. Its principal function is to recycle the H+ and Na+ that are transported during amino acid uptake, e.g., phenylalanine. This uptake is mediated, in part, by a voltage-driven, Na+-coupled, nutrient amino acid transporter (AeNAT8). The voltage is generated by an H+ V-ATPase. All three components, V-ATPase, antiporter, and nutrient amino acid transporter (VAN), are present in brush border membrane vesicles isolated from whole larvae of A. aegypti. By omitting ATP and amino acids, Na+/H+ antiport was measured by fluorescence quenching of acridine orange (AO) caused by acidification of either the internal vesicle medium (Na+in > Na+out) or the external fluid-membrane interface (Na+in < Na+out). Vesicles with 100 micromolar Na+ inside and 10 micromolar Na+ outside or with 0.01 micromolar Na+ inside and 100 micromolar Na+ outside quenched fluorescence of AO by as much as 30%. Acidification did not occur in the absence of AeBBMVw. Preincubation of AeBBMVw with antibodies to NHA1 inhibit Na+/H+ antiport dependent fluorescence quenching, indicating that AeNHA1 has a significant role in Na+/H+ exchange.

Rapid differentiation of the human RPE cell line, ARPE-19, induced by nicotinamide.

Human RPE cell lines, especially the ARPE-19 cell line, are widely-used in eye research, as well as general epithelial cell studies. In comparison with primary RPE cells, they offer relative convenience and consistency, but cultures derived from these lines are typically not well differentiated. We describe a simple, rapid method to establish cultures from ARPE-19 cells, with significantly improved epithelial cell morphology and cytoskeletal organization, and RPE-related functions. We identify the presence of nicotinamide, a member of the vitamin B family, as an essential factor in promoting the observed differentiation, indicating the importance of metabolism in RPE cell differentiation.

Three-dimensional localization of T-cell receptors in relation to microvilli using a combination of superresolution microscopies.

Leukocyte microvilli are flexible projections enriched with adhesion molecules. The role of these cellular projections in the ability of T cells to probe antigen-presenting cells has been elusive. In this study, we probe the spatial relation of microvilli and T-cell receptors (TCRs), the major molecules responsible for antigen recognition on the T-cell membrane. To this end, an effective and robust methodology for mapping membrane protein distribution in relation to the 3D surface structure of cells is introduced, based on two complementary superresolution microscopies. Strikingly, TCRs are found to be highly localized on microvilli, in both peripheral blood human T cells and differentiated effector T cells, and are barely found on the cell body. This is a decisive demonstration that different types of T cells universally localize their TCRs to microvilli, immediately pointing to these surface projections as effective sensors for antigenic moieties. This finding also suggests how previously reported membrane clusters might form, with microvilli serving as anchors for specific T-cell surface molecules.

Ultrastructure and Nanomorphology of the American Mink (Mustela vison) Kidney.

The ultrastructure of the nephron subcellular organelles was studied in healthy mink kidneys. The data obtained were compared with the results of transmission electron microscopy. The renal cell nanomorphology proved to be similar when electronograms and the atomic force microscopy images were analyzed. The methods used enabled us to visualize the glomerular capillary endotheliocytes with cytolemma pits in the area of fenestrae that provide blood filtration; in the proximal nephron part, on the apical pole of the epithelial cells, brush-border soft microvilli were observed. The microvilli were characterized by a well-organized structure along their entire length and the membrane integrity. The data obtained show morphological parameters of the healthy mink organ and can be helpful in diagnosing of nephropathology.

FOXO1 Inhibits Tumor Cell Migration via Regulating Cell Surface Morphology in Non-Small Cell Lung Cancer Cells.

BACKGROUND/AIMS: Cell surface morphology plays pivotal roles in malignant progression and epithelial-mesenchymal transition (EMT). Previous research demonstrated that microvilli play a key role in cell migration of non-small cell lung cancer (NSCLC). In this study, we report that Forkhead box class O1 (FOXO1) is downregulated in human NSCLC and that silencing of FOXO1 is associated with the invasive stage of tumor progression. METHODS: The cell proliferation, migration, and invasion were characterized in vitro, and we tested the expression of the Epithelial-mesenchymal transition (EMT) marker by immunofluorescence staining and also identified the effect of FOXO1 on the microvilli by scanning electron microscopy (SEM). RESULTS: Functional analyses revealed that silencing of FOXO1 resulted in an increase in NSCLC cell proliferation, migration, and invasion; whereas overexpression of FOXO1 significantly inhibited the migration and invasive capability of NSCLC cells in vitro. Furthermore, cell morphology imaging showed that FOXO1 maintained the characteristics of epithelial cells. Immunofluorescence staining and western blotting showed that the E-cadherin level was elevated and Vimentin was reduced by FOXO1 overexpression. Conversely, the E-cadherin level was reduced and Vimentin was elevated in cells silenced for FOXO1. Furthermore, scanning electron microscopy (SEM) showed that FOXO1 overexpression increased the length of the microvilli on the cell surface, whereas FOXO1 silencing significantly reduced their length. CONCLUSIONS: FOXO1 is involved in human lung carcinogenesis and may serve as a potential therapeutic target in the migration of human lung cancer.

Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing.

A canine model of experimental infection with Cryptosporidium canis.

Cryptosporidium is a genus of protozoal parasites that affects the gastrointestinal epithelium of a variety of hosts. Several models of experimental infection have been described to study the susceptibility, infectivity and pathogenicity among different Cryptosporidium species and isolates. This study aimed to establish an experimental infection of Cryptodporidium canis in canids. Infectivity and pathogenicity have been measured by evaluating the clinical status, pattern of oocyst excretion and histological examination. Results showed that C. canis was not infective for immunocompetent dogs or mice with severe combined immunodeficiency syndrome (SCID). Oocysts were first detected in the feces of immunosuppressed dogs on day 3 post-infection (p.i.), with levels peaking twice on days 10 and 17 p.i. during the patent period. cryptosporidial developmental stages were found in the duodenum and jejunum of dogs in histological sections stained with hematoxylin and eosin (H & E) and using scanning electron microscopy (SEM). Histopathological changes in the intestinal tract of infected dogs were characterized by epithelial metaplasia and dilatation; the integrity of intestinal mucosal epithelial cells was distinctly damaged with whole sheets of cilia sloughed away. Ultrastructural observation data were consistent with histological observations. Based on these findings, the canine model described in this work will be useful to evaluate clinical, parasitological and histological aspects of C. canis infection and will be useful for the further understanding of cryptosporidiosis, drug development, and vaccine development.