Decreased corneal subbasal nerve fiber length and density in diabetic dogs with cataracts using in vivo confocal microscopy.

OBJECTIVE: To determine whether there is a difference in corneal sensitivity and corneal subbasal nerve plexus (CSNP) morphology in cataractous dogs with diabetes mellitus (DM) versus without DM. ANIMALS STUDIED: Twenty six domestic dogs with cataracts of various breeds presented for phacoemulsification, 13 with DM and 13 without DM. PROCEDURE: The inclusion criteria for the study were dogs with bilateral cataracts and no clinical evidence of corneal disease. The diabetic group had documented hyperglycemia and was currently treated with insulin. The non-diabetic group had no evidence of DM on examination and bloodwork. Complete ophthalmic examination, corneal esthesiometry, and in vivo confocal microscopy of the CSNP was performed for both eyes of each dog. The CSNP was evaluated using a semi-automated program and statistically analyzed. RESULTS: The mean (±SD) CSNP fiber length was significantly decreased in diabetic (3.8 ± 3.0 mm/mm2 ) versus non-diabetic (6.7 ± 1.9 mm/mm2 ) dogs. Likewise, the mean (±SD) fiber density was significantly decreased in diabetic (8.3 ± 3.1 fibers/mm2 ) versus non-diabetic (15.5 ± 4.9 fibers/mm2 ) dogs. The corneal touch threshold was significantly reduced in diabetic (2.1 ± 0.8 cm) versus non-diabetic (2.8 ± 0.4 cm) dogs. There was a non-significant trend towards subclinical keratitis in diabetic (9/13) versus non-diabetic (4/13) dogs. CONCLUSIONS: Morphological and functional abnormalities of the CSNP were present in dogs with DM, including decreased fiber length, fiber density, and corneal sensitivity. These findings are consistent with diabetic neuropathy and could contribute to clinically significant corneal complications after cataract surgery.

Nocardia and Streptomyces keratitis in dogs: In vivo detection of filamentous bacteria by confocal microscopy.

OBJECTIVE: To describe the clinical features of dogs with Nocardia and Streptomyces keratitis, including the results of in vivo confocal microscopy examinations. ANIMAL STUDIED: A 15-year-old, male-castrated, miniature Schnauzer was presented with a multilobulated, cystic, pink, ulcerated corneal mass with surrounding dense leukocyte infiltrates. Cytologic evaluation of a corneal scraping identified pyogranulomatous inflammation and filamentous bacteria. Nocardia nova was cultured from corneal samples. Anterior lamellar keratectomy was performed to excise the affected corneal region and histopathologic evaluation confirmed the diagnosis of pyogranulomatous keratitis. A 10-year-old, male-castrated, Yorkshire terrier was presented for evaluation of a chronic anterior stromal corneal ulcer associated with a brown corneal plaque. Cytologic evaluation of a corneal scraping identified suppurative inflammation and filamentous bacteria. A Streptomyces sp. was cultured from corneal samples. The keratitis in both dogs resolved with therapy. PROCEDURES: In vivo confocal microscopy examination of the corneal lesions in both dogs revealed dense accumulations of leukocytes and clusters of hyperreflective, slender, branching bacterial structures that were approximately 1.5-2.0 µm in diameter and 25-50 µm in length. Confocal microscopy imaging of the Nocardia isolate in vitro, and ex vivo canine corneas experimentally infected with the bacteria, was performed to corroborate the in vivo findings. The morphology of the filamentous bacteria was similar between the in vivo, in vitro, and ex vivo confocal microscopy examinations. CONCLUSIONS AND CLINICAL RELEVANCE: Nocardia and Streptomyces spp. can be associated with infectious keratitis in dogs. In vivo detection of filamentous bacteria in the cornea can be accomplished by confocal microscopy.

Applications of in vivo confocal microscopy in the management of infectious keratitis in veterinary ophthalmology.

