Development of an oral nanovaccine for dogs against Echinococcus granulosus.

Dogs are the main source of animal and human cystic echinococcosis caused by the Cestode parasite Echinococcus granulosus. Dog vaccination seems to be a good strategy to control this parasitic disease. Here we present the development of a polymeric nanoparticle-based oral vaccine for dogs against Echinococcus granulosus delivered in enteric-coated capsules. To achieve our target, we encapsulated two recombinant antigens into biodegradable polymeric nanoparticles in the presence of Monophosphoryl lipid A as an adjuvant to ensure efficient delivery and activation of a protective mucosal immune response. The formulated delivery system showed a nanoparticle size less than 200 nm with more than 80 % antigen encapsulation efficiency and conserved integrity and immunogenicity. The nanoparticle surface was coated with chitosan to enhance adhesion to the gut mucosa and a subsequent antigen delivery. Chitosan-coated nanoparticles showed a higher cell internalization in murine macrophages and dendritic cells as well as a higher penetration into Caco-2 cells in vitro. Antigen-loaded nanoparticles were freeze-dried and enteric-coated capsules were filled with the obtained powder. The obtained results show a promising nanoparticles delivery system for oral vaccination.

Implication of Netrin-1 Gain of Expression in Canine Nodal Lymphoma.

Netrin-1 is a member of the laminin superfamily, and is known to interact with specific receptors, called dependence receptors. While upon netrin-1 binding these receptors initiate positive signaling, in absence of netrin-1, these receptors trigger apoptosis. Tumor cells can avoid apoptosis by inactivating these receptors or by gaining ligand expression. The aim of the present study was to investigate the expression of netrin-1, the ligand of dependence receptors, in canine healthy lymph nodes (LN), and in lymphomas and to evaluate efficiency of a netrin-1 interfering compound in cell cultures from canine lymphoma. Thirty-two control LN and 169 lymphomas were analyzed through immunohistochemistry. Netrin-1 was expressed in the nucleoli of lymphoid and non-lymphoid cells in controls. Acquisition of a cytoplasmic expression was present in B-cell lymphomas (23.1 % in low-grade and 50.6% in high-grade) and T-cell lymphomas (50.0 % in low-grade and 78.8 % in high-grade), with a significant difference between the high- and low-grade in B-cell lymphomas. Through flow cytometry, we showed a significant increase in netrin-1 expression in either high-grade B-cell and T-cell lymphomas (19 and 5, respectively) compared with healthy LN (5), likewise an RT-qPCR analysis demonstrated a significant increase in netrin-1 expression level in 14 samples of lymphomas compared with eight samples of healthy LN. A T-cell aggressive canine lymphoma cell line and four primary canine nodal lymphomas cell cultures were treated with a netrin-1 interfering antibody. Apoptosis by measuring caspase 3 activity was significantly increased in the cell line and viability was decreased in three of the four primary cell cultures. Together, these data suggest that netrin-1 expression is increased in lymphoma, and more specifically in high-grade lymphomas, and that netrin-1 can act as a survival factor for the neoplastic cells, and so be a therapeutic target.

Long-term successful treatment of a donkey with cutaneous lupus erythematous with methotrexate.

Cutaneous lupus erythematosus (CLE) is a rare immune-mediated dermatitis. To the best of the authors' knowledge it has not been described in donkeys. A 5-year-old male neutered donkey, living in south-east France, was diagnosed with CLE. Clinical signs included generalized symmetrical areas of alopecia, erythema, crusting and scales. Diagnostic tests included examination of skin biopsy samples by histopathological and immunohistochemical analysis which demonstrated an interface dermatitis with CD8+ T cells. The skin condition was successfully treated initially with glucocorticoids and methotrexate; successful long-term maintenance was associated with administration of methotrexate.

Le lupus cutané érythémateux (CLE) est une dermatite à médiation immune rare. A la connaissance des auteurs, il n'a pas été décrit chez le singe. Un singe mâle castré de 5 ans, vivant dans le sud-est de la France a été diagnostiqué avec CLE. Les signes cliniques incluaient des zones symétriques généralisées d'alopécie, d'érythème, de croûtes et de pellicules. Les tests diagnostics comprenaient un examen histopathologique et immunohistochimique de biopsies cutanées qui ont révélé une dermatite d'interface avec cellules T CD8+. La dermatose a été traitée avec succès initialement avec des corticoïdes et du méthotrexate; un traitement efficace au long cours a été associé avec l'administration de méthotrexate.

