Effects of glucagon‐like peptides 1 and 2 and epidermal growth factor on the epithelial barrier of the rumen of adult sheep

Epidermal growth factor (EGF) and glucagon‐like peptides (GLP) modulate the tight junctions (TJ) of the intestinal epithelial barrier (EB) of monogastric animals. This work tried to elucidate whether GLP‐1, GLP‐2 and EGF can affect the EB of the rumen. Ovine ruminal epithelia were incubated in Ussing chambers for 7 hr with 25 or 250 nM of either GLP‐1 or GLP‐2 on the serosal side, with 2.5 nM of EGF on the serosal side or with 0.25 or 2.5 nM EGF on the mucosal side. No treatment affected tissue conductance. Short‐circuit current (I_sc) was affected by time and treatment and their interactions. Only 250 nM of either GLP‐1 or GLP‐2 decreased I_sc in certain periods compared with 25 nM GLP‐1 or 0.25 nM mucosally applied EGF; however, not when compared to control epithelia. Fluorescein flux rates (J_fluor) of ruminal epithelia were affected by treatment, time and time × treatment interaction. The time × treatment interaction was based on an increase in J_fluor between the first and last hour in epithelia incubated with 25 nM GLP‐1 or GLP‐2 and in epithelia incubated with EGF. After 7 hr incubation, claudin‐7 mRNA expression was downregulated in all treatments. Claudin‐1 mRNA was upregulated after incubation with 2.5 nM EGF on the serosal side, claudin‐4 mRNA was downregulated by 2.5 nM EGF on the mucosal side, and occludin mRNA was increased after incubation with 250 nM GLP‐2. The protein abundance of all tested TJ proteins was not influenced by treatment. We conclude that GLP‐1, GLP‐2, and EGF have no obvious acute effects on the EB of ruminal epithelia under simulated physiological conditions ex vivo. However, by decreasing the mRNA expression of claudin‐7 and partly affecting other TJ proteins, they may modulate EB in the longer term or under certain conditions.     

N‐Acetyl‐d‐galactosamine prevents soya bean agglutinin‐induced intestinal barrier dysfunction in intestinal porcine epithelial cells

Soya bean agglutinin (SBA) is a glycoprotein and the main anti‐nutritional component in most soya bean feedstuffs. It is mainly a non‐fibre carbohydrate‐based protein and represents about 10% of soya bean‐based anti‐nutritional effects. In this study, we sought to determine the effects of N‐Acetyl‐D‐galactosamine (GalNAc or D‐GalNAc) on the damage induced by SBA on the membrane permeability and tight junction proteins of piglet intestinal epithelium (IPEC‐J2) cells. The IPEC‐J2 cells were pre‐cultured with 0, 0.125 × 10^?4, 0.25 × 10^?4, 0.5 × 10^?4, 1.0 × 10^?4 and 2.0 × 10^?4 mmol/L GalNAc at different time period (1, 2, 4 and 8 hr) before being exposed to 0.5 mg/ml SBA for 24 hr. The results indicate that pre‐incubation with GalNAc mitigates the mechanical barrier injury as reflected by a significant increase in trans‐epithelial electric resistance (TEER) value and a decrease in alkaline phosphatase (ALP) activity in cell culture medium pre‐treated with GalNAc before incubation with SBA as both indicate a reduction in cellular membrane permeability. In addition, mRNA levels of the tight junction proteins occludin and claudin‐3 were lower in the SBA‐treated groups without pre‐treatment with GalNAc. The mRNA expression of occludin was reduced by 17.3% and claudin‐3 by 42% (p < 0.01). Moreover, the corresponding protein expression levels were lowered by 17.8% and 43.5% (p < 0.05) respectively. However, in the GalNAc pre‐treated groups, occludin and claudin‐3 mRNAs were reduced by 1.6% (p > 0.05) and 2.7% (p < 0.01), respectively, while the corresponding proteins were reduced by 4.3% and 7.2% (p < 0.05). In conclusion, GalNAc may prevent the effect of SBA on membrane permeability and tight junction proteins on IPEC‐J2s.     

