LEUCOCYTE BINDING TO THE UTERINE EPITHELIAL CELL SURFACE DURING LECTIN-INDUCED DECIDUALIZATION

Intra-uterine injection of the lectin Concanavalin A (ConA) on day 5 of pseudopregnancy induced a rapid and persistent infiltration of leucocytes into the rat uterine stroma. Although the infiltration of leucocytes was seen along the entire length of the uterine horn, areas of stromal oedema, indicative of decidualization (as indicated by a positive Pontamine Sky Blue reaction), were only associated with regions in which leucocytes had crossed the uterine epithelium and were present in the uterine lumen. Ultrastructural evaluation of the interaction of the luminal leucocytes with the apical surface of the uterine epithelium appeared strikingly similar to that of the blastocyst and the uterine epithelium during normal implantation. It is proposed that leucocytes, induced by ConA, may initiate a decidual response in a manner analogous to that of the blastocyst through surface epithelial interaction.     

Varying effects of an IUD on decidualization in mice.

Chronically implanted IUDs consisting of silk suture threads induced decidualization in regions of the uterus remote from the suture site in ovariectomized mice treated with a regimen of progesterone and oestrogen which sensitizes the uterus to a decidual stimulus. In these conditions the IUDs did not inhibit decidualization induced by instilled oil, although they did so in pregnant animals of the same strain. Varying the dose of progesterone and oestrogen did not produce conditions in which IUD's inhibited oil-induced decidualization in ovariectomized mice and progesterone treatment did not prevent IUDs inhibiting decidualization in pregnant animals. However, when ovariectomized mice, sensitized as before, were primed repeatedly with oestrogen to simulate continuing oestrous cycles after IUD insertion, the IUD's inhibited oil-induced decidualization. This involved the premature loss of instilled oil from the uterine lumen and was associated with heavy infiltration of leucocytes into the luminal epithelium. Numbers of leucocytes free in the uterine lumen did not appear to be critical. It appears that contact between the oil and the luminal epithelial surface must be sustained for some length of time to induce a decidual reaction; brief contact is not sufficient to trigger the response.     

Suppressor cell activity of intestinal mucosal leucocytes from buffalo (Bubalus bubalis arni)

Suppressor activity of buffalo intestinal intraepithelial leucocytes and lamina propria leucocytes was induced by Concanavalin A, and was assayed against the mitogenic response of autologous and allogenic leucocytes to mitogens. Appreciable suppression was observed with 25 micrograms ConA/ml on the proliferative activity of the responder cells cocultured at a ratio of < 2:1 (suppressor:responder cell). Mitomycin C treatment of intestinal leucocytes did not totally vanish the viability and functionality of leucocytes.     

Lectin-induced deciduoma formation in the pseudopregnant rat

Decidual cell induction in the pseudopregnant rat was examined in this study using the lectin concanavalin A (ConA). The histochemical binding of the lectin to the uterine cell surface at the time of deciduomatic induction was also studied. ConA was found to induce significant deciduomata (decidual-like tissue) in the uterine horn when injected intraluminally on day 5 of pseudopregnancy (PSP). ConA-induced deciduomata appeared as a series of discrete nodules in the uterine horn, reminiscent of the anatomical appearance of normal embryo implantation sites. Deciduoma induction by ConA was greatly reduced by pre-absorption of the lectin with its competitive sugar. Lectin histochemistry revealed binding of ConA to the cell surface on day 5 of PSP. Pre-absorption of the lectin with its competitive sugar also significantly reduced surface binding of the lectin, and this finding may be correlated with the greatly reduced ability of the pre-absorbed lectin to induce deciduomata. Possible mechanisms for the induction of deciduomata by lectins are considered.     

Studies on the vaginal plug of the Japanese greater horseshoe bat, Rhinolophus ferrumequinum nippon

The vaginal plug of the Japanese greater horseshoe bat is composed of an outer, thick, hard and homogeneous layer originating from the stratum disjunctum of the vaginal mucosa and a thin, soft and opaque central core containing accidentally trapped dead spermatozoa. The vaginal plug appeared to be expelled just before or after ovulation in spring. Immediately after copulation in autumn, uterine spermatozoa were dead but few leucocytes were present. As time passes after copulation, however, the infiltration of leucocytes into the uterus increased gradually, and extensive leucocytic phagocytosis of dead spermatozoa occurred during hibernation.     