El lupus eritematoso cutáneo (CLE) es una rara dermatitis inmunomediada. A entender de los autores, esta enfermedad no se ha descrito en burros. Un burro castrado macho de 5 años de edad, que vive en el sureste de Francia fue diagnosticado con CLE. Los signos clínicos incluyeron áreas simétricas generalizadas de alopecia, eritema, costras y escamas. Las pruebas de diagnóstico incluyeron el examen de muestras de biopsia de piel mediante análisis histopatológico e inmunohistoquímico que demostró una dermatitis de interfase con células T CD8+. La condición de la piel se trató con éxito inicialmente con glucocorticoides y metotrexato; el control exitoso a largo plazo de la enfermedad se obtuvo con la administración de metotrexato.

O lúpus eritematoso cutâneo (LEC) é uma dermatite imunomediada rara. De acordo com os conhecimentos do autor, a doença ainda não foi descrita em jumentos. Um jumento macho castrado de cinco anos de idade, habitante do sul da França, foi diagnosticado com LEC. Os sinais clínicos incluíram alopecia, eritema, crostas e descamação generalizadas e simétricas. Os testes diagnósticos utilizados foram avaliação de amostras de biópsia por análise histopatológica e imunohistoquímica, que demonstraram dermatite de interface com células T CD8+. A dermatopatia foi tratada satisfatoriamente inicialmente com glicocorticoide e metotrexato; a manutenção satisfatória a longo prazo foi associada à administração de metotrexato.

In vitro evaluations on canine monocyte-derived dendritic cells of a nanoparticles delivery system for vaccine antigen against Echinococcus granulosus.

Since dogs play a central role in the contamination of humans and livestock with Echinococcus granulosus, the development of an effective vaccine for dogs is essential to control the disease caused by this parasite. For this purpose, a formulation based on biodegradable polymeric nanoparticles (NPs) as delivery system of recombinant Echinococcus granulosus antigen (tropomyosin EgTrp) adjuved with monophosphoryl lipid A (MPLA) has been developed. The obtained nanoparticles had a size of approximately 200 nm in diameter into which the antigen was correctly preserved and encapsulated. The efficiency of this system to deliver the antigen was evaluated in vitro on canine monocyte-derived dendritic cells (cMoDCs) generated from peripheral blood monocytes. After 48 h of contact between the formulations and cMoDCs, we observed no toxic effect on the cells but a strong internalization of the NPs, probably through different pathways depending on the presence or not of MPLA. An evaluation of cMoDCs activation by flow cytometry showed a stronger expression of CD80, CD86, CD40 and MHCII by cells treated with any of the tested formulations or with LPS (positive control) in comparison to cells treated with PBS (negative control). A higher activation was observed for cells challenged with EgTrp-NPs-MPLA compared to EgTrp alone. Formulations with MPLA, even at low ratio of MPLA, give better results than formulations without MPLA, proving the importance of the adjuvant in the nanoparticles structure. Moreover, autologous T CD4+ cell proliferation observed in presence of cMoDCs challenged with EgTrp-NPs-MPLA was higher than those observed after challenged with EgTrp alone (p<0.05). These first results suggest that our formulation could be used as an antigen delivery system to targeting canine dendritic cells in the course of Echinococcus granulosus vaccine development.

In situ synthesis of Fe3O4 nanoparticles coated by chito-oligosaccharides: physico-chemical characterizations and cytotoxicity evaluation for biomedical applications.

Fe3O4 nanoparticles coated with chito-oligosaccharides (COS) were prepared in situ by a simple co-precipitation method through a mixing of iron ions (Fe3+ and Fe2+) and COS aqueous solutions followed by precipitation with ammonia. The impact of COS with different degree of polymerization (DP 10, 24 and 45) and degree of N-acetylation (DA) âˆ¼ 24% and 50% (exhibiting high solubility) on the synthesis and physical properties of the coated magnetic nanoparticles was evaluated. Several advantages were found when the magnetic nanoparticles were prepared in the presence of the studied COS, such as: preparation of functionalized magnetic nanoparticles with narrower size distributions and, consequently, higher saturation magnetization (an increase of up to 22%); and an expressive increasing in the concentration of COS-coated magnetic nanoparticles (up to twice) in the cell viability test in comparison with pure Fe3O4 nanoparticles. Furthermore, among the analyzed samples, the magnetic nanoparticles coated by COS with DA âˆ¼ 50% present a higher cytocompatibility. Our results allow envisioning various biomedical applications, valorizing the use of coated-magnetic nanoparticles for magnetic-field assisted drug delivery, enzyme or cell immobilization, or as a marker for specific cell tracking, among others.

