On the safety assurance of cell processing carried out in medical institutions for autologous immuno-cell therapy

Autologous immuno-cell therapy of cancer has been mainly carried out in university hospitals as therapeutic research. Recently, private clinics specialized in the therapy are appearing in Japan. At present, cell processing necessary for the therapy is carried out not by pharmaceutical companies but by medical institutions. In this case, there is no government regulation on the safety assurance of the procedure. For the wholesome popularization of the therapy, however, it is of utmost importance to carry out cell processing under safety assurance, which could be done as self-regulation by medical institutions. A guideline for safety assurance tentatively set by Seta Clinic Group, which is a group of clinics specialized in the therapy and its usefulness in protecting patients from hazardous accidents are shown.     

In vivo and in vitro interactions of the Bombyx mori chymotrypsin inhibitor b1 with Escherichia coli

Various chymotrypsin inhibitors occur in the hemolymph of silkworm larvae. Interaction of chymotrypsin inhibitor b1 (CI-b1) with Escherichia coli was examined from the viewpoint of action against invading bacteria. Injection of dead E. coli cells into larva reduced the CI-b1 content of the hemolymph, suggesting in vivo binding of CI-b1 to the outer membrane of the cell. Results from incubation of E. coli in cell-free hemolymph in the presence or absence of lipopolysaccharide indicated that CI-b1 is the only CI bound to E. coli and that it interacts with lipopolysaccharide. CI-b1 formed a complex with lipopolysaccharide in vitro; the value of the dissociation constant was relatively large. Inhibitory activity of CI-b1 changed insignificantly in mixture with lipopolysaccharide. CI-b1 affected the growth of E. coli but never worked lethally. CI-b1 is speculated to be a mediator that scavenges intruding bacteria rather than a direct anti-bacterial factor. This is the first report confirming that CI-b1 is a lipopolysaccharide binding protein.     

Is death-feigning adaptive? Heritable variation in fitness difference of death-feigning behaviour.

The adaptation of death-feigning (thanatosis), a subject that has been overlooked in evolutionary biology, was inferred in a model prey-and-predator system. We studied phenotypic variation among individuals, fitness differences, and the inheritance of death-feigning behaviour in the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Two-way artificial selections for the duration of death-feigning, over 10 generations, showed a clear direct response in the trait and a correlated response in the frequency of death-feigning, thus indicating variation and inheritance of death-feigning behaviour. A comparison of the two selected strains with divergent frequencies of death-feigning showed a significant difference in the fitness for survival when a model predator, a female Adanson jumper spider, Hasarius adansoni Audouin (Araneomophae: Salticidae), was presented to the beetles. The frequency of predation was lower among beetles from strains selected for long-duration than among those for short-duration death-feigning. The results indicate the possibility of the evolution of death-feigning under natural selection.     

Sex-specific effects of carotenoid intake on the immunological response to allografts in guppies (Poecilia reticulata)

Rarely are the evolutionary origins of mate preferences known, but, recently, the preference of female guppies (Poecilia reticulata) for males with carotenoid-based sexual coloration has been linked to a sensory bias that may have originally evolved for detecting carotenoid-rich fruits. If carotenoids enhance the immune systems of these fishes, as has been suggested for other species, this could explain the origin of the attraction to orange fruits as well as the maintenance of the female preference for orange males. We used the classic immunological technique of tissue grafting to assay a component of the immune response of guppies raised on two different dietary levels of carotenoids. Individual scales were transplanted between pairs of unrelated fishes, creating reciprocal allografts. Transplanted scales were scored on a six-point rejection scale every day for 10 days. Five days later, the same pairs of fishes received a second set of allografts and were scored again. Compared with low-carotenoid-diet males, high-carotenoid-diet males mounted a significantly stronger rejection response to the second allograft but not to the first allograft. High-carotenoid-diet females, however, showed no improvement in graft rejection compared with low-carotenoid-diet females. To our knowledge, this is the first experimental evidence for sex-specific effects of carotenoid consumption on the immune system of a species with carotenoid-based sexual coloration. These results are consistent with the hypothesis that the mate preference for carotenoid coloration is maintained by the benefits to females of choosing healthy mates, but they cast doubt on the idea that the benefits of carotenoid consumption, per se, could account for the origin of the preference. The sex-specificity of carotenoid effects on allograft rejection in guppies provides indirect support for the general hypothesis that males pay an immunological cost for sexual ornamentation.     

