Induction of IFNT-Stimulated Genes by Conceptus-Derived Exosomes during the Attachment Period.

Biochemical and/or physical communication between the conceptus and the uterine endometrium is required for conceptus implantation to the maternal endometrium, leading to placentation and the establishment of pregnancy. We previously reported that in vitro co-culture system with bovine trophoblast CT-1 cells, primary uterine endometrial epithelial cells (EECs), and uterine flushings (UFs) mimics in vivo conceptus attachment process. To identify molecules in UFs responsible for this change, we first characterized protein contents of UFs from day 17 cyclic (C17) and pregnant (P17) ewes through the use of two dimensional-Polyacrylamide Gel Electrophoresis (2D-PAGE), followed by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) analysis. These analyses identified 266 proteins specific for P17 UFs, from which 172 proteins were identified as exosomal proteins. Among 172 exosomal proteins, 8 proteins that had been identified as exosomal proteins were chosen for further analysis, including macrophage-capping protein (CAPG), aldo-keto reductase family 1, member B1 protein (AKR1B1), bcl-2-like protein 15 (BCL2L15), carbonic anhydrase 2 (CA2), isocitrate dehydrogenase 2 (IDH2), eukaryotic translation elongation factor 2 (EEF2), moesin (MSN), and ezrin (EZR). CAPG and AKR1B1 were again confirmed in P15 and P17 UFs, and more importantly CAPG and AKR1B1, mRNA and protein, were found only in P15 and P17 conceptuses. Moreover, exosomes were isolated from C15, C17, P15, or P17 UFs. Only P15 and P17 exosomes, originated from the conceptus, contained interferon tau (IFNT) as well as CAPG and AKR1B1, and up-regulated STAT1, STAT2, MX1, MX2, BST2, and ISG15 transcripts in EECs. These observations indicate that in addition to endometrial derived exosomes previously described, conceptus-derived exosomes are present in UFs and could function to modify endometrial response. These results suggest that exosomes secreted from conceptuses as well as endometria are involved in cell to cell interactions for conceptus implantation to the maternal endometrium.

Progesterone initiates Wnt-beta-catenin signaling but estradiol is required for nuclear activation and synchronous proliferation of rat uterine stromal cells.

Progesterone pretreatment of ovariectomized rat uteri increases the number of synchronously proliferating stromal cells in response to estradiol 17-beta. To identify the signals involved in stimulating synchronous proliferation, sexually mature ovariectomized rats were injected with progesterone (2 mg) for 3 consecutive days. Estradiol 17-beta (0.2 microg) was administered to initiate cell cycle entry. Uterine samples were removed at various times after hormone administration and changes in wingless (Wnt) pathway effectors and gene targets were identified by microarray. Progesterone pretreatment decreased glycogen synthase kinase-3beta (GSK-3beta) and increased expression of T-cell factor/lymphoid enhancer factor (TCF/LEF). GSK-3beta protein decreased markedly in the uterine stroma of progesterone-pretreated uteri with the concomitant appearance of beta-catenin in these stromal cells. Translocation of beta-catenin from the cytosol to the nuclei in progesterone-pretreated stromal cells was stimulated in response to estradiol. Beta-catenin binding to TCF/LEF increased (P<0.05) in progesterone-pretreated uteri in response to estradiol. Progesterone stimulated the expression of the Wnt target gene urokinase plasminogen activator receptor (uPA-R) in the periluminal uterine stromal cells. The expression of uPA-R increased in progesterone-pretreated stromal cells in response to estradiol administration. Together, the results indicate that progesterone initiates Wnt signaling in the uterine stroma by down-regulating GSK-3beta. However, nuclear translocation of beta-catenin and sufficient complex formation with TCF/LEF to activate stromal cell cycle entry requires estradiol. Stimulation of a uterine stromal cell line to proliferate and differentiate resulted in beta-catenin accumulation, suggesting that endocrine-dependent Wnt signaling controls proliferation and differentiation (decidualization).

Up-regulation of NOD1 and NOD2 through TLR4 and TNF-alpha in LPS-treated murine macrophages.