Milk Fat Globules Hamper Adhesion of Enterohemorrhagic Escherichia coli to Enterocytes: In Vitro and in Vivo Evidence.

Enterohemorrhagic Escherichia coli (EHEC; E. coli) are food-borne agents associated with gastroenteritis, enterocolitis, bloody diarrhea and the hemolytic-uremic syndrome (HUS). Bovine milk glycans have been shown to contain oligosaccharides which are similar to host epithelial cell receptors and can therefore prevent bacterial adhesion. This study aimed to describe interactions between EHEC O157:H7 EDL933 and O26:H11 21765 and milk fat globules (MFGs) in raw milk and raw milk cheese, and the impact of MFGs on EHEC strains adhesion to the intestinal tract in vitro and in vivo. Both EHEC serotypes clearly associated with native bovine MFGs and significantly limited their adhesion to a co-culture of intestinal cells. The presence of MFGs in raw milk cheese had two effects on the adhesion of both EHEC serotypes to the intestinal tracts of streptomycin-treated mice. First, it delayed and reduced EHEC excretion in mouse feces for both strains. Second, the prime implantation site for both EHEC strains was 6 cm more proximal in the intestinal tracts of mice fed with contaminated cheese containing less than 5% of fat than in those fed with contaminated cheese containing 40% of fat. Feeding mice with 40% fat cheese reduced the intestinal surface contaminated with EHEC and may therefore decrease severity of illness.

Fetal and neonatal exposure to the mycotoxin zearalenone induces phenotypic alterations in adult rat mammary gland.

Zearalenone is a mycotoxin that is widespread in cereal food. We questioned whether this mycotoxin, administered during known critical exposure periods such as the fetal period and the first days of life, at doses compatible with mean daily intake in humans, could have an effect on mammary gland development in rodents. Wistar female rats were exposed to zearalenone (0.2 µg/kg to 5mg/kg) during the last 14 days of fetal life and the first 5 post-natal days (PND). The mammary tissue was examined for development and maturation by morphologic analyses and immunochemistry. At PND 30, the mean length of terminal buds was significantly enhanced in all of the zearalenone-exposed females (p<0.05). The mammary tissue, as evaluated by scoring of tissue slides, was significantly more differentiated in the 1mg/kg treated group than in controls (p<0.05). At PND 180, mammary tissue was more differentiated in all of the zearalenone treated groups (p<0.05). At six months, 4 of 18 females exposed to 5mg/kg of zearalenone presented mammary hyperplasia lesions. The induction of phenotypic alterations by zearalenone administered in utero and in the neonatal period at doses as low as 0.2 µg/kg suggests that zearalenone could contribute to the induction of breast endocrine disorders.

In vitro infection of aortic endothelial cells by caprine arthritis encephalitis virus enhances in vitro transmigration of peripheral blood leukocytes and modulates their phenotypic expression.

Infection of goats by caprine arthritis-encephalitis virus (CAEV) provides a convenient example of the infiltration of various tissues by leukocytes following a natural lentiviral infection. This event is important in determining organ susceptibility and local immunity. Caprine vascular endothelial cells are susceptible to infection by CAEV in vitro, so we have investigated the consequences of this infection on the transmigration of uninfected leukocytes in an in vitro model. After in vitro infection by CAEV or stimulation by TNFalpha, the endothelial cells allowed the passage of tenfold more leukocytes from uninfected donors than did the uninfected endothelial cells. The transmigrating leukocytes were enriched in CD8+ lymphocytes, and the leukocytes appeared to have been activated during transmigration, as demonstrated by their expression of IL2R, MHC class II antigens and gamma-delta T-lymphocyte markers. CD4+, CD8+ and B-lymphocytes all proliferated in culture after transmigration. These results suggest that any possible infection or specific stimulation of endothelia in an infected animal could profoundly influence the choice of target organs and could activate the cells involved in local mucosal immune responses.