Aspartame supplementation in starter accelerates small intestinal epithelial cell cycle and stimulates secretion of glucagon‐like peptide‐2 in pre‐weaned lambs

The objective of this study was to test the hypothesis that aspartame supplementation in starter diet accelerates small intestinal cell cycle by stimulating secretion and expression of glucagon‐like peptide ?2 (GLP‐2) in pre‐weaned lambs using animal and cell culture experiments. In vivo, twelve 14‐day‐old lambs were selected and allocated randomly to two groups; one was treated with plain starter diet (Con, n = 6) and the other was treated with starter supplemented with 200 mg of aspartame/kg starter (APM, n = 6). Results showed that the lambs received APM treatment for 35 d had higher (p < .05) GLP‐2 concentration in the plasma and greater jejunum weight/live body weight (BW) and jejunal crypt depth. Furthermore, APM treatment significantly upregulated (p < .05) the mRNA expression of cyclin D1 in duodenum; and cyclin A2, cyclin D1, cyclin‐dependent kinases 6 (CDK6) in jejunum; and cyclin A2, cyclin D1, CDK4 in ileum. Moreover, APM treatment increased (p < .05) the mRNA expression of glucagon (GCG), insulin‐like growth factor 1 (IGF‐1) in the jejunum and ileum and mRNA expression of GLP‐2 receptor (GLP‐2R) in the jejunum. In vitro, when jejunal cells were treated with GLP‐2 for 2 hr, the 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide (MTT) OD, IGF‐1 concentration, and the mRNA expression of IGF‐1, cyclin D1 and CDK6 were increased (p < .05). Furthermore, IGF‐1 receptor (IGF‐1R) inhibitor decreased (p < .05) the mRNA expression of IGF‐1, cyclin A2, cyclin D1 and CDK6 in GLP‐2 treatment jejunal cells. These results suggest that aspartame supplementation in starter accelerates small intestinal cell cycle that may, in part, be related to stimulate secretion and expression of GLP‐2 in pre‐weaning lambs. Furthermore, GLP‐2 can indirectly promote the proliferation of jejunal cells mainly through the IGF‐1 pathway. These findings provide new insights into nutritional interventions that promote the development of small intestines in young ruminants.     

Eleutheroside B increase tight junction proteins and anti‐inflammatory cytokines expression in intestinal porcine jejunum epithelial cells (IPEC‐J2)

Eleutheroside B (EB) is a phenylpropanoid glycoside with anti‐inflammatory properties, neuroprotective abilities, immunomodulatory effects, antinociceptive effects, and regulation of blood glucose. The aim of this study was to investigate the effects of EB on the barrier function in the intestinal porcine epithelial cells J2 (IPEC‐J2). The IPEC‐J2 cells were inoculated into 96‐well plates at a density of 5 × 10³ cells per well for 100% confluence. The cells were cultured in the presence of EB at concentrations of 0, 0.05, 0.10, and 0.20 mg/ml for 48 hr. Then, 0.10 mg/ml was selected as the suitable concentration for the estimation of transepithelial electric resistance (TEER) value, alkaline phosphatase activity, proinflammatory cytokines mRNA expression, tight junction mRNA and protein expression. The results of this study indicated that the supplementation of EB in IPEC‐J2 cells decreased cellular membrane permeability and mRNA expression of proinflammatory cytokines, including interleukin‐6 (IL‐6), interferon‐γ (INF‐γ), and tumour necrosis factor‐α (TNF‐α). The supplementation of EB in IPEC‐J2 cells increased tight junction protein expression and anti‐inflammatory cytokines, interleukin 10 (IL‐10) and transforming growth factor beta (TGF‐β). In addition, the western blotting and real‐time quantitative polymerase chain reaction (RT‐qPCR) results indicated that EB significantly (p < 0.05) increased the mRNA and protein expression of intestinal tight junction proteins, Claudin‐3, Occludin, and Zonula Occludins protein‐1 (ZO‐1). Therefore, dietary supplementation of EB may increase intestinal barrier function, tight junction protein expression, anti‐inflammatory cytokines, and decrease proinflammatory cytokines synthesis in IPEC‐J2 cells.     

