Interleukin-33 amplifies IgE synthesis and triggers mast cell degranulation via interleukin-4 in naïve mice.

BACKGROUND: The regulation and function of IgE in healthy individuals and in antigen-naïve animals is not well understood. IL-33 administration increases serum IgE in mice with unknown mechanism. We tested the hypothesis that IL-33 provides an antigen-independent stimulus for IgE production and mast cell degranulation. METHODS: IL-33 was administered to naïve wild-type (WT), nude and ST2(-/-) , IL-4(-/-) , IL4Rα(-/-) and T-or B-cell-specific IL-4Rα(-/-) mice. IgE and cytokines were quantified by ELISA. T- and B-lymphocyte numbers and CD40L expression were determined by flow cytometry. Anaphylaxis was measured by temperature, mast cell degranulation and histamine release. RESULTS: IL-33 enhanced IgE production in naïve WT, T-IL-4Rα(-/-) but not in ST2(-/-) , IL-4(-/-) , IL-4Rα(-/-) or B-cell-specific IL-4Rα(-/-) mice, demonstrating IL-33 specificity and IL-4 dependency. Moreover, IL-4 was required for IL-33-induced B-cell proliferation and T-cell CD40L expression, which promotes IgE production. IL-33-induced IL-4 production was mainly from innate cells including mast cells and eosinophils. IL-33 increased mast cell surface IgE and triggered degranulation and systemic anaphylaxis in allergen-naïve WT but not in IL-4Rα(-/-) mice. CONCLUSION: IL-33 amplifies IgE synthesis and triggers anaphylaxis in naïve mice via IL-4, independent of allergen. IL-33 may play an important role in nonatopic allergy and idiopathic anaphylaxis.

Characterization of initial halogen adsorption on Si(111) surface by scanning tunnelling microscopy: correlation with optical measurements.

Initial adsorption processes of halogen atoms on a Si(111)-(7 × 7) surface were studied by means of scanning tunnelling microscopy (STM). The adsorption sites of halogen atoms were clarified directly with STM, and the results were compared with the partial coverage at each site, estimated previously from surface differential reflectance and thermal desorption spectroscopic analyses. The microscopic geometry of the atomic structure showed a good correspondence with the optical measurements, especially in terms of the density of the reacted sites. Bromine atoms were predominantly adsorbed near already adsorbed bromine, while chlorine atoms were almost randomly adsorbed. Polybromide formation occurred at coverage levels above 0.1 ML. Bromine atoms break the back-bonds of Si adatoms at lower levels of coverage than do chlorine atoms. The reason for the difference in adsorption behaviour between chlorine and bromine is discussed.

Locomotion and chemotaxis of lymphocytes.

The behaviour of locomotor T and B lymphocytes and the chemoattractants to which they respond in vitro are reviewed. Following activation, T cells respond by locomotion and chemotaxis to cytokine attractants including IL-15 and IL-2 and several chemokines. In activated B cells chemotaxis may be signalled through the antigen receptor. Conversely resting lymphocytes respond poorly to the above signals though their locomotion is activated by contact with high endothelial venular cells. These differences in locomotion between resting and activated lymphocytes, together with differences in adhesion, may explain why activated lymphocytes migrate preferentially into inflammatory sites while resting cells recirculate.

Spread of malignant cells in the surgical margin with stapled excision of lung cancer: comparison of aggressive clump and less traumatic jaw closure type staplers.

BACKGROUND: During stapled excision of lung cancer tissue, malignant cells can spread in the surgical margin. Stapling methods can be classified as aggressive clumping (AC) and less traumatic jaw closing (LTJC) types, thus the ratio of malignant margins may differ between stapler types. METHODS: The malignant status of the stapled margin was retrospectively investigated in 112 cases using a cytology technique. Stapler type, maximum tumor diameter, distance from surgical margin, thoracotomy type, and tumor location were used as variables. In addition, clinical results of excision cases were assessed. RESULTS: The ratio of malignant margins was 22/54 (41 %) in the AC group and 11/58 (19 %) in the LTJC group ( P = 0.01). Multivariate analysis revealed that the stapling method and tumor location were an independently significant factor. Surgical margin recurrence occurred only in 4 (57 %) of 7 cases with malignant margin. CONCLUSIONS: The AC type method showed a greater potential to spread malignant cells, thus there seems to be a higher possibility of regional relapse with that technique.