Endocrinology of pregnancy: consequences for the diagnosis and treatment of pregnancy disorders

Placental human chorionic gonadotropin and corpus luteum secretion of progesterone and oestradiol are the main endocrine events at the beginning of pregnancy, whilst the luteo-placental shift is an important step during the later stages. Progesterone not only affects decidualisation, but is the major immunological determinant and controls uterine contractibility and cervical competence. These properties all contribute considerably towards the correct development of pregnancy and delivery at term.     

A basal membrane-like structure surrounding tumour nodules may prevent intraepithelial leucocyte infiltration in colorectal cancer

Epithelial tumours consist of an epithelial compartment and a stromal compartment, which are sometimes separated by a basal membrane-like structure. We sought to determine whether these factors have prognostic value in 84 curatively resected stage II and III colorectal cancer by immunohistochemically staining tumours for leucocytes (CD45) and extracellular matrix, and to assess the presence of a basal membrane-like structure. Leucocyte infiltration was also assessed in hematoxylin-eosin (HE) stained sections. Most leucocytes were located in the tumour stroma. A relatively high intraepithelial leucocyte infiltration was significantly correlated with a lower level of tumour recurrence (P=0.03) and a longer disease-free survival (P=0.05), whereas leucocytes located in the tumour stroma (P=0.92) or at the advancing margin (p=0.06) were not. Intraepithelial leucocyte infiltration was also significantly correlated with leucocyte infiltration in the tumour stroma (P=0.02) and at the advancing tumour margin (P=0.005), and as assessed in HE-stained tumour sections (P=0.05), but each of these parameters on its own did not have a prognostic value in predicting disease-free survival. Moreover, the presence of a basal membrane-like structure surrounding the tumour epithelium was inversely correlated with the number of intraepithelial leucocytes (P=0.05), suggesting that this membrane-like structure functions as a barrier to intraepithelial leucocyte infiltration. We conclude that leucocytes must be in the direct vicinity of tumour cells to affect tumour growth. The presence of an extracellular matrix barrier seems to prevent this interaction.     

Lectin binding sites as markers of neonatal porcine uterine development.

To identify lectin binding sites and to determine if lectin binding patterns change with age in developing neonatal porcine uterine tissues, gilts (n = 3/day) were hysterectomized on Day 0 (birth), 7, 14, 28, 42, or 56. Lectin binding was visualized in Bouin's-fixed uterine tissues with seven biotinylated lectins (ConA, DBA, PNA, RCA-I, SBA, UEA-I, and WGA) and avidin-peroxidase staining procedures. Lectin specificities were demonstrated by pre-incubating lectins with appropriate inhibitory sugars (0.2 M). Staining intensity was evaluated visually (absent, weak, moderate, or strong) for three endometrial tissues; luminal epithelium, glandular epithelium, and stroma. Staining intensities for DBA, PNA, SBA, and WGA were not affected by neonatal age. Staining with these lectins was greater in uterine epithelium (moderate or strong) than in stroma (weak). In contrast, binding patterns for ConA, UEA-I, and RCA-I were affected by neonatal age. Strong epithelial staining associated with ConA binding was observed on all days, whereas stromal ConA staining decreased in intensity from moderate to weak after Day 14. Epithelial staining with UEA-I increased from moderate to strong after Day 28, whereas stromal UEA-I staining decreased from moderate to weak after day 28. Staining with RCA-I was homogeneous for luminal epithelium and stroma but variegated for glandular epithelium on and after Day 7. These observations indicate that a variety of lectin binding sites are present in developing neonatal porcine endometrial tissues and that developmentally related alterations in the distribution and/or orientation of glycoconjugates containing alpha-D-mannose, beta-D-galactose, beta-D-acetyl-N-galactosamine, and alpha-L-fucose residues occur between birth and Day 56 as these tissues mature.     