Accumulation of pathogenic DeltamtDNA induced deafness but not diabetic phenotypes in mito-mice

Mito-mice carrying various proportions of deletion mutant mtDNA (DeltamtDNA) were generated by introduction of the DeltamtDNA from cultured cells into fertilized eggs of C57BL/6J (B6) strain mice. Great advantages of mito-mice are that they share exactly the same nuclear-genome background, and that their genetic variations are restricted to proportions of pathogenic DeltamtDNA. Since accumulation of DeltamtDNA to more than 75% induced respiration defects, the disease phenotypes observed exclusively in mito-mice carrying more than 75% DeltamtDNA should be due to accumulated DeltamtDNA. In this study, we focused on the expressions of hearing loss and diabetic phenotypes, since these common age-associated abnormalities have sometimes been reported to be inherited maternally and to be associated with pathogenic mutant mtDNAs. The results showed that accumulation of exogenously introduced DeltamtDNA was responsible for hearing loss, but not for expression of diabetic phenotypes in mito-mice.     

Importance of the converter region for the motility of myosin as revealed by the studies on chimeric Chara myosins

A long alpha-helix in myosin head constitutes a lever arm together with light chains. It is known from X-ray crystallographic studies that the first three turns of this lever arm alpha-helix are inserted into the converter region of myosin. We previously showed that chimeric Chara myosin in which the motor domain of Chara myosin was connected to the lever arm alpha-helix of Dictyostelium myosin had motility far less than that expected for the motor domain of Chara myosin. Here, we replaced the inserted three turns of alpha-helix of Dictyostelium myosin with that of the Chara myosin and found that the replacement enhanced the motility 2.6-fold without changing the ATPase activity so much. The result clearly showed the importance of interaction between the converter region and the lever arm alpha-helix for the efficient motility of myosin.     

Factors affecting morphogenesis of rabbit gallbladder epithelial cells cultured in collagen gels

Although peptide growth factors play an important role in the morphogenesis of gallbladder, little is known about how they effect the morphogenesis of gallbladder epithelial cells. Rabbit gallbladder epithelial cells (RGEC) were isolated and cultured in monolayer or collagen gels. Epidermal growth factor (EGF), hepatocyte growth factor (HGF), epimorphin, transforming growth factor-beta 1 (TGF-beta 1), and fibroblast-conditioned medium (FCM) were added to the cultured cells to clarify the effects of these peptides and FCM on morphogenesis of RGEC. RGEC suspended in collagen gels form spherical cysts with morphologic polarity. EGF, HGF, epimorphin, and FCM promoted cyst maturation by accelerating the proliferation and aggregation of clear, polarized vesicles. In contrast, TGF-beta 1 markedly inhibited DNA synthesis in both monolayer and collagen gel cultures and promoted formation of branching structures in collagen gels. Furthermore, in the presence of EGF, TGF-beta 1 induced a drastic change in morphogenesis, with the formation of branching networks that showed cell-cell contact only at sites where branches touched. RGEC-forming multicellular cysts did not express vimentin but expressed significant amounts of cytokeratin and regained junctional complexes. In contrast, TGF-beta 1-treated cells strongly expressed vimentin along with branching structures and showed decreases in cytokeratin expression and junctional complexes. Thus, TGF-beta 1 induces a mesenchyme-like cell shape accompanied by cytoskeletal molecular changes, with loss of both epithelial polarization and junctional complexes. These results suggest that the morphogenetic program of RGEC is likely to be determined by the interaction of these peptides and the timing of their presence.     

Ovarian follicular development stimulated by leuprorelin acetate plus human menopausal gonadotropin in chimpanzees

We attempted ovarian stimulation using gonadotropins in 14 chimpanzees. Subjects were given a single administration of leuprorelin acetate, followed by repeated administration of human menopausal gonadotropin (hMG) for 16-21 days. During the dosing period, the ovarian follicle diameter and count were measured by transvaginal ultrasonography. The hormone administration induced the development of multiple follicles, and multiple oocytes were subsequently retrieved. However, the follicle count was decreased, suggesting atresia, in some subjects. Statistically, the final follicle diameter was dependent on the dosing duration and the hMG dose in the late stage, while the maximum follicle count during hMG administration was dependent on age and the hMG dose in the early stage. Five subjects showed mild ovarian hyperstimulation syndrome (OHSS)-like symptoms with a high serum estradiol (E2) concentration. These results suggest that leuprorelin acetate plus hMG administration successfully stimulates the development of multiple ovarian follicles for oocyte retrieval and that the serum E2 concentration is predictive of OHSS-like symptoms in chimpanzees.