NOD1 (Card4) and NOD2 (Card15) are thought to be responsible for cytoplasmic defense against bacterial entry. To gain further knowledge about how their expressions are regulated in murine macrophages, we investigated the expression of NOD1 and NOD2 mRNAs after stimulation with various endotoxins, lipopolysaccharide, lipoteichoic acid and peptidoglycan. In macrophage RAW264.7 cells, the first and second rises in NOD1 and NOD2 mRNAs were observed at 2 hr and at 8-12 hr after endotoxin treatment. Increases in NOD1 and NOD2 mRNAs at 2 hr in lipopolysaccharide-treated RAW264.7 cells were reduced with the use of NF-kappaB inhibitor, caffeic acid phenethyl ester. In RAW264.7 cells, lipopolysaccharide-induced increases in NOD1 and NOD2 mRNAs were inhibited with anti-TLR4 antibody, and partially reduced in peritoneal macrophages obtained from TLR4-deficient mice. Furthermore, NOD1 and NOD2 mRNA expressions in RAW264.7 cells were increased by the treatment with proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), or IL-6. In TNF-alpha deficient macrophages, the expression of NOD molecules was minimal at 12 hr, and the second rise in NOD mRNA seen in lipopolysaccharide-treated RAW264.7 cells was inhibited with anti-TNF-alpha, but not with anti-IL-1beta or anti-IL-6 antibody. These observations suggest that immediate response of NODs to endotoxins could result from NF-kappaB activation via TLR signaling, whereas the second rise in NOD mRNAs might have resulted from TNF-alpha production possibly through NF-kappaB, TLR, and/or NOD signalings.

Intestinal gene expression in TNBS treated mice using genechip and subtractive cDNA analysis: implications for Crohn's disease.

So far it has proven difficult to identify a causative gene(s) or gene product initiating the events that lead to inflammation of the intestinal mucosa and, ultimately, progression to Crohn's disease (CD), an inflammatory bowel disease. However, gene transcripts identified in the intestine of trinitrobenzene sulfonic acid (TNBS)-treated mice might suggest a clue, and even represent candidate genes leading to inflammation and mucosal damage, and to subsequent fibrosis. In the present study, DNA microarray (13000 transcripts) methodology was applied to mucosal RNA extracted from TNBS-treated mice, some transcripts of which were validated via cDNA subtraction and RT-PCR analyses. Intestinal biopsy samples from CD patients were then analyzed using cDNA mini-array (1300 cDNAs), focusing on gene transcripts associated with cancer and immunity. Mini-array results revealed transcript changes similar and also dissimilar to those found from the DNA microarray analysis. These changes, previously known or newly identified, possibly occurring during the initial and progressive stages of inflammatory conditions may provide a clue to identify marker transcripts and/or targets for the development of future gene therapy.

Effects of progranulin on blastocyst hatching and subsequent adhesion and outgrowth in the mouse.

Using cDNA microarray methodology, we have shown previously that transcripts of progranulin gene (Grn, also known as acrogranin), a recently identified autocrine growth factor, were upregulated in mouse blastocysts adhered to the filter membrane in an in vitro-culture system. In the present study, we investigated the expression and effects of progranulin on blastocyst hatching, adhesion, and embryo outgrowth during the peri-implantation period in the mouse. During this period, substantial amounts of Grn mRNA were present in both inner cell mass (ICM) and trophectoderm. Progranulin was localized exclusively to the surface of the trophectoderm in early and pre- and postadhesion blastocysts as well as in trophoblast cells and ICM of outgrowth embryos, being secreted as a single, 88-kDa form into the surrounding medium. NIH3T3 cells that had been transfected with a progranulin expression construct secreted the 88-kDa form of the protein, from which a 68-kDa form could be generated by deglycosylation. In vitro treatment of blastocysts with recombinant progranulin promoted blastocyst hatching, adhesion, and outgrowth, whereas rabbit anti-mouse progranulin immunoglobulin G reduced the incidence of blastocyst hatching, adhesion, and outgrowth. Studies of bromodeoxyuridine incorporation and immunodissection of the ICM revealed that progranulin was effective on the trophectoderm but not on the ICM. These results indicate that progranulin is an important factor for the processes of blastocyst hatching, adhesion, and outgrowth, and they suggest that the effects of progranulin on blastocyst adhesion and outgrowth may have been triggered by the previous action of progranulin to induce hatching of the blastocysts.