Involvement of PKA signalling in anti‐inflammatory effects of chitosan oligosaccharides in IPEC‐J2 porcine epithelial cells

Weaning is characterized by intestinal inflammation, which is a big challenge in pig industry. Control of intestinal inflammation is important for improvement of growth performance and health. Therefore, the study was focused on the anti‐inflammatory activity of low‐molecular‐weight chitosan oligosaccharide (LCOS) in a porcine small intestinal epithelial cell line (IPEC‐J2). The results showed that TNF‐α, as inflammation inducer, significantly upregulated the mRNA expression of IL‐8 and MCP‐1. Afterwards, LCOS significantly attenuated mRNA expression of IL‐8 and MCP‐1 induced by TNF‐α in the cells. Mannose (MAN), as ligand of mannose receptor, had no effect on the anti‐inflammatory activity of LCOS, which suggested that mannose receptor may not involve in the anti‐inflammatory activity of LCOS in IPEC‐J2 cells. Interestingly, N‐[2‐(p‐bromocinnamylamino)ethyl]‐5‐isoquinolinesulfonamide 2HCl hydrate (H89), as PKA (protein kinase A)‐specific inhibitor, reversed the mRNA expression of IL‐8 when co‐cultured with LCOS. Furthermore, LCOS concentration dependent downregulated the mRNA expression of claudin‐1 compared with TNF‐α treatment. However, the trans‐epithelial electric resistance (TEER) was not affected by LCOS when co‐cultured with TNF‐α in 3 hr. In conclusion, LCOS have a potent anti‐inflammatory activity, and as a feed additives, may be useful for the inhibition of inflammatory process in weaning period of pigs with intestinal inflammation occurring.     

Pharmacokinetics of enrofloxacin HCl‐2H2O (ENRO‐C), PK/PD,and Monte Carlo modeling vs. Leptospira spp. in cows

The pharmacokinetics, PK/PD ratios, and Monte Carlo modeling of enrofloxacin HCl‐2H₂O (Enro‐C) and its reference preparation (Enro‐R) were determined in cows. Fifty‐four Jersey cows were randomly assigned to six groups receiving a single IM dose of 10, 15, or 20 mg/kg of Enro‐C (Enro‐C₁₀, Enro‐C₁₅, Enro‐C₂₀) or Enro‐R. Serial serum samples were collected and enrofloxacin concentrations quantified. A composite set of minimum inhibitory concentrations (MIC) of Leptospira spp. was utilized to calculate PK/PD ratios: maximum serum concentration/MIC (C_max/MIC₉₀) and area under the serum vs. time concentration of enrofloxacin/MIC (AUC_0‐24/MIC₉₀). Monte Carlo simulations targeted C_max/MIC = 10 and AUC_0‐24/MIC = 125. Mean C_max obtained were 6.17 and 2.46 μg/ml; 8.75 and 3.54 μg/ml; and 13.89 and 4.25 μg/ml, respectively for Enro‐C and Enro‐R. C_max/MIC₉₀ ratios were 6.17 and 2.46, 8.75 and 3.54, and 13.89 and 4.25 for Enro‐C and Enro‐R, respectively. Monte Carlo simulations based on C_max/MIC₉₀ = 10 indicate that only Enro‐C₁₅ and Enro‐C₂₀ may be useful to treat leptospirosis in cows, predicting a success rate ≥95% when MIC₅₀ = 0.5 μg/ml, and ≥80% when MIC₉₀ = 1.0 μg/ml. Although Enro‐C₁₅ and Enro‐C₂₀ may be useful to treat leptospirosis in cattle, clinical trials are necessary to confirm this proposal.     

β‐carotene attenuates lipopolysaccharide‐induced inflammation via inhibition of the NF‐κB,JAK2/STAT3 and JNK/p38 MAPK signaling pathways in macrophages

β‐carotene is one of the most abundant carotenoids, has potential anti‐inflammatory effect, it has been reported that β‐carotene could suppress LPS‐induced inflammatory responses by inhibiting nuclear factor kappa B (NF‐κB) translocation, but the more detailed molecular mechanisms underlying the anti‐inflammatory action of β‐carotene remain to be fully understood. In this study, we investigated the influence of β‐carotene on the activation of JAK2/STAT3, MAPK, and NF‐κB signaling pathway induced by LPS in RAW264.7 cells and peritoneal macrophages. Cells were treated with different concentrations of β‐carotene for 3 hr after LPS treatment for 24 hr. The mRNA expression and the release of IL‐1β, IL‐6, and TNF‐α were evaluated by RT‐PCR and ELISA, and the level of signaling proteins of JAK2/STAT3, MAPK, and NF‐κB signaling pathway were detected by Western blot. The results showed that β‐carotene significantly suppressed (p < 0.05) LPS‐induced release of IL‐1β, IL‐6, and TNF‐α and their mRNA expression. LPS‐induced JAK2/STAT3, IκB/NF‐κB p65, JNK/p38 MAPK signal activation were significantly attenuated (p < 0.05) by β‐carotene in a dose‐dependent manner. In conclusion, β‐carotene could attenuate LPS‐induced inflammation via inhibition of the NF‐κB, JAK2/STAT3, and JNK/p38 MAPK signaling pathways in macrophages.     