In vivo confocal microscopy (IVCM) is a relatively new ocular imaging technique that permits morphological and quantitative assessment of the living cornea on the cellular level. The applications for IVCM in clinical ophthalmology are numerous and diverse. There are several advantages inherent to IVCM over standard diagnostic techniques currently used to confirm a diagnosis of infectious keratitis in veterinary ophthalmology. With IVCM, images can be viewed in real-time providing immediate diagnostic information. Traumatic corneal sampling techniques are avoided, and the procedure can be repeated as frequently as is clinically indicated without risk of corneal tissue damage. Both superficial and deep corneal lesions can be evaluated by IVCM in an atraumatic fashion. Microorganism viability is not required for their detection and specialized diagnostic laboratory assay procedures are not necessary. Many larger infectious agents can be directly identified within corneal lesions by IVCM, including fungi and parasites such as Acanthamoeba spp. In other situations, such as bacterial infectious crystalline keratopathy, the biological systems associated with the microorganism can be detected within the cornea. The current resolution of IVCM is inadequate to directly visualize some corneal infectious agents, such as herpesviruses, but host responses and virus-infected epithelial cells can be identified. This review summarizes the current knowledge and applications of IVCM in the management of infectious keratitis in veterinary ophthalmology, including its use in animals with bacterial, fungal, parasitic, and viral keratitis.

Retinal structural and microvascular abnormalities in retinal dysplasia imaged by OCT and OCT angiography.

OBJECTIVE: To describe the in vivo structural characteristics of multifocal and geographic retinal dysplasia visualized with advanced retinal imaging including confocal scanning laser ophthalmoscopy (cSLO), optical coherence tomography (OCT), en face OCT, and the novel vascular imaging technique OCT angiography (OCTA). DOGS STUDIED AND PROCEDURES: Two dogs were diagnosed with unilateral multifocal or geographic retinal dysplasia and underwent advanced retinal imaging under general anesthesia at the Retinal Disease Studies Facility of the University of Pennsylvania. RESULTS: In both cases, the morphological pattern of the lesions was similar including outer retinal folds that invaginated and formed tubular retinal rosettes, surrounding a central inner retinal thickening (multifocal) or plaque (geographic). The two dogs had multiple vascular anomalies in the lesions such as increased tortuosity, abnormal change of vessel diameter including aneurysms and capillary network disruption. We also identified increased autofluorescence by AF cSLO with short wavelength light source (488 nm and barrier filter at 500 nm), and several areas of photoreceptor loss associated with the lesions. CONCLUSION: The use of OCTA allowed the identification of microvascular abnormalities associated with multifocal and geographic retinal dysplasia in two dogs. To our knowledge, this is the first report where the dye-free OCTA technique is used to study vascular lesions in canine retinas.

Assessment of colorectal anastomosis perfusion with confocal laser endomicroscopy − an experimental study

Introduction: Early diagnosis of complicated healing of colorectal anastomosis can increase the chance for salvage surgery and thus reduce overall morbidity. Confocal laser endomicroscopy (CLE) enables in vivo assessment of tissue perfusion without disturbing its integrity. This experimental study evaluates the potential of CLE for postoperative monitoring of colorectal anastomosis. Methods: A hand-sewn colorectal anastomosis was performed in 9 pigs. The animals were subsequently divided into groups with normal (N=3) and ischemic anastomosis (N=6). Microscopic signs of hypoperfusion were evaluated postoperatively at regular intervals using CLE. Results: Uneven saturation of the images was evident in the group with ischemic anastomosis. The epithelium had inhomogeneous edges and more numerous crypt branching was visible. Tissue oedema quantified as the number of crypts per visual field was already more extensive at the first measurement after induction of ischemia. There was also a significant difference between the values measured before and 10 minutes after ischemia ­ 8.7±1.9 vs. 6.0±1.1 (p=0.013). Conclusion: Postoperative monitoring of the colorectal anastomosis using CLE enables prompt detection of perfusion disorders.

Proteomic Analysis of Lysosomal Membrane Proteins in Bovine Mammary Epithelial Cells Illuminates Potential Novel Lysosome Functions in Lactation.

