Non-controlled, open-label trial to assess clinical and immunological parameters in atopic dogs feeding monoprotein grain free diet versus a standard grain diet.

Canine atopic dermatitis (cAD) is a common inflammatory skin disease that is treated with medicines or allergen-specific immunotherapy. An improvement diet can help treatment of cAD. The purpose of this study was compare two diets on clinical and immunological parameters in atopic dogs without food hypersensitivity. Diet A, a commercial based on rice, was offered to 22 atopic dogs during 30 days and Diet B (grain free, rich in salmon) was given to 8 atopic dogs. Clinical scores were assessed by CADESI-4 and PVAS at the beginning (T0) and at the end of the study (T30). CD4+ and CD8+ were measured in PBMCs, and serum cytokines (TNF-α, IL-10, IL-31 and IL-34) were determined. Both diets decreased CADESI-4 score and Diet A decreased PVAS score (p < 0.05). There were no statistical significant differences between diets at T30 for CD4+ and CD8+. A decrease in the IL-31 concentrations and increase in IL-10 levels (p < 0.05) was observed with Diet A at T30. There were no differences between any of the two diets when the other results at T0 and T30 were compared for any of the parameters analysed. In conclusion, the results indicate that dietary intervention had not influence on cellular component of the immune system, but a positive effect was observed on IL-31, IL-10 serum levels for Diet A. Further studies are needed to enrich dietary components of the food for atopic dogs without food hypersensibility to help improvement the management of the cAD.

Comparison of circulating CD4+, CD8+ lymphocytes and cytokine profiles between dogs with atopic dermatitis and healthy dogs.

INTRODUCTION: Canine atopic dermatitis (cAD) is an inflammatory skin disease characterized by impaired immune function. Changes in the proportions of CD4+ and CD8+ lymphocytes and the serum concentrations of cytokines in the pathogenesis of cAD have been described. OBJECTIVES: To assess whether the changes in the ratio of CD4+ and CD8+ lymphocytes in the peripheral blood of atopic dogs at the time of diagnosis are related to the severity of the disease. Furthermore, we determined whether the changes in the serum concentrations of the cytokines IL-31, IL-34, IL-10, IFN-γ, and TNF-α were different between atopic and control dogs. PROCEDURES: Fifty-six client-owned dogs with atopic dermatitis and 53 healthy control dogs were used. The percentages of CD4+ and CD8+ lymphocytes were determined by imaging flow cytometry. The index of CADESI-03 was calculated. Serum cytokine levels were analyzed using ELISA. RESULTS: Atopic dogs showed a higher percentage of CD8+ lymphocytes, a lower CD4+/CD8+ ratio than healthy dogs, and a positive correlation with CADESI-03. Atopic dogs also showed higher serum IL-31 and IL-34 levels and lower IL-10 levels. A moderate positive correlation was found between serum IL-31 and CADESI-03. CONCLUSIONS: The CD4+/CD8+ ratio may be a sensitive parameter that positively correlates with the severity of cAD, and elevated serum levels of IL-31 and IL-34 may facilitate diagnosis of the disease.

Author Correction: Enabling cell recovery from 3D cell culture microfluidic devices for tumour microenvironment biomarker profiling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Effects of allergen-specific immunotherapy on peripheral blood regulatory T cells and serum concentrations of cytokines and immunoglobulins in horses with allergic dermatitis.

The aim of this study was to assess the effect of allergen-specific immunotherapy (ASIT) on the immunological responses of horses. Blood samples were taken from thirty-two horses with allergic dermatitis treated with ASIT and 10 healthy control horses at 0, 3, 6, 9 and 12 months to investigate the evolution of the percentage of regulatory T cells (Treg) in the peripheral blood and the serum levels of cytokines and immunoglobulins. Clinical improvement was appreciated by the majority of the horses' owners (56.6%). No effect of ASIT on CD4+CD25High Treg cells was found during the one year treatment period. No differences in the percentage of CD4+ T cells were observed between the groups, and no effects of ASIT over time were observed. The percentage of CD25+ T cells was always higher in the ASIT group (17.9 ±â€¯11.3%) than in the control group (7.3 ±â€¯4.4%, p < 0.001). We did not detect any effect of ASIT on the serum levels of TGF-ß, IL-10 and IFN-γ or on the serum concentrations of IgA and IgG4. A reduction in the serum levels of total IgE in the horses with allergic dermatitis was observed at the 6th month (p < 0.05), but increased again at the end of the study. The results indicate that immunotherapy was insufficient to induce significant changes that could indicate T cell tolerance, a shift in cytokine production to more protective Th1 cells. More studies are needed with new vaccine compositions and administration protocols to improve the immunological responses of the horses with allergic dermatitis.

