Effect of Senecio scandens ethanol extract on gut microbiota composition in mice.

The gut microbiota inhabits the animal intestinal tract, and dysbiosis of the gut microbiota may result in disease. Senecio scandens has pharmaceutical antibacterial activities and is regarded as a broad-spectrum antibiotic in traditional Chinese medicine. Extracts of S. scandens are reported to show strong antimicrobial activity, and quercetin significantly decreases some species in the caecal microflora. However, the bactericidal effects of the extracts on the gut microbiota remain obscure. Here, we supplied ethanol extract of S. scandens, which might possibly be used as an alternative for chemical antibiotics, to mice to investigate the state of the intestinal microbiota. Our studies included a control group, low-, moderate-, and high-dose ethanol extract groups, and cefixime capsule group. The ethanol extract groups did not present reduced diversity or differences in the gut microbiota balance. There were significant differences between the ethanol extract and cefixime capsule groups in terms of the gut microbiota. The control and ethanol extract groups contained similar bacteria, which suggested that the ethanol extract has no inhibitory effect on the gut microbiota in vivo. Bifidobacteriales and Lactobacillus acidophilus were significantly increased in the high-dose group. Both secretory immunoglobulin A and mucin 2 concentrations increased as the dose of ethanol extract increased. The functional prediction differences between the control and ethanol extract groups decreased with increasing extract doses, which indicated that the low-dose and high-dose extract treatments might regulate different pathways and functions of the gut microbiota. The results also highlighted the prevention of bacterial drug resistance in the ethanol extract groups.

Influence of Lacto-Immuno-Vital on growth performance and gene expression of IgA, MUC-2, and growth factor IGF-2 in the jejunum of broiler chickens.

The effects of Lacto-Immuno-Vital synbiotic preparation on gene expression of IgA, MUC-2, and growth factor IGF-2 in the jejunum and on BW gain in broiler chickens were studied. A flock of 64,400 1-day-old Hybrid ROSS 308 chickens was inducted in the 42-day experiment. The chickens were divided into 2 equally size groups in separate halls. The chickens in the experimental (E) group received 500 g of Lacto-Immuno-Vital in 1,000 L of drinking water. The preparation was administered daily from the first day (day 1) to day 7 of the experiment. From day 7 to day 22, it was given in pulsed manner (every third day) at a dose of 300 g in 1,000 L of drinking water. The broiler chickens in the E group gained more weight (P < 0.001) compared with control from day 10 to day 42. Death of animals during feeding period was 1,078 chickens in the E group compared with 1,115 dead chickens in the control group. Feed conversion ratio was 1.61 kg of supplemented diet/kg of BW in the E group compare with 1.67 kg of nonsupplemented diet/kg of BW in control. The relative expression of IgA gene in the jejunum was upregulated on day 22 in the E group compared with control (P < 0.05), whereas relative expression of MUC-2 gene was upregulated in the E group compared with control on day 8 and day 22 (P < 0.05; P < 0.001). Similarly, relative expression of IGF-2 gene was upregulated in the E group compared with control on both samplings (P < 0.01). The composition of Lacto-Immuno-Vital synbiotic preparation showed beneficial effects on growth performance, feed conversion ratio, morbidity, mortality, and selected parameters of mucosal immunity in the chicken jejunum.

Effects of supplementation with ß-carotene on the growth performance and intestinal mucosal barriers in layer-type cockerels.

ß-carotene is a robust modulator of mucosal barriers, and it can amplify the immunoglobulin A (IgA) response via the retinoic acid (RA)-mediated pathway. We investigated the influence of ß-carotene on intestinal barriers in layer-type cockerels. In this study, ß-carotene has a positive influence on growth performance and intestinal morphology. ß-carotene remarkably enhanced serum secretory immunoglobulin A (sIgA) levels, jejunal mucosal sIgA, and IgA concentrations. ß-Carotene significantly enhanced mRNA expression levels of IgA, CC chemokine receptor-9 (CCR9), polymeric immunoglobulin receptor (pIgR), and retinoic acid receptor α (RARα) in the ileal tissues and pIgR in the jejunal tissues. ß-Carotene improves mRNA expression of intestinal barrier-related proteins including: mucin-2 (MUC-2), zonula occludens-2 (ZO-2), occludins (OCLN), and zonula occludens-1 (ZO-1) in the ileal tissues. Moreover, ß-carotene decreased the levels of Escherichia coli and elevates the levels of Lactobacillus. The results indicate that ß-carotene can promote growth performance and contribute to the gradual development of intestinal barriers in Hyline Brown chicks. This study enriches our knowledge about the effects of ß-carotene on intestinal barrier and highlights a theoretical basis of ß-carotene application in the poultry industry.