Locomotor properties of human germinal centre B cells: activation by anti-CD40 and IL-4 allows chemoattraction by anti-immunoglobulin.

The locomotor properties of B cells isolated from the germinal centres (GC) of human tonsils were studied using polarization, collagen gel invasion and micropore filter assays. The proportion of motile GC cells in the freshly isolated population was small. During culture in interleukin-4 (IL-4)+anti-CD40, but not in control medium, the proportion of polarized cells increased and these cells migrated actively into collagen gels. After 24 hr culture, most of the surviving population was in locomotor morphology. The locomotor population consisted mainly of centrocytes in the G1 phase of growth. More locomotor cells than spherical cells took up [3H]uridine, but locomotor cells did not take up [3H]thymidine. After culture for 6 hr in IL-4+anti-CD40, GC B cells were tested in short-term polarization assays and filter assays for their response to chemoattractants. In both assays, a proportion of the cells responded to anti-IgA and to anti-IgA F(ab')2 fragments at 1 ng/ml., or to anti-IgG, anti-IgM and F(ab')2 fragments of these antibodies at 100 ng-1 microgram/ml. A checkerboard filter assay showed a good chemokinetic response and a weaker chemotactic response of GC cells to anti-IgA. Expression of Fc gamma RII (CD32) was increased after culture in IL-4+anti-CD40, and these cultured cells responded in filter and polarization assays to anti-CD32. Thus culture in IL-4 and anti-CD40 not only rescues GC B cells, but also increases their locomotor capacity and allows them to respond in chemotaxis assays to anti-immunoglobulin.

Chemotaxis of human B lymphocytes to anti-IgD.

The resting population of small surface IgM+ and surface IgD+ B cells from the human tonsil can be preactivated by overnight culture in interleukin-4 (IL-4) to show locomotor responses to anti-IgM and anti-IgD at between 10 ng and 1 microgram/ml. Because this locomotion is activated through the antigen receptor and may simulate a response to antigen, we set out to establish whether this was a chemotactic response using a checkerboard filter assay with a range of concentrations and concentration gradients of anti-IgD. At high concentrations (100 ng/ml to 1 microgram/ml), a chemokinetic response, but no chemotaxis, to anti-IgD was seen. However, in concentration gradients set up at lower concentrations (0-50 ng/ml) a chemotactic response was demonstrable. During the period of culture in anti-IgD at 1 microgram/ml, a progressive loss of surface IgD from the cells was seen, but there was no loss at 10 ng/ml. This receptor loss from the cell surface may account for the lack of chemotactic effect of the anti-IgD at higher concentrations.

TGF-beta stimulates but IFN-gamma inhibits growth-related activation of locomotion of human B cells.

Effects of TGF-beta and IFN-gamma on locomotion of high density B cells from human tonsils were studied using polarization and filter assays. Culture with TGF-beta (10 pg to 1 ng/ml) induced a gradual increase in locomotor morphologies in 40 to 50% of B cells during overnight culture. This was not immediate (<30 min) suggesting that TGF-beta is not a chemotactic factor. The time course suggests that B cells acquired a locomotor phenotype during the first few hours of culture. B cells cultured in TGF-beta increased in size, but to a lesser extent than those cultured in IL-4 used as a control. More cells were polarized in TGF-beta + IL-4 than in either alone. Following culture in TGF-beta, B cells showed vigorous responses to anti-IgD used as a chemoattractant in short-term assays. In contrast, addition of IFN-gamma (20-100 U/ml) to B cells in culture in IL-4, anti-CD40, or TGF-beta inhibited activation of locomotor capacity by the latter agents, and IFN-gamma-cultured B cells showed an even lower response to anti-IgD in a short-term polarization or filter assay than those cultured in medium alone. IFN-gamma also inhibited uridine incorporation by cultured B cells, and cells cultured in IFN-gamma showed no size increase. We suggest that IFN-gamma prevents locomotor activation by inhibiting progress of B cells into the G1 phase of growth.