Enabling cell recovery from 3D cell culture microfluidic devices for tumour microenvironment biomarker profiling.

The tumour microenvironment (TME) has recently drawn much attention due to its profound impact on tumour development, drug resistance and patient outcome. There is an increasing interest in new therapies that target the TME. Nonetheless, most established in vitro models fail to include essential cues of the TME. Microfluidics can be used to reproduce the TME in vitro and hence provide valuable insight on tumour evolution and drug sensitivity. However, microfluidics remains far from well-established mainstream molecular and cell biology methods. Therefore, we have developed a quick and straightforward collagenase-based enzymatic method to recover cells embedded in a 3D hydrogel in a microfluidic device with no impact on cell viability. We demonstrate the validity of this method on two different cell lines in a TME microfluidic model. Cells were successfully retrieved with high viability, and we characterised the different cell death mechanisms via AMNIS image cytometry in our model.

In-vitro toxicity of carbon nanotube/polylysine colloids to colon cancer cells.

Single-walled carbon nanotubes (SWCNTs) are thoroughly purified and dispersed in an aqueous solution of high molecular weight poly-L-lysine (pLlys). Human intestinal epithelial Caco-2/TC7 cells are incubated with the SWCNT dispersions in pLlys, and their effects on cell viability are studied by image flow cytometry. No significant changes are observed in the cell culture wells up to pLlys concentrations of 10 µg ml-1. However, high mortality is detected at pLlys concentrations of 100 µg ml-1. The presence of oxygen-free SWCNTs does not modify the effects of pLlys on cell cultures at any of the tested concentrations (≤1 µg ml-1). In addition, SWCNTs having an 8 wt.% of surface oxygen are tested with identical results. Thus, purified SWCNTs, even bearing oxygen functional groups, act as inert particles in the cell culture medium. This result supports the applicability of SWCNTs as carriers in pharmacological formulations against digestive tract diseases.

Protein kinase A signalling is involved in the relaxant responses to the selective ß-oestrogen receptor agonist diarylpropionitrile in rat aortic smooth muscle in vitro.

OBJECTIVES: The oestrogen receptor ß (ERß) selective agonist diarylpropionitrile (DPN) relaxes endothelium-denuded rat aorta, but the signalling mechanism is unknown. The aim of this study was to assess whether protein kinase A (PKA) signalling is involved in DPN action. METHODS: cAMP was measured by radioimmunoassay, HSP20 phosphorylation by 2D gel electrophoresis with immunoblotting, and membrane potential and free cytosolic calcium by flow cytometry. KEY FINDINGS: DPN increased cAMP content and hyperpolarised cell membranes over the same range of concentrations as it relaxed phenylephrine-precontracted aortic rings (10-300 µM). DPN-induced vasorelaxation was largely reduced by the PKA inhibitors Rp-8-Br-cAMPS (8-bromoadenosine-3', 5'-cyclic monophosphorothioate, Rp-isomer) and H-89 (N-(2-bromocynnamyl(amino)ethyl)-5-isoquinoline sulfonamide HCl) (-73%) and by the adenylate cyclase inhibitor MDL12330A (cis-N-(2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine)) (-65.5%). Conversely, the PKG inhibitor Rp-8-Br-cGMP was inactive against DPN vasorelaxation. In aortic smooth muscle segments, DPN increased PKA-dependent HSP20 phosphorylation, an effect reversed by H-89. Relaxant responses to DPN were modestly antagonised (-23 to -48% reduction; n=12 per compound) by the potassium channel inhibitors iberiotoxin, PNU-37883A, 4-aminopyridine, or BaCl(2) . All four potassium channel inhibitors together reduced DPN relaxation by 86±9% (n=12) and fully blocked DPN hyperpolarisation. CONCLUSIONS: ERß-dependent relaxation of rat aortic smooth muscle evokes an adenylate cyclase/cAMP/PKA signalling pathway, likely activating the cystic fibrosis transmembrane conductance regulator chloride channel and at least four potassium channels.

