Dietary bile acid supplementation reveals beneficial effects on intestinal healthy status of tongue sole (Cynoglossus semiliaevis).

The aim of this study was to investigate the effects of dietary bile acids (BAs) on intestinal healthy status of tongue sole in terms of immunity, antioxidant status, digestive ability, mucosal barrier-related genes expression and microbiota. Three experimental diets were prepared with BA levels at 0 mg/kg (CT), 300 mg/kg (BA1) and 900 mg/kg (BA2) in a commercial basal diet. Each diet was fed to three replicates with 120 fish (10.87 ± 0.32 g) in each tank. After an 8-week feeding trial, growth parameters were significantly enhanced in both BAs supplementary groups (P < 0.05), and compared with CT group, survival rate in BA2 group was significantly improved (P < 0.05). Intestinal lysozyme activity and contents of immunoglobulin M and complement 3 were significantly increased in both BAs supplementary groups (P < 0.05), suggesting an enhancement effect on the non-specific immune response. BAs inclusion also significantly improved intestinal antioxidant capabilities by increasing antioxidase activities and decreasing malondialdehyde levels. In addition, compared with CT group, intestinal digestive ability was substantially enhanced as indicated by the significantly increased lipase activity in BA2 group (P < 0.05) and significantly increased amylase activity in BA1 and BA2 groups (P < 0.05). Coincidentally, BAs inclusion significantly upregulated the relative expression of intestinal mucosal barrier-related genes (P < 0.05). Further, dietary BAs distinctly remodeled intestinal microbiota by decreased the abundance of some potential pathogenic bacteria. In conclusion, dietary BAs supplementation is an effective way to improve the intestinal healthy status of tongue sole.

Baicalein inhibits inflammatory response and promotes osteogenic activity in periodontal ligament cells challenged with lipopolysaccharides.

BACKGROUND: Periodontitis is a chronic infection initiated by oral bacterial and their virulence factors, yet the severity of periodontitis is largely determined by the dysregulated host immuno-inflammatory response. Baicalein is a flavonoid extracted from Scutellaria baicalensis with promising anti-inflammatory properties. This study aims to clarify the anti-inflammatory and osteogenic effects of baicalein in periodontal ligament cells (PDLCs) treated with lipopolysaccharides (LPS). METHODS: Human PDLCs were incubated with baicalein (0-100 µM) for 2 h prior to LPS challenge for 24 h. MTT analysis was adopted to assess the cytoxicity of baicalein. The mRNA and protein expression of inflammatory and osteogenic markers were measured by real-time polymerase chain reaction (PCR), western blot and enzyme-linked immunosorbent assay (ELISA) as appropriate. Alkaline phosphatase (ALP) and Alizarin red S (ARS) staining were performed to evaluate the osteogenic differentiation of PDLCs. The expression of Wnt/ß-catenin and mitogen-activated protein kinase (MAPK) signaling related proteins was assessed by western blot. RESULTS: MTT results showed that baicalein up to 100 µM had no cytotoxicity on PDLCs. Baicalein significantly attenuated the inflammatory factors induced by LPS, including interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), matrix metalloprotein-1 (MMP-1), MMP-2 and monocyte chemoattractant protein 1 (MCP-1) at both mRNA and protein level. Moreover, MAPK signaling (ERK, JNK and p38) was significantly inhibited by baicalein, which may account for the mitigated inflammatory response. Next, we found that baicalein effectively restored the osteogenic differentiation of LPS-treated PDLCs, as shown by the increased ALP and ARS staining. Accordingly, the protein and gene expression of osteogenic markers, namely runt-related transcription factor 2 (RUNX2), collagen-I, and osterix were markedly upregulated. Importantly, baicalein could function as the Wnt/ß-catenin signaling activator, which may lead to the increased osteoblastic differentiation of PDLCs. CONCLUSIONS: With the limitation of the study, we provide in vitro evidence that baicalein ameliorates inflammatory response and restores osteogenesis in PDLCs challenged with LPS, indicating its potential use as the host response modulator for the management of periodontitis.

The effect of Pediococcus acidilactici on mucosal immune responses, growth, and reproductive performance in zebrafish (Danio rerio).

