A Potent CD1d-binding Glycolipid for iNKT-Cell-based Therapy Against Human Breast Cancer

Background/Aim: Invariant natural killer T-cells (iNKT) stimulated by CD1d-binding glycolipids have been shown to exert antitumor effects by a number of studies in a mouse model. Breast cancer is a devastating disease, with different types of breast cancer recurring locally or distant as metastatic/advanced disease following initial treatment. The aim of this study was to examine the tumoricidal effect of a CD1d-binding glycolipid, called 7DW8-5, against a highly invasive human breast cancer cell line both in vitro and in vivo. Materials and Methods: Parental MDA-MB-231 cells and MDA-MB-231 cells transduced with human CD1d were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), followed by loading with glycolipids. After co-culturing with human iNKT cells, the cells were permeabilized and stained with Alexa Flour 647-conjugated antibody to active caspase-3, and analyzed using a BD LSR II. For the in vivo tumoricidal effect, MDA-MB-231 cells transduced with human CD1d and luciferase genes were injected into the mammary fat pad of female NOD/SCID/IL2rγnull (NSG) mice, followed by the injection of human iNKT cells with or without 7DW8-5, and the levels of luminescence were analyzed with whole-body imaging. Results: Human iNKT cells could kill CD1d-expressing human breast cancer cells in vitro in the presence of 7DW8-5, but not α-GalCer. As for in vivo, the adoptive transfer of human iNKT cells into tumor-challenged NSG mice significantly inhibited the growth of CD1d+ MDA-MB-231 human breast cancer cells in the presence of 7DW8-5. Conclusion: CD1d-binding, glycolipid-based iNKT-cell therapy is suggested as a potent and effective treatment against breast cancer in humans

Influence of Circumferential Flaw Length on Internal Burst Pressure of a Wall-Thinned Pipe

This paper examines the effect of the circumferential angle of a flaw θ on the internal burst pressure pf of pipes with artificial wall-thinned flaws. The effect of θ has conventionally been regarded as unimportant in the evaluation of the pf of wall-thinned straight pipes. Therefore, a burst pressure equation for an axial crack inside a cylinder (Fig. 1, left), such as Kiefner’s equation (Kiefner et al., 1973), has been widely applied (ANSI/ASME B31.G., 1991; Hasegawa et al., 2011). However, the following implicit assumptions notably exist when applying the equation to planar flaws in situations with non-planar flaws. 1) The fracture mode of the non-planar flaw under consideration is identical to that of the crack. 2) The effect of θ on pf, which is not considered for an axial crack, is small or negligible. However, the experimental results from the systematic burst tests for carbon steel pipes with artificial wall-thinned flaws examined in this paper showed that these implicit assumptions may be incorrect. In this paper the experimental results are evaluated in further detail. The purpose of the evaluation was to clarify the effect of θ on pf. Specifically, the significance of the flaw configuration (axial length δz and wall-thinning ratio t1/t) was studied for its effects on θ　and pf. In addition, a simulation of this effect was conducted using a large strain elastic-plastic Finite Element Analysis (FEA) model. As observed from the experimental results, θ　tended to affect pf in cases with large δz, and t1/t was also correlated with a decrease in pf with an increase in θ. These tendencies were successfully simulated by the large strain elastic-plastic FEA model. The observed effects demonstrate that the burst pressure predicted for a crack with identical ligament thickness decreases with an increase in θ, so that the effect of θ on pf should be taken into consideration when evaluating pf

Influence of Circumferential Flaw Length on Internal Burst Pressure of a Wall-Thinned Pipe

This paper examines the effect of the circumferential angle of a flaw θ on the internal burst pressure pf of pipes with artificial wall-thinned flaws. The effect of θ has conventionally been regarded as unimportant in the evaluation of the pf of wall-thinned straight pipes. Therefore, a burst pressure equation for an axial crack inside a cylinder (Fig. 1, left), such as Kiefner’s equation (Kiefner et al., 1973), has been widely applied (ANSI/ASME B31.G., 1991; Hasegawa et al., 2011). However, the following implicit assumptions notably exist when applying the equation to planar flaws in situations with non-planar flaws. 1) The fracture mode of the non-planar flaw under consideration is identical to that of the crack. 2) The effect of θ on pf, which is not considered for an axial crack, is small or negligible. However, the experimental results from the systematic burst tests for carbon steel pipes with artificial wall-thinned flaws examined in this paper showed that these implicit assumptions may be incorrect. In this paper the experimental results are evaluated in further detail. The purpose of the evaluation was to clarify the effect of θ on pf. Specifically, the significance of the flaw configuration (axial length δz and wall-thinning ratio t1/t) was studied for its effects on θ　and pf. In addition, a simulation of this effect was conducted using a large strain elastic-plastic Finite Element Analysis (FEA) model. As observed from the experimental results, θ　tended to affect pf in cases with large δz, and t1/t was also correlated with a decrease in pf with an increase in θ. These tendencies were successfully simulated by the large strain elastic-plastic FEA model. The observed effects demonstrate that the burst pressure predicted for a crack with identical ligament thickness decreases with an increase in θ, so that the effect of θ on pf should be taken into consideration when evaluating pf

Cytoskeletal inhibitors, anti-adhesion molecule antibodies, and lectins inhibit hepatocyte spheroid formation.