Endothelium-independent vasorelaxation by the selective alpha estrogen receptor agonist propyl pyrazole triol in rat aortic smooth muscle.

OBJECTIVES: This study investigated the signalling mechanism of the relaxant responses to the estrogen receptor (ERalpha) agonist PPT (propyl pyrazole triol) in endothelium-denuded rat aortic rings. METHODS: Several compounds, including protein kinase G (PKG) inhibitors and potassium channel inhibitors, were tested against PPT-dependent rat aortic relaxation. Cyclic GMP and cytosolic calcium responses to PPT in isolated aortic smooth muscle were investigated in parallel. KEY FINDINGS: PPT vasorelaxation was largely reduced by the selective ERalpha antagonist methyl-piperidinopyrazole (MPP; -91.6+/-2.5%), by the selective PKG inhibitor Rp-8-Br-cGMP (-78.6+/-4.9%), by the specific soluble guanylyl cyclase inhibitor ODQ (1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one; -85.3+/-5.2%) and to a lesser extent by the selective BKCa (large-conductance calcium- and voltage-activated potassium channel) inhibitor iberiotoxin (-59.3%), the selective IKCa (intermediate-conductance calcium-activated potassium channel) inhibitor TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole; -50.7%) and the voltage-gated potassium channel inhibitor 4-aminopyridine (-40.8%). In isolated aortic smooth muscle, PPT strongly enhanced the cyclic GMP content (+144%) and Rp-8-Br-cGMP largely reduced the PPT-dependent calcium signal (-80.8%). CONCLUSIONS: ERalpha receptor stimulation in rat aortic smooth muscle evokes a PKG-signalling pathway, likely triggering relaxation by BKCa and IKCa channel opening.

Role of chloride transport proteins in the vasorelaxant action of nitroprusside in isolated rat aorta.

Chloride ions play a key role in smooth muscle contraction, but little is known concerning their role in smooth muscle relaxation. Here we investigated the effect of chloride transport inhibitors on the vasorelaxant responses to nitroprusside in isolated and endothelium-denuded rat aorta, precontracted with phenylephrine 1 muM. Incubation of aortic rings in NO(3)(-) media strongly potentiated the vasorelaxant responses to nitroprusside. Bumetanide, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) and acetazolamide strongly potentiated the vasorelaxant responses to nitroprusside (by 70-100%). EC(50) were 2.3+/-0.5 microM for bumetanide, 26+/-15 microM for DIDS and 510+/-118 microM for acetazolamide (n=6 for condition). Niflumic acid, a selective inhibitor of ClCa (calcium-activated chloride channels), potentiated nitroprusside relaxation to a similar extent as chloride transport inhibitors, in a non-additive manner. Zinc and nickel ions, both modestly potentiated nitroprusside vasorelaxation (by 20-30%). Cobaltum had negligible effect on nitroprusside vasorelaxation. CPA (p-chlorophenoxy-acetic acid), an inhibitor of volume-sensitive chloride channels (ClC), slightly potentiated nitroprusside vasorelaxation (by 15%), and the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel inhibitors CFTR(inh)172 (5-[(4-Carboxyphenyl)methylene]-2-thioxo-3-[(3-trifluoromethyl)phenyl-4-thiazolidinone), DPC (diphenylamine-2,2'-dicarboxylic acid) and glibenclamide were without significant effect. In conclusion, inhibition of chloride transport proteins strongly potentiates the vasorelaxant responses to nitroprusside in isolated rat aorta. This effect seems mediated by chloride depletion and inhibition of a chloride channel activated by both, calcium and cyclic GMP (cGMP).