A completely randomized experimental design carried out to investigate the effects of different levels of Pediococcus acidilactici (PA) including 0 (basal diet as a control diet), 1 × 106, 2 × 106, 4 × 106, and 8 × 106 colony-forming unit (CFU) per gram of the diet for 60 days on the mucosal immunity responses, growth, and reproductive performance, in zebrafish, Danio rerio (with mean weigh ± SE: 120 ± 10 mg). The obtained results revealed that the best growth and reproduction indices were related to the concentration of 4 × 106 CFU PA g-1 diet (P < 0.05). The maximum activities of mucosal immune responses including total protein, alternative complement system, IgM, and lysozyme were observed in the fish fed with 4 × 106 CFU PA g-1 diet (P < 0.05). Furthermore, the maximum alkaline phosphatase activity of skin mucus was recorded in the fish fed with 8 × 106 CFU PA g-1 diet (P < 0.05). Fish fed with 4 × 106 CFU PA g-1 diet had the highest villus length and width of the intestine (P < 0.05). Supplementing the diet with 4 × 106 CFU PA g-1 diet more significantly enhanced Cyp19a gene expression in comparison with this in other groups. Hence, PA with a concentration of 4 × 106 CFU g-1 diet can be considered as a proper level of probiotic for improving the health, growth, and reproductive performance of the D. rerio.

Effects of dietary Lactobacillus plantarum (KC426951) in biofloc and stagnant-renewal culture systems on growth performance, mucosal parameters, and serum innate responses of Nile tilapia Oreochromis niloticus.

Effects of dietary Lactobacillus plantarum (KC426951) on growth and innate responses of Nile tilapia Oreochromis niloticus were evaluated in biofloc technology system and stagnant-renewal culture system (SRCS). The 90-day-long experiment contained four treatments: SRCS without probiotic (T1), SRCS with probiotic (T2), biofloc without probiotic (T3), and biofloc with probiotic (T4). The administration dose of probiotic was 2 × 108 CFU kg-1 diet. At the end of experiment, the mean final weights, specific growth rates, feed conversion ratios, and total biomass were significantly (P < 0.05) better in BFT treatments, with no significant effect of probiotic on these parameters in both culture systems. Meanwhile, skin mucosal parameters including total protein (TP), lysozyme (LYZ), alkaline phosphatase (ALP), and protease (PRO) activity were significantly enhanced following probiotic supplementation. T4 treatment displayed a significantly higher LYZ and ALP activity in mucus versus other treatments. Also, serum alternative complement activity was significantly heightened in probiotic-supplemented fish. Superoxide dismutase activity in T4 was detected higher than that of SRCS groups. The results of the current study demonstrated the enhancement of some mucosal and serum innate responses of Nile tilapia in both culture systems upon L. plantarum (KC426951) supplementation.

Dietary supplementation of drumstick tree, Moringa oleifera, improves mucosal immune response in skin and gills of seabream, Sparus aurata, and attenuates the effect of hydrogen peroxide exposure.

In aquatic animals, the mucosal barrier is the first line of innate immune defence against external chemicals and pathogens. In this study, the effects of dietary Moringa oleifera leaf (MOL) supplementation on skin and gill mucosal immunity, antioxidants and stress responses were evaluated in seabream (Sparus aurata) fingerlings exposed to hydrogen peroxide (H2O2). A total of 144 specimens (10.11 ± 0.41 g) were divided into four treatments (three replicates per treatment contained 12 specimens each) and fed a non-supplemented control diet or a 1, 2.5 or 5% MOL-supplemented diet. After three weeks of feeding, six specimens from each aquarium were sampled for blood, mucus and tissues. The other six fish in each aquarium were subjected to H2O2 exposure. The results revealed that MOL did not negatively affect either cortisol or glucose levels. MOL supplementation significantly (P < 0.05) improved skin mucosal immunity-related characteristics, including phosphatase, peroxidase and lysozyme activity and IgM levels. Additionally, MOL upregulated the expression of antioxidant genes (sod and cat), an anti-inflammatory gene (tgf-ß), tight junction protein genes (occludin and zo-1), c3, and igm in both the skin and gills. However, H2O2 exposure significantly (P < 0.05) increased both cortisol and glucose levels and disrupted skin mucosal immune function by significantly (P < 0.05) decreasing phosphatase, peroxidase, protease, antiprotease and lysozyme activity and IgM levels. H2O2 exposure severely decreased the mRNA levels of the studied genes. MOL dietary supplementation at the 5% level successfully attenuated the negative effects of H2O2 on the mucosal immune response in both the skin and gills. In conclusion, dietary MOL supplementation at the 5% level is recommended to improve S. aurata mucosal immune function under both normal and stress conditions. Additionally, exposure to H2O2 disrupts the mucosal immunity of fish. This contributes knowledge on the routes involved in mucosal innate immunity and could help to understand the fish resistance against chemicals exposure. Graphical abstract.