We investigated the role of cytoskeletons, adhesion molecules, membrane-glycosylations, and proteoglycans in forming the shape of adult rat hepatocyte spheroids. Isolated hepatocytes were cultured on dishes coated with chondroitin sulfate phosphatidyl ethanolamine (CS-PE). Spheroid-forming ability was observed after adding cytoskeletal inhibitors (cytochalasin D, colchicine, okadaic acid, mycalolide B), anti-adhesion molecule antibodies (anti-E-cadherin, anti-connexin 32, anti-zo-1), a glycosphingolipid synthetic inhibitor (N-butyldeoxynojirimycin), a proteoglycan synthetic inhibitor (p-nitrophenyl-beta-D-xylopyranoside), and several lectins. Localization of actin was studied using confocal microscopy after rhodamine-phalloidin staining. Adding cytoskeletal inhibitors on the initial day resulted in weakly clustered cell aggregates rather than smoothly formed spheroids. These effects disappeared at lower reagent concentrations. When reagents were added on day 3, after the formation of spheroids, only mycalolide B was associated with an irregular spheroid surface; the others had no effect. Adding the anti-E-cadherin, anti-connexin 32 on the initial day showed inhibition of spheroid formation, but anti-zo-1 and proteoglycan synthetic inhibitor had no effects. Among the several lectins, only Wheat Germ Agglutinin (WGA), Ricinus communis Agglutinin I (RCA-I), and Concanavalin A (ConA) showed inhibition. These results suggest that cytoskeletal conformation and some adhesion molecules are necessary to form spheroids. Based on the interactions between lectins and hepatocytes in the present study, hepatocytes appear to contain an N-linked complex or N-linked hybrid glycosylated chains

Synthesis of C6′′-modified α-C-GalCer analogues as mouse and human iNKT cell agonists

alpha-GalCer analogues that combine known Th1 polarizing C6''-modifications with a C-glycosidic linkage were synthesized. We employed a protecting group strategy that allowed the preparation of both saturated and unsaturated derivatives with variable C6''-substituents. Selected analogues demonstrate promising activity in mice. Interestingly, the introduction of a 6''-O-pyridinylcarbamoyl substituent to alpha-C-GalCer restores its antigenicity in human iNKT cells

Combination of epidermal growth factor and insulin is required for multicellular spheroid formation of rat hepatocytes in primary culture.

We showed that the combination of epidermal growth factor (EGF) and insulin is an essential supplement to Williams' #E medium for the formation of floating multicellular spheroids in primary culture of rat hepatocytes. Isolated hepatocytes assembled to form floating multicellular spheroids within 96 h through transient assembly of monolayer islands within the initial 24 h in dishes coated with liver-derived proteoglycans. However, the assembly of multicellular spheroids was severely suppressed in the absence of either EGF or insulin. The reduction of spheroid assembly was correlated with decreased attachment and subsequent decreased formation of monolayer islands within 24 h. The minimum amounts of EGF and insulin required for the formation of floating spheroids were 1 ng/ml and 0.4 microgram/ml, respectively. These results suggest that the enhancement of hepatocyte attachment provided by the combination of EGF and insulin during the early phase of culture is required for the formation of floating spheroids

Efficient divergent synthesis of new immunostimulant 4″-modified α-galactosylceramide analogues

A synthesis strategy for the swift generation of 4"-modified alpha-galactosylceramide (alpha-GalCer) analogues is described, establishing a chemical platform to comprehensively investigate the structure activity relationships (SAR) of this understudied glycolipid part. The strategy relies on a late-stage reductive ring-opening of a p-methoxybenzylidene (PMP) acetal to regioselectively liberate the 4"-OH position. The expediency of this methodology is demonstrated by the synthesis of a small yet diverse set of analogues, which were tested for their ability to stimulate invariant natural killer T cells (iNKT) in vitro and in vivo. The introduction of a p-chlorobenzyl ether yielded an analogue with promising immunostimulating properties, paving the way for further SAR studies