Comparative study to evaluate the voluntary acceptance of two liquid oral formulations of ciclosporin in dogs.

BACKGROUND: The purpose of this study was to determine and compare the voluntary acceptance of two oral liquid formulations of ciclosporin, investigational Atopica® oral solution (Elanco Animal Health) and Cyclavance® Oral Solution (Virbac), when given orally via syringe or offered freely after mixing with food to dogs.Twenty-five adult mixed breed dogs were selected for this two-phase study. In Phase 1, 12 (Group I) and 13 (Group II) dogs received Atopica® oral solution and Cyclavance® Oral Solution, respectively, daily for 7 days via an oral syringe. After a 3-day washout period, the dosing was switched for a further 7 days. For Phase 2, dosing was by acceptance from freely offered test article mixed in a small amount of food, approximately 6 h after the routine morning feeding. During the first part of this phase, normal daily ration of food offered in the morning was continuously left in the cage. Group I was offered Atopica® oral solution and Group II was offered Cyclavance® Oral Solution mixed with ~ 25 g of food for 3 days. After another 2-day washout period, the test articles were switched for another 3 days but the animals received food for only 1 h in the morning. Five hours after the food was removed, the test articles with food were offered in the same manner as in the first part of Phase 2. Animals were also monitored for adverse events (AEs). RESULTS: During Phase I, voluntary acceptance rates of 100 and 98.9% were noted for Atopica® oral solution and Cyclavance® Oral Solution, respectively. The corresponding immediate prehension rates during Phase 2 (Period 1) were 61.1 and 56.4%, respectively. During Phase 2 (Period 2), the immediate prehension rates were 69.2, 69.4 and 92.0% for Atopica® oral solution, Cyclavance® Oral Solution and the positive control (DYNE®; High Calorie Liquid Dietary Supplement), respectively. Two adverse events of diarrhea and vomiting, with a probable relationship to the test articles, were reported. CONCLUSION: There was no significant difference in acceptance of the two oral ciclosporin solutions, the investigational Atopica® oral solution (Elanco) and Cyclavance® (Virbac) for dogs.

Induction of monocyte chemotactic protein 1 in colonic epithelial cells by Entamoeba histolytica is mediated via the phosphatidylinositol 3-kinase/p65 pathway.

The role intestinal epithelial cells play in the pathogenesis of amebic colitis is poorly understood. Herein, we demonstrate that secreted and soluble ameba (Entamoeba histolytica) proteins (SAP) induce expression of the chemoattractant monocyte chemotactic protein (MCP) in the colonic epithelial cell lines Caco-2, T84, and LS174T. MCP-1 mRNA induction was both dose and time dependent, with peak induction occurring at 8 h and with 100 mug/ml of SAP. Significant increase in MCP-1 protein expression was observed after 12 h. SAP failed to activate any of the mitogen-activated protein kinase pathways or IkappaB kinase activity. Moreover, inhibiting the classical pathway of NF-kappaB activation did not affect SAP-induced MCP-1 expression. Instead, we find that SAP-induced MCP-1 expression is dependent on posttranslational modification of the NFkappaB p65 subunit. SAP induced phosphorylation of p65 and enhanced NF-kappaB transcriptional activity, which are phosphatidylinositol 3-kinase (PI3 kinase) dependent. Treatment with PI3 kinase inhibitor LY290004 significantly abrogated the activation of Akt, p65, and MCP-1 mRNA induction. We conclude that colonic epithelial cells play a role in the initiation of inflammation by secreting chemokines in response to soluble ameba components.

Suppression of NF-kappaB activation by Entamoeba histolytica in intestinal epithelial cells is mediated by heat shock protein 27.