Effects of astaxanthin-enriched yeast on mucosal IgA induction in the jejunum and ileum of weanling mice.

The present study was conducted to clarify the effects of astaxanthin-enriched yeast on the concentration of immunoglobulin A (IgA), the numbers of IgA antibody-secreting cells (ASC) and the messenger RNA (mRNA) expression of IgA C-region in the jejunum and ileum of weanling mice. Weanling mice were fed rodent feed or astaxanthin-enriched yeast-supplemented rodent feed for 7, 14 or 21 days. Supplemental astaxanthin-enriched yeast increased the numbers of IgA ASC in the jejunum and ileum after 7, 14 and 21 days of treatment. Supplemental astaxanthin-enriched yeast increased IgA concentrations in the jejunum after 21 days of treatment, but IgA concentrations in the ileum were not affected by the treatment. The mRNA expressions of IgA C-region in the jejunum after 14 and 21 days of treatment and the ileum after 14 days of treatment were enhanced by supplementation of astaxanthin-enriched yeast. These results indicate that supplementation of astaxanthin-enriched yeast is effective to enhance the numbers of IgA ASC in the jejunum and ileum and IgA concentrations in the ileum of weanling mice.

Supplemental ß-carotene increases IgA-secreting cells in mammary gland and IgA transfer from milk to neonatal mice.

Mortality of neonates continues to be a major problem in humans and animals. IgA provides protection against microbial antigens at mucosal surfaces. Although ß-carotene supplementation has been expected to enhance retinoic acid-mediated immune response in neonates, the exact mechanism by which ß-carotene enhances IgA production is still unclear. We investigated the effect of supplemental ß-carotene for maternal mice during pregnancy and lactation on IgA antibody-secreting cells (ASC) in mammary gland and guts and on IgA transfer from milk to neonatal mice. Pregnant mice were fed untreated or 50 mg/kg ß-carotene-supplemented diets from 6·5 d postcoitus (dpc) to 14 d postpartum (dpp). Supplemental ß-carotene increased the numbers of IgA ASC in mammary gland (P < 0·05) and ileum (P < 0·001), and also mRNA expression of IgA C-region in ileum (P < 0·05) of maternal mice at 14 dpp, but few IgA ASC were detected in mammary gland at 17·5 dpc. IgA concentration in stomach contents, which represents milk IgA level, was significantly higher (P < 0·01) in neonatal mice born to ß-carotene-supplemented mothers at 7 and 14 dpp, and IgA concentration in serum, stomach contents and faeces increased (P < 0·001) drastically with age. These results suggest that ß-carotene supplementation for maternal mice during pregnancy and lactation is useful for enhancing IgA transfer from maternal milk to neonates owing to the increase in IgA ASC in mammary gland and ileum during lactation.

Use of virus vectors for the expression in plants of active full-length and single chain anti-coronavirus antibodies.

To extend the potential of antibodies and their derivatives to provide passive protection against enteric infections when supplied orally in crude plant extracts, we have expressed both a small immune protein (SIP) and a full-length antibody in plants using two different plant virus vectors based on potato virus X (PVX) and cowpea mosaic virus (CPMV). The alphaSIP molecule consisted of a single chain antibody (scFv) specific for the porcine coronavirus, transmissible gastroenteritis virus (TGEV) linked to the alpha-CH3 domain from human IgA. To express the full-length IgA, the individual light and heavy chains from the TGEV-specific mAb 6A.C3 were inserted into separate PVX constructs and plants were co-infected with both constructs. Western blot analysis revealed the efficient expression of both the SIP and IgA molecules. Analysis of crude plant extracts revealed that both the plant-expressed alphaSIP and IgA molecules could bind to and neutralize TGEV in tissue culture, indicating that active molecules were produced. Oral administration of crude extracts from antibody-expressing plant tissue to 2-day-old piglets showed that both the alphaSIP and full-length IgA molecules can provide in vivo protection against TGEV.