Influences of protein ingestion on glucagon‐like peptide (GLP)‐1‐immunoreactive endocrine cells in the chicken ileum

Influences of a specific dietary nutrient on glucagon‐like peptide (GLP)‐1‐containing cells in the chicken intestine are not yet clear. Significance of dietary protein level on GLP‐1‐containing cells in the chicken ileum was investigated. Chickens fed control or experimental diets of varying protein levels were examined using immunohistochemical and morphometrical techniques. We show that the protein ingestion had an impact on the activities of GLP‐1‐immunoreactive cells in the chicken ileum. Weight gains declined with decreasing dietary crude protein (CP) levels, but no significant differences were detected in the daily feed intake and villous height. GLP‐1‐immunoreactive cells with a round or oval shape were frequently observed in the lower CP level groups (4.5% and 0%). Frequencies of occurrence of GLP‐1‐immunoreactive cells were 41.1 ± 4.1, 38.5 ± 4, 34.8 ± 3.1 and 34.3 ± 3.7 (cells/mm², mean ± SD) for dietary CP level of 18%, 9%, 4.5% and 0% groups, respectively and significant differences were recognized between the control and lower CP level groups (P < 0.05). Multiple regression analysis indicated a significant correlation between the daily protein intake and frequencies of occurrence of GLP‐1‐immunoreactive cells. The protein ingestion is one of the signals that influence GLP‐1‐containing cells in the chicken small intestine.     

Pharmacokinetics and bioavailability after intramuscular injection of the 5‐HT1A serotonin agonist R‐8‐hydroxy‐2‐(di‐n‐propylamino) tetralin (8‐OH‐DPAT) in domestic goats (Capra aegagrus hircus)

To determine the bioavailability and pharmacokinetic properties of the serotonin 5‐HT_1A receptor agonist R‐8‐OH‐DPAT in goats, and 0.1 mg kg^?1 R‐8‐OH‐DPAT hydrobromide was administered intramuscularly (i.m.) and intravenously (i.v.) to six goats in a two‐phase cross‐over design experiment. Venous blood samples were collected from the jugular vein 2, 5, 10, 15, 20, 30, 40 and 60 min following treatment and analysed by liquid chromatography tandem mass spectrometry. Bioavailability and pharmacokinetic parameters were determined by a one‐compartment analysis. Mean bioavailability of R‐8‐OH‐DPAT when injected i.m. was 66%. The mean volume of distribution in the central compartment was 1.47 L kg^?1. The mean plasma body clearance was 0.056 L kg^?1 min^?1. All goats injected i.v. and two of six goats injected i.m. showed signs of serotonin toxicity. In conclusion, R‐8‐OH‐DPAT is well absorbed following i.m. injection and the observed pharmacokinetics suggest that administration via dart is feasible. Administration of R‐8‐OH‐DPAT hydrobromide, at a dosage of 0.1 mg kg^?1, resulted in the observation of clinical signs of serotonin toxicity in the goats. It is suggested that dosages for the clinical use of the compound should be lower in order to achieve the desired clinical effect without causing serotonin toxicity.     