Injury to a duplicated cystic duct during laparoscopic cholecystectomy.

Duplication of the cystic duct is a rare variation of the biliary system. It is important to pay attention to this variation to avoid intraoperative biliary system injury, especially during laparoscopic cholecystectomy. In a 66-year-old woman with gallstones, endoscopic retrograde cholangiopancreatography (ERCP) revealed no anomaly of the biliary system. At laparoscopic cholecystectomy, although intraoperative cholangiography demonstrated the second cystic duct, we misdiagnosed the duplication of cystic duct because preoperative ERCP had demonstrated normal anatomy. It was thought that the second cystic duct was dissected during the operation and the patient suffered from postoperative bile leakage. After reoperation, the patient recovered well. The diagnostic and therapeutic problems of such an anomaly are discussed below with the review of the literature.

Masking and reduction of the Galactose-alpha1,3-Galactose (alpha-Gal) epitope, the major xenoantigen in swine, by the glycosyltransferase gene transfection.

The alpha-Gal epitope (Gal-alpha1-3Gal-beta1-4-GlcNAc-R), which is biosynthesized by the UDP-Gal:alpha1-3-galactosyltransferase (alpha1, 3GT), is highly associated with hyperacute rejection in swine to human xenotransplantation. A variety of strategies have been pursued to reduce or eliminate this epitope from swine tissues. Since swine ES cells are not available at present, the targeted knock out of the alpha1,3GT is restricted. Other strategies, such as enzyme competition of the alpha1,3GT with other glycosyltransferases and/or control of sugar processing by the glycosyltransferases, provide a new insight into the downregulation of the alpha-Gal epitope. This review will focus on this type of strategy, which involves a gene transfection of variety of glycosyltransferases as competitors against alpha1,3GT.

Interactions between IL-4, anti-CD40, and anti-immunoglobulin as activators of locomotion of human B cells.

The locomotor properties of small, surface IgM+ and surface IgD+ B cells from the human tonsil were studied using polarization assays and collagen gel invasion assays. These cells gave poor locomotor responses when freshly isolated from the tonsil; but 30 to 40% of the cells polarized and invaded collagen gels after overnight culture in IL-4 or anti-CD40. IL-13 had a similar but weaker effect. Culture with anti-CD40 and IL-4 together gave a higher proportion of polarized cells than either alone, and culture in anti-CD40, IL-4, and anti-IgM gave a still higher proportion (> 60% of B cells polarized). Polarization increased gradually, during hours of culture, unlike the typical rapid response to chemoattractants. We also studied the immediate (< 30 min) effects of chemoattractants on B cell polarization. B cells cultured overnight then washed, but not B cells fresh from the tonsil, polarized immediately in response to anti-CD40. Similar responses to anti-IgM and anti-IgD, both pre- and postculture were also observed, but the response of cultured cells was stronger. IL-4-cultured B cells invaded collagen gels incorporating anti-IgM, anti-IgD, or anti-CD40 in higher numbers than control gels. Most of the invading cells were surface IgM+. These results suggest that locomotor activation in B cells requires two steps. The capacity for locomotion is growth-related and is first activated by IL-4 or by anti-CD40, enhanced by the presence of anti-IgM. Following activation, the cells respond rapidly to chemoattractants such as anti-Ig or anti-CD40.

Antigen-specific chemotaxis of B cells.