Regulation of embryo outgrowth by a morphogenic factor, epimorphin, in the mouse.

Conceptus implantation to the uterine endometrium represents a complex series of events, including synchronized development of conceptus and uterus through up- and/or down-regulation of numerous gene products. In a previous study using the DNA microarray technique, we had discovered evidence that increase in a transcript for mesenchymal morphogen, epimorphin, was noted as the conceptus attached to the matrix in vitro (Qin et al., 2003). In the present study, the expression and potential function of epimorphin in developing conceptuses was investigated through the use of reverse transcription-polymerase chain reaction (RT-PCR), whole mount in situ hybridization/immunohistochemistry, and in vitro blastocyst culture. RT-PCR and in situ hybridization analysis revealed that epimorphin mRNA was expressed weakly in murine conceptuses during early developmental stages (1 cell to post-adhesion blastocyst stages) and higher levels of epimorphin transcripts were observed in both inner cell mass (ICM) and trophectoderm of outgrowing blastocysts. Immunohistochemical analysis confirmed that epimorphin was localized in outgrowing trophoblast cells and ICM. Treating blastocysts in culture with a 115 kDa form of recombinant epimorphin promoted trophoblast outgrowth (P < 0.05), but a 34 kDa form of recombinant epimorphin had no effect. Treatment with a function inhibitor, rat anti-mouse epimorphin IgM, reduced the number of embryos progressing to blastocyst outgrowth to the levels similar to those observed with plain culture medium. Reverse transcription-polymerase chain reaction (RT-PCR) analysis also revealed that epimorphin increased the expression of a trophoblast cell differentiation marker, placental lactogen-1 (PL-1), mRNA (P < 0.01). These results suggest that epimorphin is involved in trophoblast outgrowth, a process required for conceptus implantation into the endometrium.

[Inter-laboratory evaluation studies for development of notified ELISA methods for allergic substances (milk)].

Extracts of sausage, sauce, cookie, cereal and pasta sauce spiked with milk standard protein at a level of 5-20 ng/mL as sample solutions were analyzed in replicate in 10 laboratories. Coefficients of variation (CVs) of the three ELISA methods using a Milk Protein Casein ELISA Kit (Casein kit), a Milk Protein Beta-Lactoglobulin ELISA Kit (Beta-Lactoglobulin kit) and a FASTKIT Milk ELISA Kit (Milk ELISA kit) were mostly below 10%. Mean recoveries of the milk standard protein from the food extracts were over 40% in the three ELISA methods with a few excertions. The recoveries of milk standard protein from the sauce extract in Casein kit were improved by adjusting the extract to neutrality before the Casein kit assay. The recoveries of milk standard protein from cookie, cereal and pasta sauce were improved by the increasing the amount of antibody coated in the Milk ELISA kit. The detection limits of all the ELISA methods were 1 ng/mL in sample solutions. These results suggested that the notified ELISA methods are reliable and reproducible for the inspection of milk protein levels in extracts of sausage, sauce, cookie, cereal and pasta sauce.

[Inter-laboratory evaluation studies for development of notified ELISA methods for allergic substances (wheat)].

Extracts of sausage, sauce, pasta sauce, fish paste and cereal spiked with wheat standard protein at a level of 5-20 ng/mL as sample solutions were analyzed in replicate in 10 laboratories. Coefficients of variation (CVs) of both ELISA methods using a Wheat Protein ELISA Kit (Gliadin kit) and a FASTKIT Wheat ELISA Kit (Wheat ELISA kit) were mostly below 10%. Mean recoveries of the wheat standard protein from the food extracts were over 40% in the two ELISA methods except those from cereal extract determined using the Wheat ELISA kit. Repeatability relative standard deviations of wheat standard protein in the five food extracts were in the ranges of 16-26.9% and 3.7-36.2% for the Gliadin kit and the Wheat ELISA kit, respectively. Reproducibility relative standard deviations of wheat standard protein in the five food extracts were 21.6-38.5%, 29.7-53.8% for the Gliadin kit and the Wheat ELISA kit, respectively. The recoveries of wheat standard protein from the cereal extract were improved by the increasing the amount of antibody coated on the plate in the Wheat ELISA kit. The detection limits of both ELISA methods were 1 ng/mL in sample solutions. These results suggested that the notified ELISA methods are reliable and reproducible for the inspection of wheat protein levels in extracts of sausage, sauce, pasta sauce, fish paste and cereal.