Mobility modulation by local concanavalin A binding. Selectivity toward different membrane proteins

We have employed fluorescence photobleaching recovery to demonstrate selective immobilization of lymphocyte membrane proteins by localized concanavalin A (ConA) binding to the cell surface. Localized ConA binding was achieved by the binding of ConA coupled to paraformaldehyde-fixed platelets to mouse spleen lymphocytes. The effect of the localized cross-linking of ConA receptors on the lateral mobility of specific membrane proteins at regions distal to the ConA platelets was investigated. The diffusion of surface immunoglobulins and ConA receptors was inhibited above a threshold coverage (12%) of the upper lymphocyte surface by ConA platelets. In contrast, no effect was observed on the diffusion and aggregation of mouse histocompatibility antigens (H-2Kk) labeled with a fluorescent monoclonal antibody. Since the ConA modulation was shown to propagate through the cytoskeleton, these results indicate specificity in the interactions of membrane proteins with the cytoskeleton. This specificity enables a selective response of different membrane proteins to the ConA anchorage modulation.     

Inhibition of ConA erythroagglutination by alkali-treated lipopolysaccharide

Bacterial lipopolysaccharide treated by hydrolysis in basic solution (hLPS) and then added to suspensions of human erythrocytes markedly inhibited erythroagglutination by concanavalin A (ConA), soybean agglutinin (SBA), and Phaseolus vulgaris phytohemagglutinin (PHA). Likewise, red cells presensitized by exposure to hLPS and then suspended in hLPS-free buffer were not agglutinated by ConA. However, when hLPS was not added to buffer both SBA and PHA strongly agglutinated erythrocytes presensitized by hLPS. Also, the binding of ³H-labeled ConA to red cells was unaffected by presensitization of the cells with hLPS. It appears, therefore, that membrane-associated hLPS localizes in the ConA receptor regions of erythrocyte membranes and inhibits ConA erythroagglutination by disruption of receptor responses to bound ConA or alteration of the active subunit structure of bound ConA.     

SD大鼠血管内皮细胞的凝集素组织化学研究

为进一步了解大鼠血管内皮细胞表面糖基的分布, 本文应用５ 种生物素化凝集素（ＧＳ－Ｉ、ＲＣＡ－Ｉ、ＷＧＡ、ＣｏｎＡ和ＵＥＡ－Ｉ） 对大鼠心脏、胸主动脉、肝脏和子宫的血管内皮细胞采用ＡＢＣ法标记。结果表明,光镜下, ＧＳ－Ｉ对心脏、胸主动脉、子宫的血管内皮染色阳性; ＲＣＡ－Ｉ对所检组织内血管内皮细胞染色阳性,其中对心脏和肝的血管内皮与血管外组织细胞的染色强度有显著不同; ＣｏｎＡ对肝血窦内皮与对肝细胞的染色强度有明显不同; ＵＥＡ－Ｉ对各脏器血管内皮细胞未见阳性标记。电镜下ＧＳ－Ｉ阳性反应产物位于心肌毛细血管内皮细胞表面。提示, 血管内皮细胞表面存在的某些糖复合物含有Ｄ－半乳糖、Ｎ－乙酰氨基半乳糖、Ｎ－乙酰氨基葡萄糖、Ｄ－甘露糖。ＧＳ－Ｉ可视为心脏、胸主动脉、子宫的血管内皮细胞的特异性标记物。ＲＣＡ－Ｉ既可作为心脏的血管内皮, 亦可作为肝血窦内皮的特异性标记物, ＣｏｎＡ可作为肝血窦内皮的特异性标记物。     

Application of Concanavalin A during immune responsiveness skin‐swelling tests facilitates measurement interpretation in mammalian ecology

The skin‐swelling test is a simple and widespread method used in field ecological research to estimate cellular immune responsiveness in animals. This immunoecological test is based on measuring the magnitude of tissue swelling response at specific times following subcutaneous application of an experimental pro‐inflammatory stimulant. In the vast majority of studies across vertebrate taxa, phytohemagglutinin (PHA) is used as a universal stimulant. Given the complexity of immune response activation pathways of PHA, however, interpretation of test results can be ambiguous. Goal of this study was to improve methodology of the skin‐swelling test to decrease this ambiguity. Here, we present an alternative protocol aimed at facilitating interpretation of skin‐swelling data for mammals. Based on previous evidence suggesting that mammalian T cells are readily activated by Concanavalin A (ConA) in vitro, we compared cellular immune responses in vivo to PHA and ConA as an alternative pro‐inflammatory stimulant in mice. We measured magnitude of tissue swelling and compared it with intensity of blood cell infiltration into tissue over a 72‐hour interval. Our results corroborate that PHA and ConA show important differences in both dynamics and response amplitude in rodents. ConA induces stronger swelling with a distinct leukocyte activity pattern and higher pro‐inflammatory cytokine (interleukin 6 [IL‐6] and interferon gamma[IFN‐γ]) expression than PHA during peak response (24‐h post‐treatment). Furthermore, unlike PHA, magnitude of swelling was positively associated with cellular activity (number of neutrophils infiltrating tissue) following ConA injection. We conclude that ConA is the more suitable stimulant for skin‐swelling tests in mammals. This is because of the molecular binding specificity in the two lectins, that is, ConA specifically activates T cells while PHA also triggers erythroagglutination. We propose that ConA be used in all future ecological testing in mammals as it exhibits better performance and its application facilitates immunological interpretation of skin‐swelling test results.     