The chemoattractant effect of soluble protein antigens for B cells from immunized mice was examined. Mice were immunized either via the footpad with ovalbumin (OVA) in complete Freund's adjuvant (CFA) or with CFA alone; or intraperitoneally with OVA incorporated in immune-stimulating complexes (OVA-ISCOM) or with saline. After 8-14 days B cells were purified from the spleens or the draining popliteal nodes and tested in vitro for locomotor responses to antigen using polarization (shape-change) and filter assays. Cells obtained by both routes of immunization, but not cells from control mice, gave locomotor responses to OVA. Responses were seen in B cells directly after preparation (5-8% of cells responding) but were enhanced if the cells were cultured overnight in the presence of interleukin-4 (IL-4) before testing (10-12% of cells responding). Checkerboard filter assays suggested that the response to OVA was chemotactic. The response was antigen specific since cells from OVA-immunized mice did not respond to bovine serum albumin (BSA), and cells from BSA-immunized mice responded to BSA but not to OVA. The response to OVA was inhibited by preincubation of OVA with anti-OVA but not with anti-BSA. Many of the cells that polarized in response to antigen were larger than any B cells found in control populations suggesting that the responsive cells are those that had been stimulated to enter cell cycle following immunization.

Reduction of the major swine xenoantigen, the alpha-galactosyl epitope by transfection of the alpha2,3-sialyltransferase gene.

alpha2,3-Sialyltransferase represents a putative enzyme that reduces the Galalpha1-3Gal beta1-4GlcNAc-R (the alpha-galactosyl epitope) by intracellular competition with alpha1,3-galactosyltransferase for a common acceptor substrate. This study demonstrates that the overexpression of the alpha2,3-sialyltransferase gene suppresses the antigenicity of swine endothelial cells to human natural antibodies by 77% relative to control cells and by 30% relative to cells transfected with alpha1,2-fucosyltransferase, and in addition, it reduces the complement-mediated cell lysis by 75% compared with control cells and by 22% compared with cells transfected with alpha1, 2-fucosyltransferase. The mechanism by which the alpha-galactosyl epitope was reduced was also studied. Suppression of alpha1, 3-galactosyltransferase activity by 30-63% was observed in the transfectants with alpha2,3-sialyltransferase, and mRNA expression of the alpha1,3-galactosyltransferase gene was reduced as well. The data suggest that the alpha2,3-sialyltransferase effectively reduced the alpha-galactosyl epitope as well as or better than the alpha1, 2-fucosyltransferase did and that the reduction of the alpha-galactosyl epitope is due not only to substrate competition but also to an overall reduction of endogenous alpha1, 3-galactosyltransferase enzyme activity.

Down-regulation of the alpha-Gal epitope expression in N-glycans of swine endothelial cells by transfection with the N-acetylglucosaminyltransferase III gene. Modulation of the biosynthesis of terminal structures by a bisecting GlcNAc.

The down-regulation of the alpha-Gal epitope (Galalpha1,3Galbeta-R) in swine tissues would be highly desirable, in terms of preventing hyperacute rejection in pig-to-human xenotransplantation. In an earlier study, we reported that the introduction of the beta1,4-N-acetylglucosaminyltransferase (GnT) III gene into swine endothelial cells resulted in a substantial reduction in the expression of the alpha-Gal epitope. In this study, we report on the mechanism for this down-regulation of the alpha-Gal epitope by means of structural and kinetic analyses. The structural analyses revealed that the amount of N-linked oligosaccharides bearing the alpha-Gal epitopes in the GnT-III-transfected cells was less than 10% that in parental cells, due to the alteration of the terminal structures as well as a decrease in branch formation. In addition, it appeared that the addition of a bisecting GlcNAc, which is catalyzed by GnT-III, leads to a more efficient sialylation rather than alpha-galactosylation. In vitro kinetic analyses showed that the bisecting GlcNAc has an inhibitory effect on alpha-galactosylation, but does not significantly affect the sialylation. These results suggest that the bisecting GlcNAc in the core is capable of modifying the biosynthesis of the terminal structures via its differential effects on the capping glycosyltransferase reactions. The findings may contribute to the development of a novel strategy to eliminate carbohydrate xenoantigens.