From transcriptome to immunome: identification of DTH inducing proteins from a Phlebotomus ariasi salivary gland cDNA library.

Delayed-type hypersensitivity (DTH) response to arthropod vector salivary proteins is associated with protection against pathogen transmission. Massive cDNA sequencing, high-throughput DNA plasmid construction and DNA immunisation were used to identify twelve DTH inducing proteins isolated from a Phlebotomus ariasi salivary gland cDNA library. Additionally, nine P. ariasi DNA plasmids produced specific anti-saliva antibodies, four of these showed a Th1 immune response while the other two exhibited a Th2 profile as determined by IgG2a and IgG1 isotype switching, respectively. In order to validate the specificity of sand fly DNA plasmids, mice previously exposed to sand fly saliva were intradermally injected once with selected P. ariasi plasmids and a specific DTH response consisting of infiltration of mononuclear cells in varying proportions was observed at 24 and 48 h. This approach can help to identify DTH inducing proteins that may be related to host protection against vector-borne diseases or other disease agents where cellular immune response is protective.

Antibody repertoire development in fetal and neonatal piglets. IV. Switch recombination, primarily in fetal thymus, occurs independent of environmental antigen and is only weakly associated with repertoire diversification.

The epitheliochorial placenta of swine is considered a barrier to Ag and selective transport of IgG, so this species should be an excellent model with which to determine whether switch recombination is Ag dependent. Analysis of Ig levels and Ig isotype profiles in >150 normal and virus-infected fetuses from 38-110 days of gestation (DG) suggested that IgG, IgA, and IgM were most likely the result of de novo fetal synthesis. Although transcripts for IgM could be recovered at DG 50 (114 DG is full gestation) in all major fetal lymphoid tissues, those for IgG and IgA first became prominent at 60 DG in thymus, and transcription and spontaneous secretion became especially pronounced in this organ in older fetuses. Data on transcription, secretion, and serum isotype profiles suggest that although all fetal IgA and IgM may result from de novo synthesis, some IgG may result from low-level selective transport. The complementarity-determining region 3 spectratypes of thymic IgA and IgG transcripts at 70 and 90 days, respectively, were as polyclonal as that of IgM, indicating a broad repertoire of switched B cells although the VDJs transcribed with these switched isotypes in normal fetuses were not diversified in comparison to those from animals exposed to environmental Ags such as age-matched, virus-infected fetuses, colonized isolator piglets, and conventional adults. However, VDJs expressed with switched isotypes were more diversified than those expressed with IgM. Thus, switch recombination in fetal life does not appear to be driven by environmental Ag and is only weakly coupled to VDJ diversification. These findings, and the fact that the oligoclonal IgA and IgM repertoires in a noninductive site of the mucosal immune system (parotid gland) become polyclonal in piglets reared germfree, suggest that initial expansion of the switched cells in the B cell compartment of fetal and neonatal piglets is not driven by environmental Ag.

TPN decreases IL-4 and IL-10 mRNA expression in lipopolysaccharide stimulated intestinal lamina propria cells but glutamine supplementation preserves the expression.