Lactation of bovine mammary epithelial cells (BMEC) is a complex biological process that involves in various organelles. Studies have shown that lysosome and lysosomal membrane proteins (LMP) plays an important role in lactation of BMEC. But the LMP of BMEC remains poorly understood. To obtain a global view of the LMP of BMEC and the affect of lysosome on lactation, the LMP of BMEC was identified using sequential windowed acquisition of all theoretical mass spectra (LC-SWATH/MS). 1214 LMP were identified and 559 were reported to be localized on lysosomal membrane for the first time in BMEC. Gene ontology annotation of these identified proteins showed that both previously reported casein synthesis-related LMP, such as LAMTOR1, 2, 3, and rRagC, and newly identified casein and milk fat synthesis-related LMP, such as EIF4E and ACAA1, were found. KEGG pathway analysis of these identified proteins showed that some pathways involved in lactation, such as PI3K-Akt, mTOR, insulin, PPAR, and JAK-STAT pathway, were found. The lysosomal location of five proteins (PRKCA, EIF4E, ACAA1, HRAS, and THBS1) was analyzed by laser confocal microscopy, and all five were associated with the lysosomal membrane. These findings help to elucidate lysosome functions in the regulation of lactation. The results implicate lysosomes as important organelles in regulation of lactation of BMEC that have been previously undervalued.

R-Phycoerythrin-labeled Mannheimia haemolytica for the simultaneous measurement of phagocytosis and intracellular reactive oxygen species production in bovine blood and bronchoalveolar lavage cells.

The present study aimed to validate the use of R-phycoerythrin (R-PE)-labeled Mannheimia haemolytica to simultaneously stimulate phagocytosis and intracellular production of reactive oxygen species (ROS) by blood phagocytes in bronchoalveolar lavage (BAL) fluid. Initially, R-PE-labeled M. haemolytica was inactivated using a water bath at 60 °C for 60 min. Afterwards, R-PE labelling of bacteria was confirmed by flow cytometry. The geometric mean fluorescence intensity of R-PE-labeled bacteria (FL2 detector, 585 ±â€¯42 nm) was analyzed by flow cytometry and was 41.5-fold higher than the respective unlabeled controls, confirming the success of bacterial conjugation to R-PE. Phagocytosis and intracellular production of ROS by blood neutrophils and monocytes, and by BAL CD14+ macrophages, in 12 healthy 6-month-old male calves were then performed using R-PE-labeled bacteria and 2',7'-dichlorofluorescein diacetate (DCFH-DA) as probes. Confocal microscopy was used to confirm phagocytosis of R-PE-labeled M. haemolytica by phagocytes and to exclude erroneous measurements of bacteria adhering to the leukocyte membrane. The present study showed that there is no difference in the ROS production without stimulus and in the presence of M. haemolytica by peripheral blood neutrophils and monocytes, in contrast to the increased ROS production by local alveolar macrophages upon stimulation by M. haemolytica. This emphasizes the importance of alveolar macrophages in the maintenance of homeostasis and health of the respiratory system, which can be supported during the inflammatory process by the rapid recruitment of neutrophils with high microbicidal and phagocytic capacity. The method described here provides an easy and feasible tool to measure phagocytosis and intracellular ROS production by phagocytes, especially when commonly used probes for intracellular ROS production were used, such as DCFH-DA and dihydrorhodamine 123.

In vivo confocal microscopy for detection of subconjunctival Onchocerca lupi infection in a dog.

A seven-year-old male castrated mixed-breed dog was diagnosed with bilateral subconjunctival masses. In vivo confocal microscopy facilitated visualization of Onchocerca lupi adult nematodes and their characteristic cuticular morphology. Long, thin, white nematodes were extracted during excisional biopsy. Histopathologic and parasitologic evaluation confirmed the diagnosis of O. lupi. In addition to surgical debulking of the parasitic granulomas, the dog received systemic doxycycline, prednisone, and ivermectin therapy. In vivo confocal microscopy was repeated one year after initial diagnosis, and no remaining nematodes were visible. To the authors' knowledge, this is the first report of use of in vivo confocal microscopy as a noninvasive diagnostic and monitoring tool for canine onchocerciasis.

Long-term culture and differentiation of porcine red bone marrow hematopoietic cells co-cultured with immortalized mesenchymal cells.