IFN-gamma/STAT1 acts as a proinflammatory signal in T cell-mediated hepatitis via induction of multiple chemokines and adhesion molecules: a critical role of IRF-1

We have previously shown that IFN-gamma/STAT1 plays an essential role in concanavalin A (ConA)-induced T cell hepatitis via activation of apoptotic signaling pathways. Here we demonstrate that IFN-gamma/STAT1 also plays a crucial role in leukocyte infiltration into the liver in T cell hepatitis. After injection of ConA, leukocytes were significantly infiltrated into the liver, which was suppressed in IFN-gamma(-/-) and STAT1(-/-) mice. Disruption of the IFN regulatory factor-1 (IRF-1) gene, a downstream target of IFN-gamma/STAT1, abolished ConA-induced liver injury and suppressed leukocyte infiltration into the liver. Additionally, ConA injection induced expression of a wide variety of chemokines and adhesion molecules in the liver. Among them, expression of ICAM-1, VCAM-1, monokine induced by IFN-gamma (Mig), CC chemokine ligand-20, epithelial cell-derived neutrophil-activating peptide (ENA)-78, IFN-inducible T cell-alpha chemoattractant (I-TAC), and IFN-inducible protein-10 (IP-10) was markedly attenuated in IFN-gamma(-/-), STAT1(-/-), and IRF-1(-/-) mice. In primary mouse hepatocytes, Kupffer cells, and endothelial cells, in vitro treatment with IFN-gamma activated STAT1, STAT3, and IRF-1, and induced expression of VCAM-1, ICAM-1, Mig, ENA-78, I-TAC, and IP-10 mRNA. Induction of these chemokines and adhesion molecules was markedly diminished in STAT1(-/-) and IRF-1(-/-) hepatic cells compared with wild-type hepatic cells. These findings suggest that in addition to induction of apoptosis, previously well documented, IFN-gamma also stimulated hepatocytes, sinusoidal endothelial cells, and Kupffer cells partly via an STAT1/IRF-1-dependent mechanism to produce multiple chemokines and adhesive molecules responsible for promoting infiltration of leukocytes and, ultimately, resulting in hepatitis.     

Application of fluorescently labelled lectins for the visualization and biochemical characterization of polysaccharides in biofilms of Pseudomonas aeruginosa

Fluorescently labelled lectins were used in combination with epifluorescence microscopy and confocal laser scanning microscopy to allow the visualization and characterization of carbohydrate-containing extracellular polymeric substances (EPS) in biofilms of Pseudomonas aeruginosa. A mucoid strain characterized by an overproduction of the exopolysaccharide alginate, and an isogenic, non-mucoid strain were used. Model biofilms grown on polycarbonate filters were treated with lectins concanavalin A (ConA) and wheat germ agglutinin (WGA) that were fluorescently labelled with fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate. Fluorescently labelled ConA yielded cloud-like regions that were heterogeneously distributed within mucoid biofilms, whereas these structures were only rarely present in biofilms of the non-mucoid strain. The bacteria visualized with the fluorochrome SYTO 9 were localized both within and between the ConA-stained regions. In WGA-treated biofilms, the lectin was predominantly associated with bacterial cells. Alginate seemed to be involved in the interaction of ConA with the EPS matrix, since (i) pre-treatment of biofilms with an alginate lyase resulted in a loss of ConA biofilm staining, and (ii) using an enzyme-linked lectinsorbent assay (ELLA), ConA was shown to bind to purified alginate, but not to alginate that was degraded by alginate lyase. The application of fluorescently labelled lectins in combination with ELLA was found to be useful for the visualization and characterization of extracellular polysaccharide structures in P. aeruginosa biofilms.     