Total parenteral nutrition (TPN) decreases intestinal IgA and levels of Th2 cytokines, interleukin (IL)-4, and IL-10 within the supernatants of intestinal homogenates. These cytokines are known to stimulate IgA production in vitro by cells of the gut-associated lymphoid tissue (GALT). Glutamine (GLN) supplementation of TPN normalizes GALT mass and cytokine levels. Because intestinal homogenates contain mucosa which itself is a source of cytokines, it was unclear whether cytokines change within the GALT itself. This study investigates dietary effects on IL-4 and IL-10 cytokine mRNA expression within isolated GALT lamina propria cells after lipopolysaccharide (LPS) stimulation. Prospective randomized experimental trials were used in this study. Fifty-nine mice were randomized to chow, intravenous TPN (IV-TPN), intragastric TPN (IG-TPN), complex enteral diet (CED), or 2% GLN-supplemented TPN (GLN-TPN). In experiment 1, animals were fed chow, IV-TPN, IG-TPN, or CED for 5 days and received intraperitoneal LPS (100 microg/kg BW), and then were sacrificed 1 h later. Intestine was harvested for GALT lamina propria. Total RNA was extracted from lamina propria cells and cytokine mRNA for IL-4, and IL-10 was measured by reverse transcriptase polymerase chain reaction. IgA levels of intestinal washing were also measured with ELISA. In experiment 2, mRNA for IL-4 and IL-10, and intestinal IgA levels were measured in mice fed chow, IV-TPN, or GLN-TPN as in experiment 1. Both IL-4 and IL-10 mRNA expression decreased significantly in IV-TPN mice compared to chow or CED feeding. IG-TPN resulted in IL-10 mRNA expression significantly lower than chow or CED but significantly better than IV-TPN. GLN preserved IL-4 and IL-10 mRNA levels, which correlated with intestinal IgA levels. Route and type of nutrition as well as GLN influence message for the Th2 type IgA-stimulating cytokines, IL-4 and IL-10, within the primary site of GALT IgA production, the lamina propria.

Polymeric IgA binding to the human pIgR elicits intracellular signalling, but fails to stimulate pIgR-transcytosis.

The intracellular pathway of polymeric immunoglobulin receptor (pIgR) is governed by multiple signals that lead to constitutive transcytosis. In addition, in transfected polarized MDCK cells, polymeric immunoglobulin A (pIgA) binding stimulates rabbit pIgR-transcytosis, owing to phospholipase-C gamma 1 activation and increase of intracellular calcium. Transcytosis of rat pIgR across hepatocytes is similarly accelerated by pIgA injection. In contrast we show here that human Madrin-Darby Canine Kidney (pIgR)-transcytosis, in human Calu-3 and human pIgR-transfected MDCK cells, is not promoted by pIgA, as monitored by a continuous apical release of its secreted ectodomain. However, the incubation of cells expressing human or rabbit pIgR with pIgA induces a comparable IP3 production, and pIgR-transcytosis of either species is accelerated by the protein kinase C (PKC)-activator phorbol myristate acetate. Without pIgA, mimicking phospholipase-C activation by combining low concentrations of phorbol myristate acetate with ionomycin, or high concentrations of ionomycin alone, stimulates the rabbit, but not the human, pIgR transcytosis. These data suggest that the species difference in pIgA-induced pIgR-transcytosis does not stem from the defective production of second messengers, but from a different sensitivity of pIgR to intracellular calcium. Our results outline the danger of extrapolating to humans the abundant data obtained from mucosal vaccination of laboratory animals.

Two stages of increased IgA transfer during lactation in the marsupial, trichosurus vulpecula (Brushtail possum).

The polymeric Ig receptor (pIgR) and J chain molecules are involved in the transfer of IgA across the mammary gland epithelia into milk. The J chain binds two IgA molecules to form dimeric IgA, and the pIgR transports this complex through epithelial cells. We report here the cloning of the first marsupial homologues for the pIgR and J chain from the brushtail possum. Marsupial young are born after a short gestation and are less developed than eutherian newborn. The pouch young is completely dependent on milk as its sole source of nutrition during early lactation and this phase can be considered to be equivalent to an external gestation. Two periods of increased expression of pIgR, J chain, and IgA heavy chain mRNAs were observed in the mammary gland during lactation. The first occurs for a brief period after birth of the pouch young and is likely to reflect IgA transfer via the colostrum. The second period of increased expression, which is unique to marsupials, occurs after the early lactation period and just before young exit the pouch. We propose that this represents a second colostral-like phase at the end of the external gestation.

Enhanced allergic responses to house dust mite by oral exposure to carbaryl in rats.

