Delta-like ligand (DLL)1 expression in early mouse decidua and its localization to uterine natural killer cells.

Uterine vascular changes, critical for pregnancy success, occur at each implant site during endometrial decidualization. Mesometrial decidualization recruits high numbers of angiogenic, uterine Natural Killer (uNK) cells that trigger midpregnancy spiral arterial remodeling. We postulated that uNK cells contribute to early decidual angiogenesis as endothelial-cell extrinsic sources of Delta-like ligand 1 (DLL1), a molecule that induces endothelial tip cell differentiation and orthogonal vascular growth in other tissues. Virgin uteri expressed Dll1 mesometrially and anti-mesometrially and relative expression increased in both anatomic regions as pregnancy progressed. Analyses of transcripts from gd10.5 uNK cells flow sorted on the basis of expression of Dolichos biflorus agglutinin (DBA) lectin revealed that DBA+ but not DBA- uNK cells expressed Dll1. Immunostaining at gd4.5 found DLL1-expressing cells rare. At gd6.5, DBA+ uNK cells at all stages of maturation expressed DLL1. By gd10.5, DLL1 immunoreactivity was strongly expressed by some but not all DBA+ uNK cells and more weakly by DBA- cells. DLL1+ cells were mesometrially stratified and concentrated within central decidua basalis. Our data suggest that uNK cells have the potential to induce endothelial tip cell differentiation and to promote non-planar vascular growth within early decidua basalis

Uterine Natural Killer Cells Are Immunogenic In Syngeneic Male Mice.

Uterine natural killer (uNK) cells expand rapidly during endometrial decidualization and account for 70% of leukocytes in early gestational uteri of humans and rodents. These cells make unique contributions to pregnancy, contributing to the success of embryo implantation and maintenance of decidual tissue that supports placental and fetal development. We postulated that uNK cells express molecules that are not shared by circulating NK (cNK) cells or other leukocytes and, therefore, would be immunogenic for male mice. We isolated viable uNK cells from gestation day 9 pregnant mice and inoculated them into syngeneic males. This induced antibodies reactive with mouse uNK cells but not with cNK cells or other lymphocytes. The antibodies reacted identically with uNK cells in tissue sections from five different mice strains from gestational day 7-12 and in pregnant rat uterus, suggesting that the recognized antigen should be a specific marker of uNK cell. Spleen cells from inoculated males were used subsequently to produce a monoclonal antibody reactive to a uNK cell surface antigen. These experiments confirm that uNK cells are a pregnancy-specific subset of NK cells expressing distinct surface antigen from those found in other tissues.7918-2

Quantitative realtime PCR analyses of Dll1 in mesometrial and anti-mesometrial uterine samples and in flow sorted uNK cells.

<p>Relative mRNA expression of <i>Dll1</i> by the mesometrial side (M) of the virgin, gd4.5, gd5.5 and gd6.5 uterus of of the gd10.5 mesometrial lymphoid aggregate of pregnancy (MLAp) and decidua basalis (DB) is shown in (A). Relative mRNA expression of <i>Dll1</i> by anti- mesometrial (AM) virgin uterus and by gd4.5, gd5.5 and gd6.5 anti-mesometrial decidua is shown in (B). Relative mRNA expression of <i>Dll1</i> at gd10.5 by CD3-CD122+DBA- (DBA-) and CD3-CD122+DBA+ (DBA+) CD1 decidual cells normalized to <i>Hprt1</i>(C) is shown in (C). Data are means±SEM from all replicate analyses of two independent experiments. * P<0.05, ** P<0.01, *** P<0.001.</p

Histological analysis of gd6.5 B6 decidua basalis for expression of DLL1.

<p>Photomicrographs of gd6.5 B6 decidua basalis stained with DBA lectin-FITC (green), anti-DLL1-PE (red) and DAPI (blue) demonstrate in (A) DBA lectin-reactive small, agranular uNK cells and immature uNK cells with a few cytoplasmic granules. In (B), the same field is imaged showing cells reactive with DLL1. In the merged image (C), the co-expression of DBA lectin and DLL1 is shown (B and C; arrows mark representative cells). Additional cells that were DBA- and not identified expressed DLL1. The 6.5gd DBA+DLL1+uNK cells were found in the mesometrial decidua basalis (Meso DB) a region indicated as above the horizontal line in drawing (D). BV, entry of major blood vessel branches from the uterine artery; C conceptus, including ectoplacental cone. The area enclosed by dashed lines represents the residual uterine lumen. Bars: A, B and C are 40 µm.</p

Whole mount in situ immunohistochemistry of gd7.5 B6 implantation sites co-stained with PE-CD31 (red) and FITC-CD45 (green).