Quantification of Atlantic salmon type-I interferon using an Mx1 promoter reporter gene assay

We here describe an assay for the detection of interferon-like activity in Atlantic salmon based on the transient transfection of chinook salmon embryo cells (CHSE-214 cells) with a rainbow trout Mx1 promoter linked to a luciferase reporter. A beta-galactosidase gene under the control of a constitutively expressed beta-actin promoter was used as a transfection standard, and luciferase and beta gal expression were measured by a commercially available kit. Interferon containing supernatants from poly I:C- or CpG-stimulated leucocytes added to transfected CHSE-cells induced high luciferase expression (>60-fold induction compared to supernatants from non-stimulated cells). There was no response to supernatants from LPS- and ConA/PMA-stimulated leucocytes, demonstrating the specificity for type I interferon-like activity. Duplicate samples analysed using a cell protection assay for detection of antiviral activity correlated well with levels obtained by the Mx1 promoter reporter gene assay (R2=0.97), confirming the reporter assay as a reliable substitute for the standard antiviral assay. The Mx reporter gene assay also has advantages in terms of sensitivity, high dynamic range and reliability over the conventional cell protection assay.     

Bacteriological investigation of the postpartum uterus: Relationship to involution and histopathological findings

A bacteriological investigation of the postpartum uteri of suckled and nonsuckled Bunaji (White Fulani) cows was undertaken to determine the microflora, the effect of suckling on the flora and the influence of the flora on uterine involution and histopathology. Uterine contamination by bacteria was highest between days 10 and 21 postpartum with Staphylococcus aureus being the commonest organism isolated. There was no consistent isolation of one type of bacterial organism from a cow throughout the sampling period. Suckling did not affect the incidence of uterine contamination by bacteria. Foci of leucocytes (mainly lymphocytes) were found in uterine sections in an increasing frequency with days postpartum. Bacteria were isolated from only 3 of the 12 uteri that had the leucocytic foci. Uterine involution was normal and complete by 25 days after calving irrespective of the presence or absence of bacteria or leucocytic foci. We concluded that postpartum uterine contamination by bacteria was not influenced by suckling and that puerperal uterine contamination by bacteria did not interfere with the normal process and duration of uterine involution.     

Dilated thin-walled blood and lymphatic vessels in human endometrium: a potential role for VEGF-D in progestin-induced break-through bleeding

Progestins provide safe, effective and cheap options for contraception as well as the treatment of a variety of gynaecological disorders. Episodes of irregular endometrial bleeding or breakthrough bleeding (BTB) are a major unwanted side effect of progestin treatment, such that BTB is the leading cause for discontinued use of an otherwise effective and popular medication. The cellular mechanisms leading to BTB are poorly understood. In this study, we make the novel finding that the large, dilated, thin walled vessels characteristic of human progestin-treated endometrium include both blood and lymphatic vessels. Increased blood and lymphatic vessel diameter are features of VEGF-D action in other tissues and we show by immunolocalisation and Western blotting that stromal cell decidualisation results in a significant increase in VEGF-D protein production, particularly of the proteolytically processed 21 kD form. Using a NOD/scid mouse model with xenografted human endometrium we were able to show that progestin treatment causes decidualisation, VEGF-D production and endometrial vessel dilation. Our results lead to a novel hypothesis to explain BTB, with stromal cell decidualisation rather than progestin treatment per se being the proposed causative event, and VEGF-D being the proposed effector agent.     

N-acetyl-beta-D-glucosaminidase activity in bovine uterine fluid and blood serum during the postpartum period

Uterine fluid and serum N-acetyl-beta-D-glucosaminidase (NAGase) was determined in cows during the first 32 d post partum following normal and abnormal parturitions. Both uterine fluid and serum NAGase activities were elevated after calving and then started to decline gradually toward the 32nd day after calving, when they reached their lowest concentrations. No significant differences were found between the mean NAGase concentrations in uterine fluid of the two groups, although significant differences were found between the mean values of the combined groups between days. With serum NAGase concentrations, significant differences (P<0.01) were found between the mean values of the normal and abnormal puerperium groups. The major part of the enzyme detected in postpartum uterine fluid is probably contributed by epithelial cells present in the fluid. Uterine leucocytes and endometrial cell damage caused by bacterial infection may also contribute to the total NAGase activity in uterine fluid.