Raf‐ERK1/2 signalling pathways mediate steroid hormone synthesis in bovine ovarian granulosa cells

Steroid hormones are required for normal reproductive function of female. The aim of this study was to investigate the role of Raf‐ERK1/2 on steroid hormone synthesis in bovine ovarian granulosa cells. Immunohistochemistry assay showed that both B‐Raf and C‐Raf were expressed in granulosa cells, theca cells and Sertoli cells. The protein expression of Raf or ERK1/2 was clearly decreased by Raf inhibitor GSK2118436 or ERK1/2 inhibitor SCH772984, respectively (p < 0.05). In addition, western blotting was performed for investigating the crosstalk between Raf and ERK1/2, the data showed that Raf positively regulated ERK1/2, whereas ERK1/2 had a negative feedback effect on Raf. The biosynthesis of oestradiol or testosterone was significantly decreased by treatment with GSK2118436 or SCH772984 (p < 0.05). Conversely, the progesterone biosynthesis was clearly increased by treatment with those inhibitors (p < 0.05). Furthermore, the mRNA expression of STAR, aromatase and CYP17 was blocked by Raf‐ERK1/2 signalling inhibition, which oppositely induced the mRNA expression of CYP11. Together, these findings suggested that Raf‐ERK1/2 signalling pathways mediate steroid hormone synthesis via affecting the expression of steroidogenic enzymes.     

miR‐26a suppresses autophagy in swine Sertoli cells by targeting ULK2

A large number of microRNAs (miRNAs) have been detected from porcine testicular tissues thanks to the development of high‐throughput sequencing technology. However, the regulatory roles of most identified miRNAs in swine testicular development or spermatogenesis are poorly understood. In our previous study, ULK2 (uncoordinated‐51‐like kinase 2) was predicted as a target gene of miR‐26a. In this study, we aimed to investigate the role of miR‐26a in swine Sertoli cell autophagy. The relative expression of miR‐26a and ULK2 levels has a significant negative correlation (R² = .5964, p ≤ .01) in nine developmental stages of swine testicular tissue. Dual‐luciferase reporter assay results show that miR‐26a directly targets the 3′UTR of the ULK2 gene (position 618–624). In addition, both the mRNA and protein expression of ULK2 were downregulated by miR‐26a in swine Sertoli cells. These results indicate that miR‐26a targets the ULK2 gene and downregulates its expression in swine Sertoli cells. Based on the expression of marker genes (LC3, p62 and Beclin‐1), overexpression of miR‐26a or knock‐down of ULK2 inhibits swine Sertoli cell autophagy. Taken together, these findings demonstrate that miR‐26a suppresses autophagy in swine Sertoli cells by targeting ULK2.     

The effect of tributyrin supplementation to milk replacer on plasma glucagon‐like peptide 2 concentrations in pre‐weaning calves

The objective of this study was to evaluate the effect of tributyrin (TB) supplementation to milk replacer (MR) on performance, health, and blood concentrations of metabolite and glucagon‐like peptide (GLP‐2) in pre‐weaning calves. Twenty Holstein heifer calves were raised on an intensified nursing program using MR supplemented with either palm oil (CON) or TB (TB) at 0.3% (as fed basis) for 7 weeks starting 1 week after birth. Calves were fed a calf starter and kleingrass from the beginning of the study. Blood samples were obtained weekly to measure blood glucose, serum β‐hydroxybutyric acid (BHBA), insulin‐like growth factor 1 (IGF‐1), and plasma GLP‐2 concentrations. Starter DMI and metabolizable energy (ME) intake were lower in TB calves at 46, 47, from 49 to 55 days after birth compared with the CON calves. However, any growth parameters were not affected by TB treatment. Blood glucose, serum BHBA, and IGF‐1 concentrations were not affected by TB supplementation. On the other hand, mean plasma GLP‐2 concentration among whole experimental period was higher for TB (0.60 ng/ml) compared with CON (0.41 ng/ml). In conclusion, feeding MR supplemented with TB increases plasma GLP‐2 concentration, which might counterbalance the growth performance of TB calves despite the decreased ME intake.     

