Takuya Tashiro

The specialized iNKT cell system recognizes glycolipid antigens and bridges the innate and acquired immune systems with potential applications for cancer therapy

Invariant NKT (iNKT) cells bridge innate and acquired immunity and play an important role in both protective and regulatory responses. The nature of the response is dictated by the initial cytokine environment: interaction with IL-10-producing cells induces negative regulatory T(h)2/regulatory T cell-type iNKT cells, while that with IL-12-producing cells results in pro-inflammatory T(h)1-type responses. Particularly, in the anti-tumor response, iNKT cells mediate adjuvant activity by their production of IFN-gamma, which in turn activates both innate and acquired immune systems. Thus, upon activation of iNKT cells, both MHC(-) and MHC(+) tumor cells can be efficiently eliminated. On the basis of these mechanisms, iNKT cell-targeted adjuvant cell therapies have been developed and have shown great promise in initial clinical trials on cancer patients.

Spatial Arrangement of Glomerular Molecular-Feature Clusters in the Odorant-Receptor Class Domains of the Mouse Olfactory Bulb

The glomerular layer of the mammalian olfactory bulb (OB) forms odorant receptor (OR) maps. Each OR map is structurally and functionally compartmentalized into zones (dorsal and ventral) and domains (DI and DII in the dorsal zone). We previously reported that glomeruli with similar molecular receptive range properties formed molecular feature clusters at stereotypical positions in the rat OB. However, the spatial arrangement of the molecular feature clusters with regard to the OR zones and domains has not been systematically examined. In this study, we optically mapped the molecular feature clusters of glomeruli within the domain and zone framework of the OB using domain-visible class II GFP transgenic mice. In all mice examined, fatty acid-responsive cluster A was located in the lateral part of domain DI, whereas clusters B, C, and D were arranged in an anterior to posterior order within domain DII. We also found a new cluster of glomeruli that respond to fox odor trimethyl-thiazoline and its structural analogs (heterocyclic odorants that contain sulfur and nitrogen atoms within the ring). This cluster (named cluster J) was located posterior to cluster D within the DII domain. These results show that molecular feature clusters correspond to specific subsets of glomeruli in selective domains of the OR map, suggesting that the molecular feature clusters represent specific ORs that have similar molecular receptive range properties and functional roles.

Synthesis and biological activity of ester and ether analogues of α-galactosylceramide (KRN7000)

Alpha-Galactosylceramide (alphaGalCer, KRN7000) has been identified as a modulator of immunological processes through its capacity to bind iNKT cells mediated by CD1d molecules. Some analogues in while the amide group in alphaGalCer is replaced with ester or ether groups were synthesized from d-arabinitol or l-ribose to evaluate their ability to activate iNKT cells. Ester analogues 30a, 31a, and 61 showed activity for IFNgamma and IL-4 production of iNKT cells, while ether (31b) and 4-methoxy ester (76) analogues of alpha-galactosylceramide were not active for iNKT cells.

The Identification of Attractive Volatiles in Aged Male Mouse Urine

In many species, older males are often preferred mates because they carry good genes that account for their viability. In some animals, including mice, which rely heavily on chemical communication, there is some indication that an animals age can be determined by its scent. In order to identify the attractants in aged male mouse urine, chemical and behavioral studies were performed. We herein show that aged mice have higher levels of 3,4-dehydro-exo- brevicomin (DB), 2-sec-butyl-4,5-dihydrothiazole (BT), and 2-isopropyl-4,5-dihydrothiazole (IT) and a lower level of 6-hydroxy-6-methyl-3-heptanone relative to adult male mice. We also demonstrate that the attraction of females to the odor of male mouse urine is greater when the urine is from aged males. However, the attraction of aged urine odor was offset by the ultrafiltration of adult and aged mouse urine. When DB, BT, and IT were added to adult urine, the attraction of the urine was enhanced. Our results suggest that inbred aged male mice develop an aging odor that is attractive to female mice in an experimental setting and that this attraction is due to increased mouse pheromone signaling.

Structure-activity relationship studies of novel glycosphingolipids that stimulate natural killer T-cells.

KRN7000, an anticancer drug candidate developed by Kirin Brewery Co. in 1995, is an α-galactosyl ceramide. It is a ligand making a complex with CD1d protein, and it stimulates invariant natural killer T (NKT) cells, which are one of the lineages of immunocytes. NKT cells activated by recognition of the CD1d/KRN7000 complex with its invariant T-cell receptor (TCR) can induce both protective and regulatory immune responses. To determine the recognition and activation mechanisms of NKT cells and to develop drug candidates more effective than KRN7000, a large number of analogs of KRN7000 have been synthesized. Some of them show potent bioactivities and have the potential of being utilized as therapeutic agents. In this review, structure-activity relationship studies of novel glycolipids which stimulate NKT cells efficiently are summarized.