Little is known about the pathogenesis of Entamoeba histolytica and how epithelial cells respond to the parasite. Herein, we characterized the interactions between E. histolytica and colonic epithelial cells and the role macrophages play in modulating epithelial cell responses. The human colonic epithelial cell lines Caco-2 and T84 were grown either as monoculture or co-cultured in transwell plates with differentiated human THP-1 macrophages for 24 h before stimulation with soluble amebic proteins (SAP). In naive epithelial cells, prolonged stimulation with SAP reduced the levels of heat shock protein (Hsp) 27 and 72. However in THP-1 conditioned intestinal epithelial cells SAP enhanced Hsp27 and Hsp72, which was dependent on the activation of ERK MAP kinase. Hsp synthesis induced by SAP conferred protection against oxidative and apoptotic injuries. Treatment with SAP inhibited NF-kappaB activation induced by interleukin-1beta; specifically, the NF-kappaB-DNA binding, nuclear translocation of p65 subunit, and phosphorylation of IkappaB-alpha were reduced. Gene silencing by small interfering RNA confirmed the role of Hsp27 in suppressing NF-kappaB activation at IkappaB kinase (IKK) level. By co-immunoprecipitation studies, we found that Hsp27 interacts with IKK-alpha and IKK-beta, and this association was increased in SAP-treated conditioned epithelial cells. Overexpression of wild type Hsp27 amplified the effects of SAP, whereas a phosphorylation-deficient mutant of Hsp27 abrogated SAP-induced NF-kappaB inhibition. In conditioned epithelial cells, Hsp27 was phosphorylated at serine 15 after prolonged exposure to SAP. This mechanism may explain the absence of colonic inflammation seen in the majority of individuals infected with E. histolytica.

Regulation of Toll-like receptor-2 expression by the Gal-lectin of Entamoeba histolytica.

The Gal/GalNAc lectin (Gal-lectin) of Entamoeba histolytica is a surface molecule involved in parasite adherence to host cells and is the most promising subunit vaccine candidate against amoebiasis. As macrophages are the major effector cells in host defense against amoebas, we studied the molecular mechanisms by which Gal-lectin activates macrophage. Microarray analysis showed that Gal-lectin up-regulated mRNAs of several cytokines and receptor genes involved in proinflammatory responses. The mechanism whereby the Gal-lectin regulates Toll-like receptor 2 (TLR-2) expression in macrophages was studied. Native Gal-lectin increased TLR-2 mRNA expression in a dose- and time-dependent fashion; peak response occurred with 1 microg/ml after 2 h stimulation. By immunoflourescence, enhanced surface expression of TLR-2 was observed after 12 h. With the use of nonoverlapping anti-Gal-lectin monoclonal antibodies that map to the carbohydrate recognition domain, amino acid 596-1082 was identified as the TLR-2 stimulating region. The Gal-lectin increased TLR-2 gene transcription, and the half-life of the mRNA transcripts was 1.4 h. Inhibition of nuclear factor (NF)-kappaB suppressed TLR-2 mRNA induction by the Gal-lectin. Moreover, cells pretreated with an inhibitor of p38 kinase (SB 208530) inhibited Gal-lectin induced TLR-2 mRNA expression by 40%. We conclude that the Gal-lectin activates NF-kappaB and MAP kinase-signaling pathways in macrophages culminating in the induction of several genes including TLR-2 and hypothesize that this could have a significant impact on macrophage activation and contribute to amoebic pathogenesis.

Mucin and Toll-like receptors in host defense against intestinal parasites.

Gastrointestinal mucin is a constituent of luminal barrier function and is the first line of host defense against invading pathogens. Mucin carbohydrates and amino acids, as well as trapped soluble host defense molecules, serve as substrates for colonization and control or deter pathogen invasion to the underlying mucosal epithelial cells. Toll-like receptors on the surface of epithelial cells act as sensors for invading pathogens, and the ensuing host response limits parasite invasion and leads to adaptive immunity. The latest work in the field and the use of parasite model systems to illustrate the delicate host-parasite interaction at the mucosal surface of the gut are discussed here.