Insulin‐independent actions of glucagon‐like peptide‐1 in wethers

Insulin‐independent actions of glucagon‐like peptide‐1 (GLP‐1) are not yet clear in ruminants. Four Suffolk mature wethers (60.0 ± 6.7 kg body weight (BW)) were intravenously infused with insulin (0.5 mU/kg BW/min; from 0 to 90 min) and GLP‐1 (0.5 μg/kg BW/min; from 60 to 150 min) with both hormones co‐administered from 60 to 90 min, in a repeated‐measure design under euglycemic clamp for 150 min, to investigate whether GLP‐1 has insulin‐independent actions. Jugular blood samples were taken at 15‐min intervals for plasma hormones and metabolites analysis. Compared to baseline concentrations (at 0 min), insulin infusion decreased (P < 0.05) plasma concentrations of glucagon, non‐esterified fatty acids (NEFA), lactate, nonessential amino acids (NEAA), branched‐chain amino acids (BCAA), total amino acids (TAA) and urea nitrogen (UN). Insulin plus GLP‐1 infusion induced a greater increase (P < 0.05) in plasma concentrations of insulin and triglyceride (TG), but decreased (P < 0.05) glucagon, total cholesterol (T‐Cho), NEAA and UN plasma concentrations. GLP‐1 infusion increased (P < 0.05) NEFA, β‐hydroxybutyrate and TG, but decreased (P < 0.05) glucagon, T‐Cho, NEAA, BCAA and UN plasma concentrations. In conclusion, GLP‐1 exerts extrapancreatic roles in ruminants not only insulin‐independent but probably, in contrast to non‐ruminants, antagonistic to insulin effects.     

Inhibition of PI3K‐Akt‐mTOR signal pathway dismissed the stimulation of glucose on goose liver cell growth

In the formation of goose fatty liver induced by a high‐carbohydrate diet, it is characterized by the quick cell growth of liver. The carbohydrate is mostly digested and absorbed in the small intestine by the form of glucose. Recent studies have suggested a crucial role for PI3K‐Akt‐mTOR pathway in regulating cell proliferation, and then we speculate that PI3K‐Akt‐mTOR pathway may mediate glucose‐induced liver cell proliferation. Goose primary hepatocytes were isolated and incubated in either no addition as a control or glucose or PI3K‐Akt‐mTOR pathway inhibitors or cotreatment with glucose and PI3K‐Akt‐mTOR pathway inhibitors. The results firstly showed that 35 mmol/l glucose stimulated the mRNA level and protein content of factors involved in PI3K‐Akt‐mTOR signal pathway in goose primary hepatocytes. Secondly, 35 mmol/l glucose evidently changed the cell cycle PI index and protein expression of cyclin D1. Meanwhile, the upregulation of 35 mmol/l glucose on the DNA synthesis rate, cell cycle PI index, the mRNA expression, protein content and protein expression of factors involved in the cell proliferation was decreased significantly by the inhibitors of PI3K‐Akt‐mTOR pathway, LY294002, rapamycin or NVP‐BEZ235. In summary, glucose could stimulate the cell proliferation, and the PI3K‐Akt‐mTOR pathway inhibitors could dismiss glucose‐induced the upregulation of cell proliferation in goose primary hepatocyte.     

Lactobacillus rhamnosus GG promotes M1 polarization in murine bone marrow‐derived macrophages by activating TLR2/MyD88/MAPK signaling pathway

Lactobacillus rhamnosus GG (LGG) is increasingly applied in functional food products and acts as a probiotic model in nutritious and clinical studies. Increasing evidences have revealed the immune modulation of LGG on macrophages. The aim of this study is to investigate the effect of LGG on macrophage polarization of murine bone marrow‐derived macrophages (BMDMs). BMDMs were treated with 10⁸ colony‐forming units (CFU)/ml LGG for 1.5, 3, and 6 hr. Results showed that LGG obviously upregulated the mRNA expression of M1‐associated cytokines (p < .05), including interleukin‐1 beta (IL‐1β), IL‐6, tumor necrosis factor‐alpha (TNF‐α), and inducible nitric oxide synthase (iNOS), whereas had no effect on the expression of M2‐associated markers (p > .05), including arginase 1 (Arg1), mannose receptor, and chitinase‐like protein 3 (YM1). Furthermore, LGG markedly increased the expression of pro‐inflammatory cytokines (IL‐12p40, cyclooxygenase‐2 [COX‐2], and interferon‐γ [IFN‐γ]) (p < .05) and anti‐inflammatory cytokines (IL‐10, IL‐4, and transforming growth factor‐β [TGF‐β]) (p < .05). In addition, we also found that TLR2/MyD88/MAPK signaling pathway was required for LGG‐induced M1 macrophage polarization and M1‐related cytokines expression. Together, these findings demonstrate that probiotic LGG facilitates M1 polarization of BMDMs, suggesting that LGG may have an immunotherapeutic potential in regulating the host defense against pathogen invasion.     