Mesenchymal cells are multipotent stromal cells with self-renewal, differentiation and immunomodulatory capabilities. We aimed to develop a co-culture model for differentiating hematopoietic cells on top of immortalized mesenchymal cells for studying interactions between hematopoietic and mesenchymal cells, useful for adequately exploring the therapeutic potential of mesenchymal cells. In this study, we investigated the survival, proliferation and differentiation of porcine red bone marrow hematopoietic cells co-cultured with immortalized porcine bone marrow mesenchymal cells for a period of five weeks. Directly after collection, primary porcine bone marrow mesenchymal cells adhered firmly to the bottom of the culture plates and showed a fibroblast-like appearance, one week after isolation. Upon immortalization, porcine bone marrow mesenchymal cells were continuously proliferating. They were positive for simian virus 40 (SV40) large T antigen and the mesenchymal cell markers CD44 and CD55. Isolated red bone marrow cells were added to these immortalized mesenchymal cells. Five weeks post-seeding, 92±6% of the red bone marrow hematopoietic cells were still alive and their number increased 3-fold during five weekly subpassages on top of the immortalized mesenchymal cells. The red bone marrow hematopoietic cells were originally small and round; later, the cells increased in size. Some of them became elongated, while others remained round. Tiny dendrites appeared attaching hematopoietic cells to the underlying immortalized mesenchymal cells. Furthermore, weekly differential-quick staining of the cells indicated the presence of monoblasts, monocytes, macrophages and lymphocytes in the co-cultures. At three weeks of co-culture, flow cytometry analysis showed an increased surface expression of CD172a, CD14, CD163, CD169, CD4 and CD8 up to 37±0.8%, 40±8%, 41±4%, 23±3% and 19±5% of the hematopoietic cells, respectively. In conclusion, continuous mesenchymal cell cultures were successfully established and characterized and they supported the proliferation of red bone marrow hematopoietic cells, which finally differentiated into monocytic cells and CD4+ and CD8+ cells.

Superficial Keratectomy and Conjunctival Advancement Hood Flap (SKCAHF) for the Management of Bullous Keratopathy: Validation in Dogs With Spontaneous Disease.

PURPOSE: To evaluate the efficacy of superficial keratectomy and conjunctival advancement hood flap (SKCAHF) for the treatment of bullous keratopathy in canine patients. METHODS: Nine dogs (12 eyes) diagnosed with progressive corneal edema underwent superficial keratectomy followed by placement of conjunctival advancement hood flaps. The canine patients were examined pre- and postoperatively using in vivo confocal microscopy, ultrasonic pachymetry (USP), and Fourier-domain optical coherence tomography (FD-OCT). All owners responded to a survey regarding treatment outcomes. RESULTS: Mean central corneal thickness (CCT) as measured by FD-OCT was 1163 ± 290 µm preoperatively and significantly decreased postoperatively to 795 ± 197 µm (P = 0.001), 869 ± 190 µm (P = 0.005), and 969 ± 162 µm (P = 0.033) at median postoperative evaluations occurring at 2.2, 6.8, and 12.3 months, respectively. Owners reported significant improvement (P < 0.05) in vision and corneal cloudiness at 6.8 and 12.3 months postoperatively. The percentage of cornea covered by the conjunctival flap was correlated (P = 0.0159) with a reduction in CCT by USP at 12.3 months postoperatively. All canine patients were comfortable pre- and postoperatively. CONCLUSIONS: SKCAHF results in a reduction of corneal thickness in canine patients with bullous keratopathy. The increase in corneal thickness over time, after performing SKCAHF, is likely because of progressive endothelial decompensation. This surgery is a potentially effective intervention for progressive corneal edema in dogs that may have value in treatment of human patients with bullous keratopathy.

Comparison of in vivo confocal endomicroscopy with other diagnostic modalities to detect intracellular helicobacters.

Intracellular colonisation may serve as a protected niche where Helicobacter spp. organisms evade effective treatment. In dogs, non-Helicobacter pylori-helicobacters are frequently intracellular. Confocal endomicroscopy allows in vivo gastrointestinal imaging and has aided real-time identification of Helicobacter pylori and other intracellular and mucosally associated bacteria. The objectives of this study were: (1) to determine the utility of confocal endomicroscopy to identify non-Helicobacter pylori-helicobacters compared with other diagnostic modalities, and (2) to assess its ability to identify intracellular organisms. Fourteen clinically healthy dogs underwent standard gastroduodenoscopy followed by confocal endomicroscopy using topical acriflavine. Confocal images were obtained from at least five gastric sites. Endoscopic biopsies were obtained for histopathology, PCR and fluorescence in situ hybridisation (FISH). Methodologies were compared for their ability to determine the presence and spatial distribution of gastric helicobacters in dogs. Confocal endomicroscopy provided high quality images allowing in vivo identification of non-Helicobacter pylori-helicobacters in 13 dogs. Histopathology identified helicobacters in 11 dogs. Organisms were identified within the superficial gastric mucus and within gastric pits, and distribution throughout the stomach was diffuse and multi-focal. Confocal endomicroscopy findings correlated with PCR and FISH post-procedure analysis. Only FISH identified intracellular organisms, which were present in 13/14 dogs. Confocal endomicroscopy provided in vivo histology images and was capable of identifying non-Helicobacter pylori-helicobacters during gastroscopy, but was unable to identify intracellular organisms using the current fluorophore protocol.