<p>Low power image (A) is a for orientation. The mesometrial side of the uterus is uppermost; the central black region (*) represents the embryonic crypt. More CD31 expression is present mesometrially than anti-mesometrially or in lateral (L) decidua. Images B, D, F are of the same field (represented by the boxed M) of the mesometrial uterus. Images C, E, G are of the same field (represented by the boxed AM) from the anti-mesometrial uterus. Image (B) illustrates the larger, web-like, less linked vessels of the decidua basalis. These are distinctly different to the narrower, more frequently linked anti-mesometrial vessels shown in (C). (B) but not (C) contains high numbers of CD31+ leukocytes. Image (D) shows the enrichment of leukocytes in decidua basalis while image (E) shows that leukocytes present anti-mesometrially are less frequent and more heterogeneous in shape and size. Red and green are merged in images (F) and (G). Image (F) shows that many cells with co-expression of CD31+ and CD45+ are present mesometrially while image (G) shows their absence anti-mesometrially. Magnification bar in (A) is 200 µm; in B-G 50 µm.</p

Histological analysis of DLL1 expression in mesometrial sections of gd10.5 B6 and CD1 implant sites.

<p>Photomicrographs A-L and O show gd10.5 implant site cryosections co-stained with DBA lectin-FITC (green) to identify uNK cells and anti-DLL1-PE (red). Panel M(i) is stained with only DBA lectin-FITC (green). Panels A–M(i) are B6 implant sites and panel O is a CD1 implant site. Panel M(ii) provides a diagram of the regions images were collected from for the other panels and has been marked to show the banding pattern seen for DLL1 expression. This should be compared to low power image M(i) that shows DBA lectin-stained uNK cell distribution in each banded area. These are labeled region “1” for the MLAp, region 2 for decidua basalis distal to the placenta (pdDB) and region 3 for decidua basalis proximal to the placenta (ppDB). Region 4, the AM decidua, is at the bottom of the image. “C” represents the conceptus, including the placenta and the green ring around “C” is DBA lectin-stained yolk sac endothelium surrounding the fetus. The black space between the yolk sac and the region labeled “3” is the placenta which is uNK cell deficient. Expression of DLL1 by gd10.5 DBA+ cells was stratified within the mesometrial side of the implant sites in both strains and no significant differences were noted. In B6 MLAp (A–C; labeled “1” in M(i) and diagrammed in M(ii)), DLL1 was strongly expressed only infrequently (B) and not by the smaller, immature uNK cells that proliferate in this region (arrow heads in A, C). In decidua basalis of B6 that was distal to the placenta (D–F) and CD1 (O), DBA+ uNK cells brightly expressed DLL1 (arrows in D–F; O). DBA+DLL1+ uNK cells appeared to surrounded vessels (*). Additional perivascular DLL1 staining was present that was not associated with DBA+ cells. The decidual region proximal to the placenta was devoid of DLL1+ cells but abundantly populated by DBA+ uNK cells (G). Neither DLL1+ nor DBA+ uNK cells were present in the highly regressed anti-mesometrial decidua (A-Meso; J-L). DBA-stained yolk sac endothelium was present in this region (arrows in J, L). N is a photomicrograph of the placenta distal decidua basalis in a section from an archived paraffin-embedded gd10.5 B6 implant site double stained using DBA lectin-horseradish peroxidase and Periodic Acid Schiff’s reagent <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052037#pone.0052037-Zhang1" target="_blank">[25]</a>. The latter stain reveals all granulated uNK cells and shows cells of the DBA-PAS+ subset (yellow circle). This image shows the typical strong association of uNK cells with arterioles and with microvessels, including intravascular positions and supports interpretations of the fluorescence images. In M(ii), BV indicates entry of major blood vessel branches from the uterine artery. Bars: A, B, C, J, K, L, O: 40 µm; D, E, F, G, H, I: 20 µm; M: 200 µm.</p

Effect Of Ph And Interaction Between Egg White Protein And Hydroxypropymethylcellulose In Bulk Aqueous Medium On Foaming Properties.