Recent advances in intestinal alkaline phosphatase, inflammation, and nutrition.

In recent years, much new data on intestinal alkaline phosphatase (IAP) have been published, and major breakthroughs have been disclosed. The aim of the present review is to critically analyze the publications released over the last 5 years. These breakthroughs include, for example, the direct implication of IAP in intestinal tight junction integrity and barrier function maintenance; chronic intestinal challenge with low concentrations of Salmonella generating long-lasting depletion of IAP and increased susceptibility to inflammation; the suggestion that genetic mutations in the IAP gene in humans contribute to some forms of chronic inflammatory diseases and loss of functional IAP along the gut and in stools; stool IAP as an early biomarker of incipient diabetes in humans; and omega-3 fatty acids as direct inducers of IAP in intestinal tissue. Many recent papers have also explored the prophylactic and therapeutic potential of IAP and other alkaline phosphatase (AP) isoforms in various experimental settings and diseases. Remarkably, nearly all data confirm the potent anti-inflammatory properties of (I)AP and the negative consequences of its inhibition on health. A simplified model of the body AP system integrating the IAP compartment is provided. Finally, the list of nutrients and food components stimulating IAP has continued to grow, thus emphasizing nutrition as a potent lever for limiting inflammation.

Nanochannels Photoelectrochemical Biosensor.

Nanochannels have brought new opportunities for biosensor development. Herein, we present the novel concept of a nanochannels photoelectrochemical (PEC) biosensor based on the integration of a unique CuxO-nanopyramid-islands (NPIs) photocathode, an anodic aluminum oxide (AAO) membrane, and alkaline phosphatase (ALP) catalytic chemistry. The CuxO-NPIs photocathode possesses good performance, and further assembly with AAO yields a designed architecture composed of vertically aligned, highly ordered nanoarrays on top of the CuxO-NPIs film. After biocatalytic precipitation (BCP) was stimulated within the channels, the biosensor was used for the successful detection of ALP activity. This study has not only provided a novel paradigm for an unconventional nanochannels PEC biosensor, which can be used for general bioanalytical purposes, but also indicated that the new concept of nanochannel-semiconductor heterostructures is a step toward innovative biomedical applications.

Influence of protein conformation and adjuvant aggregation on the effectiveness of aluminum hydroxide adjuvant in a model alkaline phosphatase vaccine.

The mechanism(s) of the enhancement of the immune response by addition of aluminum salt adjuvants to parenterally administered protein-based vaccines is still the subject of debate. It has been hypothesized, however, that destabilization of the antigen structure on the surface of the adjuvant may be important for eliciting immune response. Also, it has been suggested that immune response to adjuvanted vaccines is reduced if the adjuvant particles become aggregated before administration because of processing steps such as freeze-drying. In this study, we tested these hypotheses and examined the immune response in a murine model to various liquid, freeze-dried, and spray freeze-dried formulations of a model vaccine, bovine intestinal alkaline phosphatase adsorbed on aluminum hydroxide. Enzymatic activity of the alkaline phosphatase was used as a sensitive indicator of intact native antigen structure. By manipulating the secondary drying temperature during lyophilization, vaccines were produced with varying levels of alkaline phosphatase enzymatic activity and varying degrees of adjuvant aggregation, as assessed by particle size distribution. Anti-alkaline phosphatase titers observed in immunized mice were independent of both the antigen's retained enzymatic activity and the vaccine formulation's mean particle diameter.

Monoclonal antibodies against tissue-nonspecific alkaline phosphatase. Report of the ISOBM TD9 workshop.