Anti‐allergic effects of Lactobacillus crispatus KT‐11 strain on ovalbumin‐sensitized BALB/c mice

In this study, we investigated the effects of oral ingestion of Lactobacillus crispatus KT‐11 strain (KT‐11) on the immune response in an allergic rhinitis mouse model, ovalbumin (OVA)‐sensitized BALB/c mice. Sneezing activity in mice that were administered a KT‐11‐supplemented diet was significantly lower than that in mice administered a KT‐11‐free diet (control diet) at age 11 weeks. We found that serum OVA‐specific immunoglobulin E (IgE) levels and total number of interleukin (IL)‐4⁺CD4⁺ spleen cells in mice that were administered a KT‐11‐supplemented diet were significantly lower than in mice administered a control diet. The ratio of spleen interferon‐γ⁺CD4⁺/IL‐4⁺CD4⁺ cells was higher in the mice administered the KT‐11‐supplemented diet compared to that in mice administered the control or L. rhamnosus GG‐supplemented diet. In contrast, the number of CD11b⁺CD80⁺ and FcεRIα⁺CD117⁺ cells was significantly lower in mice administered the KT‐11‐supplemented diet. These results suggested that KT‐11 reduced OVA‐induced allergic symptoms in BALB/c mice via the adjustment of the T helper type 1/T helper type 2 balance, and a decrease in the number of antigen‐presenting cells and high affinity IgE receptor‐positive mast cells.     

Extended‐Spectrum β‐lactam Resistance in the Enteric Flora of Patients at a Tertiary Care Medical Centre

The dissemination of Enterobacteriaceae expressing resistance to extended‐spectrum cephalosporins, which are therapeutically used in both human and veterinary medicine, is of critical concern. The normal commensal flora of food animals may serve as an important reservoir for the zoonotic food‐borne transmission of Enterobacteriaceae harbouring β‐lactam resistance. We hypothesized that the predominant AmpC and ESBL genes reported in US livestock and fresh retail meat products, bla_CMY‐2 and bla_CTX‐M, would also be predominant in human enteric flora. We recovered enteric flora from a convenience sample of patients included in a large tertiary medical centre's Clostridium difficile surveillance programme to screen for and estimate the frequency of carriage of AmpC and ESBL resistance genes. In‐ and outpatient diarrhoeic submissions (n = 692) received for C. difficile testing at the medical centre's clinical diagnostic laboratory from July to December, 2013, were included. Aliquoted to a transport swab, each submission was inoculated to MacConkey broth with cefotaxime, incubated at 37°C and then inoculated to MacConkey agars supplemented with cefoxitin and cefepime to select for the AmpC and ESBL phenotypes, with bla_CMY and bla_CTX‐M genotypes confirmed by PCR and sequencing. From the 692 diarrhoeic submissions, our selective culture yielded 184 isolates (26.6%) with reduced susceptibility to cefotaxime. Of these, 46 (6.7%) samples harboured commensal isolates carrying the AmpC bla_CMY. Another 21 (3.0%) samples produced isolates harbouring the ESBL bla_CTX‐M: 19 carrying CTX‐M‐15 and 2 with CTX‐M‐27. Our results indicate that β‐lactam resistance genes likely acquired through zoonotic food‐borne transmission are present in the enteric flora of this hospital‐associated population at lower levels than reported in livestock and fresh food products.     

The effect of M‐phase stage‐dependent kinase inhibitors on inositol 1,4,5‐trisphosphate receptor 1 (IP3R1) expression and localization in pig oocytes