Egg white protein (EW) is used as surface-active ingredient in aerated food and hydroxypropylmethylcellulose (HPMC) is a polysaccharide that behaves as a surfactant. This study aimed at investigating the effects of process parameters biopolymer concentration (2.0-5.0%, w/w), EW:HPMC ratio (2:1-18:1), pH (3.0-6.0), and the influence of biopolymers' behavior in aqueous solution at different pH on the foaming properties (overrun, drainage, and bubble growth rate). Process parameters had effect on foaming properties. The pH was the major factor influencing the type of EW/HPMC interaction and affected the foaming properties of biopolymer mixture. At pH 3.0, EW and HPMC showed thermodynamic compatibility leading to better foaming properties, higher foaming capacity, and stability than without HPMC addition whereas at pH 4.5 and 6.0, EW and HPMC are incompatible that causes lower stability concerning the disproportionation comparing to foam without HPMC. At pH between 3.0 and 4.5, HPMC improves foaming properties of aerated products.12526-3

Intake of protein hydrolysates and phenolic fractions isolated from flaxseed ameliorates tnbs‐induced colitis

In the attempt to develop new therapeutic treatments for colitis, fractions containing phenolic compound isolate (Phi) and phenolic reduced‐flaxseed protein hydrolysate (phr‐FPH) from flaxseed are evaluated for their effects on the in vitro production of pro‐inflammatory mediators and on the course of experimental colitis. Methods and results The anti‐inflammatory effects of Phi and phr‐FPH from flaxseeds are studied in RAW264.7 cells and in trinitrobenzene sulphonic acid (TNBS) colitis model. It is observed that the incubation with Phi or phr‐FPH result in lower levels of tumor necrosis factor α and nitric oxide in macrophages stimulated with bacterial lipopolysaccharide + interferon‐γ. Prophylactic and therapeutic treatments with Phi and phr‐FPH, respectively, greatly contribute to the prevention of weight loss and colon inflammation in colitic BALB/c mice. T cell proliferation, expansion of TH1 and TH17 cells, and pro‐inflammatory cytokines are lower, whereas Treg cells are higher in spleen cell cultures from Phi‐treated mice. In addition, therapeutic phr‐FPH treatment is able to reduce the expansion of TH17 in splenic cell cultures. Conclusion The consumption of phenolic and protein compounds extracted from flaxseeds has a protective effect on TNBS‐induced colitis, and may be useful in the control of other inflammatory disorders6217CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPSem informação2014/ 08591-0; 2010/52680-7; 2013/20258-

Oral tolerance induced by OVA intake ameliorates TNBS-induced colitis in mice

Introduction Literature data have shown that the consumption of dietary proteins may cause modulatory effects on the host immune system, process denominated oral tolerance by bystander suppression. It has been shown that the bystander suppression induced by dietary proteins can improve inflammatory diseases such as experimental arthritis. Here, we evaluated the effects of oral tolerance induced by ingestion of ovalbumin (OVA) on TNBS-induced colitis in mice, an experimental model for human Crohn's disease. Methods and Results Colitis was induced in BALB/c mice by instilling a single dose of TNBS (100 mg/kg) in ethanol into the colon. Tolerized mice received OVA (4mg/mL) dissolved in the drinking water for seven consecutive days, prior to or concomitantly with the intrarectal instillation. Control groups received protein-free water and ethanol by intrarectal route. We observed that either the prior or concomitant induction of oral tolerance were able to reduce the severity of colitis as noted by recovery of body weight gain, improvement of clinical signs and reduction of histological abnormalities. The in vitro proliferation of spleen cells from tolerant colitic mice was lower than that of control mice, the same as the frequencies of CD4(+) T cells secreting IL-17 and IFN-gamma. The frequencies of regulatory T cells and T cells secreting IL-10 have increased significantly in mice orally treated with OVA. The levels of inflammatory cytokines (IL-17A, INF-alpha, IL-6 and IFN-gamma) were lower in supernatants of cells from tolerant colitic mice, whereas IL-10 levels were higher. Conclusion Our data show that the modulation of immune response induced by oral tolerance reduces the severity of experimental colitis. Such modulation may be partially attributed to the increase of Treg cells and reduction of pro-inflammatory cytokines in peripheral lymphoid organs of tolerant mice by bystander suppression121COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2013/20258-2; 2014/086192; 2015/09326-1; 2014/167010; 2014/08591-0