[Inter-laboratory evaluation studies of notified ELISA methods for allergic substances (egg)].

Inter-laboratory evaluation studies were conducted for the notified ELISA methods for allergic substances (Egg). Standard extracts of egg spiked in extracts of sausage, sauce, cookie, bread and cereal at a level of 5-20 ng/mL as the sample solution were analyzed in replicate in 10 laboratories. Coefficients of variation (CVs) of all three ELISA methods using an Egg Protein ovalbumin ELISA Kit (ovalbumin kit), an Egg Protein ovomucoid ELISA Kit (ovomucoid kit) and a FASTKIT Egg ELISA kit (Egg ELISA kit) were mostly less than 10%. Mean recoveries of the standard extract of egg were over 40% in the three ELISA methods. Repeatability relative standard deviations of egg standard solution in five food extracts were in the ranges of 18.7-25.5%, 18.6-41.8%, 21.3-43.3% for the ovalbumin kit, the ovomucoid kit and the Egg ELISA kit, respectively. Reproducibility relative standard deviations of egg standard solution in five food extracts were 16.8-35.1%, 19.6-35.8%, 24.7-51.1% for the ovalbumin kit, the ovomucoid kit and the Egg ELISA kit, respectively. The detection limits of all the ELISA methods were 4-5 ng/mL in sample solutions. These results suggested that the notified ELISA methods are reliable and reproducible for the inspection of egg protein levels in extracts of sausage, sauce, cookie, bread and cereal.

[A detection method of recombinant DNA from genetically modified potato (NewLeaf Plus potato) and detection of NewLeaf Plus potato in snack].

A detection method using polymerase chain reaction (PCR) was developed to detect the genetically modified (GM) potato (NewLeaf Plus potato; NL-P), which has not been authorized as safe in foods in Japan. The potato sucrose synthase gene was used as an internal control. The DNA from NL-P specifically provided an amplified band using PCR with a primer pair recognizing PLRV-rep gene. In addition, to prevent false-positive results in processed potato foods infected with PLRV, we designed a primer pair recognizing sequences derived from two organisms to detect specifically NL-P in processed potato. The PCR product obtained using the designed primer pair was specific for NL-P. The DNA introduced into NL-P could be detected from potato powder samples containing 0.05% NL-P. The proposed method was applied to the detection of NL-P in 25 processed potato foods. NL-P was detected in 3 snack products.

Effect of subchronic feeding of genetically modified corn (CBH351) on immune system in BN rats and B10A mice.

Subchronic animal feeding studies to examine the effect on the immune system of genetically modified corn CBH351, which contains the Cry9C protein derived from Bacillus thuringiensis subspecies tolworthi, were conducted in female BN rats and B10A mice. The studies were designed to compare the effect of a line of genetically modified corn CBH351 (GM corn) with that of isoline corn (non-GM corn). Heat-treated corn meal was incorporated into the diets of the rats and mice at a concentration of 50%. The study duration was 13 weeks. Growth, food intake, and organ weights of the thymus, spleen, and liver were compared between animals fed the non-GM and GM lines. The histological findings in thymus, spleen, mesenteric lymph nodes, Peyer's patches, small intestines, liver, kidney, and bone marrow, and the presence of Cry9C-specific IgE, IgG, IgG1 and IgA antibodies in serum were also compared. The results showed no significant differences in growth, feeding value, or the histological findings in immunity-related organs between the animals fed the GM and non-GM lines. Production of Cry9 C-specific IgE and IgA was not detected in the serum of either group. Production of Cry9C-specific IgG and IgG1 was slightly increased in the 50% GM groups of BN rats. No Cry9C-specific IgG or IgG1 was detected in the serum of BN rats fed the diet containing 5% GM-corn In conclusion, no immunotoxic activity was detected in the GM-corn-fed rats and mice in this subchronic dietary study.