Brucella pinnipedialis in hooded seal (Cystophora cristata) primary epithelial cells.

BACKGROUND: Marine Brucella spp. have been isolated from numerous pinniped and cetacean species, but pathological findings in association with infection with Brucella pinnipedialis in pinnipeds have been sparse. The capacity of brucellae to survive and replicate within host macrophages underlies their important ability to produce chronic infections, but previous work has shown that B. pinnipedialis spp. are rapidly eliminated from hooded seal (Cystophora cristata) alveolar macrophages. RESULTS: To investigate if multiplication could take place in other hooded seal cell types, primary epithelial cells were isolated, verified to express the epithelial marker cytokeratin and challenged with three different strains of B. pinnipedialis; B. pinnipedialis sp. nov., B. pinnipedialis hooded seal strain B17, and B. pinnipedialis hooded seal strain 22F1. All strains were steadily eliminated and the amounts of intracellular bacteria were reduced to less than one-third by 48 h post infection. Intracellular presence was verified using immunocytochemistry. CONCLUSIONS: So far, intracellular multiplication in seal cells has not been documented for B. pinnipedialis. The lack of intracellular survival in macrophages, as well as in epithelial cells, together with the fact that pathological changes due to B. pinnipedialis infection is not yet identified in seals, suggests that the bacteria may only cause a mild, acute and transient infection. These findings also contribute to substantiate the hypothesis that seals may not be the primary host of B. pinnipedialis and that the transmission to seals are caused by other species in the marine environment.

Evaluation of ABCG2 and p63 expression in canine cornea and cultivated corneal epithelial cells.

OBJECTIVE: To examine the expressions of ABCG2 and p63 in canine corneal epithelia and to evaluate their significance in corneal regeneration. PROCEDURES: Canine corneal and limbal epithelial cells were obtained from five healthy beagle dogs. We analyzed the morphological properties of cultivated limbal and corneal epithelial cells. We compared the expressions of ABCG2 and p63 in the limbus and central cornea by immunohistochemistry and real-time quantitative PCR. We analyzed the expression of these markers in cultivated cells by immunocytochemistry and real-time quantitative PCR. RESULTS: The limbal epithelial cells were smaller and proliferated more rapidly than the corneal epithelial cells in primary cultures. The corneal cells failed to be subcultured, whereas the limbal cells could be subcultured with increasing cell size. ABCG2 was localized in the basal layer of the limbal epithelium, and p63 was widely detected in the entire corneal epithelia. ABCG2 expression was significantly higher, and p63 was slightly higher in the limbus compared with the central cornea. ABCG2 was detected only in limbal cells in primary culture, not in corneal cells or passaged limbal cells. p63 was detected in both limbal and corneal cells and decreased gradually in the limbal cells with the cell passages. CONCLUSIONS: ABCG2 was localized in canine limbal epithelial cells, and p63 was widely expressed in canine corneal epithelia. ABCG2 and p63 could prove to be useful markers in dogs for putative corneal epithelial stem cells and for corneal epithelial cell proliferation, respectively.

Interaction between Campylobacter and intestinal epithelial cells leads to a different proinflammatory response in human and porcine host.

Campylobacter jejuni and Campylobacter coli are recognized as the leading causes of human diarrheal disease throughout the development world. Unlike human beings, gastrointestinal tract of pigs are frequently colonized by Campylobacter to a high level in a commensal manner. The aim of this study was to identify the differences underlying the divergent outcome following Campylobacter challenge in porcine versus human host. In order to address this, a comparative in vitro infection model was combined with microscopy, gentamicin protection assay, ELISA and quantitative PCR techniques. Invasion assays revealed that Campylobacter invaded human cells up to 10-fold more than porcine cells (p<0.05). In addition, gene expression of proinflammatory genes encoding for IL1α, IL6, IL8, CXCL2 and CCL20 were strongly up-regulated by Campylobacter in human epithelial cell at early times of infection, whereas a very reduced cytokine gene expression was detected in porcine epithelial cells. These data indicate that Campylobacter fails to invade porcine cells compared to human cells, and this leads to a lack of proinflammatory response induction, probably due to its pathogenic or commensal behavior in human and porcine host, respectively.