At fertilization, inositol 1,4,5‐trisphosphate receptor type 1 (IP₃R1) has a crucial role in Ca²⁺ release in mammals. Expression levels, localization and phosphorylation of IP₃R1 are important for its function, but it still remains unclear which molecule(s) regulates IP₃R1 behavior in pig oocytes. We examined whether there was a difference in localization of IP₃R1 after in vitro or in vivo maturation of pig oocytes. In mouse oocytes, large clusters of IP₃R1 were formed in the cortex of the oocyte except in a ring‐shaped band of cortex adjacent to the spindle. However, no such clusters of IP₃R1 were observed in pig oocytes and there was no difference in its localization between in vitro and in vivo matured oocytes. We next tried to clarify which factor(s) regulates IP₃R1 localization, phosphorylation and expression using M‐phase stage‐dependent kinase inhibitors. Our results show that treatments with roscovitine (p34^cdc2 kinase inhibitor) or U0126 (mitogen‐activated protein kinase inhibitor) did not affect IP₃R1 expression or localization in pig oocytes, although the latter strongly inhibited phosphorylation. However, treatment with BI‐2536, an inhibitor of polo‐like kinase 1 (Plk1), dramatically decreased the expression level of IP₃R1 in pig oocytes in a dose‐dependent manner. From these results, it is suggested that Plk1 is involved in the regulation of IP₃R1 expression in pig oocytes.     

Ruminal epithelial insulin‐like growth factor‐binding proteins 2, 3, and 6 are associated with epithelial cell proliferation

The aim of this study was to identify factors that regulate ruminal epithelial insulin‐like growth factor‐binding protein (IGFBP) expression and determine its role in rumen epithelial cell proliferation. Primary bovine rumen epithelial cells (BREC) were incubated with short‐chain fatty acids (SCFAs) at pH 7.4 or 5.6, lactate, lipopolysaccharide (LPS), insulin‐like growth factor‐I (IGF‐I), ‐II (IGF‐II), or recombinant bovine IGFBP2 (rbIGFBP2). The mRNA expression levels of IGFBP in BREC were analyzed using quantitative real‐time polymerase chain reaction (qRT‐PCR). The proliferation rate of BREC was analyzed using a WST‐1 assay. IGFBP2 gene expression tended to be lower with SCFA treatment (p < .1), and IGFBP6 gene expression was significantly lower with SCFA treatment (p < .05). IGFBP3 and IGFBP6 gene expression tended to be higher with d ‐Lactate treatment (p < .1). IGFBP3 gene expression was significantly higher (p < .05) with LPS treatment. BREC treated with IGF‐I grew more rapidly than vehicle control‐treated cells (p < .01); however, recombinant bovine rbIGFBP2 inhibited IGF‐I‐induced proliferation. IGF‐II and/or rbIGFBP2 did not affect BREC proliferation. Taken together, SCFA treatment decreased IGFBP2 and IGFBP6 expression in rumen epithelial cells, and lower expression of these IGFBP might promote rumen epithelial cell proliferation by facilitating IGF‐I.     

Pharmacokinetics of florfenicol in blunt‐snout bream (Megalobrama amblycephala) at two water temperatures with single‐dose oral administration

The pharmacokinetics and residue elimination of florfenicol (FFC) and its metabolite florfenicol amine (FFA) were studied in healthy blunt‐snout bream (Megalobrama amblycephala, 50 ± 10 g). The study was conducted with a single‐dose (25 mg/kg) oral administration at a water temperature of 18 or 28°C, while in the residue elimination study, fish were administered at 25 mg/kg daily for three consecutive days by oral gavage to determine the withdrawal period (WDT) at 28°C. The FFC and FFA levels in plasma and tissues (liver, kidneys and muscle) were analysed using high‐performance liquid chromatography (HPLC). A no‐compartment model was used to analyse the concentration versus time data of M. amblycephala. In the two groups at 18 and 28°C, the maximum plasma concentration (C_max) of FFC was 5.89 and 6.21 μg/ml, while the time to reach C_max (T_max) was 5.97 and 2.84 hr, respectively. These suggested that higher temperature absorbed more drug and more quickly at M. amblycephala. And the elimination half‐life (T_1/2kβ) of FFC was calculated as 26.75 and 16.14 hr, while the total body clearance (CL) was 0.09 and 0.15 L kg^?1 hr^?1, and the areas under the concentration–time curves (AUCs) were 265.87 and 163.31 μg hr/ml, respectively. The difference demonstrated that the elimination rate of FFC in M. amblycephala at 28°C was more quickly than that at 18°C. The results of FFA showed the same trend in tissues of M. amblycephala. After multiple oral doses (25 mg/kg daily for 3 days), the k (eliminate rate constant) of FFA in M. amblycephala muscle was 0.017, the C₀ (initial concentration) was 3.07 mg/kg, and the WDT was 10 days (water temperature 28°C).