Epidemiological studies have demonstrated an association between use of carbamate insecticides, including carbaryl, and increased incidence of allergic asthma in farmers. In this study the effect of oral carbaryl exposure on the development of allergic responses to house dust mite (HDM) was examined in female Brown Norway rats. Rats were gavaged for 2 weeks with 0, 2, 10, or 50 mg/kg/day of carbaryl. They were sensitized with a subcutaneous injection of HDM in aluminum hydroxide adjuvant 3 days after the beginning of carbaryl exposure and challenged with antigen via the trachea 1 day after the final carbaryl ingestion. Two days after challenge, antigen-specific cell proliferation in pulmonary lymph nodes was significantly higher in the 50 mg/kg group than in controls, while antigen-specific splenocyte proliferation was decreased in groups dosed with 2, 10, and 50 mg/kg carbaryl. Total protein and lymphocyte number in bronchoalveolar lavage (BAL) fluid were also increased in the 50 mg/kg group. By 7 days after challenge, immune-mediated pulmonary inflammation (eosinophils), antigen-specific immunoglobulin (Ig) E level in serum, and antigen-specific IgE and IgA levels in BAL fluid were significantly elevated in the 50 mg/kg group. No apparent change was observed for lactate dehydrogenase and eosinophil peroxidase in BAL fluid, while the number of BAL macrophages were decreased in groups dosed with 10 and 50 mg/kg carbaryl. The results suggest that carbaryl may cause systemic immune suppression, while enhancing pulmonary allergic responses to house dust mite antigen.

Endogenous cytokine expression profiles in retinoic acid-induced IgA production by LPS-stimulated murine splenocytes.

All-trans-retinoic acid (RA) enhances IgA production by LPS-stimulated murine splenocytes. After stimulation by RA and LPS, or by LPS alone, total RNA was extracted from cultured cells on Days 1 to 4, and the kinetics of expression of various cytokine mRNAs were analyzed by the RT-PCR method. RA induced the expression of IL-5 and TGF-beta 2 mRNAs in the LPS-stimulated cells. In addition, the expression of IL-6 and IL-2 mRNAs was more intensive in RA-stimulated cells than in unstimulated cells. TGF-beta 1 and TGF-beta 3 mRNAs were constitutively expressed in both culture groups. RA enhanced IgA production by LPS-stimulated spleen cells but not that by LPS-stimulated mu(+) naive splenic B-cells. For RA-induced IgA production, the B-cells required T-cells or the culture supernatant from RA-stimulated T-cells. Furthermore, exogenous IL-5 replaced the T-cell requirement, at least in part, in RA-induced IgA production by LPS-stimulated B-cells. This reaction was partially inhibited by anti-TGF-beta-neutralizing antibodies. These findings suggest that RA induces IgA production by (IL-5 + LPS)-stimulated B-cells in TGF-beta-independent and TGF-beta-dependent manners.

Scintigraphic imaging of oncogenes with antisense probes: does it make sense?

Based on the specificity of the Watson-Crick base pairing formation, antisense deoxyoligonucleotides have been used to inhibit the expression of oncogenes in various cancer cells. Activation of an oncogene by means of amplification leads to an increased, detectable amount of the mRNA transcript in the cytoplasm. The aim of this study was to demonstrate that cells which are expressing a particular mRNA transcript do preferentially and specifically retain the antisense probe targeting that mRNA. Using a mouse plasmacytoma cell line (MOPC315) which produces high levels of IgA heavy chain mRNA, a control mouse pre B cell line (7OZ/3B), a human mammary cell line (MCF7) which expresses the erbB2 or neu oncogene, MOPC315 cells as neu-negative controls, and antisense DNA oligonucleotides complementary to the 5' region of the mRNAs and the sense sequence, we have shown that there is a preferential, specific retention of the IgA and neu antisense sequence in MOPC315 and MCF7 cells, respectively. We have further demonstrated that this retention is time and concentration dependent with a maximum at 24 h. We conclude that cancer cells which express a particular oncogene are suitable targets for radiolabeled antisense deoxyoligonucleotides directed toward the oncogene transcript. This work and recent developments in the antisense field lead to the expectation of a new class of radiopharmaceuticals with unique specificity.