Characterization of SET/I2PP2A isoforms in dogs.

SET is an endogenous protein phosphatase 2A (PP2A) inhibitor and is associated with a poor prognosis in human leukemia. Previously, we reported increased SET protein levels in canine lymphoma cell lines and the potential therapeutic application of SET antagonists in canine lymphoma. Here, we found that canine cells express several isoforms of the SET protein. We cloned 4 isoforms of SET, named SETα, ß, γ and δ. Genomic BLAST showed that the SET genes are located on chromosomes X, 7, 1 and 8, respectively. An immunofluorescent study showed nuclear localization of SETα and ß, and nuclear and cytosolic localization of SETγ and δ. We confirmed that SETα and ß possess the ability to associate with PP2A. Our data reveal the existence of unique SET isoforms that should be taken into account in SET-targeting drug development studies in dogs.

Antimicrobial activity of trout hepcidin.

Hepcidin is an antimicrobial peptide and a hormone produced mostly the liver. It is a cysteine-rich peptide with a highly conserved ß-sheet structure. Recently, we described the hepcidin expression in liver of rainbow trout and its inducibility by iron overloading and lipopolysaccharide (LPS). Thus, in this work, we focused in analyzing the importance of the peptide conformation associated to its oxidative state in the antimicrobial activity. This peptide showed a α-helix conformation in reduced state and the characteristic ß-sheet conformation in the oxidized state. Antimicrobial activity assays showed that the oxidized peptide is more effective than the reduced peptide against Escherichia coli and the important salmon fish pathogen Piscirickettsia salmonis. In addition, confocal analysis of P. salmonis culture exposed to trout hepcidin coupled with rhodamine revealed the intracellular location of this peptide and Sytox permeation assay showed that membrane disruption is not the mechanism of its antimicrobial action. Moreover, a conserved ATCUN motif was detected in the N-terminus of this peptide. This sequence has been described as a small metal-binding site that has been implicated in DNA cleavage. In this work we proved that this peptide is able to induce DNA hydrolysis in the presence of ascorbate and CuCl2. When the same experiments were carried out using a variant with truncated N-terminus no DNA hydrolysis was observed. Our results suggest that correct folding of hepcidin is required for its antimicrobial activity and most likely the metal-binding site (ATCUN motif) present in its N-terminus is involved in the oxidative damage to macromolecules.

In vivo histologically equivalent evaluation of gastric mucosal topologic morphology in dogs by using confocal endomicroscopy.

BACKGROUND: Confocal endomicroscopy (CEM) is an endoscopic technology permitting in vivo cellular and subcellular imaging. CEM aids real-time clinical assessment and diagnosis of various gastrointestinal diseases in people. CEM allows in vivo characterization of small intestinal mucosal morphology in dogs. OBJECTIVE: To determine the feasibility of CEM to evaluate gastric mucosal morphology in dogs and to characterize the appearance in healthy dogs. ANIMALS: Fourteen clinically healthy research colony dogs. METHODS: Experimental study. Under general anesthesia, dogs underwent standard endoscopic evaluation and CEM of the gastric mucosa. In the initial 6 dogs, fluorescent contrast was provided with the fluorophore acriflavine (0.05% solution), applied topically. Subsequently, 8 dogs were assessed using a combination of fluorescein (10% solution, 15 mg/kg IV), followed by acriflavine administered topically. For each fluorophore, a minimum of 5 sites were assessed. RESULTS: Confocal endomicroscopy provided high quality in vivo histologically equivalent images of the gastric mucosa, but reduced flexibility of the endoscope tip limited imaging of the cranial stomach in some dogs. Intravenous administration of fluorescein allowed assessment of cellular cytoplasmic and microvasculature features. Topical application of acriflavine preferentially stained cellular nucleic acids, allowing additional evaluation of nuclear morphology. Identification of Helicobacter-like organisms was possible in 13 dogs. CONCLUSION AND CLINICAL IMPORTANCE: Confocal endomicroscopy provides in vivo images allowing assessment of gastric mucosal morphology during endoscopy, potentially permitting real-time diagnosis of gastrointestinal disease.