Nineteen monoclonal antibodies (MAbs) against tissue-nonspecific (liver/bone/kidney) alkaline phosphatase (TNALP) were investigated in the ISOBM TD-9 Workshop. These MAbs were generated with antigens obtained from human bone tissue (n = 9), human osteosarcoma cell lines (SaOS-2 and TPX; n = 7) and human liver tissue (n = 3). The evaluation included the following antigen forms: (a) commercially available preparations of human bone ALP (BALP) and liver ALP (LALP); (b) human BALP isoforms, B/I, B1 and B2; and (c) soluble secreted epitope-tagged recombinant human TNALP (setTNALP) expressed in COS-1, osteosarcoma (SaOS-2) and hepatoma (Huh2) cell lines. In addition, 16 TNALP mutant cDNAs corresponding to a wide spectrum of reported hypophosphatasia mutations were used in an attempt to map specific immunoreactive epitopes on the surface of the TNALP molecule. The TD-9 MAbs were evaluated by immunoradiometric (IRMA) assays, cross-inhibition and different enzyme immunoassay designs. No indications of explicit tissue discriminatory immunoreactivities of the investigated MAbs against TNALP were found. However, certain IRMA combinations of MAbs increased the specificity of BALP measurements. All MAbs bound to the three BALP isoforms B/I, B1 and B2, but none of the investigated MAbs were specific for any of the isoforms. Significant differences were, however, found in immunoreactivity between these isoforms, with cross-reactivities ranging from 21 to 109% between the two major BALP isoforms B1 and B2. Desialylation with neuraminidase significantly increased the MAb affinity for the BALP isoforms B/I, B1 and B2, and also decreased the observed differences in cross-reactivity between these isoforms. We suggest, therefore, that the MAb affinity is dependent on the amount/number of terminal sialic acid residues located at the five putative N-glycosylation sites. Based on the overall results, we present a putative three-dimensional model of the TNALP molecule with positioning of the four major antigenic domains (designated A-D) of the investigated MAbs. The TNALP molecule is depicted as a homodimer, hence most, but not necessarily all, epitopes are displayed twice. The antigenic domains were positioned with the following assumptions: domain A was positioned close to the active site since most of these MAbs interfered with the catalytic activity. Interestingly, both MAbs included in the commercial BALP kits were grouped with domain A. Moreover, 4 of the 5 putative N-glycosylation sites (with terminal sialic acid residues) are located within, or with close proximity to, domain A. Domain B was localized at the top flexible loop (crown domain) of the TNALP molecule. Domain C was clearly defined by the IRMA assay combinations and by site-directed mutants of TNALP to be close to residue E281, which is located near the fourth metal binding site, likely to be occupied by a calcium ion. Domain D was positioned close to residues A115, A162 and E174, but this domain was also close to the GPI anchor site. In conclusion, none of the 19 investigated TD-9 MAbs were entirely specific for BALP or LALP, thus indicating that all MAbs bind mainly to epitopes on the common protein core of BALP and LALP and/or common glycosylated epitopes. However, some MAbs (either single or in combination with other MAbs) work sufficiently well to measure BALP when the assayed samples do not contain elevated levels of LALP.

Expression of IL-4, Cepsilon RNA, and Iepsilon RNA in the nasal mucosa of patients with seasonal rhinitis: effect of topical corticosteroids.

BACKGROUND: Nasal allergen provocation has demonstrated that allergen-induced rhinitis is associated with an increase in local IL-4 mRNA and IgE heavy chain (Cepsilon) and IgE heavy chain promoter (Iepsilon) RNA and that pretreatment with topical glucocorticosteroids inhibits the increase in these transcripts. OBJECTIVE: This study was undertaken to determine whether observations made after acute allergen provocation can be extended to the case of chronic exposure experienced during the pollen season. METHODS: Biopsy specimens were obtained from the inferior turbinate of 33 pollen-sensitive subjects with allergic rhinitis before and during pollen season. Patients were randomized in a double-blind fashion and treated with either topical steroids (200 microg fluticasone propionate twice daily; n = 16) or matched placebo nasal spray (n = 17) before the pollen season. Alkaline phosphatase anti-alkaline phosphatase immunocytochemistry was used to identify B cells (CD20+), and in situ hybridization was used to detect IL-4, Cepsilon, and Iepsilon RNA+ cells. RESULTS: Baseline examination revealed IL-4 and Cepsilon RNA but virtually no Iepsilon RNA+ cells in the nasal mucosa. Analysis revealed a significant difference in the expression of Cepsilon and Iepsilon RNA+ cells (p < 0.001). Biopsy specimens taken after antigen exposure exhibited highly significant increases in placebo-treated (p < 0.001) but not steroid-treated patients. In both groups, the number of CD20+ cells was unchanged when preexposure and postexposure biopsy specimens were compared. CONCLUSIONS: These results show strong support for the hypothesis that IgE class switching occurs locally within the nasal mucosa of subjects with seasonal allergic rhinitis and that this response can be inhibited through strategies directed against local IgE production.