Evidence that (+)- endo -Brevicomin is a male-produced component of the Southern Pine Beetle aggregation pheromone

Previous research indicated that the aggregation pheromone of the southern pine beetle, Dendroctonus frontalis, is produced only by females, the sex that initiates attacks. We provide evidence indicating that secondarily arriving males augment mass aggregation by releasing the attractive synergist (+)-endo-brevicomin. Healthy pines artificially infested with both sexes of D. frontalis were significantly more attractive to conspecifics than trees infested solely with females. Coupled gas chromatography-electroantennographic detection (GC-EAD) analyses of volatiles isolated from male beetles revealed substantially greater olfactory sensitivity by D. frontalis to endo-brevicomin than to any other component. The threshold of detection of both sexes for (+)-endo-brevicomin was four orders of magnitude lower than for its antipode and at least one order of magnitude lower than for either enantiomer of frontalin, the major female-produced aggregation pheromone component. Pairing with a female in a gallery stimulated individual male beetles to produce hundreds of nanograms of (+)-endo-brevicomin. (+)-endo-Brevicomin was detected in a small percentage of female D. frontalis, whereas (−)-endo-brevicomin was never detected in either sex. In field trapping bioassays, we confirmed that (+)-endo-brevicomin is a potent synergist for attractive combinations of frontalin and pine turpentine. However, (+)-endo-brevicomin failed to attract D. frontalis either when presented alone or in combination with turpentine. We postulate that mass colonization of host trees by D. frontalis is mediated by distinct semiochemicals from both sexes rather than females alone. Our discovery of a key aggregation pheromone component in such an apparently well-studied species implies that the pheromone models of other bark beetles could benefit from systematic reexamination using newer technologies. Additionally, baits fortified with (+)-endo-brevicomin may enhance pest management strategies that exploit attractants for D. frontalis.

Synthesis of the Enantiomers of 2‐sec‐Butyl‐4,5‐dihydrothiazole and (1R,5S,7R)‐3,4‐Dehydro‐exo‐brevicomin, Pheromone Components of the Male Mouse, Mus musculus

Two components [2-sec-butyl-4,5-dihydrothiazole (1) and 3,4-dehydro-exo-brevicomin (2)] of a male-produced pheromone of the mouse Musmusculus have been synthesized in optically active forms. The enantiomers of 1 were obtained with an enantiomeric purity of ca. 92% ee and were found to be readily racemizable. Asymmetric dihydroxylation was employed as the key reaction (1516) allowing the preparation of (1R,5S,7R)-2 with ca. 94% ee.

Generation of functional NKT cells in vitro from embryonic stem cells bearing rearranged invariant Vα14-Jα18 TCRα gene

Establishment of a system with efficient generation of natural killer T (NKT) cells from embryonic stem (ES) cells would enable us to identify the cells with NKT-cell potential and obtain NKT cells with desired function. Here, using cloned ES (NKT-ES) cells generated by the transfer of nuclei from mature NKT cells, we have established a culture system that preferentially developed functional NKT cells and also identified early NKT progenitors, which first appeared on day 11 as a c-kit(+) population in the cocultures on OP9 cells with expression of Notch ligand, delta-like1 (OP9/Dll-1) and became c-kit(lo/-) on day 14. Interestingly, in the presence of Notch signals, NKT-ES cells differentiated only to thymic CD44(lo) CD24(hi) NKT cells producing mainly interleukin-4 (IL-4), whereas NKT cells resembling CD44(hi) CD24(lo) liver NKT cells producing mainly interferon gamma (IFN-gamma) and exhibiting strong adjuvant activity in vivo were developed in the switch culture starting at day 14 in the absence of Notch. The cloned ES culture system offers a new opportunity for the elucidation of the molecular events on NKT-cell development and for the establishment of NKT-cell therapy.

Induction of Th1-biased cytokine production by α-carba-GalCer, a neoglycolipid ligand for NKT cells

NKT cells are characterized by their production of both T(h)1 and T(h)2 cytokines immediately after stimulation with alpha-galactosylceramide (alpha-GalCer), which is composed of alpha-galactopyranose linked to ceramide (itself composed of sphingosine and fatty-acyl chains); the chain length of the ceramide varies and this affects the ability of alpha-GalCer to stimulate cytokine production. However, the contribution of its galactopyranose sugar moiety remains unclear. We synthesized alpha-carba-GalCer, which has an alpha-linked carba-galactosyl moiety; here, the 5a-oxygen atom of the D-galactopyranose ring of alpha-GalCer is replaced by a methylene group. The alpha-carba-GalCer was more stable and showed higher affinity to the NKT receptor. It thus enhanced and prolonged production of IL-12 and IFN-gamma compared with alpha-GalCer, resulting in augmented NKT cell-mediated adjuvant effects in vivo. The alpha-carba-GalCer, which has an ether linkage, was more resistant to degradation by liver microsomes than was alpha-GalCer, which has an acetal bond. Modulation of the sugar moiety in glycolipids might therefore provide optimal therapeutic reagents for protective immune responses against tumor or pathogens.

Useful Reactions in Modern Pheromone Synthesis

Various aspects of pheromone synthesis are reviewed by analyzing examples published between 1990 and early 2003. Syntheses executed with new methodologies such as organoborane reactions, organotransition metal chemistry including olefin metathesis, asymmetric epoxidation, asymmetric dihydroxylation, other asymmetric chemical processes, and stereoselective biocatalysis are selected to illustrate the usefulness of new reactions in pheromone synthesis.