Change in ß-catenin localization suggests involvement of the canonical Wnt pathway in Boxer dogs with arrhythmogenic right ventricular cardiomyopathy.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited myocardial disease with high prevalence in the Boxer dog population. It is characterized by replacement of the myocardium with fatty or fibro-fatty tissue. Several mechanisms for the development of ARVC have been suggested, including dysfunction of the canonical Wnt pathway, which is linked to many cellular functions, including growth and differentiation of adipocytes. HYPOTHESIS: Wnt pathway dysfunction is involved in the development of ARVC in the Boxer as evidenced by mislocalization of ß-catenin, an integral Wnt pathway modulator, and striatin, a known Wnt pathway component. ANIMALS: Five dogs without ARVC and 15 Boxers with ARVC were identified by 24-hour Holter monitoring and histopathologic examination of the heart. METHODS: Right ventricular samples were collected and examined using confocal microscopy, Western blots, and quantitative (q) PCR. RESULTS: Confocal microscopy indicated that ß-catenin localized at sites of cell-to-cell apposition, and striatin localized in a diffuse intracellular pattern in hearts without ARVC. In hearts affected with ARVC, both ß-catenin and striatin were colocalized with the endoplasmic reticulum (ER) marker calreticulin. Western blots indentified a 50% increase in the amount of ß-catenin in ARVC samples. No change in ß catenin mRNA was detected using qPCR. CONCLUSIONS: Our data suggest that trafficking of Wnt pathway proteins from the ER to their proper location within the cell is inhibited in Boxers with ARVC. These results suggest that disturbances in the Wnt pathway may play a role in the development of ARVC in the Boxer.

Enteral leptin administration affects intestinal autophagy in suckling piglets.

Leptin has been shown to play an integral role in the endocrine regulation of metabolism. Moreover, a substantial amount of this peptide has been found in colostrum and milk. The aim of the study was to investigate the effects of exogenous leptin, administered intragastrically, on the process of autophagy and the changes in cell hyperplasia and hypertrophy in the small intestine mucosa. Three groups (n = 6) of neonatal piglets were used in the study. The pigs were fed either by their sows (sow-reared piglets) or with only milk formula, or with milk formula together with leptin administered via a stomach tube (10 µg/kg BW) every 8 h for 6 d. We have shown that pure milk formula feeding significantly elevates (P < 0.05) autophagy compared with that observed in sow-reared piglets. Compared with the control group, feeding milk formula supplemented with leptin resulted in a significant decrease (P < 0.05) in immunodetection of microtubule-associated protein 1 light chain 3, as well as significantly accelerated epithelial cell renewal (P < 0.05). We demonstrated that autophagy is involved in the remodeling of the small intestine mucosa and that leptin, when administered enterally, may be an important factor for its regulation.

Analysis of reactive oxygen species, Ca²+ , and Hsp70 in the gill and mantle of clams Ruditapes philippinarum exposed in cadmium.

In this study, the probes 2',7'-dichlorofluorescein diacetate (H2 DCF-DA) and Fluo-3 AM were used to investigate the instantaneous change of reactive oxygen species (ROS) and Ca(2+) in the gill and mantle of clams Ruditapes philippinarum exposed in 0.05 mg L(-1) Cd(2+) with the laser-scanning confocal microscopy. The results indicated that Ca(2+) level was declined in the gill and slightly increased in the mantle. The level of ROS was declined in the gill, while the oscillation of ROS level was observed in the mantle. These data revealed that Ca(2+) could stimulate mitochondrial activity and enhance the respiratory chain in the gill and mantle. In addition, the expression of Hsp70 was increased in the gill and mantle of clams exposed in 0.05 mg L(-1) Cd(2+) . The change of Ca(2+) and ROS level affected the expression of Hsp70 in the gill and mantle. An appropriate method was established to analyze the effects of Cd(2+) on ROS, Ca(2+) , and Hsp70 in the gill and mantle of clams with confocal microscopy. Both confocal microscopy and chemical fluorescent are valuable tools for measurement of time-dependent intracellular ROS and Ca(2+) signals.