A scFv-alkaline phosphatase fusion protein which detects potato leafroll luteovirus in plant extracts by ELISA.

A single chain Fv antibody fragment (scFv) was obtained from a synthetic phage-antibody library after four rounds of selection against purified preparations of potato leafroll luteovirus (PLRV). Nucleotide sequence analysis showed that the scFv belongs to the human V(H)3 family. DNA encoding the scFv was sub-cloned into pDAP2 such that a scFv-alkaline phosphatase fusion protein was produced by transformed bacteria following induction by isopropyl-beta-D-thiogalactopyranoside (IPTG). The fusion protein was obtained at concentrations of 10 mg/l of Escherichia coli culture medium and these fusion protein preparations were used directly in ELISA to detect PLRV in sap extracts from infected plants. Our work is the first report of the selection of a scFv specific for a luteovirus from a synthetic phage-display library and the production of a fusion protein with alkaline phosphatase for the detection of PLRV in infected plants. The results demonstrate the potential of scFv and enzyme-scFv fusion proteins in routine testing for plant virus infection.

Radioimmunoscintigraphy of gynecologic tumors with 131I-labeled anti-PLAP monoclonal antibodies.

Radioimmunoscintigraphy (RIS) was performed in 20 patients with gynecologic tumors, 14 ovarian, 5 cervical, and one endometrial carcinoma. One murine monoclonal antibody (mab) against placental alkaline phosphatase (H7) was used after radiolabeling with 131I. The labeling procedure yielded antibodies with specific activity varying between 60 and 73 MBq/mg mab. Each patient received 57 to 100 MBq of the preparation. RIS was performed 7 to 35 days later. Patients with ovarian adenocarcinoma had an accumulation of activity on RIS at tumor sites (79%, 11/14) verified by ultrasonography, CT, and clinical examination. A low or absent accumulation of activity was seen in patients with cervical tumors. The patient with an endometrial adenocarcinoma was seen to have an activity accumulation at RIS corresponding to tumor sites determined by ultrasound and/or CT. It is concluded that RIS using monoclonal antibodies against placental alkaline phosphatase can provide information which will supplement that gained from other investigations of patients with ovarian adenocarcinomas.

The prenatal development of alkaline phosphatase activity in the hypothalamus of the rat.

The localization of alkaline phosphatase (E.C. 3.1.3.1.) positivity during prenatal development of the hypothalamus of the rat is described. At E12 all layers of the prosencephalon display alkaline phosphatase (AP) positivity. The AP positivity increases from dorsal to ventral. Within the hypothalamic area a second, rostro-ventral gradient exists from E14 onwards. At E18 both gradients have decreased. At E20 almost all AP positivity has disappeared from the hypothalamus, with the exception of some reaction product in the dorsal ventricular matrix of the hypothalamus. The significance of this pattern in relation to the differentiation of the hypothalamus and to the formation of hypothalamic connections is discussed. It is suggested that AP activity is related to the formation of connections.

The detection of viruses by enzyme-linked immunosorbent assay (ELISA).

The use of enzyme-linked antibodies for the detection of two morphologically different plant viruses is described. The technique is extremely sensitive, enabling assay of the viruses at concentrations as low as 10 to 100 ng/ml both in purified preparations and in crude plant extracts.

Characterization of some of the enzymes in ragweed pollen.

Ragweed pollen contains 11 esterase, 5 acid phosphatase, 2 alkaline phosphatose, 2 hexokinase, 2 glucose-6-phosphate dehydrogenase isozymes and one leucine amino peptidase band which can be separated by starch gel electrophoresis. The isozymes were distinguished from one another by their electrophoretic mobility, heat inactivation temperatures and antigenic differences.